One-Month-Old Rabbits Exhibit a Longer Postoperative Remodeling in Extraocular Muscles Compared to 3-Month-Old Rabbits
Grouping of Animals and EOM Treatment The experimental groups consisted of 32 New Zealand White rabbits, 1 month old and 3 months, with an additional four rabbits at each age serving as controls. The experimental protocol was approved by the Ethics Committee of Wenzhou Medical University (Approval No. wydw2021-0309), and all experiments adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. For the experimental group, half of the 1-month-old and 3-month-old rabbits were randomly selected to undergo either superior rectus (SR) muscle recession or resection surgery on the right eye. The rabbits were preoperatively anesthetized with an injection of xylazine (10 mg/kg) into the thigh muscles. In the recession procedure, the right SR was detached from its insertion and reattached 6 mm posterior to the original insertion site using an 8-0 suture (polyglactin 910; Ethicon, Raritan, NJ, USA). In the resection procedure, a 4-mm segment of the right SR near its insertion was excised, and the remaining muscle was sutured to the original insertion with an 8-0 suture. Postoperatively, 0.5% levofloxacin eye drops (Santen Pharmaceutical, Osaka, Japan) and 0.1% fluorometholone eye drops (Santen Pharmaceutical) were administered three times daily for 7 days. The rabbits were maintained under standard care until tissue collection. At 1 week and 4 weeks post-surgery, four rabbits from each age group and surgical group were euthanized using xylazine (20 mg/kg). Following carotid artery exsanguination, the full length of the right SR was harvested under a microscope, rinsed with normal saline, and fixed for 24 hours. The right SR was then extracted from the fixative solution and prepared in 2-µm paraffin sections. We prepared tissue sections from the proximal end and recorded the number and the thickness of the sections to determine their location. Because MyHC expression levels vary along the length of the EOM, we selected five consecutive sections located approximately 5 mm from the proximal end for analysis, with one designated for hematoxylin and eosin (H&E) staining and the remaining four for immunohistochemical staining. Hematoxylin and Eosin Staining The sections were sequentially immersed in xylene solution (10023418; Sinopharm, Shanghai, China) and graded ethanol (10009218; Sinopharm) at 100%, 85%, and 75% for deparaffinization and hydration, followed by staining with hematoxylin (HK1024; HaoKebio, Hangzhou, China) and eosin (HK2051; HaoKebio). They were then dehydrated in absolute ethanol and xylene before being mounted for the observation of centrally nucleated myofibers, which appear during the repair of EOMs. Immunohistochemical Staining MyHC is integral to the function of EOMs. This study aimed to investigate the representative embryonic and neonatal MyHC, as well as fast 2X-MyHC. Muscle SCs are responsible for the repair of EOMs and specifically express MyoD when activated. Therefore, MyoD was used in this study to label activated SCs. As mentioned above, paraffin sections were subjected to washes with xylene solution and graded ethanol, followed by heating in citric acid antigen retrieval solution (HKI0001, pH 6.0; HaoKebio) for antigen retrieval. After cooling naturally, the sections were rinsed three times with PBS solution (HK2022; HaoKebio), each rinse lasting 5 minutes. Subsequently, a 3% hydrogen peroxide solution (73113760; Sinopharm) was employed to block endogenous peroxidase for 25 minutes, followed by another PBS rinse (three times, 5 minutes each). Antigen blocking was performed using 3% BSA (HK5021; HaoKebio) at room temperature for 30 minutes. Following removal of the BSA solution, diluted primary antibodies, including mouse anti-fast 2X-MyHC (67299-1-Ig, 1:3000; Proteintech, Wuhan, China), mouse anti-embryonic MyHC (ab264038, 1:300; Abcam, Cambridge, UK), rabbit anti-neonatal MyHC (NBP2-41309, 1:200; Bio-Techne, Minneapolis, MN, USA), and rabbit anti-MyoD (18943-1-AP, 1:200; Proteintech), were added, followed by incubation overnight at 4°C in a wet box. After rinsing with PBS three times for 5 minutes each, the secondary antibody solution (I20012C; BioLynx, Hangzhou, China) was added and incubated at room temperature for 50 minutes. Following another PBS wash, 3,3 -diaminobenzidine (DAB) substrate solution (PR30010; Proteintech) was applied for color development, and hematoxylin was used to counterstain the nuclei. Finally, the sections were sequentially dehydrated using graded ethanol and xylene solutions before being mounted for observation. Data Analysis For each section, at least three random fields were selected to observe MyHC expression, activated SCs, the number of centrally nucleated myofibers, and myofiber mean cross-sectional area. Muscle fibers expressing each MyHC isoform were counted, and the proportion of myofibers expressing each isoform relative to the total number of muscle fibers was calculated. For activated SCs, the number of MyoD + nuclei attached to the sarcolemma was counted, and the ratio of MyoD + nuclei to muscle fibers in each field was used to determine the number of activated SCs. The ratio of centrally nucleated myofibers to the total number of muscle fibers was calculated. The cross-sectional area of myofibers was measured using ImageJ (National Institutes of Health, Bethesda, MD, USA) from H&E-stained images. Data for the orbital layers (OLs) and global layers (GLs) were analyzed separately except for the count of centrally nucleated myofibers, which was recorded as a combined value. Statistical analysis was conducted using SPSS Statistics 26.0 (IBM Corporation, Chicago, IL, USA), and the Shapiro–Wilk test was applied to assess normality. Normally distributed data are presented as mean ± SD, and abnormally distributed data are expressed as median (interquartile range). Independent t-tests or ANOVA tests were applied to normally distributed data with homogeneity of variance, and the Mann–Whitney U test or Kruskal–Wallis test was used for abnormally distributed data or data with unequal variance. Bonferroni corrections were applied for post hoc comparisons. 

