| Objectives:1.To explore the water content of brain tissue at 8 h,24 h,2 d,3 d,5 d and 7 d after traumatic brain injury(TBI)and determine the peak of brain edema;2.To explore the intervention effect of cannabinoid(CBD)gradient dose(5 mg/kg,10 mg/kg,20 mg/kg,30 mg/kg)on TBI rats,and to determine the optimal intervention dose of CBD;3.To explore the effect of the best intervention dose of CBD on the permeability of blood brain barrier(BBB)in TBI rats.Methods:(1)Using the modified "Feeney free fall impact method to establish the rat TBI model",they were randomly divided into groups at 8 h,24 h,2 d,3 d,5 d,7 d after TBI,and a Sham group(n=3),by calculating the dry/wet weight ratio to determine the peak period of cerebral edema.(2)The peak period of cerebral edema was 24 hours after TBI.Sham group,TBI+vehicle group,TBI+CBD 5 mg/kg group,TBI+CBD 10 mg/kg group and TBI+CBD 20 mg/kg group(n=3)were set to explore the best intervention dose of CBD,through behavior test,morphological experiment,molecular biology and ELISA experiments.(3)After the optimal intervention dose of CBD was determined to be 10 mg/kg,Sham group,TBI+vehicle group,and TBI+CBD 10 mg/kg group(n=3)were set to explore the effect of CBD 10 mg/kg on the permeability of BBB after TBI,via morphological experiment,molecular biology and Evans Blue staining experiments.Results:1 Explore the peak period of brain edemaThe results of dry/wet weight ratio showed that,compared with Sham group,the water content of brain tissue was significantly increased at 8 h,24 h,2 d,3 d,5 d and 7 d after TBI(P<0.05).The brain water content reached the peak at 24 h after TBI.2 Explore the best intervention dose of CBD2.1 The modified neurological severity scores(mNSS)(1)Compared with Sham group,the mNSS of TBI group was significantly higher(P<0.01).(2)Compared with TBI group,in different doses of CBD intervention groups,the mNSS of CBD 10 mg/kg group was the lowest(P<0.05).2.2 ELISA assay(S100-β is a marker of brain damage)(1)Compared with Sham group,the content of S100-β in TBI group was significantly increased(P<0.001).(2)Compared with TBI group,in different doses of CBD intervention groups,the content of S100-β in CBD 10 mg/kg group was the lowest(P<0.001).2.3 Dry/wet weight ratio(1)In Sham group,there was no significant change in the water content between left brain tissue(contralateral side to the injury)and right brain tissue(injured side).(2)In TBI group,the water content of the ipsilateral side to the injury was higher than that of the contralateral side to the injury(P<0.05).(3)In the right(injured side)brain tissue of rats,①Compared with Sham group,the water content of brain tissue in TBI group was significantly increased(P<0.05);②Compared with TBI group,in different doses of CBD intervention groups,the water content of brain tissue in CBD 10 mg/kg group was the lowest(P<0.05).2.4 HE staining(the injured cortex of rats)(1)In Sham group,neurons were in normal shape,the staining was uniform,the structure was complete,the outline was clear,the cytoplasm was rich,the nucleus was clear,large and round at the center of cells,and the nucleolus was obvious.(2)Compared with Sham group,the neurons in TBI group showed pathological changes,such as cell swelling,deep staining of nucleus,the outline was unclear,cytoplasmic dissolution and vacuoles.(3)Compared with TBI group,there were still some pathological changes such as swelling of neuronal cell body,deep staining of nucleus and cytolysis in CBD 5 mg/kg intervention group,and slight swelling in CBD 10 mg/kg and CBD 20 mg/kg intervention groups.2.5 Nissl staining(the injured cortex of rats)(1)In Sham group,the neurons were in normal shape,the outline was clear,the Nissl bodies were abundant,and evenly stained.The nucleus was light purple,round,and located in the center of the cells,with obvious nucleoli.(2)Compared with Sham group,the nucleus and nucleolus of neurons in TBI group disappeared,the cell body condensed into a triangle,and a large number of pyknotic necrosis appeared(P<0.01).