Roberto Farina de Almeida

The Potential Therapeutic Effect of Guanosine after Cortical Focal Ischemia in Rats

Background and Purpose Stroke is a devastating disease. Both excitotoxicity and oxidative stress play important roles in ischemic brain injury, along with harmful impacts on ischemic cerebral tissue. As guanosine plays an important neuroprotective role in the central nervous system, the purpose of this study was to evaluate the neuroprotective effects of guanosine and putative cerebral events following the onset of permanent focal cerebral ischemia. Methods Permanent focal cerebral ischemia was induced in rats by thermocoagulation. Guanosine was administered immediately, 1 h, 3 h and 6 h after surgery. Behavioral performance was evaluated by cylinder testing for a period of 15 days after surgery. Brain oxidative stress parameters, including levels of ROS/RNS, lipid peroxidation, antioxidant non-enzymatic levels (GSH, vitamin C) and enzymatic parameters (SOD expression and activity and CAT activity), as well as glutamatergic parameters (EAAC1, GLAST and GLT1, glutamine synthetase) were analyzed. Results After 24 h, ischemic injury resulted in impaired function of the forelimb, caused brain infarct and increased lipid peroxidation. Treatment with guanosine restored these parameters. Oxidative stress markers were affected by ischemic insult, demonstrated by increased ROS/RNS levels, increased SOD expression with reduced SOD activity and decreased non-enzymatic (GSH and vitamin C) antioxidant defenses. Guanosine prevented increased ROS/RNS levels, decreased SOD activity, further increased SOD expression, increased CAT activity and restored vitamin C levels. Ischemia also affected glutamatergic parameters, illustrated by increased EAAC1 levels and decreased GLT1 levels; guanosine reversed the decreased GLT1 levels and did not affect the EAAC1 levels. Conclusion The effects of brain ischemia were strongly attenuated by guanosine administration. The cellular mechanisms involved in redox and glutamatergic homeostasis, which were both affected by the ischemic insult, were also modulated by guanosine. These observations reveal that guanosine may represent a potential therapeutic agent in cerebral ischemia by preventing oxidative stress and excitotoxicity.

Effects of chronic guanosine treatment on hippocampal damage and cognitive impairment of rats submitted to chronic cerebral hypoperfusion

Chronic cerebral hypoperfusion contributes to a cognitive decline related to brain disorders. Its experimental model in rats is a permanent bilateral common carotid artery occlusion (2VO). Overstimulation of the glutamatergic system excitotoxicity due to brain energetic disturbance in 2VO animals seems to play a pivotal role as a mechanism of cerebral damage. The nucleoside guanosine (GUO) exerts extracellular effects including antagonism of glutamatergic activity. Accordingly, our group demonstrated several neuroprotective effects of GUO against glutamatergic excitotoxicity. Therefore, in this study, we evaluated a chronic GUO treatment effects in rats submitted to 2VO. We evaluated the animals performance in the Morris water maze and hippocampal damage by neurons and astrocytes immunohistochemistry. In addition, we investigated the cerebrospinal fluid (CSF) brain derived neurotrophic factor (BDNF) and serum S100B levels. Additionally, the purine CSF and plasma levels were determined. GUO treatment did not prevent the cognitive impairment promoted by 2VO. However, none of the 2VO animals treated with GUO showed differences in the hippocampal regions compared to control, while 20% of 2VO rats not treated with GUO presented loss of pyramidal neurons and increased glial labeling cells in CA1 hippocampal region. In addition, we did not observe differences in CSF BDNF nor serum S100B levels among the groups. Of note, both the 2VO surgery and GUO treatment changed the purine CSF and plasma profile. In conclusion, GUO treatment did not prevent the cognitive impairment observed in 2VO animals, but our data suggest that GUO could be neuroprotective against hippocampal damage induced by 2VO.

