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Reversible middle cerebral Artery occlusion without craniectomy in rats
Abstract
To develop a simple, relatively noninvasive small-animal model of reversible regional cerebral ischemia, we tested various methods of inducing infarction in the territory of the right middle cerebral artery (MCA) by extracranial vascular occlusion in rats. In preliminary studies, 60 rats were anesthetized with ketamine and different combinations of vessels were occluded; blood pressure and arterial blood gases were monitored. Neurologic deficit, mortality rate, gross pathology, and in some instances, electroencephalogram and histochemical staining results were evaluated in all surviving rats. The principal procedure consisted of introducing a 4-0 nylon intraluminal suture into the cervical internal carotid artery (ICA) and advancing it intracranially to block blood flow into the MCA; collateral blood flow was reduced by interrupting all branches of the external carotid artery (ECA) and all extracranial branches of the ICA. In some groups of rats, bilateral vertebral or contralateral carotid artery occlusion was also performed. India ink perfusion studies in 20 rats documented blockage of MCA blood flow in 14 rats subjected to permanent occlusion and the restoration of blood flow to the MCA territory in six rats after withdrawal of the suture from the ICA. The best method of MCA occlusion was then selected for further confirmatory studies, including histologic examination, in five additional groups of rats anesthetized with halothane. Seven of eight rats that underwent permanent occlusion of the MCA had resolving moderately severe neurologic deficits (Grade 2 of 4) and unilateral infarcts averaging 37.6 +/- 5.5% of the coronal sectional area at 72 hours after the onset of occlusion.(ABSTRACT TRUNCATED AT 250 WORDS)
... Hence, in this study, we aimed to investigate and compare the neuroprotective effects of RJ and 10-HDA in a rat model of ischemic stroke using the transient intraluminal filament MCAo method [24]. For this purpose, the effects of these compounds on the pathophysiological consequences of ischemic damage such as inflammation and apoptosis were investigated by analyzing brain levels of poly-(ADP-ribose)-polymerase-1 (PARP-1) and active caspase-3, respectively. ...
... Right MCAo was induced as described previously [24]. Briefly, the rats were anesthetized with sevoflurane and a midline neck incision was made under the operating microscope; right common carotid artery (CCA), external carotid artery (ECA) and internal carotid artery (ICA) were exposed. ...
... We employed a widely accepted MCAo model [24] and supplemented the rats orally with RJ or 10-HDA (at an amount that was included in RJ) for 7 days. Supplementation with RJ or 10-HDA significantly reduced brain infarct volume (10.1 ± 1.7% or 11 ± 1.8%, respectively vs. 15.7 ± 1.3% in control group) and weight loss (35 or 36 g, respectively vs. 68 g loss in control group) of animals, indicating that 10-HDA is the major component of RJ which reproduces its effects on brain ischemia/reperfusion injury. ...
Objectives:
This study aimed to compare the efficacy of royal jelly (RJ) and its major fatty acid 10-hydroxy-2-decenoic acid (10-HDA) on ischemic stroke-related pathologies using histological and molecular approaches.
Methods:
Male rats were subjected to middle cerebral artery occlusion (MCAo) to induce ischemic stroke and were supplemented daily with either vehicle (control group), RJ or 10-HDA for 7 days starting on the day of surgery. On the eighth day, rats were sacrificed and brain tissue and blood samples were obtained to analyze brain infarct volume, DNA damage as well as apoptotic, inflammatory and epigenetic parameters.
Results:
Both RJ and 10-HDA supplementation significantly reduced brain infarction and decreased weight loss when compared to control animals. These effects were associated with reduced levels of active caspase-3 and PARP-1 and increased levels of acetyl-histone H3 and H4. Although both RJ and 10-HDA treatments significantly increased acetyl-histone H3 levels, the effect of RJ was more potent than that of 10-HDA. RJ and 10-HDA supplementation also alleviated DNA damage by significantly reducing tail length, tail intensity and tail moment in brain tissue and peripheral lymphocytes, except for the RJ treatment which tended to reduce tail moment in lymphocytes without statistical significance.
Conclusions:
Our findings suggest that neuroprotective effects of RJ in experimental stroke can mostly be attributed to 10-HDA.
... According to the previous description, the MCAO/R model was developed (Longa et al., 1989). In short, we anesthetized the rats by injecting sodium pentobarbital (30 mg/kg) intraperitoneally. ...
... Neurologic deficit scores were obtained 7 days after EA invention based on Zea Longa scores (Longa et al., 1989). The scoring system was defined as follows: 0, no deficit; 1, unable to stretch the contralateral forelimb fully; 2, circling to the contralateral side; 3, falling over to the contralateral side; and 4, did not walk spontaneously. ...
Background
Electroacupuncture (EA) has been shown to facilitate brain plasticity‐related functional recovery following ischemic stroke. The functional magnetic resonance imaging technique can be used to determine the range and mode of brain activation. After stroke, EA has been shown to alter brain connectivity, whereas EA's effect on brain network topology properties remains unclear. An evaluation of EA's effects on global and nodal topological properties in rats with ischemia reperfusion was conducted in this study.
Methods and results
There were three groups of adult male Sprague‐Dawley rats: sham‐operated group (sham group), middle cerebral artery occlusion/reperfusion (MCAO/R) group, and MCAO/R plus EA (MCAO/R + EA) group. The differences in global and nodal topological properties, including shortest path length, global efficiency, local efficiency, small‐worldness index, betweenness centrality (BC), and degree centrality (DC) were estimated. Graphical network analyses revealed that, as compared with the sham group, the MCAO/R group demonstrated a decrease in BC value in the right ventral hippocampus and increased BC in the right substantia nigra, accompanied by increased DC in the left nucleus accumbens shell (AcbSh). The BC was increased in the right hippocampus ventral and decreased in the right substantia nigra after EA intervention, and MCAO/R + EA resulted in a decreased DC in left AcbSh compared to MCAO/R.
Conclusion
The results of this study provide a potential basis for EA to promote cognitive and motor function recovery after ischemic stroke.
... Interestingly, in rats subjected to middle cerebral artery ischemia-reperfusion, collagen deposition in the ischemic hemisphere peaks [31,32]. The permanent MCAO model induces more severe pathological changes in the rat brain than the reperfusion model [33]. Based on these findings, we propose that large infarcts in the rat brain induce severe inflammatory responses, promoting collagen deposition and accelerating the development of fibrosis. ...
The development of fibrosis after injury to the brain or spinal cord limits the regeneration of the central nervous system in adult mammals. However, the extent of fibrosis in the injured brain has not been systematically investigated in mammals in vivo. This study aimed to assess whether [¹⁸F]AlF-FAPI-42-based cerebral positron emission tomography (PET) can be utilized to assess the extent of fibrosis in ischemic regions of the brain in vivo. Sprague–Dawley rats underwent permanent occlusion of the right middle cerebral artery (MCAO). On days 3, 7, 14, and 21 after MCAO, the uptake of [¹⁸F]AlF-FAPI-42 in the ischemic region of the brain in the MCAO groups surpassed that in the control group (day 0). The specific expression of fibroblast activation protein-α (FAP) in ischemic regions of the brain was also confirmed in immunohistofluorescence experiments in vitro. [¹⁸F]AlF-FAPI-42 intensity correlated with the density of collagen deposition in the ischemic hemisphere (p < 0.001). [¹⁸F]AlF-FAPI-42 PET/CT imaging demonstrated a specific uptake of radioactivity in the infarcted area in an ischemic stroke patient. PET imaging by using [¹⁸F]AlF-FAPI-42 offers a promising non-invasive method for monitoring the progression of cerebral fibrosis caused by ischemic stroke and may facilitate the clinical management of stroke patients. Trial registration: chictr.org.cn ChiCTR2200059004. Registered April 22, 2022.
... After surgery, the rats in other groups except the sham group were evaluated for the modeling results by the modified Zea-Longa method [9] (0 point, no symptoms of nerve damage; 1 point, inability to extend the other front paw; 2 points, turning laterally; 3 points, tipping to the opposite side; 4 points, inability to walk spontaneously, loss of consciousness; 5 points, death). After surgery, rats scored 0 and 5 points were excluded, and the remaining rats were regrouped. ...
