Topographic anatomy and vascularization of the glandula thyroidea in rats

Topographical anatomy and detailed measurements of the glandula thyroidea (thyroid gland) and the glandula parathyroidea (parathyroid gland) were determined in rats, with significant differences identified between the sexes. In the rats (N = 10 male, 10 female), the glandula thyroidea were positioned at the level of the C1 and C2 vertebrae. One glandula parathyroidea was present in each glandula thyroidea lobe, localized in the cranial part of the lateral lobes in 60% of the animals. There was no glandula thyroidea left lobe in one female and no isthmus in two females. Both the A. thyroidea cranialis and the A. pharyngea ascendens originated from the A. carotis externa, which acted as a common trunk. On the left, the A. thyroidea caudalis originated from the truncus brachiocephalicus in all rats and on the right side was found to originate from both the truncus costocervicalis and the A. subclavia dextra in three females, and only from the truncus costocervicalis in seven females. The V. thyroidea cranialis opened into the V. jugularis interna in the neck region and at the level of the apertura thoracis cranialis, and the V. jugularis interna united with the V. thyroidea caudalis. In addition, on the right, the V. thyroidea cranialis joined the V. jugularis interna, at the level of the A. subclavia. The veins on both sides opened into the V. cava cranialis. Significant differences were observed between the sexes and detailed anatomical analysis of the glandula thyroidea and the glandula parathyroidea, and related vasculature and innervation, have been described in this paper.


| Glandula thyroidea
The glandula thyroidea has long attracted the attention of the scientific world due to the number of disorders relating to it such as hypoand hyperthyroidism. The anatomical and functional properties of this gland have not been fully elucidated and are still being investigated. The glandula thyroidea has been phylogenetically characterized as one of the oldest and largest of the endocrine glands in vertebrate species (Onwuaso & Nwagbo, 2014).
In rats, the glandula thyroidea are two-lobed structures at the C2-C3 level in Sprague-Dawley male rats, located lateral to the trachea near the base of the laryngeal cartilages (Hadie et al., 2013).
The guinea-pig glandula thyroidea reaches the 6th trachea ring in the caudal of the larynx (Yamasaki, 1995). The cranial margin of the glandula thyroidea is at the junction of the cricoid and thyroid cartilages. There is no correlation between the glandula thyroidea and body volumes in albino rats (Hall & Kaan, 1942). According to Nakamura et al. (2019), the glandula thyroidea may have differing volumes in the different lobes and in the rat sometimes the left lobe, or one lobe, is absent. Laterally and ventrally, the glandula thyroidea is covered by the M. sternohyoideus, immediately dorsal lies the A. carotis communis, the V. jugularis interna and the N. laryngeus caudalis (Mense & Boorman, 2018).
The glandula thyroidea has an oval cranial end and presents as a butterfly-shaped organ in the lower part of the larynx cartilages and does not join dorsal part of the trachea (La Perle et al., 2018;Tadjalli & Faramarzi, 2016;Yavru & Yavru, 1996). In rats, the glandula thyroidea is generally an oval-shaped long reddish body with smooth surfaces (Enemali et al., 2016) but in wild rats, it is long, thin and brownish, while in domesticated rats it is short, rounded and pinkred (Mosier & Richter, 1967). In the adult mongoose, the glandula thyroidea is dark brown (Tadjalli & Faramarzi, 2016). In addition, the isthmus the may not be present. For example Mota and Serkiz (2019) reported that there was no isthmus in rats, and guinea pigs do not always have one either (Yamasaki, 1995).

| Glandula parathyroidea
Although it is generally reported that rats, mice and hamsters only have one pair of glandulae parathyroidea, increased numbers have been reported in some hamsters and mice (Kittel et al., 1996b). In rats, two glandulae parathyroidea are localized in each thyroid lobe usually located bilaterally (Chiasson, 1978), under the capsule, near the dorsolateral border of each thyroid gland lobe, or on the craniolateral side of the glandula thyroidea in mice (Kittel et al., 1996b).The glandula parathyroidea can also be localized more superficial and subcapsularly. In mice, it is an oval or lens-shaped organ (Jones et al., 1983), small and variable in size (0.7-1 mm × 0.3-0.5 mm), usually at the anterolateral edge of the glandula thyroidea but this position can vary (Fox et al., 2007;Krinke, 2004). In the female mongoose, the glandula parathyroidea is positioned medial to the ventral border of the middle portion of the lobes of the glandula thyroidea, whereas in males they are in the caudal and lateral sides of each glandula thyroidea lobe (Tadjalli & Faramarzi, 2016). On the contrary, according to La In rats, the glandula parathyroidea is supplied by the same arteries that supply the glandula thyroidea (Allen & Fingeret, 2022).
Therefore, there is a direct correlation between the vascular bed and the thyroid parenchyma (Abdreshov et al., 2019). In mice, the glandula parathyroidea blood supply is provided by the A. thyroidea cranialis (Murakami et al., 1987), with venous drainage provided by the V. thyroidea cranialis (Murakami et al., 1995).

