Agmatine attenuates brain edema through reducing the expression of aquaporin-1 after cerebral ischemia

Brain edema is frequently shown after cerebral ischemia. It is an expansion of brain volume because of increasing water content in brain. It causes to increase mortality after stroke. Agmatine, formed by the decarboxylation of -arginine by arginine decarboxylase, has been shown to be neuroprotective in trauma and ischemia models. The purpose of this study was to investigate the effect of agmatine for brain edema in ischemic brain damage and to evaluate the expression of aquaporins (AQPs). Results showed that agmatine significantly reduced brain swelling volume 22 h after 2 h middle cerebral artery occlusion in mice. Water content in brain tissue was clearly decreased 24 h after ischemic injury by agmatine treatment. Blood–brain barrier (BBB) disruption was diminished with agmatine than without. The expressions of AQPs-1 and -9 were well correlated with brain edema as water channels, were significantly decreased by agmatine treatment. It can thus be suggested that agmatine could attenuate brain edema by limitting BBB disruption and blocking the accumulation of brain water content through lessening the expression of AQP-1 after cerebral ischemia.     

Tight junctions in the blood–brain barrier promote edema formation and infarct size in stroke – Ambivalent effects of sealing proteins

The outcome of stroke is greatly influenced by the state of the blood–brain barrier (BBB). The BBB endothelium is sealed paracellularly by tight junction (TJ) proteins, i.e., claudins (Cldns) and the redox regulator occludin. Functions of Cldn3 and occludin at the BBB are largely unknown, particularly after stroke. We address the effects of Cldn3 deficiency and stress factors on the BBB and its TJs. Cldn3 tightened the BBB for small molecules and ions, limited endothelial endocytosis, strengthened the TJ structure and controlled Cldn1 expression. After middle cerebral artery occlusion (MCAO) and 3-h reperfusion or hypoxia of isolated brain capillaries, Cldn1, Cldn3 and occludin were downregulated. In Cldn3 knockout mice (C3KO), the reduction in Cldn1 was even greater and TJ ultrastructure was impaired; 48 h after MCAO of wt mice, infarct volumes were enlarged and edema developed, but endothelial TJs were preserved. In contrast, junctional localization of Cldn5 and occludin, TJ density, swelling and infarction size were reduced in affected brain areas of C3KO. Taken together, Cldn3 and occludin protect TJs in stroke, and this keeps the BBB intact. However, functional Cldn3, Cldn3-regulated TJ proteins and occludin promote edema and infarction, which suggests that TJ modulation could improve the outcome of stroke.     

Increased cerebral protein ISGylation after focal ischemia is neuroprotective

Addition of a small peptide called ISG15 is known as ISGylation, which is an ubiquitin (ub)-like posttranslational modification. We currently show that focal ischemia induced by transient middle cerebral artery occlusion (MCAO) in adult mice significantly induces cortical protein ISGylation between 6 and 24 hours reperfusion. With two-dimensional western blotting, 45 proteins were observed to be significantly increased in ISGylation (by 1.8- to 9.7-fold) after focal ischemia compared with sham control. Immunochemistry showed that ISGylated proteins are localized in neurons within the ipsilateral striatum and in astroglia within the peri-infarct cortex of ischemic mice. When subjected to transient MCAO, ISG15^−/− mice showed increased mortality, exacerbated infarction, and worsened neurologic recovery than did wild-type controls. In addition, mice lacking UBE1L (ub-activating enzyme E1-like protein, the first enzyme of the ISGylation cycle) also showed bigger infarcts when subjected to transient MCAO. Regional cerebral blood flow or other physiologic parameters were not significantly different in both knockouts compared with wild-type controls. These studies indicate that increased protein ISGylation might be an endogenous neuroprotective adaptation to minimize poststroke brain damage.     

