肝细胞部分肝切除术后脾移植。小鼠

For treating patients having acute hepatic failure hepatocyte transplantation is an alternative to whole organ transplantation. Partial hepatectomy is used as a technique for creating AHF. Besides it is also utilized to investigate cell engraftment and proliferation during cell based therapy.

Mouse is one of the most researched models for performing hepatectomy for such objectives. In this video we will show a procedure for partial hepatectomy followed by transplantation of hepatocytes in the spleen of a NOD. SCID mouse.

Before you begin make sure that you have necessary approval from appropriate animal ethics committee for performing the experiment. In this procedure we will make two small incisions. The first incision is for partial liver hepatectomy which will be around one centimeter long made just below the xiphoid to expose the liver.

For transplanting cells another smaller incision is made on the left lateral portion to expose the spleen of the animal. The advantages are that it leads to less trauma and pain to the animal faster recovery and nearly hundred percent survival. Moreover this procedure does not require any specialized surgical skills and can be completed quickly in five to seven minutes.

In this video we will demonstrate the transplantation of hepatocyte like cells called as NeoHep and hepatocytes isolated from a transgenic GFP mouse in a partially hepatectomized NOD. SCID mouse. The procedure does not restrict the type of cells for transplantation.

All surgical tools and PBS were duly autocleaned and kept ready prior to surgery. Weigh the mouse.NOD. SCID mouse of six to eight week age weighing between fourteen to eighteen grams are included in this procedure.

Shave the central, upper, and left portion of the mouse abdomen. In order to remove trimmed hair, apply hair removing cream over the shaved region. Wait for four to five minutes and gently remove the hair.

Unlock the oxygen cylinder valve. Place the mouse in the Isoflurane chamber. Adjust the oxygen flow at four liter per minute.

Adjust the isoflurane vaporization at four percent for inducing the anesthesia. Ensure that the mouse has been anesthetized properly by gentle toe pinching. Place a surgery board inside a biosafety cabinet.

Place the animal on the surgery board such that the ventral portion of the mouse is facing up and anterior portion of the mouse is placed inside the nose cone connected to the Isoflurane and oxygen supply. Reduce the Isoflurane vaporization to two percent for maintaining the anesthesia. Disinfect the skin by wiping it with sterile cotton soaked in seventy percent ethanol.

Make a transverse incision of around one centimeter in the skin just below the sternum perpendicular to xiphoid parallel to ribcage. Gently separate the skin and peritoneum near the incision. Make a transverse incision through the peritoneal layer just below the xiphoid to expose the left lobe of the liver.

Two moistened cotton tip swill be used. Place one of the cotton tips on the abdominal side of the cut and place another cotton tip on the diaphragm side. Now gently press the tip placed towards the diaphragm and give a sliding push by the other tip to lift the left lobe of the liver.

Place a loop of silk thread on the base of the left lateral lobe close to the liver hilum using the micro-dissecting forceps. After slightly closing the knot, slide the silk thread as much possible to the base of the liver lobe. Tighten the knot and place two additional knots on the other side.

Cut the tied lobe using a microsurgery scissor. This image shows remaining part of the left lobe after resection. Sew the peritoneum by continuous suturing with a four zero Catgut absorbable suture.

Close the skin by discontinuous or interrupted suturing using the same four zero Catgut absorbable suture. Apply Betadine in the vicinity of sutures. Keep the hepatocytes or NeoHep suspended in fifty micro-liter IMDM ready in a one ML insulin syringe with twenty-nine gauge needle.

Place the mouse such that the left lateral portion would face up. Identify the spleen region and transversely dissect the skin. Make a two to three millimeter cut in the peritoneal layer to expose the spleen.

Place the spleen between two moist cotton tips and gently lift it. Pierce the needle in the spleen cortex gently and release the cell suspension into the spleen. Close the peritoneum by continuous suturing with a four zero suture and close the skin by discontinuous suturing.

Apply Betadine on sutures. Inject hundred micro liter saline intraperitoneally containing twelve milligram antibiotic Cefotaxime. Inject hundred micro liter saline intraperitoneally containing twelve micro gram of analgesic Meloxicam.

This is to be given daily for three days post-surgery. Close the isoflurane vaporizer and place the mouse in the cage. The animal will gain consciousness within seconds.

The proliferation of hepatocytes in the liver post twenty-four hours of thirty percent hepatectomy was examined by immunohistochemical staining of liver sections with Ki-67 which is a cell proliferation marker. After staining these sections with anti-Ki-67 horseradish peroxidase conjugated secondary antibody was used for detection and Di-Amino Benzadine was used as substrate to develop brown color. The nucleus was counter stained with hematoxylin.

In this image brown colored cells are Ki-67 positive proliferating hepatocytes. Ten days post transplantation the mouse was euthanized and the liver was examined. A representative image is shown here.

From the regenerated liver the right lateral lobe the right medial lobe and the left medial lobes were excised and five micron sections were obtained. These sections were stained with primary rabbit polyclonal anti-GFP and secondary anti-rabbit Alexa Fluor 594 antibodies. The nucleus was counterstained with DAPI.

This image shows the homing of hepatocytes expressing green fluorescent protein which is visible as red. When hepatocyte like cells of human origin called NeoHep were transplanted, we observed the expression of human Albumin and human Connexin 32 in the liver sections of recipient hepatectomized mouse. Ten days post surgery the biochemical analysis of liver secreted ensymes in serum confirmed that the levels of aspartate aminotransferase, alanine amintransferase, and alkaline phosphatase were restored to normal.

Similarly the histological examination of liver sections by haematoxylin and eosin staining showed no abnormalities ten days post-surgery indicating proper recovery of the liver. It is important to take a few precautions. Try to place the knot of nylon thread at the base of the liver lobe.

Do not forget to make two additional tight knots in the opposite side and do not attempt to cut very close to the thread. For transplanting cells into the spleen, pierce the needle exactly vertical and it should not go deeper than two millimeters. For closing skin avoid using wound clips.

Instead close it by a four zero suture. Wound clips restrict the natural movement of the mouse and often clips become loose and come out within no time. After placing the animal back into the cage, you may place a few pieces of sterile corn flakes in the cage and provide sterile water having five percent glucose.

If you have no earlier exposure to animal surgery, practice separately the partial liver hepatectomy and transplantation in normal immuno-competent mice and then perform both the procedutes in a single animal. Perform the procedures in immunodeficient NOD. SCID mouse, only after attaining hundred percent survival rate in immuno-competent mice.