INTRODUCTION

The placenta is one of our favorite specimens. This is not because it is particularly attractive. Most would agree that other organs, such as the heart, would win a beauty contest from a placenta. Nor is the placenta one of the most difficult specimens to plastinate. If you're looking for challenge, we would not hesitate to recommend the kidney. The reason for our appreciation of the placenta is because it is one of the few plastination projects in which the person who provided you with the specimen will be able to share your enjoyment of the results.

We will review the process of plastination of the placenta in the sequence in which the actual procedure would be carried out. In doing so, we hope that even the novice plastinator will then be able to follow the recommended steps and arrive at an acceptable teaching specimen.

SELECTION OF SPECIMENS

If you have a relationship with an obstetrician, in which he or she is willing to provide you with a constant supply of placentas, you are in an ideal position. You may then become very picky and select only the most charming examples.

In Heidelberg, where a relationship such as this does exist, we have developed
standards to which placentas must conform before they will be accepted for processing. First and foremost, they must be intact and free of injury. Punctured vessels or damaged placental lobes will cause you nothing but misery. You'll be plagued by leaking fluids and experience an awful mess during color injection. Lamentably, most placentas are just not worth the trouble.

Second, we appreciate specimens that are extraordinary in some way. For example, we greatly esteem those which

- have elongated umbilical cords,
- exhibit deviant features, like a velamentous insertion of the umbilical cord,
- are of large size,
- represent unusual (but otherwise normal) conditions, like twin-placentas.

There is one, conspicuous exception to these rules, however. No matter how ordinary, damaged or even unattractive a placenta may be, we'll go to the very frontier of our ability to transform it into an exquisite specimen if we're doing it for personal reasons. Nobody can be objective about the placenta of his or her own offspring! This may seem like a weird preoccupation, but sometimes we   wonder why  people  who  bronze  their  children's first  shoes,  record  their  first  words   and take dozens of pictures of every stage  of their development, will simply discard their infant's placenta  as  if  it  was  an  empty cigar wrapper! Of course, not everyone is aware of  the  personal/sentimental possibilities of plastination. But we would suggest to you, the insider, that you never tolerate this kind of callous negligence.

REMOVAL OF BLOOD

Once we have decided which  are  the favored  specimens,  we  can  "go  for  it." Let's  start  by  getting  rid   of   the   blood. The best  way  to  do  this  is  by  simply rinsing the vascular system. This  can  be done very easily if you cannulate the umbilical vein with plastic tubing, and connect    it   to   a   water    tap.     This "cannulation" is not meant to be a sophisticated  procedure.  Just  insert a piece of tubing into the vein and tie a thread around the whole  umbilical  cord to hold  it in  place.  Be  sure  to  use   an   overflow device to keep the  hydrostatic  pressure below 2 meters. Rinsing will take about  12 hours, so it's wise to do it overnight.

Incidentally,  rinsing  provides  you  with  a fine opportunity to check the vessels for damage. Veins or arteries that spray water will make  you  wet  and  attract  your attention  immediately.

COLOR INJECTION

This brings  us to the most complicated step in  the  whole  procedure,  color  injection. The goal of color injection is to arrive at a specimen that demonstrates  the  course  of the umbilical and placental vessels and exhibits something close to a natural hue.

With the removal of blood, the placenta turns   into   a   very   dejected-looking,   pale thing. Here is where we will use our cosmetic skills to restore its natural beauty. In order to inject the vessels of the placenta with colored resin we will have to cannulate them. Cannulation of the vein is a mere trifle, as we pointed out before . Unfortunately we cannot say the same about the arteries. These little devils are so tiny that cannulation will take considerable effort and skill. We have developed two methods to get it done.

1. FREE-HANDED CANNULATION:

We will call the first method, "free­ handed cannulation," because it does not require complex preparation. Instruments and materials needed for this step are as follows:

- scalpel
- scissors
- elastic string
a tiny plastic cannula

Take the end of the umbilical cord between your fingers, look for an artery and cut across this artery with the scalpel. The best place to do this is just below the thread you used to hold the venous cannula in place for rinsing. This same venous cannula and vein will be used for color injection so don't cut them.

Once the selected artery is bisected, you will be able to see its lumen in cross-section. Position the cannula in the lumen, hold it with your fingers and tie the elastic string around the whole umbilical cord to keep it in place.

