Oral Surgery Oral Medicine Oral Pathology Oral Radiology and Endodontics

Objective. This work evaluated sciatic nerve regeneration after cryotherapy. Study design. Rats underwent surgical access of the sciatic nerve and subsequent cryotherapy, crush lesion or no manipulation. Walking-track, electroneuromyographic and histomorphometric analyses were performed at 15, 30 and 70 postoperative days. Results. At 15 days, the crush and cryotherapy groups showed significant morphofunctional impairment. At 30 days, functional loss was significant in the walking track group, but at 70 days, there were no significant differences between the groups. Amplitude was near zero for the crush group at 15 and 30 days, and zero for the cryotherapy group. Measurement of latency was not possible in this latter group. Crush and cryotherapy groups showed greater amounts of myelinated fibers (by 30 day), with axonal diameter and width of the myelin sheath being less than in controls. Conclusion. Sciatic nerve lesion by application of liquid nitrogen is classified as axonotmesis, which is reversible. INTRODUCTION The jaws can be the site of some benign lesions such as ameloblastoma, odontogenic keratocystic tumor, odontogenic myxoma and central giant cell lesion, whose locally aggressive behavior leads to potential recurrences. These recurrences are related to tissue remnants of the lesion in the bone margins. Therefore, the conventional treatment in these cases is surgery with safety margins or resection, which can result in mutilation and difficult esthetic and functional rehabilitation. The therapeutic approach can be conservative, through surgical enucleation or curettage, if these procedures are complemented by applying to the residual bone cavity an agent capable of eliminating the cellular remnants. Cryotherapy is an efficient method of tissue destruction by freezing, which induces bone necrosis, where the inorganic scaffold remains intact, allowing osteoconduction. For this reason, it has been used as complementary therapy for locally aggressive bone lesions. It produces safety margins without esthetic and functional sequelae, with the major advantage of a conservative treatment, preserving important anatomical structures such as the inferior dental nerve and infraorbital nerve. However, in cases of extensive lesions, low temperature can also exert some effects on nerve branches causing injury to peripheral nervous tissue. Some clinical studies suggest the reversibility of neurosensory changes associated with cryotherapy applied near or directly to peripheral nervous tissue. Nevertheless, the issue is controversial and most studies supporting this idea are clinical, with no experimental in vivo studies reported. Research involving humans generally shows high variability, whereas the rat sciatic nerve lesion is the best experimental model to test the morphophysiologic effects of different protocols such as crushing and cryotherapy. The present work investigated rat sciatic nerve regeneration after cryotherapy with liquid nitrogen by means of walking-track, electroneurophysiologic and histomorphometric analyses. MATERIAL AND METHODS The present study was approved by the Ethics Committee for Animal Use of the Pontifical Catholic University of Rio Grande do Sul. The sample comprised 54 adult male Wistar rats weighing an average of 272 g. The animals were randomly allocated into 9 groups of 6 rats each, and classified according to the type of lesion produced in the peripheral nerve and the time of analysis. Groups 1 (15 days), 2 (30 days) and 3 (70 days) comprised rats without sciatic nerve lesion (controls); groups 4 (15 days), 5 (30 days) and 6 (70 days) comprised rats subjected to a sciatic nerve crush lesion; and groups 7 (15 days), 8 (30 days) and 9 (70 days) comprised rats subjected to cryotherapy lesion of the sciatic nerve. Sciatic nerve approach The animals were anesthetized intraperitoneally with a solution of 5% ketamine (90 mg / kg, Vetbrands, Jacareí, SP, Brazil) and 2% xylazine (15 mg/kg, Vetbrands, Jacareí, SP, Brazil). The right sciatic nerve was surgically accessed and exposed at its emergence from the distal bifurcation. A styrofoam device was positioned between the sciatic nerve and dissected muscles, in order to isolate the area receiving cryotherapy. The lesions (crush and cryotherapy) were