The experimental groups consisted of 32 New Zealand White rabbits, 1 month old and 3 months, with an additional four rabbits at each age serving as controls. The experimental protocol was approved by the Ethics Committee of Wenzhou Medical University (Approval No. wydw2021-0309), and all experiments adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. For the experimental group, half of the 1-month-old and 3-month-old rabbits were randomly selected to undergo either superior rectus (SR) muscle recession or resection surgery on the right eye. The rabbits were preoperatively anesthetized with an injection of xylazine (10 mg/kg) into the thigh muscles. In the recession procedure, the right SR was detached from its insertion and reattached 6 mm posterior to the original insertion site using an 8-0 suture (polyglactin 910; Ethicon, Raritan, NJ, USA). In the resection procedure, a 4-mm segment of the right SR near its insertion was excised, and the remaining muscle was sutured to the original insertion with an 8-0 suture. Postoperatively, 0.5% levofloxacin eye drops (Santen Pharmaceutical, Osaka, Japan) and 0.1% fluorometholone eye drops (Santen Pharmaceutical) were administered three times daily for 7 days. The rabbits were maintained under standard care until tissue collection. At 1 week and 4 weeks post-surgery, four rabbits from each age group and surgical group were euthanized using xylazine (20 mg/kg). Following carotid artery exsanguination, the full length of the right SR was harvested under a microscope, rinsed with normal saline, and fixed for 24 hours. The right SR was then extracted from the fixative solution and prepared in 2-µm paraffin sections. We prepared tissue sections from the proximal end and recorded the number and the thickness of the sections to determine their location. Because MyHC expression levels vary along the length of the EOM, we selected five consecutive sections located approximately 5 mm from the proximal end for analysis, with one designated for hematoxylin and eosin (H&E) staining and the remaining four for immunohistochemical staining. 

The sections were sequentially immersed in xylene solution (10023418; Sinopharm, Shanghai, China) and graded ethanol (10009218; Sinopharm) at 100%, 85%, and 75% for deparaffinization and hydration, followed by staining with hematoxylin (HK1024; HaoKebio, Hangzhou, China) and eosin (HK2051; HaoKebio). They were then dehydrated in absolute ethanol and xylene before being mounted for the observation of centrally nucleated myofibers, which appear during the repair of EOMs. 

MyHC is integral to the function of EOMs. This study aimed to investigate the representative embryonic and neonatal MyHC, as well as fast 2X-MyHC. Muscle SCs are responsible for the repair of EOMs and specifically express MyoD when activated. Therefore, MyoD was used in this study to label activated SCs. As mentioned above, paraffin sections were subjected to washes with xylene solution and graded ethanol, followed by heating in citric acid antigen retrieval solution (HKI0001, pH 6.0; HaoKebio) for antigen retrieval. After cooling naturally, the sections were rinsed three times with PBS solution (HK2022; HaoKebio), each rinse lasting 5 minutes. Subsequently, a 3% hydrogen peroxide solution (73113760; Sinopharm) was employed to block endogenous peroxidase for 25 minutes, followed by another PBS rinse (three times, 5 minutes each). Antigen blocking was performed using 3% BSA (HK5021; HaoKebio) at room temperature for 30 minutes. Following removal of the BSA solution, diluted primary antibodies, including mouse anti-fast 2X-MyHC (67299-1-Ig, 1:3000; Proteintech, Wuhan, China), mouse anti-embryonic MyHC (ab264038, 1:300; Abcam, Cambridge, UK), rabbit anti-neonatal MyHC (NBP2-41309, 1:200; Bio-Techne, Minneapolis, MN, USA), and rabbit anti-MyoD (18943-1-AP, 1:200; Proteintech), were added, followed by incubation overnight at 4°C in a wet box. After rinsing with PBS three times for 5 minutes each, the secondary antibody solution (I20012C; BioLynx, Hangzhou, China) was added and incubated at room temperature for 50 minutes. Following another PBS wash, 3,3 -diaminobenzidine (DAB) substrate solution (PR30010; Proteintech) was applied for color development, and hematoxylin was used to counterstain the nuclei. Finally, the sections were sequentially dehydrated using graded ethanol and xylene solutions before being mounted for observation. 

