Vasoactivity of Intraluminal and Extraluminal Agonists in Perfused Retinal Arteries
Dao-Yi Yu, Valerie A. Alder, Stephen J. Cringle, Er-Ning Su, and Paula K. Yu
Purpose. To evaluate the vasoactive response of isolated perfused arteries of the pig to K+ and adrenergic agonists and to compare the effects of intraluminal (IL) and extraluminal (EL) drug delivery. Methods. A new microperfusion system was developed, in which short lengths of porcine retinal arteries (outer diameter 90.4 ± 2.7 fim) were cannulated at both ends and perfused at a controlled rate (5 /xl/min) with outflow through a single side branch. The diameter of the vessel and the intraluminal pressure were monitored, and the effect of intraluminally and extraluminally applied agonists was determined. Endothelial cell function and the integrity of the blood retinal barrier was verified. Results. Consistent vasoactive responses were obtained from most vessels. The resting diameter of the vessel was not greatly influenced by changes in flow rate or intraluminal pressure over the physiological range. Adrenaline and noradrenaline caused dose-dependent contractions, which were larger when applied intraluminally than they were when applied extraluminally. The largest contraction for adrenaline was 19.0% ± 2.1% (n = 13) IL and 8.4% ± 1.5% (n = 13) EL, and for noradrenaline, 17.8% ± 1.9% (n = 13) IL and 6.8% ± 1.1% (n = 13) EL. The IL contraction to 124-mM K+, 19.0% ± 1.6% (n = 21), was also greater than that for EL application, 5.0% ± 1.0% (n = 13). We found that the existence of myogenic contractions was restricted to the special case in which vessels with no branches were pressurized under zero flow conditions. Conclusions. Pig retinal arteries exhibited asymmetry in their responses to adrenergic agonists and K+, with contractions significantly larger when the drug was applied to the intraluminal surface rather than the extraluminal surface. This asymmetry may reflect an important prop- erty of retinal vessels. Microperfusion systems of this type may prove valuable in developing a better understanding of control mechanisms in retinal circulations. Invest Ophthalmol Vis Sci. 1994; 35:4087-4099.
A he retinal circulation is a highly specialized vascular autonomic innervation ceases at the optic nerve head,1 bed where the requirement of guaranteeing a good and there is no evidence of parasympathetic or peptid- optical image appears to have taken precedence over ergic innervation in retinal arteries,2 so the dynamic the provision of a plentiful blood supply. It is relatively process of matching local tissue blood flow to local sparse, and even under normal conditions it operates metabolic needs must be controlled by other factors. with a large arteriovenous oxygen difference. The Likely candidates for such control mechanisms are: main arteries divide to form two to three capillary beds smooth muscle myogenic responses to blood pressure in most holangiotic mammalian retinas. Adrenergic and intraocular pressure changes, release of metabolic signalers from neural and glial tissue, blood-borne fac- tors, autocoids such as nitric oxide and endothelin, From the Lions Eye Institute, University of Western Australia, Nedlands, Australia. which may be released from endothelial cells, and Supported by the Juvenile Diabetes Foundation International, the Medical Research Fund of Western Australia, and the National Health and Medical Research Council feedback communication between the venous and ar- of Australia. terial sides of the circulation. It is possible that consid- Submitted for publication October 19, 1993; revised June 7, 1994; accepted June 10, 1994. erable heterogeneity exists in the vasoactive response Proprietary interest category: N. of different segments of the retinal circulation from Reprint requests: Dr. Dao-Yi Yu, Lions Eye Institute, University of Western Australia, Nedlands, WA 6009, Australia. first order arteriole to capillary and retinal veins. Loca-
Investigative Ophthalmology & Visual Science, November 1994, Vol. 35, No. 12 Copyright © Association for Research in Vision and Ophthalmology 4087
Downloaded from iovs.arvojournals.org on 10/02/2021 4088 Investigative Ophthalmology & Visual Science, November 1994, Vol. 35, No. 12 tion-specific heterogeneity has already been reported MATERIALS AND METHODS in in vitro investigations of the ophthalmociliary ar- Animals tery,3 and the observation of a differential shunting of the red cell moiety of blood between the superficial A total of 60 pig eyes was used in this study. All proce- and deep capillary beds in the retina4 also implies lo- dures adhered to the ARVO Statement for the Use of calized control mechanisms. If such heterogeneity is Animals in Ophthalmic and Vision Research. The eyes a consistent feature of the retinal circulation, then in were obtained either from a local abattoir immediately vitro preparations could provide the means to help after slaughter or from animals under Halothane anes- unravel this differential control. The local control of thesia in our own surgical facility. In both instances retinal arteries has already been explored in in vitro the eyes were enucleated and placed in a sealed bottle preparations of the larger (>200 //m diameter) bo- of oxygenated Krebs solution containing 0.6% dia- vine retinal arteries, using a ring segment technique.5" lyzed albumin (BSA) and kept on ice for transfer to 12 The ring segment technique that we and others the laboratory (10 to 45 minutes). have used has the limitation that candidate vasoactive Histology of the Retinal Artery agents act on both sides of the vessel wall simultane- ously, and that small diameter vessels such as those In a preliminary histologic study, two freshly enucleated found in the human cannot be used. The recent pro- pig eyes were used for structural studies of the retinal liferation of papers demonstrating differential re- artery. The ophthalmic artery was cannulated, and the sponses to intraluminal (IL) and extraluminal (EL) whole eye was perfused initially with Krebs solution for application of vasoactive candidates in vessels from 10 minutes at an input pressure of 50 to 70 mm Hg, other organs such as the brain,1314 ear,!5 tail,16 mesen- followed by 2.5% gluteraldehyde for 30 minutes. After tery,17 and cheek pouch,1819 as well as the carotid ar- making an entry hole into the vitreous at pars plana tery,20 has driven us to question whether