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The influence of opioids on local cerebral glucose utilization in the newborn pig
Brain Research, 571 (1992) 97-102
97
Elsevier BRES 17379
The influence of opioids on local cerebral glucose utilization in the newborn pig William M. Armstead, Robert Mirro, Samuel Zuckerman, David W. Busija and Charles W. Leffler Laboratory for Research in Neonatal Physiology, Departments of Physiology~Biophysics, Pediatrics and Obstetrics~Gynecology, University of Tennessee, Memphis, TN 38163 (U.S.A.) (Accepted 10 September 1991)
Key words: Opioid; Newborn; Cerebral circulation; Cerebral metabolic rate
Topical methionine enkephalin, leucine enkephalin, and dynorphin (10-6 M) previously have been observed to produce prominent p i a l arteriolar dilation. Dilation to these opioids could be caused directly by opioids acting on vascular receptors, or indirectly, as a consequence of increased metabolism. Therefore, we examined this possibility by determining the influence of opioids on cerebral glucose utilization in piglets with closed cranial windows using the [14C]deoxyglucose method. Qualitatively, the autoradiographic images expressed as a change in relative optical density from vehicle were unchanged by these opioids. Quantitatively, the opioids similarly had no effect on cerebral glucose utilization (53 + 5, 70 + 8, 63 + 5, and 52 + 3,/zmol.100 g-l.min-1 for vehicle, methionine enkephalin, leueine enkephalin, and dynorphin, respectively). In contrast, topical glutamate (10-3 M) produced similar dilation but increased cerebral glucose utilization (41 + 3 vs 89 + 8 /~mol.100 g-Lmin-1 for vehicle and glutamate, respectively). Therefore, these opioids do not appear to produce vascular effects through a change in cerebral metabolic utilization of glucose. INTRODUCTION
O p i o i d peptides were discovered in the brain in 197511 and recent observations indicate that these peptides could contribute to the regulation of cerebral hemodynamics, R e c e p t o r binding for opioids has b e e n d e m o n s t r a t e d on cerebral microvessels 17, and e n k e p h a l i n immunoreactivity, indicative o f innervation, has b e e n o b s e r v e d in large cerebral arteries of the pig 2~. Opioids have b e e n d e t e c t e d in the CSF, and C S F opioid concentrations are in the vasoactive range u n d e r control conditions in the n e w b o r n pig 2,a. H o w e v e r , opioids have been o b s e r v e d to either have p r o m i n e n t dilator, constrictor, o r minimal cerebrovascular activity~'a's'24. The p o t e n t dilation o b s e r v e d after the topical administration of methionine e n k e p h a -
lin, leucine enkephafin and dynorphin in the newborn pig 2'3 could result from either direct effects on vascular smooth muscle o r be due to increased cerebral m e t a b o lism 24. The [14C]deoxyglucose m e t h o d provides a means to measure the rates of cerebral glucose utilization 19. Because functional activity and energy metabolism a p p e a r to be closely c o r r e l a t e d in the brain, local alterations in glucose utilization reflect local changes in functional brain activities 19'2°.
The present study was designed to address the hypothesis that o p i o i d - m e d i a t e d cerebrovascular dilation is associated with changes in cerebral glucose utilization. In o r d e r to investigate this hypothesis, the effects of topical methionine enkephalin, leucine e n k e p h a l i n and dynorphin on pial arteriolar d i a m e t e r and cerebral glucose utilization were d e t e r m i n e d . MATERIALS AND METHODS The animal protocols used were reviewed and approved by the Animal Care and Use Committee of the University of Tennessee, Memphis. Newborn pigs (1-7 days old, 0.9-2.9 kg) of either sex were used in these experiments. They were anesthetized with ketamine hydrochloride (33 mg/kg, i.v.) and acepromazine (3.3 mg/kg, i.v.). Anesthesia was maintained with a-chloralose (30-50 mg/kg initially, supplemented with 5 mg/kg per h i.v.). The trachea was cannulated and the animals were ventilated with room air. Body temperature was maintained at 37-38"C with an overhead radiant heater. Catheters were placed in both femoral arteries and both femoral veins. One set of catheters was used to monitor blood pressure, sample arterial blood, and provide venous access as necessary. The other femoral artery and vein were connected together with a 5-era length of silicon tubing to form an arteriovenous shunt. This shunt allowed for rapid, sequential sampling of arterial blood. Just prior to shunt sampling, heparin (50 U/kg) was given intravenousiy to maintain shunt patency. For insertion of the cranial windows, the scalp was removed and openings were made in the skull over both the left and right parietal cortex. The dura was cut and retracted over the cut bone edge. The cranial windows were placed in the holes and cemented in place
Correspondence: W.M. Armstead, Department of Physiology & Biophysics, University of Tennessee, Memphis, TN 38163, U.S.A.
