microRNAs in stroke pathogenesis.

Stroke is one of the leading causes of death and disability worldwide. There are two major types of stroke: cerebral ischemia caused by obstruction of blood vessels in the brain and haemorrhagic stroke that is triggered by the disruption of blood vessels. Thrombolytic therapy involving recombinant tissue plasminogen activator (rtPA) has been shown to be beneficial only when used within 4.5 hours of onset of acute ischemic stroke. rtPA treatment beyond this time window has been found to be unsuitable and usually resulting in haemorrhagic transformation. Stroke is a multifactorial disease that forms a possible end state for majority of patients suffering from diabetes, atherosclerosis and hypertension which are known risk factors. Although the biochemistry of stroke and related diseases is quite well understood, the knowledge on the molecular mechanisms underlying these diseases is still at its infancy. microRNAs that form a unique class of endogenous riboregulators of gene function, offer tremendous potential in unraveling the mechanisms underlying stroke pathogenesis. microRNA expression also reflects the response of individuals to drugs and therapy. Several microRNAs and their target genes, known to be involved in endothelial dysfunction, dysregulation of neurovascular integrity, edema formation, pro-apoptosis, inflammation and extra-cellular matrix remodeling contribute to the critical processes in the pathogenesis of stroke. In this review, we will also be discussing the role of microRNAs as possible diagnostic and prognostic biomarkers as well as potential therapeutic targets in stroke pathogenesis.

Hydrogen sulfide protects neurons against hypoxic injury via stimulation of ATP-sensitive potassium channel/protein kinase C/extracellular signal-regulated kinase/heat shock protein 90 pathway.

Cerebral hypoxia is one of the main causes of cerebral injury. This study was conducted to investigate the potential protective effect of H(2)S in in vitro hypoxic models by subjecting SH-SY5Y cells to either oxygen-glucose deprivation or Na(2)S(2)O(4) (an oxygen scavenger) treatment. We found that treatment with NaHS (an H(2)S donor, 10-100 microM) 15 min prior to hypoxia increased cell viability in a concentration-dependent manner. Time-course study showed that NaHS was able to exert its protective effect even when added 8 h before or less than 4 h after hypoxia induction. Interestingly, endogenous H(2)S level was markedly reduced by hypoxia induction. Over-expression of cystathionine-beta-synthase prevented hypoxia induced cell apoptosis. Blockade of ATP-sensitive K(+) (K(ATP)) channels with glibenclamide and HMR-1098, protein kinase C (PKC) with its three specific inhibitors (chelerythrine, bisindolylmaleide I and calphostin C), extracellular signal-regulated kinase 1/2 (ERK1/2) with PD98059 and heat shock protein 90 (Hsp90) with geldanamycin and radicicol significantly attenuated the protective effects of NaHS. Western blots showed that NaHS significantly stimulated ERK1/2 activation and Hsp90 expression. In conclusion, H(2)S exerts a protective effect against cerebral hypoxia induced neuronal cell death via K(ATP)/PKC/ERK1/2/Hsp90 pathway. Our findings emphasize the important neuroprotective role of H(2)S in the brain during cerebral hypoxia.

Chronic stimulation of corticotropin-releasing factor receptor 1 enhances the anxiogenic response of the cholecystokinin system.

Corticotropin-releasing factor (CRF) and cholecystokinin (CCK), two highly colocalized neuropeptides, have been linked to the etiology of stress-related anxiety disorders. Recent evidence points to the possibility that some of the anxiogenic effects of the central CCK system take place through interplay with the CRF system. The aim of the present study was to examine the effects of chronic, mild activation of CRF receptor 1 (CRF(1)) on the central CCK system of the C57BL/6J mouse. As shown by in situ hybridization, real-Time PCR and immunohistochemistry, 5 days of intracerebroventricular (i.c.v.) injections of a subeffective dose (2.3 pmol) of cortagine, a CRF(1)-selective agonist, resulted in an increase in CCK mRNA levels and CCK(2) receptor immunoreactivity in several brain regions, such as amygdala and hippocampus, known to be involved in the regulation of anxiety. Mice with elevated endogenous central CCK tone exhibited significantly higher anxiety-like behaviors in the open-field task and elevated plus maze, and enhanced conditioned fear. These behavioral changes were reversed by i.c.v. administration of the CCK(2)-selective antagonist LY225910, after 5 days of priming with cortagine. Under the same conditions, the intraperitoneal administration of the CRF(1) antagonist antalarmin was ineffective. This result indicated that once the CCK system was sensitized by prior CRF(1) activation, it exhibited its anxiogenic effects, without influence by CRF(1), possibly because of its observed downregulation. In sum, our results provide a novel model for the interaction of the CRF and CCK systems contributing to the development of hypersensitive emotional circuitry.