retinal vessels ciliaris to allow easy diffusion into the ocular tissues, the display a preference for IL or EL application of candi- whole eyeball was further fixed in 2.5% gluteraldehyde date vasoactive agents. To differentiate between IL- for 24 hours. Pieces of major arteries and adjacent retina and EL-induced responses, it is necessary to perfuse were excised, post fixed in osmium tetroxide, dehy- drated in graded ethanol's, and embedded in epoxy the vessel segment with a physiological solution so that resin for light and electron microscopy. the agents can be added either to the perfusate or to the bathing solution separately. This constant perfu- Preparation of Vessel for In Vitro Study sion technique is arguably closer to the true physiolog- Using a dissecting microscope (Zeiss, Oberkochen, Ger- ical situation than can be achieved with the ring seg- many), the eyes were sectioned at pars plana ciliaris, ment preparation or with vessels that are pressurized 21 separating the anterior segment and adherent vitreous under no flow conditions. Indeed, perfusion may be body from the posterior pole. The retina, choroid, and functionally important to vasoactivity, because sclera were divided into quadrants using a razor blade, changes in flow through vessels have been shown to 22 taking care not to section any major retinal arteries. mediate vessel responses both in vivo and in vitro. The retina was carefully separated from the underlying We have therefore performed pilot studies that choroid and sclera using an iris speculum. A quadrant have allowed us to develop a perfusion-based tech- of retina was placed on a hollowed glass slide containing nique for retinal vessels based on the work of Chonko Krebs solution plus 0.6% dialyzed albumin (BSA) kept et al23 in the kidney, and Duling and Rivers24 for mes- at less than 4°C. Using a combination of transmitted enteric vessels. It also owes some of its features to the light with a stereo microscope (Wild, M3Z, Heerbrugg, microperfusion system used by Murta et al25 for blood Switzerland), individual first-order retinal arteries (out- retinal barrier studies in rabbit retinal vessels. Al- side diameter 60 to 120 //m) were dissected from retinal though this study relates exclusively to retinal vessels tissue with a fire-polished micropipette. A segment of from pigs, the same system has proved suitable for artery approximately 800 to 1500 //m long was selected; retinal vessels from human, cat, and dog. We initially in most experiments, care was taken to include a side concentrated on optimizing and validating the perfu- branch of outside diameter 15 to 30 fim, although in a sion technique itself and then investigated the differ- few cases, a segment with no side branch was chosen. ential sensitivity of in vitro pig retinal arteries to IL and This arterial segment was then transferred to the incuba- EL application of catecholamines and K+. Histology of tor chamber. these vessels has been performed in parallel so that the structure of the vessel wall may be related to vascular Incubator Chamber responses. We regard this as a necessary first step in The incubator chamber (PDMI-2, Medical Systems, the process of determining controlling factors for the New York, NY) was mounted on the stage of an in- retinal circulation. verted microscope (Nikon Diaphot-TMD, Tokyo, Ja-
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FIGURE l. Schematic of the configuration used in the pilot study of vessels with no side branches. The heights of the reservoirs HL and HR produce pressures PL and PR in the left- hand and right-hand pipettes.
pan). The chamber contained 5 ml Krebs solution and, secondly, a vessel that is perfused but has a similar maintained at 37°C using a bipolar temperature con- intraluminal pressure shows little myogenic activity and troller (Model TC-202, Medical Systems). The incubat- has a larger resting diameter. This flow dependency at ing solution was equilibrated with 95% O2, 5% CO2 low flow rates makes it particularly important that the gas flowing over the surface of the chamber to main- perfusate flow rate should be a known and controlled tain PO2, PCO2, and pH of the incubating solution. parameter. We also reasoned that continuous perfusion This was verified by occasional aspiration of samples is a more physiological condition, and consequently we for blood gas analysis (Ciba-Corning, Essex, UK). 110 Pilot Study Initially, a modified form of the perfusion system of Duling and Rivers23 was set up as shown schematically 100 in Figure 1. Pieces of vessel without any branches were selected, each end of the arterial segment was sealed into the specially made cannulae (described later), 90 and hydrostatic pressure heads HR and HL were con- nected to the two ends, creating pipette pressures PR 80 and PL, respectively. Two configurations were used. 70 PR>PL, in which case perfusate flowed from right to left with an unknown flow rate. Alternatively, the con- nection to the left-hand reservoir was closed, in which 60 case the vessel was pressurized at a value equal to PR with no flow during equilibrium conditions. The effect on diameter of these two procedures for one vessel is 40 shown in Figure 2, where both outside diameter in microns and luminal pressure are plotted as a function 30 of time. Initially, a flow condition was used with 20 PR>PL, and the vessel diameter was 104 fj,m. Then, Flow No Flow when the vessel was pressurized at the same value (33 mm Hg), but with no flow, the diameter of the vessel FIGURE 2. Demonstration of flow sensitivity and a myogenic dropped to 95 fim. Furthermore, when the HR pres- sure head was increased to 65 mm Hg, a clear myo- response of a sealed vessel with no side branch. Arterial genic contraction was seen, with a further decrease in diameter (OD fxm, upper panel), and luminal pressure PL (PP mm Hg, lower panel), are shown. Initially, there was an diameter to 82 jum. On lowering the pressure once unknown flow due to the height difference between the more, the vessel dilated back to 95 /im. two reservoirs. When the vessel was pressurized to the same These data demonstrate two important points that extent but with no flow (stopcock (S) closed, HR adjusted) were verified on many occasions. Firstly, a pressurized the diameter was reduced. Raising of the pressure head HR vessel with no flowexhibit s a clear myogenic contraction, then produced a myogenic constriction.
