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A Special Construction of Subepidermal Capillary Loops in the Hippopotamus(Hippopotamus amphibius)Author(s): Wilfried MeyerSource: Zoological Science, 29(7):458-462. 2012.Published By: Zoological Society of JapanDOI: http://dx.doi.org/10.2108/zsj.29.458URL: http://www.bioone.org/doi/full/10.2108/zsj.29.458

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2012 Zoological Society of JapanZOOLOGICAL SCIENCE 29: 458–462 (2012)

A Special Construction of Subepidermal Capillary Loops in the

Hippopotamus (Hippopotamus amphibius)

Wilfried Meyer

Institute for Anatomy, University of Veterinary Medicine Hannover Foundation,30173 Hannover, Germany

Based on LM, TEM, and histochemical methods, the study describes the specific structure of sub-

epidemal capillary loops in the integument of the hippopotamus (Hippopotamus amphibius). At 25–

60 μμm, the diameter of the capillaries was more than twenty times larger than those found in other

mammals, as was the diameter of the epidermal contact area of the hairpin turn, which had

enlarged up to 200–400 μμm2. At about 13,400, the number of loops per cm2 was three times higher

than in the few other mammalian species measured to date. The remarkable sheath (thickness 2–

20 μμm) of the capillary loops consists of a multitude of fine collagen IV fibres, which were in direct

contact with the epidermal stratum (str.) basale, emphasizing an origin from the lamina fibroretic-

ularis of the basement membrane. Additionally, the sheath contained many regions filled with free

fatty acids. All observations confirmed the view that the walls of the subepidermal capillaries in the

hippopotamus are adapted to withstand high blood pressure, permitting a high rate of blood vessel-

based heat transfer from the periphery of the body. Until now this function is only known as an

important thermoregulatory response in highly active mammals, e.g. dolphins. However, under hot

climatic conditions but without strong exercise for cooling, such ability could be an effective and

energy-saving procedure in semi-aquatic mammals.

Key words: hippopotamus, subepidermal capillary loop, integument, thermoregulation, aquatic mammals

INTRODUCTION

The epidermis of the hippopotamus appears rather

smooth and free of keratin flakes. Its thickness without the

deep epidermal papillae varies about 0.2 mm, including the

papillae it varies between 0.7 and 1.0 mm at the dorsum,

and 0.5 and 0.9 mm at the ventrum. The str. corneum nor-

mally is compact but with about 100 μm relatively thin, and

the deeper part, i.e., the thick vital epidermis, generally has

a homogeneous character (Luck and Wright, 1964; Meyer

et al., 2011a, b). Recent studies have demonstrated that the

cells of the latter epidermal part accumulate free fatty acids,

whereas the corneal layer shows such lipids only in the

intercellular spaces (Meyer et al., 2011a). Contrary to these

observations, a typical barrier region between the str. cor-

neum and the str. granulosum containing glycolipids, nor-

mally present in haired mammals from different groups, is

missing in the hippopotamus (Meyer et al., 2011b). The spe-

cific functions of the epidermal system of this animal regard-

ing its adaptation to the aqueous medium are still for the

most part unknown or controversial. One problem is the

thermoregulation of the species in water and on land,

whereby varying water temperatures lead to variations in

behavioural thermoregulation, i.e., the skin temperatures

vary with the environmental temperature (Cena, 1964), the

exposure of the animals lasts longer in cool water, and sun-

bathing occurs during the hottest hours (Noirard et al.,

2008). Thermoregulation on land, however, results in an

increase of no more than 1°C in core temperature under hot

environmental conditions during the day. Additionally, the

rising adverse radiation and convection heat load is met by

an increase in evaporative water loss from the skin, still

attributed to higher activities of the tubular apocrine skin

glands (Olivier, 1975; Wright, 1987). Realizing that the apo-

crine skin glands in wild mammals—in contrast to the

eccrine tubular glands of humans and hominids—cannot be

defined as sweat glands, but rather as glands the secretion

of which has several important protective functions on the

epidermal surface (e.g., Meyer et al., 1994, 2008b; Yasui

et al., 2007; for review see Meyer, 2010a, b). Thus, intense

water movement through the skin of the hippopotamus must

have another structural basis. In this context, the present

study tries to give a biologically relevant explanation of the

phenomenon mentioned above, using different histological,

histochemical and electron microscopical techniques.

