Variations in Origin of Arteries Supplying the Brain in Rabbit and Their Impact on Total Cerebral Ischemia

1.1 Laboratory animals Animal testing, also known as animal experimentation, is the use of non-human animals in experiments. Worldwide it is estimated that the number of vertebrate animals—from zebrafish to non-human primates—ranges from the tens of millions to more than 100 million used annually (Cohn, 2010). The number of mice and rats used in the United States alone in 2001 was 80 million. Most animals are euthanized after being used in an experiment (Carbone, 2004). It includes pure research such as genetics, developmental biology, behavioral studies, as well as applied research such as biomedical research, xenotransplantation, drug testing and toxicology tests, including cosmetics testing. Animals are also used for education, breeding and defense research. The practice is regulated to various degrees in different countries. The earliest references to animal testing are found in the writings of the Greeks in the 2nd and 4th centuries BCE. Aristotle (384–322 BCE) and Erasistratus (304–258 BCE) were among the first to perform experiments on living animals (Cohen & Loew, 1984). The ability of humans to change the genetics of animals took a large step forwards in 1974 when Rudolf Jaenisch was able to produce the first transgenic mammal, by integrating DNA from the SV40 virus into the genome of mice (Jaenisch & Mintz, 1974). This genetic research progressed rapidly and in 1996 Dolly the sheep was born, the first mammal to be cloned from an adult cell (Wilmut et al., 1997). Toxicology testing became important in the 20th century. In the 19th century laws regulating drugs were more relaxed. For example, in the U.S. the government could only ban a drug after a company had been prosecuted for selling products that harmed customers. In the 1960s, in reaction to the Thalidomide tragedy, further laws were passed requiring safety testing on pregnant animals before a drug can be sold (Burkholz, 1997). Albino rabbits are used in eye irritancy tests because rabbits have less tear flow than other animals and the lack of eye pigment in albinos make the effects easier to visualize. Rabbits are also frequently used for the production of polyclonal antibodies.


Anatomy of the arteries supplying the brain in the rabbit 1.3.1 Aorta ascendens
The aorta ascendens arises almost linearly and dorsally in the midline.It extends from the cranial margin of the second rib cartilage to the cranial margin of the first rib cartilage.At first runs inside the pericard, left to beginning of the pulmonary trunk, to the left and cranially to the dorsal part of the right auricle and to the left and cranially to the right ventricle.Then it runs inside the thymus dorsally to the left side of the v. cava cranialis dextra (vena) and continues to the arcus aortae (Nejedlý, 1965).

Arcus aortae
The arcus aortae is running from the right to the left transversally and also a little caudally from the point of the second thoracic vertebra.Its dorsal wall is convex, the ventral wall is concave.It lies to the left from the v. cava cranialis dextra.The left part turns around the bronchus principalis sinister and ends on the left side of the third thoracic vertebra behind the v. cava cranialis sinistra.Very close to the midline of the body from its arise to the right the truncus brachiocephalicus and to the left the a. subclavia sinistra (arteria) (Popesko et al., 1990; Fig. 1).The truncus brachiocephalicus, the a. carotis communis sinistra and the a. subclavia dextra as branches of the arcus aortae were described by Nejedlý (1965).Nellie (1930) described the a. subclavia dextra and the a. subclavia sinistra as branches of the arcus Variations in Origin of Arteries Supplying the Brain in Rabbit and Their Impacton Total Cerebral Ischemia 67 aortae.Ding (2006) found the origin of the a. subclavia dextra in 1.5% of cases and the origin of the a. carotis communis sinistra in 4% of cases from the arcus aortae.White (1893) by studying 700 rabbits found in one case the a. carotis communis dextra et sinistra and the a. subclavia dextra et sinistra as independent branches of the arcus aortae.

Arteria carotis communis
In the neck region the a. carotis communis is covered by m. sternohyoideus (musculus), more cranially also by the m.sternothyroideus.It lies on the lateral surface of the trachea.The a. carotis communis lies dorsally to the esophagus, then laterally to the larynx.It reaches the maxillar angle laterally from the cranial end of the m.sternohyoideus and under the parotid gland.In this area it divides into the a. carotis interna and a. carotis externa (Nejedlý, 1965).

