Effect of ACTH4-10Pro8-Gly9-Pro10 on anti-inflammatory cytokine (IL-4, IL-10, IL-13) expression in acute spinal cord injury models (male Sprague Dawley rats)

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Its final neurological outcome is determined by both primary and secondary injury processes. A key component of secondary injury mechanisms after initial trauma is neuroinflammation. A neuroprotective compound, ACTH4-10Pro8-Gly9-Pro10 (ACTH4-10) also known as semax, has shown neuroprotective and anti-inflammatory properties. ACTH4-10 has also been actively used in the treatment of brain ischemia without serious complication reported. Here, we analyzed the effects of ACTH4-10 at regulating the inflammatory cascade in SCI by looking at anti-inflammatory cytokine (IL-4, IL-10 and IL-13) levels after acute SCI. Method We carried out laminectomies in male Sprague Dawley rats at the second thoracic vertebrae. After laminectomy, we exposed the myelum and created mild SCI models with 20-g, and severe SCI with 35-g aneurysm clips. ACTH4-10 was administered intranasally to the treatment group and 0.9% NaCl to the control group (placebo). Both groups were kept alive and terminated at 3 and 6 hours. The tissue sample preparations were fixed in formalin and examined for immunohistochemistry. Quantitative measurement of the cytokines was done in the posterior horn area with specific associated anti-monoclonal antibodies. Results Rats with mild SCI that were given ACTH4-10 showed greater anti-inflammatory levels at 3 hours post-compression but only IL-10 and IL-13 were elevated significantly at 6 hours. Rats with severe compression in ACTH4-10 group showed greater levels of IL-10, IL-13 at 3 hours and IL-4, IL-10 at 6 hours compared with the placebo group. Conclusions Administration of ACTH4-10Pro8-Gly9-Pro10 intranasal can increase anti-inflammatory cytokine expression in Sprague Dawley rat models with mild and severe SCI. Expression of anti-inflammatory cytokines was greater in mild compression and 3-hour termination. Further research is needed to determine the optimal dose and clinical outcome in vivo. 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F1000Research 2025, 12 :194 ( https://doi.org/10.12688/f1000research.127413.2 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. Close Copy Citation Details Export Export Citation Sciwheel EndNote Ref. Manager Bibtex ProCite Sente EXPORT Select a format first Track Share ▬ ✚ Research Article Revised Effect of ACTH4-10Pro8-Gly9-Pro10 on anti-inflammatory cytokine (IL-4, IL-10, IL-13) expression in acute spinal cord injury models (male Sprague Dawley rats) [version 2; peer review: 2 approved, 1 not approved] Asadullah Asadullah https://orcid.org/0000-0002-9368-4717 1,2 , Abdul Hafid Bajamal 1 , Muhammad Arifin Parenrengi 1 , [...] Agus Turchan 1 , Budi Utomo https://orcid.org/0000-0001-6060-9190 3 , I Ketut Sudiana 4 , Eko Agus Subagio https://orcid.org/0000-0003-4526-0300 1 Asadullah Asadullah https://orcid.org/0000-0002-9368-4717 1,2 , Abdul Hafid Bajamal 1 , [...] Muhammad Arifin Parenrengi 1 , Agus Turchan 1 , Budi Utomo https://orcid.org/0000-0001-6060-9190 3 , I Ketut Sudiana 4 , Eko Agus Subagio https://orcid.org/0000-0003-4526-0300 1 PUBLISHED 22 Sep 2025 Author details Author details 1 Neurosurgery Department, Dr. Soetomo Hospital, Surabaya, East Java, 60286, Indonesia 2 Neurosurgery Residency Program, Airlangga University, Surabaya, East Java, 60132, Indonesia 3 Public Health and Preventive Medicine Department, Airlangga University, Surabaya, East Java, 60132, Indonesia 4 Anatomical Pathology Department, Airlangga University, Surabaya, East Java, 60132, Indonesia Asadullah Asadullah Roles: Conceptualization, Data Curation, Methodology, Project Administration, Resources, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing Abdul Hafid Bajamal Roles: Conceptualization, Methodology, Project Administration, Supervision, Validation, Writing – Original Draft Preparation, Writing – Review & Editing Muhammad Arifin Parenrengi Roles: Conceptualization, Methodology, Visualization, Writing – Review & Editing Agus Turchan Roles: Conceptualization, Methodology, Visualization, Writing – Review & Editing Budi Utomo Roles: Conceptualization, Methodology, Visualization, Writing – Review & Editing I Ketut Sudiana Roles: Conceptualization, Methodology, Visualization, Writing – Review & Editing Eko Agus Subagio Roles: Conceptualization, Methodology, Project Administration, Resources, Supervision, Validation, Writing – Review & Editing OPEN PEER REVIEW DETAILS REVIEWER STATUS This article is included in the Cell & Molecular Biology gateway. Abstract Background Spinal cord injury (SCI) is a damage to the spinal cord caused mainly by trauma resulting in major motor, sensory and autonomic dysfunctions. Its final neurological outcome is determined by both primary and secondary injury processes. A key component of secondary injury mechanisms after initial trauma is neuroinflammation. A neuroprotective compound, ACTH 4-10 Pro 8 -Gly 9 -Pro 10 (ACTH 4-10 ) also known as semax, has shown neuroprotective and anti-inflammatory properties. ACTH 4-10 has also been actively used in the treatment of brain ischemia without serious complication reported. Here, we analyzed the effects of ACTH 4-10 at regulating the inflammatory cascade in SCI by looking at anti-inflammatory cytokine (IL-4, IL-10 and IL-13) levels after acute SCI. Method We carried out laminectomies in male Sprague Dawley rats at the second thoracic vertebrae. After laminectomy, we exposed the myelum and created mild SCI models with 20-g, and severe SCI with 35-g aneurysm clips. ACTH 4-10 was administered intranasally to the treatment group and 0.9% NaCl to the control group (placebo). Both groups were kept alive and terminated at 3 and 6 hours. The tissue sample preparations were fixed in formalin and examined for immunohistochemistry. Quantitative measurement of the cytokines was done in the posterior horn area with specific associated anti-monoclonal antibodies. Results Rats with mild SCI that were given ACTH 4-10 showed greater anti-inflammatory levels at 3 hours post-compression but only IL-10 and IL-13 were elevated significantly at 6 hours. Rats with severe compression in ACTH 4-10 group showed greater levels of IL-10, IL-13 at 3 hours and IL-4, IL-10 at 6 hours compared with the placebo group. Conclusions Administration of ACTH 4-10 Pro 8 -Gly 9 -Pro 10 intranasal can increase anti-inflammatory cytokine expression in Sprague Dawley rat models with mild and severe SCI. Expression of anti-inflammatory cytokines was greater in mild compression and 3-hour termination. Further research is needed to determine the optimal dose and clinical outcome in vivo. READ ALL READ LESS Keywords Spinal Cord Injury, Anti inflammatory cytokine, Neuroprotector, Experimental, Laminectomy Corresponding Author(s) Eko Agus Subagio ( [email protected] ) Close Corresponding author: Eko Agus Subagio Competing interests: No competing interests were disclosed. Grant information: The author(s) declared that no grants were involved in supporting this work. Copyright: © 2025 Asadullah A et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. How to cite: Asadullah A, Bajamal AH, Parenrengi MA et al. Effect of ACTH4-10Pro8-Gly9-Pro10 on anti-inflammatory cytokine (IL-4, IL-10, IL-13) expression in acute spinal cord injury models (male Sprague Dawley rats) [version 2; peer review: 2 approved, 1 not approved] . F1000Research 2025, 12 :194 ( https://doi.org/10.12688/f1000research.127413.2 ) First published: 20 Feb 2023, 12 :194 ( https://doi.org/10.12688/f1000research.127413.1 ) Latest published: 22 Sep 2025, 12 :194 ( https://doi.org/10.12688/f1000research.127413.2 ) Revised Amendments from Version 1 Correction and addition of incorrect terms in the Semax dosage section (Researchers clarified that ACTH4-10Pro8-Gly9-Pro10 and semax were administered intranasally at a dose of 300 mcg/kg, sourced from PT Axomedica, one hour after laminectomy). Significant improvements were made to the visual presentation of results. Figure 1 now clearly shows myelum samples without ACTH4-10Pro8-Gly9-Pro10 administration to demonstrate injury severity differences1. New figures (Figures 2 and 3) display immunohistochemical staining for anti-inflammatory cytokines IL-4, IL-10, and IL-13 in both mild and severe SCI groups1. Statistical data previously presented in multiple tables was consolidated into a comprehensive ANOVA chart (Figure 4). In the Conclusion section, it is further explained that Administration of ACTH 4-10Pro 8-Gly 9-Pro 10 can increase anti-inflammatory cytokine levels in Sprague Dawley rat models with mild and severe SCI. The optimal effect of ACTH 4-10Pro 8-Gly 9-Pro 10 can increase anti-inflammatory cytokines occurs in the third hour after SCI injury. Correction and addition of incorrect terms in the Semax dosage section (Researchers clarified that ACTH4-10Pro8-Gly9-Pro10 and semax were administered intranasally at a dose of 300 mcg/kg, sourced from PT Axomedica, one hour after laminectomy). Significant improvements were made to the visual presentation of results. Figure 1 now clearly shows myelum samples without ACTH4-10Pro8-Gly9-Pro10 administration to demonstrate injury severity differences1. New figures (Figures 2 and 3) display immunohistochemical staining for anti-inflammatory cytokines IL-4, IL-10, and IL-13 in both mild and severe SCI groups1. Statistical data previously presented in multiple tables was consolidated into a comprehensive ANOVA chart (Figure 4). In the Conclusion section, it is further explained that Administration of ACTH 4-10Pro 8-Gly 9-Pro 10 can increase anti-inflammatory cytokine levels in Sprague Dawley rat models with mild and severe SCI. The optimal effect of ACTH 4-10Pro 8-Gly 9-Pro 10 can increase anti-inflammatory cytokines occurs in the third hour after SCI injury. See the authors' detailed response to the review by Mawaddah Ar Rochmah READ REVIEWER RESPONSES Introduction Spinal cord injury (SCI) is a pathological condition that is known to cause severe neurological deterioration resulting in physical dependency, morbidity, mortality and financial burden for decades. 1 Even tough significant research has been conducted to develop effective treatments for SCI, until now none of these treatments has shown any meaningful effect to improve functional outcome after injury. 2 Demographic data suggest that SCI mainly affects the male population with over 80% cases occurring in males between 25 to 45 year of age. 3 Interestingly, not only do males have a higher incidence rate of SCI, but some studies have also shown males tend to have worse recovery rates compared with females. 4 This phenomenon is thought to be caused by female sex hormone such as progesterone and estrogen, that have shown modest neuroprotective behaviour in brain injury. 5 In general, pathophysiology of traumatic SCI comprises primary and secondary injury. The primary injury is caused by the initial traumatic event such as compression, distraction, laceration and transection; this kind of injury causes irreversible damage to the spinal cord. In contrast, secondary injury, including processes such as neuroinflammation, spinal cord ischemia, and cellular excitotoxicity which begin soon after the initial trauma, give a window of opportunity to prevent further damage of the spinal cord. Therefore, managing secondary injuries remains the main target for SCI treatment. 6 , 7 Above all the potential threats from secondary SCI, neuroinflammation is thought to be the key component causing many local and systemic consequences. It is known that injury to the spinal cord initiates an inflammatory-mediated response that could alter the repair processes within the nervous system and cause more damage to the nerves. Therefore, managing inflammation is thought to be an important factor for optimizing the outcome of SCI. 8 Inflammation response in the spinal cord begins after macrophages activation and recruitment at the site of injury; activation of these macrophages could work in different pathways promoting, but also inhibiting the inflammation. Macrophages initiate inflammation by releasing pro-inflammatory cytokines (IL-1, IL-6, IL-8, IFN-γ and TNF-α) and other chemical mediators. After three or four days, macrophages that initiate and maintain the inflammation will then be deactivated by anti-inflammatory cytokines (IL-4, IL-10, IL-13 and TGF-β) that are also mainly produced by macrophages. The way that macrophages could regulate the inflammatory process is through the activation and counterbalance of pro- and anti-inflammatory cytokines. This balance determines the net effect of the inflammatory response in SCI. 9 , 10 ACTH 4-10 Pro 8 -Gly 9 -Pro 10 is an adrenocorticotropic hormone analogue that is known to have an anti-inflammatory effect. 11 It has also been used in many conditions such as stroke, traumatic brain injury and Alzheimer disease as a neuroprotective agent with no report of serious complications. 