Reduction of Brain Injury After Stroke in Hyperglycemic Rats via Fasudil Pretreatment

Abstract

Abstract:Diabetes is usually associated with cerebrovascular disease, especially stroke. In practice, fasudil is widely accepted to be applied for the treatment of vascular disease. This article demonstrates the study concentrating on the effects of fasudil pretreatment on the prognosis of diabetic stroke. 250—300 g Sprague- Dawley rats were randomly divided into three groups, non-diabetic stroke group, diabetic stroke group, and fasudil pretreatment group. The rats of diabetes group were treated with intraperitoneal injection of streptozotocin (60 mg/kg), in the meantime the same dose of citrate buffer was injected into those of the control group. The rats of the fasudil group received daily fasudil intraperitoneal injection at 10 mg/kg for three consecutive weeks. After four weeks, all the rats of the experimental group were treated with middle cerebral artery occlusion for 90 min. After sacrifice, the fresh brain samples were collected for following experiments, including infarct volume, edema volume, blood-brain barrier (BBB), which were detected by immunohistochemistry. Inflammatory factors were examined by real-time polymerase chain reaction (RT-PCR) using tissue Ribonucleic Acid (RNA). The concentration of blood glucose is 15 mmol/L or more, which proved that the diabetes model was a success. Fasudil pretreatment decreases the percentage of stroke mortality of diabetes from 43.75% to 31.25%, while the infarction volume decreases from 52.95% ± 12.7% to 45.97% ± 6.7%. Gap formation of tight junction and Immunoglobulin G (IgG) leakage were reduced (P < 0.05), and the expression of inflammatory factors decreases (P < 0.05) in fasudil pretreatment after diabetic stroke. Diabetes aggravates the mortality of cerebral ischemic rats. Prolonged fasudil pretreatment can reduce mortality of diabetic stroke, decrease cerebral infarction volume and undermine inflammatory factors expression, and protect the BBB.

Key words:diabetes| stroke| fasudil pretreatment| blood-brain barrier (BBB)

摘要：Diabetes is usually associated with cerebrovascular disease, especially stroke. In practice, fasudil is widely accepted to be applied for the treatment of vascular disease. This article demonstrates the study concentrating on the effects of fasudil pretreatment on the prognosis of diabetic stroke. 250—300 g Sprague- Dawley rats were randomly divided into three groups, non-diabetic stroke group, diabetic stroke group, and fasudil pretreatment group. The rats of diabetes group were treated with intraperitoneal injection of streptozotocin (60 mg/kg), in the meantime the same dose of citrate buffer was injected into those of the control group. The rats of the fasudil group received daily fasudil intraperitoneal injection at 10 mg/kg for three consecutive weeks. After four weeks, all the rats of the experimental group were treated with middle cerebral artery occlusion for 90 min. After sacrifice, the fresh brain samples were collected for following experiments, including infarct volume, edema volume, blood-brain barrier (BBB), which were detected by immunohistochemistry. Inflammatory factors were examined by real-time polymerase chain reaction (RT-PCR) using tissue Ribonucleic Acid (RNA). The concentration of blood glucose is 15 mmol/L or more, which proved that the diabetes model was a success. Fasudil pretreatment decreases the percentage of stroke mortality of diabetes from 43.75% to 31.25%, while the infarction volume decreases from 52.95% ± 12.7% to 45.97% ± 6.7%. Gap formation of tight junction and Immunoglobulin G (IgG) leakage were reduced (P < 0.05), and the expression of inflammatory factors decreases (P < 0.05) in fasudil pretreatment after diabetic stroke. Diabetes aggravates the mortality of cerebral ischemic rats. Prolonged fasudil pretreatment can reduce mortality of diabetic stroke, decrease cerebral infarction volume and undermine inflammatory factors expression, and protect the BBB.

