Injection Of Autologous Blood Research Articles

Activating transcription factor 6 alleviates secondary brain injury by increasing cystathionine γ-lyase expression in a rat model of intracerebral hemorrhage.

Intracerebral hemorrhage (ICH) comprises primary and secondary injuries, the latter of which induces increased inflammation and apoptosis and is more severe. Activating transcription factor 6 (ATF6) is a type-II transmembrane protein in the endoplasmic reticulum (ER). ATF6 target genes could improve ER homeostasis, which contributes to cryoprotection. Hence, we predict that ATF6 will have a protective effect on brain tissue after ICH. The ICH rat model was generated through autologous blood injection into the right basal ganglia, the expression of ATF6 after ICH was determined by WB and IF. The expression of ATF6 was effectively controlled by means of intervention, and a series of measures was used to detect cell death, neuroinflammation, brain edema, blood-brain barrier and other indicators after ICH. Finally, the effects on long-term neural function of rats were measured by behavioral means. ATF6 was significantly increased in the ICH-induced brain tissues. Further, ATF6 was found to modulate the expression of cystathionine γ-lyase (CTH) after ICH. Upregulation of ATF6 attenuated neuronal apoptosis and inflammation in ICH rats, along with mitigation of ICH-induced brain edema, blood-brain barrier deterioration, and cognitive behavior defects. Conversely, ATF6 genetic knockdown induced effects counter to those aforementioned. This study thereby emphasizes the crucial role of ATF6 in secondary brain injury in response to ICH, indicating that ATF6 upregulation may potentially ameliorate ICH-induced secondary brain injury. Consequently, ATF6 could serve as a promising therapeutic target to alleviate clinical ICH-induced secondary brain injuries.

Microglial SLC25A28 Deficiency Ameliorates the Brain Injury After Intracerebral Hemorrhage in Mice by Restricting Aerobic Glycolysis.

The microglia overactivation-induced neuroinflammation is a significant cause of the brain injury after intracerebral hemorrhage (ICH). Iron homeostasis is crucial for microglia activation, but the mechanism and causality still need further study. This study aimed to explore the roles and mechanism of the mitochondrial iron transporter SLC25A28 in microglia activation after ICH. Intrastriatal injection of autologous blood was used to establish ICH model, and the neuroinflammation, iron metabolism and brain injuries were assessed in wildtype or microglia-specific SLC25A28 knockout mice after ICH. Mitochondria iron levels and microglial function were determined in SLC25A28 overexpressed or deleted microglia. The extracellular acidification rate (ECAR), lactate production, and glycolytic enzyme levels were used to determine aerobic glycolysis. The results showed that ICH stimulated mitochondrial iron overload, and synchronously upregulated the SLC25A28 expression. In vitro, SLC25A28 overexpression increased mitochondrial iron levels in microglia. Interestingly, microglial SLC25A28 deficiency ameliorated neuroinflammation, brain edema, blood-brain barrier injury and ethological alterations in mice after ICH. Mechanically, SLC25A28 deficiency inhibited microglial activation by restricting the aerobic glycolysis. Moreover, zinc protoporphyrin could reduce SLC25A28 expression and mitigated brain injury. SLC25A28 plays crucial roles in mitochondrial iron homeostasis and microglia activation after ICH, and it might be a potential therapeutic target for ICH.

Clinical efficacy and safety of autologous serum intramuscular injection in patients with mild-to-moderate atopic dermatitis: a prospective, open-label, uncontrolled study

Background: Autologous blood therapy (ABT) has been used to treat atopic dermatitis (AD) for over a century, even though evidence supporting its efficacy is lacking. We aimed to investigate the effectiveness and safety of autologous serum intramuscular injection (ASIM), which is a modified form of ABT, in treating mild-to-moderate AD. Methods: This study was a 12-week, open-label, prospective, uncontrolled trial. Following a 4-week run-in period, 22 out of 25 screened patients received ASIM once a week for 4 weeks in conjunction with standard treatment. The primary outcome measure was the Eczema Area and Severity Index (EASI), while the secondary outcomes included the Scoring Atopic Dermatitis (SCORAD) score, Dermatologic Life Quality Index (DLQI), and patient ratings of pruritus, sleep difficulty, disease status, and treatment effectiveness. Safety parameters were also assessed. Results: EASI scores showed a non-statistically significant trend toward improvement during ASIM intervention. Patients with at least a 50% improvement in the EASI score at 4 weeks were older and had lower peripheral eosinophil counts (p<0.05). Secondary endpoints, including the SCORAD score, pruritus, sleep difficulty, and DLQI, demonstrated statistically significant improvements at week 4 compared to baseline (p<0.05). No significant adverse reactions were observed. Conclusions: This pioneering study suggests that repeated ASIM may improve the clinical symptoms of mild-to-moderate AD, particularly in terms of pruritus and overall quality of life. However, further research with a larger sample size is required to establish the clinical significance of these findings.

