A study on tacrolimus combined with hyaluronic acid gel to inhibit scar proliferation at nerve anastomoses

A study on tacrolimus combined with hyaluronic acid gel to inhibit scar proliferation at nerve anastomoses | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article A study on tacrolimus combined with hyaluronic acid gel to inhibit scar proliferation at nerve anastomoses Shi Lexiang, Zhang Yinan, zhang wanli This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9261730/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Objective : To investigate the effect of additional application of hyaluronic acid (HA) gel containing tacrolimus (FK506) at the nerve anastomosis site after acute peripheral nerve injury treated with xenogeneic nerve transplantation on inhibiting the growth and formation of scar around the anastomosis. Methods : A total of 60 healthy male Sprague-Dawley (SD) rats weighing 250 g to 350 g were selected. Among them, 20 rats were used as nerve donors, and the remaining 40 rats were randomly divided into 4 groups. The groups were as follows: combination group (acellular xenogeneic nerve transplantation + application of HA gel containing FK506 at the anastomosis site), tacrolimus group (FK506 group, acellular xenogeneic nerve transplantation + application of FK506 at the anastomosis site), hyaluronic acid group (HA group, acellular xenogeneic nerve transplantation + application of HA at the anastomosis site), and control group (only acellular xenogeneic nerve transplantation was performed). Experimental procedures: 1. Donor nerve sampling; 2. Establishment of sciatic nerve defect and nerve transplantation model. 3. Sampling and detection: Tissue samples were collected 12 weeks after surgery, and analyzed by Peterson score, Masson staining, immunohistochemical analysis of type I and type III collagen, and counting of myelinated nerve fibers in the middle segment of the transplanted nerve. SPSS 23.0 statistical software was used for data processing and analysis. This study has been approved by the Animal Experimental Ethics Committee of Mudanjiang Medical University (Approval No.202009015). Confirming that all experiments were performed in accordance with relevant guidelines and regulations. Results :The results of Peterson score, Masson staining, immunohistochemical analysis of type I and type III collagen, and counting of myelinated nerve fibers in the middle segment of the transplanted nerve showed that: 1. The degree of adhesion between the nerve anastomosis and surrounding tissue was lighter in the combination group, FK506 group and HA group, all of which were better than that in the control group, and the differences were statistically significant (P < 0.05). 2. The amount of scar formation was the least in the combination group, intermediate in the FK506 group and HA group, and the largest in the control group. 3. The number of myelinated nerve fibers in the middle segment of the transplanted nerve in the combination group was higher than that in the other three groups (FK506 group, HA group, control group), and the difference between the combination group and the control group was highly statistically significant (P < 0.01). Conclusion :After acellular xenogeneic nerve transplantation, the effect of applying HA gel containing FK506 at the anastomosis site on inhibiting scar formation is better than that of applying FK506 alone or HA gel alone. HA gel containing FK506 can inhibit scar growth at the nerve anastomosis site and promote nerve regeneration. Health sciences/Medical research Biological sciences/Neuroscience nerve transplantation nerve repair tacrolimus hyaluronic acid nerve scar tissue Figures Figure 1 Figure 2 Introduction Repair of peripheral nerve injury is highly challenging, and autologous nerve transplantation is considered the gold standard for repair of peripheral nerve injury [ 1 – 3 ] . Nerve transplantation mainly addresses two major difficulties:1.How to completely remove the major histocompatibility complex (MHC) from allogeneic nerves, and 2. How to reduce perineural scar formation after transplantation to promote nerve regeneration. Autologous nerve transplantation contains autologous Schwann cells, native extracellular matrix and a variety of neurotrophic factors, and is currently widely used in the treatment of peripheral nerve injury with good therapeutic effects [ 4 ] . However, this method still has many limitations in practical application [ 5 ] . Allogeneic nerve transplantation can well compensate for the defects of autologous nerve transplantation and is an alternative material for autologous nerve transplantation [ 6 – 14 ] . Existing studies have confirmed that nerve scar formation is an important inhibitory factor in the process of nerve regeneration. Once new nerve axons encounter obstacles such as inflammatory granulation tissue or collagen scar tissue, it may lead to the failure of nerve repair [ 15 – 16 ] . In recent years, numerous studies have reported the properties of the local immunosuppressant tacrolimus (FK506) and synthetic hyaluronic acid, and both have been applied in multiple fields with good results [ 17 – 20 ] . In this study, FK506 and hyaluronic acid (HA) were selected as research factors to investigate the effect of their combined application on inhibiting scar growth around the nerve anastomosis. Materials and Methods 1.1 Design: A randomized controlled animal experiment. 1.2 Time and location: The experiment was completed at the Medical Research Center of Mudanjiang Medical University from November 2021 to April 2023. 1.3 Materials 1.3.1 Experimental animals: Forty healthy adult male Sprague-Dawley rats (divided into 4 groups) weighing 250–350 g were selected and provided by the Laboratory of Mudanjiang Medical University. The disposal of animals during the experiment complied with animal ethics requirements. 1.3.2 Main experimental reagents and instruments: Tacrolimus (25 mg), purchased from Shanghai Beyotime Biotechnology Co., Ltd.; Hyaluronic acid (1 g), purchased from Shanghai Macklin Biochemical Technology Co., Ltd.; Masson trichrome staining kit, purchased from Fuzhou Maixin Biotech Co., Ltd.; PBS phosphate buffer dry powder (for 2 L preparation, 500 mL specification), purchased from Aladdin; Beijing Solarbio Science & Technology Co., Ltd.; Upright microscope, purchased from Leica Visualization Systems (Shanghai) Co., Ltd.; Image analysis system (CIAS-1000), purchased from Beijing Daheng Image Vision Co., Ltd.; Paraffin microtome (RM2235), purchased from Leica Camera AG, Germany. 1.4 Research Methods 1.4.1 Experimental animals and grouping: Sixty healthy adult male Sprague-Dawley (SD) rats weighing 250–350 g were selected and provided by the Medical Research Center of Mudanjiang Medical University. Twenty of them were used as nerve donors (a total of 40 sciatic nerves were harvested); the remaining 40 rats were randomly divided into 4 groups with 10 rats in each group. The grouping was as follows: control group (simple acellular allogeneic nerve transplantation), hyaluronic acid group (HA group, HA was applied to the anastomotic stoma after acellular allogeneic nerve transplantation), tacrolimus group (FK506 group, FK506 was applied to the anastomotic stoma after acellular allogeneic nerve transplantation), oral tacrolimus group (oral administration of FK506) and combined group (HA gel containing FK506 was applied to the anastomotic stoma after acellular allogeneic nerve transplantation). 1.4.2 Collection of allogeneic nerves and preparation of acellular allogeneic nerves Collection of allogeneic nerves: Twenty healthy adult male SD rats were selected, and their bilateral sciatic nerves were used as donors for allogeneic nerve transplantation. SD rats were anesthetized by intraperitoneal injection of 3% pentobarbital sodium (30 mg/kg). A longitudinal incision was made on the dorsal side of both lower limbs under sterile conditions, the space between biceps femoris and vastus lateralis was bluntly separated to expose the sciatic nerve. A 15 mm-long sciatic nerve segment was sharply cut between the lower edge of pectineus and the bifurcation of the sciatic nerve. Fat and connective tissue on the peripheral surface of the nerve were removed, rinsed with sterile normal saline, and placed in a sterile culture dish for later use. Preparation of acellular allogeneic nerves: All 30 donor allogeneic nerves were prepared using the decellularization method described by Sondell [ 21 ] . 1.4.3 Establishment of animal model: Forty SD rats were fasted for 4–6 hours before surgery, and anesthetized by intraperitoneal injection of 3% pentobarbital sodium (30 mg/kg), then underwent allogeneic sciatic nerve transplantation. Under sterile conditions, a longitudinal incision was made along the course of the right lower extremity sciatic nerve, and the sciatic nerve was bluntly separated and freed along the intermuscular space. The sciatic nerve was sharply resected starting from the lower edge of the perineal muscle, and a 6-mm-long sciatic nerve segment was excised distally to form a 10-mm nerve defect. The sharply dissected 10-mm acellular allograft was anastomosed and transplanted to the nerve defect. Under an operating microscope, 9 − 0 non-invasive sutures were used, and 4 stitches were placed at four equally divided positions of the epineurium to complete the sciatic nerve anastomosis. In the HA group, a micro-syringe (10 µl) was used to inject HA gel at multiple points around the nerve anastomosis site via microinjection, with an average injection volume of about 15 µL [ 15 ] ; in the FK506 group, FK506 solution was injected at the nerve anastomosis site [ 22 – 23 ] , with an injection volume of about 15 µl; in the combination group, HA gel containing FK506 was injected at the nerve anastomosis site, with an injection ratio of 1:1 (15 µl each of HA and FK506); the control group received no treatment. After completion of treatment in all groups, the incision was sutured layer by layer. Gentamicin (40 mg/ml) 1 ml was injected intramuscularly daily for 3 consecutive days to prevent infection. Animals were housed in individual cages with unrestricted activity. 