MOG35-55诱发C57BL/6小鼠实验性自身免疫性脑脊髓炎模型轴索损害可能并非继发于炎性脱髓鞘

采用MOG35-55加完全弗氏佐剂诱发实验性自身免疫性脑脊髓炎C57BL/6小鼠模型,其发病潜伏期约12 d,发病率100%,呈慢性非复发性病程经过。其神经组织病理改变表现为：光镜下见小血管周围炎细胞浸润,呈袖套状改变;Luxol-fast-blue染色可见髓鞘脱失;TUNEL染色和Bielshowsky银染法发现神经元变性和轴索损害;电镜下可见线粒体肿胀,细胞器消失,髓鞘结构松散、断裂或融合, 并可见不同程度的髓鞘重建。且轴索损害与髓鞘脱失的分布并不一致,轴索损伤的程度与髓鞘脱失亦不成比例,部分甚至与脱髓鞘无关。提示实验性自身免疫性脑脊髓炎的轴索损害并非继发于炎性脱髓鞘。     

实验性轴索型GBS坐骨神经病理学研究

目的：探讨轴索型格林巴利综合征（GBS）动物模型坐骨神经的病理学改变。结果：对发病兔坐骨神经进行分离单神经、LFB镀银染色、HE染色及电镜检查。结果：分离单神经显示严重Wallerian样变性，串珠样改变；HE染色炎性细胞浸润少见；LFB镀银染色坐骨神经轴索肿胀、增粗、粗细不均、扭曲、部分破坏、断裂、消失，呈严重的轴索变性；电镜检查坐骨神经轴索固缩，体积变小，微丝、微管致密、集聚成团、线粒体固缩。严重者可见轴索膜破坏溶解，呈大小不等、形态不规则的空泡样结构、轴索移向一边或消失，髓鞘相对完整。结论：本实验证实了有轴索型GBS亚型的存在，同时也证实AMAN型GBS为一种原发性轴索变性疾病。     

脑缺血再灌注后白质损伤研究

目的 观察大鼠脑缺血再灌注后不同时间髓鞘和轴突损伤的病理变化,明确脑缺血后白质损伤情况.方法 利用线栓法制备雄性SD大鼠大脑中动脉闭塞(MCAO)模型.将大鼠随机分为假手术组和缺血2 h再灌注6 h、12 h、1 d、3 d、7 d、14 d和21 d组,每组4只.于规定时间点处死大鼠取脑,行常规HE染色观察皮层区神经元病理变化,行Luxol fast blue-periodic acid Schiff(LFB-PAS)染色法标记大脑神经髓鞘,Bielschowsky银染法标记轴突,计算缺血侧与健侧髓鞘染色的积分吸光度(integrated optical densities,IODs)比值代表白质受损程度.结果 MCAO再灌注6 h皮层区即已出现细胞间质水肿,核深染、固缩,细胞变性坏死.随再灌注时间延长损伤加重,神经元进一步减少.再灌注14 d及21 d有大量胶质细胞增生.与假手术组比较,再灌注6 h时髓鞘染色IODs比值亦开始下降(P<0.01);12 h时有空洞形成,再灌注14 d髓鞘损伤达高峰,IODs比值降到最低,髓鞘脱失明显;21 d时髓鞘IODs比值与14 d比较差异无统计学意义(P>0.05).轴突变化规律与髓鞘基本相同.结论 脑白质对缺血同样敏感,在急性脑缺血早期即已存在白质损伤,并随再灌注时间延长逐渐加重,提示脑缺血后应及早对白质损伤进行干预.     

