蛇毒神经生长因子促进周围神经再生的诱发电位观察

目的：研究蛇毒神经生长因子（sNGF)对大鼠坐骨神经损伤后诱发电位的影响,评价蛇毒神经生长因子在促进周围神经再生中的作用。方法：建立大鼠坐骨神经钳夹模型,局部滴加药物和术后肌注sNGF,通过脊髓诱发电位（SEP）,运动诱发电位（MEP）评定,观察坐骨神经修复情况。结果：sNGF治疗后可使伤后SEP,MEP提早出现,结论：蛇毒提取的NGF对大鼠坐骨神经损伤修复具有促进作用。     

Changes in CLIP3 expression after sciatic nerve injury in adult rats

CLIP3 (cytoplasmic linker protein 3) is a 547 amino acid residue cytoplasmic protein that localises to Golgi stacks and tubulovesicular elements juxtaposed to Golgi cisternae. Composed of three Ank (ankyrin) repeats and two CAP-Gly (cytoskeleton-associated protein-glycine) domains, CLIP3 may function as a cytoplasmic linker protein that is involved in TGN–endosome dynamics. To define the expression and role of CLIP3 during peripheral nervous system degeneration and regeneration, we created an acute sciatic nerve injury (SNI) model in adult rats. Western blot analyses revealed prominent up-regulation of CLIP3 and PCNA (proliferating cell nuclear antigen) protein levels at 3?days after SNI. Immunohistochemistry displayed that the expression of CLIP3 was noticeably increased in the injured nerve. Immunofluorescence further revealed that the CLIP3 and PCNA proteins colocalised respectively with S100 in the cytoplasm of Schwann cells. The expression profile of the SC/neuron co-cultures demonstrated that CLIP3 and PCNA protein levels were markedly expressed during the early stage of myelination. These results suggest that CLIP3 is likely associated with the myelination of proliferating Schwann cells, and nerve tissue regeneration after peripheral nerve injury. CLIP3 and PCNA expression during early myelination may be related to the direct uptake and transport of lipids and cholesterol, which were derived from the degenerating myelin, by Schwann cells to prepare for the formation of myelin sheath-like structures around regenerated axons after SNI.     

Preparation of carboxymethyl chitosan in aqueous solution under microwave irradiation

Carboxymethyl chitosan was prepared by reacting chitosan with chloroacetic acid in water under microwave irradiation. The effect of the reaction conditions was investigated and optimal conditions were identified. The influence of mass ratio of chloroacetic acid to chitosan, microwave power and pH on the degree of substitution or intrinsic viscosity were further studied. The degree of substitution of the carboxymethyl chitosan synthesized exceeded 0.85.     

Lentiviral-mediated transfer of CDNF promotes nerve regeneration and functional recovery after sciatic nerve injury in adult rats

Peripheral nerve injury is often followed by incomplete and unsatisfactory functional recovery and may be associated with sensory and motor impairment of the affected limb. Therefore, a novel method is needed to improve the speed of recovery and the final functional outcome after peripheral nerve injuries. This report investigates the effect of lentiviral-mediated transfer of conserved dopamine neurotrophic factor (CDNF) on regeneration of the rat peripheral nerve in a transection model in vivo. We observed notable overexpression of CDNF protein in the distal sciatic nerve after recombinant CDNF lentiviral vector application. We evaluated sciatic nerve regeneration after surgery using light and electron microscopy and the functional recovery using the sciatic functional index and target muscle weight. HE staining revealed better ordered structured in the CDNF-treated group at 8 weeks post-surgery. Quantitative analysis of immunohistochemistry of NF200 and S-100 in the CDNF group revealed significant improvement of axonal and Schwann cell regeneration compared with the control groups at 4 weeks and 8 weeks after injury. The thickness of the myelination around the axons in the CDNF group was significantly higher than in the control groups at 8 weeks post-surgery. The CDNF group displayed higher muscle weights and significantly increased sciatic nerve index values. Our findings suggest that CDNF gene therapy could provide durable and stable CDNF protein concentration and has the potential to enhance peripheral nerve regeneration, morphological and functional recovery following nerve injury, which suggests a promising strategy for peripheral nerve repair.     

