Gender differences in alcohol-induced neurotoxicity and brain damage
Introduction
It has long since been known that women are more vulnerable than men to many medical consequences of alcohol abuse. For example, alcoholic women develop cirrhosis (Loft et al., 1987), alcohol-induced damage of the heart muscle (i.e., cardiomyopathy) (Fernandez-Sola et al., 1997) and nerve damage (i.e., peripheral neuropathy) (Ammendola et al., 2000) after fewer years of heavy drinking than alcoholic men. Studies comparing men and women's sensitivity to alcohol-induced brain damage, however, have not proved conclusive. Some studies using imaging with computerized tomography (Mann et al., 1992) have compared brain shrinkage, a common indicator of brain damage, in alcoholic men and women, and have reported that male and female alcoholics showed significantly greater brain shrinkage than control subjects. Similarly, other studies have revealed that both men and women present similar learning and memory problems as a result of heavy drinking (Nixon et al., 1995). Nevertheless, one important difference is that alcoholic women have reported that they had been drinking for shorter periods than alcoholic men, suggesting that women's brains might be more vulnerable to alcohol-induced damage than men's (Hommer, 2003). Clearly, more research on this topic is necessary, especially as alcoholic women have received much less research-based attention than alcoholic men despite clear evidence showing that women may be particularly vulnerable to the effects of alcohol on many key organ systems.
More recently, several studies have demonstrated the differential neurotoxic effects of ethanol on male and female adolescents with binge drinking. Gender differences have been described in prefrontal cortex volumes of adolescents with alcohol use disorders, where females and males respectively present smaller and larger volumes than controls (Medina et al., 2008). In addition, limited frontal responses to a spatial working memory task and reduced grey matter volume in females with alcohol use disorders as compared to males suggest that female adolescents may be more vulnerable to impairing effects of alcohol (Caldwell et al., 2005, Schweinsburg et al., 2003). Likewise, binge drinking during adolescence is associated with gender-specific differences in frontal, temporal and cerebellar brain activation during spatial working memory tasks which, in turn, relate to cognitive performance. Under such effects, adolescent females have been found to be more vulnerable to the neurotoxic effects of heavy alcohol use than adolescent males (Squeglia et al., 2011b). Magnetic resonance imaging (MRI) has also shown that female adolescents participating in binge drinking had thicker cortices in left frontal regions than non-drinker females, which has been linked to worse visuospatial, inhibition and attention performance than binge-drinking males, and they presented thinner cortices in these areas than non-drinking males (Squeglia et al., 2012).
Although the mechanisms underlying the structural and damaged effects of ethanol are presently unknown, we have reported that binge-ethanol drinking during adolescence (Pascual et al., 2007) and chronic alcohol abuse in adulthood (Alfonso-Loeches et al., 2010) induces brain damage by neuroinflammation. We have shown that ethanol activates the innate immune system by acting as an agonist of Toll-like receptor 4 (TLR4) in glial cells (Blanco et al., 2005, Fernandez-Lizarbe et al., 2009), leading to TLR4 signalling, production of inflammatory mediators, such as iNOS and COX-2 along with cytokines (IL-1β, TNF-α, IL-6), causing neuroinflammation, brain injury, white matter disarrangements, and even neurodegeneration (Alfonso-Loeches et al., 2010, Alfonso-Loeches et al., 2012). The role of the TLR4 function has been further demonstrated by showing that chronic ethanol intake does not cause neuroinflammation, demyelination and brain damage in TLR4-deficient mice (Alfonso-Loeches et al., 2010, Alfonso-Loeches et al., 2012). However, whether there are gender differences in alcohol-induced neuroinflammation and brain injury are presently unknown. Sex-related differences have been described to be related to the incidence and severity of diverse neurological disorders, and also in sepsis processes in humans (Johansson et al., 2012). A sexual dimorphism in systemic inflammation and in the progression of some demyelinating diseases, such as multiple sclerosis, has been observed to affect females more than males (Johansson et al., 2012). In addition, differential sex effects on neuroinflammation associated with dementia (Miller et al., 2010), and the influence of estradiol on glial pro-inflammatory responses to lipopolysaccharide, have been suggested to underlie the greater prevalence of neuroinflammatory diseases in females than in males (Loram et al., 2012).
Therefore by considering women's vulnerability to the toxic effects of ethanol, and the gender differences in neuroinflammation, the present study aims to evaluate the effects of chronic ethanol treatment on ethanol-induced neuroinflammation and brain damage in male and female mice. We herein demonstrate that chronic ethanol consumption up-regulates inflammatory mediators (iNOS and COX-2) and cytokines (IL-1β, TNF-α), increases caspase-3 cleavage in the cerebral cortex of both female and male mice, and that the levels of inflammatory mediators, and even the apoptotic cleavage of caspase-3, tend to be higher in female than in male mice. These differential effects correlate with increased astrogliosis along with neuronal reduction assessed by in vivo imaging studies. Our results also support the role of TLR4 in alcohol-induced neuroinflammation and brain injury since elimination of TLR4 mostly abolishes ethanol-induced brain damage in both males and females.
Section snippets
Mice
C57BL/6 WT (n = 40, 20 male and 20 female, Harlan Ibérica S.L., Barcelona) and TLR4 knockout mice (n = 40, 20 male and 20 female, C57BL/6 background, kindly provided by Dr. S. Akira, Osaka University, Japan) were used. The animals were kept under controlled light and dark conditions, at a temperature of 23 °C, with 60% humidity. All the animal experiments were carried out in accordance with the guidelines established by European Communities Council Directive (86/609/ECC) and Spanish Royal Decree
Chronic ethanol treatment induces inflammatory mediators, cytokines production and causes apoptosis in cerebral cortices of male and female mice: role of TLR4
We have previously demonstrated that ethanol consumption in female mice induces neuroinflammation by the activation of the TLR4 signalling pathway in the brain, leading to the release of inflammatory mediators and cytokines (Alfonso-Loeches et al., 2010). Therefore, in order to assess the possible gender differences in ethanol-induced neuroinflammation, the iNOS and COX-2 protein expressions were evaluated in the cerebral cortices of male and female WT mice. The results in Fig. 1A demonstrate
Discussion
The present study evidences females’ vulnerability to alcohol-induced neuroinflammation and brain damage. In agreement with previous findings (Alfonso-Loeches et al., 2010), we reveal that chronic ethanol intake causes neurotoxicity and brain damage by activating the neuroimmune response in glial cells, and by triggering the induction of inflammatory mediators, such as iNOS and COX-2, caspase-3 cleavage and cytokines, events which might trigger neuroinflammation, gliosis and neuronal cell
Conclusion
Studies in humans suggest that women are more vulnerable than men to the many medical consequences of alcohol abuse. However, studies into possible gender differences in alcohol-induced brain damage in adults have proven inconclusive. The findings presented herein evidence that during the same period of chronic ethanol treatment, female mice are more susceptible than male mice to ethanol-induced neuroinflammation, astrogliosis and reduced MAP-2 positive neurons. These findings further evidence
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgments
We would like to thank M. March for her excellent technical assistance and D. Hervás for his help in the statistical analysis from the Biostatistics Unit Department at the IIS, La Fe Hospital (Valencia). We are also thankful to Dr. S. Akira who provided us with the TLR4−/− knockout mice. This work has been supported by grants from the Spanish Ministry of Science and Innovation (SAF 2009-07503), the Spanish Ministry of Health: The Institute Carlos III and FEDER funds (RTA-Network) and PNSD (Ex.
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The authors contributed equally to this work.