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. 2020 Oct 15:11:540144.
doi: 10.3389/fneur.2020.540144. eCollection 2020.

Sex-Specific Differences in Rodents Following a Single Primary Blast Exposure: Focus on the Monoamine and Galanin Systems

Lizan Kawa  1 , Ulf P Arborelius  1 , Tomas Hökfelt  1 , Mårten Risling  1
Affiliations

Sex-Specific Differences in Rodents Following a Single Primary Blast Exposure: Focus on the Monoamine and Galanin Systems

Lizan Kawa et al. Front Neurol. .

Abstract

Most blast-induced traumatic brain injuries (bTBI) are mild in severity and culpable for the lingering and persistent neuropsychological complaints in affected individuals. There is evidence that the prevalence of symptoms post-exposure may be sex-specific. Our laboratory has focused on changes in the monoamine and the neuropeptide, galanin, systems in male rodents following primary bTBI. In this study, we aimed to replicate these findings in female rodents. Brainstem sections from the locus coeruleus (LC) and dorsal raphe nuclei (DRN) were processed for in situ hybridisation at 1 and 7 days post-bTBI. We investigated changes in the transcripts for tyrosine hydroxylase (TH), tryptophan hydroxylase two (TPH2) and galanin. Like in males, we found a transient increase in TH transcript levels bilaterally in the female LC. Changes in TPH2 mRNA were more pronounced and extensive in the DRN of females compared to males. Galanin mRNA was increased bilaterally in the LC and DRN, although this increase was not apparent until day 7 in the LC. Serum analysis revealed an increase in corticosterone, but only in exposed females. These changes occurred without any visible signs of white matter injury, cell death, or blood-brain barrier breakdown. Taken together, in the apparent absence of visible structural damage to the brain, the monoamine and galanin systems, two key players in emotional regulation, are activated deferentially in males and females following primary blast exposure. These similarities and differences should be considered when developing and evaluating diagnostic and therapeutic interventions for bTBI.

Keywords: anxiety; dorsal raphe (DR); locus caeruleus; neuropeptide; noradrenaline; post concussive disorder; post traumatic stress disorder (PSTD); serotonin.

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Figures

Figure 1
Figure 1
Basic parameters. Average weight of female rats, the week before and the week following blast exposure (A). Serum analysis of CORT levels in female vs. males at 1 day following blast exposure (B). The levels of the hormones estradiol (C) and progesterone (D) in females at 1 and 7 days as determined in serum using ELISA. No differences in body weight are encountered between the exposed and sham group. CORT levels are increased, but only in females. The female hormones vary among animals. CORT, corticosterone; ELISA, enzyme-linked immunosorbent assay. Data are presented as mean ± SEM. (**p < 0.01). Female n numbers included: at 1 day = 6 + 5, and at 7 days = 6 + 6 exposed and sham, respectively. For males sacrificed at 1 day post-exposure (n = 5, 5 exposed and sham). One of the shams from both the male and female group were excluded from the CORT analysis as they were outliers.
Figure 2
Figure 2
ISH analysis of transcript levels of TH and galanin in the LC following exposure to a single mbTBI in female rats. Representative dark-field ISH photomicrographs of emulsion-dipped sections showing the distribution and levels of TH mRNA in exposed (a) and sham (b) animals; galanin mRNA in the exposed (d) and sham (e) LC at 1 day post-exposure. Quantification of transcript levels indicates that TH mRNA levels are significantly increased bilaterally in the LC at 1 day and return to sham levels by 7 days post-exposure (c). Galanin transcript only reaches statistically significant levels by day 7 post-exposure (f). There were no differences in the left vs. right LC, so these groups were collapsed. The same is true for 1-day and 7-day shams, so these groups were also collapsed. All transcript levels have been normalised to their respective shams. ISH, in situ hybridisation; LC, locus coeruleus; TH, tyrosine hydroxylase. Data are presented as mean ± SEM. (*p < 0.05, ****p < 0.0001). Female n numbers included: at 1 day = 6 + 5, and at 7 days = 6 + 6 exposed and sham, respectively.
Figure 3
Figure 3
ISH analysis of transcript levels of TPH2 and galanin in the DRN following exposure to a single mbTBI in female rats. Representative dark-field ISH photomicrographs of emulsion-dipped sections showing the distribution and levels of TPH2 in the mid/caudal (a,b), and rostral (c,d) DRN, and galanin in the mid/caudal (f,g) at 1 day post-exposure. Quantification of TPH2 mRNA in the DRN (e) indicate that TPH2 is significantly increased in both the mid/caudal and rostral DRN at 1 day post-exposure, and remains elevated even at 7 days post-exposure, relative to sham levels. Quantification of galanin mRNA in the DRN was only explored in the mid/caudal region (h) and is significantly elevated already at 1 day post-exposure, and remains elevated at 7 days post-exposure, relative to sham levels. The sham groups of the two different time points were not statistically significantly different, so these groups were also collapsed. All transcript levels have been normalised to their respective shams. Data are presented as mean ± SEM. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). ISH, in situ hybridisation; DRN, dorsal raphe nucleus; TPH2, tryptophan hydroxylase 2. Female n numbers included: at 1 day = 6 + 5, and at 7 days = 6 + 6 exposed and sham, respectively.
Figure 4
Figure 4
Comparison between female and male rats. Changes in transcripts for TH, TPH2, and galanin in females (present study) and for already published findings TH, TPH2, and galanin in males (47, 48) in the LC (A,D) and DRN (B,C,E) are shown. The increase in TH mRNA levels is more pronounced in males as compared to females, and is only seen at 1 day. The galanin transcript levels increase faster in males than females, but are still elevated at 7 days in both sexes. In the rostral DRN, TPH2 mRNA levels are increased at 1 and 7 days in females, sharply contrasting males. Galanin mRNA shows gradual and parallel increases in the Mid/Caudal DRN. All transcript levels have been normalised to their respective shams. LC, locus coeruleus; TH, tyrosine hydroxylase; DRN, dorsal raphe nucleus; TPH2, tryptophan hydroxylase 2.
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