Homeostatic regulation of the endoneurial microenvironment during development,aging and in response to trauma,disease and toxic insult

The endoneurial microenvironment, delimited by the endothelium of endoneurial vessels and a multi-layered ensheathing perineurium, is a specialized milieu intérieur within which axons, associated Schwann cells and other resident cells of peripheral nerves function. The endothelium and perineurium restricts as well as regulates exchange of material between the endoneurial microenvironment and the surrounding extracellular space and thus is more appropriately described as a blood–nerve interface (BNI) rather than a blood–nerve barrier (BNB). Input to and output from the endoneurial microenvironment occurs via blood–nerve exchange and convective endoneurial fluid flow driven by a proximo-distal hydrostatic pressure gradient. The independent regulation of the endothelial and perineurial components of the BNI during development, aging and in response to trauma is consistent with homeostatic regulation of the endoneurial microenvironment. Pathophysiological alterations of the endoneurium in experimental allergic neuritis (EAN), and diabetic and lead neuropathy are considered to be perturbations of endoneurial homeostasis. The interactions of Schwann cells, axons, macrophages, and mast cells via cell–cell and cell–matrix signaling regulate the permeability of this interface. A greater knowledge of the dynamic nature of tight junctions and the factors that induce and/or modulate these key elements of the BNI will increase our understanding of peripheral nerve disorders as well as stimulate the development of therapeutic strategies to treat these disorders.     

Ultrastructural and histophysiological studies on the blood-nerve barrier and perineurial barrier in leprosy neuropathy

Summary Onset and nature of ultrastructural changes in endoneurial vasa nervorum during the pathogenesis of leprosy neuropathy and possibly associated alterations in the blood-nerve barrier were investigated, together with perineurial barrier functioning, in mice infected 20–28 months previously withMycobacterium leprae and in (ageing) non-infected mice. Barriers were tested by i.v. administration of markers (Trypan blue and ferritin) 1–4 days before killing the mice.Twenty-eight months after infection, histopathology of sciatic nerves was comparable to that seen in sensory nerves in clinically early human (borderline-) lepromatous leprosy. Schwann cells and endoneurial macrophages were bacillated, endothelia of endoneurial vessels not, and the perineurium rarely.Many infected mice and all (ageing) controls possessed ultrastructurally and functionally normal endoneurial vessels. Their continuous endothelium with close junctions had prevented marker passage, even when surrounding endoneurial tissue cells were quite heavily bacillated. The perineurium was also normal.By contrast, in infected mice showing hind limb paralysis serious histopathologic involvement and large globi of bacilli intrafascicularly in sciatic nerves, endoneurial blood vessels were abnormal. Open endothelial junctions, extreme attenuation, fenestrations, and luminal protrusions were all features comparable to neural microangiopathy encountered in leprosy patients (Boddingius 1977a, b). The blood-nerve barrier clearly had become defective allowing excessive exudation of Trypan blue and ferritin, via four pathways from the vessel lumen, deep into surrounding endoneurial tissues but halted by a normal perineurial barrier. Markers in such blue nerves were not found in bacillated or non-bacillated Schwann cells, thus denying significant phagocytotic and lysosomal activities of Schwann cells at this stage of neuropathy. Possible implications of barrier performances for anti-leprosy drug treatment of patients are discussed.Supported by the British Medical Research Council (MRC), the British Leprosy Relief Association (LEPRA), the Wellcome Trust, and the Netherlands Leprosy Relief Association (NSL)     

Pure neuritic leprosy: Resolving diagnostic issues in acid fast bacilli (AFB)-negative nerve biopsies: A single centre experience from South India

Background and Purpose:

Demonstration of lepra bacilli is essential for definite or unequivocal diagnosis of pure neuritic leprosy (PNL) on nerve biopsy. However, nerves always do not show bacilli owing to the changes of previous therapy or due to low bacillary load in tuberculoid forms. In absence of granuloma or lepra bacilli, other morphologic changes in endoneurium and perineurium can be of help in making a probable diagnosis of PNL and treating the patient with multidrug therapy.

Materials and Methods:

Forty-six biopsies of PNL were retrospectively reviewed and histologic findings were compared with 25 biopsies of non leprosy neuropathies (NLN) including vasculitic neuropathy and chronic inflammatory demyelinating polyneuropathy (CIDP). The distribution of endoneurial infiltrate and fibrosis, perineurial thickening, and myelin abnormalities were compared between PNL and NLN biopsies and analyzed by Chi-square test.

