Rosfaiizah Siran

Preconditioning effect of (S)-3,5-dihydroxyphenylglycine on ischemic injury in middle cerebral artery occluded Sprague-Dawley rats

Glutamate receptors are the integral cellular components associated with excitotoxicity mechanism induced by the ischemic cascade events. Therefore the glutamate receptors have become the major molecular targets of neuroprotective agents in stroke researches. Recent studies have demonstrated that a Group I metabotropic glutamate receptor agonist, (S)-3,5-dihydroxyphenylglycine ((S)-3,5-DHPG) preconditioning elicits neuroprotection in the hippocampal slice cultures exposed to toxic level of N-methyl-d-aspartate (NMDA). We further investigated the preconditioning effects of (S)-3,5-DHPG on acute ischemic stroke rats. One 10 or 100μM of (S)-3,5-DHPG was administered intrathecally to Sprague-Dawley adult male rats, 2h prior to induction of acute ischemic stroke by middle cerebral artery occlusion (MCAO). After 24h, neurological deficits were evaluated by modified stroke severity scores and grid-walking test. All rats were sacrificed and infarct volumes were determined by 2,3,5-triphenyltetrazolium chloride staining. The serum level of neuron-specific enolase (NSE) of each rat was analyzed by enzyme-linked immunosorbent assay (ELISA). One and 10μM of (S)-3,5-DHPG preconditioning in the stroke rats showed significant improvements in motor impairment (P<0.01), reduction in the infarct volume (P<0.01) and reduction in the NSE serum level (P<0.01) compared to the control stroke rats. We conclude that 1 and 10μM (S)-3,5-DHPG preconditioning induced protective effects against acute ischemic insult in vivo.

REM sleep deprivation induces changes of Down Regulatory Antagonist Modulator (DREAM) expression in the ventrobasal thalamic nuclei of Sprague–Dawley rats

REM sleep is a crucial component of sleep. Animal studies indicate that rapid eye movement (REM) sleep deprivation elicits changes in gene expression. Downregulatory antagonist modulator (DREAM) is a protein which downregulates other gene transcriptions by binding to the downstream response element site. The aim of this study is to examine the effect of REM sleep deprivation on DREAM expression in ventrobasal thalamic nuclei (VB) of rats. Seventy-two male Sprague–Dawley rats were divided into four major groups consisting of free-moving control rats (FMC) (n = 18), 72-h REM sleep-deprived rats (REMsd) (n = 18), 72-h REM sleep-deprived rats with 72-h sleep recovery (RG) (n = 18), and tank control rats (TC) (n = 18). REM sleep deprivation was elicited using the inverted flower pot technique. DREAM expression was examined in VB by immunohistochemical, Western blot, and quantitative real-time polymerase chain reaction (qRT-PCR) studies. The DREAM-positive neuronal cells (DPN) were decreased bilaterally in the VB of rats deprived of REM sleep as well as after sleep recovery. The nuclear DREAM extractions were increased bilaterally in animals deprived of REM sleep. The DREAM messenger RNA (mRNA) levels were decreased after sleep recovery. The results demonstrated a link between DREAM expression and REM sleep deprivation as well as sleep recovery which may indicate potential involvement of DREAM in REM sleep-induced changes in gene expression, specifically in nociceptive processing.

Ursodeoxycholic acid upregulates ERK and Akt in the protection of cardiomyocytes against CoCl2.

