Phenotypes of Motor Deficit and Pain after Experimental Spinal Cord Injury.

Motor disability is a common outcome of spinal cord injury (SCI). The recovery of motor function after injury depends on the severity of neurotrauma; motor deficit can be reversible, at least partially, due to the innate tissue capability to recover, which, however, deteriorates with age. Pain is often a comorbidity of injury, although its prediction remains poor. It is largely unknown whether pain can attend motor dysfunction. Here, we implemented SCI for modelling severe and moderate neurotrauma and monitored SCI rats for up to 5 months post-injury to determine the profiles of both motor deficit and nociceptive sensitivity. Our data showed that motor dysfunction remained persistent after a moderate SCI in older animals (5-month-old); however, there were two populations among young SCI rats (1 month-old) whose motor deficit either declined or exacerbated even more over 4-5 weeks after identical injury. All young SCI rats displayed changed nociceptive sensitivity in thermal and mechanical modalities. The regression analysis of the changes revealed a population trend with respect to hyper- or hyposensitivity/motor deficit. Together, our data describe the phenotypes of motor deficit and pain, the two severe complications of neurotrauma. Our findings also suggest the predictability of motor dysfunction and pain syndromes following SCI that can be a hallmark for long-term rehabilitation and recovery after injury.

Measurement of intracellular concentration of fluorescently-labeled targets in living cells.

Estimations of intracellular concentrations of fluorescently-labeled molecules within living cells are very important for guidance of biological experiments and interpretation of their results. Here we propose a simple and universal approach for such estimations. The approach is based upon common knowledge that the dye fluorescence is directly proportional to its quantum yield and the number of its molecules and that a coefficient of proportionality is determined by spectral properties of the dye and optical equipment used to record fluorescent signals. If two fluorescent dyes are present in the same volume, then a ratio of their concentrations is equal to a ratio of their fluorescence multiplied by some dye- and equipment-dependent coefficient. Thus, if the coefficient and concentration of one dye is known then the concentration of another dye can be determined. Here we have demonstrated how to calculate this coefficient (called a ratio factor) and how to use it for concentration measurements of fluorescently tagged molecules. As an example of how this approach can be used, we estimated a concentration of exogenously expressed neuronal Ca2+ sensor protein, hippocalcin, tagged by a fluorescent protein in a dendritic tree of rat hippocampal neurons loaded via a patch pipette with Alexa Fluor dye of known concentration. The new approach should allow performing a fast, inexpensive and reliable quantitative analysis of fluorescently-labeled targets in different parts of living cells.