Pododermal angioarchitecture in the equine hoof wall: A light and scanning electron microscopic study of the wall proper

2.1 Micro-corrosion casting and SEM examination

Six fore and six hind limb hooves, respectively, of four adult hoof-healthy warm-blooded horses (Equus caballus forma domestica) were submitted to micro-corrosion casting. All limbs were cast employing the improved micro-corrosion-casting technique described by Hirschberg et al. [27]. The following injection media were used: Tensolcement^® No. 7 (ICI Chemicals & Polymers Ltd., Darwen, Great Britain), diluted with ethylacetate (Fluka, Buchs, Switzerland) on 8 hoof specimens, and Mercox^® (Japan Vilene Hospital Co. Ltd., Tokyo, Japan), diluted with methylmethacrylate (Fluka) on 4 hoof specimens. The casts were subsequently corroded in sodium hydroxide (Fluka), detergent (Fairy Ultra^®, Procter & Gamble, Mainz, Germany) and enzyme (Biozym^® SE; Spinnrad, Gelsenkirchen, Germany) solutions at 40°C complemented by rinsing in warm tap water at regular intervals in between. Finally, the casts were rinsed in distilled water and dried in an incubator at 45°C.

The corrosion casts were then dissected using a thermocausticus and fine forceps and scissors, removing 1×1×1 cm cubes from all regions that showed good vascular filling after stereomicroscopic examination. If possible, proximal, middle and distal samples were taken from the four different regions in the circumference of the wall proper, i.e., from toe, lateral/medial wall, quarter and bar. From each site, if possible at least two samples were taken; one mounted parallel, the other rectangular to the dermo-epidermal border line, in order to allow a complete insight into the three-dimensional angioarchitecture of the parietal pododerma. The specimens were mounted with conductive glue (Leit-C^®; Plano, Marburg, Germany) onto aluminium stubs and coated with gold for 3 minutes (30 – 45 nm) using a sputter coater (Polaron, Watford, Great Britain). Scanning electron microscopic (SEM) examination (Nanolab^® 2000, Bausch & Lomb, Canada) was carried out at an accelerating voltage of 5 to 10 kV.

Angle and spacing of vascular structures and occurrence of non-segment-typical formations such as vascular equivalents of branching lamellae or side-lamellae etc. (see below) were assessed in a qualitative analysis during SEM-examination.

2.2 Histological examination

In order to verify the results gained by SEM examination of the corrosion casts, a concomitant histological investigation of the equine pododerma of the coronary segment, the proximal and distal wall segment (toe, quarter and bar regions only) was added to the scope of the study. Paraffin-embedded specimens taken from the listed localisations of seven hooves (fore or hind limbs) of five adult hoof-healthy warm-blooded horses were sectioned and stained with resorcin-fuchsin-thiazine-red-picric acid according to the standard protocol given by Romeis [55]. In order to display the presumable vasomotor properties, immuno-histochemistry (IHC) employing a primary mouse antibody (MCA1905HT, Serotec, Oxford, UK) against human alpha-Smooth-Muscle-Actin (SMA), an appropriate biotinylated secondary antibody (ShpXMsFab’2 Ig, Chemicon, Ternecula, USA) and chromogen detection system (StreptABComplex/HRP, Dako; 3,3’-diaminobenzidine tablets, Sigma-Immuno Chemicals, St. Louis, USA) was carried out according to the manufacturer’s specifications after protein blocking (X0909, Dako). Appropriate positive (mouse intestinal and porcine kidney tissue, due to the specified cross reactivity of the primary antibody with mouse and porcine tissue) and negative controls (mouse IgG control serum X0931, Dako; buffer control) were included.

2.3 Assessment of histochemical and immuno-reactivity

Histochemical and immuno-reactivity within the whole histological section of each specimen was assessed semi-quantitatively according to staining intensity. No staining was assessed as non-reactive (–), very slight staining as marginal reactivity (±), noticeable staining as reactive (+), strong staining as strong reactivity (++).

2.4 Digital image recording

Representative images from the sections of each specimen were digitally recorded employing a light microscope (Axioskop, Carl Zeiss, Oberkochen, Germany), a digital camera (DS-Ri1, Nikon, Düsseldorf, Germany) and a PC-based laboratory imaging programme (NIS-Elements, Version 3.0, Nikon, Düsseldorf, Germany).

3.1 Filling degree of the corrosion-casts

The employed micro-corrosion-casting technique rendered nearly complete casts of the pododerma in all segments of the hoof (Figs. 1b, c,). In some casts localised filling defects occurred at the junction of coronet and wall proper in the dorsal and lateral regions of both segments. Others showed filling defects in the sole of the hoof. Only 6 of the originally 12 hoof casts showed sufficiently good filling of dermal lamellae of the wall at mesoscopic examination. Thus, suitable well-filled samples from all four regions of the wall proper were obtained for the SEM-examination, but not always from the same individual hooves.

3.2 Comparison of light and scanning electron microscopic findings

Vessel identity, i.e., arterial, capillary or venous, and localisation of the pododermal vessels with regard to the adjacent dermal and epidermal structures of the hoof was established by light microscopy of the histological sections and compared to the findings obtained from the micro-corrosion casts. The findings of both methods were in accordance, as exemplarily demonstrated e.g. in Fig. 4 for the terminal papillae.