The NLRP3 inhibitor, OLT1177 attenuates brain injury in experimental intracerebral hemorrhage

Background and PurposeIt has been reported activation of NLRP3 inflammasome after intracerebral hemorrhage (ICH) ictus exacerbates neuroinflammation and brain injury. We hypothesized that inhibition of NLRP3 by OLT1177 (dapansutrile), a novel NLRP3 inflammasome inhibitor, could reduce brain edema and attenuate brain injury in experimental ICH. MethodsICH was induced by injection of autologous blood into basal ganglia in mice models. Sixty-three C57Bl/6 male mice were randomly grouped into the sham, vehicle, OLT1177 (Dapansutrile, 200 mg/kg intraperitoneally) and treated for consecutive three days, starting from 1 h after ICH surgery. Behavioral test, brain edema, brain water content, blood–brain barrier integrity and vascular permeability, cell apoptosis, and NLRP3 and its downstream protein levels were measured. ResultsOLT1177 significantly reduced cerebral edema after ICH and contributed to the attenuation of neurological deficits. OLT1177 could preserve blood–brain barrier integrity and lessen vascular leakage. In addition, OLT1177 preserved microglia morphological shift and significantly inhibited the activation of caspase-1 and release of IL-1β. We also found that OLT1177 can protect against neuronal loss in the affected hemisphere. ConclusionsOLT1177 (dapansutrile) could significantly attenuate the brain edema after ICH and effectively alleviate the neurological deficit. This result suggests that the novel NLRP3 inhibitor, OLT1177, might serve as a promising candidate for the treatment of ICH.

M6A reader YTHDC2 mediates NCOA4 mRNA stability affecting ferritinophagy to alleviate secondary injury after intracerebral haemorrhage

ABSTRACT Oxidative stress and neuronal dysfunction caused by intracerebral haemorrhage (ICH) can lead to secondary injury. The m6A modification has been implicated in the progression of ICH. This study aimed to investigate the role of the m6A reader YTHDC2 in ICH-induced secondary injury. ICH models were established in rats using autologous blood injection, and neuronal cell models were induced with Hemin. Experiments were conducted to overexpress YTH domain containing 2 (YTHDC2) and examine its effects on neuronal dysfunction, brain injury, and neuronal ferritinophagy. RIP-qPCR and METTL3 silencing were performed to investigate the regulation of YTHDC2 on nuclear receptor coactivator 4 (NCOA4). Finally, NCOA4 overexpression was used to validate the regulatory mechanism of YTHDC2 in ICH. The study found that YTHDC2 expression was significantly downregulated in the brain tissues of ICH rats. However, YTHDC2 overexpression improved neuronal dysfunction and reduced brain water content and neuronal death after ICH. Additionally, it reduced levels of ROS, NCOA4, PTGS2, and ATG5 in the brain tissues of ICH rats, while increasing levels of FTH and FTL. YTHDC2 overexpression also decreased levels of MDA and Fe2+ in the serum, while promoting GSH synthesis. In neuronal cells, YTHDC2 overexpression alleviated Hemin-induced injury, which was reversed by Erastin. Mechanistically, YTHDC2-mediated m6A modification destabilized NCOA4 mRNA, thereby reducing ferritinophagy and alleviating secondary injury after ICH. However, the effects of YTHDC2 were counteracted by NCOA4 overexpression. Overall, YTHDC2 plays a protective role in ICH-induced secondary injury by regulating NCOA4-mediated ferritinophagy.

Attenuation of neuronal ferroptosis in intracerebral hemorrhage by inhibiting HDAC1/2: Microglial heterogenization via the Nrf2/HO1 pathway.