1.4.4 Criteria for successful animal model establishment: reliable nerve anastomosis, no tension on the nerve after anastomosis, and no deep infection or abscess formation after surgery. 1.5 Methods for implementing euthanasia: Drugs: Potassium chloride Anesthesia: Induce deep anesthesia in the animal through inhalation anesthesia (isoflurane) to ensure the animal feels no pain. Precise injection: After opening the chest to expose the heart, use a fine needle to insert into the left ventricle or right atrium along the axis of the heart from the apex, and slowly inject the drug. Confirm death: After injection, observe the cessation of the animal's heartbeat and breathing, and dilation of the pupils to confirm death. 1.6 Main outcome measures 1.6.1 Gross observation: Three months after surgery, the incision was opened under anesthesia, and whether there was bulging in the surgical area and the adhesion between the nerve graft segment and surrounding tissues were observed. Evaluation was performed according to the grading standard score of Peterson et al [ 24 ] , as detailed in Table 1. 1.6.2 Detection of epineurial scar: Masson trichrome staining: In Masson trichrome staining, cell nuclei are stained black-blue, collagen fibers are stained blue, and glial fibers, muscle fibers and red blood cells are stained red. 1.6.3 Analysis of the relative contents of type I and type III collagen: Specimens were collected 12 weeks after surgery, and detection was performed at the proximal nerve anastomosis. The immunohistochemical SABC method was used, and the operation procedure was performed in accordance with the reagent instructions. Images of type I and type III collagen were collected under a 100× optical microscope, and image analysis was performed using the CIAS-1000 image analysis system. In a visual field at 640× magnification, positive regions were selected with a mouse tool to measure the gray value. In this system, the gray value varies according to the staining depth and reaction intensity of positive regions. The gray value is divided into 256 levels in total, with level 0 having the darkest color indicating a strong positive reaction, and level 255 having the lightest color indicating a weak positive reaction. Collagen content is negatively correlated with the gray value. 1.6.4 Counting analysis of myelinated nerve fibers in the middle segment of the transplanted nerve: Three months after surgery, toluidine blue staining was used to observe the growth of myelinated nerve fibers in each group. 1.6.5 Statistical analysis: SPSS 25.0 statistical analysis software and GraphPad Prism 8.0 were used to perform statistical analysis on the data and generate graphs. Measurement data are expressed as (X ± S). Grouped data were analyzed by one-way analysis of variance, and the q-test for multiple comparisons was performed on groups with statistical differences indicated by analysis of variance. P < 0.05 was considered a significant difference, and P < 0.01 was considered a highly significant difference. Results 2.1 Quantitative analysis of experimental animals: A total of 40 SD rats were included and randomly divided into 4 groups, with 10 rats in each group. All rats were included in the result analysis, with no dropout. 2.2 Gross observation through surgical windowing: After anesthesia, all rats were incised to observe the adhesion between the nerve anastomosis and surrounding tissues. Postoperatively, the skin, muscles, and fascia of most SD rats in each group did not dehisce, and partial dehiscence of skin, muscles, and fascia occurred in a few SD rats, with no infection. Varying degrees of nerve adhesion existed in all 4 groups. The Peterson scores (data results are ranked categorical data) were analyzed for quantitative data using analysis of variance and q-test for multiple comparisons. The adhesion between nerve anastomosis and surrounding tissues was less in the combination group, tacrolimus group and hyaluronic acid group, while nerve adhesion was the most severe in the control group. The difference between the control group and the other three groups was statistically significant (P < 0.05). Multiple comparisons of the remaining indicators among the four groups showed no statistically significant difference among the four groups. The results of gross observation of Peterson scores in each group are shown in Table 2. 2.3 Masson staining: After Masson staining, observation under a 400× light microscope revealed collagen deposition in the epineurium of the nerve anastomosis in all groups (see Fig. 1 ). Figure 1 A shows that the combination group had the least collagen content, with tightly arranged fibers, small curling angles, and clear boundaries with surrounding tissues. Figure 1 B shows the tacrolimus group, which had less collagen fiber content with very tight arrangement. Figure 1 C shows that the hyaluronic acid group had a slightly higher content of collagen fibers in the epineurium, relatively sparse arrangement, larger curling angles of collagen fibers, and clear boundaries with surrounding tissues. Figure 1 D shows that the control group had the highest content of collagen fibers, disordered arrangement, and unclear boundaries with surrounding tissues. 2.4 Gray value analysis of relative contents of type Ⅰ and type Ⅲ collagen Total collagen fiber gray value: The difference between the combination group and the FK506 group and HA group was statistically significant (P < 0.05), and the difference between the combination group and the control group was highly statistically significant (P < 0.01). There was no statistically significant difference between the FK506 group and the HA group, and both the FK506 group and the HA group showed statistically significant differences compared with the control group (P < 0.05). Gray value of type Ⅰ collagen: The combination group, FK506 group and HA group all showed statistically significant differences compared with the control group (P < 0.05), and there was no statistically significant difference between the FK506 group and the HA group. Gray value of type Ⅲ collagen: The combination group showed statistically significant differences compared with the FK506 group and HA group (P < 0.05), and there was no statistically significant difference between the combination group and the control group; there was no statistically significant difference between the FK506 group and the HA group and the control group, and no statistically significant difference between the HA group and the control group. In conclusion, the amount of scar formation at the nerve anastomosis in the combination group was the least, which was better than the other three groups (FK506 group, HA group and control group), and the difference was statistically significant compared with the other three groups. The amount of scar formation at the nerve anastomosis in the FK506 group and HA group was less, which was better than that in the control group (the control group had the largest amount of scar formation), and both groups showed statistically significant differences compared with the control group (P < 0.05). There was no statistically significant difference in collagen content analysis between the FK506 group and the HA group. (The results of immunohistochemical gray analysis of type Ⅰ and type Ⅲ collagen are shown in Table 3.) 2.5 Analysis of counting results of myelinated nerve fibers in the middle segment of nerve grafts Three months after surgery, the number of myelinated nerve fibers in the middle segment of the grafted nerve in the combination group was (1145.8 ± 29.6) per unit visual field, which was superior to that of the other three groups (tacrolimus group, hyaluronic acid group and control group). Compared with the tacrolimus group (894.2 ± 46.1 myelinated nerve fibers per unit visual field in the middle segment of the grafted nerve), the hyaluronic acid group (892.1 ± 37.3 myelinated nerve fibers per unit visual field in the middle segment of the grafted nerve), and the control group (697.6 ± 29.6 myelinated nerve fibers per unit visual field in the middle segment of the grafted nerve), the differences were all statistically significant (p < 0.05); the difference between the combination group and the control group (697.6 ± 21.7 myelinated nerve fibers per unit visual field) was highly statistically significant (p < 0.01). There was no statistically significant difference in the number of myelinated nerve fibers in the middle segment of the grafted nerve between the tacrolimus group and the hyaluronic acid group, while the number of myelinated nerve fibers in the middle segment of the grafted nerve in both the tacrolimus group and the hyaluronic acid group was higher than that in the control group, with statistically significant differences (p < 