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FIGURE 3. Schematic of the cannulation and perfusion system for the isolated retinal arteries. Both ends of the vessel were cannulated and sealed by gentle squeezing the vessel wall between the inner and outer pipettes. Syringe pumps delivered perfusate at a controlled rate. Pressure transducers PI, P2, P3, and P4 measured the resulting pressures at the points indicated. Typically, the right-hand pipette system was used for the main perfusate (5 jj,\/ min) and drug delivery, whereas the left-hand pipette was used to measure the intravascular pressure. The only perfusate outflow path (arrow) is through the side branch of the main vessel. Test agents were introduced into the perfusate stream through a sample injector valve, VI. The vessel was maintained in an incubation bath on the stage of an inverted microscope (M), and video images were output to a monitor and automated vessel diameter measuring system. All relevant parameters were recorded on a chart recorder, and data were streamed directly to computer disk when required.
devised a perfusion system in which the flow could be B and to separate it from the inner wall of A. Pipette controlled and the pressures and the vessel diameter D delivered the perfusate and IL drugs directly at the continuously monitored. Because this is a new tech- shoulder of the perfusing pipette B, close to the vessel nique, we describe it in some detail. to minimize the delay between injection and arrival of the drug in the vessel. Pipette C also delivered per- Cannula and Perfusion System fusate at a rate one fifth of that in D. This ensured that there was no back flow of drugs into the main General. The principle used in these experiments body of B that could later serve as a pocket of contami- was that vessels with a side branch were cannulated at nation. The resistance of pipette B was minimized and both ends and perfused through one end (proximal) offered less resistance to perfusate flow than that pre- with a flow of 5 fxl/min in the orthograde direction, sented by a typical second-order branch. with the side branch acting as the exit route, as shown in Figure 3. This choice of 5 fi\/min as a baseline Perfusion and Mounting System, Perfusate flow was perfusion flow rate was based on in vivo measurements delivered by a computer-controlled syringe pump using laser Doppler velocimetry.26 The other end (dis- (Model 22, Harvard Apparatus, South Natick, MA) con- tal) was generally left with a small flow rate (0.3 yul/ taining one 1-ml and one 5-ml gas-tight syringe (Activon; min). This residual flow helped ensure that drugs de- Pennant Hills, New South Wales, Australia) connected livered from the other pipette did not collect in the to die C and D pipettes, respectively. A specially manu- distal end of the vessel. The vessel diameter to the factured pipette holding system was developed that al- right-hand side of the branch was monitored continu- lowed relative movement between pipettes A and B while ously. The diameter changes in response to intralumi- maintaining a pressure seal and minimal compliance. nal or extraluminal delivery of drugs were compared. The whole assembly was mounted on a joystick-con- trolled KYZ micro drive (Fine Science Tools, Foster City, Cannula. Each cannula consisted of four concen- CA) and angled at 35° to the horizontal. An equivalent tric pipettes (Fig. 3). The outer or holding pipette A system of pipettes and manipulators was used for the was shaped on its innermost surface to present two left-hand side and perfused by a second, independently constrictions. The constriction closer to the open end controlled pump. of the pipette served as a surface against which the inner pipette B, the pipette that entered the vessel Vessel Cannulation lumen, squeezed and sealed the vessel wall. The inner- The vessel was positioned horizontally in the incuba- most constriction served to centralize the tip of pipette tion bath close to the bottom of the dish. By conven-
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tion, the proximal end of the vessel was mounted on Intraluminal and Extraluminal Drug Delivery the right-hand pipette system. The XYZ drive was ma- Drugs to be delivered intraluminally were adminis- nipulated to place the tip of pipette A adjacent to tered as a 5-//1 bolus into the perfusate stream through the proximal end of the vessel. Relative movement the sample injector valves. The intrinsic design of this between pipettes A and B was achieved using a hydrau- injector system permitted inclusion of the drug bolus lic micro drive. Pipette B was retracted into the can- without any pressure artifact or problems with air bub- nula and gentle suction was applied to pipette A to bles. A built-in switch allowed a signal to be generated draw the end of the vessel into the cavity between the to indicate loading and injection phases of the proce- two constrictions. Pipette B was then advanced into dure, and this signal was recorded by the chart re- the vessel lumen such that it squeezed the vessel wall corder and the computer. At a typical perfusion rate against the constriction. This procedure was per- of 5 //1/min, the drug arrived at the vessel approxi- formed while a continuous perfusion of 5 //1/min was mately 90 seconds after injection. This was determined flowing. It was found that this helped keep the vessel by visual observation of dye boli. Spreading of the lumen open. Once this end of the vessel was sealed, original bolus in transit results in a slight dilution of the perfusate flow flushed out the red blood cells from the drug, but this is small compared to the log unit the lumen of the vessel, and a similar procedure was increments in concentration. The size, and hence the repeated on the other end. Once both ends were can- duration of the bolus, was sufficient for the vasoactive