Excitotoxicity is the major contributor to the pathophysiological damage after acute spinal cord injury which is often incomplete, yet it produces paralysis with uncertain outcome for gait recovery despite early intensive care support. Neuroprotecting the spinal cord during the early phase of injury is an important goal to determine a favourable outcome to suppress delayed pathological events that extend the primary damage and amplify the loss of motor function often with irreversible consequences. While intensive care and neurosurgical intervention remain mainstay treatments, effective neuroprotection requires further focused experimental studies under controlled conditions. To better understand the pathophysiological mechanism of spinal lesion an in vitro model of rat spinal cord has been developed by our laboratory whereby injury is mimicked by a moderate excitotoxic insult. Such an injury suppresses the locomotor networks together with partial loss of motoneuronal population. The present thesis explores if the volatile general anesthetic methoxyflurane can protect spinal locomotor networks from kainate induced excitotoxicity and whether motoneuronal survival after excitotoxicity relies on cell expression of heat shock protein 70 (HSP70), a cytosolic neuroprotective protein binding and sequestering metabolic distress-generated proteins. The protocols involved 1 h excitotoxic stimulation on day 1 followed by electrophysiological and immunohistochemical testing after 24 h. A time-limited (1 h), single administration of methoxyfluorane together with kainate (or with 30 min or 60 min delay), prevented any depression of spinal reflexes, loss of motoneuron excitability, and histological damage. Methoxyfluorane per se temporarily decreased synaptic transmission and motoneuron excitability. These effects were readily reversible on washout. When methoxyfluorane was applied with or after kainate, spinal locomotor activity recorded as alternating electrical discharges from lumbar motor pools was fully preserved after 24 h. Furthermore to test the second hypothesis, the motoneurons were investigated for their expression of apoptosis inducing factor (AIF; a known biomarker of cell death) which became preferentially localized to the nucleus in pyknotic cells after excitotoxicity. The surviving motoneurons showed strong expression of HSP70 with no nuclear AIF. The sham preparations did not show any AIF nuclear translocation whereas the preparations treated with kainate (100 µM) were the most affected. VER155008, a pharmacological inhibitor of HSP70, per se induced neurotoxicity comparable to that of kainate. Electrophysiological recording indicated depression of motoneuron field potential with strong decrease in excitability and impaired synaptic transmission following kainate or VER155008. Their combined application elicited more intense neurotoxicity. Interestingly, motoneurons in the spinal cord (24 h in vitro) showed large expression of HSP70 compared to freshly dissected tissue, suggesting that HSP70 up-regulation was critical for spinal cord preparation survival in vitro. These data suggest that a volatile general anesthetic could provide strong electrophysiological and histological neuroprotection that enabled retention of locomotor network activity even one day after the excitotoxic challenge. Our study also showed that HSP70 is important for motoneuronal survival. It is hypothesized that the benefits of early neurosurgery for acute SCI might be enhanced if, in addition to injury decompression and stabilization, the protective role of general anesthesia is maximized. Another potential future strategy to neuroprotect motoneurons could be the upregulation of HSP70 activity by either using its pharmacological enhancers or by inducing its over-expression.
|Titolo:||Study of the neuroprotection mechanisms in a model of spinal cord injury in vitro|
|Data di pubblicazione:||26-gen-2015|
|Appare nelle tipologie:||8.1 PhD thesis|