A Preliminary Study on the Functionality of the Carotid-Vertebral Anastomotic Artery in the Regulation of Blood Flow in the Giraffe ( Giraffa camelopardalis ) by Duplex Ultrasound Examination

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Introduction
Te order Artiodactyla is recognized as very successful ungulates inhabiting a wide array of diferent and often characteristically challenging environments [1]. Te girafe (Girafa camelopardalis) is not only challenged in its external environment but is also physiologically challenged. An extremely long neck, with the heart located 2 meters below the brain, high blood pressure of 200-400 mmHg [2][3][4], and long thin legs describe their unique build and highlight cardiovascular challenges faced by this species. Critical physiological issues result at this typical high blood pressure [2][3][4]. Continuous blood supply and subsequent oxygen supply to the brain, without any disruption in fow, are of utmost importance to prevent fainting and brain damage [1,[5][6][7]. Fainting is preceded by the dilation of blood vessels with the concomitant drop in blood pressure resulting in an inadequate fow of blood to the brain [8]. Tis is exacerbated by the fact that brain tissue is incapable of storing the fuel supply source, glucose [9]. Te gravitational force exerted on the 2 m blood column from the heart to the head, when the animal reaches down to drink, renders it susceptible to protrusion of a wall of an artery, also known as a brain aneurysm or stroke [10]. High blood pressure in the body, neck, and head is overwhelmed by even higher blood pressures in the lower legs [11]. Te combined infuence of high blood pressure and the extreme gravitational efect on the extended blood column from the heart to the legs should, under normal circumstances, be physiologically ideal for the development of oedema in the lower extremities [11]. With blood pressures in the legs, recorded well above 400 mmHg, oedema is expected [12]. Several studies have explored possible adaptations to the physiological challenges girafes face [11,[13][14][15]. One possible adaption to cerebral blood fow control is the unique carotid-vertebral arterial connection observed in girafes [15]. Te anastomosis between the two arterial pathways known to supply blood to the brain is signifcant when taken into consideration that the basilar artery in girafe is reduced in size and not functional with regard to cerebral blood supply [13,14]. Te functionality of this connection in girafes is unknown and not well researched. A study conducted on the efects of gravity on blood circulation in snakes indicated structural and functional countermeasures work in combination to adapt to body form [11]. We, therefore, aimed to confrm the location of the anastomotic arterial connection between the common carotid artery and vertebral artery in girafes and the functionality thereof. We also investigated the fow of blood within the anastomosis, during head movements from upright, to ground level, and back to a fully erect position. Few studies describe the unifed concept of how girafes successfully circulate blood, avoid fainting and blackouts, avert brain aneurysms, and prevent oedema in the legs.

Experimental Model and Subject Details.
Two mature girafe heads were obtained. No animals were specifcally sacrifced for use in this study. Unfortunately, these girafes were culled by the reserve management due to overstocking densities and food scarcity. We only collected biological samples during the culling and were not involved in the actual process at all. Most reserves and game ranches either sell, translocate, or cull the excess number of animals every year. Tis is considered a common and frequent practice in Southern Africa Wildlife Industry to limit herbivore numbers to exceed the actual carrying capacity and to prevent death due to starvation.
A third, living adult girafe male was used for the second experiment.
Experiment part 1: Two adult girafe (G. camelopardalis) heads n � 1 male and n � 1 female were obtained. Experiment part 2: One male girafe (G. camelopardalis) was used for this experiment. Te experiment was conducted on only one girafe due to the following factors: (1) High incidence of fatality of a girafe during chemical immobilization (2) Availability of a highly specialized ultrasound device suitable for use in the feld (3) Availability of human cardiac radiologists to conduct a feld operation Experiment part 3: Two adult girafes (G. camelopardalis) n � 1 male and n � 1 female were obtained. Tese are the same animals used in experiment 1.

