models of hypertension

7/31/2019 Models of Hypertension

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UNIT 5.53Models of Cardiovascular Disease:Measurement of AntihypertensiveActivity in the Conscious Rat (SHR,DOCA-Salt, and Goldblatt HypertensionModels)

Philippe Guillaume,

1

Daniel Provost,

1

Pamela Legrand,

1

Michael Godes,

2

andPierre Lacroix1

1Porsolt and Partners Pharmacology, Boulogne-Billancourt, France2Center for Cardiovascular Research/Institute of Pharmacology, Berlin, Germany

ABSTRACT

The protocols described in this unit are used to assess the effects of new chemical entities

on hypertension in conscious rats. In the spontaneously hypertensive rat (SHR) model,

the results obtained with the reference compounds clonidine, prazosin, propranolol,

and captopril are provided for illustration. All compounds demonstrate antihypertensive

activity, with captopril and prazosin being the least and the most active, respectively. In

the deoxycorticosterone acetate (DOCA)-salt model in the rat, the test substance shownas an example (a potential endothelin ETA-receptor antagonist) prevents the development

of hypertension in the first phase. However, the effects of treatment disappear in the very

last phase of the study, suggesting the development of a malignant hypertension resistant

to treatment in this model. In the Goldblatt hypertension rat model (renal artery stenosis),

losartan prevents the development of hypertension. It does not modify the weight of the

right and left kidneys but slightly reduces the degree of cardiac hypertrophy. Curr. Protoc.

Pharmacol. 44:5.53.1-5.53.15. C 2009 by John Wiley & Sons, Inc.

Keywords: cardiovascular disease r hypertension r SHR r DOCA-salt r

Goldblatt hypertension

INTRODUCTION

There are several rat models of hypertension that are suitable for the detection of antihy-

pertensive agents, and this unit describes three of them: the spontaneously hypertensive

rat (SHR) model, the deoxycorticosterone acetate (DOCA)-salt model, and the Goldblatt

model. Each of these models induces hypertension by a different mechanism and the

models are sensitive to different therapeutic agents.

The spontaneously hypertensive rat (SHR) model (Okamoto, 1972) is particularly useful

since it requires no surgery or pharmacological agent to induce hypertension, and it

is sensitive to most of the clinically active antihypertensive drugs (Roba, 1976). Basic

Protocol 1 can be used to screen for potential antihypertensive activity in the SHR.

The DOCA-salt-induced model of hypertension in rats is a standard example of pharma-

cologically induced hypertension (Seymour et al., 1991). The combination of deoxycor-

ticosterone acetate (DOCA), saline drinking fluid, and unilateral nephrectomy results in

severe hypertension with cardiac and renal hypertrophy. Basic Protocol 2 can be used to

detect the antihypertensive potential of compounds. However, angiotensin I converting

enzyme (ACE) inhibitors and angiotensin II receptor blockers are ineffective in reducing

Current Protocols in Pharmacology 5.53.1-5.53.15, March 2009

Published online March 2009 in Wiley Interscience (www.interscience.wiley.com).

DOI: 10.1002/0471141755.ph0553s44

Copyright C 2009 John Wiley & Sons, Inc.

Animal Modelsof Disease

5.53.1

Supplement 44


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AntihypertensiveActivity in theConscious Rat

5.53.2

Supplement 44 Current Protocols in Pharmacology

arterial blood pressure in this model of low-renin DOCA-salt hypertension. In contrast,

aldosterone receptor blockers and diuretics are effective in this model.

A renal-impairment induced experimental hypertension model (Dussaule et al., 1986) is

also described in this unit (see Basic Protocol 3). In this case, the one-clip, two kidney

hypertensive rat model (2K1C Goldblatt hypertension) is utilized. This model is useful

for studying hypertension resulting from activation of the renin-angiotensin system, an

important component in the regulation of blood pressure (Leenen and De Jong, 1975).

This model is also of value for studying the long-term effects of treatments in preventing

cardiovascular and renal injury (Wenzel et al., 2003).

NOTE: All protocols using live animals must first be reviewed and approved by an

Institutional Animal Care and Use Committee (IACUC) or must conform to governmental

regulations regarding the care and use of laboratory animals.

