Subcortical Infarct Volumes Research Articles

A modified murine photothrombotic stroke model: a minimally invasive and reproducible cortical and sub-cortical infarct volume and long-term deficits.

Ischemic stroke is one of the major causes of devastating neurological disabilities and mortality worldwide. Despite extensive research for treatment approaches, there remains limited therapy in the stroke field. Therefore, more research is required for reproducibility to understand stroke pathology in pre-clinical studies. In the current modified method, mice were subjected to photothrombotic stroke (pt-MCA; proximal-middle cerebral artery was exposed with a 532nm laser beam for 4min) by retro-orbital injection of photosensitive dye, Rose Bengal (15mg/kg) before the laser light exposure. Sensorimotor deficits were assessed by rotarod and catwalk test at 72h following post-pt-MCAO, and brain samples were collected for infarct volume and hemorrhagic transformation (HT) assessments. Cognitive impairments were assessed by a novel objective recognition and Morris's water maze tests at the end of the follow-up. pt-MCAO animals significantly reduced body weight and impaired motor and cognitive functions. Furthermore, pt-MCAO animals showed apparent infarction, brain edema, and increased HT compared to the sham animals. Additionally, this method enables concurrent measurement of short-term and long-term neurological dysfunction with relatively larger cortical and sub-cortical infarct volume following pt-MCAO. With respect to the other models, this modified model offers enhanced reproducibility regarding infarct volume and cognitive/functional outcomes and avoids complications associated with critical surgeries and craniotomy. In conclusion, this modified model helps to understand stroke pathogenesis and minimize the animals' numbers which help to increase the scientific and statistical potential in pre-clinical studies.

Subcortical infarcts on admission CTP predict poor outcome despite excellent reperfusion in delayed time windows.

The effect of pretreatment infarct location on clinical outcome after successful mechanical thrombectomy is not understood. Our aim was to evaluate the association between computed tomography perfusion (CTP)-based ischemic core location and clinical outcome following excellent reperfusion in late time windows. We retrospectively reviewed patients who underwent thrombectomy for acute anterior circulation large vessel occlusion in late time windows from October 2019 to June 2021 and enrolled 65 patients with visible ischemic core on admission CTP who had received excellent reperfusion (modified thrombolysis in cerebral infarction grade 2c/3). Poor outcome was defined as a modified Rankin scale score of 3-6 at 90 days. The ischemic core infarct territories were classified into the cortical and subcortical areas. Multivariate logistic regression and receiver operating characteristic (ROC) curve analyses were used in this study. Of the 65 patients analyzed, 38 (58.5%) had a poor outcome. Multivariable logistic analysis showed that the subcortical infarcts (OR 11.75; 95% CI 1.79-77.32; P = 0.010) and their volume (OR 1.17; 95% CI 1.04-1.32; P = 0.011) were independently associated with poor outcome. The ROC curve indicated the capacity of the subcortical infarct involvement (areas under the curve (AUC) = 0.65; 95% CI, 0.53-0.77, P < 0.001) and subcortical infarct volume (AUC = 0.72; 95% CI, 0.60-0.83, P < 0.001) in predicting poor outcome accurately. Subcortical infarcts and their volume on admission CTP are associated with poor outcome after excellent reperfusion in late time windows, rather than cortical infarcts.

Smaller baseline subcortical infarct volume predicts good outcomes in patients with a large core in early acute ischemic stroke after endovascular treatment.

Patients with acute ischemic stroke (AIS) and a large core may benefit from endovascular treatment (EVT) in the early time window. To examine the prognostic factors for good outcomes in patients with a large core (70-130 ml) after EVT. We retrospectively reviewed 40 patients who met the criteria from October 2019 to April 2021. Based on the modified Rankin Score (mRS) at 90 days, the patients were divided into a good outcome group (mRS 0-2) and a poor outcome group (mRS 3-6). Baseline and procedural characteristics were collected for unilateral and multivariate regression analyses to explore the factors that influence good outcomes. In particular, the infarct territories were quantified as subcortical infarct volume (SIV) and cortical infarct volume (CIV). Of the 40 patients included, good outcomes were observed in 11 (27.5%) patients. Younger age, smaller SIV and larger mismatch volume were noted in the good outcome group than in the poor outcome group (all P < 0.05). Multivariate logistic regression analysis showed that only a smaller SIV [odds ratio (OR) 0.801; 95% CI 0.644-0.996; P = 0.046] was an independent predictor for good outcomes. The receiver operating characteristic curve indicated a moderate value of SIV for predicting good outcomes, with an area under the receiver operating characteristic curve of 0.735 (95% CI 0.572-0.862; P = 0.007). Subcortical infarct volume was a potential useful predictor of good outcomes in patients with a large core after EVT in the early time window.

Association between Infarct Location and Hemorrhagic Transformation of Acute Ischemic Stroke following Successful Recanalization after Mechanical Thrombectomy.

