Efficacy of Rectal Systemic Administration of Mesenchymal Stem Cells to Injury Sites via the CXCL12/CXCR4 Axis to Promote Regeneration in a Rabbit Skeletal Muscle Injury Model

Mesenchymal stem cells (MSCs) have been transplanted directly into lesions or injected intravenously. The administration of MSCs using these delivery methods requires specialized knowledge, techniques, and facilities. Here, we describe intrarectal systemic administration of MSCs, a simple, non-invasive route for homing to the injury sites to promote the regeneration of skeletal muscle injuries. Using a cardiotoxin (CTX)-induced rabbit skeletal muscle injury model, homing to the site of muscle injury was confirmed by intrarectal administration of MSCs; the time required for homing after intrarectal administration was approximately 5 days. In addition, the C-X-C chemokine ligand 12 (CXCL12)/C-X-C chemokine receptor-4 (CXCR4) axis was found to be involved in the homing process. Histopathological examinations showed that skeletal muscle regeneration was promoted in the MSCs-administered group compared to the CTX-only group. Myosin heavy polypeptide 3 (Myh3) expression, an indicator of early muscle regeneration, was detected earlier in the intrarectal MSCs group compared to the CTX-only group. These findings indicate that intrarectal administration of MSCs is effective in homing to the injured area, where they promote injury repair. Since intrarectal administration is a simple and non-invasive delivery route, these findings may be valuable in future research on stem cell therapy.


Introduction
Skeletal muscles are tissues in which regeneration is actively performed. Muscle satellite cells (skeletal muscle tissue stem cells) are known to play a central role in the regeneration of injured skeletal muscle tissues [1,2]. When the skeletal muscle is injured, satellite cells are activated and proliferate, either fusing with the injury site to repair damaged muscle cells or fusing to form new muscle cells [3]. Therefore, the activation of muscle satellite cells has been shown to be an important step in the regeneration of injured skeletal muscle tissues. However, age-related decreases in the number of muscle satellite cells have been reported [1], as well as decreases in self-replication functions [4], and proliferative capacity [5]. Moreover, satellite cell abnormalities are related to the pathology of Duchenne muscular dystrophy (DMD) in mdx mice, an animal model of DMD [6].
Following pathologic or mechanical damage, there is upregulation of cytokines, such as the insulin-like growth factor, which aids muscle repair [7]. C-X-C chemokine ligand

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To confirm the absorption and homing of Green Fluorescent Protein (GFP)-labeled MSCs to the injury sites after intrarectal administration by immunofluorescence (IF) staining and Western blotting (WB).

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To evaluate the effect of MSCs homing on muscle regeneration by histopathology and immunohistochemical (IHC) examination for myosin heavy polypeptide 3 (Myh3), a marker of early muscle differentiation. This study could provide novel insights into the mechanisms and regulation of MSCs migration and differentiation for skeletal muscle regeneration, and could have a significant impact on the field of stem cell therapy and regenerative medicine, especially for skeletal muscle diseases.

Experimental Animals
A total of 46 eight-week-old male Japanese white rabbits (Sankyo Labo Service, Tokyo, Japan) were used in this study. The rabbits were maintained at the Laboratory Animal Center of Kanazawa Medical University. Each experimental animal was housed in a separate cage under standard laboratory conditions (temperature: 24 • C, light/dark cycle: 12-h/12-h) and provided with food and water ad libitum. The experimental protocol for this study was approved by the Animal Research Committee of Kanazawa Medical University (#2022-15).

Cell Culture and GFP-Labeling of MSCs
Rabbit MSCs (DS Pharma Biomedical, Osaka, Japan and Cyagen, Silicon Valley, CA, USA) were maintained as a subconfluent monolayer culture in MSC growth medium (Cyagen, DS Pharma Biomedical) at 37 • C under 5% CO 2 . The medium was exchanged every 3 days. Passaging was routinely performed when the culture reached 70% confluence. The rabbit MSCs were transfected with pCAG-GFP (Addgene, Watertown, MA, USA), encoding GFP, using the Effectene Transfection Reagent (QIAGEN, Venlo, The Netherlands). The transfected GFP-labeled rabbit MSCs (GFP-MSCs) were harvested when the cultures reached 80% confluence [42].

