Effect of Calcitriol and Vitamin D Receptor Modulator 2 on Recovery of Injured Skeletal Muscle in Wistar Rats

Abstract

Muscle injuries often result in functional limitations due to insufficient healing. This study assessed the influence of calcitriol and vitamin D Receptor Modulator 2 (VDRM2) on muscle regeneration in male Wistar rats following open blunt muscle injury. The injured left soleus muscle of the rats was treated for the first four days after trauma with local injections of either calcitriol, VDRM2, or a 10% ethanol solution (control). Although muscle strength significantly decreased post-injury, all groups showed gradual improvement but did not achieve full recovery. By the 14th day, calcitriol-treated rats significantly outperformed the control group in the incomplete tetanic force, with VDRM2-treated rats showing muscle strength values that fell between the control and calcitriol groups. Similar trends were observed in complete tetanic contractions and were confirmed histologically via muscle cell width quantification. Additionally, histological analysis showed increased cellular turnover on the fourth postoperative day in the calcitriol group, as indicated by elevated cell proliferation rates and fewer apoptotic cells. VDRM2-treated animals showed only an increased proliferative activity on day 4 after injury. No noticeable differences between the groups for CAE-positive cells or visible muscle tissue area were found. In conclusion, predominantly calcitriol positively influenced post-trauma muscle recovery, where VDRM2 had substantially lower biological activity.

Keywords: calcitriol; cellular turnover; healing; muscle injury; vitamin D receptor modulator 2 (VDRM2).

Figure 1

(A): Experimental design. Calcitriol, VDRM2, or 10% ethanol solution was injected i.p. on the day of trauma induction (T; green square) and on each of the following three days (red arrows). The final analysis (A; brown square) took place on the 1st, 4th, and 14th postoperative days. 48 h before the final analysis, the i.p. injection of 50 mg/kg bw BrdU (B; blue square) was applied. (B): Graphical representation of the soleus muscle (arrow) with the supplying vessels and nerve (circle) (a). Trauma induction of the left soleus muscle is performed using the instrumented clamp (b). Shown in (b) is the first of the seven contusions. The macroscopic result directly after traumatization of the muscle by seven successive contusions (c). The muscle area with the central nerve vessel bundle (arrow) is left out of the trauma (c).

Figure 2

Method for measurement of the muscle fiber width in HE-stained muscle sections. The width of each visible muscle fiber was measured twice at perpendicular points (white arrows with straight ends), recorded in μm, and averaged. Tissue sample: injured soleus muscle; magnification: ×200.

Figure 3

Quantitative analysis of the incomplete tetanic force (A) and the complete tetanic force (B) of the injured left soleus muscle, expressed relative to the force of the contralateral non-injured soleus muscle (in %). The animals underwent after-muscle injury treatment with VDRM2, calcitriol, or equivalent volumes of 10% ethanol (control). The data are presented as box plots (median, 1st, and 3rd quartile) with whiskers (minimum and maximum values). Statistical analysis: one-way ANOVA; * p < 0.05 vs. control.

Figure 4

Quantitative analysis of the BrdU-positive cells (left panel) and exemplary images of BrdU staining at day 4 (right panel) of the injured left soleus muscle. Positive cell nuclei are stained brown and are pointed out by red arrows. Deep blue cell nuclei did not incorporate BrdU; muscle cells are illustrated in a light blue-brownish color. The animals underwent after-muscle injury treatment with VDRM2, calcitriol, or equivalent volumes of 10% ethanol (control). The data are presented as box plots (median, 1st, and 3rd quartile) with whiskers (minimum and maximum values). Statistical analysis: one-way ANOVA; * p < 0.05 vs. control.

Figure 5

Quantitative analysis of the TUNEL-positive cells (left panel) and exemplary images of TUNEL staining at day 4 (right panel) of the injured left soleus muscle. Positive cell nuclei are stained deep red and are pointed out by red arrows. Blue cell nuclei did not incorporate BrdU. The animals underwent after-muscle injury treatment with VDRM2, calcitriol, or equivalent volumes of 10% ethanol (control). The data are presented as box plots (median, 1st, and 3rd quartile) with whiskers (minimum and maximum values). Statistical analysis: one-way ANOVA; * p < 0.05 vs. control.

Figure 6

Quantitative analysis of the CAE-positive cells (A), myofiber coverage (B), and myofiber width (C) of the injured left soleus muscle. The animals underwent after-muscle injury treatment with VDRM2, calcitriol, or equivalent volumes of 10% ethanol (control). The data are presented as box plots (median, 1st, and 3rd quartile) with whiskers (minimum and maximum values). Statistical analysis: one-way ANOVA; * p < 0.05 vs. control, ^# p < 0.05 vs. VDRM2.