Mechanisms of Ageing and Development, 65 (1992) 257-276

257

Elsevier Scientific Publishers Ireland Ltd.

AGE RELATED ALTERATIONS IN THE RESPONSE OF THE PIAL ARTERIOLES TO ADENOSINE IN THE RAT

HONG-XIN JIANG a, PETER C.-Y. CHEN a, SIDNEY S. SOBIN a'c and STEVEN L. GIANNOTTA b aThe American Heart Association Greater Los Angeles Affiliate, University of Southern California Cardiovascular Research Laboratory, Department of Physiology and Biophysics and bthe Department of Neurosurgery, University of Southern California School of Medicine, Los Angeles, California and Cthe Department of Bioengineering, University of California, San Diego, La Jolla, California (USA) (Received March 25th, 1992)

SUMMARY

To evaluate the effects of aging on vasoreactivity of pial arterioles to adenosine and barium chloride, an hydraulically intact cranial window preparation was developed in the rat. The microvasculature of anesthetized 3- and 24-month-old Fischer-344 rats was studied during superfusion with artificial cerebrospinal fluid with and without test agents and results determined by videomicroscopy techniques. In both cohorts, the response of pial arterioles to adenosine was both dose and vessel size dependent: arteriolar dilation increased with increasing concentrations of adenosine and at any given concentration the percent increase in diameter was greater in the smaller vessels. During adenosine superfusion the absolute changes and percent increase in vessel caliber were greater in the young rats. Arteriolar vasoconstriction due to barium chloride was vessel size dependent but there were no significant differences in response between young and aged rats. The results indicated an attenuated cerebrovascular response in aged rats to adenosine, but not to barium chloride. This may be due to a difference in the mode of action in these two compounds. Venules did not respond to adenosine at any concentration.

Key words: Adenosine; Barium chloride; Pial vessel; Closed cranial window; Rat; Aging; Cerebral microvasculature

Correspondence to: Sidney S. Sobin, Department of Physiology and Biophysics, University of Southern California School of Medicine, 2025 Zonal Avenue, Los Angeles, CA 90033, USA. 0047-6374/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

258 INTRODUCTION Of all the tissues of the body, the brain most rigorously requires continuously oxygenated blood for normal function and even survival. Blood flow to the brain can be physiologically expressed as cerebral blood flow (CBF), a measurable quantity. CBF normally changes little over a wide range of arterial blood pressures [1,2], yet changes quickly and appropriately in response to a change in brain metabolism [3,4] and in oxygen supply [5,6]: with an increase in brain metabolism and a reduction in arterial oxygen tension, CBF promptly increases. Tissue blood flow is usually under dual control by [1] the peripheral nervous system (neurotransmitters) and [2] by locally produced chemical substances (metabolites) that originate in the surrounding parenchymal tissues. In the brain the metabolic mechanism predominates [71. Kontos et al. [8] reported that cerebrovascular resistance changes during hypoxia are controlled entirely by local mechanism(s). Although the mechanism(s) responsible for local metabolic regulation of CBF is unknown and controversial, considerable evidence supports the hypothesis that adenosine plays a major controlling role. Adenosine fulfils many of the criteria of a metabolic regulator of CBF [9-10]: (1) it is a potent dilator of cerebral resistance vessels [1 I-15]; (2) adenosine concentrations measured in brain tissue [16-22] and in cerebral interstitial fluid [23-24], in which cerebral resistance vessels are bathed, appear to be within the vasoactive range; (3) when there is a mismatch between local blood flow and local metabolism in the brain, i.e., a decrease in oxygen supply or increase in oxygen demand, brain cells accelerate adenosine production to compensate for the discrepancy [11,16-23] and (4) the increase in adenosine concentration temporally parallels the change in CBF [18,19,21,22]. Pharmacological studies with dipyridamole [14] and theophylline [6,13] support the concept that adenosine is a local regulator of CBF. Dipyridamole inhibits the cellular uptake of adenosine and should result in an increase in endogenous adenosine concentration; thus, as expected, intracarotid infusion of dipyridamole results in an increase in CBF [14]. Theophylline [6,13], an adenosine receptor blocker and adenosine deaminase [25], which deaminates adenosine to the nonvasoactive inosine, effectively inhibit pial vasodilatation caused by topically applied adenosine and hypoxia. Theophylline also attenuates the increase in CBF induced by hypoxia [6,26]. With both direct and indirect evidence that adenosine plays an important regulatory role in CBF, it is important to know whether the reactivity of the cerebral microvasculature to adenosine is altered in aging. Changes in cerebrovascular reactivity to adenosine with aging may affect the elderly individual's ability to respond to reduced blood oxygen tension under physiological stress as well as hypoxicischemic challenges which may occur during stroke and further increase the risk of hypoxic-ischemic brain damage. To our knowledge there are no direct in situ studies on the cerebrovascular reactivity to adenosine in aging. The present study was

