Efficacy of nonviral gene transfer of human hepatocyte growth factor (HGF) against ischemic-reperfusion nerve injury in rats.

Ischemic neuropathy is common in subjects with critical limb ischemia, frequently causing chronic neuropathic pain. However, neuropathic pain caused by ischemia is hard to control despite the restoration of an adequate blood flow. Here, we used a rat model of ischemic-reperfusion nerve injury (IRI) to investigate possible effects of hepatocyte growth factor (HGF) against ischemic neuropathy. Hemagglutinating virus of Japan (HVJ) liposomes containing plasmids encoded with HGF was delivered into the peripheral nervous system by retrograde axonal transport following its repeated injections into the tibialis anterior muscle in the right hindlimb. First HGF gene transfer was done immediately after IRI, and repeated at 1, 2 and 3 weeks later. Rats with IRI exhibited pronounced mechanical allodynia and thermal hyperalgesia, decreased blood flow and skin temperature, and lowered thresholds of plantar stimuli in the hind paw. These were all significantly improved by HGF gene transfer, as also were sciatic nerve conduction velocity and muscle action potential amplitudes. Histologically, HGF gene transfer resulted in a significant increase of endoneurial microvessels in sciatic and tibial nerves and promoted nerve regeneration which were confirmed by morphometric analysis. Neovascularization was observed in the contralateral side of peripheral nerves as well. In addition, IRI elevated mRNA levels of P2X3 and P2Y1 receptors, and transient receptor potential vanilloid receptor subtype 1 (TRPV1) in sciatic nerves, dorsal root ganglia and spinal cord, and these elevated levels were inhibited by HGF gene transfer. In conclusion, HGF gene transfer is a potent candidate for treatment of acute ischemic neuropathy caused by reperfusion injury, because of robust angiogenesis and enhanced nerve regeneration.

Pathological Findings of Hourglass-Like Constriction in Spontaneous Posterior Interosseous Nerve Palsy.

We report the pathological findings of hourglass-like fascicular constriction (HLFC) under optical and electron microscopy. A 24-year-old man with spontaneous posterior interosseous nerve palsy was treated by interfascicular neurolysis at 29 weeks after onset. One fascicle in the radial nerve presented severe HLFC with torsion at 5 cm proximal to the elbow. Functional recovery was achieved by resection of the enlarged fascicle including HLFC and sural nerve grafting. Proximal to the HLFC, the endoneurium was filled with clusters of regenerating nerve fibers. At the level of the HLFC, a complete loss of myelinated nerve fibers and vascular occlusion of endo- and perineurial vessels were found. Few regenerating nerve fibers were observed. Distal to the HLFC, severe endoneurial edema, a complete loss of myelinated and unmyelinated nerve fibers, and bands of Büngner were noted. These electron microscopic findings demonstrated a detailed pathology of the nerve around the HLFC.

Hypertension Contributes to Neuropathy in Patients With Type 1 Diabetes.

BACKGROUND: Diabetic peripheral neuropathy (DPN) can lead to foot ulceration and amputation. There are currently no disease-modifying therapies for DPN. The aim of this study was to determine if hypertension contributes to DPN in patients with type 1 diabetes mellitus (T1DM). METHODS: Subjects with T1DM (n = 70) and controls (n = 78) underwent a comprehensive assessment of DPN. RESULTS: Hypertension was present in 40 of 70 T1DM subjects and 20 of 78 controls. Hypertension was associated with abnormal nerve conduction parameters (P = 0.03 to <0.001), increased vibration perception threshold (P = 0.01) and reduced corneal nerve fiber density and length (P = 0.02) in subjects with T1DM. However, after adjusting for confounding factors only tibial compound motor action potential and nerve conduction velocity were associated with hypertension (P = 0.03) and systolic blood pressure (P < 0.01 to <0.0001). Hypertension had no effect on neuropathy in subjects without diabetes. CONCLUSIONS: This study shows that hypertension is associated with impaired nerve conduction in T1DM. It supports previous small trials showing that angiotensin-converting enzyme inhibitors improve nerve conduction and advocates the need for larger clinical trials with blood pressure lowering agents in DPN.

