Abstract
Objective
We aimed to determine the effects of methylprednisolone and thymoquinone on nerve healing in a traumatic facial nerve paralysis animal model.
Subjects and methods
Twenty-four rabbits were randomly divided into 4 groups: group I: control group received no medication and no trauma; group II: sham group received no medication after facial nerve trauma group III: 5 mg/kg/day thymoquinone administered; group IV: 1 mg/kg/day methylprednisolone administered. An initial electrophysiological assessment was performed in all the animals. The buccal branch of the facial nerve was then clipped to form a traumatic facial paralysis model. The drugs were administered for two weeks once a day. At the end of the second month, the electrophysiological assessments were performed and the distal part of the traumatic facial nerve were dissected and examined under light microscopy.
Results
Best nerve regeneration was observed in the control and the thymoquinone groups, respectively, whereas the weakest regeneration was determined in the sham group. Thymoquinone and methylprednisolone significantly increased nerve recovery, as measured by histopathological scores and electrophysiological assessment. In the thymoquinone group, due to postoperative amplitude, axon diameter and thickness of myelin sheath values were significantly further increased nerve regeneration compared to that of the methylprednisolone group and these values were close to those of the values of the control group.
Conclusion
Thymoquinone was slightly better than methylprednisolone for functional nerve recovery. The neuroprotective effect of thymoquinone was attributed to its antioxidant and anti-inflammatory effects. Thymoquinone can have a new treatment option to ameliorate the nerve injury.
1
Introduction
Facial expressions, controlled by fine movements of the facial muscles, are essential features that serve important functional and aesthetic roles. Innervation of all muscles involved in facial expression is controlled by the facial, or seventh cranial nerve . Facial nerve paralysis is the most commonly encountered cranial neuropathy, and develops for various reasons . After Bell’s palsy, trauma is the second most common cause of facial nerve paralysis. During post-traumatic recovery, inflammation can lead to extensive fibroconnective tissue formation on the nerve tissue and scar formation .
Although many methods are used to treat traumatic peripheric facial nerve paralysis (TPFNP), the functional outcomes and complications after such treatments are not always satisfactory . Therefore, additional studies on alternative treatment methods for better functional and cosmetic recovery are ongoing. Due to their anti-inflammatory and anti-edema effects, corticosteroids are the most frequently used agents in the treatment of both traumatic and idiopathic facial paralysis . However, several studies have reported that corticosteroids have harmful effects on wound healing .
Thymoquinone (TQ), a bioflavonoid, can be isolated from Nigella sativa (NS), which is widely grown in Mediterranean countries and belongs to the Ranunculaceae family. Many studies have shown that TQ (2-isopropyl-5-methyl-1,4-benzoquinone; C10H12O2) has beneficial antioxidant, anti-inflammatory, anti-hyperlipidemic, anti-diabetic, anti-allergic, gastroprotective, hepatoprotective, and anti-carcinogenic effects . In addition, recent studies have shown that TQ has neuroprotective effects by acting as a free radical scavenger for radicals released after traumatic nerve injury and the subsequent production of arachidonic acid and other metabolites from membrane lipid peroxidation . However, a review of the literature did not uncover any studies on the effect of TQ on traumatic peripheric facial nerve injury.
The purpose of this study was to investigate the effect of TQ on peripheral nerve regeneration using a rabbit facial nerve model. Assessment of nerve regeneration was based on electrophysiological measurement and histopathological assessment.
2
Material and methods
This study was performed in the Experimental Animal Research Center of XXXXXXXX University. In total, 24 male albino New Zealand rabbits, each weighing 2–3 kg and aged 6 months, were used. The study protocol was approved by the XXXXXXX University Faculty of Medicine Ethics Committee (B.30.2.ABÜ.0.05.05–050.01.04–78). All animals were housed in an air-conditioned room at constant temperature (18–20 °C) with a 12-h light/dark cycle, and given food and water ad libitum throughout the experiment.