259

undertaken to determine whether a difference in cerebrovascular reactivity to adenosine exists between young and aged rats. The blood vessels to the brain ultimately travel and anastomose freely within the pia-arachnoid membrane, from which they directly enter the surface of the brain [27]. Thus the microvasculature of the pia-arachnoid membrane is viewed as equivalent to the microvasculature of the brain proper and is directly accessible for physiological and pharmacological study. For these studies we have modified a closed cranial window method for the rat [28] and made it facile in installation and operation, to provide a stable physiologicpharmacologic preparation in which we now demonstrate that with respect to adenosine, the arterioles vasodilate in a dose-dependent and size-dependent manner with a reduced response in the old rat. METHODS

Cranial window We were unable to find in the literature an individual cranial window designed for superfusion studies of the rat cerebral (pia-pia-arachnoid) microvasculature and accordingly constructed a separate window and devised a methodology for its installation. Figure 1 is an expanded view of the cranial window in place on the skull. The rectangular frame is 10 x 7 x 1.5 nun, constructed from a single piece of No. 303 stainless steel, perforated at its midlevel by three holes into each of which a 7-mm length of stainless steel tubing, cut from a 20-gauge stainless steel needle stock, is silver-soldered. The diameter of the tubing accepts a length of PE-60 tubing; tube 2 is connected to a pressure transducer (Gould P23d) for continuous measurement of intracranial pressure (Beckman 511 Dynograph); tube 1 is for perfusate inflow; tube 3 is for perfusate outflow and by height of the distal end controls intracranial pressure. The rectangular configuration of the frame is selected because it affords a satisfactory view of a relatively large area of the surface of the brain. The parietal bone of the rat is rectangular in shape. The superior sagittal sinus on the medial sagittal side and the temporal crest on the outer sagittal side limit the width of the window. The window is installed on the skull prior to opening the meninges. The dynamics of fluid flow in this window preparation are important. The inflow tube 1 is arranged at the proximal end of the medial sagittal side, far from the outflow tube, in order to ensure that the drug solution used flows entirely over the exposed cortical surface and is thoroughly rinsed out of the window. The window volume is approximately 0.11 cm 3. In preliminary experiments we demonstrated that an indigo carmine solution (blue) fills the window entirely in 20 s at a superfusion rate of 0.5 ml/min and chamber (intracranial) pressure of 5 mmHg. The solution is virtually rinsed out in the same period of time and completely cleared within 1 min. This provides for a relatively rapid fluid turnover and maintains Po2, Pco2 and temperature of the solution in a narrow range.

260

Fig. 1. Illustration of the cranial window superimposed on an outline of the skull, including landmarks. The entry ports to the chamber are indicated and discussed in the text.

Surgical preparation and installation of the window The skin over the dorsum of the head was shaved and the rat placed in a prone position. The head was secured in a head holder• A strip of skin in the midline from the interparietal bone to the frontal bone was removed to provide a convenient working area and the external periosteum flushed with saline. Bleeding from soft tissues and the surface of the skull was controlled by bipolar cautery (Solid-State Electrosurgery, model Surgistat); bleeding from the cut edges of the skull was controlled by drilling. An electric drill (Dremel Moto-tool 270-5) with Revelation dental burrs 577 (S.S. White Dental Mfg. Co.) was used to cut a rectangular bone flap (4 m m x 6 mm) on the right parietal bone, previously marked with a felt pen. Care was taken to assure even deepening of the four border grooves. Heat generated by

261

the drilling process was minimized by frequent withdrawal of the drill and by wetting and blotting the bone groove with saline between each cut. Periodic placement of the burr tip on the flap was used as an estimate of the depth of penetration. The bone flap was gently pried open with a microcurette (Fine Science Tools No. 10081-10) and under direct microscopy with fine forceps the remainder of the inner table of the skull was removed or pried upward. Bleeding from the exposed dural surface was controlled with minute pledgets of gelfoam. The clean surface of the skull was wiped dry with small pieces of filter paper and the rectangular stainless steel window was then cemented to the skull with cyanoacrylate over the opening. Next, dental acrylic cement was applied to the outside of the frame at the bony surface. Care was taken at this stage to leave enough space on both sagittal sides to hold dental acrylic (1.5 mm lateral to the temporal crest) and to avoid injury and bleeding from the sagittal sinus (0.5 mm lateral to the sagittal suture).

