The Effect Of Hyaluronic Acid In Tissue Injury After Intracerebral Hemorrhage

The clinical and experimental studies about intracerebral hemorrhage(ICH)had illustrated that some components of extracellular matrix(ECM)like matrix metalloproteinases(MMPs), cell-fibronectin all were related to the prognosis of ICH. Hyaluronic acid or hyaluronan(HA) is a major ECM component. Hyaluronan forms specific stable tertiary structures in aqueous solution. It has multiple functions, such as space filling, hydration, and so on. HA can regulate cell differentiation, proliferation, migration, and also participate in activating inflammatory response after tissue injury.Al’ Qteishat A and his colleagues found that the production of total HA and low molecular mass3-10disaccharides of HA (o-HA) was increased in post-mortem tissue and in the serum of patients after ischaemic stroke. Hyaluronidase activity was also increased in serum. Affinity-histochemical staining showed enhanced deposition of HA in blood vessels and intracellularly as well as in the nuclei of peri-infarcted neurons. HA synthases (HAS1and2) and hyaluronidases (HYAL1and2) upregulated in peri-infarcted regions of the brain. Receptor for HA-mediated motility (RHAMM), CD44all increased in inflammatory regions. The results demonstrate that HA breakdown is a feature of the acute stage of stroke injury. Increased o-HA production soon after stroke may be detrimental through enhancing of the inflammatory response, whilst activation of HA and/or o-HA induced cellular signalling pathways in neurons and microvessels may impact on the remodelling process by stimulating angiogenesis and revascularization, as well as the survival of susceptible neurons.We deduce that similar changes in brain should be resulted from ICH, HA would be decomposed into low-molecular weight HA. HAS, hyaluronidases and HA receptors all would be altered, which may make important influences on edema formation and tissue damage after ICH. So, we first made autologous blood injection model of ICH with HAS1knock out mice at Aichi Medical University, to study neurological scale and water content of brain after ICH, and the changes of total of HA, different molecular weight HA, HAS, hyaluronidase. We also detected cell’s necrosis and/or apoptosis, cytokines expression in HAS1knock out mice and widetype mice. All of these works is in order to explore the role of HA in tissue injury after ICH, which would become the bases for further researches about HA in brain damage and expand our knowledge about tissue injury and repair after ICH.Part Ⅰ:The Difference of Edema and Neurological Deficiency after ICH between HAS1Knock Out Mice and WT MiceObjective and Background:Before exploring the mechanism of one molecule in stroke, we should know whether it will affect the prognosis of stroke. So, at the first part, we investigated if the change of HA would affect the prognosis of ICH, that is, if HAS1ko disturbanced the edema and neurological scale.Methods:Intracerebral hemorrhage mice model were made of male HAS1ko mice by injected25ul autologous blood into right caudate nucleus. The control group was C57BL/6J mice. We investigated the neurological scale and water content of right hemisphere at the point of72h after ICH. Different ages of WT and HAS1group included2-months old,3-months old and9-months old, every group had no less than6mice. The neurological scale was evaluated by28-points method, higher scale means more serious deficiency. In order to determine the brain water content by the wet/dry ratio, the brains were removed from the skull and were treated by vacuum freeze drying for48h after weighing, then were dried further in a incubator at100℃for24h and reweighed to give the dry weight. Water content was calculated by (wet weight-dry weight)/wet weight.Results:(1) Behavioral testing were performed in all71mice (WT were33mice, HAS1were38mice). The neurological scale of WT group of2-months old,3-months old,9-months old mice was respectively4.7±3.0,4.3±1.8,7.1±4.5, the same age of HAS1ko mice’s neurological scale was7.8±4.6,7.7±4.2,10.8±2.8(all p<0.05). It showed that HAS1group all had higher scale at72h of ICH than WT group, which illustrated more severe neurological function deficits in HAS1ko mice after ICH.