Microvascular Corrosion Casting Research Articles

Cilioretinal anastomoses in the anterior optic nerve of a healthy subject

With the use of a modified microvascular corrosion casting technique in healthy eye-bank eyes from a 77-year-old woman 9 hours post mortem, a large communication between the central retinal and posterior ciliary arterial systems was demonstrated. The communication was seen within the retrobulbar optic nerve bilaterally. These types of vascular anatomical variations may explain part of the variability of blood flow patterns within the anterior optic nerve.

Bifurcation Patterns and Branching Exponents in Branchial Vessels in Tadpoles of Xenopus laevis Daudin during Metamorphosis-3D-Measurements from Microvascular Corrosion Casts

IntroductionFluid transport systems of organisms [1] in general and the blood vascular system in particular are considered to be optimally designed. From Murray's laws [2] it is concluded that an arterial bifurcation where the diameter of the parent vessel (d0) relates to the larger (d1) and the smaller daughter vessel (d2) according to d03 = d13 + d23 is optimal. Interestingly, existing data predominantly refer to arterial branchings of the fully developed circulatory system of mammals. to the best of our knowledge there are no data available on arterial bifurcations and venous mergings in an initially growing but then regressing tubular system of blood vessels as it is found in the gill filter apparatus of the anuran tadpole where the highly complex three-dimensional vascular network totally disappears at the end of metamorphosis.

3D microvascular architecture of pre-cancerous lesions and invasive carcinomas of the colon.

Despite the significance of tumour neoangiogenesis and the extensive knowledge on the molecular basis of blood vessel formation currently no quantitative data exist on the 3D microvascular architecture in human primary tumours and their precursor lesions. This prompted us to examine the 3D vascular network of normal colon mucosa, adenomas and invasive carcinomas by means of quantitative microvascular corrosion casting. Fresh hemicolectomy specimens from 20 patients undergoing cancer or polyposis coli surgery were used for corrosion casting, factor VIII and VEGF immunostaining. In addition, immunostaining was done on colorectal tissue from 33 patients with metastatic and non-metastatic carcinomas, polyposis coli and adenomas. This first quantitative analysis of intervessel and interbranching distances, branching angles and vessel diameters in human cancer specimens revealed distinct patterns of the microvascular unit in the tumour centre and periphery. Irrespective of the tumour localization and grading all individual tumours displayed qualitatively and quantitatively the same vascular architecture. This gives further evidence for the existence of a tumour type-specific vascular architecture as recently demonstrated for experimental tumours. Metastatic tumours displayed different vascular architectures only within hot spots, in terms of smaller intervascular distances than in non-metastatic tumours. Pre-cancerous lesions have in part virtually the same vascular architecture like invasive carcinomas. Comparison of VEGF immunostaining also suggests that angiogenesis sets in long before the progress towards invasive phenotypes and that the so-called angiogenic switch is more likely a sequence of events. © 2001 Cancer Research Campaign www.bjcancer.com

Effects of thrombin and of the phospholipase C inhibitor, D609, on the vascularity of the chick chorioallantoic membrane

Microvascular corrosion casting was used to assess the effects of thrombin and D609, a phospholipase C inhibitor, on the vascularity of the chick embryo chorioallantoic membrane (CAM). Discs containing vehicle, thrombin or D609 were placed on the CAM of fertilized white Leghorn eggs on Day 9 of gestation and vascularity was assessed on Day 11. Thrombin caused significant increases in the numbers (43%), diameters (5%) and lengths (17%), of both pre- and postcapillaries (first-order vessels by centripetal ordering). Conversely, D609 caused a decrease in the numbers (27%), lengths (12%) and diameters (8%) of first-order vessels. D609 decreased the total vascular volume of first- to third-order vessels by 32%, whereas thrombin increased vascular volume by 27%. Additionally, thrombin increased capillary plexus density by 6%, whereas D609 decreased capillary plexus density by 3%. These findings provide a quantitative assessment of changing vascularity in the chick CAM—a model assay system in the development of pro- and antiangiogenic agents.

Anterior optic nerve microvascular changes in human glaucomatous optic neuropathy

The microvascular changes in the anterior optic nerve in human glaucomatous eyes were examined by selective methylmethacrylate microvascular corrosion castings following cannulation of the central retinal artery and posterior ciliary arteries in 11 normal eyes and 9 glaucomatous eyes. The resulting castings were examined with scanning electron microscopy. Microvascular changes were found in the anterior optic nerves of all the glaucomatous eyes with visual function loss. These findings include areas of capillary filling defects within the anterior optic nerve and a decreased numbers of feeding arteriolar vessels to the anterior optic nerve. In the prelaminar and laminar regions, the typical capillary patterns are lost and laminar striations are not present. Juxtapapillary choroidal and retinal avascular areas were also identified in two of the glaucomatous eyes. Selective microvascular corrosion casting is an excellent method to examine the three-dimensional microvasculature of the anterior optic nerve. Microvascular changes in the anterior optic nerve may play a role in the development of glaucomatous optic neuropathy.