Exposure of the pia-arachnoid Anatomical considerations. The cerebral meninges are thin in the rat, especially in the very young animal and close to the surface of the brain. Both thick dura and the thin pia have microvessels that may be a source of bleeding during surgical preparation. The avascular arachnoid is in contact with the pia a.t some points making it difficult to remove the arachnoid without damage to the pial vessels. Venous tributaries of the sagittal sinus, large pial veins and the dura are closely adherent some distance from the sinus and removal of the medial portion of the dura often leads to tearing of these pial vessels. There are other variations in structure of the dura, arachnoid and pia. Sometimes the dura and arachnoid form a complex continuous membrane and are removed together. Under an operating microscope, after hemostasis was secured, the dura mater and arachnoid were torn open carefully by means of a dural hook, constructed by bending a finely-tipped micro-needle at a right angle and a RS-5000 micro-dissecting tweezers (Roboz Surgical Instrument), to expose the pial vessels. With the tip tangential to the surface, the dura and arachnoid, in the low median portion of the surgical aperture, are pricked and elevated. A slit is made with the micro-dissecting tweezers. The opening is extended quickly because the brain bulges out as soon as a slit is made and deteriorates with time. The brain presses up tightly against the broken edge of the dura. This creates great difficulty in picking up the edge. In this step a very fine glass rod with a smooth tip is very helpful. It is used to push the dural edge off the brain's surface so the dural edge can be picked up without damage to the brain. Blood from the edges of the dura mater was rinsed away promptly with artificial CSF. If the bleeding continued, it was cauterized with minimum electrical current; the cautery needles were kept sufficiently distant from the brain to avoid injury to the pial vessels. The superfluous dural strip was removed with microscissors. During exposure of the pia mater, the brain surface was kept continually moist with artificial CSF. The CSF was removed just before placement of

262

the cover glass. A no. 1 cover glass was put on the frame and pressed down with a micromanipulator and embedded in dental acrylic. As soon as the dental acrylic solidified, about one minute, the space under the window was filled with artificial CSF. The volume under the window was approximately 0.11 cm 3. The three tubes from the cranial window ports were connected as described above; tube 3 was adjusted in height initially to provide an intracranial pressure of 10 mmHg then lowered to decrease to 5 mmHg pressure where it was maintained for the duration of the experiment.

General methods Sixteen male Fisher-344 rats (Harlan Sprague-Dawley) at 3 months, and 22 rats at 24 months of age were studied. They were anesthetized intraperitoneally (i.p.) with sodium Brevital (Lilly) as follows: the 3-month-old rats, 275-300 g, were given Brevital 90 mg/kg initially and supplemented as required with doses of 30 mg/kg; the 24-month-old rats, 425-475 g, were given Brevital 50 mg/kg initially and supplemented as required with 15 mg/kg. Supplemental sodium Brevital during the experiment was infused intravenously (Sage model 352 syringe pump) with infusion rate individualized for each animal based on depth of anesthesia, blood pressure and arterial blood oxygen. To maintain a clear airway, a tracheotomy was performed and intubated with PE 260 tubing. The right femoral artery was cannulated with PE 50 tubing for arterial blood pressure, blood gas (Po2, Pco2), pH (Radiometer ABL 30 analyzer) and hematocrit (micromethod) determination. The right femoral vein was cannulated for administration of supplemental anesthetic agent. The femoral artery and vein catheters were filled with lactated Ringer's solution in 5% dextrose to prevent blood clotting at the tips when not used for recording, sampling or infusion. Blood gases and other parameters were measured at the beginning, mid-point and at the end of each experiment. To ensure animal temperature stability, rectal temperature was measured with an Astrotemp 4 temperature probe and body temperature maintained at 37°C with an overhead infrared lamp. The PE 60 infusion tubing connecting the syringe and the window was warmed by enclosing it in a larger plastic jacket through which 37°C water circulated. A 3-way connector was used for flushing and removal of air bubbles in the tubing before introduction of fresh solutions into the window. The infusion rate of test experimental solutions was 0.5 ml/min.

Preparation of artificial CSF solutions The artificial CSF had the following composition: Na ÷ 156.50 mequiv, K ÷ 2.95 mequiv/1, Ca +÷ 2.50 mequiv/l, Mg ÷÷ 1.33 mequiv/l, CI- 138.70 mequiv/l, HCO 324.60 mequiv/1, dextrose 665 mg/l and urea 402 mg/l [28] and refrigerated. To maintain proper gas tensions and pH, a small volume of the artificial CSF was first warmed to 37°C for 5 minutes and was then equilibrated in a tonometer (MOD. R-400, MK. 2 tonometer, Marquest Med. Products, Englewood) at 10 p.s.i, for 5 min/ml

