Chronic intrinsic transient tracheal occlusion elicits diaphragmatic muscle fiber remodeling in conscious rodents

Abstract

Background: Although the prevalence of inspiratory muscle strength training has increased in clinical medicine, its effect on diaphragm fiber remodeling is not well-understood and no relevant animal respiratory muscle strength training-rehabilitation experimental models exist. We tested the postulate that intrinsic transient tracheal occlusion (ITTO) conditioning in conscious animals would provide a novel experimental model of respiratory muscle strength training, and used significant increases in diaphragmatic fiber cross-sectional area (CSA) as the primary outcome measure. We hypothesized that ITTO would increase costal diaphragm fiber CSA and further hypothesized a greater duration and magnitude of occlusions would amplify remodeling.

Methodology/principal findings: Sprague-Dawley rats underwent surgical placement of a tracheal cuff and were randomly assigned to receive daily either 10-minute sessions of ITTO, extended-duration, 20-minute ITTO (ITTO-20), partial obstruction with 50% of cuff inflation pressure (ITTO-PAR) or observation (SHAM) over two weeks. After the interventions, fiber morphology, myosin heavy chain composition and CSA were examined in the crural and ventral, medial, and dorsal costal regions. In the medial costal diaphragm, with ITTO, type IIx/b fibers were 26% larger in the medial costal diaphragm (p<0.01) and 24% larger in the crural diaphragm (p<0.05). No significant changes in fiber composition or morphology were detected. ITTO-20 sessions also yielded significant increases in medial costal fiber cross-sectional area, but the effects were not greater than those elicited by 10-minute sessions. On the other hand, ITTO-PAR resulted in partial airway obstruction and did not generate fiber hypertrophy.

Conclusions/significance: The results suggest that the magnitude of the load was more influential in altering fiber cross-sectional area than extended-duration conditioning sessions. The results also indicated that ITTO was associated with type II fiber hypertrophy in the medial costal region of the diaphragm and may be an advantageous experimental model of clinical respiratory muscle strength training.

Figure 1. Illustration of ITTO experimental setup.

(A) ITTO was administered in a plethysmograph to permit limited animal movement. The tracheal cuff was inflated by injection of sterile saline into the actuating line. The timing and magnitude of cuff pressure was monitored with a polygraph. (B) Cut-out illustration of the tracheal cuff. The trachea was fully patent with the cuff deflated. With cuff inflated, the trachea was fully and reversibly occluded.

Figure 2. Regional remodeling of the diaphragm, following ITTO or SHAM training.

(A) CSA. Type IIx/b CSA in the medial costal (#) and crural (*) regions was significantly greater in ITTO animals than SHAM animals. In the ITTO group, type IIx/b medial costal diaphragm fibers were significantly larger than dorsal, ventral and crural segments (†). In both groups, type IIx/b fibers were significantly larger than oxidative fibers (‡). (B) Fiber proportions. In the SHAM group, the ventral region contained significantly fewer type IIx/b fibers than the dorsal region (*). In all groups, type IIx/b fibers were significantly less prevalent than type IIa or I fibers (‡). (C) MHC A_A. The type I fiber A_A of the medial region was significantly larger in ITTO animals than SHAM animals (*). The A_A of type IIx/b fibers of the SHAM ventral diaphragm was significantly lower than the SHAM medial diaphragm (^#) and the ITTO ventral diaphragm (*). (Error bars are ± 1SD, *p<0.05; ^# p<0.01; ‡ p<0.001; † p<0.05 vs other regions).

Figure 3. Immunofluorescent analysis of myosin heavy chain in the medial costal diaphragm.

(A) Diaphragm from ITTO animal. (B) Diaphragm from SHAM animal. Type I fibers fluoresced blue, type IIa illuminated green, and type IIx/b fibers remained free of fluorescence. Images were captured with 100X magnification. Scale bar represents 100 µm.

Figure 4. Remodeling of the medial costal diaphragm after 10- or 20-minute ITTO conditioning sessions.

(A) CSA in type IIx/b fibers was significantly greater for ITTO (# p<0.01) and ITTO-20 (* p<0.05) animals, compared to SHAM animals. In all conditions, type IIx/b fibers were significantly larger than type I or IIa fibers (‡ p<0.001). (B) Type IIx/b fibers were significantly less prevalent than type I or type IIa (‡ p<0.001). The duration of ITTO sessions did not influence fiber type proportions. (C) Type IIx/b fibers occupied the largest A_A (‡ p<0.001), but there were no significant group variations in MHC A_A.

Figure 5. Medial costal diaphragm remodeling after complete or partial ITTO.

(A) CSA was significantly larger in type IIx/b fibers for ITTO, compared to ITTO-PAR and SHAM animals (‡ p<0.001). In addition, SHAM animals had significantly larger type IIx/b fibers than ITTO-PAR animals (* p<0.05). Type IIa fibers were significantly larger in ITTO animals, compared to ITTO-PAR (# p<0.01). (B) Although there were significantly fewer type IIx/b fibers in all groups (‡ p<0.001), training did not affect fiber type proportions. (C) Type IIx/b fibers occupied the largest A_A, regardless of group (‡ p<0.001).