Ursodeoxycholic acid (UDCA) is used to treat liver diseases and demonstrates cardioprotective effects. Accumulation of the plasma membrane sphingolipid sphingomyelin in the heart can lead to atherosclerosis and coronary artery disease. Sphingomyelinases (SMases) break down sphingomyelin, producing ceramide, and inhibition of SMases activity can promote cell survival. We hypothesized that UDCA regulates activation of ERK and Akt survival signaling pathways and SMases in protecting cardiac cells against hypoxia. Neonatal cardiomyocytes were isolated from 0- to 2-day-old Sprague Dawley rats, and given 100 μM CoCl2, 150 μM H2O2, or placed in a hypoxia chamber for 24 h. The ameliorative effects of 100-μM UDCA treatment for 12 h were then assessed using MTS, QuantiGene Plex (for Smpd1 and Smpd2), and SMase assays, beating rate assessment, and western blotting (for ERK and Akt). Data were analyzed by the paired Student t-tests and one-way analyses of variance. Cell viability decreased significantly after H2O2 (85%), CoCl2 (50%), and hypoxia chamber (52%) treatments compared to the untreated control (100%). UDCA significantly counteracted the effects of chamber- and CoCl2- induced hypoxia on viability and beating rate. However, no significant differences were observed in acid SMase gene and protein expression between the untreated, CoCl2, and UDCA-CoCl2 groups. In contrast, neutral SMase gene and protein expression did significantly differ between the latter two groups. ERK and Akt phosphorylation was higher in hypoxic cardiomyocytes treated with UDCA than those given CoCl2 alone. In conclusion, UDCA regulates the activation of survival signaling proteins and SMases in neonatal rat cardiomyocytes during hypoxia.

Ursodeoxycholic acid protects cardiomyocytes against cobalt chloride induced hypoxia by regulating transcriptional mediator of cells stress hypoxia inducible factor 1α and p53 protein

In hepatocytes, ursodeoxycholic acid (UDCA) activates cell signalling pathways such as p53, intracellular calcium ([Ca2+]i), and sphingosine‐1‐phosphate (S1P)‐receptor via Gαi‐coupled‐receptor. Recently, UDCA has been shown to protect the heart against hypoxia‐reoxygenation injury. However, it is not clear whether UDCA cardioprotection against hypoxia acts through a transcriptional mediator of cells stress, HIF‐1α and p53. Therefore, in here, we aimed to investigate whether UDCA could protect cardiomyocytes (CMs) against hypoxia by regulating expression of HIF‐1α, p53, [Ca2+]i, and S1P‐Gαi‐coupled‐receptor. Cardiomyocytes were isolated from newborn rats (0‐2 days), and hypoxia was induced by using cobalt chloride (CoCl2). Cardiomyocytes were treated with UDCA and cotreated with either FTY720 (S1P‐receptor agonist) or pertussis toxin (PTX; Gαi inhibitor). Cells were subjected for proliferation assay, beating frequency, QuantiGene Plex assay, western blot, immunofluorescence, and calcium imaging. Our findings showed that UDCA counteracted the effects of CoCl2 on cell viability, beating frequency, HIF‐1α, and p53 protein expression. We found that these cardioprotection effects of UDCA were similar to FTY720, S1P agonist. Furthermore, we observed that UDCA protects CMs against CoCl2‐induced [Ca2+]i dynamic alteration. Pharmacological inhibition of the Gαi‐sensitive receptor did not abolish the cardioprotection of UDCA against CoCl2 detrimental effects, except for cell viability and [Ca2+]i. Pertussis toxin is partially effective in inhibiting UDCA protection against CoCl2 effects on CM cell viability. Interestingly, PTX fully inhibits UDCA cardioprotection on CoCl2‐induced [Ca2+]i dynamic changes. We conclude that UDCA cardioprotection against CoCl2‐induced hypoxia is similar to FTY720, and its actions are not fully mediated by the Gαi‐coupled protein sensitive pathways. Ursodeoxycholic acid is the most hydrophilic bile acid and is currently used to treat liver diseases. Recently, UDCA is shown to have a cardioprotection effects; however, the mechanism of UDCA cardioprotection is still poorly understood. The current data generated were the first to show that UDCA is able to inhibit the activation of HIF‐1α and p53 protein during CoCl2‐induced hypoxia in cardiomyocytes. This study provides an insight of UDCA mechanism in protecting cardiomyocytes against hypoxia.

The neuroprotective effects of (S)-3,5-dihydroxyphenylglycine preconditioning in middle cerebral artery occluded rats: a perspective as a contrivance for stroke.

