CONTENTS EDITORIAL................................................................................................................4 INTRODUCTION - DEPARTMENT OF NEUROLOGY..........................................................................5 ■ ORIGINAL RESEARCH THE PROMOTOR POLYMORPHISM OF MACROPHAGE METALLOELASTASE IN MULTIPLE SCLEROSIS..........................11 Hladíková M., Štourač P., Benešová Y.Tschoplová S. PAINFUL SENSORY NEUROPATHIES INTHE ELDERLY.......................................................................16 Vlčková-Moravcová E., BednaříkJ. THE RESULTS AND CONTRIBUTION OF ELECTROPHYSIOLOGICAL EXAMINATION IN PATIENTS WITH LUMBAR SPINAL STENOSIS.............................................................................38 Mičánková Adamová B.,Voháňka S. THE VALUE OF INTRATHECAL MRZ REACTION AND OLIGOCLONAL IgG BANDS FOR DISCRIMINATION BETWEEN THE PRIMARY PROGRESSIVE AND RELAPSING REMITTING COURSES OF MULTIPLE SCLEROSIS...............................46 Štourač P., Bednářová J., Hladíková M., Praksová P., Benešová Y, Kontrová I. ■ REVIEWS SPONDYLOTIC CERVICAL MYELOPATHY: WHAT IS THE BEST APPROACH TO TREATMENT?.....................................6 Kadaňka Z., BednaříkJ. CRITICAL ILLNESS POLYNEUROMYOPATHY - A WELL-KNOWN BUT STILL CONTROVERSIAL ENTITY...........................27 BednaříkJ., Vondráček P. ■ CASE REPORTS NON-JEWISH CHILD WITH CANAVAN DISEASE.............................................................................52 Slouková E., Ošlejšková H., Šoukalová J., Masaříková H. ■ AUTHOR AND REVIEWER PACK 57 VíERS^ \ fr ^ NA ** MASARYK UNIVERSITY J ALUMNI DAY 30.5.™ ÖGETHER SENIOR PARTNERS: Bvn Cesmqmoiumský ja Horavostav Brno (j£) s^fi&fíľ DNES ©«ÄÍÄ. ^:p OHLZS www.absolventi.muni.cz/en EDITOR'S OFFICE SCRIPTA MEDICA is indexed in SCRIPTA MEDICA Editor-in-Chief M. Korcová Faculty of Medicine Masaryk University Kamenice 5 CZ-625 00 Brno, Czech Republic Tel.: +420 549 493 998 E-mail: scriptme@med.muni.cz mkorcova@med.muni.cz muni PRESS Publisher: Faculty of Medicine, Masaryk University, Brno Periodicity: 4 issues per volume The journal Scripta Medica is printed in 500 copies. ri helbichtiskárna Printed by Tiskárna Helbich, a.s., Brno I me | to | da. Art studio: Metoda, s.r.o., Brno Photos: LiborTeplý (pages 2,3,57) The Faculty of Medicine uses the traditional symbol of the serpent-entwined staff of Asclepius. This also forms the head of its mace. Photos: Jaroslav Winkler, Faculty of Medicine, Masaryk University Advertising is accepted in the journal. Further information including rates, specification, etc. is available on request. ISSN 1211-3395 MKČRE4996 Scripta Medica is available free online at http:/www.med. muni.cz/biomedjournal/ EMBASE/Excerpta Medica DIALOG DataStar MEDLINE LEBSCO ProQuest Scopus PubMed Bibliographia Medica Čechoslovaca Online Computer Library Center, Inc. Deutsches Institut für Medizinische Dokumentation und Information SCRIPTA MEDICA/Volume 82/No. 1/2009 http://www.med.muni.cz/biomedjournal/ EDITORIAL BOARD Editorial Assistant Jitka Vavrouchová Zdeňka Dohnálková Nataša Honzíkova Martina Kukletová Daniel Schwarz i 'JindřichVomela International Advisory Board Section Experimental Medicine F. Čiampor, Bratislava, Slovakia K. Javorka, Martin, Slovakia E. Kellerová, Bratislava, Slovakia M. V. Nermut, Herts., South Mimms, UK J. Stingl, Praha, Czech Republic Surgical Medicine J. Dominik, Hradec Králové, Czech Republic S. Haruštiak, Bratislava, Slovakia V. Král, Olomouc, Czech Republic J. Šnajdauf, Praha, Czech Republic Internal Medicine M. Benedeková, Bratislava, Slovakia Z. Pelikán, Breda,The Netherlands J. Rovenský, Piešťany, Slovakia F. Stožický, Plzeň, Czech Republic Foreign Language Editor Ladislav Červený ZdeněkAdam Petr Dubový Hana Hrstková . Jindřich Lokaj ' Jaromír Sláma Editor-in-Chief Marie Korcová Managing Editor Svatopluk Čech Online Presentation Zbyněk Diviš EDITORIAL BOARD Svatopluk Čech PetrGál Roman Janisch Marie Nováková Anna Vašků http://www.med.muni.cz/biomedjournal/ SCRIPTA MEDICA/Volume 82/No. 1/2009 3 EDITORIAL Ladies and gentlemen, Scripta Medica, the scientific journal of the Faculty of Medicine of Masaryk University, has been published since 1922. The journal has been an important source of scientific literature based on exchange services with medical schools and other scientific institutes abroad. The history of the journal is too long to be able to describe everything important. During the history of the journal we have learnt that a good communication strategy is an absolute condition of success for a scientific journal. Today the journal Scripta Medica enters a new period of its existence. We have learnt from the mistakes and found out that we have established tradition, research experience, work skills and practice, and reasonable chances to be successful. We are aware of the heritage of our past and we choose the best of it. We do not want to focus on publishing scientific information only, but we want to pay attention to scientific communication, scientific research, scientific congresses.and all events leading up to better understanding of medical science research. We appreciate your comments as well as we appreciate good communication and cooperation with you. We believe in closer cooperation in the international field of science. It is with great pleasure that we introduce to you a new layout ofthe journal butalsoa new communication strategy.We wish you were our readers, authors, reviewers, and critics.The journal is quarterly, with four issues in a year. The scientific standard of published papers is guaranteed by the Editorial Board. The journal is addressed to scientists not only in the Czech Republic butall over the world. We use the English language as a scientific language and we believe that we will be able to speak to scientists from different disciplines in all aspects. We wish to be a journal widely opened to all research workers for scientific investigation - medical and biomedical research, laboratory research, and experimental work. We are prepared to become a communication channel joining research workers from every branch of biological medicine. Our targets are ambitious. Our journal is now changing into a modern scientific journal in all aspects, with changes in its title page, size and graphic layout, taking heed ofthe quality of its papers and reviews, and also changing its distribution. The journal is distributed free again, but we have given priority to explicit addressing. The journal Scripta Medica is printed in a number of 500 copies. Ladies and gentlemen, We are honoured by the fact that in 2008 the journal Scripta Medica Facultatis Medicae Universitatis Brunensis Masaryki-anae was included bythe Research and Development Council in the List of Reviewed Non-impacted Periodicals published in the Czech Republic. Marie Korcová 4 SCRIPTA MEDICA/Volume 82/No. 1/2009 http://www.med.muni.cz/biomedjournal/ Prof. MUDr. Josef Bednařík, CSc. Department of Neurology: Twenty years' history and present state The Department of Neurology was founded and started its work on 1st September 1989 as one of the core departments of the new Faculty Hospital of Brno-Bohunice, when the new 17-floor building was opened. The first head of the department was Assoc. Prof. MUDr. Pavel Strnad, CSc. The new department was equipped with modern technology, especially for electrodiagnostics. An intensive care unit as part of a neurological department was very rare at that time and its focus on vascular neurology was pioneering. After a short period of provisional heading by the deputy head, MUDr. Ladislav Ondroušek, the department had been headed by Prof. MUDr. Zdeněk Kadaňka, CSc, since 1st January 1993, and after his retirement it has been headed by Prof. MUDr. Josef Bednařík, CSc, since September 2007. The current deputy heads are MUDr. Stanislav Voháňka, CSc. (deputy for treatment) and Assoc Prof. MUDr. Pavel Štourač, Ph.D. (deputy for science and education). The Department of Neurology nowadays has one 6-bed intensive care unit, one 6-bed stroke unit and two general wards, each with 24 beds, specialised electrophysiological INTRODUCTION - DEPARTMENT OF NEUROLOGY MUDr. Stanislav Voháňka, CSc, MBA laboratories for electromyography, evoked potentials, electroencephalography, ultrasonography and sleepanalysis.and an outpatient neurological department with 5 ambulances. Specialised care is concentrated into several dedicated centres for the treatment of multiple sclerosis, sleep disorders, neuromuscular disorders, spondylogenic disorders, and bot-ulotoxin treatment for movement disorders. The working team consists of 28 doctors, 2 psychologists, one speech therapist-aphasiologist, 53 nurses, 9 hospital attendants, 2 physiotherapists, 2 secretaries, and 1 data manager. The Department of Neurology is involved in several national and international grants and research projects. The neurologists are members of several society committees including the committee of the Czech Neurological Society (Prof. Bednařík is the vice-chairman). Dr. Voháňka is the current chairman of the Czech Neuromuscular Society and Prof. Bednařík is the editor-in-chief of the Czech and Slovak Neurology and Neurosurgery (the impacted official journal of the Czech and Slovak Neurological and Neurosurgical Societies). Prof. MUDr. Josef Bednařík, CSc. MUDr. StanislavVoháňka, CSc, MBA http://www.med.muni.cz/biomedjournal/ SCRIPTA MEDICA/Volume 82/No. 1/2009 5 00 REVIEW SPONDYLOTIC CERVICAL MYELOPATHY: WHAT IS THE BEST APPROACH TO TREATMENT? Kadaňka Z., Bednařík J. Department of Neurology, Faculty of Medicine, Masaryk University and Faculty Hospital Brno Received after revision February 2009 KEYWORDS Spondylotic cervical myelopathy Spinal cord compression MR hyperintensities Cross-sectional transverse area ABSTRACT The authors address the daunting challenge of the treatment of patients with spondylotic cervical myelopathy (SCM). It is emphasised that clinical diagnosis must be made in the light of all available information from the history of the disease and from objective neurological investigation, and that everything must be done with reference to the paraclinical findings (imaging, electrophysiological, serological, etc.). Decision-making must then be based on evaluation of the cross-sectional transverse area of the compressed spinal cord, the number of compressed levels and its significance, hyperintensities in the spinal cord at the level of maximal compression, duration of symptoms, and the overall clinical status (including mental condition). Practical recommendations for the treatment of SCM conclude the article. H CORRESPONDING AUTHOR Kadaňka Z. Department of Neurology Faculty of Medicine Masaryk University and Faculty Hospital Brno Jihlavská 20 625 00 Brno Czech Republic INTRODUCTION Although a large amount of information has been published about spondylotic cervical myelopathy (SCM), in everyday clinical practice many doctors are still hesitant to commit themselves to a treatment regime.The radiological finding of a narrow spinal canal, or even spinal cord compression, often proves too strong a temptation to solve the problem radically "as soon as possible","not wasting valuable time" regardless of any facts arising out of evidence-based medicine, or even of common sense. Overemphasis on one finding irrespective of a non-corresponding clinical picture often verges on desperate endeavour to disclose immediately the cause of the patient's signs and symptoms. How is this challenge best addressed? The clinical diagnosis must take into full account all the information contained in the history of the disease and the objective neurological investigation, and everything must be done in concordance with the paraclinical findings (imaging, electrophysiological, serological, etc.). If this is done, however, other difficulties must be overcome in the process of making a decision as to which treatment to opt for. The following items should be evaluated: SCRIPTA MEDICA/Volume 82/No. 1/2009 http://www.med.muni.cz/biomedjournal/ 1. The cross-sectional transverse area of the compressed spinal cord 2. The number of compressed levels and its significance 3. Hyperintensities versus normal MR spinal cord image at the level of maximum compression 4. Duration of symptoms 5. Overall clinical status (including mental condition) Clinical features Symptoms of spondylotic cervical myelopathy (SCM) most frequently begin in patients between 50 and 70 years of age, but may occur much earlier or in more advanced years. The patients tend to visit their doctor at 50-55 (1-4). Men are affected more frequently (2-3:1) than women [1,3,4], Symptoms may develop progressively or in stepwise fashion, with remissions between periods of deterioration. Symptoms and signs may arise for the first time or be aggravated following injuries such as carrying an excessive weight, a fall on a slippery surface, a motor vehicle accident, or forced hyper-extension of the neck. The most frequent and characteristic symptoms are gait disorders (82 %), clumsy hands (84 %), and neck and back pain (95 %). Pain in the neck (67 %), shoulder, and arms is a common presenting complaint [1]. Pain may radiate in a radicular distribution and is usually dermatomic. However, it may occasionally occur in the distribution of the affected myotome. Paresthesias (78 %), fasciculations in the upper extremities (32 %), and muscle weakness in the distribution of the affected nerve roots (30%) are often encountered. Reduction in the reflexes may be observed in the upper extremities, but in the lower extremities it may be associated with hyperactive reflexes (60%) [1 ]. Although the patient may complain of only unilateral lower extremity symptoms, neurological examination usually reveals signs of bilateral disturbance of long tract function. Spasticity (62%) is one typical sign, and suddenly stretching legs may be reported. Sensory complaints, especially pain in the lower extremities, may be misleading. Lhermitte's sign is very specific, though of low sensitivity (14-27%) [5,1]. Disturbances of sphincter function (24%) are late phenomena; they are usually mild and generally do not occur in the absence of advanced spinal cord dysfunction, which manifests itself earlier by dysfunction of other modalities [1], The duration of signs and symptoms varies considerably with personal history. It may last from several hours through many (25-35) years, but typically occurs over one or several years (1-4). Hyperintensities MRI is an excellent means to visualise the narrowed spinal canal associated with spinal cord compression due to degeneration of the spine (Figure 1). New technology, such as SPONDYLOTIC CERVICAL MYELOPATHY... Figure 1 An MR proton density image in the sagittal plane; spondylogenic multistage spinal cord compression of CD-7 by massive protrusion of intervertebral disc multi-array coils, provides high-resolution sagittal images of the entire cord in a single scan and fast spin-echo sequences produce high-resolution T2WI within a short time [12], MRI can reveal cord signal abnormalities on T2WI at (or near) the site of compression, which may signal evidence of oedema, demyelinisation, gliosis, or myelomalacia. The clinical significance of increased signal intensity on MR scans is controversial. Some studies have found a negative correlation between the outcome of surgery or conservative treatment and increased signal intensity (6-9), but others have not [11], or have even noted a positive correlation (11). Some found this relation predictive only in cases with multilevel manifestation [13] or when a low signal on T1WI and a high signal onT2WI were combined [14]. Those that found no correlation consider the reason to be that hyperintensities chiefly indicate lesions in the grey matter that do not give rise to spastic paralysis and are, therefore, not significantly related to patients'disabilities. Decompressive surgery can result in rapid http://www.med.muni.cz/biomedjournal/ SCRIPTA MEDICA /Volume 82/No. 1/2009 7 SPONDYLOTIC CERVICAL MYELOPATHY.. Inpatient clinic - doctors room resolution of imaging abnormalities, but clinical improvement does not always accompany these changes [15]. In our studies comparing conservative treatment with surgery the appearance of increased signal intensity in the cervical spinal cord did not predict the outcome in either type of treatment [16,17]. Cross-sectional transverse area at the level of maximal compression In some cases, the spinal cord does not lose its function even under severe compression. It has been well known from animal experiments that spinal cord function may be considerably resistant to compression [18]. Moreover, the critical degree of spinal cord compression required to induce pathological changes in clinical practice remains unknown [19]. This renders matters difficult, since radiological findings for spinal cord compression, particularly if mild, may be clinically relevant or merely a coincidental anomaly that has little or nothing to do with the symptoms reported by the individual patient. This situation is far from unfamiliar in healthy subjects aged 18 to 72 years; degenerative changes, especially in the cervical region, are present on MRI in 64% of them and are associated with cord impingement in only 11 % [12]. If only subjects aged 64 or more are taken into account, cord impingement has been observed in 26%, and cord compression in 7% [12]. The percentage of cord area reduction, however, has never exceeded 16%. No significant difference in cord cross-sectional areas between those with degenerative changes and those without them has been observed. This could mean that direct and pronounced compression is necessary to evoke signs of spinal cord dysfunction [12], To identify a clinically significant degree of compression, already associated with corresponding clinical symptoms or for the identification of patients suitable for active treatment, it is essential to establish the critical value of such compression, or other circumstances such as hyperintensities, number of compressed levels, etc. Although in population studies there exists a correlation between a narrow sagittal diameter of the spinal canal and SCM, there is a considerable degree of overlap between the frequency histograms for the minimum anteroposterior diameter of the asymptomatic population and those with SCM [21]. An acquired anteroposterior diameter of the spinal SCRIPTA MEDICA/Volume 82/No. 1/2009 http://www.med.muni.cz/biomedjournal/ ■ SPONDYLOTIC CERVICAL MYELOPATHY... canal of less than 11-12mm results in deformation of the cord (the normal sagittal diameter of the spinal cord at C5 segment is around x = 9.6mm (range 8.5-11) taken from myelograms and from cadaver cord specimens). A more versatile measurement is, however, the cross-sectional area of the cord. The spinal cord is reported to lose its functional tolerance if the transverse area, as measured by computed tomographic myelography and MRI, is less than 55-75% of the normal value [22,23]. These studies were first performed in 12 post-mortem specimens and then in animal models. However, a minimum spinal cord cross-sectional area of 0.44 cm2 in a non-myelopathic group and a maximum measurement of 0.86 cm2 in a myelopathic group (20 patients with cervical spondylosis) has been demonstrated [24]. The cross-sectional area at the level of maximum compression is not the only factor in the estimation of myelopathy, but is very important and easy to measure. In one group of 20 patients it has been demonstrated that narrowness of the cross-sectional area of the spinal cord was an independent prognosticator for the severity of myelopathy (p<0.05), accounting for 63% of the variations in the myelopathy score [24], We studied the impact of the degree of spinal cord compression on clinical picture in a group of 246 patients with spondylogenic cervical cord compression. A significant difference in mJOA score was found in those with a transverse spinal cord area of below 50 mm2 in comparison with patients with a transverse spinal cord area of above 60 mm2. The critical degree of spinal cord compression needed to induce clinically pathological changes is between 50 and 60 mm2.This relationship is valid for compression accompanied by the presence of hyperintensities on MR T2WI and was not found in patients with small cross-sectional area lacking hyperintensities in the spinal cord. The duration of symptoms is frequently considered a significant factor with predictive value for long-term results [25-27]. However, it is necessary to approach the evaluation of this factor with care, because recognition of the precise onset of the disease may prove to be a difficult task. The number of compression levels can affect the outcome of treatment or the natural history. A single compression level produces a mild functional deficit in comparison with multiple compression, as has been shown experimentally [28], According to some studies, the results of surgery are better in single-level than in multilevel compressions [29-30], The overall clinical status (including mental condition) For surgical treatment to be indicated, the patient must be of a status that allows such an approach, i.e. normal internal (cardiopulmonary and metabolic) condition combined with a good mental approach to co-operation and the willpower necessary for rehabilitation in the postoperative period. Psychological condition is important and it is necessary to be aware of the fact that depression is not a significant factor in the pathogenesis of the neurological clinical picture. Patients must be informed that, often contrary to expectations, surgery may stop the progression of the disease but not actually ameliorate their symptoms. Conclusions for clinical practice The crucial question in the treatment of mild and moderate non-progressing SCM is not "to operate or not to operate", because both the conservative and the surgical approaches are potentially useful. Considering the results of our studies of SCM and taking the relevant literature into account, we feel that we can recommend the following guidelines: 1. Surgery is indicated in patients with clinically severe (mJOA -modified Japanese Orthopaedic Association- score less than 12 points -Score) or progressing clinical compressive spondy-lotic cervical myelopathy (a minority of the patients). 2. Surgery is indicated in patients with severe spinal cord compression (50% or more) in spite of minimal symptoms and signs. 