| Arterial supply
The arterial blood supply for the glandula thyroidea is provided by the A. thyroidea cranialis and the A. thyroidea caudalis, both of which mainly originate from the A. carotis externa (Yamasaki, 1990).
The origin of these arties has been debated in the literature but it has been reported that the A. thyroidea cranialis originates from the A. carotis externa (Stathatos, 2019) or the A. carotis interna (Monroe & Turner, 1946), whereas Vdoviaková et al. (2022) indicate that the A. thyroidea cranialis and the A. thyroidea caudalis originate from the A. carotis communis.
According to Yamasaki (1990), in more than 70% of animals, on both sides of the neck, the A. thyroidea cranialis forms a common trunk with the A. pharyngea ascendens. The main vessel feeding the glandula thyroidea, the A. thyroidea cranialis, also branches into the larynx (McLaughlin & Chiasson, 1990). The A. thyroidea caudalis is a branch of the A. subclavia, or one of its subsiduaries, and courses with the N. laryngeus caudalis (Stathatos, 2019). According to Yamasaki (1990) the A. thyroidea caudalis does not originate from the A. cervicalis profunda but instead originates from the A.
phrenicopericardiaca. It has also been reported that the A. thyroidea caudalis springs from the truncus costocervicalis and the truncus cervicalis supplies the muscles of the neck and shoulder (Maynard & Downes, 2019).
Previous research has shown that the A. thyroidea cranialis gives a branch to the isthmus and anastomoses with the A. thyroidea caudalis (Green, 1955). Green (1955) also reported that the A. thyroidea caudalis runs along the lateral edge of the trachea in a cranial direction, accompanied by the N. laryngeus caudalis, and anastomoses with the A. thyroidea cranialis and terminates in the glandula thyroidea. Together, the A. thyroidea cranialis and the A. thyroidea caudalis maintain a rich anastomosis network within the glandula thyroidea (Policeni et al., 2012).

| Venous drainage
The veins originate from the glandula thyroidea parenchyma and consist of three groups of veins, the cranial, middle and caudal thyroid veins, to form a plexus alongside the V. jugularis interna and the V. brachiocephalicus (Policeni et al., 2012). The V. thyroidea cranialis and the V. thyroidea medialis in the rat glandula thyroidea open into the V. jugularis interna (Monroe & Turner, 1946). The V. thyroidea caudalis opens into the V. brachiocephalicus (Hadie et al., 2013) or the V. cava cranialis (Green, 1955).

| Innervation
Both the glandula thyroidea and the glandula parathyroidea receive their sympathetic fibres from the ganglion cervicale craniale, and their parasympathetic fibres from the N. vagus via the N. laryngeus cranialis and the N. laryngeus caudalis (Wells, 1968). In mice and rats, the sympathetic fibres are more numerous in younger individuals compared to older animals (Domeij, 1990;Kumar et al., 2018;Romeo et al., 1986).
Although there are many studies relating to the glandula thyroidea and the glandula parathyroidea anatomical structures in rats, there are no detailed studies regarding the topography of the region.
In addition, to date, the circulatory and nervous systems of these regions have been depicted using hand-drawn pictures or described but not shown using photographs or corrosion casting images. This present manuscript provides topographical descriptions, measurements, and detailed anatomical images of the glandula thyroidea and the glandula parathyroidea in both male and female rats.

| Samples and perfusion
In total, 10 male and 10 female deceased Wistar albino rats, 10-11 weeks old, weighing 180-220 g, were used. The healthy rats were obtained from the Erciyes University Experimental Research and Application Center according to institutional, national and international ethical guidelines. There was no trauma, infection or drug administration present in the rats used that would affect metabolism.
The thoracic cavities of the rats were opened and 1 mL (25,000 IU) of heparin (Nevparin/Istanbul) in a 10 mL saline mixture was administered via the apex of the heart into the circulatory system until clean liquid emerged from the V. cava cranialis, thereafter processing of the latex injection and corrosion casts commenced. A digital 1/100 calliper was used for all measurements.

| Latex injection
Following perfusion, 6 mL of red latex was injected into the apex of the heart with the aid of a catheter. N = 6 male and 6 female rats.

