Overview

This protocol describes how to use the Karl Storz Coloview system to perform a colonoscopy on a mouse. Parts of this protocol are based on a protocol written by Jamie Hadac, Linda Clipson, and Richard Halberg.

Warning! The telescope is very delicate and costs $6,000. If you drop it or bend it your lab will have to pay for a replacement. Always keep either the telescope sheath or the plastic protector covering the telescope. Never set the telescope down unprotected! Other expensive delicate parts include the camera ($25,000) and the biopsy forceps ($1,000). Always make sure the camera will not slide off of a table due to its cord weight and keep the lens cap on the camera when not in use.

The xenon light source has a life of approximately 350 hours. The light source is running, even when the rheostat is turned all the way down. Do not repeatedly turn the light on and off, but you do not need to wait a specified amount of time after turning the light off to turn the light back on.

Equipment

The basic setup of the Coloview system is shown in Figure 1.

The unassembled sheath, telescopes and camera are shown in Figure 2. The sheath is a 3-French sheath, which means any accessories that are 3-French in size or below will fit into the sheath along with the telescope. French is a system of measurement of non-circular tubes.

The injection needle, biopsy forceps and needle sheath are shown in Figure 3.

The Plastic Telescope Guard and Obdurator are shown in Figure 4.

The air infuser (not pictured) is a simple fish tank air pump with plastic tubing used to connect to the port on the sheath.

Cleaning the equipment

All equipment (Telescope, Telescope Sheath, Plastic Telescope Guard, Obdurator, Light Cable, Camera Head, Injection Needle, and Biopsy Forceps) are autoclavable and fully submersible (EXCEPTION: The plug at the end of the cord attached to the camera head cord is NOT submersible and the cord is NOT detachable from the camera head). Equipment can also be gassed. Only use DI water with an enzymatic cleaner (ask Lee Pribyl which enzymatic cleaners are acceptable), as regular water and soap will damage the telescope and other equipment. The tip of the telescope can be cleaned with a kim wipe or other tissue such as gauze during the procedure.

Equipment setup

• Remove the plastic guard (Figure 4B) from the telescope
• Slide the telescope sheath gently over the telescope. Line up the two lines on the sheath with the line on the telescope. Secure the sheath to the telescope by gently turning the red lever 1/4 turn.
• Remove the camera lens cap and insert the black end of the the telescope into the camera by slightly twisting the black end of the camera and then releasing.
• Plug camera cord into HD slot (left slot) on camera capture device. Do not plug into right slot, which only works on older cameras.
• Plug light cable into Xenon Light source box. Cord must be inserted completely into hole, hold box to avoid moving the entire box.
• Screw the other end of the light cable onto the telescope port. Avoid kinking the light cable.
• Connect the plastic tube from the air infuser to one of the 90º ports on the telescope sheath (Figure 5A). Open both of the 90º ports on the telescope sheath by positioning the small lever parallel to the port. The three small levers on the ports can be positioned in the open (parallel) position (Figure 6C) or the closed (perpendicular) position, or in between for variable opening. The choice of which port to connect the air infuser depends on which hand you will use to operate the telescope. For right-handed manipulation, put the air infuser tube on the left-side port
• Optional: Turn the lever to the parallel position on the 30º port (Figure 5B) to open the port, then insert the injection needle or the biopsy forceps into the 30º port on the telescope sheath (Figure 5B). To avoid air leakage, cover the port hole with the whtie rubber cap (Figure 6A) that has a small hole in the middle and insert the injection needle or the biopsy forcep through the small hole in the rubber cover (Figure 6B). The forceps and injection needle are very long (Figure 7) because they are made for multiple uses including exotic birds. Unfortunately, this makes them less convenient for mice, and it will require two people to operate.
• Plug the cart electric cord into an electrical socket
• Turn on all devices (monitor, computer, light source, camera capture device, air infuser). The computer takes a while to connect to all devices.

