The influence of peripheral nerve graft's predegeneration stage on the regrowth of hippocampal injured neurites and concomitant changes in submicrosomal fraction proteins of grafts.

The present work has a twofold aim: 1. To ascertain whether the stimulative influence of peripheral nerve grafts on injured hippocampal neurons depends on the time lapse after transection and; 2. To examine whether the mentioned effect runs parallel to the time-dependent changes of proteins contents and composition in the submicrosomal fraction from transected rat sciatic nerves. Fluorescence microscope examination revealed that FITC-HRP labeled cells extending their neurites into the implanted peripheral nerve segments were particularly numerous among the hippocampal neurons when 7- and 35-day-old predegenegated distal stumps were used as grafts. Discontinuous SDS-slab polyacrylamide gel electrophoresis of submicrosomal fraction proteins obtained from distal stumps of rat sciatic nerves was performed at the 7, 14, 21 and 35 days after transection. Among the obtained protein fractions the most interesting seem to be the ones of 47 and 54 kDa, which reached maximal levels at the 7th day and the 50 kDa fraction with a maximum at the 35th experimental day. It is possible that the growth promoting power of the employed grafts depends on the presence of proper proteins.     

Role of macrophage migration inhibitory factor (MIF) in peripheral nerve regeneration: anti-MIF antibody induces delay of nerve regeneration and the apoptosis of Schwann cells

BACKGROUND: Macrophage migration inhibitory factor (MIF) is a pluripotent cytokine involved in inflammation and immune responses as well as in cell growth. Although we previously demonstrated the presence of MIF in peripheral nerves, and MIF mRNA expression was up-regulated after axotomy, the role of MIF in nerve injury and regeneration has not been evaluated. MATERIALS AND METHODS: To examine the potential role of MIF in nerve regeneration, we locally administered an anti-MIF polyclonal antibody into regenerating rat sciatic nerves using the silicone chamber model. The effect of the anti-MIF antibody on nerve regeneration was evaluated using an axonal reflex test. In addition, we carried out a terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) assay and immunohistochemical analysis of the damaged nerve segments with regard to apoptosis-related proteins such as p53 to evaluate the effects of anti- MIF antibodies on apoptosis during the regeneration process. RESULTS: The regeneration length of the nerve in the anti-MIF antibody-treated group was significantly shorter than that in the non-immune rabbit IgG-treated group at weeks 2, 4 and 6 after surgery. TUNEL assay showed that a large number of apoptotic cells, mostly Schwann cells, were observed in the intratubal and distal nerve segments at weeks 4 and 6 after surgery by the anti-MIF antibody treatment. Consistent with these results, Ki-67-positive cells were significantly decreased by the anti-MIF antibody treatment. Immunohistochemical analyses revealed that p53 and, to a lesser extent, Fas were more up-regulated in the anti-MIF antibody-treated nerves than in the controls. CONCLUSION: Taken together, these results suggest that MIF plays an important role in acceleration of peripheral nerve regeneration and in prevention of Schwann cell apoptosis, mainly through overcoming the apoptotic effect of p53.     

Expression of the neural cell adhesion molecules L1 and N-CAM and their common carbohydrate epitope L2/HNK-1 during development and after transection of the mouse sciatic nerve

The expression of the neural cell adhesion molecules L1 and N-CAM and of their shared carbohydrate epitope L2/HNK-1 was studied during the development and after the transection of mouse sciatic nerves. During development, L1 and N-CAM were detectable on most, if not all, Schwann cells at embryonic day 17, the earliest stage tested. With increasing age, the immunoreactivity was reduced being confined to non-myelinating Schwann cells by post-natal day 10, at which stage the staining pattern resembled that seen in adult sciatic nerves. Double-immunolabelling experiments revealed a complete overlap between L1 and N-CAM antibodies. The L2/HNK-1 epitope was not detectable in developing sciatic nerves until the end of the 2nd post-natal week, when it appeared to be associated with the outer profiles of thick myelin sheets, as also seen in adult sciatic nerves. Three days after the transection of adult sciatic nerves, L1 antigen and N-CAM was detectable in more Schwann cells in the distal nerve end than in untreated control nerves. The peak level of the reappearance of L1 antigen and N-CAM in Schwann cells occurred between 2 and 4 weeks after transection. The reduction of L1-antigen expression to its normal adult level took more than a year, thus recapitulating normal development, but on a more protracted time scale. Similarly, the L2/HNK-1 epitope remained undetectable until the transected nerve had returned to its normal state of myelination, i.e. approximately 1 year after transection.     