BASIC

PROTOCOL 1

MEASURING ANTIHYPERTENSIVE ACTIVITY IN THE SPONTANEOUSLYHYPERTENSIVE RAT (SHR)

This protocol describes a method for identifying the antihypertensive potential of test

compounds following oral administration in the spontaneously hypertensive rat (SHR).

A device to measure blood pressure and heart rate is surgically implanted to allow

measurement of the effects of test compounds in non-anesthetized animals.

Materials

Male spontaneous hypertensive rats (SHR/Kyo; Elevage Janvier) 16 weeks old and340 to 360 g

30 mg/ml chloral hydrate (Cooper, Rhone-Poulenc Rorer)

5000 IU/ml sodium heparin (Sanofi-Winthrop) in isotonic saline (LaboratoireAguettant)

Penicillin G Diamant (Rhone-Poulenc Rorer)

Isotonic saline (Laboratoire Aguettant)

Test compound(s)

0.5% (w/v) carboxymethylcellulose (Cooper, Rhone-Poulenc Rorer)

6% (w/v) sodium pentobarbital in 0.9% (w/v) NaCl (physiological saline)

Polypropylene cages with 191-in.2 (1232-cm2, 28 44cm) floor areaAnimal balance accurate to 1 g (e.g., Sartorius)

Scalpel

0.86-mm i.d., 1.27-mm o.d. catheter

Surgical thread (4.0 silk ligature)

Autoclavable stainless steel button (15-mm diameter; Instech Solomon)

Tether assembly (Instech Solomon)

Single-channel cannula swivel (Instech Solomon)

Harvard-lock sutures with needles (Harvard Apparatus)

Blood pressure recorder (TA 240 graphic recorder; Gould Instrument Systems)equipped with:Pressure processor

Isolated physiologic pressure transducer5-ml syringes

25-G, 5/8-in. Luer-lock needles

25 12/10mm Luer gastric probes with olive extremity

Prepare animals

1. House male spontaneously hypertensive rats in groups of six in 191-in.2 (1232-cm2,

28 44cm) polypropylene cages under standard conditions for 5 days prior to

starting the experiment.


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5.53.4


6. Randomly assign six rats each to four experimental groups and mark the tails using

a different color waterproof marker for each group.

For each test compound, four groups of six animals are requiredcontrols (vehicle),

treated (dose 1), treated (dose 2), and treated (dose 3).

Captopril at 120 mg/kg or prazosin at 50 mg/kg can be administered p.o. as positive

controls. In this case, five groups can be used.

Administer test compounds

7. Fill a syringe with isotonic saline and inject sequential boluses of 0.15 ml in 1-sec

(total 0.9 ml) into the catheterized carotid artery. Wait 20 min to allow the arterialblood pressure to stabilize before the test compound is injected in step 10.

The injection of isotonic saline is used to maximize the pulse pressure amplitude and to

ensure that the catheter is not partially obstructed.

8. Prepare the test compound in distilled water or 0.5% (w/v) carboxymethylcellulose

to deliver the desired dose in a volume of 0.5 ml/100 g body weight.

Examine the test compound at three different oral doses, which are determined on the

basis of other pharmacological data.

9. Connect a recorder to the catheterand record thepulse pressure at a rate of 0.5 mm/sec.

10. Use a 25 12/10mm Luer gastric probe with an olive extremity to administer the

test compound (or vehicle) orally in a volume of 0.5 ml/100 g body weight.

11. Take 10-sec recordings at 10, 20, 30, 40, 50, 60, 120, 180, 240 min, and 8 and 24 hr

after treatment.

Four animals are prepared and tested at a time. All four animals are connected to the

same arterial blood pressure transducer, but hemostatic clamps are used for the inactive

connections so only one arterial blood pressure recording is taken at a time. Recordings

from the different animals are made at staggered 2-min intervals, which require that the

test compound be administered at 2-min intervalsto for rat 1, to + 2 min for rat 2, to +

4 min for rat 3, and to + 6 min for rat 4.

12. At the end of the experiment, sacrifice the animal by administering 6% (w/v) sodium

pentobarbital in 0.9% (w/v) NaCl or by cervical dislocation.

