Screening methods of anti hypertensive agents

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SCREENING METHODS OF
ANTI HYPERTENSIVE AGENTS
Dr.Swaroopa, 1st year pg,
Department of pharmacology

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CONTENTS
 INTRODUCTION
 REGULATION OF HYPERTENSION
 SCREENING MODELS
 CONCLUSION
 REFERENCES

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INTRODUCTION
 Hypertension is a complex multifactorial disease and one of the
leading causes of mortality and morbidity (92 million disability-
adjusted life years worldwide were attributable to high blood
pressure)
 Hypertension doubles the risk of cardiovascular diseases, including
coronary heart disease (CHD), congestive heart failure (CHF),
ischemic and haemorrhagic stroke, renal failure, and peripheral
arterial disease.
 The prevalence of hypertension increases with age, among
individuals age ≥60, it is 65.4%.
Harrison’s principles of Internal medicine, 20th Edition, Hypertensive Vascular Disease,
SK Gupta, Drug screening methods, Antihypertensive agents.

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Contd……
 As the etiology of essential hypertension is not specific and is
multifactorial, the use of experimental animal models may provide
valuable information regarding many aspects of the disease,
 Which include etiology, pathophysiology, complications and treatment.

HYPERTENSION
 Hypertension is defined as a sustained increase in blood
pressure of 140/90 mmHg or higher, a criterion that
characterizes a group of patients whose risk of hypertension-
related cardiovascular disease is high enough to merit
medical attention.
 Isolated systolic hypertension : defined as systolic blood
pressure greater than 140mmHg with diastolic blood
pressure less than 90 mmHg, is largely confined to people
older than 60 years.

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Classification
Blood
Systolic BP
pressure(mmHg)
Diastolic BP
Normal <120 <80
Prehypertension 120-139 80-89
Hypertension, stage1 140-159 90-99
Hypertension, stage 2 >160 >100
Hypertensive crisis >180 >110
Goodman and Gilman, 13th edition, The Pharmacological Basis of Therapeutics, Section III,
Treatment of Hypertension, pg 507 – 526
AMERICAN HEART ASSOCIATION CRITERIA FOR HYPERTENSION IN
ADULTS

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Bertram G Katzung, Antihypertensive agents. Basics & Clinical Pharmacology, McGraw Hill,
New Delhi, 14th edition;2018, pg-173 – 193

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Screening methods of Antihypertensive agents:
 The animal models of hypertension share many features which are common to
human Hypertension.
 Many of these models have been developed by utilizing the etiological factors
that are presumed to be responsible for human hypertension like excessive salt
intake, Hyperactivity of Renin-Angiotensin-Aldosterone-System(RAAS) and
genetic factors.

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ANIMALS USED
 Earlier, most studies on experimental hypertension were
carried out on Dogs.
 Currently, Rat is the preferred species.
 Spontaneous Hypertensive Rat(SHR), the genetic strain of
hypertensive rat, is the animal of choice.

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SCREENING MODELS
IN VIVO MODELS:-
 Acute renal hypertension in Rats
 Chronic Renal Hypertension in Rats
 Chronic Renal Hypertension in Dogs
 Neurogenic Hypertension in Dogs
 DOCA-salt Induced Hypertension in Rats
 Fructose Induced Hypertension in Rats
 Genetic Hypertension in Rats
 Hypertension Induced by Chronic NO-synthase Inhibition
 Portal Hypertension in Rats
Vogel & Vogel, drug discovery and evaluation ,chapter A2, methods to induce experimental
Hypertension, pg. no -239 -250

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 Pulmonary Hypertension Induced by Monocrotaline
 Endothelin Receptor Antagonism in Porcine Isolated Hearts
IN VITRO MODELS:

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Methods of inducing renovascular hypertension
Goldblatt Method :- Goldblatt et.al (1934)
Three variants of hypertension are produced by
this method.
1. Two kidney one clip method(2K1C)
2. One kidney one clip method(1K1C)
3. Two kidney two clip method(2K2C)

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Acute Renal Hypertension in Rats/2K1C
Principle used :
Ischemia of the kidneys causes elevation of blood pressure by
activation of renin-angiotensin system.
Clamping of renal artery for 4h
Accumulated renin released into
circulation after releasing the vessel
Protease renin catalyses the first & rate
limiting step in the formation of
angiotensin2
Acute hypertension Vogel & Vogel, drug discovery and evaluation ,chapter A2, methods to induce
experimental Hypertension, pg no -239 .

