Mouse Model Of Cerebral Ischemia Research Articles

In vivo Imaging of Cannabinoid Type 2 Receptors: Functional and Structural Alterations in Mouse Model of Cerebral Ischemia by PET and MRI

PurposeStroke is one of the most prevalent vascular diseases. Non-invasive molecular imaging methods have the potential to provide critical insights into the temporal dynamics and follow alterations of receptor expression and metabolism in ischemic stroke. The aim of this study was to assess the cannabinoid type 2 receptor (CB2R) levels in transient middle cerebral artery occlusion (tMCAO) mouse models at subacute stage using positron emission tomography (PET) with our novel tracer [18F]RoSMA-18-d6 and structural imaging by magnetic resonance imaging (MRI).ProceduresOur recently developed CB2R PET tracer [18F]RoSMA-18-d6 was used for imaging neuroinflammation at 24 h after reperfusion in tMCAO mice. The RNA expression levels of CB2R and other inflammatory markers were analyzed by quantitative real-time polymerase chain reaction using brain tissues from tMCAO (1 h occlusion) and sham-operated mice. [18F]fluorodeoxyglucose (FDG) was included for evaluation of the cerebral metabolic rate of glucose (CMRglc). In addition, diffusion-weighted imaging and T2-weighted imaging were performed for anatomical reference and delineating the lesion in tMCAO mice.ResultsmRNA expressions of inflammatory markers TNF-α, Iba1, MMP9 and GFAP, CNR2 were increased to 1.3–2.5 fold at 24 h after reperfusion in the ipsilateral compared to contralateral hemisphere of tMCAO mice, while mRNA expression of the neuronal marker MAP-2 was markedly reduced to ca. 50 %. Reduced [18F]FDG uptake was observed in the ischemic striatum of tMCAO mouse brain at 24 h after reperfusion. Although higher activity of [18F]RoSMA-18-d6 in ex vivo biodistribution studies and higher standard uptake value ratio (SUVR) were detected in the ischemic ipsilateral compared to contralateral striatum in tMCAO mice, the in vivo specificity of [18F]RoSMA-18-d6 was confirmed only in the CB2R-rich spleen.ConclusionsThis study revealed an increased [18F]RoSMA-18-d6 measure of CB2R and a reduced [18F]FDG measure of CMRglc in the ischemic striatum of tMCAO mice at subacute stage. [18F]RoSMA-18-d6 might be a promising PET tracer for detecting CB2R alterations in animal models of neuroinflammation without neuronal loss.

Sustained Baclofen-Induced Activation of GABA B Receptors After Cerebral Ischemia Restores Receptor Expression and Function and Limits Progressing Loss of Neurons.

One important function of GABAB receptors is the control of neuronal activity to prevent overexcitation and thereby excitotoxic death, which is a hallmark of cerebral ischemia. Consequently, sustained activation of GABAB receptors with the selective agonist baclofen provides neuroprotection in in vitro and in vivo models of cerebral ischemia. However, excitotoxic conditions severely downregulate the receptors, which would compromise the neuroprotective effectiveness of baclofen. On the other hand, recent work suggests that sustained activation of GABAB receptors stabilizes receptor expression. Therefore, we addressed the question whether sustained activation of GABAB receptors reduces downregulation of the receptor under excitotoxic conditions and thereby preserves GABAB receptor-mediated inhibition. In cultured neurons subjected to oxygen and glucose deprivation (OGD), to mimic cerebral ischemia, GABAB receptors were severely downregulated. Treatment of the cultures with baclofen after OGD restored GABAB receptor expression and reduced loss of neurons. Restoration of GABAB receptors was due to enhanced fast recycling of the receptors, which reduced OGD-induced sorting of the receptors to lysosomal degradation. Utilizing the middle cerebral artery occlusion (MCAO) mouse model of cerebral ischemia, we verified the severe downregulation of GABAB receptors in the affected cortex and a partial restoration of the receptors after systemic injection of baclofen. Restored receptor expression recovered GABAB receptor-mediated currents, normalized the enhanced neuronal excitability observed after MCAO and limited progressive loss of neurons. These results suggest that baclofen-induced restoration of GABAB receptors provides the basis for the neuroprotective activity of baclofen after an ischemic insult. Since GABAB receptors regulate multiple beneficial pathways, they are promising targets for a neuroprotective strategy in acute cerebral ischemia.

