Neovascularization after ischemic nerve injury

Because angioneogenesis is associated with wound repair, neovascularization may play an important role in regeneration after nerve injury. This hypothesis was tested in the present study in which the number and distribution of endoneurial capillaries were determined 6 weeks after ischemic nerve injury. The density of capillaries was increased in the central fascicular regions of the distal sciatic and proximal tibial nerves at sites of ischemic injury. This was accompanied by a decrease in intercapillary distance. Neovascularization may be a response to ischemia or a consequence of nerve degeneration or regeneration, the mechanisms of which are poorly understood.     

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.     

Arachidonate-induced experimental nerve infarction

Despite the clinical importance of ischemia in the pathogenesis of many human neuropathies, little is known about the effect of circulatory compromise on the structure of peripheral nerves. This results in part from the lack of an entirely satisfactory model in which to study ischemic neuropathy. We therefore injected arachidonic acid, a potent stimulus to platelet aggregation and vasoconstriction, into the femoral artery of normal rats. This resulted in the rapid onset of focal infarction of the proximal posterior tibial nerve in all animals. Distally there was evidence of Wallerian degeneration but not of primary ischemic damage. The site and nature of the infarct and the temporal sequence of the pathological changes were highly consistent. This new method is a simple and highly reproducible means of producing experimental nerve infarction.     

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.     

Aggravated reperfusion injury in STZ-diabetic nerve

The streptozocin (STZ)-diabetic nerve manifests increased morphological susceptibility to a superimposed acute ischemic injury, and reperfusion injury exaggerates ischemic nerve pathology. To determine whether STZ-diabetic nerves are susceptible to reperfusion, we evaluated the pathological consequences after 2.5 hours of ischemia followed by 3 and 24 hours of reperfusion in a 20-week STZ-diabetic rat sciatic nerve. After 3 hours of reperfusion, endoneurial edema developed in diabetic nerves, whereas non-diabetic controls showed mild or no edema. Morphometric analysis of endoneurial edema, quantified by the total transverse fascicular area and the point-count score of endoneurial structureless space, confirmed significantly more reperfusion-induced edema at thigh and knee levels in diabetic nerves than in controls. Reperfusion caused a significant increase in the number of endoneurial mast cells at the thigh level in diabetic nerves. After 24 hours of reperfusion, there were striking morphological anomalies of myelinated nerve fibers in diabetic nerves, without any observable changes in control nerves. In conclusion, we have demonstrated that STZ-diabetes exacerbates the morphological change to reperfusion. Diabetes therefore renders the microvasculature more vulnerable to the deleterious effects of ischemia/reperfusion.     

Post-traumatic endoneurial neovascularization and nerve regeneration: a morphometric study

Neovascularization would be expected to play an important role in regeneration after nerve injury, but its mechanism is poorly understood. Quantitative investigations of endoneurial capillaries and myelinated fibers 5 and 15 mm distal to different types of nerve injury have therefore been performed. This study demonstrated that numbers of endoneurial capillaries were significantly increased at the 5 mm level 2, 4, 6 and 8 weeks after crush, transection and ischemic lesions, but not following permanent axotomy. Late neovascularization associated with delayed nerve regeneration was found following nerve ischemia. These results suggest that neovascularization following nerve injury is dependent on two variables, the degree of nerve regeneration and the severity of ischemia. Axonal outgrowth appears to be an important determinant of post-traumatic new capillary formation, while nerve ischemia causes both delayed neovascularization and nerve regeneration.     

Correlation between the number of epineurial and endoneurial blood vessels in diseased human sural nerves

In a preceding study it was shown that changes in the number of epineurial blood vessels may be a prominent feature in angiopathic and other peripheral neuropathies, for instance in vasculitis, diabetes mellitus, or cerebral autosomal dominant angiopathy with multiple infarcts and leukoencephalopathy (CADASIL). Endoneurial blood vessels usually may also show significant structural alterations in a broad spectrum of neuropathic conditions, although these are not as prominent as in the epineurium. However, the relationship between changes in the number of epineurial and endoneurial blood vessels in diseased human sural nerves, and the impact of the loss of myelinated nerve fibers on the number of endoneurial blood vessels has thus far not been determined. Therefore, we investigated and compared the number of epineurial and endoneurial blood vessels in 50 human sural nerve biopsy specimens, representing a variety of peripheral neuropathies. We found that despite a significant increase of the number of epineurial blood vessels in cases with vasculitic neuropathy (P<0.05) and neuropathy with other types of microangiopathy (P<0.01), the number and density of the endoneurial blood vessels remained remarkably constant. In cases with an axonal type of neuropathy, severe neuropathic changes were associated with a decreased epineurial blood vessel number and a simultaneous, relative increase in the endoneurial blood vessel density. No significant correlation was found between (1) the number of epineurial and endoneurial blood vessels, and (2) the severity of the neuropathy and the number or density of epineurial and endoneurial blood vessels.     

