Intranasal guanosine administration presents a wide therapeutic time window to reduce brain damage induced by permanent ischemia in rats

In addition to its intracellular roles, the nucleoside guanosine (GUO) also has extracellular effects that identify it as a putative neuromodulator signaling molecule in the central nervous system. Indeed, GUO can modulate glutamatergic neurotransmission, and it can promote neuroprotective effects in animal models involving glutamate neurotoxicity, which is the case in brain ischemia. In the present study, we aimed to investigate a new in vivo GUO administration route (intranasal, IN) to determine putative improvement of GUO neuroprotective effects against an experimental model of permanent focal cerebral ischemia. Initially, we demonstrated that IN [³H] GUO administration reached the brain in a dose-dependent and saturable pattern in as few as 5 min, presenting a higher cerebrospinal GUO level compared with systemic administration. IN GUO treatment started immediately or even 3 h after ischemia onset prevented behavior impairment. The behavior recovery was not correlated to decreased brain infarct volume, but it was correlated to reduced mitochondrial dysfunction in the penumbra area. Therefore, we showed that the IN route is an efficient way to promptly deliver GUO to the CNS and that IN GUO treatment prevented behavioral and brain impairment caused by ischemia in a therapeutically wide time window.     

Pituitary adenylate cyclase-activating polypeptide is up-regulated in cortical pyramidal cells after focal ischemia and protects neurons from mild hypoxic/ischemic damage

The protective effect of pituitary adenylate cyclase-activating polypeptide (PACAP) in stroke models is poorly understood. We studied patterns of PACAP, vasoactive intestinal peptide, and the PACAP-selective receptor PAC1 after middle cerebral artery occlusion and neuroprotection by PACAP in cortical cultures exposed to oxygen/glucose deprivation (OGD). Within hours, focal ischemia caused a massive, NMDA receptor (NMDAR)-dependent up-regulation of PACAP in cortical pyramidal cells. PACAP expression dropped below the control level after 2 days and was normalized after 4 days. Vasoactive intestinal peptide expression was regulated oppositely to that of PACAP. PAC1 mRNA showed ubiquitous expression in neurons and astrocytes with minor changes after ischemia. In cultured cortical neurons PACAP27 strongly activated Erk1/2 at low and p38 MAP kinase at higher nanomolar concentrations via PAC1. In astrocyte cultures, effects of PACAP27 on Erk1/2 and p38 were weak. During OGD, neurons showed severely reduced Erk1/2 activity and dephosphorylation of Erk1/2-regulated Ser112 of pro-apoptotic Bad. PACAP27 stimulation counteracted Erk1/2 inactivation and Bad dephosphorylation during short-term OGD but was ineffective after expanded OGD. Consistently, PACAP27 caused MEK-dependent neuroprotection during mild but not severe hypoxic/ischemic stress. While PACAP27 protected neurons at 1–5 nmol/L, full PAC1 activation by 100 nmol/L PACAP exaggerated hypoxic/ischemic damage. PACAP27 stimulation of astrocytes increased the production of Akt-activating factors and conferred ischemic tolerance to neurons. Thus, ischemia-induced PACAP may act via neuronal and astroglial PAC1. PACAP confers protection to ischemic neurons by maintaining Erk1/2 signaling via neuronal PAC1 and by increasing neuroprotective factor production via astroglial PAC1.     

Effects of PACAP on in vitro and in vivo neuronal cell death, platelet aggregation, and production of reactive oxygen radicals

Pituitary adenylate cyclase activating polypeptide (PACAP) exerts neuroprotective effects in various in vitro and in vivo models of cerebral pathologies. It has been shown that PACAP protects neurons in rat models of both global and focal ischemia. In the present study, we investigated factors that may play a role in the neuroprotective effects of PACAP. PACAP strongly reduced the anisomycin-induced apoptosis of PC12 cells, which was abolished in a PKA-deficient PC12 cell line (A126). This effect was also observed in vivo, in permanent occlusion of the middle cerebral artery, where the number of TUNEL-positive neurons was significantly reduced in the ischemic core of PACAP-treated animals. Our results show that PACAP has a minor antioxidant effect in a non-cellular in vitro system, and has considerable antioxidant effects in an in vitro red blood cell filtration model. PACAP had no effect on platelet aggregation induced by collagen, ADP or epinephrine. Our results demonstrate that the effects of PACAP on delayed neuronal death may play a significant role in the reduction of the infarct size in vivo, but the antioxidant effect could only be observed at concentrations higher than that used in the model of focal ischemia.     

