Mild hypothermia therapy attenuates early BBB leakage in acute ischaemic stroke.

Reperfusion therapy inevitably leads to brain-blood barrier (BBB) disruption and promotes damage despite its benefits for acute ischaemic stroke (AIS). An effective brain cytoprotective treatment is still needed as an adjunct to reperfusion therapy. Here, we explore the potential benefits of therapeutic hypothermia (HT) in attenuating early BBB leakage and improving neurological outcomes. Mild HT was induced during the early and peri-recanalization stages in a mouse model of transient middle cerebral artery occlusion and reperfusion (tMCAO/R). The results showed that mild HT attenuated early BBB leakage in AIS, decreased the infarction volume, and improved functional outcomes. RNA sequencing data of the microvessels indicated that HT decreased the transcription of the actin polymerization-related pathway. We further discovered that HT attenuated the ROCK1/MLC pathway, leading to a decrease in the polymerization of G-actin to F-actin. Arachidonic acid (AA), a known structural ROCK agonist, partially counteracted the protective effects of HT in the tMCAO/R model. Our study highlights the importance of early vascular protection during reperfusion and provides a new strategy for attenuating early BBB leakage by HT treatment for ischaemic stroke.

Regulating Second-Shell Coordination in Cobalt Single-Atom Catalysts toward Highly Selective Hydrogenation.

Manipulating the local coordination environment of central metal atoms in single-atom catalysts (SACs) is a powerful strategy to exploit efficient SACs with optimal electronic structures for various applications. Herein, Co-SACs featured by Co single atoms with coordinating S atoms in the second shell dispersed in a nitrogen-doped carbon matrix have been developed toward the selective hydrogenation of halo-nitrobenzene. The location of the S atom in the model Co-SAC is verified through synchrotron-based X-ray absorption spectroscopy and theoretical calculations. The resultant Co-SACs containing second-coordination shell S atoms demonstrate excellent activity and outstanding durability for selective hydrogenation, superior to most precious metal-based catalysts. In situ characterizations and theoretical results verify that high activity and selectivity are attributed to the advantageous formation of the Co-O bond between p-chloronitrobenzene and Co atom at Co1N4-S moieties and the lower free energy and energy barriers of the reaction. Our findings unveil the correlation between the performance and second-shell coordination atom of SACs.

Method validation and dissipation kinetics of the novel HPPD-inhibiting herbicide cypyrafluone in winter wheat using QuEChERS method coupled with UPLC-MS/MS.

Cypyrafluone, a novel hydroxyphenylpyruvate dioxygenase (HPPD)-inhibiting herbicide, can successfully control a wide species of grass and broadleaf weed in wheat fields. However, the dissipation behaviors and terminal residues of cypyrafluone in wheat fields remain unclear. Here, a simple, accurate, and dependable approach for the analysis of cypyrafluone in soil, wheat plant, and grain was constructed utilizing an adapted QuEChERS extraction combined with UPLC-MS/MS. For accurate quantification, matrix-matched calibrations with high linearity (R2 >0.99) were employed to eliminate matrix interference. The method possessed high accuracy with recoveries in the range of 85.5%- 100.6% and precision with relative standard deviations < 14.3%, as well as high sensitivity with limits of quantifications of 0.001 mg kg-1 in the three matrixes. The dissipation kinetics and terminal residues of cypyrafluone were determined at two separate locations with different climates, soil types and cropping systems in 2018. The half-lives of cypyrafluone in soil and wheat plant were 1.47-1.55 d and 1.00-1.03 d, respectively. At harvest, the terminal residue values of cypyrafluone detected in wheat plants were 0-0.0025 mg kg-1 and 0.0044-0.0057 mg kg-1 at the recommended dose and 1.5 times of the recommended dose, respectively, and 0.0049 mg kg-1 of this herbicide was detected in grain at 1.5 times of the recommended dose, which was below the maximum residue limit (MRL). Finally, the risk quotient for cypyrafluone ranged from 0.33% to 0.81% (<1) for different age groups in China, indicating that the impact of residues from the cypyrafluone application on wheat was acceptable. These findings above will offer scientific guidelines for cypyrafluone application in the wheat field ecosystem.

