Human umbilical cord blood stem cells and brain-derived neurotrophic factor for optic nerve injury:a biomechanical evaluation

Treatment for optic nerve injury by brain-derived neurotrophic factor or the transplantation of human umbilical cord blood stem cells has gained progress, but analysis by biomechanical indicators is rare. Rabbit models of optic nerve injury were established by a clamp. At 7 days after injury, the vitreous body received a one-time injection of 50 μg brain-derived neurotrophic factor or 1 × 106 human umbilical cord blood stem cells. After 30 days, the maximum load, maximum stress, maximum strain, elastic limit load, elastic limit stress, and elastic limit strain had clearly improved in rabbit models of optical nerve injury after treatment with brain-derived neurotrophic factor or human umbilical cord blood stem cells. The damage to the ultrastructure of the optic nerve had also been reduced. These findings suggest that human umbilical cord blood stem cells and brain-derived neurotrophic factor effectively repair the injured optical nerve, improve biomechanical properties, and contribute to the recovery after injury.     

Transplantation of human amniotic epithelial cells repairs brachial plexus injury： pathological and biomechanical analyses

A brachial plexus injury model was established in rabbits by stretching the C6 nerve root. Imme- diately after the stretching, a suspension of human amniotic epithelial cells was injected into the injured brachial plexus. The results of tensile mechanical testing of the brachial plexus showed that the tensile elastic limit strain, elastic limit stress, maximum stress, and maximum strain of the injured brachial plexuses were significantly increased at 24 weeks after the injection. The treatment clearly improved the pathological morphology of the injured brachial plexus nerve, as seen by hematoxylin eosin staining, and the functions of the rabbit forepaw were restored. These data indicate that the injection of human amniotic epithelial cells contributed to the repair of brachial plexus injury, and that this technique may transform into current clinical treatment strategies.     

Human umbilical cord blood-derived stem cells and brain-derived neurotrophic factor protect injured optic nerve：viscoelasticity characterization

The optic nerve is a viscoelastic solid-like biomaterial.Its normal stress relaxation and creep properties enable the nerve to resist constant strain and protect it from injury.We hypothesized that stress relaxation and creep properties of the optic nerve change after injury.Moreover,human brain-derived neurotrophic factor or umbilical cord blood-derived stem cells may restore these changes to normal.To validate this hypothesis,a rabbit model of optic nerve injury was established using a clamp approach.At 7 days after injury,the vitreous body received a one-time injection of 50 μg human brain-derived neurotrophic factor or 1 × 106 human umbilical cord blood-derived stem cells.At 30 days after injury,stress relaxation and creep properties of the optic nerve that received treatment had recovered greatly,with pathological changes in the injured optic nerve also noticeably improved.These results suggest that human brain-derived neurotrophic factor or umbilical cord blood-derived stem cell intervention promotes viscoelasticity recovery of injured optic nerves,and thereby contributes to nerve recovery.     

Stress and strain analysis on the anastomosis site sutured with either epineurial or perineurial sutures after simulation of sciatic nerve injury

The magnitude of tensile stress and tensile strain at an anastomosis site under physiological stress is an important factor for the success of anastomosis following suturing in peripheral nerve injury treatment. Sciatic nerves from fresh adult cadavers were used to create models of sciatic nerve injury. The denervated specimens underwent epineurial and perineurial suturing. The elastic modulus (40.96 ± 2.59 MPa) and Poisson ratio (0.37 ± 0.02) of the normal sciatic nerve were measured by strain electrical measurement. A resistance strain gauge was pasted on the front, back, left, and right of the edge of the anastomosis site after suturing. Strain electrical measurement results showed that the stress and strain values of the sciatic nerve following perineurial suturing were lower than those following epineurial suturing. Scanning electron microscopy revealed that the sciatic nerve fibers were disordered following epineurial compared with perineurial suturing. These results indicate that the effect of perineurial suturing in sciatic nerve injury repair is better than that of epineurial suturing.     

