Injury models are indispensable to study regeneration in vivo, however, investigating regeneration in the intestine is proven to be a technical challenge. The lack of longitudinal imaging protocols has prevented deeper insight into the cell and tissue scale dynamics that orchestrate intestinal regeneration. In this protocol, we describe an intravital microscopy method that locally induces tissue damage at single crypt scale, and follows the regenerative response of the intestinal epithelium in the living mouse to photon based laser ablation damage of single crypt or larger intestinal fields in a time and space controlled manner.
Subsequent, long-term, repetitive intravital imaging enables the tracking of damaged area over time, and allows for the monitoring of crypt dynamics during tissue recovery over a period of multiple weeks. Induce mice by injecting tamoxifen dissolved in sunflower oil into four to six weeks prior to surgery. Apply analgesia, inject 200 microliter of buprenorphine subcutaneously 30 minutes before surgery.
Administer carprofen in drinking water 24 hours pre-surgery. Introduce autoclaved sterile surgical tools in the biohazard cabinet. Turn on the heating pad, and set the temperature at 37 degrees Celsius.
Turn on the temperature control chamber of the microscope at least four hours before imaging. Keep the temperature inside the climate chamber stable at 37 degrees Celsius. Turn on the two photon microscope, the scanner, and the laser.
Launch the imaging software. Tune the wavelength of the laser to 960 nanometer and open the shutter. Anesthetize the mouse using two to 3%isoflurane, and place it on a heating pad covered with a sterile cloth.
Check the depth of anesthesia by assessing the frequency and the quality of breathing one breath per second, and by checking the reflex of the mouse. Cover the eyes of the mouse with eye ointment. Inject 200 microliter of pre-warmed sterile saline subcutaneously.
Shave the abdomen and remove the hair. Change the sterile cloth in the surgical area. Insert rectal probe to monitor the temperature of the mouse.
Temperature should be approximately at 37 degrees Celsius. Wear a new pair of sterile gloves. Clean the surgical area in a circular way with alternating scrubs of antiseptic solution followed by 80%ethanol three times.
Cover the mouse with a sterile surgical drape. Check the reflex of the mouse. Make a 10 millimeter vertical midline incision through the skin using a sterile scalpel.
Incise the linea alba using the scissors to separate the rectus abdominal muscles and open the abdomen. Find the cecum of the mouse using a sterile cotton swab drenched in pre-warmed saline to use it as a reference point. Make a small cut in piece of gauze, wet it in preheated sterile saline, and place it above the incision.
Take out the intestine with sterile cotton swabs drenched in pre-warmed sterile saline. Keep the intestine hydrated by adding sterilized pre-warmed saline. Transfer the mouse to a preheated sterile imaging box.
Place the intestine on the sterile glass. Place the head of the mouse inside the inhalation tube of the imaging box. If necessary, secure the mouse with sterile flexible film and tape.
Place the imaging box containing the mouse in the microscope chamber. Monitor the mouse during the imaging by checking the frequency and depth of breathing, and temperature via a rectal probe every 15 minutes. Isoflurane should be kept between one to 2%Find a region in the intestine using the IPs of the microscope.
Obtain a wide field view of the region of interest using the microscope's internal camera. Image the region of interest using the 960 nanometer laser of multiphoton microscope. Adjust the laser power and wavelength according to the fluoroforms used in the experiment.
In this example, crypts expressed membrane is red fluorescent protein, and are stochastically labeled with green fluorescent protein upon induction with low end dose of tamoxifen. Acquire a 10 to 20 step Z-stack of three micron of the region of interest. Pick a single position or multiple positions from the tile image previously.
Use the breach point calibration function in the imaging software at zoom 32 and 124 by 124 pixel resolution with a scan speed 400 hertz using the bidirectional scanning property for three to 10 seconds, depending on the size of damage aimed for. The initiation of damage in the crypt area can be recognized by an increase in autofluorescence in both the green and red channel. After ablation, acquire Z-stacks of the damaged regions to confirm the location and the extent of the damage.
Repeat the previous two steps for multiple regions in the same mouse. Place the mouse while still under anesthesia on a sterile suturing area and cover with a sterile drape. Insert the exposed intestine back into the abdomen using sterile cotton swabs drenched in preheated sterile saline.
Suture the linea alba by performing simple continuous suture using absorbable suture. Close the extremities of the suture with surgical knots. Repeat the same step with the skin layer.
Switch off the isoflurane station, clean and sterilize the imaging box and inlay. Let the mouse recover from surgery while the cage is placed on the heating pad for one hour. Inject 200 microliter of buprenorphine subcutaneously six to 12 hours post-surgery.
Administer carprofen in drinking water for 72 hours post-surgery. Weigh the mouse and monitor the welfare every day for one week post-surgery. On the second time point at least one week after the first imaging session, repeat surgery and intravital imaging.
Use the pattern of blood vessels to find back the same regions of interest image on the first time point. If the mouse is not meant to be imaged on another time point, sacrifice the mouse by performing cervical dislocation under anesthesia. Otherwise, continue with the closure of surgical site and post-surgery care.
K19-Cre ERT mTmG mice were injected with tamoxifen to induce stochastic labeling of cells with GFP four to six weeks before surgery and first imaging session. After surgically exposing the intestine of the mouse and acquiring camera and fluorescent images of the region of interest, breach point settings of the multiphoton laser is used to ablate crypts. The initiation of damage can be recognized by an increase in autofluorescence in both the green and the red channel.
The same procedure is repeated one month later, and the vasculature is used as a landmark to find back the same region. Using the Lgr5-eGFP confetti model, crypts labeled with different colors can be followed over time to map the dynamics of recovery. In this example, crypts in green are expressing the Lgr5 Cre, and a crypt in magenta is labeled with confetti color.
Different modes of regeneration are observed two weeks after laser ablation. Some regions remain unchanged, while other regions exhibit remodeling in forms of crypt fission, so the division of one crypt into two, or fusion, merging of two crypts into one, or crypt disappearance. Laser ablation combined with intravital microscopy method restricts the damage to a defined region of interest.
This enables us to control the location of the damage as well as the damage extent. Damage severity can be modulated to ablate crypts or entire intestinal fields to inform us about the regenerative response at the crypt scale. In addition to spatial control, laser ablation also allows to precisely time the onset of damage in addition to imaging the same organ under both homeostatic and regenerating conditions in the same mouse, thereby surpassing the precision of previous injury models.
The application of laser ablation and repetitive intravital imaging can be utilized as a platform for a multitude of research questions and diverse scientific areas that span regeneration, immunology, and cancer research.
