Anatomical variations in the cerebral arterial circle of the Saimaa (Pusa hispida saimensis) and Baltic ringed seals (Pusa hispida botnica)

The intracranial arterial vascularization of the Saimaa ringed seals (Pusa hispida saimensis; Nordquist, 1899) and Baltic ringed seals (Pusa hispida botnica; Gmelin, 1788) disclosed patterns of anatomical architecture comparable to that of other pinniped species. Arterial silicone casts on skull scaffolds, and magnetic resonance imaging (MRI) showed that the besides joining the caudal communicating arteries upon entering the cerebral arterial circle, the bilateral internal carotid arteries bifurcated as laterally oriented rostral choroidal arteries and rostral cerebral arteries. The latter arteries almost immediately gave off the laterally oriented middle cerebral arteries. Numerous individual variations were evident in differences in the exact branching sites of bilateral vessels or the size or number of arterial branches. Two Saimaa ringed seals had only a tiny foramen for the left internal carotid artery to enter the intracranial space, and the intracranial part of this vessel was short. It did not reach the cerebral arterial circle. The intracranial part of the right internal carotid artery is bifurcated and also supplied the left side of the cerebral arterial circle. Both specimens had aplasia of the left rostral cerebral artery. The intracranial arterial arrangement of Saimaa and Baltic ringed seals reflects the arterial architecture of this body region in terrestrial mammals with little evidence for aquatic adaptations or changes related to thermoregulation.


| INTRODUCTION
Two arterial systems supply the vertebrate brain.The anterior carotid system derives from the internal carotid artery and irrigates the forebrain.The posterior vertebral system derives from the vertebral arteries and mainly supplies the brainstem (Fenrich et al., 2021).In mammals and other amniotes (birds and reptiles), the arteries at the base of the brain form a polygonal structure, cerebral arterial circle, anatomically also known as the circle of Willis (Lo & Ellis, 2010; Figure 1).It is thought to provide constant and steady blood flow to the brain and protect cerebral arteries and the brain barrier from hemodynamic stress (Caldwell et al., 2011;Gunnal et al., 2014).The most recent morphological change in the evolutionary history of the cerebral arterial circle was the fusion of the basilar artery with the vertebral arteries, forming the vertebra-basilar arterial system of birds and mammals (Rahmat & Gilland, 2014).
The basic vascular architecture of the vertebrate brain is highly conserved among species, and its evolutionary history is well known and likely to result from a combination of developmental and functional constraints (Allis, 1908(Allis, , 1912;;De Vriese, 1905;Rahmat & Gilland, 2014;Tandler, 1899).There is, however, variation among species in the arterial sources that supply the cerebral arterial circle and the direction in which blood flows in the main vessels.A number of early papers described the cerebral blood supply in humans and a few other mammal species (Bugge, 1972;Duvernoy, 1978;Hofmann, 1900;Padget, 1948;Tandler, 1899), and more recent papers have focused on the vascular variation in the brain of the Ruminantia (Ashwini et al., 2008;Brudnicki, 2011;Frąckowiak et al., 2015;Frąckowiak & Jakubowski, 2008;Kiełtykaka-Kurz et al., 2015;Zdun et al., 2019), various pet animals (Kapoor et al., 2003;Kier et al., 2019;Tanaka et al., 2018), rodents (Steele et al., 2006), and a marsupial species (Zdun et al., 2022).For zoology and anatomy students, introduction to the anatomy of cerebral arterial circle in animals is often through the vessel arrangements in a dog, which is relatively simple, well-studied, and a common arrangement among mammals.
The emergence of the rostral communicating artery (also known as the anterior communicating artery) of the cerebral arterial circle is intriguing because it does not significantly contribute to brain vascularization, at least in humans (Alastruey et al., 2007;Moore et al., 2006;Ren et al., 2015;Ujiie et al., 1996;Zhu et al., 2015) but it has persisted in different taxa for hundreds of millions of years (Rahmat & Gilland, 2014).The rostral communicating artery is a single vessel in humans, and its absence appears to be among the rarest of the cerebral arterial circle variations in our species (Alahmari, 2020).Of domestic animals, the rostral communicating artery is always present in swine (which appears to have few variations in the cerebral arterial circle in general; Ashwini et al., 2008) but variably present in Carnivora and Ruminantia (Ashwini et al., 2008; International Committee on Veterinary Gross Anatomical Nomenclature (ICVGAN) 2017; Tanaka et al., 2018).Among members of domestic and wild Ruminantia, there is a great deal of variation in the course of the rostral communicating artery and the manner of its anastomosis with the bilateral rostral communicating arteries (Ashwini et al., 2008;Brudnicki, 2011;Kiełtykaka-Kurz et al., 2015;Zdun et al., 2019).The rostral communicating artery was recently argued to be in a critical location to facilitate the forebrain dehydration sensing and faster behavioral response to water loss (Fenrich et al., 2021).The risk of dehydration was a source of intense selection pressure in early amniotes, and one of the primary adaptations in vertebrates is the ability to counteract fluctuations in the osmolarity of body fluids (Takei, 2000).
