Lesion-associated expression of transforming growth factor-beta-2 in the rat nervous system: evidence for down-regulating the phagocytic activity of microglia and macrophages

The mechanisms that control the phagocytic activities of microglia and macrophages during disorders of the nervous system are largely unknown. In the present investigation, we assessed the functional role of transforming growth factor (TGF)beta2 in vitro and studied TGFbeta-2mRNA and protein expression in two CNS lesion paradigms in vivo characterized by fundamental differences in microglia/macrophage behaviour: optic nerve crush exhibiting slow, and focal cerebral ischemia exhibiting rapid phagocytic transformation. Furthermore, we used sciatic nerve crush injury as a PNS lesion paradigm comparable to brain ischemia in its rapid phagocyte response. In normal and degenerating optic nerves, astrocytes strongly and continuously expressed TGF-beta2 immunoreactivity. In contrast, TGF-beta2 was downregulated in Schwann cells of degenerating sciatic nerves, and was not expressed by reactive astrocytes in the vicinity of focal ischemic brain lesions during the acute phagocytic phase. In line with its differential lesion-associated expression pattern, exogenous TGF-beta2 suppressed spontaneous myelin phagocytosis by microglia/macrophages in a mouse ex vivo assay of CNS and PNS Wallerian degeneration. In conclusion, we have identified TGF-beta2 as a nervous system intrinsic cytokine that could account for the differential regulation of phagocytic activities of microglia and macrophages during injury.     

Comparison of matrix metalloproteinase expression during Wallerian degeneration in the central and peripheral nervous systems

The matrix metalloproteinases (MMPs) are a large family of zinc-dependent enzymes which are able to degrade the protein components of the extracellular matrix. They can be placed into subgroups based on structural similarities and substrate specificity. Aberrant expression of these destructive enzymes has been implicated in the pathogenesis of immune-mediated neuroinflammatory disorders. In this study we investigate the involvement of MMPs, from each subgroup, in Wallerian degeneration in both the central and peripheral nervous systems. Wallerian degeneration describes the process initiated by transection of a nerve fibre and entails the degradation and removal of the axon and myelin from the distal stump. A similar degenerative process occurs as the final shared pathway contributing to most common neuropathies. MMP expression and localisation in the peripheral nervous system are compared with events in the CNS during Wallerian degeneration. Within 3 days after axotomy in the peripheral nervous system, MMP-9, MMP-7 and MMP-12 are elevated. These MMPs are produced by Schwann cells, endothelial cells and macrophages. The temporospatial expression of activated MMP-9 correlates with breakdown of the blood-nerve barrier. In the CNS, 1 week after optic nerve crush, four MMPs are induced and primarily localised to astrocytes, not microglia or oligodendrocytes. In the degenerating optic nerve, examined at later time points (4, 8, 12 and 18 weeks), MMP expression was down-regulated. The absence of MMPs in oligodendrocytes and mononuclear phagocytes during Wallerian degeneration may contribute to the slower removal of myelin debris observed in the CNS. The low level of the inactive pro-form of MMP-9 in the degenerating optic nerve may explain why the blood-brain barrier remains intact, while the blood-nerve barrier is rapidly broken down. We conclude that the difference in the level of expression, activation state and cellular distribution of MMPs may contribute to the different sequence of events observed during Wallerian degeneration in the peripheral compared to the CNS.     

