Injury-induced activation of ERK 1/2 in the sciatic nerve of healthy and diabetic rats

Phosphorylation of extracellular-signal-regulated kinase 1/2 (p-ERK 1/2) was investigated by immunohistochemistry at 30 min, 1 h, and 48 h after nerve transection in the sciatic nerve of healthy and diabetic [streptozotocin (STZ)-induced diabetes mellitus and BioBreeding (BB; i.e. DR.lyp/lyp or BBDP)] rats. Transection injury increased the intensity of p-ERK 1/2 in nerve stumps at all time points. Staining was confined to Schwann cells with occasional faint staining in single axons. In diabetic rats, a lower intensity of p-ERK 1/2 was found at 1 and 48 h in the distal and proximal nerve stumps compared with healthy rats. STZ-induced diabetic rats were not different from BB rats. p-ERK 1/2 is activated differentially in Schwann cells after nerve injury in diabetic rats, whereas activation in STZ-induced diabetic rats did not differ from BB rats.     

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.     

Transport of acid phosphatase in normal and transected rat sciatic nerve

Acid phosphatase accumulates at the severed ends of transected peripheral nerve or spinal cord. We examined the temporal sequence of enzyme accumulation from 1 to 14 days after transecting or applying colchicine to the rat sciatic nerve in order to ascertain whether or not this accumulation is related to axonal transport. Enzyme activity was observed in the proximal and distal stumps by 1 day after transection, and by 1 week activity in the proximal stump exceeded that in the distal stump. In some experiments, colchicine was applied to the nerve proximal to the site of nerve transection. This procedure resulted in an accumulation of acid phosphatase proximal to the site of drug application and not immediately proximal to the site of transection. These results are consistent with the hypothesis that bidirectional transport of acid phosphatase is a normal feature of peripheral nerves.     

Local administration of vasoactive intestinal peptide after nerve transection accelerates early myelination and growth of regenerating axons

Our goal was to determine whether local injections of vasoactive intestinal peptide (VIP) promote early stages of regeneration after nerve transection. Sciatic nerves were transected bilaterally in 2 groups of 10 adult mice. In the first group, 15 microg (20 microL) of VIP were injected twice daily into the gap between transected ends of the right sciatic nerve for 7 days (4 mice) or 14 days (6 mice). The same number of mice in the second group received placebo injections (20 microL of 0.9% sterile saline) in the same site, twice daily, for the same periods. After 7 days, axon sizes, relationships with Schwann cells and degree of myelination were compared in electron micrographs of transversely sectioned distal ends of proximal stumps. Fourteen days after transection, light and electron microscopy were used to compare and measure axons and myelin sheaths in the transection gap, 2-mm distal to the ends of proximal stumps. Distal ends of VIP-treated proximal stumps contained larger axons 7 days after transection. More axons were in 1:1 relationships with Schwann cells and some of them were surrounded by thin myelin sheaths. In placebo-treated proximal stumps, axons were smaller, few were in 1:1 relationships with Schwann cells and no myelin sheaths were observed. In VIP-treated transection gaps, measurements 14 days after transection showed that larger axons were more numerous and their myelin sheaths were thicker. Our results suggest that in this nerve transection model, local administration of VIP promotes and accelerates early myelination and growth of regenerating axons.     

