Glymphatic Drainage Blocking Aggravates Brain Edema, Neuroinflammation via Modulating TNF-α, IL-10, and AQP4 After Intracerebral Hemorrhage in Rats

Abstract

Objective: The "Glymphatic" system, a network of perivascular tunnels wrapped by astrocyte endfeet, was reported to be closely associated with the diseases of the central nervous system. Here, we investigated the role of the glymphatic system in intracerebral hemorrhage (ICH) and its protective mechanism. Method: Experimental ICH model was induced by type IV collagenase in rats. Cerebral lymphatic blockage was induced by ligation and removal of cervical lymph nodes. The experimental rats were divided into sham-operated (SO) group, ICH group, and cerebral lymphatic blocking and ICH (ICH + CLB) group. Neurological scores were measured using the Garcia scoring system on the third and seventh day after ICH. Active caspase-3 was immunostained to evaluate neuronal apoptosis. Brain water content was calculated using the dry-wet specific gravity method. The expression of inflammatory factors TNF-α, IL-1β, and IL-10 were detected using ELISA. Aquaporins-4 (AQP-4) and glial fibrillary acidic protein (GFAP) were detected using western blot analysis. Results: The neurological scores of rats in the CLB + ICH group were significantly lower than those in the in ICH group. The number of active caspase-3 neurons was significantly higher in the CLB + ICH group compared to the ICH group. CLB significantly aggravated ICH-induced brain edema 3 d after ICH. There was an increase in the expression of TNF-α, IL-1β, IL-10, AQP-4, GFAP after ICH. The expression of TNF-α was significantly higher in the CLB + ICH group compared to ICH group 3 d after ICH while there was no difference 7 d after ICH. There was no statistical difference in the expression of IL-1β between the ICH group and CLB + ICH group. However, the expression of IL-10 in the CLB + ICH group was significantly lower than that in the ICH group. Lastly, AQP-4 expression was significantly lower in the CLB + ICH group compared to the ICH group while the expression of GFAP was higher in the CLB + ICH group compared to the ICH group. Conclusion: CLB exacerbated cerebral edema, neuroinflammation, neuronal apoptosis and caused neurological deficits in rats with ICH via down-regulating AQP-4, up-regulating inflammatory TNF-α and inhibiting IL-10 expression. The glymphatic drainage system protects against neurologic injury after ICH induction in rats under normal physiological conditions.

Keywords: aquaporins-4; brain edema; glymphatic; intracerebral hemorrhage; neuroinflammation; neuronal apoptosis.

FIGURE 1

Neurological deficit score after cerebral lymphatic blocking (CLB) in intracerebral hemorrhage (ICH) rats. The scores of the rats in the CLB + ICH group were significantly lower than those of rats in the ICH group 7 days after ICH (△, P < 0.05). There was a significant increase in the scores for the rats 7 days after ICH compared to 3 day after ICH (#, P < 0.05). On the contrary, there was a decrease in the scores 7 days after ICH compared to 3 days after ICH for rats in the CLB + ICH group (#, P < 0.05). SO group, n = 10. ICH group, n = 10; and CLB + ICH group, n = 10, 3, or 7 days after ICH.

FIGURE 2

Results of neuronal apoptosis 3 days after ICH in three groups. (A) Representative immunofluorescent images of Active caspase-3 (green) and DAPI (blue) staining (×200). Scale bar: 40 μm. (B) Quantitative analysis showed more apoptotic cells 3 days in ICH and CLB + ICH groups than in SO group (*P < 0.05). The numbers of active caspase-3cells in hemorrhagic rat brain tissues of CLB + ICH group were further increased comparing with those in ICH group (△, P < 0.05). SO group, n = 5; ICH group, n = 5; and CLB + ICH group, n = 5.