0.05). See Fig. 2 and Table 4. Discussion Peripheral nerve injury is a common clinical condition. After peripheral nerve injury, there are many factors affecting nerve regeneration and repair, including scar formation, nerve anastomosis technique, and the microenvironment for nerve regeneration. Among these factors, scar is a key factor affecting nerve regeneration after nerve bridging. After scar formation, local tissue motor, sensory and information conduction functions are impaired, which will also lead to incomplete postoperative functional recovery in patients. In clinical practice, it is extremely common that patients with peripheral nerve injury experience incomplete postoperative functional recovery or even loss of function due to scar formation around the nerve. Therefore, inhibiting scar hyperplasia around the nerve anastomosis is crucial for nerve regeneration. After body trauma, healing usually occurs through scar repair, but the specific repair mechanism of scar formation has not been fully elucidated and still requires in-depth research. Existing studies believe that the mechanism of scar repair is as follows: a series of inflammatory reactions (also known as inflammatory cascade reaction) occur rapidly at the injury site, and the inflammatory cascade reaction affects the transformation of fibroblasts and the synthesis and degradation of collagen fibers in the body [ 25 ] . During this reaction, inflammatory cells aggregate and release inflammatory mediators, stimulating fibroblasts to synthesize a large amount of type I and type III collagen and matrix, leading to abnormal arrangement of collagen and matrix, prompting the tissue to repair the wound through scar healing to achieve the purpose of wound repair. With the progress of research, some researchers and scholars have put forward the conjecture whether wound healing can achieve scar-free healing. Researchers in this field have also carried out in-depth research on nerve injury repair, exploring whether minimal scarring or even scar-free repair can also be achieved during nerve defect repair. Therefore, researchers have tried to add a variety of different drugs and cytokines at the nerve anastomosis site to explore their effects on scar formation at the nerve anastomosis. In recent years, tacrolimus (FK506) and hyaluronic acid (HA) have often been used as research factors to explore their effects on peripheral nerve regeneration. Hyaluronic acid can inhibit scar growth through mechanisms such as inhibiting lymphocyte proliferation, phagocytosis, and hindering granulocyte movement [ 26 ] . Relevant research results show that hyaluronic acid can selectively stimulate fibroblasts to synthesize type III collagen, reduce the ratio of type I collagen to type III collagen, thereby reducing scar formation [ 27 – 28 ] . Tacrolimus is a natural macrolide immunosuppressant, which can block the activity of immune cells and inhibit autoimmune reactions, thereby alleviating the symptoms of patients' autoimmune diseases. In clinical practice, tacrolimus has been widely used in many fields such as dermatology, ophthalmology, and orthopedics [ 29 ] , and has achieved good therapeutic effects. In addition, tacrolimus also has a significant curative effect in promoting the repair of nerve injury. Clear research conclusions show that the mechanism by which tacrolimus promotes nerve injury repair is as follows: tacrolimus mainly forms a relevant complex with FK506 binding protein 12 (FKBP12) through its binding region, then binds to the functional effector region, thereby up-regulating the expression of growth-associated protein-43 (GAP-43) and promoting the growth of nerve growth cones [ 30 – 31 ] , exerting a neurotrophic effect. According to the experimental results, we observed the following four characteristics: 1. In terms of Petersen scoring results, adhesion between the nerve anastomosis and surrounding tissues was the mildest in the combination group, mild in the FK506 group and HA group, and the most severe in the control group. Compared with the control group, the combination group, FK506 group and HA group all showed statistically significant differences in Petersen nerve adhesion scores (P < 0.05), while there were no statistically significant differences in Petersen scores for skin and myofascia among all groups. 2. Regarding the Masson staining results of total collagen fibers in the epineurium of the anastomosis, the combination group had the smallest total number of collagen fibers, with tightly arranged fibers, small curling angles, and clear boundaries with surrounding tissues. The FK506 group had a smaller total number of collagen fibers around the nerve, looser arrangement, larger curling angles of collagen fibers, and clear boundaries with surrounding tissues. The HA group had a smaller total number of collagen fibers with very dense arrangement. The control group had the largest total number of collagen fibers, disordered arrangement, and clear boundaries with surrounding tissues. 3. In the results of immunohistochemical analysis of type I and type III collagen, for the gray value of total collagen fibers: the combination group had the lowest gray value, the FK506 group had a lower gray value, the HA group had a slightly higher gray value than the FK506 group, and the control group had the highest gray value. The differences between the combination group and the FK506 group, and between the combination group and the HA group were both statistically significant (P < 0.05), and the difference between the combination group and the control group was statistically significant (P < 0.01). There was no statistically significant difference between the FK506 group and the HA group, and both the FK506 group and the HA group showed statistically significant differences compared with the control group (P < 0.05). For the gray value of type I collagen: the combination group had the lowest gray value, the HA group had a lower gray value, the FK506 group had a slightly higher gray value than the HA group, and the control group had the highest gray value. The difference between the combination group and the FK506 group was statistically significant (P < 0.05), the difference between the combination group and the HA group was statistically significant (P < 0.05), and the difference between the combination group and the control group was statistically significant (P < 0.01); there was no statistically significant difference between the FK506 group and the HA group, the difference between the FK506 group and the control group was statistically significant (P < 0.05), and the difference between the HA group and the control group was statistically significant (P < 0.05). For the gray value of type III collagen: the combination group had the lowest collagen gray value, the control group had a lower gray value, the FK506 group had a slightly higher gray value than the control group, and the HA group had the highest gray value. The differences between the combination group and the FK506 group, and between the combination group and the HA group were both statistically significant (P < 0.05), while there was no statistically significant difference between the combination group and the control group; there were no statistically significant differences between the FK506 group and the HA group or the control group, and there was no statistically significant difference between the HA group and the control group. Previous studies have confirmed that local application of HA at the anastomosis of acellular allogeneic nerve grafts has significant effects in reducing adhesion between nerves and surrounding tissues and inhibiting scar formation around nerves [ 15 ] , and the same results were obtained in this experiment. The possible reasons for the above results in points 1, 2 and 3 are as follows: after repair of nerve defects with acellular allogeneic nerve transplantation, the addition of FK506-containing HA gel around the graft anastomosis, or the addition of FK506 or HA alone can all regulate the proportion of type I and type III collagen fibers, or reduce the total content of collagen fibers, and ultimately inhibit scar formation around the nerve anastomosis. Existing studies have shown [ 15 ] that HA can reduce scar formation by inhibiting the inflammatory reaction around the nerve anastomosis; the reason for this result in the FK506 group may be that FK506 binds to the FKBP12 receptor in the cytoplasm to form a functional complex, which blocks the dephosphorylation process of T cells, thereby inhibiting the expression of subsequent inflammatory factors (IL2, IL3, rINF) and reducing the aggregation of fibroblasts. The process of fibroblast transformation into scar tissue is thus inhibited, and an anti-scar effect is ultimately exerted [ 32 – 36 ] . The occurrence of this result in the combination group may be due to the additive or synergistic effect produced by the combined application of FK506 and HA. 4. Regarding the count of myelinated nerve fibers in the middle segment of the transplanted nerve: the combination group had the largest number of myelinated nerve fibers in the middle segment of the transplanted nerve, followed by the FK506 group, the HA group had fewer myelinated nerve fibers than the FK506 group, and the control group had the smallest number of myelinated nerve fibers. Compared with the FK506 group and the HA group, the combination group showed statistically significant differences in the total