James D. Schmelzer

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.

Effect of ischemia and reperfusion in vivo on energy metabolism of rat sciatic-tibial and caudal nerves

Our model of severe nerve ischemia consistently results in extinction of the compound nerve and muscle action potentials (NAP; CMAP) within 30 min. Since impulse transmission may depend on nerve energy metabolism (NEM), we studied the effects of ischemia with reperfusion on sciatic-tibial nerve NEM in vivo and compared these results with NEM of this nerve in deoxygenated Ringers solution in vitro and postmortem. Ischemia for 30 min postmortem or in deoxygenated Ringers solution resulted in marked depletion of adenosine triphosphate (ATP) and creatine phosphate (CP) and an increase in lactate (LAC) of sciatic-tibial nerve of adult male Sprague-Dawley rats. In vivo ischemia for up to 3 h to sciatic-tibial nerve was sufficient to extinguish CMAP but not NAP and did not deplete ATP, CP, or GLU nor did it increase LAC. Ischemia sufficient to extinguish NAP resulted in reduction of energy substrates to about 50% of resting. Muscle fails to conduct impulses before nerve and in vivo reductions of energy substrates are milder than in vitro changes. These changes are explainable in terms of energy requirements and supply. These findings support an energetic basis of ischemic conduction failure.

Efficacy of limb cooling on the salvage of peripheral nerve from ischemic fiber degeneration

Since peripheral nerve has a large ischemic safety factor, hypothermia, by reducing metabolic demands, it is potentially an efficacious technique to rescue nerve from ischemic fiber degeneration (IFD). We therefore evaluated the influence of temperature on the severity of IFD resulting from a standard ischemic stress. Ischemia to the left sciatic nerve in the rat was produced by embolization of 2 × 106 microspheres (14 μm) into its supplying arteries. The limb was embolized at three temperatures, 37°C, 32°C, and 28°C and was maintained at each temperature for an additional 4 h. End points, evaluated 7 days after embolization, for the embolized limb were: (1) behavioral scores, 0–11 in increasing limb function; (2) compound nerve action potential of sciatic‐tibial nerve; (3) sciatic nerve blood flow (NBF, in mL/100 g/min); and (4) histologic grade, expressed as percentage of fibers undergoing IFD (0 = < 5% 1 = 5–25% 2 = 26–50% 3 = 51–75% 4 = >75%). NBF was reduced in all groups, varying with temperature, and all indices of nerve structure and function were significantly improved with hypothermia. We conclude that hypothermia, easily achievable in a limb nerve, is highly efficacious in the rescue of nerve from IFD. These findings are of clinical relevance.

Effect of cilostazol on experimental diabetic neuropathy in the rat

SummaryTwo 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.

Altered vasoreactivity to angiotensin II in experimental diabetic neuropathy: role of nitric oxide.

We evaluated the effects of angiotensin II and an angiotensin‐converting enzyme inhibitor (cilazapril) on nerve blood flow (NBF) and electrophysiology in control and diabetic rats. When applied locally to the sciatic nerve, the dose–response curve of angiotensin II was more potent in experimental diabetic neuropathy (EDN) than control rats. No difference existed in plasma angiotensin II levels between EDN and controls. The rats were given typical rat pellets or pellets treated with 10 mg/kg per day cilazapril for 4 weeks. Diabetes caused a significant reduction in NBF, nerve conduction velocity, and compound muscle action potential (CMAP) amplitudes. NBF was significantly increased in diabetic rats supplemented with cilazapril diet, and nerve conduction velocity and amplitudes of the CMAP were also improved after 4 weeks on this diet. Direct application 10−3 mol/L cilazapril on sciatic nerve did not increase NBF in normal and EDN rats. We topically applied the nitric oxide synthase (NOS) inhibitor, NG‐nitro‐L‐arginine, on sciatic nerve and observed reduced inhibition of NBF in EDN, which was correctable with a cilazapril diet. These results suggest that diabetic neuropathy may have an increasing vasopressor action with angiotensin II and this is likely to be the mechanism of NOS inhibition. Angiotensin II‐converting enzyme inhibitors may have potential in the treatment of diabetic neuropathy.

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.

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

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

Aortic occlusion and reperfusion and conduction, blood flow, and the blood-nerve barrier of rat sciatic nerve.

We studied the effect of ischemia and reperfusion on blood flow, electrophysiology, and the blood-nerve barrier of the sciatic nerve of the rat. Ischemia for 10 to 60 min, with up to 3 h of reperfusion, was produced by closure and then release of a noose around the abdominal aorta. Nerve blood flow and function were measured serially using microelectrode-hydrogen polarography and electrophysiologic recordings, respectively. The integrity of the blood-nerve barrier was assessed using the permeability surface area product to [14C]sucrose. Ischemia of up to 30 min did not impair reperfusion. One hour of ischemia resulted in reperfusion abnormalities that affected about half of the nerves. The ischemic and reperfusion stresses did not disrupt the blood-nerve barrier to [14C]sucrose nor produce conduction block. Possible mechanisms for this resistance to ischemic and reperfusion injury are discussed.

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.

A small dose of the immunosuppressive agent FK506 (tacrolimus) protects peripheral nerve from ischemic fiber degeneration

The immunosuppressant agent FK506 (tacrolimus) has proven to be neuroprotective against brain ischemia, but there are no data on potential neuroprotective effects of FK506 in peripheral nerve ischemia. We examined the potential effects of two doses of FK506 in protecting peripheral nerve from ischemic fiber degeneration. Ischemia in the left sciatic nerve of the rat was produced by injecting 2 × 106 microspheres (14 μm) into the left femoral, hypogastric, and superior gluteal arteries in proportions of 47%, 37%, and 17%, respectively. After embolization, FK506 was injected into the left femoral, hypogastric, and superior gluteal arteries in doses of 9.4, 7.4, and 3.4 μg, respectively, for the high‐dose group and 4.7, 3.7, and 1.7 μg, respectively, for the low‐dose group. The control rats were injected with saline. FK506 treatment resulted in dramatic behavioral improvement in nerve function, in the number of functioning nerve fibers, and in the salvage of a majority of nerve fibers from ischemic fiber degeneration in a dose‐dependent fashion. These results suggest that a small dose of FK506 protects peripheral nerve from ischemic fiber degeneration and that it may have potential in the treatment of ischemic neuropathy.