- Email: [email protected]
The effect of tacrolimus on facial nerve injury: Histopathological findings in a rabbit model
AM ER IC AN JOURNAL OF OT OLARYNGOLOGY–H E A D A N D NE CK M E D IC IN E A ND S U RGE RY 3 7 (2 0 1 6) 3 93–3 9 7
Available online at www.sciencedirect.com
ScienceDirect www.elsevier.com/locate/amjoto
Original contributions
The effect of tacrolimus on facial nerve injury: Histopathological findings in a rabbit model☆,☆☆,★ Kamil Gokce Tulaci, MD a,⁎, Arzu Tuzuner, MD b , Hatice Karadas Emir, MD b , İlkan Tatar, MD c , Mustafa F. Sargon, MD c , Tugba Tulaci, MD a , Yunus Karadavut, MD d , Ethem Erdal Samim, MD b a
Department of Otorhinolaryngology — Head and Neck Surgery, Ministry of Health, Kirikkale Yüksek İhtisas Hospital, Kirikkale, Turkey Department of Otorhinolaryngology — Head and Neck Surgery, Ministry of Health, Ankara Training and Research Hospital, Ankara, Turkey c Hacettepe University, Faculty of Medicine, Department of Anatomy, Ankara, Turkey d Department of Otorhinolaryngology — Head and Neck Surgery, MediBafra Hospital, Samsun, Turkey b
ARTI CLE I NFO
A BS TRACT
Article history:
Hypothesis: Tacrolimus helps healing of facial nerve injury.
Received 24 March 2016
Background: Positive effects of tacrolimus on axon regeneration and healing of injured peripheral nerves (eg. sciatic nerve) have been reported in the literature. Tacrolimus may be an additional treatment method that could improve the nerve healing after surgical treatment of cut injury of facial nerve. Methods: 20 New Zealand rabbits were randomly separated into control and study groups of 10. In control group, no medical treatment was given after facial nerve anastomosis, and the animals were followed up for 2 months. In the study group rabbits were given 1 mg/kg/day tacrolimus subcutaneously for 2 months after the facial nerve anastomosis. The histopathologic findings of axon regeneration like axon myelination were analyzed in both groups under electron and light microscopy. The data obtained in the groups were compared. Results: Greater axon diameters, thicker myelin sheaths, and higher total number of myelinated axons were found in the tacrolimus group, suggesting better regeneration in this group when compared to the control group. There was less vacuolar degeneration in the study group. All these findings suggest that tacrolimus positively affects healing after facial nerve anastomosis. Conclusion: The results of this study indicate that tacrolimus has favorable effects on the healing process of the facial nerve after end-to-end anastomosis. Tacrolimus may be a promising agent in the future for nerve regeneration following traumatic facial paralysis surgery. © 2016 Elsevier Inc. All rights reserved.
☆
This study was performed in the Ministry of Health Animal Experiments Laboratory at Ankara Training and Research Hospital. Obtaining the specimens, and their preparation and examination were performed at Hacettepe University, Faculty of Medicine, Department of Anatomy. ☆☆ We do not have a financial support for this study. ★ This paper was presented as oral presentation at the 36th Turkish National Otorhinolaryngology — Head and Neck Surgery Meeting, Antalya, Turkey (5th–9th of November 2014). ⁎ Corresponding author at: Etlik Cad Kutluhan Apt. 173/b No. 5 Etlik/Keçiören, Ankara, Turkey. Tel.: +90 532 5947384. E-mail addresses: [email protected], [email protected] (K.G. Tulaci). http://dx.doi.org/10.1016/j.amjoto.2016.06.003 0196-0709/© 2016 Elsevier Inc. All rights reserved.
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AM ER IC AN JOURNAL OF OT OLA RYNGOLOGY–H E A D A N D NE CK M E D ICI N E AN D S U RGE RY 3 7 (2 0 1 6) 3 93 –3 9 7
1.
Introduction
Traumatic facial nerve paralysis must be treated surgically immediately if a complete cut or injury of the facial nerve is suspected [1]. Surgical approaches include facial nerve decompression, end-to-end anastomosis of the cut nerve [2], end-to-end anastomosis after re-routing of the facial nerve when there is a loss of a segment of the nerve, and use of a free nerve graft obtained from the greater auricular nerve or sural nerve if an end-to-end anastomosis is not possible [3–5]. In traumatic facial nerve paralysis, the probability for sequela-free healing is low with the use of current surgical options [6]. To prevent functional, cosmetic, and sociological problems in patients with peripheral facial paralysis, additional treatment methods that could improve the nerve healing have been investigated, and a number of methods have been used in addition to surgery. These include gangliocytes, hormones, electromagnetic field studies [7,8], hyperbaric oxygen treatment [9], and corticosteroids [10]. Tacrolimus (Prograf®) is usually used after organ transplantations, and it is an 822-Da, lipophilic, macrolide antibiotic [11]. It was first identified as the fermentation product of Streptomyces tsukubaensis, in 1984. Cyclosporine and tacrolimus are FDA approved strong immunosuppressive agents, called calcineurin inhibitors [12,13]. Some studies have shown the neuroprotective and neurotrophic characteristics of tacrolimus, and its positive effects have been shown on axon regeneration and nerve healing in peripheral nerve injuries [2,14–16]. In the light of the aforementioned information, the aim of this study was to investigate the efficacy of tacrolimus on facial nerve healing after surgical treatment of experimental total peripheral facial nerve paralysis.
