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2 의학박사학위청구논문 흰쥐좌골신경에서탈수초병변의전기생리학적소견 Electrophysiologic findings in Demyelinating Lesion of Rat Sciatic Nerve 2008 년월 인하대학교대학원 의학과재활의학전공 김일수

3 지도교수정한영 이논문을박사학위논문으로제출함

4 이논문을김일수의박사학위논문으로인정함 2008 년월일 주심 부심 위원 위원 위원

5 Electrophysiologic findings in Demyelinating Lesion of Rat Sciatic Nerve by Il-Soo Kim A THESIS Submitted to the faculty of INHA UNIVERSITY in partial fulfilment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Rehabilitation Medicine February 2008

6 목차 List of figures and table i 국문요약 iv 영문요약 vi 서론 1 연구방법 3 결과 15 고찰 32 결론 36 참고문헌 37 - i -

7 List of figures and table Figure 1. Picture of the new compression instrumen 9 Figure 2. Schematic illustration of cross-sectional view of the nerve compressed by the same pneumatic pressure 10 Figure 3. Sciatic nerve injury using the new miniature compression instrument 11 Figure 4. Direct stimulation on sciatic nerve to evoke the compound muscle action potential (CMAP) of flexor digitorum brevis 12 Figure 5. Overview of entire experimental process 13 Figure 6. Illustration of sciatic functional index 14 Figure 7. Serial changes of amplitude of CMAP in Group A (5 min injury group) 19 Figure 8. Examples of serial sciatic motor nerve conduction study in one rat of group A 20 Figure 9. Findings of needle EMG in group A 21 Figure 10. Serial changes of amplitude of CMAP in Group B (10 min injury group) 22 - ii -

8 Figure 11. Examples of serial sciatic motor nerve conduction study in one rat of group B 23 Figure 12. Findings of needle EMG in group B 24 Figure 13. Serial changes of amplitude of CMAP in Group C (30 min injury group) 25 Figure 14. Examples of serial sciatic motor nerve conduction study in one rat of group C 26 Figure 15. Findings of needle EMG in group C 27 Figure 16. Immunohistochemical staining for neurofilament in rat sciatic nerves at 36 hours after compression injury 28 Figure 17. Luxol fast blue staining in rat sciatic nerves at 36 hours after compression injury 29 Figure 18. Immunohistochemical staining for neurofilament in rat sciatic nerves at 1 week after compression injury 30 Figure 19. Luxol fast blue staining in rat sciatic nerves at 1 week after compression injury 31 Table 1. Temporal Changes of Sciatic Functional Index (SFI) 18 - iii -

9 국문요약 말초신경의손상후신경손상의정도와회복에대한예후는축삭손상과수초손상의유무및정도에의하여많은영향을받는다. 근전도검사는이러한병태생리적신경손상의정도를객관적으로평가할수있는좋은도구이나, 탈수초병변에서비정상자발전위의발생여부에관해서는많은논란이있다. 따라서본연구에서는압박손상에의하여탈수초병변을정량적으로유발하고이러한탈수초병변에서전기생리학적검사소견을분석하고자하였다. 먼저흰쥐의좌골신경에미세한압박손상을만들수있게손상정도를정량화할수있는새로운압박손상기구를개발하였다. 폴리에틸렌재질의매우얇은필름 (film) 이신경을감싸게하고필름이커프역할을하여신경의모든면에동일한기압 (pneumatic pressure) 이가해지도록하였다. 예비실험을통하여좌골신경의신경전도검사상경도의전도차단 (mild conduction block) 을일으키는압박손상의정도를확인하였으며, 이를통하여총 30 마리의 Sprague-Dawley 수컷흰쥐를세군으로나누어압박손상의정도를달리하는본실험을진행하였다. 각군당 10마리씩 160 mmhg의동일한압력을 5분 (A 군 ), 10분 (B 군 ), 30분 (C 군 ) 동안각각가하였다. 압박손상을가하기직전과손상후에신경전도검사를시행하였으며, 각실험군중 5마리는 36 시간후에, 나머지 5마리는 1주일후에신경전도검사를재시행한후짧은발가락굽힘근에서침근전도검사를시행하였다. 이중 3 마리씩은침근전도검사후신경조직표본을추출하였다. 조직검사로는축삭을관찰하기위하여 neurofilament에대한면역조직화학염색을시행하였고, 수초를관찰하기위하여 luxol fast blue 염색을시행하였다. 압박손상직후시행한신경전도검사상, A 군은압박손상부위를기준으로통계적으로유의하지는않았지만경도의전도차단이확인되었으며, 손상 36 시간및 1 주후에도비슷한소견을보였다. 침근전도검사에서는 36 시간후에삽입전위가증가하였으며 1 주후에는양성예각파가관찰되었으나세동전위는전혀관찰되지않았다. A 군의조직검사에서는경도의수초손상소견이관찰되었으며축삭손상의증거는발견할수없었다. B 군의경우조직검사 - iv -

10 에서수초손상과함께일부축삭손상이관찰되었다. 신경전도검사소견은 A 군과마찬가지로통계적유의성은없었지만압박손상부위에서전도차단의정도는더컸으며, 손상 1주후에는원위부자극시에도복합근활동전위의진폭이경도로감소되어축삭손상이일부동반되었음을확인할수있었다. B 군의침근전도검사에서는양성예각파와세동전위가모두관찰되었다. C 군의신경전도검사에서는압박손상직후전도차단소견과함께손상 1주일째원위부의복합근활동전위의진폭이감소되는소견이함께관찰되었으며, 조직검사에서도축삭손상의소견이 B 군보다는뚜렷하게관찰되어신경전도검사와부합되는결과를보였다. 침근전도검사에서는양성예각파와세동전위가비교적풍부하게관찰되었다. 새로운압박손상모델을이용하여좌골신경에의탈수초병변을유발할수있었으며, 신경전도검사와조직학적검사를통하여순수탈수초병변을확인할수있었다. 탈수초병변의침근전도검사에서비정상자발전위는유발되었으나, 양성예각파만을확인할수있었고세동전위는관찰할수없었다. 이는양성예각파와세동전위가서로다른전기생리학적기전으로발생하며임상적으로서로다르게해석되어야함을제시하는결과로생각된다. 주요어 : 말초신경, 수초손상, 축상손상, 근전도, 신경전도검사, 비정상자발 전위 - v -

