INTRODUCTION

Plastination is a groundbreaking invention that ensures the anatomical preservation of realistic, odorless, tangible, and durable specimens. This technique was developed by Gunther von Hagens in 1977, revolutionizing museological exhibition, teaching, and research in morphological sciences. It involves replacing tissue fluids with a curable polymer through dehydration, forced impregnation with the curable polymer, and finally curing the anatomical piece. This method has introduced new perspectives in the field of anatomy and has been well-documented in the scientific literature (von Hagens, 1979, 1986; Ottone et al., 2015; von Horst et al., 2019; Monteiro et al., 2022; Ottone, 2023).

The most commonly used polymers in plastination techniques are silicone, epoxy resin, and polyester resin (von Hagens, 1979, 1986; Ottone et al., 2015; Ottone, 2023). Depending on the polymer used during impregnation, the optical quality of the samples will change (Sora et al., 2019). Polyester allows the creation of semi-transparent slices with a thickness ranging from 1 to 4 millimeters (Henry & Latorre, 2007). Semi-transparent slices make it easier to study structures or body regions in a non-collapsed or dislocated state (Sora et al., 2019; Ottone, 2023). Therefore, the sheet plastination technique with polyester resin can be used to understand and interpret tomography and magnetic resonance imaging (Latorre et al., 2004; Sora et al., 2019; Ottone, 2023). In the late 1980s, Prof. Gunther von Hagens developed P35 polyester resin (von Hagens, 1994), which allowed for the production of opaque brain slices with excellent differentiation between gray and white matter, becoming the gold standard for sheet plastination (Henry & Latorre, 2007; Ottone, 2023). Despite its challenges, von Hagens continued his research and developed P40 polyester resin (Baptista et al., 2019; Ottone, 2023). Based on Latorre et al. (2004), P40 polyester resin was found to be suitable for preservation in all types of tissues. Therefore, it could not only be used for preserving brain sections, but also for preserving thin sections from other parts of the body. Additionally, the sheet plastination technique with polyester resin has been used as a practical method to obtain teaching resources from limbs, joints, and organs such as the heart, kidney, and especially the brain (Henry & Latorre, 2007; Riederer, 2014; Baptista et al., 2019). In fact, the technique of plastination in sections with polyester resin is ideal for teaching neuroanatomy, as it allows the visualization and identification of structures that are not possible to observe in three-dimensionally plastinated organs, such as the gray and white matter (Ottone, 2023).

The aim of this work was to create teaching materials for studying the bovine brain using the sheet plastination technique with polyester resin (P40 plastination technique), specifically using Biodur® P40. The Animal Anatomy Laboratory of the Faculty of Veterinary Medicine and Zootechnics (FMVZ) at Universidad Central del Ecuador (UCE) had been using the cold silicone plastination technique since 2019. This project marked the first time we applied the P40 sheet plastination technique. Notably, the project involved three FMVZ students, aiming to provide immersive training in morpho-functional studies.

MATERIALS AND METHODS

In the present work, the sheet plastination technique was applied using polyester resin Biodur® P40 (Henry & Latorre, 2007; Baptista et al., 2019; Ottone, 2023) with modifications specific to the Plastination Laboratory of the FMVZ of the UCE. The process involved five phases: 1) fixation and cutting of the organ; 2) dehydration; 3) forced impregnation; 4) inclusion of slices in chambers, and 5) curing/polymerization. Prior to the procedure, a bovine brain was obtained from a grocery store certified by the local municipality. As the organ was sourced from a deceased animal previously subjected to euthanasia, ethical approval from the Faculty Research Commission (COIF) was not required, as the commission only grants ethical approval for research involving live vertebrate animals.

Before the brain was fixed in formalin, a dissection was performed to remove the meninges and superficial blood vessels. The brain was then weighed and measured for length, width, and thickness. It was fixed in 20% formalin for two months, then washed with running water. The brain was sliced into 4 mm sections using a deli slicer (Bizerba, BJ2014), and transverse slices were made. To aid in handling during sectioning, the brain was embedded in natural unflavored gelatin and expanding polyurethane foam. The 4 mm slices were placed individually on plastic meshes with 4 mm holes.

The brain slice meshes were grouped and tied together to maintain their sequential order, and then immersed in 100 % acetone for dehydration at -20 °C. The acetone was changed every 48 hours until it was verified that ≥ 99.5 % concentration was maintained in two consecutive measurements. The dehydration process took 15 days.