3. Conservative treatment is appropriate for patients with a stable, non-progressing, mild-to-moderate clinical picture (mJOA score >12 points) and mild spinal cord compression (30% or less), regardless of the presence of hyperintensities at the level of maximum compression. It is, however, necessary to check them at regular intervals (6-12 months), both clinically and with imaging (MR every 3-4 years) and electrophysiological approaches (every 3-4 years), and to reconsider indications for surgery in the event of progression. Our results may serve as a contribution to the theory that conservative treatment has some advantages over surgery in a carefully selected group of patients. The most promising candidates for high predictive values for good results in either conservative treatment or surgery could be the transverse area of the stenotic cord, duration of the disease [30], the osseous or cartilaginous compression, the developmental diameter of the canal, positivity of electrophysiological findings, low-signal intensity changes on T1-weighted sequences [18], and severity of the neurological deficit and its dynamics. ACKNOWLEDGEMENT This study was supported by the Czech Ministry of Education Research Plan No. MSM0021622404. REFERENCES 1. Kadaňka Z, Bednařík J, Voháňka S, Vlach O. Spondylo-tic cervical myelopathy: a clinical update. Scripta Medica 1996;65:157-172. http://www.med.muni.cz/biomedjournal/ SCRIPTA MEDICA/Volume 82/No. 1/2009 9 SPONDYLOTIC CERVICAL MYELOPATHY. 2. Brain WR, Northfield DWC, Wilkinson M. The neurological manifestations of cervical spondylosis. Brain 1952; 75: 187-225. 3. Sadasivan KK, Reddy RP, Albright JA. The natural history of cervical spondylotic myelopathy. Yale J Biol 1993; 66: 235-242. 4. Gregorius FK, Estrin T, Crandal PH. Cervical spondylotic radiculopathy and myelopathy. Arch Neurol 1976; 33: 618-625. 5. Crandal PH, Batzdorf U. Cervical spondylotic myelopathy. J Neurosurg 1966; 24: 57-66. 6. Okada Y, Ikata T, Hamada H, Sakamoto R, Katoh S. Magnetic resonance imaging study on the results of surgery for cervical compression myelopathy. Spine 1993; 18: 2024-2029. 7. Kohno K, Kumon Y, Oka Y, Matsui S, Ohue S, Sasaki S. Evaluation of prognostic factors following expansive lamino-plasty for cervical spinal stenotic myelopathy. Surg Neurol 1997; 48: 237-245. 8. Bucciero A, Vizioloi L, Carangelo B, Tedeschi G. MR signal enhancement in cervical spondylotic myelopathy. Correlation with surgical results in 35 cases. J Neurosurg Sei 1993;37:217-222. 9. Takahashi M, Yamashita Y, Sakamoto Y, Kojima R. Chronic cervical cord compression: clinical significance of increased signal intensity on MR images. Radiology 1989; 173:219-224. 10. Matsumoto M.Toyama Y, Ishikawa M, Chiba K, Suzuki N, Fujimura Y. Increased signal intensity of the spinal cord on magnetic resonance images in cervical compressive myelopathy. Does it predict the outcome of conservative treatment? Spine 2000; 25:677-682. 11. Singh A, Crocard HA, Platts A, Stevens J. Clinical and radiological correlates of severity and surgery-related outcome in cervical spondylosis. J Neurosurg 2001; 94:189-198. 12. Thorpe JW, Kidd D, Kendall BE, et al. Spinal cord MRI using multi-array coils and fast spin echo. I. Technical aspects and findings in healthy adults. Neurology 1993; 43: 2625-2631. 13. Wada E, Yonenobu K, Suzuki K. Can intramedullary signal changes on MRI predict surgical outcome in cervical spondylotic myelopathy? Spine 1999; 24: 455-461. 14. Morio Y, Teshima R, Nagashima H, et al. Correlation between operative outcomes of cervical compression myelopathy and MRI of the spinal cord. Spine 2001; 26: 1238-1245. 15. Ratliff J, Voorhies R. Increased MRI signal intensity in association with myelopathy and cervical instability: case report and review of the literature. Surg Neurol 2000; 53: 8-13. 16. Kadaňka Z, Mareš M, Bednařík J, et al. Predictive factors for mild forms of spondylotic cervical myelopathy treated conservatively or surgically. EurJ Neurol 2005:12 [1]: 16-24. 17. Kadaňka Z, Keřkovský M, Bednařík J, Jarkovský J. Cross-sectional transverse area and hyperintensities on magnetic resonance imaging in relation to the clinical picture in cervical spondylotic myelopathy. Spine 2007; 32 [23]: 2573-2577. 18. Al-Mefty O.Harkey HL.Marawi I, et al. Experimental chronic compressive cervical myelopathy. J Neurosurg 1993; 79: 550-561. 19. Iwabuchi M, Kikuchi S, Sato K. Pathoanatomic investigation of cervical spondylotic myelopathy. Fukushima J Med Sei 2004; 50: 47-54. 20. Teresi LM, Lufkin RB, Reicher MA, et al. Asymptomatic degenerative disc disease and spondylosis of the cervical spine: MR imaging. Radiology 1987; 164: 83-88. 21. Nurick S. The pathogenesis of the spinal cord disorder associated with cervical spondylosis. Brain 1972; 95: 87-100. 22. Fujiwara K, Yonenobu K, Hiroshima K, etal. Morphometry of the cervical spinal cord and its relation to pathology in cases with compression myelopathy. Spine 1988; 13: 1212-1216. 23. Hukuda S.Wilson CB. Experimental cervical myolepathy: effect of compression and ischemia on the canine cervical cord. Neurosurg 1972;37:631-652. 24. Golash A, Birchall D, Laitt RD, etal. Significance of CSF area measurements in cervical spondylotic myelopathy. Brit J Neurosurg 2001; 15:17-21. 25. Fujiwara K, Yonenobu K, Ebara S, Yamashita K, Ono K. The prognosis of surgery for cervical compression myelopathy. An analysis of the factors involved. J Bone Joint Surg 1989; 71B: 393-398. 26. Koyanagi T, Hirabayashi K, Satomi K, Toyama Y, Fujimura Y. Predictability of operative results of cervical compression myelopathy based on preoperative computed tomographic myelography. Spine 1993; 18: 1958-1963. 27. Yonenobu K, Okada K, Fuji T, Fujiwara K. Causes of neurologic deterioration following surgical treatment of cervical myelopathy. Spine 1985; 11:818-823. 28. Shinomiya K, Mutih N, Furuya K. Study of experimental cervical spondylotic myelopathy. Spine 1992; 17: 383-387. 29. Hirabayashi K, Uzawa M. Multilevel anterior cervical interbody fusion. A new method of subcortical binding to prevent graft discolation. Neuroorthop 1995; 17/18: 21-28. 30. Irwine GB, StrachanWE.The long-term results of localized anterior cervical decompression and fusion in spondylotic myelopathy. Paraplegia 1987; 25:18-22. 10 SCRIPTA MEDICA /Volume 82/No. 1/2009 http://www.med.muni.cz/biomedjournal/ ORIGINAL RESEARCH THE PROMOTOR POLYMORPHISM OF MACROPHAGE METALLOELASTASE IN MULTIPLE SCLEROSIS Hladíková M.1, Štourač P.1, Benešová Y.1,Tschoplová S.2 1 Department of Neurology, Faculty of Medicine, Masaryk University, Faculty Hospital Brno 2 Department of Pathological Physiology, Faculty of Medicine, Masaryk University, Brno Received after revision February 2009 KEYWORDS Matrix metalloproteinases Macrophage metalloelastase Multiple sclerosis Polymorphism H CORRESPONDING AUTHOR Hladíková M. Department of Neurology Faculty of Medicine Masaryk University and Faculty Hospital Brno Jihlavská 20 625 00 Brno Czech Republic ABSTRACT Multiple sclerosis is a chronic demyelinating disease of the central nervous system in which the genetic background plays an important role. Its pathophysiology is characterised by two major processes: neuroinflammation and neurode-generation. Matrix metalloproteinases are involved in both of them. Macrophage metalloelastase is one of the three matrix metalloproteinases the common elevation of which has been confirmed in multiple sclerosis and also in animal models with experimental allergic encephalomyelitis. To assess the association between its promotor polymorphism and demyelinating disease we genotyped a total of 92 patients (23 men, 69 women, mean age 37 years) with definite multiple sclerosis (according to the McDonald criteria) and 51 healthy controls (17 men, 34 women) matched for age and sex. Genotyping was performed by means of polymerase chain reaction with restriction analysis. We observed no statistically significant differences in genotype or allele distribution of-82 A/G polymorphism between the groups examined (OR=2.6, p=0.026, pcorr=0.078). Due to insufficient numbers of patients with the progressive form [9], no statistically significant differences in genotype or allele frequencies emerged among the patients with variant forms of multiple sclerosis. Nevertheless, all patients with the progressive form (which is associated with a more severe course and higher disability) were of the same genotype: homozygotes AA. This genotype is connected with higher promotor activity and a higher expression of the final protein. It may represent a variant genotype base for a different course of multiple sclerosis in the polymorphism under investigation. ABBREVIATIONS USED MS-multiple sclerosis MMP - matrix metalloproteinase CNS - central nervous system EAE - experimental allergic encephalomyelitis http://www.med.muni.cz/biomedjournal/ SCRIPTA MEDICA /Volume 82/No. 1 / 2009 11 00 00 00 00 00 THE PROMOTOR POLYMORPHISM OF MACROPHAGE.. Figure 1 Final genotypes visualised in UV light after ELFO on 2% Serva agarose gel with ethidium bromide RR - relapsing remitting SP - secondary progressive PP - primary progressive DNA - deoxyribonucleic acid PCR - polymerase chain reaction ELFO - electrophoresis AP-1 -activator protein-1 HW - Hardy-Weinberg equilibrium INTRODUCTION Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system (CNS). It is one of the most frequent causes of young adult disability in our geographical zone [1]. MS is a multifactorial disease in which the genetic background plays an important role. Its pathophysiology is characterised by neuroinflammation and neurodege-neration. Matrix metalloproteinases (MMPs) - a family of Independent endopeptidases [2] - are involved in both these processes. Some 26 MMPs are known to date, of which 23 may be present in humans. The expression of MMPs is usually low in the adult body and is typically observed in a local manner in the course of specific processes [2-4]. In the pathogenesis of multiple sclerosis, MMPs participate in blood-brain barrier disruption, leukocyte perivascular infiltration, myelin destruction, and conversion of the proforms of inflammatory molecules; they may also be responsible for neurotoxicity [4], Because of their heavy destructive effect, their activity is under very tight regulation [2, 3]. The first step in this path is regulation through the gene polymorphisms. Elevated levels of nine MMPs have been found in spinal cord samples taken at the peak of disease in experimental allergic encephalomyelitis (EAE), the accepted animal model of MS [5-7]. One of them was macrophage metalloelastase (MMPl 2), which up-regulated by a factor of more than three in one of these studies [8]. Increased expression of MMP12 was also confirmed in vitro in cultured rat astrocytes [9]. It has been found that the macrophages of MM12-deficient mice have a markedly diminished capacity to degrade extracellular matrix components. In addition, these macrophages were essentially unable to penetrate reconstituted basement membranes both in vitro and in vivo. MMPl 2 is therefore required for macrophage-mediated extracellular matrix proteolysis and tissue invasion [10]. Elevated MMPs have also been 12 SCRIPTA MEDICA/Volume 82/No. 1/2009 http://www.med.muni.cz/biomedjournal/ 00 7795 THE PROMOTOR POLYMORPHISM OF MACROPHAGE.. Table 1 Primers and PCR conditions Left primer (5-3T Right primer (5'-3T PCR (temperature °C, time) GAGATAGTCAAGGGATGATATCAGC AAGAGCTCCAGAAGCAGTGG 95°C/5' 95745"-55730"-72745" (3Ox) 7277' 10710 Table 2 Restriction analysis Restriction enzyme Conditions Fragment length (bp) Pvu II, 5'CAG4-CTG 3'GTClGAC 37°C/4h: PCR product 15ul, H20 2.7ul, buffer NEB 2 2ul, Pvu II 3U AA199/199bp GG 24,175/24,175 bp AG 199/24,175 bp Table 3 Genotypes and alleles MMPl 2 -82 A/G PATIENTS number (frequence %) CONTROLS number (frequence %) Genotypes All (n=92) Men (n=23) Women (n=69) All (n=51) Men (n=17) Women (n=34) AA 64 (69.57) 16 48 42 (82.35) 13 29 AG 27 (29.35) 7 20 7(13.73) 3 4 GG 1 (1.09) 0 1 2 (3.92) 1 1 Alleles A 155(0.84) 39 116 91 (0.89) 29 62 G 29(0.16) 7 22 11 (0.11) 5 6 detected in serum and cerebrospinal fluid and the brains of MS patients on autopsy. Prominent MMPl 2 staining has been reported in macrophages found in active demyelinating MS lesions [11]. A lower proportion of phagocytes was positive for MMPl 2 in chronic active and inactive plaques [11]. Thus, MMPl 2 is one of the three MMPs (together with MMP9 and 3) the common elevation of which has been confirmed in MS and also in mouse and rat EAE models [4,12], The MMPl 2 gene is located on the eleventh chromosome (11q22.2-q22.3) [13]. By single-strand polymorphism confirmation analysis of deoxyribonucleic acid (DNA) from healthy individuals a common polymorphism within the MMPl 2 gene promotor has been detected. This -82 A/G polymorphism of MMPl 2 presents a functional polymorphism in which allele A shows a higher affinity for the transcription factor activator protein-1 (AP-1) and is thus associated with a higher promo-tor activity and a higher MMPl 2 expression in assays [14]. In the present study we examined the relationship between -82 A/G MMPl 2 polymorphism and MS. MATERIALS AND METHODS Patients and control subjects In a case-control study a total of 92 unrelated patients (23 men and 69 women, mean patient age 37.3 ± 9.0years, mean EDSS score 3.71 ± 1.4, and mean disease duration 9.9 ± 5.1 http://www.med.muni.cz/biomedjournal/ SCRIPTA MEDICA/Volume 82/No. 1/2009 13 THE PROMOTOR POLYMORPHISM OF MACROPHAGE... years) with definite MS according to the McDonald criteria were recruited from the MS centre at the Department of Neurology, Faculty Hospital and Faculty of Medicine, Masaryk University, Brno. The cohort included 83 patients with the remitting relapsing form of MS - RRMS, 8 patients with the secondary progressive form - SPMS (5 women and 3 men, mean EDSS score 5.8 ± 1.4, mean disease duration 10.4 ± 4.3 years), and one patient with the primary progressive form -PPMS (a man, EDSS 4.0, disease duration 4 years). The control group consisted of 51 healthy controls (17 men and 34 women) with no history of MS or other autoimmune disease, and the subjects were matched for age and sex. All patients and controls were of Czech ancestry. Written informed consent was obtained from all the subjects examined. The study was approved by the Committee for the Ethics of Medical Experiments on Human Subjects, Faculty of Medicine, Masaryk University, Brno [ref. No. 48/ 2003]. Genotyping Genomic DNA was isolated from peripheral blood leukocytes by a standard technique using proteinase Kdigestion of cells. The resulting DNA was used as a template for polymerase chain reaction (PCR). The method used to type the MMP12 -82A/G promotor polymorphism has been described previously [14]. Primers and conditions for amplification are shown in Table 1. PCR was performed at a final volume of 25ul containing 3ul of genomic DNA, 0.5ul dNTP, 1.25ul of each primer, 2.5ul buffer, 0.14ul Taq polymerase, and 2ul 25mM MgCI2. Equal amounts of 15ul of the PCR products were digested with specific endonucleases to obtain final fragments (Table 2). The final genotypes were visualised in UV light after ELFO on 2% Serva agarose gel with ethidium bromide (Figure 1). Statistical analysis Hardy-Weinberg equilibria (HW) of the study polymorphism in all cases and controls were chi-square tested. Comparisons of the allele variant frequencies as well as comparisons of genotype incidence in the case-control study were calculated using the Fisher exact test. Statistical significance was considered as p<0.05. The Holm test for multiple comparisons was employed where appropriate. RESULTS Differences were evident in genotype distribution between our study groups, in which the AG heterozygotes were more frequent in MS patients (OR=2.6, p=0.026). Using the Holm test for multiple comparison, the results indicated only a trend, lacking statistical significance (pcorr=0.078). Even after the stratification of the subjects by sex, no allele or genotype differences were observed between the groups examined. The representation of the appropriate genotypes and alleles is shown in Table 3. The control group was not in HW equilibrium. No differences were found in genotypes or alleles among MS patients with variant forms of the disease. Nevertheless, all the patients with the progressive form (which is associated with a more severe course and higher disability) were of the same genotype: homozygotes AA (data not shown). DISCUSSION It is assumed that different genetic backgrounds exist for the various forms of MS and also that genetic backgrounds vary from population to population. The Czech Republic is a country with both a high genetic homogeneity and a high prevalence of MS. The group examined therefore constitutes a proper and representative sample. On the other hand, the study group is too small to assess susceptibility to the different forms of MS. Despite this, all the patients with the progressive form of MS were of the same genotype, homozygotes AA.This genotype is associated with a higher promotor activity, a higher expression, and a higher final MMP12 plasma level. This corresponds with the idea of different genetic backgrounds for the various courses of MS. In animal models elevated MMP12 has repeatedly been observed not only at the peak disease activity but also in the late phase of infection [11,15-17]. MMP12 protein has been localised byimmunohistochemistryinintralesional microglia/ macrophages and astrocytes and might account for ongoing demyelination [15]. One of these studies demonstrated that MMP12 was the most highly up-regulated MMR However, in contrast to previously published findings, this increase was associated with protection, as MMP12-null mice had a significantly worse maximum severity and EAE disease burden compared with wild-type controls [17]. MMP12 can therefore play both negative and positive roles in the pathophysiology of MS. At the peak phase of the disease during relapses, it may participate in blood-brain barrier disruption and potentiation of leukocyte infiltration, but in the late phase of the neuroinflammation the increased MMP12 level may be connected, for example, with some remyelination. Elevated levels of MMP12 have been observed at the time of myelina-tion in mouse brain maturation [18]. CONCLUSIONS There can be no doubt about MMP participation in the pathophysiology of MS, but the exact function of specific MMPs is not sufficiently understood. This results from the high degree of interaction among most MMPs, not only at the gene level but 14 SCRIPTA MEDICA/Volume 82/No. 1/2009 http://www.med.muni.cz/biomedjournal/ 00 00 also at further stages, where they may activate or inhibit each other. Forthis reason,furtherand more extensive investigations into the genotypes.and especially the haplotypes of MMPs.are indicated.They may well disclose more profound effects on susceptibility to MS or on tendencies to different courses of MS. ACKNOWLEDGEMENT This study was supported by the Czech Ministry of Education Research Plan No. MSM0021622404. REFERENCES 1. Havrdová E. Neuroimunologie [Neuroimmunology]. Praha: MAXDORF, 2001: 231 pp. 2. Wikipedia.org [homepage on the Internet], the free encyclopedia. Available at: http://en.wikipedia.org/wiki/ Metalloproteinase 3. Nagase H, Woessner JF. Matrix metalloproteinases. J Biol Chem 1999; 274: 21491-21494. 4. Yong VW, Power C, Forsyth P, Edwards DR. Metalloproteinases in biology and pathology of the nervous system. Nat Rev Neurosci 2001; 2(7): 502-511. 5. Pagenstecher A, Stalder AK, Kincaid CL, Shapiro SD, Campbell IL Differential expression of matrix metalloproteinase and tissue inhibitor of matrix metalloproteinase genes in the mouse central nervous system in normal and inflammatory states. Am J Pathol 1998; 152(3): 729-741. 6. Kieseier BC, Kiefer R, Clements JM, et al. Matrix metallopro-teinase-9 and -7 are regulated in experimental autoimmune encephalomyelitis. Brain 1998; 121(1): 159-166. 7. Nygárdas PT, Hinkkanen AE. Up-regulation ofMMP-8 and MMP-9 activity in the BALB/c mouse spinal cord correlates with the severity of experimental autoimmune encephalomyelitis. Clin Exp Immunol 2002; 128(2): 245-254. 8. Toft-Hansen H, Nuttall RK, Edwards DR, Owens T. Key metalloproteinases are expressed by specific cell types in experimental autoimmune encephalomyelitis. J Immunol 2004; 173(8): 5209-5218. 9. Wells GM, Catlin G, Cossins JA, et al. Quantitation of matrix metalloproteinases in cultured rat astrocytes using the polymerase chain reaction with a multi-competitor cDNA standard. Glia 1996; 18(4): 332-340. 10. Shipley JM.Wesselschmidt RL, Kobayashi DK, LeyTJ, Shapiro SD. Metalloelastase is required for macrophage-me-diated proteolysis and matrix invasion in mice. Proc Natl Acad Sei USA 1996; 93(9): 3942-3946. 11. Vos CM, van Haastert ES, de Groot CJ, van der Valk P, de Vries HE. Matrix metalloproteinase-12 is expressed in phagocytotic macrophages in active multiple sclerosis lesions. J Neuroimmunol 2003; 138(1-2): 106-114. THE PROMOTOR POLYMORPHISM OF MACROPHAGE... 12. Maeda A, Sobel RA. Matrix metalloproteinases in the normal human central nervous system, microglial nodules, and multiple sclerosis lesions. J Neuropathol Exp Neurol 1996; 55(3): 300-309. 13. MEROPS the Peptidase Database [homepage on the Internet]. Available at: http://merops.sanger.ac.uk/ 14. Jormsjö S, Ye S, Moritz J, et al. Allele-specific regulation of matrix metalloproteinase-12 gene activity is associated with coronary artery luminal dimensions in diabetic patients with manifest coronary artery disease. Circ Res 2000; 86(9): 998-1003. 15. Kurzepa J, Bartosik-Psujek H, Suchozebrska-Jesionek D, et al. Role of matrix metalloproteinases in the pathogenesis of multiple sclerosis. Neurol Neurochir Pol 2005; 39(1): 63-67. 16. Ulrich R, Baumgärtner W, Gerhauser I, et al. MMP-12, MMP-3,andTIMP-1 are markedly upregulated in chronic demyelinating Theiler murine encephalomyelitis. J Neuropathol Exp Neurol 2006; 65(8): 783-793. 17. Weaver A, Goncalves da Silva A, Nuttall RK, et al. An elevated matrix metalloproteinase (MMP) in an animal model of multiple sclerosis is protective by affecting Thl/ Th2 polarization. FASEBJ 2005; 19(12): 1668-1670. 