2.1
Preparation of drugs
Thymoquinone (TQ), obtained in pure form, was dissolved in 0.1% dimethyl sulfoxide (DMSO) to a concentration of 6.25 mg/ml, and 1 mg/kg/day/2 ml was administered intraperitoneally once a day for 14 days. Methylprednisolone was dissolved in the pure water provided in its commercial package, and 5 mg/kg/day/2 ml was administered intramuscularly once a day for 14 days.
Group 1 (Control group): No trauma or medication, facial nerve was exposed and then the skin was sutured.
Group 2 (Sham group): Received no medication after facial nerve trauma.
Group 3 (Thymoquinone group): TQ (Thymoquinone, Sigma–Aldrich Chemie, Steinheim, Germany), 5 mg/kg/day, was administered intraperitoneally once a day for 2 weeks beginning immediately after facial nerve trauma.
Group 4 (Methylprednisolone group): Methylprednisolone (Prednol L 1, Mustafa Nevzat Drug Ind, Turkey), 1 mg/kg/day, was administered intramuscularly once a day for 2 weeks after facial nerve trauma.
2.2
Surgical procedure
All operations were performed by same surgeon using the same standard surgical technique. The rabbits were anesthetized with 5 mg/kg xylazine hydrochloride (Rompun, Bayer Drugs, Turkey) and 35 mg/kg ketamine hydrochloride (Ketalar, Eczacibasi Drugs, Turkey). Skin on the facial nerve trace was shaved, cleaned with 70% ethanol and povidone iodine, and then dried. The procedure was performed on the left side under sterile conditions using 3 × magnification surgical eye loupes. An oblique incision approximately 4 cm in length was made over the rabbit zygoma, from posterior–superior to anterior–inferior under the eye. The skin and the subcutaneous tissue were dissected until the superficial fascia was revealed, and the buccal branch of the facial nerve was identified ( Fig. 1 a ). The buccal branch innervates the rabbit’s quadratus labii superioris muscle, which plays a major role in movement of the upper lip and whiskers . An initial electrophysiological assessment was then performed in all the animals for basal measurement, as described in the electrophysiological assessment part of the “Materials and Methods” section. The buccal branch of the facial nerve was then clipped for 3 min using a mosquito clamp . Monofilament polydioxanone sutures (7–0) (PDS II; Ethicon, Germany) were then used in the subcutaneous tissue near the destroyed section of the facial nerve to mark the area for the follow-up surgery performed 2 months later. The rabbits received 20–40 mg/kg Cephazolin sodium (Iespor, I.E. Ulagay, Turkey) for prophylaxis one hour prior to, and two hours after, the surgical procedure. Following surgery, all rabbits, except the control and sham groups, were given medications for 2 weeks as described above.
2.3
Electrophysiological assessment
Electrophysiological analysis was performed following anesthesia. Skin-surface temperature was maintained at 32–36 °C using a heat lamp as necessary. The rabbits underwent electromyography (EMG) and electrophysical testing (Synergy On Nicolet EDX, Care Fusion model) of their facial nerves preoperatively and postoperatively at week 8. After surgically exposing the buccal branch of the facial nerve, stimulus electrodes were applied to the exposed nerve. Stimulation was given using two standard silver chloride cup electrodes with the cathode and anode placed 1 cm apart (Ag–AgCl, 10-mm diameter, Nihon Kohden, NM-312S). The recording was made using two subdermal needle electrodes inserted 1 cm apart into the ipsilateral quadratus labii superioris muscle (SN-SNE0915, Rhythmlink 13-mm-long 0.4-mm-diameter needle) ( Fig. 1 b). After a single-pulse stimulus was applied, the intensity was slowly increased until maximal compound muscle action potentials (cMAPs) were obtained. Maximal response was sequentially recorded with supramaximal stimuli. The amplitude (mV) and latency (ms) from the stimulus to the onset of the response were measured. Peak-to-peak amplitude measurements were performed, and the supramaximal value was accepted. The same electrophysiological assessments were performed before surgery and at 8 weeks after surgery. All basal and 8-week values were compared in detailed.
