Neuroprotective Effect of Chrysophanol as a PI3K/AKT/mTOR Signaling Inhibitor in an Experimental Model of Autologous Blood-induced Intracerebral Hemorrhage

Objective Intracerebral hemorrhage(ICH)refers to predominant,sporadic,and non-traumatic bleeding in the brain parenchyma.The PI3K/AKT/mTOR signaling pathway is an important signal transduction pathway regulated by enzyme-linked receptors and has many biological functions in mammals.It plays a key role in neuronal metabolism,gene expression regulation,and tissue homeostasis in the healthy and diseased brain.Methods In the present study,the role of the PI3K/AKT/mTOR pathway inhibitor chrysophanol(CPH)(10 mg/kg and 20 mg/kg,orally)in the improvement of ICH-associated neurological defects in rats was investigated.Autologous blood(20µL/5 min/unilateral/intracerebroventricular)mimics ICH-like defects involving cellular and molecular dysfunction and neurotransmitter imbalance.The current study also included various behavioral assessments to examine cognition,memory,and motor and neuromuscular coordination.The protein expression levels of PI3K,AKT,and mTOR as well as myelin basic protein and apoptotic markers,such as Bax,Bcl-2,and caspase-3,were examined using ELISA kits.Furthermore,the levels of various neuroinflammatory cytokines and oxidative stress markers were assessed.Additionally,the neurological severity score,brain water content,gross brain pathology,and hematoma size were used to indicate neurological function and brain edema.Results CPH was found to be neuroprotective by restoring neurobehavioral alterations and significantly reducing the elevated PI3K,AKT,and mTOR protein levels,and modulating the apoptotic markers such as Bax,Bcl-2,and caspase-3 in rat brain homogenate.CPH substantially reduced the inflammatory cytokines like interleukin(IL)-1β,IL-6,and tumor necrosis factor-α.CPH administration restored the neurotransmitters GABA,glutamate,acetylcholine,dopamine,and various oxidative stress markers.Conclusion Our results show that CPH may be a promising therapeutic approach for overcoming neuronal damage caused by the overexpression of the PI3K/AKT/mTOR signaling pathway in ICH-induced neurological dysfunctions in rats.

Treatment of refractory giant macular hole by vitrectomy with internal limiting membrane transplantation and autologous blood

AIM：To investigate the effect of internal limiting membrane transplantation and autologous blood on treating refractory giant macular hole.METHODS：Thirty-seven eyes with giant macular hole of the smallest hole diameter 〉700 μm,the maximum diameter of the substrate 〉1000 μm and hole formation factor 〈0.6 underwent surgical treatment.The patients were randomly divided into two groups.Nineteen eyes with surgical flip of the internal limiting membrane in group A,18 eyes with internal limiting membrane transplantation in group B who underwent the tamponade of internal limiting membrane into the hole,autologous plasma was used to seal the hole.The patients were followed up for 3 mo,optical coherence tomography and best corrected visual acuity（BCVA） were recorded before and after operation,and the results were statistically analyzed.RESULTS：At 3 mo after operation,BCVA of the two groups was significantly improved compared with that before operation（tA=4.192,tB=4.374,P〈0.05）.But there was no significant difference in visual acuity between the two groups（χ^2=0.128,P〉0.05）.At 3 mo after operation,the closure rate of group A was 68.4%,and 100% in group B.（χ^2=5.628,P〈0.05）.The defect diameter of inner segment/outer segment at 3 mo after the operation was significantly lower than that before operation（tA=12.287,tB=15.481,P〈0.05）,and the difference was statistically significant（t=2.552,P〈0.05）.CONCLUSION：Internal limiting membrane transplantation combined with autologous whole blood can improve the postoperative closure rate of the refractory large aperture,and can effectively improve the postoperative visual acuity.