2. DISSECTION CANNULATION:

We call the second method "dissection­ cannulation". This is intended to mean that one must dissect the artery out of the umbilical cord before inserting a cannula. It may sound easy but, alas, it isn't! Sure, you'll get the artery

separated from the cord without damage but, once you have inserted the cannula, any ill-considered move could ruin its delicate wall. In contrast to the artery used in the first method, this one has been removed from its adventitia and is very fragile. As you will notice, more tools will be needed to get the job done. We would suggest the following, in addition to those needed for the first method:

- a frame to hold the cannula in place during injection. This must be locally constructed.
- clamps and hemostats

Once the artery is dissected free of the rest of the umbilical cord, the cannula can be inserted and ligated into this vessel, using the elastic string. The string should be tied around the artery, leaving a generous length of loose ends.

After injection, these ends can be used to stabilize the vessel and keep it from moving and becoming damaged. This, of course, would result in a very undesirable

leakage of the resin you have just injected. The . frame and clamps are used to hold the cord and the cannulated artery while injection is under way.

Now that you are familiar with both methods, the time has come to discuss when to apply each. The rule of thumb is that one uses free-handed cannulation whenever possible. There are circumstances, however, in which this method simply will not work. For example,  what if the vessels in the umbilical cord are so spiraled, that you can't find a piece of artery long enough to put the full length of the cannula in? The only possibility is dissection. And what if the umbilical cord happens to have "false knot" along its course? You would have to put so much pressure on the syringe that you would loose your hold on the slippery umbilical cord. The cannula would come flying out of the artery, and there you are, a colored resin mess. To avoid this misery you had better employ dissection cannulation in this case as well.

Now let's move on to something simple. What quantity of which mixture should be injected into which vessel in what sequence? Well, we start by injecting the artery -- but we inject it with BLUE resin. Remember that the oxygenation of blood in this organ is ·opposite that in most others. Arteries are blue and veins are red. Once the artery is adequately injected and tied off, we may turn our attention to the vein. Two kinds of resin material are available for use in this step, epoxy and silicone rubber. We will briefly outline the composition the of injection mixtures associated with each (in parts per weight).

1- EPOXY INJECTION MIXTURE:
BIODUR E 20 red/blue:   100 ppw
methyl ethyl ketone:            40 ppw
BIODUR E 2:                            45 ppw

These components should be added in the order given and thoroughly stirred for at least 5 minutes. Because BIODUR E 2 is a hardener, these mixtures have a limited pot life. Don't worry, you should have enough time to get your specimens injected. But we would advise you to mix and inject the blue resin before starting on the red.

2. SILICONE INJECTION MIXTURE:

BIODUR S 10 red/blue :   100 ppw
methyl ethyl ketone:             40 ppw
BIODUR S 3:                            3 ppw
BIODUR S 6:                             1 ppw
BIODUR S 2:                             1 ppw

Again, the components should be added in the order shown. Precautions regarding pot life and injection sequence apply here as well.

Whether you select epoxy or silicone mixture is a matter of personal preference. Epoxy resins have been used successfully for a long time in this role. But they are more rigid and more allergenic than the silicone. The silicone injection mixture is a rather new development. It seems to be very promising, but you might say that it's "just beyond the experimental stage." We hope that a lot of brave researchers will elect to try it. About 10-20 ml of blue injection mixture and 100-200 ml of red injection mixture is required per placenta.

One more thing about arterial injection. We have been talking of cannulation and injection of just one artery. But, as we know, the standard umbilical cord contains two arteries. You needn't bother about the second one. The hyo communicate in such an· extensive collateral network that the second artery will be filled by retrograde flow in almost 100% of the specimens. If not, you can always cannulate it separately.

When you have completed arterial (blue) injection in the free-handed method all you have to do is remove the cannula from the injected artery and the elastic string will pull itself tight around the umbilical cord constricting the injected artery. This serves very nicely to prevent resin leakage without injury to the vessel. In the dissection method, leave the cannula in place and put a stopper on it to get the same result.

Now that we have cannulation under control, we must look at the instruments and material needed to get the venous injection process started.

• injection resin (epoxy or silicone) acetone, to be in use as a cleaning agent
• scissors
• cotton thread
• syringe
• adapters to connect the syringe to the plastic tubing
• an in-line shut-off valve or faucet
• a rather strong hemostat
• a rubber band

The in-line valve will enable us to remove the syringe from the adapter without spilling any resin. We need that because we'll have to refill the syringe several times.