In some mammals, the internal carotid artery does not supply the brain, but they developed an alternative cerebral vascularization source.In this system, the external carotid artery supplies the brain via the maxillary artery with a specialized vascular plexus called the rete mirabilia located intra-or extracranially (Carlton & McKean, 1977;Kier et al., 2019).This vascular arrangement serves as a water-conserving mechanism, especially in artiodactyls, and it also helps to cool the brain (O'Brien, 2015(O'Brien, , 2020;;Strauss et al., 2017).Thought initially to protect the brain from thermal damage (Baker & Hayward, 1967;Mitchell et al., 1987), the rete mirabile system became more recently viewed as a contributor to the selective brain cooling that reduces evaporative water loss (Jessen et al., 1998;Kuhnen, 1997;Strauss et al., 2017).
Other than for the vessels forming the cerebral arterial circle, there is a deficit of information on the cranial vasculature of many representatives of major taxa inhabiting aquatic habitats with different salinity (Rahmat & Gilland, 2014).Some marine mammals possess only vestigial remnants of the original cerebral arterial circle (Ridgway, 1988).Specifically, cetaceans appear to lack connecting vessels that complete the cerebral arterial circle in most mammals (Ridgway, 1988).Only a few species representing another large group of marine mammals, pinnipeds, have been studied in this respect (Frąckowiak & Godynicki, 1996;Hafferl, 1938;Tandler, 1899).Based on the relatively scarce information available, the arterial arrangement of the cerebral arterial circle and the main arteries leading to it in harbor, gray and Baikal seals appear similar to those of terrestrial mammals (Frąckowiak & Godynicki, 1996;Hafferl, 1938;Tandler, 1899).Of noted differences, harbor, harp, gray, and Baikal seals (Folkow et al., 1988;Frąckowiak & Godynicki, 1996;Hafferl, 1938;Tandler, 1899) do not possess intracranial carotid retia commonly found in the even-toed ungulates (located extracranially in the cat; Kier et al., 2019).Of the previously studied seals, the Baikal ringed seal inhabits a freshwater environment similar to Saimaa ringed seal.Other than naming the arteries forming the cerebral arterial circle, the authors gave little information on the intracranial vasculature of the Baikal ringed seal (Frąckowiak & Godynicki, 1996).
The purpose of this article was to describe the intracranial arterial arrangement of Saimaa (Pusa hispida saimensis; Nordquist, 1899) and Baltic ringed seals (Pusa hispida botnica; Gmelin, 1788), which occupy habitats differing in the availability of fresh water.The endangered Saimaa ringed seal is an endemic subspecies to Lake Saimaa in Finland.The Baltic ringed seal inhabits the brackish water Baltic Sea, which is almost entirely landlocked.Both subspecies may originally derive from a seal population physically separated into different water basins by geological changes during and after the last ice age (Ukkonen et al., 2014).Small and isolated populations are often subject to loss of genetic variability (Nyman et al., 2014).However, new research shows that the Saimaa and Baltic ringed seals are genetically even further away from each other than the latter is from the other ringed seal subspecies (Löytynoja et al., 2023).To document the transportation routes of blood to the cranial region and the intracranial arterial arrangement in Saimaa and Baltic ringed seals, we used silicone casts of cranial arteries on skeletal scaffolds and magnetic resonance imaging (MRI) techniques.
The arterial arrangement of the cerebral arterial circle and the main arteries leading to it in Saimaa and Baltic ringed seals was predicted to be similar to those of harbor, gray, and Baikal seals (Frąckowiak & Godynicki, 1996;Hafferl, 1938;Tandler, 1899).Alternatively, the geographic and, in the case of the Saimaa ringed seal, also the genetic isolation may manifest as variation in the arrangement of the cerebral arterial circle and the arteries leading to it.As the number of individuals studied here was small, caution should be used in the interpretation of any variations detected, especially as individual anatomical variations in the cerebral arterial circle are relatively common in mammals (Ashwini et al., 2008;Brudnicki, 2011;Tanaka et al., 2018).