3.3 Pododermal angioarchitecture of the wall proper – SEM of micro-corrosion casts

Comparable to the results in a study on micro-corrosion casts of the bovine pododerma [27–29], the surface of the equine micro-corrosion casts in the present study resembled the known surface of the pododermal surface (or dermo-epidermal interface) of the hoof wall (e.g. [8, 11]) as a vascular equivalent in great detail.

The present study follows the anatomical terminology for the equine hoof complemented by the former “Digital End Organ” group at the Berlin Institute of Veterinary Anatomy (reviewed in: [8, 11]).

Thus, at the junction of coronary and wall segment, vascular equivalents of the distal coronary papillae arose from a common basal ledge that was continuous with the vascular equivalent of the primary lamellae of the wall segment. The vascular equivalent of smaller papillae originating from these ledges at the junction area of coronet and wall proper were regarded as vascular equivalent of the proximal cap papillae (Figs. 3h, 4g). Vascular equivalents of small papillae arising from the vessels at the ridge of the primary (and, in case, secondary) lamellae in the distal half of the wall proper were regarded as distal cap papillae vessels (Fig. 3g, 4a), whereas vessels resembling papillae originating from the distal aspect of the primary lamellae at the wall-sole junction were identified as vascular equivalents of terminal papillae (Fig. 4b). The terms for these different papillary and lamellar pododermal surface/dermo-epidermal interface structures were also used for their vascular equivalents in the more general descriptions of the microvasculature as revealed by SEM of micro-corrosion casts.

Notwithstanding region-specific differences in orientation and size of the primary pododermal lamellae as well as of the cap and terminal papillae originating from them, the basic microvascular pattern of these lamellae and papillae was comparable in all examined regions of the wall segment. Incidence, orientation, size and vascular pattern of the secondary pododermal lamellae were varying in the different wall regions.

Arterial and venous sides of the pododermal microvascularisation were distinguished by the typical pattern of endothelial cell nuclei imprints (e.g. Figs. 3f, g): the arterial side of the circulation was characterised by regular elongated, spindle-shaped endothelial cell nuclei imprints orientated along the longitudinal axis of the vessel, whereas in the venous side of the circulation, the cell nuclei imprints appeared more irregular and ovoid to polygonal.

Venous valves were identified by their typical bicuspid imprints in well-filled venous casts or by typically shaped filling defects resulting when the casting media did not overcome the valve (Fig. 2a, insert).

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Fig.2

Angioarchitecture of pododermal lamellae, light microscopy. a-g: SMA immunohistochemistry (positive SMA-reaction, DAB: brown colour in the online version), h: RF-staining 2a: Overview of angioarchitecture of lamellar body in the proximal dorsal wall region, note the wide vessels of the superficial pododermal plexus at the base of the lamellae. Insert: SEM of micro-corrosion cast depicting typical blind ending of filled venous valve, superficial plexus at lamellar base. 2b: Overview of lamellar body in the distal quarter region, the axial lamellar vessels are ascending/descending from the parietal vessels that appear as cross sectioned bundles with accompanying nerves. Note the high secondary lamellae and distinct SMA-reactivity also of intimal cells. 2c: Artery (left) and muscular vein (right) within the deep pododermal plexus, proximal wall segment. Insert: Note the slight SMA-reactivity within the media of the vein. 2d: Two muscular veins at the base of dermal lamellae within the proximal wall segment, left: venous valve, right: intimal protrusion. The insert shows the venous valve in higher magnification. 2e: Lamellar body, proximal wall, toe region: Note the axially ascending/descending lamellar vessels and the short AVA at the base of the middle lamella (arrow), shown in higher magnification within the insert. 2f: Higher magnification of axial lamellar vessels giving rise to the abaxial capillary legs (arrows) for the narrow secondary lamellae of the proximal toe region. 2g: Abaxial capillary legs of high secondary lamellae of the distal toe region. Note the distinct SMA-reactivity of these capillaries. 2h: Higher magnification of abaxial lamellar arteriole with distinct internal elastic laminae delineated by RF-reactivity (reddish-brown in the online version). Also note the elastic elements transgressing the high secondary lamellae to reach the basal membrane at the ridge of the secondary lamellae (arrow).

Differentiation of vascular sprouts from incomplete vascular filling in the micro-corrosion casts was also based on the occurrence and distribution pattern of endothelial cell nuclei imprints (as described by [14]; review e.g.: [50]; for bovine pododerma: [29]): angiogenic sprouts were defined as slender extensions arising from capillaries and small venules, whereby endotheliocytic nuclei imprints appeared more numerous than in non-angiogenic regions of the microvasculature, while pointed blind endings without endothelial cell nuclei imprints were regarded as artefacts resulting from incomplete filling.

Intussusceptive angiogenic figures were identified by their typical non-filled “core” structures within microcorrosion-casts (e.g. Figs. 3f-h, 4f) in early stage intussusception, or by typically shaped branching vascular channels within the microvascularisation (e.g. Fig. 3b) in later stages of intussusception (reviewed e.g. by [12, 60]; for the bovine pododerma, by [29]).

