The phosphodiesterase type 2 inhibitor BAY 60-7550 reverses functional impairments induced by brain ischemia by decreasing hippocampal neurodegeneration and enhancing hippocampal neuronal plasticity.

Cognitive and affective impairments are the most characterized consequences following cerebral ischemia. BAY 60-7550, a selective phosphodiesterase type 2 inhibitor (PDE2-I), presents memory-enhancing and anxiolytic-like properties. The behavioral effects of BAY 60-7550 have been associated with its ability to prevent hydrolysis of both cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) thereby interfering with neuronal plasticity. Here, we hypothesize that PDE2-I treatment could promote functional recovery after brain ischemia. Mice C57Bl/6 were submitted to bilateral common carotid artery occlusion (BCCAO), an experimental model of transient brain ischemia, for 20 min. During 21 days after reperfusion, the animals were tested in a battery of behavioral tests including the elevated zero maze (EZM), object location task (OLT) and forced swim test (FST). The effects of BAY 60-7550 were evaluated on neuronal nuclei (NeuN), caspase-9, cAMP response element-binding protein (CREB), phosphorylated CREB (pCREB) and brain-derived neurotrophic factor (BDNF) expression in the hippocampus. BCCAO increased anxiety levels, impaired hippocampus-dependent cognitive function and induced despair-like behavior in mice. Hippocampal neurodegeneration was evidenced by a decrease in NeuN and increase incaspase-9 protein levels in BCCAO mice. Ischemic mice also showed low BDNF protein levels in the hippocampus. Repeated treatment with BAY 60-7550 attenuated the behavioral impairments induced by BCCAO in mice. Concomitantly, BAY 60-7550 enhanced expression of pCREB and BDNF protein levels in the hippocampus of ischemic mice. The present findings suggest that chronic inhibition of PDE2 provides functional recovery in BCCAO mice possibly by augmenting hippocampal neuronal plasticity.

4-hydroxy-3-methoxy-acetophenone-mediated long-lasting memory recovery, hippocampal neuroprotection, and reduction of glial cell activation after transient global cerebral ischemia in rats.

4-Hydroxy-3-methoxy-acetophenone (apocynin) is a naturally occurring methoxy-substitute catechol that is isolated from the roots of Apocynin cannabinum (Canadian hemp) and Picrorhiza kurroa (Scrophulariaceae). It has been previously shown to have antioxidant and neuroprotective properties in several models of neurodegenerative disease, including cerebral ischemia. The present study investigates the effects of apocynin on transient global cerebral ischemia (TGCI)-induced retrograde memory deficits in rats. The protective effects of apocynin on neurodegeneration and the glial response to TGCI are also evaluated. Rats received a single intraperitoneal injection of apocynin (5 mg/kg) 30 min before TGCI and were tested 7, 14, and 21 days later in the eight-arm aversive radial maze (AvRM). After behavioral testing, the hippocampi were removed for histological evaluation. The present results confirm that TGCI causes memory impairment in the AvRM and that apocynin prevents these memory deficits and attenuates hippocampal neuronal death in a sustained way. Apocynin also decreases OX-42 and glial fibrillary acidic protein immunoreactivity induced by TGCI. These findings support the potential role of apocynin in preventing neurodegeneration and cognitive impairments following TGCI in rats. The long-term protective effects of apocynin may involve inhibition of the glial response.

NLX-101, a 5-HT1A receptor-biased agonist, improves pattern separation and stimulates neuroplasticity in aged rats.

