Neuronotrophic activities accumulate in vivo within silicone nerve regeneration chambers

Rat sciatic nerves can be transected and their proximal and distal stumps sutured into the openings of cylindrical silicone chambers. Anatomical regeneration has been demonstrated across 10 mm long chambers containing both stumps, although little or no axonal outgrowth occurs in chambers omitting the distal stump or exceeding the 10 mm length. We have previously shown that chambers containing both proximal and distal stumps accumulate within days of implantation a clear fluid containing neuronotrophic factors (NTFs) directed to neurons from neonatal mouse dorsal root ganglia. We report here that these chamber fluids also have considerable neuronotrophic activity for chick embryo neurons from embryologic day 4 (E4) lumbar spinal cord, E12 sympathetic ganglia, E12 (but not E8) dorsal root ganglia and E8 ciliary ganglia. Thus, the neuronal types supported by trophic factors of these fluids include all those which contribute axons to the sciatic nerve, namely sensory, spinal motor, and sympathetic. In fluid collected 1 week after implantation, NTF levels directed to different neurons varied independently from one another in chambers with different nerve insertions, suggesting that these activities reside in separate factors. Fluid collected from chamber arrangements allowing little proximal fiber regrowth did not always contain correspondingly lower titers of NTFs. However, generally higher titers of all NTFs were found in chambers containing either or both nerve stumps that in nerve-free chambers. Fluids collected from nerve-containing chambers were subjected to heat, dialysis or trypsin treatments. The behavior of their neuronotrophic activities suggests their association with proteins.     

Transport of acid phosphatase in normal and transected rat sciatic nerve

Acid phosphatase accumulates at the severed ends of transected peripheral nerve or spinal cord. We examined the temporal sequence of enzyme accumulation from 1 to 14 days after transecting or applying colchicine to the rat sciatic nerve in order to ascertain whether or not this accumulation is related to axonal transport. Enzyme activity was observed in the proximal and distal stumps by 1 day after transection, and by 1 week activity in the proximal stump exceeded that in the distal stump. In some experiments, colchicine was applied to the nerve proximal to the site of nerve transection. This procedure resulted in an accumulation of acid phosphatase proximal to the site of drug application and not immediately proximal to the site of transection. These results are consistent with the hypothesis that bidirectional transport of acid phosphatase is a normal feature of peripheral nerves.     

Nerve regeneration model and trophic factors in vivo

The proximal stump of a transected rat sciatic nerve has been observed to regenerate through a cylindrical silicone chamber across a 10 mm gap to the distal stump. The fluid filling such in vivo chambers contains trophic factors that ensure in vitro survival and growth of at least sensory neurons from rodent dorsal root ganglia — as already demonstrated for fluid generated in vitro from Schwann and other cell cultures.     

Axonal growth in mesothelial chambers: Effects of a proximal preconditioning lesion and/or predegeneration of the distal nerve stump

Preformed, autologous mesothelial chambers were utilized to study axonal growth following selective predegeneration of the distal nerve stump and/or preconditioning of the proximal nerve stump. The left and/or right sciatic nerve of rats was exposed and transected in the thigh. Two weeks after transection, the left proximal nerve stump was cross-anastomosed with the right distal nerve stump by using a mesothelial chamber leaving a 15-mm gap between the two nerve stumps. Previous studies have shown that axonal overgrowth normally does not occur over this gap distance to the distal stump. Three months after cross-anastomosing, regeneration across the 15-mm gap was evaluated by muscle action potential recordings and light microscopical examination. In experiments in which a distal nerve stump was selectively degenerated and the proximal segment was freshly cut, axons had bridged the 15-mm gap in six of seven rats. When a proximal preconditioned nerve stump was matched with a freshly cut distal stump, axonal overgrowth occurred in only 4 of 10 experiments. In experiments including a proximal preconditioned nerve stump and a distal predegenerated stump, axons bridged the gap in 6 of 8 experiments. We concluded that a priming lesion, including manipulation with proximal and/or distal stump, enhances axonal growth in mesothelial chambers.     

