The isolated-perfused rabbit oviduct: A simple experimental preparation

The isolated-Perfused Rabbit Oviduct: A Simple Experimental Preparation

BEATU. RAESSAND FRANK F. VINCENZI

A new, simple method to study several physiological parameters in the isolated rabbit oviduct is described in detail. The whole oviduct with its supporting membranes, the ovary and part of the uterine horn, and part of the systemic blood supply were excised and perfused in vitro through cannulated blood vessels with a blood-physiological solution mixture. Autoregulation of the vasculature was demonstrated, suggesting that the preparation was viable and well oxygenated. The frequency of contractions of the ampullary and isthmic parts was decreased Key Words:

investigators

by prostaglandin Oviduct;

of oviductal

ductal smooth

muscle

ES added to the perfusing

Perfusion;

function

(Vincenzi

Prostaglandins;

have usually

and Raess,

1972;

solution.

Egg transport.

used isolated Higgs

segments

and Moawad,

of ovi-

1974) or in

situ whole animal preparations (Baling and Blandau, 1971). The preparation described in this paper is an isolated, but intact, perfused oviduct. It is a very simple experimental With

this

animal

approach

and offers

preparation

there

or complications

several

distinct

is no concern

arising

from

for

advantages the

anesthesia,

over other

cardiovascular

as in whole

systems.

status

animal

of the

preparations.

Homologous blood and a physiological solution are used to make up a perfusate mixture, the compositjon of which can be controlled reasonably well. Drug metabolism

and accumulation

perfusion

mixture

The method

of physiologic

is usually

of preparing

metabolites

are mjnimized

since the blood

not recirculated. the in vitro preparation

is emphasized

vitro autoregulation of the vasculature of the oviduct effects of prostaglandin Ez are demonstrated.

and

in this report.

some

In

pharmacologic

METHODS Primiparous New Zealand rabbits, S-10 lb in weight were isolated and caged separately for at least 30 days. For reasons described by Boling and Blandau (1971), the animals were kept on a restricted but complete diet. The animals were killed From

the

department

of

Pharmacology,

University

of

Washington,

School

of

Medicine,

Seattle,

Washington. Address Washington, Received

reprint

requests

School April

to:

of Medicine,

23, 1979;

revised

Dr.

Frank

Seattle,

F. Vincenzi,

Department

of

Pharmacology,

University

of

WA 98195.

and accepted September

11, 1979. 267

lourn. of Pharmacoio~cal Methods 3,267-273 (1980) @1380 Elswier North Holland, Inc., 52 Vanderbilt Avenue,

New York, NY 10017

Ol~~~~m3026~7~2.25

268

B. U. Raessand F. F. Vincenzi by a blow on the neck. Exsanguination arteries. Blood was collected immediately

was performed and defibrinated

wooden probes. Through a midline abdominal incision, transversely at its upper fourth and (carefully avoiding ductal smooth

muscle) the mesosalpinx,

ovary, and the major blood vessels in preheated NaCI,

(35°C) oxygenated

5.4 mM

KCI,

the entire

by cutting the carotid by gentle stirring with

the uterine horn was cut any stretching of the ovi-

oviduct,

the mesotubarium,

were excised as a unit and immediately

Earle’s solution.

Earle’s solution

1.8 mM CaC&, 0.81 mM MgS04,

I.0

mM

contains NaH,PO,,

the

placed

116.4 mM 26.2 mM

NaHCO,, and 5.55 mM D-glucose. When oxygenated with a mixture of 95% O2 and 5% COz, Earle’s solution has a pH of 7.4 and because of high bicarbonate content has better buffering capacity than some other physiological solutions (Parker, 1961). The specially constructed

tissue

bath, illustrated

Earle’s solution and provided a mounting grid (manufactured locally from a block

in Figure

1, contained

oxygenated

surface for the preparation. The Teflon of Teflon) on which the preparation is

placed allows the few erythrocytes that may escape from the tissue to fall to the bottom of the funnel-shaped bath. They may be washed out of the system, if desired.

Preheated

new solution

was dripped

into the chamber

near the oviduct

at

a rate of 2-3 mllmin. This solution bathed the tissue but was not in direct communication with the vascular bed of the tissue (see below). The tissue bath level was maintained with a suction pump device. Once the excised tissue was positioned on the grid to simulate

E

I

E

an in vivo position,

E

F

FlGURE 1 Schematic diagram of the isolated perfused oviduct preparation: B- physiological Earle’s solution; C-constant temperature water bath; D-02-COZ inlet; E-arterial perfusate; F-fresh Earle’s solution; G-suction; l-venous cannula; K4rain; L-Teflon support grid.

Isolated Perfused Oviduct the ovarian

artery

and vein were freed

branes and cannulated

with

from

fine (PE-60-90)

adipose

tissue

polyethylene

and supporting

tubing

mem-

at the mesosalpinx

where vessel branching occurs. Cut free ends on the uterine horn and other major blood vessels were ligated or clamped. A catheter was placed in the lumen of the uterine

horn as a drain for accumulation

by a ligature

around

The arterial

inflow

cannula was connected

sure head of 65 mm Hg unless solution

otherwise

and checked for possible

should

nonetheless

of luminal

secretions.