12 This experimental study was intended to show the effect of ACTH 4-10 administration on anti-inflammatory cytokine levels in the spinal cord after acute SCI. Methods In this study we used 12-week-old male Sprague Dawley rats weighing 250-300 g to reproduce homogenous sample for SCI models and eliminate confounding factor such as sex hormone. This animal study obtained ethical clearance by the Airlangga University animal care and use committee number 2.KE.094.07.2021 to ensure all effort was taken to reduce animal suffering. After randomization, 27 samples were divided into nine groups of three samples each. Laminectomy at the level of second thoracic vertebra was performed on all the samples. We took one group with the spinal cord left uninjured as a baseline control. The other samples became the treatment groups, in which SCI was performed by compressing the spine using an aneurysm clip in one minute with a clamping force of 20 g to reproduce mild SCI (four groups) and 35 g for severe SCI models (four groups). 13 The laminectomy site was then closed with sutures and the Sprague Dawley rats were kept alive. After closing the suture, all treatment groups were divided into two subgroups. Placebo control groups were given 0.9 % NaCl intranasally, while the positive treatment groups were given ACTH 4-10 Pro 8 -Gly 9 -Pro 10 intranasally at a 300 mg/kg dose. Each group was divided again into two groups to be respectively terminated at 3 hours and 6 hours after compression. Myelum transection was performed at the level of injured myelum after the termination. SCI myelum samples were fixed in 10% formalin and examined for IHC with specific monoclonal antibodies for each of IL-4, IL-10 and IL-13. Examination was done on the posterior horn of myelum to ensure homogenous sampling area in each group. Then anti-inflammatory cytokines were counted per 100 cells using associated anti-monoclonal antibodies and viewed with a light microscope at 1000× magnification. Cells with a brown colour in the cytoplasm showed positive expression of each anti-inflammatory cytokine. The result of cytokine level measurements in each group are shown in a relative expression graph. Normality data analysis was performed using a Shapiro-Wilk test. Normally distributed data were analyzed with ANOVA while the data with non-normal distribution were analyzed using a Kruskal-Wallis test, followed by a non-parametric Mann-Whitney U test. Results We carried out an experimental study in 27 male Sprague Dawley rats weighing 250-300 g, we severed the spinal cords in the area of injury, then we fixed the specimen with formalin. The specimen was then cut into 4-6 μm-thick slices with rotatory microtome and mounted on glass slides before further staining as seen in Figure 1 . Figure 1. Myelum sample from control group (A), mild spinal cord injury (SCI, B), severe SCI (C). In Figure 1 , the picture is without ACTH 4-10 Pro 8 -Gly 9 -Pro 10 administration after injury. This is expected so that the picture of myelum injury/damage looks different from mild to severe. After immunohistochemistry staining with each specific antibody we examined the posterior horn area and counted stained cell number per 100 cells with a light microscope at 1000× magnification. Positive cells appeared brown as seen in Figure 2 . Figure 2. Cells were counted to assess anti-inflammatory cytokine levels in SCI models. 37 Immunoreacted neutrophil cytoplasm that stained brown after using monoclonal antibodies are shown. Red arrows mark positive stained neutrophils for IL-4 (A), IL-10 (B) and IL-13 (C) in 3 hours observed mild spinal cord injury (SCI) group. Figure 3. Cells were counted to assess anti-inflammatory cytokine levels in SCI models. 37 Immunoreacted neutrophil cytoplasm that stained brown after using monoclonal antibodies are shown. Red arrows mark positive stained neutrophils for IL-4 (A), IL-10 (B) and IL-13 (C) in 3 hours observed severe spinal cord injury (SCI) group. The results Table 1 shows higher IL-4 levels in both mild and severe SCI models with ACTH 4-10 Pro 8 -Gly 9 -Pro 10 administration that were terminated after three and six hours, compared to the placebo group. Table 2 shows higher IL-10 levels in both mild and severe SCI models with ACTH 4-10 Pro 8 -Gly 9 -Pro 10 administration and who were terminated after three and six hours compared to the placebo group. Table 3 shows higher IL-13 levels in both mild and severe SCI models with ACTH 4-10 Pro 8 -Gly 9 -Pro 10 administration and who were terminates after three and six hours compared to the placebo group. Table 1. ANOVA study of IL-4 levels. SCI: spinal cord injury. Group IL-4 expression Mean Standard deviation p Control 5.96 2.52 0.005 * Mild SCI NaCl 0.9% 3 hours 6.29 3.40 Mild SCI NaCl 0.9% 6 hours 5.61 2.45 Mild SCI ACTH 4-10 3 hours 10.39 1.67 Mild SCI ACTH 4-10 6 hours 8.05 1.62 Control 5.75 2.20 0.001 * Severe SCI NaCl 0.9% 3 hours 7.32 2.26 Severe SCI NaCl 0.9% 6 hours 5.29 2.16 Severe SCI ACTH 4-10 3 hours 9.36 1.98 Severe SCI ACTH 4-10 6 hours 9.21 1.37 * p < 0.05. Table 2. ANOVA study of IL-10 levels. Group IL-10 expression Mean Standard deviation p Control 5.57 1.573 <0.001 * Mild SCI NaCl 0.9% 3 hours 6.86 1.848 Mild SCI NaCl 0.9% 6 hours 4.71 1.741 Mild SCI ACTH 4-10 3 hours 14.64 2.893 Mild SCI ACTH 4-10 6 hours 11.46 1.350 Control 5.36 1.587 Severe SCI NaCl 0.9% 3 hours 7.07 2.344 Severe SCI NaCl 0.9% 6 hours 5.71 1.629 Severe SCI ACTH 4-10 3 hours 13.25 3.086 Severe SCI ACTH 4-10 6 hours 9.54 2.138 * p < 0.05. Table 3. ANOVA study of IL-13 level. Group IL-13 expression Mean Standard deviation p Control 3.86 1.682 <0.001 * Mild SCI NaCl 0.9% 3 hours 7.86 2.445 Mild SCI NaCl 0.9% 6 hours 6.25 2.046 Mild SCI ACTH 4-10 3 hours 13.36 1.676 Mild SCI ACTH 4-10 6 hours 11.57 3.313 Control 4.50 1.893 Severe SCI NaCl 0.9% 3 hours 5.79 1.410 Severe SCI NaCl 0.9% 6 hours 5.68 1.718 Severe SCI ACTH 4-10 3 hours 10.18 3.570 Severe SCI ACTH 4-10 6 hours 9.00 1.893 * p < 0.05. Using Shapiro-Wilk test, we found our data had a normal distribution; further statistical analysis was carried out with a post hoc study using the Tukey method (Tables 4-9 in attachments). Mean levels of IL4, IL-10 and IL-13 were higher in the mild SCI models group that was given ACTH 4-10 Pro 8 -Gly 9 -Pro 10 for both three- and six-hour termination groups, compared with placebo. IL-14 levels in models given ACTH 4-10 Pro 8 -Gly 9 -Pro 10 at three and six hours were 10.39 and 8.05 respectively, and 6.29 and 5.61 respectively in the placebo group. IL-10 levels in ACTH 4-10 Pro 8 -Gly 9 -Pro 10 at three and six hours were 14.64 and 11.46 respectively, while in the placebo group they were 6.86 and 4.71 respectively. IL-13 levels in ACTH 4-10 Pro 8 -Gly 9 -Pro 10 at three and six hours were 13.36 and 11.57 respectively, and 7.86 and 6.25 respectively in the placebo group. Post hoc analysis with Tukey test showed significant differences in IL-4, IL-10 and IL-13 at three hours, and in IL-10 and IL-13 at six hours. Figure 4 show results from severe SCI models that were given ACTH 4-10 Pro 8 -Gly 9 -Pro 10 also showed higher mean IL-4, IL-10 and IL-13 levels compared with placebos in the three-hour and six-hour groups. IL-4 levels in ACTH 4-10 Pro 8 -Gly 9 -Pro 10 at three and six hours were 9.36 and 9.21 respectively, and 7.82 and 5.29 respectively in the placebo. IL-10 levels in ACTH 4-10 Pro 8 -Gly 9 -Pro 10 at three and six hourswas 13.25 and 9.54 respectively, while in placebos they were 7.07 and 5.71, respectively. IL-13 levels in models administered ACTH 4-10 Pro 8 -Gly 9 -Pro 10 at three and six hours were 10.18 and 9.00 respectively, while in placebo they were 5.79 and 5.68 respectively. Post hoc analysis with Tukey test showed significant differences in IL-10 and IL-13 levels at three hours, and in IL-4 and IL-10 at six hours. Overall, level of anti-inflammatory cytokines were higher in each treatment group compared with placebo; the levels were significantly higher in models with mild SCI and terminated three hours after injury. Figure 4. Chart of Annova Study. Discussion Inflammation following the primary spinal cord injury is known to cause further spinal cord damage, resulting in apoptosis of oligodendrocyte, demyelination and degradation of axons, and neuronal cell death. Many attempts were made to reduce this deleterious effect caused by inflammation after initial injury occurrs. 14 Neuroinflammation following the primary injury is predominantly regulated by the balance of macrophage activation. 15 In order to regulate this inflammation cascade, macrophages play a key role by releasing pro- or anti-inflammatory cytokine. Studies in SCI models have shown that some pro-inflammatory cytokines like TNF-α, IL-1, and IL-6 are found upregulated within an hour after injury, leading to further inflammatory response causing neuronal apoptosis. 16 In contrast, anti-inflammatory cytokines that are capable of altering macrophage activity, such as IL-4, IL-13 16 – 19 and IL-10 which can directly inhibiting other pro-inflammatory cytokines, are generally present at low levels after SCI. 20 , 21 We realize sex hormone like estrogen and progesterone have some immunosuppressive profile in SCI 22 ; to minimize this potentially confounding factor that might bias the outcome we only used male Sprague Dawley rats in this study. Our study found significantly higher IL-4, IL-10 and IL-13 levels in the mild SCI group that was terminated after three hours, and higher IL-10, IL-13 levels in the six-hour termination group that was given ACTH 4-10 . In the severe SCI group that were given ACTH 4-10 , we found higher IL-10, IL-13 levels in the group that was terminated after three hours, and higher IL-4 and IL-10 levels after six hours. Compared with mild SCI, in the severe SCI group inflammation was more pronounced and abundant in pro-inflammatory cytokines. These pro-inflammatory cytokines in turn could suppress the release of anti-inflammatory cytokines. In addition, inflammation that occurs in SCI rat models is known to begin one hour after injury and reach its peak at six hours after injury. 23 This is in accordance with our study, that the anti-inflammatory cytokine group from the mild or severe injury group showed an increase in the ratio of inflammatory cytokines at 3 to 6 hours. However, in the type of inflammatory cytokine IL-10, the peak cytokine ratio was at 3 hours, and decreased at 6 hours. The decrease in the inflammatory cytokine IL-10 at 6 hours is possibly due to a decrease in the influence of ACTH 4-10 Pro 8 -Gly 9 -Pro 10 at 6 hours. After the strongest influence to increase cytokines at 3 hours. ACTH 4-10 Pro 8 -Gly 9 -Pro 10 is an analogue of adrenocorticotropic hormone and is created from a fragment of ACTH(4–7) combined with C-terminal tripeptide Pro-Gly-Pro, which acts as a neuroprotective compound. 24 Similar to its analogue, ACTH 4-10 Pro 8 -Gly 9 -Pro 10 is said to bind at the melanocortin receptor promoting anti-inflammatory and neuroprotective effects. Activated melanocortin receptors could supress pro-inflammatory cytokine release and promote the anti-inflammatory cytokine release. Together, this condition could supress inflammation. 26 In another study, when compared with the administration of steroids, that Methylprednisolone could attenuate astrocyte cell death, decrease microglia activation, suppress A1 astrocytes activation, and promote functional recovery after acute SCI in mouse models. 25 Methylprednisolone sodium succinate (MP) became the most discussed treatment in the last three to four decades for reducing the deleterious effect caused by inflammation after initial injury occurs. The first study that was conducted in the early 1980s showed a promising potential neuroprotective effect of MP in reducing inflammation after acute SCI in a preclinical setting. This study has led many clinicians to do more extensive research (NASCIS I and II). 27 , 28 This large-scale randomized control trial showed that there was a statistical improvement in neurological outcomes in the treatment group compared with the placebo if MP was administered within less than eight hours after initial injury. 28 , 29 However, with an average increase of just five points in the American Spinal Injury Association (ASIA) impairment score compared with the placebo, this was said to not be clinically significant in the overall quality of life of the patient. Moreover, along with the limited overall improvement, a high dose of MP came with several complications, such as increased rate of infection along with gastrointestinal haemorrhages. 30 , 31 Considering the limited benefit and potential morbidity or even life-threatening complications, in 2013 the American Association of Neurological Surgeon (AANS) ruled out MP administration from the current recommendations for managing patients with SCI. 32 , 33 Our finding showing increased levels of anti-inflammatory cytokines in the treatment group complements previous studies that revealed ACTH 4-10 Pro 8 -Gly 9 -Pro 10 could decrease pro-inflammatory cytokines in SCI rats model. 