重组腺相关病毒神经肽Y基因转染对癫大鼠海马病理变化的影响

目的观察重组腺相关病毒介导人源性神经肽Y(rAAV-hNPY-EGFP)基因转染对癫大鼠海马病理变化的影响。方法 28只Wistar大鼠随机分为点燃组(n=20)和正常对照组(n=8)。正常对照组不进行特殊处理,点燃组以大鼠海马内多次注射红藻氨酸(KA)建立慢性癫模型,造模成功16只,其随机分为模型组和神经肽Y(NPY)治疗组,每组各8只大鼠。NPY治疗组大鼠转染rAAV2/1-hNPY-EGFP基因,模型组未转染。转染4周后,每组取6只大鼠海马行苏木精-伊红染色,2只行电镜观察。结果苏木精-伊红染色显示:正常对照组大鼠海马CA3区神经元形态正常;模型组海马CA3区神经元丢失,胶质细胞增生;NPY治疗组基因转染后神经元丢失减少。模型组神经元数目为(10.67±7.87)个/视野,正常对照组为(81.42±5.63)个/视野,明显多于模型组(P<0.05);而NPY治疗组神经元数目为(65.73±2.81)个/视野,明显多于模型组(P<0.05)。电镜显示:正常对照组神经元结构正常;模型组神经元固缩,线粒体肿胀;NPY治疗组神经元线粒体结构完整。结论 rAAV-hNPY-EGFP基因转染可减轻大鼠癫发作引起的病理改变,发挥抑制癫的作用。     

癫痫大鼠海马神经元和星形胶质细胞的病理演变

目的　探讨癫痫大鼠海马神经元和星形胶质细胞在点燃后各期的病理特点、时序及机制。方法　针对匹罗卡品癫痫大鼠模型,行Nissl、免疫组化和HE染色,观察海马神经元及星形胶质细胞的病理变化。结果　癫痫持续状态后超急性期（4h）,CA3区神经元呈嗜酸性变性、胞浆深染;急性期（24h）,嗜酸性变性最为显著,神经元固缩、核仁消失、突起断裂,星形胶质细胞水肿;缄默期（7d）,CA3、CA1区及门区神经元大量坏死、脱失,胶质增生肥大,海马构筑紊乱;慢性期（6w）,CA3、CA1区出现胶质瘢痕,遗有形态正常的神经元,且颗粒细胞层增厚。结论　癫痫时海马神经元先于星形胶质细胞发生病理改变,二者均参与癫痫发生。     

鸡甲胺磷中毒性神经肌病的神经病理及药物干预治疗

目的　研究甲胺磷中毒迟发性神经病的周围神经病理特点 ,并探讨三磷酸胞苷二钠 (CTP)、地塞米松对有机磷中毒迟发性神经病 (OPIDN)是否有保护作用。方法　通过甲胺磷染毒制备鸡 OPIDN模型 ,观察小、大剂量 CTP[1.5 mg/ (kg· d) ,3mg/ (kg· d) ]和地塞米松 [1mg/ (kg· d) ]不同作用时间 (1、2、3、4、8周 )的神经病理改变。结果　甲胺磷染毒后光镜下甲苯胺兰染色第 2周末即出现髓鞘轻微改变 ,第 3周末出现部分神经纤维变性 ,髓鞘深染、形状不规则 ,神经轴索肿胀 ,第 4周末达高峰 ,第 8周末病变明显好转。电镜下第 3周末髓鞘增厚、板层松解、轴索肿胀。大剂量 CTP组和地塞米松组神经病理改变相对减轻 ,但有髓神经纤维横切面积与正常组相比具有显著性差异 (P<0 .0 5 )。大剂量 CTP组郎飞结间的距离与其余各组相比均具有显著性差异 (P<0 .0 5 )。结论　甲胺磷染毒后周围神经的病理改变为髓鞘脱失和轴索肿胀 ,大径神经纤维更易感。应用大剂量 CTP、地塞米松可减轻病损 ,促进恢复 ,但不能防止 OPIDN的发生。     

