The previous protocols for mouse models of peritoneal dialysis suffer from limitations precluding their long-term use. These limitations, such as catheter coiling and dislodgement, are addressed in this study. This technique prevents PD catheter malfunction for mechanical reasons, allowing their use for a longer time to study pathologies, such as peritoneal fibrosis.
This technique will provide a better mouse model to probe molecular pathogenesis and novel therapeutics of peritoneal fibrosis. A key limitation of PD in humans. PD is the mainstay of renal replacement therapy in pediatric patients with ESKD.
This optimized model will provide a better test bed to investigate all the issues with PD in humans. Generation of an animal model has a learning curve. This step-by-step video will assist an individual in implementing this technique to generate a durable model for peritoneal dialysis successfully.
To begin place the fully anesthetized eight to 12 weeks old C57 black/6J mouse in a left lateral position. After shaving the mouse as specified in the manuscript, disinfect the surgical area by exposing the right flank of the mouse to the heating blanket. Then place the sterile surgical drape over the mouse.
Assign the access port pocket one centimeter above the mouse tail. Hold the installation segment with the non-dominant index and thumb finger over the assigned area near the tail. Place the catheter above the skin, and estimate the place for the catheter's tube insertion within the abdominal cavity.
Then mark the assigned place for tube insertion, respecting the minimal bending of the tube near the anterior midline. Punch a side hole over the frame of the reservoir section with the mouse ear tagger. Make a horizontal one centimeter wide skin incision one centimeter above the tail.
Then bluntly dissect the subcutaneous plane from the underlying muscular layer to make a pouch for the catheter placement to ensure the installation port freely resides in the ideal port pocket. Past the 3-0 suture from the customized side hole. Fix the access port to the muscular bed by tightening the passed suture, keeping the tubing course cephalad.
Make a one centimeter incision over the formally marked area near the midline. Place a loose purse string suture with 4-0 round absorbable suture around the incised abdominal wall muscle. Make a one centimeter incision over the muscular layer close to the right midline and pass the proximal felt of the catheter inside the incision.
Pass the catheter tube through the prepared tract. Tighten the prepared purse string suture around the tube, while keeping the second felt outside the purse string over the muscular layer, and close the skin with 3-0 absorbable sutures. Ensure that the placed catheter is functional.
Check the function with a one milliliter syringe attached to the specific Huber needle for the port. Inject normal saline into the installation port. After confirming a smooth flow with a zero tolerance for resistance, close the skin incisions around the port reservoir with 3-0 absorbable sutures.
Enter the port with the Huber needle and inject 100 microliters of normal saline into the port to confirm the patent course. Then inject 200 microliters of lipopolysaccharide, followed by 100 microliters of saline for tube irrigation and ensure that there is no resistance. After seven days of LPS injections and two weeks of catheter implantation, plan for the peritoneal biopsy.
After confirming successful anesthesia, make a midline skin incision from bladder to sub xiphoid. Then perfuse the subfascial plane with cold PBS. Make sure the plane is completely dissected without disturbing the integrity of the peritoneum.
Analysis of the hematoxylin and eosin stained sections showed a substantial increase in the extracellular matrix, or ECM, in the subperitoneal space. The average ECM in the sub peritoneal space of the control mice was half of that in the LPS exposed mice. The Masson's Trichrome staining detects fibrosis, measured as intensity density normalized to the surface area.
Altered vascularity and widening of the subperitoneal space were analyzed using endothelial cell marker CD31, and measured as integrated density in randomly selected high-power field images. Compared to the control, LPS-induced mice showed a threefold increase in subperitoneal fibrosis, approximately eight to ninefold increase in the vascularity, and a twofold increase in the subperitoneal space, marked as SP, were observed. Place the catheter above the skin and mark the point for the catheter insertion, while avoiding the bending of the tube.
The insertion should be near the interior midline. Providing the more durable murine PD catheter placement method will allow the researchers to design and execute longer term experiments to probe and target peritoneal membrane failure.
