Structural specificity in demyelination induced by lysophospholipids.

The demyelinating activity of lysophosphatidylcholine (lysoPC) and various structural analogs in rat sciatic nerve was evaluated by following electrophysiologic changes within the first hour and 1 week after intraneural injection. The lysophospholipids tested included 1-O-hexadecanoyl-sn-glycero-3-phosphocholine (1-acyl-GPC), 3-O-hexadecanoyl-sn-glycero-1-phosphocholine (3-acyl-GPC), 1-O-hexadecanoylpropanediol-3-phosphocholine (acyl-PPC), 1-O-hexadecylpropanediol-3-phosphocholine (alkyl-PPC) and 1-acyl-sn-glycero-3-phosphoethanolamine (1-acyl-GPE). Changes in conduction velocity, width, amplitude and time integral percentage were measured. Within 1 hour, the highest demyelinating activity was observed for alkyl-PPC, followed by 3-acyl-GPC, 1-acyl-GPC and acyl-PPC. Hydrolysis products of lysoPC (glycerophosphocholine, fatty acid), lysophosphatidylethanolamine (1-acyl-GPE), biradyl choline phospholipids (1,2-di-O-alkyl-rac-glycero-3-phosphocholine, dialkyl-GPC) or sodium deoxycholate proved ineffective in these short-term experiments. One week after intraneural injection, all lysophospholipids tested caused severe electrophysiologic changes, although dialkyl-GPC and sodium deoxycholate did not. Our data suggest (i) that differences in early demyelinating activity by the choline lysophospholipids are related to their rate of turnover, as highest activity was associated with the agents that are not metabolized by lysophospholipase (e.g., alkyl-PPC) or lysolecithin acyltransferase (e.g., 3-acyl-GPC), (ii) that the lysoPC molecule as such and not its products of catabolism causes demyelination, and (iii) that demyelinating activity is not due to the general detergent action of lysoPC, but rather that specific interactions appear to trigger the processes of demyelination induced by lysophospholipids.

Alpha-lipoic acid: effect on glucose uptake, sorbitol pathway, and energy metabolism in experimental diabetic neuropathy.

The peripheral nerve of experimental diabetic neuropathy (EDN) is reported to be ischemic and hypoxic, with an increased dependence on anaerobic metabolism, requiring increased energy substrate stores. When glucose stores become reduced, fiber degeneration has been reported. We evaluated glucose uptake, nerve energy metabolism, the polyol pathway, and protein kinase C (PKC) activity in EDN induced by streptozotocin. Control and diabetic rats received lipoic acid (0, 10, 25, 50, 100 mg/kg). Duration of diabetes was 1 month, and alpha-lipoic acid was administered intraperitoneally 5 times per week for the final week of the experiment. Nerve glucose uptake was reduced to 60, s 37, and 30% of control values in the sciatic nerve, L5 dorsal root ganglion, and superior cervical ganglion (SCG), respectively, in rats with EDN. Alpha-lipoic acid supplementation had no effect on glucose uptake in normal nerves at any dose, but reversed the deficit in EDN, with a threshold between 10 and 25 mg/kg. Endoneurial glucose, fructose, sorbitol, and myo-inositol were measured in sciatic nerve. Alpha-lipoic acid had no significant effect on either energy metabolism or polyol pathway of normal nerves. In EDN, endoneurial glucose, fructose, and sorbitol were significantly increased, while myo-inositol was significantly reduced. Alpha-lipoic acid had a biphasic effect: it dose-dependently increased fructose, glucose, and sorbitol, peaking at 25 mg/kg, and then fell beyond that dose, and it dose-dependently increased myo-inositol. Sciatic nerve cytosolic PKC was increased in EDN. ATP, creatine phosphate, and lactate were measured in sciatic nerve and SCG. Alpha-lipoic acid prevented the reduction in SCG creatine phosphate. We conclude that glucose uptake is reduced in EDN and that this deficit is dose-dependently reversed by alpha-lipoic acid, a change associated with an improvement in peripheral nerve function.

