Rabbit Elastase Aneurysm Model Mimics the Recurrence Rate of Human Intracranial Aneurysms following Platinum Coil Embolization

BACKGROUND AND PURPOSE: Intracranial aneurysms treated with coils have been associated with incomplete occlusion, particularly in large or wide-neck aneurysms. This study aimed to validate the accuracy of the rabbit elastase model in predicting aneurysm recurrence in humans treated with platinum coils. MATERIALS AND METHODS: Elastase-induced saccular aneurysms were induced in rabbits and embolized with conventional platinum coils. The recurrence rates of aneurysms were retrospectively analyzed. Morphologic characteristics of aneurysms, angiographic outcomes, and histologic healing were evaluated. RESULTS: A total of 28 (15.3%) of 183 aneurysms recurred. The aneurysm recurrence rate observed in this study (15.3%) is similar to those reported in multiple analyses of aneurysm recurrence rates in humans (7% – 27%). The rate of recurrence was higher in aneurysms treated without balloon assistance (19/66, 28.8%) compared with those treated with balloon assistance (9/117, 7.7%). Aneurysms treated with bal-loon-assisted coiling had a lower recurrence rate (OR = 0.17; 95% CI, 0.05 – 0.47; P = .001) and higher occlusion rate (OR = 6.88; 95% CI, 2.58 – 20.37; P , .001) compared with those treated without balloon-assisted coiling. In this rabbit elastase – induced aneurysm model, packing density and aneurysm volume were weak predictors of aneurysm recurrence; however, the packing density was a good predictor of the occlusion rate (OR = 1.05; 95% CI, 1.02 CONCLUSIONS: The rabbit elastase aneurysm model may mimic aneurysm recurrence rates observed in humans after platinum coil embolization. Moreover, balloon assistance and high packing densities were signi ﬁ cant predictors of aneurysm recurrence and occlusion.

T he prevalence of intracranial aneurysms in healthy adults is estimated to be 3%-5%. 1,2 Endovascular embolization with platinum coils is the preferred treatment for unruptured and ruptured intracranial aneurysms because it is associated with lower morbidity and mortality rates compared with surgical clipping. 3,4 However, coils have been associated with incomplete occlusion, leading to compaction and aneurysm recanalization, particularly in large aneurysms (diameter of $ 10 mm) or those with a wide neck (.4 mm). The recurrence rate increases from 5% for aneurysms with a neck size between 4 and 10 mm to 20% for those with wider necks (.10 mm). Similarly, the recurrence rate of large aneurysms is 35%-50% compared with an overall recurrence rate of 20% in a heterogeneous population. 5,6 To lower the recurrence rate of large and wide-neck aneurysms, a plethora of endovascular devices have been developed and tested in preclinical animal models. 7 Preclinical trials of endovascular devices are necessary to evaluate their safety and efficiency. An ideal model requires aneurysm hemodynamic and histologic healing characteristics similar to those seen in humans. In models with surgical creation of aneurysms such as in both canine and rabbit venous pouch models, the presence of sutures at the aneurysm neck causes healing and fibrotic response, making it difficult to analyze healing after coil embolization. The canine venous pouch model, though widely used, cannot simulate the histologic and hemodynamic characteristics of humans. 7 The swine model of intracranial aneurysms shows progressive occlusion and complete healing of aneurysms. 8 This outcome makes the model irrelevant for testing aneurysm recurrence rates and the long-term efficiency of devices. In contrast, the rabbit aneurysm model has some advantages compared with clinical tissue. For instance, excising the aneurysm sac during the operation results in small segments of the aneurysm dome, while the rabbit model provides the opportunity to study the entire sac. 9 Moreover, the rabbit model can be used to investigate the progressive wall degeneration of the aneurysm at multiple time points. 9 Furthermore, the elastase-induced model has shown a similarity to the human intracranial aneurysm regarding geometric and hemodynamic aspects, including anatomy, size, long-term patency, and molecular characteristics. 7 Nevertheless, as for all animal models, this model cannot account for the complex pathophysiology of human aneurysms, such as genetic predisposition, previous aneurysm history, comorbidities, wall inflammation, and individual lifestyle differences.
Typically, the elastase-induced aneurysm is created at the origin of right common carotid artery (RCCA) using a combination of open and endovascular techniques. 10 Other modifications such as adjusting the position of the ligation, adjusting the position of the inflated balloon, injuring the aneurysm neck, and using the left common carotid artery (CCA) instead of the RCCA have been proposed as techniques to alter the volume, neck size, and configuration of the aneurysm, respectively. 7,[11][12][13][14] This model has been extensively used to study the healing response, occlusion rates, and recanalization rates after coil embolization. However, data are limited about the recurrence rate of aneurysms after platinum coil embolization in the rabbit elastase-induced aneurysm model. Therefore, the objective of this study was to evaluate the aneurysm recurrence rate in association with platinum coils in this aneurysm model.