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Fig.3

SEM of microcorrosion casts of dermal lamellae 3a: Overview of lamellae in the toe region; right: proximal, left: distal; note the longitudinally arranged abaxial capillary legs of the secondary lamellae and the capillary loops at the lamellar ridges. Insert: incomplete cast of dermal lamellae, displaying the central axial arterioles and venules of the primary lamellae. The axial lamellar arterioles (arrows) are embedded in the dense axial capillary and venular network of the primary lamellae, the first level of arteriolar arcadiform branching within the lamellae is shown. 3b: Detail showing the abaxial capillary legs of the secondary lamellae. Note second-tier capillary levels (arrow) and small intussusceptive ‘islands’ (asterisk). 3c: Detailed view (insert: overview) onto capillaries of secondary lamellae displaying an ascending afferent capillary leg. Note circularly arranged endothelial cell imprints indicating a capillary sphincter (arrow). 3d: Detail of a capillary sphincter with distinct circular endothelial cell imprints (arrows) within the abaxial capillary legs of the secondary lamellae. 3e: View onto a primary lamella at the buttress of the heel, subdivided by several high secondary lamellae (height indicated by black lines). Each secondary lamella is supplied by a moderately branching two-dimensional microvascular networks, with capillary loops at the lamellar ridges (taken with permission from [30]). 3f: Detail of microvascular networks of higher secondary lamellae at the buttress of the heel. Note intussusceptive “holes” (asterisks) within the peripheral capillary loops at the ridge of the lamellae. 3g: Higher magnification of the ridge of a high secondary lamellae at the buttress of the heel (insert: overview): note the capillary loops displaying sprouting and intussusceptive angiogenesis. 3h: Detail of the marginal network and capillary loops at the ridge of a primary lamella: Note the capillary blind ends indicating sprouting angiogenesis (bottom left) and also the ‘holes’ within the capillaries (arrows) indicating intussusceptive remodelling.

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Fig.4

Cap and terminal papillae. a-d: SEM of microcorrosion casts 4a: View onto the ridge of a primary lamella, proximal wall segment. Distally orientated capillary loops (asterisks) originate from the axial marginal network, supplying the proximal cap papillae. 4b: Distal wall: Overview of terminal papillae originating from the primary lamellae at the weight-bearing margin. Some terminal papillae are incompletely cast, displaying the blind ends of the peripheral vascular loop within the centre of the terminal papillae (asterisks). Note the lower side-lamellae (arrows) in-between the rows of terminal papilla, displaying a two-dimensional microvascular network without abaxial secondary lamellae (taken with permission from [30]). 4c: Origin of a terminal papillae from the distal ‘end’ of a lamella. Note that the capillary legs of the secondary lamellae (arrows) continue into the peripheral capillary network of the terminal papilla. 4d: Detail of the peripheral capillary network of a terminal papilla. Note the dense tortuous peripheral capillary network arranged in rows on the body of the papillae, resembling ‘cannelures’. The capillary network displays numerous intussusceptive “holes” (arrows). Fig. 4e+f: light microscopy, SMA immunohistochemistry (positive SMA-reaction, DAB: brown colour in the online version) 4e: Cross sections of cap papillae in the proximal dorsal wall region. Note the central papillary vessels surrounded by peripheral capillaries. Most intimal cells of the microvasculature display distinct SMA-reactivity. 4f: Cross sections of terminal papillae originating from the distal ‘end’ of the primary lamellae, distal wall region, quarter. Note the marginal axial vessels of the lamella (A = arteriole, V = venule) continuing into the central papillary vessels of the terminal papillae. Also note the short AVA at the base of a terminal papilla (arrow). Most intimal cells including capillaries are distinctly SMA-reactive, while arterioles and the AVA also display SMA-reactivity of media cells.

3.3.1 Pars dorsalis (toe) and lateralis/medialis (quarter) of the wall segment, SEM and light microscopy

The angioarchitecture of the pododermal lamellae and papillae in pars dorsalis and pars lateralis/medialis of the wall proper was comparable. Both regions differed only regarding orientation and spacing of the pododermal lamellae: Whereas in the toe region, the primary lamellae originated radially from the deeper dermal tissue layers and thus were arranged rectangular to the surface of the distal phalanx, in the lateral respectively medial region the primary lamellae were tilted towards the palmar respectively plantar aspect of the hoof2. The spacing of the vascular equivalents of the primary lamellae appeared slightly denser in the dorsal area of the wall proper (Fig. 2a, e).

The angioarchitecture of the primary dermal lamellae consisted of basal parietal3 vessels that gave rise to the lamellar vessels, i.e., arterioles and venules ascending, or descending, respectively, within the axis of the primary lamellae from base to ridge (arterioles) or vice versa (venules) (Figs. 2a, b). The parietal vessels ran proximo-distally within the base of the lamellae and were supplied by the basal dermal blood vessels. This basal vascular plexus was arranged parallel to the surface of the distal phalanx and consisted of a network of proximo-distally orientated veins and arteries connected by transverse collaterals, comparable to ladders and their rungs. Whereas the parietal veins ran continuously within the base of the lamellae, the parietal arteries of each lamella were supplied by several ascending branches and were thus discontinuous throughout the whole proximodistal length of the lamella. In the interlamellar area, both, parietal arteries and veins were connected to the neighbouring lamellar parietal vessels via horizontally orientated transverse connections(‘rungs’).