For each section, at least three random fields were selected to observe MyHC expression, activated SCs, the number of centrally nucleated myofibers, and myofiber mean cross-sectional area. Muscle fibers expressing each MyHC isoform were counted, and the proportion of myofibers expressing each isoform relative to the total number of muscle fibers was calculated. For activated SCs, the number of MyoD + nuclei attached to the sarcolemma was counted, and the ratio of MyoD + nuclei to muscle fibers in each field was used to determine the number of activated SCs. The ratio of centrally nucleated myofibers to the total number of muscle fibers was calculated. The cross-sectional area of myofibers was measured using ImageJ (National Institutes of Health, Bethesda, MD, USA) from H&E-stained images. Data for the orbital layers (OLs) and global layers (GLs) were analyzed separately except for the count of centrally nucleated myofibers, which was recorded as a combined value. Statistical analysis was conducted using SPSS Statistics 26.0 (IBM Corporation, Chicago, IL, USA), and the Shapiro–Wilk test was applied to assess normality. Normally distributed data are presented as mean ± SD, and abnormally distributed data are expressed as median (interquartile range). Independent t-tests or ANOVA tests were applied to normally distributed data with homogeneity of variance, and the Mann–Whitney U test or Kruskal–Wallis test was used for abnormally distributed data or data with unequal variance. Bonferroni corrections were applied for post hoc comparisons. 

Preoperatively, the expression of embryonic MyHC in the GL and the number of activated SCs in the SRs of 1-month-old and 3-month-old rabbits differed significantly (all P < 0.001) . These findings suggest variations in EOM maturity between two groups, aligning with the developmental stage variations identified in our previous study. shows the summary of changes post-surgery compared to pre-surgery. Impact of Recession on MyHC Expression Alteration in Fast 2X-MyHC Expression In the SRs of 1-month-old rabbits, fast 2X-MyHC expression decreased 1 week after surgery in the OL ( P < 0.001) ( A, D) or remained unchanged in the GL ( P = 1) ( A, D). At 4 weeks post-surgery, fast 2X-MyHC expression further decreased in both layers (both P < 0.01) ( A, E, A, E). In the SRs of 3-month-old rabbits, fast 2X-MyHC expression decreased 1 week after surgery (both P < 0.01) ( A, I, A, I); by 4 weeks post-surgery, expression returned to preoperative levels (both P = 1) ( A, J, A, J; ). Alteration in Embryonic MyHC Expression In the SRs of 1-month-old rabbits, embryonic MyHC expression showed no significant change 1 week post-surgery (OL, P = 0.06; GL, P = 1) ( A, D, A, D). At 4 weeks post-surgery, embryonic MyHC expression increased in both layers (both P < 0.001) ( A, E, A, E). In the SRs of 3-month-old rabbits, expression in the GL increased 1 week after surgery ( P < 0.001) ( A, I) but returned to preoperative levels by 4 weeks post-surgery ( P = 0.952) ( A, J). In contrast, expression in the OL remained stable at both 1 and 4 weeks post-surgery, showing no significant differences compared to preoperative levels ( P = 0.239) ( A, I, J; ). Impact of Resection on MyHC Expression Alteration in Fast 2X-MyHC Expression Fast 2X-MyHC expression was not significantly different compared with pre-surgery levels in all groups ( B, B). However, in the SRs of 1-month-old rabbits, fast 2X-MyHC expression in the OL exhibited a declining trend relative to pre-surgery levels ( B, F, G). In the SRs of 3-month-old rabbits, fast 2X-MyHC expression decreased 1 week after surgery and returned to preoperative levels by 4 weeks after surgery ( B, K, L; ). Alteration in Embryonic MyHC Expression In the SRs of 1-month-old rabbits, expression in the OLs increased compared to preoperative levels, with statistically significant differences observed at 4 weeks post-surgery (1 week post-surgery, P = 1; 4 weeks post-surgery, P = 0.004) ( B, F, G). Expression in the GLs decreased compared to preoperative levels (both P < 0.001) ( B, F, G). In the SRs of 3-month-old rabbits, expression in the OLs also increased compared to preoperative levels (both P < 0.05); however, the expression at 4 weeks post-surgery showed a decreasing trend compared to that of 1 week post-surgery ( B, K, L). Expression in the GLs decreased 1 week after surgery compared to preoperative levels ( P = 0.004) ( B, K) but returned to preoperative levels by 4 weeks post-surgery ( P = 0.574) ( B, L; ). Neonatal MyHC Expression Neonatal MyHC was expressed in all myofibers both preoperatively and postoperatively, with no differences noted in the SRs of 1-month-old and 3-month-old rabbits . Myofiber Mean Cross-Sectional Area The mean cross-sectional area of myofibers in the SRs of 1-month-old rabbits increased after surgery (all P < 0.001), except for the myofibers in the GL at 1 week post-recession ( P < 0.001) ( A). In 3-month-old rabbits, the mean cross-sectional area of myofibers in the OL decreased 4 weeks after recession compared to pre-surgery levels ( P < 0.001). The remaining showed an increase in the mean cross-sectional area post-surgery (all P < 0.001) ( B; ). Number of Activated Satellite Cells and Centrally Nucleated Myofibers In the SRs of 1-month-old rabbits, there was a significant increase in activated SCs in both the OLs and GLs 1 week post-surgery compared to preoperative levels (all P < 0.05, except in the OL post-recession, where P = 0.348) ( B; B, D, B, D), and this increase was maintained at 4 weeks post-surgery (all P < 0.001, except in the OL post-resection, where P = 0.079) ( B; C, E, C, E; ). In the SRs of 3-month-old rabbits, the number of activated SCs showed no significant change 1 week post-surgery compared to preoperative levels ( C; G, I, G, I). At 4 weeks post-recession, the number of activated cells in both the OLs and GLs decreased compared to preoperative levels ( C; H, H) and in the GL at 4 weeks post-resection ( C; J) (all P < 0.01). In contrast, the number of activated SCs in the OLs remained at preoperative levels post-resection ( P = 0.36) ( C, J, ). Additionally, we observed that the number of centrally nucleated myofibers in the SRs of both age groups showed no significant difference before surgery and at 1 week post-surgery. However, at 4 weeks post-surgery, the number of centrally nucleated myofibers in the SRs of 1-month-old rabbits was significantly higher than that in 3-month-old rabbits . 

