Anti - hypertensive activity 26.7.2024.pptx

anti - hypertensive activity screening in rodents

1. SCREENING OF ANTIH Y PE R TENS I V E A GENTS 12/21/2020 Sc r eeni ng of a n t i h ype r ten s i v e a g ents 1 Dr.S.Santhosh Post - Graduate

2. Goldblatt method 12/21/2020 Sc r eeni ng of a n t i h ype r ten s i v e a g ents 2 Acute renal hypertension in rats Principle Since the experiments of Goldblatt et al. (1934) there is evidence that the ischemia of the kidneys causes elevation of blood pressure by activation of the renin-angiotensin system . In rats acute renal hypertension is induced by clamping the left renal artery for 4 h . After reopening of the vessel, accumulated renin is released into circulation. The protease enzyme renin catalyzes the first and rate-limiting step in the formation of angiotensin II leading to acute hypertension.

3. Goldblatt method 12/21/2020 Sc r eeni ng of a n t i h ype r ten s i v e a g ents 3 Procedure Male Sprague-Dawley rats are anesthetized → A PVC-coated special clip is placed onto the left hilum of the kidney and fixed to t h e back m uscles → T h e r en al a r te r y i s occluded f or 3 . 5 – 4 h. 3.5 h following the surgery, the animals are anesthetized → The trachea is cannulated to facilitate spontaneous respiration → To measure systolic and diastolic blood pressure, the cannula in the ca r ot i d a r te r y i s c o n n ected to a p r ess u r e transduce r . For administration of the test compound, jugular vein is cannulated.

4. Goldblatt method Following a stable blood pressure state, ganglionic blockade is done with pentolinium (i.v.) → After obtaining stable reduced blood pressure, the renal arterial clip is removed → This leads to a rise in blood pressure due to elevated plasma renin level → Within 15 min a stable hypertension is achieved. The test substance is th en administered b y i. v r out e . B l o od p r ess u r e i s moni t o r ed until a renewed increase to the starting level is obtained. 12/21/2020 Sc r eeni ng of a n t i h ype r ten s i v e a g ents 4

5. Goldblatt method 12/21/2020 Sc r eeni ng of a n t i h ype r ten s i v e a g ents 5 Evaluation Increase in blood pressure after reopening of the renal artery and reduction in blood pressure after administration of the test drug are determined [mm Hg]. 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.

6. Goldblatt method 12/21/2020 Sc r eeni ng of a n t i h ype r ten s i v e a g ents 6 B) Chronic renal hypertension in rats Principle On the basis of the findings of Goldblatt et al. (1934) that ischemia of the kidneys induces hypertension, various modifications of the tec h n i que h a v e been desc ri b ed f or s e v eral a n i mal species. One of the most effective modifications in rats is the 1-kidney-1- clip (1K1C) method.

7. Goldblatt method 12/21/2020 Sc r eeni ng of a n t i h ype r ten s i v e a g ents 7 B) Chronic renal hypertension in rats Male Sprague-Dawley rats are anaesthetized → The fur on the back i s s h a v ed and t h e skin disi n f ected. In the left lumbar area a flank incision is made parallel to the long ax i s of t h e rat → T h e r en al pe dicel i s ex p osed with t h e kid n e y retracted to the abdomen → The renal artery is dissected and a U- shaped clip is slipped around it near the aorta → Using forceps the size of t h e cl i p i s a dj u sted → T h e r ig h t kid n e y i s r em o v ed t h r ou g h a f l ank i n cision after ty i ng off t h e r en al pe dicl e → T h e s kin i n cisio n s are closed by wound clips.

8. Goldblatt method 12/21/2020 Sc r eeni ng of a n t i h ype r ten s i v e a g ents 8 4–5 weeks after clipping blood pressure is measured and rats with values >150 mm Hg selected for the experiments → Blood pressure readings are taken on each of 3 days prior to drug treatment. Drugs are administered orally → The rats are divided into 4 an i mals pe r dos e and e a ch an i mal i s used as hi s o w n cont r ol. Drugs are administered for 3 days and predrug and 2 h post-drug blood pressure readings are taken.

9. Goldblatt method 12/21/2020 Sc r eeni ng of a n t i h ype r ten s i v e a g ents 9 EVALUATION Changes i n s y stolic b l o o d p r ess u r e a r e exp r ess ed i n mm Hg. 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.