小鼠实验性自身免疫性脑脊髓炎的病理变化

目的用髓鞘少突胶质细胞糖蛋白多肽(MOG35-55)诱发实验性自身免疫性脑脊髓炎(experi-m ental autoimmune encephalomyelitis,EAE)小鼠模型。方法应用MOG35-55抗原加完全弗氏佐剂免疫C57BL/6小鼠,利用光镜、电镜观察小鼠组织学改变。结果光镜下可见小血管周围炎细胞浸润,呈袖套状改变、血管周围明显脱髓鞘及神经元变性,B ieschowsky银染显示大量轴索肿胀和轴索卵形体的形成,电镜下可见髓鞘结构松散、断裂或融合,包括不同程度的髓鞘重建,脊髓病变广泛,程度重于脑部。结论EAE的病理改变为血管周围炎性细胞浸润、白质脱髓鞘及髓鞘重建。     

多重脑震荡对大鼠空间认知行为的影响

目的探讨多重脑震荡（MCC）对大鼠认知行为的影响。方法成年SD大鼠24只，应用自制金属单摆式闭合性脑损伤机械打击装置复制MCC大鼠模型，随机分为对照组和MCC组，伤后应用Morris水迷宫实验，评价大鼠的空间学习记忆功能。结果在Morris水迷宫测试中，与对照组（8～14d测试结果分别为：64．74±23、43，33．16±17．70，36．59±27．53，37、60±18．20，37．14±26．03，16．85±10．63，10、02±6．51）相比，MCC大鼠伤后8～14d找到平台的时间明显延长，且在伤后的第9d（71．74±35．76）、第10d（63．14±31．02）、第13d（47．51±40．93）、第14d（42、39±43．33）差异具有显著性。在无平台探测实验中，与对照组（23．89±11．82）相比，MCC后第14d（15．12±6．56）大鼠在原有平台第三象限停留时间明显减少，差异显著（P〈0．05）。结论实验性多重脑震荡大鼠在Morris水迷宫实验中，出现较为严重的空间认知行为障碍。     

大鼠轻型颅脑损伤后神经胶质病理改变与血清GFAP水平

目的研究轻型颅脑损伤的病理变化和胶质纤维酸性蛋白(GFAP)的表达。方法收集32只成年大鼠,随机分为假手术组(n=8)、伤后1 d组(n=8)、伤后3 d组(n=8)和伤后7 d组(n=8)。损伤组采用液压冲击设备建立大鼠轻型闭合性颅脑损伤模型,假手术组仅切开头皮并钻孔。免疫组化方法检测脑组织中星形细胞特异性标志物GFAP的表达,Fluoro-Jade B(FJ-B)免疫荧光检测脑组织中神经元急性损伤程度,ELISA法测定尾静脉的血清GFAP水平,观察损伤后脑组织神经元和星形细胞改变,并分析血清GFAP水平和神经元损伤程度间的关系。结果与假手术组比较,伤后1、3、7 d邻近顶叶皮质GFAP阳性染色的星形细胞数量减少(P0.05);伤后1、3、7 d可见星形细胞肿胀增生。伤后1 d皮质FJ-B阳性染色神经元轻度增加,伤后3、7 d明显增加(P0.05)。伤后1 d血清GFAP水平较假手术组明显增加(P0.05),伤后3、7 d回落至正常水平;伤后1 d血清GFAP水平与伤后7 d皮质区损伤神经元计数呈正相关(r=0.8095,P0.05)。结论轻型颅脑损伤后急性期发生星形细胞破坏伴胶质反应及血清标志物GFAP增加,亚急性期发生神经元变性损伤。急性期血清GFAP水平可以预测中枢神经元损伤程度。     

快速眼动睡眠剥夺后大鼠脑内神经元骨架蛋白及超微结构的变化

目的探讨经历不同时间快速眼动（REM）睡眠剥夺对大鼠皮质及海马各区神经元形态结构的影响。方法选择微管相关蛋白（MAP2）和神经丝（NF）作为正常神经元结构的标识物，利用免疫组织化学法和Western blot技术观察REM睡眠剥夺1、3、5、7 d4个时间点大鼠皮质及海马MAP2和NF表达的时空变化规律。同时运用电镜技术观察睡眠剥夺后神经元超微结构的变化。我们的实验是用改良的多平台睡眠剥夺模型进行REM睡眠剥夺，结合免疫组织化学染色技术和蛋白质电泳以及电镜超微结构分析。结果REM睡眠剥夺后5d大鼠皮质、海马CA1及齿状回神经元结构蛋白MAP2和NF表达较对照组明显减少（P〈0．05）；电镜神经元核仁偏位，胞质中出现少量肿胀的线粒体和内质网；部分神经轴突的髓鞘溶解与浓集。环境对照组、REM睡眠剥夺5d和7d组，皮质中超微结构改变的神经元所占比例分别为1．2％、3．6％和5．8％。结论REM睡眠剥夺能够导致大鼠脑内神经元的超微结构发生异常变化。     

