Effect of Melatonin Administration on Nerve Regeneration after Recurrent Laryngeal Nerve Injury

Abstract

Recurrent Laryngeal Nerve (RLN) injury is a complication in neck surgery. The aim of this study is to evaluate the effect of primary suture repair with melatonin treatment on nerve regeneration after RLN damage. After the RLN damage, nerve repair was performed in the first and fourth groups. The third and fourth groups were given intraperitoneal melatonin therapy daily for six weeks. EMG was applied to all subjects and vocal cord movements were evaluated endoscopically. At the end of the sixth week, all subjects were sacrificed, and their larynx were examinedhistologically. Vocal cord paralysis (VCP) was observed in all subjects after RLN damage. In the sixth week, improvement was observed in the first and fourth group who underwent nerve repair, whereas none in the second and third group, who did not undergo nerve repair, improved. With EMG, the highest MUP was in the fourth group. Histologically, an increase in Schwann cells, a decrease in axon damage, and cytoplasmic vacuolization were in the fourth group. Myelin protein zero and Ki-67 staining were the most in the fourth group. In our study, laryngoscopic, electrophysiological and histopathological findings show that melatonin contributes to nerve healing but this could not translate into functional recovery.

Key words
Melatonin; nerve regeneration; Vagus; larynx

INTRODUCTION

Vocal cord paresis, also known as recurrent laryngeal nerve paralysis or vocal fold paralysis, is an injury to one or both recurrent laryngeal nerves (RLNs), which control all intrinsic muscles of the larynx except for the cricothyroid muscle. The RLN is important for speaking, breathing and swallowing.

The primary larynx-related functions of the mainly efferent nerve fiber RLN, include the transmission of nerve signals to the muscles responsible for regulation of the vocal folds’ position and tension to enable vocalization, as well as the transmission of sensory nerve signals from the mucous membrane of the larynx to the brain.

A unilateral injury of the nerve typically results in hoarseness caused by a reduced mobility of one of the vocal folds. It may also cause minor shortages of breath as well as aspiration problems especially concerning liquids. A bilateral injury causes the vocal folds to impair the air flow resulting in breathing problems, stridor and snoring sounds, and fast physical exhaustion. This strongly depends on the median or paramedian position of the paralyzed vocal folds. Hoarseness rarely occurs in bilaterally paralyzed vocal folds.

Hence the Recurrent laryngeal nerve damage signs and symptoms vary, and unilateral injuries may present with dysphagia, dysphonia, and dyspnea or they may be asymptomatic. Early intervention is vital for full functional recovery (Mok et al. 2020, Wang et al. 2016).

Current treatment options for VCP are: injection laryngoplasty, medialization thyroplasty, arytenoid adduction, and laryngeal reinnervation techniques. In the near future, reinnervation techniques will be needed more, as they have advantages such as preventing loss of muscle tone with changes in vocal cord position and preserving both the anatomy and the voice quality of the larynx (Lal & Clark 2005).

In recent years, melatonin, the pineal gland’s primary hormone, has attracted researchers’ attention because of its free radical scavenger quality, antioxidant, anti-inflammatory and analgesic properties, and neuroprotective effects. However, further experimental and clinical studies are needed to determine the clinical benefit of melatonin (Kandemir & Sarikcioglu 2015).

This study aims to evaluate the effect of melatonin on peripheral nerve regeneration by combining reinnervation techniques, both histologically and electrophysiologically, and investigate its effects on vocal cord movements by visualizing the vocal cords laryngoscopically which is novel in the literature.

MATERIALS AND METHODS

This study was carried out at Sakarya University Experimental Medicine Research and Application Unit, with the permission of the Animal Experiments Local Ethics Committee, between 10/12/2020-21/01/2021 (SUHADYEK-05-02-2020-16). The study was funded by the Sakarya University Scientific Research Projects Unit. Twenty-eight Wistar albino male rats with normal laryngeal functions, weighing between 250-300 grams were used in the study. Four groups, with seven animals in each group were created.

Subjects were put under general anesthesia via administration of intraperitoneal Xylazine (7.5 mg/kg), and Ketamine (100 mg/kg) and intramuscular Buprenorphine (0.3 mg/kg) with all efforts made to minimize suffering. Oral Enrofloxacin (5 mg/kg) was used for surgical wound infection prophylaxis. Surgical Microscope was used for exploration, incision and suturing of the recurrent laryngeal nerve. The RLNs in the first group were incised, and then underwent primary suture repair with one epineural suture each. The subjects in the second group underwent RLN incisions but did not undergo primarily suture repair. The subjects in the third group did not undergo primary suture repair after nerve incision but 10 mg/kg intraperitoneal melatonin was administered for six weeks. The last group received both primary suture repair with one epineural suture each and 10 mg/kg intraperitoneal melatonin was administered for six weeks.

