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.     

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.     

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.     

How useful is plastination in learning anatomy?

In recent years plastination has begun to revolutionize the way in which human and veterinary gross anatomy can be presented to students. The study reported here assessed the efficacy of plastinated organs as teaching resources in an innovative anatomy teaching/learning system. The main objective was to evaluate whether the use of plastinated organs improves the quality of teaching and learning of anatomy. For this purpose, we used an interdepartmental approach involving the departments of Veterinary Anatomy, Human Anatomy, Veterinary Surgery, and Education Development and Research Methods. The knowledge base of control and experimental student groups was examined before and after use of the fixed or plastinated resources, respectively, to gather information evaluating the effectiveness of these teaching resources. Significant differences (p < 0.001) between control and experimental groups of Human and Veterinary Anatomy were observed in the post-test results. The Veterinary Surgery students had the most positive opinion of the use of plastinated specimens. Using these data, we were able to quantitatively characterize the use of plastinated specimens as anatomy teaching resources. This analysis showed that all the plastinated resources available were heavily used and deemed useful by students. Although the properties of plastinated specimens accommodate student needs at various levels, traditional material should be used in conjunction with plastinated resources.     

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).     

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.     

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.     

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.     

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.     

塑化标本在畜禽解剖学教学中的应用

生物塑化技术是目前国际上最先进的生物标本保存技术,其原理是利用液态高分子化合物或多聚物代替生物标本中的水分,并通过硬化处理达成组织塑化,制成的塑化标本具有无味、无毒、可用手直接触摸、无需防腐液保存、持久耐用等特点。黑龙江八一农垦大学动物科技学院购置了一批畜禽塑化标本并已在课堂教学中应用,使师生体验到塑化标本所具有的浸泡标本和模型所无法替代的优点。塑化标本能够清晰呈现一些细微的解剖结构特征,在教学过程中无论是教师使用塑化标本示教还是学生自己辨认,均不会因标本具有刺激性和毒性而影响观察。使用塑化标本示教更直观,使学生更易接受,从而提高了教学质量。     

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.     

Mast cells of the bovine trachea: staining characteristics, dispersion techniques and response to secretagogues.

Sections of the lower trachea of cattle, fixed in either Carnoy''s or formalin, were stained with toluidine blue, alcian blue, or alcian blue and safranin O to study the mast cell population. After toluidine blue staining, about twice as many cells in tissue fixed in Carnoy''s contained dark blue granules compared with tissue fixed in formalin. In addition, for the first time in cattle, a population of cells containing red granules was identified after staining with alcian blue and safranin O. Most of these red granules were formalin sensitive. An enzymatic dispersal technique for mast cells is described that yielded 9.4+/-0.4% mast cells (percentage of nucleated cells) with a viability of 92.3+/-0.6%. Spontaneous histamine release was 3.3+/-0.8%. Dispersed mast cells were challenged with various immunological and nonimmunological secretagogues. The calcium ionophores, A23187, ionomyocin, and BrX537A, were effective in releasing up to 94% of histamine in mast cells in a dose-response relationship. Pasteurella haemolytica culture supernate caused about 10% histamine release at a dose of 0.5 mg/mL after correction for spontaneous release. The average histamine content of the mast cells was 6.6+/-1.0 pg/cell. Cytospins of dispersed cells fixed in Carnoy''s and stained with alcian blue and safranin O contained mast cells with blue and red granules, and a few cells with a mixture of both granule types. Based on the effects of type of fixation, staining characteristics and histamine content, a mix of subtypes of mast cells is present in the bovine trachea. However, functionally they respond to secretagogues differently than rodent mast cells. Without an immunological secretagogue, studies to determine compounds that will be effective in blocking mast cell degranulation will be limited.     

Effects of delayed or prolonged fixation on immunohistochemical detection of bovine viral diarrhea virus type I in skin of two persistently infected calves.

