Scanning Microscopy

Volume 5 Number 3 Article 25

6-25-1991

Scanning Electron Microscopy of Corrosion Casting in Medicine

M. A. Konerding University of Essen

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Recommended Citation Konerding, M. A. (1991) "Scanning Electron Microscopy of Corrosion Casting in Medicine," Scanning Microscopy: Vol. 5 : No. 3 , Article 25. Available at: https://digitalcommons.usu.edu/microscopy/vol5/iss3/25

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SCANNING ELECTRON MICROSCOPY OF CORROSION CASTING IN MEDICINE

M. A. Konerding*

Institute of Anatomy, University of Essen, 4300 Essen 1, Germany

(Received for publication November 27 , 1990, and in revised form June 25, 1991)

Abstract Introduction

The aims of this review are: 1. to provide a biblio­ The vascular system plays an important role in graphy of the publications that have used the corrosion numerous proliferative , inflammatory and casting technique; 2. to describe the advantages and degenerative diseases as well as neoplastic limitations of the methodology ; 3. to illustrate possible processes . The central significance of the vascular applications in the field of medicine , and 4. to highlight system as a target , mediator and release mechanism the significance of this method in the teaching of medi­ of physiological and pathological processes cal students . Thus , this paper is primarily focused on necessitates further, detailed studies of the normal as the scanning electron microscopical examination of well as the pathologically altered vascular system. For vascular corrosion casts. this, various methods must be chosen depending on The unsurpassed three-dimensionality of the cor­ the problem involved . In this respect , this review is rosion casting technique compared to any other means concerned with the possibilities and lim its of stands out in particular . This can be especially useful microvascular corrosion casting in medicine. when complex vascular-anatomical relationships are The corrosion casting technique is a relatively present. This applies not only to the portrayal of the old method of preparation which has been used since modes of branching and varying vascular densities but the middle ages, particularly after the advent of the sy­ also to regulatory arrangements , such as sphincter s ringe , for imaging the inner aspects of hollow organs and arteriovenous anastomoses . and vascular systems . Although, initially, this was used Between 1966 and 1990, a total of 549 publica­ almost exclusively for macroscopic purposes , it has tions were found in the Medline literature data bank, experienced a renaissance in the last few years fol­ containing the key words "corrosion casting" , "micro­ lowing the introduction of plastic materials. These ma­ vascular cast" , or "vascular cast" (as of August , 1990). terials can better withstand physical noxious agents Of those publications , most dealt with applications to than previously used casting media. The history of cor­ experimental animals. By contrast , only 142 reports rosion casting , both at the macroscopic as well as the were mainly or partially concerned with human in­ microscopic level (microcorrosion casting) , has been vestigational material. described in detail in previous publications (20, 21, 61, The normal vascular system of nearly all organs , 73, 74, 111 ). Nowadays , corrosion anatomy is used for insofar as this is of direct medical relevance , has been macroscopic purposes in exceptional cases only, so largely resolved. In our opinion , one of the most impor­ that this aspect will not be dealt with further in this re­ tant potential applications of the corrosion casting view. Both microscopic and scanning electron micros­ technique lies in the investigation of gastrointestinal , copic procedures have achieved priority . This was renal or hepatic ailments, which coincide with the re­ only made possible through the introduction of low­ construction or rarefication of the vascular bed, e.g., in viscosity plastics such as Mercox® or Batson No. 17, ulcers , ileitis terminalis , colitis ulcerosa , cirrhosis or which, at least in part, satisfy the requirements set up glo meru lonephritis. for a suitable casting medium (61, 71, 73, 74, 94, 126). With these plastics, it is possible to obtain technically KEY WORDS: microvascular corrosion casting , cor­ sound casts with respect to informationa l detail; rosion casting technique, applications, advantages , however, the ideal casting medium still needs to be human material , vessels , vascular architecture , vascu­ found. In this connection, two opposed limiting lar structure, lymphatics, angiogenesis . parameters exist: viscosity and shrinkage. The physical-technical parameters of nearly all of *Address for correspondence: todays commercially used casting media have been M.A . Konerding investigated systematically so that each investigator is lnstitut fur Anatomie, Universitatsklinikum Essen, Hufe­ aware of the artifacts and limitations of such media landstraBe 55, D-4300 Essen 1, Germany and able to take these into consideration in his FAX No. (-201) 723-5916, Phone No. (-201) 723-4285 interpretations, especially in morphometric,