Camera and Computer setup

• Light intensity setting: Turn the rheostat to maximum using the knob on the xenon light source box. Generally you will want to use the maximum amount of light when performing a colonoscopy, however it is adjustable. Warning: if the light cable is not connected to the telescope, the tip of the cable will become extremely hot and will ignite clothing or paper. If the cable is connected to the telescope the heat will dissipate. You should be able to point the telescope at objects and see them on the monitor.
• Focus and zoom: Use the gold ring on the camera for focus and the blue ring for zooming. The camera does not have a digital zoom. Once the camera is focused, it should not need to be refocused after zooming.
• Camera settings: Press the triangular button on the camera head (Figure 8A) to activate the Camera User Menu (Figure 8B). Use the previous/next buttons on the camera head to select different items in the User Menu. Every time you set up the Coloview system you should check the Enhancement and Brightness settings and then set the White Balance. The other options on the User Menu are not currently used with our setup. The camera does have the ability to be operated by the buttons on the camera head, but we have purposefully not attached the necessary cables because using these buttons usually results in poorer image quality.
• Enhancement: Generally the "low" setting is optimal, although you can try the "high" setting (Figure 8C). We currently do not have Fiberscope capabilities.
• Brightness: You can experiment with low, medium, high and peak to determine which setting results in the optimal image. Generally start with medium.
• White Balance: Set the white balance by pointing the telescope/camera directly at a white object a few millimeters away from the telescope. Select the white balance option from the User Menu and the camera will automatically adjust the White Balance. Remember that the white balance needs to be set everytime the camera is turned on.

Operating the acquisition software

• The touch screen for the computer will slide in and out and can be tilted to an optimal position.
• Use the icons on the bottom right of the screen to begin a session. Generally a "session" will be a set of images and/or videos associated with a single animal during the day of the experiment. The software was designed for colonoscopies performed on human patients, so once a session is closed nothing can be changed later. This means it is not advisable to image several animals in a single session, and it is not possible to re-open the session at a later date to take more images of the same animal. This also means a single experiment on one day will consist of as many sessions as there are animals to image.
• Session settings (Figure 9A): Click on the "Patient Info" icon on the bottom right of the touch screen to activate the session set-up screen. The three boxes outlined in yellow are required, the rest are optionial. Fill in Surgeon (name of key person performing the experiment that session), Surgical Specialty (put whatever you want), and Procedure (generally this will be colonoscopy, but you can edit this field and put in anything you want). At a minimum, enter the Mouse ID # in the Patient ID field and check that the Date of Treatment field contains the correct date. You can use other fields if you want.
• Capture screen (Figure 9B): Click on "Capture" icon on the bottom right of the touch screen. To capture a single image press the "Still" icon. To start and stop a video press the "Video" icon.
• Review screen: Use the review screen (Figure 9C) to review any images or videos. You can also delete images and videos from this screen.
• Finish screen: Select the Finish screen (Figure 9D) to finish the session and start a new session, or shut down the computer. Note: We purposefully have not set up different users, because that would require a password for each user and would complicate matters. In general, always bring a thumb drive or USB external hard drive and copy your images/videos after closing the session to your own personal storage device. The computer only has a 40 GB hard drive, so any sessions that are left on the hard drive will be deleted by the next user.
• System settings: To alter system settings select the wrench icon on the top right of the screen (Figure 10A). Generally you will not need to change any of these settings. Consult the user manuals for more information on these fields.
• To review or download any sessions that are still on the hardrive, use the filing cabinet icon (Figure 10B).
• To access help documents, click on the ? icon (Figure 10C).

Preparing the mouse for colonoscopy

• Anesthetize the mouse using IACUC approved methods.
• Currently using isofluorane. For colonoscopy set isoflourane tank at 4 and pressure gauge at 3.Procedure should last less than 5 minutes. For colonoscopy with injection, reduce isflourane tank to 3.5
• Set up heating pad, set to medium
• Test the airflow of the insufflator by sticking endoscopy in 50 ml conical with water. Adjust the flow rate by tightening the clamp attached to the air tube. Ideal flow rate should produce about 2 - 3 bubbles per second.
• Secure the anesthetized mouse to the surgical platform using straps around the hind legs with the head placed in tube taped to platform.
• Place the mouse near the edge of a working surface to facility telescope manipulation
• Connect an oral gavage needle to a 10ml syringe and then fill the syringe with with 1X PBS. Alternatively use an enema preparation of 1.5% w/v sodium dihydrogen phosphate (NaH2PO4) and 11% w/v disodium hydrogen phosphate (Na2HPO4).
• Flush the colon with PBS or the enema solution (~2 ml) to remove fecal pellets. Do not force the gavage needle pass fecal pellets because this could easily perforate the bowel. In addition, avoid using too much PBS because if PBS fills the cecum the mouse may aspirate during insufflation of the colon. Insert gavage needle fully into mouse, but do not insert if you feel any resistance.

Performing the Colonoscopy

• Enter Patient ID and other necessary fields in the Patient Info screen.
• Grasp the camera head in a manner that will allow you to cover the port opposite the air infusion intake with a finger in order to insufflate the colon as the telescope is inserted. Alternatively, set an appropriate flow rate for the insufflator and close the port that you would cover with your thumb. The flow rate should be ~2 to 3 bubbles per second when the endoscope is inserted into a 50 ml conical of H2O. There are different methods of handling the camera depending upon the user. If there are no accessories in the 30º port make sure that port's lever is in the perpendicular (off) position to block air escape. Both of the horizontal port levers should be in the parallel (open) position.

