Mannan-Binding Lectin Is Involved in the Protection against Renal Ischemia/Reperfusion Injury by Dietary Restriction

Preoperative fasting and dietary restriction offer robust protection against renal ischemia/reperfusion injury (I/RI) in mice. We recently showed that Mannan-binding lectin (MBL), the initiator of the lectin pathway of complement activation, plays a pivotal role in renal I/RI. Based on these findings, we investigated the effect of short-term DR (30% reduction of total food intake) or three days of water only fasting on MBL in 10–12 weeks old male C57/Bl6 mice. Both dietary regimens significantly reduce the circulating levels of MBL as well as its mRNA expression in liver, the sole production site of MBL. Reconstitution of MBL abolished the protection afforded by dietary restriction, whereas in the fasting group the protection persisted. These data show that modulation of MBL is involved in the protection against renal I/RI induced by dietary restriction, and suggest that the mechanisms of protection induced by dietary restriction and fasting may be different.     

Cardioprotective effect of intermittent fasting is associated with an elevation of adiponectin levels in rats

It has been reported that dietary energy restriction, including intermittent fasting (IF), can protect heart and brain cells against injury and improve functional outcome in animal models of myocardial infarction (MI) and stroke. Here we report that IF improves glycemic control and protects the myocardium against ischemia-induced cell damage and inflammation in rats. Echocardiographic analysis of heart structural and functional variables revealed that IF attenuates the growth-related increase in posterior ventricular wall thickness, end systolic and diastolic volumes, and reduces the ejection fraction. The size of the ischemic infarct 24 h following permanent ligation of a coronary artery was significantly smaller, and markers of inflammation (infiltration of leukocytes in the area at risk and plasma IL-6 levels) were less, in IF rats compared to rats on the control diet. IF resulted in increased levels of circulating adiponectin prior to and after MI. Because recent studies have shown that adiponectin can protect the heart against ischemic injury, our findings suggest a potential role for adiponectin as a mediator of the cardioprotective effect of IF.     

Delayed activin A administration attenuates tissue death after transient focal cerebral ischemia and is associated with decreased stress-responsive kinase activation

Focal cerebral ischemia and reperfusion initiates complex cellular and molecular interactions that lead to either cell repair or destruction. In earlier work, we found that activin A is an early gene response to cerebral ischemia and supports cortical neuron survival in vitro. In this study, the ability of exogenous activin A to attenuate injury from transient middle cerebral artery occlusion was tested in adult mice. Intracerebroventricular administration of activin A prior to middle cerebral artery occlusion reduced infarct volume apparent 1 day after experimental stroke. A single activin A administration at 6 h following ischemia/reperfusion reduced lesion volumes at 1 and 3 days and led to improved neurobehavior. Moreover, activin A treatment spared neurons within the ischemic hemisphere and led to a concomitant reduction in microglial activation. Activation of the stress-responsive kinases p38 and c- jun N-terminal kinase implicated in neuronal apoptosis after stroke was reduced following activin A treatment. Together these findings suggest that activin A promotes tissue survival after focal cerebral ischemia/reperfusion with an extended therapeutic window.     

Direct Angiotensin AT2 Receptor Stimulation Using a Novel AT2 Receptor Agonist,Compound 21, Evokes Neuroprotection in Conscious Hypertensive Rats

Background

In this study, the neuroprotective effect of a novel nonpeptide AT2R agonist, C21, was examined in a conscious model of stroke to verify a class effect of AT2R agonists as neuroprotective agents.

Methods and Results

Spontaneously hypertensive rats (SHR) were pre-treated for 5 days prior to stroke with C21 alone or in combination with the AT2R antagonist PD123319. In a separate series of experiments C21 was administered in a series of 4 doses commencing 6 hours after stroke. A focal reperfusion model of ischemia was induced in conscious SHR by administering endothelin-1 to the middle cerebral artery (MCA). Motor coordination was assessed at 1 and 3 days after stroke and post mortem analyses of infarct volumes, microglia activation and neuronal survival were performed at 72 hours post MCA occlusion. When given prior to stroke, C21 dose dependently decreased infarct volume, which is consistent with the behavioural findings illustrating an improvement in motor deficit. During the pre-treatment protocol C21 was shown to enhance microglia activation, which are likely to be evoking protection by releasing brain derived neurotrophic factor. When drug administration was delayed until 6 hours after stroke, C21 still reduced brain injury.

