Sex-specific responses of the pubertal neuroimmune axis in CD-1 mice.

The mechanistic relationship between the sexually dimorphic neuroimmune system and the sex-specific outcomes of a pubertal immune challenge is unclear. Therefore, we examined sex differences in the progression of cytotoxic microglial responses and blood-brain barrier (BBB) disruption to a peripubertal lipopolysaccharide (LPS) treatment in brain regions relevant to stress responses and cognitive function. Six-week-old (i.e., stress-sensitive pubertal period) male and female CD-1 mice were treated with LPS (1.5 â€‹mg/kg body weight, ip) or 0.9% saline (LPS-matched volume, ip). Sex and treatment differences in microglial (Iba1+) and apoptotic neuronal (caspase-3+/NeuN+) and non-neuronal (caspase-3+/NeuN-) expression were examined in the hippocampus, medial prefrontal cortex (mPFC), and paraventricular nucleus 24 â€‹h (sickness), one week (symptomatic recovery) and four weeks (early adulthood) post-treatment (n â€‹= â€‹8/group). Microglial morphology was quantified with fractal analyses. Group differences in BBB permeability to 14C-sucrose were examined 24 â€‹h (whole-brain, hippocampus, prefrontal cortex, hypothalamus, and cerebellum) and one week (whole-brain) post-treatment. The acute effects of pubertal LPS were specific to females (i.e., global BBB disruption, altered microglial expression and morphology in the mPFC and hippocampus, increased hippocampal apoptosis). The residual effects of pubertal LPS-induced sickness observed in microglia persisted into adulthood in a sex- and region-specific manner. In addition to highlighting these sex-specific responses of the pubertal neuroimmune system, we report baseline region-specific sex differences in microglia spanning puberty through adulthood. We propose that these sex differences in neuroimmune-neurovascular interactions during the stress-sensitive pubertal period create sex biases in stress-related disorders of brain and behaviour.

Pubertal LPS treatment selectively alters PSD-95 expression in male CD-1 mice.

Puberty includes a highly stress-sensitive period with significant sex differences in the neurophysiological and behavioural outcomes of a peripheral immune challenge. Sex differences in the pubertal neuroimmune network's responses to systemic LPS may explain some of these enduring sex-specific outcomes of a pubertal immune challenge. However, the functional implications of these sex-specific neuroimmune responses on the local microenvironment are unclear. Western blots were used to examine treatment- and sex-related changes in expression of regulatory proteins in inflammation (NFκB), cell death (AIF), oxidative stress (SOD-1), and synaptic plasticity (PSD-95) following symptomatic recovery (i.e., one week post-treatment) from pubertal immune challenge. Across the four examined brain regions (i.e., hippocampus, PFC, hypothalamus, and cerebellum), only PSD-95 levels were altered one week post-treatment by the pubertal LPS treatment. Unlike their female counterparts, seven-week-old males showed increased PSD-95 expression in the hippocampus (p < .05). AIF, SOD-1, and NFκB levels in both sexes were unaffected by treatment (all p > .05), which suggests appropriate resolution of NFκB-mediated immune responses to pubertal LPS without stimulating AIF-mediated apoptosis and oxidative stress. We also report a significant male-biased sex difference in PSD-95 levels in the PFC and in cerebellar expression of SOD-1 during puberty (all p < .05). These findings highlight the sex-specific vulnerability of the pubertal hippocampus to systemic LPS and suggest that a pubertal immune challenge may expedite neurodevelopment in the hippocampus in a sex-specific manner.

Pubertal immune stress transiently alters spatial memory processes in adulthood.