count of myelinated nerve fibers (P < 0.05), and the difference between the combination group and the control group was statistically significant (P < 0.01). The counts of myelinated nerve fibers in both the FK506 group and the HA group were higher than that in the control group, and the differences were both statistically significant (P < 0.05), while there was no statistically significant difference between the FK506 group and the HA group. The possible reason for the above result in point 4 is that after acellular allogeneic nerve transplantation, adding FK506-containing HA gel around the anastomosis, or adding FK506 or HA alone can all inhibit scar formation around the nerve anastomosis, reduce the barrier that affects the regeneration of myelinated nerve fibers, and thereby promote nerve regeneration. In conclusion, FK506-containing HA gel can inhibit scar growth around nerve anastomosis and promote nerve regeneration. However, the mechanisms underlying its inhibition of scar growth and promotion of nerve regeneration remain very complex. Whether these two effects are additive or synergistic in scar inhibition has not been clarified in this study. Therefore, further research is needed to elucidate the specific mechanism of scar inhibition, and it is also necessary to deeply explore whether HA and FK506 have a direct effect on promoting nerve regeneration. Conclusion Based on the results of this study, we can draw the following conclusion: in acellular allogeneic nerve transplantation, the effect of FK506-containing HA gel on inhibiting scar formation is superior to that of single application of FK506 and single application of HA gel. The promoting effect of FK506-containing HA gel on nerve regeneration may be related to its ability to promote nerve regeneration by regulating the distribution of type I and type III collagen, reducing total collagen content, and decreasing perineural scar formation. Declarations Conflict of Interest All authors declare that there is no conflict of interest in this study. Funding This study received no funding. Author Contribution Dr. LX, and Dr. YN conceived and designed the research, drafted the initial manuscript, and reviewed and revised the manuscript. Dr.WL collected the data, performed a preliminary analysis. All authors have read and approved the manuscript. Data Availability All data generated or analysed during this study are included in this published article and its supplementary information files. Ethical Compliance Statement Ethical Requirements This study complies with all ethical Requirements. This study complies with all ethical requirements. References Pollins, A. C. et al. Mass spectrometric comparison of decellularization processes for nerve allotransplantation [J]. J. Mater. Science: Mater. Med. 28 (1), 20 (2017). Carvalho, C. R. et al. Cell adhesion study of chitosan membranes for peripheral nerve regeneration [J]. J. Mater. Sci. Eng. C: Mater. Biol. Appl. 71 , 1122–1134 (2017). Zhang Li, Z. et al. 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Neurotrauma . 21 (5), 627–635 (2004). Guo, J. et al. Zhao Xiao-wei, Fan Guo-hua,. Observation on the efficacy of tacrolimus in preventing scar adhesion after hip arthroplasty in the elderly [J]. Chinese Journal of Hospital Pharmacy, 41(12): 1255–1257 + 1263. (2021). Que Jun. Study on the effect and mechanism of FK506 in promoting peripheral nerve regeneration by reducing scar formation [D] (Nanjing Medical University, 2012). Mekaj, A. Y. et al. Effects of hyaluronic acid and tacrolimus on the prevention of perineural scar formation and nerve regeneration after sciatic nerve repair in a rabbit model [J]. Eur. J. Trauma. Emerg. Surg. August . 43 (4), 497–504 (2017). Mekaj, A. Y. et al. Electrophysiological and functional evaluation of rabbit common peroneal nerve regeneration after local application of hyaluronic acid or tacrolimus following nerve repair. Nigerian J. Postgrad. Med. 22 , 179–184 (2015). Mekaj, A. Y. et al. Biomechanical properties of sciatic nerve after repair: the effect of local application of hyaluronic acid or tacrolimus. Int. J. Clin. Exp. Med. 8 , 20218–20226 (2015). Tables Table 1 to 4 are available in the Supplementary Files section. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9261730","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":629668810,"identity":"56fefc67-660a-4c67-8750-43b1c28e5aab","order_by":0,"name":"Shi Lexiang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuUlEQVRIie3QsQrCMBCA4QuBdCnOEcW8QsTBpYOPckVop66SsVJIlj6Dj+GckjX4Ai71LepmRxfx3BzyzffD3QEkyT/KgI1oYCMy54kJB67HCLtFHpGciOXDQnmRB00rVJdrWdpQWwkIk7l+T3QQqPFWNXZ19qyPd0LCuUc8FY1de+TMEhLVsdajkLWQqGkJBD60aAukJ/MtR8BYbe385IF0i3L9/jmZoJRzwzgZymLv/I/zSZIkyScvFWw6Onh5X8gAAAAASUVORK5CYII=","orcid":"","institution":"Northwest University","correspondingAuthor":true,"prefix":"","firstName":"Shi","middleName":"","lastName":"Lexiang","suffix":""},{"id":629668811,"identity":"6293e7e9-0204-4bc9-8964-9e870fd2b759","order_by":1,"name":"Zhang Yinan","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Zhang","middleName":"","lastName":"Yinan","suffix":""},{"id":629668812,"identity":"55fab7ac-eeb9-4b06-86ad-fe50d03e1d42","order_by":2,"name":"zhang wanli","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"zhang","middleName":"","lastName":"wanli","suffix":""}],"badges":[],"createdAt":"2026-03-30 02:38:03","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9261730/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9261730/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108494458,"identity":"897f3a2f-97f6-4f44-a91c-277e91f93034","added_by":"auto","created_at":"2026-05-05 10:05:28","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":354243,"visible":true,"origin":"","legend":"\u003cp\u003eMasson staining of the three groups (400x light microscopy): the combined group had the least amount of collagen fibers compared to the other three groups. The hyaluronic acid and tacrolimus groups had more collagen fibers than the combined group collagen fiber content. The control group had the most collagen content. (A Combined group ; B Tacrolimus group . ; C. Hyaluronic acid group; D. Control group)\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9261730/v1/8f008edc905e0c8b4f8ede67.jpeg"},{"id":108494478,"identity":"1c388548-d204-4ed5-a779-c421cc8c699a","added_by":"auto","created_at":"2026-05-05 10:05:34","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":962062,"visible":true,"origin":"","legend":"\u003cp\u003eToluidine blue staining results of the middle section of the nerve three months after allogeneic nerve grafting (400x light microscope, field size: 430x320um2 ). The highest number of myelinated nerve fibers in the cross-section was found in the combined group. The tacrolimus group had a higher number of myelinated nerve fibers in the cross section. The hyaluronic acid group had a higher number of myelinated nerve fibers in the cross-section. The control group had the lowest number of myelinated nerve fibers in the cross section. (A. Combined group; B. Tacrolimus group; C. Hyaluronic acid group; D. Control group)\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9261730/v1/fd1eade9cd3683d2d474fb42.jpeg"},{"id":108804037,"identity":"607cc967-35f3-43ad-9998-c4ac39bd6bff","added_by":"auto","created_at":"2026-05-08 15:14:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1550174,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9261730/v1/517cae5e-61e0-4a4d-8e2d-a5fa30e8d29d.pdf"},{"id":108494663,"identity":"b402dfec-76d8-4e10-b3b0-d7866330ed74","added_by":"auto","created_at":"2026-05-05 10:06:22","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":95102,"visible":true,"origin":"","legend":"","description":"","filename":"3.30table.docx","url":"https://assets-eu.researchsquare.com/files/rs-9261730/v1/c91960a652e0ef25a89db4d1.docx"},{"id":108493684,"identity":"d02bbead-7d33-4894-a91c-12482b269f00","added_by":"auto","created_at":"2026-05-05 10:01:17","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":6694557,"visible":true,"origin":"","legend":"","description":"","filename":"fulluncroppedGelsandBlotsimages.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9261730/v1/7e93bf636916eb0eb72df769.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"A study on tacrolimus combined with hyaluronic acid gel to inhibit scar proliferation at nerve anastomoses","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRepair of peripheral nerve injury is highly challenging, and autologous nerve transplantation is considered the gold standard for repair of peripheral nerve injury \u003csup\u003e[\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Nerve transplantation mainly addresses two major difficulties:1.How to completely remove the major histocompatibility complex (MHC) from allogeneic nerves, and 2. How to reduce perineural scar formation after transplantation to promote nerve regeneration. Autologous nerve transplantation contains autologous Schwann cells, native extracellular matrix and a variety of neurotrophic factors, and is currently widely used in the treatment of peripheral nerve injury with good therapeutic effects\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. However, this method still has many limitations in practical application\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Allogeneic nerve transplantation can well compensate for the defects of autologous nerve transplantation and is an alternative material for autologous nerve transplantation\u003csup\u003e[\u003cspan additionalcitationids=\"CR7 CR8 CR9 CR10 CR11 CR12 CR13\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Existing studies have confirmed that nerve scar formation is an important inhibitory factor in the process of nerve regeneration. Once new nerve axons encounter obstacles such as inflammatory granulation tissue or collagen scar tissue, it may lead to the failure of nerve repair\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. In recent years, numerous studies have reported the properties of the local immunosuppressant tacrolimus (FK506) and synthetic hyaluronic acid, and both have been applied in multiple fields with good results\u003csup\u003e[\u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. In this study, FK506 and hyaluronic acid (HA) were selected as research factors to investigate the effect of their combined application on inhibiting scar growth around the nerve anastomosis.