脊髓損傷大鼠遠端神經(jīng)元及骨骼肌變化的基礎研究

解金三

中國平煤神馬醫(yī)療集團總醫(yī)院，河南 平頂山 467000

·論著臨床診治·

脊髓損傷大鼠遠端神經(jīng)元及骨骼肌變化的基礎研究

解金三

中國平煤神馬醫(yī)療集團總醫(yī)院，河南 平頂山 467000

目的觀察脊髓損傷大鼠遠端神經(jīng)元及骨骼肌變化情況。方法20只大鼠隨機分為2組，每組10只，分別為假手術組和脊髓損傷組，假手術組行椎板切除術，脊髓損傷組行胸10完全脊髓損傷，在制成模型后1、2、4、12、24周觀察大鼠坐骨神經(jīng)-運動終板-內(nèi)側腓腸肌形態(tài)變化情況。結果脊髓損傷組電鏡下坐骨神經(jīng)術后12周有髓神經(jīng)纖維髓鞘崩解，其板層結構清晰，有髓神經(jīng)纖維髓鞘于術后24周模糊、碎裂髓鞘變多，12周后無髓神經(jīng)纖維及薄髓增多；術后12周腓腸肌光鏡下局部肌細胞多數(shù)模糊，但邊界清楚，結締組織增生明顯，肌細胞核相對聚集；肌細胞于術后24周融合，融合細胞間有空隙，細胞核密集，結締組織增生明顯；術后12周電鏡下運動終板突觸前后及皺褶膜不可分辨，肌纖維明暗帶清晰，突觸結構紊亂，z線不連續(xù)，高倍鏡下突觸前后膜不可辨，突觸皺褶未見，可見類圓形細小顆粒及突觸小泡，肌板結構清晰。結論大鼠脊髓損傷后在損傷平面以下周圍神經(jīng)、運動終板、骨骼肌在形態(tài)上會發(fā)生規(guī)律性變化，12周后顯著變化，24周后則毀損性改變。

脊髓損傷；大鼠；遠端神經(jīng)元；骨骼肌變化

脊髓損傷是臨床上診斷和治療較為復雜的疾病，一般治療目的均為恢復損傷平面以下的排泄及運動相關功能。但這與腦和周圍神經(jīng)肌肉功能、脊髓傳導性等關系十分密切，三者只有統(tǒng)一協(xié)調(diào)才能恢復滿意。若遠端神經(jīng)或肌肉不可逆損傷則脊髓傳導恢復幾乎不可能。相關研究[1]稱，通過切斷胸12脊髓，制造的完全性截癱動物模型，將下肢全部神經(jīng)干在傷后3、6周切除，病理檢查髓鞘、神經(jīng)纖維等變化不明顯。而另有學者研究[2]稱，在大鼠胸10完全脊髓橫斷損傷后，周圍神經(jīng)在損傷平面部分會產(chǎn)生跨神經(jīng)元變性。因此，本研究特制作脊髓損傷大鼠模型，并觀察模型鼠遠端神經(jīng)元及骨骼肌變化情況。

1 資料與方法

1.1臨床資料選取20只大鼠隨機分組，每組10只，分別為假手術組和脊髓損傷組。均為雄性，近交系，平均體質(zhì)量(200±10)g。

1.2方法先制備動物模型，術前大鼠禁食12 h，10%水合氯醛3～4 mL/kg于稱重后腹腔內(nèi)注射麻醉，備皮消毒后行背側正中切口，以胸10棘突為中心長約3 cm，將椎旁肌肉和軟組織緊貼棘突骨面縱向分離，顯露出胸9-11椎板和棘突，將胸10脊髓用剪刀迅速橫斷，切除脊髓斷端頭側2 mm脊髓組織，移除后在脊髓端明膠海綿置入，逐層縫合。研究期間，每日人工排尿2～3次直至恢復正常排尿為止。手術組不損傷脊髓，僅行椎板及棘突切除。取右側坐骨神經(jīng)1 cm，腓腸肌肌腹中部神經(jīng)入肌處取0.8 cm肌肉和小腿內(nèi)側腓腸肌肌腹1 cm，固定12 h后進行處理，石蠟打包后將內(nèi)側腓腸肌標本進行連續(xù)橫向切片，厚度為10 μm，進行蘇木精-伊紅染色，將坐骨神經(jīng)和運動終板行半薄切片后在光鏡下進行定位，超薄切片后行電子染色進行電鏡觀察。