GHB analogs confer neuroprotection through specific interaction with the CaMKIIα hub domain

Ca2+/calmodulin-dependent protein kinase II alpha subunit (CaMKIIα) is a key neuronal signaling protein and an emerging drug target. The central hub domain regulates the activity of CaMKIIα by organizing the holoenzyme complex into functional oligomers, yet pharmacological modulation of the hub domain has never been demonstrated. Here, using a combination of photoaffinity labeling and chemical proteomics, we show that compounds related to the natural substance γ-hydroxybutyrate (GHB) bind selectively to CaMKIIα. By means of a 2.2-Å x-ray crystal structure of ligand-bound CaMKIIα hub, we reveal the molecular details of the binding site deep within the hub. Furthermore, we show that binding of GHB and related analogs to this site promotes concentration-dependent increases in hub thermal stability believed to alter holoenzyme functionality. Selectively under states of pathological CaMKIIα activation, hub ligands provide a significant and sustained neuroprotection, which is both time and dose dependent. This is demonstrated in neurons exposed to excitotoxicity and in a mouse model of cerebral ischemia with the selective GHB analog, HOCPCA (3-hydroxycyclopent-1-enecarboxylic acid). Together, our results indicate a hitherto unknown mechanism for neuroprotection by a highly specific and unforeseen interaction between the CaMKIIα hub domain and small molecule brain-penetrant GHB analogs. This establishes GHB analogs as powerful tools for investigating CaMKII neuropharmacology in general and as potential therapeutic compounds for cerebral ischemia in particular.

Sex-Specific Differences in Autophagic Responses to Experimental Ischemic Stroke.

Ischemic stroke triggers a series of complex pathophysiological processes including autophagy. Differential activation of autophagy occurs in neurons derived from males versus females after stressors such as nutrient deprivation. Whether autophagy displays sexual dimorphism after ischemic stroke is unknown. We used a cerebral ischemia mouse model (middle cerebral artery occlusion, MCAO) to evaluate the effects of inhibiting autophagy in ischemic brain pathology. We observed that inhibiting autophagy reduced infarct volume in males and ovariectomized females. However, autophagy inhibition enhanced infarct size in females and in ovariectomized females supplemented with estrogen compared to control mice. We also observed that males had increased levels of Beclin1 and LC3 and decreased levels of pULK1 and p62 at 24 h, while females had decreased levels of Beclin1 and increased levels of ATG7. Furthermore, the levels of autophagy markers were increased under basal conditions and after oxygen and glucose deprivation in male neurons compared with female neurons in vitro. E2 supplementation significantly inhibited autophagy only in male neurons, and was beneficial for cell survival only in female neurons. This study shows that autophagy in the ischemic brain differs between the sexes, and that autophagy regulators have different effects in a sex-dependent manner in neurons.

Cognitive Dysfunction in a Mouse Model of Cerebral Ischemia Influences Salivary Metabolomics.

Vascular dementia, caused by cerebrovascular disease, is associated with cognitive impairment and reduced hippocampal metabolite levels. Specifically, cognitive impairment can be induced by decreased hippocampal brain-derived neurotrophic factor (BDNF) expression. The development of low or non-invasive biomarkers to characterize these diseases is an urgent task. Disturbance of metabolic pathways has been frequently observed in cognitive impairment, and salivary molecules also showed the potentials to reflect cognitive impairment. Therefore, we evaluated salivary metabolic profiles associated with altered hippocampal BDNF expression levels in a cerebral ischemia mouse model using metabolomic analyses. The effect of tacrine (a cholinesterase inhibitor) administration was also examined. The arteries of ICR mice were occluded with aneurysm clips to generate the cerebral ischemia model. Learning and memory performance was assessed using the elevated plus maze (EPM) test. Hippocampal and blood BDNF levels were quantified using an enzyme-linked immunosorbent assay. Glutamate decarboxylase 1 (GAD1) mRNA expression, is associated with cognitive impairment, was quantified by a real-time polymerase chain reaction. The EPM test revealed impaired spatial working memory in the cerebral ischemia mouse model; tacrine administration ameliorated this memory impairment. Cerebral ischemia suppressed GAD1 expression by decreasing hippocampal BDNF expression. In total, seven salivary metabolites, such as trimethylamine N-oxide and putrescine, were changed by cognitive impairment and tacrine administration. Our data suggest that salivary metabolite patterns were associated with cognitive function.