The effects of delayed or prolonged fixation on immunohistochemical detection of bovine viral diarrhea virus (BVDV) antigen were evaluated in skin. Ear-notch specimens from 2 calves persistently infected with BVDV type 1 were handled in 1 of 3 ways: 1) fixed in formalin promptly and processed for immunohistochemistry (IHC) after 3-176 days; 2) held at 3-4degreesC in plastic bags up to 10 days, then fixed in formalin for 2-5 days before processing; or 3) exposed to room air and temperature for 1-5 days before formalin fixation. Immunohistochemical staining intensity was evaluated without the knowledge of specimen handling. Staining of specimens that had been promptly fixed in formalin was moderate to strong at all fixation periods through 36 days, weak or no staining was evident in specimens fixed for 176 days. Refrigerated specimens typically had moderate to strong immunohistochemical staining. Even after 10 days of refrigeration before fixation, all immunohistochemical reactions were positive. However, no immunohistochemical staining was detected in any specimen that was exposed to room air. Results indicate that prompt formalin fixation is optimal for BVDV IHC. Samples can be held in formalin at least 36 days, without loss of reactivity. A 1-day delay in fixation caused no loss of reactivity, provided the specimen was refrigerated and protected from desiccation.     

A retrospective study of the clinical, histological, and immunohistochemical manifestations of 5 dogs originally diagnosed histologically as necrotizing scleritis

Purpose To describe the clinical, histological, and immunohistochemical manifestations of canine necrotizing scleritis. Methods A retrospective examination of the clinical records and samples of ocular tissues from five dogs with a histological diagnosis ‘necrotizing scleritis’ was completed. Archived, formalin‐fixed, paraffin‐embedded samples and two control globes were stained with hematoxylin and eosin, Gram, periodic acid–Schiff (PAS) and Masson trichrome stains, and they were immunohistochemically labeled for CD3, CD18, and CD20. Results Of the five cases reviewed, only two could be confirmed as idiopathic necrotizing scleritis. The other three cases were retrospectively diagnosed as unilateral focal, non‐necrotizing scleritis, one as episcleritis and the third was scleritis secondary to a proptosed globe based on our retrospective clinical, histological, and immunohistochemical evaluations. In these two cases, idiopathic necrotizing scleritis manifested as a bilateral, progressive, inflammatory disease of the sclera and cornea that induces significant uveitis. Light microscopic examination confirmed collagen degeneration and granulomatous inflammation. There was no evidence for an infectious etiology based on Gram’s and PAS stainings. Immunohistochemical labeling revealed a predominance of B cells in idiopathic, bilateral necrotizing scleritis. Tinctorial staining abnormalities with Masson’s trichrome stain were present in scleral collagen of the two cases with idiopathic necrotizing scleritis as well as a case of secondary traumatic scleritis. Conclusions Based on a limited number of cases, idiopathic canine necrotizing scleritis shares similar histopathological features with non‐necrotizing scleritis and episcleritis; however, necrotizing scleritis is B‐cell‐dominated and bilateral, and significant collagen alterations manifest with Masson’s trichrome stain.     

Estructura del estroma conjuntivo del epidídimo del bovino cebú (Bos indicus)

Six adult Zébu cattle were used for a study of the histologic structure of the epididymus. Tissues were fixed in neutral formalin or in Bouin's solution. Tissues were prepared with parafin sectioning and stained with hematoxylin and eosin, Masson and Mallory's trichrome stain and with aldehyde fuchsin and Gomori's argentamine stains. The structure of the connective tissue stroma is described and compared with that of other mammals. The presence of smooth muscle within the stroma and within the tunica albuginea is described for the Zébu.     

High field magnetic resonance imaging anatomy of feline carpal ligaments is comparable to plastinated specimen anatomy

Feline carpal ligament injuries are often diagnosed indirectly using palpation and stress radiography to detect whether there is instability and widening of joint spaces. There are currently no reports describing normal feline carpal ligament anatomy and the magnetic resonance imaging (MRI) appearance of the carpal ligaments. The objective of this prospective, anatomic study was to describe normal feline carpal ligament anatomy using gross plastinated specimens and MRI. We hypothesized that MRI could be used to identify the carpal ligaments as previously described in the dog, and to identify species specific variations in the cat. The study was conducted using feline cadaver antebrachii that were radiographed prior to study inclusion. Three limbs were selected for MRI to confirm repeatability of anatomy between cats. The proton density weighted pulse sequence was used and images were acquired in transverse, dorsal, and sagittal planes. Following MRI, the limbs were plastinated and a collagen stain was used to aid in identification of carpal ligament anatomy. These limbs were sliced in sagittal section, and a further six paired limbs were included in the study and sliced in transverse and dorsal planes. Anatomic structures were initially described using MRI and then subjectively compared with gross plastinated specimens. Readers considered the transverse MRI plane to be most useful for visualizing the majority of the carpal ligaments. Findings indicated that MRI anatomy of the carpal ligaments was comparable to plastinated anatomy; therefore MRI appears to be a beneficial imaging modality for exploration of feline carpal pathology.     

