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Pre-clinical development of therapy for acute ischemic stroke requires robust animal models; the rodent middle cerebral artery occlusion (MCAo) model using a nylon filament inserted into the internal carotid artery is the most popular. Drug screening requires targeted delivery of test substance in a controlled manner. To address these needs, we dev...
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... ICA but the knot was not tightened. A loop of silk was placed around the ECA immediately above the bifurcation of the CCA however the knot was not pulled tight. The weight of hemostats at the ends of the sutures occluded the vessels without having to tighten knots or use vascular clamps. Gentle dissection rostrally along the ICA exposed the ptery- gopalatine artery (PPA), which courses more laterally, thus allowing better visualization of the more medially placed ICA. This rostral dissection along the ICA aided in the insertion of the occlusion filament later. All distal vessels should be identified and retracted before clamping the CCA while the vessels are still distended with blood. A cotton tipped applicator was used to gently free the vagus nerve from the fascia surrounding the CCA. To temporarily occlude the CCA, a Micro Serrefine vessel clamp (Fine Science Tools; Cat. No. 18055-04) was placed as proximally as possible to ensure the deepest seating of the intra-arterial catheter. The vagus nerve was visualized thoroughly to ensure that it had not been included in the clamps’ teeth. Vagal stimulation will cause the animals to gasp for air or cardiac arrest (Heiser, 2007). If gasping, bradycardia, or arrest occurred, the clamp was removed, the vagus nerve was more carefully freed from the surrounding fascia and the clamp was replaced. At this point there were two sutures with tails cut (the superior thyroid and the occipital artery) and three with tails weighted with hemostats (occluded ECA, ICA, and the loop above the bifurcation on the ECA) on the carotids arteries (Fig. 3). Using Vannus Spring scissors (3 mm cutting edge; Fine Science Tools; Cat. No. 15000-00), an arteriotomy was made in the ECA distal to the bifurcation of the CCA between the two suture loops (one was tightened and one was loose at that point). A small amount of blood oozed from the incision. The arteriotomy was not made extremely close to the bifurcation because fitting a secure suture proximal to the incision but distal to the bifurcation would prove difficult. If excessive or pulsing blood was observed, the ICA was checked to ensure that it was properly occluded. The occlusion monofilament was inserted into the ECA pointing toward the CCA and then curved up toward the ICA. Loosening the loop around the ECA aided in the passage through the bifurcation. Using a finely pointed cotton tip applicator, the filament was gently guided up the ICA (Fig. 4) and the loop on the ECA was re-tightened (not tied). The loop on the ICA was loosened slightly to let the filament pass and this allowed some blood to flow out of the incision in the ECA. Once the filament passed the loop on the ICA, cotton tipped applicators were used to absorb any blood to maintain a clear view of the course of the filament in the ICA. A small narrow cotton tipped applicator was used to gently occlude the origin of the PPA and guide the filament medially up the ICA (Fig. 4). The filament was advanced until the mark (1.7 cm) reached the bifurcation of the ICA and ECA. Resistance was felt as the tip entered the proximal segment of the anterior cerebral artery and thus blocked the ostium of the middle cerebral artery (MCA). If the filament could only be advanced 0.5–0.7 cm, it had entered the PPA, and was withdrawn until the tip could be visualized at the loop on the ICA. A cotton tipped applicator was placed a bit more ventrally to block the PPA and the filament was again advanced up the ICA. Once the filament had been advanced the correct distance, the occlusion time was noted. The filament was trimmed so that 5–7 mm was extending out of the ECA. In the group of animals that were to receive jugular infusions, the suture surrounding the monofilament and ECA was then tightened to secure the monofilament. Catheters were then placed in the jugular vein as described previously (Heiser, 2007). Briefly, on the same side of the occlusion, the jugular vein was identified, dissected and ligated. Through a small venotomy, the silicone catheter was advanced approximately 12 mm and secured with two sutures; one distal to the venotomy and one proximal. The catheter was then tunneled around the side of the neck and secured as described for the arterial catheter. The sham IV group did not receive MCAo, but did receive the jugular catheter. If the animal was in the intra- arterial group (both MCAo and sham), a catheter was placed in the common carotid as described below. A catheter was placed in the CCA for both the group that received the MCAo and the sham group that received the IA infusion. First, the suture loop around the ECA just above the bifurcation was loosened. The catheter was then introduced into the incision in the ECA. Inserting the catheter