Background
Stroke is one of the leading causes of death around the world. The sequelae of ischemic stroke cause drastic effects on the quality of life for patients. Sanwujiaowan (SWJW) is a mixture prepared with 5 herbal medicines (Aconiti Lateralis Radix Praeparata, Aconiti Kusnezoffii Radix, Polygoni Multiflori Radix, Aconiti Radix, and Olibanum), with a long history of application in treating the sequelae of stroke.
Objectives
To provide evidence and decipher the mechanism of SWJW in alleviating stroke.
Materials and Methods
In this article, we expanded the indicators of SWJW by an integrated strategy based on signature metabolomics, target proteins, and bioinformatics and probed into the mechanism of SWJW intervention in ischemic stroke in a rat model.
Results
The results indicated that SWJW protected rats from nerve damage during the acute phase of ischemic stroke by regulating tau phosphorylation via the PI3K/Akt pathway.
Conclusions
This study, for the first time, proved that the reduction of phosphorylated tau was harmful for the neural function in the acute phase of ischemic stroke. Meanwhile, the pathological changes of phosphorylated tau proteins were detected in stroke and recalled by SWJW. This finding may provide a new reference for formulating treatment strategies for the acute phase of ischemic stroke.
... Mice in the MCAO group at 0 h, 12 h, and 72 h after reperfusion were examined. The Longa scoring system [24] was used for evaluation: 0, no symptoms of nerve injury; 1, the right forepaw could only be slightly straightened when the mouse tail was lifted; 2, turned right when walking (moderate); 3, barely walking, right paralysis (severe); and 4, unable to walk spontaneously. ...
Ischemic stroke is a devastating disease in which mitochondrial damage or dysfunction substantially contributes to brain injury. Mitochondrial uncoupling protein-2 (UCP2) is a member of the UCP family, which regulates production of mitochondrial superoxide anion. UCP2 is reported to be neuroprotective for ischemic stroke–induced brain injury. However, the molecular mechanisms of UCP2 in ischemic stroke remain incompletely understood. In this study, we investigated whether and how UCP2 modulates neuroinflammation and regulates neuronal ferroptosis following ischemic stroke in vitro and in vivo. Wild-type (WT) and UCP2 knockout (Ucp2−/−) mice were subjected to middle cerebral artery occlusion (MCAO). BV2 cells (mouse microglial cell line) and HT-22 cells (mouse hippocampal neuronal cell line) were transfected with small interfering (si)-RNA or overexpression plasmids to knockdown or overexpress UCP2 levels. Cells were then exposed to oxygen–glucose deprivation and reoxygenation (OGD/RX) to simulate hypoxic injury in vitro. We found that UCP2 expression was markedly reduced in a time-dependent manner in both in vitro and in vivo ischemic stroke models. In addition, UCP2 was mainly expressed in neurons. UCP2 deficiency significantly enlarged infarct volumes, aggravated neurological deficit scores, and exacerbated cerebral edema in mice after MCAO. In vitro knockdown of Ucp2 and in vivo genetic depletion of Ucp2 (Ucp2−/− mice) increased neuronal ferroptosis-related indicators, including Fe²⁺, malondialdehyde, glutathione, and lipid peroxidation. Overexpression of UCP2 in neuronal cells resulted in reduced ferroptosis. Moreover, knockdown of UCP2 exacerbated neuroinflammation in BV2 microglia and mouse ischemic stroke models, suggesting that endogenous UCP2 inhibits neuroinflammation following ischemic stroke. Upregulation of UCP2 expression in microglia appeared to decrease the release of pro-inflammatory factors and increase the levels of anti-inflammatory factors. Further investigation showed that UCP2 deletion inhibited expression of AMPKα/NRF1 pathway-related proteins, including p-AMPKα, t-AMPKα, NRF1, and TFAM. Thus, UCP2 protects the brain from ischemia-induced ferroptosis by activating AMPKα/NRF1 signaling. Activation of UCP2 represents an attractive strategy for the prevention and treatment of ischemic stroke.
Graphical Abstract
Ischemic stroke suppresses UCP2 induction of lipid peroxidation, leading to neuronal ferroptosis via direct interaction with GPX4. In contrast, low levels of UCP2 activate the AMPKα/NRF1 signaling axis and lead to microglia release of IL-1β, TNF-α, and IL-6. UCP2, mitochondrial uncoupling protein 2; GPX4, glutathione peroxidase 4; MDA, malondialdehyde; GSH, glutathione; LIPID ROS, lipid peroxide reactive oxygen species; IL-1β, interleukin-1β; TNF-α, tumor necrosis factor-α; IL-6, interleukin-6; IL-4, interleukin-4; IL-10, interleukin-10; IL-13, interleukin-13; AMPK, adenosine 5′-monophosphate (AMP)-activated protein kinase; NRF1, nuclear respiratory factor 1.
... Neurological impairment was evaluated using Zea Longa score: 0 (no symptoms), 1 (mild focal nerve dysfunction), 2 (severe focal neurologic deficit), 3 (severe focal neurologic deficits), and 4 (inability to ambulate, decreased consciousness) [21]. ...
Ischemic stroke is a leading cause of death and disability worldwide, with limited treatment options available. Recent studies have shown that B cells play a critical role in the pathogenesis of ischemic stroke through their interaction with T cells in the meninges. In this study, we investigated whether temporary inhibition of B-cell activity through anti-CD19 antibody (aCD19 Ab) treatment could alleviate ischemic brain injury in a stroke mouse model by suppressing meningeal immunity.
Methods
Male C57BL/6 mice were subjected to transient middle cerebral artery occlusion (tMCAO) to induce focal ischemia. aCD19 Ab or Iso Ab was administered intraperitoneally 3 days prior to tMCAO. The mice were euthanized 24h after tMCAO for histological and immunological analyses. Other outcomes were assessed during the 7-day period.
Results
Compared to the MCAO/R group, treatment with Anti-CD19 Ab significantly reduced infarct size and brain edema, prolonged post-MCAO survival, and improved behavioral outcomes. Transmission electron microscope (TEM) and head Computed Tomography Angiography (CTA) results demonstrated that temporary B cell depletion led to improvements in microvascular endothelial edema, mitochondrial protection, reduced apoptosis, and enhanced post-MCAO cerebral vascular network reconstruction. Additionally, immune cells other than B cells decreased both in the brains (including brain parenchyma and meninges) and peripheral circulation of MCAO mice following B cell blockade, accompanied by decreased levels of cerebral parenchymal pro-inflammatory factors, including interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-10 (IL-10), and tumor necrosis factor-alpha (TNF-α). The immune response alterations observed in the MCAO/R group were consistent with the trends indicated by stroke patient data.
Conclusion
Temporary inhibition of B-cell activity via aCD19 antibody injection may mitigate ischemic brain injury in a mouse model of stroke by suppressing systemic immune reactions. Changes in immune cells within the meninges play a role, and further investigation is needed to understand the mechanisms involved. These findings suggest that post-stroke local and systemic immune responses contribute to the pathogenesis of ischemic stroke, and temporary B cell depletion may represent a potential therapeutic target for stroke therapy.
... The neurological score of each group was evaluated 3 days after surgery using the longa neural scoring method 66 . An open-field test was employed to detect the motor ability of mice. ...
Injuries to the brain result in tunable cell responses paired with stimulus properties, suggesting the existence of intrinsic processes that encode and transmit injury information; however, the molecular mechanism of injury information encoding is unclear. Here, using ATP fluorescent indicators, we identify injury-evoked spatiotemporally selective ATP dynamics, Inflares, in adult mice of both sexes. Inflares are actively released from astrocytes and act as the internal representations of injury. Inflares encode injury intensity and position at their population level through frequency changes and are further decoded by microglia, driving changes in their activation state. Mismatches between Inflares and injury severity lead to microglia dysfunction and worsening of injury outcome. Blocking Inflares in ischemic stroke in mice reduces secondary damage and improves recovery of function. Our results suggest that astrocytic ATP dynamics encode injury information and are sensed by microglia.
... Neurological evaluations were performed on all rats 30 min after MCA occlusion using a five-tiered grading system based on the Zea-Longa scale [4,12] as follows: 0, no neurological deficit; 1, failure to fully extend the left forepaw; 2, circling or walking to the left; 3, falling to the left; and 4, unable to walk spontaneously. ...