Effect of middle cerebral artery occlusion on the passage of pituitary adenylate cyclase activating polypeptide across the blood-brain barrier in the rat

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been shown to be a potent neuroprotective agent in global and focal ischemia. We demonstrated that PACAP could cross the blood-brain barrier (BBB) by a saturable transport system, and a systemic administration of PACAP reduced the infarct induced by unilateral middle cerebral artery occlusion (MCAO). Therefore, we studied whether this transport system is affected by MCAO in the rat. The entry of PACAP38 into the brain was compared in five groups: control, 4, 6, 24, and 48 h after MCAO. [(125)I]PACAP38 was injected intravenously and serum and various brain regions were collected 3 min later. The rate of entry into the brain of PACAP38 was also determined. We showed that PACAP entered the rat brain via a rapid transport system when the BBB is intact. After transient (2 h) unilateral MCAO, all regions of the brain, showed a selective increase in the passage of PACAP38 across the BBB after 4 h after the occlusion, which was not related to any generalized change in the permeability of the BBB, as measured with albumin. A significant decrease in the amount of PACAP38 entering the brain was observed in the 6- and 24-h groups, but it returned to the baseline level in the 48-h group. These results suggest that focal cerebral ischemia can selectively modify the passage of PACAP38 across the BBB, in both damaged and undamaged sides of the brain, and that these changes in influx are not solely due to the disruption of BBB. These findings imply the necessity of adjusting the dose of intravenously administered PACAP38 in order to maximize its therapeutic effect on the brain damage resulting from focal ischemia     

Neuroprotection with the CXCL8 inhibitor repertaxin in transient brain ischemia

Infiltration of polymorphonuclear neutrophils (PMNs) is thought to play a role in ischemic brain damage. The present study investigated the effect of repertaxin, a new noncompetitive allosteric inhibitor for the receptors of the inflammatory chemokine CXC ligand 8 (CXCL8)/interleukin-8 (IL-8), on PMN infiltration and tissue injury in rats. Cerebral ischemia was induced by permanent or transient occlusion of the middle cerebral artery and myeloperoxidase activity, a marker of PMN infiltration, and infarct volume were evaluated 24 h later. Repertaxin (15 mg/kg) was administered systemically at the time of ischemia and every 2 h for four times. In permanent ischemia repertaxin reduced PMN infiltration by 40% in the brain cortex but did not limit tissue damage. In transient ischemia (90-min ischemia followed by reperfusion), repertaxin inhibited PMN infiltration by 54% and gave 44% protection from tissue damage. Repertaxin had anti-inflammatory and neuroprotective effects also when given at reperfusion and even at 2 h of reperfusion. The protective effect of repertaxin did not interfere with brain levels of the chemokine. Since the PMN infiltration and its inhibition by repertaxin were comparable in the two models we conclude that reperfusion induces PMN activation, and inhibition of CXCL8 by repertaxin might be of pharmacological interest in transient ischemia.     

Valproic acid reduces brain damage induced by transient focal cerebral ischemia in rats: potential roles of histone deacetylase inhibition and heat shock protein induction

Growing evidence from in vitro studies supports that valproic acid (VPA), an anti-convulsant and mood-stabilizing drug, has neuroprotective effects. The present study investigated whether VPA reduces brain damage and improves functional outcome in a transient focal cerebral ischemia model of rats. Subcutaneous injection of VPA (300 mg/kg) immediately after ischemia followed by repeated injections every 12 h, was found to markedly decrease infarct size and reduce ischemia-induced neurological deficit scores measured at 24 and 48 h after ischemic onset. VPA treatment also suppressed ischemia-induced neuronal caspase-3 activation in the cerebral cortex. VPA treatments resulted in a time-dependent increase in acetylated histone H3 levels in the cortex and striatum of both ipsilateral and contralateral brain hemispheres of middle cerebral artery occlusion (MCAO) rats, as well as in these brain areas of normal, non-surgical rats, supporting the in vitro finding that VPA is a histone deacetylase (HDAC) inhibitor. Similarly, heat shock protein 70 (HSP70) levels were time-dependently up-regulated by VPA in the cortex and striatum of both ipsilateral and contralateral sides of MCAO rats and in these brain areas of normal rats. Altogether, our results demonstrate that VPA is neuroprotective in the cerebral ischemia model and suggest that the protection mechanisms may involve HDAC inhibition and HSP induction.     

