Laparoscopische techniek voor seriële Verzameling van lever en mesenteriale lymfeklieren in makaken

The overall goal of this minimally invasive laparoscopic procedure is to improve assessment of the effects of microbial translocation from the GI tract by collection of mesenteric lymph nodes and liver biopsies at multiple time points while improving animal welfare. This method can aid in answering key questions in the field of HIV biology about immunological consequences of infection and microbial translocation on the mesenteric lymph nodes and liver, and the pathology of these tissues. The main advantage of this technique is that it allows for sampling at multiple time points in a study with minimal impact to the animal or to the parameters that are being evaluated.

This method can provide samples critical to furthering our understanding of HIV. It can also be applied to other models where GI inflammation and microbial translocation are important. Doing the procedure will be Dr.Jeremy Smedley and Dr.Cassie Moats, veterinary surgeons at the Washington National Primate Research Center.

After preparing a macaque for the surgery as indicated in the text protocol, cover it with a sterile fenestrated drape. Next, using a sterile camera drape, tightly cover the digital camera and plug it into the tower. Connect the rigid scope with the camera head.

Then attach a sterile light cable to the rigid scope and connect it to the source of light. Finally, mount a sterile insufflation tube on the insufflator. To begin laparoscopy, position a 15 scalpel blade approximately one to two centimeters to the left of the umbilicus and make an approximately 5 millimeter stab incision in the skin that reaches the abdominal muscles.

With the nondominant hand, lift up the abdominal wall on the cranial side of the cut. Then using the other hand, introduce through the incision the tip of a varus needle angled toward the animal's head. Hold the varus needle by the shaft and firmly push it until it clicks through the abdominal wall and the resistance against the needle decreases in the abdominal cavity.

Then attach the tube of the CO2 insufflator to the needle and open the stopcock to release the gas into the abdominal cavity. Create a pneumoperitoneum by reaching the pressure of up to 10 to 12 millimeters of mercury. Now that the abdomen is fully insufflated, position the 15 scalpel blade over the left cranial abdomen, approximately two to four centimeters caudal to the last rib, and make an approximately five to seven millimeter skin incision.

Next, insert through the cut a five millimeter trocar cannula assembly. Introduce the assembly to a depth of one to two centimeters, directing it at approximately 45 to 60 degrees caudomedially. Then with a rotating motion, slide the cannula forward reaching a depth of 1.5 to three centimeters to screw the cannula into the abdomen.

Afterward, remove the trocar. Disconnect the insufflator tube from the varus needle and after, turn the stopcock off. Connect the tube to the cannula and open the stopcock to maintain pneumoperitoneum.

Once verified that the pressure is being maintained successfully, remove the varus needle. Next, through the cannula, insert the rigid scope into the abdominal cavity. Under the control of the camera, deepen the skin incision remaining after varus needle removal, cutting through the abdominal muscles and peritoneum.

Lastly, position the animal with the feet elevated at approximately 15 to 30 degrees above the head. To collect a biopsy from the mesenteric lymph node, introduce a solid probe to the abdominal cavity through the prepared incision. Position the probe between the omentum and the bowel, and with a gentle sweeping-like motion expose the bowel from the omentum to visualize mesentery that is hidden below.

Continue to move the probe from caudal to cranial direction to push the omentum away from the operative field. Then using the probe, move the descending colon toward the abdominal wall. Examine the mesentery with the probe to locate mesenteric lymph nodes located along the colon and mesenteric vessels.

Once the lymph node is identified, replace the probe in the abdominal cavity with Maryland ratcheted forceps and grab the mesentery adjacent to the node. Pull the mesentery toward the incision and remove the scope from the cannula. Then turn off the insufflator and leave the cannula stopcock open to release gas from the abdomen, which will ease the mesentery exteriorization.

Then pull the mesentery through the incision. Grasp the exteriorized mesentery with forceps to ensure access through the incision. Palpate or observe the mesentery to identify the lymph node that should appear as a firmer nodule or a grayer area.

Next, using curved hemostats, make a three to five millimeter opening in the mesentery adjacent to the node, and bluntly dissect any attachments to the tissue. If required, ligate the vasculature with a 4-0 nonabsorbable monofilament suture, and using the scalpel, dissect the lymph node. Then, place the biopsy in RPMI 1640 medium on ice.

Finally, release the mesentery and insufflate the abdomen once more to enable pulling back the tissue to the abdominal cavity. Return table to a horizontal position. To collect a liver biopsy, insert a threaded cannula into the abdominal cavity through the incision used for lymph node collection.

Then using the cannula, slide in the rigid scope directed toward the cranial abdominal cavity to visualize the liver. Under the control of the camera, introduce biopsy forceps through the cannula previously used to insufflate the abdominal cavity. Position the forceps below the designated liver lobe margin so that their jaws face the tissue, and open the forceps allowing the margin to fall into the jaws.

After adjusting their position, close the forceps and maintain the pressure for approximately 20 to 30 seconds. Then remove the sample by gently pulling the forceps through the cannula. Place the biopsy in RPMI medium on ice.

Finally, make sure no hemorrhage is observed in the site of the biopsy, and close the incisions. Presented here are cellular yields obtained from mesenteric lymph node and liver biopsies collected from two healthy female mecaques at two different time points. As expected, lymph node biopsies yielded a significantly higher number of the isolated cells when compared to the liver biopsies.

Regardless of the source tissue, over 85%of the isolated cells were viable. Then the isolated cell samples were analyzed for CD45+leukocytes and CD45+CD3+lymphocyte populations. Mesenteric lymph nodes had greater frequencies of CD45+leukocytes and CD3+lymphocytes when compared to liver derived cells.

Detailed characterization of the isolated populations showed that mesenteric lymph nodes contained significantly higher proportions of CD3+T cells and CD4+T cells, as well as an elevated number of CD8+T cells. Conversely, the liver was significantly enriched in CD14+leukocytes. Both organs demonstrated similar abundances of CD20+B cells.

Once mastered, this technique can be performed in 30 to 40 minutes. Animals are typically recovered from anesthesia, active and eating within a couple of hours, and the incisions are so small they can be hard to see cage side. When attempting this procedure, it's important to maintain animals in a body condition that will facilitate success.

Significant mesentery fat can be a challenging obstacle as it obscures nodes and increases fryability and bleeding. Following this procedure, other methods such as phlebotomy, peripheral lymph node biopsy, upper and lower GI endoscopy and rectal biopsy can be performed in order to evaluate systemic and mucosal immune responses, status of microbes and epithelial barriers, as well as viral loads. After its development, this technique paved the way for researchers in our lab to explore microbial populations in inflammation longitudinally in tissues from macaques that were previously only available by necropsy.

The implications of this technique extend toward therapy for HIV, as microbial translocation is a major cause of inflammation which drives HIV pathogenesis. Evaluating this in a serial fashion can provide potential targets and allow for evaluation of therapeutic interventions. Don't forget that working with any virus infected macaques can be extremely hazardous.

Precautions such as proper training for biohazards including macaquesin herpesvirus 1, proper personal protective equipment, bite and scratch kits, and proper medical followup should be available.