Lipoic acid improves nerve blood flow, reduces oxidative stress, and improves distal nerve conduction in experimental diabetic neuropathy.

OBJECTIVE: To determine whether lipoic acid (LA) will reduce oxidative stress in diabetic peripheral nerves and improve neuropathy. RESEARCH DESIGN AND METHODS: We used the model of streptozotocin-induced diabetic neuropathy (SDN) and evaluated the efficacy of LA supplementation in improving nerve blood flow (NBF), electrophysiology, and indexes of oxidative stress in peripheral nerves affected by SDN, at 1 month after onset of diabetes and in age-matched control rats. LA, in doses of 20, 50, and 100 mg/kg, was administered intraperitoneally five times per week after onset of diabetes. RESULTS: NBF in SDN was reduced by 50%; LA did not affect the NBF of normal nerves but improved that of SDN in a dose-dependent manner. After 1 month of treatment, LA-supplemented rats (100 mg/kg) exhibited normal NBF. The most sensitive and reliable indicator of oxidative stress was reduction in reduced glutathione, which was significantly reduced in streptozotocin-induced diabetic and alpha-tocopherol-deficient nerves; it was improved in a dose-dependent manner in LA-supplemented rats. The conduction velocity of the digital nerve was reduced in SDN and was significantly improved by LA. CONCLUSIONS: These studies suggest that LA improves SDN, in significant part by reducing the effects of oxidative stress. The drug may have potential in the treatment of human diabetic neuropathy.

Aging differentially modifies sensitivity of nerve blood flow to vasocontractile agents (endothelin-1, noradrenaline and angiotensin II) in sciatic nerve.

This study examined the influence of the vaso-constricting agents (noradrenaline, endothelin-1 and angiotensin II) in Sprague-Dawley rats aged 2, 6 and 24 months by evaluating epineurial arteriolar vasoreactivity in response to superfused teat agents. Nerve blood flow (NBF) was measured using microelectrode H2 polarography. In 24-month-old rats, NBF was decreased and vascular resistance (VR) was increased compared with 2- and 6-month-old rats. All of the constricting agents reduced NBF in the 2-, 6- and 24-month groups, however, the effects of the constricting agents reduced significantly with age. These results suggest that during aging, there is a decline of vasoconstrictive responses to noradrenaline, endothelin-1 and angiotensin II in peripheral nerve and that these changes may be due to altered function of receptors.

Pyridoxine neuropathy in rats: specific degeneration of sensory axons.

When rats received pyridoxine in doses large enough to cause neuropathy in humans, the animals developed gait ataxia that subsided after the toxin was withdrawn. By using quantitative histologic techniques, we found axonal degeneration of sensory system fibers and that the fibers derived from the ventral root were spared. Although the degeneration approached the dorsal root ganglion, neurons in the ganglion did not degenerate. We found no early decrease in oxygen consumption of nerve, suggesting that impaired oxidative metabolism was not the primary event.

Results of endoneural injection of Guillain-Barré serum in Lewis rats.

We previously studied the neurophysiologic effect of endoneural injection of serum from patients with acute inflammatory-demyelinating polyradiculoneuropathy into the sciatic nerve of Sprague-Dawley rats and did not observe a statistically significant difference between the results with that serum and control serum at 1 week. Because of potential strain susceptibility to acute inflammatory-demyelinating polyradiculoneuropathy serum after endoneural injection, the syngeneic Lewis rat--which is more susceptible than other strains of rats to experimental allergic neuritis (considered to be an experimental model of acute inflammatory-demyelinating polyradiculoneuropathy)--was studied. We used sera from five severely affected patients and also used a more sensitive (compared with our earlier studies) electrophysiologic approach that included in vivo monophasic compound action potential recordings and paired pulses. No statistically significant differences in conduction velocity, amplitude, indices of dispersion, or time-integral percentage were found between disease and control sera at 1 week. We conclude that the human acute inflammatory-demyelinating polyradiculoneuropathy serum tested did not contain measurable demyelinating activity for rat nerve in excess of that of control serum.