Parafoveal retinal massage combined with autologous blood cover in the management of giant,persistent or recurrent macular holes

AIM:To assess the efficacy and safety of parafoveal retinal massage combined with autologous whole blood cover in the treatment of refractory macular holes(MHs)and present the surgical procedure.METHODS:Patients with giant(minimum diameter>800 pm),recurrent or persistent MHs who underwent PPV combined with parafoveal retinal massage and autologous whole blood cover using C3F8 as tamponade agent from February 2018 to May 2019 were enrolled in this retrospective study.After surgery,all patients were informed to maintain a prone position for at least 7d.Preoperative and postoperative best-corrected visual acuities(BCVAs)were compared and MH closure rate was measured as the main outcome.RESULTS:A total of 13 MH patients consisted of 6 giant MHs,4 persistent holes and 3 recurrent holes(5 men and 8 women;average age was 56.40±11.72y)were enrolled in this study.MH closure was achieved in 11 eyes by this modified surgical technique while 2 eyes failed.Revitrectomy with autologous neurosensory retinal patch transplantations was applied for those 2 patients and then both holes were closed.No intraoperative complications were observed.BCVA improved from 1.73 IogMAR to 0.74 IogMAR at 6mo postoperation.There was significant difference in BCVA before versus after the surgery(P<0.05).There were no adverse events occurred during the follow-up period.CONCLUSION:With easier surgical procedure,parafoveal retinal massage combined with autologous whole blood cover is an effective addition to the surgical options for the management of refractory MHs.

Effects of Intraoperative Autologous Blood Donation and Tepid Temperature Cardiopulmonary Bypass on Blood System

The effects of the intraoperative autologous bind donation and tepid temperature cardiopulmonary bypass (CPB) on blood system were investigated. Twenty-four patients with rheumatic heart valve diseases scheduled for open heart surgery were selected and divided randomly into group A (intraoperative autologous blood donation and tepid temperature, nasopharyngeal temperature was at 32-34 ℃ during CPB) and group B (control, nasopharyngeal temperature was at 25-28 ℃ during CPB). The plasmatic concentrations of GMP-140 and D-Dimer and the plasmatic activities of 6- ketoPGF1α and AT- Ⅲ were measured by using ELISA or substrate luminescence techniques before operation, at the end of CPB, after discontinuation of CPB and postoperatively. Red blood cell count, platelet count, hematocrit, the amount of blood drainage and the amount of blood transfusion needed were measured or recorded postoperatively. The results showed the plasmatic concentrations of GMP140 and D-Dimer in group A were significantly less (P<0. 05) than those in group B during and after operation. The activity of 6-keto--PGF1α in group A was higher (P<0. 05) than that in group B during and after operation. The AT- Ⅲ activity in group A was less (P<0. 05) during CPB but higher 30 min after discontinuation of CPB than that in group B. The amount of postoperative blood loss (283± 166 versus 722± 194 ml, P<O. 01) and amount of blood transfusion (816±126 versus 1443± 678 ml, P<0. 01) in group A were significantly less than those in group B, respectively. The red blood cell count, platelet count and hematocrit in group A were significantly higher than those in group B after operation. The results suggests intraoperative autologous blood donation and tepid temperature have a good protection on blood system and can reduce postoperative non-surgical bleeding.

Vitrectomy with residual internal limiting membrane covering and autologous blood for a secondary macular hole:A case report

BACKGROUND Myopic foveoschisis(MF)is a common complication of pathological myopia.A macular hole(MH)usually results from the natural progression of MF and is a common complication of vitrectomy.Vitrectomy combined with residual internal limiting membrane(ILM)covering and autologous blood was effective for closing a secondary MH.CASE SUMMARY A 52-year-old woman presented to our clinic with a complaint of blurred vision in the right eye for 7 years.Her best corrected visual acuity(BCVA)was 20/100,axial length was 25.79 mm and standard equivalent refractive error was-10.5 dioptres.Preoperative optical coherence tomography revealed foveoschisis in the right eye.Vitrectomy with fovea-sparing ILM peeling was performed.An MH developed and gradually expanded 5 mo after the initial vitrectomy.Vitrectomy with residual ILM covering and autologous blood was performed.The MH closed 3 wk after the second vitrectomy.CONCLUSION Fovea-sparing ILM peeling can provide residual ILM for the treatment of MH secondary to vitrectomy for MF.Vitrectomy combined with residual ILM covering and autologous blood is effective for closing secondary MH and improving BCVA.