Chicken NGF and non-NGF trophic factor synthesis and release by sciatic nerves in vitro

The time course of production and release of nerve growth factor (NGF) and non-NGF neuronotrophic factors for sympathetic neurons by chicken and rat sciatic nerves in culture was examined. These tissues actively synthesize and release neuronotrophic activity as metabolically poisoning nerves with azide dramatically reduced the amount of trophic activity released into the culture medium. The sustained release of this activity also was shown to be dependent on the presence of low-molecular-weight dialysable molecules present in foetal calf serum and amniotic fluid from day 11 chicken embryos. Affinity-purified antimouse NGF antibodies were used to show that sciatic nerves in culture release both NGF and non-NGF trophic factors. These antibodies inhibited all bioactivity of both mouse NGF and of a partially purified preparation of chicken NGF. Immunoblot studies confirm that the antibodies recognize both rodent and avian NGF. Excess antibody inhibited only about 50% of the trophic activity in media conditioned over rat or chicken nerves for the first 24 hr. Relatively similar amounts of this non-NGF trophic activity were released throughout 6 days in culture, and this trophic activity kept sympathetic neurons alive in culture in the absence of NGF for more than 4 days. NGF levels were quantified with a two-site enzyme-linked immunoassay and found to parallel changes in NGF bioactivity. Rat nerves released increasing amounts of NGF with time in culture. Whole chicken sciatic nerves, however, released decreasing amounts of NGF with time in culture, but when these nerves were desheathed by removal of the epineurium and attached tissue, the pattern of NGF release was similar to that observed in the smaller rat sciatic nerves. These studies therefore characterize antibodies recognizing chicken NGF, demonstrate that peripheral nerve tissue synthesize trophic factors other than NGF, and identify factors that influence NGF synthesis.     

Bioinformatic analysis of cytokine expression in the proximal and distal nerve stumps after peripheral nerve injury

In our previous study, we investigated the dynamic expression of cytokines in the distal nerve stumps after peripheral nerve injury using microarray analysis, which can characterize the dynamic expression of proteins. In the present study, we used a rat model of right sciatic nerve transection to examine changes in the expression of cytokines at 1, 7, 14 and 28 days after injury using protein microarray analysis. Interleukins were increased in the distal nerve stumps at 1–14 days post nerve transection. However, growth factors and growth factor-related proteins were mainly upregulated in the proximal nerve stumps. The P-values of the inflammatory response, apoptotic response and cell-cell adhesion in the distal stumps were higher than those in the proximal nerve stumps, but the opposite was observed for angiogenesis. The number of cytokines related to axons in the distal stumps was greater than that in the proximal stumps, while the percentage of cytokines related to axons in the distal stumps was lower than that in the proximal nerve stumps. Visualization of the results revealed the specific expression patterns and differences in cytokines in and between the proximal and distal nerve stumps. Our findings offer potential therapeutic targets and should help advance the development of clinical treatments for peripheral nerve injury. Approval for animal use in this study was obtained from the Animal Ethics Committee of the Chinese PLA General Hospital on September 7, 2016(approval No. 2016-x9-07).     

Neurofilament mRNAs are present and translated in the normal and severed sciatic nerve

Local protein synthesis within axons has been studied on a limited scale. In the present study, several techniques were used to investigate this synthesis in sciatic nerve, and to show that it increases after damage to the axon. Neurofilament (NF) mRNAs were probed by RT-PCR, Northern blot and in situ hybridization in axons of intact rat sciatic nerve, and in proximal or distal stumps after sciatic nerve transection. RT-PCR demonstrated the presence of NF-L, NF-M and NF-H mRNAs in intact sciatic nerve, as well as in proximal and distal stumps of severed nerves. Northern blot analysis of severed nerve detected NF-L and NF-M, but not NF-H. This technique did not detect the three NFs mRNAs in intact nerve. Detection of NF-L and NF-M mRNA in injured nerve, however, indicated that there was an up-regulation in response to nerve injury. In situ hybridization showed that NF-L mRNA was localized in the Schwann cell perinuclear area, in the myelin sheath, and at the boundary between myelin sheath and cortical axoplasm. RNA and protein synthesizing activities were always greater in proximal as compared to distal stumps. NF triplet proteins were also shown to be synthesized de novo in the proximal stump. The detection of neurofilament mRNAs in nerves, their possible upregulation during injury and the synthesis of neurofilament protein triplet in the proximal stumps, suggest that these mRNAs may be involved in nerve regeneration, providing a novel point of view of this phenomenon.     

