The overall goal of this procedure is to provide a minimally invasive approach for real-time monitoring of N-A-D-P-H oxidase derived reactive oxygen species or ROS generation in three different inflammatory mouse models. The first model is intra tracheal administration of Xan in which the pro-inflammatory molecule Xan is injected into the trachea of anesthetized mice. Xan is a fungal cell wall derived product that can activate N-A-D-P-H oxidase.
The second model is se a ligation and puncture or CLP. Here the abdomen of the mouse is opened and the secum is ligated with the silk suture and then punctured using a needle to induce intraabdominal sepsis and secondary acute lung inflammation and injury. The third model is administration of carbon tetrachloride by oral gavage.
Carbon tetrachloride causes hepatocellular necrosis and can be used to model both acute liver injury and hepatic fibrosis. Bioluminescent imaging is performed on model mice to assess ROS production. The resulting data indicate the N-A-D-P-H oxidase is the major source of ROS generation in response to inflammatory stimuli.
The major advantage for this technique over existing method, for example, Florence probe, is that this method provides minimally invasive approach for real time monitoring of RS generation during inflammation in vivo. This method can help us answer key question in post defense inflammation and injury such as the rule of RS in lung inflammation and injury. We first had the idea for this method when we started the role of reactive oxygen species in hosted defense For all three animal models of ROS generation shown in this video.
Use P 47 Fox null mice and age and sex matched C 57 BL six DBA mice. Obtain approval for experiments from institutional animal care and use committee in preparation for intratracheal administration of xin. Sterilize the surgical platform with 70%ethanol and cover it with a sterile pad.
The surgical procedure should be performed wearing a clean surgical gown, gloves, mask, and using sterile instruments for anesthesia. Fill the vaporizer of a continuous isof fluorine administration system with two to 3%isof fluorine and turn it on. Then place the mice in the anesthetic chamber after two to three minutes, confirm that the mice are fully anesthetized by performing a toe pinch and observing respiration and movement.
The respiratory rate should be reduced by about 50%Next, using clippers, shave the incision site on the front of the mouse's neck. Place the mouse on the surgery platform in a ine position and place a nose cone to maintain anesthesia. Alternate application of Betadine and 70%ethanol to the neck three times.
Next, using straight sharp scissors, cut the front neck skin about 0.5 centimeters. Then using curved forceps, dissect the muscle around the trachea to expose it to administer Xan Intratracheally. Use a syringe equipped with a 27 gauge needle to pierce the trachea in the middle part between cartilaginous rings.
Then direct the needle anteriorly and slowly inject one microgram per gram of body weight of Xan. Close the incision with five oh sterile nylon sutures postoperatively. Place the mouse in a recovery cage covered with flat paper and place half of the recovery cage on a heating pad.
Monitor the mice until they're awake and moving freely. To perform a seco ligation and puncture, shave the abdomen of an anesthetized mouse with clippers and place the mouse on a surgical platform in the supine position. Then disinfect the surgical area with Betadine and 70%ethanol.
As before using a scalpel, make a 1.5 centimeter incision in the abdominal midline skin using scissors. Cut the peritoneum and open the abdominal cavity with a retractor locate and expose the secum, which is located on the left side of the abdominal cavity. Using non crushing forceps exteriorize.
The secum then ligate the distal 50%of exposed secum with four oh silk suture. Use a 21 gauge needle to puncture the seum distal to the ligation. Replace the secum in the abdomen.
Use four O sterile nylon sutures to close the abdomen wall. Then close the incision with five sterile nylon sutures. Administer 0.5 milliliters of saline with 2.5 micrograms of buprenorphine subcutaneously and perform postoperative observation as before.
Oral carbon tetrachloride is a model of reactive oxygen species dependent hepatic injury to administer carbon tetrachloride by oral gavage, scruff, and anesthetized mouse to hold the head in vertical alignment with the esophagus. Then insert the tip of a 22 gauge feeding and dosing needle containing carbon tetrachloride behind the incisors, directing it towards the back of the throat and down the esophagus to press the plunger to administer the carbon tetrachloride. Monitor the mouse for recovery as before bioluminescent images should be acquired prior to and four and 20 hours after inducing ROS production.
To do this open live image 4.2 software. Next to initialize the ivus 200 imaging system. Click initialize and set the system to luminescence mode.
Wait for the charge coupled device temperature to lock, which will be indicated on the screen in the software. Set the exposure time to 40 seconds. Then set the Benning to medium and the F-stop to one eighth.
Place an anesthetized mouse in the imaging chamber with the temperature controlled stage and place it in the supine position. Place a nose cone to administer anesthesia as before two minutes prior to each image capture. Use a one milliliter syringe with a 27 gauge needle to administer 50 microliters of the luminal derivative L 0 1 2 intravenously.
Be a retroorbital injection. Finally, acquire images and analyze them as described. In the next section of the video, analyze the data collected using living image software version 4.2 in the software.
Open an image and click ROI tools in the tool palette. Next in the ROI tools menu, select ROI shape and click the circle on the dropdown list. Select the number of ROIs that will be added to the image.
Adjust the ROI position over the chest and or abdomen to quantify the signal intensity or photon flux from the region of interest in the ROI tools menu. Click on the measure button to open the ROI measurements table, which displays the data for all of the ROIs created in the images. Use a statistical analysis software package to perform two-way a novo with Bon Ferone post testing at individual time points to compare ROS generation in the lungs of wild type and N-A-D-P-H oxidase deficient.
P 47 Fox null mice following Xan challenge. Xan was administered as described in this video at baseline four and 24 hours. The mice were imaged as shown here.
There was a significant increase in photo emission over the chest in wild type, but not P 47. Fox sno mice at four hours and 24 hours as compared to baseline. This suggests that N-A-D-P-H oxidase is the major source of ROS generation in the lungs following XUS Sand Administration.
Next ROS production was assessed in the CLP induced sepsis model. Sepsis is a life-threatening syndrome associated with end organ injury, including acute lung injury and acute respiratory distress syndrome. R os mediated injury has been considered to be an important factor driving sepsis induced multi-organ function.
As shown in this graph, ROS were significantly increased over the chest four hours following CLP and over the abdomen 24 hours following CLP in wild type mice. However, ROS levels in P 47 Fox null mice were similar to baseline levels at both time points. These results show that ROS generation in the CLP induced sepsis mouse model is N-A-D-P-H oxidase dependent.
Finally, bioluminescent imaging was used to detect ROS production in a carbon tetrachloride induced liver injury model. In contrast to the previously described models of inflammation and injury, ROS production in the abdomen was modestly about 35%increased over baseline in P 47, Fox null mice four hours following carbon tetrachloride administration. However, the magnitude of carbon tetrachloride induced ROS generation was significantly greater in wildtype mice than in P 47 Fox null mice.
These data suggest that both P 47 Fox containing N-A-D-P-H oxidase dependent and independent ROS generation occur in acute carte chloride induced liver injury. Bio luminance image result that demonstrated increased ROS level in Wildtime mice compared to P 47. Knock Mized indicated that a DP H oxidase is a major source of RS generation in response to inflammatory stimulus.
This method provides a minimally invasive approach for real time monitoring of RS generation during inflammation in vivo.