98 with dental acrylic. The space under the windows was filled with artificial CSF of the following composition: 220 mg KC1, 123 mg MgC12, 221 mg CaC12, 7710 mg NaC1, 402 mg urea, 665 mg dextrose and 2066 mg NaHCO3/I, pH 7.33, pCO 2, 46 mmHg and pO 2 43 mmHg. We have previously shown that this artificial CSF does not affect resting pial arteriolar diameter in piglets2. Pial arteriolar diameter was determined through the use of a microscope and an optical image shearing device, Protocol
Experiments were designed to obtain parallel control and experimental data from a single animal using the two cranial windows, CSF or CSF containing a drug was injected under the window at the start of the experiment and replenished every 10 rain for the duration of the protocol. Responses to topical vehicle (0.9% saline), methionine enkephalin, leucine enkephalin, dynorphin, and glutam a t e (10-3 M Glut, 10-6 M for all others; Sigma Chemical Co., St. Louis, MO) in CSF were obtained. Although several dynorphin peptides are known to exist, we have chosen to investigate the dynorphin peptide containing 13 amino acids ('13jr-Gly-Gly-PheLeu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys). Throughout the text, this form is referred to simply as dynorphin. The effect of only one opioid and its vehicle were observed per each animal. Each animal had two closed cranial windows. One contained the test substance, the other contained the vehicle control. The effect of the test substance was determined by comparing the [14C]deoxyglucose uptake by the two windows (see below). Animals were divided into 5 groups according to which substance was placed under each of the two cranial windows labeled as a and b in each of the following groups: (1) (a) artificial CSF (aCSF), (b) vehicle in aCSF, (2) (a) vehicle in aCSF, (b) methionine enkephalin, (3) (a) vehicle in aCSF, (b) leucine enkephalin, (4) (a) vehicle in aCSF, (b) dynorphin and (5) (a) vehicle in aCSF, (b) glutamate (n = 6 for each group), Therefore, autoradiographic measurements were made for each agent and its corresponding vehicle in each animal. Data for the calculation of cerebral glucose utilization in vehicle animals were obtained from group 1. The design of the experiments allowed for a comparison to this baseline autoradiographic data to be made in each group. All drug solutions were made fresh on the day of use.