Results:

Out of 46 PNL casses, 24 (52.17 %) biopsies were negative for acid fast bacilli (AFB). In these cases, the features which favor a diagnosis of AFB-negative PNL were endoneurial infiltrate (51.1%), endoneurial fibrosis (54.2%), perineurial thickening (70.8%), and reduced number of myelinated nerve fibers (75%).

Interpretation and Conclusion:

Nerve biopsy is an efficient tool to diagnose PNL and differentiate it from other causes of NLN. In absence of AFB, the diagnosis of PNL is challenging. In this article, we have satisfactorily evaluated the various hisopthological features and found that endoneurial inflammation, dense fibrosis, and reduction in the number of myelinated nerve fibers are strong supportive indicators of PNL regardless of AFB positivity.     

Perineurial cell basement membrane thickening and myelinated nerve fibre loss in diabetic and nondiabetic peripheral nerve

Diabetic neuropathy is associated with changes in the extracellular matrix of the perineurium, including thickening of the basement membrane of the perineurial cells. Peripheral vascular disease (PVD) is a common vascular condition that can occur in the absence or presence of diabetes. Thickening of the vascular basement membrane of the vasa nervorum is associated with both diabetes and nondiabetic peripheral vascular disease. However, perineurial cell basement membrane (PCBM) thickening in the nondiabetic PVD state has not, until now, been investigated. In this study, 36 nerve fascicles were examined from three patient groups: a diabetic group, a nondiabetic PVD group, and a group free of both PVD and diabetes (control group). PCBM thickness, fascicle size, and myelinated nerve fibre (MNF) density were measured in all three groups. Endoneurial blood vessels were also observed for evidence of morphological changes. The results showed that the thickness of the PCBM is significantly greater in the diabetic group in comparison with both the control and the nondiabetic PVD group, and this increase in thickness is linearly related to fascicle size. The thickness of the PCBM was not significantly different between the nondiabetic PVD and control groups. Although both the nondiabetic PVD and diabetic groups showed a loss of myelinated nerve fibres in comparison with the control group, this loss was statistically greater in the diabetic group. The endoneurial blood vessels of both the diabetic and nondiabetic PVD groups showed evidence of endothelial cell hyperplasia, hypertrophy, and basement membrane reduplication.     

Licht- und elektronenmikroskopische sowie cytometrische Untersuchungen an peripheren Nerven beim Morbus Tangier

Summary A sural nerve biopsy was performed in a 55-year-old male patient with Tangier disease (familiallipoprotein deficiency). Light-microscopy showed an increase in the endoneural connective tissue and a loss of nerve fibers indicating a chronic peripheral neuropathy. Electron-microscopy revealed an accumulation of lipid droplets within Schwann cells of myelinated and unmyelinated nerve fibers. When compared with age-matched controls the myelinated fiber density was reduced with a relative preponderance of small myelinated fibers. In addition, distributional cytometric studies of nerve vibers in relation to the perineurium and endoneurial capillaries showed: Contrary to 4.6–7.5 m thick nerve fibers, which accumulated in the center of the nerve fascicle, small (0.5–4.5 m) and large (7.6–10.0 m) fibers lay nearby the perineurium. The measured increase in small myelinated nerve fibers around endoneurial capillaries may be explained as a sign of regeneration.

     

THE BLOOD-NERVE BARRIER AND RECONSTITUTION OF THE PERINEURIUM FOLLOWING NERVE GRAFTING

Sural nerve autografts were performed on intact rat sural nerves and on sural nerves excised proximal to the site of grafting. The effect of the presence or absence of regenerating axons upon reconstitution of the perineurium at the graft junctions and upon re-establishment of the blood-nerve barrier to horseradish peroxidase (HRP) were studied over the succeeding 3--24 weeks. Compartmentation of the nerve fascicle occurred at the graft junctions where the perineurium was damaged. Each compartment contained Schwann cells with or without axons and was surrounded by elongated fibroblast-like cells which resembled perineurial cells in the longer surviving animals. It was concluded that, (a) compartments form in a nerve at the site of perineurial damage even in the absence of axons; (b) although compartmentation may be a mechanism for perineurial regeneration and reconstitution of the blood-nerve barrier, blood vessels and the cell layers forming compartments at graft junctions remain permeable to HRP for at least 6 months; and (c) the intact perineurium around the distal stump of a denervated nerve is permeable to HRP but the endoneurial blood vessels are not.     