5-HT1A serotonin receptors may play a role in cognitive function changes related to advanced age. Here, we investigated the effects of acute and repeated treatment with NLX-101 (F15599), a postsynaptic 5-HT1A receptor-biased agonist, and F13714, a presynaptic 5-HT1A receptor-biased agonist on spatial object pattern separation (OPS) in aged (22-24 months) rats. Neuroplasticity markers including brain-derived neurotrophic factor, PSD95, synaptophysin, and doublecortin were evaluated in the hippocampus. Unlike younger rats, aged rats were incapable of discriminating any new position of the objects in the arena, reflecting the detrimental effect of aging on pattern separation. However, aged animals treated with NLX-101 showed a significant cognitive improvement in the OPS test, accompanied by increases in hippocampal brain-derived neurotrophic factor and PSD95 protein levels. In contrast, no improvement in OPS performance was observed when aged rats received F13714. Both F13714 and NLX-101 increased the number of newborn neurons in the hippocampi of aged rats. These findings provide a rationale for targeting post-synaptic 5-HT1A as a treatment for cognitive deficits related to aging.

Activation of 5-HT1A postsynaptic receptors by NLX-101 results in functional recovery and an increase in neuroplasticity in mice with brain ischemia.

Pharmacological interventions that selectively activate serotonin 5-hydroxytryptramine-1A (5-HT1A) heteroreceptors may prevent or attenuate the consequences of brain ischemic episodes. The present study investigated whether the preferential 5-HT1A postsynaptic receptor agonist NLX-101 (a.k.a. F15599) mitigates cognitive and emotional impairments and affects neuroplasticity in mice that are subjected to the bilateral common carotid artery occlusion (BCCAO) model of brain ischemia. The selective serotonin reuptake inhibitor escitalopram (Esc) was used for comparative purposes because it is able to decrease morbidity and improve recovery in stroke patients and ischemic rodents. Sham and BCCAO mice received daily doses of NLX-101 (0.32 mg/kg, i.p) or Esc (20 mg/kg, i.p) for 28 days. During this period, they were evaluated for locomotor activity, anxiety- and despair-related behaviors and hippocampus-dependent cognitive function, using the open field, elevated zero maze, forced swim test and object location test, respectivelly. The mice's brains were processed for biochemical and histological analyses. BCCAO mice exhibited high anxiety and despair-like behaviors and performed worse than controls in the cognitive assessment. BCCAO induced neuronal and dendritic spine loss and decreases in the protein levels of neuronal plasticity markers, including brain-derived neurotrophic factor (BDNF), synaptophysin (SYN), and postsynaptic density protein-95 (PSD-95), in prefrontal cortex (PFC) and hippocampus. NLX-101 and Esc attenuated cognitive impairments and despair-like behaviors in BCCAO mice. Only Esc decreased anxiety-like behaviors due to brain ischemia. Both NLX-101 and Esc blocked the increase in plasma corticosterone levels and, restored BDNF, SYN and PSD-95 protein levels in the hippocampus. Moreover, both compounds impacted positively dentritic remodeling in the hippocampus and PFC of ischemic mice. In the PFC, NLX-101 increased the BDNF protein levels, while Esc in turn, attenuated the decrease in the PSD-95 protein levels induced by BCCAO. The present results suggest that activation of post-synaptic 5-HT1A receptors is the molecular mechanism for serotonergic protective effects in BCCAO. Moreover, post-synaptic biased agonists such as NLX-101 might constitute promising therapeutics for treatment of functional and neurodegenerative outcomes of brain ischemia.

Roflumilast promotes memory recovery and attenuates white matter injury in aged rats subjected to chronic cerebral hypoperfusion.

Chronic cerebral hypoperfusion (CCH) has been associated with aging-related vascular dementia, including Alzheimer's disease. It can be induced by the four-vessel occlusion/internal carotid artery (4VO/ICA) model in aged rats, resulting in persistent memory deficits, white matter injury, and significant neuronal loss in the hippocampus and cerebral cortex. The phosphodiesterase type 4 inhibitor (PDE4-I) roflumilast has been reported to have pro-cognitive effects in several behavioral paradigms. The present study evaluated the effects of repeated roflumilast treatment in aged rats that were subjected to CCH. After surgery, roflumilast (0.003 and 0.01 mg/kg) was administered intraperitoneally once per day for 29 days. Memory performance was assessed in the aversive radial maze (AvRM) 7, 14, and 21 days after CCH. The effects of roflumilast on hippocampal neurodegeneration and white matter injury were investigated using Nissl and Kluver-Barrera staining, respectively. Western blot and RT-qPCR were used to explore microglial polarization using M1 (Iba-1 and iNOS) and M2 (Arginase-1) markers. Chronic cerebral hypoperfusion caused persistent memory deficits, hippocampal neurodegeneration, and vacuolization and fiber disarrangement in white matter. Repeated roflumilast treatment restored CCH-induced cognitive impairments in aged rats but in the absence of the rescue of hippocampal neurons. Attenuation of white matter injury was detected in the optic tract in aged CCH rats that were treated with roflumilast. In vitro, roflumilast increased Arg-1 gene expression in myelin-laden primary microglia. The present data suggest that roflumilast might be useful for the treatment of cognitive sequelae associated with CCH.