2.
Materials and methods
2.1.
Animals
This study was performed in the Ministry of Health Animal Experiments Laboratory at Ankara Training and Research Hospital. Approval for the study was obtained from the Local Ethics Committee (ref. no.: 0016/243). The study comprised 20 male New Zealand rabbits weighing 2500–3000 g. The facial functions of all the animals were examined, and those with normal facial nerve functions were included in the study. The criteria for normal facial functions were symmetrical movements of the whiskers during chewing, and the presence of the eye blinking reflex when pressurized air was administered with a syringe. The animals were housed in separate cages under standard laboratory conditions and fed with special pellets and water ad libitum.
2.2.
Surgical procedure
The same standard surgical procedure was applied to all 20 rabbits by the same surgeon. The rabbits were anesthetized with 10 mg/kg xylazine hydrochloride (Rompun, Bayer İlaç, Turkey) and 50 mg/kg ketamine hydrochloride (Ketalar,
Eczacıbaşı İlaç, Turkey). Surgery was performed on the left side of all the animals. The surgical field corresponding to the course of the facial nerve was shaved, rinsed with 70% ethanol and povidone iodine, and dried. The procedure was performed under sterile conditions, using a surgical microscope. A 2 cm long, modified Blair incision type incision was performed starting from the inferoposterior of the eye and anteroinferior of the ear, running parallel to the mandible. The skin and subcutaneous tissues were dissected, and the superficial fascia was exposed. The facial nerve trunk was identified with the help of a facial nerve stimulator, and it was dissected from the surrounding tissues. The nerve was cut using a scalpel blade no. 11 (Aesculap, Germany). Two sutures were placed to suture the epineurium of the distal and the proximal ends of the nerve, using an 8-0 monofilament suture (Ethicon, Germany). After suturing subcutaneous tissues, the skin was sutured using 4-0 silk (Ethicon, Germany). The rabbits were administered prophylactic 20–40 mg/kg cefazolin sodium (Cefozin, Bilim İlaç, Turkey) one hour before and one hour after the operation. All the animals were seen to have total facial paralysis on the left side. The rabbits were randomly separated into 2 groups of 10. Group 1: Control group: No medical treatment was given after facial nerve anastomosis, and the animals were followed up for 2 months. Group 2: Study group (tacrolimus group): After the facial nerve anastomosis was performed on the left side of the face, the rabbits were given 1 mg/kg/day tacrolimus subcutaneously for 2 months.
2.3. Obtaining the specimens, and their preparation and examination The rabbits were administered intramuscular 10 mg/kg xylazine hydrochloride, and 50 mg/kg ketamine hydrochloride in the postoperative 8th week, and the site of facial anastomosis was exposed through the previous incision site. The site of anastomosis was identified by the sutures previously placed on the nerve. The nerve was dissected free from the surrounding tissues, and approximately 1 cm of the nerve was excised distal to the anastomosis suture. The nerve tissue was fixed in 2.5% glutaraldehyde for 24 h. Later, the specimens were washed with SPB (Sorenson phosphate buffer) which had a pH of 7.4, and a post-fixation procedure was performed with 1% osmium tetroxide. Then, the specimens were washed with SPB again. The dehydration procedure was performed using low-to-high concentrations of alcohol (25%, 50%, and 75% absolute alcohol). The specimens were washed twice with propylene oxide before embedding. The first stage of preparation for embedding was the mixing of propylene oxide and epoxy resin embedding material in equal amounts (1:1 mixture), and the specimens were placed in this for 1 h. At the end of 1 h, the same amount of epoxy resin was added to the previously prepared mixture, to create a 1:3 mixture. The specimens were left in the rotator for one night, and the preparation for embedding was finished. Later, the specimens that were embedded in epoxy resin embedding material using plastic capsules were put into
AM ER IC AN JOURNAL OF OT OLARYNGOLOGY–H E A D A N D NE CK M E D IC IN E A ND S U RGE RY 3 7 (2 0 1 6) 3 93–3 9 7
the incubator for 48 h, at 60 degrees Celsius. After 48 h, the specimens were taken out of the incubator, and semi-thin sections were obtained with a LKB Nova ultramicrotome device (Sweden). The 2-μm-thick sections were stained with methylene blue to identify the fields suitable for obtaining thinner sections. The fields suitable for thinner sections were trimmed, and a tissue surface that could be sectioned for transmission electron microscopy was obtained. Then, the same ultramicrotome was used to prepare thin sections, with a thickness of approximately 60 nm. Those thin sections were stained using the double contrast method with uranyl acetate and lead citrate, and were then examined under the transmission electron microscope (Jeol JEM 1200 EX, Japan) by two authors, and photographed. The examinations of the study and the control groups were performed by two independent investigators. The axon myelination, normality of the myelin structure, the diameters of the myelinated axons, the thickness of the myelin sheath of the axons, and vacuolar degeneration were analyzed in both groups under the electron microscope. The number of total myelinated axons was determined by light microscopy. The data obtained in the groups were compared.
2.3.1.
Statistical analysis
The data were analyzed using SPSS 15.0 statistics package program. The descriptive statistics were presented as mean ± standard deviation, median (minimum; maximum), frequency distribution, and percentage. The Chi square test with Yates' correction was also applied. The comparisons of the variables were made with the Mann–Whitney U test. A value of p < 0.05 was accepted as statistically significant.
