In vivo imaging of Α7 nicotinic receptors as a novel method to monitor neuroinflammation after cerebral ischemia

In vivo positron emission tomography (PET) imaging of nicotinic acetylcholine receptors (nAChRs) is a promising tool for the imaging evaluation of neurologic and neurodegenerative diseases. However, the role of α7 nAChRs after brain diseases such as cerebral ischemia and its involvement in inflammatory reaction is still largely unknown. In vivo and ex vivo evaluation of α7 nAChRs expression after transient middle cerebral artery occlusion (MCAO) was carried out using PET imaging with [¹¹C]NS14492 and immunohistochemistry (IHC). Pharmacological activation of α7 receptors was evaluated with magnetic resonance imaging (MRI), [¹⁸F]DPA‐714 PET, IHC, real time polymerase chain reaction (qPCR) and neurofunctional studies. In the ischemic territory, [¹¹C]NS14492 signal and IHC showed an expression increase of α7 receptors in microglia and astrocytes after cerebral ischemia. The role played by α7 receptors on neuroinflammation was supported by the decrease of [¹⁸F]DPA‐714 binding in ischemic rats treated with the α7 agonist PHA 568487 at day 7 after MCAO. Moreover, compared with non‐treated MCAO rats, PHA‐treated ischemic rats showed a significant reduction of the cerebral infarct volumes and an improvement of the neurologic outcome. PHA treatment significantly reduced the expression of leukocyte infiltration molecules in MCAO rats and in endothelial cells after in vitro ischemia. Despite that, the activation of α7 nAChR had no influence to the blood brain barrier (BBB) permeability measured by MRI. Taken together, these results suggest that the nicotinic α7 nAChRs play a key role in the inflammatory reaction and the leukocyte recruitment following cerebral ischemia in rats.     

Suppression of hypoxia‐inducible factor‐1α and its downstream genes reduces acute hyperglycemia‐enhanced hemorrhagic transformation in a rat model of cerebral ischemia

We evaluated a role of hypoxia‐inducible factor‐1α (HIF‐1α) and its downstream genes in acute hyperglycemia‐induced hemorrhagic transformation in a rat model of focal cerebral ischemia. Male Sprague‐Dawley rats weighing 280–300 g (n = 105) were divided into sham, 90 min middle cerebral artery occlusion (MCAO), MCAO plus HIF‐1α inhibitors, 2‐methoxyestradiol (2ME2) or 3‐(5′‐hydroxymethyl‐2′‐furyl)‐1‐benzylindazole (YC‐1), groups. Rats received an injection of 50% dextrose (6 ml/kg intraperitoneally) at 15 min before MCAO. HIF‐1α inhibitors were administered at the onset of reperfusion. The animals were examined for neurological deficits and sacrificed at 6, 12, 24, and 72 hr following MCAO. The cerebral tissues were collected for histology, zymography, and Western blot analysis. The expression of HIF‐1α was increased in ischemic brain tissues after MCAO and reduced by HIF‐1α inhibitors. In addition, 2ME2 reduced the expression of vascular endothelial growth factor (VEGF) and the elevation of active matrix metalloproteinase‐2 and ?9 (MMP‐2/MMP‐9) in the ipsilateral hemisphere. Both 2ME2 and YC‐1 reduced infarct volume and ameliorated neurological deficits. However, only 2ME2 attenuated hemorrhagic transformation in the ischemic territory. In conclusion, the inhibition of HIF‐1α and its downstream genes attenuates hemorrhagic conversion of cerebral infarction and ameliorates neurological deficits after focal cerebral ischemia. © 2010 Wiley‐Liss, Inc.     

Tumor necrosis factor alpha expression produces increased blood-brain barrier permeability following temporary focal cerebral ischemia in mice.