851 M.A. Konerding quantitative studies (55, 64, 109, 111). In comparison to other , more sophisticated mor­ phological investigations of the micro-angioarchitec ­ ture , corrosion casting is relatively simple to learn, an efficient method that is cost-effective . The number of scanning electron microscopes in universities and other research institutes is so great nowadays that this method can be practically utilized universally. The aim of this review is to achieve a better com­ prehension of this efficaceous method and to serve as a stimulus for further research . Particular emphasis is placed on the following: 1. to provide a summary of the publicat ions involving human investigational material that have used the corrosion casting technique ; 2. to describe the advantages and limitations of the metho­ dology ; 3. to illustrate possible applications in the field of medicine , and 4. to highlight the significance of this method in the teaching of medical students. A presentation of individual results on studies of various organs and a comprehensive description about what casting has taught about each organ sy­ stem are dispensed with, since this would be beyond the scope of this review.

What are the advantages of this method?

In principle, investigations of vessels in sectional preparations can be adequately conducted using light­ ' transmission-, or scanning electron microscopic pro­ cedures as well as immunological staining techniques depending on the problem in question. However , the corrosion casting technique has numerous advanta­ ges both as an autonomous and complementary me­ thod compared to other techniques . The complex registration of the microangioarchi ­ tecture is useful in the understanding of organ functi ­ ons. After complete filling of the section of the vessel involved, which , as a rule, can be achieved with sui­ table preparation , the whole terminal blood stream from the arterial to the venous segment including the capillary bed can be clearly shown (fig. 1) . The unsurpassed three-dimensionality of this method compared to other means stands out in parti­ cular and can be especially useful when comple x vascular-anatomical relationships are present. This applies not only to the portrayal of the modes of bran­ ching (figs. 2, 4) and varying vascular densities (fig. 3) but also to regulatory arrangements (fig. 5), such as sphincters and arteriovenous anastomoses . The three­ dimensionality can, if necessary, be further increased by stereoscopic monitoring techniques . In view of the fact that the individual vascular sections can be distinguished by the shapes of their

~ Vascular corrosion cast of peripheral vessels around a subcutaneously implanted liver fragment (rat) with a newly-formed, sinusoidal capillary bed stretching from an artery (a) to a vein (v) with typical endothelial cell impressions (~). Note the dilated, im­ mature capillaries (c). Bar= 100 µm.

Fig. 2 a and b: Vascular corrosion cast of the rat cochlea as seen cranially (a) and, in detail, laterally laryngology, Osaka City University, Japan. Bar= 500 (b). Courtesy of Prof. Dr. Y. Nakai, Department of Oto- µm in a, 300 µm in b.

852 SEM of Corrosion Casting in Medicine

rangement. Arrowheads point to endothelial cell nuclei impressions indicat ing the venous origin of the arca­ des . v = main collecting vein , c = choriocapillaris , A = arcades. Courtesy of Dr. F. Koch, Clinic of Ophthalmo ­ logy , University of Bonn , Germany . Bar= 200 µmin a, 50 µm in b.

Fig. 5 a - c: Regulatory arrangements seen by Fig. 3: Branching pattern of the peripheral vessels of scann ing electron microscopy of vascular corrosion a xenotransplanted human melanoma on a nude casts : Circular contraction profiles (a) , and shunt mouse, forming the tumor vascular envelope . Bar = connection (arrows) of two arterioles with sphincter ­ 100 µm . like impressions (b) in chicken sciatic nerve and arte­ rial sphincter with a typical endothelial cushion (ti) in Fig. 4 a and b: Loop and arcade formation in the the periphery of a xenografted human spindle -cell sar­ outer periphery of the rat retina. Several loops merge coma (c). a = artery , v = vein. Bar in a = 20 µm , in b = to form a main collecting vein (a). Detail (b): loop ar- 10 µm , inc= 30 µm.