Within the primary lamellae, the lamellar arterioles rose in a relatively direct course towards the ridge of the lamellae where they divided into proximal or distal arcuate branches, respectively, and then resolved into the peripheral capillary net of the lamellar ridge. Whereas in the proximal third of the lamella, the proximo-dorsally aimed lamellar vessels rose in an acute angle (with regard to the surface of the distal phalanx), in the middle third of the lamella these vessels rose at an approximately rectangular angle, i.e., straight from the base to the ridge of the lamellae. In the distal third of the lamella, this changed again to an obtuse angle aimed distally (Fig. 1a).

Approximately in the middle of the lamellar height4, proximal or distal arcadiform branches, respectively, arose from the lamellar arteriole (Fig. 3a, insert), further branching within the middle of the lamellae and finally giving rise to the middle axial capillary net (Figs. 2f, 3h, 4h). At the base of the lamella, the basal axial capillary net arose from the lamellar arteriole. The basal and middle capillary net drained directly into the heavily branching and irregular network of the lamellar venules (Fig. 3a). At the ridge of the lamella, small distally orientated capillary loops were arising from the distal arteriolar arcades that either drained into the proximo-distally orientated marginal vein running within the lamellar ridge, or directly into the lamellar venules that ran from the ridge to the base of the lamellae. These outer capillary loops of the lamellar ridge often displayed angiogenic figures, i.e. either capillary sprouts or intussusceptive remodelling as indicated by characteristic ‘holes’ and ‘islands’ (non-filled areas) within the capillaries (Fig. 3h)

The abaxial capillary net of the primary lamellae provided the vascular equivalent of the secondary dermal lamellae and consisted of long and straight proximo-distally orientated capillary legs at the base of or in-between the bases of two neighbouring secondary lamellae (Figs. 2f-h, 3b-d). The spacing of these parallel longitudinal capillaries was dense in the ridge-ward periphery of the primary lamellae (Fig. 3a), and more wide-spaced towards the base of the lamella (Fig. 3e). Additionally, the capillary system of the secondary lamellae increased both in density and vascular diameter in proximo-distal orientation. In contrast to the axial capillary network of the primary lamellae, the longitudinal abaxial capillary legs of the secondary lamellae were very straight and regular (Fig. 3b-d). The arterial branch of circulation of these abaxial capillaries arose from the axial lamellar arterioles, and they drained into the axial lamellary venular network (Fig. 2f, g). In micro-corrosion casts, the lamellar capillary network displayed sparse endothelial imprints throughout afferent and efferent legs that were not indicative for preferential flow direction (Fig. 3c). The course of the afferent and efferent capillaries was characteristic for the respective branch of capillary circulation: whereas the afferent capillaries arose and branched in the form of a gentle ‘Y’-type ramification (Figs. 2g, 3c), the efferent venous capillaries drained rectangularly (‘T’-type ramification) into the underlying venular net of the lamella. The site of origin of an abaxial capillary from a lamellar arteriole was often characterised by circular long and narrow endothelial cell nuclei imprints in micro-corrosion casts, indicating precapillary sphincter mechanisms (Fig. 3c, d). Such sphincters were also detected at ramification points within the abaxial capillary network. In most cases, the abaxial capillary system of a secondary lamella consisted of a ‘single tier’ longitudinal capillary leg. In cases of higher secondary lamellae – hence particularly in the distal half of the wall segment - this capillary system developed a ‘second tier’, i.e., the afferent arterial capillary branches gave off a longitudinal proximal and distal branch (first tier), ran further towards the abaxial periphery of the secondary lamella and then again ramified to form the second tier proximo-distally orientated capillary leg (Fig. 3b). The capillary net of the secondary lamellae capillaries frequently showed angiogenic figures, i.e., intussusceptive remodelling of the capillaries (‘holes’ and ‘islands’, see above, Fig. 3b). Distally, at the wall-sole-junction, the paraxial capillary legs of the lamellae were continued by the peripheral capillary net of the distal cap and terminal papillae, i.e., they were prolonged by rib-like capillary loops that were arranged proximo-distally on top of each other at the surface of the papillary body(Fig. 4b, c, h).

Focal dilations were frequent in the axial capillary and venular networks of the primary lamellae, more seldom also in the network of the secondary lamellae. Focal dilations were often delimited by bottleneck-like constrictions on which sometimes sphincter-like circular endothelial cell imprints were detectable.

Short direct and indirect arteriovenous anastomoses between lamellar arterioles and venules occurred irregularly at the bases of the primary lamellae (Figs. 2e, 5a). In the distal wall segment, the axial lamellar vessels continued into the central vessel of the terminal papillae, where often a short direct AVA was situated (Fig. 5b).

