Experimental And Clinical Research Of The Mechanism Of Brain Tissue Fibrosis In Hemorrhagic Stroke

Background, Objective and SignificanceTransforming growth factor beta 1(TGF-β₁)was the main cytokine found inrecently ten years which participated in many patho-physiology process, primingand end tissues damage and recovery. The synthesize and sediment of collagen wasbasic pathology of tissues and organ in human, TGF-β₁ played a key role in fibrousdegeneration by multiple mechanism, such as stimulating collagen gene toover-expression and it's autocrine regulation.Many studies on peripheral tissues damage showed that, if TGF-β₁ in normallow level over-expressed, the extracellular matrix (ECM) would over produced anddeposit to be the causative agent of many chronic disease such as glomerularsclerosis, hepatic fibrosis, pulmonary fibrosis, scleroderma, etc. TGF-β₁over-expression played an important role in inflammatory reaction, tissues and fibrillahyper-plasy and cicatrisation after central neural system(CNS) damaged, In recentlyyears, researchists gradually payed much attention to this discovery, especially tocommunicating hydrocephalus after subarachnoid hemorrhage(SAH).The mechanism of communicating hydrocephalus was still unknown, except forinborn and unknown aetiology, most of them complicated by SAH and central nervous system infection. Hemorrhage and inflammation could make meningesgenerally fibrous degeneration and conglutination with the result that thecerebrospinal fluid recrement was stop, arachnoid granulation degenerated so that thechannels of cerebrospinal fluid recirculated to veins was blocked, these were the twoapproved pathomechanisms, but the mechanism of meninges fibrosis and the pathwayof cerebrospinal fluid circulation and reabsorption were still unknown. Maillotconsidered that drainage of cerebrospinal fluid had ascending pathway (cranio-subarachnoid space pathway), lymph and intravenous pathways, the latter twopathways had no exact morphology evidence, but in the study, the medicine injectedin canalis spinalis could soon appeared in venous system with the high density, itdemonstrated that there may be the preferential channel from cerebrospinal fluidsystem to venous system.The meninges tissues in subarachnoid space was the basic structure wherecerebrospinal fluid circulating and reabsorbing between the cerebrospinal fluidsystem and venous system, but now we have not known too much aboutphysiological and pathological mechanisms of them, especially after bleeding orunder inflammation. With the progress of study in fibrosis mechanism of peripheraltissues, a series of signal transduction mechanisms of tissues impairment andreparation with TGF-β₁ participated in were gradually discovered, the important roleof TGF-β₁ in central nervous system disease was beginning to be focused on bypeople. Some studies had discovered that TGF-β₁ obviously presented abnormally incerebral hemorrhage, SAH, cerebral ischemia and hypoxia, etc. there were reportsshown that drainage disturbance of cerebrospinal fluid would happened in intrathecalinjection with recombined TGF-β₁ and transgenic rats, this indicated that TGF-β₁played an important role in hydrocephaly.Colloid scar was the universalistic result of central nervous system damaged or pathologic course, it's pykno-barrier interfered with the nervus axon regeneration andfunctional restoration, this was the main course of residual disability. With thelucubrating of nervus stem cells transplant, there were more and more problems to besolved, the regenerate hindrance of nervus cells was gradually focused on by people.Astrocytes were the main gliocytes to make up for colloid scars, when damaged,they could bring about all kinds of growth factors and cell regulatory factorsincluding TGF-β₁, there would be many negative effects when repressed directlybecause of the multiplicity of physiological functions with TGF-β₁. The connectivetissues growth factor (CTGF) was the main downstream factor who mediated TGF-β₁to participate in fibrous degeneration, because it's biological effect was single, theside effect after blocked in upstream of TGF-β₁ might be avoided if we directlyrepressed CTGF. So we can see, CTGF could be a target to inhibit negative affectwhen we use nervus stem cells transplant to treat nervous system diseases.Thrombin was the most special substance in haematoma of the hemorrhagicstroke, it has been taken as an extracellular signaling molecule and has extensivecytobiological effects. Pretreatment with low dose(1～2u/ml) thrombin couldobviously protect neuron and gliocyte from cell death after damaged byhypoglycemia, ischemia and hypoxia, thrombin with higher