Effect of brimonidine on optic nerve blood flow in rabbits

PURPOSE: Brimonidine is a highly selective α 2-adrenoreceptor agonist that lowers intraocular pressure. The aim of the present study was to analyze in vivo the vasomotor effects and the influence of brimonidine on blood flow within the optic nerve, by means of intraluminal microvascular corrosion casting technique and intravascular injection of colored microspheres. METHODS: New Zealand white rabbits received either brimonidine tartrate 0.2% or placebo (vehicle) topical drops in one eye for 4 weeks. Intraocular pressures were measured at baseline and 4 weeks. The anterior optic nerve microvasculature of four rabbits was examined with corrosion castings for regions of focal vasoconstriction. Optic nerve blood flow was determined in 16 rabbits by means of nonradioactive colored microspheres. RESULTS: The vasoconstriction values of the short posterior ciliary arterial branches in the brimonidine eyes were 16.7% ± 3.7%. In the fellow untreated eyes, the mean vasoconstriction was 16.6% ± 2.4%. In the placebo-treated eyes, the average constriction was 15.9% ± 3.2%; the fellow eyes showed a mean constriction value of 16.1% ± 5.3%. There was no statistical difference between any of the groups ( P= .2). The optic nerve blood flow in the brimonidine-treated rabbits was 0.18 ± 0.06 ml/mg/min and 0.17 ± 0.04 ml/mg/min in the treated and the fellow eyes, respectively. The difference between the optic nerve blood flow in the brimonidine-treated eyes and the optic nerve blood flow in all of the untreated eyes (0.19 ± 0.06 ml/mg/min) also was not statistically different ( P = .82). CONCLUSIONS: Long-term application of brimonidine 0.2% does not affect the blood flow or vasomotor activity of the anterior optic nerve.

Effect of anticoagulation and lavage prior to casting of postmortem material with Mercox and Batson 17.

In this study we compare the quality of vascular casts, obtained from organs of several animal species from different sources and sacrificed under different conditions. Organs from healthy animals were injected with two different polymers such as Mercox and Batson No. 17. When the specimens were observed under a scanning electron microscope structural elements such as endothelial nuclear impressions on vessels and capillaries, endothelial cell borders, venous valves, imprints of smooth muscle cells and intra-arterial cushions were identified. Organs excised post mortem from large animals can be used for microvascular corrosion casting studies with optimal results.

Microvasculature of the bovine claw demonstrated by improved micro-corrosion-casting technique.

A new and improved technique for microvascular corrosion casting was developed and verified by examination of corrosion casts of 90 bovine limbs. The described technique renders a complete filling of the vasculature of the claw even in regions that hitherto proved to be difficult regarding completeness of filling such as the dorsal area of the claw. This is demonstrated by an exemplary examination of all regions of the claw. The advantages and disadvantages of the new method are discussed.

Evidence for characteristic vascular patterns in solid tumours: quantitative studies using corrosion casts.

The vascular architecture of four different tumour cell lines (CaX, CaNT, SaS, HEC-1B) transplanted subcutaneously in mice was examined by means of microvascular corrosion casting in order to determine whether there is a characteristic vascular pattern for different tumour types and whether it differs significantly from two normal tissues, muscle and gut. Three-dimensional reconstructed scanning electron microscope images were used for quantitative measurements. Vessel diameters, intervessel and interbranch distances showed large differences between tumour types, whereas the branching angles were similar. In all tumours, the variability of the vessel diameters was significantly higher than in normal tissue. The quantitative data provide strong evidence for a characteristic vascular network determined by the tumour cells themselves. © 1999 Cancer Research Campaign

The vascular architecture of the chick chorioallantoic membrane: sequential quantitative evaluation using corrosion casting.