Rabbit Aneurysm Creation
Some of the rabbits used in this study were initially used as part of other investigations, in which we studied aneurysm healing mechanisms, 15 developed a histologic healing scale, 16 analyzed the relationship between aneurysm volume and healing, 17 and compared the occlusion rates of platinum and modified coils. 17,18 However, the original investigations were unrelated to this study. For this study, saccular aneurysms were created as described by Altes et al. 10 By means of a sterile surgical technique, the RCCA was exposed to create a 1-to 2-mm beveled arteriotomy, and a 5F sheath was inserted retrograde in the midportion of the RCCA. Through this sheath, a 3F Fogarty balloon (Baxter Healthcare) was pushed to the origin of the RCCA and inflated to cause occlusion. Elastase (100 U/mL) mixed with equal amounts of iodinated contrast was intubated for 20 minutes, after which the balloon was deflated, the sheath was removed, and the RCCA was ligated. A 4-0 running Vicryl suture (Ethicon) was used to close the skin before sending the rabbits to recovery.

Embolization Procedure
The embolization procedure was performed as described previously. 18 Animals were anesthetized with the same procedure used during aneurysm creation. Under sterile conditions, the right common femoral artery was surgically exposed to place a 5F sheath and administer an injection of 500 U of heparin. Using the coaxial technique with continuous flushing with a heparin and normal saline solution, we advanced a 2 marker microcatheter into the aneurysm cavity. Radiopaque sizing devices were used to assess the size of the aneurysm during the 2D DSA. The aneurysm was embolized with $1 coil, depending on aneurysm diameter, and packing density was calculated as described in Herting et al. 18 All the aneurysms were embolized as densely as possible using bare conventional platinum coils. A final control DSA was performed after coil placement and embolization, followed by removal of the catheters and sheath, ligation of femoral artery, and incision closure using a 4-0 Vicryl suture. Aneurysm occlusion was evaluated per the Raymond-Roy method: class 1, complete occlusion; class 2, near-complete occlusion; class 3, incomplete occlusion. 19 We performed this evaluation on 2 occasions; postoperative and before sacrifice. We defined recanalization as any increased aneurysm filling in the follow-up angiography compared with the postoperative 2D DSA result. In contrast, recurrence was defined as a recurring or larger persistent filling defect on the follow-up angiography studies than the defect identified at the initial posttreatment and imaging (Figs 1 and 2).

Animal Sacrifice and Tissue Harvest
Angiographic follow-up was performed 15 days (n = 23), 1 month (n = 100), 1.5-2 months (n = 9), 3 months (n = 8), 4 months (n = 10), and 6-12 months (n = 33) after embolization. DSA was performed after deeply anesthetizing the animals, and aneurysm occlusion was evaluated as described before: class 1, complete occlusion; class 2, near-complete occlusion; and class 3, incomplete occlusion. 19 As described earlier, to determine the durability of the embolization posttreatment and follow-up, we categorized the angiograms into 3 categories: stable, recurrent, and progressive aneurysm. 17,18 A lethal injection of pentobarbital was used to euthanize animals and harvest the aneurysms and parent arteries. The tissue samples were immediately fixed in 10% neutral buffered formalin.

