Intrathecal administration of nerve growth factor delays GAP 43 expression and early phase regeneration of adult rat peripheral nerve

Whether nerve growth factor (NGF) promotes peripheral nerve regeneration in vivo, in particular in adults, is controversial. We therefore examined the effect of exogenous NGF on nerve regeneration and the expression of GAP 43 (growth-associated protein 43) in adult rats. NGF was infused intrathecally via an osmotic mini-pump, while control rats received artificial cerebrospinal fluid. Two days after the infusion was initiated, the right sciatic nerves were transected or crushed, and the animals allowed to survive for 3 to 11 days. The right DRG, the right proximal stump of the transected sciatic nerve, and the posterior horn of the spinal cord were examined by Western blotting, immunohistochemistry, and electron microscopy. GAP 43 immunoreactivity in the NGF-treated animals was significantly lower than in the aCSF-treated controls. Electron microscopy showed that the number of myelinated and unmyelinated axons decreased significantly in the NGF-treated rats as compared with the controls. These findings are indicative that exogenous NGF delayed GAP 43 induction and the early phase of peripheral nerve regeneration and supports the hypothesis that the loss of NGF supply from peripheral targets via retrograde transport caused by axotomy serves as a signal for DRG neurons to invoke regenerative responses. NGF administered intrathecally may delay the neurons' perception of the reduction of the endogenous NGF, causing a delay in conversion of DRG neurons from the normal physiological condition to regrowth state.     

Regulation of myelination: axons not required for the biosynthesis of basal levels of the major myelin glycoprotein by Schwann cells in denervated distal segments of the adult cat sciatic nerve

The adult cat sciatic nerve was examined for Schwann cell biosynthesis of the major myelin glycoprotein (P0) in the distal segments after permanent nerve transection, where there is no axonal regeneration or myelin assembly. Endoneurial slices (intrafascicular tissue) from the distal segment of the desheathed cat sciatic nerves at 10 wk after transection and from normal adult desheathed brachial nerves were incubated with radioactive mannose; [3H]mannose incorporation into P0 was observed by fluorography after sodium dodecyl sulphate-pore gradient electrophoresis (SDS-PGE). Analysis of immune precipitates by SDS-PGE after incubation of an aliquot of an endoneurial fraction with rabbit antichick P0 gamma globulin verified that the [3H]mannose-labeled glycoprotein was P0. The level of incorporation of [3H]mannose into P0 and into other endoneurial glycoproteins in the normal brachial nerve from the adult cat was at substantially reduced levels compared with the transected nerve. Such incorporation was detectable by fluorography only after prolonged exposure to X-ray film (15 days). As a result, the level of biosynthesis of P0 in the normal adult cat is substantially reduced, suggesting that the extent of active myelination in the adult cat nerve is at a low level. Furthermore, Schwann cells are capable of continued synthesis of P0 in the adult, permanently transected nerve in the absence of axonal influence, suggesting that axonal association is not an absolute requirement for specifying myelin protein synthesis.     

Nerve growth activities in rat peripheral nerve

Nerve growth activities in rat sciatic nerves were assayed by recording the neuritic outgrowth from chick embryonic ganglia cultured in collagen gels beside nerve fragments for two days. Living nerve explants released activity that resembled nerve growth factor (NGF) in its effect on sympathetic ganglia and that was almost totally blocked by an antiserum to 2.5 S mouse NGF. Frozen and thawed specimens from normal nerves elicited responses from sympathetic ganglia that were only partially suppressed by anti-NGF and also induced neuritic outgrowth from ciliary ganglia. Thus, from observations on normal nerves, at least two agents promoting axonal extension in vitro were deduced to exist; one substance similar to NGF plus another, non-NGF factor. The level of NGF-like activity was low in killed segments of normal nerves but higher in autologous nerve grafts and degenerating nerves two days after grafting or cutting. However, one or two weeks after nerve transection, distal nerve segments contained little nerve growth activity of either kind. Furthermore, when endoneurial fragments from chronically denervated stumps were cultured, they appeared to have lost some of their capacity to produce NGF-like activity in vitro although the production of activity had, if anything, increased in the perineurial region. In summary, rat peripheral nervous tissue releases two or more soluble substances that stimulate neuritic outgrowth. The level of one or both activities in the endoneurium can be altered by manipulation of nerves in vivo.     

