J. Anat. (1998) 193, pp. 73–79, with 6 figures Printed in the United Kingdom 73

Morphological study by an ‘ in vivo cryotechnique’ of the

shape of erythrocytes circulating in large blood vessels

MEI XUE, YASUKO KATO, NOBUO TERADA, YASUHISA FUJII, TAKESHI BABA

AND SHINICHI OHNO

Department of Anatomy, Yamanashi Medical University, Yamanashi, Japan

(Accepted 23 March 1998)

Changes in the shape of erythrocytes circulating in large blood vessels of mice were examined by our ‘ in

vivo cryotechnique’. The abdominal aorta and inferior vena cava (IVC) were cut vertically with a precooled

knife and simultaneously an isopentane–propane mixture (®193 °C) was poured over them for freezing.

They were freeze-substituted in acetone containing 2% osmium tetroxide. Some specimens were embedded

in Quetol-812, and thick or ultrathin sections were examined by light or transmission electron microscopy.

Serial ultrathin sections were used to reconstruct 3-dimensional images of native erythrocytes. Others were

transferred into t-butyl alcohol and freeze-dried for scanning electron microscopy. The tissue surfaces were

sufficiently frozen to prevent large ice crystal formation, and erythrocyte shapes were also preserved. The

shapes of circulating erythrocytes appeared to be varied in the abdominal aorta but typical biconcave

discoid shapes were rarely observed. Conversely, erythrocytes were approximately biconcave discoid in shape

in the IVC. Our in vivo cryotechnique was useful for clarifying the in vivo morphology of erythrocytes

circulating in large blood vessels.

Key words : Vasculature ; freeze-substitution; circulating erythrocytes.

It is well known that mammalian erythrocytes

maintain their biconcave discoid shape in vitro, but

that they change their form under complicated

dynamic conditions in blood capillaries (Maeda,

1996). The deformability of erythrocytes is an im-

portant factor in such a situation. Some investigators

have studied the changes in shape of erythrocytes

affected by various drugs (Nishiguchi et al. 1995),

inhibitors of glucose or anion transporters (Blank &

Diedrich, 1990) and calcium (Kon et al. 1993). Others

have also examined the behaviour of erythrocytes in

narrow tubes as a model of the microcirculation

(Kubota et al. 1996) to clarify their deformability.

However, the morphological appearances of erythro-

cytes in large blood vessels are still unknown because

of limitations in preparative techniques for electron

microscopy.

It has been difficult to observe directly the behaviour

of erythrocytes in large blood vessels with their thick

walls. Conventional morphological studies, therefore,

Correspondence to Prof. Shinichi Ohno, Department of Anatomy, 1110 Shimokato, Tamaho, Yamanashi 409-3898, Japan. Fax: ­81-552-

73-6743; Tel : ­81-552-73-6743.

using immersion or perfusion fixation methods, have

not revealed the true morphology of circulating

erythrocytes under normal blood flow conditions.

Recently, a new preparative technique, referred to as

the ‘ in vivo cryotechnique’, was developed for

freezing tissues and cells in vivo without arresting the

blood supply (Ohno et al. 1996). The in vivo

cryotechnique could reduce time-dependent morpho-

logical changes of tissues and cells, as usually seen

when they are conventionally fixed. We have already

reassessed the glomerular ultrastructure of function-

ing renal glomeruli without arresting the blood supply

(Ohno et al. 1996). In the present study, we used the

same in vivo cryotechnique to examine the behaviour

of circulating erythrocytes in large blood vessels.

A total of 16 female BALB}c mice, each weighing

15–20 g, were used for transmission (TEM) or

scanning (SEM) electron microscopy. They were

anaesthetised with sodium pentobarbital (50 µg}g

c

b

a

Fig. 1. Schematic representation of the ‘ in vivo cryotechnique’. (a)

Cutting line of the abdominal aorta and inferior vena cava. (b)

Cryoknife. (c) Liquid isopentane-propane cryogen (®193 °C).

body weight). The abdomen was opened and the

abdominal aorta and inferior vena cava (IVC)

identified under a stereoscopic microscope (Fig. 1).

Thin aluminium foil was put under the blood vessels

without disturbing the circulation. The in vivo

cryotechnique was performed under normal blood

flow conditions, as previously described (Ohno et al.