The association between infarct location and hemorrhagic transformation of acute ischemic stroke after mechanical thrombectomy is not understood. We aimed to evaluate the association between CTP-based ischemic core variables at admission and hemorrhagic transformation after a successful thrombectomy. We retrospectively analyzed patients who underwent endovascular thrombectomy for acute anterior circulation large-vessel occlusion between October 2019 and June 2021. We enrolled 146 patients with visible ischemic core on pretreatment CTP who had successful reperfusion. The ischemic core infarct territories were classified into the cortical and subcortical areas and then qualitatively and quantitatively analyzed by CTP. Logistic regression and receiver operating characteristic curve analyses were performed to determine the association between ischemic core variables and hemorrhagic transformation. Of the 146 patients analyzed, 72 (49.3%) had hemorrhagic transformation and 23 (15.8%) had symptomatic intracerebral hemorrhage. Multivariate analysis showed that subcortical infarcts were independently associated with hemorrhagic transformation (OR, 8.06; 95% CI, 2.31-28.10; P = .001) and subcortical infarct volume was independently linked to symptomatic intracerebral hemorrhage (OR, 1.05; 95% CI, 1.01-1.09; P = .039). The receiver operating characteristic curve indicated that subcortical infarcts can predict hemorrhagic transformation accurately (area under the curve = 0.755; 95% CI, 0.68-0.82; P < .001) and subcortical infarct volume can predict symptomatic intracerebral hemorrhage (area under the curve = 0.694; 95% CI, 0.61-0.77; P = .002). Subcortical infarcts seen on CTP at admission are associated with hemorrhagic transformation in patients after successful thrombectomy, and subcortical infarct volume may influence the risk of symptomatic intracerebral hemorrhage.

Abstract TP240: Novel Lipid Mediators C-36:6 And C-38:6 Administered Intranasally Are Neuroprotective In Experimental Ischemic Stroke

Objective: Ischemic stroke induces multiple pathophysiological processes involving oxidative stress, loss of homeostasis, excitotoxicity, disruption of the blood-brain barrier and neurovascular unit, contributing to neuronal death. In the early phases following the onset of ischemic damage, neuroinflammation contributes to tissue damage and enlargement of the infarct area. Elovanoids (ELVs) are a class of pro-homeostatic lipid mediators that are derivatives of very long-chain polyunsaturated fatty acids (VLC-PUFAs). VLC-PUFAs are synthesized by ELOVL4 an enzyme primarily expressed in neurons. ELVs have been shown to reduce infarct volume and promote cell survival when administered IV following 2h of middle cerebral artery occlusion (MCAo). This study aimed to test the neuroprotective efficacy of these VLC-PUFA ELV precursors when administered intranasally (IN) in a model of experimental ischemic stroke. Methods: Male Sprague-Dawley rats received 2h of the middle cerebral artery occlusion (MCAo). Treatment with C-36:6, C-38:6 (1μg/μl, 20 μg) or vehicle (saline + ethanol) was administered at 1, 24, and 48h after onset MCAo. Behavior was evaluated using a composite neurological battery; a grading scale of 0-12 was employed (normal score=0, maximal deficit=12). Behavior testing was performed on days 1, 2, 3, and 7, followed by ex vivo MRI for lesion volumes and edema (T2WI) and immunohistochemistry on day 7. Results: Following drug administration, no behavioral side effects were observed. Neurologic function was improved on days 1, 3, and 7 by 24%, 37%, and 28% in animals receiving C-36:6 and by 26%, 26%, and 29% in the group receiving C-38:6. Total, core, and penumbra lesion volumes, computed using T2WI, were reduced with C-36:6 by 60%, 68%, and 53%, and C-38:6 by 35%, 43% 25% vs vehicle. Total, cortical and subcortical infarct areas and volumes were reduced by treatments with C-36:6 (by 42% 48% and 34%) and C-38:6 (by 28% 31% and 20%) vs vehicle. Conclusion: IN delivered ELV precursors demonstrated neuroprotective efficacy improving behavior and reducing infarct volume compared to vehicle. We are exploring neuroprotective molecular mechanisms exerted by intranasally delivered VLC-PUFAs as an experimental therapeutic approach.