Diabetes does not accelerate neuronal loss following nerve injury

Abstract   To determine the resistance of neuronal dorsal root ganglion (DRG) cells in experimental diabetes, we studied the neuronal cell loss after severe axonal injury in streptozotocin (STZ) diabetic rats with unilateral transection of the L5 spinal nerve for 12 weeks. Fifty 18-week-old inbred male Wistar rats were randomly allocated to three study groups. In study group 1 without spinal nerve injury, STZ diabetes was induced in 9 and 10 rats were kept as nondiabetic controls. In study group 2, spinal nerve injury was performed in 10 diabetic rats and in 10 nondiabetic controls. In study group 3, six nondiabetic control rats at 18 weeks and five nondiabetic control rats at 30 weeks were included to determine whether DRG cell changes occur without nerve injury during the study period. In group 1, the stereologically determined number of all neuronal DRG cells was unchanged after 12 weeks of diabetes. The mean perikaryal volume of neuronal DRG cells of the A and B subtypes was reduced by 10% each (p < 0.05). In group 2, spinal nerve injury led to neuronal cell loss, chromatolysis, and perikaryal shrinkage but without any acceleration of cell loss after 12 weeks of diabetes. In group 3, there were no changes indicating that the reduction of perikaryal volume in diabetic rats without nerve injury represents shrinkage. We conclude that neuronal DRG cells in rats are resistant to diabetes per se and that addition of diabetes for 12 weeks to spinal nerve injury does not further accelerate the cell loss.     

Temporal changes of neuronotrophic activities accumulating in vivo within nerve regeneration chambers

The presence of neuronotrophic factors (NTFs) in noninjured sciatic nerve extract and the course of their accumulation from 3 h to 30 days after nerve transection was examined. Rat sciatic nerves were transected and their proximal and distal stumps sutured into the openings of cylindrical silicone chambers leaving a 10-mm interstump gap. Previous studies had shown that regeneration occurs in chambers containing both stumps but is absent in chambers lacking the distal stump. Chambers became completely filled with fluid 10 to 12 h after implantation. Fluid from chambers without nerve stumps (open-ended) implanted adjacent to nerve-containing chambers had markedly lower trophic activities than those containing one or both stumps. In fluid collected from chambers containing both proximal and distal nerve stumps, the highest titers of NTFs directed to sensory neurons were measured at 3 h posttransection whereas the highest titers of NTFs directed to sympathetic and spinal cord neurons were detected at 1 and 3 days, respectively. Chambers containing only the proximal or only the distal stumps showed similar temporal dynamics for sensory and sympathetic NTFs. Sensory and sympathetic neuronotrophic activity in extracts of proximal and distal stumps followed a similar temporal course to those in chamber fluid. Extracts of nonlesion nerve segments 5 mm from the transection site contained higher sensory and lower sympathetic trophic activity than extracts including the transection site. Spinal cord activity was undetectable in all extracts. Antiserum to nerve growth factor had no effect on fluid or extracts containing high sensory or sympathetic activities. These observations suggested that (i) some NTFs may be present in normal nerves and others may be synthesized or accumulated in response to nerve injury, (ii) sensory, sympathetic, and spinal cord NTFs are separate agents and immunochemically distinct from nerve growth factor, (iii) NTFs predominantly originate from nerve stumps rather than from surrounding fluid, and (iv) proximal and distal nerve stumps accumulate and release NTFs at similar rates.     

Retina-derived growth-promoting extract supports axonal regeneration in vivo

The ability of a growth-promoting extract derived from bovine retina was assessed for its ability to support nerve fiber regeneration from proximal stumps of transected sciatic nerves. Proximal stumps of transected rat peripheral nerves were inserted into the single inlet end of a 6 mm long Y-shaped Silastic implant. One of the paired outlets was attached to an Elvax pellet containing the retina-derived growth extract, and the other outlet to a pellet containing an equivalent amount of tissue extract-free Hank's balanced salt solution. At 3 weeks postoperatively, the number of axons and blood vessels in the midportion of implant forks was assessed. The extent of axonal regeneration and blood vessel formation induced were similar to each other and dose-dependent. Results indicate preferential and dose-dependent growth of axons toward pellets containing the retina-derived growth-promoting extract.     

