The effect of ACTH4-10Pro 8 -Gly 9 -Pro 10 on neurotrophin-3 expression in Sprague Dawley rat on acute spinal cord injury

Background: Neurotrophic factors such as NT-3 play an important role in spinal cord regeneration, contributing to spinal cord regeneration assisting functional improvement in SCI. ACTH4-10Pro 8 -Gly 9 -Pro 10 , an analog of the N-terminal fragment (4-10) adrenocorticotropic hormone, is one of the neuroprotective compounds that can increase NT-3 levels in brain ischemia. Therefore, we conducted a study with experimental animals to determine the effect of NT-3 expression on ACTH4-10Pro 8 Gly 9 -Pro 10 administration in acute compression SCI. Method: We used Sprague Dawley rats with acute compression SCI model using 20 gr and 35 gr aneurysm clips. ACTH4-10Pro 8 -Gly 9 -Pro 10 was administered intranasally to the treatment group, and 0.9% NaCl was administered intranasally to the positive control group. Both of the groups then terminated at 3 and 6 hours. Results: In rats with mild SCI that were given ACTH4-10Pro 8 -Gly 9 -Pro 10 , the NT -3 expression after 3 hours and 6 hours was 14 (12-17) and 10 (7-13). In rats with severe SCI given ACTH4-10Pro 8 -Gly 9 -Pro 10 , the NT-3 expression after 3 hours and 6 hours was 9 (6-11) and 8 (7-10). Intranasal administration of ACTH4-10Pro 8 -Gly 9 -Pro 10 showed higher NT-3 expression than the group with 0.9% NaCl in mild and severe SCI. Conclusion: Intranasal administration of ACTH4-10Pro 8 -Gly 9 -Pro 10 can increase NT-3 expression in mild and severe acute SCI at 3 and 6 hours. Expression of NT-3 in the group with ACTH4-10Pro 8 -Gly 9 -Pro 10 mild acute SCI was higher than in the group with severe acute SCI. Further research needs to be done to determine the neuroregeneration effect of ACTH4-10Pro 8 -Gly 9 Pro 10 on SCI.


INTRODUCTION
Spinal cord injury (SCI) is an injury responsible for most disabilities, ranging from damage to sensory and motor functions to multiorgan dysfunction. The incidence of SCI in developing countries ranges from 13.1 to 163.4 per million population with prevalence rates between 490 to 560 per million population. 1 In Indonesia, there were 104 SCI cases registered at Fatmawati General Hospital in 2014, with the most common etiology being traffic accidents and falling from a height. 2 The highest incidence of traumatic SCI occurs in men of productive age with an average age at the time of injury between 32 -55.4 years, 37 -47.9 years, and 26.8-56.6 years, for North America, Europe, and Asia, respectively. 3 The high incidence of SCI at a young age certainly impacts the emergence of serious economic loss for families, communities, and countries. Functional defects caused by SCI significantly impact decreasing quality of life and patient life expectancy. 4 Nerve cells in the central nervous system (CNS) in humans have a very limited regeneration response that does not allow for complete healing after injury. 5 However, it is possible to develop neuroprotective therapies to maximize the functional integrity of the spinal cord remaining after injury. 6 The most important thing in the treatment of spinal cord injury is how to achieve axonal regeneration in the damaged area. 7 NT-3 is important for developing and maintaining neuronal populations and promotes differentiation. Also, NT-3 is important in forming the substantia nigra and pyramidal pathways responsible for motor activity and have the best response in corticospinal axon regeneration compared to other neurotrophins, even considered superior. 8 A neuroprotective compound, ACTH4-10Pro 8 -Gly 9 -Pro 10 , also known as heptapeptide Semax an analog of the N-terminal fragment (4-10) of adrenocorticotropic hormone, is known to enhance the transcription of NT-3 mRNA 24 hours after the ischemia of the rat brain cortex. 9 ACTH4-10Pro 8 -Gly 9 -Pro 10 has also been shown to be effective in treating vascular disease, allergic and toxic inflammation, and partial atrophy  of the human optic nerve. 10 Intranasal administration of ACTH4-10Pro 8 -Gly 9 -Pro 10 in rats with SCI showed an antiinflammatory response expected to prevent secondary injury to nerve. 11, 12 Our study was aimed to determine the effect of ACTH4-10Pro 8 -Gly 9 -Pro 10 administration on the expression of NT-3 in the spinal cord after mild and severe compression SCI.