Enhanced Herbicide Metabolism and Target-Site Mutations Confer Multiple Resistance to Fomesafen and Nicosulfuron in Amaranthus retroflexus L.

Amaranthus retroflexus L. is a highly competitive broadleaf weed of corn-soybean rotation in northeastern China. In recent years, the herbicide(s) resistance evolution has been threatening its effective management in crop fields. One resistant A. retroflexus (HW-01) population that survived the protoporphyrinogen oxidase (PPO) inhibitor fomesafen and acetolactate synthase (ALS) inhibitor nicosulfuron applied at their field-recommended rate was collected from a soybean field in Wudalianchi City, Heilongjiang Province. This study aimed to investigate the resistance mechanisms of fomesafen and nicosulfuron and determine the resistance profile of HW-01 to other herbicides. Whole plant dose-response bioassays revealed that HW-01 had evolved resistance to fomesafen (50.7-fold) and nicosulfuron (5.2-fold). Gene sequencing showed that the HW-01 population has a mutation in PPX2 (Arg-128-Gly) and a rare mutation in ALS (Ala-205-Val, eight/twenty mutations/total plants). In vitro enzyme activity assays showed that ALS extracted from the HW-01 plants was less sensitive to nicosulfuron (3.2-fold) than ST-1 plants. Pre-treatment with the cytochrome P450 inhibitors malathion, piperonyl butoxide (PBO), 3-amino-1,2,4-triazole (amitrole), and the GSTs inhibitor 4-chloro-7-nitrobenzofurazan (NBD-Cl) significantly increased fomesafen and nicosulfuron sensitivity in the HW-01 population compared with that of the sensitive (S) population ST-1. Moreover, the rapid fomesafen and nicosulfuron metabolism in the HW-01 plants was also confirmed via HPLC-MS/MS analysis. Furthermore, the HW-01 population showed multiple resistance (MR) to PPO, ALS, and PSII inhibitors, with resistance index (RI) values ranging from 3.8 to 9.6. This study confirmed MR to PPO-, ALS-, and PSII-inhibiting herbicides in the A. retroflexus population HW-01, as well as confirming that the cytochrome P450- and GST-based herbicide metabolic along with TSR mechanisms contribute to their multiple resistance to fomesafen and nicosulfuron.

Branched-chain amino acids and risk of stroke: A Mendelian randomization study.

Background: The causality between plasma branched-chain amino acids (BCAAs) levels and stroke remains uncertain and the stratified research on the association between BCAAs levels and subtypes of stroke is not well studied. Therefore, the association of genetically proxied circulating BCAA levels with the risks of stroke and its subtypes was explored by Mendelian randomization (MR) in this study. Methods: Summary-level data derived from the published genome-wide association studies (GWAS) were employed for analyses. Data for plasma BCAA levels (n = 16,596) were obtained from a meta-analysis of GWAS. The MEGASTROKE consortium provided data for ischemic stroke (n = 440,328) and its subtypes and data for hemorrhagic stroke were available from 2 meta-analyses of GWAS of European-ancestry groups (intracerebral hemorrhage, n = 3,026; subarachnoid hemorrhage, n = 77,074). The inverse variance weighted (IVW) method was selected as the primary MR analysis. Supplementary analysis used included the weighted median, MR-Egger regression, Cochran's Q statistic, MR Pleiotropy Residual Sum and Outlier global test, and leave-one-out analysis method. Results: According to IVW analysis, 1-SD increment in genetically determined circulating isoleucine was associated with increased risks of cardioembolic stroke (CES) (OR: 1.56, 95% CI: 1.21-2.20, P = 0.0007), but not with risks of other stroke subtypes. We could not discover any proof that leucine and valine levels could increase risk of any stroke subtype. All heterogeneity tests produced stable findings, and there was no concrete evidence to indicate the perturbation of horizontal multiplicity. Conclusion: Increasing plasma isoleucine level had a causal effect on the risk of CES but not on the risk of other stroke subtypes. Further research is needed to identify the mechanisms of the causal associations between BCAAs and stroke subtypes.