Conclusion

These results indicate that centrally administered C21 confers neuroprotection against stroke damage. This benefit is likely to involve various mechanisms, including microglial activation of endogenous repair and enhanced cerebroperfusion. Thus, we have confirmed the neuroprotective effect of AT2R stimulation using a nonpeptide compound which highlights the clinical potential of the AT2R agonists for future development.     

Oral administration of grape polyphenol extract ameliorates cerebral ischemia/reperfusion-induced neuronal damage and behavioral deficits in gerbils: comparison of pre- and post-ischemic administration

Oxidative stress has been regarded as an important underlying cause for the delayed neuronal death (DND) after cerebral ischemia. In this study, the effects of short-term oral administration of grape polyphenol extract (GPE) on ischemia/reperfusion (I/R) injury in a gerbil global ischemia model were determined. Ischemia was induced by occlusion of the common carotid arteries for 5 min. GPE (30 mg/ml)-containing formula or formula without GPE was administered daily via gavage for 4 days prior to and/or for 4 days after I/R. I/R resulted in hyperlocomotion, extensive DND, oxidative and fragmented DNA damage, and an increase in reactive astrocytes and microglial cells in the hippocampal CA1 region. GPE administration for 4 days prior to I/R and for 4 days after I/R attenuated DND, DNA damage and glial cell activation. However, neuroprotection was more pronounced when GPE was administered for 4 days after I/R than when administered for 4 days prior to I/R. GPE administration after I/R attenuated I/R-induced hyperlocomotion. These findings indicate that oral GPE intake may confer protection against I/R injury and emphasize that early intervention may be an effective therapeutic measure for ameliorating brain injury in stroke.     

Continuous oral administration of atorvastatin ameliorates brain damage after transient focal ischemia in rats

Aims

Pre-treatment with statins is known to ameliorate ischemic brain damage after experimental stroke, and is independent of cholesterol levels. We undertook pre- vs post-ischemic treatment with atorvastatin after focal cerebral ischemia in rats.

Main methods

Male Sprague–Dawley rats underwent transient 90-min middle cerebral artery occlusion (MCAO). Atorvastatin (20 mg/kg/day) or vehicle was administered orally. Rats were divided into vehicle-treated, atorvastatin pre-treatment, atorvastatin post-treatment, and atorvastatin continuous-treatment groups. In the pre-treatment, rats were given atorvastatin or vehicle for 7 days before MCAO. In the post-treatment, rats received atorvastatin or vehicle for 7 days after MCAO. Measurement of infarct volume, as well as neurological and immunohistochemical assessments, were done 24 h and 7 days after reperfusion.

Key findings

Each atorvastatin-treated group demonstrated significant reductions in infarct and edema volumes compared with the vehicle-treated group 24 h after reperfusion. Seven days after reperfusion, infarct volumes in the post-treatment group and continuous-treatment group (but not the pre-treatment group) were significantly smaller than in the vehicle-treated group. Only the continuous-treatment group had significantly improved neurological scores 7 days after reperfusion compared with the vehicle group. Post-treatment and continuous-treatment groups had significantly decreased lipid peroxidation, oxidative DNA damage, microglial activation, expression of tumor necrosis factor-alpha, and neuronal damage in the cortical ischemic boundary area after 7 days of reperfusion.

Significance

These results suggest that continuous oral administration (avoiding withdrawal) with statins after stroke may reduce the extent of post-ischemic brain damage and improve neurological outcome by inhibiting oxidative stress and inflammatory responses.     