METHOD
This study used a spinal cord injury model of male Sprague Dawley rats, weighing 250-300 g. Forty-five samples were divided into nine groups of five samples each. One group became the control group with the spinal cord was left uninjured as a baseline. The rest became the treatment group in which laminectomy at the level of 2 nd thoracic vertebra was performed, followed by spinal cord compression using an aneurysm clip with a clamping force of 20 g for mild SCI (4 groups) and 35 g for severe SCI (4 groups) in 1 minute. The laminectomy site was then closed with sutures. Each treatment group was divided into two subgroups. Positive control groups were given 0.9% NaCl intranasally, while the treatment groups were given ACTH4-10Pro 8 -Gly 9 -Pro 10 intranasally at a 300 mg/kg dose. Then each group was divided again into two groups to be terminated, and then myelum transection was performed at 3 hours and 6 hours after compression. The preparations were fixed in formalin and examined for IHC. The NT-3 was calculated per 100 cells using associated anti-monoclonal antibodies and viewed with a light microscope using a 1000x magnification. Cells that showed positive expression gave the results of a brown color image in the cell cytoplasm ( Figure 1).
Data collection is carried out in a controlled environment, assuming that all conditions are managed equally and controlled. Data of NT-3 level was presented in a relative expression graph. Data normality analysis was performed using Shapiro-Wilk, and then the collected data were analyzed using the nonparametric Kruskal-Wallis test with Mann-Whitney difference test.
Mann-Whitney test was carried out to pinpoint the differences between groups. Overall NT-3 expressions are shown in figure 2. Except between the control group and group administered with Nacl 0,9% 3 hours, the median expression of NT-3 was significantly different between all comparison groups.
Overall, it appears that administration of ACTH4-10Pro 8 -Gly 9 -Pro 10 induced higher NT-3 expression compared to Nacl 0,9%. Also, the expression of NT-3 appeared to increase over time in groups treated with Nacl 0,9% bit significantly decreased overtime in groups treated with ACTH4-10Pro 8 -Gly 9 -Pro 10 .
Higher expression of NT-3 was also achieved significantly in ACTH4-10Pro 8 -Gly 9 -Pro 10 groups with severe SCI compared to NaCl 0,9% group at 3 hours and 6 hours after compression (p<0,05) as shown in table 3. However, the time factor appears to be less significant in severe SCI because no difference was found when comparing the group with Nacl 0,9% at 3  hours and 6 hours and ACTH4-10Pro 8 -Gly 9 -Pro 10 at the same time frame. Our study found significantly higher NT-3 expression in the ACTH4-10Pro8-Gly9-Pro10 group with mild SCI at 3 hours compared to groups with severe SCI at both 3 hours and 6 hours shown in table 4. However, at 6 hours, the differences were not significant anymore. Therefore, it seems that the NT-3 inducing effect of ACTH4-10Pro 8 -Gly 9 -Pro 10 is optimum at 3 hours after administration in the mild SCI, and the effect is sustained or slightly reduced over time.