All subjects underwent a laryngoscopic examination before the procedure. In addition, transoral laryngeal Electromyography (EMG) was recorded to be evaluated as the baseline value of the healthy larynx. For the surgical procedure, the surgical area was prepared aseptically for surgery. Subjects breathed in the room air throughout the entire surgical procedure. A vertical incision of approximately 2 cm was made from the mentum to the sternum in the midline of the neck with a 15-blade scalpel. After passing through the skin and subcutaneous tissue, the salivary glands were gently retracted laterally for clear visualization of the surgical site, and the trachea was reached in the midline between the strap muscles (Figure 1a). After this stage, the left RLN was seen in the tracheoesophageal groove and was dissected (Figure 1b). The nerve was cut in a straight line with the 11-blade scalpel, and the subjects in the first and fourth groups underwent primary epineural nerve suture repair with one 10.0 polypropylene suture (Figure 1c). After suturing the strap muscles and skin, the surgical field was closed, and the operation was terminated (Figure 1d). In order to control the effect on the vocal cord immediately after the surgery, laryngoscopic examination of all animals was performed again, and it was observed that the left vocal cord was paralyzed.

Figure 1
Surgical steps in order of a, b, c and d. a) A vertical incision of approximately 2 cm was made from the mentum to the sternum in the midline of the neck with a 15-blade scalpel. After passing through the skin and subcutaneous tissue, the salivary glands were gently retracted laterally for clear visualization of the surgical site, and the trachea was reached in the midline between the strap muscles. b) The left RLN was seen in the tracheoesophageal groove and was dissected. c) The nerve was cut in a straight line with the 11-blade scalpel, and the subjects in the first and fourth groups underwent primary epineural nerve suture repair with 10.0 polypropylene sutures. d) After suturing the strap muscles and skin, the surgical field was closed, and the operation was terminated.

For laryngoscopy procedures, all subjects were placed on their backs under anesthesia and then inclined to 15 degrees in reverse Trendelenburg position. A 3.0 silk suture was passed through the anterior middle 1/3 of the tongue, and the tongue was pulled and suspended to provide a better view of the field. Vocal cord movements were evaluated with a grade 3.0 otoendoscope (R11573A; Karl Storz (Karl Storz Endoscope, Tuttlingen, Germany). Vocal cord movements were scored for standardization: 0; completely immobile, 1; decreased motion in the vocal fold, 2; evaluated as normal vocal fold motion.

All subjects underwent transoral laryngeal EMG under general anesthesia during the unstimulated respiratory cycle. After the vocal cord examination under general anesthesia, the EMG electrode was placed in a way that coincides with the left posterior cricoarytenoid muscle. A ground electrode was placed on the right pectoralis major muscle of the subject. The screen sweep rate was set to 10 ms/division; the screen sensitivity was set to 100 mV. EMG recordings were taken from all subjects before and after the surgical procedure, in the third week and in the sixth week (Figures 2a, b and c).

Figure 2
EMG procedure steps in order of a, b and c. a) Vocal cord movements were evaluated with a grade 3.0 otoendoscope (R11573A; Karl Storz (Karl Storz Endoscope, Tuttlingen, Germany). b) Vocal cords were examined under general anesthesia. Vocal cord movements were scored for standardization: 0; completely immobile, 1; decreased motion in the vocal fold, 2; evaluated as normal vocal fold motion. c) The EMG electrode was placed in a way that coincides with the left posterior cricoarytenoid muscle. A ground electrode was placed on the right pectoralis major muscle of the subject. The screen sweep rate was set to 10 ms/division; the screen sensitivity was set to 100 mV.

After a six-week recovery period, all subjects were sacrificed using the cervical dislocation method, and the larynx and the tracheal tissues were removed. Tissue samples were embedded in paraffin. From the prepared paraffin blocks, 4 µm thick sections were taken on polylysine slides using a Thermo Scientific HM 355S microtome (Thermo Fisher Scientific, Waltham, MA, USA). For hematoxylin-eosin (Bio-Optica, Milano, Italy) staining, the sections taken with a microtome were placed on lapped slides and kept at room temperature to dry until the staining process. Tissue samples were also examined for immunofluorescence for Ki-67 and Myelin Protein Zero.

