Nature of signals that initiate the immune response during Wallerian degeneration of peripheral nerves

Monocyte chemoattractant protein-1 is produced by Schwann cells during Wallerian degeneration of a peripheral nerve and contributes to a selective accumulation of macrophages in the degenerating segment. An in vitro preparation has been developed to analyze the molecules from axons and non-neuronal cells in nerves that stimulate an increased production of monocyte chemoattractant protein-1 mRNA by Schwann cells. For this purpose, Schwann cells obtained from neonatal rats were maintained in culture, exposed to putative molecular stimuli and analyzed for their content of monocyte chemoattractant protein-1 mRNA. Under basal conditions, the concentration of monocyte chemoattractant protein-1 in Schwann cells was low. Freeze-killed fragments or homogenates of nerve (or brain) but not viable nerve or freeze-killed muscle were effective in inducing monocyte chemoattractant protein-1 mRNA. The inductive activity was abolished by heating. Results of dialysis of supernatants of nerve homogenates indicate that a protein or proteins of 1-10 kDa were capable of stimulating synthesis of monocyte chemoattractant protein-1 by Schwann cells. Also, the activity in nerve homogenates was partially inhibited by antibodies to Toll-like receptor-4. The observations suggest that a non-secreted protein is released from disintegrating axons to initiate the innate immune response that characterizes Wallerian degeneration.     

The role of TNF-alpha during Wallerian degeneration

The role of TNF-alpha in the course of Wallerian degeneration of the sciatic nerve was studied in control and TNF-alpha deficient mice. In control animals, the characteristic phenomena of Wallerian degeneration such as axon and myelin degeneration as well as macrophage recruitment with subsequent myelin removal were observed. In TNF-alpha deficient mice, in contrast, macrophage recruitment into the degenerating nerves was impaired resulting in a delayed myelin removal. However, the myelin phagocytic capacity of macrophages was not affected as it could be demonstrated by a similar myelin load of control and TNF-alpha deficient macrophages. These data indicate that the main function of TNF-alpha during Wallerian degeneration is the induction of macrophage recruitment from the periphery without affecting myelin damage or phagocytosis.     

Evidence that the Rate of Wallerian Degeneration is Controlled by a Single Autosomal Dominant Gene

In a substrain of C57BL mice, C57BL/Ola, Wallerian degeneration in the distal segment of the severed sciatic nerve is extremely slow when compared to other mice. Despite this very slow degeneration in the distal segment regeneration of the motor nerves is not impaired. From suitable genetic outcrosses and backcrosses, the authors provide evidence that the rate of Wallerian degeneration in this strain is controlled by a single autosomal gene product. The authors have also shown that the rate of degeneration, in C57BL/Ola mice, is influenced by the environment in which the animals were bred and housed. Wallerian degeneration in the sciatic nerves of mice raised in isolators is slower than in those raised in a conventional animal house. This strain of mouse may prove to be of value in the understanding of nerve degeneration and regeneration.     

The effects of claudin 14 during early Wallerian degeneration after sciatic nerve injury

Claudin 14 has been shown to promote nerve repair and regeneration in the early stages of Wallerian degeneration(0–4 days) in rats with sciatic nerve injury, but the mechanism underlying this process remains poorly understood. This study reported the effects of claudin 14 on nerve degeneration and regeneration during early Wallerian degeneration. Claudin 14 expression was up-regulated in sciatic nerve 4 days after Wallerian degeneration. The altered expression of claudin 14 in Schwann cells resulted in expression changes of cytokines in vitro. Expression of claudin 14 affected c-Jun, but not Akt and ERK1/2 pathways. Further studies revealed that enhanced expression of claudin 14 could promote Schwann cell proliferation and migration. Silencing of claudin 14 expression resulted in Schwann cell apoptosis and reduction in Schwann cell proliferation. Our data revealed the role of claudin 14 in early Wallerian degeneration, which may provide new insights into the molecular mechanisms of Wallerian degeneration.     