Abstract TP274: Post-stroke Treatment With Docosanoids Promote Functional Recovery, Neurogenesis and Angiogenesis, Attenuate Blood-brain Barrier After Experimental Ischemic Stroke

Objective: Ischemic injury induces neurogenesis in the subventricular zone (SVZ) of the lateral ventricles and subgranular zone (SGZ) of dentate gyrus (DG) promotes the migration of neuroblasts, guided by blood vessels, into the ischemic damaged area. Neurogenesis leads the functional and histological recovery in the ischemic-damaged brain through the proliferation, migration, differentiation and integration of newly generated neural cells into the existing neural circuit. Recently, we have shown that Docosahexaenoic acid (DHA) and Neuroprotectin D1 (NPD1) therapy improves functional and histological outcomes following experimental stroke. It has also been shown that DHA promotes neurite outgrowth of immature neurons, such as embryonic hippocampal cells and embryonic cortical cells. The objective of this study was to explore underlying mechanisms of docosanoid neuroprotection after cerebral ischemia. Methods: Male Sprague-Dawley rats were anesthetized with isoflurane/nitrous oxide, underwent 2h of middle cerebral artery occlusion (MCAo) and treated with DHA (5mg/kg, IV) or NPD1 (5μg/per rat, ICV) and vehicles 1h after. Neuro-behavioral assessments were conducted on days 1, 2, 3 and on weeks 1, 2, 3 or 4. BrdU was injected on days 4, 5 and 6, immunohistochemistry was performed on week 2 or 4, MRI on day 7 and lipidomic analysis at 4 and 5h after onset of stroke. Results: DHA improved short- and long-term behavioral functions and reduced cortical, subcortical and total infarct volumes (by 42%, 47% and 31%, respectively) after 2 weeks and reduced tissue loss by 50% after 4 weeks. DHA increased the number of BrdU + /Ki-67 + , BrdU + /DCX + and BrdU + /NeuN + cells in the cortex, subventricular zone and dentate gyrus and potentiated NPD1 synthesis in the penumbra at 5h after MCAo. NPD1 improved behavior, reduced lesion volumes, protected ischemic penumbra, increased NeuN, GFAP, SMI-71 positive cells and vessels, axonal regeneration in the penumbra and attenuated blood-brain barrier (BBB) after MCAo. Conclusion: We conclude that docosanoid administration increases neurogenesis, angiogenesis, activates NPD1 synthesis in the penumbra and diminishes BBB, which correlates to long-term neurobehavioral recovery after experimental ischemic stroke.

Argon Inhalation for 24 Hours After Onset of Permanent Focal Cerebral Ischemia in Rats Provides Neuroprotection and Improves Neurologic Outcome.

We tested the hypothesis that prolonged inhalation of 70% argon for 24 hours after in vivo permanent or temporary stroke provides neuroprotection and improves neurologic outcome and overall recovery after 7 days. Controlled, randomized, double-blinded laboratory study. Animal research laboratories. Adult Wistar male rats (n = 110). Rats were subjected to permanent or temporary focal cerebral ischemia via middle cerebral artery occlusion, followed by inhalation of 70% argon or nitrogen in 30% oxygen for 24 hours. On postoperative day 7, a 48-point neuroscore and histologic lesion size were assessed. After argon inhalation for 24 hours immediately following "severe permanent ischemia" induction, neurologic outcome (neuroscore, p = 0.034), overall recovery (body weight, p = 0.02), and infarct volume (total infarct volume, p = 0.0001; cortical infarct volume, p = 0.0003; subcortical infarct volume, p = 0.0001) were significantly improved. When 24-hour argon treatment was delayed for 2 hours after permanent stroke induction or until after postischemic reperfusion treatment, neurologic outcomes remained significantly improved (neuroscore, p = 0.043 and p = 0.014, respectively), as was overall recovery (body weight, p = 0.015), compared with nitrogen treatment. However, infarct volume and 7-day mortality were not significantly reduced when argon treatment was delayed. Neurologic outcome (neuroscore), overall recovery (body weight), and infarct volumes were significantly improved after 24-hour inhalation of 70% argon administered immediately after severe permanent stroke induction. Neurologic outcome and overall recovery were also significantly improved even when argon treatment was delayed for 2 hours or until after reperfusion.

Docosanoids Promote Neurogenesis and Angiogenesis, Blood-Brain Barrier Integrity, Penumbra Protection, and Neurobehavioral Recovery After Experimental Ischemic Stroke.

Docosahexaenoic acid (DHA) and neuroprotectin D1 (NPD1) are neuroprotective after experimental ischemic stroke. To explore underlying mechanisms, SD rats underwent 2h of middle cerebral artery occlusion (MCAo) and treated with DHA (5mg/kg, IV) or NPD1 (5μg/per rat, ICV) and vehicles 1h after. Neuro-behavioral assessments was conducted on days 1, 2, and 3, and on week 1, 2, 3, or 4. BrdU was injected on days 4, 5, and 6, immunohistochemistry was performed on week 2 or 4, MRI on day 7, and lipidomic analysis at 4 and 5h after onset of stroke. DHA improved short- and long-term behavioral functions and reduced cortical, subcortical, and total infarct volumes (by 42, 47, and 31%, respectively) after 2weeks and reduced tissue loss by 50% after 4weeks. DHA increased the number of BrdU+/Ki-67+, BrdU+/DCX+, and BrdU+/NeuN+ cells in the cortex, subventricular zone, and dentate gyrus and potentiated NPD1 synthesis in the penumbra at 5h after MCAo. NPD1 improved behavior, reduced lesion volumes, protected ischemic penumbra, increased NeuN, GFAP, SMI-71-positive cells and vessels, axonal regeneration in the penumbra, and attenuated blood-brain barrier (BBB) after MCAo. We conclude that docosanoid administration increases neurogenesis and angiogenesis, activates NPD1 synthesis in the penumbra, and diminishes BBB permeability, which correlates to long-term neurobehavioral recovery after experimental ischemic stroke.