The expression of nuclear 3,5,3' triiodothyronine receptors is induced in Schwann cells by nerve transection.

The effects of thyroid hormones on the nervous system are mediated by the presence of nuclear T3 receptors (NT3R). In this study, the expression of NT3R was investigated in spinal cord, dorsal root ganglia (DRG), or sciatic nerve of adult rats after immunostaining with a 2B3-NT3R monoclonal antibody which recognizes both alpha and beta types of NT3R. The specificity of this monoclonal antibody was confirmed by Western blots. The 2B3-NT3R monoclonal antibody recognized one band corresponding to a molecular weight of 57 kDa in extract of spinal cord or DRG. No staining was observed on immunoblot of intact sciatic nerve. In the spinal cord, the nuclei of the neurons and glial cells including both astrocytes and oligodendrocytes exhibited 2B3-NT3R immunoreactivity. While all the nuclei of the DRG sensory neurons expressed the NT3R, all the nuclei of the satellite and Schwann cells were devoid of any immunoreaction. In the sciatic nerve, the nuclei of the Schwann cells also lacked 2B3-NT3R-immunoreactivity. After sciatic nerve transection in vivo, Schwann cell nuclei, which never expressed NT3R in intact nerves of adult rats, displayed a clear 2B3-NT3R immunoreaction in proximal and distal stumps adjacent to the section. Double immunostaining with antibodies raised to 3-sulfogalactosylceramide or S100 confirmed that most of the NT3R containing nuclei belong to Schwann cells. In dissociated cell cultures grown in vitro from sciatic nerves, Schwann cells exhibited 2B3-NT3R immunoreactivity. These data suggest that the inhibition of NT3R expression in Schwann cells ensheathing axons in intact nerve is reversed when the axons are degenerating or lacking.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification of adult rat sciatic nerve ciliary neuronotrophic factor

The ciliary neuronotrophic factor (CNTF), a protein required for the survival of cultured avian embryonic parasympathetic ciliary ganglionic neurons, was recently purified from extracts of selected chick intraocular tissues. Here we report the purification of a mammalian CNTF activity from extracts of adult rat sciatic nerve using a fractionation procedure similar to that employed for isolating chick eye CNTF. About 2 micrograms of CNTF protein can be obtained from each 1.5 g batch of nerve tissue. Like the chick CNTF, the mammalian factor displays trophic activity for dorsal root and sympathetic as well as ciliary ganglionic neurons. The nerve CNTF activity differs from its chick counterpart in molecular weight and chromatographic behavior on ion-exchange columns. Unlike purified nerve growth factor (NGF), nerve CNTF activity is insensitive to anti-NGF antibodies and is unable to support the survival of 8-day chick embryo dorsal root ganglion neurons.     

Transganglionic regulation of central terminals of dorsal root ganglion cells by nerve growth factor (NGF)

Blockade of axonal transport or transection of the rat sciatic nerve results in transganglionic degenerative atrophy (TDA) of nerve terminals containing fluoride-resistant acid phosphatase (FRAP) in the Rolando substance of the spinal cord. Application of vinblastine (9 micrograms) in a cuff around the sciatic nerve of adult rats blocked the retrograde transport of [125I]NGF in sensory fibers; this amount of vinblastine is identical to the threshold amount that induces TDA. Conversely, application of NGF to the proximal stump of the transected sciatic nerve prevented or delayed the occurrence of TDA as reflected by the maintenance of FRAP in the upper dorsal horn, that otherwise would inevitably disappear following the peripheral nerve lesion. These results suggest that endogenous NGF transported retrogradely in peripheral sensory fibers of the adult rat under normal conditions may be responsible for the regulation of the structural and functional integrity of the central terminals of these FRAP-containing primary sensory neurons and that TDA may be the consequence of the failure of NGF to reach the perikarya of these neurons.     