一次性与三重性脑震荡鼠八臂迷宫逆行性遗忘变化研究

目的运用八臂迷宫实验比较研究一次性与三重性脑震荡大鼠的逆行性遗忘变化情况,以探讨不同损伤次数脑震荡大鼠的认知变化情况。方法用金属单摆闭合性脑损伤打击装置,复制大鼠一次性脑震荡和三重性脑震荡模型,检测脑震荡后大鼠在八臂迷宫中的食物摄取量、进入有食物臂次数、进入有食物臂臂数和重复进人无食物臂的次数变化情况,以反映大鼠损伤前后参考记忆与工作记忆的改变。结果与正常组比较,一次性脑震荡和三重性脑震荡组在伤后食物摄取量、进入有食物臂次数、进入有食物臂臂数和重复进入无食物臂的次数均呈下降趋势,且三重性脑震荡组比一次性脑震荡组下降幅度更大,恢复时间更长。结论脑震荡后,大鼠出现逆行性遗忘,且随着脑震荡次数增加,遗忘恢复时间延长。     

脑出血大鼠锥体束显微和超微结构改变

目的研究脑出血大鼠锥体束病理变化规律及特点。方法使用Ⅳ型胶原酶．肝素诱导大鼠基底节脑出血,采用劳克坚牢蓝（LFB）染色、神经丝蛋白（NF）免疫组化和电镜对内囊后肢进行观察。结果Ⅳ型胶原酶-肝素能成功建立具有典型神经功能缺损的大鼠脑出血模型。光镜发现锥体束髓鞘损伤在脑出血1～3d逐渐加重,7d开始再生修复,1～7d轴突损伤持续加重,14d轴突光度值（0．09±0．01）有所增加,但与7d（0．10±0．02）相比无显著性差异（P〉0．05）。电镜显示脑出血1d锥体束髓鞘松解,轴突水肿,部分无髓轴突崩解坏死消失,3d髓鞘松解、空泡样变性,局部髓鞘消失、厚薄不均,轴突水肿严重,甚至坏死崩解。结论大鼠脑出血后1w内锥体束损伤呈进行性加重,提示应早期和超早期予以干预治疗以减轻损伤和促进修复。     

L-carnitine alleviates sciatic nerve crush injury in rats：functional and electron microscopy assessments

Several studies have demonstrated that L-carnitine exhibits neuroprotective effects on injured sciatic nerve of rats with diabetes mellitus. It is hypothesized that L-carnitine exhibits neuro-protective effects on injured sciatic nerve of rats. Rat sciatic nerve was crush injured by a forceps and exhibited degenerative changes. After intragastric administration of 50 and 100 mg/kg L-carnitine for 30 days, axon area, myelin sheath area, axon diameter, myelin sheath diameter, and numerical density of the myelinated axons of injured sciatic nerve were similar to normal, and the function of injured sciatic nerve also improved signiifcantly. These ifndings suggest that L-carnitine exhibits neuroprotective effects on sciatic nerve crush injury in rats.     

How do axons control myelin formation? The model of 6-aminonicotinamide neuropathy.

Injection of 6-aminonicotinamide into young rats produces a peculiar neuropathy characterized by selective swelling and disruption of the layer of Schwann cell cytoplasm lining the inner surface of the myelin sheath. This layer increases greatly in volume, compressing the axon and distending the myelin sheath. Morphometry of such swollen fibers discloses that the amount of myelin in the distended sheaths is considerably greater than would correspond to the size of the axons, even if axonal compression is accounted for. The data favor the concept that sheath growth is stimulated by non-specific distension of the myelin sheath from inside.     