Depletion of 5 hydroxy-triptamine (5-HT) affects the antidepressant-like effect of neuronal nitric oxide synthase inhibitor in mice.

Clinical and experimental evidence indicates that nitric oxide (NO) is involved in the genesis of depression as well as in antidepressant drug effects. Inhibitors of nitric oxide synthases (NOS) exert antidepressant-like effect in several animal models, but also interfere with the locomotor activity. The involvement of different isoforms of NOS in the antidepressant-like effects is not clearly established. The objective of this study was to investigate the effects of acute or repeated administration of selective inhibitors of neuronal NOS (nNOS) and induced NOS (iNOS), 7 nitroindazole (7NI) and 1400W, respectively, in mice subjected to open field (OF) and forced swim test (FST). We also investigated if the antidepressant-like effect of nNOS inhibitor, 7NI, was dependent on hippocampal serotonin. The results demonstrated that single or repeated (3 and 7days) administration of 7NI resulted in antidepressant-like effects in mice, evidenced by a significant decrease in immobility time in the FST. However, antidepressant-like effects of the iNOS inhibitor, 1400W, were only identified after repeated administration for 3 or 7days. The effects of both inhibitors were comparable to those obtained with the classical antidepressant fluoxetine. It was also demonstrated that the effect of 7NI was dependent of hippocampal serotonin. We concluded that inhibition of nNOS and iNOS result in antidepressant-like effects, and that these effects hold up after repeated administration.

Cannabidiol reduces neuroinflammation and promotes neuroplasticity and functional recovery after brain ischemia.

This study investigated the effects of cannabidiol (CBD), a non-psychotomimetic phytochemical present in Cannabis sativa, on the cognitive and emotional impairments induced by bilateral common carotid artery occlusion (BCCAO) in mice. Using a multi-tiered behavioral testing battery during 21days, we found that BCCAO mice exhibited long-lasting functional deficits reflected by increase in anxiety-like behavior (day 9), memory impairments (days 12-18) and despair-like behavior (day 21). Short-term CBD 10mg/kg treatment prevented the cognitive and emotional impairments, attenuated hippocampal neurodegeneration and white matter (WM) injury, and reduced glial response that were induced by BCCAO. In addition, ischemic mice treated with CBD exhibited an increase in the hippocampal brain derived neurotrophic factor (BDNF) protein levels. CBD also stimulated neurogenesis and promoted dendritic restructuring in the hippocampus of BCCAO animals. Collectively, the present results demonstrate that short-term CBD treatment results in global functional recovery in ischemic mice and impacts multiple and distinct targets involved in the pathophysiology of brain ischemic injury.

Postischemic fish oil treatment confers task-dependent memory recovery.

