TrkB-mediated sustained neuroprotection is sex-specific and \(\text{ER}\alpha\)-dependent in adult mice following neonatal hypoxia ischemia

Abstract

Neonatal hypoxia ischemia (HI) related brain injury is one of the major causes of life-long neurological morbidities that result in learning and memory impairments. Evidence suggests that male neonates are more susceptible to the detrimental effects of HI, yet the mechanisms mediating these sex-specific responses to neural injury in neonates remain poorly understood. We previously tested the effects of treatment with a small molecule agonist of the tyrosine kinase B receptor (TrkB), 7,8-dihydroxyflavone (DHF) following neonatal HI and determined that females, but not males exhibit increased phosphorylation of TrkB and reduced apoptosis in their hippocampi. Moreover, these female-specific effects of the TrkB agonist were found to be dependent upon the expression of $$\text{ER}\alpha$$ . These findings demonstrated that TrkB activation in the presence of $$\text{ER}\alpha$$ comprises one pathway by which neuroprotection may be conferred in a female-specific manner. The goal of this study was to determine the role of $$\text{ER}\alpha$$ -dependent TrkB-mediated neuroprotection in memory and anxiety in young adult mice exposed to HI during the neonatal period. In this study, we used a unilateral hypoxic ischemic (HI) mouse model. $$\text{ER}\alpha$$ +/+ or $$\text{ER}\alpha$$ −/− mice were subjected to HI on postnatal day (P) 9 and mice were treated with either vehicle control or the TrkB agonist, DHF, for 7 days following HI. When mice reached young adulthood, we used the novel object recognition, novel object location and open field tests to assess long-term memory and anxiety-like behavior. The brains were then assessed for tissue damage using immunohistochemistry. Neonatal DHF treatment prevented HI-induced decrements in recognition and location memory in adulthood in females, but not in males. This protective effect was absent in female mice lacking $$\text{ER}\alpha$$ . The female-specific improved recognition and location memory outcomes in adulthood conferred by DHF therapy after neonatal HI tended to be or were $$\text{ER}\alpha$$ -dependent, respectively. Interestingly, DHF triggered anxiety-like behavior in both sexes only in the mice that lacked $$\text{ER}\alpha$$ . When we assessed the severity of injury, we found that DHF therapy did not decrease the percent tissue loss in proportion to functional recovery. We additionally observed that the presence of $$\text{ER}\alpha$$ significantly reduced overall HI-associated mortality in both sexes. These observations provide evidence for a therapeutic role for DHF in which TrkB-mediated sustained recovery of recognition and location memories in females are $$\text{ER}\alpha$$ -associated and dependent, respectively. However, the beneficial effects of DHF therapy did not include reduction of gross tissue loss but may be derived from the enhanced functioning of residual tissues in a cell-specific manner. Periods of low oxygen delivery and blood flow to the brains of newborns are known to cause life-long impairments to their cognitive ability as adults. Interestingly, male newborns are more susceptible to this injury than females. The mechanisms causing this sex difference are poorly understood. Here we test the role of the nerve growth factor receptor tyrosine kinase B (TrkB) in providing long-term neuroprotection following neonatal hypoxia–ischemia (HI) in mice. We have previously shown that when mice are treated with the TrkB agonist 7,8-dihydroxyflavone (DHF) in the days following neonatal HI, the result is short-term neuroprotection only in females and this protection is dependent on the presence of the estrogen receptor alpha receptor ( $$\text{ER}\alpha$$ ). In this study, we extend these observations by subjecting mice either with or without $$\text{ER}\alpha$$ to HI. Some of the mice were then treated with DHF immediately after HI. As adults, we performed tests to assess the mice’s memory and anxiety-like behavior. At the end of these tests, we assessed the brains for tissue loss. Our results show that as adults the DHF treatment following HI in neonatal mice preserved memory only in females and this effect was dependent on the presence of $$\text{ER}\alpha$$ . In addition, DHF therapy triggered anxiety-like behavior in mice lacking $$\text{ER}\alpha$$ . We also show that this neuroprotection is not dependent on preservation of brain tissue following the injury. These results provide insight into the mechanisms behind the female resistance to hypoxic ischemic episodes as newborns.