Linking Alterations in Circulating Sex-steroid Hormones to Sex-dependent Blood-brain Barrier Dysfunction after Traumatic Brain Injury

Introduction::
Traumatic Brain Injury (TBI) is initiated by a blow or jolt to the head and leads to a complex cascade of events including a primary and secondary injury phase. The primary injury phase occurs at acute timepoints after the initial insult and encompasses the direct axonal shearing, neuronal cell death, and hemorrhage caused by the insult. The secondary injury phase includes the dysfunction of the blood-brain barrier (BBB) and neuroinflammation, which can last years after the initial injury. Despite efforts by physicians to manage the symptoms of TBI, clinically approve therapies that focus on neural regeneration to improve functional outcomes are nonexistent. Many researchers, including our lab, are developing nanoparticle-based therapeutic delivery systems to transport drugs across the BBB to address this problem. Interestingly, our pre-clinical studies have shown that there is a significant difference in the nanoparticle deposition profile in the female brain compared to the male brain [1]. This finding suggests that biological sex influences BBB dysfunction seen after TBI, and therefore impacts the subsequent nanoparticle deposition in the injured brain. Prior literature reports that sex steroid hormones such as progesterone, estradiol and testosterone impact the permeability of the BBB [2,3,4,5]. Here, our goal is to assess how alterations in the levels of circulating sex steroid hormones in male and female mice impact BBB dysfunction after TBI.




Materials and Methods::
We used a well-established mouse controlled cortical impact (CCI) model to induce a moderate TBI over the primary somatosensory cortex in C57BL/6J mice. Male (n=10) and female (n=12) cohorts were injured at 9-10 weeks of age. Naïve cohorts served as baseline controls and injured cohorts were sacrificed at 24hrs and 72hrs post-CCI. Females began estrus cycle tracking 8 days prior to injury and continued until sacrifice. Vaginal smears were used to record phase on day of CCI and sacrifice. At the specified endpoint, animals were sacrificed to collect plasma and tissue samples. Plasma samples were analyzed with an LC-MS/MS hormone panel for testosterone, androstenedione, estradiol, estrone, progesterone, DHEA and corticosterone at the University of Wisconsin National Primate Research Center. Brain tissue was collected for immunohistochemistry (IHC) or RNA-sequencing analysis. For IHC, the animals were transcardially perfused with PBS, followed by 4% paraformaldehyde PBS solution. Brain tissue was collected and immersed in 4% paraformaldehyde overnight, followed by immersion in 15% and 30% sucrose PBS solutions. The tissue was cryopreserved in optimal cutting temperature (OCT) medium and stored at -80°C until. For the RNA-sequencing preparation, the animals were transcardially perfused with diethyl pyrocarbonate (DEPC) treated PBS. The brain was isolated, and a 4 mm cortical punch was taken directly over the injury penumbra and contralateral hemisphere. Cortical punches were placed in the Qiagen RNAprotect reagent for storage at -20°C. Qiagen’s RNeasy Lipid Tissue Mini Kit was used to extract the RNA from the samples, which were sent to Novogene Co for sequencing.




Results, Conclusions, and Discussions:: Results:
At both injury timepoints, both males and females exhibited significant changes in circulating sex steroid hormone levels compared to the naïve controls (fig. 1). For females there was significant reduction in androstenedione(p< 0.0001), progesterone(p=0.0468), and testosterone(p=0.0090) at 24-hours post-injury. Additionally for females, at 72-hours post-injury there was significant reduction in estrone(p=0.0017), androstenedione(p=0.0245), and testosterone(p=0.0183). Conversely for the males, no significant changes were observed 24-hours post-injury. However, at 72-hours post-injury there was a significant reduction in estradiol (p=0.0045) and estrone(p=0.0002) at 72-hours post injury. Conversely, there was an increase in DHEA for males at 72-hours post-injury(p=0.0175).
Discussion:
Here, we present novel findings regarding the changes in circulating sex-steroid hormones after TBI at 24- and 72-hours post-injury. Notably, the observed alterations in hormone levels were sex-specific, with males and females displaying distinct hormone profiles. Each of these hormones is involved in the steroidogenesis pathway and play a role in the biosynthesis of another hormone in the pathway [6]. For example, androstenedione is the precursor hormone for testosterone, estrone, and estradiol. These findings support the hypothesis that changes in sex-steroid hormone levels may underlie the disparate nanoparticle extravasation patterns observed in prior pre-clinical studies. Additionally, preliminary differential gene expression analysis suggest sex-specific gene dysregulation following TBI – additional analysis is currently underway to identify potential correlations between circulating sex hormone profiles and DEGs.


Conclusions:
The present studies demonstrate the existence of sex-specific alterations in circulating sex-steroid hormone levels that may have an impact on the injury-induced permeability of the blood-brain barrier (BBB). To further investigate this phenomenon, we plan to correlate hormone levels with RNA-seq and IHC analysis of tight junctions and adherens junctions within the injury penumbra. Incorporating the 72-hours post-injury RNA-sequencing data will provide a comprehensive view of the gene expression analysis and shed light on the sex-specific gene dysregulation at different time points post-injury. These data will determine potential influence of biological sex and circulating sex hormones on BBB permeability following TBI. Ultimately, we aim to develop personalized therapeutics that promote neural regeneration after TBI, specifically delivery of the right therapy to the right person at the right time.







Acknowledgements (Optional): :

References (Optional): :
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[2] McConeghy, et al. CNS Drugs. 2012, 26(7):613-36




[3] Atallah, et al. J Cereb Blood Flow Metab, 2017, 37: 3161-3175




[4] Sárvári, et al. J of Neuroinflammation, 2011, 8:82




[5] Si, et al. Exp Ther Med, 2014, 8: 1010-1014




[6] Chakraborty, et al. Genes & Immunity, 2021, 22;3: 125-140