Bcl-2 deficiency deprives peripheral nerves of neurotrophic activity against injured optic nerve

Optic nerve injury leads to retinal ganglion cell apoptosis, thus preventing fiber regeneration. Peripheral nerve grafts are known to promote survival and regeneration in injured adult mammalian central nervous system, including optic nerve, but the mechanisms of their activity remain unclear. It is likely that they attenuate the apoptotic cascade triggered by axotomy in retinal ganglion cells. The aim of this work was to examine the role of the antiapoptotic gene bcl-2 in the optic nerve regeneration induced by such grafts. Experiments were carried out on bcl-2-deficient and wild-type mice. We have reported previously that predegeneration markedly enhances neurotrophic activity of peripheral nerve grafts, so we applied both predegenerated and non-predegenerated implants to the transected optic nerves. We studied the neurotrophic effects of bcl-2-deficient grafts on wild-type and bcl-2 knock-out optic nerves, as well as wild-type grafts on both strains of mouse optic nerves. After application of fluorescent dye to the end of the graft, we counted the stained retinal ganglion cells. Predegenerated wild-type grafts promoted survival and outgrowth of retinal ganglion cells axons in both types of mice. By contrast, non-predegenerated and predegenerated bcl-2-deficient grafts induced little or no regeneration in the optic nerves. These results indicate that the lack of bcl-2 gene does not deprive retinal ganglion cells of their regenerative potential. At the same time, we found that bcl-2 knock-out dispossesses peripheral nerves of their neurotrophic activity.     

Peripheral nerve-stimulated macrophages simulate a peripheral nerve-like regenerative response in rat transected optic nerve

We have previously demonstrated that the failure of the mammalian central nervous system (CNS) to regenerate following axonal injury is related to its immunosuppressive nature, which restricts the ability of both recruited blood-borne monocytes and CNS-resident microglia to support a process of repair. In this study we show that transected optic nerve transplanted with macrophages stimulated by spontaneously regenerating nerve tissue, e.g., segments of peripheral nerve (sciatic nerve), exhibit axonal regrowth at least as far as the optic chiasma. Axonal regrowth was confirmed by double retrograde labeling of the injured optic axons, visualized in their cell bodies. Transplanted macrophages exposed to segments of CNS (optic) nerve were significantly less effective in inducing regrowth. Immunocytochemical analysis showed that the induced regrowth was correlated with a wide distribution of macrophages within the transplanted-transected nerves. It was also correlated with an enhanced clearance of myelin, known to be inhibitory for regrowth and poorly eliminated after injury in the CNS. These results suggest that healing of the injured mammalian CNS, like healing of any other injured tissue, requires the partnership of the immune system, which is normally restricted, but that the restriction can be circumvented by transplantation of peripheral nerve-stimulated macrophages. GLIA 24:329–337, 1998. © 1998 Wiley-Liss, Inc.     

神经再生素和神经生长液联合应用对成年兔视神经挫伤后修复的影响

目的研究中药神经再生素（NRF）和神经生长液对成年兔视神经挫伤后修复的影响。方法16只成年兔随机分成实验组和对照组．每组8只。建立兔右眼视神经挫伤模型后．分别将载有0．06mL NRF（浓度为2g／L，实验组）或等量磷酸盐缓冲液（PBS）（对照组）的组织工程化神经移植于视神经损伤处；并向右眼玻璃体腔内注入0．02mL NRF（浓度为2g／L，实验组）或等量PBS（对照组）。实验组兔术后每日喂服神经生长液（5mL／kg），共6周。伤后1d、2周、8周进行闪光视觉诱发电位（FVEP）检查。挫伤后8周时作光镜和电镜检查观察视网膜神经节细胞（RGC）、视网膜神经纤维层和视神经的改变，同时用计算机图像处理系统作视神经纤维计数。结果术后8周时实验组致伤眼与未致伤眼FVEP幅值比为0．774±0．184，对照组为0．409±0．119，差异有显著性（P〈0．01）。术后8周时的光镜和电镜检查示：实验组RGC、视神经纤维的退变较对照组轻。两组视神经纤维计数分别为（15045±716．2）根／mm^2（实验组）和（7898±608．8）根／mm^2（对照组），差异有显著性（P〈0．01）。结论NRF和神经生长液联合应用能够增加RGC的存活，促进轴突的再生，因而对视神经挫伤后的修复、视功能的恢复具有一定的促进作用。     

