2012 Boletn Latinoamericano y del Caribe de Plantas Medicinales y Aromticas 11 (1): 35 - 45

ISSN 0717 7917

www.blacpma.usach.cl

Artculo Tcnico | Tecnical Article
35


Isolated aorta model and its contribution to phytopharmacology

[Modelo de aorta aislada y su contribucin a la Fitofarmacologa]


Ral VI NE T
1,2
, Marcela K NOX
1
, Dieter MASCHER
3
, Cristina PAREDES-CARBAJAL
3
& Jos L MARTI NE Z
4


1
Laboratorio de F armacolog a y Bioqumica, Facultad de F armacia, Universidad de Valparaso, 2360102 Valparaso, Chile
2
Centro Regional de Estudios en Alimentos Saludables (CREAS), 2362696 Valparaso, Chile
3
Departamento de F isiologa, F acultad de Medicina, Universidad Nacional Autnoma de Mxico, Mexico D. F. 04510, Mexico
4
Editor BLACPMA, Universidad de Santiago de Chile, Santiago, Chile
Correspondencia | Correspondence: Ral Vinet
Contactos | Contacts: Ral Vinet - E-mail address: rvinet@uv.cl
Contactos | Contacts: Cristina Paredes-Carbajal - E-mail address: cparedes54@yahoo.com
Contactos | Contacts: Jos L. Martnez - E-mail address: editor.blacpma@usach.cl

Abstract
Since the early 50's until now the isolated thoracic aorta has been a traditional and productive model for pharmacological studies. This experimental model
has been closely related to Dr. Robert Furchgott`s research. The discovery of the role of endothelium in the vasorelaxation induced by acetylcholine (ACh),
represented a milestone in biological sciences and also had an important consequence on the isolated aorta preparation. In this work, we describe the isolated
aorta technique and the improvements made in Dr. Penna`s laboratory at Facultad de Medina, Universidad de Chile, as well as the Mexican contribution.
Since endothelium plays a key role on vascular relaxation and its dysfunction is one of first indicators (biomarker) of cardiovascular diseases, the isolated
aorta model is a valuable preparation. Considering the great amount of phytochemical present in many natural sources, like vegetables, fruits and medinal
plants, we expect this model to continue delivering significant contributions to the knowledge in pharmacology and phytopharmacology.

Keywords: aorta, aortic rings, phytochemicals, phytopharmacology.

Resumen
Desde principios de los aos 50 hasta ahora la aorta torcica aislada ha sido un modelo tradicional y productivo para estudios farmacolgicos. Este modelo
experimental ha estado estrechamente relacionado con la investigacin realizada por el Dr. Robert Furchgott. El descubrimiento de la funcin del endotelio en
la vasodilatacin inducida por la acetilcolina (ACh), represent un hito en las ciencias biolgicas y tambin tuvo una consecuencia importante en la
preparacin de aorta aislada. En este trabajo se describe la tcnica de aorta aislada y las mejoras realizadas en el laboratorio del Dr. Penna en la Facultad de
Medicina, Universidad de Chile, as como la contribucin de investigadores mexicanos. Puesto que el endotelio juega un papel clave en la relajacin vascular
y su disfuncin es uno de los primeros indicadores (biomarcadores) de enfermedad cardiovascular, el modelo de aorta aislada es una valiosa preparacin.
Teniendo en cuenta la gran cantidad de fitoqumicos presentes en muchas fuentes naturales como verduras, frutas y plantas medicinales, podemos esperar que
este modelo contine entregando importantes aportes al conocimiento en farmacologa y fitofarmacologa.

Palabras Claves: aorta, anillos de aorta, fitoqumicos, fitofarmacologa.