Calcitonin gene-related peptide prevents blood-brain barrier injury and brain edema induced by focal cerebral ischemia reperfusion

Cerebral ischemia is one of the diseases that most compromise the human species. Therapeutic recovery of blood-brain barrier (BBB) disruption represents a novel promising approach to reduce brain injury after stroke. To determine the effects of calcitonin gene-related peptide (CGRP) on the BBB participate in stroke progression, rat cerebral ischemia reperfusion injury was induced by a 2-hour left transient middle cerebral artery occlusion (MCAO) using an intraluminal filament, followed by 46h of reperfusion. CGRP (1μg/ml) at the dose of 3μg/kg (i.p.) was administered at the beginning of reperfusion. Subsequently, 48h after MCAO, arterial blood pressure, infarct volume, water content, BBB permeability, BBB ultrastructure, levels of aquaporin-4 (AQP4) and its mRNA were evaluated. CGRP could reduce arterial blood pressure (P<0.001), infarct volume (P<0.05), cerebral edema (P<0.01), BBB permeability (P<0.05), AQP4 mRNA expression (P<0.05) and AQP4 protein expression (P<0.01). Furthermore, CGRP treatment improved ultrastructural damage of capillary endothelium cells and decreased the loss of the tight junction observed by transmission electronic microscopy (TEM) after 46h of reperfusion. Our findings show that CGRP significantly reduced postischemic increase of brain edema with a 2-hour therapeutic window in the transient model of focal cerebral ischemia. Moreover, it seems that at least part of the anti-edematous effects of CGRP is due to decrease of BBB disruption by improving ultrastructural damage of capillary endothelium cells, enhancing basal membrane, and inhibiting AQP4 and its mRNA over-expression. The data of the present study provide a new possible approach for acute stroke therapy by administration of CGRP.     

Protective effect of resveratrol against oxidative stress in middle cerebral artery occlusion model of stroke in rats

Free radicals have been implicated in neuronal injury during ischemia reperfusion in stroke. Trans resveratrol, a potent antioxidant, polyphenolic compound found in grapes and wines has recently been shown to have neuroprotective activity against oxidative stress in in vitro studies. In the present study the effect of chronic treatment of trans resveratrol was evaluated in focal ischemia induced by middle cerebral artery [MCA] occlusion in rats. Male Wistar rats were pretreated with trans resveratrol 20 mg/kg i.p. for 21 days and were subjected to focal ischemia by occlusion of MCA using intraluminal thread. After two hours of MCA occlusion reperfusion was allowed by retracting the thread. Animals were assessed for motor performance after 24 hours and subsequently rats were sacrificed for estimation of markers of oxidative stress [malondialdehyde [MDA] and reduced glutathione] and for evaluation of volume of infarction. Control group received vehicle and similar protocol was followed. Significant motor impairment, with elevated levels of MDA and reduced glutathione was observed in the vehicle treated MCA occluded rats. Treatment with trans resveratrol prevented motor impairment, rise in levels of MDA and reduced glutathione and also significantly decreased the volume of infarct as compared to control. The study provides first evidence of effectiveness of trans resveratrol in focal ischemia most probably by virtue of its antioxidant property.     

原花青素对脑缺血再灌损伤大鼠模型的影响

目的研究原花青素对脑缺血/再灌损伤(ischemia/reperfusion,I/R)大鼠神经功能评分(neurologicaldeficit score,NDS)、脑梗死体积、脑含水量等指标的药理作用。方法采用大鼠大脑中动脉阻断(middle cerebralartery occlusion,MCAO)法复制类似人类缺血性卒中的I/R损伤模型。结果该模型各时间点内均有程度不同的神经功能缺失,原花青素给药组神经功能评分明显低于对照组(P0.05),假手术组大鼠均无神经功能缺失,脑水肿情况均较对照组明显改善(P0.05),脑梗死体积与盐水对照组相比差异有显著性(P0.05),而假手术组均未见有梗死灶。结论原花青素具有一定的保护大鼠I/R后受损脑组织的作用,可供后续研究,并可为缺血性卒中使用原花青素治疗提供确凿的理论依据。     

Perspectives on reperfusion-induced damage in rodent models of experimental focal ischemia and role of gamma-protein kinase C

Ischemic stroke represents the leading cause of death and disability among elderly people. Most stroke survivors are left with lifelong disability. With the exception of tissue-type plasminogen activator (t-PA), no effective therapy exists for the management of acute stroke. Understanding the role of various extrinsic and intrinsic pathogenic factors of ischemic damage represents a prime objective of ongoing stroke research. An important variable affecting stroke outcome is the presence or absence of reperfusion (recanalization of the occluded vessel) following an ischemic event. It appears that early reperfusion after a stroke is beneficial and capable of reversing the majority of ischemic dysfunctions. However, in some instances, late reperfusion may contrarily trigger deleterious processes and lead to more ischemic damage. Examples of ischemia/reperfusion damage using an experimental model of focal ischemia in rodents are provided, along with evidence that the brain-enriched gamma-isoform of protein kinase C may represent an important mediator of reperfusion-induced brain injury in mutant mice.     

