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Thrombolysis decreased malondialdehyde (MDA) contents after MCAO. Quantification of MDA contents in the sham group, conventional group and thrombolytic group. * indicates P < 0.05
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Recombinant tissue plasminogen activator (rt‐PA) is the first‐line drug for revascularization in acute cerebral infarction (ACI) treatment. In this study, an improved rat embolic middle cerebral artery occlusion model for ischaemic stroke was used and the rats were killed on the first, third and seventh day after model establishment. Increases in i...
Citations
... Adenosine 5'-monophosphateactivated protein kinase (AMPK), which functions as a cellular energy sensor, can protect against cerebral ischemic injury by activating catabolic pathways and inactivating ATP-consuming processes [36]. Excessive levels of intracellular free radicals and NO: Superoxide dismutase is an important antioxidant enzyme in brain tissue and can protect the brain by removing excessive free radicals in cells [37]. NO plays a crucial role in information transmission and neuroprotection in the central nervous system, but excessive release of NO, as an active gaseous free radical, can lead to neuron damage [11]. ...
Stroke is a main cause of disability and death among adults in China, and acute ischemic stroke accounts for 80% of cases. The key to ischemic stroke treatment is to recanalize the blocked blood vessels. However, more than 90% of patients cannot receive effective treatment within an appropriate time, and delayed recanalization of blood vessels causes reperfusion injury. Recent research has revealed that ischemic postconditioning has a neuroprotective effect on the brain, but the mechanism has not been fully clarified. Long non-coding RNAs (lncRNAs) have previously been associated with ischemic reperfusion injury in ischemic stroke. LncRNAs regulate important cellular and molecular events through a variety of mechanisms, but a comprehensive analysis of potential lncRNAs involved in the brain protection produced by ischemic postconditioning has not been conducted. In this review, we summarize the common mechanisms of cerebral injury in ischemic stroke and the effect of ischemic postconditioning, and we describe the potential mechanisms of some lncRNAs associated with ischemic stroke.
Background: Ischemic stroke (IS) is one of the leading causes of death and disability worldwide. The narrow therapeutic window (within 4.5 h) and severe hemorrhagic potential limits therapeutic efficacy of recombinant tissue type plasminogen activator (rt-PA) intravenous thrombolysis for patients. Xingnao Kaiqiao (XNKQ) acupuncture is an integral part of traditional Chinese medicine, specifically designed to address acute ischemic stroke by targeting key acupoints such as Shuigou (GV26) and Neiguan (PC6). In this study, we explored the therapeutic potential of XNKQ acupuncture in extending the time window for thrombolysis and interrogated the molecular mechanisms responsible for this effect. Methods: The effect of extending the thrombolysis window by acupuncture was evaluated via TTC staining, neuronal score evaluation, hemorrhagic transformation assay, and H&E staining. RNA sequencing (RNA-seq) technology was performed to identify the therapeutic targets and intervention mechanisms of acupuncture. Evans blue staining and transmission electron microscopy were used to assess blood–brain barrier (BBB) integrity. Immunofluorescence staining and co-immunoprecipitation were performed to evaluate the level of autophagy and apoptosis and validate their interactions with BBB endothelial cells. Results: Acupuncture alleviated infarction and neurological deficits and extended the thrombolysis window to 6 h. The RNA-seq revealed 16 potential therapeutic predictors for acupuncture intervention, which related to suppressing inflammation and restoring the function of BBB and blood vessels. Furthermore, acupuncture suppressed BBB leakage and preserved tight junction protein expression. The protective effect was associated with regulation of the autophagy–apoptosis balance in BBB endothelial cells. Acupuncture intervention dissociated the Beclin1/Bcl-2 complex, thereby promoting autophagy and reducing apoptosis. Conclusion: XNKQ acupuncture could serve as an adjunctive therapy for rt-PA thrombolysis, aiming to extend the therapeutic time window and mitigate ischemia–reperfusion injury. Acupuncture suppressed BBB disruption by regulating the autophagy–apoptosis balance, which in turn extended the therapeutic window of rt-PA in IS. These findings provide a rationale for further exploration of acupuncture as a complementary candidate co-administered with rt-PA.