11 Abstracts Functional recovery after peripheral nerve injury mainly depends on the degree of axonotmesis and demyelination, which are commonly assessed by electromyography. There has been a debate over whether abnormal spontaneous activity is observed in pure demyelinating lesion. The purpose of the study is to develop the new compression injury model in rat sciatic nerve, and to identify the needle electromyographic findings in demyelinating lesion induced by new injury model. The new compression instrument, consisted of flexible polyethylene film wrapping the sciatic nerve and two metal caps, was developed to apply the same pneumatic pressure at all contact area of the sciatic nerve. After the pilot study to determine the appropriate pressure and injury duration to induce pure demyelinating lesion, total 30 rats were allocated to three groups. Compression by pneumatic pressure of 160 mmhg were applied to rat sciatic nerve for 5 min (group A), 10 min (group B), and 30 min (group C). Compound muscle action potentials were obtained by direct nerve stimulation on distal and proximal sciatic nerve before and immediately after injury. Needle electromyographic studies were performed at 36 hours after injury in 5 rats in each group, and then sciatic nerve specimen were taken for histologic examination in 3 rats among these 5 rats. One week later from injury, the other 5 rats were sacrificed after sampling of the sciatic nerve specimen following needle elctromyographic studies. In group A, electromyographic study revealed very mild conduction block and positive sharp waves (PSWs) without any fibrillation potential, and histologic examination showed mild demyelinationg lesion without axonal injury. On the other hand, combined lesion of axonotmesis and demyelination were found on histologic examination and needle electromyographic study revealed fibrillation potentials as well as PSWs in group B and C. - vi -

12 Pure demyelinating lesion, which was confirmed by nerve conduction study and histologic examination, could be induced using the new compression injury model in rat sciatic nerve. The needle electromyographic study revealed PSWs without any fibrillation potential in pure demyelinating lesion of sciatic nerve. These findings suggest that PSW might differ electrophysiologically from fibrillation potential in the pathomechanism and clinical significance. Key Words: Peripheral nerve, Demyelination, Axonotmesis, Electromyography, Nerve conduction study, Abnormal spontaneous activity. - vii -

13 서론 말초신경이손상되었을때손상의정도및부위를적절히평가하는것은치료계획을수립할때매우중요하다. 특히손상정도를병태생리적으로평가하는것은향후신경회복의정도와이를통한기능적회복을예측하는데필수적이다. Seddon(1943) 은조직학적손상정도와기능적회복에기초하여신경손상을다음과같이분류하였다. 먼저생리적신경차단 (neurapraxia) 은수초가국소적으로손상되어신경전도가차단되는것으로수주에서몇개월에걸쳐회복되며, 두번째로는축삭손상 (axonotmesis) 으로손상원위부로왈러변성 (wallerian degeneration) 이발생하며주위의신경내막 (endoneurium) 과신경주위막 (perineurium) 의손상정도에따라다소의차이는있지만일반적으로생리적신경차단보다는나쁜예후를보인다. 마지막으로신경절단 (neurotmesis) 은신경전체가완전히절단되어자연회복은거의기대할수없으며대부분수술적치료를필요로하는경우이다. 대부분의신경손상과손상후회복에대한예후는축삭손상과수초손상의정도에많은영향을받으며, 일반적으로축삭손상과수초손상의정도를예측하는가장객관적인방법으로는신경전도검사와침근전도검사가사용된다. 그중신경의병태생리적인손상정도에관한침근전도검사소견에있어서축삭손상병변에서의비정상자발전위의유발에는이견이없으나, 수초손상병변에서비정상자발전위의발생여부는아직까지많은논란에싸여있다. 이는사람에서손상된신경을직접적출하여조직학적으로확인할수없기때문에발생하는당연한논란으로이를보완하기위하여몇가지동물실험이시도되었고, 이들연구에서는알레르기신경염 (allergic neuritis) 을면역학적으로유발시킨후침근전도검사에서양성예각파 (positive sharp wave) 의발생을보고하였다 (Kraft, 1971; 1973; 1975). 하지만이중에서하나의연구에서만탈수초병변을조직학적으로확인하였고 (Kraft, 1973), 이경우에도심한탈수초병변으로축삭손상의동반가능성은완전히배제하기어려우며특히면역학적으로유발된탈수초병변이므로일반적으로흔하게접하게되는압박이나허혈에의한신경손상과는병태생리적으로부합되지않는문제점이있다

14 한편, 동물에서말초신경의압박손상을일으키는여러가지방법들이사용되고있으며, 대표적인방법으로압박커프 (tourniquet) 를손상을가하고자하는신경이포함된사지 (limb) 를감싸서직접압박을가하는방법 (Lundborg, 1970; Fowler 등, 1972; Ochoa와 Fowler, 1972) 이사용되고있지만손상의부위와손상의정도를정량화하기매우어려운단점이있으며, 이를보완하기위하여 miniature inflatable cuff로신경조직을직접압박하거나 (Rydevik과 Lundborg, 1977; Dahlin과 McLean, 1986; Dyck 등, 1990) clip이나 clamp를이용하여손상을유발하는방법 (Denny-Brown과 Brenner 1944; Horiuchi, 1983; Nemoto, 1983) 들이사용된다. 하지만이러한방법들모두신경조직에압궤손상 (crushing injury) 을유발하므로수초병변 (demyelinating lesion) 만을유발하는것은불가능하다. 따라서본연구는흰쥐의말초신경에서신경손상정도를정량화할수있는새로운손상모델을개발하고이를통하여탈수초병변을유발하며, 궁극적으로는흰쥐의탈수초말초신경병변에서침근전도소견을확인하여향후사람에있어서근전도소견으로신경손상정도를평가하고예후를예측하는데기초자료를제공하고자한다

