Background
Hypoxic-ischemic encephalopathy (HIE) is a common cause of long-term neurological sequelae and hemiplegic cerebral palsy in children [
1]. Male sex is a risk factor for neonatal HIE [
2]. Clinical evidence demonstrates that among infants at risk for HIE, females are at a quite significant advantage over males. Males are two times more likely to experience prenatal anoxia, hemorrhage, and infection and 1.8 times more likely to suffer cerebral birth trauma suggesting sex differences in the incidence of HIE [
3-
5]. Overall, childhood ischemic injury appears to be more common in boys regardless of age, stroke subtype, or history of trauma [
1,
6]. Notably, boys also experience worse recovery once an injury occurs. Male infants suffer more long-term cognitive deficits compared to their female counterparts with comparable hypoxic-ischemic injury [
7]. The mechanisms underlying this sexual dimorphism in HIE remain elusive. It is well established that estrogen plays a neuroprotective role in adult ischemic stroke [
8]. However, hormones may not play a significant role in the HIE sexual dimorphism as hormone levels are equivalently low between male and female neonates [
9].
Cerebral ischemia induces both a central and a peripheral inflammatory response which contributes to secondary neuronal damage [
10]. The immune response is primarily initiated by activation of microglia, the major resident immune cells in the brain [
11,
12]. Once activated, microglia develop macrophage-like capabilities including phagocytosis, cytokine production, antigen presentation, and the release of matrix metalloproteinases (MMPs) that disrupt the blood brain barrier (BBB) [
13]. As a result, peripheral leukocytes infiltrate into the brain and the normally immune-privileged brain environment is exposed to systemic responses that further exacerbate inflammation and brain damage. Recent studies report a sexual dimorphism in microglia numbers and expression of activation markers in neonatal brains under normal conditions [
14-
16]. How these basal sex differences in microglia affect stroke phenotypes and inflammation triggered by HIE remains largely unexplored. In this study, we utilized the Rice-Vanucci model (RVM) in male and female wild-type littermate mice to induce HIE at post-natal day 10 (P10) to investigate the sex difference in immune responses and HIE outcomes.
Discussion
Sexual dimorphism in HIE has long been recognized clinically; nevertheless, the underlying mechanism remains elusive. The present study employed a widely utilized HIE model (RVM) to study these sex differences and revealed several important new findings. Firstly, hypoxic-ischemic insults lead to an equivalent primary injury in male and female neonatal brains. However, this ischemic damage evolves differently in each sex with males showing significantly worse histological damage at later time points. Circulating hormone levels are not responsible for this sex difference, as the serum levels of testosterone and estradiol were equivalent in males and females. Secondly, HIE induces microglial activation in a time-dependent manner, as microglia were significantly activated at 3 days but not at 1 day after HIE. Thirdly, consistent with HIE outcomes, a delayed sex difference in both central and peripheral immune responses exists evidenced by activation of microglia, infiltration of peripheral leukocytes, and expression of inflammatory cytokines (Figures
2,
3, and
4). Finally, the sex difference in neonatal HIE outcomes was not attenuated by development as these difference extended to the chronic stages of HIE.
Cerebral ischemia is a sexually dimorphic disease throughout the life span [
34]; however, the underlying mechanisms responsible for these differences may differ with age. Sexual dimorphism in adult stroke has been largely attributed to the protective effects of estrogen [
35,
36]. In children, estradiol levels are equivalently low in males and females until adolescence [
37]. Male neonatal mammals (rat, mouse, horse, human, etc.) undergo a testosterone surge within 24 h of birth, and then the levels decrease and approaches that of females during the remainder of the neonatal period [
38]. Consistent with previous studies, our data showed that serum testosterone and estradiol levels are equivalent between males and females, suggesting that circulating gonadal hormones do not mediate the sexually dimorphic phenotype of HI-induced neonatal brain damage. The testosterone surge in newborn males figures prominently in the development of mechanisms controlling gonadotropin, sexual behavior, and also promotes the functional differentiation of the accessory sex glands [
38]. Whether the sex difference in HIE is related to the early organizational effects of hormones remains unknown. However, in our study, the HI insult did not induce a sex difference in outcomes until 3 days after HIE (Figure
1), suggesting a differential down-stream mechanistic signaling in males vs. females may exist secondary that is triggered by the primary HI damage. Of note, the principal form of hypoxia-ischemic brain injury in the immature brain involves cerebral white matter [
39]. The hypoxic-ischemic lesion induced by RVM in the present study includes both the white and gray matter which cannot be distinguished with TTC staining. Therefore, the sex differences revealed in the study reflect morphological and functional changes after HIE in the whole brain instead of the white matter only.