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Procedure: Male Sprague-Dawley rats weighing 300 g are
anesthetized by intraperitoneal injection of 100
mg/kg hexobarbital sodium.
A PVC-coated clip is placed onto the left hilum
of the kidney and fixed to the back muscles.
The renal artery is occluded for 3.5–4 h.
The animals are anesthetized by intraperitoneal
injection of 30–40 mg/kg pentobarbital sodium.
The trachea is cannulated to facilitate
spontaneous respiration.
Voge l& vogel, drug discovery and evaluation ,chapter A2, methods to induce experimental
Hypertension, pg no -239

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The cannula in the carotid artery is connected to a pressure
transducer to measure systolic and diastolic blood pressures.
For administration of the test compound, a jugular vein is cannulated.
After obtaining stable reduced blood pressure values, ganglionic blockade is
performed with pentolinium(10mg/kg i.v.) and the renal arterial clip is removed.
This leads to a rise in blood pressure as a consequence of elevated
plasma renin level. Within 15 min a stable hypertension is
achieved (control = 100%).
Voge l& vogel, drug discovery and evaluation ,chapter A2, methods to induce experimental

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The test substance is then administered by intravenous
injection at doses of 10 and 100 µg/kg.
Blood pressure is monitored continuously until an
increased level to the starting level is obtained.
Ten–twelve
animals are
used per
compound.
Vogel & vogel, drug discovery and evaluation ,chapter A2, methods to induce experimental

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 Increase in blood pressure is determined after reopening of the
renal artery.
 Reduction in blood pressure is determined after administration
of the test drug.
 Percent inhibition of hypertensive blood pressure values under
drug treatment are calculated as compared to pretreatment
hypertension values.
 Duration of the effect is determined [min].
 Statistical significance is assessed by the paired t-test.
EVALUATION :

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MODIFICATIONS OF THE METHOD:
cardiogenic hypertensive chemo reflex
Sharp and transient increase in systemic arterial blood
pressure associated with reflex bradycardia can be elicited by
injection of 5-hydroxytryptamine, cyanide, nicotine or
lobeline into the coronary artery blood stream of dogs
(Berthold et al. 1989)
5-HT proved to be the
most powerful agent
for its initiation

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Chronic Renal Hypertension in Rats/1K1C method:
This is One of the most effective modifications in rats.
Procedure:
Male Sprague-Dawley rats weighing 200–250 g are
anaesthetized with 50 mg/kg i.p. pentobarbital.
The fur on the back is shaved and the skin disinfected. In the left
lumbar area a flank incision is made parallel to the long axis of the rat.
The renal pedicel is exposed with the kidney retracted to the abdomen.
The renal artery is dissected clean and a U-shaped silver clip is slipped
around it near the aorta.

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The right kidney is removed through a flank incision after tying off
the renal pedicle. The skin incisions are closed by wound clips.
4–5 weeks after clipping blood pressure is measured and rats with
values higher than 150 mm Hg are selected for the experiments.
Blood pressure readings are taken on each of 3 days prior to drug
treatment.
Drugs are administered orally in volumes of 10 ml/kg.
Compounds are administered for 3 days and predrug and
postdrug(2h) blood pressure readings are taken.
The rats are
divided into 4
animals per dose
and each animal
is used as his
own control

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 Activity is determined by comparing treatment blood pressure
values with the control blood pressure value (Day 1, predrug
blood pressure).
 Comparisons are made using the paired t-test for evaluation of
statistical significance.
EVALUATION

MODIFICATIONS OF THE METHOD
Duan et al. (1996) induced renal hypertension in male Hartley
guinea pigs by a two-step procedure consisting of ligation of
the left caudal renal artery and right nephrectomy.
Arterial blood pressure and heart rate were monitored in
conscious animals.
ACE-inhibitors reduced blood pressure in sham-operated and in
renal hypertensive guinea pigs,
whereas renin inhibitors were effective only in renal hypertensive
animals

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Chronic Renal Hypertension in Dogs
• first described by Goldblatt et al. (1934) in dogs.
• The method has been modified as the “wrapping” technique (Abram and Sobin 1947).
PROCEDURE
Dogs weighing 8–12 kg are anesthetized with i.v. injection of 15 mg/kg
thiopental. Anesthesia is maintained with a halothane-oxygen mixture.
Under aseptic conditions, a midline abdominal incision is made
One kidney is exposed and wrapped in cellophane and then replaced.
The contralateral kidney is exposed.