Construction of a model of autologous blood intracerebral hemorrhage in rats with a double injection and double needle withdrawal and the characteristics of the operative technique

BACKGROUND ： Experimental animal models of intracerebral hemorrhag （ICH） are greatly needed, so the process of establishment should be ideal in hematoma formation and easy to operate. OBJECTIVE ： To construct model of ICH in rats with double injection of autologous blood taken from the cut tail cut and double withdrawal of the needle （shortened as two-step injection model）, and compare with those induced by single and double injections. DESIGN ： A randomized controlled tria SETTING： Department of Neurology, General Hospital of Military Area Command of Chinese PLA. MATERIALS： Thirty male Wistar rats of 10 to 12 months, weighing （400±25） g, provided by the Experimental Center of Medical Animals, General Hospital of Shenyang Military Area Command of Chinese PLA, were divided randomly into 3 groups with 10 rats in each group： two-step injection group, single injection group, double injection group. METHODS ： The experiment was carried out in the Department of Neurology, General Hospital of Military Area Command of Chinese PLA from March to June in 2004. Autologous blood ICH model in rats were established as follows： In the two-step injection group, 50 μL unclotted autologous blood was taken from the rat tail cut, then injected with microsyringe into the caudate nucleus, 10 μL injected at first, paused for 2 minutes, and then the rest 40 μL injected slowly and continuously within 2 minutes. After the injection, the needle was kept immovable for about 4 minutes, withdrawn 2.0 mm, again kept immovable for about 4 minute, and then removed wholly at a slow speed. In the single injection group, 50 μL unclotted tail blood was injected slowly and continuously all within 2 minutes and the needle was slowly removed;（4） In the double injection group, 10 mL blood was injected at first, paused for 2 minutes, the rest 40 μL injected evenly within 2 minutes, and then the needle was withdrawn slowly and uninterruptedly. Neurologic findings were scored in accordance with Longa＇s five-point scale （0-4 scores, the higher the score, the severer the neurological dysfunction）. The rats were killed to remove and sections were prepared, the morphological features of hematomas were grossly observed, the maximal diameter and size of hematomas in each slice were measured with the imaging analytical system, and the volume was calculated. Meanwhile, the conveniences of the techniques were compared. MAIN OUTCOME MEASURES： The morphological features and volume of hematomas, neurologic deficit score （NDS）, and the convenience of the techniques were compared. RESULTS： All the 30 rats were involved in the analysis of results without deletion. （1） Results of the morphological observation of volume of hematoma： In the two-step injection group, hematomas located in the right caudate nucleus area regularly in circular or analogously circular shape in each slice. The formation rate of hematomas in the single injection group and double injection group were lower than those in the two-step injection group [60% （6/10）, 80% （8/10）, 100% （10/10）, P〈 0.01, 0.05]. The volume of hematomas in the single injection group and double injection group were smaller than those in the two-step injection group [（28.5±14.8）, （33.4±7.4）, （41.6±3.9） mm3, P〈 0.01, 0.05]. （2） NDS results： The NDS scores in the single injection group and double injection group were smaller than that in the two-step injection group （0.90±0.83, 1.30±0.78, 1.90±0.57, P〈 0.05）. （3）Comparison of the convenience of the techniques： The double injection method allowed generating reproducible hematomas in rats with shortcomings that it needed autologous arterial blood from femoral artery, and precision instruments such as microinfusion pump. The two-step injection injected fresh unclotting blood taken directly from the tail cut with microsyringe into the rat brain, and it has the advantages of easy operation, no influence on the activity of thrombase, shorter duration for model establishment, and higher rate of hematoma formation, which could generate ideal and economical models of ICH. The two-step injection induced hemotoma regularly in circular or analogously circular shape in each slice, but those induced by single and double injections were mostly in strip or fusiform shapes and extending along the needle tracks or into the ventricle or subarachnoid space. CONCLUSION ： The autologous blood ICH model induced by the two-step injection method is a reproducible and reliable one in regular shape, which is better than those induced by double and single injections.