Pubertal immune challenge can permanently alter hippocampus-dependent memory processes in a sex-specific manner. Although gonadal hormones can influence various cognitive processes, their role in regulating the cognitive sequelae to pubertal immune challenge has not been thoroughly assessed. We examined whether a pubertal immune challenge could affect hippocampus-dependent memory functions in adulthood and whether these effects are regulated by gonadal steroid hormones. We hypothesized that exposure to an immune challenge during puberty would induce sex-specific deficits in the behavioral and cellular correlates of hippocampus-dependent memory during adulthood. At six weeks of age, during the stress-vulnerable pubertal period, male and female CD-1 mice were injected with either saline or the bacterial endotoxin lipopolysaccharide (LPS). Three weeks later, mice underwent either gonadectomy or sham-surgery. At ten weeks of age (i.e., in adulthood), mice began behavioral testing in an open field, Barnes maze, and Morris water maze. Brain tissue was collected at 17 weeks of age and stained for doublecortin and Ki67 to examine migrating neuronal progenitor cells and cellular proliferation in the neurogenic subgranular zone (SGZ) and the cornus ammonis (CA)1 and CA3 regions of the hippocampus. Pubertal LPS treatment impaired learning during adulthood in both sexes and increased cellular proliferation in the CA1 region in castrated males only. Although adult sex hormones did not reliably modulate these changes, gonadectomy impaired learning during the Morris water maze in both sexes. Learning deficits were more prominent during the Barnes maze, which suggests a stress-dependent expression of LPS-induced cognitive deficits. Neurogenesis in the SGZ and cellular proliferation in the CA3 were not affected by pubertal LPS treatment or gonadectomy. These novel findings emphasize the sensitivity of developing cognitive processes during puberty to immune challenges and suggest a possible mechanism for learning-based difficulties in adulthood.

Sex differences in the peripheral and central immune responses following lipopolysaccharide treatment in pubertal and adult CD-1 mice.

Puberty is a critical developmental period that is characterized by significant brain development. Exposure to stress during this time can alter brain functioning setting the stage for long-lasting behavioural outcomes. The objective of this study was to investigate age and sex differences in the peripheral and central immune responses, along with sickness behaviour, following immune stress. The results showed that LPS treatment increased serum cytokine levels and sickness symptoms in all mice. Pubertal males displayed increased IL-1ß concentrations at 2 h and increased IL-6 concentrations at 8 h post-treatment whereas increased concentrations of TNFα, IL-10, IL-12, IL-1ß, IFNγ, and IL-6 persisted at 8 and 24 h in adult females. Consistent with peripheral cytokines, pubertal males displayed greater IL-1ß, TNFα, and IL-6 mRNA expression in the prefrontal cortex at 2 h, whereas adult males expressed more of the aforementioned cytokines at 8 h compared to saline controls. Adult males also displayed greater IL-1ß mRNA expression compared to their female counterparts, and adult females displayed greater TNFα mRNA expression compared to their male counterparts. These results not only provide a better understanding of the age and sex differences in acute immune response, but also show important region- and time-specific differences in the response to an immune challenge, and that the peripheral immune response differs from the central response. This highlights the need to examine immune markers in both the periphery and the central nervous system for an accurate depiction of acute immune response following an immune challenge.

Monitoring Pathogen-Induced Sickness in Mice and Rats.

Sickness behavior monitoring, a technique for examining the development of sickness symptomatology following infection, is necessary in experiments studying neurochemical and physiological changes associated with pathogen-induced immune activation. However, the results of sickness behavior monitoring are difficult to reconcile due to inconsistencies in protocol methods and rater bias. The protocol described herein offers a non-invasive and unbiased approach to assess the progression of pathogen-induced sickness behaviors. This simple, straightforward method uses a five-point scale to assess animals for the presence of four sickness behaviors (i.e., '"0" = no sickness behaviors; "4" = four sickness behaviors) at various time points following exposure to a pathogen. This approach removes the ambiguity and bias inherent to other methods of sickness behavior monitoring that rely on subjective ratings of severity for individual symptoms. This protocol has been successfully applied to male and female rodents injected intraperitoneally with lipopolysaccharide and polyinosinic:polycytidylic acid, and has been effective in pubertal and adult populations. Protocols for changes in body temperature and weight are also provided as physiological markers of sickness. © 2017 by John Wiley & Sons, Inc.