The effect of hyperbaric oxygenation and hypoxia on the blood-nerve barrier.

We measured the permeability coefficient--surface area product (PA) of peripheral nerve to [14C]sucrose in rat sciatic nerve in experimental chronic hypoxic hypoxia of up to 8 weeks and following hyperbaric oxygenation for 1 to 4 weeks. Chronic hypoxia caused an increase in PA presumably indicating impairment of the blood-nerve barrier at 4 weeks with restoration to normal by 8 weeks. Hyperbaric oxygenation (100% oxygen at 2.5 ata for 120 min/day 5 days per week) caused a mild but time dependent increase in PA reaching statistical significance by 4 weeks.

Slow axonal transport in experimental hypoxia and in neuropathy induced by p-bromophenylacetylurea.

The slow axonal transport of proteins radiolabeled by incorporation of [35S]methionine was studied in motor nerves of rats subjected to chronic hypoxia. The conditions involved exposure to an atmosphere of 8-10% oxygen for periods of 3, 5, or 10 weeks. An experimentally verified computer model predicted a drop in mean endoneurial oxygen tension from 30.5 to 19 mm Hg, despite a measured increase in circulating hemoglobin from 16 to 22 g%. Nerve conduction velocity was unaffected during the early stages of hypoxia. After 10 weeks of hypoxia, conduction velocity still appeared normal in the sciatic nerve but was reduced in the caudal nerve by 2.5-4.5 m/s. At no time, however, was there evidence of impaired slow axonal transport, which proceeded with a mean velocity between 1 and 2 mm/day. Another set of experiments was performed to evaluate slow axonal transport in motor nerves of rats with peripheral neuropathy induced by the toxicant, p-bromophenylacetylurea. The results suggested a lower transport velocity in rats showing total hind-limb paralysis as compared with rats showing only mild to moderate motor dysfunction. The difference, however, could have reflected accelerated transport in mild neuropathy. In our view, the observations in experimental hypoxia- and toxicant-induced neuropathy are noteworthy for the resistance of slow transport to perturbation of the neuronal environment.

Comparison of nerve regeneration in vascularized and conventional grafts: nerve electrophysiology, norepinephrine, prostacyclin, malondialdehyde, and the blood-nerve barrier.

We compared the efficacy of vascularized (VASC) and conventional (CONV) sciatic nerve grafts in restoring nerve blood flow (NBF), conduction, the blood-nerve barrier, norepinephrine (NE), and 6-keto prostaglandin F1 alpha (6-KPGF; the stable prostacyclin metabolite) in the sciatic nerve of the rat. We also measured malondialdehyde (MDA) content. NBF was much greater in VASC grafts, but the increase was confined to non-nutritive flow. There was a statistically non-significant increase in nerve action potential amplitude in the grafted segments of VASC nerves at 1 and 2 months post graft. The [14C]sucrose permeability surface area (PA) product was increased in both CONV and VASC at 1 and 3 months and was not different to each other. NE and 6-KPGF, the major vasoconstrictor and dilator of nerve microvessels were better restored in VASC than CONV reaching statistically significance for 6-KPGF (P less than 0.001). MDA used as an index of oxygen free radical generation was not significantly different in the 3 groups. The better restoration of 6-KPGF and perhaps NE suggest that VASC grafts may be more effective in restoring vasoreactivity of peripheral nerve following graft.

Different reinnervation patterns in the celiac/mesenteric and superior cervical ganglia following guanethidine sympathectomy in adult rats.

