E12 technique: Conventional epoxy resin sheet plastination

Epoxy plastination techniques were developed to obtain thin transparent body slices with high anatomical detail. This is facilitated because the plastinated tissue is transparent and the topography of the anatomical structures well preserved. For this reason, thin epoxy slices are currently used for research purposes in both macroscopic and microscopic studies. The protocol for the conventional epoxy technique (E12) follows the main steps of plastination—specimen preparation, dehydration, impregnation and curing/casting. Preparation begins with selection of the specimen, followed by freezing and slicing. Either fresh or fixed (embalmed) tissue is suitable for epoxy plastination, while slice thickness is kept between 1.5 and 3 mm. Impregnation mixture is made of epoxy E12 resin plus E1 hardener (100 ppw; 28 ppw). This mixture is reactive and temperature sensitive, and for this reason, total impregnation time under vacuum at room laboratory temperature should not last for more than 20–24 hr. Casting of impregnated slices is done in either flat chambers or by the so‐called sandwich method in either fresh mixture or the one used for impregnation. Curing is completed at 40°C to allow a complete polymerization of the epoxy‐mixture. After curing, slices can be photographed, scanned or used for anatomical study under screen negatoscope, magnification glass or fluorescent microscope. Based on epoxy sheet plastination, many anatomical papers have recent observations of and/or clarification of anatomical concepts in different areas of medical expertice.     

Plastination—A scientific method for teaching and research

Over the last four decades, plastination has been one of the best processes of preservation for organic tissue. In this process, water and lipids in biological tissues are replaced by polymers (silicone, epoxy, polyester) which are hardened, resulting in dry, odourless and durable specimens. Nowadays, after more than 40 years of its development, plastination is applied in more than 400 departments of anatomy, pathology, forensic sciences and biology all over the world. The most known polymers used in plastination are silicone (S10), epoxy (E12) and polyester (P40). The key element in plastination is the impregnation stage, and therefore depending on the polymer that is used, the optical quality of specimens differs. The S10 silicone technique is the most common technique used in plastination. Specimens can be used, especially in teaching, as they are easy to handle and display a realistic topography. Plastinated silicone specimens are used for displaying whole bodies, or body parts for exhibition. Transparent tissue sections, with a thickness between 1 and 4 mm, are usually produced by using epoxy (E12) or polyester (P40) polymer. These sections can be used to study both macroscopic and microscopic structures. Compared with the usual methods of dissection or corrosion, plastinated slices have the advantage of not destroying or altering the spatial relationships of structures. Plastination can be used as a teaching and research tool. Besides the teaching and scientific sector, plastination becomes a common resource for exhibitions, as worldwide more and more exhibitions use plastinated specimens.     

Ultra‐thin sectioning and grinding of epoxy plastinated tissue

With classical sheet plastination techniques such as E12, the level and thickness of the freeze‐cut sections decide on what is visible in the final sheet plastinated sections. However, there are other plastination techniques available where we can look for specific anatomical structures through the thickness of the tissue. These techniques include sectioning and grinding of plastinated tissue blocks or thick slices. The ultra‐thin E12 technique, unlike the classic E12 technique, starts with the plastination of a large tissue block. High temperatures (30–60°C) facilitate the vacuum‐forced impregnation by decreasing the viscosity of the E12 and increasing the vapour pressure of the intermediary solvent. By sectioning the cured tissue block with a diamond band saw plastinated sections with a thickness of <300 μm can be obtained. The thickness of plastinated sections can be further reduced by grinding. Resulting sections of <100 µm are suitable for histological staining and microscopic studies. Anatomical structures of interest in thick plastinate slices can be followed by variable manual grinding in a method referred to as Tissue Tracing Technique (TTT). In addition, the tissue thickness can be adapted to the transparency or darkness of tissue types in different regions of the same plastinated section. The aim of this study was to evaluate the advantages of techniques based on sectioning and grinding of plastinated tissue (E12 ultra‐thin and TTT) compared to conventional sheet‐forming techniques (E12).     