Histologic Evaluation
Harvested tissue samples were processed as described by Dai et al. 15 Samples were dehydrated in increasing concentrations of alcohol, followed by clearing with xylene. Specimens were embedded in paraffin blocks and sectioned in a coronal orientation at 1000mm intervals using an IsoMet Low Speed saw (NCI MICRO).
Metallic coil fragments were removed under a dissecting microscope before reembedding sections in paraffin blocks. These blocks were then sectioned at 4-mm intervals using a microtome with disposable blades. The sections were floated in a 42°C water bath, mounted on Superfrost Plus slides (Cardinal Health), and dried overnight in an oven at 37°C. Slides were deparaffinized and hydrated in water before staining with H&E. An ordinal grading system was used to evaluate histologic healing as described earlier. 18 The total score was calculated by adding together the neck average, microcompaction score, and healing score.

Statistical Analysis
Categoric variables were presented as frequencies and percentages, with x 2 tests (or the Fisher exact test) used for testing the difference. Continuous variables were expressed as means (SD), with testing differences evaluated using a t test or Mann-Whitney U test based on the data distribution (normally distributed or not). Logistic regression was used to identify any possible predictors of aneurysm recurrence and occlusion. Regression results were expressed as ORs and 95% CIs. Receiver operating characteristic curves were also generated. The area under the curve was calculated; building the associated receiver operating characteristic curve to provide aggregate values of significant predictors correctly classified the occlusion status of each rabbit. All data were analyzed using R statistical and computing software (http://www.r-project. org/software), Version 4.1.1, using the "rcmdr" and "glm2" packages. P , .05 was considered significant for all statistical tests.

Aneurysm Characteristics
A total of 183 rabbits were included in this study (aneurysm characteristics are shown in Table 1), with a mean follow-up point of 2.3 (SD, 2.4) months, and balloon-assisted coiling was used in 63.9% (117/183) of aneurysms. Angiography before sacrifice showed complete occlusion in 79.8% of the subjects, while nearcomplete and incomplete occlusions were found in 11.5% and  8.7% of the subjects, respectively. Comparing posttreatment angiographic occlusion with the presacrifice angiography showed a stable course in 79.8% of the subjects (146/183), recurrence in 15.3% (28/183), and a progressive course in 4.9%. The mean packing density was 28.5 (SD, 14.6), and the average histologic healing score was 5.5 (SD, 2.5). The recurrence rate was 7.7% (9/ 117) when a balloon was used compared with 28.8% (19/66) when a balloon was not used. The presence or absence of balloon-assisted coiling was a significant predictor of different outcomes ( Table 2).

Predictors of Aneurysm Recurrence
Univariate and multivariate analyses of the potential predictors of the recurrence risk were performed.  Table 3).

Packing Density Cutoff Values
Packing density cutoff values for predicting occlusion were obtained after analyzing the receiver operating characteristic