纤溶酶原激活剂及其相关分子在小鼠视神经损伤再生中的变化

目的:检测细胞外基质(ECM)中各蛋白酶在视神经损伤后的变化,分析ECM蛋白酶活性的变化与小鼠视神经损伤和损伤后再生之间的关系。方法:本实验采用建立小鼠视神经钳夹伤的动物模型,用WesternBlot方法检测小鼠视神经损伤后不同时间点神经丝(NF)、金属基质蛋白酶-9(MMP-9)、IgG的表达变化。同时采用原位酶谱分析法检测纤溶酶原激活剂(PA)活性在视神经损伤后各阶段的变化,并分析这种变化与纤维蛋白(原)沉积、髓鞘碎片清除等影响神经再生的因素之间的关系。结果:小鼠视神经损伤后发生进行性Wallerian变性,血-神经屏障(BNB)修复迟缓,沉积的纤维蛋白(原)于损伤后第2d清除。MMP-9在损伤后2d达到高峰,以后仍呈现高水平的表达,且均以前体形式出现。PA活性在损伤后第7d达到高峰,并持续至第28d。结论:视神经损伤后,损伤部位BNB重建、PA激活、纤维蛋白(原)的清除以及MMP-9的表达与周围神经截然不同,正是由于微环境的迥然差异,导致了中枢神经系统(CNS)髓鞘碎片清除不利、轴突再生障碍。     

Differential recruitment of CD8+ macrophages during Wallerian degeneration in the peripheral and central nervous system

The strong macrophage response occurring during Wallerian degeneration in the peripheral but not central nervous system has been implicated in tissue remodeling and growth factor production as key requirements for successful axonal regeneration. We have previously identified a population of CD8+ phagocytes in ischemic brain lesions that differed in its recruitment pattern from CD4+ macrophages/microglia found in other lesion paradigms. In the present study we show that crush injury to the sciatic nerve induced strong infiltration by CD8+ macrophages both at the crush site and into the degenerating distal nerve stump. At the crush site, CD8+ macrophages appeared within 24 hours whereas infiltration of the distal nerve parenchyma was delayed to the second week. CD8+ macrophages were ED1+ and CD11b+ but always MHC class II-. Most CD8+ macrophages coexpressed CD4 while a significant number of CD4+/CD8-macrophages was also present. Expression of the resident tissue macrophage marker ED2 was largely restricted to the CD4+/CD8- population. Following intraorbital crush injury to the optic nerve, infiltration of CD8+ macrophages was strictly confined to the crush site. Taken together, our study demonstrates considerable spatiotemporal diversity of CD8+ macrophage responses to axotomy in the peripheral and central nervous system that may have implications for the different extent of axonal regeneration observed in both systems.     

Microglia in tadpoles ofXenopus laevis: Normal distribution and the response to optic nerve injury

Summary We have studied the distribution of microglia in normalXenopus tadpoles and after an optic nerve lesion, using a monoclonal antibody (5F4) raised againstXenopus retinas of which the optic nerves had been cut 10 days previously. The antibody 5F4 selectively recognizes macrophages and microglia inXenopus. In normal animals microglia are sparsely but widely distributed throughout the retina, optic nerve, diencephalon and mesencephalon (other regions were not examined). After crush or cut of an optic nerve, or eye removal, there occurs an extensive microglial response along the affected optic pathway. Within 18 h an increase in the number of microglial cells in the optic tract and tectum can be detected. This response increases to peak at around 5 days after the lesion. At this time the nerve distal to the lesion contains many microglial cells; the entire optic tract is outlined by microglia, extended along the degenerating fibres; and the affected tectum shows a heavy concentration of microglia. This microglial response thereafter decreases and has mostly gone by 34 days. We conclude that the microglial response to optic nerve injury inXenopus tadpoles starts early, peaks just before the regenerating optic nerve axons enter the brain, and is much diminished by the time the retinotectal projection is re-established. The timing is such that the microglial response could play a major role in facilitating regeneration.     

