Screening Methods of Anti Hypertension Agents on Animal Model

High blood pressure - Blood forced through the arteries at an increased rate. It is measured as two numbers. 
e.g. 120/80 mmHg. 
Normal blood pressure is below this value. The first number is the systolic blood pressure. This is the maximum pressure in the arteries when heart contracts/beats. The second number is the diastolic blood pressure. This is the minimum pressure in the arteries when heart is at rest between beats. Until it becomes extreme, there are no symptoms. Hence, it is called the “Silent Killer.”

Severe hypertension may cause headache, sleepiness, confusion or coma.

Blood Pressure and Its procedure For Measurement 

Mechanism of hypertension 

In vivo models for anti-hypertension 

1. Angiotensin converting enzyme inhibition in rat (ACE Inhibition in rat)
2. Goldblatt Hypertension
3. Chronic renal hypertension in rats
4. Tail cuff method
5. Salt- sensitive Dahl Rats
6. Renin inhibition in Monkeys
7. Chronic renal hypertension in Dogs.
8. Antihypertensive and vasodilator action in rats

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Angiotensin converting enzyme inhibition in rat  

Mechanism of angiotensin converting enzyme inhibitor

Angiotensin converting enzyme inhibition in rat

• Male Sprague- Dawley rats (200-225gm)  are selected.
• They are anesthetized with 60mg/kg of phenobarbitone sodium i.v.
• The intubated trachea is artificially respirated  with 30strokes/min and a stroke volume of  6-8ml.
• The right artery is cannulated for recording the pressure.
• The jujular vein is cannulated for i.v. test injection.
• The B.P is diminished by the administration of 5 mg/kg of pentolinium i.p.
• Atropine(40 µg/kg) is also injected i.m to inhibit the mucous secretion.
• Now 310 ng/kg of Angiotensin I is injected i.v. in 0.1ml saline.
• The injection is repeated in 5 minute interval until an identical pressure is attained.
• The test drug is administered at a dose of  10 mg/kg intravenously or 25 mg/kg intradermally.
• Again Angiotensin I is injected as the similar dose above.
• The diminution of the pressure after the administration of potent  ACE inhibitors is compared .

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Goldblatt hypertension ( 2 kidney 1 clip method) 

Acute hypertension 

Procedure of goldblatt hypertension 

• Sprague – Dawley Rats (300gm) are anesthetized Hexobarbital sodium (100mg/kg) Intra peritonially
• Cannulate the trachea for respiration and the Jugular vein for test compound administration.
• A transducer is connected to the carotid artery for recording the pressure.
• A PVC coated clip is placed in the left hilum of the kidney by fixing with the back muscle for 3.5 - 4 hour.
• Pentolinium is administered for ganglionic blockage.
• Release the clip and record the rise in blood pressure. 
• Administer the test drug through I.V. and monitor the pressure continuously.
• Increase in blood pressure after releasing the clip and reduction after the drug administration is determined. 
• Compare using percentage values

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Chronic renal hypertension (1 kidney 1 clip method)

• Sprague – Dawley Rats (200 - 300gm) are anesthetized phenobarbitone sodium (100mg/kg) Intra peritonially.
• A flank parallel incision is made in the left lumbar area.
• Renal artery is dissected, cleaned and ‘U’ shaped silver clip is slipped around near the aorta.
• The internal gap b/w the clip is adjusted to 0.25 – 0.38 nm.
• The right kidney is removed after tying off the renal pedicel.
• After 4-5 weeks the B.P is measured and the animals are divided into groups of different doses.
• Test drug is administered for 3 days
• Pressure before and after drug administration(3minute) are recorded.
• Percent reduction in pressure is calculated and compare to the Standard. 

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Tail Cuff Method

Tail cuff method also know as Blood Pressure in Conscious rats.

• This method without any surgical procedure, Similar to sphygomanometry in humans (systolic pressure).
• Widely used to evaluate the anti-hypertensive drugs in experimentally induced animals.Charles River male rats(300-350 g) are anesthetized using I.P injection of 0.8 ml of 4% of chloral hydrate.
• Both the kidneys are exposed, hypertension is induced by placing a silver clip on both renal arteries,(0.2 mm dm & 4 mm l).
• After 5 to 6 weeks operated animals attain renal hypertension with systolic B.P of 170 to 200mmHg.
• A tubular inflatable cuff is placed around the base of tail and a Pizo- electric pulse detector is positioned distal to the cuff.
• The cuff is inflated to approximately 300mmHg.
• As the pressure in the cuff is released slowly, the systolic pressure is detected and recorded in the poly graph.
• Test substance is administered intra-peritonially  for alternative days in three times.
• Decrease in the systolic pressure is determined by the following steps.    

                     Day 1 : Predose & 2 hours postdrug

                     Day 3 : Predose & 2 hours postdrug

                     Day5  : Predose , 2 hours post drug & 4 hours postdrug

Salt Sensitive Dahl Rats

These rats Develop severe fatal hypertension when fed high – salt diets, whereas salt resistance rats cant.

• Sprague – Dawley Rats (250 - 300gm) are used in this study.
• Drinking water is replaced with 8% NaCl solution high salt diet is prepared by mixing salt with regular diet.
• The animals are fed with the above diet and saline.
• The test group rats are administered the drug orally for 1 month.
• Blood pressure changes are recorded.
• After the experiment the animals (both control & test) are sacrificed.
• The hearts are isolated, the mass, weight of  left ventricle & right ventricle is compared and measured.
• Upon salt feeding animals B.P rises steeply (up to 36%).
• The ability of the drug to reverse these changes is studied.
• Cardiac failure occurs at 4 to 5 months of age in these kind of rats.