Nogo-A expression in the intact and injured nervous system

The expression of Nogo-A mRNA and protein in the nervous system of adult rats and cultured neurons was studied by in situ hybridisation and immunohistochemistry. Nogo-A mRNA was expressed by many cells in unoperated animals, including spinal motor, DRG, and sympathetic neurons, retinal ganglion cells, and neocortical, hippocampal, and Purkinje neurons. Nogo-A protein was strongly expressed by presumptive oligodendrocytes, but not by NG2+glia and was abundant in motor, DRG, and sympathetic neurons, retinal ganglion cells, and many Purkinje cells, but was difficult to detect in dentate gyrus neurons and some neocortical neurons. Cultured fetal mouse neocortical neurons and adult rat DRG neurons strongly expressed Nogo-A in their perikarya, growth cones, and axonal varicosities. All axons in the intact sciatic nerve contained Nogo-A and many but not all regenerating axons were strongly Nogo-A immunopositive after sciatic nerve transection. Ectopic muscle fibres that developed among the regenerating axons were also Nogo-A immunopositive. Following injury to the spinal cord, Nogo-A mRNA was upregulated around the lesion and Nogo-A protein was strongly expressed in injured dorsal column fibres and their sprouts which entered the lesion site. Following optic nerve crush, Nogo-A accumulated in the proximal and distal stumps bordering the lesions.     

Categories of axons in the inferior cardiac nerve of the cat

Myelinated and unmyelinated axons in the inferior cardiac nerve of the cat were examined to determine how many axons were (1) sensory, (2) preganglionic sympathetic, and (3) postganglionic sympathetic. In one group of cats, a segment was removed from the middle of the inferior cardiac nerve as a control, and the proximal and distal stumps of the nerve were examined one week later. In another group of cats, the control segment of nerve was removed and the first thoracic white ramus communicans and sympathetic trunk were cut proximal to the stellate ganglion, followed in one week by examination of the proximal and distal stumps of the inferior cardiac nerve. In still another group of cats, the first five thoracic spinal nerves were cut just distal to the dorsal root ganglion. The counts of myelinated and unmyelinated axons after these surgical procedures indicated that, in the cat inferior cardiac nerve, all or almost all of the approximately 30,000 unmyelinated axons and 10 percent of the myelinated axons are postganglionic sympathetic fibers, and that approximately 90 percent of the myelinated axons are sensory.     

Local administration of vasoactive intestinal peptide after nerve transection accelerates early myelination and growth of regenerating axons

Our goal was to determine whether local injections of vasoactive intestinal peptide (VIP) promote early stages of regeneration after nerve transection. Sciatic nerves were transected bilaterally in 2 groups of 10 adult mice. In the first group, 15 microg (20 microL) of VIP were injected twice daily into the gap between transected ends of the right sciatic nerve for 7 days (4 mice) or 14 days (6 mice). The same number of mice in the second group received placebo injections (20 microL of 0.9% sterile saline) in the same site, twice daily, for the same periods. After 7 days, axon sizes, relationships with Schwann cells and degree of myelination were compared in electron micrographs of transversely sectioned distal ends of proximal stumps. Fourteen days after transection, light and electron microscopy were used to compare and measure axons and myelin sheaths in the transection gap, 2-mm distal to the ends of proximal stumps. Distal ends of VIP-treated proximal stumps contained larger axons 7 days after transection. More axons were in 1:1 relationships with Schwann cells and some of them were surrounded by thin myelin sheaths. In placebo-treated proximal stumps, axons were smaller, few were in 1:1 relationships with Schwann cells and no myelin sheaths were observed. In VIP-treated transection gaps, measurements 14 days after transection showed that larger axons were more numerous and their myelin sheaths were thicker. Our results suggest that in this nerve transection model, local administration of VIP promotes and accelerates early myelination and growth of regenerating axons.     

