Fig. 12.1
Scanning electron micrographs of flowing erythrocyte s in abdominal aorta (a) and inferior vena cava (c) of living mice, in addition to both images reconstructed by serial ultrathin sections (b, d) under the normal blood flow condition, as prepared by IVCT. Bars: 5 μm. (a) and (b) Flowing erythrocytes in the abdominal aorta appear to be various in shape (R), and some of them are stretched into ellipsoidal shapes (arrows). (c) and (d) Flowing erythrocytes in the inferior vena cava are mostly resembling the typical biconcave discoid shape s (R). (e) and (f) Scanning electron micrographs of erythrocytes in hepatic sinusoid s of mice under normal blood flow (e) or heart-arrest (f) condition, as prepared by IVCT. Bars: 1 μm. (e) In a freeze-fractured sinusoid , flowing erythrocytes are seen to be variously shaped (R). Asterisks, open space of Disse. (f) Under the heart-arrest condition , erythrocytes are aggregated in the collapsed sinusoid to form the typical biconcave discoid shapes (R). Asterisks, collapsed space of Disse
In response to the fluid shear forces, erythrocytes were easily changed from the resting biconcave discoid shape into an ellipsoid form and aligned with their long axes parallel to the fluid stream. Such temporary erythrocyte deformability in large blood vessel s was clearly examined by the present IVCT. It is tempting to conclude that the erythrocyte deformability completely differs between the abdominal aorta and the inferior vena cava, because hematocrit, plasma viscosity, and erythrocyte aggregation are significantly higher in venous blood than in arterial blood [7]. Therefore, the erythrocyte deformability in the blood vessel s should be adapted to the blood flow conditions and in relation to their functions in oxygen delivery. It has been apparent that the shape and elasticity of flowing erythrocyte s in human diseases are important for explaining the etiology of certain pathological conditions [8, 9]. Some hemolytic anemias, for example, are closely related to increased mechanical fragility of erythrocyte membranes [10], which would be confirmed in vivo by the IVCT.
12.3 Dynamically Changing of Flowing Erythrocyte Shapes in Hepatic Sinusoids of Living Mice
We have also applied the IVCT to examining the flowing erythrocyte shapes in hepatic sinusoid s of living mouse liver s (Fig. 12.1e), as reported before [3]. Their morphological features were different from those flowing in the abdominal aorta and inferior vena cava, as described above. Some wide spaces between flowing erythrocytes with various shapes were usually observed in the hepatic sinusoids with open spaces of Disse (Fig. 12.1e, asterisk). After the artificial heart arrest, however, they were changed to be congested in the sinusoidal lumen (Fig. 12.1f). Such erythrocyte shapes were completely different from those in the sinusoids of living mouse livers, and most of them appeared to be typical biconcave discoid shape s . Moreover, hepatic sinusoids and spaces of Disse between hepatocytes and endothelial cell s were completely collapsed (Fig. 12.1f, asterisk).
The flowing erythrocyte s kept various shapes in hepatic sinusoid s , not typical biconcave discoid shape s , probably responding to hemodynamic stresses. In the isotonic physiological solution, erythrocyte shapes are generally known to be regular and uniform to form the biconcave discoid shapes. Some distance between the erythrocyte surface and the endothelium has the physiological significance of erythrocyte-capillary relationship, resulting in reduction of the diffusion distance and increased shear stresses. Moreover, the erythrocyte deformation to allow its passage through networks of narrow blood capillaries also increases the contact surface of the endothelium and erythrocytes themselves and effectively broadens their surface area for gas diffusion . With the erythrocyte deformation, the whole surface structure of erythrocytes, including membrane-skeletal proteins under the erythrocyte lipid membranes, must be responsible for constantly flowing through the narrow blood vessel s [11, 12].
12.4 Element Detection of Flowing or Congesting Erythrocytes in Hepatic Sinusoids by X-Ray Microanalysis
The IVCT was already developed for examining the erythrocyte shapes flowing in large blood vessel s and hepatic sinusoid s of living mice, as described above [2, 3]. However, there has been no report about electrolyte concentration s of erythrocytes in vivo under blood flow ing conditions. Recently, the variable pressure SEM has been often used to observe hydrated and uncoated biological specimens. In the present study by using the IVCT combined with the common freeze-drying method , we have examined the uncoated erythrocyte morphology and electrolyte elements of sinusoidal erythrocytes under the normal blood flow or heart-arrest condition by the variable pressure SEM equipped with X-ray microanalysis system.
The mice were anesthetized with sodium pentobarbital, and their liver organs were routinely prepared by the IVCT, as described before [3]. The frozen liver tissue surface was routinely freeze-fractured with another cryoknife in the liquid nitrogen, as reported before [3]. The specimens were then freeze-dried at −95 °C in a freeze-etching apparatus (10−5 Pa) for 24 h, as reported before [13, 14]. The freeze-dried specimens were gradually warmed up to room temperature. They were attached onto carbon plates by using graphite-containing resin, and some were coated with carbon alone. They were finally analyzed by Hitachi S-4300 SEM or S-3000 N variable pressure SEM equipped with X-ray microanalysis system (accelerating voltage; 10 kV, vacuum condition; 30 Pa, illumination current; 50 μA, analytical time; 950 s), or conventional S-4500 SEM (Hitachi High-Technologies Corporation, Tokyo, Japan) at an accelerating voltage of 10 kV. The analyzed elements were Na (sodium), P (phosphorus), S (sulfur), Cl (chloride), and K (potassium).
Stay updated, free articles. Join our Telegram channel
Full access? Get Clinical Tree
