A Murine Model of Irreversible and Reversible Unilateral Ureteric Obstruction

This video presents a mouse model of obstructive neuropathy induced by unilateral ureteric obstruction that can either be irreversible or reversible histological data highlighting the resulting structural injury and its recovery following the Reversal is presented. This schematic Cartoon illustrates the major steps of this model. Here we see the left kidney ureter and bladder.

A standard unilateral ureteric obstruction is induced by ligation of the ureter. The ureter becomes dis standard and the kidney becomes hydro nephrotic over time. To perform a reversible unilateral ureteric obstruction, the ureter is ligated close to the bladder and a soft wall plastic tubing shown in gray is applied around the ureter at the point of obstruction.

In order to limit excessive ureteric dilation, the kidney becomes hydro nephrotic over time and is then de obstructed at a time point appropriate for the study. Seven days in this experiment, the soft wall tubing is removed and the ureter is severed just above the level of ligation. The unobstructed ureter is an anastomosis into the bladder to permit urine flow, and the kidney will typically decompress over several days.

The unilateral ureteric obstruction model referred to as UUO can be used to study the development of tissue injury and fibrosis following obstructive nephropathy. The reversible ureteric obstruction model abbreviated to RUUO can be used to gain an insight into the tissue remodeling that may follow injury. Here is a representation of a typical experiment incorporating both UUL and our UUL procedures.

Mice undergo UUL on day zero and seven days later. The mice are either sacrifice to assess the level of injury or the ureters are de obstructed to perform an RUUR. The kidneys are then allowed to decompress for seven days before tissue is harvested.

The tissue can be assessed for structural injury, macrophage, infiltration, myofibroblast accumulation, and Fibrosis. Here is a typical Surgical setup. The procedure is performed using sterile autoclave instruments and consumables positioned within a sterile surgical field.

If multiple animals are operated upon instruments should be sterilized between procedures by a bead, sterilizer, or a new set of instruments should be used for each procedure. Operators wear a sterile gown, mask, hat, and gloves. This male eight week old C 57 black six mouse has been anesthetized by an intraperitoneal injection of ketamine, hydrochloride, and meine.

The depth of anesthesia has been confirmed by loss of reaction to a toe pinch. The surgical area has been shaved and sanitized using a dilute chlorhexidine solution. Eye lubrication has been applied to prevent corneal drying.

The analgesic buprenorphine hydrochloride has been administered by a subcutaneous injection. The mouth has been placed on a sterile draped heated surgical pad, and the limbs have been immobilized using tissue separating scissors and midline laparotomy is performed and an incision along the avascular linear elbow is made. This next chapter will detail how to perform an irreversible UUOA calibra retractor is inserted into the incision and the mouse is covered with sterile drapes.

The intestines are displaced and covered with moisture sterile gauze to expose the left ureter. Ureter is exposed and isolated using angled forceps to induce obstruction. The ureter is ligated twice using six oh black braided silk suture.

The ureter can be obstructed anywhere between the bladder and the renal pelvis. The ureter is then divided between the sutures. This next chapter will detail how to perform a ureteric obstruction that can be reversed.

This is the first stage of an RUUL. The ureter is exposed and isolated using angled forceps to induce obstruction. The ureter is ligated twice using 6.0 black braided silk suture.

The ureter should be obstructed close to the bladder in order to preserve a suitable length of ureter for anastomosis to the bladder. During the reversal procedure, the sutures are trimmed, but one is left long for later use, and approximately five millimeter length of soft wall plastic tubing is cut. Longitudinal slit is then made such that it can be splayed open to allow it to be applied around the ureter.

The soft wall plastic tubing should have an internal diameter of one millimeter and an external diameter of two millimeters. The soft wall plastic tubing is then placed carefully around the ureter ensuring that once closed, the long suture emerges from the center of the slit. Next, a length of six oh black braided silk suture is passed around the tubing and is tied.

Once the long suture emerging from the center of the tubing is then incorporated into the next tie. This is performed in order to securely anchor the ureter and tubing in place. The tubing around the ureter will prevent excessive ureteric Dilation at the end of Both UUO and RUUO procedures.

The intestines are replaced and the peritoneum is sutured and the skin is closed with metallic clips. Iodine is also applied to the surgical area. Anesthesia is partially reversed with a subcutaneous injection of aamaz.

Hydrochloride and fluids are administered by a subcutaneous injection of one mil warmed saline. Mice are monitored until they have recovered consciousness and they're then allowed to recover any heat box kept at 29 degrees for 24 hours. The kidneys become hydro nephrotic in the days following uter obstruction.