Effect of Baicalin on Matrix Metalloproteinase-9 Expression and Blood–Brain Barrier Permeability Following Focal Cerebral Ischemia in Rats

Focal cerebral ischemia results in an increased expression of matrix metalloproteinase-9 (MMP-9), which induces vasogenic brain edema via disrupting the blood–brain barrier (BBB) integrity. Recent studies from our laboratory showed that baicalin reduces ischemic brain damage by inhibiting inflammatory reaction and neuronal apoptosis in a rat model of focal cerebral ischemia. In the present study, we first explored the effect of baicalin on the neuronal damage, brain edema and BBB permeability, then further investigated its potential mechanisms. Sprague–Dawley rats underwent permanent middle cerebral artery occlusion (MCAO). Baicalin was administrated by intraperitoneally injected twice at 2 and 12 h after the onset of MCAO. Neuronal damage, brain edema and BBB permeability were measured 24 h following MCAO. Expression of MMP-9 protein and mRNA were determined by western blot and RT–PCR, respectively. Expression of tight junction protein (TJP) occludin was detected by western blot. Neuronal damage, brain edema and BBB permeability were significantly reduced by baicalin administration following focal cerebral ischemia. Elevated expression of MMP-9 protein and mRNA were significantly down-regulated by baicalin administration. In addition, MCAO caused the decreased expression of occludin, which was significantly up-regulated by baicalin administration. Our study suggested that baicalin reduces MCAO-induced neuronal damage, brain edema and BBB permeability, which might be associated with the inhibition of MMP-9 expression and MMP-9-mediated occludin degradation.     

Neuroprotective roles of pituitary adenylate cyclase-activating polypeptide in neurodegenerative diseases

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic bioactive peptide that was first isolated from an ovine hypothalamus in 1989. PACAP belongs to the secretin/glucagon/vasoactive intestinal polypeptide (VIP) superfamily. PACAP is widely distributed in the central and peripheral nervous systems and acts as a neurotransmitter, neuromodulator, and neurotrophic factor via three major receptors (PAC1, VPAC1, and VPAC2). Recent studies have shown a neuroprotective role of PACAP using in vitro and in vivo models. In this review, we briefly summarize the current findings on the neurotrophic and neuroprotective effects of PACAP in different brain injury models, such as cerebral ischemia, Parkinson’s disease (PD), and Alzheimer’s disease (AD). This review will provide information for the future development of therapeutic strategies in treatment of these neurodegenerative diseases. [BMB Reports 2014; 47(7): 369-375]     

Protections of SMND-309, a novel derivate of salvianolic acid B,on brain mitochondria contribute to injury amelioration in cerebral ischemia rats

SMND-309, a novel compound named (2E)-2-{6-[(E)-2-carboxylvinyl]-2,3-dihydroxyphenyl}-3-(3,4-dihydroxyphenyl) propenoic acid, is a new derivate of salvianolic acid B. The present study was conducted to investigate whether SMND-309 has a protective effect on brain injury after focal cerebral ischemia, and if it did so, to investigate its effects on brain mitochondria. Adult male SD rats were subjected to middle cerebral artery occlusion (MCAO) by bipolar electro-coagulation. Behavioral tests and brain patho-physiological tests were used to evaluate the damage to central nervous system. Origin targets including mitochondria production of reactive oxygen species, antioxidant potentia, membrane potential, energy metabolism, mitochondrial respiratory enzymes activities and mitochondria swelling degree were evaluated. The results showed that SMND-309 decreased neurological deficit scores, reduced the number of dead hippocampal neuronal cells in accordance with its depression on mitochondria swelling degree, reactive oxygen species production, improvements on mitochondria swelling, energy metabolism, membrane potential level and mitochondrial respiratory chain complex activities. All of these findings indicate that SMND-309 exerted potent neuroprotective effects in the model of permanent cerebral ischemia, contributed to its protections on brain mitochondrial structure and function.     

