Schwann cell changes and demyelination in chronic galactose neuropathy

Dystrophic changes of Schwann cells and demyelination occurred in rats with chronic nerve edema induced by feeding a galactose-rich diet for two years. The mechanism for edema is the sorbitol pathway which generates osmotically active polyols from galactose or glucose. The blood-nerve barrier impedes diffusion of macromolecules from peripheral nerves, and endoneurial fluid pressure (EFP) becomes elevated. After 24-26 months of feeding with 40% galactose diet, myelinated nerve fibers showed segmental demyelination with bubbly disintegration of myelin sheaths, axonal degeneration, and remyelination. These pathologic changes were significantly more common than similar abnormalities in age-matched controls. Massive glycogen accumulation in Schwann cells, a unique morphologic finding, appeared only in experimental rats. Since edema and increased EFP are the earliest pathologic changes and are present for months before demonstrable nerve fiber injury, we suggest that they are responsible for the changes of myelinated fibers in chronic galactose neuropathy.     

Ischemic neuropathy

Ischemia plays an important role in the development of neuropathies associated with various disorders, such as peripheral vascular occlusive diseases, necrotizing vasculitides, diabetes mellitus and nerve compression or trauma. Although a multiple mononeuropathy or an asymmetrical polyneuropathy is the usual clinical presentation of ischemic neuropathy, some patients present with a neuropathy that is mainly distal and symmetrical. Pathologically, nerve ischemia results in focal or multifocal central fascicular or sector fiber degeneration. These ischemic lesions tend to begin at mid-upper arm or midthigh level, which is the watershed zone of poor perfusion, and become more diffuse distally. Nerve ischemia at the level of distal small fascicles often induces sub-perineurial crescent lesion rather than central fascicular fiber degeneration. Physiologically, reduced nerve blood flow with endoneurial hypoxia has been demonstrated in experimental diabetic and galactose neuropathies. Endoneurial ischemia/hypoxia in galactose neuropathy appears to be due to increased intercapillary distances and constriction of trans-perineurial vessels resulting from endoneurial edema. Although acute ischemic neuropathy has been well investigated, little is known about functional or structural responses of peripheral nerve to chronic ischemia.     

Reduced nerve blood flow in hexachlorophene neuropathy: relationship to elevated endoneurial fluid pressure

To test the hypothesis that increased endoneurial fluid pressure (EFP) causes a reduction in nerve blood flow (NBF) in the vasa nervorum, we adapted a noninvasive method for measurement of nerve blood flow which was originally developed for measurement of local cerebral blood flow. This technique measures tissue distribution to the radioisotope, 14C-iodoantipyrine, and was used to compare NBF in sciatic nerves of rats with increased EFP induced by feeding them hexachlorophene (HCP), a neurotoxin which causes edema exclusively to the nervous system and confined to the myelin sheath. Elevation of interstitial fluid pressure in peripheral nerves from control values of 2.0 +/- 1.0 cm H2O to over approximately 6 cm H2O was associated with a statistically significant reduction in nerve blood flow from 14.8 +/- 5.9 to 7.8 +/- 2.5 ml/100 g of tissue/minute (min). These results support the hypothesis that increased endoneurial fluid pressure exacerbates the neuropathy by diminishing local blood flow.     

Galactose neuropathy

Summary Galactose neuropathy is characterized by progressive endoneurial edema manifested by a gradual increase in endoneurial fluid pressure. Edema accumulates via a unique mechanism of osmotic force generated by products of the polyol pathway, synthesized within the endoneurial compartment. This paper presents morphologic findings showing firstly, that blood nerve barrier permeability to horseradish peroxidase complexes appears unchanged and secondly, peripheral nerve edema in this condition is restricted to extraganglionic endoneurium sparing the spinal ganglia and adjacent roots. Thirdly, mast cells accumulated in significant numbers and electron microscopy revealed degranulation. There was no evidence of edema in Schwann cell cytoplasm, the putative site of galactitol accumulation via the sorbitol pathway. These findings are discussed with respect to diabetic neuropathy for which galactose intoxication is a useful experimental model.Supported by National Institute of Health, NINCDS 14162, the Veterans Administration Research Service, and aided by a Basil O'Connor Starter Research Grant from the March of Dimes, Birth Defects Foundation     

Chronic elevation of endoneurial fluid pressure is associated with low-grade fiber pathology