15 연구방법 1. 실험동물 g의 Sprague-Dawley 수컷흰쥐를실험동물로사용하였다. 1주간사육장에대한적응기간을가지게하였고모든실험동물은실험기간동안섭씨 22 도실온에서항온, 항습이가능한사육장에서 12시간채광, 12시간차광의조건에서식수와사료를자유롭게섭취하도록하였다. 일주일정도실험실환경에적응시킨후사용하였다. 2. 좌골신경의노출 흰쥐를 Telazol (40 mg/kg) 과 Xylazine (5-10 mg/kg) 를혼합하여복강내마취한후, 우측하지의둔부이하슬관절부위까지체모를제거하였으며, potadine 을이용하여무균소독하였다. 좌골절흔부위에서부터슬관절근위부까지종절개하였으며둔부근육군과슬와부근육군을조심스럽게박리하여좌골신경을노출시켰다. 좌골절흔부위의둔부근육과슬관절상부의슬와부근육을절개하여좌골신경을되도록길게노출시키고압박기구를위치할수있는공간을확보하였다. 3. 새로운신경손상기구개발 실험동물의좌골신경손상을위하여좌골신경을감쌀수있도록소형으로제작한압박기구 (miniature compression instrument, Figure 1) 를개발제작하였고, 현재의료용으로사용되고있는수은식혈압계를이용하여압박정도를계측하였다. 소형압박기구는금속으로제작된두개의덮개 (two caps) 로구성되어있으며, 각덮개의안쪽으로는폴리에틸렌재질의랩필름 (wrap film) 이점착되어있어서손상을가하고자하는신경의위아래를두개의덮개로감싸서덮개안 - 3 -

16 으로일정한기압을가할수있도록설계하였다. 신경과접촉하게되는랩필름 (film) 은두께 9.5 um로매우유연하기때문에공기의압력 (pneumatic pressure) 이가해졌을때신경을완전히감싸게되고, 신경과닿는모든부분에서동일한압력이가해지도록하였다 (Figure 2). 소형압박기구는공기를집어넣을수있는수동식펌프와수은기둥에함께연결되어있어서가해지는압력을실시간으로모니터할수있으며, 따라서압력과시간을달리하여실험동물의좌골신경에손상정도를정량적으로조절하였다 (Figure 3). 4. 신경전도검사및침근전도검사 짧은발가락굽힘근 (flexor digitorum brevis) 에활동전극을붙이고, 두번째부터네번째발가락에기준전극을붙여좌골신경에대한운동신경전도검사를시행하였다. 근전도기기는 2 channel EMG/NCV portable system (Medelec Synergy, Oxford Instrument Medical, UK) 을사용하였으며, 자극시간 0.1 ms 의전류를초최대자극강도 (supramaximal stimulation) 로자극하였고여과주파수는 10 Hz 10 khz, 기록감응도는 5 mv/div, 기록속도는 1 ms/div로하였다. 기록전극으로는정사각형의표면전극 (1cm 1cm) 을이용하였으며접지전극은작위신호 (artifact signal) 를감소시키기위하여 SSEP(somatosensory evoked potential) 의기록에사용되는침전극을사용하여자극위치와기록전극사이의비복근에삽입하였다. 본연구에서는균일한압박에의하여탈수초병변을유발하여야하므로신경손상의정도가미약할수밖에없고, 따라서압박부위의근위부와원위부를자극할때미미한전도차단 (conduction block) 의정도가일관되게기록될수있도록신경을직접자극하는방법을택하였다 (Figure 4). 음극 (cathode) 과양극 (anode) 이 1cm 정도떨어져있는소형전기자극기를제작하여사용하였다. 신경전도검사에서는압박부위의근위부와원위부를자극하여복합근활동전위 (compound muscle action potential) 의진폭을각각계측하였다. 근위부자극은좌골절흔부위에서, 원위부자극은오금부위 (popliteal area) 에서시행하여되도록두자극간에거리를멀리하였다. 근육손상이침근전도검사결과에영향을미칠수있으므로, 가장원위부근 - 4 -

17 육인짧은발가락굽힘근에서동심전극 (concentric needle) 을이용하여침근전도검사를시행하였다. 침근전도검사에서는삽입전위의증가여부, 양성예각파와세동전위의유무및정도를확인하였으며비정상자발전위가최소 2 부위이상에서관찰되는경우 +, 3 부위이상에서중등도로관찰되는경우 ++, 모든부위에서풍부하게관찰되는경우 +++의등급으로구분하였다 (Daniel과 Machiel, 2002). 5. 좌골신경의탈수초병변유발을위한예비실험 먼저 1단계로새로운압박기구를이용하여신경에가해지는압력을달리하여손상의정도를신경전도검사를통하여확인하였다. 100 mmhg, 120 mmhg, 140 mmhg, 160 mmhg의압력으로각 3마리씩시행하였으며, 모두 30분간압력을가하였다. 압력을가하기전, 그리고압력을가한후에는압박기구를통하여압력을가하고있는상태에서 5분간격으로총 30 분까지신경전도검사를시행하였다. 압박부위를기준으로원위부와근위부의좌골신경을직접자극하여복합근활동전위를각각기록하였다. 100 mmhg와 120 mmhg에서는 30분의압박을가하는동안근위부자극시복합근활동전위의진폭감소소견이전혀관찰되지않았으며, 140 mmhg에서는 15 분정도에서원위부에비해근위부에서 10% 정도의진폭감소가관찰되었고이후 20% 정도까지의진폭감소가관찰되었으나, 더이상의진폭감소는관찰되지않았다. 160 mmhg에서는 5 분에서 10% 정도의진폭감소가, 10 분에서 % 의진폭감소가관찰되었으며, 20 분부터진폭감소가더진행하여 30 분정도에서는약 40-50% 의진폭감소가관찰되었다. 신경전도검사후절개한근육을봉합하고이후피부를봉합하였다. 압박손상 2일, 4일, 1주일후각각 1마리씩짧은발가락굽힘근에서침근전도를시행하여비정상자발전위의유발유무및정도를확인하였으며, 침근전도검사를시행하기전에봉합부위를다시노출시켜신경전도검사를재시행하였다. 침근전도검사상, 100 mmhg와 120 mmhg에서 4일째와 1주일째에삽입전위의증가현상이관찰되었으나비정상자발전위는확인되지않았으며, 140 mmhg에서는 4일째와 1주일째에양성예각파가각각관찰되었고, 160 mmhg에서는 4일째와 1주일째에양성예각파와세동전위가모두관찰되었고 1-5 -