| MATERIALS AND METHODS
All Saimaa ringed seals that are found dead are collected by the staff of the Parks and Wildlife of a state enterprise Metsähallitus and stored at À20 C until necropsy to determine the cause of death and to collect various samples for research and conservation purposes at the facilities of the Finnish Food Authority pathology unit in Oulu.Specimens are usually in various stages of decomposition, but five individuals collected between 2017 and 2019 were in relatively good condition (Table 1), enabling a more detailed anatomical examination either in Oulu or at the Veterinary Faculty of the University of Helsinki in Helsinki.The seals examined were by-catch found in gill nets.Metsähallitus and the University of Helsinki share a research agreement that permits using Saimaa ringed seal material.In addition, two Baltic ringed seals (Table 1) shot by a hunter in the Bothnian Bay (the northernmost part of the Baltic Sea) in 2018 and in 2019 were shipped frozen to the Veterinary Faculty of the University of Helsinki for anatomical examination.Baltic ringed seals are legally hunted in Finland, and the studied individuals were hunted for reasons that were unrelated to this research.The ringed seals examined were age grouped according to body weight: under 30 kg individuals were pups, 30-42 kg individuals were subadults, and those with body weight over 42 kg were adults.
The carcasses were allowed to thaw for the silicone injections and magnetic resonance imaging (MRI).Silicone casts of the arterial arrangement of the head and neck region of both ringed seal subspecies were created by injecting silicone (3 M Express™ 2 Light Body Standard Quick or 3 M Express™ 2, Light Body Flow Quick) as described in detail by Laakkonen and Kivalo (2013) into the right or both common carotid or arteries at the level of atlas (except in two cases where the injection site was close to the thorax region).If only one side of the head was injected, this was due to damage to the other side, or the material was used for other research purposes (the availability of the Saimaa ringed seal material is limited due to the low population number of this endangered subspecies ($430-440 individuals in 2022; Metsähallitus, 2023).Even if arteries were injected on one side of the head, in most cases, silicone reached the internal carotid artery on the opposite side, allowing examination of all intracranial arteries.For three specimens, two types of silicone differing in color were used to allow visualization of the possible flow of silicone to the opposite side.Once the silicone was hardened (in <5 min), two of the specimens (2704 and 2747) were transported by car to the nearby Veterinary Teaching Hospital of the University of Helsinki for MRI of the entire animal (see below).For the other specimens, the head was separated from the body from the atlantooccipital joint (Part of the neck was also removed in one specimen) with a scalpel, and the skin and blubber from the head were removed.All but two of the heads were separately boiled in tap water for 2-3 h, after which the major muscles and other large soft tissue parts were removed by hand.For the removal of the remaining soft tissue parts in hard-to-reach cavities and around the smallest vessels of the silicone model (which are difficult to remove without breaking the tiny branches of the model), the heads were placed in sodium hypochlorite solution (14%, Sigma-Aldrich, Switzerland) in a horizontal position for soft tissue digestion.During maceration, which took up to 10 days, the specimen was rotated daily, and an effort was made to limit damage to the bony structures.After the chemical maceration process, the skulls were thoroughly washed under running water for 15 min and then placed separately in clean tap water in 5-L buckets for 3 days, during which the water was changed twice a day.
The two heads (specimens 668 and 2704) were not processed chemically.Soft tissue removal was carried out at the Finnish Museum of Natural History by an enzymatic maceration technique (a detailed comparison of the two maceration processes is reported elsewhere).Maceration with papain followed the process described by Niederklopfer and Troxler (2001).In brief, the ratio of weight of T A B L E 1 Demographic data for the ringed seals examined for the cranial arterial arrangement.

Species
Specimen specimen to water: 1:20-1:40; Enzyme: 5 g/L papain MERCK Nro.107,147; NaCl: 15 g/L; Disinfectant and wetting agent: 0.5 g/L Supralan 80; Emulsifier: 0.5 g/L SUPRALAN UF; pH: 8.0-8.5;controlled with sodium carbonate (Na 2 CO 3 ); Temperature: 40-45 C; Agitation: 80-s run/5-min break, with circulatory pumps.Preprocessing consisted of boiling in water with 10 g/L NaCl and 1 g/L SUPRALAN UF to denature the proteins.After maceration, the specimens were thoroughly rinsed and briefly immersed in hot water with 10 g/L NaCl and 1 g/L SUPRALAN UF to denature the remaining papain in the bone.The duration of the entire process was 24-36 h.Upon completion of the soft tissue removal, the roof of the cranium (calvaria) of two specimens was removed with a bone saw to allow a dorsal view of the intracranial parts of the silicone model.In all specimens, some of the bone was fractured or became partly separated during the maceration process, allowing a view of the intracranial vessels.The bones of one individual (specimen 2745) separated during the chemical maceration process, leaving the silicone model without any bony reference landmarks (except where it went through the right carotid canal of the temporal bone).Upon completion, this cast was examined and immersed in water to allow for better 3D visualization.
