A Comparative Study of Variables Influencing Ischemic Injury in the Longa and Koizumi Methods of Intraluminal Filament Middle Cerebral Artery Occlusion in Mice.
ABSTRACT: The intraluminal filament model of middle cerebral artery occlusion (MCAO) in mice and rats has been plagued by inconsistency, owing in part to the multitude of variables requiring control. In this study we investigated the impact of several major variables on survival rate, lesion volume, neurological scores, cerebral blood flow (CBF) and body weight including filament width, time after reperfusion, occlusion time and the choice of surgical method. Using the Koizumi method, we found ischemic injury can be detected as early as 30 min after reperfusion, to a degree that is not statistically different from 24 h post-perfusion, using 2,3,5-Triphenyltetrazolium chloride (TTC) staining. We also found a distinct increase in total lesion volume with increasing occlusion time, with 30-45 min a critical time for the development of large, reproducible lesions. Furthermore, although we found no significant difference in total lesion volume generated by the Koizumi and Longa methods of MCAO, nor were survival rates appreciably different between the two at 4 h after reperfusion, the Longa method produces significantly greater reperfusion. Finally, we found no statistical evidence to support the exclusion of data from animals experiencing a CBF reduction of <70% in the MCA territory following MCAO, using laser-Doppler flowmetry. Instead we suggest the main usefulness of laser-Doppler flowmetry is for guiding filament placement and the identification of subarachnoid haemorrhages and premature reperfusion. In summary, this study provides detailed evaluation of the Koizumi method of intraluminal filament MCAO in mice and a direct comparison to the Longa method.
Project description:In order to improve clinical trial design and translation of normobaric oxygen (NBO) treatment of ischemic stroke, NBO treatment parameters need to be better understood. This study investigated NBO treatment efficacy at two different stroke severities and two NBO treatment durations in rats. For the 60-min middle cerebral artery occlusion (MCAO), NBO treatment for 25 min and 150 min were studied. For the 90-min MCAO, NBO treatment for 55 min and 150 min were studied. Cerebral blood flow (CBF), apparent diffusion coefficients (ADC) and T2 MRI were acquired during occlusion prior to treatment, after reperfusion, and 48h after MCAO. The effects of NBO treatment on lesion volumes, and CBF, ADC and T2 of ischemic core, perfusion-diffusion mismatch and normal tissue were analyzed longitudinally. The major findings were: i) NBO treatment was effective in both groups of stroke severities, salvaging similar percentage of initial abnormal ADC tissue, and ii) NBO treatments continued after reperfusion were more beneficial than NBO treatment during occlusion alone for both MCAO groups. These findings underscore the importance of the effects of NBO duration and stroke severity on treatment outcomes.
Project description:Recanalization of an occluded vessel with recombinant tissue plasminogen activator is an effective strategy for treating acute ischemic stroke. Recombinant tissue plasminogen activator is administered as alteplase, a formulation containing many excipients including L-arginine, the substrate for nitric oxide production. Most studies fail to compare the effects of alteplase on brain injury to its L-arginine carrier solution. This study aimed to verify the previously reported detrimental effects of alteplase after cerebral ischemia and delineate the contribution of L-arginine. Male Wistar rats, subjected to 90?minutes of intraluminal middle cerebral artery occlusion (MCAO), were administered alteplase, the carrier solution or saline upon reperfusion. Neither alteplase nor the carrier affected cerebral blood flow (CBF) restoration throughout the first 60?minutes of reperfusion. Alteplase treatment was associated with increased mortality after MCAO. Twenty-four hours after MCAO, neurologic function and infarct volume did not differ between rats treated with alteplase, the carrier solution, or saline. Irrespective of treatment group, infarct volume was correlated with CBF during reperfusion, neuroscore, and peri-infarct depolarizations. These results suggest that alteplase treatment, independent of thrombolysis, does not cause increased ischemic injury compared with its appropriate carrier solution, supporting the continued use of alteplase in eligible ischemic stroke patients.