MHC-positive, ramified macrophages in the normal and injured rat peripheral nervous system

Summary Resident endoneurial macrophages form a prominent, but little recognized component of the PNS. We have studied immunocytochemically the distribution, morphology and immunophenotype of endoneurial macrophages in several normal peripheral nerves of the rat. In addition, we investigated the macrophage response following crush injury of the sciatic nerve.Resident endoneurial macrophages had a ramified morphology with processes oriented parallel to the long axis of nerve fibres. They were positive for several monocyte/macrophage markers such as ED1, ED2 and the recently-described MUC 101 and MUC 102 antibodies. They furthermore expressed the complement type three receptor, the CD4 antigen and MHC class I and II molecules. These results were consistent in all the peripheral nerves studied. In addition, 1000 rad of -irradiation led to a strong reduction in the number of MHC class II-positive ramified cells in the peripheral nerves similar to that observed in other peripheral organs such as the heart. A considerable percentage of resident macrophages in the PNS and/or their precursor cells are therefore radiosensitive and could be related to the lineage of dendritic cells.Following crush injury, ED1-3-, OX-42-, MUC 101- and MUC 102-positive round macrophages were observed from 24 h postlesion onward at the site of trauma. In the distal part, they were observed to form strings of round, foamy macrophages probably involved in myelin phagocytosis. In contrast, the number of MHC class II-positive resident macrophages was only slightly increased at the site of trauma and in the distal part. These cells transformed from a ramified to a round morphology, but did not appear as typical strings of foamy macrophages.These results demonstrate that the PNS is provided with a resident macrophage population analogous in many respects to microglial cells in the CNS. These constitutively MHC class II-positive PNS microglial-like cells could act as the major antigen-presenting cells in the peripheral nerve. They may thus constitute a local immune defense system of the PNS with a function similar to that of microglial cells in the CNS.On leave of absence from the Institute of Neurology, University of Verona, Italy.     

用于中枢神经系统Waller变性研究的新型大鼠视神经夹伤模型

本实验旨在建立可用于中枢神经系统Waller变性研究的大鼠视神经(ON)损伤模型。成年SD大鼠12只,用夹持力为148g、镊端宽1mm的小号动脉阻血镊于球后2mm处夹持左侧视神经,致伤时间10s。术后随机分为两组(每组6只):一组动物术后立即于双侧上丘表面放置逆行荧光染料荧光金(FG),72h后处死。取视网膜铺片,ON冰冻连续切片。另一组动物术后72h常规灌注固定,取ON冰冻连续切片,分别行β-tubulin及NF-160免疫组化染色和HE染色。结果显示:左侧ON夹伤后3d,同侧视网膜内未见FG逆行标记的视网膜神经节细胞,ON夹伤部位形成一条宽达1mm的损伤带,ON基质连续,FG标记的神经纤维被阻断于损伤远侧,损伤远侧的β-tublin阳性纤维多已崩溃,NF-160阳性纤维数量较多,部分纤维末梢形成膨大的回缩球。上述结果表明ON夹伤后神经元轴突完全离断,中枢神经基质的连续性得以保持,损伤远侧段轴突发生典型的Waller变性,从而为中枢神经Waller变性及其药物治疗研究的筛选和评估提供了简单实用的实验模型。     

Wallerian degeneration in the peripheral nervous system: participation of both Schwann cells and macrophages in myelin degradation

Summary This study examined the role of Schwann cells and hematogenous macrophages in myelin degradation and Ia antigen expression during Wallerian degeneration of rodent sciatic nerve. To identify and distinguish between macrophages and Schwann cells we used, in addition to electron microscopy, immunocytochemical staining of teased nerve fibres and 1 m thick cryosections. Before the appearance of adherent macrophages the myelin sheath fragmented into ovoids, small whorls of myelin debris appeared within Schwann cell cytoplasm and the Schwann cell displayed numerous lipid droplets. However, at least in large fibres most myelin degradation and removal was accomplished or assisted by macrophages, identified by their expression of the ED1 marker. These cells began entering the nerve from blood vessels by day 2, migrated to degenerating nerve fibres and adhered to nerve fibres in the regions of the ovoids. There they penetrated the Schwann cell basal lamina to occupy an intratubal position and phagocytose myelin.During Wallerian degeneration a subpopulation of ED1-positive monocytes/macrophages expressed Ia antigen; Schwann cells were Ia-negative. Ia expression by monocytes/macrophages appeared to be a transient event and was not seen in post-phagocytic macrophages, as indicated by the fact that ED1-positive phagocytes with large vacuoles were Ia-negative.Our data show that both Schwann cells and macrophages play important roles in degrading and removing myelin during Wallerian degeneration. The expression of Ia antigen during Wallerian degeneration indicates that Ia expression need not necessarily reflect specific immune events but in some instances can represent a nonspecific response to PNS damage.     

