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)

Asadullah Asadullah; Abdul Hafid Bajamal; Muhammad Arifin Parenrengi; Agus Turchan; Budi Utomo; I Ketut Sudiana; Eko Agus Subagio

doi:10.12688/f1000research.127413.1

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Research Article

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 1; peer review: 1 approved with reservations]

Asadullah Asadullah^1,2, Abdul Hafid Bajamal¹, Muhammad Arifin Parenrengi¹, [...] Agus Turchan¹, Budi Utomo³, I Ketut Sudiana⁴, Eko Agus Subagio¹

Asadullah Asadullah^1,2, Abdul Hafid Bajamal¹, [...] Muhammad Arifin Parenrengi¹, Agus Turchan¹, Budi Utomo³, I Ketut Sudiana⁴, Eko Agus Subagio¹

PUBLISHED 20 Feb 2023

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

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_4-10Pro⁸-Gly⁹-Pro¹⁰(ACTH_4-10) also known as semax, has shown neuroprotective and anti-inflammatory properties. ACTH_4-10has also been actively used in the treatment of brain ischemia without serious complication reported. Here, we analyzed the effects of ACTH_4-10at 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_4-10was 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_4-10showed 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_4-10group 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_4-10Pro⁸-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.

Keywords

Spinal Cord Injury, Anti inflammatory cytokine, Neuroprotector, Experimental, Laminectomy

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: © 2023 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 1; peer review: 1 approved with reservations]. F1000Research 2023, 12:194 (https://doi.org/10.12688/f1000research.127413.1) First published: 20 Feb 2023, 12:194 (https://doi.org/10.12688/f1000research.127413.1) Latest published: 20 Feb 2023, 12:194 (https://doi.org/10.12688/f1000research.127413.1)

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_4-10Pro⁸-Gly⁹-Pro¹⁰ 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 ACTH4-10Pro⁸-Gly⁹-Pro¹⁰ 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).

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. White arrows mark positive stained neutrophils for IL-4 (A), IL-10 (B) and IL-13 (C) in mild spinal cord injury (SCI) group.

Table 1 shows higher IL-4 levels in both mild and severe SCI models with ACTH4-10Pro⁸-Gly⁹-Pro¹⁰ administration that were terminated 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
Group	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 ACTH4-10 3 hours	10.39	1.67
Mild SCI ACTH4-10 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 ACTH4-10 3 hours	9.36	1.98
Severe SCI ACTH4-10 6 hours	9.21	1.37

* p < 0.05.

Table 2 shows higher IL-10 levels in both mild and severe SCI models with ACTH4-10Pro⁸-Gly⁹-Pro¹⁰ administration and who were terminated after three and six hours compared to the placebo group.

Table 2. ANOVA study of IL-10 levels.

Group	IL-10 expression
Group	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 ACTH4-10 3 hours	14.64	2.893
Mild SCI ACTH4-10 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 ACTH4-10 3 hours	13.25	3.086
Severe SCI ACTH4-10 6 hours	9.54	2.138

* p < 0.05.

Table 3 shows higher IL-13 levels in both mild and severe SCI models with ACTH4-10Pro⁸-Gly⁹-Pro¹⁰ administration and who were terminates after three and six hours compared to the placebo group.

Table 3. ANOVA study of IL-13 level.

Group	IL-13 expression
Group	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 ACTH4-10 3 hours	13.36	1.676
Mild SCI ACTH4-10 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 ACTH4-10 3 hours	10.18	3.570
Severe SCI ACTH4-10 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).

