The mdx TS muscle exhibits fiber loss, regeneration, cellular infiltration, hypercontraction, and fibrosis
The nondystrophic TS exhibits a rather uniform thickness ranging from about 3 or 4 fibers thick at the ends of the section to about 2 fibers thick in the center of the middle TS. The fibers were healthy in appearance and had relatively uniform diameters (). In contrast, the middle region of the mdx TS was highly irregular with some areas appearing to be up to 5 or 6 fibers thick and other areas devoid of muscle fibers (). Low power micrographs also revealed discrete areas of cellular infiltration and widespread fibrosis (). Fiber cross sections in the middle mdx TS were highly variable, with the largest fiber diameters approaching those seen in nondystrophic muscle, but with most fibers exhibiting extremely small cross sectional profiles ().
Longitudinal sections through the nondystrophic TS showed uniform-diameter striated fibers (). In contrast, the mdx TS exhibited numerous hypercontracted fibers () and an increase in muscle mass in the caudal region () that was due to an increase in the density of muscle fibers. At higher magnification, the nondystrophic TS exhibited tightly packed and relatively uniform fiber cross sections (). In contrast, the middle mdx TS showed extensive fibrosis between individual fibers (), cellular infiltration (), hypercontraction (), and fiber loss ().
Figure 2 The mdx TS muscle is characterized by a preponderance of hypercontracted fibers, fibrosis, and substantial increases in fiber density in the caudal region. (A) Longitudinal section through a portion of the caudal TS of an adult nondystrophic mouse showing (more ...)
Figure 3 The middle region of the TS muscle in the mdx mouse exhibits substantial fibrosis, cellular infiltration, reduced fiber diameters, and centronucleation. (A) Cross section through the middle region of the nondystrophic TS showing uniform and packed fiber (more ...)
The mdx TS exhibits hypercontraction with adjacent remnants of empty sarcoplasm, and plasma membrane abnormalities
Further examination of hypercontracted fibers using transmission electron microscopy revealed amorphous areas of sarcoplasmic rarefaction adjacent to more dense sarcoplasmic areas (). These relatively mild areas of hypercontraction lacked striations and were characterized by clumps of highly disorganized sarcoplasm (). More discrete areas of hypercontraction were characterized by sharp boundaries separating dense sarcoplasmic plugs from relatively empty areas that were devoid of organized sarcoplasm (; ). The empty areas contained either scattered filamentous structures which appeared to be diffuse remnants of myofibrils (), or were packed with abnormal, swollen mitochondrial profiles (). In some cases, adjacent areas of hypercontraction appeared to surround a central empty area ().
Figure 4 Hypercontraction of mdx TS muscle fibers. (A) Low power transmission electron micrograph of a fiber exhibiting relatively mild hypercontraction (calibration bar = 2 μm). Note the inhomogeneous sarcoplasmic density characterized by clumps of sarcoplasm (more ...)
In areas devoid of organized sarcoplasm that were adjacent to hypercontracted areas, isolated portions of the plasma membrane were often extended inwards away from the continuous external basal lamina layer to form long membrane strands that projected towards the cell interior (). This apparent stripping of the membrane was seen in association with the formation of internal and external vacuoles of assorted shapes and sizes (). In addition, organelles resembling swollen mitochondria were seen in association with these large areas of membrane disruption. These areas of membrane stripping and vacuole formation were seen only in empty fiber areas devoid of any sarcomeric structure.
Figure 5 Plasma membrane abnormalities occur in regions lacking sarcoplasm in mdx TS fibers. (A) Plasma membrane stripping in an empty sarcoplasmic area adjacent to a hypercontraction plug (calibration bar = 500 nm). Note that the sarcolemma and the basal lamina (more ...)
The density and diameter of fibers in the TS are a function of position
Adult (7- 10 mos) nondystrophic and mdx TS muscles exhibited similar regional differences in fiber density and diameter. In each case, the fiber density (), Feret's diameter (), average cross-sectional fiber area (), and total working area per unit length (fiber density × average cross sectional fiber area; ) increased in the cephalad to caudal direction along the TS. Analyses of variance indicated a significant regional effect on fiber diameter in the mdx TS (; τ − p<0.05, ANOVA, Holm-Sidak) and a similar statistical trend in nondystrophic TS. The fiber density (; τττ − p<0.001; ANOVA, Holm-Sidak) and the total working area per unit length of mdx TS muscle (; ττ − p<0.01, ANOVA, Holm-Sidak) each exhibited significant cephalad to caudal gradients. Nondystrophic TS muscle also exhibited a significant cephalad to caudal gradient in working area per unit length (; τ, p<0.05, ANOVA, Holm-Sidak) and a similar gradient in fiber density that just failed to reach significance (; p=0.06, ANOVA).