Establishing the Model and MSCs Administration
A skeletal muscle injury model was created by injecting 10 µmol (1 mL) of CTX (Latoxan, Valence, France) into the right vastus lateralis femoris of the rabbit; CTX is widely used in skeletal muscle repair and regeneration experiments because it induces immediate inflammation and injury [43][44][45]. To confirm MSCs homing, a solution containing GFP-MSCs in an MSCs growth medium (1.0 × 10 7 cells, 1 mL) was administered intrarectally through the anus, using a 24-gauge catheter one day after CTX injection (after muscle injury and inflammation set in). In order to prevent the leakage of administered MSCs, the anus was sealed tightly with Steri-Strip tape for 1 h after the intrarectal administration.
First, we confirmed the absorption of intrarectally administered MSCs into the rectal tissue. The two experimental groups consisted of rabbits treated with CTX and administered GFP-MSCs (intrarectal administration group [IAG]; n = 3) and untreated rabbits (total control group, rabbits not treated with CTX and to which MSCs were not administered, [TC]; n = 3). IAG rabbits were sacrificed 10 min after GFP-MSC administration and rectal tissues were promptly harvested to confirm the absorption of GFP-MSCs. Rectal tissues from TC rabbits served as the control.
Next, to confirm homing to the site of muscle injury, IAG rabbits were sacrificed 1, 3, 5, and 7 days after GFP-MSCs administration and the right vastus lateralis was harvested (n = 5, each). The groups at these time points were CTX 2 days/MSCs 1 day, CTX 4 days/MSCs 3 days, CTX 6 days/MSCs 5 days, and CTX 8 days/MSCs 7 days. The left vastus lateralis muscle of the femur to which CTX was not administered was the control for MSCs homing (non-CTX group).
Additionally, the regeneration of skeletal muscle was compared with that in the MSCs administration group by administering only CTX to the same site and sacrificing the animals after 2, 4, 6, and 8 days to collect the right vastus lateralis muscle (n = 3, each). The CTX-only groups were CTX 2 days, CTX 4 days, CTX 6 days, and CTX 8 days. In addition, to evaluate the effect of MSCs administration on promoting skeletal muscle regeneration, rabbits were prepared, in which only CTX was injected into the right vastus lateralis (non-MSCs administration). These rabbits were sacrificed 2, 4, 6, and 8 days after CTX administration, and the right vastus lateralis was harvested. The groups were designated as CTX 2 days, CTX 4 days, CTX 6 days, and CTX 8 days, respectively (n = 3, each). The right vastus lateralis of untreated rabbits (using the TC group in the rectal study, n = 3) was used as a control group.
In all experiments, rabbits were sacrificed by rapid injection of sodium thiopental (Nipro Pharma, Osaka, Japan) via an auricular vein. After collection, the samples were immediately snap-frozen in liquid nitrogen for WB analysis and cryopreserved in OCT (Sakura Finetek Japan Co., Tokyo, Japan) at −80 • C for IF staining. Additionally, a part of each collected sample was immersed in formalin for histopathological and IHC examination.

Western Blotting
For the quantification of GFP expression, immunoblotting was performed on rabbit skeletal muscles and rectum tissues. Protein was extracted using a protein extraction solution (PRO-PREP, iNtRON Biotechnology, Seongnam, Republic of Korea). Equal amounts of protein (20 µg) were electrophoresed on a 10% polyacrylamide gel and transferred to a nitrocellulose membrane (Wako, Tokyo, Japan). The membranes were incubated with anti-GFP (1.0 µg/mL; Cell Signaling, Danvers, MA, USA) overnight at 4 • C. After incubation with peroxidase-labeled, goat anti-mouse or anti-rabbit secondary antibodies (0.7 µg/mL; Dako Cytomation) for 1 h at room temperature and vigorous washing; the nitrocellulose membrane was incubated with Chemiluminescence Luminol Reagent (Immuno Star LD, Fujifilm Wako, Japan) and photographed digitally using Image Quant LAS 4000 mini (GE healthcare Japan Co., Tokyo, Japan). Immunoblotting using the anti-actin monoclonal antibody (Sigma Chemical Co., St. Louis, MO, USA) was used for standardization. The intensity was measured using the Multi Gauge v3.1 (Fujifilm, Tokyo, Japan). Experiments were repeated at least three times.