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
			ACTH4-10 3 hours	10.39	1.67	0.015^*
			ACTH4-10 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
			ACTH4-10 3 hours	10.39	1.67	0.027^*
			ACTH4-10 6 hours	8.05	1.62	0.663
NaCl 0.9% 6 hour	5.61	2.45	ACTH4-10 3 hours	10.39	1.67	0.007^*
NaCl 0.9% 6 hour	5.61	2.45	ACTH4-10 6 hours	8.05	1.62	0.353
ACTH4-10 3 hour	10.39	1.67	ACTH4-10 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
			ACTH4-10 3 hours	14.64	2.893	<0.001^*
			ACTH4-10 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
			ACTH4-10 3 hours	14.64	2.893	<0.001^*
			ACTH4-10 6 hours	11.46	1.350	<0.001^*
NaCl 0.9% 6 hour	4.71	1.741	ACTH4-10 3 hours	14.64	2.893	<0.001^*
NaCl 0.9% 6 hour	4.71	1.741	ACTH4-10 6 hours	11.46	1.350	<0.001^*
ACTH4-10 3 hour	14.64	2.893	ACTH4-10 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
			ACTH4-10 3 hours	13.36	1.676	<0.001^*
			ACTH4-10 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
			ACTH4-10 3 hours	13.36	1.676	0.001^*
			ACTH4-10 6 hours	11.57	3.313	0.040^*
NaCl 0.9% 6 hour	6.25	2.046	ACTH4-10 3 hours	13.36	1.676	<0.001^*
NaCl 0.9% 6 hour	6.25	2.046	ACTH4-10 6 hours	11.57	3.313	0.001^*
ACTH4-10 3 hour	13.36	1.676	ACTH4-10 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
			ACTH4-10 3 hours	9.36	1.98	0.018^*
			ACTH4-10 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
			ACTH4-10 3 hours	9.36	1.98	0.349
			ACTH4-10 6 hours	9.21	1.37	0.421
NaCl 0.9% 6 hour	5.29	2.16	ACTH4-10 3 hours	9.36	1.98	0.006^*
NaCl 0.9% 6 hour	5.29	2.16	ACTH4-10 6 hours	9.21	1.37	0.009^*
ACTH4-10 3 hour	9.36	1.98	ACTH4-10 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
			ACTH4-10 3 hours	13.25	3.086	<0.001^*
			ACTH4-10 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
			ACTH4-10 3 hours	13.25	3.086	<0.001^*
			ACTH4-10 6 hours	9.54	2.138	0.258
NaCl 0.9% 6 hour	5.71	1.629	ACTH4-10 3 hours	13.25	3.086	<0.001^*
NaCl 0.9% 6 hour	5.71	1.629	ACTH4-10 6 hours	9.54	2.138	0.024^*
ACTH4-10 3 hour	13.25	3.086	ACTH4-10 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
			ACTH4-10 3 hours	10.18	3.570	<0.001
			ACTH4-10 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
			ACTH4-10 3 hours	10.18	3.570	0.007^*
			ACTH4-10 6 hours	9.00	1.893	0.078
NaCl 0.9% 6 hour	5.68	1.718	ACTH4-10 3 hours	10.18	3.570	0.006^*
NaCl 0.9% 6 hour	5.68	1.718	ACTH4-10 6 hours	9.00	1.893	0.064
ACTH4-10 3 hour	10.18	3.570	ACTH4-10 6 hours	9.00	1.893	0.858

* p < 0.05.

Mean levels of IL4, IL-10 and IL-13 were higher in the mild SCI models group that was given ACTH_4-10Pro⁸-Gly⁹-Pro¹⁰ for both three- and six-hour termination groups, compared with placebo. IL-14 levels in models given ACTH_4-10Pro⁸-Gly⁹-Pro¹⁰ 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_4-10Pro⁸-Gly⁹-Pro¹⁰ 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_4-10Pro⁸-Gly⁹-Pro¹⁰ 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.

The results from severe SCI models that were given ACTH_4-10Pro⁸-Gly⁹-Pro¹⁰ 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_4-10Pro⁸-Gly⁹-Pro¹⁰ 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_4-10Pro⁸-Gly⁹-Pro¹⁰ 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_4-10Pro⁸-Gly⁹-Pro¹⁰ 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.

