Aline de Souza Pagnussat

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.

Resveratrol prevents CA1 neurons against ischemic injury by parallel modulation of both GSK‐3β and CREB through PI3‐K/Akt pathways

Accumulating evidence indicates that resveratrol potently protects against cerebral ischemia damage due to its oxygen free radicals scavenging and antioxidant properties. However, cellular mechanisms that may underlie the neuroprotective effects of resveratrol in brain ischemia are not fully understood yet. This study aimed to investigate the potential association between the neuroprotective effect of resveratrol and the apoptosis/survival signaling pathways, in particular the glycogen synthase kinase 3 (GSK‐3β) and cAMP response element‐binding protein (CREB) through phosphatidylinositol 3‐kinase (PI3‐K)‐dependent pathway. An experimental model of global cerebral ischemia was induced in rats by the four‐vessel occlusion method for 10 min and followed by different periods of reperfusion. Nissl staining indicated extensive neuronal death at 7 days after ischemia/reperfusion. Administration of resveratrol by i.p. injections (30 mg/kg) for 7 days before ischemia significantly attenuated neuronal death. Both GSK‐3β and CREB appear to play a critical role in resveratrol neuroprotection through the PI3‐K/Akt pathway, as resveratrol pretreatment increased the phosphorylation of Akt, GSK‐3β and CREB in 1 h in the CA1 hippocampus after ischemia/reperfusion. Furthermore, administration of LY294002, an inhibitor of PI3‐K, compromised the neuroprotective effect of resveratrol and decreased the level of p‐Akt, p‐GSK‐3β and p‐CREB after ischemic injury. Taken together, the results suggest that resveratrol protects against delayed neuronal death in the hippocampal CA1 by maintaining the pro‐survival states of Akt, GSK‐3β and CREB pathways. These data suggest that the neuroprotective effect of resveratrol may be mediated through activation of the PI3‐K/Akt signaling pathway, subsequently downregulating expression of GSK‐3β and CREB, thereby leading to prevention of neuronal death after brain ischemia in rats.

Resveratrol preconditioning modulates inflammatory response in the rat hippocampus following global cerebral ischemia.

Considerable evidence has been accumulated to suggests that blocking the inflammatory reaction promotes neuroprotection and shows therapeutic potential for clinical treatment of ischemic brain injury. Consequently, anti-inflammatory therapies are being explored for prevention and treatment of these diseases. Induction of brain tolerance against ischemia by pretreatment with resveratrol has been found to influence expression of different molecules. It remains unclear, however, whether and how resveratrol preconditioning changes expression of inflammatory mediators after subsequent global cerebral ischemia/reperfusion (I/R). Therefore, we investigated the effect of resveratrol pretreatment on NF-κB inflammatory cascade, COX-2, iNOS and JNK levels in experimental I/R. Adult male rats were subjected to 10 min of four-vessel occlusion and sacrificed at selected post-ischemic time points. Resveratrol (30 mg/kg) pretreatment was injected intraperitoneally 7 days prior to I/R induction. We found that resveratrol treatment before insult remarkably reduced astroglial and microglial activation at 7 days after I/R. It greatly attenuated I/R-induced NF-κB and JNK activation with decreased COX-2 and iNOS production. In conclusion, the neuroprotection of resveratrol preconditioning may be due in part to the suppression of the inflammatory response via regulation of NF-κB, COX-2 and iNOS induced by I/R. JNK was also suggested to play a protective role through in neuroprotection of resveratrol, which may also be contributing to reduction in neuroinflammation. The study adds to a growing literature that resveratrol can have important anti-inflammatory actions in the brain.

Skilled reaching training promotes astroglial changes and facilitated sensorimotor recovery after collagenase-induced intracerebral hemorrhage.

Spontaneous intracerebral hemorrhage (ICH) is the most devastating type of stroke and a leading cause of disability and mortality worldwide. Although rehabilitation improves recovery after ICH the cellular mechanisms involved are poorly understood. We decided to examine if skilled (SK) and unskilled (US) training after sham or intracerebral hemorrhage (ICH) surgeries would induce GFAP+ astrocytic changes and whether these modifications can be associated with functional improvement. A 4-week course of motor training, involving either skilled and unskilled training began seven days after surgery; sensorimotor recovery was evaluated using Staircase, ladder walk and cylinder tests. Histological and morphometric analyses were used to assess GFAP+ cell bilaterally in forelimb sensorimotor cortex and dorsolateral striatum. All behavioral tests showed that ICH-SK rats experienced a greater degree of recovery when compared to ICH no task or ICH-US groups; no behavioral differences were found among all sham groups. Astrocytic density was increased in all analyzed structures for ICH no task, ICH-SK and ICH-US rats. Morphological analysis revealed an increased number of primary processes in ipsilateral (to lesion) sensorimotor cortex for all ICH groups. Present results also revealed that both ICH and SK induced an increased length of GFAP+ primary process; there was a further increase in length processes for ICH-SK group in sensorimotor cortex and ipsilateral striatum. We suggest that skilled reaching is an effective intervention to promote astrocytic plasticity and recovery after ICH.