Cell Culture and Transfection with Stealth siRNA against CXCR4
Rabbit MSCs were cultured as before. At about 60% confluence, MSCs were transfected with the stealth siRNA duplex oligoribonucleotides (Invitrogen Life Technologies, Carlsbad, CA, USA). The sequence of the stealth siRNA duplex oligoribonucleotides against CXCR4 is 5 -GCCCUCAAGACUACGGUCAUCCUUA -3 , and its corresponding complementary strand is 5 -UAAGGAUGACCGUAGUCUUGAGGGC -3 (Invitrogen Life Technologies). A negative stealth siRNA sequence was used as a control. The CXCR4 siRNA was transfected transiently using LipofectamineTM RNAiMAX (Invitrogen Life Technologies) according to the manufacturer's instructions. Briefly, siRNA plasmid (150 pmol) was diluted in 245 µL of Opti-MEMI (Gibco, Waltham, MA, USA) and the solution was gently mixed. Lipofectamine™ RNAiMAX (Invitrogen Life Technologies) was gently mixed and 4 µL of the reagent was then diluted in 246 µL of Opti-MEMI. Then, the diluted siRNA and the diluted LipofectamineTM RNAiMAX were combined for 20 min at room temperature to allow the formation of transfection complexes. The final volume of these solutions was 0.5 mL, and the final concentration of RNA was 40 nM. The complexes were then added to each well containing cells in six-well plates while they were in the quiescent state, and all were swirled gently to ensure uniform distribution. After incubation at 37 • C for 48 h, IF staining and WB were performed.

Confirmation of Homing Suppression by CXCR4 Knockdown in MSCs Expressing GFP (siCXCR4/GFP-MSCs)
To confirm the involvement of the CXCL12/CXCR4 axis, 1.0 × 10 7 siCXCR4/GFP-MSCs were administered intrarectally through the anus 1 day after CTX administration. Since this protocol showed a significant increase in homing at the muscle injury site 5 days after MSCs administration, we evaluated the area of muscle injury 5 days after siCXCR4/GFP-MSCs administration (n = 4) and compared the results with the newly created GFP-MSCs 5-day group (n = 4).

Histopathology and Immunohistochemistry
For histological evaluations, the rabbit femoral skeletal muscle was removed and fixed in 10% buffered formalin for 24 h. Next, the sample was decalcified by immersion in 5% formic acid for 48 h, after which, paraffin-embedded specimens were prepared. The prepared paraffin block was sliced at a thickness of 3 µm and examined using hematoxylin and eosin (H & E) staining.
To investigate the skeletal muscle regenerative effects of MSCs homing in vivo in a CTX-induced rabbit skeletal muscle injury model, immunohistochemical studies were performed using Myh3, which is expressed during the early stages of skeletal muscle regeneration [46,47].
For all groups, skeletal muscle sections were prepared. Sections obtained from the femoral proximal medial diaphysis were deparaffinized with xylene and ethanol. For antigen retrieval, the sections were autoclaved at 121 • C for 15 min. Endogenous peroxidase was eliminated using 0.3% H 2 O 2 and blocking was performed using mouse or goat normal serum. Finally, the sections were incubated with the anti-Myh3 mouse monoclonal antibody (5.0 µg/mL; Santa Cruz Biotechnologies, Dallas, TX, USA) at 4 • C overnight in a cool dark room. Next, the sections were incubated with a secondary antibody (biotin) that was reacted with an enzymatic agent (streptavidin). After a 5 min immersion in 3,3 Diaminobenzidine (DAB) solution to allow for color development, the nuclei were stained and studied under a light microscope.

Statistical Analysis
Data are expressed as the mean ± standard error (SE). One-way analyses of variance followed by Bonferroni's post-hoc tests were used to compare GFP expression after administration of GFP-MSCs. The Mann-Whitney U test was used to compare GFP expression between the siCXCR4/GFP-MSCs group and the GFP-MSCs group. Significance was defined at p < 0.05. Data analysis was performed using IBM SPSS Statistics 28.

