A Cell Culture Model for Studying the Role of Neuron-Glia Interactions in Ischemia

Gava-Junior, Genilso; Roque, Cláudio; Mendes-Oliveira, Julieta; Bernardino, Ana  C; Serrenho, Inês; Pires, Joel; Baltazar, Graça

doi:10.3791/61388

Cited by 4 publications

(3 citation statements)

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“…This fact indicates that the results obtained in biochemical tests, as well as in experiments in the T-maze, are stipulated precisely by the change in the concentration of deuterium. This is also consistent with the results of experiments on cerebellar cell cultures, which showed that placing neurons in a medium containing δ 2 H equal to −357‰ increases their resistance to glucose deprivation, which was used to model pathological processes in cerebral ischemia [ 66 , 67 ]. In this case, the response of cultured neurons is generally comparable to the response of brain cells in vivo, when intensification of free radicals’ synthesis, lipid peroxidation, and changes in the activity of antioxidant enzymes are observed [ 68 , 69 ].…”

Section: Discussionsupporting

confidence: 87%

Reduction of Deuterium Level Supports Resistance of Neurons to Glucose Deprivation and Hypoxia: Study in Cultures of Neurons and on Animals

et al. 2021

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The effect of a reduced deuterium (D) content in the incubation medium on the survival of cultured neurons in vitro and under glucose deprivation was studied. In addition, we studied the effect of a decrease in the deuterium content in the rat brain on oxidative processes in the nervous tissue, its antioxidant protection, and training of rats in the T-shaped maze test under hypoxic conditions. For experiments with cultures of neurons, 7–8-day cultures of cerebellar neurons were used. Determination of the rate of neuronal death in cultures was carried out using propidium iodide. Acute hypoxia with hypercapnia was simulated in rats by placing them in sealed vessels with a capacity of 1 L. The effect on oxidative processes in brain tissues was assessed by changes in the level of free radical oxidation and malondialdehyde. The effect on the antioxidant system of the brain was assessed by the activity of catalase. The study in the T-maze was carried out in accordance with the generally accepted methodology, the skill of alternating right-sided and left-sided loops on positive reinforcement was developed. This work has shown that a decrease in the deuterium content in the incubation medium to a level of −357‰ has a neuroprotective effect, increasing the survival rate of cultured neurons under glucose deprivation. When exposed to hypoxia, a preliminary decrease in the deuterium content in the rat brain to −261‰ prevents the development of oxidative stress in their nervous tissue and preserves the learning ability of animals in the T-shaped maze test at the level of the control group. A similar protective effect during the modification of the 2H/1H internal environment of the body by the consumption of DDW can potentially be used for the prevention of pathological conditions associated with the development of oxidative stress with damage to the central nervous system.

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Section: Discussionsupporting

confidence: 87%

Reduction of Deuterium Level Supports Resistance of Neurons to Glucose Deprivation and Hypoxia: Study in Cultures of Neurons and on Animals

et al. 2021

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“…For astrocyte‐enriched cultures, the cells were cultured at a density of 0.26 × 10 6 cells/cm 2 in Minimum Essential Medium Eagle (MEM, Sigma‐Aldrich, Cat: M0268) supplemented with insulin from bovine pancreas 5 mg/L (Sigma‐Aldrich, Cat: I5500), 45% anhydrous D‐glucose 3.375 g/L (Fisher Scientific, Cat: G/0450/60), penicillin/streptomycin 12 U/ml (Biochrom, Cat: A2213), 0.026 M NaHCO3 (Sigma‐Aldrich, Cat: S5761), 0.49 mM glutamine (Sigma‐Aldrich, Cat: G3126), and 10% of FBS. In both cases, cell density was defined based on previous reports from our group (Gava‐Junior et al, 2020). Cells were plated on poly‐ d ‐lysine‐coated (Sigma‐Aldrich, Cat: P1024) dishes and maintained in a 5% humidified CO 2 incubator at 37°C.…”

Section: Methodsmentioning

confidence: 99%

“…The experimental procedures started at 7 days in vitro. The cellular composition of these cultures was previously characterized by our group (Gava‐Junior et al, 2020; Roque & Baltazar, 2017).…”

Section: Methodsmentioning

confidence: 99%

High‐frequency repetitive magnetic stimulation rescues ischemia‐injured neurons through modulation of glial‐derived neurotrophic factor present in the astrocyteʼs secretome

Gava-Junior

Ferreira

Roque

et al. 2022

Journal of Neurochemistry

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Due to its ability to improve the most frequent clinical sequelae left by ischemia, repetitive transcranial magnetic stimulation has been considered a promising therapeutic strategy for stroke. Those improvements are associated with changes in neurons and their synaptic liaisons. However, the hypothesis that this technique modulates astrocytes, potentiating their neuroprotective capabilities, was also raised. This study aims to identify the effects triggered by high‐frequency repetitive magnetic stimulation (HF‐rMS) on astrocytes that contribute to its neuroprotective effects. Neuron–glia and astrocyte cortical cultures subject to oxygen and glucose deprivation were used as an in vitro model of ischemia. Neuroprotection promoted by HF‐rMS was evaluated by analysis of markers of neuronal activity and morphometric analysis of neurons. Glial reactivity was determined by immunocytochemistry. The levels of growth factors in the astrocyte‐conditioned medium (CM) were assessed through a Growth Factor Array and glial‐derived neurotrophic factor (GDNF) expression was analyzed by RT‐PCR and Western blot. Our results show that neurons injured by ischemia can be rescued through the modulation of astrocytes by HF‐rMS. This modulation helps to maintain the number and length of neurites and increases the number of neurons expressing ERK1/2 and c‐Fos. Analysis of the astrocyte‐CM showed that HF‐rMS stimulated the release of several trophic factors by astrocytes. Moreover, GDNF was one of the released factors that contributed to the recovery mechanisms triggered by HF‐rMS. Our results show that modulation of astrocytes by HF‐rMS effectively rescues neurons injured by ischemia and suggest that by targeting astrocytes this approach can also be used to promote neuroprotection in other brain lesions.

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In Vitro Model for Ischemic Stroke: Functional Analysis of Vascular Smooth Muscle Cells

et al. 2021

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A Cell Culture Model for Studying the Role of Neuron-Glia Interactions in Ischemia

Cited by 4 publications

References 0 publications

Reduction of Deuterium Level Supports Resistance of Neurons to Glucose Deprivation and Hypoxia: Study in Cultures of Neurons and on Animals

Reduction of Deuterium Level Supports Resistance of Neurons to Glucose Deprivation and Hypoxia: Study in Cultures of Neurons and on Animals

High‐frequency repetitive magnetic stimulation rescues ischemia‐injured neurons through modulation of glial‐derived neurotrophic factor present in the astrocyteʼs secretome

In Vitro Model for Ischemic Stroke: Functional Analysis of Vascular Smooth Muscle Cells

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