(2) Water content was not different between WT and HAS1group in2-months old mice. However, HAS1group’s water content were lower man WT in3-months and9-months mice. So, HAS1ko would result in water content declining after ICH.(3) Correlation analysis with all mice showed that neurological scale had no correlation with water content.Conclusions:HAS1ko resulted in more severe neurological deficits after ICH but the brain water content declined.Part Ⅱ:The Changes of HA, HAS and Hyaluronidases due to HAS1KO before and after ICHObjective and Background:The first part of our research initiated that the neurological deficits exacerbated in HAS1ko mice after ICH but did not accompanying with edema increased. So, we should know how HA, HAS and hyaluronidases altered before and after ICH due to HAS1ko.Methods:WT and HAS1group were divided to normal control group,24h and72h after ICH group. Each group had6mice. HA content(ug/g wet tissue) of hemorrhagic hemisphere were measured with ELISA method. Then,0.4ug sample of each brain in one group are mixtured (total content was2.4ug) to do sephadex gel chromatographic separation in order to separate different molecular weight HA. The curve of elution tube-HA concentration was drawed and the elution positions of HA standard samples with average molecular masses of2130,850,150,5.6kDa also determined by same procedure. Finally, high molecular weight HA(>850kDa)/total HA ratio and low molecular weight HA(<850kDa)/total HA ratio were caculated. HAS1-3and hyaluronidase1-3(Hyall-3) mRNA expression after ICH were detected by real time PCR.Results:(1) HA content in brain decreased25.6%due to HAS1ko. After ICH, HA content decreased, especially in HAS1ko mice.(2) Gel filtration showed HA still was native high molecular mass polymer in normal HAS1mice brain. However, low molecular weight HA was increasing and high molecular mass HA was decreasing after ICH, and this trend was more dominant in HAS1ko mice.(3) MRNA expression of HAS1-3, Hyall-3all were growing after ICH both of two groups, only HAS3expression may be higher in HAS1group than WT(p=0.05). Conclusions:HAS1ko resulted in25.6%decreased of total HA content in brain. Increased hyaluronidases and HAS3expression brought about HA content declining further and low molecular weight HA increasing after ICH.Part Ⅲ:The Effect of HAS1KO for Cell Apoptosis/Necrosis and cytokine expression after ICHObjective and Background:It was known that HA content decreased due to HAS1ko and low molecular weight HA increased after ICH, and this change was accompanied by neurological deficits exacerbated. Therefore, we wanted to know if there was different apoptosis/necrosis between HAS1and WT groups, and if cytokine expression was also different after ICH.Methods:WT and HAS1mice all divided into three groups including sham,24h,72h after ICH. Each group had five mice. Nissle, DAPI, TUNEL staining with frozen section of brain and Caspase-3activity fraction(17KDa) with Western Blot all were done for illustrating whether the apoptosis/necrosis of cell were more severe in HAS1ko group than WT mice. The RayBiotech protein chip were used to measure the changes of62kinds of cytokines expression after ICH both in two kinds of mice. Results:(1) Frozen section staining and Caspase-3expression all showed that apoptosis/necrosis of cell were deteriorated in HAS1group.(2)Although EL-13, TNF-a, Axl, IL-10, GM-CSF, IGFBP5-6expression in HAS1group were higher after ICH, there was no difference between HAS1and WT groups.Conclusions:Cell apoptosis/necrosis was more dominant in HAS1ko mice, this may be result of cytokine expressing higher after ICH.To sum up, HAS1ko resulted in HA content of brain decreased1/4. After ICH, low molecular weight HA increased especially in HAS1ko group and these changes may be trigger inflammatory response and finally lead to more severe cell apoptosis/necrosis, so the neurological scale worsen after ICH. Cerebral water content declined in HAS1ko mice after ICH revealed edema had no correlation to neurological deficits of ICH. At the same time, these results also initiated high molecular weight HA may be have protective effect to brain tissue but low molecular weight HA aggravate tissue injury of ICH.