Ultrastructural changes in blood vessels of peripheral nerves in leprosy neuropathy

Summary Radial or superficial peroneal nerve biopsies of 6 patients with tuberculoid or borderline-tuberculoid leprosy and 6 control nerve biopsies were examined by electron microscopy.Endoneurial blood vessels showed histopathology in all the leprosy patients. Changes, in particular, involved the basement membrane in postcapillary venules and venules. Multilayered parellel basement membranes, with collagen and ground substance, formed a thick coat (hyaline zone) around the vessels. It is suggested that the zone inhibits passage of nutrients and metabolites and, thus, contributes to or is the main cause of the local destruction of (unmyelinated) nerve fibres and the lack of nerve fibre regeneration observed in this type of leprosy. The perivascular zone, presumably, is produced by pericytes in response to defects in the blood-nerve barrier of endoneurial vessels. In granulomata of leprosy skin lesions, a perivascular zone was not present.The endothelium of endoneurial vessels, in affected nerves, generallywas normal. Occasionally, however, gaps and fenestrations were seen and there were histological indications that leakage of blood plasma had occurred through the gaps and through the basement membrane of the endothelium.Occlusion of endoneurial vessels was found only in the oldest patient and the degeneration of nerve fibres generally observed thus is considered not to be caused by ischaemia.Histopathology in epi-and perineurial vessels was definitely less pronounced than in endoneurial vessels.     

A comparison of perineurial and vascular basal laminal changes in diabetic neuropathy

Measurements were made of the thickness of the basal lamina of perineurial cells in the sural nerve in a series of patients with diabetic neuropathy and compared with a group of patients with type I hereditary motor and sensory neuropathy (HMSN) and with organ donor control cases. The thickness was significantly greater in the diabetic patients as compared both with the HMSN cases and the organ donor controls. This was most obvious for the intermediate layers of the perineurium. Perineurial basal laminal thickness was only slightly greater in the HMSN cases than in the organ donor controls and the difference was not statistically significant. The thickening of the perineurial cell basal laminae was compared with the thickening of the basal laminal zone around the endoneurial microvessels. No significant correlation was found either for the diabetic neuropathy or HMSN cases or for the organ donor controls. As had been observed previously, the basal laminal zone around the endoneurial capillaries was of increased thickness both in the diabetic neuropathy and the HMSN cases and, although it was greater for the diabetic neuropathy patients, the difference was not statistically significant. Taken together, these findings indicate that the thickening of the basal lamina of the perineurial cells in a more characteristic feature of diabetic neuropathy than is thickening of the basal laminal zone around the endoneurial capillaries. The results suggest that the causative mechanisms are likely to differ, a conclusion supported by the morphological appearances: the basal laminal thickening around the perineurial cells is uniform, whereas that around the capillaries consists of basal laminal reduplication. Atrophy and necrosis of perineurial cells were observed in patients with diabetic neuropathy but rarely in the cases with HMSN and not in the organ donor cases. This may be similar to the degeneration of endoneurial fibroblasts that has been described as a non-specific finding in neuropathies.Supported by the British Diabetic Association and in part by the Medical Research Council and grants from the Stanley Thomas Johnson Foundation, Ciba Geigy Ltd., Basel and Action Research     

Inner perineurial cell vulnerability in ischemia

The perineurium of peripheral nerve plays important roles in anatomical organization of fiber groups, in endoneurial fluid homeostasis and in maintenance of tensile strength but little is known about the functional and structural alterations of the perineurium with injury. Large arteries of supply to lower limb of Sprague-Dawley rats were ligated to study the structural reactions of perineurium at 36 h and at 7 days after induction of ischemic injury. Lipid droplets were found to be an early reactive change to ischemia in multiple cell types including perineurial, endothelial and Schwann cells. In peripheral nerve levels showing early myelinated fiber injury the inner perineurial sheath was widened and was undergoing degeneration. The inner layers of perineurial cells showed swelling, organelle disruption and membrane dissolution while outer layers remained intact. Inner perineurial cell degeneration is a prominent early feature of ischemic injury and may be an important mechanism of altered endoneurial homeostasis, fiber function and structure.     

Innervation of the vasa nervorum: changes in human diabetics.