Long-term NMDAR antagonism correlates reduced astrocytic glutamate uptake with anxiety-like phenotype

[This corrects the article on p. 219 in vol. 9, PMID: 26089779.].The role of glutamate N-methyl-D-aspartate receptor (NMDAR) hypofunction has been extensively studied in schizophrenia; however, less is known about its role in anxiety disorders. Recently, it was demonstrated that astrocytic GLT-1 blockade leads to an anxiety-like phenotype. Although astrocytes are capable of modulating NMDAR activity through glutamate uptake transporters, the relationship between astrocytic glutamate uptake and the development of an anxiety phenotype remains poorly explored. Here, we aimed to investigative whether long-term antagonism of NMDAR impacts anxiety-related behaviors and astrocytic glutamate uptake. Memantine, an NMDAR antagonist, was administered daily for 24 days to healthy adult CF-1 mice by oral gavage at doses of 5, 10, or 20 mg/kg. The mice were submitted to a sequential battery of behavioral tests (open field, light–dark box and elevated plus-maze tests). We then evaluated glutamate uptake activity and the immunocontents of glutamate transporters in the frontoparietal cortex and hippocampus. Our results demonstrated that long-term administration of memantine induces anxiety-like behavior in mice in the light–dark box and elevated plus-maze paradigms. Additionally, the administration of memantine decreased glutamate uptake activity in both the frontoparietal cortex and hippocampus without altering the immunocontent of either GLT-1 or GLAST. Remarkably, the memantine-induced reduction in glutamate uptake was correlated with enhancement of an anxiety-like phenotype. In conclusion, long-term NMDAR antagonism with memantine induces anxiety-like behavior that is associated with reduced glutamate uptake activity but that is not dependent on GLT-1 or GLAST protein expression. Our study suggests that NMDAR and glutamate uptake hypofunction may contribute to the development of conditions that fall within the category of anxiety disorders.

Systemic administration of GMP induces anxiolytic-like behavior in rats.

The glutamatergic system has received considerable attention over the last few years as potential target to develop anxiolytic drugs. Guanine based purines (GBPs) play an important neurmodulatory effect in the glutamatergic system. Several studies have shown the ability of the GBPs to reduce glutamatergic activity. In the present study, we investigated the anxiolytic effect of GBPs - by Guanosina Monophosphate (GMP) administration - in rodents. Adult male Wistar rats were pretreated with GMP (10, 25, 50, 100 and 150mg/kg: i.p.); or saline (NaCl 0.9%; i.p.) (control); or, diazepam (2mg/kg: i.p.) (positive control). One hour after the injection, the anxiety-related behaviors for each animal was evaluated in the light/dark, elevated plus-maze, and open field tasks. Additionally, purines concentration in the cerebrospinal fluid (CSF) was verified. The administration of 25 and 50mg/kg GMP was able to promote anxiolytic-like behavior, in the light/dark and elevated plus-maze task, similar to diazepam effect. However, no changes in the open field task, or CSF purines concentration were found for either GMP or diazepam treated animals, when compared with saline group. Thus, this study suggests that acute administration of GMP was able to decrease the levels of anxiety in classical behavioral tasks.

Intracerebroventricular administration of inosine is anticonvulsant against quinolinic acid-induced seizures in mice: an effect independent of benzodiazepine and adenosine receptors.

Inosine (INO) has an anticonvulsant effect against seizures induced by antagonists of GABAergic system. Quinolinic acid (QA) is an agonist NMDA receptors implicated in the neurobiology of seizures. In the present study, we investigated the anticonvulsant effect of intracerebroventricular (i.c.v.) INO administration against QA-induced seizures in adult mice. We also investigated whether the benzodiazepines (BZ) or adenosine (ADO) receptors were involved in the INO effects. Animals were pretreated with an i.c.v. injection of either vehicle or INO before an i.c.v. administration of 4 μl QA (36.8 nmol). All animals pretreated with vehicle followed by QA presented seizures. INO protected against QA-induced seizures in a time and dose dependent manner (up to 60% at 400 nmol, 5 min before QA injection). Diazepam (DZ) and ADO (i.c.v.) also exhibited anticonvulsant effect against QA induced seizures. Additionally, i.p. administration of either flumazenil, a BZ receptor antagonist, or caffeine, an ADO receptor antagonist, did not change the anticonvulsant potency of INO i.c.v. injection, but completely abolished the DZ and ADO anticonvulsant effects, respectively. In conclusion, this study demonstrated that INO exert anticonvulsant effect against hyperactivity of the glutamatergic system independently of BZ or ADO receptors activation.