Concentration-dependent effects of the esterase inhibitor BNPP on macrophage migration and myelin phagocytosis

Wallerian degeneration of a peripheral nerve is mainly characterized by axon and myelin degradation and is paralleled by a massive invasion of peripheral macrophages into the nerve. These cells enter the nerve attracted by a cascade of chemokines and cytokines but require proteolytic and enzymatic factors which enables them to cross the blood-nerve barrier. Here we investigated whether alpha-naphthyl (alpha-NA) esterases -- which have been shown to be exclusively expressed in human monocytes -- play a role during Wallerian degeneration. These enzymes were blocked by the specific inhibitor bis(4-nitrophenyl)-phosphate (BNPP) in an established in vitro model of Wallerian degeneration. Sciatic nerve segments of mice were co-cultured with peritoneal macrophages and BNPP was added to the cultures in various concentrations and at different timepoints. The macrophage numbers and myelin density in the nerve segments and the myelin load of macrophages were evaluated. After BNPP treatment the macrophage number within the nerve was significantly diminished and the myelin load within the macrophages was decreased, resulting in elevated levels of preserved myelin within the nerves. These experiments clearly showed a double effect of the alphaNA esterase inhibitor BNPP on macrophages. First, it suggests a role for alphaNA esterases on the migratory potential of macrophages since their invasion into the nerves was diminished. Second, the reduced myelin uptake is due to the inhibition of phagocytic capacity of these cells by BNPP. The therapeutical use of this inhibitor for treatment of autoimmune diseases such as multiple sclerosis or Guillain-Barré syndrome remains to be investigated.     

Macrophage function during Wallerian degeneration of rat optic nerve: clearance of degenerating myelin and Ia expression

This study examined Wallerian degeneration (WD) of rat optic nerve (ON) with 2 goals: to determine which cell types are involved in myelin degradation and clearance and to evaluate the extent to which Ia antigen, a product of the immune response genes, is expressed. We examined immunostained 1-micron and ultrathin cryosections of rat ON at 1, 8, and 16 weeks after nerve transection. Serial 1-micron cryosections were stained with monoclonal antibodies to rat Ia antigen (Ox6) and with antibodies that identify astrocytes (GFAP) and monocytes/macrophages (ED1). In normal ON, ED1-positive cells were not found. A few ED1-positive monocytes/macrophages were present in the transected ON at one week. Macrophages were prominent throughout the ON at 8 weeks; by 16 weeks their number was decreasing. These cells contained myelin debris in various stages of digestion. GFAP-positive astrocytes did not contain myelin debris in their cytoplasm. At all 3 times a subpopulation of the ED1-positive monocytes/macrophages expressed Ia antigen. Ia antigen was not detected in endothelial cells or GFAP-positive astrocytes. In ultrathin cryosections stained by immunogold procedures, Ia immunoreactivity was found exclusively in cells containing multiple vacuoles and myelin debris and lacking intermediate filaments. The same cell type was labeled by ED1 antibodies. Our results indicate that macrophages remove the vast majority of debris during Wallerian degeneration in the CNS; a proportion of these macrophages concomitantly express Ia antigen. These results suggest that the Ia expression by macrophages observed in other CNS disorders does not necessarily reflect specific local immune events, but in some instances can represent a nonspecific response to CNS damage.     

Ascorbic acid accelerates Wallerian degeneration after peripheral nerve injury

Wallerian degeneration occurs after peripheral nerve injury and provides a beneficial microenvironment for nerve regeneration. Our previous study demonstrated that ascorbic acid promotes peripheral nerve regeneration, possibly through promoting Schwann cell proliferation and phagocytosis and enhancing macrophage proliferation, migration, and phagocytosis. Because Schwann cells and macrophages are the main cells involved in Wallerian degeneration, we speculated that ascorbic acid may accelerate this degenerative process. To test this hypothesis, 400 mg/kg ascorbic acid was administered intragastrically immediately after sciatic nerve transection, and 200 mg/kg ascorbic acid was then administered intragastrically every day. In addition, rat sciatic nerve explants were treated with 200 μM ascorbic acid. Ascorbic acid significantly accelerated the degradation of myelin basic protein-positive myelin and neurofilament 200-positive axons in both the transected nerves and nerve explants. Furthermore, ascorbic acid inhibited myelin-associated glycoprotein expression, increased c-Jun expression in Schwann cells, and increased both the number of macrophages and the amount of myelin fragments in the macrophages. These findings suggest that ascorbic acid accelerates Wallerian degeneration by accelerating the degeneration of axons and myelin in the injured nerve, promoting the dedifferentiation of Schwann cells, and enhancing macrophage recruitment and phagocytosis. The study was approved by the Southern Medical University Animal Care and Use Committee(approval No. SMU-L2015081) on October 15, 2015.     