Figure 6 Morphometric comparisons between untreated adult nondystrophic and mdx TS muscles. (A) Regional differences in fiber density (number of fibers per μm length TS) in nondystrophic (black histobars) and mdx (gray histobars) TS muscles. Note the cephalad (more ...)
Mdx TS muscles exhibit elevated fiber densities and reduced fiber diameters
The fiber density in the middle mdx TS was significantly greater (; * - p< 0.05, Mann Whitney Rank Sum Test) than the corresponding region of the nondystrophic TS at this age (7 to 10 months). Other regions of the mdx TS tended towards higher fiber densities than corresponding nondystrophic regions, but the results did not reach statistical significance (). Muscle fiber diameter () and cross sectional area () in the mdx TS was significantly (** - p<0.01, *** - p<0.001, t test or Mann Whitney Rank Sums test) smaller than in the age-matched nondystrophic TS throughout all regions of the muscle. The combined effects of the increased fiber density and smaller fiber cross-sectional area in the mdx TS produced significant reductions in the working area per unit length for the cephalad and middle regions (; ** - p<0.01, t test) and a reduction in the caudal region that just failed to reach statistical significance (p=0.06, t test).
The effects of PDTC and UDCA on fiber density and diameter in the mdx TS muscle
Fiber density in the caudal TS region of PDTC treated adult mdx mice () was significantly (p<0.05; Mann Whitney Rank Sum test) increased from approximately 0.02 ± 0.01 (SEM; N = 7) in the vehicle treated preparations to 0.08 ± 0.03 (N=6) fibers/μm. PDTC treatment did not influence fiber density in either the cephalad or middle TS regions. As in the PDTC studies, UDCA had no effect on fiber density in either the cephalad or middle TS regions. In the caudal region, UDCA increased fiber density from 0.28 ± 0.03 (N=6) to 0.38 ± 0.04 (N=7) fibers/μm, an effect which just failed to reach statistical significance (p=0.07).
Figure 7 The effect of PDTC (A,B) or UDCA (B,C) treatment on the TS muscle. Staining is H&E (20 μm calibration). Cross-sections obtained from caudal TS muscles from mature adult mdx mice treated chronically with vehicle (A) or PDTC (B). (A) Severely (more ...)
PDTC significantly increased fiber diameter in the middle region and decreased diameter in the caudal region (; * - p<0.05; ** - p<0.01, t tests). A second smaller study examining the action of PDTC in young adult (1 month old) mdx mice indicated a similar statistical trend in which a 30 day PDTC treatment period reduced fiber diameter by 12 % in the caudal region and increased diameter by 14 % in the cephalad region. The UDCA experiments on young adult mdx mice () also indicated a significant drug-induced increase in Feret's diameter in the middle region (; p < 0.01, t test), and a reduction that just failed to reach significance in the caudal region (; p=0.07, t test). Similar effects of UDCA were observed on the minor diameter and the fiber cross-sectional area, but these effects did not reach statistical significance.
Figure 8 Treatment with two distinct inhibitors of the NF-κB pathway produced similar effects on fiber diameter in the mdx TS muscle. Black histobars represent vehicle treated mdx mice and gray histobars mdx mice treated with either PDTC (A) or UDCA (B). (more ...)
The effect of PDTC and UDCA on percent centronucleation
In the vehicle treated mature mdx mice used in the PDTC investigations, there were no significant regional differences in percent centronucleation, and the data from all regions were combined. PDTC treatment produced a significant 32 % reduction in the percentage of centrally located nuclei from 27.6 ± 2.1 (SEM) % (vehicle-treated) to 18.7 ± 1.1% (; εε - p< 0.05; Mann- Whitney Rank Sum test).
Figure 9 Treatment with two distinct inhibitors of the NF-κB pathway produced similar effects on centronucleation in the mdx TS muscle. (A) Treatment of mature mdx mice with PDTC reduced percent centronucleation (εε - p<0.01). Both (more ...)
In the 2 month old mdx TS preparations, percent centronucleation was a function of region with the highest percentage observed in the caudal TS (; μμ - p< 0.01, μμμ - p<0.001, ANOVA, Holm-Sidak). In the middle TS region, UDCA produced a significant (; * - p< 0.05, Mann-Whitney Rank Sum test) 23% reduction in percent centronucleation from 31.8 ± 2.0 in the vehicle treated mice to 24.6 ± 1.5%. In contrast, UDCA produced a 19% increase in percent centronucleation (; ** - p< 0.01, Mann-Whitney Rank Sum test) in the caudal TS, increasing this measure from 33.0 ± 1.2 to 39.3 ± 1.5 %. Similarly, the 30 day PDTC treatment of 1 month old mdx mice produced significant (p<0.01; p<0.001, t tests) reductions in centronucleation in the middle (by 42%) and cephalad regions (by 34%), respectively, but failed to influence centronucleation in the caudal TS region.