Cerebral Vascular Structure And Ischemic Brain Injury Changes In Mice With Metabolic Dysfunction And Corresponding Prevention And Treatment Study

BackgroundStroke related prevention and treatment strategies have always been a concentration inmedical research field. There are more than9,000,000cases of stroke around the worldper year and the prevalence is as high as30,000,000cases. The fact that more then onethird of the patients developed moderate to severe disability makes stroke the number onecause of disability, cognitive dysfunction and death. What makes it more noticeable is thatof the death and disability cases are from countries with low to middle average income,suggests that medical care standard plays a pivotal role in it.In decades, promising compounds and therapies has been springing up in basicresearch against ischemic stroke. But once proceed onto clinical trials, hardly any of them could repeat the effects gained in basic research. Severe complications such bleeding (tPA)or lack of effectiveness is the major problem for their clinical application. One of thenotable problems is that the actual status of patients is of huge difference from the healthytested subjects in basic research. Stroke patients usually have fundamental heath problemssuch as metabolic disorders or cardiovascular diseases.Advanced age, obesity,hyperglycemia, hyperlipidemia, hypertension, atherosclerosis etc. could all be risk factorsfor stroke. But in the past years of basic research about stroke, young healthy animals havealways been main research subjects [1]. The pathophysiological difference could be one ofthe causes for the failure of translational studies. Among all the risk factors for stroke,obesity is of the highest prevalence. There are more then7,000,000people in China thatare obese, which accounts for20%of the world’s obese population and the number isincreasing rapidly. Another major risk factor of stroke-diabetes-also has a highprevalence of9.7%in China. There are a lot of pandemic research suggests that metabolicdiseases such as hyperlipidemia and diabetes are all independent risk factors forcardiovascular disease. Metabolic dysfunction does not only incresase the risk fordeveloping ischemic stroke[2], it is also related to deterioration of neurologic outcome [3;4]. However, conclusions draw from clinical researches are more likely to be correlativeanalysis rather then direct causality evidence.“Vascular remodeling” is a concept created in1987by Glagov, representing failureof the arterial wall to maintain the appropriate lumen size needed to permit normal bloodflow under physiological needs [5].Early studies about vascular remodeling mostlyconcentrate on coronary artery disease (cardiovascular) and hypertension (systemicarterial). More recently, researchers started to point out that metabolic disease can alsocause vascular remodeling in the cerebral arterial [6;7]. ET1and MMP-1have beensuggested to involve in the progress of vascular remodeling in the brain. However, thedirect evidence leading metabolic disorders to cerebral vascular remodeling andaggravation of ischemic stroke injury is still weak. In this study, we proposed that high fatdiet could induce obesity, hyperglycemia and hyperlipidemia in mice and cause cerebralvascular remodeling as well as aggravation of ischemic stroke injury and diabetes could do the same. And that MMPs activity and blood glucose level play important roles in thetwo different types of pathophysiological changes, respectively.In addition to how metabolic diseases affect the cerebral vascular structure andischemic stroke injury, we also explored the effect of a novel small molecular compound,trans-sodium crocetinate (TSC) on cerebral ischemic reperfusion injury in high-fat dietinduced obese mice and the effect of early high dose pioglitazone injection–derived bloodglucose control against the cerebral vascular remodeling and aggravation of cerebralischemic injuryin diabetic db/db mice. TSC is a trans-sodium salt compound. It changesthe hydrogen bound among water molecules in fluid that promotes small size molecules(like oxygen and glucose) to diffuse in the fluid. It has been proved that TSC inducesneuroprotection in healthy rats and rabbits. Since its mechanism-affecting hydrogen bondbetween water molecules-does not involve affecting any pathophysiological changesinduced by high fat diet, we speculate that it could also provide neuroprotection againstcerebral ischemic reperfusion injury in obese mice and tested the theory in the second partof this study.Part One: The Effect of High