Correction of nerve conduction and endoneurial blood flow deficits by the aldose reductase inhibitor, tolrestat, in diabetic rats

Increased activation of the first half of the polyol pathway, the conversion of glucose to sorbitol by aldose reductase, has been implicated in aldose reductase inhibitor-preventable neurochemical changes that may contribute to the aetiology of diabetic neuropathy. Tolrestat has been used as a standard aldose reductase inhibitor to dissect out polyol pathway-dependent mechanisms in many experimental studies; however, doubt has been cast upon its ability to prevent nerve conduction velocity deficits in diabetic rats. Nerve dysfunction has also been linked to abnormal endoneurial blood flow and oxygenation via increased vasa nervorum polyol pathway flux. The aim of this study was to test whether tolrestat could correct sciatic conduction velocity and perfusion defects in diabetic rats. Sciatic motor conduction velocity, 21% reduced by 1 month of streptozotocin-induced diabetes, was corrected by 23% and 84% with 1 month of tolrestat treatment at doses of 7 and 35 mg/kg/day respectively. Endoneurial blood flow, 44-52% reduced by untreated diabetes, was within the nondiabetic range with high-dose tolrestat treatment and the flow deficit was 39% corrected by the low dose. Sciatic sorbitol and fructose concentrations were approximately 13-fold and approximately 4-fold elevated by untreated diabetes. This was 32-50% attenuated by low-dose tolrestat and sorbitol and fructose content was suppressed below the nondiabetic level by high dose treatment. A 58% nerve myo-inositol deficit was partially (32%) corrected by high-dose tolrestat treatment. We conclude that tolrestat restores defective conduction and blood flow in diabetic rats and is a good pharmacological tool for studies on polyol pathway effects in peripheral nerve.     

Peptide accumulations in proximal endbulbs of transected axons

Axons proximal to a transection develop into enlarged, but presumed 'passive' endbulb structures. In previous studies, we observed that proximal stumps of transected sciatic nerves accumulate discrete and striking deposits of calcitonin gene-related peptide (CGRP) that have apparent direct and local actions on nearby microvessels. In this work, we provide evidence that CGRP, in the company of several additional peptides, are deposited through 'arrested' anterograde transport into axon endbulbs that develop after transection. In proximal stump tips of rat sciatic nerves transected 48 h earlier, CGRP accumulation colocalized with a label for neurofilament that was accentuated at axon tips, but was prevented by a concurrent more proximal sciatic section. Similarly, interruption of CGRP deposition eliminated its apparent actions on local microvessels following injury. CGRP accumulation was also observed in sural nerve proximal stump tips, indicating its presence in sensory axons despite the known declines in the sensory neuronal synthesis of CGRP that occur following axotomy. Peptide accumulation was not unique to CGRP, with a similar pattern of anterograde accumulation observed for substance P (SP), neuropeptide Y (NPY) and galanin. Deposited peptides and perhaps other axonal constituents in the milieu of a peripheral nerve injury may be associated with important local physiological actions in the regenerative microenvironment.     

Regional blood flow in resting and stimulated sciatic nerve of diabetic rats

The role of ischemia in the pathogenesis of diabetic peripheral neuropathy remains uncertain. We used the distribution of [14C]butanol to measure resting regional sciatic nerve blood flow in normal, anesthetized rats and in rats with acute experimental diabetes from streptozotocin administration. Regional flows in hind limb biceps femoris muscle and skin were simultaneously measured. In additional diabetic rats, these blood flows were compared in both limbs after proximal electrical stimulation of one sciatic trunk (10 shocks/s) for 15 min. One month after streptozotocin administration, 8 of 11 test rats were hyperglycemic. Resting nerve blood flow in the hyperglycemic rats--5.6 +/- 3.07 ml.min-1.100 g-1--was significantly less than that in the controls (9.4 +/- 3.9 ml.min-1.100 g-1, P = 0.002). Muscle blood flow was normal and skin blood flow decreased in these rats. Calculated tissue vascular resistances were elevated in all three tissues. Stimulation of one sciatic trunk in five other diabetic rats resulted in a stimulated nerve blood flow of 15.7 +/- 7.7 ml.min-1.100 g-1, and nerve blood flow in the resting control limb was 7.7 +/- 4.3 ml.min-1.100 g-1 (P = 0.009). Muscle blood flow increased approximately fourfold on the stimulated side but skin blood flow did not increase. Resting sciatic nerve blood flow is modestly decreased in acute streptozotocin-induced diabetes, but the neural blood vessels are still responsive to the increase in nerve metabolic activity associated with nerve stimulation.     