853 M.A. Konerding

endothelial cells and nuclei , they can, thus, be identi­ fied quickly and reliably after replication (fig. 6). An additional advantage is that the direction of blood flow can be determined indirectly from the length-breadth relation of the nuclear impressions. With increasing distance from the arterial bed, where endothelial cell nuclei show an elongated form , the nuclear impressi­ ons in the capillary bed become increasingly more oval.and the length-breadth relationship is smaller (20, 21, 22) . This is almost certainly due to a flow-depen­ dent orientation of endothelial cells and their nuclei . The pre-requisite for this indirect display of the direc­ tion of blood flow in an individual capillary is the posi­ tive identification of associated arterioles and venoles. If there is no corresponding hierarchical vascular sys­ tem, e.g. as in tumors, where capillaries frequently originate from venous vessels and again end in such after formation of a rete mirabilis (69, 70), no inferences as to the direction of blood flow can be drawn. Otherwise , this parameter can only be determined through the use of comparatively laborious serial sec­ tions from light or transmission electron microscopic specimens. The small size of specimens used in transmission electron microscopy , however , only permits the determination of short stretches of vessels. Again, scann ing electron microscopy of freeze-frac­ tured tissues only affords an insight into the superficial structure of endothelial cells and nuclei over short dis­ tances. Indeed, angiogenetic processes can also be def­ initely determined by means of transmission electron microscopy , but through the corrosion casting prepa­ ration it was possible to characterize angiogenetic features comprehensively (20, 45; fig . 7). In addition , a quantification of casted structures is possible (42, 55, 62, 64,109,111).

What are the putative and real disadvantages of this method?

Each method of preparation is beset with more or less major disadvantages and limitations, and must be compensated for by the introduction of complementary methods. Thus , the corrosion cast specimen is only able to yield information about the differentiation of lu­ men forming endothelial cells with a distinctly lower si­ gnificance than light or electron microscopy . No repro­ ducible results whatsoever can be expected concer­ ning the configuration and structure of subendothelial cells. Hence , structural characteristics in general can be better determined using other methods. However, this is not a real disadvantage of corrosion casting, since the fundamental value of this method lies not in the clarification of vascular structures , but their archi- 1.e..Q1.u.r.e.. Nevertheless , disadvantages can also arise in the case of correct indications. Most of the problems encountered in the prepa­ Fig. 6 a - c: Endothelial cell nuclei impressions in ration of corrosion cast specimens are concerned with small and large calibre vessels. Elongated impres­ the complete filling of the vessel of interest. This can sions in an arteriole (a) and round-to -oval shaped im­ pressions in a venular vessel (b). Inset in a : constric­ sels, CH = choriocapillaris. a and b are micrographs of tion caused by an arteriolar sphincter (arrow). Com­ corrosion casts of rat retinal vessels, c of a human xe­ pare these imprints with those of large calibre arterial nografted sarcoma on a nude mouse. Bar in a = 10 and venous vessels in c. A = arterial , V = venous ves- µm, in b = 10 µm, inc= 50 µm.

854 SEM of Corrosion Casting in Medicine possibly be rendered more difficult by arteriovenous anastomoses, extremely large drops in pressure or an unfavourable choice of the access route. A one­ hundred percent filling of all capillaries of any one or­ gan thus appears unlikely. Even with an injection of aqueous barium sulphate , not all capillary regions and arterioles can be completely shown despite the low viscosity . However, this is not a grave disadvantage since , in general, only a limited section of a vascular system can be examined by scanning electron micros­ copy anyway ("pars pro toto") , and scanning electron microscopy directly shows whether filling is complete or not. However , in the case of autoradiographic or immunological methods , for example , one can only be sure whether an avascular region was artifactual or in­ deed deficient or free of vessels only after convention­ al histological counterstaining . In studies on pathologi ca l vascular systems, it must be especially borne in mind that the vascular density in the corrosion cast specimen can deviate considerably from that of the histological specimen. On the one hand , artificial thrombi , which can only be avoided by adequate pretreatment (if necessary , he­ parinisation , flushing etc ; for details see 73, 74) , can falsely bring about a too low vascular density . On the other hand , partially or completely compressed ves­ sels may no longer be pervious to the casting medium. Thus , only functional , open vessels can be determi­ ned. This is of no disadvantage since these vessels cannot be of any nutritive service . Conversely , howe­ ver, it must not be concluded that all vessels seen in the corrosion cast specimens fulfil a nutritve function . Tumor casts with extremely high vascular densities in part, but still with large amounts of necroses , indicate that the existence of corresponding vascular densities is only the requirement but not the guarantee for suffi­ cient functionalism and nutrition . The significance of corrosion cast specimens with respect to the functio­ nalism of the vascular system under investigation can only be enhanced by including parameters such as branching pattern , vessel length, or distance to or from arterioles, etc .. However, these parameters can be well determined with scanning electron microscopy of vascular corrosion casts . The shrinkage that occurs during the polymerisa­ tion of the casting medium cannot, in our opinion , be considered to be a grave disadvantage even in mor­ phometric studies of vascular diameters . On the one hand , the shrinkage is a constantly occurring feature, for which a correction factor can be introduced, and on

