Role of melatonin in the dynamics of acute spinal cord injury in rats

Abstract Melatonin is well‐documented to have the ability of reducing nerve inflammation and scavenging free radicals. However, the therapeutic effect of melatonin on spinal cord injury has not been fully described. In this study, we assessed the effect of melatonin on T9 spinal cord injury established by Allen method in rats. Melatonin deficiency significantly delayed the recovery of sensory and motor functions in SCI rats. Treatment with melatonin significantly alleviated neuronal apoptosis and accelerated the recovery of spinal cord function. These results suggest that melatonin is effective to ameliorate spinal cord injury through inhibition of neuronal apoptosis and promotion of neuronal repair.

Melatonin (MT) possesses various neuroprotective effects and has been characterized at enriched levels in cerebrospinal fluid. 13,14 As an endogenous substance, MT is degraded by metabolic pathway related processes and lacks subsequent accumulation within the body. 15 MT also was identified as effective in the treatment of SCI because of beneficial effects upon neuronal cell bodies and synapses. In particular, MT has been shown as effective in protecting mitochondria and other subcellular organelles. 16,17 Numerous studies have found that protective effects of MT upon the spinal cord were mainly manifested through scavenging of oxygen free radicals hence protecting against ischaemia and hypoxia of nerve tissue. 14,18 MT also promoted regeneration of peripheral nerve myelin sheath and stimulated various anti-apoptosis signalling pathways. 19,20 However, the underlying mechanisms by which MT acts in such manners, and with respect to spinal cord injuries have not been fully clarified. We tried to explore the effect of melatonin on spinal cord injury, whether melatonin is a participating factor in spinal cord repair, and the impact of melatonin inhibition on spinal cord repair. Therefore, we sought to investigate functions and mechanisms of MT with respect to its use as a treatment for spinal cord injury.

| Animals
We purchased N = 144 9-week-old Sprague-Dawley (SD) rats weighing about 250 g were from the SPF Biotechnology Company for experimentation. Rats were grown using a 12-hour light-dark cycle at a constant temperature of 25°C ± 5°C and with a constant atmosphere of 60-70% humidity. Free access to water and food was provided ad-libitum. All animal experimental protocols were reviewed and approved by the Ethics Committee of Peking Union Medical College Hospital (China).

| Rat spinal cord injury establishment and intervention
The rat SCI model was established as previously reported. 21 Rats were anaesthetized using 1% sodium pentobarbital (50 mg/kg). A longitudinal midline incision was made with T9 as the centre, and the spinous process and lamina of T8-10 were exposed and rinsed with normal saline. Rats were injured by dropping a 10 g hammer from a height of 25 mm upon the T9 portion of the spinal cord followed by a manual application of a large amount of pressure upon the spinal cord for 1 minutes. Small amounts of muscle and skin were cut out, and tissues were sutured. Animals completely paralysed below the injury site were included in subsequent experiments. Urinary bladders were pressed thrice a day until the bladder regained urination reflex. The control group (CTR) was anaesthetized followed by exposure of T9 segment and suturing of the skin layer by layer.
Successfully modelled rats were randomly divided into treatment groups as follows: (i) negative control (SCI); (ii) Luzindole at 15 mg/ kg, i.p. (LUZ); and (iii) melatonin, 15 mg/kg, i.p (MT). Rats in the CTR and SCI groups were injected with 1 mL of 1% ethanol saline solution. Movement and sensory recovery in all animals were examined at 1, 3, 7 and 14 days after initiation of the modelling experiment.
Six rats from each group were killed for testing on each of these days upon whence T8-T9 spinal cord segments were preserved for subsequent experiments.

| Rehabilitation assessment
Motor function was evaluated using the BBB scale, and tail tenderness tests were performed using an electronic tenderness tester (ZS Dichuang Corporation).

| Nissl staining
Spinal cord T8-T9 segments were fixed in 4% formaldehyde for 4 hours and embedded in paraffin. Sections were stained in 1% cresyl violet for 10 minutes, differentiated in 95% alcohol, and subsequent Nissl bodies observed. Three 100× visual fields were randomly selected and photographed, and neurons in each visual field were counted from which averages and measures of error were determined.

