Anatomical organization and neural pathways of the ovarian plexus nerve in rats

The experiment protocol numbered VIEP/0118/2014 to use rats was approved by the Care and Use of Laboratory Animals Committee at Benemérita Universidad Autónoma de Puebla. Technical specifications related to productions, Care and Use of Animals are Specified in the Guidelines of the Mexican Council on Laboratory Animals Care (NOM-062-Z00-1999).

Nineteen 3-month-old virgin rats of the CIIZ-V strain (250–350 g body weight) were studied. The rats were maintained on a 12/12-light/dark cycle with food (LabDiet 500, Rodent diet) and water provided ad libitum. Estrous cycles were monitored by daily vaginal smears. Only rats showing at least two consecutive 4-day cycles were used in the experiment. Rats were randomly distributed to one of the following experiments: gross anatomy (n = 8), histological processing (n = 3), and retrograde-neuron tracer (n = 8). The surgeries were performed under anesthesia between 9:00 and 10:00 h on diestrus. The diestrus period in the rat, has the longest duration of the estrous cycle, besides the neuronal activity is the most stable [19]. All rats were sacrificed during the diestrus day a cycle after with an overdose of sodium pentobarbital (60 mg/kg, i.p. Anestesal, Smith Kline, Mexico City, Mexico).

The rats were anesthetized with an intraperitoneal injection of urethane (ethyl carbamate, 1.2 g/kg; Sigma-Aldrich, Toluca, Mexico). For exposure of the OPN, a 3 cm paramedian skin incision was made. The OPN was dissected and the SMG was located attached to the inferior vena cava. Drawings were made using a stereoscopic microscope (Carl Zeiss Stemi 2000C, USA). The digital photographs were taken and managed in Imagen Pro Plus, version 6.3 for Windows (Media Cybernetics, Inc.).

Three rats were sacrificed with sodium pentobarbital. The skin and the abdominal muscle were cut. Using a stereoscopic microscope, the superior mesenteric ganglion (SMG) was located attached to the inferior vena cava, and it was dissected. The tissue was placed in Böuin Duboscq’s fixative solution (glacial acetic acid, formalin, and picric acid) for 12 h. The fixed tissues were rinsed, dehydrated in graded ethanol, and embedded in paraffin. Paraffin sections at 7-μm thickness were cut with a microtome (Leica RM2125RT, Germany) and collected on gelatin-coated slides. The sections were deparaffinized in xylene, rehydrated through graded ethanol, and stained with Nissl stain (used for the detection of Nissl bodies in the cytoplasm of neuron). All slides were subjected to histological analysis.

Eight rats in diestrus were anesthetized with an intraperitoneal injection of ketamine (90 mg/kg) and xylazine (15 mg/kg, i.p.). A unilateral incision was made 3 cm below the last rib, affecting the skin, muscle and peritoneum. Under a stereoscopic microscope, the left or right ovarian bursa was injected with 5 μl of True Blue (TB; Sigma, St. Louis, Missouri, USA), which was diluted at 1%, of distilled water; This procedure was previously reported by Lawrence and Burden [1] where they made an immersion of the cut superior ovarian nerve in the TB solution. In this study we injected the TB into the ovarian bursa [19, 20]. To prevent a leakage of the tracer, the needle was kept in the bursa for 5 min after injection. The ovary was carefully cleaned, dried, and returned to the abdominal cavity. The rats were kept in a warm chamber until their recovery from anesthesia, and they were provided with antibiotics and analgesia. After that, they were returned to their bioterio.

Four days later, the rats were anesthetized, sacrificed by transcardial perfusion with 200 mL of cold saline solution, followed by 200 mL of fixative solution (4% paraformaldehyde in phosphate buffer at pH 7.3). After perfusion the right SMG, including lumbar ganglion of the sympathetic trunk (LGST) was kept in the fixative solution (approximately 2 h). The tissue nervous was cryoprotected in sucrose solution of increased concentration (10, 20 and 30% sucrose in phosphate buffer 1%). The nervous tissue was stored for 24–48 h in sucrose buffer solution at 4 °C, embedded in Tissue-Tek medium for frozen tissue specimens (Sakura Finetek USA, Torrence, CA) and cryostat sectioned (Micron HM 505 N Cryostat, Walldorf, Gemany) at 20 μm. The sections were mounted on poly-L-lysine (Slide adhesive solution, Sigma-Aldrich, St, Louis, MO, USA) and placed onto clean microscope glass slides.

