Increased brain water in the clock gene Bmal1 knockout rat

Abstract

The modern lifestyle can lead to many circadian rhythm and sleep disturbances through factors such as artificial lighting and shiftwork. Central fluid flow follows a strong circadian rhythm with increased CSF flow and interstitial clearance during sleep. Disruption of this rhythm in central fluid flow has been implicated in numerous neurodegenerative diseases. However, how circadian central fluid homeostasis is regulated is incompletely understood. The essential clock gene, Bmal1, regulates many circadian processes. Whole body knockout of Bmal1 in mice increases blood brain barrier (BBB) permeability and eliminates many 24-hour rhythms. The recently developed Bmal1 knockout (Bmal1KO) rat has preserved activity and blood pressure rhythms, but central fluid regulation has not been explored. We hypothesized that Bmal1KO rats have altered central fluid control and blunted circadian rhythms of genes involved in central fluid homeostasis. We harvested brains from male and female Bmal1KO and wildtype (WT) littermate control rats and subtracted the dry weight from the wet weight to determine brain water content. Bmal1KO had elevated brain water compared to WT (1.54 ± 0.03 vs 1.44 ± 0.03 g, n = 8-11; p = 0.04). We then evaluated BBB permeability by injecting FITC-dextran (3-5 kDa) intravenously followed by transcardiac perfusion of saline after 30 minutes. Brain sections were dissected and homogenized, and the amount of FITC-dextran was measured by spectrometry. Bmal1KO rats had more FITC-dextran in the brain stem (261.3 ± 18.8 vs 196.4 ± 10.6 ng/mg tissue, n = 4-5; p = 0.03) and in the spinal cord (331.9 ± 17.1 vs 239.2 ± 31.7 ng/mg, p = 0.03) indicating increased BBB permeability. We measured gene expression via RT-qPCR from prefrontal cortex samples collected every four hours throughout the day. We then fit a Cosinor curve to the results in order to evaluate the circadian rhythmicity, and differences were established by extra sum-of-squares F test. We measured aquaporin 4 (aqp4) expression because it is a key component of BBB permeability and glymphatic flux in the brain. Bmal1KO had a blunted aqp4 amplitude (1.2 ± 0.2 vs 0.8 ± 0.2 Ct; p = 0.004 comparison of fit). Bmal1 is also known to contribute to endothelin-1 (edn1) expression rhythms, and we found a dramatic blunting of edn1 amplitude in Bmal1KO rats (0.41 ± 0.19 vs 1.31 ± 0.39 Ct; p < 0.001 comparison of fit) and a phase shift in peak edn1 expression (10.71 ± 0.48 vs 19.72 ± 0.30 zeitgeber time). These data indicate that loss of the circadian gene Bmal1 in rats causes increased BBB permeability and disrupted circadian rhythms of genes involved in central fluid homeostasis. These results suggest that circadian genes are integral in maintaining proper central fluid dynamics and that alterations in these rhythms may play a role in neurodegenerative diseases linked with circadian disruption. NIH K01HL159047 This is the full abstract presented at the American Physiology Summit 2023 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.