Preparation and properties of alginate/carboxymethyl chitosan blend fibers

Alginate/carboxymethyl chitosan blend fibers, prepared by spinning their mixture solution through a viscose-type spinneret into a coagulating bath containing aqueous CaCl₂, were studied for structure and properties with the aid of infrared spectroscopy (IR), X-ray diffraction (XRD) and scanning electron micrography (SEM). The analyses indicated a good miscibility between alginate and carboxymethyl chitosan, because of the strong interaction from the intermolecular hydrogen bonds. The best values of the dry tensile strength and breaking elongation were obtained when carboxymethyl chitosan content was 30 and 10 wt%, respectively. The wet tensile strength and breaking elongation decreased with the increase of carboxymethyl chitosan content. Introduction of CM-chitosan in the blend fiber improved water-retention properties of blend fiber compared to pure alginate fiber. Antibacterial fibers, obtained by treating the fibres with aqueous solution of N-(2-hydroxy)-propyl-3-trimethylammonium chitosan chloride and silver nitrate, respectively, exhibited good antibacterial activity to Staphylococcus aureus.     

Effects of carboxymethyl chitosan on the blood system of rats

Carboxymethyl chitosan (CM-chitosan), a derivative of chitosan, was extensively studied in the biomedical materials field for its beneficial biological properties of hemostasis and stimulation of healing. However, studies examining the safety of CM-chitosan in the blood system are lacking. In this study CM-chitosan was implanted into the abdominal cavity of rats to determine blood indexes at different times and to evaluate the effects of CM-chitosan on the blood system of rats. Coagulation function was reflected by thrombin time (TT), prothrombin time (PT), activated partial thromboplatin time (APTT), fibrinogen (FIB) and platelet factor 4 (PF4) indexes; anti-coagulation performance was assessed by the index of antithrombinIII (ATIII); fibrinolytic function was reflected by plasminogen (PLG) and fibrin degradation product (FDP) indexes; and blood viscosity (BV) and plasma viscosity (PV) indexes reflected hemorheology. Results showed that CM-chitosan has no significant effects on the blood system of rats, and provides experimental basis for CM-chitosan to be applied in the field of biomedical materials.     

Simvastatin对大鼠坐骨神经crush损伤修复作用研究

目的：探讨他汀类（statins）药物Simvastatin在大鼠坐骨神经损伤修复中的作用及可能的作用机制。方法：制作SD大鼠标准坐骨神经钳夹损伤（crush）模型后,分别予Simvastatin和溶媒对照干预2周。手术前后不同时间点进行趾展功能指数测定、神经电生理学、血脂水平、血清IL-6检测和组织学评价。结果：Simvastatin干预组与对照组比较,趾展功能指数在术后5d和8d显著增大（P〈0．05）,足趾展开速度快;2周肌肉复合动作电位幅度高,4周神经传导速度快;组织学显示有髓神经纤维数量多,髓鞘厚,排列相对整齐。各组手术前血脂水平无差异,手术后2周均有不同程度的降低,但Simvastatin干预组总胆固醇降低程度最轻,与对照组比较有显著差异（P〈0．05）;Simvastatin干预组手术后5d,血清IL-6水平明显低于对照组（P〈0．05）。结论：本研究发现,Simvastatin可能通过抑制免疫炎症反应,维持神经损伤后胆固醇的平衡,促进大鼠坐骨神经损伤的修复和再生。     

Preparation and antimicrobial activity of some carboxymethyl chitosan acyl thiourea derivatives