Fig. 7 a - c: Vascular sprout ing and the result of an­ giogenic activities in human tumors as seen by corro­ sion casting. (a) new sprout and loop formation origi­ nating from venous vessels in front of a large collecting vein (v) in the background. The furrows and flaps (arrows) represent new sprouts. Note the changes in diameter (+). (b) Corkscrew-l ike tortuous vessel with several globular outpouchings representing further bud formations (arrowheads) as a result of redundant angiogenesis . (c) Glomeruloidal arrangement of newly formed vessels in the tumor bed. Bar in a= 25 µm, in b = 30 µm, inc= 100 µm.

855 M.A. Konerding the other hand , the percent of shrinkage is very small material , 97, i.e. 68.3 %, can be assigned to eight fields (about 6 % for Mercox®) compared with that of biologi­ of research (graph 1b). These concern the brain, eye , cal tissues in light microscopic or scanning electron lung, placenta , kidney, liver, spleen and tumors . The microscopic preparations , e.g., critical point drying, remainder of the publications more or less homo­ where one must anticipate about 50% shrinkage in geneously deals with other organs . volume (71, 126). Thus, in principle , valid morphome­ At first glance, the above mentioned eight fields of tric studies on corrosion cast specimens with respect interest concern regions that are easy to remove and to vascular densities , vessel diameters, intercapillary nourished by a few major vessels and , thus , can be distances and vascular mass can also be carried out readily filled. For example, this applies , particularly, to despite the existing problems in the software , for the spleen, kidney, eye , and liver (Table 2). On the gathering of data and for spatial resolution (55, 64, other hand, numerous organs, whose vascular sys­ 109, 111). tems up to now have not or only been sporadically subjected to vascular corrosion casting, can be remov­ When and what has been published up to ed and prepared just as easily . In particular , this ap­ now? plies to the complete gastrointestinal tract, peripheral nervous system, as well as, the endocrine organs. This Between 1966 and 1990 , a total of 549 publica­ is inconceivable insofar as that these organs are fre­ tions were found in the Medline literature data bank , quently resected during surgery and can thus be sub­ containing the key words "corrosion casting" , jected to direct study with a relatively low degree of "microvascular cast", or "vascular cast" (as of August artifacts. Against this background , the question arises 1990). Of those publications, the greatest number , 426 as to why this method is not used more often. publications (77.6 %), dealt with applications to experi­ mental animals . By contrast , only 142 reports were mainly , or at least , partially concerned with human in­ vestigational material (graph 1a) . If one subdivides the total number of publications according to years (Table 1), it can be seen that corro­ Table1: Publications per Year : sion casting was used to an appreciable extent only after 1976, i.e. after the introduction of suitable plastics ~ Animal Man Year Animal Man Year Animal Man for electron microscopy . However , it must be taken into 1966 1 3 197 4 0 1 1982 22 9 consideration that some of the journals , that played a 1967 2 0 1975 4 0 1983 31 7 significant role in the propagation of this method , were 1968 0 1 1976 1 8 5 1984 33 9 not listed in several data banks over the entire period 1969 1 0 1977 1 3 7 1985 20 11 of time ; even so , the trend mentioned here would 1970 0 0 1978 1 5 3 1986 45 1 3 seem to be correct . For example, the first report on 1971 2 0 1979 19 7 1987 43 15 scanning electron microscopy of corros ion casts by 1 972 1 0 1980 1 8 8 1988 53 14 Nowell et al. (1970 ; 94) is not listed in any literature 1973 4 1 1981 1 8 7 1989 55 20 data bank . In addition , it should be taken into account 1990 (7)* (1 )* that, apart from publications prior to 1970, even some *final numbers for 1990 are not yet available after this date restricted themselves to the use of light microscopy . On listing the publications according to organs or organ systems , a very heterogeneous distribution as well as several research fronts become apparent. Of Graph 1 b: What has been published ? the 142 publications involving human investigational - Fronts of research involving human material -