Objective: Citicoline can be used to reduce acute ischemic stroke injury via venous infusion, however, its protective effects in the brain extracellular space remain largely unknown. Herein, we investigated the brain protective effects of citicoline administered via the brain extracellular space and sought precise effective dosage range that can protect against ischemic injury after experimental ischemic stroke in rats.
Methods: Fifty-six Sprague-Dawley rats were randomly divided into control, intraperitoneal (IP), caudate-putamen (CPu)-25, CPu-40, CPu-50, CPu-60 and CPu-75 groups based on the infusion site and concentration of citicoline. Two hours after the administration of citicoline, the rats were subjected to a permanent middle cerebral artery occlusion to mimic acute ischemic stroke. Then, the brain infarct volume in rats after stroke was measured and their neurological deficiency was evaluated to explain the protective effects and effective dosage range of citicoline.
Results: Compared to the control and IP groups, brain infarct volume of rats in CPu-40, CPu-50, and CPu-60 groups is significant smaller. Furthermore, the brain infarct volume of rats in CPu-50 is the least.
Conclusions: Here, we showed that citicoline can decrease the brain infarct volume, thus protecting the brain from acute ischemic stroke injury. We also found that the appropriate effective citicoline dose delivered via the brain extracellular space is 50 mM. Our study provides novel insights into the precise treatment of acute ischemic stroke by citicoline via the brain extracellular space, further guiding the treatment of brain disease.
... Rat cerebral ischemia model was established using the modified Zea longa bolus method [7]. An intraperitoneal injection of 1.5% pentobarbital sodium (2 ml/kg) was administered to anesthetize the rat. ...
Traditional Chinese medicine (TCM) has long been used to treat various diseases, including cerebral ischemia. The specific molecular mechanism of TCM in the treatment of cerebral ischemia, however, is still unclear. This study investigated the effects of gastrodin, electroacupuncture and their combination on cerebral ischemic rats. We used Nissl staining, immunohistochemical staining and immunoblotting to detect the expression changes of brain-derived neurotrophic factor (BDNF) and interleukin-6 (IL-6) in the frontal cortex. The results showed that the combination therapy of gastrodin and electroacupuncture significantly increased the number of Nissl-positive neurons and improved cell morphology compared with other groups. Mechanistically, we found that the combination of gastrodin and electroacupuncture treatment group can restore the abnormal morphology of neuronal cells caused by cerebral ischemia by rebalancing the expression levels of BDNF and IL-6. Our research indicates that gastrodin combined with electroacupuncture has a significant protective effect on cerebral ischemic injury in rats, possibly by regulating the expression of BDNF and IL-6. This combination therapy is superior to single-drug or electroacupuncture therapy.
... The transient I/R model induction was performed as described by Longa et al. (Longa et al., 1989). The animals were anesthetized with an intraperitoneal injection of with 10% chloral hydrate at a dose of 350 mg/kg (Merck, Germany, Cat No. 102425). ...
... The modified Longa method was used to induce MCAO surgery 22 . The animals in the Disease and DS groups were weighed and anesthetized with pentobarbital sodium (40 mg·kg −1 ) after 12 h of fasting (water allowed). ...
A research model combining a disease and syndrome can provide new ideas for the treatment of ischemic stroke. In the field of traditional Chinese medicine, blood stasis and toxin (BST) syndrome is considered an important syndrome seen in patients with ischemic stroke (IS). However, the biological basis of IS-BST syndrome is currently not well understood. Therefore, this study aimed to explore the biological mechanism of IS-BST syndrome. This study is divided into two parts: (1) establishment of an animal model of ischemic stroke disease and an animal model of BST syndrome in ischemic stroke; (2) use of omics methods to identify differentially expressed genes and metabolites in the models. We used middle cerebral artery occlusion (MCAO) surgery to establish the disease model, and utilized carrageenan combined with active dry yeast and MCAO surgery to construct the IS-BST syndrome model. Next, we used transcriptomics and metabolomics methods to explore the differential genes and metabolites in the disease model and IS-BST syndrome model. It is found that the IS-BST syndrome model exhibited more prominent characteristics of IS disease and syndrome features. Both the disease model and the IS-BST syndrome model share some common biological processes, such as thrombus formation, inflammatory response, purine metabolism, sphingolipid metabolism, and so on. Results of the “gene–metabolite” network revealed that the IS-BST syndrome model exhibited more pronounced features of complement-coagulation cascade reactions and amino acid metabolism disorders. Additionally, the “F2 (thrombin)–NMDAR/glutamate” pathway was coupled with the formation process of the blood stasis and toxin syndrome. This study reveals the intricate mechanism of IS-BST syndrome, offering a successful model for investigating the combination of disease and syndrome.
... tMCAO surgery was performed to induce focal cerebral ischemia as reported previously [18,19]. Briefly, rats were anesthetized with intraperitoneal (i.p.) injection of ketamine and xylazine at the dose of 75 mg/kg b.wt. ...
AT1 receptor blockers (ARBs) are commonly used drugs to treat cardiovascular disease and hypertension, but research on their impact on brain disorders is unattainable. Valsartan (VAL) is a drug that specifically blocks AT1 receptor. Despite the previous evidence for VAL to provide neuroprotection in case of ischemic reperfusion injury, evaluation of their potential in mitigating mitochondrial dysfunction that causes neuronal cell death and neurobehavioral impairment remains unknown. The aim of this study was to evaluate the therapeutic effect of repurposed drug VAL against ischemic reperfusion injury–induced neuronal alternation. tMCAO surgery was performed to induce focal cerebral ischemic reperfusion injury. Following ischemic reperfusion injury, we analyzed the therapeutic efficacy of VAL by measuring the infarct volume, brain water content, mitochondrial oxidative stress, mitochondrial membrane potential, histopathological architecture, and apoptotic marker protein. Our results showed that VAL administrations (5 and 10 mg/kg b.wt.) mitigated the brain damage, enhanced neurobehavioral outcomes, and alleviated mitochondrial-mediated oxidative damage. In addition to this, our findings demonstrated that VAL administration inhibits neuronal apoptosis by restoring the mitochondrial membrane potential. A follow-up investigation demonstrated that VAL induces BDNF expression and promoted ischemic tolerance via modulating the Akt/p-Creb signaling pathway. In summary, our results suggested that VAL administration provided neuroprotection, ameliorated mitochondrial dysfunction, preserved the integrity of neurons, and lead to functional improvement after ischemic reperfusion injury.
Graphical Abstract
... All rats were given free access to food and water. Animals were operated with slightly modified MCAO model as described by Longa et al. [37]. Anesthesia was induced by 4% isoflurane and maintained by 2% isoflurane during MCAO operation. ...
Dexmedetomidine displays multiple mechanisms of neuroprotection in ameliorating ischemic brain injury. In this study, we explored the beneficial effects of dexmedetomidine on blood-brain barrier (BBB) integrity and neuroinflammation in cerebral ischemia/reperfusion injury. Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO) for 1.5 h and reperfusion for 24 h to establish a rat model of cerebral ischemia/reperfusion injury. Dexmedetomidine (9 μg/kg) was administered to rats 30 min after MCAO through intravenous injection, and SB203580 (a p38 MAPK inhibitor, 200 μg/kg) was injected intraperitoneally 30 min before MCAO. Brain damages were evaluated by 2,3,5-triphenyltetrazolium chloride staining, hematoxylin-eosin staining, Nissl staining, and brain water content assessment. BBB permeability was examined by Evans blue staining. Expression levels of claudin-5, zonula occludens-1, occludin, and matrix metalloproteinase-9 (MMP-9) as well as M1/M2 phenotypes-associated markers were assessed using immunofluorescence, RT-qPCR, Western blotting, and gelatin zymography. Enzyme-linked immunosorbent assay was used to examine inflammatory cytokine levels. We found that dexmedetomidine or SB203580 attenuated infarct volume, brain edema, BBB permeability, and neuroinflammation, and promoted M2 microglial polarization after cerebral ischemia/reperfusion injury. Increased MMP-9 activity by ischemia/reperfusion injury was inhibited by dexmedetomidine or SB203580. Dexmedetomidine inhibited the activation of the ERK, JNK, and p38 MAPK pathways. Moreover, activation of JNK or p38 MAPK reversed the protective effects of dexmedetomidine against ischemic brain injury. Overall, dexmedetomidine ameliorated brain injury by alleviating BBB permeability and promoting M2 polarization in experimental cerebral ischemia/reperfusion injury model by inhibiting the activation of JNK and p38 MAPK pathways.
