Scanning Electron Microscopy of Vascular Corrosion Casts - Technique and Applications: Updated Review

The present paper states very briefly the main steps leading to the technique of scanning electron microscopy (SEM) of vascular corrosion casts. From the terms presently used (injection method, microcorrosion cast, injection replica, vascular corrosion cast, vascular cast) the use of vascular corrosion cast for lymphatic and blood vessels is recommended. Specification and pretreatment (kind, volume, dosage of anticoagulants, vasoactive substances and spasmolytica used) of the animals examined are referenced as they are available from the literature. The recommendation is given to pay more attention to these parameters than done so far. The steps necessary for producing reasonable and suitable vascular corrosion casts are critically described. Special attention is paid to the physical and chemical properties of the casting media and their significance for polymerization, shrinkage, casting quality, corrosion resistance, and thermal and spatial stability. Emphasis is also focused on the advantages of cutting the vascular corrosion casts embedded in an ice block by a band saw, a self constructed multi-blade cutting device or a mini wheel-saw placed in the chamber of a cryomicrotome. From the drying methods presently used freeze-drying is stressed because of minimal specimen damage. To render casts conductive in most cases sputter-coating is sufficient. It is recommended to run the SEM with 5 - 10 kV since the resolution received still reveals all details the casting media presently can replicate. Further the application of scanning electron microscopy of vascular corrosion casts in fully differentiated normal tissue, in pathologic tissue as well as in developing tissues and organs is stated. Lastly possibilities and conditions are discussed under which SEM of vascular corrosion casts can serve to quantify vascular structures in order to make the technique more than pure descriptive

Microvascular Corrosion Casting of the Rat Mandibular Joint: A Technical Approach

The rat mandibular joint is a ginglymoarthrodial joint deeply hidden under the zygomatic process of the squamosal bone. This joint was studied by scanning electron microscopy (SEM) of microvascular corrosion casts in 47 adult rats. Six additional rats were injected with India ink and thick sections of the joint area were examined by light microscopy. The small size of the joint (2.5 mm x 1.5 mm in occlusion), the existence of two slit-like joint compartments, the close proximity of bones and soft tissues in a mobile structure, and the intimate relationships of the joint with adjacent skeletal muscles made the vascular mapping difficult. Five different technical procedures (protocols) were tested. The most satisfactory approach is, briefly, as follows: each rat was injected simultaneously via both common carotid arteries with 9.5 ml of casting medium (Mercox-CL-2B diluted 4:1 with monomeric methyl methacrylate), and with an additional volume of 0.5 ml of the same medium after ligation of the external jugular veins used for outflow. After polymerization of the injected resin, the rats were frozen, and the heads were severed and spliced sagittally into two halves. These specimens were cut in the transversal (coronal) and sagittal planes into small blocks. Each block was then cut systematically either in the transversal or parasagittal plane running through the joint. Afterwards thick slices were macerated in warm (45°C) 15% KOH, rinsed, (some were decalcified in 2% HCl), rinsed again in tap water, frozen in distilled water, freeze-dried, mounted, dissected, coated and examined under the SEM at 5 kV

The Vascularization of the Skin of the Atlantic Hagfish, Myxine glutinosa L. as Revealed by Scanning Electron Microscopy of Vascular Corrosion Casts

The vascularization of three different (A, B, C) skin regions (from the level of the heart to the cloaca including dorsal, lateral and ventral skin areas) of the Atlantic hagfish, Myxine glutinosa L. was studied by scanning electron microscopy of vascular corrosion casts. Vessel variables were measured either from semithin sections (diameters) or from vascular corrosion casts (diameters, lengths) and total blood capacities as well as vessel surfaces per unit skin area (mm2) were calculated. There are no significant differences in the number of subepidermal capillary meshes (ranging from 164 to 185 meshes per micrograph) in areas A, B or C nor in vessel lengths. The average vessel length per mm2 is 32 mm. Assuming an average diameter of 22.3 μm these vessels have an average surface of 2.24 mm2 and a volume of 12.5 nanoliters (nl). In contrary weighing two pieces ( 5 mm times 5 mm in size) of the whole skin vascular bed - knowing the density of the casting medium -results in only one fifth of that volume. Overestimation of vessel lengths and diameters by measuring casted structures from micrographs on the one hand and inaccuracies in weighing or dissection of casted skin pieces on the other hand are discussed as sources of observed differences

