6. Discussion
6.1 COX-2 KI mice demonstrate increased microglial density over development
We began by examining microglial density within the E19 and P25 mouse brain within our regions of interest, selected based on their associations to ASD. To our knowledge this is the first time these time points have been examined within any of the given brain areas, in both the healthy WT and COX-2-KI mouse. We found sex differences in microglial density within the WT mice, demonstrating that postnatally males had greater microglial density compared to females in the hippocampus and olfactory bulb. These sex differences were not maintained for either region of the COX-2-KI mouse. This is likely due to the significant increase in density in the COX-2-KI females compared to the WT control within the hippocampus, and that within the olfactory bulb we found a decrease in COX-2-KI male densities compared to the control. The changes in the olfactory bulb were not significant. These findings suggest that the COX-2/PGE2 pathway may play a role within the sex-differences we found in the postnatal brain in a region- specific manner.
123 When looking to the effect on microglial density due to the disruption of COX-2
compared to the WT, we found in a region, developmental stage and sex specific manner, that the COX-2-KI mice had increased microglial density. Specifically, we found within the prenatal brain at E19 the female olfactory bulb had greater density values compared to the WT female. At P25 we found that the male COX-2-KI cerebellum and prefrontal cortex, and female COX-2-KI hippocampus all showed significant increases compared to their sex-matched control. In summary, this implicates that the lack of the COX-2 enzyme results in increases in microglial density in a sex, time and region-specific manner.
In the healthy brain microglia are well spread-out throughout all regions, with varying densities from one region to the next. (Lawson et al, 1990; Yang et al, 2012). Within our density study we found for the first time for these developmental time points that each region we studied presented differences in microglial density from one area to the next, following the trends of previously shown in literature (Lawson et al, 1990; Yang et al, 2013; Keller et al, 2018).
Alterations of microglial density from healthy brains have been found in a variety of diseases and disorders and are recognized as a sign of pathology (Furube et al, 2018). Typically, microglial proliferation in both the adult rodent and human brain is slow with a stable density maintained throughout adulthood (Askew et al, 2017). Increased microglial density has been noted in a variety of pathologies in the brain such as traumatic brain injuries, prion diseases, schizophrenia, Alzheimer’s disease, and ASD in humans (Uranova et al, 2018; Morgan et al, 2010; Tetreault et al, 2012). Post mortem studies of human ASD patients have shown increased microglial density in a variety of areas including the prefrontal cortex and cerebellum (Vargas et al, 2005; Morgan et al, 2010; Suzuki et al, 2013). The increase in microglial density poses concern to the developing brain as studies have correlated the disruption of neural progenitor
124 proliferation as well as the inhibition of neuronal differentiation to increased microglial densities (Smith et al, 2014; Appel et al, 2018, Cunningham et al, 2013). The exact mechanisms are not yet known for how the increase in density directly results in such abnormalities in progenitor proliferation and neuronal differentiation, although it has been suggested that the phagocytic activity of microglia and the wide range of soluble factors microglia can secrete may play a role (Appel et al, 2018; VanRyzin et al, 2018).
Interestingly, while we found an increase in density beyond the healthy brain in both sexes of the COX-2-KI animals, we saw greater changes in the females. Within the healthy brain we typically found that males had greater density than females, but in our COX-2-KI model the exaggerated density values beyond the healthy brain were similar between the sexes. This may relate to the Extreme Male Brain (EMB) theory of autism (Ferri et al, 2018). EMB is the theory that the “autistic brain” is identified by having more extreme versions of the typical male brain, exhibited through differences in brain morphology seen in both males and females. This agrees with our findings in the male brains as well, where COX-2-KI mice exhibited increased density compared to the control. A relationship between the masculinization of the brain and the COX- 2/PGE2 pathway has been demonstrated in the past (Amateau and McCarthy, 2004; Wright et al, 2008). It has been found that in the healthy male mouse brain that during development there is an upregulation of the COX-2 enzyme, which will ultimately result in high levels of PGE2
(Amateau and McCarthy, 2004). As well, new born female mice when injected with PGE2 were found to display male sexual behaviors, implying the importance of the COX-2/PGE2 pathway to the masculinization of the brain.
One possible explanation for the increases in microglia density we demonstrate across development is the long-term effect of PGE2 on microglia recently discovered. While PGE2 is a
125 known microglial activator, discussed in further detail in the next section, it has been recently reported that PGE2 also induces apoptosis in microglia after stimulation in cell models (Nagano et al, 2014; Fu et al, 2015; Yang et al, 2006). In particular, the prostaglandin E2 receptor (EP2) was found induce apoptotic effects on the cell once activated, while EP1, EP3 and EP4 had no effect on cell viability (Fu et al, Nagano et al, 2014). The downstream effect of PGE2 activation of EP2 is increased intracellular levels of cyclic monophosphate (cAMP) and is believed to induce caspase-3 activity in microglia resulting in apoptosis (Narumiya et al, 1999, Fu et al 2015). Due to the disruption in the production of PGE2 in our COX-2-KI model we can speculate that decreased EP2 activation potentially results in less activity-driven apoptosis within our ASD mouse model.