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RENAL CAPSULE GRAFTING

Animals are weighed and anesthetized by avertin or some other agent (see avertin). Anesthesia is checked by applying pressure to the hind foot. If the animal responds to pressure, more time is allowed for the anesthesia to take effect. Alternatively, additional anesthetic is administered (for avertin usually no more than an additional an 0.05 to 0.1 ml for a 20-30 gram mouse). Once the animal no longer responds to pressure, the back of the skin is wetted with 70% ethanol. The back skin is lifted with a pair of blunt forceps and cut along the midline with a pair of scissors creating an incision approximately an inch in length (incision). The dermis is separated from the underlying body wall on both sides of the incision for bilateral grafting or on one side only (blunt dissection). The animal is placed on its side, and the location of the kidney is identified by viewing it through the body wall. A small incision is made in the body wall just slightly longer than the long axis of the kidney. A small blunt tipped scissors is used for this procedure and care is taken to avoid major vessels and nerves. The kidney is popped out of the hole in the body wall by applying pressure on either side of the kidney using the forefinger and thumb (exteriorization). The exteriorized kidney is allowed to rest on the body wall. In the female hosts, removal of the kidney from the body cavity can be facilitated by pulling on the fat pad associated with the ovary (at the base of the kidney) with a pair of blunt forceps and gently guiding the kidney through the opening in the body wall. The pressure of the cut edges of the body wall is sufficient to keep the exteriorized kidney in place. A pair of fine forceps (#5) is used to gently pinch and lift the capsule from the parenchyma of the kidney so that a 2 to 4 mm incision is made in the capsule with fine spring-loaded scissors (opening the kidney capsule). The size of the incision in the capsule is determined by the size of the graft. A glass Pasteur pipette which has been drawn thin and fire-polished with a rounded closed end is manipulated under the capsule tangential to the surface of the kidney (opening capsule pocket). By blunt dissection a pocket is created between the capsule and kidney parenchyma. Great care is taken not to damage the kidney parenchyma which if damaged will bleed. Grafts are transferred to the surface of the kidney using a blunt scalpel. The cut edge of the kidney capsule is lifted with the pair of fine forceps, and the graft is inserted into the pocket under the capsule using the polished glass pipette. Several grafts can be placed under the kidney capsule with no apparent ill effect to the mouse. If during the course of grafting the capsule becomes dehydrated, it can be easily and quickly moistened with PBS. When grafting is complete, the kidney is gently eased back into the body cavity. The operation can be repeated using the other kidney. After grafting the edges of the back skin are aligned and closed with the aid of wound clips. The animal is allowed to recover in the absence of non-anesthetized animals. While grafts of any size smaller than the surface area of the kidney can be implanted, one dimension of the graft should not exceed 2 mm. Implantation of grafts greater than 2 mm will result in central necrosis of the graft prior to establishment of vascularization.



		RENAL CAPSULE GRAFTING
	Animals are weighed and anesthetized by avertin or some other agent (see avertin). Anesthesia is checked by applying pressure to the hind foot. If the animal responds to pressure, more time is allowed for the anesthesia to take effect. Alternatively, additional anesthetic is administered (for avertin usually no more than an additional an 0.05 to 0.1 ml for a 20-30 gram mouse). Once the animal no longer responds to pressure, the back of the skin is wetted with 70% ethanol. The back skin is lifted with a pair of blunt forceps and cut along the midline with a pair of scissors creating an incision approximately an inch in length (incision). The dermis is separated from the underlying body wall on both sides of the incision for bilateral grafting or on one side only (blunt dissection). The animal is placed on its side, and the location of the kidney is identified by viewing it through the body wall. A small incision is made in the body wall just slightly longer than the long axis of the kidney. A small blunt tipped scissors is used for this procedure and care is taken to avoid major vessels and nerves. The kidney is popped out of the hole in the body wall by applying pressure on either side of the kidney using the forefinger and thumb (exteriorization). The exteriorized kidney is allowed to rest on the body wall. In the female hosts, removal of the kidney from the body cavity can be facilitated by pulling on the fat pad associated with the ovary (at the base of the kidney) with a pair of blunt forceps and gently guiding the kidney through the opening in the body wall. The pressure of the cut edges of the body wall is sufficient to keep the exteriorized kidney in place. A pair of fine forceps (#5) is used to gently pinch and lift the capsule from the parenchyma of the kidney so that a 2 to 4 mm incision is made in the capsule with fine spring-loaded scissors (opening the kidney capsule). The size of the incision in the capsule is determined by the size of the graft. A glass Pasteur pipette which has been drawn thin and fire-polished with a rounded closed end is manipulated under the capsule tangential to the surface of the kidney (opening capsule pocket). By blunt dissection a pocket is created between the capsule and kidney parenchyma. Great care is taken not to damage the kidney parenchyma which if damaged will bleed. Grafts are transferred to the surface of the kidney using a blunt scalpel. The cut edge of the kidney capsule is lifted with the pair of fine forceps, and the graft is inserted into the pocket under the capsule using the polished glass pipette. Several grafts can be placed under the kidney capsule with no apparent ill effect to the mouse. If during the course of grafting the capsule becomes dehydrated, it can be easily and quickly moistened with PBS. When grafting is complete, the kidney is gently eased back into the body cavity. The operation can be repeated using the other kidney. After grafting the edges of the back skin are aligned and closed with the aid of wound clips. The animal is allowed to recover in the absence of non-anesthetized animals. While grafts of any size smaller than the surface area of the kidney can be implanted, one dimension of the graft should not exceed 2 mm. Implantation of grafts greater than 2 mm will result in central necrosis of the graft prior to establishment of vascularization.