Arteria carotis interna
The a. carotis interna as poorly developed artery it arises by the division of the a. carotis communis into the a. carotis externa and the a. carotis interna.From the a. carotis externa it is divided by m. styloglossus and m. stylopharyngeus.It is running dorsally on the medial side of the bulla tympanica ossis temporalis.It enters the canalis caroticus ossis temporalis and inside this canal continues into the skull cavity.It is running rostrally on the medial surface of the n.trigeminus (nervus) and on the lateral surface of the corpus ossis basisphenoidalis is directed into the sulcus caroticus.It turns dorsally on the medial surface on the place of the entrance of the n.oculomotorius into the fissura orbitalis.It crosses the n.oculomotorius to the right.In this way it makes three bends.After this it enters the ventral end of the canalis caroticus.Here it gives off the a. communicans caudalis and a. ophtalmica dorsalis and subsequently is divided into the a. carebri rostralis and a. cerebri media (Nejedlý, 1965).

Arteria vertebralis
The a. vertebralis enters the foramen transversarium of the sixth cervical vertebra.It gives off rr. musculares (rami) and rr.spinales.It continues inside the canalis transversarius of the cervical vertebrae cranially.It passes through the foramen tranversarium of the atlas, courses medially, then cranially and a little dorsally.It runs through the foramen vertebrale laterale of the atlas and gives off the a. spinalis dorsalis and ventralis.After this it penetrates the dura mater and continues to the cranial part of the medulla oblongata.On the caudal margin of the dorsal surface of the pars basilaris ossis occipitalis it is fused together with the contralateral a. vertebralis.This fusion forms the a. basilaris (Fig. 3). A. basilaris continues rostrally on the ventral surface of the medulla oblongata and pons.This artery gives off some branches which praticipate on the formation of the circle of Willis and by this way it participates on the arterial supplying of the brain (Nejedlý, 1965).(Popesko et al., 1990) f. A. cerebri rostralis is the continuation of the a. carotis interna after the origin of the a.
cerebri media.It passes to the rostral portion of the ventral surface of the cerebral hemisphere and to the olfactory bulb.

www.intechopen.com
Variations in Origin of Arteries Supplying the Brain in Rabbit and Their Impacton Total Cerebral Ischemia 71 The a. cerebri rostralis unites with that of the other side to form a short common trunk between the hemispheres, which redivides into the paired vessels supplying the medial surfaces.A complete anastomic loop is thus formed round the hypothalamus by the a. carotis interna, a. cerebri rostralis, a. communicans caudalis and a. cerebri caudalis.This is the circle of Willis or the circulus arteriosus cerebri (Popesko et al., 1990).Nejedlý (1965) described the a. communicans rostralis as a connection between the bilateral aa.cerebri rostrales.

Rabbit as experimental animal of the brain ischemia
Over 20,000 rabbits were used for animal testing only in the UK in 2004.Examples include restricting blood flow to the brain to induce cerebral ischemia (Tolwani et al., 1999).This is most often carried out by ligation of major vessels, e.g. the truncus brachiocephalicus and the a. subclavia sinistra, in their place of origin (Hossmann 1998;Iwama et al., 2000;Pluta 1987).Harukuni and Bhardwaj (2006) reported ligation of the truncus brachiocephalicus and the a. subclavia sinistra as one way to induce total cerebral ischemia.Ischemia within the arteries branching from the vertebral arteries i n t h e b a c k o f t h e b r a i n m a y r e s u l t i n symptoms such as dizziness, vertigo, double vision, or weakness on both sides of the body.
Other symptoms include, difficulty speaking, slurred speech and the loss of coordination (Beers et al., 2003).The aim of this study was to verify whether experimentally induced total cerebral ischemia in rabbits actually corresponds to total ischemia on the basis of the origin of certain vessels.We observed morphological variations in the origin and course of the arteries supplying the brain with blood in the rabbit.