Alteration of blood-brain barrier (BBB) function occurs in both permanent and temporary cerebral ischemia. Studies in vivo and in vitro have shown that tumor necrosis factor-alpha (TNFalpha) is involved in changes of BBB permeability. However, the relationship between TNFalpha expression and BBB disruption during reperfusion is unclear. The aim of this study is to find the cell source of TNFalpha and to determine the relationship between TNFalpha expression and BBB disruption following temporary focal cerebral ischemia in mice. Adult CD-1 mice received 1 h middle cerebral artery occlusion (MCAO) followed by 2 h, 6 h, 12 h, 24 h, and 48 h of reperfusion. MCAO was achieved using an intraluminal suture technique and reperfusion was performed by the suture withdrawal. Neutralizing monoclonal anti-mouse TNFalpha antibody was administrated intraventricularly immediately after reperfusion. TNFalpha expression was determined by double labeling immunohistochemistry. BBB permeability was determined by albumin immunostaining. TNFalpha immunoreactivity (IR) was observed in the ipsilateral hemisphere from 1 h MCAO with 2 h reperfusion. TNFalpha positive cells included neurons, astrocytes, and ependymal cells. BBB disruption was detected beginning at 6 h reperfusion but was not present at 2 h of reperfusion. The areas of BBB disruption were significantly enlarged at 12 h reperfusion and plateaued at 24 h to 48 h reperfusion. BBB disruptions were significantly attenuated in the anti-TNFalpha antibody treated mice (p<0.05). Our results demonstrate that TNFalpha IR existed in neurons, astrocytes, and ependymal cells during reperfusion. TNFalpha IR following temporary focal cerebral ischemia precedes increased BBB permeability. Treatment with TNFalpha antibody reduces BBB disruption, suggesting TNFalpha may be an important mediator in altering BBB permeability during reperfusion.     

Endothelin-1 overexpression leads to further water accumulation and brain edema after middle cerebral artery occlusion via aquaporin 4 expression in astrocytic end-feet.

Stroke patients have increased levels of endothelin-1 (ET-1), a strong vasoconstrictor, in their plasma or cerebrospinal fluid. Previously, we showed high level of ET-1 mRNA expression in astrocytes after hypoxia/ischemia. It is unclear whether the contribution of ET-1 induction in astrocytes is protective or destructive in cerebral ischemia. Here, we generated a transgenic mouse model that overexpress ET-1 in astrocytes (GET-1) using the glial fibrillary acidic protein promoter to examine the role of astrocytic ET-1 in ischemic stroke by challenging these mice with transient middle cerebral artery occlusion (MCAO). Under normal condition, GET-1 mice showed no abnormality in brain morphology, cerebrovasculature, absolute cerebral blood flow, blood-brain barrier (BBB) integrity, and mean arterial blood pressure. Yet, GET-1 mice subjected to transient MCAO showed more severe neurologic deficits and increased infarct, which were partially normalized by administration of ABT-627 (ET(A) antagonist) 5 mins after MCAO. In addition, GET-1 brains exhibited more Evans blue extravasation and showed decreased endothelial occludin expression after MCAO, correlating with higher brain water content and increased cerebral edema. Aquaporin 4 expression was also more pronounced in astrocytic end-feet on blood vessels in GET-1 ipsilateral brains. Our current data suggest that astrocytic ET-1 has deleterious effects on water homeostasis, cerebral edema and BBB integrity, which contribute to more severe ischemic brain injury.     

MicroRNA‐365 modulates astrocyte conversion into neuron in adult rat brain after stroke by targeting Pax6