Controlled release of nerve growth factor from a polymeric implant

Recent studies suggest that neurotrophic factors applied directly to brain tissue may enhance regeneration in the central nervous system. Biocompatible polymeric implants providing a controlled release of nerve growth factor (NGF) for over one month were developed. The released nerve growth factor stimulated neurite sprouting in cultured PC12 cells. While a model polymer with demonstrated biocompatibility was used for the present study, the methods can be extended to other polymer systems. Controlled release implants may be useful in the treatment of Alzheimer's disease.     

A new transorbital surgical approach to the rabbit's optic nerve

This work describes a surgical approach which establishes the rabbit's visual system as an experimental model for studying CNS regeneration. Using this model, the optic nerve, its cell bodies, and the axons, are easily accessible through an orbital approach, without the need for craniotomy and brain retraction. This surgical approach allows transplantation and 'wrap around' implantations of nerve segments from xenogeneic and syngeneic systems and diffusible substances derived from them, respectively. Furthermore, it enables studies aimed at determining deficiencies in mammalian CNS and investigating methods of augmentating mammalian CNS regeneration.     

A double-transgenic mouse used to track migrating Schwann cells and regenerating axons following engraftment of injured nerves

We propose that double-transgenic thy1-CFP(23)/S100-GFP mice whose Schwann cells constitutively express green fluorescent protein (GFP) and axons express cyan fluorescent protein (CFP) can be used to serially evaluate the temporal relationship between nerve regeneration and Schwann cell migration through acellular nerve grafts. Thy1-CFP(23)/S100-GFP and S100-GFP mice received non-fluorescing cold preserved nerve allografts from immunologically disparate donors. In vivo fluorescent imaging of these grafts was then performed at multiple points. The transected sciatic nerve was reconstructed with a 1-cm nerve allograft harvested from a Balb-C mouse and acellularized via 7 weeks of cold preservation prior to transplantation. The presence of regenerated axons and migrating Schwann cells was confirmed with confocal and electron microscopy on fixed tissue. Schwann cells migrated into the acellular graft (163+/-15 intensity units) from both proximal and distal stumps, and bridged the whole graft within 10 days (388+/-107 intensity units in the central 4-6 mm segment). Nerve regeneration lagged behind Schwann cell migration with 5 or 6 axons imaged traversing the proximal 4 mm of the graft under confocal microcopy within 10 days, and up to 21 labeled axons crossing the distal coaptation site by 15 days. Corroborative electron and light microscopy 5 mm into the graft demonstrated relatively narrow diameter myelinated (431+/-31) and unmyelinated (64+/-9) axons by 28 but not 10 days. Live imaging of the double-transgenic thy1-CFP(23)/S100-GFP murine line enabled serial assessment of Schwann cell-axonal relationships in traumatic nerve injuries reconstructed with acellular nerve allografts.     

Regeneration of axons from the adult rat optic nerve: influence of fetal brain grafts, laminin, and artificial basement membrane