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Fig.5

Histology of specialised angioadaptive pododermal features, light microscopy. Figs. 5a, f, g: SMA immunohistochemistry; Figs 5b-e, h-1: RF staining 5a: short and mildly convolutet AVA at base of dermal lamella, note the distinct SMA-reactivity of the media cells. Insert: AVA, RF-staining. Note single light epitheloid cells within the media (arrow). 5b: Intermediary section of short AVA at base of terminal papilla displaying prominent epitheloid media cells. 5c: Arterial segment of convolutet AVA, base of dermal lamella. Note the VSMCs of the media arranged in longitudinal strands rather than circularly (arrows). 5d: Artery within the deep pododermal plexus, displaying intima thickening with increased subendothelial material as well as VSMCS longitudinal strands beneath the internal elastic membrane and towards the outer elastic membrane (arrows). 5e: Artery within the deep pododermal plexus, displaying strong intimal thickening, Insert: Higher magnification of vessel wall, depicting RF-positive elastic material within the subendothelial layer. 5f: Deep pododermal plexus of the wall, displaying artery with non-SMA- reactive intimal cushion flanked by thin-walled vein (or lymphatic vessel). Also note the distinct SMA-reactivity of the intima of the vein/lymphatic. 5g: Muscular vein within deep pododermal plexus of the proximal wall region displaying intimal thickening with very discrete SMA-reactivity and slight reactivity of the media cells. 5h: Small muscular vein with intimal cushions, superficial layer of the wall pododerma. Note the fragementary internal elastic membrane as delineated by RF-reactivity. 5i: Small muscular vein with high intimal cushion containing a strand of longitudinally arranged VSMCs, superficial layer of the wall pododerma.

Besides the secondary-lamellae-bearing main, i.e., primary lamellae, few lower, non-segment-typical lamellae appeared that were regarded as side lamellae. Their angioarchitecture consisted of rather simple, two-dimensional arcuate axial networks with distinct capillary loops at the ridge of the lamella (Fig. 4b).

The basic angioarchitecture of the proximal and distal cap papillae, respectively, as well as of the terminal papillae (Fig. 4), consisted of a centrally situated papillary arteriole and venule surrounded by a network of subepidermal capillaries and venules. At the tip of the papillae, the arteriole drained directly into the venule, thus forming a peripheral capillary loop.

The subepidermal network ensheathed the afferent and efferent central papillary vessels (Fig. 4b). Particularly in the distal cap and the terminal papillae, this capillary network was heavily convoluted (Fig. 4d) - reminiscent of tortuous vessels [39] -, and formed rib-like loops, one upon the other in proximo-distal direction, as vascular equivalent of longitudinal micro-ridges of the papillae (“cannelures” 5) (Figs. 4b-d). At the base of these papillae, the course of the abaxial longitudinal capillary legs of the secondary lamellae continued by the rib-like loops of the terminal papillae was evident (Fig. 4c). In the proximal part of the terminal papillae, this rib-like formation of convoluted capillary loops was confined to the dorsal ridge of the papillae only, whereas the sides of the papillae displayed comparably straighter capillary legs. The whole peripheral capillary net of the terminal papillae displayed features of intussusceptive remodelling (‘holes’ and ‘islands’, see above), particularly at the base of these papillae(Fig. 4d).

The angioarchitecture of the mobile part of the wall segment generally resembled that of the dorsal part. Differences were only related to the varying shape and spacing of the dermal lamellae in these parts of the wall segment: The spacing of the vascular equivalent of the primary lamellae was less dense compared to that of the lateral respectively medial region. Branching primary lamellae were more common than in pars dorsalis and pars lateralis/medialis of the wallsegment.

3.5 Pars inflexa (bar) of the wall segment

The pars inflexa of the wall segment is comprised of the buttress of the heel (reverse point of the wall) and the medial respectively lateral bar. As in the other regions of the wall segment, the lamellae of the pars inflexa were continuous with the basal ridges of the coronary segment. Proximally, the ridges of the lamellae were fringed by distinct and numerous (proximal) cap papillae (Fig. 3g).

In the bar area, the surface of the primary lamellae was subdivided into numerous secondary lamellae far proximally. These secondary lamellae were distinctly higher than in the dorsal, lateral and mobile region of the wall segment, and were vascularised by an already moderately branching capillary system (Figs. 3e, 3f). Very high secondary lamellae in the bar were characterised by the same microvascularisation pattern as the primary lamellae in the other wall regions, i.e., arcuate arterioles and venules giving rise to a peripheral capillary net (Fig. 3f). These high secondary lamellae were in part further subdivided into tertiary lamellae, which in turn showed the same microvasculature as low secondary lamellae in the other wall regions, i.e., paraxial longitudinal straight capillary legs. Like the ridge of the primary lamellae, the secondary lamellae gave rise to small capillary loops as vascular equivalent of (proximal) cap papillae in the proximal area of the bar (Fig. 3e-g).

Low to medium height side lamellae were much more frequent in the bar than in the remaining pododermal areas of the wall segment. In-between the main lamellae, low vascular arcades, i.e., side lamellae, were detected that gave rise to extended capillary loops. Very low side lamellae gave rise to short convoluted capillary loops that were mainly non-ramifying.