18 주일째에발생빈도는더욱증가하는양상이었다. 침근전도검사직전에시행한신경전도검사상, 160 mmhg에서는 4일째원위부자극시에진폭감소소견이관찰되었으며, 1주일째에는진폭감소가더욱진행하였다. 1단계예비연구를통하여 160 mmhg에서압박시간이 5분, 10분, 30분되었을때전도차단의정도가정량적으로증가됨이확인되어 160 mmhg에서압박시간을달리하는 2단계예비연구를시행하였다. 가장경미한전도차단의정도를확인하기위하여 4분간의압박시간을추가하여, 160 mmhg에서 4 min, 5 min, 10 min, 30 min 간의압박시간으로각각 4 마리씩손상을가하고 4일째, 7일째, 9일째, 14일째각각신경전도검사와침근전도검사를확인하였다. 비정상자발전위가가장많이나오는시기를확인하기위하여 14일째까지실험을진행하였다. 2단계예비연구에서 4 min간압박을가한경우에는신경전도검사와침근전도검사모두에서이상소견은관찰되지않았으며, 5 min, 10 min, 30 min간압박을가한경우에는 4일째부터비정상자발전위가모두관찰되었으며, 1주일째정점을이루어 14일째까지비슷한유발빈도를보였다. 군간비정상자발전위의유발빈도는압박시간에비교적비례하는양상이었다. 6. 연구디자인 (Figure 5) 실험군은세군으로나누어각군당 10마리씩총 30마리의쥐를사용하였다. 예비실험의결과에따라, 각군당 10마리씩 160 mmhg의동일한압력을 5분 (A 군 ), 10분 (B 군 ), 30분 (C 군 ) 동안각각가하였다. 6마리의흰쥐는대조군으로하였으며, 이중 3마리는 sham 조작대조군으로하였으며나머지 3마리는아무런조작을가하지않은 true 대조군으로하였다. sham 조작은실험군과동일한수술과처치를받고압박기구로신경을 30분간감싸놓지만압력은가하지않은것으로압력이외에신경에손상을주는요인이있는지를확인하였다. 좌골신경의기능평가는압박손상전, 손상 36 시간후, 그리고손상 1 주후좌골신경에대한시술이나근전도검사를위하여마취를하기전에각각시행하였다. 실험군의우측좌골신경에압박손상을가하였으며, 압박손상을가한부위는신경주위막 (epineurium) 에봉합사를이용하여표시하였다. 압박손 - 6 -

19 상을가하기직전과손상후에신경전도검사를시행하였으며, 각실험군중 5 마리는 36 시간후에, 나머지 5마리는 1주일째봉합을풀고신경을노출시켜신경전도검사를시행한후짧은발가락굽힘근에서침근전도검사를시행하였다. 이중 3 마리씩은침근전도검사후신경조직을추출하여표본을추출하였다. 손상된우측좌골신경의표본을추출하는날실험군의좌측좌골신경에서도신경을노출시켜신경전도검사와침근전도검사를시행한후조직검사를위하여신경을추출하였다. 6마리의대조군역시우측좌골신경에서신경전도검사와침근전도검사를시행한후좌골신경을추출하였다. 7. 좌골신경기능지수 (Sciatic functional index) 좌골신경의기능평가는압박손상전, 손상 36 시간후, 그리고손상 1 주후각각시행하였다. 길이 50 cm, 폭 10 cm의통속에화선지를바닥에깔고흰쥐의양측뒤꿈치 (heel) 부터발가락끝까지잉크를바른다음, 화선지위를흰쥐가걷게하였다. 족문이뚜렷이찍힐때까지반복하여시행하였다 (Varejão 등, 2001). SFI (Sciatic functional index) 의산출공식은 Bain 등 (1989) 의방법을사용하였으며, 3개측정치의평균값을사용하였다 (Figure 6). 0 값에가까울수록정상이며, -100 에가까울수록기능저하가심함을의미한다. 대문자 E와 N은좌골신경손상측과비손상측을의미하며, 손상측다리를들고서걷거나발을질질끌어족문을확인할수없는경우에는 -100으로점수화하였다 (Dijkstra, 2000). Print length factor (PLF) = (EPL - NPL) / NPL Toe spread factor (TSF) = (ETS - NTS) / NTS Intermediary toe spread factor (ITF) = (EIT - NIT) / NIT SFI = (-38.3 PLF) + (109.5 TSF) + (13.3 ITF)

20 8. 조직검사 각실험동물에대하여근전도검사를시행한후손상된좌골신경의압박부위에서표본을추출한후안락사시켰으며, 추출한신경으로파라핀블록을제작하였다. 축삭을관찰하기위하여 neurofilament에대한면역조직화학염색을시행하였다. 파라핀블록에서 4 um 두께의절편을얻어자일렌으로파라핀을제거한후 100% 알콜로 10초간 3회처리하였다. 10% citrate buffer에서고압멸균기를이용하여항원을복원시킨후 3% 과산화수소용액으로내인성과산화효소를제거하고일차항체 neurofilament (Zymed, San Francisco, CA, USA) 를 1:100으로희석하여 90분간반응시켰다. peroxidase가부착된이차항체 (biotynylated anti-mouse immunoglobin antibody, Dako Co., Ltd., Kyoto, Japan) 를 30분간반응시킨후 DAB(3,3'-diaminobenzidine tetrahydrochloride solution, Research Genetics, USA) 로 10분간발색하였고, Mayer's hematoxylin (Zymed, San Francisco, CA, USA) 으로대조염색하였다. 좌골신경에서수초를관찰하기위해서 luxol fast blue 염색을시행하였다. 파라핀블록에서 4 um 두께의절편을얻어 95% 알콜로처리하였으며 luxol fast blue 용액으로 37 C에서 overnight 염색을시행하고, cresyl violet 용액으로 5분간염색하였다. 9. 통계분석 통계분석은 SPSS for Windows, 12.0 버전을이용하였다. 좌골신경기능지수에서독립된비모수집단간평균치비교를위하여, 통계적유의수준을 0.05 로하는 Kruskal-Wallis test를사용하였고, 사후다중비교에는유의수준 0.017의 Mann-Whitney U test with bonferroni correction를이용하였다. 근전도검사시기에따른복합근활동전위 (compound muscle action potential) 의진폭비교및원위부와근위부자극시의진폭을비교하기위하여 Wilcoxon signed-rank test를이용하였다