Abstract WP118: Docosahexaenoic Acid Provides Neuroprotection by Increasing Neurogenesis After Experimental Stroke

Introduction: Ischemic injury induces neurogenesis in the subventricular zone (SVZ) of the lateral ventricles and subgranular zone (SGZ) of dentate gyrus (DG), and it promotes the migration of neuroblasts, guided by blood vessels, into the ischemic damaged area. Recently, we have shown that docosahexaenoic acid (DHA; 22:6n-3) therapy improves functional and histological outcomes following experimental stroke. The objective, hence, is to determine whether DHA administration enhances endogenous neurogenesis after cerebral ischemia. Methods: Twenty-one male SD rats were anesthetized with isoflurane and subjected to 2 h of middle cerebral artery occlusion (MCAo) by intraluminal filament. DHA (5 mg/kg, n=10) or saline (n=11) was administered intravenously at 3 h after onset of MCAo (n = 7-10 per group). BrdU was injected on days 4, 5 and 6. DCX (doublecortin), NeuN (a marker for mature neurons), ki-67 (marker for cell proliferation) have been used to test the proliferation, migration and differentiation of neural precursor cells. Behavioral tests were conducted on days 1, 2 and 3 and weeks 1 and 2. Immunohistochemistry with BrdU and ki-67, DCX or NeuN, and histopathology were performed on day 14. Results: DHA-treated animals showed improved neurologic scores compared to the saline group during the two-week survival period. Total, cortical and subcortical infarct areas in the DHA-treated group were significantly attenuated at multiple bregma levels as well as total, cortical and subcortical infarct volumes by 42.4 %, by 47.5 %, and by 31.2 % compared to the vehicle-treated group. The number of BrdU + /ki-67 + cells in DHA-treated rats was increased in cortex by 88 %, SVZ by 40 % and DG by 270 % compared to saline-treated rats. DHA treatment increased the number of BrdU + /DCX + cells in the cortex by 65 %, the SVZ by 29 % and the DG (by 58 %) compared to saline-treated group. In addition, the numbers of BrdU + /NeuN + cells were increased in the cortex by 49 %, SVZ by 28 % and DG by 48% compared to vehicle treatment. Conclusion: DHA increased neurogenesis by proliferation, differentiation and migration of neural stem cells in the DG, SVZ and peri-infarct area two weeks after ischemic stroke. In addition, DHA promoted neurobehavioral recovery and reduced infarct volume.

The Neuroprotective Effect of Rosemary (Rosmarinus officinalis L.) Hydro-alcoholic Extract on Cerebral Ischemic Tolerance in Experimental Stroke.

The prevention of BBB breakdown and the subsequent vasogenic edema are important parts of the medical management of ischemic stroke. The purpose of this study was to investigate the ischemic tolerance effect of Rosmarinus officinalis leaf hydro-alcoholic extract (RHE). Five groups of animals were designed: sham (underwent surgery without MCAO) and MCAO groups, the MCAO groups were pretreated orally by gavages with RHE (50, 75, and 100 mg/Kg/day), daily for 30 days. Two hours after the last dose, serum lipid levels were determined and then the rats were subjected to 60 min of middle cerebral artery occlusion followed by 24 h of reperfusion. Subsequently, brain infarct size, brain edema and Evans Blue dye extravasations were measured and neurological deficits were scored. Dietary RHE could significantly reduce cortical and sub-cortical infarct volumes (211.55 ± 24.88 mm3vs. 40.59 ± 10.04 mm3vs. 29.96 ± 12.19 mm3vs. 6.58 ± 3.2 mm3), neurologic deficit scores, cerebral edema (82.34 ± 0.42% vs. 79.92 ± 0.49% vs. 79.45 ± 0.26% vs. 79.30 ± 0.19%), blood-brain barrier (BBB) permeability (7.73 ± 0.4 μg/g tissue vs. 4.1 ± 0.23 μg/g tissue vs. 3.58 ± 0.3 μg/g tissue vs. 3.38 ± 0.25 μg/g tissue) in doses of 50, 75 and 100 mg/Kg/day as compared with the control group in the transient model of focal cerebral ischemia. Although pretreatment with RHE plays an important role in the generation of tolerance against cerebral I/R injury, further studies are needed to clarify the mechanism of the ischemic tolerance.