Alteration in Fast 2X-MyHC Expression In the SRs of 1-month-old rabbits, fast 2X-MyHC expression decreased 1 week after surgery in the OL ( P < 0.001) ( A, D) or remained unchanged in the GL ( P = 1) ( A, D). At 4 weeks post-surgery, fast 2X-MyHC expression further decreased in both layers (both P < 0.01) ( A, E, A, E). In the SRs of 3-month-old rabbits, fast 2X-MyHC expression decreased 1 week after surgery (both P < 0.01) ( A, I, A, I); by 4 weeks post-surgery, expression returned to preoperative levels (both P = 1) ( A, J, A, J; ). Alteration in Embryonic MyHC Expression In the SRs of 1-month-old rabbits, embryonic MyHC expression showed no significant change 1 week post-surgery (OL, P = 0.06; GL, P = 1) ( A, D, A, D). At 4 weeks post-surgery, embryonic MyHC expression increased in both layers (both P < 0.001) ( A, E, A, E). In the SRs of 3-month-old rabbits, expression in the GL increased 1 week after surgery ( P < 0.001) ( A, I) but returned to preoperative levels by 4 weeks post-surgery ( P = 0.952) ( A, J). In contrast, expression in the OL remained stable at both 1 and 4 weeks post-surgery, showing no significant differences compared to preoperative levels ( P = 0.239) ( A, I, J; ). 

In the SRs of 1-month-old rabbits, fast 2X-MyHC expression decreased 1 week after surgery in the OL ( P < 0.001) ( A, D) or remained unchanged in the GL ( P = 1) ( A, D). At 4 weeks post-surgery, fast 2X-MyHC expression further decreased in both layers (both P < 0.01) ( A, E, A, E). In the SRs of 3-month-old rabbits, fast 2X-MyHC expression decreased 1 week after surgery (both P < 0.01) ( A, I, A, I); by 4 weeks post-surgery, expression returned to preoperative levels (both P = 1) ( A, J, A, J; ). 

In the SRs of 1-month-old rabbits, embryonic MyHC expression showed no significant change 1 week post-surgery (OL, P = 0.06; GL, P = 1) ( A, D, A, D). At 4 weeks post-surgery, embryonic MyHC expression increased in both layers (both P < 0.001) ( A, E, A, E). In the SRs of 3-month-old rabbits, expression in the GL increased 1 week after surgery ( P < 0.001) ( A, I) but returned to preoperative levels by 4 weeks post-surgery ( P = 0.952) ( A, J). In contrast, expression in the OL remained stable at both 1 and 4 weeks post-surgery, showing no significant differences compared to preoperative levels ( P = 0.239) ( A, I, J; ). 

Alteration in Fast 2X-MyHC Expression Fast 2X-MyHC expression was not significantly different compared with pre-surgery levels in all groups ( B, B). However, in the SRs of 1-month-old rabbits, fast 2X-MyHC expression in the OL exhibited a declining trend relative to pre-surgery levels ( B, F, G). In the SRs of 3-month-old rabbits, fast 2X-MyHC expression decreased 1 week after surgery and returned to preoperative levels by 4 weeks after surgery ( B, K, L; ). Alteration in Embryonic MyHC Expression In the SRs of 1-month-old rabbits, expression in the OLs increased compared to preoperative levels, with statistically significant differences observed at 4 weeks post-surgery (1 week post-surgery, P = 1; 4 weeks post-surgery, P = 0.004) ( B, F, G). Expression in the GLs decreased compared to preoperative levels (both P < 0.001) ( B, F, G). In the SRs of 3-month-old rabbits, expression in the OLs also increased compared to preoperative levels (both P < 0.05); however, the expression at 4 weeks post-surgery showed a decreasing trend compared to that of 1 week post-surgery ( B, K, L). Expression in the GLs decreased 1 week after surgery compared to preoperative levels ( P = 0.004) ( B, K) but returned to preoperative levels by 4 weeks post-surgery ( P = 0.574) ( B, L; ). 

Fast 2X-MyHC expression was not significantly different compared with pre-surgery levels in all groups ( B, B). However, in the SRs of 1-month-old rabbits, fast 2X-MyHC expression in the OL exhibited a declining trend relative to pre-surgery levels ( B, F, G). In the SRs of 3-month-old rabbits, fast 2X-MyHC expression decreased 1 week after surgery and returned to preoperative levels by 4 weeks after surgery ( B, K, L; ). 