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e1.1 Design: A randomized controlled animal experiment.\u003c/p\u003e\n\u003cp\u003e1.2 Time and location: The experiment was completed at the Medical Research Center of Mudanjiang Medical University from November 2021 to April 2023.\u003c/p\u003e\n\u003cp\u003e1.3 Materials\u003c/p\u003e\n\u003cp\u003e1.3.1 Experimental animals: Forty healthy adult male Sprague-Dawley rats (divided into 4 groups) weighing 250\u0026ndash;350 g were selected and provided by the Laboratory of Mudanjiang Medical University. The disposal of animals during the experiment complied with animal ethics requirements.\u003c/p\u003e\n\u003cp\u003e1.3.2 Main experimental reagents and instruments: Tacrolimus (25 mg), purchased from Shanghai Beyotime Biotechnology Co., Ltd.; Hyaluronic acid (1 g), purchased from Shanghai Macklin Biochemical Technology Co., Ltd.; Masson trichrome staining kit, purchased from Fuzhou Maixin Biotech Co., Ltd.; PBS phosphate buffer dry powder (for 2 L preparation, 500 mL specification), purchased from Aladdin; Beijing Solarbio Science \u0026amp; Technology Co., Ltd.; Upright microscope, purchased from Leica Visualization Systems (Shanghai) Co., Ltd.; Image analysis system (CIAS-1000), purchased from Beijing Daheng Image Vision Co., Ltd.; Paraffin microtome (RM2235), purchased from Leica Camera AG, Germany.\u003c/p\u003e\n\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e1.4 Research Methods\u003c/h2\u003e\n \u003cp\u003e1.4.1 Experimental animals and grouping: Sixty healthy adult male Sprague-Dawley (SD) rats weighing 250\u0026ndash;350 g were selected and provided by the Medical Research Center of Mudanjiang Medical University. Twenty of them were used as nerve donors (a total of 40 sciatic nerves were harvested); the remaining 40 rats were randomly divided into 4 groups with 10 rats in each group. The grouping was as follows: control group (simple acellular allogeneic nerve transplantation), hyaluronic acid group (HA group, HA was applied to the anastomotic stoma after acellular allogeneic nerve transplantation), tacrolimus group (FK506 group, FK506 was applied to the anastomotic stoma after acellular allogeneic nerve transplantation), oral tacrolimus group (oral administration of FK506) and combined group (HA gel containing FK506 was applied to the anastomotic stoma after acellular allogeneic nerve transplantation).\u003c/p\u003e\n \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e\n \u003ch2\u003e1.4.2 Collection of allogeneic nerves and preparation of acellular allogeneic nerves\u003c/h2\u003e\n \u003cp\u003eCollection of allogeneic nerves: Twenty healthy adult male SD rats were selected, and their bilateral sciatic nerves were used as donors for allogeneic nerve transplantation. SD rats were anesthetized by intraperitoneal injection of 3% pentobarbital sodium (30 mg/kg). A longitudinal incision was made on the dorsal side of both lower limbs under sterile conditions, the space between biceps femoris and vastus lateralis was bluntly separated to expose the sciatic nerve. A 15 mm-long sciatic nerve segment was sharply cut between the lower edge of pectineus and the bifurcation of the sciatic nerve. Fat and connective tissue on the peripheral surface of the nerve were removed, rinsed with sterile normal saline, and placed in a sterile culture dish for later use.\u003c/p\u003e\n \u003cp\u003ePreparation of acellular allogeneic nerves: All 30 donor allogeneic nerves were prepared using the decellularization method described by Sondell\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cspan\u003e1.4.3 Establishment of animal model: Forty SD rats were fasted for 4\u0026ndash;6 hours before surgery, and anesthetized by intraperitoneal injection of 3% pentobarbital sodium (30 mg/kg), then underwent allogeneic sciatic nerve transplantation. Under sterile conditions, a longitudinal incision was made along the course of the right lower extremity sciatic nerve, and the sciatic nerve was bluntly separated and freed along the intermuscular space. The sciatic nerve was sharply resected starting from the lower edge of the perineal muscle, and a 6-mm-long sciatic nerve segment was excised distally to form a 10-mm nerve defect. The sharply dissected 10-mm acellular allograft was anastomosed and transplanted to the nerve defect. Under an operating microscope, 9\u0026thinsp;\u0026minus;\u0026thinsp;0 non-invasive sutures were used, and 4 stitches were placed at four equally divided positions of the epineurium to complete the sciatic nerve anastomosis. In the HA group, a micro-syringe (10 \u0026micro;l) was used to inject HA gel at multiple points around the nerve anastomosis site via microinjection, with an average injection volume of about 15 \u0026micro;L\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e; in the FK506 group, FK506 solution was injected at the nerve anastomosis site\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e, with an injection volume of about 15 \u0026micro;l; in the combination group, HA gel containing FK506 was injected at the nerve anastomosis site, with an injection ratio of 1:1 (15 \u0026micro;l each of HA and FK506); the control group received no treatment. After completion of treatment in all groups, the incision was sutured layer by layer. Gentamicin (40 mg/ml) 1 ml was injected intramuscularly daily for 3 consecutive days to prevent infection. Animals were housed in individual cages with unrestricted activity.\u003cbr\u003e\u003c/span\u003e \u003cspan\u003e\n \u003cp\u003e1.4.4 Criteria for successful animal model establishment: reliable nerve anastomosis, no tension on the nerve after anastomosis, and no deep infection or abscess formation after surgery.\u003c/p\u003e\n \u003c/span\u003e\n \u003c/div\u003e\n \u003ch2\u003e1.5 Methods for implementing euthanasia:\u003c/h2\u003e\n \u003cp\u003eDrugs: Potassium chloride\u003c/p\u003e\n \u003cp\u003eAnesthesia: Induce deep anesthesia in the animal through inhalation anesthesia (isoflurane) to ensure the animal feels no pain.\u003c/p\u003e\n \u003cp\u003ePrecise injection: After opening the chest to expose the heart, use a fine needle to insert into the left ventricle or right atrium along the axis of the heart from the apex, and slowly inject the drug.\u003c/p\u003e\n \u003cp\u003eConfirm death: After injection, observe the cessation of the animal\u0026apos;s heartbeat and breathing, and dilation of the pupils to confirm death.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e1.6 Main outcome measures\u003c/h2\u003e\u003cspan\u003e1.6.1 Gross observation: Three months after surgery, the incision was opened under anesthesia, and whether there was bulging in the surgical area and the adhesion between the nerve graft segment and surrounding tissues were observed. Evaluation was performed according to the grading standard score of Peterson et al\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e, as detailed in Table\u0026nbsp;1.\u003cbr\u003e\u003c/span\u003e \u003cspan\u003e\n \u003cp\u003e1.6.2 Detection of epineurial scar: Masson trichrome staining: In Masson trichrome staining, cell nuclei are stained black-blue, collagen fibers are stained blue, and glial fibers, muscle fibers and red blood cells are stained red.\u003c/p\u003e\n \u003c/span\u003e \u003cspan\u003e\n \u003cp\u003e1.6.3 Analysis of the relative contents of type I and type III collagen: Specimens were collected 12 weeks after surgery, and detection was performed at the proximal nerve anastomosis. The immunohistochemical SABC method was used, and the operation procedure was performed in accordance with the reagent instructions. Images of type I and type III collagen were collected under a 100\u0026times; optical microscope, and image analysis was performed using the CIAS-1000 image analysis system. In a visual field at 640\u0026times; magnification, positive regions were selected with a mouse tool to measure the gray value. In this system, the gray value varies according to the staining depth and reaction intensity of positive regions. The gray value is divided into 256 levels in total, with level 0 having the darkest color indicating a strong positive reaction, and level 255 having the lightest color indicating a weak positive reaction. Collagen content is negatively correlated with the gray value.\u003c/p\u003e\n \u003c/span\u003e \u003cspan\u003e\n \u003cp\u003e1.6.4 Counting analysis of myelinated nerve fibers in the middle segment of the transplanted nerve: Three months after surgery, toluidine blue staining was used to observe the growth of myelinated nerve fibers in each group.