1 -Department of Anatomy, Dalian Medical University, Dalian, 116027, China.
2 -Department of Comparative Medicine, College of Veterinary Medicine, University of Tennessee, 2407 River Drive, Knoxville, TN, 37996-4543, USA.
Plastination has become the gold standard for preservation of biological specimens. Plastination is applicable to many allied areas: anatomy, biology, clinical medicine and art. The polyester technique produces 2-3mm semi-transparent to translucent slices which display anatomy within its normal anatomical environs. Polyester slices are an excellent modality for understanding modern diagnostic images: Computed tomography, magnetic resonance and ultra sound. Polyester was developed for preservation of brain tissue. In recent years, polyester is being used for presentation of numerous tissues.
plastination; polyester method; body slices; polyester resin; P45
H. J. Sui -Department of Anatomy, Dalian Medical University, Dalian, 116027, China. Telephone: (86) 411 - 8480 - 0916; Fax: (86) 411 - 8472 - 4558; E-mail: firstname.lastname@example.org
Sheet plastination, developed for preservation of brain tissue in a 4-8mm slice format, has been used for anatomical study or research using the flat chamber technique (Barnett, 1997, Henry and Weiglein, 1999, Sora et al., 1999; Latorre et al., 2002). Polyester plastination incorporates the general principals of the classic plastination techniques (von Hagens, 1979; 1986; von Hagens et al., 1987) and tissue fluid is removed from the slices and replaced with a curable polyester resin. The P35 resin, developed in the late 1980's, is the gold standard for brain slice production of translucent brain slices with exquisite differentiation of white and gray matter. Head slices have been produced with the P35 resin (de Boer-van et al., 1993). P40 brain slices yield good white/gray differentiation. Generally the P40 process utilizes thinner slices (2-3mm) (von Hagens, 1994). More recently, P40 polyester resin is used also for body slices (Latorre et al., 2004) and to plastinate gross anatomical structures (Sora, 1998). A newer polyester (P45) technique will be presented (Gao et al., 2006). All of these resins utilize forced impregnation and casting between glass plates.
Chemicals used in polyester-plastination include:
The Hoffen products for polyester plastination are:
The basic steps of plastination are: Specimen prep, dehydration, impregnation and curing.
Note for production of P45 body slices, the steps of specimen preparation, slicing and dehydration are similar to the "Biodur™ El 2 Epoxy Technique". Please refer to that section of the E12 epoxy process for a more detailed description .
Specimen preparation equipment:
More detail may also be found in the P35 and P40 techniques which precede this manuscript. The non fixed specimen is positioned for anatomical alignment and frozen preferably in an ultra-cold (-70°C) deep freezer for two days (longer for larger specimens) for best slice production. Fresh tissue may be preferred . However, tissue should be fixed in formalin to decrease the potential for exposure to biohazards (Smith and Holladay , 2001). Tissue color preservation is a prime reason for formalin not to be used. Hairy specimens should be clipped.
Slicing: Large specimens should be divided into smaller manageable portions which will also prevent thawing. Set the guide stop at the desired specimen thickness (2- 3mm) and saw serial sections. Cooling the guide stop and saw table prevents premature thawing of the specimen and slices. Square the end of the tissue block and commence sawing. Slices are placed on an acetone resistant grid and the saw dust is removed by scraping it off with a knife and/or running a small-brisk stream of tap water across the surface. Caution: Do not thaw the slice. The grids with their cleaned slices are stacked, tied together with twine and placed in either the first cold acetone (-25°C) bath or in a fixative bath.
Fixation and Bleaching - Optional : Depending on the specimen, it may be necessary to fix the slices, as well as bleach them. Slices can be submerged in 10% formalin for one or two weeks. Once fixation is completed the fixative can be rinsed out in running tap water over night. If brightening of the slices is desirable, immerse them in 5% dioxigen (bleach) overnight or until the desired brightening is completed. Flush with running water for one hour and precool (5°C) to prevent ice crystal formation when submerged in the cold acetone.
