Marta Regueiro-Purriños

Transient global ischemia in rat brain promotes different NMDA receptor regulation depending on the brain structure studied.

The mRNA expression of the major subunits of N-methyl-d-aspartate receptors (NR1, NR2A and NR2B) following ischemia-reperfusion was studied in structures with different vulnerabilities to ischemic insult in the rat brain. The study was performed using quantitative real-time PCR on samples from 3-month-old male Sprague-Dawley rats after global transient forebrain ischemia followed by 48h of reperfusion. Expression of NMDA receptor subunits mRNAs decreased significantly in all structures studied in the injured animals as compared to the sham-operated ones. The hippocampal subfields (CA1, CA3 and dentate gyrus) as well as the caudate-putamen, both reported to be highly ischemic-vulnerable structures, showed outstandingly lower mRNA levels of NMDA receptor subunits than the cerebral cortex, which is considered a more ischemic-resistant structure. The ratios of the mRNA levels of the different subunits were analyzed as a measure of the NMDA receptor expression pattern for each structure studied. Hippocampal areas showed changes in NMDA receptor expression after the insult, with significant decreases in the NR2A with respect to the NR1 and NR2B subunits. Thus, the NR1:NR2A:NR2B (1:1:2) ratios observed in the sham-operated animals became (2:1:4) in insulted animals. This modified expression pattern was similar in CA1, CA3 and the dentate gyrus, in spite of the different vulnerabilities reported for these hippocampal areas. In contrast, no significant differences in the expression pattern were observed in the caudate-putamen or cerebral cortex on comparing the sham-operated animals with the ischemia-reperfused rats. Our results support the notion that the regulation of NMDA receptor gene expression is dependent on the brain structure rather than on the higher or lower vulnerability of the area studied.

Global ischemia-induced modifications in the expression of AMPA receptors and inflammation in rat brain

Alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors (AMPAR) and inflammatory processes have been related to ischemia-induced damage, but there are few studies addressing their response in different brain areas. Here we compare AMPAR expression after ischemia in several brain areas (hippocampus, cerebral cortex and caudate-putamen) in an attempt to correlate it with their different vulnerabilities. We found outstanding decreases in GluR1 and GluR2 mRNA levels after global ischemia and 48 h reperfusion (I/R) in all the areas studied, however, protein levels maintained in some areas such as CA3, suggesting different post-transcriptional control in different areas of the brain. To characterize the inflammatory response in these areas, we measured the mRNA levels of CD11b/CD18 membrane integrin (a reactive microglia marker), which showed an important but similar up-regulation in all brain areas studied, which was confirmed by immunohistochemistry. We conclude that the down-regulation of AMPAR gene expression following I/R does not explain differences in the vulnerability of different areas. Additionally, our data indicate that the level of inflammation is independent of the vulnerability of the different brain areas and does not explain differences in the AMPAR expression observed in the brain areas studied.

Closed-chest experimental porcine model of acute myocardial infarction-reperfusion

INTRODUCTION Progress in cardiovascular regenerative medicine research requires the availability of appropriate experimental animal models that are as close to humans as feasible. Our objective was to assess the validity of a porcine endovascular model of myocardial infarction and reperfusion. METHODS Fifteen domestic pigs (Large White race) were anesthetized and pre-medicated with amiodarone. Endovascular fluoroscopy-guided coronary procedures were performed to occlude the mid-left anterior descending artery using a coronary angioplasty balloon. Occlusion was confirmed by angiography and electrocardiography. After 75 min the balloon catheter system was withdrawn and the presence of reperfusion flow was verified. The animals were sacrificed after 1 and 2 weeks of follow-up, the hearts were explanted, and the extent of myocardial infarction with respect to the left ventricle was quantified. RESULTS Overall survival rate was 67%. Five animals died prematurely: 3 showing signs of heart failure, 1 had reperfusion failure (final TIMI flow grade 1) and 1 succumbed to acute stress. The most common adverse event was ventricular fibrillation (87% of the animals) and defibrillation was effective in all affected animals. The extent of myocardial infarct in the animals followed-up for 1 and 2 weeks was similar (20.4+/-4.3% vs. 20.9+/-2.8%, respectively; p=0.8) but was significantly greater in the animals that died prematurely (29.5+/-3.6%, p=0.02). CONCLUSIONS The endovascular porcine model we have explored constitutes a feasible and reproducible alternative for the evaluation of human myocardial infarction and reperfusion.

Age and meloxicam attenuate the ischemia/reperfusion-induced down-regulation in the NMDA receptor genes

This study describes the effect of global brain ischemia followed by 48 h reperfusion, when delayed neuronal death can be already observed. We quantified the mRNA levels of the N-methyl-D-aspartate receptor (NMDAR) subunits and those of the astroglia (glial fibrilar acidic protein, GFAP) and microglia (CD11b) markers using real time PCR on the cerebral cortex and hippocampus of 3- and 18-month-old Sprague-Dawley rats. Data show an ischemia/reperfusion-induced decrease in the mRNA levels of the NMDAR NR1, NR2A and NR2B subunits genes, which contrasts with the increase in the CD11b and GFAP mRNA levels. These effects are attenuated in all the genes studied in 18-month-old animals, suggesting that this mechanism of response is less efficient in aged animals. Western blot assays of NR1, NR2A and NR2B show parallels with the real time PCR data, indicating that the down-regulation of these genes is controlled at the transcriptional level. We suggest that a decrease in the efficiency in the control of the NMDAR transcription could account for the higher vulnerability in aged animals, but it cannot explain by itself differences in the vulnerability to ischemia in different areas of the brain. In the assays of ischemia/reperfusion followed by a treatment with the anti-inflammatory agent meloxicam, we observed that ischemic insult was unable to elicit changes in the NMDAR transcription, thus suggesting that inflammation plays a crucial role in the transcriptional control of these genes.