After transection of the optic nerve of adult rats, most of the axons in the proximal stump die and the surviving ones are unable to regenerate into the distal optic nerve. Since the fetal brain has an inherent capacity to regenerate axons, we investigated whether fetal (E16) target regions of optic axons (thalamus and tectum) transplanted to the completely transected optic nerve of adult rats would promote axon regeneration. In control operated rats, axon growth beyond the site of transection was restricted to a few fibers that grew irregularly within the connective tissue scar. By contrast, in grafted animals directed outgrowth of optic axons toward the transplant started at 6 days postoperation (p.o.) and reached its maximum 15 days p.o. and later, when numerous single optic fibers and small axon fascicles had grown toward and into the graft, where they formed arborizations and terminal varicosities. Regenerating optic axons were further advanced than GFAP-positive strands of astroglia that emanated from the proximal optic nerve stump. Laminin immunoreactivity appeared at 6 days p.o. in the zone of reactive astroglia in the terminal part of the optic nerve stump. Later it showed a distribution complementary to the pattern of GFAP immunoreactivity, which it seemd to circumscribe. There was no unequivocal codistribution of laminin immunoreactivity with regenerating axons. In further experiments, target regions from different ontogenetic stages (E14 to neonate and adult) and nontarget regions (E16, cerebral cortex or spinal cord) were grafted to the optic nerve stump. With the exception of the adult grafts, all transplants had effects on axon regeneration comparable to those of E16 target regions. In order to test the effects of extracellular matrix molecules on axon regeneration, a basement membrane gel reconstituted from individual components of the Engelbreth-Holm-Sarcoma (EHS) sarcoma was implanted between proximal and distal optic nerve stumps. No axons were induced to regenerate by this matrix. Likewise, laminin adsorbed to nitrocellulose paper and implanted at the lesion site did not stimulate axon growth from the proximal optic nerve stump. These results indicate that fetal brain is able to induce and direct regrowth of axons from the optic nerve toward the graft across a substrate that is not composed of astroglia or basement membrane components like laminin. The directed growth of axons in the absence of a preformed substrate implies a chemotactic growth response along a concentration gradient mediated by neurotropic molecules released from the graft.     

Mononuclear cell proliferation and hyperplasia during Wallerian degeneration in the visual system of the goldfish in the presence or absence of regenerating optic axons

Patterns of proliferation and changes in non-neuronal cell number in the visual system of the goldfish have been quantitatively examined during optic axon regeneration after an optic nerve crush (ONC). In addition, in order to examine the effect of the regenerating axons on cellular responses in the visual pathways, we did a similar analysis of animals with the right eye removed (ER). Finally, we used double labeling protocols to demonstrate that the proliferating cells that we were counting were mostly phagocytic cells of the mononuclear lineage. In animals with an ONC, we observed an early burst of proliferation that peaked between 7 and 14 days after surgery in all parts of the visual system. In the optic tract, there was also a secondary rise that peaked at 21 days. Levels of proliferation returned to normal by 32 days postoperative in the tract and tectum, while they remained somewhat elevated in the optic nerve for at least 93 days. The total number of non-neuronal cells in the visual paths also rose to peak values between 7 and 14 days after ONC surgery. In the optic tract and tectum, the values fell rapidly after this time, while in the optic nerve, there was a secondary peak at 32 days after which values remained elevated for the duration of the experiment. As compared to animals with an ONC, enucleation resulted in elevated proliferation and hyperplasia at early postoperative intervals. However, because these differences occurred when axons had not yet regenerated into the affected structures, these data do not provide strong evidence for a direct effect of regenerating optic axons on the early cellular responses during Wallerian degeneration in the goldfish. In addition, in the tectum, there was an early increment in cell number that was not associated with elevated levels of proliferation. We believe that this increment represents immigration of resident microglia from other regions of the brain.     

Detection of molecules with nerve growth factor binding activity in medium conditioned by L-929 fibroblasts

L-929 fibroblasts (L cells) secrete a high molecular weight form of nerve growth factor (NGF) that is non-covalently bound and contains as part of its structure a molecule similar, if not identical, to beta-NGF in mouse submandibular glands. The other components of the NGF complex have not been characterized. In this study we used radiolabeled beta-NGF as a probe to detect molecules with NGF binding activity in L cell conditioned medium. The L cell NGF complex was dissociated at low pH, or with denaturants or detergents, and allowed to reassociate in the presence of 125I-beta-NGF. Radioactivity became associated with a complex that eluted in a high molecular weight volume on columns of Sephadex G-200 and Sephacryl S-500. Incorporation was saturable and did not occur under non-dissociating conditions. The complex was affinity cross-linked and studied by SDS gel electrophoresis. Radiolabeled molecules were observed with molecular weights of 151,000, 56,000 and 53,000. Labeling did not occur in the presence of excess unlabeled NGF or when cross-linking was done with fetal bovine serum, indicating that binding is specific and that binding activity is not derived from serum added to tissue culture medium. Solutions containing 7S NGF from mouse salivary glands were cross-linked by similar procedures but different banding patterns were observed. The data show that NGF binding molecules dissimilar from those in salivary glands are present in L cell conditioned medium.     