Chronic treatment with simvastatin upregulates muscarinic M1/4 receptor binding in the rat brain.

Statins are increasingly being used for the treatment of a variety of conditions beyond their original indication for cholesterol lowering. We previously reported that simvastatin affected the dopaminergic system in the rat brain. This study aims to investigate regional changes of muscarinic M1/4 receptors in the rat brain after 4-week administration of simvastatin (1 or 10 mg/kg/day). M1/4 receptor distribution and alterations in the post-mortem rat brain were detected by [(3)H]pirenzepine binding autoradiography. Simvastatin (1 mg/kg/day) increased [(3)H]pirenzepine binding, predominantly in the prefrontal cortex (171%, P<0.001), primary motor cortex (153%, P=0.001), cingulate cortex (109%, P<0.001), hippocampus (138%, P<0.001), caudate putamen (122%, P=0.002) and nucleus accumbens (170%, P<0.001) compared with controls; while lower but still significant increases of [(3)H]pirenzepine binding were observed in the examined regions following simvastatin (10 mg/kg/day) treatment. Our results also provide strong evidence that chronic simvastatin administration, especially at a low dosage, up-regulates M1/4 receptor binding, which is likely to be independent of its muscarinic agonist-like effect. Alterations in [(3)H]pirenzepine binding in the examined brain areas may represent the specific regions that mediate the clinical effects of simvastatin treatment on cognition and memory via the muscarinic cholinergic system. These findings contribute to a better understanding of the critical roles of simvastatin in treating neurodegenerative disorders, via muscarinic receptors.

Loss of [3H]4-DAMP binding to muscarinic receptors in the orbitofrontal cortex of Alzheimer's disease patients with psychosis.

RATIONALE: Neuropsychiatric behaviours in Alzheimer's disease (AD) patients have been associated with neocortical alterations of presynaptic cholinergic and muscarinic M2 receptor markers. In contrast, it is unclear whether non-M2 muscarinic receptors have a role to play in AD behavioural symptoms. OBJECTIVES: To correlate the alterations of neocortical postsynaptic muscarinic receptors with clinical features of AD. MATERIALS AND METHODS: [(3)H]4-DAMP were used in binding assays with lysates of Chinese hamster ovary (CHO) cells stably transfected with M1-M5 receptors. [(3)H]4-DAMP was further used to measure muscarinic receptors in the postmortem orbitofrontal cortex of aged controls and AD patients longitudinally assessed for cognitive decline and behavioural symptoms. RESULTS: [(3)H]4-DAMP binds to human postmortem brain homogenates and M1-, M3-, M4- and M5-transfected CHO lysates with subnanomolar affinity. Compared to the controls, the [(3)H]4-DAMP binding density is reduced only in AD patients with significant psychotic symptoms. The association between reduced [(3)H]4-DAMP binding and psychosis is independent of the effects of dementia severity or neurofibrillary tangle burden. CONCLUSIONS: This study suggests that the loss of non-M2 muscarinic receptors in the orbitofrontal cortex may be a neurochemical substrate of psychosis in AD and provides a rationale for further development of muscarinic receptor ligands in AD pharmacotherapy.

Hydrogen sulfide: neurochemistry and neurobiology.

Current evidence suggests that hydrogen sulfide (H2S) plays an important role in brain functions, probably acting as a neuromodulator as well as an intracellular messenger. In the mammalian CNS, H2S is formed from the amino acid cysteine by the action of cystathionine beta-synthase (CBS) with serine (Ser) as the by-product. As CBS is a calcium and calmodulin dependent enzyme, the biosynthesis of H2S should be acutely controlled by the intracellular concentration of calcium. In addition, it is also regulated by S-adenosylmethionine which acts as an allosteric activator of CBS. H2S, as a sulfhydryl compound, has similar reducing properties as glutathione. In neurons, H2S stimulates the production of cAMP probably by direct activation of adenylyl cyclase and thus activate cAMP-dependent processes. In astrocytes, H2S increases intracellular calcium to an extent capable of inducing and propagating a "calcium wave", which is a form of calcium signaling among these cells. Possible physiological functions of H2S include potentiating long-term potentials through activation of the NMDA receptors, regulating the redox status, maintaining the excitatory/inhibitory balance in neurotransmission, and inhibiting oxidative damage through scavenging free radicals and reactive species. H2S is also involved in CNS pathologies such as stroke and Alzheimer's disease. In stroke, H2S appears to act as a mediator of ischemic injuries and thus inhibition of its production has been suggested to be a potential treatment approach in stroke therapy.