CXCR4 Expression in Cultured MSCs
The clinical efficacy of MSCs depends on their ability to home to lesions. The chemokine/receptor axis is thought to play an important role in homing. Therefore, we first confirmed the expression of CXCR4, a chemokine receptor involved in homing, was maintained in cultured MSCs ( Figure 1A). Next, to confirm the homing of intrarectally administered MSCs to the site of injury, we used cultured MSCs that were engineered to express GFP ( Figure 1B). okine/receptor axis is thought to play an important role in homing. Therefore, we first confirmed the expression of CXCR4, a chemokine receptor involved in homing, was maintained in cultured MSCs ( Figure 1A). Next, to confirm the homing of intrarectally administered MSCs to the site of injury, we used cultured MSCs that were engineered to express GFP ( Figure 1B).

Migration of MSCs in Rectal Tissues after Intrarectal Administration
To investigate the absorption of intrarectally administered MSCs into tissues in vivo, GFP fluorescence in rectal tissues was examined after administration. To study homing, we used a skeletal muscle injury model in which CTX was injected into the right vastus lateralis femoris of Japanese white rabbits. To ensure that MSCs home to the injury site,

Migration of MSCs in Rectal Tissues after Intrarectal Administration
To investigate the absorption of intrarectally administered MSCs into tissues in vivo, GFP fluorescence in rectal tissues was examined after administration. To study homing, we used a skeletal muscle injury model in which CTX was injected into the right vastus lateralis femoris of Japanese white rabbits. To ensure that MSCs home to the injury site, they were administered intrarectally one day after CTX administration, allowing time for the induction of inflammation and injury.
No GFP-positive cells were observed in the rectal tissues in the TC (total control) group ( Figure 1C). Conversely, in the group that received intrarectal administration of GFP-MSCs (IAG group), GFP-positive cells were observed in the subepithelial mucosal and submucosal tissue, as well as the surface of the rectal epithelium 10 min after GFP-MSCs administration, as revealed by IF staining and WB ( Figure 1D,E), confirming that intrarectally administered MSCs migrated into rectal tissues promptly after administration.

Homing of MSCs to Injury Sites after Intrarectal Administration
Homing of MSCs after intrarectal administration was examined by analyzing GFPpositive cells and GFP expression (using IF and WB) at the site of skeletal muscle injury. GFP-positive cells were absent in the healthy skeletal muscle tissues of the non-CTXtreated side of the rabbits and the CTX-alone group (CTX 6 days) (Figure 2A,B). In the CTX 2 days/MSCs 1 day group and CTX 4 days/MSCs 3 days group, GFP-positive cells were absent at the injury site in all groups. However, in the CTX 6 days/MSCs 5 days group and CTX 8 days/MSCs 7 days group, GFP-positive cells were observed and converged into clusters at the injury site in all groups ( Figure 2C,D). Moreover, WB showed significantly stronger GFP expression in the CTX 6 days/MSCs 5 days and CTX 8 days/MSCs 7 days groups compared with the non-CTX-treated side (p < 0.0001 and p = 0.0002, respectively) ( Figure 2E,F). These results confirmed that intrarectally administered MSCs migrate to the site of injury within approximately five days. To our knowledge, this is the first evidence of the homing of intrarectally administered MSCs to lesions. These findings could be useful for future research and clinical applications involving intrarectal administration of cellular therapeutics. positive cells and GFP expression (using IF and WB) at the site of skeletal muscle injury. GFP-positive cells were absent in the healthy skeletal muscle tissues of the non-CTXtreated side of the rabbits and the CTX-alone group (CTX 6 days) (Figure 2A,B). In the CTX 2 days/MSCs 1 day group and CTX 4 days/MSCs 3 days group, GFP-positive cells were absent at the injury site in all groups. However, in the CTX 6 days/MSCs 5 days group and CTX 8 days/MSCs 7 days group, GFP-positive cells were observed and converged into clusters at the injury site in all groups ( Figure 2C,D). Moreover, WB showed significantly stronger GFP expression in the CTX 6 days/MSCs 5 days and CTX 8 days/MSCs 7 days groups compared with the non-CTX-treated side (p < 0.0001 and p = 0.0002, respectively) ( Figure 2E,F). These results confirmed that intrarectally administered MSCs migrate to the site of injury within approximately five days. To our knowledge, this is the first evidence of the homing of intrarectally administered MSCs to lesions. These findings could be useful for future research and clinical applications involving intrarectal administration of cellular therapeutics.

