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JoVE Journal Biology

Biology

Tail Vein Transection Bleeding Model in Fully Anesthetized Hemophilia A Mice

Published: September 30, 2021 doi: 10.3791/62952
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1,2, 1, 1, 3, 1, 4, 5, 1, 6, 1
1Global Drug Discovery, Novo Nordisk A/S, 2Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, 3Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, 4Independent consultant, 5Catalyst Biosciences, 6Værløse Dyreklinik

Summary

The refined tail vein transection (TVT) bleeding model in anesthetized mice is a sensitive in vivo method for the assessment of hemophilic bleeding. This optimized TVT bleeding model uses blood loss and bleeding time as endpoints, refining other models and avoiding death as an endpoint.

Abstract

Tail bleeding models are important tools in hemophilia research, specifically for the assessment of procoagulant effects. The tail vein transection (TVT) survival model has been preferred in many settings due to sensitivity to clinically relevant doses of FVIII, whereas other established models, such as the tail clip model, require higher levels of procoagulant compounds. To avoid using survival as an endpoint, we developed a TVT model establishing blood loss and bleeding time as endpoints and full anesthesia during the entire experiment. Briefly, anesthetized mice are positioned with the tail submerged in temperate saline (37°C) and dosed with the test compound in the right lateral tail vein. After 5 min, the left lateral tail vein is transected using a template guide, the tail is returned to the saline, and all bleeding episodes are monitored and recorded for 40 min while collecting the blood. If no bleeding occurs at 10 min, 20 min, or 30 min post-injury, the clot is challenged gently by wiping the cut twice with a wet gauze swab. After 40 min, blood loss is quantified by the amount of hemoglobin bled into the saline. This fast and relatively simple procedure results in consistent and reproducible bleeds. Compared to the TVT survival model, it uses a more humane procedure without compromising sensitivity to pharmacological intervention. Furthermore, it is possible to use both genders, reducing the total number of animals that need to be bred, in adherence with the principles of 3R's. A potential limitation in bleeding models is the stochastic nature of hemostasis, which can reduce the reproducibility of the model. To counter this, manual clot disruption ensures that the clot is challenged during monitoring, preventing primary (platelet) hemostasis from stopping bleeding. This addition to the catalog of bleeding injury models provides an option to characterize procoagulant effects in a standardized and humane manner.

Introduction

Animal models are essential for understanding the pathogenesis of hemophilia and developing and testing treatment regimens and therapies. The Factor VIII knock-out mouse (F8-KO) is a widely used model for the study of hemophilia A1,2. These mice recapitulate key features of the disease and have been widely used for development of treatments, such as recombinant FVIII products3,4,5 and gene therapy strategies6,7.

There are various bleeding injury models for evaluating the pharmacological effects of different hemostatic compounds in vivo. One of these coagulation models is the tail vein transection survival model in mice8,9,10,11,12,13,14, measuring the ability of hemophilic mice to survive exsanguination after tail transection. This method was introduced more than four decades ago15 and is still used9,16,17. However, the model utilizes survival as an endpoint and requires observation of the animals over a period of up to 24 h, during which the animals are conscious and hence can experience pain and distress.

Bleeding models of shorter duration and under full anesthesia have been described previously, such as the tail clip model (also known as the tail tip)8,18,19,20,21,22,23,24,25,26,27,28. Nevertheless, for a complete normalization of blood loss after the bleeding challenge, these models require doses of procoagulant compounds (e.g., FVIII) far higher than those administered clinically29. A different injury model under anesthesia, the vena saphena bleeding method, is sensitive to lower doses of procoagulant compounds30 but requires a high level of experimenter intervention since the clots must be disrupted frequently (as opposed to 3 times in the presented model).

Standardization towards a common protocol to test new procoagulant compounds would greatly facilitate data comparison between laboratories31,32,33. In TVT models, there is not yet a common agreement on studied endpoints (blood loss7,26, bleeding time9,34, and survival rate35,36), and experimental length varies between studies13.

Our primary objective is to describe and characterize an optimized model with high reproducibility, the possibility to study on-demand as well as a prophylactic treatment, sensitivity to pharmacological intervention equivalent to the survival model, yet not using death or near-death as endpoints. In order to reduce pain and distress, the animals should not be conscious during bleeding and a more ethical endpoint needs to be implemented37.