nulated, perfusion delivery at the distal end was re- response to stabilize. Extraluminal drug delivery was duced to a residual level, typically 0.3 //1/min. The achieved by direct pipetting into the incubating solu- vessel was left to stabilize for 30 minutes. tion to achieve the required concentration. The bath- ing solution was flushed frequently. Experiment Control and Diameter and Pressure Measurement Blood Retinal Barrier All of the data recording and much of the instrument control was under computer control. The software was The retinal circulation normally has a tight blood reti- developed using the graphical programming language nal barrier. Sodium fluorescein (0.05%) was injected LabView (National Instruments, Austin, TX) and run intraluminally in the manner described above. Using on a 486DX PC with the appropriate serial and IEEE the epifluorescence attachment (TMD-EF, Nikon), communications cards added. The inverted micro- the sodium fluorescein could be seen arriving at the scope image of the vessel was captured with a CCD vessel, and we looked carefully for leakage through camera (MW-BL602, Panasonic, Tokyo, Japan), was the vessel wall and for the presence of fluorescein displayed on a color video monitor (Trinitron; Sony, within or between the mural cells of the vessel. Osaka, Japan), and could be recorded using a video- Solutions cassette recorder (VO-9850P, Sony; Tokyo, Japan). Vessel diameter was measured by a second computer Vessels were usually bathed and perfused with normal (386SX) using a commercially produced software Krebs solution with the following composition: NaCl package and frame grabbing card (DIAMTRAK, Din- 119 mM, KC1 4.6 mM, CaCl2 1.5 mM, MgCl2 1.2 mM, dima; Ringwood, Victoria, Australia), which produced NaHCO3 15 mM, NaH2PO4 1.2 mM, Glucose 6 mM. + an analog output proportional to vessel diameter. The High potassium solution (124 mM K ) was produced + + pressures PI, P2, P3, and P4 (Fig. 3) were measured by by equimolar substitution of Na with K . Solutions conventional transducers (Cobe, Arvada, Colorado), were equilibrated with 95% O2, 5% CO2. each connected to a bridge amplifier (5B38-02, Ana- log Devices; Norwood, MA). The monitoring of pres- Agonists sure at two points in each pipette system was found Agonists, (+/-)-noradrenaline HC1 (NA) and (-)- to be a valuable aid in detecting small leaks or partial adrenaline (A) were obtained from Sigma Chemicals blockages in the system. These pressure signals, to- (St. Louis, MO) and dissolved in 0.9% NaCl. Stock gether with vessel diameter, the flow rate of the two solutions of all drugs were stored at — 70°C, and fresh pumps, and injection signals from the sample injector dilutions were made daily. valves (V! and V2, 7725i Rheodyne; Cotati, California) were all recorded on a chart recorder (LR8100, Yoko- Experimental Protocol gawa; Tokyo, Japan). Using IEEE-GPIB communica- After an equilibration time of 30 minutes, three re- tion between the host computer and the chart re- peated IL injections of 124-mM K+ were performed corder, all eight channels were sampled every 2 sec- to test for vessel viability and stability. After successful onds, and the data were reproduced on the computer completion of this stage, frequently, but not always, a screen and streamed directly to a spreadsheet file as 10~8-M Ach dilatation response was checked to ensure required. viability of the endothelial cells. Then IL dose re-
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sponse curves were measured for the appropriate ago- artery in situ in the incubation chamber, showing the nist. For each IL injection, the valve loop was loaded cannulation pipette at the proximal end. The artery with the 5 fi\ of the appropriate concentration. The has an external diameter of 83.3 fim and a side branch 5-/il bolus was switched into the perfusion line, passing of diameter 16.7 //m. A magnified photograph of the through the pipette into the artery. As soon as the same vessel in a different focal plane is shown in Fig- response stabilized, replacement of the incubating so- ure 5B. Note the thin muscle (media) endothelial cell lution was begun to ensure that drug concentrations layer, and the clear outer edge free of neural and glial in the bath remained low. After at least 10 minutes, tissue. With still higher magnification, it is possible to the time required for the vessel to return to its preacti- distinguish the intact endothelial and smooth muscle vated state, the process was repeated with a higher cells by changing the focal plane of the inverted micro- concentration of intraluminal drug. Dose concentra- scope. The average diameter of arteries used in this tion was usually increased in log units from 10~10 to study was 90.4 ± 2.7 /im (n = 42). 10~3 M. Before and after each type of agonist, the valve was flushed and loaded with Krebs solution, and Effects of Perfusion Pressure and Flow on the a control IL injection was performed. A positive con- Vessel trol response meant that further flushing of the valve The effect of increasing and then decreasing flow and was required. Then, cumulative EL dose response hence intravascular pressure over a wide range is dem- curves were measured by adding a stepwise increasing onstrated in the left and right panels of Figure 6, dose to the incubating solution, whereas the vessel was respectively, for a vessel with a small-diameter side perfused IL by Krebs solution. On some occasions, as branch, so that lumen pressures are relatively high. In a direct confirmation of IL and EL agonist sensitivities, the bottom panel are the stepwise increases in flow the agonist was first applied EL and left in the bath, from 1 //1/min to 10 /xl/min. The two traces in the and the same dose was perfused intraluminally; com- middle panel are the pressure values P2 (upper) and parisons of diameter changes on the proximal and