Effects on the growth of damaged ganglion cell axons after peripheral nerve transplantation in adult hamsters

After transplantation of autologous sciatic nerve segments into the retina of adult hamsters for 1-2 months, retrograde labelling with horseradish peroxidase demonstrated a population of ganglion cells situated peripheral to the graft. If an additional lesion was placed between the insertion of the graft and the optic disc at the same time as transplantation, in addition to labelled cells situated peripheral to the graft, retrograde labelling with horseradish peroxidase demonstrated a population of labelled neurons located between the graft and the optic disc which was not observed in animals without the additional lesion. Since the axons of this population of cells would have to turn around away from their normal course towards the optic disc and travel for about 1.5 mm in order to grow into the graft, it suggests that the peripheral nerve graft might play an active role in attracting and/or guiding damaged ganglion cell axons to grow into it.     

Temporal parameters of low energy laser irradiation for optimal delay of post-traumatic degeneration of rat optic nerve

Compression injury of a central nerve results in its degeneration with irreversible loss of function due to the inability of the mammalian central nervous system (CNS) to regenerate. In contrast, the CNS of lower vertebrates has a high capacity to regenerate. Recently, low energy laser irradiation was shown to attenuate degeneration in injured CNS nerves. The optic nerves of rats were subjected to moderate crush, calibrated so that some electrophysiological activity was preserved. The nerves were then subjected to low energy laser irradiation (10.5 mW, 2 min daily) for various periods. The electrical activity of the nerves, distal to the site of injury, was determined by measuring the compound action potential at the termination of the experiment. Two weeks of irradiation begun immediately after injury and continued daily thereafter, resulted in a compound action potential which was significantly higher (mean ± S.E.M. 1856 ± 535 μV) than that of non-irradiated injured nerves (351 ± 120 μV). The effect was temporary and subsided within a week. This two-week irradiation was slightly more effective than a treatment lasting one week (1406 ± 225 μV) and was significantly more effective than 4 days of irradiation (960 ± 133 μV). The number of treatments is therefore important. The time at which the treatment commences relative to the injury is also critical. Irradiation initiated two hours after the crush was about half as effective as immediate irradiation (810 ± 42 μV). No apparent effect was evident when the laser was applied for the first time 5 h, or longer, after the crush. Pretreatment with a laser irradiation immediately before the crush was an effective (1430 ± 281 μV) as irradiation soon after the injury. It is important to note that the laser effect is demonstrable only in moderately injured nerves. Severely injured nerves are not affected by treatment even after prolonged (3 and 4 weeks) irradiation. Our results further suggest that low energy laser irradiation only delays degenerative processes but does not prevent them. Optimal results are obtained when nerve crush is moderate, and the irradiation is initiated immediately before or soon after the injury and continued for at least one week.     

Remodeling of adult sensory axons in the superior cervical ganglion in response to exogenous nerve growth factor

In previous studies, we found that a 2-week in vivo intracerebroventricular infusion of nerve growth factor (NGF) elicited a sprouting response by sympathetic perivascular axons associated with the intradural segment of the internal carotid artery. We hypothesized that NGF infused into the ventricular system would be internalized by responsive sympathetic cerebrovascular axons, retrogradely transported to parent cell bodies in the superior cervical ganglion (SCG), and subsequently released into the local ganglionic environment. Because fibers exhibiting immunoreactivity for calcitonin gene related peptide (CGRP) have been localized in the SCG, we used immunohistochemical methods to investigate whether a response by CGRP-immunoreactive axons in the SCG occurred following the proposed transport to and release of exogenous NGF in the ganglion. In consecutive tissue sections of the SCG stained for either CGRP or NGF, we found CGRP pericellular 'baskets' surrounding identified NGF-immunoreactive cell bodies. Nerve growth factor infusion resulted in a significant increase both in the number of CGRP pericellular baskets and in NGF-immunoreactive cell bodies. A significant positive correlation (r=0.95, P<0.05) between the pericellular baskets and NGF-immunoreactive cell bodies was observed, suggesting that intracranial projection neurons in the SCG released infused NGF (or possibly a converted signal) into the local ganglionic environment to elicit remodeling of CGRP fibers to form pericellular baskets. These findings were confirmed in sections double labeled for NGF and CGRP immunoreactivity. This remodeling suggests that exogenous NGF may mediate retrograde transneuronal plasticity, allowing for future in vivo examinations of the mechanisms involved in neurotrophin transport and release.     