The Role of Macrophages in Wallerian Degeneration

The present review focuses on macrophage properties in Wallerian degeneration. The identification of hematogenous phagocytes, the involvement of cell surface receptors and soluble factors, the state of activation during myelin removal and the signals and factors leading to macrophage recruitment into degenerating peripheral nerves after nerve transection are reviewed. The main effector cells in Wallerian degeneration are hematogenous phagocytes. Resident macrophages and Schwann cells play a minor role in myelin removal. The macrophage complement receptor type 3 is the main surface receptor involved in myelin recognition and uptake. The signals leading to macrophage recruitment are heterogenous and not yet defined in detail. Degenerating myelin and axons are suggested to participate. The relevance of these findings for immune-mediated demyelination are discussed since the definition of the role of macrophages might lead to a better understanding of the pathogenesis of demyelination.     

Wallerian degeneration in ICAM-1-deficient mice.

Wallerian degeneration of the peripheral nervous system was studied in ICAM-1-deficient mice and compared with the phenomena observed in C57BL wild-type animals. There was a decrease in myelin density in both mice strains 4 and 6 days after transection of the sciatic nerve. The degenerating nerves were invaded by Mac-1-, LFA-1-, and F4/80-positive macrophages; significantly lower numbers of macrophages were present in ICAM-1-deficient nerves. Myelin loss decreased after nerve transection with a more prominent loss in ICAM-1-deficient animals. Schwann cells revealed a much higher myelin load in these animals when compared with wild-type nerves, and there was an increased proliferation of endoneurial cells in ICAM-1-deficient mice. These data indicate that ICAM-1 is involved in macrophage recruitment to injured peripheral nerves as well as in the proliferative and phagocytic response of Schwann cells after peripheral nerve transection.     

Comparison of blood-nerve barrier disruption and matrix metalloprotease-9 expression in injured central and peripheral nerves in mice

To investigate the involvement of blood-born factors and extracellular proteases in axonal degeneration and regeneration in both PNS and CNS, we directly compared the differences of blood-nerve barrier (BNB) disruption and matrix metalloprotease-9 (MMP-9) induction between the sciatic nerve and optic nerve after crush injury in the same animal. In sciatic nerve, BNB disruption, fibrin(ogen) deposition and MMP-9 expression were observed only in the first week following injury. Neurofilament (NF) immunoreactivity dramatically decreased in the first 2 days, gradually recovered to the normal levels by day 28. In contrast, the immunoglobulin G deposits spanned from 4 h to 28 days in crushed optic nerves. Fibrin(ogen) deposition was only observed in the first 2 days, while MMP-9 induction did not occur until a week after injury but lasted for 3 weeks in the crushed optic nerves. The NF immunoreactivity did not change much until day 7 and almost completely disappeared on day 28. The decrease of NF immunoreactivity coincided with the induction of MMP-9 after optic nerve crush. These results show that BNB disruption and MMP-9 induction are differentially regulated in the PNS and CNS after injuries, and they may contribute to the different regeneration capacities of the two systems.     