Utility of nerve conduction studies for diagnosis of injury to the medial branch of the superficial radial nerve.

INTRODUCTION: The clinical utility of nerve conduction study (NCS) for the distal medial branch of the superficial radial nerve (SRN) has not yet been clarified. Therefore, we investigated the clinical utility of NCS in patients with suspected SRN injury and compared the results with those in healthy control subjects. METHODS: Bilateral NCS of the medial branch of the SRN was performed in two patients with suspected injury of the medial branch of the SRN, and in 20 healthy control subjects. A surface recording electrode was placed at the medial side of the metacarpophalangeal joint of the thumb. The SRN was then stimulated at a location 12 cm proximal from the recording electrode. RESULTS: The mean sensory nerve action potential in the two patients was significantly lower than that of the controls (6.75 ± 0.92 vs. 23.8 ± 8.2 µV, P < 0.05). The side-to-side differences in sensory nerve action potential in the two patients were significantly higher than in the controls (55 ± 7.1 vs. 11 ± 7.8%, P < 0.05). CONCLUSIONS: NCS may be useful for diagnosing injury of the medial branch of the SRN.

Transplantation of dental pulp stem cells improves long-term diabetic polyneuropathy together with improvement of nerve morphometrical evaluation.

BACKGROUND: Although previous reports have revealed the therapeutic potential of stem cell transplantation in diabetic polyneuropathy, the effects of cell transplantation on long-term diabetic polyneuropathy have not been investigated. In this study, we investigated whether the transplantation of dental pulp stem cells (DPSCs) ameliorated long-term diabetic polyneuropathy in streptozotocin (STZ)-induced diabetic rats. METHODS: Forty-eight weeks after STZ injection, we transplanted DPSCs into the unilateral hindlimb skeletal muscles. Four weeks after DPSC transplantation (i.e., 52 weeks after STZ injection) the effects of DPSC transplantation on diabetic polyneuropathy were assessed. RESULTS: STZ-induced diabetic rats showed significant reductions in the sciatic motor/sensory nerve conduction velocity, increases in the current perception threshold, and decreases in capillary density in skeletal muscles and intra-epidermal nerve fiber density compared with normal rats, all of which were ameliorated by DPSC transplantation. Furthermore, sural nerve morphometrical analysis revealed that the transplantation of DPSCs significantly increased the myelin thickness and area. DPSC-conditioned media promoted the neurite outgrowth of dorsal root ganglion neurons and increased the viability and myelin-related protein expression of Schwann cells. CONCLUSIONS: These results indicated that the transplantation of DPSCs contributed to the neurophysiological and neuropathological recovery from a long duration of diabetic polyneuropathy.

Neuropathy in the spontaneously hypertensive rat: An electrophysiological and histological study.

INTRODUCTION: Hypertension is identified as a risk factor for development of polyneuropathy. In this study we examined nerve conduction and morphological alteration of peripheral nerves in spontaneously hypertensive rats (SHR). METHODS: Motor nerve conduction velocity (MNCV) in the sciatic-tibial nerve and sensory nerve conduction velocity (SNCV) in the sural nerve were measured. Pathological investigations included spinal cord, dorsal root ganglion, and hindlimb nerves in SHR and Wistar-Kyoto rats (WKY) aged 4-64 weeks. RESULTS: Blood pressure was significantly higher in SHR than WKY animals at 4 weeks and elevated further with aging. MNCV and SNCV were significantly slower in SHR compared with WKY after age 24 weeks. Prominent morphological changes in SHR nerves included axonal atrophy and myelin splitting. SHR also had endoneurial microangiopathy with reduplication of basement membrane. CONCLUSIONS: SHR showed slowed nerve conduction velocity and pathological abnormalities of hindlimb nerves. Sustained severe hypertension may cause axonal atrophy and endoneurial microangiopathy. Muscle Nerve 54: 756-762, 2016.

Ischemia and diabetic neuropathy.

