体外培养的大鼠皮层神经元氧糖剥夺后Sema3A、Nrp-1 mRNA表达上调与轴突网络损伤的关系

【目的】研究体外培养的大鼠皮层神经元氧糖剥夺(OGD)处理后,Sema3A、Nrp-1mRNA的表达变化及其与神经元轴突网络损伤的关系。【方法】体外培养新生SD大鼠皮层神经元,随机分为正常对照组和OGD处理组:噻唑蓝(MTT)比色法测定神经元活性;βⅢ-tubulin免疫荧光染色观察轴突网络形态学变化,Real-timePCR检测Sema3A及其受体Nrp-1mRNA的表达;βⅢ-tubulin免疫荧光染色观察外源性Sema3A蛋白对神经元轴突生长的影响。【结果】随着OGD时间的延长,神经元活性呈梯度下降(P<0.01);OGD处理4h可导致神经元轴突网络发生明显的崩解损伤;OGD处理4h、6h后,Sema3AmRNA表达水平显著上调(P<0.01);OGD处理6h后,Nrp-1mRNA表达水平显著上调(P<0.01);经外源性Sema3A(5mg/mL)蛋白处理,可观察到神经元的轴突生长及延伸受到明显抑制(P<0.01)。【结论】OGD处理可诱导Sema3A、Nrp-1mRNA表达上调,并且外源性Sema3A蛋白可在体外抑制轴突生长,提示Sema3A/Nrp-1可能参与OGD处理后轴突网络崩解、细胞凋亡等病理生理过程。

重组人Semaphorin3A诱导体外培养的大鼠大脑皮层神经元轴突损伤的实验研究

目的探讨外源性Semaphorin3A（Sema3A）对于体外培养神经元轴突生长及神经元生长活性的影响。方法体外培养新生SD大鼠皮质神经元,随机分为正常对照组和Sema3A不同浓度处理组,倒置相差显微镜及微管相关蛋白-2（MAP-2）荧光染色分别观察生长锥及轴突形态学变化;噻唑蓝（MTT）法检测神经元存活率。结果 Sema3A（0.5mg/ml）处理可诱发神经元生长锥崩解;与正常对照组相比,Sema3A（5mg/ml）处理组轴突平均长度缩短（P〈0.001）;经不同浓度Sema3A处理后,神经元存活率呈剂量依赖性下降,其中浓度范围为0.5-5.0mg/ml的处理组与正常组比较均有统计学意义（P〈0.001）。结论 Sema3A在体外可发挥明显的促生长锥崩解及抑制轴突生长的作用,并且具有一定的神经元毒性。

Regulation of axonal regeneration following the central nervous system injury in adult mammalian

It has been well established that the recovery ability of central nervous system （CNS） is very poor in adult mammals. As a result, CNS trauma generally leads to severe and persistent functional deficits. Thus, the investigation in this field becomes a ＂hot spot＂. Up to date, accumulating evidence supports the hypothesis that the failure of CNS neurons to regenerate is not due to their intrinsic inability to grow new axons, but due to their growth state and due to lack of a permissive growth environment. Therefore, any successful approaches to facilitate the regeneration of injured CNS axons will likely include multiple steps： keeping neurons alive in a certain growth-state, preventing the formation of a glial scar, overcoming inhibitory molecules present in the myelin debris, and giving direction to the growing axons. This brief review focused on the recent progress in the neuron regeneration of CNS in adult mammals.

