The Study Of Tolvaptan On The Treatment Of Brain Edema After Intracerebral Hemorrhage In Rats And The Effect Of Iron Ions On The Viability Of Nerve Cells

Intracerebral hemorrhage refers to hemorrhage of the brain caused by cerebral vascular rupture caused by a variety of reasons,and is a common and frequently-occurring disease in the clinic.According to statistics,stroke is one of the leading causes of death and disability among the elderly in the world.It has risen to the second leading cause of death in the world.About 16 million people have their first stroke each year,resulting in nearly 6 million deaths.Intracerebral hemorrhage accounts for 10% to 30% of all strokes [1].Although the incidence of Intracerebral hemorrhage is lower than that of cerebral infarction,the mortality rate is much higher than the latter.With the advancement of medical technology,although the mortality rate of Intracerebral hemorrhage has decreased,the mortality rate in the acute phase can still be as high as 30% ~ 40%,and only 12% to 39% of the survivors have no residual disability,but many patients also make them basically lose their ability to work,causing a heavy burden on the patient's family.Although the research on Intracerebral hemorrhage has never stopped,the mechanism of its development has not been clear so far,and there are few methods for clinically selected treatment of Intracerebral hemorrhage.There is still no specific drug or surgical treatment to significantly improve the prognosis of patients.Therefore,it is of great significance to further study the mechanism and prevention of Intracerebral hemorrhage and explore new therapeutic targets for Intracerebral hemorrhage.In this study,we investigated the effects of the V2 receptor antagonist tolvaptan on brain edema after Intracerebral hemorrhage(ICH)in rats and the effects of iron ions on nerve cells.Animals were randomized to take tolvaptan or saline at 12 hours,36 hours and 60 hours after ICH surgery by oral gavage.Brain swelling(%),brain water content(BWC),neurological score,Evans blue fluorescence and blood brain barrier(BBB)tight junction protein were measured to determine the effect of tolvaptan after ICH.We found that tolvaptan reduced brain swelling(%),decreased BWC growth,and attenuated neurological deficits after ICH(p < 0.05,compared to vehicle).More importantly,tolvaptan increased the expression of ZO-1 and occludin(p < 0.05 compared to vehicle)and the results indicated that tolvaptan may have the effect of down-regulating MMP-9.At the same time,the experiment of simulating Intracerebral hemorrhage with single factor iron ion in vitro showed that iron ions are nutrients to nerve cells at low concentration,but they will produce cytotoxicity after a certain concentration.The experimental results provide strong data support for tolvaptan for ICH-induced cerebral edema.At the same time,it is necessary to use the experimental data of animals closer to the human body structure to further study the efficacy and principle mechanism of tolvaptan in the treatment of ICH.1.The role of tolvaptan in the treatment of cerebral edema after Intracerebral hemorrhage in ratsPurposeBased on the ICH model of SD rat collagenase Intracerebral hemorrhage,12 hours after model establishment,oral administration was given to the tolvaptan group and the saline group,and the rats were given once every 24 hours,and the rats were measured.After 3 days of body weight,BBB permeability,brain water content(BWC)and behavioral behavior,the effect of the drug tolvaptan on cerebral edema swelling in experimental rats after ICH surgery was observed.MethodBefore the ICH operation,40 rats were randomly divided into two groups: ICH group and sham group,32 rats in ICH group,and 5 sham group in sham operation.In the ICH group,the ICH group was injected with collagenase in the basal ganglia.The sham operation group only injected the needle into the experimental animals without injecting collagenase.After 12 hours,the rats were intragastrically administered every 24 hours.Three times in the stomach,16 rats in the ICH group were randomly selected as the drug-administered group,and 10 mg of Tolvaptan was administered per kg of rats.The tablets were directly dissolved in drinking water for oral feeding;the remaining 16 As a control group,the control group and the sham group were orally gavaged with an equal volume of water.On the fourth day after surgery,all experimental animals were subjected to a corner angle test and a forelimb placement test to conduct a behavioral test score,and then an MRI scan was performed to measure the volume and extent of brain edema.After the MRI measurement was completed,8 experimental samples were randomly selected from each group to measure the BWC to measure the degree of brain edema,and the remaining 6 experimental animals were subjected