Objective:
To investigate the effects of alteplase on neurological deficits, as well as on the expressions of glial fibrillary acidic protein (GFAP) and growth-associated protein-43 (GAP-43) in brain tissues of rats with acute cerebral infarction (ACI).
Methods:
Sprague Dawley (SD) rats (n = 50) were enrolled in a trial to establish a ACI rat model; of these, 48 rats were succeeefully modeled and were randomized into either the model or alteplase group, whereas another 24 SD rats were included in the sham-operated group.
Findings:
No significant difference in scores was observed between the model and alteplase groups at T1 (P > 0.05); however, rats in the alteplase group demonstrated lower scores than those in the model group at T2, T3, and T4 (P < 0.05). Rats in the model group showed a larger cerebral infarction volume than those in the alteplase group (P < 0.05), and the infarction volume on day 1, 3, 6, and 9 was higher in rats in the alteplase group than those in the sham-operated group (P < 0.05).
Conclusion:
Treatment with alteplase can be effective in reducing cerebral infarction volume and moderating neurological deficits in ACI modeled rats within a 6-h time window, which may be correlated with the regulation of GFAP and GAP-43 expressions by alteplase.
To determine the curative effect and prognosis of Solitaire FR stent thrombectomy integrated with the suction thrombus on the treatment of acute middle cerebral artery occlusion (AMCAO). Based on the treatment, patients suffering from AMCAO were separated into the Solitaire FR group (Solitaire FR stent + suction thrombus) and suction group (suction thrombus). Modified thrombolysis in cerebral infarction grading, National Institutes of Health Stroke Scale (NIHSS) score, modified Rankin Scale score, and safety performance were compared between the two groups. The operation time in the suction group was obviously shorter than the Solitaire FR group ( P < 0.05 ). Significant differences were observed in the NIHSS scores 1 week and 4 weeks after the operation between the Solitaire FR group and the suction group ( P < 0.05 ). The NIHSS scores 1 week and 4 weeks after operation were significantly lower than those before operation ( P < 0.05 ). NIHSS scores 1 week after operation did not show obvious difference ( P > 0.05 ). The Solitaire FR group showed obvious lower NIHSS scores than the suction group 4 weeks after surgery ( P < 0.05 ). Statistically obvious difference in cerebral infarction grading of modified thrombolysis between the Solitaire FR group and the suction group were observed ( P < 0.05 ). The recanalization rate of the Solitaire FR group was obviously higher than the suction group ( P < 0.05 ). The difference in the monthly modified Rankin Scale score was obvious ( P < 0.05 ). The good prognosis rate of the Solitaire FR group was obviously higher than the suction group ( P < 0.05 ). No obvious differences in the incidence of internal bleeding, reocclusion, and 3-month postoperative mortality were observed ( P > 0.05 ). These results showed that the treatment of the Solitaire FR stent + suction thrombus in AMCAO patients has a good thrombus recanalization rate and is helpful in improving the prognosis and safety performance.
Objective
Ischemic stroke seriously threatens human health, characterized by the high rates of incidence, disability, and death. Developing a reliable animal model that mimics most of the features of stroke is critical for pathological studies and clinical research. In this study, we aimed to establish and examine a model of middle cerebral artery occlusion (MCAO) guided by digital subtraction angiography (DSA) in cynomolgus monkeys.
Materials and Methods
In this study, 15 adult male cynomolgus monkeys were enrolled. Under the guidance of DSA, a MCAO model was established by injecting an autologous venous clot into the middle cerebral artery (MCA) via femoral artery catheter. Thrombolytic therapy with alteplase (rt-PA) was given to eight of these monkeys at 3 h after the occlusion. Blood test and imaging examination, such as computed tomography angiography (CTA), CT perfusion (CTP), brain magnetic resonance imaging (MRI), and brain magnetic resonance angiography (MRA), were performed after the operation to identify the post-infarction changes. The behavioral performance of cynomolgus monkeys was continuously observed for 7 days after operation. The animals were eunthanized on the 8th day after operation, and then the brain tissues of monkeys were taken for triphenyltetrazolium chloride (TTC) staining.