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-13¹⁶^–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 SCI²²; 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 may explain our finding that in the group with severe SCI and termination six hours after injury, anti-inflammatory cytokine expression ratio between the ACTH4-10Pro⁸- Gly⁹-Pro¹⁰ and placebo groups was not as high as in the mild SCI and three-hour group.

ACTH_4-10Pro⁸-Gly⁹-Pro¹⁰ 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_4-10Pro⁸-Gly⁹-Pro¹⁰ is said to bind at the melanocortin receptor promoting anti-inflammatory and neuroprotective effects.²⁵ Activated melanocortin receptors could supress pro-inflammatory cytokine release and promote the anti-inflammatory cytokine release. Together, this condition could supress inflammation.²⁶

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_4-10Pro⁸-Gly⁹-Pro¹⁰ could decrease pro-inflammatory cytokines in SCI rats model.³⁴^,³⁵ Those studies might provide a fundamental basis showing ACTH4-10Pro⁸- Gly⁹-Pro¹⁰ could reduce inflammation and potentially prevent secondary injury in SCI. To date, ACTH4-10Pro⁸- Gly⁹-Pro¹⁰ 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, we see potential benefits from ACTH4-10Pro⁸- Gly⁹-Pro¹⁰ to reduce inflammation and its deleterious effect on secondary injury after SCI.

Conclusions

Administration of ACTH_4-10Pro⁸-Gly⁹-Pro¹⁰ can increase anti-inflammatory cytokine levels in Sprague Dawley rat models with mild and severe SCI. Rats with mild compression that were terminate after three hours showed an increase in IL-4, IL-10 and IL-13; after six hours, only IL-10 and IL-13 were increased. Rats with severe compression that were terminate after three hours showed an increase in IL-10 and IL-13; after six hours, only IL-4, IL-10 were increased. Further studies are needed to determine the optimal dose and functional outcome in rats with SCI.