6.3 % kidney Graph I a: What has bee n published?

7 % tumor s 31. 7 other orga ns

7 % lung

cemed with erial: 77.6 % 7.8 % brain

8.5 % spleen 13.4 % eye

12 % liver

856 SEM of Corrosion Casting in Medicine

Table 2: Corrosion Casting Papers Involving Why aren't there more papers involving hu­ Human Material man investigational material?

Area of Interest Reference No. A major reason for the more frequent use of ma­ terial from experimental animals is its easier procure­ Nervous System: ment and handling. In smaller animals, the casting brain/CNS : 2,4, 12, 28, 29,30 , 57,82, medium can be directly injected via the heart or 86, 105, 117, 118, 134,143 nearby vessels without the need for elaborate choroid plexus : 89 preparation of the main vessels of the organ in Eye/Choriocapillaris /Ciliary body/Uvea: 15, 32, 33, 34, question to be filled. The vascular system can be 35,36 , 37, 38, 98, 99, 100, adequately treated prior to preparation depending on 127, 128, 129, 135, 136, the requirements (e.g., application of vasoactive 140,141,142 substances or anticoagulants) and rinsed or fixed (cf. Peripheral Endocrine Oroans: lit in 61, 73, 74, 112). These steps are considerably pancreas: 1 8 more complex in the case of human material, although Female Reproductive Organs: particularly good fillings can be achieved with excision ovary: 11 and surgical material from an appraisal of nutritive placenta: 16, 17, 54, 75, 76, 77, 78, vessels and ligature of drainage vessels. 115,121 In larger specimens where no satisfactory vaso­ Male Reproductive Oroans: anatomical resolution could be achieved prior to injec­ testis/pamp . plexus : 119 tion, there is according to our experience nevertheless penis: 7,31,125 the possibility of preventing extravasations by immer­ Heart/Coronaries: 83, 92, 93, 101, 103, 139 sing non-investigational relevant sectional- or organ ischemia : 1 O1 edges in liquid nitrogen and so obtain an adequate fil­ Kidney: 1, 8, 13, 60, 80, 81, 90, 107 ling of the vascular system . uropathies: 1 3 Even though it is technically possible in principle Respiratory System: to use autopsy material , the question must be raised lung: 5, 23, 26, 27, 84, 109, 113, whether , the same resu Its cannot be obtained by use 123 of a suitably relevant animal model because of the pulmon . diseases : 84, 108 higher probability of artifact induction . On the other Oral oroaos· hand, valid results can be achieved even through the tongue: 139 use of autopsy material for periods of up to 12 hours, tonsils : 39, 97 as long as the vascular system has been effectively Gastrointestinum : flushed with physiological saline (117, 118, 134). In stomach : 3, 40, 95,9 6, 104,124 general, however , biopsy or resection material ought appendix : 58 to be more effective . The investigator , however , must Liver: 46 , 48, 49, 50, 51 , 52, 53, realise that by using fresh human material, the sur­ 55, 85, 91, 96, 106, 116, 120, geon and the surgical techniques , but not himself , 133 dictate the time schedule . Overall , the time spent on cirrhosis : 59, 120, 132 the use of experimental animals is much less, since, in Skin: 43 general , we can dispense with the elaborate or even skin graft: 130, 131 microscopic preparation of supplying vessels prior to Spleen : 9, 10,24 , 25, 41, 47, 63, 65, filling because the casting medium can be injected di­ 66, 67, 68,110 rectly into a larger vessel such as the aorta or their Vessels : main branches in smaller laboratory animals. malformations /shunts: 3, 9, 57 However, apart from the objections based on sub­ venous valves: 88 optimal perfusion , there are two major arguments ductus venosus: 6 against the use of surgical human material : 1. interfer­ atherosclerosis : 52 ence to histopathology, and 2. the pathological chan­ diabetes : 33, 34, 36, 38, 127 ges of the vascular system. hypertension: 33, 115 In our opinion , the first point should not represent Bone Musculature & Connective Tissue: a handicap, because the injection of methylme­ bone : 19, 122 thacrylate should not greatly impair a subsequent his­ meniscus : 56 topathological appraisal. If one removes a part of the Cancer/Tumor Angiogenesis: specimen for histopathological examination immedia­ larynx carcinoma: 72, 87 tely after injection and hardening of the casting me­ M. Paget: 19 dium, extravasations can mostly be avoided and a sa­ basal cell tumor: 44 tisfactory histology can be carried out since the plastic glioma : 102 is largely removed by the ascending alcohols. This liver cell tumor: 79 procedure cannot be applied only in those cases liver metastases: 116 where cryosections are required for direct diagnosis or renal cell ca.: 14 histochemical studies . xenografts: 69, 70 The second contention , i.e. the pathological