Plasma from healthy donors protects blood–brain barrier integrity via FGF21 and improves the recovery in a mouse model of cerebral ischaemia

BackgroundHealthy plasma therapy reverses cognitive deficits and promotes neuroplasticity in ageing brain disease. However, whether healthy plasma therapy improve blood–brain barrier integrity after stroke remains unknown.MethodsHere, we intravenously injected healthy...

Deficiency in Neuroserpin Exacerbates CoCl2 Induced Hypoxic Injury in the Zebrafish Model by Increased Oxidative Stress.

Protective strategy against hypoxic-ischemic (H/I) induced injury has been intensively discussed. Neuroserpin, an inhibitor for tissue plasminogen activator (tPA), has been proved a vital neuroprotective agent in cerebral ischemia mouse model and oxygen-glucose deprivation and reoxygenation (OGD/R) cell model. Neuroserpin is a promising therapeutic hint for neonatal hypoxic-ischemia injury. Here, we established a neuroserpin deficient zebrafish to study its role in CoCl2 chemically induced hypoxic injury. CoCl2 exposure was beginning at the embryonic stage. Development defects, neuronal loss, and vascular malformation was assessed by imaging microscopy. Neuroserpin deficient zebrafish showed more development defects, neuronal loss and vascular malformation compared to wide-type. Apoptosis and oxidative stress were evaluated to further identify the possible mechanisms. These findings indicate that neuroserpin could protective against CoCl2 induced hypoxic injury by alleviating oxidative stress.

Neuroprotective effects of zonisamide on cerebral ischemia injury via inhibition of neuronal apoptosis

It is known that neuronal apoptosis contributes to pathology of cerebral ischemia injury. Zonisamide (ZNS) has shown anti-apoptosis effects in recent studies. The present study investigated whether the anti-apoptotic effect can account for the neuroprotective action of ZNS on cerebral ischemia. Neuronal cells were maintained under oxygen-glucose deprivation conditions to simulate cerebral ischemia and treated with ZNS simultaneously. The apoptosis of the cells and expression of apoptosis-related proteins were investigated by flow cytometry and western blot analysis, respectively. A cerebral ischemia mouse model was created via middle cerebral artery occlusion, and the mice were treated with ZNS. Neurological deficit scores and infarct volumes of the cerebral ischemia mice were measured. The apoptosis status of the neuronal cells was evaluated by TUNEL staining. In vitro, the ZNS treatment inhibited both the apoptosis of the neuronal cells and apoptosis-related protein expression (caspase-3, caspase-8, and calpain-1) induced by the oxygen-glucose deprivation. The anti-apoptosis effect of ZNS could occur through the blocking of reactive oxygen species. Moreover, ZNS treatment significantly ameliorated neurological deficits and reduced infarct volumes in the cerebral ischemia mice model. In this study, ZNS exerted neuroprotective effects by inhibition of apoptosis in neuronal cells in cerebral ischemia. Therefore, ZNS might be a promising therapy for cerebral ischemia.

Imaging and optogenetic modulation of vascular mural cells in the live brain.

Mural cells (smooth muscle cells and pericytes) are integral components of brain blood vessels that play important roles in vascular formation, blood-brain barrier maintenance, and regulation of regional cerebral blood flow (rCBF). These cells are implicated in conditions ranging from developmental vascular disorders to age-related neurodegenerative diseases. Here we present complementary tools for cell labeling with transgenic mice and organic dyes that allow high-resolution intravital imaging of the different mural cell subtypes. We also provide detailed methodologies for imaging of spontaneous and neural activity-evoked calcium transients in mural cells. In addition, we describe strategies for single- and two-photon optogenetics that allow manipulation of the activity of individual and small clusters of mural cells. Together with measurements of diameter and flow in individual brain microvessels, calcium imaging and optogenetics allow the investigation of pericyte and smooth muscle cell physiology and their role in regulating rCBF. We also demonstrate the utility of these tools to investigate mural cells in the context of Alzheimer's disease and cerebral ischemia mouse models. Thus, these methods can be used to reveal the functional and structural heterogeneity of mural cells in vivo, and allow detailed cellular studies of the normal function and pathophysiology of mural cells in a variety of disease models. The implementation of this protocol can take from several hours to days depending on the intended applications.

