Caspase-mediated cleavage of actin and tubulin is a common feature and sensitive marker of axonal degeneration in neural development and injury.

BACKGROUND: Axon degeneration is a characteristic feature of multiple neuropathologic states and is also a mechanism of physiological neurodevelopmental pruning. The vast majority of in vivo studies looking at axon degeneration have relied on the use of classical silver degeneration stains, which have many limitations including lack of molecular specificity and incompatibility with immunolabeling methods. Because Wallerian degeneration is well known to involve cytoskeletal disassembly and because caspases are recently implicated in aspects of this process, we asked whether antibodies directed at caspase-generated neoepitopes of beta-actin and alpha-tubulin would be useful immunohistochemical markers of pathological and developmental axon degeneration. RESULTS: Here we demonstrate that several forms of axon degeneration involve caspase-mediated cleavage of these cytoskeletal elements and are well-visualized using this approach. We demonstrate the generation of caspase-induced neoepitopes in a) an in vitro neuronal culture model using nerve growth factor-deprivation-induced degeneration and b) an in vivo model using ethanol-induced neuronal apoptosis, and c) during normal developmental pruning and physiological turnover of neurons. CONCLUSIONS: Our findings support recent experimental data that suggests caspase-3 and caspase-6 have specific non-redundant roles in developmental pruning. Finally, these findings may have clinical utility, as these markers highlight degenerating neurites in human hypoxic-ischemic injury. Our work not only confirms a common downstream mechanism involved in axon degeneration, but also illuminates the potential utility of caspase-cleavage-neoepitope antibodies as markers of neurodegeneration.

Independent contributions of alcohol and stress axis hormones to painful peripheral neuropathy.

Painful small-fiber peripheral neuropathy is a debilitating complication of chronic alcohol abuse. Evidence from previous studies suggests that neuroendocrine mechanisms, in combination with other, as yet unidentified actions of alcohol, are required to produce this neuropathic pain syndrome. In addition to neurotoxic effects of alcohol, in the setting of alcohol abuse neuroendocrine stress axes release glucocorticoids and catecholamines. Since receptors for these stress hormones are located on nociceptors, at which they can act to cause neuronal dysfunction, we tested the hypothesis that alcohol and stress hormones act on the nociceptor, independently, to produce neuropathic pain. We used a rat model, which allows the distinction of the effects of alcohol from those produced by neuroendocrine stress axis mediators. We now demonstrate that topical application of alcohol and exposure to unpredictable sound stress, each alone, has no effect on the nociceptive threshold. However, when animals that had previous exposure to alcohol were subsequently exposed to stress, they rapidly developed mechanical hyperalgesia. Conversely, sound stress followed by topical alcohol exposure also produced mechanical hyperalgesia. The contribution of stress hormones was prevented by spinal intrathecal administration of oligodeoxynucleotides antisense to ß(2)-adrenergic or glucocorticoid receptor mRNA, which attenuates receptor level in nociceptors, as well as by adrenal medullectomy. These experiments establish an independent role of alcohol and stress hormones on the primary afferent nociceptor in the induction of painful peripheral neuropathy.

Illicit alcohol consumption and neuropathy--a preliminary study in Sri Lanka.

AIMS: To compare the effects on peripheral and autonomic nerve functions of Sri Lankan illicitly distilled alcohol consumption versus legal spirit consumption. METHODS: Peripheral nerve conduction and autonomic nerve functions were assessed in 40 healthy control subjects and two groups of chronic heavy drinkers: 41 illicit spirit drinkers and 17 legal spirit drinkers. RESULTS: Lower-limb motor and sensory nerve conduction parameters were affected in both groups of alcoholics. When compared with controls, in illicit spirit drinkers the mean heart rate indexes of all parasympathetic tests were lower while in legal spirit drinkers the heart rate response to standing was affected. There were no differences in the results of the above tests when the two groups of heavy drinkers were compared. CONCLUSIONS: Though chronic alcoholism results in peripheral and autonomic nerve damage, the damage caused by consumption of illicitly distilled spirit is not worse than the damage caused by consumption of legal spirits.

mGlu5 receptor and protein kinase C implicated in the development and induction of neuropathic pain following chronic ethanol consumption.

The central mechanisms of neuropathic pain following chronic ethanol consumption are poorly understood. We previously reported that the levels of metabotropic glutamate 5 (mGlu5) receptor and phosphorylated-protein kinase C (PKC) were significantly increased in the spinal cord following chronic ethanol consumption. The aim of this study was to investigate whether mGlu5 receptor and PKC inhibitors directly attenuate the neuropathic pain-like state induced by chronic ethanol treatment in rats. A significant decrease in the mechanical nociceptive threshold was observed 5 weeks of chronic ethanol consumption. This hyperalgesia was significantly attenuated by repeated i.p. injection of (S)-2,6-diamino-N-[[1-(oxotridecyl)-2-piperidinyl]methyl] hexanamide dihydrochloride (NPC15437), a selective PKC inhibitor, once a day for a week after 4 weeks of ethanol treatment. Furthermore, this hyperalgesia was also significantly attenuated by repeated i.p. injection of 6-methyl-2-[phenylethynyl]-pyridine (MPEP), a selective mGlu5 receptor inhibitor, once a day for a week after 4 weeks of ethanol treatment. Furthermore, the hyperalgesia that developed after 5 weeks of ethanol treatment was significantly suppressed by a single i.p. post-injection with either NPC15437 or MPEP. These findings constitute direct evidence that spinal mGlu5 receptor and PKC play substantial roles in the development and maintenance of an ethanol-dependent neuropathic pain-like state in rats.