behind the filament eased the insertion. Care was taken not to pull or push on the filament as it was not secured. The catheter was advanced and a cotton tipped applicator was used to guide the tip down the CCA oriented retro- grade (Fig. 5). The catheter had a tendency to follow the course of the filament up the ICA anterograde. The catheter was advanced as far down the CCA as possible. The placement of the proximal vessel clamp limited the extent of the catheter advancement down the CCA. Next the loop of suture around the ECA (that also encircled the filament and the catheter) was tightened, but not excessively as this would occlude the catheter. This suture must be proximal to the incision in the ECA or bleeding would occur. Rubber-shod hemostats (hemostat with plastic tubing cov- ering the teeth; Supplementary Fig. S2) were clamped on the catheter close to the syringe. The loop of suture from the ICA was then removed. To ensure the catheter had not been occluded by the suture on the ECA, the rubber-shod hemostats were briefly unclamped from the catheter and the syringe pulled to check if blood could be withdrawn into the line. The blood was then slowly flushed back through the line. The rubber-shod hemostats were re-clamped on the catheter. Once the first suture had been tightened, the first retention bead was moved down the catheter so that it sat immediately above the incision in the ECA and above the first suture. The tails from the suture that occluded the ECA were then looped back around the ECA to now include the catheter and another knot was made (Fig. 6). This suture was positioned right above the retention bead. Carefully and slowly, the clamp from the CCA was removed. The ECA incision was observed for bleeding. Any bleeding indicated that the first suture around the ECA was insuf- ficient. If this was the case, another loop of 4-0 silk suture was passed around the ECA/catheter/filament, ensuring that the suture is proximal to the incision, and tightened (but not overly tight as to occlude the catheter). A small segment of 4-0 silk suture was passed through the super- ficial fascia in the neck lateral to catheter exit site and was used to secure the catheter to the body wall. This suture was positioned right above the second retention bead. Due to the direction of the catheter exiting from the ECA, it was usually best to tunnel the catheter around the opposite side of the body. Care was taken to ensure that there was enough slack so that when the animal flexed its neck there would not be excessive force on the catheter or pres- sure on the trachea. The animal was then turned over to lateral recumbency (for a left MCAo the animal would be turned to left recumbency, i.e. the catheter would be tunneled out the right side of the neck). A small cut was then made on the nape of the neck. Sharp hemostats, starting closed and staying close to the skin, were then pushed through to the ventral incision. The end of the catheter was grabbed with the hemostats and rubber-shod hemostats were unclamped. The catheter was removed from the stub adaptor and pulled through the tunnel to back of neck and out through the small skin incision. Care was taken not to pull the catheter too tight as this would either occlude the trachea or pull the catheter from the CCA; considerable slack was left in the catheter to allow free movement. The rubber-shod hemostats were replaced on the catheter and the terminal segment, where the hemostats were clamped (the silicone was possibly punctured), was trimmed. The catheter was then reattached to the adaptor on the syringe. The animal was turned to dorsal recumbency and the skin incision was closed with 4-0 Prolene being careful to avoid the catheter with the suture needle while closing. A single suture was placed in the skin (and encircled the catheter as well) to close the incision on the nape of the neck. Care was taken to not tighten the knot excessively. The entire procedure took approximately (MCAo and catheter placement) 20–25 min. The animal was removed from anesthesia and transferred to a pre-warmed recovery chamber. 5 cc Lactated ringers saline was administered subcutaneously. A syringe containing 4% Evans blue was attached to the catheter and the syringe plunger was slowly depressed until the Evans blue filled the entire catheter line. The syringe was placed into a syringe pump (Braintree Scientific, Inc.; Cat. No. BS-300) and the desired rate was set (here 0.2 mL/h or 26.67 mg/kg/h). The animal remained tethered in the recovery cage receiving a continuous infusion of Evans blue during the entire 4 h MCAo. A piece of tubing stretched across the top of the recovery cage provided a support to wrap the catheter up and over on its way to the syringe pump (Fig. 7). MCAo animals will often circle and the torque from the twisting on the catheter may dislodge the catheter, therefore the animals were monitored closely, especially immediately upon waking up. At the end of the 4 h occlusion, the pump was stopped and the catheter was clamped with the rubber-shod hemostats. The adaptor was removed from the syringe and a cap (Becton Dickinson Vascular Access; Cat. No. 388161) was placed on the adaptor. The ...