Resveratrol promoted the M2 polarization of microglia and reduced neuroinflammation after cerebral ischemia by inhibiting miR-155

Purpose: Neuroinflammation was demonstrated to play an important role in the brain injury induced by cerebral ischemia, which was mainly mediated by microglia. MicroRNA-155 (MiR-155) was reported to promote the M1 polarization of microglia and increase neuroinflammation. Resveratrol was identified to have the ability to promote the M2 polarization of microglia and reduce inflammation. Whether resveratrol can promote the M2 polarization of microglia and further inhibit neuroinflammation after cerebral ischemia, and its correlation with miR-155 is unclear. To clarify this, we conducted this study to explore the potential of resveratrol as an effective strategy to treat cerebral ischemia induced neuroinflammation.Materials and methods:The cerebral ischemia mouse model was first constructed by middle cerebral artery occlusion (MCAO). Then resveratrol was intraperitoneally injected at 0 h, 8 h and 18 h after cerebral ischemia. Subsequently, the relative expression of miR-155 and the signature genes of M1 and M2 microglia in injured brain were measured by RT-PCR, and the concentration of pro-inflammatory and anti-inflammatory cytokines were detected by ELISA. Further, the in vitro experiments were also conducted to explore the effect of resveratrol on the inflammation mediated by LPS activated BV2 microglia.Results: Results indicated that the relative expression of miR-155 in ischemia brain and activated BV2 microglia was elevated, while resveratrol reduced the expression of miR-155. Resveratrol promoted the M2 polarization of microglia and reduced neuroinflammation in injured brain and activated BV2 microglia.Conclusions: In conclusion, this research indicated that resveratrol promoted the M2 polarization of microglia and reduced neuroinflammation after cerebral ischemia by inhibiting miR-155.

Role of Metallothionein-1 and Metallothionein-2 in the Neuroprotective Mechanism of Sevoflurane Preconditioning in Mice.

This study investigated the protective effects and mechanisms of sevoflurane preconditioning (SPC) on neurons in ischemic mice. After SPC, mice were subjected to middle cerebral artery occlusion (MCAO). Cerebral infarction area, cell apoptosis, and metallothionein-1 (MT-1) and metallothionein-2 (MT-2) expressions in MCAO mice were analyzed. Mouse primary neurons were isolated and cultured to determine the location of metallothioneins (MTs) using immunofluorescence. Neurons transfected with MT-siRNA, exogenous MTs, or sh-MTF-1 were subjected to SPC and/or oxygen-glucose deprivation (OGD), and MT-1/MT-2 expression and neurotoxin release were assayed. Meanwhile, neurons were treated with the nitric oxide donor SNAP, degraded SNAP, or the peroxide initiator paraquat, and alterations in MT-1/MT-2 expression and neurotoxicity release were observed. SPC attenuated neuronal injury and apoptosis in MCAO mice. SPC could protect neurons against OGD injury and resulted in upregulated MT-1/MT-2 expression. MT-siRNA transfection led to the downregulated expression of MT-1/MT-2 and increased neurotoxicity, and the expression patterns of these neurons were different from those of neurons transfected with exogenous MTs. The knockdown of MTs could hinder the protective effect of SPC against OGD. Pretreatment with SNAP or paraquat could increase MTF-1 expression in the nucleus of neurons, protecting against OGD injury. The inhibition of nitric oxide and peroxide inhibited the protective role of SPC in OGD by downregulating MTF-1 expression. sh-MTF-1 transfection downregulated MT-1/MT-2 expression and enhanced neurotoxicity in neurons. SPC confers neuroprotection in focal cerebral ischemia mouse models by upregulating the expression of MT-1 and MT-2 by activating NO and peroxide and increasing MTF-1 expression in the nucleus.

