Corticotropin‐releasing hormone–mediated blood pressure elevation during acute stress in male rats

Figures

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  FIGURE 1. Schematic overview of experimental process. (A) Establish an acute restraint stress model; (B) Acute stress following CRISPR/Cas9‐medicated CRH knockdown in the PVN. (C) Acute stress following CRHR1 antagonist intervention. i.p., intraperitoneal injection (n = 10) unless otherwise indicated.

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  FIGURE 2. Acute restraint stress increases blood pressure, heart rate, and circulating stress hormones. (A) Continuous recordings of systolic blood pressure (SBP; a), diastolic blood pressure (DBP; b), mean arterial pressure (MAP; c), and heart rate (HR; d) before and during restraint (SBP, DBP, and MAP: N = 10; HR: N = 5). Time courses were analyzed separately by one‐way repeated‐measures ANOVA with the Geisser–Greenhouse correction. Graph labels show two‐sided paired t‐tests comparing each rat's final 30‐min baseline mean with its mean during restraint (5–120 min). “Stress” marks the restraint period. (B) Serum corticosterone (CORT; a) and norepinephrine (NE; b) measured in the same rats at 0, 0.5, 1, 1.5, and 2 h (n = 6; 0 h, pre‐restraint). Each analyte was analyzed using the same ANOVA and Dunnett's comparisons with the matched 0‐h value without assuming sphericity. Graph labels show Dunnett‐adjusted p values. Data are mean ± SD.

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  FIGURE 3. CRH knockdown selectively reduced CRH protein expression in the PVN. (A) Diagram of the rAAV9‐U6‐sasgRNA(rCrf)‐CMV‐NLS‐SaCas9‐3xHA‐WPRE‐BGHpA knockdown vector and control construct. (B) Schematic of bilateral PVN microinjection. (C) CRH protein levels in the LC (a), NTS (b), and PVN (c) at 21 days post‐injection (n = 3). Comparisons between the CON and KD groups were performed using unpaired t‐tests. Exact p values for significant comparisons are shown directly on the graphs; ns indicates no significant difference. Data are expressed as mean ± SD. Full‐length Western blot images with labeled molecular weight marker positions are provided in Supplementary Figure S1.

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  FIGURE 4. CRH knockdown attenuated acute stress‐induced cardiovascular and hormonal responses. (A) Cardiovascular responses to restraint stress in CRH knockdown (KD) and control (CON) rats (n = 6): systolic blood pressure (SBP; a), diastolic blood pressure (DBP; b), mean arterial pressure (MAP; c), and heart rate (HR; d). (B) Serum corticosterone (CORT; a) and norepinephrine (NE; b) responses (n = 3). Baseline and stress‐period measurements were averaged for each rat. All outcomes were analyzed using two‐way repeated‐measures ANOVA with group (CON vs. KD) as the group factor and stress condition (baseline vs. stress) as the repeated factor, followed by Šídák's multiple‐comparisons test. Graph labels show Šídák‐adjusted p values for within‐group baseline–stress comparisons in panel A and CON–KD comparisons during stress in panel B. Data are mean ± SD.

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  FIGURE 5. CRHR1 antagonism attenuated acute stress‐induced HR and hormonal responses. (A) Effects of intraperitoneal antalarmin or vehicle treatment on systolic blood pressure (SBP; a), diastolic blood pressure (DBP; b), mean arterial pressure (MAP; c), and heart rate (HR; d) during restraint stress (n = 5). (B) Serum corticosterone (CORT; a) and norepinephrine (NE; b) before and after restraint. Baseline and stress‐period measurements were averaged for each rat. All outcomes were analyzed using two‐way repeated‐measures ANOVA with treatment (vehicle vs. antalarmin) as the group factor and stress condition (baseline vs. stress) as the repeated factor, followed by Šídák's multiple‐comparisons test. Graph labels show Šídák‐adjusted p values for within‐group baseline–stress comparisons in panel A and vehicle–antalarmin comparisons under stress in panel B. Data are mean ± SD.

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  FIGURE 6. Glucocorticoid receptor blockade did not significantly alter serum NE levels. (A) Changes in blood pressure during mifepristone treatment (n = 5), analyzed using two‐way ANOVA. Exact p values for significant comparisons are shown directly on the graph. (B, C) Serum CORT (B) and NE (C) concentrations following treatment (n = 3 biologically independent animals per group). Comparisons between the control and mifepristone groups were performed using unpaired t‐tests. The exact p value for the significant CORT comparison is shown directly on the graph; ns indicates no significant difference in NE concentration. Data are expressed as mean ± SD. CORT, corticosterone; NE, norepinephrine.

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  FIGURE 7. Proposed mechanism model. Schematic illustrating the role of PVN‐derived CRH in activating ACTH release, CORT secretion, LC‐NE signaling, and blood pressure regulation during acute stress. ACTH, adrenocorticotropic hormone; BP, blood pressure; CORT, corticosterone; CRH, corticotropin‐releasing hormone; NE, norepinephrine; PVN, paraventricular nucleus.