density could makeaxon and dendron of neuron to degenerate, the axon minificated, cells aggregated andgliocyte astro-transmutated, the interaction among cells and connection among bloodcapillaries were destroyed, a micromole thrombin could make neuron and gliocyte invitro culture to die in procedure, but if some specific inhibitors such as hirudin andprotease nexin-1 were added in, the effect would be reversed. So the dual effects ofthrombin which protect and toxic to neurocytes were close correlated to volume ofhemorrhage, this was extremely focused on in cerebral hemorrhage. In peripheraltissues, thrombin together with TGF-β₁ could mediate SMalphA genetic expression and result in overdifferentiation of fibroblast, overdifferentiated fibroblast stimulatedkidney proximal tubule to secrete and produce metalloprotease, so synthesis of typeâ…£collagen was promoted. There were few study reports about central nervus tissues.So we prepare to investigate the correlation of thrombin and TGF-β₁ in hemorrhagiccerebrovascular diseases and the effects of thrombin in nervus reparation after centralnervus damaged, and give active suppression in this underlaying, this can find notonly the effective target to prevent communicating hydrocephalus after SAH, butalso the common effective target to treat brain cell edema, inflammation andcicatrisation.Some study about periphery tissue had shown that TGF-β₁ cytokine played an importantrole in hyperplastic scarring of skin, it made for transcription of collagen gene and synthesis ofcollagen, glucocorticoids could inhibit transcription and synthesis of collagen by many ways. Buttill now, we have not cleared the molecular biology mechanism about glucocorticoids' actions andinterrelation between TGF-β₁ and glucocorticoids. Ghahary studied regulation sequence ofcollogen gene, he investigated the dexamethasone's interaction with TGF-β₁, and the influenceto al(â… ) precollagen gene priming activity of human. In experiment, TGF-β₁ couldup-regulate the priming activity of al(â… ) precollagen gene, but dexamethasone could inhibitsignificantly the priming activity of al(â… ) precollagen gene in normal fibroblast, this indicatedthat they could regulate transcription of al(â… ) precollagen gene in transcriptional level, itcoincidence to their roles in mRNA level of collogen gene. Meanwhile, dexamethasone couldrivalry the up-regulation of TGF-β₁ to the priming activity of al(â… ) precollagen gene, it furtherconfirmed Meisler's study in transcriptional level, it can be seen that in transcriptional level,dexamethasone not only could directly inhibit priming transcription of precollagen gene, but alsorivalry the fibering effect of TGF-β₁.Generally fibering, gliosis and scarring in central nervus tissues were the signalsafter central nervous system was damaged, we planned to investigate mechanism of dys-expression with TGF-β₁ in hemorrhagic cerebrovascular diseases and fibrosisreparation after tissues damaged in central nervous system; to research themorphologic change of rat's astrocytes and meningina cell, meningina fibering,CTGF expression of astrocytes and glial fibrillary acidic protein (GFAP) expressionwith molecular biologic technology such as RT-PCR, Western-blotting, RNAinterference, transfection and so on; to analyze correlation factors of over-expressionwith TGF-β₁ in hemorrhagic cerebrovascular diseases; to produce a theory thatthrombin participates in over-expression of TGF-β₁ as a promoter, and TGF-β₁induces, CTGF mediates astrocytes to reactiveness gumming, the mechanism oftissues fibering will be revealed, then we can attempt to solve the key problem ofregenerate impediment and tissues fibering adherence in damaged nervus repairprocess, look for a new thought to prevent central nervus tissues adhesivenessdiseases such as communicating hydrocephalus, furthermore, in the following nervusstem cell transplant, we can search effective intervention site to promote axonregenerating and repairing, inhibit negative effect of glial scar, maintain and recovernervus functions utmostly.Objective1. To evaluate the relationship between thrombin and over-expression oftransforming growth factor-β₁ and to explore the mechanism of communicatinghydrocephalus following subarachnoid hemorrhage (SAH).2. Cellular model with brain injured was established by rat cerebral corticalastrocytes in vitro culture stimulated by TGF-β₁, a stimulating factor.Then, thechange of astrocytes morph was observed. The change of quantity of GFAP(cytoskeletal protein of astrocytes) and CTGF(downstream factor mediated TGF-β₁to promote fibrosis)were investigated from gene translational level and proteinsynthetic level. Generate