The CaCl2 aneurysm model involves an anaesthetic time of ~30 minutes, a laparotomy, exposure of the aorta (through blunt dissection) and insult to the main vessel of the abdomen. Although initial pilot work had not shown any significant problems with the CaCl2 model in terms of bleeding, wound healing, abdominal complications or mortality, the majority of this work was undertaken in C57Bl/6 and TG2-/- mice because of delays in breeding the FXIII-A-/- and DKO transgenic mice.
As further experiments were undertaken it became apparent that mice lacking FXIII-Ahad an obviously decreased survival rate following apparently normal recovery from the injury procedure.
In general, the mice appeared to recover normally but were then either found dead in the subsequent 1-5 days, or failed to gain weight post-operatively, became increasingly ill and were terminated (by a Schedule-1 approved method) as per Home Office guidelines. Overall the mortality rate for all operations (from which the mouse was recovered from anaesthetic) was 8.6% in the C57Bl/6 group, 6.8% in the TG2-/- group, 41.1% in the FXIII-A-/- group (p<0.0001) and 62.3% (p<0.0001) in the DKO mice.
In response to this unexpected high mortality rate, we initiated closer monitoring of the post-op animals, held a low threshold for termination if there was evidence of suffering and undertook detailed recording of cause of death and post-mortem findings where possible. Table 7 outlines operating numbers, mortality rates and causes of death for the 6 week CaCl2 injury model and highlights that there is a low level of mortality (8.6%) associated with this model in C57Bl/6 mice. The causes of death in these mice included bleeding problems (presumably from unidentified injury at the time of dissection which resulted in continued bleeding) and bowel ischaemia (due to torsion of bowel loops on return to the abdominal cavity) as well as one case of post-op fighting. A similar mortality rate was seen in the TG2-/- mice (6.8%). It should be noted that the majority of these deaths occurred within the first few months of setting up the model and hence may reflect the initial “learning curve”
of this technique.
The FXIII-A-/- and DKO mice exhibited very different appearances post-mortem to those animals with normal FXIII-A gene function. These mice showed more episodes of bleeding (which might be expected from their impaired clot function) but also, a significant number of mice (approximately 30-40% of post-op deaths), showed features of cardiac pathology (with blackened, thrombus filled atria) as well as multiple, discrete areas of focal necrosis (for example in the bowel, spleen and
liver). This bowel ischaemia showed a different pattern to that seen with simple bowel torsion/injury and examples of the post-mortem pathology seen are shown in Figure 46. Although it is obviously difficult to conclusively determine cause of death in these mice, we hypothesise that these animals, deficient in FXIII-A, could be forming non-stable clots which are embolised to visceral arteries. The source of these clots may be the site of aortic injury (although this is unlikely given the observed sites of embolisation) or more likely the heart, secondary to some undetermined cardiac pathology. We have not seen additional deaths in our aged, un-operated FXIII-A and DKO mice when compared to WT, hence suggesting that the physiological stress of surgery is required to “trigger” this pathology. Deaths were also seen in sham operated DKO animals (but not in FXIII-A-/- sham operated mice) possibly hinting at a more severe phenotype in this group.
Post-operative weight was measured in all animals as an adjunct to close monitoring of appearance and behaviour in the assessment of animal welfare. As can be seen from Figure 47, the FXIII-A-/- and DKO mice had a lower average weight over the 6 weeks between operation and harvest (AUC data; C57 871.4 ± 235.2, TG2-/- 892.9 ± 239.1, FXIII-A-/- 576.2 ± 413.1, DKO 456.3 ± 408.7) which is attributable to a greater weight loss in the immediate post-op period. When only data from those animals that survived to harvest were analysed (panel C) there was no significant difference in weight between genotypes (p=0.265), suggesting that weight loss is a surrogate for probability of survival. Panel D shows the weight loss in the first 7-days after surgery in DKO mice, sub-grouped into surviving and non-surviving animals. It is clear that as early as day 2, weight loss can be used to identify mice that are unlikely to survive and hence, in the future, we would suggest that weight loss on day 2 of >12% is an indication for termination by a Schedule 1 approved method.
Genotype Operations Deaths Mortality Rate Causes of Death
Table 7. Mortality Rates – CaCl
2Injury
Table outlines operating numbers, mortality rates and causes of death for the CaCl2 injury model. The mortality rate for all operations (from which the mouse was recovered from anaesthetic) was 8.6% in the C57Bl/6 group, 6.8% in the TG2-/- group, 41.1% in the FXIII-A-/- group and 62.3% in the DKO mice. Causes of death are listed in the table and are discussed in detail in the main text.
Figure 46. Post-mortem pathology in DKO mice
Representative images from post-mortem examination of DKO mice. Panel A.
shows a DKO mouse found dead on day 5 post op and is typical of the appearances seen with post-op bleeding. There is bruising to the abdomen with blood and clots found in the abdomen and pelvis. The most common site of bleeding was from the pelvic vessels or along the posterolateral aspect of the aorta.
Panel B. shows the heart of a DKO mouse that underwent sham operation and was found dead on day 2 post-op. There were no problems with the wound and no evidence of bleeding into the abdomen. Blood stained serous fluid was found in the thoracic cavity and the heart appeared as shown here with a black, clot filled atrium and dusky appearance to the anterior surface. Panels C and D are images taken of a DKO mouse found dead at day 5 post-op. Again, there was no evidence of bleeding however the abdominal contents had patchy areas of necrosis with discrete ischaemia shown here in the bowel (C) and stomach (D).
A.
Figure 47. Weight change in mice post-injury
All data is shown as mean % weight change (from pre-op) with 95% confidence intervals. Panel A shows weight change in all operated mice after 0.5M CaCl2 injury. Panel B shows AUC data for the curves in panel A. One-way ANOVA testing revealed a significant difference in weight between groups (p<0.0001) due to lower weights in the FXIII-A-/- and DKO groups, however, when mice that died post-operatively are excluded from analysis (C), there is no difference between groups (p=0.265). Panel D plots weight change in the first 7-days in surviving vs non-surviving DKO mice and shows that by post-op day 2, weight loss can be used to predict survival.