18. Larsen PH, DaSilva AG, Conant K, Yong VW. Myelin formation during development of the CNS is delayed in matrix metalloproteinase-9 and -12 null mice. J Neurosci 2006; 26(8): 2207-2214. http://www.med.muni.cz/biomedjournal/ SCRIPTA MEDICA/Volume 82/No. 1/2009 15 00 00 00 ORIGINAL RESEARCH PAINFUL SENSORY NEUROPATHIES IN THE ELDERLY Vlčková-Moravcová E., Bednařík J. Department of Neurology, Masaryk University and Faculty Hospital Brno Received after revision February 2009 KEYWORDS Elderly Intraepidermal nerve fibre density Painful neuropathy Quantitative sensory testing Small and large nerve fibres H CORRESPONDING AUTHOR Vlčková-Moravcová E. Department of Neurology Faculty of Medicine Masaryk University and Faculty Hospital Brno Jihlavská 20 625 00 Brno Czech Republic ABSTRACT Introduction: Painful sensory neuropathies are frequently encountered in elderly individuals. The pathophysiology of peripheral nerve dysfunction as well as the clinical picture and applicable diagnostic methods in senescence may be at least partly different from those in the general neuropathy population with respect to the potential role of age-related neurodegeneration as well as to some specific features of the elderly population in general. Patients and methods: Thermal quantitative sensory testing (T-QST), evaluation of intra- (IENFD) and subepidermal nerve fibre densities in skin biopsy samples, nerve conduction studies, autonomic nervous system testing, and clinical neurological examination including detailed assessment of neuropathic symptoms and pain intensity were performed in a group of 25 elderly (>65 years) and 74 non-elderly patients with painful sensory neuropathy. For comparison, data from 37 age-matched healthy individuals (10 elderly and 27 non-elderly) were used. Results: The involvement of small nerve fibres documented by T-QST or IENFD was almost invariably found in both elderly and non-elderly patients and the sensitivity as well as applicability of both these methods was similar between the age groups. Nevertheless, an obvious trend to decrease in nerve fibre counts with age was observed in the healthy control group. The dysfunction of large nerve fibres (assessed by nerve conduction studies or clinical examination) as well as of the autonomic ones was significantly more frequent in elderly neuropathy patients compared to younger age groups. Moreover, evaluation of the autonomic nervous system could frequently not be performed in elderly patients with respect to associated heart diseases or medication. Conclusions: Painful neuropathy patients almost invariably display involvement of small nerve fibres regardless of the patient's age. In elderly painful neuropathy patients, however, a more frequent and more extensive dysfunction of large myelinated fibres and autonomic fibres was found suggesting the increasing role of age-related neurodegeneration in the development of peripheral neuropathies in old age and 16 SCRIPTA MEDICA/Volume 82/No. 1/2009 http://www.med.muni.cz/biomedjournal/ ■ PAINFUL SENSORY NEUROPATHIES IN THE ELDERLY implying the need of age-stratified reference data of most of the diagnostic tests of small- and large-fibre dysfunction in peripheral neuropathies. ABBREVIATIONS USED AUDIT - alcohol use disorders identification test IENFD - intraepidermal nerve fibre density MNSI - Michigan neuropathy screening instrument questionnaire (subscales i and ii) NDS - neuropathy disability score NSS - neuropathy symptom score PGP - protein gene product T-QST - thermal quantitative sensory testing SENPD - subepidermal nerve plexus density SNAP - sensory nerve action potential VAS - visual analogue scale INTRODUCTION Painful sensory neuropathies are frequently encountered in elderly individuals and may have an important impact on their sleep and quality of life. Besides an increasing incidence of neuropathy risk factors in senescence, degeneration of the peripheral nervous system in old age has repeatedly been shown in humans as well as in animal models of nervous system aging, and seems to play a role in the development of peripheral neuropathies in the elderly [1,2]. Both the age-related degeneration and the involvement of peripheral nerves by some pathological processes may involve various types of nerve fibres, and the pattern and proportion (and probably also the time sequence) of the fibres affected are crucial for the clinical picture of peripheral neuropathy. Experimental models [1, 2, 3] suggest that, in contrast to e.g. metabolic neuropathies, where initial and more pronounced involvement of so-called small non-myelinated and low myelinated nerve fibres of the classes A-delta and C is usually described, the age-related degeneration process affects predominantly large myelinated fibres of A-beta class. The suggested different pattern of the affected nerve fibres in the elderly therefore indicates that the resulting clinical picture and diagnostic methods of peripheral neuropathies in senescence may be at least partly different from those in younger age groups. Besides more frequent affection of large nerve fibres in senescence, age-related loss of small nerve fibres has also been described in rodents [2, 3] as well as in some of the human studies on epidermal innervation reflecting the age-related changes of small nerve fibre status [4, 5]. The involvement of these fibres (mediating pain and temperature, and also serving autonomic functions) has repeatedly been shown to play a n important role in the pathophysiology of neuropathic pain [6, 7], and their dysfunction has been found in most of the painful neuropathies [8, 9, 10]. A-delta and C fibres can even be the only nerve fibres affected in some of the painful neuropathy patients (so-called small fibre neuropathies) [8,9, 10]. Their involvement, however, is usually underdiagnosed in clinical practice, because common clinical examination and nerve conduction studies fail in the verification of their dysfunction [8], and special diagnostic methods (e.g. examination of thermal and/or pain sensation on quantitative sensory testing (T-QST) or quantification of intraepidermal nerve fibre density (IENFD)) have to be used for this purpose [8], Both of these methods, however, may have some limitations, particularly in elderly patients. Together with some specific characteristics of senescence in general, all these facts point out the possibility that the pathophysiology, the clinical picture and applicable diagnostic methods of neuropathies in the elderly are at least partly different from the general neuropathy population, in particular when pain is a leading clinical symptom. Only few studies, however, deal with clinical, neurophysiological, and morphological characteristics of painful peripheral neuropathies in senescence. The aim of our study was therefore to evaluate selected clinical, morphological, and electro- and psychophysical findings in older adults with painful sensory neuropathy compared to younger patients to reveal possible specific features of this diagnostic unit in the elderly and to assess the diagnostic validity of several methods used to confirm a peripheral nerve dysfunction in these patients. PATIENTS AND METHODS Twenty-five elderly patients (older than 65 years) with painful sensory neuropathy and a prominent complaint of "burning feet" were included in the study (Table 1). For comparison, data from seventy-four non-elderly individuals with painful neuropathy complying with the same inclusion and exclusion criteria (see below) were used (Table 1). All the patients from both these groups were prospectively recruited from the Peripheral Neuropathy Outpatient Clinic of the Brno Faculty Hospital between September 1999 and March 2005. The protocol was approved by the institutional ethics committee of the University of Brno; written informed consent was obtained from all patients and volunteers before inclusion into the study. The following inclusion criteria had to be met: [1] Positive sensory symptoms (pain or painful dysesthesias described as electric shock-like, burning, cold, prickling, tingling, or itching) in a distal symmetrical distribution in the lower extremities for more than 3 months and with an intensity of at least 3 ona visual analogue scale (VAS) of 0-10. [2] Abnormal thresholds for at least one thermal modality as assessed by T-QST. http://www.med.muni.cz/biomedjournal/ SCRIPTA MEDICA/Volume 82/No. 1/2009 17 PAINFUL SENSORY NEUROPATHIES IN THE ELDERLY Table 1 Clinical characteristics of patients and controls. Abbreviations are defined in the text Demographic characteristics Neuropathy group Controls Elderly Non-elderly Elderly Non-elderly Number of subjects studied 25 74 10 27 Males 13 45 6 11 Signs of large fibre affection 17 24 Age Median (min/max) (years) 71 (65/83) 55 (25/64) 70 (67/86) 54 (27/64) Symptom duration Median (min/max) (years) 1.50 (0.25/6) 2.00 (0.25/13) Associated relevant diseases (number of si jbjects) Diabetes mellitus or impaired glucose tolerance 5 27 Alcohol abuse 2 13 Others1 7 14 Undetermined 11 20 'Others: potential aetiological factors or co-factors include toxins (anticancer chemotherapy or antituberculous drugs), paraneoplastic syndromes, monoclonal gammopathy, hyperlipidaemia, and amyloidosis Exclusion criteria were signs of central (brain or spinal cord) involvement and any overt clinical motor signs (weakness, distal muscle atrophy, fasciculations). Other diseases or conditions leading to foot pain (both neuropathic and non-neuropathic) such as plantar fasciitis, Charcot's joints, osteoarthritis, peripheral vascular disease, central nervous system dysfunction, tarsal tunnel syndrome, and other peripheral mononeuropathies were excluded by history and clinical examination. For comparison of skin nerve fibre densities with the non-neuropathy population, data from a group of thirty-seven healthy volunteers matched for age and body mass index (10 elderly and 27 non-elderly subjects) were used (Table 1). These individuals were recruited at the Brno Faculty Hospital, mainly from among hospital employees and their relatives, and the details about their history and clinical status as well as their laboratory findings of the parameters with a potential aetiological relation to peripheral neuropathies were published previously in detail [9,10], Data from the patients and controls included into the present study were reported in part in a previous study evaluating in detail the diagnostic value of skin biopsy (including a newly validated parameter - subepidermal nerve plexus density) [9]. The previous study, however, dealt mainly with morphological findings and was focused neither on the specifications of painful neuropathies in the elderly nor on the differences between elderly patients and those of younger age groups. In contrast, the current study deals only with characteristic features of painful peripheral neuropathy in senescence, which are suggested to be partly different from the general neuropathy population. A detailed medical history was taken from all patients, who underwent a thorough clinical neurological examination, including assessment of tendon reflexes, muscle strength, trophism, sensation to touch, pinprick, warm and cold stimuli, proprioception, and vibration sense. Associated diseases with potential aetiological relevance were explored by history and routine biochemical, haematological, and immunological blood tests including thyroid hormones, triglycerides, vitamin B12 and folate levels, serum protein electrophoresis, carbohydrate-deficient transferrin, and a set of autoantibodies (ANA, anti-Ro, anti-La, anti-DNA). Diabetes mellitus was confirmed by fasting plasma glucose levels and oral glucose tolerance tests. Quantification of alcohol consumption as well as further confirmation of harmful alcohol useand possible alcohol dependence was performed according to the WHO AUDIT (Alcohol Use Disorders Identification Test) manual [11], For quantification of the presence and severity of clinical symptoms and neurological impairment, the Neuropathy Symptom Score (NSS) [12], the Neuropathy Disability Score (NDS) [12], and the Michigan Neuropathy Screening Instrument Questionnaire (MNSI) [13] were employed. The mean intensity of neuropathic pain during the week before clinical 18 SCRIPTA MEDICA/Volume 82/No. 1/2009 http://www.med.muni.cz/biomedjournal/ PAINFUL SENSORY NEUROPATHIES IN THE ELDERLY Figures la, b Images depicting the method for evaluation of theintraepidermal nerve fibre density (IENFD) and subepidermal nerve plexus density (SENPD) on PGP 9.5 immunoreacted 40-um cryosections of the skin. SENPD was measured using a density threshold for PGP 9.5 immunoreactive structures and expressed as a percentage of the whole subepidermal area analysed (200x50 urn adjacentto the dermoepidermal junction in each optical field) A: Original digitised image. Bar = 50 urn 1 + 2 = epidermis (1 = stratum corneum, 2 = stratum basale, spinosum, granulosum, and lucidum) 3 = dermis 4 = subepidermal nerve plexus 5 = intraepidermal nerve fibre B: Density threshold set for morphometric determination of nerve fibre area. Bar = 50 urn http://www.med.muni.cz/biomedjournal/ SCRIPTA MEDICA/Volume 82/No. 1/2009 19 PAINFUL SENSORY NEUROPATHIES IN THE ELDERLY and electrophysiological testing was assessed using a 10-cm VAS, in which 0 cm represented "no pain"and 10 cm "the worst pain I can imagine". All the clinical and electrophysiological tests were performed at the time of admission to the clinic and no specific treatment for neuropathic pain had been delivered to these patients previously. Quantitative sensory thermal threshold testing (T-QST) was performed using thermal sensory analyser software (Medoc Thermal Sensory Analyser 2001). Thermal thresholds were examined on the dorsum of the right foot; both the method of limits and levels were used. For evaluation we used our own normal limits [14]. The methodology was described elsewhere in detail [9,14]. Electrophysiological examinations, sympathetic skin response, and cardiovascular tests based on heart rate variability examination were performed using a Keypoint type II electromyograph system (Dantec, Skovlunde, Denmark), following the published recommendations [15]. The results were processed according to the reference values related to age and height at the Brno laboratory. Skin punch biopsies were taken from the distal calf. The details of skin specimen removal and further processing were published previously [9,16] and follow standard recommendations [17,18]. Sections of 40 urn thickness were immunos-tained with rabbit polyclonal antibodies to human protein gene product (PGP) 9.5 (Ultraclone.Wellow, UK; 1:800) as a primary antibody and goat anti-rabbit IgG labelled with a cya-nine fluorescent probe as a secondary antibody (Amersham, Biosciences, Piscataway, NJ; 1:100). Using Image-Pro Plus 4.0 software (Media Cybernetics, Leiden, The Netherlands), the intraepidermal nerve fibre density (IENFD) was quantified by counting the nerve endings within an accurately measured length of epidermis [17,18] (Figure 1). Subepidermal nerve plexus density (SENPD) was measured using a density threshold for PGP9.5 immunoreactive structures and expressed as a percentage of the whole subepidermal area analysed (200 x 50 um adjacent to the dermoepi-dermal junction in each optical field) [9] (Figure 1).The IENFD was defined as abnormal if lower than the cut-off value of 8.8 fibres/mm [9]. Similarly, the SENPD was defined as abnormal if lower than the cut-off value of 7.25% [9]. The normal limits were established from the skin biopsy results in the group of normal individuals and the method settings were described in our previous publication [9], Standard descriptive statistics were used to summarise the distribution of the data. Comparison of continuous data between both the subgroups was performed by a univariate t-test, while categorical data were compared using a chi-square test. The Pearson's correlation coefficient was used in correlation analyses. The value a <0.05 was taken as the universal limit for statistical significance. RESULTS As a matter of course, the age of elderly patients was higher in comparison with non-elderly ones (p<0.001), while the other demographic characteristics (i.e. sex and duration of symptoms) did not differ between the groups (Table 1). Prominent aetiological factors of both groups represented impaired glucose metabolism and chronic harmful alcohol use or possible alcohol dependence. The incidence and proportion of the particular aetiological factors did not differ significantly between the groups, though a trend towards lower incidence of glucose dysmetabolism (p=0.12) and a higher proportion of patients with undetermined (i.e. idiopathic) aetiology in elderly patients (p=0.11) was found (Table 1). Clinically, impaired proprioception and vibration sense as signs of large-fibre sensory dysfunction were significantly more frequent in elderly patients (p=0.001 and 0.002, respectively; data not shown). For other sensory modalities, a trend towards higher incidence of abnormities in the elderly group was also found, though without statistical significance (p= 0.07, 0.05, and 0.10 for sensation to pinprick and pain and thermal perception, respectively; data not shown). In accordance with these findings, highervaluesand higher sensitivity of NDS and a second part of the MNSI questionnaire (as the scales reflecting the presence of clinical abnormities and in particular large-fibre function) were found in our group of elderly patients, while only less obvious and less significant differences were found in the Neuropathy Symptom Score values (reflecting both the small and large nerve fibre function) (Table 2). The intensity of neuropathic pain as assessed by VAS as well as the presence of clinical symptoms of sensory dysfunction (evaluated either as particular items or as a summary score reflecting mainly the dysfunction of small nerve fibres, i.e. the first part of the MNSI questionnaire) were similar in both groups of neuropathy patients (Table 2). Accordingly, the sensitivity of this questionnaire was also comparable in elderly and non-elderly individuals. Absolute values of cold and warm detection thresholds, the number of abnormities of thermal sensation found by the particular testing algorithms using the age-related normal values, and also the variability of the responses did not differ significantly between the groups (Table 2). The results obtained by the three T-QST methods were consistent and values did not differ significantly between the methods. The reduction of sensory nerve action potential (SNAP) amplitudes represented the most significant difference of nerve conduction studies between the groups (Table 2). In general, electrophysiological signs of large fibre sensory neuropathy were more frequent in elderly patients (p = 0.002) (data not shown). The elicitability of sympathetic skin response was similar in both groups of patients. In the elderly group, however, slightly 20 SCRIPTA MEDICA/Volume 82/No. 1/2009 http://www.med.muni.cz/biomedjournal/ PAINFUL SENSORY NEUROPATHIES IN THE ELDERLY Table 2 Absolute values and number of abnormities (if applicable) revealed by selected psychophysical, neurophysiological, and clinical tests in elderly and non-elderly neuropathy patients. Values are mean ± standard deviation. Abbreviations are defined in the text Absolute values Number of abnormities revealed Test Elderly Non-elderly p-value (t-test) Elderly Non-elderly p-value (chi2-test) Thermal thresholds (dorsum of the right foot, °C) MLI-RCS 20.2 ±10.4 21.5 ±8.8 0.54 14 37 0.60 WS 47.0 ± 3.4 46.8 ± 2.8 0.73 22 56 0.19 MLE - CS 22.3 ±10.6 23.3 ± 9.3 0.66 13 38 0.96 WS 44.4 ± 4.1 44.2 ± 3.5 0.76 22 67 0.72 Nerve conduction studies Sural SNAP amplitude (uV) 3.7 ±3.3 8.7 ±5.5 | < 0.001 | 16 20 < 0.001 Sympathetic skin response (lower extremities) Mean latency (ms)* 2.23 ± 0.28 2.11 ±0.22 0.04 Mean amplitude (mV) * 0.41 ± 0.25 0.73 ± 0.72 0.04 Elicitability(%) 58 66 0.27 4 8 0.50 Heart rate variability Heart-rate variability to |deep breathing (%) 11.9 ±5.8 18.8 ±10.3 0.04 8 22 0.04 Clinical findings VAS* 6.14 ±2.56 6.38 ±2.0 0.62 MNSI1 7.60 ± 2.00 7.93 ± 2.29 0.52 19 57 0.91 MNSI 2 2.48 ±1.41 1.35 ±1.33 < 0.001 20 26 < 0.001 NSS* 2.56 ±1.16 2.04 ± 0.83 0.02 NDS* 15.7 ±13.7 6.9 ±7.9 < 0.001 *No limit data for discrimination of normal and abnormal findings of this parameter are available MLI - Method of Limits, MLE - Method of Levels, CS - cold sensation, WS - warm sensation, R - random variant of the test); other abbreviations are defined in the text higher latencies and lower amplitudes of the response were found in the lower extremities (Table 2). The heart rate variability testing could not be performed or evaluated in more than a half of the elderly group because of associated heart disease or medication with potential influence on the heart rate (56% compared to 24% of non-elderly individuals, p = 0.003). From the evaluable tests, the vast majority was abnormal in elderly patients, while in the non-elderly group abnormal results were much less frequent (72 and 36%, respectively, p = 0.02). In most of the tests performed, significantly lower heart rate variability values were thus obtained were thus obtained in elderly individuals in comparison to younger ones (Table 2). In the group of healthy individuals, a non-significant trend to the correlation of the IENFD and SENPD values with age as well as to higher values in the subgroup of elderly controls compared to non-elderly ones was found (Table 3). Comparison of healthy individuals and painful neuropathy patients demonstrated a clear reduction in both intra-and subepidermal nerve fibre/plexus density in both the elderly and non-elderly subgroups of patients with neuropathies compared to the particular age-related subgroups of healthy controls (Table 3). In neuropathy patients, lower values of the SENPD were found in elderly patients than in non-elderly ones, while the IENFD values did not differ significantly between the age groups (Table 3). A clear correlation of the IENFD values with warm thresholds in T-QST examination and with the scales reflecting particularly small nerve fibre involvement (i.e. MNSI I) and lower intraepider-mal nerve fibre counts in the presence of clinical signs of small fibre dysfunction were found in both the age subgroups of neuropathy patients, while the SENPD correlated better with SNAP amplitude and MNSI II and NDS (as the scales showing mainly large nerve fibre function) in both elderly and non-elderly individuals (p from 0.02 to <0.001; data not shown). DISCUSSION The principal finding of this prospective study is that the involvement of small nerve fibres is almost invariably present in elderly patients with painful neuropathies. Despite this fact, http://www.med.muni.cz/biomedjournal/ SCRIPTA MEDICA/Volume 82/No. 1/2009 21 86 PAINFUL SENSORY NEUROPATHIES IN THE ELDERLY Table 3 Skin biopsy findings. IENFD (fibres/mm) and SENPD (% of the subepidermal area of the size 200 x 50 um adjacent to the dermoepidermal junction): basic summary statistics, comparison of the neuropathy and control groups and elderly and non-elderly individuals, and correlation with age in the control group of healthy individuals. Abbreviations are defined in the text Groups of patients Comparison of the groups Correlation with age in control group Elderly Non-elderly p,-value r p-value IENFD (fibres/mm) Group of patients 4.95 ± 3.02 5.69 ± 4.08 0.40 Healthy individuals 9.81 ±1.61 11.80 ±3.30 0.08 -0.325 0.06 Comparison of the groups (p^-value) < 0.001 < 0.001 SENPD (%) Group of patients 3.19 ± 1.91 4.88 ± 2.75 0.006 Healthy individuals 8.20 ±1.31 9.76 ± 2.76 0.10 -0.237 0.16 Comparison of the groups (p^-value) < 0.001 < 0.001 p,-value - comparison of nerve fibre density between elderly and non-elderly individuals in a group of patients (first row) or in a group of healthy individuals (second row) p2-value - comparison of nerve fibre density between elderly neuropathy patients and controls (first column) or non-elderly patients and controls (second column) a partly different pattern of involvement of the particular nerve fibre types with a more severe and more frequent dysfunction of large nerve fibres compared to younger age groups was found in these individuals showing a probable role of age-related neurodegeneration in the development of peripheral