CXCL12 and CXCR4 Expression in the Injury Area
Next, using IF staining, we evaluated the expression and localization of factors associated with MSC homing-CXCL12 and CXCR4-at the site of muscle injury. On the TC (total control) group, CXCL12 and CXCR4 were observed only sporadically. However, CXCL12 expression was observed in the muscle injury site in the CTX 6 days group. In the CTX 6 days/MSCs 5 days group, where MSCs homing was confirmed; staining for GFP and CXCL12 expression showed that GFP-MSCs were concentrated around CXCL12positive areas. Similar to CXCL12, CXCR4 was expressed in the CTX 6 days/MSCs 5 days group. Since both MSCs and endogenous migratory cells express the homing factor CXCR4, CXCL12 could have induced the migration of endogenous CXCR4-positive cells other than the intrarectally administered MSCs to the site of muscle injury. Therefore, when we examined MSCs by staining GFP and CXCR4, some CXCR4-expressing cells merged with GFP-MSCs, confirming that CXCR4 was strongly expressed on the surface of MSCs (Figure 3).

Suppression of Homing by Knocking down CXCR4 in MSCs
We confirmed the involvement of the CXCL12/CXCR4 axis in homing by using siCXCR4/GFP-MSCs generated by knocking down CXCR4 using siRNA ( Figure 4A-C).
These siCXCR4/GFP-MSCs were administered intrarectally to investigate homing to the site of skeletal muscle injury. Homing of siCXCR4/GFP-MSCs to the site of muscle injury was examined 5 days after MSC administration, since our earlier results showed that GFP-positive cells significantly increased at injury sites by day 5. The appearance of GFP-positive cells at the injury site was significantly lower (p = 0.0209) for intrarectally administered siCXCR4/GFP-MSCs than for intrarectally administered GFP-MSCs ( Figure 4D-F), suggesting that the CXCL12/CXCR4 axis is involved in homing.

Suppression of Homing by Knocking down CXCR4 in MSCs
We confirmed the involvement of the CXCL12/CXCR4 axis in homing by using siCXCR4/GFP-MSCs generated by knocking down CXCR4 using siRNA ( Figure 4A-C). These siCXCR4/GFP-MSCs were administered intrarectally to investigate homing to the site of skeletal muscle injury. Homing of siCXCR4/GFP-MSCs to the site of muscle injury was examined 5 days after MSC administration, since our earlier results showed that GFPpositive cells significantly increased at injury sites by day 5. The appearance of GFP-positive cells at the injury site was significantly lower (p = 0.0209) for intrarectally administered siCXCR4/GFP-MSCs than for intrarectally administered GFP-MSCs ( Figure 4D-F), suggesting that the CXCL12/CXCR4 axis is involved in homing. The expression of GFP was normalized to that of β-actin. The expression at the muscle injury site after knockdown of CXCR4 is significantly decreased. Each dot represents the data from an individual rabbit (n = 4 per group). Data represent the mean ± SE. p values were determined using the Mann-Whitney U test.

Staining of Nucleated Fibers
H&E-stained specimens were examined to confirm the efficacy of rectally administered MSCs on the injury site. From 2 to 4 days after CTX injection, several inflammatory (G) The expression of GFP was normalized to that of β-actin. The expression at the muscle injury site after knockdown of CXCR4 is significantly decreased. Each dot represents the data from an individual rabbit (n = 4 per group). Data represent the mean ± SE. p values were determined using the Mann-Whitney U test.

Staining of Nucleated Fibers
H&E-stained specimens were examined to confirm the efficacy of rectally administered MSCs on the injury site. From 2 to 4 days after CTX injection, several inflammatory cells infiltrated the injured muscle in both groups. For the CTX-only administration groups, centrally nucleated fibers started to appear on day 6 (CTX 6 days). In contrast, in the MSCstreated group, fibers with central nuclei began to appear on day 4 (CTX 4 days/MSCs 3 days) and increased on days 6 and 8 (CTX 6 days/MSCs 5 days, CTX 8 days/MSCs 7 days) ( Figure 5). cells infiltrated the injured muscle in both groups. For the CTX-only administration groups, centrally nucleated fibers started to appear on day 6 (CTX 6 days). In contrast, in the MSCs-treated group, fibers with central nuclei began to appear on day 4 (CTX 4 days/MSCs 3 days) and increased on days 6 and 8 (CTX 6 days/MSCs 5 days, CTX 8 days/MSCs 7 days) ( Figure 5).