The persistence and possible externalization of axonal debris during Wallerian degeneration.

The sequence of changes in transected rat sciatic nerves were subjected to parallel ultrastructural and biochemical studies. Widespread granular disintegration of axoplasmic microtubules and neurofilaments occurred in the 24--72 hour interval following nerve transection. These changes were associated with a loss of neurofilament proteins and a marked enhancement of 53,000, 70--73,000 and 85,000 MW proteins in transected nerve. The emergence of prominent nerve proteins during the aftermath of axonal degeneration supports their derivation from axonal sources. These three proteins remained prominent components in transected nerves and comprised the major proteins found in 34-day transected nerves. Amorphous granular breakdown products from myelinated and unmyelinated nerve fibers were encountered with progressively decreasing frequency after 48 hours. This diminution of intracellular axonal debris was accompanied by the appearance and increasing prominence of amorphous granular deposits within the endoneurium of transected nerves. These endoneurial deposits became closely associated with collagen fibers and persisted as a prominent component in 34-, 80- and 120-day transected nerves. It is suggested that the amorphous endoneurial granular deposits arise in part from the externalization of granular axoplasmic breakdown products. Externalized axonal components could have important implications for tissue reaction to injury.     

Axonal signals regulate expression of glia maturation factor-beta in Schwann cells: an immunohistochemical study of injured sciatic nerves and cultured Schwann cells

Glia maturation factor-beta (GMF-beta) is a 17 kDa protein purified and sequenced from bovine brains. Using the monoclonal antibody G2-09 directed against GMF-beta, we previously demonstrated endogenous GMF-beta in astroblasts, Schwann cells, and their tumors in culture. In the present study, we have used indirect immunofluorescence microscopy with G2-09 to examine the effects of transection, crush, and regeneration of sciatic nerve on the expression of GMF-beta in Schwann cells in situ and to study the time course of GMF-beta induction in Schwann cells in vitro. For comparison, a parallel study was carried out with monoclonal antibodies directed against nerve growth factor (NGF) receptor. We found that (1) neither GMF-beta nor NGF receptor was detectable in intact sciatic nerves, (2) all Schwann cells of the distal segment of the transected nerve expressed GMF-beta as early as 3 d after axotomy that persisted up to 3 weeks, (3) axonal regeneration repressed the Schwann cell expression of GMF-beta, (4) isolated Schwann cells derived from rat sciatic and adult human sural nerves developed intracellular GMF-beta in culture following an initial lag period, and (5) the induction of Schwann cell NGF receptor coincided temporally with that of GMF-beta in the transected nerve and in culture. These results show that the expression of GMF-beta in Schwann cells, as is the case with the NGF receptor, is induced by the loss of the normal axon-Schwann cell contact. We propose that the induction of GMF-beta, as well as NGF receptor, in Schwann cells after nerve injury plays a role in axonal regeneration.     

Endoneurial microvasculature of chronically transected sciatic nerves in diabetic rats

To characterize the morphology of the endoneurial microvasculature of degenerating nerves under hyperglycemia, the morphology of endoneurial microvessels in transected sciatic nerves was examined in normal and streptozotocin-induced diabetic rats. Three months after transection, the fascicular area and median vascular luminal area at the proximal level of the distal stump were significantly larger in diabetic than in control animals, whereas the number of vessels per fascicle was the same in the two groups. Arterioles in various stages of development were found in the centrifascicular region in some transected nerves. Serial sections revealed that these vessels originated from transperineural arterioles. The frequency and magnitude of vascular wall thickening were both greater in diabetic rats. These results suggest that the endoneurial microvasculature responds abnormally to nerve injury under hyperglycemia.     

P0 gene expression in Schwann cells is modulated by an increase of cAMP which is dependent on the presence of axons.