The stock opioid solution (10-3 M) was diluted appropriately and aliquoted for experimentation. These aliquots were stored at -20°C until the day of use when small aliquots were then added to CSF for topical application. For measurement of cerebral glucose metabolism 19, piglets were administered a bolus injection of 40/tCi/kg of 2 [1-14C]deoxy-Dglucose (NEN, Boston, MA) in saline via the femoral vein. Blood samples (300 #1) were taken from the arteriovenous shunt at 0, 10, 15, 30, 45, 60, 120, 180, 300, 450, 600, 900, 1500, 2100 and 3600 s and the plasma separated by centrifugation. 10-#1 Aliquots of plasma were used for glucose analysis and 50-/A aliquots were used for scintillation counting. Corrections for quench were made from quenched standards and data handled as dissociations per min per ml of blood. Animals were sacrificed at 60 min by an intravenous dose of KCI. Brain sections beneath the cranial window were stained with a 5% Evans blue solution for orientation, rapidly removed, frozen in isopentane at -38°C, and stored at -60°C until further processing. The tissue was equilibrated to -20°C before sectioning in a cryostat. Coronal sections (20-/zm thick) were collected onto glass coverslips, dried at 50°C, and mounted on Bainbridge board. Sequential sections were collected on glass slides and stained with hemotoxilin and eosin for histological identification of brain structures. The mounted tissue sections were placed in x-ray cassettes with [14C]methylmethacrylate standards (American Radiolabeled Chemicals, St. Louis, MO) and OMC-1 film (Kodak, Rochester, NY) for 12 days. Local tissue concentrations of [14C] were determined using the standard curve and a computer-based image analyzer. The image analyzer used was a Joyce-Loebl Magiscan 2A, installed in the Neuroscience Imaging Center at the University of Tennessee, Memphis. The components and use of this system have been previously described TM. Briefly, this system digitizes 64 gray levels (63 the lightest, 0 the darkest) and automatically subtracts the background for image analysis. Typical autoradiographs with relative optical density determined at particular points were obtained. We were careful to choose points that appeared to be representative of a particular area. Since an agent could have variable effects on relative optical density depending on where the determination was made, we averaged the optical density for the whole surface area.
Fig. 1. Typical autoradiograph with relative optical density indication (0 dark, 63 light). Left panel: influence of topical CSF o n [14C] brain content. Right panel: influence of topical 0.9% saline (vehicle) on brain [14C] content.
99
Fig. 2. Typical autoradiograph with relative optical density indication (0 dark, 63 light). Left panel: influence of topical vehicle on [14C] brain content. Right panel: influence of topical methionine enkephalin (10-6 M) on [~4C] brain content.
This area was somewhat variable but the average size was 180 mm 2. Therefore, data were normalized to account for this fact. Local rates of glucose metabolism were computed from the autoradiographic and plasma concentrations of [14C] according to the operational equation of the methods TM. The lumped constant of 0.401 previously determined for the neonatal sheep 1 was used in the present study.
Statistical analysis Data were analyzed by t-test. An a level of P < 0.05 was considered significant in all statistical tests. Values are represented as mean + S.E.M. of absolute value or as percentage of changes from control values.
Fig. 3. Typical autoradiograph with relative optical density indication (0 dark, 63 light). Left panel: influence of topical vehicle on [14C] brain content. Right panel: influence of topical glutamate (10-3 M) on [14C] brain content.
100 100
...................
X
]
80 t
oJ
, CSF V
V ME V LEU V DYN V GLU
Fig. 5. Influence of opioids and glutamate on glucose metabohsm. V, vehicle; MF., methionine enkeph~n (I0 -~ M); LEU, Leucine enkephalin (10-6 M), DYN, dynorphin (10- 6 M); GLU, glutamate (10-3 M); glucose metabolism expressed as/zmol/100g/min, n = 6; *P < 0.05 compared to corresponding vehicle.
, Fig. 4. Typical hemotoxilin- and eosin-stained coronal brain section.
RESULTS Qualitatively, topical vehicle (0.9% saline) and methionine enkephalin (10 -6 M) (right panels) had no effect on the amount of [14C] in brain tissue as compared to control (left panels) when the data were expressed as a change in relative optical density from vehicle at specific points in the autoradiograph (Figs. 1, 2). In contrast, glutamate (10 -3 M) produced a large change in relative optical density point determination (right panel) from the corresponding vehicle treated autoradiograph (left panel) that extended approximately 500/~m into the brain parenchyma (Fig. 3). When comparable sections were stained with hemotoxilin and eosin, the area associated with a change in gray level was anatomically determined to constitute the pia and a portion of layer I in the cerebrum (Fig. 4).
