scholarly journals Hippocampal Neurons Become More Vulnerable to Glutamate after Subcritical Hypoxia: An in vitro Study

1990 ◽  
Vol 10 (6) ◽  
pp. 877-884 ◽  
Author(s):  
Eiji Kohmura ◽  
Kazuo Yamada ◽  
Toru Hayakawa ◽  
Akira Kinoshita ◽  
Katsumi Matsumoto ◽  
...  
Keyword(s):  
Nmda Receptor ◽  
Hippocampal Neurons ◽  
Neuronal Loss ◽  
In Vitro Study ◽  
Defined Medium ◽  
High Dose ◽  
Mk 801 ◽  
Plating Density ◽  
Increased Susceptibility

The neurotoxicity of glutamate and hypoxia was investigated in vitro on hippocampal neurons, which were obtained from 18-day-old rat fetuses and were maintained for 3 days in culture. Chemically defined medium without glutamate was used and the plating density was low enough that the effect of exogenously added glutamate could be directly evaluated. In the normal culture condition 1 m M glutamate was necessary to cause significant neuronal loss in the following 24 h. In marked contrast, when glutamate was added after subcritical hypoxic stress, a dose of glutamate as low as 10 μ M could exhibit neurotoxicity. Administration of MK-801, a selective noncompetitive antagonist of the N-methyl-d-aspartate (NMDA) receptor, could in part reverse this increased susceptibility to low-dose glutamate after hypoxia, although MK-801 could not protect hippocampal neurons from high-dose glutamate. Therefore, both the NMDA receptor and other subclasses of the glutamate receptor may be involved in this neurotoxicity of glutamate. Different mechanisms of glutamate neurotoxicity with high and low doses are discussed. Our results showed that hippocampal neurons exposed to subcritical hypoxia become more vulnerable to glutamate than those without hypoxia. This increased susceptibility is of great interest to understanding the mechanism of slowly ongoing neuronal loss caused by ischemia or epilepsy.

Autoradiographic analysis of 3H-MK-801 (dizocilpine) in vivo uptake and in vitro binding after focal cerebral ischemia in the rat

1992 ◽  
Vol 76 (1) ◽  
pp. 127-133 ◽  
Author(s):  
Michael C. Wallace ◽  
Graham M. Teasdale ◽  
James McCulloch
Keyword(s):  
Nmda Receptor ◽  
Caudate Nucleus ◽  
Intravenous Administration ◽  
Nmda Receptor Antagonists ◽  
Contralateral Hemisphere ◽  
Receptor Antagonists ◽  
Isotopic Tracer ◽  
Mk 801 ◽  
Ischemic Tissue

✓ The clinical utility of N-methyl-D-aspartate (NMDA) receptor antagonists is now being assessed in ischemic brain injury in humans. The uptake and retention of NMDA receptor antagonists in ischemic tissue will influence the design of clinical trials. The effects of permanent occlusion of the middle cerebral artery, induced 15 minutes prior to isotope administration, on the uptake of 3H-MK-801 (dizocilpine) have been assessed in the rat with quantitative autoradiography. In a group of three rats at 15 minutes after the intravenous administration of 3H-MK-801, the level (mean ± standard error of the mean) of isotopic tracer in the ischemic cortex and striatum was markedly less than that in the contralateral hemisphere (ipsilateral vs. contralateral caudate nucleus: 22 ± 4 vs. 84 ± 11 pmol/gm, p < 0.01). In contrast, in a group of five rats at 60 minutes after the intravenous administration of 3H-MK-801, the level of isotopic tracer in the ischemic cortex and striatum was greater than that in the contralateral hemisphere (ipsilateral vs. contralateral caudate nucleus: 52 ± 8 vs. 32 ± 4 pmol/gm, p < 0.05). There were no significant alterations in the specific binding of 3H-MK-801 in vitro in ischemic tissue at equivalent times. The early uptake of 3H-MK-801 into the central nervous system is dominated by the level of cerebral blood flow, whereas at later times after administration enhancement of MK-801 binding by elevated extracellular glutamate concentrations appears to be more important in determining the level of the drug in ischemic tissue.

scholarly journals Influence of Cannabinoid on Interleukin-1β Treated Cartilage: An In Vitro Study