Coumestrol has neuroprotective effects before and after global cerebral ischemia in female rats

Global ischemia arising during cardiac arrest or cardiac surgery causes highly selective, delayed death of hippocampal CA1 neurons. Phytoestrogens are naturally occurring plant-derived compounds that are present in the human diet and are considered selective estrogen receptor (ER) modulators. The phytoestrogen coumestrol is a potent isoflavonoid, with binding affinities for both ER-α and ER-β that are comparable to those of 17 b-estradiol. The present study examined the hypothesis that coumestrol protects hippocampal neurons in ovariectomized rats in a model of cerebral global ischemia. Ovariectomized rats were subjected to global ischemia (10 min) or sham surgery and received a single intracerebroventricular or peripheral infusion of 20 μg of coumestrol, 20 μg of estradiol or vehicle 1h before ischemia or 0 h, 3h, 6h or 24h after reperfusion. Estradiol and coumestrol afforded significant neuroprotection in all times of administration, with the exception of estradiol given 24h after the ischemic insult. Animals received icv infusion of the broad-spectrum ER antagonist ICI 182,780 (50 μg) or vehicle into the lateral ventricle just before the E2 or coumestrol administration. The ER antagonist abolished estradiol protection, consistent with a role of classical ERs. In contrast, ICI 182,780 effected only partial reversal of the neuroprotective actions of coumestrol, suggesting that other cellular mediators in addition to classical ERs may be important. Additional research is needed to determine the molecular targets mediating the neuroprotective action of coumestrol and the therapeutic potential of this phytoestrogen in the mature nervous system.

Effects of skilled and unskilled training on functional recovery and brain plasticity after focal ischemia in adult rats.

Stroke is a leading cause of morbidity and mortality worldwide. Recovery of motor function after stroke can be modified by post-injury experience, but most of surviving patients exhibit persistence of the motor dysfunctions even after rehabilitative therapy. In this study we investigated if skilled and unskilled training induce different motor recovery and brain plasticity after experimental focal ischemia. We tested this hypothesis by evaluating the motor skill relearning and the immunocontent of Synapsin-I, PSD-95 and GFAP (pre and post-synaptic elements, as well as surrounding astroglia) in sensorimotor cortex of both hemispheres 6 weeks after endothelin-1-induced focal brain ischemia in rats. Synapsin-I and PSD-95 levels were increased by skilled training in ischemic sensorimotor cortex. The content of GFAP was augmented as a result of focal brain ischemia in ischemic sensorimotor cortex and that was not modified by rehabilitation training. Unexpectedly, animals remained permanently impaired at the end of motor/functional evaluations. Significant modifications in protein expression were not observed in undamaged sensorimotor cortex. We conclude that skilled motor activity can positively affect brain plasticity after focal ischemia despite of no functional improvement in conditions here tested.

An ultrastructural study of cell death in the CA1 pyramidal field of the hippocapmus in rats submitted to transient global ischemia followed by reperfusion

In the course of ischemia and reperfusion a disruption of release and uptake of excitatory neurotransmitters occurs. This excitotoxicity triggers delayed cell death, a process closely related to mitochondrial physiology and one that shows both apoptotic and necrotic features. The aim of the present study was to use electron microscopy to characterize the cell death of pyramidal cells from the CA1 field of the hippocampus after 10 min of transient global ischemia followed by short reperfusion periods. For this study 25 adult male Wistar rats were used, divided into six groups: 10 min of ischemia, 3, 6, 12 and 24 h of reperfusion and an untouched group. Transient forebrain ischemia was produced using the 4‐vessel occlusion method. The pyramidal cells of the CA1 field from rat hippocampus submitted to ischemia exhibited intracellular alterations consistent with a process of degeneration, with varied intensities according to the reperfusion period and bearing both apoptotic and necrotic features. Gradual neuronal and glial modifications allowed for the classification of the degenerative process into three stages: initial, intermediate and final were found. With 3 and 6 h of reperfusion, slight and moderate morphological alterations were seen, such as organelle and cytoplasm edema. Within 12 h of reperfusion, there was an apparent recovery and more ‘intact’ cells could be identified, while 24 h after the event neuronal damage was more severe and cells with disrupted membranes and cell debris were identified. Necrotic‐like neurons were found together with some apoptotic bodies with 24 h of reperfusion. Present results support the view that cell death in the CA1 field of rat hippocampus submitted to 10 min of global transient ischemia and early reperfusion times includes both apoptotic and necrotic features, a process referred to as parapoptosis.