Adrenergic innervation in the tibial and vagus nerves of rats with streptozotocin-induced diabetes

Adrenergic innervation of tibial and vagus nerves was studied after 1-16 weeks duration of streptozotocin (STZ)-induced diabetes in rats. Sucrose-phosphate glyoxylic acid (SPG) histochemistry and the formaldehyde-induced fluorescence (FIF) method were used to demonstrate adrenergic nerve fibers in the epi-perineurial and endoneurial compartments. Densities of innervation were quantitated with fluorescence microscopy. The density of periarteriolar adrenergic innervation in the epi-perineurium of the tibial and vagus nerves was increased 5 and 12 weeks after STZ injections as compared with control. At 16 weeks, mean densities of periarteriolar innervation in epi-perineurium had returned to or below control levels in both nerve types. In the endoneurium, however, the mean density of adrenergic nerve fibers decreased gradually at 5 weeks after induction of diabetes in both nerves, and was totally absent at 12 weeks. At 16 weeks no sign of recovering innervation in the endoneurium was seen. In conclusion, adrenergic innervation goes through similar pathological alterations both in tibial and vagus nerves shortly after the induction of streptozotocin diabetes. These changes may contribute to diabetic peripheral neuropathy by impairing the regulation of nerve blood flow.     

Bioinformatic analysis of cytokine expression in the proximal and distal nerve stumps after peripheral nerve injury

In our previous study, we investigated the dynamic expression of cytokines in the distal nerve stumps after peripheral nerve injury using microarray analysis, which can characterize the dynamic expression of proteins. In the present study, we used a rat model of right sciatic nerve transection to examine changes in the expression of cytokines at 1, 7, 14 and 28 days after injury using protein microarray analysis. Interleukins were increased in the distal nerve stumps at 1–14 days post nerve transection. However, growth factors and growth factor-related proteins were mainly upregulated in the proximal nerve stumps. The P-values of the inflammatory response, apoptotic response and cell-cell adhesion in the distal stumps were higher than those in the proximal nerve stumps, but the opposite was observed for angiogenesis. The number of cytokines related to axons in the distal stumps was greater than that in the proximal stumps, while the percentage of cytokines related to axons in the distal stumps was lower than that in the proximal nerve stumps. Visualization of the results revealed the specific expression patterns and differences in cytokines in and between the proximal and distal nerve stumps. Our findings offer potential therapeutic targets and should help advance the development of clinical treatments for peripheral nerve injury. Approval for animal use in this study was obtained from the Animal Ethics Committee of the Chinese PLA General Hospital on September 7, 2016(approval No. 2016-x9-07).     

Reactions of unmyelinated nerve fibers to injury. An ultrastructural study

Reactions of unmyelinated nerves to injury were studied in the distal stumps of rabbit anterior mesenteric nerves following transection. These nerves, chosen because they are almost exclusively unmyelinated, were examined by phase contrast and electron microscopy at intervals from 12 h to 2 weeks after transection. Swollen axons containing mitochondria and other organelles were prominent in the proximal few mm of the distal stump of anterior mesenteric nerve trunks during the first 4 days after transection. As early as 6 days after injury, regenerative changes consisting of numerous small axons with an increased axon-Schwann cell ratio were observed; there was little trace of degenerating axons, or their debris. Thus the capacity of unmyelinated nerve fibers for rapid regeneration has been demonstrated. It is anticipated that this delineation of reactions in unmyelinated nerves will contribute to a greater understanding of functional and morphologic abnormalities in disorders of peripheral nerves.     