Acetyl, chloroacetyl and benzoyl thiourea derivatives of carboxymethyl chitosan (ATUCMCS, CATUCMCS, and BZTUCMCS) with comparable grafting degree were synthesized and their structures were characterized by FTIR spectroscopy and elemental analyses. The antimicrobial behaviors of CMCS and its derivatives against three types of bacteria [Bacillis subtilis (B. subtilis), Staphylococous aureus (S. aureus) and Escherichia coli (E. coli)] and three crop-threatening pathogenic fungi [Aspergillus fumigate (A. fumigate), Geotrichum candidum (G. candidum) and Candida albicans (C. albicans)] were investigated. The results indicated that the antibacterial and the antifungal activities of the acyl thiourea derivatives are much higher than that of the parent CMCS. The acyl thiourea derivatives were more potent in case of Gram-positive bacteria than Gram-negative bacteria. This is illustrated for example by the values of minimum inhibitory concentration (MIC) of the ATUCMCS, CATUCMCS and BZTUCMCS against B. subtilis were 3.9, 15.6 and 62.5, respectively, while the MIC values of these derivatives against E. coli were 62.5, 125 and 500. Moreover, the antifungal activity of the CATUCMCS is higher than that of the acetyl and benzoyl thiourea derivatives. This may be due to the presence of chlorine atom.     

Increased expression of Gem after rat sciatic nerve injury

Gem belongs to the Rad/Gem/Kir subfamily of Ras-related GTPases, whose expression is induced in several cell types upon activation by extracellular stimuli. Two functions of Gem have been demonstrated, including regulation of voltage-gated calcium channel activity and inhibition of Rho kinase-mediated cytoskeletal reorganization, such as stress fiber formation and neurite retraction. Because of the essential relationship between actin reorganization and peripheral nerve regeneration, we investigated the spatiotemporal expression of Gem in a rat sciatic nerve crush (SNC) model. After never injury, we observed that Gem had a significant up-regulation from 1 day, peaked at day 5 and then gradually decreased to the normal level. At its peak expression, Gem expressed mainly in Schwann cells (SCs) and macrophages of the distal sciatic nerve segment, but had few colocalization in axons. In addition, the peak expression of Gem was in parallel with PCNA, and numerous SCs expressing Gem were PCNA positive. Thus, all of our findings suggested that Gem may be involved in the pathophysiology of sciatic nerve after SNC.     

Alginate/chitosan hydrogel containing olfactory ectomesenchymal stem cells for sciatic nerve tissue engineering

Regeneration and functional recovery after peripheral nerve damage still remain a significant clinical problem. In this study, alginate/chitosan (alg/chit) hydrogel was used for the transplantation of olfactory ectomesenchymal stem cells (OE-MSCs) to promote peripheral nerve regeneration. The OE-MSCs were isolated from olfactory mucosa biopsies and evaluated by different cell surface markers and differentiation capacity. After creating sciatic nerve injury in a rat model, OE-MSCs were transplanted to the injured area with alg/chit hydrogel which was prepared and well-characterized. The prepared hydrogel had the porosity of 91.3 ± 1.27%, the swelling ratio of 379% after 240 min, weight loss percentages of 80 ± 5.56% after 14 days, and good blood compatibility. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, 4′,6-diamidino-2-phenylindole, and LIVE/DEAD staining were done to assay the behavior of OE-MSCs on alg/chit hydrogel and the results confirmed that the hydrogel can provide a suitable substrate for cell survival. For functional analysis, alg/chit hydrogel with and without OE- MSCs was injected into a 3-mm sciatic nerve defect of Wistar rats. The results of the sciatic functional index, hot plate latency, electrophysiological assessment, weight-loss percentage of wet gastrocnemius muscle, and histopathological examination using hematoxylin–eosin and Luxol fast blue staining showed that utilizing alg/chit hydrogel with OE-MSCs to the sciatic nerve defect enhance regeneration compared to the control group and hydrogel without cells.     

Allografts of the acellular sciatic nerve and brain-derived neurotrophic factor repair spinal cord injury in adult rats

Objective

We aimed to investigate whether an innovative growth factor-laden scaffold composed of acellular sciatic nerve (ASN) and brain-derived neurotrophic factor (BDNF) could promote axonal regeneration and functional recovery after spinal cord injury (SCI).