Context 2
... the bifurcation was loosened. The catheter was then introduced into the incision in the ECA. Inserting the catheter behind the filament eased the insertion. Care was taken not to pull or push on the filament as it was not secured. The catheter was advanced and a cotton tipped applicator was used to guide the tip down the CCA oriented retro- grade (Fig. 5). The catheter had a tendency to follow the course of the filament up the ICA anterograde. The catheter was advanced as far down the CCA as possible. The placement of the proximal vessel clamp limited the extent of the catheter advancement down the CCA. Next the loop of suture around the ECA (that also encircled the filament and the ...
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Background:
Middle cerebral artery (MCA) ischemic stroke poses a major threat to human beings and prompts intravenous thrombolytic and/or thrombectomy management remains the gold standard treatment. However, not all MCA stroke patients fit in the inclusion and exclusion criteria that many patients only receive conventional medical therapy. We atte...
Citations
... in the iR and Mt + iR groups, McaO was applied to the right Mca of rats in accordance with the procedures described by Koizumi et al. [16]. Monofilaments were prepared by us using 4-0 ethilon Nylon suture (ethicon comp., G7143, somerville, NJ) as previously described in the literature [17]. Prepared monofilaments were photographed under a 2× objective with millimetric paper using a surgical microscope, and the tip diameters of the monofilaments were measured from these photographs using a digital image analysis software (imageJ, 1.53k, Bethesda, MD) [18]. ...
Purpose: Neurological impairments are the leading cause of post-stroke mortality, while stroke-related cardiovascular diseases rank second in significance.This study investigates the potential protective effects of MitoTEMPO (2,2,6,6-tetramethyl-4-[[2-(triphenylphosphonio) acetyl] amino]-1-piperidinyloxy, monochloride, monohydrate), a mitochondria-specific antioxidant, against cardiac and neurological complications following stroke. The objective is to assess whether MitoTEMPO can be utilized as a protective agent for individuals with a high risk of stroke.
Materials and Methods: 17-week-old male Wistar Albino rats were randomly assigned to three groups: SHAM, ischemia-reperfusion (IR), and MitoTEMPO+ischemia-reperfusion (MT+IR; MitoTEMPO injection 0.7 mg/kg/day for 14 days). The SHAM group underwent a sham operation, while the IR group underwent 1-hour middle cerebral artery occlusion (MCAO) followed by 3 days of reperfusion. Afterwards, noninvasive thoracic electrical bioimpedance (TEB) and electrocardiography (ECG) measurements were taken, and sample collection was performed for histological and biochemical examinations.
Results: Our TEB and ECG findings demonstrated that MitoTEMPO exhibited a protective effect on most parameters affected by IR compared to the SHAM group. Furthermore, our biochemical and histological data revealed a significant protective effect of MitoTEMPO against oxidative damage.
Conclusions: The findings suggest that both IR-induced cardiovascular abnormalities and the protective effect of MitoTEMPO may involve G-protein coupled receptor (GPCR)-mediated signaling mechanisms. This study was conducted with limitations including a single gender, a uniform age group, a specific stroke model limited to MCA, and pre-scheduled only one IR period. In future studies, addressing these limitations may enable the implementation of preventive measures for individuals at high risk of stroke.
... The advent of intravascular mechanical thrombectomy caused renewed interest in testing putative cerebroprotectants because recanalization can be confirmed angiographically in each patient (14,17). In preclinical modeling, confirmed recanalization can be replicated with a widely accepted model of transient middle cerebral artery occlusion (tMCAo) in both mice and rats (18). Thrombectomy in patients, and tMCAo in animals, ensures that the test drug or intervention fully reaches the brain after ischemia and confirmed reperfusion (14,19). ...
