Abstract
Addressing potential sex differences in pre-clinical studies is crucial for developing therapeutic interventions. Although sex differences have been reported in epidemiological studies and from clinical experience, most pre-clinical studies of neuroinflammation use male rodents; however, sexual dimorphisms in microglia might affect the CNS inflammatory response. Developmental changes are also important and, in rodents, there is a critical period of sexual brain differentiation in the first 3 weeks after birth. We compared rat microglia from sex-segregated neonates (P1) and at about the time of weaning (P21). To study transitions from a basal homeostatic state (untreated), microglia were subjected to a pro-inflammatory (IFNγ + TNFα) or anti-inflammatory (IL-4) stimulus. Responses were compared by quantifying changes in nitric oxide production, migration, and expression of nearly 70 genes, including inflammatory mediators and receptors, inflammasome molecules, immune modulators, and genes that regulate microglial physiological functions. No sex differences were seen in transcriptional responses in either age group but the IL-4-evoked migration increase was larger in male cells at both ages. Protein changes for the hallmark molecules, NOS2, COX-2, PYK2 and CD206 correlated with mRNA changes. P1 and P21 microglia showed substantial differences, including expression of genes related to developmental roles. That is, P21 microglia had a more mature phenotype, with higher basal and stimulated levels of many inflammatory genes, while P1 cells had higher expression of phagocytosis-related molecules. Nevertheless, cells of both ages responded to IL-4 and IFNγ + TNFα. We examined the Kv1.3 potassium channel (a potential target for modulating neuroinflammation) and the Kir2.1 channel, which regulate several microglia functions. Kv1.3 mRNA (Kcna3) was higher at P21 under all conditions and male P21 cells had higher mRNA and Kv currents in response to IFNγ + TNFα. Overall, numerous transcriptional and functional responses of microglia changed during the first 3 weeks after birth but few sex-dependent changes were seen.
Highlights
It is recognized that sex can profoundly influence both the susceptibility and response to disease (Ober et al, 2008; Regitz-Zagrosek, 2012)
We previously found that this method was preferable to a single reference protein (e.g., β actin) because such ‘housekeeping’ proteins can change with microglial activation states (Lam et al, 2017)
Sex differences are recognized in brain development, adult brain structure and chemistry (Cosgrove et al, 2007); little is known about sex differences in specific brain cells
Summary
It is recognized that sex can profoundly influence both the susceptibility and response to disease (Ober et al, 2008; Regitz-Zagrosek, 2012). Developmental and hormonal changes can contribute to sex differences in the brain. The testosterone surge influences development of neural cells and plays important organizational roles in establishing sexual dimorphisms in neural circuitry, notably in areas responsible for sexually divergent behaviors (Clarkson and Herbison, 2016). Not enough is known about whether there are sex differences in responses of individual CNS cell types. Both genetic differences and early exposure to sex hormones have the potential to alter microglial responses to inflammatory stimuli, and it is possible that such changes are sustained after they are removed from the brain
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