Mice can either be sacrificed at various time points during the injury phase or following reversal of the obstruction. This next chapter details how to perform a reversal, which is the second stage of the RUUO procedure. The mouse is anesthetized and prepared as previously described.

The metallic skin clips are removed and a laparotomy is performed. The peritoneal sutures are removed and the peritoneum can be gently opened with rat tooth forceps. The mouse is then prepared and the left ureter is exposed as previously described using angled forceps.

The ureter and soft walled plastic tubing is isolated from surrounding tissue. The sutures holding the soft wall tubing in place are cut with the scalpel and then removed. The soft wall plastic tubing is then carefully removed and a dilated ureter is evident.

The ureter is fragile at the point of obstruction and may well break above the obstruction as occurred in this clip. However, the sutures previously placed close to the bladder during the first stage of the UUO will remain intact, and this will ensure that no urine will leak from the bladder into the peritoneal cavity. A piece of sterile gze is placed underneath the ureter to collect any urinary sediment that will drain from the previously obstructed ureter.

The remaining length of de obstructed ureter must be allowed to drain until clear urine is produced to ensure that the ureter lumen is cleared of any debris. Once the ureter and renal pelvis have been drained, a long six oh black braided silk suture is applied to the end of the ureter. This is used to aid the subsequent ureter to bladder anastomosis.

The ureter is then turned such that it lies anterior to its original position and lies over the kidney. A single nano polyamide monofilament TAing suture is placed at the end of the ureter. Care is taken to ensure that the suture remains in the muscular coat and does not enter the ureteric lumen.

The suture is used to anchor the ureter in the bladder later to prepare the bladder for anastomosis, a channel is created through the bladder by passing a 21 gauge needle diagonally through the bladder. The 21 gauge needle is then used to guide an eye needle through the bladder. The Nino suture previously placed in the ureter is then passed through the first incision in the bladder and out through the bladder wall adjacent to the entry point to pass the ureter through the bladder.

The long six oh suture placed at the end of the ureter is passed through the eye of the needle. Following this, the eye needle is then slowly withdrawn through the bladder whilst ensuring that the ureter is also pulled through the bladder. Once the ureter is located within the bladder, the nano sutures placed in the ureter and bladder wall are tied in order to anchor the ureter in place to securely anchor the ureter.

A second otax suture is placed. This is again passed through the ureter, ensuring that it remains within the muscular coat, then through the bladder incision and out through the bladder wall. This is then tied in a similar manner.

A third nano tax suture is also placed at the rear of the bladder. Next, the backs of open scissors are used to push the bladder wall back slightly to expose the length of ureter. The ureter is then divided above the lung suture.

The painted ureter will then retract into the bladder angled. Four tips are used to ensure that the ureter has fully retracted into the bladder. Finally, the exit wound in the bladder is closed with a nine oh polyamide monofilament suture before closure, ensure that the ureter is firmly anchored within the bladder close.

The peritoneum and skin as previously described, provide postoperative care as previously described. All UUO mice are allowed to recover for seven days in this study to allow the de obstructed kidney to decompress, although earlier or later time points may be studied.Presented. Next is data that illustrates the appearance of obstructed and de obstructed kidneys and the resulting injury.

This figure illustrates the appearance of native U-U-L-U-U-O kidneys, renal pelvis, and ureters in comparison to the native kidney. The UUO kidney that's been obstructed for seven days appears hydro nephrotic and pale in color. Following seven days of de obstruction, the RUO kidney has become decompressed with the renal pelvis and ureter returning to similar size and color of the native kidney.

Note that the IUO ureter is shorter than the ureters of the UUO and native kidneys due to the destructuring technique and anastomosis into the bladder. This figure demonstrates representative images of H and E stained kidney sections from native UUO and RUUO kidneys. The healthy interstitium of the normal kidney comprises of cross sections of compact healthy tubules.

A number of tubules indicated by the black arrows exhibit dramatic dilatation at day seven. Following QUL in contrast tube, the dilatation is markedly reduced at seven days following Reversal of UUL. The mouse model Of obstructive nephropathy presented here is induced by unilateral ureteric obstruction, which can either be irreversible or reversible.

The reversible model detailed here allows removal of the ureteric obstruction and the anastomosis of the ureter to the bladder to fully restore urinary flow. The resultant kidney decompression enables the study of tissue remodeling post-injury experiments. Incorporating both irreversible and reversible obstructive nephropathy models, provides researchers with a powerful insight into disease processes and how therapeutic interventions modulate both the development of injury and the subsequent tissue remodeling.