Blood Transfusion and Traumatic Brain Injury
Cham : Springer International Publishing AG, 2021
MILRINONE ATTENUATES THE CARDIOMYOCYTE APOPTOSIS INDUCED BY HYPOXIA [Meeting Abstract]
Isoflurane, but Not the Nonimmobilizers F6 and F8, Inhibits Rat Spinal Cord Motor Neuron CaV1 Calcium Currents
BACKGROUND: Volatile anesthetics decrease Ca entry through voltage-dependent Ca channels. Ca 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 Ca 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. CONCLUSIONS: The findings that isoflurane, but not nonimmobilizers, inhibited CaV1 Ca 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 is sufficient or whether actions on other channels/proteins contribute to isoflurane-induced immobility.
Preparation of adult spinal cord motor neuron cultures under serum-free conditions
Spinal cord motor neuron cultures are an important tool for the study of mechanisms involved in motor neuron survival, degeneration and regeneration, volatile anesthetic-induced immobility, motor neuron disorders such as amyotrophic lateral sclerosis or spinal muscular atrophy as well as in spinal cord injury. Embryonic spinal cord motor neurons derived from rats have been successfully cultured; unfortunately, the culture of adult motor neurons has been problematic due to their short-term survival. Recently, by using a cocktail of target-derived factors, neurotrophins (brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor) and a permeable cyclic adenosine monophosphate analog, we have established a reproducible protocol for long-term cultures of healthy and functional adult motor neurons (Exp Neurol 220:303-315, 2009). Here, we now describe in detail the steps that we used for the optimization of the process of isolation and maintenance of adult rat ventral horn motor neurons in vitro.
A Role for the Cannabinoid 1 Receptor in Neuronal Differentiation of Adult Spinal Cord Progenitors in vitro is Revealed through Pharmacological Inhibition and Genetic Deletion
In contrast to the adult brain, the adult spinal cord is a non-neurogenic environment. Understanding how to manipulate the spinal cord environment to promote the formation of new neurons is an attractive therapeutic strategy for spinal cord injury and disease. The cannabinoid 1 receptor (CB1R) has been implicated as a modulator of neural progenitor cell proliferation and fate specification in the brain; however, no evidence exists for modulation of adult spinal cord progenitor cells. Using adult rat spinal cord primary cultures, we demonstrated that CB1R antagonism with AM251 significantly decreased the number of Nestin(+) cells, and increased the number of betaIII tubulin(+) and DCX(+) cells, indicative of neuronal differentiation. AM251's effect was blocked by co-application of the CB1R agonists, WIN 55, 212-2, or ACEA. Consistent with our hypothesis, cultures, and spinal cord slices derived from CB1R knock-out (CB1-/-) mice had significantly higher levels of DCX(+) cells compared to those derived from wild type (CB1+/+) mice, indicative of enhanced neuronal differentiation in CB1-/- spinal cords. Moreover, AM251 promoted neuronal differentiation in CB1+/+, but not in CB1-/- cultures. Since CB1R modulates synaptic transmission, and synaptic transmission has been shown to influence progenitor cell fate, we evaluated whether AM251-induced neuronal differentiation was affected by chronic inactivity. Either the presence of the voltage-dependent sodium channel blocker tetrodotoxin (TTX), or the removal of mature neurons, inhibited the AM251-induced increase in DCX(+) cells. In summary, antagonism or absence of CB1R promotes neuronal differentiation in adult spinal cords, and this action appears to require TTX-sensitive neuronal activity. Our data suggest that the previously detected elevated levels of endocannabinoids in the injured adult spinal cord could contribute to the non-neurogenic environment and CB1R antagonists could potentially be used to enhance replacement of damaged neurons.