In addition to hyperglycemia and ensuing metabolic changes, vascular abnormalities and ischemia driven by hyperglycemia and metabolic change plays a paramount role for the development of diabetic polyneuropathy. Endothelial dysfunction plays a key role in the initiation of cellular events evolving into the development of vascular complications of diabetes, and is the common denominator between the metabolic and vascular abnormalities detected in diabetes. Diminished production and function of endothelium-derived vasodilators, and the exaggerated production of vasoconstrictors, lead to endothelial dysfunction, resulting in elevated vascular tone, culminating in macro- and microvascular damage. There is microvascular pathology in human diabetic polyneuropathy: basement membrane thickening, pericyte degeneration, and endothelial cell hyperplasia in endoneurial microvessels. These vascular changes strongly correlate with clinical defects and nerve pathology. Studies in human and animal models have shown endoneurial hypoxia caused by a reduction in nerve blood flow and increased endoneurial vascular resistance. There is strong evidence that the nerve fiber degeneration and a loss of nerve fibers are ischemic in diabetic polyneuropathy. Diabetic nerves reveal a paradoxical contrast between their physiological resistance to ischemia and increased morphological susceptibility to ischemia. Diabetic nerves are particularly vulnerable to reperfusion injury. This chapter will review the response of diabetic nerves to ischemia and reperfusion injury, and the evidence of ischemia and hypoxia in diabetic neuropathies.

Therapeutic effect of exendin-4, a long-acting analogue of glucagon-like peptide-1 receptor agonist, on nerve regeneration after the crush nerve injury.

Glucagon-like peptide-1 (GLP-1) is glucose-dependent insulinotropic hormone secreted from enteroendocrine L cells. Its long-acting analogue, exendin-4, is equipotent to GLP-1 and is used to treat type 2 diabetes mellitus. In addition, exendin-4 has effects on the central and peripheral nervous system. In this study, we administered repeated intraperitoneal (i.p.) injections of exendin-4 to examine whether exendin-4 is able to facilitate the recovery after the crush nerve injury. Exendin-4 injection was started immediately after crush injury and was repeated every day for subsequent 14 days. Rats subjected to sciatic nerve crush exhibited marked functional loss, electrophysiological dysfunction, and atrophy of the tibialis anterior muscle (TA). All these changes, except for the atrophy of TA, were improved significantly by the administration of exendin-4. Functional, electrophysiological, and morphological parameters indicated significant enhancement of nerve regeneration 4 weeks after nerve crush. These results suggest that exendin-4 is feasible for clinical application to treat peripheral nerve injury.

Increased susceptibility to ischemia and macrophage activation in STZ-diabetic rat nerve.

Ischemic vulnerability in diabetic nerve plays a paramount role in the development of diabetic neuropathy, yet little is known of the underlying mechanism. Diabetes enhances the inflammatory response to ischemia and reperfusion. We investigated pathological characteristics of nerve fibers and endoneurial macrophages along the length of sciatic-tibial nerves before and after ischemia (60 to 90 min) and reperfusion (6h to 7 days) in 8 weeks of STZ-induced diabetic rats. Without ischemia, diabetic nerves revealed significantly increased the density of Iba-1-positive endoneurial macrophages when compared with controls. Most of macrophages appeared slim and triangular in shape, but in diabetic nerves, some were rounded with bromodeoxyuridine (BrdU) incorporation, suggesting proliferating macrophages. Seventy-five minutes of ischemia is the minimal ischemic time to cause pathological changes in diabetic nerves. Following 90 min of ischemia and 6h of reperfusion in diabetic rats, the number of Iba-1-positive endoneurial macrophages was increased significantly at the thigh level of sciatic nerve when compared with those before ischemia. Endoneurial macrophages in diabetic nerves increased in number further significantly after 24 and 48 h of reperfusion and underwent morphological alterations; swollen and rounded including phagocytosis. After 90 min of ischemia and 7 days of reperfusion, severe pathological alterations, e.g., demyelination and endoneurial edema at proximal nerves and axonal degeneration distally, were observed in diabetic nerves, while control nerves showed normal morphology. We conclude that macrophage proliferation occurs in STZ-diabetic nerves. The acute inflammatory response after ischemia and reperfusion was intensified in diabetic nerves. Activation of resident macrophages and infiltration by recruited macrophages could be casually linked to ischemic susceptibility in diabetic nerve.

