The techniques illustrated in this video are useful in experiments, evaluating hormonal regulation of carcinogenesis and benign growth of the prostate. First appellate presses used to make hormone pellets that will allow for continuous administration. Next glass pipettes with rounded tips are prepared after the kidney is exteriorized.
The pipette tip is used to aid in graft insertion. The compressed hormone pellets are then implanted subcutaneously. Ultimately, results are obtained that show the effects of hormone treatment on the growth of benign tissue xenografts.
The techniques presented in this video are simple to perform and reproducible. This method can help answer key questions in the field, such as how hormones are implicated in the transformation of benign cells to malignancy, as well as how benign growth of the prostate is regulated by hormones. The implications of this technique extend toward potential new therapies and prevention strategies for benign prosthetic hyperplasia and prostate cancer.
Generally, individuals that are new to this technique will struggle with the technical aspect of renal capsule. Xenograft Pellets are prepared in a chemical safety hood while wearing proper protective equipment to begin clean the pellet making equipment thoroughly with 70%ethanol to prevent cross-contamination. Next, weigh the desired amount of hormone powder, allowing for approximately 5%extra material to accommodate for loss during pressing.
Some hormones require a binding agent or filler, which is mixed in prior to pellet manufacturer. Here estradiol is combined with cholesterol filler resulting in a 25 milligram pellet. Once everything is weighed and combined, transfer the powder into the dset.
Place the dye under the press and push the lever down firmly to compress the pellet. Use consistent pressure to maintain constant surface area to volume ratio. Next, reverse the dye holder and push the lever down again to release the pellet.
Inspect the pellet for integrity and determine if the final mass is within the desired range. Pellets can be made prior to surgery and stored according to the stability of the hormone or drug used while wearing appropriate protection. Pass the tip of a glass pastier pipette through the flame, a bunsen burner to create a fire polished, rounded tip.
Maintain constant and slight movement of the pipette to avoid hotspots. Next, place the pipette into the flame at approximately a 60 degree angle to create a curved rounded tip. This tip will be used to create a pocket for graft under the renal capsule.
Before beginning the surgical procedure, sterilize all necessary instruments and prepare the work area. Once the animal is properly anesthetized, disinfect the surgical site with repeated applications of Betadine and alcohol. After sterile drapes are applied to the area, a two to three centimeter dorsal midline incision is performed.
Next, separate the underlying dermis from the body wall. Separate both sides of the incision for bilateral grafting, or just one for unilateral grafting. Once separated, place the mouse into a lateral position and locate the kidney by viewing the renal profile through the muscle wall.
Applying gentle pressure with the thumb and index finger may assist with visualizing the internal organs using fine iris scissors and taking care to avoid major vessels and spinal nerves. Make a one centimeter incision in the body wall parallel to the spine. Widen this incision to one and a half to two centimeters by gently opening the scissors wider.
After placing them in the initial incision, exteriorize the kidney by applying gentle pressure outside the muscle wall on either side of the kidney. Next, tuck the skin edges below the exteriorized kidney, which will rest on the body wall. If other structures are also present, they can be gently tucked in while the kidney is exteriorized.
Maintain hydration of the renal capsule by applying sterile saline. Gently lift the kidney capsule and to make a two to four millimeter incision. The size of the incision is determined by the size of the graft, but should be minimized in order to maintain the integrity of the capsule.
Insert the rounded closed end of the pipette prepared earlier under the capsule tangential to the surface of the kidney. Once inserted, gently open a small pocket for the grafts using great care not to damage the kidney. Parenchyma tissue grafts are prepared from fresh prostate tissue obtained during surgical resection.
Lift the cut edge of the kidney capsule and insert the graft into the pocket. Using the pipette tip, several grafts can be placed under the kidney capsule and evenly spaced on the kidney surface. When grafting is complete, gently lift the sides of the muscle wall incision to replace the kidney back into the body cavity.
Make sure that the graft do not slip out from under the capsule. A single suture is used to close the muscle wall before proceeding with subcutaneous pellet implantation. When grafting is complete, separate the underlying dermis from the body wall in a cranial direction.
Gently lift the cranial aspect of the skin incision to create a pocket while holding the hormone pellet Gently with straight serrated forceps, insert the pellet at the scruff of the neck. Using toothed forceps, align the skin edges and apply two to three surgical wound clips to close the incision. In this example, primary tissue xenografts from patients with benign prostatic hyperplasia were supplemented with exogenous testosterone.
For one month, the arrowheads denote the xenograft locations. Renal capsule grafting can also be used to evaluate hormonal carcinogenesis. Here, cell recombinants containing benign prostate epithelium and inductive urogenital mechy form benign growths in untreated mice.
In this example, the same graft is grown in a host that received subcutaneous testosterone and estradiol pellets for four months and chose prostate carcinoma that invades the renal capsule. After watching this video, you should have a good understanding of how to manufacture and implant compressed hormone pellets, as well as how to create a fire polished glass pipette for surgery and perform renal capsule xenograft. When attempting this procedure, it's important to remember to gently handle the renal capsule, maintain moisture, and to avoid damaging the renal parenchyma Following this procedure.
Other methods such as histology, immunohistochemistry, and molecular techniques to detect R-N-A-D-N-A or proteins of interest can be performed. This will allow for a better assessment of the nature of the benign or malignant growth observed. Don't forget that hormones utilized in these experiments are potentially teratogenic and carcinogenic.
Compressed hormone pellet manufacturer should always be performed in a chemical safety hood. Proper personal protective equipment should always be worn for each step of the procedure outlined in this video.