Method Details
Experiment part 1: Te two girafe heads were obtained. Te specimens included the head and neck, which were severed at the position of the fourth cervical vertebra. Te vascular system was manually rinsed out with warm water. Te specimens were elevated with the nasal region pointing upwards and rinsed until water drainage ceased.
Two diferent mixtures were prepared for the arterial and venous systems, respectively. Barium sulphate (BaSO 4 ) (X-ray grade, Kyron Powder, Kyron, SA), Latex moulding rubber (A. Shak (Pty) Ltd, SA), and red and blue pigments were used. Arteries were colored red (Stamp ink, Ofce mate, SA) and veins were colored blue (Print ink, Treeline, SA) for diferential purposes. Te mixture used for arteries included BaSO 4 powder of 40 percent volume, 58 percent latex volume, and 2 percent volume of red ink. Te mixture used for veins included BaSO 4 powder of 20 percent volume, 78 percent latex volume, and 2 percent volume blue ink.
After fushing with water, the vascular system was infused with the variable latex mixtures by means of a pipe (10 mm PVC tubing), held 2 meters above the girafe head, thus creating a pressure gradient. Tubing was fxed with adhesives and ligatures. Te red latex mixture was infused into the common carotid artery. Te blue latex mixture was infused into the jugular vein. Following latex infusion, all arteries and veins were ligated with catgut (Catgut 3, Kyron, SA). Te specimen heads were placed at a 30°angle for 24 hours, allowing the latex to set. Specimens were stored frozen until computerized tomography (CT) scanning could be performed. Te heads were CT scanned at Netcare Kroon Hospital, Radiology Department, South Africa. Te CT scan was performed by a qualifed radiologist using a Siemens SOMATOM Emotion 6, CT scanner. Specimens were scanned with a resolution of 1.25/6x100, p0.7 slice thickness, 130 kV, and 79 mA. In both the heads, the arterial and venous systems were injected. Te General Electronic Computerised Tomography with associated software was used to take the CT scan illustrating the latex-flled arterial and venous system. A medically associated photoshop program (Photoshop version CS.6) allows the radiologist to remove, for instance, bone showing only soft tissue. In this case, the photo was manipulated to remove the venous system and enhance the arterial system. We used two different colorants in the latex, a typical red colorant was used for the arterial system and a blue colorant was used for the venous system. Tat made it possible afterward to only select one set of systems at a time using the photoshop program. With the help of Dr. Christopher Basu at the Royal Veterinary College of London, we used the Sketchfab software to digitally process the scans. Te Sketchfab software is a 3D program typically used in the medical world for explaining complex anatomical structures to teach people about physiology and anatomy. Following the CT scanning of specimens, girafe heads were manually dissected to expose the area where the anastomosis was located according to the CT scan results. Te anastomosis, a prominent artery, between the common carotid artery and the vertebral artery, was exposed and photographed with a Canon D5 camera, with 24-105 Canon lens. Experiment part 2: One male girafe (G. camelopardalis) was used for this experiment.
A young girafe bull aged 7-8 years was located and was immobilised with a trained and experienced girafe capture team for the cardiology radiologists to perform a sonar. Te girafe was darted in the rump, with a dart gun (Pneu-Dart, 389). Te dart contained a mixture of A3080 (Tiafentanil) and Stresnil (Azaperone). After 10 minutes, the typical immobilizing efect of the drugs was seen in the girafe, whereafter it became recumbent. Te ears and eyes were closed immediately to prevent stimulation by sight and sound from subsequently causing stress. A partial antagonist drug, M5050 (Diprenorphine), was used as the antidote against the immobilizing drug (A3080). Tis allows for the calm regaining of consciousness. Te blood pressure of the girafe is highly sensitive to the drugs used to immobilize the animal, and the drugs must, therefore, be reversed immediately after the animal becomes recumbent in order to prevent death [16]. Te girafe was held in a recumbent position, with the head and neck moved to simulate a head upright (Figure 1(a)) and head-down position (Figure 1 A SonoScape S8 Exp Ultrasound device, equipped with a 3.6-5 MHz convex probe, was used to determine the fow direction in the anastomotic artery between the common carotid artery and the vertebral artery. Te hair was shaved in all the areas where measurements were taken. Coupling gel was applied, and the probe was placed posterior to the sternocleidomastoid muscle. After locating the carotidvertebral anastomosis, the Doppler sample volume was placed in the anastomotic artery approximately 2 cm from its confuence with the carotid artery ( Figure 2). A Doppler waveform was obtained to determine the direction of fow in the anastomotic artery (away from or towards the carotid artery), whilst in a head upright as well as a head-down position. Te ultrasound device was also used to measure the diameter of the proximal and distal common carotid artery and the internal jugular vein in a head upright and headdown position.
After completion of the measurements, the girafe was given the full dose of antidote intravenously. Te ear and head covers were removed, and the animal regained complete consciousness. Experiment part 3: Samples were retrieved from the two girafes. Te neck of the specimens was severed at the position of the fourth cervical vertebra. Te neck was opened from this point along the length of the neck up to the connection between the seventh cervical and the frst thoracic vertebra, exposing the jugular vein and carotid artery. Te jugular vein and carotid artery were removed and manually rinsed out with warm water. Both were cut open to expose the lumen, to confrm the presence of valves along the entire length of the jugular vein and the absence of valves along the length of the carotid artery. Experiment three confrmed that speculations on possible arterial valves are not present [17].