The class I histone deacetylases (HDACs) implicate in microglial heterogenization and neuroinflammation following Intracerebral hemorrhage (ICH). Ferroptosis has also been reported in the ICH model. However, the relationship between HDAC1/2's role in microglial heterogenization and neuronal ferroptosis remains unclear. In both invivo and invitro models of ICH, we used Romidepsin (FK228), a selective HDAC1/2 inhibitor, to investigate its effects on microglial heterogenization and neuronal ferroptosis. In the invitro ICH model using Hemin, a transwell system was utilized to examine how microglia-driven inflammation and ICH-triggered neuronal ferroptosis interact. Immunostaining, Western blotting and RT-qPCR were used to evaluate the microglial heterogenization and neuronal ferroptosis. Microglial heterogenization, neuronal ferroptosis, and neurological dysfunctions were assessed invivo ICH mice model performed by autologous blood injection. HDAC1/2 inhibition altered microglial heterogenization after ICH, as showing the reducing neuroinflammation and shifting microglia towards an anti-inflammatory phenotype by immunostaining and qPCR results. HDAC1/2 inhibition reduced ferroptosis, characterized by high ROS and low GPx4 expression in HT22 cells, and reduced iron and lipid deposition post-ICH invivo. Additionally, the Nrf2/HO1 signaling pathway, especially acetyl-Nrf2, activated in the invivo ICH model due to HDAC1/2 inhibition, plays a role in regulating microglial heterogenization. Furthermore, HDAC1/2 inhibition improved sensorimotor and histological outcomes post-ICH, offering a potential mechanism against ICH. Inhibition of HDAC1/2 reduces neuro-ferroptosis by modifying the heterogeneity of microglia via the Nrf2/HO1 pathway, with a particular focus on acetyl-Nrf2. Additionally, this inhibition aids in the faster removal of hematomas and lessens prolonged neurological impairments, indicating novel approach for treating ICH.

Intracerebral hemorrhage-induced brain injury in mice: The role of peroxiredoxin 2-Toll-like receptor 4 inflammatory axis.

Peroxiredoxin 2 (Prx2), an intracellular protein that regulates redox reactions, released from red blood cells is involved in inflammatory brain injury after intracerebral hemorrhage (ICH). Toll-like receptor 4 (TLR4) may be crucial in this process. This study investigated the role of the Prx2-TLR4 inflammatory axis in brain injury following experimental ICH in mice. First, C57BL/6 mice received an intracaudate injection of autologous arterial blood or saline and their brains were harvested on day 1 to measure Prx2 levels. Second, mice received an intracaudate injection of either recombinant mouse Prx2 or saline. Third, the mice were co-injected with autologous arterial blood and conoidin A, a Prx2 inhibitor, or vehicle. Fourth, the mice received a Prx2 injection and were treated with TAK-242, a TLR4 antagonist, or saline (intraperitoneally). Behavioral tests, magnetic resonance imaging, western blot, immunohistochemistry/immunofluorescence staining, and RNA sequencing (RNA-seq) were performed. Brain Prx2 levels were elevated after autologous arterial blood injection. Intracaudate injection of Prx2 caused brain swelling, microglial activation, neutrophil infiltration, neuronal death, and neurological deficits. Co-injection of conoidin A attenuated autologous arterial blood-induced brain injury. TLR4 was expressed on the surface of microglia/macrophages and neutrophils and participated in Prx2-induced inflammation. TAK-242 treatment attenuated Prx2-induced inflammation and neurological deficits. Prx2 can cause brain injury following ICH through the TLR4 pathway, revealing the Prx2-TLR4 inflammatory axis as a potential therapeutic target.

Edaravone Maintains AQP4 Polarity Via OS/MMP9/β-DG Pathway in an Experimental Intracerebral Hemorrhage Mouse Model.