Fat Diet on Cerebral VesselRemodeling and Ischemic Brain Injury in Mice and Role ofMMP-9in these changesExperiment One: Effect of High Fat Diet on Cerebral VascularRemodeling and Ischemic Brain Injury in CD1Mice Objectives To observe the effect of high fat diet on the weight, blood glucose, bloodlipid level changes and cerebral vascular remodeling and to compare the differences ofbrain ischemia reperfusion injury between mice fed on regular diet and high fat diet.Methods Six week old CD1mice were given regular diet (RD, n=9) or high fat diet(HF, n=9) for10weeks. Body weight, blood glucose and glycosylated hemoglobin(HbA1c) of all animals were evaluated before sacrificed for vascular casting. Number ofcollateralsbetween middle cerebral artery (MCA) and anterior cerebral artery (ACA) orposterior cerebral artery (PCA), tutoriosity index of MCA middle size branches andinterior diameter of MCA root were calculated. Another two groups of animals (n=8each)were perfused at age16wk and brains were processed intocoronary paraffin slices andimmunofluorescence staining for vWF factor (an marker for vascular endothelia cells) wasperformed to observe microvascular density in cerebral cortex and wall thickness of MCAvessel root. The third set of animals (n=11for RD and n=10for HF group) were given90min middle cerebral artery occlusion and reperfusion injury at16wk old. Brain edema,hemorrhagic transformation and infarct volume ratio were evaluated at72h afterreperfusion. Blood lipid profile was tested with blood collected just before sacrifice. Andcorrelations of blood lipid profile with brain edema index or with infarct volume ratiowere evaluated.Results High fat diet could induce obesity, hyperlipidemia and hyperglycemia in CD1mice. Number of collaterals didn’t change but tutoriosity of MCA branches was increasedin HF group. The interior diameter of MCA root was thinner and vessel wall was thicker.HF animals also have increased density of microvessels in the cerebral cortex andaggravated brain ischemia reperfusion injury. Edema index, HT rate and infarct volumeratio were higher in HF animals and motor coordinate function was poorer (P<0.05vs.RD). Brain edema index and infarct volume ratio were positively correlated to blood LDLlevel, LDL/cholesterol ratio and LDL/HDL ratio.Conclusions High fat diet could induce obesity, hyperlipidemia and hyperglycemia in CD1mice. It could also induce cerebral vascular remodeling and more severe brainischemic injury. And the level of brain ischemic injury was correlated to blood lipidcontent.Experiment Two: Role of MMP-9in cerebral vascular remodeling andischemic injury change in high fat diet fed mice Objectives To explore the mechanism that cause cerebral vascular remodeling andbrain ischemic injury aggravation in high fat diet fed mice.Methods Six wk old CD1mice were fed on regular diet (RD) or high fat diet (HF) for10weeks before middlecerebral artery occlusion (Con) or sham surgery (Sham, n=6-11).At6h after reperfusion or sham surgery, mice were perfused and cerebral cortex wasdissected as frontal1(Fr1, penumbra), cortex and striatum (ischemic core). Western blotwas employed to assess MMP-9expression as well as claudin-5and occluding expressionin whole cell lysates. We also employed zymography to evaluate MMP-2and MMP-9activity in the cytoplasm. To observe the role of MMP-9in the change of cerebral vascularstructure and the severity of ischemic brain injury, we employed MMP-9-/-mice and theirgene background animal-C57BL/6J mice. The effect of high fat diet on body weight,blood lipid, blood glucose, HbA1c as wellas cerebral vascular remodeling and brainischemic reperfusion injury was evaluated in both kinds of animals. We also tested theeffect of high fat diet on brain IgG leakage at24h after reperfusion in C57BL/6J mice.Results High fat diet could increase MMP-9activity in both ischemic brain and shambrain tissue without affecting the expression of claudin-5and occludin. High fat diet alsoinduced weight gain, hyperglycemia and lipid dysregulation in C57BL/6J and MMP-9-/- animals. In C57BL/6J mice, high fat diet affects the tutoriosity of MCA branches and itsinterior diameter as it did in CD1mice. Brain edema, IgG leakage, HT, infarct volume andneurologic function were also deteriorated in high fat diet C57BL/6J mice compare totheir lean mates. Although body weight, blood glucose and blood lipid were elevated inHF MMP-9-/-animals, cerebral vascular remodeling and brain damage aggravation werenot observed in these animals compared to their lean mates fed with RD.Conclusions These results