Tail clip models are generally conducted in one of two variants, either amputating the tip of the tail, e.g., amputation of 1-5 mm18,19,20,21,23,24 or, in a more severe variant, transected at a tail diameter around 1-3 mm8,22,25. This causes a combined arteriovenous bleed, as the lateral and dorsal veins and ventral artery are usually severed, and in general, the larger the amputation, the lower the sensitivity to a procoagulant compound. Furthermore, since the tail tip is amputated, the arteriovenous injury is exposed without any opposing tissue; thus, at least in theory, it is dissimilar to the most common hemophilic bleeds.

As the name implies, only the vein is injured in tail vein transection models such as described in this paper, thus resulting in an exclusively venous bleed. Since the vessel is not fully severed, the injury is expected to be smaller than in the amputation models, and the tissue around the cut, which a clot may adhere to, is retained. In addition, there is lower blood pressure in the vein as opposed to the artery. These factors contribute to an increased sensitivity relative to amputation models, such that normalization of bleeding can be achieved with clinically relevant doses of replacement therapy, e.g., with rFVIII in hemophilia A, which is useful for evaluating the magnitude and durability of effects of procoagulant treatment26,38,39.

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Protocol

All procedures described in this protocol have been approved by the Animal Welfare Body at Novo Nordisk A/S, and the Danish Animal Experiments Inspectorate, The Danish Ministry of Food, Agriculture, and Fisheries. The optimized 40 minutes method includes anesthesia and dosing time in the design (Figure 1). Hemophilic mice of both genders between 10-16 weeks of age are required for this procedure.

1. Preparations before the study

  1. Prepare the dosing solutions in the correct concentrations.
  2. Start the water bath and heat to 37 °C. Fill the 15 mL centrifuge tubes for blood collection with saline (0.9% NaCl).
  3. Place the 15 mL saline tubes in the holes in the warmed base plate at least 15 min prior to the start of the experiment.
  4. Identify the mice and record their weight. Avoid handling mice more than necessary as this can cause stress and affect the study.
  5. Prepare the workstation in the fume hood before proceeding so that everything is within reach: napkins, tail holder, gauze, syringes, scalpels, stopwatches, and blood flow notation paper.
  6. Place the tail mark and cutting blocks on the heating plate - cold blocks will make the veins contract and thereby affect the bleeding.

2. Anesthesia

  1. Conduct the isoflurane anesthetic procedure inside a fume hood.
  2. Set the gas vaporizer to initially 5% isoflurane in 30% O2/70% N2O in the anesthesia chamber with 1 L/min flow. Allow sufficient time for the anesthesia chamber to fill (about 5 min depending on chamber volume and gas flow rate). Ensure rapid induction (less than one minute).
  3. Place the mice in the anesthesia chamber until they lose consciousness.
    NOTE: This should occur within a minute or less if the chamber is sufficiently filled.
  4. Ensure proper anesthetization by the absence of painful response to pedal reflex (firm toe pinch).
  5. Place the mice on the heating plate, making sure that the nose is in the nose cone.
  6. Reduce the anesthesia to a maintenance level of 2% isoflurane in 30% O2/70% N2O and place a plastic cover above the mice to reduce the loss of heat. Apply a suitable eye ointment to prevent dryness while under anesthesia.
  7. Mark the tail at a diameter of 2.5 mm using the tail mark block. Do not force the tail into the slit in the block - it must fit snugly (Supplemental Figure 1)
  8. Place the tail in the saline tube for at least 5 min to ensure a warm tail vein that is optimal for intravenous (i.v.) dosing.

3. Dosing of test solution

  1. Place one mouse in the tail holder with the nose in an anesthesia mask.
  2. Dose the animal with the compound of interest (in this case, rFVIII) and immediately start the stopwatch (t = 0).
  3. Place the mouse back on the heating plate with the tail in the saline tube. Repeat the procedure with the other mice.

4. Performing tail vein transection

  1. Perform the tail vein transection exactly 5 min after dosing. Place the tail in the cutting block and turn 90° to expose the vein (Supplemental Figure 2).
  2. Perform the cut on the opposite side/vein from where the test solution was dosed.
  3. Draw the #11 scalpel blade through the slit of the cutting block holding the tail to create bleeding. Reset the stopwatch and return the tail immediately to the saline.

