CGRP as a potential mediator for the sexually dimorphic responses to traumatic brain injury

Abstract

Background: The outcomes of traumatic brain injury (TBI) exhibit variance contingent upon biological sex. Although female sex hormones exert neuroprotective effects, the administration of estrogen and progesterone has not yielded conclusive results. Hence, it is conceivable that additional mediators, distinct from female sex hormones, merit consideration due to their potential differential impact on TBI outcomes. Calcitonin gene-related peptide (CGRP) exhibits sexually dimorphic expression and demonstrates neuroprotective effects in acute brain injuries. In this study, we aimed to examine sex-based variations in TBI structural and functional outcomes with respect to CGRP expression.

Methods: Male and female Sprague Dawley rats were exposed to controlled cortical impact to induce severe TBI, followed by interventions with and without CGRP inhibition. In the acute phase of TBI, the study centered on elucidating the influence of CGRP on oxidative stress, nuclear factor erythroid 2-related factor 2 (Nrf2) and endothelial nitric oxide synthase (eNOS) signaling in the peri-impact tissue. Subsequently, during the chronic phase of TBI, the investigation expanded to evaluate CGRP expression in relation to lesion volume, microvascular dysfunction, and white matter injury, as well as working and spatial memory, anxiety-like, and depression-like behaviors in subjects of both sexes.

Results: Female rats exhibited elevated levels of CGRP in the peri-impact brain tissue during both baseline conditions and in the acute and chronic phases of TBI, in comparison to age-matched male counterparts. Enhanced CGRP levels in specific brain sub-regions among female rats correlated with superior structural and functional outcomes following TBI compared to their male counterparts. CGRP inhibition induced heightened oxidative stress and a reduction in the expression of Nrf2 and eNOS in both male and female rats, with the observed alteration being more pronounced in females than in males.

Conclusions: This study marks the inaugural identification of CGRP as a downstream mediator contributing to the sexually dimorphic response observed in TBI outcomes.

Keywords: Anxiety; Calcitonin gene-related peptide; Depression; Memory; Microvascular dysfunction; Oxidative stress; Sex hormones; Sexual dimorphism; Traumatic brain injury.

Fig. 1

A Timeline of the experimental procedures. Controlled cortical impact (CCI) was induced on D0, at the same time as CGRP_8-37 administration, for animals in CGRP_8-37 group. Animals in the acute group were collected on D1, while animals in the chronic group were collected on D30 (red boxes indicate time of behavior assessment). B Definition of impact core and peri-impact brain tissue for analysis

Fig. 2

Sex differences in lesion volume development correlate with CGRP levels at the peri-impact cortex. A Representative H&E stained images for lesion volume. Due to variations in lesional development between males and females, “0” indicates lesion center, rather than bregma. B Quantified lesion volume at 30 d after CCI. C Representative immunofluorescent stained images for CGRP and NeuN expression at the impact core and peri-impact brain tissue. D Quantified CGRP levels at the impact core tissue. E Quantified CGRP levels at the peri-impact tissue. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 3

Sex differences in oxidative stress depend on CGRP levels. A Quantified CGRP levels after TBI with and without CGRP inhibition. B Quantified nitrotyrosine levels after TBI with and without CGRP inhibition. C Quantified reduced glutathione levels after TBI with and without CGRP inhibition. D Quantified pNrf2 levels after TBI with and without CGRP inhibition. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 4

Sex differences in microvascular dysfunction depend on CGRP levels. A Quantified eNOS levels after TBI with and without CGRP inhibition. B Representative H&E stained images for pial arterioles at 30 d after CCI. Red arrows indicate pial arterioles. C Representative H&E stained images for parenchymal arterioles at 30 d after CCI. Red arrows indicate opened parenchymal arterioles. Black arrow heads indicate constricted parenchymal arterioles. D Quantified wall thickness of pial arterioles. E Quantified vessel diameter of pial arterioles. F Quantified wall thickness to vessel diameter ratio of pial arterioles. G Quantified constricted vessels of parenchymal arterioles. C Representative immunofluorescent stained images for CGRP surrounding parenchymal arterioles. I Quantified CGRP levels around the parenchymal vessels after 30 d TBI. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 5

Sex differences in white matter and hippocampal injury depend on CGRP levels. A Representative immunofluorescent stained images for CGRP and MBP at the corpus callosum. B Quantified MBP levels at 30 d after TBI. C Quantified CGRP levels in the corpus callosum at 30 d after TBI. D Representative H&E stained and immunofluorescent stained images for CGRP and NeuN expression at dental gyrus of hippocampus. Blue arrow heads indicate injured cells. E Quantified unhealthy cell counts at dental gyrus. F Quantified CGRP levels at dental gyrus. G Quantified spontaneous alternation for working spatial memory assessment. H Quantified novel arm entrance for long-term spatial memory assessment. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 6

Sex differences in anxiety- and depression-like behavior depend on CGRP levels. A Representative H&E stained and immunofluorescent stained images for CGRP and NeuN at amygdala and thalamus. B Quantified unhealthy cell counts at amygdala at 30 d after TBI. C Quantified CGRP levels at amygdala at 30 d after TBI. D Quantified unhealthy cell counts at thalamus at 30 d after TBI. E Quantified CGRP levels at thalamus at 30 d after TBI. F Quantified open arm entry for anxiety-like behavior assessment. G Quantified immobile percent for depression-like behavior assessment. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 7

Conceptual diagram illustrating CGRP as a potential mediator for the sexually dimorphic responses to traumatic brain injury