... mRNA and protein levels of NLRP3, ASC, Caspase-1, IL-1β and IL-18 were all increased after ischemia and reperfusion and all significantly reversed by C + P at normothermic and hypothermic condition. Data are presented as mean ± SEM. et al. [31]and Belayev et al. [32]before surgery for baseline, after 2 h MCA occlusion (immediately before reperfusion), and after 48 h reperfusion. Higher scores indicate more severe deficits in both scoring systems. ...
Background
Inflammation and subsequent mitochondrial dysfunction and cell death worsen outcomes after revascularization in ischemic stroke. Receptor-interacting protein kinase 1 (RIPK1) activated dynamin-related protein 1 (DRP1) in a NLRPyrin domain containing 3 (NLRP3) inflammasome-dependent fashion and Hypoxia-Inducible Factor (HIF)-1α play key roles in the process. This study determined how phenothiazine drugs (chlorpromazine and promethazine (C + P)) with the hypothermic and normothermic modality impacts the RIPK1/RIPK3-DRP1 and HIF-1α pathways in providing neuroprotection.
Methods
A total of 150 adult male Sprague-Dawley rats were subjected to 2 h middle cerebral artery occlusion (MCAO) followed by 24 h reperfusion. 8 mg/kg of C + P was administered at onset of reperfusion. Infarct volumes, mRNA and protein expressions of HIF-1α, RIPK1, RIPK3, DRP-1, NLRP3-inflammation and cytochrome c-apoptosis were assessed. Apoptotic cell death, infiltration of neutrophils and macrophages, and mitochondrial function were evaluated. Interaction between RIPK1/RIPK3 and HIF-1α/NLRP3 were determined. In SH-SY5Y cells subjected to oxygen/glucose deprivation (OGD), the normothermic effect of C + P on inflammation and apoptosis were examined.
Results
C + P significantly reduced infarct volumes, mitochondrial dysfunction (ATP and ROS concentration, citrate synthase and ATPase activity), inflammation and apoptosis with and without induced hypothermia. Overexpression of RIPK1, RIPK3, DRP-1, NLRP3-inflammasome and cytochrome c-apoptosis were all significantly reduced by C + P at 33 °C and the RIPK1 inhibitor (Nec1s), suggesting hypothermic effect of C + P via RIPK1/RIPK3-DRP1pathway. When body temperature was maintained at 37 °C, C + P and HIF-1α inhibitor (YC-1) reduced HIF-1α expression, leading to reduction in mitochondrial dysfunction, NLRP3 inflammasome and cytochrome c-apoptosis, as well as the interaction of HIF-1α and NLRP3. These were also evidenced in vitro, indicating a normothermic effect of C + P via HIF-1α.
Conclusion
Hypothermic and normothermic neuroprotection of C + P involve different pathways. The normothermic effect was mediated by HIF-1α, while hypothermic effect was via RIPK1/RIPK3-DRP1 signaling. This provides a theoretical basis for future precise exploration of hypothermic and normothermic neuroprotection.
... % of infract volume = vol of control contralateral hemisphere − vol of non − infracted tissue ∕vol of control contralateral hemisphere × 100% Neurological deficit scoring was performed before euthanizing the animals. The behavioral analysis in rats was performed in a single-blind manner using the method of Longa et al. [28], and the rats were scored based on their performance from 0 to 4. Zero: healthy rats with no deficit; the rats with spontaneous activity were scored as 1; the rats that were unable to extend their contralateral forelimb and walk straight were scored as 2, and the rats that walked in a circle with contralateral forelimb flexion were scored as 3. The rats walked in a circle towards the contralateral side and scored 4. ...
Cedrol is a major bioactive compound present in the Cedrus atlantica with numerous biological properties. In this study, we elucidated the neuroprotective properties of cedrol against ischemic infarction in animal and in vitro studies. A cerebral ischemic/reperfusion model was induced in adult Wistar rats, and oxygen–glucose deprivation/reperfusion was induced in SH-SY5Y neuronal cells and treated with different concentrations of cedrol. The percentage of water content, cerebral infarct, and neurological deficit score was assessed in experimental rats. The acetylcholinesterase activity and inflammatory cytokines were quantified to analyze the anti-inflammatory potency of cedrol. Oxidative stress marker malondialdehyde and antioxidants were quantified to evaluate the antioxidant potency of cedrol in an ischemic condition. The neuroprotective potency of cedrol was confirmed by histopathological analysis of the brain tissue of cedrol-treated I/R-induced rats. In in vitro studies, the MTT and LDH assays were performed in cedrol-treated OGD/R SH-SY5Y cells to analyze the cytoprotective effect of cedrol. The anti-inflammatory property of cedrol was confirmed by quantifying the pro-inflammatory cytokine levels in OGD/R-induced cedrol-treated SH-SY5Y cells. The results obtained prove that cedrol significantly prevents brain edema, neurological deficits, acetylcholinesterase activity, and oxidative damage in ischemic-induced rats. It inhibited neuroinflammation in ischemic-induced rats and also in in vitro models. The neuroprotective effect of cedrol during an ischemic condition was authentically established with histological analysis in an animal model and cell survival assays in an in vitro model. Overall, our results confirm that cedrol is a potent alternative drug to treat cerebral ischemia in the future.
... The right MCAO method was performed using the intraluminal filament method as previously described [19,20]. Rats were anesthetized with chloral hydrate at a dose of 400 mg/kg (i.p.). ...
Aim: The study was designed to develop and analyze curcumin nanoparticles. Methods: Curcumin nanoparticles were formulated and evaluated. Their efficacy in protecting against brain damage was investigated in a rat model of ischemic stroke, considering motor function, muscle strength and antioxidant enzyme activity. Results: Curcumin nanoparticles displayed a zeta potential of -55 ± 13.5 mV and an average particle size of 51.40 ± 21.70 nm. In ischemic stroke rat models, curcumin nanoparticle treatment significantly improved motor functions, and muscle strength and increased the activities of antioxidant enzymes like glutathione peroxidase, glutathione, glutathione S-transferase, superoxide dismutase and catalase, reducing oxidative stress and inflammation. Conclusion: Curcumin nanoparticles showed significant neuroprotective effects in ischemic stroke models.
... Interestingly, in rats subjected to middle cerebral artery ischaemia-reperfusion, collagen deposition in the ischaemic hemisphere peaks on day 14 postreperfusion [28,29]. The permanent MCAO model induces more severe pathological changes in the rat brain than the reperfusion model [30]. Based on these ndings, we propose that large infarcts in the rat brain induce severe in ammatory responses, promoting collagen deposition and accelerating the development of brosis. ...
The development of fibrosis after injury to the brain or spinal cord limits the regeneration of the central nervous system in adult mammals. However, the extent of fibrosis in the injured brain has not been systematically investigated in mammals in vivo. This study aimed to assess whether [¹⁸F]AlF-FAPI-42-based cerebral positron emission tomography (PET) can be utilized to assess the extent of fibrosis in ischaemic regions of the brain in vivo. Sprague-Dawley rats underwent permanent occlusion of the right middle cerebral artery (MCAO) or sham surgery (control). On days 3, 7, 14, and 21 post-MCAO, the uptake of [¹⁸F]AlF-FAPI-42 in the ischaemic region of the brain in the MCAO groups surpassed that in the control group. Specificity to FAP was confirmed through immunofluorescence staining. Histopathological analysis revealed higher collagen deposition in the ischaemic hemisphere of the rats in the MCAO group than the control level. [¹⁸F]AlF-FAPI-42 intensity correlated with the density of collagen fibres in the ischaemic hemisphere (p < 0.001). [¹⁸F]AlF-FAPI-42 PET/CT imaging revealed high FAP in the infarct zone of ischemic stroke patients. PET imaging by using [¹⁸F]AlF-FAPI-42 offers a promising non-invasive method for monitoring the progression of cerebral fibrosis caused by ischaemic stroke and may facilitate the clinical management of stroke patients.
Trial registration: chictr.org.cn ChiCTR2200059004. Registered April 22, 2022.
... Cerebral ischemia was induced by focal middle cerebral artery occlusion according to previous methods (Longa et al. 1989). Briefly, rats were deeply anaesthetized with intraperitoneal injection of ketamine (60 mg/kg) and xylazine (15 mg/kg) under a stereo dissecting microscope (Nikon, Japan), and the right common carotid artery (CCA) and carotid bifurcation were exposed through midline neck incision (1.5-2 cm) freed from surrounding nerves and fascia without damaging the muscles or the vagus nerve or its collaterals. ...