Results
Among the 15 cynomolgus monkeys, 12 of them were successfully modeled, as confirmed by the imaging findings and staining assessment. One monkey died of brain hernia resulted from intracranial hemorrhage confirmed by necropsy. DSA, CTA, and MRA indicated the presence of an arterial occlusion. CTP and MRI showed acute focal cerebral ischemia. TTC staining revealed infarct lesions formed in the brain tissues.
Conclusion
Our study may provide an optimal non-human primate model for an in-depth study of the pathogenesis and treatment of focal cerebral ischemia.
Teriparatide is a commonly used drug indicated for the treatment of osteoporosis in postmenopausal women. Teriparatide can also upregulate Ang-1 expression through the AC/PKA signaling pathway to promote angiogenesis. At present, promoting angiogenesis is a promising but unrealized strategy for the treatment of ischemic cerebral infarction. However, there are few studies on the application of teriparatide in the treatment of cerebral infarction. We used teriparatide to treat ischemic cerebral infarction in rats and obtained three major findings. First, teriparatide can promote angiogenesis, reduce cerebral infarct size, and increase cerebral perfusion by upregulating Ang-1 expression. Second, teriparatide can promote the expression of HO1, SOD2 and inhibit the production of pro-inflammatory cytokines IL-1β, IL-6 by upregulating Nrf2 expression. Third, we further found that teriparatide can mitigate blood-brain barrier disruption and brain edema by downregulating the expressions of MMP9, Ang-2 and AQP4. Our results indicate that teriparatide is neuroprotective through multiple mechanisms of action that include promoting angiogenesis, inhibiting oxidative stress and neuroinflammation, protecting blood-brain barrier, and reducing brain edema.
Despite recent advances in recanalization therapy, mechanical thrombectomy will never be a treatment for every ischemic stroke because access to mechanical thrombectomy is still limited in many countries. Moreover, many ischemic strokes are caused by occlusion of cerebral arteries that cannot be reached by intra-arterial catheters. Reperfusion using thrombolytic agents will therefore remain an important therapy for hyperacute ischemic stroke. However, thrombolytic drugs have shown limited efficacy and notable hemorrhagic complication rates, leaving room for improvement. A comprehensive understanding of basic and clinical research pipelines as well as the current status of thrombolytic therapy will help facilitate the development of new thrombolytics. Compared with alteplase, an ideal thrombolytic agent is expected to provide faster reperfusion in more patients; prevent re-occlusions; have higher fibrin specificity for selective activation of clot-bound plasminogen to decrease bleeding complications; be retained in the blood for a longer time to minimize dosage and allow administration as a single bolus; be more resistant to inhibitors; and be less antigenic for repetitive usage. Here, we review the currently available thrombolytics, strategies for the development of new clot-dissolving substances, and the assessment of thrombolytic efficacies in vitro and in vivo .
Objective
Acute cerebral infarction (ACI) is susceptible to cause disability or death of people. Astaxanthin (ATX) possesses the protective effect of organ injury. Therefore, the study was to explore the potential mechanism of protective effect with ATX on ACI.
Methods
30 SD rats were divided into Sham, ACI, and ATX groups. The rats in the ATX group were pretreated with ATX by gavage for three days before surgery, while the rats in the other two groups were pretreated with saline. The model of ACI was established by thread embolization. 24 h after the operation, the neurological function was scored, and cerebral infarct area and pathological morphology of brains were measured; the edema of the brain was detected by dry/wet method; Western blot was applied to measure the translocation of Nrf-2 and the protein expression of HO-1, Bax and BCL-2; Brain cell apoptosis was assessed through TUNEL; ELISA was used to detect the oxidative stress factors of catalase (CAT) superoxide dismutase (SOD), glutathione peroxidase (GPX) and malondialdehyde (MDA), and the inflammatory factors of TNF-α, IL-1β, IL-6.
