Background and Purpose Pressure-induced myogenic responses have been demonstrated in cerebral resistance arteries isolated from a number of species. In the present study, we determined the response of human isolated cerebral resistance arteries to a pressure stimulus.
Methods Arteries were set up in a pressure myograph and exposed to alterations in intravascular pressure.
Results Human isolated cerebral resistance arteries developed spontaneous intrinsic tone in response to a pressure stimulus over the pressure range of 20 to 90 mm Hg that was not apparent in the absence of extracellular calcium. This intrinsic tone may be an inherent property of the vascular smooth muscle, since it remained after functional removal of the endothelium.
Conclusions Human isolated cerebral resistance arteries spontaneously contract when exposed to raised intravascular pressure. This pressure-induced myogenic response may contribute to cerebral autoregulation of blood flow.
The first suggestion that arterial smooth muscle contracted in response to an increase in intraluminal pressure was made by Bayliss1 almost a century ago. It has only been in recent years, however, with the development of systems that allow assessment of the properties of small arteries in vitro that the mechanisms involved in transducing a pressure stimulus into vascular contraction have been examined.2 3 Of particular interest has been the role of the endothelium in mediating responses to an increased intravascular pressure. The majority of studies, using resistance arteries isolated from animals (such as rat cerebral resistance arteries,4 porcine coronary arterioles,5 rat skeletal muscle arterioles,6 and rat mesenteric resistance arteries7 ), have reported that pressure-induced responses were independent of the endothelium, ie, truly myogenic. These observations contrast with earlier reports that suggested that the pressure-induced responses in canine renal arteries8 9 and feline cerebral resistance arteries10 11 were dependent on an intact and functional endothelium. To date, there have been no reports on the responses of human isolated resistance arteries to alterations in intraluminal pressure. In the present study, therefore, we examined the effect of pressure on human isolated cerebral resistance arteries and examined the dependence of pressure-induced responses on the presence of a functional endothelium.
Materials and Methods
Cerebral artery segments were obtained from the pia mater of biopsy specimens from the temporal, parietal, or frontal cortex of patients undergoing neurosurgical resection for cerebral tumors. The specimens did not contain visible evidence of the diseased tissue or tumor vessels. The biopsy specimens were placed in oxygenated physiological salt solution (PSS) at 4°C and brought to the laboratory on ice almost immediately. All patients (aged 13 to 78 years; mean, 48±11 years) were normotensive and had no indications of other cardiovascular diseases. This study was approved by the local ethics committee.
Preparation of Arteries
Arteries were dissected from the pial surface of brain biopsy specimens removed during surgery, as far away as possible from the diseased area, and placed in a dissecting dish containing cold pregassed PSS. Thereafter, arterial segments were cleaned of surrounding connective tissue and set up in a Halpern perfusion myograph as previously described.12 Briefly, vessel segments were secured between two cannulas with single strands of braided nylon suture. Both cannulas were connected to pressure-servo units, which allowed intraluminal pressure to be precisely controlled and altered as required. The myograph was a 10-mL vessel chamber with input and output channels to allow superfusion of PSS. The blood vessel was imaged using a video camera and analyzed with a video dimension analyzer (Living Systems Instrumentation) that was linked in turn to a MACLAB data acquisition system in conjunction with a Macintosh Performa LC475 computer.
The myograph was connected to a 200-mL reservoir of PSS that was bubbled with a 5% CO2/95% O2 gas mixture and circulated by use of a Masterflex pump (Cole-Parmer) at a rate of approximately 10 mL/min. This ensured that the arteriograph volume was exchanged once every minute and that when drugs were added to the external reservoir, equilibrium response could be achieved within 10 minutes. Temperature was maintained at 37°C in the organ bath, with a pH of between 7.2 and 7.4.
On the basis of our previous experience with equivalently sized arteries isolated from animals, an initial intraluminal pressure of 60 mm Hg permits the development of myogenic tone, without damaging the vessels. Therefore, the human isolated vessels were placed under an initial intraluminal pressure of 60 mm Hg, and the temperature in the myograph was slowly raised to 37°C. Arteries were superfused with fresh PSS over a period of 90 minutes to ensure washout of any chemicals remaining from the surgical procedure. During this time, human isolated resistance arteries spontaneously contracted. Thereafter, the pressure-diameter relationship was determined for human isolated cerebral resistance arteries. To achieve this, pressure was initially reduced to 10 mm Hg. Thereafter, pressure was increased in increments of 10 mm Hg (up to a maximum of 90 mm Hg), and diameter was monitored at each pressure until a maintained response was observed.