13. Repeat steps 7 to 12 for each animal in each group.

Analyze data

14. For each group of rats, express the results in absolute values for mean, systolic, and

diastolic arterial blood pressure (BP) in millimeters mercury (mmHg), and heart rate

(HR) in beats per minute (bpm).

Data can be presented in grouped data tables as means the (SEM) or graphically (see

Fig. 5.53.2).

All differences are considered statistically significant when the null hypothesis can be

rejected at the risk = 0.05.

15. Verify homogeneity between groups before treatment using a one-way analysis ofvariance with group as factor.

16. Perform intra-group comparisons using a two-way analysis of variance (rat, time),

with repeated measures at each time.

This is performed for the control group only.

17. Perform inter-group comparisons using a two-way analysis of variance (time, group),

with repeated measures at each time, followed by Dunnetts t-test where group effect

is significant.


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5.53.5

Current Protocols in Pharmacology Supplement 44

control

clonidine

prazosin

propranolol

captopril

100

150

200

250

A

B

400

300

200

0 50 100 150 200 250 300 500 1400 1500

Time (min)

)gHmm(erusserpd

oolblairetranaeM

)mpb(etartraehnaeM

Figure 5.53.2 Effects of test compounds on (A) mean arterial blood pressure and (B) heart ratein the conscious spontaneously hypertensive rat following a single oral administration of 0.5 mg/kgclonidine, 1 mg/kg prazosin, 100 mg/kg propranolol, 120 mg/kg captopril, or vehicle control. Mean SEM with six animals per group.

18. Perform a one-way analysis of variance (group) at each time where the interaction

time group is significant.

BASIC

PROTOCOL 2

MEASURING ANTIHYPERTENSIVE ACTIVITY IN THEPHARMACOLOGICALLY INDUCED HYPERTENSIVE RAT (DOCA-SALT)

This protocol describes a method for identifying the antihypertensive potential of testcompounds following oral administration in the deoxycorticosterone acetate (DOCA)-

salt hypertensive rat. Systolic blood pressure is measured by the indirect tail cuff method.

Materials

Male Crl:CD (SD) Sprague Dawley rats (6 weeks old, 150 to 200 g; Charles River)

Powdered food (SAFE 113)

Isoflurane (Minerve)

Carprofen (Rimadyl; Pfizer)


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5.53.6


Amoxicillin (Clamoxyl LA; Pfizer)

Test compound(s)

NaCl (Sigma), dissolved in drinking water

KCl (Sigma), dissolved in drinking water

25-mg deoxycorticosterone acetate (DOCA) pellet (21-day slow release,Innovative Research of America cat. no. M-121)


Macrolon cages with 250-in.2 (1610-cm2, 35 46cm) floor area

Animal balance accurate to 1 g (e.g., Sartorius)

Surgical thread (4.0 silk ligature)

Tail cuff apparatus (e.g., BP-2000, Visitech Systems)

Prepare animals

1. House male rats in groups of three in 250-in.2 (1610-cm2, 35 46cm) macrolon

cages under standard conditions for 5 days prior to commencing the experiment.

Standard conditions for housing these animals are at room temperature (21C), 12-hr

on/12-hr off light cycle, with illumination from 0700 to 1900, and water and food available

ad libitum.

2. At the end of the acclimatization period (day 0), weigh and anesthetize the rat using

inhalant anesthesia (3% isoflurane in air). Wait until the animal no longer responds

to a tail pinch and administer 7.5 mg/kg carprofen subcutaneously.

3. Place the animal on its left side and make a 2-cm dorsal flank incision, ligate the

renal veins and arteries and then remove the right kidney (Fig. 5.53.3).

4. In other animals, perform a 2-cm dorsal flank incision, but without uninephrectomy.

Maintain this sham-operated group as a control on standard rat chow and tap water.

5. Suture the wound with surgical thread, administer 100 mg/kg amoxicillin subcuta-

neously, and return the animal to its cage.

6. Randomly assign rats to experimental groups and mark the tails using a different

color waterproof marker for each group.

7. One week later (day 6), measure arterial blood pressure using the indirect tail cuffmethod (e.g., BP-2000, Visitech Systems).

2-cm dorsalflank incision

right kidney

ligated vein and artery

Figure 5.53.3 Illustration of a uninephrectomy following dorsal flank incision in the rat.