3.
Results
Electron and light microscopic examinations of the study and the control groups were analyzed independently. The electron microscopic examination of the tacrolimus group showed increased myelinization and thickened endoneurium
(Figs. 1 and 2). The myelin structure, axon numbers and axon cytoplasms were observed to be normal. Some unmyelinated nerves were seen in the fields examined, although the unmyelinated nerve ratios were similar in the two groups (Fig. 3). The axon diameters, myelin sheath thickness and number of total myelinated axons in the study and the control groups are presented in Table 1. The axon diameters were statistically significantly different when the study and the control groups were compared (p = 0.0001). The median axon diameter was statistically significantly greater in the study group. The median myelin sheath thickness was 0.83 (0.63–2.33) micrometers in the study group, and 0.38 (0.33–0.46) micrometers in the control group, with a statistically significant difference between the groups (p = 0.0001). The myelin sheaths were statistically significantly thicker in the study group. The median of total myelinated axon number was 570 (460–588) in the study group, and 470 (455–490) in the control group. The total myelinated axon numbers were statistically significantly higher in the study group when compared to the control group (p = 0.0001). In the study group, the number of the cells containing 1 or less vacuolar degeneration was 8 (80%) while the number of cells containing 2 or more vacuolar degenerations was 2 (20%). Those numbers were 2 (20%), and 8 (80%) in the control group, respectively. A statistically significant difference was determined between the groups in respect of vacuolar degeneration (p =0.025) (Chi square test with Yates' correction). The number of cells containing 1 or less vacuolar degeneration was significantly higher in the study group when compared to the control group. Statistically significantly greater axon diameters, thicker myelin sheaths, and higher total number of myelinated axons were found in the tacrolimus group suggesting better regeneration in this group when compared to the control group. There was less vacuolar degeneration in the study group. All those findings suggest that tacrolimus positively affects healing after facial nerve anastomosis.
4.
Fig. 1 – The electron microscopic photograph of the study group: Schwann cells are highly increased in number, and regenerated myelinated axons can be seen (original magnification ×3000).
395
Discussion
With increased understanding of anatomical, histological, and pathological processes and advances in the surgical techniques used in the treatment, major improvements have been made in the repair of peripheral nerve injuries that occur due to trauma, surgery, malignancy, inflammatory conditions or infections. Even when optimum conditions are provided, facial functions do not heal 100% in any cases after primary nerve repair [17]. To date, a number of methods have been studied in a number of clinical and experimental studies to increase healing and to improve functional results after traumatic facial nerve injury. Exploration, decompression, end-to-end anastomosis, free nerve graft, and lateral nerve anastomosis have been used as surgical treatment methods. Primary endto-end anastomosis must be performed if possible [2–5]. The possibility of complete, sequela-free healing is low in traumatic total peripheral facial nerve paralysis with current
396
AM ER IC AN JOURNAL OF OT OLA RYNGOLOGY–H E A D A N D NE CK M E D ICI N E AN D S U RGE RY 3 7 (2 0 1 6) 3 93 –3 9 7
Fig. 2 – The electron microscopic photograph of the study group: Myelinated axons showing lamellar degeneration, and one ultrastructurally normal, regenerated, myelinated axon (original magnification ×3000).
Fig. 3 – The electron microscopic photograph of the control group: Ultrastructurally normal myelinated axons, normal unmyelinated axons, and a normal Schwann cell number (original magnification ×3000).
treatment options [6]. To prevent functional, cosmetic, and sociological problems in patients with peripheral facial paralysis, additional treatment methods that could improve nerve healing are needed. Tacrolimus is mainly used as an immunosuppressant agent in solid organ transplantations, such as liver and kidney, and it is particularly effective in steroid-resistant rejections [18]. The neuroprotective effect of tacrolimus has been reported in focal and extensive cerebral ischemia [19]. Some studies have shown the positive effects of tacrolimus on axon regeneration and nerve healing in peripheral nerve injury [2,15,16]. Li et al. investigated the efficacy of tacrolimus on healing of rat sciatic nerve, and found that the axons regenerated in various sizes and shapes and that myelin sheath thickness healed better in the tacrolimus group. The authors also showed that the axon diameters were significantly greater in the tacrolimus-chitosan guide group [13]. In the current study, the axon diameters of the nerves were also measured using electron microscopy, and the axon diameters were found to be significantly greater in the study group than in the control group. Just et al. [21] showed that tacrolimus had positive effects on healing of posterior tibial nerve injury in rats. In 2009, Chen et al. [3] investigated the effect of tacrolimus on end-to-end and end-to-side anastomosis of peroneal nerve, and reported that the tacrolimus group was superior for myelin sheath thickness and myelinated axon numbers. Similarly, the current study group was found to be statistically significantly superior to the control group in respect of total number of myelinated axons, which was used as the healing parameter in the study. There are few studies in the literature that have investigated the effect of tacrolimus on the facial nerve. The studies in the literature have investigated the immunosuppressive effect of tacrolimus for rejection of allografts and its effect on crush injury, rather than investigating its effect on the regeneration of the facial nerve in the event of cut injury. Lee et al. [20] found favorable functional effects of tacrolimus on healing of the rat posterior tibial nerve crush injury. That study showed the effect of tacrolimus on a peripheral nerve. The nerve was not cut completely but crushed and the crush injury improved with this agent. In the current