Experimental animals
The study was carried out on 50 adult (age=140 days) New Zealand white rabbits (breed HY+), females (n=25) and males (n=25) of weight range 2.5-3 kg in an accredited experimental laboratory at the University of Veterinary Medicine in Kosice, Slovak Republic.The animals were kept in cages under standard conditions (temperature 15-20 °C, relative humidity 45 %, 12 hours light period) and fed granular mixed feed (O-10 NORM TYP, Spišské krmné zmesi, Spišské Vlachy, Slovak Republic).Drinking water was provided ad libitum.

Surgical preparation of the rabbit
The animals were injected intravenously with heparine (50, 000 UI/kg) 30 minutes before they were sacrified with intravenous injection of Embutramide (T-61, 0.3 mL/kg).The skin was subsequently removed as far as possible to prevent it from sticking to the corrosive cast in the maceration process.The thoracic cavity was opened from the left side by removing of the ribs.After the opening of the pericardial cavity a ligature was introduced to the ascending aorta.The aorta was cannulated through the left ventricle.The perfusion started after the fixation of the cannula in the ascending aorta with the ligature.The right vestibule was opened to lower the pressure in the vessels to ensure good injection.The vascular network was manually perfused through the fixed cannula in the ascending aorta for approximately 15-20 minutes with 2.5-3 l of warm (37 °C), 0.9 % NaOH in 0.01 M phosphate, pH 7.3 (Hossler & Monson, 1995).

Preparation of the casting medium
The red pigment was added to the Base solution A prior to mixing the catalyst and promoter.The pigment was added in the amount of 5 %.It was mixed and divided into two equal parts (each part=25 mL).To the first half Catalyst in amount of 12 mL was added and mixed.To the second half Promoter C in amount of 12 drops was added and mixed.Then these two parts were mixed together.

Application of the casting medium
The arterial network was filled with the casting medium manually through the same cannula inserted in the ascending aorta.Adequate filling was determined by the visualization of an even distribution of the casting medium (red) throughout the superficial vessels of the body.After the vascular casting is complete, the animals must not be manipulated for at least 30 minutes and then must be submersed in water at a temperature ranging from 40 °C to 60 °C for a period of 24 hours for full polymerization of the casting medium (Lametschwandtner et al., 1990).

Corrosion
The corrosion as the dissolution of tissues surrounding the cast was performed by potassium hydroxide (KOH) at the concentration of solution 2-4 % for a period of 2 days.
For the corrosion to be faster, the solution must remain at a constant temperature of 40 °C (Lametschwandtner et al., 1990).The solution for the corrosion was changed every 12 hours.After the surrounding tissue was dissolved vascular castings were rinsed in running water for removing the rests of the soft tissues.Specimens were dried at the room temperature by air exposure (Flešárová et al., 2003).

Variations in origin 3.1.1 A. carotis communis
Vascular corrosion cast of the rabbit aortic arch displaying the origin of the truncus brachiocephalicus and the a. subclavia sinistra from the arcus aortae in 92 % of cases (46 animals).a. Arteria.Ventral view.Macroscopic image Vascular corrosion cast of the rabbit aortic arch displaying the origin of the truncus brachiocephalicus, the a. subclavia sinistra and the a. carotis communis sinistra from the arcus aortae in 6 % (3 animals).a. Arteria.Ventral view.Macroscopic image In 92 % of cases (46 animals) the a. carotis communis sinistra originated as the first branch from the truncus brachiocephalicus.The a. carotis communis dextra arise together with the a. subclavia dextra by terminal division of truncus brachiocephalicus (Fig. 5).The a. carotis communis sinistra originated from arcus aortae in 6 % (3 animals; Fig. 6).