The effects of chronic elevations of endoneurial fluid pressure (EFP) on mammalian nerve fibers were studied using a modified model of experimental galactose neuropathy. Fiber pathology was monitored by the sensitive method of grading the pathologic abnormalities of single teased fibers. Prominent edema was produced in rats poisoned with combined oral and parenteral galactose. EFP and fascicular size were markedly elevated and tibial nerve conduction velocity was reduced. We have demonstrated, for the first time, the presence of fiber degeneration in galactose fed rats. Possible mechanisms of nerve fiber damage include: (1) impaired capillary circulation and increased fiber separation secondary to increased EFP and edema, respectively; (2) increased endoneurial hyperosmolarity, which is known to cause changes in fiber shape; and (3) unknown metabolic derangement of axons. Because much higher rates and different types of fiber pathology are encountered in lead and hexachlorophene intoxication having comparable degrees of EFP, one cannot attribute the fiber pathology directly to the raised EFP in these latter neuropathies.     

Changes in endoneurial fluid pressure, permeability, and peripheral nerve ultrastructure in experimental lead neuropathy

The dynamics of endoneurial edema were studied by quantifying endoneurial fluid pressure (EFP) during the development of lead neuropathy and correlating these data with changes in blood-nerve barrier permeability and with morphological alterations in nerves, capillaries, and Schwann cells. EFP measured from the sciatic nerve in control Long-Evans rats was 2.1 +/- 1.0 cm H2O. EFP was significantly elevated 7 weeks after animals were started on a diet containing 6% lead carbonate, and it increased progressively until a plateau in pressure was reached between weeks 9 and 11. Thereafter, EFP gradually returned to normal values. The progressive increase in EFP was highly correlated with the extravasation of osmotically active macromolecules, traced by fluorescein isothiocyanate-dextran compounds of graded molecular weight and by horseradish peroxidase (HRP). Electron microscopy revealed extravasation of HRP between endothelial cells, intranuclear inclusions characteristic of lead poisoning in Schwann cell nuclei, demyelination, and remyelination. The observation of intranuclear inclusions consistent with lead deposition in Schwann cells strengthens the hypothesis that extravasated lead in the interstitial fluid causes direct injury to Schwann cells, giving rise to demyelination. Nerve compliance was determined.     

Axonal caliber and neurofilaments are proportionately decreased in galactose neuropathy

Feeding galactose to rats induces nerve conduction abnormalities, increased levels of nerve galactitol, endoneurial edema, elevated pressure and hypoxia of endoneurial fluid, and pathological abnormalities of nerve fibers. To investigate the cellular mechanisms of the fiber lesions and their possible relationship to alterations in the nerve microenvironment, rat peroneal nerves were morphometrically evaluated eight months after the commencement of galactose feeding. Whereas the density of neurofilaments (NF/micron2) in the transverse axonal area of myelinated fibers was not significantly different between the nerves of galactose-fed and control rats, axonal areas and the number of NF/axon, when related to myelin spiral length, were significantly less in nerves of galactose-fed rats. Myelin alterations, characteristic of axonal atrophy, were also significantly increased. The present data provide evidence of a proportionate decrease in axonal caliber and the number of NF/axon in myelinated fibers in experimental galactose neuropathy, suggesting that galactose induces fibers in experimental galactose neuropathy, suggesting that galactose induces either decreased NF synthesis, assembly or transport. The possible role of microenvironmental alterations, including endoneurial hypoxia and hyperosmolarity, in the production of this axonal atrophy is discussed.     

Experimental germinaium dioxide-induced neuropathy in rats

We report and experimental model of germanium dioxide (GeO₂)-induced neuropathy in rats. More than 6 months administration of GeO₂ to young rats produced neuropathy characterized by segmental demyelination/remyelination and nerve edema. Electron microscopic studies demonstrated that changes in Schwann cells, such as an increased cytoplasmic volume or disintegration of the cytoplasm, were the earliest pathological findings. Schwann cell mitochondria contained high electron-dense materials. Subsequent removal of necrotic Schwann cell debris and myelin by invading macrophages was evident. These findings suggested that the Schwann cells themselves are the primary target of the toxin. The deposition of electron-dense granules in the intra-axonal vesicles, which was suggestive of glycogen granules in mitochondria, was observed in the advanced stage of the neuropathy. The findings of endoneurial edema with splitting of myelin lamellae were noted at the early stage of demyelination. Nerve edema may be the result of GeO₂-induced endothelial cell injury.Supported by a grant from the National Center of Neurology and Psychiatry of the Ministry of Health and Welfare, Japan     

Dose-dependence of endoneurial fluid sodium and chloride accumulation in galactose intoxication