study, the effect of tacrolimus, which is an immunosuppressive and also a neuroregenerative agent for the peripheral nerves, was investigated for healing of iatrogenic cut injury of the rabbit facial nerve. The regeneration parameters used were the number of the total myelinated axons in a nerve bundle wrapped by the perineurium, the axon diameter and the myelin sheath thickness of the axons. Vacuolar degeneration was used as the parameter of degeneration. The myelinated axon number, myelin sheath thickness and myelinated axon diameters were found to be significantly higher in the tacrolimus group than the control group. Various agents such as insulin growth factor II have been continuously infused to the site of injury to increase nerve regeneration. Gold et al. [14] reported that the first agent which showed favorable results after systemic use was tacrolimus. The results of the current study have also shown that systemic administration of tacrolimus increased nerve regeneration. As it has immunosuppressive effects and immunosuppression related side effects, the minimal doses of tacrolimus for various organ transplantations are well studied for human in the literature. At this point, a very important limitation of this study is that the dose-dependent effects and the standard dose of tacrolimus are not clearly determined for animals in the literature. Before stating a dose for tacrolimus, we scanned literature and found some animal studies on neuroprotective effect of tacrolimus. Yeh et al. [16] used tacrolimus at a dose of 2 mg/kg/day, Gold et al. a dose of 1 mg/ kg/day [14], and Udina et al. [22] a dose of 5 mg/kg/day, which are high doses for human in the condition of organ transplantation. Also there are several animal studies examined the effect of subimmunosuppressive doses of tacrolimus on neuroregeneration. In the study of Yang et al. [23], the subimmunosuppressive doses of FK506 were determined as the doses below 2.0 mg/kg by examining skin allograft survival in a rat model in which the animals that received 2.0 mg/kg FK506 per day exhibited complete skin graft take, whereas all other groups that received FK506 below the doses of 2 mg/kg demonstrated complete rejection. In the second part of the same study statistically significant improvements in neuroregeneration with subimmunosuppressive FK506 doses
AM ER IC AN JOURNAL OF OT OLARYNGOLOGY–H E A D A N D NE CK M E D IC IN E A ND S U RGE RY 3 7 (2 0 1 6) 3 93–3 9 7
397
Table 1 – Axon diameters, myelin sheath thickness and number of total myelinated axons in the study and the control groups. Study Group (n = 10)
Axon Diameter Myelin Sheath Thickness Total Myelinated Axon Number
Control Group (n = 10)
Median (min; max)
Mean ± SD
Median (min; max)
Mean ± SD
5.22 (3.97; 7.76) 0.83 (0.63; 2.33) 570 (460; 588)
5.55 ± 1.24 1.04 ± 0.51 539.40 ± 56.58
2.88 (2.73; 3.43) 0.38 (0.33; 046) 470 (455; 490)
2.96 ± 0.27 0.39 ± 0.05 472 ± 14.37
p Value 0.0001 ⁎ 0.0001 ⁎ 0.0001 ⁎
min; max: minimum; maximum, SD: standard deviation. ⁎ Mann–Whitney U Test.
of 0.5 and 1.0 mg/kg per day are reported [23]. Further studies are needed to determine the optimal dose for various animals. In our study, tacrolimus was used at a dose of 1 mg/kg/day.
5.
Conclusion
The results of this study indicate that tacrolimus has favorable effects on the healing process of the facial nerve after end-toend anastomosis. Tacrolimus may be a promising agent in the future for nerve regeneration following traumatic facial paralysis surgery. As this is a preliminary animal study, further studies are needed to evaluate the functional results of the facial nerve repair with tacrolimus and investigate the optimal dose and dose-dependent effects of tacrolimus.
REFERENCES
[1] Akyildiz N. Microsurgery and diseases of ear. Bilimsel Tip Press; 2002 215–332. [2] Chen B, Song Y, Liu Z. Promotion of nerve regeneration in peripheral nerve by short-course FK506 after end-to-side neurorrhaphy. J Surg Res 2009;152:303–10. [3] Davis RE, Telischi FF. Traumatic facial nerve injuries: review of diagnosis and treatment. J Craniomaxillofac Trauma 1995;1:30–41. [4] Hu M, Zhang L, Niu Y, et al. Repair of whole rabbit facial nerve defects using facial nerve allografts. J Oral Maxillofac Surg 2010;68:2196–206. [5] Costa HJ, Ferreira Bento R, Salomone R, et al. Mesenchymal bone marrow stem cells within polyglycolic acid tube observed in vivo after six weeks enhance facial nerve regeneration. Brain Res 2013;1510:10–21. [6] Briggs R, Mattox DE. Management of the facial nerve in skull base surgery. Otolaryngol Clin North Am 1991;24:653–62. [7] Lundborg G. Nerve injury and repair. Edinburgh: Churchill Livingstone; 1988 1–22. [8] Longo FM, Yang T, Hamilton S, et al. Electromagnetic fields influence NGF activity and levels following sciatic nerve transection. J Neurosci Res 1999;55:230–7. [9] Haapaniemi T, Nylander G, Kanje M, et al. Hyperbaric oxygen treatment enhances regeneration of the rat sciatic nerve. Exp Neurol 1998;149:433–8.