... Modern stroke therapy includes thrombectomy with documented reperfusion, which is well represented by the standard nylon filament occlusion model in rodents (19). For stage 1 of SPAN, a filament tMCAo with reperfusion after 60 min was performed in young C57BL6/J mice (n = 895 from the Jackson Laboratory, n = 18 bred in-house), closely following published protocols (18). Anesthesia was induced with 4% isoflurane in mixed oxygen:nitrous oxide 30:70 and maintained with 1 to 2% isoflurane in the same gas mixture. ...
Human diseases may be modeled in animals to allow preclinical assessment of putative new clinical interventions. Recent, highly publicized failures of large clinical trials called into question the rigor, design, and value of preclinical assessment. We established the Stroke Preclinical Assessment Network (SPAN) to design and implement a randomized, controlled, blinded, multi-laboratory trial for the rigorous assessment of candidate stroke treatments combined with intravascular thrombectomy. Efficacy and futility boundaries in a multi-arm multi-stage statistical design aimed to exclude from further study highly effective or futile interventions after each of four sequential stages. Six independent research laboratories performed a standard focal cerebral ischemic insult in five animal models that included equal numbers of males and females: young mice, young rats, aging mice, mice with diet-induced obesity, and spontaneously hypertensive rats. The laboratories adhered to a common protocol and efficiently enrolled 2615 animals with full data completion and comprehensive animal tracking. SPAN successfully implemented treatment masking, randomization, prerandomization inclusion and exclusion criteria, and blinded assessment of outcomes. The SPAN design and infrastructure provide an effective approach that could be used in similar preclinical, multi-laboratory studies in other disease areas and should help improve reproducibility in translational science.
... Através de uma pequena arteriotomia ou venotomia, a cânula é inserida em aproximadamente 12 mm e fixada com dois pontos de sutura, um distal e outro proximal à incisão realizada no vaso. Após a sutura de fixação, deve-se atentar ao fluxo sanguíneo ideal, evitando compressão e redução da luz do vaso com a cânula (VAN WINKLE et al., 2013).Visando manter a estabilidade e funcionalidade da cânula, é preciso fixá-la de modo a permitir a movimentação normal do animal e, ao mesmo tempo, longe do seu alcance, evitando o seu acesso. Com esse propósito, é comum fixar a cânula através da fáscia superficial no pescoço, lateralmente à parede corporal (Figura 3), com um fio de seda 4.0. ...
... Para isso, deve ser garantida folga suficiente para flexão do pescoço do animal. A cânula é levada até a porção posterior do pescoço, na região da nuca, onde é realizada uma pequena incisão para fixação com uma sutura e possibilitando o acesso externo com adaptador para conexão de seringa (VAN WINKLE et al., 2013). ...
... In the ischemic stroke model studies performed with the monofilament insertion technique, researchers use selfdeveloped monofilaments. We also made monofilament from nylon suture as in the literature (15)(16)(17). Sterile 4-0 nylon suture (Ethilon Nylon Monofilament Suture, Ethicon Inc.) was cut 4-5 cm long and blunted by bringing the tip close to the heat source ( Figure 1-A) (12). It is known that there is an MCA bifurcation of the ICA at a distance of about 1.8 cm from the arteriotomy site shown in Figure 1-E. ...
Abstract
Aim: In this experiment, we aimed to convey our experience of the temporary stroke model created with the monofilament technique in the middle cerebral artery and to characterize the histopathological changes in the brain tissue caused by ischemia-reperfusion at the microscopic level.
Material-Method: Eleven adult (27 weeks-old) Wistar-Albino female rats were used in the experiments. Experimental animals were randomly divided into 2 groups. The first group tagged as SHAM underwent the mock operation. The other group, which underwent 3 days of reperfusion after 1 hour of ischemia, was labeled as IR. Histological examinations were performed from the isolated brain tissues at the end of the reperfusion period.
Results: Our histological examinations showed that the cortical structure was highly irregular, vacuolization in the molecular layer and homogeneous cytoplasm and paler nuclei were observed in some Purkinje cells in the images belonging to the IR group. In addition, our experience with surgical procedures has shown that animal deaths in studies with the monofilament technique are mostly due to vagus nerve damage.