The sections were analyzed with a fluorescence microscope (Olympus BX 41, Olympus Corporation, Tokio, Japan). A positive neuron to TB was seen through the ultraviolet light (340–380 nm excitation filters). The raw number of labeled neurons was adjusted using the formula for 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 the images were analyzed and measured using Image-Pro Plus 6 (Version 6.3 for Windows, Media Cybernetics, Bethesda, MD, USA) with automatic adjustment of brightness with the purpose to prevent the auto-fluorescence by a fixative solution. The results of the number of TB labeled neurons in the SMG and LGST were presented as mean ± standard error of the mean (SEM) for the eight rats. The area of the soma was presented as mean ± SEM of the 10 neurons per rat by tracing the outline of the labeled cell body and calculating the enclosed area.

The OPN is a bilateral nerve and it has a length of 2.5 to 3 cm from the ovary to the first prevertebral ganglion. The right and left OPN runs adjacently and along the ovarian artery and bifurcates for approximately 3 mm before it gets to the ovary, the other branch goes to the uterus (Figs. 1 and 5).

The right OPN (R-OPN) has a dual origin: one branch arises from the SMG and the second one from the mesenteric plexus (Fig. 1, number 5). The postganglionic fibers of the R-OPN show two arrangements in relation to its origin (Fig. 2a, b). In 63% of the rats, one plexus under the renal vein (Fig. 2a) was found ventrolaterally attached to the inferior vena cava. This plexus consists of two nerves: lumbar (Fig. 2, number 2) and celiac branch (Fig. 2, number 1). The first one arises from the LGST, while the second one from the celiac ganglion (CG). The lumbar branch joins the anastomotic one (Fig. 2, number 3) whereas the celiac branch follows two paths: the anastomoses to the SMG and the other one joins the lumbar branch (Fig. 2, number 2). On the other hand, in 37% of the rats, the SMG and the anastomotic branches that originate the R-OPN come directly from the SMG. The lumbar (Fig. 2b, number 2) and celiac branch (Fig. 2b, number 1) go directly to the cranial part of the SMG (Fig. 2c).

The left OPN (L-OPN) postganglionic fibers originate from the anastomotic branch of the LGST (Fig. 1, number 6), and the anastomotic branch from the splanchnic nerve (ABSN) (Fig. 1, number 7). Before the L-OPN innervates the ovary for approximately 3 mm before it gets to the ovary, the other branch goes to the uterus (Fig. 1).

The SMG is the only ganglion located in the right side, and it is attached to the inferior vena cava. Three nerves arise from the SMG: two cranial that anastomose in the celiac, and the lumbar branch. The last two join the anastomotic branch and they form right OPN. The intermesenteric nerve arises caudally from the SMG, and it goes through the ventrolateral wall of the inferior vena cava and joins a small ganglion located in the inferior vena cava. The fibers of this ganglion reach the inferior mesenteric ganglion (IMG; Fig. 2a, b).

The SMG has a spindle shape (area 3824 μm²), and it contains 177 ± 26 neurons (numbers adjusted using Abercrombie’s formula) (Fig. 3a). It was observed that the shape of the SMG neurons was oval. The majority of the nuclei were localized in the center of the perikarya (Fig. 3b). The area of the majority of the neurons was 84 ± 4 μm².

When the TB tracer was injected into the right ovary, labeled postganglionic neurons were observed in the SMG (30 ± 8 neurons; Fig. 4a). In contrast, when the TB was injected into the left ovary, labeled postganglionic neurons were not observed in the SMG (Fig. 4b); however, labeled postganglionic neurons were observed in the first LGST (58 ± 17.4 neurons; Fig. 4c).