General Strategy toward Hydrophilic Single Atom Catalysts for Efficient Selective Hydrogenation.

Well dispersible and stable single atom catalysts (SACs) with hydrophilic features are highly desirable for selective hydrogenation reactions in hydrophilic solvents towards important chemicals and pharmaceutical intermediates. A general strategy is reported for the fabrication of hydrophilic SACs by cation-exchange approach. The cation-exchange between metal ions (M = Ni, Fe, Co, Cu) and Na+ ions introduced in the skeleton of metal oxide (TiO2 or ZrO2 ) nanoshells plays the key role in forming M1 /TiO2 and M1 /ZrO2 SACs, which efficiently prevents the aggregation of the exchanged metal ions. The as-obtained SACs are highly dispersible and stable in hydrophilic solvents including alcohol and water, which greatly facilitates the catalysis reaction in alcohol. The Ni1 /TiO2 SACs have been successfully utilized as catalysts for the selective C=C hydrogenation of cinnamaldehyde to produce phenylpropanal with 98% conversion, over 90% selectivity, good recyclability, and a turnover frequency (TOF) of 102 h-1 , overwhelming most reported catalysts including noble metal catalysts.

A clinically relevant model of focal embolic cerebral ischemia by thrombus and thrombolysis in rhesus monkeys.

Over decades of research into the treatment of stroke, nearly all attempts to translate experimental treatments from discovery in cells and rodents to use in humans have failed. The prevailing belief is that it might be necessary to pretest pharmacological neuroprotection in higher-order brains, especially those of nonhuman primates (NHPs). Over the past few years, chemical thrombolysis and mechanical thrombectomy have been established as the standard of care for ischemic stroke in patients. The spotlight is now shifting towards emphasizing both focal ischemia and subsequent reperfusion in developing a clinically relevant stroke model in NHPs. This protocol describes an embolic model of middle cerebral artery occlusion in adult rhesus monkeys. An autologous clot is combined with a microcatheter or microwire through endovascular procedures, and reperfusion is achieved through local intra-artery thrombolysis with tissue plasminogen activator. These NHP models formed relatively stable infarct sizes, delivered predictable reperfusion and survival outcomes, and recapitulated key characteristics of patients with ischemic stroke as observed on MRI images and behavioral assays. Importantly, treated animals could survive 30 d after the surgery for post-stroke neurologic deficit analyses. Thus far, this model has been used in several translational studies. Here we describe in detail the teamwork necessary for developing stroke models of NHPs, including the preoperation preparations, endovascular surgery, postoperation management and histopathological analysis. The model can be established by the following procedures over a 45-d period, including preparation steps (14 d), endovascular operation (1 d) and evaluation steps (30 d).

Phenotype Shifting in Astrocytes Account for Benefits of Intra-Arterial Selective Cooling Infusion in Hypertensive Rats of Ischemic Stroke.

The translational failure of neuroprotective therapies in stroke may be influenced by the mismatch of existing comorbidities between animal models and patients. Previous studies found that single-target neuroprotective agents reduced infarction in Sprague-Dawley but not in spontaneously hypertensive rats. It is of great interest to explore whether multi-target neuroprotectants and stroke models with comorbidities should be used in further translational researches. Ischemic stroke was induced in normotensive or hypertensive rats by 90- or 120-min middle cerebral artery occlusion (MCAO) and reperfusion. Intra-Arterial Selective Cooling Infusion (IA-SCI) was started at the onset of reperfusion for 30 minutes. Acute neurological deficits, infarct volumes, gene expression and markers of A1-like and A2-like astrocytes were evaluated. In further analysis, TNFα and IL-1α were administrated intracerebroventricularly, phenotype shifting of astrocytes and infarct volumes were assessed. Normobaric oxygen treatment, as a negative control, was also assessed in hypertensive rats. IA-SCI led to similar benefits in normotensive rats with 120-min MCAO and hypertensive rats with both 90-min and 120-min MCAO, including mitigated functional deficit and reduced infarct volumes. IA-SCI shifted astrocyte phenotypes partly by downregulating A1-like astrocytes and upregulating A2-like astrocytes in both RNA and protein levels. Upregulated A1-type astrocyte markers levels, induced by intracerebroventricular injection of TNFα and IL-1α, were closely related to increased infarct volumes in hypertensive rats, despite receiving IA-SCI treatment. In addition, infarct volumes and A1/A2-like genes were not affected by normobaric oxygen treatment. IA-SCI reduced infarction in both normotensive and hypertensive rats. Our results demonstrated the neuroprotective effects of IA-SCI in hypertensive rats may be related with phenotype shifting of astrocytes.