[10] Becker KW, Kienecker EW, Andrae I. Effect of locally applied corticoids on the morphology of peripheral nerves following neurotmesis and microsurgical suture. Neurochirurgia (Stuttg) 1987;30:161–7. [11] Hooks MA. Tacrolimus, a new immunosuppressant — a review of the literature. Ann Pharmacother 1994;28:501–11. [12] Venkataramanan R, Jain A, Warty VW, et al. Pharmacokinetics of FK 506 following oral administration: a comparison of FK 506 and cyclosporine. Transplant Proc 1991;23: 931–3. [13] Li X, Wang W, Wei G, et al. Immunophilin FK506 loaded in chitosan guide promotes peripheral nerve regeneration. Biotechnol Lett 2010;32:1333–7. [14] Gold BG, Katoh K, Storm-Dickerson T. The immunosuppressant FK506 increases the rate of axonal regeneration in rat sciatic nerve. J Neurosci 1995;15:7509–16. [15] Udina E, Rodríguez FJ, Verdú E, et al. FK506 enhances regeneration of axons across long peripheral nerve gaps repaired with collagen guides seeded with allogeneic Schwann cells. Glia 2004;47:120–9. [16] Yeh C, Bowers D, Hadlock TA. Effect of FK506 on functional recovery after facial nerve injury in the rat. Arch Facial Plast Surg 2007;9:333–9. [17] Al Abri R, Kolethekkat AA, Kelleher MO, et al. Effect of locally administered ciliary neurotrophic factor on the survival of transected and repaired adult sheep facial nerve. Oman Med J 2014;29:208–13. [18] Jensik SC. Tacrolimus (FK 506) in kidney transplantation: three-year survival results of the US multicenter, randomized, comparative trial. FK 506 kidney transplant study group. Transplant Proc 1998;30:1216–8. [19] Takamatsu H, Tsukada H, Noda A, et al. FK506 attenuates early ischemic neuronal death in a monkey model of stroke. J Nucl Med 2001;42:1833–40. [20] Lee M, Doolabh VB, Mackinnon SE. FK506 promotes functional recovery in crushed rat sciatic nerve. Muscle Nerve 2000;23:633–40. [21] Jost SC, Doolabh VB, Mackinnon SE, et al. Acceleration of peripheral nerve regeneration following FK506 administration. Restor Neurol Neurosci 2000;17:39–44. [22] Udina E, Gold BG, Navarro X. Comparison of continuous and discontinuous FK506 administration on autograft or allograft repair of sciatic nerve resection. Muscle Nerve 2004;29: 812–22. [23] Yang RK, Lowe III JB, Sobol JB, et al. Dose-dependent effects of FK506 on neuroregeneration in a rat model. Plast Reconstr Surg 2003;112:1832–40.
Available online at www.sciencedirect.com
ScienceDirect www.elsevier.com/locate/amjoto
Original contributions
The effect of tacrolimus on facial nerve injury: Histopathological findings in a rabbit model☆,☆☆,★ Kamil Gokce Tulaci, MD a,⁎, Arzu Tuzuner, MD b , Hatice Karadas Emir, MD b , İlkan Tatar, MD c , Mustafa F. Sargon, MD c , Tugba Tulaci, MD a , Yunus Karadavut, MD d , Ethem Erdal Samim, MD b a
Department of Otorhinolaryngology — Head and Neck Surgery, Ministry of Health, Kirikkale Yüksek İhtisas Hospital, Kirikkale, Turkey Department of Otorhinolaryngology — Head and Neck Surgery, Ministry of Health, Ankara Training and Research Hospital, Ankara, Turkey c Hacettepe University, Faculty of Medicine, Department of Anatomy, Ankara, Turkey d Department of Otorhinolaryngology — Head and Neck Surgery, MediBafra Hospital, Samsun, Turkey b
ARTI CLE I NFO
A BS TRACT
Article history:
Hypothesis: Tacrolimus helps healing of facial nerve injury.
Received 24 March 2016
Background: Positive effects of tacrolimus on axon regeneration and healing of injured peripheral nerves (eg. sciatic nerve) have been reported in the literature. Tacrolimus may be an additional treatment method that could improve the nerve healing after surgical treatment of cut injury of facial nerve. Methods: 20 New Zealand rabbits were randomly separated into control and study groups of 10. In control group, no medical treatment was given after facial nerve anastomosis, and the animals were followed up for 2 months. In the study group rabbits were given 1 mg/kg/day tacrolimus subcutaneously for 2 months after the facial nerve anastomosis. The histopathologic findings of axon regeneration like axon myelination were analyzed in both groups under electron and light microscopy. The data obtained in the groups were compared. Results: Greater axon diameters, thicker myelin sheaths, and higher total number of myelinated axons were found in the tacrolimus group, suggesting better regeneration in this group when compared to the control group. There was less vacuolar degeneration in the study group. All these findings suggest that tacrolimus positively affects healing after facial nerve anastomosis. Conclusion: The results of this study indicate that tacrolimus has favorable effects on the healing process of the facial nerve after end-to-end anastomosis. Tacrolimus may be a promising agent in the future for nerve regeneration following traumatic facial paralysis surgery. © 2016 Elsevier Inc. All rights reserved.