Project description:Two classical surgical approaches for intraluminal filament middle cerebral artery occlusion (MCAO), the Longa et al. (LM) and Koizumi et al. methods (KM), are used as alternatives in preclinical studies to induce stroke in rodents. Comparisons of these MCAO models in mice showed critical differences between them along with similarities (Smith et al. 2015; Morris et al. 2016). In this study, a direct comparison of MCAO-KM and MCAO-LM in rats was performed. Three days after MCAO, infarct volume, mortality rate, neurological deficit, and weight loss were similar in these models. MCAO-LM rats showed an increase in ACTH levels, while MCAO-KM rats demonstrated elevated corticosterone and interleukin-1β in blood serum. Corticosterone accumulation was detected in the frontal cortex (FC) and the hippocampus of the MCAO-KM group. IL1β beta increased in the ipsilateral hippocampus in the MCAO-KM group and decreased in the contralateral FC of MCAO-LM rats. Differences revealed between MCAO-KM and MCAO-LM suggest that corticosterone and interleukin-1β release as well as hippocampal accumulation is more expressed in MCAO-KM rats, predisposing them to corticosterone-dependent distant neuroinflammatory hippocampal damage. The differences between two models, particularly, malfunction of the hypothalamic-pituitary-adrenal axis, should be considered in the interpretation, comparison, and translation of pre-clinical experimental results.
Project description:Selective brain hypothermia is considered an effective treatment for neuronal injury after stroke, and avoids the complications of general hypothermia. However, the mechanisms by which selective brain hypothermia affects mitochondrial fission remain unknown. In this study, we investigated the effect of selective brain hypothermia on the expression of fission 1 (Fis1) protein, a key factor in the mitochondrial fission system, during focal cerebral ischemia/reperfusion injury. Sprague-Dawley rats were divided into four groups. In the sham group, the carotid arteries were exposed only. In the other three groups, middle cerebral artery occlusion was performed using the intraluminal filament technique. After 2 hours of occlusion, the filament was slowly removed to allow blood reperfusion in the ischemia/reperfusion group. Saline, at 4°C and 37°C, were perfused through the carotid artery in the hypothermia and normothermia groups, respectively, followed by restoration of blood flow. Neurological function was assessed with the Zea Longa 5-point scoring method. Cerebral infarct volume was assessed by 2,3,5-triphenyltetrazolium chloride staining, and apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining. Fis1 and cytosolic cytochrome c levels were assessed by western blot assay. Fis1 mRNA expression was assessed by quantitative reverse transcription-polymerase chain reaction. Mitochondrial ultrastructure was evaluated by transmission electron microscopy. Compared with the sham group, apoptosis, Fis1 protein and mRNA expression and cytosolic cytochrome c levels in the cortical ischemic penumbra and cerebral infarct volume were increased after reperfusion in the other three groups. These changes caused by cerebral ischemia/reperfusion were inhibited in the hypothermia group compared with the normothermia group. These findings show that selective brain hypothermia inhibits Fis1 expression and reduces apoptosis, thereby ameliorating focal cerebral ischemia/reperfusion injury in rats. Experiments were authorized by the Ethics Committee of Qingdao Municipal Hospital of China (approval No. 2019008).