Acute Treatment with Docosahexaenoic Acid Complexed to Albumin Reduces Injury after a Permanent Focal Cerebral Ischemia in Rats

Docosahexaenoic acid complexed to albumin (DHA-Alb) is highly neuroprotective after temporary middle cerebral artery occlusion (MCAo), but whether a similar effect occurs in permanent MCAo is unknown. Male Sprague-Dawley rats (270–330 g) underwent permanent MCAo. Neurological function was evaluated on days 1, 2 and 3 after MCAo. We studied six groups: DHA (5 mg/kg), Alb (0.63 or 1.25 g/kg), DHA-Alb (5 mg/kg+0.63 g/kg or 5 mg/kg+1.25 g/kg) or saline. Treatment was administered i.v. at 3 h after onset of stroke (n = 7–10 per group). Ex vivo imaging of brains and histopathology were conducted on day 3. Saline- and Alb-treated rats developed severe neurological deficits but were not significantly different from one another. In contrast, rats treated with low and moderate doses of DHA-Alb showed improved neurological score compared to corresponding Alb groups on days 2 and 3. Total, cortical and subcortical lesion volumes computed from T2 weighted images were reduced following a moderate dose of DHA-Alb (1.25 g/kg) by 25%, 22%, 34%, respectively, compared to the Alb group. The total corrected, cortical and subcortical infarct volumes were reduced by low (by 36–40%) and moderate doses (by 34–42%) of DHA-Alb treatment compared to the Alb groups. In conclusion, DHA-Alb therapy is highly neuroprotective in permanent MCAo in rats. This treatment can provide the basis for future therapeutics for patients suffering from ischemic stroke.

Docosahexaenoic Acid Signaling Modulates Cell Survival in Experimental Ischemic Stroke Penumbra and Initiates Long-Term Repair in Young and Aged Rats

BackgroundDocosahexaenoic acid, a major omega-3 essential fatty acid family member, improves behavioral deficit and reduces infarct volume and edema after experimental focal cerebral ischemia. We hypothesize that DHA elicits neuroprotection by inducing AKT/p70S6K phosphorylation, which in turn leads to cell survival and protects against ischemic stroke in young and aged rats.Methods and ResultsRats underwent 2 h of middle cerebral artery occlusion (MCAo). DHA, neuroprotectin D1 (NPD1) or vehicle (saline) was administered 3 h after onset of stroke. Neurological function was evaluated on days 1, 2, 3, and 7. DHA treatment improved functional recovery and reduced cortical, subcortical and total infarct volumes 7 days after stroke. DHA also reduced microglia infiltration and increased the number of astrocytes and neurons when compared to vehicle on days 1 and 7. Increases in p473 AKT and p308 AKT phosphorylation/activation were observed in animals treated with DHA 4 h after MCAo. Activation of other members of the AKT signaling pathway were also observed in DHA treated animals including increases in pS6 at 4 h and pGSK at 24 h. DHA or NPD1 remarkably reduced total and cortical infarct in aged rats. Moreover, we show that in young and aged rats DHA treatment after MCAo potentiates NPD1 biosynthesis. The phosphorylation of p308 AKT or pGSK was not different between groups in aged rats. However, pS6 expression was increased with DHA or NPD1 treatment when compared to vehicle.ConclusionsWe suggest that DHA induces cell survival, modulates the neuroinflammatory response and triggers long term restoration of synaptic circuits. Both DHA and NPD1 elicited remarkable protection in aged animals. Accordingly, activation of DHA signaling might provide benefits in the management of ischemic stroke both acutely as well as long term to limit ensuing disabilities.

Influence of exercise training on ischemic brain injury in type 1 diabetic rats

While exercise training (ExT) appears to influence cerebrovascular function during type 1 diabetes (T1D), it is not clear whether this beneficial effect extends to protecting the brain from ischemia-induced brain injury. Thus our goal was to examine whether modest ExT could influence transient focal ischemia-induced brain injury along with nitric oxide synthase (NOS)-dependent dilation of cerebral (pial) arterioles during T1D. Sprague-Dawley rats were divided into four groups: nondiabetic sedentary, nondiabetic ExT, diabetic (streptozotocin; 50 mg/kg ip) sedentary, and diabetic ExT. In the first series of studies, we measured infarct volume in all groups of rats following right MCA occlusion for 2 h, followed by 24 h of reperfusion. In a second series of studies, a craniotomy was performed over the parietal cortex, and we measured responses of pial arterioles to an endothelial NOS (eNOS)-dependent, a neuronal NOS (nNOS)-dependent, and a NOS-independent agonist in all groups of rats. We found that sedentary diabetic rats had significantly larger total, cortical, and subcortical infarct volumes following ischemia-reperfusion than sedentary nondiabetic, nondiabetic ExT, and diabetic ExT rats. Infarct volumes were similar in sedentary nondiabetic, ExT nondiabetic, and ExT diabetic rats. In contrast, ExT did not alter infarct size in nondiabetic compared with sedentary nondiabetic rats. In addition, ExT diabetic rats had impaired eNOS- and nNOS-dependent, but not NOS-independent, vasodilation that was restored by ExT. Thus ExT of T1D rats lessened ischemic brain injury following middle cerebral artery occlusion and restored impaired eNOS- and nNOS-dependent vascular function. Since the incidence of ischemic stroke is increased during T1D, we suggest that our finding are significant in that modest ExT may be a viable preventative therapeutic approach to lessen ischemia-induced brain injury that may occur in T1D subjects.