History and development of plastination techniques

Plastination was a game‐changing invention for macroscopic anatomical preparation. The method yielded dry, odourless, tangible and durable specimens which allowed new exhibition and teaching set‐ups and paved the way for sophisticated preparations and spectacular positioning of specimens. Despite the impact of the new method, there have been similar techniques in place before. Exsiccation techniques, polymer embeddings and specimen impregnation with hardening substances were earlier methods which already included the main concepts that were later combined and refined in plastination. S10 silicone plastination, the technique most commonly known and applied, was followed by plastination methods suitable for research and sectional anatomy teaching. Numerous variations of sheet plastination techniques allow research applications and new ways of presenting topographic relations and mesoscopic insights. Besides the development of plastination techniques in sensu stricto, related techniques had a renaissance with new applications and developments, including corrosion casting and diaphonization methods. This brief review shall provide a historical context of plastination including some anecdotal spotlights on the ideas and innovations that lead to nowadays plastination techniques.     

Multiple polymer plastination: Combining different types of polymers in teaching and exhibition plastinates

Vacuum forced tissue impregnation is the signature step of the plastination process. It requires polymers with a low vapour pressure, low viscosity and a long pot life. Plastination polymers are a compromise between these mandatory requirements on the one hand and various secondary demands such as specimen stability, resistance to UV light and defined light refraction index on the other hand. Combining different polymers in one plastinate instead of using one plastination polymer alone can result in improved specimens for exhibitions and teaching including hands‐on use for students. The aim of this study was to assess the range of possible sheet plastinate modifications and how the resulting multiple polymer plastinates can fulfil the secondary requirements of user‐friendly plastinates. Adding sub‐steps of tissue impregnation and processing to the standard plastination protocol allows combining different polymer properties including the use of substances which are not suitable for conventional plastination as such but have better properties than plastination polymers. Advantages like resistance to UV light and mechanical stability can be combined and characteristic disadvantages of plastination polymers can be avoided. Acrylic protection layers (APL) offer a complete protection of the specimen in combination with advanced presentation possibilities and the option of completely refurbishing valuable specimens. Hybrid sheet plastinates provide lower preparation cost and polymer–tissue interactions for an improved visualization of fat, nerves and brain tissue. Selective impregnation is a promising approach for the clearer differentiation of various structures and tissue types.     

Room temperature/Corcoran/Dow Corning™—Silicone plastination process

For 20 years, the cold temperature/S10/von Hagens' plastination technique was used to preserve biological specimens without challenge. It became the “gold standard” for preservation of beautiful, dry biological specimens. Near the end of the 21st century, a group from the University of Michigan and environs and Dow Corning?, USA, combined silicone ingredients, similar to the von Hagens' plastination products, however in a different sequence. The new polymer (Cor‐tech) was combined with the cross‐linker to design the “impregnation mix” which would invade the cellular structure of the specimen and yet was stable at room temperature. Later, curing would be by application of the catalyst onto the impregnated specimen. This unique sequencing of products would become the “Room temperature/Dow Corning?/Corcoran—Silicone plastination technique.” The results of this room temperature technique provided similar plastinates, beautiful and practical for demonstration, containing no toxic chemical residues and forever preserved. As the name implies, impregnation of this silicone mix could be done at room temperature, without having to be kept cold. Both processes (cold and room temperature) required the same four basic steps for plastination. As well, both processes used similar basic polymers and additives to produce plastinates. However, they were combined in a different sequence. Cold temperature combines polymer and catalyst/chain extender, which is not stable and therefore must be kept colder than ?15°C, while room temperature combines polymer with cross‐linker which is stable, and likely forever.     

Plastinated heart slices aid echocardiographic interpretation in the dog

Our aim was to compare plastinated sections of the canine heart with corresponding two-dimensional (2D) echocardiographic images. Thirteen dog hearts were fixed by dilation and then processed by the S10 silicon plastination method (Biodur). Two dogs without evidence of cardiac disease were imaged using 2D echocardiography so as to obtain a complete series of the standard right and left parasternal images, which were compared with corresponding plastinated slices obtained by knife sectioning of the hearts. The plastinated slices revealed the internal anatomy of the heart with great detail and were particularly useful to display the spatial relationship between complex anatomic structures. The plastinated slices corresponded accurately with the echocardiographic images. Because of the dilation of the right heart during the fixation process, it was not possible to obtain plastinated specimens in ventricular systole. This paper may be a reference atlas for assisting 2D echocardiography interpretation.     