2 結果

2.1坐骨神經(jīng)電鏡觀察手術組脊髓損傷變化規(guī)律，有髓神經(jīng)纖維外形正常，術后2周有髓神經(jīng)纖維髓鞘正常，術后4周有髓神經(jīng)纖維崩解、板層分離，術后12周有髓神經(jīng)纖維崩解增多、碎裂、崩解髓鞘板層清晰，新生神經(jīng)纖維髓鞘細壁薄，無髓神經(jīng)纖維增多，術后24周有髓神經(jīng)纖維髓鞘已模糊、碎裂髓鞘增多，薄髓和無髓神經(jīng)纖維較12周后增多。 對照組有髓神經(jīng)纖維、無髓神經(jīng)纖維一直存在，髓鞘外形正常。

2.2光鏡下觀察腓腸肌手術組術后2周肌細胞肌束和肌內(nèi)膜邊界清楚，脊髓損傷術后2周肌細胞略縮小，術后4周肌細胞截面積縮小，局部肌細胞破壞明顯，術后12周局部肌細胞邊界清楚，多數(shù)模糊，肌細胞核相對聚集，結締組織增生明顯；術后24周肌細胞融合，細胞核密集，融合細胞間有空隙，結締組織增生明顯。對照組細胞肌束膜和肌內(nèi)膜邊界清楚，肌細胞核位于肌膜下方。

2.3運動終板電鏡下觀察脊髓損傷術后4周突觸皺褶結構清晰，突觸內(nèi)可見細顆粒狀物質(zhì)，高倍鏡下觀察可見突觸前后膜清晰，突觸間隙、皺褶清晰，各個結構內(nèi)見細小顆粒，術后12周突觸皺褶和前后膜清晰，突觸內(nèi)可見細小顆粒，肌纖維明暗可見，z線不連續(xù)。高倍鏡下突觸皺褶清晰，突觸前后膜層次分明，結構內(nèi)可見細小顆粒，肌板清晰；術后12周突觸皺褶和突觸前后膜不可辨，結構紊亂，肌纖維明暗帶清晰，z線不連續(xù)，高倍鏡下突觸皺褶未見，突觸前后膜不可辨，可見類圓形突觸小泡和細小顆粒，肌板結構清晰。

3 討論

結果顯示，大鼠脊髓橫斷性損傷4周后坐骨神經(jīng)出現(xiàn)有髓神經(jīng)纖維髓鞘退變，且會隨著時間推移而變化，12周后髓鞘出現(xiàn)崩解碎解現(xiàn)象，24周后最明顯。這和手術、正常大鼠比較有明顯不同，這可推測出脊髓損傷后遠端神經(jīng)元發(fā)出軸突構成周圍神經(jīng)發(fā)生退變，且會隨著時間進行性加重。結合相關報道[3]，是因為存在跨神經(jīng)元變性。報道稱[4-5]，脊髓損傷后發(fā)生跨神經(jīng)元變性時間窗為20周左右。在運動終板形態(tài)上，24周后則無法找到運動終板。與相關報道[6-7]結果類似，報道稱，脊髓橫斷損傷8周后運動終板會出現(xiàn)明顯退變，染色區(qū)空白，神經(jīng)肌肉突觸出現(xiàn)選擇性分解[8-9]。在腓腸肌形態(tài)變化上，結果顯示，脊髓損傷4周后腓腸肌肌細胞截面積縮小，且截面積和觀察時間呈負相關。12周肌細胞萎縮明顯，大量結締組織增生，24周后肌細胞大片融合，無法確認肌細胞結構，且結締組織增生明顯，而手術組和正常組則無此變化。推測脊髓損傷后肌細胞會發(fā)生進行性退變，肌細胞截面積縮小，后期表現(xiàn)為肌纖維形態(tài)結構破壞和結締組織填充[10]。與相關報道[11-12]結果類似，脊髓損傷后大鼠肌纖維數(shù)目減少，肌纖維萎縮，表現(xiàn)為肌肉截面積縮小，肌肉質(zhì)量下降，肌間脂肪堆積。大鼠比目魚肌和內(nèi)側腓腸肌在脊髓損傷后1周下降22%和27%，脊髓損傷后3周下降15%和18%，脊髓損傷10周和脊髓損傷3周在肌肉濕重量上相近[13]。

