Plastinated body slices for verification of magnetic resonance tomography images

ANNALS OF ANATOMY

Plastinated body slices for verification of magnetic resonance tomography images Hanno Steinke Department of Anatomy, University of Leipzig, Liebigstrasse 13, D-04103 Leipzig, Germany

Summary. Plastinated body slices provide a higher resolution than photos taken from frozen objects as obtained for the "Visible H u m a n Project". The impregnation with curable polymer gives those slices transparency. Since the thickness of a plastinated slice determines its optical resolution, very thin plastinated slices are necessary for comparison with MRT-images (MRIs) of high quality. The present technical note describes how very thin plastinated slices can be prepared by modification of the classical plastination technique and for comparison with MRI. Having labelled the object's planes recorded as MRIs, we, for the first time, plastinated 800 tam thick body slices superior in resolution to the corresponding MRIs, confirming and extending their structures. Thus, very thin plastinated body slices can be regarded as a helpful tool for radiological studies.

Key words: Plastination technique - M R I - Macroscopy

Introduction The anatomical specimen provides the basis against which the correct interpretation of the M R I must be assessed. For this reason, many attempts have been made throughout the world to compare the one to the other, these attempts being exemplified by the famous "Visible H u m a n Project" of the National Library of Medicine, where serial sections of a frozen human body were compared with M R I and CT images obtained before slicing. Body sections prepared with the plastination technique

Correspondence to: H. Steinke E-mail: [email protected] Ann Anat (2001) 183:275-281 © Urban & Fischer Verlag http:llwww'urbanfischer'delj°urnalslannanat

provide a much higher resolution than photos taken from a frozen specimen as for the "Visible H u m a n Project", or a formaldehyde fixed one as described e.g. by Andermahr et al. (1998). The precise value of the resolution depends on the thickness of the plastination slice. Since the resolution of plastinated body slices stays the same (Hagens et al. 1987; Entius et al. 1997), whereas the quality of M R I exploring is still improving by the rapid progress in radiological diagnostics, continued improvement in the plastination technique is called for. If successful in preparation, plastinated slices of an anatomical specimen can be directly compared with previously obtained MRIs, and then the same structures can be identified (Brizzi et al. 1994; Magiros et al. 1997). The preparation of thin slices by the classical plastination technique is unfortunately associated with a wide variety of technical problems. For this reason the limit in thickness for plastinating whole body slices is at 2 m m at this time. We have modified the plastination technique so as to produce thin plastinated 800 pm slices of the whole unfixed body and obtained a better native quality for optimal comparison with previously acquired MRIs.

Material and methods We have prepared plastination slices from the human elbow carrying out the following consecutive steps: preparation, slicing, dehydration, embedding, and hardening. Preparation: To reduce the number of artefacts appearing in the MRIs as a result of fixation, we perfused each specimen with a specially prepared buffer solution. To prepare this, 62.6 g sodium chloride (Merck), 4.48 g potassium chloride (Sigma), 4.38 g calcium chloride (Sigma), 3.05 g magnesium chloride (Sigma) and 44.88 g sodium acetate (Sigma) were dissolved in 1 1 aqua bidest and the pH adjusted to 7.2. 0940-96021011183•3-275 $I 5.00•0

A tube was inserted into either the brachial or the popliteal artery, and another was placed into the veins. The fluid was then injected into the arteries by means of an infusion system, so as for the blood to be rinsed through the veins. As soon as the specimen turned pale, it was examined with a powerful MRI machine (Siemens Magnetom at the Department of Radiology of the University of Leipzig) by a method already described by Magiros (1997). The margin of each 5 mm slice was marked with a tissue pencil on the upper surface of specimen, so that the respective plane for slicing could be reliably identified. This margin was continued on the back surface also, and for this we installed a mirror to reflect a scanning light beam from the front to the back surface (Fig. 1). The MRI exploration were adjusted as follows: at first T R = 600; TE = 20/1, TA = 7 : 44, thickness SL = 5 mm, at last T R = 770; T E = 11/1; TA = 4:45, thickness SL = 5 ram. In order to eliminate the formation of clefts, we precooled the specimens at 5 °C according to yon Hagens (1985). Since delicate clefts continued to appear after immediate removal of the specimens from an environment controlled at 5 °C into a deep freezer, we had to modify the described freezing process. The onset of freezing was controlled by a ventilator mounted in the ultra-deep freezer. As a further precaution, "Hot/Cold Packs" (E & M, Essex) which had been cooled to -85 °C were laid around the specimen and changed after five minutes. After a further five minutes, the specimen undergoing freezing was immersed in 85% acetone at -5 °C. To accelerate the shock freezing, liquid nitrogen was poured into the acetone solution, thus bringing it down to below -100 °C. After 30 minutes the specimen was removed from the freezing solution, placed in a deep freezer (modified as described above) at -85 °C for a further day, and then transferred back into a freezer at -25 °C and stored foil packed for preventing damage by freezing until required for slicing. Slicing: We decided to saw in series from frozen specimens (yon Hagens et al. 1987; Entius et al. 1997; Peschers et al.