Poly（lactic-co-glycolic acid） conduit for repair of injured sciatic nerve A mechanical analysis

Tensile stress and tensile strain directly affect the quality of nerve regeneration after bridging nerve defects by poly（lactic-co-glycolic acid） conduit transplantation and autogenous nerve grafting for sciatic nerve injury. This study collected the sciatic nerve from the gluteus maximus muscle from fresh human cadaver, and established 10-mm-long sciatic nerve injury models by removing the ischium, following which poly（lactic-co-glycolic acid） conduits or autogenous nerve grafts were transplanted. Scanning electron microscopy revealed that the axon and myelin sheath were torn, and the vessels of basilar membrane were obstructed in the poly（lactic-co-glycolic acid） conduit-repaired sciatic nerve following tensile testing. There were no significant differences in tensile tests with autogenous nerve graft-repaired sciatic nerve. Following poly（lactic-co-glycolic acid） conduit transplantation for sciatic nerve repair, tensile test results suggest that maximum tensile load, maximum stress, elastic limit load and elastic limit stress increased compared with autogenous nerve grafts, but elastic limit strain and maximum strain decreased. Moreover, the tendencies of stress-strain curves of sciatic nerves were similar after transplantation of poly（lactic-co-glycolic acid） conduits or autogenous nerve grafts. Results showed that after transplantation in vitro for sciatic nerve injury, poly（lactic-co-glycolic acid） conduits exhibited good intensity, elasticity and plasticity, indicating that poly（lactic-co-glycolic acid） conduits are suitable for sciatic nerve injury repair.     

Chemically extracted aceUular allogeneic nerve graft combined with ciliary neurotrophic factor promotes sciatic nerve repair

A chemically extracted acellular allogeneic nerve graft can reduce postoperative immune rejection, similar to an autologous nerve graft, and can guide neural regeneration. However, it remains poorly understood whether a chemically extracted acellular allogeneic nerve graft combined with neurotrophic factors provides a good local environment for neural regeneration. This study investigated the repair of injured rat sciatic nerve using a chemically extracted acellular allogeneic nerve graft combined with ciliary neurotrophic factor. An autologous nerve anastomosis group and a chemical acellular allogeneic nerve bridging group were prepared as controls. At 8 weeks after repair, sciatic functional index, evoked potential amplitude of the soleus muscle, triceps wet weight recovery rate, total number of myelinated nerve fibers and myelin sheath thickness were measured. For these indices, values in the three groups showed the autologous nerve anastomosis group 〉 chemically extracted acellular nerve graft ＋ ciliary neurotrophic factor group 〉 chemical acellular allogeneic nerve bridging group. These results suggest that chemically extracted acellular nerve grafts combined with ciliary neurotrophic factor can repair sciatic nerve defects, and that this repair is inferior to autologous nerve anastomosis, but superior to chemically extracted acellular allogeneic nerve bridging alone.     

Chemically extracted acellular allogeneic nerve graft combined with ciliary neurotrophic factor promotes sciatic nerve repair

A chemically extracted acellular allogeneic nerve graft can reduce postoperative immune rejection, similar to an autologous nerve graft, and can guide neural regeneration. However, it remains poorly understood whether a chemically extracted acellular allogeneic nerve graft combined with neurotrophic factors provides a good local environment for neural regeneration. This study investigated the repair of injured rat sciatic nerve using a chemically extracted acellular allogeneic nerve graft combined with ciliary neurotrophic factor. An autologous nerve anastomosis group and a chemical acellular allogeneic nerve bridging group were prepared as controls. At 8 weeks after repair, sciatic functional index, evoked potential amplitude of the soleus muscle, triceps wet weight recovery rate, total number of myelinated nerve fibers and myelin sheath thickness were measured. For these indices, values in the three groups showed the autologous nerve anastomosis group chemically extracted acellular nerve graft + ciliary neurotrophic factor group chemical acellular allogeneic nerve bridging group. These results suggest that chemically extracted acellular nerve grafts combined with ciliary neurotrophic factor can repair sciatic nerve defects, and that this repair is inferior to autologous nerve anastomosis, but superior to chemically extracted acellular allogeneic nerve bridging alone.     