\u003c/p\u003e\n \u003c/span\u003e \u003cspan\u003e\n \u003cp\u003e1.6.5 Statistical analysis: SPSS 25.0 statistical analysis software and GraphPad Prism 8.0 were used to perform statistical analysis on the data and generate graphs. Measurement data are expressed as (X\u0026thinsp;\u0026plusmn;\u0026thinsp;S). Grouped data were analyzed by one-way analysis of variance, and the q-test for multiple comparisons was performed on groups with statistical differences indicated by analysis of variance. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered a significant difference, and P\u0026thinsp;\u0026lt;\u0026thinsp;0.01 was considered a highly significant difference.\u003c/p\u003e\n \u003c/span\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e2.1 Quantitative analysis of experimental animals: A total of 40 SD rats were included and randomly divided into 4 groups, with 10 rats in each group. All rats were included in the result analysis, with no dropout.\u003c/p\u003e\n\u003cp\u003e2.2 Gross observation through surgical windowing: After anesthesia, all rats were incised to observe the adhesion between the nerve anastomosis and surrounding tissues. Postoperatively, the skin, muscles, and fascia of most SD rats in each group did not dehisce, and partial dehiscence of skin, muscles, and fascia occurred in a few SD rats, with no infection. Varying degrees of nerve adhesion existed in all 4 groups. The Peterson scores (data results are ranked categorical data) were analyzed for quantitative data using analysis of variance and q-test for multiple comparisons. The adhesion between nerve anastomosis and surrounding tissues was less in the combination group, tacrolimus group and hyaluronic acid group, while nerve adhesion was the most severe in the control group. The difference between the control group and the other three groups was statistically significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Multiple comparisons of the remaining indicators among the four groups showed no statistically significant difference among the four groups. The results of gross observation of Peterson scores in each group are shown in Table 2.\u003c/p\u003e\n\u003cp\u003e2.3 Masson staining: After Masson staining, observation under a 400\u0026times; light microscope revealed collagen deposition in the epineurium of the nerve anastomosis in all groups (see Fig. \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Figure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA shows that the combination group had the least collagen content, with tightly arranged fibers, small curling angles, and clear boundaries with surrounding tissues. Figure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB shows the tacrolimus group, which had less collagen fiber content with very tight arrangement. Figure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC shows that the hyaluronic acid group had a slightly higher content of collagen fibers in the epineurium, relatively sparse arrangement, larger curling angles of collagen fibers, and clear boundaries with surrounding tissues. Figure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD shows that the control group had the highest content of collagen fibers, disordered arrangement, and unclear boundaries with surrounding tissues.\u003c/p\u003e\n\u003cp\u003e2.4 Gray value analysis of relative contents of type Ⅰ and type Ⅲ collagen\u003c/p\u003e\n\u003cp\u003eTotal collagen fiber gray value: The difference between the combination group and the FK506 group and HA group was statistically significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and the difference between the combination group and the control group was highly statistically significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). There was no statistically significant difference between the FK506 group and the HA group, and both the FK506 group and the HA group showed statistically significant differences compared with the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Gray value of type Ⅰ collagen: The combination group, FK506 group and HA group all showed statistically significant differences compared with the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and there was no statistically significant difference between the FK506 group and the HA group. Gray value of type Ⅲ collagen: The combination group showed statistically significant differences compared with the FK506 group and HA group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and there was no statistically significant difference between the combination group and the control group; there was no statistically significant difference between the FK506 group and the HA group and the control group, and no statistically significant difference between the HA group and the control group. In conclusion, the amount of scar formation at the nerve anastomosis in the combination group was the least, which was better than the other three groups (FK506 group, HA group and control group), and the difference was statistically significant compared with the other three groups. The amount of scar formation at the nerve anastomosis in the FK506 group and HA group was less, which was better than that in the control group (the control group had the largest amount of scar formation), and both groups showed statistically significant differences compared with the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). There was no statistically significant difference in collagen content analysis between the FK506 group and the HA group. (The results of immunohistochemical gray analysis of type Ⅰ and type Ⅲ collagen are shown in Table 3.)\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e2.5 Analysis of counting results of myelinated nerve fibers in the middle segment of nerve grafts\u003c/h2\u003e\n \u003cp\u003eThree months after surgery, the number of myelinated nerve fibers in the middle segment of the grafted nerve in the combination group was (1145.8\u0026thinsp;\u0026plusmn;\u0026thinsp;29.6) per unit visual field, which was superior to that of the other three groups (tacrolimus group, hyaluronic acid group and control group). Compared with the tacrolimus group (894.2\u0026thinsp;\u0026plusmn;\u0026thinsp;46.1 myelinated nerve fibers per unit visual field in the middle segment of the grafted nerve), the hyaluronic acid group (892.1\u0026thinsp;\u0026plusmn;\u0026thinsp;37.3 myelinated nerve fibers per unit visual field in the middle segment of the grafted nerve), and the control group (697.6\u0026thinsp;\u0026plusmn;\u0026thinsp;29.6 myelinated nerve fibers per unit visual field in the middle segment of the grafted nerve), the differences were all statistically significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05); the difference between the combination group and the control group (697.6\u0026thinsp;\u0026plusmn;\u0026thinsp;21.7 myelinated nerve fibers per unit visual field) was highly statistically significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). There was no statistically significant difference in the number of myelinated nerve fibers in the middle segment of the grafted nerve between the tacrolimus group and the hyaluronic acid group, while the number of myelinated nerve fibers in the middle segment of the grafted nerve in both the tacrolimus group and the hyaluronic acid group was higher than that in the control group, with statistically significant differences (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). See Fig. \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Table 4.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePeripheral nerve injury is a common clinical condition. After peripheral nerve injury, there are many factors affecting nerve regeneration and repair, including scar formation, nerve anastomosis technique, and the microenvironment for nerve regeneration. Among these factors, scar is a key factor affecting nerve regeneration after nerve bridging. After scar formation, local tissue motor, sensory and information conduction functions are impaired, which will also lead to incomplete postoperative functional recovery in patients. In clinical practice, it is extremely common that patients with peripheral nerve injury experience incomplete postoperative functional recovery or even loss of function due to scar formation around the nerve. Therefore, inhibiting scar hyperplasia around the nerve anastomosis is crucial for nerve regeneration.\u003c/p\u003e \u003cp\u003eAfter body trauma, healing usually occurs through scar repair, but the specific repair mechanism of scar formation has not been fully elucidated and still requires in-depth research. Existing studies believe that the mechanism of scar repair is as follows: a series of inflammatory reactions (also known as inflammatory cascade reaction) occur rapidly at the injury site, and the inflammatory cascade reaction affects the transformation of fibroblasts and the synthesis and degradation of collagen fibers in the body\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. During this reaction, inflammatory cells aggregate and release inflammatory mediators, stimulating fibroblasts to synthesize a large amount of type I and type III collagen and matrix, leading to abnormal arrangement of collagen and matrix, prompting the tissue to repair the wound through scar healing to achieve the purpose of wound repair. With the progress of research, some researchers and scholars have put forward the conjecture whether wound healing can achieve scar-free healing. Researchers in this field have also carried out in-depth research on nerve injury repair, exploring whether minimal scarring or even scar-free repair can also be achieved during nerve defect repair. Therefore, researchers have tried to add a variety of different drugs and cytokines at the nerve anastomosis site to explore their effects on scar formation at the nerve anastomosis.