doi: 10.3967/bes2018.055
An Experimental Study on the Optimal Timing for the Repair of Incomplete Facial Paralysis by Hypoglossal-facial 'Side'-to-side Neurorrhaphy in Rats
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Abstract:
Objective To investigate the optimal timing for the repair of persistent incomplete facial paralysis by hypoglossal-facial 'side'-to-side neurorrhaphy in rats. Methods A total of 30 adult rats with crushed and bulldog-clamped facial nerve injury were randomly divided into 5 groups (n=6 each) that were subjected to injury without nerve repair or with immediate repair, 2-week-delayed repair, 4-week-delayed repair, or 8-week-delayed repair. Three months later, the effects of repair in each rat were evaluated by facial symmetry assessment, electrophysiological examination, retrograde labeling, and axon regeneration measurement. Results At 3 months after injury, the alpha angle significantly increased in the group of rats with 4-week-delayed repair compared with the other four groups. Upon stimulation of the facial nerve or Pre degenerated nerve, the muscle action potentials MAPs were recorded in the whisker pad muscle, and the MAP amplitude and area under the curve in the 4-week-delayed repair group were significantly augmented at 3 months post-injury. Similarly, the number of retrograde-labeled motor neurons in the facial and hypoglossal nuclei was quantified to be significantly greater in the 4-week-delayed repair group than in the other groups, and a large number of regenerated axons was also observed. Conclusion The results of this study demonstrated that hemiHN-FN neurorrhaphy performed 4 weeks after facial nerve injury was most effective in terms of the functional recovery of axonal regeneration and activation of facial muscles. -
Key words:
- Facial paralysis /
- Hypoglossal-facial nerve anastomosis /
- Nerve autograft /
- Optimal time
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Figure 1. Schematic drawings showing the surgical procedures for facial nerve (FN) injury and repair in the rat. (A) A crush- and bulldog clamp-induced injury was caused at the beginning of the FN. A hypoglossal-facial 'side'-to-side neurorrhaphy with a pre-degenerated nerve graft (PNG) was then performed after the injury using 10-0 nylon microsutures. (B) The anastomosis between the FN and the PNG. A window was unilaterally created on the FN trunk, which was anastomosed with the distal site of the PNG in an end-to-side fashion. (C) The anastomosis between the PNG and the hypoglossal nerve (HN). The HN was hemi-sectioned, and the proximal site of the PNG was anastomosed with the sectioned site of the HN in an end-to-'side' fashion.
Figure 2. Assessment of the alpha angle to indicate facial symmetry. The alpha angle was defined as the acute angle between the line extending from the fold on the bridge of the nose and the line linking the two outer canthi. (A) The alpha angle of a normal rat was 88.50°. (B) The alpha angle of a control group rat was 74.82°. (C) The alpha angle of an immediate HemiHN-FN neurorrhaphy group rat was 76.17°. (D) The alpha angle of a 2-WK Delayed Neurrph group rat was 77.89°. (E) The alpha angle of a 4-WK Delayed Neurrph group rat was 84.15°. (F) The alpha angle of an 8-WK Delayed Neurrph group rat was 80.28°. (G) Measurements of the alpha angle 1 week after facial nerve injury and 3 months after neurorrphy. The results are presented as the means ± SEM. The differences between groups 3 months after repair treatment were significant based on one-way ANOVA: F (5, 25) = 13.39. Bonferroni's post hoc test for multiple comparisons was performed for comparison between groups. ***P < 0.001, compared to groups with mean values at or below the dotted line or as specifically indicated, as well as for the alpha angle in the Control group vs. that of the 4-WK Delayed Neurrph group. **P < 0.01, the alpha angle in the 2-WK Delayed Neurrph group vs. that in the 4-WK Delayed Neurrph group. *P < 0.05, the alpha angle in the 8-WK Delayed Neurrph group vs. that in the 4-WK Delayed Neurrph group.
Figure 3. The evaluation of muscle action potentials (MAPs) recorded from the paralyzed whisker pad muscle in response to electro-stimulation of the PNG at 3 months after FN neurorrhaphy. The MAP amplitude and area under the curve were measured to reveal the quantity of conductive axons and their capability of synchronous conduction. (A) The MAP amplitude and area under the curve of the IMM Neurrph group. (B) The MAP amplitude and area under the curve of the 2-WK Delayed Neurrph group. (C) The MAP amplitude and area under the curve of the 4-WK Delayed Neurrph group. (D) The MAP amplitude and area under the curve of the 8-WK Delayed Neurrph group. (E) The MAP amplitude of each group is presented as the mean ± SEM. The differences between groups at 3 months after repair treatment were significant based on one-way ANOVA: F (3, 14) = 3.210. Bonferroni's post hoc test for multiple comparisons was performed for comparisons between groups. *P < 0.05, the MAP amplitude in the 8-WK Delayed Neurrph group vs. that in the 4-WK Delayed Neurrph group. **P < 0.01, the MAP amplitude in the IMM Neurrph and 2-WK Delayed Neurrph groups vs. that in the 4-WK Delayed Neurrph group. (F) The MAP area under the curve of each group at different time points is presented as the mean ± SEM. The difference between repair treatment groups was not significant based on one-way ANOVA: F (3, 13) = 2.077. Bonferroni's post hoc test for multiple comparisons was performed for comparisons between groups. **P < 0.01, the MAP areas under the curve of the IMM Neurrph and 2-WK Delayed Neurrph groups vs. that of the 4-WK Delayed Neurrph group.