Glial cells in transected optic nerves of immature rats. II. An immunohistochemical study

Summary The glial response to Wallerian degeneration was studied in optic nerves 21 days after unilateral enucleation (PED21) of immature rats, 21 days old (P21), using immunohistochemical labelling. Nerves from normal P21 and P42 nerves were also studied for comparison. At PED21, there was a virtual loss of axons apart from a few solitary fibres of unknown origin. The nerve comprised a homogeneous glial scar tissue formed by dense astrocyte processes, oriented parallel to the long axis of the nerve along the tracks of degenerated axons. Astrocytes were almost perfectly co-labelled by antibodies to glial fibrillary acid protein and vimentin in both normal and transected nerves. However, there was a small population of VIM⁺GFAP^– cells in normal P21 and P42 nerves, and we discuss the possibility that they correspond to O-2A progenitor cells describedin vitro. Significantly, double immunofluorescence labelling in transected nerves revealed a distinct population of hypertrophic astrocytes which were GFAP⁺VIM^–. These cells represented a novel morphological and antigenic subtype of reactive astrocyte. It was also noted that the number of oligodendrocytes in transected nerves did not appear to be less than in normal nerves, on the basis of double immunofluorescence staining for carbonic anhydrase II, myelin oligodendrocyte glycoprotein, myelin basic protein, glial fibrillary acid protein and ED-1 (for macrophages), although it was not excluded that a small proportion may have been microglia. A further prominent feature of transected nerves was that they contained a substantial amount of myelin debris, notwithstanding that OX-42 and ED1 immunostaining showed that there were abundant microglia and macrophages, sufficient for the rapid and almost complete removal of axonal debris. In conclusion, glial cells in the immature P21 rat optic nerve reacted to Wallerian degeneration in a way equivalent to the adult CNS, i.e. astrocytes underwent pronounced reactive changes and formed a dense glial scar, oligodendrocytes persisted and were not dependent on axons for their continued survival, and there was ineffective phagocytosis of myelin possibly due to incomplete activation of microglia/macrophages.     

Tenascin-C expression during Wallerian degeneration in C57BL/Wlds mice: possible implications for axonal regeneration

Summary Schwann cells in the distal stumps of lesioned peripheral nerves strongly express the extracellular matrix glycoprotein tenascin-C. To gain insights into the relationship between Wallerian degeneration, lesion induced tenascin-C upregulation and regrowth of axons we have investigated C57BL/Wld^s (C57BL/Ola) mice, a mutant in which Wallerian degeneration is considerably delayed. Since we found a distinct difference in the speed of Wallerian degeneration between muscle nerves and cutaneous nerves in 16-week-old C57BL/Wld^s mice, as opposed to 6-week-old animals in which Wallerian degeneration is delayed in both, we chose the older animals for closer investigation. Five days post lesion tenascin-C was upregulated in the muscle branch (quadriceps) but not in the cutaneous branch (saphenous) of the femoral nerve in 16-week-old animals. In addition myelomonocytic cells displaying endogenous peroxidase activity invaded the muscle branch readily whereas they were absent from the cutaneous branch at this time. We could further show that it is only a subpopulation of axon-Schwann cell units (mainly muscle efferents) in the muscle branch which undergo Wallerian degeneration and upregulate tenascin-C at normal speed and that the remaining axon-Schwann cell units (mainly afferents) displayed delayed Wallerian degeneration and no tenascin-C expression. Regrowing axons could only be found in the tenascin-C-positive muscle branch where they always grew in association with axon-Schwann cell units undergoing Wallerian degeneration. These observations indicate a tight relationship between Wallerian degeneration, upregulation of tenascin-C expression and regrowth of axons, suggesting an involvement of tenascin-C in peripheral nerve regeneration.     

Macrophage response to peripheral nerve injury: the quantitative contribution of resident and hematogenous macrophages

