Systemic pro-inflammatory response facilitates the development of cerebral edema during short hypoxia

Abstract

Background: High-altitude cerebral edema (HACE) is the severe type of acute mountain sickness (AMS) and life threatening. A subclinical inflammation has been speculated, but the exact mechanisms underlying the HACE are not fully understood.

Methods: Human volunteers ascended to high altitude (3860 m, 2 days), and rats were exposed to hypoxia in a hypobaric chamber (5000 m, 2 days). Human acute mountain sickness was evaluated by the Lake Louise Score (LLS), and plasma corticotrophin-releasing hormone (CRH) and cytokines TNF-α, IL-1β, and IL-6 were measured in rats and humans. Subsequently, rats were pre-treated with lipopolysaccharide (LPS, intraperitoneal (ip) 4 mg/kg, 11 h) to induce inflammation prior to 1 h hypoxia (7000 m elevation). TNF-α, IL-1β, IL-6, nitric oxide (NO), CRH, and aquaporin-4 (AQP4) and their gene expression, Evans blue, Na(+)-K(+)-ATPase activity, p65 translocation, and cell swelling were measured in brain by ELISA, Western blotting, Q-PCR, RT-PCR, immunohistochemistry, and transmission electron micrography. MAPKs, NF-κB pathway, and water permeability of primary astrocytes were demonstrated. All measurements were performed with or without LPS challenge. The release of NO, TNF-α, and IL-6 in cultured primary microglia by CRH stimulation with or without PDTC (NF-κB inhibitor) or CP154,526 (CRHR1 antagonist) were measured.

Results: Hypobaric hypoxia enhanced plasma TNF-α, IL-1β, and IL-6 and CRH levels in human and rats, which positively correlated with AMS. A single LPS injection (ip, 4 mg/kg, 12 h) into rats increased TNF-α and IL-1β levels in the serum and cortex, and AQP4 and AQP4 mRNA expression in cortex and astrocytes, and astrocyte water permeability but did not cause brain edema. However, LPS treatment 11 h prior to 1 h hypoxia (elevation, 7000 m) challenge caused cerebral edema, which was associated with activation of NF-κB and MAPKs, hypoxia-reduced Na(+)-K(+)-ATPase activity and blood-brain barrier (BBB) disruption. Both LPS and CRH stimulated TNF-α, IL-6, and NO release in cultured rat microglia via NF-κB and cAMP/PKA.

Conclusions: Preexisting systemic inflammation plus a short severe hypoxia elicits cerebral edema through upregulated AQP4 and water permeability by TLR4 and CRH/CRHR1 signaling. This study revealed that both infection and hypoxia can cause inflammatory response in the brain. Systemic inflammation can facilitate onset of hypoxic cerebral edema through interaction of astrocyte and microglia by activation of TLR4 and CRH/CRHR1 signaling. Anti-inflammatory agents and CRHR1 antagonist may be useful for prevention and treatment of AMS and HACE.

Figures

Fig. 1

High-altitude hypoxia induced increased levels of TNF-α, IL-1β, IL-6, and CRH in humans and rats. a Human SpO2 was measured by fingertip pulse oximeter at low and high altitudes. b The levels of CRH in plasma of all human volunteers was determined by ELISA (n = 74). c The correlation between LLS score data and cytokines (TNF-α, IL-1β, and IL-6) level at high altitude by Spearman correlation analysis. d–f The levels of TNF-α, IL-1β, and IL-6 in plasma of all human volunteers was determined by ELISA (n = 74). ***P < 0.001 compared with control (low altitude, 540 m); #P < 0.05, ###P < 0.001 compared with no-AMS group at high altitude (3860 m). g–i The rats in hypoxia group were exposed to hypoxia at altitude of 5000 min a hypobaric chamber for 2 days. The levels of CRH, TNF-α, and IL-1β in plasma were measured (n = 7). j, k Hypoxia-increased TNF-α and IL-1β levels in plasma were blocked by CRHR1 antagonist CP154,526 (n = 7). **P < 0.01; ***P < 0.001 compared with control, &P < 0.05 (hypoxia vs hypoxia + CRHR1 antagonist); the data are presented as mean ± SD