Recibido | Received: December 20, 2011.
Aceptado en versin cor regi da | Accepted i n revised form: January 2, 2012.
Publicado en l nea | Published onli ne: January 30, 2012.
Declaracin de i ntereses | Declaration of i nterests: a This work was supported in part by Grants CREAS-I12R3007 and FONDEF D07I1136.
Este articulo puede ser citado como / This article must be cited as: Ral Vinet, Marcelo Knox, Dieter Mascher, Cristina Paredes-Carbajal, Jos L Martnez. 2012. Isolated aorta
model and its contribution to phytopharmacology. Bol Lati noam Caribe Plant Med Aromat 11(1): 35 - 45.
Vinet et al. Isolated aorta model and its constribution to phytopharmacology


Boletin Latinoamericano y del Caribe de Plantas Medicinales y Aromticas/36

I NTRODUCCI N
We would like to dedicate this work to the memory of
Dr. Mario Penna (1924 1994) (Martinez, 1995) and
Dr. Gianni Pinardi (1929 2009) for their invaluable
contribution to Chilean Pharmacology.
In 1987, Dr. Pinardi introduced the isolated
aorta preparation in the laboratory of Dr. Penna
allowing the generation of several investigations
(Vinet and Pinardi, 1988; Vinet et al., 1991a, Vinet et
al., 1991b; Brieva et al., 1992; Illanes et al., 1993;
Lutz et al., 1994; Lutz et al., 1995; Zamorano et al.,
1995; Martinez and Andai, 1996; Martnez et al.,
1997).
Since the early 50's until now isolated aorta
has been a traditional and productive model for
pharmacological studies. The story of isolated thoracic
aorta as an experimental model has been closely
related to Robert Furchgott`s research. Furchgott was
awarded the Nobel Prize 1998 in Physiology or
Medicine, shared with Louis Ignarro and Ferid Murad
for their findings concerning nitric oxide (NO) as a
signalling molecule in the cardiovascular system. The
discovery of the role of endothelium in the
vasorelaxation induced by acetylcholine (ACh),
considered 'an accidental finding represented a
milestone in biological sciences and also had an
important consequence on isolated aorta preparation
(Furchgott and Zawadzki, 1980; Furchgott, 1998).
What does explain ACh failed to induce
relaxation of isolated thoracic aorta preparations?
Now, the answer is well known: any kind of
preparation (strip or ring), gentle rubbing of the
intimal surface (endothelium), whether deliberately or
accidentally eliminated the relaxing response to ACh
and other muscarinic agonists. Strips as well as rings
show good relaxation when caution is taken not to rub
the endothelial surface. We will describe both
preparations, as well as the improvements made in the
laboratory of Dr. Mario Penna at Facultad de Medina,
Universidad de Chile.
Furchgott and Bhadrakom (1953) introduced
the helical strip of the rabbit aorta as a useful
preparation for quantitative pharmacological studies
on vasoactive drugs and drug-receptor interactions.
They showed that strips never exhibit rhythmic
contractions and when is mounted in muscle chambers
they undergo a gradual elongation and increase in
sensitivity to stimulating drugs over a period of about
2 to 3 hr. Drugs, which were found to give only
contraction of aortic strips fall in the following order
of potency: norepinephrine and epinephrine >
histamine > ACh. A surprising finding was that ACh,
well recognized as a very potent vasodilator in whole
animal and perfused organ studies, elicited no
relaxation but only contraction of the aortic strip,
whether or not the strip was tested at rest or
precontracted with a vasoconstrictor like
norepinephrine.

Preparation of rabbit aortic strips
Here we describe the original method used for
Furchgott and Bhadrakom (1953) in rabbit. For each
experiment a rabbit weighing 2.5 to 3.5 k is rapidly
decapitated. The descending thoracic aorta is removed
and placed in a Petri dish containing Krebs
bicarbonate solution at room temperature. Excess fat
and connective tissue are trimmed off. The whole
length of aorta is then cut along a close spiral. During
the cutting, which is done with a small, sharp-pointed
scissors, the uncut portion of the aorta, held gently
between the thumb and fingers of the operator's free
hand, is gradually rotated and moved forward toward
the scissors in such a manner as to permit a continuous
spiral incision. The resulting strip is usually about 0.4
mm thick, 2 mm wide and 20 cm long, and contained
smooth muscle fibers (the circular fibers of the intact
aorta) oriented at about 15 degrees relative to its long
axis. From this strip shorter strips of 2 to 4 cm length
are cut for use in experiments. During the whole
procedure of preparing the strips, the tissue is kept
moistened with Krebs solution. Then strips are
mounted in organ chambers of 20-30 mL working
volume with the aid of stainless steel S-hooks, under 4
g of basal tension. The Krebs bicarbonate solution is
bubbled with 95% O
2
and 5% CO
2
and maintain at 37
C. The isotonic recording is carried out with an
adequate force transducer attached to a polygraph.
When aortic strips are first attached under 4 g
tension they immediately stretch about 40 per cent
(Figure 1). They then undergo a further gradual
increase in length, which usually amounts to about 5
per cent, but varies from 2 to 10 per cent. This gradual
increase approximately follows an exponential curve
with a half-time of 10 to 25 minutes, so that elongation
is essentially complete in 2 to 3 hours.