10. Dietary hypertension 12/21/2020 Sc r eeni ng of a n t i h ype r ten s i v e a g ents 10 Fructose induced hypertension in rats PRINCIPLE Increases in dietary carbohydrate intake can raise blood pressure in experimental animals. Hwang et al. (1987 ) first reported that hypertension could be induced in normal rats by feeding a high-fructose diet. Fructose feeding was also found to cause insulin resistance, hyperinsulinemia, and hypertriglyceridemia in normal rats . Dai and McNeill (1995) studied the concentration- and duration- dependence of fructose-induced hypertension in rats.

11. Dietary hypertension 12/21/2020 Sc r eeni ng of a n t i h ype r ten s i v e a g ents 11 PROCEDURE G r oups of male Wistar rats a r e used. They are housed in cages on a 12-h light 12-h dark cycle and are allowed access to standard laboratory diet and drinking fluid. Drinking fluid consists either of tap water or 10%-fructose solution. 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. Blood samples are collected before and every 2nd week during treatment f or d eterminat i on of plas m a gluc o s e , insu l in , and trig l y c eri d es.

12. EVALUATION Since maximum effects on the chosen parameters are achieved after 6 weeks, the duration of treatment can be limited to this time. Statistical analysis is performed using a one-way or two-way analysis of variance.

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14. Endocrine hypertension 12/21/2020 Sc r eeni ng of a n t i h ype r ten s i v e a g ents 14 Mineralocorticoid induced hypertension DOCA induced hypertension PRINCIPLE Mineralocorticoid-induced hypertension is thought to be due to the sodium retaining properties of the steroid causing increases in plasma and extracellular volume. The hypertensive effect is increased by salt loading and unilateral nephrectomy in rats.

15. Endocrine hypertension 12/21/2020 Sc r eeni ng of a n t i h ype r ten s i v e a g ents 15 PROCEDURE M a l e Spra g ue D a wl e y rats a r e anest h et i zed. Th r ough a fl ank i ncision t h e lef t kidn e y i s r em o v ed. The rats are injected twice weekly with 20 mg/kg s.c. desoxycorticosterone-acetate in olive oil for 4 weeks . Drinking water is replaced with a 1% NaCl solution. Blood pressure starts to rise after 1 week and reaches systolic values between 160 and 180 mm Hg after 4 weeks. EVALUATION Blood pressure is measured before and after treatment every week. Statist ica l ana l ysis i s perf ormed.

16. Genetic hypertension 12/21/2020 Sc r eeni ng of a n t i h ype r ten s i v e a g ents 16 In 1963, Okamoto and Aoki introduced a new model of experimental hypertension that serves as a counterpart for clinical essential hypertension as well as model for complications of hypertension. The so called „ spontaneous hypertensive rat (SHR ) ‟ was developed by genetic inbreeding that uniformly resulted in 100% of the progeny having naturally occurring hypertension. In SHRs, BP gradually increases until it is maintained at a markedly elevated level after approx. 12 weeks of age. Estimates have ranged from a single gene to as many as 6 major genes that determine high blood pressure in these rats.

17. In unrestrained male SHRs, mean arterial pressure is approximately 190-200 mm Hg as c o m p a r ed to 11 5 - 130 mm Hg i n n o r m al ra t s . 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)”. Two strains of rats with inbred dissimilar sensitivity to DOCA-salt hypertension ( “Sabra strain”, SBH, SBN) have been separated by Ben- Ishay et al. (1972, 1984) in Israel.

18. Transgenic rat (TGR) models Several transgenic rat lines expressing candidate genes for h ype r te n sion h a v e been p r oduced. Introducing an additional renin gene, the murine Ren-2 gene , into the germ line of rats results in transgenic hypertensive rat strain, TGR (mREN2) 27, with an overexpression of renin , described by Peters et al. (1993). 12/21/2020 Sc r eeni ng of a n t i h ype r ten s i v e a g ents 18

19. Transgenic rat (TGR) Models 12/21/2020 Sc r eeni ng of a n t i h ype r ten s i v e a g ents 19 Genetic linkage studies have shown that the components of RAAS are associated with this type of hypertension. L i nk a g e has been desc ri b ed f or t h e angiot en sin o gen gene i n h u man hypertension. TGR model may be useful for studying the role of local RAAS system in hypertension.