大鼠脊髓損傷后在損傷平面以下周圍神經(jīng)、運動終板、骨骼肌在形態(tài)上會發(fā)生規(guī)律性變化，12周后顯著變化，24周后則毀損性改變。但本次研究中也存在缺陷，主要是未將神經(jīng)電生理學相關評價標準納入，這樣就不能從量化程度上反應神經(jīng)肌肉運動功能變化情況。但大鼠脊髓損傷和人脊髓損傷在運動神經(jīng)元和骨骼肌上有一定相近性，對治療時間窗有一定的作用。

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(收稿2017-04-11)

責任編輯：王喜梅

Basicresearchofthechangesindistalneuronsandskeletalmusclesinratswithspinalcordinjury

XieJinsan

GeneralHospitalofChinaPingmeiShenmaMedicalGroup，Pingdingshan467000，China

ObjectiveTo investigate the changes in distal neurons and skeletal muscles in rats with spinal cord injury (SCI).Methods20 rats were randomly selected as the research objects and were divided into sham-operated group in which the rats

laminectomy and SCI group in which the rats suffered from 10th thoracic cord injury，10 rats in each group.The changes in sciatic nerve，motor end-plate and inside gastrocnemius muscle were observed in the 1st，2nd，4th，12th and 24th week after successful model.Results12 weeks after sciatic nerve operation in spinal cord injury rats under the electron microscope，the myelin disintegration occurred in myelinated nerve fibers where the lamellar structure was clear，and 24 weeks after successful model the myelinated nerve fibers became indistinct and showed more cataclastic myelin sheaths.In addition，12 weeks later，the unmyelinated nerve fibers and thin myelin sheaths were increased.12 weeks after operation at the light microscopic level the most partial muscle cells turned into being blurred but the periphery was clear，connective tissues presented obvious hyperplasia and muscular cell nucleus aggregated.Furthermore，the muscle cells merged together and the lacuna appeared between them accompanied by hugely hyperplastic connective tissues and intensive cell nucleus.In the 12th week，it was difficult to distinguish presynaptic membrane and postsynaptic membrane of motor end-plate as well as ruffle membrane observed by electron microscope.Although synaptic structure disorder and z-line discontinuity were found，the parazone and diazone of muscle fiber were clear.Similarly，we couldn’t make out the presynaptic membrane and postsynaptic membrane by using high-power microscope，found out no synapsis folds and clearly discovered round-like tiny particles and synaptic vesicles，but the muscular plate structure was distinct.ConclusionThe peripheral nerves，motor end plates and skeletal muscles can change beneath the injured plane after spinal cord injury in rats，and the significant changes occur in the 12th week and completely damaged changes appear in the 24th week.

Spinal cord injury；Rats；Distal neurons；Changes in skeletal muscle

10.3969/j.issn.1673-5110.2017.16.029

解金三(1980-)，碩士，主治醫(yī)師。研究方向：關節(jié)及運動醫(yī)學。Email：172289221@qq.com

R-332

A

1673-5110(2017)16-0092-04