Result
Compared with the ACI group, ATX pretreatment can significantly improve neurological function; reduce the edema index of the brain, cerebral infarct area, cerebral pathological damage and apoptosis of brain cells. Moreover, ATX also can increase the protein expression of nuclear Nrf-2, HO-1, BCL-2, CAT, SOD, and GPX by decreasing the content of TNF-α, IL-1β, IL-6, MDA, Bax and cytosolic Nrf-2.
Conclusion
ATX might have a protective effect of acute cerebral infarction, and the mechanism is probably associated with suppressing oxidative stress, inflammation, and apoptosis by activating Nrf-2/HO-1signalling.
Background. Recombinant tissue plasminogen activator (rtPA) is the only recommended pharmacological treatment for acute ischemic stroke, but it has a restricted therapeutic time window. When administered at time points greater than 4.5 h after stroke onset, rtPA disrupts the blood-brain barrier (BBB), which leads to serious brain edema and hemorrhagic transformation. Electroacupuncture (EA) exerts a neuroprotective effect on cerebral ischemia; however, researchers have not clearly determined whether EA increases the safety of thrombolysis and extends the therapeutic time window of rtPA administration following ischemic stroke. Objective. The present study was conducted to test the hypothesis that EA extends the therapeutic time window of rtPA for ischemic stroke in a male rat model of embolic stroke. Methods. SD rats were randomly divided into the sham operation group, model group, rtPA group, EA+rtPA group, and rtPA+MEK1/2 inhibitor group. An injection of rtPA was administered 6 h after ischemia. Rats were treated with EA at the Shuigou (GV26) and Neiguan (PC6) acupoints at 2 h after ischemia. Neurological function, infarct volume, BBB permeability, brain edema, and hemorrhagic transformation were assessed at 24 h after ischemia. Western blotting and immunofluorescence staining were performed to detect the levels of proteins involved in the ERK1/2 signaling pathway (MEK1/2 and ERK1/2), tight junction proteins (Claudin5 and ZO-1), and MMP9 in the ischemic penumbra at 24 h after stroke. Results. Delayed rtPA treatment aggravated hemorrhagic transformation and brain edema. However, treatment with EA plus rtPA significantly improved neurological function and reduced the infarct volume, hemorrhagic transformation, brain edema, and EB leakage in rats compared with rtPA alone. EA increased the levels of tight junction proteins, inhibited the activation of the ERK1/2 signaling pathway, and reduced MMP9 overexpression induced by delayed rtPA thrombolysis. Conclusions. EA potentially represents an effective adjunct method to increase the safety of thrombolytic therapy and extend the therapeutic time window of rtPA administration following ischemic stroke. This neuroprotective effect may be mediated by the inhibition of the ERK1/2-MMP9 pathway and alleviation of the destruction of the BBB.
1. Introduction
Stroke is a leading cause of mortality and disability worldwide [1]; approximately 13.7 million new stroke cases, 5.5 million deaths, and 116.4 million disability-adjusted life-years due to stroke were reported in 2016 [2]. Acute ischemic stroke, the most common subtype, accounts for 87% of all strokes [3] and primarily results from occlusion of the cerebral arteries by thrombosis or embolism [4]. Currently, intravenous thrombolysis with recombinant tissue plasminogen activator (rtPA) has been proven to be the most effective pharmacological treatment for acute ischemic stroke when administered within 3-4.5 h after ischemia onset [5]. Unfortunately, the use of rtPA is restricted by its narrow thrombolytic time window, because it may cause thrombolytic complications, such as brain edema and hemorrhagic transformation, particularly when delayed thrombolysis is initiated after 4.5 h [6, 7]. Due to these limitations, only 3.8-8% of patients with ischemic stroke benefit from rtPA-mediated thrombolysis [8]. Therefore, any neuroprotective strategy designed to reduce complications and extend the thrombolytic time window will be very important.