Stroke is one of the fearsome causes of death that leads to high mortality and morbidity worldwide. Apparently, the management of choice for ischemic stroke is either by thrombolysis or thrombectomy. The paramount challenge in most clinical cases is that those interventions are time-dependent as they need to be administered between 3 to 5 hours after the onset of an ischemic event in order to reduce the number of damaged neurons (Ahmed et al., 2013). In fact that a neuron is incapable of regeneration requires the understanding of the pathophysiology of ischemia which may probe an alternative neuroprotective strategy against an ischemic insult. The concept of excitotoxicity by Olney and Sharpe (1969) has garnered a great number of basic researches focusing on neuroprotection against ischemia. This paradigm illustrates the fundamental mechanism related to the ischemic cell injury. In general, this concept explains that during an ischemic event, there are excessive releases of presynaptic glutamate neurotransmitter. Upon these, two families of glutamate receptors (GluRs): the ligand-gated ion channels (ionotropic, iGluRs) and the G protein-coupled receptors (metabotropic, mGluRs) are activated by the toxic level of the glutamate. Over-activation of the postsynaptic glutamate receptors results in massive excitations which may lead to the damage then death of the brain cells. One example of the most studied iGluRs is the N-methyl-D-aspartate (NMDA) receptors (NMDARs) which are endowed with high permeability to calcium ions after being activated by the glutamate. The increase in free calcium ions into the neurons has a major impact on the cellular functions. Obviously, direct inhibition of the NMDARs seems to be the best option in order to arrest the excitotoxic event. However, this option disrupts the physiological roles of NMADRs as the mediators of the synaptic transmission. Throughout the years, the researchers have focused particularly to minimize the side effects resulted from the inhibition of the NMDARs (Xu et al., 2013). Fascinatingly, in contrast, moderate levels of glutamate agonists protect neurons from damage when subsequently exposed to the toxic levels of glutamate. The term preconditioning was first coined in the ischemic myocardial cells (Murry et al., 1986). Ischemic preconditioning involves a brief exposure of certain level of ischemic insult which results in a fundamental response of protection against injury after subsequent severe ischemic attack. However, due to the fact that neurons are irreversible to damage, ischemic preconditioning is a less favorable approach for ischemic stroke patients. Pharmacological preconditioning poses similar protective effects as ischemic preconditioning without the need of any brief ischemic insults (Balzan et al., 2014). Pharmacological preconditioning appears to be an appealing avenue for the patients who have a higher risk toward suffering of ischemic injury after a brain surgery such as endarterectomy or cerebral aneurysm repair. One of the Group I mGluRs selective agonists, (S)-3,5-dihydroxyphenylglycine ((S)-3,5-DHPG) has been shown to elicit neuroprotection by preconditioning during an ischemic event in vitro. Using hippocampal slice cultures, it has been demonstrated that 2 hours of (S)-3,5-DHPG preconditioning before exposing the cells to the excitotoxic level of NMDA has elicited reduction in the propidium iodide uptakes, prevention of cell nucleus fragmentations and decrement in caspase-3 activities which is associated with the depression of the NMDA induced inward currents (Blaabjerg et al., 2003). Furthermore, those significant observations were evidenced in the 10 and 100 μM as compared to 1 μM (S)-3,5-DHPG preconditioning. Our present work confirms the neuroprotective effects of (S)-3,5-DHPG preconditioning by using the middle cerebral artery occluded Sprague-Dawley rat as an acute ischemic stroke model (Nik Ramli et al., 2015). In general, we observed that 1 and 10 μM (S)-3,5-DHPG preconditioning significantly reduced the severity of the ischemic injuries after 24 hours of the arterial occlusion. Nevertheless, no significant difference in ischemic injury observed in the 100 μM (S)-3,5-DHPG preconditioning. In fact that the lower dose of (S)-3,5-DHPG preconditioning results in neuroprotection, indicating that the outcomes may facilitate the body systems to keep the constant internal environment via nervous, hormonal, vascular, immune and other systems as a whole. Stroke results in neuronal death which is associated with the major impairment of sensory and motor functions. It is imperative to carry out the sensory and motor assessments in the stroke experimental design in order to monitor the outcomes. Neurological Stroke Scales (NSS) is one of the most common scoring tests used to measure various degrees of neurological impairments such as