Peripheral Administration of IL-13 Induces Anti-inflammatory Microglial/Macrophage Responses and Provides Neuroprotection in Ischemic Stroke

Neuroinflammation is strongly induced by cerebral ischemia. The early phase after the onset of ischemic stroke is characterized by acute neuronal injury, microglial activation, and subsequent infiltration of blood-derived inflammatory cells, including macrophages. Therefore, modulation of the microglial/macrophage responses has increasingly gained interest as a potential therapeutic approach for the ischemic stroke. In our study, we investigated the effects of peripherally administered interleukin 13 (IL-13) in a mouse model of permanent middle cerebral artery occlusion (pMCAo). Systemic administration of IL-13 immediately after the ischemic insult significantly reduced the lesion volume, alleviated the infiltration of CD45+ leukocytes, and promoted the microglia/macrophage alternative activation within the ischemic region, as determined by arginase 1 (Arg1) immunoreactivity at 3 days post-ischemia (dpi). Moreover, IL-13 enhanced the expression of M2a alternative activation markers Arg1 and Ym1 in the peri-ischemic (PI) area, as well as increased plasma IL-6 and IL-10 levels at 3 dpi. Furthermore, IL-13 treatment ameliorated gait disturbances at day 7 and 14 and sensorimotor deficits at day 14 post-ischemia, as analyzed by the CatWalk gait analysis system and adhesive removal test, respectively. Finally, IL-13 treatment decreased neuronal cell death in a coculture model of neuroinflammation with RAW 264.7 macrophages. Taken together, delivery of IL-13 enhances microglial/macrophage anti-inflammatory responses in vivo and in vitro, decreases ischemia-induced brain cell death, and improves sensory and motor functions in the pMCAo mouse model of cerebral ischemia.

Neuroprotective Effects of Diets Containing Olive Oil and DHA/EPA in a Mouse Model of Cerebral Ischemia.

Stroke is one of the leading causes of death worldwide and while there is increasing evidence that a Mediterranean diet might decrease the risk of a stroke, the effects of dietary fat composition on stroke outcomes have not been fully explored. We hypothesize that the brain damage provoked by a stroke would be different depending on the source of dietary fat. To test this, male C57BL/6J mice were fed for 4 weeks with a standard low-fat diet (LFD), a high-fat diet (HFD) rich in saturated fatty acids (HFD-SFA), an HFD containing monounsaturated fatty acids (MUFAs) from olive oil (HFD-OO), or an HFD containing MUFAs from olive oil plus polyunsaturated fatty acids (PUFAs) docosahexaenoic acid/eicosapentaenoic acid (DHA/EPA) (HFD-OO-ω3). These mice were then subjected to transient middle cerebral artery occlusion (tMCAo). Behavioural tests and histological analyses were performed 24 and/or 48 h after tMCAo in order to elucidate the impact of these diets with different fatty acid profiles on the ischemic lesion and on neurological functions. Mice fed with HFD-OO-ω3 displayed better histological outcomes after cerebral ischemia than mice that received an HFD-SFA or LFD. Furthermore, PUFA- and MUFA-enriched diets improved the motor function and neurological performance of ischemic mice relative to those fed with an LFD or HFD-SFA. These findings support the use of DHA/EPA-omega-3-fatty acid supplementation and olive oil as dietary source of MUFAs in order to reduce the damage and protect the brain when a stroke occurs.

Siponimod (BAF312) Treatment Reduces Brain Infiltration but Not Lesion Volume in Middle-Aged Mice in Experimental Stroke.

Background and Purpose- The contribution of neuroinflammation and, in particular, the infiltration of the brain by lymphocytes is increasingly recognized as a substantial pathophysiological mechanism after stroke. The interaction of lymphocytes with endothelial cells and platelets, termed thromboinflammation, fosters microvascular dysfunction and secondary infarct growth. Siponimod is an S1PR (sphingosine-1-phosphate receptor) modulator, which blocks the egress of lymphocytes from lymphoid organs and has demonstrated beneficial effects in multiple sclerosis treatment. We investigated the effect of treatment with siponimod on stroke outcome in a mouse model of cerebral ischemia. Methods- Transient middle cerebral artery occlusion was induced in middle-aged wild-type mice. Animals were either treated with siponimod (3 mg/kg; intraperitoneal) or vehicle for 6 days. Stroke outcome was assessed by magnetic resonance imaging (spleen volume: prestroke, day 3, and day 7; infarct volume: days 1, 3, and 7) and behavioral tests (prestroke, day 2, and day 6). Immune cells of the peripheral blood and brain-infiltrating cells ipsilateral and contralateral were analyzed by VETScan and by flow cytometry. Results- Siponimod significantly induced lymphopenia on day 7 after transient middle cerebral artery occlusion and reduced T-lymphocyte accumulation in the central nervous system. No effect was detected for lesion size. Conclusions- For siponimod administered at 3 mg/kg in transient middle cerebral artery occlusion mouse model, our findings do not provide preclinical evidence for the use of S1PR1/5 modulators as neuroprotectant in stroke therapy.

