The experimental protocol for the use of rats was evaluated and approved by the Committee for the Care and Use of Laboratory Animals, at Benemérita Universidad Autónoma de Puebla (100–310-955-UALVIEP-19/2). Technical specifications related to the production, care, and use of animals are specified in the guidelines of the Mexican Council on Laboratory Animal Care (NOM-062-Z00-1999).
Experimental designThree-month-old, virgin rats of the CIIZ-V strain (250–350 g body weight) were studied. The rats were maintained on a 12/12-h light/dark cycle with food (Lab-Diet 5001, Rodent diet) and water, provided ad libitum. Estrous cycles were monitored with daily vaginal smears. Only rats that showed at least two consecutive four-day cycles were used in the experiment.
Rats were randomly assigned to one of the following bilateral studies: Gross anatomy (n = 10), acetylcholinesterase histochemistry (n = 4), or scanning electron microscopy (SEM; n = 4). Additionally, rats were analyzed unilaterally (right side) with a fluorescent retrograde tracer (n = 4).
Surgery was performed under anesthesia with ketamine (90 mg/kg) and xylazine (15 mg/kg) at 9:00–10:00 am, on diestrus. All rats were sacrificed with an overdose of sodium pentobarbital (60 mg/kg, i.p.; Anestesal, Smith Kline, Mexico City, Mexico).
Gross anatomyRats were anesthetized with an intraperitoneal injection of urethane (ethyl carbamate, 1.2 g/kg; Sigma-Aldrich, St. Louis, MO). To expose the SON, a 3-cm paramedian skin incision was made. The SON was located and dissected. Images viewed through a stereo microscope (LEICA M80, GERMANY) were captured in hand drawings. Additionally, digital photographs were acquired with a digital camera (PROGRES GRYPHAX® SUBRA, Germany) and managed in PROGRES GRYPHAX® Microscope Camera Software.
Acetylcholinesterase histochemistryThe acetylcholinesterase histochemistry is a technique useful in the diagnosis, gross anatomy and recognition of nervous fibers of the peripheral nervous system [8, 15,16,17]. In this work it is used it to localize very thin fibers.
Rats were sacrificed with an overdose of sodium pentobarbital, then were perfused transcardially with 300 ml saline solution (0.9% NaCl). The SON was dissected from the uterus to the diaphragm, and the dissection included the ovaries, kidneys, and the suspensory ligament of the ovary. A longitudinal incision was made in the abdominal-pelvic regions to extend it flat, and fat tissue was removed. Thereafter, the tissue was fixed by immersing in 10% formalin for at least 48 h. The tissue was washed in phosphate buffer (PBS) 1X, then incubated for 2.5–3 h at room temperature in a solution of acetylthiocholine-iodide dissolved in 0.1 M sodium-hydrogen-maleate, 0.1 M sodium-citrate, 30 mM CuSO4, 5 mM potassium-ferricyanide, and 0.1% Triton X-100 (pH 6.0). Next, the tissue was, dehydrated with ascending alcohol concentrations, cleared with xylene, and observed and photographed under a stereo microscope (LEICA M80, GERMANY). Digital photographs were taken with digital camera (PROGRES GRYPHAX® SUBRA, Germany).
Electron microscopyThe micromorphology of the suspensory ligament of the ovary was observed and analyzed with a Tescan Vega SEM (TS 51365B, Tescan, Brno, Czech Republic). Briefly, the suspensory ligament of the ovary tissue was fixed in a 4% glutaraldehyde solution for 24 h and washed with PBS (pH = 7.1). The tissue was then dehydrated in a series of ethanol solutions (30%, 50%, 70%, 85%, 96%, and absolute ethanol). Subsequently, the tissue was dried with liquid CO2 for 10 min in a Samdri-795 point dryer (Tousimis, Rockville, MD), incorporated into aluminum pin stubs, and sputter-coated with a 10-nm gold layer, with a Vacuum Desk II sputter coater (Denton Vacuum, LLC, Moorestown, NJ). The scanning was performed with an accelerating voltage of 20 kV.
Fluorescent retrograde tracerFour rats in proestrus were anesthetized with an intraperitoneal injection of ketamine (90 mg/kg) and xylazine (15 mg/kg, i.p.). Surgery was performed with a unilateral incision, starting at 3 cm below the lower-most rib, under a stereomicroscope (LEICA M80, GERMANY). The right OPN was surgically denervated, and the right SON remained intact. The right ovarian bursa was then injected with 5 µl of True Blue tracer (TB, diluted 1% with distilled water). The ovary was carefully cleaned, dried, and returned to the abdominal cavity. The rats were maintained in a warm chamber until they recovered from anesthesia, and they were given antibiotics and analgesia. Then, the rats were returned to the bioterium for maintenance.
Seven days later, the rats were anesthetized and sacrificed by performing transcardial perfusion with 200 mL cold saline solution, followed by 200 mL fixative solution (4% paraformaldehyde in PBS at pH 7.1). After perfusion, the CG, SG, and superior mesenteric ganglion (SMG) were stored in the fixative solution (approximately 2 h). The nervous tissue was cryoprotected by immersing in a series of sucrose solutions (10%, 20%, and 30% sucrose in PBS 1X); the tissue was stored for 24–48 h at 4 °C in each solution. Next, the tissue was embedded in Tissue-Tek medium for frozen tissue samples (Sakura Finetek USA, Torrance, CA) and sectioned in 20-µm slices with a cryostat (77,210,163, Thermo Scientific, Waltham, MA). The sections were mounted in poly-L-lysine (adhesive slide solution, Sigma-Aldrich, St. Louis, MO) and placed on clean, glass microscope slides.
The sections were analyzed with epifluorescence microscopy (Olympus BX 41, Olympus Corporation, Tokyo, Japan). TB-positive neurons were observed with UV light (340–380 nm excitation filters). The raw number of labeled neurons was adjusted with the Abercrombie´s correction factor. The sections were photographed with a digital camera (Evolutions VF, Media Cybernetics, Canada). Digital photomicrographs were saved as TIFF files, and images were analyzed and measured with Image-Pro Premier 9.2 software (Media Cybernetics, Bethesda, MD), after contrast and brightness were automatically adjusted. The number of neurons labeled with TB in the CG, the SG, and the SMG were counted, and results are presented as the mean ± standard error of the mean for the four rats.
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