Conclusion: In this study, we reported the histopathological changes in the brain tissue caused by stroke at the microscopic level. We also presented our experience in the surgical processes of an animal model used in the study of stroke. As a result, we concluded that it is important to gain manual dexterity by making preliminary work due to the difficulty of creating a stroke model with the monofilament technique.
... In this study, we described a new method to induce IS and then investigated the effects of high LDL-C or low HDL-C on IS using human-like genetically engineered hamster models. Utilizing c-MCAO, hamsters exhibited IS of greater severity with larger infarct sizes and more BBB disruption compared to the d-MCAO hamster model, which is also consistent with murine results [29,30]. This protocol allows researchers to better study ischemia/reperfusion in animals without need for permanent occlusion and thus could be used conveniently for applications to assess drug efficacy in future IS studies. ...
Rationale:
While high low-density lipoprotein cholesterol (LDL-C) and low high-density lipoprotein cholesterol (HDL-C) levels are positively associated with cardiovascular events, it is still unclear whether familial hypercholesterolemia (FH) and Tangier's disease (TD), caused by mutations in LDLR and ABCA1, respectively, influence ischemic stroke (IS) in humans.
Objective:
We sought to establish an easier, more effective, and time-saving method to induce IS, then studied the precise effects of different types of lipoproteins on IS.
Methods and results:
A new technique termed contralateral middle cerebral artery occlusion (c-MCAO) was introduced to human-like hamster models to induce IS. Compared to traditional distal MCAO (d-MCAO) induced by electrocoagulation, c-MCAO resulted in a more severe IS with larger infarct sizes and more blood-brain barrier (BBB) disruption after 24 h. It was shown that c-MCAO markedly elicited an increase in brain infarct volume and BBB leakage in both homozygous LDLR (LDLR-/-) and ABCA1 knockout (ABCA1-/-) hamsters, but not in heterozygous LDLR knockout (LDLR+/-) hamsters when compared to wild-type (WT) controls.
Conclusions:
Using human-like genetically engineered hamsters, our findings demonstrated that both high LDL-C level caused by homozygous LDLR deficiency and severe low HDL-C level caused by deleting ABCA1 were risk factors of IS. As such, we believe the development of this novel IS hamster model is suitable for future ischemic/reperfusion studies.
... All methods used have been published and used extensively in our laboratory (Van Winkle et al., 2013). Male Sprague Dawley rats weighing 290-310 g (young adult) were purchased from Harlan Laboratories. ...
... We occluded the left MCA for 2 h or 4 h using our method as described previously (Chen et al., 2012;Van Winkle et al., 2013). In brief, animals were anesthetized with 4% isoflurane induction and maintained on 1.5-2% isoflurane mixed in oxygen and nitrous oxide (30:70) and received tail-vein injections of 0.3 ml FITC-conjugated 2 MDa dextran in PBS at onset of surgery. ...
Thrombin and activated protein C (APC) are known coagulation factors that exhibit profound effects in brain by acting on the protease activated receptor (PAR). The wild type (WT) proteases appear to impact cell survival powerfully, and therapeutic forms of APC are under development. Engineered recombinant thrombin or APC were designed to separate their procoagulant or anticoagulant effects from their cytoprotective properties. We measured vascular disruption and neuronal degeneration after a standard rodent filament stroke model. For comparison to a robust anticoagulant, we used a GpIIb/IIIa inhibitor, GR144053. During 2 h MCAo both WT murine APC and its mutant, 5A-APC, significantly decreased neuronal death 30 min after reperfusion. During 4 h MCAo, only 5A-APC significantly protected neurons but both WT-APC and 5A-APC exacerbated vascular disruption during 4 h MCAo. Human APC mutants appeared to reduce 24 h neuronal injury significantly when given after 2 h delay after MCAo. In contrast, 24 h vascular damage was worsened by high doses of WT and mutant APCs, although only statistically significantly for high dose 3K3A-APC. Mutated thrombin worsened vascular damage significantly without affecting neuron damage. GR144053 failed to ameliorate vascular disruption or neuronal injury despite significant anticoagulation. Differential effects on neurons and the vasculature were demonstrated using wild-type and mutated proteases. The mutants murine 3K3A-APC and 5A-APC protected neurons in this rodent model but in high doses worsened vascular leakage. Cytoactive effects of plasma proteases may be separated from their coagulation effects. Further studies should explore impact of dose and timing on cytoactive and vasculoactive properties of these drugs.
