Hitoshi Nukada

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.

Spatial Distribution of Nerve Injury after Occlusion of Individual Major Vessels in Rat Sciatic Nerves

In an attempt to better understand the spatial distribution of ischemic injury secondary to occlusion of major arteries, we measured nerve blood flow (NBF) and studied morphologic changes at various levels distal to the ligature site. Arterial ligation of the femoral, internal iliac, or superior gluteal artery was preceded and followed by measurement of NBF using laser Doppler flowmetry which helped identify “watershed areas” and guided the sampling process as nerves were examined pathologically and areas of injury were identified. Femoral artery ligation produced the most severe ischemia, focally reducing NBF by 80% in the tibial nerve at a level just below the knee. Within these ischemic nerve segments there were degenerative changes of nerve fibers seen mainly in the subperineurial region. Ligation of the internal iliac artery caused an approximately 60% reduction in NBF at the upper and mid-thigh levels of the sciatic nerve which resulted only in endoneurial edema in tissue taken at this level. Following superior gluteal artery ligation, NBF was reduced by only 20% at the pelvic level of the sciatic nerve and there was neither endoneurial edema nor fiber abnormalities. This study demonstrates the watershed pattern of ischemic injury associated with single vessel ligation by correlating neuropathologic change with quantitative measures of local nerve blood flow. The data further support the concept that mild levels of ischemia cause endoneurial edema, while moderate levels of ischemia produce demyelination and severe ischemia produces Wallerian degeneration.

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. Ann Neurol 2000; 47:71–79

Protein carbonyl formation and tyrosine nitration as markers of oxidative damage during ischaemia-reperfusion injury to rat sciatic nerve

We have investigated the role of oxidative damage in peripheral nerve ischaemia-reperfusion injury using a rat sciatic nerve model. After 5 h ischaemia blood flow to the sciatic nerve was restarted and markers of oxidative damage measured after various times of reperfusion. As a marker of protein oxidative damage, protein carbonyl formation was measured using a sensitive enzyme-linked immunosorbent assay. Protein carbonyl content was unaffected by ischaemia alone, but increased by 55% after 12-18 h reperfusion, correlating with the onset of nerve pathology. Pretreatment with the xanthine oxidase inhibitor allopurinol prevented these abnormalities, suggesting that xanthine oxidase activity is proximal to oxidative damage during reperfusion injury. To determine whether formation of the potent oxidant peroxynitrite from nitric oxide and superoxide contributed to ischaemia-reperfusion injury, we measured the accumulation of 3-nitrotyrosine residues in proteins. Only one protein of 49,000 mol. wt contained significant amounts of 3-nitrotyrosine residues which was shown to be glial fibrillary acidic protein, an abundant cytoskeletal protein in Schwann cells. However glial fibrillary acidic protein contained 3-nitrotyrosine residues prior to ischaemia-reperfusion, and the amount of nitrated tyrosine residues in total glial fibrillary acidic protein did not increase significantly during reperfusion, therefore it was not possible to draw conclusions about the role of peroxynitrite in nerve reperfusion injury.

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.

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.

Perineurial window : demyelination in nonherniated endoneurium with reduced nerve blood flow

The perineurial window, created by surgical incision of the perineurial sheath allowing its contents to herniate into the epineurial space, provides an experimental model of primary demyelination, the cause of which is unclear. Because the injury is localized and involves distortion of tissue at the lesion site, ischemia is suspected as a cause of demyelination. To study the mechanism of demyelination in the perineurial window model, we measured nerve blood flow (NBF) with a laser Doppler flowmeter before and after perineurial rupture in rat sciatic nerve and assessed the spatial distribution of demyelinated fibers, particularly in the nonhemiated portion of the endoneurium. Nerve blood flow at the site of the perineurial window was reduced significantly with an average level of NBF approximately 50% of presurgical values 10 minutes, 60 minutes and 6 hours after surgery. By light microscopic examination, most nerve fibers that herniated through the perineurial window underwent demyelination by 7 days. In addition, focal lesions of subperineurial demyelination were found in the nonhemiated endoneurium in the adjacent subperineurial region and proximally and distally to the perineurial window. Endoneurial vessels adjacent to the perineurial incision appeared to be compressed. We suggest that ischemia contributes to the process of demyelination in the perineurial window model.