Observation on the Efficacy of Autologous Blood Transfusion Combined with Controlled Hypotension in Patients with Spinal Internal Fixation during the Outbreak of New Coronavirus Pneumonia in Western Guangxi (in Guixi)

Objective: To explore the therapeutic effect of autologous blood reinfusion combined with controlled hypotension in surgical spinal fixation during the outbreak of COVID-19. Methods: 30 patients with spinal internal fixation autologous blood transfusion combined with controlled hypotension were selected as the subjects during the epidemic period from December 2019 to June 2020 in our hospital and during the operation, on the basis of routine blood pressure reduction, the American Haemonetice Corporation autologous blood continuous reinfusion system was used to infuse the blood recovered during the operation to the patient through filtration and other procedures. Results: 30 patients had no complications such as fever and hemolysis;And after the operation, the tube was dialed according to the drainage volume, the cervical thoracic and lumbar brace was customized, and the patient walked on the ground for one week;After no abnormalities, the patient was discharged. Intraoperative comparison of white blood cells (WBC), red blood cells (RBC), red blood cell pressure (HCT), hemoglobin (HGB), and coagulation time (PT) of patients with autologous blood before and after transfusion showed statistically significant differences before and after surgery (P < 0.001). Conclusion: During the new coronavirus pneumonia epidemic, the internal fixation of spinal surgery used a recovery machine to collect intraoperative blood for reinfusion. Intraoperative antihypertensive drugs were used to control blood pressure within a certain safe range. The postoperative clinical observation effect was significant and safe;especially at present the clinical significance during the epidemic was significant.

The safety of autologous peripheral blood stem cell transplantation by intracoronory infusionin in patients with acute myocardial infarction

Objective Bone-marrow stem-cell transplantation has been shown to improve cardiac function in patients with AMI, but the safety of intracoronory infusion of autologous peripheral blood stem-cell(PBSCs) in patients with AMI is unknown. For this reason, we observe the feasibility and safety of PBSCs transplantation by intracoronory infusion in such patients.Method Fourty one patients with AMI were allocated to receive Granulocyte Colony-Stimulating Factor (G-CSF:Filgrastim,300 μg) with the dose of 300 μg-600 μg/day to mobilize the stem cell, and the duration of applying G-CSF was 5 days . On the sixth day, PBSCs were separated by Baxter CS 3000 blood cell separator into suspend liquid 57 ml. Then the suspend liquid was infused into the infarct related artery (IRA)by occluding the over the wire balloon and infusing artery through balloon center lumen. In the process of the intracoronary infusion of PBSCs, the complications should be observed, which were arrhythmias including of bradycardia, sinus arrest or atrial ventricular block, premature ventricular beats ,ventricular tachycardia, ventricular fibrillation; and hypotention, etc. Results There were total 10 cases with complications during the intracoronary infusion of PBSCs. The incidence of complications was 24.4%(10/41), including bradycardia is 2.4 %(1/41), sinus arrest or atrial ventricular block is 4.9%(2/41), ventricular fibrillation is 2.4 %( 1/41), hypotention is14.6 % (6 /41).Conclusions In patients with AMI, intracoronary infusion of PBSCs is feasible and safe.

The Safety of Autologous Peripheral Blood Stem Cell Transplantation by Intracoronory Infusion in Patients with Acute Myocardial Infarction

Objectives Bone-marrow stem-cell transplantation has been shown to improve cardiac function in patients with acute myocardial infarction (AMI) , but the safety of intracoronory infusion of autologous peripheral blood stem-cell (PBSCs) in patients with AMI is unknown. For this reason, we observe the feasibility and safety of PBSCs transplantation by intracoronory infusion in such patients. Methods 41 patients with AMI were allocated to receive granulocyte colony-stimulating factor (G- CSF: Filgrastim,300μg) with the dose of 300μg~ 600μg/day to mobilize the stem cell, and the duration of applying G-CSF was 5 days. On the sixth day, PBSCs were separated by Baxter CS 3000 blood cel 1 separator into suspend liquid 57 ml. Then the suspend liquid was infused into the infarct related artery (IRA) by occluding the over the wire balloon and infusing artery through balloon center lumen. In the process of the intracoronary infusion of PBSCs, the complications should be observed, which were arrhythmias including of bradycardia, sinus arrest or atrial ventricular block, premature ve. ntricular beats ,ven~icular tachycardia, ventricular fibrillation; and hypotention, etc. Results There were total 10 cases with complications during the intracoronary infusion of PBSCs. The incidence of complications was 24.4% (10/41), including bradyca- rdia was 2.4 % (1/41), sinus arrest or atrial ventri- cular block was 4.0% (2/41), ventricular fibrillation was 2.4 % (1/41), hypotention was 14.6 % (6/41). Conclusions In patients with AMI, intracoronary infusion of PBSCs is feasible and safe.

