Carlos Alexandre Netto

Preconditioning Prevents the Inhibition of Na+,K+-ATPase Activity after Brain Ischemia

Application of single transient forebrain ischemia (ISC) in adult Wistar rats, lasting 2 or 10 min, caused inhibition of Na+,K+-ATPase activity in cytoplasmic membrane fractions of hippocampus and cerebral cortex immediately after the event. In the 2-min ISC group followed by 60 min of reperfusion, the enzyme inhibition was maintained in the cortex, while there was an increase in hippocampal enzyme activity; both effects were over 1 day after the event. However, in the 10-min ISC group enzyme inhibition had been maintained for 7 days in both cerebral structures. Interestingly, ischemic preconditioning (2-min plus 10-min ISC, with a 24-hour interval in between) prevented the inhibitory effect of ischemia/reperfusion on Na+,K+-ATPase activity observed either after a single insult of 2 min or 10 min ischemia. We suggest that the maintenance of Na+,K+-ATPase activity afforded by preconditioning be related to cellular neuroprotection.

Global ischaemia: Hippocampal pathology and spatial deficits in the water maze

Spatial deficits were assessed in male Wistar rats which had undergone 4 vessel occlusion for 5, 10, 15 or 30 min. Relationships between the extent of brain damage, the duration of 4-vessel occlusion, and the behavioural impairment consequent upon ischaemia were investigated. Starting 13-18 days after occlusion, rats were trained to find a hidden platform in a Morris water maze. All ischaemic groups were impaired on some performance indices relative to controls, in both acquisition and retention of the platform location. Increasing the duration of ischaemia increased behavioural deficits on some measures, but there was no clear-cut evidence that longer durations of ischaemia resulted in increased behavioural impairments. Histological assessment, at two coronal levels in hippocampus and four coronal levels in cortex and striatum, revealed CA1 cell loss in all ischaemic groups, which varied between 10-100% across the range of durations employed. CA1 cell loss increased as both a linear and quadratic function of increasing the duration of ischaemia. In rats subjected to 5-15 min ischaemia, cell loss was almost exclusively confined to the CA1 area. In rats subjected to 30 min ischaemia there was additional, variable damage in hippocampal areas CA2, 3 and 4, substantial cell loss in the striatum (50-70%) and some neuronal damage in the cortex (largely in layer III). However correlations between CA1 cell loss in ischaemic rats and indices of spatial ability were non-significant, despite avoiding bias in the analysis by ensuring that only those rats with submaximal CA1 cell loss estimates and behavioural impairments were included. Given the lack of correlation between damage to the CA1 region and behaviour, it is suggested that CA1 cell loss may not be the only determinant of the water maze deficits displayed by 4-vessel occlusion ischaemic rats.

Repeated Restraint Stress Induces Oxidative Damage in Rat Hippocampus

It has been shown that emotional stress may induce oxidative damage, and considerably change the balance between pro-oxidant and antioxidant factors in the brain. The aim of this study was to verify the effect of repeated restraint stress (RRS; 1 h/day during 40 days) on several parameters of oxidative stress in the hippocampus of adult Wistar rats. We evaluated the lipid peroxide levels (assessed by TBARS levels), the production of free radicals (evaluated by the DCF test), the total radical-trapping potential (TRAP) and the total antioxidant reactivity (TAR) levels, and antioxidant enzyme activities (SOD, GPx and CAT) in hippocampus of rats. The results showed that RRS induced an increase in TBARS levels and in GPx activity, while TAR was reduced. We concluded that RRS induces oxidative stress in the rat hippocampus, and that these alterations may contribute to the deleterious effects observed after prolonged stress.

