**2. Selection of the animal species**

#### **2.1. Concept of experimental animal**

The terms "laboratory animal" or "experimental animal" are somewhat inappropriate because in theory, any animal can be used in laboratory experiments. Nevertheless, both terms are frequently used in scientific literature to refer to animals exhibiting (natural or induced) diseases in which the mechanisms are similar to human diseases.

#### **2.2. Types of experimental models with animals**

Experimental models with animals are classified as: 1) induced, 2) spontaneous, 3) negative and 4) orphan[18]. In the induced animal models, the investigated condition is induced experimentally, which can be highly advantageous because these models allow for free selection of the animal species, for example, intake of beta-aminopropionitrile combined with arterial hypertension induces intracranial aneurysm formation in rats [19]. Induced animal models are very important in the development of novel surgical procedures, to assess the viability of procedures and their physiological consequences, and in therapeutic assays, for instance, the surgical creation of aneurysms on the lateral wall of the common carotid artery of dogs [20] and pigs [21]. In spontaneous models, the investigated disease occurs naturally, such as with prostatic hypertrophy in dogs and some diseases in animals with genetic mutations. The spontaneous occurrence of intracranial saccular aneurysms in animals is rare. Negative models involve a particular disease that does not develop in a particular species and, thus, these models are ideal to study mechanisms of resistance or a lack of reactivity to a given stimulus. For example, rabbits do not develop gonorrhea and vultures do no exhibit neoplasms. In orphan models, a disease (or condition) that occurs naturally in non-human species is "adopted" when a similar human disease is identified at a later time (e.g., bovine spongiform encephalopathy, which is also known as mad cow disease) [18].

#### **2.3. Principles for animal selection**

Experimental animals should only be used when there are limitations to the research with humans. In therapeutic assays, the use of animals is mandatory and constitutes an essential phase of the preclinical testing of embolization devices or materials. In general, small animals are the most frequently used for research purposes; mice, rats, rabbits, and guinea pigs correspond to 90% of scientific studies [22]. Larger animals such as dogs [20], pigs [21], or monkeys [23] are also used for research purposes, albeit less frequently. Such diversity of species that exhibit different characteristics makes it difficult to select a particular species for experimental aneurysm production. Although there are no specific guidelines on how to perform such a selection, three general principles must be considered: 1) the type of animal that will be used, 2) the type of aneurysm one seeks to simulate, and 3) the aims of the study.

Regarding the animal type, researchers should be thoroughly aware of its biological characteristics, behavior, vascular anatomy, and phylogenetic similarity with humans.

44 Aneurysm

disease) [18].

study.

**2.3. Principles for animal selection** 

**2. Selection of the animal species** 

**2.1. Concept of experimental animal** 

**2.2. Types of experimental models with animals** 

The terms "laboratory animal" or "experimental animal" are somewhat inappropriate because in theory, any animal can be used in laboratory experiments. Nevertheless, both terms are frequently used in scientific literature to refer to animals exhibiting (natural or

Experimental models with animals are classified as: 1) induced, 2) spontaneous, 3) negative and 4) orphan[18]. In the induced animal models, the investigated condition is induced experimentally, which can be highly advantageous because these models allow for free selection of the animal species, for example, intake of beta-aminopropionitrile combined with arterial hypertension induces intracranial aneurysm formation in rats [19]. Induced animal models are very important in the development of novel surgical procedures, to assess the viability of procedures and their physiological consequences, and in therapeutic assays, for instance, the surgical creation of aneurysms on the lateral wall of the common carotid artery of dogs [20] and pigs [21]. In spontaneous models, the investigated disease occurs naturally, such as with prostatic hypertrophy in dogs and some diseases in animals with genetic mutations. The spontaneous occurrence of intracranial saccular aneurysms in animals is rare. Negative models involve a particular disease that does not develop in a particular species and, thus, these models are ideal to study mechanisms of resistance or a lack of reactivity to a given stimulus. For example, rabbits do not develop gonorrhea and vultures do no exhibit neoplasms. In orphan models, a disease (or condition) that occurs naturally in non-human species is "adopted" when a similar human disease is identified at a later time (e.g., bovine spongiform encephalopathy, which is also known as mad cow

Experimental animals should only be used when there are limitations to the research with humans. In therapeutic assays, the use of animals is mandatory and constitutes an essential phase of the preclinical testing of embolization devices or materials. In general, small animals are the most frequently used for research purposes; mice, rats, rabbits, and guinea pigs correspond to 90% of scientific studies [22]. Larger animals such as dogs [20], pigs [21], or monkeys [23] are also used for research purposes, albeit less frequently. Such diversity of species that exhibit different characteristics makes it difficult to select a particular species for experimental aneurysm production. Although there are no specific guidelines on how to perform such a selection, three general principles must be considered: 1) the type of animal that will be used, 2) the type of aneurysm one seeks to simulate, and 3) the aims of the

induced) diseases in which the mechanisms are similar to human diseases.