neuropathies in the elderly. These findings imply an increasing significance of clinical examination and nerve conduction studies in the diagnostic algorithm of painful neuropathies in senescence. Almost all the elderly painful neuropathy patients however present an involvement of small nerve fibres and in a remarkable part of them (about 1/3) these fibres were shown to be the only affected ones. Appropriate diagnostic methods of small nerve fibre involvement (i.e. thermal quantitative sensory testing and examination of intraepidermal nerve fibre density) thus still play a pivotal role in the battery of diagnostic tests of painful neuropathies in the elderly, and our findings prove their applicability in older adults as well as their similar diagnostic validity compared to younger age groups, though some particularities (e.g. the need of age-related normal limit data) should be taken into account. Painful sensory neuropathies are frequently encountered in older adults. As in the other types of peripheral nerve affections, both the clinical picture and applicable diagnostic methods of painful neuropathies in senescence depend in particular on the pattern and proportion of the affected types of nerve fibres. In accordance with the observations in the general painful neuropathy population [9,19], dysfunction of small nerve fibres was almost invariably present in our group of elderly painful neuropathy patients. A comparison of the age groups showed a similar extent of small-fibre affection (proved both by examination of IENFD andT-QST) as well as an almost identical spectrum of corresponding clinical symptoms and pain intensity (as assessed by VAS) in elderly and non-elderly patients in our study. These findings suggest that, regardless of the patients'age, a similar extent of small fibre damage leads to a similar clinical picture, and thus indirectly confirm the role of small nerve fibre dysfunction in the development of pain and appropriate clinical symptoms of painful neuropathies. In contrast to these findings, most of the diagnostic methods (in particular clinical examination and nerve conduction studies) proved a more severe and more frequent affection of large myelinated nerve fibres in our group of elderly neuropathy patients compared to the younger ones. A similar pattern of nerve fibre involvement was repeatedly shown in animal models of age-related degeneration of the peripheral nervous system [1, 2]. In aging rats, more symptoms of disturbed mechanosensa-tion compared to disturbed nociception were found [2], and a more severe loss of large and myelinated fibres compared to smaller ones was shown in sural nerve studies of peripheral nervous system aging [1]. Our data thus confirm the observation of more severe age-related degeneration of large and myelinated fibres compared to smaller ones and imply that neurodegeneration plays an important role in the development of painful peripheral neuropathies in the elderly. Despite these facts, age-related loss of epidermal and dermal innervation, involving both sensory and autonomic 22 SCRIPTA MEDICA/Volume 82/No. 1/2009 http://www.med.muni.cz/biomedjournal/ 54 42 ■ PAINFUL SENSORY NEUROPATHIES IN THE ELDERLY components, was also described in rodents [2, 3]. In humans, some of the studies [4, 5] suggest a similar decrease of intraepidermal innervation with age, but there is no clear agreement on this field and not all the studies show such a significant negative correlation [20], probably due to the considerable interindividual variability of epidermal nerve counts in normal humans. Despite such controversial findings, only few studies are focused on normal skin innervation in senescence and provide separated limit data of intraepidermal nerve fibre counts in the elderly [21], which may complicate the evaluation and the validity of skin biopsy examination in older adults. In our control group of healthy individuals, an obvious trend to inversed correlation of IENFD/SENPD with age was observed, as well as a trend to lower fibre counts in the subgroup of elderly controls compared to the younger ones. Both of these trends were apparent, but none of them was statistically significant, probably with respect to the small size of our control group and in particular to the small number of elderly individuals among our healthy controls (10 out of 37). Our findings nevertheless seem to confirm the age-related degeneration of intra- and subepidermal nerve fibres and suggest the need of setting particular normal limits for elderly and non-elderly individuals. An increased number of healthy controls and in particular a higher proportion of elderly individuals are therefore needed in future studies. In comparison with the age-related subgroups of healthy controls, a highly significant decrease in IENFD/SENPD values was found in both the elderly and non-elderly painful neuropathy patients, confirming the high sensitivity of this method, regardless of the patients'age.The examination was well tolerated by all the individuals and no complications of wound healing were observed in any one of the elderly or non-elderly patients. Together with a good correlation of the IENFD with warm thermal thresholds in T-QST examination and with MNSII as the methods reflecting mainly small fibre dysfunction, these findings show that the examination of the IENFD from skin biopsy is an applicable method in the evaluation of small fibre damage in older adults, and we can recommend the inclusion of this method to the diagnostic algorithm of painful neuropathies in the elderly. Another method of evaluation of the small nerve fibre dysfunction involved in our study was the examination of thermal thresholds on quantitative sensory testing [8,22]. In contrast to the morphological character of the previous test, T-QST is a psychophysiological method and therefore requires concentration, attention, and the ability of fast response [22], which may be decreased in the elderly. However, when using the age-stratified reference data, the number of abnormities revealed by the T-QST and the response variability of the methods used did not differ between younger and older neuropathy patients and the results obtained by the three T-QST methods were consistent and values did not differ significantly between the particular algorithms. These findings suggest that when using the age-stratified reference data, psychophysiological methods are useful in elderly patients and provide reliable and reproducible results, fully comparable with younger individuals. Besides the methods evaluating sensory small nerve fibres, assessment of autonomic nervous system functions can also be used for the evaluation of small nerve fibre status in polyneuropathy patients. Our previous findings [9], however, suggest that the autonomic nervous system testing is less sensitive compared to T-QST and skin biopsy in the evaluation of painful neuropathies in general. In elderly patients, the use of these methods (in particular examination of heart rate variability) is furthermore complicated by the increasing number of patients with coincidental heart rate abnormalities (e.g. atrial fibrillation) and those with implanted pacemakers or using antiarrhythmics (e.g. beta-blockers) in their regular medication. Due to these conditions, the heart rate variability testing could not be performed or evaluated in up to 60% of elderly patients in our study (but only in about 1/4 of younger individuals). Among the evaluable tests, a significantly higher proportion of abnormalities was found in older patients, suggesting that the autonomic nerves are more frequently involved in painful neuropathies in the elderly as compared with younger age groups. Despite this fact, the sensitivity of autonomic nervous system assessment did not reach the value of the T-QST or the IENFD examination and autonomic tests thus still remain only complementary methods in the diagnostic algorithm of painful neuropathies in senescence and we recommend their use in particular for the verification of autonomic dysfunction in patients with relevant clinical symptoms. The signs of more severe dysfunction of autonomic fibres in our elderly neuropathy patients compared to younger age groups are in agreement with the observation of a significant age-related degeneration of autonomic nerve fibres in experimental models of nervous system aging [3] and may again support the hypothesis of the important pathophysiological role of age-related degeneration in the development of peripheral neuropathies in the elderly, which is also corroborated by the diverse pattern of large and small nerve fibre affection and by the higher proportion of idiopathic neuropathies in senescence.Thus, in younger patients the development of peripheral nerve dysfunction seems to be in particular the result of peripheral nerve damage caused by various aetiological factors, while in the elderly, age-related nerve degeneration is suggested to play an increasing role in the development of peripheral neuropathies. Finally we conclude that painful neuropathies in the elderly display a slightly different pattern of involvement of the http://www.med.muni.cz/biomedjournal/ SCRIPTA MEDICA/Volume 82/No. 1/2009 23 614676 PAINFUL SENSORY NEUROPATHIES IN THE ELDERLY particular nerve fibre types with a more frequent and more extensive involvement of large myelinated fibres as well as of the autonomic ones in comparison with younger age groups, suggesting the increasing role of age-related neu-rodegeneration in the development of peripheral neuropathies in senescence. The involvement of small nerve fibres, however, represents the most frequent and most remarkable abnormality in elderly painful polyneuropathy patients, confirming the role of small nerve fibre dysfunction in the development of painful neuropathy symptoms regardless of the patient's age. The assessment of thermal thresholds on the T-QST and the examination of the IENFD on skin biopsy as methods of evaluation of small fibre status proved to be applicable and sensitive in older adults and continue to play a key role in the diagnostic algorithm of painful neuropathies in senescence, despite the increasing sensitivity of nerve conduction studies and autonomic nervous system testing in these age groups. ACKNOWLEDGEMENTS The study was supported by the Czech Ministry of Education Research Plan No. MSM0021622404. Dr. Vlčková-Moravcová performed the morphological studies at the University of Würzburg, supported by a Fellowship from the European Neurological Society (ENS). REFERENCES 1. Jeronimo A, Jeronimo CA, Filho OA, Sanada LS, Fazan VP. A morphometric study on the longitudinal and lateral symmetry of the sural nerve in mature and aging female rats. Brain Res 2008; 1222: 51-60. 2. Ulfhake B, Bergman E, Fundin BT Impairment of peripheral sensory innervation in senescence. Auton Neurosci 2002; 96(1): 43-49. Review. 3. Fundin BT, Bergman E, Ulfhake B. Alterations in mysta-cial pad innervation in the aged rat. Exp Brain Res 1997; 117(2): 324-340. 4. Goransson LG, Mellgren SI, Lindal S, Omdal R.The effect of age and gender on epidermal nerve fiber density. Neurology 2004; 62: 774-777. 5. Umapathi T, Tan WL, Loke SC, et al. Intraepidermal nerve fiber density as a marker of early diabetic neuropathy. Muscle Nerve 2007; 35: 591-598. 6. Kramer HH, Rolke R, Bickel A, Birklein F. Thermal thresholds predict painfulness of diabetic neuropathies. Diabetes Care 2004; 27: 2386-2391. 7. Polydefkis M, Yiannoutsos CT, Cohen BA, et al. Reduced intraepidermal nerve fiber density in HIV-associated sensory neuropathy. Neurology 2002; 58:115-119. 8. Lacomis D. Small-fiber neuropathy. Muscle Nerve 2002; 26:173-188. 9. Vlčková-Moravcová E, Bednařík J, Dušek L.Toyka K, Sommer C. Diagnostic validity of epidermal nerve fiber densities in painful sensory neuropathies. Muscle Nerve 2008; 37: 50-60. 10. Vlčková-Moravcová E, Bednařík J, Bělobrádková J, Sommer C. Small-fibre involvement in diabetic patients with neuropathic foot pain. Diabet Med 2008; 25:692-699. 11. Saunders JB, Aasland OG, BaborTF, de la Fuente JR, Grant M. Development of the Alcohol Use Disorders Identification Test (AUDIT): WHO Collaborative Project on Early Detection of Persons with Harmful Alcohol Consumption-ll. Addiction 1993; 88: 791-804. 12. Dyck PJ, Sherman WR, Hallcher LM, et al. Human diabetic endoneurial sorbitol, fructose, and myo-inositol related to sural nerve morphometry. Ann Neurol 1980; 8:590-596. 13. Feldman EL, Stevens MJ,Thomas PK, et al. A practical two-step quantitative clinical and electrophysiological assessment for the diagnosis and staging of diabetic neuropathy. Diabetes Care 1994; 17:1281 -1289. 14. Moravcová E, Bednařík J. Computer-assisted thermal threshold testing - normative data and sources of variability. Cesk Slov Neurol N 2003; 66/99: 263-269. 15. Proceedings of a consensus development conference on standardized measures in diabetic neuropathy. Electrodi-agnostic measures. Muscle Nerve 1992; 15:1150-1154. 16. Moravcová E, Bednařík J, Feit J, Sommer C. The evaluation of intraepidermal nerve fiber density in skin biopsy of polyneuropathy patients. Cesk Slov Neurol N 2005; 68/101:219-226. 17. Lauria G, Cornblath DR, Johansson O, et al. EFNS guidelines on the use of skin biopsy in the diagnosis of peripheral neuropathy. Eur J Neurol 2005; 12: 747-758. 18. Kennedy WR, Wendelschafer-Crabb G, Polydefkis M, McArthur JC. Pathology and quantitation of cutaneous innervation. In: Dyck PJ, Thomas PK, eds. Peripheral neuropathy. Philadelphia: Elsevier, 2005: 869-895. 19. Sorensen L, Molyneaux L, Yue DK. The relation among pain, sensory loss and small nerve fibers in diabetes. Diabetes Care 2006; 29: 883-887. 20. McArthur JC, Stocks EA, Hauer P, Cornblath DR, Griffin JW. Epidermal nerve fiber density: normative reference range and diagnostic efficiency. Arch Neurol 1998; 55: 1513-1520. 21. Lauria G, Holland N, Hauer P, et al. Epidermal innervation: changes with aging, topographic location, and in sensory neuropathy. J Neurol Sei 1999; 164:172-178. 22. Yarnitsky D. Quantitative sensory testing. Muscle Nerve 1997; 20:198-204. 24 SCRIPTA MEDICA/Volume 82/No. 1/2009 http://www.med.muni.cz/biomedjournal/ Department of Neurology http://www.med.muni.cz/biomedjournal/ SCRIPTA MEDICA /Volume 82/No. 1 / 2009 25 Sleep research laboratory 26 SCRIPTA MEDICA/Volume 82/No. 1/2009 http://www.med.muni.cz/biomedjournal/ REVIEW CRITICAL ILLNESS POLYNEUROMYOPATHY - A WELL-KNOWN BUT STILL CONTROVERSIAL ENTITY Bednařík J.1, Vondráček P.2 1 Department of Neurology, Faculty of Medicine, Masaryk University and Faculty Hospital Brno 2 Department of Paediatric Neurology, Faculty of Medicine, Masaryk University and Faculty Hospital Brno ABSTRACT Received after revision February 2009 KEYWORDS Critical illness Polyneuropathy Myopathy Two main clinical, pathological and electrophysiological types of acquired neuromuscular involvement in critically ill patients have been described in the past two decades: critical illness polyneuropathy and critical illness myopathy. However, there, still exist many controversies and unresolved questions regarding definition, terminology, diagnosis, and differentiation of what appears to be a spectrum of more or less overlapping neuromusculardisorders ratherthan distinct entities, and recently a new term - critical illness polyneuromyopa-thy (CIPM) has been coined. CIPM may present with muscle weakness and failure to wean from mechanical ventilation, but is discovered more often and earlier by electrophysiological examination. In this review, the incidence, clinical, electrophysiological and histopathological features, and risk factors of CIPM will be described. Among the most important risk factors for CIPM are sepsis or systemic inflammatory response syndrome and the severity of multi-organ failure. Acquired neuromuscular weakness in critically ill patients should be regarded as a part ratherthan a complication of critical illness — dysfunction or failure of a further (neuromuscular) system. H CORRESPONDING AUTHOR Bednařík! Department of Neurology Faculty of Medicine Masaryk University and Faculty Hospital Brno Jihlavská 20 625 00 Brno Czech Republic THE ICU WEAKNESS:TERMINOLOGY, DIAGNOSTIC CRITERIA AND DIFFERENTIAL DIAGNOSIS Neuromuscular weakness is often encountered in patients in the intensive care unit (ICU). Patients can be admitted to the ICU because of increasing muscle weakness and threatening respiratory failure due to an underlying neuromuscular disorder, such as Guillain-Barré syndrome (GBS) and myasthenia gravis (MG). More frequently, a new weakness appears during the ICU stay. If weakness of central origin due to encephalopathy and rarely to myelopathy is excluded, a neuromuscular weakness could be caused by exacerbation of an underlying neuromuscular disease (such as MG, motor neuron disease or muscular dystrophy), or a weakness due to persistent neuromuscular blockade after administration http://www.med.muni.cz/biomedjournal/ SCRIPTA MEDICA /Volume 82/No. 1 / 2009 27 CRITICAL ILLNESS POLYNEUROMYOPATHY of non-depolarising muscle blocking agent (NDMBA) must be taken into consideration. By far the most frequent cause of newacquired weakness of neuromuscular origin in ICU patients is, however, critical illness polyneuropathy (CIP), critical illness myopathy (CIM), or both [1,2]. In this review, we will focus mainly on those aspects of acquired neuromuscular weakness in critically ill patients that we addressed in our previous research: epidemiology, diagnosis, and aetiology. CIP was first systematically described in the early 1980s [3], while myopathy in critically ill patients was first reported in 1977 in association with the administration of corticosteroids [4]. The first paper focused on this problem in the Czech or Slovak literatures was published in 2000 [5], and subsequently also in paediatric patients [6]. The differentiation between CIP and CIM is based on the typical clinical, electrophysiological and histopathological signs of acute axonal sensorimotor polyneuropathy and myopathy, and on the assumption that most cases can be categorised as one type or the other [7,8], There is, however, increasing evidence that myopathy may co-exist with neuropathy [9,10], and that the final diagnosis - CIP, CIM or both - is critically dependent on the method used [10]. Another factor complicating diagnosis is the fact that neither CIP nor CIM are homogenous entities. Although CIP is typically sensorimotor acute axonal polyneuropathy, pure motor and pure sensory forms of CIP have also been described [10-12]. CIM is an even more heterogeneous group of muscle disorders. At least four main types of CIM have been reported: myopathy with selective loss of myosin filaments ('myosin loss myopathy', 'thick filament myopathy1), acute necrotising myopathy, non-necrotising 'cachectic' myopathy, and decreased sarcolemmic excitability [13,14].The term 'critical illness myopathy' has been coined for myopathy with loss of myosin [8]; for non-necrotising 'cachectic myopathy' with dominant type II atrophy [15]; or as a descriptive counterpart to CIP covering all types of myopathy in critically ill patients [16]. It is not known whetherall these changes share the same aetiology and represent one disease. CIP has been thought of as an easy diagnosis to make. Clinical diagnosis of CIP is based on the presence of flaccid are-flexic quadriparesis with sensory involvement; this presence is, however, difficult to prove in comatose or unco-operative patients. Although some authors have reported different distributions of muscle weakness in CIP and CIM, differentiation between neuropathy and myopathy based on clinical grounds in comatose, sedated or encephalopathic critically ill patients is highly unreliable [8,17], ELECTROPHYSIOLOGY An electrophysiological pattern of acute axonal sensorimotor polyneuropathy is not difficult to discern, but differentiation between CIP and CIM is difficult or even impossible for numerous reasons notassociated in other conditions [11,18], Routine conduction studies and needle electromyography (EMG) provide only non-specific data. Abnormal spontaneous activity and decreased amplitudes of compound muscle action potentials can occur in different lesions of a motor unit, including myopathy. Diffuse tissue oedemas of the extremities of critically ill patients could lead to false-positive sensory conduction abnormalities. The assessment of recruitment and interference in the voluntary EMG pattern is obscured by severe weakness or poor voluntary effort in most patients. Some authors have reported the usefulness of sophisticated electrophysiological methods, such as single-fibre EMG [19], quantitative electromyography (QEMG), and motor unit number estimation [18,20]. CIM is a difficult diagnosis to make using electrophysiological methods [16]. If such diagnosis is based only on electrophysiological criteria, it is considerably underdiagnosed [10]. There are several reasons for this underestimation. Motor involvement in acute axonal neuropathy and myopathy shares the same pattern onconventionalelectroneurography(ENG)and needle EMG. In the presence of an abnormal sensory neurogram, motor abnormalities have invariably been attributed to neuropathy, irrespective of the possibility of concomitant myopathy associated with sensory or sensorimotor polyneuropathy [12]. Some additional techniques useful in the detection of myopathy, such as quantitative EMG, require patient cooperation, which is lacking in most cases. Nevertheless, some studies utilising these techniques have reported predominant myopathic involvement in critically ill patients [13,18,20].Tro-jaborg et al. [18] carried out electrophysiological studies on 22 consecutive patients with critical illness-associated weakness and found electrophysiological signs of myopathy in all cases (confirmed by biopsy in nine of them), while only one patient showed electrophysiological signs of polyneuropathy. Lacomis et al. [20], studying 100 patients with new weakness, reported that electrophysiological signs of acute myopathy were three times more frequent (42%) than those of acute polyneuropathy (13 %). Rich et al. [13] reported direct muscle stimulation (DMS) findings, indicative of decreased sarcolemmic excitability, in 11 out of 14 critically ill weak patients. The newly introduced DMS method [5,13,21] enables the investigator to distinguish decreased muscle membrane excitability from other causes of muscle weakness. It has, however, some methodological pitfalls [18,21]. One of them is the use of the ratio of compound muscle action potentials (CMAPs) obtained with direct muscle stimulation (dmCMAP) and nerve stimulation (neCMAP); where the ratio ne/dmCMAP is >0.5 it is seen as a sign of decreased muscle membrane excitability [13]. This value can be found in normal muscle, and electro-clinical correlation would be rendered difficult or impossible by lack of co-operation or central palsy. The absolute value of 28 SCRIPTA MEDICA /Volume 82/No. 1/2009 