MATERIALS AND METHODS

Skin material from the dorsolateral or lateroventral body

regions of one adult female and one adult male (Hippopotamus amphibius), fixated in 4% formol and stored in 70% ethanol, was

obtained from the collections of the Institute for Anatomy and the

Institute for Zoology (University of Veterinary Medicine Hannover

Foundation, Hannover, Germany). Additionally, a series of HE

stained sagittal paraffin sections from the dorsolateral body region

of one juvemile hippopotamus (sex not known) could be evaluated

regarding basic structural information; this material had been found

in the collection of the Institute for Anatomy.

Standard histology and lipid staining: Small tissue blocks were

* Corresponding author. Tel. : +49-511-856-7214;

Fax : +49-511-856-7683;

E-mail: wilfried.meyer@tiho-hannover.de

doi:10.2108/zsj.29.458

Subepidermal Capillaries of Hippopotamus 459

prepared, carefully dehydrated with graded ethanol and embedded in

the water-soluble and rather shrinkage-free 2-hydroxy-methacrylate

Technovit® 7100 (Heraeus-Kulzer, Wehrheim/Ts, Germany)

(Hanstede and Gerrits, 1983). 3 μm plastic sections were cut with

a motor driven rotation microtome (Autocut, Reichert-Jung, Nussloch,

Germany) and transferred to slides. In order to evaluate the quality

of tissue preservation and for general structural analysis, sections

were stained with hematoxylin and eosin (H & E, hematoxylin accord-

ing to Delafield; Boeck, 1989). Free fatty acids (and triglycerides)

were stained with the very sensitive red fluorescence dye BODIPY®

665/676 [(E,E)-3,5-bis-(4-phenyl-1,3-butadienyl)-4,4-difluoro-4-bora-

3a,4a-diaza-s-indacene] (Molecular Probes Inc., Eugene, OR,

USA), according to Meyer et al. (2009). For control, the material

was treated in two ways: a) plastic sections were immersed at room

temperature for 48 and 72 hrs. in acetone (abs.) prior to staining

with the two dyes; b) small skin samples were immersed in a mix-

ture of chloroform (abs.) and methanol (abs.) for 24, 36, and 48 hrs

at room temperature, afterwards embedded via ethanol (abs.) in

Technovit® 7100, whereby control sections and normal plastic sec-

tions were treated together as described beforehand to achieve rel-

evant lipid staining results of both probes.

Immunohistochemistry: Skin samples of the adult animals were

embedded in paraffin wax (Paraplast plus, Covidien, Neustadt,

Germany) according to standard protocols, 8 μm paraffin sections

were deparaffinized, carefully hydrated and used for the determina-

tion of (a) collagen IV (dilutions 1:50 and 1:100; anti-bovine, from

rabbit, polyclonal; Biologo, Kronshagen, Germany), and nidogen-1

(dilutions 1:50, 1:100; anti-human from goat, polyclonal; R&D

Systems, Wiesbaden-Nordenstadt, Germany). Following incubation

over night at 4°C, the reaction was detected by the EnVision® sys-

tem (Dako, Hamburg, Germany), using peroxidase-based very sen-

sitive dextran-polymer visualization. One part of the sections was

also digested for 30–60 min with 0.1% trypsin (from porcine pan-

creas, type II, crude; Sigma-Aldrich, Deisenhofen, Germany)

(Hautzer et al., 1980), or incubated for 30 min in TEC buffer at 90°C

prior to the reaction. Control sections were incubated without the

antibody and/or the visualization system.

The light microscopical and histochemical results were docu-

mented with a Zeiss Axioskop equipped with an epifluorescense

device (FITC filter combination, BP450-490, FT510, LP520) and a

digital camera (Olympus DP70; software Olympus DP-SOFT, ver-

sion 3.1 and 3.2). The epifluorescence device was also used for an

autofluorescence analysis of the plastic sections, as autofluores-

cence can rise from structural proteins, in particular collagen and

elastin, which can be considered the most important fluorophores in

the extracellular matrix (Monici, 2005; Hagiwara et al., 2011).