Four micron thick tissue samples were cut from paraffin embedded blocks and deparaffinized using a decreasing alcohol series. Citrate buffer was heated in the microwave for 20 minutes. Endogenous peroxidase activity was blocked with 3% H2 O2. The primary antibodies used was anti-Ki-67 (1/400 dilution, GeneTex; Cat. No: GTX16667; USA) and anti-P0 (1/100 dilution Thermo Fisher Scientific, Waltham, MA, USA Cat. No: PA5-72541.

Tissue samples were incubated overnight at +4 degrees. The secondary antibody [Ultra Vision Large Volume Detection System Anti-rabbit by LabVision, conjugated with horse radish peroxidase (HRP)] was used in accordance with the manufacturer’s instructions. DAB (3,3’-diaminobenzidine) was used for immunohistochemical staining of HRP conjugated secondary antibody-labeled proteins in tissues. Mayer’s hematoxylin was used as the counterstain. The prepared slides were covered in mounting medium (Aqueous Mounting Medium by ScyTek). Anti-P0 and anti Ki-67 acitivity was assessed with semi-quantitative analysis (h-score) by selecting 10 random fields, and 200 cells were photographed in each area. The Ki-67, P0 indexes were calculated as the percentage of positively stained cells among the total cells assessed (Yokoyama et al. 1998, McCampbell et al. 2008). Immunohistochemically positive cell percentages of Ki-67 and P0 proteins belonging to the study groups are seen in (Table II).

Table I
Comparison of Third- and Sixth-Week Vocal Cord Recovery Rates Between Groups.

Time	Recovery	1st Group n (%)	2nd Group n (%)	3th Group n (%)	4th Group n (%)	p
3rd week results	Partially Recovery	1 (%14.3)	0	0	3 (%42.9)	0.237
	Total Recovery		0	0	0	-
	Partially/Total Recovery	1 (%14.3)	0	0	3 (%42.9)	0.237
6th week results	Partially Recovery	2 (%28.6)	0	0	2 (%28.6)	0.788
	Total Recovery	1 (%14.3)	0	0	2 (%28.6)	-
	Partially/Total Recovery	3 (%42.9)	0	0	4 (%57.2)	0.593
p		0.097	-	-	0.039*

• Chi-square test;
  n: number, %: Percentage, * the significance level was set at p=0.05.

Table II
Histological and Immunohistochemical Results of All Groups.

Parameter	1th Group Mean±SD Median [QL-QU]	2nd Group Mean±SD Median [QL-QU]	3th Group Mean±SD Median [QL-QU]	4th Group Mean±SD Median [QL-QU]	p
Schwann cells +	14.43±1.27 15.00 [14.00-15.00]	9.00±0.82 9.00 [8.00-10.00]	11.71±0.76 12.00 [1.00-12.00]	18.00±2.24 17.00 [16.00-20.00]	<0,001* p1-2=0.002 p2-4<0.001 p3 -4=0.002
Axonal degeneration +	8.00±0.82 8.00 [7.00-9.00]	15.29±1.11 15.00 [14.00-16.00]	11.86±1.34 12.00 [11.00-13.00]	4.14±0.69 4.00 [3.00-4.00]	<0,001** p1-2=0.002 p2-4<0.001 p3-4=0.001
Cytoplasmic Vacuolization +	5.43±0.98 5.00 [5.00-6.00]	11.71±1.50 12.00 [10.00-13.00]	9.29±1.80 9.00 [8.00-11.00]	3.86±0.69 4.00 [3.00-4.00]	<0,001** p1-2=0.002 p1-3=0.050 p2-4<0.001 p3-4=0.001
Ki-67 on vocal cord α	%20.43±1.71 20.00 [19.00-22.00]	%11.29±1.25 12.00 [10.00-12.00]	%13.43±0.80 14.00 [13.00-14.00]	%22.14±1.46 22.00 [21.00-23.00]	<0,001** p1-2=0.001 p1-3=0.038 p2-4<0.001 p3-4=0.003
P0 protein α	%18.29±1.89 18.00 [17.00-19.00]	%13.86±1.21 14.00 [13.00-15.00]	%13.86±1.21 14.00 [13.00-15.00]	%26.57±1.81 26.00 [25.00-28.00]	<0,001** p1-2=0.017 p1-3=0.018 p2-3=0.016 p2-4<0.001 p3-4<0.001
Ki-67 on connective tissue α	%11.71±1.11 12.00 [11.00-13.00]	%7.43±0.98 7.00 [7.00-8.00]	%8.29±0.95 8.00 [8.00-9.00]	%13.71±1.50 14.00 [12.00-15.00]	<0,001*** p1-2=0.003 p1-3=0.026 p2-4<0.001 p3-4=0.001

• *Welch Anova Test, **Anova Test,*** Kruskal Wallis H Test, The significance level was set at p = 0.05. SD: Standart Deviation, Q _L : Lower Quartile, Q _U : Upper Quartile. +: Number of positive cells per squared mm. α : Percentage of positively stained cells among the total cells assessed.