High-Frequency Electrical Stimulation Can Be a Complementary Therapy to Promote Nerve Regeneration in Diabetic Rats

The purpose of this study was to evaluate whether 1 mA of percutaneous electrical stimulation (ES) at 0, 2, 20, or 200 Hz augments regeneration between the proximal and distal nerve stumps in streptozotocin diabetic rats. A10-mm gap was made in the diabetic rat sciatic nerve by suturing the stumps into silicone rubber tubes. Normal animals were used as the controls. Starting 1 week after transection, ES was applied between the cathode placed at the distal stump and the anode at the proximal stump every other day for 3 weeks. At 4 weeks after surgery, the normal controls and the groups receiving ES at 20, and 200 Hz had a higher success percentage of regeneration compared to the ES groups at 0 and 2 Hz. In addition, quantitative histology of the successfully regenerated nerves revealed that the groups receiving ES at a higher frequency, especially at 200 Hz, had a more mature structure with more myelinated fibers compared to those in the lower-frequency ES groups. Similarly, electrophysiology in the ES group at 200 Hz showed significantly shorter latency, larger amplitude, larger area of evoked muscle action potentials and faster conduction velocity compared to other groups. Immunohistochemical staining showed that ES at a higher frequency could significantly promote calcitonin gene-related peptide expression in lamina I-II regions in the dorsal horn and recruit a higher number of macrophages in the diabetic distal sciatic nerve. The macrophages were found that they could stimulate the secretion of nerve growth factor, platelet-derived growth factor, and transforming growth factor-β in dissected sciatic nerve segments. The ES at a higher frequency could also increase cutaneous blood flow in the ipsilateral hindpaw to the injury. These results indicated that a high-frequency ES could be necessary to heal severed diabetic peripheral nerve with a long gap to be repaired.     

Cold Blockade of Axonal Transport Activates Premitotic Activity of Schwann Cells and Wallerian Degeneration

Between 3 and 4 days after transection of cat sciatic nerve, Schwann cell-associated premitotic activity spreads anterogradely along degenerating distal nerve stumps at a rate of approximately 200 mm/day. We investigated whether fast anterograde axonal transport contributes to the initiation of this component of Wallerian degeneration. Axonal transport was blocked in intact and transected cat sciatic nerves by focally chilling a proximal segment to temperatures below 11 degrees C for 24 hr. Incorporation of [3H]thymidine (a marker of premitotic DNA synthesis) was then measured 3 and 4 days posttransection in cold blocked- and control-degenerating nerves. Effects of cold block prior to and concomitant with nerve transection were studied. Results failed to support the hypothesis that Schwann-cell premitotic activity after axotomy is associated with entry into the axon of mitogenic substances and their anterograde fast transport along the distal stump. Instead, data suggested that progressive anterograde failure of fast anterograde transport distal to transection serves to effect the Schwann-cell premitotic response to axotomy.     

Differential expression of mRNAs for neurotrophins and their receptors after axotomy of the sciatic nerve

