Response of the axon and barrier endothelium to experimental allergic neuritis induced by autoreactive T cell lines

Summary Experimental allergic neuritis was induced in Lewis rats by inoculation with autoreactive T cell lines senisitized to residue 57–81 of P₂ myelin protein. Control rats received cells derived from immunization to complete Freund's adjuvant alone. Endoneurial fluid pressure (EFP) was measured in both sciatic nerves at 0, 3, 5, 7, 9, and 11 days post-inoculation (PI). The temporal evolution of inflammatory disease was studied by correlating EFP with a morphometric analysis of the nerve microenvironment and with electron microscopic observations. Both edema, as evidenced by increased endoneurial extracellular space, and inflammation paralleled the time course of the EFP increase, reaching peak values at 7 days PI and declining to near-normal values after 11 days. Wallerian degeneration was detectable at 7 days and increased 9 days after inoculation. Axonal damage appeared at the height of the inflammatory process, when edema and increased EFP were maximal. Evidence of demyelination was apparent by 7 days and persisted through 11 days. The onset of edema was associated with changes in venular endothelial cells which tended to lose their normal scaphoid appearance and assumed rhomboid configurations reminiscent of high endothelial venules. At that point, the barrier endothelium was visibly disrupted with the loss of tight junctions and separation of adjacent cells. Specific cell-cell interactions took place between endothelial cells and infiltrating leukocytes as they immigrated into the endoneurial compartment. There was evidence of altered perineurial permeability with fibrin deposition and leukocyte infiltration between the layers of the perineurial sheath.Supported in part by NIH grants NS11867, NS14162, NS18715, the Juvenile Diabetes Foundation International and the Veterans Administration Research Service     

Endoneurial fluid pressure in wallerian degeneration.

Endoneurial fluid pressure (EFP) was recorded by an active, servo-null pressure system after a glass micropipette was inserted into rat sciatic nerve undergoing wallerian degeneration. The lesions were produced by crushing the left sciatic nerve of the anesthetized animal at its point of entry into the thigh. Eighty-four animals were employed in this experiment, in which EFP was recorded from sham-operated rats and other controls as well as from rats with wallerian degeneration. The experiment was designed so that EFP could be recorded from 2 or more experimental animals at daily intervals starting at day 0 and concluding on day 28. Pressure progressively increased during the first week, reaching a peak elevation four to five times normal. The subsequent decline in EFP was more gradual, with values approaching normal during the third week after injury. Linear regression analysis showed the progressive increase in EFP to be statistically significant (p less than or equal to 0.01). To determine the time at which EFP was maximum, we used the Marquardt computer algorithm for lease-squares estimation of nonlinear variables. By this procedure the peak value for EFP occurred at six days. These biophysical observations were correlated with subsequent microscopic examination of 1 mu thick sections of Araldite-embedded sciatic nerve. Microscopy confirmed the presence of wallerian degeneration associated with edema, which was observed in every instance of elevated EFP.     

Endoneurial pressure in hexachlorophene neuropathy

Summary Increased endoneurial pressure of up to 17.0 cm H₂O was recorded in the peripheral nerves of rats fed hexachlorophene in their laboratory diet. The pressure was measured using a micropressure transducer developed for recording pressure in the microcirculation. The results were correlated with morphologic findings. Teased nerve fibers and aralditeembedded specimens of hexachlorophene damaged sciatic nerve revealed the characteristic severe intramyelinic edema due to splits in the minor dense lines of compact myelin giving rise to wide interlamellar spaces as shown in previous studies. The endoneurial pressure of rats exposed to hexachlorophene for 11 days and subsequently fed a normal diet returned to normal (0.2–3.0cm H₂O) after 12 days, and morphologic examination showed few residual abnormalities. Prolonged exposure to hexachlorophene for up to 4 weeks caused widespread axonal degeneration in addition to intramyelinic edema. Animals treated with hexachlorophene for 21 days followed by a normal diet for 14 days showed degenerated axons, phagocytosis of myelin as well as interstitial edema and elevated endoneurial pressure. It is suggested that axonal degeneration in hexachlorophene neuropathy is caused by increased endoneurial pressure.Supported in part by NS 09053 and by NS 14162 from National Institute of Neurological Disorders and Communicative Disorders and Stroke and HL 10881 from the National Heart and Lung Institute     

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.     

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.     

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.     

Laser injury of peripheral nerve: a model for focal endoneurial damage.

