The Establishment Of Evaluation Index System Of Rat Brain Edema Model Based On Living Imaging And Mechanism Of Borneol On The Improvement Of Brain Edema Induced By Lipopolysaccharide In Rats

Background The animal model of brain edema is an important carrier for studying the causes of brain edema in clinic.At present,the evaluation indexes of animal models of infectious cerebral edema are mostly prepared by injection of lipopolysaccharide to detect brain water content which need to kill the animals or to just detect body temperature and autonomous activities which pertinence and reliability are poor,lack of evaluation system.As a non-invasive method for quantitative detection of morphological,living imaging has not been applied to the study of animal models of brain edema.The famous Chinese medicine borneol has modern pharmacological effects such as anti-inflammatory and anti-brain edema.In recent years,it has been found that the clathrin and caveolin of cerebrovascular endothelial cells are involved in the degradation of tight junction proteins between cerebral vascular endothelial cells,thereby enhancing the permeability of the blood-brain barrier and leading to cerebral edema,which called the endocytosis of brain vascular endothelial cells.However,whether borneol can regulate endocytosis and whether to protect the blood-brain barrier by endocytosis has not been reported yet.Purpose To established model induced by lipopolysaccharide evaluation index system based on living imaging.To observe the mechanism of borneol on the brain edema induced by lipopolysaccharide is related to the protection of blood-brain barrier and the inhibition of endocytosis of cerebral vascular endothelial cells.Methods 1.Modeling method for examining and improving rat model of brain edema induced by lipopolysaccharide Male Sprague-Dawley rats were injected with lipopolysaccharide to study the modeling conditions include the lipopolysaccharide dosage(5 mg·kg-1,10 mg·kg-1,20 mg·kg-1),the injection(tail vein injection,intraperitoneal injection),the modeling time(6 h,12 h,24 h,36 h),and the number of injections(single,twice,three times),with brain water content as a test index,and then the brain edema model induced by lipopolysaccharide was optimized.2.The Method for establishing evaluation index system of brain edema rat model based on living imaging To observe the following indicators and screen the criteria for determining the success of modeling: 24-hour video recording method is used to record whether the animal has: a.body hair straight,lack of spirit;b.bow back buried action signs;To observe within 18 h if there are: c.diarrhea;d.the eyes,mouth and nose bleeding;e.At 0.5 h,1 h,3 h,6 h,12 h,and 24 h,the body temperature was measured using a rat thermometer to plot the body temperature curve.f.The FX7 in vivo imager is used to detect of average fluorescence intensity of the head at 18 h 3.The study of borneol improving effect on brain edemas of test animals and the administration group After modeling for 18 h in the above method,the model rats were randomly divided into model group,Angong Niuhuang Pill 270 mg·kg-1 group,and borneol according to the method of grasping sputum.100 mg·kg-1 group,borneol 200 mg·kg-1 group,borneol 400 mg·kg-1 group,10 rats in each group,another 10 rats are in the normal control group After 24 hours of modeling,Angong Niuhuang Pill group was intragastrically administered with 270 mg·kg-1 Angong Niuhuang Pill,and three doses of borneol were intragastrically administered with 100,200,400 mg·kg-1 borneol,the control group and the model group were intragastrically administered with an equal volume of 0.5% sodium carboxymethylcellulose,and were taken 1 hour after gavage.4.Observation index of borneol on brain edema and the determination method After 30 min of intragastric administration in each group,2.3 mg·kg-1 indocyaninegreen was injected into the tail vein,and the fluorescence of the brain was observed by in vivo imaging to calculate the average fluorescence.After 1 h of intragastric administration,the animals were divided into 4 batches and tested separately:(1)The heart was perfused with paraformaldehyde to take the brain and prepare the paraffin section to observe of its tissue morphology by HE staining.(2)The heart was perfused with 2.5% glutaraldehyde,and the brain was taken and prepared for electron microscopy.The degree of edema around the brain microvascular was observed.(3)The heart is perfused with normal saline,and then perfused with 4% Evans blue(mg/ml)75 ml,then perfused with normal saline,and the brain is divided into two halves,called wet weight: The left brain was freeze-dried for 12 h and then weighed dry,and the dry and wet weight method was used to calculate brain water content;The right brain was chopped,and 350 ?l of formamide was added per 100 mg of brain tissue,and leached at 56 ° C for 12 h,then the supernatant was taken for absorbance.(4)The brain tissue was taken and the expression of tumor necrosis factor alpha(TNF-?)and interleukin-6(IL-6)was determined by enzyme-linked immunosorbent assay.Aquaporin 4(AQP4)and inducible nitric oxide synthase(i NOS)were detected by immunoblotting.The expression of nitric oxide(NO)was determined by nitrate reductase method 5.The Observation index of borneol on blood-brain barrier and its determination method(1)Taking the above electron microscopy,it was observed of the morphology,organelle and tight connection integrity of brain endothelial cells and astrocytes by transmission electron microscopy.