(3)Compared with TBI group,in different doses of CBD intervention groups,the number of pyknotic necrotic neurons in CBD 10 mg/kg group was the least(P<0.01).2.6 Single immunofluorescence staining(the injured cortex of rats)(1)The marker of astrocyte,GFAP.1)Morphological changes of GFAP positive cells,①In Sham group,the astrocytes positive expression had smaller cell bodies and elongated processes;②Compared with Sham group,the astrocytes in TBI group had bigger cell body;more protrusions and coarser processes,which were activated;③Compared with TBI group,in different doses of CBD intervention groups,the activation of astrocytes in the CBD 5 mg/kg intervention group was decreased,the activation of astrocytes in the CBD 10 mg/kg intervention group was much lower,the morphological changes of astrocytes in the CBD 20 mg/kg intervention group were similar to those in the CBD 10 mg/kg intervention group.2)The mean fluorescence intensity of GFAP positive expression.①The fluorescence intensity of GFAP in TBI group was higher than that in Sham group(P<0.05);②Compared with TBI group,in different doses of CBD intervention groups,the mean fluorescence intensity of GFAP in CBD 10 mg/kg group was significantly lower than that in TBI group(P<0.05).(2)Aquaporin AQP4.1)The morphological changes of AQP4 positive expression,①In Sham group,AQP4 positive expression was on the cell membrane,showing a hollow tubular structure;②Compared with Sham group,the morphology of AQP4 positive expression in TBI group had little change;③Compared with TBI group,the positive expression of AQP4 in different doses of CBD intervention groups was still hollow small tube.2)The mean fluorescence intensity of AQP4.①The fluorescence intensity of AQP4 in TBI group was higher than that in Sham group(P<0.05);②Compared with TBI group,in different doses of CBD intervention groups,the mean fluorescence intensity of AQP4 in CBD 10 mg/kg group was significantly lower than that in TBI group(P<0.05).2.7 Western blotting assay(the injured cortex of rats)(1)The protein expression of GFAP、AQP4、TNF-α and IL-1β,①Compared with Sham group,the expression of GFAP,TNF-α and IL-1β in TBI group was higher(P<0.05),he expression of AQP4 protein was not statistical significance(P>0.05);②Compared with TBI group,in different doses of CBD intervention groups,except TNF-α,the expression of GFAP,AQP4 and IL-1β in CBD 10 mg/kg group was the lowest(P<0.05).(2)The protein expression of Claudin-5 and Occludin,①Compared with Sham group,there was no significant difference in the protein expression of Claudin-5 and Occludin in TBI group(P>0.05);②Compared with TBI group,in different doses of CBD intervention groups,the protein expression of Claudin-5 and Occludin had no significant difference(P>0.05).2.8 RT-PCR assay(the injured cortex of rats)(1)The mRNA expression of GFAP、AQP4、TNF-α and IL-1β,①Compared with Sham group,the mRNA expression of GFAP、AQP4、TNF-α and IL-1β mRNA in TBI group was significantly increased(P<0.05);②Compared with TBI group,in different doses of CBD intervention groups,except TNF-α,the mRNA expression of GFAP,AQP4 and IL-1β in CBD 10 mg/kg group was the lowest(P<0.05).(2)The mRNA expression of Claudin-5 and Occludin,①Compared with Sham group,the mRNA expression of Claudin-5 and Occludin in TBI group decreased(P<0.05);②Compared with TBI group,the mRNA expression of Claudin-5 and Occludin in CBD 10 mg/kg group was the highest(P<0.05).3 Explore the improvement of CBD to BBB permeability3.1 Single immunofluorescence staining(the injured cortex of rats)(1)The marker of astrocyte,GFAP.1)The morphological changes of GFAP positive astrocytes were as follows,①In Sham group,astrocytes had small cell body,slender processes and foot plates attached to the vascular wall;②Compared with Sham group,astrocytes in TBI group were activated,with large cell body,more protrusions,swelling of foot plate at the end of protrusions,and rupture of adhesion to blood vessels;③Compared with TBI group,the activation of astrocytes in CBD 10 mg/kg group was decreased.2)The mean fluorescence intensity of GFAP.①the fluorescence intensity of GFAP in TBI group was higher than that in Sham group(P<0.05);②The fluorescence intensity of GFAP in CBD 10 mg/kg intervention group was lower than that in TBI group(P<0.05).(2)Tight junction protein Claudin-5.1)Morphological changes of Claudin-5 positive expression,①In Sham group,the positive expression of Claudin-5 was mostly linear;②Compared with Sham group,the positive expression of Claudin-5 in TBI group was broken in line and scattered in dots;③Compared with TBI group,the positive expression of Claudin-5 in CBD 10 mg/kg intervention group was restored to the normal line shape.2)The mean fluorescence intensity of Claudin-5.①the fluorescence intensity of Claudin-5 in TBI group was significantly weakened than that in Sham group(P<0.01);②The fluorescence intensity of Claudin-5 in CBD 10 mg/kg intervention group was significantly increased than that in TBI group(P<0.05).