Transperineurial and epineurial vessels are innervated by plexuses of unmyelinated axons. Human sural nerve biopsies were examined ultrastructurally and immunocytochemically with an antibody which recognizes a neuronal and neuroendocrine protein, PGP 9.5, to characterize perivascular axons of these plexuses. Diabetics exhibited a greater degree of abnormal innervation of the vasa nervorum than nondiabetics with and without neuropathy. Abnormal innervation included: a reduction in the percentage of vessels exhibiting perivascular axons and a concomitant increase in the percentage of vessels having denervated Schwann cell units, particularly around vessels confined to perineurial compartments, and remaining axons in nerves from diabetics exhibited fewer varicosities. Denervated arterioles of diabetics also displayed structural changes indicating injury. The arteriolar structural defects and loss of neurogenic control of neural blood flow may lead to or aggravate endoneurial ischemia or hypoxia. The patchy, focal endoneurial fiber loss that is prominent in proximal nerves and associated with the distal myelinated fiber loss of some diabetic patients may be due in part to perivascular denervation of the vasa nervorum.     

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.     

Perineurial cells filled with collagen in ‘atypical’ Cogan’s syndrome

Cogan’s syndrome is a rare clinical entity characterized by non-infectious interstitial keratitis with vestibuloauditory dysfunction. The clinical course is extremely variable. In the majority of patients, there appears to be an underlying systemic process, often a “vasculitis”. We were able to study for the first time a sural nerve biopsy of a 38-year-old female with clinically suggested Cogan’s syndrome associated with a severe multiplex type of neuropathy. There were unusual cells in or below the perineurium and along perineurial extensions into the endoneurium which were usually associated with blood vessels and which have thus far not been described in association with any type of peripheral neuropathy. The unusual cells were identified as perineurial cells because (1) they were frequently associated with the perineurium and its endoneurial extensions; (2) they were immunoreactive for antibodies against epithelial membrane antigen (EMA) but did not react with antibodies against protein S100, GFAP, and CD 68; and (3) they showed focally accumulated pinocytotic vesicles and hemidesmosomes. Some of these cells were clearly immunoreactive with antibodies against collagen VI. Electron microscopic examination revealed numerous intracellular bundles of collagen fibers which were surrounded by an amorphous basal lamina-like material, indicating that they were located within intracellular projections of the surface membrane. The number of myelinated and unmyelinated nerve fibers was severely reduced corresponding to the clinical manifestation of the neuropathy and to the atrophy, especially of the distal arm and leg muscles. It is concluded that the changes were caused by a special type of autoimmune reaction involving blood vessels and perineurial cells of peripheral nerves.     

Nerve regeneration through the cryoinjured allogeneic nerve graft in the rabbit

Summary To examine whether the 34-cm-long allogeneic basal lamina tubes of Schwann cells serve as conduits for regenerating axons in rabbits, allogeneic saphenous nerve, which had been predenervated and pretreated by freezing, were transplanted from Japanese White rabbits (JW) to New Zealand White rabbits (NW). Animals were killed 1, 2, 6, 8, and 14 weeks after transplantation, and the cytology at the mid-portion of the grafts was examined by electron microscopy. The distal portion of the host saphenous nerves was also examined 14 weeks after grafting. Myelin sheath debris was phagocytosed by macrophages, while the basal lamina of Schwann cells were left intact in the form of tubes. Regenerating axons were first found in such basal lamina tubes 2 weeks after grafting, and gradually increased in number. Host Schwann cells accompanied the regenerating axons behind their growing tips, separating them into individual fibers and forming thin myelin sheaths on thick axons by 6 weeks after grafting. Regenerating nerves were divided into small compartments by new perineurial cells. Newly formed blood vessels were situated outside the compartment 8 weeks after grafting. The percentage of myelinated fibers in the regenerating nerves was roughly 10% at 8 weeks and 30% at 14 weeks after grafting. The diameter of the regenerating axons, both myelinated and unmyelinated, was less than that of normal axons at all the stages examined. Numerous regenerating axons, some of which were fully myelinated, were found at the site 10 mm distal to the distal end of the graft 14 weeks after grafting. These results indicate that the Schwann cell basal lamina tubes of cryoinjured allogeneic nerves can serve as conduits for regenerating nerves in the 34-cm-long graft in the rabbit.     

The effect of extraction of the intrafascicular contents of peripheral nerve trunks on perineurial structure

Summary The intrafascicular contents have been extracted from the tibial nerve of the rabbit through perineurial incisions. Within 6–8 days following this procedure, the perineurial cells separate from one another, become dissociated from their basement membranes and assume a fibroblast-like appearance. The intrafascicular space becomes populated with endoneurial fibroblasts. With the ingrowth of regenerating axons, bundles of axons and associated Schwann cells become surrounded by cells of fibroblastic appearance which undergo perineurial transformation resulting in the development of multiple small fascicles. The cells of the surrounding perineurium appear to reassume a lamellar organization and to reestablish contacts with each other with the formation of junctional complexes. It is therefore suggested that neural structures may be responsible for the development and maintenance of the structural organization of the perineurium.     