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5.53.7


Administer test compounds

8. Administer test compound orally (mixed with food) from day 6 (i.e., 1 day before

DOCA pellet implantation) until day 42. Incorporate the test substance in standard

powdered diet using a mixer.

The amount of test substance is adjusted two times weekly according to body weight and

food intake, which are monitored daily. Thus, the actual dose is calculated accordingly.

9. On day 7, anesthetize the animal with inhalant anesthesia (3% isoflurane in air)

and implant the uninephrectomized rats in the neck area with a slow-release 25-mg

DOCA pellet. From this point, give these rats a mixture of 1% NaCl and 0.2% KClsolution in drinking water ad libitum.

Mineralocorticoids associated with excess salt induce severe hypertension. KCl solution

is given to avoid hypokalemia.

An experiment includes the following groups:

Non-nephrectomized control group: sham-operated+ tap water, vehicle control group

(six rats)

Uninephrectomized control group: DOCA+ salts+ uninephrectomy, non-treated control

group (twelve rats)

Uninephrectomized treated groups: DOCA + salts + uninephrectomy, treated group

(twelve rats)

Reference compounds are usually not used. They can however be included, depending

on the specific strategy or mechanism of action of the test compound (i.e., ET-receptor

antagonist).

10. Three weeks later (day 28), anesthetize the animals with inhalant anesthesia (3%

isoflurane in air) and implant them with a second 25-mg DOCA pellet in the opposite

neck area.

11. Measure arterial systolic blood pressure (SBP in mmHg) and heart rate (HR in

beats/minute) once weekly for 5 weeks (on day 6, day 13, day 20, day 27, day 34,

and day 41) by the indirect tail cuff method in the morning or early in the afternoon

(between 0900 and 1500).

Note that arterial diastolic blood pressure cannot be accurately determined using thismethod and is therefore not assessed. Heart rate is derived from the pressure waveform.

12. Record body weight (g) once daily.

13. At the end of the study (i.e., on day 42) exsanguinate the animals, rapidly remove the

heart and the left kidney, rinse the organs in saline and blot them dry. Weigh the left

kidney, the left ventricle (including the septum), and the right ventricle. Normalize

each organ weight by the body weight (express organ weights in mg/100 g body

weight).

Analyze data

14. For each group of rats, express the results in absolute values for systolic blood

pressure (SBP) in mmHg, heart rate (HR) in beats per minute (bpm), body weight(g), and organ weight (mg).

Data can be presented in grouped data tables as means the standard error of the mean

(SEM) or as graphs (see Fig. 5.53.4).




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5.53.8


non-treated (sham-operated rat)

non-treated (DOCA uninephrectomized rat)

ETA-receptor antagonist (50 mg/kg/day p.o.)

non-treated (sham-operated rat)non-treated (DOCA uninephrectomized rat)

ETA-receptor antagonist (50 mg/kg/day p.o.)

100

150

200

175

125

250

225

A

6 13 20 27 34 41

(a)

*

(a)

***

(a)

***

(b)

*

(a)

***

(a)

***

***(a)

*(b)

*(b)

** (b)

Days

Systolicarterialbloodp

ressure(mmH

g)

0

500

1000

750

250

1500

1200

B

Left kidney

mg/100gbodyweight

(a)

***(b)

0

200

400

300

100

600

500

Heart

mg/100gbodyweight

Figure 5.53.4 Effects of a potential ETA-receptor antagonist on (A) systolic arterial blood pres-sure, and (B) left kidney and heart weight in the conscious DOCA-salt hypertensive rat followingchronic oral treatment (36-day period). Mean SEM with twelve animals per group (six in sham-operated rats). (a) Compared to sham-operated group: *, p 0.05; **, p 0.01;***, p 0.001.(b) Compared to non-treated group: *, p0.05; **, p0.01;***, p0.001.

15. Verify homogeneity between groups before treatment using a one-way analysis of

variance with group as factor.



is significant.

17. Perform a one-way analysis of variance (group) at each time where the interaction

time group is significant.


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5.53.9


BASIC

PROTOCOL 3

MEASURING ANTIHYPERTENSIVE ACTIVITY IN THE GOLDBLATTHYPERTENSION (RENAL ARTERY STENOSIS) RAT MODEL

This protocol describes a method for identifying the antihypertensive potential of test

compounds following p.o. administration in the two-kidney, one-clip (2K1C) hyperten-

sive rat. Systolic blood pressure is measured by the indirect tail cuff method.