Schwann cells in the peripheral nervous system play a pivotal role in several aspects of nerve repair such as degeneration, remyelination, and axonal growth. Myelin Protein Zero (P0) is a major structural component of the myelin sheath in the peripheral nervous system hence, the Schwann cells. A high P0 density is an indicator for remyelination and regeneration in these cells. Furthermore, Ki-67 is a protein that is found only in cells that are dividing. A high Ki-67 proliferation index means many cells are regenerating rapidly. Therefore, tissue samples were also examined for immunofluorescence for Ki-67 and Myelin Protein Zero (P0) to analyse nerve regeneration.

SPSS (Statistical Package for Social Sciences) v.26 program was used for statistical analysis. Mean, ± standard deviation values were used for continuous variables, and percentage values were used for categorical variables. Compliance with the normal distribution was checked with the Kolmogorov Smirnov test in the follow-up of the comparison of the numerical data. As a result, it was decided to use non-parametric tests. Kruskal Wallis tests were used for numerical comparisons between groups and Chi-square tests were used for categorical comparisons. Post-Hoc analysis was performed in case of statistical significance in multiple comparisons. p<0.05 was considered sufficient for statistical significance.

RESULTS

All subjects participating in the study before the procedure underwent laryngoscopic examination, and vocal cord examinations were observed to be normal. In the laryngoscopic examination performed after the left RLN incision, left VCP was observed in all subjects, as expected.

Vocal cord movements were observed in more subjects in the fourth group than other groups in the third and sixth-week evaluations. However, the differences were insignificant (p=0.237, p=0.593) (Table I). When the results of the third and sixth months were compared separately within the groups, the difference between the third and sixth months did not constitute statistical significance. However, the improvement rate increased numerically in the first group over time (p=0.097). In the second and third groups, since no vocal cord movement was observed in any of the subjects for six weeks, these groups were not included in the statistical evaluation. In the fourth group, it was observed that the total of subjects with partial and complete vocal cord movement in the sixth week increased at a statistically significant level (p=0.039) (Table I).

All subjects in the experimental groups had normal motor unit potential (MUP) amplitude values in the transoral laryngeal EMG tests performed before the procedure. In the EMG recordings of all subjects after the procedure, MUP amplitudes disappeared as expected. In the EMG recordings made in the third week, MUP amplitude was detected in one subject with endoscopic left vocal cord movement in the first group. Vocal fold movement was not observed in other subjects in the first group, and MUP could not be detected in EMG. In the fourth group, MUP amplitude was detected in three subjects with endoscopic left vocal cord movement, while MUP could not be detected in four subjects in this group whose left vocal cord was immobile. MUP could not be obtained in any subjects in the second and third groups. In the EMG recording made in the sixth week, In the first group, MUP was found in three subjects with endoscopic left vocal cord movement, while MUP amplitudes became more frequent and voltage values increased in the subject with MUP in the third week. In the fourth group, MUP was detected in four subjects with endoscopic left vocal cord movement, while MUPs became more frequent, and voltage values increased in subjects with MUP in the third week. No statistically significant difference was found in the statistical comparison of the MUP amplitude values of the first and fourth groups with MUP amplitude in the sixth week. However, the number of subjects with MUP was high in the fourth group, and the amplitude values were high (p=0.248). In the EMG examination, when the temporal variation of the amplitude values in the fourth group were examined, a statistically significant difference was observed between the MUP amplitude values calculated in the third and sixth weeks (p=0.021) (Figure 3).

Figure 3
Time Variable MUP Amplitude Values of the Groups. MUP: Motor Unit Potential.

Histopathological examinations were examined under a light microscope at 40X, 100X, and 200X magnifications. Eight linked sections were carefully selected from each experimental group, and the mean values were numerically determined by counting six consecutive areas of each cross-section. When the RLN surround was examined by hematoxylin-eosin staining, pycnotic neuron cells and occasionally Schwann cells were observed in the first group, and it was observed that the epineurium layer started to heal. In the second group, nerve tissue fragmentation was observed, Schwann cells were less common than in the first group, and damaged pycnotic neuron cells were observed. In the third group, Schwann cell counts were low. In the third group, the epineurium layer was enlarged. In the fourth group, a large number of Schwann cells and very few pycnotic neuron cells were seen. The epineurium layer was in its usual arrangement around the nerve tissue (Figure 4). The number of Schwann cells was evaluated and compared between the groups, a statistically significant difference was observed (p<0.001).