Plastination of larger and massive specimens—With Silicone

In 1977, plastination was unveiled, which replaced tissue fluid with a curable polymer. Today preservation via plastination of various animal and plant tissues, organs, and whole bodies is an extremely useful technique to display such and help educate vast arrays of both allied science students and the lay public across the planet. The diversity of applications of plastination techniques seems to be without limits. In fact, the only real limitation to plastination is one's imagination! The size of plastinates during the early years of plastination was comparatively small and dictated primarily by the size of the available plastination kettle/chamber, 35 L Heidelberg plastination kettle (49 cm H × 34.5 cm diam.). In the 1990s larger chambers were designed and slowly became available:150–210 cm (long) × 65–80 cm (wide) and 83–92 cm (high). Today a few large vacuum chambers are in service which will accommodate whole bodies of man and domestic or exotic animals. Today, at least two gigantic chambers are available to impregnate massive specimens. These are 3.5 m × 2 m × 1.5 m (Dalian) and 4 m × 3 m × 2.2 m (Guben). Also, the need for larger quantities of acetone and impregnation mix, not to mention the great increase in specimen preparation time, makes this a major investment. The “cold temperature process” is used to impregnate these massive creations. The room temperature technique could be used. The same four plastination steps are necessary for larger and massive specimens. Besides their tremendous size, the slippery silicone polymer is a reckoning force.     

Corrosion casting in anatomy: Visualizing the architecture of hollow structures and surface details

Corrosion casting is the technique by which a solid, negative replica is created from a hollow anatomical structure and liberated from its surrounding tissues. For centuries, different types of hardening substances have been developed to create such casts, but nowadays, thermosetting polymers are mostly used as casting medium. Although the principle and initial set‐up are relatively easy, producing high‐quality casts that serve their intended purpose can be quite challenging. This paper evaluates some of the more popular casting resins that are currently available and provides a step‐by‐step overview of the corrosion casting procedure, including surface casts of anatomical structures. Hurdles and pitfalls are discussed, along with possible solutions to circumvent them, based on personal experience by the authors.     

Computed tomography of the abdomen in Saanen goats: I. reticulum, rumen and omasum

Computed tomography (CT) of the reticulum, rumen and omasum was carried out in 30 healthy goats and the images were compared to corresponding body sections obtained at postmortem. A multidetector CT was used to examine goats in sternal recumbency. A setting of 120 KV and 270 mA was used to produce 1.5-mm transverse slices from the fifth thoracic vertebra to the sacrum. Soft tissue structures were assessed in a soft tissue with a window width (W) of 400 Hounsfield Units (HU), and a window level (L) of 40 HU. The layering of the ruminal contents was assessed in an ingesta window with a W of 1500 HU and an L of 30 HU. After subjective evaluation, the size of the rumen and omasum, the thickness of the walls of the reticulum, rumen and omasum and the height of the gas cap and fibre and liquid phases of the rumen were measured. Fifteen goats were euthanised after CT examination, placed in sternal recumbency and frozen at -18 oC for three to 10 days. Thirteen goats were then cut into 1.0- to 1.5-cm-thick transverse slices. One goat was cut in dorsal-plane slices and another in sagittal slices. The structures in the CT images were identified by using the corresponding anatomical slices.     

Cold temperature/Biodur®/S10/von Hagens'—Silicone plastination technique

Plastination is a late 20th century preservation methodology which replaces tissue fluid within a specimen with a curable polymer, such as silicone. Plastination yields superb, beautiful, well‐preserved specimens each with their own unique qualities. Silicone polymer is used around the world to preserve macroscopic cadavers or portions/organs thereof. Plastination was conceived by Dr. Gunther von Hagens, Universität Heidelberg, Heidelberg, Germany prior to 1977. Silicone polymer was the primary polymer which emerged initially for plastination. The Biodur^® line of silicone polymer and additives was chosen and manufactured because it has consistently produced the best plastinates since the inception of plastination. Since the discovery of silicone, generic and similar silicone polymers are known and used around the World by many industries and used in numerous products. The plastination process has four steps: Specimen preparation, Specimen dehydration and degreasing, Vacuum‐forced impregnation of specimens and Specimen hardening.     