Vascular permeability associated with axonal regeneration in the optic system of the goldfish

Summary An association between axonal regeneration and failure of the blood-brain barrier to plasma proteins has been studied in the goldfish. Vascular permeability was examined by fluorescence microscopy following injection of rhodamine B-labelled bovine serum albumin. Axonal regeneration was studied in adjacent silver-stained sections. Following transection of axons by crushing one optic nerve, it was found that a zone of increased vascular permeability accompanied the advancing front of regenerating axons through the optic nerve, chiasma and tract and into the stratum opticum of the tectum.These observations lend support to a hypothesis in which it is postulated that axons are able to regenerate only when plasma proteins are available to their growth-cones. However, it is also possible that the increased permeability is a consequence rather than a cause of the presence of regenerated axons.     

Growth stimulation and chemotropic attraction of rat retinal ganglion cell axons in vitro by co-cultured optic nerves, astrocytes and astrocyte conditioned medium

The effects of explants of optic nerves of different ontogenetic ages (P0-P14, adult), and of cultured astrocytes of various ages on the neurite regeneration of rat retinal ganglion cells (RGC) were assessed in vitro, using a three-dimensional culture system which allows the co-cultivation of various explants. Both co-cultured P0-P12 optic nerves and astrocyte cultures from P2 cerebral cortex stimulated the regeneration of neurites from the retinal explants after 3 days in culture. By contrast, P14 and older explants of the optic nerve, astrocytes from P17 optic nerve and astrocytes that had previously been grown in culture for more than 6 weeks had no effect on RGC neurite outgrowth. Moreover, both the P0–P12 optic nerve explants and the astrocytes from P2 cerebral cortex also seemed to have a chemotropic effect on the regenerating neurites, because the latter were longer on the side facing the co-explantat. The absence of a cellular bridge between retinal and optic nerve explants suggests that the effects are mediated by astroglia-derived diffusible neurite growth promoting factors. Accordingly, astrocyte-conditioned medium from P2 astrocytes also stimulated the outgrowth of neurites from the retinal explants. These findings show that immature astrocytes of a limited ontogenetic period release as yet unknown diffusible neurite growth-promoting factors which stimulate the regeneration of neurites from retinal explants.     

Distribution of the phosphorylated form of microtubule associated protein 1B in the fish visual system during optic nerve regeneration

Microtubule associated proteins are a heterogeneous group of proteins that have been implicated in regulating microtubule stability. They play an important role in the organisation of the neuronal cytoskeleton during neurite outgrowth, plasticity and regeneration. The fish visual system presents a considerable degree of plasticity. Thus, the retina grows continually throughout life and the optic nerve regenerates after crush. In the present study, we compared the distribution of the microtubule associated protein 1B in its phosphorylated form (MAP1B-phos) in the normal adult fish visual system with that observed during optic nerve regeneration after adult optic nerve crush using a specific monoclonal antibody mAb-150. Expression of MAP1B-phos was observed in some ganglion cell somata and in developing, growing axons within the control optic nerve. Few immunoreactive terminals were seen in the control optic tectum. After optic nerve crush, we found additional MAP1B-phos expression in regenerating axons throughout the visual system. Our results demonstrate that MAP1B-phos is present in growing and regenerating axons of fish retinal ganglion cells, which suggests that the phosphorylated form of MAP1B may play an important role in developmental and regeneration processes within the fish central nervous system.     