A. vertebralis
In 86 % of cases (43 animals) the a. vertebralis sinistra originated directly from the a. subclavia sinistra (Fig. 8) in 10 % of cases (5 animals) it originated from the arcus aortae as an independent branch (Fig. 9) and in 4 % of cases (2 animals) it arose from the arcus aortae as a common trunk with the a. scapularis descendens.The a. vertebralis dextra originated from the a. subclavia dextra in 98 % (49 animals) of cases.In that case we observed two aa.vertebralis dextrae (arteriae) with two different origins.The a. vertebralis dextra I originated from the a. subclavia dextra and the a. vertebralis dextra II arose from the common trunk with the a. cervicalis superficialis dextra that originated from the a. carotis communis dextra.After a short distance, they merged between the fifth and sixth cervical vertebrae into a single a. vertebralis dextra, which then entered the canalis transversarius at the level of the fifth cervical vertebra (Fig. 10).In summary, the origin of both aa.vertebrales varied in 16 % of cases (8 animals).In 8 % (4 animals) we found a bypass between the a. vertebralis sinistra and the a. basilaris (Fig. 11).This a. basilaris was also formed by the fusion of the a. vertebralis dextra et sinistra.Vascular corrosion cast of the cephalic and neck region displaying the origin of the a. carotis interna from the common trunk with the a. occipitalis in 6 % (3 animals).a. Arteria.Caudoventral view.Macroscopic image In 94 % (47 animals) the a. carotis interna arised by terminal division of the a. carotis communis together with the a. carotis externa (Fig. 12).In 6 % (3 animals) the a. carotis interna originated from a common trunk with the a. occipitalis (Fig. 13).The trunk is a branch of the a. carotis communis.

The arteries of the brain
In 40 % (20 animals) the a. cerebelli caudalis dextra et sinistra originated at the same level (Fig. 14).In 40 % (20 animals) the a. cerebelli caudalis dextra originated from the a. basilaris more rostrally than the a. cerebelli caudalis sinistra (Fig. 15).In 20 % (10 animals) the a. cerebelli caudalis sinistra originated more rostrally than the a. cerebelli caudalis dextra.The a. cerebri caudalis dextra et sinistra originated from the a. basilaris at the same level in all studied animals (100 %).The a. cerebri media dextra et sinistra originated from the a. carotis interna at the same level in all studied animals (100 %).The a. cerebri rostralis dextra et sinistra as the direct continuation from the a. carotis interna originated at the same level in all studied animals (100 %).These all arteries were divided into the r.rostralis and r. caudalis.In 10 % (5 animals) the a. cerebri caudalis sinistra was divided into the r.rostralis, r. medius and r. caudalis.

carotis communis
In 92 % of cases (46 animals) the a. carotis communis sinistra originated as first branch from the truncus brachiocephalicus.The a. carotis communis dextra arise together with the a. subclavia dextra by the terminal division of the truncus brachiocephalicus.Popesko et al. (1990) described a. carotis communis dextra et sinistra as branches arising from truncus bicaroticus by its terminal division.Truncus bicaroticus was described as the direct continuation of the truncus brachiocephalicus.The a. carotis communis sinistra as branch of the arcus aortae in 4 % (2 animals) was also described by Ding (2006).By Nejedlý (1965) the origin of the a. carotis communis sinistra from the arcus aortae was described as a typical arrangement of branches of the arcus aortae.In 2 % (1 animal) originated from the arcus aortae the truncus bicaroticus.Nellie (1930) described this arrangement in all studied animals.White (1893) by studying 700 rabbits found in one case the a. carotis communis dextra et sinistra as independent branches of the arcus aortae.

A. subclavia
In 98 % (49 animals) the a. subclavia dextra arise together with the a. carotis communis dextra by the terminal division of the truncus brachiocephalicus.By some authors the a.
subclavia dextra was described as the first branch arising from the truncus brachiocephalicus (Popesko et al., 1990).Nellie (1930) described the a. subclavia dextra and the a. subclavia sinistra as branches of the arcus aortae.Ding (2006) found the origin of the a. subclavia dextra in 1.5% of cases from the arcus aortae and Nejedlý (1965) described it as typical arrangement of the branches of the arcus aortae.White (1893) by studying 700 pieces rabbits found in one case a. subclavia dextra et sinistra as independent branches of the arcus aortae.

A. vertebralis
Until now the scientific literature has cited almost exclusively the uniform origin of the a. vertebralis from the a. subclavia (Nejedlý, 1965;Popesko et al., 1990).It was not described independent origin from the arcus aortae or the doubled a. vertebralis like in our cases.In 8 % (4 animals) we found the bypass between the a. vertebralis sinistra and the a. basilaris at the place of fusion of the a. vertebralis dextra et sinistra to the a. basilaris.