2.4
Histopathological evaluation
All rabbits were examined 8 weeks post-surgery and anesthetized by the same technique. The previously cut skin was incised in the same region, and the destroyed site was identified by locating the previously implanted sutures. The nerve was dissected from the adjacent tissues and released. The buccal branch of the facial nerve, including regions 5 mm distal from the destroyed region, was then excised. The distal part of the facial nerve was fixed in a 2.5% glutaraldehyde in 0.1 M PBS solution; post fixation was performed using osmium tetroxide for 1 h at 4 °C after washing with 0.1 M PBS solution . Nerve tissues were embedded in araldite. After polymerizing the araldite, tissue blocks were prepared, and 1-μm semi-thin sections were cut, stained with 1% Toluidine Blue in 1% sodium borate for 30 s at 60 °C, and studied with light microscopy. Histologic assessment was performed on 24 specimens, and the following features were recorded: axonal degeneration, hyperplasia of Schwann cells, axon diameter, vacuolization, myelin sheath thickness, and axon count .
2.5
Statistical analysis
Statistical analyses were made by use of IBM SPSS Statistics for Windows, version 20.0 (Armonk, NY: IBM Corp.). Continuous variables were tested for normality by the Kolmogorov–Smirnov test. Normally distributed data are presented as mean ± standard deviations. The rates and proportions of discrete variables were performed using the chi-square test. For data not normally distributed, median with data range (minimum to maximum) was used. The Wilcoxon test was used to compare the change in latance and amplitude. One-way ANOVA was used to compare normally distributed variables. The Kruskal–Wallis test is used for comparing ordinal and non-normal variables for more than two groups. The independent samples t-test and Mann–Whitney U test were used to test the significance of pairwise differences using Bonferroni corrections to adjust for multiple comparisons. A “ p ” value < 0.05 was considered as significant.
2
Material and methods
This study was performed in the Experimental Animal Research Center of XXXXXXXX University. In total, 24 male albino New Zealand rabbits, each weighing 2–3 kg and aged 6 months, were used. The study protocol was approved by the XXXXXXX University Faculty of Medicine Ethics Committee (B.30.2.ABÜ.0.05.05–050.01.04–78). All animals were housed in an air-conditioned room at constant temperature (18–20 °C) with a 12-h light/dark cycle, and given food and water ad libitum throughout the experiment.
2.1
Preparation of drugs
Thymoquinone (TQ), obtained in pure form, was dissolved in 0.1% dimethyl sulfoxide (DMSO) to a concentration of 6.25 mg/ml, and 1 mg/kg/day/2 ml was administered intraperitoneally once a day for 14 days. Methylprednisolone was dissolved in the pure water provided in its commercial package, and 5 mg/kg/day/2 ml was administered intramuscularly once a day for 14 days.
Group 1 (Control group): No trauma or medication, facial nerve was exposed and then the skin was sutured.
Group 2 (Sham group): Received no medication after facial nerve trauma.
Group 3 (Thymoquinone group): TQ (Thymoquinone, Sigma–Aldrich Chemie, Steinheim, Germany), 5 mg/kg/day, was administered intraperitoneally once a day for 2 weeks beginning immediately after facial nerve trauma.
Group 4 (Methylprednisolone group): Methylprednisolone (Prednol L 1, Mustafa Nevzat Drug Ind, Turkey), 1 mg/kg/day, was administered intramuscularly once a day for 2 weeks after facial nerve trauma.