The neurotrophin family includes NGF, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4). Previous studies have demonstrated that expression of NGF and its low-affinity receptor is induced in nonneuronal cells of the distal segment of the transected sciatic nerve suggesting a role for NGF during axonal regeneration (Johnson, E. M., M. Taniuchi, and P. S. DeStefano. 1988. Trends Neurosci. 11:299-304). To assess the role of the other neurotrophins and the members of the family of Trk signaling neurotrophin receptors, we have here quantified the levels of mRNAs for BDNF, NT-3, and NT-4 as well as mRNAs for trkA, trkB, and trkC at different times after transection of the sciatic nerve in adult rats. A marked increase of BDNF and NT-4 mRNAs in the distal segment of the sciatic nerve was seen 2 wk after the lesion. The increase in BDNF mRNA was mediated by a selective activation of the BDNF exon IV promoter and adrenalectomy attenuated this increase by 50%. NT-3 mRNA, on the other hand, decreased shortly after the transection but returned to control levels 2 wk later. In Schwann cells ensheathing the sciatic nerve, only trkB mRNA encoding truncated TrkB receptors was detected with reduced levels in the distal part of the lesioned nerve. Similar results were seen using a probe that detects all forms of trkC mRNA. In the denervated gastrocnemius muscle, the level of BDNF mRNA increased, NT-3 mRNA did not change, while NT-4 mRNA decreased. In the spinal cord, only small changes were seen in the levels of neutrophin and trk mRNAs. These results show that expression of mRNAs for neurotrophins and their Trk receptors is differentially regulated after a peripheral nerve injury. Based on these results a model is presented for how the different neurotrophins could cooperate to promote regeneration of injured peripheral nerves.     

Changes of nerve growth factor synthesis in nonneuronal cells in response to sciatic nerve transection

The intact sciatic nerve contains levels of nerve growth factor (NGF) that are comparable to those of densely innervated peripheral target tissues of NGF-responsive (sympathetic and sensory) neurons. There, the high NGF levels are reflected by correspondingly high mRNANGF levels. In the intact sciatic nerve, mRNANGF levels were very low, thus indicating that the contribution of locally synthesized NGF by nonneuronal cells is small. However, after transection an increase of up to 15-fold in mRNANGF was measured in 4-mm segments collected both proximally and distally to the transection site. Distally to the transection site, augmented mRNANGF levels occurred in all three 4-mm segments from 6 h to 2 wk after transection, the longest time period investigated. The augmented local NGF synthesis after transection was accompanied by a reexpression of NGF receptors by Schwann cells (NGF receptors normally disappear shortly after birth). Proximal to the transection site, the augmented NGF synthesis was restricted to the very end of the nerve stump that acts as a "substitute target organ" for the regenerating NGF-responsive nerve fibers. While the mRNANGF levels in the nerve stump correspond to those of a densely innervated peripheral organ, the volume is too small to fully replace the lacking supply from the periphery. This is reflected by the fact that in the more proximal part of the transected sciatic nerve, where mRNANGF remained unchanged, the NGF levels reached only 40% of control values. In situ hybridization experiments demonstrated that after transection all nonneuronal cells express mRNANGF and not only those ensheathing the nerve fibers of NGF-responsive neurons.     

Laminin B1 and Collagen Type IV Gene Expression in Transected Peripheral Nerve: Reinnervation Compared to Denervation

The expression of B1 laminin and type IV collagen was followed in the microsurgically isolated endoneurium of transected rat sciatic nerves from 3 days until 8 weeks. Northern hybridizations revealed that after nerve transection the proximal stumps of denervated, as well as freely regenerating, nerves showed a markedly increased expression of laminin and type IV collagen which lasted from 3 days up to 8 weeks. In the distal stumps, close to the site of transection (2-7 mm), the expression of laminin, and to a certain extent that of type IV collagen, seemed to be enhanced if free axonal reinnervation was allowed. Further distally (10-15 mm), the patterns of B1 laminin and type IV collagen expression were similar in both experimental groups, so that an increased expression was noticed during the first 2 weeks. The present results suggest that laminin and type IV collagen gene expression is markedly different in different parts of transected rat sciatic nerve. During peripheral nerve regeneration, there is a long-lasting basement membrane gene expression in the proximal stump. In the distal part of the transected nerve, the axonal reinnervation possibly up-regulates, but is not essential for, the expression of B1 laminin and type IV collagen.     

