Diego Pérez-Rodríguez

Unfolded protein response to global ischemia following 48 h of reperfusion in the rat brain: the effect of age and meloxicam

The unfolded protein response (UPR) in the hippocampal regions Cornu Ammonis 1 hippocampal region, Cornu Ammonis 3 hippocampal region, and dentate gyrus, as well as in the cerebral cortex of 3‐month‐old and 18‐month‐old rats were studied in a model of 15 min of global cerebral ischemia followed by 48 h of reperfusion. UPR was measured by quantifying the protein disulfide isomerase (PDI), C/EBP‐homologous protein (CHOP), GRP78 and GRP94 transcripts using qPCR and the amounts of PDI and GRP78 by western blot. The study shows how the mRNA levels of these genes were similar in 3‐month‐old and 18‐month‐old sham‐operated animals, but the ischemic insult elicited a noticeable increase in the expression of these genes in young animals that was scarcely appreciable in older animals. The striking increase in the mRNA levels of these genes in 3‐month‐old animals was abolished or even reverted by treatment with meloxicam, an anti‐inflammatory agent. Western blot assays showed that the UPR was still detectable 48 h after ischemia in some of the studied areas, and provided evidence that the UPR is different between young and older animals. Western blot assays carried out in young animals also showed that meloxicam elicited different effects on the levels of PDI and GRP78 in the cerebral cortex and the hippocampus. We conclude that the UPR response to ischemic/reperfusion insult is age‐ and probably inflammation‐dependent and could play an important role in ischemic vulnerability. The UPR appears to be strongly decreased in aged animals, suggesting a reduced ability for cell survival.

Hippocampus and cerebral cortex present a different autophagic response after oxygen and glucose deprivation in an ex vivo rat brain slice model

To evaluate the neuroprotective role of autophagy in the cerebral cortex and hippocampus using an ex vivo animal model of stroke in brain slices.

Post‐ischemic salubrinal treatment results in a neuroprotective role in global cerebral ischemia

This study describes the neuroprotective effect of treatment with salubrinal 1 and 24 h following 15 min of ischemia in a two‐vessel occlusion model of global cerebral ischemia. The purpose of this study was to determine if salubrinal, an enhancer of the unfolded protein response, reduces the neural damage modulating the inflammatory response. The study was performed in CA1 and CA3 hippocampal areas as well as in the cerebral cortex whose different vulnerability to ischemic damage is widely described. Characterization of proteins was made by western blot, immunofluorescence, and ELISA, whereas mRNA levels were measured by Quantitative PCR. The salubrinal treatment decreased the cell demise in CA1 at 7 days as well as the levels of matrix metalloprotease 9 (MMP‐9) in CA1 and cerebral cortex at 48 h and ICAM‐1 and VCAM‐1 cell adhesion molecules. However, increases in tumor necrosis factor α and nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) inflammatory markers were observed at 24 h. Glial fibrillary acidic protein levels were not modified by salubrinal treatment in CA1 and cerebral cortex. We describe a neuroprotective effect of the post‐ischemic treatment with salubrinal, measured as a decrease both in CA1 cell demise and in the blood–brain barrier impairment. We hypothesize that the ability of salubrinal to counteract the CA1 cell demise is because of a reduced ability of this structure to elicit unfolded protein response which would account for its greater ischemic vulnerability. Data of both treated and non‐treated animals suggest that the neurovascular unit present a structure‐dependent response to ischemia and a different course time for CA1/cerebral cortex compared with CA3. Finally, our study reveals a high responsiveness of endothelial cells to salubrinal in contrast to the limited responsiveness of astrocytes.

Neuroprotective effect of 2-hydroxy arachidonic acid in a rat model of transient middle cerebral artery occlusion

Stroke modifies the composition of cell membranes by eliciting the breakdown of membrane phospholipids whose products, such as arachidonic acid (AA), are released in the cytosol. The action of enzymes such as cyclooxygenases on AA leads to inflammatory stimuli and increases the cell oxidative stress. We report here the neuroprotective effect of 2-hydroxyarachidonic acid (2OAA), a cyclooxygenase inhibitor derived from AA, as a promising neuroprotective therapy against stroke. The effect of a single dose of 2OAA, administered intragastrically 1h after the ischaemic insult, in a rat model of transient middle cerebral artery occlusion (tMCAO) was tested after 24h of reperfusion. Infarct volume was measured by TTC method to evaluate the neuroprotective effect. Levels of phospholipids and neutral lipids were measured by thin-layer chromatography. The expression of cPLA2 and sPLA2 phospholipases responsible for the cleavage of membrane phospholipids, as well as the expression of antioxidant enzymes, was measured by qPCR. Lipid peroxidation was measured as the concentration of malondialdehyde and 4-hydroxynonenal. The treatment with 2OAA reduced the infarct volume and prevented ischaemia-induced increases in transcription levels of free fatty acid (FFAs), as well as in both phospholipases A2 (cPLA2 and sPLA2). The lipid peroxidation and the transcription levels of antioxidant enzymes induced by ischaemia were also decreased by this treatment. We conclude that 2OAA treatment results in a strong neuroprotective effect that seems to rely on a decrease in PLA2 transcriptional activity. This would reduce their action on the membrane phospholipids reducing reactive oxygen and nitrogen species generated by FFAs. Based on the transcriptional activity of the antioxidant enzymes, we conclude that the treatment prevents oxidative stress rather than promoting the antioxidant response. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá.