/

MRI explored and labelled specimen

1, Marking ~e MRP planes on the fr0ntside during MR[ exploration

/ Light poi.terfixedtea

I longitudinal axis

Bo. . . . .

tthe

,og a,oog

transversal p ane

Mirror for beam

h

reflection

2, Scanning the label with a

3. Extendingthe iabei 4. L~belii~g the serrationplane on from the front to the

pointer

backside

the backside.

Fig. 1. Method of labelling the slicing planes of the specimen for the MRI.

1997). In order to obtain very thin body slices we had to improve our standard plastination saw ( B I O D U R ©, Heidelberg). We used Weber's experience (1994). The diamond saw-blade was cooled with CO2 liquid gas and continuously cleaned during the sectioning. A nozzle was fixed below the saw-table and connected to a CO2 bottle. The evaporating gas cooled the saw and its surroundings, and guaranteed the continuous removal of sawdust from the blade. We isolated the fence with polyurethane foam, attached a polystyrene plate to the upper surface and cooled it from the inside with deep-frozen bricks. These were easily removed and refrozen with liquid nitrogen. The optimal temperature for the slicing touch was around -30°C, and for the specimens, about -35 °C. While cutting in the plane of the previously applied label, it was helpful to check the corresponding MRIs, which were mounted for comparison on a light box. A brain knife, frozen to -20 °C, was placed between the specimen and the slice during cutting. In this way the touch fixed the slice while removing it from the frozen specimen. We found that moistened blotting-paper of 4 °C is ideal for removing the very thin slices from the saw by freezing it on the slice fixed by the brain knife at the sawing touch. Although the room temperature was below -5 °C and the laboratory was full of carbon dioxide, we succeeded in obtaining intact slices and collected them in wire boxes. Dehydration: The wire boxes with the slices were put into freezer at -85 °C, and then carefully immersed in a dehydrating solution of 85% acetone at the same temperature (Steinke and Schmidt 1992). 8% peracetic acid was added to produce mild bleaching during the freeze substitution according to the personal recommendation of von Hagens. We tried to accelerate bleaching by raising the temperature to -1 °C. However, this caused the slices to fragment, and so we returned to the standard temperature of -25 °C, which produced the best results. After two months of treatment with the bleaching solution, the slices had become pale. They were then treated for another two weeks by pure acetone at -25 °C. After that, allowing them to remain in a new pure acetone bath, at room temperature for a further two weeks, was effective in removing the lipids from the adipose tissue. Embedding: Before the final embedding, methylene chloride was used as intermedium, and this was carefully extracted by placing the treated slices under vacuum. 10-3 Torr was slowly generated over a matter of hours, and the exsiccator gently refilled with air when no more gas bubbles could be seen. The slices were then embedded at room temperature in a special resin mixture: 95 parts of B I O D U R E12, 8 parts of B I O D U R AT30, 26 parts of B I O D U R E1 (all from B I O D U R , Heidelberg). The "sandwich method" was used as described by von Hagens (1985) and yon Hagens et al. (1996). Hardening and evaluation: The resin mixture polymerised at room temperature. The bleached slices had undergone a marked decrease in pigmentation, so that hardly any detectable structure could be recognised. To skip the staining, which was not available at this time, the polymerisation was prolonged for several weeks. Following our experience with the modified Spalteholz technique (Steinke and Schmidt 1991), the slices were left to turn brown by being kept at 50 °C in a drying chamber, and then by being exposed for several weeks to U V radiation in a light-curing unit (Biodur). We documented the slices with a high resolution scanner (1200 dpi, Canon Tokyo) for comparison with the corresponding MRI. TIFF- or DICOM-files were acquired for further processing in databases (Mashuda et al. 1997).

