HIF-2α and CRLF3 expression changes following mild repeated hypoxia

Ischemic stroke remains a leading cause of long-term disability and death worldwide. Current treatments restore cerebral blood flow but do not prevent reperfusion injury. Repeated exposure to mild hypoxia before stroke onset has shown in rats to reduce infarct volume and neuronal damage, suggesting activation of endogenous neuroprotective pathways. One such pathway is mediated by hypoxia-inducible factor 2α (HIF-2α), known to promote erythropoietin (EPO) and its receptor (EPOR), contributing to neuronal survival. Recent studies have highlighted cytokine receptor-like factor 3 (CRLF3) as an alternative EPOR mediating neuroprotection. This study aims to describe CRLF3 expression in the rat brain and whether repeated mild hypoxia upregulates HIF-2α and CRLF3 and compare these molecular responses in male versus female rats to guide sex-specific therapeutic development. Male and female Sprague-Dawley rats (8-10 weeks old) were randomly assigned to receive a daily 2-hour exposure of either normoxia (CTL, 21% O 2 /79% N 2 ; ~760 mmHg) or hypoxia (HX, 10% O 2 /90% N 2 ; ~760 mmHg) for either 3 (n = 6 males and 2 females/group) or 5 (n = 4/group/sex) consecutive days using a whole cage hypoxia chamber (BioSperix). Naïve rats were not exposed to the chambers (n = 2/group/sex). In a subset of males, we recorded breathing responses using plethysmography chambers (3 day-exposure; n = 5/group). Western blot was used to measure protein expression changes for HIF-2α and CRLF3 in forebrain sections. Data were analyzed using GraphPad Prism 9. First, we analyzed the physiological response of male rats to repeated mild hypoxia episodes. Rats increased minute ventilation from baseline during the 2-hour period of hypoxia, but responses did not differ between the 3 days (Two-way ANOVA; minute ventilation: ptime > 0.0001, pday = 0.782). This study is the first to analyze CRLF3 expression in rat brain tissue. Western blots of naïve tissue showed a single protein band at 55kDa, resembling the predicted molecular weight of 58kDa and expression was similar between both different brain regions and between male and female rats in each region. After 3 days of episodic hypoxia, neither HIF-2α nor CRLF3 expression differed between HX and CTL in either sex. After 5 days of episodic hypoxia HIF-2α and CRLF3 expression increased mildly in males, but not females, resulting in a significant difference between male-HX and female-HX groups for both proteins (One-way ANOVA; pHIF2a = 0.002; pCRLF3 = 0.0212). Physiological responses to hypoxia did not differ across the 3 days of episodic hypoxia, indicating that the mild hypoxia regimen did not induce a pathological stress response. CRLF3 is broadly expressed across rat brain in both sexes, suggesting its role in general cell function. The 3 days of episodic hypoxia was insufficient to alter their expression. However, 5 days mildly elevated HIF 2α and CRLF3 in males only. These findings suggest that HIF-2α-dependent mechanisms may contribute to CRLF3 regulation and that males exhibit greater molecular sensitivity to mild hypoxia than females. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.