1996). First, a cryoknife edge was precooled in liquid

nitrogen and reached a low temperature (®196 °C). It

was then placed over the abdominal aorta and IVC of

the mice. While the heart was beating normally, both

the abdominal aorta and IVC were cut vertical to their

longitudinal axes as rapidly as possible (Fig. 1), and

simultaneously frozen by the cryoknife edge. A cooled

liquid isopentane–propane mixture (®193 °C) was

immediately poured over the vessels, as reported

before (Ohno et al. 1996). Some of the applied

cryogen remained along the cryoknife edge and

participated in the freezing process, followed by liquid

nitrogen (®196 °C) to prevent a rise of temperature.

The additional liquid nitrogen was needed to maintain

the blood vessels at a low temperature. The frozen

aorta and IVC specimens were preserved in liquid

nitrogen after being separated from other abdominal

organs and submitted to the routine freeze-substi-

tution as follows. They were transferred to absolute

acetone containing 2% osmium tetroxide at ®80 °Cfor 20 h, then at ®20 °C for 2 h and 4 °C for 2 h.

Finally, they were allowed to warm to room tem-

perature and washed in pure acetone 3 times. Some

specimens were embedded in Quetol-812. Thick

Fig. 2. Light micrographs of toluidine blue-stained thick sections,

prepared by freeze-substitution method after the in vivo cryo-

technique. (a) The abdominal aorta (A) and IVC (V) are seen. Bar,

100 µm. (b) Higher magnification of a part of the aorta shown in

(a). Well-frozen erythrocytes are seen outside (large arrow) and

inside the aorta (small arrow). Asterisk, open part of aorta. Bar,

10 µm. (c) Higher magnification of a part of the IVC. Bar, 10 µm.

74 M. Xue and others

sections (1 µm) were stained with toluidine blue and

examined by light microscopy. Serial ultrathin

sections were routinely prepared, contrasted with

uranyl acetate and lead citrate and observed in an H-

600 transmission electron microscope. In addition,

serial ultrathin sections were used to reconstruct 3-

dimensional (3-D) images of erythrocytes in the

abdominal aorta and IVC. Serial electron micro-

graphs were scanned into a Macintosh computer and

their 3-D images reconstructed using NIH image

software. Other freeze-substituted specimens were

transferred to t-butyl alcohol, freeze-dried in an ES-

2030 apparatus (Hitachi, Japan), ion-sputtered with

platinum}palladium (10–15 nm) and examined in an

S-4500 scanning electron microscope (Hitachi, Japan)

at an accelerating voltage of 5 kV.

Results were obtained from 8 mice for TEM and 8

mice for SEM with consistent findings in both sets. In

toluidine blue-stained thick sections, the abdominal

aorta and IVC were clearly evident (Fig. 2a). The

lumen of the aorta was partially open but that of the

IVC was compressed, probably because of pressure

from the cryoknife. The region of initial contact with

the precooled cryoknife edge was better preserved,

which was not far from the cutting tissue surface at

light microscopic level (Fig. 2b). At high magnifi-

cation, aortic erythrocytes were divided into 3 groups.

The 1st group (Fig. 2b, large arrow) was located in

well-frozen areas outside the aorta; the erythrocytes

Fig. 3. Electron micrograph of erythrocytes in the abdominal aorta under normal blood flow conditions, prepared by freeze-substitution

method after the in vivo cryotechnique. Large arrows, cutting tissue surface. Ice crystal damage is observed in deep areas (large asterisk).

Erythrocyte shape is rarely seen to be biconcave discoid (small arrows). Bar, 5 µm.

had a variety of shapes and showed no fixed

alignment. The 2nd group (Fig. 2b, asterisk) had been

frozen as they flowed from the aorta. The well-frozen

areas reflected the behaviour of erythrocytes circu-

lating in the blood vessels, closer to the living state. In

such areas, erythrocyte shapes were irregular, and

biconcave discoid shapes were rarely observed.

Although their preservation was not as satisfactory

within the aorta (group 3), the shape of these

erythrocyte (Fig. 2b, small arrow) was also irregular

and elongated in the direction of blood flow. In

contrast, in the IVC most erythrocytes exhibited an

approximately biconcave discoid shape, as revealed at

a higher magnifications (Fig. 2c). They showed a

tendency to be packed together with no obvious flow

direction.