MRI was performed at the Veterinary Teaching Hospital of the University of Helsinki using a 1.5 Tesla scanner (Philips, Ingenia 1.5 TS, Philips Medical System).Sequences designed for imaging living tissues had to be adjusted for the thick blubber layer of ringed seals.The carcass was imaged while lying on its back, and a head coil was used for the head region.The protocol and parameters for imaging were as follows: Turbo-Spin-Echo T2-weighted and T1-weighted dorsal, sagittal, and transversal plane MRI; T2: Imaging parameters for sagittal imaging TR 13,123 ms; TE 110, TR for transversal imaging 12,788 ms; TE 110 ms, for dorsal imaging TR100; TE4040, flip angle 90 , slice thickness 2,5 mm; T1: Imaging parameters for sagittal, transversal and dorsal imaging TR 633 ms; TE 15, flip angle 90 , slice thickness 2,5 mm.For one specimen (2704), the injected arteries were visualized with an additional head series with black blood flip angle 10, TE 34, TR 24, and slice thickness 1.5 mm.All image analyses were performed with Imaios DICOM viewer.Once the imaging was completed, the heads were removed, and soft tissues macerated with either the chemical (specimen 2747) or the enzyme maceration process (specimen 2704) as described above.The hunted Baltic ringed seal specimens could not be imaged because of the bullet fragments in their body (strong magnetic fields can remove the metal pieces causing tissue and equipment damage).
During the examination of the completed silicone casts, a digital caliper (Digitronic caliper, Polycal series, Moore & Wright) was used to measure the luminal diameter (in millimeters, Table 2) of the main arteries described.Smaller arteries were excluded from measurements because of the potential inaccuracy of measurements due to technical factors such as the dilation status of the vessel at the time of death, the potential effect of the injection site, and pressure applied during the injection.The larger vessels' arterial measurements were not exact for the same reasons.Also, the low number of specimens available prevented any quantitative analyses of the size differences of the cranial vessels.Any incomplete vessel filling was monitored during the gradual maceration process (whether a vessel or a branch was visible in the specimen but not filled with silicone).In the finished model, this was manifested as interruptions to vessels' smooth or tapering sides where the missing branches should originate.
Attempts were made to produce casts of venous circulation, but complete and reliable venous casts were challenging due to the valves blocking the silicone flow.The anatomical terminology is in accordance with the International Committee on Veterinary Gross Anatomical nomenclature (ICVGAN, 2017).

| RESULTS
The basic configuration of the cranial arteries is described first, followed by the presentation of the major individual variations in the branching of vessels and anomalies detected in the silicone specimens.
The ascending pharyngeal artery left the common carotid artery between the bifurcation of the external and internal carotid artery on the caudomedial side (not shown).The common carotid artery ascended to the level of the atlanto-occipital joint, where it branched into internal and external carotid arteries (Figure 2).The latter vessel gave rise to the occipital artery slightly lateral but close to that of the origin of the internal carotid artery (Figure 2).As the occipital artery reached the occipital condyle, it gave rise to a ramus that ascended over the cranial border of the wing of the atlas to the dorsal surface where it anastomosed with a terminal branch of the vertebral artery (Figure 3; see below).
The external carotid artery continued past the tympanic bulla as it ascended at the level of the temporomandibular joint to give rise to the caudal auricular artery (Figure 2).rostral to this level, the external carotid artery terminated by dividing into superficial temporal and maxillary arteries (Figure 2).Superficial temporal artery continued caudal the zygomatic arch to the temporal region.Continuations of the maxillary artery were the main suppliers of the ocular, facial, and mandibular regions (reported elsewhere).