CCL2 (C-C Motif Chemokine Ligand 2) Biomarker Responses in Central Versus Peripheral Compartments After Focal Cerebral Ischemia.

Background and Purpose: Inflammatory mediators in blood have been proposed as potential biomarkers in stroke. However, a direct relationship between these circulating factors and brain-specific ischemic injury remains to be fully defined. Methods: An unbiased screen in a nonhuman primate model of stroke was used to find out the most responsive circulating biomarker flowing ischemic stroke. Then this phenomenon was checked in human beings and mice. Finally, we observed the temporospatial responsive characteristics of this biomarker after ischemic brain injury in mice to evaluate the direct relationship between this circulating factor and central nervous system­specific ischemic injury. Results: In a nonhuman primate model, an unbiased screen revealed CCL2 (C-C motif chemokine ligand 2) as a major response factor in plasma after stroke. In mouse models of focal cerebral ischemia, plasma levels of CCL2 showed a transient response, that is, rapidly elevated by 2 to 3 hours postischemia but then renormalized back to baseline levels by 24 hours. However, a different CCL2 temporal profile was observed in whole brain homogenate, cerebrospinal fluid, and isolated brain microvessels, with a progressive increase over 24 hours, demonstrating a mismatch between brain versus plasma responses. In contrast to the lack of correlation with central nervous system responses, 2 peripheral compartments showed transient profiles that matched circulating plasma signatures. CCL2 protein in lymph nodes and adipose tissue was significantly increased at 2 hours and renormalized by 24 hours. Conclusions: These findings may provide a cautionary tale for biomarker pursuits in plasma. Besides a direct central nervous system response, peripheral organs may also contribute to blood signatures in complex and indirect ways.

Quantitative Proteomic Analysis of Plasma after Remote Ischemic Conditioning in a Rhesus Monkey Ischemic Stroke Model.

BACKGROUND: Animal and clinical studies have shown that remote ischemic conditioning (RIC) has protective effects for cerebral vascular diseases, with induced humoral factor changes in the peripheral blood. However, many findings are heterogeneous, perhaps due to differences in the RIC intervention schemes, enrolled populations, and sample times. This study aimed to examine the RIC-induced changes in the plasma proteome using rhesus monkey models of strokes. METHODS: Two adult rhesus monkeys with autologous blood clot-induced middle cerebral artery (MCA) occlusion underwent RIC interventions twice a week for five consecutive weeks. Each RIC treatment included five cycles of five minutes of ischemia alternating with five minutes of reperfusion of the forearm. The blood samples were taken from the median cubital vein of the monkeys at baseline and immediately after each week's RIC stimulus. The plasma samples were isolated for a proteomic analysis using mass spectrometry (MS). RESULTS: Several proteins related to lipid metabolism (Apolipoprotein A-II and Apolipoprotein C-II), coagulation (Fibrinogen alpha chain and serpin), immunoinflammatory responses (complement C3 and C1), and endovascular hemostasis (basement membrane-specific heparan sulfate proteoglycan) were significantly modulated after the RIC intervention. Many of these induced changes, such as in the lipid metabolism regulation and anticoagulation responses, starting as early as two weeks following the RIC intervention. The complementary activation and protection of the endovascular cells occurred more than three weeks postintervention. CONCLUSIONS: Multiple protective effects were induced by RIC and involved lipid metabolism regulation (anti-atherogenesis), anticoagulation (antithrombosis), complement activation, and endovascular homeostasis (anti-inflammation). In conclusion, this study indicates that RIC results in significant modulations of the plasma proteome. It also provides ideas for future research and screening targets.