Transmission electron microscopy: In view of the fact that stan-

dard TEM fixation (e.g., Karnovsky’s solution) could not be performed

as fresh material was not available, the formalin-fixed material had

to be used. This was washed in PBS and postfixed in buffered 1%

osmium tetroxide (Millonig, 1961). After careful dehydration in

graded ethanol, all samples were embedded in Epon 812 (Serva

Electrophoresis) (Luft, 1961), and cut with a diamond knife on the

ultramicrotome Ultracut E (Leica Microsystems). Semithin sections

were stained with 0.2% toluidine blue O (Richardson et al., 1960);

thin sections (< 100 nm) were contrasted with methanolic uranyl

acetate (Stempak and Ward, 1964) and lead citrate (Reynolds,

1963) and viewed in the electron microscopes EM10 and EM10C

(Carl Zeiss, Jena, Germany) operated at 60 kV.

RESULTS

In all animals studied, dermal papillae inserting in the

epidermal ridge pattern were centrally occupied by small

blood vessels, which typically formed one capillary loop per

papilla (Figs. 1, 3A). These vessels arose from a terminal

arteriole more or less in the superficial horizontal plexus, as

followed by an ascending capillary limb having a hairpin turn

very close to the epidermal basement membrane, and a

descending capillary limb that was connected with a postcap-

illary venule in the horizontal plexus. In the adult animals, the

number per capillary loops per cm2 at the dorsolateral body

region was 13,400 (± 1810), with somewhat, but not signifi-

cantly, lower numbers at the ventral body region, or better,

a reduction of loops per cm2 from dorsum to ventrum

betweeen 10 and 20%. On evaluation by both light and elec-

tron microscopy, the very sparsely haired hippopotamus

[hair density at the dorsum, measured in the adult animals

studied: 110 (± 40) primary hairs/cm2, no wool hairs] showed

remarkable, and even peculiar, structural variations of the

dermal or subepidermal blood capillaries, including the fact

that the hairpin turn was not only close to the epidermal

basement membrane but also more or less “penetrating”

into the vital epidermis [thickness 158.9 (± 25.1)] until it was

very near to the epidermal surface (Fig. 1A, B). The dis-

tances in question here were between 40 and 75 μm (e.g.,

Fig. 1B), although the basement membrane was not pierced

but the number of cell layers was reduced, as, for example,

one layer of the str. granulosum and three or for corneal lay-

ers. Moreover, the ascending and the descending capillary

Fig. 1. Light microscopical demonstration of the collagen IV

sheath of the subepidermal capillary loops, using Technovit plastic

sections and autofluorescence (yellow); (A) overview of the general

construction, (B) hairpin turn region of a capillary loop with high

amounts of red blood corpuscles, below a thin part of the vital epi-

dermis, (C) lower region of the capillary loop system, demonstrating

collagen sheath thickness and the lumina of the ascending (one

asterisk) and the descending (two asterisks) loop part; CAP = capil-

lary loop, E = vital epidermis, E-SB = stratum basale of the epider-

mis, SC = str. corneum, all examples are longitudinal sections.

W. Meyer460

limbs had lumina with diameters between 25–45 μm, which

pertained also to most parts of the hairpin turn.

Electron microscopical analysis clearly demonstrated

that each of the capillary loops was surrounded by a distinct

collagenous cover, although formalin-fixed skin material had

to be used (Fig. 2). This cover sheath had a thickness of 3–

5 μm in the apex region and 10–25 μm in the lower region

of the dermal papillae, and was composed of a multitude of

thin collagen fibres (φ20–30 nm). The capillary lumen was

densely filled with red blood corpuscles, particularly near the

hairpin turn. The endothelial cells had a thickness of 1–2

μm, and sometimes showed exocytotic activities. Near to

and within the collagenous cover, dark longitudinal or

rounded areas could be regularly observed (Fig. 2A, B). The

use of the red fluorescence dye BODIPY® 665/676 on plas-

tic sections proved that these dark homogenous structures

consisted of free fatty acids (Fig. 3B). The control with two

lipid extraction procedures before embedding or staining

confirmed this finding by negative results.