Novel aspirin-triggered neuroprotectin D1 attenuates cerebral ischemic injury after experimental stroke

Acute ischemic stroke triggers complex neurovascular, neuroinflammatory and synaptic alterations. Aspirin and docosahexaenoic acid (DHA), an omega-3 essential fatty acid family member, have beneficial effects on cerebrovascular diseases. DHA is the precursor of neuroprotectin D1 (NPD1), which downregulates apoptosis and, in turn, promotes cell survival. Here we have tested the effect of aspirin plus DHA administration and discovered the synthesis of aspirin-triggered NPD1 (AT-NPD1) in the brain. Then we performed the total chemical synthesis of this molecule and tested in the setting of 2h middle cerebral artery occlusion (MCAo) in Sprague–Dawley rats. Neurological status was evaluated at 24h, 48h, 72h, and 7days. At 3h post-stroke onset, an intravenous administration of 333μg/kg of AT-NPD1 sodium salt (AT-NPD1-SS) or methyl-ester (AT-NPD1-ME) or vehicle (saline) as treatment was given. On day 7, ex vivo magnetic resonance imaging (MRI) of the brains was conducted on 11.7T MRI. T2WI, 3D volumes, and apparent diffusion coefficient (ADC) maps were generated. In addition, infarct volumes and number of GFAP (reactive astrocytes), ED-1 (activated microglia/macrophages) and SMI-71-positive vessels were counted in the cortex and striatum at the level of the central lesion. All animals showed similar values for rectal and cranial temperatures, arterial blood gases, and plasma glucose during and after MCAo. Treatment with both AT-NPD1-SS and AT-NPD1-ME significantly improved neurological scores compared to saline treatment at 24h, 48h, 72h and 7days. Total lesion volumes computed from T2WI images were significantly reduced by both AT-NPD1-SS and AT-NPD1-ME treatment in the cortex (by 44% and 81%), striatum (by 61% and 77%) and total infarct (by 48% and 78%, respectively). Brain edema, computed from T2WI in the cortex (penumbra) and striatum (core), was elevated in the saline group. In contrast, both AT-NPD1 decreased water content in the striatum on day 7. 3D volumes, computed from T2WI, were dramatically reduced with both AT-NPD1 and the lesion was mostly localized in the subcortical areas. Treatment with both AT-NPD1-SS and AT-NPD1-ME significantly reduced cortical (by 76% and 96%), subcortical (by 61% and 70%) and total (69% and 84%, respectively) infarct volumes as defined by histopathology. In conclusion, a novel biosynthetic pathway that leads to the formation of AT-NPD1 mediator in the brain was discovered. In addition, administration of synthetic AT-NPD1, in either its sodium salt or as the methyl ester, was able to attenuate cerebral ischemic injury which leads to a novel approach for pharmaceutical intervention and clinical translation.

Abstract 115: Aspirin-triggered Neuroprotectin D1 Protects The Penumbra In Focal Cerebral Ischemia In Rats