Laminin A, B1, and B2 Chain Gene Expression in Transected and Regenerating Nerves: Regulation by Axonal Signals

Abstract: Laminin A, B1, and B2 chain mRNA levels in degenerating and regenerating mouse sciatic nerves were examined using northern blot analysis. In normal intact nerves, B1 and B2 mRNA steady-state levels were high, but when the nerves were crushed, the steady-state levels of B1 and B2 mRNA per milligram wet tissue weight of the distal segments of the nerves increased five- to eightfold over that of control levels as the total RNA and β-actin mRNA levels increased, suggesting that these increases were the consequence of Schwann cell proliferation after axotomy. When the steady-state levels of B1 and B2 mRNA were normalized as the ratio to total RNA or β-actin mRNA levels, however, they drastically decreased to about 20% of the normal nerve levels in the nerve segments distal to both the crush and transaction sites 1 day after injury. In the crushed nerves, B1 and B2 mRNA levels gradually increased as the regenerating nerves arrived at the distal segments and reestablished normal axon–Schwann cell contact, and then returned to normal levels on the 21 st day. In the transected nerves, where Schwann cells continued to be disconnected from axons, both B1 and B2 mRNA levels remained low. Cultured Schwann cells expressed detectable levels of B1 and B2 chain mRNA which significantly increased when the cells were cocultured with sensory neurons. However, mRNA for A chain was not detectable in the normal, axotomized nerves or in cultured Schwann cells. These data indicate that Schwann cells express laminin B1 and B2 chain mRNA that are up-regulated by axonal or neuronal contact, but they do not express A chain mRNA.     

Immunoelectron microscopic localization of neural cell adhesion molecules (L1, N-CAM, and myelin-associated glycoprotein) in regenerating adult mouse sciatic nerve

The localization of the neural cell adhesion molecules L1, N-CAM, and the myelin-associated glycoprotein was studied by pre- and postembedding staining procedures at the light and electron microscopic levels in transected and crushed adult mouse sciatic nerve. During the first 2-6 d after transection, myelinated and nonmyelinated axons degenerated in the distal part of the proximal stump close to the transection site and over the entire length of the distal part of the transected nerve. During this time, regrowing axons were seen only in the proximal, but not in the distal nerve stump. In most cases L1 and N-CAM remained detectable at cell contacts between nonmyelinating Schwann cells and degenerating axons as long as these were still morphologically intact. Similarly, myelin-associated glycoprotein remained detectable in the periaxonal area of the degenerating myelinated axons. During and after degeneration of axons, nonmyelinating Schwann cells formed slender processes which were L1 and N-CAM positive. They resembled small-diameter axons but could be unequivocally identified as Schwann cells by chronical denervation. Unlike the nonmyelinating Schwann cells, only few myelinating ones expressed L1 and N-CAM. At the cut ends of the nerve stumps a cap developed (more at the proximal than at the distal stump) that contained S-100-negative and fibronectin-positive fibroblast-like cells. Most of these cells were N-CAM positive but always L1 negative. Growth cones and regrowing axons expressed N-CAM and L1 at contact sites with these cells. Regrowing axons of small diameter were L1 and N-CAM positive where they made contact with each other or with Schwann cells, while large-diameter axons were only poorly antigen positive or completely negative. 14 d after transection, when regrowing axons were seen in the distal part of the transected nerve, regrowing axons made L1- and N-CAM-positive contacts with Schwann cells. When contacting basement membrane, axons were rarely found to express L1 and N-CAM. Most, if not all, Schwann cells associated with degenerating myelin expressed L1 and N-CAM. In crushed nerves, the immunostaining pattern was essentially the same as in the cut nerve. During formation of myelin, the sequence of adhesion molecule expression was the same as during development: L1 disappeared and N-CAM was reduced on myelinating Schwann cells and axons after the Schwann cell process had turned approximately 1.5 loops around the axon. Myelin-associated glycoprotein then appeared both periaxonally and on the turning loops of Schwann cells in the uncompacted myelin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Transport of muscarinic cholinergic receptors in the sciatic nerve of rat