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Figure 1


Change in length and sensitivity of rabbit aortic strips with time. Abscissa is time after strip was attached to lever exerting 4 g
tension. Ordinate is change in length in per cent of initial length under tension. Typical experiment showing gradual increase in
sensitivity to epinephrine with time, with attainment of maximal sensitivity in 2 to 3 h. At each horizontal bar, the strip was
exposed to 3 x 10
-9
epinephrine (modified from Furchgott and Bhadrakom, 1953).


Preparation of rat aortic rings
We describe a modified method for the determination
of aorta contractility (Vinet and Pinardi, 1988; Vinet et
al., 1991a; Illanes et al., 1993). Rats from 250 to 290 g
are killed by decapitation and the thoracic aorta is
carefully excised and placed in a Petri dish containing
Krebs-Henseleit modified buffer (in mM: NaCl 122;
KCl 4.7; NaHCO
3
15.5; KH
2
PO
4
1.2; MgCl
2
1.2;
CaCl
2
2.0; glucose 11.5; EDTA 0.026; pH 7.4) at
room temperature. Aorta is dissected, clean of
connective tissue and divided into 5 mm rings
segments.
The rings are suspended between two L-
shaped stainless steel hooks and placed in a 20-30 mL
organ chambers containing modified Krebs-Henseleit
buffer, maintained at 37 C and oxygenated
continuously with a 95% O
2
- 5% CO
2
gas. Isometric
tensions are measured using a force displacement
transducer connected to a polygraph. The rings are
allowed to equilibrate in the tissue bath for 60 min
under an optimal resting tension of 1.5 g. Rings are
progressively stretched at least three times with a
depolarizing 70 mM KCl solution (in mM: NaCl 52;
KCl 70.0; NaHCO
3
15.5; KH
2
PO
4
1.2; MgCl
2
1.2;
CaCl
2
2.0; glucose 11.5; EDTA 0.026; pH 7.4) until
the contractile response is maximal (optimal and
reference tension).
Aortic rings are repeatedly washed and
allowed to re-equilibrate for an additional 30 min. The
preparation is ready to evaluate relaxation or
contraction activities. The analysis of the effect of
drug or extract on aortic reactivity included maximal
relaxation (Rmax) and the concentration causing 50%
of the maximal response (EC
50
), expressed as pD
2
(-
log EC
50
). Integrity of endothelium may be assessed
by testing the relaxation produced by the addition of
acetylcholine (1 M) in phenylephrine (0.1-1 M)
precontracted rings. When relaxation is evaluated,
rings are usually precontracted with an alpha
1

adrenergic agonist (i.e. phenylephrine 0.1-1 M) and
once a stable contraction is achieved, cumulative
concentration-response curve is obtained by a stepwise
increase in the drug or extract concentration (usually
in the range from 10
-9
to 10
-4
M). Response is
measured as a percentage of relaxation from the
precontracted level, considering the baseline as 100%
relaxation.
When contraction is evaluated, drug or extract
is added directly on aortic rings under basal tension by
cumulative addition to obtain a concentration-response
curve. The analysis of the effect of drug or extract also
included maximal contraction (Cmax) and EC
50
, as
previously described.
The experiments were performed in male rat
because females in response to differents drugs
depends on the rat estrous cycle (Zamorano et al.,
1994).