P4 (lower). P4 represents the IL pressure at the distal distal sides of the branch vessel were made (see later). end of the vessel. P4 increases stepwise with flow. Ves- Any vessel in which the diameter did not return to sel outside diameter (OD, top panel) increases with within 10% of baseline after exposure to an agonist pressure and flow from 97 fim to 112 fim. With step- was rejected. wise reduction in flow, diameter and pressure return to similar values. The diameter of the main vessel is not greatly influenced by changes in intraluminal RESULTS pressure or flow over what one might expect to be the normal physiological range. Histology and Visualization of Perfused Vessel However, luminal flow and pressure were im- Figure 4A is a light micrograph of a pig retinal section portant parameters in determining the magnitude of containing a retinal artery. Perfusion fixation was per- drug responses. Figure 7 shows outside diameter formed at close to in vivo pressure. The retinal artery changes in response to repeated injections of 10~5 M (4 to 5 mm peripheral to the disk edge) lies close to noradrenaline (arrow) IL (upper panel) as luminal the internal limiting membrane and protrudes into pressure/flow was increased (lower panel). The vessel the vitreous body; endothelial cells and smooth mus- constricts from 108 fim to 95 fim at a pressure of 35 cle cells are clearly visible. Figure 4B is an electron mm Hg (A), with an accompanying small increase in micrograph of a small section of the same arterial wall. pressure caused by an increase in branch resistance. The endothelial cell (E) is supported by a peripherally For increasing values of pressure and flow, the nor- located basement membrane (*). External to this adrenaline response decreases (B) and is then lost basement membrane is one smooth muscle cell of the altogether between 65 (C) and 110 (D) mm Hg. Re- media (SM) and its nucleus. Also present is the muscle turning to lower values of pressure and flow allows cell process (P) of another smooth muscle cell. Other the vessel to constrict once more in response to nor- similar sections showed that the arterial media consists adrenaline (E). If the luminal pressure was left high of no more than two cell bodies and a few cell pro- for extended periods, vasoactivity did not recover on cesses at any location. Each smooth muscle cell is sur- . return to normal pressures. rounded not only by its own plasma membrane but also by a basement membrane that often connects Drug Responses with the endothelial basement membrane of similar A raw dose response sequence for IL and EL delivery structure and width. External to the smooth muscle of noradrenaline is presented in Figure 8 for a vessel cells are loosely arranged collagen fibers that make of external resting diameter 105 fim. The top panel up the meager adventitia (A). shows the diameter response for IL delivery. There Figure 5A is a photograph of a perfused retinal is a control injection causing no change in arterial
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imwmmww FIGURE 4. (A) Light micrograph of a pig retinal section showing a superficial retinal artery lying in the nerve fiber layer and protruding into the vitreous body (stained with toiuidine blue, X420). (B) Electron micrograph of a section through the wall of the same artery, showing an endothelial cell (E), a smooth muscle cell (M), and processes of another smooth muscle cell (P), and the lumen (L). Basement membrane (*) separates endothelial cells and smooth muscle cells. Note the insubstantial adventitia (A) (original magnification, XI 8,000).
diameter, followed by increasing noradrenaline doses Plateau values were 17.8% ± 1.9% IL and 6.8% ± of 10~8 to 1CT3 M producing an increasingly large 1.1% EL. contraction. This was then followed by a control injec- The possibility that this differential response was tion. At 10~3 M, the contraction was 22% of resting caused by the cumulative nature of the EL drug deliv- external diameter. Recovery times were considerably ery compared with the transient nature of the IL deliv- longer for the higher concentrations, and it was neces- ery was ruled out as follows. A single EL dose of 10~5 sary to ensure a prompt bath washout of any accumu- M noradrenaline was administered, and the response lated drug in the bathing solution to guarantee a re- was allowed to plateau. At this stage, the same concen- turn to baseline. For cumulative EL delivery (lower tration of IL noradrenaline was administered. The IL panel), it is clear that all contractions are smaller, with dose always caused a further contraction in addition the contraction at 10~3 M only 9.4% of the original to the sustained EL induced contraction. A second external diameter. Results from several such experi- possible explanation for the smaller EL responses is ments are averaged and plotted in Figure 9, which that the flowing perfusate continuously flushed away shows the mean ± SEM, n = 7 to 13, dose response the EL delivered drug so that its effective concentra- curves for IL (open circles) and EL (filled circles) tion at the smooth muscle cell was reduced. To investi- noradrenaline. They have similar thresholds between gate this possibility, the vessel diameter responses were 10"9 and 1(T8 M and rise to saturate at 10~4 M, but measured on both the distal and proximal sides of the the IL response is significantly larger (Student's Hest, branch point after the drug was added to the bath P < 0.05) at concentrations of 10~6 M and greater. (EL) with no flow in the distal end so there could be
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(A) (B) (C) (D) (E)
FIGURE 7. Contraction responses (OD p, upf>er panel) to single intraluminal doses of 10~5 M noradrenaline {arrow), as luminal pressure (PP mm Hg, lower panel) is increased and then returned to control value. PP = Perfusion pres- sure.
