Abstract
Objective
The aim of this experimental study was to evaluate the effects of hyperbaric oxygen, methylprednisolone and combined hyperbaric oxygen–methylprednisolone treatments on traumatic facial nerve regeneration in rats.
Subjects and methods
After exposure to facial nerve injury, four groups of rats were created with five subjects in each group: Group 1 (hyperbaric oxygen), group 2 (control), group 3 (combined hyperbaric oxygen–methylprednisolone), group 4 (methylprednisolone). Facial nerve specimens from sacrificed animals were examined for axonal degeneration, vascular congestion, macro vacuolization, axon diameter and thickness of myelin sheath.
Results
There were significant differences with regard to axonal degeneration, vascular congestion and axon diameter between group 3 and the control group. In addition to lower axonal degeneration and vascular congestion, a larger diameter of axons was observed in group 3. There were significant differences with regard to vascular congestion and axon diameter between group 4 and the control group. We observed thicker myelin and lower axonal degeneration in group 3 compared with group 4.
Conclusion
The combination therapy with hyperbaric oxygen and methylprednisolone had an additive beneficial effect on regeneration of the facial nerve and may provide better treatment outcomes than methylprednisolone or hyperbaric oxygen therapy alone.
1
Introduction
Traumatic facial nerve injury is a common clinical disorder with significant morbidity. It is the most frequent cause of facial paralysis during childhood and is the second most frequent in adults, followed by Bell’s palsy . In addition to its serious functional deficits, it can cause psychological trauma owing to facial asymmetry. Although the facial nerve motor fibers have the ability to regenerate their injured axons, not all patients with facial nerve paralysis will have complete functional recovery. Earlier studies have also demonstrated that spontaneous recovery of facial nerve follows a predictable time course after a crush injury in rats .
Wallerian degeneration, which follows nerve injury, involves degeneration of axons, myelin phagocytosis and proliferation of Schwann cells. Injuries to the peripheral nerve might be complicated as a result of thrombosis, endothelial swelling, endoneural edema, granulocyte plugging of the vasa nervorum or interruption of microvessels at the site of injury . In both traumatic and infectious neuritis of the facial nerve, neural edema within the bone canal of the facial nerve provokes compression, causing ischemia and axonal degeneration . There are studies introducing substances (neurotrophic factors, androgens, nimodipine) as therapeutic alternatives for the paralysis caused by a full section of the facial nerve . Steroids have been the classical treatment for facial nerve paralysis on the basis of their anti-inflammatory and immunosuppressive effects . Hyperbaric oxygen (HBO) has been reported to reduce ischemic injuries in various tissues and is a standard adjunctive treatment method for acute traumatic ischemic-reperfusion injuries, refractory wounds, compromised tissue flaps and grafts, and osteoradionecrosis . HBO is thought to decrease injury related edema, decrease the concentration of oxygen free radicals after ischemia with reperfusion and increase local tissue oxygen levels . Several studies have investigated the effect of HBO on facial nerve regeneration .
Since there are studies reporting the efficacy of HBO or steroid treatment alone in the management of ischemic-reperfusion tissue injuries , it occurred to us that combination therapy with HBO and methylprednisolone ( CS ) might have an additive beneficial effect on regeneration of the facial nerve. Therefore, in this experimental study, we aimed to evaluate the effects of HBO, CS , and combined HBO- CS treatments on traumatic facial nerve regeneration by histopathological examination of the facial nerve in rats.
2
Materials and methods
Approval was obtained from Haydarpaşa Numune Education and Research Hospital Ethics Committee. Twenty Sprague–Dawley rats weighing between 350 and 450 g were used in the experiment. Animals were kept under a 12-h light/dark cycle and fed with standard diet and water ad libitum.
2.1
Surgical procedure
The procedures were carried out by two surgeons. For facial nerve crush injury, all rats were anesthetized with intraperitoneal injections of Ketamine (100 mg/mL; 0.1 mL/100 g body weight) and Xylazine (20 mg/mL; 0.025 mL/100 g body weight). The right facial nerve was exposed at its exit from the stylomastoid foramen by a postauricular approach. The main trunk of the nerve before branching was compressed with a hemostat for 1 min. Complete loss of facial function on the right side was achieved confirming the crush injury in all rats.
2.2
Treatment protocols
The animals were divided into four experimental groups based on the type of treatment:
- Group 1:
HBO therapy for 60 min under 2.4 atm pressure each day for 5 days (twice a day only on the first day)
- Group 2:
Control animals receiving no therapy
- Group 3:
HBO therapy for 60 min under 2.4 atm pressure each day for 5 days (twice a day only on the first day) together with 30 mg/kg methylprednisolone ( CS )
- Group 4:
Methylprednisolone 30 mg/kg via the intraperitoneal route.
The animals were sacrificed with high-dose general anesthesia 1 week after the trauma, followed by removal of a 1 cm nerve segment from the crush site to the distal end.
2.3
Histopathological evaluation
The facial nerve specimens were fixed in formaldehyde with tamponade. After fixation, the specimens were embedded in paraffin blocks and 4 μm sections were obtained. The specimens were colored with hematoxylin-eosin and Giemsa stain.