Results
Experiment part 1: Te CT scans performed on both male and female girafe illustrated the position of the anastomotic artery, which provides a direct connection between the common carotid artery and the vertebral artery (Figure 3(a)). Te anastomotic artery is situated at the center of the atlas, where it is observed as a prominent vessel that branches of the common carotid artery. At the location where the anastomotic artery branches from the common carotid artery, it transits through a foramen, known as the alar fossa [11,15]. Two canals are evident within the alar fossa, with the medially situated, larger canal allowing a notable link from the anastomotic artery straight into the vertebral arteries (Figure 3(a)). In the girafe, it is evident that blood supply from the vertebral arteries does not contribute to the cerebral arterial circle, due to an underdeveloped basilar artery (Figure 3(b)) and that the vertebral arteries course within the vertebral column through the axis and atlas bones where they connect to the anastomotic artery.
Experiment part 2: Te duplex ultrasound examination showed blood fow direction during head upright and head-down positions. Te Doppler waveform with color fow images demonstrated a preferential blood fow in the anastomotic artery in the direction away from the vertebral artery towards the common carotid artery when the head is in an upright position (Figure 4(a)). Te blue color in the anastomotic artery and the Doppler waveform below the basis line indicate fow within the anastomotic artery, which is away from the sample volume. Tus, the fow direction in the anastomotic artery is away from the vertebral artery towards the common carotid artery. Te blood fow direction as observed on the ultrasound device is demonstrated in the illustrations of the dissected specimens where the common carotid artery, the anastomotic artery, and the vertebral artery are present (Figure 4(b)). Supplementary Figures 1(a) and 1(b) show additional illustrations of raw dissected specimens and specimens with red and blue colors added, respectively, to distinguish arteries and veins.
Te Doppler waveform with color fow images demonstrated a preferential fow in the anastomotic artery directed away from the common carotid artery towards the vertebral artery, in the head-down position ( Figure 5(a)). Te red color in the anastomotic artery and the Doppler waveform above the basis line indicate that fow is towards the sample volume. Tus, the fow direction within the anastomotic artery is away from the common carotid artery towards the vertebral artery. Te blood fow direction as observed on the ultrasound device is demonstrated in the illustrations of the dissected specimens where the common carotid artery, the anastomotic artery, and the vertebral artery are present ( Figure 5(b)). Tis equates to a summary confrmation of blood fow within the anastomotic artery by means of a CT scan, physical dissection of the anastomotic and its associated arteries, and the Doppler fow experiment. Te CT scan illustrates the potential for blood fow direction. Te physical dissection of latex-flled arteries proved the existence and possible blood fow direction within the International Journal of Zoology anastomotic artery in diferent head positions. Most importantly, the Doppler experiment provides evidence on the blood fow direction within the anastomotic artery in diferent head positions.
Experiment part 3: Te jugular vein in the girafe contains several valves throughout the length of the neck. However, no valves were observed anywhere along the length of the carotid artery throughout the length of the neck ( Figure 6). Blood fow can, therefore, occur bidirectionally in arteries, but in veins, it can only occur in a single direction due to the presence of valves.
Blood movement within the anastomotic artery is thus posture dependent, as illustrated diagrammatically in Figure 7. With posture changing to fully erect, the blood supply to the brain route entails blood fow from the vertebral artery through the anastomoses into the common carotid artery. Blood then follows the normal route via the maxillary artery supplying the rostral epidural rete mirabile with subsequent brain perfusion.   International Journal of Zoology Te blood fow direction associated with posture change where the head is at ground level is altered fowing from the common carotid artery into the vertebral artery. Possible brain damage associated with blood moving towards the head as a result of the force of gravity is prevented by the diversion in fow out of the common carotid artery via the anastomoses, into the vertebral artery.