Oxidative stress (OS) is the main cause of secondary damage following intracerebral hemorrhage (ICH). The polarity expression of aquaporin-4 (AQP4) has been shown to be important in maintaining the homeostasis of water transport and preventing post-injury brain edema in various neurological disorders. This study primarily aimed to investigate the effect of the oxygen free radical scavenger, edaravone, on AQP4 polarity expression in an ICH mouse model and determine whether it involves in AQP4 polarity expression via the OS/MMP9/β-dystroglycan (β-DG) pathway. The ICH mouse model was established by autologous blood injection into the basal nucleus. Edaravone or the specific inhibitor of matrix metalloproteinase 9 (MMP9), MMP9-IN-1, called MMP9-inh was administered 10min after ICH via intraperitoneal injection. ELISA detection, neurobehavioral tests, dihydroethidium staining (DHE staining), intracisternal tracer infusion, hematoxylin and eosin (HE) staining, immunofluorescence staining, western blotting, Evans blue (EB) permeability assay, and brain water content test were performed. The results showed that OS was exacerbated, AQP4 polarity was lost, drainage function of brain fluids was damaged, brain injury was aggravated, expression of AQP4, MMP9, and GFAP increased, while the expression of β-DG decreased after ICH. Edaravone reduced OS, restored brain drainage function, reduced brain injury, and downregulated the expression of AQP4, MMP9. Both edaravone and MMP9-inh alleviated brain edema, maintained blood-brain barrier (BBB) integrity, mitigated the loss of AQP4 polarity, downregulated GFAP expression, and upregulated β-DG expression. The current study suggests that edaravone can maintain AQP4 polarity expression by inhibiting the OS /MMP9/β-DG pathway after ICH.

Does Autologous Blood Injection Following Dextranomer/Hyaluronic Acid Copolymer Implantation in Treating Vesicoureteral Reflux Affect the Microsphere Particle Leakage?

Objectives: It has been shown that concomitant autologous blood and dextranomer/hyaluronic acid (Deflux®) injection, hydrodistension autologous blood injection technique (HABIT), had a better mound preservation and treatment success compared to the hydrodistension injection technique (HIT) in vesicoureteral reflux (VUR) correction. In this study, we aimed to show microscopically whether the concomitant injection of autologous blood decreases the leakage of Deflux® particles. Methods: Children with VUR who underwent HIT or HABIT between March 2020 and January 2023 were enrolled. Following the completion of the procedure on each ureter, the bladder was irrigated for 3 to 5 min, and the retrieved sample of irrigation fluid was evaluated for dextranomer particle count as “immediate leakage”. A Foley catheter was placed, and a urine sample after 12 h was collected as “early leakage”. Results: A total of 86 children with a median age of 3.0 years (interquartile range = 4.6) were included. Overall, 66 patients underwent HABIT, and 20 children underwent HIT. Rupture was observed in five patients during the procedure, and re-injection was conducted successfully in these cases. Immediate, early, and total particle leakage in the first 12 h of the injection were significantly less in the HABIT group compared to the HIT group. In the regression analysis, only the injection technique (HIT/HABIT) and rupture were significantly associated with the total particle leakage in the first 12 h. Conclusions: Immediate injection of autologous blood into the mound following an endoscopic correction of VUR in children is associated with significantly less Deflux® particle leakage from the injection site regardless of the VUR grade. We hypothesize that a concomitant blood injection into the Deflux® mound will create a blood clot while the needle is kept in situ and help to stabilize the mound and decrease treatment failure by minimizing particle leakage from the injection site.

Recombinant soluble form of receptor for advanced glycation end products ameliorates microcirculation impairment and neuroinflammation after subarachnoid hemorrhage

Impaired cerebral microcirculation after subarachnoid hemorrhage (SAH) has been shown to be related to delayed ischemic neurological deficits (DIND). We previously demonstrated the involvement of the receptor for advanced glycation end products (RAGE) in the pathogenesis of SAH related neuronal death. In the present study, we aimed to investigate the therapeutic effects of a recombinant soluble form of RAGE (sRAGE) on microcirculation impairment following SAH. Intrathecal injection of autologous blood in rats, mixed primary astrocyte and microglia cultures exposed to hemolysates and endothelial cells ​(ECs) from human brain microvascular exposed to glia-conditioned medium or SAH patient's CSF were used as experimental SAH models in vivo and in vitro. The results indicated that intrathecal administration of recombinant sRAGE significantly ameliorated the vasoconstriction of cortical arterioles and associated perfusion impairment, brain edema, reduced cell death, endothelial dysfunction, and improved motor performance at 24 and 48 ​h after SAH induction in rats. The in vitro results further showed that recombinant sRAGE significantly reduced astrocyte swelling and microglia activation, in parallel with decreased mRNA expression levels of pro-inflammatory cytokines including interleukin-6 (IL-6) and interleukin-1β (IL-1β) in vitro. Moreover, the in vitro model of SAH-induced p-eNOS and eNOS suppression, along with stress fiber formation in brain microvascular ECs, was effectively reversed by sRAGE treatment and led to a decrease in cleaved-caspase 3 expression. In summary, recombinant sRAGE effectively lessened microcirculation impairment and vascular injury after SAH via the mechanism of anti-inflammation, which may provide a potential therapeutic strategy for SAH.