In the SRs of 1-month-old rabbits, expression in the OLs increased compared to preoperative levels, with statistically significant differences observed at 4 weeks post-surgery (1 week post-surgery, P = 1; 4 weeks post-surgery, P = 0.004) ( B, F, G). Expression in the GLs decreased compared to preoperative levels (both P < 0.001) ( B, F, G). In the SRs of 3-month-old rabbits, expression in the OLs also increased compared to preoperative levels (both P < 0.05); however, the expression at 4 weeks post-surgery showed a decreasing trend compared to that of 1 week post-surgery ( B, K, L). Expression in the GLs decreased 1 week after surgery compared to preoperative levels ( P = 0.004) ( B, K) but returned to preoperative levels by 4 weeks post-surgery ( P = 0.574) ( B, L; ). 

Neonatal MyHC was expressed in all myofibers both preoperatively and postoperatively, with no differences noted in the SRs of 1-month-old and 3-month-old rabbits . 

The mean cross-sectional area of myofibers in the SRs of 1-month-old rabbits increased after surgery (all P < 0.001), except for the myofibers in the GL at 1 week post-recession ( P < 0.001) ( A). In 3-month-old rabbits, the mean cross-sectional area of myofibers in the OL decreased 4 weeks after recession compared to pre-surgery levels ( P < 0.001). The remaining showed an increase in the mean cross-sectional area post-surgery (all P < 0.001) ( B; ). 

In the SRs of 1-month-old rabbits, there was a significant increase in activated SCs in both the OLs and GLs 1 week post-surgery compared to preoperative levels (all P < 0.05, except in the OL post-recession, where P = 0.348) ( B; B, D, B, D), and this increase was maintained at 4 weeks post-surgery (all P < 0.001, except in the OL post-resection, where P = 0.079) ( B; C, E, C, E; ). In the SRs of 3-month-old rabbits, the number of activated SCs showed no significant change 1 week post-surgery compared to preoperative levels ( C; G, I, G, I). At 4 weeks post-recession, the number of activated cells in both the OLs and GLs decreased compared to preoperative levels ( C; H, H) and in the GL at 4 weeks post-resection ( C; J) (all P < 0.01). In contrast, the number of activated SCs in the OLs remained at preoperative levels post-resection ( P = 0.36) ( C, J, ). Additionally, we observed that the number of centrally nucleated myofibers in the SRs of both age groups showed no significant difference before surgery and at 1 week post-surgery. However, at 4 weeks post-surgery, the number of centrally nucleated myofibers in the SRs of 1-month-old rabbits was significantly higher than that in 3-month-old rabbits . 