Characterization of the optic nerve and retinal ganglion cell layer in the dysmyelinated adult Long Evans Shaker rat: evidence for axonal sprouting

Myelin in the central nervous system (CNS) is hypothesized to help guide the growth of developing axons by inhibiting sprouting of aberrant neurites. Previous studies using animal models lacking CNS myelin have reported that increasing capacity for sprouting axons is negatively correlated with the degree of myelination. In the present study, we investigated the optic nerves of the recently identified Long Evans Shaker (LES) rat with prolonged dysmyelination of adult axons to determine whether the lack of myelin basic protein (MBP) in adult LES rats could manifest as increases in the population of CNS axons. We observed numerous small, unmyelinated axon profiles (<0.3 microm in diameter) clustered in bundles alongside normal caliber axons in dysmyelinated LES rats but not in normal myelinated Long Evans (LE) rats. These putative axon profiles resembled sprouting axons previously described in the CNS. Moreover, the high number of small putative axon profiles could not be accounted for by any significant increases in the number of ganglion cells and displaced amacrine cells in the ganglion cell layer when compared with normal rats as evaluated by using a variety of techniques. This finding suggests that the observed clusters of putative axon profiles were not due to developmental abnormalities in the retina but to the lack of myelin in the optic nerves of LES rats. The adult LES rat, therefore, may serve as a useful model to study the role of myelin in regulating axon development or axon regeneration after CNS injury in the adult mammalian system.     

Further observations on the response of the peripheral and central nervous system of the rabbit to sodium diethyldithiocarbamate

Summary Rabbits were given NaDDC by mouth. Groups of 2 treated and 2 control animals were killed after 4 and 6 weeks and 3 treated and 3 control rabbits were killed after 9 weeks of administration. The peripheral and central nervous system and muscles were examined. Nerve fibre degeneration was seen in the long spinal tracts after 4 weeks of NaDDC administration and was severe after 9 weeks. Nerve fibre diameter measurements showed that there was loss of large diameter fibres in the peripheral nervous system after 9 weeks of administration. The relationship of these changes to those produced by organophosphorous compounds is discussed.     

Herpes simplex virus,type 1 invasion of the rabbit and mouse nervous systems revealed by in situ hybridization

Summary Using a³H-labelled virion DNA probe applied to tissue sections, we have previously identified the precise microscopic anatomical localtion of herpes simplex virus (HSV) during the acute and latent stages of infection of the mouse trigeminal ganglia and central nervous system (CNS). In the present investigation, we compared the mouse and the rabbit with respect to their ability to support acute and latent infections of trigeminal ganglionic and central nervous system neurons. We found that HSV-1, strain F, produced acute and latent infection of trigeminal ganglion cells in both mice and rabbits; however, lower levels of HSV-1 RNA were expressed in rabbit neurons as compared to mouse neurons, and many fewer neurons of the rabbit supported an acute infection than in the mouse. Studies of the trigeminal system within the CNS revealed that HSV-1 established latency more readily in the mouse than in the rabbit. The histopathology observed in acutely infected rabbit brain was less intense and less widespread than in mouse brain.Supported by the Medical Research Service of the Veterans Administration and by Institutional and Biomedical Research Support Funds awarded by the Dean of the Medical School, University of Utah     

Role of locus coeruleus heme oxygenase-carbon monoxide-cGMP pathway during hypothermic response to restraint

Central heme oxigenase-carbon monoxide (HO-CO) pathway has been shown to play a pyretic role in the thermoregulatory response to restraint. However, the specific site in the central nervous system where CO may act modulating this response remains unclear. LC is rich not only in sGC but also in heme oxygenase (HO; the enzyme that catalyses the metabolism of heme to CO, along with biliverdin and free iron). Therefore, the possible role of the HO-CO-cGMP pathway in the restraint-induced-hypothermia by LC neurons was investigated. Body temperature dropped about 0.7 degrees C during restraint. ZnDPBG (a HO inhibitor; 5 nmol, intra-LC) prevented the hypothermic response during restraint. Conversely, induction of the HO pathway in the LC with heme-lysinate (7.6 nmol, intra-LC) intensified the hypothermic response to restraint, and this effect was prevented by pretreatment with ODQ (a sGC inhibitor; given intracerebroventricularly, 1.3 nmol). Taken together, these data suggest that CO in the LC produced by the HO pathway and acting via cGMP is implicated in thermal responses to restraint.