Angiotensin - II antagonism 

• Male Sprague- Dawley rats (200-225gm)  are selected.
• They are anesthetized with 60 mg/kg of phenobarbitone sodium i.v.
• One carotid artery is cannulated & connected with a Statham transducer and the B.P is recorded in a polygraph.
• Both the jujular veins are cannulated to administer the test drug.
• Pentolinium (10mg/kg) is injected to block the ganglionic activity.
• Atleast 5 animals are used  for the evaluation of test drug.
• In 10 min interval doses of  0.5,1.0, and 2µg/kg of angiotensin II are injected to establish the dose response curves.
• After 10 min a continuous infusion is started of the Ang II blocker in a dose of  10µg/kg/ 0.1ml/ml.
• Again doses of 0.5,1.0, and 2µg/kg of angiotensin II are injected .
• Intensity and duration of fall in B.P. is recorded.
• The results are compared with the known std. drug.

Antihypertensive and vasdilator activity in ganglion blocked angiotensiongen II supported rats 

• To demonstrate the direct vasodilator activity of antihypertensive agent.
• This method is an anesthetized ganglion blocked rat whose B.P is maintained by an i.v. infusion of Ang II. 
• 7-9 nos of Wister Rats (250-300g) are anesthetized using a combination of Urethrane & Chlorolase (60mg/kg).
• Chlorisondamine is administered to block the ganglion (both sympathetic and para sympathetic nerve activity i.p)
• The right faemoral artery is cannulated to record the blood pressure. 
• Artificial respiration is supported during the experiment.
• Ang II is infused at a rate of  3.5µg/min in a volume equivalent to 0.05ml/min using Harvard  infusion pump.
• The steady state pressure is established with in 15-20 minute.
• Test drugs are injected i.v. at an interval of 3 minute (2ml/kg).
• Arterial pressure is recorded on a polygraph at 5,10, 15, 20 & 30 min.
• Blood pressure response for graded dose of phenylephrine can be obtained 15min before test administration.
• Data is obtained from 5-6 animals.
• A fall in blood pressure indicates the vasodilative action in turn anti hypertensive action of the test drug.

In vitro models of antihypertension agents 

1. Monocrotaline induced pulmonary hypertension 
2. Angiotensin converting enzyme inhibition in guinea pig ileum 
3. Beta 1 sympatholytic activity in guinea pig atria 

Monocrotaline induced pulmonary hypertension 

MONOCROTALINE, a pyrrolizidine alkaloids derived from Crotaloria spectabillis.

• Sprague- Dawley rats (200-225gm) of either sex are selected.
• Animals are fed with test drug for one week prior to S.C injection of MONOCROTALINE 100mg/kg.
• Animals are sacrificed 7 or 14 days later and their hearts and lungs are excised.
• Left ventricle and lung are weighed, along with main, extra and intrapulmonary, artery are isolated.
• After 1 hour arteries are made to contract with KCl.
• Contractions are recorded using lever transducer.
• Maximum force generated by an artery is plotted as a function of applied force and recorded on polygraph.
• Contraction and relaxation of agonist responses of artery are assessed. 
• Cumulative concentration. response of KCl, Angiotension II, and NOR-Epinephrine are plotted.
• Both the responses are plotted as a function of negative log of agonist concentration. 
• To compare the differences in mean responses, t-test is applied. 

Angiotensin converting enzyme inhibition in guinea pig ileum

The Guinea Pig Ileum responds powerfully for both Angiotensin II, and Bradykinin.

Mechanism of angiotensin converting enzyme 

Procedure of angiotensin converting enzyme inhibition in guinea pig ileum

• Guinea Pig of either sex (300 – 500gm) is selected.
• Animals are sacrificed by stunning, and abdomen is opened.
• A chord is tied around the starting of intestine.
• The intestine is gradually removed from the bottom and the mesentry is cut away.
• When reached the colon, the intestine is cut halfway by passing Tyrode solution to clean the surface.
• The distal pieces (more sensitive)  are fixed in the tissue clamp and brought into organ bath of 37 degree centigrade (oxygenated).
• Angiotensin I is added (10ng/ml) after 30mts of equilibrium in bath solution and contraction is recorded.
• 5 minute after the addition of ACE Inhibitor (test drug), the diminished contraction is recorded.
• The opposite response can be observed while using Bradykinin.

β1 sympatholytic activity in guinea pig atria

β1 receptor blocking activity can be evaluated by this technique.

Beta sympatholytic activity

Procedure of β1 sympatholytic activity in guinea pig atria

• Guineapig of either sex (250-300gm) is used.
• The animal is sacrificed by stunning and exsanguination.
• Heart is removed and right/ left atrium is mounted in 50ml organ bath containing Krebs – Henseleit buffer with aeration (95%O2, 5% CO2) at 37 degree centigrade.
• Contractions are recorded using lever transducer.

Right atrium

• Isoprenaline is administered in the organ bath after 30 minutes to induce ionotropy.
• Cumulative dose is maintained starting from 0.5µgm/ml and consecutive doses at 3 minute intervals.
• The bath is flushed for 3-5 times after the stable max plateau is achieved. 
• The test drug is added into the organ bath.
• 5 minute later, again isoprenaline is added in above Concentrations & observe the response.

Observation

Beta receptor blocking activity 

Left  atrium (Electricaly stimulated)

• Left Atrium is stimulated by square wave stimulator (2 impulses at 15V, duration - 1 minute).
• After equilibrium, repeat the same as above using Isoprenaline at concentrations of   0.05-0.1 5µgm/ml.
• The bath is flushed for 3-5 times after the stable max. plateau is achieved.
• The test drug is added into the organ bath, 3 minute later again isoprenaline is added in above same doses.
• If it has β1 receptor blocking activity, the ISP induced activity is inhibited.
• IC50 values are determined from the individual dose response.