Materials

Male Rj: Sprague Dawley rats (6 to 7 weeks old, 200 to 250 g; Janvier)

Powdered food (SAFE 113)Ketamine 1000 (Virbac Sante Animale)

Xylazine (Rompum, Bayer)

Carprofen (Rimadyl, Pfizer)


Losartan (Cozaar-50 mg, GlaxoSmithKline)

Amoxicillin (Clamoxyl LA, Pfizer)

Test compound(s)

Isoflurane (Minerve)

3-ml EDTA glass tubes (Venoject)

Macrolon cages with 250-in.2 (1610-cm2, 35 46cm) floor area

Animal balance accurate to 1 g (e.g., Sartorius)

U-shaped silver clip (0.20-mm i.d., Fine Science Tools)Surgical thread (4.0 silk ligature)

Tail cuff apparatus (BP-2000, Visitech Systems)

Microcapillary tubes

Laboratory ultracentrifuge (3 to 16K, Sigma)

Prepare animals

1. House male rats in groups of three in 250-in.2 (1610-cm2, 35 46cm) cages under

standard conditions for 5 days prior to commencing the experiment.

Standard conditions for housing these animals are room temperature (21C), 12-hr on/

12-hr off light cycle, with illumination from 0700 to 1900, and water and food ad libitum.

2. At the end of the acclimatization period (day 0), weigh and anesthetize a rat using100 mg/kg ketamine i.m. and 100 mg/kg xylazine i.m. Wait until the animal no longer

responds to a tail pinch and administer 7.5 mg/kg carprofen s.c.

3. Place the animal on its right side and make a 2-cm dorsal flank incision. Place a

U-shaped silver clip (0.20-mm i.d.) around the left renal artery (Fig. 5.53.5; Wenzel

et al., 2003). Take care to leave the contralateral kidney untouched.

4. Perform a 2-cm dorsal flank incision in other animals, but do not place a U-shaped

silver clip. Maintain these sham-operated animals on standard rat chow and tap water,

as a control group for organ weight, biochemistry, and blood pressure measurements.

5. Suture the wound with a 4.0 surgical thread, administer 100 mg/kg amoxicillin s.c.,

and return the animal singly to its cage.An experiment includes the following groups:

a. Non-clipped control group: sham, non-treated control group (six rats),

b. Clipped control group: 2K1C, non-treated control group (twelve rats),

c. Clipped positive treated group: 2K1C losartan-treated group (20 mg/kg/day)(twelve rats).

6. Measure arterial systolic blood pressure (mmHg) and heart rate (beats/min) 2 weeks

after surgery (week 0) using the indirect tail cuff method.

Only 2K1C rats with a systolic blood pressure >150 mmHg are included in the study.


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5.53.10


abdominalaorta

vena cava

clip

ureter

kidney

Figure 5.53.5 Illustration of the position of the silver clip around the left renal artery.

7. Randomly assign rats to experimental groups and mark the tails using a different

color waterproof marker for each group.Administer test compounds

8. Administer a test substance orally (mixed with food) from week 0 (i.e., 2 weeks after

clipping the renal artery) until week 12.

9. Record body weight (g) and food intake (g) twice weekly throughout the 12-week

treatment period.

10. Measure arterial systolic blood pressure (mmHg) and heart rate (beats/min) at weeks

4, 8, and 12 by the indirect tail cuff method in the morning or early in the afternoon

(between 0900 and 1500; be consistent).

Note that arterial diastolic blood pressure cannot be accurately determined using this

method and is therefore not assessed. Heart rate is derived from the pressure waveform.

11. At 6 and 12 weeks after starting treatment, anesthetize rats in the morning with

inhalant anesthesia (3% isoflurane in air) and collect from the retrobulbar plexus

1 ml of blood into EDTA-tubes using a microcapillary tube. Gently push the

microcapillary tube through the conjunctiva to the back wall of the orbit until it

punctures the venous sinus (blood transfers into the EDTA-tube by capillary action).