263 with a gas mixture containing 10% oxygen, 6% carbon dioxide in nitrogen at 37°C. The solution was withdrawn into a plastic 10-cm3 syringe, mounted on and sampled from an infusion pump (Harvard syringe-pump model 2681). The pH, Po2 and Pco 2 after equilibration were 7.31 ± 0.01, 89.05 4- 5.79 mmHg and 45.51 4- 0.78 mmHg (mean 4- S.D.), respectively. The osmolarity was 308.70 4- 5.42 mOsmol/l (mean 4- S.D.). CSF prepared with adenosine (Sigma grade purity) was treated as above. Barium chloride (Sigma) was prepared as a 0.5% solution in distilled water and sodium chloride added to obtain an average osmolarity of 305.75 4- 4.36 mOsmol/l (mean 4- S.D.); before use the solution was equilibrated with oxygen and carbon dioxide in nitrogen, as above.

Image processing system The videomicroscopy system consisted of an Olympus BH2 microscope, a Cuda 1-150 fiberoptics illuminator, a Dage-MTI NC-67M high resolution newvicon video camera with a sensitivity of 0.016 FC, an Odetics G-77 time code generator with a 1/100 s clock imprint, a Panasonic AG 6300MD VHS video recorder and a SONY PVM-1271Q monitor. The microscope was modified to hold a two-component-table animal board for holding the skull and adjustable positioning of the board on the microscope table. The magnification of the system was adjusted to 120 x at the eyepiece of the camera and calibrated with a Bureau of Standards B&L stage micrometer. The measurement system consisted of an IBM-AT computer equipped with AT&T Targa M8 image processing hardware and a digitizing table. JAVA software (Jandel Scientific, Corte Madera, CA) which provided menu driven options including image and color enhancement, as well as x2, x4 and x8 magnification functions, was used. A frame grabber could freeze any single frame of interest. Vessels of any orientation could be measured with this system, so that several vessels in the field could be measured simultaneously. The width of the red cell column was measured as an index of the blood vessel diameter. Contrast enhancement routines were used to improve recognition of cell column boundary. Single video frames were selected for vessel diameter measurement. Six measurements were made in a predetermined area and averaged to obtain the mean: we previously validated the use of pooling measurements in a single field by measurements of 6 arterioles in two separate fields in 3 rats under conditions of adenosine and barium chloride challenge; variations between the two fields were not significantly different. The audio commentary and time code on the tape provided rapid access to any frames during playback. The maximal change in diameter was measured and expressed as a percent of baseline diameter.

Experimental protocol With the window prepared, we studied the reactivity of pial vessels to adenosine. Before each experiment, adenosine was dissolved in artificial CSF at 10-7, 10-6, 10-5, 10-4, 10-3 molar and maintained at 3°C until used. Immediately prior to

264

TABLE I RESPONSE O F 29.8 -4- 2.7/zM ( R A N G E 25-35/~ M) P I A L A R T E R I O L E S (N = 16) TO IN C R EA S I N G C O N C E N T R A T I O N S OF A D E N O S I N E A N D 0.5% BaCI 2 S U P E R F U S I O N IN 3-MONTHOLD RATS

Diameter (#m) Adenosine 10 -7 Adenosine 10 -6 Adenosine 10 -5 Adenosine 10 -4 Adenosine 10 -3 BaCI 2 0.5%

M M M M

35.9 38.9 42.3 46.0 51.0 6.9

± ± ± ± ± ±

3.1 3.7 3.9 4.4 5.5 4.4

Percent change in diameter (%) 20.3 30.4 42.2 54.6 71.0 -77.1

± 3.5 ± 5.4 ± 9.3 ± 12.2 ± 11.7 ± 14.6

perfusion, the solutions were warmed to 37°C, equilibrated with gas mixture and connected to the infusion system as described above. The pH, P02 and P c o 2 of 10 -3 M adenosine solutions after equilibration were 7.30 4- 0.01, 87.89 4- 5.63 mmHg and 45.23 4- 0.52 mmHg, respectively; the osmolarity was 307.40 + 4.25 mOsmo/1. Starting at 10 -7 M, the adenosine solutions in increasing concentrations were superfused consecutively through the cranial window at 0.5 ml/min. With intracranial pressure maintained at 5 mmHg, each concentration was superfused for 5 minutes. Preliminary studies showed that a peak response was achieved during this interval. At the end of each concentration run the window was superfused with artificial CSF for 20 rain; when vessels did not return to control diameter after superfusion with control CSF, the study was terminated. After observations with adenosine were finished, normal cerebrovascular reactivity was tested by superfusion of BaCI2. It required 20 s to achieve maximum constriction with the barium chloride. Only arterioles that showed at least a 30% constriction to BaC12 were included in the data. Observation of the preparation through the cranial window was carried out on a TV monitor. Blood vessel diameters were recorded by videomicroscopy as TABLE II RESPONSE OF 30.4 ± 2.6 #M ( R A N G E 25-35/~ M) P IA L A R T E R I O L E S (N = 22) TO INCREASI N G C O N C E N T R A T I O N S OF A D E N O S I N E A N D 0.5% BaCI 2 S U P E R F U S I O N IN 24-MONTHOLD RATS