Fig. 2

Systemic inflammation facilitates the onset of brain edema under transient hypoxia. a, b TNF-α and IL-1β level in serum and in brain cortex of rats were tested after hypoxia exposure (1–8 h) and increased after 4 h. c Hypoxia-increased TNF-α and IL-1β mRNA expression in brain cortex (n = 6). *P < 0.05; **P < 0.01; ***P < 0.001 compared with control. &P < 0.05; & &P < 0.01; &&&P < 0.001 (4 h hypoxia vs 1 h hypoxia). d, e TNF-α and IL-1β levels in serum and cortex of rats were measured by ELISA and increased, following the LPS treatments indicated (n = 6). *P < 0.05; **P < 0.01; ***P < 0.001 compared with control. f Brain water content (BWC) was determined by wet/dry weight ratio at the time points indicated after LPS treatment (n = 6–12). g, h TNF-α and IL-1β levels in serum and cortex of rats were measured by ELISA and increased, following LPS or hypoxia alone or combination of both treatments indicated (n = 6). *P < 0.05; **P < 0.01; compared with control. +P < 0.05; ++P < 0.01, hypoxia + LPS vs hypoxia. i BWC was determined by wet/dry weight ratio after LPS, hypoxia or both (n = 6–12), ***P < 0.001 compared with control, +++P < 0.001, compared with LPS or hypoxia alone. j BBB permeability was determined by detecting the extravasations of Evans blue dye (n = 8). **P < 0.01 compared with control, ++P < 0.001, compared with LPS or hypoxia alone. k Na+/K+-ATPase activity in brain cortex was measured following the indicated treatments (n = 6). **P < 0.01 compared with control, ++P < 0.01, compared with hypoxia or hypoxia + LPS. All data are presented as mean ± SD. l Representative transmission electron micrographs of perivascular astrocytes and mitochondria. Scale bar, 1 μm, arrow indicates enlarged astrocyte foot processes

Fig. 3

LPS and cytokines increase AQP4 mRNA and protein expression in the brain cortex and astrocytes, and LPS increases water permeability of astrocytes. a The expression of AQP4 mRNA in brain cortex tested by Q-PCR following hypoxia exposure for the time points indicated (n = 6). **P < 0.01, ***P < 0.001 compared with control; & &P < 0.01 (hypoxia 4 h vs hypoxia 1 h). b The expression of AQP4 mRNA in the brain cortex of rats determined by Q-PCR following injection of LPS for the time points indicated (n = 6). ***P < 0.001 compared with control. c Hypoxia + LPS and LPS increased AQP4 mRNA expression in brain cortex (by Q-PCR), ***P < 0.001 compared with control. d AQP4 protein expression in brain cortex was detected by western blot after LPS treatment at the time points indicated (n = 6). ***P < 0.001compared with control, +++P < 0.001 vs LPS 10 h. e AQP4 mRNA expression and f AQP4 protein expression in astrocytes were detected by RT-PCR and western blot after LPS challenge (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001, compared with control; ++P < 0.01, compared with 8 h. g AQP4 mRNA expression in astrocytes was determined by RT-PCR after LPS, TNF-α, or IL-1β treatment for 8 h (n = 3). **P < 0.01, ***P < 0.001, compared with control. h Water permeability of cells increased by LPS challenge (n = 100). ***P < 0.001, compared with control. All the data are presented as mean ± SD

