Search

The inflammatory response plays an important role in neuroprotection and regeneration after ischemic insult. The use of non-steroidal anti-inflammatory drugs has been a matter of debate as to whether they have beneficial or detrimental effects. In this context, the effects of the anti-inflammatory agent meloxicam have been scarcely documented after stroke, but its ability to inhibit both cyclooxygenase isoforms (1 and 2) could be a promising strategy to modulate post-ischemic inflammation. This study analyzed the effect of the anti-inflammatory agent meloxicam in a transient focal ischemia model in rats, measuring its neuroprotective effect after 48 hours and 7 days of reperfusion and the effects of the treatment on the glial scar and regenerative events such as the generation of new progenitors in the subventricular zone and axonal sprouting at the edge of the damaged area. We show that meloxicam’s neuroprotective effects remained after 7 days of reperfusion even if its administration was restricted to the two first days after ischemia. Moreover, meloxicam treatment modulated glial scar reactivity, which matched with an increase in axonal sprouting. However, this treatment decreased the formation of neuronal progenitor cells. This study discusses the dual role of anti-inflammatory treatments after stroke and encourages the careful analysis of both the neuroprotective and the regenerative effects in preclinical studies.Abstract Figure

Ischemic stroke results from the temporary or permanent lack of blood supply in the brain due to the occlusion of a brain blood vessel. Around 85% of patients with cerebrovascular accidents suffer from ischemic strokes. Although cerebrovascular accidents represent the major cause of death and permanent disability worldwide, thus far, only processes addressed at eliminating the vessel obstruction (chemical or mechanical) have been successfully developed. Many neuroprotective strategies have been tested in preclinical studies, but clinical trials have, so far, failed to result in beneficial effects. These issues may be due to the very complex pathophysiology of ischemic stroke, which involves the integration of multiple signaling pathways ultimately resulting in neuronal loss.

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.

141 p. Cerebrovascular accident (stroke) represents one of the most devastating diseases in elderly since it is the main cause of death and permanent disability in adults and the second most common cause of dementia. A very active search for effective neuroprotective therapies exists in this field as a consequence of the lack of effective treatments, the high incidence of this pathology and its social and medical costs. In this study, the post-ischemic effect of celecoxib, a selective cyclooxygenase-2 inhibitor, was evaluated in a rat model of transient middle cerebral artery occlusion (tMCAO), followed by two different times of reperfusion (12 h and 48 h). A standard time of ischemia of 1 h was used. In the first aim of this study, the neuroprotective effects of celecoxib were evaluated measuring both tissue damage and neurological deficits. Triple immunostaining, labelling neurons, microglia and astrocytes, was also carried out to analyze the effect of celecoxib on neuronal demise as well as on microglial and astroglia activation. Treatment with celecoxib reduced the infarct volume, neuronal demise, selective neuronal loss (SNL) areas and glial activation. Accordingly, celecoxib improved the neurological deficit. Data of celecoxib treatment suggest the idea that non-SNL areas progress toward SNL slower in the piriform cortex (Pir) than in the frontoparietal cortex, somatosensory area (FrPaSS), suggesting higher neuronal vulnerability to ischemia in the neocortex (FrPaSS) than in paleocortex (Pir). The second aim was to analyze the effect of celecoxib on ER stress. The study showed that treatment with celecoxib reduced GRP78 levels, increased XBP-1s mRNA levels, did not modify eIF2α phosphorylation or ATF4 transcript levels and reduced the Caspase 12, CHOP and polyubiquitinated protein levels. Moreover, celecoxib increased the transcription for catalytic proteasome subunits (β1, β2 and β5) and BAG1, decreased p62/SQSTM1 levels and BAG3 transcripts levels and did not modify the ratio of LC3BII/I. These results led to the conclusion that celecoxib treatment reduces ER-stress by igniting IRE1 pathway and UPS degradation pathway, and it does not seem to modify autophagy. In the third aim of this study, the effect of celecoxib on oxidative stress was analyzed. Treatment with celecoxib reduced lipid peroxidation and cPLA2 phosphorylation. Celecoxib increased superoxide dismutase (SOD) and total antioxidant capacity (TAC) activities, while it did not modify glutathione peroxidase (GPx) activity. Celecoxib also increased transcript levels of heme oxygenase 1 (HO-1) and glutamate-cysteine ligase modifier subunit (GCLM) but did not modify SOD transcript levels. These data allow the conclusion that celecoxib decreases the oxidative state of the cell by modifying the activity of some antioxidant enzymes and increasing the amount of some antioxidants such as glutathione.

Ischemic stroke is one of the most important causes of death and disability worldwide. Subroutines underlying cell death after stroke are largely unknown despite their importance in the design of novel therapies for this pathology. Necroptosis, a recently described form of regulated cell death, has been related with inflammation and, in some models, with endoplasmic reticulum (ER) stress. We hypothesize that alleviation of ER stress following a salubrinal treatment will reduce the ischemic-dependent necroptosis. To probe the hypothesis, we measured, at 48 and 72 h after transient global cerebral ischemia in rat, in cerebral cortex and cornu ammonis 1, the main hallmarks of necroptosis: mRNA levels and phosphorylation of mixed lineage kinase domain like pseudokinase as well as receptor interacting serine/threonine protein kinase 3, along the years 2017-2018. Selective neuronal loss after 7 days of the ischemic insult, and other markers related with the inflammatory response were also measured. This study shows that necroptosis in cerebral cortex can be detected after 72 h of the insult and seems to be elicited before 48 h of reperfusion. The type of necroptosis here observed seems to be tumor necrosis factor receptor 1 independent. Necroptotic response is less evident in the cornu ammonis 1 hippocampal area than in cerebral cortex. The treatment with salubrinal administered 1 and 24 h after the ischemia, decreased the necroptotic marker levels and reduced the areas of selective neuronal loss, supporting the presence of ischemic-dependent necroptosis, and the notion that ER stress is involved in the necroptotic response. Open Science: This manuscript was awarded with the Open Materials Badge For more information see: https://cos.io/our-services/open-science-badges/.

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.

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. The alleviation of ER stress by enhancing UPR with salubrinal treatment reduces the ischemic damage. This effect varies across the different neurovascular unit cell types. The salubrinal neuroprotective effect on CA1 supports differences in neurovascular unit for different brain regions and involves the inflammatory response and its time course. Thus, UPR modulation could be a therapeutic target in cerebral ischemia.