A model of endoneurial oedema is presented which produces localised nerve fibre injury including Wallerian degeneration, endoneurial oedema and increased endoneurial fluid pressure. A carbon dioxide laser was used to irradiate rat sciatic nerve and quantitative results were collected 2 days and 6 days after irradiation. The increase in endoneurial fluid pressure is compared with other models of Wallerian degeneration in which the entire fascicle was involved to aid in a comparative study of the role of oedema in neuropathy.     

Changes in the blood-nerve barrier after sciatic nerve cold injury:indications supporting early treatment

Severe edema in the endoneurium can occur after non-freezing cold injury to the peripheral nerve, which suggests damage to the blood-nerve barrier. To determine the effects of cold injury on the blood-nerve barrier, the sciatic nerve on one side of Wistar rats was treated with low temperatures(3–5°C) for 2 hours. The contralateral sciatic nerve was used as a control. We assessed changes in the nerves using Evans blue as a fluid tracer and morphological methods. Excess fluid was found in the endoneurium 1 day after cold injury, though the tight junctions between cells remained closed. From 3 to 5 days after the cold injury, the fluid was still present, but the tight junctions were open. Less tracer leakage was found from 3 to 5 days after the cold injury compared with 1 day after injury. The cold injury resulted in a breakdown of the blood-nerve barrier function, which caused endoneurial edema. However, during the early period, the breakdown of the blood-nerve barrier did not include the opening of tight junctions, but was due to other factors. Excessive fluid volume produced a large increase in the endoneurial fluid pressure, prevented liquid penetration into the endoneurium from the microvasculature. These results suggest that drug treatment to patients with cold injuries should be administered during the early period after injury because it may be more difficult for the drug to reach the injury site through the microcirculation after the tissue fluid pressure becomes elevated.     

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     

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     

Peripheral nerve ischemia: reperfusion injury and fiber regeneration

We continued our studies of ischemia-reperfusion (IR) injury, extending the reperfusion duration to 42 days to capture the fiber regeneration process. We used a rat model for IR injury produced by ligation and release of nooses around supplying vessels to the sciatic nerve. Fifty-six rats were used. One group (control N = 8) underwent sham ischemia; the other six groups (N = 8 each) underwent complete hind limb ischemia for 4 h followed by reperfusion durations of 0 h (ischemia alone), 3 h, 7 days, 14 days, 28 days, and 42 days. Behavioral and electrophysiological data were obtained immediately before euthanasia. Pathologically, three phases were identifiable: Phase 1 (0-3 h)-minimal pathological changes, minimal edema; phase 2 (7 days, 14 days)-prominent fiber degeneration, endoneurial edema; phase 3 (28 days, 42 days)-abundant small regenerating fiber clusters, minimal edema. Compound muscle action potential (CMAP) was the most sensitive index of neural deficits and recovery, showing progressive recovery beyond 14 days. Severe functional deficits developed immediately and persisted with a trend to recovery at the 42-day time-point. It was concluded that reperfusion, by oxidative injury, worsened nerve function and aggravated fiber degeneration, but in the longer time frame, permitted fiber regeneration to occur.     

Orchestration of the inflammatory response in ischemia-reperfusion injury

Ischemia to nerve can cause fiber degeneration and reperfusion following ischemia [ischemia-reperfusion (IR)] adds the additional insult of an inflammatory response and oxidative injury. Limited information is available on the molecular mediators and their endoneurial targets. In this study, using a highly reproducible animal model of IR injury to nerve and selective immunolabeling methods [for nuclear factor kappa B (NF-kappaB), intercellular adhesion molecule-1 (ICAM-1), cytokines, and inflammatory cells] over an expanded time frame, we evaluated the temporal pattern and localization of mediators of the inflammatory response. Sixty rats were used. Nine groups (N=6 each) underwent complete hind limb ischemia for 4 h, followed by reperfusion durations of 0, 3, 12, 24, and 48 h, and 7, 14, 28, and 42 days. One group underwent sham operation (N=6). The earliest change was ICAM-1 expression in the microvessel (endothelial cell) followed almost immediately by NF-kappaB activation with axonal expression (24 and 48 h), followed by endoneurial edema and ischemic fiber degeneration (7 and 14 days). Granulocytic infiltration was followed by endoneurial infiltration of mononuclear phagocytes (14 days), expression of interleukin 6 (IL-6) (microvessels), and subsequent Schwann cell NF-kappaB expression. Granulocytes, tumor necrosis factor alpha, and IL-6-positive cells were observed primarily within the epineurium. IR results in changes in a number of interacting networks of targets and inflammatory mediators. NF-kappaB activation has a central orchestrating role involving both the axon and the Schwann cell in effecting the inflammatory response.     

