Adenosine deaminase reduces hypoxic and hypercapnic dilatation of rat pial arterioles: evidence for mediation by adenosine

Brain Research, 553 (1991) 305-308 © 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 ADONIS 000689939124725E

305

BRES 24725

Adenosine deaminase reduces hypoxic and hypercapnic dilatation of rat pial arterioles: evidence for mediation by adenosine Richard E. Simpson and John W. Phillis Department of Physiology, Wayne State University School of Medicine, Detroit, MI 48201 (U.S.A.) (Accepted 26 March 1991) Key words: Adenosine deaminase; Adenosine; Arteriole; Dilatation; Hypercapnia; Hypoxia

Rat pial arteries were observed through a closed cranial window during hypercapnic and hypoxic episodes whilst the cerebral cortex was superfused at 37 *(2 first with artificial cerebrospinal fluid (CSF) and subsequently with adenosine deaminase (ADA, 0.5-2.0 U/n-d) in CSE The results indicate that ADA attenuated hypercapnic and hypoxic dilatatory arteriolar responses by 64% and 56% respectively. Recovery was obtained by superfusing with ADA-free CSF for 1 h. We conclude that adenosine is involved in hypereapnia- and hypoxia-evoked dilation of pial arteries. Adenosine, a vasodilator, is thought to play a major role in the cerebral vascular response to both hypoxia and possibly hypercapnia 11. Cerebral blood flow (CBF) in the rat responds rapidly to conditions of hypoxia 7'13 and hypercapnia s'12. Large amounts of adenosine are released into rat brain cortical superfusates during hypoxia 14 and Morii et al. s have used various physiologically active levels of exogenous adenosine in cortical superfusates to dilate rat pial arterioles. Furthermore, the adenosine antagonist caffeine decreases hypoxia and hypercapnia evoked increases in CBF 12A3. However, conclusions about rat cerebral vascular reactivity have been largely based upon indirect observations through the measurement of CBF. Our previous observations of rat cerebral hypoxic and hypercapnic dilatatory responses were on CBF measurement, utilizing a venous outflow technique via cannulation of the retroglenoid vein which limited the pharmacological studies to agents which cross the blood-brain barrier 12,13. In the present study we have utilized a closed window technique for the rat, as described by Morii et al. 8, to directly observe the in vivo reactivity of rat pial arterioles to challenges of hypoxia or hypercapnia and the effect of adenosine deaminase, which does not cross the blood-brain barrier, and which converts adenosine to its inactive metaboljte inosine 1°, on rat pial dilatatory responses to these challenges. Male Sprague-Dawley rats (350-450 g) were anesthetized with 2.5-3% halothane. The animals were then tracheostomized and the left femoral artery was cannu-

lated for the purpose of monitoring mean arterial blood pressure (MABP). Body temperature was maintained at 37 °C with a heating pad actuated by a rectal probe. The animal was then placed in a Narashige SH-8 nontraumatic head holder. A longitudinal incision was made along the entire midline of the top of the head in order to expose the skull. The periosteum of the exposed skull was scraped off and the underlying bone was kept clean and dry. A thin circular line of cyanoacrylate adhesive (Dap Inc., Dayton, O H ) was applied to the exposed bone followed immediately by a layer of dental acrylic (Kerr, Romulus, MI). One tube for a temperature probe (PE-50), one tube for efferent artificial cerebral spinal fluid (CSF) (PE-160) and two tubes for afferent CSF (PE-60) were placed in the dental acrylic. A glass slide was then pressed onto the acrylic doughnut before it dried in order to create a flat surface onto which a cover glass would eventually be mounted. The bone inside the acrylic doughnut was then thinned around the edges with a dental drill and removed with fine forceps. Bone bleeding was controlled with bone wax and dural bleeding was controlled with the temporary application of cotton pledgets. The dura-arachnoid complex was incised, cut with microscissors and reflected. Bleeding from the reflected dura mater was controlled with an ophthalmic cautery while the dura was pressed against the bone. The size of the cranial window was approximately 4 mm x 4 mm. A cover glass was mounted to the dental acrylic around the well using cyanoacrylate glue and the result-

Correspondence: J.W. Phillis, Department of Physiology, Wayne State University, School of Medicine, 540 E. Canfield, Detroit, MI 48201, U.S.A.