关键词:diabetes| stroke| fasudil pretreatment| blood-brain barrier (BBB)

CLC Number:

R 743.31

LIU Jianyu (刘健宇), MU Zhihao (木志浩), WANG Liping (王丽萍), WEN Ruoxue (闻若雪), WANG Yongting (王永亭), YANG Guoyuan (杨国源), ZHANG Zhijun (张志君) . Reduction of Brain Injury After Stroke in Hyperglycemic Rats via Fasudil Pretreatment[J]. Journal of Shanghai Jiao Tong University (Science), 2019, 24(6): 723-731.

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[32]	PRAKASH R, LI W G, QU Z, et al. Vascularizationpattern after ischemic stroke is different in control versusdiabetic rats: Relevance to stroke recovery [J].Stroke, 2013, 44(10): 2875-2882.
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[48]	SIMPKINS A N, DIAS C, LEIGH R. Identification ofreversible disruption of the human blood-brain barrierfollowing acute ischemia [J]. Stroke, 2016, 47(9): 2405-2408.
[34]	YAN T, CHOPP M, CHEN J L. Experimental animalmodels and inflammatory cellular changes in cerebralischemic and hemorrhagic stroke [J]. Neuroscience Bulletin,2015, 31(6): 717-734.
[49]	GIBSON C L, SRIVASTAVA K, SPRIGG N, et al. Inhibitionof Rho-kinase protects cerebral barrier fromischaemia-evoked injury through modulations of endothelialcell oxidative stress and tight junctions [J].Journal of Neurochemistry, 2014, 129(5): 816-826.
[35]	LIUY, TANGGH, SUNYH, et al.The protective roleof Tongxinluo on blood-brain barrier after ischemiareperfusion brain injury [J]. Journal of Ethnopharmacology,2013, 148(2): 632-639.
[50]	FUJIIM, DURIS K, ALTAY O, et al. Inhibition of Rhokinase by hydroxyfasudil attenuates brain edema aftersubarachnoid hemorrhage in rats [J]. NeurochemistryInternational, 2012, 60(3): 327-333.
[51]	DEJANA E. Endothelial cell-cell junctions: Happytogether [J]. Nature Reviews Molecular Cell Biology,2004, 5(4): 261-270.
[36]	ZHANG Y Q,WU S J. Effects of fasudil on pulmonaryhypertension in clinical practice [J]. Pulmonary Pharmacology& Therapeutics, 2017, 46: 54-63.
[52]	WISPELWEY B, LESSE A J, HANSEN E J, etal. Haemophilus influenzae lipopolysaccharide-inducedblood brain barrier permeability during experimentalmeningitis in the rat [J]. The Journal of Clinical Investigation,1988, 82(4): 1339-1346.
[37]	HUANG J, LI Y N, TANG Y H, et al. CXCR4 antagonistAMD3100 protects blood-brain barrier integrityand reduces inflammatory response after focal ischemiain mice [J]. Stroke, 2013, 44(1): 190-197.
[53]	QUAGLIARELLO V J, WISPELWEY B, LONG WJ, et al. Recombinant human interleukin-1 inducesmeningitis and blood-brain barrier injury in the rat:Characterization and comparison with tumor necrosisfactor [J]. The Journal of Clinical Investigation, 1991,87(4): 1360-1366.
[38]	GASCHE Y, COPIN J C, CHAN P H. The role ofmetalloproteinases on blood-brain barrier breakdownafter ischemic stroke [M]//FEUERSTEIN G Z. Inlammationand stroke. Basel, Switzerland: Birkh¨auserBasel, 2001: 265-274.
[39]	LAZAREVIC-PASTI T, LESKOVAC A, VASIC V.Myeloperoxidase inhibitors as potential drugs [J]. CurrentDrug Metabolism, 2015, 16(3): 168-190.