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. J. Neurosurg. Spine. February 2019; 30: 683–699. PubMed Abstract | Publisher Full Text
2. Hellenbrand DJ, Quinn CM, Piper ZJ, et al.: Inflammation after spinal cord injury: a review of the critical timeline of signaling cues and cellular infiltration. J. Neuroinflammation. 2021; 18(1): 284. PubMed Abstract | Publisher Full Text | Free Full Text
3. Chen Y, He Y, DeVivo MJ: Changing Demographics and Injury Profile of New Traumatic Spinal Cord Injuries in the United States, 1972-2014. Arch. Phys. Med. Rehabil. 2016; 97(10): 1610–1619. PubMed Abstract | Publisher Full Text
4. Wilson JR, Cadotte DW, Fehlings MG: Clinical predictors of neurological outcome, functional status, and survival after traumatic spinal cord injury: a systematic review. J. Neurosurg. Spine. 2012; 17(1 Suppl): 11–26. PubMed Abstract | Publisher Full Text
5. Stein DG, Hoffman SW: Estrogen and progesterone as neuroprotective agents in the treatment of acute brain injuries. Pediatr. Rehabil. 2003; 6(1): 13–22. Publisher Full Text
6. Eckert MJ, Martin MJ: Trauma: Spinal Cord Injury. Surg. Clin. North Am. 2017; 97(5): 1031–1045. Publisher Full Text
7. Witiw CD, Fehlings MG: Acute Spinal Cord Injury. J. Spinal Disord. Tech. 2015; 28(6): 202–210. Publisher Full Text
8. Popovich PG, Stuckman S, Gienapp IE, et al.: Alterations in immune cell phenotype and function after experimental spinal cord injury. J. Neurotrauma. 2001; 18(9): 957–966. PubMed Abstract | Publisher Full Text
9. Murray PJ, Wynn TA: Protective and pathogenic functions of macrophage subsets. Nat. Rev. Immunol. 2011; 11(11): 723–737. PubMed Abstract | Publisher Full Text | Free Full Text
10. Fujiwara N, Kobayashi K: Macrophages in inflammation. Curr. Drug Targets Inflamm. Allergy. 2005; 4(3): 281–286. Publisher Full Text
11. Faris M, Utomo B, Al Fauzi A , et al.: ACTH_4-10PRO⁸-GLY⁹-PRO¹⁰ improves Neutrophil profile in spinal cord injury of rat models. J Adv Pharm Educ Res. 2021; 11(2): 61–65. Publisher Full Text
12. Kolomin T, Shadrina M, Slominsky P, et al.: A New Generation of Drugs: Synthetic Peptides Based on Natural Regulatory Peptides. Neurosci Med. 2013; 04: 223–252. Publisher Full Text
13. Poon PC, Gupta D, Shoichet MS, et al.: Clip compression model is useful for thoracic spinal cord injuries: histologic and functional correlates. Spine (Phila Pa 1976). 2007; 32(25): 2853–2859. PubMed Abstract | Publisher Full Text
14. Fleming JC, Norenberg MD, Ramsay DA, et al.: The cellular inflammatory response in human spinal cords after injury. Brain. 2006; 129(Pt 12): 3249–3269. Publisher Full Text
15. Gensel JC, Zhang B: Macrophage activation and its role in repair and pathology after spinal cord injury. Brain Res. 2015; 1619: 1–11. PubMed Abstract | Publisher Full Text
16. DiSabato DJ, Quan N, Godbout JP: Neuroinflammation: the devil is in the details. J. Neurochem. 2016; 139(Suppl 2): 136–153. PubMed Abstract | Publisher Full Text | Free Full Text
17. McCormick SM, Heller NM: Commentary: IL-4 and IL-13 receptors and signaling. Cytokine. 2015; 75(1): 38–50. PubMed Abstract | Publisher Full Text | Free Full Text
18. Gordon S, Martinez FO: Alternative activation of macrophages: mechanism and functions. Immunity. 2010; 32(5): 593–604. Publisher Full Text
19. Junttila IS: Tuning the Cytokine Responses: An Update on Interleukin (IL)-4 and IL-13 Receptor Complexes. Front. Immunol. 2018; 9: 888. PubMed Abstract | Publisher Full Text | Free Full Text
20. Hellenbrand DJ, Reichl KA, Travis BJ, et al.: Sustained interleukin-10 delivery reduces inflammation and improves motor function after spinal cord injury. J. Neuroinflammation. 2019; 16(1): 93. PubMed Abstract | Publisher Full Text | Free Full Text
21. Thompson CD, Zurko JC, Hanna BF, et al.: The therapeutic role of interleukin-10 after spinal cord injury. J. Neurotrauma. 2013; 30(15): 1311–1324. PubMed Abstract | Publisher Full Text
22. Elkabes S, Nicot AB: Sex steroids and neuroprotection in spinal cord injury: a review of preclinical investigations. Exp. Neurol. 2014; 259: 28–37. PubMed Abstract | Publisher Full Text
23. Taoka Y, Okajima K, Uchiba M, et al.: Role of neutrophils in spinal cord injury in the rat. Neuroscience. 1997; 79(4): 1177–1182. Publisher Full Text
24. Medvedeva EV, Dmitrieva VG, Povarova OV, et al.: The peptide semax affects the expression of genes related to the immune and vascular systems in rat brain focal ischemia: genome-wide transcriptional analysis. BMC Genomics. 2014; 15: 228. PubMed Abstract | Publisher Full Text | Free Full Text
25. Bertolini A: Drug-induced activation of the nervous control of inflammation: A novel possibility for the treatment of hypoxic damage. Eur. J. Pharmacol. 2012; 679(1): 1–8. PubMed Abstract | Publisher Full Text
26. Wang W, Guo D, Lin Y, et al.: Melanocortin Regulation of Inflammation. Front Endocrinol (Lausanne). Published online. 2019; 10. PubMed Abstract | Publisher Full Text | Free Full Text
27. Bracken MB, Shepard MJ, Collins WF, et al.: A randomized, controlled trial of methylprednisolone or naloxone in the treatment of acute spinal-cord injury. Results of the Second National Acute Spinal Cord Injury Study. N. Engl. J. Med. 1990; 322(20): 1405–1411. Publisher Full Text
28. Bracken MB, Collins WF, Freeman DF, et al.: Efficacy of methylprednisolone in acute spinal cord injury. JAMA. 1984; 251(1): 45–52. Publisher Full Text
29. Bracken MB, Shepard MJ, Holford TR, et al.: Administration of methylprednisolone for 24 or 48 hours or tirilazad mesylate for 48 hours in the treatment of acute spinal cord injury. Results of the Third National Acute Spinal Cord Injury Randomized Controlled Trial. National Acute Spinal Cord Injury Study. JAMA. 1997; 277(20): 1597–1604. PubMed Abstract | Publisher Full Text
30. Hurlbert RJ, Hadley MN, Walters BC, et al.: Pharmacological therapy for acute spinal cord injury. Neurosurgery. 2013; 72: 93–105. Publisher Full Text
31. Eck JC, Nachtigall D, Humphreys SC, et al.: Questionnaire survey of spine surgeons on the use of methylprednisolone for acute spinal cord injury. Spine (Phila Pa 1976). 2006; 31(9): E250–E253. PubMed Abstract | Publisher Full Text
32. Fehlings MG, Wilson JR, Tetreault LA, et al.: A Clinical Practice Guideline for the Management of Patients With Acute Spinal Cord Injury: Recommendations on the Use of Methylprednisolone Sodium Succinate. Glob spine J. 2017; 7(3 Suppl): 203S–211S. PubMed Abstract | Publisher Full Text | Free Full Text
33. Frangen TM, Ruppert S, Muhr G, et al.: The beneficial effects of early stabilization of thoracic spine fractures depend on trauma severity. J. Trauma. 2010; 68(5): 1208–1212. PubMed Abstract | Publisher Full Text
34. 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