Among the biological characteristics, the size and metabolism of the animals exert a direct influence on the selection. Large animals are more difficult to handle and require more complex infrastructure (lodging, feeding, care, anesthesia, and specialized human resources), which increases the cost of research. In addition, size also influences the number of animals used in experiments. Thus, for ethical reasons, studies that use large animals such as dogs and monkeys restrict their number to the bare minimum needed to ensure the validity of the results. A reduced number of animals influences the statistical methods applied to the analysis, because small samples can reduce the statistical power of tests and lead researchers to infer inaccurate conclusions. In addition, the calculation of the minimum number of animals is difficult because unpredictable losses can also occur as a function of the initial training and pilot study.

With regard to metabolism, different animal species also exhibit different patterns of metabolic rate; for instance, the metabolism of rodents is often faster than that of humans. This metabolic power (also known as metabolic body weight) interferes with the effects of drugs on the organism, as well as with its processing, distribution across organic fluids and tissues, and modes of excretion. Thus, the calculation of experimental doses should be performed according to the metabolic weight rather than the absolute body weight of the animals. In surgical studies, different metabolic rates (influenced by factors such as age, gender, diet, and circadian rhythm) interfere with wound healing and regeneration of tissues and organs, thus encouraging researchers to learn the principles of veterinary anesthesia that correspond to the involved animals, the characteristics of the drugs that will be used, and more specifically, the potential interference of medications with the parameters analyzed in the study[18].

In addition to the biological characteristics, researchers must also be familiar with the intracranial and cervical arterial anatomy of each animal species, and the histology, diameters, flow patterns, and anastomoses of the vessels, because these are essential factors in the selection of the aneurysm construction technique.

The phylogenetic similarity between animals and humans is also important in species selection, but it does not suggest that the extrapolation of the results to humans will be reliable. For example, human immunodeficiency virus (HIV) does not induce immunodeficiency in monkeys, and thus, does not represent the ideal animal model to study acquired immunodeficiency syndrome (AIDS). Transgenic animals have been increasingly used in research studies; however, caution is needed because such animals might exhibit unknown disorders that may interfere with the extrapolation of the results to humans[18].

Once the animal model has been selected, the experiment performed, and the data selected, the stage of explaining the phenomena by means of induction begins. This process consists of verifying a particular fact and its adequation to a known general law. This mode of reasoning has inherent odds of error; thus, one must be cautious in the extrapolation of the

results of experiments performed with non-human species to humans. In other words, compounds that might be noxious to a given non-human species might be innocuous or even beneficial to humans. For example, penicillin is lethal for guinea pigs, but is well tolerated and even beneficial for humans. In addition, aspirin is teratogenic in cats, dogs, rats, guinea pigs, mice, and monkeys, but it is innocuous in pregnant women. Thalidomide is teratogenic in human beings and monkeys, but innocuous in rats and other species. Therefore, phylogenetic proximity is not a fully reliable measure of similarity between the physiological phenomena of animals and humans [18].

To reduce the odds of selecting an inappropriate animal model for a given experiment, the *multispecies approach* is recommended. At least two different species including nonrodents must be used in studies employing drugs, whereas the use of more than one animal species is rare in studies of surgical techniques. Accordingly, some animal species have become traditional standard models for specific surgical procedures. However, surgical studies focusing on the physiological features of a disease require more than one animal species, which despite its usefulness, does not ensure the absolute reliability of the extrapolation of the results from animals to humans [18].

Regarding the aneurysm model, a comprehensive awareness of the available models is required, in addition to their construction techniques, advantages and disadvantages, and more specifically, which features of human aneurysms one seeks to simulate, that is, their histological, geometric, physiopathological, and hemodynamic characteristics (e.g., ruptured or not, small, medium-sized, large or giant, with or without thrombus, on the lateral wall or at a bifurcation, high or low hemodynamic tension, etc.).