Nerve cables formed in silicone chambers reconstitute a perineurial but not a vascular endoneurial permeability barrier.

The passage of molecules into the endoneurial environment of the axons of normal peripheral nerve is regulated by two permeability barriers, the perineurial-nerve barrier and the endoneurial blood-nerve barrier. These barriers exist because of the presence of tight junctions between adjacent perineurial cells and adjacent endothelial cells. In the present study we investigated whether permeability barriers form in nerve cables, which develop inside silicone chambers. The sciatic nerves of adult rats were cut, and the proximal and distal ends sutured into opposite ends of silicone chambers that were filled with dialyzed plasma. The presence of barriers was determined with the tracer horseradish peroxidase (HRP), which was injected intravenously and detected histochemically in tissues by light and electron microscopy. At four weeks, a regenerated nerve cable extended across the 10 mm length of each chamber. However, no permeability barriers were present since the reaction product for HRP was visible throughout the cable. At twenty-six weeks, all the axons in cables were gathered into minifascicles. Each minifascicle of axons was surrounded by perineurial cells. Blood vessels were excluded from the minifascicles by the perineurial cells and the vessels were permeable to HRP, thus indicating that their endothelial cells had not formed tight junctions. Despite the leakage of HRP from the excluded vessels, the tracer did not reach the axons because the perineurial cells encircling the minifascicles developed tight junctions. In some animals, the chambers were removed at four weeks to determine whether the chamber influenced barrier development. This manipulation had no effect since cables, with or without chambers, exhibited similar findings at twenty-six weeks. Our results indicate that nerve cables regenerate a perineurial but not an endoneurial permeability barrier. We conclude that axons in long-term cables are protected by only a perineurial permeability barrier.     

Endoneurial proliferation of perineurial cells in leprosy

Summary In leprous neuropathy the perineurium has very often an abnormal multilayered appearance and is infiltrated by many different types of inflammatory cells. We report here 13 cases characterized by an abnormal endoneurial proliferation of fibroblasts which seems to differentiate in perineurial cells. In several instances there is formation of many intrafascicular microcompartments. Such aspects have been described in various, but infrequent, cases of experimental and human neuropathies. It seems that severe Wallerian degeneration, diffuse endoneurial macrophage infiltration and lesion of the perineurium might lead to such a process.Supported in part by La Fondation pour la Recherche Médicale     

A study of the perineurium in peripheral nerve pathology

Summary The response of the perineurium to the following experimental systems was investigated by light and electron microscopy: nerve crush, cold lesion and microinjection of (a) histamine liberator, (b) potassium cyanide, (c) lysophosphatidyl choline (LPC). Where myelin breakdown occurred, lipid globules were seen within Schwann cells, macrophages and also perineurial cells. Where increased vascular permeability occurred, proteinaceous material leaked from endoneurial vessels into the endoneurial space and later appeared between perineurial laminae. It is suggested that the normal homeostatic function of the perineurium is extended in pathology to the removal of protein and lipid debris. In this way the perineurium contributes to the restoration of the normal microenvironment of peripheral nerve fibres.A synopsis of this paper was presented at the VIIth International Congress of Neuropathology, Budapest.     

Microangiopathy of endoneurial vessels in hypoxemic chronic obstructive pulmonary disease (COPD)

Summary A peripheral neuropathy has been reported in patients with chronic respiratory insufficiency due to chronic obstructive pulmonary disease (COPD). It is mainly characterized by axonal degeneration, secondary demyelination and abnormal endoneurial vessels. The pathogenesis of these lesions remains obscure. To investigate whether relationships exist between neuritic and vascular lesions, a qualitative and quantitative ultrastructural study was performed on nerve biopsies in 13 patients with chronic respiratory insufficiency due to COPD, and in 9 normal controls without pulmonary lesions. A computer-assisted multiple regression analysis taking into account clinical, electrophysiological, biological and morphometric parameters was performed. Statistically significant differences in the endoneurial structure of microvessels were: (1) thickening of the basement membrane; (2) narrowing of the lumen; (3) mural pericytic debris deposits, occurring in the COPD group. In the latter, hypercapnia correlated positively with nerve fibers lesions (P=0.03) and endothelial area (P=0.03). No correlations were found between age and other parameters. These findings highlight the fact that the microangiopathy in peripheral nerves in patients with COPD may be diffuse and essentially due to hypoxia and reduction in blood flow, as in diabetic neuropathy.