Centrifuge tubes 15 min at 1500 g, 4C, collect the resultant plasma and store the

sample frozen at approximately 80C until analysis within 6 months for plasma

renin activity (PRA, pg/ml) using a radioimmunoassay.

12. At the end of the study (i.e., at week 12) exsanguinate the animals, rapidly remove the

heart and the left kidney, rinse the organs in saline and blot them dry. Weigh kidneys,

the left ventricle (including the septum) and right ventricle. Normalize each organweight by the body weight (organ weights are expressed in mg/100 g body weight).

Analyze data

13. For each group of rats, express the results in absolute values for systolic blood

pressure in mmHg, heart rate (bpm), body weight (g), and organ weight (mg).

Data can be presented in grouped data tables as means the standard error of the mean

(SEM) or graphically (see Fig. 5.53.6).


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5.53.11




14. Verify homogeneity between groups before treatment using a one-way analysis of

variance with group as factor.



is significant.

16. Perform a one-way analysis of variance (group) at each time where the interactiontime group is significant.


non-treated (renovascular hypertensive rat)

losartan (20 mg/kg/day p.o.)


non-treated (renovascular hypertensive rat)

losartan (20 mg/kg/day p.o.)

50

100

200

150

300

250

75

125

225

175

275

A

0 84 12

(a)

***(a)

***

(a)

** (b)

(a)

***

(a)

***

** (b)

Weeks

Systolicarterialbloodpressu

re(mmH

g)

0

150

250

200

100

50

350

300

B

Left kidney

mg/100gbodyweight (a)

***(b)

0

200

400

300

100

600

500

Right kidney

mg/100gbodyweight

(a)

*** (b)*

0

100

300

200

500

400

Heart

mg/100gbodyweight

Figure 5.53.6 Effects of losartan (20 mg/kg/day) on (A) systolic arterial blood pressure, and(B) kidneys and heart weight in the conscious 2K1C-salt hypertensive rat following chronic oraltreatment (12-week period). Mean SEM with twelve animals per group (six in sham-operatedrats). (a) Compared to SHAM-operated group: *, p0.05; **, p0.01;***, p0.001. (b) Comparedto non-treated group: *, p0.05; **, p0.01;***, p0.001.


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5.53.12


COMMENTARY

Background InformationThe spontaneously hypertensive rat (SHR)

model for studying humanhypertension is well

documented (Okamoto, 1972; Stoepel et al.,

1981; Clapham et al., 1991; Chaouche-Teyara

et al., 1997). Pfeffer and Frohlich (1973) note

that these hemodynamic characteristics dur-

ing the development of spontaneous hyper-

tension provide the first direct evidence fora sequential transition from a hyperkinetic cir-

culatory state with inappropriately normal re-

sistance to that of a normal cardiac output and

a sustained increase in vascular resistance in

an experimental model closely akin to human

essential hypertension.

A number of reports have documented the

mechanisms leading to hypertension in this rat

strain. The hypertension of SHRs is heredi-

tary, and its mode of transmission indicates a

polygenic mechanism (Louis et al., 1969). De-

velopment of hypertension is not connected to

either diet or salt intake. However, if SHRsare given a salt-containing diet (up to 4%

NaCl), the level of hypertension is increased

proportionally to the ingested salt dose. The

SHRs have a marked preference for salt wa-

ter over distilled water, and this preference is

significantly greater than in the normal strain

(Catalanoto et al., 1972). Furthermore, SHRs

are more sensitive to stress than normal rats,

and stress can increase the level of hyperten-

sion (Yamori et al., 1969). While the level

of noradrenalin in various organs of SHRs is

normal, catecholamine turnover is reduced in

these animals (Louis et al., 1970). There is no

transmissible hypertensive substance in SHRs

(Ebihara, 1972), and the plasma level of renin

and kidney activity are normal and even de-

crease with age. However, a transitory increase

in renin is found in young SHRs during the

development phase of hypertension (Koletsky

et al., 1970).

The deoxycorticosterone acetate (DOCA)-

salt-induced model of hypertension in rats has

also been well documented. A high dose of

DOCA is required to induce hypertension and

isotonic saline is the sole drinking fluid. This

model is thus salt-dependent in its initiation.