Skin Disinfection with 2% Chlorine Gluconate-Adine in Autologous Peripheral Hematopoietic Stem Cell Transplantation Patients

Background: Autologous peripheral blood hematopoietic stem cell transplantation is widely used in the treatment of malignant lymphoma. Patients are prone to infection during the transplantation immune deficiency period. There has been a lot of clinical research into how to better manage this period of vulnerability. Objective: This study aims to investigate the efficacy of 2% chlorhexidine gluconate (CHG) for skin disinfection in patients undergoing autologous hematopoietic stem cell transplantation (HSCT) and observe any adverse reactions. Methods: A total of 106 patients receiving autologous hematopoietic stem cell transplantation from November 2019 to December 2020 in our district were selected as the control group. From January 2021 to January 2022, 106 patients with autologous hematopoietic stem cells were included in the experimental group. The control group used the immersion bath method. The experimental group was treated with an improved scrub bath method (including 3M 2% chlorhexidine gluconate medical sanitary wipes to wipe the whole skin once). Results: The bacteria-carrying rate of the improved method (37.74%) was significantly better than that of the traditional soaking method (72.64%), and the difference was statistically significant (P Conclusion: The improved bath/wipe method has a significant positive effect on skin disinfection for patients undergoing HSCT.

Establishment of a rat model of severe spontaneous intracerebral hemorrhage

Background:Severe intracerebral hemorrhage(ICH)is the most devastating subtype of stroke resulting in high mortality and disability.At present,the development of targeted treatments to minimize the high morbidity and mortality is limited partly due to the lack of a severe ICH animal model.In this study,we aimed to establish an accurate severe ICH model in rats and examine the pathological and physiological changes associated with ICH.Methods:A rat model of severe ICH model was established by intrastriatal injection of autologous blood using different blood volumes(ICH 100μL group,ICH 130μL group,ICH 160μL group,ICH 170μL group,and ICH 180μL group).The mortality was assessed during the 28-day post-ICH period.Short-and long-term neurological deficits were evaluated using the Longa method,foot fault,falling latency,and Morris water maze tests.Brain water content,hematoma volume,hemoglobin content,and magnetic resonance imaging were assessed to determine the extent of brain injury.Immunofluorescence staining was conducted to examine microglial activation and neuronal apoptosis.Hematoxylin and eosin(H&E)staining,lung water content,and western blotting were used to assess lung injury following ICH.Results:The mortality of ICH rats increased significantly with an increase in autologous blood injection.The 28-day mortality in the 100μL,130μL,160μL,170μL,and 180μL ICH groups were 5%,20%,40%,75%,and 100%,respectively.A significantly higher 28-day mortality was observed in the ICH 160μL group compared to the ICH 100μL group.The ICH 160μL group exhibited significantly increased neurological deficits,brain edema,hematoma volume,and hemoglobin content compared to the sham group.Compared with the sham operation group,the activation of microglia and neuronal death in ICH 160μL rats increased.The use of H&E staining and western blotting demonstrated that disruption of the intra-alveolar structure,alveolar edema,and infiltration of inflammatory cells and cytokines into the lung tissue were more severe in the ICH 160μL group than the sham group.Conclusions:A severe ICH model in rats was successfully established using an injection of autologous blood at a volume of 160μL.This model may provide a valuable tool to examine the pathological mechanisms and potential therapeutic interventions of severe ICH.