The endothelium was removed by use of an air bolus, as described by Falloon et al.13 Briefly, intraluminal pressure was reduced to 0 mm Hg, and one end of the vessel was untied. An air bolus was drawn into one of the two cannulas. The vessel was retied to this cannula and loosened from the opposite cannula. The air bolus was then slowly perfused through the arterial segment at a pressure not exceeding 20 mm Hg over a short period (<15 seconds). The arterial segment was then reattached to both cannulas. Endothelium removal was confirmed by obtaining contractile responses to the thromboxane A2 mimetic U46619 to reduce vascular diameter to approximately 50% of the initial diameter and by exposing tissues to the endothelium-dependent vasodilator substance P (1 to 3×10−7 mol/L) before and after removal of the endothelium, at an intraluminal pressure of 60 mm Hg. Substance P was used because it has previously been shown to produce endothelium-dependent relaxations in larger cerebral arteries isolated from humans.14
After removal of the endothelium, we reassessed the relationship between vessel diameter and intraluminal pressure by increasing intraluminal pressure between 10 and 90 mm Hg (in increments of 10 mm Hg). At the end of each experiment, diameter measurements were made in a calcium-free PSS solution containing 0.5 mmol/L EGTA to determine the passive characteristics of the artery segment through the same pressure range.
Where appropriate, results are shown as mean±SEM (number of vessels). Differences between means were considered significant at a value of P<.05 using Student's paired or unpaired t test.
Drugs and Solutions
The following drugs and chemicals were used: 9,11-dideoxy-9α,11α-methanoepoxy prostaglandin F2α (U46619; Cascade Biochem) and substance P acetate (Sigma Chemical Co). The composition of the PSS was (mmol/L) NaCl 119, NaHCO3 24, KCl 4.7, KH2PO4 1.17, MgSO4 1.17, disodium EDTA 0.023, CaCl2 1.25, and glucose 5.5. Ca2+-free PSS was prepared by omitting Ca2+ and adding 0.5 mmol/L EGTA.
Human isolated cerebral resistance arteries (pressurized internal diameter at 60 mm Hg, 295±30; n=7), set up in the pressure myograph, spontaneously reduced their diameter by 23.4±4.2% (n=7) when exposed to an intraluminal pressure of 60 mm Hg. Fig 1⇓ shows a representative trace recording of the effects on the diameter of a human cerebral artery of altering intraluminal pressure between 30 and 60 mm Hg. This vessel had spontaneously contracted in response to an intraluminal pressure of 60 mm Hg. Thereafter, reducing pressure to 30 mm Hg resulted in an initial passive decrease in vascular diameter, followed by an active vasodilatation to an equilibrium diameter that was greater than the original diameter at 60 mm Hg. Conversely, increasing the pressure from 30 to 60 mm Hg resulted in an initial passive increase in vascular diameter, followed by an active constriction, and then a return to the original diameter (at 60 mm Hg) over a period of 2 to 3 minutes (Fig 1⇓).
Fig 2⇓ shows the normalized data for the responses of seven human isolated resistance arteries in response to stepwise increases of 10 mm Hg in intraluminal pressure, between 10 and 90 mm Hg, in calcium (1.6 mmol/L)–containing PSS and calcium-free PSS (containing 0.5 mmol/L EGTA). There was a significant difference in vascular diameter in the presence and absence of calcium at all pressures examined with the exception of 10 mm Hg, indicating the presence of active tone through the pressure range of 20 to 90 mm Hg. This pressure-induced tone was manifest to the greatest degree between 30 mm Hg and 60 to 70 mm Hg. At pressures lower than 30 mm Hg, vascular diameter tended to increase with increasing pressure to some extent, whereas the ability to maintain a high degree of intrinsic tone was modest at pressures greater than 70 mm Hg, as shown by the gradual increase in vascular diameter with further pressure increments.