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Version 1

VERSION 1 PUBLISHED 20 Feb 2023

Author details Author details

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.

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Published: 20 Feb 2023, 12:194

https://doi.org/10.12688/f1000research.127413.1

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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 1; peer review: 1 approved with reservations]. F1000Research 2023, 12:194 (https://doi.org/10.12688/f1000research.127413.1)

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Reviewer Report 13 Mar 2023

Mawaddah Ar Rochmah, Department of Anatomy, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta, Indonesia

Approved with Reservations

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 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.

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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
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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

Comments on this article Comments (0)

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VERSION 1 PUBLISHED 20 Feb 2023

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Mawaddah Ar Rochmah, Universitas Gadjah Mada, Yogyakarta, Indonesia

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13 Mar 2023 | for Version 1

Mawaddah Ar Rochmah, Department of Anatomy, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta, Indonesia

24 Views Cite this report Responses(1)

Approved With Reservations

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.

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

Respond to this report

Responses (1)

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.

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

[1] 1. Khorasanizadeh M, Yousefifard M, Eskian M, et al.: Neurological recovery following traumatic spinal cord injury: a systematic review and meta-analysis. J. Neurosurg. Spine. February 2019; 30: 683–699. PubMed Abstract | Publisher Full Text

[2] 2. Hellenbrand DJ, Quinn CM, Piper ZJ, et al.: Inflammation after spinal cord injury: a review of the critical timeline of signaling cues and cellular infiltration. J. Neuroinflammation. 2021; 18(1): 284. PubMed Abstract | Publisher Full Text | Free Full Text

[3] 3. Chen Y, He Y, DeVivo MJ: Changing Demographics and Injury Profile of New Traumatic Spinal Cord Injuries in the United States, 1972-2014. Arch. Phys. Med. Rehabil. 2016; 97(10): 1610–1619. PubMed Abstract | Publisher Full Text