Myelin phagocytosis in Wallerian degeneration of peripheral nerves depends on silica-sensitive,bg/bg-negative and Fc-positive monocytes

Previous experiments with nerves enclosed in millipore diffusion chambers had shown that myelin degradation during Wallerian degeneration depends on invasion by non-resident cells. The present study was aimed at a more precise identification of the invading cell population. Monoclonal antibody studies of degenerating nerves showed many cells with the Fc marker; cells having the Lyt-1, Lyt-2, Ia or Mac-1 markers were sparse or absent. Nerves transplanted into mice of the Chediak-Higashi bg/bg strain were invaded by cells lacking the bg/bg marker (giant lysosomes), while cotransplanted muscle tissue was invaded by cells with the bg/bg marker. Blocking monocytes with silica reduced both cell invasion and myelin degradation in degenerating nerves. These observations show that Wallerian degeneration of peripheral nerve fibers involves a subset of monocytes which are silica-sensitive and have Fc receptors but no bg/bg giant lysosomes.     

Critical signaling pathways during Wallerian degeneration of peripheral nerve

Wallerian degeneration is a critical biological process that occurs in distal nerve stumps after nerve injury. To systematically investigate molecular changes underlying Wallerian degeneration, we used a rat sciatic nerve transection model to examine microarray analysis out-comes and investigate significantly involved Kyoto Enrichment of Genes and Genomes (KEGG) pathways in injured distal nerve stumps at 0, 0.5, 1, 6, 12, and 24 hours, 4 days, 1, 2, 3, and 4 weeks after peripheral nerve injury. Bioinformatic analysis showed that only one KEGG pathway (cytokine-cytokine receptor interaction) was significantly enriched at an early time point (1 hour post-sciatic nerve transection). At later time points, the number of enriched KEGG pathways initially increased and then decreased. Three KEGG pathways were studied in further detail: cytokine-cytokine receptor interaction, neuroactive ligand-receptor interaction, and axon guidance. Moreover, temporal expression patterns of representative differentially expressed genes in these KEGG pathways were validated by real time-polymerase chain reaction. Taken together, the above three signaling pathways are important after sciatic nerve injury, and may increase our understanding of the molecular mechanisms underlying Wallerian degeneration.     

Axonal autophagy during regeneration of the rat sciatic nerve**★

BACKGROUND: The removal of degenerated axonal debris during Wallerian degeneration is very important for nerve regeneration. However, the mechanism by which debris is removed is not been completely understood. Considerable controversy remains as to the clearance pathway and cells that are involved. OBJECTIVE: To investigate axonal autophagy during removal of degenerated axonal debris by transecting the sciatic nerve in a rat Wallerian degeneration model. DESIGN, TIME AND SETTING: Experimental neuropathological analysis. The experiment was conducted at the Laboratory Animal Service Center of the Southern Medical University between January and June 2005. MATERIALS: Fifty-four adult, Wistar rats of either sex, weighing 180-250 g, were obtained from the Laboratory Animal Service Center of the Southern Medical University. Animals were randomly divided into nine groups of six rats. METHODS: Wallerian degeneration was induced by transecting the rat sciatic nerve, and tissue samples from the distal stump were obtained 0.2, 0.4, 1, 2, 3, 4, 7, 10, and 15 days post-transection. Ultrathin sections were prepared for electron microscopy to study ultrastructure and enzyme cytochemistry staining. MAIN OUTCOME MEASURES: Ultrastructure (axon body, autophagic body, and cystoskeleton) of axons and myelin sheaths observed with electron microscopy; acidic phosphatase activity detected by Gomori staining using electron microscopy. RESULTS: The major changes of degenerating axons after transection were axoplasm swelling and separation of axons from their myelin sheath between five hours and two days post-transection. At four days post-transection, the axoplasm condensed and axons were completely separated from the myelin sheath, forming dissociative axon bodies. Vacuoles of different sizes formed in axons during the early phase after lesion. Larger dissociative axon bodies were formed when the axons were completely separated from the myelin sheath during a late phase. The axolemma surrounding the axon body was derived from the neuronal cell membrane; the condensed axoplasm contained many autophagic vacuoles at all levels. A large number of neurofilaments, microtubules, and microfilaments were arranged in a criss-cross pattern. The autophagic vacuoles exhibited acidic phosphatase activity. Axonal bodies were absorbed after degradation from day 7 onwards, and macrophages were observed rarely in the formative cavity. CONCLUSION: The degenerating axons were cleared mainly by axonal autophagy and Schwann cell phagocytosis during regeneration of the rat sciatic nerve, and macrophages exhibited only an assisting function. Key Words: axon; autophagy; nerve regeneration     