orientation, motor strength and verbal communication in stroke patients. This test has been modified for assessing neurological impairments in stroke animals, and is named as the modified neurological severity scores (mNSS). mNSS is a general composite test that evaluates several aspects of stroke impairment including motor, sensory, coordination and reflexes. We observed that 1 and 10 μM (S)-3,5-DHPG preconditioning significantly improved the mNSS of the stroke when compared to control ischemic rats (Nik Ramli et al., 2015). On the other hand, 100 μM (S)-3,5-DHPG preconditioning did not show any significant improvement in the mNSS with stroke when compared to control ischemic rats. Infarct volume is an essential indicator for the level of severity of the ischemic stroke. 2,3,5-Triphenyltetrazolium chloride (TTC) staining is frequently used to determine the degree of infarct induced by focal cerebral ischemia in rats or mice. Using this technique, we observed that the brains of 1 and 10 μM (S)-3,5-DHPG preconditioned rats depicted lesser infarct volume whilst the 100 μM (S)-3,5-DHPG preconditioned rats posed increments in the infarct volume when compared to control ischemic rats. The importance of the significant reduction of the ischemic volumes in 1 and 10 μM (S)-3,5-DHPG preconditioned rats indicates that the penumbra areas have been successfully salvaged and protected by the (S)-3,5-DHPG preconditioning. One of the main outcomes following ischemic insult is the perturbation of blood brain barriers permeability which consequently leads to edema of the brain, infiltration of leukocytes and increases risks towards spontaneous hemorrhagic events. To further confirm the effect of (S)-3,5-DHPG in reducing the ischemic severity, we quantitated the neuron specific enolase (NSE) of the rats blood serum. It has been reported that NSE is one of the specific biomarkers for prediction of ischemic severity in both experimental and clinical cases (Bharosay et al, 2012). We observed that there were significant reductions in the NSE levels of the 1 and 10 μM (S)-3,5-DHPG as compared to 100 of μM (S)-3,5-DHPG preconditioned rats which implied that the disruption of blood brain barriers were minimal during the ischemic insult when the rats were preconditioned with lower doses. Many researchers have suggested the potential effects of specific glutamate receptor agonists as the neuroprotective agents which were achieved by direct activations of the protective cellular pathways via the pharmacological preconditioning (Pellegrini-Giampietro, 2003). Nevertheless, the fates of the cells depend on the intensity of the stimuli applied which are evidenced by our findings. The medium intensity stimulus such as 1 or 10 μM (S)-3,5-DHPG preconditioning is committed to the cell protection which resulted in reduction of brain damages, while extreme intensity as such 100 μM (S)-3,5-DHPG preconditioning leads to apoptosis and necrosis which were similar in those of ischemic rats without preconditioning (Figure 1). Figure 1 Dose dependent preconditioning effects of (S)-3,5-DHPG against ischemia Group I mGluRs represent important sites for interactions between numerous drugs, whereas (S)-3,5-DHPG is known to modulate several pathways via various types of neurotransmitters. The stimulation of phospholipase C by group I mGluRs is known to escalate both phosphoinositide turnover and endoplasmic reticulum (ER) stores of Ca2+ which relatively effects both neuronal development and necrosis. In addition, group 1 mGluRs activities also lead to diacylglycerol (DAG) formation, a cofactor that remains at cell membrane and further activates protein kinase C. Extended cell culture and animal studies of hypoxia and preconditioning with low concentration of general anesthetic had discussed the effects of moderate release of Ca2+ into cytosolic space from ER via IP3, resulting in NMDA receptor inhibition and internalization while initiating endogenous neuroprotective mechanism such as the PI3-AKT pathway (Wei and Inan, 2013). On the contrary, application of high dose with prolonged duration of stimulation causes overactivation of IP3 receptors and excessive release of Ca2+ from ER, eventually results in apoptosis and protein trafficking. Due to the nature of neuron which is irreversible to damage, ischemic preconditioning is unlikely to give any benefit to ischemic stroke patients. However, preconditioning is an appealing avenue for surgical procedures which predisposed the patients with higher risk of ischemic brain injury such as endarterectomy and cerebral aneurysm surgery. We demonstrated that preconditioning with lower doses of (S)-3,5-DHPG is protective against subsequent ischemic insult in the acute ischemic stroke rats. Therefore, understanding of mechanisms underlying the preconditioning effect of (S)-3,5-DHPG is a critical point of salvation in this area.