Background and Purpose: Acute ischemic stroke triggers complex neurovascular, neuroinflammatory and synaptic alterations. Aspirin and the omega-3 essential fatty acid, docosahexaenoic acid (DHA), have beneficial effects on cerebrovascular diseases. Unlike most anti-inflammatory agents, aspirin jumpstarts resolution mainly due to acetylation of cyclooxygenase enzymes. Both neuroprotectin D1 (NPD1) and its precursor, DHA, participate in neuroprotection. Here we have tested the effect of aspirin plus DHA administration and discovered the synthesis of aspirin-triggered NPD1 (AT-NPD1) in the penumbra. Then we performed the total chemical synthesis of this molecule and, upon systemic administration 1h after 2h of middle cerebral artery occlusion (MCAo), uncovered remarkable protection. Methods: Sprague-Dawley rats were anesthetized with isoflurane and received 2h MCAo. Neurological status was evaluated at 24h, 48h, 72h, and 7 days. Treatment with 333µg/kg of AT-NPD1 sodium salt (AT-NPD1-SS) or AT-NPD1 methyl-ester (AT-NPD1-ME) or vehicle (saline) (n=7-8 per group) was administered i.v. at 3h after stroke. On day 7, ex vivo magnetic resonance imaging (MRI) of the brains was conducted on 11.7T MRI. T2WI, 3D volumes, and apparent diffusion coefficient (ADC) maps were generated. In addition, infarct volumes and number of GFAP (reactive astrocytes), ED-1 (activated microglia/microphages) and NeuN (neurons)-positive cells were counted in the cortex and striatum at the level of the central lesion. Results: All animals showed similar values for rectal and cranial temperatures, arterial blood gases, and plasma glucose during and after MCAo. Treatment with both AT-NPD1-SS and AT-NPD1-ME significantly improved neurological scores compared to saline treatment at 24h, 48h, 72h and 7 days. Total lesion volumes computed from T2WI images were significantly reduced by both AT-NPD1-SS and AT-NPD1-ME treatment in the cortex (by 44% and 81%, respectively), striatum (by 61% and 77%) and total infarct (by 48% and 78%, respectively). Brain edema, computed from T2WI in the cortex (penumbra) and striatum (core), was elevated in the saline group. In contrast, both AT-NPD1-SS and AT-NPD1-ME decreased water content in the striatum on day 7. 3D volumes, computed from T2WI, were dramatically reduced with AT-NPD1-SS and AT-NPD1-ME, and the lesion was mostly localized in the subcortical areas. Treatment with both AT-NPD1-SS and AT-NPD1-ME significantly reduced cortical (by 76% and 96%), subcortical (by 61% and 70%) and total (69% and 84%) infarct volumes as defined by histopathology. Conclusions: Ex vivo MRI, histopathology, and behavioral testing shows neuroprotective efficacy of novel AT-NPD1 therapy in focal cerebral ischemia and uncover a novel approach for pharmaceutical intervention and clinical translation.

Superior Neuroprotective Efficacy of LAU-0901, a Novel Platelet-Activating Factor Antagonist, in Experimental Stroke

Platelet-activating factor (PAF) accumulates during cerebral ischemia, and inhibition of this process plays a critical role in neuronal survival. Recently, we demonstrated that LAU-0901, a novel PAF receptor antagonist, is neuroprotective in experimental stroke. We used magnetic resonance imaging in conjunction with behavior and immunohistopathology to expand our understanding of this novel therapeutic approach. Sprague–Dawley rats received 2 h middle cerebral artery occlusion (MCAo) and were treated with LAU-0901 (60 mg/kg) or vehicle 2 h from MCAo onset. Behavioral function, T2-weighted imaging (T2WI), and apparent diffusion coefficients were performed on days 1, 3, and 7 after MCAo. Infarct volume and number of GFAP, ED-1, and NeuN-positive cells were conducted on day 7. Behavioral deficit was significantly improved by LAU-0901 treatment compared to vehicle on days 1, 3, and 7. Total lesion volumes computed from T2WI were significantly reduced by LAU-0901 on days 1, 3, and 7 (by 83%, 90%, and 96%, respectively), which was consistent with decreased edema formation. Histopathology revealed that LAU-0901 treatment resulted in significant reduction of cortical and subcortical infarct volumes, attenuated microglial infiltration, and promoted astrocytic and neuronal survival. These findings suggest LAU-0901 is a promising neuroprotectant and provide the basis for future therapeutics in patients suffering ischemic stroke.

The site of embolization related to infarct size, oedema and clinical outcome in a rat stroke model - further translational stroke research

Background and purposeReliable models are essential for translational stroke research to study the pathophysiology of ischaemic stroke in an effort to find therapies that may ultimately reduce oedema, infarction and mortality in the clinic. The purpose of this study was to investigate the relation between the site of arterial embolization and the subsequent oedema, infarction and clinical outcome in a rat embolic stroke model.MethodsThirty-six male Sprague-Dawley rats were thromboembolized into the internal carotid artery. The site of occlusion was demonstrated by arteriography. Following histological preparation and evaluation, the size of the hemispheres and the infarcts were measured by quantitative histology and planimetry. Another parallel stroke model study was subsequently examined to investigate if the conclusions from the first study could be applied to the second study.ResultsThe median size of the infarct was 40% of the ipsilateral hemisphere in both the 19 animals with occlusion localised to the intracranial part of the internal carotid artery and in the 11 animals where the main trunk of the middle cerebral artery was occluded. In 5 animals, occlusion of the extracranial part of the internal carotid artery resulted in significantly smaller infarcts compared to other groups (p < 0.01). Another independent study re-confirmed these results. Furthermore, significant correlations (R > 0.76, p < 0.0001) were found between 1) cortical, subcortical, and total infarct volumes, 2) oedema in percent of the left hemisphere, 3) clinical score before termination and 4) postoperative weight loss.ConclusionsDistal occlusions of the intracranial part of the internal carotid or middle cerebral arteries resulted in comparable large sized infarctions and oedema. This indicates that investigators do not need a similar number of such occlusions in each experimental group. Contrary to observations in the clinic, distal internal carotid artery occlusions did not result in worse outcome than middle cerebral stem occlusions, but this finding may be explained by the controlled emboli size in this experimental stroke model.

