Stress Uniformly Increases the Ratio of Nkcc1:Kcc2 Expression in the Brain

Abstract

Introduction: We demonstrated traumatic brain injury (TBI)-dependent increases in cardiovascular reactivity are associated with increased transcription ratio of the genes controlling intraneuronal chloride concentrations, the sodium chloride cotransporter-1 (Nkcc1) and the potassium chloride cotransporter-2 (Kcc2). We also found that in an effort to maintain constant intraneuronal chloride concentrations, greater Nkcc1:Kcc2 ratios are inversely correlated with the expression of the neuronal chloride/bicarbonate exchanger (Ae3). Hypothesis: Enhancement of Nkcc1:Kcc2 represents a generalized response to hazards confronting the milieu interior, and other challenges such as post-traumatic stress (PTS) would similarly increase Nkcc1:Kcc2 and correspondingly diminish Ae3 expression. Conversely, reductions in the expression of neuronal Ae3 could raise the Nkcc1:Kcc2 ratio and alter cardiovascular reactivity after TBI. Methods: PTS was induced in C57Bl/6 mice with intermittent, unpredictable foot shocks and noise exposure, and mRNA from brain tissue was extracted and quantified for Nkcc1, Kcc2, and Ae3 with RT-PCR. In a separate cohort of FVB/N mice, blood pressure (SBP) and heart rates (HR) were assessed noninvasively in awake, control mice and those with targeted disruption of Ae3 after TBI was induced with mild cortical impact; transcription levels of Nkcc1 and Kcc2 were measured similarly. Results: Compared to control mice, PTS increased the Nkcc1:Kcc2 ratio from (values in percent ± SEM), 1.70 ± 0.14 vs. 4.79 ± 0.53, p < 0.0001, n ≥ 10, and decreased the expression of Ae3 (values expressed as 2^[-Ct] ± SEM) from 5.78 x 10−8 2.21 x 10−8, p < 0.0001. However, the Nkcc1:Kcc2 ratio correlated significantly with Ae3 expression only in the mice without PTS, r = -0.41, p = 0.026. In our cohort of mice with targeted disruption of Ae3, Nkcc1:Kcc2 ratios were unchanged between our inbred FVB/N control mice and knockout animals on an FVB/N background (0.51 ± 0.02 vs. 0.63 ± 0.02, p = n.s.) before surgery, but these values were much lower than the ratios from commercially obtained FVB/N inbred mice (1.03 ± 0.07, p < 0.0001). After TBI, mice with targeted disruption of the Ae3 did not have different HR or blood pressure when compared to our inbred FVB/N mice. TBI increased HR only in the commercially obtained FVB/N mice compared to the knockout mice, but this is likely due to lower resting HR of the commercially obtained FVB/N mice. Conclusions: Similarly to what is found after the stress of TBI, mice with PTS had an increased Nkcc1:Kcc2 ratio, but the relationship between the Nkcc1:Kcc2 ratio and the chloride/bicarbonate exchanger is lost. When compared to commercially inbred FVB/N mice, loss of Ae3 increases HR after TBI, however, whether this relationship will be confirmed with truly inbred FVB/N mice remains to be determined. Henry M. Jackson Foundation for the Advancement of Military Medicine. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.