The Improvement Of Rat Model For The Chronic Subdural Hematoma And The Regulatory Effect Of CXCR4 On The Absorption Of The Hematoma

Objective As one of the mainly secondary brain injury, chronic subdural hematoma(CSDH) is liable to attack the elderly especially with the risk factors of, history of traumatic brain injury, administration of anticoagulants, or older than 65 years. The therapy of CSDH is mainly based on drilling drainage and craniotomy, but the operation will influence the patients’ survival condition, and the neurological and respiratory complication on the patients will deteriorate the mortality and morbidity. Due to the limitation of the animal model of CSDH, the pathogenesis and treatment of the disorder have not achieved the substantial progress. Chemokine receptor 4(CXCR4) is an important regulator in multiple a variety of physiological and pathophysiological processes including inflammation, angiogenesis, wound healing and etc., which is closely related to the occurrence and development of hemorrhagic and ischemic disease. Therefore, utilizing a CSDH rat model which could preferably imitate the human CSDH process on human and the observation of clinical CSDH patients, this study focus on the pathogenesis of CSDH and the role of CXCR4 on the absorption of cranial hematoma, so as to explore the scientific evidence for the clinical therapy and drug promoting the absorption and the rehabilitation.Methods The design of the Research included basic and clinical parts. The basic research included: Establishing the Wistar rat model of CSDH with stereotaxic frame and injector. The rats were randomly divided into 3 groups: the sham control, CSDH and AMD3100(the high selective CXCR4 antagonist) pretreated CSDH group, and the dynamic observation will be performed on 1, 3, 10, 17 and 24 day( acute stage, 1~10 day; subacute stage 10~17 day; chronic stage, 17~24 day). The blood perfusion into the subdural space was performed twice with the gap of 72 hours in CSDH group; the rats in AMD3100 group were pretreated with the 2.5 ug/kg AMD3100 in the subdural space 2 hours prior to blood perfusion, and the following procedures were same to the CSDH group. The rats in the sham group were treated with the same procedure except blood perfusion(1) The Magnetic Resonance Imaging(MRI) examination was performed to detected whether the CSDH model is successful and measure the volume of hematoma;(2) The H&E staining was used to observe the morphological change in the hematoma and its peripheral region;(3) ELISA was performed to test the level Interleukin(IL)-6, IL-8, IL-10, IL-13, TNF-a and VEGF in the membrane and its peripheral tissue;(4) The quantification of endothelial progenitor cells(EPC) and regulated T cells(Treg) was detected by Flow Cytometry;(5) The expression of v WF as a vascular endothelial cell marker and CXCR4 were tested by Western blot;(6) The neurological function was estimated by m NSS scale, Footfault accessing and Morris Water Maze;(7) Statistical analysis is performed to observe the pathophysiological relationship between the hematoma absorption and inflammation, angiogenesis, and explore the role of CXCR4 in the two courses. The clinical observation: 24 CSDH patients and 30 healthy volunteers were enrolled and the information were collected as follow: the general information, the hematoma volume tested by MRI, the neurological function evaluated with the GOSE method, Treg and EPC level tested with Flow Cytometry, the correlation between Treg, EPC and hematoma volume, neurological function.Results(1) Imitating the multiple hemorrhage during the CSDH on human, we succeed to establish model of CSDH maintaining 24 days with repeated blood perfusion with gradient speed, and dynamically observed the change of the hematoma during 24 days.(2) The coronal sections of the brain burdened with CSDH and its H&E staining showed that the hematoma rose up to the peak on 3 day and gradually shrinked, the absorption of CSDH pretreated with AMD3100 delayed and was obviously slower than the ones in CSDH group on 10~24 day(P<0.05,P<0.05,P<0.05).(3) The result of the neurological function in CSDH group were worse than then sham control ones at the same stage, and AMD3100 further deteriorated it.(4) As the pro-inflammatory factors, IL-6, IL-8 and TNF-α significantly increased vs. sham group(P<0.05), and got to peak on 3~10 day; compared with CSDH group, the IL-6、IL-8 and TNF-α in AMD3100 group rose up more obviously, especially the IL-8 and TNF-α on 17~24 day. The anti-inflammatory factor IL-10、IL-13 in CSDH group increased vs. sham control and peak on 17 day, significant difference appears on 10~24 day(P<0.05). IL-10、IL-13 in AMD3100 group decreased on 17~24 day(P<0.05) while appeared no difference on 1~10 day vs. CSDH group(P>0.05). It showed positive correlation between volume hematoma、degeneration of neurological function and IL-6、IL-8、TNF-α, while negative with IL-10、IL-13.(5) The EPC level decreased on the primary stage of 1and 3 day, and rose to the highest level on 10 day and kept the high level with trend of down-regulation on 10~24 day(P<0.05),AMD3100 decreased the EPC level in the same time vs. CSDH ones(P<0.05);Treg was reduced compared with the sham control on 1~10 day(P<0.05), and increased on 17~24 day(P<0.05), AMD3100 down-regulated the Treg level on 10~24 day(P<0.05). Level of Treg and EPC show negative correlation to the hematoma volume and degeneration of neurological function.(6) The expression of v WF in CSDH group increased from 3 ~ 24 day and get to the peak on 24 day(P<0.05), AMD3100 decreased its level during the whole course(P<0.05); the expression of CXCR4 was up-regulated at 10 day and peaked on 17 day(P<0.05)in CSDH group, AMD3100 blocked its expression.(7) The level of EPC and Treg in the CSDH patients’ peripheral blood decreased before operation and gradually increased after bur-hole drainage, there was negative correlation between Treg 、EPC and the hematoma volume, degeneration of neurological function.Conclusion CXCR4 promote promoted absorption of CSDH via regulating the inflammation and angiogenesis, and benefit the improvement and recovery of neurological function during CSDH.