Technical Parameters of Plastics (Mercox Cl-2B and Various Methylmethacrylates) Used in Scanning Electron Microscopy of Vascular Corrosion Casts

The most frequently used resins for vascular corrosion casting Mercox Cl-2B, Mercox Cl-2B diluted with methylmethacrylate (MMA) monomer and various self prepared MMA and hydroxyproyl-methacrylate mixtures were tested with regard to their thermostability, shrinkage, viscosity and replication quality. It was found that tempering of the plastics improves their thermostability with the exception of Mercox Cl-2B and that shrinkage depends on the amount of monomers a resin contains. In detail; Mercox Cl-2B has the lowest shrinkage (8.018%) whereas a hydroxypropyl-methacrylate mixture possessed the highest (20.408%). But, on the other hand, viscosity decreases with the quantity of monomers. All resins tested were able to replicate structures of 260nm height but the resins\u27 quality of replication was found to be limited by the effects of shrinkage. Finally, a method to estimate the blood volume of organs and tissues with the help of vascular corrosion casts is given

Microvascularization of the Pleura in Rats and Guinea Pigs

The microvascularization of the visceral and parietal pleura was studied in rats and guinea pigs using vascular corrosion casts and scanning electron microscopy. The visceral pleura was shown to be devoid of a vascular bed of its own. The capillary meshwork observed on the surface of the lung belongs to the pulmonary parenchyma. The parietal pleura, by contrast, possesses its own capillary network with an appropriate arterial supply and a venous drainage. The parietal pleural capillaries cover the costal regions completely, whereas the intercostal spaces are only provided by interspersed small patches of capillaries. That the feeding arteries of the parietal pleura are connected to the systemic circulatory system, supports the well-known fact that the parietal pleura is the main site for production of pleural fluid

The Characteristic Structural Features of the Blood Vessels of the Lewis Lung Carcinoma (A Light Microscopic and Scanning Electron Microscopic Study)

Vascular corrosion casts of Lewis lung carcinomas (LLC) grown subcutaneously in C57BL/6-mice are correlated with histological sections and with tumor tissue prepared for scanning electron microscopy (SEM). By making low, medium and high pressure cast preparations we studied the influence of perfusion and injection pressure on the resulting cast sample. Three types of vascular proliferations are distinguishable in LLC: 1) Small globular outgrowths on sinusoidal dilated tumor capillaries, caused by proliferation of their endothelial cells. 2) New sprouts on surrounding host vessels, invading the small, still avascular implant. 3) Superficially located, centrifugally running sprouts in peripheral regions of large tumors. They invade the surrounding host tissue. Vascular sprouts are of venous origin, have a fragmentary endothelium and are rather leaky if casted. High pressure preparations of large tumors reveal central avascular cavities surrounded by centripetally running, compressed and blind ending tumor vessels. Irrespective of the applied injection pressure, the casts always exhibit extravasal channels caused by degeneration of the endothelium of central tumor vessels. We show that SEM of vascular corrosion casts combined with histology not only demonstrates such contrary processes as the development of tumor blood vessels and the simultaneously occurring vascular degeneration, but also elucidates all other morphological characteristics of the tumor vascular system

The Secondary Blood Vessel System of Segmental Arteries and Dorsal Aorta in Blennius pavo and Zosterisessor ophiocephalus. Histology, Fine Structure and SEM of Vascular Corrosion Casts.

The secondary blood vessel system of the segmental arteries and of the dorsal aorta of the teleost fish Blennius pavo and Zosterisessor ophiocephalus are examined by light microscopy, transmission electron microscopy and by scanning electron microscopy of appropriately processed tissue and of vascular corrosion casts. Dorsal, lateral and ventral segmental arteries and the caudal portions of the dorsal aorta have a secondary vessel system. The secondary vessels are formed by capillaries which arise from the proximal portions of the segmental arteries and from the caudal parts of the dorsal aorta. In Z. ophiocephalus these capillaries are strongly dilated at their origin, in B. pavo they are less dilated, but more intensively coiled. The secondary vessels establish veins which run parallel to the corresponding primary arteries. At the ultrastructural level the secondary vessels consist of a thin endothelium and a longitudinal and a transversal orientated layer of collagen fibres. Secondary veins lack a basal membrane. A possible function of the secondary vessel system of B. pavo and Z. ophiocephalus as a modified drainage system is discussed

