Neuroprotectants attenuate hypobaric hypoxia-induced brain injuries in cynomolgus monkeys
摘要: 高原是地球上最恶劣的环境之一，最主要的特点是低气压所导致的缺氧。暴露在低压缺氧环境下，未习服人群会出现脑损伤，在严重情况下甚至会发展成为致命性的高原脑水肿。长期以来，人们应用一些遗传背景和人类差异较大的动物研究高原相关疾病，这可能延缓了防治这些疾病的研发进展。在这项研究中，我们应用非人灵长类动物食蟹猴为研究对象，通过对白细胞和大脑皮层进行转录组测序的方法来探索低压缺氧引起脑损伤的机制，并评价两种具有神经保护功能的甾体——孕酮和5α-androst-3β,5,6β-triol （TRIOL）在这个模型上的药效与可能分子机制。暴露在急性低压缺氧环境下，食蟹猴出现了与人类高原脑水肿相似的症状，包括行为失调、脑含水量升高、神经元受损以及血管源性脑水肿等。白细胞转录组数据分析揭示了经典的缺氧反应通路HIF-1信号，以及一些新发现的关键性通路如维生素D受体信号，这些通路可能共同调控了低压缺氧所导致的炎症反应。急性低压缺氧也改变了大脑的转录调控模式，激活了血管发生的调控通路，并抑制了有氧呼吸和蛋白折叠相关的通路，而这些通路很有可能与脑损伤的病理机制相关。整体上两种甾体的药效相似，都可以显著有效减轻急性低压缺氧所造成的脑损伤和大脑转录谱的改变，但功能分析显示两种药物却作用在不同的通路上，孕酮可以有效的提高红细胞生成，而TRIOL可以减轻谷氨酸兴奋性毒性。总之，本研究加深了我们对急性低压缺氧诱发脑损伤病理机制的理解，并为缺氧性脑损伤的药物研发提供了潜在的候选化合物。Abstract: Hypobaric hypoxia (HH) exposure can cause serious brain injury as well as life-threatening cerebral edema in severe cases. Previous studies on the mechanisms of HH-induced brain injury have been conducted primarily using non-primate animal models that are genetically distant to humans, thus hindering the development of disease treatment. Here, we report that cynomolgus monkeys (Macaca fascicularis) exposed to acute HH developed human-like HH syndrome involving severe brain injury and abnormal behavior. Transcriptome profiling of white blood cells and brain tissue from monkeys exposed to increasing altitude revealed the central role of the HIF-1 and other novel signaling pathways, such as the vitamin D receptor (VDR) signaling pathway, in co-regulating HH-induced inflammation processes. We also observed profound transcriptomic alterations in brains after exposure to acute HH, including the activation of angiogenesis and impairment of aerobic respiration and protein folding processes, which likely underlie the pathological effects of HH-induced brain injury. Administration of progesterone (PROG) and steroid neuroprotectant 5α-androst-3β,5,6β-triol (TRIOL) significantly attenuated brain injuries and rescued the transcriptomic changes induced by acute HH. Functional investigation of the affected genes suggested that these two neuroprotectants protect the brain by targeting different pathways, with PROG enhancing erythropoiesis and TRIOL suppressing glutamate-induced excitotoxicity. Thus, this study advances our understanding of the pathology induced by acute HH and provides potential compounds for the development of neuroprotectant drugs for therapeutic treatment.
- Acute hypobaric hypoxia /
- Cynomolgus monkeys /
- Brain injury /
- Neuroprotectant /
- Gene regulatory networks
Figure 1. Acute HH-induced behavioral and cerebral impairments were attenuated by PROG and TRIOL treatments
A: Experimental procedure for HH treatment of cynomolgus monkeys (HH group, n=6). Vertical bars along y-axis indicate medical definitions of high, very high, and extreme altitudes. Horizontal bars represent duration spent at each altitude. Blood drop and brain icons indicate altitudes and time points at which blood and brain samples, respectively, were collected for RNA-seq analyses. B: Experimental procedure for drug treatment (HH+ PROG and HH+TRIOL groups, each n=6). Needle tubes indicate altitudes and time points at which drugs were injected; blood drop and brain icons indicate altitudes and time points at which blood and brain samples were collected, respectively. Drugs were injected after blood collection at each stage. Samples from stages highlighted in red were selected for RNA-seq analyses. C: Chemical structures of two neuroprotectants (PROG and TRIOL) used in this study. D: Mean skeletal muscle coordination scores for each experimental group, with higher scores representing a lower degree of coordination, assessed after 2 h at 7 500 m. Each group was compared with HH group (*: Kruskal-Wallis test P<0.05). Data are presented with error bars indicating means±SD, n=6 per group. E: Mean brain water content for each experimental group, measured as 1–dry/wet weight of left hemisphere. Each group was compared with HH group (*: one-way ANOVA P<0.05; **: P<0.01, HH n=5, each other group n=6). Error bars represent SD. F: Representative images showing ultrastructure of capillaries in frontal cortex of each experimental group, taken after 48 h at 7 500 m. Blood-brain barrier (BBB) disruption and vasogenic edema evident in HH group but not in other groups, as characterized by severely shrunken capillaries with fluid penetrating into pericapillary space. Red arrowheads indicate capillary boundary. Scale bar: 2 μm. G: Representative images showing Nissl staining of frontal cortex tissues of each experimental group. Neuronal injuries evident in HH group and markedly attenuated in drug-treated groups. Black squares in first row denote areas magnified in second row. Red arrowheads indicate injured cells with dark staining, condensed nuclei, shrunken cell bodies, or weak staining with irregular shapes. Red scale bar: 75 μm, black scale bar: 20 μm. H: Percentage of injured neurons in frontal cortex of each experimental group based on Nissl staining shown in panel G. Each group was compared with HH group (**: one-way ANOVA P≤0.01; ***: P≤0.001, NN n=5, all other groups n=4). Error bars represent SD.