A series of our previous studies demonstrated that fish oil (FO), equivalent to 300mg/kg docosahexahenoic acid (DHA), facilitates memory recovery after transient, global cerebral ischemia (TGCI) in the aversive radial maze (AvRM). The present study sought to address two main issues: (i) whether the memory-protective effect of FO that has been observed in the AvRM can be replicated in the passive avoidance test (PAT) and object location test (OLT) and (ii) whether FO at doses that are lower than those used previously can also prevent TGCI-induced memory loss. In Experiment 1, naive rats were trained in the PAT, subjected to TGCI (4-vessel occlusion model), and tested for retrograde memory performance 8 and 15days after ischemia. Fish oil (300mg/kg/day DHA) was given orally for 8days. The first dose was delivered 4h postischemia. In Experiment 2, the rats were subjected to TGCI, treated with the same FO regimen, and then trained and tested in the OLT. In Experiment 3, the rats were trained in the AvRM, subjected to TGCI, administered FO (100, 200, and 300mg/kg DHA), and tested for memory performance up to 3weeks after TGCI. At the end of the behavioral tests, the brains were examined for neurodegeneration and neuroblast proliferation. All of the behavioral tests (PAT, OLT, and AvRM) were sensitive to ischemia, but only the AvRM was able to detect the memory-protective effect of FO. Ischemia-induced neurodegeneration and neuroblast proliferation were unaffected by FO treatment. These results suggest that (i) the beneficial effect of FO on memory recovery after TGCI is task-dependent, (ii) doses of FO<300mg/kg DHA can protect memory function in the radial maze, and (iii) cognitive recovery occurs in the absence of neuronal rescue and/or hippocampal neurogenesis.

Rolipram improves cognition, reduces anxiety- and despair-like behaviors and impacts hippocampal neuroplasticity after transient global cerebral ischemia.

Cognitive impairment, anxiety- and depressive-like symptoms are well recognized outcome of cerebral ischemia in clinical and preclinical settings. Rolipram, a phosphodiesterase-4 (PDE-4) inhibitor, improves cognition and produces anxiolytic- and antidepressant-like effects in rodents. Rolipram also exerts anti-inflammatory effects and enhances survival of newborn hippocampal neurons in mice subjected to transient global cerebral ischemia. Here, we evaluated the effects of chronic rolipram treatment in mice subjected to transient global brain ischemia. C56B6/7 mice were subjected to bilateral common carotid artery occlusion (BCCAO) and were then tested in a multi-tiered behavioral battery including the elevated zero maze (EZM), open field (OF), object location test (OLT), and forced swim test (FST). We also investigated the effects of rolipram on hippocampal neurodegeneration and the expression of the neuronal plasticity markers doublecortin (DCX) and microtubule-associated protein (MAP-2). Ischemic mice exhibited memory deficits OLT, higher levels of anxiety EZM and behavioral despair FST. BCCAO caused neuronal loss in the CA3 hippocampal subfield and basolateral amygdala (BLA). In the hippocampus of BCCAO mice, a disrupted neuronal plasticity was evidenced by decreased DCX expression. Chronic treatment with rolipram attenuated the behavioral effects of BCCAO. Rolipram also decreased neurodegeneration in the CA3 while it increased dendritic arborization of DCX-immunoreactive (DCX-IR) neurons and microtubule associate MAP-2 expression in the hippocampus of BCCAO mice. These data suggest that chronic inhibition of PDE-4 can be a useful therapeutic strategy to improve the emotional and cognitive outcomes of transient global cerebral ischemia.

Phosphodiesterase Inhibition as a Therapeutic Target for Brain Ischemia.

Preclinical studies have shown that phosphodiesterase inhibitors (PDE-Is) represent a potential pharmacological strategy for the treatment of brain ischemia sequelea. PDE-Is 3, 4 and 5 have been tested in several brain ischemia models. All the three PDE-Is after acute or chronic treatment decreased the degree of neurodegeneration and most of them improved functional recovery after brain injury by specific cellular and molecular mechanisms mainly involving an anti-inflammatory and/or neuroprotection action. In contrast to the large number of investigations using PDE-Is in experimental brain ischemia research, the number of clinical studies is still limited. The purpose of this review is to summarize the data currently available on the effects of PDE-Is in experimental models of cerebral ischemia.

Protective effects of cannabidiol against hippocampal cell death and cognitive impairment induced by bilateral common carotid artery occlusion in mice.

