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Abstract: Overactivation of NMDA-Rs may mediate excitotoxic cell death associated with epileptic seizures, and hypoxic–ischemic conditions. We assessed whether repeated subcutaneous administration of l-glutamate to neonatal rats affects the subunit composition of NMDA-Rs. Accordingly, cortical and hippocampal tissue from 14-day-old rats was analyzed by Western blotting and RT-PCR to quantify the protein and mRNA expression of different NMDA-R subunits. In addition, tissue sections were Nissl stained to assess the cell damage in this tissue. Early exposure of neonatal rats to l-glutamate differentially affects the expression of mRNA transcripts for NMDA-R subunits in the cerebral cortex and hippocampus. In the cerebral cortex, a decrease in NR2B subunit mRNA expression was observed, as well as a loss of NR1 and NR2A protein. By contrast, neonatal l-glutamate administration augmented the transcripts encoding the NR1, NR2B, and NR2C subunits in the hippocampal formation. The expression of mRNA encoding the NR2A subunit was not affected by neonatal l-glutamate administration in either of the brain regions examined. This differential expression of NMDA-R subunits following neonatal exposure to l-glutamate may represent an adaptive response of the glutamate receptors to overactivation in order to reduce the effect of high l-glutamate during the early period of life when the animal is more vulnerable to excitotoxicity. [Copyright &y& Elsevier]

Abstract: Kainic acid receptor (KA-R) subunits are differentially expressed during brain development, and they modulate both neural growth and survival. High concentrations of glutamate in the brain can induce neuronal injury through these receptors, altering normal development. However, it is unclear whether KAR subunit expression itself is also modified by neonatal exposure to high glutamate. To analyze this, monosodium glutamate (4mg/g of body weight) was subcutaneously administered on postnatal days 1, 3, 5 and 7, and the expression of GluR5, GluR6, KA1 and KA2, as well as [3H]-kainic acid (KA-R) binding, was evaluated on postnatal days 14, 21, 30 and 60 in different regions of rat brain. As a result, high levels of GluR5 expression associated with strong [3H]-kainic acid binding were observed on postnatal days 30 and 60 in the cerebral cortex of rats exposed to glutamate. Similarly, the changes induced by glutamate administration in the expression of the KA1 and KA2 subunits were paralleled by those of [3H]-kainic acid binding in the striatum at postnatal days 21 and 30. In contrast, while KAR subunits were over expressed in the hippocampus, no changes were observed in [3H]-kainic acid binding in adult rats that had been exposed to glutamate. Therefore, glutamate modifies both the expression of kainic acid receptor subunits and kainic acid binding in a determined spatial and temporal manner, which may be indicative of a regional susceptibility to glutamate neurotoxicity. [Copyright &y& Elsevier]

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Olfactory ensheathing cells (OECs) and mesenchymal stem cells (MSCs) differentiated towards Schwann-like have plasticity properties. These cells express the Glial fibrillary acidic protein (GFAP), a type of cytoskeletal protein that significantly regulates many cellular functions, including those that promote cellular plasticity needed for regeneration. However, the expression of GFAP isoforms (α, β, and δ) in these cells has not been characterized. We evaluated GFAP isoforms (α, β, and δ) expression by Polymerase Chain Reaction (PCR) assay in three conditions: (1) OECs, (2) cells exposed to OECs-conditioned medium and differentiated to Schwann-like cells (dBM-MSCs), and (3) MSC cell culture from rat bone marrow undifferentiated (uBM-MSCs). First, the characterization phenotyping was verified by morphology and immunocytochemistry, using p75, CD90, and GFAP antibodies. Then, we found the expression of GFAP isoforms (α, β, and δ) in the three conditions; the expression of the GFAPα (10.95%AUC) and GFAPβ (9.17%AUC) isoforms was predominantly in OECs, followed by dBM-MSCs (α: 3.99%AUC, β: 5.66%AUC) and uBM-MSCs (α: 2.47%AUC, β: 2.97%AUC). GFAPδ isoform has a similar expression in the three groups (OEC: 9.21%AUC, dBM-MSCs: 11.10%AUC, uBM-MSCs: 9.21%AUC). These findings suggest that expression of different GFAPδ and GFAPβ isoforms may regulate cellular plasticity properties, potentially contributing to tissue remodeling processes by OECs, dBM-MSCs, and uBM-MSCs.