Project description:Ischemia/hypoxia induces de novo expression of the sulfonylurea receptor 1-regulated NC(Ca-ATP) channel. In rodent models of ischemic stroke, early postevent administration of the sulfonylurea, glibenclamide, is highly effective in reducing edema, mortality, and lesion volume, and in patients with diabetes presenting with ischemic stroke, pre-event plus postevent use of sulfonylureas is associated with better neurological outcome. However, the therapeutic window for treatment with glibenclamide has not been studied.We examined the effect of low-dose (nonhypoglycemogenic) glibenclamide in 3 rat models of ischemic stroke, all involving proximal middle cerebral artery occlusion (MCAo): a thromboembolic model, a permanent suture occlusion model, and a temporary suture occlusion model with reperfusion (105 minutes occlusion, 2-day reperfusion). Treatment was started at various times up to 6 hours post-MCAo. Lesion volumes were measured 48 hours post-MCAo using 2,3,5-triphenyltetrazolium chloride.Glibenclamide reduced total lesion volume by 53% in the thromboembolic MCAo model at 6 hours, reduced corrected cortical lesion volume by 51% in the permanent MCAo model at 4 hours, and reduced corrected cortical lesion volume by 41% in the temporary MCAo model at 5.75 hours (P<0.05 for all 3). Analysis of pooled data from the permanent MCAo and temporary MCAo series indicated a sigmoidal relationship between hemispheric swelling and corrected cortical lesion volume with the half-maximum cortical lesion volume being observed with 10% hemispheric swelling.Low-dose glibenclamide has a strong beneficial effect on lesion volume and has a highly favorable therapeutic window in several models of ischemic stroke.
Project description: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 developed a novel method for delivering substances directly into the ischemic brain during MCAo in the awake rat. An indwelling catheter is placed in the common carotid artery ipsilateral to the occlusion at the time of the surgical placement of the occluding filament. The internal and common carotid arteries are left patent to allow superfusion anterograde. The surgeries can be completed quickly to allow rapid recovery from anesthesia; tests substances can be infused at any given time for any given duration. To simulate clinical scenarios, the occluding filament can be removed minutes or hours later (reperfusion) followed by therapeutic infusions. By delivering drug intra-arterially to the target tissue, "first pass" loss in the liver is reduced and drug effects are concentrated in the ischemic zone. To validate our method, rats were infused with Evans blue dye either intra-arterially or intravenously during a 4 h MCAo. After a 30 min reperfusion period, the dye was extracted from each hemisphere and quantitated with a spectrophotometer. Significantly more dye was measured in the ischemic hemispheres that received the dye intra-arterially.
Project description:Cytochrome P450 epoxygenases (CYP) metabolize arachidonic acid to epoxyeicosatrienoic acids (EETs), which exhibit vasodilatory, anti-inflammatory and neuroprotective actions in experimental cerebral ischemia. We evaluated the effect of endothelial-specific CYP overexpression on cerebral blood flow, inflammatory cytokine expression and tissue infarction after focal cerebral ischemia in transgenic mice.Male and female wild-type and transgenic mice overexpressing either human CYP2J2 or CYP2C8 epoxygenases in vascular endothelium under control of the Tie2 promoter (Tie2-CYP2J2 and Tie2-CYP2C8) were subjected to 60-min middle cerebral artery occlusion (MCAO). Microvascular cortical perfusion was monitored during vascular occlusion and reperfusion using laser-Doppler flowmetry and optical imaging. Infarct size and inflammatory cytokines were measured at 24h of reperfusion by TTC and real-time quantitative PCR, respectively. Infarct size was significantly reduced in both Tie2-CYP2J2 and Tie2-CYP2C8 transgenic male mice compared to corresponding WT male mice (n=10 per group, p<0.05). Tie2-CYP2J2, but not Tie2-CYP2C8 male mice maintained higher blood flow during MCAO; however, both Tie2-CYP2J2 and Tie2-CYP2C8 had lower inflammatory cytokine expression after ischemia compared to corresponding WT males (n=10 per group for CBF and n=3 for cytokines, p<0.05). In females, a reduction in infarct was observed in the caudate-putamen, but not in the cortex or hemisphere as a whole and no differences were observed in blood flow between female transgenic and WT mice (n=10 per group).Overexpression of CYP epoxygenases in vascular endothelial cells protects against experimental cerebral ischemia in male mice. The mechanism of protection is in part linked to enhanced blood flow and suppression of inflammation, and is both sex- and CYP isoform-specific.