Role of CCL2 (MCP-1) in traumatic brain injury (TBI): evidence from severe TBI patients and CCL2?/? mice

Cerebral inflammation involves molecular cascades contributing to progressive damage after traumatic brain injury (TBI). The chemokine CC ligand-2 (CCL2) (formerly monocyte chemoattractant protein-1, MCP-1) is implicated in macrophage recruitment into damaged parenchyma after TBI. This study analyzed the presence of CCL2 in human TBI, and further investigated the role of CCL2 in physiological and cellular mechanisms of secondary brain damage after TBI. Sustained elevation of CCL2 was detected in the cerebrospinal fluid (CSF) of severe TBI patients for 10 days after trauma, and in cortical homogenates of C57Bl/6 mice, peaking at 4 to 12 h after closed head injury (CHI). Neurological outcome, lesion volume, macrophage/microglia infiltration, astrogliosis, and the cerebral cytokine network were thus examined in CCL2-deficient (−/−) mice subjected to CHI. We found that CCL2−/− mice showed altered production of multiple cytokines acutely (2 to 24 h); however, this did not affect lesion size or cell death within the first week after CHI. In contrast, by 2 and 4 weeks, a delayed reduction in lesion volume, macrophage accumulation, and astrogliosis were observed in the injured cortex and ipsilateral thalamus of CCL2−/− mice, corresponding to improved functional recovery as compared with wild-type mice after CHI. Our findings confirm the significant role of CCL2 in mediating post-traumatic secondary brain damage.     

The role of bradykinin B1 and B2 receptors for secondary brain damage after traumatic brain injury in mice

Inflammatory mechanisms are known to contribute to the pathophysiology of traumatic brain injury (TBI). Since bradykinin is one of the first mediators activated during inflammation, we investigated the role of bradykinin and its receptors in posttraumatic secondary brain damage. We subjected wild-type (WT), B₁-, and B₂-receptor-knockout mice to controlled cortical impact (CCI) and analyzed tissue bradykinin as well as kinin receptor mRNA and protein expression up to 48 h thereafter. Brain edema, contusion volume, and functional outcome were assessed 24 h and 7 days after CCI. Tissue bradykinin was maximally increased 2 h after trauma (P<0.01 versus sham). Kinin B₁ receptor mRNA was upregulated up to four-fold 24 h after CCI. Immunohistochemistry showed that B₁ and B₂ receptors were expressed in the brain and were significantly upregulated in the traumatic penumbra 1 to 24 h after CCI. B₂R^−/− mice had significantly less brain edema (−51% versus WT, 24 h; P<0.001), smaller contusion volumes (∼50% versus WT 24 h and 7 d after CCI; P<0.05), and better functional outcome 7 days after TBI as compared with WT mice (P<0.05). The present results show that bradykinin and its B₂ receptors play a causal role for brain edema formation and cell death after TBI.     

Mast cells promote blood brain barrier breakdown and neutrophil infiltration in a mouse model of focal cerebral ischemia

Blood brain barrier (BBB) breakdown and neuroinflammation are key events in ischemic stroke morbidity and mortality. The present study investigated the effects of mast cell deficiency and stabilization on BBB breakdown and neutrophil infiltration in mice after transient middle cerebral artery occlusion (tMCAo). Adult male C57BL6/J wild type (WT) and mast cell-deficient (C57BL6/J Kit^Wsh/Wsh (Wsh)) mice underwent tMCAo and BBB breakdown, brain edema and neutrophil infiltration were examined after 4 hours of reperfusion. Blood brain barrier breakdown, brain edema, and neutrophil infiltration were significantly reduced in Wsh versus WT mice (P<0.05). These results were reproduced pharmacologically using mast cell stabilizer, cromoglycate. Wild-type mice administered cromoglycate intraventricularly exhibited reduced BBB breakdown, brain edema, and neutrophil infiltration versus vehicle (P<0.05). There was no effect of cromoglycate versus vehicle in Wsh mice, validating specificity of cromoglycate on brain mast cells. Proteomic analysis in Wsh versus WT indicated that effects may be via expression of endoglin, endothelin-1, and matrix metalloproteinase-9. Using an in vivo model of mast cell deficiency, this is the first study showing that mast cells promote BBB breakdown in focal ischemia in mice, and opens up future opportunities for using mice to identify specific mechanisms of mast cell-related BBB injury.     