[4] 4. Wilson JR, Cadotte DW, Fehlings MG: Clinical predictors of neurological outcome, functional status, and survival after traumatic spinal cord injury: a systematic review. J. Neurosurg. Spine. 2012; 17(1 Suppl): 11–26. PubMed Abstract | Publisher Full Text

[5] 5. Stein DG, Hoffman SW: Estrogen and progesterone as neuroprotective agents in the treatment of acute brain injuries. Pediatr. Rehabil. 2003; 6(1): 13–22. Publisher Full Text

[6] 6. Eckert MJ, Martin MJ: Trauma: Spinal Cord Injury. Surg. Clin. North Am. 2017; 97(5): 1031–1045. Publisher Full Text

[7] 7. Witiw CD, Fehlings MG: Acute Spinal Cord Injury. J. Spinal Disord. Tech. 2015; 28(6): 202–210. Publisher Full Text

[8] 8. Popovich PG, Stuckman S, Gienapp IE, et al.: Alterations in immune cell phenotype and function after experimental spinal cord injury. J. Neurotrauma. 2001; 18(9): 957–966. PubMed Abstract | Publisher Full Text

[9] 9. Murray PJ, Wynn TA: Protective and pathogenic functions of macrophage subsets. Nat. Rev. Immunol. 2011; 11(11): 723–737. PubMed Abstract | Publisher Full Text | Free Full Text

[10] 10. Fujiwara N, Kobayashi K: Macrophages in inflammation. Curr. Drug Targets Inflamm. Allergy. 2005; 4(3): 281–286. Publisher Full Text

[11] 11. Faris M, Utomo B, Al Fauzi A , et al.: ACTH_4-10PRO⁸-GLY⁹-PRO¹⁰ improves Neutrophil profile in spinal cord injury of rat models. J Adv Pharm Educ Res. 2021; 11(2): 61–65. Publisher Full Text

[12] 12. Kolomin T, Shadrina M, Slominsky P, et al.: A New Generation of Drugs: Synthetic Peptides Based on Natural Regulatory Peptides. Neurosci Med. 2013; 04: 223–252. Publisher Full Text

[13] 13. Poon PC, Gupta D, Shoichet MS, et al.: Clip compression model is useful for thoracic spinal cord injuries: histologic and functional correlates. Spine (Phila Pa 1976). 2007; 32(25): 2853–2859. PubMed Abstract | Publisher Full Text

[14] 14. Fleming JC, Norenberg MD, Ramsay DA, et al.: The cellular inflammatory response in human spinal cords after injury. Brain. 2006; 129(Pt 12): 3249–3269. Publisher Full Text

[15] 15. Gensel JC, Zhang B: Macrophage activation and its role in repair and pathology after spinal cord injury. Brain Res. 2015; 1619: 1–11. PubMed Abstract | Publisher Full Text

[16] 16. DiSabato DJ, Quan N, Godbout JP: Neuroinflammation: the devil is in the details. J. Neurochem. 2016; 139(Suppl 2): 136–153. PubMed Abstract | Publisher Full Text | Free Full Text

[17] 17. McCormick SM, Heller NM: Commentary: IL-4 and IL-13 receptors and signaling. Cytokine. 2015; 75(1): 38–50. PubMed Abstract | Publisher Full Text | Free Full Text

[18] 18. Gordon S, Martinez FO: Alternative activation of macrophages: mechanism and functions. Immunity. 2010; 32(5): 593–604. Publisher Full Text

[19] 19. Junttila IS: Tuning the Cytokine Responses: An Update on Interleukin (IL)-4 and IL-13 Receptor Complexes. Front. Immunol. 2018; 9: 888. PubMed Abstract | Publisher Full Text | Free Full Text

[20] 20. Hellenbrand DJ, Reichl KA, Travis BJ, et al.: Sustained interleukin-10 delivery reduces inflammation and improves motor function after spinal cord injury. J. Neuroinflammation. 2019; 16(1): 93. PubMed Abstract | Publisher Full Text | Free Full Text