Designing of bacterial cellulose-based superhydrophilic/underwater superoleophobic membrane for oil/water separation.

The oil/water (o/w) separation is a global challenge because of the increasing water contamination by oil spill accidents, and oil-containing wastewater produced by food, textile, and petrochemical industries. In this study, we have developed bacterial cellulose (BC) based superhydrophilic/underwater superoleophobic (SUS) membrane for o/w separation. The membrane was designed through a facile method by blending BC nanofibers with silica microparticles (SiO2-MPs), which was further modified by bio-inspired polydopamine (PDA) coatings. The composite membrane exhibited SiO2-MPs dependent o/w separation with a high separation efficiency of >99.9 % and a high flux rate of ∼10,660 Lm-2 h-1 while applying a small negative pressure (0.3-0.5 bar). The membrane with different content of SiO2-MPs also showed the potential to separate oil-in-water emulsion with the highest oil rejection of 98.2 % and the highest flux rate of ∼1250 Lm-2 h-1 on an ultra-low pressure <0.1 bar. Moreover, the membrane showed antifouling properties, recyclability, and stability in harsh conditions.

Selective intra-arterial brain cooling improves long-term outcomes in a non-human primate model of embolic stroke: Efficacy depending on reperfusion status.

Nearly all stroke neuroprotection modalities, including selective intra-arterial cooling (SI-AC), have failed to be translated from bench to bed side. Potentially overlooked reasons may be biological gaps, inadequate attention to reperfusion states and mismatched attention to neurological benefits. To advance stroke translation, we describe a novel thrombus-based stroke model in adult rhesus macaques. Intra-arterial thrombolysis with tissue plasminogen activator leads to three clinically relevant outcomes - complete, partial, and no recanalization based on digital subtraction angiography. We also find reperfusion as a prerequisite for SI-AC-induced benefits, in which models with complete or partial reperfusion exhibit significantly reduced infarct volumes, mitigated neurological deficits, improved upper limb motor dysfunction in both acute and chronic stages; however, no further neuroprotection is observed in those without reperfusion. In summary, we discover reperfusion as a crucial regulator of SI-AC-induced neuroprotection and provide insights of long-term functional benefits in behavior and imaging levels. Our findings could be important not only for the translational prerequisite and potential molecular targets, but also for this thrombus-thrombolysis model in monkeys as a powerful tool for further translational stroke studies.

New Endovascular Approach for Hypothermia With Intrajugular Cooling and Neuroprotective Effect in Ischemic Stroke.

Background and Purpose- Induction of hypothermia as a stroke therapy has been limited by logistical challenges. This study was designed to determine the hypothermic and neuroprotective efficacy of infusing cold saline directly into the internal jugular (IJ) vein and compare the effects of IJ hypothermia to those achieved by intracarotid artery hypothermia in an ischemic stroke model. Methods- The right middle cerebral artery was occluded in rats using an intraluminal filament. Immediately following reperfusion, hypothermia was achieved by infusing isotonic saline through microcatheter into the right IJ or right intracarotid over 30 minutes. Infarct sizes, neurological deficits, blood-brain barrier damage, edema volume, blood-brain barrier associated molecules (MMP-9 [matrix metallopeptidase 9] and AQP-4 [aquaporin 4]), and apoptosis-associated proteins (Bcl-2 and cleaved Caspase-3) were measured. Results- We found that both IJ- and intracarotid-based infusion cooled the brain robustly with a minimal effect on rectal temperatures. This brain cooling led to significantly reduced infarct volumes at 24 hours after reperfusion, as well as decreased expression of the proapoptotic protein cleaved Caspase-3 and increased expression of the antiapoptotic protein Bcl-2. Intracarotid and IJ cooling also aided in blood-brain barrier maintenance, as shown by decreased edema volumes, reduced Evans Blue leakage, and decreased expression of edema-facilitating proteins (MMP-9 and AQP-4). Both cooling methods then translated to preserved neurological function as determined by multiple functional tests over a 28-day observation period. Most importantly, the cooling and neuroprotective efficacy of IJ cooling was comparable to intracarotid cooling by almost every metric evaluated. Conclusions- Compared with intracarotid infusion, IJ infusion conferred a similar degree of hypothermia and neuroprotection following ischemic stroke. Given the ease of establishing vascular access via the internal jugular vein and the powerful neuroprotection that hypothermia provides, IJ brain cooling could be used as a promising hypothermia-induction modality going forward.