Repair of peripheral nerve defects with chemically extracted acellular nerve allografts loaded with neurotrophic factors-transfected bone marrow mesenchymal stem cells

Chemically extracted acellular nerve allografts loaded with brain-derived neurotrophic factor-transfected or ciliary neurotrophic factor-transfected bone marrow mesenchymal stem cells have been shown to repair sciatic nerve injury better than chemically extracted acellular nerve allografts alone, or chemically extracted acellular nerve allografts loaded with bone marrow mesenchymal stem cells. We hypothesized that these allografts compounded with both brain-derived neurotrophic factor- and ciliary neurotrophic factor-transfected bone marrow mesenchymal stem cells may demonstrate even better effects in the repair of peripheral nerve injury. We cultured bone marrow mesenchymal stem cells expressing brain-derived neurotrophic factor and/or ciliary neurotrophic factor and used them to treat sciatic nerve injury in rats. We observed an increase in sciatic functional index, triceps wet weight recovery rate, myelin thickness, number of myelinated nerve fibers, amplitude of motor-evoked potentials and nerve conduction velocity, and a shortened latency of motor-evoked potentials when allografts loaded with both neurotrophic factors were used, compared with allografts loaded with just one factor. Thus, the combination of both brain-derived neurotrophic factor and ciliary neurotrophic factor-transfected bone marrow mesenchymal stem cells can greatly improve nerve injury.     

Synergetic effects of ciliary neurotrophic factor and olfactory ensheathing cells on optic nerve reparation (complete translation)

At present, there is no effective treatment for the repair of the optic nerve after injury, or improvement of its microenvironment for regener-ation. Intravitreally injected ciliary neurotrophic factor (CNTF) and olfactory ensheathing cells (OECs) promote the long-distance regrowth of severed optic nerve ifbers after intracranial injury. Here, we examined the efifcacy of these techniques alone and in combination, in a rat model of optic nerve injury. We injected condensed OEC suspension at the site of injury, or CNTF into the vitreous body, or both simulta-neously. Retrograde tracing techniques showed that 4 weeks postoperatively, the number of surviving retinal ganglion cells and their axonal density in the optic nerve were greater in rats subjected to OEC injection only than in those receiving CNTF injection only. Furthermore, combined OEC + CNTF injection achieved better results than either monotherapy. These ifndings conifrm that OECs are better than CNTF at protecting injured neurons in the eye, but that combined OEC and CNTF therapy is notably more effective than either treatment alone.     

视神经损伤后睫状神经营养因子的神经保护效应

目的 研究睫状神经营养因子(CNTF)不同给药途径对猫视神经不全损伤后的神经保护效应.方法 完成猫视神经不全损伤模型的制作;实验组和空白对照组分别给予静脉、前房、玻璃体内CNTF注射和生理盐水玻璃体注射;术后15 d双侧上丘、外侧膝状体显微注射DiI逆行神经示踪;不同时间点行视觉诱发电位(PVEP)检测;术后30 d原位心脏灌注;视网膜铺片,视网膜神经节细胞计数;取术侧视神经组织,光镜和电镜观察各组视神经形态变化.结果 空白对照组术侧眼PVEPP100较实验各组振幅明显减低,潜伏期明显延长,差异有统计学意义(P＜0.01);与空白对照组比较,实验组视网膜神经节细胞存活数量明显增加(P＜0.01),神经结构更加完整.结论 视神经不全损伤后CNTF不同途径给药具有视神经保护效应,但效应存在差异.

Abstract：

Objective To research the nerve protective effect of ciliary neurotrophic factor (CNTF) after cat optic nerve not entire injury.Methods Completes the cat optic nerve not entire damage model.The experimental group gives CNTF by in the vein,the anterior chambe,the intravitreal to inject separately,the control group gives the physiological saline.Operation latter 15 days of two-sided anterior colliculi,lateral geniculate body microinjection DiI retrogradation nerve tracing.The different time spot completes the examination of visual evoked potential (PVEP).Operation latter 30 days complete the situ heart dabbling;Retina stretched preparation,retina ganglion cell counting.Takes the optic nerve of operation side,the light microscope and the electron microscope observation morphologic change of optic nerve.Result Controlled group PVEP compares the experimental groups oscillation amplitude of P100 to decrease obviously,incubation period of P100 obvious extension,difference remarkable (P ＜ 0.0l ).Compares with the controlled group,the survival number of retina ganglion cell of experimental groups increases obviously(P＜0.01 ),the nerve structure is more complete.Conclusion After optic nerve not entire damage,the CNTF different way injection has the optic nerve protective effect,but effect existence difference.     