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The artery, vein and ureter are ligated and the kidney is
removed. The abdomen is closed by sutures and clips
On the day of surgery and for 3 days following, the dogs are given
antibiotics. Body temperature is measured twice daily for 4 days
following surgery.
Six weeks following surgery, blood pressure is measured using a tail-cuff
method. For recording, the tail cuff is attached to a polygraph.
Only animals with a systolic blood pressure higher than 150 mm Hg are
considered to be hypertensive and can participate in studies evaluating
potential antihypertensive compounds.

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Blood pressure is recorded either by the indirect tail-cuff
method or by direct measurement via an implanted arterial
cannula.
On day 1 readings are made every 2 h, just before, and 2
and 4 h after oral treatment with the potential
antihypertensive compound. Drug administration is
repeated for 5 days.
On days 3 and 5 blood pressure readings are taken before
and 2 and 4 h after treatment.
At least 3
dogs are
used per
dose and
compound.

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EVALUATION
 The starting value is the average of the 2 readings
before application of the drug.
 Each of the following readings is subtracted from
this value and recorded as fall of blood pressure at
the various recording times.

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1. Technique according to Grollman (1944).
• The kidney is exposed through a lumbar incision,
• The renal capsule is removed by gentle traction,
• A figure-8ligature is applied being tight enough to deform the
kidney but not tight enough to cut the tissue.
2. Technique according to Abrams and Sobin 1947
• Renal hypertension may be induced in the rat by encapsulating
both kidneys with latex rubber capsules

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• Moulds are formed from plastic using a rat kidney as a model.
• The capsules are prepared by dipping the moulds in liquid latex
allowing them to dry in the air.
• Three applications of latex are applied before the capsules are
toughened by placing them under warm running tap water.
• The kidney is exposed by lumbar incision, the renal capsule gently
removed and the capsule applied.

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Vasodilator and depressor reflexes, originating in the
baroreceptor areas of the carotid sinus and aortic arch, play
an important part in the regulation of blood pressure.
Neurogenic Hypertension in Dogs
Stimulation of the afferent buffer fibres exerts an inhibitory
influence on the vasomotor center, and their sectioning leads to a
persistent rise in blood pressure.
Acute neurogenic hypertension can be induced in dogs
Principle :

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PROCEDURE Adult dogs of either sex weighing 10–15 kg are anesthetized using 15
mg/kg sodium thiopental, 200 mg/kg sodium barbital and 60 mg/kg
sodium pentobarbital i.v.
A femoral vein and artery are cannulated using polyethylene
tubing to administer compounds i.v and record arterial
pressure and heart rate, respectively
Left ventricular pressure is recorded via the left common carotid
artery (post-deafferentiation) using a Millar micro tip pressure
transducer.

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Pmax is recorded by speeding up the chart paper.
Cardiac output is determined by introducing a Swan-Ganz catheter
into the right heart and pulmonary artery via a jugular vein.
5 ml of cold 5% dextrose is injected into the right atrium and
an Edwards Cardiac output computer is used to calculate the
cardiac output from the temperature change in the
pulmonary artery.
All recordings are made with a polygraph.

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Both of the carotid arteries are cleared up to the bifurcation of the
internal and external carotid arteries.
The carotid sinus nerves are isolated, ligated and sectioned and a
bilateral vagotomy is performed to produce neurogenic hypertension
(mean arterial pressure more than 150 mm Hg).
The dog is allowed to equilibrate for approximately 30
min and a bolus of the test compound is administered by
intravenous injection.
Heart rate, arterial pressure, left ventricular pressure,
Pmax and are monitored for 90 min.
minimum of
3 dogs are
used for
each
compound.

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EVALUATION
Changes of the cardiovascular parameters are expressed as
percentage of the values before administration of the drug.
Neurogenic hypertension through baroreceptor denervation has
also been described in rabbits (Angell James 1984) and in rats
(Krieger 1984).

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CRITICAL ASSESSMENT OF THE METHOD
• The neurogenic hypertension is useful for acute
experiments.
• It is less useful for chronic experiments since the
elevated blood pressure caused by buffer nerve section is
more labile than that caused by renal ischemia.

