Sheyla Montori

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.

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.

EARLY MODIFICATIONS IN N-METHYL-D-ASPARTATE RECEPTOR SUBUNIT mRNA LEVELS IN AN OXYGEN AND GLUCOSE DEPRIVATION MODEL USING RAT HIPPOCAMPAL BRAIN SLICES

Glutamatergic N-methyl-d-aspartate NMDA receptors (NMDAR) are considered to play a key role in ischemia-induced damage. Long-term (hours) changes in their expression upon ischemia have been shown. Here we report short-term changes in the mRNA levels of the major hippocampal NMDAR subunits (NR1, NR2A and NR2B), as well as c-fos, in an ex vivo ischemia model using hippocampal slices. This effect can be observed also in a calcium free incubation solution. Striking early decreases in the NMDAR subunit mRNA levels were observed after 30 min of oxygen and glucose deprivation (OGD) as well as a partial recovery when the tissues were returned to the balanced salt solution (reperfusion-like period) for 3 h. Since OGD-induced damage has been reported to be a consequence of the increase in OGD-related glutamate release, we also analyzed NMDAR mRNA levels following increased glutamate levels in hippocampal sections in which no significant effects on NMDAR subunit mRNA levels were detected. Furthermore, we describe that the presence of MK-801 (a selective NMDAR antagonist), CNQX (a selective AMPA/kainate receptor antagonist) or their combined action in the incubation solution is able to induce a significant decrease in NMDAR expression but in these conditions the OGD does not induce further decreases in mRNA levels. We suggest that the mechanisms triggered during OGD to downregulate mRNA levels of NMDAR subunits could be the same than those induced by glutamate receptor antagonists.

AMPA receptor downregulation induced by ischaemia/reperfusion is attenuated by age and blocked by meloxicam

S. Montori, S. Dos_Anjos, M. A. Ríos‐Granja, C. C. Pérez‐García, A. Fernández‐López and B. Martínez‐Villayandre (2010) Neuropathology and Applied Neurobiology36, 436–447
AMPA receptor downregulation induced by ischaemia/reperfusion is attenuated by age and blocked by meloxicam

Age-dependent modifications in the mRNA levels of the rat excitatory amino acid transporters (EAATs) at 48 hour reperfusion following global ischemia

This study reports the mRNA levels of some excitatory amino acid transporters (EAATs) in response to ischemia-reperfusion (I/R) in rat hippocampus and cerebral cortex. The study was performed in 3-month-old and 18-month-old animals to analyze the possible role of age in the I/R response of these transporters. The I/R resulted in a reduced transcription of both the neuronal EAAC1 (excitatory amino acid carrier-1) and the neuronal and glial GLT-1 (glial glutamate transporter 1), while the glial GLAST1a (l-glutamate/l-aspartate transporter 1a) transcription increased following I/R. The changes observed were more striking in 3-month-old animals than in 18-month-old animals. We hypothesize that increases in the GLAST1a mRNA levels following I/R insult can be explained by increases in glial cells, while the GLT-1 response to I/R mirrors neuronal changes. GLAST1a transcription increases in 3-month-old animals support the hypothesis that this transporter would be the main mechanism for extracellular glutamate clearance after I/R. Decreases in EAAC1 and GLT-1 mRNA levels would represent either neuronal changes due to the delayed neuronal death or a putative protective down-regulation of these transporters to decrease the amount of glutamate inside the neurons, which would decrease their glutamate release. This study also reports how the treatment with the anti-inflammatory agent meloxicam attenuates the transcriptional response to I/R in 3-month-old rats and decreases the survival of the I/R-injured animals.

Quantitative gene expression analysis in a brain slice model: Influence of temperature and incubation media

We describe the RNA integrity (28S/18S ratio) and the messenger RNA (mRNA) expression of genes encoding glyceraldehyde 3-phosphate dehydrogenase (GAPDH), microtubule-associated serine/threonine kinase 2 (Mast2), and beta-actin in cortical brain slices incubated for up to 24h in Ringers solution and Dulbeccos modified Eagles medium (DMEM) at 25 and 37 degrees C. Our data reveal an optimal temporal working window between 1 and 6h when slices are incubated in Ringers solution at 25 degrees C that allows experiments related to gene expression dynamics to be performed more suitably than those carried out at 37 degrees C. In addition, we show that reference gene expression may be modified in dynamic experiments and may compromise studies of gene expression.

