Exploration Of Pathophysiological Mechanism And Conservative Treatment Of Chronic Subdural Hematoma

Objective: There are high concentration of inflammatory and pro-angiogenesis cytokines in the hematoma of Chronic subdural hematoma(CSDH)patients.However where these cytokines come from and how they are modulated are still unknown.Bur-hole drainage is the first-line therapy for CSDH.Due to the high recurrence rate after surgery,increased underlying disease in the aged and the widely use of antithrombotic drugs,some patients may suffer bad outcomes after the surery.Therefore,the conservative drug therapy is drawing more and more attention.Atorvastatin is the only conservative drug therapy which has obtained the level I evidence of Evidence Based Medicine.It has the potential to be the first-line treatment for CSDH.However,there were still 11.2 percent patients who showed no response to the Atorvastatin therapy and needed the surgerical treatment.In addition,the Atorvastatin therapy may take 8 weeks or even longer to be effective.Therefore,further studies are necessary to improve the conservative drug therapy.Exosomes,one kind of endosome-derived membrane-bound vesicles with diameters of ~30–150 nm,exit widely in the serum,urine and cerebral spinal fluid and other body fluid modulating the intercellular communication.Whether there are exosomes in the hematoma and their function is still unknown.This study consists of the basic research and the clinical trial.The objevtice of the basic research part is to verify whether there is exosome in the hematoma,compare the diference of mi-RNA carried by the serum-derived exosomes and hematoma-derived exosomes and study how the hematoma-derived exosomes affect the SDH rat and modulate the angiogenesis of HUVEC,which will deepen our understanding of the physiopathological mechanism of CSDH.In the clinical trial part,we are aiming to compare the therapeutic efficiency of Atorvastatin and the combined therapy of Atorvastatin and low dose of Dexamethasome on CSDH patiens.Their effects on angiogenesis and inflammation will also be evaluated,which may provide new thoughts for the improvement of conservative treatment for CSDH.Method: The basic research part:(1)The hematoma of CSDH patients was collected during the surgery.The surpernant of the hematoma was treated with ultracentrifugation.Western Blot(WB),transmission electron microscope(TEM)and Nanosight were used to identify whether the extractive were exosome;(2)After the rat SDH model was established,the volume of hematoma and neurological deficits were evaluated with magnetic resonance imaging(MRI)and modified neurological severity score(mNSS)score respectively;(3)The PKH26,PKH67 and immunoflourance were used to study whether HUVEC could intake the hematoma-derived exosome;(4)After the HUVEC were treated with the serum-derived exosome and hematoma derived exosome,the effect of hematoma-derived exosome on the tube formation and angiogenesis related cytokines were evaluated with cytokine array,RT-PCR;(5)The concentration of ANG-1 and ANG-2 in the control serum,CSDH patient serum and CSDH patient hematoma were evaluated with ELISA.The basic information and labatory results of these CSDH patients were also compared with the healthy control;(6)The difference of mi-RNA carried by the serum-derived exosomes and hematoma-derived exosomes were studied with high throughput mi-RNA sequencing and the selected mi-RNA were verified with RT-PCR;(7)Whether the hematoma-derived exosomes ccould increase the concentration of mir-144-5p in HUVEC was examined with RT-PCR;(8)The concentration of mir-144-5p in HUVEC was upregulated with siRNA-mate transfecting kit and mir-144-5p mimics.Then the effects of mir-144-5p on angiogenesis,permeability and ANG-1/ANG-2 expression were evaluated with tube formation assay,In Vitro Vascular Permeability Assay,WB and RT-PCR;The clinical trial part: After the CSDH patients was recruited,the neurological function improvement and hematoma absorbation were evaluated with MGS-GCS,ADL-BI,GOSE and imageological examination.Circulating EPC,Treg and T lymphocyte subset were also examined with Flow Cytometry to evaluate the effect of combined therapy on angiogenesis and inflammation.Results: The basic research part:(1)There exit exosomes in the hematoma of CSDH patients,which express the common marker of exosome including CD9,CD63 and TSG101.The diameter of hematoma-derived exosomes range from 30 nm to 150 nm with a peak at 74nm;(2)Hematoam-derived exosomes inhibit the hematoma absorpation and aggravate the