2020 ◽  
Vol 5 (4) ◽  
pp. 2473011420S0043
Author(s):  
Jiangyinzi Shang ◽  
Yuning Hu ◽  
Peter Alexander ◽  
MaCalus V. Hogan ◽  
Hang Lin ◽  
...  
Keyword(s):  
Real Time ◽  
Joint Damage ◽  
In Vitro Study ◽  
Interleukin 1Β ◽  
Human Chondrocytes ◽  
High Dose ◽  
Engineer Cartilage ◽  
Anti Inflammatory ◽  
Win 55

Category: Basic Sciences/Biologics Introduction/Purpose: Cannabinoids have been reported to possess the analgesic, immunomodulatory and anti-inflammatory properties. Recent studied further shown that cannabinoids attenuated joint damage in animal models of arthritis. However, the underlying mechanism has been completely understood. Interleukin-1β (IL-1β), a proinflammatory cytokine that can result in the degradation of cartilage, is associated with the pathogenesis of osteoarthritis. In this study, we hypothesize that cannabinoid can mitigate the detrimental effect of IL-1β on cartilage, thus reduce the progression of osteoarthritis. To test the hypothesis, we insulted human chondrocyte-derived cartilage with IL-1 β for 48 hours and then applied a synthetic cannabinoid agonist, Win- 55,212-2(Win-55), into the culture. The tissue phenotypes were assessed by real time polymerase chain reaction (PCR), histology and immunostaining. Methods: With the approval from CORID, human chondrocytes were isolated from healthy articular cartilage. P2 cells were used. MTS assay were employed to test the half-maximal (50%) inhibitory concentration (IC50). To generate cartilage in vitro, chondrocytes were pelleted and subjected to 14 days chondrogenic culture. The engineered cartilages were stimulated with 10 ng/ml IL-1β for 48 hours and then treated with different concentration of Win-55 (0.01, 0.1, or 1 µM) for another 48 hours. The tissue phenotype was assessed by glycosaminoglycan (GAG) assay, real-time PCR and histology. Results: We tested 10 doses, from 0.001µM up to 10 µM, and determined that the IC 50 of Win-55 on human chondrocytes for 2 days was ˜ 2 µM. Interestingly, this dose is significantly lower than the doses reported in similar studies. As shown in Figure 1, treatment with 2µM Win-55 causes the complete loss of GAG from engineer cartilage. In a relatively safe dose (<=1 µM), we did not observe obvious changes in all tested genes after the treatments of Win-55 (Figure 2). Conclusion: High dose of Win-55 may directly cause the degeneration of cartilage, while low dose of Win-55 doesn’t show beneficial influence on the phenotype of IL1-β-insulted cartilage. The reported anti-inflammatory effect of Win 55 on chondrocytes may due to the cytotoxicity or global inhibition of high dose Win 55 on cell activities. Therefore, if cannabinoid can be used to treat OA requires further investigation.

scholarly journals Ascorbic acid attenuates activation and cytokine production in sepsis-like monocytes

2021 ◽  
Author(s):  
Tobias Schmidt ◽  
Robin Kahn ◽  
Fredrik Kahn
Keyword(s):  
Flow Cytometry ◽  
Ascorbic Acid ◽  
Cytokine Production ◽  
In Vitro Study ◽  
Surface Expression ◽  
High Dose ◽  
Functional Assay ◽  
Dependent Manner ◽  
Dose Dependent

Objective To investigate the effects of high dose ascorbic acid (AA) on monocyte polarization and cytokine production in vitro Design Experimental in vitro study of cells from healthy subjects and patients with sepsis Setting University research laboratory and academic hospital Subjects Six healthy controls and three patients with sepsis Interventions Monocytes were isolated from whole blood of healthy donors (n=6) and polarized in vitro for 48hrs using LPS or LTA. Polarization was confirmed by surface marker expression using flow cytometry. As a comparison, monocytes were also isolated from septic patients (n=3) and analyzed for polarization markers. The effect of AA on monocyte polarization was evaluated. As a functional assay, AA-treated monocytes were analyzed for cytokine production of TNF and IL-8 by intracellular staining and flow cytometry following activation with LPS or LTA. Measurements and Main Results Both LPS and LTA induced polarization in healthy monocytes in vitro, with increased expression of both pro- (CD40 and PDL1, p<0.05) and anti-inflammatory (CD16 and CD163, p<0.05) polarization markers, with non-significant effects on CD86 and CD206. This pattern resembled, at least partly, that of monocytes from septic patients. Treatment with AA significantly inhibited the upregulation of surface expression of CD16 and CD163 (p<0.05) in a dose dependent manner, but not CD40 or PDL-1. Finally, AA attenuated LPS or LTA-induced cytokine production of IL-8 and TNF in a dose-dependent manner (both p<0.05). Conclusions AA inhibits upregulation of anti-, but not pro-inflammatory related markers in LPS or LTA polarized monocytes. Additionally, AA attenuates cytokine production from in vitro polarized monocytes, displaying functional involvement. This study provides important insight into the immunological effects of high dose AA on monocytes, and potential implications in sepsis.