Stroke-induced brain parenchymal injury drives blood–brain barrier early leakage kinetics: a combined in vivo/in vitro study

The disappointing clinical outcomes of neuroprotectants challenge the relevance of preclinical stroke models and data in defining early cerebrovascular events as potential therapeutic targets. The kinetics of blood–brain barrier (BBB) leakage after reperfusion and the link with parenchymal lesion remain debated. By using in vivo and in vitro approaches, we conducted a kinetic analysis of BBB dysfunction during early reperfusion. After 60 minutes of middle cerebral artery occlusion followed by reperfusion times up to 24 hours in mice, a non-invasive magnetic resonance imaging method, through an original sequence of diffusion-weighted imaging, determined brain water mobility in microvascular compartments (D*) apart from parenchymal compartments (apparent diffusion coefficient). An increase in D* found at 4 hours post reperfusion concurred with the onset of both Evans blue/Dextran extravasations and in vitro BBB opening under oxygen-glucose deprivation and reoxygenation (R). The BBB leakage coincided with an emerging cell death in brain tissue as well as in activated glial cells in vitro. The co-culture of BBB endothelial and glial cells evidenced a recovery of endothelium tightness when glial cells were absent or non-injured during R. Preserving the ischemic brain parenchymal cells within 4 hours of reperfusion may improve therapeutic strategies for cerebrovascular protection against stroke.     

Lipocalin-2 deficiency attenuates neuroinflammation and brain injury after transient middle cerebral artery occlusion in mice

Lipocalin-2 (LCN2) is a secreted protein of the lipocalin family, but little is known about the expression or the role of LCN2 in the central nervous system. Here, we investigated the role of LCN2 in ischemic stroke using a rodent model of transient cerebral ischemia. Lipocalin-2 expression was highly induced in the ischemic brain and peaked at 24 hours after reperfusion. After transient middle cerebral artery occlusion, LCN2 was predominantly expressed in astrocytes and endothelial cells, whereas its receptor (24p3R) was mainly detected in neurons, astrocytes, and endothelial cells. Brain infarct volumes, neurologic scores, blood–brain barrier (BBB) permeabilities, glial activation, and inflammatory mediator expression were significantly lower in LCN2-deficient mice than in wild-type animals. Lipocalin-2 deficiency also attenuated glial neurotoxicity in astrocyte/neuron cocultures after oxygen-glucose deprivation. Our results indicate LCN2 has a critical role in brain injury after ischemia/reperfusion, and that LCN2 may contribute to neuronal cell death in the ischemic brain by promoting neurotoxic glial activation, neuroinflammation, and BBB disruption.     

Platelet-leukocyte-endothelial cell interactions after middle cerebral artery occlusion and reperfusion.

The adhesion of both leukocytes and platelets to microvascular endothelial cells has been implicated in the pathogenesis of ischemia/reperfusion (I/R) injury in several vascular beds. The objectives of this study were to (1) assess the platelet-leukocyte-endothelial cell interactions induced in the cerebral microvasculature by middle cerebral artery occlusion (MCAO)/reperfusion, and (2) define the molecular determinants of the prothrombogenic and inflammatory responses in this model of focal I/R. MCAO was induced for 1 hour in wild-type (WT) mice, WT mice treated with a monoclonal antibody (mAb) to either P-selectin or GPIIb/IIIa, and in P-selectin-/-(P-sel-/-) chimeras. Isolated platelets labeled with carboxyfluorescein diacetate succinimidyl ester (CFDASE) were administered intravenously and observed with intravital fluorescence microscopy. Leukocytes were observed after intravenous injection of rhodamine 6G. One hour of MCAO followed by 1 hour of reperfusion resulted in the rolling and adhesion of leukocytes in venules, and after 4 hours of reperfusion, the adhesion of both leukocytes and platelets was detected. Although both the P-selectin and GPIIb/IIIa mAbs significantly reduced the adhesion of leukocytes and platelets at 4 hours of reperfusion, the antiadhesive effects of the P-selectin mAb were much greater. The leukocyte and platelet adhesion responses were significantly attenuated in both P-sel-/- --> WT and WT --> P-sel-/- bone marrow chimeras, compared with WT --> WT chimeras. Neutropenia, induced by antineutrophil serum treatment, also reduced the recruitment of leukocytes and platelets after cerebral I/R. These findings implicate a major role for both platelet-associated and endothelial cell-associated P-selectin, as well as neutrophils in the inflammatory and prothrombogenic responses in the microcirculation after focal cerebral I/R.     