Project description:Occlusion of the middle cerebral artery (MCAo) is among the most common causes of ischemic stroke in humans. Cerebral ischemia leads to brain lesions existing of an irreversibly injured core and an ischemic boundary zone, the penumbra, containing damaged but potentially salvageable tissue. Using a transient occlusion (30 min) of the middle cerebral artery (tMCAo) mouse model in this cross-institutional study we investigated the neurorestorative efficacy of a dietary approach (Fortasyn) comprising docosahexaenoic acid, eicosapentaenoic acid, uridine, choline, phospholipids, folic acid, vitamins B12, B6, C, and E, and selenium as therapeutic approach to counteract neuroinflammation and impairments of cerebral (structural+functional) connectivity, cerebral blood flow (CBF), and motor function. Male adult C57BL/6j mice were subjected to right tMCAo using the intraluminal filament model. Following tMCAo, animals were either maintained on Control diet or switched to the multicomponent Fortasyn diet. At several time points after tMCAo, behavioral tests, and MRI and PET scanning were conducted to identify the impact of the multicomponent diet on the elicited neuroinflammatory response, loss of cerebral connectivity, and the resulting impairment of motor function after experimental stroke. Mice on the multicomponent diet showed decreased neuroinflammation, improved functional and structural connectivity, beneficial effect on CBF, and also improved motor function after tMCAo. Our present data show that this specific dietary intervention may have beneficial effects on structural and functional recovery and therefore therapeutic potential after ischemic stroke.
Project description:Ticagrelor is a direct acting and reversibly binding P2Y12 antagonist approved for the prevention of thromboembolic events. Its potential benefits in ischemic stroke have not been investigated sufficiently. Mice were subjected to 2 hours of transient middle cerebral artery occlusion (MCAO). Mice were orally treated with ticagrelor (10 or 30 mg/kg), aspirin (60 mg/kg), or vehicle at 3 and 24 hours before MCAO and 0 and 6 hours after reperfusion. The infarct volume and neurological deficits 22 hours after reperfusion were evaluated. Cerebral blood flow (CBF) within 24 hours after MCAO was monitored. We performed western blotting and in vitro analysis using oxygen-glucose deprivation (OGD) stress in human brain microvessel endothelial cells (HBMVECs) to investigate the protective effects of ticagrelor. Ticagrelor (30 mg/kg) improved neurological deficits, reduced the infarct volume, and improved CBF. It promoted the phosphorylation of endothelial nitric oxide synthase (eNOS) and extracellular signal-regulated kinase 1/2 (ERK1/2) during the early phase after reperfusion. Increased phosphorylation of eNOS and ERK1/2 were also observed in HBMVECs after OGD stress. Ticagrelor attenuate ischemia reperfusion injury possibly via phosphorylation of eNOS and ERK1/2 in endothelial cells. This suggests that ticagrelor has neuroprotective effects via mechanisms other than its antiplatelet action.
Project description:Cerebral blood flow and oxygenation in the first few hours after reperfusion following ischemic stroke are critical for therapeutic interventions but are not well understood. We investigate changes in oxyhemoglobin (HbO2) concentration in the cortex during and after ischemic stroke, using multispectral optical imaging in anesthetized mice, a remote filament to induce either 30 minute middle cerebral artery occlusion (MCAo), sham surgery or anesthesia alone. Immunohistochemistry establishes cortical injury and correlates the severity of damage with the change of oxygen perfusion. All groups were imaged for 6 hours after MCAo or sham surgery. Oxygenation maps were calculated using a pathlength scaling algorithm. The MCAo group shows a significant drop in HbO2 during occlusion and an initial increase after reperfusion. Over the subsequent 6 hours HbO2 concentrations decline to levels below those observed during stroke. Platelets, activated microglia, interleukin-1?, evidence of BBB breakdown and neuronal stress increase within the stroked hemisphere and correlate with the severity of the delayed reperfusion deficit but not with the ?HbO2 during stroke. Despite initial restoration of HbO2 after 30 min MCAo there is a delayed compromise that coincides with inflammation and could be a target for improved stroke outcome after thrombolysis.