Anoxia-induced LTP of isolated NMDA receptor-mediated synaptic responses

1993 ◽  
Vol 69 (5) ◽  
pp. 1774-1778 ◽  
Author(s):  
V. Crepel ◽  
C. Hammond ◽  
K. Krnjevic ◽  
P. Chinestra ◽  
Y. Ben-Ari
Keyword(s):  
Nmda Receptor ◽  
Neuronal Death ◽  
Hippocampal Neurons ◽  
Input Resistance ◽  
Long Term Potentiation ◽  
Term Potentiation ◽  
Bath Application ◽  
Synaptic Responses

1. The effects of an anoxic-aglycemic episode (1-3 min) on the pharmacologically isolated N-methyl-D-aspartate (NMDA)-mediated responses were examined in CA1 pyramidal hippocampal neurons in vitro. 2. An anoxic-aglycemic episode induced a long term potentiation (LTP) of the NMDA receptor-mediated field excitatory post-synoptic potentials (EPSPs). This LTP, referred to as anoxic LTP, was observed in the presence of 1) a normal Mg2+ concentration [+40.1 +/- 5% (mean +/- SE)], 2) a low Mg2+ concentration (+52.2 +/- 10%), or 3) a Mg2+ free (+49 +/- 11%), 1 h after anoxia. 3. Bath application of D-2-amino-5-phosphonovaleric acid (D-APV, 20 microM, 15-21 min) before, during, and after the anoxic-aglycemic episode, which transiently blocked the synaptic NMDA receptor mediated response, prevented the induction of anoxic LTP. 4. The intracellularly recorded NMDA receptor-mediated EPSP was also persistently potentiated by anoxia-aglycemia (+47 +/- 4%). This potentiation was not associated with changes in membrane potential or input resistance. 5. These findings provide the first evidence that an anoxic-aglycemic episode induces an LTP of NMDA receptor-mediated responses. This potentiation may participate in the cascade of events that lead to delayed neuronal death.

Drug Elution From High-Dose Antibiotic-Loaded Acrylic Cement: A Comparative, In Vitro Study

Orthopedics ◽  
2014 ◽  
Vol 37 (11) ◽  
pp. e999-e1005 ◽  
Author(s):  
Giorgio Gasparini ◽  
Marco De Gori ◽  
Giovanni Calonego ◽  
Tommaso Della Bora ◽  
Benedetto Caroleo ◽  
...  
Keyword(s):  
In Vitro Study ◽  
Vitro Study ◽  
High Dose ◽  
Acrylic Cement ◽  
Drug Elution

scholarly journals Dose and Temporal Pattern of Estrogen Exposure Determines Neuroprotective Outcome in Hippocampal Neurons: Therapeutic Implications

Endocrinology ◽  
2006 ◽  
Vol 147 (11) ◽  
pp. 5303-5313 ◽  
Author(s):  
Shuhua Chen ◽  
Jon Nilsen ◽  
Roberta Diaz Brinton
Keyword(s):  
Intracellular Calcium ◽  
Temporal Pattern ◽  
Hippocampal Neurons ◽  
Estrogen Therapy ◽  
Therapeutic Benefit ◽  
High Dose ◽  
Therapeutic Implications ◽  
Estrogen Exposure ◽  
The Impact