Diameter (l~m) Adenosine 10 -7 Adenosine 10 -6 Adenosine 10 -5 Adenosine 10 -4 Adenosine 10 -3 BaCI 2 0.5%

M M M M M

33.9 36.8 40.1 43.3 46.6 8.6

4aa± ± ±

2.6 2.9 3.8 4.1 4.5 3.6

Percent change in diameter (%) 11.9 21.2 32.1 42.5 53.6 -72.1

± ± a± ± ±

3.2 4.2 6.8 7.5 7.8 10.8

265

described above and subsequently measured. Control diameter measurements were obtained during superfusion with artificial CSF alone. Data are presented as mean 4- S.D. Statistical significance was determined by Student's t-test, both for absolute values and for percent changes in diameter. Significance was accepted at P < 0.05. Correlation coefficients and linear regression were computed for control arteriole diameter and the percent diameter change during adenosine and BaCI2 superfusion.

80-

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60-

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young rats ( n : 1 6 ) old rats ( n : 2 2 )

I

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I

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10 -7

10 - s

10-5

10 - 4

I 10 - 3

Adenosine concentration (M) Fig. 2. Response of pial arterioles 25-35 tan diameter of young and old rats, expressed as percent increase in caliber from control value, to increasing concentrations of adenosine superfusion. Bars indicate S.E.

266

RESULTS

Reactivity of pial arterioles to adenosine in the young (3 months) and old (24 months) adult Fischer-344 rat The pial arterioles responded to adenosine with concentration-dependent vasodilation in both young and aged rats: the diameter of pial arterioles increased with increasing concentrations of adenosine (Tables I and II and Fig. 2). The absolute value and percent increase in caliber was greater in young than in aged rats. The control diameters of the young (29.8 ± 2.7 am) and aged (30.4 ± 2.6 am) rats were not statistically different. These responses to adenosine, expressed as % change in diameter in relation to control diameter, were statistically significantly different except at lowest concentration of 10-7. Except for arterial Po2 values, the arterial pH, PCO2 and Hct determined at the beginning of each test sequence were not different in young and aged rats, Table III: none of these values changed during an experimental run when measured at the start, mid-point and the end of the run. Figures 3-7 illustrate the reactivity of pial arterioles to increased concentrations of adenosine in young and aged rats expressed as % diameter change in relation to the control diameter. The response to adenosine was vessel size dependent both in young and aged rats, with increased response in the smaller arterioles. Correlation coefficients for linear regression were statistically significant for both cohorts. The slope for linear regression increased with increasing adenosine concentration except at 10-3 M in young rats. The slope increased from 0.37 for 10-7 M to 0.89 for 10-4 M in young rats, from 0.48 for 10 -7 M to 1.3 for 10-3 M in aged rats. At all concentrations of adenosine there was a strong indication of significant change in magnitude of response associated with age that is shown in the regression lines. Reactivity of pial arterioles to 0.5°0 BaCI2 (Fig. 8) The arterioles responded to 0.5% BaCI 2 with vasoconstriction. The absolute values and percent decrease in caliber were not different between young and aged rats. Figure 8 illustrates the reactivity of pial arterioles to BaCI2 compared with control diameter. The response was size dependent both in young and aged rats, with

TABLE III A R T E R I A L BLOOD GASES, pH A N D Hct IN Y O U N G A N D OLD RATS

Young rats Aged rats

pH

Pco 2 mmHg

Po 2 mmHg

Hct %

7.389 ± 0.032 7.375 ± 0.042*

44.4 ± 4.6 48.5 + 5.4*

86.1 4. 5.6 73.5 a- 7.5**

44.7 + 4.6 43.5 4- 10.0"

Values are mean ± S.D. (n = 10 for both young and aged rats). *Indicates P > 0.05 between young and aged rats (unpaired t-test, two tailed). **Indicates P < 0.001 between young and aged rats (unpaired t-test, two tailed).

