J.F. Gordon

Confocal Microscopic Characterization of a Lesion in a Cerebral Vessel of the Stroke-Prone Spontaneously Hypertensive Rat

BACKGROUND AND PURPOSE Hypertension is a major risk factor for stroke and is associated with alterations in vascular structure and function. The aim of this study was to determine vascular function, wall morphology, and vascular smooth muscle cell (VSMC) arrangement in basilar arteries from stroke-prone spontaneously hypertensive rats (SHRSP) and normotensive control strain Wistar-Kyoto rats (WKY). The effect of perindopril treatment on SHRSP structure and function was also assessed. METHODS VSMC orientation was determined with laser-scanning confocal microscopy and computer-assisted image processing in basilar arteries stained with 5(6)-carboxyfluorescein (wavelengths: excitation, 488; emission, 515) or propidium iodide (excitation, 529; emission, 550). Measurements of wall morphology and functional responses to serotonin and KCl were assessed with wire myography. RESULTS In the WKY basilar arteries, VSMCs were uniformly oriented perpendicular to the longitudinal axis of the vessel, whereas in the SHRSP there were localized foci of VSMC geometric disorganization, with a significant deviation from 90 degrees. The SHRSP basilar arteries also showed structural remodeling and reduced contractile responses to serotonin and KCl. Perindopril treatment normalized blood pressure, prevented wall morphology alterations, and improved function but had no effect on VSMC disorganization. CONCLUSIONS This is the first demonstration of lesions of VSMC geometric disorganization in a cerebral artery from a stroke-prone genetically hypertensive rat strain. These structural abnormalities are independent of blood pressure. Their functional sequel may play a role in the pathogenesis of stroke in this model.

The use of fluorescent nuclear dyes for the study of blood vessel structure and function: novel applications of existing techniques

We have used nuclear fluorescent dyes to develop a technique for the study of vascular structure and function. Nuclear stained blood vessels, viewed with the appropriate filter sets, can be studied in great detail. Only the nuclei of the cells which form the walls are visible and so their positions relative to one another as well as their viability can be quickly assessed. The dyes are not toxic, therefore when the vessel contracts or relaxes, the changes in position of the nuclei can be monitored. In this paper we describe two original applications of fluorescent nuclear dyes in vascular research.

The effects of perindopril on vascular smooth muscle polyploidy in stroke-prone spontaneously hypertensive rats

Objective To quantify vascular smooth muscle polyploidy and growth kinetics in aortic cells from stroke-prone spontaneously hypertensive rats (SHRSP) and from normotensive Wistar-Kyoto (WKY) rats, and to examine the effects of treatment with the angiotensin converting enzyme (ACE) inhibitor perindopril on these parameters. Design The following experimental groups were used: young (age <20 weeks) and old (age > 20 weeks) untreated WKY rats and untreated SHRSP; SHRSP treated with perindopril, and age- and sex-matched control SHRSP; and SHRSP treated with hydralazine and hydrochlorothiazide and age- and sex-matched control SHRSP. The effects of treatment of the SHRSP with perindopril for 30 days on vascular smooth muscle polyploidy and growth kinetics were measured and compared with the effects of equivalent antihypertensive doses of hydralazine and hydrochlorothiazide. Methods Vascular smooth muscle polyploidy was measured using flow-cytometry DNA analysis of freshly harvested cells. Growth curves were performed on cultured aortic cells. Plasma renin activity was measured by an antibody-trapping method, plasma angiotensin II (Ang II) by radioimmunoassay and plasma ACE activity by a colorimetric method. Cardiac hypertrophy was evaluated by measuring the heart weight: body weight and left ventricle + septum weight: body weight ratios. Results The SHRSP had markedly and significantly elevated G2 + M phase of the cell cycle. Treatment with perindopril resulted in a significant reduction in polyploidy in the SHRSP, whereas treatment with hydralazine and hydrochlorothiazide had no effect on the percentage of cells in the G2 + M phase of the cell cycle. The regression of polyploidy after treatment with perindopril was associated with a significant reduction in the concentration of Ang II and ACE activity, and with a significant regression of cardiac hypertrophy. Increased mitogenesis of cultured vascular smooth muscle cells from the SHRSP was not altered by treatment with perindopril. Conclusions ACE inhibition reduces vascular smooth muscle polyploidy in large conduit arteries. This type of vascular protection is mediated by the reduced Ang II and possibly by increased kinins level, rather than by the hypotensive effect alone.

Endogenous nitric oxide modulates sympathetic neuroeffector transmission in the isolated rabbit lateral saphenous vein

Summary: The rabbit isolated lateral saphenous vein (RLSV) has a heterogeneous population of &agr;‐adrenoceptors. Responses to electrical field stimulation, in the presence of cocaine, exhibit both &agr;1‐ and &agr;2‐adreno‐ceptor‐mediated components. The present study examined sympathetic neuroeffector transmission and the response to exogenous catecholamines after inhibition of nitric oxide (NO) synthesis with N&ohgr;‐nitro‐L‐arginine methyl ester (l‐NAME). A comparison of the response in the presence and absence of a functional endothelium was also carried out. l‐NAME potentiated the first and second components of the response to nerve stimulation on the order of 300 and 500%, respectively. l‐NAME also significantly potentiated responses to norepinephrine (NE), phenylephrine (PE), and UK 14304. Selective antagonism of the first phase was seen with prazosin (&agr;1‐antagonist, 0.1 μM) and the second phase with rauwolscine (&agr;2‐antagonist, 1 μM). In the presence of l‐NAME, the remaining (uninhibited) components were potentiated. Removal of endothelial function induced by gentle rubbing of the intimal surface abolished potentiation to exogenous NE, PE, and UK14304 by l‐NAME. However, a significant degree of potentiation of the neurogenic response was observed in the rubbed tissues in response to l‐NAME. This suggests that there may be a nonendothelial source of NO that can modulate the neurogenic response to electrical field stimulation.

Confocal Myography: Applications for the Study of Resistance Arteries

The development of confocal microscopes has enabled the collection and reconstruction of serial ‘optical’ sections of labelled unfixed specimens (1). Using fluorescent nuclear dyes and conventional fluorescence microscopy it is possible to identify cell type, location and viability within living myograph-mounted resistance arteries (2). We have now extended this technique using confocal microscopy and a variety of intracellular and extracellular stains. Many in-vitro experiments on resistance arteries rely on the integrated response of all of the cells in an isolated segment. This response may be either a change in diameter, isometric force or total fluorescence of a given indicator. It has been our aim to develop techniques which will allow visualisation of individual cells within a myograph mounted vessel in order to assess each cell’s activity. We have now identified a number of possible methods which may help to answer some of the questions which cannot be addressed using currently available techniques.