Subarachnoid hemorrhage (SAH) is a devastating cerebrovascular event that often is followed by permanent brain impairments. It is necessary to explore the pathogenesis of secondary pathological damages in order to find effective interventions for improving the prognosis of SAH. Blockage of brain lymphatic drainage has been shown to worsen cerebral ischemia and edema after acute SAH. However, whether or not there is persistent dysfunction of cerebral lymphatic drainage following SAH remains unclear. In this study, autologous blood was injected into the cisterna magna of mice to establish SAH model. One week after surgery, SAH mice showed decreases in fluorescent tracer drainage to the deep cervical lymph nodes (dcLNs) and influx into the brain parenchyma after injection into the cisterna magna. Moreover, SAH impaired polarization of astrocyte aquaporin-4 (AQP4) that is a functional marker of glymphatic clearance and resulted in accumulations of Tau proteins as well as CD3⁺, CD4⁺, and CD8⁺ cells in the brain. In addition, pathological changes, including microvascular spasm, activation of glial cells, neuroinflammation, and neuronal apoptosis were observed in the hippocampus of SAH mice. Present results demonstrate persistent malfunction of glymphatic and meningeal lymphatic drainage and related neuropathological damages after SAH. Targeting improvement of brain lymphatic clearance potentially serves as a new strategy for the treatment of SAH.
Animals ; Apoptosis ; Aquaporin 4 ; Astrocytes ; Brain ; Brain Ischemia ; Cisterna Magna ; Drainage ; Edema ; Hippocampus ; Lymph Nodes ; Mice ; Neuroglia ; Neurons ; Prognosis ; Spasm ; Subarachnoid Hemorrhage ; tau Proteins

ObjectiveTo validate the efficacy of Yunpi Huatan Tongqiao prescription （YHTP） in down-regulating glycolysis to modulate microglia phenotype and improve inflammation and cognitive memory deficits in obstructive sleep apnea （OSA） mice. MethodForty-eight male Balb/C mice were randomly divided into a normal group， a model group， a montelukast sodium group （30 mg·kg^-1）， and low， medium， and high dose groups of YHTP （8.28， 16.56， and 33.12 g·kg^-1）， with 8 mice in each group. All groups， except the normal group， received intraperitoneal injections of lipopolysaccharide （LPS） and underwent chronic intermittent hypoxia （CIH） modeling for 4 weeks. Subsequently， the mice were treated with medications for 4 weeks and then sampled. Animal behavioral tests assessed memory impairment due to hypoxia. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to measure mRNA expression levels of M1-associated inflammatory factors interleukin-1β （IL-1β）， tumor necrosis factor-α （TNF-α）， and markers such as T lymphocyte activation antigen （CD86） and inducible nitric oxide synthase （iNOS）， as well as M2-associated inflammatory factors interleukin-10 （IL-10）， transforming growth factor-β （TGF-β）， and the marker mannose receptor （CD206） in hippocampal tissue. Western blot was employed to detect differences in the expression of M1 and M2 microglia phenotypic markers （CD86， CD206） and glycolysis-related proteins glucose transporter type 1 （GLUT1）， hexokinase 2 （HK2）， phosphofructokinase （PFKM）， pyruvate kinase 2 （PKM2）， and monocarboxylic acid transporter 1 （MCT1）. ResultBehavioral tests showed that compared to the results in the normal group， the Y-maze autonomous alternation rate was significantly reduced in the model group （P<0.01）. The latency time for the target hole in the Barnes' maze during the training period （days 2， 3， 4） and testing period （days 5， 12） was significantly increased （P<0.05， P<0.01）. M1 glial cell markers CD86 and iNOS， as well as inflammatory factors IL-1β and TNF-α mRNA， were significantly elevated （P<0.01）. In contrast， the mRNA expression of M2 glial cell markers IL-10， CD206， and TGF-β was significantly reduced （P<0.01）. The protein expression of glycolytic proteins HK2， PFKM， PKM2， MCT1， and the M1 marker CD86 was significantly increased （P<0.05， P<0.01）， while M2 marker CD206 protein expression was significantly decreased （P<0.01）. Compared to the results in the model group， the Y-maze autonomous alternation rate was significantly increased in the medium and high dose groups of YHTP （P<0.05， P<0.01）. The latency time for the target hole during the training （day 4） and testing periods （days 5， 12） was significantly reduced （P<0.01）. Real-time PCR results indicated that mRNA expression levels of M1-related pro-inflammatory factors in the hippocampal tissue were significantly reduced in the low， medium， and high dose groups of YHTP （P<0.01）， while M2-related inflammatory factors' mRNA expression was significantly increased （P<0.01）. Western blot results showed that in the medium and high dose groups of YHTP， the expression of the M1 marker CD86 in the hippocampus was reduced， whereas the expression of the M2 marker CD206 was significantly increased （P<0.01）， with a significant decrease in the expression of glycolysis-related proteins （P<0.01）. ConclusionYHTP can improve inflammation and cognitive impairment induced by hypoxia in OSA model mice. This is achieved by downregulating glycolysis in brain microglia， inhibiting M1 activation， reducing pro-inflammatory factor release， and promoting M2 activation， thereby exerting a therapeutic effect on inflammation and cognitive impairment caused by OSA.