The evaluation of the autofluorescence spectrum of the

plastic sections indicated that the cover of the large capillary

loops was based on collagen. The application of immunohis-

tochemistry corroborated this view and revealed by a

strongly positive reaction labeling that the collagen type

present was collagen IV (Fig. 3C). In the control experi-

ments performed by incubation with PBS without primary

antibodies or exposure of sections of the PO-DAB system

without primary or secondary antibodies, all structures

reacted negatively (Fig. 3D). In contrast to this staining, it

was not possible to achieve positive immunohistochemcial

reactions for nidogen-1.

DISCUSSION

It seems that capillaries are the most crucial distributive

blood vessels for substance exchange necessary for the

epidermis, inasmuch as the basic structure of the capillary

loops and their cellular components found in this study did

not differ from those described for other mammals, including

humans. Interestingly, these elements of dermal microcircu-

lation are normally distinguished by a relatively thick vessel

wall, consisting of endothelial cells with a nucleus narrowing

the capillary lumen, and more or less regularly a pericyte, in

some cases, additionally, a veil cell (adventitial cell) (e.g.,

Imayama, 1981; Braverman, 1989, 2000; Ryan, 1991;

Pavelka and Roth, 2005; Meyer et al., 2007).

However, regarding the hippopotamus skin, several

interesting structural variations could be observed. First of

all, the diameters of the capillaries including the hairpin turn

and of the vessel lumen were more than twenty times larger

than those found in the other mammals studied until now

(e.g., Braverman, 2000; Meyer et al., 2007, 2008a). More-

Fig. 2. TEM demonstration of specific features of the collagen IV

sheath of the capillary loop; (A) and (B) emphasize high amounts of

fine collagen fibres forming the basic structural sheath system, (C)

shows an endothelial cell of the capillary loop and exocytotic vesi-

cles (asterisk) of a neighbouring cell; dark fat containing regions are

found in all parts of the capillary loop system (see Fig. 2C, arrow);

C4 = collagen IV, CAP = capillary loop, EC = endothelial cell, RBC =

red blood corpuscles.Fig. 3. Demonstration of specific histochemical features of the col-

lagen IV sheath of the capillary loop; (A) two parts of the capillary

loop are shown (marked by asterisks), toluidine blue staining, hori-

zontal semithin section, (B) demonstration of free fatty acids in the

epidermis and the capillary loop using the very sensitive red fluores-

cence dye BODIPY® 665/676 (asterisk marks the collagen IV

sheath), longitudinal Technovit section, (C) immunohistochemical

demonstration of collagen IV (dark brown colour, marked by an

arrow), longitudinal paraffin section, (D) negative control reaction of

the immunohistochemical collagen IV staining, longitudinal paraffin

section; E = vital epidermis, C4 = collagen IV, D = dermis, SS = str.

spinosum.

Subepidermal Capillaries of Hippopotamus 461

over, the normally homogeneous pattern of punctiform con-

tacts of the apex of the hairpin turn with the epidermal base-

ment membrane (Meyer et al., 2007) has been lost and

appears irregular. Additionally, the diameter of the contact

area of the loop apex had enlarged from about 8–10 μm2 as

in smaller or larger mammals (Imayama, 1981; Meyer et al.,

2007) to 200–400 μm2. Likewise the number of loops per

cm2 was about three times higher than in the few other

mammalian species measured until now (see Meyer et al.,

2007).