Endoneurial blood supply to peripheral nerves is not uniform.

Peripheral nerves are not uniformly susceptible to the effects of ischemia in human and experimental ischemic neuropathies. Since endoneurial blood flow is directly proportional to the number of endoneurial capillaries, we studied endoneurial capillary density at multiple levels of the peripheral nerves of normal rats. Capillary density was lowest in the sciatic and proximal tibial nerves and significantly higher in dorsal and ventral roots and distal tibial and plantar nerves. Endoneurial capillary density corresponds to the hierarchy of susceptibility to ischemic nerve damage in human and experimental ischemic neuropathies. These findings suggest that susceptibility of peripheral nerves to ischemia is determined, at least in part, by the density of endoneurial capillaries.     

Streptozotocin-induced diabetes in rats causes neuronal deficits in tyrosine hydroxylase and 5-hydroxytryptamine specific to mesenteric perivascular sympathetic nerves and without loss of nerve fibers.

The aim of this study was to investigate the possibility that inadequate venous return to the heart in diabetes is the result of a neuropathy which affects autonomic nerves supplying the splanchnic vasculature. Mesenteric veins from rats with streptozotocin-induced diabetes were markedly dilated in vivo compared to veins from control animals. Dilation appeared to be the result of loss of muscle tone rather than hypertrophy or hyperplasia of the vessel wall. Using quantification by image analysis and double-labeling immunohistochemistry on mesenteric veins, significant reductions in the density of nerve plexuses staining for 5-hydroxytryptamine (5-HT) and tyrosine hydroxylase (TH) were shown in vessels from diabetic rats compared to controls. No reductions were observed in the density of nerve plexuses stained for the neuronal marker, PGP 9.5, or for substance P (SP), a marker for afferent nerve fibers. These results indicate neurochemical deficits in experimentally induced diabetes which are specific to perivascular noradrenergic nerves and which, within the time-scale of our experiments, do not involve loss of nerve fibers. These deficits may contribute to an increase in venous pooling of blood in the splanchnic vasculature of diabetic rats and thus to inadequate venous return to the heart.     

An in vivo method to prepare normal Schwann cells free of axons and myelin

A viable population of undifferentiated Schwann cells may be prepared from chronically denervated peripheral nerves. Nerve transection stimulates a sequence of cellular events in distal stumps leading to removal of axons and myelin, and proliferation of Schwann cells. Sealing the ends of nerve stumps prevents reinnervation and leaves daughter Schwann cells residing in longitudinal columns. Schwann cells may be harvested from the endoneurial tissue of the nerve stumps 5-12 weeks after nerve transection. Unlike myelinating cells prepared from intact tissue, where function has been specified by associated axons, Schwann cells obtained from denervated stumps are functionally naive. Their usefulness in analyzing axonal regulation of myelinogenesis and mitosis is therefore suggested.     

Peripheral motor neuropathy in spontaneously diabetic WBN/Kob rats: a morphometric and electron microscopic study

Summary The morphology and function of peripheral motor nerves of WBN/Kob rats of the newly identified strain with late onset and long-lasting diabetes were studied in comparison with those of nondiabetic age-matched rats. The most conspicuous and frequent change seen by electron microscopy in the diabetic rats was myelinopathy, initiated by vesicle formation in granular material accumulated in the myelin lamella. Initial changes developed into myelin blebbing or distention, demyelination and remyelination. A decreased population of nerve fibers in diabetic rats of advanced age suggested that the final stage of these changes was neuronal loss resulting from complete destruction of the neurons. The myelinopathy was consistently more frequent and conspicuous in diabetic rats than in age-matched nondiabetic rats, but the difference was not significant in rats of over 28 months old, because of the age-dependent increase of the change in older rats. The conduction velocity was significantly less in diabetic rats than in age-matched nondiabetic animals at 20 months but not at 28 months of age, because at the older age nondiabetic rats also showed a reduced conduction velocity. These findings showed that male WBN/Kob rats develop diabetic motor neuropathy, but that the essential changes in diabetic neuropathy are indistinguishable morphologically and functionally from those in age-dependent neuropathy.     