Abstract TP133: GSNO Pre- and Post-Conditioning Blocks Blood-Brain Barrier Disruption and Improves Endothelial Function in a Mouse Model of Cerebral Ischemia and Reperfusion

Background: Endothelial dysfunction is a major risk factor of stroke, and stroke injury itself causes endothelial dysfunction. Endothelial pathology following stroke blocks the mechanisms of neuroprotection and brain repair. Aberrant endothelial nitric oxide synthase (eNOS) activity and its consequent disturbed nitric oxide (NO) metabolome are mainly responsible for endothelial dysfunction. The major NO metabolite S-nitrosoglutathione (GSNO) maintains endothelial function via S-nitrosylation and invokes neuroprotection via activation of Akt. We investigated whether exogenous administration of GSNO mimics beneficial functions of eNOS-derived NO in protecting the neurovascular unit using wild-type and eNOS-deficient mouse models of stroke. Methods: Transient cerebral ischemic injury was induced by middle cerebral artery occlusion (MCAO) for 60 minutes in male adult wild-type and eNOS-null mice. GSNO (0.1 mg/kg bw, iv) and GSNO reductase inhibitor N6022 (5mg/kg bw, iv) were administered 30 minutes before MCAO in pre-injury studies and at reperfusion in post-injury studies. Brain infarctions, edema, and neurobehavioral functions were evaluated 24 h after reperfusion. Akt activity was determined by Western blot and immunohistochemistry. Results: eNOS-null mice had a higher degree (p<0.05) of injury than wild-type. Pre-MCAO treatment with GSNO significantly reduced infarct volume, decreased edema and improved neurological score, and increased tactile strength in both wild-type and eNOS-null mice compared with untreated, injured animals. Post-ischemic injury GSNO treatment also significantly reduced infarct volume and improved neurological score. N6022 also had effects similar to exogenously administered GSNO. Conclusions: Reduced brain infarction and edema, and improved neurobehavioral function by pre-injury exogenous/endogenous GSNO treatment indicate that GSNO-mediated pre-conditioning protects against IR, likely by mimicking the eNOS-derived NO-dependent functions involving Akt activity. Neurovascular protection by GSNO and N6022 in both pre- and post-ischemic injury events support GSNO and/or N6022 as a promising candidate to be evaluated for prevention and protection of humans from stroke injury.

Amelioration of ischaemia reperfusion-induced cerebral injury in mice by liposomes containing Allium cepa fraction administered intranasally

Neuroprotection in ischaemic stroke prevents neuronal injury and subsequent death. Our earlier work revealed the neuroprotective effect of ethylacetate fraction (EF) obtained from Allium cepa outer scales in a mouse model of cerebral ischaemia–reperfusion (I–R) injury. The present study was designed to develop and optimize a liposomal delivery system for EF, along with its biological assessment. Thin film hydration method was used for the preparation of liposomal formulation. The prepared liposomes were optimized with respect to particle size, size distribution and encapsulation efficiency (EE) and characterised on the basis of zeta potential, in vitro release, morphology (TEM) and physical stability. The biological activity of the optimized liposomal formulation (EF-L; 8.5 mg/kg, intra-nasal) was evaluated after induction of cerebral injury in the experimental animals. Neuroprotective effects were assessed in terms of improvement of cognitive/sensorimotor functions and reduction of cerebral infarct size and brain oxidative stress. EF-L (particle size of 204.93 ± 7.96 nm; EE of 88.02 ± 2.09%; zeta potential of –20.8 ± 1.24 mV) showed controlled release pattern; spherical shape and were physically stable for 60 days at 4 °C. Intra-nasal administration of EF-L produced significant neuroprotection in mice at 1/10th the oral dose. Thus, EF-L may be developed as a neuroprotective formulation.