[54]	DELI M A, DESCAMPS L, DEHOUCK M P, et al.Exposure of tumor necrosis factor-α to luminal membraneof bovine brain capillary endothelial cells coculturedwith astrocytes induces a delayed increaseof permeability and cytoplasmic stress fiber formationof actin [J]. Journal of Neuroscience Research, 1995,41(6): 717-726.
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[55]	BANKS W A, KASTIN A J. The interleukins-1α, -1β,and -2 do not acutely disrupt the murine blood-brainbarrier [J]. International Journal of Immunopharmacology,1992, 14(4): 629-636.
[56]	TARKOWSKI E, ROSENGREN L, BLOMSTRANDC, et al. Intrathecal release of pro- and antiinflammatorycytokines during stroke [J]. Clinical &Experimental Immunology, 1997, 110(3): 492-499.
[41]	ZAKRZESKA A, GROMOTOWICZ-POPWSKA A,SZEMRAJ J, et al. Eplerenone reduces arterial thrombosisin diabetic rats [J]. Journal of the Renin-Angiotensin-Aldosterone System, 2015, 16(4): 1085-1094.
[57]	ZHANG W D, SMITH C, SHAPIRO A, et al. Increasedexpression of bioactive chemokines in humancerebromicrovascular endothelial cells and astrocytessubjected to simulated ischemia in vitro [J]. Journalof Neuroimmunology, 1999, 101(2): 148-160.
[58]	HE Y, XU H S, LIANG L Q, et al. Antiinflammatoryeffect of Rho kinase blockade via inhibition of NF-κB activation in rheumatoid arthritis [J]. Arthritis &Rheumatism, 2008, 58(11): 3366-3376.
[42]	DEMIRY¨UREK S, KARA A F, C?ELIK A, et al. Effectsof fasudil, a Rho-kinase inhibitor, on myocardialpreconditioning in anesthetized rats [J]. EuropeanJournal of Pharmacology, 2005, 527(1/2/3): 129-140.
[59]	THORLACIUS K, SLOTTA J E, LASCHKE M W, etal. Protective effect of fasudil, a Rho-kinase inhibitor,on chemokine expression, leukocyte recruitment, andhepatocellular apoptosis in septic liver injury [J]. Journalof Leukocyte Biology, 2006, 79(5): 923-931.
[43]	RIKITAKE Y, KIM H H, HUANG Z H, et al. Inhibitionof Rho kinase (ROCK) leads to increased cerebralblood flow and stroke protection [J]. Stroke, 2005,36(10): 2251-2257.
[44]	VENNIN C, RATH N, PAJIC M, et al. TargetingROCK activity to disrupt and prime pancreatic cancerfor chemotherapy [J]. Small GTPases, 2017: 1-8. DOI:10.1080/21541248.2017.1345712.
[45]	ALLEN C, SRIVASTAVA K, BAYRAKTUTAN U.Small GTPase RhoA and its effector Rho kinase mediateoxygen glucose deprivation-evoked in vitro cerebralbarrier dysfunction [J]. Stroke, 2010, 41(9): 2056-2063.
[60]	CHENG T, PETRAGLIA A L, LI Z, et al. Activatedprotein C inhibits tissue plasminogen activatorinducedbrain hemorrhage [J]. Nature Medicine, 2006,12(11): 1278-1285.
[46]	THOMPSON B J, RONALDSON P T. Drug deliveryto the ischemic brain [J]. Advances in pharmacology,2014, 71: 165-202.
[61]	ZLOKOVIC B V. Remodeling after stroke [J]. NatureMedicine, 2006, 12(4): 390-391.
[47]	LATOUR L L, KANG D W, EZZEDDINE M A, et al.Early blood-brain barrier disruption in human focalbrain ischemia [J]. Annals of Neurology, 2004, 56(4):468-477.
[62]	KEMPURAJ D, THANGAVEL R, SELVAKUMAR G P, et al. Brain and peripheral atypical inflammatorymediators potentiate neuroinflammation and neurodegeneration[J]. Frontiers in Cellular Neuroscience,2017, 11: 216.