Upregulation of dihydropyrimidinase-related protein 2, spectrin alpha II chain, heat shock cognate protein 70 pseudogene 1 and tropomodulin 2 after focal cerebral ischemia in rats--a proteomics approach.

In recent years, there are an increasing number of proteomics studies that investigated the alterations in the protein expression relevant to human diseases but none for stroke. We, therefore, attempted such a study in a paradigm of focal cerebral ischemia in rat. Rats were subjected to cerebral ischemia by unilateral occlusion of the middle cerebral artery. Global protein analysis was performed after 24h on the lesioned and sham-control cerebral cortex using two-dimensional gel electrophoresis. Protein spots with more than a 3-fold change in intensity were identified by mass spectrometry. Middle cerebral artery occlusion (MCAO) caused infarct volume of 18-22% predominantly in the cortex of the lesioned hemisphere. Two-dimensional gel electrophoresis resolved about 1500 protein spots of which only 12 were significantly upregulated by 3-46-fold. Three spots were identified to be dihydropyrimidinase-related protein 2 (DRP-2, also known as collapsin response mediator protein 2 (CRMP-2) or turned on after division, 64 kD protein (TOAD-64)). The spots varied in pI values only and this may reflect different phosphorylation status of the same protein. Two spots were identified as spectrin alpha II chain (rat fragment, also known as alpha-fodrin or non-erythroid alpha chain, SPNA-2); and one spot each for heat shock cognate protein 70 pseudogene 1 (HSC70-ps1, also known as heat shock protein 8 pseudogene 1), and tropomodulin 2 (Tmod2). The upregulation of protein expression was corroborated by observed upregulation of mRNA expression. The remaining five spots were not identified satisfactorily. As DRP-2, spectrin, and Tmod2 are involved in axonal and neurite growth as well as synaptic plasticity and maturation, the presently observed upregulation of the expression of these proteins may indicate active neuroregeneration and repair at 24h after the induction of cerebral ischemia.

Simvastatin reverses the downregulation of dopamine D1 and D2 receptor expression in the prefrontal cortex of 6-hydroxydopamine-induced Parkinsonian rats.

Sprague-Dawley rats with unilateral lesion of the medial forebrain bundle by 6-hydroxydopamine showed marked decrease in the expression of dopamine D1 and D2 receptors in the prefrontal cortex. Simvastatin (10 mg/kg/day for 4 weeks) restored receptor expression to control levels. Given the association of dopaminergic dysfunction in the prefrontal cortex and cognitive deficits in Parkinson's disease, these findings may have implication in the treatment of cognitive decline in advanced Parkinson's disease.

Cytokine changes in the horizontal diagonal band of Broca in the septum after running and stroke: a correlation to glial activation.

The relationship between running, glial cell activation and pro-inflammatory cytokines was studied in the context of neuroprotection against ischemic stroke induced by middle cerebral artery occlusion (MCAO). This was investigated in four groups of rats, namely, (1) nonrunner, (2) runner after 12 weeks of treadmill running, (3) nonrunner with MCAO and (4) runner with MCAO. The horizontal diagonal band of Broca (HDB) in the septum was scrutinized for qualitative cum quantitative changes in the microglia and astrocytes. Reverse transcription-polymerase chain reaction and immunoblot work were carried out in the forebrain homogenate to determine, respectively, the gene and protein expression of several pro-inflammatory cytokines. Our results indicated that the runner exhibited less immunoreactivity and reduced numbers of glial cells within the HDB compared with the nonrunner. Interestingly, the mRNA and protein levels of tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-6 and interferon-gamma, were significantly downregulated in the runner. Our data also suggest albeit with some inconsistency that the runner/MCAO rats had benefited from running. These observations suggest that running can result in changes to the microenvironment, in which the microglia and astrocytes exist in a state of quiescence concomitant with a reduced expression of pro-inflammatory cytokines, that may lead to beneficial effects seen in ischemic stroke induced by MCAO.