FIGURE 3

The hematoma volume of the three groups. (A) The brain hematoma were successfully induced 3 day after collagenase IV injection in ICH and CLB + ICH group. (B) There was no significant difference in hematoma volume between the ICH and CLB + ICH group 3 days after ICH induction (p > 0.05). *P < 0.05, compare to SO group. SO group, n = 5; ICH group, n = 5; and CLB + ICH group, n = 5.

FIGURE 4

Brain water content of the three groups. There was a significant increase in the brain water content of ipsilateral cortex of the ICH group and CLB + ICH group compared to that in the sham group (*P < 0.05). Seven days after ICH, CLB significantly inhibited regression of ICH-induced cerebral edema (△, P < 0.05). There was a significant decrease in the brain water content for rats in the ICH group (#, P < 0.05), but there was no significant difference in the CLB + ICH group (P > 0.05). SO group, n = 5. ICH group, n = 5; and CLB + ICH group, n = 5, 3 or 7 days after ICH.

FIGURE 5

HE staining result of three groups. (A) Histopathological changes in perihematomal tissues of each group 3 days after ICH induction (HE staining, ×400). In the SO group, cerebral hematoma, edema, and liquefactive necrosis were not found. In the ICH group, tissues surrounding the cerebral hematoma showed edema, liquefactive necrosis, deposition of hemosiderin, inflammatory cells (mainly neutrophils). In the CLB + ICH group, there was greater edema and higher infiltration of immune cells (predominant monocytic and a low-degree neutrophilic granulocyte) in the perihematomal region compared to that in ICH group. Scale bar: 100 μm. (B) The number of inflammatory cells in the ICH group was significantly higher than those in control group (^∗P < 0.05). Compared to ICH groups, numbers of inflammatory cells in CLB + ICH group was further increased (△, P < 0.05). SO group, n = 5; ICH group, n = 5; and CLB + ICH group, n = 5.

FIGURE 6

Levels of TNF-α, IL-1β, and IL-10 in the perihematoma tissue using ELISA. (A) Compared to SO group, TNF-α significantly increased in ICH and CLB + ICH groups (*P < 0.05) 3 days after ICH. The levels of TNF-α expression 7 days after ICH decreased significantly compared to the levels at day 3 days, in the ICH and CLB + ICH groups (#, P < 0.05). Compared to that in ICH group, TNF-α was significantly up-regulated in the CLB + ICH group 3 days after ICH (△, P < 0.05). There were no statistical difference between ICH group and CLB + ICH group 7 days after ICH. (B) The expression levels of IL-1β in both ICH and CLB + ICH groups were significantly higher compared to the SO group (*P < 0.05). IL-1β was significantly decreased 7 days after ICH compared to 3 days after ICH in the ICH and CLB + ICH groups (#, P < 0.05). There were no statistical differences in the levels of IL-1β between ICH group and CLB + ICH group 3 and 7 d after ICH (P > 0.05). (C) The expression levels of IL-10 in the CLB + ICH group were significantly lower than those in the ICH group 3 and 7 d after ICH (△, P < 0.05). There was a statistically significant decrease in the expression levels of IL-10 in the ICH group between 3 and 7 d after ICH (#, P < 0.05). However, the levels were only slightly decrease in the CLB + ICH group (P > 0.05). SO group, n = 5. ICH group, n = 5; and CLB + ICH group, n = 5, 3, or 7 days after ICH.

FIGURE 7

Western blot results of AQP4 and glial fibrillary acidic protein (GFAP) of three groups 3 d after ICH. (A) Representative result of Western Blot. (B) The expression of AQP4 in the ICH and CLB + ICH groups was significantly higher compared to SO group (^∗P < 0.05). AQP-4 level was significantly decreased in the CLB + ICH group compared to the ICH group (△, P < 0.05). (C) The expression of GFAP was significantly increased in the ICH and CLB + ICH group as compared to the SO group (^∗P < 0.05). The expression of GFAP was further increased in the CLB + ICH group compared to that in ICH group (△, P < 0.05). SO group, n = 5; ICH group, n = 5; and CLB + ICH group, n = 5.