Discussion
Te distinctive build of the girafe results in unique cardiovascular and circulatory challenges. Several aspects of individual challenges have evoked the interest of scientists worldwide. Most articles focus on a specifc structure as a single solution. Te information shared in this paper also specifcally discusses a single structure, but the authors believe that a group of mechanisms work in unison to allow the girafe to successfully overcome these physiological challenges.
An exceptional mechanism for cerebral blood fow control lay within the diversion of large blood volumes via the anastomosis characteristic of artiodactyls, between the common carotid artery and the vertebral artery [5,15,[18][19][20]. Tis anastomotic artery is also known as the alar artery. Te occurrence of blood volumes shunted away from the cranium provides the support of the carotidvertebral anastomosis that exerts a meaningful infuence during the movement of the girafe when in the upright International Journal of Zoology position and at ground level, in altering blood pressure [15,20]. In a girafe, blood fow to the head is described as uncontrolled, during the head-down position [20]. Is the fow uncontrolled or specifcally controlled by means of shunting blood rushing to the brain from the common carotid artery through the anastomosis into the vertebral artery? Furthermore, blood fow occurs bidirectionally in arteries and is not, as in veins, inhibited or directed towards a specifc direction, by valves [15]. When the girafe lifts its head, extracranial vasoconstriction is the mechanism, together with the movement of common carotid blood within the carotid-vertebral anastomosis towards the brain, that prohibits fainting [20]. Te anatomical structure of this anastomosis that is exceptionally well developed in girafes [19,21] in comparison to its presence in other artiodactyls can be a part of the list of structures that in conjunction aid in efective blood circulatory control in long-necked girafes. Similarly, a study showed that in Alpaca (Vicugna pacos), this anastomosis known as the alar artery is present [22]. Terefore, the anastomosis exists in girafes, camels, and alpacas, all animals with characteristically long necks. In a descriptive analysis of the cerebral arterial vascular system of the dromedary camel (Camelus dromedarius), the anastomotic artery called the occipital artery was also described as a notable vessel [18]. Te alar canal through which the anastomotic artery connects the two cerebral arterial pathways is a relatively short canal diagonally through the atlas wing acting as a link between the ventral and dorsal openings. Te anastomotic artery exits the common carotid artery passing into the ventral opening of the alar canal. Te course of the vertebral artery runs through the transverse foramen situated at the wing of the atlas following through the transverse canal. Within the alar canal, the connection with the vertebral artery occurs. Tis course is similar to dromedary camels [18] and is also evident in our study, on girafes. In camels, however, the vertebral arteries branch several times to meet the basilar artery, which contributes signifcantly to the cerebral arterial circle. In a girafe, the basilar artery is rudimentary and does not contribute to the supply of blood towards the cerebral arterial circle [13,15,[23][24][25][26]. Te functionality of the anastomotic artery between both long-necked dromedary camels and girafes shares similarities. However, the degree of importance of this anastomosis in the longer-necked girafe, lacking basilar contribution to cerebral supply, might be extensive in exerting control on blood movement to and from the brain during alteration of head position at head upright and head down. A study on dromedary camels described the internal carotid-rostral epidural rete mirabile joining, the anastomotic connection of the common carotid, and vertebral artery, as well as a third connection between the vertebral and basilar arteries-as diversions employing an essential part in both blood pressure and blood fow control mechanisms during head movements of long-necked dromedaries [18,27]. Removing the vertebral-basilar connection, our study also supports this idea in girafes to an even greater extent. Te connection of the common carotid artery and the vertebral artery in the girafe plays an essential role in blood pressure and blood fow control during the movement of the head from the head down, to the upright position and again to the head-down position. In combination with other structures, such as the rostral epidural rete mirabile, large blood volumes rushing to the brain, when the head moves down, are diverted from the common carotid artery towards the vertebral arteries, away from the cerebral supply. It has also been hypothesized although not yet proven that the rostral epidural rete mirabile meshwork ofers resistance to fow, due to its structure [5,[27][28][29] adding to the slowing down of the blood column rushing down with gravitational force, towards the brain. Furthermore, blood fow is directed from the vertebral arteries through the anastomotic artery, into the common carotid artery, supplying essential blood that can be moved quickly to the rostral epidural rete mirabile directly to the circle of Willis. Tis occurs at a time when a centripetal force causes the blood to fow downwards, away from the brain during head movement to an upright position. As previously mentioned, the vertebral veins run through the transverse foramina, closely associated with connective tissue and the back muscles, and run through the axis and atlas to connect to the anastomotic artery. Unlike the common carotid artery that expands when the head moves to a fully erect position, the tight connective tissue and bony structures enclosing the vertebral veins prevent expansion. Less efort is thus required from the heart to pump blood towards the brain via the nonexpandable vertebral artery route, through the anastomotic artery, into the common carotid artery and the brain. Fainting is thus prevented by the supply of blood instantly to the brain from the vertebral artery via the anastomotic artery into the common carotid artery, as well as a small amount of blood that is already within the meshwork of rostral epidural rete mirabile arteries.