\u003c/p\u003e \u003cp\u003eIn recent years, tacrolimus (FK506) and hyaluronic acid (HA) have often been used as research factors to explore their effects on peripheral nerve regeneration. Hyaluronic acid can inhibit scar growth through mechanisms such as inhibiting lymphocyte proliferation, phagocytosis, and hindering granulocyte movement\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Relevant research results show that hyaluronic acid can selectively stimulate fibroblasts to synthesize type III collagen, reduce the ratio of type I collagen to type III collagen, thereby reducing scar formation\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. Tacrolimus is a natural macrolide immunosuppressant, which can block the activity of immune cells and inhibit autoimmune reactions, thereby alleviating the symptoms of patients' autoimmune diseases. In clinical practice, tacrolimus has been widely used in many fields such as dermatology, ophthalmology, and orthopedics\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e, and has achieved good therapeutic effects. In addition, tacrolimus also has a significant curative effect in promoting the repair of nerve injury. Clear research conclusions show that the mechanism by which tacrolimus promotes nerve injury repair is as follows: tacrolimus mainly forms a relevant complex with FK506 binding protein 12 (FKBP12) through its binding region, then binds to the functional effector region, thereby up-regulating the expression of growth-associated protein-43 (GAP-43) and promoting the growth of nerve growth cones \u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e, exerting a neurotrophic effect.\u003c/p\u003e \u003cp\u003eAccording to the experimental results, we observed the following four characteristics: 1. In terms of Petersen scoring results, adhesion between the nerve anastomosis and surrounding tissues was the mildest in the combination group, mild in the FK506 group and HA group, and the most severe in the control group. Compared with the control group, the combination group, FK506 group and HA group all showed statistically significant differences in Petersen nerve adhesion scores (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while there were no statistically significant differences in Petersen scores for skin and myofascia among all groups. 2. Regarding the Masson staining results of total collagen fibers in the epineurium of the anastomosis, the combination group had the smallest total number of collagen fibers, with tightly arranged fibers, small curling angles, and clear boundaries with surrounding tissues. The FK506 group had a smaller total number of collagen fibers around the nerve, looser arrangement, larger curling angles of collagen fibers, and clear boundaries with surrounding tissues. The HA group had a smaller total number of collagen fibers with very dense arrangement. The control group had the largest total number of collagen fibers, disordered arrangement, and clear boundaries with surrounding tissues. 3. In the results of immunohistochemical analysis of type I and type III collagen, for the gray value of total collagen fibers: the combination group had the lowest gray value, the FK506 group had a lower gray value, the HA group had a slightly higher gray value than the FK506 group, and the control group had the highest gray value. The differences between the combination group and the FK506 group, and between the combination group and the HA group were both statistically significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and the difference between the combination group and the control group was statistically significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). There was no statistically significant difference between the FK506 group and the HA group, and both the FK506 group and the HA group showed statistically significant differences compared with the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). For the gray value of type I collagen: the combination group had the lowest gray value, the HA group had a lower gray value, the FK506 group had a slightly higher gray value than the HA group, and the control group had the highest gray value. The difference between the combination group and the FK506 group was statistically significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), the difference between the combination group and the HA group was statistically significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and the difference between the combination group and the control group was statistically significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01); there was no statistically significant difference between the FK506 group and the HA group, the difference between the FK506 group and the control group was statistically significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and the difference between the HA group and the control group was statistically significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). For the gray value of type III collagen: the combination group had the lowest collagen gray value, the control group had a lower gray value, the FK506 group had a slightly higher gray value than the control group, and the HA group had the highest gray value. The differences between the combination group and the FK506 group, and between the combination group and the HA group were both statistically significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while there was no statistically significant difference between the combination group and the control group; there were no statistically significant differences between the FK506 group and the HA group or the control group, and there was no statistically significant difference between the HA group and the control group. Previous studies have confirmed that local application of HA at the anastomosis of acellular allogeneic nerve grafts has significant effects in reducing adhesion between nerves and surrounding tissues and inhibiting scar formation around nerves\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e, and the same results were obtained in this experiment. The possible reasons for the above results in points 1, 2 and 3 are as follows: after repair of nerve defects with acellular allogeneic nerve transplantation, the addition of FK506-containing HA gel around the graft anastomosis, or the addition of FK506 or HA alone can all regulate the proportion of type I and type III collagen fibers, or reduce the total content of collagen fibers, and ultimately inhibit scar formation around the nerve anastomosis. Existing studies have shown\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e that HA can reduce scar formation by inhibiting the inflammatory reaction around the nerve anastomosis; the reason for this result in the FK506 group may be that FK506 binds to the FKBP12 receptor in the cytoplasm to form a functional complex, which blocks the dephosphorylation process of T cells, thereby inhibiting the expression of subsequent inflammatory factors (IL2, IL3, rINF) and reducing the aggregation of fibroblasts. The process of fibroblast transformation into scar tissue is thus inhibited, and an anti-scar effect is ultimately exerted\u003csup\u003e[\u003cspan additionalcitationids=\"CR33 CR34 CR35\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. The occurrence of this result in the combination group may be due to the additive or synergistic effect produced by the combined application of FK506 and HA. 4. Regarding the count of myelinated nerve fibers in the middle segment of the transplanted nerve: the combination group had the largest number of myelinated nerve fibers in the middle segment of the transplanted nerve, followed by the FK506 group, the HA group had fewer myelinated nerve fibers than the FK506 group, and the control group had the smallest number of myelinated nerve fibers. Compared with the FK506 group and the HA group, the combination group showed statistically significant differences in the total count of myelinated nerve fibers (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and the difference between the combination group and the control group was statistically significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The counts of myelinated nerve fibers in both the FK506 group and the HA group were higher than that in the control group, and the differences were both statistically significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while there was no statistically significant difference between the FK506 group and the HA group. The possible reason for the above result in point 4 is that after acellular allogeneic nerve transplantation, adding FK506-containing HA gel around the anastomosis, or adding FK506 or HA alone can all inhibit scar formation around the nerve anastomosis, reduce the barrier that affects the regeneration of myelinated nerve fibers, and thereby promote nerve regeneration.