VALIDATION OF A NOVEL TECHNIQUE FOR CREATING SIMULATED RADIOGRAPHS USING COMPUTED TOMOGRAPHY DATASETS

Understanding radiographic anatomy and the effects of varying patient and radiographic tube positioning on image quality can be a challenge for students. The purposes of this study were to develop and validate a novel technique for creating simulated radiographs using computed tomography (CT) datasets. A DICOM viewer (ORS Visual) plug‐in was developed with the ability to move and deform cuboidal volumetric CT datasets, and to produce images simulating the effects of tube‐patient‐detector distance and angulation. Computed tomographic datasets were acquired from two dogs, one cat, and one horse. Simulated radiographs of different body parts (n = 9) were produced using different angles to mimic conventional projections, before actual digital radiographs were obtained using the same projections. These studies (n = 18) were then submitted to 10 board‐certified radiologists who were asked to score visualization of anatomical landmarks, depiction of patient positioning, realism of distortion/magnification, and image quality. No significant differences between simulated and actual radiographs were found for anatomic structure visualization and patient positioning in the majority of body parts. For the assessment of radiographic realism, no significant differences were found between simulated and digital radiographs for canine pelvis, equine tarsus, and feline abdomen body parts. Overall, image quality and contrast resolution of simulated radiographs were considered satisfactory. Findings from the current study indicated that radiographs simulated using this new technique are comparable to actual digital radiographs. Further studies are needed to apply this technique in developing interactive tools for teaching radiographic anatomy and the effects of varying patient and tube positioning.     

自发性急性犬瘟热的原发性脱髓性脑病

为了进一步观察犬瘟热病毒引起的原发性脑损伤和包涵体形成的特点，调查脑组织的损伤与神经症状的关系，对10只急性犬瘟热病犬的脑组织进行了详细的病理学研究。为了仔细地观察病变，本试验按照解剖学关系将脑组织分成3个大部分和11个切面，即大脑(4个切面)，脑干(5个切面)和小脑(2个切面)。组织切片经HE、LFB和免疫组织化学染色后进行检查，结果表明：在大脑，脱髓呈弥漫性发生，程度较轻；脑干的周围或靠近第三脑室的白质脱髓较重；小脑在轻度或中度脱髓的基础上常出现严重的多发性脱髓灶。脱髓部呈空泡或海绵状，有少量胶质细胞存在，但无炎性反应。脱髓性病损是非时称性发生，对神经束没有特殊的亲和力。在脑室的室管膜细胞内发现有较多的嗜酸性胞浆或核内包涵体。用抗犬瘟热病毒抗体染色，带有包涵体的室管膜细胞呈现强阳性反应。部分锥体细胞，神经核细胞和漓氏细胞变性、溶解或胞浆深染。胞核浓缩。这种变化以小锥体神经细胞表现得最为明显。根据此研究结果，作者认为由犬瘟热病毒引起的原发性脑组织损伤是一种脱髓性脑病，而不是脑炎变化；位于室管膜细胞内的包涵体对于脑组织犬瘟热的确诊具有重要的作用；由于犬瘟热病毒引起神经细胞的损伤是非特异性的，对脑组织的侵害是非对称性的。对神经束的作用无特殊的亲和力，所以患犬瘟热的犬在临床上可出现不同的神经症状。     

Comparison of aspiration and nonaspiration techniques for obtaining cytologic samples from the canine and feline spleen