(2)The brain tissue was taken and the expression of the tight junction protein claudin-5,occluding,and ZO-1 was determined by immunoblotting.6.The observation index of borneol on endocytosis of cerebral vascular endothelial cells and its determination method(1)Taking the above electron microscopy,it was observed of the number and volume of endocytic vesicles in cerebrovascular endothelial cells by transmission electron microscopy(2)The brain tissue was taken and the expression of the endocytic protein caveolin 1,CHC,Autophagy-associated protein LC3,becline 1,p-Akt,Akt,p-ERK 1/2,ERK 1/2 was determined by immunoblotting.The expression of p-MEK 1/2,MEK 1/2,MMP-2,MMP-9 was determined by enzyme-linked immunosorbent assay.Results 1.The lipopolysaccharide-induced brain edema rat model improved successfully Compared with the normal control group,male rats were injected intraperitoneally with lipopolysaccharide 5 mg·kg-1 for 24 h at 0 and 12 h,and the brain water content was significantly increased(P<0.05).,indicating successful modeling.2.The evaluation index system of rat model of cerebral edema based on living imaging was successfully constructed.After trial and screening,it is determined that the following(1),(2),and(3)items are met and any of(4),(5),and(6)is satisfied,which is regarded as successful modeling:(1)body hair straight,lack of spirit;(2)diarrhea;(3)living body imaging head average Fluorescence intensity is not lower than 3800;(4)after 0.5 h after modeling,the body temperature is higher than before modeling,3 h body temperature is lower than before modeling;(5)eyes,nose and nose can be seen bleeding;(6)have arched back head movement signs.3.Borneol can significantly improve the degree of brain edema induced by lipopolysaccharide in model rats.(1)Modeling degree: Compared with the model,the mean fluorescence intensity difference in the brain of rats with borneol 200 and 400 mg·kg-1(post-dose-administration)significantly reduced(both P < 0.05).(2)Brain water content: Compared with the model,the brain water content of rats in the borneol 400 mg·kg-1 group was significantly decreased(P<0.05).(3)Brain pathological morphology: Compared with the model group,the degree of brain edema in the borneol group improved to varying degrees.(4)Blood-brain barrier permeability: Compared with the model,the content of Evans blue in the brain tissue of rats with borneol 100,200,400 mg·kg-1 was significantly increased(P<0.05).(5)Indicative molecular markers of cerebral edema: Compared with the model,the TNF-?,IL-6,AQP4,i NOS and NO content in the brain tissue of rats with borneol was significantly decreased(all P<0.05 or P<0.01).4.Borneol can protect brain blood edema model rat blood brain barrier(1)Blood brain barrier morphology: Compared with the model group,the extent of tight junction open and the degree of astrocyte edema of the borneol group were alleviated.(2)Tightly connected observation indicators: compared with the model group,borneol group of brain tissue of content of claudin-5,occluding and ZO-1 was significantly increased(both P<0.05).5 Borneol can significantly inhibit the endocytosis of endothelium in rat brain edema model.(1)The morphological observation of endocytic vesicles: Compared with the model group,the number of endocytic vesicles formed by endothelial cells in the borneol group was reduced.(2)The observation of endocytic protein: Compared with the model group,the caveolin-1 content in the brain tissue of the rats in the borneol group was significantly decreased(P<0.01);the CHC content in the brain tissue of the rats in the 400 mg·kg-1 borneol group was significantly increased(P<0.05).(3)The observation of LC3 and becline 1 protein: The LC3 and becline 1 content in the brain tissue of the rats in the borneol group was significantly decreased(P<0.05).(4)The observation of caveolin 1—Akt/MEK/ERK—MMP-9 passageway:The p-Akt/Akt,p-ERK/ERK,p-MEK/MER,MMP-2 and MMP-9 content in the brain tissue of the rats in the borneol group was significantly decreased(all P<0.05 or P<0.01).Conclusions 1.The improved lipopolysaccharide modeling method model was successful.Male SD rats were injected intraperitoneally with 10 mg·kg-1 lipopolysaccharide for 24 h,and the brain edema model was successfully constructed.The model not only increased brain water content,blood-brain barrier permeability and conventional brain edema,but also showed that the average fluorescence intensity of the brain was significantly increased,the caveolin-1,CHC,LC3 and becline 1 content was significantly increased,and the claudin-5,occludin and ZO-1 content was significantly increased.reduce.2.The established evaluation criteria for rats with cerebral edema induced by lipopolysaccharide.The standard is as follows: a.body hair straightening,lack of spirit;b.diarrhea;c.average fluorescence intensity of the living imaging head is not less than 3800;d.0.5 h after modeling is higher than before modeling while 3 h is lower;e.Bleeding can be seen in the eyes,nose and nose;f.There are signs of movement of the head behind the bow.It is necessary to satisfy a,b,and c at the same time,and satisfy any one of d,e,and f,which is regarded as successful modeling.3.Borneol can significantly improve the degree of cerebral edema in model rats,including the reduction of brain edema under light microscope and transmission electron microscope,significantly reducing brain water content,AQP4 expression,significantly reducing the expression of TNF-?,IL-6,i NOS,extremely significant reducing brain tissue the content of NO.4.Borneol can resist brain edema by inhibiting endocytosis and its downstream pathways.Borneol can protect the blood-brain barrier structure and up-regulates the expression of tight junction protein claudin-5,occludin,and ZO-1.The mechanism may be related to inhibition of endocytosis,down-regulation of endocytosis,inhibition of caveolin 1-Akt/MEK/ERK-MMP-9 pathway,thereby reversing the degradation of tight junction proteins under pathological conditions.