ATF5-Mediated Mitochondrial Unfolded Protein Response (UPRmt) Protects Neurons Against Oxygen-Glucose Deprivation and Cerebral Ischemia.

BACKGROUND: The mitochondrial unfolded protein response (UPRmt) is an evolutionarily conserved mitochondrial response that is critical for maintaining mitochondrial and energetic homeostasis under cellular stress after tissue injury and disease. Here, we ask whether UPRmt may be a potential therapeutic target for ischemic stroke. METHODS: We performed the middle cerebral artery occlusion and oxygen-glucose deprivation models to mimic ischemic stroke in vivo and in vitro, respectively. Oligomycin and meclizine were used to trigger the UPRmt. We used 2,3,5-triphenyltetrazolium chloride staining, behavioral tests, and Nissl staining to evaluate cerebral injury in vivo. The Cell Counting Kit-8 assay and the Calcein AM Assay Kit were conducted to test cerebral injury in vitro. RESULTS: Inducing UPRmt with oligomycin protected neuronal cultures against oxygen-glucose deprivation. UPRmt could also be triggered with meclizine, and this Food and Drug Administration-approved drug also protected neurons against oxygen-glucose deprivation. Blocking UPRmt with siRNA against activating transcription factor 5 eliminated the neuroprotective effects of meclizine. In a mouse model of focal cerebral ischemia, pretreatment with meclizine was able to induce UPRmt in vivo, which reduced infarction and improved neurological outcomes. CONCLUSIONS: These findings suggest that the UPRmt is important in maintaining the survival of neurons facing ischemic/hypoxic stress. The UPRmt mechanism may provide a new therapeutic avenue for ischemic stroke.

SLC22A17 as a Cell Death-Linked Regulator of Tight Junctions in Cerebral Ischemia.

BACKGROUND: Beyond neuronal injury, cell death pathways may also contribute to vascular injury after stroke. We examined protein networks linked to major cell death pathways and identified SLC22A17 (solute carrier family 22 member 17) as a novel mediator that regulates endothelial tight junctions after ischemia and inflammatory stress. METHODS: Protein-protein interactions and brain enrichment analyses were performed using STRING, Cytoscape, and a human tissue-specific expression RNA-seq database. In vivo experiments were performed using mouse models of transient focal cerebral ischemia. Human stroke brain tissues were used to detect SLC22A17 by immunostaining. In vitro experiments were performed using human brain endothelial cultures subjected to inflammatory stress. Immunostaining and Western blot were used to assess responses in SLC22A17 and endothelial tight junctional proteins. Water content, dextran permeability, and electrical resistance assays were used to assess edema and blood-brain barrier (BBB) integrity. Gain and loss-of-function studies were performed using lentiviral overexpression of SLC22A17 or short interfering RNA against SLC22A17, respectively. RESULTS: Protein-protein interaction analysis showed that core proteins from apoptosis, necroptosis, ferroptosis, and autophagy cell death pathways were closely linked. Among the 20 proteins identified in the network, the iron-handling solute carrier SLC22A17 emerged as the mediator enriched in the brain. After cerebral ischemia in vivo, endothelial expression of SLC22A17 increases in both human and mouse brains along with BBB leakage. In human brain endothelial cultures, short interfering RNA against SLC22A17 prevents TNF-α (tumor necrosis factor alpha)-induced ferroptosis and downregulation in tight junction proteins and disruption in transcellular permeability. Notably, SLC22A17 could repress the transcription of tight junctional genes. Finally, short interfering RNA against SLC22A17 ameliorates BBB leakage in a mouse model of focal cerebral ischemia. CONCLUSIONS: Using a combination of cell culture, human stroke samples, and mouse models, our data suggest that SLC22A17 may play a role in the control of BBB function after cerebral ischemia. These findings may offer a novel mechanism and target for ameliorating BBB injury and edema after stroke.

Bioinformatics to analyze the differentially expressed genes in different degrees of Alzheimer's disease and their roles in progress of the disease.

Employing bioinformatics approaches, this investigation pinpointed pivotal differentially expressed genes (DEGs) across the spectrum of Alzheimer's disease (AD), from incipient to severe stages, using the GSE28146 dataset from the GEO repository. Analytical methods included DEG identification via the limma package in R, coupled with GO and KEGG pathway analyses through clusterProfiler, to discern biological processes and pathway involvements. Key findings spotlighted the roles of proteasome subunits PSMB4, PSMB8, PSMC4, and PSMD6 in the early stage, ribosomal proteins RPS3 and RPL11 during moderate AD, and mitochondrial components COX5B, COX6B2, and COX7A2 in severe AD, underscoring their importance in the disease's pathogenesis. Conclusively, these results not only delineate the dynamic genetic shifts accompanying AD progression but also propose critical biomarkers for potential therapeutic targeting, offering a consolidated basis for future AD research and treatment development. This offered a novel idea for analyzing the pathogenesis and development of AD and investigation of targeted drugs.

