Ketamine Reduces Inflammation Pathways in the Hypothalamus and Hippocampus Following Transient Hypoxia in the Late-Gestation Fetal Sheep

Abstract

The physiological response to hypoxia in the fetus has been extensively studied with regard to redistribution of fetal combined ventricular output and sparing of oxygen delivery to fetal brain and heart. Previously, we have shown that the fetal brain is capable of mounting changes in gene expression that are consistent with tissue inflammation. The present study was designed to use transcriptomics and systems biology modeling to test the hypothesis that ketamine reduces or prevents the upregulation of inflammation-related pathways in hypothalamus and hippocampus after transient hypoxic hypoxia. Chronically catheterized fetal sheep (122 ± 5 days gestation) were subjected to 30 min hypoxia (relative reduction in P_aO₂∼50%) caused by infusion of nitrogen into the inspired gas of the pregnant ewe. RNA was isolated from fetal hypothalamus and hippocampus collected 24 h after hypoxia, and was analyzed for gene expression using the Agilent 15.5 k ovine microarray. Ketamine, injected 10 min prior to hypoxia, reduced the cerebral immune response activation to the hypoxia in both brain regions. Genes both upregulated by hypoxia and downregulated by ketamine after hypoxia were significantly associated with gene ontology terms and KEGG pathways that are, themselves, associated with the tissue response to exposure to bacteria. We conclude that the results are consistent with interruption of the cellular response to bacteria by ketamine.

Keywords: fetal brain; fetal hypoxia; inflammation; ketamine; transcriptomics.

FIGURE 1

As shown in top panels, analysis of gene expression using Agilent ovine 15.5 k array revealed significant up- and down-regulation of gene expression by hypoxia compared to normoxia [HC-NC, previously reported (Zarate et al., 2017)], as well as significant up- and down-regulation of gene expression in hypoxic animals by ketamine (HK-HC). Network inference and statistical modeling of gene ontology terms revealed that hypoxia upregulated pathways related to inflammation, and that ketamine was effective at decreasing the gene expression in those pathways in the hypoxic animals. Transcriptomic modeling indicated a high likelihood of the entire inflammation pathway from TLR2 and TLR4 being involved in the response. HC-NC plots were previously published under a Creative Commons Attribution 4.0 International License in Zarate et al. (2017).

FIGURE 2

Top panels, significant up- and down-regulation of gene expression in hippocampus by hypoxia compared to normoxia [HC-NC, previously reported (Zarate et al., 2017)], as well as significant up- and down-regulation of gene expression in hypoxic animals by ketamine (HK-HC). Network inference and statistical modeling of gene ontology terms were as described in legend to Figure 1. HC-NC plots were previously published under a Creative Commons Attribution 4.0 International License in Zarate et al. (2017).

FIGURE 3

Gene expression (mRNA abundance) in fetal hypothalamus measured by real-time qPCR for genes in the toll-like receptor inflammation pathway. Values are represented as negative delta cycle threshold (–ΔC_t) compared to the NC group. Open bars represent experiments in which ketamine was not administered. Filled bars represent experiments in which ketamine was administered before normoxia or hypoxia. The criterion for statistical significance was P < 0.05 (Student’s t-test). Data are presented as means ± SEM, and the y-axis scale varies between plots. ^∗Statistically significant difference of hypoxia group compared to normoxia control. ∧Statistically significant difference of hypoxia control group compared to hypoxia + ketamine group.

FIGURE 4

Gene expression (mRNA abundance) in fetal hippocampus measured by real-time qPCR for genes in the toll-like receptor inflammation pathway. Values are represented as negative delta cycle threshold (–ΔC_t) compared to the NC group. Open bars represent experiments in which ketamine was not administered. Filled bars represent experiments in which ketamine was administered before normoxia or hypoxia. The criterion for statistical significance was P < 0.05 (Student’s t-test). Data are presented as means ± SEM, and the y-axis scale varies between plots. ^∗Statistically significant difference of hypoxia group compared to normoxia control. ∧Statistically significant difference of hypoxia control group compared to hypoxia + ketamine group.

FIGURE 5

Ketamine reduces the number of microglia and macrophages in the hypothalamus and hippocampus 24 h after transient hypoxia. For each brain region, the Iba-1 positive cells counted per 40X field (average of 7 fields analyzed for each animal) were averaged from 4 animals per group. Data from hippocampus are average measurements from regions CA1, CA2, CA3, CA4, and Dentate Gyrus. Data are expressed as mean ± SEM. Different letters indicate statistically significant difference (P < 0.05). NC, normoxia control; NK, normoxia + ketamine; HC, hypoxia control; HK, hypoxia + ketamine.

FIGURE 6

Ketamine reduces the number of broken or leaky blood vessels in the fetal hypothalamus and cerebral cortex 24 h after transient hypoxia. Number of broken blood vessels were counted in 10X field images and averaged for each animal per brain region: hypothalamus, cerebral cortex, and medulla. Data are expressed as means ± SEM (n = 4/group). Different letters indicate statistically significant difference (P < 0.05). NC, normoxia control; NK, normoxia + ketamine; HC, hypoxia control; HK, hypoxia + ketamine.

FIGURE 7

Effect of ketamine on the expression of 16S bacterial rRNA in hypothalamus and hippocampus 24 h after transient hypoxia. Data are presented as –ΔC_t values relative to the NC group for each brain region (n = 4 animals per group). Data are expressed as mean ± SEM.