Membrane flow within the myelin sheath in IDPN neuropathy

This report describes some aspects of beta,beta'-iminodipropionitrile (IDPN) neuropathy in rats as observed by ultrastructural methods and X-ray diffraction. Light microscopy shows gross swelling of the axons in proximal lumbar spinal roots 8 days after intraperitoneal injection of IDPN. Mean axon cross-sectional area and mean axon perimeter increased to 280% and 160% of their control values, respectively. At the same time, myelin membrane packing was not visibly disturbed. In addition, X-ray diffraction patterns, recorded under physiological conditions, demonstrate that the myelin lipid bilayer thickness and widths of the aqueous spaces between bilayers did not change. Related observations are made on posterior tibial nerve (PNS myelin) and ventral spinal cord (CNS myelin). The various observations together are interpreted in terms of a fluid myelin membrane. It is proposed that the myelin membrane flows during axon swelling even though normal membrane-membrane contacts are maintained within the sheath. Membrane flow and slippage between membranes are explained in terms of a molecular model of the myelin multilayer.     

Spinal radiculoneuropathy in aging rats: Demyelination secondary to neuronal dwindling?

Temporal development of radicular demyelination was studied in male albino rats examined sequentially throughout the lifespan of the animals. The rats were perfusion-fixed with paraformaldehyde and glutaraldehyde and areas of their nervous system including the lumbar spinal roots, the spinal cord, and the peripheral sciatic nerve, were embedded in epoxy resin and submitted to microscopic examination in semithin and ultrathin sections. In addition, a vital fat stain, teasing of single nerve fibers, and estimates of axon diameter and fiber number were obtained. Degenerative changes occurred earlier in the distal portions of nerve fibers than in the spinal roots. The radicular lesion consisted of swelling of myelin and demyelination possibly secondary to shrinkage of axons, resulting in focal accumulation of lipid debris within the spinal roots of old rats. Although the causation of senile neuronal atrophy affecting rat peripheral neurons is not fully obvious, this condition may be exacerbated by such factors as pressure on the nerves and hypoactivity.     

Cation-binding sites in trigeminal ganglia and maxillary nerve: unusual reactivity of perikarya, stem axons and satellite cells

We have used the cupric/ferrocyanide reaction to study cation-binding in trigeminal ganglia and maxillary nerve of adult rats. Unmyelinated axons did not react, whereas myelinated axons were stained at nodal, paranodal or cleft sites. At 'nodal' sites, metallic deposits were found in the axoplasm, along the axolemma, and at the extracellular interfaces of the paranodal myelin. At 'paranodal' sites, particles were concentrated in the paranodal axoplasm and in the intracellular spaces of the myelin loops. Most maxillary axons examined at successive sites had all nodal or all paranodal staining, but 13 of 51 had a mixture. In trigeminal ganglia there was no staining of perineurial sheath, endoneurial cells or mast cells. Satellite cells and their basal laminae were prominently stained, with those around small neurons more reactive than those of large neurons. Patches of neuronal membrane on cell bodies were stained, more often for small than large neurons. The axon hillock and proximal stem axon were not stained in some cases, but approximately half the neurons had staining of perikaryal cytoplasm at the axon hillock or a dense asymmetric band in the proximal stem axon. Strong intraaxonal staining was found at the junction between unmyelinated proximal and myelinated distal stem axon. In distal stem axons, staining was found at the first myelin segment and at each successively thicker myelin segment; staining was mostly weak and paranodal, with intensity proportional to myelin thickness. The T-junction between stem and main myelinated axon had nodal or paranodal patterns; unmyelinated T-junctions were not stained. The varied cation-binding patterns in trigeminal ganglia show unusual properties of satellite cells and important differences between stem and main axons. The results that the cell membrane of axon hillock and proximal stem regions of many sensory large and small neurons may have numerous sodium channels and could affect signal propagation.     