OVEREXPRESSION OF HUMAN DOWN SYNDROME CANDIDATE REGION 1 (DSCR1) GENE IS PROTECTIVE IN STROKE

The Down syndrome candidate region 1 (DSCR1) gene is found on chromosome 21. DSCR1 protein is highly expressed in the brain, and following middle cerebral artery occlusion (MCAO) in mice DSCR1 mRNA and protein increases in the peri‐infarct region. We examined the effect of overexpression of DSCR1 on outcome following stroke. DSCR1 transgenic (Tg) mice were produced by the overexpression of the human DSCR1 gene, only in cells that normally express DSCR1, on a C57Bl6/J X CBA background. Cerebral ischemia was induced by MCAO for 0.5 h followed by reperfusion for 23.5 h (ischemia‐reperfusion; I‐R) in 8–14 week old male DSCR1 Tg (n=14) and wild‐type (Wt; n=9) mice. After 24 h, neurological impairment was assessed (neurological score and hanging wire test). Brain infarct and edema volume were then measured in thionin‐stained coronal sections. After I‐R, Tg mice had less neurological impairment than Wt, with a lower neurological score (2.4±0.3 vs 3.9±0.1; P&lt;0.05) and a longer hanging‐wire time (38±5 vs 9±3 s; P&lt;0.01). After I‐R, Tg mice had a significantly smaller subcortical infarct volume than Wt mice (15±2 vs 32±5 mm3; P&lt;0.01). In addition, total infarct volume tended to be smaller in Tg than Wt mice (26±5 vs. 36±6 mm3; P=0.26). Furthermore, edema volume was smaller in Tg than Wt mice (17±3 vs 31±6 mm3; P&lt;0.05). Thus, overexpression of DSCR1 improves outcome following stroke.

Intracellular mechanisms of N-acylethanolamine-mediated neuroprotection in a rat model of stroke

N-acyl ethanolamines (NAEs) are endogenous lipids that are synthesized in response to tissue injury, including ischemia and stroke, suggesting they may exhibit neuroprotective properties. We hypothesized that NAE 16:0 (palmitoylethanolamine) is neuroprotective against ischemia-reperfusion injury in rats, a widely employed model of stroke, and that neuroprotection is mediated through an intracellular mechanism independent of known NAE receptors. Administration of NAE 16:0 from 30 min before to 2 h after stroke significantly reduced cortical and subcortical infarct volume, and correlated with an improvement of the neurological phenotype, as assessed by the neurological deficit score. We here show that NAE 16:0-mediated neuroprotection was independent of cannabinoid (CB1) and vanilloid (VR1) receptor activation, known NAE receptors on the plasma membrane, as determined by inclusion of specific inhibitors. The inclusion of an NAE uptake inhibitor (AM404), however, completely reversed NAE 16:0-mediated neuroprotection, suggesting that NAE 16:0s effects are through an intracellular mechanism. NAE 16:0 produced a significant reduction in the number of cells undergoing apoptosis and reversed ischemia-induced upregulation of several proteins, including inducible nitric oxide synthase and transcription factor NFκB. Our findings suggest that NAE 16:0-mediated neuroprotection is due to the reduction of neuronal apoptosis and inflammation in the brain.

Neuroprotective effect of 4-hydroxybenzyl alcohol against transient focal cerebral ischemia via anti-apoptosis in rats

4-hydroxybenzyl alcohol (4-HBA), one of the major active phenolic constituents of Gastrodia elata Blume, a very important traditional Chinese medicinal herb, has been shown to be an effective agent against the central and peripheral nervous disorders. In this study, we attempted to explore the possible mechanisms underlying the neuroprotection against transient focal cerebral ischemia by 4-HBA.4-HBA (25, 50 mg/kg) was given 30 min before focal ischemia in rats caused by middle cerebral artery occlusion (1 h of occlusion, 24 h of reperfusion). Under the treatment of 50 mg/kg 4-HBA, total (100.76±2.90 mm3), cortical (64.91±1.46 mm3), and sub-cortical (38.77±2.78 mm3) infarct volumes were significantly decreased in comparison to ischemia-reperfusion values. Neurological evaluation and Nissl-staining of the 4-HBA group were improved significantly compared to the untreated ischemia group. TUNEL-positive cells were reduced significantly in 4-HBA treated group. Results of immunofluorescence staining analysis and Western immunoblot indicated that 4-HBA increased the expression of Bcl-2 and inhibited the activation of caspase-3 ultimately inhibiting apoptosis. These results suggested that 4-HBA ameliorated ischemic injury induced by transient focal cerebral ischemia in rats, and this neuroprotective effect may be partly related to attenuate apoptosis pathway.