Expression of Thy–1 Antigen in Reactive Endoneurial Capillaries of the Rat Following Peripheral Nerve Damage

Immunohistochemical staining of Thy-1 antigen was studied in rat peripheral nerves shortly after a complete crush lesion and after an epineural depot injection of lysophosphatidylcholine-palmitate (lysoPC). The Thy-1 antigen was expressed in endoneurial capillaries after 20-30 h and was visible previous to alterations in the axolemma or other signs of neural damage with the exception of endoneurial oedema formation. Thy-1 positive capillaries were restricted to the vicinity of degenerating nerve fibres in partially damaged nerves following epineural depot injection of lysoPC. It is therefore concluded that expression of the Thy-1 antigen in response to peripheral nerve damage in reactive endoneurial capillaries is an active process and not due to diffusion from degenerating axolemmata. Thy-1 staining could be used as a marker of reactive endoneurial capillaries in pathological conditions of peripheral nerves.     

Acute endothelial swelling is induced in endoneurial microvessels by ischemia

Structural alterations of endoneurial microvessels occur in diabetic neuropathy and are statistically associated with severity of nerve fiber loss and teased fiber abnormality. It is therefore hypothesized that the microvessel alterations may cause or contribute to pathologic alterations of nerve fibers in diabetic neuropathy, possibly through hypoxic injury. The mechanism of the microvessel change in diabetic neuropathy is unknown. The role of microvessels and details of microvessel structure in other possible ischemic neuropathies has not been studied completely. Already there is evidence that hypoxia induces endothelial swelling but this has not been characterized or quantitated in nerve. To determine the acute morphologic effect of ischemia on ultrastructural features of transverse profiles of endoneurial microvessels major pelvic arteries were ligated in rats. At 36 h mean lumen and mural areas were greater in ischemic than in control nerves. All components (endothelium, pericytes and basement membrane) were on average greater in ischemic than controls. The greatest increase was in endothelial cells. In these cells swollen mitochondria were abundant. This study demonstrates that acute ischemia induces swelling of the cells and organelles of endoneurial microvessels.     

Peripheral nerve endoneurial microangiopathy and necrosis in rats with insulinoma

Peripheral nerve pathology related to chronic hyperinsulinemia and hypoglycemia has yet to be fully explored. Here we conducted a systematic quantitative analysis of morphological alterations in peripheral sensory and motor nerve fibers and endoneurial microvasculature in longstanding insulinoma-carrying rats (I-rats; n=12). Age-matched normal rats (n=6) served as controls. Over the 15-month observation period, two of I-rats developed paresis of the hind limbs when their blood glucose level fell below 1.7 mmol/l. These animals showed a massive myelinated fiber loss associated with active degeneration of residual myelinated fibers and multiple endoneurial microvascular occlusions at the sciatic nerve level. The rest of the non-paretic I-rats showed a decreased density of large myelinated fibers with axonal degeneration in the peroneal nerve and an increased density of small myelinated fibers with preserved morphology in the sural nerve. This was associated with endoneurial microangiopathic changes indicative of endoneurial ischemia/hypoxia in the sciatic and peroneal nerves, and an increase in endoneurial microvascular density in the sciatic and sural nerves. In conjunction with previous data, these findings suggest that the observed increase in endoneurial microvascular density may be a compensatory response to endoneurial ischemia/hypoxia induced by chronic hyperinsulinemia in I-rats without paresis. In conclusion, the present study showed characteristic morphological alterations in peripheral sensory and motor nerve fibers associated with microangiopathy indicative of endoneurial ischemia/hypoxia in the sciatic and peroneal nerves, and provides the first evidence for the occurrence of endoneurial necrosis in the sciatic nerve, to which the hind limb paresis can be ascribed in I-rats.     