| Corrosion cast
Following perfusion, 4 male and 4 female rats underwent corrosion casting using cold acrylic (Trade name, Takilon). Red Carmine (0.5 g, Sigma-Aldrich) was added to a mixture of powdered polymethylmethacrylate (1 g) and liquid monomethylacrylate (5 mL) and injected into the apex of the heart (Nur, 1992).
The cranial part of the body, from the level of the heart, was kept at +4°C for 24 h. The cadavers were then placed in 250 mL of distilled water with pancreatin 50 g (350 FIP-U/g protease, 6000 FIP-U/g lipase), ensuring coverage of the entire tissue. Each specimen was then incubated at 37°C and washed gently with running tap water every 4-5 h each day, after which the pancreatin water was replaced. The soft tissues were removed within 3 days, and images were taken following natural drying.

| Statistics
Statistical analysis was conducted using SPSS v26.0 (IBM Corporation). Levene's test for Equality of Variances was undertaken, and all data were normally distributed; therefore, independent samples t-tests were conducted, and significance was determined at p > 0.05. Statistical analysis was conducted for all quantitative measurements which included the glandula thyroidea weight, right and left lobe length, width and breadth (termed thickness), right cranial and caudal pole widths, and the isthmus length, width and breadth (thickness), and the glandula parathyroidea length and width, alongside the diameters of the A. thyroidea cranialis, A. thyroidea caudalis, V. thyroidea cranialis, V. thyroidea caudalis, V. jugularis interna and the A. carotis communis.

| Glandula thyroidea, glandula parathyroidea, isthmus and vascular measurements, anatomical locations and appearance
In the study, both the borders of the glandula thyroidea and its relationship with the anatomical structures in the region were investigated. The measurements created using the latex and corrosion casting, and accompanying statistical analysis, are provided in Table 1.

| Topographic anatomy of the glandula thyroidea
In the supine position, the skin was cut along the median line from the apertura thoracis cranialis to the chin; then, the skin and underlying connective tissue were removed. The muscles on the ventral aspect of the neck were also exposed, and the glandula mandibularis and the The rat glandula thyroidea weights did not differ between males and females at 6.6 ± 3.7 mg in males and 5.5 ± 2.2 mg in females (Table 1; p > 0.575), despite most of the length, width and thickness measurements being significantly larger in males (Table 1; p < 0.05).
Each glandula thyroidea was a reddish-brownish coloured organ consisting of two lobes connected by a narrow isthmus, located ventrolateral to the trachea just behind the larynx. The glandula thyroidea was located at the C1 and C2 vertebral levels in both male and female Wistar albino rats. The glandula thyroidea consisted of two lobes (the lobus dexter and the lobus sinister).The cranial ends of the glandulae thyroidea were pointed. It also extended to the caudolateral edge of the cartilago thyroidea. The caudal ends of the glandulae thyroidea extended to the level of the 3rd or 4th cartilage rings of the trachea, forming a blunter shape compared to the cranial ends.
In rats, the lobus dexter and lobus sinister did not converge dorsal to the trachea; there was no lobus sinister present in one female.
The lobus dexter and the lobus sinister were connected together by a thin, narrow and transparentisthmus (Figures 1-3). The transparent nature of the isthmus made it difficult to identify but it was positioned at the level of 1st and 2nd trachea ring. There was no isthmus present in two females.  In both sexes, 60% of the glandulae parathyroidea was situated on the lobus dexter and sinister, while 30% was at the level of the isthmus, and 10% was in the ventrolateral part of the lateral lobe.
The glandula parathyroidea and glandula thyroidea both had dense vascular networks.

| Blood supply and innervation of the glandula thyroidea
The glandula thyroidea arterial blood supply consisted of branches The ventral branch was larger in diameter than the dorsal branch.  (Figures 4-7).

| Venous drainage
The origin and course of the V. thyroidea cranialis and the V. thyroidea caudalis on the right and left differed from each other (Figures 8-10). On the right side, the V. thyroidea cranialis origi- Thereafter, it opened into the V. subclavian dextra as a single vessel. On the left side, the V. thyroidea cranialis opened into the V. jugularis interna. The V. thyroidea caudalis originated from the caudal end of the lobus sinister and opened 1 cm later into the V.
jugularis interna at the level of the apertura thoracis cranialis. The V.
jugularis interna also opened to the V. cava cranialis.
The V. thyroidea caudalis was larger diameter in males compared to females (Table 1; p = 0.011), whereas no sex differences were observed between the diameters of the cranial veins in relation to the sexes (Table 1; p = 0.291).