http://www.med.muni.cz/biomedjournal/ 17 ■ CRITICAL ILLNESS POLYNEUROMYOPATHY dmCMAP amplitude is a more valuable indicator of decreased sarcolemmic excitability [18,21], but it is an extremely variable parameter, prone to false-positive findings [21,22]. Another pitfall in the interpretation of DMS signs of decreased muscle membrane excitability is that the histopathological correlates of this condition are unknown in man. Recently, decreased muscle membrane excitability caused by increased fast inactivation of sodium channels was documented in an animal model of myosin loss myopathy in steroid-denervated rats [23]. In our material [21], decreased muscle membrane excitability was associated with various histopathological myopathic changes in all seven patients with this particular DMS pattern in whom muscle biopsy was performed. One can speculate that decreased muscle membrane excitability is probably a part or a phase of a complex pathological process involving muscle fibres during critical illness that does not usually remain isolated as a cause of muscle weakness. The interpretation of isolated electrophysiological motor abnormalities has been a matter of discussion for the last 15 years. A substantial proportion of weak, critically ill patients (46% in our study sample, 30% in a group studied by Coakley et al. [11]) show a pure motor abnormality on conventional ENG/ EMG examination. Coakley [11] introduced the term'pure motor syndrome'for this electrophysiological pattern. There are several reports of'pure motor neuropathy', mostly attributed to the administration of non-depolarising muscle-blocking agents [23-25], but the evidence of neuropathy is based on non-specific neurophysiological motor abnormalities [24], on the absence of myopathic changes [23], or on the presence of neurogenic atrophy on muscle biopsy [25]. Interpretation of histopathological signs of'denervation atrophy'is, however, somewhat controversial (see discussion below). Lacomis et al. [20] interpreted isolated motor abnormalities as signs of myopathy and we confirmed this assumption in our study: myopathic changes on muscle biopsy were found in all six patients with pure motor syndrome chosen for biopsy and decreased muscle excitability DMS pattern in eight out of 12 cases with pure motor syndrome in our group [21]. We can thus conclude that a motor form of critical illness polyneuropathy has not been adequately proved by pathological or electrophysiological studies. Recently, early electrophysiological detection of neuromuscular involvement in critically ill patients has been reported [26], BIOPSY Histopathological changes on muscle biopsy are used as a benchmark in the diagnosis of critical illness myopathy [4] and as a gold standard for evaluation of the validity of electrophysiological parameters [7,8,10,11,18]. Latronico et al. [10] studied 24 acutely ill neurological patients with clinical and ENG-EMG signs of CIP with muscle and nerve biopsy. They found that 23 patients (96%) had a myopathy, and that 15 of these'would have been diagnosed as having only a critical illness polyneuropathy', had they not performed muscle biopsy. Similar results were obtained in a study by DeJonghe et al. [27], in which all 10 patients with an ENG-EMG diagnosis of CIP also had signs of myopathy on muscle biopsy. The pathological features of myopathy in critically ill patients are somewhat complex, but most authors classify them into three main types: • non-necrotising changes with atrophy of myofibres predominantly involving type II fibres (Figure 1), abnormal variation of muscle fibre size, angulated fibres, internalised nuclei, rimmed vacuoles, fatty degeneration of muscle fibres and fibrosis; • necrotic changes with signs of regeneration (Figure 2); • selective loss of thick myosin myofilaments (Figure 3) [8,10,15]. This classification is, however, somewhat arbitrary, with significant overlap of different changes in the same patient [8,18,28]. In contrast to the loss of myosin, considered to be a change that is pathognomic for CIM, atrophy of myofibres is less specific. Selective atrophy of type II myofibres is reported in myopathy, neuropathy, and disuse atrophy. Angular atrophic myofibres found multifocally or in small groups are traditionally interpreted as signs of denervation (Figure 4) [7,29,30]. Bolton and Breuer [7] stated that muscle biopsy in critically ill patients usually discloses 'denervation atrophy and mild muscle necrosis'and they interpret these findings as signs of critical illness polyneuropathy. Others, however, have described atrophic angular fibres as being characteristic signs of critical illness myopathy [15,31 ]. These discrepancies have resulted in striking differences in the classification of muscle biopsy changes in critically ill patients. Most authors who have systematically examined histopathological muscle changes in critically ill patients reported dominant myopathic changes [10,18,21,28,30]. The study carried out by Sander et al. [32] in eight quadriplegic areflexic patients with electrophysiological findings suggestive of CIP found normal nerve biopsy, but myopathic changes on muscle biopsy including myosin loss in all cases. Moreover, a significant proportion of cases examined has been interpreted as having a 'mixed' myopathic and neuropathic pattern. In particular, non-specific changes with dominant type II fibre atrophy were reported to be associated with CIP [15], but we found all types of myopathic changes to be associated with neuropathy [21]. Histopathological and immunopathological approaches are extremely valuable in the detection of various myopathic changes, but are less specific in the detection of acute denervation, especially in association with myopathic changes. The presence of muscle histopathological changes indicative of myosin loss has been proposed as one of four http://www.med.muni.cz/biomedjournal/ SCRIPTA MEDICA /Volume 82/No. 1/2009 29 CRITICAL ILLNESS POLYNEUROMYOPATHY major diagnostic features necessary to meet research diagnostic criteria for the diagnosis of definite critical illness myopathy [4]. Biopsy is, however, not a practical screening tool for larger observational epidemiological studies [18,21,33], The methodological difficulties and reported coincidence of both types of neuromuscular involvement in the same critically ill patients led to the introduction of the descriptive term'critical illness polyneuromyopathy'(CIPM) [5,6], critical illness polyneuropathy and myopathy [10,28,34], or CRIMYNE [35]. Some authors even maintain that the concept of critical illness polyneuropathy is outdated and have suggested the use of the clinical descriptive term'critical illness weakness' followed by a description of myopathic, neuropathic, neuromuscular junction, metabolic and encephalopathic components [36]. Others have used a further descriptive term, 'ICU-acquired paresis'[27], EPIDEMIOLOGY At the beginning, the CIP-CIM interface seemed no more than a scientific curiosity, but research over the past 20 years has shown that CIP and CIM are the most frequent acute polyneuropathies and myopathies encountered in critically ill patients [37]. Although the exact incidence is unknown, due to wide variation in diagnostic criteria and patient case-mix, available data regarding the incidence of neuromuscular involvement in critically ill patients are rather impressive (Table 1): 25-57% of critically ill patients (usually defined as >7 days in ICU (most or all mechanically ventilated, most or all with associated sepsis and multiple organ failure)) show clinically symptomatic weakness [21,28,34,38,39], and electrophysiological methods reveal abnormalities suggestive of CIP orCIPM in 21-100% of these patients [11,21,26,34,38-43]. Critical illness myopathy is a primary myopathy that has only been characterised in recent years [10]. Data on its incidence are lacking; evidence is, however, mounting that CIM is at least as frequent as CIP [10,13,18,27,28]. Douglass et al. [44] reported myopathy detected clinically in 36% of patients with status asthmaticus treated with corticosteroids and mostly with non-depolarising muscle-blocking agents (NDMBA). Campellone et al. [45], in a series of 100 consecutive patients after liver transplantation, detected clinical and electrophysiological signs of myopathy in seven cases (7 %); biopsy confirmed myosin loss myopathy in five cases. AETIOLOGY The concept of critical illness myopathy and neuropathy leads to a dichotomy in aetiological considerations of both types of involvement. Recently some authors have tended to replace this approach with the more descriptive concept of'critical illness polyneuromyopathy'or'ICU-acquired paresis'[27,28,34], and significant risk factors or predictors of CIPM have been sought. Sepsis or systemic inflammatory response syndrome (SIRS) is thought to be a leading cause of CIP [3,34,43,46,47], Factors responsible for the systemic effects of sepsis, i.e. release of tumour necrosis factor (TNF), histamines and arachi-donicacid metabolites.activation ofthecomplementand cell adhesion systems, and formation of local free radicals may cause axonal degeneration [48]. As a mechanism, Bolton [48] suggested disturbances in the microcirculation of peripheral nerves. Using a bioassay by which toxic effects of patients' sera on motor neurons could be determined quantitatively, Hund etal. [49] demonstrated the presence of a low-molecular weight factor (<3 kDa) in the serum of patients with CIP that kills cultured motor neurons. There exists a growing body of evidence that sepsis could also be responsible, at least in association with other factors, for the development of CIM or a myopathic component of CIPM [10,50-52]. Recently, the presence of SIRS has been found to increase the risk of clinically symptomatic CIPM development leading to artificial ventilation and this, together with the APACHE III score, has been used to estimate the risk [34]. Critical illness myopathy was originally reported as a complication of corticosteroid administration, either alone or in association with NDMBA [4,53,54]. Corticosteroids and NDMBA may serve as triggers, especially in necrotising and myosin-loss myopathies [15]. NDMBA may play a potentiating role, by virtue of a pharmacological denervation, that facilitates the toxic effect of other agents such as corticosteroids or inflammatory mediators. The functional denervation in CIP may provide a link between CIP and CIM. Pure motor neuropathy, on the other hand, has been attributed to the administration of NDMBA [55].The causal role of NDMBA and corticosteroids in the development of CIM has been supported by numerous case reports, as well as by experimental data [56,57]. Recently, prospective studies reported controversial data on the significant independent influence of corticosteroids or NDMBA upon CIPM or ICU-acquired paresis development [27,34,40,58,59], The differences between the studies might be explained by variable dosage of the corticosteroids administered, depending on the spectrum of diagnoses leading to critical illness. Another more general pitfall in the assessment of the aetiological influence of corticosteroids (in neuromuscular involvement of critically ill patients) could be a possibly predominant impact on the development of a myopathic component that is difficult to assess reliably in larger studies. Although administration of corticosteroids and NDMBAs has come to be avoided - or the drugs have been administered at the lowest possible doses - this practice does not seem to reduce the occurrence of neuromuscular involvement in critically ill patients. The influence of these drugs upon the development of CIPM and particular pathological subtypes of neuromuscular involvement thus remains to be established. 30 SCRIPTA MEDICA /Volume 82/No. 1/2009 http://www.med.muni.cz/biomedjournal/ 00 ■ CRITICAL ILLNESS POLYNEUROMYOPATHY Table 1 Incidence of acquired neuromuscular involvement in critically ill patients (other than prolonged neuromuscular blockade): prospective observational studies Incidence of neuromuscular involvement (%) Group definition (inclusion criteria) Diagnostic criteria Study sample (No of cases) Reference 57% 28% >7 days in ICU, failure of at least 2 organs Electrophysiological criteria of CIPMafter4 weeks Clinical signs of flaccid quadriparesis + electrophysiological criteria of CIPM after 4 weeks 61 Bednariketal. 2005 [63] 84% >7 days in ICU, failure of at least 1 organ Electrophysiological abnormalities 44 Coakley etal. 1998 [11] 25% Mechanical ventilation >7 days Severe muscle weakness on day 7 95 DeJongheetal. 2002 [27] 75% Mechanical ventilation, SIRS, MOF Electrophysiological criteria of CIP after 3 weeks 73 Garnacho-Montero etal. 2001 [40] 33% Mechanical ventilation Clinical and electrophysiological criteria of CIPM 98 DeLetter etal. 2001 [34] 57% Mechanical ventilation > 3 days Clinical and electrophysiological criteria of CIP on day 14 28 Druschky etal. 2001 [39] 100% Mechanical ventilation, sepsis, MOF Electrophysiological abnormality (decrease in CMAP amplitudes and/or fibrillations on days 2-5 9 Tennilä etal. 2000 [26] 21% MOF Electrophysiological signs of CIP on discharge from ICU 33 Mohretal. 1997 [41] 70% Sepsis, MOF Electrophysiological signs of CIP 43 Wittetal. 1991 [42] 41% 82% Sepsis, MOF Clinical signs of CIP Electrophysiological signs of CIP 22 Bereketal. 1996 [38] 52% (conventional treatment) 29% (treated with insulin) > 7 days in ICU Electrophysiological signs of CIPM 206 157 Van den Berghe etal. 2001 [43] 36% Status asthmaticus, corticosteroid treatment Clinical signs of CIM 25 Douglass etal. 1992 [44] 7% Liver transplantation Clinical and electrophysiological signs of CIM 100 Campellone et al. 1998 [45] 30% > 10 days in ICU Electrophysiological signs of CRIMYNE 92 Latronicoetal. 2007 [35] SIRS = systemic inflammatory response syndrome MOF = multiple organ failure CRIMYNE = critical illness myopathy and/or neuropathy http://www.med.muni.cz/biomedjournal/ SCRIPTA MEDICA /Volume 82/No. 1/2009 31 00 CRITICAL ILLNESS POLYNEUROMYOPATHY Figure 1 Figure 2 Myosin-ATPase: Atrophy oftype 2 muscle fibres (darkfibres of Hematoxylin-eosin staining: necrotising myopathy with retype 2, light fibres oftype 1) generating muscle fibres Figure 3 Figure 4 Electron microscopy from tibial anterior muscle: thick fila- Hematoxylin-eosin staining: atrophic, polygonal, partially re- ment loss with relative preservation of thin filaments and Z- generating muscle fibres in groups as signs of denervation lines (arrows) Multiple organ failure is a hallmark of systemic critical illness and failures of individual organ systems have also been proposed as possible causative factors for the development of neuromuscular involvement. A significant association between CIP development and the duration of mechanical ventilation and neurological failure was found in a cohort of 73 critically ill patients with multiple organ dysfunction [40]. In contrast, Campellone et al. [45] found a significant association between CIM development and the severity of critical illness with the presence of renal failure. These associations between CIP or CIM and other organ failures, however, raise questions as to whether neuromuscular involvement is not simply a part of systemic critical illness and whether or not it represents just another organ failure. Among other variables reported as significantly and independently associated with increased risk of CIP development were hyperosmolality, parenteral nutrition [40], hypoalbuminaemia, hyperglycaemia [27,42,43], hyperpyrexia [60], NDMBA [40,55,61], aminoglycoside antibiotics [37], catecholamines/vasopressors [43,58], increased age [40], and female sex [27]. Most of these factors are intrinsically 32 SCRIPTA MEDICA /Volume 82/No. 1/2009 http://www.med.muni.cz/biomedjournal/ ■ CRITICAL ILLNESS POLYNEUROMYOPATHY related to sepsis and severity of critical illness and their causal relationship to CIPM is unclear [62,63]. In a recent prospective study [34], the development of critical illness polyneu-romyopathy (CIPM) was significantly associated with APACHE III score (as a quantitative index of disease severity) and the presence of SIRS. These factors were used to estimate the risk of developing CIPM. In a prospective cohort study [63], we have demonstrated an increased risk of CIPM development in the presence of SIRS and especially in its longer duration. Another variable significantly associated with CIPM development was the severity and duration of multiple and some individual organ failures, namely neurological, cardiovascular and respiratory failures (and closely correlated duration of ventilation support). The discrepancy between studies on the aetiology of neuromuscular disorders in critically ill patients could be explained by the varying severity of neuromuscular involvement in the cases studied and by different criteria for CIP, CIM or CIPM. Recent comparable prospective studies focused on clinically symptomatic CIPM in patients on mechanical ventilation [27,34], while others relied on more sensitive electrophysiological or histological criteria that are able somewhat to extend differentiation between neuropathy and myopathy. CONCLUSIONS Current knowledge of the incidence and pathophysiology of acquired neuromuscular involvement in critically ill patients is limited, which is a fact that has restrictive implications for clinical practice. Studies published to date are limited by a low number of analysed patient cases; however, inconsistent eligibility criteria and inconsistent case definitions preclude the combination of results by meta-analysis. Bet-teranimal models and improved epidemiological studies on the incidence of acquired neuromuscular involvement and on all potential risk factors with complete evaluation of all ICU patients at risk are required. Acquired neuromuscular weakness as a result of critical illness is more common than previously recognised and may result in substantial excess morbidity, mortality, and costs. It is questionable whether forthcoming aetiological studies on neuromuscular involvement in critically ill patients should adhere to the concept of CIPM or try to differentiate different components of neuromuscular component and assess them separately. The possibility of achieving reliable differentiation of myopathic and neuropathic components in larger prospective studies is limited not only by the inherent limitations of every method, but also by the frequent association between myopathy and neuropathy in critically ill patients. The neuromuscular system is probably diffusely, although patchily, involved in critical illness. Neuromuscular involvement in critically ill patients probably represents a continuum of neurogenic and myogenic changes of varying severity and progression overtime. Studies utilising clinical and electrophysiological techniques should avoid strict categorisation of cases into CIP or CIM, but mightdo betterto employ description of the main electrophysiological syndromes: isolated sensory neuropathy, isolated motor syndrome, mixed sensorimotor involvement, and decreased sarcolemmic excitability. Forced categorisation into myopathy and polyneuropathy frequently leads to misinterpretation of abnormalities and discrepancies between studies. Muscle biopsy,as an invasive technique, is not a suitable diagnostic tool for large epidemiological studies. Nevertheless, it would be helpful to design a large multicentre study employing biopsy and differentiating all the histopathological types of CIM to address the dilemmas of the incidence of CIP and different types of CIM; of the correlation between electrophysiological findings and histopathological subtypes; and of the risk factors for different pathological subtypes of neuromuscular involvement. Acquired neuromuscular weakness in critically ill patients should be regarded as dysfunction or failure of a further (neuromuscular) system that could be detected early during critical illness. Records of neuromuscular function should be included as measures of outcome in all interventional ICU studies. Measurements to detect early signs of neuromuscular dysfunction (such as, possibly, decrease of CMAP amplitude, as shown recently by Latronico et al. [35], and decrease of sarcolemmic excitability) should be sought and validated. 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Critical illness polyneuropathy: clinical findings and cell culture assay of neurotoxicity assessed by a prospective study. Intensive Care Med 2001; 27:686-693. 40. Garnacho-Montero J, Madrazo-Osuna J, Garcia-Garmen-dia JL, et al. Critical illness polyneuropathy: risk factors and clinical consequences. A cohort study in septic patients. Intensive Care Med 2001; 27:1288-1296. 41. Mohr M, Englisch L, Roth A, Burchardi H, Zielmann S. Effects of early treatment with immunoglobulin on critical illness polyneuropathy following multiple organ failure and gram-negative sepsis. Intensive Care Med 1997; 23: 1144-1149. 42. Witt NJ, Zochodne DW, Bolton CF, et al. Peripheral nerve function in sepsis and multiple organ failure. Chest 1991; 99:176-184. 43. Van den Berghe G, Wouters P, Weekers F, et al. Intensive insulin therapy in the critically ill patients. N Engl J Med 2001;345:1359-1367. 44. Douglass JA.Tuxen DV, Horne M, et al. Myopathy in severe asthma. Am RevRespir Dis 1992; 146:517-519. 45. Campellone JV, Lacomis D, Kramer DJ, van Cott AC, Giuliani MJ. Acute myopathy after liver transplantation. Neurology 1998; 50: 46-53. 46. Zochodne DW, Bolton CF, Wells GA, et al. Critical illness polyneuropathy. A complication of sepsis and multiple organ failure. Brain 1987; 110: 819-841. 47. Visser LH. Critical illness polyneuropathy and myopathy: clinical features, risk factors and prognosis. Eur J Neurol 2006,13:1203-1212. 48. Bolton CF. Sepsis and the systemic inflammatory response syndrome: neuromuscular manifestations. Crit Care Med 1996;24:1408-1416. 49. Hund E, Herkert M, Becker C-M, Hacke W. A humoral neurotoxic factor in sera of patients with critical illness polyneuropathy. Ann Neurol 1996; 40: 539. 50. Bolton CF, Ramsay DA, Rutledge F. Acute quadriplegic myopathy (AQM), sepsis and the systemic inflammatory response syndrome (SIRS). Neurology 1998; 50: A242. 51. Showalter CJ, Engel AG. Acute quadriplegic myopathy: analysis of myosin isoforms and evidence of calpain-me-diated proteolysis. Muscle Nerve 1997; 20: 316-322. CRITICAL ILLNESS POLYNEUROMYOPATHY 52. Friedrich O. Critical illness myopathy: sepsis-mediated failure of the peripheral nervous system. Eur J Anaesthe-siol Suppl 2008; 42: 73-82. 53. Hirano M, Ott BR, Raps EC, et al. Acute quadriplegic myopathy: a complication of treatment with steroids, nondepolarizing blocking agents, or both. Neurology 1992; 42: 2082-2087. 54. Segredo V, Caldwell JE, Matthay MA, Sharma ML, Gruenke LD, Miller RD. Persistent paralysis in critically ill patients after long-term administration of vecuronium. N Engl J Med 1992; 327: 524-528. 