Our findings suggest that gonadal hormone deficiency causes an inflammatory response, followed initially by microgial proliferation and activation, and later by microglial dystrophy and decline. These effects preferentially target the hippocampus and lead to a shrinking of the microglia population as well as smaller microglia somata. These effects may therefore contribute to the observed decline of hippocampal volume in depressive patients . We found that hormone replacement at a testosterone/estradiol ratio of 35:1 suppressed inflammation and microglial activation in gonadectomized mice and reduced depressive behavior. These findings suggest a model in which an over-expression of Traf6 contributes to gonadal hormone deficiency-induced microglial activation or immunoreaction leading to the development of depressive-like behaviors.
We found that short-term gonadectomy induced the activation of Traf6/TAK1 signaling, corresponding to inflammatory response as reported in previous work [46, 47]. At the same time, short-term gonadectomy stimulated inflammation, similar to previous results , and subsequently triggered microglial proliferation and activation. Consistent with our results, previous work showed that exposing mice to lipopolysaccharide activates microglia within 6 h , exposing animals to live Escherichia coli activates microglia after 2 days , and exposing leptomeningeal cells to heat-killed bacteria activates microglia after 2 weeks . These results indicate that gonadal hormone deficiency compromises the blood-brain barrier , triggering the release of peripheral inflammatory cytokines into the brain followed by microglial proliferation and activation. The role of inflammatory cytokines in activating microglia and stimulating their proliferation is supported by our observation that hormone replacement with the 35:1 testosterone/estrogen ratio reversed the microglial activation induced by gonadectomy. This initial activation and proliferation of microglia subsequently leads to microglial decline, decreased synaptic remodeling, suppressed neurogenesis, and depressive behaviors, which we were able to reverse in our study through hormone replacement with the testosterone/estrogen ratio. Our work is consistent with several previous studies documenting the neuroprotective effects of androgens and estrogen specifically [53–58]. In all these cases, it appears that the sex hormone inhibits microglial proliferation and activation, albeit through different mechanisms.
Earlier studies in rodents have suggested that pathogenic bacteria, aging, brain injury, and gonadal hormone deficiency stimulate an immunoreactive population of microglia [59–62], whose activation can be observed as increased Iba-1 and MHC-II staining, shortening of cell processes, increase in cell soma area, and acute production of inflammatory cytokine after immunosuppression. The present study extends that literature by showing that gonadal hormone deficiency leads to a large number of activated microglia that develop and persist in the hippocampus for at least 7 days after gonadectomy. Interestingly, by day 14, microglial Iba-1 staining and soma area had returned to baseline, although cell processes remained shorter. This time-dependent effect echoes a previous study in which unpredictable stress caused microglia in the dentate gyrus to become hyperactive on day 2, yet by day 4 their activation status had reverted to baseline . In the present study, we found that at 30 days after gonadectomy, microglial apoptosis increased and astrocyte number fell, followed by onset of depressive behaviors. Our results demonstrate that using microglial inhibitors to block microglial activation and subsequent neuronal apoptosis can alleviate depressive behaviors. In support of this notion, 1-week gonadal hormone replacement inhibited microglial activation, and 5-week replacement caused gonadectomy-induced neuroinflammation, neurogenesis suppression, neuronal apoptosis and depressive behavior to revert to baseline.
Studies suggest that microglia are not passive, simply waiting to be activated by various stimulus, but rather that they are active even when "at rest": they interact with neurons and astrocytes via tight intercellular junctions to regulate physiological processes such as nutrient transport and synaptic formation [64–66]. Our results suggest that depressed symptomatology associated with gonadal hormone deficiency is related to microglial suppression, rather than activation. Thus, analysis at 30 days after gonadectomy showed microglial decline, increased immobile time in the FST and TST, and decreased sucrose preference. Gonadal hormone deficiency was also associated with elevated levels of the neuroinflammatory factors IL-6 and TNF-α.
Our observations of synergism between testosterone and estradiol in the development of depression and in their therapeutic effects is consistent with our previous report that the 10:1 ratio of testosterone/estradiol synergistically inhibits apoptosis in PC12 cells via the inhibition of Traf6/TAK1 signaling (43). Traf6 regulates TLRs-mediated pro-inflammatory cytokine production . This study also demonstrated that the 35:1 ratio of testosterone/estradiol synergistically suppresses the microglial priming via decreasing Traf6 expression and the levels of inflammatory cytokines, suggesting that Traf6 is a key mediator for regulating the activation of microglia in gonadectomized mice. Similar synergistic therapeutic effect has been reported in early-stage atherosclerosis, proceptive behavior, prostatic hyperplasia, and abnormal spermatogenesis [15, 68–69]. Our results suggest that this synergism exerts anti-depressant effects at least in part by inhibiting microglial activation. In contrast, testosterone on its own has previously been shown to exert anti-depressant effects by undergoing aromatization, and then the resulting derivative induces over-expression of extracellular signal-regulated kinase 2 (ERK2) within the dentate gyrus [14, 23]. Our findings appear to be linked to suppression of hippocampal inflammatory cytokines, and induction of neurogenesis.
The neuroprotective roles of testosterone and estradiol were well demonstrated in previous publications according to clinical retrospective studies and depressive model investigations [5–14, 70]. Although the molecular mechanisms of depression remain unclear, the pathophysiology that excessive induction of the hypothalamic-pituitary-adrenal cortical (HPA) axis leading to increased glucocorticoid abundances and decreased feedback suppression of the axis . Moreover, our findings display that the pathophysiology, gonadal hormone deficiency, results in microglial activation in hippocampus followed by the development of depression. Through the microglail activation, subsequent excess decline of microglia may contribute to sex hormone deficiency-induced structural alterations in the hippocampus [72, 73], a phenomenon often related to the decreased releasing of BDNF. The neurotrophic theory of depression refers to the decreased of hippocampal BDNF level, which can be ameliorated through the treatment with antidepressants and replacements of gonadal hormone in our study . Accordingly, it is likely that testosterone and estradiol can induce the expression level of BDNF. Testosterone and estradiol were demonstrated to have neuroprotective roles through different molecular mechanisms, respectively [21–24], whereas our findings show that they have a significant synergistic antidepressant-like effects in gonadectomized male mice via suppressing the microglial activation. However, the molecular mechanisms in suppressed microglial after treatment with sex hormone need to be further studied.