ryanodine and Spinal-Cord-Injuries

Ryanodine receptors (RyRs) mediate calcium release from calcium stores and have been implicated in axonal degeneration. Here, we use an intravital imaging approach to determine axonal fate after spinal cord injury (SCI) in real-time and assess the efficacy of ryanodine receptor inhibition as a potential therapeutic approach to prevent intra-axonal calcium-mediated axonal degeneration. Adult 6-8 week old

Topics: Animals; Axons; Calcium; Disease Models, Animal; Intravital Microscopy; Mice; Mice, Transgenic; Ryanodine; Ryanodine Receptor Calcium Release Channel; Spinal Cord; Spinal Cord Injuries

Transected axons of the central nervous system fail to regenerate and instead die back away from the lesion site, resulting in permanent disability. Although both intrinsic (eg, microtubule instability, calpain activation) and extrinsic (ie, macrophages) processes are implicated in axonal dieback, the underlying mechanisms remain uncertain. Furthermore, the precise mechanisms that cause delayed "bystander" loss of spinal axons, that is, ones that were not directly damaged by the initial insult, but succumbed to secondary degeneration, remain unclear. Our goal was to evaluate the role of intra-axonal Ca(2+) stores in secondary axonal degeneration following spinal cord injury.. We developed a 2-photon laser-induced spinal cord injury model to follow morphological and Ca(2+) changes in live myelinated spinal axons acutely following injury.. Transected axons "died back" within swollen myelin or underwent synchronous pan-fragmentation associated with robust Ca(2+) increases. Spared fibers underwent delayed secondary bystander degeneration. Reducing Ca(2+) release from axonal stores mediated by ryanodine and inositol triphosphate receptors significantly decreased axonal dieback and bystander injury. Conversely, a gain-of-function ryanodine receptor 2 mutant or pharmacological treatments that promote axonal store Ca(2+) release worsened these events.. Ca(2+) release from intra-axonal Ca(2+) stores, distributed along the length of the axon, contributes significantly to secondary degeneration of axons. This refocuses our approach to protecting spinal white matter tracts, where emphasis has been placed on limiting Ca(2+) entry from the extracellular space across cell membranes, and emphasizes that modulation of axonal Ca(2+) stores may be a key pharmacotherapeutic goal in spinal cord injury.

Topics: Animals; Axons; Bacterial Proteins; Boron Compounds; Caffeine; Calcium; Disease Models, Animal; Endoplasmic Reticulum; Enzyme Inhibitors; Laser Therapy; Luminescent Proteins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mutation; Nerve Degeneration; Purinergic P1 Receptor Antagonists; Ryanodine; Ryanodine Receptor Calcium Release Channel; Spinal Cord Injuries; Thapsigargin; Time Factors

In rat tail artery (RTA), spinal cord injury (SCI) increases nerve-evoked contractions and the contribution of L-type Ca2+ channels to these responses. In RTAs from unoperated rats, these channels play a minor role in contractions and Bay K8644 (L-type channel agonist) mimics the effects of SCI. Here we investigated the mechanisms underlying the facilitatory actions of SCI and Bay K8644 on nerve-evoked contractions of RTAs and the hypothesis that Ca2+ entering via L-type Ca2+ channels is rapidly sequestered by the sarcoplasmic reticulum (SR) limiting its role in contraction. In situ electrochemical detection of noradrenaline was used to assess if Bay K8644 increased noradrenaline release. Perforated patch recordings were used to assess if SCI changed the Ca2+ current recorded in RTA myocytes. Wire myography was used to assess if SCI modified the effects of Bay K8644 and of interrupting SR Ca2+ uptake on nerve-evoked contractions. Bay K8644 did not change noradrenaline-induced oxidation currents. Neither the size nor gating of Ca2+ currents differed between myocytes from sham-operated (control) and SCI rats. Bay K8644 increased nerve-evoked contractions in RTAs from both control and SCI rats, but the magnitude of this effect was reduced by SCI. By contrast, depleting SR Ca2+ stores with ryanodine or cyclopiazonic acid selectively increased nerve-evoked contractions in control RTAs. Cyclopiazonic acid also selectively increased the blockade of these responses by nifedipine (L-type channel blocker) in control RTAs, whereas ryanodine increased the blockade produced by nifedipine in both groups of RTAs. These findings suggest that Ca2+ entering via L-type channels is normally rapidly sequestered limiting its access to the contractile mechanism. Furthermore, the findings suggest SCI reduces the role of this mechanism.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Calcium; Calcium Channels; Calcium Channels, L-Type; Cytoplasm; Electrochemistry; Female; Indoles; Muscle Cells; Muscle Contraction; Nifedipine; Norepinephrine; Rats; Rats, Sprague-Dawley; Ryanodine; Sarcoplasmic Reticulum; Spinal Cord Injuries; Tail