B conclude that any removal of the EL-delivered drug by the IL perfusate flow does not account for the dif- FIGURE 5. (A) Photograph of a perfused retinal artery taken through the inverted microscope. Note the cannulation pi- ference in response between IL and EL applied ago- pette and the side branch (15 fim in diameter) through nists. which the perfusate exits (X120). (B) High-power photo- Adrenaline shows an IL-EL response pattern sim- graph of the same artery to show the mural cells in focus ilar to that observed for noradrenaline. Raw data sets (see Results) (original magnification, X480). for IL (top panel) and EL (bottom panel) adrenaline delivery for a 92-//m vessel are shown in Figure 10. The contraction at 10~3-M IL was 26% of the original no "flushing" effect in this region (see Fig. 3). In this diameter, whereas the contraction with 10~3-M EL de- situation, both segments contracted a similar amount, livery was only 8%. The averaged dose response curves with the proximal end (with flow) contracting slightly (n = 7 to 13) shown in Figure 11 tell the same story, less than the distal end (no flow). These contractions with IL delivery producing significantly larger re- were much smaller than those produced by subse- sponses than EL delivery (P < 0.05) at concentrations quent IL application in the proximal end. Thus, we of 10~5 to 10~3 M. For adrenaline, the IL responses
t 100 -3 ioo 8
50 50 2S0 250
200 200 f 150 ^ 150 100 •* 100 i L 50 L 50 0 0 12 12 a I 8
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0 0
FIGURE 6. Graph of perfusion flow (bottom panel), P2 mm Hg {upper trace), and P4 mm Hg {lower trace) in the middle panel, and outside arterial diameter (OD /j.m, top panel) as a function of time in minutes.
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Control -4 Control 110 -8 -7 -6 -5 ^ too "V "\ r § 90 V (A) 5 min V V BO
-10 -9 -6 5 -4 -3 110 r » I I I 1 100
a O 90 (B) 5 min
B0
FIGURE 8. Intraluminal dose responses to increasing doses of noradrenaline from 10 H M to 10~* M (upper panel) compared with extraluminal cumulative dose responses from 10~'" M to 10 ^ M (loxverpanel).
have a lower threshold (10 8 M) than do the EL re- caused only 5.0% ± 1.0% (n = 13). These contraction sponses (10~7 M). The maximum percentage constric- percentages are significandy different (P < 0.001). tions, 19.0% ± 2.1% 1L and 8.4% ± 1.5% EL with adrenaline, are not significantly different (P > 0.05) Blood Retinal Barrier from those for noradrenaline. + Using IL sodium fluorescein with fluorescent micros- K -induced contractions also caused differential copy, visualization of the perfusion of a fluorescent responses for IL and EL application. Intraluminal in- + marker through the vessel lumen was observed with- jection of 124-mM K caused an average contraction out leakage from either cannulation point. This indi- of 19.0% ± 1.6% (n= 21) of the vessel's resting diame- cated a tight cannulation widi all perfusate and drugs ter, whereas EL application of the same concentration passing through the vessel lumen. Furthermore, move- ment of the marker across the vessel wall or between
25 r endothelial cells to smooth muscle cells was never ob- served. Therefore, an intact blood retinal barrier to sodium fluorescein can be assumed to exist in tfiis retinal artery preparation. c o "u o DISCUSSION o o This technique of continuous IL perfusion confers several important advantages over the more com- o 3 27 30 -+-J monly used ring segment preparation that we ' " c and others5"x>i have previously used to investigate the U l_ vasoactivity of ocular arteries. A perfused artery corre- 0) a. 5 - sponds more closely to the in vivo situation, allows differentiation of IL and EL responses, may cause less endothelial cell damage, especially for small vessel of outside diameter less than 100 //m, and is more readily -10 -9 -8 -7 -6 -5 -4 -3 applied to smaller vessels such as the human or por- Log ([M]) cine retinal arteries and arterioles. Moreover, the use of a controlled perfusion technique means that the FIGURE 9. Average percentage contraction for increasing log flow rate is known, and the intraluminal pressure and ([M]) concentrations of noradrenaline for intraluminal vessel diameter can be measured. Drug-induced (open circles) and extraluminal (filled circles) application, n = changes in this pressure provide information about 7 to 13. the combined vascular resistance of the main vessel
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and its single side branch, through which the perfu- those with larger side branches. The vessels with sate exits. higher pressures did not respond to noradrenaline, Interestingly, our comparison of pressurized ves- whereas those with larger side branches and lower sels with no flow and pressurized vessels with flow un- intravascular pressures did respond. covered a strong myogenic contraction with increas- In parallel with these quantitative measurements, ing pressure for vessels in which there was no flow. we were able to visualize clearly and observe the However, with flow, but at the same pressure, the ves- smooth muscle and endothelial cells lining the lumen sel dilates. Bevan et al22 and Kuo et al31 also observed through the microscope and video camera, because flow-induced dilatation in isolated vessels. This effect of the thin nature of the vessel walls and the minimal is currently the subject of further investigations in adventitia (Figs. 4, 5). With change of focus, individual which the