267 a greater decrease in diameter in the smaller arterioles. Linear regression correlation coefficients were significant at the 0.05 level for y o u n g rats, a n d at the 0.01 level for

aged rats. There were n o differences in m a g n i t u d e o f response a n d slope between y o u n g a n d aged rats. The control diameters before BaCI2 application were n o t

10 - 7 M Adenosine 100

-

• young rats & old r a t s 80-

z# 0

0~ 6 0 all to

E o ~

~40-

°

•

20-

I

I

i

20

40

60

Control

diameter

(/~m)

Fig. 3. Relationship between the diameter of pial arterioles of different sizes and percent change in diameter during 10-7 M adenosine superfusion in young and old rats. Each point represents the response of a singlearteriole. There is a significantdifference(P < 0.001) in magnitude of response associated with age, but the difference in slope is not significant (P > 0.6). y = 31.9 - 0.37x young y = 26,1 - 0.48x old.

268

10 - s M Adenosine 100 -

•

young rats old rats

80-

• 60 c-

o @

E co i5 40-

20-

I

I

I

20

40

60

Control d i a m e t e r (/~m) Fig. 4. Relationship between the diameter of pial arterioles of different sizes and percent change in diameter during 10 -6 M adenosine superfusion in y o u n g and old rats. Each point represents the response of a single arteriole. There is a significant difference (P < 0.001) in magnitude of response associated with age but the difference in slope is not significant (P > 0.5). y = 46.4 - 0.53x young y = 43.5 - 0.72x old.

269

different from the original control values at the beginning of the experimental run, prior to the initial superfusion of adenosine. Arterial blood gases and pH in the young and old rat: effect on vessel reactivity to adenosine and BaCI2 Comparison was made of blood gases, pH and hematocrit for the 3-month and 24-month-old Fischer-344 male rat. Only resting (control) arterial P02 in these 10 - 5 M Adenosine 100 -

• young r a t s & old r a t s

80-

eo

6O

im

I

m

e

40

2O

I 20

I 40 Control d i a m e t e r (%)

I 60

Fig. 5. Relationship between the diameter of pial arterioles of different sizes and percent change in diameter during 10 -s M adenosine superfusion in young and old rats. Each point represents the response of a single arteriole. There is a significant difference (P < 0.01) in magnitude of response associated with age but the difference in slope is not significant (P > 0.7). y = 69.9 - 0.87x young y = 63.5 - 1.00x old.

270 Brevital anesthetized rats, tracheal i n t u b a t e d a n d b r e a t h i n g s p o n t a n e o u s l y , was significantly different in these two g r o u p s o f animals: 86.1 4- 5.0 m m H g in the 3m o n t h - a n d 73.5 4- 7.5 m m H g in the 2 4 - m o n t h - o l d . Despite the difference in resting c o n t r o l P02 in the y o u n g a n d aged rats, d u r i n g the a d e n o s i n e a n d BaC12 sequences the arterial gases, p H a n d h e m a t o c r i t did n o t change in either the y o u n g or aged rats. H o w e v e r it was necessary to evaluate the effect o f arterial b l o o d P02 on a d e n o s i n e a n d BaCI2 vasoreactivity: when the responses o f arterioles in the two age groups

10 - 4 M Adenosine 100

-

• young rats A old r a t s

80-

60

==

! N

40

20-

I

I

I

20

40

60

Control diameter

......

(#m)

Fig. 6. Relationship between the diameter of pial arterioles of different sizes and percent change in diameter during 10-4 M adenosine superfusion in young and old rats. Each point represents the response of a single arteriole. There is a significant difference (P < 0.001) in magnitude of response associated with age but the difference in slope is not significant (P > 0.5). y = 83.4 - 0.89x young y = 77.5 - 1.12x old.

271

with significant differences only in P02 were compared, no significant differences in adenosine and BaCI2 responses were found. Response of venules to adenosine Venules did not respond to adenosine, even at the highest concentration (10_ 3 M), regardless of the vessel size studied (29-62 gin). However, venules did respond to BaC12 with constriction: the control diameter before 0.5% BaCI2 superfusion was

10 - 3 M Adenosine

100 •

• young rats & old rats

•

80 A

o e J= ¢.1

60

4.o

Q

E a 40

20

I,

20

I

I

40 60 Control diameter (#m)

Fig. 7. Relatioaship hetwecn the diameter of pial arterioles of different sizes and percent change in diameter during l0 -3 M adenosine supeffusion in young and old rats. Each point represents the response of a single arteriole. There is a significant differences (P < 0.001) in magnitude of response associated with age but the difference in slope is not significant (P > 0.3). y = 95.8 - 0.77x young y = 93.7 - 1.30x old.

272

47.1 4- 2.1 /zm (mean 4- S.E.). During 0.5% BaC12 superfusion the mean diameter decreased to 40.3 4- 2.1 #m (P < 0.001), the percent change in diameter was 15.0 4- 1.4% (mean 4- S.E.). There was no relationship between control diameter of venules and the percent diameter change during 0.5% BaCI2 superfusion (P > 0.05). _ 0.5%

BaCl2

•

-20

-

- 40

-

young rats old r a t s

A

A •

o~

old

~

young

¢1l ta

- 60

-

- 80

-

@

o II.