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Figure 2

Typical recording showing rat aortic tension (mg) with time. After equilibration at 1.5 g of tension, aotic ring was exposed
three times to 70 mM KCl and then a cumulative concentration-curve was constructed using phenylephrine. Finally, on the
precontracted ring a cumulative concentration-curve was constructed using acetylcholine (Flores et al., 2011).

If there is interest in assessing the influence of
the endothelium in relation to a particular agonist-
induced response, endothelium removal is a good
alternative. It is possible by gently rubbing the intimal
surface of the vessel with a stainless steel rod and
rolling the vessel on Whatman filter paper moistened
with cold MKHB (Vinet et al., 1991a).
In order to to better understand the mechanism
involved in endothelium-dependent responses, is very
frequent to evaluate if NO and/or prostaglandins are
involved. For this purpose aortic preparation may be
incubated with NG-nitro-L-arginine methyl ester (L-
NAME), a selective inhibitor of nitric oxide synthase
(eNOS) and/or indomethacin, a nonselective inhibitor
of cyclooxygenase (COX) (Mishra et al., 2000;
Paredes-Carbajal et al., 2001).

The Mexican experience in aortic rings

The experiments are performed on aorta rings from male rats of the Wistar strain, weighing 250-300 g
(males are used in order to avoid hormonal changes during the estral cycle, but, obviously, female rats should be
used in studies analyzing the effects of plants acting on the reproductive system). All animals are kept in individual
cages at room temperature, exposed to 12-h light-dark cycle and with free access to food and water. However, in
studies analyzing the effects of acute or chronic oral ingestion of a plant or its extract either the usual rat chow or
the water is accordingly substituted. After 12 h fasting, animals are killed by cervical dislocation and following an
immediate thoracotomy the thoracic aorta is removed and placed in a bath with oxygenated Tyrodes solution.
Under a dissection microscope the aorta is then cleaned of connective and adipose tissue and cut into 2 mm long
rings. Special care is taken to avoid damage to the endothelium. Afterwards in every other ring, the endothelium is
removed by gently rubbing the rings.
For each experiment a pair of rings from the central portion of the same aorta (one with intact endothelium,
the other without a functional endothelium) is used. Each of these rings is suspended horizontally in the same
miniature organ chamber (volume 0.5 ml) between a pole fixed to the bottom of the bath and a hook attached to an
isometric force transducer. Both, the poles and the hooks are made from thin (0.5 mm) stainless steel insect needles.
With this setup, it is possible to maintain the two rings under the same experimental conditions and record
simultaneously responses mediated by a direct effect on the vascular smooth muscle and those where the
endothelium is involved. The vessels are continuously superfused (2 ml/min) with prewarmed (37
0
C) aerated (95%
O
2
and 5% CO
2
) modified Tyrode`s solution (composition in mM: NaCl, 137; KCl, 2.7; MgCl
2
, 0.69; NaHCO
3
,
11.9; NaH
2
PO
4
, 0.4; CaCl
2
, 1.8 and glucose, 10; pH was adjusted to 7.4).
The rings are initially stretched until resting tension reaches 2 g and allowed to equilibrate for one hour;
during this period the resting tension is continuously monitored and, if needed, readjusted to 2 g by further
stretching.
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Before starting an actual experiment, responsiveness of each pair of rings to the alfa
1
adrenoceptor agonist
phenylephrine and to the stable cholinergic agonist carbachol (carbamoylcholine) is tested. This is achieved by
switching the superfusing Tyrode`s solution for 10 min to one containing phenylephrine (10
-5
M) and, thereafter, to
one containing, in addition to phenylephrine, carbachol (10
-5
M). Development of a vigorous (2.0 -3.0 g) and
sustained contracture in response to phenylephrine evidences the functional integrity of the smooth muscle layer.
Carbachol-induced relaxation of the phenylephrine precontracted vessels is taken as evidence for the preservation
of an intact endothelium whereas lack of relaxation confirms the absence of a functional endothelium.