Time Course of Reendothelialization of Stents in a Normal Coronary Swine Model Characterization and Quantification

Late thrombosis of coronary drug-eluting stents is an infrequent but serious complication of percutaneous transluminal coronary angioplasty. The best predictor of this event is the lack of endothelialization of stent struts. The objective of this study is to characterize and quantify the time course of endothelialization of different stents implanted in nonatherosclerotic swine coronary arteries. Thirty-three Carbofilm-coated stents were implanted percutaneously in 11 anesthetized domestic, crossbred pigs (weight 25 ± 3 kg, 2 months old). Each animal received 1 stainless steel stent (SS), 1 cobalt-chromium stent (CCS), and 1 tacrolimus-eluting stent (TES) in each coronary artery. Follow-up periods were 1 day (n = 9 stents), 3 days (n = 9 stents), and 7 days (n = 15 stents). Longitudinal sections of the stented vessels were examined using scanning electron microscopy. At 1 day, there was scarce, patchy endothelialization with areas of fibrin; the endothelialization rate was similar for all the stents (SS, 29% ± 23%; CCS, 29% ± 24%; TES, 31% ± 25%; P = .9). At 3 days, there were more endothelial cells but with immature features and giant cells over fibrin; the endothelialization was greater in SS and CCS than in TES (SS, 79% ± 14%; CCS, 81% ± 17%; TES, 46% ± 9%; P = .007). At 7 days, arteries showed better endothelialization with few giant cells; the endothelialization was greater in SS and CCS than in TES (SS, 95% ± 4%; CCS, 98% ± 4%; TES, 79% ± 9%; P = .01). In conclusion, the described model is useful for the analysis of endothelialization of coronary stents and facilitates measurement of its rate of formation and characterization of the involved cell types.

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.

Análisis de la inflamación luminal inducida por distintos tipos de stent coronario en el modelo coronario animal mediante microscopía electrónica de barrido

There is histological evidence that drug-eluting stents are associated with delayed endothelialization and a persistent inflammatory state. Moreover, clusters of inflammatory cells have been observed on luminal surfaces by scanning electron microscopy. With the aim of quantifying this inflammatory response, we implanted one bare-metal stent and two drug-eluting stents containing different doses of vinblastine embedded in the same polymer into the coronary arteries of 12 domestic pigs. The density of inflammatory cells in a representative area (100 x 100 μm) was quantified at 3 and 7 days. Endothelialization was more complete in bare-metal stents than in drug-eluting stents at both 3 days (P=.016) and 7 days (P=.0001). The degree of inflammation induced by the drug-eluting stents was higher than that induced by the bare-metal stents at both 3 days (11.8±3.5% vs. 4.5±2%; P=.001) and 7 days (26.3±4.4% vs. 1.2±1.5%; P=.0001). In addition, the time sequence was inverted: the inflammatory response increased over time with the drug-eluting stents, while the opposite occurred with the bare-metal stents.

Histology and immunochemistry evaluation of autologous translocation of retinal pigment epithelium‐choroid graft in porcine eyes

Purpose:  To evaluate structure and cellular functionality of retinal pigment epithelium (RPE)‐choroid grafts after autologous translocation in porcine eyes.

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.

Safety and Efficacy of New Sirolimus-eluting Stent Models in a Preclinical Study

INTRODUCTION AND OBJECTIVES Initial preclinical studies are required during the process of improving polymers, platforms, and drug-eluting systems for new coronary stent designs. Our objective was to analyze the efficacy and safety of new drug-eluting stent models compared with a conventional stent and commercialized drug-eluting stents in an experimental model with healthy porcine coronary arteries. METHODS Sixty stents (conventional stent, new sirolimus-eluting stents: drug-eluting stents 1, 2 and 3; Cypher(®) and Xience(®)) were randomly placed in the coronary arteries of 20 Large White domestic pigs. Angiographic and histomorphometric studies were done 28 days later. RESULTS The stents were implanted at a stent/artery ratio of 1.34±0.15, with no significant differences between groups. The new stents showed less late loss and angiographic restenosis than conventional stents (P=.006 and P<.001, respectively). Histologically, restenosis and neointimal area were lower with all the new platforms than with the conventional stents (P<.001 for each variable), and no differences were found vs the drug-eluting stents on the market. Safety data showed that endothelialization was lower with drug-eluting stents than with conventional stents, except for drug-eluting stent 3 (P=.084). Likewise, inflammation was lower with drug-eluting stent 3 than with other stents. CONCLUSIONS The new drug-eluting stent platforms studied are associated with less restenosis than conventional stents and showed no significant differences in safety or efficacy vs commercialized drug-eluting stents.