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DOCA-salt Induced Hypertension in Rats
(desoxycorticosteroneacetate)
Mineralocorticoid-induced hypertension
sodium retaining properties of the steroid
increases in plasma and extracellular volume
The hypertensive effect
is increased by salt
loading and unilateral
nephrectomy in rats.

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Male Sprague Dawley rats weighing 250–300 g
are anesthetized with ether.
PROCEDURE
Through a flank incision the left kidney is removed.
The rats are injected twice weekly with 20 mg/kg s.c.
desoxycorticosteroneacetate in olive oil for 4 weeks.
Drinking water is replaced with a 1% NaCl solution. B
Blood pressure starts to rise after one week and reaches
systolic values between 160 and 180 mm Hg after 4
weeks

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 The regimen to induce DOCA-salt hypertension modified by
many authors (Stanton 1971).
 DOCA-salt hypertension can also be achieved without
nephrectomy (Bockman et al. 1992).
 Using kininogen-deficient Brown Norway Katholiek (BN-Ka) rats,
Majima et al. (1991, 1993) showed suppression of rat
desoxycorticosterone-salt hypertension by the kallikrein-kinin
system.
 Li et al. (1996) examined small-artery structure on a wire myograph
and quantified endothelin-1 messenger RNA by Northern blot analysis
in DOCA-salt hypertensive rats after administration of an ACE-
inhibitor, a calcium channel antagonist and a nitric oxide synthase
inhibitor.

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Fructose Induced Hypertension in Rats
The increased intake of either sucrose or glucose was shown to enhance the
development of either spontaneous hypertension or salt hypertension in rats
(Hall and Hall 1966).
Hwang et al. (1987) first reported that hypertension could be induced in
normal rats by feeding a high-fructose diet. F
Fructose feeding was also found to cause insulin resistance,
hyperinsulinemia, and hypertriglyceridemia in normal rats (Zavaroni et al.
1980; Tobey et al. 1982).
Tobey et al. 1982). Dai and McNeill (1995) studied the concentration- and
duration-dependence of fructose induced hypertension in rats.

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PROCEDURE
Groups of 8 male Wistar rats weighing 210–250 g are used.
The are housed two per cage on a 12-h light 12- h dark cycle and are
allowed free access to standard laboratory diet (Purina rat chow) and
drinking fluid
Drinking fluid consists either of tap water or 10%- fructose solution

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Blood samples are collected before and every second
week during treatment for determination of plasma
glucose, insulin, and triglycerides.
Body weight, food intake and fluid intake of each rat are
measured every week during treatment.
Using the tail-cuff method, systolic blood pressure and pulse
rate is measured before and every week during treatment.

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EVALUATION
The duration of treatment 6 weeks.
Statistical analysis is performed using a one-way or two-
way analysis of variance, followed by the Newman-Keuls
test.

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• Reaven et al. (1988, 1989) found an attenuation of fructose-induced
hypertension by exercise training and an inhibition by somatostatin
treatment.
• Brands et al. (1991, 1992) found an increase of arterial
pressure during chronic hyperinsulinemia in conscious rats.
• Hall et al. (1995) reported the effects of 6 weeks of a high-fat
diet on cardiovascular, renal, and endocrine functions in
chronically instrumented conscious dogs. Body weight
increased by approximately 16.9 kg, whereas MAP, cardiac
output, and heart rate increased by 28%, 77%, and 68%,
respectively

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Genetic Hypertension in Rats
Inherited hypertension in rats has been described by Smik and Hall
1958;
Phelan 1968 as genetically hypertensive (GH) rats (Simpson and
Phelan 1984).
Okamoto et al. (1963, 1966) reported the development
of a strain of spontaneously hypertensive rats from
mating one Wistar male rat with spontaneously
occurring high blood pressure with a female with
slightly elevated blood pressure.