mechanism of gliosis after CNS injured was investigate via those changes.3. To study the prevention effect of the inflammation inhibition in subarachnoidon the occurrence of communicating hydrocephalus after SAH.Methods1. Male Wistar rats weight range from 200g to 300g were randomized intoexperiment group and compare group.There are 21 Wistar rats in experiment groupand 15 rats in compare group. The method of infusing medicine into subarachnoidspace via foramen magnum was adopted.The rat in experiment group was placed inthe side-lied and low-headed pose under 10% chloral hydrate (3ml/kg) injectedintraperitoneally anesthesia. After succeeded-puncture via foramen magnum and0.2ml cerebrospinal fluid (CSF) was drawn out, the rat thrombin (3u, 0.3ml) wasinfused into subarachnoid space.The puncture point was pressed 3 minutes and thelow-headed pose was held 30 minutes after the acus was drawn away.Then the ratwas put back to cage to be observed carefully.The rat thrombin was substituted with0.3ml saline in the compare group.The experiment group and the compare group were randomized into 3 groupsagain, 1 day, 10 days and 20 days, respectively by the different reperfusion time. Allof the animals were anesthetized with an intraperitoneal injection of chloral hydrate(3ml/kg of body weight) according to different time point and taken 200μl CSFrespectively into centrifugal machine in 2000r/min for 20 minutes and then put intorefrigerator of—60℃to be preserved.Then rats were perfused transcardially with saline until the fluid was clear, then4% paraformaldehyde in 0.1 M sodium phosphate (4℃, pH 7.4) for 30 minutes, andkilled by decapitation. Brains were postfixed in 4% paraformaldehyde overnight andrinsed with 75%, 85%, 95%, and 100% ethanol. The issueswere transparented with xylene after air drying and embedded in paraffin. Paraffin-embedded frontal,parietal and occipital coronal sections were cut at a thickness of 5μm and stained byMasson's technique.Tissue samples were fixed under the optical microscope of 40×10 times tomeasure the thickness of meningina fibril by the software of micrograph collectionand analysis computer system.TGF-β₁ in CSF was measured through ELISA.2. Rat cerebral cortical astrocytes in vitro culture: was in accordance withMcCarthy's cultural method and improved.through repetat treatment per cellsadhering by different speed and passage.After thrice passages, cells were accreditedby immunofluorescence.Cultured astrocytes were incubation in TGF-β₁ of different concentration for 24hours.Then, GFAP expression was examined by reverse transcription-polymerasechain reaction (RT-PCR)and Western-blotting analysis (WB) while the change ofastrocytes morphous was observed by phase-contrast microscopy. Astrocyte ofGliosis induced by TGF-β₁ was observed from above.Connective tissue growth factor (CTGF)expression in astrocytes of gliosis byincubation in TGF-β₁ of different concentration was examined by reversetranscription-polymerase chain reaction (RT-PCR) and Western-blotting analysis(WB). Effect of CTGF induced by TGF-β₁ was study from point of transcription andtranslation.3. Reference to Hijidro's method, we scored on the haemorrhage amount insubarachnoid from some patients in acute stage CT scan. The patients whose totalscores exceeded 4 grades were selected for research, being divided into thecontrast group (ie regular treatment group) and the therapy group (ie regulartreatment plus injection of Dexamethasone into subarachnoid). The venticle Huckman datum, the third ventricle width and the lateral ventricle width weremeasured from the skull CT scans in the initial stage and after 20 days respectively.4. SPSS13.0 was adopted to do statistical analysis. Data of every group wererepresented by (?)±s and treated by Factorial-ANOV. One-way ANOV was used tocompare data of every time-point in experiment group and control group.Independent-Samples T Test was used to compare data of every time-point betweentwo groups. Linear correlation analysis was used to compare index of theconcentration of TGF-β₁(pg/ml) in cerebrospinal fluid and the average thickness ofcerebral pia mater. Independent-Samples T Test and Paired-Samples T Test, betweenintra-groups and between two groups, was respectively used to compare data of CTscan with SAH cases between early stage and later stage of in experiment group andcontrol group. Size of test a=0.05.Results1. The results of the concentration of TGF-β₁(pg/ml) in CSFThe difference of the concentration of TGF-β₁(pg/ml) in cerebrospinal fluidbetween experiment group and control group was significant by Factorial-ANOV (F=60.743, P=0.000) .The difference of the concentration of TGF-β₁(pg/ml) incerebrospinal