The brain slices were placed in P40 polyester resin (Biodur®) for 24 hours without applying vacuum pressure. After that, the pressure was gradually reduced based on the absence of bubbles on the polymer surface. The impregnation was done at room temperature in a vacuum chamber (Biodur®, model HD22A1.0) covered with a black plastic lid to block light. Once the process was complete at 24 hours with 10 mmHg, the vacuum pump (Slaykov u Brna®, TYP model, 1.5m3/h) was turned off and allowed to sit for a further 24 hours.

As the bovine brain slices were resting in the vacuum chamber, we assembled the curing chambers (“flat chamber”) where they would be placed (Fig. 1). Each chamber was made of two 15 x 15 cm glass plates separated by a 6 mm (¼ inch) PVC hose. The hose was positioned 2 cm from the outer edge of the glass and secured with three butterfly clips on each side, leaving only the top side accessible for inserting the slices and the polyester resin (P40, Biodur®).

Two slices were placed into each flat chamber and secured with a metal clip and a magnet. P40 (Biodur®) was poured to close the open side of the chamber. After ensuring there were no air bubbles, the chamber was sealed with modeling clay. The sealed glass chambers were exposed to shaded sunlight to promote polymerization (Fig. 2). Once the brain slices were cured, the chambers were taken apart, and the samples were individually placed in airtight Ziploc®-type bags. After 15 days, the edges of the plates were trimmed, leaving about 3 cm around the brain slices (Fig. 3). Finally, the brain sections were placed sequentially and orderly on a wooden support (Fig. 4).

Figure 1. Flat chamber assembly prior to the polymerization process, using 6 mm (¼ inch) PVC hose	Figure 2. The process of curing/polymerization was conducted in a shaded area, away from direct sunlight
Figure 3. Final results of the bovine brain slices preserved using the P40 sheet plastination technique	Figure 4. Assembly of polyester plates on a wooden support

RESULTS

Using the P40 sheet plastination technique, twenty-four 4 mm cross sections were obtained from a bovine brain that was originally 10 cm long, 9.9 cm wide, and 5.4 cm thick. All the slices obtained were of very good quality with rigid consistency and showed no apparent alterations in the morphology of the anatomical structures (Fig. 3). It is worth noting that the brain sections were arranged sequentially on a wooden stand (Fig. 4) to make storage and the teaching-learning process of the sectional anatomy of the bovine brain easier.

DISCUSSION

Regarding the methodology used, it is important to note that the sheet plastination technique with P40 (Biodur®) followed the basic steps of the plastination process developed by Professor Gunther von Hagens (von Hagens, 1979, 1986). This technique was easily carried out in the Animal Anatomy Laboratory of the FMVZ-UCE, as previous work had been done there on plastination with silicone (Toaquiza et al., 2023). Additionally, following the protocols of Henry & Latorre (2007), Baptista et al. (2019), and Ottone (2023), we were able to achieve solid and easy-to-orientate brain slices, with some methodological adaptations.

Baptista et al. (2019) suggested using 10% formalin for fixation for a minimum of 6 months, while Henry & Latorre (2007) recommended fixation with 10% formalin for 2 months. In our study at the Animal Anatomy laboratory of the FMVZ-UCE, we used 20 % formalin for 2 months, as recommended by Guerrero et al. (2019) and Ottone (2023), to ensure the brain had the appropriate consistency and to prevent excessive shrinkage during the plastination process. Additionally, in the P40 (Biodur®) sheet plastination technique, the organs are embedded in 20% gelatin, and the solid block is then sectioned or cut before dehydration (Baptista et al., 2019). In our study, we utilized expanding polyurethane foam in addition to gelatin to form the block, thus making the cutting of the bovine brain easier.

Another adjustment in the protocol involved using 6 mm diameter PVC hose instead of silicone tubes in assembling the chambers. This change was made for practical reasons because silicone tubes longer than 15 cm are not available in Ecuador. Similarly, the chambers were sealed using plasticine, as Biodur® sealant was not available. According to Valenzuela et al. (2012), using these materials did not cause any issues when separating the glass plates. Additionally, there are no UV lamps in the animal anatomy laboratory, therefore, indirect sunlight was used for curing, as recommended by Henry & Latorre (2007), Baptista et al. (2019), and Ottone (2023).

CONCLUSION

The aim of this work was to obtain brain slices suitable for teaching sectional anatomy. The sections preserved anatomical details and effectively differentiated gray and white matter, making them ideal for teaching macroscopic anatomy. Arranging the bovine brain slices sequentially on a wooden support will make it easier to correlate with sectional anatomy images. Finally, the sheet plastination technique with polyester resin (P40, Biodur®) successfully preserved bovine brain sections for educational purposes, allowing long-term preservation of anatomical complexity.