As second astonishing feature detected during the

course of this study, autofluorescence and TEM analysis

emphasized a rather compact accumulation of fine collage-

nous fibres, the diameter of which with about 20–30 nm

being in the same range as observed in such fibres of the

lamina fibroreticularis of the epidermal basement membrane

in pig (Meyer et al., 2007). In this way, a very specific strong

variation of the lamina fibroreticularis became obvious as a

part of the basement membrane of the subepidermal capil-

laries of the hippopotamus, revealing a thickness of about

2–5 μm. This means that it was at least 20 times thicker than

in the normal epidermal basement membrane of mammals

(e.g., Imayama, 1981; Braverman, 1989; Pavelka and Roth,

2005). The immunohistochemical findings corroborated

such view, confirming that the thick sheath surrounding the

capillary loop parts in the hippopotamus consisted of colla-

gen IV. This collagen type is still regarded to be an exclusive

member of basement membranes, where it forms flexible

networks that can extend and retract these structures, and

helps to achieve physiologically-relevant compliance (Gao

et al., 2008; Khoshnoodi et al., 2008). Thus, the walls of the

subepidermal capillaries in the hippopotamus are better

equipped to withstand high blood pressure. In general, the

results confirmed that basement membranes and their com-

ponents in skin are unique specialized matrix structures that

can fulfill diverse functions, such as respond to varying

mechanical stress (Breitkreutz et al., 2009; Kruegel and

Miosge, 2010). The negative results of this study concerning

the demonstration of nidogen-1 may be based on the fact

that this ubiquitous component of the specialized extracellu-

lar matrix is not required for the overall architecture of the

basement membrane. Only in development it plays an

important role in BM stabilization, especially in tissues

undergoing rapid growth or turnover (Ho et al., 2008;

Hashmi and Marinkovich, 2011).

The finding of free fatty acids around and within the col-

lagenous cover, as demonstrated histochemically, should be

regarded as important for energy supply, but particularly for

special protective functions of the epidermal and subepider-

mal layers. In the hippopotamus, intense lipid accumulation

begins already in the stratum spinosum and is less obvious

in the cells of the corneal layer system (Meyer et al., 2011a).

Such variation is probably due to the fact that the not very

compact corneal layer system of this semiaquatic fresh

water species has to endure stronger mechanical stress with

the need to defend against rapid microbial invasion (Drake

et al., 2008; Meyer et al., 2011a).

The final aspect of this study is to evaluate the obser-

vations made from a general biological point of view as

related to the ecology and behaviour of the hippopotamus.

The only reasonable explanation of the structural peculiari-

ties found, which are not known from other aquatic mam-

mals (Reidenberg, 2007), could be interlinked with intense

thermoregulative events of the species, such as behavioral

thermoregulation (Cena, 1964; Olivier, 1975; Noirard et al.,

2008). This view is relevant particularly to restriction of the

animal to the land under hot environmental conditions during

the day, resulting in an increase of no more than 1°C in core

temperature. The rising adverse radiation and convection

heat load is met by an increase in evaporative water load

from the skin, avoiding thermal stress on land (Cena, 1964;

Wright, 1987). The unsubmerged dorsal body region, partic-

ularly, has the highest heat load (Luck and Wright, 1959), with

the effect that the animals may stay immersed in water for

about 16 hrs during the day until the early evening (Fraedrich,

1967). The specific subepidermal capillary loop structure in

the hippopotamus demonstrated in this study emphasizes

and permits a very high rate of such, non-gland-related

transepidermal water loss due to improvements of normal

capillary structure and size to counter high blood pressure.

Such integumental blood vessel-based heat transfer from

the periphery of the body is normally an important thermo-

regulatory response in highly active mammals, such as dol-

phins (Meyer, 2010c). Nevertheless, the use of skin-related

vascular adaptations under hot climatic conditions but with-

out strong exercise for cooling seems to be an effective and

energy-saving procedure in semi-aquatic mammals.

ACKNOWLEDGMENTS

For help with the skin material, the author is greatly indebted

to the late Prof. Dr. Manfred Roehrs, Institute for Zoology, and the

late Prof. Dr. Helmut Wilkens, Institute for Anatomy, University of

Veterinary Medicine Hannover Foundation, Hannover, Germany.

The excellent technical assistance of Marion Gaehle and Kerstin

Rohn, and the support of Prof. Dr. Marion Hewicker-Trautwein,

Institute for Pathology, is also gratefully acknowledged; the latter

three colleagues are also active at the University of Veterinary

Medicine Hannover Foundation.

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(Received January 16, 2012 / Accepted February 14, 2012)