In summary, we conclude that the well-developed anastomotic artery greatly assists in blood fow and blood supply to the brain of the girafe during the movement of the head from fully erect to ground level and back to fully erect. Te anastomotic artery allows for adequate cerebral perfusion at the stage where blood fow, against gravity, away from the brain. Tis is by means of an alternative route for blood fow from the heart via the vertebral arterial pathway, which is protected from both collapse and expansion by surrounding structures. Blood fow from this point follows the usual route from hereon, via fowing from the common carotid into the maxillary and subsequently through the rostral epidural rete mirabile network, to reach the brain. In addition, the anastomotic artery also assists in the prevention of brain damage by providing the route change as head posture is altered moving towards the ground. Te large volume of blood fowing with gravity and centripetal force from the heart to the brain is diverted from the common carotid artery through the anastomotic artery into the vertebral artery, preventing the large volume from entering the rostral epidural rete mirabile and circle of Willis. In other species, the blood shunted toward the vertebral arteries would have still entered the brain via the basilar artery, but uniquely in the girafe, the basilar artery is rudimentary and does not contribute blood to the brain.
What is the possible reason and advantages for this evolutionary development in a girafe? Te efectiveness of the anastomotic artery in girafes in aiding in the control of blood pressure is critical in the following naturally occurring situations: (a) when the head is moved in a relatively fast motion to ground level for the animal to drink, (b) when the head is moved in a relatively fast motion from ground level to fully erect, and (c) during the fghting when the males swing their heads and necks rapidly, hitting the rival with the full force of the head. Furthermore, the anastomotic artery assists in blood pressure control during darting, when the animal not only experiences a sudden drop to the ground when it loses consciousness due to the efect of the morphine drug on the neuroreceptors in the brain but also the additive efect of the alpha-2 agonist that is used in combination with the morphine drug during darting, on blood pressure, International Journal of Zoology causing a primary rise and then a quick fall in blood pressure due to the drop in cardiac output [16]. In this emergency circumstance, the mechanism of the anastomotic artery prevents death.
Another question arises as to when this anastomotic artery evolved. Was the cerebral circulation of extant girafe similar to that of extinct girafe of 5 million years ago? Preliminary work in our study showed that, unlike extant girafes, extinct girafds do have a fully functional basilar artery, contributing to the supply of blood to the brain. Whether a connection existed between the common carotid and vertebral systems may only be speculated. Te mechanism would have been less efective in blood pressure control because the diversion of large blood volumes in the head-down position would not be possible due to blood that could still move to the brain through the basilar artery. Why would a girafe living 5 million years ago, not have to regulate its blood pressure by means of the anastomotic artery mechanism? Did these girafes not fght by means of head butting or was it because of the shorter heart-to-head distance in comparison to extant girafes? Te results of this preliminary study are of major signifcance, contributing to the understanding of the concurrent evolution of circulatory control in parallel to neck elongation in girafes and stimulating further research on larger groups to confrm the fndings.

Conclusions
Tis is the frst study that indicates blood fow direction within the anastomotic artery in the girafe, by means of Doppler fow waveforms by duplex ultrasound examination, CT scans of the anastomotic arterial connection, and physical dissections to prove our fndings. We conclude that the anastomosis, connecting the common carotid artery and the vertebral artery, in combination with other signifcant structures, is of great importance in helping to overcome the specifc physiological challenges faced by girafes.

Data Availability
All data are available from the main author upon request.

Additional Points
Direct communication between the common carotid artery and the vertebral artery at the midpoint of the atlas called anastomotic artery. Blood fow in anastomotic artery infuenced by posture: head-upright and head-down positions. Head upright: fow direction away from vertebral artery towards the common carotid artery. Head down: fow direction away from the common carotid artery towards the vertebral artery

Ethical Approval
Tis study obtained the approval from the Animal Ethics Research Committee at the UFS, SPCA, and DESTEA, Ethics approval no. UFS-AED2020/0083.

Conflicts of Interest
Te authors declare that there are no conficts of interest.