Reusable ternary PVA films containing bacterial cellulose fibers and ε-polylysine with improved mechanical and antibacterial properties.

The combination of high mechanical properties, antibacterial activity and a green synthesis of the polyvinyl alcohol (PVA) based films remains challenging. This study presents a ternary system of PVA films containing bacterial cellulose (BC) and epsilon-polylysine (ε-PL) by a green solution casting method. The prepared composite films showed more than 99% antibacterial properties against both Staphylococcus aureus and Escherichia coli bacteria. Moreover, the films were collected after a single use and were reused twice, which still exhibited strong antibacterial activity. The films showed thermal stability and higher mechanical properties as compared to pure PVA films. In addition, the cytotoxicity of the films was evaluated by MTT assay against NIH 3T3 mouse fibroblast cells. The results showed no toxicity of the films towards tested cells. We believe that these antibacterial films may find applications in active food packaging and biomedical fields.

Neuroprotection by mesenchymal stem cell (MSC) administration is enhanced by local cooling infusion (LCI) in ischemia.

OBJECTIVE: The present study aimed to determine if hypothermia augments the neuroprotection conferred by MSC administration by providing a conducive micro-environment. METHODS: Sprague-Dawley rats were subjected to 1.5 h middle cerebral artery occlusion (MCAO) followed by 6 or 24 h of reperfusion for molecular analyses, as well as 1, 14 and 28 days for brain infarction or functional outcomes. Rats were treated with either MSC (1 × 105), LCI (cold saline, 0.6 ml/min, 5 min) or both. Brain damage was determined by Infarct volume and neurological deficits. Long-term functional outcomes were evaluated using foot-fault and Rota-rod testing. Human neural SHSY5Y cells were investigated in vitro using 2 h oxygen-glucose deprivation (OGD) followed by MSC with or without hypothermia (HT) (34 °C, 4 h). Mitochondrial transfer was assessed by confocal microscope, and cell damage was determined by cell viability, ATP, and ROS level. Protein levels of IL-1ß, BAX, Bcl-2, VEGF and Miro1 were measured by Western blot following 6 h and 24 h of reperfusion and reoxygenation. RESULTS: MSC, LCI, and LCI + MSC significantly reduced infarct volume and deficit scores. Combination therapy of LCI + MSC precipitated better long-term functional outcomes than monotherapy. Upregulation of Miro1 in the combination group increased mitochondrial transfer and lead to a greater increase in neuronal cell viability and ATP, as well as a decrease in ROS. Further, combination therapy significantly decreased expression of IL-1ß and BAX while increasing Bcl-2 and VEGF expression. CONCLUSION: Therapeutic hypothermia upregulated Miro1 and enhanced MSC mitochondrial transfer-mediated neuroprotection in ischemic stroke. Combination of LCI with MSC therapy may facilitate clinical translation of this approach.

A facile construction of bacterial cellulose/ZnO nanocomposite films and their photocatalytic and antibacterial properties.