Salubrinal and robenacoxib treatment after global cerebral ischemia. Exploring the interactions between ER stress and inflammation

Background: Blood reperfusion of the ischemic tissue after stroke promotes increases in the inflammatory response as well as accumulation of unfolded/misfolded proteins in the cell, leading to endoplasmic reticulum (ER) stress. Both Inflammation and ER stress are critical processes in the delayed death of the cells damaged after ischemia. The aim of this study is to check the putative synergic neuroprotective effect by combining anti‐inflammatory and anti‐ER stress agents after ischemia. Methods: The study was performed on a two‐vessel occlusion global cerebral ischemia model. Animals were treated with salubrinal one hour after ischemia and with robenacoxib at 8 h and 32 h after ischemia. Parameters related to the integrity of the blood–brain barrier (BBB), such as matrix metalloproteinase 9 and different cell adhesion molecules (CAMs), were analyzed by qPCR at 24 h and 48 h after ischemia. Microglia and cell components of the neurovascular unit, including neurons, endothelial cells and astrocytes, were analyzed by immunofluorescence after 48 h and seven days of reperfusion. Results: Pharmacologic control of ER stress by salubrinal treatment after ischemia, revealed a neuroprotective effect over neurons that reduces the transcription of molecules involved in the impairment of the BBB. Robenacoxib treatment stepped neuronal demise forward, revealing a detrimental effect of this anti‐inflammatory agent. Combined treatment with robenacoxib and salubrinal after ischemia prevented neuronal loss and changes in components of the neurovascular unit and microglia observed when animals were treated only with robenacoxib. Conclusion: Combined treatment with anti‐ER stress and anti‐inflammatory agents is able to provide enhanced neuroprotective effects reducing glial activation, which opens new avenues in therapies against stroke.

A role for lipids as agents to alleviate stroke damage: the neuroprotective effect of 2-hydroxy arachidonic acid

Cerebrovascular accident (CVA) or stroke is one of the world’s leading causes of death and permanent disability. The high social and medical costs associated with this pathology mean there is an urgent need to find effective therapies. Occlusion of the middle cerebral artery (MCAO), mainly by clots, is the origin of most CVAs in humans. The vessel occlusion (ischemic stroke) presents as a region with a significant reduction in blood flow, known as the ischemic core. This is surrounded by a region called the penumbra, where the blood flow is only partially reduced and neurons can still survive if they are able to recover their homeostatic balance (Durukan and Tatlisumak, 2007). Both the ischemic core and penumbra can be reproduced in experimental transient MCAO (tMCAO), a focal cerebral ischemia model widely used to analyze the neuroprotective role of molecules with the potential to become therapeutic drugs. Inflammation and oxidative stress are considered promising pathways in the search for useful targets to reduce stroke-induced damage and to be useful in a clinical context. In this regard, some anti-inflammatory drugs have exhibited neuroprotective effects in different animal models of ischemia (Candelario-Jalil and Fiebich, 2008). Putative therapies for stroke based on decreasing reactive oxygen species (ROS) and reactive nitrogen species (RNS), either by inhibiting their formation or by increasing the cell antioxidant power are also being studied, and some of them have reached advanced phases of clinical trials (Shirley et al., 2014).

Celecoxib treatment improves neurological deficit and reduces selective neuronal loss and glial response in rats following transient middle cerebral artery occlusion

Areas of selective neuronal loss (SNL) represent the first morphologic signs of damage in the penumbra region and are considered putative targets for ischemic stroke therapy. We performed a novel assessment of measuring the effects of the anti-inflammatory agent celecoxib by analyzing simultaneously the different neural populations (neurons, astrocytes, and microglia cells) in SNL and non-SNL areas. Rats were subjected to 1 hour of middle cerebral artery occlusion (MCAO) and treated with celecoxib 1 and 24 hours after ischemia. Infarct volume measurements and triple immunostaining of neurons (neuronal nuclear antigen), microglia (ionized calcium-binding adaptor molecule 1), and astroglia were performed after 12 and 48 hours of reperfusion. Motor response was tested by standard behavioral assays at 3, 12, 24, and 48 hours. Confocal analysis revealed that the percentage of SNL areas, microglia densities, and glial activation increased at 48 hours of reperfusion. Celecoxib treatment improved the neurologic deficit, reduced the infarct volume by 50% after 48 hours of reperfusion, and resulted in a reduced percentage of SNL areas and microglia and astroglia reactivity after 48 hours of reperfusion. This study proves, for the first time, that celecoxib presents postischemic neuroprotective effects in a transient MCAO model, prevents or delays the presence of SNL areas, and reduces glial activation.

Brain-derived neurotrophic factor alleviates the oxidative stress induced by oxygen and glucose deprivation in an ex vivo brain slice model: GONZÁLEZ-RODRÍGUEZ et al.

Brain‐derived neurotrophic factor (BDNF) is considered as a putative therapeutic agent against stroke. Since BDNF role on oxidative stress is uncertain, we have studied this role in a rat brain slice ischemia model, which allows BDNF reaching the neural parenchyma. Hippocampal and cerebral cortex slices were subjected to oxygen and glucose deprivation (OGD) and then returned to normoxic conditions (reperfusion‐like, RL). OGD/RL increased a number of parameters mirroring oxidative stress in the hippocampus that were reduced by the BDNF presence. BDNF also reduced the OGD/RL‐increased activity in a number of antioxidant enzymes in the hippocampus but no effects were observed in the cerebral cortex. In general, we conclude that alleviation of oxidative stress by BDNF in OGD/RL‐exposed slices relies on decreasing cPLA2 activity, rather than modifying antioxidant enzyme activities. Moreover, a role for the oxidative stress in the differential ischemic vulnerability of cerebral cortex and hippocampus is also supported.