Cannabinoid CB(2) receptor activation decreases cerebral infarction in a mouse focal ischemia/reperfusion model.

Cannabinoid CB(2) Receptor (CB(2)) activation has been shown to have immunomodulatory properties without psychotropic effects. The hypothesis of this study is that selective CB(2) agonist treatment can attenuate cerebral ischemia/reperfusion injury. Selective CB(2) agonists (O-3853, O-1966) were administered intravenously 1 h before transient middle cerebral artery occlusion (MCAO) or 10 mins after reperfusion in male mice. Leukocyte/endothelial interactions were evaluated before MCAO, 1 h after MCAO, and 24 h after MCAO via a closed cranial window. Cerebral infarct volume and motor function were determined 24 h after MCAO. Administration of the selective CB(2) agonists significantly decreased cerebral infarction (30%) and improved motor function (P<0.05) after 1 h MCAO followed by 23 h reperfusion in mice. Transient ischemia in untreated animals was associated with a significant increase in leukocyte rolling and adhesion on both venules and arterioles (P<0.05), whereas the enhanced rolling and adhesion were attenuated by both selective CB(2) agonists administered either at 1 h before or after MCAO (P<0.05). CB(2) activation is associated with a reduction in white blood cell rolling and adhesion along cerebral vascular endothelial cells, a reduction in infarct size, and improved motor function after transient focal ischemia.     

Association between pre-treatment perfusion profile and cerebral edema after reperfusion therapies in ischemic stroke

The relationship between reperfusion and edema is unclear, with experimental and clinical data yielding conflicting results. We investigated whether the extent of salvageable and irreversibly-injured tissue at baseline influenced the effect of therapeutic reperfusion on cerebral edema. In a pooled analysis of 415 patients with anterior circulation large vessel occlusion from the Tenecteplase-versus-Alteplase-before-Endovascular-Therapy-for-Ischemic-Stroke (EXTEND-IA TNK) part 1 and 2 trials, associations between core and mismatch volume on pre-treatment CT-Perfusion with cerebral edema at 24-hours, and their interactions with reperfusion were tested. Core volume was associated with increased edema (p < 0.001) with no significant interaction with reperfusion (p = 0.82). In comparison, a significant interaction between reperfusion and mismatch volume (p = 0.03) was observed: Mismatch volume was associated with increased edema in the absence of reperfusion (p = 0.009) but not with reperfusion (p = 0.27). When mismatch volume was dichotomized at the median (102 ml), reperfusion was associated with reduced edema in patients with large mismatch volume (p < 0.001) but not with smaller mismatch volume (p = 0.35). The effect of reperfusion on edema may be variable and dependent on the physiological state of the cerebral tissue. In patients with small to moderate ischemic core volume, the benefit of reperfusion in reducing edema is related to penumbral salvage.     

