Preparation of Adult Spinal Cord Motor Neuron Cultures Under Serum-Free Conditions

Montoya-Gacharna, Jose V.; Sutachán, Jhon-Jairo; Chan, Wai Si; Sideris, Alexandra; Blanck, Thomas J. J.; Recio-Pinto, Esperanza

doi:10.1007/978-1-61779-536-7_10

Cited by 12 publications

(8 citation statements)

References 34 publications

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“…The generation of C2C12 myocyte-conditioned medium was performed like previously described by Montoya-Gacharna et al 2012 [ 40 ]. In brief, C2C12 myocytes were cultured in a culture bottle containing proliferation medium (20% FCS, 1% Pen/Strep, 4.5 g/L D-glucose in Dulbecco’s modified eagle medium (DMEM)) until the settlement attained a confluence of 30%.…”

Section: Methodsmentioning

confidence: 99%

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Caffeine and NAD+ Improve Motor Neural Integrity of Dissociated Wobbler Cells In Vitro

2020

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Amyotrophic lateral sclerosis (ALS) is a common degenerative disease of the central nervous system concerning a progressive loss of upper and lower motor neurons. While 5%–10% of patients are diagnosed with the inherited form of the disease, the vast majority of patients suffer from the less characterized sporadic form of ALS (sALS). As the wobbler mouse and the ALS show striking similarities in view of phenotypical attributes, the mouse is rated as an animal model for the disease. Recent investigations show the importance of nicotinamide adenine dinucleotide (NAD+) and its producing enzyme nicotinic acid mononucleotide transferase 2 (Nmnat2) for neurodegeneration as well as for the preservation of health of the neuronal cells. Furthermore, it is newly determined that these molecules show significant downregulations in the spinal cord of wobbler mice in the stable phase of disease development. Here, we were able to prove a positive benefit on affected motor neurons from an additional NAD+ supply as well as an increase in the Nmnat2 level through caffeine treatment in cells in vitro. In addition, first assumptions about the importance of endogenous and exogenous factors that have an influence on the wellbeing of motor nerve cells in the model of ALS can be considered.

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Section: Methodsmentioning

confidence: 99%

“…To study the growth and regeneration of motor neurons ex vivo, a dissociated culture of the ventral horn was performed according to Montoya-Gacharna et al 2012 [ 40 ] and Brewer and Torricelli 2007 [ 41 ].…”

Section: Methodsmentioning

confidence: 99%

Caffeine and NAD+ Improve Motor Neural Integrity of Dissociated Wobbler Cells In Vitro

2020

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“…Spinal cord neurons of Sprague-Dawley rats at postnatal days 3–5 were purchased from the Shanghai Animal Center, Chinese Academy of Sciences (Shanghai, China) and were harvested and cultured in vitro using a modified method ( 18 ). Rats were anesthetized with diethyl ether (60297; Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) and total spinal cord tissues were isolated.…”

Section: Methodsmentioning

confidence: 99%

MicroRNA-21 promotes neurite outgrowth by regulating PDCD4 in a rat model of spinal cord injury

Jiang

Ding

et al. 2017

Molecular Medicine Reports

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Altered expression levels of microRNA-21 (miRNA-21) have been observed in a series of pathological processes, including cancer and central nervous system injury; however, the involvement of miRNA-21 in the molecular pathophysiology of spinal cord injury (SCI) has not been well documented. The present study examined the expression levels of miRNA-21 and its predicted target genes, programmed cell death 4 (PDCD4) and phosphatase and tensin homolog (PTEN), in rats using quantitative polymerase chain reaction and western blotting to further understand the role of miRNA-21 and the mechanisms underlying repair following SCI. The present study demonstrated that compared with uninjured spinal cords, miRNA-21 expression levels were significantly downregulated in injured spinal cords 4 and 8 h, and 1 day post-SCI, and were significantly upregulated after 3 and 7 days. Conversely, expression levels of PDCD4 and PTEN were significantly decreased at days 3 and 7 post-SCI compared with the control group. miRNA-21 overexpression in monolayer-cultured postnatal rat spinal cord neurons promoted neurite outgrowth and downregulated protein expression levels of PDCD4; however, PTEN protein expression levels were unaltered. To confirm that miRNA-21 directly targets PDCD4, a pRL-CMV luciferase reporter construct was used to detect miRNA-21 interactions with the PDCD4 3′-untranslated region. The results demonstrated that miRNA-21 decreased luciferase activity compared with a rat PDCD4 control reporter. The results of the present study suggested that increased miRNA-21 expression levels following SCI may promote the repair of injured spinal cords by inhibiting the expression of its target gene PDCD4.

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“…Primary cultures of adult rat DRGN and SCMN were prepared and maintained as previously described. 23,24 DRGN were studied within 48-72 hrs; SCMN were studied after 2-6 weeks. SCMN were identified by their morphology.…”

Section: Methodsmentioning

confidence: 99%

Isoflurane, but Not the Nonimmobilizers F6 and F8, Inhibits Rat Spinal Cord Motor Neuron CaV1 Calcium Currents

Recio-Pinto

Montoya-Gacharna²,

Xu³

et al. 2016

Anesthesia &Amp; Analgesia

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Background Volatile anesthetics decrease Ca2+ entry through voltage-dependent Ca2+ channels. Ca2+ influences neurotransmitter release and neuronal excitability. Because volatile anesthetics act specifically on the spinal cord to produce immobility, we examined the effect of isoflurane and the nonimmobilizers F6 (1, 2- dichlorohexafluorocyclobutane) and F8 (2, 3- dichlorooctafluorobutane) on CaV1 and CaV2 Ca2+ channels in spinal cord motor neurons and dorsal root ganglion neurons. Methods Using patch clamping, we compared the effects of isoflurane with those of F6 and F8 on CaV1 and CaV2 channels in isolated, cultured adult rat spinal cord motor neurons and on CaV1 and CaV2 channels in adult rat dorsal root ganglion sensory neurons. Results In spinal cord motor neurons, isoflurane, but not F6 or F8, inhibited currents through CaV1 channels. Isoflurane and at least one of the nonimmobilizers inhibited currents through CaV1 and CaV2 channels in dorsal root ganglion neurons and Cav2 in spinal cord motor neurons Conclusion The findings that isoflurane, but not nonimmobilizers, inhibited CaV1 Ca2+ channels in spinal cord motor neurons are consistent with the notion that spinal cord motor neurons might mediate isoflurane-induced immobility. Additional studies are required to examine whether inhibition of CaV1 calcium currents in spinal cord motor neurons are sufficient, or whether actions on other channels/proteins also contribute to isoflurane-induced immobility.

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Preparation of Adult Spinal Cord Motor Neuron Cultures Under Serum-Free Conditions

Cited by 12 publications

References 34 publications

Caffeine and NAD+ Improve Motor Neural Integrity of Dissociated Wobbler Cells In Vitro

Caffeine and NAD+ Improve Motor Neural Integrity of Dissociated Wobbler Cells In Vitro

MicroRNA-21 promotes neurite outgrowth by regulating PDCD4 in a rat model of spinal cord injury

Isoflurane, but Not the Nonimmobilizers F6 and F8, Inhibits Rat Spinal Cord Motor Neuron CaV1 Calcium Currents

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