306 ing cavity was filled with artificial CSF routed through a heat exchanger before it entered one of the afferent tubes. The temperature of the superfusate in the chamber was continuously monitored by the temperature probe and the heat exchanger was adjusted to maintain chamber temperature at 36.5-37.0 °C. Superfusate flow rate was maintained at 2.0 ml/min by monitoring the drop rate from the efferent tube into a waste container. Brain herniation was avoided by maintaining intracranial pressure (ICP) at 3-5 mm Hg through adjustment of the height of the outflow tube. Following the beginning of superfusion the animal was immobilized by the i.v. administration of Pavulon (pancuronium bromide, 1 mg/kg), placed on a mechanical ventilator (Harvard, Model 666) and respired with a gas mixture of 30% oxygen in nitrogen. The respirator was adjusted to maintain blood gases within the normal range. Periodic small arterial blood samples (0.3 ml), were used to measure pH, pCO 2 and pO 2. Anesthesia was maintained with 0.4% halothane. Upon completion of surgery, the animal was given a 2 h rest period to stabilize before blood vessel diameter measurements were begun. Small blood samples were taken prior to beginning and periodically throughout the experiment to ensure that pH and blood gases remained essentially unchanged in the basal condition. MABP was continuously monitored. Superfused sterile artificial CSF had the following composition: Na ÷, 155.8 mEq/l; K ÷, 2.95 mEq/l; Ca 2÷, 2.5 mEq/l; Mg 2÷, 1.8 mEq/l; Cl ÷, 141.13 mEq/1; HCO3-, 22 mEq/l; dextrose, 66.5 mg/dl; and urea, 40.2 mg/dl. This CSF was bubbled with a gas mixture of 5% carbon dioxide (CO2) in 95% nitrogen (N2) for several hours (PO 2 = 29.1 + 2.9 mm Hg) prior to and during superfusion. Artificial CSF was superfused through one afferent tube during the control challenges of 8% oxygen in nitrogen or 10% carbon dioxide and 30% oxygen in nitrogen. The administration of 2-3 control challenges at 10-15 min intervals to each animal confirmed the reproducibility of the dilatatory response for a given vessel. All vessels were allowed to recover to basal condition (10-15 min) before further challenges were administered or before adenosine deaminase was added to the superfused artificial CSE After completion of the control challenges, flow through the first afferent tube was occluded and adenosine deaminase (Sigma Type V) in artificial CSF (ADA, 0.5-2.0 U/ml) was superfused through an identical second afferent tube for 15-30 min prior to the administration of the 10% carbon dioxide (CO2) (1-2 min) and 8% oxygen (02) (1 min) challenges. A D A superfusion alone had no effect on vessel diameter at the concentrations used. After the ADA-influenced challenges were completed, the second afferent tube

(ADA) was occluded and the cranial opening was superfused with ADA-free artificial CSF from the first afferent tube for a minimum of 1 h in order to obtain recovery. Following recovery, the first afferent tube (ADA-free CSF) was again occluded and A D A (2-5 U/ml) that had been inactivated by boiling 19 in CSF was superfused through the second afferent tube for 30 rain prior to the administration of additional challenges of 10% CO2 (1-2 min) and 8% 02 (1 min) so as to confirm the role of ADA in the attenuation of pial arteriolar dilatatory responses. The microscope used was a Zeiss (Jena, Germany) Technival model with an additional x 2 objective lens and trinocular tube for the video camera. The overall magnification of this system was 200x. The microscope field was illuminated by a high intensity lamp with a flexible fiberoptic extension (Cole Parmer Instrument Co., Model No. 9741-00, Chicago, IL) which provided a cool source of light to prevent heating of the cranial window. An image contrast enhancer (University of Tennessee) was used to reduce the illuminating light requirements and enhance black-white contrast on the video monitor (Sony PVM 122). A Newvicon video camera (Dage-MTI Inc., Model No. NC67M, Michigan City, IN) was mounted on the microscope body and video signals were transmitted to and recorded by an 8-track video recorder (Sony model EV-S350). Vessel diameter was measured with an IPM video photometric analyzer (Model 204) and an IPM Image Shearing Monitor, Model 907 (San Diego, CA), with a horizontal image splitter function. Although the IPM monitor permitted continuous monitoring of blood vessel diameter, our data were obtained from recordings of the experiments as the continuous play, stop action and rewind features of the video recorder allowed us to view a single vessel's reactivity several times. Mean values and standard errors of the mean (S.E.M.) were calculated for all data. Differences between the means were determined by the Student's t-test for paired data. Animals which received control challenges of 8% 02 (1 rain) (n = 6 animals, 18 arterioles) or 10% CO 2 (1-2 rain) (n = 8 animals, 26 arterioles) exhibited significant arteriolar diameter increases to 8% 02 of 61% (P < 0.001) and to 10% CO2 of 42% (P < 0.001) over the prechallenge condition. A D A superfusion significantly reduced the increases in mean rat pial arteriolar dilatation in response to 8% 02 by 56% (P < 0.001; a 34% diameter increase) and to 10% CO2 by 64% (P < 0.001; a 27% diameter increase); with the 10% CO2 challenge more effectively attenuated by lower concentrations of ADA than the 8% 02 challenge. Recovery of the dilatatory responses was obtained following 1 h of

307 TABLE I Changes in mean arterial blood pressure (MABP), p H and blood gases I rain before and at the end of a 1-2 rain 10% CO 2 challenge Values are means _+ S.E.M. (n = 8). Control Basal