34 , 35 Those studies might provide a fundamental basis showing ACTH 4-10 Pro 8 -Gly 9 -Pro 10 could reduce inflammation and potentially prevent secondary injury in SCI. To date, ACTH 4-10 Pro 8 -Gly 9 -Pro 10 has been used as a neuroprotective agent for stroke and Alzheimer patients with no report of serious complications. It has also been given through the intranasal route and has a rapid onset within minute, allowing this drug to be given early on after the initial injury. Finally, Increased anti-inflammatory mediators can be triggered by ACTH 4-10 Pro 8 -Gly 9 -Pro 10 , which is expected to suppress inflammatory events that can trigger further secondary injury. Conclusions Administration of ACTH 4-10 Pro 8 -Gly 9 -Pro 10 can increase anti-inflammatory cytokine levels in Sprague Dawley rat models with mild and severe SCI. The optimal effect of ACTH 4-10 Pro 8 -Gly 9 -Pro 10 can increase anti-inflammatory cytokine occurs in the third hour after SCI injury. Further studies are needed to determine the optimal dose and functional outcome in rats with SCI. Table 4. Post hoc study of IL-4 level in mild SCI. Group Mean Standard deviation Group Mean Standard deviation p Control 5.96 2.52 NaCl 0.9% 3 hours 6.29 3.40 0.999 NaCl 0.9% 6 hours 5.61 2.45 0.999 ACTH 4-10 3 hours 10.39 1.67 0.015 * ACTH 4-10 6 hours 8.05 1.62 0.510 NaCl 0.9% 3 hour 6.29 3.40 NaCl 0.9% 6 hours 5.61 2.45 0.984 ACTH 4-10 3 hours 10.39 1.67 0.027 * ACTH 4-10 6 hours 8.05 1.62 0.663 NaCl 0.9% 6 hour 5.61 2.45 ACTH 4-10 3 hours 10.39 1.67 0.007 * ACTH 4-10 6 hours 8.05 1.62 0.353 ACTH 4-10 3 hour 10.39 1.67 ACTH 4-10 6 hours 8.04 1.62 0.382 * p < 0.05. Table 5. Post hoc study of IL-10 level in mild SCI. Group Mean Standard deviation Group Mean Standard deviation p Control 5.57 1.573 NaCl 0.9% 3 hours 6.86 1.848 0.734 NaCl 0.9% 6 hours 4.71 1.741 0.922 ACTH 4-10 3 hours 14.64 2.893 <0.001 * ACTH 4-10 6 hours 11.46 1.350 <0.001 * NaCl 0.9% 3 hour 6.86 1.848 NaCl 0.9% 6 hours 4.71 1.741 0.268 ACTH 4-10 3 hours 14.64 2.893 <0.001 * ACTH 4-10 6 hours 11.46 1.350 <0.001 * NaCl 0.9% 6 hour 4.71 1.741 ACTH 4-10 3 hours 14.64 2.893 <0.001 * ACTH 4-10 6 hours 11.46 1.350 <0.001 * ACTH 4-10 3 hour 14.64 2.893 ACTH 4-10 6 hours 11.46 1.350 0.036 * * p < 0.05. Table 6. Post hoc study of IL-13 level in mild SCI. Group Mean Standard deviation Group Mean Standard deviation p Control 3.86 1.682 NaCl 0.9% 3 hours 7.86 2.445 0.023 * NaCl 0.9% 6 hours 6.25 2.046 0.322 ACTH 4-10 3 hours 13.36 1.676 <0.001 * ACTH 4-10 6 hours 11.57 3.313 <0.001 * NaCl 0.9% 3 hour 7.86 2.445 NaCl 0.9% 6 hours 6.25 2.046 0.694 ACTH 4-10 3 hours 13.36 1.676 0.001 * ACTH 4-10 6 hours 11.57 3.313 0.040 * NaCl 0.9% 6 hour 6.25 2.046 ACTH 4-10 3 hours 13.36 1.676 <0.001 * ACTH 4-10 6 hours 11.57 3.313 0.001 * ACTH 4-10 3 hour 13.36 1.676 ACTH 4-10 6 hours 11.57 3.313 0.605 * p < 0.05. Table 7. Post hoc study of IL-4 level in severe SCI. Group Mean Standard deviation Group Mean Standard deviation p Control 5.75 2.20 NaCl 0.9% 3 hours 7.32 2.26 0.600 NaCl 0.9% 6 hours 5.29 2.16 0.993 ACTH 4-10 3 hours 9.36 1.98 0.018 * ACTH 4-10 6 hours 9.21 1.37 0.025 * NaCl 0.9% 3 hour 7.32 2.26 NaCl 0.9% 6 hours 5.29 2.16 0.349 ACTH 4-10 3 hours 9.36 1.98 0.349 ACTH 4-10 6 hours 9.21 1.37 0.421 NaCl 0.9% 6 hour 5.29 2.16 ACTH 4-10 3 hours 9.36 1.98 0.006 * ACTH 4-10 6 hours 9.21 1.37 0.009 * ACTH 4-10 3 hour 9.36 1.98 ACTH 4-10 6 hours 9.21 1.37 1.000 * p < 0.05. Table 8. Post hoc study of IL-10 level in severe SCI. Group Mean Standard deviation Group Mean Standard deviation p Control 5.36 1.587 NaCl 0.9% 3 hours 7.07 2.344 0.607 NaCl 0.9% 6 hours 5.71 1.629 0.998 ACTH 4-10 3 hours 13.25 3.086 <0.001 * ACTH 4-10 6 hours 9.54 2.138 0.012 NaCl 0.9% 3 hour 7.07 2.344 NaCl 0.9% 6 hours 5.71 1.629 0.784 ACTH 4-10 3 hours 13.25 3.086 <0.001 * ACTH 4-10 6 hours 9.54 2.138 0.258 NaCl 0.9% 6 hour 5.71 1.629 ACTH 4-10 3 hours 13.25 3.086 <0.001 * ACTH 4-10 6 hours 9.54 2.138 0.024 * ACTH 4-10 3 hour 13.25 3.086 ACTH 4-10 6 hours 9.54 2.138 0.030 * * p < 0.05. Table 9. Post hoc study of IL-13 level in severe SCI. Group Mean Standard deviation Group Mean Standard deviation p Control 4.50 1.893 NaCl 0.9% 3 hours 5.79 1.410 0.816 NaCl 0.9% 6 hours 5.68 1.718 0.858 ACTH 4-10 3 hours 10.18 3.570 <0.001 ACTH 4-10 6 hours 9.00 1.893 0.006 * NaCl 0.9% 3 hour 5.79 1.410 NaCl 0.9% 6 hours 5.68 1.718 1.000 ACTH 4-10 3 hours 10.18 3.570 0.007 * ACTH 4-10 6 hours 9.00 1.893 0.078 NaCl 0.9% 6 hour 5.68 1.718 ACTH 4-10 3 hours 10.18 3.570 0.006 * ACTH 4-10 6 hours 9.00 1.893 0.064 ACTH 4-10 3 hour 10.18 3.570 ACTH 4-10 6 hours 9.00 1.893 0.858 * p < 0.05. Data availability Underlying data DRYAD: Effects of ACTH4-10Pro8-Gly9-Pro10 on anti-inflammatory cytokine (IL-4, IL-10, IL-13) expression in acute spinal cord injury model (Sprague Dawley rats), https://doi.org/10.5281/zenodo.7446188 . 36 Extended data Zenodo: Effects of ACTH4-10Pro8-Gly9-Pro10 on anti-inflammatory cytokine (IL-4, IL-10, IL-13) expression in acute spinal cord injury model (Sprague Dawley rats), https://doi.org/10.5281/zenodo.7568360 . 37 Reporting guidelines Zenodo: ARRIVE checklist for “Effect of ACTH4-10Pro8-Gly9-Pro10 on anti-inflammatory cytokine (IL-4, IL-10, IL-13) expression in acute spinal cord injury models (male Sprague Dawley rats)”, https://doi.org/10.5281/zenodo.7568360 . 37 Data are available under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0). References 1. Khorasanizadeh M, Yousefifard M, Eskian M, et al. : Neurological recovery following traumatic spinal cord injury: a systematic review and meta-analysis. 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Azzam M, Fahmi A, Utomo B, et al. : The Effect of ACTH(4-10) PRO8-GLY9-PRO10 Administration on the Expression of IL-6 and IL-8 in Sprague Dawley Mice with Spinal Cord Injury. J. Neurosci. Rural. Pract. 2022; 13 (3): 370–375. PubMed Abstract | Publisher Full Text | Free Full Text 35. Ardananurdin A, Subagio EA, Utomo B: Spinal Cord’ s IL-1, TNF-α and NF-κB Expression in Sprague-Dawley Rat on Acute Spinal Cord. Injury. 2020; 3 (3): 109–114. Publisher Full Text 36. Asadullah A, et al. : Effects of ACTH4-10Pro8-Gly9-Pro10 on anti-inflammatory cytokine (IL-4, IL-10, IL-13) expression in acute spinal cord injury model (Sprague Dawley rats). Dataset. Dryad. 2023. Publisher Full Text 37. Asadullah A, Subagio EA, Bajamal AH, et al. : Effects of ACTH4-10Pro8-Gly9-Pro10 on anti-inflammatory cytokine (IL-4, IL-10, IL-13) expression in acute spinal cord injury model (Sprague Dawley rats). Zenodo. 2023. Publisher Full Text Comments on this article Comments (0) Version 2 VERSION 2 PUBLISHED 20 Feb 2023 ADD YOUR COMMENT Comment Author details Author details 1 Neurosurgery Department, Dr. Soetomo Hospital, Surabaya, East Java, 60286, Indonesia 2 Neurosurgery Residency Program, Airlangga University, Surabaya, East Java, 60132, Indonesia 3 Public Health and Preventive Medicine Department, Airlangga University, Surabaya, East Java, 60132, Indonesia 4 Anatomical Pathology Department, Airlangga University, Surabaya, East Java, 60132, Indonesia Asadullah Asadullah Roles: Conceptualization, Data Curation, Methodology, Project Administration, Resources, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing Abdul Hafid Bajamal Roles: Conceptualization, Methodology, Project Administration, Supervision, Validation, Writing – Original Draft Preparation, Writing – Review & Editing Muhammad Arifin Parenrengi Roles: Conceptualization, Methodology, Visualization, Writing – Review & Editing Agus Turchan Roles: Conceptualization, Methodology, Visualization, Writing – Review & Editing Budi Utomo Roles: Conceptualization, Methodology, Visualization, Writing – Review & Editing I Ketut Sudiana Roles: Conceptualization, Methodology, Visualization, Writing – Review & Editing Eko Agus Subagio Roles: Conceptualization, Methodology, Project Administration, Resources, Supervision, Validation, Writing – Review & Editing Competing interests No competing interests were disclosed. Grant information The author(s) declared that no grants were involved in supporting this work. Article Versions (2) version 2 Revised Published: 22 Sep 2025, 12:194 https://doi.org/10.12688/f1000research.127413.2 version 1 Published: 20 Feb 2023, 12:194 https://doi.org/10.12688/f1000research.127413.1 Copyright © 2025 Asadullah A et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Download Export To Sciwheel Bibtex EndNote ProCite Ref. Manager (RIS) Sente metrics Views Downloads F1000Research - - PubMed Central info_outline Data from PMC are received and updated monthly. - - Citations open_in_new 0 open_in_new 0 open_in_new SEE MORE DETAILS CITE how to cite this article Asadullah A, Bajamal AH, Parenrengi MA et al. Effect of ACTH4-10Pro8-Gly9-Pro10 on anti-inflammatory cytokine (IL-4, IL-10, IL-13) expression in acute spinal cord injury models (male Sprague Dawley rats) [version 2; peer review: 2 approved, 1 not approved] . F1000Research 2025, 12 :194 ( https://doi.org/10.12688/f1000research.127413.2 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS track receive updates on this article Track an article to receive email alerts on any updates to this article. TRACK THIS ARTICLE Share Open Peer Review Current Reviewer Status: ? Key to Reviewer Statuses VIEW HIDE Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Version 2 VERSION 2 PUBLISHED 22 Sep 2025 Revised Views 0 Cite How to cite this report: Ar Rochmah M. Reviewer Report For: Effect of ACTH4-10Pro8-Gly9-Pro10 on anti-inflammatory cytokine (IL-4, IL-10, IL-13) expression in acute spinal cord injury models (male Sprague Dawley rats) [version 2; peer review: 2 approved, 1 not approved] . F1000Research 2025, 12 :194 ( https://doi.org/10.5256/f1000research.177886.r416146 ) The direct URL for this report is: https://f1000research.com/articles/12-194/v2#referee-response-416146 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 31 Oct 2025 Mawaddah Ar Rochmah , Department of Neurology Faculty of Medicine, Public Health, and Nursing, Universitas Gadjah Mada, Yogyakarta, Indonesia Approved VIEWS 0 https://doi.org/10.5256/f1000research.177886.r416146 The authors have made the ... Continue reading READ ALL The authors have made the necessary revisions in the current manuscript. Competing Interests: No competing interests were disclosed. Reviewer Expertise: Neurology, Genetic I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Ar Rochmah M. Reviewer Report For: Effect of ACTH4-10Pro8-Gly9-Pro10 on anti-inflammatory cytokine (IL-4, IL-10, IL-13) expression in acute spinal cord injury models (male Sprague Dawley rats) [version 2; peer review: 2 approved, 1 not approved] . F1000Research 2025, 12 :194 ( https://doi.org/10.5256/f1000research.177886.r416146 ) The direct URL for this report is: https://f1000research.com/articles/12-194/v2#referee-response-416146 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Yao X. Reviewer Report For: Effect of ACTH4-10Pro8-Gly9-Pro10 on anti-inflammatory cytokine (IL-4, IL-10, IL-13) expression in acute spinal cord injury models (male Sprague Dawley rats) [version 2; peer review: 2 approved, 1 not approved] . F1000Research 2025, 12 :194 ( https://doi.org/10.5256/f1000research.177886.r423811 ) The direct URL for this report is: https://f1000research.com/articles/12-194/v2#referee-response-423811 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 29 Oct 2025 Xue Yao , Tianjin Key Laboratory of Spine and Spinal Cord, Tianjin, China Not Approved VIEWS 0 https://doi.org/10.5256/f1000research.177886.r423811 This article focuses on the core link after spinal cord injury - neuroinflammation. It selects ACTH analogues with no serious complications reported and adopts the non-invasive, fast-acting intranasal administration method suitable for early intervention in acute injury, which has high ... Continue reading READ ALL This article focuses on the core link after spinal cord injury - neuroinflammation. It selects ACTH analogues with no serious complications reported and adopts the non-invasive, fast-acting intranasal administration method suitable for early intervention in acute injury, which has high clinical transformation feasibility. However, some issues may be addressed to improve the current manuscript. The preface section describes that the pro-inflammatory macrophages are inactivated by anti-inflammatory cytokines IL-4 and IL-10 three to four days after spinal cord injury. Why the experimental design does not choose to continue the repeated detection of IL-4, IL-10 and IL-13 until three to four days, but only selects 3 hours and 6 hours? It is suggested to provide supplementary explanations. Figure 1 is blurry and of poor quality. It is recommended to replace it with a clearer image. It is suggested that the tables in Table 1-3 be transformed into bar charts and placed on Figures 2 and 3, which will enhance the readability of the article for readers. In the sixth paragraph of the results section, the author mistakenly wrote IL4 as IL14. There was an error in the spelling of a proper noun. It is recommended that you read the entire text carefully and conduct a thorough review of the entire article. In the writing of research papers, * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001. It is recommended to provide supplementary statistical results for the tables in the article. The sample size of the experiment was relatively small, with only 3 rats in each group. Although the differences were confirmed to be significant through statistical tests, the stability and generalizability of the results require verification with a larger sample size. Without excluding the indirect influence of gender-related confounding factors, although male rats were used to avoid the interference