MyHC is a key component of myosin, primarily responsible for the contractile properties of muscle and critical for the precise control of eye positioning by EOMs. , The presence of activated SCs and centrally nucleated myofibers indicates the postoperative repair of EOMs. Our study revealed marked differences in the recovery of SRs between 1-month-old and 3-month-old rabbits following surgery. The 1-month-old rabbits exhibited a constant higher expression of embryonic MyHC isoforms with more activated SCs in SRs 4 weeks post-surgery, but the MyHC expression pattern of the 3-month-old rabbits generally returned to preoperative levels, and activated SCs decreased to a relatively low level by 4 weeks post-surgery. These findings suggest that 1-month-old rabbits may experience a longer remodeling period and demonstrate greater plasticity in their EOMs following surgical intervention compared to 3-month-old rabbits. MyHC Expression With advancing age, the percentage of myofibers expressing embryonic and neonatal isoforms gradually decreases, whereas the proportion of myofibers expressing fast and slow isoforms, which are more closely associated with the contractile properties of EOMs (i.e., muscle strength and contraction speed), increases. Although this study found no statistically significant difference in fast 2X-MyHC expression between 1-month-old and 3-month-old rabbits, our previous research identified notable differences between 2-week-old rabbits and those 1 month or 3 months old, consistent with findings reported by Brueckner et al. Our prior study also identified differences in muscle morphology, embryonic MyHC expression, and the number of activated SCs between 1-month-old and 3-month-old rabbits. This suggests that the EOMs of these two age groups are at different developmental stages. During the remodeling of EOMs post-surgery, we observed an increase in the expression of embryonic isoforms in the SRs of 1-month-old rabbits, whereas the expression of fast 2X isoforms continuously decreased. For example, at 4 weeks post-recession, the expression of fast 2X-MyHC remained significantly lower than preoperative levels, with a more pronounced decrease compared to 1 week post-surgery. The remodeling of the OL post-resection showed a similar trend, although the difference was not statistically significant. Embryonic MyHC showed a significant increase post-surgery, continuing to rise and remaining above preoperative levels at 4 weeks post-surgery. In contrast, although the early postoperative phase in 3-month-old rabbits mirrored that of 1-month-old rabbits, characterized by increased expression of embryonic isoforms and decreased expression of fast 2X isoforms, by 4 weeks post-surgery expression had essentially returned to preoperative levels. For example, fast 2X-MyHC demonstrated a decrease in expression at 1 week post-surgery but returned to preoperative levels by 4 weeks. Similarly, although embryonic MyHC expression was elevated at 1 week post-surgery compared to pre-surgery, it reverted to preoperative levels by 4 weeks post-surgery. Previous research has not investigated the postoperative remodeling of EOMs in immature rabbits. Christiansen et al. examined the expression changes of fast MyHC isoforms in the SRs of adult rabbits at 1 and 2 weeks post-recession. Their findings indicated a decrease in fast isoform expression at 1 week post-surgery, analogous to the fast isoform (fast 2X-MyHC) observed in this study. At 2 weeks post-surgery, the expression remained lower than preoperative levels, although there was a trend toward recovery. Our findings suggest that the postoperative recovery of EOMs in 3-month-old rabbits may resemble that of adult rabbits. Furthermore, we found that the postoperative remodeling in 1-month-old rabbits appears to be prolonged compared to that in 3-month-old rabbits. It has been suggested that the emergence of embryonic and neonatal isoforms facilitates adaptation to the regenerative environment of muscles. In contrast to the reduction in embryonic MyHC expression associated with developmental changes, a sustained postoperative increase in embryonic MyHC expression (except in GLs after resection) was observed in the EOMs of 1-month-old rabbits. This suggests that the changes in MyHC expression are predominantly a consequence of the postoperative remodeling observed in the current study rather than developmental modifications. The differences in embryonic MyHC expression post-surgery between 1-month-old and 3-month-old rabbits may indicate a greater impact of surgery on the development of EOMs in early developing rabbits. Changes in the MyHC composition of EOMs during the postoperative remodeling may affect their contraction properties, such as eye movement speed and precise control of eye position, ultimately affecting the long-term outcomes of strabismus surgery. The changes of fast 2X-MyHC and embryonic MyHC expression in the SRs of 1-month-old rabbits after surgery appear to be more enduring than those in 3-month-old and adult rabbits. This suggests a longer remodeling period following surgical intervention in the EOMs of early developing rabbits, necessitating additional time to achieve stable postoperative outcomes. We observed similar changes in MyHC expression in both the OLs and GLs of SRs following recession. In contrast, post-resection changes differed, with embryonic MyHC expression increased in the OL but decreased in the GL of the SRs in 1-month-old rabbits. A similar pattern was observed in the SRs of 3-month-old rabbits, although these changes tended to return to pre-surgery levels over time. We proposed two potential explanations for this discrepancy. First, the unique structure of the OL, which does not extend to the insertion as observed in humans and monkeys, may contribute. This structural distinction means that resection affects both the OLs and GLs whereas recession predominantly impacts the GL. Second, these differences may stem from variations in innervation between the OL and GLs, as these layers adapt to distinct functional requirements. Although we did not directly measure muscle force, we assessed myofiber cross-sectional area (MCSA), which can partially reflect muscle force. A decrease in MCSA was observed post-recession, likely resulting from muscle unloading, as reported in previous study. The differences between 1-month-old and 3-month-old rabbits may represent a combination of ongoing muscle development and the effects of muscle unloading. Future studies incorporating comparison with age-matched normal controls could provide clearer insight into the impact of surgery on muscle development and muscle force. Activated Satellite Cells and Centrally Nucleated Myofibers The activation of SCs is intricately linked to the compensatory repair ability of EOMs. , And centrally nucleated myofibers, which typically increase following muscle injure, are generally recognized as regenerated myofibers or markers of muscle repair process. This study identified differences in the activation of SCs in the SRs of 1-month-old and 3-month-old rabbits following surgery. In 1-month-old rabbits, the number of activated SCs in the SRs showed a continuous increase from 1 week to 4 weeks post-surgery. In contrast, 3-month-old rabbits exhibited a high number of activated SCs at 1 week post-surgery, which decreased by 4 weeks post-surgery, even falling below preoperative levels. Christiansen et al. reported that the number of MyoD + activated SCs increased to approximately 10% in the SRs of adult rabbits 1 to 2 weeks after resection. In the current study, the quantity of MyoD + activated SCs in the SRs of 3-month-old rabbits at 1 week post-surgery was comparable to the reported levels for adult rabbits following surgery. Christiansen et al. did not assess the activation status of SCs at 4 weeks post-surgery, but our study found a declining trend in the number of activated SCs in the SRs of 3-month-old rabbits at this time point. Accordingly, the quantity of centrally nucleated myofibers in the SRs of 1-month-old rabbits at 4 weeks post-surgery was also higher than that in 3-month-old rabbits. The findings indicate a more intense and prolonged postoperative repair process in early developing rabbits. There is broad consensus that early surgical intervention is essential for promoting the development of binocular vision and cortical function in strabismus, particularly in cases of congenital esotropia. Previous research has explored various factors influencing surgical outcomes, such as binocular function, age of strabismus onset, and refractive status, demonstrating that early surgery often yields satisfactory results. Currently, clinical decisions regarding the timing of strabismus surgery are often based on the development of patients’ binocular visual function. , The results of this study indicate that, in addition to possessing greater visual plasticity, young children exhibit pronounced plasticity and an extended remodeling period in the postoperative recovery of EOMs. Such postoperative remodeling alterations might play a role in influencing surgical results. The regulatory mechanisms underlying EOM remodeling remain unclear, yet research indicates that alterations in the neuromuscular drive from the central nervous system may be involved in this process. Moreover, it is still uncertain whether surgery impacts the development of EOMs in early childhood. Therefore, elucidating the postoperative recovery process and remodeling characteristics of EOMs in strabismus patients at different developmental stages is relevant for clinical decision-making regarding surgical timing and for achieving more stable surgical results. To our knowledge, this is the first study to investigate the postoperative recovery of EOMs in rabbits at different developmental stages. We found that EOMs in 1-month-old rabbits may have a longer remodeling period and greater plasticity compared to those in 3-month-old rabbits. However, this study has several limitations. First, the follow-up duration was somewhat short, and extending it to adulthood while including an adult group would enhance the persuasiveness of the results. However, the 1-month-old and 3-month-old rabbits chosen for this study correspond to the early developing and adolescent periods in humans, the primary age groups for strabismus surgery, making this study of significant practical relevance. Second, we did not measure muscle strength or contraction velocity, which could provide a more direct reflection of the functional changes in muscles due to postoperative MyHC alterations. However, we measured MCSA, which can partially reflect muscle force. Third, changes in neural impulses during the postoperative recovery process were not investigated, despite their role in connecting the central nervous system with the execution of movements by the EOMs. Research in this area would aid in better understanding the mechanisms underlying EOM remodeling and the difference between the OL and GL after strabismus surgery. In addition, although rabbits are a commonly used model in strabismus research, they are lateral-eyed animals, differing anatomically and functionally from humans, who are frontal-eyed species. Furthermore, we conducted surgeries on normal rabbits, whereas studies have shown that the EOMs in strabismus patients differ significantly from those in normal populations. , These distinctions suggest that the postoperative responses observed in the EOMs of non-strabismic rabbits may not be fully applicable to the postoperative remodeling in humans with strabismus. In conclusion, the postoperative repair responses of EOMs vary across developmental stages. EOMs in early developing rabbits exhibit greater plasticity and a longer remodeling during postoperative recovery compared to those in adolescent rabbits. The findings suggest that the effects of postoperative recovery in EOMs should be taken into consideration when planning surgeries for strabismus patients of different ages. 