Electron micrographs (Fig. 3) of the same region as

shown in Figure 2b indicated that the tissue surface

was adequately frozen to prevent ice crystal for-

mation, although tissue damage was observed in

deeper areas (Fig. 3, large asterisk). The shape of each

erythrocyte varied and they showed no tendency to

aggregate. A biconcave discoid shape was rarely

observed. Although erythrocyte structure was de-

stroyed in inadequately frozen areas, circulating

erythrocytes within the aorta did not exhibit a

biconcave discoid shape. In order to obtain the 3-D

image of such erythrocytes (Fig. 4), we reconstructed

several erythrocytes in the aorta, as shown in Figure

3, which were taken from the serial ultrathin sections.

The reconstructed shapes clearly differed from the

typical biconcave discoid form. They appeared to be

Erythrocyte shapes in blood vessels 75

(a)

(b)

Fig. 4. (a) Two erythrocytes in the aorta are reconstructed, based on serial ultrathin sections (small asterisks in Fig. 3). ¬12000. (b) Half

of a reconstructed erythrocyte observed from 4 different angles (small asterisks in Fig. 3). A biconcave discoid shape is not apparent. ¬7500.

elongated, with their long axes parallel to the direction

of blood flow, resulting in a near ellipsoid con-

figuration (Fig. 4a). Moreover, half of another

erythrocyte was similarly reconstructed and observed

from 4 different directions (Fig. 4b). These 3-D

reconstructions definitely showed that no biconcave

discoid shapes appeared in the aorta under normal

blood circulation conditions. Reconstructed erythro-

cytes in the IVC (Fig. 5) were also analysed in a

similar way to those in the abdominal aorta. Half of

an erythrocyte was reconstructed from serial ultrathin

sections (Fig. 5, inset) and viewed from 2 different

angles. It appeared to have a biconcave discoid shape.

According to the SEM images, the 3-D structure of

erythrocytes flowing in the aorta varied in shape, and

stretched erythrocytes were also observed (Fig. 6a).

Concavities on both sides of an erythrocyte were

rarely detected. Compared with those in the aorta,

most erythrocytes in the IVC showed an approxi-

mately biconcave discoid shape (Figs 6b, c).

The ‘ in vivo cryotechnique’ (Ohno et al. 1996),

performed directly on blood vessels, is a new method

for simultaneously cutting and freezing them to avoid

morphological changes in erythrocytes due to cess-

ation of the blood circulation. Some points concerning

the cryofixation technique have been discussed before,

including aspects of the spatial and dynamic time

resolution (Plattner & Bachmann, 1982). The time

resolution for cryofixation can be estimated to be

76 M. Xue and others

Fig. 5. Half of a reconstructed erythrocyte in the IVC viewed from

2 different angles. It appears to have a biconcave discoid shape.

¬6000. Inset : sectioned erythrocyte circulating in the IVC.

between 0±1 ms and 1 ms, as usually obtained with

some available cryofixation methods (Plattner &

Bachmann, 1982). The cryotechnique using liquid

nitrogen permits rates of freezing of up to

10%–10& K}s. In our study, the acceptably frozen areas

were obtained along the tissue surface layer, where ice

crystal sizes were up to almost 30–50 nm. Biconcave

discoid shaped erythrocytes are 6–8 µm in diameter

and 2–3 µm in thickness (Fung, 1977). The well-frozen

areas on the tissue surface were therefore used to

examine the erythrocyte shapes. One of the drawbacks

in our cryofixation method is the compression of

blood vessels caused by the cryoknife edge. However,

some erythrocytes within the cut aorta are considered

to reflect the in vivo morphology. The in vivo

cryotechnique, followed by freeze-substitution and

freeze-drying methods for SEM, has demonstrated the

various shapes for erythrocytes flowing in the ab-

dominal aorta.

Erythrocytes in stored blood are known to have the

typical biconcave discoid shape. Moreover, a change

in shape has not been observed in isotonic solutions,

although some different types of erythrocyte shape

are seen when in artificial solutions (Simpson, 1993).