21 A B Figure 1. Picture of the new compression instrument. A; photograph of entire instruments setting B; enlarged photograph of the new miniature compression metal caps

22 Figure 2. Schematic illustration of cross-sectional view of the nerve compressed by the same pneumatic pressure

23 Figure 3. Sciatic nerve injury using the new miniature compression instrument. The clamp was used for holding the two caps of the miniature compression instrument

24 Figure 4. Direct stimulation on sciatic nerve to evoke the compound muscle action potential (CMAP) of flexor digitorum brevis

25 Figure 5. Overview of entire experimental process

26 Figure 6. Illustration of sciatic functional index. The parameter measured in the normal (left) and injured (right) footprints. NIT: Normal intermediary toe spread; Distance between 2nd and 4th toes, NPL: Normal paw length, NTS: Normal toe spread; Distance between 1st and 5th toes, EIT: Experimental intermediary toe spread, EPL: Experimental paw length, ETS: Experimental toe spread

27 결과 1. 좌골신경기능지수 (Sciatic functional index) 각군별로손상이후시간에따른좌골신경의기능을비교하였을때, A 군에서압박손상 36시간후 (-33.59, ) 와 1 주후 (-20.87, ) 에평가한좌골신경기능지수는손상전 (-7.33, ) 과비교하여각각유의하게감소하였으며 (Wilcoxon signed-rank test, P=0.005 and P=0.007), 손상 36시간후와 1 주후에평가한좌골신경기능지수간에유의한차이는보이지않았다 (Wilcoxon signed-rank test, P=0.16). B 군과 C 군역시동일한양상을보여손상전 (-7.88, in B group; -4.38, in C group) 에비해 36 시간후 (-32.28, in B group; , in C group) 및 1 주후 (-24.50, in B group; , in C group) 에좌골신경기능지수는유의하게감소하였으며 (Wilcoxon signed-rank test, P=0.009 and P=0.005 in B group, both P=0.005 in C group), 손상 36시간후와 1 주후간에유의한차이는보이지않았다 (Wilcoxon signed-rank test, P=0.17 in B group and P=0.40 in C group)(table 1). 세군간에좌골신경의기능을비교하였을때좌골신경의압박손상전과손상후 36 시간후에평가한좌골신경기능지수에서세군간에차이는관찰되지않았으며 (Kruskal-Wallis test, P=0.55 and P=0.089), 1 주후좌골신경기능지수에서만유의한차이가관찰되었다 (Kruskal-Wallis test, P=0.046). 그러나 1 주후좌골신경기능지수에서사후다중비교를하였을때의미있는차이를확인할수는없었다 (Mann-Whitney U test with bonferroni correction, P=0.029 between group B and C, P=0.043 between group A and C, and P=0.971 between group A and B)(Table 1)

28 2. 신경전도검사및침근전도검사 신경전도검사상좌골신경의복합근활동전위 (compound muscle action potential) 의진폭은 A 군에서압박손상직후원위부자극 (4.80±1.03 mv) 에비해근위부자극 (4.34±1.09 mv) 시에평균 9.5 % 정도감소하였지만통계적으로유의한차이를보이지는않았다 (Wilcoxon signed-rank test, P=0.11)(Figure 7 and 8). 손상 36 시간및 1 주후에도원위부자극 (4.78±1.36 mv and 4.76±1.42 mv) 에비해근위부자극시 (4.54±1.48 mv and 4.53±1.51 mv) 에유의한진폭감소소견은관찰되지않았으며손상전의진폭과각각비교하였을때에도통계적으로의미있는감소소견은관찰되지않았다 (Wilcoxon signed-rank test, all P>0.05)(Figure 7, 8). A 군에서시행한침근전도검사상, 36 시간후에 2 마리의흰쥐에서삽입전위가증가하였으며 1 주후에는모든흰쥐에서삽입전위의증가와양성예각파가 +의등급으로관찰되었고세동전위는모든흰쥐에서관찰되지않았다 (Figure 9). B 군역시손상직후근위부진폭 (3.74±1.21 mv) 이원위부 (5.19±1.13 mv) 에비해 28 % 정도의감소소견을보였으나통계적으로유의하지는않았고 (Wilcoxon signed-rank test, P=0.08), 손상 36 시간및 1 주후에도각검사시점에서근위부 (3.98±1.43 mv and 4.01±1.46 mv) 와원위부 (5.02±1.33 mv and 4.63±1.41 mv) 진폭간차이는보이지않았으며, 손상전복합근활동전위의진폭과비교하였을때에도의미있는차이는발견되지않았다 (Wilcoxon signed-rank test, all P>0.05)(Figure 10, 11). 손상 36시간후침근전도검사에서는 3마리의흰쥐에서삽입전위가증가하였으며 1 마리의쥐에서는짧은시간동안양성예각파 (brief PSW) 가한차례관찰되었다. 1 주후에는 3 마리의쥐에서삽입전위의증가소견이관찰되었고모든쥐에서양성예각파와세동전위를관찰할수있었다. 이중 2마리의쥐는 ++, 3마리는 +의등급으로양성예각파가관찰되었고세동전위는모두 +의등급으로관찰되었다 (Figure 12). C 군에서압박손상직후, 근위부진폭 (2.12±1.18 mv) 이원위부 (5.11±1.11 mv) 에비해 58 % 의의미있는감소소견을보였으며 (Wilcoxon signed-rank test, P=0.043), 손상 36 시간후에는근위부진폭 (2.53±1.46 mv) 이원위부 (4.97±1.39 mv) 에비해 50 % 감소하여다소간의 (marginal significance) 통계