Total antioxidant capacity is impaired in different structures from aged rat brain

Our data support a disproportion between free radicals levels and scavenging systems activity in different cerebral regions of the aging rat. We investigated the total reactive antioxidant potential and reactivity levels, which represent the total antioxidant capacity, in different cerebral regions of the aging rat (cortex, striatum, hippocampus and the cerebellum). In addition, we have determined several oxidative stress parameters, specifically the free radicals levels, the macromolecules damage (lipid peroxidation and carbonyl content), as well as the antioxidant enzymes activities in different cerebral areas from young (2 months‐old), mature adult (6 months‐old) and old (24 months‐old) male Wistar rats. Free radicals levels, determined by 2′,7′‐dichlorofluorescein diacetate probe, were higher in striatum, cerebellum and hippocampus from aged rats. There was an age‐related increase in lipoperoxidation in hippocampus and cerebral cortex. In the cerebellum, a high activity of superoxide dismutase and a decrease of catalase activity were observed. The striatum exhibited a significant catalase activity decrease; and glutathione peroxidase activity was diminished in the hippocampus of mature and aged rats. There was a marked decrease of total antioxidant capacity in hippocampus in both reactivity and potential levels, whereas striatum and cerebral cortex displayed a reduction on reactivity assay. We suggest that age‐related variations of total antioxidant defenses in brain may predispose structures to oxidative stress‐related neurodegenerative disorders.

Novelty causes time-dependent retrograde amnesia for one-trial avoidance in rats through NMDA receptor- and CaMKII-dependent mechanisms in the hippocampus.

Exposure to a novel environment (an open field) for 2 min, 1 h after one‐trial inhibitory avoidance training, hindered memory of the avoidance task measured 24 h later. The effect was seen regardless of the intensity of the avoidance training footshock. The effect was not seen if the exposure to novelty was carried out 5 min before, or 6 h after, the avoidance training, or if the animals did not perceive the open field as new and react accordingly. The amnesic effect of the novelty presented 1 h after avoidance training was blocked by the intrahippocampal infusion of d‐2‐amino‐5‐phosphono‐pentanoic acid (AP5, 25 nmoles per side) or 1‐(N,O‐bis‐[5‐isoquinolinylsulphonyl]‐N‐methyl‐l‐tyrosyl)‐4‐phenylpiperazine (KN62, 100 μmoles per side) but not by that of C32H25N3O6 (KT5720, 90 μmoles per side) given 5 min before the novelty. In the open field there was habituation, measured by the decrease in exploration between the first and second minute. AP5 and KN62 impaired this habituation, but not KT5720. Exploration of the open field was similar in the groups exposed to the avoidance task 5 min later, or 1 h or 6 h before. Therefore, there was no reciprocity between the effect of the two tasks: novelty was amnesic for the one‐trial avoidance task, but the opposite was not true. The amnesic effect of novelty appears to rely on N‐methyl‐d‐aspartate (NMDA) receptor‐ and calcium/calmodulin‐dependent protein kinase II (CaMKII)‐dependent, but not on PKA‐dependent, aspects of its habituation learning.

Neonatal cerebral hypoxia-ischemia causes lateralized memory impairments in the adult rat.

Neonatal hypoxia-ischemia (HI) has been extensively studied in a rat model characterized by unilateral brain damage (Rice-Vannucci Model). However, as well as in humans, each rat brain hemisphere is distinctly involved in cognitive functions, as for example retrieval of emotionally based memory, and neurochemical asymmetries have been described. In this paper we investigated whether hypoxia-ischemia could cause distinct cognitive deficits depending on which hemisphere is damaged. Seven-day-old male Wistar rats were submitted to permanent occlusion of left or right common carotid artery and were exposed to a mixture of 8% oxygen-92% nitrogen for 2.5 h. On adulthood, these rats were trained in step-down inhibitory avoidance and in two tasks in the Morris water maze. Both experimental groups (right and left lesioned) showed a deficit of retrieval in the inhibitory avoidance task compared to controls, although rats with right hemisphere lesion showed a significantly greater deficit than the left damaged group (P<0.05). In the Morris maze, both damaged groups presented cognitive deficits in the reference memory task (P<0.05), however only the right damaged group had an impairment in the working memory task. Brain coronal areas, at levels +1.20 and -3.30 mm from bregma of both HI groups were smaller than those of control, with no differences between the right and left damaged groups (P<0.05). These results show that cerebral hypoxia-ischemia in neonatal rats causes asymmetric behavioral outcomes depending on which of the hemispheres is lesioned and support the hypothesis of lateralization of cognitive functions in the rodent brain.

Changes in heat shock protein 27 phosphorylation and immunocontent in response to preconditioning to oxygen and glucose deprivation in organotypic hippocampal cultures.