Two routes supplied the intracranial arterial system via internal carotid and vertebral arteries.Bilaterally, the internal carotid artery entered medially the tympanic part of the temporal bone through the carotid canal (Figure 2).Upon emerging from the canal intracranially T A B L E 2 Morphometric data of the main neck and cranial arteries of Saimaa and Baltic ringed seals examined in this study.at the bottom of the cranial cavity (Figure 1), the internal carotid artery turned caudally before ascending slightly to reach the cerebral arterial circle at approximately of its midpoint (Figures 1 and 4).The vertebral arteries (diameter 2.2-2.4 mm at the level of the atlas, visible only in two specimens due to the injection site) ascended cranially in the transverse foramina of the cervical vertebrae until they turned dorsally through the alar groove (incisura) to enter the vertebrate canal via the lateral vertebrate foramen of the atlas (Figure 3).The surface of the vertebral arteries gave rise to cervical spinal branches, which entered the vertebrate canal through the cervical foramina intervertebralia and united through the ventral spinal artery (Figure 3).The diameter of the spinal branches was approximately half a millimeter but was double of that between axis and atlas.Vertebral arteries gave rise to lateral branches, which had various configurations (Figure 3).Often, two rami branched right next to each other.Despite the configuration, the lateral rami almost immediately gave rise to numerous smaller vessels.The vertebral arteries entered the foramen magnum and, together with the ventral spinal artery, formed a complete spinal arterial circle (Figure 3).The caudal cerebellar arteries arose from this circle or the basilar artery (Figures 3 and 5).After continuing $1.5 cm to the rostral direction, the vertebral arteries combined to form the basilar artery almost exactly at the ventral edge of the foramen magnum.

Species
The caudal cerebellar arteries (<1 mm in diameter) arising from the spinal arterial circle or the basilar artery had a rostrolateral orientation towards the caudal end of the tympanic bulla (Figure 5).Next, the basilar artery gave rise to the rostrolaterally oriented labyrinthine arteries (<1 mm in diameter), which continued to the internal ear canal opening on the petrous part of the temporal bone (Figure 5).Between caudal cerebellar arteries and the cerebral arterial circle, the basilar artery gave rise to several small pontine arteries with <0.5 mm in diameter.
The basilar artery ended by dividing into right and left caudal communicating arteries, each joining the ipsilateral internal carotid artery (Figures 1 and 4).The rostral cerebellar and the caudal cerebral arteries branched off at the laterocaudal corners of the cerebral arterial circle almost side by side (Figure 1).These vessels were <1 mm in diameter except in one Saimaa ringed seal specimen (2744) that had slightly larger caudal cerebral arteries (1.32 mm on the right side and 1.31 mm on the left side), and in the larger Baltic ringed seal specimen (668) whose corresponding arteries were just over 2 mm in diameter.Caudal cerebral arteries almost immediately led to one large and several smaller vessels in all specimens.The choroidal and rostral cerebral arteries originated at the point where ipsilateral internal carotid arteries entered the cerebral arterial circle.The rostral cerebral arteries gave almost immediate rise to the middle cerebral arteries.The internal ophthalmic arteries arose from the middle cerebral arteries or rostral cerebral arteries (Figure 1).Rostral cerebral arteries united at the rostral end of the circle and formed a dorsally arched vessel into the longitudinal fissure of the brain.The dorsally arched united rostral cerebral artery gave bilaterally rise to laterally oriented vessels before dividing into two large vessels.The middle and caudal cerebral arteries ascended and branched to the lateral and dorsal side of the brain (Figure 6).On both sides of the dorsally arched vessel, internal ethmoidal arteries left the rostral cerebral arteries (Figure 1).
In the silicone casts of one Saimaa ringed seal (specimen 2704) and one Baltic ringed seal (specimen 668), the caudal communicating and rostral cerebral arteries were joined by small arteries that formed a round, smaller circle inside the cerebral arterial circle (Figure 1).Such small arteries were visible in all specimens.However, they did not form the described circle in other specimens.
Numerous individual variations in the size (diameter) of the vessels or their exact point of branching were recorded (Table 3).Typically one of the bilateral arteries was larger than the corresponding artery on the other side, or one of the pair left the main artery earlier than the other.There was no predilection to one side having larger vessels than the other.In some cases, instead of leaving the main artery as one branch, either side left the main artery as a group of several smaller arteries.
A major anomaly occurred in the supply of the cerebral arterial circle in two Saimaa ringed seals (Figure 1).
In specimen 2698, the left internal carotid artery entering the carotid canal was short and small (0.66 mm in diameter) compared with the corresponding right one (3.57mm).Inside the cranium, there was only a small bony foramen for the left internal carotid artery to enter, and a tiny vessel did not reach the cerebral arterial circle (at least in the cast; Figure 1).At the caudal level of the cerebral arterial circle, the right internal carotid artery divided and the additional vessel entered the cerebral arterial circle on the left side of the circle.Most vessels branching from the cerebral arterial circle appeared similar to those in other specimens, but the rostral cerebral and internal ophthalmic arteries were missing from the left side.Both of the internal ethmoidal arteries were visible, but the right rostral cerebral artery alone formed the dorsally oriented continuation of this vessel.