Regeneration of sciatic nerves of adult rats induced by extracts from distal stumps of pre-degenerated peripheral nerves

Despite numerous experimental and clinical attempts to reconstruct injuries of peripheral nerves, the methods developed until now have not been sufficiently effective. We examined the influence of extracts (postmicrosomal fractions) obtained from non-pre-degenerated or 7-day-pre-degenerated distal segments of peripheral nerves on the regeneration of injured sciatic nerves of male adult rats. The extracts were introduced to the site of injury with autologous connective tissue chambers filled with fibrin. Reference groups were treated with brain-derived neurotrophic factor (BDNF) or fibrin only. We examined DiI-labeled motoneurons, toluidine blue-labeled myelinated fibers in the mid-part of the chambers, and AChE-positive nerve endings to assess the regeneration intensity. In addition, the length of fibers regrowing within the chambers was measured. We found that extracts obtained from distal stumps of 7-day-pre-degenerated peripheral nerves enhanced nerve regeneration as strongly as BDNF.     

Increased rate of peripheral nerve regeneration using bioresorbable nerve guides and a laminin-containing gel

The sciatic nerve of adult mice was transected and proximal and distal nerve stumps were sutured into a nontoxic bioresorbable nerve guide. Nerve guide lumens were either empty or filled with a gel containing 80% laminin and additional extracellular matrix components. Two weeks later cells in the L3 through L5 dorsal root ganglia and the ventral horn of the spinal cord were retrogradely filled with horseradish peroxidase. All animals with the laminin-containing gel but none with empty nerve guides displayed labeled cells. This suggests that the laminin-containing gel significantly hastened axonal regeneration in vivo.     

Modification of fibrin matrix formation in situ enhances nerve regeneration in silicone chambers

The spatial-temporal progress of nerve regeneration was examined in silicone chambers of three different volume capacities: 11, 25, and 75 microliter. In all chambers, the stumps of a transected rat sciatic nerve were sutured into the ends of the chamber leaving a 10 mm gap between the stumps. Chambers were implanted empty (E chambers) or prefilled with saline (PF chambers). A coaxial and continuous fibrin matrix had formed in all chambers by 1 week. In E chambers, the matrices had a proximal-distal taper that was more pronounced in E25 and E75 chambers due to significantly larger matrix diameters in the proximal region. At 3 weeks, vascular and Schwann cell migration and axonal regeneration were less advanced in the E25 and E75 than in the control E11 chambers. The retardation correlated with the presence of an avascular organization of circumferential cells. Saline prefilling affected the caliber and density of fibrin fibers in the 1 week matrices of PF25 and PF75 chambers. The matrices did not have a prominent taper and diameters were progressively larger with increasing chamber volume. Saline prefilling did not affect regeneration progress in 3 week PF11 chambers but did enhance regeneration in the PF25 chambers; a 1.5-fold larger diameter nerve formed at 3 weeks that contained 2.6-fold more axons. Progress in the PF75 chamber was retarded. We conclude that the volume, timing, and nature of the fluid filling a silicone chamber have significant influence on the formation of fibrin matrices. Alterations in matrix formation correlate with substantial changes in the subsequent progress of intrachamber regeneration events.     

Calcitonin gene-related peptide (CGRP) in the regenerating rat sciatic nerve

Calcitonin-gene related peptide (CGRP) is a neuromodulatory peptide present in motoneurons and a subpopulation of sensory neurons of the adult peripheral nervous system. Here we have investigated the changes in axonal transport of CGRP and CGRP receptor expression in the injured and regenerating rat sciatic nerve using CGRP-immunocytochemistry, radioimmunoassay and quantitative in situ receptor autoradiography techniques. Axotomy led to a gradual and prolonged, 2.5- to 3.5-fold increase in specific CGRP binding to the distal part of the crushed sciatic nerve, beginning 4-6 days after axotomy. An even stronger, up to 30-fold increase was observed after 30-42 day denervation in the distal part of the transected sciatic nerve, where neurite reinnervation was prevented by retroversion and ligation of the proximal nerve stump. Reconnection of the proximal and distal nerve stumps 21 days after transection did not lead to a major reduction in specific CGRP binding but prevented a further increase that occurred between 21 and 42 days after transection without reconnection. In contrast, the anterograde axonal transport of CGRP decreased after axotomy to 40-50% of the control values 6-8 days after nerve crush but recovered towards normal levels during successful regeneration. Interestingly, the retrograde axonal transport of CGRP appeared to amount to only 10-20% of the anterograde transport, suggesting that the peptide may be released by the regenerating neurites into the endoneurium of the injured peripheral nerve. In view of the persistent upregulation in endoneural CGRP binding after axotomy these data indicate that axonal CGRP could play a regulatory role in mediating axonal-endoneural cell interaction during peripheral nerve regeneration.     