Knockdown of LCN2 Attenuates Brain Injury After Intracerebral Hemorrhage via Suppressing Pyroptosis.

The aims of this study are to screen novel differentially expressed genes (DEGs) for intracerebral hemorrhage (ICH) and reveal the role of Lipocalin-2 (LCN2) in ICH. We constructed the ICH model by injection of autologous whole blood into the right basal ganglia in rats. RNA-sequencing and bioinformatics analyses were performed to identify the DEGs between ICH and sham rats, and some important ones were confirmed using quantitative real-time PCR (qRT-PCR). LCN shRNA was used to knockdown of LCN2 in ICH rats. Pathological examination was carried out using 2,3,5-triphenyltetrazolium chloride (TTC) staining and Hematoxylin-eosin (HE) staining. Immunohistochemistry detected Caspase-3, and co-staining of Terminal dUTP nick end labeling (TUNEL) and NEUN staining were performed for neuron apoptosis assessment. Western blot analysis was performed to quantify pyroptosis-related proteins. Enzyme-linked immunosorbent assay (ELISA) was used to measure inflammatory cytokine levels. ICH rats exhibited significant hematomas, higher brain water content, obvious interstitial edema, and inflammatory infiltration, as well as more apoptotic cells in brain tissues. RNA-seq analysis identified 103 upregulated and 81 downregulated DEGs. The expression of LCN2, HSPB1, CXCL10, and MEF2B were upregulated in ICH rats. ICH triggered the release of interleukin (IL)-1β, tumor necrosis factor-α (TNF-α), and IL-18, and promoted the expression of pyroptosis-related proteins Caspase-1, GSDMD, NLRP3, and ASC. LCN2 knockdown attenuated the pathological characteristics of ICH, and also reduced pyroptosis in brain tissues. Inhibition of LCN2 attenuates brain injury after ICH via suppressing pyroptosis, which provide guidance for ICH management.

A Review of Lateral Epicondylitis Injection: Drugs Used, Injection Techniques and Guidance Method

Abstract This article aimed to study the most suitable drug for injection, injection techniques and guidance method for treating tennis elbow. The words ‘injection technique’, ‘lateral epicondylitis’ and ‘tennis elbow’ were keyed into PubMed search base, and 145 full-text English language articles were obtained, from which 22 articles were deemed suitable for review based on content and relevance. One article was added from the author’s collection. The eligibility was (a) patients with tennis elbow or lateral epicondylitis, (b) patients who had undergone injection therapy and (c) studies that had done a comparison between two or more agents or two techniques or used comparison between using or not using imaging guidance for injection. It was found that corticosteroid injections in tennis elbow produce a short-term improvement in symptoms, while prolotherapy, autologous blood and platelet-rich plasma (PRP) injections may show more efficacy in the long term. In terms of technique, peppering seems to be a better alternative to tender point injections. The use of ultrasound guidance does not seem to have any effect on the overall efficacy of the injection treatment. The evidence in this study shows that it is better to use corticosteroids for short-term improvement, whereas bioactive agents like PRP injections may work better in the long term.

Clearance of Neutrophils From ICH-Affected Brain by Macrophages Is Beneficial and Is Assisted by Lactoferrin and CD91.