The neuroprotective effects of PACAP in monosodium glutamate-induced retinal lesion involve inhibition of proapoptotic signaling pathways

Pituitary adenylate cyclase activating polypeptide (PACAP) and its receptors are present in the retina and exert several distinct functions. PACAP has well-known neuroprotective effects in neuronal cultures in vitro and against different insults in vivo. Recently we have shown that PACAP is neuroprotective against monosodium glutamate (MSG)-induced retinal degeneration. In the present study we investigated the possible signal transduction pathways involved in the protective effect of intravitreal PACAP administration against apoptotic retinal degeneration induced by neonatal MSG treatment. MSG induced activation of proapoptotic signaling proteins and reduced the levels of antiapoptotic molecules in neonatal retinas. Co-treatment with PACAP attenuated the MSG-induced activation of caspase-3 and JNK, inhibited the MSG-induced cytosolic translocation of apoptosis inducing factor (AIF) and cytochrome c, and increased the level of phospho-Bad. Furthermore, PACAP treatment alone decreased cytosolic AIF and cytochrome c levels, while PACAP6-38 increased cytochrome c release, caspase-3 and JNK activity and decreased phospho-Bad activity. In summary, our results show that PACAP treatment attenuated the MSG-induced changes in apoptotic signaling molecules in vivo and suggest that also endogenously present PACAP has neuroprotective effects. These results may have further clinical implications in reducing glutamate-induced excitotoxicity in several ophthalmic diseases.     

Autophagy activation is associated with neuroprotection in a rat model of focal cerebral ischemic preconditioning

Several recent studies have showed that autophagy is involved in ischemic brain damage, but it may also play a pro-survival role in ischemic preconditioning. This study was taken to determine the role of autophagy in an animal model of cerebral ischemic preconditioning (IPC). Focal cerebral IPC was produced in rats by a brief ischemic insult followed by permanent focal ischemia (PFI) 24 h later using the suture occlusion technique. The rats were pretreated with intracerebral ventricle infusion of the autophagy inhibitors 3-methyladenine (3-MA) and bafliomycin A1 (Baf A1) or the autophagy inducer rapamycin to evaluate the contribution of autophagy to IPC-induced neuroprotection. The results from electron microscopic examinations and immunofluorescence showed that both IPC and PFI induced autophagy activation, but the extent and persistence of autophagy activation were varied. IPC treatment significantly reduced infarct volume, brain edema and motor deficits after subsequent PFI, whereas 3-MA and Baf A1 suppressed the neuroprotection induced by IPC. 3-MA pretreatment also significantly attenuated upregulation of LC3-II, beclin 1 and HSP70 and downregulation of p62. To further determine if autophagy induction is responsible for IPC-induced neuroprotection, rats were treated with rapamycin 24 h before the onset of PFI. The results showed that rapamycin reduced infarct volume, brain edema and motor deficits induced by PFI. Rapamycin pretreatment also increased the protein levels of LC3-II and beclin 1. These results demonstrate that autophagy activation during IPC offers a remarkable tolerance to a subsequent fatal ischemic insult, and IPC's neuroprotective effects can be mimicked by autophagy inducers.     

Protective effect of melatonin against transient global cerebral ischemia-induced neuronal cell damage via inhibition of matrix metalloproteinase-9

Aims

Melatonin possesses various pharmacological effects including neuroprotective effects against brain ischemia. Post-ischemic increases in matrix metalloproteinase-9 (MMP-9) expression and activity mainly contribute to neuronal damage by degradation of the extracellular matrix. This study was designed to examine whether melatonin has a neuroprotective effect and an influence on MMP-9 in transient global brain ischemia.

Main methods

Mice were subjected to 20 min of global brain ischemia and sacrificed 72 h later. Melatonin (30 mg/kg) was administered 30 min before and 2 h after ischemia as well as once daily until sacrifice.

Key findings

Hippocampal pyramidal cell damage after ischemia was significantly decreased by melatonin. As observed by zymography, melatonin inhibited the increase of MMP-9 activity after ischemia. In the brain sections, the increased gelatinase activity was mainly observed in the hippocampus after ischemia and melatonin also reduced gelatinase activity. The laminin and NeuN expression levels were reduced in the hippocampal CA1 and CA2 regions after ischemia, and melatonin reduced laminin degradation and neuronal loss. A TUNEL assay demonstrated that there were TUNEL-positive cells in the hippocampus and the number of TUNEL-positive cells was significantly decreased by melatonin. There was no difference in the ischemia-induced hippocampal neuronal damage between the vehicle- and melatonin-treated groups of MMP-9 knock-out mice.

Significance

These data demonstrate that melatonin suppressed the occurrence of neuronal injury, which might be partly due to its inhibitory effects on MMP-9 in addition to its anti-oxidative effects. MMP-9 may be an important key target of melatonin in neuroprotection against global ischemia.     