Reactive astrocytes induced by ischemia can transdifferentiate into mature neurons. This neurogenic potential of astrocytes may have therapeutic value for brain injury. Epigenetic modifications are widely known to involve in developmental and adult neurogenesis. PAX6, a neurogenic fate determinant, contributes to the astrocyte‐to‐neuron conversion. However, it is unclear whether microRNAs (miRs) modulate PAX6‐mediated astrocyte‐to‐neuron conversion. In the present study we used bioinformatic approaches to predict miRs potentially targeting Pax6, and transient middle cerebral artery occlusion (MCAO) to model cerebral ischemic injury in adult rats. These rats were given striatal injection of glial fibrillary acidic protein targeted enhanced green fluorescence protein lentiviral vectors (Lv‐GFAP‐EGFP) to permit cell fate mapping for tracing astrocytes‐derived neurons. We verified that miR‐365 directly targets to the 3′‐UTR of Pax6 by luciferase assay. We found that miR‐365 expression was significantly increased in the ischemic brain. Intraventricular injection of miR‐365 antagomir effectively increased astrocytic PAX6 expression and the number of new mature neurons derived from astrocytes in the ischemic striatum, and reduced neurological deficits as well as cerebral infarct volume. Conversely, miR‐365 agomir reduced PAX6 expression and neurogenesis, and worsened brain injury. Moreover, exogenous overexpression of PAX6 enhanced the astrocyte‐to‐neuron conversion and abolished the effects of miR‐365. Our results demonstrate that increase of miR‐365 in the ischemic brain inhibits astrocyte‐to‐neuron conversion by targeting Pax6, whereas knockdown of miR‐365 enhances PAX6‐mediated neurogenesis from astrocytes and attenuates neuronal injury in the brain after ischemic stroke. Our findings provide a foundation for developing novel therapeutic strategies for brain injury.     

Immunohistochemical detection of Ndrg2 in the mouse nervous system

NDRG2, a member of the N-myc downstream-regulated gene (NDRG) family, is involved in cell differentiation and development. However, the distribution and function of Ndrg2 in the central nervous system remains unclear. Here, we analyzed the expression and distribution of Ndrg2 in the mouse brain and explored the potential physiological functions of Ndrg2. Ndrg2 was expressed in different regions of the brain, including the cerebral cortex, olfactory bulb, midbrain, hippocampus, and thalamus, with high levels in the midbrain and thalamus. Immunohistochemistry assay revealed that Ndrg2-positive cells distributed widely in the adult mouse brain and some of them showed nuclear staining. Indirect immunofluorescence and confocal microscopy studies showed that Ndrg2 protein colocalized with glial fibrillary acidic protein, indicating that Ndrg2 is expressed in astrocytes. Furthermore, Ndrg2 expression increased in glioma cells that were differentiating into astrocytes. Taken together, these findings suggest that Ndrg2 is possibly associated with glial cell proliferation and differentiation based on its immunolocalization in this study.     

A Lipoxin A<Subscript>4</Subscript> Analog Ameliorates Blood–Brain Barrier Dysfunction and Reduces MMP-9 Expression in a Rat Model of Focal Cerebral Ischemia–Reperfusion Injury

LXA₄ methyl ester (LXA₄ME), a lipoxin A₄ analog, reduces ischemic insult in the rat models of transient or permanent cerebral ischemic injury. We investigated whether LXA₄ME could ameliorate blood–brain barrier (BBB) dysfunction after stroke by reducing matrix metalloproteinase (MMP)-9 expression. Adult male rats were subjected to 2-h middle cerebral artery occlusion (MCAO) followed by 24-h reperfusion. Brain infarctions were detected by triphenyltetrazolium chloride (TTC) staining. BBB dysfunction was determined by examining brain edema and Evans Blue extravasation. Temporal expression of MMP-9 was determined by zymography and Western blot. The presence of tissue inhibitors of metalloproteinase-1 (TIMP-1) was also determined by Western blot in tissue protein sample. Brain edema and Evans Blue leakage were significantly reduced after stroke in the LXA₄ME group and were associated with reduced brain infarct volumes. MMP-9 activity and expression were inhibited by LXA₄ME after stroke. In addition, LXA₄ME significantly increased TIMP-1 protein levels. Our results indicate that LXA₄ME reduces brain injury by improving BBB function in a rat model of MCAO, and that a relationship exists between BBB permeability and MMP-9 expression following ischemic insult. Furthermore, these results suggest that LXA₄ME-mediated reduction of MMP-9 following stroke are attributed to increased TIMP-1 expression.     