Rational
Patients experience post-stroke cognitive impairment during aging. To date, no specific treatment solution has been reported for this disorder.
Objective
The purpose of this study was to evaluate the effects of exercise training and coenzyme Q10 supplementation on middle cerebral artery occlusion (MCAO) induced behavioral impairment, long-term potentiation inhibition and cerebral infarction size in aging rats.
Methods
Fifty aging male rats underwent MCAO surgery and were randomly distributed in to the following groups: 1-Sham, 2- control, 3- Coenzyme Q10, 4- Exercise training and 5- Exercise training with Q10 supplementation (Ex + Q10). Aerobic training groups were allowed to run on a treadmill for 12 weeks. Q10 (50 mg/kg) was administered intragastrically by gavage. Morris water maze, shuttle box and elevated plus maze tests were used to evaluate cognitive function. The population spike (PS) amplitude and slope of excitatory postsynaptic potentials (EPSP) in the dentate gyrus area were recorded as a result of perforant pathway electrical stimulation.
Results
Our study showed that Q10 and aerobic training alone ameliorate spatial memory in the acquisition phase, but have no effect on spatial memory in the retention phase. Q10 and exercise training synergistically promoted spatial memory in the retention phase. Q10 and exercise training separately and simultaneously mitigated cerebral ischemia-induced passive avoidance memory impairment in acquisition and retention phases. The EPSP did not differ between the groups, but exercise training and Q10 ameliorate the PS amplitude in hippocampal responses to perforant path stimulation. Exercising and Q10 simultaneously reduced the cerebral infarction volume.
Conclusion
Collectively, the findings of the present study imply that 12 weeks of aerobic training and Q10 supplementation alone can simultaneously reverse cerebral ischemia induced neurobehavioral deficits via amelioration of synaptic plasticity and a reduction in cerebral infarction volume in senescent rats.
... Neurological deficits in rats were evaluated in a singleblind fashion after reperfusion by Longa et al. [13]. The scoring criteria were as follows: 0 points: no nerve damage symptoms; 1 point: incomplete extension of the right forepaw; 2 points: rightward turn; 3 points: rightward collapse; 4 points: impaired walking and unconsciousness. ...
Background
Shenma Jingfu Granule, a traditional Chinese medicine formula, has been used clinically for the treatment of cerebral circulation insufficiency. However, the mechanism involved in alleviating cerebral ischemia has not yet been fully elucidated.
Methods
An integrated approach involving network pharmacology and transcriptomics was utilized to clarify the potential mechanisms of SMJF Granule. Molecular docking and surface plasmon resonance (SPR) were employed to identify potential targets and ingredients of SMJF Granule. The anti-CI effect of SMJF Granule was determined on the middle cerebral artery occlusion (MCAO) model by using hematoxylin–eosin (H&E) and Nisslʼs staining, as well as triphenyl tetrazolium chloride (TTC) staining, and the potential targets involved in the mechanisms were validated by RT-qPCR and western blotting.
Results
Integrated analysis revealed the mechanism of SMJF Granule intervening in CI injury might be related to the HIF-1 signaling pathway and angiogenesis. Molecular docking and SPR assays demonstrated robust binding interactions between key compounds like salvianolic acid A and naringenin with the core target HIF-1α protein. The experiment confirmed that SMJF Granule lowered neurological scores, diminished infarct volume, and alleviated histopathological changes in vivo. The possible mechanism of SMJF Granule was due to regulating HIF-1 pathway, which contributed to up-regulating expression of VEGF and vWF in the penumbral region, showing a significant promotion of angiogenesis.
Conclusion
SMJF Granule promoted angiogenesis through HIF-1α pathway, thereby alleviating cerebral ischemia injury. In addition, our findings provide some evidence that SMJF Granule is a candidate compound for further investigation in treating CI in the clinical.
Graphical Abstract
Excessive reactive oxygen species (ROS) generated during cerebral ischemic reperfusion (CIRI) are crucial for subsequent tissue damage. However, despite the potential benefits of antioxidants reported in clinical applications, few have proven effective in treating CIRI, particularly in the elderly. Epicatechin (EC) is a catechol flavonoid monomer derived from natural tea plants. Multiple phenolic hydroxyl groups give it strong antioxidant properties, which can not only degrade ROS through chemical reactions between hydroxyl and ROS but also enhance the activity of antioxidant enzymes in cells, and it is easy to penetrate the blood–brain barrier. But its antagonistic effect on age‐related CIRI and potential medicinal value are still unknown. Nuclear factor erythroid 2‐related factor 2 (Nrf2) is the most important transcription factor regulating the expression of antioxidant proteins in the body. This study first compared the pathological differences of the Nrf2 system in CIRI between 2‐month‐old and 12‐month‐old Sprague–Dawley (SD) rats. Subsequently, EC was administered to 12‐month‐old rat models of middle cerebral artery occlusion and reperfusion (MCAO/R) and senescent SH‐SY5Y cell models subjected to oxygen glucose deprivation/reoxygenation (OGD/R). EC treatment improved cerebral morphology and function; increased p‐Nrf2, heme oxygenase‐1 (HO‐1), superoxide dismutase (SOD), and glutathione (GSH) expression; reduced infarct volume; and neuronal apoptosis in senescent rats. Moreover, EC enhanced cellular activity and the expression of p‐Nrf2, HO‐1, and quinone oxidoreductase‐1 (NQO‐1) while decreasing ROS and malondialdehyde (MDA) levels and mitigating apoptosis in senescent SH‐SY5Y cells. These effects were reversed upon si‐Nrf2. In sum, we confirm that EC exerts neuroprotective effects by upregulating Nrf2/ARE and reducing oxidative stress, suggesting that EC may be a promising drug for the treatment of senile cerebral apoplexy. This study also provides a scientific basis for the development and selection of new drugs for ischemic stroke in elderly patients.
Ischemic stroke is a common cerebrovascular disease with high mortality, high morbidity, and high disability. Cerebral ischemia/reperfusion injury seriously affects the quality of life of patients. Luteolin-7-O-β-d-glucuronide (LGU) is a major active flavonoid compound extracted from Ixeris sonchifolia (Bge.) Hance, a Chinese medicinal herb mainly used for the treatment of coronary heart disease, angina pectoris, cerebral infarction, etc. In the present study, the protective effect of LGU on cerebral ischemia/reperfusion injury was investigated in an oxygen–glucose deprivation/reoxygenation (OGD/R) neuronal model and a transient middle cerebral artery occlusion (tMCAO) rat model. In in vitro experiments, LGU was found to improve the OGD/R-induced decrease in neuronal viability effectively by the MTT assay. In in vivo experiments, neurological deficit scores, infarction volume rates, and brain water content rates were improved after a single intravenous administration of LGU. These findings suggest that LGU has significant protective effects on cerebral ischemia/reperfusion injury in vitro and in vivo. To further explore the potential mechanism of LGU on cerebral ischemia/reperfusion injury, we performed a series of tests. The results showed that a single administration of LGU decreased the content of EB and S100B and ameliorated the abnormal expression of tight junction proteins ZO-1 and occludin and metalloproteinase MMP-9 in the ischemic cerebral cortex of the tMCAO 24-h injury model. In addition, LGU also improved the tight junction structure between endothelial cells and the degree of basement membrane degradation and reduced the content of TNF-α and IL-1β in the brain tissue. Thereby, LGU attenuated cerebral ischemia/reperfusion injury by improving the permeability of the blood–brain barrier. The present study provides new insights into the therapeutic potential of LGU in cerebral ischemia.
Background
The inflammatory response is a key factor in the pathogenesis of cerebral ischemia/reperfusion injury (CIRI), and anti-inflammatory interventions may offer a promising therapeutic strategy. Forsythoside B (FB) is a phenylethanoid glycoside isolated from Forsythiae fructus, which has been reported to have anti-inflammatory effects. However, the mechanism of the neuroprotective effect of FB on CIRI remains unclear.