Microvascular corrosion casting was used for evaluating qualitatively and quantitatively angiogenesis in the chick chorioallantoic membrane (CAM). Series of CAMs from day 8 to 18 were examined. The density of plexus capillaries increases rapidly until day 10 and then remains constant. The vessels connected to the plexus (first order vessels) increase in number and length between days 10 and 12. The vessels initiating from first-order vessels (second order) also increase in number but remain nearly the same in lengths and diameters. Compared to previous studies using stereomicroscopy, significant differences in vessel numbers and lengths exist, which can be explained by the low resolution and magnification resulting in too low vascular densities of the pre- and post-capillaries. The third-order vessels increase in number until day 12 when they reach a plateau, whereas higher-order vessels increase in number. These results give an insight into the vascular development of this organ, and provide the basis for assessing the targets and effects of angiogenic or antiagiogenic agents.

Angiogenesis in cultured and cryopreserved pancreatic islet grafts.

The ability of rat pancreatic islets to revascularize after transplantation was examined via in vitro and in vivo imaging of the microvasculature using laser scanning confocal microscopy (LSCM). Cultured or cryoprocessed islets were transplanted at the renal subcapsular site in rats. At various time intervals after transplantation, three-dimensional imaging of the graft was performed by LSCM. In vitro studies were conducted via microvascular corrosion casting of the grafted kidney in situations where it was difficult to obtain in vivo confocal data due to surgical complications. The vascular morphology of the islet grafts was evaluated quantitatively via digital image analysis algorithms to determine the morphology of the neovascular ingrowth and the rate of revascularization. In cultured islet grafts, the initiation of angiogenesis was observed within 1 week, characterized by the presence of capillary sprouts, tortuous vessels, and blood vessels with blind ends. The revascularization of the graft was typically completed within 2 weeks and could be distinguished as a network of completely perfused blood vessels consisting of intertwining capillaries, with surrounding arterioles and venules. The angiogenesis process in cryopreserved islet grafts required a longer time period to initiate (approximately 2 weeks), and the revascularization was completed in 1 week after the initiation. These results successfully demonstrate the potential of the described in vivo and in vitro LSCM techniques to measure the angiogenesis process in pancreatic islet grafts.

Optic nerve vasomotor effects of topical apraclonidine hydrochloride.

To examine, in vivo, the anterior optic nerve vasomotor effects of chronic apraclonidine hydrochloride in rabbits. After local treatment in one randomly chosen eye with apraclonidine hydrochloride 0.5% over 21 days, the microvasculature of the optic nerve was examined in five rabbits using an intraluminal microvascular corrosion casting technique. The investigators were masked as to which eye was treated. The vasoconstriction near the branching point of arterioles supplying the optic nerve was calculated as a percentage of the downstream vessel calibre. An average constriction was calculated and compared between the treated and the contralateral, untreated, eyes by means of a two tailed t test for paired variables. Constriction values of a total of 72 arterioles supplying the optic nerve were obtained for the five rabbits. The average constriction in the treated and the control eyes was comparable (p = 0.96). Chronic administration of apraclonidine hydrochloride 0.5% produces no observable optic nerve vasomotor effects in the rabbit eye.

Optic Nerve Vasomotor Effects of Topical Beta-Adrenergic Antagonists in Rabbits

To examine the anterior optic nerve vasomotor effects of nonselective and relatively beta-1-selective beta-adrenergic antagonists in rabbits, because different influences on optic nerve blood flow with these medications have been suggested. After topical therapy for 30 days with either timolol maleate 0.5% (six rabbits), betaxolol hydrochloride 0.5% (six rabbits), or placebo (two rabbits), the microvasculature of the optic nerve was examined with an intraluminal microvascular corrosion casting technique. The investigators were masked to both the medication group and the treated eye. The constriction, in percent of the downstream vessel caliber, was measured at the vascular branching point of arterioles supplying the anterior optic nerve. An average constriction was calculated and compared between the medication groups and between the treated and the contralateral, untreated eyes. Constriction values from a total of 218 arterioles supplying the anterior optic nerve were obtained for the 14 rabbits. The means of the average constriction on the treated side were comparable between the groups treated with timolol maleate, betaxolol hydrochloride, and placebo (one-way analysis of variance, P = .64), as well as between the treated and untreated eyes (two-tailed t-test for paired variables, P = .68 for timolol maleate and P = .42 for betaxolol hydrochloride). The statistical power to find a difference of 5% or more average constriction was at least 90%. Both relatively selective and nonselective beta-adrenergic antagonists produce no observable optic nerve vasomotor effects in the rabbit eye.