Based on accumulating evidence from clinical and animal studies, the disruption of the blood-brain barrier (BBB) is the key event that leads to brain edema and hemorrhagic transformation during thrombolysis for ischemic stroke [9–11]. The BBB is composed of endothelial cells, tight junctions (TJs), pericytes, astrocytic endfeet, and extracellular matrix (ECM) [12]. Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that are best known for their role in the degradation and remodeling of ECM components [13]. The level of the matrix metalloproteinase 9 (MMP9) protein has consistently been shown to increase after ischemic stroke, and it plays an important role in BBB destruction by degrading ECM and TJ proteins [14–16]. More importantly, rtPA may cross the BBB, enter the brain parenchyma, and thereby damage the neurovascular matrix by promoting MMP9 production and activation [17, 18]. Consequently, selective inhibition of MMP9 reduces brain injury, particularly the degradation of the BBB, after rtPA thrombolysis for ischemic stroke [14, 19].
Extracellular signal-regulated kinase 1/2 (ERK1/2), a critical member of mitogen-activated protein kinase (MAPK) cascades, is activated by dual phosphorylation catalyzed by MAPK kinase (MAPKK, also known as MEK1/2). The ERK1/2 signaling pathway is involved in the inflammatory response and apoptosis and plays an important role in the repair of the BBB after brain injury [20, 21]. MMP9 expression is induced by ERK1/2 signaling, and inhibition of ERK1/2 signaling reduces the hemorrhagic transformation and brain edema caused by overexpression of MMP9 following cerebral ischemia [14, 22–24].
Electroacupuncture (EA), a type of acupuncture with electronic stimulation, is a well-known complementary and alternative medical treatment for ischemic stroke in China. Based on both clinical and experimental studies, EA, a safe and effective treatment, significantly reduces the infarct volume and neurological deficit score following cerebral ischemia [25–27]. EA stimulation exerts a neuroprotective effect by increasing the expression of TJ proteins (Claudin5 and ZO-1), reducing MMP9 expression and protecting the BBB integrity in various animal models of ischemic stroke [28–30]. In addition, EA alleviates cerebral ischemia and reperfusion injury by modulating the ERK1/2 signaling pathway [31]. However, researchers have not yet clearly determined whether EA improves the safety of thrombolysis and extends the therapeutic time window during rtPA thrombolysis for ischemic stroke. Therefore, the present study was conducted to test the hypothesis that EA represents an adjunct therapy that will extend the therapeutic time window of rtPA for ischemic stroke by alleviating BBB damage and reducing the risk of complications induced by delayed rtPA thrombolysis. Moreover, we further elucidated whether this neuroprotective effect was associated with the modulation of the ERK1/2-MMP9 signaling pathway, as well as the underlying mechanisms.
2. Materials and Methods
2.1. Animals
Adult male Sprague-Dawley (SD) rats weighing g were supplied by Shanghai Xipuer-Bikai Experimental Animal Co., Ltd. (Shanghai, China; license no. SCXK (Hu): 2018-0006). All rats were housed in a temperature- and humidity-controlled room on a 12 h light/dark cycle at the Experimental Animal Centre of Nanjing University of Chinese Medicine. This study was approved by the Institutional Animal Care and Use Committee of Nanjing University of Chinese Medicine, and all procedures were strictly conducted in accordance with the guidelines of the National Institutes of Health Animal Care and Use Committee. The experiments reported here were performed in accordance with the ARRIVE guidelines.