29 적인차이를보였다 (Wilcoxon signed-rank test, P=0.061). 1 주후에는원위부진폭 (3.98±1.58 mv) 이감소하여근위부 (2.51±1.62 mv) 와비교하였을때의미있는차이는관찰되지않았다 (Wilcoxon signed-rank test, P=0.09). 손상전복합근활동전위의진폭과비교하였을때에는손상직후의근위부진폭에서의미있는차이를확인할수있었으며 (Wilcoxon signed-rank test, P=0.045), 손상 36 시간후의근위부진폭 (2.53±1.46 mv) 역시손상전근위부진폭 (4.98±0.99 mv) 에비해경계값의통계적유의성 (marginal significance) 을갖는차이를발견할수있었다 (Wilcoxon signed-rank test, P=0.059)(Figure 13 and 14). 침근전도검사상, 손상 36 시간후에대부분의쥐에서삽입전위는증가되었으며 2마리의쥐에서짧은시간동안양성예각파 (brief PSW) 가관찰되었으나 + 등급이상의비정상자발전위는모든쥐에서관찰되지않았다. 1 주후에는모든쥐에서양성예각파와세동전위가관찰되었으며 4 마리는 1+ 에서 2+ 의등급으로나머지 1 마리는 3+ 등급으로관찰되었다 (Figure 15). 3. 조직검사 축삭과수초관찰을위하여시행한면역조직화학염색과 Luxol 염색에대한광학현미경소견상, true 대조군과 sham 조작대조군그리고각실험군의비손상측좌골신경모두에서축삭및수초의손상소견은관찰되지않았으며, 축삭이나수초의염색정도및두께등에서대조군과실험군의비손상측좌골신경간에차이를보이지않았다. A 군의경우손상 36 시간후의면역조직화학염색에서모든축삭이정상적으로염색되었으나 Luxol 염색검사에서는경도의수초손상소견이관찰되었으며, 1주후의조직검사에서도동일한소견을보여수초의재생소견은관찰되지않았다. B 군의조직검사상, 손상 36 시간후에는극소수의축삭손상이관찰되었으나뚜렷하지않았고수초손상소견은 A 군에비해증가되었다. 축삭손상은 1 주후다소증가하였으나그외뚜렷한변화는관찰되지않았다. C 군에서는수초손상이관찰되는빈도와축삭손상의소견도 B 군에비해증가되어관찰되었으며이러한소견은 1 주후검사소견에서더뚜렷하였다 (Figure 16 19)

30 Table 1. Temporal Changes of Sciatic Functional Index (SFI) Group Pre Post-36hr Post-1week A -7.33( ) ( ) ( ) B -7.88( ) ( ) ( ) C -4.38( ) ( ) ( ) Values are median (interquartile range). Pre; baseline SFI score before injury, Post-36hr; SFI score at 36 hours after injury, Post-1week; SFI score at 1 week after injury

31 Figure 7. Serial changes of amplitude of CMAP in Group A (5 min injury group). Each point represents the mean, and the vertical bar indicates the standard deviation. There are no significant differences from baseline CMAP amplitude and between distal and proximal CMAP amplitudes. Pre; baseline CMAP amplitude before injury, Post-imm; CMAP amplitude assessed immediately after injury, Post-36hr; CMAP amplitude at 36 hours after injury, Post-1week; CMAP amplitude at 1 week after injury

32 Figure 8. Examples of serial sciatic motor nerve conduction study in one rat of group A. Upper and lower graphs were obtained at distal and proximal stimulation, respectively. Pre; proximal CMAP shows no reduction of amplitude compared with distal CMAP before injury. Post-imm, Post-36hr, and Post-1week; proximal CMAPs show very mild reduction of amplitude compared with distal CMAPs immediately after, at 36 hours, and at 1 week after injury, respectively

33 Figure 9. Findings of needle EMG in group A. Needle-36hr; the EMG graph shows increased insertional activity without any abnormal spontaneous activity at 36 hours after injury. Needle-1week; the EMG graph shows positive sharp waves without any fibrillation potential at one week after injury

34 Figure 10. Serial changes of amplitude of CMAP in Group B (10 min injury group). Each point represents the mean, and the vertical bar indicates the standard deviation. There are no significant differences from baseline CMAP amplitude and between distal CMAP amplitude and proximal CMAP amplitude. Pre; baseline CMAP amplitude before injury, Post-imm; CMAP amplitude assessed immediately after injury, Post-36hr; CMAP amplitude at 36 hours after injury, Post-1week; CMAP amplitude at 1 week after injury

35 Figure 11. Examples of serial sciatic motor nerve conduction study in one rat of group B. Upper and lower graphs were obtained at distal and proximal stimulation, respectively. Pre; proximal CMAP shows no reduction of amplitude compared with distal CMAP before injury. Post-imm and Post-36hr; proximal CMAPs show mild reduction of amplitude compared with distal CMAPs immediately after and at 36 hours after injury, respectively. Post-1week; distal CMAP show very mild reduction of amplitude at 1 week after injury

36 Figure 12. Findings of needle EMG in group B. Needle-36hr; the EMG graph shows increased insertional activity and brief PSWs at 36 hours after injury. Needle-1week; the EMG graph shows PSWs and fibrillation potentials at one week after injury

37 ** * ** * * Figure 13. Serial changes of amplitude of CMAP in Group C (30 min injury group). Each point represents the mean, and the vertical bar indicates the standard deviation. Double asterisks indicate significant difference between distal and proximal CMAP amplitude and between baseline CMAP amplitude and each proximal CMAP amplitude by Wilcoxon signed-rank test (P<0.05). Single asterisks indicate marginal significance by Wilcoxon signed-rank test. Pre; baseline CMAP amplitude before injury, Post-imm; CMAP amplitude assessed immediately after injury, Post-36hr; CMAP amplitude at 36 hours after injury, Post-1week; CMAP amplitude at 1 week after injury