The N-methyl-d-aspartate-evoked cytoplasmic calcium increase in adult rat dorsal root ganglion neuronal somata was potentiated by substance P pretreatment in a protein kinase C-dependent manner
The involvement of substance P (SP) in neuronal sensitization through the activation of the neurokinin-1-receptor (NK1r) in postsynaptic dorsal horn neurons has been well established. In contrast, the role of SP and NK1r in primary sensory dorsal root ganglion (DRG) neurons, in particular in the soma, is not well understood. In this study, we evaluated whether SP modulated the NMDA-evoked transient increase in cytoplasmic Ca(2+) ([Ca(2+)](cyt)) in the soma of dissociated adult DRG neurons. Cultures were treated with nerve growth factor (NGF), prostaglandin E(2) (PGE(2)) or both NGF+PGE(2). Treatment with NGF+PGE(2) increased the percentage of N-methyl-d-aspartate (NMDA) responsive neurons. There was no correlation between the percentage of NMDA responsive neurons and the level of expression of the NR1 and NR2B subunits of the NMDA receptor or of the NK1r. Pretreatment with SP did not alter the percentage of NMDA responsive neurons; while it potentiated the NMDA-evoked [Ca(2+)](cyt) transient by increasing its magnitude and by prolonging the period during which small- and some medium-sized neurons remained NMDA responsive. The SP-mediated potentiation was blocked by the SP-antagonist ([D-Pro(4), D-Trp(7,9)]-SP (4-11)) and by the protein kinase C (PKC) blocker bisindolylmaleimide I (BIM); and correlated with the phosphorylation of PKCepsilon. The Nk1r agonist [Sar(9), Met(O(2))(11)]-SP (SarMet-SP) also potentiated the NMDA-evoked [Ca(2+)](cyt) transient. Exposure to SP or SarMet-SP produced a rapid increase in the labeling of phosphorylated-PKCepsilon. In none of the conditions we detected phosphorylation of the NR2B subunit at Ser-1303. Phosphorylation of the NR2B subunit at Tyr1472 was enhanced to a similar extent in cells exposed to NMDA, SP or NMDA+SP, and that enhancement was blocked by BIM. Our findings suggest that NGF and PGE(2) may contribute to the injury-evoked sensitization of DRG neurons in part by enhancing their NMDA-evoked [Ca(2+)](cyt) transient in all sized DRG neurons; and that SP may further contribute to the DRG sensitization by enhancing and prolonging the NMDA-evoked increase in [Ca(2+)](cyt) in small- and medium-sized DRG neurons
Isoflurane inhibits cyclic adenosine monophosphate response element-binding protein phosphorylation and calmodulin translocation to the nucleus of SH-SY5Y cells
BACKGROUND: Calmodulin (CaM) activation by Ca(2+), its translocation to the nucleus, and stimulation of phosphorylation of cyclic adenosine monophosphate response element-binding protein (CREB) (P-CREB) are necessary for new gene expression and have been linked to long-term potentiation, a process important in memory formation. Because isoflurane affects memory, we tested whether isoflurane interfered with the translocation of CaM to the neuronal cell nucleus and attenuated the formation P-CREB. METHODS: SH-SY5Y cells, a human neuroblastoma cell line, were cultured. Cells were depolarized with KCl and the phosphorylation of CREB examined by Western blotting, enzyme-linked immunosorbant assay, and immunocytochemistry. The translocation of CaM from the cytosol to the nucleus was also examined after depolarization. Cells were depolarized and lysed and fractionated by centrifugation to determine the amount of CaM translocated to the nucleus. CaM was localized by immunocytochemistry and quantitated by Western blotting and imaging. Before and during KCl depolarization, cells were exposed to isoflurane, isoflurane plus Bay K 8644, nitrendipine, and omega-conotoxin GVIa, respectively. RESULTS: P-CREB increased after KCl depolarization. The increase of P-CREB peaked at depolarization duration of 30 s. The increase in P-CREB formation was inhibited by nitrendipine, but not omega-conotoxin, and by isoflurane in a concentration-dependent fashion. Pretreatment with the L-type Ca(2+) channel agonist, Bay K 8644, attenuated the inhibition of P-CREB formation by isoflurane. CaM presence in the nucleus occurred after KCl depolarization. CaM translocation was inhibited by nitrendipine and attenuated by isoflurane. Bay K 8644 pretreatment decreased the isoflurane inhibition of CaM translocation to the nucleus. CONCLUSIONS: Our data demonstrate that isoflurane inhibits CaM translocation and P-CREB formation. This most likely occurs through isoflurane inhibition of Ca(2+)entry through L-type Ca(2+) channels