Short-term effect of weight loss through restrictive bariatric surgery on serum levels of vaspin in morbidly obese subjects

The aims of this study were to evaluate the short-term effects of laparoscopic restrictive bariatric surgery (LRBS) on plasma levels of vaspin and the potential associations of changes in vaspin levels with changes in anthropometric indices, insulin-resistance and dietary intake. Thirty, severely obese subjects (21 female; mean age, 32.5 years) with a mean body mass index (BMI) of 44.1 ± 4.9 kg/m(2) underwent LRBS. Measurements of anthropometric indices, dietary intakes, physical activity and plasma vaspin concentrations were performed prior to, and six weeks after LRBS. Insulin-sensitivity was estimated using the homeostasis model assessment of insulin-resistance (HOMA-IR). Six weeks after LRBS, BMI decreased to a mean of 38.4 ± 4.9 kg/m(2). Significant reductions were also observed in waist circumference (WC), daily intakes of calorie, fat and protein, and plasma concentrations of triglyceride. No significant change was observed in fasting levels of insulin, blood sugar or HOMA-IR. Vaspin decreased significantly (0.26 ± 0.17 vs 0.36 ± 0.20, p=0.048) following surgery. While the percentage change of vaspin was not correlated with percent changes in anthropometric indices and HOMA-IR, it correlated positively with the percentage change in intake of calories, fat and protein: this correlation remained significant even after adjustment for sex and changes in WC and HOMA-IR. Our study suggests that LRBS decreases the serum vaspin concentrations in parallel with the restriction of dietary intake. Furthermore, decreased levels of vaspin early after LRBS seem more likely to result from decreased dietary intake rather than weight-loss-induced insulin sensitivity improvement.     

Edaravone ameliorates oxidative stress associated cholinergic dysfunction and limits apoptotic response following focal cerebral ischemia in rat

Stroke is a life-threatening disease with major cause of mortality and morbidity worldwide. The neuronal damage following cerebral ischemia is a serious risk to stroke patients. Oxidative stress and apoptotic damage play an important role in cerebral ischemic pathogenesis and may represent a target for treatment. The objective of this study was to test the hypothesis that administration of edaravone (Edv) maintains antioxidant status in brain, improves the cholinergic dysfunction and suppresses the progression of apoptosis response in rat. To test this hypothesis, male Wistar rats were subjected to middle cerebral artery occlusion (MCAO) of 2 h followed by reperfusion for 22 h. Edv was administered (10 mg/kg bwt) intraperitoneally 30 min before the onset of ischemia and 1 h after reperfusion. After reperfusion, rats were tested for neurobehavioral activities and were sacrificed for the infarct volume, estimation of oxidative damage markers. Edv treatment significantly reduced ischemic lesion volume, improved neurological deficits, contended oxidative loads, and suppressed apoptotic damage. In conclusion, treatment with Edv ameliorated the neurological and histological outcomes with elevated endogenous anti-oxidants status as well as reduced induction of apoptotic responses in MCA occluded rat. We theorized that Edv is among the pharmacological agents that reduce free radicals and its associated cholinergic dysfunction and apoptotic damage and have been found to limit the extent of brain damage following stroke.     

Genetic Variation at the BDNF Locus: Evidence for Association with Long-Term Outcome after Ischemic Stroke

Background and Purpose

Rates and extent of recovery after stroke vary considerably between individuals and genetic factors are thought to contribute to post-stroke outcome. Brain-derived neurotrophic factor (BDNF) plays important roles in brain plasticity and repair and has been shown to be involved in stroke severity, recovery, and outcome in animal models. Few clinical studies on BDNF genotypes in relation to ischemic stroke have been performed. The aims of the present study are therefore to investigate whether genetic variation at the BDNF locus is associated with initial stroke severity, recovery and/or short-term and long-term functional outcome after ischemic stroke.