5. Observation time and challenges

  1. Observe the bleeding and annotate the start and stop of the bleeding throughout 40 min; annotate it on the blood flow notation paper.
    NOTE: This visual assessment of bleeding may vary slightly due to subjectivity.
  2. The primary bleeding must stop within 3 min after the cut is made. If this is not the case, disqualify the mouse, euthanize, and replace (failure to stop the primary bleed can indicate a too severe injury or lacking primary hemostasis, as in vWF KO mice).
  3. If there is no bleeding at 10 min, 20 min, and 30 min post-injury, challenge the tail cut as described in steps 4-5.
  4. Use a gauze swab soaked in warm saline from a separate tube kept in the water bath. Lift the tail out of the saline and softly wipe twice with the wet gauze in a distal direction over the tail cut.
  5. After each challenge, immediately re-submerge the tail into the saline tube again.
  6. At t=40 min, stop the blood collection by removing the tail from the saline tube.

6. Blood sampling

  1. After t = 40 min, obtain blood samples from the supraorbital vein.

7. Euthanasia

  1. Euthanize the mice by cervical dislocation while still under full anesthesia.

8. Treatment of samples

  1. Centrifuge the 15 mL blood collection tubes with saline at 4000 x g for 5 min at room temperature.
  2. Discard the supernatant from the 15 mL tubes, resuspend the pellet in 2-14 mL of erythrocytes (RBC) lysing solution, and then dilute it until it reaches a light coffee color.
  3. Note the total volume (volume of blood + volume of erythrocytes (RBC) lysing solution added using the graduation marks on the tube).
  4. Transfer 2 mL of the dilution to a hemoglobin tube and refrigerate it until the hemoglobin analysis.
  5. Determine the blood loss by measuring the hemoglobin concentration in the saline. Measure the absorbance at 550 nm on a microplate reader (Table of Materials).
  6. Convert the absorbance to nmol hemoglobin using a standard curve prepared from human hemoglobin (Table of Materials) and correct for the dilution with RBC lysing solution.

9. Statistical analyses

  1. Analyze the data using appropriate software. Here GraphPad Prism software was used. Over a range of studies, the following statistical methods were found to perform well.
    NOTE: To analyze blood loss, bleeding time, exposure, platelet counts, and hematocrit; Brown-Forsythe and Welch ANOVA test was used, (as the data were continuous but without variance homogeneity of the residuals) applying Dunnett's test to adjust for multiple comparisons. A Pearson's test was used to test for correlations between bleeding time, blood loss, and doses. To determine ED50 values, a four-parameter inverse log (dose) response equation was fitted to bleeding- and blood loss data. To analyze the gender effect, a two-way ANOVA test was used, applying Bonferroni correction to adjust for multiple comparisons. The significance level was defined as P < 0.05. Data are displayed as means ± SEM.

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Representative Results

To assess the applicability of the optimized model, a study was performed in F8-KO (C57BL genetic background) mice administered with a commercially available recombinant factor VIII replacement therapy (rFVIII); four different doses were tested: 1 IU/kg, 5 IU/kg, 10 IU/kg, and 20 IU/kg. Furthermore, we tested the corresponding vehicle (negative) control in F8-KO mice and wild-type (WT) group using C57BL mice as a positive control group to assess the response range in the model.

Following the optimized protocol, there was a significant reduction in blood loss for rFVIII treatment groups compared to the vehicle group. Additionally, a reduction in bleeding time was observed in the 5 IU/kg, 10 IU/kg, and 20 IU/kg treatment groups compared to the vehicle group (Figure 2). In WT mice, total blood loss ranged from 201.8-841.9 nmol Hgb (95% CI) and in vehicle mice ranged from 5335 to 7148 nmol Hgb (95% CI). Following the approximate equivalence of 1000 nmol Hgb ~125 µL of whole blood, the mean bleeding in vehicle-treated mice was 780.25 µL, while on the 20 IU/kg group was 89.95 µL. Thus, a dose of 20 IU/kg completely normalized the bleeding, and administration of 10 IU/kg caused a significant effect, reducing blood loss nearly to the upper limit of the WT range (Figure 3). The bleeding time of WT mice ranged from 0.98-9.16 min (95% CI), and dose levels of 10 IU/kg and 20 IU/kg reduced bleeding time to within this range. A strong correlation between blood loss and bleeding time was observed in the combined data (r = 0.9357, P < 0.0001) (Figure 4).