Auditory and inspiratory gasp-evoked sympathetic skin response: age effects.

The sympathetic skin response (SSR) has been employed to assess peripheral neuropathy as an index of sympathetic sudomotor activity. A variety of stimuli can be used to elicit the SSR, but their relative ease of use and reliability have not been studied. In addition, the extent to which age affects the SSR remains unresolved. We compared two different stimuli, a sudden loud noise and an inspiratory gasp, whilst recording SSRs from the hand and foot. We also investigated the effects of age on SSR amplitude and latency in 58 healthy volunteers (ages 13-79). SSRs evoked by the auditory stimulus were recorded in all subjects, while gasp-induced SSRs were not elicited in two subjects. We found that SSRs evoked by the auditory stimulus had less inter- and intra-subject latency and waveform variability than the gasp-induced response. The increased latency variability associated with the inspiratory gasp technique was probably due to triggering errors. Our results confirmed that the amplitude of the SSR is extremely variable and appears to be affected by many factors. Auditory-evoked SSR latencies revealed a significant non-linear increase with age, while SSRs evoked by an inspiratory gasp did not demonstrate age dependence. We conclude that an auditory stimulus is superior to an inspiratory gasp in evoking SSRs, both in terms of consistent appearance and reduced latency variability. As the SSR latency increases significantly with age, this effect should be carefully considered when interpreting the response.

Is ischemia implicated in chronic multifocal demyelinating neuropathy

We describe a patient with chronic multifocal demyelinating neuropathy associated with persistent conduction block. Multifascicular lesions in sural nerve included a complete loss of myelinated fibers, demyelination, remyelination, onion bulb formation, and axonal attenuation. On the basis of these morphometric results we hypothesize that nerve ischemia may be involved in the pathogenesis of chronic multifocal demyelinating neuropathy

Carbonyl histochemistry in rat reperfusion nerve injury

Free radical mediated, site-specific lipid and protein oxidation has been implicated in the pathophysiology of an ischaemic/reperfusion injury. The aim of the present study was to determine whether carbonyl formation could be detected histochemically in reperfused rat sciatic nerves. We also examined the effects of preischaemic alpha-tocopherol supplementation on carbonyl formation in reperfused nerves. Seven hours of near-complete ischaemia was induced in rat right hindlimb by occlusion of major arteries using microvascular clips. Histochemical detection of carbonyl compounds, applying naphthoic acid hydrazide (NAH) and Fast Blue B (FBB), was undertaken at thigh, knee and calf levels of sciatic, tibial and peroneal nerves. NAH-FBB reactivity was confined to vessels in reperfused nerves. Positively stained epi-, peri- and endoneurial vessels were invariably observed after 2 h of reperfusion at all levels examined. After 24 and 48 h and 7 days of reperfusion, NAH-FBB-positive vessels were more frequently found at knee and calf levels than at the thigh level. Following preischaemic alpha-tocopherol supplementation, no vessels were stained positively with NAH-FBB, except for some epineurial vessels at knee and calf levels after 2 h of reperfusion. Morphometry in endoneurial vessels at the knee level revealed that endothelial cell area in alpha-tocopherol-treated reperfused nerves was significantly less when compared with those in reperfused nerves without alpha-tocopherol. In conclusion, we have demonstrated histochemical evidence of carbonyl formation in vessels, but not with nerve fibres, in ischaemic/reperfused rat sciatic nerves. These abnormalities were prevented with preischaemic supplementation of alpha-tocopherol.