We sought to determine whether chronic guanethidine (Gu) treatment in adult rats produces depletion of sympathetic neurons and hyperinnervation by sensory neuropeptides in the celiac/superior mesenteric (C/SMG) ganglion. Rats received Gu 40 mg/kg per day i.p or saline for 5 weeks. Upon completion of treatment, the C/SMG and the superior cervical ganglion (SCG) were examined for neuropeptide Y (NPY), substance P (SP), calcitonin gene-related peptide (CGRP) and vasoactive intestinal polypeptide (VIP), both by immunocytochemistry (ICC) and radioimmunoassay (RIA). Gu produced marked depletion of NPY-containing neurons and NPY content in the C/SMG, similar to that in the SCG (-89 +/- 2 vs. -92 +/- 4%, respectively). SP and CGRP immunoreactivities were significantly higher in control C/SMG as compared with SCG; after Gu treatment, there was no significant increase in either SP or CGRP in the C/SMG, however, both increased in the SCG. In contrast, VIP levels were similar in the SCG and C/SMG in controls and increased in the C/SMG but not in the SCG after Gu treatment. Thus, in adult rats, the C/SMG is as susceptible as the SCG to Gu treatment; the different pattern of hyperinnervation by SP, CGRP and VIP of the C/SMG as compared with the SCG may reflect the different sources for these neuropeptides in prevertebral as compared with paravertebral ganglia.

Guanethidine sympathectomy increases substance P concentration in the superior sympathetic ganglion of adult rats.

Adult rats received intraperitoneal injections of guanethidine or saline for 5 weeks. Six to 8 weeks following completion of treatment, concentrations of substance P and neuropeptide Y (NPY) were measured by radioimmunoassay in the superior cervical ganglion (SCG) and thoracic spinal cord. The SCG was also immunostained for NPY and substance P. No differences were observed in thoracic spinal cord content of either NPY or substance P. We observed depletion of NPY immunoreactive neurons and NPY levels in the SCG, and pharmacologic evidence of postganglionic denervation in guanethidine-treated rats. In guanethidine-treated rats, there was a marked increase of substance P levels in the SCG, where substance P was localized in fibers, but not cell bodies. Thus, sprouting of substance P-containing sensory fibers in the sympathetic ganglia occurs following postganglionic sympathectomy in adult rats.

Hypothermic neuroprotection of peripheral nerve of rats from ischemia-reperfusion injury: intraischemic vs. reperfusion hypothermia.

The pathophysiology of ischemic fiber degeneration (IFD) is not known, but mechanisms involved during nerve ischemia differ from those during reperfusion. We have previously demonstrated hypothermic neuroprotection of peripheral nerve from IFD. We now evaluate the efficacy of hypothermia in the intraischemic vs. the reperfusion period, using our established model of ischemia-reperfusion injury. Intraischemic hypothermia resulted in significant recovery of all indices (behavior score, electrophysiology and histology, P<0.01 or 0.05) while hypothermia during reperfusion period showed less improvement, significant only for the histological score compared to normothermia group (IFD index, P<0.05). Once hypothermia was applied in the ischemic period, the resultant neuroprotection continued into the reperfusion period, even if nerve temperature was then raised during the reperfusion period. These results indicate that hypothermic neuroprotection is more efficacious during the intraischemic period than during reperfusion, when a lesser degree of neuroprotection ensued.

The expression of proinflammatory cytokine mRNA in the sciatic-tibial nerve of ischemia-reperfusion injury.

We evaluated the proinflammatory cytokines, TNF-alpha and IL-1beta, mRNA expression in the rat sciatic and tibial nerves following ischemia-reperfusion (IR) injury, using competitive RT-PCR, to explore the role of cytokines in IR injury. The expressions of both TNF-alpha and IL-1beta mRNA were related to severity of ischemia and occurred with reperfusion rather than ischemia alone. TNF-alpha gene expression peaked at 24 h of reperfusion, while that of IL-1beta peaked at 12 h. These data support the notion that the proinflammatory cytokines TNF-alpha and IL-1beta are involved in the inflammatory response of IR injury to the peripheral nervous system and may be involved in the pathophysiology of ischemic fiber degeneration.

Endoneurial oxygen tension and radial topography in nerve edema.