On the basis of the specific [³H]quinuclidinyl-benzilate binding, the transport of muscarinic cholinergic receptors has been demonstrated in the ventral horn, sciatic nerve and in the 3 mm segments proximal and distal to the ligature of rat sciatic nerves ligated for 24 h (a) without electrolytic lesion, (b) six days after lesion of the spinal ganglia, (c) six days after lesion of the motoric axons, and (d) six days after transection of the sciatic nerve. The distribution of these receptors was also studied in the ventral spinal horn, dorsal root sensory axons, spinal ganglia and sciatic nerve of rabbit.Our results suggest that the receptors are transported in the sciatic nerve of rat. This transport consists of a large anterograde, and a discrete retrograde flow of muscarinic cholinergic receptors. Most of the receptors are possibly synthesized in the motoneuron cell bodies and migrate in the motoric axons; to a lesser extent they may also be synthesized in the cell bodies of the dorsal root ganglia and migrate in the sensory axons of the sciatic nerve.     

Levels of myo-inositol in normal and degenerating peripheral nerve

—Free inositol was measured in peripheral nerves of the monkey, rabbit, rat, frog and lobster; levels in mammalian nerve were similar, and two to three times greater than in the other species. Concentrations of myo-inositol in rabbit tibial nerve increased from proximal to distal segments; in optic nerve the concentrations decreased with greater distance from the retina. In the early stages of Wallerian degeneration rabbit tibial nerve contained 25 per cent less free myo-inositol, rat nerve 50 per cent less. Rabbit nerves were analysed at 2 and 5 weeks after section; by 5 weeks levels of myo-inositol had increased to 50 per cent above normal. Similar changes were found in degenerating rabbit optic nerve. The combination of galactose feeding and nerve section resulted in reduction of the myo-inositol in rat sciatic nerve to one-fifth of the control value; galactitol in the nerve decreased by 50 per cent after section. The evidence suggests that myo-inositol in nerve is located mainly in Schwann cells or glia.     

Absence of 'superfast' axonal transport in rat sciatic nerve.

Axonal transport of labelled protein was studied in rat sciatic nerve by analyzing nerve segments at intervals after injection of L-[3H]leucine into the lumbar spinal cord. Some nerves were sectioned before injection so that material in transit accumulated proximal to the section. The segments distal to the section served as controls for incorporation into the nerve of blood-borne label. An analysis of TCA-soluble and TCA-insoluble activity in cut and intact nerve segments was also made. No evidence was found for the existence of a 'superfast' component of axonal transport (velocity 2000 mm/day). Results showed that the most rapidly transported protein derived from the neuron soma had a conventional 'fast' velocity of 350-420 mm/day. There was no transport of TCA-soluble material. It is suggested that 'superfast' transport, detected in mice by other investigators, is an artefact resulting from failure to control for incorporation of circulating label into the sciatic nerve.     

Altered Expression of Inducible Nitric Oxide Synthase After Sciatic Nerve Injury in Rat

Nitric oxide is known to contribute to neuronal damage as well as to peripheral neuronal regeneration following injury. Sciatic nerve injury is a common and serious complication of intramuscular injections. In order to ascertain the role of inducible nitric oxide synthase (iNOS) in the injured sciatic nerve, we studied the expression of this enzyme by RT-PCR and immunohistochemistry, in a rat model of sciatic nerve injury. In sham-operated control rats iNOS expression was undetectable by immunohistochemistry and its mRNA level was also very low. In contrast, in the experimental group that was subjected to sciatic nerve injury, both mRNA and protein of iNOS were found to be significantly elevated. The protein level of iNOS, as revealed by positive immunostaining, peaked at 7 days post-surgery followed by a decrease. Similarly, the iNOS mRNA levels remained elevated at 1, 3, 7 days but declined to very low level by day 21, after surgery. This study indicates that the increased expression of iNOS after sciatic nerve injury in rats may contribute to nerve regeneration. Thus our results suggest that excessive expression of iNOS after nerve injury is not conducive to nerve regeneration.     