All specimens were examined in terms of the degree of axonal degeneration, vascular congestion, macro vacuolization, axon diameter and thickness of myelin sheath. Axonal degeneration, vascular congestion, and macro vacuolization were evaluated as none, mild, moderate or severe. Axon diameter was assessed as very small, small or normal. The thickness of the myelin sheath was evaluated as very thin, thin or normal.
2.4
Statistical analysis
All statistical calculations were performed with NCSS (Number Cruncher Statistical System) 2007&PASS (Power Analysis and Sample Size) 2008 Statistical Software (UT, USA). In addition to standard descriptive statistical calculations (median, frequency and ratio), the Kruskal–Wallis test and Mann–Whitney U -test were used in the assessment of parameters. The statistical significance level was established at p < 0.05.
2
Materials and methods
Approval was obtained from Haydarpaşa Numune Education and Research Hospital Ethics Committee. Twenty Sprague–Dawley rats weighing between 350 and 450 g were used in the experiment. Animals were kept under a 12-h light/dark cycle and fed with standard diet and water ad libitum.
2.1
Surgical procedure
The procedures were carried out by two surgeons. For facial nerve crush injury, all rats were anesthetized with intraperitoneal injections of Ketamine (100 mg/mL; 0.1 mL/100 g body weight) and Xylazine (20 mg/mL; 0.025 mL/100 g body weight). The right facial nerve was exposed at its exit from the stylomastoid foramen by a postauricular approach. The main trunk of the nerve before branching was compressed with a hemostat for 1 min. Complete loss of facial function on the right side was achieved confirming the crush injury in all rats.
2.2
Treatment protocols
The animals were divided into four experimental groups based on the type of treatment:
- Group 1:
HBO therapy for 60 min under 2.4 atm pressure each day for 5 days (twice a day only on the first day)
- Group 2:
Control animals receiving no therapy
- Group 3:
HBO therapy for 60 min under 2.4 atm pressure each day for 5 days (twice a day only on the first day) together with 30 mg/kg methylprednisolone ( CS )
- Group 4:
Methylprednisolone 30 mg/kg via the intraperitoneal route.
The animals were sacrificed with high-dose general anesthesia 1 week after the trauma, followed by removal of a 1 cm nerve segment from the crush site to the distal end.
2.3
Histopathological evaluation
The facial nerve specimens were fixed in formaldehyde with tamponade. After fixation, the specimens were embedded in paraffin blocks and 4 μm sections were obtained. The specimens were colored with hematoxylin-eosin and Giemsa stain.
All specimens were examined in terms of the degree of axonal degeneration, vascular congestion, macro vacuolization, axon diameter and thickness of myelin sheath. Axonal degeneration, vascular congestion, and macro vacuolization were evaluated as none, mild, moderate or severe. Axon diameter was assessed as very small, small or normal. The thickness of the myelin sheath was evaluated as very thin, thin or normal.
2.4
Statistical analysis
All statistical calculations were performed with NCSS (Number Cruncher Statistical System) 2007&PASS (Power Analysis and Sample Size) 2008 Statistical Software (UT, USA). In addition to standard descriptive statistical calculations (median, frequency and ratio), the Kruskal–Wallis test and Mann–Whitney U -test were used in the assessment of parameters. The statistical significance level was established at p < 0.05.
3
Results
Evaluations of axonal degeneration, vascular congestion, macro vacuolization, axon diameter and thickness of the myelin sheath for each group are presented in Tables 1 and 2 .
| HBO (n = 5) | Control (n = 5) | CS + HBO (n = 5) | CS (n = 5) | p | ||
|---|---|---|---|---|---|---|
| N | n | N | n | |||
| Axonal Degeneration | None | – | – | – | – | |
| Mild | 1 | 0 | 4 | 0 | ||
| Moderate | 4 | 3 | 1 | 5 | ||
| Severe | 5 | 5 | 5 | 5 | ||
| Median | 2.00 | 2.00 | 1.00 | 2.00 | 0.011 ⁎ | |
| Vascular congestion | None | – | – | – | – | |
| Mild | 2 | 0 | 5 | 4 | ||
| Moderate | 3 | 4 | 0 | 1 | ||
| Severe | 0 | 1 | 0 | 0 | ||
| Median | 2.00 | 2.00 | 1.00 | 1.00 | 0.008 ⁎⁎ | |
| Macro vacuolization | None | 0 | 0 | 3 | 0 | |
| Mild | 2 | 3 | 2 | 3 | ||
| Moderate | 3 | 2 | 0 | 2 | ||
| Severe | – | – | – | – | ||
| Median | 2.00 | 1.00 | 0.00 | 1.00 | 0.040 ⁎ | |
| Axon Diameter | Very small | 3 | 5 | 1 | 0 | |
| Small | 2 | 0 | 5 | 1 | ||
| Normal | – | – | – | – | ||
| Median | 1.00 | 1.00 | 2.00 | 2.00 | 0.010 ⁎⁎ | |
| Myelin thickness | Very thin | 3 | 4 | 0 | 4 | |
| Thin | 2 | 0 | 5 | 1 | ||
| Normal | 0 | 1 | 0 | 0 | ||
| Median | 1.00 | 1.00 | 2.00 | 1.00 | 0.089 | |
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