Endoneurial edema in galactose neuropathy was studied in a colony of Sprague-Dawley rats fed diets containing 0%, 10%, 20% or 40% D-galactose for approx. 200 days. Endoneurial fluid was analyzed by X-ray microanalysis for electrolyte concentration, by microgravimetry of whole nerve segments for water content, by measurement of endoneurial fluid pressure and by morphometry in transverse sections of nerve. Galactose intoxication resulted in dose-dependent increases in endoneurial fluid sodium and chloride that were directly associated with increases in nerve water content and endoneurial fluid pressure. The presence of edema and its dose-dependence was also confirmed by morphometric analysis of sciatic nerves at the light microscopic level. The data demonstrate that electrolyte-induced osmotic imbalances in endoneurial fluid are dependent on the amount of galactose ingested and suggest that the dose-related accumulation of sodium and chloride in endoneurial fluid contributes substantially to the pathogenesis of galactose neuropathy.     

Temporal relationship of blood-nerve barrier breakdown to the metabolic and morphologic alterations of tellurium neuropathy

The appearance of endoneurial edema early in the evolution of tellurium neuropathy raises the possibility that a breakdown of the blood-nerve barrier (BNB) plays a role in the pathogenesis of the tellurium-induced demyelination. To investigate this possibility, we correlated the temporal onset of breakdown of the BNB with inhibition of cholesterol synthesis and ultrastructural abnormalities in sciatic nerve of weanling Long-Evans rats fed a diet containing 1.1% elemental tellurium. Permeability of the BNB was assessed with [125I]-albumin and horseradish peroxidase (HRP); cholesterol synthesis was assessed by incubating segments of sciatic nerve in vitro with [1-14C]acetate. Cholesterol synthesis was severely inhibited and labeled squalene was accumulating in sciatic nerve at 12 hr of tellurium exposure. The permeability of the BNB progressively increased between 24 hr and 72 hr of tellurium exposure. Membrane-delimited vacuoles, lipid droplets and cytoplasmic excrescences appeared in myelinating Schwann cells at 24 hr; demyelinating axons appeared at 48 hr of tellurium exposure. These observations suggest that factors other than BNB breakdown and vasogenic endoneurial edema are responsible for the initial Schwann-cell injury in tellurium neuropathy. However, the early onset of BNB breakdown may have a synergistic role in the pathogenesis of tellurium-induced demyelination.     

Cellular pathology of the nerve microenvironment in galactose intoxication

The effect of chronic hyperglycemia and polyol pathway activation on the Schwann cell has not been resolved although injury to this cell has long been suspected in diabetic neuropathy. Hyperglycemia, resulting from galactose intoxication of four months duration, induces dose-dependent accumulations of endoneurial fluid sodium and chloride that are linked to polyol pathway activity and associated with dose-dependent increases in sciatic nerve water content, endoneurial fluid pressure and (Na+, K+)-ATPase activity. In order to understand the impact of these changes on the nerve microenvironment, cellular elements of the endoneurium were quantitatively and qualitatively assessed in rats receiving 0%, 10%, 20% or 40% galactose diets. After four months of galactose intoxication, dose-dependent changes in the size distribution of myelinated nerve fibers were apparent. A shift in size-frequency histograms of galactose-intoxicated animals towards smaller fibers was accompanied by a decrease in axon diameter and the volume fraction ratio of axon to myelinated nerve fibers. In the sciatic nerve of all 40% galactose-fed rats examined by electron microscopy, Schwann cells of myelinated fibers showed both reactive and degenerative changes. Demyelination was preceded by splitting at the intraperiod line. Remyelination was identified by axons with disproportionately thin myelin sheaths. Axonal dystrophy and degeneration were infrequently seen, but there was axonal regeneration. Dose-dependent increases in mast cell number were observed with degranulation apparent in rats receiving 20% and 40% galactose. Endothelial cell number and basal lamina thickness were increased in the endoneurial vessels of galactose-intoxicated rats. Increased cytoplasmic area and degenerative changes in pericytes were also noted. These observations indicate that significant morphologic changes accompany the hyperosmotic imbalance resulting from galactose intoxication of four months duration. Schwann cell injury and demyelination are present in a disorder linked to polyol metabolism since aldose reductase, the anabolic enzyme of the polyol pathway, is localized to this myelin-forming cell.     

