Abstract
Introduction
Experimental studies of the spinal cord before and after injury caused by trauma are necessary to evaluate the new therapeutic options, such as stem cell therapy. In those experiments, it is mandatory to measure motor and sensitive function adequately to the bias and increase true results. The objective is to describe the technique used to obtain the compound and extracellular action potentials in the spinal cord.
Materials and Methods
To obtain the spinal cord compound action potentials (CAPs), the spinal cord of the rat spine was immediately placed in a Perspex chamber covered with Sylgard and superfused at (2 mL/min) with Krebs solution and glucose. The solution was maintained at pH 7.2 and 5% CO2/95% O2. The Ag/AgCl needle electrodes fixed the tissue in the chamber. The needle electrodes were connected to an operational amplifier and to a programmable signal conditioner model. The electrical stimulation was done by suction bipolar electrode placed in the inferior segment of the spinal cord. The electrode was connected to an analogical stimulator, which delivered stimulus at 01 Hz for a 0.2 milliseconds duration. The data always began to be recorded after 15 minutes of stimulation to ensure the stability of the preparation. The recordings of the CAPs were made during 30 minutes directly to an AxoScope software. The events were analyzed by the Clampfit software. To acquire extracellular action potential, the spinal cord was cooled in Krebs solution, previously gassed with a 95% O2 and 5% CO2 mixture to obtain a pH value of 7.2 to 7.4. A 400 µm thick coronal section was made. The spinal cord slice was recovered during at least 30 minutes at 32°C in Krebs solution and transferred to a submersion-type recording chamber with Krebs solution, 95% O2 and 5% CO2. Extracellular recordings were made using 5 to 10 MΩ resistance glass pipettes. An iridium stimulation electrode was connected to an analogical stimulator and positioned at the proximal side of the tissue to deliver electrical stimuli (0.1 Hz, 0.2 ms). The recording electrode was placed in the opposite or distal side. The recordings were performed using an amplifier connected to a programmable signal conditioner. The experiments were recorded during 1 hour. The data were retrieved by an AxoScope software and analyzed by the Clampfit software.
Results
The compound and extracellular action potential could be generated in both experiments indicating that the functional analysis of spinal cord neurons can be assessed by in vitro stimulation of the spinal cord tissue or its slice. The control values for the amplitude of CAPs was 4.4 ± 0.3 mV (n = 3). The rise time of the CAPs was 1.73 ± 03 ms; the decay time was 4.2 ± 2 ms, and the duration was 4.2 ± 0.8 ms. The analysis of the extracellular action potentials showed that the mean amplitude, rise time, decay time, and duration were 2.17 ± 0.3 mV, 0.4 ± 0.2, 4 ± 1, and 2.1 ± 0.2 ms, respectively.
Conclusion
The analysis of compound and extracellular action potential can be used as outcome measurement of spinal cord functionality. This can measure more precisely, for example, the neuronal repopulation and motor and sensitive recovery of spinal cord injury.