An in vivo model for studying the dynamics of intracellular free calcium changes in slow- and fast-twitch muscle fibres.

The understanding of the regulation of the free cytosolic [Ca2+] ([Ca2+]i) in skeletal muscle is hampered by the lack of techniques for quantifying free [Ca2+]i in muscle fibres in situ. We describe a model for studying the dynamics of free [Ca2+]i in the fast-twitch extensor digitorum longus (EDL) and the slow-twitch soleus (SOL) muscles of the rat in vivo using caffeine superfusion to induce changes in free [Ca2+]i. We assumed that differences in sensitivity between the two muscle types for this substance reflect differences in intracellular Ca2+ handling in the fibres of which these muscles consist. The Indo-1 ratiometric method, using intravital microscopy with incident light, was adapted to measure free [Ca2+]i in vivo. Fluorescence images were collected by means of a digital camera. Caffeine superfusion at 37 degrees C for 2 min, at concentrations of 1, 2, 5, 10 or 20 mmol/l, induced a concentration-dependent increase in free [Ca2+]i and revealed differences in caffeine sensitivity between the muscle types, with the SOL being more sensitive. In a separate set of experiments the contracture threshold, as assessed by topical application of caffeine, was determined in both muscle types. EDL had a higher threshold for developing contracture than SOL. These finding are in agreement with previous in vitro studies. We may conclude that the dynamics of free [Ca2+]i can be assessed reliably in intact mammalian muscle in vivo.

The functional and morphological damage of ischemic reperfused skeletal muscle.

Skeletal muscle is frequently damaged by ischemia-reperfusion both caused by direct injury and also by surgery. The purpose of the present experiments was to examine how the different types of skeletal muscles (fast and slow) react functionally and morphologically after 1 and 2 h of ischemia followed by different periods of reperfusion. The fast-twitch (musculus extensor digitorum longus, EDL) and the slow-twitch (musculus soleus, SOL) muscle of Wistar rats were prepared. They were stimulated in vivo, either directly or indirectly at different reperfusion times following tourniquet ischemia, and the contraction force of the muscles was recorded. The morphological changes were examined by light microscopy. At early reperfusion times, the contraction force of the EDL muscle was reduced by 40 and 90% after 1 and 2 h of ischemia, respectively. The contraction force was about 50% at the end of a 2-week reperfusion period in the 1-hour ischemia group and it increased significantly (from 5 to 38%) during the second week if the ischemia lasted for 2 h. Reduction of contraction force in the SOL muscle was over 50 and 90% following 1 and 2 h of ischemia, respectively, and it started to improve from the 2nd week. Morphological changes of the two types of muscle were identical. At early reperfusion times granulocytes were seen in the blood vessels adhering to the endothelium. 24 h later neutrophil granulocytes migrated into the endomysium and thereafter into the perimysium. One week after 1 h of ischemia both muscles showed normal histology. However, the structural regeneration process only started at the end of the 1st week of reperfusion after 2 h of the ischemic damage. The following conclusions can be drawn. (1) There is functional morphological evidence of ischemic and reperfusion injury in both muscles after 24 h and also after 1 week of reperfusion. (2) Functionally, the two types of muscles regenerate differently, i.e. the SOL starts to regenerate earlier than the EDL. (3) Morphologically the two types of muscle show the same reactions. An increase in the time of ischemia from 1 to 2 h delays the regeneration processes.

The functional damages of ischemic/reperfused skeletal muscle.

Skeletal muscle is frequently damaged by ischemia-reperfusion when exposed to direct injury or in the surgical practice. The purpose of the present experiments was to examine how the different types of skeletal muscles (fast & slow) react functionally to one and two hours of ischemia followed by two weeks of reperfusion. The fast-twitch (m. extensor digitorum longus/EDL) and the slow-twitch (m. soleus/SOL) muscle were prepared. They were stimulated, in vivo, either directly or indirectly at different reperfusion times following tourniquet ischemia, and the contraction force (CF) was recorded. CF of the EDL was reduced over 40% and 90% of the control value during the first 24 hours of reperfusion after 1 and 2 hours of ischemia, respectively. It was about 50% at the end of the 2nd week in the one-hour group. CF increased significantly during the second week if ischemia lasted for two hours. Reduction of CF in the SOL muscle was over 50% and 90% following one and two hours of ischemia, respectively. It further decreased in the 1-hour group, and it started to regenerate from the second week after 2 hours of ischemia. It is concluded that 1. two hours of ischemia causes significantly more severe damages in both types of skeletal muscles than one hour. 2. There is a reperfusion injury in both muscles during the first week of reperfusion. 3. The two types of muscles regenerate differently, i.e. the SOL starts to regenerate earlier than the EDL.