38 Figure 14. Examples of serial sciatic motor nerve conduction study in one rat of group C. Upper and lower graphs were obtained at distal and proximal stimulation, respectively. Pre; proximal CMAP shows no reduction of amplitude compared with distal CMAP before injury. Post-imm; proximal CMAP shows severe reduction of amplitude compared with distal CMAP immediately after injury. Post-36hr; proximal CMAP shows moderate reduction of amplitude and distal CMAP shows very mild reduction of amplitude at 36 hours after injury. Post-1week; distal CMAP shows mild reduction of amplitude at 1 week after injury

39 Figure 15. Findings of needle EMG in group C. Needle-36hr; the EMG graph shows brief PSWs at 36 hours after injury. Needle-1week; the EMG graph shows relatively profound PSWs and fibrillation potentials at one week after injury

40 Figure 16. Immunohistochemical staining for neurofilament in rat sciatic nerves at 36 hours after compression injury. A; control, every axon reveals normal endoneural structure, B; group A (5 min injury), and C; group B (10 min injury), axonal loss is not revealed, D; group C (30 min injury), some axons show axonal loss (arrow). (X200)

41 Figure 17. Luxol fast blue staining in rat sciatic nerves at 36 hours after compression injury. A; control, every axon reveals normal axon-myelin architecture, B; group A (5 min injury), and C; group B (10 min injury), some axons reveal myelin collapse (arrow head), D; group C (30 min injury), definite collapse of myelin is shown. (X400)

42 A B C D Figure 18. Immunohistochemical staining for neurofilament in rat sciatic nerves at 1 week after compression injury. A; control, every axon reveals normal endoneural structure, B; group A (5 min injury), no axonal loss is revealed, C; group B (10 min injury), some axons show axonal loss (arrow), D; group C (30 min injury), increased number of axons show axonal loss. (X200)

43 Figure 19. Luxol fast blue staining in rat sciatic nerves at 1 week after compression injury. A; control, normal axon-myelin architecture, B; group A (5 min injury), some axons reveal myelin collapse (arrow head), C; group B (10 min injury), and D; group C (30min injury), definite collapse of myelin is shown. (X400)

44 고찰 말초신경손상의정도를객관적으로평가하고예후를예측하기위하여임상적으로시행되고있는침근전도검사에있어서탈수초 (demyelination) 병변에서의비정상자발전위의발생여부는아직까지많은논란에싸여있다. 하지만사람에있어서침근전도검사와손상된말초신경의조직검사를병행하여서로간에연관성을확인하기는불가능하며, 이에대한동물실험으로는기니피그 (guinea pig) 에서실험적으로유발된알레르기신경염 (experimental allergic neuritis) 에서본침근전도소견이유일하다 (Kraft, 1973). 하지만알레르기신경염이탈수초병변을주로유발하지만축삭손상없이탈수초병변만을일으켰다고보기에는무리가있으며, 무엇보다도면역학적인방법으로유발된좌골신경병변이므로임상에서흔히접하게되는압박이나허혈로인한탈수초병변과는병태생리적으로차이를보인다할수있다. 본연구에서는균일한압박에의하여매우미약하지만정량적인탈수초병변을유발하였고, 이러한탈수초병변에서는양성예각파만이관찰되었으며, 조직학적으로축삭손상이증가함에따라세동전위의유발정도도증가됨을침근전도검사로확인하였다. 따라서침근전도검사에서축상손상의유무를예측하기위한비정상자발전위로는양성예각파보다는세동전위의유발유무를중요한척도로제시하는바이다. 동물에서말초신경손상을유발하기위해대표적으로사용되었던압박커프는신경이포함된사지 (limb) 를감싸서직접압박을가하기때문에 500 mmhg 에서 1000 mmhg의매우큰압력을가하게되어 (Lundborg, 1970; Fowler 등, 1972; Ochoa와 Fowler, 1972), 손상의부위를정확히확인할수없고손상의정도역시정량화하기어려운단점이있었다. 이를보완하여좀더정량화된신경손상을가하기위하여 miniature inflatable cuff를이용하여신경조직을직접압박하는방법이사용되었는데 (Rydevik과 Lundborg, 1977; Dahlin과 McLean, 1986; Dyck 등, 1990), 이방법역시 2 개의커프가알루미늄판을밀어서알루미늄판사이의신경에손상이가해지도록제작하였기때문에일종의압궤손상을가하게되어역시경도의신경손상을유발하거나정량화된신경손상을만드는데에는제한이있었다. 만성적인압박손상을유발하기위해서는금속

45 clip이나 clamp, 실리콘이나폴리에틸렌재질의튜브등을이용하여지속적인압박이가해지도록하는방법이사용되나 (Dahlin과 Kanje, 1992; Kajander 등, 1996; Kanje 등, 1995; Weisl과 Osborne, 1964) 압박되는정도를조절할수없고염증이유발되는단점이있어사람에서수근관증후군과같은만성압박손상의병태생리와는맞지않는제한점을가지고있다. 본연구에서사용하였던압박기구는신경조직을완전히감쌀정도의유연한재질을사용하여신경을직접감싸는커프를이용하였고, 따라서비교적정량적인손상을가할수있었다. 일반적으로말초신경에 분간의허혈이발생하면일시적인전도차단이발생하지만, 바로회복되는것으로알려져있으며, 이러한허혈은 mmhg의압력이가해졌을때발생한다. 따라서예비연구에서는 30 분간의동일시간동안 100 mmhg부터 160 mmhg의압박압력을비교하여가장실현가능성이있는 (feasible) 압력으로 160 mmhg를택하였고, 그다음단계에서는 160 mmhg에서압박시간을달리하는예비연구를진행하여본실험에부합하는압박시간을 5 분, 10분, 30 분으로설정하였다. 예비연구결과 100 mmhg 와 120 mmhg에서는 30분간아무런변화가관찰되지않았고, 140 mmhg에서는 15 분정도부터, 160 mmhg에서는 5분정도부터전도차단이발생하는것으로확인되어, 압박압력을낮게하고압박시간을길게한다면본압박기구를이용하여허혈에의한신경손상을연구하는데에도사용할수있을것으로생각된다. 말초신경의탈수초병변을유발하는기존의방법으로는기니피그의좌골신경을갈아서화학처리를하고, 여기서추출한항원을주사하여주로수초를파괴하는알레르기신경염모델이사용되며, 이와같은모델에서도양성예각파만이관찰되어 (Kraft, 1973) 본연구와부합되는결과를보였다. 또다른탈수초병변의유발모델로는원래중추신경의탈수초병변유발을위하여사용하였던염색약의일종인 ethium bromide를말초신경에반복적으로주사하는방법 (Riet-Correa 등, 2002) 과역시중추신경의탈수초병변유발을위하여사용하였던 lysolecithin을국소적으로말초신경에도포하는방법 (Wallace 등, 2003) 이사용되나순수한탈수초병변만을유발하는지는확실하지않다. 그동안탈신경 (denervation) 시발생하는비정상자발전위에대한연구들은대부분동물에서세동전위의체외 (in vitro) 전기생리학적연구에집중되어왔