857 M.A. Konerding

changes in the vascular system , does not argue against but m.rinvestigations with the corrosion casting technique, because the normal vascular system of nearly all organs , insofar as this is of direct medical rel­ evance , has been largely resolved. In our opinion , one of the most important future applications of the corro­ sion casting technique lies precisely in the investi ­ gation of pathological alterations of the vessels (fig. 8). This aspect has systematically been taken into consideration in only exceptional cases : actually , only extensive reports from the field of tumor vascularisation exist , and which form an ideal supplement and extension to experimental animal studies. As before , the question still remains to what extent can animal data be extrapolated to the human situation. Hence, the question of further applications arises as a matter of course , and which may be of particular relevance .

Possible further medically relevant fields of application

In the following , those areas of application are addressed , which, up to now , have either been in­ completely investigated or not at all. 1. Studies invol­ ving the gastrointestinal tract could show qualitative and quantitative changes in the vasculature in ulcers and chronic illnesses such as ileitis terminalis or colitis IQ ' ~..--:a ~ • r f ulcerosa . Equally possible are studies on changes in . I vascularisation following irradiation. 2. Vascular chan­ ges, e .g. arising during diabetes mellitus or atherosclerosis , also have only been insufficiently studied . 3. All hepatic ailments , which coincide with the reconstruction or rarefication of the vascular bed such as cirrhosis or cholestasis , could be determined in experimental animals or, posthumously , in man. 4. Up to now , reports on uropathies including -~­t·•'? glomerulonephrites are lacking completely. 5. An area of great promise would appear to lie in the studies on ' stimulation of wound healing processes , which is intimally associated with neovascularisation . Publications on neoangiogenesis have already shown that angiogenetic processes can be excellently differentiated by means of the corrosion casting technique (14, 20, 21, 45, 70).

How can Microvascular Corrosion Casting be learned?

Detailed reports concerning the application and technique are amply documented in the case of ex­ perimental animal research (16, 61, 71, 73, 74,107 , 111, 112, 114, 137), so that the investigator not yet fa­ mi liar with this method will be able to learn these Fig. 8 a and b: Corrosion casts of pathologically altered ocular vasculature. (a) 68 year old female with insulin-dependent diabetes mellitus of 26 years dura­ Venous filling defects (arrows) indicate areas tion . Posterior view of the midperiphery of the choroid. occupied by plaques or thrombi . Retinal vessels are Note the vascular irregularity , tortuosity, changes in seen in front of the choriocapillaris (CH). a= artery, V = diameter and drop-out of capillaries in the vein . background . Arterio-venous crossing in the Courtesy of Prof. Dr. A.W . Fryczkowski , Dept. of Oph­ foreground. (b) 79 year old male with retinal vein thalmology, The Ohio State University, Columbus , OH. branch occlusion . Anterior view of the posterior pole. Bar in a= 50 µm, in b = 30 µm.