Amyloid-β oligomers are a cytotoxic structure that is key for the establishment of the beginning stages of Alzheimer's disease (AD). These structures promote subcellular alterations that cause synaptic dysfunction, loss of cell communication, and even cell death, generating cognitive deficits. The aim of this study was to investigate the cytotoxic effects of amyloid-β1-42 oligomers (AβOs) on the membranous organelles involved in protein processing: the endoplasmic reticulum (ER) and Golgi apparatus (GA). The results obtained with 10 μM AβOs in SH-SY5Y neuroblastoma cells showed that oligomeric structures are more toxic than monomers because they cause cell viability to decrease as exposure time increases. Survivor cells were analyzed to further understand the toxic effects of AβOs on intracellular organelles. Survivor cells showed morphological alterations associated with abnormal cytoskeleton modification 72-96 h after exposure to AβOs. Moreover, the ER and GA presented rearrangement throughout the cytoplasmic space, which could be attributed to a lack of constitutive protein processing or to previous abnormal cytoskeleton modification. Interestingly, the disorganization of both ER and GA organelles exposed to AβOs is likely an early pathological alteration that could be related to aberrant protein processing and accumulation in AD., Competing Interests: Conflicts of InterestThe authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results., (© 2024 by the authors.)

Background: Mesenchymal stem cells (MSCs) are multipotent precursor cells with the ability to self-renew and differentiate into multiple cell linage, including the Schwann-like fate that promotes regeneration after lesion. Raman spectroscopy provides a precise characterization of the osteogenic, adipogenic, hepatogenic and myogenic differentiation of MSCs. However, the differentiation of bone marrow mesenchymal stem cells (BMSCs) towards a glial phenotype (Schwann-like cells) has not been characterized before using Raman spectroscopy., Method: We evaluated three conditions: 1) cell culture from rat bone marrow undifferentiated (uBMSCs), and two conditions of differentiation; 2) cells exposed to olfactory ensheathing cells-conditioned medium (dBMSCs) and 3) cells obtained from olfactory bulb (OECs). uBMSCs phenotyping was confirmed by morphology, immunocytochemistry and flow cytometry using antibodies of cell surface: CD90 and CD73. Glial phenotype of dBMSCs and OECs were verified by morphology and immunocytochemistry using markers of Schwann-like cells and OECs such as GFAP, p75 NTR and O4. Then, the Principal Component Analysis (PCA) of Raman spectroscopy was performed to discriminate components from the high wavenumber region between undifferentiated and glial-differentiated cells. Raman bands at the fingerprint region also were used to analyze the differentiation between conditions., Results: Differences between Raman spectra from uBMSC and glial phenotype groups were noted at multiple Raman shift values. A significant decrease in the concentration of all major cellular components, including nucleic acids, proteins, and lipids were found in the glial phenotype groups. PCA analysis confirmed that the highest spectral variations between groups came from the high wavenumber region observed in undifferentiated cells and contributed with the discrimination between glial phenotype groups., Conclusion: These findings support the use of Raman spectroscopy for the characterization of uBMSCs and its differentiation in the glial phenotype., Competing Interests: The authors declare no conflict of interest., (© 2023 The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V.)

Vascular endothelial growth factor (VEGF) exerts neuroprotective or proinflammatory effects, depending on what VEGF forms (A-E), receptor types (VEGFR1-3), and intracellular signaling pathways are involved. Neonatal monosodium glutamate (MSG) treatment triggers neuronal death by excitotoxicity, which is commonly involved in different neurological disorders, including neurodegenerative diseases. This study was designed to evaluate the effects of VEGFR-2 inhibition on neuronal damage triggered by excitotoxicity in the cerebral motor cortex (CMC) and hippocampus (Hp) after neonatal MSG treatment. MSG was administered at a dose of 4 g/kg of body weight (b.w.) subcutaneously on postnatal days (PD) 1, 3, 5, and 7, whereas the VEGFR-2 inhibitor SU5416 was administered at a dose of 10 mg/kg b.w. subcutaneously on PD 5 and 7, 30 min before the MSG treatment. Neuronal damage was assessed using hematoxylin and eosin staining, fluoro-Jade staining, and TUNEL assay. Additionally, western blot assays for some proteins of the VEGF-A/VEGFR-2 signaling pathway (VEGF-A, VEGFR-2, PI3K, Akt, and iNOS) were carried out. All assays were performed on PD 6, 8, 10, and 14. Inhibition of VEGFR-2 signaling by SU5416 increases the neuronal damage induced by neonatal MSG treatment in both the CMC and Hp. Moreover, neonatal MSG treatment increased the expression levels of the studied VEGF-A/VEGFR-2 signaling pathway proteins, particularly in the CMC. We conclude that VEGF-A/VEGFR-2 signaling pathway activation could be part of the neuroprotective mechanisms that attempt to compensate for neuronal damage induced by neonatal MSG treatment and possibly also in other conditions involving excitotoxicity., (© 2023 Wiley Periodicals LLC.)