Methods
Adult male Sprague-Dawley rats were subjected to transient middle cerebral artery occlusion/reperfusion (MCAO/R). FB was administered intraperitoneally for 3 days prior to MCAO/R. Cerebral infarct volume and neurological deficit score were used as indices to evaluate MCAO/R injury. The serum levels of inflammatory factors and antioxidant enzymes were measured. The activation of silent information regulator 2 homolog 1 (Sirt1) and the inhibition of the nucleotide-binding oligomerization domain-like receptor with a pyrin domain 3 (NLRP3) pathway were assessed through western blot and immunohistochemistry analysis. Furthermore, the rats were treated with Sirt1 shRNA 3 days before MCAO/R by stereotactical injection into the ipsilateral hemispheric region to assess the impact of Sirt1 knockdown on the protection of FB during MCAO/R.
Results
FB reduced cerebral infarct volume and neurological deficit score in MCAO/R rats. FB reduced pathological changes and cell apoptosis in the hippocampal CA1 region and cortex on the ischemic side of rats. FB inhibited the serum levels of inflammatory factors and increased the activities of antioxidant enzymes. Further study showed that FB inhibited the activation of the NLRP3 pathway and induced Sirt1 activation.
Conclusion
FB demonstrated neuroprotective and anti-inflammatory effects by inhibiting the NLRP3 pathway through Sirt1 activation in CIRI.
ApTOLL, a TLR4 modulator aptamer, has demonstrated cerebroprotective effects in a permanent ischemic stroke mouse model, as well as safety and efficacy in early phase clinical trials. We carried out reverse translation research according to STAIR recommendations to further characterize the effects and mechanisms of ApTOLL after transient ischemic stroke in rats and to better inform the design of pivotal clinical trials. Adult male rats subjected to transient middle cerebral artery occlusion were treated either with ApTOLL or the vehicle intravenously at different doses and time-points. ApTOLL was compared with TAK-242 (a TLR4 inhibitor). Female rats were also studied. After neurofunctional evaluation, brains were removed for infarct/edema volume, hemorrhagic transformation, and histologic determinations. Peripheral leukocyte populations were assessed via flow cytometry. ApTOLL showed U-shaped dose-dependent cerebroprotective effects. The maximum effective dose (0.45 mg/kg) was cerebroprotective when given both before reperfusion and up to 12 h after reperfusion and reduced the hemorrhagic risk. Similar effects occurred in female rats. Both research and clinical ApTOLL batches induced slightly superior cerebroprotection when compared with TAK-242. Finally, ApTOLL modulated circulating leukocyte levels, reached the brain ischemic tissue to bind resident and infiltrated cell types, and reduced the neutrophil density. These results show the cerebroprotective effects of ApTOLL in ischemic stroke by reducing the infarct/edema volume, neurofunctional impairment, and hemorrhagic risk, as well as the peripheral and local immune response. They provide information about ApTOLL dose effects and its therapeutic time window and target population, as well as its mode of action, which should be considered in the design of pivotal clinical trials.
Aims
The main objective of this study was to investigate the role and mechanism of acupuncture on anti-nerve injury in the acute phase by regulating mitochondrial energy metabolism via monophosphate-activated protein kinase (AMPK)/peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) axis in rat ischemic stroke.
Main methods
Middle cerebral artery occlusion (MCAO) was established by middle cerebral artery occlusion/reperfusion. One-week of acupuncture was performed during the acute phase of ischemic stroke. The neurological function and brain tissue integrity were evaluated. Mitochondrial function (intracellular ATP level and the activity of mitochondrial respiratory chain complex I) and the level of NADH oxidase (NOX) were detected by enzymatic chemistry. Next, the potential molecular mechanisms were explored by western blotting, fluorescence quantitative PCR and immunohistochemistry method.
Key findings
(1) Acupuncture treatment for MCAO/R rats showed a significant improvement in the infarcted tissue accompanied by functional recovery in Zea-Longa score and balance beam score outcomes, motor function performances. (2) Acupuncture increased the levels of ATP and mitochondrial respiratory chain complex I, decreased the NOX levels in cerebral ischemia established by suture-occluded method. (3) Acupuncture reduced the necrosis dissolution of neuronal cells and meningeal edema, while promoting angiogenesis. (4) Quantitative immunohistochemical staining results showed acupuncture can increase the expression of AMPK, p-AMPK and the mitochondrial transcription factor PGC-1α, NRF2, TFAM and uncoupling protein 2 (UCP2). Meanwhile, acupuncture treatment up-regulated the expression of the corresponding protein. (5) Subsequently, acupuncture enhanced AMPK phosphorylation as well as the expression of PGC-1α, NRF2, TFAM and UCP2, implicated in mitochondrial synthesis and cellular apoptosis. (6) Finally, injections of AMPK antagonists and activators confirmed AMPK as a therapeutic target for the anti-nerve damage effects of acupuncture.
Significance
Acupuncture intervention relieved ischemic stroke progression in MCAO rats by promoting energy metabolism and mitochondrial biogenesis in the brain and alleviating neuronal apoptosis, which was mediated by eliciting AMPK/PGC-1α axis, among them AMPK is a therapeutic target.
Mitochondria play a crucial role in maintaining cellular energy supply and serve as a source of energy for repairing nerve damage following a stroke. Given that exercise has the potential to enhance energy metabolism, investigating the impact of exercise on mitochondrial function provides a plausible mechanism for stroke treatment. In our study, we established the middle cerebral artery occlusion (MCAO) model in Sprague–Dawley rats and implemented early exercise intervention. Neurological severity scores, beam-walking test score, and weight were used to evaluate neurological function. The volume of cerebral infarction was measured by MRI. Nerve cell apoptosis was detected by TUNEL staining. Mitochondrial morphology and structure were detected by mitochondrial electron microscopy. Mitochondrial function was assessed using membrane potential and ATP measurements. Western blotting was used to detect the protein expression of AMPK/PGC-1α/GLUT4. Through the above experiments, we found that early exercise improved neurological function in rats after MCAO, reduced cerebral infarction volume and neuronal apoptosis, promoted the recovery of mitochondrial morphology and function. We further examined the protein expression of AMPK/PGC-1α/GLUT4 signaling pathway and confirmed that early exercise was able to increase its expression. Therefore, we suggest that early exercise initiated the AMPK/PGC-1α/GLUT4 signaling pathway, restoring mitochondrial function and augmenting energy supply. This, in turn, effectively improved both nerve and body function in rats following ischemic stroke.
The study aimed to assess 𝛾-Terpinene’s (𝛾-TER) neuroprotective potential in acute cerebral ischemia, characterized by reduced cerebral blood flow in rats. Middle cerebral artery occlusion (MCAO), a standard method for inducing cerebral ischemia, was employed in male Wistar rats. 𝛾-TER at varying doses (5, 10, and 15 mg/kg) were intraperitoneally administered during reperfusion onset. Neurological outcomes, cerebral infarct size, edema, and enzymatic activities (SOD, GPx, and catalase) in the brain were evaluated using diverse techniques. The study examined gene expression and pathways associated with neuroinflammation and apoptosis using Cytoscape software, identifying the top 10 genes involved. Pro-inflammatory and pro-apoptotic factors were assessed through real-time PCR and ELISA, while apoptotic cell rates were measured using the TUNEL and Flow cytometry assay. Immunohistochemistry assessed apoptosis-related proteins like Bax and bcl-2 in the ischemic area. 𝛾-TER, particularly at doses of 10 and 15 mg/kg, significantly reduced neurological deficits and cerebral infarction size. The 15 mg/kg dose mitigated TNF-α, IL-1β, Bax, and caspase-3 gene and protein levels in the cortex, hippocampus, and striatum compared to controls. Furthermore, Bcl-2 levels increased in these regions. 𝛾-TER show cased neuroprotective effects by suppressing inflammation, apoptosis, and oxidation. In conclusion, 𝛾-TER, possessing natural anti-inflammatory and anti-apoptotic properties, shields the brain against ischemic damage by reducing infarction, edema, oxidative stress, and inflammation. It modulates the expression of crucial genes and proteins associated with apoptosis in diverse brain regions. These findings position 𝛾-TER as a potential therapeutic agent for ischemic stroke.