Microvascular Corrosion Casting in the Study of Tumor Vascularity: A Review

Tumor blood flow is dependent on the structure and three-dimensional (3-D) architecture of the vascular network. The latter can be best studied by scanning electron microscopy of microvascular corrosion casts. However, literature reviews show that nearly all studies using this technique render comparisons of different tumors more difficult since they are mainly based on descriptive terms that might lead to misunderstandings. Qualitative comparisons of 13 experimental and 3 human primary tumors of different origin show a high degree of similarity in the vasculature. Quantitative analysis of these casts reveals similar ranges of parameters such as diameters, intervascular and interbranching distances. Diameters of vessels with capillary wall structure range from 6 μm to 55 μm in the human primary tumors (renal clear cell carcinoma, basalioma), and from 5 μm to 80 μm in xenografted tumors (sarcomas, colon carcinoma). Intervascular distances in the human primary tumors range from 2 μm to 52 μm, and from 11 μm to 105 μm in the xenografts. Interbranching distances range from 34 μm to 258 μmin the former, and from 11 μm to 160 μmin the latter. Both qualitative and quantitative analyses of tumor microvascular corrosion casts enable pathophysiological conclusions to be drawn and contribute to a better understanding of tumor vascularity

The Vascularization of the Digestive Tract Studied by Scanning Electron Microscopy with Special Emphasis on the Teeth, Esophagus, Stomach, Small and Large Intestine, Pancreas, and Liver

The periodontal vessels in adult rats show a ladder-like pattern; in guinea pigs molars, by contrast, they present a honey-comb pattern. The vascular architecture in human teeth seems to be similar to that of rabbits. In guinea pigs, rats, rabbits and humans esophagus circumferential vessels give off perforating vessels. In human esophagus the number and diameter of the vessels in the submucous venous plexus decrease from proximal to distal. In the stomach the subepithelial capillary network shows a honey-comb pattern reflecting the arrangement of the gastric pits. A local portal system between the gastric glands and the surface mucosal cells for the transport of HCO3- ions has been suggested. In the small intestine of humans and rabbits the existence of a dual blood supply of the villus has meanwhile been established. It consists of pericryptal capillaries for the lower portion of the villus (tuft pattern) and a direct arterial supply up to the villus tip (fountain pattern). The colonic microvasculature closely resembles that of the stomach. In the pancreas the insulo-acinar portal system is physiologically significant in that it connects the venules draining the islets with the acini. Venous sphincters in the vascular system of the exocrine pancreas of the rat are of particular functional importance. The hepatic sinusoids are supplied both by the hepatic artery and the branches of the portal vein. The peribiliary plexus is supplied by the afferent vessels of the hepatic artery, the efferent vessels drain the plexus either into the sinusoids or into the lobular vein

Diluted and Undiluted Mercox Severely Destroy Unfixed Endothelial Cells. A Light and Electron Microscopic Study Using Cultured Endothelial Cells and Tadpole Tail Fin Vessels

Mercox is a methylmethacrylate-based resin which is widely used for vascular corrosion casting with subsequent scanning electron microscopic analysis. In the present study the effect of undiluted and diluted Mercox (4+1; volume + volume; Mercox: monomeric methylmethacrylate (MMA); 0.02 g catalyst MA/ml Mercox) and methylmethacrylate with and without catalyst MA (0.625 g/10 ml MMA) on fixed and unfixed endothelial cells was studied. Light microscopy (LM) of cultured capillary endothelial cells (ECs), which were replicated with diluted or undiluted Mercox shows degranulation and membrane perturbation of ECs, while no morphological changes occur in glutaraldehyde-prefixed ECs. Scanning electron microscopy (SEM) of replicas ( = resin blocks) polymerized on prefixed ECs reveals unchanged ECs and replicas show many details. Unfixed ECs are destroyed and replicas reveal aberrant features. Transmission electron microscopy (TEM) of prefixed and unfixed ECs (cultured endothelial cells, endothelial cells of perfusion prefixed and of unfixed tadpole tail fin vessels) substantiates LM and SEM findings. Prefixed ECs resist Mercox without fine structural changes, while unfixed cells undergo destruction. It is recommended to fix vessels prior to casting. Extravasations in microvessels are considered to be caused by focal chemical destruction of endothelial cells