Abstract P332: Hesperidin in Citrus Fruit Juice Plays a Role in Preventing Cognitive Impairment Induced by Ischemic Brain Damage

Recently we reported that drinking Citrus iyo juice (CI) inhibited more effectively vascular remodeling in the inflammation-induced vascular injury mouse model than Citrus unshiu juice (CU). These results led us to explore the possibility that citrus fruits juice drinking could attenuate cognitive decline in transient cerebral ischemia mouse model, focusing on the effects of flavanone, hesperidin, which is more abundantly contained in CI compared with CU and has antioxidant activity. Eight-week-old male C57BL/6 mice were administrated 10 % CI or CU in drinking water or 100 mg/kg/day hesperidin orally by gavage. Two weeks after administration, brain ischemia was induced by bilateral common carotid artery occlusion (BCCAO) for 18 minutes. The Y maze task was performed 2 weeks after BCCAO operation. After cognitive task, cerebral blood flow (CBF) was measured by a laser speckle flowmetry. Morphological changes in the hippocampus were examined. Administration of CI, CU or hesperidin did not influence systolic blood pressure, body weight and brain weight. Cognitive function was significantly impaired (sham, 71% (16 of 23) vs BCCAO, 55% (14 of 27) in Y maze) with the increase in superoxide anion production after BCCAO. The cognitive impairment was more effectively attenuated by the administration of CI than CU (CI, 66% (13 of 23); CU, 61% (17 of 27)) with the significant increase in CBF. Interestingly, we also observed that the treatment with hesperidin significantly prevented cognitive decline (67% (13 of 21)) after BCCAO. The increase in superoxide anion production 24 hours after BCCAO (expressed as fold-increase compared to sham) was attenuated by CI or hesperidin, not by CU (BCCAO, 4.0; CI, 1.2; hesperidin, 1.1; CU, 3.0). The fold-increase in TNF-α mRNA in the hippocampus 24 hours after BCCAO was prevented by hesperidin (BCCAO 5.0; hesperidin, 2.8). Cell number in CA1 region of hippocampus decreased in BCCAO-operated mice. Hesperidin treatment attenuated this decrease (sham, 163±5; BCCAO, 136±4; hesperidin, 154±4). These results suggest that the intake of hesperidin in citrus fruits juice should prevent cognitive decline after brain ischemia at least in part due to reduction of oxidative stress, inflammatory cytokine and an increase in CBF.

Importance of Angiogenin and Endothelial Progenitor Cells After Rehabilitation Both in Ischemic Stroke Patients and in a Mouse Model of Cerebral Ischemia.

Background: Rehabilitation therapy is the only available treatment for stroke survivors presenting neurological deficits; however, the underlying molecules and mechanisms associated with functional/motor improvement during rehabilitation are poorly understood.Objective: Our aim is to study the modulation of angiogenin and endothelial progenitor cells (EPCs) as repair-associated factors in a cohort of stroke patients and mouse models of rehabilitation after cerebral ischemia.Methods: The clinical study included 18 ischemic strokes admitted to an intensive rehabilitation therapy (IRT) unit, 18 non-ischemic controls and brain samples from three deceased patients. Angiogenin and EPCs were measured in blood obtained before and up to 6 months after IRT together with an extensive evaluation of the motor/functional status. In parallel, C57BL/6 mice underwent middle cerebral artery occlusion, and the pasta matrix reaching-task or treadmill exercises were used as rehabilitation models. Angiogenin RNA expression was measured after 2 or 12 days of treatment together with cell counts from EPCs cultures.Results: Brain angiogenin was identified in both human and mouse tissue, whereas serum levels increased after 1 month of IRT in association with motor/functional improvement. EPC populations were increased after stroke and remained elevated during follow-up after IRT. The mouse model of rehabilitation by the task-specific pasta matrix exercise increased the number of EPCs at 2 days and increased angiogenin expression after 12 days of rehabilitation.Conclusions: Angiogenin and EPCs are modulated by rehabilitation after cerebral ischemia, suggesting that both angiogenin and EPCs could serve as biomarkers of improvement during rehabilitation or future therapeutic targets.