Figure 5.
Hematoxylin and eosin staining in the skeletal muscle. TC is the total control group (rabbits not treated with CTX and to which MSCs were not administered). After CTX administration, skeletal muscle fiber necrosis and mononuclear cell infiltration peaked in the CTX 4 days group.
Repair started in the CTX 6 days group, central nucleation occurred (arrows on representative parts), and interstitial fibrosis and mild regenerative changes in skeletal muscle fibers were observed (n = 3 per group). Meanwhile, in the MSCs administration group, central nucleation appeared in the CTX 4 days/MSCs 3 days group (arrows on representative parts), where the extent of skeletal muscle fiber necrosis was decreased and regeneration was promoted. In the CTX 6 days/MSCs 5 days and CTX 8 days/MSCs 7 days groups, there was a presence of increased central nucleation (arrows on representative parts) at the margin (n = 5 per group). Scale bar, 100 µm (×20).
Next, we investigated Myh3, an indicator of early muscle regeneration [46,47], using IHC examination. No Myh3 expression was observed in the TC (total control), CTX 2 days, and CTX 2 days/MSCs 1 day groups. For the CTX-only administration groups, Myh3-positive cells began to appear in the CTX 6 days group. Meanwhile, for the MSCs administration groups, Myh3 expression was observed in the CTX 4 days/MSCs 3 days group. Additionally, compared to the CTX-only administration groups, the MSCs administration groups had more Myh3-expressing cells in the CTX 6 days/MSCs 5 days group and CTX 8 days/MSCs 7 days group ( Figure 6). Histopathological examination and Myh3 expression analysis showed that intrarectal administration of MSCs promoted tissue repair and regeneration. It has also been reported that MSCs administered remotely have beneficial effects similar to endocrine actions [48,49]. In this study, the tissue repair tendencies and scattered regenerated skeletal muscles that began to appear in the CTX 4 days/MSCs 3 Figure 5. Hematoxylin and eosin staining in the skeletal muscle. TC is the total control group (rabbits not treated with CTX and to which MSCs were not administered). After CTX administration, skeletal muscle fiber necrosis and mononuclear cell infiltration peaked in the CTX 4 days group. Repair started in the CTX 6 days group, central nucleation occurred (arrows on representative parts), and interstitial fibrosis and mild regenerative changes in skeletal muscle fibers were observed (n = 3 per group). Meanwhile, in the MSCs administration group, central nucleation appeared in the CTX 4 days/MSCs 3 days group (arrows on representative parts), where the extent of skeletal muscle fiber necrosis was decreased and regeneration was promoted. In the CTX 6 days/MSCs 5 days and CTX 8 days/MSCs 7 days groups, there was a presence of increased central nucleation (arrows on representative parts) at the margin (n = 5 per group). Scale bar, 100 µm (×20).
Next, we investigated Myh3, an indicator of early muscle regeneration [46,47], using IHC examination. No Myh3 expression was observed in the TC (total control), CTX 2 days, and CTX 2 days/MSCs 1 day groups. For the CTX-only administration groups, Myh3-positive cells began to appear in the CTX 6 days group. Meanwhile, for the MSCs administration groups, Myh3 expression was observed in the CTX 4 days/MSCs 3 days group. Additionally, compared to the CTX-only administration groups, the MSCs administration groups had more Myh3-expressing cells in the CTX 6 days/MSCs 5 days group and CTX 8 days/MSCs 7 days group ( Figure 6). Histopathological examination and Myh3 expression analysis showed that intrarectal administration of MSCs promoted tissue repair and regeneration. It has also been reported that MSCs administered remotely have beneficial effects similar to endocrine actions [48,49]. In this study, the tissue repair tendencies and scattered regenerated skeletal muscles that began to appear in the CTX 4 days/MSCs 3 days group before homing was thought to be involved in endocrine action by the systemic administration of MSCs. days group before homing was thought to be involved in endocrine action by the systemic administration of MSCs. Figure 6. Immunohistochemical staining of Myh3 expression. Myh3 expression was not observed in the TC (total control, rabbits not treated with CTX and to which MSCs were not administered) group, while it was observed in the CTX 6 days group, increasing in the CTX 8 days group (arrows on representative parts) (n = 3 per group). In the MSCs administration group, Myh3 expression was observed in the CTX 4 days/MSCs 3 days group, with expression increasing gradually in the CTX 6 days/MSCs 5 days and CTX 8 days/MSCs 7 days groups, with the cell diameter also increasing (arrows on representative parts) at the margin (n = 5 per group). Scale bar, 100 µm (×20).