[48]	SIMPKINS A N, DIAS C, LEIGH R. Identification ofreversible disruption of the human blood-brain barrierfollowing acute ischemia [J]. Stroke, 2016, 47(9): 2405-2408.
[49]	GIBSON C L, SRIVASTAVA K, SPRIGG N, et al. Inhibitionof Rho-kinase protects cerebral barrier fromischaemia-evoked injury through modulations of endothelialcell oxidative stress and tight junctions [J].Journal of Neurochemistry, 2014, 129(5): 816-826.
[50]	FUJIIM, DURIS K, ALTAY O, et al. Inhibition of Rhokinase by hydroxyfasudil attenuates brain edema aftersubarachnoid hemorrhage in rats [J]. NeurochemistryInternational, 2012, 60(3): 327-333.
[51]	DEJANA E. Endothelial cell-cell junctions: Happytogether [J]. Nature Reviews Molecular Cell Biology,2004, 5(4): 261-270.
[52]	WISPELWEY B, LESSE A J, HANSEN E J, etal. Haemophilus influenzae lipopolysaccharide-inducedblood brain barrier permeability during experimentalmeningitis in the rat [J]. The Journal of Clinical Investigation,1988, 82(4): 1339-1346.
[53]	QUAGLIARELLO V J, WISPELWEY B, LONG WJ, et al. Recombinant human interleukin-1 inducesmeningitis and blood-brain barrier injury in the rat:Characterization and comparison with tumor necrosisfactor [J]. The Journal of Clinical Investigation, 1991,87(4): 1360-1366.
[54]	DELI M A, DESCAMPS L, DEHOUCK M P, et al.Exposure of tumor necrosis factor-α to luminal membraneof bovine brain capillary endothelial cells coculturedwith astrocytes induces a delayed increaseof permeability and cytoplasmic stress fiber formationof actin [J]. Journal of Neuroscience Research, 1995,41(6): 717-726.
[55]	BANKS W A, KASTIN A J. The interleukins-1α, -1β,and -2 do not acutely disrupt the murine blood-brainbarrier [J]. International Journal of Immunopharmacology,1992, 14(4): 629-636.
[56]	TARKOWSKI E, ROSENGREN L, BLOMSTRANDC, et al. Intrathecal release of pro- and antiinflammatorycytokines during stroke [J]. Clinical &Experimental Immunology, 1997, 110(3): 492-499.
[57]	ZHANG W D, SMITH C, SHAPIRO A, et al. Increasedexpression of bioactive chemokines in humancerebromicrovascular endothelial cells and astrocytessubjected to simulated ischemia in vitro [J]. Journalof Neuroimmunology, 1999, 101(2): 148-160.
[58]	HE Y, XU H S, LIANG L Q, et al. Antiinflammatoryeffect of Rho kinase blockade via inhibition of NF-κB activation in rheumatoid arthritis [J]. Arthritis &Rheumatism, 2008, 58(11): 3366-3376.
[59]	THORLACIUS K, SLOTTA J E, LASCHKE M W, etal. Protective effect of fasudil, a Rho-kinase inhibitor,on chemokine expression, leukocyte recruitment, andhepatocellular apoptosis in septic liver injury [J]. Journalof Leukocyte Biology, 2006, 79(5): 923-931.
[60]	CHENG T, PETRAGLIA A L, LI Z, et al. Activatedprotein C inhibits tissue plasminogen activatorinducedbrain hemorrhage [J]. Nature Medicine, 2006,12(11): 1278-1285.
[61]	ZLOKOVIC B V. Remodeling after stroke [J]. NatureMedicine, 2006, 12(4): 390-391.
[62]	KEMPURAJ D, THANGAVEL R, SELVAKUMAR G P, et al. Brain and peripheral atypical inflammatorymediators potentiate neuroinflammation and neurodegeneration[J]. Frontiers in Cellular Neuroscience,2017, 11: 216.