Superfusion and bath temperature control
Rings are continuously superfused with either Tyrode`s or test solutions at a rate of 2 ml/min driven by a peristaltic
pump (although gravity may equally be used). Before entering at the bottom of the miniature organ bath, solutions
flow through a spiral immersed in water at a thermostatically controlled temperature. When a solution is changed,
the arrival of the new solution at the bottom of the bath may be monitored by the small air bubble preceding its
inflow. Solutions are drained by overflow assisted by a cellulose wick to prevent small volume changes (see
Figure).

Tension recording.
Tension developed by each ring is picked-up by an isometric force transducer (Grass FT03), and following
appropriate amplification and filtration, continuously recorded analogically on a Grass model 79 polygraph system.
Additionally, with the aid of a PowerLab/200 (ADInstruments) A/D converter, the amplified analog tension signal
is digitized and stored on a hard disk, for further analysis

Experimental protocol
The basic experimental protocol used by us to test the effects of a given substance on the vasoreactivity of aortic
rings is as follows: after confirming the functional integrity of the pair of ring (as described above) we induce a
contracture with a solution containing 10
-5
M phenylephrine (maximal tension development or T max), after
stabilization of this contracture (approximately 10 min) we start a concentration response curve to the test
compound by switching the solution to solutions containing in addition to phenylephrine successively increasing
concentrations of the test compound. The optimal concentration determined by the procedure is then used to
compare the concentration-response curves to phenylephrine (10
-9
to 10
-5
M) in the presence or absence of the test
compound. In order to analyze the plausible involvement of nitric oxide and/or cyclooxigenase metabolites in the
observed effects we repeat, thereafter, the procedures in the presence of L-NAME or indomethacin respectively.
Obviously, this protocol can be adapted to the use of a great variety agonists, antagonists or enzymatic inhibitors

Data analysis.
The contractile responses induced by phenylephrine are expressed as tension increment in grams above the basal
tension (imposed on the vessel throughout the experiment). Carbachol-induced relaxations are expressed either as
the percent reduction in tension relative to maximal tension developed in response to phenylephrine (10
-5
M) or as
the percentage of that maximal tension.
All data are expressed as means S.D. PD
2
(-Log of the mean molar concentration of agonist producing
50% of the maximal response) is determined with the software package Graph Pad Prism (San Diego, CA., USA).
Comparisons of means are made by One Way Analysis of Variance (ANOVA) and differences among groups are
evaluated using Student-Newman-Keuls Method (Sigma Stat software; St. Louis, MO., USA). A P value of 0.05 or
less is considered significant.







Vinet et al. Isolated aorta model and its constribution to phytopharmacology


Boletin Latinoamericano y del Caribe de Plantas Medicinales y Aromticas/40

Figure 3







A schematic diagram showing the different components of the system used for recording aortic rings mechanical activity.


DISCUSSI ON
The mechanical response i.e. tension increase or decrease, oI 'in vitro superIused vascular segments to natural
products or their extracts is widely used to analyze their presumptive vasoactive actions. Must such studies use a
vascular ring (generally an arterial rings) suspended in a rather large organ bath (volume 10 ml or more) with no
continuous flow. Substances to be tested are either added as a bolus or by bulk solution exchange, and washout of
the substance is achieved by repeated bulk solution exchange. Since bulk solution exchange cause mechanical
artifacts continuous tension recordings are thus not possible which precludes the detection of plausible short lasting
transient responses.
To our knowledge, in none of the studies performed to test whether or not the endothelium is involved in
the response to a given substance, the experiments were performed with a pair of rings (one with and one without a
functional endothelium) suspended in the same bath and, hence, simultaneously exposed to the test substance. It is,
obviously, a valid approach to perform the experiments on rings superfused separately but we consider that the
Vinet et al. Isolated aorta model and its constribution to phytopharmacology


Boletin Latinoamericano y del Caribe de Plantas Medicinales y Aromticas/41

simultaneous exposure of a pair of rings gives more direct evidence regarding the role of the endothelium in
theobserved responses.
The rather high flow rate (2 ml/min) combined with the small bath volume (0.5 ml) guarantees a
rapidsolution change which allows the detection of early plausible brief transient responses and on the other hand,
avoids uncontrolled alterations of the extracellular medium (by either accumulation or depletion of molecules)
which mayinterfere with the effect produced by a given challenge.
Our experience with this model, in testing the effects of synthetic estrogens, Spirulina extracts,
Rutachalepensis infusion, psithacantus callyculatus extract, thrombin analogues, endo- and synthetic
cannabinoids,Ranolazine and some of its derivatives and different types of tea, has been always successful.