The present study investigated whether cannabidiol (CBD), a major non-psychoactive constituent of marijuana, protects against hippocampal neurodegeneration and cognitive deficits induced by brain ischemia in adult mice. Male Swiss mice were subjected to a 17 min of bilateral common carotid artery occlusion (BCCAO) and tested in the Morris water maze 7 days later. CBD (3, 10, and 30 mg/kg) was administered 30 min before and 3, 24, and 48 h after BCCAO. After behavioral testing, the brains were removed and processed to evaluate hippocampal cell survival and degeneration using Nissl staining and FluoroJade C histochemistry, respectively. Astroglial response was examined using immunohistochemistry for glial fibrillary acidic protein (GFAP). CBD (3-30 mg/kg) improved spatial learning performance in BCCAO mice. The Nissl and FJC staining results showed a decrease in hippocampal neurodegeneration after CBD (10 and 30 mg/kg) treatment. GFAP immunoreactivity was also decreased in ischemic mice treated with CBD (30 mg/kg). These findings suggest a protective effect of CBD on neuronal death induced by ischemia and indicate that CBD might exert beneficial therapeutic effects in brain ischemia. The mechanisms that underlie the neuroprotective effects of CBD in BCCAO mice might involve the inhibition of reactive astrogliosis.

Anxiolytic-like and proneurogenic effects of Trichilia catigua ethyl-acetate fraction in mice with cerebral ischemia

Abstract Trichilia catigua A. Juss., Meliaceae, known as "catuaba" in Brazil, has been popularly used as a tonic for fatigue, impotence and memory deficits. Previously, we have demonstrated that T. catigua ethyl-acetate fraction exerted antidepressive-like effects in mice. Affective-like symptoms are also well recognized outcome of cerebral ischemia in clinical and preclinical settings. Therefore, here we evaluated the effects of ethyl-acetate fraction on the emotional outcomes and its relation with hippocampal neurogenesis in ischemic mice. Male Swiss mice were subject to the bilateral common carotid occlusion during 20 min. The animals received ethyl-acetate fraction (400 mg/kg, orally) 30 min before and once per day during 7 days after reperfusion. Emotional outcomes were assessed using the open field test, elevated zero maze, and the tail suspension test. After the behavioral testing, the animals were sacrificed and their brains were processed to immunohistochemistry and Nissl staining. Ischemic mice exhibited anxiogenic-like behaviors in the elevated zero maze, hippocampal neurodegeneration and decreased hippocampal neurogenesis. The anxiogenic-like effect was counteracted by ethyl-acetate fraction administration. Furthermore, ethyl-acetate fraction restored the number of newborn neurons in the dentate gyrus of hippocampus of ischemic mice. In conclusion, T. catigua ethyl-acetate fraction promoted functional recovery and restored hippocampal neurogenesis in ischemic mice.

Cognitive impairment and persistent anxiety-related responses following bilateral common carotid artery occlusion in mice.

The present study examined the behavioral and neurohistological changes induced by the bilateral common carotid artery occlusion (BCCAO) model of brain ischemia in Swiss mice. The post-ischemic behavioral effects of 17min BCCAO were recorded 7, 14, and 28 days after reperfusion in the Morris water maze, open field, and elevated plus maze to assess spatial learning and memory, general locomotor activity, and levels of anxiety-like behavior, respectively. After behavioral testing, the brains were removed and processed to evaluate hippocampal neurodegeneration using Nissl staining and Fluoro-Jade C histochemistry and hippocampal neurogenesis using doublecortin immunohistochemistry. BCCAO induced memory impairment 7 and 14 days after reperfusion, with apparent functional recovery 28 days later. Anxiety-related behaviors remained elevated in ischemic compared to sham mice tested 28 days after reperfusion. Hippocampal neurodegeneration was detected in all hippocampal subfields (CA1-CA4) from day 7 to day 28. Decreased hippocampal neurogenesis was observed 14 and 28 days after BCCAO. The effects of BCCAO on spatial memory were transient, whereas anxiety-like behavior was persistent and might be related to CA3 hippocampal injury induced by BCCAO in mice.