Microvascular pattern in the metaphysis during bone growth

Little is known about the three-dimensional micromorphology of vessels in the growth zone of long bones, where significant vasculogenesis occurs. Therefore, we examined the microvascular pattern of the femoral metaphysis. Six-week-old normal rats of either sex were used. We cast the femurs of 14 rats with Mercox for scanning electron microscopy (SEM), and in 10 rats we prepared tissue sections of femurs for light (LM) and transmission electron microscopy (TEM). In the LM, calcified cartilage was found to define cylindrical compartments beneath the last row of hypertrophied chondrocytes of the metaphyseal growth plate. These compartments ran in the bone's longitudinal axis and contained a single capillary profile. Endothelial cells of these capillaries often showed increased cytoplasmic volume and loose texture of nuclear chromatin. Cast metaphyses by SEM showed numerous parallel vascular loops with nodular protrusions 10-12 microns in diameter at their tips. The loops had ascending and descending limbs with a luminal diameter of 10-14 microns. Small projections 4-5 microns in diameter and delicate crests were sometimes found on the tip of the larger nodes. In a 100 x 100 microns area, there were 14-17 large nodes. By TEM, capillary sprouts were identified at the level beneath the last row of hypertrophied chondrocytes. These capillaries had voluminous endothelial cells rich in free ribosomes and rough endoplasmic reticulum. Endothelial cell nuclei were rounded and showed loose chromatin texture. Endothelial cells were connected by intermediate junctions and there was no basal lamina. Deeper into the metaphysis, arterioles and sinusoids were present. We conclude that the metaphyseal plate of the growing rat offers an optimal model to study vasculogenesis. Capillary sprouts can be readily identified, measured, and counted because they are located within a plane bordering against avascular cartilage. In addition, by using microvascular corrosion casting in SEM not only capillary sprouting per se but also different stages of neovascularization, indicated by differently sized nodular projections at the tip of vascular loops, can be studied in the growing long bone.

An in vivo model of chronic optic nerve ischemia: the dose-dependent effects of endothelin-1 on the optic nerve microvasculature.

The purpose of this study was to evaluate the effects induced by chronic microapplication of endothelin-1 on the anterior optic nerve microvasculature and to determine the dose-response characteristics of endothelin-1 on this vascular bed. Daily dosages between 4.69 x 10(-4) and 9.0 x 10(-1) micrograms/day of endothelin-1 were delivered continually over 3 days, and at a constant flow rate, to the perineural region of the anterior optic nerve of 15 albino rabbits via osmotically-driven minipumps. The vasomotor effect of local endothelin-1 on the microvasculature of the optic nerve was examined using intraluminal microvascular corrosion casting technique. The vasomotor effects were quantified by measuring the relative amount of vasoconstriction of the arterioles supplying the anterior optic nerve (primary and secondary branches of the short posterior ciliary arteries). The average constriction was calculated for the endothelin-treated eyes and the untreated, contralateral eyes. The mean vasoconstriction in the endothelin treated eyes ranged from 14.7% to 30.0% and was highly correlated with the logarithmic value of the daily dose of endothelin-1 (R2 = 0.59, p = 0.00083). The interocular difference (between treated and untreated eyes) of the optic nerve vasoconstriction ranged from 0-19% (mean +/- SD: 7.23 +/- 5.7%). This interocular difference also correlated highly with the log of the daily endothelin-1 dosage (R2 = 0.80; p < 0.0001). By additionally accounting for the weight and sex in a multiple linear regression function, the correlation was markedly improved (R2 = 0.92; p < 0.0001). In conclusion, the microvasculature supplying the anterior optic nerve of the rabbit demonstrates a dose-dependent vasoconstriction with chronic local application of endothelin-1. This in vivo, experimental model offers a titratable method with which the effects of chronic vasoconstriction and vascular insufficiency on the optic nerve can be examined.

New aspects of microvascular corrosion casting: a scanning, transmission electron, and high-resolution intravital video microscopic study.