脊髓等柔软组织石蜡组织切片制作技术探讨

以家兔脊髓组织为实验材料,探讨制作大批量脊髓组织教学标本简便、可行的方法。结果表明,用常规方法制作优质的脊髓组织切片时,在10%的中性福尔马林固定液中固定7 d,进行H.E染色时,苏木精染色的时间为30 min,伊红染色时间6 min,制作的切片细胞核嗜碱性、呈蓝紫色,细胞质和细胞间质嗜酸性、呈粉红色,胞质内尼氏体为蓝紫色颗粒,两者间颜色对比度好,容易区分,是制作大批量教学标本的可选方法。     

用台盼兰—姬姆萨染色检测家畜精子顶导反应的研究

本文探讨了用台盼兰—姬姆萨染色检测家畜精子顶体反应的可行性。用肝素或钙离子载体诱发精子顶体反应。根据染色结果将精子分为四类:a)核后帽部不着色或淡青色,顶体不着色或部分紫红色(有顶体反应活精子);b)核后帽部暗青色.顶体部不着色或部分暗红色(有顶体反应死精子);c)核后帽部不着色或淡青色,顶体部紫红色(无顶体反应活精子);d)核后帽部暗青色,顶体部暗紫红色(无顶体反应死精子)。有顶体反应活精子百分率与仓鼠卵穿透率呈强正相关。从而证明台盼兰—姬姆萨染色是检测家畜精子顶体反应的有效手段,并能预测获能处理后精子的受精能力。     

Mohs micrographic surgery: a technique for total margin assessment in veterinary cutaneous oncologic surgery

Mohs micrographic surgery (MMS) is the gold standard for the excision of locally invasive cutaneous malignancies in human dermatological surgery. Using a unique horizontal sectioning technique, MMS enables 100% surgical margin assessment and provides the lowest recurrence rates for locally invasive tumours. The purposes of this preliminary study were to explore the feasibility of application of MMS in the veterinary setting and to establish practical advantages and limitations of its use in a pilot programme. It was hypothesized that MMS technique could provide 100% tumour margin assessment using frozen and/or formalin‐fixed horizontal histopathologic sections. Tumour excision and colour‐coded mapping were performed, and specimen tissue was fixed using either frozen sections or formalin‐fixed sections. Horizontal sections were assessed for quality and presence and location of neoplastic cells based on the mapped orientation. The MMS technique was used in the excision of six squamous cell carcinomas and five mast cell tumours. In all cases, the MMS permitted 100% tumour margins examination.     

Characterization of Lesions Caused by a South American Virulent Isolate (‘Quillota’) of the Hog Cholera Virus

In this study, macroscopic and histopathological lesions produced by a virulent South American isolate (‘Quillota’) of hog cholera virus were studied. The virus was inoculated in doses of 10⁵ TCID₅₀ in each of 35 pigs of 20 kg live weight. The animals were slaughtered from 4 to 18 days post‐inoculation. The presence of virus antigens in lymphatic tissue was confirmed by both direct immunofluorescence and Avidin‐Biotin‐Peroxidase techniques in formalin‐embedded tissue samples. Histological sections were stained with haematoxylin‐eosin and Mallory's phosphotungstic acid haematoxylin methods. The ‘Quillota’ isolate used in this study caused a disease characterized by vascular lesions (splenic infarcts, haemorrhages in the lymph nodes and the urinary system and disseminated microthrombosis), and necrosis of lymphocytes, particularly in the B‐areas of the lymphoid organs, lesions that are characteristic of the acute form of the disease. Other lesions observed were a non‐purulent meningoencephalitis, the necrosis of the epithelial cells of tonsils, the presence of fibrin nets in the red pulp and a marked thickening of the alveolar septa.