to evans blue(EB)exudation.This experiments were conducted to test the BBB permeability of experimental animals.Result1.Magnetic resonance imaging results showed that the volume of cerebral edema in the experimental rats fed with oral tolvaptan was reduced(p < 0.05).And BWC results showed that rats injected with tolvaptan reduced brain water content and reduced brain edema compared with control rats.2.Although m NSS score test data and forelimb placement test data showed that the effect of tolvaptan on the improvement of neurological function in rats did not reach statistical difference(p>0.05),but we can draw from the test data of the corner experiment.Tolvaptan helped restore some of the neurological function of rats after ICH(p<0.05).3.From the results of EB immunofluorescence and EB extravasation quantification,tolvaptan effectively reduced the extravascular exudation of EB dye,which indirectly showed that BBB permeability was on the top of the taflucan after stabilization of rat ICH.It has a positive effect.ConclusionOral administration of tolvaptan can effectively reduce the volume of cerebral edema after ICH in rats,and has a certain protective effect on the blood-brain barrier after ICH.2.Mechanism of the protection of blood-brain barrier after Intracerebral hemorrhage in rats with tolvaptanPurposeEstablishment of rat ICH model,expression of MMP-9 in BBB-associated tight junction proteins(occludin,ZO-1)and matrix metalloproteinases after oral administration,analysis of tolvaptan to BBB The protective effect and internal mechanism.MethodBefore the ICH operation,25 rats were randomly divided into two groups: ICH group and sham group,ICH group 20 rats,and sham-operated sham group 4,other spare.The modeling process of the ICH group was consistent with the previous one.After 12 hours after surgery,10 of the ICH group were randomly selected as the drug-administered group,and the remaining 10 were used as the control group.The dose and number of administrations were the same.One chapter was consistent,and the control group and the sham group were orally gavaged with an equal volume of water.On the third day after surgery,5 experimental animals in each group of ICH administration group and ICH experimental group were randomly selected for immunofluorescence staining to detect the expression of BBB-associated compact protein(ZO-1,occludin),and each group was taken.Five western blots(WB)were used to quantitatively compare ZO-1,occludin and MMP-9 proteins.Result1.By detecting the expression of ZO-1 and occludin around the hematoma(immunofluorescence,WB),it was found that tolvaptan can up-regulate the expression of these two BBB-associated tight junction proteins.The results show that tolvaptan maintains the integrity of BBB.The drugs have positive effects.2.Immunofluorescence and WB experiments showed that the decrease of MMP-9 expression in oxilaptan experimental group after ICH indicated that tolvaptan had a certain protective effect on BBB.ConclusionAlthough the study on the mechanism of action of V2 receptor antagonist tolvaptan on cerebral edema reduction after oral administration of ICH in rats is not very deep,from the overall effect,tolvaptan has a certain protective effect on BBB..3.A preliminary study on the effect and influence of iron ions on nerve cellsPurposeThe in vitro model cells of rat adrenal pheochromocytoma PC12 cells were used as neurons.After a certain concentration of iron ions was given in the medium,the absorbance of cck8 was detected 48 h later to detect PC12 under a certain drug concentration gradient.Proliferation of proliferation,and detection of apoptosis by flow cytometry to explore the approximate range of concentrations of iron ions that cause damage to nerve cellsMethodSet the concentration of ferrous sulfate to 1000 ?M group,800 ?M group,400 ?M group,200 ?M group,100 ?M group,80 ?M group,50 ?M group,20 ?M group,10 ?M group,5 ?M group,control group and blank group.After the cells were cultured for 48 hours,CCK8 detection reagent(10 ?L/well)was added,and the optical density value [D(450)] was measured at a wavelength of 450 nm using a multi-function microplate reader,and 1000 ?M group and 800 ?M group were picked.400 ?M group,200 ?M group,100 ?M group,80 ?M group,after 48 hours of treatment,Annexin V and PI were double stained with apoptosis kit,and the fluorescence signals of two dyes were detected by flow cytometry at 488 nm channel.Intensity to distinguish between yin and yang cellsResultAt low concentrations,iron ions promote cell proliferation,and apoptosis begins at a concentration of 200 umol/L.Macroscopic death can be seen at a concentration of 800 umol/L.ConclusionIron ions are nutrients to nerve cells at low concentrations,but after a certain concentration,they produce cytotoxic effects,causing apoptosis and necrosis of nerve cells.