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Hypertension in these rats is clearly hereditary and genetically
determined, thus comparable to primary hypertension in humans.
Cardiac hypertrophy (Sen et al. 1974) and cellular ionic transport
abnormalities have been observed (Yamori 1984).
By inbreeding over several generations a high incidence of
hypertension with blood pressure values of 200 mm Hg or more
was achieved.
These strains were called “Spontaneously hypertensive rats
(Akamoto-Aoki)” = SHR or “Wistar–Kyoto rats” =WKY

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• Inbred strains being salt-hypertension-sensitive and salt-hypertension-
resistant (RD) have been developed by Dahl et al.
• Another hypertensive strain derived from Wistar rats was produced by
brother-sister mating in the group of Bianchi et al. (1974, 1986) at the
University of Milan called “Milan hypertensive strain” = MHS.
• These rats show a cell membrane defect resulting in abnormal kidney
function. Salvati et al. (1990) studied the diuretic effect of bumetanide
in isolated perfused kidneys of Milan hypertensive rats

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• Several substrains of spontaneous hypertensive rats were separated
by the group of Okamoto et al. (1974) including the stroke-prone
strain SHR = SHRSP.
• These rats have an increased sympathetic tone and show a high
incidence of hemorrhagic lesions of the brain with motor
disturbances followed by death

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• Bohlender et al. (1996) reconstructed the human renin-angiotensin
system in transgenic rats overexpressing the human angiotensin gene
TGR(hOGEN) 1623 by chronically injecting human recombinant renin
intravenously using Alzet pumps.
• Zolk et al. (1998) described the effects of quinapril, losartan and
hydralazine on cardiac hypertrophy and β-adrenergic neuroeffector
mechanisms in transgenic TGR(mREN2)27 rats.

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CRITICAL ASSESSMENT OF THE METHOD
On the basis of available data no preference can be given to a particular
strain
Transgenic rats with well defined genomes are gaining more importance.

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• Pijl et al. (1994) described streptozotocin-induced diabetes mellitus
in spontaneously hypertensive rats as a pathophysiological model for
the combined effects of hypertension and diabetes.
• Rosenthal et al. (1997) used rats of the CohenRosenthal diabetic
hypertensive strain to examine the effects of an ACE-inhibitor, an
ATII antagonist and a calcium antagonist on systolic pressure and
spontaneous blood glucose levels.
• Holycross et al. (1997) used hypertensive SHHF/ Mcc-facp rats to
study plasma renin activity during development of heart failure

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• Linz et al. (1997) compared the outcome of lifelong treatment with
the ACE inhibitor Ramipril in young prehypertensive stroke-prone
spontaneously hypertensive rats and age-matched normotensive
Wistar–Kyoto rats.
• Lifelong ACE inhibition doubled the lifespan in hypertensive rats
matching that of normotensive
• Ohkubo et al. (1990) generated transgenic mice with elevated blood
pressure by introduction of the rat renin and angiotensinogen genes.

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Hypertension Induced by Chronic NO-Synthase Inhibition
Chronic blockade of NO synthesis in the rat produces systemic
hypertension and glomerular damage (Baylis et al. 1992). This
was recommended by Ribeiro et al. (1992) as a model of
hypertension.
The detrimental sequels of chronic NO synthase inhibition in rats can
be inhibited by treatment with ACE inhibitors.

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PROCEDURE Male Wistar rats at an age of 7–8 weeks weighing
210± 10 g were placed at random in metabolic
cages, divided in four to six groups of six to eight
rats each.
Group 1 (control) had free access to tap water
and food.
Groups 2–4 were treated with 0.02% L-NAME
water solution for 6 weeks in a daily dose of 25
mg/kg.

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Groups 3 and 4 received the angiotensin receptor antagonists
fonsartan (10 mg/kg) or lorsatan (30 mg/kg) for 6 weeks daily
per stomach tube.
Groups 5 and 6 received fonsartan and lorasatan alone.
At the end of the study, 24-h urine samples were collected and
retrobulbar blood samples were taken in short inhalation
anesthesia.

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For clearance evaluation rats were anesthetized with 50 mg/kg thiopentone i.p.
In order to determine glomerular filtration rate and renal plasma flow, clearances
of inulin and para-aminohippurate were performed.
EVALUATION
Results are presented as arithmetical means ± SEM.
A one-way ANOVA was calculated with SYSTAT for Windows (SYSTAT,
Evanston, Ill., USA) followed by multiple pairwise comparisons according to
Tukey.