[21] 21. Thompson CD, Zurko JC, Hanna BF, et al.: The therapeutic role of interleukin-10 after spinal cord injury. J. Neurotrauma. 2013; 30(15): 1311–1324. PubMed Abstract | Publisher Full Text

[22] 22. Elkabes S, Nicot AB: Sex steroids and neuroprotection in spinal cord injury: a review of preclinical investigations. Exp. Neurol. 2014; 259: 28–37. PubMed Abstract | Publisher Full Text

[23] 23. Taoka Y, Okajima K, Uchiba M, et al.: Role of neutrophils in spinal cord injury in the rat. Neuroscience. 1997; 79(4): 1177–1182. Publisher Full Text

[24] 24. Medvedeva EV, Dmitrieva VG, Povarova OV, et al.: The peptide semax affects the expression of genes related to the immune and vascular systems in rat brain focal ischemia: genome-wide transcriptional analysis. BMC Genomics. 2014; 15: 228. PubMed Abstract | Publisher Full Text | Free Full Text

[25] 25. Bertolini A: Drug-induced activation of the nervous control of inflammation: A novel possibility for the treatment of hypoxic damage. Eur. J. Pharmacol. 2012; 679(1): 1–8. PubMed Abstract | Publisher Full Text

[26] 26. Wang W, Guo D, Lin Y, et al.: Melanocortin Regulation of Inflammation. Front Endocrinol (Lausanne). Published online. 2019; 10. PubMed Abstract | Publisher Full Text | Free Full Text

[27] 27. Bracken MB, Shepard MJ, Collins WF, et al.: A randomized, controlled trial of methylprednisolone or naloxone in the treatment of acute spinal-cord injury. Results of the Second National Acute Spinal Cord Injury Study. N. Engl. J. Med. 1990; 322(20): 1405–1411. Publisher Full Text

[28] 28. Bracken MB, Collins WF, Freeman DF, et al.: Efficacy of methylprednisolone in acute spinal cord injury. JAMA. 1984; 251(1): 45–52. Publisher Full Text

[29] 29. Bracken MB, Shepard MJ, Holford TR, et al.: Administration of methylprednisolone for 24 or 48 hours or tirilazad mesylate for 48 hours in the treatment of acute spinal cord injury. Results of the Third National Acute Spinal Cord Injury Randomized Controlled Trial. National Acute Spinal Cord Injury Study. JAMA. 1997; 277(20): 1597–1604. PubMed Abstract | Publisher Full Text

[30] 30. Hurlbert RJ, Hadley MN, Walters BC, et al.: Pharmacological therapy for acute spinal cord injury. Neurosurgery. 2013; 72: 93–105. Publisher Full Text

[31] 31. Eck JC, Nachtigall D, Humphreys SC, et al.: Questionnaire survey of spine surgeons on the use of methylprednisolone for acute spinal cord injury. Spine (Phila Pa 1976). 2006; 31(9): E250–E253. PubMed Abstract | Publisher Full Text

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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)

Abstract

Keywords

Introduction

Methods

Results

Figure 1. Myelum sample from control group (A), mild spinal cord injury (SCI, B), severe SCI (C).

Figure 2. Cells were counted to assess anti-inflammatory cytokine levels in SCI models.37

Table 1. ANOVA study of IL-4 levels. SCI: spinal cord injury.

Table 2. ANOVA study of IL-10 levels.

Table 3. ANOVA study of IL-13 level.

Table 4. Post hoc study of IL-4 level in mild SCI.

Table 5. Post hoc study of IL-10 level in mild SCI.

Table 6. Post hoc study of IL-13 level in mild SCI.

Table 7. Post hoc study of IL-4 level in severe SCI.

Table 8. Post hoc study of IL-10 level in severe SCI.

Table 9. Post hoc study of IL-13 level in severe SCI.

Discussion

Conclusions

Data availability

Underlying data

Extended data

Reporting guidelines

References

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Figure 2. Cells were counted to assess anti-inflammatory cytokine levels in SCI models.³⁷