HIF-1α Induced by Hypoxia Promotes Peripheral Nerve Injury Recovery Through Regulating Ferroptosis in DRG Neuron.

Peripheral nerve injury (PNI) usually has a poor effect on functional recovery and severely declines the patient's quality of life. Our prior findings indicated that hypoxia remarkably promoted nerve regeneration of rats with sciatic nerve transection. However, the underlying molecular mechanisms of hypoxia in functional recovery of PNI still remain elusive. In this research, we tried to explain the functional roles and mechanisms of hypoxia and the hypoxia-inducible factor-1α (HIF-1α) in PNI. Our results indicated that hypoxia promoted proliferation and migration of dorsal root ganglia (DRG) and increased the expression of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF). Mechanistically, hypoxia suppressed ferroptosis through activating HIF-1α in DRG neurons. Gain and loss of function studies were performed to evaluate the regulatory roles of HIF-1α in ferroptosis and neuron recovery. The results revealed that up-regulation of HIF-1α enhanced the expression of solute carrier family membrane 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) and increased the contents of cysteine and glutathione, while inhibiting the accumulation of reactive oxygen species (ROS). Our findings provided novel light on the mechanism of ferroptosis involved in PNI and manifest hypoxia as a potential therapeutic strategy for PNI recovery.

HIF-1α-induced upregulation of m6A reader IGF2BP1 facilitates peripheral nerve injury recovery by enhancing SLC7A11 mRNA stabilization.

The recovery of peripheral nerve injury (PNI) is not ideal in clinic. Our previous study revealed that hypoxia treatment promoted PNI repair by inhibiting ferroptosis. The aim of this study was to investigate the underlying molecular mechanism of HIF-1α in hypoxia-PNI recovery. M6A dot blot was used to determine the total level of m6A modification. Besides, HIF-1α small interfering RNA (siRNA) or IGF2BP1 overexpression vector was transfected into dorsal root ganglion (DRG) neurons to alter the expression of HIF-1α and IGF2BP1. Subsequently, MeRIP-PCR analysis was applied to validate the m6A methylation level of SLC7A11. We demonstrated the hypoxia stimulated HIF-1α-dependent expression of IGF2BP1 and promoted the overall m6A methylation levels of DRG neurons. Overexpression of HIF-1α increased the expressions of neurotrophic factors including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and glial-derived neurotrophic factor (GDNF), which could be effectively reversed by siRNA knockdown of IGF2BP1. Moreover, upregulation of HIF-1α contributed to the m6A methylation level and mRNA stabilization of SLC7A11. This study revealed that the HIF-1α/IGF2BP1/SLC7A11 regulatory axis facilitated the recovery of injured DRG neurons. Our findings suggest a novel insight for the m6A methylation modification in PNI recovery.

Endothelial cells regulate astrocyte to neural progenitor cell trans-differentiation in a mouse model of stroke.

The concept of the neurovascular unit emphasizes the importance of cell-cell signaling between neural, glial, and vascular compartments. In neurogenesis, for example, brain endothelial cells play a key role by supplying trophic support to neural progenitors. Here, we describe a surprising phenomenon where brain endothelial cells may release trans-differentiation signals that convert astrocytes into neural progenitor cells in male mice after stroke. After oxygen-glucose deprivation, brain endothelial cells release microvesicles containing pro-neural factor Ascl1 that enter into astrocytes to induce their trans-differentiation into neural progenitors. In mouse models of focal cerebral ischemia, Ascl1 is upregulated in endothelium prior to astrocytic conversion into neural progenitor cells. Injecting brain endothelial-derived microvesicles amplifies the process of astrocyte trans-differentiation. Endothelial-specific overexpression of Ascl1 increases the local conversion of astrocytes into neural progenitors and improves behavioral recovery. Our findings describe an unexpected vascular-regulated mechanism of neuroplasticity that may open up therapeutic opportunities for improving outcomes after stroke.

The effects of acrylamide-mediated dorsal root ganglion neurons injury on ferroptosis.