Axonal autophagy during regeneration of the rat sciatic nerve**★

BACKGROUND: The removal of degenerated axonal debris during Wallerian degeneration is very important for nerve regeneration. However, the mechanism by which debris is removed is not been completely understood. Considerable controversy remains as to the clearance pathway and cells that are involved. OBJECTIVE: To investigate axonal autophagy during removal of degenerated axonal debris by transecting the sciatic nerve in a rat Wallerian degeneration model.DESIGN, TIME AND SETTING: Experimental neuropathological analysis. The experiment was conducted at the Laboratory Animal Service Center of the Southern Medical University between January and June 2005. MATERIALS: Fifty-four adult, Wistar rats of either sex, weighing 180-250 g, were obtained from the Laboratory Animal Service Center of the Southern Medical University. Animals were randomly divided into nine groups of six rats. METHODS: Wallerian degeneration was induced by transecting the rat sciatic nerve, and tissue samples from the distal stump were obtained 0.2, 0.4, 1, 2, 3, 4, 7, 10, and 15 days post-transection. Ultrathin sections were prepared for electron microscopy to study ultrastructure and enzyme cytochemistry staining. MAIN OUTCOME MEASURES: Ultrastructure (axon body, autophagic body, and cystoskeleton) of axons and myelin sheaths observed with electron microscopy; acidic phosphatase activity detected by Gomori staining using electron microscopy. RESULTS: The major changes of degenerating axons after transection were axoplasm swelling and separation of axons from their myelin sheath between five hours and two days post-transection. At four days post-transection, the axoplasm condensed and axons were completely separated from the myelin sheath, forming dissociative axon bodies. Vacuoles of different sizes formed in axons during the early phase after lesion. Larger dissociative axon bodies were formed when the axons were completely separated from the myelin sheath during a late phase. The axolemma surrounding the axon body was derived from the neuronal cell membrane; the condensed axoplasm contained many autophagic vacuoles at all levels. A large number of neurofilaments, microtubules, and microfilaments were arranged in a criss-cross pattern. The autophagic vacuoles exhibited acidic phosphatase activity. Axonal bodies were absorbed after degradation from day 7 onwards, and macrophages were observed rarely in the formative cavity. CONCLUSION: The degenerating axons were cleared mainly by axonal autophagy and Schwann cell phagocytosis during regeneration of the rat sciatic nerve, and macrophages exhibited only an assisting function.     

The Effectiveness of the Gene Which Slows the Rate of Wallerian Degeneration in C57BL/Ola Mice Declines With Age

The rate of Wallerian degeneration is unusually slow in severed axons of mice of the C57BL/Ola strain. Within mice of that strain we have now found that the rate of degeneration increases with the age of the animal. In 4-week-old mice nerve stimulation evokes muscle contractions even 5 days after sciatic nerve section and compound action potentials can be recorded in the distal nerve stump up to 3 weeks after section. In 1-year-old animals no action potentials can be excited 5 days after nerve section. Heterozygous mice carrying only one copy of the dominant gene show the same age-related decline in viability of the distal nerve stump after axotomy, and the rate of decline is no greater than for homozygous mice. The more rapid rate of degeneration of severed axons of mice of the C57BL/6J strain was affected in the opposite way by age, degeneration occurring more slowly in older animals.     