Fig. 4

Inflammation induced increased AQP4 expression via the NF-κB and MAPKs pathway. a Astrocytes pre-treated with or without PDTC were treated with LPS, TNF-α, or IL-1β for 2 h, followed by staining with specific p65 antibody (n = 3). b p65 translocation into cell nuclei (%) after treatment with LPS, TNF-α, or IL-1β for 2 h. c AQP4 protein levels in astrocytes treated with LPS for 12 h after pre-treatment with or without PDTC (n = 3). d Water permeability of cells after LPS or LPS + PDTC treatment (n = 100). **P < 0.01; ***P < 0.001 compared with control; ##P < 0.01;###P < 0.001 compared with LPS 12 h. e Phosphorylated levels of ERKs and p38 in astrocytes treated with LPS, TNF-α, or IL-1β for 0.5 h were increased (n = 3). ***P < 0.001, compared with control. f AQP4 mRNA changes measured by RT-PCR in astrocytes treated with LPS for 8 h after pre-treatment with p38 inhibitor, ERK1/2 inhibitor, JNK inhibitor (n = 3). ***P < 0.001 compared with control; ##P < 0.01 compared with LPS treatment. g Water permeability changes of astrocytes pre-treated with p38 inhibitor, ERK inhibitor, JNK inhibitor, p300 inhibitor, or transfected with p300 siRNA then exposure to LPS for 12 h (n = 100). ***P < 0.001 compared with control; ###P < 0.001 compared with LPS 12 h. All the data are presented as mean ± SD

Fig. 5

CRH stimulates microglia and triggers an immune-inflammatory response. a, b Increase in TNF-α and IL-6 release in cultured primary cortical microglia induced by CRH or LPS or CRH+LPS challenge for 24 h. **P < 0.01, compared with control, #P < 0.05; ##P < 0.01, LPS + CRH vs LPS. c Increase in NO production in cultured primary cortical microglia challenged by CRH, LPS or CRH+LPS. **P < 0.01, compared with control; &P < 0.01, compared between LPS + CRH and LPS; @@P < 0.01 compared between CRH + LPS and CRH + LPS + CP154,526; ###P < 0.01, compared between CRH and CRH + CP154,526. CRH stimulated increased NO production, was blocked by PKA inhibitor (inset). ***P < 0.001 compared with control, +++P < 0.001 compared between CRH and CRH + PKA inhibitor. All the data are presented as mean ± SD. d Hypoxia and LPS increased IL-6 protein levels in brain cortex. Increased IL-6 protein in rat brain cortex following either hypoxia or LPS (8 h) was blocked by CRHR1 antagonist, n = 6, **P < 0.01 compared with control, ++P < 0.01 compared between hypoxia and hypoxia + CP154,526, ##P < 0.01 compared between LPS and LPS + CP154,526, the data are presented as mean ± SD. e Hypoxia stimulated expression of mRNAs of CRH, TNF-α, and IL-6 in brain cortex (n = 6) **P < 0.01, compared with control, the data are presented as mean ± SD. f Hypoxia-increased CRH release in circulation **P < 0.01, compared with control; ++P < 0.01, compared between hypoxia 2 and 8 h, the data are presented mean ± SD

Fig. 6

Integrated results of this study to diagrammatically show how LPS-induced systemic inflammation facilitates the onset of cerebral edema under short-term hypoxia in rats by (1) LPS-induced pro-inflammatory pathway which activates AQP4 and contributes to water permeability of astrocytes via TLR4 signaling MAPKs and NF-κB, (2) short-term hypobaric hypoxia which reduced Na+-K+-ATPase activity resulting in water inflow in astrocytes, (3) LPS + hypoxia-induced leakage of BBB resulting in vasogenic edema. All together, these factors contribute to brain edema. Under prolonged hypoxia, systemic inflammation may worsen the cerebral edema by (1) LPS plus hypoxia-induced astrocyte cytotoxic swelling; (2) LPS plus hypoxia-activated cortical microglia release of cytokines, which further stimulate astrocyte swelling; and (3) hypoxia further reducing Na+-K+-ATPase activity, and LPS + hypoxia triggering leakage of BBB. This mechanism in rats may describe the onset and deterioration of HACE in human