Whereas local microglial cells of the CNS rapidly respond to injury, little is known about the functional role of resident macrophages of the peripheral nervous system in nerve pathology. Using bone marrow chimeric rats, we recently identified individual resident endoneurial macrophages that rapidly became activated after nerve injury. However, the extent of local macrophage activation and its quantitative contribution to the total macrophage response is unknown. We now have created chimeric mice by transplanting bone marrow from green fluorescent protein (GFP)-transgenic mice into irradiated wild-type mice, allowing easy differentiation and quantification of hematogenous and resident endoneurial macrophages. After sciatic nerve crush injury, both GFP(-) and GFP(+) resident macrophages, the latter having undergone physiological turnover from the blood before injury, rapidly underwent morphological alterations and increased in number. Proliferating GFP(-) and GFP(+) resident macrophages were abundant and peaked 3 days after injury. A major lesion-induced influx of hematogenous macrophages with a disproportionate increase of GFP(+) macrophages was not observed until Day 4. Throughout all time points examined, GFP(-) resident macrophages were strikingly frequent, reaching maximum numbers 9.5-fold above baseline. There was also a notable proportion of GFP(-) resident endoneurial macrophages phagocytosing myelin and expressing major histocompatibility complex class II. Our results demonstrate for the first time that the rapid response of resident endoneurial macrophages to nerve injury is quantitatively important and that local macrophages contribute significantly to the total endoneurial macrophage pool during Wallerian degeneration.     

Microenvironmental changes during axonal regrowth in the optic nerve of the myelin deficient rat. Immunocytochemical and ultrastructural observations

Summary Lesion-induced regenerative sprouting of CNS axons is accompanied by reactions of the supporting glia and vascular and connective tissue which may influence the extent of regeneration. In a previous report, it was shown that after crush injury, the amyelinated optic nerve of the myelin deficient (md) mutant rat contains greater numbers of regrowing axons proximal to the site of crush than that of normally myelinated littermates. The present study was designed to compare the response of the microenvironment, i.e. glial cells and vascular and connective tissue, in md and normally myelinated optic nerves 2, 4 and 6 days after crush injury. In unoperated normal optic nerves monoclonal antibodies to the HNK-1 carbohydrate labelled astrocytic processes at the ultrastructural level whereas in unoperated md mutants HNK-1 staining was restricted to axonal surfaces. Immunoreactivity with monoclonal antibodies to stage-specific embryonic antigen-1 (SSEA-1) was confined to astrocytic surfaces in both md and wildtype animals. After axotomy of md optic nerves regrowing axons were more numerous in the proximal site of the crush and extended further into the lesion than in wildtype animals. In both md and wildtype rats regrowing axons were HNK-1-positive. In md rats strong reaction with antibodies to laminin and fibronectin was only seen in 6-day-old lesions of md rats whereas immunoreactivity was less distinct in operated littermate controls. Immunolabelling was obviously associated with blood vessels, since crush lesions in both md and wildtype rats were Schwann cell-free as assessed by electron microscopy and immunocytochemistry. In both operated md and normal littermates crush lesions contained degenerating astrocytes as well as reactive astrocytes in which the intermediate filaments of the perikarya failed to stain immunocytochemically for GFAP, vimentin, desmin, and a common determinant of intermediate filaments. In contrast, reactive astrocytes in the lesion site of normally myelinated rats expressed the SSEA-1 antigen intracytoplasmically whereas in md mutants astrocytes were completely SSEA-1-negative. Infiltration of crush lesions by macrophages was less extensive in md rats than in normal littermates. However the overall content of macrophages in the peritoneal cavity was also reduced. The present study demonstrates that (1) md optic nerves lack HNK-1-reactive astrocytes; (2) in the axotomized wildtype optic nerve impaired axonal regrowth may be associated with distinct immuno-phenotypes of the supporting glial cells, i.e. SSEA-1-positive astrocytes; (3) laminin and fibronectin seem not to be essential for improved axonal regrowth in md rats.     