Selective knockout of astrocytic Na+/H+ exchanger isoform 1 reduces astrogliosis,BBB damage,infarction, and improves neurological function after ischemic stroke

Stimulation of Na⁺/H⁺ exchanger isoform 1 (NHE1) in astrocytes causes ionic dysregulation under ischemic conditions. In this study, we created a Nhe1^flox/flox (Nhe1^f/f) mouse line with exon 5 of Nhe1 flanked with two loxP sites and selective ablation of Nhe1 in astrocytes was achieved by crossing Nhe1^f/f mice with Gfap‐Cre^ERT2 Cre‐recombinase mice. Gfap‐Cre^ERT2+/?;Nhe1^f/f mice at postnatal day 60–90 were treated with either corn oil or tamoxifen (Tam, 75 mg/kg/day, i.p.) for 5 days. After 30 days post‐injection, mice underwent transient middle cerebral artery occlusion (tMCAO) to induce ischemic stroke. Compared with the oil‐vehicle group (control), Tam‐treated Gfap‐Cre^ERT2+/?;Nhe1^f/f (Nhe1 KO) mice developed significantly smaller ischemic infarction, less edema, and less neurological function deficits at 1–5 days after tMCAO. Immunocytochemical analysis revealed less astrocytic proliferation, less cellular hypertrophy, and less peri‐lesion gliosis in Nhe1 KO mouse brains. Selective deletion of Nhe1 in astrocytes also reduced cerebral microvessel damage and blood–brain barrier (BBB) injury in ischemic brains. The BBB microvessels of the control brains show swollen endothelial cells, opened tight junctions, increased expression of proinflammatory protease MMP‐9, and significant loss of tight junction protein occludin. In contrast, the Nhe1 KO mice exhibited reduced BBB breakdown and normal tight junction structure, with increased expression of occludin and reduced MMP‐9. Most importantly, deletion of astrocytic Nhe1 gene significantly increased regional cerebral blood flow in the ischemic hemisphere at 24 hr post‐MCAO. Taken together, our study provides the first line of evidence for a causative role of astrocytic NHE1 protein in reactive astrogliosis and ischemic neurovascular damage.     

Neuroprotective effect of L‐serine against temporary cerebral ischemia in rats

To investigate the neuroprotective effect of L‐serine and its underlying mechanisms, focal cerebral ischemia was induced in rats by occlusion of middle cerebral artery (MCAO) with a suture, and reperfusion was given by filament withdrawal 2 hr later. Meanwhile, rat hippocampal neurons were primarily cultured, and incubated in serum‐free medium in an incubator containing 1% O₂ for hypoxic exposure of 5 hr, or incubated in serum‐free medium containing 1 mM glutamate for glutamate exposure of 2 hr. Brain tissue injury and cell damage were then measured. L‐serine dose‐dependently decreased the neurology deficit score and infarct volume, elevated the cell viability and inhibited the leakage of lactate dehydrogenase. These effects were blocked by strychnine in both MCAO rats and cultured hippocampal neurons. Furthermore, L‐serine (168 mg·kg^‐1) reduced the brain water content, permeability of blood‐brain barrier, neuronal loss and the expression of activated caspase‐3 in the cortex. In addition, L‐serine effectively protected the brain from damage when it was administered within 6 hr after the end of MCAO. It is suggested that L‐serine could exert a neuroprotective effect on the ischemic‐reperfused brain and on the hypoxia‐ or glutamate‐exposed hippocampal neurons, which may be mediated by activating glycine receptors. © 2010 Wiley‐Liss, Inc.     

Effects of green tea polyphenols on caveolin-1 of microvessel fragments in rats with cerebral ischemia

Abstract

Objective: This study was designed to investigate the effects of green tea polyphenols (GTPs) on the permeability of blood–brain barrier (BBB), and the expression of caveolin-1 and extracellular signal-regulated kinase ½ (ERK1/2) after cerebral ischemia.