Methods

Following complete transection at the thoracic level (T9), we immediately transplanted the grafts between the stumps of the severed spinal cords. We evaluated the functional recovery of the hindlimbs of the operated rats using the BBB locomotor rating scale system every week. Eight weeks after surgery, axonal regeneration was examined using the fluorogold (FG) retrograde tracing method. Electrophysiological analysis was carried out to evaluate the improvement in the neuronal circuits. Immunohistochemistry was employed to identify local injuries and recovery.

Results

The results of the Basso-Beattie-Bresnahan (BBB) scale indicated that there was no significant difference between the individual groups. The FG retrograde tracing and electrophysiological analyses indicated that the transplantation of ASN-BDNF provided a permissive environment to support neuron regeneration.

Conclusion

The ASN-BDNF transplantation provided a promising therapeutic approach to promote axonal regeneration and recovery after SCI, and can be used as part of a combinatory treatment strategy for SCI management.     

周围神经损伤后外源性GDNF对神经元的保护作用

采用硅管套接大鼠切断的坐骨神经模型 ,局部给予胶质细胞源性神经营养因子 (GDNF) ,应用尼氏染色、酶组织化学染色方法 ,观察到外源性GDNF能减少脊髓修复侧前角运动神经元死亡的数目 ,降低脊髓前角运动神经元及脊神经节感觉神经元中胆碱酯酶 (CHE)及酸性磷酸酶 (ACP)变化的幅度。这表明外源性GDNF能保护周围神经切断后引起的神经元损伤。     

Effect of FK506 in reducing scar formation by inducing fibroblast apoptosis after sciatic nerve injury in rats

We previously demonstrated that FK506, a generally applied immunosuppressant in organ transplantation, could promote peripheral nerve regeneration through reducing scar formation. However, little is known about how FK506 reduces scar formation. Herein we investigated the influence of FK506 on fibroblast proliferation and its correlation with scar formation after sciatic nerve injury in rats, and further explored the effect of FK506 on fibroblast proliferation and apoptosis in vitro. Masson staining and immunohistochemistry revealed that scar area and fibroblast number in the nerve anastomosis of sciatic nerve-injured rats were significantly reduced after FK506 administration. The scar area had a significant positive correlation with the fibroblast number, as detected by linear correlation analysis. CCK-8 assay and flow cytometry indicated that FK506 also inhibited proliferation and induced apoptosis of fibroblasts in vitro. It was primarily phosphorylation of JNK and ERK that were activated during the apoptosis of fibroblast. Pretreatment of cells with JNK inhibitor, SP600125, or ERK inhibitor, PD98059, could inhibit FK506-induced fibroblast apoptosis, respectively. Moreover, simultaneous application of both inhibitors had additive roles in cell protection from apoptosis. These results suggest that FK506-induced fibroblast apoptosis contributes to the suppression of fibroblast proliferation and then results in the reduction of scar formation in sciatic nerve-injured rat, and that JNK and ERK are involved in FK506-induced fibroblast apoptosis.     

Safety evaluation of formulations containing carboxymethyl derivatives of starch and chitosan in albino rats

Two composite formulations, based on carboxymethyl derivatives of starch (formulation I) and chitosan (formulation II), used in the preparation of coating formulations to enhance post harvest shelf-life of fruits and vegetables, were evaluated for safety by single dose dietary (formulation I, coating on feed pellet-1.3% w/w and formulation II, coating on feed pellet-1% w/w) and oral (1 ml, 2% aqueous solution) administration to albino rats. Experiment was carried out for 4 weeks. No significant changes were observed in gain in weekly body weight, weight of vital organs and in parameters of haematology and histopathology among experimental groups, thus indicating safety (and non-toxicity) of the coating formulations.     