2.2
Surgical procedure
All operations were performed by same surgeon using the same standard surgical technique. The rabbits were anesthetized with 5 mg/kg xylazine hydrochloride (Rompun, Bayer Drugs, Turkey) and 35 mg/kg ketamine hydrochloride (Ketalar, Eczacibasi Drugs, Turkey). Skin on the facial nerve trace was shaved, cleaned with 70% ethanol and povidone iodine, and then dried. The procedure was performed on the left side under sterile conditions using 3 × magnification surgical eye loupes. An oblique incision approximately 4 cm in length was made over the rabbit zygoma, from posterior–superior to anterior–inferior under the eye. The skin and the subcutaneous tissue were dissected until the superficial fascia was revealed, and the buccal branch of the facial nerve was identified ( Fig. 1 a ). The buccal branch innervates the rabbit’s quadratus labii superioris muscle, which plays a major role in movement of the upper lip and whiskers . An initial electrophysiological assessment was then performed in all the animals for basal measurement, as described in the electrophysiological assessment part of the “Materials and Methods” section. The buccal branch of the facial nerve was then clipped for 3 min using a mosquito clamp . Monofilament polydioxanone sutures (7–0) (PDS II; Ethicon, Germany) were then used in the subcutaneous tissue near the destroyed section of the facial nerve to mark the area for the follow-up surgery performed 2 months later. The rabbits received 20–40 mg/kg Cephazolin sodium (Iespor, I.E. Ulagay, Turkey) for prophylaxis one hour prior to, and two hours after, the surgical procedure. Following surgery, all rabbits, except the control and sham groups, were given medications for 2 weeks as described above.
2.3
Electrophysiological assessment
Electrophysiological analysis was performed following anesthesia. Skin-surface temperature was maintained at 32–36 °C using a heat lamp as necessary. The rabbits underwent electromyography (EMG) and electrophysical testing (Synergy On Nicolet EDX, Care Fusion model) of their facial nerves preoperatively and postoperatively at week 8. After surgically exposing the buccal branch of the facial nerve, stimulus electrodes were applied to the exposed nerve. Stimulation was given using two standard silver chloride cup electrodes with the cathode and anode placed 1 cm apart (Ag–AgCl, 10-mm diameter, Nihon Kohden, NM-312S). The recording was made using two subdermal needle electrodes inserted 1 cm apart into the ipsilateral quadratus labii superioris muscle (SN-SNE0915, Rhythmlink 13-mm-long 0.4-mm-diameter needle) ( Fig. 1 b). After a single-pulse stimulus was applied, the intensity was slowly increased until maximal compound muscle action potentials (cMAPs) were obtained. Maximal response was sequentially recorded with supramaximal stimuli. The amplitude (mV) and latency (ms) from the stimulus to the onset of the response were measured. Peak-to-peak amplitude measurements were performed, and the supramaximal value was accepted. The same electrophysiological assessments were performed before surgery and at 8 weeks after surgery. All basal and 8-week values were compared in detailed.
2.4
Histopathological evaluation
All rabbits were examined 8 weeks post-surgery and anesthetized by the same technique. The previously cut skin was incised in the same region, and the destroyed site was identified by locating the previously implanted sutures. The nerve was dissected from the adjacent tissues and released. The buccal branch of the facial nerve, including regions 5 mm distal from the destroyed region, was then excised. The distal part of the facial nerve was fixed in a 2.5% glutaraldehyde in 0.1 M PBS solution; post fixation was performed using osmium tetroxide for 1 h at 4 °C after washing with 0.1 M PBS solution . Nerve tissues were embedded in araldite. After polymerizing the araldite, tissue blocks were prepared, and 1-μm semi-thin sections were cut, stained with 1% Toluidine Blue in 1% sodium borate for 30 s at 60 °C, and studied with light microscopy. Histologic assessment was performed on 24 specimens, and the following features were recorded: axonal degeneration, hyperplasia of Schwann cells, axon diameter, vacuolization, myelin sheath thickness, and axon count .
2.5
Statistical analysis
Statistical analyses were made by use of IBM SPSS Statistics for Windows, version 20.0 (Armonk, NY: IBM Corp.). Continuous variables were tested for normality by the Kolmogorov–Smirnov test. Normally distributed data are presented as mean ± standard deviations. The rates and proportions of discrete variables were performed using the chi-square test. For data not normally distributed, median with data range (minimum to maximum) was used. The Wilcoxon test was used to compare the change in latance and amplitude. One-way ANOVA was used to compare normally distributed variables. The Kruskal–Wallis test is used for comparing ordinal and non-normal variables for more than two groups. The independent samples t-test and Mann–Whitney U test were used to test the significance of pairwise differences using Bonferroni corrections to adjust for multiple comparisons. A “ p ” value < 0.05 was considered as significant.