Ischemic stroke causes a lack of oxygen and glucose supply to brain, eventually leads to severe neurological disorders. Retinoic acid is a major metabolic product of vitamin A and has various biological effects. The PI3K-Akt signaling pathway is an important survival pathway in brain. Phosphorylated Akt is important in regulating survival and apoptosis. We examined whether retinoic acid has neuroprotective effects in stroke model by regulating Akt and its downstream protein, Bad. Moreover, we investigated the relationship between retinoic acid and Bcl-2 family protein interactions. Animals were intraperitoneally administered vehicle or retinoic acid (5 mg/kg) for four days before surgery and ischemic stroke was induced by middle cerebral artery occlusion (MCAO) surgery. Neurobehavioral tests were performed 24 h after MCAO and cerebral cortical tissues were collected. Cresyl violet staining and TUNEL histochemistry were performed, Western blot and immunoprecipitation analysis were performed to elucidate the expression of various proteins. Retinoic acid reduced neurological deficits and histopathological changes, decreased the number of TUNEL-positive cells, and alleviated reduction of phospho-PDK1, phospho-Akt, and phospho-Bad expression caused by MCAO damage. Immunoprecipitation analysis showed that MCAO damage reduced the interaction between phospho-Bad and 14-3-3, which was attenuated by retinoic acid. Furthermore, retinoic acid mitigated the increase in Bcl-2/Bad and Bcl-xL/Bad binding levels and the reduction in Bcl-2/Bax and Bcl-xL/Bax binding levels caused by MCAO damage. Retinoic acid alleviated MCAO-induced increase of caspase-3 and cleaved caspase-3 expression. We demonstrate that retinoic acid prevented apoptosis against cerebral ischemia through phosphorylation of Akt and Bad, maintenance of phospho-Bad and 14-3-3 binding, and regulation of Bcl-2 family protein interactions.
Background
This study investigates the potential of diffusion tensor imaging (DTI) in identifying penumbral volume (PV) compared to the standard gadolinium-required perfusion–diffusion mismatch (PDM), utilizing a stack-based ensemble machine learning (ML) approach with enhanced explainability.
Methods
Sixteen male rats were subjected to middle cerebral artery occlusion. The penumbra was identified using PDM at 30 and 90 min after occlusion. We used 11 DTI-derived metrics and 14 distance-based features to train five voxel-wise ML models. The model predictions were integrated using stack-based ensemble techniques. ML-estimated and PDM-defined PVs were compared to evaluate model performance through volume similarity assessment, the Pearson correlation analysis, and Bland–Altman analysis. Feature importance was determined for explainability.
Results
In the test rats, the ML-estimated median PV was 106.4 mL (interquartile range 44.6–157.3 mL), whereas the PDM-defined median PV was 102.0 mL (52.1–144.9 mL). These PVs had a volume similarity of 0.88 (0.79–0.96), a Pearson correlation coefficient of 0.93 (p < 0.001), and a Bland–Altman bias of 2.5 mL (2.4% of the mean PDM-defined PV), with 95% limits of agreement ranging from -44.9 to 49.9 mL. Among the features used for PV prediction, the mean diffusivity was the most important feature.
Conclusions
Our study confirmed that PV can be estimated using DTI metrics with a stack-based ensemble ML approach, yielding results comparable to the volume defined by the standard PDM. The model explainability enhanced its clinical relevance. Human studies are warranted to validate our findings.
Relevance statement
The proposed DTI-based ML model can estimate PV without the need for contrast agent administration, offering a valuable option for patients with kidney dysfunction. It also can serve as an alternative if perfusion map interpretation fails in the clinical setting.
Key points
• Penumbral volume can be estimated by DTI combined with stack-based ensemble ML.
• Mean diffusivity was the most important feature used for predicting penumbral volume.
• The proposed approach can be beneficial for patients with kidney dysfunction.
Graphical Abstract
Supplementary Information
The online version contains supplementary material available at 10.1186/s41747-024-00455-z.
Stroke remains the 3rd leading cause of long-term disability globally. Over the past decade, mesenchymal stem cell (MSC) transplantation has been proven as an effective therapy for ischemic stroke. However, the mechanism of MSC-derived exosomal lncRNAs during cerebral ischemia/reperfusion (I/R) remains ambiguous. The oxygen–glucose deprivation/reoxygenation (OGD/R) and middle cerebral artery occlusion (MCAO) rat model were generated. MSCs were isolated and characterized by flow cytometry and histochemical staining, and MSC exosomes were purified and characterized by transmission electron microscopy, flow cytometry and Western blot. Western blot, RT-qPCR and ELISA assay were employed to examine the expression or secretion of key molecules. CCK-8 and TUNEL assays were used to assess cell viability and apoptosis. RNA immunoprecipitation and RNA pull-down were used to investigate the direct association between krüppel-like factor 3 antisense RNA 1 (KLF3-AS1) and musashi-1(MSI1). Yin Yang 1 (YY1)-mediated transcriptional regulation was assessed by chromatin immunoprecipitation and luciferase assays. The histological changes and immunoreactivity of key molecules in brain tissues were examined by H&E and immunohistochemistry. MSCs were successfully isolated and exhibited directionally differential potentials. MSC exosomal KLF3-AS1 alleviated OGD/R-induced inflammation in SK-N-SH and SH-SY5Y cells via modulating Sphk1. Mechanistical studies showed that MSI1 positively regulated KLF3-AS1 expression through its direct binding to KLF3-AS1. YY1 was identified as a transcription activator of MSI1 in MSCs. Functionally, YY1/MSI1 axis regulated the release of MSC exosomal KLF3-AS1 to modulate sphingosine kinase 1 (Sphk1)/NF-κB pathway, thereby ameliorating OGD/R- or cerebral I/R-induced injury. MSCs promote the release of exosomal KLF3-AS1 to regulate Sphk1 through YY1/MSI axis and improve cerebral I/R injury.
In translational animal study aimed at evaluation of the effectiveness of innovative methods for treating cerebral stroke, including regenerative cell technologies, of particular importance is evaluation of the dynamics of changes in the volume of the cerebral infarction in response to therapy. Among the methods for assessing the focus of infarction, MRI is the most effective and convenient tool for use in preclinical studies. This review provides a description of MR pulse sequences used to visualize cerebral ischemia at various stages of its development, and a detailed description of the MR semiotics of cerebral infarction. A comparison of various methods for morphometric analysis of the focus of a cerebral infarction, including systems based on artificial intelligence for a more objective measurement of the volume of the lesion, is also presented.
Ischemia/reperfusion (IR) can induce deleterious responses such as apoptosis, inflammation, and oxidative stress; however, there are currently no efficient therapeutics to treat IR brain injury. Dragon’s blood (DB) plays a significant role in treating ischemic stroke in China. Borneol (B) is an upper ushering drug that guides drugs to the target organs, including the brain. Therefore, we hypothesized that the combination of DB and B (DB + B) would provide cooperative therapeutic benefits for IR brain injury. To confirm this, we first investigated the protective effect of DB + B in an IR brain injury rat model using the modified neurological severity score (mNSS), infarction size measure, HE staining, and laser speckle contrast imaging. Then, we comprehensively evaluated the mechanism of DB + B in ameliorating IR brain injury based on RNA sequencing, serum untargeted metabolomics, and 16S rRNA sequencing. We have confirmed that DB + B enhanced the efficacy of the ischemic stroke treatment compared to DB or B alone for the first time. Our study provisionally confirms that the mechanism by which DB + B prevents IR brain injury is related to the maintenance of intestinal microecological balance and regulation of metabolic dysfunction, thereby suppressing inflammation and regulating immunity. DB + B may effectively regulate intestinal flora including o_Pseudomonadales, s_Bacteroides_caecimuris, o_unidentified_Bacilli, f-Pseudomonadaceae, and g-Pseudomonas, mainly regulate serum metabolites including improve the protective benefit of IR brain injury lysoPCs and lysoPEs, thus inhibiting TLR4/MyD88/NF-κB and IL-17 signing pathway to reduce inflammatory reactions. hat this mechanism is associated with the maintenance of intestinal flora balance and the regulation of metabolic dysfunction, thereby suppressing inflammation and regulating immunity. This provides scientific support for the clinical translation of DB + B in the prevention and treatment of ischemic stroke and establishes a basis for further investigation of its therapeutic mechanism.