A blind trial of silicone chamber fluid in nerve regeneration in the rat

The nerve growth-promoting effects of the fluid accumulating in vivo from 1 to 28 days within silicone chambers were tested on the sciatic nerves of rats. We measured the elongation of sensory axons following nerve transection repaired by means of fibrin sealant allowing local application of the fluid. We found no difference between transections treated with chamber fluid and controls.     

Fine structural and immunohistochemical identification of perineurial cells connecting proximal and distal stumps of transected peripheral nerves at early stages of regeneration in silicone tubes

Perineurial cells are specialized connective tissue cells that form a barrier between endoneurium and epineurium in normal nerves. In the present study, the formation of the perineurium after transection of rat sciatic nerves was investigated. The cord bridging the gap between proximal and distal stumps through silicone tubes was studied 3, 7, 12, 18, and 21 days after surgery using electron microscopy and antibodies against epithelial membrane antigen (EMA), a marker for perineurial cells that has thus far not been applied to the study of differentiating cells in nerve tubulation systems. Initially, a thin cord consisting of fibrin bridged the gap between the stumps. At 7 days, longitudinal cells had migrated from both stumps toward the center of the tubes on the surface of the fibrin cord. These cells were immunoreactive with anti-EMA. At 12 days, ultrastructural features of perineurial cells (desmosomes, tight junctions, actin filaments with dense bodies, tonofilaments) were prominent in these cells. Subsequently, the gap was bridged through the perineurial tube by endothelial cells, pericytes, fibroblasts, Schwann cells, and axons. At 21 days, a single large nerve fascicle ensheathed by a mature perineurium was found between the stumps. Thus, the first cells to connect proximal and distal stumps in the investigated nerve regeneration silicon chamber system are perineurial cells. Through the tube formed by these cells, blood vessels and nerve fibers bridge the gap. Therefore, establishment of a perineurial connection between nerve stumps appears to be important in the sequence of events during nerve regeneration.The results of this study were presented in part at the Post Graduate Boerhaave Course: Brachial Plexus Injury, Leyden, March 25 and 26, 1993 [17] and at the 38th Annual Meeting of the Deutsche Gesellschaft für Neuropathologie and Neuroanatomie, Berlin, October 6–9, 1993 [27]     

INFLUENCE OF EXPERIMENTAL DIABETES ON THE MICROCIRCULATION OF INJURED PERIPHERAL NERVE - FUNCTIONAL AND MORPHOLOGICAL ASPECTS

Regeneration of diabetic axons has delays in onset, rate and maturation. It is possible that microangiopathy of vasa nervorum, the vascular supply of the peripheral nerve, may render an unfavorable local environment for nerve regeneration. We examined local nerve blood flow proximal and distal to sciatic nerve transection in rats with long-term (8 month) experimental streptozotocin diabetes using laser Doppler flowmetry and microelectrode hydrogen clearance polarography. We then correlated these findings, using in vivo perfusion of an India ink preparation, by outlining the lumens of microvessels from unfixed nerve sections. There were no differences in baseline nerve blood flow between diabetic and nondiabetic uninjured nerves, and vessel number, density, and area were unaltered. After transection, there were greater rises in blood flow in proximal stumps of nondiabetic nerves than in diabetic animals associated with a higher number, density, and caliber of epineurial vessels. Hyperemia also developed in distal stumps of nondiabetic nerves but did not develop in diabetic nerves. In these stumps, diabetic rats had reduced vessel numbers and smaller mean endoneurial vessel areas. Failed or delayed upregulation of nerve blood flow after peripheral nerve injury in diabetes may create a relatively ischemic regenerative microenvironment.