Effects of cold stress on the blood-brain barrier in Plectropomus leopardus

Abstract

Background: The leopard coral grouper (Plectropomus leopardus) is a commercially valuable tropical marine fish species known to be sensitive to low temperatures. A comprehensive understanding of the molecular mechanisms governing its response to acute cold stress is of great importance. However, there is a relative scarcity of fundamental research on low-temperature tolerance in the leopard coral grouper.

Methods: In this study, a cooling and rewarming experiment was conducted on 6-month-old leopard coral groupers. Within 24 h, we decreased the ambient temperature from 25 °C to 13 °C and subsequently allowed it to naturally return to 25 °C. During this process, a comprehensive investigation of serum hormone levels, enzyme activity, and brain transcriptome analysis was performed.

Results: P. leopardus displayed a noticeable adaptive response to the initial temperature decrease by temporarily reducing its life activities. Our transcriptome analysis revealed that the differentially expressed genes (DEGs) were primarily concentrated in crucial pathways including the blood-brain barrier (BBB), inflammatory response, and coagulation cascade. In situ hybridization of claudin 15a (cldn15a), a key gene for BBB maintaining, further confirmed that exposure to low temperatures led to the disruption of the blood-brain barrier and stimulated a pronounced inflammatory reaction within the brain. Upon rewarming, there was a recovery of BBB integrity accompanied by the persistence of inflammation within the brain tissue.

Conclusions: Our study reveals the complex interactions between blood-brain barrier function, inflammation, and recovery in P. leopardus during short-term temperature drops and rewarming. These findings provide valuable insights into the physiological responses of this species under cold stress conditions.

Keywords: Blood brain barrier; Brain; Claudin; Low temperature; Transcriptome.

Fig. 1

Experimental design. A The leopard coral groupers (n = 50) were cultured in the 100 L tank: temporary cultured in 25 °C for 48 h before cooling at rate of 0.5/h from 25 °C to 13 °C, and naturally rewarmed to 25 °C. A control group (animals at 25 °C before cooling), two low temperature group (animals at 19 °C and 13 °C) and a rewarming group (animals at 25 °C after rewarming). B Blood and brain tissue samples were collected in triplicate from relevant groups for further tests, including ACTH and Cortisol measurement, SOD activity detection, transcriptome sequencing and in situ hybridization experiments

Fig. 2

Hormone and enzyme detection results in serum of P. leopardus. The abscissa represents the group, 25 °C (animals at 25 °C before cooling), 19 °C and 13 °C (animals at 19 °C and 13 °C), R- 25 °C (animals at 25 °C after rewarming). The vertical axis represents the unit of hormone level and enzyme activity. The values are expressed as mean ± SEM. n = 3. Significant differences are shown when P < 0.05

Fig. 3

Analysis of differentially expressed genes in brain transcriptome of P. leopardus. A principal component analysis plot of 25 °C (animals at 25 °C before cooling), 13 °C (animals at 13 °C) and R- 25 °C (animals at 25 °C after rewarming). B Venn diagram of the distribution of the DEGs of groups. C Statistical table for the number of differentially expressed genes (log2FoldChange > = 2, P < = 0.05) pairwise comparison between groups. D The heatmap of the differentially expressed genes. E Soft clustering plots of DEGs in the time series: Each line in graph represents the expression profile of an individual gene

Fig. 4

Functional enrichment analysis of differentially expressed genes in brain transcriptome of P. leopardus.A-C Enhanced volcano plots with annotated partial genes that enriched in significant pathways. The x-axis represents log2 fold change (FC) in expression levels, the y-axis shows -log10(p-value) to indicate statistical significance. D-F KEGG bubble plot illustrating the top 10 pathway enrichment analysis of DEGs. The x-axis indicates the enrichment score, reflecting the degree of enrichment in each pathway. The y-axis lists the KEGG pathways, and the size of the bubbles corresponds to the number of genes mapped to each pathway. The color gradient indicates the statistical significance (-log10(p-value)) of the enrichment

Fig. 5

Quantitative PCR and FISH results. A Relative expression level of cldn15a, cebpd, chac1, cyp1a1 and tjp1 in 13 °C, 25 °C and R-25 °C groups. B Co-localization of cldn15a and tjp1 antisense probe in brain at 25 °C, 13 °C and R-25 °C groups. a, e and i Nucleus was counterstained with DAPI. b, f and j The fluorescent signal of cldn15a antisense probe. c, g and k The fluorescent signal of tjp1 antisense probe. d, h and l Merged images. Scale bars = 50 μm. Arrows indicate capillaries