Objective
Ischaemic stroke is a leading cause of death and disability in individuals worldwide. Cerebral ischaemia reperfusion injury (CIRI) usually leads to severe secondary injury and complications following reperfusion therapy. Microglia play critical roles in the inflammatory reaction of CIRI. However, less attention has been given to microglial death in this process. Our study aimed to explore microglial death in CIRI and the effect and mechanism of minocycline treatment on microglia
Methods
A middle cerebral artery occlusion (MCAO) model was applied to induce CIRI in rats. At 0 h, 24 h and 48 h postoperation, 45 mg/kg minocycline was intraperitoneally injected into the rats. Neurological deficit scoring, 2,3,5-triphenyltetrazolium chloride (TTC) staining, activated microglia and mitochondrial structure were observed and checked at 72 h after reperfusion. Moreover, an in vitro model of oxygen-glucose deprivation/reperfusion (OGD/R) model was established. BV-2 cells were treated with either various pharmacological inhibitors of cell death or minocycline. Cell viability, lipid peroxidation, mitochondrial structure and functioning, and labile Fe²⁺ and ferroptosis-associated gene/proteins levels were measured. Hemin was used for further validation after transcriptome analysis.
Results
In the MCAO and OGD/R models, ferroptosis was identified as a major form of microglial death. Minocycline inhibited microglial ferroptosis by reducing HO-1 expression. In addition, minocycline improved mitochondrial membrane potential, mitochondrial structures and microglial survival in vivo. Minocycline also decreased labile Fe²⁺ levels, lipid peroxidation, and ferritin heavy chain (FTH) expression and improved mitochondrial structure and functioning in vitro. HO-1 overexpression counteracted the protective effect of minocycline.
Conclusion
Ferroptosis is a major form of microglial death in CIRI. The mechanism of the protective role of minocycline in CIRI is partly dependent on its ability to effectively ameliorate microglial ferroptosis by reducing HO-1 expression. Therefore, targeting microglial ferroptosis is a promising treatment for CIRI.
Background
As a leading cause of mortality and long-term disability, acute ischemic stroke can produce far-reaching pathophysiological consequences. Accumulating evidence has demonstrated abnormalities in the lower motor system following stroke, while the existence of Transsynaptic degeneration of contralateral spinal cord ventral horn (VH) neurons is still debated.
Methods
Using a rat model of acute ischemic stroke, we analyzed spinal cord VH neuron counts contralaterally and ipsilaterally after stroke with immunofluorescence staining. Furthermore, we estimated the overall lower motor unit abnormalities after stroke by simultaneously measuring the modified neurological severity score (mNSS), compound muscle action potential (CMAP) amplitude, repetitive nerve stimulation (RNS), spinal cord VH neuron counts, and the corresponding muscle fiber morphology. The activation status of microglia and extracellular signal-regulated kinase 1/2 (ERK 1/2) in the spinal cord VH was also assessed.
Results
At 7 days after stroke, the contralateral CMAP amplitudes declined to a nadir indicating lower motor function damage, and significant muscle disuse atrophy was observed on the same side; meanwhile, the VH neurons remained intact. At 14 days after focal stroke, lower motor function recovered with alleviated muscle disuse atrophy, while transsynaptic degeneration occurred on the contralateral side with elevated activation of ERK 1/2, along with the occurrence of neurogenic muscle atrophy. No apparent decrement of CMAP amplitude was observed with RNS during the whole experimental process.
Conclusions
This study offered an overview of changes in the lower motor system in experimental ischemic rats. We demonstrated that transsynaptic degeneration of contralateral VH neurons occurred when lower motor function significantly recovered, which indicated the minor role of transsynaptic degeneration in lower motor dysfunction during the acute and subacute phases of focal ischemic stroke.
Cerebral ischemia-reperfusion injury (CIRI) is one of the most difficult challenges in cerebrovascular disease research. It is primarily caused by excessive autophagy induced by oxidative stress. Previously, a novel compound X5 was found, and the excellent antioxidant activity of it was verified in this study. Moreover, network pharmacological analysis suggested that compound X5 was closely associated with autophagy and the mTOR pathway. In vitro, X5 could significantly inhibit the expression of autophagy proteins Beclin-1 and LC3-β, which are induced by H2O2, and promote the expression of SIRT1. In vivo, compound X5 significantly reduced the infarct size and improved the neurological function scores in the middle cerebral artery occlusion (MCAO) model of rats. In conclusion, ROS-induced autophagy is closely related to mTOR, SIRT1 and others, and X5 holds promise as a candidate for the treatment of CIRI.
Objectives
Astragaloside IV (AST IV) and ligustrazine (Lig), the main ingredients of Astragali Radix and Chuanxiong Rhizoma respectively, have demonstrated significant benefits in treatment of cerebral ischemia ‐reperfusion injury (CIRI); however, the mechanisms underlying its benificial effects remain unclear. SUMO‐1ylation and deSUMO‐2/3ylation of dynamin‐related protein 1 (Drp1) results in mitochondrial homeostasis imbalance following CIRI, which subsequently aggravates cell damage. This study investigates the mechanisms by which AST IV combined with Lig protects against CIRI, focusing on the involvement of SUMOylation in mitochondrial dynamics.
Methods
Rats were administrated AST IV and Lig for 7 days, and middle cerebral artery occlusion was established to mimic CIRI. Neural function, cerebral infarction volume, cerebral blood flow, cognitive function, cortical pathological lesions, and mitochondrial morphology were measured. SH‐SY5Y cells were subjected to oxygen–glucose deprivation/reoxygenation (OGD/R) injury. Mitochondrial membrane potential and lactic dehydrogenase (LDH), reactive oxygen species (ROS), and adenosine triphosphate (ATP) levels were assessed with commercial kits. Moreover, co‐immunoprecipitation (Co‐IP) was used to detect the binding of SUMO1 and SUMO2/3 to Drp1. The protein expressions of Drp1, Fis1, MFF, OPA1, Mfn1, Mfn2, SUMO1, SUMO2/3, SENP1, SENP2, SENP3, SENP5, and SENP6 were measured using western blot.
Results
In rats with CIRI, AST IV and Lig improved neurological and cognitive functions, restored CBF, reduced brain infarct volume, and alleviated cortical neuron and mitochondrial damage. Moreover, in SH‐SY5Y cells, the combination of AST IV and Lig enhanced cellular viability, decreased release of LDH and ROS, increased ATP content, and improved mitochondrial membrane potential. Furthermore, AST IV combined with Lig reduced the binding of Drp1 with SUMO1, increased the binding of Drp1 with SUMO2/3, suppressed the expressions of Drp1, Fis1, MFF, and SENP3, and increased the expressions of OPA1, Mfn1, Mfn2, SENP1, SENP2, and SENP5. SUMO1 overexpression promoted mitochondrial fission and inhibited mitochondrial fusion, whereas SUMO2/3 overexpression suppressed mitochondrial fission. AST IV combined with Lig could reverse the effects of SUMO1 overexpression while enhancing those of SUMO2/3 overexpression.
Conclusions
This study posits that the combination of AST IV and Lig has the potential to reduce the SUMO‐1ylation of Drp1, augment the SUMO‐2/3ylation of Drp1, and thereby exert a protective effect against CIRI.
Rhubarb (RR), Chinese name Dahuang, is commonly used in the treatment of ischemic stroke (IS). However, its potential mechanism is not fully elucidated. This study intended to verify the effect of RR on IS and investigate the possible mechanism of RR in preventing IS. IS in male rats was induced by embolic middle cerebral artery occlusion (MCAO) surgery, and drug administration was applied half an hour before surgery. RR dramatically decreased the neurological deficit scores, the cerebral infarct volume, and the cerebral edema rate, and improved the regional cerebral blood flow (rCBF) and histopathological changes in the brain of MCAO rats. The 16S rRNA analysis showed the harmful microbes such as Fournierella and Bilophila were decreased, and the beneficial microbes such as Enterorhabdus, Defluviitaleaceae, Christensenellaceae, and Lachnospira were significantly increased, after RR pretreatment. 1H-nuclear magnetic resonance (1H-NMR) was used to detect serum metabolomics, and RR treatment significantly changed the levels of metabolites such as isoleucine, valine, N6-acetyllysine, methionine, 3-aminoisobutyric acid, N, N-dimethylglycine, propylene glycol, trimethylamine N-oxide, myo-inositol, choline, betaine, lactate, glucose, and lipid, and the enrichment analysis of differential metabolites showed that RR may participate in the regulation of amino acid metabolism and energy metabolism. RR exerts the role of anti-IS via regulating gut bacteria and metabolic pathways.
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