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Effects of a Transient Increase in Blood Pressure Upon Cell Injury Markers in the Hypertensive Rat
Martin José F. Vilela; Barbieri Neto
Lachat João José; Furtado Mozart R. Fortes.
University of São Paulo, Ribeirão Preto, São Paulo, Brazil.
Material and Methods
Introduction: In a hypertensive crisis, vasoconstriction might occur and could be followed by a relative ischemia of tissues. In sequence, a vascular relaxing phase ensues and leads to a return to lower levels of the blood pressure. This second phase of reperfusion could, conceivably, be followed by tissue reoxygenation which is capable of leading to formation of oxygen free radicals (OFRs).
Objective: Aiming to demonstrate this possibility (the formation of oxygen free radicals), "hypertensive crises" were provoked in spontaneously hypertensive rats (SHRs) by the infusion of phenylephrine (PHE).
Material and Methods: Male animals weighing 230-300g, with mean arterial pressure of 186,06± 2,99 mmHg (n=61) were used. Control rats (n=10) were infused with isotonic saline solution. In the experimental group (n=10), rats were infused intravenously with PHE (15 m g/min) for 30 min. Blood was collected for enzyme determinations at 1, 5, and 10 min after stopping PHE infusion: glutamic-oxalacetic transaminase (GOT), creatine-phosphokinase (CPK), and lactic-dehydrogenase (LDH) were studied. Blood, hearts, kidneys, and brains were also collected for the study of OFRs formation by means of electronic-paramagnetic resonance (EPR), and malondialdehyde (MDA) determination.
Results: The experimental group showed MAP of 231,35± 4,51 mmHg , and a significant increase in GOT, CPK, and LDH was observed for every sample of the experimental group. Free radical spectra were not observed in the EPR studies. In addition, there was not any difference among the MDA values for the two groups.
Conclusions: We concluded that although the elevated enzymatic profile is suggestive of tissue lesion during the ischemia/reperfusion episode of a "hypertensive crisis", the absence of both the alterations in MDA values, and of free radical spectra in the EPR records does not support the intervention of oxy-radicals in the genesis of the catecholaminogenic cardiac lesions found in the present study. In times of orthomolecular histeria, these results sound as a caution signal against overenthusiastic interpretations.
The medical literature has been showing that a bigger variability of blood pressure (BP) counts with a worse prognostic of arterial hypertension. But a question to be answered would be which mechanisms and organic lesions would be acting as the responsibles for this worse prognostic. In a hypertensive crisis, vasoconstriction might occur and could be followed by a relative ischemia of tissues. In sequence, a vascular relaxing phase ensues and leads to a return to lower levels of the blood pressure. This second phase of reperfusion could, conceivably, be followed by tissue reoxygenation which is capable of leading to formation of oxygen free radicals (OFRs) (figure 1). Under such conditions, there can be microvascular oxidative injury (mainly in target organs). This condition resembles the ischemic/reperfusion model talked about in studies of isolated hearts  and of occlusions of coronarian blood flow , that counts with changes caused by oxygen free radicals (OFRs) generated during reperfusion: a) anaerobic metabolism, b) intracellular ultra structural changes and c) destruction of cellular membranes with enzimatic liberation and consequent cellular death . Oxygen free radical is a molecule or oxygen atom with one or more electrons in its outerest orbite non parallel with other of opposite rotations (spins) in the same orbital layer, fact which gives it high reactivity and makes it unsteady, with a short life. The OFRs can be the mediators of vascular injury in several conditions, including acute hypertension , situations of ischemia/reperfusion , atherosclerotic process , senility. Concerning arterial hypertension, therre is no sign of the direct participation of OFRs in the tissue lesions, but only indirect evidence of their action [7,8].
The present study employed the variability of BP as a model of ischemia/reperfusion and had as aims: 1-Investigate the generation of OFRs during the phase of reperfusion of a hypertensive crises, indirectly by enzimatic changes and through the evaluation of the lipidic peroxidation. 2-Investigate the generation of OFRs directly through their detection by eletronic paramagnetic resonance (EPR).
|Representative diagram of the ischemia/reperfusion episode during a bigger variability of blood pressure registered in blood pressure monitoring.|
Material and Methods:
Hypertensive crises were provoked in spontaneously hypertensive rats
(SHRs) by the infusion of phenylephrine (PHE). Male animals weighing 230-300g, with mean
arterial pressure of 186.06± 2.99 mmHg (n=61) were used. Control rats were infused with
isotonic saline solution.
