Background. The basis for the function of the nervous system is signalling among the nerve cells through the synapses. Normal signalling requires precise synthesis and localisation of synaptic structures within the cell. There is a lot of work done in this field and the most often used model for the research is the neuromuscular junction because of its accessibility. The enzyme acetylcholinesterase (AChE) plays a crucial role in it by degrading acetylcholine and by that stopping the electrical signal among the nerve cells. The activity and localisation within the cell are very important for the proper function of the AChE. They depend on the expression of the AChE gene, which is further dependant on many factors and their interrelationships. Among them, the most important are electromechanical activity and local neural mediators. Denervation of the muscle causes a lack of the factors mentioned above and consequently leads to atrophic changes of the muscle, decrease of the AChE activity in neuromuscular junction and the level of AChE mRNA in the muscle cells.
Aim and hypothesis. Our observations were based on the measurement of the AChE activity and the level of AChE mRNA in the fast rat extensor digitorum longus (EDL) muscle. We used different stimulation patterns on the animal model. The results were then compared in order to test the next hypothesis:
Change in the level of AChE mRNA and the activity of AChE in the EDL muscle after denervation can be partially prevented by a specific simple pattern of electrical muscle stimulation.
Rationale for hypothesis. Electromechanic activity caused by the nerve is crucial for the regulation of AChE metabolism. Denervation of the muscle causes a drop of AChE activity and level of AChE mRNA within the first week. Electrical stimulation can affect many post- denervational processes and can stop them to a certain extent. However, a stimulation pattern that would prevent the postdenervational changes of AChE activity and level of AChE mRNA in the EDL muscle has not been discovered yet. By using different patterns of stimulation we can expect to find the most appropriate one, that would cause the least change comparing to the normal innervated muscle.
Methods. The experiments were performed on female Wistar rat strains. Animals were divided in four groups. In the first group of animals the EDL muscles were denervated and then stimulated with different electrical patterens. In the second group muscles were stimulated through the nerve. In the third group muscles were denervated and the electrodes were placed as usually, but there was no stimulation. And in the fourth group we only placed the electrodes on usual places without denervation and stimulation of the muscles. After four days of stimulation the isolation of the EDL muscles was performed. We measured the AChE activity in the muscles with a spectrophotometrical method and the level of AChE mRNA with the Northern-blot method. The results were statistically evaluated with Kruskal-Wallis test and unpaired Wilcoxon’s test.
Results. In all groups of denervated EDL muscles we measured a significant drop of the AChE activity comparing to the normally innervated muscle of the other leg. Different patterns of electrical stimulation had various success in preventing the postdenervational drop of AChE activity. However, the highest AChE activity was obtained with the stimulation pattern with frequency of impulses 150 Hz, duration of impulse trains 0.2 s and interval between trains of 15min. This AChE activity was significantly different from the activities of other groups of denervated muscles (p<0,01). Similar results were obtained with the measurement of the level of AChE mRNA. The same pattern proved to be the most successful in preventing AChE mRNA level drop.
Conclusions. The above mentioned results proved our hypothesis. There does exist a simple stimulation pattern that can partially prevent the postdenervational decrease of AChE mRNA level and AChE activity. However, due to inability to completely prevent that decrease, we can conclude that there must be other mechanisms involved in maintenance of normal level of AChE mRNA and activity of AChE. These are most probably more complex muscle activation patterns, separate activation of motor units within the muscle with different activation patterns and local mediators from the peripheral nerve.