To evaluate the sensitivity of the model, a four-parameter inverse log(dose) response equation was fitted to blood loss and bleeding time observations, and a clear dose-dependent effect of rFVIII administration on blood loss and for the bleeding time was observed (Figure 3). The estimated ED50 values for blood loss and bleeding time were 2.41 ± 1.69 IU/kg and 2.55 ± 2.80 IU/kg, respectively.

To illustrate how bleeding occurs in the model, all recorded bleeding episodes have been plotted to provide a visualization of the length and number of bleeds for by each individual mouse (Figure 5). The primary bleeding is very similar in all the groups. Most of the hemophilic vehicle group starts to re-bleed before the second challenge at 20 min, and for about half of these animals, the bleeding does not stop after the first challenge. Finally, as described, rFVIII treatment reduced the length of the bleeding episodes in treated F8-KO mice compared to vehicle, with already an observable change at the lowest dose. At the highest dose levels, most of the mice only bled briefly after being challenged.

Plasma rFVIII concentration was measured by Luminescent oxygen channeling assay (LOCI) detecting hFVIII and analogs to verify that the effect observed in reducing blood loss and bleeding time was concentration-dependent (Figure 6). There is variability within each group (mean CV 46%), but still, significant differences between groups can be observed, thus corroborating that the effect observed in blood loss and bleeding time is dependent on the plasma concentration of FVIII. All vehicle-treated mice measured below the lower limit of quantitation for the assay (2 U/L) and are represented with this value. WT mice were not measured since the applied FVIII assay is specific for detecting human FVIII.

Platelet counts and hematocrit were determined for all groups using the blood samples collected post-bleeding (Figure 7 and measured with a hematological analyzer (Table of Materials). There was no variation between groups in platelet counts, indicating that platelet numbers are not affected, and the mice, therefore, remain capable of primary hemostasis. For hematocrit measurements, normal levels were observed in animals receiving moderate and higher FVIII doses (5 IU/kg, 10 IU/kg, and 20 IU/kg), whereas significantly lower levels were observed in the vehicle and 1 IU/kg treated groups (compared to WT animals). This is a frequent observation in hemophilic animals after heavy bleeding.

Classically, only one gender of animals (typically male) has been used in animal studies, and this is also described for TVT survival models8,9,11,12. Striving to reduce the total numbers of haemophilic mice needed in future studies (for breeding and the study), both genders were used. To evaluate the effect of gender in this optimized model (Figure 8), both blood loss and bleeding time results were subjected to two-way ANOVA analysis with gender and dose as factors. In this analysis, the effect of rFVIII dose was statistically significant (P < 0.0001), but mouse gender did not significantly affect the results (P 0.35), and no significant interaction was found between the parameters, indicating that responses to treatment did not differ between genders.

Figure 1
Figure 1: Temporal design of the experimental setup. The phase prior to tail vein transection (TVT) includes anesthesia induction, warming the tail in saline, and dosing. After the TVT injury, 40 minutes of monitoring the bleeding under anesthesia, with subsequent challenges every 10 min, is performed. The experimental procedure is concluded by the collection of blood samples and euthanasia. Please click here to view a larger version of this figure.

Figure 2
Figure 2: Effect of rFVIII after intravenous administration. Total blood loss (left panel) and total bleeding time (right panel) of F8-KO mice. Each mouse is shown as individual observations and mean ± SEM. The different test doses of rFVIII are represented in the x-axis. Data were analyzed by Brown-Forsythe and Welch ANOVA applying Dunnett's test to adjust for multiple comparisons. **** P < 0.0001. Please click here to view a larger version of this figure.

Figure 3
Figure 3: Dose-response curves for blood loss and bleeding time. Blood loss (left panel) and bleeding time (right panel). The grey area represents the 95% CI from the values from 6 untreated wild-type C57BL/6 mice. Please click here to view a larger version of this figure.

Figure 4
Figure 4: Correlation plot for blood loss and bleeding time. R2 = 0.8755, P < 0.0001. Please click here to view a larger version of this figure.

Figure 5
Figure 5: Individual bleeding profiles in wild-type, vehicle, 1 IU/kg, 5 IU/kg, 10 IU/kg, and 20 IU/kg rFVIII treated mice. Each line represents the bleeding profile of a single mouse, whereas each dotted bar represents a bleeding episode. Please click here to view a larger version of this figure.