\u003c/p\u003e \u003cp\u003eIn conclusion, FK506-containing HA gel can inhibit scar growth around nerve anastomosis and promote nerve regeneration. However, the mechanisms underlying its inhibition of scar growth and promotion of nerve regeneration remain very complex. Whether these two effects are additive or synergistic in scar inhibition has not been clarified in this study. Therefore, further research is needed to elucidate the specific mechanism of scar inhibition, and it is also necessary to deeply explore whether HA and FK506 have a direct effect on promoting nerve regeneration.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eBased on the results of this study, we can draw the following conclusion: in acellular allogeneic nerve transplantation, the effect of FK506-containing HA gel on inhibiting scar formation is superior to that of single application of FK506 and single application of HA gel. The promoting effect of FK506-containing HA gel on nerve regeneration may be related to its ability to promote nerve regeneration by regulating the distribution of type I and type III collagen, reducing total collagen content, and decreasing perineural scar formation.\u003c/p\u003e"},{"header":"Declarations","content":" \u003ch2\u003eConflict of Interest\u003c/h2\u003e \u003cp\u003eAll authors declare that there is no conflict of interest in this study.\u003c/p\u003e \u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis study received no funding.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eDr. LX, and Dr. YN conceived and designed the research, drafted the initial manuscript, and reviewed and revised the manuscript. Dr.WL collected the data, performed a preliminary analysis. All authors have read and approved the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data generated or analysed during this study are included in this published article and its supplementary information files.\u003c/p\u003e\n\u003ch3\u003eEthical Compliance Statement\u003c/h3\u003e\n\u003cp\u003eEthical Requirements This study complies with all ethical Requirements. This study complies with all ethical requirements.\u003c/p\u003e \n"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePollins, A. C. et al. Mass spectrometric comparison of decellularization processes for nerve allotransplantation [J]. \u003cem\u003eJ. Mater. Science: Mater. Med.\u003c/em\u003e \u003cb\u003e28\u003c/b\u003e (1), 20 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarvalho, C. R. et al. Cell adhesion study of chitosan membranes for peripheral nerve regeneration [J]. \u003cem\u003eJ. Mater. Sci. Eng. C: Mater. Biol. 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(2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQue Jun. \u003cem\u003eStudy on the effect and mechanism of FK506 in promoting peripheral nerve regeneration by reducing scar formation [D]\u003c/em\u003e (Nanjing Medical University, 2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMekaj, A. Y. et al. Effects of hyaluronic acid and tacrolimus on the prevention of perineural scar formation and nerve regeneration after sciatic nerve repair in a rabbit model [J]. \u003cem\u003eEur. J. Trauma. Emerg. Surg. August\u003c/em\u003e. \u003cb\u003e43\u003c/b\u003e (4), 497\u0026ndash;504 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMekaj, A. Y. et al. Electrophysiological and functional evaluation of rabbit common peroneal nerve regeneration after local application of hyaluronic acid or tacrolimus following nerve repair. \u003cem\u003eNigerian J. Postgrad. Med.\u003c/em\u003e \u003cb\u003e22\u003c/b\u003e, 179\u0026ndash;184 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMekaj, A. Y. et al. Biomechanical properties of sciatic nerve after repair: the effect of local application of hyaluronic acid or tacrolimus. \u003cem\u003eInt. J. Clin. Exp. Med.\u003c/em\u003e \u003cb\u003e8\u003c/b\u003e, 20218\u0026ndash;20226 (2015).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 to 4 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"nerve transplantation, nerve repair, tacrolimus, hyaluronic acid nerve, scar tissue","lastPublishedDoi":"10.21203/rs.3.rs-9261730/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9261730/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cb\u003eObjective\u003c/b\u003e: To investigate the effect of additional application of hyaluronic acid (HA) gel containing tacrolimus (FK506) at the nerve anastomosis site after acute peripheral nerve injury treated with xenogeneic nerve transplantation on inhibiting the growth and formation of scar around the anastomosis.\u003c/p\u003e \u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e: A total of 60 healthy male Sprague-Dawley (SD) rats weighing 250 g to 350 g were selected. Among them, 20 rats were used as nerve donors, and the remaining 40 rats were randomly divided into 4 groups. The groups were as follows: combination group (acellular xenogeneic nerve transplantation\u0026thinsp;+\u0026thinsp;application of HA gel containing FK506 at the anastomosis site), tacrolimus group (FK506 group, acellular xenogeneic nerve transplantation\u0026thinsp;+\u0026thinsp;application of FK506 at the anastomosis site), hyaluronic acid group (HA group, acellular xenogeneic nerve transplantation\u0026thinsp;+\u0026thinsp;application of HA at the anastomosis site), and control group (only acellular xenogeneic nerve transplantation was performed). Experimental procedures: 1. Donor nerve sampling; 2. Establishment of sciatic nerve defect and nerve transplantation model. 3. Sampling and detection: Tissue samples were collected 12 weeks after surgery, and analyzed by Peterson score, Masson staining, immunohistochemical analysis of type I and type III collagen, and counting of myelinated nerve fibers in the middle segment of the transplanted nerve. SPSS 23.0 statistical software was used for data processing and analysis. This study has been approved by the Animal Experimental Ethics Committee of Mudanjiang Medical University (Approval No.202009015). Confirming that all experiments were performed in accordance with relevant guidelines and regulations.\u003c/p\u003e \u003cp\u003e \u003cb\u003eResults\u003c/b\u003e:The results of Peterson score, Masson staining, immunohistochemical analysis of type I and type III collagen, and counting of myelinated nerve fibers in the middle segment of the transplanted nerve showed that: 1. The degree of adhesion between the nerve anastomosis and surrounding tissue was lighter in the combination group, FK506 group and HA group, all of which were better than that in the control group, and the differences were statistically significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). 2. The amount of scar formation was the least in the combination group, intermediate in the FK506 group and HA group, and the largest in the control group. 3. The number of myelinated nerve fibers in the middle segment of the transplanted nerve in the combination group was higher than that in the other three groups (FK506 group, HA group, control group), and the difference between the combination group and the control group was highly statistically significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003e \u003cb\u003eConclusion\u003c/b\u003e:After acellular xenogeneic nerve transplantation, the effect of applying HA gel containing FK506 at the anastomosis site on inhibiting scar formation is better than that of applying FK506 alone or HA gel alone. HA gel containing FK506 can inhibit scar growth at the nerve anastomosis site and promote nerve regeneration.\u003c/p\u003e","manuscriptTitle":"A study on tacrolimus combined with hyaluronic acid gel to inhibit scar proliferation at nerve anastomoses","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-04 05:50:52","doi":"10.21203/rs.3.rs-9261730/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-02T12:07:09+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-30T13:59:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"289511877690940739313135797627665540822","date":"2026-04-23T02:49:30+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-21T12:43:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"191084152410753322749574771150323289506","date":"2026-04-21T09:44:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"113896174468971918751325363561697841990","date":"2026-04-21T06:40:19+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-20T22:12:33+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-20T22:08:53+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-04-20T19:08:03+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-17T03:42:31+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-04-17T03:37:29+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b9a746aa-d138-4dc9-a648-d736fadba5c2","owner":[],"postedDate":"May 4th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-02T12:07:09+00:00","index":49,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-30T13:59:34+00:00","index":48,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":67021048,"name":"Health sciences/Medical research"},{"id":67021049,"name":"Biological sciences/Neuroscience"}],"tags":[],"updatedAt":"2026-05-04T05:50:52+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-04 05:50:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9261730","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9261730","identity":"rs-9261730","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}