3
Results
3.1
Electrophysiological test results
3.1.1
Intragroup evaluation
For all groups, the preoperative and postoperative latency and amplitude values and the percentage of change in latency and amplitude values are shown in Table 1 . The median, minimum, and maximum values are shown in Table 2 . The percentage of change in latency and amplitude values was calculated as the difference between pre- and post-surgery process by dividing to pre-surgery value and then multiplying this value with a hundred.
Group 1: The preoperative and postoperative 2-month latency and amplitude values, and the percentage of change in latency and amplitude values were not significantly different.
Group 2: The postoperative amplitude values were significantly lower compared to the preoperative values ( p < 0.001 ). Similarly, the postoperative latency values were significantly higher compared to the preoperative values ( p < 0.001 ).
Group 3: The postoperative amplitude values were significantly lower than the preoperative values ( p < 0.001 ). The preoperative latency values were significantly higher compared to the postoperative values ( p < 0.05 ).
Group 4: The postoperative 2-month amplitude values were significantly lower compared to the preoperative ones ( p < 0.001 ). The postoperative latency values were significantly higher compared to postoperative ones ( p < 0.05 ).
| Groups | Presurgery amplitude (mV) | Postsurgery amplitude (mV) | Percentage of change in amplitude (%) | Presurgery latency (ms) | Postsurgey latance (ms) | Percentage of change in latency (%) |
|---|---|---|---|---|---|---|
| Group 1 (control) | ||||||
| 1 | 15.62 | 15.63 | 0.06 | 1.00 | 1.00 | 0.00 |
| 2 | 16.78 | 16.80 | 0.12 | 0.80 | 1.10 | 37.5 |
| 3 | 17.32 | 17.32 | 0.00 | 1.00 | 1.10 | 10.0 |
| 4 | 16.02 | 16.02 | 0.00 | 1.00 | 1.00 | 0.00 |
| 5 | 15.67 | 15.68 | 0.06 | 1.00 | 1.00 | 0.00 |
| 6 | 16.21 | 16.23 | 0.12 | 1.10 | 1.50 | 36.4 |
| Group 2 (sham) | ||||||
| 1 | 14.53 | 1.35 | 91.00 | 1.10 | 3.50 | 218.2 |
| 2 | 13.08 | 1.17 | 91.00 | 1.10 | 3.60 | 227.3 |
| 3 | 18.96 | 1.56 | 92.00 | 1.00 | 3.50 | 250.0 |
| 4 | 18.42 | 1.62 | 91.00 | 1.10 | 3.50 | 218.2 |
| 5 | 21.35 | 1.64 | 92.00 | 1.00 | 3.60 | 260.0 |
| 6 | 19.01 | 1.61 | 92.00 | 1.00 | 4.10 | 310.0 |
| Group 3 (TQ ⁎ ) | ||||||
| 1 | 18.21 | 11.99 | 34.00 | 1.10 | 1.30 | 18.2 |
| 2 | 18.60 | 12.45 | 33.00 | 1.00 | 1.40 | 40.0 |
| 3 | 15.72 | 11.40 | 27.00 | 1.00 | 1.50 | 50.0 |
| 4 | 15.99 | 14.71 | 8.00 | 1.10 | 1.30 | 18.2 |
| 5 | 16.15 | 15.26 | 6.00 | 1.10 | 1.30 | 18.2 |
| 6 | 16.68 | 13.52 | 19.00 | 1.00 | 1.30 | 30.0 |
| Group 4 (MP ⁎ ) | ||||||
| 1 | 15.16 | 8.58 | 43.00 | 1.20 | 1.40 | 16.7 |
| 2 | 16.17 | 12.48 | 23.00 | 1.10 | 1.30 | 18.2 |
| 3 | 15.95 | 10.71 | 33.00 | 1.10 | 1.60 | 45.5 |
| 4 | 15.03 | 10.51 | 30.00 | 1.20 | 1.70 | 41.7 |
| 5 | 14.84 | 10.67 | 28.00 | 1.10 | 1.70 | 54.5 |
| 6 | 14.71 | 10.16 | 31.00 | 1.10 | 1.60 | 45.5 |
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