Galactose neuropathy: impact of chronic endoneurial edema on nerve blood flow

Peripheral neuropathy induced by galactose feeding results in endoneurial edema with increased tissue pressure and ultimate demyelination of nerve fibers. To assess the role of ischemia in the pathogenesis of this neuropathy, nerve blood flow was measured 6 months after the start of galactose ingestion, between the onset of edema and nerve fiber changes. Measurement was carried out by a noninvasive technique involving the use of [14C]iodoantipyrine as a radioactive tracer of tissue perfusion. A significant decline in nerve blood flow was found, which correlated positively with increased nerve water content as quantified by a microgravimetric technique. We sought to establish the pathogenic role of nerve edema in the development of schwannopathy and demyelination by morphological examination. It was found that Schwann cells appeared normal in areas of the peripheral nervous system in which edema does not occur, such as spinal ganglia and roots, whereas in regions in which there was edema, massive glycogen accumulation in Schwann cells and demyelination occurred. These findings suggest that edema, rather than hyperactivity in the sorbitol pathway, is responsible for the pathological changes in galactosemic neuropathy and that ischemia resulting from edema and increased endoneurial fluid pressure is the mechanism responsible for fiber injury.     

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.     

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.     

Endoneurial sodium accumulation in galactosemic rat nerves

Microdrop samples of endoneurial fluid and serum from galactosemic rats were analyzed with energy dispersive spectrometry (EDS). The sodium concentration of endoneurial fluid from galactose-fed rats (295 meq/liter) was nearly twice that of age-matched controls (152 meq/liter) and indicates that sodium is present in an osmotically significant concentration that is capable of causing the edema and increased endoneurial fluid pressure (EFP) associated with galactose neuropathy. It is suggested that accumulation of endoneurial sodium is linked to the movement of sugar into the endoneurium.     

Mammalian peripheral nerve sheath has unique responses to chronic elevations of endoneurial fluid pressure

In acute experiments with fibroelastic tubes such as blood vessels there is a linear ΔP:ΔV relationship followed by a steeper ΔP:ΔV relationship for further increments of volume. Endoneurial fluid pressure (EEP) of peripheral nerve also increases with increases in endoneurial fluid volume. We monitored the effects of volume changes on ΔEFP during a protracted period of time (6 weeks to 1 year on 16 control and 16 experimental rats) to study if a similar relationship occurred in nerve. Nerves were rendered edematous using parenteral and oral galactose administration, EFP was monitored using an active servonull system, and endoneurial volume and subperineurial area (SPA) was measured on fixed tissue. Marked endoneurial edema was produced but EFP did not exceed 6 mm Hg at any time. There was a linear ΔP:ΔV relationship for a limited range of volumes followed by a reduced ΔP:ΔV slope. Because these changes evolved over a long time course we examined the response of $\text{ΔEFP}\text{ΔSPA}$ as a function of time. There was an exponential reduction with time, thus underlying the importance of time-dependent processes in the production of a reduced ΔP:ΔV slope. We conclude that neuropathic changes are unlikely to be due to ischemia by compression of capillaries. Instead, in edematous states there is a very low shear modulus (i.e., a small ΔEFP produces a major volume change with time) and certain types of deformations appear very likely to cause demyelination.     

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     

The experimental production of Renaut bodies

Renaut bodies are loosely-textured whorled, cell-sparse structures found in the subperineurial space of peripheral nerves. Although described in 1881, their significance is still debated. Rats were placed in wire-mesh cages for 4 days to 6 weeks and the lateral and medial plantar nerves were sequentially removed. The initial change was the presence of endoneurial edema which dissected and displaced nerve fibers producing an endoneurial cleft. With the influx of fibroblasts, these clefts became discretely separated by circumferentially oriented processes. Over time the clefts enlarged and became filled with loosely-textured amorphous and fibrillar material as well as collagen. The Renaut bodies ranged from 15 to 80 microns in diameter. In this model the Renaut bodies formed at the maximum site of compression of the lateral plantar nerve. The fibroblasts appeared to be derived from the endoneurial connective tissue and were not the result of degenerating endoneurial structures. Renaut body formation was independent of axonal degeneration. The present study strongly suggests that Renaut bodies are a response to repeated mechanical stress.