Endothelial Dependence of Pressure-Induced Responses
We assessed the role of the endothelium in mediating pressure-induced responses in human isolated cerebral resistance arteries by passing an air bolus through the lumen of vessels to remove the endothelium, according to the method of Falloon et al.13 Before this procedure, vascular diameter was reduced to approximately 50% of the initial diameter with U46619. The concentration of U46619 required to do this varied between 30 and 100 nmol/L. Subsequently, the endothelium-dependent vasodilator substance P (1 to 3×10−7 mol/L) caused an increase in artery diameter equivalent to 45.8±9.2% (n=5) of the maximum possible dilatation (Fig 3b⇓). In two preparations, passing air through the vessel abolished pressure-induced myogenic responses. However, these preparations did not subsequently respond to U46619, and these vessels were therefore excluded from subsequent analysis. In five other arteries, passing an air bolus through the artery successfully removed the endothelium, as assessed by the virtual abolition of responses to substance P (Fig 3b⇓) (maximum vasodilatation, 4.8±4.0%; n=5), without excessive damage to the underlying vascular smooth muscle, as assessed by subsequent responses to U46619 (in a similar concentration range to that used before endothelium removal) and the presence of pressure-induced myogenic tone (Figs 3 and 4⇓⇓). The −log molar concentrations of U46619 producing a 10% reduction in artery diameter were 7.68±0.40 and 7.45±0.50 before and after removal of the endothelium, respectively (n=5).
After removal of the endothelium, vessels were exposed to alterations in intraluminal pressure, in increments of 10 mm Hg, between 10 and 90 mm Hg. Fig 4⇑ shows that responses to alterations in intraluminal pressure in calcium-containing PSS did not differ in the presence or absence of a functional endothelium, ie, the pressure-induced myogenic response was not attenuated by removing the endothelium.
The development of technology that allows assessment of the properties of small resistance arteries in vitro has permitted studies that have demonstrated the importance of intrinsic factors, such as pressure and flow, in controlling vascular diameter. In recent years, pressure-induced myogenic tone has been demonstrated in isolated resistance arteries and arterioles from a number of species and a range of vascular beds, particularly those that display a high degree of autoregulation in vivo, such as the cerebral circulation (for review see Reference 2). A pressure stimulus has been shown to cause vasoconstriction in cerebral resistance arteries isolated from rats,4 12 rabbits,15 cats,10 11 and calves.16 Similarly, in patients undergoing craniotomies, small cerebral arteries from humans have been shown to constrict in response to an increase in blood pressure.17 Under in vivo conditions, however, it is impossible to discriminate whether this constriction results from the increased pressure per se or some other factor, such as an alteration in intraluminal flow characteristics (see Reference 15) or a cardiovascular reflex in response to the alteration in pressure. Thus, the present study represents the first demonstration of myogenic responses induced by alterations in pressure alone in isolated resistance-sized arteries from the human cerebral circulation. Small arteries (295±30 μm) obtained from humans (aged 13 to 78 years; mean, 48±11 years) displayed intrinsic tone over the pressure range of 20 to 90 mm Hg compared with their diameter in calcium-free PSS. This dependence of myogenic tone on extracellular calcium has also been shown in the majority of studies with small animal tissue (see Reference 2).
Although vessels displayed intrinsic myogenic tone at all pressures between 20 and 90 mm Hg, the ability of human isolated cerebral arteries of this size (≈300 μm) to maintain a constant diameter over a wide pressure range was relatively modest. Vascular diameter either decreased or remained constant only between 30 and 60 mm Hg. At pressures exceeding 60 to 70 mm Hg, vessels tended to increase in diameter in the face of an increased pressure (to prevent any irreversible effects, arteries were not exposed to pressures >90 mm Hg). Because it is not known what pressure range vessels of this size experience in vivo, it is difficult to be certain of the physiological role for this pressure-induced myogenic tone. However, it seems likely that the pressure response contributes to the autoregulation of cerebral blood flow in humans that is observed in the face of alterations to arterial pressure.18 19