☆
This study was performed in the Ministry of Health Animal Experiments Laboratory at Ankara Training and Research Hospital. Obtaining the specimens, and their preparation and examination were performed at Hacettepe University, Faculty of Medicine, Department of Anatomy. ☆☆ We do not have a financial support for this study. ★ This paper was presented as oral presentation at the 36th Turkish National Otorhinolaryngology — Head and Neck Surgery Meeting, Antalya, Turkey (5th–9th of November 2014). ⁎ Corresponding author at: Etlik Cad Kutluhan Apt. 173/b No. 5 Etlik/Keçiören, Ankara, Turkey. Tel.: +90 532 5947384. E-mail addresses: [email protected], [email protected] (K.G. Tulaci). http://dx.doi.org/10.1016/j.amjoto.2016.06.003 0196-0709/© 2016 Elsevier Inc. All rights reserved.
394
AM ER IC AN JOURNAL OF OT OLA RYNGOLOGY–H E A D A N D NE CK M E D ICI N E AN D S U RGE RY 3 7 (2 0 1 6) 3 93 –3 9 7
1.
Introduction
Traumatic facial nerve paralysis must be treated surgically immediately if a complete cut or injury of the facial nerve is suspected [1]. Surgical approaches include facial nerve decompression, end-to-end anastomosis of the cut nerve [2], end-to-end anastomosis after re-routing of the facial nerve when there is a loss of a segment of the nerve, and use of a free nerve graft obtained from the greater auricular nerve or sural nerve if an end-to-end anastomosis is not possible [3–5]. In traumatic facial nerve paralysis, the probability for sequela-free healing is low with the use of current surgical options [6]. To prevent functional, cosmetic, and sociological problems in patients with peripheral facial paralysis, additional treatment methods that could improve the nerve healing have been investigated, and a number of methods have been used in addition to surgery. These include gangliocytes, hormones, electromagnetic field studies [7,8], hyperbaric oxygen treatment [9], and corticosteroids [10]. Tacrolimus (Prograf®) is usually used after organ transplantations, and it is an 822-Da, lipophilic, macrolide antibiotic [11]. It was first identified as the fermentation product of Streptomyces tsukubaensis, in 1984. Cyclosporine and tacrolimus are FDA approved strong immunosuppressive agents, called calcineurin inhibitors [12,13]. Some studies have shown the neuroprotective and neurotrophic characteristics of tacrolimus, and its positive effects have been shown on axon regeneration and nerve healing in peripheral nerve injuries [2,14–16]. In the light of the aforementioned information, the aim of this study was to investigate the efficacy of tacrolimus on facial nerve healing after surgical treatment of experimental total peripheral facial nerve paralysis.
2.
Materials and methods
2.1.
Animals
This study was performed in the Ministry of Health Animal Experiments Laboratory at Ankara Training and Research Hospital. Approval for the study was obtained from the Local Ethics Committee (ref. no.: 0016/243). The study comprised 20 male New Zealand rabbits weighing 2500–3000 g. The facial functions of all the animals were examined, and those with normal facial nerve functions were included in the study. The criteria for normal facial functions were symmetrical movements of the whiskers during chewing, and the presence of the eye blinking reflex when pressurized air was administered with a syringe. The animals were housed in separate cages under standard laboratory conditions and fed with special pellets and water ad libitum.
2.2.
Surgical procedure
The same standard surgical procedure was applied to all 20 rabbits by the same surgeon. The rabbits were anesthetized with 10 mg/kg xylazine hydrochloride (Rompun, Bayer İlaç, Turkey) and 50 mg/kg ketamine hydrochloride (Ketalar,
Eczacıbaşı İlaç, Turkey). Surgery was performed on the left side of all the animals. The surgical field corresponding to the course of the facial nerve was shaved, rinsed with 70% ethanol and povidone iodine, and dried. The procedure was performed under sterile conditions, using a surgical microscope. A 2 cm long, modified Blair incision type incision was performed starting from the inferoposterior of the eye and anteroinferior of the ear, running parallel to the mandible. The skin and subcutaneous tissues were dissected, and the superficial fascia was exposed. The facial nerve trunk was identified with the help of a facial nerve stimulator, and it was dissected from the surrounding tissues. The nerve was cut using a scalpel blade no. 11 (Aesculap, Germany). Two sutures were placed to suture the epineurium of the distal and the proximal ends of the nerve, using an 8-0 monofilament suture (Ethicon, Germany). After suturing subcutaneous tissues, the skin was sutured using 4-0 silk (Ethicon, Germany). The rabbits were administered prophylactic 20–40 mg/kg cefazolin sodium (Cefozin, Bilim İlaç, Turkey) one hour before and one hour after the operation. All the animals were seen to have total facial paralysis on the left side. The rabbits were randomly separated into 2 groups of 10. Group 1: Control group: No medical treatment was given after facial nerve anastomosis, and the animals were followed up for 2 months. Group 2: Study group (tacrolimus group): After the facial nerve anastomosis was performed on the left side of the face, the rabbits were given 1 mg/kg/day tacrolimus subcutaneously for 2 months.