Laminin-incorporated nerve conduits made by plasma treatment for repairing spinal cord injury

To better direct the repair of damaged axons following spinal cord injury (SCI), we designed a nerve conduit (NC) modeled after the intact spinal cord, which would enable the axons to cross the lesioned area to rejoin on the other side. The NC consisted of a porous chitosan scaffold and was incorporated with laminin (LN) on the inner surface through oxygen plasma treatment. According to the BBB, CBS, and treadmill analyses, we found that following the implantation of the laminin-coated NC (LN-NC) the rats showed a tendency towards behavior improvement and functional recovery. Histology and immunocytochemical analyses indicated that the NC groups were capable of leading the damaged axons through the lesioned area without triggering inflammation or apoptosis. Together with the significantly enhanced expression of local GAP-43 in the LN-NC groups, as evidenced by western blot analysis, axon re-growth mediated by LN-NC was found to compare better than that by NC group. These results suggest a new possible approach to repairing SCI and, in general, a model which will be useful for other multidisciplinary procedures for complex neurological situations.     

周围神经损伤后外源性GKNF对神经元的保护作用

采用硅管套接大鼠切断的坐骨神经模型,局部给予胶质细胞源性神经营养因子（ＧＤＮＦ）,应用尼氏染色、酶组织化学染色方法,观察到外源性ＧＤＮＦ能减少脊髓修复侧前角运动神经元死亡的数目,降低脊髓前角运动神经元及脊神经节感觉神经元中胆碱酯酶（ＣＨＥ）及酸性磷酸酶（ＡＣＰ）变化的幅度。这表明外源性ＧＤＮＦ能保护周围神经切断后引起的神经元损伤．     

Faster nerve regeneration after sciatic nerve injury in mice over-expressing basic fibroblast growth factor

Basic fibroblast growth factor (FGF-2) is expressed in the peripheral nervous system and is up-regulated after nerve lesion. It has been demonstrated that administration of FGF-2 protects neurons from injury-induced cell death and promotes axonal regrowth. Using transgenic mice over-expressing FGF-2 (TgFGF-2), we addressed the importance of endogenously generated FGF-2 on sensory neuron loss and sciatic nerve regeneration. After sciatic nerve transection, wild-type and transgenic mice showed the same degree of cell death in L5 spinal ganglia. Also, the number of chromatolytic, eccentric, and pyknotic sensory neurons was not changed under elevated levels of FGF-2. Morphometric evaluation of intact nerves from TgFGF-2 mice revealed no difference in number and size of myelinated fibers compared to wild-type mice. One week after crush injury, the number of regenerated axons was doubled and the myelin thickness was significantly smaller in transgenic mice. After 2 and 4 weeks, morphometric analysis and functional tests revealed no differences in recovery of sensory and motor nerve fibers. To study the role of FGF-2 over-expression on Schwann cell proliferation during the early regeneration process, we used BrdU-labeling to mark dividing cells. In transgenic mice, the number of proliferating cells was significantly increased distal to the crush site compared to wild-types. We propose that endogenously synthesized FGF-2 influences early peripheral nerve regeneration by regulating Schwann cell proliferation, axonal regrowth, and remyelination.     

Drebrin在坐骨神经慢性压迫性损伤大鼠远位触液神经元的表达

为观察坐骨神经慢性压迫性损伤(chronic constriction injury,CCI)后大鼠远位触液神经元(distal cerebrospinal fluid con-tacting neurons,dCSF-CNs)中drebrin的表达,探讨免疫荧光技术用于dCSF-CNs研究的可能性,将雄性Sprague-Dawley大鼠随机分为空白对照组、假手术组和CCI组,采用侧脑室注射霍乱毒素亚单位B(choleratoxin subunit B,CB)与辣根过氧化物酶(horseradish peroxidase,HRP)结合物(CB-HRP)示踪标记大鼠dCSF-CNs,观测三组大鼠行为学评分,并应用免疫荧光双标记和激光共聚焦显微镜技术比较各组dCSF-CNs中drebrin的表达.结果显示,三组中仅CCI组大鼠痛阈下降,三组大鼠dCSF-CNs均显示清晰,空白对照组和假手术组dCSF-CNs胞浆内无drebrin表达,CCI组dCSF-CNs胞浆内drebrin表达较多.结果表明,应用免疫荧光双标记观察dCSF-CNs,形态清晰,技术可靠.dCSF-CNs可能参与了神经病理性疼痛的信息传递.