INTRODUCTION

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:

• Acetone
• Methylene chloride
• Polyester resin

The Hoffen products for polyester plastination are:

• P45: Epoxy resin
• P45A & P45C: Polyester plasticizers
• P45B: Hardener

MATERIALS AND METHODS

The  basic  steps  of plastination  are:  Specimen  prep, dehydration, impregnation  and curing.

Specimen preparation

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:

• Bandsaw
• Grids
• Grid/specimen basket

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.

Dehydration equipment:

• Acetonometer
• Specimen/slice basket
• Chemical resistant acetone reservoirs

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.

Impregnation equipment:

• Vacuum chamber with a transparent lid
• Vacuum pump (oil pump is preferred)
• Vacuum tubing and fme adjustment needle-valves
• Vacuum gauge
• Bennert mercury or digital manometer

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).

Curing equipment:

• 40°C water bath and circulation

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.

RESULTS

The P45 sections are semi-transparent (Fig. 1), durable, and correlate well with radiographic, CT and MR images.

Figure 1. Cured p45 sagittal body slices of human.

DISCUSSION

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.