Bursting the unfolded protein response accelerates axonal regeneration

Peripheral neuropathies refer to a group of conditions in which the peripheral nervous system(PNS)is damaged.These pathological state are are associated with weakness,pain,and loss of motor and sensory control.More than 100 types of peripheral neuropathies have been identified,with distinct symptoms and prognosis classified according to the type of damage to the nerves.Injury to peripheral nerves results in disabling loss of sensory and motor func-

Protective effect of Tanreqing injection on axon myelin damage in the brain of mouse model for experimental autoimmune encephalomyelitis

OBJECTIVE: To evaluate the effect of Tanreqing injection on axon myelin in the mouse brain of experimental autoimmune encephalomyelitis(EAE).METHODS: An EAE model was established by myelin oligodendrocyte glycoprotein(MOG)35-55 immunization in C57BL/6 mice. Mice were randomly divided into the following groups: normal, model,prednisone acetate(PA)(6 mg/kg), Tanreqing high dose(5.14 m L/kg), Tanreqing low dose(2.57 m L/kg). On the day of immunization, both Tanreqing groups were treated by intraperitoneal injection,with the PA group treated by intragastrical perfusion after T cell response, and the other groups treated with saline. Changes in body weight, neurological deficit score, incidence rate, mortality rate,and course of disease were observed for all mice.Brain tissue was isolated and stained with hematoxylin-eosin, and pathological investigations performed to evaluate axon myelin damage by transmission electron microscopy(TEM). Myelin basic protein and microtubule associated protein-2 were analyzed by immunohistochemistry.RESULTS: Tanreqing injection significantly prolonged EAE latency and decreased the neurological deficit score, alleviated infiltration of inflammatory cells in the focus area, up-regulated hippocampal MBP expression at the acute stage and the remission stage, and increased microtubule associated protein-2 expression in the EAE brain to varying degrees in the acute stage. TEM analysis indicated that Tanreqing injection alleviates myelin damage in the EAE mouse and maintains the integrity of circular layer structures and alleviates axon mitochondrial swelling.CONCLUSION: Tanreqing injection alleviates EAE symptoms.

Influences of olfactory ensheathing cells transplantation on axonal regeneration in spinal cord of adult rats

Objective: To observe whether olfactory ensheathing cells could be used to promote axonal regeneration in a spontaneously nonregenerating system.Methods: After laminectomy at the lower thoracic level, the spinal cords of adult rats were exposed and completely transected at T10. A suspension of ensheathing cells was injected into the lesion site in 12 adult rats, and control D/F 12 ( 1∶1 mixture of DMEM and Hams F 12) was injected in 12 adult rats. Six weeks and ten weeks after cell transplantation, the rats were evaluated by climbing test and motor evoked potentials (MEPs) monitoring. The samples were procured and studied with histologicl and immunohistochemical methods.Results: At the 6th week after cell transplantation, all the rats in both the transplanted and control groups were paraplegic and the MEPs could not be recorded. At the 10th week after cell transplantation, of 7 rats in the control group, 2 rats had muscles contraction of the lower extremities, 2 rats had hips and/or knees active movement; and 5 rats MEPs could be recorded in the hind limbs in the transplanted group (n=7). None of the rats in the control group had functional improvement and no MEPs recorded (n=7). Numerous regenerating axons were observed through the transplantation and continued to regenerate into the denervated host tract. Cell labelling using anti Myelin Basic Protein (MBP) and anti Nerve Growth Factor Receptor (anti NGFR) indicated that the regenerated axons were derived from the appropriate neuronal source and that donor cells migrated into the denervated host tract. But axonal degeneration existed and regenerating axons were not observed within the spinal cords of the adult rats with only D/F 12 injection. Conclusions: The axonal regeneration in the transected adult rat spinal cord is possible after ensheathing cells transplantation.

Controlled study of nimodipine in treatment of patients with diffuse axonal injury

Objective: To determine the efficacy and safety of early intervention with nimodipine treatment in diffuse axonal injury. Methods: Based on the characteristic radiological signs and criteria for diffuse axonal injury (DAI), 89 patients with the diagnosis of DAI were enrolled in this randomized, double blind, placebo controlled trial. Results: Nimodipine proved to be safe and well tolerated. With TCD sonography we found that there was a higher incidence of cerebral vasospasm in this series ( 38.2 %). Overall, Nimodipine produced a better clinical result than placebo, but there was no statistically significant difference in favorable outcome at 3 months after injury (P= 0.11 ) between the two groups. A trend toward a favorable effect was suggested by the analyses in two small subgroups, either in the patients suffering from clinical Grade Ⅲ DAI (P= 0.04 ), or in those with the TCD evidence of cerebral vasospasm during clinical observation (P= 0.049 ). Conclusions: We postulate that a clinically valuable benefit is possible with nimodipine treatment in DAI patients. However, the effects on outcome should be verified by further controlled study.