It was held in place

the horn. to a reservoir

noted. The tissue

blood clots, an extremely

not be overlooked.

Heparin

with

a hydrostatic

was flushed

pres-

with Earle’s

rare complication

which

was given to some animals before

exsanguination in an unsuccessful attempt to improve the yield of blood. In all preparations where heparin was administered, poor blood flow and extensive extravasation

of blood

containing

perfusate

start of perfusion.

Heparin

of Earle’s

was established

solution

blood-containing

occurred

use was therefore

perfusate.

When

in a given tissue, this

in the tissue

discontinued.

was done,

shortly

after the

Once a vigorous

a switch

could

the preparation

changed

pale white to a somewhat blood-engorged but normal-appearing oviduct appears much as oviducts appear in vivo (Baling and Blandau, 1971). Within a spontaneous tissue

reduction

in perfusate

flow

then autoregulated

its perfusate

flow at the mean arterial

Hg. At the same time,

the smooth

typically

(soling

seen

in vivo

and engorg~ment

1971).

pressure

Consequently,

and these can be clamped or tied off as shown

(which 1 min, The

of 65 mm of motility

we believe

tissue obtained sufficient oxygen to meet its needs under these conditions. perfusing with blood containing perfusate, one can observe “leaks” from blood vessels

from

was observed.

muscle appeared to take on patterns

and Blandau,

flow

be made to a

the

When minor

in Figure 2. Elimination

of these “bleeders” maintains clarity of the bath solution. This aided viewing of the preparation but did not appear to significantiy influence the overall perfusion or activity of the oviductal muscle. The

last stage of the tissue

preparation

consisted

of mounting

mutual

induction

FtCURE 2 Photograph of perfused preparation. Arrows indicate direction of flow of the perfusate. A-ampulla, optical cuff not shown; t-isthmus; F-fimbria. The ovary lies underneath the fimbria in this photograph. Optical transducer not present in this photograph.

269

270

B. U. Raess and F. F. Vincenzi

coils (Duff et al., 1972) or optical cuff transducers (Halbert et al., 1975) for measuring and monitoring motility. Only one optical cuff was fastened to each isthmus and ampulla. However, more cuffs could easily have been employed. A “tissue glue”’ was used to attach the transducers. Preparation of the Perfusate Mixture Blood collected as noted above was immediately defibrinated by stirring with a wooden stick. A fibrin clot formed readily on the stick. The blood was then filtered through several layers of surgical gauze. Typically, we obtained about 70 ml of defibrinated blood from each rabbit. This was then diluted 1 :2 with oxygenated Earle’s solution (70 ml blood and 140 ml Earle’s solution). In this form, the perfusate was stored in an oxygenated (95% 02:5% CO,) reservoir from which it could drain through a Travenol Blood Administration set (serves as bubble trap) to the inflow cannula. Measuring

Venous Outflow and Return

There was minimal leakage of blood into the tissue bath. Practically all the blood flows out through the ovarian venous cannula, and therefore blood flow through the isolated organ was estimated by monitoring venous outflow. This was measured automatically by “weighing“ (with a Statham pressure transducer, O-5 cm Hg, P 23 BB) the venous effluent in an overflow-siphon cylinder. The pressure of the column of blood perfusate building up was sensed by the pressure transducer, amplified, and recorded. The system was easily calibrated by adding known amounts of Earle’s solution in a stepwise fashion. All recordings were made on a Beckman (Offner Type R) ~ynograph. Contraction Quantitation of oviductal motility was made with a specially developed frequency discriminator. The discriminator “recognizes” any single contraction seen by the optical cuff and is able to differentiate it from system noise signals. The discriminator operates by continuous sampling (3isec) of the slope of the signal coming from the optical cuff. A contraction is indicated by a signal from the optical cuff which rises above a certain threshold, reaches a peak, and then declines to a value below the peak. In other words, a contraction is defined as a positive slope followed by a negative slope, disregarding slow baseline shifts. Therefore, system noise can be disregarded and the potential biasing of manual discrimination of actual contractions is eliminated. RESULTS Autoregulation

in the Oviduct

The isolated perfused oviduct, prepared as described, appears to autoregulate” oxygenation of the perfusate, pH, etc., are kept constant, the If temperature, * 2-ethyl-hexyl-2-cyanoa~~late.

Isolated Perfused Oviduct imposed

changes of arterial

resistance

changes

pressure,

within

that tend to maintain

a certain

constant

range, are met with vascular

blood

This apparent autoregulation of blood flow is illustrated reservoir was moved up and down to exert different pressed

in mm Hg on the abscissa in Fig. 3). Between

was relatively

constant

and therefore,

were

through

the organ.

55 and 70 mm Fig, blood flow

for all subsequent

pressure of 65 mm was adopted. fn vitro egg-transport experiments

flow

in Figure 3. The perfusate hydrostatic pressures (ex-

performed

experiments, using

stained

a perfusion rabbit

ova in

their cumulus masses. Ova were transported through the ampulla of the isolated perfused oviduct at average velocities ranging from 0.09-0.12 mmisec. These rates are in good agreement

with

by Boling

(1971), Verdugo

and Blandau

data from

in vivo egg-transport

experiments

et al. (1975), and Halbert

reported

et al. (1976).