55. Geller TJ, Kaiboriboon K, Fenton GA, Hayat GR. Vecuro-nium-associated axonal motor neuropathy: a variant of critical illness polyneuropathy? Neuromuscul Disord 2001;11:579-582. 56. Massa R, Carpenter S, Holland P, Karpati G. Loss and renewal of thick myofilaments in glucocorticoid treated rat soleus after denervation and reinnervation. Muscle Nerve 1992;15:1290-1298. 57. DuBois DC, Almon RR. A possible role for glucocorticoids in denervation atrophy. Muscle Nerve 1981; 4: 370-373. 58. Thiele Rl, Jakog H, Hund E, et al. Sepsis and catecholamine support are the major risk factors for critical illness polyneuropathy after open heart surgery. Thorac Cardio-vascSurg 2000;48:145-150. 59. Leatherman JW, Fluegel LW, Davis WS, Davies SF, Iber C. Muscle weakness in mechanically ventilated patients with severe asthma. Am J Respir Crit Care Med 1996; 153: 1686-1690. 60. Wilmshurst PT, Treacher DF, Lantos PL, Wiles CM. Critical illness polyneuropathy following severe hyperpyrexia. QJM 1995; 88: 351-355. 61. Hund E. Neuromuscular complications in the ICU: the spectrum of critical illness-related conditions causing muscular weakness and weaning failure. J Neurol Sei 1996;136:10-16. 62. Deem S, Lee CM, Curtis JR. Acquired neuromuscular disorders in the intensive care unit. Am J Respir Crit Care Med 2003;168:735-739. 63. Bednarik J, Vondráček P, Dusek L, Moravcova E, Cundrle I. Risk factors for critical illness polyneuromyopathy. J Neurol 2005; 252: 343-351. http://www.med.muni.cz/biomedjournal/ SCRIPTA MEDICA /Volume 82/No. 1/2009 35 00 A meeting of the Department of Neurology management Sonography laboratory 36 SCRIPTA MEDICA/Volume 82/No. 1/2009 http://www.med.muni.cz/biomedjournal/ Lecture hall Speech therapy examination http://www.med.muni.cz/biomedjournal/ SCRIPTA MEDICA /Volume 82/No. 1 / 2009 37 ORIGINAL RESEARCH THE RESULTS AND CONTRIBUTION OF ELECTROPHYSIOLOGICAL EXAMINATION IN PATIENTS WITH LUMBAR SPINAL STENOSIS Mičánková Adamová B., Voháňka S. Department of Neurology, Faculty of Medicine, Masaryk University, and Faculty Hospital Brno Received after revision February 2009 8-TT KEYWORDS Lumbar spinal stenosis Electrophysiological examination Radiculopathy H CORRESPONDING AUTHOR Mičánková Adamová B. Department of Neurology Faculty of Medicine Masaryk University and Faculty Hospital Brno Jihlavská 20 625 00 Brno Czech Republic ABSTRACT Monoradicular or polyradicular lumbosacral involvement is typical of lumbar spinal stenosis. Needle electrode examination is considered the most useful procedure for the evaluation of patients with suspected radiculopathy. The clinical relevance of motor and somatosensory evoked potentials is, however, uncertain. The aim of this study was to evaluate the results of electrophysiological tests in patients with lumbar spinal stenosis and to assess the contribution of electrophysiological tests made to diagnosis. The study group consisted of 102 patients (44 males, 58 females, aged 62 +/-13 years) with clinically symptomatic lumbar spinal stenosis documented by computed tomography scan, all of whom volunteered to participate in the study. All patients underwent electrophysiological examination, which included needle electromyography and nerve conduction studies of the lower extremities, somatosensory evoked potentials of tibial, sural and superficial sensory peroneal nerves, and motor evoked potentials to the abductor hallucis muscle and the tibialis anterior muscle. On the basis of nerve conduction studies and needle electromyography, the presence of radiculopathy was established in 70.6% of patients with lumbar spinal stenosis; polyradicular involvement (46.1 % of the patients) was more common than monoradicular involvement (24.5% of the patients). Involvement of the L4 root was established in 37.2% of the patients, L5 root in 51.9%, and SI root in 50.9% of the patients. Abnormal motor evoked potentials were found in 30.7% of the patients and abnormal somatosensory evoked potentials in 58.8% of the patients with lumbar spinal stenosis. Normal needle electromyography and nerve conduction studies were recorded in 18.6% of the patients. When abnormalities of any evoked potentials were considered, the number of patients with normal electrophysiological findings was reduced to 12.7%. Nerve conduction studies and needle electromyography are the most useful electrophysiological examinations for the 38 SCRIPTA MEDICA/Volume 82/No. 1/2009 http://www.med.muni.cz/biomedjournal/ THE RESULTS AND CONTRIBUTION OF ELECTROPHYSIOLOGICAL EXAMINATION IN PATIENTS WITH LUMBAR SPINAL STENOSIS evaluation of suspected radiculopathies in patients with lumbar spinal stenosis. Involvement of L5 and SI roots is the most common.The diagnostic contribution of evoked potentials is of limited value in patients with lumbar spinal stenosis. ABBREVIATIONS USED LSS - lumbar spinal stenosis NC - neurogenic claudication MEP - motor evoked potential SEP - somatosensory evoked potential CT - computed tomography SD - standard deviation CMAP - compound muscle action potential CMCT- central motor conduction time MUAP - motor unit action potential SNAP - sensory nerve action potential EMG - electromyography NCS - nerve conduction study AH muscle-abductor hallucis muscle TA muscle - tibialis anterior muscle INTRODUCTION Lumbar spinal stenosis is defined as any type of narrowing of the spinal canal, nerve root canals, or intervertebral foramina [1]. In the pathogenesis of LSS, the degenerative process in the lumbar spine is the main component producing compression of neural tissue in the spinal and/or nerve root canal [2], LSS can give rise to several clinical syndromes: neurogenic claudication, low back pain and/or radiculopathy, and chronic cauda equina syndrome. Postural dependency is a hallmark of the symptoms of LSS. Spinal extension narrows the spinal canal and exacerbates symptoms, whereas spinal flexion increases the dimensions of the spinal canal and reduces symptoms [3,4, 5]. NC is typical of LSS and is characterised by intermittent pain and paresthesia of the leg(s), most often in a lumbosacral root distribution, followed by weakness apparent on walking or standing [6,7]. The incidence of NC is reported at 11 %-100% in patients with LSS; the mean calculated from 32 studies is 62%. LSS is currently the most common diagnosis for individuals over the age of 65 undergoing spinal surgery [8], The diagnosis of LSS is based on the results of clinical examination and radiological documentation of narrowing of the lumbar spinal canal [9, 10]. Whereas clinical examination, even in severe LSS, has shown no specific sensory-motor deficit, electrophysiological recordings have indicated a neurogenic disorder within the lumbar spine of a majority of the patients. Electrophysiological recordings thus supplement neurological examination when the clinical relevance of a ra-diologically suspected LSS needs to be confirmed [11], Electrophysiological examination in LSS is intended to detect lumbosacral root involvement (radiculopathy). EMG, especially needle electrode examination, is considered the most useful procedure in the detection of radiculopathy. Nerve conduction studies may yield abnormal results only rarely in radiculopathies, but are essential to the elimination of other conditions that may produce similar symptoms and signs [12]. Further, examination of SEPs and MEPs is used in different degree, but consensus on the contribution of evoked potentials in the diagnostics of LSS has not been established and the clinical relevance of MEPs and SEPs is uncertain [13], Initially, the contribution of evoked potentials to the diagnostics of radiculopathy was overestimated. Electrophysiological examination in LSS patients is also used to exclude other peripheral neurogenic lesions (for example diabetic polyneuropathy). The clinical differentiation between lumbosacral polyradicular disease (typical of LSS) and generalised peripheral neuropathies of the distal-axonal type (typical of diabetic patients) can sometimes be difficult [14]. Electro-diagnostic testing is quite specific and reasonably sensitive in diagnosing spinal stenosis in comparison with low back pain or asymptomatic persons, and can detect neuromuscular diseases mimicking stenosis [15], EMG findings may vary in patients with LSS. The most frequent finding (approximately 50% of the patients) is bilateral multiple lumbosacral radiculopathy (cauda equina lesion). Polyradicular lumbosacral lesions are often asymmetrical, and usually involve the lower lumbosacral roots, especially SI. Chronic neurogenic MUAP changes tend to be prominent in needle EMG, whereas fibrillation potentials are often restricted to the more distal muscles of the myotomes. In some, two distinct lumbosacral radiculopathies occur either symmetrically or asymmetrically. In others.an isolated radiculopathy, almost always either an L5 or an SI lesion, is found. Non-diagnostic abnormalities are encountered in some patients. Bilaterally absent H waves associated with normal sural SNAPs and a normal needle EMG of the lower extremities are common. Fibrillation potentials in a single limb muscle, most often one innervated by the SI root, are sometimes encountered. In the remaining patients, extensive EMG examination is normal [16]. Magnetic transcranial and spinal stimulation of the motor pathway is a painless and safe neurophysiological technique for the examination of the central and proximal peripheral motor pathways [17]. Transcranial brain stimulation and paravertebral magnetic root stimulation are reliable diagnostic methods for the investigation of patients with radiculopathies. Their advantage is that disturbances of the motor functions in the nerve roots can be detected and, because of the different MEP patterns, localisation of the compression can often be established [18]. The clinical relevance of this examination is uncertain. False negative findings in MEP http://www.med.muni.cz/biomedjournal/ SCRIPTA MEDICA /Volume 82/No. 1/2009 39 THE RESULTS AND CONTRIBUTION OF ELECTROPHYSIOLOGICAL EXAMINATION IN PATIENTS WITH LUMBAR SPINAL STENOSIS examination in patients with LSS may be as high as 75% [19, 20]. One source of such errors may be the fact that it is difficult to stimulate the nerve roots supra maximally. No electrophysiological examination has attracted as much attention, or for that matter evoked so much controversy, as SEPs in radiculopathy. The theoretical advantage is that SEPs evaluate a sensitive part of the nerve root, including the preganglionic part of the sensitive pathway; abnormality is immediately present at the start of radiculopathy and images are provided of both axonal and demyelinating involvement. The normative data of latencies and amplitudes are heterogeneous. False-negative findings in SEP examination in patients with LSS may be as high as 65% [19, 20]. In contrast, altered tibial SEPs were observed in 61.5% of patients with LSS in another study [21], The aim of the study was to evaluate the results of electrophysiological tests in patients with LSS and to assess the contribution of electrophysiological tests made to diagnosis. MATERIALS AND METHODS Patients with LSS One hundred and two patients (44 males, 58 females, aged 62 +/-13 years) were recruited consecutively from a total of 132 patients with LSS treated and observed in the Department of Neurology, Faculty Hospital, Brno, between 1998 and 2001. Inclusion criteria for patients with LSS: clinically symptomatic LSS (NC and/or low back pain) at least one level LSS documented by CT scans absence of hip and/or knee joint degenerative arthritis limiting walking absence of diabetes mellitus or other disease causing polyneuropathy no serious cardiac disease Electrophysiological examination All patients underwent electrophysiological examination, which included: • Needle electromyography and nerve conduction studies of the lower extremities (Table 1). Needle EMG centred on the muscles of L4, L5, SI myotomes bilaterally, with evaluation of abnormal spontaneous activity and analysis of action potentials of motor units. A conventional EMG device was used to perform the nerve conduction studies from temperature-checked skin. Motor conduction studies of the peroneal nerve (muscle responses recorded via surface electrodes placed over the extensor digitorum brevis muscle) and the tibial nerve (muscle responses recorded via surface electrodes placed over the abductor hallucis muscle) were performed bilaterally. Sensory conduction studies of the sural and superficial peroneal nerves were carried out employing an antidromic surface technique. The F-wave responses of the tibial and peroneal nerves and the H-reflex of the soleus muscle were recorded bilaterally. Submaximal stimuli with increasing voltage were delivered and facilitation was used to provide the maximum H-reflex amplitude. • Somatosensory evoked potentials of tibial, sural, and superficial sensory peroneal nerves. Electrical stimulation of the tibial nerve was applied posterior to the medial ankle, the sural nerve was stimulated posterior to the lateral ankle, and the superficial peroneal nerve was stimulated in the join of both ankles at a distance of one third from the lateral ankle. Scalp registration was used in all examined nerves, while registration over the spinosus process of vertebra LI was used for the tibial nerve. The latency of potentials P40 and N45 and the amplitude of P40/N45 were evaluated in all examined nerves, while the latency of potential N22 was evaluated in the tibial nerve. Our electrophysiological laboratory has generated our own reference values for all the parameters, and latencies are correlated to body height. Prolongation of latencies of more than 3 SD above average, unelicited responses, side-to-side differences in latencies N22 or P40 of more than 3 SD above average, and reduction of amplitude P 40/N45 of more than 3 SD below average (after logarithmic transformation) were considered abnormal. • Motor evoked potentials to the abductor hallucis muscle and tibialis anterior muscle. A Magstim 200 was used for transcranial and spinal stimulation. In the course of spinal stimulation, the coil was located one centimetre to one side of the centre of the low lumbar spine; for transcranial stimulation, the coil was located above the motor cortex and facilitation was used. Supramaximal stimulation was applied. Spinal latency, cortical latency, CMCT, and amplitude of cortical response were determined. Unelicited responses upon transcranial and spinal stimulation, prolongation of latencies of more than 3 SD above average, and an amplitude of cortical response of less than six per cent of the amplitude of CMAP (our own reference values) were considered abnormal. Radiological examination The patients were radiologically examined according to the following protocol: 1 A plain radiograph of the lumbar spine was taken, with assessment for the presence of spondylarthrosis, scoliosis, and degenerative or isthmic spondylolisthesis. 2. CT axial scans atthree levels (L3-S1) were performed.The following standard parameters of the spinal canal were measured: 40 SCRIPTA MEDICA /Volume 82/No. 1/2009 http://www.med.muni.cz/biomedjournal/ THE RESULTS AND CONTRIBUTION OF ELECTROPHYSIOLOGICAL EXAMINATION IN PATIENTS WITH LUMBAR SPINAL STENOSIS Table 1 Nerve conduction studies (NCS) of the lower extremities Nerve conduction studies Nerve Motor conduction studies peroneal nerve tibial nerve Sensory conduction studies superficial peroneal nerve sural nerve Late responses F-wave of tibial nerve F-wave of peroneal nerve soleus H-reflex Table 2 Evaluation of the nerve conduction studies and needle EMG Type of involvement Normal finding Radiculopathy Polyneuropathy monoradiculopathy polyradiculopathy together Number of patients 19.0 25.0 47.0 72.0 12.0 In % of cases 18.6 24.5 46.1 70.6 11.7 • Anteroposterior diameter of the spinal canal at the level of the middle of the L3, L4, and L5 vertebrae. • Transverse interarticular diameter (between ventral margins of facet joints) at the level of the upper margins of the L3/4, L4/5, and L5/S1 discs. • Lateral recess diameter bilaterally at the same levels as the transverse diameter. CT criteria of spinal stenosis were based on our own normal data [22]: • Central stenosis: anteroposterior diameter <11.7mm and/or transverse diameter<16.0mm • Lateral stenosis: lateral recess diameter <5.2 mm RESULTS Nerve conduction studies and needle EMG The presence of radiculopathy and/or polyneuropathy in patients with LSS was established on the basis of NCS and needle EMG. The criteria for radiculopathy included the presence of abnormal spontaneous activity or chronic neurogenic MUAP changes in two or more muscles that receive innervation from the same root, preferably via different peripheral nerves. Abnormal values for the soleus H-reflex amplitude were also used to assess SI radiculopathy. If clinical suspicion of radiculopathy existed and needle EMG of the muscles of this myotome was normal but the H-reflex of the soleus muscle was abnormal, it was considered sufficient to confirm radiculopathy SI. If the H-reflex proved impossible to elicit, or a side-to-side difference in the amplitude of more than 50% in comparison with the healthy side emerged, the findings were considered abnormal. The criteria for polyneuropathy were non-elicitability or reduced amplitude in the sensory neurogram of the lower extremities, bilaterally abnormal values of the soleus H-reflex, and abnormal EMG needle findings in only the distal muscles of the lower extremities. The presence of radiculopathy was established in 70.6% of the patients with LSS; polyradicular involvement (46.1 % of the patients) was more common than monoradicular involvement (24.5% of the patients). Normal needle EMG and NCS were recorded in 18.6% of the LSS patients and polyneuropathy was established in 11.7% of the patients (Table 2). The involvement of the particular roots was also established in patients with LSS (number of patients with radiculopathy in at least one lower extremity). Involvement of the L4 root emerged in 37.2% of the patients, L5 root in 51.9%, and SI root in 50.9% (Table 3). Evoked potentials (MEPs, SEPs) Motor evoked potentials were evaluated in 101 patients with LSS (one patient was exempted from this examination because he had a pacemaker). Abnormal MEPs to the abductor hallucis muscle and/or to the tibialis anterior muscle were found in 31 patients (30.7%) (Table 4). On detailed analysis of MEPs it was found that prolongation of spinal latency or non-elicitability of spinal response to AH muscle and/or TA http://www.med.muni.cz/biomedjournal/ SCRIPTA MEDICA /Volume 82/No. 1/2009 41 THE RESULTS AND CONTRIBUTION OF ELECTROPHYSIOLOGICAL EXAMINATION IN PATIENTS WITH LUMBAR SPINAL STENOSIS Table 3 The presence of involvement of lumbosacral roots based on nerve conduction studies and needle EMG Distribution of radiculopathy L4 L5 s, other Number of patients with radiculopathy in at least one lower extremity 38.0 53.0 52.0 0.0 In % of cases 37.2 51.9 50.9 0.0 Table 4 Results of motor evoked potential examination Type of MEP examination MEP to AH muscle MEP to TA muscle Number of patients with abnormal MEP in at least one lower extremity 18.0 21.0 In % of cases 17.8 20.8 Table 5 Results of somatosensory evoked potential examination Type of SEP examination SEP of tibial nerve SEP of superficial sensory peroneal nerve SEP of sural nerve Number of patients with abnormal SEP in at least one lower extremity 48.0 36.0 40.0 In % of cases 47.1 35.3 39.2 muscle had occurred in 27 patients. Three patients exhibited abnormal spinal response and an abnormal value of CMCT contemporaneously. One patient exhibited a low amplitude of cortical response in comparison with the CMAP amplitude. No isolated abnormality of CMCT to AH and/orTA muscle appeared. Contemporaneous abnormality of MEP to TA muscle and to AH muscle was found in 8 patients, only to TA muscle in 13 patients, and only to AH muscle in 10 patients. Abnormal SEPs were found in 60 patients with LSS (58.8%) (abnormality of SEPs in at least one of the three nerves evaluated) (Table 5). Unambiguous damage to the peripheral part of the somatosensory pathway predominated. Abnormal SEPs of the tibial nerve in at least one lower extremity were found in 47.1 % of the patients, abnormal SEPs of the superficial sensory peroneal nerve in 35.3%, and SEPs of the sural nerve in 39.2%. Contemporaneous abnormality of all three types of SEP was found in 26 patients (25.5%). Of 19 patients with normal EMG, one patient showed abnormal MEPs, 4 patients abnormal SEPs, and one patient abnormal SEPs as well as MEPs. When abnormalities of any evoked potential (MEP, SEP) were considered, the number of patients with normal electrophysiological findings was reduced from 18.6% to 12.7% (Figure 1). DISCUSSION It is believed that the presence of radicular or (possibly) polyradicular involvement is a typical electrophysiological finding in patients with LSS. In this study, the presence of radiculopathy was established in 70.6% of the patients with LSS, with polyradicular involvement predominating (46.1 % of the patients). These results do not disagree with the literature. In a prior study, needle EMG and nerve conduction studies revealed pathology in 75% of patients with LSS [23], The most frequent findings in LSS patients are considered bilateral, multiple lumbosacral radiculopathies (in about 50% of the patients), and in approximately 20% of the patients the presence of monoradiculopathy is determined. Lower lumbosacral roots, especially L5 and SI, are afflicted most often in patients with LSS [16]. In a study involving 200 patients with lumbosacral radiculopathy, L5 radiculopathy was recorded in 47.6% of the patients, SI radiculopathy in 30%, 42 SCRIPTA MEDICA /Volume 82/No. 1/2009 http://www.med.muni.cz/biomedjournal/ 00 THE RESULTS AND CONTRIBUTION OF ELECTROPHYSIOLOGICAL EXAMINATION IN PATIENTS WITH LUMBAR SPINAL STENOSIS 100 90 80 OJ TO Q_ 70 60 GO GO _l 50 4— O 40 30 20 10 0 86.3 87.3 ■ 81.4 1-------------1-------------' 1-------------1-------------' EMG/NCS EMG/NCS+SEP EMG/NCS+SEP+MEP Figure 1 Diagnostic contribution of electrophysiological tests in patients with LSS and L4 radiculopathy in 17.2% [24]. This study found L5 radiculopathy in 51.9% of the patients and SI radiculopathy in 50.9% of them; L4 root lesion was less common, in 37.2% of the patients. The higher percentage of the incidence of L4, L5 and SI radiculopathy in this paper may be due to the selected patients with LSS, in whom the presence of multiple radiculopathy is supposed. One patient was unsuitable for MEP examination because of a pacemaker. Abnormal MEPs to the AH muscle and/or to the TA muscle were found in 31 patients (30.7 %). A study evaluating MEPs to the TA muscle in patients with LSS demonstrated abnormal findings in 42.3% of its subjects [21]. In our study, abnormal MEPs to the TA muscle (separately or in combination with abnormal MEPs to the AH muscle) were found in 20.8% of the patients. In another study, 65% of the patients (of 43 patients