Substantial evidence in the literature demonstrates the pleiotropic effects of the administration of recombinant human erythropoietin (rhEPO) and its molecular variants in different tissues and organs, including the brain. Some of these reports suggest that the chemical properties of this molecule by itself or in combination with other agents (e.g., growth factors) could provide the necessary pharmacological characteristics to be considered a potential protective agent in neurological disorders such as Alzheimer's disease (AD). AD is a degenerative disorder of the brain, characterized by an aberrant accumulation of amyloid β (Aβ) and hyperphosphorylated tau (tau-p) proteins in the extracellular and intracellular space, respectively, leading to inflammation, oxidative stress, excitotoxicity, and other neuronal alterations that compromise cell viability, causing neurodegeneration in the hippocampus and the cerebral cortex. Unfortunately, to date, it lacks an effective therapeutic strategy for its treatment. Therefore, in this review, we analyze the evidence regarding the effects of exogenous EPOs (rhEPO and its molecular variants) in several in vivo and in vitro Aβ and tau-p models of AD-type neurodegeneration, to be considered as an alternative protective treatment to this condition. Particularly, we focus on analyzing the differential effect of molecular variants of rhEPO when changes in doses, route of administration, duration of treatment or application times, are evaluated for the improved cellular alterations generated in this disease. This narrative review shows the evidence of the effectiveness of the exogenous EPOs as potential therapeutic molecules, focused on the mechanisms that establish cellular damage and clinical manifestation in the AD.

The blood-brain barrier (BBB) maintains the optimal microenvironment for brain function. Tight junctions (TJs) allow endothelial cells to adhere to each other, leading to the formation of a barrier that prevents the penetration of most molecules via transcellular routes. Evidence has indicated that seizure-induced vascular endothelial growth factor (VEGF) type 2 receptor (VEGFR-2) pathway activation weakens TJs, inducing vasodilatation and increasing vascular permeability and subsequent brain injury. The present study focused on investigating the expression levels of VEGF-related (VEGF-A and VEGFR-2) and TJ-related proteins (claudin-5, occludin and ZO-1) in the neocortical microvasculature of patients with drug-resistant temporal lobe epilepsy (TLE). The results obtained from hippocampal sclerosis TLE (HS-TLE) patients were compared with those obtained from patients with TLE secondary to lesions (lesion-TLE) and autopsy samples. The Western blotting and immunofluorescence results showed that VEGF-A and VEGFR-2 protein expression levels were increased in HS-TLE and lesion-TLE patients compared to autopsy group. On the other hand, claudin-5 expression was higher in HS-TLE patients and lesion-TLE patients than autopsies. The expression level of occludin and ZO-1 was decreased in HS-TLE patients. Our study described modifications to the integrity of the BBB that may contribute to the pathogenesis of TLE, in which the VEGF system may play an important role. We demonstrated that the same modifications were present in both HS-TLE and lesion-TLE patients, which suggests that seizures modify these systems and that they are not associated with the establishment of epilepsy., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Glutamate-mediated excitatory synaptic signalling is primarily controlled by excitatory amino acid transporters (EAATs), such as EAAT1 and EAAT2, which are located mostly on astrocytes and, together, uptake more than 95 % of extracellular glutamate. Alterations in the functional expression levels of EAATs can lead to excessive extracellular glutamate accumulation, potentially triggering excitotoxicity and seizures, among other neurological disorders. Excitotoxicity induced in early developmental stages can lead to lasting changes in several neurotransmission systems, including the glutamatergic system, which could make the brain more susceptible to a second insult. In this study, the expression levels of EAAT1 (GLAST) and EAAT2 (GLT-1) proteins were assessed in the cerebral motor cortex (CMC), striatum, hippocampus and entorhinal cortex (EC) of male adult rats following the neonatal excitotoxic process triggered by monosodium glutamate (MSG)-treatment (4 g/kg of body weight at postnatal days 1,3,5 and 7, subcutaneously). Western blot analysis showed that neonatal MSG-treatment decreased EAAT1 expression levels in the CMC, striatum and hippocampus, while EAAT2 levels were increased in the striatum and EC and decreased in the CMC. Immunofluorescence staining confirmed the changes in EAAT1 and EAAT2 expression induced by neonatal MSG-treatment, which were accompanied by an increase in the glial fibrillary acidic protein (GFAP) immunofluorescence signalthat was particularly significant in the hippocampus. Our results show that a neonatal excitotoxic processes can induce lasting changes in the expression levels of EAAT1 and EAAT2 proteins and suggest that although astrogliosis occurs, glutamate uptake could be deficient, particularly in the CMC and hippocampus., (Copyright © 2020 Elsevier B.V. All rights reserved.)