Rivaroxaban does not increase hemorrhage after thrombolysis in experimental ischemic stroke

The management of acute ischemic stroke during anticoagulation with a novel oral anticoagulant (NOAC) is challenging because intravenous thrombolysis is contraindicated because of a putative increased risk of intracerebral hemorrhagic complications. We examined the risk of secondary postischemic hemorrhage after thrombolysis in rodents pretreated with rivaroxaban or warfarin. Mice were pretreated with either rivaroxaban (30 mg/kg), warfarin (target international normalized ratio 2 to 3) or vehicle. After 2 or 3 hours, middle cerebral artery occlusion (MCAO), mice received 9 mg/kg recombinant tissue plasminogen activator. Twenty-four hours after MCAO, secondary hemorrhage was quantified using a macroscopic hemorrhage score and hemoglobin spectrophotometry. Blood–brain barrier (BBB) permeability was measured by Evans Blue spectrofluorometry. To increase the validity of our findings, experiments were also performed using a thromboembolic model in anticoagulated rats. Infarct size did not differ among groups. Pretreatment with warfarin led to significantly more secondary hemorrhage compared with rivaroxaban and nonanticoagulated controls after 2- and 3-hour ischemia in mice as well as in rats. Blood–brain barrier permeability was significantly higher in the warfarin group compared with rivaroxaban and control. Thus, rivaroxaban in contrast to warfarin does not increase secondary hemorrhage after thrombolysis in experimental cerebral ischemia. Less effects of rivaroxaban on postischemic BBB permeability may account for this difference.     

Human urinary kallidinogenase suppresses cerebral inflammation in experimental stroke and downregulates nuclear factor-��B

The purpose of this study is to investigate the possible mechanism and the neuroprotective effect of human urinary kallidinogenase (HUK) in cerebral ischemia. The mouse middle cerebral artery occlusion (MCAO) model was used. Mice were treated with HUK (20 PNAU/g per day, intravenous) or saline as control, from the beginning of reperfusion to 72 h. Neurological deficits, infarct size, and BWC were measured at 6, 24, 48, and 72 h after MCAO, respectively. Pathological changes of brain were observed by TUNEL assay. Inflammatory factors were measured by real-time PCR and western blotting. Activation of MAPKs, Akt, and nuclear factor-κB (NF-κB) was detected by western blotting. Our results indicated that HUK significantly improved neurofunction, decreased infarct size, and suppressed edema, as well as inflammatory mediators as compared with the vehicle group. Furthermore, HUK inhibited the NF-κB pathway and activated the MAPK/ERK pathway in this neuroprotection.     

Thrombopoietin protects the brain and improves sensorimotor functions: reduction of stroke-induced MMP-9 upregulation and blood–brain barrier injury

This study was conducted to determine the protective efficacy and mechanisms of thrombopoietin (TPO) intervention in experimental focal stroke. Male rats underwent 2 hours of left middle cerebral artery occlusion (MCAO) followed by 22 hours of reperfusion. Vehicle or TPO (0.03 to 1.00 μg/kg) was administered intravenously immediately after reperfusion. Brain infarct and swelling, neurologic deficits, matrix metalloproteinase-9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1), TPO and c-Mpl (TPO receptor) mRNA, MMP-9 enzyme activity and protein expression, and the integrity of the blood–brain barrier (BBB) were subsequently measured. MCAO reperfusion produced a large infarct and swelling after stroke. Thrombopoietin significantly reduced these in a dose-dependent manner. The most effective TPO dose, 0.1 μg/kg, when administrated immediately or 2 hours after reperfusion, significantly reduced infarct and swelling and ameliorated neurologic deficits after stroke. Stroke-induced increases in cortical MMP-9 mRNA, enzyme activity and protein expression, TIMP-1 mRNA, and Evans blue extravasation were reduced by TPO intervention. Thrombopoietin did not alter cortical TPO or c-Mpl mRNA expression, blood pressure, heart rate, blood hematocrit, or platelets. This is the first demonstration of TPO''s efficacy in reducing ischemic brain injury and improving functional outcome, partly by inhibiting the stroke-induced increase in MMP-9 and the early, negative effects on the BBB.     