Endoneurial edema occurs in numerous human and experimental neuropathies. We tested the hypothesis that the resultant increase in intercapillary distance (ICD) may result in endoneurial hypoxia. Experimental galactose neuropathy (EGN) was chosen since in this model, edema is due to the accumulation of galactitol, which does not directly damage nerve fibers, so that it was possible to study the role of endoneurial edema alone. We measured endoneurial oxygen tensions (PnO2) using oxygen-sensitive microelectrodes and related PnO2 radial topography to ICD. We also determined local oxygen consumption (VLO2) and critical PnO2(PcritO2). EGN and age-matched controls were studied at 4 months. (1) Caudal nerve conduction velocity was reduced in EGN. (2) The PnO2 values were reduced in EGN and the PnO2 histogram was shifted into the hypoxic range. These changes were paralleled by a significant increase in ICD in EGN. (3) The radial topography of PnO2 in EGN differed from the relatively uniform distribution in control nerves. In EGN the subperineurial PnO2 was significantly lower than the PnO2 at the center of the fascicle. These changes were paralleled by a significantly greater increase in ICD in the periphery. (4) That the PnO2 reduction in EGN was significant is suggested by the marked reduction in VLO2 and the large percentage (greater than 75%) of intrafascicular regions that fell below PcritO2 in EGN.

Peripheral nerve conduction studies in galactose-poisoned rats. Demonstration of increased resistance to ischemic conduction associated with endoneurial edema due to sugar alcohol accumulation.

Edematous nerves of galactose-poisoned rats had an increased resistance to ischemic conduction block when compared with control animals. Ischemia was caused by cardiac arrest due to intracardiac air embolism in 1 group and by occlusion of the abdominal aorta in another. In these 2 groups of galactose-fed rats, time to 50% conduction block was increased by 27 and 42%, respectively and the difference between control and galactose rats was significant (P less than 0.01 and P less than 0.001).

Effect of alpha-tocopherol deficiency on indices of oxidative stress in normal and diabetic peripheral nerve.

We tested the hypothesis that oxidative stress can cause neuropathy by evaluating the effect of alpha-tocopherol depletion in normal and streptozotocin (STZ) diabetic peripheral nerve (known to be subject to oxidative stress). The end points were nerve electrophysiology and indices of oxidative stress. Studies were done on 6 groups of rats at 1 and 3 months: (1) Controls, normal alpha-tocopherol (Con[N]). (2) Controls, alpha-tocopherol-deficient (Con[-]) (3) Controls, alpha-tocopherol supplemented (Con[+]); (4) Diabetic, normal alpha-tocopherol (STZ[N]); (5) Diabetic, alpha-tocopherol-deficient (STZ[-]) (6) Diabetic, alpha-tocopherol supplemented (STZ[+]). An alpha-tocopherol-deficient diet resulted in a rapid depletion of the vitamin in plasma and sympathetic neurones (superior cervical ganglion), and a slower depletion in sensory neurones (dorsal root ganglion) and nerve. The depletion was associated with a reduction in reduced glutathione and an increase in conjugated dienes and hydroperoxides in normal rats, and resulted in similar changes, or accentuated the abnormalities, in diabetic nerves. Changes were more pronounced at 1 than 3 months and alpha-tocopherol supplementation, for the most part, did not prevent the abnormalities. alpha-Tocopherol depletion induced or worsened nerve conduction abnormalities in both sciatic-tibial and caudal nerves. Sensory fibers were more affected than motor fibers and the changes were more pronounced at 3 than 1 month. These findings support the notion that oxidative stress may cause neuropathy and that it might be mechanistically implicated in experimental diabetic neuropathy (STZ-EDN).

Motor and somatosensory evoked potentials in mice infected with Theiler's murine encephalomyelitis virus.

We used an in vivo technique to record spinal motor and somatosensory evoked potentials in SJL/J and B10 mice chronically (4-10 months) infected with Daniel's strain of Theiler's murine encephalomyelitis virus (TMEV). SJL/J mice demonstrated primary spinal cord demyelination with chronic TMEV infection, whereas B10 mice were resistant to TMEV induced demyelination. Analysis based on the velocity of the initial peak of evoked responses demonstrated significantly slower conduction velocities in infected SJL/J mice as compared to age-matched uninfected SJL/J controls (p < 0.01) and infected B10 mice (p < 0.01). We noted no significant differences in conduction velocities of spinal evoked potentials recorded between uninfected SJL/J mice, uninfected B10 mice and infected B10 mice. Chronic infection with TMEV in susceptible SJL/J mice is associated with slowed conduction of spinal motor and somatosensory evoked potentials. This sensitive electrophysiologic assay will provide an in vivo method to test therapeutic regimens to inhibit demyelination or promote remyelination.