The DOCA-salt model is a low renin and vol-

ume overloaded form of hypertension. Argi-

nine vasopressin (AVP) plays a role in both

the development and maintenance of this con-

dition and it is believed that AVP is involved

as a vasopressin hormone in the pathogene-

sis of DOCA-salt hypertension (Crofton et al.,

1978). Studies also indicate that the endothelin

system plays an important role in the patho-

genesis of this model (Kowala et al., 2004).

One of the major advantages of this model is

its value for investigating the role of sodium

in the development of hypertension (Fujisawa,

2003).

The kidneys, through the production of

urine, regulate the volume and concentration

of body fluids and electrolytes. Urine is pro-duced in a process called glomerular filtra-

tion, which is the removal of waste products,

minerals, and water from the blood. The kid-

neys maintain the volume and concentration

of urine by filtering waste products and re-

absorbing useful substances and water from

the blood. The kidney also secretes renin, an

important component of the renin-angiotensin

system. As a result, the kidney is critically

involved in regulating arterial blood pressure

and in the development of hypertension. In the

experimental conditions described in this unit,

plasma renin activity measured at week 6 andweek 12 was significantly increased in reno-

vascular hypertensive rats as compared with

sham-operated animals (at week 6: 9.6 1.1

versus 4.0 0.3 ng/ml/hr in the sham con-

trol group, i.e., +140%, p < 0.01 inter-group

comparison; on week 12: 18.0 2.0 versus

2.5 0.5 ng/ml/hr in the sham control group,

i.e., +620%, p < 0.001, inter-group compar-

ison) demonstrating that the one-clip, two-

kidney hypertensive rat is a model of renin-

dependent hypertension.

Critical Parameters andTroubleshooting

In the SHR model, 24 hr after surgical im-

plantation of the instrumentation, the ampli-

tude of the arterial blood pressure pulse should

be the same as when it was recorded imme-

diately after introduction of the catheter into

the carotid artery, indicating no obstruction.

An isotonic saline push into the carotid artery

may help maintain catheter function, although

no more than 1 ml should be injected before

commencing the experiment. The most critical

factor is the placement and attachment of the

catheter between the point of emergence of the

catheter from the carotid artery and the nape of

the neck. Care must be taken to avoid extreme

curvature of the catheter around the neck. To

this end, two sutures can be made along the

path of the catheter from the carotid artery to

the button (6 cm), e.g., at 2 and 4 cm, to hold

the catheter in proper position.


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5.53.13


As described in Basic Protocol 2, the

DOCA-salt treatment is longer (5 weeks ver-

sus 3 to 4 weeks) and produces greater hyper-

tension than in most published studies using

this model (Pu et al., 2005), with blood pres-

sure eventually reaching values >200 mmHg.

This could be regarded as a malignant hyper-

tension. The fact that in the present investiga-

tion the extent of the antihypertensive effects

of test substance was greatest in the phase pre-

ceding the full development of this malignant

hypertension indicates that, under these ex-

perimental conditions, the hypertension devel-

ops through several stages, the final one being

less sensitive to treatments. Thus, a shorter

protocol is recommended

In the Goldblatt model (see Basic Proto-

col 3), 2 weeks after surgery, typically 60%

to 80% of clipped rats display systolic blood

pressures >150 mmHg and are included in the

study. In addition, the mortality rate by the end

of the study can reach 20% in the renovascu-

lar hypertensive control group. No mortality istypically noted in the losartan-treated group.

As both models use the tail cuff method

to measure blood pressure, rats must be ha-

bituated to the blood pressure measurement

procedure to avoid excessive movement of the

tail that could interfere with the blood pressure

reading.

Anticipated ResultsFigure 5.53.2 shows results of a study

of several different potential antihypertensive

compounds on the arterial blood pressure and

heart rate of the spontaneously hypertensiverat. All of these agents displayed antihyper-

tensive activity, with captopril and prazosin

being the least and the most active in reduc-

ing arterial blood pressure and with prazosin

inducing reflex tachycardia.

Figure 5.53.4 shows the results of a study

with a potential antihypertensive drug (ETAreceptor antagonist) showing the systolic ar-

terial blood pressure and end organ weight

(left kidney and heart) of the DOCA-salt rat.