Temporary colonization of the site of lesion by macrophages is a prelude to the arrival of regenerated axons in injured goldfish optic nerve

Summary. In crushed goldfish optic nerve, regenerating axons cross the site of lesion within 10 days following injury. Some 30 days later, Schwann cells accumulate at the lesion, where they myelinate the new axons. In this study, we have used immunohistochemistry and electron microscopy to examine the cellular environment of the crush site prior to the establishment of Schwann cells in order to learn more about the early events that contribute to axonal regeneration. During the first week following injury, macrophages enter the site of lesion and efficiently phagocytose the debris. The infiltration of macrophages precedes the arrival of regenerating axons that abut and surround these phagocytes. Based on EM morphology and phagocytic capacity, macrophages of the type observed at the site of lesion are not present in the degenerating distal nerve segment, where debris clearance is shared between conventional microglia and astrocytes over a period of several weeks. During this period, axon bundles emerging distally from the injury zone become enwrapped by astrocyte processes, thereby re-establishing the characteristic fascicular cytoarchitecture of the optic nerve. The process of fasciculation also leads to the displacement of myelin debris to the margins of the fiber bundles, where it is trapped by the astrocytes. Our results suggest that the early robust appearance of macrophages at the lesion, and their effectiveness as phagocytes compared with the microglia distally, may contribute to the vigorous axonal regeneration across the crush, beyond which axons<197>excepting the pioneers<197>extend through newly formed debris-free channels delineated by astrocyte processes.     

Central and peripheral nerve regeneration by transplantation of Schwann cells and transdifferentiated bone marrow stromal cells

In contrast to the peripheral nervous system (PNS), little structural and functional regeneration of the central nervous system (CNS) occurs spontaneously following injury in adult mammals. The inability of the CNS to regenerate is mainly attributed to its own inhibitorial environment such as glial scar formation and the myelin sheath of oligodendrocytes. Therefore, one of the strategies to promote axonal regeneration of the CNS is to experimentally modify the environment to be similar to that of the PNS. Schwann cells are the myelinating glial cells in the PNS, and are known to play a key role in Wallerian degeneration and subsequent regeneration. Central nervous system regeneration can be elicited by Schwann cell transplantation, which provides a suitable environment for regeneration. The underlying cellular mechanism of regeneration is based upon the cooperative interactions between axons and Schwann cells involving the production of neurotrophic factors and other related molecules. Furthermore, tight and gap junctional contact between the axon and Schwann cell also mediates the molecular interaction and linking. In this review, the role of the Schwann cell during the regeneration of the sciatic (representing the PNS) and optic (representing the CNS) nerves is explained. In addition, the possibility of optic nerve reconstruction by an artificial graft of Schwann cells is also described. Finally, the application of cells not of neuronal lineage, such as bone marrow stromal cells (MSCs), in nerve regeneration is proposed. Marrow stromal cells are known as multipotential stem cells that, under specific conditions, differentiate into several kinds of cells. The strategy to transdifferentiate MSCs into the cells with a Schwann cell phenotype and the induction of sciatic and optic nerve regeneration are described.     

Peripheral nerve explants grafted into the vitreous body of the eye promote the regeneration of retinal ganglion cell axons severed in the optic nerve