Dehydration and degreasing of body slices
Freeze substitution in -25°C acetone is the recommended dehydration procedure for plastination.
The precooled, cleaned stack of slices is placed into the first cold (-25°C) acetone bath for one week. Next the stack of slices is placed into the next fresh acetone at -l 5°C for seven days. The third change is into 100% acetone at room temperature for one week for degreasing. If more transparency of fat is desired, dehydrated slices may be placed into methylene chloride (dichloromethane) (MeCl) for one or two days. Monitor degreasing in MeCl daily. When body slices are appropriately degreased, transfer the slices from their solvent bath (acetone or MeCl) into the impregnation resin.
Forced impregnation of body slices
Forced impregnation, replacement of solvent with a curable resin, is based on a difference of vapor pressure of the solvent and the resin.
Preparing the impregnation -mixture: The polyester resin impregnation bath is made by thoroughly mixing : 1000 ml Hoffen polyester P45 resin with 10g of P45A, 30ml P45B and 5g of P45C.
Immersion into P45 resin of brain slices: Flat chambers will be used to immerse the dehydrated slices into the p45 resin-mix .
Preparing flat (glass) casting chambers for forced impregnation of body slices: Casting chambers are built for casting of the slices prior to impregnation. The flat chambers are constructed of two plates of 5mm tempered glass, 4mm flexible latex tubing and large fold back clamps. The glass and tubing are clamped together around the perimeter of the bottom and sides of the glass. The gasket end, which is left longer, will be used to close the top prior to curing. Once the casts are assembled, the impregnation resin mixture is prepared. 1000 ml Hoffen polyester P45 resin is mixed with 1Og of P45A, 30ml P45B and 5g of P45C. P45A and P45C are plasticizers and P45B is a hardener. After preparation of this impregnation reaction-mixture , a dehydrated slice is removed from the acetone and placed in the chamber. Immediately the chamber is filled with the impregnation-mixture using a funnel. The filled chamber is placed upright in the room temperature vacuum chamber for impregnation. Large bubbles trapped in the casting chamber are manually removed using a Imm stainless steel wire. The vacuum chamber is sealed and pressure is lowered slowly to 20mm Hg while monitoring for slow bubble release . Similarly pressure is lowered incrementally and slowly through 10mm Hg, 5mm Hg and finally to 0 mm Hg while maintaining slow bubble production and release . Pressure is maintained at 0 mm Hg until bubbling ceases. Duration of impregnation is eight plus hours.
Heat curing of body slices
After impregnation is complete, the chamber is returned to atmospheric pressure and the chambers checked for trapped bubbles which are removed with the aid of a wire. Slice alignment is checked and corrected using the stainless steel wire. The gasket is closed across the top and clamped in place in preparation for curing (Table 1).
The slices in their casting chambers, are placed upright in the 40°C water bath for three days. A circulation pump is used to keep water around the chambers the constant 40°C.
Finishing: After curing, the sheets in their flat chambers are removed from the water bath and cooled to room temperature. The flat chamber is dismantled by removing the clamps, gasket and glass. The specimen is wrapped in light weight foil to prevent any uncured resin and debris from contacting the surface of the slice. After curing, release and wrapping is complete, the excess cured resin is trimmed on a band saw. The edges may be smoothed using a wool sander and new foil is placed on the slice which is ready for use.
The P45 sections are semi-transparent (Fig. 1), durable, and correlate well with radiographic, CT and MR images.
The dehydrated slices placed in the open topped flat chambers for impregnation is a potential time saver. As well, heat curing in a water bath in the same chamber after closure of the top is unique and a time saver. As with the other polyester techniques, the impregnated slice is surrounded by polyester resin-mixture (P45) while it is curing. Hence, the plastinated slices are incorporated as a part of a single cured sheet of the resin. They are not merely embedded in the resin. The specimens in the slice are durable and show good anatomical detail.
The main advantage of the P45 sheet plastination method is the decreased volume of resin used. As with P40 the impregnation resin is used as the casting resin. The process is not complicated and less equipment and time is needed.
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