Alpha-lipoic acid provides neuroprotection from ischemia-reperfusion injury of peripheral nerve.

BACKGROUND: Reperfusion aggravates nerve ischemic fiber degeneration, likely by the generation of reduced oxygen species. We therefore evaluated if racemic alpha-lipoic acid (LA), a potent antioxidant, will protect peripheral nerve from reperfusion injury, using our established model of ischemia-reperfusion injury. METHODS: We used male SD rats, 300+/-5 g. Ischemia was produced by the ligature of each of the supplying arteries to the sciatic-tibial nerve of the right hind-limb for predetermined periods of time (either 3 or 5 h), followed by the release of the ligatures, resulting in reperfusion. LA was given intraperitoneally daily for 3 days for both pre- and post-surgery. Animals received either LA, 100 mg/kg/day, or the same volume of saline intraperitoneally. Clinical behavioral score and electrophysiology of motor and sensory nerves were obtained at 1 week after ischemia-reperfusion. After electrophysiological examination, the sciatic-tibial nerve was fixed in situ and embedded in epon. We evaluated for ischemic fiber degeneration (IFD) and edema, as we described previously. RESULTS: Distal sensory conduction (amplitude of sensory action potential and sensory conduction velocity (SCV) of digital nerve) was significantly improved in the 3-h ischemia group, treated with LA (P<0.05). LA also improved IFD of the mid tibial nerve (P=0.0522). LA failed to show favorable effects if the duration of ischemia was longer (5-h ischemia). CONCLUSION: These results suggest that alpha-lipoic acid is efficacious for moderate ischemia-reperfusion, especially on distal sensory nerves.

Electrophysiological studies on the effect of age on caudal nerve of the rat.

The effect of age on electrophysiologic parameters was studied on the caudal nerve of 374 male Sprague-Dawley rats (age 24 to 630 days) and on 83 Fischer 344 rats (age 29 to 900 days). For Sprague-Dawley rats nerve conduction velocity and nerve action potential amplitude were both a parabolic function of age, increasing to 1 year and then declining. For Fischer 344 rats the same parameters increased progressively to 1.5 years and then declined. In contrast, for both Sprague-Dawley and Fischer 344 rats, resistance to ischemic conduction failure was a hyperbolic function of age reaching an asymptote at 1.5 to 2 years of age. These age-related changes in caudal nerve parallel the electrophysiologic alterations with age in human peripheral nerve more closely than previous reports in rodent limb nerves.

Effect of cilostazol on experimental diabetic neuropathy in the rat.

Two proposed mechanisms of diabetic neuropathy are microvascular ischaemia and a reduction in Na,K-ATPase activity. We evaluated the effect of cilostazol, a drug that is both a potent phosphodiesterase inhibitor that normalizes nerve Na,K-AT-Pase and a vasodilator, on nerve blood flow (NBF) to determine whether it would improve experimental diabetic neuropathy. We examined whether epineurally applied cilostazol acted as a vasodilator on the peripheral nerve of normal and diabetic rats, and whether feeding the rats a cilostazol-supplemented diet could improve diabetic neuropathy. Cilostazol increased nerve blood flow (NBF) in a dose-dependent fashion with an EC50 of 10(-5.74) mol/l. Cilostazol also normalized NBF in experimental diabetic neuropathy with a 10(-4) mol/l local application on the sciatic nerve. In diabetic neuropathy, a cilostazol-supplemented diet improved both NBF and nerve conduction in a dose- and time-dependent fashion. Potential mechanisms of action of cilostazol on the nerve include its effect on NBF, Na, K-ATPase, and restoration of the thromboxane:prostacyclin ratio. Cilostazol may have potential in the treatment of diabetic neuropathy.