Murine Prostate Micro-dissection and Surgical Castration

The overall goal of these procedures is to allow the study of the prostate, as well as varying hormonal conditions within the mouse. This method can help answer key questions in the prostate cancer field, such as, what are the effects on hormone deprivation on prostate carcinogenesis. The main advantage of this technique is that the effects of various pathological processes and hormonal changes can be studied in individual prostate glands.

Begin by pinning a mouse to a dissection board in the supine position through the foot-pad of each paw and spraying the abdomen with 70%ethanol. Next, holding the skin of the lower abdomen with forceps, use scissors to cut through the skin and peritoneum approximately one centimeter anterior to the opening of the penis. Continue to cut the skin and peritoneum from the lower abdomen up both sides of the abdomen to the rib cage, exposing the peritoneal cavity.

After identifying the location of the bladder and urogenital tract, or UGT, gently move the fat and other organs to the side to expose the urinary tissues. Then, using forceps, grasp a vas deferens at its base near the urethra, and tear away the vessel. After removing the other duct, use forceps to carefully hold and pull up the bladder, while simultaneously using scissors to cut through the urethra below the ventral prostate and approximately one centimeter below the base of the bladder.

Transfer the harvested UGT into a 60 to 100 millimeter petri dish containing approximately five to 10 milliliters of PBS, and place the dish under a dissection microscope. Next, pick up one pair of fine forceps in each hand, holding the instruments like pencils, and rest the forearms on the bench top, on either side of the microscope, to keep them steady. Position the UGT with the forceps, such that the bladder is facing up, the urethra is pointed down, and the seminal vesicles are positioned on either side of the bladder.

Grasping the urethra with one forceps, use the other forceps to carefully pull away the fat without removing any prostate tissue. When all of the fat has been removed, tear the connective tissue between the two ventral lobes of the prostate to separate them. Then, holding one of the ventral lobes near the tip with one pair of forceps, grasp the base of the lobe as close to the urethra as possible.

Now, move the other pair of forceps to restrain the urethra while pulling the lobe away from the vessel with a firm, smooth motion. Confirm that no prostate tissue remains attached to the urethra. Then, place lobe into the appropriate medium, according to the next experimental step.

After isolating the other ventral lobe in the same manner, use forceps to gently pull one of the anterior lobes away from the seminal vesicle, taking care not to puncture the seminal vesicle. Then, once the lobe is independent of the seminal vesicle, remove it in the same fashion as just demonstrated for the ventral lobe. After isolating the second anterior lobe, flip over the remaining UGT, such that the dorsal prostate is visible, and use forceps to tear the connective tissue between the two dorsal lateral lobes and between the lobes and the posterior region of the seminal vesicles.

Then, remove the individual dorsal lateral lobes, as just demonstrated. To perform the castration, begin by placing an anesthetized mouse onto a clean, sterile surface in the supine position and confirm the appropriate level of sedation by toe pinch. If the mouse does not react, use an electric razor to shave the surgical area and sterilize the abdomen.

Next, use sterile surgical scissors to make a one centimeter vertical incision through the midline skin of the lower abdomen, approximately one point five centimeters anterior to the penis, followed by a smaller incision through the peritoneum, taking care not to cut any organs. Using sterile forceps, gently lift the cut edge of the peritoneum to visualize the peritoneal cavity and use another sterile forceps to pull out one of the testicular fat pads until the testes appears. Then, use a cautery pen to slowly cut through the fat pad, holding the testes until the testicular artery has been severed.

If the artery is cut too fast, the vessel may bleed too much, requiring euthanization of the mouse. I recommend heating the cautery pen in spurts, and not letting it get too hot, then, moving the instrument slowly through the artery so that the animal does not bleed out. Once the fat pad and artery have been cut, discard the testes and fat pad and repeat the castration for the other testes.

After the second testes has been removed, close the peritoneum with sterile silk sutures, and staple the skin with surgical wound clips. Then, administer analgesia to manage the pain and place the mouse in a clean cage, on a cage warmer, for 5 to 10 minutes with monitoring, until it is fully recovered. The complete urogenital tract is composed of all six prostate lobes, the bladder, the seminal vesicles, and the urethra.

The vas deferens attaches to the urethra, but is unnecessary for the prostate miro-dissection and can thus be detached before the removal of the UGT. The urogenital tract will remain intact once it has been removed from the abdomen;covered in fatty tissue that needs to be removed to gain access to the prostate lobes. In this representative experiment, the castration was followed by two cycles of hormonal regeneration, and the testosterone levels in the serum were monitored by ELISA.

As expected, under castration conditions, the testosterone levels virtually disappeared. In the presence of tamoxifen, a selective estrogen receptor modulator, no significant difference to the hormonally normal condition was observed. After castration, however, testosterone reintroduction caused a dramatic spike in the serum testosterone levels.

After micro-dissection, the prostate lobes of castrated mice are significantly diminished, while the lobes from testosterone-treated animals, recover to their original size. Further, hematoxylin and eosin staining of the anterior prostate tissue under normal, castrated, and regenerated conditions, reveal a dramatic reduction in robust tissue growth in the sections from the castrated animals. Once mastered, this technique can be completed in five minutes per mouse, if it is performed properly.

While attempting the castration, it is important to remember to keep everything sterile and to make sure to control the pain felt by the mouse. Following castration, other methods like androgen reintroduction, can be performed to answer additional questions about the effects of testosterone on prostate tumors. After its development, this technique paved the way for researchers in the field of prostate cancer to explore carcinogenesis, development, and castration resistance in mice.