Nociceptin/Orphanin FQ (N/OFQ) is neuroprotective in an ex vivo mouse model of cerebral ischaemia

Cerebral ischemia is a leading cause of death and disability in the UK with limited treatment options.1 The Nociceptin/Orphanin FQ (N/OFQ) receptor, NOP, and its endogenous ligand N/OFQ, are non-classical members of the opioid receptor family.2 Central activation of NOP results in general inhibition of neurotransmitter release, in particular glutamate,2 which contributes to cell death following cerebral ischemia. Activation of NOP by N/OFQ or other small molecule agonists would therefore be predicted to be neuroprotective. Here, we have assessed NOP expression in hippocampal and subcortical regions of the mouse brain by PCR and assessed the effects of activation in an ex vivo model of cerebral ischaemia. We used C57/BL6j neonatal mice, aged 6-9 days. For NOP expression studies, mRNA was extracted from hippocampal and subcortical regions using a tri-reagent methodology, and cDNA formed via reverse transcription. cDNA was probed for NOP relative to the house-keeper gene Beta-actin, using TaqMan probes via QPCR.3 To investigate the neuroprotective properties of NOP activation, an ex vivo model of cerebral ischemia was used where cortical brain slices were exposed to oxygen and glucose deprivation (OGD) to mimic ischemic damage, in the presence/absence of N/OFQ. Brain slices were immersed in artificial cerebral spinal fluid (aCSF) to recover from slicing prior to OGD exposure in the presence/absence of N/OFQ (1μM) for 40 minutes. Slices were incubated with 2% 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) to visualise infarcted tissue.4 Area of cell death including hippocampus and sub-cortex but excluding the outer cortex was measured using Imagej software 1.51. NOP receptor mRNA was expressed in both tissues with deltaCt, relative to Beta-actin of 10.52 ∗ 0.25 and 9.62 ∗ 0.65 in hippocampus and sub-cortex respectively (mean + SEM from 9 mice across 3 litters). Subcortical tissue expressed 1.87 fold more NOP than hippocampal tissue. In an acute model of cerebral ischaemia OGD increased the area of cell death from 25.92 ± 4.09% (in normoxic condition) to 66.02 ± 9.99% (Data are mean ± SEM from 12 neonatal mice, across 4 litters; p<0.01). Treatment with N/OFQ significantly (p<0.01) reduced this ischaemic damage to 39.47 ± 7.82%. The vehicle for N/OFQ, dH2O, was ineffective; 72.09 ± 11.87% compared to OGD control. These data show a neuroprotective role for NOP activation and suggest this pathway as a potential target in acute ischaemic stroke. Further studies, with more metabolically stable NOP agonists are underway and a series of in vivo experiments planned. BF is funded by a British Journal of Anaesthesia/Royal College of Anaesthetists PhD studentship. 1.Gibson, C.L and Bath, P.M. J. Cereb. Blood Flow Metab. 2016; 36: 487–491.2.Lambert, D.G. Nat. Rev. Drug Discov. 2008; 7: 694–710.3.McDonald, J., Leonard, A.D., Serrano-Gomez et al. Br. J. Anaesth. 2010; 104: 698–704.4.Zille, M., Farr, T.D., Przesdzing, I. et al . J. Cereb. Blood Flow Metab. 2012; 32: 213–231.

Noninvasive detection of acute cerebral hypoxia and subsequent matrix-metalloproteinase activity in a mouse model of cerebral ischemia using multispectral-optoacoustic-tomography.

Oxygen metabolism and matrix metalloproteinases (MMPs) play important roles in the pathophysiology of cerebral ischemia. Using multispectral optoacoustic tomography (MSOT) imaging, we visualized in vivo changes in cerebral tissue oxygenation during 1h of transient middle cerebral artery occlusion (tMCAO) and at 48h after reperfusion together with MMP activity using an MMP-activatable probe. The deoxyhemoglobin, oxyhemoglobin, and MMP signals were coregistered with structural magnetic resonance imaging data. The ipsi-/contralateral ratio of tissue oxygen saturation ([Formula: see text]) was significantly reduced during 1h of tMCAO and recovered after 48h of reperfusion in tMCAO compared with sham-operated mice ([Formula: see text] to 10 per group). A higher ipsi-/contralateral MMP signal ratio was detected at 48h after reperfusion in the lesioned brain regions of tMCAO compared with the sham-operated animal ([Formula: see text] to 6 per group). Ex vivo near-infrared fluorescence imaging of MMP signal in brain slices was used to validate in vivo MSOT measurements. In conclusion, noninvasive MSOT imaging can provide visualization of hemodynamic alterations and MMP activity in a mouse model of cerebral ischemia.