Olfactory bulbectomy in mice triggers transient and long-lasting behavioral impairments and biochemical hippocampal disturbances.

&NA; Major depressive disorder (MDD) is a neuropsychiatric disease that is associated with profound disturbances in affected individuals. Elucidating the pathophysiology of MDD has been frustratingly slow, especially concerning the neurochemical events and brain regions associated with disease progression. Thus, we evaluated the time‐course (up to 8 weeks) behavioral and biochemical effects in mice that underwent to a bilateral olfactory bulbectomy (OBX), which is used to modeling depressive‐like behavior in rodents. Similar to the symptoms in patients with MDD, OBX induced long‐lasting (e.g., impairment of habituation to novelty, hyperactivity and an anxiety‐like phenotype) and transient (e.g., loss of self‐care and motivational behavior) behavioral effects. Moreover, OBX temporarily impaired hippocampal synaptosomal mitochondria, in a manner that would be associated with hippocampal‐related synaptotoxicity. Finally, long‐lasting pro‐oxidative (i.e., increased levels of reactive oxygen species and nitric oxide and decreased glutathione levels) and pro‐inflammatory (i.e., increased levels of pro‐inflammatory cytokines IL‐1, IL‐6, TNF‐&agr; and decreased anti‐inflammatory cytokine IL‐10 levels) effects were induced in the hippocampus by OBX. Additionally, these parameters were transiently affected in the posterior and frontal cortices. This study is the first to suggest that the transient and long‐lasting behavioral effects from OBX strongly correlate with mitochondrial, oxidative and inflammatory parameters in the hippocampus; furthermore, these effects show a weak correlation with these parameters in the cortex. Our findings highlight the underlying mechanisms involved in the biochemical time course of events related to depressive behavior. HighlightsOlfactory bulbectomy in mice triggers to a transient and long‐lasting behavioral disturbances.Olfactory bulbectomy in mice induces a transient impairment in synaptosomal mitochondria mass and &Dgr;&PSgr;.Olfactory bulbectomy in mice leads to a long‐lasting hippocampal imbalance in redox and inflammatory homeostasis.

Long-term effect of uncomplicated Plasmodium berghei ANKA malaria on memory and anxiety-like behaviour in C57BL/6 mice

BackgroundCerebral malaria, the main complication of Plasmodium falciparum infection in humans, is associated with persistent neurocognitive sequels both in human disease and the murine experimental model. In recent years, cognitive deficits related to uncomplicated (non-cerebral) malaria have also been reported in chronically exposed residents of endemic areas, but not in some murine experimental models of non-cerebral malaria. This study aimed at evaluating the influence of uncomplicated malaria on different behavioural paradigms associated with memory and anxiety-like parameters in a murine model that has the ability to develop cerebral malaria.MethodsPlasmodium berghei ANKA-infected and non-infected C57BL/6 mice were used. Development of cerebral malaria was prevented by chloroquine treatment starting on the fourth day of infection. The control group (non-infected mice) were treated with PBS. The effect of uncomplicated malaria infection on locomotor habituation, short and long-term memory and anxious-like behaviour was evaluated 64 days after parasite clearance in assays including open field, object recognition, Y-maze and light/dark tasks.ResultsPlasmodium berghei ANKA-infected mice showed significant long-lasting disturbances reflected by a long-term memory-related behaviour on open field and object recognition tasks, accompanied by an anxious-like phenotype availed on open field and light-dark tasks.ConclusionsLong-term neurocognitive sequels may follow an uncomplicated malaria episode in an experimental model prone to develop cerebral malaria, even if the infection is treated before the appearance of clinical signs of cerebral impairment.

Corrigendum: Long-term NMDAR antagonism correlates reduced astrocytic glutamate uptake with anxiety-like phenotype

In the subsection “Glutamate Uptake Assay,” the greek letter micro (symbol μ) was accidentally deleted twice probably due to software incompatibility. The correct sentence should read: “Afterwards, 0.66 and 0.33 μCi ml−1L-[3H]glutamate were added to a final 100 μM concentration of glutamate for incubation with hippocampal and cortical samples, respectively.” This correction does not affect the scientific relevance of the results.