Within hours after intracerebral hemorrhage (ICH) onset, masses of polymorphonuclear neutrophils (PMNs) infiltrate the ICH-affected brain. After degranulation involving controlled release of many toxic antimicrobial molecules, the PMNs undergo rapid apoptosis and then are removed by phagocytic microglia/macrophages (MΦ) through a process called efferocytosis. Effective removal of PMNs may limit secondary brain damage and inflammation; however, the molecular mechanisms governing these cleanup activities are not well understood. We propose that scavenger receptor CD91 on myeloid phagocytes especially in presence of CD91 ligand, LTF (lactoferrin, protein abundant in PMNs), plays an important role in clearance of dead apoptotic PMNs (ANs). Mice/rats were subjected to an autologous blood injection model of ICH. Primary cultured microglia were used to assess phagocytosis of ANs. Immunohistochemistry was employed to assess CD91 expression and PMN infiltration. CD91 knockout mice selectively in myeloid phagocytes (Mac-CD91-KO) were used to establish the CD91/LTF function in phagocytosis and in reducing ICH-induced injury, as assessed using behavioral tests, hematoma resolution, and oxidative stress. Masses of PMNs are found in ICH-affected brain, and they contain LTF. MΦ at the outer border of hematoma are densely packed, expressing CD91 and phagocytosing ANs. Microglia deficient in CD91 demonstrate defective phagocytosis of ANs, and mice deficient in CD91 (Mac-CD91-KO) subjected to ICH injury have increased neurological dysfunction that is associated with impaired hematoma resolution (hemoglobin and iron clearance) and elevated oxidative stress. LTF that normally ameliorates ICH injury in CD91-proficient control mice shows reduced therapeutic effects in Mac-CD91-KO mice. Our study suggests that CD91 plays a beneficial role in improving ANs phagocytosis and ultimately post-ICH outcome and that the beneficial effect of LTF in ICH is in part dependent on presence of CD91 on MΦ.

INJECTION OF AUTOLOGOUS WHOLE BLOOD VERSUS CORTICOSTEROID FOR LATERAL EPICONDYLITIS: A COMPARATIVE STUDY

Objective: The objective is to compare the efficacy, in terms of pain relief and functional improvement, of autologous whole blood and corticosteroid injection in managing lateral epicondylitis (Tennis elbow). Methods: This was a comparative study conducted in Department of Orthopedics, National Institute of Medical Sciences Jaipur, Rajasthan, India. Eighty patients diagnosed to be having lateral epicondylitis were included in this study on the basis of predefined inclusion and exclusion criteria. The patients were divided into 2 groups on the basis of whether they were given autologous whole blood (Group B) or corticosteroid injection (Group S). Efficacy of both the treatment modality was compared in terms of Pain relief (as assessed by reduction in visual analogue score [VAS] score) and functional improvement (as assessed by improvement in Mayo Elbow performance score). SSPS 22.0 software was used for statistical analysis and p<0.05 was taken as statistically significant. Results: A total of 80 patients were included in this study out of which there were 37 (46.25%) males and 43 (53.75%) females with a M: F ratio of 1:1.16. The mean age of patients in group B and group S was found to be found to be 52.36±9.98 years and 48.46±10.12 years, respectively. The mean age of patients in both the groups was found to be comparable with no statistically significant difference (p=0.0866). At the time of 4 weeks, 8 weeks, and 12 weeks follow–up, the mean VAS score in Group B was less as compared to group S and the difference was found to be statistically highly significant (p<0.0001). At the time of 4 weeks, 8 weeks, and 12 weeks’ follow-up, the mean MEPS in Group B was more as compared to group S, and the difference was found to be statistically significant (p<0.05). Conclusion: For lateral epicondylitis, whole blood injection demonstrated superior efficacy in terms of pain relief and functional improvement as compared to injection of corticosteroid.

Granulocyte colony-stimulating factor induces the migration of non-resident cells to intracerebral hemorrhage

The aim of the study is to determine the effect of granulocyte colony-stimulating factor (rHuG-CSF) on the migration of non-resident cells into the region of intracerebral hemorrhage (ICH), which can potentially be considered as mesenchymal stem cells. Materials and methods. In the experiment, unilateral ICH was simulated in rats by injection of autologous blood into the right hemisphere. Immunohistochemical method was used to study perihematomal area on day 1, 3, 10, 30, and 60 after ICH modeling. The appearance of cells expressing CD44, CD68, and CD146 markers was evaluated on a 3-point scale after penetrating trauma (PT) of the brain, ICH, and ICH with the growth factor injection (ICH/rHuG-CSF). Results. In contrast to PT, ICH intensified the infiltration of CD44+ cells in the perihematomal area of the brain and caused the appearance of CD68+ and CD146+ cells in hemorrhage. The specific density of CD44+ cells was decreased on day 60 after ICH, and the effect of rHuG-CSF consisted in reducing the density of CD44+ cells on days 30 and 60. The appearance of CD68+ and CD146+ cells in the perihematomal area was seen after 10 days and further their detection was rare, while after the rHuG-CSF injection, the infiltration by cells with a similar immunophenotype was detected on day 3 ICH with a tendency to accumulate. Conclusions. Infiltration by cells expressing CD44, CD68, and CD146 into the perihematomal area of the rat brain was heterogeneous as to the time after brain injury. The appearance of cells with specified immunophenotype could be associated with hematoma elimination, angiogenesis processes, and other mechanisms of the perihematomal brain region remodeling, in which mesenchymal stem cells were involved. rHuG-CSF modulated the migration of CD68+ and CD146+ cells, which consisted in their earlier and more intense migration to the damaged area of the brain after ICH.