Pathology of local anesthetic-induced nerve injury

Summary Nerve fiber injury and endoneurial edema were induced by the injection of the local anesthetic 2-chloroprocaine, tetracaine, procaine, etidocaine or mepivacaine into the soft tissue and fascia surrounding the sciatic nerve of Sprague-Dawley rats. Light microscopy demonstrated that the perineurial barrier was not mechanically damaged by the surgical procedure but, at 48 h post-injection, perineurial permeability was increased. Previous observations of leakage of horseradish peroxidase and the present report of neutrophils and eosinophils in the endoneurium indicate a disruption of blood-nerve barrier systems. Endoneurial edema was observed in the subperineurial, interstitial and perivascular regions. Axonal degeneration and demyelination occurred; the latter associated with accumulation of large lipid droplets in Schwann cells. Degranulation of mast cells, proliferation of fibroblasts and macrophage activity were noteworthy in affected areas. The findings are remarkable in that this is the first model of endoneurial edema by a neurotoxin which penetrates the perineurium, disrupting barrier system and inducing nerve fiber injury.Supported in part by USPHS NS18715, NS14162 and NS07078 and the Veteran's Administration Research Service     

Characterization of nerve and microvessel damage and recovery in type 1 diabetic mice after permanent femoral artery ligation

Neuropathy is the most common complication of the peripheral nervous system during the progression of diabetes. The pathophysiology is unclear but may involve microangiopathy, reduced endoneurial blood flow, and tissue ischemia. We used a mouse model of type 1 diabetes to study parallel alterations of nerves and microvessels following tissue ischemia. We designed an easily reproducible model of ischemic neuropathy induced by irreversible ligation of the femoral artery. We studied the evolution of behavioral function, epineurial and endoneurial vessel impairment, and large nerve myelinated fiber as well as small cutaneous unmyelinated fiber impairment for 1 month following the onset of ischemia. We observed a more severe hindlimb dysfunction and delayed recovery in diabetic animals. This was associated with reduced density of large arteries in the hindlimb and reduced sciatic nerve epineurial blood flow. A reduction in sciatic nerve endoneurial capillary density was also observed, associated with a reduction in small unmyelinated epidermal fiber number and large myelinated sciatic nerve fiber dysfunction. Moreover, vascular recovery was delayed, and nerve dysfunction was still present in diabetic animals at day 28. This easily reproducible model provides clear insight into the evolution over time of the impact of ischemia on nerve and microvessel homeostasis in the setting of diabetes. © 2015 Wiley Periodicals, Inc.     

Edema and increased endoneurial sodium in galactose neuropathy. Reversal with an aldose reductase inhibitor

Galactose neuropathy was produced in rats by feeding a diet containing 30% D-galactose. After 12 weeks of galactose ingestion, all rats developed bilateral cataracts, polydypsia and polyuria. These galactose-intoxicated animals were divided into two groups that both continued with the galactose diet: animals that were treated with the aldose reductase inhibitor, ICI 128,436, for 4-6 weeks, and a control group of animals that received just excipient. At the end of the study, endoneurial fluid pressures, nerve water contents and endoneurial fluid electrolyte concentrations were determined from sciatic nerves of treated and untreated animals. The extent of neuropathy in each animal was evaluated by light microscopy. Treatment of galactose-intoxicated rats with ICI 128,436 restored to normal levels the elevated endoneurial sodium concentration, increased water content and interstitial fluid pressure characteristic of galactose neuropathy. These results, obtained with an agent that blocks the sorbitol pathway, associate elevated sodium with an osmotic force contributing to edema and increased endoneurial fluid pressure in galactose neuropathy and suggest that endoneurial sodium levels are linked to blood-sugar concentration.     

Chronic constriction model of rat sciatic nerve: nerve blood flow,morphologic and biochemical alterations

We evaluated the nerve blood flow (NBF), light and electron microscopy, and adrenergic innervation of rat sciatic nerve at 2–45 days after the application of four loose ligatures. Ischemia developed at the lesion edge, creating an endoneurial dam. Calcitonin gene-related peptide, norepinephrine and NBF were increased within the lesion. Morphologic alterations consisted of early endoneurial edema, followed by myelinated fiber degeneration, with relative sparing of small myelinated and unmyelinated nerve fibers, and leukocyte adhesion to microvessels. Axonal degeneration predominated over demyelination. At 45 days, profuse regeneration of small myelinated fibers was seen. The mechanism of lesional sensitization is discussed. Received: 30 May 1996 / Revised, accepted: 13 August 1996     

Hypertension-induced peripheral neuropathy and the combined effects of hypertension and diabetes on nerve structure and function in rats