2.2. Establishment of the Embolic Stroke Model
An embolic stroke model was induced by placing a blood clot into the middle cerebral artery (MCA) using the methods described by Zhang et al. [32].(a)Preparation of Embolus. The external carotid artery (ECA) of the donor rat was catheterized; blood was transferred into 20 cm long PE-50 tubing, allowed to clot for 2 h at 37°C, and then stored at 4°C for 22 h. A 5 cm segment of clot-filled PE-50 tubing was cut, and the clot was then drawn into a PE-10 tubing connected to a saline-filled syringe via a 30 G needle. The clot was drawn into and flushed out of the PE-10 tubing repeatedly to remove the red blood cells. A 4 cm segment of clot was cut and transferred to a modified PE-50 catheter (outer diameter of 0.35 mm) connected to a 100 μl syringe and the clot was then injected into the MCA(b)Embolic Stroke Model Establishment. Rats were anesthetized with isoflurane (5% for induction and 1.5-2% for maintenance), and then, an embolic stroke was induced. The rectal temperature (°C) was maintained throughout surgery with an electric blanket. The right common carotid artery (CCA), internal carotid artery (ICA), and ECA were exposed via a midline cervical incision. The distal end and branches of the ECA were ligated, and the CCA and the ICA were temporarily clamped with a microvascular clip. Immediately thereafter, a partial arteriotomy on the ECA was performed, and the tip of a modified PE-50 catheter containing the clot was inserted into the ECA lumen and advanced 19-22 mm from the ECA into the lumen of the ICA until it reached the origin of the MCA. Then, the catheter was retracted 1-2 mm, and the clot was slowly injected with 5-10 μl of saline at a rate of 10 μl/min. The catheter was withdrawn from the arteriotomy 5 min after the injection. The cerebral blood flow (CBF) was monitored with laser Doppler flowmetry (LDF, moorVMS-LDF1) by gently attaching an LDF probe to the dura mater. The successful obstruction of CBF by the thrombus was defined as a reduction in perfusion greater than 70% of the baseline CBF [33] (Supplementary Figure 1). The successful establishment of the model was judged based on the obstruction of CBF and neurological deficit score at 2 h after stroke. For sham-operated rats, the same surgery was performed, except that 5-10 μl saline was injected into the MCA
2.3. Experimental Design and Groups
Two sets of experiments were performed, and the animals were randomly divided into various group.
In the first experiment, rats were randomly assigned to the following groups: (1) sham, (2) model, (3) rtPA, and (4) rtPA+EA. A tail vein injection of rtPA (Boehringer Ingelheim, Germany) was administered at 6 h after stroke induction, and the doses of rtPA (10 mg/kg) were determined based on previous studies [34, 35]. An equal volume of normal saline was injected intravenously at the corresponding times in rats that did not receive rtPA. These animals were used to measure the infarct volume, brain edema, neurological deficit score, hemorrhagic transformation, BBB permeability, and expression of ZO-1, Claudin5, ERK1/2, and MMP9. In this experiment, we determined that EA attenuated delayed rtPA-induced BBB disruption and hemorrhagic transformation and improved neurological function by preventing the activation of ERK1/2 and MMP9.
We conducted a second experiment to determine whether ERK1/2 signaling affects the expression of MMP9 in rats with embolic stroke. The rats were randomly divided into the sham group, rtPA group, and rtPA+MEK1/2 inhibitor (U0126). U0126 (5 μl, 0.2 μg/μl) [36] or vehicle (0.1 M PBS containing 0.4% DMSO) was administered intracerebroventricularly (ICV) 30 min prior to embolic stroke. The ICV injection site was chosen at the following coordinates from the bregma according to the rat brain in stereotaxic coordinates: anteroposterior, 1.2 mm; lateral, 2.0 mm; and depth, 3.8 mm.
At 24 h after stroke, all animals were euthanized, and their brains were harvested for further experiments.
2.4. Electroacupuncture Treatment
Rats in the rtPA+EA group received EA at the Shuigou (GV26) and left Neiguan (PC6) acupoints at 2 h postembolic stroke. GV26 was located at the junction of the upper 1/3 and middle 1/3 of the upper lip. PC6 was located approximately 3 mm proximal to the palm crease above the median nerve. Stainless steel acupuncture needles (outer diameter 0.3 mm) were inserted 2-3 mm into GV26 and PC6. Then, the needles were connected to an electrical stimulator (HANS-200, Nanjing Jisheng Medical Technology Company, China) with an intensity of 1 mA and frequency of 2/15 Hz for 30 min.
2.5. Measurement of Neurological Function
The neurological deficit score was recorded at 2 h and 24 h after embolic stroke and determined with a modified 6-point scoring system [37, 38] as follows: 0, no apparent deficits; 1, contralateral forelimb flexion; 2, decreased grip of the contralateral forelimb while pulled by tail; 3, spontaneous movement in all directions, contralateral circling only if pulled by tail; 4, spontaneous contralateral circling; and 5, death. The successful establishment of the embolic stroke model was confirmed by the obstruction of CBF and a neurological deficit score of no less than 2 points at 2 h after stroke.