Figure 2. Transcriptomic dynamics of WBCs in response to acute HH.
A: Principal component analysis (PCA) using genes robustly expressed in at least one of six HH stages (A–F). Colors denote stages of HH at which WBC samples were collected, as described in Figure 1A. B: Numbers of differentially expressed genes (DEGs) between stage A and each of the other five stages. C: Expression patterns of DEGs in three HH-responding modules, shown as log2-transformed reads per kilobase of transcript per million mapped reads (RPKM) fold changes of DEGs in each stage compared to stage A. Red and light blue lines indicate expression patterns of individual DEGs up- and down-regulated by HH, respectively. Bold lines show mean expression levels of all up- or down-regulated DEGs, with error bars indicating SD. UP and DOWN indicate exact number of up- or down-regulated DEGs by HH in each module. D: Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enriched in three HH-responding modules. Only significantly enriched pathways with a false discovery rate (FDR)-adjusted P-value of <0.05 were plotted. Dot color denotes P-value after FDR correction, and dot size denotes ratio of DEGs versus all expressed genes in each pathway. Different background colors denote classification of pathways. E: Interaction of pathways and symptoms according to reported articles. Blue color denotes pathways of impulse module; red color denotes pathways of late-responding module. Solid arrow line denotes reported direct interaction; dashed arrow line denotes reported associated relationship. F: Local co-expression network revealing genes in vitamin D receptor (VDR) complex as hub genes. Dark blue circles denote DEGs in impulse module; red circles denote genes in VDR complex and HIF1A as hub genes.
Figure 3. Regulation of WBC transcriptomic dynamics by PROG and TRIOL
A: Principal component analysis (PCA) using genes robustly expressed in at least one of nine sample groups (i.e., no-drug treated group, PROG-treated group, and TRIOL-treated group in stages A, D, and F, respectively). Colors denote HH stages at which WBC samples were collected during HH+drug experiments, as described in Figure 1B, whereas shapes denote treatments. B: Number of differentially expressed genes (DEGs) between drug-treated and untreated groups in each HH stage studied. Stage A samples from both PROG and TRIOL groups were collected prior to drug injection; thus, DEGs of A/PROG*-A and A/TRIOL*-A reflect random variations between two groups of experimental monkeys rather than drug-specific effects. C: Erythrocyte-associated DEGs up-regulated by PROG. Red color and arrows denote genes up-regulated by PROG. D: Expression patterns and functions of TRIOL-induced DEGs in three HH-responding modules, shown as log2-transformed reads per kilobase of transcript per million mapped reads (RPKM) fold changes of DEGs in each sample group compared to stage A samples without TRIOL treatment. Light red and blue lines indicate expression patterns of individual DEGs in TRIOL-treated and untreated groups, respectively. Bold lines show mean expression levels of all TRIOL up- or down-regulated DEGs in each group, with error bars representing SD. UP and DOWN indicate exact number of up- or down-regulated DEGs by TRIOL in each module. Text below each module outlines functions associated with TRIOL-induced DEGs, with up and down arrows denoting gene-associated functions up- or down-regulated by TRIOL, respectively.
Figure 4. Transcriptomic changes in frontal cortex in response to acute HH, PROG, and TRIOL
A: Principal component analysis (PCA) using genes robustly expressed in at least one of four sample groups (normobaric normoxia [NN], HH, HH+ PROG, and HH+TRIOL). Colors denote sample group. B: Expression changes of HH-induced differentially expressed genes (DEGs) after PROG or TRIOL treatment, shown as log2-transformed reads per kilobase of transcript per million mapped reads (RPKM) fold changes of DEGs in each sample group compared to NN group. HH-induced DEGs are categorized into strongly responsive genes (SRGs), weakly responsive genes (WRGs), and non-responsive genes (NRGs) according to their degree of expression level recovery after drug treatment. Red and light blue lines denote expression patterns of individual DEGs up- and down-regulated by HH, respectively. Bold lines show mean expression levels of all HH up- or down-regulated DEGs in each group, with error bars representing SD. Percentages of SRGs and WRGs versus all HH-induced DEGs are presented above each plot. C: Expression changes of drug DEGs after PROG or TRIOL treatment, shown as log2-transformed RPKM fold changes of DEGs in each sample group compared to NN group. Red and light blue lines denote expression patterns of individual DEGs up- or down-regulated by drugs, respectively. Bold lines show mean expression levels of all DEGs up- or down-regulated by drugs in each group, with error bars representing SD. D: Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enriched in HH-induced or drug-recovered DEGs. Only significantly enriched pathways with a false discovery rate (FDR)-adjusted P-value of <0.05 are plotted. In HH DEGs, SRGs, WRGs, and NRGs, red and blue dots denote pathways enriched in DEGs up- and down-regulated by HH, respectively. In drug DEGs (PROG DEGs and TRIOL DEGs), red and blue dots denote pathways enriched in DEGs up- and down-regulated by drugs, respectively. Color intensity of dot indicates level of significance, and size of dot denotes ratio of DEGs versus all expressed genes in each pathway. E: Postulated regulation of excitatory glutamate signaling by TRIOL. Rectangles represent proteins of TRIOL DEGs involved in glutamate signaling pathway. Blue colors denote down-regulation by HH. Gene symbols of DEGs associated with each protein in the pathway are listed below. F: Time-course showing intraneuronal calcium [Ca2+]i in primary cultured rat cortical neurons stimulated by glutamate with different doses of TRIOL. Glutamate stimulation was added at the fiftieth second in the experiment.
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