The role of cAMP in the regulation of P0 gene expression was investigated in Schwann cells of normal, regenerated, and permanently transected rat sciatic nerve. Forskolin treatment of endoneurial segments of rat sciatic nerve resulted in increased cAMP and P0 mRNA levels in normal and regenerated nerves but not in permanently transected nerves, where axonal regeneration is prevented. This increase of cAMP and P0 mRNA occurred within 30 and 90 min, respectively. P0 mRNA levels in the endoneurial segment of the permanently transected nerve were not increased with dibutyryl cAMP. The Schwann cells of the permanently transected nerve, however, retained the ability to myelinate 15 embryonic day (E15) dorsal root ganglia (DRG) neuron and neurite networks cultured in vitro. P0 mRNA levels increased within 4 days in transected endoneurium segments cocultured with E15 DRG neurons and neurites and further increased in 21 day myelinating cocultures. Although cAMP was not detectable in 4 day cocultures, it increased to detectable levels in 21 day cultures, suggesting that cAMP is involved in the myelinating process. These results indicate that the presence of the axon is required for the observed increase of cAMP and P0 mRNA levels and suggest that the increase of cAMP occurs within the axon which then presumably activates a different Schwann cell second messenger pathway to induce P0 gene expression.     

Nerve growth factor and proprotein convertases furin and PC7 in transected sciatic nerves and in nerve segments cultured in conditioned media: Their presence in Schwann cells,macrophages, and smooth muscle cells

Synthesis of proteins such as nerve growth factor (NGF) is induced after nerve lesion. The NGF precursor (pro-NGF) requires a posttranslational processing by proprotein convertases to become active. In this report, we re-examine the localization of NGF protein and mRNA in injured nerve and show that the candidate pro-NGF convertases furin and PC7 colocalize with NGF in non-neuronal cells in nerve. By Northern blot analysis, 1.5-kb and 1.3-kb NGF mRNAs were shown to be increased in distal and immediately proximal nerve segments on days 1, 4, and 14 after lesion; by Western blot analysis, NGF proteins of high molecular weight were detected after injury. In vivo, two phases of NGF immunopositivity were observed, in macrophages and perivascular cells shortly after lesion and in endoneurial cells on day 1 and 4. To identify the cells containing NGF, nerve segments were incubated in serum-containing medium with or without conditioning by white blood cells isolated from the circulation. Both hybridization and immunoreactivity signals for NGF were elevated after incubation of nerve segments for 4 hours in conditioned media, so that cells with NGF immunoreactivity could be identified by antibodies to specific cell markers. In these nerve fragments, Schwann cells, perivascular smooth muscle cells, and macrophages contained NGF immunoreactivity. The concentration of furin and PC7 mRNA also increased in lesioned nerves. By immunocytochemical investigation of nerve explants, furin and PC7 were detected in endoneurial cells, macrophages and perivascular cells and were colocalized with NGF. These in vitro and in vivo findings suggest that both furin and PC7 are associated with NGF in several cell types of the sciatic nerve and, hence, may be implicated in intracellular processing of pro-NGF. J. Comp. Neurol. 403:471–485, 1999. © 1999 Wiley-Liss, Inc.     

Cultured rat Schwann cells express low affinity receptors for nerve growth factor

Schwann cell cultures prepared from postnatal Sprague-Dawley rat sciatic nerves were used to demonstrate the presence of specific receptors for the beta-subunit of nerve growth factor (NGF) on rat Schwann cells. Indirect immunofluorescence microscopy with a monoclonal antineuronal NGF receptor (NGFR) antibody indicated that NGFR antigen was expressed on the surface of Schwann cells but not of endoneurial fibroblasts. Studies with 125I-NGF confirmed this distribution of NGFR in the cultures and showed that the Schwann cell NGFR had a single NGF binding affinity (Kd of 1.8 x 10(-9) M). 125I-NGF binding by the cultured Schwann cells increased with time in vitro, reaching a plateau level on the 4th day, but decreased with increasing age, reaching 40% of the neonatal value in Schwann cells isolated from 12-day-old rats. Treatment of the cultures with NGF did not alter Schwann cell phenotype, survival or proliferation.     