2.6. Measurement of the Infarct Volume and Brain Edema
TTC staining was performed 24 h after ischemia onset to determine the infarct volume. Rats were euthanized, and the brains were rapidly removed and frozen at -30°C for 15 min. Then, brain tissues were sectioned into 2-mm-thick coronal slices and immersed in a 2% solution of TTC (Sigma) at 37°C in the dark for 30 min. Normal regions were stained red, whereas infarct regions appeared white. Finally, the stained slices were fixed with a 4% paraformaldehyde solution for 24 hours and scanned to measure the ratio of infarct area to the whole brain using an image analysis system (ImageJ software).
Brain edema was assessed at 24 h after ischemia onset by measuring the brain water content using the wet-dry weight technique. The cerebellum and olfactory bulb were removed. Then, the injured right hemisphere was weighed to obtain its wet weight and subsequently dried at 100°C for 24 hours to obtain the dry weight. The percentage of the brain water content was calculated as .
2.7. Evaluation of BBB Permeability
BBB permeability was assessed by measuring the extravasation of Evans Blue (EB, Sigma) dye in the rat brain at 24 h after embolic stroke [39, 40]. Two hours before decapitation, 2% EB was injected into rats via the tail vein at 4 mL/kg (body weight). The rats were deeply anesthetized and transcardially perfused with normal saline through the left ventricle until an outflow of clear perfusion fluid from the right atrium was observed. After decapitation, the brain tissue was removed, and the right hemisphere was weighed. The brain tissue was homogenized in a formamide solution (1 mL/100 mg) and then incubated in a water bath (60°C) for 24 h before being centrifuged at 1000 × g for 30 min. Finally, the absorbance of EB in the supernatants was measured at 620 nm using a spectrophotometer. The EB content was reported as micrograms per gram of brain tissue and was calculated from a standard curve.
2.8. Measurement of Hemorrhagic Transformation
Hemorrhagic transformation was determined by detecting hemoglobin levels on the ischemic side of the brain using a method reported in a previous study [41]. At 24 h after embolic stroke, rats were anesthetized and perfused transcardially with 0.1 M phosphate-buffered saline (PBS). The ischemic hemisphere was separated, homogenized in 0.1 M PBS, and then centrifuged for 30 min (13000 rpm). Thereafter, the supernatant was collected, and the hemoglobin level was measured with a hemoglobin assay kit (QuantiChrom™ Hemoglobin Assay Kit, Hayward, USA) according to the manufacturer’s protocol. The optical density value of each sample was measured at 400 nm using a microplate reader.
2.9. Western Blot Analysis
All rats were sacrificed at 24 h poststroke, and the ischemic penumbra was separated based on a previous study [42]. Total protein was extracted using a protein extraction kit (Beyotime Biotech) according to the manufacturer’s protocol. The protein content was determined using the quantitative BCA protein assay. Equal amounts of protein (30 μg) were separated by electrophoresis on 10% SDS-PAGE gels and transferred to PVDF membranes. The membranes were blocked with 5% BSA for 2 h at room temperature, followed by an overnight incubation at 4°C with the following specific primary antibodies: ERK1/2 (1 : 1000, ab17942, Abcam), MEK1/2 (1 : 1000, ab178876, Abcam), MMP9 (1 : 2000, ab76003, Abcam), p-MEK1/2 (1 : 1000, CST9154, CST), p-ERK1/2 (1 : 2000, CST4370, CST), Claudin5 (1 : 2000, ab131259, Abcam), and ZO-1 (1 : 1000, 21773-1-AP, Proteintech). After washes with TBST, the membranes were incubated with HRP-labeled goat anti-rabbit IgG (1 : 1000, A0208, Beyotime Biotech) at room temperature for one hour. The bands were detected using an enhanced chemiluminescent substrate (ECL, Thermo Scientific) and visualized using a Bioshine ChemiQ4800 imaging system (Shanghai Bioshine Scientific Instrument Co., Ltd). Finally, the gray level ratio of target proteins was obtained using the ImageJ software, and β-tubulin (1 : 2000, 30302ES20, Yeasen) was used as the internal control.