Regenerative axonal sprouting in the cat trochlear nerve

Following peripheral trochlear nerve axotomy in the cat, the normal number of myelinated axons is restored despite significant motor neuron death, suggesting regulation of the number of myelinated axons in the regenerated nerve. In this study we used light and electron microscopy to examine the production and maintenance of axonal sprouts at different locations in the nerve and at different postoperative intervals. Despite proliferative sprouting and an overproduction of nonmyelinated axons in the regenerating trochlear nerve, the number of myelinated axons was strictly regulated. Only ～1,000 regenerated axons were eventually remyelinated, but many nonmyelinated axons were still present 6–8 months postaxotomy. Regenerated axons were remyelinated in a proximal-to-distal direction between 3 and 4 weeks postaxotomy. We also examined the maturation of regenerated myelinated axons by measuring axon diameter and myelin index (an expression of myelin thickness). Mean myelinated axon diameter remained significantly below normal in long-term regenerated nerves. Mean myelin index was not different from normal at 4 weeks postaxotomy but was significantly decreased at long postoperative intervals, reflecting a slightly thicker myelin sheath relative to the axon diameter. This relative increase in mean myehn thickness could serve to restore normal conduction velocity despite the decrease in mean axon diameter. We suggest that the regulation of the number of myelinated axons at the normal number despite cell death and the increase in mean myelin thickness may both be compensatory mechanisms that function to restore preoperative conditions and maximize functional recovery. © 1995 Wiley-Liss, Inc.     

Axonal autophagy during regeneration of the rat sciatic nerve**★

BACKGROUND: The removal of degenerated axonal debris during Wallerian degeneration is very important for nerve regeneration. However, the mechanism by which debris is removed is not been completely understood. Considerable controversy remains as to the clearance pathway and cells that are involved. OBJECTIVE: To investigate axonal autophagy during removal of degenerated axonal debris by transecting the sciatic nerve in a rat Wallerian degeneration model. DESIGN, TIME AND SETTING: Experimental neuropathological analysis. The experiment was conducted at the Laboratory Animal Service Center of the Southern Medical University between January and June 2005. MATERIALS: Fifty-four adult, Wistar rats of either sex, weighing 180-250 g, were obtained from the Laboratory Animal Service Center of the Southern Medical University. Animals were randomly divided into nine groups of six rats. METHODS: Wallerian degeneration was induced by transecting the rat sciatic nerve, and tissue samples from the distal stump were obtained 0.2, 0.4, 1, 2, 3, 4, 7, 10, and 15 days post-transection. Ultrathin sections were prepared for electron microscopy to study ultrastructure and enzyme cytochemistry staining. MAIN OUTCOME MEASURES: Ultrastructure (axon body, autophagic body, and cystoskeleton) of axons and myelin sheaths observed with electron microscopy; acidic phosphatase activity detected by Gomori staining using electron microscopy. RESULTS: The major changes of degenerating axons after transection were axoplasm swelling and separation of axons from their myelin sheath between five hours and two days post-transection. At four days post-transection, the axoplasm condensed and axons were completely separated from the myelin sheath, forming dissociative axon bodies. Vacuoles of different sizes formed in axons during the early phase after lesion. Larger dissociative axon bodies were formed when the axons were completely separated from the myelin sheath during a late phase. The axolemma surrounding the axon body was derived from the neuronal cell membrane; the condensed axoplasm contained many autophagic vacuoles at all levels. A large number of neurofilaments, microtubules, and microfilaments were arranged in a criss-cross pattern. The autophagic vacuoles exhibited acidic phosphatase activity. Axonal bodies were absorbed after degradation from day 7 onwards, and macrophages were observed rarely in the formative cavity. CONCLUSION: The degenerating axons were cleared mainly by axonal autophagy and Schwann cell phagocytosis during regeneration of the rat sciatic nerve, and macrophages exhibited only an assisting function. Key Words: axon; autophagy; nerve regeneration