DISCUSSION
SCI causes an imbalance in the microenvironment that can exacerbate and accelerate nerve damage, impairing regeneration and functional recovery. 13 The balance between proneurotrophins and neurotrophins is disrupted after SCI, resulting in increased proneurotrophins and decreased neurotrophins, leading to cellular apoptosis, reducing synaptic plasticity, promoting the inflammatory response, and degeneration. 14 NT-3, BDNF, and NGF expression decrease significantly as early as 6 hours after spinal cord contusion in rat model. 15 Our study showed that NT-3 expression in rats with mild SCI was significantly higher in ACTH4-10Pro 8 -Gly 9 -Pro 10 groups compared to Nacl 0,9% group at 3 hours and 6 hours after compression (p<0,05), as shown in table 2. Higher expression of NT-3 was also achieved significantly in ACTH4-10Pro 8 -Gly 9 -Pro 10 groups with severe SCI compared to NaCl 0,9% group at 3 hours and 6 hours after compression (p<0,05), as shown in table 3. ACTH4-10Pro 8 -Gly 9 -Pro 10 is known to act by binding to the melanocortin receptor, such as MC4R found in the spinal cord. 16 In vitro studies on astrocytes showed that activation of the MC4R receptor can increase the expression of BDNF through the cAMP-PKA-CREB. 17 A previous study in rats showed that administration of ACTH4-10Pro 8 -Gly 9 -Pro 10 intranasally can increase BDNF expression in 3 hours after SCI. 18 The mechanism for increasing NT-3 expression in this study has not been elucidated, but it is possible to have the same mechanism as the increase in BDNF.
Our study found significantly higher NT-3 expression in the ACTH4-10Pro 8 -Gly 9 -Pro 10 group with mild SCI than in the ACTH4-10Pro 8 -Gly 9 -Pro 10 group with severe SCI at 3 hours after injury (p<0,05) as shown in table 4. Nerve damage that occurs after SCI can result from primary or secondary injury. More severe injury would result in more severe primary damage, causing a more severe imbalance at the tissue, cellular, and molecular levels, which could affect the expression of NT-3. Intranasal administration of ACTH4-10Pro 8 -Gly 9 -Pro 10 in rats with SCI model also showed anti-inflammatory effects by reducing the expression of IL-1, TNF-⍺, NF-KB, neutrophil, and induced reduction in the apoptotic mechanism through increased ratio of Bcl-2/HSP70. 11,12, 19 Our study's peak of NT-3 expression was shown in the ACTH4-10Pro 8 -Gly 9 -Pro 10 group 3 hours after mild and severe SCI. Intranasal administration of ACTH4-10Pro 8 -Gly 9 -Pro 10 is known to reach the rat brain within 2 minutes postadministration and has a concentration 10-15 times higher than intravenous administration 20 . Another study in rats said that the nootropic and analgesic effects after intranasal administration of ACTH4-10Pro 8 -Gly 9 -Pro 10 could be observed 15-30 minutes after administration. 21 Human studies have shown that ACTH4-10 concentrations in spinal CSF increase within 10 minutes and peak within 30 minutes after intranasal administration, suggesting the possibility of using an extraneuronal pathway for the peptide to reach its target, pass through the intercellular gap in the olfactory epithelium and then diffuse into the subarachnoid space. 22 In this study, no locomotor assessment was carried out in rats, so the difference in the functional recovery effect of mild and severe SCI is not known after ACTH4-10Pro 8 -Gly 9 -Pro 10 treatment. However, one study showed improved BBB scores in rats with the SCI model after intranasal administration of ACTH4-10Pro 8 -Gly 9 -Pro 10 . 2 The receptor NT-3 also changes after the occurrence of SCI, so research on the TrkA, TrkB, TrkC, p75NTR, proneurotrophin, PC1, PC2, and furin receptors is also necessary to understand more clearly the action of ACTH4-10Pro 8 -Gly 9 -Pro 10 . 15

CONCLUSIONS
Intranasal administration of ACTH4-10Pro 8 -Gly 9 -Pro 10 increased NT-3 expression in mild and severe acute SCI at 3 and 6 hours. Expression of NT-3 was higher in the mild acute SCI group after administration of ACTH4-10Pro 8 -Gly 9 -Pro 10 than in the severe acute SCI group. Further studies are needed to determine the neuroregeneration effect of ACTH4-10Pro 8 -Gly 9 -Pro 10 on SCI.

CONFLICT OF INTEREST
No competing interests were declared.

ETHICS CONSIDERATION
Ethics approval has been obtained from Animal Care and Use Committee (ACUC), Faculty of Veterinary Medicine, Airlangga University, Surabaya, with EC number 325-KE.