With advancing age, the percentage of myofibers expressing embryonic and neonatal isoforms gradually decreases, whereas the proportion of myofibers expressing fast and slow isoforms, which are more closely associated with the contractile properties of EOMs (i.e., muscle strength and contraction speed), increases. Although this study found no statistically significant difference in fast 2X-MyHC expression between 1-month-old and 3-month-old rabbits, our previous research identified notable differences between 2-week-old rabbits and those 1 month or 3 months old, consistent with findings reported by Brueckner et al. Our prior study also identified differences in muscle morphology, embryonic MyHC expression, and the number of activated SCs between 1-month-old and 3-month-old rabbits. This suggests that the EOMs of these two age groups are at different developmental stages. During the remodeling of EOMs post-surgery, we observed an increase in the expression of embryonic isoforms in the SRs of 1-month-old rabbits, whereas the expression of fast 2X isoforms continuously decreased. For example, at 4 weeks post-recession, the expression of fast 2X-MyHC remained significantly lower than preoperative levels, with a more pronounced decrease compared to 1 week post-surgery. The remodeling of the OL post-resection showed a similar trend, although the difference was not statistically significant. Embryonic MyHC showed a significant increase post-surgery, continuing to rise and remaining above preoperative levels at 4 weeks post-surgery. In contrast, although the early postoperative phase in 3-month-old rabbits mirrored that of 1-month-old rabbits, characterized by increased expression of embryonic isoforms and decreased expression of fast 2X isoforms, by 4 weeks post-surgery expression had essentially returned to preoperative levels. For example, fast 2X-MyHC demonstrated a decrease in expression at 1 week post-surgery but returned to preoperative levels by 4 weeks. Similarly, although embryonic MyHC expression was elevated at 1 week post-surgery compared to pre-surgery, it reverted to preoperative levels by 4 weeks post-surgery. Previous research has not investigated the postoperative remodeling of EOMs in immature rabbits. Christiansen et al. examined the expression changes of fast MyHC isoforms in the SRs of adult rabbits at 1 and 2 weeks post-recession. Their findings indicated a decrease in fast isoform expression at 1 week post-surgery, analogous to the fast isoform (fast 2X-MyHC) observed in this study. At 2 weeks post-surgery, the expression remained lower than preoperative levels, although there was a trend toward recovery. Our findings suggest that the postoperative recovery of EOMs in 3-month-old rabbits may resemble that of adult rabbits. Furthermore, we found that the postoperative remodeling in 1-month-old rabbits appears to be prolonged compared to that in 3-month-old rabbits. It has been suggested that the emergence of embryonic and neonatal isoforms facilitates adaptation to the regenerative environment of muscles. In contrast to the reduction in embryonic MyHC expression associated with developmental changes, a sustained postoperative increase in embryonic MyHC expression (except in GLs after resection) was observed in the EOMs of 1-month-old rabbits. This suggests that the changes in MyHC expression are predominantly a consequence of the postoperative remodeling observed in the current study rather than developmental modifications. The differences in embryonic MyHC expression post-surgery between 1-month-old and 3-month-old rabbits may indicate a greater impact of surgery on the development of EOMs in early developing rabbits. Changes in the MyHC composition of EOMs during the postoperative remodeling may affect their contraction properties, such as eye movement speed and precise control of eye position, ultimately affecting the long-term outcomes of strabismus surgery. The changes of fast 2X-MyHC and embryonic MyHC expression in the SRs of 1-month-old rabbits after surgery appear to be more enduring than those in 3-month-old and adult rabbits. This suggests a longer remodeling period following surgical intervention in the EOMs of early developing rabbits, necessitating additional time to achieve stable postoperative outcomes. We observed similar changes in MyHC expression in both the OLs and GLs of SRs following recession. In contrast, post-resection changes differed, with embryonic MyHC expression increased in the OL but decreased in the GL of the SRs in 1-month-old rabbits. A similar pattern was observed in the SRs of 3-month-old rabbits, although these changes tended to return to pre-surgery levels over time. We proposed two potential explanations for this discrepancy. First, the unique structure of the OL, which does not extend to the insertion as observed in humans and monkeys, may contribute. This structural distinction means that resection affects both the OLs and GLs whereas recession predominantly impacts the GL. Second, these differences may stem from variations in innervation between the OL and GLs, as these layers adapt to distinct functional requirements. Although we did not directly measure muscle force, we assessed myofiber cross-sectional area (MCSA), which can partially reflect muscle force. A decrease in MCSA was observed post-recession, likely resulting from muscle unloading, as reported in previous study. The differences between 1-month-old and 3-month-old rabbits may represent a combination of ongoing muscle development and the effects of muscle unloading. Future studies incorporating comparison with age-matched normal controls could provide clearer insight into the impact of surgery on muscle development and muscle force. 

