Ethical statement
The experimental procedures followed the guidelines for the ethical treatment of experimental animals and were approved by Mustansiriyah University, Iraqi Center for Cancer and Medical Genetic Research, Animal Care, and Use Ethics Committee. The date of ethical approval is 30 July 2010, and reference number was 5.
Experimental animals
The animals used were 8 New Zealand White rabbits (Oryctolagus cuniculus), adult male (weight 2.8–3.9 kg) and 3 months old. The animals were randomly divided into two experimental groups, four in each: first group treated with LLLT at dose of irradiation (31.5) J/cm2. The second group was control non-irradiated. Both groups received a standard diet and water during the study and were kept under standard lighting, temperature, and humidity environment.
Anesthesia
All rabbits were anesthetized by xylazine hydrochloride 2% (5 mg/kg body weight) and ketamine hydrochloride 10% (50 mg/kg body weight) through intramuscular injection.
Surgical procedure
The experimental surgical procedure was performed according to La et al. [15] under the typical aseptic conditions via exposing the sciatic nerve through midline incision at the back of the right leg and longitudinal separation of the muscles. Focal crushing of right and left sciatic nerve of each rabbit was done for 60 s by straight hemostat. The wound was closed using silk simple sutures. The surgical procedures were performed by a veterinary surgeon.
Postoperatively, 40.0 mg gentamycin were given to the rabbits for 4 days to prevent infection. Clinical assessment was measured postoperatively via recording the daily examination of ability onset to walk after operation until 2 months.
Laser irradiations
The source of laser used in this study was Helium-Neon (He-Ne) gas laser (Model DL30, LG Lasers) with output power approximately 20 mW as a continuous wave irradiation (Fig. 1a). The wavelength of light that emitted from this laser and output power were determined by using a portable power meter (LaserCheck, Coherent CA, USA) (Fig. 1b), according to the measurements, the wavelength was 630 nm, penetration of 0.6 cm. The irradiation area was (0.125) cm2, while the exposure time of irradiation was 240 s, Dose of irradiation was 31.5 J/cm2. The treatment by laser was started first day post-surgical, where all the rabbits in the treatment group were exposed to the identical technique with once a day application for 10 consecutive days and observed for 30 days.
Rabbits were handled softly, and laser treatment did not yield any painful impression to the treated group. Control group were exposed to the same technique, but with no laser treatment.
Gross examination
Clinical assessment was measured postoperatively via recording the daily examination of ability onset to walk after operation until 30 days.
Euthanasia
All animals were sacrificed 30 days post-surgery using overdose of anesthesia, and the healed wound nerve segment of the operated location was collected from each sciatic nerve.
Histopathological examination
Every 7 days of treatment, one longitudinal section was made through the site of repair and was stained with hematoxylin and eosin. They were then examined under light microscopy for qualitative assessment of the repair process. Transverse sections were obtained for quantitative assessment of the diameters of the axons.
Directly after harvesting the repaired nerves, they were fixed in 10% neutral buffer formalin, and the specimens were sent for histological processing. The samples were treated with xylene, dehydrated using graded ethanol, nerve tissue samples were paraffin embedded, sectioned at 5 μm longitudinally, via microtome and stained with hematoxylin-eosin. The injured nerve area was observed under a light microscope (Leica-microsystems, Germany).
Histopathological images quantitative analysis
The histopathological sections were photographed at _× 200 magnification at four randomly designated fields at the histological sections, using the light microscope (Leica-microsystems, Germany) and a digital color camera (Motic, Hong Kong). The pictures were analyzed using ImageJ software (http://rsb.info.nih.gov/ij/). The longitudinal and transverse sections of the nerve samples were analyzed by pathologist. The histopathology examiner was unaware about the groups. For statistical analysis, the quantitative measurement of each picture was taken at least three times. Nerve nuclei and vacuoles (%) in sections stained by hematoxylin-eosin (H&E) were measured. This was done according to Yang et al. [30].