Organotypic hippocampal cultures have been recently used to study in vitro ischaemic neuronal death. Sub-lethal periods of ischaemia in vivo confer resistance to lethal insults and many studies have demonstrated the involvement of heat shock proteins in this phenomenon. We used organotypic hippocampal cultures to investigate the involvement of heat shock protein (HSP) 27 in preconditioning to oxygen and glucose deprivation. Neuronal damage was assessed using propidium iodide fluorescence; HSP27 phosphorylation and immunocontent were obtained using (32)Pi labelling followed by sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotting. We observed that immunocontent of HSP27 was increased after lethal or sub-lethal treatment, indicating it is a response to metabolic stress. Treatments with 5 or 10 min of oxygen and glucose deprivation (OGD) or 1- microM N-methyl-D-aspartate (NMDA) induced tolerance to 40 min of OGD associated with an increase in HSP27 immunocontent and phosphorylation. These data suggest that, in vitro, phosphorylated HSP27 might be involved in preconditioning, probably acting as a modulator of actin filaments or by the blockage of neurodegenerative processes.

Chronic brain hypoperfusion causes early glial activation and neuronal death, and subsequent long-term memory impairment

Reduction of cerebral blood flow is an important risk factor for dementia states and other brain dysfunctions. In present study, the effects of permanent occlusion of common carotid arteries (2VO), a well established experimental model of brain ischemia, on memory function were investigated, as assessed by reference and working spatial memory protocols and the object recognition task; cell damage to the hippocampus, as measured through changes in immunoreactivity for GFAP and the neuronal marker NeuN was also studied. The working hypothesis is that metabolic impairment following hypoperfusion will affect neuron and glial function and result in functional damage. Adult male Wistar rats were submitted to the modified 2VO method, with the right common carotid artery being occluded first and the left one week later, and tested seven days, three and six months after the ischemic event. A significant cognitive deficit was found in both reference and working spatial memory, as well as in the object recognition task, three and six months after surgery. Neuronal death and reactive astrogliosis were already present at 7 days and continued for up to 3 months after the occlusion; interestingly, there was no significant reduction in hippocampal volume. Present data suggests that cognitive impairment caused by brain hypoperfusion is long - lasting and persists beyond the time point of recovery from glial activation and neuronal loss.

Ketogenic diet increases glutathione peroxidase activity in rat hippocampus.

Ketogenic diets have been used in the treatment of refractory childhood epilepsy for almost 80 years; however, we know little about the underlying biochemical basis of their action. In this study, we evaluate oxidative stress in different brain regions from Wistar rats fed a ketogenic diet. Cerebral cortex appears to have not been affected by this diet, and cerebellum presented a decrease in antioxidant capacity measured by a luminol oxidation assay without changes in antioxidant enzyme activities—glutathione peroxidase, catalase, and superoxide dismutase. In the hippocampus, however, we observed an increase in antioxidant activity accompanied by an increase of glutathione peroxidase (about 4 times) and no changes in lipoperoxidation levels. We suggest that the higher activity of this enzyme induced by ketogenic diet in hippocampus might contribute to protect this structure from neurodegenerative sequelae of convulsive disorders.

Effect of various behavioral training and testing procedures on brain β-endorphin-like immunoreactivity and the possible role of β-endorphin in behavioral regulation

Beta-Endorphin-like immunoreactivity is reduced in the rat diencephalon after the animals are exposed for the first time to any of the following behavioral situations: 50 tones (habituation), 50 tone-footshock shuttle avoidance trials, one step-down inhibitory avoidance trial, simple exposure to the avoidance apparatus with no footshocks, or inescapable shock. The effect is not observed when animals are exposed to any of these situations for a second time. The reduction of brain beta-endorphin-like immunoreactivity is attributable to release and subsequent metabolism of the substance, and correlates with the novelty inherent in the diverse training or test situations. The role of beta-endorphin in behavior is discussed in the light of these and previous results which showed that it causes both retrograde amnesia and a facilitation of retrieval. The substance would appear to serve an adaptive function when animals are exposed to a new experience, by inducing a temporary forgetting of the experience together with (or leading to) a state of alertness or preparedness for what may happen next.