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 Arnal et al. (1993) measured cardiac weight of rats in
hypertension induced by NO synthase blockade.
 Linz et al. (1999) reviewed the interactions between ACE, kinins
and NO.
 Sampaio et al. (2002) reported that hypertension plus diabetes
mimics the cardiomyopathy induced by NO inhibition in rats.
 Rossi et al. (2003) found that chronic inhibition of NO synthase
induces hypertension and cardiomyocyte mitochondrial and
myocardial remodeling in the absence of hypertrophy

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Portal Hypertension in Rats
Portal hypertension is associated with hyperdynamic splanchnic
circulation and reduced vascular resistance (Vorobioff et al. 1983).
Tanoue et al. (1991) developed a method for inducing portal
hypertension and esophageal varices in rats – partial ligation of
the portal vein after devascularization of the circumference of
the left renal vein and complete ligation of the portal vein on
the fifth day thereafter.
Tsugawa et al. (2000) used this model to study the role of nitric
oxide and endothelin-1 in rat portal hypertension.

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Male Sprague-Dawley rats were anesthetized
with 50 mg/kg Nembutal intraperitoneally.
PROCEDURE
The portal vein was isolated and stenosis created by a single ligature of
3–0 silk placed around the portal vein and a 20-gauge blunt-tipped
needle after devascularization of the left renal vein.
This devascularization is indispensable in preventing the development
of excess collateral vessels, which inhibit the formation of esophageal
varices and the portal hypertensive state.
3–0 silk was also placed at the area of partial ligation (loose ligation),
and both ends were then drawn out through the abdominal wall.

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Five days after the operation, the ends of the silk that had been
placed in the flank were simultaneously pulled to induce
complete portal vein ligation.
Two weeks later, this portal hypertension model was completed.
Portal venous pressure, blood flow volume in the intra-
abdominal viscera, plasma NO and plasma endothelin-1 were
measure
EVALUATION
Results were expressed as mean ± standard deviation. The Student’s t-test
was used to determine significance between portal hypertension rats and
sham-operated controls.

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• Portal hypertension by portal vein ligation without devascularization of the left
renal vein was used by Lee et al. (1985), Braillon et al. (1986), Oren et al. (1995),
Fernandez et al. (1996), Moreno et al. (1996),
• Dieguez et al. (2002) used a surgical technique based on the development of a
triple stenosing ligation to worsen the complications inherent to the prehepatic
chronic portal hypertension.
• Jaffe et al. (1994) and Li et al. (1998) used injection of different sized
microspheres into the portal vein of male Wistar rats to induce portal
hypertension.

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METHODS TO MEASURE BP IN RATS
Direct method:
• It is an invasive method .
• Femoral/carotid cannula is inserted into an anaesthetized animal.
• On day of screening, cannula is connected to a pressure transducer
then to the pre-amplifier.
• BP is recorded on the polygraph.

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Indirect /Non invasive method:
Tail cuff method:
• It is a common and convenient method.
• Tail cuff is inflated and deflated
• Pulsations disappear when cuff is inflated.
• Pulsations start appearing when pressure
in the cuff equals systolic pressure while
deflating.
• The cuff is attached to a tail cuff
sphygmomanometer or pressure
transducer and BP is recorded on a chart.

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Effects of antihypertensive agents
Antihypertensive drugs, according to their mode of action, will affect the blood
pressure in certain types of experimental hypertension, and not in all.
Vasodilators like Minoxidil, Hydralazine and Diazoxide are effective in Renal
hypertensive rats.
Calcium channel blockers, ACE Inhibitors and AT-1 antagonists decrease BP in
nephrectomised SHR.
Diuretics, are active in mineralocorticoid or salt induced hypertension.
Sympathomimetic Drugs decrease BP in both endocrine and neurogenic
hypertension.

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CONCLUSION
The most important point from direct comparison of these animal
models is that, despite the well-known heterogeneity of
hypertension, the outcome of hypertension can be similar in some
respects.
Not all classes of antihypertensives are equally effective in all rat
models of hypertension.
These models of hypertension provide ample opportunity not only
to investigate the mechanisms involved in the pathogenesis of
hypertension, but also to learn about the critical balance between
stress and its overcoming which eventually determines prognosis.

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REFERENCES
• Vogel & Vogel, 3rd edition, Drug discovery and evaluation ,chapter A2, methods to induce
experimental Hypertension, pg. no -239 -250.
• SK. Gupta, Drug screening methods, chapter16, Antihypertensive agents, pg.no 266-276.
• www.slideshare.net

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