2.3. Obtaining the specimens, and their preparation and examination The rabbits were administered intramuscular 10 mg/kg xylazine hydrochloride, and 50 mg/kg ketamine hydrochloride in the postoperative 8th week, and the site of facial anastomosis was exposed through the previous incision site. The site of anastomosis was identified by the sutures previously placed on the nerve. The nerve was dissected free from the surrounding tissues, and approximately 1 cm of the nerve was excised distal to the anastomosis suture. The nerve tissue was fixed in 2.5% glutaraldehyde for 24 h. Later, the specimens were washed with SPB (Sorenson phosphate buffer) which had a pH of 7.4, and a post-fixation procedure was performed with 1% osmium tetroxide. Then, the specimens were washed with SPB again. The dehydration procedure was performed using low-to-high concentrations of alcohol (25%, 50%, and 75% absolute alcohol). The specimens were washed twice with propylene oxide before embedding. The first stage of preparation for embedding was the mixing of propylene oxide and epoxy resin embedding material in equal amounts (1:1 mixture), and the specimens were placed in this for 1 h. At the end of 1 h, the same amount of epoxy resin was added to the previously prepared mixture, to create a 1:3 mixture. The specimens were left in the rotator for one night, and the preparation for embedding was finished. Later, the specimens that were embedded in epoxy resin embedding material using plastic capsules were put into
AM ER IC AN JOURNAL OF OT OLARYNGOLOGY–H E A D A N D NE CK M E D IC IN E A ND S U RGE RY 3 7 (2 0 1 6) 3 93–3 9 7
the incubator for 48 h, at 60 degrees Celsius. After 48 h, the specimens were taken out of the incubator, and semi-thin sections were obtained with a LKB Nova ultramicrotome device (Sweden). The 2-μm-thick sections were stained with methylene blue to identify the fields suitable for obtaining thinner sections. The fields suitable for thinner sections were trimmed, and a tissue surface that could be sectioned for transmission electron microscopy was obtained. Then, the same ultramicrotome was used to prepare thin sections, with a thickness of approximately 60 nm. Those thin sections were stained using the double contrast method with uranyl acetate and lead citrate, and were then examined under the transmission electron microscope (Jeol JEM 1200 EX, Japan) by two authors, and photographed. The examinations of the study and the control groups were performed by two independent investigators. The axon myelination, normality of the myelin structure, the diameters of the myelinated axons, the thickness of the myelin sheath of the axons, and vacuolar degeneration were analyzed in both groups under the electron microscope. The number of total myelinated axons was determined by light microscopy. The data obtained in the groups were compared.
2.3.1.
Statistical analysis
The data were analyzed using SPSS 15.0 statistics package program. The descriptive statistics were presented as mean ± standard deviation, median (minimum; maximum), frequency distribution, and percentage. The Chi square test with Yates' correction was also applied. The comparisons of the variables were made with the Mann–Whitney U test. A value of p < 0.05 was accepted as statistically significant.
3.
Results
Electron and light microscopic examinations of the study and the control groups were analyzed independently. The electron microscopic examination of the tacrolimus group showed increased myelinization and thickened endoneurium
(Figs. 1 and 2). The myelin structure, axon numbers and axon cytoplasms were observed to be normal. Some unmyelinated nerves were seen in the fields examined, although the unmyelinated nerve ratios were similar in the two groups (Fig. 3). The axon diameters, myelin sheath thickness and number of total myelinated axons in the study and the control groups are presented in Table 1. The axon diameters were statistically significantly different when the study and the control groups were compared (p = 0.0001). The median axon diameter was statistically significantly greater in the study group. The median myelin sheath thickness was 0.83 (0.63–2.33) micrometers in the study group, and 0.38 (0.33–0.46) micrometers in the control group, with a statistically significant difference between the groups (p = 0.0001). The myelin sheaths were statistically significantly thicker in the study group. The median of total myelinated axon number was 570 (460–588) in the study group, and 470 (455–490) in the control group. The total myelinated axon numbers were statistically significantly higher in the study group when compared to the control group (p = 0.0001). In the study group, the number of the cells containing 1 or less vacuolar degeneration was 8 (80%) while the number of cells containing 2 or more vacuolar degenerations was 2 (20%). Those numbers were 2 (20%), and 8 (80%) in the control group, respectively. A statistically significant difference was determined between the groups in respect of vacuolar degeneration (p =0.025) (Chi square test with Yates' correction). The number of cells containing 1 or less vacuolar degeneration was significantly higher in the study group when compared to the control group. Statistically significantly greater axon diameters, thicker myelin sheaths, and higher total number of myelinated axons were found in the tacrolimus group suggesting better regeneration in this group when compared to the control group. There was less vacuolar degeneration in the study group. All those findings suggest that tacrolimus positively affects healing after facial nerve anastomosis.
4.
Fig. 1 – The electron microscopic photograph of the study group: Schwann cells are highly increased in number, and regenerated myelinated axons can be seen (original magnification ×3000).
395
Discussion
With increased understanding of anatomical, histological, and pathological processes and advances in the surgical techniques used in the treatment, major improvements have been made in the repair of peripheral nerve injuries that occur due to trauma, surgery, malignancy, inflammatory conditions or infections. Even when optimum conditions are provided, facial functions do not heal 100% in any cases after primary nerve repair [17]. To date, a number of methods have been studied in a number of clinical and experimental studies to increase healing and to improve functional results after traumatic facial nerve injury. Exploration, decompression, end-to-end anastomosis, free nerve graft, and lateral nerve anastomosis have been used as surgical treatment methods. Primary endto-end anastomosis must be performed if possible [2–5]. The possibility of complete, sequela-free healing is low in traumatic total peripheral facial nerve paralysis with current
396
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Fig. 2 – The electron microscopic photograph of the study group: Myelinated axons showing lamellar degeneration, and one ultrastructurally normal, regenerated, myelinated axon (original magnification ×3000).
