Table 1.
Experimental groups.
Figure 1.
NADH-tetrazolium reductase stained sections from the ischemic experiment.
The figure shows representative pictures taken from the anterior tibialis muscle of untreated control animals (A), as well as after 4 (B), 6 (C), 8 (D) and 9 hours (E) of ischemia (induced by infrarenal aortic occlusion) without reperfusion. Typical members of Type I and Type IIb fibers are marked on each picture as I or IIb, respectively. Bar: 35 µm.
Figure 2.
Changes in muscle fiber viability with the progression of ischemia regarding Type IIb, Type I fibers as well as total fiber viability.
Muscle fiber viability decreased continuously over the time of ischemia, Pearson's analysis showed high correlation between the length of ischemia and muscle fiber viability in all three measurements. Type IIb fibers suffered greater damage compared to Type I fibers, which became significant after 6 hours of ischemia (P6h<.05, P8h<.01, P9h<.05). No significant differences (P>.05) could be found between total fiber viability and any fiber types (Two-way ANOVA with Scheffe's post-hoc correction). n = 6 per group.
Figure 3.
Morphology of muscle fibers in control animals and after 8 and 9 hours of ischemia.
Normal morphology of muscle fibers, semithin sections from control muscle (A). After 8 hours of ischemia (B), mostly mitochondria-rich fibers seemed to be affected, while other fibers appeared to be normal. After 9 hours of ischemia (C), almost all fibers showed mild or moderate degree of damage. Toluidine blue staining. Bar: 20 µm.
Figure 4.
Ultrastructural changes in muscle fibers after 8 or 9 hours of ischemia.
Fibers from control muscle displayed normal morphology both in mitochondria-poor (A) and mitochondria-rich muscle fibers (the latter were assumed to be red fibers) (B). After 8 (C, D) and 9 hours of ischemia (E, F) signs of morphological damages were already evident, several lipid-like homogeneous droplets with a peripheral zone of electron-dense margin could be seen (*), especially in mitochondria-rich fibers (D, F, right column). Bar: 2 µm.
Figure 5.
Viability results after long-lasting ischemia and reperfusion.
Muscle fiber viability showed significant reduction after 8-hour-ischemia-only group regarding all three fiber measurements. Also, viability after 9 hours of ischemia and 2 hours of reperfusion decreased significantly compared to the 9-hour-ischemia-only group. Significant difference could be found between the two ischemia-only groups, nevertheless 8 hours ischemia followed by 2 hours reperfusion and 9 hours ischemia alone resulted in a similar loss of viability in all fiber measurements. Type I fibers showed significantly greater loss of viability compared to Type IIb fibers in the 8I, 8IR and 9I groups. Values are given as means ±s.e.m. Between group and within fiber comparison by Two-way ANOVA: P<.001; P<.001 respectively. Differences between the groups by Scheffe's post-hoc test were as follows: # P<.001 vs. corresponding 8I group; & P<.05 vs. corresponding 8IR group; § P<.001 vs. corresponding 9I group; NS P>.05 vs. corresponding 9I group. Differences within fibers by Scheffe's post-hoc test were: $ P<.05 vs. corresponding Type IIb fibers; @ P<.001 vs. corresponding Type IIb fibers; n = 6 per group.
Figure 6.
Morphological signs of ischemic-reperfusion damage of muscle fibers as demonstrated on semithin sections.
Marked morphological changes were visible in all fibers even after 8(A), while after 9 hours of ischemia followed by 2 hours of reperfusion (B) necrosis (double arrow) was already evident in the majority of the fibers. Toluidine blue staining. Bar: 20 µm.
Figure 7.
High magnification images of damaged muscle fibers from the 8IR group.
Lipid-like droplets (*) and myelin figures (arrowheads) were frequently seen (A). Disrupted mitochondria with dense granules of various sizes appeared in the same position (B - arrow). Bar: 1 µm on figure A and 2 µm on figure B.
Figure 8.
Reperfusion augments ultrastructural damage after 8 or 9 hours ischemia and 2 hours reperfusion.
In the 8IR group, signs of degenerative changes similar to those observed in 8I group became more prominent on electron microscopic samples. Several lipid-like droplets were visible, being present in almost all fibers, both with lower (A) or higher amounts of mitochondria (B). Prominent swelling of the sarcoplasmic reticulum was also observed. In the 9IR group, several necrotic fibers were seen (D), while the surviving fibers belonged exclusively to the mitochondria-poor group (C). Bar: 2 µm.