ADA 10% C02 challenge

Basal

10% CO 2 challenge

pH 7.32_+0.02 7.10_+0.08 7.31_+0.02 7.06_+0.11 pCO 2 (mm Hg) 34.0_+0.6 50.8_+3.0 33.9_+0.7 49.0_+1.0 pO 2(mmHg) 120.0_+4.0 137.2_+5.7 117.2_+3.9 130.8_+5.5 MABP (ram Hg) 113_+3 115_+2 107_+4 113_+3

superfusion with ADA-free artificial CSE Subsequent superfusion with inactivated A D A failed to reduce the dilatatory responses. Prechallenge MABP's were found to be insignificantly different from those of the challenges as were the MABPs during the control and A D A superfusion periods during challenges, pH and blood gases varied insignificantly between the control CSF and A D A containing CSF challenges for both hypoxia and hypercapnia (Tables I and II). The closed cranial window technique used in these experiments offers two major advantages over an open window. Firstly, the pH of artificial CSF in an open window preparation is subject to CO 2 loss and will therefore become alkaline due to gradual diffusion of the CO2 in the CSF into the atmosphere 9. A further disadvantage of the open window preparation is that ICP is uncontrolled, resulting in cortical herniation through the opening in the cranium. This may be especially pronounced during hypoxia and hypercapnia where vascular engorgement of the brain causes herniation in the absence of a physiologic ICE Brain herniation results in the compression of pial vessels at the bone edge and a possible reduction in blood volume flowing through these vessels which may influence experimental observations 8.

TABLE II Changes in mean arterial blood pressure (MABP), p H and blood gases I rain before and at the end of a I rain 8% 02 challenge Values are means + S.E.M. (n = 6). Control Basal pH 7.34+0.03 pCO~(mmHg) 34.5+0.3 pO 2 (ram Hg) 119.1+3.9 MABP (ram Hg) 119_+3.0

ADA 8% 02 challenge

Basal

8% 02 challenge

7.39+0.08 31.8+1.9 29.4+4.9 118_+5

7.32+0.05 7.38+0.03 34.3+0.3 32.3+1.9 117.4+5.5 33.2+6.8 106+3 111_+2

The vessels measured in this study were in the 20-60 #m range. No significant differences were observed in the reactivity of small and large arteries to hypoxic or hypercapnic challenges. This finding is in apparent contrast to the observation s that during hypercapnia vessels of less than 15 gm diameter dilated more than the larger arteries. However, in the present experiments a trend was observed towards a greater challenge-induced percentage dilatation in the smallest over the largest vessels measured and it is possible that a larger arteriolar sample, over a wider range of vessel sizes, would have resulted in the observation of significantly higher arteriolar diameter percent increases in smaller vessels during both hypoxia and hypercapnia. Greater arteriolar dilatatory responses to a CO 2 challenge in small vessels have also been observed in the cat 3A6'2° and it has been suggested that small arterioles, when exposed to adenosine, dilate more than larger ones 1. A similar observation has been made in the heart, where small arterioles were found to be more sensitive to adenosine administration than large ones 18. One explanation for the differences in arteriole reactivity may be related to vessel size. If one assumes that the volume of blood which flows through the many small arterioles is equal to the volume of blood which flows through the fewer large arterioles, it can be argued that the percent diameter difference in the reactivity of small vs large vessels to an 8% 0 2 or 10% CO 2 challenge may in part be due to the fact that blood flow through a vessel is directly proportional to the fourth power of its radius (r4). Therefore, it would be necessary for the many smaller vessels to increase their radius in a geometric manner over the fewer large vessels in order to maintain an equal rate of blood flow. Consequently, smaller vessels would be expected to have greater percent diameter increases over large arterioles. Our results show that A D A superfusion attenuated rat pial arteriolar dilatation to both hypoxic and hypercapnic challenges. A reduction by A D A of hypoxia-evoked dilation of pial vessels has previously been reported 6 and adenosine antagonists attenuate the increases in CBF during b o t h h y p o x i a 7,11,13,15,17,21 and hypercapnia 12. However, other observers have reported conflicting findings. Infusion of the adenosine antagonist theophylline produced no significant effect on hypercapnia induced increases in CBF in rats s'7 and the administration of theophylline to cats, either topically or systemically, failed to attenuate hypoxia-induced increases in CBF 2 or vasodilatation 4. The reasons for these differences are uncertain, but may be due to differences in animal species and the duration and/or severity of the Challenges. The arteriolar dilatatory responses returned to control

308 levels after superfusion with A D A - f r e e C S F and since this recovery was unaffected by subsequent superfusion with inactivated A D A , we conclude that adenosine plays a significant role in the rat pial arteriolar dilatatory response to both hypoxia and hypercapnia. F u r t h e r support of adenosine as a m e d i a t o r of the dilatatory response in hypoxia and hypercapnia was o b t a i n e d when higher concentrations of A D A (5 U/ml) in C S F were superfused into the cranial window. R a t pial arteriolar responses to both the hypercapnic and the

hypoxic challenge were substantially r e d u c e d with increases of only 10-15% over the prechallenge basal diameters. These data were not used in o u r experimental results as it was not possible to obtain recovery after this high dose of A D A . Since hypoxia- or hypercapniae v o k e d dilatation was never totally abolished it is important to acknowledge that o t h e r dilatatory mechanisms, in addition to those m e d i a t e d by adenosine, are also involved. Future research will be necessary to identify these alternate dilatatory mechanisms.

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