Ultraviolet blood irradiation and oxygenation affects free radicals and antioxidase after rabbit spinal cord injury

To investigate the effects of ultraviolet blood irradiation and oxygenation (UBIO) on free radicals and antioxidase after spinal cord injury in rabbits Methods Totally, 186 rabbits were used and divided randomly into four experimental groups: control (n=6), blood transfusion (n=24), injured (n=96) and treatment (n=60) groups The relative intensity of free radical (FR) signals, malondialdehyde (MDA) content, as well as the activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH PX) were compared among the four groups at 6, 24, 48, and 72 hours and 6 days after injury Results The relative intensity of FR signals in spinal cord tissue in the injured group increased at 48 hours and showed a striking difference compared with the control group; in the treatment group, it decreased and showed a striking difference compared with the injured group MDA content in blood in the injured group increased and showed a striking difference at 6, 24 and 48 hours and showed a significant difference at 72 hours and 6 days after injury compared with the control group In the treatment group, MDA content in blood decreased and showed a significant difference at 48 hours compared with the injured group MDA content in spinal cord tissue increased in the injured group and showed a striking difference compared with the control group; in the treatment group, it decreased and showed a striking difference compared with the injured group at the corresponding times The activity of SOD in blood and spinal cord tissue decreased in the injured group and showed a striking difference compared with the control group; in the treatment group, it increased and showed a striking difference compared with the injured group at the corresponding times The changes in activity of GSH PX in blood and spinal cord tissue were similar to that in SOD No significant difference was observed between the blood transfusion and control groups Conclusion UBIO can ease free radical damages and elevate the activity of antioxidases after spinal cord injury in rabbits

Treatment of chronic recurrent dislocation of temporomandibular joint by autologus blood injection

Aim:The aim of this study was to evaluate the efficacy of autologus blood injection in the management of recurrent temporomandibular joint(TMJ)dislocation.Methods:A total of 11 patients,4 males,7 females,mean age of 58.6 years and suffering from recurrent dislocation of TMJ,were included in the study.In all the patients the procedure included anesthesia-local or sedation,arthrocentesis which was followed by autologous blood injection in the upper joint cavity.The peri-articular tissues were also infiltrated with autologous blood.Post procedure advice included restricted mouth activity and liquid diet for a month.Results:The results indicate that success rate of treatment of recurrent dislocation of TMJ is 72.8%which can be considered as impressive.The recurrence was noticed in 27.2%cases after one year follow up.Conclusion:Autologus blood injection is an effective,simple,non-invasive,and safe procedure for treatment of recurrent dislocation of TMJ and can be performed on outpatient basis.

Autotransfusion of shed mediastinal blood after open heart surgery

Objective To determine the safety and effectiveness of autotransfusion of shed mediastinal blood after open heart surgery. Methods Sixty patients undergoing coronary artery bypass grafting (CABG) were selected randomly to receive either nonwashed shed mediastinal blood (Group 1,n=30) or banked blood (Group 2, n=30). Drainage and transfusion volume were determined after the operation. Hb,RBC,HCT and PLT were detected immediately before and after the operation,as well as 24 hours and 7 days after the operation. Data were analyzed using Fisher’s exact test. A P <0.05 was considered significant. Results There were no significant differences in Hb,HCT,PLT or length of cardiopulmonary bypass (CPB) ( P >0.05). In the two groups,no significant difference in the mean blood loss was observed during 24 hours after the operation (660±300 ml in Group 1 and 655±280 ml in Group 2, P >0.05). In Group 1,the mean volume autotransfused was 280±160 ml,and the patients required 360±80 ml banked blood compared with 660±120 ml in Group 2. In other words,the banked blood requirement in Group 1 was 40% lower. Conclusions Autotransfusion of shed mediastinal blood after an open heart operation is safe and effective.

Spontaneous intracranial hypotension complicated with cerebral venous thrombosis and subdural effusion:a case report

Spontaneous intracranial hypotension treatment can be complicated by concomitant cerebral venous thrombosis and subdural hematoma.A 48 years old male,presenting orthostatic headache and neck pain for 1 month displayed sagittal sinus thrombosis and bilateral subdural effusions,as well as extradural fluid collection at T3-T8 level,upon magnetic resonance imaging.Cerebrospinal fluid opening pressure was 50 mmH2O,and a leak was confirmed at C2-C3 level by computed tomography(CT)myelogram.The presence of subdural hematoma precluded anticoagulation treatments.An autologous epidural blood patch at C2-C3 level under CT guidance improved the patient’s condition,remaining free of residual symptoms or recurrence at six-month follow-up.