(3)Tight junction protein Occludin.1)Morphological changes of Occludin positive expression,①In Sham group,Occludin positive expression appeared between cells in a linear shape;②Compared with Sham group,the morphology of Occludin positive expression in TBI group changed,the line shape was no longer continuous,and it was broken or dotted;③Compared with TBI group,the morphology of Occludin positive expression was restored in CBD 10 mg/kg intervention group.2)The mean fluorescence intensity of Occludin.①the fluorescence intensity of Occludin in TBI group was significantly weakened than that in Sham group(P<0.01);②The fluorescence intensity of Occludin increased in the CBD 10 mg/kg intervention group than that in TBI group(P<0.05).3.2 Double immunofluorescence staining(the injured cortex of rats)(1)The marker of astrocyte,GFAP.1)Morphological changes of GFAP positive cells,①In Sham group,GFAP positive astrocytes in blood vessels had small cell bodies,slender processes and foot plates attached to the vascular wall;②Compared with Sham group,astrocytes in TBI group were activated,with large cell body,more protrusions,swelling of foot plate at the end of protrusions,and rupture of adhesion on blood vessels;③Compared with TBI group,the activation of astrocytes in CBD 10 mg/kg group was decreased.(2)Aquaporin AQP4.1)The morphological changes of AQP4 positive expression,①In Sham group,AQP4 positive expression was on the cell membrane,showing a hollow tubular structure;②Compared with Sham group,the morphology of AQP4 positive expression in TBI group had little change;③Compared with TBI group,the positive expression of AQP4 in CBD 10 mg/kg group was still hollow and small tubular.(3)The mean fluorescence intensity of GFAP and AQP4 positive co-expression.①In Sham group,there was co-expression(yellow)of GFAP(green)and AQP4(red);②The co-expression of GFAP and AQP4 in TBI group was stronger than that in Sham group(P<0.01);③The co-expression of GFAP and AQP4 in CBD 10 mg/kg intervention group was weaker than that in TBI group(P<0.05).3.3 Western blotting assay(the injured cortex of rats)(1)The protein expression of GFAP、TNF-α and IL-1β,①the protein expression of GFAP and IL-1β in TBI group was higher than that in Sham group(P<0.05);②Compared with TBI group,the protein expression of GFAP、INF-α and IL-1β in CBD 10 mg/kg intervention group was significantly decreased(P<0.05).(2)The protein expression of Claudin-5 and Occludin,①Compared with Sham group,the protein expression of Claudin-5 and Occludin in TBI group was lower(P<0.05);②Compared with TBI group,the protein expression of Claudin-5 and Occludin increased in CBD 10 mg/kg intervention group(P<0.05).3.4 RT-PCR assay(the injured cortex of rats)(1)The mRNA expression of GFAP、TNF-α and IL-1β,①The mRNA expression of GFAP,TNF-α and IL-1β in TBI group was significantly higher than that in Sham group(P<0.05);②Compared with TBI group,the mRNA expression of GFAP,TNF-α and IL-1β in CBD 10 mg/kg intervention group were significantly decreased(P<0.05).(2)The mRNA expression of Claudin-5 and Occludin,①Compared with Sham group,the mRNA expression of Claudin-5 in TBI group had no significant difference(P>0.05),but the mRNA expression of Occludin was decreased(P<0.05);②Compared with TBI group,the mRNA expression of Claudin-5 and Occludin in CBD 10 mg/kg intervention group was significantly increased(P<0.05).3.5 Evans Blue(EB)staining(the injured cortex of rats)(1)The appearance of brain tissue was observed,there was no blue staining was found in the Control group;there was a little blue staining in Sham group;the blue staining was obvious in TBI group;the blue staining was obvious in the CBD 10 mg/kg intervention group.(2)The content of EB in the injured cortex was detected,①Compared with the Control group,the EB content in Sham group increased slightly,but there was no statistical significance(P>0.05);②Compared with Sham group,the content of EB in TBI group was significantly higher(P<0.01);③Compared with TBI group,the content of EB in CBD 10 mg/kg intervention group was significantly decreased(P<0.05).Conclusions:1.The peak of brain edema in TBI rats occurred 24 hours after TBI injury;2.In the gradient dose of CBD,CBD 10 mg/kg was the best intervention dose;3.CBD 10 mg/kg can improve the neural function defect,decrease the activation of astrocytes and increase the expression of tight junction protein in TBI rats,so as to improve the structural integrity and permeability of BBB and slow down the secondary brain edema after TBI. |