Improvement of osteogenesis in dental pulp pluripotent-like stem cells by oligopeptide-modified poly(β-amino ester)s

Controlling pluripotent stem cell differentiation via genetic manipulation is a promising technique in regenerative medicine. However, the lack of safe and efficient delivery vehicles limits this application. Recently, a new family of poly(β-amino ester)s (pBAEs) with oligopeptide-modified termini showing high transfection efficiency of both siRNA and DNA plasmid has been developed. In this study, oligopeptide-modified pBAEs were used to simultaneously deliver anti-OCT3/4 siRNA, anti-NANOG siRNA, and RUNX2 plasmid to cells from the dental pulp with pluripotent-like characteristics (DPPSC) in order to promote their osteogenic differentiation. Results indicate that transient inhibition of the pluripotency marker OCT3/4 and the overexpression of RUNX2 at day 7 of differentiation markedly increased and accelerated the expression of osteogenic markers. Furthermore, terminally-differentiated cells exhibited higher matrix mineralization and alkaline phosphatase activity. Finally, cell viability and genetic stability assays indicate that this co-delivery system has high chromosomal stability and minimal cytotoxicity. Therefore, we conclude that such co-delivery strategy is a safe and a quick option for the improvement of DPPSC osteogenic differentiation. STATEMENT OF SIGNIFICANCE Controlling pluripotent stem cell differentiation via genetic manipulation is a promising technique in regenerative medicine. However, the lack of safe and efficient delivery vehicles limits this application. In this study, we propose the use of a new family of oligopeptide-modified pBAEs developed in our group to control the differentiation of dental pulp pluripotential stem cells (DPPSC). In order to promote their osteogenic differentiation. The strategy proposed markedly increased and accelerated the expression of osteogenic markers, cell mineralization and alkaline phosphatase activity. Finally, cell viability and genetic stability assays indicated that this co-delivery system has high chromosomal stability and minimal cytotoxicity. These findings open a new interesting path in the usage of non-viral gene delivery systems for the control of pluripotential stem cell differentiation.

Differential effect of transient global ischaemia on the levels of γ-aminobutyric acid type A (GABAA) receptor subunit mRNAs in young and older rats

S. Montori, S. Dos_Anjos, A. Poole, M. M. Regueiro‐Purriños, I. L. Llorente, M. G. Darlison, A. Fernández‐López and B. Martínez‐Villayandre (2012) Neuropathology and Applied Neurobiology38, 710–722

Dental pulp pluripotent-like stem cells (DPPSC), a new stem cell population with chromosomal stability and osteogenic capacity for biomaterials evaluation

BackgroundBiomaterials are widely used to regenerate or substitute bone tissue. In order to evaluate their potential use for clinical applications, these need to be tested and evaluated in vitro with cell culture models. Frequently, immortalized osteoblastic cell lines are used in these studies. However, their uncontrolled proliferation rate, phenotypic changes or aberrations in mitotic processes limits their use in long-term investigations. Recently, we described a new pluripotent-like subpopulation of dental pulp stem cells derived from the third molars (DPPSC) that shows genetic stability and shares some pluripotent characteristics with embryonic stem cells. In this study we aim to describe the use of DPPSC to test biomaterials, since we believe that the biomaterial cues will be more critical in order to enhance the differentiation of pluripotent stem cells.MethodsThe capacity of DPPSC to differentiate into osteogenic lineage was compared with human sarcoma osteogenic cell line (SAOS-2). Collagen and titanium were used to assess the cell behavior in commonly used biomaterials. The analyses were performed by flow cytometry, alkaline phosphatase and mineralization stains, RT-PCR, immunohistochemistry, scanning electron microscopy, Western blot and enzymatic activity. Moreover, the genetic stability was evaluated and compared before and after differentiation by short-comparative genomic hybridization (sCGH).ResultsDPPSC showed excellent differentiation into osteogenic lineages expressing bone-related markers similar to SAOS-2. When cells were cultured on biomaterials, DPPSC showed higher initial adhesion levels. Nevertheless, their osteogenic differentiation showed similar trend among both cell types. Interestingly, only DPPSC maintained a normal chromosomal dosage before and after differentiation on 2D monolayer and on biomaterials.ConclusionsTaken together, these results promote the use of DPPSC as a new pluripotent-like cell model to evaluate the biocompatibility and the differentiation capacity of biomaterials used in bone regeneration.