2.10. Immunofluorescence Staining
The animals were perfused transcardially with a 4% paraformaldehyde solution, and the brains were dissected and fixed with paraformaldehyde for 24 h at 4°C. After dehydration with a 40% sucrose solution and embedding in OTC, the brain tissues were cut into 12-μm frozen sections for staining using a cryostat (Leica CM1950, Germany). Next, tissue sections were washed three times with PBS, permeabilized, and blocked with PBS containing 0.1% Tween 20, 0.3% Triton X-100, and 5% BSA. Then, the sections were incubated overnight at 4°C with the following specific primary antibodies: MMP9 (1 : 300, ab76003, Abcam), Claudin5 (1 : 500, ab131259, Abcam), and ZO-1 (1 : 50, 21773-1-AP, Proteintech). Sections were then washed with PBS and incubated for 1 h with secondary antibodies (Alexa Fluor 594, ab150080, Abcam and Alexa Fluor 488, ab150077, Abcam) at room temperature. Nuclear counterstaining was performed using DAPI (C0065, Solarbio, China). Images of ischemic penumbral sections were randomly captured using an Olympus BX63 fluorescence microscope (Olympus, Tokyo, Japan). Fluorescence intensity was quantified using the ImageJ software, and the results are presented as average optical density (AOD) values.
2.11. Statistical Analysis
All data were analyzed using the SPSS 24 and GraphPad Prism 7 software, and the data are reported as . Data with a normal distribution and homogeneity of variance were analyzed using analysis of variance (ANOVA). The nonparametric Mann–Whitney test was used to analyze nonnormally distributed data. Statistical significance was defined as , and a high level of statistical significance was defined as .
3. Results
3.1. EA Improved Brain Injury following Delayed rtPA Thrombolysis for Ischemic Stroke
We evaluated neurological deficits and the cerebral infarct volume at 24 hours after stroke to determine the effects of EA on delayed rtPA thrombolysis for ischemic stroke. The neurological deficit score and infarct volume in rats with rtPA thrombolysis did not differ from the model group (, Figures 1(a) and 1(c)). However, the combination of EA and 6 h rtPA resulted in significant reductions in the neurological deficit score and infarct volume compared to the model group and to the group treated with 6 h rtPA alone (, , Figures 1(a) and 1(c)). Based on the results, EA improved brain injury following delayed rtPA thrombolysis for ischemic stroke.
(a)
Objectives: To investigate the thrombolysis with recombinant human prourokinase (rhPro-UK) on thromboembolic stroke in rats at different therapeutic time windows (TTW).
Methods: Rats were subjected to embolic middle cerebral artery occlusion. RhPro-UK and positive control drugs rt-PA,UK were administered 3 h, 4.5 h, 6 h after inducing thromboem-bolic stroke. Neurological deficit scoring (NDS) was evaluated at 6 h and 24 h after the treatment. The lesion volume in cerebral hemispheres was measured by MRI scanning machine after 6 h of thrombolysis, and the infarct volume was measured by TTC stain, together with hemorrhagic volume quantified by a spectrophotometric assay after 24 h of thrombolysis.
Results: RhPro-UK 10, 20 × 10⁴ U/kg significantly improved the NDS after cerebral thromboembolism in rats at 3 h, 4.5 h TTW, and at the 6 h TTW, the NDS was improved by 28.0% (P = 0.0690) and 29.2% (P = 0.0927) at 6 h and 24 h after rhPro-UK 20 ×10⁴ U/kg administration, respectively. RhPro-UK 10, 20 × 10⁴ U/kg significantly reduced the brain lesions measured by MRI at 3 h and 4.5 h TTW. RhPro-UK 10, 20 × 10⁴ U/kg significantly reduced the cerebral infarction measured by TTC at 3 h, 4.5 h TTW. There was no increase in cerebral hemorrhage compared with untreated group after rhPro-UK administration.
Conclusions: RhPro-UK had an obvious therapeutic effect on ischemic stroke caused by thrombosis, and could be started within 4.5 h TTW with less side effects of cerebral hemorrhage than that of UK.