Acute inflammatory demyelination in reperfusion nerve injury

We investigated the pathological appearance of acute inflammation and its role in the development of demyelination in reperfused rat sciatic, tibial, and peroneal nerves after a 5-hour period of near-complete ischemia. Polymorphonuclear neutrophil migration was seen early in the endoneurial lesion. After 18 hours of reperfusion, there was maximal intercellular adhesion molecule-1 expression on endoneurial vessels, and polymorphonuclear neutrophil accumulation was then prominent, reaching a peak 24 hours after reperfusion. Endoneurial mononuclear macrophages increased nearly fourfold after 48 to 72 hours of reperfusion. Macrophages were observed invading Schwann cells and myelin lamellae with associated demyelination. Thus, this study provides evidence of macrophage-associated demyelination after reperfusion similar to that seen in inflammatory neuropathies.     

Patterns of change in endoneurial capillary permeability and vascular space during Wallerian degeneration

In degenerating frog sciatic nerves an in situ perfusion technique was used to measure the permeability coefficient-surface area product (PA) of endoneurial capillaries to [14C]sucrose, and the endoneurial vascular space (V). Both PA and V started to increase after the 3rd day of degeneration. The increase in PA peaked around the 14th day of degeneration and then declined to reach near normal levels at 6 weeks post-transection. V increased until about the end of the 3rd week of degeneration and then declined to near normal levels at 6 weeks after transection. The delayed increase in capillary permeability may reflect an adaptive reorganization of endoneurial capillary structure and function in response to altered conditions of the endoneurial microenvironment, and it is suggested that this permeability increase is induced by breakdown products of axons or chemical signals from Schwann cells enveloping transected axons. Fluid extravasation from these leaking capillaries is probably responsible for the endoneurial oedema observed in Wallerian degeneration. The recovery of endoneurial capillary tone to near-normal levels at 6 weeks after transection leads to the intriguing conclusion that healthy nerve fibers are not essential for the maintenance of normal endoneurial capillary permeability.     

Improvement of endoneurial lipid abnormalities in experimental diabetic neuropathy by oxygen modification

Endoneurial hypoxia and a high frequency of closed capillaries have been found in chronic experimental diabetes and human diabetic sural nerve, respectively. These findings have led to the hypothesis that the pathogenesis of diabetic neuropathy is due to endoneurial hypoxia. To evaluate the role of endoneurial hypoxia in experimental diabetic neuropathy, the effects of supplementation and deprivation of oxygen on peripheral nerve lipid biosynthesis were studied in normal control and streptozotocin-induced diabetic rats. Defective lipid biosynthesis in diabetic nerve was partially prevented by oxygen supplementation. When normal rats were placed in a hypoxic chamber, lipid abnormalities similar to those observed in diabetic nerves were demonstrated in the absence of changes in nerve free sugars. These findings suggest that endoneurial hypoxia may underlie some key biochemical abnormalities encountered in experimental diabetic neuropathy.     

Mild ischemia causes severe pathological changes in experimental diabetic nerve.

Nerve ischemia is considered one of etiological factors in the development of structural changes in peripheral nerves associated with diabetes mellitus. To assess the effect of mild ischemia on diabetic nerve, a subthreshold dose of polystyrene microspheres was injected intraarterially to occlude microvessels of the sciatic nerve and its branches in 20-week streptozotocin-induced diabetic and control rats. Diabetic sciatic and tibial nerves showed severe pathological change of myelinated fibers, whereas nondiabetic nerves were normal or had minor structural abnormalities. Morphometrical evaluation confirmed a greater frequency of abnormal myelinated fibers in diabetic nerves especially in central fascicular regions. The results indicate that diabetic nerve has an increased morphological susceptibility to nerve ischemia. Endoneurial hypoxia, which may result from hemorheological and vascular abnormalities, is likely to cause a lowered threshold to ischemic tolerance in diabetic nerve. This increased vulnerability to ischemia may render diabetic nerve unduly susceptible to hyperglycemia-induced systemic tissue abnormalities.     

Reversible conduction block in human ischemic neuropathy after ergotamine abuse.

