The microscopic anatomy of the equine dental pulp: Histomorphometric and stratigraphic analysis of the dental pulp in equine incisors and molars. The developing field of equine dentistry requires detailed knowledge of equine-specific tooth morphology and anatomy in order to apply complex treatment techniques such as endodontic therapies. The fundamental differences between hypsodont teeth of horses and brachydont teeth of humans and small animals (dogs, cats) preclude the transfer of knowledge gained on brachydont teeth. Accordingly, studies on equine specimens are required to generate morphological basics for the development of equine-specific therapies. The permanent dental wear of the equine tooth requires a continuous production of dentin, even in older horses, to prevent occlusal exposure of the vital pulp tissue. However, the pulp of brachydont teeth of humans is considered to develop a less active and mature status with age which is indicated – on a morphological level – by a stratigraphic arrangement containing different zones in the pulp periphery. Therefore, the question arises whether the functional differences of a lifelong dentin production in the hypsodont tooth reflects morphological differences in the endodontic system of equine teeth. The aim of this study was to perform a detailed anatomical description and analysis of the equine dental pulp using histological and immunohistochemical examination methods. Special attention was paid not only to age-related histomorphometric changes (length, width and area in cross-section), but also histomorphological characteristics and their changes in relation to age were analyzed (odontoblasts, mineralization of predentin, blood vessels, nerve fibers and cellular components). In total, 10 incisors (including 4 deciduous incisors) from the lower jaw and 10 cheek teeth (including 4 deciduous cheek teeth) from the upper jaw were used for this study. Postmortem, a macroscopic examination of all teeth was performed, and only clinically healthy teeth were used for further histological and immunohistochemical examinations. All specimens were stored in 10% buffered formalin. Subsequently, horizontal sections were taken from three defined levels (subocclusal, central, apical) using a diamond-coated, water-cooled micro-band saw. After 8 weeks of decalcification in EDTA solution, the specimens were embedded in paraffin wax and histological sections were prepared and stained in toluidine blue. A subset was examined by immunohistochemical staining techniques for blood vessels (von-Willebrand factor VIII for the detection of blood vascular endothelial cells) and neuronal structures (neurofilament protein for the detection of nerve fibers). In contrast to adult horses, the horizontal expansion of the dental pulp (length, width and total area) in deciduous incisors and cheek teeth of foals (AG 1) was significantly enlarged. In deciduous incisors a nearly oval shape in the subocclusal area was observed, tapering in apical direction. In permanent incisors of adult and older horses (AG 2 and AG 3) the horizontal expansion of the pulp remained nearly constant, forming a straw-like shape. The same phenomenon was found in cheek teeth of older horses (AG 3). In contrast to the latter finding, the horizontal expansion of cheek teeth of horses of AG 1 and AG 2 was enlarged in apical direction, forming a cone-like shape. Although the expansion of the equine dental pulp changed with age, the width of the odontoblastic layer (20–40µm) and the width of the predentin (7–14µm) remained constant in incisors as well as in cheek teeth. Furthermore, no age-related changes of the shape of the odontoblasts were observed. Additionally, two thirds of the examined teeth showed a linear as well as a globular mineralisation front, displaying a wave-like and ongoing mineralisation. Immunohistochemically stained sections were used to evaluate the blood vessel and nerve fiber density. In general, the density of blood vessels showed an increasing trend with age in incisors as well as in cheek teeth. The highest density of blood vessels with a diamter < 10 µm was detected in the periphery of the equine dental pulp, adjacent to the odontoblastic layer forming a nutritive capillary network. Blood vessels with a diameter up to 50 µm or even larger were not obtained in the periphery but in the central area of the dental pulp. The lowest density of nerve fibers was recorded in deciduous incisors as well as in deciduous cheek teeth of foals (AG 1), displaying a more or less uniform distribution. In contrast, an accumulation of nerve fibers was observed in permanent incisors and cheek teeth of adult and older horses (AG 2 and AG 3) in the periphery of the dental pulp, where heavily branched nerve fibers were obtained within the subocclusal area, suggesting the presence of a subodontoblastic nerve fiber plexus. Additionally, an overall decrease of nerve fiber density in incisors and cheek teeth was recorded from subocclusal to apical. In incisors, the density of fibroblastic cells remained almost constant in all age groups, whereas cheek teeth showed a significant decrease. The highest density of fibroblastic cells was detected in the periphery of the dental pulp, similar to the distribution pattern of blood vessels and nerve fibers. A stratigraphic arrangement as described for brachydont teeth of humans (cell-free and a cell-rich zone in the periphery of the dental pulp), indicating a mature and less active status was not observed within the equine dental pulp. The presented results illustrate massive histomorphometrical as well as histomorphological differences between the equine dental pulp and the dental pulp of brachydont teeth. The histological characteristics found in the equine dental pulp indicate an age-independent and highly active status of the dental pulp, maintaining a continuous production of secondary dentin. This specific characteristic of the equine pulpal tissue is considered as an evolutionary adaptation to the lifelong occlusal wear of the tooth. Thus, under physiological conditions occlusal exposure of the sensitive pulpal tissue due to abrasion is avoided; under pathological conditions the pulpal tissue is able to react quickly and efficiently to pathogenic stimuli. Further investigations are needed to evaluate the potential use of the high productivity of the equine dental pulp for the development of equine specific therapeutically strategies.