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Table 1. Similarities and differences of the classical and modified plastination technique for the preparation of 800 pm body slices Steps

Similarities

Classical procedure

Modified procedure

preparation

precooling at 5 °C

slicing

plastination sectioning cooling the slice

formaldehyde fixation freezing at -25 °C removing sawdust from slice sectioning in a cool room cooling specimens to between -10 °C and -20 °C

dehydration

freeze substitution methylene chloride as intermedium

rinsing with pure acetone

buffer perfusion labelled MRI-planes shock-freezing at -85 °C in 85% acetone fence isolated, cooling with CO2 and N2 blowing sawdust from blade to avoid scraping cooling specimen to below <-35 °C freezing specimen onto blotting paper (1) 85% acetone with 8%0 peracetic acid removing saw dust from slice manually (2) 100% acetone

embedding

Biodur E12

standard mixture

hardening

polymerisation

heat polymerisation

Results Table 1 shows both the classical steps of the procedure and our modification. Fig. 2 additionally depicts the quality of an 800 gm slice in comparison to a 3 m m thick plastinated one. Figures 3 and 4 show that 800 gm plastinated slices are superior in resolution to MRIs of 5 m m thickness. In the following, the points of consideration are presented for each step of our technique. Preparation: The advantages of the perfusion recommended here are: (1) The blood clots were rinsed away; (2) the M R I accurately reflected the true anatomical structure; (3) the bleaching time was reduced; (4) the specimens turned pale after perfusion. The recorded planes were labelled on the specimen's front surface at the acquisition time of the M R I and were adequately continued to the unlabeled back surface when the specimen had been shock-frozen. Slicing: During the production of 800 gm slices most of the sawdust was blown away by the evaporating CO2. The section passed correctly through the plane of the label. It was immediately verified by comparing the slice with its corresponding MRI. Slices could be removed from the saw touch when it had been cooled down to -35 °C. Moistened blotting paper stuck to the slices and thus reinforced them during handling. If the blotting paper was used, there was no need to scrape away the sawdust and damage to the surface was avoided. Any remaining sawdust could be washed away manually during dehydration. Dehydration: When using an 85% acetone solution at -85 °C for starting the dehydration of very thin body slices, and continuing the processing at -25 °C, the slices preserved their original shape. A further increase of the temperature to -1 °C caused shrinkage, although this temperature was suitable for bleaching. Nevertheless, the slices were sufficiently bleached at -25 °C as personally recommended by von Hagens. At the end of the final dehydration in 100% acetone, the slices did not show any signs of clefts or shrinkage, or the blotting paper, which is no longer adherent to the slices (when the water has been

increasing the amount of softener (AT 30) gently adaption of vacuum polymerising by heat and UV radation

removed). They were transferred from the dehydration chamber and immersed in the resin mixture. Embedding: By increasing the percentage of the softener AT 30 in the resin mixture flexible and unbreakable

Fig. 2. A plastinated body slice of 800 gin thickness (a) is compared with a slice of 3 mm (b). Magnification x 1.

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m

Fig. 3. A n 800 gm slice from the region of the h u m a n elbow region: Comparison of the M R I with the corresponding plastinated slice at the horizontal level. a: T2-MRI. Reduction in size x 2. b: Corresponding 800 gm plastinated slice. Reduction in size x 2. e: T1-MRI. Reduction in size x 2. d: Enlarged area of (a), medial part of the elbow joint, AM: anconeus muscle, T: trochlea, U: ulna. Magnification x2.5. e: The same area in the 800 gm plastinated specimen (b). In addition to (d) the cartilage and the synovial folds are apparent. Magnification x 2.5. f: The same area at a magnification of the T1-MRI (c). This M R I does not achieve the resolution as in (e), but describes the containing free water into the articulation slit. 278

Fig. 4. 800 gm slice from the region of the human elbow: Comparison of the MRI to the corresponding plastinated slice of the sagittal plane. a: T2-MRI. Reduction in size x 2. b: The MRI explored specimen of (a) as an 800 pm plastinated slice. Reduction in size x 2. c: Enlarged area of (a), insertion of the brachial muscle, the trochlea of the humerus, the trochlearis groove of the ulna. Magnificationx2.5, d- Enlarged area of (b), in addition to (c) the interosseus vessels are distinguishable as two veins and the brachial interosseous artery (arrow). Magnification. x 2.5. slices were obtained. After careful evacuation, the impregnated slices remained intact and were e m b e d d e d until the resin started polymerisation. The sandwich technique was sufficient (yon Hagens 1985). Hardening: The thin slices were nearly uncoloured and hard to differentiate. O u r experience in browning of Spalteholz-cleared specimen (Steinke and Schmidt 1991) resulted in prolongation of the hardening procedure. W h e n the slices remained in a heating chamber for 3-4 weeks, they turned brown. A n additional U V radiation was helpful as depicted in figures 3 and 4.

W h e n the scanner of high resolution was used, the resulting images resembled an artist's drawing. The scanner's quality of up to 1200 dpi surpassed the quality of the optical resolution of the M R I s obtainable with 70-100 dpi.