Diabetic neuropathy includes damage to neurons, Schwann cells and blood vessels. Rodent models of diabetes do not adequately replicate all pathological features of diabetic neuropathy, particularly Schwann cell damage. We, therefore, tested the hypothesis that combining hypertension, a risk factor for neuropathy in diabetic patients, with insulin-deficient diabetes produces a more pertinent model of peripheral neuropathy. Behavioral, physiological and structural indices of neuropathy were measured for up to 6 months in spontaneously hypertensive and age-matched normotensive rats with or without concurrent streptozotocin-induced diabetes. Hypertensive rats developed nerve ischemia, thermal hyperalgesia, nerve conduction slowing and axonal atrophy. Thinly myelinated fibers with supernumerary Schwann cells indicative of cycles of demyelination and remyelination were also identified along with reduced nerve levels of myelin basic protein. Similar disorders were noted in streptozotocin-diabetic rats, except that thinly myelinated fibers were not observed and expression of myelin basic protein was normal. Superimposing diabetes on hypertension compounded disorders of nerve blood flow, conduction slowing and axonal atrophy and increased the incidence of thinly myelinated fibers. Rats with combined insulinopenia, hyperglycemia and hypertension provide a model for diabetic neuropathy that offers an opportunity to study mechanisms of Schwann cell pathology and suggests that hypertension may contribute to the etiology of diabetic neuropathy.     

Class II antigen expression in peripheral neuropathies.

The expression of class II antigen was studied in sural nerve biopsies from patients with peripheral neuropathies. These included patients with chronic demyelinating polyradiculoneuropathy (CIDP), non-immune mediated neuropathies of diverse etiologies and controls without evidence of neuropathy. The major finding in CIDP was a marked increase in class II expression on Schwann cells. Endoneurial Schwann cell staining to the same degree as in CIDP was seen in diabetic symmetric proximal motor neuropathy, neuropathies associated with monoclonal gammopathies and hereditary sensory and autonomic neuropathy type 1. In the control nerves and the other non-immune mediated neuropathies class II expression was mainly restricted to endothelial and perineurial cells. Increased endoneurial expression of class II antigen was found to correlate with elevated cerebrospinal fluid (CSF) protein levels but not with other clinical variables or demyelination as defined by electrophysiologic criteria or teased fiber analysis. The increased expression of class II antigen on Schwann cells may be indicative of a breakdown in immunological tolerance but should not be used as a diagnostic marker for dysimmune neuropathies due to overlap with non-immune mediated neuropathies.     

Peripheral neuropathy of dietary riboflavin deficiency in chickens

A strain of rapidly growing meat-type chickens was fed a diet deficient in riboflavin from 1-40 days of age. Diminished growth rate, progressive gait abnormality and reluctance to move were noted beginning on day 8. Neurologic abnormalities were related to peripheral neuropathy characterized by Schwann cell hypertrophy and degeneration with cytoplasmic lipid droplets' and segmental demyelination. Lesions were initially detected on day 10, and in concert with clinical signs became more profound between days 14 and 21. Sequestration of myelin debris within Schwann cells was common. Other features of the neuropathy included the presence of endoneurial edema and axonal degeneration involving small numbers of fibers. Remyelination of peripheral nerve fibers in birds on the deficient diet was occasionally seen on day 10, became progressively more prominent, and was marked by day 37. There was an associated, variable but incomplete, clinical improvement evident in later stages of the study. Liver concentrations of riboflavin in deficient birds were significantly reduced on day 13 but not on day 26. This neuropathy may be related to diminished tissue levels of the riboflavin-based coenzymes flavin-adenine dinucleotide (FAD) and flavin mononucleotide (FMN) leading to reduced cellular energy levels and profoundly affecting Schwann cells at some critical point in growth.     

Peripheral nerve pathological findings in familial amyloid polyneuropathy: a correlative study of proximal sciatic nerve and sural nerve lesions

To analyze the peripheral nerve pathological abnormalities in familial amyloid polyneuropathy, a correlative pathological study was carried out on the spinal nerve roots, proximal sciatic nerves, sural nerves, and brachial plexuses from 3 patients with the disease in Japan. The spinal nerve roots appeared to be unaffected except for amyloid deposition on the epineurium. In sciatic nerves and brachial plexuses the nerve lesions had a multifocal distribution, showing prominent interstitial edema in the endoneurium frequently adjacent to deposits of amyloid; in these regions the nerve fibers were severely depleted. A teased-fiber study revealed that segmental demyelination was the predominant type of nerve fiber abnormality. However, these findings were not seen in the sural nerves; instead a diffuse fiber loss with axonal degeneration was observed. It is suggested that multifocal lesions in the proximal portions of the long extremity nerves could summate distally to produce a symmetrical polyneuropathy in the disease. In addition to a space-occupying effect of amyloid deposits in the endoneurium, severe endoneurial edema associated with amyloid deposition in blood vessels and the endoneurial interstitium may induce ischemia in nerve fibers, thus causing the progressive polyneuropathy in this disorder.     

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.