Laminin in rat sciatic nerve undergoing Wallerian degeneration. Immunofluorescence study with laminin and neurofilament antisera

Immunofluorescence with laminin antisera revealed a striking change in the localization of this basal membrane glycoprotein in rat sciatic nerve as a result of Wallerian degeneration. The staining was confined to the endoneurium in normal sciatic nerve and during the first days of degeneration. On day 11 endoneurial tubes were no longer identified in the distal stump of crushed nerves or of nerves that had been transected and tightly ligated to prevent regeneration. In both crushed and ligated nerves proliferating Schwann cells forming the cell-bands of Büngner were intensely laminin positive. With double-labeling experiments, laminin and neurofilament antisera revealed similar but not identical staining patterns in crushed nerves, which suggests a close relation between laminin and regenerating axons. Crushed nerves had recovered their normal appearance 18 days after operation while anti-laminin reactivity was decreased in parts of ligated nerves undergoing fibrosis. The localization of laminin in reactive Schwann cells was confirmed by electron microscopy using the indirect immunoperoxidase procedure. Axons did not contain reaction product.     

Macrophages contribute to the maintenance of stable regenerating neurites following peripheral nerve injury

Normal adult uninjured nerve is unable to support axonal regeneration. We have studied the mechanisms underlying the regeneration of peripheral nerve by culturing adult mouse dorsal root ganglia (DRG) explants on unfixed, longitudinal cryosections of either the uninjured sciatic nerve or the distal segment of the transected sciatic nerve. We found that, initially, DRG grew vigorously on cryosections of both uninjured and postinjury sciatic nerves. However, the neurites began to degenerate shortly after contact with the uninjured nerve, whereas those growing on postinjury nerve substrate remained healthy for up to 9 days in culture. This ability to support stable outgrowth peaked at 8 days, gradually decreased by 10 days, and disappeared by 20 days after injury. Macrophages appeared in the distal segment by 4 days postinjury and had infiltrated its entire length by 8 days. Uninjured nerve cryosections could be rendered supportive of stable outgrowth by preincubation with macrophage-conditioned medium or by brief trypsinization. The activity of the macrophage-conditioned medium was augmented upon activation of macrophages. Together these findings suggest that the environment of the sciatic nerve undergoes a transformation during Wallerian degeneration such that it becomes transiently supportive of the stable outgrowth of neurites. This transformation may be mediated by a proteolytic activity, generated by activated macrophages, that removes a putative "degeneration signal" protein normally present in the adult nerve and thus contributes to the maintenance of stable regenerating neurites.     

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.     

Long-term endoneurial changes after nerve transection

Summary Long-term endoneurial changes in the distal stump of transected rat sciatic nerve were examined from 8 to 50 weeks after nerve transection. The morphological alterations were followed both in nerves which were allowed to regenerate and in nerves in which regeneration was prevented by suturing. The nerves prevented from regenerating showed markedly atrophied Schwann cell columns after 20 weeks and a disappearance of some Schwann cell columns after 30 weeks. The surrounding endoneurial fibroblast-like cells gradually lost their delicate cytoplasmic extensions and formed rough fascicles around numerous shrunken Schwann cell columns or around areas from which Schwann cells had apparently disappeared. Inside the fascicles, the Schwann cell loss was replaced by collagen fibrils or occasionally, by a dense accumulation of microfibrils. The loss of endoneurial cytoplasmic processes continued up to 50 weeks, leaving behind patches of thin fibrils around numerous shrunken Schwann cell columns or around collagenous areas where Schwann cells were lost. The endoneurial matrix showed presence of thin 25- to 30-nm collagen fibrils close to shrunken Schwann cell columns up to 50 weeks but in areas with advanced degeneration a shift towards regular 50- to 60-nm collagen fibrils occurred. The degenerated areas resembled those described in Renaut bodies and neurofibromas. Despite suturing of transected nerves to prevent sprouting, occasional regenerating sprouts were noted in the Schwann cell columns. These axons were surrounded in a sheath-like fashion by pre-existing endoneurial cell fascicles covered by a basal lamina. In the reinnervating nerves the endoneurial space gradually lost its compartmentized structures consisting of collagen fibrils and endoneurial fibroblast-like cells. After 20 weeks the endoneurial cells were inconspicuous and the extracelluar matrix consisted mainly of 50- to 60-nm collagen fibrils. During axonal growth and maturation, Schwann cells containing unmyelinated axons surrounded large, myelinated axons in a collar-like fashion. Close to these collars of Schwann cells, thin 25- to 30-nm collagen fibrils were noted in focal areas, even after 50 weeks. Occasionally, numerous clusters of regenerating axonal sprouts were noted in the perineurium. These were surrounded by multiple layers of cells possessing basal lamina. The present results show that after nerve transection the distal stump of the severed nerve shows dynamic changes in the endoneurial space, especially in nerves where reinnervation is prevented. The endoneurial fascicles around occasional axonal sprouts in sutured nerves, representing possibly a delayed type of regeneration, show that axons have a strong ability to grow but on the other hand endoneurial structures are unable to respond normally to axonal growth after advanced degeneration.     