Finally, the aims of the study are essential in the selection of the animal species and the techniques that will be used in aneurysm construction, e.g., verification of the physiopathological mechanisms, therapeutic assays, creation of novel surgical/endovascular techniques, or training of doctors in these therapeutic modalities. Regarding the latter issue, medical training using animals is justified as training on humans exposes patients to medical error. Thus, practical training using animal models is indispensable for medical education because it contributes to the development of psychomotor skills and enables physicians to safely perform invasive techniques.

### **2.4. Main animal species used in the construction of experimental saccular aneurysms**

Despite all of the considerations above, the selection of the ideal animal species for studies on experimental saccular aneurysms is not yet well established. As spontaneous intracranial aneurysms rarely occur in animals, most studies employ induced models, which have the advantage of allowing for the free selection of species. Animals such as rats[19], rabbits[24], dogs[20], pigs[21], and monkeys[23] have been used in studies on physiopathology [25, 26], hemodynamics[27-31], and the training of surgical[32, 33] and endovascular techniques, in addition to the testing of embolization devices and new materials[21, 34-38]. In studies aimed at developing surgical/endovascular techniques, it is rare that more than one animal species is used in the same experimental model; therefore, there are no systematic comparative studies seeking to define which is the ideal animal species for the experimental production of intracranial saccular aneurysms. Nevertheless, in recent years, rabbits (*Oryctolagus cuniculus*) have been preferred for these studies because their coagulation system is very similar to that of humans. Rabbits are easy to handle, and the diameters of their extracranial carotid arteries are very similar to those of humans [39-44].

#### **2.5. Cervicocerebral vascular anatomy of rabbits**

46 Aneurysm

results of experiments performed with non-human species to humans. In other words, compounds that might be noxious to a given non-human species might be innocuous or even beneficial to humans. For example, penicillin is lethal for guinea pigs, but is well tolerated and even beneficial for humans. In addition, aspirin is teratogenic in cats, dogs, rats, guinea pigs, mice, and monkeys, but it is innocuous in pregnant women. Thalidomide is teratogenic in human beings and monkeys, but innocuous in rats and other species. Therefore, phylogenetic proximity is not a fully reliable measure of similarity between the

To reduce the odds of selecting an inappropriate animal model for a given experiment, the *multispecies approach* is recommended. At least two different species including nonrodents must be used in studies employing drugs, whereas the use of more than one animal species is rare in studies of surgical techniques. Accordingly, some animal species have become traditional standard models for specific surgical procedures. However, surgical studies focusing on the physiological features of a disease require more than one animal species, which despite its usefulness, does not ensure the absolute reliability of the

Regarding the aneurysm model, a comprehensive awareness of the available models is required, in addition to their construction techniques, advantages and disadvantages, and more specifically, which features of human aneurysms one seeks to simulate, that is, their histological, geometric, physiopathological, and hemodynamic characteristics (e.g., ruptured or not, small, medium-sized, large or giant, with or without thrombus, on the lateral wall or

Finally, the aims of the study are essential in the selection of the animal species and the techniques that will be used in aneurysm construction, e.g., verification of the physiopathological mechanisms, therapeutic assays, creation of novel surgical/endovascular techniques, or training of doctors in these therapeutic modalities. Regarding the latter issue, medical training using animals is justified as training on humans exposes patients to medical error. Thus, practical training using animal models is indispensable for medical education because it contributes to the development of psychomotor skills and enables

**2.4. Main animal species used in the construction of experimental saccular** 

Despite all of the considerations above, the selection of the ideal animal species for studies on experimental saccular aneurysms is not yet well established. As spontaneous intracranial aneurysms rarely occur in animals, most studies employ induced models, which have the advantage of allowing for the free selection of species. Animals such as rats[19], rabbits[24], dogs[20], pigs[21], and monkeys[23] have been used in studies on physiopathology [25, 26], hemodynamics[27-31], and the training of surgical[32, 33] and endovascular techniques, in addition to the testing of embolization devices and new materials[21, 34-38]. In studies aimed at developing surgical/endovascular techniques, it is rare that more than one animal

physiological phenomena of animals and humans [18].

extrapolation of the results from animals to humans [18].

at a bifurcation, high or low hemodynamic tension, etc.).

physicians to safely perform invasive techniques.

**aneurysms** 

Regarding the vascular anatomy of rabbits, knowledge of the cervicocerebral vessels and their connections is essential in the construction of experimental saccular aneurysms. Below, we present a summary of the cervical and intracranial vascular anatomy of rabbits together with their main anastomoses.