Kv1.3 Blockade Alleviates White Matter Injury through Reshaping M1/M2 Phenotypes via the NF-κB Signaling Pathway after Intracerebral Hemorrhage.

White matter injury (WMI) in basal ganglia usually induces long-term disability post intracerebral hemorrhage (ICH). Kv1.3 is an ion channel expressed in microglia and induces neuroinflammation after ICH. Here, we investigated the functions and roles of Kv1.3 activation-induced inflammatory response in WMI and the Kv1.3 blockade effect on microglia polarization after ICH. Mice ICH model was constructed by autologous blood injection. The expression of Kv1.3 was measured using immunoblot, real-time quantitative polymerase chain reaction (RT-qPCR), and immunostaining assays. Then, the effect of administration of 5-(4-Phenoxybutoxy) psoralen (PAP-1), a selectively pharmacological Kv1.3 blocker, was investigated using open field test (OFT) and basso mouse score (BMS). RT-qPCR, immunoblot, and enzyme-linked immunosorbent assay (ELISA) were taken to elucidate the expression of pro-inflammatory or anti-inflammatory factors around hematoma. PAP-1's function in regulating microglia polarization was investigated using immunoblot, RT-qPCR, and immunostaining assays. The downstream PAP-1 signaling pathway was determined by RT-qPCR and immunoblot. Kv1.3 expression was increased in microglia around the hematoma significantly after ICH. PAP-1 markedly improved neurological outcomes and the WMI by reducing pro-inflammatory cytokine accumulation and upregulating anti-inflammatory factors. Mechanistically, PAP-1 reduces NF-κB p65 and p50 activation, thus facilitating microglia polarization into M2-like microglia, which exerts this beneficial effect. PAP-1 reduced pro-inflammatory cytokines accumulation and increased anti-inflammatory factors by facilitating M2-like microglia polarization via the NF-κB signaling pathway. Thus, the current study shows that the Kv1.3 blockade is capable of ameliorating WMI by facilitating M2-like phenotype microglia polarization after ICH.

IL-10 protects against OPC ferroptosis by regulating lipid reactive oxygen species levels post stroke

Accumulation of reactive oxygen species (ROS), especially on lipids, induces massive cell death in neurons and oligodendrocyte progenitor cells (OPCs) and causes severe neurologic deficits post stroke. While small compounds, such as deferoxamine, lipostatin-1, and ferrostatin-1, have been shown to be effective in reducing lipid ROS, the mechanisms by which endogenously protective molecules act against lipid ROS accumulation and subsequent cell death are still unclear, especially in OPCs, which are critical for maintaining white matter integrity and improving long-term outcomes after stroke. Here, using mouse primary OPC cultures, we demonstrate that interleukin-10 (IL-10), a cytokine playing roles in reducing neuroinflammation and promoting hematoma clearance, significantly reduced hemorrhage-induced lipid ROS accumulation and subsequent ferroptosis in OPCs. Mechanistically, IL-10 activated the IL-10R/STAT3 signaling pathway and upregulated the DLK1/AMPK/ACC axis. Subsequently, IL-10 reprogrammed lipid metabolism and reduced lipid ROS accumulation. In addition, in an autologous blood injection intracerebral hemorrhagic stroke (ICH) mouse model, deficiency of the endogenous Il-10, specific knocking out Il10r or Dlk1 in OPCs, or administration of ACC inhibitor was associated with increased OPC cell death, demyelination, axonal sprouting, and the cognitive deficits during the chronic phase of ICH and vice versa. These data suggest that IL-10 protects against OPC loss and white matter injury by reducing lipid ROS, supporting further development of potential clinical applications to benefit patients with stroke and related disorders.