of neuroprotective effects of estrogen and progesterone, the influence of gender differences on the ACTH 4-10 effect was not explored. The results are only applicable to the male model, and additional verification with female animals is required for clinical application. The mechanism of action has not been thoroughly investigated. It is only clear that ACTH 4-10 can upregulate anti-inflammatory factors, but the molecular mechanism has not been revealed: such as whether it functions by binding to the melanocyte-stimulating hormone receptor (MC receptor), whether it regulates downstream signaling pathways (such as NF-κB), whether the increase in anti-inflammatory factors is directly induced by the drug rather than a compensatory response after injury, etc. The mechanism analysis remains at the level of phenomenon description. Is the work clearly and accurately presented and does it cite the current literature? No Is the study design appropriate and is the work technically sound? No Are sufficient details of methods and analysis provided to allow replication by others? No If applicable, is the statistical analysis and its interpretation appropriate? No Are all the source data underlying the results available to ensure full reproducibility? No Are the conclusions drawn adequately supported by the results? No Competing Interests: No competing interests were disclosed. Reviewer Expertise: spinal cord injury I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Yao X. Reviewer Report For: Effect of ACTH4-10Pro8-Gly9-Pro10 on anti-inflammatory cytokine (IL-4, IL-10, IL-13) expression in acute spinal cord injury models (male Sprague Dawley rats) [version 2; peer review: 2 approved, 1 not approved] . F1000Research 2025, 12 :194 ( https://doi.org/10.5256/f1000research.177886.r423811 ) The direct URL for this report is: https://f1000research.com/articles/12-194/v2#referee-response-423811 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Gulsever CI. Reviewer Report For: Effect of ACTH4-10Pro8-Gly9-Pro10 on anti-inflammatory cytokine (IL-4, IL-10, IL-13) expression in acute spinal cord injury models (male Sprague Dawley rats) [version 2; peer review: 2 approved, 1 not approved] . F1000Research 2025, 12 :194 ( https://doi.org/10.5256/f1000research.177886.r423815 ) The direct URL for this report is: https://f1000research.com/articles/12-194/v2#referee-response-423815 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 25 Oct 2025 Cafer Ikbal Gulsever , İstanbul University, İstanbul, Turkey Approved VIEWS 0 https://doi.org/10.5256/f1000research.177886.r423815 Scientific merit: The study addresses a relevant and novel topic — the anti-inflammatory role of ACTH4-10Pro8-Gly9-Pro10 (Semax) in acute spinal cord injury. The experimental design using Sprague-Dawley rats is appropriate, and the methodology (clip-compression model, ... Continue reading READ ALL Scientific merit: The study addresses a relevant and novel topic — the anti-inflammatory role of ACTH4-10Pro8-Gly9-Pro10 (Semax) in acute spinal cord injury. The experimental design using Sprague-Dawley rats is appropriate, and the methodology (clip-compression model, immunohistochemistry, ANOVA analysis) is sound. Data and presentation: Figures and statistical results are clear and well-organized after revision. The inclusion of IL-4, IL-10, and IL-13 as markers strengthens the biological interpretation. Revisions adequately addressed: The authors have responded thoroughly to the previous reviewer’s comments—clarifying dosage, source of reagents, timing of administration, and improving figures and statistical visualization. Writing and clarity: The manuscript is generally well written. Minor linguistic polishing may further improve readability, but does not affect scientific quality. Recommendation: The article is scientifically solid, revisions are satisfactory, and data are consistent with conclusions. Recommendation: Accept (minor editorial corrections only). Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: Spinal cord injury, emergency neurosurgery I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Gulsever CI. Reviewer Report For: Effect of ACTH4-10Pro8-Gly9-Pro10 on anti-inflammatory cytokine (IL-4, IL-10, IL-13) expression in acute spinal cord injury models (male Sprague Dawley rats) [version 2; peer review: 2 approved, 1 not approved] . F1000Research 2025, 12 :194 ( https://doi.org/10.5256/f1000research.177886.r423815 ) The direct URL for this report is: https://f1000research.com/articles/12-194/v2#referee-response-423815 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Version 1 VERSION 1 PUBLISHED 20 Feb 2023 Views 0 Cite How to cite this report: Ar Rochmah M. Reviewer Report For: Effect of ACTH4-10Pro8-Gly9-Pro10 on anti-inflammatory cytokine (IL-4, IL-10, IL-13) expression in acute spinal cord injury models (male Sprague Dawley rats) [version 2; peer review: 2 approved, 1 not approved] . F1000Research 2025, 12 :194 ( https://doi.org/10.5256/f1000research.139917.r164062 ) The direct URL for this report is: https://f1000research.com/articles/12-194/v1#referee-response-164062 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 13 Mar 2023 Mawaddah Ar Rochmah , Department of Neurology Faculty of Medicine, Public Health, and Nursing, Universitas Gadjah Mada, Yogyakarta, Indonesia Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.139917.r164062 This study explored the use of ACTH4-10Pro8-Gly9-Pro10 in the rat model of spinal cord injury (SCI). The study focused on the role of the above-mentioned agent in ameliorating neuroinflammation within the acute phase of the SCI by measuring the inflammatory ... Continue reading READ ALL This study explored the use of ACTH4-10Pro8-Gly9-Pro10 in the rat model of spinal cord injury (SCI). The study focused on the role of the above-mentioned agent in ameliorating neuroinflammation within the acute phase of the SCI by measuring the inflammatory cytokines after the treatments. The study is novel and well-written. However, there are some issues that might be addressed to improve the current manuscript. The introduction is quite informative to provide the background of the study. However, in the introduction section, the authors are suggested to elaborate on the introduction of the ACTH4-10Pro8-Gly9-Pro10 in the treatment of SCI. There is information that the authors need to provide in the materials & methods section. In the methods section, it would be clearer if the authors completed the following pieces of information: Why was the ACTH4-10Pro8-Gly9-Pro10 intranasally at a 300 mg/kg dose chosen? Any previous study that suggested the dose? If any, please provide the reference and citation. How long was the period between treatment initiation after finishing the laminectomy procedure? Please provide the companies which produce the materials (reagents, software, etc) for the purpose of reproducibility. Please kindly provide the information if the measurement was performed at least in duple for each sample. These are some suggestions for the results section (including figures and tables): The authors are suggested to provide more description of Figure 1, either in Figure Legend of Figure 1 or as a narration in the text. Figure 1 is the evidence to differentiate the spinal cord in healthy, mild SCI, and severe SCI. Please provide the information on whether the histological figure of the spinal cord SCI groups was with or without the administration of ACTH4-10Pro8-Gly9-Pro10 intranasally. The authors need to provide the readers with what to expect in the microscopic changes of the spinal cord after mild and severe SCI. In Figure 2, the white arrows are not quite readily visible, the authors are suggested to pick a more contrasting color for the arrow. Why did the authors show only the mild SCI groups here? The authors are suggested to present at least one representative figure for each IHC staining for every group. Tables 1-3 are best presented in bar diagrams between groups in a figure. This will give the readers a better description and impression of the results of the study. In addition, the authors can explain more detailed data in the text. This will avoid the repetition of data explanation in figures/tables in the text. Tables 4-9, in my opinion, can be submitted as supplementary data. In the results section, the authors are suggested to explain the core findings of the posthoc analysis in the text. The authors are suggested to make sure that figures and tables are self-explanatory. Any repetition of the data from the figure/tables in the text should be avoided. In the discussion section, the authors have explained and compared the results of the study to other previous studies. Please kindly focus on the difference between the follow up period of 3 hrs and 6 hrs in the study, particularly in the level of the anti-inflammatory cytokines and the SCI severity. In my opinion, I found the fifth paragraph of this section a little odd since the authors did not mention that the study using methylprednisolone was performed in humans. If the authors would like to compare the efficacy of the currently used treatment of SCI, in this case, is methylprednisolone, to the agent used here in the study, I would prefer to use the studies with the same subjects (in this case, the authors should also add any study regarding the use of methylprednisolone treatment in rodents. The study in humans should come as an additional information), the same studied objects/variables (any agents that may increase or decrease anti-inflammatory cytokine in SCI, neurological deficit severity, etc), or other agents that have similar mechanisms of action (in this case, any other agent that binds to melanocortin receptor). In the last sentence of the discussion section, the authors mention “Finally, we see potential benefits from ACTH4-10Pro8- Gly9-Pro10 to reduce inflammation and its deleterious effect on secondary injury after SCI”. Please kindly provide the “deleterious effect of the SCI” that was studied here, since the anti-inflammatory cytokines studied here, in my understanding, are not the deleterious effects of the SCI. The authors are suggested to make the conclusion brief indicating that anti-inflammatory cytokines might be increased after the administration of ACTH4-10Pro8-Gly9-Pro10 intranasally in the acute SCI model. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: Neurology, Genetic I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Ar Rochmah M. Reviewer Report For: Effect of ACTH4-10Pro8-Gly9-Pro10 on anti-inflammatory cytokine (IL-4, IL-10, IL-13) expression in acute spinal cord injury models (male Sprague Dawley rats) [version 2; peer review: 2 approved, 1 not approved] . F1000Research 2025, 12 :194 ( https://doi.org/10.5256/f1000research.139917.r164062 ) The direct URL for this report is: https://f1000research.com/articles/12-194/v1#referee-response-164062 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 08 Oct 2024 Asadullah Asadullah , Neurosurgery Residency Program, Airlangga University, Surabaya, 60132, Indonesia 08 Oct 2024 Author Response Why was the ACTH4-10Pro8-Gly9-Pro10 intranasally at a 300 mg/kg dose chosen? Any previous study that suggested the dose? If any, please provide the reference and citation. >This is ... Continue reading Why was the ACTH4-10Pro8-Gly9-Pro10 intranasally at a 300 mg/kg dose chosen? Any previous study that suggested the dose? If any, please provide the reference and citation. >This is previous study https://www.scirp.org/journal/paperinformation.aspx?paperid=40560 . Dose is 300 mcg / kg. not mg. thank you. How long was the period between treatment initiation after finishing the laminectomy procedure? > administration of drugs after 1 hour of laminectomy and spinal cord clip with aneurysm clip to reproduce spinal cord injury. Please provide the companies which produce the materials (reagents, software, etc) for the purpose of reproducibility. > The medicine is from PT Axomedica with the trade name Semax Why was the ACTH4-10Pro8-Gly9-Pro10 intranasally at a 300 mg/kg dose chosen? Any previous study that suggested the dose? If any, please provide the reference and citation. >This is previous study https://www.scirp.org/journal/paperinformation.aspx?paperid=40560 . Dose is 300 mcg / kg. not mg. thank you. How long was the period between treatment initiation after finishing the laminectomy procedure? > administration of drugs after 1 hour of laminectomy and spinal cord clip with aneurysm clip to reproduce spinal cord injury. Please provide the companies which produce the materials (reagents, software, etc) for the purpose of reproducibility. > The medicine is from PT Axomedica with the trade name Semax Competing Interests: The authors declare that they have no conflicts of interest. Close Report a concern Author Response 10 Sep 2025 Asadullah Asadullah , Neurosurgery Residency Program, Airlangga University, Surabaya, 60132, Indonesia 10 Sep 2025 Author Response Note Of revision: Revision on Method: • Why was the ACTH4-10Pro8-Gly9-Pro10 intranasally at a 300 mg/kg dose chosen? Any previous study that suggested the dose? If any, please provide ... Continue reading Note Of revision: Revision on Method: • Why was the ACTH4-10Pro8-Gly9-Pro10 intranasally at a 300 mg/kg