DISCUSSION
The aneurysm recurrence rate observed in this study (15.3%) is similar to those reported in multiple analyses of aneurysm recurrence rates in humans (7%-27%). [20][21][22][23][24][25][26] We also observed that the rate of aneurysm recurrence was significantly reduced in procedures with balloon-assisted coiling and in aneurysms showing complete occlusion immediately after coil embolization. In this rabbit elastase-induced aneurysm model, packing density and aneurysm volume were weak predictors of aneurysm recurrence; however, the packing density was a good predictor of the occlusion rate. Previous effort had been made to compare the histologic changes in human cerebral aneurysms with the findings in animal models. A retrospective histopathologic evaluation of 27 elastase-induced aneurysms in rabbits was conducted, 9 matching the findings to the 4-point scale described by Frösen et al. 27 The authors found similarities between the 2 models in terms of underlying aneurysm wall degeneration mechanisms such as lack of an intact elastic lamina, loss of the endothelium, and hypocellular aneurysm walls. 9 In the elastase-induced model, the progression and distribution of the histologic subtypes of the aneurysm wall were similar to those previously described in the Frösen et al model. Aneurysm recurrence is a significant limitation of endovascular coiling procedures, with recurrence rates reported as high as 50%. [20][21][22][28][29][30] Additionally, large aneurysms and wide-neck aneurysms have been identified as significant risk factors for early recurrence and are also vulnerable to early recurrence. 31 In a single-center retrospective study, complete aneurysm occlusion was obtained in 31.7% of patients who underwent endovascular coiling procedures, and at #24 months, aneurysm recurrence occurred in 35.9% of this patient population. 32 The study-level recurrence rate of the present study is comparable with that found in the human study, thus indicating the utility of the rabbit elastase model to mimic what is found in humans.
Aneurysm recurrence rates after coil embolization have been associated with different factors in humans. Aneurysms with a small neck have a lower recurrence rate compared with those with wider necks. 21 Similarly, smaller aneurysms have a lower recurrence rate compared with large or giant aneurysms. 21,22,33 Furthermore, the degree of aneurysm occlusion after treatment has also been related to recurrence. Aneurysms with initial complete occlusion were less likely to recur at follow-up. 22 Our observation of a zero recurrence rate in aneurysms with complete occlusion is in line with this finding.
Aneurysm recurrence may occur through multiple mechanisms. Lower packing density or aneurysm growth may lead to coil compaction, 34 affecting histologic healing and subsequently causing aneurysm recanalization. In clinical settings, a large aneurysm volume is associated with low packing density and higher compaction rates. It is reported that a packing density of 24% is required to prevent compaction in aneurysms with a volume of ,600 mm. 3,35 Our analysis showed that aneurysm volume and packing density were weak predictors of aneurysm recurrence. This result may be due to specific differences found in this model and to our embolization procedure. While it is known that the range in values of hemodynamic factors such as pressure, oscillatory shear index, and wall shear stress found in the rabbit elastase-induced aneurysm model is similar to that seen in humans, 36 the model is also known for its aneurysm patency. One report observed no changes in aneurysm geometry during 24 months. 37 The mean volume of aneurysms created in this study was less than that found in humans, 35 while the mean packing density was higher than 24%. A higher packing density has been associated with better histologic healing in the rabbit elastase aneurysm model. 38 This model also showed a mild biologic response, with studies reporting poor healing with the formation of loose connective tissue, the absence of contractile cells, and a lack of collagen deposition. 15,39,40 In this rabbit elastase aneurysm model, the curvature of the parent vessel causes substantial inertia-driven flow, similar to that in humans, which may also contribute to aneurysm growth and recurrence. 41 The development of more efficient endovascular devices for the treatment of intracranial aneurysms relies on preclinical testing in animal models. The rabbit aneurysm model has been considered a criterion standard for preclinical testing of various neurointerventional devices. 17,[42][43][44][45] Our results show that the aneurysm occlusion rates in rabbit aneurysms are comparable with those of human aneurysms following the use of endoluminal and intrasaccular flow-diverting devices, 45,46 making this an ideal model for testing endovascular devices.

Limitations
Our study has limitations. The follow-up times in this study were limited to 1 year, which should be extended in future studies. Additionally, this is an extracranial aneurysm model with thick aneurysm walls, making it difficult to assess potential complications that may arise in treating human intracranial aneurysms. 7 Moreover, our model has the inherent limitation of animal models, with their inability to account for all factors involved in the human complex pathophysiology, including various cellular phenotypes in the aneurysm wall. Although the coils were all of the same type, they were not the same brand and the operator was not the same in all cases, possibly introducing some bias. Finally, Raymond et al 22 demonstrated that almost half of all recurrences were present by 6 months after coiling. Unfortunately, typical follow-up imaging of patients would be done at 3-6 months after coiling, but there is not much literature available to compare humans versus rabbits. We believe that evaluating the aneurysm occlusion stability and recurrence at early (,1 month), subchronic (1-3 months), and chronic (.3 months) stages would provide valuable insight into the timing of the recurrence.

CONCLUSIONS
The rabbit elastase aneurysm model may be a mosaic piece in the evaluation process of aneurysm treatments and may mimic aneurysm recurrence rates in humans. However, a comparative study against a human sample is necessary to confirm this finding.