The activation of SCs is intricately linked to the compensatory repair ability of EOMs. , And centrally nucleated myofibers, which typically increase following muscle injure, are generally recognized as regenerated myofibers or markers of muscle repair process. This study identified differences in the activation of SCs in the SRs of 1-month-old and 3-month-old rabbits following surgery. In 1-month-old rabbits, the number of activated SCs in the SRs showed a continuous increase from 1 week to 4 weeks post-surgery. In contrast, 3-month-old rabbits exhibited a high number of activated SCs at 1 week post-surgery, which decreased by 4 weeks post-surgery, even falling below preoperative levels. Christiansen et al. reported that the number of MyoD + activated SCs increased to approximately 10% in the SRs of adult rabbits 1 to 2 weeks after resection. In the current study, the quantity of MyoD + activated SCs in the SRs of 3-month-old rabbits at 1 week post-surgery was comparable to the reported levels for adult rabbits following surgery. Christiansen et al. did not assess the activation status of SCs at 4 weeks post-surgery, but our study found a declining trend in the number of activated SCs in the SRs of 3-month-old rabbits at this time point. Accordingly, the quantity of centrally nucleated myofibers in the SRs of 1-month-old rabbits at 4 weeks post-surgery was also higher than that in 3-month-old rabbits. The findings indicate a more intense and prolonged postoperative repair process in early developing rabbits. There is broad consensus that early surgical intervention is essential for promoting the development of binocular vision and cortical function in strabismus, particularly in cases of congenital esotropia. Previous research has explored various factors influencing surgical outcomes, such as binocular function, age of strabismus onset, and refractive status, demonstrating that early surgery often yields satisfactory results. Currently, clinical decisions regarding the timing of strabismus surgery are often based on the development of patients’ binocular visual function. , The results of this study indicate that, in addition to possessing greater visual plasticity, young children exhibit pronounced plasticity and an extended remodeling period in the postoperative recovery of EOMs. Such postoperative remodeling alterations might play a role in influencing surgical results. The regulatory mechanisms underlying EOM remodeling remain unclear, yet research indicates that alterations in the neuromuscular drive from the central nervous system may be involved in this process. Moreover, it is still uncertain whether surgery impacts the development of EOMs in early childhood. Therefore, elucidating the postoperative recovery process and remodeling characteristics of EOMs in strabismus patients at different developmental stages is relevant for clinical decision-making regarding surgical timing and for achieving more stable surgical results. To our knowledge, this is the first study to investigate the postoperative recovery of EOMs in rabbits at different developmental stages. We found that EOMs in 1-month-old rabbits may have a longer remodeling period and greater plasticity compared to those in 3-month-old rabbits. However, this study has several limitations. First, the follow-up duration was somewhat short, and extending it to adulthood while including an adult group would enhance the persuasiveness of the results. However, the 1-month-old and 3-month-old rabbits chosen for this study correspond to the early developing and adolescent periods in humans, the primary age groups for strabismus surgery, making this study of significant practical relevance. Second, we did not measure muscle strength or contraction velocity, which could provide a more direct reflection of the functional changes in muscles due to postoperative MyHC alterations. However, we measured MCSA, which can partially reflect muscle force. Third, changes in neural impulses during the postoperative recovery process were not investigated, despite their role in connecting the central nervous system with the execution of movements by the EOMs. Research in this area would aid in better understanding the mechanisms underlying EOM remodeling and the difference between the OL and GL after strabismus surgery. In addition, although rabbits are a commonly used model in strabismus research, they are lateral-eyed animals, differing anatomically and functionally from humans, who are frontal-eyed species. Furthermore, we conducted surgeries on normal rabbits, whereas studies have shown that the EOMs in strabismus patients differ significantly from those in normal populations. , These distinctions suggest that the postoperative responses observed in the EOMs of non-strabismic rabbits may not be fully applicable to the postoperative remodeling in humans with strabismus. In conclusion, the postoperative repair responses of EOMs vary across developmental stages. EOMs in early developing rabbits exhibit greater plasticity and a longer remodeling during postoperative recovery compared to those in adolescent rabbits. The findings suggest that the effects of postoperative recovery in EOMs should be taken into consideration when planning surgeries for strabismus patients of different ages. 

Supplement 1 