Figure 4. The evaluation of muscle action potentials (MAPs) recorded from the paralyzed whisker pad muscle in response to electro-stimulation of the FN trunk at 3 months after FN neurorrhaphy. (A) The MAP amplitude and area under the curve in the Control group. (B) The MAP amplitude and area under the curve in the IMM Neurrph group. (C) The MAP amplitude and area under the curve in the 2-WK Delayed Neurrph group. (D) The MAP amplitude and area under the curve in the 4-WK Delayed Neurrph group. (E) The MAP amplitude and area under the curve in the 8-WK Delayed Neurrph group. (F) The MAP amplitude of each group is presented as the mean ± SEM. The differences between groups 3 months after repair treatment were significant based on one-way ANOVA: F (4, 14) = 15.59. Bonferroni's post hoc test for multiple comparisons was performed for comparisons between groups. **P < 0.01, MAP amplitude in the IMM Neurrph and 2-WK Delayed Neurrph groups vs. that in the 4-WK Delayed Neurrph group. *P < 0.05, MAP amplitude in the Control group vs. that in the 4-WK Delayed Neurrph group. (G) The MAP area under the curve of each group at the different time points is presented as the mean ± SEM. The differences between groups at 3 months after repair treatment were significant based on one-way ANOVA: F (4, 14) = 12.45. Bonferroni's post hoc test for multiple comparisons was performed for comparisons between groups. ***P < 0.001, MAP area under the curve in the 2-WK Delayed Neurrph group vs. that in the 4-WK Delayed Neurrph group. **P < 0.01, MAP area under the curve in the Control and IMM Neurrph groups vs. that in the 4-WK Delayed Neurrph group. *P < 0.05, MAP area under the curve in the 4-WK Delayed Neurrph group vs. that in the 8-WK Delayed Neurrph group.
Figure 5. Observation and quantification of retrogradely labeled motor neurons in the facial nucleus 5 days after multipoint injection of Alexa CTB-555 into the paralyzed whisker pad muscle. (A) The CTB-555-labeled motor neurons (in red) in the facial nucleus of the control group 3 months after nerve neurorrhaphy. (B) The CTB-555-labeled motor neurons in the IMM Neurrph group. (C) The CTB-555-labeled motor neurons in the 2-WK Delayed Neurrph group. (D) The CTB-555-labeled motor neurons in the 4-WK Delayed Neurrph group. (E) The CTB-555-labeled motor neurons in the 8-WK Delayed Neurrph group. Bar = 50 µm. (F) The CTB-555-labeled neurons in the facial nucleus were quantified for each group at 3 months after the FN injury, and the counts are presented as the means ± SEM. The difference between groups 3 months after repair treatment were significant based on one-way ANOVA: F (4, 15) = 66.08. Bonferroni's post hoc test for multiple comparisons was performed for comparisons between groups. ***P < 0.001, the number of labeled neurons in the IMM Neurrph group vs. that in the 4-WK Delayed Neurrph group. **P < 0.01, the number of labeled neurons in the 4-WK Delayed Neurrph group vs. that in the 8-WK Delayed Neurrph group. *P < 0.05, the number of labeled neurons in the Control and 2-WK Delayed Neurrph groups vs. that in the 4-WK Delayed Neurrph group.
Figure 6. Observation and quantification of retrogradely labeled motor neurons in the hypoglossal nucleus 5 days after multipoint injection of Alexa CTB-555 into the paralyzed whisker pad muscle. (A) The CTB-555-labeled motor neurons (in red) were not detected in the hypoglossal nucleus of the control group after 3 months of nerve neurorrhaphy. (B) The CTB-555-labeled motor neurons in the IMM Neurrph group. (C) The CTB-555-labeled motor neurons in the 2-WK Delayed Neurrph group. (D) The CTB-555-labeled motor neurons in the 4-WK-Delayed Neurrph group. (E) The CTB-555-labeled motor neurons in the 8-WK Delayed Neurrph group. Bar = 50 µm. (F) Quantification of labeled neurons in the hypoglossal nucleus of each group. The results are presented as the means ± SEM, and one-way ANOVA: F (3, 13) = 28.67 revealed significant differences between groups. Bonferroni's post hoc test for multiple comparisons was performed for comparisons between groups. ***P < 0.001, the number of labeled neurons in the IMM Neurrph group and the 8-WK Delayed Neurrph group vs. that in the 4-WK Delayed Neurrph group. **P < 0.01, the number of labeled neurons in the 2-WK Delayed Neurrph group vs. that in the 4-WK Delayed Neurrph group.
Figure 7. Representative photomicrographs of myelinated axons on thionin-stained sections at a level distal to the neurorrhaphy site of the FN and the intermediate part of the PNG 3 months after nerve neurorrhaphy. (A) Myelinated axons in the PNG of a rat subjected to immediate HemiHN-FN neurorrhaphy. (B) Myelinated axons in the PNG of a rat subjected to a 2-WK Delayed Neurrph. (C) Myelinated axons in the PNG of a rat subjected to a 4-WK Delayed Neurrph. (D) Myelinated axons in the PNG of a rat subjected to 8-WK Delayed Neurrph. Scale bars: 200 μm. (E) Myelinated axons were quantified at a level distal to the neurorrhaphy site of the FN for each group. The differences between groups 3 months after repair treatment were not significant based on one-way ANOVA: F (3, 11) = 1.946. Myelinated axons were quantified in the PNG. The differences between groups 3 months after repair treatment were significant based on one-way ANOVA: F (3, 8) = 5.332. n.s, no significant difference. *P < 0.05, myelinated axon count in the IMM Neurrph group, the 2-WK Delayed Neurrph group and the 8-WK Delayed Neurrph group vs. that in the 4-WK Delayed Neurrph group.
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