To address controversies of estrogen therapy, in vitro models of perimenopause and prevention vs. treatment modes of 17β-estradiol (E2) exposure were developed and used to assess the neuroprotective efficacy of E2 against β-amyloid-1–42 (Aβ1–42)-induced neurodegeneration in rat primary hippocampal neurons. Low E2 (10 ng/ml) exposure exerted neuroprotection in each of the perimenopausal temporal patterns, acute, continuous, and intermittent. In contrast, high E2 (200 ng/ml) was ineffective at inducing neuroprotection regardless of temporal pattern of exposure. Although high E2 alone was not toxic, neurons treated with high-dose E2 resulted in greater Aβ1–42-induced neurodegeneration. In prevention vs. treatment simulations, E2 was most effective when present before and during Aβ1–42 insult. In contrast, E2 treatment after Aβ1–42 exposure was ineffective in reversing Aβ-induced degeneration, and exacerbated Aβ1–42-induced cell death when administered after Aβ1–42 insult. We sought to determine the mechanism by which high E2 exacerbated Aβ1–42-induced neurodegeneration by investigating the impact of low vs. high E2 on Aβ1–42-induced dysregulation of calcium homeostasis. Results of these analyses indicated that low E2 significantly prevented Aβ1–42-induced rise in intracellular calcium, whereas high E2 significantly increased intracellular calcium and did not prevent Aβ1–42-induced calcium dysregulation. Therapeutic benefit resulted only from low-dose E2 exposure before, but not after, Aβ1–42-induced neurodegeneration. These data are relevant to impact of perimenopausal E2 exposure on protection against neurodegenerative insults and the use of estrogen therapy to prevent vs. treat Alzheimer’s disease. Furthermore, these data are consistent with a healthy cell bias of estrogen benefit.

Brain monoamine oxidase A in hyperammonemia is regulated by NMDA receptors

2009 ◽  
Vol 4 (3) ◽  
pp. 321-326
Author(s):  
Elena Kosenko ◽  
Yury Kaminsky
Keyword(s):  
Oxidative Stress ◽  
Nmda Receptor ◽  
Monoamine Oxidase ◽  
Nmda Receptors ◽  
Monoamine Oxidase A ◽  
Mk 801 ◽  
Mao A ◽  
Vitro Effect

AbstractMitochondrial enzyme monoamine oxidase A (MAO-A) generates hydrogen peroxide (H2O2) and is up-regulated by Ca2+ and presumably by ammonia. We hypothesized that MAO-A may be under the control of NMDA receptors in hyperammonemia. In this work, the in vivo effects of single dosing with ammonia and NMDA receptor antagonist MK-801 and the in vitro effect of Ca2+ on MAO-A activity in isolated rat brain mitochondria were studied employing enzymatic procedure. Intraperitoneal injection of rats with ammonia led to an increase in MAO-A activity in mitochondria indicating excessive H2O2 generation. Calcium added to isolated mitochondria stimulated MAO-A activity by as much as 84%. MK-801 prevented the in vivo effect of ammonia, implying that MAO-A activation in hyperammonemia is mediated by NMDA receptors. These data support the conclusion that brain mitochondrial MAO-A is regulated by the function of NMDA receptors. The enzyme can contribute to the oxidative stress associated with hyperammonemic conditions such as encephalopathy and Alzheimer’s disease. The attenuation of the oxidative stress highlights MAO-A inactivation and NMDA receptor antagonists as sources of novel avenues in the treatment of mental disorders.

Effect of nutrient composition on the in vitro growth of urogenital lactobacilli and uropathogens

1998 ◽  
Vol 44 (9) ◽  
pp. 866-871 ◽  
Author(s):  
Gregor Reid ◽  
Foaud Soboh ◽  
Andrew W Bruce ◽  
Marc Mittelman
Keyword(s):  
Lactobacillus Rhamnosus ◽  
Skim Milk ◽  
In Vitro Study ◽  
Defined Medium ◽  
Nutrient Composition ◽  
Vaginal Fluid ◽  
Natural Substances ◽  
Uropathogenic Bacteria ◽  
Better Than

Previous clinical studies have shown that nutrients and probiotic agents can alter the composition of the vaginal flora. The present in vitro study has shown that uropathogens have a growth advantage over lactobacilli, but potentially there are natural substances that could be applied vaginally to stimulate lactobacilli growth to the detriment of the pathogens. When chemically defined medium representative of vaginal fluid at pH 5.5 was supplemented with skim milk, it acted as a better substrate for Lactobacillus rhamnosus GR-1 than for uropathogenic bacteria and Candida albicans. Lactobacillus MRS medium, even at pH 4.5, supports the growth of pathogens, but when supplemented with ascorbic acid or EDTA, Lactobacillus growth was significantly higher. When L. rhamnosus GR-1 was coincubated in a combined nutrient composition of vitamins and lactose, it survived better than Escherichia coli and Enterococcus faecalis. These in vitro results provide a basis for testing nutritional supplements to alter the urogenital flora in an attempt to enhance restoration and maintenance of a normal disease-free state.Key words: nutrients, lactobacilli, uropathogens, growth.

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