Histometric effects of ciliary neurotrophic factor in wobbler mouse motor neuron disease

We investigated the histological effects of ciliary neurotrophic factor on degenerating motor neurons, their axons, and skeletal muscles in 68 wobbler mice with motor neuron disease. Treatment consisted of recombinant rat or human ciliary neurotrophic factor (or a vehicle solution), 1-mg/kg subcutaneous injection, three times per week for 4 weeks after the clinical diagnosis. The number of motor neurons immunoreactive for calcitonin gene--related peptide was higher in mice receiving rat ciliary neurotrophic factor (p < 0.03), although the number of choline acetyltransferase-reactive neurons was the same in both treated and untreated control groups. Treatment did not prevent vacuolar degeneration of motor neurons. In mice treated with human ciliary neurotrophic factor, the percentage of axons undergoing acute axonal degeneration (myelin ovoids) was smaller in the entire C5 ventral root (p < 0.02) and in the musculocutaneous nerve (p < 0.04), and the number of myelinated nerve fibers was 30% higher in both nerves (p < 0.01 and p < 0.04, respectively) than in controls. In ciliary neurotrophic factor-treated mice, the biceps muscle weight was 20% greater, the mean muscle fiber diameter was 30% larger, and the number of atrophied muscle fibers was 75% lower than that in the vehicle-treated wobbler mice (p < 0.001 for all three results). The number of terminal axonal branching points and the mean length of motor end-plates were also higher in the ciliary neurotrophic factor-treated mice (p < 0.001 and p < 0.02, respectively). Our study thus suggests that ciliary neurotrophic factor slowed neuronal degeneration, enhanced axonal regeneration at both the proximal and distal motor axons, and reduced muscle atrophy in this motor neuron disease.     

视神经损伤与组织工程修复技术*★

视神经由视网膜神经节细胞轴索组成，因其周围无许旺细胞，属于中枢神经，故损伤后不能再生。1985年So和Aguayo进行周围神经视网膜移植成功,彻底改变了视神经损伤后不能再生的观念。目前神经损伤修复的方法有以下几种：①采用神经营养因子，如神经生长因子、睫状神经营养因子、脑源性神经营养因子等，这些因子具有促进视网膜神经节细胞再生和修复的作用。②基因矫正治疗与基因调控治疗通过改变宿主基因的表达，减少疾病所产生的效应，减缓疾病病程进展或提供对疾病的保护。③神经干细胞移植与组织工程化许旺细胞移植。但这些治疗方法尚处于动物实验阶段，如何及时有效地减少节细胞的凋亡和提高节细胞的存活率，在此基础上进一步促进神经的再生与修复，至今还没有一个完善的方法。     

The modification of the pupillary light reflex by chlorpromazine, diazepam, and pentobarbital

Single light flashes or electrical stimulation of the optic nerve evoke a bi- or triphasic potential in the short ciliary nerves. The central conduction and transmission time (from the optic nerve to the emergence of the preganglionic autonomic oculomotor fibers) is estimated to be about 12 msec. After prior conditioning stimuli, evoked responses to subsequent test shocks are inhibited when the interval is between 15 and 30 msec. Recovery takes 120 msec or longer. This inhibitory period is prolonged by pentobarbital. CPZ, which constricts the pupil in man, enhances optically evoked ciliary potentials, whereas diazepam and pentobarbital decrease them. The CPZ effect is interpreted as a diminution of inhibitory influences on the pupilloconstrictor outflow.     