Group 1 (n=10) ® Rats had their BP elevated by the infusion of PHE at the rate of /30min for two periods 24 hours apart. Blood was collected for enzyme determinations at 1, 5, and 10 min after stopping PHE infusion: glutamic-oxalacetic transaminase (GOT), creatine-phosphokinase (CPK), and lactic-dehydrogenase (LDH) were studied. After collecting the blood the rats were sacrified and hearts, brains, and kidneys were also collected for the study for determination of malondialdehyde (M.D.A.), product of lipidic peroxidation. The lipidic peroxidation is a complex lipidic that can occur in biological membranes constituted of polinsatured fatty acid reacting with the molecular oxigen, leading to the production of lipidic hydroperoxide and their metabolism products. Most of the cases envolving lipidic peroxidation starts with a chain reaction that spreads, mediated by the presence of OFRs. The lipidic hydroperoxides gather in the membrane stopping their receptors and enzymes, impairing their functions, leading to its distabilization making it permeable to ions . A very simple and highly sensitive method widely used as a lipid peroxidation marker is the reaction of reactive substances to the thiobarbituric acid (TBA), among them the derivations of lipidic hydroperoxides as the MDA. The colorimetric determination of a substance reactive to the TBA is carried out with a reading in spectrophotometer 535 nm.
Group 2 (n=10) ® Aiming at investigating the presence of OFRs through EPR, the SHRs were infused intravenously with PHE (15 m g/min) for 30 min associated with a spin trap agent PBN (N-tert-butyl-alfa-phenylnitrone) 50 mM in order to keep a steady concentration of the spin trap, able to catch OFRs and allow their detection. All the infused solution was protected from light, since the PNB agent is photosensitive. Immediately after the infusion of PHE and PBN, that is, by the end of the hypertensive crisis, the blood was collected and put in 0,4 ml tolueno solution, shaken by 20 seconds and centrifuged for 20 minutes, at 5000 rpm. After that, the upper part was immediately frozen in liquid nitrogen and underwent espectrometric analysis in order to detect OFRs. The EPR is a direct method of evaluation of the OFRs, able to supply energy, in this case microwaves, to the isolated electrons. The microwaves on the electrons can change its orientation and its energy slate. The assorption of microwaves by the electrons is detected by a crystal in the EPR. When the blood samples are placed in the wave tracks and an external magnetic field is applied, it is tried to detect the presence of paramagnetic centers with an odd number of electrons with isolated spins (OFRs). At this moment, a current in the detector falls, being the fall equivalent to the energy absorbed by the sample (figure 2 ) .
Group 3 (n=10) ® The control SHRs were infused with isotonic saline solution.
|Representative diagram of the spectrophotometer (EPR)|
The experimental group showed MAP of 231.35+4.51 mmHg(figure 3).
|Result of the blood pressure during a hypertensive crisis.|
|Results of the enzymatic profile after a hypertensive crisis.
A - Glutamic-oxalacetic transaminase (GOT)
B - Creatine-phosphokinase (CPK)
C - Lactic dehydrogenase (LDH)
|Free radical spectra in the eletronic paramagnetic resonance records|
The absence of oxidant substances in the EPR does not exclude the possible formation of OFRs during the moments of intense pressoric variation, because they may be occuring in concentrations so low that the sensibility of the instrument was not able to detect them. Moreover, it is also possible that the manipulation of tissues before the measuring of the OFRs may have destroyed the radicals.
We concluded that although the elevated enzymatic profile is suggestive of tissue lesion during the ischemia/reperfusion episode of a "hypertensive crisis", the absence of both the alterations in MDA values, and of free radical spectra in the EPR records does not support the intervention of oxy-radicals in the genesis of the catecholaminogenic cardiac lesions found in the present study. In times of orthomolecular histeria, these results sound as a caution signal against overenthusiastic interpretations.
References1. Garlick PB, Davies MJ, Hearse DJ, Slater TF. Direct detection of free radicals in the reperfused rat heart using electron spin resonance spectroscopy. Circ Res 1987; 61: 757-760.
Technical Assistance: MA Arantes, AV Verceze, MM Bernardes, EM Russo. Supported by CAPES
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