dose chosen? Any previous study that suggested the dose? If any, please provide the reference and citation. Rev: After closing the suture, all treatment groups were divided into two subgroups. Placebo control groups were given 0.9 % NaCl intranasally, while the positive treatment groups were given ACTH4-10Pro 8-Gly 9-Pro 10 or semax (The medicine is from PT Axomedica) intranasally at a 300 mcg/kg dose.38 38. Kolomin, T., Shadrina, M., Slominsky, P., Limborska, S., & Myasoedov, N. (2013). A New Generation of Drugs: Synthetic Peptides Based on Natural Regulatory Peptides. Neuroscience and Medicine, 04(04), 223–252. https://doi.org/10.4236/nm.2013.44035 • How long was the period between treatment initiation after finishing the laminectomy procedure? Rev: Administration of semax after 1 hour of laminectomy and spinal cord clip with aneurysm clip to reproduce spinal cord injury. • Please provide the companies which produce the materials (reagents, software, etc) for the purpose of reproducibility. Rev: The medicine is from PT Axomedica with the trade name Semax • Please kindly provide the information if the measurement was performed at least in duple for each sample. Rev: Each group was divided again into two groups to be respectively terminated at 3 hours and 6 hours after compression. Myelum transection was performed at the level of injured myelum after the termination. These are some suggestions for the results section (including figures and tables): • The authors are suggested to provide more description of Figure 1, either in Figure Legend of Figure 1 or as a narration in the text. Figure 1 is the evidence to differentiate the spinal cord in healthy, mild SCI, and severe SCI. Please provide the information on whether the histological figure of the spinal cord SCI groups was with or without the administration of ACTH4-10Pro8-Gly9-Pro10 intranasally. The authors need to provide the readers with what to expect in the microscopic changes of the spinal cord after mild and severe SCI. link of figure 1 : https://drive.google.com/file/d/1dIOgMAQP9lsSUdSw8y3vYRLLfnjYeIGs/view?usp=sharing Rev: In Figure 1, the picture is without ACTH4-10Pro8-Gly9-Pro10 administration after injury. This is expected so that the picture of myelum injury/damage looks different from mild to severe. link of figure 1 : https://drive.google.com/file/d/1dIOgMAQP9lsSUdSw8y3vYRLLfnjYeIGs/view?usp=sharing Rev: Figure 2. Cells were counted to assess anti-inflammatory cytokine levels in SCI models. 37 Immunoreacted neutrophil cytoplasm that stained brown after using monoclonal antibodies are shown. White arrows mark positive stained neutrophils for IL-4 (A), IL-10 (B) and IL-13 (C) in 3 hours observed mild spinal cord injury (SCI) group. Link of figure 2: https://drive.google.com/file/d/1zxPR-is_fgwmhZ_BKLKsHjCAd8jgLaAs/view?usp=sharing Figure 3. Cells were counted to assess anti-inflammatory cytokine levels in SCI models. 37 Immunoreacted neutrophil cytoplasm that stained brown after using monoclonal antibodies are shown. White arrows mark positive stained neutrophils for IL-4 (A), IL-10 (B) and IL-13 (C) in 3 hours observed severe spinal cord injury (SCI) group. LInk of figure 3: https://drive.google.com/file/d/10G-8kJvhFzgFQYzp7uQOwu4CnPNmLv8B/view?usp=sharing • Tables 1-3 are best presented in bar diagrams between groups in a figure. This will give the readers a better description and impression of the results of the study. In addition, the authors can explain more detailed data in the text. This will avoid the repetition of data explanation in figures/tables in the text. Rev: Figure 4. Chart Of Annova Study link of figure 4: https://drive.google.com/file/d/1DcLsen6EXCZBj-na9Wpg-qkHWc9gcv1r/view?usp=sharing • Tables 4-9, in my opinion, can be submitted as supplementary data. In the results section, the authors are suggested to explain the core findings of the posthoc analysis in the text. Rev : Done. Tables had deleted, and change as as supplementary data. • The authors are suggested to make sure that figures and tables are self-explanatory. Any repetition of the data from the figure/tables in the text should be avoided. Rev : Done • In the discussion section, the authors have explained and compared the results of the study to other previous studies. • Please kindly focus on the difference between the follow up period of 3 hrs and 6 hrs in the study, particularly in the level of the anti-inflammatory cytokines and the SCI severity. • In my opinion, I found the fifth paragraph of this section a little odd since the authors did not mention that the study using methylprednisolone was performed in humans. If the authors would like to compare the efficacy of the currently used treatment of SCI, in this case, is methylprednisolone, to the agent used here in the study, I would prefer to use the studies with the same subjects (in this case, the authors should also add any study regarding the use of methylprednisolone treatment in rodents. The study in humans should come as an additional information), the same studied objects/variables (any agents that may increase or decrease anti-inflammatory cytokine in SCI, neurological deficit severity, etc), or other agents that have similar mechanisms of action (in this case, any other agent that binds to melanocortin receptor). Rev: This is in accordance with our study, that the anti-inflammatory cytokine group from the mild or severe injury group showed an increase in the ratio of inflammatory cytokines at 3 to 6 hours. However, in the type of inflammatory cytokine IL-10, the peak cytokine ratio was at 3 hours, and decreased at 6 hours. The decrease in the inflammatory cytokine IL-10 at 6 hours is possibly due to a decrease in the influence of ACTH 4-10Pro 8-Gly 9-Pro 10 at 6 hours. After the strongest influence to increase cytokines at 3 hours. In another study, when compared with the administration of steroids, that Methylprednisolone could attenuate astrocyte cell death, decrease microglia activation, suppress A1 astrocytes activation, and promote functional recovery after acute SCI in mouse models. Finally, Increased anti-inflammatory mediators can be triggered by ACTH4-10Pro 8- Gly 9-Pro 10, which is expected to suppress inflammatory events that can trigger further secondary injury. • In the last sentence of the discussion section, the authors mention “Finally, we see potential benefits from ACTH4-10Pro8- Gly9-Pro10 to reduce inflammation and its deleterious effect on secondary injury after SCI”. Please kindly provide the “deleterious effect of the SCI” that was studied here, since the anti-inflammatory cytokines studied here, in my understanding, are not the deleterious effects of the SCI. Rev: Administration of ACTH 4-10Pro 8-Gly 9-Pro 10 can increase anti-inflammatory cytokine levels in Sprague Dawley rat models with mild and severe SCI. The optimal effect of ACTH 4-10Pro 8-Gly 9-Pro 10 can increase anti-inflammatory cytokine occurs in the third hour after SCI injury. Further studies are needed to determine the optimal dose and functional outcome in rats with SCI. Note Of revision: Revision on Method: • Why was the ACTH4-10Pro8-Gly9-Pro10 intranasally at a 300 mg/kg dose chosen? Any previous study that suggested the dose? If any, please provide the reference and citation. Rev: After closing the suture, all treatment groups were divided into two subgroups. Placebo control groups were given 0.9 % NaCl intranasally, while the positive treatment groups were given ACTH4-10Pro 8-Gly 9-Pro 10 or semax (The medicine is from PT Axomedica) intranasally at a 300 mcg/kg dose.38 38. Kolomin, T., Shadrina, M., Slominsky, P., Limborska, S., & Myasoedov, N. (2013). A New Generation of Drugs: Synthetic Peptides Based on Natural Regulatory Peptides. Neuroscience and Medicine, 04(04), 223–252. https://doi.org/10.4236/nm.2013.44035 • How long was the period between treatment initiation after finishing the laminectomy procedure? Rev: Administration of semax after 1 hour of laminectomy and spinal cord clip with aneurysm clip to reproduce spinal cord injury. • Please provide the companies which produce the materials (reagents, software, etc) for the purpose of reproducibility. Rev: The medicine is from PT Axomedica with the trade name Semax • Please kindly provide the information if the measurement was performed at least in duple for each sample. Rev: Each group was divided again into two groups to be respectively terminated at 3 hours and 6 hours after compression. Myelum transection was performed at the level of injured myelum after the termination. These are some suggestions for the results section (including figures and tables): • The authors are suggested to provide more description of Figure 1, either in Figure Legend of Figure 1 or as a narration in the text. Figure 1 is the evidence to differentiate the spinal cord in healthy, mild SCI, and severe SCI. Please provide the information on whether the histological figure of the spinal cord SCI groups was with or without the administration of ACTH4-10Pro8-Gly9-Pro10 intranasally. The authors need to provide the readers with what to expect in the microscopic changes of the spinal cord after mild and severe SCI. link of figure 1 : https://drive.google.com/file/d/1dIOgMAQP9lsSUdSw8y3vYRLLfnjYeIGs/view?usp=sharing Rev: In Figure 1, the picture is without ACTH4-10Pro8-Gly9-Pro10 administration after injury. This is expected so that the picture of myelum injury/damage looks different from mild to severe. link of figure 1 : https://drive.google.com/file/d/1dIOgMAQP9lsSUdSw8y3vYRLLfnjYeIGs/view?usp=sharing Rev: Figure 2. Cells were counted to assess anti-inflammatory cytokine levels in SCI models. 37 Immunoreacted neutrophil cytoplasm that stained brown after using monoclonal antibodies are shown. White arrows mark positive stained neutrophils for IL-4 (A), IL-10 (B) and IL-13 (C) in 3 hours observed mild spinal cord injury (SCI) group. Link of figure 2: https://drive.google.com/file/d/1zxPR-is_fgwmhZ_BKLKsHjCAd8jgLaAs/view?usp=sharing Figure 3. Cells were counted to assess anti-inflammatory cytokine levels in SCI models. 37 Immunoreacted neutrophil cytoplasm that stained brown after using monoclonal antibodies are shown. White arrows mark positive stained neutrophils for IL-4 (A), IL-10 (B) and IL-13 (C) in 3 hours observed severe spinal cord injury (SCI) group. LInk of figure 3: https://drive.google.com/file/d/10G-8kJvhFzgFQYzp7uQOwu4CnPNmLv8B/view?usp=sharing • Tables 1-3 are best presented in bar diagrams between groups in a figure. This will give the readers a better description and impression of the results of the study. In addition, the authors can explain more detailed data in the text. This will avoid the repetition of data explanation in figures/tables in the text. Rev: Figure 4. Chart Of Annova Study link of figure 4: https://drive.google.com/file/d/1DcLsen6EXCZBj-na9Wpg-qkHWc9gcv1r/view?usp=sharing • Tables 4-9, in my opinion, can be submitted as supplementary data. In the results section, the authors are suggested to explain the core findings of the posthoc analysis in the text. Rev : Done. Tables had deleted, and change as as supplementary data. • The authors are suggested to make sure that figures and tables are self-explanatory. Any repetition of the data from the figure/tables in the text should be avoided. Rev : Done • In the discussion section, the authors have explained and compared the results of the study to other previous studies. • Please kindly focus on the difference between the follow up period of 3 hrs and 6 hrs in the study, particularly in the level of the anti-inflammatory cytokines and the SCI severity. • In my opinion, I found the fifth paragraph of this section a little odd since the authors did not mention that the study using methylprednisolone was performed in humans. If the authors would like to compare the efficacy of the currently used treatment of SCI, in this case, is methylprednisolone, to the agent used here in the study, I would prefer to use the studies with the same subjects (in this case, the authors should also add any study regarding the use of methylprednisolone treatment in rodents. The study in humans should come as an additional information), the same studied objects/variables (any agents that may increase or decrease anti-inflammatory cytokine in SCI, neurological deficit severity, etc), or other agents that have similar mechanisms of action (in this case, any other agent that binds to melanocortin receptor). Rev: This is in accordance with our study, that the anti-inflammatory cytokine group from the mild or severe injury group showed an increase in the ratio of inflammatory cytokines at 3 to 6 hours. However, in the type of inflammatory cytokine IL-10, the peak cytokine ratio was at 3 hours, and decreased at 6 hours. The decrease in the inflammatory cytokine IL-10 at 6 hours is possibly due to a decrease in the influence of ACTH 4-10Pro 8-Gly 9-Pro 10 at 6 hours. After the strongest influence to increase cytokines at 3 hours. In another study, when compared with the administration of steroids, that Methylprednisolone could attenuate astrocyte cell death, decrease microglia activation, suppress A1 astrocytes activation, and promote functional recovery after acute SCI in mouse models. Finally, Increased