Knockout of silent information regulator 2 (SIRT2) preserves neurological function after experimental stroke in mice

Sirtuin-2 (Sirt2) is a member of the NAD⁺-dependent protein deacetylase family. Various members of the sirtuin class have been found to be involved in processes related to longevity, regulation of inflammation, and neuroprotection. Induction of Sirt2 mRNA was found in the whole hemisphere after experimental stroke in a recent screening approach. Moreover, Sirt2 protein is highly expressed in myelin-rich brain regions after stroke. To examine the effects of Sirt2 on ischemic stroke, we induced transient focal cerebral ischemia in adult male Sirt2-knockout and wild-type mice. Two stroke models with different occlusion times were applied: a severe ischemia (45 minutes of middle cerebral artery occlusion (MCAO)) and a mild one (15 minutes of MCAO), which was used to focus on subcortical infarcts. Neurological deficit was determined at 48 hours after 45 minutes of MCAO, and up to 7 days after induction of 15 minutes of cerebral ischemia. In contrast to recent data on Sirt1, Sirt2^−/− mice showed less neurological deficits in both models of experimental stroke, with the strongest manifestation after 48 hours of reperfusion. However, we did not observe a significant difference of stroke volumes or inflammatory cell count between Sirt2-deficient and wild-type mice. Thus we postulate that Sirt2 mediates myelin-dependent neuronal dysfunction during the early phase after ischemic stroke.     

ADAMTS13 gene deletion enhances plasma high-mobility group box1 elevation and neuroinflammation in brain ischemia–reperfusion injury

Highly adhesive glycoprotein von Willebrand factor (VWF) multimer induces platelet aggregation and leukocyte tethering or extravasation on the injured vascular wall, contributing to microvascular plugging and inflammation in brain ischemia–reperfusion. A disintegrin and metalloproteinase with thrombospondin type-1 motifs 13 (ADAMTS13) cleaves the VWF multimer strand and reduces its prothrombotic and proinflammatory functions. Although ADAMTS13 deficiency is known to amplify post-ischemic cerebral hypoperfusion, there is no report available on the effect of ADAMTS13 on inflammation after brain ischemia. We investigated if ADAMTS13 deficiency intensifies the increase of extracellular HMGB1, a hallmark of post-stroke inflammation, and exacerbates brain injury after ischemia–reperfusion. ADAMTS13 gene knockout (KO) and wild-type (WT) mice were subjected to 30-min middle cerebral artery occlusion (MCAO) and 23.5-h reperfusion under continuous monitoring of regional cerebral blood flow (rCBF). The infarct volume, plasma high-mobility group box1 (HMGB1) level, and immunoreactivity of the ischemic cerebral cortical tissue (double immunofluorescent labeling) against HMGB1/NeuN (neuron-specific nuclear protein) or HMGB1/MPO (myeloperoxidase) were estimated 24 h after MCAO. ADAMTS13KO mice had larger brain infarcts compared with WT 24 h after MCAO (p < 0.05). The rCBF during reperfusion decreased more in ADAMTS13KO mice. The plasma HMGB1 increased more in ADAMTS13KO mice than in WT after ischemia–reperfusion (p < 0.05). Brain ischemia induced more prominent activation of inflammatory cells co-expressing HMGB1 and MPO and more marked neuronal death in the cortical ischemic penumbra of ADAMTS13KO mice. ADAMTS13 deficiency may enhance systemic and brain inflammation associated with HMGB1 neurotoxicity, and aggravate brain damage in mice after brief focal ischemia. We hypothesize that ADAMTS13 protects brain from ischemia–reperfusion injury by regulating VWF-dependent inflammation as well as microvascular plugging.     