46 다. 세동전위는근섬유의휴지기막전위 (resting membrane potential) 가탈분극역치에가까워지고 (Thesleff, 1974; Thesleff와 Ward, 1975) 불안정하게진동하여 (oscillating) 탈분극, 재분극이반복되면서발생하는것으로설명되어진다 (Denny-Brown과 Pennybacker, 1938; Buchthal과 Rosenfalck, 1966; Thesleff, 1974). 이러한휴지기막전위와탈분극역치의간격은종판 (endplate) 부위에서더작기때문에세동전위가종판부위에서더쉽게관찰된다 (Spence와 Guyton, 1966; Thesleff와 Ward, 1975). 이에반해양성예각파에대한전기생리학적기초연구는매우부족하며, 기본적으로는자발적근섬유수축에의한전위가근육-힘줄경계 (musculotendinous junction) 에서기록될때양성예각파로기록되므로많은연구자들은양성예각파와세동전위에임상적으로동일한의미를부여하고있다 (Lambert, 1957; Katz와 Miledi, 1965; Buchthal과 Rosenfalck, 1966). 즉세동전위의 negative sink currunt가기록전극에도달할때이것이차단되어양성예각파의모양으로관찰되며, 최근에는조직여과 (tissue filtering) 에의해세동전위와양성예각파로의모양이결정된다는연구가보고되었다 (Campbell과 Landau, 2003). 세동전위와양성예각파모두불안정한휴지기막전위를가진근육조직에서발생하는자발적인비정상전위임에는틀림없으나, 지금까지의동물실험을통한생체내 (in vivo) 및체외전기생리학적실험들은모두세동전위에만집중되어왔으며, 이는양성예각파와세동전위가하나의신경섬유에서발생하는전위로서기록되는파형만다를뿐이지동일한근원이라는기본적인이해로부터출발한것이었다. 하지만사람이나동물모두에서양성예각파가세동전위보다신경손상후먼저발생하며 (Kimura, 1989; Brown과 Bolton, 1993), 세동전위없이양성예각파만관찰되는경우가흔하고 (Wiechers와 Johnson, 1979; 1982) 양성예각파는원위부근육에서정상적으로도발생하는경우가있어 (Wiechers 등, 1976) 전자의주장과는대치되는임상적인증거들이제시되고있다. 본실험의연구에서도조직학적으로축삭손상의증거가보이는군에서만세동전위가관찰되어양성예각파와세동전위는서로다름을암시하는결과로생각된다. 본실험의제한점으로첫째, 압박손상의원위부좌골신경에대한조직검사를통하여왈러변성소견을확인하지못한것으로서이는절제가능한좌골신경의길이가충분치못하기때문이었다. 그리고신경전도검사에서복합근활

47 동전위의여러가지측정변수중진폭만을사용하고잠시 (latency), 전도속도 (conduction velocity), 면적 (area) 등의변수는본연구에서는분석하지않았다. 하지만검사기간중에는회복기가포함되지않았기때문에전도차단이나왈러변성에의한원위부진폭의감소소견등을정확히해석할수있었고, 근위부자극과원위부자극간의거리차이가너무짧기때문에잠시와전도속도에있어서는자극시마다일관된 (consistent) 데이터를얻을수없었다. 따라서흰쥐보다좀더큰동물을대상으로한실험이향후필요할것으로생각된다. 둘째, 실험대상수의부족으로압박손상전후및근위부자극시와원위부자극간에복합근활동전위의진폭차이를경도의압박손상을가하였을때에는통계적으로검증하지못하였다. 이에대한추가적인보완연구역시필요할것으로생각되며, 좌골신경기능지수에있어서도모든실험군에서손상전후로는유의한차이가관찰되었지만군간비교에서는뚜렷한차이를발견하지못하였다. 이는새로운신경손상기구가위치할수있는공간을확보하기위하여좌골절흔부위의둔부근육과슬관절상부의슬와부근육을절개하였기때문으로생각된다. 손상측다리가들리거나질질끌리면서보행하여족문채취가불가능한경우가각군당 2 3 마리로, 이경우 -100 점으로환산되었기때문에군간차이를검증할수없었던것으로추측된다. 셋째, 본실험에서는손상후 1 주이상에서근전도소견과조직검사소견을확인하지않았다. 수초재생 (remyelination) 이나축삭재생 (axonal regeneration) 에의한회복기의신경전도검사및침근전도검사는오히려본실험의목적에부합하지않으며해석에도혼란을줄수있을것으로생각되나, 동물을대상으로하여탈수초병변이나병변의중증도에따른회복기근전도검사와조직검사역시사람에서의근전도검사소견을해석하는데에많은도움을줄수있을것으로사료된다

48 결론 흰쥐의좌골신경에서신경손상정도를정량화할수있는새로운손상모델을개발하여이를통한탈수초병변을유발하였다. 탈수초병변에서세동전위는관찰되지않았으며양성예각파만일관되게 (consistently) 확인할수있었으며, 이러한침근전도소견은양성예각파의발생기전과임상적인의미가세동전위와는서로다름을시사한다. 향후말초신경손상에관한병태생리적연구나중재적실험 (intervention study) 에서새로운말초신경손상모델을이용한다면좀더정량적이고정확한연구결과와결론을도출하는데도움이될것으로생각된다

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