A. carotis interna
We found the origin of the a. carotis interna and the a. carotis externa by the terminal division of the a. carotis communis in 92 % (46 animals).The same origin was described by Nejedlý (1965) and Popesko et al. (1990).But we found except this typical arrangement the origin of the a. carotis interna from the a. occipitalis in 8 % (4 animals).

The arteries of the brain
Until now the origin of the bilateral a. cerebri rostralis et caudalis and a. cerebelli rostralis et caudalis was described at the same level from the a. basilaris and the a. carotis interna (Nejedlý, 1965;Popesko et al., 1990).We found that in 40 % (20 animals) the a. cerebelli caudalis dextra originated from the a. basilaris more rostrally than the a. cerebelli caudalis sinistra.In 20 % (10 animals) the a. cerebelli caudalis sinistra originated more rostrally than the a. cerebelli caudalis dextra.These all arteries were divided into the r.rostralis and r. caudalis (Nejedlý, 1965;Popesko et al., 1990).In 10 % (5 animals) the a. cerebri caudalis sinistra was divided into the r.rostralis, r. medius and r. caudalis.The bilateral aa.cerebri rostrales are fused together.The same arrangement was described by Popesko et al. (1990).The a. communicans rostralis as a connection between bilateral aa.cerebri rostrales was described by Nejedlý (1965).

Conclusion
The effect of various chemical substances (Cantu & Hegsted, 1970) on the brain nerve tissue damaged by ischemia as well as various pathological and pathophysiological changes induced by the total cerebral ischemia in rabbits and other laboratory animals are the subject of many studies (Ishiyama et al., 2010).
The place of origin of the truncus brachiocephalicus and the a. subclavia sinistra are most commonly used to induce the total cerebral ischemia by ligation (Hossmann 1998;Iwama et al., 2000;Pluta, 1987).Harukuni and Bhardwaj (2006) present also the ligation of the truncus brachiocephalicus and the a. subclavia sinistra as a possible way to induce the total cerebral ischemia.The question is, whether this method of induction of the total cerebral ischemia is correct.
The variations in the origin of arteries supplying the brain which we found point to the possibility of induction only a partial brain ischemia in a given set of animals.The probability of causing the partial brain ischemia may be the same as the percentage of the occurrence of found variations.One way to avoid obtaining of distorted results is the ligation of the arteries before their entering to the target organ, in this case to the brain.These arteries can be the a. basilaris or the a. carotis interna.The best results probably would have been achieved by ligation of the arteries on the ventral surface of the brain that are directly involved in the blood supply of the nerve tissue.However, this method is time consuming and surgically very difficult in.
Another possibility is the detailed preparation of arteries in the place of their origin to avoid possible variations.
With this work we tried to emphasize the need for more detailed knowledge of the circulatory system of the rabbit, which is one of the ways to achieve more objective results also in a smaller number of animals used in experiments.

Fig. 3 .
Fig. 3. Corrosion cast of the vertebral arteries A. vertebralis dextra et sinistra located inside canalis transversarius of the cervical vertebrae.The fusion of the bilateral aa.vertebrales to the a. basilaris.a. Arteria.Dorsal view

Fig. 5 .
Fig. 5. Aortic arch of the rabbit without variations

Fig. 7 .
Fig. 7. Aortic arch of the rabbit with variation in origin of a. subclavia dextra Vascular corrosion cast of the rabbit aortic arch displaying the origin of the truncus bicaroticus, the a. subclavia dextra and the a. subclavia sinistra from the arcus aortae in 2 % (1 animal).a. Arteria.Dorsolateral view.Macroscopic image

Fig. 8 .
Fig. 8. A. vertebralis with its typical origin Vascular corrosion cast displaying the origin of the a. vertebralis dextra et sinistra from the a. subclavia dextra et sinistra in 86 % of cases (43 animals).a. Arteria.Ventral view.Macroscopic image

Fig. 15 .
Fig. 15.Variation in arrangement of a. cerebelli caudalis Vascular corrosion cast.The a. cerebelli rostralis dextra et sinistra originated from the a. basilaris at the same level in all studied animals (100 %).Dorsal view.Macroscopic image