Conduction block [a significant reduction in compound muscle action potential (CMAP) amplitude after proximal compared to distal stimulation] is often found in demyelinating neuropathies, including inflammatory neuropathies and degenerative neuropathies, such as "liability to pressure neuropathy." There is experimental evidence that a transient conduction block can occur in rats after ischemic lesions of peripheral nerves are induced either by ligation of arterial vessels supplying nerve trunks, or by injection of arachidonic acid into peripheral arterial vessels. Conduction block has also recently been described in cases with necrotizing vasculitis. To date, however, no example of a reversible conduction block has been reported in human ischemic neuropathy.     

Peripheral nerve structure and function in experimental diabetes

Observations have been made on the peripheral nerves of rats in which diabetes had been induced by streptozotocin or alloxan. Motor nerve conduction velocity was found to become reduced, the reduction developing within a few days in severely diabetic animals. Conduction velocity remained diminished during survival times of up to 1 year.Histometric studies of the myelinated fibre population of the sural and tibial nerves showed no loss of fibres or reduction in their calibre. Evidence of segmental demyelination was not detected and myelin/axon ratios were normal. Cation binding at the nodes of Ranvier was unaltered. No explanation for the reduced nerve conduction velocity in morphological terms was therefore obtained.Observations on the abdominal vagus nerve revealed no loss of unmyelinated axons or reduction in their calibre. Measurements of Schwann cell and endoneurial capillary basal laminal thickness showed no differences between diabetic and control animals. Ultrastructural examination of other features of the peripheral nerves failed to define any pathological alterations in the diabetic animals. It is therefore concluded that the extrapolation of biochemical findings in experimental diabetes in an attempt to explain the origin of human diabetic neuropathy, where there are associated structural changes, should be made with caution.     

Peripheral neuropathy after chronic endoneurial ischemia

We have developed a method for producing chronic regional nerve ischemia in rats by creating proximal limb arteriovenous shunts. This procedure results in a 50 to 75% reduction in endoneurial blood flow within the distal sciatic nerve as measured by the iodoantipyrine method. Nerve conduction velocities in sciatic nerves ipsilateral to the shunt fell by 25 to 30% within 2 weeks after creation of the shunt and did not recover for up to 10 months after the procedure. Morphological studies of the ischemic nerves showed structural abnormalities at nodes of Ranvier and mild axonal atrophy. Neither segmental demyelination nor axonal degeneration were evident. These results indicate that reduced endoneurial blood flow, insufficient to cause infarction, may result in measurable functional and morphological abnormalities in peripheral nerves.     

Endoneurial capillary permeability to [14C]sucrose in frog sciatic nerve

Using an in situ perfusion technique, we measured the [14C]sucrose permeability-surface area product (PA) in endoneurial capillaries of 48 frog sciatic nerves, as 6.6 +/- 0.6 (S.E.M.) X 10(-5) s-1, and the vascular space as 1.31 +/- 0.10%. Assuming A = 30 cm2/g, P = 2.2 X 10(-6) cm/s. P for sucrose was greater than P in some barrier tissues with tight junctions, but was less than P in all capillaries examined so far except rat cerebral capillaries. These observations demonstrate that endoneurial capillaries are an effective part of the blood-nerve barrier to water-soluble non-electrolytes. The findings are consistent with capillary impermeability to microperoxidase and with capillary ultrastructure.     

Peripheral nerve abnormalities related to galactose administration in rats.

Electrophysiological, biochemical, and morphometric observations were made on the peripheral nerves of rats after galactose feeding. Motor nerve conduction velocity was found to be reduced. This was associated with an accumulation of galactitol in the peripheral nerves and a diminution in their myoinositol content. An increased water content and fascicular area, taken in conjunction with a probable increase in the area of the endoneurial spaces, indicated overhydration of the peripheral nerves. Morphometric observations on the myelinated fibre population in the tibial nerve showed no loss of fibres and although both the maximal and the average diameter of the myelinated fibres was slightly less than in age-matched controls, this was insufficient to explain the reduction in conduction velocity. Segmental demyelination was not detected and the relationship between myelin thickness and axon circumference was not altered. Electron microscope observations revealed no ultrastructural changes in the myelinated fibres and, in particular, no abnormalities at the nodes of Ranvier or indication of abnormal hydration of the Schwann cells. The relevance of these findings to the peripheral nerve changes in human and experimental diabetes is discussed.