Dissociated neurons regenerate into sciatic but not optic nerve explants in culture irrespective of neurotrophic factors

Explants of adult or 10-day-old rat sciatic and optic nerves were implanted as "bridges" through a silicon grease seal in a three-compartment chamber culture system, leading from a narrow center chamber to two adjacent side chambers. Dissociated newborn rat sympathetic or sensory neurons were plated into the center chamber and grown in the presence of optimal concentrations of nerve growth factor (NGF). By light microscopy, nerve fibers were seen to grow out of the sciatic nerve explants in the side chambers after 2 to 3 weeks. Electron microscopy showed large numbers of axons present inside the sciatic nerves, irrespective of the presence and number of living Schwann cells. Besides their tendency to fasciculate, axons grew with high preference on Schwann cell membranes and the Schwann cell side of the basal lamina, a situation identical to in vivo regeneration. In contrast to the sciatic nerves, no axons could be found under any condition in the optic nerves. This result points to the existence of extremely poor, non-permissive substrate conditions in the differentiated optic nerves which cannot be overcome by the strong fiber outgrowth-promoting effects of NGF.     

Quantitation of Schwann cells and endoneurial fibroblast-like cells after experimental nerve trauma

Summary Schwann cells and endoneurial fibroblastlike cells were quantitatied for 30 weeks in both nonregenerating and freely regenerating, transected rat sciatic nerve. Immunocytochemical recognition of S-100 protein was used as a marker for Schwann cells and other immunocytochemical and histological methods in the differentiation of S-100 protein-negative endoneurial cells in cross sections of the distal stump 10 mm distal to the site of transection. A marked increase in the total number of cells was observed during the first 4 weeks after the injury in both operative groups. The quantitative relationships between cell populations remained essentially the same as in normal nerves, although the proliferation of the S-100 protein-negative cell population was proportionately slightly stronger when compared to the number of these cells in normal nerves. After the initial proliferation, a gradual decrease occurred in the total number of cells per cross section. This was most marked in the non-regenerating nerves, whereas in the regenerating nerves the decrease in cell number ceased at 16 weeks. The number of Schwann cells was 3.5 times as high as in the control nerves in this phase. The method used in the present study is less laborious than morphometry employing electron microscopy. Furthermore, electron microscopic characteristics of endoneurial cells are not always reliable after nerve trauma, because normal anatomical relationships have become disturbed. This study demonstrates that S-100 protein immunocytochemistry is useful in the study of traumatic lesions of peripheral nerve.Supported by a grant (to V. S.) from the Research and Science Foundation of Farmos and by institutional grants from the Sigrid Jusélius Foundation and the Turku University Foundation     

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.     