The role of the polyol pathway in acute kidney injury caused by hindlimb ischaemia in mice.

The polyol pathway, a collateral glycolytic process, previously considered to be active in high glucose milieu, has recently been proposed to play a crucial role in ischaemia/reperfusion tissue injury. In this study, we explored the role of the polyol pathway in acute kidney injury (AKI), a life-threatening condition, caused by hindlimb ischaemia, and determined if inhibition of the polyol pathway by aldose reductase (AR) inhibitor is beneficial for this serious disorder. Mice 8 weeks of age rendered hindlimb ischaemic for 3 h by the clipping of major supporting arteries revealed marked muscle necrosis with accumulation of sorbitol and fructose in ischaemic muscles. Serum concentrations of blood urea nitrogen (BUN), creatinine phosphokinase (CPK), creatinine, tumour necrosis factor (TNF)-alpha as well as interleukin (IL)-6 were all elevated in these mice. Treatment with AR inhibitor (ARI) effectively suppressed muscle necrosis and accompanying inflammatory reactions and prevented renal failure. Similar to ARI-treated mice, AR-deficient mice were protected from severe ischaemic limb injury and renal failure, showing only modest muscle necrosis and significant suppression of serum markers of renal failure and inflammation. Thus, these findings suggest that the polyol pathway is implicated in AKI caused by ischaemic limb injury and that AR may be a potential therapeutic target for this condition.

Clinical screening of autonomic dysfunction in multiple sclerosis.

BACKGROUND AND PURPOSE: Multiple sclerosis (MS) can affect the autonomic nervous system. Although exercise may be beneficial for people with MS, those with autonomic dysfunction may have altered heart rate responses to exercise. We investigated the hypothesis that the pattern of increase in heart rate on commencement of a simple cycle test would be different in those participants with MS who had been shown to have autonomic dysfunction on laboratory testing compared with both control participants and MS participants not exhibiting autonomic involvement. METHOD: A controlled cohort study with a volunteer sample of 31 adults with MS (26 women, 5 men) with a mean age of 46 +/- 8.00 years (32-60 years), a median Expanded Disability Severity Scale of 3 (1-6) and a mean duration since diagnosis of 10.3 years (0.1-39 years). Thirty-one age-matched, non-disabled, sedentary but healthy adults (26 women, 5 men) with a mean age of 45 +/- 9.5 years (24-57) comprised the control group. Autonomic function was evaluated using continuous heart rate and blood pressure responses to rhythmical deep breathing, the Valsalva manoeuvre, passive postural change and a simple cycle test. RESULTS: There were no significant differences in age, height or weight (p < 0.05) between the two groups or on any of the autonomic test results (p < 0.05). Five participants with MS (16%) had abnormal autonomic function on laboratory testing, two of whom demonstrated an abnormal heart rate response to the cycle test. CONCLUSIONS: It is suggested that physiotherapists monitor the heart rate response to a dynamic exercise test in people with MS prior to prescribing an exercise programme to ensure patients' safety. Should the response appear delayed or attenuated, referral for more formal autonomic laboratory testing is recommended.

Prolonged ischemic conduction failure after reperfusion in diabetic nerve.

Diabetic nerve exhibits morphological vulnerability to ischemia and reperfusion, in contrast to its physiological resistance to ischemic conduction failure (RICF). To examine the sequence of ischemic conduction failure after reperfusion in diabetic nerve, we measured sciatic-tibial nerve conduction before and during 30-180 min of ischemia and after reperfusion for up to 1 week in streptozocin (STZ)-induced diabetic rats. RICF in diabetic rats was confirmed during ischemia. After reperfusion, control nerves showed an immediate recovery in amplitude of compound muscle action potential (CMAP) following ischemia for 120 min or less, and delayed recovery after 150 min of ischemia. In contrast, recovery in diabetic nerves was delayed even after 1 h of ischemia. Ischemia for 75 min in diabetic nerve resulted in either delayed or no recovery of the CMAP upon reperfusion. Following ischemia for 90 or 120 min, axonal degeneration was observed in diabetic nerve. Thus, severe ischemia for 60 or 75 min causes prolonged ischemic conduction failure in diabetic nerve, compared with 150 min in control nerve. In conclusion, diabetic nerve shows delayed recovery of ischemic conduction failure after brief ischemia, compared to controls, suggesting that patients with diabetic neuropathy have a worse prognosis when faced with nerve ischemia.