effects of flow and intraluminal pressure smooth muscle cells and endothelial cells could be can be studied independently. It seems likely that ves- observed. During contraction, the wall thickness was sel diameter reflects the balance of opposing forces seen to increase, leading to a proportionally larger generated by mechanisms that are not well under- decrease in luminal diameter than that monitored by stood. Direct extrapolation of these in vitro findings outside diameter measurements. to the in vivo situation should be resisted because the With experience, it was possible to judge from the balance of competing vasoactive mechanisms and the visual appearance of the vessel the viability of vaso- additional systemic influences are too complicated to active responses. Occasional problems with air emboli predict. in a few experiments not included in these data re- Under our normal experimental conditions vealed dramatic changes in cellular appearance, a pe- where flow was present, the arterial diameter was rela- riod of vasospasm, and visible detachment of endothe- tively unaffected by incremental increases in flow (Fig. lial cells. 6). The size of the side branch diameter determined With this continuous visualization, it became ap- the resultant intraluminal pressure. A small-diameter parent when there was some damage to the prepara- side branch caused high IL pressure at constant flow, tion as the endothelial cells changed appearance, and whereas a large-diameter side branch resulted in low in extreme cases detached from the smooth muscle IL pressure. cells. These changes in appearance were confirmed We conclude that the loss of agonist response at by loss of vasoactivity. Thus, in conclusion, we believe high pressures and flows (Fig. 7) was a pressure effect that, by using this side branch preparation and associ- rather than a flow effect. This conclusion is based on ated monitoring techniques, we will be able to sepa- observations from vessels of similar outside diameter rate vasoactive responses from side branches and from but with different diameter side branches. All vessels the main vessels in future studies. were subjected to the same constant flow, but those Using this technique, we provide the first evidence with small diameter side branches naturally had of differential responses of the retinal artery to IL and higher intraluminal pressure in the main vessel than EL delivery of K+, noradrenaline, and adrenaline in
Control -8 -6 -5 -3 Control 100
90 jmtmmjm
60
70
60
-10 -9 -8 -7 -6 -5 100 r
90
1 80
(B) 5 mln
FIGURE 10. Intraluminal dose responses to increasing doses of adrenaline from 10 K M to 10 ^ M (upperpanel) compared with extraluminal cumulative dose response curve from 10"' M to 10 * M (buter panel).
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25 r Intraluminal responses have also been shown to be larger than EL in the tail artery16 and the mesentery,17 where again the asymmetry was shown not to depend on the endothelial cells. However, in the cheek c o pouch,1819 EL responses exceeded IL, and this differ- -u po ence was enhanced further down the vascular bed. In this case, the difference was ascribed to the solubility "c o of the tested molecule in water, because lipid-soluble o molecules gave similar responses to EL and IL applica- 0) 20 oen tion. Finally, in the carotid artery, there was again a •*->c difference depending on which molecules were tested,
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ther low or high pressures, leading the authors to 6. Benedito S, Prieto D, Nielsen PJ, Nyborg NCB. Role of conclude that vasomotion is a normal property of cere- the endothelium in acetylcholine-induced relaxation bral arteries/4 and spontaneous tone of bovine isolated retinal small In conclusion, we have developed a sophisticated arteries. Exp Eye Res. 1991;52:575-579. and technically demanding technique that has dem- 7. Nielsen PJ, Nyborg NCB. Contractile and relaxing ef- fects of arachidonic acid derivatives on isolated bovine onstrated that heterogeneity is an important factor retinal resistance arteries. Exp Eye Res. 1990; 50:305- in the retinal circulation between intraluminal and 311. extraluminal responses. It remains to be seen whether 8. Nielsen PJ, Nyborg NCB. Calcium antagonist-induced this polarity of response is general for all drugs or relaxation of the prostaglandin-F2a response of iso- whether some are more active when applied extralu- lated calf retinal resistance arteries. Exp Eye Res. minally. One might predict that vasoactive agents re- 1989;48:329-335. leased as metabolic signalers between tissue and the 9. Nielsen PJ, Nyborg NCB. Adrenergic responses in iso- retinal vessels will be most effective on the EL side, lated bovine retinal resistance arteries. Int. Ophthalmol. but that remains to be determined. The data pre- 1989;13:103-107. sented here demonstrate that this technique is capable 10. Hoste AM, Boels PJ, Andries LJ, Brutsaert DL, De Laey of providing reliable and reproducible data and that J. Effects of beta-antagonists on contraction of bovine it has considerable potential to enhance our under- retinal microarteries in vitro. Invest Ophthalmol Vis Sri. standing of retinal vascular control. Indeed, it is al- 1990;31:1231-1237. ready clear that the first-order retinal circulation dif- 11. Nyborg NCB, Prieto D, Benedito S, Nielsen PJ. Endo- fers substantially from the ophthalmociliary artery in thelin-1-induced contraction of bovine retinal small its pharmacologic responses, and it is likely that this arteries is reversible and abolished by nitrendipirie. heterogeneity in response continues down the vascu- Invest Ophthalmol Vis Sri.. 1991;32:27-31. 