Brain-derived neurotrophic factor (BDNF) prevents lesion-induced axonal die-back in young rat optic nerve

Lesions of the optic nerve in young animals lead to rapid retrograde degeneration of the axon stumps and to death of retinal ganglion cells. We injected different neurotrophic factors into the eye at the time of an intracranial freeze-crush lesion of the optic nerve in 8 day old rats. Optic nerve axons were visualized by anterograde tracing with wheat germ agglutinin-horseradish peroxidase (WGA-HRP) and by electron microscopy. The lesion induced a rapid die-back of the axons, which could be prevented by BDNF and to a lesser extent by neurotrophin-3 (NT-3) or ciliary neurotrophic factor (CNTF). No effect was seen in animals injected with nerve growth factor (NGF) or a mixture of acidic and basic fibroblast growth factor (FGF). In contrast to this effect on the axons, none of these factors was able to counteract the rapidly progressing degeneration of the retinal ganglion cells. These results suggest a selective influence of BDNF on the mechanisms responsible for the maintenance of optic nerve axons.     

受损坐骨神经中STAT3表达和酪氨酸磷酸化的变化及重组CNTF的作用

研究周围神经修复后 ,受损周围神经中信号转导子和转录激活子 3(STAT3)表达和酪氨酸磷酸化的变化及睫状神经营养因子 (CNTF)的作用。用硅管套接切断的成年大鼠坐骨神经 ,术时在受损神经处给予重组CNTF ,用免疫组织化学ABC法结合计算机图像分析观测修复侧神经中STAT3、磷酸化酪氨酸 (PTyr)的分布和相对含量。在生理盐水组修复侧神经中 ,轴突、雪旺细胞STAT3的含量升高 ,轴突、雪旺细胞、单核细胞的PTyr含量升高 ;CNTF组修复侧神经上述细胞成分的STAT3和PTyr含量更高。提示受损坐骨神经的STAT3表达上调 ,酪氨酸磷酸化增强 ,外加重组CNTF有进一步增强的作用。     

Predegenerated peripheral nerve grafts rescue retinal ganglion cells from axotomy-induced death

The inability of axons to grow across damaged central nervous system tissue is a well-known consequence of injury to the brain and spinal cord of adult mammals. Our previous studies showed that predegenerated peripheral nerve grafts facilitate neurite outgrowth from the injured hippocampus and that this effect was particularly distinct when 7-, 28-, and 35-day-predegenerated nerve grafts were used. The purpose of the present study was to use the above method to induce and support the regrowth of injured nerve fibers as well as the survival of retinal ganglion cells (RGCs). Adult Sprague-Dawley rats were assigned to three groups. In the experimental groups transected optic nerve was grafted with peripheral nerve (predegenerated for 7 days (PD) or nonpredegenerated). In the control group, the optic nerve was totally transected. RGCs and growing fibers labeled with fluorescent tracers were examined. They were counted and the results were subjected to statistical analysis. Retinal ganglion cells survived in the groups treated with predegenerated as well as nonpredegenerated grafts; however, the number of surviving retinal ganglion cells was significantly higher in the first one. In both groups the regrowth of the transected optic nerve was observed but the distance covered by regenerating fibers was longer in the PD group. No fibers inside grafts and no labeled cells in retinas were present in the control animals. On the basis of the obtained results we can state that the predegeneration of grafts enhance their neurotrophic influence upon the injured retinal ganglion cells.     

The changes in neurotrophic properties of the peripheral nerves extracts following blocking of BDNF activity

OBJECTIVES: Retinal ganglion cells (RGCs) of adult rats are unable to regenerate their axons after optic nerve injury and soon after they enter the pathway of apoptosis. They may, however, survive and regenerate new axons in response to application of specific peripheral nerve extracts that presumably contain a range of neurotrophic substances. One of the recognized substances of proven neurotrophic activity is brain-derived neurotrophic factor (BDNF). We have investigated whether blocking the BDNF activity in post-microsomal fractions obtained from 7 day pre-degenerated peripheral nerves would affect its neurotrophic properties towards RGCs after optic nerve transection in adult rats. METHODS: Autologous connective tissue chambers sutured to the distal end of transected optic nerve served as active substances containers. Surviving RGCs were visualized using Dil. The number of myelinated outgrowing fibers within the chambers was evaluated in histologic sections. RESULTS: BDNF and 7 day pre-degenerated nerve extracts, and also extracts with blocked BDNF activity, enhanced RGC fibers outgrowth. The regeneration was significantly weaker in the control group. Blocking the BDNF activity in the 7 day pre-degenerated peripheral nerve extract reduced its neurotrophic effects but the differences were insignificant in comparison with non-blocked extracts. DISCUSSION: The regeneration intensities in groups receiving 7 day pre-degenerated peripheral nerve extracts (PD7) and BDNF were comparable. The number of surviving cells was higher in the PD7 group and there were more regenerating fibers in the BDNF group, which may be explained by the strong BDNF effect on axonal collateralization and sprouting.     