Similarly to the arterial arrangement in specimen 2698, there was only a tiny foramen for the left internal carotid artery to enter the intracranial space in specimen 2745.The cast showed no sign of the intracranial part of the left internal carotid artery, and there was an aplasia of the left rostral cerebral artery.The intracranially divided right internal carotid artery also supplied the left side of the cerebral arterial circle.The rostral part of the circle was not formed correctly in this cast because silicone had flowed out of the vessels preventing proper examination of arteries branching from the rostral part of the circle.This incompleteness of the model was manifested as interruptions to the smooth or tapering sides of vessels where the missing branches should originate.
In Saimaa ringed seal specimen 2726, the caudal cerebellar arteries arose from the spinal arterial circle (Figure 3).The right one was double in size (diameter 1.36 mm) compared with the one on the left side (diameter 0.65 mm) and had its origin more rostrally (Figure 3).The basilar artery gave rise to the labyrinthine arteries, first to the left one.The right labyrinthine artery anastomosed with the right caudal cerebellar artery (Figure 3).The left internal carotid artery anastomosed with a thick vessel (2.22 mm) from the basilar artery.In this specimen, there was an asymmetrical arterial structure at the caudal end of the cerebral arterial circle that partly joined the right and left sides of the circle (Figure 1).
In the smaller Baltic ringed seal specimen (458), the right internal carotid artery was much thicker (2.58 mm) than the left one (2.08 mm), mostly likely because the silicone had reached the left side from the right side through the cerebral arterial circle (only the right side was injected).The internal ethmoidal arteries were not properly visible in this specimen because they had become bundled with the vessels of the cerebral arterial circle during the maceration process.They were visible only from the ventral side because the calvaria was not cut open to preserve the dorsally intact brain cavity, allowing a good view and orientation of the vessels in the dorsal part of the skull.

| DISCUSSION
The overall organization of the intracranial vasculature of both ringed seal subspecies was similar to that reported from other pinnipeds (Frąckowiak & Godynicki, 1996;Hafferl, 1938;Tandler, 1899) and other carnivores (Anderson et al., 1989a;Bugge, 1978;Jewell, 1952;Tanaka et al., 2018).Based on the thickness of the arteries, the internal carotid arteries were the primary source of arterial supply to the ringed seal brain, but the vertebra-basilar route was also well developed.It should be noted, however, that the luminal diameter of a vessel may vary depending on the dilation status of the vessel when the animal dies and on the pressure applied during the silicone injection.The formation and arterial supply of the cerebral arterial circle of ringed seals was essentially the same as in other studied carnivores (e.g., seals, dogs, and bears; Anderson et al., 1989a;Bugge, 1978;Nickel & Schwarz, 1963).The arterial supply of the cerebral arterial circle in members of the Felidae differs from that of other carnivores because, in adult felids and some other representatives of the order Carnivora, the extracranial segment of the internal carotid artery is regressed (Baker & Hayward, 1967;Bugge, 1978;Frąckowiak & Godynicki, 2003;Hsieh & Takemura, 1994;Skoczylas et al., 2016;Ziemak et al., 2021; see however Nickel & Schwarz, 1963).Of other intracranial differences, the accessory middle cerebellar arteries reported to arise from the basilar artery rostral to the origin of the caudal cerebellar arteries in bears (Anderson et al., 1989a) did not occur in any of the ringed seals of this study (and are not recognized by ICVGAN [2017]).Besides caudal cerebellar and labyrinthine arteries, only small pontine arteries arose in ringed seals from the basilar artery at this region.
A delicate arterial circle around the upper pituitary stalk occurred in Saimaa and Baltic ringed seals.It was not present in all specimens, most likely because the arteries forming it were extremely thin and often filled only partially, even in otherwise complete casts.The circle structure was reported in early studies of human cadavers (Fuchs, 1924;Winterstein, 1937;Xuereb et al., 1954), which show that the pituitary body is supplied by superior and inferior hypophyseal arteries and their anastomoses.Similar to the harbor, harp, gray, and Baikal seals (Folkow et al., 1988;Frąckowiak & Godynicki, 1996;Hafferl, 1938;Tandler, 1899), the internal carotid artery supplying the brain from the ventral side was well developed in the Saimaa or Baltic ringed seals (Figure 4).Of the previously studied seals, the Baikal ringed seal inhabits a freshwater environment similarly to Saimaa ringed seal.Other than naming the arteries forming the cerebral arterial circle, the authors gave little information on the intracranial vasculature of the Baikal ringed seal (Frąckowiak & Godynicki, 1996), preventing detailed comparisons between ring seal subspecies.The rostral communicating artery was reported to be variably present in Carnivora (ICVGAN 2017;Tanaka et al., 2018) and indicated as being involved in the forebrain dehydration sensing (Fenrich et al., 2021), was not present in any of the Saimaa or Baltic ringed seal specimens.Water balance is not an issue for freshwater seals with easy access to drinking water.Some seawater seal species cope with profound dehydration by drinking seawater (How & Nordøy, 2007).