At the buttress of the heel, the primary and secondary lamellae were lower and less subdivided than in the bar proper, whereas branching primary lamellae were more frequent in this region of the wall.

3.6 Histological characterisation of specialised vascular structures

The arterial side of the pododermal vascularisation featured arteries and arterioles of distinct muscular type (Fig. 2), with well-developed internal elastic membranes (as indicated by strong RF-reactivity, see Fig. 5).

Vascular smooth muscle cells (VSMCs) within the tunica media of the arterial side of the pododermal vascularisation displayed a strong immuno-labelling employing the Smooth-Muscle-Actin (SMA) antibody. Within the lamellar microvasculature, not only the VSMCs of the arterioles were strongly immuno-reactive to SMA, but also some cells within the tunica intima (endothelial cells, pericytes) of the lamellar capillaries displayed slight to strong SMA-reactivity (Figs. 2, 5).

The venous side of the pododermal circulation displayed both, venous systems characterised by very thin vessel walls with indistinct/inconspicuous tunica media even in large calibre veins (Figs. 2, 5), and veins displaying a muscular media (Figs. 2c-d, 5g-i). Pododermal veins of the muscular type within the proximal wall areas were identified according to their distinct to moderate SMA-reactivity of media cells while displaying only fragmentary elastic elements at the border to the intima (Figs. 5h-i) (as opposed to the wholly circumferent elastic lamina in arteries/arterioles) as well as by occurrence of bicuspid valves (Fig. 2d, insert). SMA-reactivity of venous media cells was slighter than in arteries of comparable calibre, and also often with altering intensity within the vessel circumference (Figs. 2c, 5g) The muscular venous system occurred predominantly in vicinity to the coronary segment (where they are common, data not shown in this study), i.e. in the proximal regions of the wall segment.

Both venous vessel types displayed bicuspid valves. They were traced up to the border between the deep (Str. reticulare) and the superficial layer (Str. lamellatum sive lamellare) of the dermis. The ‘last’ 6bicuspid valves were detected within the veins situated at the base of the dermal lamellae (Fig. 2a, insert, 2d).

At the border of deep and superficial layer of the dermis, as well as directly beneath the base of the dermal lamellae, specialised arteries/arterioles were detected. These arteries/arterioles displayed three different modification types (sometimes even concomitantly within the same vessel region): These modifications were characterised by specialised VSMC ‘media strands’ - with the muscle cells within the strands arranged parallel to the vessel axis instead of the ‘normal’ circular arrangement of VSMCs (Figs. 5c-e). The second type was characterised by intimal ‘cushions’ and showed localised protrusion of thickened subendothelial material (Figs. 5c-e) with SMA-reactivity ranging from non to slight to distinct (Fig. 5f), which was particularly well defined with regard to the RF-positive internal elastic membrane (Fig. 5d, e).

Equivalent alterations were also detected within the muscular venous system of the proximal wall areas. Such muscular veins sometimes even displayed VSMCs within the intima, as displayed by SMA-reactivity (Figs. 5 h, i).

The third type displayed an only slight to moderate immuno-reaction against SMA within the innermost layers of the tunica media - indicative of a less-contractile differentiation of these cells, comparable to the epithelioid cells of the AVAs (Fig. 5b).

Convoluted or - more seldom - straight AVAs were detected infrequently - with no apparent regular arrangement within the vascular pattern - at the border of the deep and the superficial layer of the dermis. In cross sections, the smooth muscle cells within the intermediary section of the AVAs sometimes seemed to have an ‘epithelioid’ character (Figs. 5a, b). Most of the examined AVAs displayed a predominantly continuous (Fig. 5c), sometimes incomplete internal elastic membrane as displayed by RF-reactivity. The inner layer of the tunica media cells within the “epithelioid” media cells of the intermediary section of AVAs often displayed only slight to none SMA- reactivity.

As a secondary finding, RF-positive fibres within the pododerma were present in the perivascular material bundling larger arteries, veins and nerves, as well as within the trajectory fibre bundles of the hoof suspensory apparatus, traceable up to the dermal secondary lamellae (Fig. 2h).

4.1 Angioarchitecture in relation to dermo-epidermal interface

In the equine hoof, the pododerma of the wall segment connects the surface of the distal phalanx to the horn-producing epidermis via a region-specific modified papillary, respectively, lamellary body and a corresponding system of directional tense fibres. Thus, the pododerma is adapted to the particular forces affecting the respective regions of the wall segment, and in co-ordination with the according epidermal structures of the hoof wall forms the highly specialised weight bearing apparatus of the distal phalanx [26, 62].

Whereas in pars dorsalis and pars lateralis/medialis of the wall, the actual weight-bearing function of the dermo-epidermal apparatus prevails, pars mobilis and pars inflexa mainly brace and stabilise the hoof capsule to the palmar processes of the pedal bone [26].

The pododermal vasculature is likewise subject to the forces affecting the pododerma, therefore the angioarchitecture in the respective wall regions reflects and corresponds to the conformation of the papillary respectively lamellar body and the direction of the connective tissue fibre system.