Figure 4 illustrates a series of experiments in which prostaglandin E2 (PGE,) was added to the perfusion mixture in known concentrations with optical cuff recording transducers from

rabbits

pressed control. period

placed on both the isthmus in estrous.

Contraction

and the ampulla. frequency

All oviducts

was recorded,

as a percent change in the frequency of contractions from those of the The frequency values reported are an arithmetic mean of a IO-min sampling by the “frequency

discriminator”

after a 5-10 min equilibrating

decreased oviductal motility, in agreement with results reported and Harper, 1973). Since available evidence points to a possible tween

were derived

and the data ex-

egg transport

and frequency

of contractions

in the ampulla

1971), such perfusion experiments could help to provide investigate prostaglandins in this aspect of reproduction.

30

40

50

60 Hydrostatic

70

60

90

period.

PGEz

earlier (Spilman relationship be(Blandau

a basis

et al.,

by which

to

100

pressure (mmtig)

FIGURE 3 Perfusatemixture flow (ml/min) at different hydrostaticpressures. Oviducts from five rabbits in estrous. Values represent mean blood flow +. standarderror of the mean from at least three measurementsover a minimum of 1 minute at any particular pressure. Flow through two oviducts at 120 mm Hg (not shown) had a mean of 3.28 ml/min + SE 1.01.

271

272

8. U. Raess and F. F. Vincenzi

-lOOi/

, OI

IO

100

[P‘S?(nglml

1050

periusate 1

FIGURE 4 Effects of prostaglandin Ez on the frequency of contractions of ampullary and isthmic smooth muscle. Contractions were determined by optical cuff transducers. Isthmus control values (no drug added) were derived from twelve oviducts from estrous animals with a mean of 4.63 + 6.48 (SE) contractions per minute (= MB%). Values on the ordinate are expressed as the 96 change from the control value in four animals. Control values for the ampulla were determined in 14 animals with a mean of 6.66 2 0.74 (SE) (= 100%) contractions per minute. Values under the influence of drug were determined in six of these estrous animals.

DISCUSSION Methods

for the study

of reproductive

processes

have been recently

reviewed

by Blandau and co-workers (1975). The authors point out that gamete transport within the oviduct is influenced by smooth-muscle contractions, ciliary activity, luminal offer fused

fluids,

and the hormonal

advantages oviduct

for studying

transports

status

of the animal.

the relative

eggs through

contributions

the ampulla

An isolated of these

at rates comparable

observed in vivo. In other respects also, the isolated, perfused function as a reasonably good model of the in vivo organ. As shown

in Figure 3, the isolated,

perfused

oviduct

factors.

oviduct

oviduct autoregulates.

could

The

per-

to those appears to

Several mech-

anisms for tissue autoregulation have been proposed (Berne and Levy, 1972). An attractive mechanism is one in which blood flow is governed by oxygen supply and demand; inadequate oxygenation of the tissue leads to the production of vasodilator metabolites. This occurs when Earle’s solution only (which has a high partial pressure but low content of oxygen) is used as a perfusate. The fact that the tissue does autoregulate when supplied with blood-containing perfusate and that it exhibits reactive hyperemia after it is perfused with Earle’s solution, leads us to believe that the preparation model of the in situ organ. Prostaglandin

effects

described

on oviducts

herein

is a reasonably

in vivo have been reported

well-oxygenated by Asplund

(1947)

Isolated Perfused Oviduct and by Spilman tative

and Harper

descriptions

of

(1973). Their

prostaglandin

reports

effects

deal for the most part with

on

different

tissues.

quali-

Unfortunately,

when prostagiandins are administered in vivo, they are subject to rapid tissue (especially lung) degradation (Piper, 1973). It is, consequently, difficult to assess dose-response relationships of prostaglandins in vivo. In the isolated, perfused oviduct preparation, prostaglandins are less subject to metabolism. Similar considerations

would,

of course,

obtain for a number

of drugs metabolized

by liver,

lung,

etc. Thus,

the method

activities,

also be useful devices

for

perfusate, inated.

described

such as muscular

here is not only a tool for the evaluation

contraction

in the evaluation

measuring

At the

of the effects

responses

many uncontrolled same time,

and the passage of stained

and with

variables the

use of vascular

The author

acknowledges

This

work

the excellent

of the contraction

was supported

by NIH

With

adequate

concentrations

in the

in vivo or in situ can be elim-

perfusion

with

a blood

enriched

which is more viable and more likely to be

assistance

discriminator,

contract

drugs.

of drug

encountered

perfusate appears to offer a preparation a useful model of the in vivo organ.

and development

of various

control

of oviductal

ova, but should

of Frances and Prof.

HD3-2788

Huhndorf,

Robert

R. J. Blandau

and Program

Adams for the design

for reviewing

Project

the manuscript.

HD-03752.

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Some

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RM,

Levy NM

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MO:

Louis,

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RJ, Boling

Methods

pp 110-131.

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JL (1972) A miniature

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