Load the syringe, connect it to the adaptor on the plastic tubing and off you go. When the venous injection is finished, pull the plastic tubing out, clamp the whole cord off with the hemostat, and wrap the rubber band around the cord several times to serve as a final stopper. Now the hemostat can be removed and no leakage will occur.

HARDENING

It's hard to believe but we finally made it through that section on color injection! Now we must talk about hardening. There is really nothing special about hardening. All we have to do is to give the injection resin some time to solidify before we proceed with the plastination of the placenta. What turned out to be the best method in Heidelberg is the following:

• put the placenta in a container, making sure that the uterine surface is on top
• fill this container with just enough water to cover it
• put a cloth over the placenta to keep it from drying
• leave it standing like that overnight at refrigerator temperature

The reason for putting the uterine surface on top and filling the container with just enough water to cover it is that this will prevent the umbilical cord from floating away from the placenta. It looks very unnatural to have the umbilical cord sticking out of the placenta at an angle of 90 degrees. On the following day you can remove the resin remnants and we are ready for the next step.

FIXATION

Fixation is about as uncomplicated as hardening. All you do is immerse the placenta in a 5% solution of formalin for about 3 weeks, making sure that you keep the umbilical cord submerged.

DEHYDRATION

Dehydration is covered in great detail elsewhere, particularly in Prof. Klaus Tiedemann's article that will appear later in this volume. We will do no more than mention a few details that might come in handy whilst plastinating a placenta.

Dehydration of the placenta is performed by freeze substitution in acetone. Three baths are usually required and the acetone must be maintained at -25°C. Each bath should consist of approximately 5 times the volume of the specimen. Defatting is not necessary. The whole process will take approximately 2 weeks. How's that for brief?

FORCED IMPREGNATION

Impregnation of the placenta is performed almost exclusively with BIODUR S l 0 nowadays. This whole method has been extensively described and is known as the S 10 Standard Technique. Again, I will mention only a few details.

Our placenta, now soaked with acetone, is put into the polymer solution used for impregnation. BIODUR S l 0 is a liquid silicone rubber. It has to be mixed with l% BIODUR S 3, which is a suitable hardener. By means of a vacuum, the acetone is then extracted from the tissue. (You might say that we encourage it to evaporate.)

A pressure gradient develops between the inside and the outside of the specimen and this causes the polymer to be drawn into the tissue. For further details, please refer to the bible of plastination, the "Heidelberg Plastination Folder."

Impregnation of one placenta will actually use up about 500 grams of polymer. But, in order to immerse it properly during impregnation, more will be required. Impregnation is carried out very gradually and takes about 3 weeks. It is wise to do the impregnation at about -20°C, for this will grant you an almost unlimited pot life for the BIODUR S 10/ S 3 mixture.

CURING

Curing is a process whereby the fluid polymer, which now saturates our specimen, can be rendered solid. It is carried out by exposing the placenta to a preparation called, BIODUR GASCURE S 6. Before we start curing, however, we might want to give the fetal side of the placenta some extra gloss. This looks quite nice on the finished specimen. It makes the placenta seem all wet and natural. To accomplish this very chic "wet look", we remove the amnion from the fetal side of the placenta, and apply a layer of BIODUR S 49, mixed with 1 % BIODUR S 3.

The specimen is then put to rest in the arms of BIODUR GASCURE S 6 in a closed container for about 1 week. Don't forget to use calcium chloride as a drying medium and, of course, contrive to speed up the evaporation and circulation of the curing agent by means of aquarium pumps. And that's it!

ACKNOWLEDGEMENTS

There is nothing left now except for the first author to acknowledge those people who offered her the opportunity to show her enthusiasm about the wonderful world of plastination. Thanks are due to

• Prof. Pim van Doorenmaalen, my anatomy professor, who introduced me into this technique.
• Dr. Gunther von Hagens, who taught me all I know about it
• The "Stichting De Drie Lichten," a Netherlands foundation that
  supported my research in Heidelberg.
• Prof. Harmon Bickley, who enabled me to communicate with you by re-editing this paper and polishing my English.

and last but not least

• my husband Bas, who stuck to my side, serving as both thesaurus and therapist while I prepared this paper