In large blood vessels in vivo, rheology should also be

considered, in relation to their bulk flow (Stuart &

Nash, 1990). Arterial flow is reported to be laminar,

especially in the abdominal aorta (Robinson, 1978). A

high shear rate in a large artery is determined by a

rapid flow velocity, C 100 cm}s (Klug et al. 1974). It

thus provides the possibility for erythrocyte defor-

mation as a result of external force stress. In the

present study, erythrocyte shapes were seen to be

varied, some being stretched along the direction of

blood flow in the aorta. Conversely, a low shear rate in

venous blood flow, probably at a velocity of 30 cm}s

(Klug et al. 1974), results in erythrocyte shapes being

approximately biconcave discoid, as observed by

SEM. The deformability of erythrocytes is also related

to their internal structure, including cell geometry,

membrane properties and cytoplasmic viscosity

(Mohandas et al. 1979; Mchedlishvili, 1991; Maeda,

1996).

Erythrocyte membrane skeletons probably provide

astonishing deformability and stability (Haest, 1982;

Ohno et al. 1993; Ursitti & Wade, 1993). It is

generally accepted that the membrane skeletal

proteins are responsible for the generation of their

unique discoid shape (Mohandas & Chasis, 1993; An

et al. 1996; Jay, 1996) ; these consist mainly of

spectrin, actin, protein 4±1, band 3 and ankyrin

(Terada et al. 1996). The ultrastructure of the

membrane skeletons in fresh human erythrocytes, in

which spectrin proteins are not ordered in an

elongated, but in a condensed form, are presumably

organised through intramolecular and}or inter-

molecular interactions (Ohno et al. 1994). In the

human erythrocyte, the strong mechanical force leads

to a rearrangement of membrane skeletal proteins,

resulting in the formation of new meshworks (Liu et

al. 1993). Such changes of the erythrocyte membrane

skeleton should be examined in vivo under active

blood flow conditions.

In response to fluid shear forces, erythrocytes are

changed from the resting biconcave discoid shape into

an ellipsoid form and align with their long axes

parallel to the fluid stream. Such temporary eryth-

rocyte deformability in large blood vessels was

examined by the in vivo cryotechnique in the present

study. It is tempting to conclude that erythrocyte

deformability differs between the abdominal aorta

and the IVC because haematocrit, plasma viscosity

and erythrocyte aggregation are significantly higher in

venous than in arterial blood (Mokken et al. 1996).

Erythrocyte deformability in blood vessels is adapted

to blood flow conditions and in relation to their

function in oxygen delivery. In recent years, it has

become apparent that the shape and elasticity of

human erythrocytes are important for explaining the

aetiology of certain pathological conditions

(Athanassiou et al. 1992; Cynober et al. 1996). Some

haemolytic anaemias, for example, are related to

Erythrocyte shapes in blood vessels 77

Fig. 6. Scanning electron micrographs of erythrocytes circulating in the aorta (a) and the IVC (b, c), as prepared by freeze-substitution and

freeze-drying methods after the in vivo cryotechnique. (a) 3-dimensional images of circulating erythrocytes are varied and some are stretched

into elongated shapes (arrow). Bar, 5 µm. (b) Some erythrocytes in the IVC are freeze-fractured (small arrow) and others maintain their shape

(large arrow). Bar, 10 µm. (c) Higher magnification. Erythrocytes in the IVC are closely similar to a biconcave discoid shape. Bar, 5 µm.

increased mechanical fragility of erythrocyte mem-

branes (Rybicki et al. 1993).

Erythrocyte shape change is not only induced by

factors in the erythrocyte itself, but also blood

viscosity, shear stress, and flow volume (House &

Johnson, 1986). They are highly complicated in blood

vessels, because metabolic, osmotic, haematological

and rheological factors also exist in vivo. It is known

that the oxygenation–deoxygenation cycle in erythro-

cytes modulates membrane fluctuations that are

directly related to deformability (Tuvia et al. 1992). It

is therefore difficult to conclude which factor is the

most important. In the present study, only some

factors have been mentioned. In many experiments,

thoroughly washed erythrocyte suspensions have

often been used for erythrocyte deformability studies,

but blood rheology might be different in vitro than in

vivo (Lowe, 1987). The most important problem that

many investigators still face is the difficulty in

simulating the physiological condition in vivo. The in

vivo cryotechnique makes it possible to undertake

more physiological studies of normal or pathological

erythrocytes subjected to various stresses.

In conclusion, the present study has enabled us to

apply the in vivo cryotechnique for examining the

morphological properties of erythrocytes circulating

in large blood vessels.

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