Induction of myelin genes during peripheral nerve remyelination requires a continuous signal from the ingrowing axon

The effect of a permanent transection on myelin gene expression in a regenerating sciatic nerve and in an adult sciatic nerve was compared to establish the degree of axonal control exerted upon Schwann cells in each population. First, the adult sciatic nerve was crushed, and the distal segment allowed to regenerate. At 12 days post-crush, the sciatic nerve was transected distal to the site of crush to disrupt the Schwann cell-axonal contacts that had reformed. Messenger RNA (mRNA) levels coding for five myelin proteins were assayed in the distal segment of the crush-transected nerve after 9 days and were compared to corresponding levels in the distal segments of sciatic nerves at 21 days post-crush and 21 days post-transection using Northern blot and slot-blot analysis. Levels of mRNAs found in the distal segment of the transected and crush-transected nerve suggested that Schwann cells in the regenerating nerve and in the mature adult nerve are equally responsive to axonal influences. The crush-transected model allowed the genes that were studied to be classified according to their response to Schwann cell-axonal contact. The levels of mRNAs were (1) down-regulated to basal levels (PO and MBP mRNAs), (2) down-regulated to undetectable levels (myelin-associated glycoprotein mRNAs), (3) upregulated (mRNAs encoding 2′3′-cyclic nucleotide phosphodiesterase and β-actin), or (4) not stringently controlled by the removal of Schwann cell-axonal contact (proteolipid protein mRNAs). This novel experimental model has thus provided evidence that the expression of some of the important myelin genes during peripheral nerve regeneration is dependent on continuous signals from the ingrowing axons. © 1993 Wiley-Liss, Inc.     

Blood-nerve barrier in the frog during wallerian degeneration: Are axons necessary for maintenance of barrier function?

Blood-nerve barrier tissues (endoneurial blood vessels and perineurium) of the frog's sciatic nerve were studied during chronic Wallerian degeneration to determine whether barrier function depends on the presence of intact axons. Sciatic nerves of adult frogs were transected in the abdominal cavity; the ends were tied to prevent regeneration and the distal nerve stumps were examined. Vascular permeabilities to horseradish peroxidase and to [14C]sucrose increased to day 14, returned toward normal levels by 6 weeks, and continued at near normal levels to 9 months. Perineurial permeabilities to the tracers increased by day 10 and remained elevated at 9 months. Proliferation of perineurial, endothelial, and mast cells occurred between 3 days and 6 weeks, resulting in an increased vascular space (measured with [3H]dextran) and number of vascular profiles. The perineurium increased in thickness and the mast cells increased in number. This study indicates that during Wallerian degeneration of the frog's sciatic nerve there is 1) a transitory increase in vascular permeability distal to the lesion, that is related to changes within the endoneurium; 2) an irreversible increase in permeability of the perineurium, which begins later than that seen in the endoneurial blood vessels; and 3) proliferation of non-neuronal components in the absence of regenerating neuronal elements. The results indicate that maintenance of vascular integrity does not require the presence of axons in the frog's peripheral nerve, whereas perineurial integrity and barrier function are affected irreversibly by Wallerian degeneration.     

Nerve growth factor enhances regeneration through silicone chambers

The effect of exogenous NGF on axonal growth across a gap between sectioned ends of a sciatic nerve within silicone chambers was examined in Sprague-Dawley rats. After nerve section and surgical implantation, silicone chambers were filled with either a 1 mg/ml nerve growth factor (NGF)/saline solution (experimental) or a normal saline solution (control). Four weeks after surgery, the regenerated nerves from within the silicone chambers were dissected and fixed for histological studies at both light microscopic and ultrastructural levels. Morphological analysis of the nerves showed no difference between the NGF-treated and control groups in the size of the regenerated nerves within the chambers or in the diameters of myelinated axons. Total myelinated axonal counts were determined from within the distal chamber. NGF significantly increased the number of myelinated axons that grew into the distal end of the chamber (2126 +/- 437 NGF/saline; 1064 +/- 268 saline; P less than 0.05 Student's t test). Counts of the unmyelinated axons from the distal nerve segment from the two groups were not different. Myelin sheath thickness was 58% greater in the NGF-treated group compared with that in the saline group. There was no difference between the two groups in the size-frequency spectra of the diameters of the myelinated axons in the distal segment. The NGF/saline group showed a more mature-appearing regenerated nerve based on the percentage of myelinated axons, thickness of the myelin sheaths, and development of internal organization (e.g., amount of endoneurial collagen fibers, ensheathment of unmyelinated axons by Schwann cells, and interfascicular patterns).(ABSTRACT TRUNCATED AT 250 WORDS)