Figure 6
Figure 6: Exposure to rFVIII. FVIII concentration in plasma was measured by a Luminescent oxygen channeling assay (LOCI) for detection of human FVIII. Vehicle mice were under the lower limit of quantitation (LLOQ) and were therefore plotted with the LLOQ value (2 U/L). WT animals were not measured since the applied FVIII assay is specific for detecting human FVIII. The different test doses of rFVIII and a WT control are represented in the x-axis. ** P < 0.01, *** P < 0.001 and **** P < 0.0001. Please click here to view a larger version of this figure.

Figure 7
Figure 7: Hematological data. Platelet count (left panel) and hematocrit (right panel) of F8-KO mice are shown as individual observations mean with SEM. The different test doses of rFVIII and a WT control are represented on the x-axis. Data were analyzed by Brown-Forsythe and Welch ANOVA applying Dunnett's test to adjust for multiple comparisons. ** P < 0.01 and *** P < 0.001. Please click here to view a larger version of this figure.

Figure 8
Figure 8. Effect of gender. Blood loss (left panel) and bleeding time (right panel) classified by treatment are shown as individual observations with mean ± SEM. Data were analyzed by two-way ANOVA applying the Bonferroni correction to adjust for multiple comparisons. Please click here to view a larger version of this figure.

ED50 bleeding time (IU/kg) ED50 blood loss (IU/kg) ED50 survival (IU/kg)
Models under anesthesia Optimized tail vein transection model 2.6 ± 2.8 2.4 ± 1.7 not relevant
Vena saphena model30 8.1 ± 2.2 5.1 ± 2.1 not relevant
Moderate tail clip (L=3mm)27 not reported 4.6 ± 0.5 not relevant
Moderate tail clip (L=4mm)28 39 28 not relevant
Moderate tail clip (L=4mm)20 not reported 53 not relevant
Survival models Tail vein transection survival model36 not reported not reported 58
Tail vein transection survival model10 not reported not reported 21

Table 1: ED50 values. Comparison of ED50 values of different available bleeding models for hemophilia studies in mice. Data is extracted from cited articles [references superscript] and presented as ED50 (IU/kg)

Supplemental Figure 1: Measuring template. Produced in aluminum. Size specifications: 20 mm x 40 mm x 10 mm (L x W x H). Groove: 2.5 mm depth and 2.5 mm width; radius 1.25 mm. Please click here to download this File.

Supplemental Figure 2: Cutting template. Produced in stainless steel. Size specifications: 20 mm x 40 mm x 10 mm (L x W x H). Groove: 3 mm depth and 3 mm width; radius 1.5 mm. Please click here to download this File.

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Discussion

This optimized method of tail vein transection (TVT) has several advantages compared to the TVT survival method. The animals are fully anesthetized for the entire study duration, which makes mouse handling easier and increases animal wellbeing. Further, unlike the TVT survival model, overnight observation is not required, and this optimized model offers the possibility of measuring blood loss and observing the exact bleeding time over 40 min. Also, longer periods of bleeding in conscious animals can cause death by exsanguination, leading to pain and distress in the animals and probably stress, potentially resulting in increased variation14. Blood loss and bleeding time have been successfully characterized and validated as endpoints, replacing time to death or near-death. The actual tail injury is well defined and standardized since it uses a cutting block guide to perform the TVT, securing a reproducible cut, reducing the difficulty of the procedure and the technical variability. Hence, higher model robustness can be achieved, reducing the number of animals needed. Furthermore, data has demonstrated that it is possible to use both male and female mice, thus reducing the total number of animals to be bred, in accordance with the 3R principles. Along with these observations, there is a trend that female mice bleed slightly less and vary slightly more in high-bleeding groups, but not in low-bleeding groups. Lower blood loss measures could be associated with females having a smaller size, and therefore less blood volume compared to same-aged males.

Spontaneous bleeds in hemophilic patients are usually internal, specifically involving musculoskeletal, soft tissue, and mucocutaneous bleeding, while external injuries (such as minor cuts) are not the most common cause of extended bleeds, although more severe cuts and trauma can be life-threatening40,41. This optimized model induces venous-only bleeding, while other models, such as the tail clip, induce a mix of arterio-venous bleeding. Since the described model is a venous-only model, the procoagulant dose-effect in this TVT model might not reflect the effect in a more severe arterial injury; thus, other bleeding models should be used if such bleeds are in focus.