performed in the sciatic nerve at 1 cm proximal to its bifurcation into the tibial and common peroneal nerves. The control groups were subjected to the surgical access and placement of the styrofoam device, without producing any sciatic lesion, either crush or cryotherapy. A Cry-Ac cryogenic system (Brymill Cryogenics Systems, Ellington, CT, USA) for liquid nitrogen spray was used in the cryotherapy groups. Two applications of nitrogen, lasting 10 seconds each, with a 2.5-minute interval between them, were performed. In the crush groups, the sciatic lesion was performed with a calibrated curved Halstead mosquito forceps, also in 2 applications of 10 seconds with a 2.5-minute interval in between. The wound was closed in layers with interrupted 4-0 mononylon sutures. Walking-track analysis At 15, 30 and 70 days after the surgery, the animals underwent a postoperative walking-track analysis, based on the protocol described by De Medinaceli et al. The rats were trained to walk over a white sheet of paper covering the bottom of a wooden alley (43 x 9 x 7 cm) ending in a dark goal box. Afterward, the animals had their ventral hindpaw painted with dark dye, and then they were placed on the track to walk. The rats paw prints were used to determine the following measurements: (1) distance from the second to the fourth toe (intermediate toe spread, ITS); (2) distance from the first to the fifth toe (toe spread, TS); and (3) distance from the heel to the third toe (print length, PL). These measurements were then inserted in the sciatic functional index (SFI) formula according to Bain et al. Electroneurophysiologic analysis After the walking track, animals underwent electroneurophysiologic analysis, in which the motor neuroconduction of the sciatic nerve was determined. With the animals hand held, an electric stimulus of 0.2 milliseconds and 30 mA was applied in the proximal region of the sciatic nerve. In this way, a bipolar stimulator (Medelec Synergy, San Francisco, CA, USA) was positioned parallel to the long axis of the sciatic, and electrodes were placed on the dorsal and ventral surfaces of the hind leg. Another surface electrode was placed on the tail, serving as a ground electrode. The variables amplitude and latency of action potential of the right paw were measured. Euthanasia of animals and histologic processing After the walking-track and electroneurophysiologic analyses at the different times of observation, the animals were anesthetized and humanely killed. A 1-cm segment of the right sciatic nerve was excised at a point distally located in relation to the lesion site. The specimen was fixed by 24-h immersion in a modified Karnovsky solution (Sigma Chemicals, St Louis, MO, USA). Next, it was bathed in 0.05 M sodium cacodylate, post-fixed in 1% osmium tetroxide (Sigma Chemicals, St Louis, MO, USA) for 2 hours, dehydrated in an increasing graded series of acetone (Electron Microscopy Sciences, Hatfield, PA, USA), and embedded in epoxy resin (Araldite, Durcupan, Fluka, Buchs, Switzerland), which was then polymerized at 60°C. Semi-thin cross-sections (1 μm) were obtained, using an ultramicrotome (MT 6000-XL, RMC, AZ, USA) and stained with 1% toluidine blue (Merck, Darmstadt, Germany) in 1% sodium tetraborate (Ecibra, Curitiba, PR, Brazil). Histomorphometric analysis The histologic images were digitized using a videocamera (Axiocam-Zeiss, Zeiss, VA, USA) coupled to a light microscope (Imager A1-Zeiss, NY, USA) and to a computer (Compaq, Pentium 4, NY, USA) with x 5 and x 100 objectives. Histomorphometric analysis was performed by a calibrated blind examiner using Image Pro Plus 4.1 software (Media Cybernetics, MD, USA). Myelinated fibers were counted in each image and their density (no. of fibers/area) was calculated. Myelin sheath thickness, axonal area and axonal diameter were calculated by means of a specific software tool. Statistical analysis The results obtained were compared between the groups (control, crush and cryotherapy) at the different times evaluated. Data were analyzed by descriptive statistics, the Kruskal-Wallis test (complemented by its multiple comparison test) and one-way ANOVA (complemented by Tukey’s test), setting the level of significance at 5%. The statistics were processed by the SPSS 10.0 software (Statistical Package for the Social Sciences, Chicago, IL, USA).