The deleterious effects of glutamate excitotoxicity are well described for central nervous system gray matter. Although overactivation of glutamate receptors also contributes to axonal injury, the mechanisms are poorly understood. Our goal was to elucidate the mechanisms of kainate receptor-dependent axonal Ca(2+) deregulation.. Dorsal column axons were loaded with a Ca(2+) indicator and imaged in vitro using confocal laser-scanning microscopy.. Activation of glutamate receptor 6 (GluR6) kainate receptors promoted a substantial increase in axonal [Ca(2+)]. This Ca(2+) accumulation was due not only to influx from the extracellular space, but a significant component originated from ryanodine-dependent intracellular stores, which, in turn, depended on activation of L-type Ca(2+) channels: ryanodine, nimodipine, or nifedipine blocked the agonist-induced Ca(2+) increase. Also, GluR6 stimulation induced intraaxonal production of nitric oxide (NO), which greatly enhanced the Ca(2+) response: quenching of NO with intraaxonal (but not extracellular) scavengers, or inhibition of neuronal NO synthase with intraaxonal Nomega-nitro-L-arginine methyl ester, blocked the Ca(2+) increase. Loading axons with a peptide that mimics the C-terminal PDZ binding sequence of GluR6, thus interfering with the coupling of GluR6 to downstream effectors, greatly reduced the agonist-induced axonal Ca(2+) increase. Immunohistochemistry showed GluR6/7 clusters on the axolemma colocalized with neuronal NO synthase and Ca(v)1.2.. Myelinated spinal axons express functional GluR6-containing kainate receptors, forming part of novel signaling complexes reminiscent of postsynaptic membranes of glutamatergic synapses. The ability of such axonal "nanocomplexes" to release toxic amounts of Ca(2+) may represent a key mechanism of axonal degeneration in disorders such as multiple sclerosis where abnormal accumulation of glutamate and NO are known to occur.

Topics: Animals; Axons; Calcium; Calcium Channels, L-Type; Cysteine; Egtazic Acid; Excitatory Amino Acid Antagonists; GluK2 Kainate Receptor; Glutamic Acid; Hydroxocobalamin; Male; Microscopy, Confocal; Myoglobin; Nerve Fibers, Myelinated; Nitric Oxide; PDZ Domains; Peptides; Protein Multimerization; Rats; Rats, Long-Evans; Receptors, Kainic Acid; Ryanodine; Spinal Cord Injuries; Spinal Nerve Roots

Calcium influx and elevation of intracellular free calcium (Ca2+i), with subsequent activation of degenerative enzymes is hypothesized to cause cell injury and death after trauma. We examined the effects of traumatic compressive injury on (Ca2+)i dynamics in spinal cord white matter. We conducted electrophysiological studies with ryanodine and inositol (1,4,5)-triphosphate (IP3) receptor agonists and antagonists in an in vitro model of spinal cord injury (SCI). A 25-30-mm length of dorsal column was isolated from the spinal cord of adult rats, pinned in an in vitro recording chamber (37 degrees C) and injured with a modified clip (2-g closing force) for 15 sec. The functional integrity of the dorsal column was monitored electrophysiologically by quantitatively measuring the compound action potential (CAP) with glass microelectrodes. The CAP decreased to 55.2+/-6.8% of control (p < 0.05) after spinal cord injury (SCI). Chelation of Ca2+i with BAPTA-AM (a high-affinity calcium chelator) promoted significantly greater recovery of CAP amplitude (83.2+/-4.2% of control; p < 0.05) after injury. Infusion of caffeine (1 and 10 mM) exacerbated CAP amplitude decline (45.1+/-5.9% of control; p < 0.05; 44.6+/-3.1% of control; p < 0.05) postinjury. Blockade of Ca2+i release through ryanodine-sensitive receptors (RyRs) with dantrolene (10 microM) and ryanodine (50 microM), conferred significant (p < 0.05) improvement in CAP amplitude after injury. On the other hand, blockade of Ca2+i with inositol (1,4,5)-triphosphate receptor (IP3Rs) blocker 2APB (10 microM) also conferred significant improvement in CAP amplitude after injury (82.9+/-7.9%; p < 0.05). In conclusion, the injurious effects of Ca2+i in traumatic central nervous system (CNS) white matter injury appear to be mediated both by RyRs and through IP3Rs calcium-induced calcium release receptors (CICRs).

Topics: Action Potentials; Animals; Boron Compounds; Caffeine; Calcium; Calcium Channels; Calcium-Binding Proteins; Central Nervous System Stimulants; Chelating Agents; Dantrolene; Egtazic Acid; Electrophysiology; In Vitro Techniques; Inositol 1,4,5-Trisphosphate Receptors; Male; Models, Animal; Muscle Relaxants, Central; Neural Conduction; Rats; Rats, Wistar; Receptors, Cytoplasmic and Nuclear; Ryanodine; Ryanodine Receptor Calcium Release Channel; Spinal Cord Injuries