This is Professor WT Godbe and along with Dr.Georgina Dobe, we'd like to present a method for establishing an orthotopic model of mure bladder carcinoma. Let us begin with the steps that we take to prepare our animals for this procedure. The mice that we're showing today are female C 57 blacks purchased from the Jackson Laboratories.
We chose female because of the ease of catheterization. These miser between three and five weeks of age, and they typically weigh in the range of 16 to 20 grams, the miser and ize using a ventilator that's set to deliver isof fluorine at 2%in oxygen at a flow rate of 0.8 to one liter per minute. Respiration rates are constantly monitored to prevent corneal drying.
During this procedure that takes in excess of one and a half hours, the eyes are lubricated with an artificial tears ophthalmic ointment that's applied directly over the eyes and then rolled down into the eyes the fur, the lower back is clipped to promote contact with the metal grounding plate, which will be used during the electrocautery procedure that will produce a burn injury within the bladder. The burn sites themselves will serve as adhesion points for the delivered tumor cells after they're introduced into the bladder lumen. The remainder of the techniques used in this protocol rely heavily on catheterization for which we use 24 gauge IV catheters.
With the stylus safely removed and discarded, the sheaths of the catheters are lubricated with the substance such as KY jelly or astroglide to help produce or prevent trauma to the urethra. For catheterization, the vulva is held steady with forceps. While the catheter is inserted in, the urethra care has to be taken for the catheterization techniques, so this will be shown in detail.
We recommend the use of surgical loops to help make the field more clear. Here we see the external pelvic anatomy of the mouse. We're now indicating the vulva which fold around the external urethral opening.
The tendency for catheterization is to try to insert the catheter into the middle of the structure, but the urethral opening is at the bottom. We have found it easier to grasp the ulfa with forceps than to gently roll the structure out and up to make the urethra more easily accessible. Take care not to catheterize the vagina, which is immediately below the urethral opening.
Another parameter to be aware of is the angle of catheterization. Now the natural tendency is to go in straight parallel to the longitudinal axis of the animal. However, a steeper angle makes insertion of the catheter a little easier after it's initially inserted.
You can then reduce the angle of catheterization to help prevent trauma to the animal. Here, Dr.Beck is coming in from a steep angle. Notice the catheter will be inserted at the bottom of the vulva.
Twisting of the catheter sometimes helps to lubricate the urethra and aid with insertion, and after it is initially inserted, the angle is made less steep and we can insert directly into the bladder. Notice how long it takes a skilled user. However, not every insertion is going to take that long.
Here's how long the procedure takes in the best case scenario. Now that we're able to efficiently catheterize a mouse, let's return to the tumor cell installation protocol. The next step is to drain the bladder of most of the urine, and we do so by applying general pressure to the pelvic area where the urine is gonna be forced out through the catheter and the end of the catheter serves as a collection area.
The catheter will then be removed and drained. Next, we prepare a platinum wire, which will be used to conduct a current into the bladder to induce the burden injury. And notice that this platinum wire has a hook on the end, and that's for measurement purposes.
Following insertion completely through the catheter, we want two to three millimeters of wire exposed on the internal side of the catheter. It is this exposed portion that will come into contact with the bladder wall and deliver the burn injury. After catheterization of the mouse, we will separately insert the wire.
The catheter is pulled out slightly to allow extra space for the wire to protrude from the end of the catheter without piercing the bladder. We deliver the current via an electrocautery unit. We use a Bovie electrocautery unit set for 2.5 watts and coagulation, and we deliver the current via tapping.
The exterior portion of the wire. Electro electric conduction gel has been placed between the mouse and the grounding plate. This is an example of the amount of time we actually spend delivering the current.
It's less than 0.5 seconds. Because of the short duration, we will deliver a second burn injury. We repositioned the wire in the catheter and then tap the wire again.
Following installation of the burn injury, the M will immediately receive the tumor cells and this will be performed through yet another catheterization. Except this time the catheter will be attached to a syringe that's loaded with a hundred microliters of cell suspension. The cells that we'll be using here are MB 49 cells at a concentration of 1 million cells per milliliter for a total of 100, 000 cells to be delivered.
We pull up an excess of 100 microliters and gauge the amount that we deliver through the marks on the side of the syringe. Following loading the syringe with the cells, we lubricate the catheter and re catheterize the animal. As you can see, not every catheterization will go as smoothly as the optimal conditions we showed before.
Care must be taken not to injure the animal. Following successful catheterization of the mouse, a hundred microliters of the cell suspension will be delivered through the syringe, through the catheter, and into the lumen of the bladder. The entire unit will be kept in place for 90 minutes during which time the isof fluorine administration rate will be reduced to 1%as a maintenance dose.
Tumor cells are allowed to develop into tumors for three to five days before we commence ultrasound examinations To monitor tumor cell growth, the fur on the abdomen is clipped to improve contact between the ultrasound probe and the subject. At this point, the mice will be catheterized and a hundred microliters of sterile normal saline will be instilled into the bladders to improve bladder visibility and tumor visualization. Upon ultrasound ultrasonic conduction gel is then applied to the abdomen and we found that swabbing it in with wet gauze helps to keep it from rolling off.
During the ultrasound procedure itself, a liberal amount of ultrasonic conduction gel is also applied to the head of the probe. Ultrasounds are then performed and monitored in real time. This is an example of the data that can be obtained during the ultrasound procedure.
In this case, the probe was in a longitudinal aspect which permits visualization of the catheter within the bladder. We're now indicating some tumor tissue that's developed during the incubation time. The entire bladder is usually easy to see and is delineated by a sharp border as indicated here.
Here we can see evidence of the establishment of the tumor model.