Influence of cryopreserved olfactory ensheathing cells transplantation on axonal regeneration in spinal cord of adult rats

Objective: To observe the effects of cryopreserved olfactory ensheathing cells (OECs) transplantation on axonal regeneration and functional recovery following spinal cord injury in adult rats. Methods: Twenty-four rats were divided into experimental and control groups, each group having 12 rats. The spinal cord injury was established by transecting the spinal cord at T 10 level with microsurgery scissors. OECs were purified from SD rat olfactory bulb and cultured in DMEM (Dulbeccos minimum essential medium) and cryopreserved (-120℃) for two weeks. OECs suspension [(1-1.4)×10 5/ul] was transplanted into transected spinal cord, while the DMEM solution was injected instead in the control group. At 6 and 12 weeks after transplantation, the rats were evaluated with climbing test and MEP (moter evoked potentials) monitoring. The samples of spinal cord were procured and studied with histological and immunohisto chemical stainings. Results: At 6 weeks after transplantation, all of the rats in both transplanted and control groups were paraplegic, and MEPs could not be recorded. Morphology of transplanted OECs was normal, and OECs were interfused with host well. Axons could regrow into gap tissue between the spinal cords. Both OECs and regrown axons were immunoreactive for MBP. No regrown axons were found in the control group. At 12 weeks after transplantation, 2 rats (2/7) had lower extremities muscle contraction, 2 rats (2/7) had hip and/or knee active movement, and MEP of 5 rats (5/7) could be recorded in the calf in the transplantation group. None of the rats (7/7) in the control group had functional improvement, and none had MEPs recorded. In the transplanted group, histological and immunohistochemical methods showed the number of transplanted OECs reduced and some regrown axons had reached the end of transected spinal cord. However, no regrown axons could be seen except scar formation in the control group. Conclusions: Cryopreserved OECs could integrated with the host and promote regrowing axons across the transected spinal cord ends.

Potassium channel blockers as an effective treatment to restore impulse conduction in injured axons

Most axons in the vertebral central nervous system are myelinated by oligodendrocytes.Myelin protects and insulates neuronal processes,enabling the fast,saltatory conduction unique to myelinated axons.Myelin disruption resulting from trauma and biochemical reaction is a common pathological event in spinal cord injury and chronic neurodegenerative diseases.Myelin damage-induced axonal conduction block is considered to be a significant contributor to the devastating neurological deficits resulting from trauma and illness.Potassium channels are believed to play an important role in axonal conduction failure in spinal cord injury and multiple sclerosis.Myelin damage has been shown to unmask potassium channels,creating aberrant potassium currents that inhibit conduction.Potassium channel blockade reduces this ionic leakage and improves conduction.The present review was mainly focused on the development of this technique of restoring axonal conduction and neurological function of demyelinated axons.The drug 4-aminopyridine has recently shown clinical success in treating multiple sclerosis symptoms.Further translational research has also identified several novel potassium channel blockers that may prove effective in restoring axonal conduction.

Clinical features of diffuse axonal injury

Objective: To analyze the mechanism of diffuse axonal injury (DAI) and study the relationship between DAI and brain concussion, brain contusion, and primary brain stem injury. Methods: The clinical data and iconographic characteristics of 56 patients with DAI were analyzed retrospectively. Results: Traffic accidents were the main cause of DAI. Among the 56 cases, 34 were injured for at least twice, and 71.43% of the patients were complicated with contusion. Conclusions: It is considered that DAI is a common pattern of primary brain injury, which is often underestimated. And DAI includes cerebral concussion and primary brain injury, and is often complicated by cerebral cortex contusion. Therefore, it is very simple and practical to divide primary brain injuries into local and diffuse injuries.