Background: Ultrasound‐guided fine needle aspiration of the spleen is commonly used in the diagnostic evaluation of veterinary patients. Techniques using suction delivered through a 6–20‐cm³ syringe are the most commonly described means of obtaining cytologic samples of the spleen. Comparison studies of various human lesions have shown nonaspiration techniques to produce equal or superior cytologic specimens with less blood than specimens obtained using aspiration techniques. Objective: The purpose of this study was to compare the quality of splenic cytology specimens obtained using aspiration and nonaspiration techniques. Methods: Client‐owned dogs (n=24) and cats (n=7) receiving an abdominal ultrasound at the University of Tennessee College of Veterinary Medicine were enrolled in the study between January and June 2005. Samples were obtained from patients with and without sonographic splenic abnormalities. Two clinical pathologists, working independently and blinded to the method of sample collection, graded the cytologic specimens using a subjective scoring system for cellularity, amount of blood, and preservation of cellular morphology. Results: Agreement between the 2 independent observers was good. Direct comparison of the 2 techniques showed that samples obtained by the nonaspiration method had higher cellularity (P=.0002), less blood (P=.0023), and similar cell morphology (P=1.0000) compared with samples obtained by the aspiration method. Conclusion: These results suggest the nonaspiration technique is a superior method for obtaining a high‐quality cytologic specimen from the canine and feline spleen.     

Differences between µCT‐imaging and conventional CT for the diagnosis of possible diseases of the middle and inner cat ear

The aim of this study was to check the relevance of using in‐vivo micro computed tomography (µCT) for the diagnosis of possible diseases of the middle and inner ear of the cat. Therefore, on the one hand, differences of the detail detectability between the two imaging methods conventional computed tomography (cCT) and in‐vivo µCT were analyzed. Six healthy cat ears were dissected and scanned several times and the obtained images were compared with each other. On the other hand, histological slices of all ears were prepared and pictures of defined anatomical structures were taken and compared with the identical sectional plane of the µCT‐images. This way it was possible to evaluate the quality and clinical limitations of the in‐vivo µCT. The results show that an in‐vivo µCT is suitable to analyze even the smallest osseous structures, such as the semicircular ducts, the spiral osseous lamina or the ossicles whereas with the help of cCT it is not possible to identify such small osseous structures because of their blurred and less detailed representation. Delicate soft tissue structures as the membranous labyrinth including hearing and vestibular organ cannot be differentiated with as well in‐vivo µCT‐ as with cCT‐images. In‐vivo µCT represent a good possibility for more detailed diagnosis of extremely fine structures which cannot be detected with cCT. Histological slices can nonetheless not be replaced by in‐vivo µCT due to a too low spatial resolution and the limitations of the in‐vivo µCT with regard to the evaluation of soft tissue dense structures.     

Occlusion of the Middle Cerebral Artery: a New Method of Focal Cerebral Ischemia in Rats

The study in Wistar rats attempted to improve the occlusion technique of the middle cerebral artery (MCA) as a precise method for initiating stroke. In a first part it was necessary to study the exact anatomy of blood vessels of the brain in seven rats of 170-410 g body weight by corrosion cast. The lengths and diameters of defined locations of the blood vessels were measured. The temporary as well as the permanent methods were refined or replaced. The first one was completed in main training the physiological blood flow after temporary occlusion, while the permanent occlusion was performed by positioning a silicone cap in the MCA. A filament guide was introduced from the common carotid artery (CCA) via internal carotid artery (ICA) to guide the silicon cap at the branch of the MCA. Histological sections of the brain of rats showed 24 h after the permanent occlusion a reproducible infarct in the brain. This area corresponded very well with the supply of the MCA. The new occlusion method with a silicon cap was compared with the occlusion methods of CCA route and external carotid artery (ECA) route. The total infarct volume was significantly larger in the CCA route and ECA route groups than in the silicon cap group (means: CCA route 261 mm³; ECA route 191 mm³ vs. 128 mm³ silicon cap group; P  < 0,05). It could be demonstrated that the new silicon cap occlusion technique imitates the pathological situation of a cerebral infarct in man. Moreover it is less invasive for the animals and more precise and reproducible regarding the infarcted area in comparison to the other occlusion methods. Based on anatomical measurements of the blood vessels the described silicon cap method can be recommended for rats of a body weight between 340–370 g.