Summary We have conducted experiments in the adult rat visual system to assess the relative importance of an absence of trophic factors versus the presence of putative growth inhibitory molecules for the failure of regeneration of CNS axons after injury. The experiments comprised three groups of animals in which all optic nerves were crushed intra-orbitally: an optic nerve crush group had a sham implant-operation on the eye; the other two groups had peripheral nerve tissue introduced into the vitreous body; in an acellular peripheral nerve group, a frozen/thawed teased sciatic nerve segment was grafted, and in a cellular peripheral nerve group, a predegenerate teased segment of sciatic nerve was implanted. The rats were left for 20 days and their optic nerves and retinae prepared for immunohistochemical examination of both the reaction to injury of axons and glia in the nerve and also the viability of Schwann cells in the grafts. Anterograde axon tracing with rhodamine-B provided unequivocal qualitative evidence of regeneration in each group, and retrograde HRP tracing gave a measure of the numbers of axons growing across the lesion by counting HRP filled retinal ganglion cells in retinal whole mounts after HRP injection into the optic nerve distal to the lesion. No fibres crossed the lesion in the optic nerve crush group and dense scar tissue was formed in the wound site. GAP-43-positive and rhodamine-B filled axons in the acellular peripheral nerve and cellular peripheral nerve groups traversed the lesion and grew distally. There were greater numbers of regenerating fibres in the cellular peripheral nerve compared to the acellular peripheral nerve group. In the former, 0.6–10% of the retinal ganglion cell population regenerated axons at least 3–4 mm into the distal segment. In both the acellular peripheral nerve and cellular peripheral nerve groups, no basal lamina was deposited in the wound. Thus, although astrocyte processes were stacked around the lesion edge, a glia limitans was not formed. These observations suggest that regenerating fibres may interfere with scarring. Viable Schwann cells were found in the vitreal grafts in the cellular peripheral nerve group only, supporting the proposition that Schwann cell derived trophic molecules secreted into the vitreous stimulated retinal ganglion cell axon growth in the severed optic nerve. The regenerative response of acellular peripheral nerve-transplanted animals was probably promoted by residual amounts of these molecules present in the transplants after freezing and thawing. In the optic nerves of all groups the astrocyte, microglia and macrophage reactions were similar. Moreover, oligodendrocytes and myelin debris were also uniformly distributed throughout all nerves. Our results suggest either that none of the above elements inhibit CNS regeneration after perineuronal neurotrophin delivery, or that the latter, in addition to mobilising and maintaining regeneration, also down regulates the expression of axonal growth cone-located receptors, which normally mediate growth arrest by engaging putative growth inhibitory molecules of the CNS neuropil.     

The kinetics and morphological characteristics of the macrophage-microglial response to kainic acid-induced neuronal degeneration.

Outside the nervous system myelomonocytic cells are known to play an important role in the inflammatory response and tissue repair after injury. In this study we have examined the myelomonocytic response to neuronal destruction following unilateral injection of the excitotoxin kainic acid into the mouse hippocampus. Intrahippocampal injection of kainate induces rapid, synchronous neuronal death. There is no neutrophil recruitment and a delay of at least 48 h before macrophage-microglial cell numbers increase. The microglial reaction in the injected hippocampus consists of altered morphology, a 6-9-fold increase in mononuclear phagocyte cell numbers and enhanced expression of the macrophage-specific plasma membrane antigen, F4/80, assessed immunohistochemically and by Western blotting. Microglia also respond at distant sites related to the projection pathway and terminals of killed pyramidal cells but the reaction varies in cell numbers, kinetics and morphology. The absence of neutrophil recruitment and the delay in an increase in macrophage or microglial cells shows that the CNS differs from other sites in the body with regard to the kinetics and nature of the myelomonocytic cell inflammatory response. The role of mononuclear phagocytes in tissue repair in the CNS remains to be defined.     

Structure and nature of macrophages participating in the Wallerian degeneration of nerve fibers

The posttraumatic processes of Wallerian degeneration of nerves have been illuminated in detail. The dynamics of the breakdown of axons and the myelin sheaths of nerve fibers has been established, as have been the periods of the changes in the composition of myelin, and the reactive changes in the Schwann cells and the connective tissue structures in the makeup of the nerve as well as the formation of "foam" cells have been described. The controversial questions which have been raised in these studies regarding the role of the cellular elements (the Schwann cells, the endoneurial fibroblasts, the cells of the epi- and perineurium) during Wallerian degeneration remain unresolved until the present time. In particular, the question as to which cells participate in the cleanup of the products of the breakdown of the myelin sheaths, and as to the character of the inflammatory infiltration in Wallerian degeneration and the degree of the participation of the various cellular elements in the destructive and reparative processes, has not been elucidated. Some investigators believe that the Schwann cells accomplish the cleanup of the products of the breakdown of the myelin sheaths. There are also data suggesting that the macrophages are of considerable significance in the cleanup of the products of the breakdown of nerve fibers of both the PNS and the CNS following their injury. It has been demonstrated by means of monoclonal antibodies to macrophages, radioautography, and immunocytochemical methods that these macrophages have a hematogenous origin.(ABSTRACT TRUNCATED AT 250 WORDS)