| RNA extraction and RT-qPCR analysis
Spinal cord tissues with lengths of 1 cm from centre were harvested from the points of injury and were washed with cold PBS. Spinal cord tissues were cut into small pieces and homogenized with 1 mL of Trizol reagent. Supernatant was centrifuged at 12 000 rpm for 12 minutes, and 250 μL of chloroform was added followed by mixing and additional centrifugation. To the supernatant, 0.8 volumes of isopropanol were added and samples were allowed to stand at −20°C for 15 minutes. Precipitates were washed with 75% ethanol, and DEPC water was used to help dissolve the RNA. Reverse transcription and amplification of RNA were performed following all manufacturer protocols. Primer set sequences and reaction conditions used are presented in Table 1.
Collected supernatant was centrifuged at 12 000 × g for 10 minutes, and protein concentrations were detected using BCA assay kits following all manufacturer protocols. Then, supernatant was diluted to a uniform concentration by adding the protein extraction reagent.
Loading buffer (5X) was added to the sample at a 1:4 ratio, and samples were then boiled for 15 minutes.
Proteins were electrophoresed on polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were blocked using TBST containing 5% non-fat milk at room temperature for 2 hours. Primary antibody was added to the PVDF membranes which were then incubated at 4℃ overnight.
Membranes were thrice washed with TBST solution, and secondary antibody was added followed by incubation at 37℃ for 2 hours.
Proteins in the PVDF membranes were quantified using enhanced chemiluminescence (CW biotech) and subsequently analysed using Image Quant TL Software (GE).

| Statistical analysis
The Statistical Package for Social Scientists (SPSS, Version 26.0, IBM) was used for data analysis. Measurements for data were expressed as the mean (M) ± standard deviation (SD). One-way analysis of variance (ANOVA) was used to facilitate comparisons of differences between groups. LSD and S-N-K methods were used to facilitate comparisons among multiple groups where variances were uniform. We used Kruskal-Wallis H tests with K independent sample tests where variances were uneven. P < .050 was considered as the level of statistical significance at which the null hypothesis of no differences among comparisons would be rejected.

| MT accelerates the recovery of spinal cord injury in SCI rats
Tenderness values reflected sensory functions of rats and a decrease in tenderness indicated that sensory function was recovering. On the 14th day after SCI, tenderness values began to decrease and sensory function recovery was observed in the SCI group. Tenderness values in the MT group were reduced compared to the negative control group whereas the LUZ group showed increased tenderness values compared with the SCI group ( Figure 1A).
BBB scores were used to facilitate assessment of motor function in rats. Scores were significantly improved at days 7 and 14 following MT-based treatments. However, BBB scores of rats in the LUZ group were reduced compared with those in the negative control group upon day 14 ( Figure 1B). These results indicated that MT accelerated the recovery of motor function in spinal cord injured rats, whereas LUZ delayed the recovery of motor function.

| MT maintained Nissl body numbers and increased the stability of the internal environment
The number of Nissl bodies in the MT group was significantly higher compared with the SCI and LUZ groups upon days 7 and day 14 post-induction of SCI (Figure 2A). Only small amounts of Nissl bodies were observed in each group upon the first day. Thereafter, Nissl body numbers were significantly increased in the MT group, but not in the LUZ and SCI groups ( Figure 2B).

| MT reduces neuronal apoptosis after spinal cord injury
TUNEL staining indicated that spinal cord neurons underwent apoptosis in SCI afflicted rats. On the third day, the apoptosis rate reached its peak and then began to decline slowly. However, after 14 days of MT-based treatments, the rate of apoptosis decreased significantly ( Figure 3A,B).

| MT activates PI3K pathway
The expression of key factors in the PI3K pathway was determined by Western blotting analyses. MT treatments significantly

| D ISCUSS I ON
The spinal cord facilitates signal transmission between the brain and limbs. Injury to fragile aspects of the spinal cord can cut off all the information arriving or feedback from the injured part of the body and can paralyse the plane below the injured locale. 22 Each year, about 500 000 people globally suffer from spinal cord injury, and frequently, the central neurons are in a terminally differentiated state and cannot be regenerated after damage. 2,3 The tenderness values and BBB scores were closely related to the reception of integrated pain signals upon the ventral grey matter of the spinal cord and to the transfer of motor commands to the dorsal axon. These states can generally reflect the recovery of rat sensory and motor functions. 23

ACK N OWLED G EM ENTS
This work was supported by the National Natural Science Foundation of China (81330044 and 81772424).

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data sets used and/or analysed during the current study are available from the corresponding author on reasonable request.