Some authors have suggested that the obliteration of the extracranial segment of the internal carotid artery in ruminants is due to eliminating factors causing interference with low-frequency sounds.These factors include the change in the relative positions of the tympanic bulla and the petrous part of the temporal bone in ontogenesis resulting in the exclusion of the vessels from the middle ear region (Graczyk et al., 2022;Zedenov, 1937).Among ringed seals, there is variation in skull morphology (Amano et al., 2002; for other pinnipeds, see Jones et al., 2015), and the size (Hyvärinen & Nieminen, 1990) and relative position of tympanic bulla in relation to the articular surface of the temporomandibular joint (Laakkonen & Jernvall, 2020) with Saimaa ringed seal specimens being the most separated from the other subspecies but the variation appears not to reflect significant changes in the cranial vasculature.
The recent view suggesting that the cerebral arterial circle protects against hemodynamic stress (Vrselja et al., 2014) is interesting from the point of view of marine mammals facing changing pressure conditions while diving.The use of silicones differing in color showed that many brain areas could be reached from either side of the brain (Figure 1b).However, the rerouting of blood flow may be hindered or even obstructed in cases of severe anomalies also detected in this study.In two of the examined five Saimaa ringed seals, the internal carotid artery on the left side was missing or improperly formed inside the cranium preventing it from supplying the cerebral arterial circle.In both specimens, there was also an aplasia of the rostral cerebral artery (Figure 1).These individuals were collected from different parts of Lake Saimaa, indicating that the anomalies were not related to a geographically subdivided local population in the complex lake system (Löytynoja et al., 2023;Palo et al., 2003).It is interesting to consider in this context that Lake Saimaa is very shallow, with a mean depth of 12 meters (maximum 85 m; Kunnasranta et al., 2021).Therefore, compared with other ringed seals, the Saimaa population may experience a lesser degree of pressure changes while diving.
Variations and anomalies in cranial arteries are not particularly rare in mammals.Aplasia of the precommunicating segment of the rostral cerebral artery was observed in 14% of dogs (Tanaka et al., 2018).The absence of the anterior communicating artery was the most common anomaly among domestic artiodactyls (Ashwini et al., 2008).In fallow deer, the highest variation was seen in the branching of the caudal cerebral arteries (Brudnicki, 2011).Alternative or complementary routes of blood circulation in the form of anastomoses may allow effective brain circulation even in cases of aplasia or other anomalies.Of carnivores, anatomic evidence for anastomoses between external carotid and intracranial circulations have been described from dogs (Ellenberger & Baum, 1891;Gillilan, 1976;Jewell, 1952;Lee et al., 1986), but no connections between intra-and extracranial circulations were evident in studies of bears (Anderson et al., 1989b;Bugge, 1978).The anastomic artery between the external ophthalmic (a branch of the maxillary artery) and internal carotid arteries is considered the most crucial anastomosis between intra-and extracranial circulation (Lee et al., 1986).Anastomoses between external carotid and intracranial circulations were not detected in any of the specimens of this study.Anastomoses are challenging to demonstrate, however, purely with methods of macroscopic anatomy as silicone does not necessarily penetrate all the tiny vessels, and casts do not provide evidence for the direction of the blood flow or other functional properties of the arterial arrangement.
In mammals, the branch angles of vessels supplying the rostral segment of the brainstem are, in most cases, small.However, the basilar artery gives off branches at a nearly 90-degree angle (Figure 5).It has been suggested that the perpendicular intersections may allow the basilar artery to adapt to altered directions of blood flow depending on anatomical conditions without changing its basic branching geometry (Rahmat & Gilland, 2014).In the ringed seals of this study, the branch angles of these vessels were perpendicular or oriented slightly to the rostral direction (Figure 5).
Because of the delicate nature of brain tissues, studies of cranial vascularization are often based on casts of various substances.One of the benefits of the silicone materials used for the casts in this study was their lightness, which allowed even thin ascending vessels to preserve their actual orientation without any support from the cerebral tissue (Çirak et al., 2021).MRI of the injected heads provided important complementary information on the intracranial orientation of the blood vessels, and it enabled to demonstrate that the cerebral arterial circle occupied several dorsoventral planes in Saimaa ringed seals (Figures 1 and 4).