Endogenous PACAP acts as a stress response peptide to protect cerebellar neurons from ethanol or oxidative insult

The rodent cerebellum is richly supplied with PACAPergic innervation. Exogenous pituitary adenylate cyclase-activating polypeptide (PACAP) increases cerebellar granule cell survival and differentiation in culture, and enhances the number of neuroblasts in the molecular and internal granule cell layers (IGL) when injected postnatally into the cerebellum in vivo. Here, we have investigated the role of endogenous PACAP during cerebellar development by comparing the morphology of normal and PACAP-deficient mouse cerebellum, and the response of cerebellar granule cells from normal and PACAP-deficient mice subjected to neurotoxic insult in culture. There was no difference in cerebellar volume or granule cell number, in 11-day-old wild type versus PACAP-deficient mice. Cultured cerebellar neurons from PACAP-deficient and wild type mice also showed no apparent differences in survival and differentiation either under depolarizing conditions, or non-depolarizing conditions in the presence or absence of either dibutyryl cAMP or 100 nM PACAP. However, cultured cerebellar neurons from PACAP-deficient mice were significantly more sensitive than wild type neurons to ethanol- or hydrogen peroxide-induced toxicity. Differential ethanol toxicity was reversed by addition of 100 nM exogenous PACAP, suggesting that endogenous PACAP has neuroprotective activity in the context of cellular insult or stress. The neuroprotective action of PACAP was mimicked by dibutryl cAMP, indicating that it occurred via activation of adenylate cyclase. These results indicate that PACAP might act to protect the brain from paraphysiological insult, including exposure to toxins or hypoxia.     

Ex vivo and in vivo neuroprotection induced by argon when given after an excitotoxic or ischemic insult

In vitro studies have well established the neuroprotective action of the noble gas argon. However, only limited data from in vivo models are available, and particularly whether postexcitotoxic or postischemic argon can provide neuroprotection in vivo still remains to be demonstrated. Here, we investigated the possible neuroprotective effect of postexcitotoxic-postischemic argon both ex vivo in acute brain slices subjected to ischemia in the form of oxygen and glucose deprivation (OGD), and in vivo in rats subjected to an intrastriatal injection of N-methyl-D-aspartate (NMDA) or to the occlusion of middle-cerebral artery (MCAO). We show that postexcitotoxic-postischemic argon reduces OGD-induced cell injury in brain slices, and further reduces NMDA-induced brain damage and MCAO-induced cortical brain damage in rats. Contrasting with its beneficial effect at the cortical level, we show that postischemic argon increases MCAO-induced subcortical brain damage and provides no improvement of neurologic outcome as compared to control animals. These results extend previous data on the neuroprotective action of argon. Particularly, taken together with previous in vivo data that have shown that intraischemic argon has neuroprotective action at both the cortical and subcortical level, our findings on postischemic argon suggest that this noble gas could be administered during but not after ischemia, i.e. before but not after reperfusion has occurred, in order to provide cortical neuroprotection and to avoid increasing subcortical brain damage. Also, the effects of argon are discussed as regards to the oxygen-like chemical, pharmacological, and physical properties of argon.     

Effects of intranasal guanosine administration on brain function in a rat model of ischemic stroke

Ischemic stroke is a major cause of morbidity and mortality worldwide and only few affected patients are able to receive treatment, especially in developing countries. Detailed pathophysiology of brain ischemia has been extensively studied in order to discover new treatments with a broad therapeutic window and that are accessible to patients worldwide. The nucleoside guanosine (Guo) has been shown to have neuroprotective effects in animal models of brain diseases, including ischemic stroke. In a rat model of focal permanent ischemia, systemic administration of Guo was effective only when administered immediately after stroke induction. In contrast, intranasal administration of Guo (In-Guo) was effective even when the first administration was 3 h after stroke induction. In order to validate the neuroprotective effect in this larger time window and to investigate In-Guo neuroprotection under global brain dysfunction induced by ischemia, we used the model of thermocoagulation of pial vessels in Wistar rats. In our study, we have found that In-Guo administered 3 h after stroke was capable of preventing ischemia-induced dysfunction, such as bilateral suppression and synchronicity of brain oscillations and ipsilateral cell death signaling, and increased permeability of the blood-brain barrier. In addition, In-Guo had a long-lasting effect on preventing ischemia-induced motor impairment. Our data reinforce In-Guo administration as a potential new treatment for brain ischemia with a more suitable therapeutic window.