Figure 4
First column: Hematoxylin Eosin staining image of peripheral nerve tissue of the groups 50 scala bar, 200x images. Gray arrowhead (Schwann cells), black star (damaged neuron cells with pycnotic nuclei), black arrow (cytoplasmic vacuolization), black triangle (axon damage), crossout sign (fragmented nerve tissue damage), black arrowhead (epineurium layer). Second column: P0 protein and Ki-67 immunohistochemical staining preparations in the peripheral nerve injury area, 50 scala bar ve 200x images. In Group 4, Ki-67, and P0 intensity was the highest; in Group 1 and Group 3, the lowest Ki-67, and P0 staining intensity was observed in Group 2. Third column: Ki-67 immunohistochemistry staining preparations of the groups in the vocal cord area, 50 scala bars. 200x magnification. In Group 4, Ki-67 intensity was the highest; in Group 1 and Group 3, the lowest Ki-67 staining intensity was observed in Group 2. In addition, the epithelial layer and underlying connective tissue and muscle layer were the thickest in Group 4. The thinnest layer was in Group 2.

The groups were also compared in terms of axon damage, cytoplasmic vacuolization, P0 density, and Ki-67 density in the nerve damage area,and it was seen that the above-mentioned findings were mainly in the second group and least in the fourth group. The difference between the groups was statistically significant for all studies (p<0.001) (Figure 4).

As a result of the histopathological examination of the vocal cords, the fourth group in this area showed the highest Ki-67 staining intensity, whereas the lowest Ki-67 staining intensity was found in the second group. In addition, the epithelial layer, the underlying connective tissue, and the muscle layer were found thickest in the fourth group,whereas the thinnest layer was found in the second group. The vocal cords were found to be thicker in the groups who underwent primary suture repair. When Ki-67 was evaluated in the vocal cord, there was a statistically significant difference between the groups (p<0.001) (Figure 4) (Table II).

DISCUSSION

Treatment for RLN injury aims to protect speech, swallowing, and respiration. End-to-end anastomosis is the gold standard treatment after the nerve trauma. However, perfect results cannot be obtained even in this way because functional recovery rates are at most 50% (Ayık 2019). Although treatments such as stem cells (Saïd et al. 2021), Polyglycolic acid (PGA) collagen tube (Suzuki et al. 2016), thymoquinone, methylprednisolone (Sereflican et al. 2016), ketorolac, platelet-rich plasma, aspartame (Okasha 2016), and mitomycin (Chen & Chang 2010) have been tried as a treatment for peripheral nerve healing, research for new methods is still up-to-date.

In the study of Suzuki et al. (2016), the effectiveness of the PGA collagen tube after rat RLN injury was examined. One group was sutured, the other group’s nerve endings were left 1mm apart, and a PGA collagen tube was wrapped around it. At the end of 15 weeks, vocal cord movement, nerve conduction velocity, and histologically evaluated nerve integrity were macroscopically observed. Although laryngeal muscle atrophy was prevented, vocal cord movement was not observed, and nerve conduction velocities could not be evaluated in both groups (Suzuki et al. 2016). Motoyoshi et al. (2004) looked at the activity of FGF after RLN injury. In this ninety-day study, vocal cord movement was observed in seven of eight animals given Fibroblast Growth Factor (FGF), whereas in the other group that was given saline, only three of eight animals’ vocal cord movement was observed (Motoyoshi et al. 2004). In our study, at the end of six weeks, vocal cord movement was observed in four of the seven subjects in the fourth group, which was sutured after the RLN injury; two of the four subjects were partially mobile, while the other two were considered fully mobile. In the first group, which was sutured and did not receive any treatment, vocal cord movement was observed in three of the seven subjects, while the vocal cord was partially mobile in two of the three subjects,and fully mobile in one subject. No vocal cord movement was observed in any of the subjects in the second and third groups, which was not repaired after the nerve injury was made. Furthermore, repair with epineural suture and melatonin was found to be effective in nerve healing. Recovery was faster in the group receiving melatonin compared to the other groups.