Despite attempts to produce venous casts, complete and reliable casts of only arterial circulation were completed.Similar challenges in preparing venous circulation with various injection materials have been reported even when injections were attempted on freshly killed animals (Carlton & McKean, 1977;Folkow et al., 1988).
In conclusion, the intracranial arterial arrangement of Saimaa and Baltic ringed seals reflected the arterial architecture of terrestrial mammals with no evidence of having only vestigial remnants of the original cerebral arterial circle as found in whales or changes related to thermoregulation in the aquatic environment.

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I G U R E 1 Intracranial views of the cranium of Saimaa (a-c) and Baltic (d) ringed seals show the arteries forming the cerebral arterial circle and its major branches.(a) 2698, dorsocaudal view; aplasia of the left rostral cerebral artery (arrowhead).Scale bar = 1 cm.(b) 2704, dorsocaudal view.Scale bar = 1 cm.(c) 2744, ventral view; skull bones became separated during the maceration process.X, an additional transverse vessel in the caudal part of the cerebral arterial circle.Scale bar = 1 cm.(d) 668, dorsal view (the skull bones became separated during the maceration process); internal carotid arteries are not visible in this view.Scale bar = 1 cm.*, a ring-like anastomosis inside the cerebral arterial circle; BA, basilar artery; CCA, caudal cerebral artery; CCoA, caudal communicating artery; DA, dorsally arched united rostral cerebral arteries; ICA, internal carotid artery; IOA, internal ophthalmic artery; MCA, middle cerebral artery; RCA, rostral cerebral artery; RCBA, rostral cerebellar artery; X, additional vessel of the right internal carotid artery.
Vessel diameter measured in millimeters.a The right internal carotid artery divided into a right and left vessel supplying the circle of Willis.In specimen 2745, the rostral part of the circle of Willis was missing from the cast due to leakage of the silicone outside the vessels.b Due to leakage of silicone out of the vessels, the cast did not stay complete.F I G U R E 2 Lateral view of Saimaa ringed seal head, silicone injected arterial vessels.Left side, rostral to the left, specimen 2704.Common carotid artery (CCA), external carotid artery (ECA), internal carotid artery (ICA), occipital artery (OA), caudal auricular artery (CAA), superficial temporal artery (STA), and maxillary artery (MA).Scale bar = 1 cm.F I G U R E 3 Dorsal view of Saimaa ringed seal atlas and silicone cast of the spinal arterial circle (arrowhead).Rostral to the top, specimen 2726.The vertebrae and the skull were separated to better view the vessel arrangement.AG, alar groove (incisura); BA, basilar artery; CCA, caudal cerebellar artery; LA, labyrinthine artery; LVF, lateral vertebrate foramen of the atlas; VA, vertebral artery (the left one is broken between the alar groove and the lateral vertebrate foramen.Scale bar = 1 cm.

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I G U R E 4 Magnetic resonance image of Saimaa ringed seal head (specimen 2704), dorsal orientation, rostral to the top.(a) The bilateral internal carotid artery emerging from the carotid canals (arrows) and ascending to reach the cerebral arterial circle (not visible at this dorsoventral plane).Basilar artery (BA), caudal cerebellar artery (CCA).(b) Image taken dorsal to the plane shown in (a).Caudal communicating arteries (CC), middle cerebral arteries (MCA), and a part of the right rostral cerebral artery (RCA).(c) Image taken dorsal to the level shown in (b); most of the cerebral arterial circle is shown; caudal communicating arteries (CC), middle cerebral arteries (MCA).Scale bar = 1 cm.

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I G U R E 5 Brain vasculature cast of Saimaa ringed seal, brain tissue partly macerated, specimen 2745.Ventral view, rostral to the top.The tympanic part of the temporal bone on the right side of the figure has been turned to the side to show the entrance of the labyrinthine artery (LA) to the internal ear canal.Basilar artery (BA), caudal cerebellar arteries (CCA).Upon entering the cranium, the right internal carotid artery (ICA) is divided, and the additional vessel (x) supplied the left side of the cerebral arterial circle (incompletely formed, but the location is shown with a red ellipse).Some of the silicone spilled out of the vessels during the injection process.Scale bar = 1 cm.F I G U R E 6 Magnetic resonance image of Saimaa ringed seal head, lateral orientation, rostral to left, specimen 2704.The dorsally arched united rostral cerebral artery is divided into two large, dorsally oriented vessels (arrows), the middle cerebral artery (MCA), caudal cerebral artery (CCA).Scale bar = 1 cm.
T A B L E 3 Individual anatomical variations and anomalies in the intracranial arterial architecture of Saimaa and Baltic ringed seals.