Clinical application of axonal repair technique for treatment of peripheral nerve injury

Objective: To evaluate the efficacy of axonal repair technique for treatment of peripheral nerve injury clinically. Methods: In 1998, the authors applied axonal repair technique to treat peripheral nerve injuries in 12 patients with 13 nerves. It consists of four steps, ie, stumps of the nerve being soaked in a modified Collins fluid, freezed, trimmed, and coapted with glue, making the injured nerve repaired at the axonal level. Results: The patients were followed up for an average of 13 months. Results showed that in 4 cases of first-stage contralateral C7 transfer, regenerating axons reached to the sternoclavicular joint or axilla at 4 to 7 months, offering the timing for performing the second-stage contralateral C7 transfer. In 5 cases of accessory nerve transferred to the suprascapular nerve, the abduction of the shoulder was 40° on average. In the other 3 patients with four different nerves repaired, results were also satisfactory. Conclusions: This technique is promising in the treatment of peripheral nerve injury.

Content change of neurofilament protein subunits in experimental brain diffuse axonal injury by lateral head rotation

To explore the content change of neurofilament (NF) protein subunits in the experimental brain diffuse axonal injury (DAI) by lateral head rotation. Methods: Twenty four Sprague Dawley (SD) rats were equally divided into three injury groups (2 h, 12 h, and 24 h post injury) and one control group. The models of DAI were made in the injury groups by lateral head rotation. Western blotting technique was used to measure the content of NF68 (a kind of NF protein subunit) in the brainstem tissues among all the injured and control rats. The NF68 immunohistochemical staining was used in another six SD rats in order to observe the morphological changes in DAI. Results: The NF68 content in the brainstem tended to decrease at 2 h post injury, decreased significantly at 12 h and continued its decrease at 24 h. NF56 and NF52, as the breakdown products of NF68, had a tendency to increase at 2 12 h after the injury, and amounted to a significantly higher level at 24 h. Microscopically, there were a lot of swelling neuronal axons in the ventral part of the medullar oblongata at 2 h after the injury. Some axons were disconnected, and axonal retraction balls formed on their proximal end. Conclusions: There is an occurrence of phosphorolysis within the brainstem in DAI by lateral head rotation. These reactions cause the breakdown of NF68, which results in the decrease of NF68 in content. It suggests that the breakdown of neurofilament protein subunits is an important reason for structural destroy of neurofilaments in DAI.

Intra axonal overloading of calcium ion in rat diffuse axonal injury and therapeutic effect of calcium antagonist

Objective: Exploring the intra axonal overloading of calcium ion (Ca 2+ ) in brain diffuse axonal injury (DAI) and the therapeutic effect of calcium antagonist(Nimotop) on DAI. Methods: Fourteen SD rats were divided into injury group, treatment group and control group. The DAI model of rats was produced by using a head instant axial rotation device. Tissues from the medulla oblongata of rats were taken 2 24 h post injury and processed for electron microscopic observation by a cytochemical technique for calcium ion. Results: In the injured rats there was evidence of local disruption of myelin sheath,lucent spaces between myelin sheath lamellae, separation of axolemma from the inner layer of myelin sheath, peripheral accumulation of organellae, intra axonal formation of vacuoles and reduction of mitochondria. A large number of fine calcium deposits were seen on the affected myelin sheath. The severity of the myelin sheath lesion was related positively to the number of calcium deposits on it. In the later post injury period the coarse calcium particles appeared within the damaged axon. Neuronal somas and microvascular endotheliums showed a lot of vacuoles and some fine calcium deposits. Many microvilli formed on the luminal aspect of endothelium. In the treatment group myelin sheath tended to be injured locally, and axoplasmic mitochondria were nearly normal in number, structure, and distribution. Few calcium deposits were found in axons. Vacuolization was obviously reduced in neuronal soma and endothelium. Conclusions: In DAI there exists an intra axonal overloading of calcium ion, which is a key factor to the occurrence and development of DAI. Early use of Nimotop can alleviate DAI.