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Principal Investigator: Peter J. Syapin
Co-Investigator: Alice M. Young
Rats intoxicated with ethanol by the Majchrowicz procedure become maximally dependent after 4 days of intoxication and display maximal withdrawal signs 2-6 h after blood ethanol levels reach zero, although signs of withdrawal emerge earlier (Majchrowicz, 1975; Majchrowicz and Hunt, 1976). Concurrently, the intoxication generates a reproducible brain damage and neuroinflammation first detected after 1-2 days of exposure and persistent for up to 4 days (Collins et al., 1996; Obernier et al., 2002a; Obernier et al., 2002b, see Preliminary Studies also). Ethanol binge-induced brain damage can be reduced by concurrent treatment with compounds such as butylated hydroxytoluene (BHT) or cannabidiol, a non-psychoactive cannabinoid, and other presumed anti-oxidant neuroprotectants (Collins et al., 1998; Hamelink et al., 2005; Crews et al., 2006). Other studies show that withdrawal from ethanol dependence causes brain damage and increases the severity of subsequent withdrawal episodes (Phillips and Cragg, 1984; Sullivan et al., 1996; Becker, 1998; Mhatre and Gonzalez, 1999; Hashimoto and Wiren, 2008), presumably via greater physiological dependence (see Background). Together, these findings support our central hypothesis that the development of neuroinflammation, brain damage, and alcohol dependence are linked, and led to our target hypothesis: reducing the severity of alcohol-induced neuroinflammation and/or brain damage concomitantly will reduce the severity of alcohol dependence. Two aims will test this hypothesis:
Specific Aim 1 will test the ability of established in vivo neuroprotectants to alter severity of ethanol withdrawal symptoms when given during development of ethanol dependence or during withdrawal. These experiments will treat female and male rats with 120 mg/kg/day BHT (p.o.) and 80 mg/kg/day cannabidiol (i.p.), both of which have been shown in published studies to protect against alcohol-induced neurodegeneration in vivo.Rats will be treated with BHT, cannabidiol, or vehicle either during acquisition of ethanol dependence in the Majchrowicz binge procedure, but not during ethanol withdrawal, or during withdrawal but not during acquisition of dependence. Withdrawal severity will be monitored by both objective measures as well as by common subjective ratings, including abnormal body posture, tail stiffness, and the ventromedial distal limb flexion response. Our goal is to determine if neuroprotectants given during exposure to ethanol doses sufficient to produce physiological dependence will reduce the magnitude of dependence, as assessed by signs measured during abrupt withdrawal from ethanol. Because proposed neuroprotectants exerts anti-convulsant activity, a drug action known to reduce ethanol withdrawal severity, other groups of ethanol-dependent rats will be exposed to the neuroprotectants only during the final binge dose and subsequent withdrawal from ethanol.
Specific Aim 2 will assess whether the neuroprotectant treatments reduce ethanol-induced brain damage and/or neuroinflammation. These experiments will be performed on cohorts of rats treated with BHT, cannabidiol or vehicle in Specific Aim 1. Beginning 2 h after the final binge dose, randomly selected non-withdrawn rats will be anesthetized and their brains preserved in situ with fixative for subsequent quantification of neurodegeneration using the de Olmos amino cupric silver stain, and quantification of neuroinflammation using markers for reactive astrocytes and activated microglia. The purpose of this aim is two-fold. Firstly, neuroprotection from alcohol-induced brain damage has never been demonstrated in females, so this aim will fill this gap in knowledge and determine if treatments effective in male rats are also effective in female rats, while simultaneously confirming their efficacy in males. Secondly, these experiments will provide new data on whether these neuroprotection treatments are effective in reducing the neuroinflammation that occurs in the brain of rats exposed to neurodamaging ethanol intoxication.
When the results of aim 1 are correlated with those of aim 2, and if as predicted anti-oxidant neuroprotectants modify ethanol dependence, these specific aims can provide the groundwork for subsequent studies towards development of prototypic therapeutic agents for prevention of dependence and subsequent withdrawal. In addition, these results can open new avenues for study of unexplored biological processes that contribute to the development of ethanol dependence.