12. Nyborg NCB, Nielsen PJ. Thrombin contracts isolated lar pathway. Thus, for a complete understanding of bovine retinal small arteries in vitro. Invest Ophthalmol the sites and actions of drugs in the retinal circulation Vis Sri. 1990;31:2307-2313. in health and disease, it is essential that all regions of 13. Ngai AC, Winn HR. Effects of adenosine and its ana- the vasculature from conduit artery to capillaries and logues on isolated intracerebral arterioles. Circ Res. veins be studied. Moreover, the perfused retinal artery 1993; 73:448-457. offers an exciting system to study more fundamental 14. Ogawa R, Ohta T, Tsuji M, Mori M. Role of the endo- questions interrelating smooth muscle and endothe- thelium on extraluminal and intraluminal vasoactive lial cell function for vascular physiologists, because it mechanisms in the perfused rabbit basilar artery. Neu- is a vessel with a simple wall structure, only 1 to 2 rolRes. 1993; 15:154-159. layers of smooth muscle cells, an endothelial cell layer, 15. HeadrickJP, Northington FJ, Hynes MR, Matherne and no confounding autonomic nerve endings. GP, Berne RM. Relative responses to luminal and ad- ventitial adenosine in perfused arteries. AmJ Physiol. Key Words 1992;263:H1437-H1446. 16. Mazmanian GM, Baudet B, Pannier-Poulain C, et al. vasoactivity, perfused artery, pig, retina, agonists Pressor responses of rat isolated tail arteries to con- tractile stimulation after methylene blue treatment: Acknowledgments Effect of adventitial versus intimal entry. / Vase Res. The authors thank Dean Darcey, Michael Brown, and Peter 1993; 30:250-256. Burrows for their excellent technical assistance. 17. Tesfamariam B, Halpern W. Asymmetry of responses to norepinephrine in perfused resistance arteries. Eur References J Pharmacol. 1988; 152:167-170. 1. Laties AM. Central retinal artery innervation: Absence 18. Lew MJ, Duling BR. Access of blood-borne vasocon- of adrenergic innervation to the intraocular branches. strictors to the arteriolar smooth muscle. / Vase Res. Arch Ophthalmol 1967; 77:405-409. 1992;29:341-346. 2. Ye X, Laties AM, Stone RA. Peptidergic innervation 19. Matsuki T, Hynes MR, Duling BR. Comparison of con- of the retinal vasculature and optic nerve head. Invest duit vessel and resistance vessel reactivity: Influence Ophthlamol Vis Sri. 1990;31:l731-l737. of intimal permeability. AmJPhysiol. 1993;264:H1251- 3. Yu D-Y, Su E-N, Alder VA, Cringle SJ, Mele EM. H1258. Pharmacological and mechanical heterogeneity of cat 20. Kaul S, Waak BJ, Heistad DD. Asymmetry of vascular isolated ophthalmociliary artery. Exp Eye Res. responses of perfused rabbit carotid artery to intralu- 1992; 54:347-359. minal and abluminal vasoactive stimuli. / Physiol. 4. Thuranszky K. Der Blutkreislauf der Netshaut. Budapest: 1992;458:223-234. Ungarischen Akademie der Wissenshaft; 1957. 21. Jackson WF, Duling BR. The oxygen sensitivity of ham- 5. Hoste AM, Andries LJ. Contractile responses of iso- ster cheek pouch arterioles. Cire Res. 1983;53:515- lated bovine retinal microarteries to acetycholine. In- 525. vest Ophthalmol Vis Sri. 1991;32:1996-2005. 22. Bevan JA, Joyce EH, Wellman GC. Flow-dependent
Downloaded from iovs.arvojournals.org on 10/02/2021 Vasoactivity of Perfused Pig Retinal Arteries 4099
dilation in a resistance artery still occurs after endo- Morgan WH. Agonist response of human isolated pos- thelial removal, drc Res. 1988;63:980-985. terior ciliary artery. Invest Ophthalmol Vis Sci. 23. Chonko AM, Irish JM III, Welling DJ. Microperfusion 1992;53:48-54. of isolated tubules. In: Martinez-Maldonado M, ed. 29. Alder VA, Su E-N, Yu D-Y, Cringle SJ. Oxygen reac- Methods in Pharmacology: Vol. 4B, Renal Pharmacology. tivity of the feline isolated ophthalmociliary artery. New York: Plenum Press; 1978:221-258. Invest Ophthalmol Vis Sci. 1993; 34:49-57. 24. Duling BR, Rivers RJ. Isolation, cannulation and per- 30. Su E-N, Yu D-Y, Alder VA, Cringle SJ. Effects of fusion of microvessels. In: Baker CH, Nastuk WL, eds. extracellular pH on agonist-induced vascular tone of Microcirculatory Technology. New York: Academic Press; the cat ophthalmociliary artery. Invest Ophthalmol Vis Sci. 1994;35:998-1007. 1986. 31. Bauknight GC, Faraci FM, Heisted DD. Endothelium- 25. Murta JN, Cunha-Vaz JG, Sabo CA, Jones CW, Laski derived relaxing factor modulates noradrenergic con- ME. Microperfusion studies on the permeability of striction of cerebral arterioles in rabbits. Stroke. retinal vessels. Invest Ophthalmol Vis Sci. 1990; 31:471- 1992;23:1522-1526. 480. 32. Forster BA, Ferrari-Dileo G, Anderson DR. Adrenergic 26. Riva CE, Grunwald JE, Sinclair SH, Petrig BL. Blood alphal and alpha2 binding sites are present in bovine velocity and volumetric flow rate in human retinal retina and retinal blood vessels. Invest Ophthalmol Vis vessels. Invest Ophthalmol Vis Sci. 1985;26:1124-1132. Sci. 1987; 28:1741-1746. 27. Yu D-Y, Alder VA, Cringle SJ. In vitro characterization 33. Fujii K, Heisted DD, Faraci FM. Vasomotion of basilar of the mechanical properties of canine ophthalmic arteries in vivo. AmJPhysiol. 1990;258:H1829-H1834. artery. Exp Eye Res. 1990;51:729-734. 34. Kuo L, Davis MJ, Chilian WM. Am J Physiol. 28. Yu D-Y, Alder VA, Su E-N, Mele EM, Cringle SJ, 1990;259:H1036-H1070.
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