| ObjectiveThe complexity and heterogeneity of ischemic stroke as well as the associated co-morbidities may render neuroprotective drugs less efficacious in clinical practice.Therefore,development of targeted therapies to specific patient subsets has become a high priority in translational stroke research.Type 2 diabetes(T2D)comprises various general metabolic perturbations including: hyperglycemia,dyslipidemia and insulin resistance.Ischemic stroke with type 2 diabetes(T2D)have a nearly double mortality rate and worse neurological outcomes.However,clinical trials of post-stroke glycemic control have not been successful in improving long-term neurological outcomes,which indicates that preservation of metabolic homeostasis after stroke might be necessary but insufficient for effectively treating T2 D ischemic stroke.More recent data demonstrate that T2 D also exerts detrimental effects via other mechanisms involving increased blood-brain barrier(BBB)permeability,enhanced neuroinflammation,neurodegeneration and cognitive impairment.Peroxisome proliferator-activated receptor gamma(PPAR?)is a member of nuclear receptor superfamily.It acts as an important ligand-activated transcription factor not only mediating glucose and lipid metabolism,but also being widely expressed in various cell types in the central nervous system(CNS)with essential anti-inflammatory function.Fibroblast growth factor 21(FGF21)is an important endocrine factor with potent metabolic regulatory,tissue protective and repair functions partially via regulating the activity of PPAR?.In the present study we tested our hypothesis that recombinant human fibroblast growth factor 21(rFGF21)administration is beneficial for improving neurological outcomes of ischemic stroke with T2 D.MethodsLeptin receptor-deficient type 2 diabetes db/db and non-diabetic genetic control db/+ mice were subjected into permanent focal ischemia of distal middle cerebral artery occlusion followed by 90 min ipsilateral common carotid artery occlusion(dMCAO).We administered subcutaneously rFGF21 or saline at 6 h after stroke,followed by twice daily subcutaneous injections with an interval of,at least,8 h until sacrifice.We performed various examinations after dMCAO and they can be divided into two major parts:1.A total of 36 db/db mice were randomly divided into three groups:sham operated group(n=12),saline treated control group(n=12)andrFGF21 treated group(n=12).A total of 24 db/+ mice were randomly divided into 2 groups: sham operated group(n=12)and saline treated control group(n=12).We monitored blood glucose level and body weight of each mouse at day 0(pre-stroke baseline),1,3,5,7,10,14 after stroke.We also measured pre-stroke baseline and 14-day post stroke levels of glycated hemoglobin.We harvested serum samples for measuring insulin and adiponectin concentration.Brain samples were used for quantifying infarct volume by hematoxylin-eosin staining(HE),degree of myelin content by Luxol fast blue staining(LFB)and white matter injury and integrity by immunofluorescence staining(IF)of myelin basic protein(MBP)and non-phosphorylated neurofilament H(SMI-32),respectively.We performed adhesive removal test,grip strength test,foot fault test and Y-maze test at day 0,1,3,5,7,14 after stroke of sensorimotor and cognitive function examination.In another set of experiment,we tested the potential effects of rFGF21 treatment in non-diabetic db/+ mice after stroke using blood glucose,body weight and infarct volume measurement as well as neurological deficit examinations.2.A total of 54 db/db mice were randomly divided into three groups:sham operated group(n=18),saline treated control group(n=18)and rFGF21 treated group(n=18).A total of 36 db/+ mice were randomly divided into 2 groups: sham operated group(n=18)and saline treatedcontrol group(n=18).Peri-infarct brain tissue samples were collected for detecting day 3 post-stroke PPAR?-DNA binding activity and day 7post-stroke mRNA levels of pro-inflammatory including tumor necrosis factor-?(TNF-?),interleukin-1?(IL-1?),Toll-like receptor(TLR),C-C motif chemokine ligand 2(CCL2)and anti-inflammatory factors including IL-4,IL-10,insulin-like growth factor-1(IGF-1)and transforming growth factor-?(TGF-?).Whole brains were also obtained for quantification of M1-like and M2-like microglia in peri-infarct area by using immunofluorescence staining.Results1.Compared to non-diabetic db/+ mice,diabetic db/db mice experienced more severe systemic metabolic dysregulation,exacerbated neurological dificits,enlarged infarct size and aggravated loss of white matter integrity.rFGF21 treatment significantly improved systemic metabolic dysregulation and neurological deficits,reduced infarct size and alleviated white matter injury and integrity loss.Long-term rFGF21 treatment did not cause hypoglycemia or low body weight in non-diabeitc db/+ mice after stroke.However,neither infarct volume nor neurological deficit improvement was found after rFGF21 treatment in non-diabetic db/+ mice.2.At day 3 post-stroke,nuclear PPAR?-DNA binding activity wassignificantly reduced in peri-infarct area in both db/+ and db/db mice.This reduction was more prominent in db/db mice.At day 7 post-stroke,in peri-infarct area,significantly higher number of M1-like microglia,lower number of M2-like microglia as well as elevated mRNA levels of pro-inflammatory factors were detected in diabetic db/db mice.rFGF21 treatment rescued PPAR?-DNA binding activity,reduced number of M1-like microglia,increased M2-like microglia,lowered pro-inflammatory factors while increased anti-inflammatory factors.ConclusionTaken together,these results suggest rFGF21 might be a novel and potent candidate of the disease-modifying strategy for treating ischemic stroke with T2 D.The potential mechanisms of rFGF21 may in part consist of potent systematic metabolic regulation and PPAR?-activation promotionassociated anti-inflammatory roles in the brain. |
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