Study and Validation of a Model
of Fetoplacental Circulation

2.4. Protocollo di prova con formaldeide Test Protocol with Formaldehyde |
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Le prove effettuate con questo tipo di perfusione sono simili a quelle gią descritte nei capitoli precedenti, relative alla sperimentazione con sangue.
Rimangono validi in particolare gli accorgimenti atti a mantenere in vita la placenta, almeno nelle fasi preliminari di perfusione con soluzione fisiologica, attuata allo scopo di effettuare un primo lavaggio della placenta, verificando che la perfusione avvenga correttamente, evidenziare subito eventuali perdite, riportare le caratteristiche geometriche dell'organo ai valori fisiologici prima di fissarle definitivamente.

2.4. Test Protocol with Formaldehyde |
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The experiments with this type of perfusion are similar to the ones already described.
The placenta has to be alive, at least during the preliminary part of the perfusion with isotonic solution, that is useful to:

- wash out blood from the placenta, in order to verify that the perfusion is correct and able to reach every district;
- put in evidence leakage of any vessels;
- restore the physiological dimensions of the vessels, applying the correct pressure.

2.4.1. Fluids for Perfusion |
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The preliminary perfusion is carried out with the same solution of the thermostatic bath: isotonic solution and heparin (1cc of heparin 5000 U.I. / liter of solution) at 39.0°C.
For the fixation of the placenta a mixture composed by 1 cc of glutaraldehyde at 2.5% each 30 cc of formalin at 4% was used.
The viscosity of the two fluids according to the literature [3] at the mentioned temperature is about 0.67 cP. We did not increase this value in order to make it more similar to the one of blood because the situation of perfusion with formaldehyde, that kills the cells very quickly, is absolutely not physiological.

2.4.2. Setup of the Circuit |
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The necessary steps to prepare the system of perfusion and the placenta are similar to the ones already described for the perfusion with blood, with following differences, in succession:

- the the connections refer to figure 44;
- the tubing that were connected to the points (1) and (2) must instead be inserted into the thermostatic bath;
- the air chambers have not been used: one of connections is used to trap eventual bubbles sucked during the connection to the formaldehyde reservoir;
- the automatic filling circuit is not used;
- the measurements of pH and partial pressures of gases are not necessary.

2.4.3. Connection and Data Measurement |
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The placenta must be prepared and connected to the circuit as described in "Protocol of test with blood".
In particular the connection of the point (11) must be done after that clean physiological solution begins to exit from the vein. During this first phase it is important to verify the absence of leakages, and the venous output flow is discarded outside of the system.
After the connection of the venous cannula, the stabilization of the fluidodynamic situation must be checked. The preliminary perfusion is hold for several minutes, obtaining the results of table 8.

Duration min | Flow cc/min | Arterial P. mmHg | Venous P. mmHg
24 | 235~ 265 | 48~ 55 | 20
18 | 225 | 45 | 20
15 | 168 | 33 | 20
8 | 190~ 265 | 22~ 40 | 20
25 | 145 | 70~ 99 | 20

Tab.8: Values obtained during the preliminary perfusion.

The following steps have to be executed in order to switch to the perfusion with formaldehyde:

- to extract from the thermostatic bath the tubing of the venous output in order to discard it temporarily out of the circuit;
- to connect to the point (1) the tube from which the pump (20) sinks;
- to wait for the formaldehyde solution to reach the venous output;
- to connect the point (2);
- to examine the level of the liquid contained in the reservoir for the measurement of the leakages.

The placentas were perfused with the formaline solution for times spanning between 15 and 43 minutes, therefore they were disconnected from the circuit and placed in watertight containers, dipped in a solution having the same composition of the perfusion fluid.
The morphometric examinations was carried out after not less than 60 days.
During the perfusion, as the conditions are not physiological, low importance has been given to the pressure values: we checked the input pressure not to exceed 120 mmHg (but this never happened) and the total leakages to stay within reasonable values (lower than 30%).
Fig.49: Placenta perfused with formaldehyde, upper side.
Fig.50: Placenta perfused with formaldehyde, lower side.

Duration of perfusion minutes | Flow cc/min | Leakage cc/min (%) | Arterial pressure mmHg
15 | 100 | -- | --
17 | 250 | 20.58 (8.3) | 30
17 | 225 | 29.41 (13) | 15
22 | 168 | 56.81 (33.8) | 13
43 | 190 | 13.95 (7.3) | 20
26 | 125 | 34.61 (27.7) | 70

Tab.9: Values obtained during the perfusion with formaldehyde.

The term "leakage" includes both the fluid that flows out through lesions of the small vessels and the seepage through the semi permeable membrane of the villi, averaging about 15% of the flow also according to the literature [38].

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