Causas de la evasión tributaria

2.3.1 Characterization of the hypertensive phenotype of the TGR(mRen2)27 rat

To date, the hypertensive phenotype of the TGR(mRen2)27 rat has not been characterized over time. Such characterization was important since the effects of hypotensive agents were to be studied in animals of different age. A descriptive temporal study of the BPs of the TGR(mRen2)27 rat was thus required. Systolic BP (SBP) and body weight (Bwt) were measured thrice weekly in untreated TGR(mRen2)27 rats (TGR, n=25) and their controls- SD rats of the same strain as the TGRs (SD, n=12).

Measurement of SBP and Bwt

SBP was measured thrice weekly from weaning (age 28 days) at the same time of day (0700-0900 h) using tail cuff (3/8" and 7/16") photoplethysmography (MacLab Bioamplifier ML-130, ADInstruments Pty Ltd, Sydney, Australia, Apple Macintosh LC475 Computer, IITC Mod 29 Pulse Amplifier and tail cuff system, IITC Life Science,

Woodland Hills, California, USA). The animals were placed under light halothane

anaesthesia by exposure to 4 parts per million (ppm) of halothane in 100 % oxygen in a closed perspex box for 90 s with transfer to a heated mat (35 °C) and exposure to halothane (1.5 ppm) by nose cone (Fluovac delivery system and 1400 g scrubber cartridge.

International Medical Supplies, Brough, Bradwell, Sheffield, UK). Bwt was also

measured at this time. The recording system was two-point calibrated with an aneroid sphygmomanometer on every occasion of use. SBP was defined as the cuff pressure at which arterial pulsation was first detected (Figure 2.1). Only SBP was documented. In a validation study using TGR(mRen2)27 rats, SBP (but not diastolic BP) recorded by plethysmography correlated well with that recorded by direct carotid cannulation, with a mean difference of 8.5 mmHg (direct cannulation higher) between the two methods (C. Whitworth, personal communication). The average of five SBP readings was recorded for each animal, identified by ear marking. Animals were studied for 10 wk.

Figure 2.1: Maclab recording o f a photoplethysmographic trace, superimposed upon the

tail cuff pressure trace. Cuff pressure was allowed to fall from 300 mmHg to 40 mmHg

over a ten second period. Systolic pressure was taken as that cuff pressure at which

systolic photocell pulsations were recorded. By positioning a cursor at the precise origin

o f the first pulsation, a cursor automatically exhibited the actual BP at this time point in

a separate analogue window. Each recording was repeated five times and the mean of

these systolic BPs recorded.

Systolic Blood. Pressure 300

2.3.2 Establishing the effects of hypotensive agents on TGR(mRen2)27 rat SBP

Studies of the response of the TGR rat to hypotensive agents was required for two reasons:

(i) The cause of the hypertensive phenotype in the TGR(mRen2)27 rat is unclear

(Chapter 1, section 1.10.4). Demonstration of sensitivity to ACE inhibition and lack of sensitivity to direct vasodilator agents would support a role for tissue All generation in the genesis of hypertension.

(ii) The relative roles of the RAS and the direct effects of BP in the aetiology of the cardiovascular changes associated with hypertension are unclear. Further studies were planned to dissociate the effects of circulating or local RAS activity and systolic pressure burden on the development of LVH and cardiac fibrosis in the TGR rat (see section 2.4, below). This would require manipulation of BP by both ACE inhibition (reducing net RAS activity) and vasodilators (having no effect on RAS activity). Dose response curves for such agents have not been established and their effects with age remain unknown in the TGR rat. These issues were addressed using the calcium channel blocker, amlodipine, the vasodilator, hydralazine and the ACE inhibitor, ramipril. A dose of the agents that would reduce BP to control levels was sought. Further, to examine the role of the local RAS specifically, a maximal non-hypotensive dose of ramipril which would inhibit tissue ACE activity whilst having no effect on plasma ACE activity was sought.

AJfter weaning, drugs were administered in the drinking water (from age 28 days) and doses adjusted thrice weekly. Ramipril was the ACE inhibitor of choice since it has been shown to have high tissue binding affinity and long biological half life (Bender et al, 1990).

Four doses of ramipril were studied in order to establish a dose that would lower BP to that seen in control animals: 1, 2, 5 and 10 mg/kg/day (Rl, R2, R5 and RIO, n= 14, 10, 10

and 13, respectively). To identify a non-hypotensive dose of the ACE inhibitor, ramipril at a dose of 10 pg/kg/day (RlOU, n= 20) was initially studied as this dose has been shown to be non-hypotensive and to block tissue ACE activity in normotensive SD rats (Dr J R McEwan, personal communication).

The calcium antagonist, amlodipine and vasodilator, hydralazine were the agents chosen to lower BP through a non RAS mechanism. Amlodipine was chosen due to its long biological half life, solubility (up to 5 mg/ml in tap water), stability in tap water, lack of photosensitivity and its neutral flavour with proven acceptability to rats. It had also been shown to be highly effective in reducing SBP in SHRs: a dose of 10 mg/kg/day reduced SBP from 190 mmHg to control levels (130 mmHg) (Bennet et al, 1996). An initial dose

of 10 mg/kg/day was therefore used (AlO, n =18). Hydralazine has been used to effectively control BP in SHRs (Fuchs et al, 1996, Bennet et al, 1996) at doses o f-80-120

mg/kg/day. The following three doses were therefore studied in order to establish the most effective hypotensive dose in TGRs: 80, 160 and 200 mg/kg/day (H80, H I60 and H200, n= 12, 12 and 7, respectively).

2.4 ESTABLISHING THE RELATIVE CONTRIBUTIONS OF BP AND THE