Lesion response of long-term and recently immigrated resident endoneurial macrophages in peripheral nerve explant cultures from bone marrow chimeric mice

Resident macrophages of the peripheral nervous system have recently been shown to respond rapidly to Wallerian degeneration before the influx of blood-derived macrophages. Because resident endoneurial macrophages are slowly but incompletely exchanged from the blood within 3 months, they could potentially comprise a heterogenous cell population consisting of long-term resident cells and more mobile cells undergoing turnover. We used bone marrow chimeric mice created by transplanting bone marrow from green fluorescent protein-transgenic mice into irradiated wildtype recipients to selectively analyse the response of these two resident macrophage populations to Wallerian degeneration in sciatic nerve explant cultures. In such nerves, recently immigrated macrophages exhibit green fluorescence whereas long-term resident macrophages do not. Studies in cultures from wildtype controls revealed rapid morphological changes of resident macrophages towards a bloated phenotype, a proliferative response resulting in a 3.7-fold increase of macrophage numbers over 2 weeks, and phagocytosis of myelin basic protein-immunoreactive myelin debris. When chimeric mice were analysed, both populations of resident endoneurial macrophages participated in morphological transformation, proliferation and phagocytosis. Quantitative studies revealed a stronger proliferative and phagocytic response in long-term resident endoneurial macrophages compared with recently immigrated macrophages. Our results point towards subtle, but not principal, differences between the two macrophage populations, which might indicate different stages of macrophage differentiation rather than the existence of entirely distinct endoneurial macrophage populations. The results further underline the versatility of resident endoneurial macrophages following peripheral nerve injury, which is reminiscent of the lesion response of microglial cells within the brain.     

On the longevity of resident endoneurial macrophages in the peripheral nervous system: a study of physiological macrophage turnover in bone marrow chimeric mice

Macrophages are intimately involved in the pathogenesis of peripheral nervous system (PNS) disorders. Recently, we characterized a resident endoneurial macrophage population, which contributes rapidly to the endoneurial macrophage response in PNS diseases. Unlike microglial cells, resident macrophages undergo a physiological turnover of 50% in the sciatic nerve and 80% in dorsal root ganglia (DRG) within 12 weeks. Further information about the dynamics of this turnover is not available. This study examined the macrophage turnover in the sciatic nerve and DRGs over a longer period and addresses the question whether the turnover of resident macrophages is complete or whether there is a truly resident endoneurial macrophage population. We used chimeric mice carrying GFP(+) bone marrow and immunohistochemistry to detect hematogenous (GFP(+)) endoneurial macrophages after turnover. Non-exchanged, resident macrophages were GFP(-). Quantification of GFP(+) and GFP(-) macrophages revealed a maximal turnover of 75%, reached in DRGs after 12 weeks and in sciatic nerves after 36 weeks. GFP(-) long-term resident macrophages were further characterized after sciatic nerve injury, where they participated in the early macrophage response of Wallerian degeneration. Our results point toward a small but truly resident PNS macrophage population. These macrophages are an interesting target for further characterization and might have a distinct role in peripheral nerve disease.     

Tumor necrosis factor-α in immune-mediated demyelination and Wallerian degeneration of the rat peripheral nervous system

This study reports on the immunocytochemical localization of tumor necrosis factor-alpha (TNFα) in immune-mediated demyelination and Wallerian degeneration of the rat peripheral nervous system (PNS) using teased nerve fiber preparations. In experimental autoimmune neuritis induced by active immunization (EAN) or by adoptive transfer of autoreactive T cells (AT-EAN), macrophages passing blood vessels as well as macrophages adherent to nerve fibers were TNFα-positive. Large post-phagocytic macrophages at later stages of demyelination were TNFα-negative. Intraperitoneal application of an anti-TNFα antibody to EAN rats significantly reduced the degree of inflammatory demyelination, suggesting a pathogenic role for TNFα. After nerve transection only macrophages located within degenerating nerve fibers were TNFα-positive, while those entering and leaving nerves were negative. TNFα produced by macrophages seems to bevolved in immune-mediated demyelination and non-immune myelin degradation after axotomy. While interferon-gamma (IFNγ) is present in EAN nerves and may act as a local stimulus for TNF expression, the nature of this signal in Wallerian degeneration in the absence of IFNγ is unknown.     