Skilled forelimb reaching in Wistar rats: evaluation by means of Montoya staircase test.

Experimental animals have been used as models for several neurological disorders; their performance in behavioral tests is useful in determining the success of lesion repair procedures and assessing functional recovery. The staircase test is a behavioral test that consists in reaching for food inside a special box and allows for a sensitive measure of skilled reaching by each limb in an independent manner. In most laboratories in the south of Brazil, Wistar rats are used for the study of experimental stroke, hypoxia and peripheral neuropathy, but most studies with the staircase test have used other strains such as Sprague-Dawley and Long-Evans. Because skilled reaching, grasping and performance can differ among strains, the purpose of the present study was to characterize the performance of Wistar rats in the staircase test and determine the effect of median and ulnar nerve crush. Our results with Wistar rats on the staircase test showed that: similar to other strains, Wistar animals can display high performance after 2 weeks of training; the number of animals that attained the inclusion criterion increased by 10% with longer times of training; the stricter criterion of 15 pellets taken can be adopted as study inclusion criterion; the test has an unquestionable value in assessing lateralized deficits, as evidenced by the lack of performance deficit of the non-manipulated forelimb at any time point. These results extend the understanding about the performance of Wistar rats in the staircase test, which will be used for the best training and research using this strain.

Resveratrol treatment has neuroprotective effects and prevents cognitive impairment after chronic cerebral hypoperfusion

Abstract Objective: The present study investigated the neuroprotective effects of Resveratrol (RSV) in rats submitted to chronic cerebral hypoperfusion (CCH) in a model of permanent two-vessel occlusion (2VO). Methods: For this purpose, adult Wistar rats received daily i.p. injections of RSV (20 mg/kg) for 7 days, starting 1 hour after the 2VO procedure. Behavioral testing was run between the 30th and 45th days after the 2VO surgery. Accordingly, spatial working memory function in the Morris water maze was evaluated. At the end of the behavioral assessment (45th day post-surgery) part of experimental animals underwent transcardiac perfusion for histological analysis. Another group was euthanized on the 3rd, 14th, and 45th days post-surgery for nerve growth factor (NGF) evaluation. Results: Resveratrol treatment along 7 days after CCH significantly attenuated pyramidal cell death in the CA1 hippocampal subfield and prevented both spatial working and reference memory impairments. Our results revealed an enhancement of NGF expression 3 days after CCH in all ischemic animals. A late increase in hippocampal NGF levels was detected after 45 days only in CCH-RSV treated animals. Conclusions: Results presented here show morphological and functional neuroprotective actions of RSV treatment for CCH, as well as support the inducing effects of RSV on the expression of NGF and its possible association to the neuroprotective action in this rodent model of vascular dementia.

Transcranial magnetic stimulation combined with upper-limb training for improving function after stroke: A systematic review and meta-analysis.

BACKGROUND Several neuromodulation treatments have been developed, and their effects have been studied in recent years in order to improve neurological rehabilitation after a stroke. The association between upper-limb training and repetitive transcranial magnetic stimulation (rTMS) has provoked controversies and produced inconclusive results. OBJECTIVE The purpose of this study was to investigate the effects of rTMS combined with upper-limb training versus sham rTMS combined with upper-limb training on the upper-limb recovery after a stroke. METHODS A systematic review with meta-analysis was performed. The eligible studies were randomized controlled trials with stroke subjects, and the outcomes were related to upper-limb motor/functional status and spasticity. RESULTS A total of 3234 citations were identified, and 11 studies were included. The meta-analysis included eight studies with 199 participants and did not show any difference between groups, neither for upper-limb function nor for spasticity (upper-limb function [0.03 (95% CI: -0.25 to 0.32; I(2) 0%)] and Modified Ashworth Scale [-0.31 (95% CI: -0.78 to 0.17; I(2) 43%)]). CONCLUSION The current state of the literature is not enough to support the hypothesis that a combination of rTMS and upper-limb training has a stronger effect on upper-limb function than upper-limb training alone.