We used intravital microscopy of small intestine and pancreas in order to show dynamic interactions between vascular wall and undiluted Mercox, because previous studies of ours have shown that Mercox diluted with monomeric methylmethacrylate penetrates cells in the vascular wall. Scanning and transmission electron microscopy were used to show three-dimensional pathways and correlating tissue structures, which cannot be identified in vivo. The microvascular diameters were not altered when the vasculature was flushed with saline/dextran solution using perfusion pressures between 70 and 140 mm Hg, but, in circumscribed areas, contraction of vascular wall was observed immediately after Mercox injection. This phenomenon was carried out by endothelial cells; pericytes were never present at the site of constrictions. Extravasation, i.e., leakage of the resin into the surrounding tissue, occurred in circumscribed areas regardless of the applied perfusion pressure. The resin also filled routes, which were not perfused with blood before casting. Scanning microscopy of corresponding specimens showed flattened cast channels, with impressions of valves and endothelial cell nuclear imprints characteristic of lymphatics. These results show that undiluted Mercox is a stimulus for vascular cellular components and that it changes the vascular wall permeability, resulting in extravasation and filling of lymphatics. Transmission electron microscopy showed that large vessels were homogeneously filled with resin and that cellular structures were not infiltrated with Mercox. Cut sections of the gold-coated surface of casts showed grooves up to 20 nm wide, suggestive of minimal deformation, while the abluminal surface of the metal film was almost smooth. Another proof of minimal deformation of undiluted Mercox casts is that the diameter of vessels was not altered during and after polymerization. Obtained casts are not fragile, as are casts of diluted Mercox, and phase separation does not occur, which would result in penetration of the cells in the vascular wall. For these reasons, the use of undiluted Mercox is recommended. Mixing 10 ml Mercox with 1 g catalyst resulted in complete polymerization within 5.5-7 min. This mixture can be used for casting biological specimens.

Experimental myocardial neovascularisation with a free muscle flap in the dog

Patients with diffuse small vessel coronary artery disease are often not suitable for direct coronary artery surgery. To gain insight into this problem, the effects on myocardial revascularisation of a free skeletal muscle flap anastomosed to the internal mammary artery were studied in 6 dogs. Four weeks after production of a zone of multiple microinfarctions of the anterior wall of the heart, a free flap of pectoralis muscle was grafted onto the heart. Sixteen to 18 weeks later the animals were sacrificed, and their hearts were subjected to histological and a microvascular corrosion cast examination. The results showed extensive development of a prominent vascular network penetrating from the graft into the heart. Thus, myocardial revascularisation in the dog is possible by grafting a free skeletal muscle onto the heart.

Microvascular corrosion casting of normal tissue, transitional mucosa and adenocarcinoma in the human colon.

The microcirculatory architecture of normal tissue, transitional mucosa and adenocarcinoma of the human colon was investigated with microvascular corrosion casting (MVCC) combined with scanning electron microscopy (SEM). The study showed that the capillaries within the normal mucosa were arranged in a regular hexagonal pattern around the mucosal glands and that the microvessels of transitional mucosa mostly had lost the typical hexagonal pattern and become slightly wider in diameter. The microvessels in the tumor periphery were increased in number and disorganized, and presented large variation in morphology with claw-like formations, widened sinuses, diverticula and appendixoid patterns. Microvessels were lacking in the central areas of tumors. These morphological alterations may serve as additional indicators of tumor development.

Cooling to heat of fusion (HOF), followed by rapid rewarming, does not reduce the integrity of microvascular corrosion casts

This study utilized microvascular corrosion casting techniques to evaluate changes in the microvascular patency of rat hindpaws cooled to four different subzero temperatures. Left hindpaws of anesthetized rats in group 1 were cooled to −5 °C, in group 2 to −15 °C, in group 3 to heat of fusion (HOF), and in group 4 to HOF and then to −15 °C. Although freezing did not take place in the hindpaws of groups 1 and 2, initiation of freezing in the tissues, as indicated by HOF, did occur in groups 3 and 4. Cooled hindpaws were rapidly rewarmed. Right hindpaws served as controls. Microvascular corrosion casts were made from the left and right hindpaws of all animals. There was no significant difference when the mean cast weights of cooled hindpaws from groups 1, 2, and 3 were compared to the mean cast weights of their respective control hindpaws. In group 4, there was a significant difference ( P < 0.05) when the mean cast weight of the cooled hindpaws (47.69 ± 9.05, mg ± SEM) was compared to that of the control hindpaws (80.63 ± 12.23). Since, in this acute experiment, a loss of vascular integrity occurred when the hindpaws in group 4 were cooled to −15 °C after reaching HOF, the initiation of freezing alone was not sufficient to reduce mean cast weight.

Endothelial nuclear bulging: Morphological evidence for an intraglomerular perfusion regulating mechanism in the axolotl

Kidneys of the Axolotl ( Ambystoma mexicanum: Urodela, Amphibia) have been investigated by light and electron microscopy as well as microvascular corrosion casting. Numerous glomerular endothelial nuclei bulge into the glomerular capillary lumen. It is concluded that these cells virtually stop the blood-flow in the respective vessel by blockage of erythrocyte passage. A proposal for an intraglomerular regulation mechanism of capillary perfusion (and thus filtration) by endothelial isometric contraction in the Axolotl is made on the basis of morphological methods.