Acrylamide (ACR) is a water-soluble chemical applied in industrial and laboratory processes. The neurotoxicity induced by acrylamide involves both peripheral and central nervous system. Hence, there is a growing urgency to investigate the mechanisms of acrylamide-induced neurotoxicity and search novel therapeutic target for the nerve repair. The effects of ACR on the proliferation, reactive oxygen species (ROS) and iron production of dorsal root ganglia (DRG) neurons and Schwann cells were determined. 5-Ethynyl-2'-deoxyuridine (EDU) staining and transwell assay were applied to detect the proliferation and migration capacity of DRG cells. Ferrostatin-1 (Fer-1) was used to suppress ferroptosis induced by ACR. RT-PCR analysis was performed to examine the expression of neurotrophic factors including brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF) and glial cell line-derived neurotrophic factor (GDNF). Moreover, Iron, ROS, malondialdehyde (MDA) and glutathione (GSH) contents were measured to reveal the regulation of ferroptosis in ACR-related nerve injury. ACR inhibited the proliferation and migration of DRG neurons and the supplementation of Fer-1 reversed the effects induced by ACR. Besides, the treatment of Fer-1 effectively increased the expression of NGF, BDNF, VEGF and GDNF. Furthermore, ACR increased the iron level, MDA and ROS contents while inhibited the level of GSH. It was unveiled that ACR attenuated the proliferation, migration and neuron repair of DRG neurons through regulating ferroptosis. The modulation of ferroptosis might be a promising therapeutic strategy and provide references for future treatment of acrylamide-induced nerve damage.

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.

Excretion and Residual Concentration Correlations of Salbutamol Between Edible Tissues and Living Samples in Pigs and Goats.

Illegal use of salbutamol (SAL), a ß-adrenergic leanness-enhancing agent, has posed potential threat to human health in China. The excretion and depletion of SAL in pigs and goats were investigated, and the concentration correlations between edible tissues and living samples were analyzed to find out a suitable living sample for pre-slaughter monitoring of SAL in pigs and goats. After a single oral dosage of 1.2 mg/kg SAL, approximately 70% of the dose was excreted by pigs and goats from their excreta. When pigs and goats were supplied feed containing SAL (20 mg/kg) for 14 consecutive days, high concentrations of SAL were observed in the liver and kidneys, and the longest persistence was observed in hair. Unlike pigs, SAL was presented primarily as conjugated SAL in goats. Excellent concentration correlations of SAL were observed between urine and edible tissues both in pigs and goats, and in addition, good correlations also were found between hair and edible tissues in pigs and between feces and edible tissues in goats. Hence, urine and hair could accurately predict SAL concentrations in edible tissues of pigs, whereas feces and urine were satisfactory for predicting SAL concentrations in edible tissues of goats. These data make it possible for pre-slaughter monitoring of SAL residues in the edible tissues of pigs and goats.

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.

Author Correction: Potential circadian effects on translational failure for neuroprotection.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Potential circadian effects on translational failure for neuroprotection.

Neuroprotectant strategies that have worked in rodent models of stroke have failed to provide protection in clinical trials. Here we show that the opposite circadian cycles in nocturnal rodents versus diurnal humans1,2 may contribute to this failure in translation. We tested three independent neuroprotective approaches-normobaric hyperoxia, the free radical scavenger α-phenyl-butyl-tert-nitrone (αPBN), and the N-methyl-D-aspartic acid (NMDA) antagonist MK801-in mouse and rat models of focal cerebral ischaemia. All three treatments reduced infarction in day-time (inactive phase) rodent models of stroke, but not in night-time (active phase) rodent models of stroke, which match the phase (active, day-time) during which most strokes occur in clinical trials. Laser-speckle imaging showed that the penumbra of cerebral ischaemia was narrower in the active-phase mouse model than in the inactive-phase model. The smaller penumbra was associated with a lower density of terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL)-positive dying cells and reduced infarct growth from 12 to 72 h. When we induced circadian-like cycles in primary mouse neurons, deprivation of oxygen and glucose triggered a smaller release of glutamate and reactive oxygen species, as well as lower activation of apoptotic and necroptotic mediators, in 'active-phase' than in 'inactive-phase' rodent neurons. αPBN and MK801 reduced neuronal death only in 'inactive-phase' neurons. These findings suggest that the influence of circadian rhythm on neuroprotection must be considered for translational studies in stroke and central nervous system diseases.

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.

Brain-to-cervical lymph node signaling after stroke.

After stroke, peripheral immune cells are activated and these systemic responses may amplify brain damage, but how the injured brain sends out signals to trigger systemic inflammation remains unclear. Here we show that a brain-to-cervical lymph node (CLN) pathway is involved. In rats subjected to focal cerebral ischemia, lymphatic endothelial cells proliferate and macrophages are rapidly activated in CLNs within 24 h, in part via VEGF-C/VEGFR3 signalling. Microarray analyses of isolated lymphatic endothelium from CLNs of ischemic mice confirm the activation of transmembrane tyrosine kinase pathways. Blockade of VEGFR3 reduces lymphatic endothelial activation, decreases pro-inflammatory macrophages, and reduces brain infarction. In vitro, VEGF-C/VEGFR3 signalling in lymphatic endothelial cells enhances inflammatory responses in co-cultured macrophages. Lastly, surgical removal of CLNs in mice significantly reduces infarction after focal cerebral ischemia. These findings suggest that modulating the brain-to-CLN pathway may offer therapeutic opportunities to ameliorate systemic inflammation and brain injury after stroke.

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.

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.