Analysis of strain difference in behavior to Cholecystokinin (CCK) receptor mediated drugs in PVG hooded and Sprague-Dawley rats using elevated plus-maze test apparatus.

This study investigated the behavioral mechanisms underlying the anxiogenic, or anxiolytic mediated effects of CCK(2) receptor mediated agonist (CCK-4) and antagonist drugs (LY225910, LY288513, CR2945) in PVG hooded and Sprague-Dawley (SD) rats using the elevated plus maze test apparatus. In addition, the effects of a CCK(1) antagonist (CR1409) were investigated for its possible mediation in anxiety behavior between PVG hooded and SD rats. PVG hooded rats treated with CCK-4, decreased the time spent in the open arm and increased the time spent in the closed arm and correspondingly showed increase in the number of entries in the open arms while the number of entries in closed arm was insignificant, whereas SD rats decreased the time spent in the closed arm, while other parameters remained insignificant. PVG hooded rats administered with various CCK(2) antagonists (LY225910, LY288513, and CR2945) significantly increased the time spent in the open arm and correspondingly decreased the time spent in the closed arm, while the number of entries in the open or closed arm was insignificant, in contrast, SD rats failed to show any reliable significance. PVG hooded rats administered with the CCK(1) antagonist (CR1409), failed to show any reliable significance, in contrast, SD rats significantly increased the time spent in the open arm. The strain differences observed in this study suggests that CCK plays mainly as a neuromodulator, in which the various CCK(2) antagonists may not affect baseline anxiety state, but instead they modulate heightened states of anxiety through differential effects of CCK(1)/CCK(2) receptors.

The CCK2 agonist BC264 reverses freezing behavior habituation in PVG hooded rats on repeated exposures to a cat.

Our previous studies (NeuroReport 12 (2001) 2717) showed that PVG hooded and not Sprague-Dawley (SD) rats exhibit remarkable freezing behavior on exposure to a cat in the cat freezing test apparatus. In the present study, we further examined the differences between these two strains of rats in response to repeated daily exposure to a cat in the cat freezing test apparatus. Freezing behavior habituation was observed in both PVG hooded (days 5-7) and SD rats (days 3-7). A selective CCK(2) agonist (BC264, 0.3 microg/kg, i.p.) on day 8 reversed habituated freezing behavior and locomotor activity in PVG hooded rats, but not in SD rats. These results suggest that CCK2 receptors mediate habituation to an anxiety-inducing stimulus in PVG hooded rats and further suggest that differential expression of these CCK2 receptors underlies this strain difference.

Distinct regions of periaqueductal gray (PAG) are involved in freezing behavior in hooded PVG rats on the cat-freezing test apparatus.

The periaqueductal gray (PAG) is considered to be an exit relay for defensive responses. Studies have shown that the ventrolateral periaqueductal gray (vlPAG) plays a role in the expression of freezing behavior whereas dorsolateral periaqueductal gray (dlPAG) is involved on both freezing and active forms of defensive behaviors. To further elucidate this theory, lesioned vlPAG and dlPAG rats were exposed to a cat in the cat-freezing test apparatus. Subsequently, a 7-day repeated exposure to a cat was done on the vlPAG and dlPAG lesioned rats. Results showed that the vlPAG lesioned rats demonstrated significant decrease in freezing behavior and corresponding increase in locomotor activity, while the dlPAG lesioned rats failed to show any significance. Subsequent repeated exposure of the vlPAG lesioned rats to a cat for 7 days showed a gradual decrease in freezing behavior with significance shown at days 5, 6 and 7 while the dlPAG lesioned rats failed to show any changes. These results suggest that vlPAG regulates freezing behavior in hooded PVG rats.

Neuroprotection associated with running: is it a result of increased endogenous neurotrophic factors?

The possible neuroprotective effect of physical exercise was investigated in rats after middle cerebral artery occlusion (MCAO), a focal stroke model. It was found that physical exercise in the form of a 12-week treadmill running programme reduced the volume of infarction caused by MCAO. At the molecular level, reverse transcription polymerase chain reaction revealed that the runner had increased gene expression for nerve growth factor (NGF) over the nonrunner with or without MCAO. Expression of the NGF receptors, p75, was increased only in the absence of MCAO. In addition, runners showed a significantly higher number of cholinergic neurons, which constitutively expressed p75, in the horizontal diagonal band of Broca. The present findings suggest that neuroprotection after physical exercise may be a result of an increase in an endogenous neurotrophic factor nerve growth factor and the proliferation of its receptive cholinergic neurons.

cDNA microarray analysis of gene expression in anxious PVG and SD rats after cat-freezing test.