The rate of regeneration after nerve injury varies between species. The rate of axon regeneration is 1-2 mm/day in humans, while it is 2-3.5 mm/day in rodents. In the study of Kaya et al. (2013), when the effect of melatonin treatment on sciatic nerve crush and incision damage was evaluated after six weeks, it showed positive results in terms of nerve healing. In the study of Rateb et al. (2017), it was stated that melatonin had an anti-inflammatory effect in a six-week period. Edizer et al. (2019) evaluated the effect of melatonin and dexamethasone on neural regeneration over a twelve-week period to evaluate melatonin therapy in facial nerve injury. He stated that the latency period was shortened in the melatonin group compared to the other groups. Our study was planned to include a six-week recovery period, considering that the duration of the experiment was different in many studies, an ideal time could not be determined, productivity might decrease, and there might be loss of subjects in studies that lasted for months. As a result of the study, although we obtained histologically significant statistical results, we saw that cellular healing could not transform into functional recovery sufficiently. We think that there will be improvement in more subjects in longer-term studies, and this improvement will be reflected in both examination and electrophysiological tests.

Oxidative stress and inflammation are one of the most worrisome conditions in peripheral nerve regeneration. They usually cause neural damage after the initial injury. Schwann cells and macrophages express proinflammatory signals and lead to the generation of free oxygen radicals in the axotomized region (Kubo et al. 2002, Zochodne & Levy 2005). Melatonin after peripheral nerve injury has been shown to reduce scar formation, have a proliferative effect on Schwann cells, prevent collateral sprouting at the neuromuscular junction by affecting the motor end plate in reinnervation, provide proper nerve healing, and reduce neuronal cell death and apoptosis (Ayık 2019). MnSOD is an important antioxidant enzyme that plays a role in the control of oxidative stress. In the study of Chen & Chang (2010) it was mentioned that melatonin regulates MnSOD expression in hypoglossal nerve damage. The neuroprotective effect of melatonin, especially at high doses, has been demonstrated in sciatic nerve damage (Shokouhi et al. 2008). It has been confirmed that melatonin can increase the antioxidant and anti-inflammatory reaction in peripheral nerve damage. It has been reported that appropriate melatonin provides sustained Schwann cell proliferation, axon regeneration and axon regeneration and remyelination over a long period of time (Qian et al. 2018). The positive effects of melatonin on nerve regeneration are associated with induced Schwann cell proliferation, which provides the ideal environment for axonal growth (Chen & Chang 2010). In our study, it was shown that the group given melatonin treatment after RLN injury caused a significant increase in the number of Schwann cells and a significant decrease in axon damage, as in other studies, compared to the control group, which underwent nerve repair and did not receive any treatment. Also, less cytoplasmic vacuolization suggested that melatonin reduced cell death and oxidative stress.

Transoral endoscopic laryngeal EMG was first performed by Thumfart et al. (1979) in the human larynx with a bipolar electrode and needle electrode. Inagi et al. (1997) developed the system further and used it to evaluate vocal cord movements in the rat larynx. In our study, we followed the technique developed by Tessema et al. (2008) and performed laryngeal EMG with a 0-degree otoendoscope with the EMG electrode in our dominant hand and the endoscope in the other hand. EMG was used to monitor the regeneration of the recurrent nerve after crush and incision injuries. Polyphasic motor unit potential and recruitment in EMG before vocal cord movement was shown as evidence of reinnervation (Shiotani et al. 2001). In our study, mean MUP amplitude values obtained at the end of six weeks were well below the pre-procedural values. This is because not all subjects have recovered yet, and the MUP amplitude cannot be obtained in these unhealed subjects, which lowers the mean. At the same time, it is predicted that the regenerated nerve fibers may not reach their previous values due to misdirection in the recovery after injury, and it can be predicted that MUP values will be closer to the basal value in a more extended study.

Although Ki-67 expression does not directly assess cell proliferation, the expression has been shown to be a reliable marker of cell proliferation (Brown et al. 2014). In fact, it is routinely used in the response of cancer patients to chemotherapy today (Chen & Chang 2010). In our study, Ki-67 expression was found significantly higher in the fourth group. This supports the fact that melatonin triggers proliferation and provides faster recovery.

Myelin protein zero is the protein that makes up 60% of the structure of myelin in the peripheral nervous system. It acts as a regulator of myelination (Shapiro et al. 1996). It is an integral membrane glycoprotein synthesized by Schwann cells, represents the main glycoprotein of peripheral nerve myelin, and plays a fundamental role in the adhesion and compression of peripheral myelin. The Myelin protein zero value was higher in a group that underwent neural modulation due to chronic compression damage to the sciatic nerve (da Silva et al. 2015). Many studies have shown that it is necessary for myelin formation and plays an essential role in axonal regeneration. In our study, myelin protein zero values were found to be significantly higher in the fourth group. Since this protein shows myelination, melatonin appears to be effective in nerve healing and axonal regeneration by taking a role in myelination.