858 SEM of Corrosion Casting in Medicine techniques relatively quickly and will be able to modify comes evident that one of the major problems in His­ them accordingly, provided he takes the recommenda­ tology and Microscopic Anatomy courses is how to tions into consideration. convert the details from 2-D cross-sectional light or One matter of particular concern is the still ques­ even transmission electron microscopic specimens into tionable reproducibility and reliability of the corrosion the third dimension. However , the difficulty ex­ casting technique . Only fewer than 1O reports publish­ perienced in drawing conclusions on the 3-D ed in the last five years contain reliable data on the appearance from the 2-D representation is not a percentage number of casts that could actually be problem encountered specifically by beginners but , evaluated by scanning electron microscopy or that instead , is also frequently seen in the case of ad­ have produced useful results . Despite being able to vanced students. Among other things , this may be control numerous experimental conditions , e.g. tem­ because the human eye and visual comprehension perature , viscosit ies, pre-treatmen t, anti-coagulation, have been directed towards 3-D perception since the rinsing media , fixation etc ., the amount of usable prep­ earliest times. Not least for this reason, corrosion arations varies considerably even in the hands of ex­ anatomy was at times one of the most commonest used perienced workers under optimal methods of methods of preparing vascular-anatomical specimens preparation . However, it must be taken into since the invention of the syringe in the middle ages. consideration that this also depends on the type of Numerous computer-assisted attempts to reconstruct 3- vascular system under investigation . Thus , D pictures from 2-D sections make it plain that this differentiated , hierarchically structured vascular problem is still of topical interest. systems of parenchymous organs can be more easily Corros ion casting anatomy can be implemented displayed than disorderly ones e.g. in neoplasias . as an outstandingly useful teaching aid particularly in Basically , we can say that vascular systems in soft the case of organs where a knowledge of vascular tissues can be demonstrated more easily and with a anatomy is of the utmost importance for understanding higher rate of success than those of hard tissues e.g. their function . Four examples are given to illustrate this bones. Equally , the risk of artificial extravasation point : increases in tissues showing an increased amount of 1. The course and mode of branching in the vasa angiogenetic vascular budding. afferentia of the glomerulus become partially evident Particularly in the case of more problematic under the light microscope only after numerous expla­ vascular systems, in my opinion it is beneficial to visit a nations are given - the direct comparison with a corro­ group familiar with this methodology so that one can sion casting specimen is almost self-explanatory (fig. learn the technique and errors arising from 9). preparatory measures and interpretation on site . A 2. This also applies to the interaction of vessels typical mis interpretat ion which can be avoided and alveoli in the lung. The high vasc.ular density and relatively easily concerns the phenomenon of vessels architecture cannot be shown from histological sec­ with blind endings . tions and that is why the significance of vascularisation According to Grunt et al. (44), as a rule, this phe­ can only be inadequately described by this means (fig. nomenon is due to one of the following reasons : 1. in­ 10). complete filling of the vascula r system ; 2. broken cast 3. The importance and function of the lymphati c endings; 3. vascular compress ion, or 4. proliferating vessel system must be made clear to each student. capillary outgrowths . During the macroscopic-anatomy dissection courses , It would be desirable to bring this method , its the student will , at best , recognize the terminal stret­ applications, and possible causes of error nearer to ches such as the ductus thoracicus or the ductus persons interested through appropriate courses or lymphaticus dexter . The build-up and distribution of workshops under the auspices of the electron micros­ lymph capillaries are often not visible or overlooked in copic professional associations . A first step in this di­ histological specimens because of the slight thickness rection was already taken in 1989 with the initiation of of wall as well as fixation -induced shrinkage, so that the "First European Workshop on Scanning Electron the student gains his knowledge about the initial Microscopy of Corrosion Casting in Biology and Medi­ lymph vessel almost entirely from descriptive reports in cine" in Salzburg , Austria , and which provides a major text-books without ever knowingly having seen a supplementation to the Corrosion Casting Sessions lymph capillary . However , the corrosion casting during the Annual Meetings of "Scanning Microscopy". technique is also able to demonstrate the initial stretches very instructively (fig. 11) . The Significance of Microvascular Corrosion 4. The problem of diabetic polyneuropathies , Casting in the Teaching of Medicine which represents one of the most frequent complicati­ ons of diabetes mellitus because of microangiopathic At present , no other anatom ical imaging proce­ damage , is excellently exemplified by the vascular sy­ dure is able to surpass the three dimensionality of light stem of the peripheral nerve after appropriate prepara­ and scanning electron microscopic impressions of tion (fig. 12). vascular corrosion casts of blood vessels from numer­ The list of such examples of uses of corrosion ous organs . Hence , this instructive method should be cast anatomy for teaching purposes can be extended reintroduced into the curriculum to a much greater ex­ at will. To avoid misunderstandings : there should be tent. no intention of increasing the (already oversized) bulk From numerous talks and discussions with col­ of facts to be taught but, instead, to simplify the under­ leagues and from our own teaching experience, it be- standing of certain curricula through adequate me-