Methods

Four BDNF tagSNPs were analyzed in the Sahlgrenska Academy Study on Ischemic Stroke (SAHLSIS; 600 patients and 600 controls, all aged 18–70 years). Stroke severity was assessed using the NIH Stroke Scale (NIHSS). Stroke recovery was defined as the change in NIHSS over a 3-month period. Short- and long-term functional outcome post-stroke was assessed using the modified Rankin Scale at 3 months and at 2 and 7 years after stroke, respectively.

Results

No SNP was associated with stroke severity or recovery at 3 months and no SNP had an impact on short-term outcome. However, rs11030119 was independently associated with poor functional outcome 7-years after stroke (OR 0.66, 95% CI 0.46–0.92; P =  0.006).

Conclusions

BDNF gene variants were not major contributors to ischemic stroke severity, recovery, or short-term functional outcome. However, this study suggests that variants in the BDNF gene may contribute to poor long-term functional outcome after ischemic stroke.     

Role of matrix metalloproteinases in delayed cortical responses after stroke

Matrix metalloproteinases (MMPs) are zinc-endopeptidases with multifactorial actions in central nervous system (CNS) physiology and pathology. Accumulating data suggest that MMPs have a deleterious role in stroke. By degrading neurovascular matrix, MMPs promote injury of the blood-brain barrier, edema and hemorrhage. By disrupting cell-matrix signaling and homeostasis, MMPs trigger brain cell death. Hence, there is a movement toward the development of MMP inhibitors for acute stroke therapy. But MMPs may have a different role during delayed phases after stroke. Because MMPs modulate brain matrix, they may mediate beneficial plasticity and remodeling during stroke recovery. Here, we show that MMPs participate in delayed cortical responses after focal cerebral ischemia in rats. MMP-9 is upregulated in peri-infarct cortex at 7-14 days after stroke and is colocalized with markers of neurovascular remodeling. Treatment with MMP inhibitors at 7 days after stroke suppresses neurovascular remodeling, increases ischemic brain injury and impairs functional recovery at 14 days. MMP processing of bioavailable VEGF may be involved because inhibition of MMPs reduces endogenous VEGF signals, whereas additional treatment with exogenous VEGF prevents MMP inhibitor-induced worsening of infarction. These data suggest that, contrary to MMP inhibitor therapies for acute stroke, strategies that modulate MMPs may be needed for promoting stroke recovery.     

Intra-arterial transplantation of bone marrow mononuclear cells immediately after reperfusion decreases brain injury after focal ischemia in rats

AIMS: Transplantation of bone marrow cells has been reported to exert neuroprotection against cerebral ischemia. However, the effect of bone marrow mononuclear cells (BMMCs) administered immediately after reperfusion has rarely been investigated. The present study was designed to examine whether brain injury in response to transient focal ischemia can be ameliorated by BMMC administration immediately after reperfusion in rats, and to determine whether there are differences in the route of administration. MAIN METHODS: Autologous BMMCs were obtained from each rat. Rats were then subjected to transient focal ischemia followed by BMMC administration via the ipsilateral carotid artery (IA group) or the femoral vein (IV group) immediately after reperfusion. Control rats underwent the same procedure but received vehicle injection. Infarct volume was compared among the groups 24 h and 7 days after reperfusion. BMMCs were fluorescently labeled with PKH26 prior to administration to track transplanted cells. KEY FINDINGS: Total infarct volume decreased in the IA group, but not in the IV group, when compared to the vehicle group. In the ipsilateral hemisphere, PKH26 positive cell count was greater in the IA group than in the IV group. Motor function, assessed with a rotarod test, improved in the IA group compared to the vehicle group. SIGNIFICANCE: These results show significant neuroprotection after transient focal ischemia by 1x10(7) autologous BMMCs administered intra-arterially, but not intravenously, immediately after reperfusion in rats. The larger number of transplanted BMMCs in the brain during the early stage of reperfusion may be responsible for the protective effect.     