Fig. 3 – The electron microscopic photograph of the control group: Ultrastructurally normal myelinated axons, normal unmyelinated axons, and a normal Schwann cell number (original magnification ×3000).
treatment options [6]. To prevent functional, cosmetic, and sociological problems in patients with peripheral facial paralysis, additional treatment methods that could improve nerve healing are needed. Tacrolimus is mainly used as an immunosuppressant agent in solid organ transplantations, such as liver and kidney, and it is particularly effective in steroid-resistant rejections [18]. The neuroprotective effect of tacrolimus has been reported in focal and extensive cerebral ischemia [19]. Some studies have shown the positive effects of tacrolimus on axon regeneration and nerve healing in peripheral nerve injury [2,15,16]. Li et al. investigated the efficacy of tacrolimus on healing of rat sciatic nerve, and found that the axons regenerated in various sizes and shapes and that myelin sheath thickness healed better in the tacrolimus group. The authors also showed that the axon diameters were significantly greater in the tacrolimus-chitosan guide group [13]. In the current study, the axon diameters of the nerves were also measured using electron microscopy, and the axon diameters were found to be significantly greater in the study group than in the control group. Just et al. [21] showed that tacrolimus had positive effects on healing of posterior tibial nerve injury in rats. In 2009, Chen et al. [3] investigated the effect of tacrolimus on end-to-end and end-to-side anastomosis of peroneal nerve, and reported that the tacrolimus group was superior for myelin sheath thickness and myelinated axon numbers. Similarly, the current study group was found to be statistically significantly superior to the control group in respect of total number of myelinated axons, which was used as the healing parameter in the study. There are few studies in the literature that have investigated the effect of tacrolimus on the facial nerve. The studies in the literature have investigated the immunosuppressive effect of tacrolimus for rejection of allografts and its effect on crush injury, rather than investigating its effect on the regeneration of the facial nerve in the event of cut injury. Lee et al. [20] found favorable functional effects of tacrolimus on healing of the rat posterior tibial nerve crush injury. That study showed the effect of tacrolimus on a peripheral nerve. The nerve was not cut completely but crushed and the crush injury improved with this agent. In the current
study, the effect of tacrolimus, which is an immunosuppressive and also a neuroregenerative agent for the peripheral nerves, was investigated for healing of iatrogenic cut injury of the rabbit facial nerve. The regeneration parameters used were the number of the total myelinated axons in a nerve bundle wrapped by the perineurium, the axon diameter and the myelin sheath thickness of the axons. Vacuolar degeneration was used as the parameter of degeneration. The myelinated axon number, myelin sheath thickness and myelinated axon diameters were found to be significantly higher in the tacrolimus group than the control group. Various agents such as insulin growth factor II have been continuously infused to the site of injury to increase nerve regeneration. Gold et al. [14] reported that the first agent which showed favorable results after systemic use was tacrolimus. The results of the current study have also shown that systemic administration of tacrolimus increased nerve regeneration. As it has immunosuppressive effects and immunosuppression related side effects, the minimal doses of tacrolimus for various organ transplantations are well studied for human in the literature. At this point, a very important limitation of this study is that the dose-dependent effects and the standard dose of tacrolimus are not clearly determined for animals in the literature. Before stating a dose for tacrolimus, we scanned literature and found some animal studies on neuroprotective effect of tacrolimus. Yeh et al. [16] used tacrolimus at a dose of 2 mg/kg/day, Gold et al. a dose of 1 mg/ kg/day [14], and Udina et al. [22] a dose of 5 mg/kg/day, which are high doses for human in the condition of organ transplantation. Also there are several animal studies examined the effect of subimmunosuppressive doses of tacrolimus on neuroregeneration. In the study of Yang et al. [23], the subimmunosuppressive doses of FK506 were determined as the doses below 2.0 mg/kg by examining skin allograft survival in a rat model in which the animals that received 2.0 mg/kg FK506 per day exhibited complete skin graft take, whereas all other groups that received FK506 below the doses of 2 mg/kg demonstrated complete rejection. In the second part of the same study statistically significant improvements in neuroregeneration with subimmunosuppressive FK506 doses
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Table 1 – Axon diameters, myelin sheath thickness and number of total myelinated axons in the study and the control groups. Study Group (n = 10)
Axon Diameter Myelin Sheath Thickness Total Myelinated Axon Number
Control Group (n = 10)
Median (min; max)
Mean ± SD
Median (min; max)
Mean ± SD
5.22 (3.97; 7.76) 0.83 (0.63; 2.33) 570 (460; 588)
5.55 ± 1.24 1.04 ± 0.51 539.40 ± 56.58
2.88 (2.73; 3.43) 0.38 (0.33; 046) 470 (455; 490)
2.96 ± 0.27 0.39 ± 0.05 472 ± 14.37
p Value 0.0001 ⁎ 0.0001 ⁎ 0.0001 ⁎
min; max: minimum; maximum, SD: standard deviation. ⁎ Mann–Whitney U Test.
of 0.5 and 1.0 mg/kg per day are reported [23]. Further studies are needed to determine the optimal dose for various animals. In our study, tacrolimus was used at a dose of 1 mg/kg/day.
5.
Conclusion
The results of this study indicate that tacrolimus has favorable effects on the healing process of the facial nerve after end-toend anastomosis. Tacrolimus may be a promising agent in the future for nerve regeneration following traumatic facial paralysis surgery. As this is a preliminary animal study, further studies are needed to evaluate the functional results of the facial nerve repair with tacrolimus and investigate the optimal dose and dose-dependent effects of tacrolimus.
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