Methods: Cerebral ischemia was established by middle cerebral artery occlusion (MCAO). Rats were randomly divided into control and GTP groups, and both included four time points of interest: MCA occluded for 0 hour, 1 hour, 2 hours, and 4 hours groups. After ischemia, triphenyltetrazolium chloride staining and Longa's score were used to determine the infarct volume and neurological deficit. Evans blue (EB) content in the brain tissue was measured to observe the BBB permeability. RT-PCR, immunohistochemistry, and western blot assessment were used to detect expression of caveolin-1 in microvessel fragments of cerebral ischemic tissue. Western blot was also used to examine ERK1/2.

Results: GTPs significantly reduced infarct volume, ameliorated the neurological deficit, and reduced the permeability of BBB. GTPs also obviously reduced the levels of caveolin-1 mRNA and protein expression as well as the expression of phosphorylated ERK1/2 in microvessel fragments of cerebral ischemic tissue, which were enhanced by cerebral ischemia.

Discussion: These data were the first to show that GTPs can decrease the elevated BBB permeability in the ischemic region, and the protective effects for cerebral injury may be related to the reduced expression of caveolin-1 and phosphorylated ERK1/2.     

Bumetanide inhibition of the blood-brain barrier Na-K-Cl cotransporter reduces edema formation in the rat middle cerebral artery occlusion model of stroke.

Increased transport of Na+ across an intact blood-brain barrier (BBB) participates in edema formation during the early hours of cerebral ischemia. In previous studies, the authors showed that the BBB Na-K-Cl cotransporter is stimulated by factors present during ischemia, suggesting that the cotransporter may contribute to the increased brain Na+ uptake in edema. The present study was conducted to determine (1) whether the Na-K-Cl cotransporter is located in the luminal membrane of the BBB, and (2) whether inhibition of the BBB cotransporter reduces brain edema formation. Perfusion-fixed rat brains were examined for cotransporter distribution by immunoelectron microscopy. Cerebral edema was evaluated in rats subjected to permanent middle cerebral artery occlusion (MCAO) by magnetic resonance diffusion-weighted imaging and calculation of apparent diffusion coefficients (ADC). The immunoelectron microscopy studies revealed a predominant (80%) luminal membrane distribution of the cotransporter. Magnetic resonance imaging studies showed ADC ratios (ipsilateral MCAO/contralateral control) ranging from 0.577 to 0.637 in cortex and striatum, indicating substantial edema formation. Intravenous bumetanide (7.6-30.4 mg/kg) given immediately before occlusion attenuated the decrease in ADC ratios for both cortex and striatum (by 40-67%), indicating reduced edema formation. Bumetanide also reduced infarct size, determined by TTC staining. These findings suggest that a luminal BBB Na-K-Cl cotransporter contributes to edema formation during cerebral ischemia.     

Anemonin Alleviates Nerve Injury After Cerebral Ischemia and Reperfusion (I/R) in Rats by Improving Antioxidant Activities and Inhibiting Apoptosis Pathway

In the present study, we aimed at evaluating the potential neuroprotective effect and the underlying mechanism of anemonin against cerebral ischemia and reperfusion (I/R) injury. Anemonin was administered to rats by the intraperitoneally (i.p.) route once daily for 7 days before middle cerebral artery occlusion (MCAO). Focal cerebral ischemia was induced by 90 min of MCAO followed by 24 h of reperfusion. After that, animals were sacrificed by decapitation, brain was removed, and various biochemical estimations, neurological status, and assessment of cerebral infarct size were carried out. MCAO followed by 24 h of reperfusion caused a significant increase in infarct size, neurological deficit score, malondialdehyde (MDA) content, reactive oxygen species (ROS) level, and DNA fragmentation, as well as a decrease in the activities of superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH), glutathione peroxidase (GPx), and Na⁺, K⁺-ATPase in the brain. Furthermore, elevated Bax expression, increased caspase-3 cleavage, and decreased Bcl-2 expression were observed in nontreated rats in response to focal cerebral I/R injury. However, pretreatment with anemonin significantly reversed these levels of biochemical parameters, reduced cerebral infarct size, and improved the neurologic score in cerebral ischemic animals. Additionally, a wide distribution of anemonin in plasma and brain tissues and the brain-to-plasma partition coefficient (Ri) ratio of 0.7 at 90 min indicated that this compound could penetrate the blood-brain barrier (BBB). These results showed that pretreatment with anemonin provided a significant protection against cerebral I/R injury in rats by, at least in part, its antioxidant action and consequent inhibition of apoptosis.     