Deafferentation-associated changes in afferent and efferent processes in the guinea pig cochlea and afferent regeneration with chronic intrascalar brain-derived neurotrophic factor and acidic fibroblast growth factor

Deafferentation of the auditory nerve from loss of sensory cells is associated with degeneration of nerve fibers and spiral ganglion neurons (SGN). SGN survival following deafferentation can be enhanced by application of neurotrophic factors (NTF), and NTF can induce the regrowth of SGN peripheral processes. Cochlear prostheses could provide targets for regrowth of afferent peripheral processes, enhancing neural integration of the implant, decreasing stimulation thresholds, and increasing specificity of stimulation. The present study analyzed distribution of afferent and efferent nerve fibers following deafness in guinea pigs using specific markers (parvalbumin for afferents, synaptophysin for efferent fibers) and the effect of brain derived neurotrophic factor (BDNF) in combination with acidic fibroblast growth factor (aFGF). Immediate treatment following deafness was compared with 3-week-delayed NTF treatment. Histology of the cochlea with immunohistochemical techniques allowed quantitative analysis of neuron and axonal changes. Effects of NTF were assessed at the light and electron microscopic levels. Chronic BDNF/aFGF resulted in a significantly increased number of afferent peripheral processes in both immediate- and delayed-treatment groups. Outgrowth of afferent nerve fibers into the scala tympani were observed, and SGN densities were found to be higher than in normal hearing animals. These new SGN might have developed from endogenous progenitor/stem cells, recently reported in human and mouse cochlea, under these experimental conditions of deafferentation-induced stress and NTF treatment. NTF treatment provided no enhanced maintenance of efferent fibers, although some synaptophysin-positive fibers were detected at atypical sites, suggesting some sprouting of efferent fibers.     

Post-injury regeneration in rat sciatic nerve facilitated by neurotrophic factors secreted by amniotic fluid mesenchymal stem cells.

Amniotic fluid mesenchymal stem cells have the ability to secrete neurotrophic factors that are able to promote neuron survival in vitro. The purpose of this study was to evaluate the effects of neurotrophic factors secreted by rat amniotic fluid mesenchymal stem cells on regeneration of sciatic nerve after crush injury. Fifty Sprague-Dawley rats weighing 250-300 g were used. The left sciatic nerve was crushed with a vessel clamp. Rat amniotic fluid mesenchymal stem cells embedded in fibrin glue were delivered to the injured nerve. Enzyme-linked immunosorbent assay (ELISA) and immunocytochemistry were used to detect neurotrophic factors secreted by the amniotic fluid mesenchymal stem cells. Nerve regeneration was assessed by motor function, electrophysiology, histology, and immunocytochemistry studies. Positive CD29/44, and negative CD11b/45, as well as high levels of expression of brain-derived neurotrophic factor, glia cell line-derived neurotrophic factor, ciliary neurotrophic factor (CNTF), nerve growth factor, and neurotrophin-3 (NT-3) were demonstrated in amniotic fluid mesenchymal stem cells. Motor function recovery, the compound muscle action potential, and nerve conduction latency showed significant improvement in rats treated with amniotic fluid mesenchymal stem cells. ELISA measurement in retrieved nerves displayed statistically significant elevation of CNTF and NT-3. The immunocytochemical studies demonstrated positive staining for NT-3 and CNTF in transplanted cells. The histology and immunocytochemistry studies revealed less fibrosis and a high level of expression of S-100 and glial fibrillary acid protein at the crush site. Rat amniotic fluid mesenchymal stem cells may facilitate regeneration in the sciatic nerve after crush injury. The increased nerve regeneration found in this study may be due to the neurotrophic factors secreted by amniotic fluid mesenchymal stem cells.