... The model used by Jones et al involved surgery to expose the MCA so it could be occluded using a ligature [28]. This is an example of a broader category of models which induce ischemia by occluding the MCA, generally using either an intra-arterial catheter [29] or by exposing the MCA and applying a clip or ligature [28]. Implementing these models of acute ischemia in pigs can be problematic for several reasons; the rete mirabelle makes it impossible to use an intraarterial catheter [14], and using a clip or ligature requires very invasive surgical procedures such as an osteotomy on the orbital rim [15] or removal of an eye [16]. ...
CT Perfusion (CTP) derived cerebral blood flow (CBF) thresholds have been proposed as the optimal parameter for distinguishing the infarct core prior to reperfusion. Previous threshold-derivation studies have been limited by uncertainties introduced by infarct expansion between the acute phase of stroke and follow-up imaging, or DWI lesion reversibility. In this study a model is proposed for determining infarction CBF thresholds at 3hr ischemia time by comparing contemporaneously acquired CTP derived CBF maps to 18F-FFMZ-PET imaging, with the objective of deriving a CBF threshold for infarction after 3 hours of ischemia. Endothelin-1 (ET-1) was injected into the brain of Duroc-Cross pigs (n = 11) through a burr hole in the skull. CTP images were acquired 10 and 30 minutes post ET-1 injection and then every 30 minutes for 150 minutes. 370 MBq of 18F-FFMZ was injected ~120 minutes post ET-1 injection and PET images were acquired for 25 minutes starting ~155-180 minutes post ET-1 injection. CBF maps from each CTP acquisition were co-registered and converted into a median CBF map. The median CBF map was co-registered to blood volume maps for vessel exclusion, an average CT image for grey/white matter segmentation, and 18F-FFMZ-PET images for infarct delineation. Logistic regression and ROC analysis were performed on infarcted and non-infarcted pixel CBF values for each animal that developed infarct. Six of the eleven animals developed infarction. The mean CBF value corresponding to the optimal operating point of the ROC curves for the 6 animals was 12.6 ± 2.8 mL·min-1·100g-1 for infarction after 3 hours of ischemia. The porcine ET-1 model of cerebral ischemia is easier to implement then other large animal models of stroke, and performs similarly as long as CBF is monitored using CTP to prevent reperfusion.
... In our method, a catheter is advanced through the vasculature of the mouse neck into the ICA so the potential drug delivery can be selective. Our model is different in that we initiate a stroke, recanalize and then inject IA therapeutics without leaving an indwelling microport or catheter (Chen et al., 2009;Van Winkle et al., 2013). Direct IA administration of neuroprotective agents represents a novel method of drug delivery for acute stroke. ...
With continuing disconnect between laboratory stroke treatment models and clinical stroke therapy, we propose a novel experimental model to study stroke and vessel recanalization that mirrors acute management of large vessel stroke, with concomitant directed pharmacotherapy. Using the tandem transient ipsilateral common carotid/middle cerebral artery occlusion (MCAO) model to induce stroke in mice we then added selective intra-arterial (IA) drug administration for directed pharmacotherapy. The IA model uses micro-angio tubing placed at the bifurcation of the CCA to selectively administer the drug to the internal carotid distribution. We have shown that delivery of pharmacotherapy agents selectively through an IA injection is feasible in a mouse model, which will permit studies involving pharmacotherapy, transgenic modification, and/or a combination. Our IA model has similarities to previously published models of IA injection but differs in that we do not leave an indwelling micro-port or catheter in our animals, which is not clinically relevant as it does not reflect the human condition or current clinical management. Furthermore, we optimized our model to selectively direct therapy to the ipsilateral, stroke affected hemisphere. By developing an IA drug delivery model that mirrors clinical conditions, we are bridging the gap between basic stroke research and what is standard practice in acute ischemic stroke intervention. The IA model of drug delivery can target agents directly to the site of injury while blunting systemic effects, dose penetration issues, and administration delay that have plagued the intraperitoneal and oral drug administration models.