The innominate artery (3.5 mm in diameter) is the first branch of the aortic arch, and after running 6 mm, it divides into the right subclavian (2 mm in diameter) and right common carotid (2 mm of diameter) arteries. The left common carotid artery (2 mm in diameter) begins immediately next to or together with the innominate artery. The left subclavian artery (2 mm in diameter) is the last branch of the aortic arch, and it originates from the left vertebral (1 mm in diameter) and superficial cervical (1 mm in diameter) arteries[45]. In the second most frequent distribution type, the aortic arch can only be divided into three branches: the innominate, left common carotid, and left subclavian arteries. Lesser variations might also occur; for instance, the supreme intercostal and left vertebral arteries might originate directly from the aortic arch. The superior thyroid artery usually originates from the common carotid arteries; however, it emerges approximately between the 3rd and 6th tracheal rings and runs towards the thyroid gland, in some cases of only one common carotid artery[46]. Upon arriving at the isthmus, the superior thyroid artery divides into two branches: one ascending (cricothyroid branch) and the other descending (which runs inferiorly between the trachea and the esophagus). The bronchial branches stem from the right supreme intercostal and left common carotid arteries and lead to the tracheoesophageal branches, which run upwards between the trachea and the esophagus and anastomose with the descending branches of the superior thyroid artery[47] (figure 1). These branches rarely exhibit variations, and when they do occur, these variations are more common on the left side[48].

The common carotid artery (CCA) leads to only one branch, namely the thyroid artery, and immediately above it, the CCA divides into the internal and external carotid arteries. The main branches of the external carotid artery (ECA) are the occipital, lingual, external maxillary (facial), and anterior and posterior auricular arteries. Both the auricular and external maxillary arteries emerge separately or from a common trunk. At the level of the zygomatic arch, the ECA divides into the superficial temporal and transverse facial arteries and continues its course up to the pterygoid canal, where it divides into small branches to the posterior side of the orbit and originates the external ophthalmic artery, which in turn forms the lacrimal and frontal branches, and subsequently, the anastomose with the internal ophthalmic artery. The main branch of the internal maxillary artery is the middle meningeal artery. The intracranial internal carotid artery (ICA) divides into the ophthalmic arteries, cranial, and caudal branches. The cranial branch runs forward towards the uncus, where it divides into the anterior choroidal artery and middle cerebral artery (MCA) trunk, and then continues up to the chiasm, where it unites with the contralateral cranial branch to form a common anterior cerebral artery trunk that separates again at the level of the corpus callosum. The common anterior trunk originates from the lateral artery of the olfactory bulb, which leads to the ethmoidal branches of the cribiform plate. The MCA runs along the lateral cerebral sulcus and divides into the posterior ophthalmic artery, large posterior branch, and large anterior and middle branches, in addition to the small olfactory bulb branches. The caudal branch of the ICA supplies most of the blood flow of the basilar artery (BA) and leads to the following branches: posterior communicating artery, small medial geniculate body branches, large anterior quadrigeminal body, small branches of the posterior side of the uncus, and the posterior segment of the corpus callosum. The cerebellar artery might originate from the ending of the ICA or the BA and connects to several branches of the brainstem. The BA is formed by the fusion of the arteries of the first spinal nerves and divides (on the ventral surface of the trapezoid body) into two vessels that reunite at the upper margin of the pons. In addition, the BA gives small lateral branches, the cerebellar artery and the perforating branches. The arteries of the first spinal nerve then reunite at a lower level and form the ventral spinal artery[49].

**Figure 1.** Graphic representation of the visceral vascularization of the neck of rabbits. 1- superior laryngeal artery, 2- superior branches, 3- superior thyroid artery, 4- cricothyroid branch, 5- bronchial branch and 6- tracheoesophageal branch. Modified from Bugge, 1967[2].

Regarding the system of intracranial anastomoses in rabbits, the collateral circulation is very different from that of dogs. The internal maxillary artery originates from the orbital branches, which end at the ophthalmic branch and represents an insufficient anastomotic pathway. The anastomotic branches between the orbital and internal carotid arteries are too small or are absent. A small branch links together the ICA and BA before they unite at the circle. Finally, when an occlusion of the common carotid artery occurs, the supply of blood is provided by the contralateral ICA (**figure 2**)[3].

**Figure 2.** Graphic representation of the intracranial anastomosis system of rabbits. 1- common carotid artery, 2- internal carotid artery, 3- external carotid artery, 4- occipital artery, 5- orbital artery, 6 – internal ophthalmic artery and 7- vertebral artery. Modified from Chungcharoen, 1954[50].
