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 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.

2.1

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 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.