with LSS) exhibited abnormal MEPs (prolongation of central motor conduction time - CMCTM) [25]. The incidence of abnormal MEPs in our study was substantially lower. The difference in the results may be partially explained by the fact that the latter study evaluated MEPs in three muscles in the lower extremity (quadriceps femoris muscle, TA muscle, and extensor digitorum brevis muscle) rather than our two, and that a "normal range" of latencies was defined as mean + 2 SD (in our study mean + 3 SD), thus increasing the probability of abnormal findings. Furthermore, the difference with the results of the study that demonstrated abnormal MEPs in 52.6% of the patients with acute radiculopathy is probably generated by different criteria for the definition of abnormal MEP findings (results that exceeded the mean + 2 SD values were considered pathological and right/left difference amplitudes of more than 50% were also considered abnormal) [26], When the MEP results in our study were analysed in detail, it was established that prolongation of spinal latency or non-elicitability of responses on spinal stimulation to AH muscle and/or to TA muscle (altogether 27 patients, i.e. 87% of abnormal MEPs) predominated among the abnormal findings, which is thought of as an indicator of lateral compression of the nerve root. This finding is possibly to be expected in patients with LSS because of the frequent presence of lateral stenosis. Three patients exhibited abnormal spinal responses and contemporaneous abnormal CMCT, which may possibly be interpreted as a combination of medial and lateral compression of the nerve root. No isolated abnormality of CMCT to the AH muscle and/or to the TA muscle (implying medial compression of the root) appeared. The results of the current study did not confirm those of Bischoff et a I., which appeared to demonstrate that there are no major differences between MEPs and EMG in terms of their sensitivity in detecting nerve root compression [18]. In the current study, the sensitivity of EMG exceeded that of MEPs.The different results of our study and that of Bischoff et al. can be elucidated by the different inclusion criteria. Bischoff investigated patients with clinically symptomatic radiculopathy with frequent occurrence of muscle weakness and the correlation between muscle weakness http://www.med.muni.cz/biomedjournal/ SCRIPTA MEDICA /Volume 82/No. 1/2009 43 00 THE RESULTS AND CONTRIBUTION OF ELECTROPHYSIOLOGICAL EXAMINATION IN PATIENTS WITH LUMBAR SPINAL STENOSIS and pathological MEP latency was demonstrated (that's why the sensitivity of MEPs was high). On the contrary, in the study of Bischoff only the presence of spontaneous EMG activity in muscles was regarded as a pathological finding but in our study the change of parameters of MUAPs was also regarded as a pathological finding (that's why the sensitivity of needle EMG was high in our study). Abnormal SEPs were found in 60 patients with LSS (58.8%) in our study (implying abnormality of SEPs in at least one of three nerves evaluated). In the literature, the positive SEP figures range from 20% to 84% in patients with radiculopathy [27, 28]. The results are substantially influenced by the type of stimulation and definitions of abnormal values. The literature indicates that SEPs make a major contribution in patients with LSS in comparison with the contribution made by SEPs in isolated radiculopathy, possibly because LSS is associated with multiple radiculopathy and a concomitant higher probability of abnormal SEPs [29, 30]. The results in the current study are similar to those of Leinonen, where altered tibial SEPs were observed in 16 of 26 patients with LSS evaluated (in our study, abnormal tibial SEPs were found in 47.1 % of the patients) [21], Of 19 patients with normal EMG, abnormal MEPs appeared in one, abnormal SEPs in 4 patients, and abnormalities of both SEPs and MEPs appeared in one patient. When abnormalities of any evoked potentials (MEPs, SEPs) were considered, the number of patients with normal electrophysiological findings was reduced from 18.6% to 12.7%. We may conclude that a diagnostic contribution of evoked potentials was documented in LSS patients with normal needle EMG and conduction studies of the lower extremities. CONCLUSIONS Nerve conduction studies and needle EMG are the most useful electrophysiological examinations for the evaluation of suspected radiculopathies in patients with LSS. The involvement of L5 and SI roots is the most common. The diagnostic contribution of evoked potentials (SEPs, MEPs) has limited value in patients with LSS. REFERENCES 1. Arnoldi CC, Brodsky AE, Cachoix J, et al. Lumbar spinal stenosis and nerve root entrapment. Syndromes, definition, and classification. Clin Orthop 1976; 115: 4-5. 2. Airaksinen O, Herno A, Turunen V, Saari T, Suomlainen 0. Surgical outcome of 438 patients treated surgically for lumbar spinal stenosis. Spine 1997; 22 [19]: 2278-2282. 3. Penning L. Functional pathology of lumbar spinal stenosis. Clin Biomech 1992; 7: 3-17. 4. Schonstrom N, Lindahl S, Willen J, Hansson T. Dynamic changes in the dimensions of the lumbar spinal canal: an experimental study in vitro. J Orthop Res 1989; 7(1): 115-121. 5. Sortland 0, Magnaes B, HaugeT. Functional myelography with metrizamide in the diagnosis of lumbar spinal stenosis. Acta Radiol Suppl 1977; 355:42-54. 6. Hall S, Bartleson JD, Onofrio BM, et al. Lumbar spinal stenosis. Clinical features, diagnostic procedures, and results of surgical treatment in 68 patients. Ann Intern Med 1985; 103:271-275. 7. London SF, England JD. Dynamic F waves in neurogenic claudication. Muscle Nerve 1991; 14:457-461. 8. Turner JA, Ersek M, Herron L, Deyo R. Surgery for lumbar spinal stenosis: an attempted meta-analysis of the literature. Spine 1992; 17:1-8. 9. Getty CJ. Lumbar spinal stenosis: the clinical spectrum and the results of operation. J Bone Joint Surg Br 1980; 62:481-485. 10. Uden A, Johnsson KE, Jonsson K, Pettersson H. Myelography in the elderly and the diagnosis of spinal stenosis. Spine 1985; 10:171-174. 11. Egli D, Hausmann O, Schmid M, et al. Lumbar spinal stenosis: Assessment of cauda equina involvement by electrophysiological recordings. J Neurol 2007; 254: 741-750. 12. Fisher MA. Electrophysiology of radiculopathies. Clin Neurophysiol 2002; 113: 317-335. 13. Dvorak J, Herdmann J, Vohanka S. Neurophysiologic assessment in patients with lumbar spinal stenosis. In: Gunzburg R, Szpalski M, eds. Lumbar Spinal Stenosis. Philadelphia: Lippincott Williams and Wilkins, 2000: 125-135. 14. Adamova B, Vohanka S, Dušek L. Differential diagnostics in patients with mild lumbar spinal stenosis: the contributions and limits of various tests. Eur Spine J 2003; 12: 190-196. 15. Haig AJ, Tong HC, Yamakawa KSJ, et al. The sensitivity and specificity of electrodiagnostic testing for the clinical syndrome of lumbar spinal stenosis. Spine 2005; 30 [23]: 2667-2676. 16. Wilbourn AJ, Aminoff MJ. AAEM minimonograph 32: the electrodiagnostic examination in patients with radiculopathies. Muscle Nerve 1998; 21:1612-1631. 17. DvořákJ, Herdmann J,Thieler R, Grob D. Magnetic stimulation of motor cortex and motor roots for painless evaluation of central and proximal peripheral motor pathways. Normal values and clinical application in disorders of the lumbar spine. Spine 1991; 16 [8]: 955-961. 18. Bischoff CH, Meyer B-U, Machetanz J, Conrad B.The value of magnetic stimulation in the diagnosis of radiculopathies. Muscle Nerve 1993; 16: 154-161. 44 SCRIPTA MEDICA /Volume 82/No. 1/2009 http://www.med.muni.cz/biomedjournal/ THE RESULTS AND CONTRIBUTION OF ELECTROPHYSIOLOGICAL EXAMINATION IN PATIENTS WITH LUMBAR SPINAL STENOSIS 19. Dvořák J. Epidemiology, physical examination and neuro-diagnostics. Spine 1998; 23: 2663-2673. 20. Dvořák J. Neurophysiologic tests in diagnosis of nerve root compression caused by disc herniation. Spine 1996; 21 (24Suppl):39S-44S. 21. Leinonen V, Maatta S, Taimela S, et al. Impaired lumbar movement perception in association with postural stability and motor- and somatosensory-evoked potentials in lumbar spinal stenosis. Spine 2002; 27 [9]: 975-983. 22. Adamová B, Bednařík J, Šmardová L, et al. Asociace mezi červikálnía lumbálnístenózou páteřního kanálu [Association between cervical and lumbar spinal canal stenosis], Cesk Slov Neurol N 2000; 5: 261-267. 23. Zileli B, Ertekin C, Zileli M, Yunten N. Diagnostic value of electrical stimulation of lumbosacral roots in lumbar spinal stenosis. Acta Neurol Scand 2002; 105 [3]: 221-227. 24. Grana EA, Kraft GH. Lumbosacral radiculopathies, distribution of electromyographic findings [abstract]. Muscle Nerve 1992; 15:1204. 25. Dvořák J, Herdmann J, Thieler R, Grob D. Magnetic stimulation of motor cortex and motor roots for painless evaluation of central and proximal peripheral motor pathways. Normal values and clinical application in disorders of the lumbar spine. Spine 1991; 16(8): 955-961. 26. Linden D, Berlit P. Comparison of late responses, EMG studies, and motor evoked potentials (MEPs) in acute lumbosacral radiculopathies. Muscle Nerve 1995; 18: 1205-1207. 27. Walk D, Fisher MA, Doundoulakis SH, Hemmati M. Somatosensory evoked responses in the evaluation of lumbosacral radiculopathy. Neurology 1991; 42:1197-1202. 28. Seyal M, Sandhu LS, MackYP. Spinal segmental somatosensory evoked potentials in lumbosacral radiculopathies. Neurology 1989; 39: 801 -805. 29. Snowden ML, Haselkorn JK, Kraft GH, et al. Dermatomal somatosensory evoked potentials in the diagnosis of lumbosacral spinal stenosis: comparison with imaging studies. Muscle Nerve 1992; 15:1036-1044. 30. Kraft GH. A physiological approach to the evaluation of lumbosacral spinal stenosis. Phys Med Rehabil Clin N Am 1998; 9 [2]: 381-389. http://www.med.muni.cz/biomedjournal/ SCRIPTA MEDICA /Volume 82/No. 1/2009 ORIGINAL RESEARCH THE VALUE OF INTRATHECAL MRZ REACTION AND OLIGOCLONAL IgG BANDS FOR DISCRIMINATION BETWEEN THE PRIMARY PROGRESSIVE AND RELAPSING REMITTING COURSES OF MULTIPLE SCLEROSIS Štourač P.1, Bednářová J.2, Hladíková M.1, Praksová P.1, Benešová Y.1, Kontrová I.1 'Department of Neurology, Faculty of Medicine, Masaryk University and Faculty Hospital Brno department of Clinical Microbiology, Faculty Hospital, Brno ABSTRACT Received after revision February 2009 KEYWORDS Multiple sclerosis Cerebrospinal fluid Oligoclonal IgG bands MRZ reaction Diagnostic significance H CORRESPONDING AUTHOR Štourač P. Department of Neurology Faculty of Medicine Masaryk University and Faculty Hospital Brno Jihlavská 20 625 00 Brno Czech Republic Generally, we diagnose two different courses of multiple sclerosis: relapsing remitting course (RR-MS) and primary progressive (PP-MS) course. Differences in pathogenesis, immunology, and prognosis are supposed between these entities. A reliable surrogate marker in cerebrospinal fluid and serum for these courses is still missing. The aims of our work were to investigate the frequency of MRZ reaction and oligoclonal IgG bands (IgG-OB) in RR-MS and PP-MS subgroups of patients and to evaluate its diagnostic significance. We examined 29 patients (n-29) with RR-MS and 10 patients with PP-MS (n-IO).The intrathecal synthesis of specific antibodies (MRZ reaction) was evaluated in the form of antibody indices calculated according to the Reiber's formula. Oligoclonal IgG bands were detected by isoelectric focusing using a commercial SEBIA kit. The MRZ reaction was positive in 2/10 patients with PP-MS and in 11/29 patients with RR-MS. IgG-OB were positive in 1/10 patients with PP-MS and in 20/29 patients with RR-MS. The calculated sensitivities for MRZ reaction were 20% in PP-MS and 37% in RR-MS; for IgG-OB they were 10% in PP-MS and 69% in RR-MS. The different frequencies of both parameters in PP-MS and RR-MS support the hypothesis about different pathogenesis and can be used as reliable surrogate markers for differential diagnosis in the context of clinical settings. ABBREVIATIONS USED CSF - cerebrospinal fluid MS- multiple sclerosis IgG -immunoglobulin G OB - oligoclonal bands IEF - isoelectric focusing MRI - magnetic resonance imaging 46 SCRIPTA MEDICA/Volume 82/No. 1/2009 http://www.med.muni.cz/biomedjournal/ 00 00 ■ THE VALUE OF INTRATHECAL MRZ REACTION AND OLIGOCLONAL IgG BANDS FOR DISCRIMINATION... INTRODUCTION Multiple sclerosis (MS) is a chronic inflammatory immune-mediated disease, probably of autoimmune origin. The diagnosis according to the International Panel on the Diagnosis of Multiple Sclerosis from 2005 revised the original McDonald's Diagnostic Criteria from 2001 [1].The Criteria formally incorporated magnetic resonance imaging (MRI) into the well-established diagnostic workup that focuses on detailed neurological history and examination and a variety of paraclinical laboratory examinations. They laid particular emphasis on determining dissemination of lesions in time and space, incorporating different types of imaging criteria into the diagnostic scheme and assessing the value of cerebrospinal fluid analysis, particularly for the diagnosis of primary progressive multiple sclerosis. The incorporation of CSF (cerebrospinal fluid) findings into the McDonald's criteria has been supported by studies suggesting that CSF analysis increases diagnostic sensitivity, though at the cost of specificity. The Revised Criteria stated the belief that a positive CSF finding was preferably based on isoelectric focusing evidence of oligoclonal IgG bands with immunofixation demonstrating that bands that are different from those in the serum or an increased IgG index or both increase the "comfort level" for the diagnosis of MS in individuals with insidious progression of disease from the onset. However, such CSF findings are not specific and may be detected in patients with progressive myelopathies of other causes, especially those associated with infections [2, 3, 4]. Besides IgG-OB the MRZ reaction (intrathecal polyspe-cific immune response against neurotropic viruses: measles, rubella, varicella zoster) is a marker for differential diagnosis between MS and other demyelinating diseases of CNS because of its higher specificity for chronic autoimmune inflammation comparing oligoclonal IgG bands and the IgG index as mentioned above [5]. The specificity of MRZ to chronic inflammatory processes of autoimmune origin such as multiple sclerosis is in the range between 84 and 94%. Combinations of M+R, M+Z or R+Z that are rarely seen in other diseases (e.g. acute infections) are clues to the presence of a chronic, especially autoimmune-type, disease. In other neurological diseases (neuroborreliosis, neurosyphilis, neurotuberculosis) the frequency of MRZ is below 1 % for the single species and far below 0.1 % for M+R+Z [6]. We previously published an evaluation of both the MRZ reaction and IgG-OB in a group of patients with OND (other neurological diseases) compared to the positivity of these parameters in a group of patients with MS [7]. The combined differential diagnostic value of both parameters for diagnosis between relapsing remitting (RR-MS) and primary progressive (PP-MS) courses has not been evaluated till now, in spite of supposed principal differences in the pathogenesis of both different MS courses. It is supposed that the RR-MS course is predominantly based on inflamma-tion-demyelination and the PP-MS course on predominant neurodegeneration and less inflammation. Our objectives were to assess the presence of MRZ reaction and IgG-OB in cohorts of patients with RR-MS and PP-MS and to evaluate their differential diagnostic potential between both courses. MATERIALS AND METHODS We evaluated the CSF and the serum for MRZ reaction and IgG-OB as the most specific and sensitive parameters of neu-roinflammation in 29 patients (n-29) with RR-MS and 10 patients (n-10) with PP-MS. The clinical diagnosis of multiple sclerosis was based on Diagnostic Criteria for Multiple Sclerosis from the International Panel published in Ann Neurol 2005; 58: 840-846 [1]. All patients fulfilled diagnostic criteria demonstrating dissemination of lesions in space and time including MRI (magnetic resonance imaging) positivity. None of these patients had a clinically isolated syndrome (CIS). Serum and CSF samples were analysed in each patient. ANALYTICAL PROCEDURES Measles, rubella, and varicella zoster virus-specific IgG antibodies were detected both in the serum and CSF by sandwich enzyme immunoassay using commercial kits from Human, Germany (Measles-Virus Human ELISA IgG Antibody Test, Rubella-Virus Human ELISA IgG Antibody Test, Varicella-Zoster-Virus Human ELISA IgG Antibody Test). In this assay microtitre strip wells as a solid phase are coated with cell-culture derived Measles, Rubella,and VZVantigens. If the corresponding specific antibodies are present in a sample, they are bound to the antigens at the solid phase. After a washing step to remove unbound material, anti-human IgG peroxidase conjugate is added, which binds specifically to IgG class antibodies. After a second washing step to remove unbound conjugate, the enzyme-linked complexes are detected by incubation with a substrate solution. The subsequent development of a blue colour is changed into yellow by stopping the enzymatic reaction with sulphuric acid. Absorbances are measured at 450 nm using an ELISA microtitre plate reader. Absorbances of serum and CSF samples were converted to arbitrary units (AU) in a log/log diagram based on a standard curve derived from seven serial dilutions of a positive standard serum. The highest standard concentration (approximately 2.0) was defined as 100 arbitrary units [5], The specific antibody index (Al) was calculated according to the Reiber's formula: Al = Qspec /QIgG. Qspec is the ratio of http://www.med.muni.cz/biomedjournal/ SCRIPTA MEDICA /Volume 82/No. 1/2009 47 00 00 00 THE VALUE OF INTRATHECAL MRZ REACTION AND OLIGOCLONAL IgG BANDS FOR DISCRIMINATION.. Table 1 Prevalence of MRZ reaction and IgG-OB in cohorts of patients with PP-MS and RR-MS MRZ reaction IgG-OB RR-MS (n-29) 11/29 20/29 PP-MS (n-10) 2/10 1/10 Table 2 Sensitivity of MRZ reaction and IgG-OB in cohorts of patients with PP-MS and RR-MS MRZ reaction (%) IgG-OB RR-MS (n-29) 37% 69% PP-MS (n-10) 20% 10% specific antiviral IgG antibodies in CSF and serum. QlgG is the ratio of total IgG antibodies in CSF and serum.The upper limit (Qlim) for IgG is Qlim = 0.93 *SQRT (Alb2 + (6*106) -1.7 *10 3. If QlgG >Qlim, then Al = Qspec/Qlim. The values Al>1.4 are positive and indicate intrathecal synthesis of specific antibodies [8]. Oligoclonal IgG bands Oligoclonal IgG bands in the serum and the CSF were detected by isoelectric focusing with subsequent immunoenzy-matic staining using a commercial kit (SEBIA, France). RESULTS MRZ reaction was positive only in 2 of 10 (n-10) patients with primary progressive MS and IgG-OB were positive in 1 of 10 (n-10) patients with PP-MS. MRZ reaction was positive in 11 of 29 patients (n-29) with RR-MS. IgG-OB were positive in 20 of 29 patients (n-29) with relapsing remitting course of MS. The differences in positivity of MRZ reaction and IgG-OB between those cohorts of patients were statistically significant with a calculated sensitivity for MRZ reaction of 20% in PP-MS and 37% in RR-MS. For IgG-OB the sensitivity was 10% in PP-MS course and 69% in RR-MS. DISCUSSION We found significantly less positivity in both parameters (MRZ and IgG-OB) in the primary progressive course of multiple sclerosis. It supports the widely accepted hypothesis about different pathogenesis and distinctive prognosis between both MS courses. The report exists about IgG-OB negative patients with relatively better prognosis [9]. True IgG-OB negative clinically definite multiple sclerosis occurs and, according to our results, is more common in the PP-MS course. From the short- and middle-time observational period this is in accord with the slower accumulating disability, i.e. temporarily and concomitantly better prognosis. MRI detected fewer lesions in patients with lower inflammatory disease activity, also reflected by the negativity of IgG-OB [11]. In the clinical setting the negativity of IgG-OB rather reflects the PP-MS course, but temporary evolution of IgG-OB positivity must be taken into account, especially in the case of clinically isolated syndrome (CIS), where the initial positivity is reported to range between 40 and 70%, but when CSF examination is repeated after 1/2 to 1 year, the positivity of IgG-OB increases over 90 % [11 ]. One study described a significant delay in disability progression during the treatment with interferon-beta in the subgroup of MS patients with no IgG-OB detectable by IEF, compared to the patients with this CSF abnormality [12]. Whether IgG-OB negative MS patients have additional characteristic immune features remains to be settled. There is a need for concerted long-term follow-up studies of the subgroup of MS patients without CSF IgG OB regarding prognosis and immunological features. As regards MRZ antibodies, 84-94% of MS patients have intrathecal antibody synthesis against one, two or three of the measles, rubella, and varicella zoster viruses, and slightly more if herpes simplex is included. The frequencies and magnitudes of the antibody indices rise with increased total IgG synthesis [13]. Although much less frequent than the MRZ reaction, increased intrathecal synthesis of HSV antibodies (28% frequency in MS patients) is still higher than other antibody species in MS (e.g. intrathecal toxoplasma antibodies - 10%, or intrathecal autoantibody synthesis against dsDNA-20%). On the contrary, the MRZ reaction is strongly predictive of an autoimmune-type chronic inflammatory disease such as 48 SCRIPTA MEDICA /Volume 82/No. 1/2009 http://www.med.muni.cz/biomedjournal/ 00 00 ■ THE VALUE OF INTRATHECAL MRZ REACTION AND OLIGOCLONAL IgG BANDS FOR DISCRIMINATION... multiple sclerosis even at the time of the first clinical symptoms. In the case of acute infections the specific antibody index has a higher diagnostic sensitivity than the oligoclonal IgG [14]. The rather lower sensitivity of MRZ reaction in our cohort of RR-MS patients could be explained by the smaller number of patients comparing numbers in relevant reports. The relevance of the MRZ reaction is different from that of oligoclonal IgG for evaluating multiple sclerosis. The presence of IgG-OB in CSF has a lower specificity for MS and must be generally evaluated in the context of clinical setting and MRI findings [15]. The MRZ reaction has not been evaluated until now in the different courses of MS; we stated that the frequency is significantly lower in the PP-MS course, likely reflecting less pronounced inflammation in CNS as was similarly detected in IgG-OB. CONCLUSION If both parameters are taken together, we can conclude that the negativity of the MRZ reaction and IgG-OB does not exclude the possibility of diagnosing multiple sclerosis and rather has a better prognostic significance for both courses of MS and is much common in the PP-MS course. These significant differences in prevalence between the two courses can be used as an additional differential diagnostic marker. REFERENCES 1. Polman CH, Reingold S, Edan G, et al. Diagnostic Criteria for Multiple Sclerosis: 2005 Revisions to the "McDonald Criteria". Ann Neurol 2005; 58: 840-846. 