Involvement of mGluR5 in the ethanol-induced neuropathic pain-like state in the rat.

Alcohol neuropathy has been thought to involve decreased nerve function following chronic ethanol consumption. However, there is no reliably successful therapy, largely due to a lack of understanding of the central underlying mechanisms. The aim of this study was to investigate the mechanisms that contribute to the neuropathic pain-like state induced by chronic ethanol treatment in rats. Rats were chronically treated with ethanol diet (1.25-5% of ethanol) for over 70 days. Mechanical hyperalgesia was observed during ethanol consumption and even after ethanol withdrawal. Under these conditions, an immunohistochemical study showed an increase in metabotropic glutamate receptor 5 (mGluR5) immunoreactivity in the superficial spinal dorsal horn of chronic ethanol-fed rats. Furthermore, immunoblot analysis revealed that the protein level of mGluR5 was clearly increased following chronic ethanol consumption. These findings support the idea that the increased levels of mGluR5 in the spinal cord may be, at least in part, involved in the induction of ethanol-dependent neuropathic pain-like state.

Alcoholic neuropathy is clinicopathologically distinct from thiamine-deficiency neuropathy.

Characteristics of alcoholic neuropathy have been obscured by difficulty in isolating them from features of thiamine-deficiency neuropathy. We assessed 64 patients with alcoholic neuropathy including subgroups without (ALN) and with (ALN-TD) coexisting thiamine deficiency. Thirty-two patients with nonalcoholic thiamine-deficiency neuropathy (TDN) also were investigated for comparison. In ALN, clinical symptoms were sensory-dominant and slowly progressive, predominantly impairing superficial sensation (especially nociception) with pain or painful burning sensation. In TDN, most cases manifested a motor-dominant and acutely progressive pattern, with impairment of both superficial and deep sensation. Small-fiber-predominant axonal loss in sural nerve specimens was characteristic of ALN, especially with a short history of neuropathy; long history was associated with regenerating small fibers. Large-fiber-predominant axonal loss predominated in TDN. Subperineurial edema was more prominent in TDN, whereas segmental de/remyelination resulting from widening of consecutive nodes of Ranvier was more frequent in ALN. Myelin irregularity was greater in ALN. ALN-TD showed a variable mixture of these features in ALN and TDN. We concluded that pure-form of alcoholic neuropathy (ALN) was distinct from pure-form of thiamine-deficiency neuropathy (TDN), supporting the view that alcoholic neuropathy can be caused by direct toxic effect of ethanol or its metabolites. However, features of alcoholic neuropathy is influenced by concomitant thiamine-deficiency state, having so far caused the obscure clinicopathological entity of alcoholic neuropathy.

Key role for the epsilon isoform of protein kinase C in painful alcoholic neuropathy in the rat.

Chronic alcohol consumption produces a painful peripheral neuropathy for which there is no reliably successful therapy, attributable to, in great part, a lack of understanding of the underlying mechanisms. We tested the hypothesis that neuropathic pain associated with chronic alcohol consumption is a result of abnormal peripheral nociceptor function. In rats maintained on a diet to simulate chronic alcohol consumption in humans, mechanical hyperalgesia was present by the fourth week and maximal at 10 weeks. Thermal hyperalgesia and mechanical allodynia were also present. Mechanical threshold of C-fibers in ethanol fed rats was lowered, and the number of action potentials during sustained stimulation increased. The hyperalgesia was acutely attenuated by intradermal injection of nonselective protein kinase C (PKC) or selective PKCepsilon inhibitors injected at the site of nociceptive testing. Western immunoblot analysis indicated a higher level of PKCepsilon in dorsal root ganglia from alcohol-fed rats, supporting a role for enhanced PKCepsilon second-messenger signaling in nociceptors contributing to alcohol-induced hyperalgesia.

Impaired stimulation of intestinal glucose absorption by portal insulin via hepatoenteral nerves in chronically ethanol-intoxicated rats.

In the isolated, jointly perfused small intestine and liver of rats insulin, infused into the portal vein, induced an increase in intestinal glucose absorption via hepatoenteral cholinergic nerves. The possible loss of function of these nerves due to ethanol-induced neuropathy was investigated with 6 weeks ethanol-fed rats. Portal insulin or arterial carbachol failed to increase intestinal glucose absorption but cAMP still did so. The intact stimulatory effect of cAMP indicated an undisturbed capacity of the enterocytes. The loss of action of portal insulin and of arterial carbachol can be explained by the impairment of the hepatoenteral nerves in line with an ethanol-induced neuropathy.