... Following injections animals survived for 1 week and were then subjected to MCAo. The MCAo procedure was performed by a surgeon blind to treatment assignment, as described previously in our laboratory , Van Winkle et al., 2012. Briefly, a midline neck incision was made exposing the left common carotid artery. ...
... Animals were anesthetized with 4% isoflurane mixed in oxygen and nitrous oxide (30:70) and received 2MDa dextran conjugated with FITC in PBS (Sigma-Aldrich, FD2000S). The MCAo model as described earlier was adapted for mice , Van Winkle et al., 2012. Cerebral blood flow was monitored using laser Doppler flowmetry (LDF) by fixing a probe on the skull, 4-mm lateral to the bregma (Moor Instruments, Devon, United Kingdom). ...
Protease activated receptors (PARs) populate neurons and astrocytes in the brain. The serine protease thrombin, which activates PAR-1 during the first hours after stroke, appears to be associated with the cytotoxicity. Thrombin antagonists and PAR-1 inhibitors have been correlated with reduced cell death and behavioral protection after stroke, but no data yet supports a mechanistic link between PAR-1 action and benefit. We sought to establish the essential role of PAR-1 in mediating ischemic damage. Using a short hairpin mRNA packaged with green fluorescent protein in a lentivirus vector, we knocked downPAR-1 in the medial caudate nucleus prior to rat middle cerebral artery occlusion (MCAo) and in rat neurons prior to oxygen-glucose deprivation. We also compared aged PAR-1 knockout mice with aged PAR-3, PAR-4 mice and young wild-type mice in a standard MCAo model. Silencing PAR-1 significantly reduced neurological deficits, reduced endothelial barrier leakage, and decreased neuronal degeneration in vivo during MCAo. PAR-1 knock-down in the ischemic medial caudate allowed cells to survive the ischemic injury; infected cells were negative for TUNEL and c-Fos injury markers. Primary cultured neurons infected with PAR-1 shRNA showed increased neuroprotection during hypoxic/aglycemic conditions with or without added thrombin. The aged PAR-1 knockout mice showed decreased infarction and vascular disruption compared to aged controls or young wild types. We demonstrated an essential role for PAR-1 during ischemia. Silencing or removing PAR-1 significantly protected neurons and astrocytes. Further development of agents that act at PAR-1or its downstream pathways could yield powerful stroke therapy.
... Male Sprague-Dawley rats (n=37; 290-310 g) were assigned randomly: saline or 1.4 U/kg thrombin (Sigma) in 0.2 mL administered intra-arterially for 120 minutes or 0.45 mg argatroban (Axxora) in 0.2 mL administered intravenously for 2 hours. 2 We used our published methods for middle cerebral artery occlusion, behavior, and histology. 1,6,7 To determine the optimum therapeutic time window for treatment with argatroban, we randomly assigned 272 male Sprague-Dawley rats, as above, to receive saline intravenously or low-dose (10 mg/kg) or high-dose (18 mg/kg) argatroban for 24 hours by Alzet mini-pump (model 2001D, Durect Corp). Treatment effects were assessed using the standard quantal bioassay procedure and 2,3,5-triphenyltetrazolium chloride (TTC) staining. ...
We showed previously robust neuroprotection with the thrombin inhibitor argatroban and now sought additional support for its neuroprotective potential.
We used behavioral and histological end points; rigorously blinded the study groups; extended the treatment window to 3 hours after ischemia onset; and used 2 separate models. First, 2-hour filament middle cerebral artery occlusion in 64 male Sprague-Dawley rats was followed by learning and memory testing and quantitative histomorphometry. Randomly assigned treatment was 0.45 mg argatroban, saline, or 0.4 U thrombin. Second, we used the quantal bioassay (n=272) after 2-hour middle cerebral artery occlusion to detect the longest time delay after which therapy failed.
Argatroban powerfully and significantly reversed learning and memory deficits because of focal ischemia compared with saline or thrombin (P<0.03; ANOVA). Argatroban was significantly (P<0.05; t test with Bonferroni) protective when given immediately or after 1, 2, 3, but not 4 hours delay.
We obtained supportive evidence for argatroban protection of the neurovascular unit using behavioral and histological measurements at realistic therapeutic time windows.