suggest that HF diet induces cerebral vascular remodelingand worsens neurological outcome after transient focal brain ischemia. MMP-9activationplays a critical role in these HFdiet induced changes.Part2: Trans-sodium crocetinate provides neuroprotectionagainst cerebral ischemia reperfusion in obese mice Objectives Trans-sodium crocetinate (TSC) is a novel synthetic carotenoid compoundthat improves the diffusion of small molecules including oxygen in solutions. TSCprovides neuroprotection in healthy rats and rabbits. In this study, we determine whetherTSC could provide neuroprotection in obese mice induced by chronic feeding withhigh-fat diet.Methods Sixteen-week old male mice fed with high fat diet (45%caloric supplied byfat) for the last10weeks were subjected to a90-min middle cerebral arterial occlusion(MCAO). They received TSC by two boluses through a tail vein at10min after the onsetof MCAO and reperfusion, respectively, with the total dose at0.14,0.28and0.7mg/kg orby a bolus-infusion-bolus strategy with a total dose of0.14mg/kg during MCAO.Neurological outcome was evaluated at72h after MCAO. Brain tissues were harvested at 24h after MCAO to measure nitrotyrosine containing proteins,4-hydroxy-2-nonenal,matrix metalloproteinase (MMP)-2activity, MMP-9activity and expression, andinflammatory cytokine expressions. MMP-9localization after cerebral ischemia was alsoevaluated with immunofluorescence staining.Results TSC given by the two-bolus strategy did not improve neurological outcome.The bolus-infusion-bolus strategy significantly reduced the hemorrhagic transformationrate, brain edema and infarct volume. Neurological functions were also improved in TSCtreated animals. TSC reduced nitrotyrosine containing proteins, MMP-9activity andexpression, as well as inflammatory cytokines in the ischemic brain tissues. MMP-9wasmainly expressed in neurons, microglia and vascular endothelia cells in the ischemiccerebral cortex.Conclusions0.14mg/kg TSC delivered by the bolus-infusion-bolus strategy improvesneurological outcome after transient focal brain ischemia in obese mice. This protectionmay be achieved through oxygen diffusion and then reduction of oxidative stress, MMP-9activity, and inflammatory cytokines in the ischemic brain tissues.Part3: Type2diabetes causes cerebral vascular remodeling,brain ischemic injury aggravation and the reversal effect ofearly treatment with pioglitazone Objectives To observe whether different age of db/db mice developed cerebralvascular remodeling and has worsen neurologic outcome after ischemic stroke compare totheir same age gene back ground (C57BLKS) animals, and the effect of early treatmentwith large dose of pioglitazone on those changes. Methods Nine or fifteen wk old db/db mice and C57BLKS mice (db+) were tested forbody weight, blood glucose (BG) and HbA1c before sacrificed for cerebral vasculaturecasting (n=7-10). MCA collaterals with ACA and PCA were calculated, tutoriosity ofMCA branches were evaluated and internal diameter of MCA root was measured. Then100mg/kg pioglitazone (Actos) or its vehicle DMSO were injected intraperitoneally everyother day for3days to two separate groups of db/db mice since age5wk after the onset ofBG elevating (fasting BG>120mg/dl or random BG>200mg/dl) to assess the treatmenteffect of pioglitazone on vascular remodeling. Another four groups of animals (db+,db++A, db/db, db/db+A) were used to undergo a45min MCAO at age9wk andneurologic outcomes including neurologic coordination function (Rota-rod), HT rate,brain edema index and infarct volume ratio were assessed after brain ischemia.Results Nine wk old type2diabetic mice are obese and hyperglycemia compare totheir same age db+mates. Number of collaterals of cerebral vascular didn’t change indb/db mice compared to db+but tutoriosity of MCA branches was increased and interiordiameter of MCA root was smaller in db/db mice. Prolongation of diabetic status from9wk old to15wk old didn’t further affect number of collaterals between MCA and ACAor PCA. Pioglitazone treatment lowered the blood glucose and HbA1c level in db/db+Amice without affecting body weight or blood lipid profile. Pioglitazone also suppressed thedevelopment of cerebral vascular remodeling. Nine wk old db/db mice has more severeischemic brain damage in all the parameters evaluated and the aggravation was reversedby early treatment with pioglitazone.Conclusions Type2diabetes could induce cerebralvascular remodeling andaggravation of ischemic brain damage. Early treatment with pioglitazone to control bloodglucose level could suppress those changes.