2. Anderson M, Alverez-Cermeno J, Bernardi G, et al. Cerebrospinal fluid in the diagnosis of multiple sclerosis: a consensus report. J Neurol Neurosurg Psychiatr 1994; 57: 897-902. 3. Link H.Tibbling G. Principles of albumin and IgG analysis in neurological disorders. III. Validation of IgG synthesis within the central nervous system in multiple sclerosis. Scan J Clin Lab Invest 1977; 37: 397-401. 4. Freedman MS,Thompson EJ, Deisenhammer F, et al. Recommended standard of cerebrospinal fluid analysis in the diagnosis of multiple sclerosis. A consensus statement. Arch Neurol 2005; 62: 865-870. 5. Reiber H, Peter JB. Cerebrospinal fluid analysis: disease-related data patterns and evaluation programs. J Neurol Sei 2001; 2:101-122. 6. Tumani H, Noelker G, Reiber H. Relevance of cerebrospinal fluid variables for early diagnosis of neuroborreliosis. Neurology 1995; 45:1663-1670. 7. Bednářová J, Štourač P, Krbková L. The diagnostic significance of intrathecal occurrence of selected antiviral antibodies in multiple sclerosis. Scripta Medica 2005; 78(4): 249-254. 8. Reiber H, Lange P. Quantification of virus specific antibodies in cerebrospinal fluid and serum: sensitive and specific detection of antibody synthesis in brain. Clinical Chemistry 1991; 37:1153-1160. 9. Zeman AZJ, Kidd J, McLean BN. A study of oligoclonal band negative multiple sclerosis. J Neurol Neurosurg Psychiatr 1996; 60: 27-30. 10. Kidd D.Thompson AJ, Kendall BE, Miller DH, McDonald Wl. Benign form of multiple sclerosis: MRI evidence for less frequent and less inflammatory disease activity. J Neurol Neurosurg Psychiatr 1994; 57:1070-1072. 11. LinkH,Yu-Min Huang. Oligoclonal bands in multiple sclerosis cerebrospinal fluid: an update on methodology and clinical usefulness. J Neuroimmunol 2006; 180:17-28. 12. Annunziata P, Giorgio A, De Santi L, et al. Absence of cerebrospinal fluid bands is associated with delayed progression in relapsing-remitting MS patients treated with interferon-beta. J Neurol Sei 2006; 244: 97-102. 13. Reiber H, Ungefehr S, Jacobi C. The intrathecal, polyspe-cific and oligoclonal immune response in multiple sclerosis. Mult Scler 1998; 4:111-117. 14. Felgenhauer K, Reiber H. The diagnostic significance of antibody specificity indices in multiple sclerosis and herpes virus induced diseases of the nervous system. Clin Invest 1992; 70: 28-37. 15. Reiber H. Cerebrospinal fluid analysis: disease-related data patterns and evaluation programmes. J Neurol Sei 2001; 184(2): 101-122. http://www.med.muni.cz/biomedjournal/ SCRIPTA MEDICA /Volume 82/No. 1/2009 49 00 00 00 00 00 17 00 00 00 00 00 00 Electromyography laboratory Stroke unit - nurse staff control centre 50 SCRIPTA MEDICA/Volume 82/No. 1/2009 http://www.med.muni.cz/biomedjournal/ Inpatient clinic- nurse staff room Outpatient clinic - nurse staff room http://www.med.muni.cz/biomedjournal/ SCRIPTA MEDICA /Volume 82/No. 1 / 2009 51 CASE REPORT NON-JEWISH CHILD WITH CANAVAN DISEASE Slouková E.1, Ošlejšková H.1, Šoukalová J.2, Masaříková H.3 1 Department of Paediatric Neurology, Faculty of Medicine, Masaryk University and Faculty Hospital Brno, Centre for Epilepsies 2 Department of Medical Genetics, Faculty of Medicine, Masaryk University and Faculty Hospital Brno 3 Department of Paediatric Radiology, Faculty of Medicine, Masaryk University and Faculty Hospital Brno Received after revision February 2009 KEYWORDS Canavan disease Leukodystrophy N-acetylaspartate Developmental delay Macrocephaly H CORRESPONDING AUTHOR Slouková E., Department of Paediatric Neurology, Faculty of Medicine Masaryk University and Faculty Hospital Brno Černopolní9 625 00 Brno Czech Republic ABSTRACT Canavan disease (CD) is a rare autosomal recessive inherited disordercaused bya deficiency ofaspartoacylase,which leads to defective myelinisation and occurrence of leukodystrophy. Most frequently it occurs in the population of Ashkenazi Jews, while in the Central European non-Jewish population only about 5% of outbreaks occur. The most common symptoms are progressive spasticity, serious developmental delay, macrocephaly, blindness, and seizures. The features crucial for diagnostics include clinical history, laboratory proof of N-acetyl-L-aspartate (NAA) accumulation in urine, magnetic resonance imaging (MRI) of the brain, and molecular analysis of the genetic mutation. Genetic therapy has already been known and used, while therapy with stem cells and glyceryl triacetate is still in the phase of research. We diagnosed a six-month non-Jewish girl with leukodystrophy suspect symptoms without EEG or clinically expressed epileptic activity. NAA elevation was proved in urine. Brain MRI showed diffused affection of the brain's white matter. The diagnosis was definitively confirmed by molecular analysis where the 914C>A mutation was found, which is most frequently expressed in the Central European non-Jewish population. The genetic confirmation explained the essence of this serious neurological disorder and allowed for a better determination of prognosis, which is unfavourable. In Central Europe, only symptomatic therapy of epileptic paroxysms has been used, along with rehabilitation. Negotiations leading to the establishment of gene therapy, which has already been used elsewhere, have not yet been initiated in this region, as it is extremely demanding and rare in Central Europe. ABBREVIATIONS USED ASPA - gene for enzyme aspartoacylase BAEP - brainstem-evoked potentials CD - Canavan disease 52 SCRIPTA MEDICA/Volume 82/No. 1/2009 http://www.med.muni.cz/biomedjournal/ NON-JEWISH CHILD WITH CANAVAN DISEASE DNA - deoxyribonucleic acid EEG - electroencephalography EMG - electromyography CSF - cerebrospinal fluid MRI - magnetic resonance imaging NAA - N-acetyl-L-aspartate TORCH - intrauterine infections (toxoplasmosis, rubella, cytomegalovirus, herpetic viruses) VEP - visual evoked potentials Table 1 The severity of the disease is dependent on mutation and population Jewish population Non-Jewish population Mild disease 854 A>C(E285>A) 82.9% (G212>A) Severe disease 693C>A(tyr231>ter)14% 914C>A(A305>E)60% ASPA gene placed on the 17th chromosome has 6 exons and 5 introns. Dependence of the severity of the disease on location of the mutation and representation of the mutations in populations has been described. Up to date, there exist in the non-Jewish population over 14 mutations with different percentage substitution INTRODUCTION In Central Europe, CD is a very rare autosomal recessive inherited leukodystrophy with a known metabolic defect. It is caused by the mutation of the gene for N-acetylaspartate acid amidohydro-lase (aspartoacylase), which catalyses the transformation of N-acetylaspartate acid to acetyl and aspartate, and actively participates in myelinisation. Due to the defective enzyme, the substrate accumulates in astrocytes with the result of leukodystrophy followed by spongiform degeneration of the brain's white matter [1, 4]. There are several types of mutation of the gene for aspartoacylase, occurring predominantly either in the population of Ashkenazi Jews (854A>C) or in the non-Jewish Central European population (914C>A) (Table 1) [3]. This means that the diagnosis of CD is, besides clinical symptoms, laboratory proof of NAA elevation in urine and MRI signs of leukodystrophy, based mostly on molecular analysis of the genetic mutation characteristic of a particular population. We have described a case of a six-month-old non-Jewish child suffering from CD, which is very rare in our country. Case: The six-month-old girl, born to non-related non-Jewish parents, from uncomplicated gravidity and without perinatal risks, was brought to our department for the diagnostics of aetiology of severe developmental delay which was on the level of a pathological newborn infant with central hypotonic syndrome and beginning spasticity of the lower extremities. Epileptic seizures were not expressed yet in the infant. Brain ultrasound examination had led to the suspicion of an inherited developmental brain disorder. The objective neurological finding also noticed suspect amaurosis and macrocephaly. EEG, EMG from the legs and BAEP were normal, VEP examination confirmed lesion in the visual tract without the possibility of a closer topisation. The basic metabolic sampling was complemented by special tests for inherited metabolic disorders with the discovery of NAA elevation in the urine. After structural brain examinations, MRI was carried out, upon which diffused affection of the white matter was described along with a myelinisation disorder and reduction of the cortex, which are all signs of leukodystrophy (Figures 1, 2). Now we could express our suspicion of CD. To prove it we sent the patient's and her parents' DNA for genetic analysis. Heterozygote mutation 914C>A was proved in both parents, and upon the test of the ASPA gene (6th exon) of the patient, 914C>A (A305E) mutation was found in homozygote condition, which is characteristic of the Central European non-Jewish population (Table 1). In our conditions the only therapy available is symptomatic treatment of epileptic seizures and rehabilitation. The girl's prognosis is therefore catastrophic due to the very substance of the disorder, as well as to the therapeutic possibilities in our country.The parents were instructed about the 25% risk of the same disorder occurring in any of their children, boys or girls, and in the case of the mother's next gravidity amniocentesis would be carried out as part of the perinatal diagnostics and monitoring of this family. DiscussiomThe main symptom of our little patient was a serious developmental delayanda central hypotonic syndrome with spasticity on the lower extremities. The same symptoms are present in children with infantile poliomyelitis, which includes positive perinatal risks in the http://www.med.muni.cz/biomedjournal/ SCRIPTA MEDICA/Volume 82/No. 1/2009 53 NON-JEWISH CHILD WITH CANAVAN DISEASE Figure 1 MRIT2W imaging - hyperintensity of white matter mainly localised subcortically, signs of grey matter reduction patient's history that are negative in our case. Macrocephaly and developmental delay are also mentioned with the Pelizaeus-Merzbacher leukodystrophy, including a similar expression of the defective myelinisation in theT2-weighted brain MRI images, but in detailed biochemical examination and molecular analysis no concrete metabolic defect is found [1, 4]. In our case we found NAA elevation in the urine and, if lumbar puncture had been carried out, the increased NAA concentration would be present in the CSF as well [2]. Macrocephaly and developmental delay can also be diagnosed in hydrocephalus occurring after intrauterine infection. Laboratory proof of TORCH was negative in our patient. Clinical epileptic seizures have not yet occurred in our case, as these develop with the reduction of the brain's grey matter; EEG showed a mature curve of basic activity without any abnormities. Unlike patients described in the literature, who suffer defective development of the cochlear apparatus and deafness, there was no proof of any hearing defect or lesion in the auditory pathway of our little patient [5], Forthe purpose of the molecularanaly-sis of the ASPA gene mutation, the non-Jewish origin of both parents was considered and the analysis was carried out directly for the A305E mutation, which was confirmed [3]. This mutation is present in approximately 60% of the Central European non-Jewish population, and currently about 14 new mutations are available, whose detection would surely extend the time of the early diagnostics (Table 1). However, the prolongation of diagnostics unfortunately has no influence on the patient's prognosis. In Central Europe, no therapy other than symptomatic has been used at the moment. In 1996 gene therapy was launched in New Zealand followed by the USA; it is based on the introduction of the normal ASPA gene in the affected areas of the brain 54 SCRIPTA MEDICA/Volume 82/No. 1/2009 http://www.med.muni.cz/biomedjournal/ NON-JEWISH CHILD WITH CANAVAN DISEASE REFERENCES Figure 2 True inversion recovery-juxtacortical changes, reduction of grey matter via recombinant virus particles [6]. The prognosis of patients treated in this way is much more positive, the quality of life improves, and they survive longer. The method of replacing the missing acetate to nerve cells for normal myelinisa-tion is still in the research phase, with the chemical being glycerol triacetate [7]. Lithium citrate, on the other hand, drains the accumulating NAA from the glial cells - currently this is carried out in vitro only [8]. Studies with stem cells should be opened in the near future, which should offer even better prognoses to patients. The establishment of special treatment methods depends on the complications of the therapy, as well as on the incidence of the cases in individual states. With the increasing achievements of the research, specialised therapy should also be implemented in areas with low incidence of the disorder, thus improving the future prospects of patients and their families. 1. Online Mendelian Inheritance in Man (OMIM): http://www.ncbi.nlm. nih.gov. /Omim/ Institute of Genetic Medicine, Johns Hopkins University, Baltimore, MD: MIM #*271900 CANAVAN DISEASE. 2. Matalon R, Michals K, Sebesta D, et al. Aspartoacylase deficiency and N-acetylaspartic aciduria in patients with Canavan disease. Am J Med Genet 1999; 87: 273-275. 3. Kaul R, GaoGP, Alova M, et al. Canavan disease: mutations among Jewish and non-Jewish patients. Am J Hum Genet 1994; 55: 34-41. 4. Toft PB, Geis-Holtorff R, Rolland MO, et al. Magnetic resonance imaging in juvenile Canavan disease. Eur J Pediatr 1993; 152: 750-753. 5. Ishiyama G, Lopez I, Baloh RW, Ishi-yama A. Canavan's leukodystrophy is associated with defects in cochlear neurodevelopment and deafness. Neurology 2003; 60:1702-1704. 6. Janson C, McPhee S, Bilaniuk L, et al. Gene therapy of Canavan disease: AAV-2 vector for neurosurgical delivery of aspartoacylase gene to the human brain. Hum Gene Ther 2002; 13:1391-1412. 7. Mathew R, Arun P, Madhavarao CN, Moffett JR, Namboodiri MA. Progress toward acetate supplementation therapy for Canavan disease: glycerol triacetate administration increases acetate, but not N-acetylaspartate, levels in brain. J Pharmacol Exp Ther 2005; 315: 297-303. REpub2005Jul7. 8. Baslow MH, Kitada K, Suckow RF, Hungund BL, Serikawa T The effects of lithium chloride and other substances on levels of brain N-acetyl-L-aspartic acid in Canavan diseaselike rats. Neurochem Res 2002; 27: 403-406. http://www.med.muni.cz/biomedjournal/ SCRIPTA MEDICA/Volume 82/No. 1/2009 55 Intensive care unit- nurse staff control centre Evoked potentials laboratory 56 SCRIPTA MEDICA/Volume 82/No. 1/2009 http://www.med.muni.cz/biomedjournal/ AUTHOR & REVIEWER PACK "The most important thing in communication is to hear what isn't being said." Peter F. Drucker J AUTHORS REVIEWER PACK_______________________________________________________________________________ INSTRUCTIONS FOR AUTHORS SUBMITTING MANUSCRIPTS FOR PUBLICATION IN SCRIPTA MEDICA You may also download the following text in PDF. GENERAL REQUIREMENTS SCRIPTA MEDICA is a scientific journal published in the English language by the Faculty of Medicine, Masaryk University in Brno; 4 issues are published annually. 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Acting as a filter, it ensures that only high-quality papers are published and thus improves the quality of research as such. When you are asked to review The first item that you have to consider when asked to review, is does the article you are being asked to review truly match your expertise? If you believe that the article is relevant, do you have time to review it? If you feel inadequately qualified to judge the research reported in a manuscript, you should return it promptly to the editor. Conducting the review Reviewing needs to be conducted confidentially. The article you have been asked to review should not be disclosed to a third party. Let the editor know beforehand if you want to elicit opinion on the respective article from colleagues or students. You should not attempt to contact the author or ask the editor who the author is. You should respect the intellectual independence of the authors. Both the reviewer and the author remain anonymous! You will be asked to evaluate the article on a number of criteria. We provide detailed guidance below. You will be expected to evaluate the article according to the following criteria: 1. ORIGINALITY (a) Is the article sufficiently novel and interesting to deserve publication? (b) Does the article comply with the journal's standards? (c) Is the research question an important one? (d) If the research has been reviewed previously pass on references to such works to the editor. 2. STRUCTURE (a) Is the article clearly laid out? (b) Are all the key elements present (abstract, introduction, materials and methods, results, conclusion)? TITLE: Does it clearly describe the article? ABSTRACT: Does it reflect the content of the article? INTRODUCTION: The introduction is one to two paragraphs long, as a standard. It summarizes relevant research to provide context. It explains what findings of others, if any, are being challenged or extended. (a) It should describe the experiment, hypothesis, general experimental design, or method. (b) Does it describe what the author hoped to achieve accurately, and clearly state the problem under investigation? METHODOLOGY (a) Is the methodology appropriate? (b) Does it explain with sufficient accuracy how the data was collected? (c) Is the design suitable for answering the question posed? (d) Is there sufficient information present for you to replicate the research? 62 SCRIPTA MEDICA/Volume 82/No. 1/2009 http://www.med.muni.cz/biomedjournal/ (e) Does the article identify the procedures followed? (f) Are these procedures ordered in a meaningful way? (g) If the methods are new, are they explained in sufficient detail? (h) Was the collecting of samples appropriate? (i) Have the equipment and materials been adequately described? (j) Does the article make it clear what type of data was recorded? (k) Has the author been precise in describing measurements? RESULTS The results should be arranged clearly and in a logical sequence. Here the author should explain in words what (s)he discovered in the research; no interpretations should be included in this section. (a) Are the statistics correct? (b) Has appropriate analysis been conducted? Ifyou are not comfortable with statistics, advise the editor when submitting your review. CONCLUSION/DISCUSSION (a) Are all statements in this section supported by the respective results? (b) Do the statements seem reasonable? (c) Have the authors indicated how the results relate to expectations and to earlier research? (d) Does the article support or contradict previous theories? (e) Does the conclusion explain the contribution of the research to the body of scientific knowledge? FIGURES, TABLES, AND GRAPHS (a) Are they an important part of the paper? (b) Do the figures describe the data accurately? (c) Are they consistent, e.g. bars in charts are the same width, the scales on the axis are logical, and the like. 3. LANGUAGE Correcting English in a paper is not the role of the reviewer. 4. PREVIOUS RESEARCH (a) If the article builds upon previous research, does it refer to that work appropriately? (b) Are there any essential works that have been omitted? (c) Are the references accurate? AUTHOR & REVIEWER PACK 5. ETHICAL ISSUES (a) Plagiarism - ifyou suspect that an article is a substantial copy of any previous work, let the editor know and please cite the respective previous work. (b) Fraud - if you suspect that the results in an article are false, let the editor know explaining why you believe the results to be deceitful. (c) Other ethical concerns - if the research is medical in nature, has confidentiality been maintained? If there has been any violation of accepted norms of ethical treatment of animal or human subjects, these should be identified. The paper should contain statement on written consent being available, if any corresponding experiments were conducted on humans. YOUR REPORT TO THE EDITOR You have completed your evaluation of the article. The next step is to write up your report. Ifyou feel you might miss your deadline, please let the editor know. You will complete a form checking individual points in it. This is a newly designed form for writing a detailed reviewer's report to which you have to add all your comments. May we remind you that your report will only be considered complete when you have also included the comments. When providing your commentary you should be courteous and constructive. It should not include any personal remarks. You should explain and support your judgment so that both editors and authors are better able to understand the basis of the comments. You should indicate whether your comments are your own opinion or reflected by the data provided. When you make a recommendation regarding an article, it is worth considering the categories that an editor will be likely to use for the classification of the article. (a) Reject (due to poor quality) (b) Accept without revision (c) Accept but needs revision. Ifyou think the article needs to be revised, indicate it to the editor clearly identifying what revision is required and advise whether or not you would be pleased to review the revised article. http://www.med.muni.cz/biomedjournal/ SCRIPTA MEDICA/Volume 82/No. 1/2009 63 03 SCRIPTA MEDICA JOURNAL FOR BIOMEDICAL RESEARCH WOULD YOU LIKE TO CO-OPERATE WITH US? WOULD YOU LIKE TO BECOME OUR REVIEWER? IN SUCHA CASE, PLEASE WRITE TO US OR YOU CAN COMPLETE THE FORM BELOW FOR BETTER COMMUNICATION. IF YOU ARE UNDECIDED, PLEASE READ OUR INFORMATION FOR REVIEWERS. WE PROVIDE DETAILED GUIDANCE ON OUR WEBSITE. THE EDITOR-IN-CHIEF CAN ASSURE YOU THAT WE ONLY PROVIDE DOUBLE-BLIND REVIEWS. WHAT DOES IT MEAN? A DOUBLE-BLIND REVIEW MEANS THAT BOTH THE REVIEWER AND THE AUTHOR REMAIN ANONYMOUS. WE ARE INTERESTED IN TOPICS THAT YOU ARE WILLING TO REVIEW. PLEASE LET US KNOW YOUR SPECIALISATION (HONOURS) AND WRITE TO US WHAT KIND OF PAPERS YOU ARE INTERESTED IN. YOU WILL BE INCLUDED IN OUR LIST OF REVIEWERS AND THEN YOU WILL BE ASKED TO REVIEW. YOU WILL BE ASKED TO EVALUATE AN ARTICLE ONA NUMBER OF CRITERIA WHICH ARE DESCRIBED IN THE INFORMATION FOR REVIEWERS. (PLEASE HAVE A LOOKATOUR WEBSITE.) Step one: Please complete the form for reviewers: First name Family name Title(s) Full affiliation address with town postcode and country Current address Telephone number E-mail Type of papers Date and signature Step two: Please send us your completed form by post or by e-mail. We will contact you to check the information. Scripta Medica, mkorcova@med.muni.cz