Neurofilament mRNAs are present and translated in the normal and severed sciatic nerve

Local protein synthesis within axons has been studied on a limited scale. In the present study, several techniques were used to investigate this synthesis in sciatic nerve, and to show that it increases after damage to the axon. Neurofilament (NF) mRNAs were probed by RT-PCR, Northern blot and in situ hybridization in axons of intact rat sciatic nerve, and in proximal or distal stumps after sciatic nerve transection. RT-PCR demonstrated the presence of NF-L, NF-M and NF-H mRNAs in intact sciatic nerve, as well as in proximal and distal stumps of severed nerves. Northern blot analysis of severed nerve detected NF-L and NF-M, but not NF-H. This technique did not detect the three NFs mRNAs in intact nerve. Detection of NF-L and NF-M mRNA in injured nerve, however, indicated that there was an up-regulation in response to nerve injury. In situ hybridization showed that NF-L mRNA was localized in the Schwann cell perinuclear area, in the myelin sheath, and at the boundary between myelin sheath and cortical axoplasm. RNA and protein synthesizing activities were always greater in proximal as compared to distal stumps. NF triplet proteins were also shown to be synthesized de novo in the proximal stump. The detection of neurofilament mRNAs in nerves, their possible upregulation during injury and the synthesis of neurofilament protein triplet in the proximal stumps, suggest that these mRNAs may be involved in nerve regeneration, providing a novel point of view of this phenomenon.     

Endoneurial microvasculature of chronically transected sciatic nerves in diabetic rats

To characterize the morphology of the endoneurial microvasculature of degenerating nerves under hyperglycemia, the morphology of endoneurial microvessels in transected sciatic nerves was examined in normal and streptozotocin-induced diabetic rats. Three months after transection, the fascicular area and median vascular luminal area at the proximal level of the distal stump were significantly larger in diabetic than in control animals, whereas the number of vessels per fascicle was the same in the two groups. Arterioles in various stages of development were found in the centrifascicular region in some transected nerves. Serial sections revealed that these vessels originated from transperineural arterioles. The frequency and magnitude of vascular wall thickening were both greater in diabetic rats. These results suggest that the endoneurial microvasculature responds abnormally to nerve injury under hyperglycemia.     

Distal neuropathy in experimental diabetes mellitus

Although nerve conduction slowing is a well-accepted abnormality in rats with acute experimental diabetes, reports of neuropathological changes in diabetic rat nerves have been inconsistent. To examine this further, we studied electrophysiological and morphological features of posterior tibial nerves and their distal branches from four-week streptozotocin-induced diabetic rats and matched controls. Diabetic rat posterior tibial motor conduction was slowed (mean ± 1 SD, 34.8 ± 3.1 m/sec; controls, 41.2 ± 2.5 m/sec), and evoked muscle response amplitudes were only half of control values. Using quantitative techniques, we documented a diminution in number of the largest myelinated fibers in otherwise normal mid posterior tibial nerves, with an increase in the smaller sizes, indicating either a degree of axonal atrophy or impaired fiber growth during development. The principal pathological finding was active breakdown of myelinated fibers in the most distal motor twigs of hind foot muscles supplied by posterior tibial branches, with preservation of fibers in more proximal segments of these nerves. This anatomical lesion in diabetic nerves could account for both observed conduction slowing and lowered muscle response amplitudes. A consistent feature in both diabetic and control mid lateral plantar nerves was a zone of demyelination that apparently occurs at this natural site of nerve entrapment in rats. Taken together, the pathological abnormalities of peripheral nerve in acute experimental diabetes are best explained as resulting from a distal axonopathy.