anti-inflammatory mediators can be triggered by ACTH4-10Pro 8- Gly 9-Pro 10, which is expected to suppress inflammatory events that can trigger further secondary injury. • In the last sentence of the discussion section, the authors mention “Finally, we see potential benefits from ACTH4-10Pro8- Gly9-Pro10 to reduce inflammation and its deleterious effect on secondary injury after SCI”. Please kindly provide the “deleterious effect of the SCI” that was studied here, since the anti-inflammatory cytokines studied here, in my understanding, are not the deleterious effects of the SCI. Rev: Administration of ACTH 4-10Pro 8-Gly 9-Pro 10 can increase anti-inflammatory cytokine levels in Sprague Dawley rat models with mild and severe SCI. The optimal effect of ACTH 4-10Pro 8-Gly 9-Pro 10 can increase anti-inflammatory cytokine occurs in the third hour after SCI injury. Further studies are needed to determine the optimal dose and functional outcome in rats with SCI. Competing Interests: There is no competing interest Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 08 Oct 2024 Asadullah Asadullah , Neurosurgery Residency Program, Airlangga University, Surabaya, 60132, Indonesia 08 Oct 2024 Author Response Why was the ACTH4-10Pro8-Gly9-Pro10 intranasally at a 300 mg/kg dose chosen? Any previous study that suggested the dose? If any, please provide the reference and citation. >This is ... Continue reading Why was the ACTH4-10Pro8-Gly9-Pro10 intranasally at a 300 mg/kg dose chosen? Any previous study that suggested the dose? If any, please provide the reference and citation. >This is previous study https://www.scirp.org/journal/paperinformation.aspx?paperid=40560 . Dose is 300 mcg / kg. not mg. thank you. How long was the period between treatment initiation after finishing the laminectomy procedure? > administration of drugs after 1 hour of laminectomy and spinal cord clip with aneurysm clip to reproduce spinal cord injury. Please provide the companies which produce the materials (reagents, software, etc) for the purpose of reproducibility. > The medicine is from PT Axomedica with the trade name Semax Why was the ACTH4-10Pro8-Gly9-Pro10 intranasally at a 300 mg/kg dose chosen? Any previous study that suggested the dose? If any, please provide the reference and citation. >This is previous study https://www.scirp.org/journal/paperinformation.aspx?paperid=40560 . Dose is 300 mcg / kg. not mg. thank you. How long was the period between treatment initiation after finishing the laminectomy procedure? > administration of drugs after 1 hour of laminectomy and spinal cord clip with aneurysm clip to reproduce spinal cord injury. Please provide the companies which produce the materials (reagents, software, etc) for the purpose of reproducibility. > The medicine is from PT Axomedica with the trade name Semax Competing Interests: The authors declare that they have no conflicts of interest. Close Report a concern Author Response 10 Sep 2025 Asadullah Asadullah , Neurosurgery Residency Program, Airlangga University, Surabaya, 60132, Indonesia 10 Sep 2025 Author Response Note Of revision: Revision on Method: • Why was the ACTH4-10Pro8-Gly9-Pro10 intranasally at a 300 mg/kg dose chosen? Any previous study that suggested the dose? If any, please provide ... Continue reading Note Of revision: Revision on Method: • Why was the ACTH4-10Pro8-Gly9-Pro10 intranasally at a 300 mg/kg dose chosen? Any previous study that suggested the dose? If any, please provide the reference and citation. Rev: After closing the suture, all treatment groups were divided into two subgroups. Placebo control groups were given 0.9 % NaCl intranasally, while the positive treatment groups were given ACTH4-10Pro 8-Gly 9-Pro 10 or semax (The medicine is from PT Axomedica) intranasally at a 300 mcg/kg dose.38 38. Kolomin, T., Shadrina, M., Slominsky, P., Limborska, S., & Myasoedov, N. (2013). A New Generation of Drugs: Synthetic Peptides Based on Natural Regulatory Peptides. Neuroscience and Medicine, 04(04), 223–252. https://doi.org/10.4236/nm.2013.44035 • How long was the period between treatment initiation after finishing the laminectomy procedure? Rev: Administration of semax after 1 hour of laminectomy and spinal cord clip with aneurysm clip to reproduce spinal cord injury. • Please provide the companies which produce the materials (reagents, software, etc) for the purpose of reproducibility. Rev: The medicine is from PT Axomedica with the trade name Semax • Please kindly provide the information if the measurement was performed at least in duple for each sample. Rev: Each group was divided again into two groups to be respectively terminated at 3 hours and 6 hours after compression. Myelum transection was performed at the level of injured myelum after the termination. These are some suggestions for the results section (including figures and tables): • The authors are suggested to provide more description of Figure 1, either in Figure Legend of Figure 1 or as a narration in the text. Figure 1 is the evidence to differentiate the spinal cord in healthy, mild SCI, and severe SCI. Please provide the information on whether the histological figure of the spinal cord SCI groups was with or without the administration of ACTH4-10Pro8-Gly9-Pro10 intranasally. The authors need to provide the readers with what to expect in the microscopic changes of the spinal cord after mild and severe SCI. link of figure 1 : https://drive.google.com/file/d/1dIOgMAQP9lsSUdSw8y3vYRLLfnjYeIGs/view?usp=sharing Rev: In Figure 1, the picture is without ACTH4-10Pro8-Gly9-Pro10 administration after injury. This is expected so that the picture of myelum injury/damage looks different from mild to severe. link of figure 1 : https://drive.google.com/file/d/1dIOgMAQP9lsSUdSw8y3vYRLLfnjYeIGs/view?usp=sharing Rev: Figure 2. Cells were counted to assess anti-inflammatory cytokine levels in SCI models. 37 Immunoreacted neutrophil cytoplasm that stained brown after using monoclonal antibodies are shown. White arrows mark positive stained neutrophils for IL-4 (A), IL-10 (B) and IL-13 (C) in 3 hours observed mild spinal cord injury (SCI) group. Link of figure 2: https://drive.google.com/file/d/1zxPR-is_fgwmhZ_BKLKsHjCAd8jgLaAs/view?usp=sharing Figure 3. Cells were counted to assess anti-inflammatory cytokine levels in SCI models. 37 Immunoreacted neutrophil cytoplasm that stained brown after using monoclonal antibodies are shown. White arrows mark positive stained neutrophils for IL-4 (A), IL-10 (B) and IL-13 (C) in 3 hours observed severe spinal cord injury (SCI) group. LInk of figure 3: https://drive.google.com/file/d/10G-8kJvhFzgFQYzp7uQOwu4CnPNmLv8B/view?usp=sharing • Tables 1-3 are best presented in bar diagrams between groups in a figure. This will give the readers a better description and impression of the results of the study. In addition, the authors can explain more detailed data in the text. This will avoid the repetition of data explanation in figures/tables in the text. Rev: Figure 4. Chart Of Annova Study link of figure 4: https://drive.google.com/file/d/1DcLsen6EXCZBj-na9Wpg-qkHWc9gcv1r/view?usp=sharing • Tables 4-9, in my opinion, can be submitted as supplementary data. In the results section, the authors are suggested to explain the core findings of the posthoc analysis in the text. Rev : Done. Tables had deleted, and change as as supplementary data. • The authors are suggested to make sure that figures and tables are self-explanatory. Any repetition of the data from the figure/tables in the text should be avoided. Rev : Done • In the discussion section, the authors have explained and compared the results of the study to other previous studies. • Please kindly focus on the difference between the follow up period of 3 hrs and 6 hrs in the study, particularly in the level of the anti-inflammatory cytokines and the SCI severity. • In my opinion, I found the fifth paragraph of this section a little odd since the authors did not mention that the study using methylprednisolone was performed in humans. If the authors would like to compare the efficacy of the currently used treatment of SCI, in this case, is methylprednisolone, to the agent used here in the study, I would prefer to use the studies with the same subjects (in this case, the authors should also add any study regarding the use of methylprednisolone treatment in rodents. The study in humans should come as an additional information), the same studied objects/variables (any agents that may increase or decrease anti-inflammatory cytokine in SCI, neurological deficit severity, etc), or other agents that have similar mechanisms of action (in this case, any other agent that binds to melanocortin receptor). Rev: This is in accordance with our study, that the anti-inflammatory cytokine group from the mild or severe injury group showed an increase in the ratio of inflammatory cytokines at 3 to 6 hours. However, in the type of inflammatory cytokine IL-10, the peak cytokine ratio was at 3 hours, and decreased at 6 hours. The decrease in the inflammatory cytokine IL-10 at 6 hours is possibly due to a decrease in the influence of ACTH 4-10Pro 8-Gly 9-Pro 10 at 6 hours. After the strongest influence to increase cytokines at 3 hours. In another study, when compared with the administration of steroids, that Methylprednisolone could attenuate astrocyte cell death, decrease microglia activation, suppress A1 astrocytes activation, and promote functional recovery after acute SCI in mouse models. Finally, Increased anti-inflammatory mediators can be triggered by ACTH4-10Pro 8- Gly 9-Pro 10, which is expected to suppress inflammatory events that can trigger further secondary injury. • In the last sentence of the discussion section, the authors mention “Finally, we see potential benefits from ACTH4-10Pro8- Gly9-Pro10 to reduce inflammation and its deleterious effect on secondary injury after SCI”. Please kindly provide the “deleterious effect of the SCI” that was studied here, since the anti-inflammatory cytokines studied here, in my understanding, are not the deleterious effects of the SCI. Rev: Administration of ACTH 4-10Pro 8-Gly 9-Pro 10 can increase anti-inflammatory cytokine levels in Sprague Dawley rat models with mild and severe SCI. The optimal effect of ACTH 4-10Pro 8-Gly 9-Pro 10 can increase anti-inflammatory cytokine occurs in the third hour after SCI injury. Further studies are needed to determine the optimal dose and functional outcome in rats with SCI. Note Of revision: Revision on Method: • Why was the ACTH4-10Pro8-Gly9-Pro10 intranasally at a 300 mg/kg dose chosen? Any previous study that suggested the dose? If any, please provide the reference and citation. Rev: After closing the suture, all treatment groups were divided into two subgroups. Placebo control groups were given 0.9 % NaCl intranasally, while the positive treatment groups were given ACTH4-10Pro 8-Gly 9-Pro 10 or semax (The medicine is from PT Axomedica) intranasally at a 300 mcg/kg dose.38 38. Kolomin, T., Shadrina, M., Slominsky, P., Limborska, S., & Myasoedov, N. (2013). A New Generation of Drugs: Synthetic Peptides Based on Natural Regulatory Peptides. Neuroscience and Medicine, 04(04), 223–252. https://doi.org/10.4236/nm.2013.44035 • How long was the period between treatment initiation after finishing the laminectomy procedure? Rev: Administration of semax after 1 hour of laminectomy and spinal cord clip with aneurysm clip to reproduce spinal cord injury. • Please provide the companies which produce the materials (reagents, software, etc) for the purpose of reproducibility. Rev: The medicine is from PT Axomedica with the trade name Semax • Please kindly provide the information if the measurement was performed at least in duple for each sample. Rev: Each group was divided again into two groups to be respectively terminated at 3 hours and 6 hours after compression. Myelum transection was performed at the level of injured myelum after the termination. These are some suggestions for the results section (including figures and tables): • The authors are suggested to provide more description of Figure 1, either in Figure Legend of Figure 1 or as a narration in the text. Figure 1 is the evidence to differentiate the spinal cord in healthy, mild SCI, and severe SCI. Please provide the information on whether the histological figure of the spinal cord SCI groups was with or without the administration of ACTH4-10Pro8-Gly9-Pro10 intranasally. The authors need to provide the readers with what to expect in the microscopic changes of the spinal cord after mild and severe SCI. link of figure 1 : https://drive.google.com/file/d/1dIOgMAQP9lsSUdSw8y3vYRLLfnjYeIGs/view?usp=sharing Rev: In Figure 1, the picture is without ACTH4-10Pro8-Gly9-Pro10 administration after injury. This is expected so that the picture of myelum injury/damage looks different from mild to severe. link of figure 1 : https://drive.google.com/file/d/1dIOgMAQP9lsSUdSw8y3vYRLLfnjYeIGs/view?usp=sharing Rev: Figure 2. Cells were counted to assess anti-inflammatory cytokine levels in SCI models. 37 Immunoreacted neutrophil cytoplasm that stained brown after using monoclonal antibodies are shown. White arrows mark positive stained neutrophils for IL-4 (A), IL-10 (B) and IL-13 (C) in 3 hours observed mild spinal cord injury (SCI) group. Link of figure 2: https://drive.google.com/file/d/1zxPR-is_fgwmhZ_BKLKsHjCAd8jgLaAs/view?usp=sharing Figure 3. Cells were counted to assess anti-inflammatory cytokine levels in SCI models. 