Brain ischemia is a powerful stimulus that triggers a series of events that lead to the rapid activation of resident microglia as well as mobilization and infiltration of circulating leukocytes [
30,
40] to elicit a secondary neuronal damage. This process is modulated by several cell adhesion molecules and cytokines which, when induced, act upon the vascular endothelium to increase the expression of ICAM-1, P-selectin, and E-selectin, leading to further local accumulation and adhesion of leukocytes [
41-
43]. After gaining entry into the central nervous system (CNS) through the BBB, infiltrating leukocytes release cytokines and chemokines, amplifying the intrinsic (microglial) brain inflammatory response further over the next few days. Microglial activation and aggregation is a pathological marker for HIE in human infants [
44]. Retrospective clinical studies on postmortem examinations from neonatal brains found patients who died of HIE had a dense infiltrate of microglia in the hippocampal dentate gyrus, whereas those dying of other acute causes (trauma or sepsis) had significantly fewer microglia [
44]. The present study revealed that the level of microglial MHC II, a widely used marker of microglial activation, was not significantly increased until 3 days after HIE, suggesting the pro-inflammatory response is delayed in neonatal ischemic brains and therefore may be an attractive target for therapeutic intervention. However, microglia are not equally activated in males vs. females, as males had significantly more MHC II
+ cells than females at 3 days of HIE. Interestingly, previous studies reported that neonatal microglia exhibit sex-specific profiles even under normal conditions. For example, neonatal male mice had twice as many microglia as female mice in the preoptic area; microglial inhibition during the critical period for sexual differentiation prevented sex differences in microglia and adult copulatory behavior [
14]. In our model, we did not observe a sex difference in microglial activation at the baseline in the whole hemisphere (Figure
3D, E); however, it is likely that male microglia are primed towards activation and are more activated once an ischemic event occurs. MHC II is increasingly used as a marker of M1 microglial activation (classical activation) that is pro-inflammatory [
30]. It was found that microglia are both M1 and M2 (alternative activation; anti-inflammatory) during the acute stage of neuroinflammation induced by adult stroke [
45-
47]; however, the M2 phenotype is transient and soon transitions to a skewed M1 that persists up to 2 weeks after stroke [
46]. Therefore, the significant difference in M1 phenotype of microglia in the present study may be critically related to the sexual dimorphism seen in HIE outcomes.
In addition to the sex difference in the activation of central immune cells (microglia), peripheral immune responses also exhibited a sex-specific profile, shown by the serum levels of IL-1β and TNF-α (Figure
2), and infiltration of peripheral leukocytes (Figure
4). IL-1β, TNF-α, and IL-6 are known to be the major inflammatory mediators with increased levels in HIE [
48-
50]. These cytokines are released by peripheral monocytes and macrophages, as well as by astrocytes and microglia [
51,
52]. In the present study, both the central and peripheral immune responses showed the same pattern as seen by histological outcomes after HIE, i.e., sex-specific changes at a later time point (3 days), suggesting HIE induces an equivalent primary brain injury initially in males and females that evolves differently later due to the secondary neuronal damage exerted by the post-ischemic inflammation. Overwhelming data have demonstrated that inflammatory responses cause secondary neuronal damage following acute ischemic injury [
40,
53,
54]. Differences between males and females can be identified at many levels of the immune response (reviewed in [
55]). Research on sex differences in immunology has centered on two main influences: endocrinology (the effect of sex hormones) and genetics (the effect of the X chromosome). In adults, estrogen has been proven to suppress inflammatory responses after stroke [
56]; however, chromosomal effects may be a more important factor in post-ischemic inflammation in neonates as hormone levels are equivalent in males and females. Nevertheless, the organizational effect of hormones cannot be excluded given the fact that males undergo a testosterone surge both during embryogenesis and early after birth [
38,
57]. This can be addressed by dissociation of the chromosomal effect from hormonal organizational effect with the ‘four core genotypes’ mouse model [
58], which is an on-going project in this lab. The sexual dimorphism in acute HIE outcomes extends to chronic stages, suggesting that the post-ischemic secondary neuronal damage exert a profound impact on the development of brain function. This underlines the importance of acute inflammatory responses in the pathophysiology of HIE. Treatments aimed at the early innate immune response may improve the long-term functional disability caused by this devastating disease.
In summary, neonatal HIE leads to an equivalent level of primary hypoxic-ischemic damage in the male vs. female brains at day 1 after injury. A sex difference subsequently develops with the progression of the disease. Circulating hormones are not contributing factors to this sexual dimorphism, whereas a striking sex-specific innate immune response is closely correlated to HIE outcomes. Although the causative evidence is lacking and warrants further exploration, this study represents the initial step in targeting inflammatory responses as an effective therapeutic avenue for HIE and suggests that sex-specific strategies should be developed.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
MAM participated in the design of the study, performed the experiments and the statistical analysis, and drafted the manuscript. RR participated in the experiment of flow cytometry. YX performed ELISA, CV staining, and sample collection. LDM participated in the design of the study and helped to draft the manuscript. FL conceived the study, participated in its design, performed experiments, and helped to draft the manuscript. All authors read and approved the final manuscript.