Neuroprotective effects of nitrone radical scavenger S-PBN on reperfusion nerve injury in rats.

The nitrone-based free radical scavengers have potent neuroprotective activities in models of stroke in which oxidative stress plays a key role in its development. We examined the effects of S-PBN (sodium 4-[(tert-butylimino) methyl]benzene-3-sulfonate N-oxide), a spin trap nitrone, on reperfusion injury in rat peripheral nerves. Immediately after the onset of 4-h ischaemia in rat right hindlimb, S-PBN was administered via mini-osmotic pumps, containing 2 ml of S-PBN (1.2 M), inserted subcutaneously. S-PBN, in addition, was given by a single injection (50 mg/kg BW, i.p.). Mean plasma concentrations of S-PBN were significantly greater in S-PBN-treated rats than in controls after 24, 48 and 72 h of reperfusion. Pump and dosing solution analysis indicated that the rats received between 82 and 99% of the target S-PBN concentration. Morphology in sciatic, tibial and peroneal nerves was assessed after 4 h of ischaemia followed by 72 h and 7 days of reperfusion. After 72 h of reperfusion, saline-treated control rats showed endoneurial oedema at the thigh level and diffuse axonal degeneration of myelinated nerve fibres distally. S-PBN-treated nerves were normal or revealed less severe abnormalities in myelinated fibres after 72 h and 7 days of reperfusion, when compared with those in saline-treated control nerves. Morphometrically, the frequency of abnormal myelinated fibres at calf levels was significantly less in S-PBN-treated nerves than in controls. In conclusion, post-ischaemic administration of S-PBN exhibits substantial neuroprotective properties in ischemia/reperfusion nerve injury.

Antioxidant and pro-oxidant properties of pyrroloquinoline quinone (PQQ): implications for its function in biological systems.

Pyrroloquinoline quinone (PQQ) is a novel redox cofactor recently found in human milk. It has been reported to function as an essential nutrient, antioxidant and redox modulator in cell culture experiments and in animal models of human diseases. As mitochondria are particularly susceptible to oxidative damage we studied the antioxidant properties of PQQ in isolated rat liver mitochondria. PQQ was an effective antioxidant protecting mitochondria against oxidative stress-induced lipid peroxidation, protein carbonyl formation and inactivation of the mitochondrial respiratory chain. In contrast, PQQ caused extensive cell death to cells in culture. This surprising effect was inhibited by catalase, and was shown to be due to the generation of hydrogen peroxide during the autoxidation of PQQ in culture medium. We conclude that the reactivities of PQQ are dependent on its environment and that it can act as an antioxidant or a pro-oxidant in different biological systems.

Hypoglycaemic neuropathy: microvascular changes due to recurrent hypoglycaemic episodes in rat sciatic nerve.

Intensive diabetes treatment causes a considerable increase in the number of severe hypoglycaemic episodes which could aggravate the progression of diabetic neuropathy. However, the effect of repeated hypoglycaemic episodes on nerve morphology has never been previously investigated. The aims of the present study were: (i) to establish a rat model of recurrent episodes of severe hypoglycaemia, and (ii) to assess morphological changes after repeated hypoglycaemic episodes in rat sciatic nerves. We induced hypoglycaemic episodes, blood glucose level <3.0 mmol/l for 3 h, by injecting regular insulin intravenously on 4 consecutive days. We found endothelial swelling of endoneurial microvessels at the thigh level of sciatic and tibial nerves 24 h after four daily episodes of hypoglycaemia. Endothelial swelling was confirmed by vascular morphometry which showed significantly increased endothelial and pericyte areas. No obvious abnormalities were seen on nerve fibres. In conclusion, recurrent hypoglycaemic episodes cause early vascular anomalies in endoneurial microvessels in rat sciatic nerves without any observable changes in nerve fibres.

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.