However, this study has some limitations that bear consideration. First, this study was conducted over six weeks in a small number of rats, and thus larger-scale, longer-term studies are needed to confirm our findings. Furthermore, another limitation of this study was the failure to measure antioxidant enzyme activity and degree of membrane lipid peroxidation in the damaged RLN. Lastly, we used immunohistochemistry to document the expression of Ki-67 and Myelin Protein Zero(P0) within nerve fibers, and despite the value of this approach for detecting specific proteins in tissues, studies supported by more quantitative techniques such as protein and gene analysis should be conducted to confirm and expand our results.

CONCLUSIONS

Melatonin contributes to nerve healing process with its possible anti-inflammatory, antioxidant, and antiapoptosis effects, as it is shown by increasing the number of Schwann cells, reducing axon damage, and decreasing cytoplasmic vacuolization. Likewise, myelination was more common in the group using melatonin, which can be considered another factor showing that it contributes to nerve regeneration. The data in our study suggests that we would encounter higher functional results in longer-term studies.

ACKNOWLEDGMENTS

We appreciate the economic contributions of the Sakarya University Scientific Research Projects Unit.

REFERENCES

• AYIK G. 2019. Akut Periferik Sinir Yaralanmalarının Rejenerasyonunda Epidermal Büyüme Faktörü (EGF)’nün Etkilerinin Tavşan Modeli Üzerinde Histolojik İncelenmesi. (Published Doctoral Thesis, Hacettepe University, Department of Orthopedıcs and Traumatology, Ankara).
• BROWN JR, DIGIOVANNA MP, KILLELEA B, LANNIN DR & RİMM DL. 2014. Quantitative assessment Ki-67 score for prediction of response to neoadjuvant chemotherapy in breast cancer. Lab Invest 94(1): 98-106. DOI: 10.1038/labinvest.2013.128.
• CHEN TC & CHANG SW. 2010. Effect of mitomycin C on IL-1R expression, IL-1-related hepatocyte growth factor secretion and corneal epithelial cell migration. Invest Ophthalmol Vis Sci 51(3): 1389-1396. DOI: 10.1167/iovs.09-3494.
• DA SILVA JT, SANTOS FM, GIARDINI AC, MARTINS D DE O, DE OLIVEIRA ME, CIENA AP, GUTIERREZ VP, WATANABE IS, BRITTO LR & CHACUR M. 2015. Neural mobilization promotes nerve regeneration by nerve growth factor and myelin protein zero increased after sciatic nerve injury. Growth Factors 33(1): 8-13. DOI: 10.3109/08977194.2014.953630.
• EDIZER D, DÖNMEZ Z, GÜL M, YİĞİT Ö, YIĞITCAN B, ADATEPE T & UZUN N. 2019. Effects of Melatonin and Dexamethasone on Facial Nerve Neurorrhaphy. J Int Adv Otol 15(1): 43-50. DOI: 10.5152/iao.2018.3273.
• INAGI K, RODRIQUEZ AA, FORD CN & HEISEY DM. 1997. Transoral electromyographic recordings in botulinum toxin-injected rat larynges. Ann Otol Rhinol Laryngol 106(11): 956-964. DOI: 10.1177/000348949710601112.
• KANDEMIR Y & SARIKCIOGLU L. 2015. Melatonin and its therapeutic actions on peripheral nerve regeneration. Folia Morphol (Warsz) 74(3): 283-289. DOI: 10.5603/FM.2015.0043.
• KAYA Y, SARIKCIOĞLU L, ASLAN M, KENCEBAY C, DEMIR N, DERIN N, ANGELOV DN & YILDIRIM FB. 2013. Comparison of the beneficial effect of melatonin on recovery after cut and crush sciatic nerve injury: a combined study using functional, electrophysiological, biochemical, and electron microscopic analyses. Childs Nerv Syst 29(3): 389-401. DOI: 10.1007/s00381-012-1936-0.
• KUBO T, YAMASHİTA T, YAMAGUCHİ A, HOSOKAWA K & TOHYAMA M. 2002. Analysis of genes induced in peripheral nerve after axotomy using cDNA microarrays. J Neurochem 82(5): 1129-1136. DOI: 10.1046/j.1471-4159.2002.01060.x.
• LAL G & CLARK O. 2005. Thyroid, parathyroid and adrenal. Schwartz’s principles of surgery. 8th ed. New York (NY): McGraw-Hill, p. 1395-1470.