周围神经再生早期过程中内源性和外源性巨噬细胞数量变化的实验研究

目的：周围神经再生过程中巨噬细胞发挥了重要的作用,然而目前对于神经内内源性和外源性巨噬细胞的具体作用了解的却很少,因此本实验研究了小鼠坐骨神经损伤后早期再生过程中内源性和外源性巨噬细胞数量比例变化的情况,探索周围神经再生的规律。方法：移植CAG-EGFP转基因小鼠的全骨髓有核细胞到骨髓灭活野生型C5781／6小鼠体内建立嵌合体小鼠模型。待移植成功3个月后夹伤小鼠一侧坐骨神经,并在损伤后第2、7、14和28天取材、切片,使用巨噬细胞特异性抗体cD68进行免疫荧光染色,分析损伤神经段中内源性巨噬细胞（CD68＋／EGFP-）、外源性巨噬细胞（CD68＋／EGFP＋）的数量及其比例变化情况。结果：①夹伤骨髓移植模型小鼠坐骨神经后,参与坐骨神经损伤修复的巨噬细胞可分为两类,即内源性巨噬细胞（CD68＋／EGFP-）和外源性巨噬细胞（CD68＋／EGFP＋）;②夹伤坐骨神经后,浸润的总巨噬细胞数量从第2天开始逐渐增加,到第14天达到高峰,约为正常情况下的60倍,随后逐渐减少;③起初外、内源性巨噬细胞间的比例是1：1,差值最大出现在损伤后第14天为4：l。结论：小鼠坐骨神经夹伤后,内外源性巨噬细胞共同参与了受损神经组织远心段的修复和再生过程,损伤初期发挥作用的主要是内源性巨噬细胞,随后大量浸润的外源性巨噬细胞占主导作用。本实验首次连续观察并定量分析了神经损伤后早期内源性和外源性巨噬细胞的数量改变,证实了瓦勒氏变性过程中内源性和外源性巨噬细胞在不同阶段对巨噬细胞总量的贡献作用。     

Axonal transports of tripeptidyl peptidase II in rat sciatic nerves

Axonal transport of tripeptidyl peptidase II, a putative cholecystokinin inactivating serine peptidase, was examined in the proximal, middle, and distal segments of rat sciatic nerves using a double ligation technique. Enzyme activity significantly increased not only in the proximal segment but also in the distal segment 12-72h after ligation, and the maximal enzyme activity was found in the proximal and distal segments at 72h. Western blot analysis of tripeptidyl peptidase II showed that its immunoreactivities in the proximal and distal segments were 3.1- and 1.7-fold higher than that in the middle segment. The immunohistochemical analysis of the segments also showed an increase in immunoreactive tripeptidyl peptidase II level in the proximal and distal segments in comparison with that in the middle segment, indicating that tripeptidyl peptidase II is transported by anterograde and retrograde axonal flow. The results suggest that tripeptidyl peptidase II may be involved in the metabolism of neuropeptides in nerve terminals or synaptic clefts.     

Neural Stem Cells Enhance Nerve Regeneration after Sciatic Nerve Injury in Rats

With the development of tissue engineering and the shortage of autologous nerve grafts in nerve reconstruction, cell transplantation in a conduit is an alternative strategy to improve nerve regeneration. The present study evaluated the effects and mechanism of brain-derived neural stem cells (NSCs) on sciatic nerve injury in rats. At the transection of the sciatic nerve, a 10-mm gap between the nerve stumps was bridged with a silicon conduit filled with 5?×?10⁵ NSCs. In control experiments, the conduit was filled with nerve growth factor (NGF) or normal saline (NS). The functional and morphological properties of regenerated nerves were investigated, and expression of hepatocyte growth factor (HGF) and NGF was measured. One week later, there was no connection through the conduit. Four or eight weeks later, fibrous connections were evident between the proximal and distal segments. Motor function was revealed by measurement of the sciatic functional index (SFI) and sciatic nerve conduction velocity (NCV). Functional recovery in the NSC and NGF groups was significantly more advanced than that in the NS group. NSCs showed significant improvement in axon myelination of the regenerated nerves. Expression of NGF and HGF in the injured sciatic nerve was significantly lower in the NS group than in the NSCs and NGF groups. These results and other advantages of NSCs, such as ease of harvest and relative abundance, suggest that NSCs could be used clinically to enhance peripheral nerve repair.