Conclusions
In this study, we investigated the feasibility of intrarectal administration of MSCs to find an administration route that is non-invasive and easier to administer than local implantation or intravenous administration by injection or surgery. The findings of this study contribute to future research on the clinical application of regenerative medicine.
In this study, we focused on the CXCL12/CXCR4 axis as a factor involved in homing. CXCL12 expression increases at inflammation sites and in necrotic tissue. Recently, CXCL12 expression was shown to be elevated in patients with DMD and in mdx mice, which is known as a DMD model [50][51][52]. Elevation of CXCL12 expression in tissues suggests that the systemic administration of MSCs expressing CXCR4, which is a receptor for CXCL12, could be effective. Herein, we focused on the homing of MSCs from intrarectal administration to the lesion site; thus, we used a drug-induced local skeletal muscle injury model. In the future, it will be necessary for clinical applications to examine whether similar results can be obtained in the mdx mice, which is a severe and difficult-to-treat DMD disease model.
Although the homing of intrarectally administered MSCs was confirmed in this study, the homing rate was not examined. Recently, researchers have focused on the issue of low homing and decreased survival rates of MSCs post-transplantation [53,54]. Thus, they are attempting to promote MSCs homing and engraftment by enhancing expression Figure 6. Immunohistochemical staining of Myh3 expression. Myh3 expression was not observed in the TC (total control, rabbits not treated with CTX and to which MSCs were not administered) group, while it was observed in the CTX 6 days group, increasing in the CTX 8 days group (arrows on representative parts) (n = 3 per group). In the MSCs administration group, Myh3 expression was observed in the CTX 4 days/MSCs 3 days group, with expression increasing gradually in the CTX 6 days/MSCs 5 days and CTX 8 days/MSCs 7 days groups, with the cell diameter also increasing (arrows on representative parts) at the margin (n = 5 per group). Scale bar, 100 µm (×20).

Conclusions
In this study, we investigated the feasibility of intrarectal administration of MSCs to find an administration route that is non-invasive and easier to administer than local implantation or intravenous administration by injection or surgery. The findings of this study contribute to future research on the clinical application of regenerative medicine.
In this study, we focused on the CXCL12/CXCR4 axis as a factor involved in homing. CXCL12 expression increases at inflammation sites and in necrotic tissue. Recently, CXCL12 expression was shown to be elevated in patients with DMD and in mdx mice, which is known as a DMD model [50][51][52]. Elevation of CXCL12 expression in tissues suggests that the systemic administration of MSCs expressing CXCR4, which is a receptor for CXCL12, could be effective. Herein, we focused on the homing of MSCs from intrarectal administration to the lesion site; thus, we used a drug-induced local skeletal muscle injury model. In the future, it will be necessary for clinical applications to examine whether similar results can be obtained in the mdx mice, which is a severe and difficult-to-treat DMD disease model.
Although the homing of intrarectally administered MSCs was confirmed in this study, the homing rate was not examined. Recently, researchers have focused on the issue of low homing and decreased survival rates of MSCs post-transplantation [53,54]. Thus, they are attempting to promote MSCs homing and engraftment by enhancing expression of CXCR4 in MSCs [37,38,55], pretreating MSCs with inflammatory cytokines [16], and using MSCs spheroids [54]. Future studies are needed to improve the homing efficiency of intrarectally administered MSCs.
This study confirmed that there was homing of MSCs to the lesion from intrarectal administration, which is a non-invasive and simple administration route. Intrarectal administration of MSCs may provide a route of administration for MSCs at home, which could be very useful for future stem cell research.