• MOK ET AL. 2020. A Surgical Mouse Model for Advancing Laryngeal Nerve Regeneration Strategies. Dysphagia 35(3): 419-437. DOI: 10.1007/s00455-019-10045-6.
• MOTOYOSHI K, HYODO M, YAMAGATA T & GYO K. 2004. Restoring vocal fold movement after transection and immediate suturing of the recurrent laryngeal nerve with local application of basic fibroblast growth factor: an experimental study in the rat. Laryngoscope 114(7): 1247-1252. DOI: 10.1097/00005537-200407000-00020.
• OKASHA EF. 2016. Effect of long term-administration of aspartame on the ultrastructure of sciatic nerve. J Microsc Ultrastruct 4(4): 175-183. DOI: 10.1016/j.jmau.2016.02.001.
• QIAN Y, HAN Q, ZHAO X, SONG J, CHENG Y, FANG Z, OUYANG Y, YUAN WE & FAN C. 2018. 3D melatonin nerve scaffold reduces oxidative stress and inflammation and increases autophagy in peripheral nerve regeneration. J Pineal Res 65(4): e12516. DOI: 10.1111/jpi.12516.
• RATEB EE, AMIN SN, EL-TABLAWY N, RASHED LA & EL-ATTAR S. 2017. Effect of melatonin supplemented at the light or dark period on recovery of sciatic nerve injury in rats. EXCLI J 6(16): 138-150. DOI: 10.17179/excli2016-763.
• SAÏD Z, PAULINE C, CLAIRE B, CELIA D, JEAN-PAUL M & NICOLAS BM. 2021. Olfactory Ecto-Mesenchymal Stem Cells in Laryngeal Nerve Regeneration in Rats. J Voice 35(3): 349-359. DOI: 10.1016/j.jvoice.2019.10.012.
• SEREFLICAN M, YURTTAS V, OZYALVACLI G, TERZI EH, TURKOGLU SA, YILDIZ S, ILGAZ Y, SEYHAN S, ORAL M & DAGLI M. 2016. The histopathological and electrophysiological effects of thymoquinone and methylprednisolone in a rabbit traumatic facial nerve paralysis model. Am J Otolaryngol 37(5): 407-415. DOI: 10.1016/j.amjoto.2016.05.006.
• SHAPIRO L, DOYLE JP, HENSLEY P, COLMAN DR & HENDRICKSON WA. 1996. Crystal structure of the extracellular domain from P0, the major structural protein of peripheral nerve myelin. Neuron 17(3): 435-449. DOI: 10.1016/s0896-6273(00)80176-2.
• SHIOTANI A, NAKAGAWA H & FLINT PW. 2001. Modulation of myosin heavy chains in rat laryngeal muscle. Laryngoscope 111(3): 472-477. DOI: 10.1097/00005537-200103000-00017.
• SHOKOUHI G, TUBBS RS, SHOJA MM, HADIDCHI S, GHORBANIHAGHJO A, ROSHANGAR L, FARAHANI RM, MESGARI M & OAKES WJ. 2008. Neuroprotective effects of high-dose vs low-dose melatonin after blunt sciatic nerve injury. Childs Nerv Syst 24(1): 111-117. DOI: 10.1007/s00381-007-0366-x.
• SUZUKI H, ARAKI K, MATSUI T, TOMİFUJI M, YAMASHITA T, KOBAYASHI Y & SHIOTANI A. 2016. Value of a novel PGA-collagen tube on recurrent laryngeal nerve regeneration in a rat model. Laryngoscope 126(7): E233-239. DOI: 10.1002/lary.25750.
• TESSEMA B, PITMAN MJ, ROARK RM, BERZOFSKY C, SHARMA S & SCHAEFER SD. 2008. Evaluation of functional recovery of recurrent laryngeal nerve using transoral laryngeal bipolar electromyography: a rat model. Ann Otol Rhinol Laryngol 117(8): 604-608. DOI: 10.1177/000348940811700810.
• THUMFART W, STEİNER W & JAUMANN MP. 1979. Lupenendoskopische Elektromyographie des Musculus crico-arytaenoideus dorsalis (posticus) am wachen Patienten [Electromyography of the cricoarytenoid muscle in the unsedated patient using the zoom-endoscope (author’s transl)]. HNO 27(6): 201-206. German. PMID: 457465.
• WANG B, YUAN J, XU J, XIE J, WANG G & DONG P. 2016. Neurotrophin expression and laryngeal muscle pathophysiology following recurrent laryngeal nerve transection. Mol Med Rep 13(2): 1234-1242. DOI: 10.3892/mmr.2015.4684.
• ZOCHODNE DW & LEVY D. 2005. Nitric oxide in damage, disease and repair of the peripheral nervous system. Cell Mol Biol (Noisy-le-grand) 51(3): 255-267. PMID: 16191393.

Publication Dates

• Publication in this collection
  21 Oct 2024
• Date of issue
  2024

History

• Received
  29 Oct 2023
• Accepted
  6 May 2024