Bacterial cellulose (BC) has numerous excellent properties but the absence of antibacterial activity restricts its applications in biomedical field. Therefore, in order to introduce the antibacterial characteristics into BC; herein, a facile method for incorporation of ZnO nanoparticles (ZnO-NPs) is presented. BC films were first immersed in zinc nitrate solution, followed by treating with NaOH solution, the BC loaded ZnO nanocomposite films were dried by a sheet former instrument at 80 °C for 20 min. The obtained BC/ZnO nanocomposites were characterized by different techniques. XRD results showed the hexagonal wurtzite structure of ZnO-NPs while FE-SEM results displayed the particle size of ZnO-NPs was ranging from 70 to 100 nm. Thermogravimetric study revealed the thermal stability of nanocomposite films. The nanocomposite exhibited photocatalytic activity and revealed 91% degradation of methyl orange (MO) under UV-irradiation within 2 h. Moreover, the nanocomposites demonstrated significant UV-blocking properties and showed antibacterial activities against tested Gram-positive and Gram-negative bacterial strains. This work provides a simple and novel method for the synthesis of BC/ZnO nanocomposite as a functional biomaterial.

Short-term remote ischemic conditioning may protect monkeys after ischemic stroke.

Objective: We aimed to evaluate the safety and effectiveness of short-term remote ischemic postconditioning (RIPC) in acute stroke monkey models. Methods: Acute stroke monkeys were allocated to four groups based on the number of limbs exposed to RIPC. RIPC was initiated by 5-min cuff inflation/deflation cycles of the target limb(s) for 5-10 bouts. Vital signs, skin integrity, brain MRI, and serum levels of cardiac enzymes (myoglobin, creatine kinase [CK], CK-muscle/brain [CK-MB]), one inflammatory marker (high-sensitivity C-reactive protein [hsCRP], and one endothelial injury marker (von Willebrand factor [vWF]) were assessed. Spetzler scores were used to assess neurological function. Results: No significant differences in vital signs or local skin integrity were found. Short-term RIPC did not reduce infarct volume under any condition at the 24th hour after stroke. However, neurological function improved in multi-limb RIPC compared with sham and single-limb RIPC at the 30th day follow-up after stroke. Myoglobin, CK, and CK-MB levels were reduced after multi-limb RIPC, regardless of the number of bouts. Moreover, multi-limb RIPC produced a greater diminution in CK-MB levels, whereas two-limb RIPC was more effective in reducing serum CK levels at the 24th hour after stroke. hsCRP increased after 5 bouts of multi-limb RIPC before decreasing below baseline and single-limb RIPC levels. Serum vWF was decreased at later time points after RIPC in all RIPC groups. Conclusions: Stroke monkeys in hyperacute stage may benefit from short-term RIPC; however, whether this intervention can be translated into clinical use in patients with acute ischemic stroke warrants further study.

Inflammatory cytokines are involved in dihydrocapsaicin (DHC) and regional cooling infusion (RCI)-induced neuroprotection in ischemic rat.

OBJECTIVE: The combination of pharmacological hypothermia - dihydrocapsaicin (DHC) and intra-arterial regional cooling infusions (RCI) was found to enhance the efficiency of hypothermia and efficacy of hypothermia-induced neuroprotection in acute ischemic stroke. The aim of this study was to explore whether the combination could induce a long-term neuroprotective effects, as well as the underlying mechanism. METHODS: Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO) for 2 h using intraluminal hollow filament. The ischemic rats were randomized to receive pharmacological hypothermia by intraperitoneal (i.p.) injection of DHC, physical hypothermia by RCI of 6 ml cold saline (4 °C), the combination, and no treatment. Over a 21-day period, brain damage was determined by infarct volume with MRI, and neurological deficit with grid-walking and beam balance tests. Blood brain barrier (BBB) was assessed by Evans-Blue (EB) contents. Inflammatory cytokines were determined in peri-infarct area by antibody array and ELISA. RESULTS: The combination of DHC and RCI reduced (p < 0.05) infarct volume and neurologic deficit after stroke. BBB leakage and pro-inflammatory cytokines (IFN-γ, IL-2, and TNF-α) were significantly decreased (p < 0.05) because of the combination, while protective cytokines (IL-4 and IL-10) were increased (p < 0.05) in the peri-infarct area. CONCLUSIONS: The combination approach enhanced the efficacy of hypothermia-induced neuroprotection following ischemic stroke. Our findings provide a hint to translate the combination method from bench to bedside.