Discussion Preparation: A complete and careful preparation of the specimens is essential for preparing very thin slices of ex279

cellent quality. Other groups performing slicing from plastinated blocks have omitted to mention how their specimen were set-up for plastination (Peschers et al. 1997; Hoch et al. 1999). The slicing from plastinated blocks according to Fritsch and H/Stzinger (1993) and Hoch et al. (1999) has some major disadvantages, because (1) the corresponding layers cannot be compared at the block, (2) the skin has to be removed as a diffusion barrier to resin, (3) shrinkage is known for resin embedded blocks (Piechocki 1986), (4) specimens cannot be sawn at the MRIplanes as we did with unfixed specimens. We obtained MRIs of the cadaver specimens of nearly the same quality as native ones. The shrinkage was strongly reduced by the isotonic treatment, most blood clots were rinsed and the specimen turned pale, which is essential for the further procedure. We strongly recommend a thorough rinsing with the buffer solution which guarantees sufficient specimen preservation and satisfying MRI. The thickness of MRI slicing was reduced from 6 mm down to 5 mm by Magiros et al. (1997) and Fr6hlich et al. (1997). An authentic interpretation of the digitally averaged 5 mm MRI is here when the plastinates' thickness has been reduced down to 800 gm. If a higher resolution of the MRI can be obtained depending to the technological progress in reducing the slices thickness, an anatomical interpretation by our method could be useful too. The method of labelling the back surface according to the front label by using a light beam and mirror certainly requires further improvement. At the moment this is the only way that we know of for labelling the entire circumference of the specimen; i.e. the plane of the MRI and subsequent slicing sequence for plastination. The exact orientation is important for correct correlation of plastihated slices to CTs and MRIs (Entius et al. 1997). The standard shock-freezing technique and slicing procedure often results in either fragmentation of the slices or the entire specimen when reducing the freezing temperature. Clefts develop during dehydration, when freezing is too moderate, because ice needles occur (Piechocki 1986) which finally force the slices to crumble. To prevent artefacts such as destruction, ice needles and clefts forming, the specimen has to be deep frozen in subsequent steps and each fairly rapidly The shrinkage of the specimen is reduced, because the dehydrating solution at the onset of the freezing process still contains water in support of the quick alteration of temperature. Stepwise freezing seems to be a prerequisite for obtaining very thin slices without undesirable defects. Slicing" Cooled CO2-gas was essential when the specimen was to be sectioned serially Since it was disadvantageous for the slicing touch to be warmer than the specimen (yon Hagens, 1985), the object's temperature must be as low as -30 °C. The specimen had to remain deeply frozen, otherwise the sawdust could not be blown away We decided to plastinate the body slices after cutting the frozen specimen as recommended by Andermahr et al. (1997), and not to plastinate the specimen first and

slice it afterwards (Fritsch and Hegemann 1991; Fritsch and H6tzinger, 1993; Fr6hlich et al. 1997; Hoch et al. 1999). Their method seems to be useful for small pieces of embryological or adult specimens. We consider the here described sawing of frozen specimen as the best way to get rather unshrunken plastinates according to Brizzi et al. (1994) and Entius et al. (1997). As an alternative to cooling the saw blade by CO2, a machine may be used for the removal of the sawdust, provided that it is run with cold air. Working in a laboratory full of CO2 can be exhausting ... Dehydration: Although thinly sliced specimens have been elaborately treated until dehydration, they do not withstand the standard freeze substitution process for plastination inaugurated by von Hagens (1977, 1979). As we found out, the adaptation to 85% acetone at -85°C maintained the slices. Starting dehydration with pure acetone causes shrinkage and therefore damage to the slice (Schmidt and Steinke 1996). When the slices are immersed carefully into the viscous mixture, mechanical destruction can be avoided. Bleaching can be omitted under these circumstances. This may save time, which is necessary for browning the slices. Embedding: As a result of Schmidt's experience on the embedding of human brains (Schmidt and Steinke 1997), we assume that the slow alteration of air pressure under vacuum is responsible for the integrity of the slices. We strongly recommend Schmidt's procedure to release the vacuum slowly, and suggest that this may be improved by using a B I O D U R Vacuum Control Unit. The "sandwichmethod" (yon Hagens 1985) is sufficient for embedding thin plastinated slices also. Hardening: The slices in a heat chamber for a number of weeks allows the slices to turn brown, and facilitates the visualisation of anatomical structures by scanning them. Hardening is necessary for the slices so that they resist scratches (yon Hagens 1985). The hardening could be avoided when leaving the slice unbleached during dehydration. In this case they have to be stored plane and pressed to avoid deformation. However, thin plastination requires the development of new staining techniques for plastinated slices in such a way that structures at the limit of macroscopic resolution may be detected. This could be of importance, since improvement in plastination helps the radiologist to confirm the nature of doubtful structures.

Acknowledgements. My sincere thanks are due to Professor K. Spanel-Borowski for continued support, to Prof. G. von Hagens for some advices, and to those of our students whose help and enthusiasm have been proved a great encouragement to me.

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Accepted November 8, 2000

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