Cyclooxygenase-1 and -2 differentially modulate lipopolysaccharide-induced blood�Cbrain barrier disruption through matrix metalloproteinase activity

Cyclooxygenases (COX) -1 and -2 are key regulators of innate immune responses. We recently demonstrated that the expression of proinflammatory cytokines and chemokines is reduced in COX-1 null (^−/−), and increased in COX-2^−/− mice compared with their respective wild type controls during lipopolysaccharide (LPS)-induced innate immune activation. As chemokines are involved in leukocyte recruitment into the inflamed brain, we hypothesized that COX-1 and COX-2 deletion will differentially modulate blood–brain barrier (BBB) permeability in response to LPS. In the present study, using quantitative magnetic resonance imaging, we found that LPS-induced BBB disruption was exacerbated in COX-2^−/− versus COX-2^+/+ mice. In the hippocampus and cortex of LPS-treated mice, matrix metalloproteinase (MMP)-3 activity was significantly decreased in COX-1^−/− mice, whereas in COX-2^−/− mice the activity of both MMP-9 and MMP-3, known to mediate BBB breakdown, was increased. Brain mRNA expression of the leukocyte attracting chemokine Cxcl10, the intercellular interaction molecule Icam-1, the pan-leukocyte marker Cd45 was increased in COX-2^−/− versus COX-2^+/+ mice, whereas Cxcl10 and Cd45 mRNA expression was decreased in COX-1^−/− versus COX-1^+/+ mice after LPS. Altogether, these results indicate that COX-2 activity modulates MMP-9 and-3 activities and is necessary to maintain BBB integrity during toll-like receptor 4-dependent innate immune activation.     

A novel PET protocol for visualization of breast cancer resistance protein function at the blood–brain barrier

Breast cancer resistance protein (BCRP) is the most abundant multidrug efflux transporter at the human blood–brain barrier (BBB), restricting brain distribution of various drugs. In this study, we developed a positron emission tomography (PET) protocol to visualize Bcrp function at the murine BBB, based on the dual P-glycoprotein (P-gp)/Bcrp substrate radiotracer [¹¹C]tariquidar in combination with the Bcrp inhibitor Ko143. To eliminate the contribution of P-gp efflux to [¹¹C]tariquidar brain distribution, we studied mice in which P-gp was genetically knocked out (Mdr1a/b^(−/−) mice) or chemically knocked out by pretreatment with cold tariquidar. We found that [¹¹C]tariquidar brain uptake increased dose dependently after administration of escalating doses of Ko143, both in Mdr1a/b^(−/−) mice and in tariquidar pretreated wild-type mice. After 15 mg/kg Ko143, the maximum increase in [¹¹C]tariquidar brain uptake relative to baseline scans was 6.3-fold in Mdr1a/b^(−/−) mice with a half-maximum effect dose of 4.98 mg/kg and 3.6-fold in tariquidar (8 mg/kg) pretreated wild-type mice, suggesting that the presented protocol is sensitive to visualize a range of different functional Bcrp activities at the murine BBB. We expect that this protocol can be translated to the clinic, because tariquidar can be safely administered to humans at doses that completely inhibit cerebral P-gp.     

Overexpression of heat shock protein 27 reduces cortical damage after cerebral ischemia

Heat shock protein 27 (HSP27) has a major role in mediating survival responses to a range of central nervous system insults, functioning as a protein chaperone, an antioxidant, and through inhibition of cell death pathways. We have used transgenic mice overexpressing HSP27 (HSP27tg) to examine the role of HSP27 in cerebral ischemia, using model of permanent middle cerebral artery occlusion (MCAO). Infarct size was evaluated using multislice T₂-weighted anatomical magnetic resonance imaging (MRI) after 24 h. A significant reduction of 30% in infarct size was detected in HSP27tg animals compared with wild-type (WT) littermates. To gain some insight into the mechanisms contributing to cell death and its attenuation by HSP27, we monitored the effect of induction of c-jun and ATF3 on tissue survival in MCAO and their effects on the expression of endogenous mouse HSP25 and HSP70. It is important that, the c-jun induction seen at 4 h tended to be localized to regions that were salvageable in HSP27tg mice but became infarcted in WT animals. Our results provide support for the powerful neuroprotective effects of HSP27 in cerebral ischemia.