J - 100

eLI

•

-

I 20 Control

I

1

40

60

diameter

(/~m)

Fig, 8. Relationship between the diameter of pial arterioles of different sizes and percent change in diameter during 0.5% BaC12 superfusion in young and old rats. Each point represents the response of a single arteriole. There is no significant difference in magnitude of response (P > 0,3) and in slope (P > 0.5) associated with age. y = -101.8 + 0.78x young y = -106.7 + 1.08x old.

273 DISCUSSION

This study was designed to assess the effect of aging on the response of pial arterioles to adenosine in the rat. Our recent studies demonstrating a histochemical change in the microvasculature in the aged Fischer-344 rat suggest a structuralfunctional basis for possible change in response to a biochemical stimulus [29,30]. Male Fischer-344 rats were used because this strain is a good model for normal aging. It is available from a colony maintained under barrier husbandry conditions to minimize possible infections. Its lifespan and age-related pathology are welldocumented and aging of the brain is not associated with gross neuropathology due to severe vascular insufficiency or to primary brain disease. Three-month-old rats were chosen for the young adult group because they are sexually mature and have a nearly mature brain. Twenty-four-month-old rats, slightly younger than the average lifespan, were chosen as the aged group. The Fischer-344 rat has a life expectancy of about 29 months [31]. These carefully prepared and unique cranial window studies to determine the response of the pial microvasculature to superfused adenosine clearly demonstrated a disparate response of pial arterioles and venules: while the arteriolar response to adenosine with dilatation was both dose- and vessel-size related, venules did not respond at any dose level. Arteriolar dilation was greater with increasing concentrations of adenosine and for any given concentration the percent increase in diameter was greater for the smaller vessels. This relationship between the adenosine response and initial arteriolar diameter, greater in smaller arterioles, also increases with increasing concentrations of adenosine, indicated by the increasing slope of the regression line; the only exception to the latter statement was that the slope of the regression lines at the two highest concentrations tested in the young rats, 10-3 M and 10-4 M were similar. Our observations relative to dose and vessel size dependent response to locally applied adenosine agree with Berne et al.'s observations using the open skull preparation in cats: 'Vessel diameter increased with increasing concentrations of adenosine, and was greater for the smaller vessels' [11]. However, our results do not correspond to those of Wahl and Kuschinsky [13], Boisvert et al. [32] and Gregory et al. [12] using the open skull preparation in the cat and those of Morii et al. [28] using the closed cranial window in the rat. The latter investigators reported the response was concentration dependent and independent of control vessel diameter. The reasons for this difference in findings are not obvious. The reciprocal relationship between vessel diameter and vascular response increased with increasing concentrations of adenosine has only been previously reported by Berne and co-workers [11]. The major findings of this study relate to the effect of aging on the cerebrovascular responses. Results presented in this paper show an attenuated cerebrovascular response in aged rats to superfused adenosine compared to a young cohort. The absolute increase and percent increase in arteriolar caliber were greater in young than

274

in aged rats. The control diameters, the arterial pH, PCO 2 and Hct determined at the beginning of each test sequence were not statistically different in young and aged rats. Arterial blood pressure was maintained above 100 mmHg in both groups. The arterial blood P02 for old rats is lower than young rats (Table III). The difference in P02 does not affect our conclusions because this variation had no significant effect on vasoreactivity when tested statistically. Furthermore, the possible effect of decreased arterial blood P02 on vessel diameter as well as on the mechanisms of adenosine smooth-muscle relaxation effect may be modified by high Poz of the superfusion CSF although the permeability of these vessels to ambient oxygen is not known. Our results are consistent with Hoffman et al.'s reports of attenuated cerebral blood flow (CBF) increase in aged rats during ischemic hypoxia [10]. Subsequently, Hoffman et al. found that inhibition of adenosine receptors with theophylline significantly attenuated the CBF increase during severe hypoxia and abolished the difference in flow changes between young and aged rats; this suggested that adenosine may be a primary factor mediating CBF increase and age related difference during hypoxia [33]. However, they were unable to determine whether the difference between the CBF response to hypoxia in young and aged rats represents a difference in cerebrovascular reactivity to adenosine or a difference in adenosine production [33]. Our experiments provide direct evidence showing the existence of age related changes in vasoreactivity to adenosine. The present study was designed primarily to determine age related changes in vasoreactivity to adenosine and not to elucidate their mechanisms. The role of adenosine receptors and their secondary effects on other receptor and transmitter systems require careful investigation and are beyond the scope of this investigation. Regardless of the underlying mechanisms, an attenuated cerebrovascular response to adenosine may increase the susceptibility of aged humans to hypoxic ischemic challenges and the risk of hypoxic ischemic brain damage. Understanding the mechanisms could in turn contribute to more specific therapy in the future and aid in its treatment. Our use of barium chloride in these studies was based on the observations of Rosenblum and Zweifach [34] and Rosenblum [35] on the pial microvasculature of the mouse; we utilized it in an attempt to standardize each experiment. Thus, barium chloride was used as a standard to test the retained integrity of the pial arterioles to constrict after repeated adenosine stimulus to vasodilate. To our knowledge it has not been so used previously. The response of arterioles to barium was dose- and vessel-size dependent, with a greater decrease in diameter in the smaller arterioles. This response is similar in both the young and aged rat. It may have been preferable to test the vasculature with barium initially for vasoconstriction at the beginning of each experiment, but since the purpose of this study was to compare the adenosine response in the young and aged animal, we wished to avoid any possible compromise with adenosine effect by prior vasoconstriction. We will explore this in future studies.