Morphological and neuro-behavioral parallels in the rat model of stroke

Middle cerebral artery occlusion (MCAO) is widely used as a rat model of focal brain ischemia. Evaluation of brain damage often includes the morphological analysis of the injury area, MRI, and various scales which depend on functional tests, commonly known as neurological severity score (NSS). We determined the optimal number of NSS tests and assessed their capacity for non-invasive evaluation of brain ischemic injury in the rat MCAO model. 275 male Sprague-Dawley rats were randomly divided into five groups, given either permanent (p) MCAO or transient (t) MCAO using an uncoated 4-0 monofilament catheter or a silicone-coated monofilament. The rats’ neurological status was examined before and at 1 and 24 h following MCAO. The size of brain injury was then measured histologically and the extent of right cerebral hemisphere edema was calculated. We established a correlation between these tests and morphological data for brain injury. Adjusted R² of the prediction of total histology score was 0.7. The Hosmer-Lemeshow p-value of this model was 0.812 for total brain histology. For the brain edema the adjusted R² of the prediction model was 0.48. The Hosmer-Lemeshow p-value of this model was 0.558 for brain edema. Our methods of estimating infarct size produces reliable and well correlated results at 24 h and demonstrates to be an easy and quick way to assess infarct size soon after ischemic injury has occurred. The described method for neurological assessment could ultimately aid in assessing various treatment modalities in the early hours following stroke.     

Non-invasive vagus nerve stimulation reduces blood-brain barrier disruption in a rat model of ischemic stroke

Background

Vagus nerve stimulation (VNS) significantly reduces infarct volume in rat models of cerebral ischemia, but the mechanism of this protective effect remains open.

Neuroprotective effects of 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), an antioxidant in middle cerebral artery occlusion induced focal cerebral ischemia in rats

Abstract

Objectives: In the present study, we have investigated the neuroprotective potential of 6hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), in middle cerebral artery occlusion (MCAO) induced focal cerebral ischemia.

Methods: Sprague–Dawley rats were subjected to 2 hours of MCAO followed by 22 or 70 hours of reperfusion. After reperfusion, rats were evaluated for neurological deficits and cerebral infarction. Brain malondialdehyde (MDA) level and in situ terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL) were also estimated.

Results: Focal cerebral ischemia produced a significant infarct volume and neurological scores as compared with sham-operated animals. Cerebral ischemia reperfusion injury was associated with an increase in lipid peroxidation in ipsilateral and contralateral hemisphere of brain along with an increase in TUNEL positive cells in ipsilateral hemisphere of brain sections indicating oxidative stress and DNA fragmentation, respectively. Trolox (10 and 30 mg/kg, i.p.) treatment significantly decreased neurological damage which was evident from the reduction in infarct volume and neurological score. Trolox (30 mg/kg) also attenuated oxidative stress and DNA fragmentation.

Discussion: Oxidative stress-induced neuronal damage is implicated in the pathophysiology of cerebral ischemia. Our study suggests that Trolox is a potent neuroprotective agent in focal cerebral ischemia and its neuroprotective effects may be attributed to the reduction of lipid peroxidation and DNA fragmentation.