37 Immunoreacted neutrophil cytoplasm that stained brown after using monoclonal antibodies are shown. White arrows mark positive stained neutrophils for IL-4 (A), IL-10 (B) and IL-13 (C) in 3 hours observed severe spinal cord injury (SCI) group. LInk of figure 3: https://drive.google.com/file/d/10G-8kJvhFzgFQYzp7uQOwu4CnPNmLv8B/view?usp=sharing • Tables 1-3 are best presented in bar diagrams between groups in a figure. This will give the readers a better description and impression of the results of the study. In addition, the authors can explain more detailed data in the text. This will avoid the repetition of data explanation in figures/tables in the text. Rev: Figure 4. Chart Of Annova Study link of figure 4: https://drive.google.com/file/d/1DcLsen6EXCZBj-na9Wpg-qkHWc9gcv1r/view?usp=sharing • Tables 4-9, in my opinion, can be submitted as supplementary data. In the results section, the authors are suggested to explain the core findings of the posthoc analysis in the text. Rev : Done. Tables had deleted, and change as as supplementary data. • The authors are suggested to make sure that figures and tables are self-explanatory. Any repetition of the data from the figure/tables in the text should be avoided. Rev : Done • In the discussion section, the authors have explained and compared the results of the study to other previous studies. • Please kindly focus on the difference between the follow up period of 3 hrs and 6 hrs in the study, particularly in the level of the anti-inflammatory cytokines and the SCI severity. • In my opinion, I found the fifth paragraph of this section a little odd since the authors did not mention that the study using methylprednisolone was performed in humans. If the authors would like to compare the efficacy of the currently used treatment of SCI, in this case, is methylprednisolone, to the agent used here in the study, I would prefer to use the studies with the same subjects (in this case, the authors should also add any study regarding the use of methylprednisolone treatment in rodents. The study in humans should come as an additional information), the same studied objects/variables (any agents that may increase or decrease anti-inflammatory cytokine in SCI, neurological deficit severity, etc), or other agents that have similar mechanisms of action (in this case, any other agent that binds to melanocortin receptor). Rev: This is in accordance with our study, that the anti-inflammatory cytokine group from the mild or severe injury group showed an increase in the ratio of inflammatory cytokines at 3 to 6 hours. However, in the type of inflammatory cytokine IL-10, the peak cytokine ratio was at 3 hours, and decreased at 6 hours. The decrease in the inflammatory cytokine IL-10 at 6 hours is possibly due to a decrease in the influence of ACTH 4-10Pro 8-Gly 9-Pro 10 at 6 hours. After the strongest influence to increase cytokines at 3 hours. In another study, when compared with the administration of steroids, that Methylprednisolone could attenuate astrocyte cell death, decrease microglia activation, suppress A1 astrocytes activation, and promote functional recovery after acute SCI in mouse models. Finally, Increased anti-inflammatory mediators can be triggered by ACTH4-10Pro 8- Gly 9-Pro 10, which is expected to suppress inflammatory events that can trigger further secondary injury. • In the last sentence of the discussion section, the authors mention “Finally, we see potential benefits from ACTH4-10Pro8- Gly9-Pro10 to reduce inflammation and its deleterious effect on secondary injury after SCI”. Please kindly provide the “deleterious effect of the SCI” that was studied here, since the anti-inflammatory cytokines studied here, in my understanding, are not the deleterious effects of the SCI. Rev: Administration of ACTH 4-10Pro 8-Gly 9-Pro 10 can increase anti-inflammatory cytokine levels in Sprague Dawley rat models with mild and severe SCI. The optimal effect of ACTH 4-10Pro 8-Gly 9-Pro 10 can increase anti-inflammatory cytokine occurs in the third hour after SCI injury. Further studies are needed to determine the optimal dose and functional outcome in rats with SCI. Competing Interests: There is no competing interest Close Report a concern COMMENT ON THIS REPORT Comments on this article Comments (0) Version 2 VERSION 2 PUBLISHED 20 Feb 2023 ADD YOUR COMMENT Comment keyboard_arrow_left keyboard_arrow_right Open Peer Review Reviewer Status info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Reviewer Reports Invited Reviewers 1 2 3 Version 2 (revision) 22 Sep 25 read read read Version 1 20 Feb 23 read Mawaddah Ar Rochmah , Universitas Gadjah Mada, Yogyakarta, Indonesia Cafer Ikbal Gulsever , İstanbul University, İstanbul, Turkey Xue Yao , Tianjin Key Laboratory of Spine and Spinal Cord, Tianjin, China Comments on this article All Comments (0) Add a comment Sign up for content alerts Sign Up You are now signed up to receive this alert Browse by related subjects keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Ar Rochmah M. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 31 Oct 2025 | for Version 2 Mawaddah Ar Rochmah , Department of Neurology Faculty of Medicine, Public Health, and Nursing, Universitas Gadjah Mada, Yogyakarta, Indonesia 0 Views copyright © 2025 Ar Rochmah M. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions The authors have made the necessary revisions in the current manuscript. Competing Interests No competing interests were disclosed. Reviewer Expertise Neurology, Genetic I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (0) Ar Rochmah M. Peer Review Report For: Effect of ACTH4-10Pro8-Gly9-Pro10 on anti-inflammatory cytokine (IL-4, IL-10, IL-13) expression in acute spinal cord injury models (male Sprague Dawley rats) [version 2; peer review: 2 approved, 1 not approved] . F1000Research 2025, 12 :194 ( https://doi.org/10.5256/f1000research.177886.r416146) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/12-194/v2#referee-response-416146 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Yao X. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 29 Oct 2025 | for Version 2 Xue Yao , Tianjin Key Laboratory of Spine and Spinal Cord, Tianjin, China 0 Views copyright © 2025 Yao X. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Not Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions This article focuses on the core link after spinal cord injury - neuroinflammation. It selects ACTH analogues with no serious complications reported and adopts the non-invasive, fast-acting intranasal administration method suitable for early intervention in acute injury, which has high clinical transformation feasibility. However, some issues may be addressed to improve the current manuscript. The preface section describes that the pro-inflammatory macrophages are inactivated by anti-inflammatory cytokines IL-4 and IL-10 three to four days after spinal cord injury. Why the experimental design does not choose to continue the repeated detection of IL-4, IL-10 and IL-13 until three to four days, but only selects 3 hours and 6 hours? It is suggested to provide supplementary explanations. Figure 1 is blurry and of poor quality. It is recommended to replace it with a clearer image. It is suggested that the tables in Table 1-3 be transformed into bar charts and placed on Figures 2 and 3, which will enhance the readability of the article for readers. In the sixth paragraph of the results section, the author mistakenly wrote IL4 as IL14. There was an error in the spelling of a proper noun. It is recommended that you read the entire text carefully and conduct a thorough review of the entire article. In the writing of research papers, * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001. It is recommended to provide supplementary statistical results for the tables in the article. The sample size of the experiment was relatively small, with only 3 rats in each group. Although the differences were confirmed to be significant through statistical tests, the stability and generalizability of the results require verification with a larger sample size. Without excluding the indirect influence of gender-related confounding factors, although male rats were used to avoid the interference of neuroprotective effects of estrogen and progesterone, the influence of gender differences on the ACTH 4-10 effect was not explored. The results are only applicable to the male model, and additional verification with female animals is required for clinical application. The mechanism of action has not been thoroughly investigated. It is only clear that ACTH 4-10 can upregulate anti-inflammatory factors, but the molecular mechanism has not been revealed: such as whether it functions by binding to the melanocyte-stimulating hormone receptor (MC receptor), whether it regulates downstream signaling pathways (such as NF-κB), whether the increase in anti-inflammatory factors is directly induced by the drug rather than a compensatory response after injury, etc. The mechanism analysis remains at the level of phenomenon description. Is the work clearly and accurately presented and does it cite the current literature? No Is the study design appropriate and is the work technically sound? No Are sufficient details of methods and analysis provided to allow replication by others? No If applicable, is the statistical analysis and its interpretation appropriate? No Are all the source data underlying the results available to ensure full reproducibility? No Are the conclusions drawn adequately supported by the results? No Competing Interests No competing interests were disclosed. Reviewer Expertise spinal cord injury I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above. reply Respond to this report Responses (0) Yao X. Peer Review Report For: Effect of ACTH4-10Pro8-Gly9-Pro10 on anti-inflammatory cytokine (IL-4, IL-10, IL-13) expression in acute spinal cord injury models (male Sprague Dawley rats) [version 2; peer review: 2 approved, 1 not approved] . F1000Research 2025, 12 :194 ( https://doi.org/10.5256/f1000research.177886.r423811) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/12-194/v2#referee-response-423811 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Gulsever C. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 25 Oct 2025 | for Version 2 Cafer Ikbal Gulsever , İstanbul University, İstanbul, Turkey 0 Views copyright © 2025 Gulsever C. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Scientific merit: The study addresses a relevant and novel topic — the anti-inflammatory role of ACTH4-10Pro8-Gly9-Pro10 (Semax) in acute spinal cord injury. The experimental design using Sprague-Dawley rats is appropriate, and the methodology (clip-compression model, immunohistochemistry, ANOVA analysis) is sound. Data and presentation: Figures and statistical results are clear and well-organized after revision. The inclusion of IL-4, IL-10, and IL-13 as markers strengthens the biological interpretation. Revisions adequately addressed: The authors have responded thoroughly to the previous reviewer’s comments—clarifying dosage, source of reagents, timing of administration, and improving figures and statistical visualization. Writing and clarity: The manuscript is generally well written. Minor linguistic polishing may further improve readability, but does not affect scientific quality. Recommendation: The article is scientifically solid, revisions are satisfactory, and data are consistent with conclusions. Recommendation: Accept (minor editorial corrections only). Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests No competing interests were disclosed. Reviewer Expertise Spinal cord injury, emergency neurosurgery I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (0) Gulsever CI. Peer Review Report For: Effect of ACTH4-10Pro8-Gly9-Pro10 on anti-inflammatory cytokine (IL-4, IL-10, IL-13) expression in acute spinal cord injury models (male Sprague Dawley rats) [version 2; peer review: 2 approved, 1 not approved] . F1000Research 2025, 12 :194 ( https://doi.org/10.5256/f1000research.177886.r423815) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/12-194/v2#referee-response-423815 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2023 Ar Rochmah M. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 13 Mar 2023 | for Version 1 Mawaddah Ar Rochmah , Department of Neurology Faculty of Medicine, Public Health, and Nursing, Universitas Gadjah Mada, Yogyakarta, Indonesia 0 Views copyright © 2023 Ar Rochmah M. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (2) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions This study explored the use of ACTH4-10Pro8-Gly9-Pro10 in the rat model of spinal cord injury (SCI). The study focused on the role of the above-mentioned agent in ameliorating neuroinflammation within the acute phase of the SCI by measuring the inflammatory cytokines after the treatments. The study is novel and well-written. However, there are some issues that might be addressed to improve the current manuscript. The introduction is quite informative to provide the background of the study. However, in the introduction section, the authors are suggested to elaborate on the introduction of the ACTH4-10Pro8-Gly9-Pro10 in the treatment of SCI. There is information that the authors need to provide in the materials & methods section. In the methods section, it would be clearer if the authors completed the following pieces of information: Why was the ACTH4-10Pro8-Gly9-Pro10 intranasally at a 300 mg/kg dose chosen? Any previous study that suggested the dose? If any, please provide the reference and citation. How long was the period between treatment initiation after finishing the laminectomy procedure? Please provide the companies which produce the materials (reagents, software, etc) for the purpose of reproducibility. Please kindly provide the information if the measurement was performed at least in duple for each sample. These are some suggestions for the results section (including figures and tables): The authors are suggested to provide more description of Figure 1, either in Figure Legend of Figure 1 or as a narration in the text. Figure 1 is the evidence to differentiate the spinal cord in healthy, mild SCI, and severe SCI. Please provide the information on whether the histological figure of the spinal cord SCI groups was with or without the administration of ACTH4-10Pro8-Gly9-Pro10 intranasally. The authors need to provide the readers with what to expect in the microscopic changes of the spinal cord after mild and severe SCI. In Figure 2, the white arrows are not quite readily visible, the authors are suggested to pick a more contrasting color for the arrow. Why did the authors show only the mild SCI groups here? The authors are suggested to present at least one representative figure for each IHC staining for every group. Tables 1-3 are best presented in bar diagrams between groups in a figure. This will give the readers a better description and impression of the results of the study. In addition, the authors can explain more detailed data in the text. This will avoid the repetition of data explanation in figures/tables in the text. Tables 4-9, in my opinion, can be submitted as supplementary data. In the results section, the authors are suggested to explain the core findings of the posthoc analysis in the text. The authors are suggested to make sure that figures and tables are self-explanatory. Any repetition of the data from the figure/tables in the text should be avoided. In the discussion section, the authors have explained and compared the results of the study to other previous studies. Please kindly focus on the difference between the follow up period of 3 hrs and 6 hrs in the study, particularly in the level of the anti-inflammatory cytokines and the SCI severity. In my opinion, I found the fifth paragraph of this section a little odd since the authors did not mention that the study using methylprednisolone was performed in humans. If the authors would like to compare the efficacy of the currently used treatment of SCI, in this case, is methylprednisolone, to the agent used here in the study, I would prefer to use the studies with the same subjects (in this case, the authors should also add any study regarding the use of methylprednisolone treatment in rodents. The study in humans should come as an additional information), the same studied objects/variables (any agents that may increase or decrease anti-inflammatory cytokine in SCI, neurological deficit severity, etc), or other agents that have similar mechanisms of action (in this case, any other agent that binds to melanocortin receptor). In the last sentence of the discussion section, the authors mention “Finally, we see potential benefits from ACTH4-10Pro8- Gly9-Pro10 to reduce inflammation and its deleterious effect on secondary injury after SCI”. Please kindly provide the “deleterious effect of the SCI” that was studied here, since the anti-inflammatory cytokines studied here, in my understanding, are not the deleterious effects of the SCI. The authors are suggested to make the conclusion brief indicating that anti-inflammatory cytokines might be increased after the administration of ACTH4-10Pro8-Gly9-Pro10 intranasally in the acute SCI model. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? Yes Competing Interests No competing interests were disclosed. Reviewer Expertise Neurology, Genetic I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. reply Respond to this report Responses (2) Author Response 08 Oct 2024 Asadullah Asadullah, Neurosurgery Residency Program, Airlangga University, Surabaya, 60132, Indonesia Why was the ACTH4-10Pro8-Gly9-Pro10 intranasally at a 300 mg/kg dose chosen? Any previous study that suggested the dose? If any, please provide the reference and citation. >This is previous study https://www.scirp.org/journal/paperinformation.aspx?paperid=40560 . Dose is 300 mcg / kg. not mg. thank you. How long was the period between treatment initiation after finishing the laminectomy procedure? > administration of drugs after 1 hour of laminectomy and spinal cord clip with aneurysm clip to reproduce spinal cord injury. Please provide the companies which produce the materials (reagents, software, etc) for the purpose of reproducibility. > The medicine is from PT Axomedica with the trade name Semax View more View less Competing Interests The authors declare that they have no conflicts of interest. reply Respond Report a concern Author Response 10 Sep 2025 Asadullah Asadullah, Neurosurgery Residency Program, Airlangga University, Surabaya, 60132, Indonesia Note Of revision: Revision on Method: • Why was the ACTH4-10Pro8-Gly9-Pro10 intranasally at a 300 mg/kg dose chosen? Any previous study that suggested the dose? If any, please provide the reference and citation. Rev: After closing the suture, all treatment groups were divided into two subgroups. Placebo control groups were given 0.9 % NaCl intranasally, while the positive treatment groups were given ACTH4-10Pro 8-Gly 9-Pro 10 or semax (The medicine is from PT Axomedica) intranasally at a 300 mcg/kg dose.38 38. Kolomin, T., Shadrina, M., Slominsky, P., Limborska, S., & Myasoedov, N. (2013). A New Generation of Drugs: Synthetic Peptides Based on Natural Regulatory Peptides. Neuroscience and Medicine, 04(04), 223–252. https://doi.org/10.4236/nm.2013.44035 • How long was the period between treatment initiation after finishing the laminectomy procedure? Rev: Administration of semax after 1 hour of laminectomy and spinal cord clip with aneurysm clip to reproduce spinal cord injury. • Please provide the companies which produce the materials (reagents, software, etc) for the purpose of reproducibility. Rev: The medicine is from PT Axomedica with the trade name Semax • Please kindly provide the information if the measurement was performed at least in duple for each sample. Rev: Each group was divided again into two groups to be respectively terminated at 3 hours and 6 hours after compression. Myelum transection was performed at the level of injured myelum after the termination. These are some suggestions for the results section (including figures and tables): • The authors are suggested to provide more description of Figure 1, either in Figure Legend of Figure 1 or as a narration in the text. Figure 1 is the evidence to differentiate the spinal cord in healthy, mild SCI, and severe SCI. Please provide the information on whether the histological figure of the spinal cord SCI groups was with or without the administration of ACTH4-10Pro8-Gly9-Pro10 intranasally. The authors need to provide the readers with what to expect in the microscopic changes of the spinal cord after mild and severe SCI. link of figure 1 : https://drive.google.com/file/d/1dIOgMAQP9lsSUdSw8y3vYRLLfnjYeIGs/view?usp=sharing Rev: In Figure 1, the picture is without ACTH4-10Pro8-Gly9-Pro10 administration after injury. This is expected so that the picture of myelum injury/damage looks different from mild to severe. link of figure 1 : https://drive.google.com/file/d/1dIOgMAQP9lsSUdSw8y3vYRLLfnjYeIGs/view?usp=sharing Rev: Figure 2. Cells were counted to assess anti-inflammatory cytokine levels in SCI models. 37 Immunoreacted neutrophil cytoplasm that stained brown after using monoclonal antibodies are shown. White arrows mark positive stained neutrophils for IL-4 (A), IL-10 (B) and IL-13 (C) in 3 hours observed mild spinal cord injury (SCI) group. Link of figure 2: https://drive.google.com/file/d/1zxPR-is_fgwmhZ_BKLKsHjCAd8jgLaAs/view?usp=sharing Figure 3. Cells were counted to assess anti-inflammatory cytokine levels in SCI models. 37 Immunoreacted neutrophil cytoplasm that stained brown after using monoclonal antibodies are shown. White arrows mark positive stained neutrophils for IL-4 (A), IL-10 (B) and IL-13 (C) in 3 hours observed severe spinal cord injury (SCI) group. LInk of figure 3: https://drive.google.com/file/d/10G-8kJvhFzgFQYzp7uQOwu4CnPNmLv8B/view?usp=sharing • Tables 1-3 are best presented in bar diagrams between groups in a figure. This will give the readers a better description and impression of the results of the study. In addition, the authors can explain more detailed data in the text. This will avoid the repetition of data explanation in figures/tables in the text. Rev: Figure 4. Chart Of Annova Study link of figure 4: https://drive.google.com/file/d/1DcLsen6EXCZBj-na9Wpg-qkHWc9gcv1r/view?usp=sharing • Tables 4-9, in my opinion, can be submitted as supplementary data. In the results section, the authors are suggested to explain the core findings of the posthoc analysis in the text. Rev : Done. Tables had deleted, and change as as supplementary data. • The authors are suggested to make sure that figures and tables are self-explanatory. Any repetition of the data from the figure/tables in the text should be avoided. Rev : Done • In the discussion section, the authors have explained and compared the results of the study to other previous studies. • Please kindly focus on the difference between the follow up period of 3 hrs and 6 hrs in the study, particularly in the level of the anti-inflammatory cytokines and the SCI severity. • In my opinion, I found the fifth paragraph of this section a little odd since the authors did not mention that the study using methylprednisolone was performed in humans. If the authors would like to compare the efficacy of the currently used treatment of SCI, in this case, is methylprednisolone, to the agent used here in the study, I would prefer to use the studies with the same subjects (in this case, the authors should also add any study regarding the use of methylprednisolone treatment in rodents. The study in humans should come as an additional information), the same studied objects/variables (any agents that may increase or decrease anti-inflammatory cytokine in SCI, neurological deficit severity, etc), or other agents that have similar mechanisms of action (in this case, any other agent that binds to melanocortin receptor). Rev: This is in accordance with our study, that the anti-inflammatory cytokine group from the mild or severe injury group showed an increase in the ratio of inflammatory cytokines at 3 to 6 hours. However, in the type of inflammatory cytokine IL-10, the peak cytokine ratio was at 3 hours, and decreased at 6 hours. The decrease in the inflammatory cytokine IL-10 at 6 hours is possibly due to a decrease in the influence of ACTH 4-10Pro 8-Gly 9-Pro 10 at 6 hours. After the strongest influence to increase cytokines at 3 hours. In another study, when compared with the administration of steroids, that Methylprednisolone could attenuate astrocyte cell death, decrease microglia activation, suppress A1 astrocytes activation, and promote functional recovery after acute SCI in mouse models. Finally, Increased anti-inflammatory mediators can be triggered by ACTH4-10Pro 8- Gly 9-Pro 10, which is expected to suppress inflammatory events that can trigger further secondary injury. • In the last sentence of the discussion section, the authors mention “Finally, we see potential benefits from ACTH4-10Pro8- Gly9-Pro10 to reduce inflammation and its deleterious effect on secondary injury after SCI”. Please kindly provide the “deleterious effect of the SCI” that was studied here, since the anti-inflammatory cytokines studied here, in my understanding, are not the deleterious effects of the SCI. Rev: Administration of ACTH 4-10Pro 8-Gly 9-Pro 10 can increase anti-inflammatory cytokine levels in Sprague Dawley rat models with mild and severe SCI. The optimal effect of ACTH 4-10Pro 8-Gly 9-Pro 10 can increase anti-inflammatory cytokine occurs in the third hour after SCI injury. Further studies are needed to determine the optimal dose and functional outcome in rats with SCI. View more View less Competing Interests There is no competing interest reply Respond Report a concern Ar Rochmah M. Peer Review Report For: Effect of ACTH4-10Pro8-Gly9-Pro10 on anti-inflammatory cytokine (IL-4, IL-10, IL-13) expression in acute spinal cord injury models (male Sprague Dawley rats) [version 2; peer review: 2 approved, 1 not approved] . F1000Research 2025, 12 :194 ( https://doi.org/10.5256/f1000research.139917.r164062) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. 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