neurological deficits;(3)Hematoam-derived exosomes could be in-taken by HUVEC;(4)Hematoam-derived exosomes increase the tube formation and expression of ANG-2,EGF,Endoglin and CXCL4 in HUVEC.Meanwhile,the ANG-2 mRNA increased in the hematoma-derived exosomes treated group,while the ANG-1 mRNA decreased;(5)There was no difference of the basic information including age and sex and the laboratory results such as blood routine examination,liver function and renal function eximation between the healthy control and CSDH patients.The concentration of ANG-1 and ANG-2 in the serum of these two group also showed no difference.However,the concentration of ANG-1 was significantly lower in the hematoma comparing with the serum,while the concentration of ANG-2 was significantly higher than the serum;(6)There was no obvious difference of the miRNA in serum-derived exosomes of the healthy control and CSDH patients.The mi-RNA distribution was similar in the serum-derived exosomes and hematoma-derived exosomes.The different mi-RNA betwwen the serum-derived exosomes and hematoma-derived exosomes are involved in Primary bile acid biosynthesis,Terpenoid backbone biosynthesis pathway,Peroxisome proliferator-activated receptors signaling pathway,Renin-Angiotensin-System,Synthesis and Degradation of Ketone Bodies,Steorid homorone biosynthesis,Endocytosis,Hippo signaling pathway and Advanced glycation end products-Receptor of advanced glycation end products signaling pathway.The mir-144-5p was enriched in the hematoma derived exosome;(6)Hematoma-derived exosomes could increase mir-144-5p concentration in HUVEC;(7)Upregulation of mir-144-5p in HUVEC caused increased tube formation and permeability accompanied by the increased expression of ANG-2 and decreased expression of ANG-1;The clinical trial part: There were 60 patients who were recruited in the study and 30 patients were assigned to Atorvastatin treatment group randomly,while the other 30 patients were assigned to the Atorvastatin + Dexamathasome group randomly.The baseline demographic and clinical characteristics of patients including age and sex were comparable between the two groups of patients.Patients treated with ATO+DXM demonstrated decreased hematoma absorption than those treated with ATO after 2(23.11 ± 15.37 vs.7.02 ± 13.06 mL,p < 0.0001)and 5 weeks(43.27 ± 19.09 vs.24.23 ± 25.17 mL,p = 0.0017)of treatments.83.33% of patients on ATO+DXM and 32.14% on ATO achieved the complete recovery of neurological dysfunction(MGS-GCS=Grade 0)on 5th week(p = 0.0004).Four patients(13.3%)from the ATO group and one patient(3.3%)from the ATO+DXM group were switched to surgery on days 14,16,40 and 48 or 36 days after first dosing during the trial due to increased hematoma volumes and/or exacerbated neurological dysfunction(p=0.161).The patients with combined treatment were also presented with significantly reduced levels of circulating T cells and increased levels of circulating Treg cells and endothelial progenitor cells(EPCs).The laboratory results of erythrocyte(RBC),white blood cells(WBC),WBC differential count,Platelet(PLT)and Hemoglobin(HGB)showed no difference in the two group.No severe complication that required medical intervention such as infection,gastrointestinal bleeding and muscle soreness was recorded from patients of both groups.Conclusion:(1)There exit exosomes in the hematoma of CSDH patients,which can inhibit the hematoma absorpation and aggravate the neurological deficits;In vitro experiments we demonstrate that hematoma-derived exosomes could be in-taken by HUVEC,which results in increased tube formation accompanied by increased expression of ANG-2 and decreased expression of ANG-1;(2)There is no difference of miRNA in the serum-derived exosomes in the healthy control and the CSDH patients.mir-144-5p is encriched in hematoma-derived exosomes.Overexpression of mir-144-5p could copy the effect of hematoma-derived exosomes on angiogenesis and the expression of ANG-1/ANG-2,which suggests hematoma-derived exosomes could deliver mir-144-5p to HUVEC resulting in increased expression of ANG-2 and decreased expression of ANG-1 accompanied by increased tube formation and permeability.This may induce the rebleeding and exudation and result in the hematoma enlargement;(3)The combined treatment of ATO and low-dose of DXM was safe and more effective than ATO alone in reducing hematoma and improving neurological function of patients with CSDH,which may be caused by that the combined therapy is more effective in modulating the angiogenesis and inhibiting the inflammation.