Chrysophanol attenuates nitrosative/oxidative stress injury in a mouse model of focal cerebral ischemia/reperfusion.

Nitrosative/oxidative stress plays an important role in neuronal death following cerebral ischemia/reperfusion (I/R). Chrysophanol (CHR) has been shown to afford significant neuroprotection on ischemic stroke, however, whether its mechanism is related to attenuating nitrosative/oxidative stress is not clear. In the present study, we investigated the effect of CHR on neuronal injury related to nitric oxide (NO) production by using mouse middle cerebral artery occlusion (MCAO) model. Our results revealed that nitrite plus nitrate (NOx-) and 3-nitrotyrosine (3-NT) levels increased in ischemic brain 14 days after reperfusion, and were subsequently attenuated by CHR treatment. Moreover, 3-NT is colocalized with NeuN and TUNEL, suggesting that neuronal apoptosis following I/R is associated with 3-NT and CHR suppresses NO-associated neuronal cell death. Accordingly, cleaved caspase-3 expression in ischemic brain was decreased by CHR treatment. I/R also decreased the activity of total superoxide dismutase (SOD) and manganese-dependent SOD (MnSOD), whilst increased reactive oxygen species (ROS) production significantly. Interestingly, CHR reversed this decrease in total SOD, and MnSOD activity, and inhibited ROS generation in the ischemic brain. Taken together, our results provide direct evidence suggesting that CHR attenuates nitrosative/oxidative stress injury induced by I/R, providing a novel therapeutic target in the treatment of acute ischemic stroke.

Synergistically Induced Hypothermia and Enhanced Neuroprotection by Pharmacological and Physical Approaches in Stroke.

Hypothermia is considered as a promising neuroprotective treatment for ischemic stroke but with many limitations. To expand its clinical relevance, this study evaluated the combination of physical (ice pad) and pharmacological [transient receptor potential vanilloid channel 1 (TRPV1) receptor agonist, dihydrocapsaicin (DHC)] approaches for faster cooling and stronger neuroprotection. A total of 144 male Sprague Dawley rats were randomized to 7 groups: sham (n=16), stroke only (n=24), stroke with physical hypothermia at 31ºC for 3 h after the onset of reperfusion (n=24), high-dose DHC (H-DHC)(1.5 mg/kg, n=24), low-dose DHC (L-DHC)(0.5 mg/kg, n=32) with (n=8) or without (n=24) external body temperature control at ~38 ºC (L-DHC, 38 ºC), and combination therapy (L-DHC+ ice pad, n=24). Rats were subjected to middle cerebral artery occlusion (MCAO) for 2 h. Infarct volume, neurological deficits and apoptotic cell death were determined at 24 h after reperfusion. Expression of pro- and anti-apoptotic proteins was evaluated by Western blot. ATP and reactive oxygen species (ROS) were detected by biochemical assays at 6 and 24 h after reperfusion. Combination therapy of L-DHC and ice pad significantly improved every measured outcome compared to monotherapies. Combination therapy achieved hypothermia faster by 28.6% than ice pad, 350% than L-DHC and 200% than H-DHC alone. Combination therapy reduced (p<0.05) neurological deficits by 63% vs. 26% with L-DHC. No effect was observed when using ice pad or H-DHC alone. L-DHC and ice pad combination improved brain oxidative metabolism by reducing (p<0.05) ROS at 6 and 24 h after reperfusion and increasing ATP levels by 42.9% compared to 25% elevation with L-DHC alone. Finally, combination therapy decreased apoptotic cell death by 48.5% vs. 24.9% with L-DHC, associated with increased anti-apoptotic protein and reduced pro-apoptotic protein levels (p<0.001). Our study has demonstrated that combining physical and pharmacological hypothermia is a promising therapeutic approach in ischemic stroke, and warrants further translational investigations.