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We believe it is necessary to give in considerable detail the technique of installation of the cranial window chamber to provide a reproducible precise procedure for acute study of the pial microvasculature in the rat brain. In addition, with some modification of the window, it appears possible to develop a chronic window preparation for study of the cerebral microvasculature. A recently published closed cranial window preparation by Kawamura et al. [36] described a method to avoid bulging of the brain when opening the dura; with the techniques described in the present communication, this has not been a major problem during surgical preparation. ACKNOWLEDGMENTS

We thank Daniel J. Netto for his aid in the design, construction of the cranial window chamber and setup of the microscope system. W. Jan Checinski assisted in video recording and data analysis. We also wish to thank Hui Qin Li for her technical help in the research. This research was supported by grants from the USPHS, NIH HL42073 and AG-2970. H.-X. Jiang was a Fellow of the World Health Organisation. S.S. Sobin is a Research Career Awardee of the NIH, K506-7064. REFERENCES 1 E.T. MacKenzie, J.K. Farrar, W. Fitch, D.I. Graham, P.C. Gregory and A.M. Harper, Effects of hemorrhagic hypotension on the cerebral circulation. Part 1: cerebral blood flow and pial arteriolar caliber. Stroke, 10 (1979) 711-718. 2 H.A. Kontos, E.P. Wei, R.M. Navari, J.B. Levasseur, W.I. Rosenblum and J.L. Patterson, Responses of cerebral arteries and arterioles to acute hypotension and hypertension. Am, J. Physiol., 234 (1978) H371-H383. 3 W. Kuschinsky and M. Wahl, Local chemical and neurogenic regulation of cerebral vascular resistance. Physiol. Rev., 58 (1978) 656-689. 4 B.S. Meldrum and B. Nilsson, Cerebral blood flow and metabolic rate early and late in prolonged epileptic seizures induced in rats by bicucuiline. Brain, 99 (1976) 523-542. 5 L. Borgstrom, H. Johannsson and B.K. Siesjr, The relationship between arterial Po 2 and cerebral blood flow in hypoxic hypoxia. Acta Physiol. Scand., 93 (1975) 423-432. 6 S. Morri, A.C. Ngai, K.R. Ko and R. Winn, Role of adenosine in regulation of cerebral blood flow: effect of theophylline during normoxia and hypoxia. Am. J. Physiol., 253 (1987) HI65-HI75. 7 R.M. Berne, R.M. Knabb, S.W. Ely and R. Rubio, Adenosine in the local regulation of blood flow: a brief review. Fed. Proc., 42 (1983) 3136-3142. 8 H.A. Kontos, E.P. Wei, A.J. Raper, W.I. Rosenblum, R.M. Navari and J.L. Patterson Jr., Role of tissue hypoxia in local regulation of cerebral microcirculation. Am. J. Physiol., 234 (5) (1978) H582-H591. 9 H.R. Winn, G.R. Rubio and R.M. Berne, The role of adenosine in the regulation of cerebral blood flow. J. Cereb. Blood Flow Metab., I (1981) 239-244. 10 W.E. Hoffman, R.F. Albrecht and D.J. Miletich, The role of adenosine in CBF increases during hypoxia in young vs. aged rats. Stroke, 15 (1984) 124-129. 11 R.M. Berne, R. Rubio and R.R. Curnish, Release of adenosine from ischemic brain. Effects of cerebral vascular resistance and incorporation into cerebral adenine nucleotides. Circ. Res., 35 (1974) 262-271. 12 P.C. Gregory, D.P. Boisvert and A.M. Harper, Adenosine response on pial arteries, influence of CO 2 and blood pressure. Pflugers Arch., 386 (1980) 187-192.

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