In those areas of the wall segment exposed to high pressure loads during the weight bearing process and therefore subject to tissue compression, i.e., the weight-bearing margin (white-line-area), the buttress of the heels and the bar, the dermo-epidermal interface is particularly subdivided in order to increase the force-bearing surface (formation of cap and terminal papillae, high secondary lamellae, in part formation of tertiary lamellae). These weight-bearing forces are also transferred to the microvasculature of these tissues, which requires permanent and temporary adaption mechanisms of the blood vessels.

4.2 Microvascularisation and specialised angio-adaptive mechanisms in the lamellae and terminal papillae of the hoof wall

The systematic examination of the angioarchitecture in all regions of the wall proper combining macro- and microscopical methods enabled a thorough insight into the parietal pododermal microvasculature and thus allowed to complement and adjust the results of earlier studies.

The principal microvascular pattern of the primary dermal lamellae with arcadiform lamellar arterioles draining into the marginal vein or venular lamellar network established by earlier investigations [46, 64] was verified by the present results. Marais [46] described only the angioarchitecture in the distal portion of the dermal primary lamellae – without precise information of the localisation within the circumference of the hoof wall - and had to admit filling defects in the ‘perpendicular abaxial capillary plexus’ that had already been described by Mishra and Leach [51]. These authors reported on the venular and capillary system of the primary and secondary lamellae of quarter and heel, but had to concede filling defects in the dorsal (toe) region of the wall proper [51]. The up to now most complete insight into the lamellar micro-vascularisation of the hoof was delivered by Pollitt and Molyneux [64] who reported filling defects in the toe region, described complete lamellar filling in the heel region only and generalised the findings obtained from this region.

They reported a high density of arteriovenous anastomoses at different levels within the lamellar angioarchitecture as well as at the basis of the dermal lamellae. According to their observations, AVAs maintain the blood flow during high pressure phases in the pododerma by shunting from artery to the vein and bypassing high resistant capillaries simultaneously [64, 67]. In a micro-corrosion study combined with a concomitant histological examination, Nasu et al. [56] described the occurrence of AVAs as restricted to the basal portions of the dermal papillae and lamella, although vessel identity could not be confirmed by the typical pattern of endothelial cell nuclei due to insufficient replication quality, and histological serial sections were not described. Likewise, in our study we detected short in-/direct AVAs between lamellar arterioles and venules irregularly at the bases of the primary lamellae and terminal papillae as well as convoluted and few straight anastomoses at the border of the deep and superficial layer of the dermis. A particular form of AVA, the so-called Hoyer-Grosser-organ or glomus organ, has been described in the skin of the equine mammary gland [43] that like the hoof resembles a specific skin modification. The Hoyer-Grosser organ is a highly convoluted indirect AVA, surrounded by wide-diameter venous and lymphatic vessels within a tight connective tissue capsule [43]. The morphology of the convoluted indirect AVA described in our study at the base of the primary lamellae and the terminal papillae of the hoof resemble that of the Hoyer-Grosser-organs of the equine mammary gland. Earlier studies on histological detection of AVAs in the equine hoof also described either glomus-type AVAs [72–74] or small, simple direct AV shunts [53, 73], localised in the perioplic and solear dermis, within the coronary and terminal papillae, at the base of the dermal laminae and at the entrance to and along the length of the dermal laminae.

Due to its capillary morphology we define the peripheral vascular loop connecting central papillary arteriole and venule of the terminal papillae, and central lamellary arterioles and venules within the primary lamellae, as thoroughfare channels (as defined by [7, 35], as previously described by Hirschberg et al. [28] for the bovine pododerma) – rather than an AVA as described by Pollitt and Molyneux [64].

Beside these blood flow-regulating devices we frequently detected focal dilations in the axial capillary and venular networks of the primary lamellae. For the first time, Mishra and Leach [51] described focal enlargements within the axial venular and capillary net of the primary lamellae that were regarded as possible temporary accommodations for the retrograde flow of venous blood not drained out during the redistribution of blood which may occur in concussion. They also reported on possible vascular sphincter mechanisms related to these enlargements, presumably acting in a manner equivalent to arteriovenous shunts in regulating the perfusion of the capillary bed within the lamella [51], as supported and complemented by the results of the present study.

Focal enlargements are also observed in the microvasculature of the bovine claw [28] as well as within the microvasculature of the equine periodontal ligament [48, 49]. Like the suspensory apparatus of the equine hoof, the periodontal ligament has to transfer high mechanical load between two hard substances, i.e. the tooth and the mandibular/maxillary bone [48, 49]. The ballooned venules in the periodontal ligament are described as a part of a fibro-vascular arrangement and were also considered as a shock absorbing system in an organic system that is exposed to regular concussions [76]. Masset et al. [48] interpreted the focal enlargements alternatively as the initial step of an intussusceptive remodelling of the blood vessels characterised by an increase of the vessel diameter [44]. Like AVAs that can emerge quickly in thermoregulatory need or induced by venous congestion/hemostasis [52], vascular sprouting as well as intussusception represent further adaption processes induced by dynamic forces which can take place rapidly [12, 40]. In our study we also observed angio-adaptive figures, i.e. either capillary sprouts or intussusceptive remodelling, frequently within the capillaries of the lamellar ridge and secondary lamellae and particularly within the terminal papillae, i.e. parts of the suspensory apparatus that are exposed to strong mechanical pressure and tensile forces [26, 63] and therefore require adaptive remodelling.