In the present study, we have also shown that pressure-induced responses in human isolated cerebral resistance arteries may be independent of a functional endothelium. There has been some controversy concerning the role of the endothelium in mediating pressure-induced responses in small resistance arteries, particularly those isolated from the cerebral circulation. For example, while pressure-induced myogenic responses are independent of the endothelium in posterior and middle cerebral resistance arteries from normotensive and hypertensive rats,4 20 responses to alterations in pressure in feline cerebral resistance arteries have been reported to be dependent on an intact functional endothelium.8 11 Clearly, the results presented in this study suggest that human isolated cerebral resistance arteries behave more like vessels isolated from rats than like isolated feline vessels, possibly reflecting a species difference in the transduction process in response to a pressure stimulus. Alternatively, the dependence of the myogenic response on the endothelium may relate to the size of the blood vessels used in each study. In the present study, and in those using rat tissue, arteries ranged in size from 200 to 300 μm. In the studies using feline cerebral arteries, vessels ranged in size from 400 to 800 μm.8 11 It is also possible that analysis of the role of a functional endothelium in mediating pressure responses is influenced by the method used for removing the endothelium. Indeed, two reports have clearly demonstrated that pressure-induced myogenic responses can be shown to be independent of the endothelium only if appropriate nonchemical methods are used to remove the endothelium.5 21 We used a bolus of air to remove the endothelium from human isolated cerebral resistance arteries and demonstrated the effectiveness of this procedure, since responses to the endothelium-dependent vasodilator substance P were present only before the air bolus was passed through the lumen of the artery. This procedure, however, was effective only if the air bolus was passed through the vessel at relatively low pressures (<20 mm Hg). In two preparations, excessive pressure abolished not only myogenic responses but also responses to the vasoconstrictor U46619 and a high extracellular potassium concentration, indicating vascular smooth muscle damage. The presence of pressure-induced myogenic tone and of responses to U46619 after removal of the endothelium (by passing the air bolus at low pressure) in the remaining vessels suggests that this procedure did not result in vascular damage. However, to be certain that our procedure selectively abolishes endothelium-dependent vasodilator responses, and to confirm the endothelium independence of the pressure-induced myogenic responses, we must determine responses to an endothelium-independent vasodilator in human isolated cerebral resistance arteries before and after passing an air bolus through the lumen of the artery.
In summary, we have demonstrated for the first time that human isolated cerebral resistance arteries display pressure-induced myogenic responses. These responses may be independent of a functional endothelium.
This work was supported by a grant from the Medical Research Council.
Reprint requests to Dr William Dunn, Department of Physiology and Pharmacology, University of Nottingham Medical School, Queen's Medical Centre, Nottingham, NG7 2UH, UK. E-mail email@example.com.
- Received March 5, 1996.
- Revision received August 8, 1996.
- Accepted August 15, 1996.
- Copyright © 1996 by American Heart Association
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A process by which the brain maintains a normal rate of blood flow during changes in perfusion pressure is myogenic autoregulation. Although myogenic autoregulation is believed to be caused by an intrinsic ability of arterial smooth muscle to respond to changes in perfusion pressure,1R some investigators have suggested that the synthesis/release of substances by the endothelium may be important.2R 3R However, others have shown that myogenic autoregulation of cerebral arteries and arterioles is independent of the synthesis/release of endothelium-derived products.4R 5R The goal of the present study was to examine myogenic autoregulation of human cerebral resistance arteries and determine the role of the endothelium in myogenic autoregulation.
Using a myograph system, the authors examined contraction of human pial arteries (about 300 μm in diameter) in response to stepwise increases in intraluminal pressure. Responses were measured in a normal calcium buffer, in a calcium-free buffer, and following an injection of air to remove the endothelium. The authors report that increases in intraluminal pressure produced spontaneous intrinsic tone that was abolished by removal of calcium but was not affected by endothelial denudation. Efficacy of endothelial denudation was tested with substance P. However, a nonspecific effect of endothelial denudation on vascular smooth muscle was not examined.
The findings of this study are the first to indicate that human cerebral resistance arteries demonstrate myogenic autoregulation independent of the synthesis/release of endothelial-derived substances, These findings support previous studies conducted using animal models.
Bayliss MM. On the local reaction of the arterial wall to changes in intraluminal pressure. J Physiol.. 1902;28:220-231.
Harder DR. Pressure-induced myogenic activation of cat cerebral arteries is dependent on intact endothelium. Circ Res.. 1987;60:102-107.
Harder DR, Sanchez-Ferrer C, Kauser K, Stekiel WJ, Rubanyi GM. Pressure releases a transferable endothelial contractile factor in cat cerebral arteries. Circ Res.. 1989;65:193-198.
Faraci FM, Baumbach GL, Heisted DD. Myogenic mechanisms in the cerebral circulation. J Hypertens.. 1989;65:S61-564.
McCarron JG, Osol G, Halpern W. Myogenic responses are independent of the endothelium in rat pressurized posterior cerebral arteries. Blood Vessels.. 1989;26:315-319.