| Innervation
Parasympathetic innervation of the glandula thyroidea in the ca-

| D ISCUSS I ON AND CON CLUS I ON
The location of the glandula thyroidea in Sprague-Dawley rats had previously been reported to be at the level C2-C3 (Hadie et al., 2013). In the present study, we demonstrated that the glandula thyroidea of Wistar Albino rats were localized at the levels of the C1-C2 vertebrae in the rats regardless of their sex. Published studies have also shown that the glandula thyroidea extends to the first three or four trachea rings in mice, and the first four or five in rats (Hebel & Stromberg, 1976;Komârek et al., 2000;Krinke et al., 2000;La Perle et al., 2018;Monroe & Turner, 1946), or can even extend up to the 7th trachea ring (Yavru & Yavru, 1996). It was determined that in the present study the caudal ends of the glandulae thyroidea were at different levels compared to those stated by this previous literature, as they extended to the 3rd or 4th trachea cartilage rings ( Figure 10c). In addition, in the present study there was no left lobe in the glandula thyroidea in one female, complimenting previous reports (Alworth & Harvey, 2012;Nakamura et al., 2019).
In one previous study in 8-10 week-old male Rattus norvegicus, the glandulae thyroidea measurements were far larger than any other study at 22 × 6 mm, weighing 1.27 g (Sawsan, 2020), which differs greatly in terms of weight compared to other published studies, and was also different to the present research. In rats, the average size reported was previously reported as 7 × 3 × 3 mm, with the glandulae thyroidea weights ranging from 0.013 to 0.028 g (La Perle et al., 2018). Other rat species have shown similar measurements, including 0.0111 g in the large Neotoma (Mexican Pack Rat), 0.008 g in the medium-sized Rattus norvegicus (Gray Norway Rat) and 0.00115 g in the small Dipodomys (Kangaroo Rat) (Crile, 1937;Midgley, 1938).
Each glandula thyroidea lobe within the rat measured 3-4 mm long, 1.5 mm wide, and weighed approximately 0.005 g (Johanson et al., 1988). According to Hebel and Stromberg (1976), the glandula thyroidea was a pinkish gland on the 4th-5th tracheal rings, 3.9-5.5 mm long, 2-3 mm wide, weighing 0.0154 g in males and 0.0179 g in females. Overall, the measurements from the present study were similar to the majority of the previously published studies, the length of the glandula thyroidea was 4.02-4.34 mm, and the width was between 1.68 and 1.87 mm.
The previously published studies only looked at the combined measurements of the glandulae thyroidea rather than detailed measurements. Therefore, the present study presents novel measurement data of the right and left lobes separately, detailing length, width and depth (thickness) to ensure a more detailed study. It was previously reported that there was a 27% difference in lobe sizes (Nakamura et al., 2019). In our findings, the right lobe measured 4.34 × 1.87 × 0.86 mm in males and 4.02 × 1.68 × 0.80 mm in females, whereas the left lobe was 4.30 × 1.85 × 0.82 mm in males compared to 4.11 × 1.60 × 0.80 mm in females. Therefore, the current study supported that the right and left lobes, in both males and females, were different in size. The present study also analysed male and female glandulae thyroidea measurements individually. The right and left lobe lengths and widths and the right lobe thicknesses were larger in the males compared to females (Table 1; p < 0.05), whereas the left lobe did not differ significantly in thickness between males and females.
In the present study, the glandulae thyroidea weights in the 10to 11-week-old Wistar albino rats were 6.6 ± 3.7 mg in males and 5.5 ± 2.2 mg in females ( in weights and measurements between the studies may be due to differing rat species, ages, weights, variability between researcher measurements and of course potentially differing sexes (especially given any body weight/size differentials between the sexes in the rat). The differences due to sex were not analysed individually within the previous studies; therefore, it is notable that our present study has shown a significant difference between the two. Variability between measurements by differing researchers, and even the precision of the equipment used, is always present between differing research groups. This study reduced intra-observer variables as the measurements were done by one person using the same callipers, but naturally, this cannot reduce the intra-observer variability compared to studies by other groups.
In cats and dogs, the glandulae parathyroideais located anterior or posterior to the glandula thyroidea (Kealy et al., 2010). In the Syrian hamster, it localizes to the anterolateral part of the glandula thyroidea (Kittel et al., 1996b;Pace et al., 2003), measuring approximately 0.7-1 mm × 0.3-0.5 mm in diameter (Kittel et al., 1996b). In mice, the glandula parathyroideais reported as an oval, sometimes longitudinal oval shape (Kittel et al., 1996a), similar to the present findings in the rat. In the present rat study, 60% of the glandulae parathyroidea were located in the cranial part of the lateral  (Hebel & Stromberg, 1976).
Our study notably showed statistically significant decreases in females compared to males (Table 1; p < 0.05).