Gene expression profiling of the rat sciatic nerve in early Wallerian degeneration after injury

Wallerian degeneration is an important area of research in modern neuroscience.A large number of genes are differentially regulated in the various stages of Wallerian degeneration,especially during the early response.In this study,we analyzed gene expression in early Wallerian degeneration of the distal nerve stump at 0,0.5,1,6,12 and 24 hours after rat sciatic nerve injury using gene chip microarrays.We screened for differentially-expressed genes and gene expression patterns.We examined the data for Gene Ontology,and explored the Kyoto Encyclopedia of Genes and Genomes Pathway.This allowed us to identify key regulatory factors and recurrent network motifs.We identified 1 546 differentially-expressed genes and 21 distinct patterns of gene expression in early Wallerian degeneration,and an enrichment of genes associated with the immune response,acute inflammation,apoptosis,cell adhesion,ion transport and the extracellular matrix.Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed components involved in the Jak-STAT,ErbB,transforming growth factor-β,T cell receptor and calcium signaling pathways.Key factors included interleukin-6,interleukin-1,integrin,c-sarcoma,carcinoembryonic antigen-related cell adhesion molecules,chemokine(C-C motif) ligand,matrix metalloproteinase,BH3 interacting domain death agonist,baculoviral IAP repeat-containing 3 and Rac.The data were validated with real-time quantitative PCR.This study provides a global view of gene expression profiles in early Wallerian degeneration of the rat sciatic nerve.Our findings provide insight into the molecular mechanisms underlying early Wallerian degeneration,and the regulation of nerve degeneration and regeneration.     

The role of cytokines and adhesion molecules in axon degeneration after peripheral nerve axotomy: a study in different knockout mice

The loss of axons and axonal dysfunction has become of outstanding interest with respect to degenerative and inflammatory diseases of the central and peripheral nervous system. In particular in terms of demyelinating diseases such as multiple sclerosis it is important to know the mechanisms which are responsible for the degeneration and destruction of axons. Here we focused on the loss or preservation of axons after induction of Wallerian degeneration in transected mouse sciatic nerves. We examined the distal transected nerve segments of different knockout mice (ICAM-1; TNF-alpha; iNOS; IL-6) 6 days after axotomy. Despite a distinct number of invading macrophages which phagocytosed most of the myelin and axonal debris, we were able to demonstrate, that animals which are deficient for the cell adhesion molecule ICAM-1 and the cytokine TNF-alpha showed a significantly higher number of preserved axons within the degenerating distal nerve stump. Since macrophage invasion is known to be impaired in the absence of ICAM-1, these data indicate an essential role of these cells and their secreted factors, namely TNF-alpha, but not nitric oxide or IL-6 in the destruction of the axonal cytoskeleton in the peripheral nervous system.     

Minocycline protects Schwann cells from ischemia-like injury and promotes axonal outgrowth in bioartificial nerve grafts lacking Wallerian degeneration

Minocycline, a broad-spectrum antimicrobial tetracycline, acts neuroprotectively in ischemia. Recently, however, minocycline has been revealed to have ambiguous effects on nerve regeneration. Thus its effects in a rat sciatic nerve transplantation model and on cultivated Schwann cells stressed by oxygen glucose deprivation (OGD) were studied. The negative effect of minocycline on Wallerian degeneration, the essential initial phase of degeneration/regeneration after nerve injury, that was recently demonstrated, was excluded by using predegenerated nerve and Schwann cell-enriched muscle grafts, both free of Wallerian degeneration. They were compared with common nerve grafts. The principle findings were that in vitro minocycline provided protective effects against OGD-induced death of Schwann cells by preventing permeability of the mitochondrial membrane. It suppressed the OGD-mediated induction of HIF-1α and BAX, and stabilized/induced BCL-2. Cytochrome c release and cleavage of procaspase-3 were diminished; release and translocation of AIF and cytotoxic cleavage of actin into fractin were stopped. In common nerve grafts, minocycline, besides its direct anti-ischemic effect, hampered revascularization by down-regulation of MMP9 and VEGF prolonging ischemia and impeding macrophage recruitment. In bioartificial nerve grafts that were free of Wallerian degeneration and revealed lower immunogenicity, minocycline aided the regeneration process. Here, the direct anti-ischemic effect of minocycline on Schwann cells, which are mandatory for successful peripheral nerve regeneration, dominated the systemic anti-angiogenic/pro-ischemic effects. In common nerve grafts, however, where Wallerian degeneration is a prerequisite, the anti-angiogenic and macrophage-depressing effect is an obstacle for regeneration.     

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.