Pharmacological neuroprotection of rat spinal locomotor networks against experimental spinal cord injury in vitro

摘要

Background: Mammalian locomotor behaviour called fictive locomotion can be elicited in an isolated spinal cord in the absence of higher brain center or sensory input. This relatively simple behaviour is produced by the motoneuronal rhythmic activity which is under the control of spinal neuronal networks called central pattern generators (CPGs). Disturbance of this rhythmic motor output can occur following spinal cord injury (SCI). This elementary isolated spinal cord model gives us an opportunity to study the basic physiology of locomotion during control conditions, the pathological processes following lesion (which can be induced chemically), and eventually the application of therapeutic approaches curbing injury. udObjectives: Multiple aspects of spinal functions can be demonstrated by stimulating or/and blocking specific inputs and measuring the outputs using electrophysiological, immunohistochemical and calcium imaging tools. Using isolated neonatal rat spinal cords and organotypic spinal slices as SCI models, the basic mechanisms (such as dysmetabolic state or excitotoxicity) which can develop during the early phase of the lesion were addressed and studied. The injury was evoked chemically by applying either pathological medium (to mimic dysmetabolic/hypoxic conditions) or kainate (to produce excitotoxicity that completely abolishes fictive locomotion and network synaptic transmission) for 1 h. Fictive locomotion was examined stimulating the lumbar dorsal root and recording from the ipsilateral and ipsi-segmental ventral roots. Other network parameters were also studied such as synaptic transmission and rhythmicity. Various therapeutic drugs such as methylprednisolone sodium succinate (MPSS), propofol, nicotine and celastrol were used during or after the injury (to produce neuroprotection) and network properties were characterized during the treatment and after 24 h as well. Subsequently, the structural properties were monitored using different biomarkers (isolated spinal cord sectioned slices) and calcium imaging (here organotypic spinal slices were used). udResults and conclusions: We found that dose-dependent application of MPSS produced modest recovery of white matter damage evoked by pathological medium resulting in the emergence of sluggish chemically induced fictive locomotor patterns. However, it could not prevent damage (to gray matter) evoked by the excitotoxic agent kainate. Therefore, to provide better neuroprotection to gray matter, we tested the widely used intravenous anaesthetic propofol. This drug has shown comparatively good protection to spinal neurons and motoneurons in the gray matter. As it is an anesthetic it acted by depressing the functional network characteristics by lowering the N-methyl-D-aspartate (NMDA) and potentiating the γ aminobutyric acid (GABAA) mediated receptor responses. udThe next issue we addressed was to study the neuroprotective roles of nicotinic acetylcholine receptors (nAChRs) by using the receptor agonist nicotine. Recent studies have shown that nicotine could provide good neuroprotection to the rat brainstem. To further investigate its effect on the spinal cord, we applied nicotine at the same concentration used in previous studies in the brainstem: such a concentration was toxic to spinal ventral motoneurons. Therefore the correct dose of nicotine was optimized and was found to be ten times lower. Thus, satisfactory protective effects to spinal neurons and motoneurons and the fictive locomotor patterns were observed. These neuroprotective effects were replicated with calcium imaging by using organotypic spinal slice cultures. The mechanism of protection predominantly involved α4β2 and less α7 nAChRs. udIn addition, the subsequent goal of our study was to explore whether the motoneuron survival after excitotoxicity relies on cell expression of heat shock protein 70 (HSP70) or some other mechanisms. To test this hypothesis we used a bioactive drug, celastrol which induces the expression of HSP70. Prior application of the drug followed by kainate preserved network polysynaptic transmission and fictive locomotion, however, it could not reverse the depression of monosynaptic reflex responses. In vivo studies are necessary in the future to further investigate the long-term neuroprotective role of these drugs.