Tail Vein Transection Bleeding Model in Fully Anesthetized Hemophilia A Mice

Tail bleeding models can be important tools to assess pharmacological effects of hemostatic compounds in vivo to ensure reproducibility between experiments. Compared to other sensitive bleeding models, our model uses blood loss and bleeding time as endpoints instead of survival. Also, the model is performed under anesthesia, thus reducing pain and distress for the animals.

This technique is especially useful for the preclinical study of hemophilia and other coagulation disorders, since it measures the ability of the blood to clot and stop a bleed after an injury. We have used this model extensively, testing procoagulant compounds in our work to develop novel hemophilia therapies using hemophilia A mice. However, other hemophilic mouse strains, or mice in which hemophilia is induced with an anti-factor VIII antibody, may also be used.

Demonstrating the experiment will be Sarah and Dennis, who are expert technicians in my lab. After anesthetizing the mouse, place it on a heating plate and ensure that the nose is in the nose cone. Apply a suitable eye ointment to prevent dryness while under anesthesia.

Mark the tail at a diameter of 2.5 millimeters using the tail mark block, taking care to not force the tail into the slit and ensuring that it fits snugly. Then, place the tail in the saline tube for at least five minutes to ensure a warm tail vein that is optimal for intravenous dosing. If necessary, IV dosing can be performed after five minutes.

Then, place a plastic cover over the mouse to reduce heat loss. Leave the tails submerged for five minutes after test solution dosing, and then perform the tail vein transection by placing the tail in the cutting block and turning it 90 degrees to expose the vein. Make the cut on the opposite side from where the test solution was dosed.

Draw the number 11 scalpel blade through the slit of the cutting block, holding the tail, to create bleeding. Then, immediately return the tail to the saline and reset the stopwatch. Bleeding should be visible immediately.

Submerging the tail in saline is important for consistent bleeding, since it would quickly stop in free air. Observe the bleeding and annotate the start and stop of the bleeding throughout 40 minutes on the blood flow notation paper. Disqualify and replace the mouse if primary bleeding does not stop within three minutes after the cut is made, which may indicate that the cut is too deep or that the mouse is deficient in primary hemostasis.

Challenge the tail cut if there is no bleeding, 10, 20, and 30 minutes post-injury. Using a gauze swab soaked and warm saline, lift the tail out of the saline and softly but firmly wipe it twice with the wet gauze in a distal direction over the tail cut. Immediately resubmerge the tail into the saline tube after each challenge.

When the experiment is finished, remove the tail from saline, collect blood samples if desired, and euthanize the mice while still under full anesthesia. Centrifuge the 15-milliliter blood collection tubes with saline at 4, 000 times G for five minutes at room temperature. Discard the supernatant from the tubes.

Resuspend the pellet in two milliliters of erythrocyte lysing solution. Then, dilute it with up to 12 additional milliliters of lysing solution until it reaches a light coffee color. Note the total volume and transfer two milliliters of the dilution to a hemoglobin tube.

Refrigerate it until the hemoglobin analysis. A significant reduction in blood loss for recombinant coagulation factor VIII treatment groups was observed using the optimized protocol. A 20 international units per kilogram dose completely normalized the bleeding, and 10 IU per kilogram caused a significant effect, reducing blood loss nearly to the upper limit of the wild-type range.

Similar results were observed for bleeding time. A reduction was observed in 5, 10, and 20 IU per kilogram groups, being completely normalized in the 20 IU per kilogram group. In wild-type mice, total blood loss ranged from 200 to 840 nanomole of hemoglobin, and in vehicle mice, from 5, 300 to 7, 100 nanomole of hemoglobin.

The mean bleeding was 5, 200 nanomole in vehicle-treated mice, and 720 nanomole in the 20 IU per kilogram group. The bleeding time of wild-type mice ranged from one to nine minutes and dose levels of 10 and 20 IU per kilogram reduced bleeding time to within this range. A strong correlation between blood loss and bleeding time was observed in the combined data.

All recorded bleeding episodes were plotted to provide a visualization of the length and number of bleeds experienced for each individual mouse. Note that in the vehicle group, after the second challenge, most animals continue bleeding until the end of the experiment. Measurement of plasma factor VIII concentration confirmed that the effect observed in reducing blood loss and bleeding time was recombinant factor VIII concentration-dependent.

Platelet counts were not noticeably affected and hematocrit levels were within normal range in animals receiving moderate and higher recombinant factor VIII doses, but significantly lower in animals which bled extensively, such as in the vehicle and one IU per kilogram groups. In animals experiencing heavy bleeding, minor in impact on blood counts, particularly total hemoglobin or hematocrit, can occur. To evaluate the effect of gender in this optimized model, both blood loss and bleeding time results were subjected to two-way ANOVA analysis, with gender and dose as factors.

These results indicated that response to treatment did not differ between genders. Correct marking of the tail and creating the cut are essential steps to ensure reliability and reproducibility in the technique, as is the challenge if there's no bleeding. Furthermore, good anesthesia induction and maintenance of the animal body temperature is particularly relevant.

We have also used this model to investigate stopping of established bleeds in a more on-demand-like setting by treating with factor VIIA 10 minutes after inducing bleeding.