The substance displayed some antihyperten-

sive effects, particularly on day 34, which is

after implantation of the second DOCA pellet,as compared with the non-treated DOCA-salt

control group. As expected, a compensatory

hypertrophy of the remaining kidney and of

the heart was observed in these subjects, with

the substance slightly amplifying hypertrophy

of the remaining kidney while having no effect

on the cardiac hypertrophy.

Presented in Figure 5.53.6 are the results of

a lorsartan study on the systolic arterial blood

pressure and end organ weight (kidneys and

heart) of the 2K1C rat. Losartan, a reference

antihypertensive agent, clearly prevented the

development of hypertension. At the end of

the study, the weight of the left clipped kidney

was significantly lower in renovascular hyper-

tensive rats than in sham-operated animals. In

contrast,the weightof therightkidney was sig-

nificantly higher in renovascular hypertensive

subjects than in sham-operated rats. Losartan

did not modify the weight of the left or of the

right kidney as compared with renovascular

hypertensive rats. The weight of the heart was

also higher in renovascular hypertensive rats

than in sham rats, with losartan very slightly

reducing the weight of the heart in comparison

with renovascular hypertensive rats.

Time ConsiderationsFor measuring antihypertensive activity in

the spontaneously hypertensive rat, on the firstday (start on Monday), four animals are sur-

gically instrumented. On the second day, they

are tested simultaneously, and at the end of

that afternoon, four more animals are instru-

mented. On the third day, the first four animals

are tested for the time point at 24 hr and eutha-

nized. The next four animals are tested simul-

taneously and at the end of the afternoon, four

more animals are instrumented. On the fourth

day, the previous four animals are tested for

the time point at 24 hr and euthanized and the

next four animals tested simultaneously. On

the fifth day, the last four animals are testedfor the time point at 24 hr and euthanized.

Thus, a maximum of 12 rats can be studied

per week. The total time for a single experi-

ment with one test compound at three different

doses, one vehicle control group, and one ref-

erence compound, is 3 weeks, including data

analysis.

In the DOCA-salt rat model, surgery can

be performed on the basis of 35 to 40 rats

per technician per day. In a study including

66 rats, i.e., 6 control sham-operated, 12 con-

trol DOCA-salt, 36 (3 doses) test substance

treated, and 12 reference-treated subjects, con-sidering that 2 spare animals should be added

per group, a total of 78 rats must be sub-

mitted to surgery. Therefore, the time re-

quired is 2 days/technician. Two additional

days are needed for habituating the animals

to blood pressure measurements. Implantation

of DOCA pellets requires 0.5 day per half


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5.53.14


sub-group on day 7, i.e., 1 day, and a further

0.5 day per half sub-group on day 28, i.e., 1

day. For measuring antihypertensive activity,

1 day is required per half sub-group (i.e., 1

day) on days 6, 13, 20, 27, 34, and 41, which

is 12 days per technician. Two hr per day are

required over 6 weeks, i.e., 10 days, for daily

recording of body weight and food prepara-

tion. Two days are required for organ sampling

and measurements, and 6 additional days for

data management. The total time for a single

8-week experiment with one test compound

at three different doses, one vehicle control

group, and one reference compound is there-

fore 37 days full-time.

In the renal hypertensive rat model, surgery

can be performed at a rate of 35 rats per two

technicians each day. In a study including 66

rats, i.e., 6 control non-clipped sham-operated,

12 control clipped, 36 (3 doses) test substance

treated, and 12 reference treated rats, consid-

ering that50% more rats should be prepared

to ensure that the animals included in the studywill meet with the inclusion criteria (systolic

blood pressure >150 mmHg), a total of 100rats

must go to surgery, requiring 3 days and two

technicians. Four additional days are needed

for habituating the animals to blood pressure

measurements. For measuring antihyperten-

sive activity, 5 days are required on week 0

and then 2 days per week on weeks 4, 8, and

12, requiring 11 days from each technician.

Four hr/week are required for 12 weeks, i.e.,

6 days, for daily follow up of body weight and

food preparation. Two days per technician are

required for blood sampling at week 6 and 2other days at week 12. Two days are needed

for organ sampling and measurements, and six

additional days for data management. The to-

tal time for a single 15-week experiment with

one test compound at three different doses,

one vehicle control group, and one reference

compound is therefore 43 full days.

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models of hypertension

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