As shown by the individual bleeding profile, the primary bleeding is very similar in all the groups indicating that primary hemostasis, i.e., aggregation of activated platelets, is intact in the hemophilia A setting42. In an immobilized injury, even under severe hemophilia, platelet aggregation can be sufficient to attenuate bleeding. For that reason, through empirical studies, we have found that inducing a bleeding challenge every 10 min is a necessary step in the protocol in order to disrupt aggregates of activated platelets, which can be strong enough to prevent bleeding even without the fibrin generation from functional coagulation. Since fully anesthetized mice do not move and cannot physically challenge the injury as it happens in the non-anesthetized TVT survival model, it was necessary to introduce the challenge steps to prevent platelet aggregation alone from attenuating bleeding. Some untreated hemophilic mice re-bleed spontaneously prior to the challenge, but after the first challenge, spontaneous re-bleeds occur in most untreated hemophilic mice, and after the second challenge, many bleed continuously until the end of the experiment. As expected, there was increased bleeding in vehicle F8-KO mice compared to WT mice. Mice treated with increased doses of rFVIII showed a dose-dependent reduction in both blood loss and bleeding time. Significant effect on bleeding was observed at doses 5 IU/kg and above (compared with vehicle-treated mice). The two highest doses reduced bleeding to levels quite close to the wild-type bleeding response, indicating normalization or near-normalization of bleeding.

In Table 1, we present a comparison of different ED50 values for several bleeding models, classified by the studied endpoints. In this optimized model, we observed comparable ED50 values for blood loss and bleeding time (2.4 ± 1.7 IU/kg and 2.6 ± 2.8 IU/kg, respectively). Using the same model studying rFVIIa, the ED50 values for blood loss and bleeding time were 0.42 mg/kg and 0.39 mg/kg, respectively38. This is a greater sensitivity to pharmacological intervention than previously described bleeding models under anesthesia, such as the tail clip model, with FVIII ED50 for blood loss of 4.6 ± 0.5 IU/kg27, 28 IU/kg28, and 53 IU/kg20. Furthermore, there is a high variability of ED50 values in the different tail clip models20,27,28. Another model under anesthesia is the severe tail clip model. It is a faster method since the observation period is only 20 min, but it is less sensitive to procoagulant activity. Doses higher than 200 IU/kg of FVIII were necessary to achieve a statistically significant reduction of blood loss compared to vehicle-treated animals25. In the vena saphena model30, the ED50 value for bleeding time was 8.1 ± 2.2 IU/kg, and ED50 for average blood loss was 5.1 ± 2.1 IU/kg, also less sensitive than our refined model in regards to both parameters. Furthermore, this model requires delicate work to perform the vessel injury and multiple repeated interventions which, if not performed reproducibly, could influence the outcome.

In the traditional TVT survival model, survival is evaluated for a period of 24 h after dosing, during which bleeding or re-bleeding can occur at any time. Thus, efficacy in the TVT survival model requires the procoagulant effect of treatment to persist for at least the majority of the 24-h observation period. In the method presented here, we assess acute effects between 5 min and 40 min after dosing; thus, a direct comparison of ED50 values is difficult to establish since higher doses will be needed in the survival models in order to maintain hemostatic coverage during the latter part of the observation period. However, if so desired, the optimized TVT model can be used to evaluate the duration of hemostatic coverage by introducing a delay between dosing and the bleeding procedure. This has been described for a previous version of our optimized TVT model where performing the TVT 24 h after dosing resulted in an ED50 of roughly 10-15 IU/kg for unmodified rFVIII26. As noted in Table 1, in tail-transection models with a 24-h survival as an endpoint, ED50 values of 21 IU/kg 9 and 58 IU/kg36 of rFVIII have been reported. Similarly, tail clip survival models8 also require higher procoagulant doses than their acute counterparts.