4.3 Functional consideration - Intima and media modifications in the vasculature of the hoof pododerma

An early study on the arterial branches from the terminal arch supplying the hoof pododerma already described specialised arteries with intimal cushions and specific modification of the VSMCs of the vessel wall media [77], now complemented by our own study of the wall pododerma supplied by these branches. With regard to their morphology and SMA-reactivity, in our own study we observed three different types of specialised arteries/arterioles at the border of the deep and the superficial layer of the dermis, as well as directly beneath the base of the dermal lamellae. They were either characterised by specialised VSMC ‘media strands’, or by ‘intimal cushions’ protruding into the vessel lumen, or by less-contractile, epithelioid cells within the innermost layer of the tunica media.

Among others, epithelioid cells frequently occur in the tunica media within the intermediate segment of AVAs [20, 53]). Their function is not clearly defined yet. Depending on their localisation in different organs they have a different expression profile of smooth muscle actin as well as intermediate filaments, e.g. desmin and vimentin [21, 57], possibly indicating that they may help to stabilize the vessel wall. They also contain a high number of organelles and are able to respond to vasotonic substances like endothelin and nitric oxide [21, 57], thus indicating a role in paracrine perfusion regulation. Early studies suggested that epithelioid cells regulate the width of the arteriovenous lumen by increasing respectively reducing their intercellular volume (reviewed by [73]).

Intimal cushions in the arteries/arterioles corresponding to those detected in the present study have already been described by Schummer and Studier within the wall of digital blood vessels in the equine hoof [73, 77]. Based on his literature review, Studier found arteries with intimal cushions occurred in organs with considerable blood flow fluctuation, and thus defined them as sphincter arteries [77] – a view that we share. Regarding the question whether occurrence of these specialised vessels is physiologic rather than pathologic, Studier considered these sphincter cushions as physiologic compensatory adaptive blood flow regulation mechanisms, because they were detectable even in the vessel walls of 7-day old foals [77].

On the other hand, the more generalised intimal thickening detected in our study particularly in the pododermal arteries can also be interpreted as sclerotic angiopathy, possibly related to the high vascular load in the hoof. Comparable vessel wall alterations characterised by increased fibrous and/or elastic compounds in either intima or media are described as so-called ovulation sclerosis of the ovary, or – more intensely studied – as pregnancy-sclerosis of endometrial vessels in various species (humans, cattle, small ruminants, buffaloes, reviewed by [31]), and best characterised for mares [18, 58]. These sclerotic and/or elastotic alterations are interpreted as the result of vascular remodelling related to hemodynamic and hormonal alterations during pregnancy, often accompanied by phlebectasia and lymphangiectasia [22]. In our present study, thin-walled veins (and possibly also lymphatic vessels) with very wide lumen were detected in the deep and superficial pododermal plexus in the present study, maybe also indicative of load/hypertension-induced changes in the efferent vessels of the hoof. True differentiation of such enlarged veins and lymphatic vessels was not possible with the employed techniques, particularly because samples for histology were taken from either exsanguinated or perfusion-fixed cadavers.

In our study, equivalent alterations as reported for the pododermal arteries, i.e. intimal cushions, strands of axial VSMCs, were also detected within muscular veins, which could be differentiated from arterial vessels of the same calibre only by their fragmentary elastic elements at the border to the intima rather than the distinct and circumferent internal elastic lamina typical for arteries. Rather than possible pathological changes as described above, these veins can also be considered as sphincter veins facilitating the blood flow against a high hydrostatic pressure, as already described by Schummer [75]. Astonishingly, most hoof literature, and even more recent review articles (e.g. [36]) and editorials (e.g. [70]) state that veins within the equine digit have no valves but thick muscular walls; on the other hand, the role of venoconstriction in laminitis has been described while it is not clear whether laminar veins contain sufficient smooth muscle to significantly alter the hemodynamics of the foot [69, 71]. The profound study of Schummer distinctly described venous valves within the equine pododerma based on both corrosion casts and histological examination [74]. Likewise, Schummer showed that generally the pododermal veins had a very thin and simple wall structure with no apparent VSMCs within the media [74]. Only specialised veins within the subcutaneous layer of the coronary segment displayed localised, circularly arranged VSMCs acting as throttling devices within this venous system [73–75]. This corresponds with the finding of our study that muscular veins were detected predominantly in the proximal area of the wall segment, close to the coronet. In our study, we were able to trace venous valves up to the veins in the superficial pododermal plexus at the base of the dermal lamellae. This complies both with the profound early studies on the hoof vasculature and peripheral circulation devices [73–75], and a recent profound literature review on occurrence of venous valves within the body, suggesting the importance of venous valves and micro-valves in preventing blood reflux in small-sized veins and restriction of flow form postcapillary venules back into the capillary bed, particularly in the distal limbs [13].