• Start the video recording.
• Grasp and raise the tail, keeping mouse feet on the bottom, and insert telescope into colon. Watching both the mouse and the video monitor is helpful at this point. Place the telescope at a small angle so that the pinched end of the telescope touches the mouse first, then slide down from tail until edge of telescope hits anus, then insert telescope. Maintain airflow through the telescope when inserting to avoid collapse of the colon. In general no lubricant is used on the telescope sheath due to the chance of it getting smeared on the tip and blurring the image. Insert the telescope about 2 to 3 cm until you get to the bend in the colon. Avoid pushing into the cecum.
• Capture images and/or video, usually with the aid of an assistant, using the Capture screen on the touch panel.
• Note: If image is fuzzy, use a cotton-tipped stick (we buy these from Amazon, they are sold for gun cleaning), rub cotton with an alcohol pad and then gently clean the tip of the telescope by rubbing with the cotton tip
• To perform a needle injection. Use LOCTITe Fun-TAK mounting putty to seal the port where the needle is going in. Take a small piece and form into small sheet (1 cm x 0.5 cm - see photo).
• Use a mound of play-doh to hold the camera.
• To perform a biopsy, place the rubber cap over the 30º port and gently insert the biopsy forceps. Minimal resistance is met at the junction between the port and the endoscope. Once the forceps are visible, they can be opened to acquire a sample; the forceps must be extended fairly far so that they can be fully opened and the forceps can best be positioned by moving the camera rather than moving the forceps themselves. GENTLY close the forceps on the tumor and then quicky pull the forceps away. Withdraw the forceps from the sheath and remove tissue from forceps using a small needle. BE CAREFUL WITH THE FORCEPS BECAUSE THEY ARE VERY FRAGILE AND QUITE EXPENSIVE – DO NOT USE FORCE TO OPEN OR TO CLOSE.
• Notes regarding the procedure including tumor number, size and position can be entered in comments.
• Save the session.
• Transfer data to your own computer using the USB port or burning to a CD.

Helpful hints from Rich Halberg

• Mice can be scoped around 40 days of age and beyond. Younger animals generally do not have a large enough anus to accommodate the size of the operating sheath with the endoscope.
• If mice routinely have a large number of fecal pellets, they can be treated with GoLYTELY or NuLYTELY to help cleanse the bowel. These agents are used with humans for colonoscopy and they are available from Braintree Scientific.
• Different rigid endoscopes are available. We prefer the 0 degree scope for colonoscopy and the 30 degree scope for biopsy.
• An easy method for cleaning the endoscope while in the operating sheath is to use a small dental brush that can fit into the opening above the endoscope.
• Air is a must because it is very difficult to visualize the tumors and estimate size without insufflation.
• Occasionally, the bowel will be perforated. This is obvious because the entire mouse rapidly increases in size as air is forced into the body cavity. A mouse with a perforated bowel must be sacrificed as soon as possible.
• Tumors can be marked with India (tattoo) ink to make sure the same lesion is followed over time. However, we have not found this necessary when multiplicity is low and new tumors are not forming during the study.
• Up to 500ng of total RNA can be purified from a tumor biopsy.
• The ability to monitor the response of individually annotated tumors to a drug significantly reduces the number of mice required for the study. The reduction in number is dependent upon the particulars of the model. With B6.Min mice,
  • the number is reduced by a factor of four.
• Managing the data can be a daunting task because the amount of information grows quickly. We print a report from each visit and enter all information in a relational database using Microsoft Access. Customized forms in Access facilitate accurate data entry. We then use Access to generate a custom report displaying a photo array of each tumor at each timepoint, allowing us to easily assess each tumor’s change in size and appearance over time (below).

Clean up time!

• Disconnect and disassemble all the equipment. Wipe all equipment with kim wipes. Clean all equipment by submersion in enzymatic cleaners for 5 to 10 minutes and gentle rubbing with a soft tooth brush followed by rinsing with DI water and careful drying with kim wipes. If sterilization is required autoclave or gas all equipment. Place telescope and plastic guard, telescope sheath, and accessory items in their respective dedicated boxes. Place the camera head, light cable, telescope box and sheath box in the bottom drawer of the Coloview cabinet (Figure 12). Place the Accessory box on top of the cabinet. Return the cabinet to its storage space and lock the wheels of the cabinet.