In perspective, several different coagulation factors (FVIII, FVIIa, FIX) and derivatives, some of which are now marketed, have been evaluated with the optimized model using different hemophilic mouse strains26,38,43,44,45,46,47. We have also been able to adapt the model to the study of on-demand interventions by dosing after the transection, just prior to the first challenge. Furthermore, we have successfully used this model to evaluate bispecific antibodies with procoagulant activity (Østergaard et al., accepted for publication in Blood), overcoming species cross-reactivity challenges by dosing human FIX and human FX. This demonstrates the versatility and translational value of the model in development of new medicines in the hemophilia field, such as platelet-targeted strategies48. AAV-based or genome editing strategies can also be evaluated in cases where there is a surrogate that is pharmacologically active in mice. Therefore, this optimized TVT bleeding model is an alternative to tail vein transection and tail clip survival models, as well as a valuable alternative to other bleeding models under anesthesia. This model is more humane compared to the survival model and an example of Refinement as the animals do not experience pain and suffering. In our view, the sensitivity to clinically relevant dose levels, relative technical simplicity, and avoiding death/near-death as endpoints are significant advantages.

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Disclosures

The authors are or were employees and/or shareholders of Novo Nordisk A/S at the time this research was carried out.

Acknowledgments

Esther Bloem and Thomas Nygaard are acknowledged for support with measurements of FVIII in plasma. Bo Alsted is acknowledged for drawing and machining the template and cutting blocks.

Materials

Name Company Catalog Number Comments
#11 Scalpel blade Swann-Morton 503
15 mL centrifuge tubes Greiner Bio-One, Austria 188271
30 G needles connected to 300 µL precision (insulin) syringes for dosing BD Micro-Fine + U-100 insulin syringe 320830
Advate Takeda, Japan Recombinant factor VIII replacement therapy (rFVIII)
Alcohol pads 70% ethanol Hartmann, Soft-Zellin 999 979
Centrifuge Omnifuge 2.0 RS, Heraus Sepatech
Cutting template (Stainless steel) Self produced, you are welcomed to contact the authors for the exact drawings Supplementary Figure 2: Size specifications: 20 mm x 40 mm x 10 mm (L x B x H). Groove: 3 mm depth and 3 mm width; radius 1.5 mm
Erythrocytes (RBC) lysing solution Lysebio, ABX Diagnostics 906012
Gauze
Haematological analyser Sysmex CT-2000iv
Heating lamp on stand Phillips IR250
Heating pad with thermostat CMA model 150
Hemoglobin standards and controls - 8.81 mmol / l batch dependent HemoCue, Denmark HemoCue calibrator, 707037 Standards and controls are made from 2 different glasses of HemoCue calibrator. The value is determined against the International Reference Method for Hemoglobin (ICSH).
Isofluorane anaesthesia system complete with tubes, masks and induction box Sigma Delta Dameca
Isoflurane Baxter 26675-46-7
Magnifier with lights Eschenbach
Measuring template (Aluminum) Self produced, you are welcomed to contact the authors for the exact drawings Supplementary Figure 1: Size specifications: 20 mm x 40 mm x 10 mm (L x B x H). Groove: 2.5 mm depth and 2.5 mm width; radius 1.25 mm
Micropipettes + tips Finnpipette
Photometer Molecular Devices Corporation, CA, USA SpectraMax 340 photometer
Prism Software GraphPad, San Diego, CA, USA Version 9.0.1
Saline 0.9% NaCl Fresenius Kabi, Sweden 883264
Special tail marker block for TVT tail cut
Tail holder
Vacuum liquid suction Vacusafe comfort, IBS
Waterbath and thermostat TYP 3/8 Julabo

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References

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Tail Vein Transection Bleeding Model Fully Anesthetized Hemophilia A Mice Pharmacological Effects Hemostatic Compounds Reproducibility Bleeding Time Anesthesia Pain And Distress Reduction Preclinical Study Clotting Ability Hemophilia Therapies Procoagulant Compounds Mouse Strains Anti-factor VIII Antibody Expert Technicians Heating Plate Nose Cone Eye Ointment Tail Mark Block Saline Tube Intravenous Dosing
Tail Vein Transection Bleeding Model in Fully Anesthetized Hemophilia A Mice
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Carol Illa, A., Baumgarten, S.,… More

Carol Illa, A., Baumgarten, S., Danielsen, D., Larsen, K., Elm, T., Johansen, P. B., Knudsen, T., Lauritzen, B., Tranholm, M., Ley, C. D. Tail Vein Transection Bleeding Model in Fully Anesthetized Hemophilia A Mice. J. Vis. Exp. (175), e62952, doi:10.3791/62952 (2021).

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