Aorta model in phytochemical evaluation
As Furchgott described (Furchgott and Zawadzki,
1980), aorta model as biomarker is dependent of
endothelium functionality. In 1982, helical strips were
used to demonstrate the effect induced by extract of
Ginkgo biloba. Ginkgo biloba provoked a
concentration-dependent contraction of spirally cut
rabbit aortic strips (Auguet et al., 1982). However,
after precontracting the strips with phenylephrine,
Ginkgo biloba also induced relaxation, effect partially
endothelium-dependent (Delaflotte et al., 1984).
The popularity of the isolated aorta as in vitro
model is closely associated to endothelial function. As
we know, endothelium plays a key role on vascular
relaxation and its dysfunction is characterized by a
shift of the actions of the endothelium toward reduced
vasodilation, a proinflammatory state, and
prothrombic properties (Endemann and Schiffrin,
2004; Fltou and Vanhoutte, 2006). Dysfunction of
the endothelium has been implicated in the
pathophysiology of different forms of cardiovascular
disease, including hypertension, coronary artery
disease, chronic heart failure, peripheral artery disease,
diabetes, and chronic renal failure (Endemann and
Schiffrin, 2004; Fltou and Vanhoutte, 2006).
On the other hands, there is evidence
indicating that many phytochemical from different
natural sources may protect the endothelium. On this
basis, many extracts and isolated phytochemicals have
been tested in this model (see Table 1). Among
phytochemicals, polyphenols have a great importance
since their total dietary intake could be as high as 1
g/d, which is much higher than that of all other classes
of phytochemicals and known dietary antioxidants.
Their main dietary sources are fruits, vegetables,
cereals and plant-derived beverages such as fruit
juices, tea, coffee and red wine (Scalbert et al., 2005).
Polyphenolic compounds derived from grapes
have been evaluated in isolated aortic rings showing
relaxing properties (Fitzpatrick et al., 1993;
Andriambeloson et al., 1998) which is endothelium-
dependent. On the other hand, aqueous extracts of a
variety of vegetables, fruits, teas, nuts, herbs, and
spices show endothelium-dependent and independent
relaxing ability in vitro. These results may support that
herbal medicines and plant foods contain compounds
that, if absorbed intact and in sufficient quantities,
could conceivably be beneficial in prevention of
cardiovascular disease (Fitzpatrick et al., 1995).

Table 1

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@,#,1,4 3 A)$(B -",". 20%4#1C *%++,#-5($#,& $,471 */0%"%#,(4 D/E% +, *)89 =FFG
5HIJ: %4 %0K%0(,L )$(B /01))*#,02.
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QN%0,2($2N,4/ A)$(B 4+&)",.+* $&#".0"" 5C *%#-5($#,& $,471 3(4#$%&#,(4 Q/47 +, *)89 :;;G
@%$,(61 20%4# /"#$%&#1 *%#-5($#,& $,471 */0%"%#,(4 O,#P2%#$,&K +, *)89 :;;R
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Vinet et al. Isolated aorta model and its constribution to phytopharmacology


Boletin Latinoamericano y del Caribe de Plantas Medicinales y Aromticas/42

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Vinet et al. Isolated aorta model and its constribution to phytopharmacology


Boletin Latinoamericano y del Caribe de Plantas Medicinales y Aromticas/43


AC K NOWL E GDGE MENTS
This work was supported in part by Grants CREAS-
I12R3007 and FONDEF D07I1136 (Chile). We want
to thank to Mr. Constantino Ramrez Miranda for his
skillful technical assistance and the design of the
figure 3.

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