Validity of D-penicillamine in experimental cerebral vasospasm therapy

BackgroundCerebral vasospasm is a reversible vessel constriction following subarachnoid hemorrhage. Immunoreactive and inflammatory mechanisms induce long-term vasoconstriction and vessel dilatation inhibition. We hypothesized that D-penicillamine depresses this process given that it reduces the concentration of free oxygen (O2) radicals. We evaluated the effects of D-penicillamine on cerebral vasospasm in rabbits induced with subarachnoid hemorrhage, achieved by autologous blood injection in the basal cistern. Materials and methodsFour experimental groups were studied: Group 1: basilar angiography, Group 2: basilar angiography + D-penicillamine, Group 3: subarachnoid hemorrhage (SAH), and Group 4: SAH + D-penicillamine. Vessel diameter and cerebral vasospasm were evaluated angiographically in each group. Vascular degeneration was studied by electron microscopy of the basilar artery. Free O2 radicals were investigated based on measurements of the levels of vascular superoxide dismutase and malondialdehyde. Myasthenia gravis–like side effects of D-penicillamine application were analyzed by electromyography and electron microscopy of the orbicularis oculi muscle. ResultsWe detected a 9.03% decrease in basilar artery constriction in Group 4 compared with Group 3. In addition, levels of free O2 radicals were reduced in Groups 2 and 4. Application of D-penicillamine resulted in the prevention of basilar artery wall degeneration, without myasthenia gravis–like side effects. ConclusionOur study indicated that D-penicillamine application induced vasodilatation and had anti-inflammatory effects.

Establishment of a rat model of severe spontaneous intracerebral hemorrhage

BackgroundSevere 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. MethodsA rat model of severe ICH model was established by intrastriatal injection of autologous blood using different blood volumes (ICH 100 µL group, ICH 130 µL group, ICH 160 µL group, ICH 170 µL group, and ICH 180 µL 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. ResultsThe mortality of ICH rats increased significantly with an increase in autologous blood injection. The 28-day mortality in the 100 µL, 130 µL, 160 µL, 170 µL, and 180 µL ICH groups were 5%, 20%, 40%, 75%, and 100%, respectively. A significantly higher 28-day mortality was observed in the ICH 160 µL group compared to the ICH 100 µL group. The ICH 160 µL 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 160 µL 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 160 µL group than the sham group. ConclusionsA severe ICH model in rats was successfully established using an injection of autologous blood at a volume of 160 µL. This model may provide a valuable tool to examine the pathological mechanisms and potential therapeutic interventions of severe ICH.

The phosphokinase activity of IRE1ɑ prevents the oxidative stress injury through miR-25/Nox4 pathway after ICH.

Endoplasmic reticulum (ER) stress and oxidative stress are the major pathologies encountered after intracerebral hemorrhage (ICH). Inositol-requiring enzyme-1 alpha (IRE1α) is the most evolutionarily conserved ER stress sensor, which plays a role in monitoring and responding to the accumulation of unfolded/misfolded proteins in the ER lumen. Recent studies have shown that ER stress is profoundly related to oxidative stress in physiological or pathological conditions. The purpose of this study was to investigate the role of IRE1α in oxidative stress and the potential mechanism. A mouse model of ICH was established by autologous blood injection. The IRE1α phosphokinase inhibitor KIRA6 was administrated intranasally at 1 h after ICH, antagomiR-25 and agomiR-25 were injected intraventricularly at 24 h before ICH. Western blot analysis, RT-qPCR, immunofluorescence staining, hematoma volume, neurobehavioral tests, dihydroethidium (DHE) staining, H2O2 content, brain water content, body weight, Hematoxylin and Eosin (HE) staining, Nissl staining, Morris Water Maze (MWM) and Elevated Plus Maze (EPM) were performed. Endogenous phosphorylated IRE1α (p-IRE1α), miR-25-3p, and Nox4 were increased in the ICH model. Administration of KIRA6 downregulated miR-25-3p expression, upregulated Nox4 expression, promoted the level of oxidative stress, increased hematoma volume, exacerbated brain edema and neurological deficits, reduced body weight, aggravated spatial learning and memory deficits, and increased anxiety levels. Then antagomiR-25 further upregulated the expression of Nox4, promoted the level of oxidative stress, increased hematoma volume, exacerbated brain edema and neurological deficits, whereas agomiR-25 reversed the effects promoted by KIRA6. The IRE1α phosphokinase activity is involved in the oxidative stress response through miR-25/Nox4 pathway in the mouse ICH brain.