Iron overload,measured as serum ferritin,increases brain damage induced by focal ischemia and early reperfusion

High levels of iron, measured as serum ferritin, are associated to a worse outcome after stroke. However, it is not known whether ischemic damage might increase ferritin levels as an acute phase protein or whether iron overload affects stroke outcome. The objectives are to study the effect of stroke on serum ferritin and the contribution of iron overload to ischemic damage.Swiss mice were fed with a standard diet or with a diet supplemented with 2.5% carbonyl iron to produce iron overload. Mice were submitted to permanent (by ligature and by in situ thromboembolic models) or transient focal ischemia (by ligature for 1 or 3 h).Treatment with iron diet produced an increase in the basal levels of ferritin in all the groups. However, serum ferritin did not change after ischemia. Animals submitted to permanent ischemia had the same infarct volume in the groups studied. However, in mice submitted to transient ischemia followed by early (1 h) but not late reperfusion (3 h), iron overload increased ischemic damage and haemorrhagic transformation.Iron worsens ischemic damage induced by transient ischemia and early reperfusion. In addition, ferritin is a good indicator of body iron levels but not an acute phase protein after ischemia.     

Post-ischaemic treatment with the cyclooxygenase-2 inhibitor nimesulide reduces blood-brain barrier disruption and leukocyte infiltration following transient focal cerebral ischaemia in rats

Several studies suggest that cyclooxygenase (COX)-2 plays a pivotal role in the progression of ischaemic brain damage. In the present study, we investigated the effects of selective inhibition of COX-2 with nimesulide (12 mg/kg) and selective inhibition of COX-1 with valeryl salicylate (VAS, 12-120 mg/kg) on prostaglandin E(2) (PGE(2)) levels, myeloperoxidase (MPO) activity, Evans blue (EB) extravasation and infarct volume in a standardized model of transient focal cerebral ischaemia in the rat. Post-ischaemic treatment with nimesulide markedly reduced the increase in PGE(2) levels in the ischaemic cerebral cortex 24 h after stroke and diminished infarct size by 48% with respect to vehicle-treated animals after 3 days of reperfusion. Furthermore, nimesulide significantly attenuated the blood-brain barrier (BBB) damage and leukocyte infiltration (as measured by EB leakage and MPO activity, respectively) seen at 48 h after the initial ischaemic episode. These studies provide the first experimental evidence that COX-2 inhibition with nimesulide is able to limit BBB disruption and leukocyte infiltration following transient focal cerebral ischaemia. Neuroprotection afforded by nimesulide is observed even when the treatment is delayed until 6 h after the onset of ischaemia, confirming a wide therapeutic window of COX-2 inhibitors in experimental stroke. On the contrary, selective inhibition of COX-1 with VAS had no significant effect on the evaluated parameters. These data suggest that COX-2 activity, but not COX-1 activity, contributes to the progression of focal ischaemic brain injury, and that the beneficial effects observed with non-selective COX inhibitors are probably associated to COX-2 rather than to COX-1 inhibition.     

White Matter Reorganization and Functional Response after Focal Cerebral Ischemia in the Rat

After stroke, the brain has shown to be able to achieve spontaneous functional recovery despite severe cerebral damage. This phenomenon is poorly understood. To address this issue, focal transient ischemia was induced by 60 min middle cerebral artery occlusion in Wistar rats. The evolution of stroke was followed using two magnetic resonance imaging modalities: diffusion spectrum imaging (acquired before, one and four weeks after stroke) and functional magnetic resonance imaging (acquired before and five weeks after stroke). To confirm the imaging observations, immunohistochemical staining for myelin, astrocytes and macrophages/microglia was added. At four weeks after stroke, a focal alteration of the diffusion anisotropy was observed between the ipsilesional ventricle and the lesion area. Using tractography this perturbation was identified as reorganization of the ipsilesional internal capsule. Functional imaging at five weeks after ischemia demonstrated activation of the primary sensorimotor cortex in both hemispheres in all rats except one animal lacking a functional response in the ipsilesional cortex. Furthermore, fiber tracking showed a transhemispheric fiber connection through the corpus callosum, which-in the rat without functional recovery-was lost. Our study shows the influence of the internal capsule reorganization, combined with inter-hemispheric connections though the corpus callosum, on the functional activation of the brain from stroke. In conclusion, tractography opens a new door to non-invasively investigate the structural correlates of lack of functional recovery after stroke.