859 M.A. Konerding

Fig. 9 a and b: Light microscopic image of a glome­ rulus (a). Note the difficulties in recognizing the vascular arrangement in comparison to the vascular b = 20 µm. pattern seen in a corrosion cast (b; Podarcis pelopon­ nesiaca) . Courtesy of Dr. H. Ditrich, Inst. of Zoology, Fig. 11 a and b: Initial lymphatics (I) of the subepi­ University of Vienna, Austria . Bar in a= 100 µm, in b = thelial zone of the rat tongue after interstitial injection of 20 µm. the casting medium (a). Detail (b) : Magnification of fig. 11a . Note the endothelial cell nuclei imprints. i = casted Fig. 10 a and b: Light microscopic (a) and corro­ structures of the interstitial spaces. Courtesy of Prof. Dr. sion cast specimen {b) of rabbit pulmonary vessels . A. Castenholz, Dept. of Human Biology , University of The capillary architecture around the alveoli (al) is not Kassel, Germany . visible in the light microscopic specimen . Bar in a and Bar in a= 100 µm, in b = 30 µm.

860 SEM of Corrosion Casting in Medicine

References

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861 M.A. Konerding

sion casting. J. Microsc . 143, 313-317 . 36. Fryczkowski AW, Sato SE, Hodes BL (1988) 17. Burton GJ (1987) The fine structure of the Changes in the diabetic choroidal vasculature : scan­ human placental villus as revealed by scanning elec­ ning electron microscopy findings . Ann . Ophthalmol. tron microscopy. Scanning Microsc.1, 1811-1828. 20, 299-305 . 18. Busnardo AC , Dido LJ, Thomford NR (1988) 37. Fryczkowski AW, Sherman MD (1988) Scan­ Anatomicosurgical segments of the human pancreas . ning electron microscopy of human ocular vascular Surg. Radio!. Anat.10 , 77-82. casts : the submacular choriocapillaris . Acta Anat. 132, 19. Chappard D, Alexandre C, Laborier JC , 265-269. Robert JM, Riffat G (1984) Paget's disease of bone. A 38. Fryczkowski AW, Hodes BL, Walker J (1989) scanning electron microscopic study . J. Submicrosc. Diabetic choroidal and iris vasculature scanning elec­ Cytol. 16, 341-348. tron microscopy findings . Int. Ophtalmol. 13, 269-279 . 20 . 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Woodlief NF, Eifrig DE (1982) Initial obser ­ (146) . Have you also observed this type of capillary vations on the ocular microcirculation in man: the cho­ formation? riocapillaris. Ann. Ophthalmol. 14, 176-180. Author : We have not observed this type of capillary 130. Xu D, Wang G, Liu M, Sun B, Zhong S formation up to now. However , our experience is re­ (1987) Anatomical analysis of the cause of skin stricted to that of tumor angiogenesis . Basically , how­ necrosis of the great toe after transplantation of the ever , such a possibility is quite conceivable; recent great toe nail flap. Br. J. Plast. Surg. 40, 283-287. studies on the angiogenesis in lung and chick chorion 131. Xu DC, Zhong SZ, Kong JM, Wang GY, Liu allantoic membrane have indeed shown using either MZ, Luo LS, Gao JH (1988) Applied anatomy of the transm ission electron microscopy or corrosion casting anterolateral femoral flap. Plast. Reconstr . 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