To identify genes involved in the development of anxiety or fear, we analyzed the gene expression profiles of the cortex of anxious hooded PVG and Sprague-Dawley (SD) rats after exposure to the cat-freezing test apparatus. These two rat strains showed a marked difference in the extent of anxious behavior on the cat-freezing test; the hooded PVG rats showed highly anxious behavior while a low anxiety state was observed in SD rats. A cDNA microarray consisting of 5,931 genes was employed to investigate the global mRNA expression profiles of anxiety-related genes. According to the assumption that an abundance ratio of > or =1.5 is indicative of a change in gene expression, we detected 16 upregulated and 38 downregulated genes in PVG hooded and SD rats. Some of these genes have not yet been associated with anxiety (e.g. FGF), while other genes were recently found to be expressed in an anxious state (e.g., rat nerve growth factor-induced gene, NGFI-A). Our study also focused on the expression of some neurotransmitter receptors that have already been proven to be relevant to anxiety or fear, e.g., gamma-aminobutyric acid (GABA), cholecystokinin (CCK) and 5-HT(3) receptors. To further confirm the microarray data, the mRNA expressions of three genes: rat activity-regulated cytoskeleton-associated gene (Arc), rat NGFI-A gene and rat 5-HT(3) receptor (5-HT(3)R) mRNA, were studied by reverse transcription-polymerase chain reaction (RT-PCR). The results of RT-PCR were basically consistent with those from cDNA microarray. Our study therefore demonstrated that the microarray technique is an efficient tool for analyzing global expression profiles of anxiety-related genes, which may also provide further insight into the molecular mechanisms underlying the states of anxiety and fear.

Genetic variations in CCK2 receptor in PVG hooded and Sprague-Dawley rats and its mRNA expression on cat exposure.

This study examined differential freezing behavior, mediated by cholecystokinin-2 (CCK2) receptors (J. M. Farook et al., 2001), in PVG hooded and Sprague-Dawley (SD) rats exposed to a predator. The authors confirmed by reverse transcription polymerase chain reaction that CCK2 receptor expression in the PVG rats was increased in the hippocampus and cerebral cortex compared with that of SD rats. In addition, 4 variations in the coding region of the CCK2 receptor gene were detected between the PVG hooded and SD rats: 1 in Exon 4, 1 in Intron 2, and 2 in Intron 3. Acute treatment with a CCK2 agonist (CCK-4) or antagonist (LY225910) did not alter the level of CCK2 receptor expression, indicating no difference between the 2 strains in sensitivity of the CCK2 receptor to drugs.

Cortical expression of endothelin receptor subtypes A and B following middle cerebral artery occlusion in rats.

This work aimed to define the spatial expression of endothelin A (ET(A)) and B (ET(B)) receptors in the cerebral cortex after permanent middle cerebral artery occlusion (MCAO) and to identify the phenotype of cells expressing ET(A) and ET(B) receptors. Cortical expression of ET(A) and ET(B) receptors was determined at the mRNA level by semi-quantitative reverse transcription-polymerase chain reaction and at the protein level by immunofluorescence staining, 12, 24 and 72 h after MCAO. Cells expressing endothelin receptors were phenotyped by double labelling with antibodies, anti-protein gene product (PGP9.5) and anti-ED1, towards neurons and activated microglia/macrophages, respectively. Both ET(A) and ET(B) receptor mRNA expressions increased significantly in the ipsilateral cortex in a time-dependent manner after MCAO. Robust expression of ET(A) receptors was noted in most neurons of the ischemic core and in several neurons in laminae 3 and 4 of the peri-infarct region 24 and 72 h after MCAO. ET(B) receptor immunoreactivity was observed in activated microglia/macrophages, beginning 24 h after MCAO. These results provide the first evidence that the action of endothelin during ischemia may be mediated by neuronal ET(A) receptors and activated microglia/macrophage ET(B) receptors. This differential localization of ET(A) and ET(B) receptors suggests that endothelin is involved in some complex neuron-glial interactions in addition to its vascular modulatory activity during ischemia.