fluid between experiment group and control group in every time-pointwas not significant (F=0.128, P=0.881).No interaction lies in two factor in sametime-point between two groups. (F=1.223, P=0.309).I.e. the difference ofchanging followed time in two groups was not significant.The concentration of TGF-β₁(pg/ml) in cerebrospinal fluid of experiment groupin different time-point of 1d, 10d, 20d was 239.02±129.40, 309.07±57.30 and283.16±128.87(pg/ml). The difference of the concentrations compared with those ofcontrol group.in the same time-point was significant by Independent-Samples T Test (P＜0.05). Those of experiment group were higher than of control group.The difference of the concentration of TGF-β₁(pg/ml) in cerebrospinal fluid ofcontrol group in different time-point was significant while the change followed timeby one way ANOVA. That of 1d was higher than of 10d or 20d by multiplecomparison of LSD.The cause may be damage in pricking which result in bloodentering cavitas subarachnoidealis and TGF-β₁ delivered by platelet. (Graph1-1) Theconcentration of TGF-β₁(pg/ml) in cerebrospinal fluid of experiment group was lastin high level. (Tab1.3)2. The results of data of average thickness of cerebral pia materThe difference of data of average thickness of cerebral pia mater betweenexperiment group and control group (F=32.178, P=0.000) was significant whilethat in every time-point was also significant (F=9.763, P=0.001) byFactorial-ANOV. Interaction lies in two factor in same time—point between twogroups. (F=6.798, P=0.004).I.e. the change followed time in two groups wasdifferent.The data of average thickness of cerebral pia mater of experiment group indifferent time-point of 1d, 10d, 20d was 3.68±0.43,4.29±0.52,5.67±0.77 (μm).The difference of the concentrations compared with those of control group.in thesame time—point was significant expect that of 1d (t=0.564, P=0.585) byIndependent-Samples T Test (P＜0.001). Those of experiment group were higherthan of control group.The difference of data of average thickness of cerebral pia mater of experimentgroup in different time—point was significant (F=20.858, P=0.000) by one wayANOVA. That of 20d was higher than of 1d or 10d by multiple comparison of LSD.(Graph1-2) The data of average thickness of cerebral pia mater of experimentgroup was higher along with the longer of time. 3. Direct correlation lies in the concentration of TGF-β₁(pg/ml) in cerebrospinalfluid and the data of average thickness of cerebral pia mater by Spearman correlationanalysis (r_s=0.541, P=0.001).4. GFAP of cultured astrocytes by treated and passaged for three times weredetected by immunofluorescence and cells of GFAP(+) were majority (＞95ï¼…).5. The morphous of astrocytes incubated in TGF-β₁ was changed, the expressionsof GFAP mRNA and protein were increased, and the effect became more and moresignificant followed density increased. The expressions of CTGF mRNA and proteinof astrocytes incubated in TGF-β₁ were increased, and the effect became more andmore significant followed density increased.6. Each cerebral ventricle datum of all the patients is enlarged in differentdegrees, but it showed more obvious in the contrast group than in the therapygroup(the Huckman data, P=0.029, and the frontal horn indices, P=0.039).Conclusions1. Experimental cell model needed by the research had been established andmeliorated,and the astrocyte of high purity had been gained by cell culture,whichmade researching fibrosis in cellular level be possible.TGF-β₁ could result ingumnosis of the astrocyte of SD rat cultured in vitro,which well simulated the cellmodel of the tissue fibrosis after bleeding.2. It had been verificated that thrombin of suitable dosage infused insubarachnoid space could induce TGF-β₁ to over-express in subarachnoid space andlead to hyperplasia of soft meninges fibril morphologically. So it could be presumedthat thrombin may be an important mediating factor that lead to communicatinghydrocephalus following SAH.3. It had been found that TGF-β₁ could mediate the over-expression of mRNA of CTGF and its corresponding protein in astrocytes of gliosis and it had the dose-effectrelationship between their expression level and the concentration of TGF-β₁,whichconfirmed furthermore that CTGF is an important downstream factor of TGF-β₁ whenit promote the fibrosis of CNS and provided the important experimental proof for thetarge of suppressing adverse effect of gliocyte to treat CNS diseases.4. It had been indicated by the study of human brains in imageology that theinflammation inhibition in subarachnoid space by using dexamethasone canobviously prevent the occurrence of ventricle enlargement after SAH.