The isthmus presents as a reasonably wide band of tissue in cows, but in horses, sheep, goats, cats and dogs, it is a narrow tissue remnant and is sometimes absent (Singh & Beigh, 2013;Yamamoto et al., 1995). In the present study, the isthmus presented as a narrow band of tissue and was absent in two female rats, complimenting previous reports (Alworth & Harvey, 2012;Nakamura et al., 2019).
In addition to the locations, size measurements and weights of the glandula thyroidea and the glandula parathyroidea being investigated, the vasculature and innervation of both was also studied in rats.  (Mense & Boorman, 2018). The present study also determined that the gland blood supply was provided via vessels arising from the A.
thyroidea cranialis and the A. thyroidea caudalis, with venous drainage conducted via the thyroid veins pouring into the V. jugularis interna. In addition, a strong anastomosis was detected on the dorsal aspect of the trachea between both arteries (Figure 7c).
Yamasaki reported that in rats, the A. thyroidea cranialis originated from the A. carotis externa as a common root with the A.
pharyngea ascendens (Yamasaki, 1990). According to our findings, this was the case in five female and six male rats (55% in total), the A. thyroidea cranialis originated with the A. pharyngea ascendens, similar to the previous findings (Yamasaki, 1990). In addition, the A. thyroidea cranialis appeared to be included in the arterial supply of the oesophagus, similar to previous findings in dogs (Swenson et al., 1950). Although it was reported that the dorsal branch of the A. thyroidea cranialis was thicker than the ventral branch (Yamasaki, 1990), the dorsal branch was thinner than the dorsal branch in the present study.
One study previously indicated that the A. thyroidea caudalis could originate from the A. phrenicopericardiaca, the truncus brachiocephalicus, and the subclavian or vertebral arteries (Yamasaki, 1990). However, others have reported that it originates from the truncus costocervicalis on the right side in rats (Green, 1955;Maynard & Downes, 2019). In three female rats studied in the present study, the A. thyroidea caudalis originated as two branches from both the truncus costocervicalis and the A.
subclavian dextra on the right side, these branches later joined into a single vessel. In addition, it was determined that it gave branches to the tracheal rings, which has not been stated previously in the literature. Yamasaki also reported that in rats there was no A. thyroidea caudalis on the right side; therefore, nutrition for the glandula thyroidea came from branches of other arteries (Yamasaki, 1990).
The V. thyroidea caudalis vein opened into the V. jugularis interna, but before that, it coursed with the A. thyroidea caudalis and the N. laryngeus reccurens in the tracheoesophageal sulcus.
The cranial and caudal thyroid veins were joined at the level of the apertura thoracis cranialis on both sides of the neck. Later, as also reported in the literature (Green, 1955;Hadie et al., 2013), it was determined that the thyroid veins connected to the V. subclavia dextra on the right side and the V. cava cranialis on the left side.
On the dorsal surface of the trachea, between the lobes of the glandula thyroidea, there was anastomosis evident between the A. thyroidea cranialis and the A. thyroidea caudalis, in line with previous findings (Radlinsky, 2007). Mutuş reported that the pharyngeal veins in rabbits participated in the venous circulation of the larynx (Mutuş, 2001), and Green reported this was undertaken by the pharyngeal plexus veins in rats (Green, 1955). The findings from the present study support those reported by Green (1955).
Although size differences between male and female blood vessels and the differing vessels in general are presented in this study, there are important limitations that must be highlighted. Although injection pressure was maintained at a similar pressure for each animal throughout the process, this cannot exclude small differences and may not always represent the differing pressures exerted during differing physiological timepoints in a living animal.
These limitations also make it more complex when comparing between differing studies, individual animals or species, or even those with different ages, diseases or pathologies. Despite these complexities, it is still worth investigating the basic measurements of the differing vessels.
In conclusion, although there have been many studies on the rat glandula thyroidea, the present research extends the breadth and depth of knowledge regarding topographic anatomy. Detailed measurements of the glands and vasculature, in-depth dissections, and corrosion casts showing locations and detailing innervation and the courses of blood vessels and nerves, add to the literature. In addition, significant differences in the glandula thyroidea, glandula parathyroidea, isthmus and vasculature measurements existed between males and females. This study has also provided photographic evidence and acts as a useful guide for those wishing to understand the anatomy of the glandula thyroidea, the glandula parathyroidea and their associated structures in the rat.

ACK N O WLE D G E M ENTS
The authors would like to thank The University of Nottingham for Open access funding and to thank our universities and departments for supporting our research.