TABLE I Influence o[ methionine enkephalin, leucine enkephalin, dynorphin, and glutamate on pial arteriolar diameter in newborn pigs
DISCUSSION
n = 3. Values are given aS mean + S.E.M. Diameter (l~m)
Control Agent
Methionine enkephalin (10-6 M) 151 + 7 186 + 5* Leucine enkephalin (10 -6 M) 146 + 8 182 + 8* Dynorphin (104 M) 158 + 8 190 +_ 5* Glutamate (10- 3 M) 147 _+ 6 181 +__5* Vehicle 145 + 9 146 + 9 * P < 0.05 compared to corresponding control,
Quantitatively, the vehicle, methionine enkephalin, leucine enkephalin, and dynorphin had no effect on the amount of [14C] in brain tissue. The values were 119 _+ 9, 139 ___ 13, 121 + 5, and 109 + 9/~Ci/g for vehicle, methionine enkephalin, leucine enkephalin, and dynorphin, respectively. In contrast, glutamate increased the [14C] brain content (119 + 9 vs 223 + 16/~Ci/g for vehicle and glutamate, respectively). Rates of glucose metabolism were unchanged by the vehicle, methionine enkephalin, leucine enkephalin, and dynorphin (Fig. 5). In contrast, glutamate significantly increased glucose metabolism (Fig. 5). Topical methionine enkephalin, leucine enkephalin, dynorphin, and glutamate all increased pial arteriolar diameter (Table I). When compared on a percentage basis, all of the above agents increased pial arteriolar diameter by the same percent (Table I). The vehicle had no effect on pial arteriolar diameter. Blood gases and p H were measured at the beginning and at the end of all experiments. These values were 7.46 + 0.01 vs 7.48 + 0.01; 31 + 1 vs 32 + 1 and 85 + 2 vs 86 + 1 for pH, pCO2, and pO2, respectively (n = 30). Topical administration of all agents had no effect on systemic arterial blood pressure (65 + 1 vs 64 + 1, n = 30).
Percent Change
23 + 3 25 + 2 20 _+ 3 24 + 2 1+ 1
Results of the present study show that opioid-mediated cerebrovascular dilation does not result from a secondary change in metabolism. The topical administration of methionine enkephalin, leucine enkephalin and dynorphin did not increase the amount of [I4c] in the cerebral cortex. Using the autoradiographic and plasma concentrations of [14C] according to the operational equation of the method 19, topical opioids had no effect on cerebral
101
glucose metabolism. However, topical glutamate increased glucose metabolism. Therefore, this method could detect changes if they were to occur and glutamate served as a positive control in these experiments, The previously observed ability of topical methionine enkephalin, leucine enkephalin and dynorphin to produce potent pial arteriolar dilation in the piglet2'3 could result from secondary changes in neuronal discharge and metabolism24. Topical methionine enkephalin has been observed to decrease cerebral blood flow and tissue pO2 TM while the synthetic opioids morphine and fentanyl decreased global cerebral metabolic rate and cerebral blood flow5'21. Alternatively, the synthetic opioid agonist etorphine increased local energy metabolism in the rat parietal cortex25 while [D-Ala2]met-enkephalinamide increased neuronal discharge23. Depending on the presence or absence, as well as the level of anesthesia, opioids have been observed to increase, decrease, or have no effect on cerebral blood flow 12'13'21. Therefore, baseline metabolic rate may also affect vascular reactivity to opioids. The [14C]2-deoxyglucose method has been used in a variety of experimental conditions to measure local cerebral metabolic utilization of glucose 19'20. Glucose has been shown to be the prime substrate for cerebral energy metabolism in the fetus and newborn 1 and it is thought that all the oxygen consumed by the brain can be accounted for by the oxidation of glucose 19'20. The method employs [14C]2-deoxyglucose as a tracer for glucose metabolism and uses a quantitative autoradiographic technique to measure local [14C] concentrations. The operational equation of the method derives from a model based on the biochemical properties of 2-deoxyglucose in the brain and the kinetics of the exchange of glucose and 2-deoxyglucose between plasma and brain tissue and their phosphorylation by hexokinase 19'2°. In the present study, quantitative changes in cerebral glucose utilization were obtained through the use of a standard curve relating optical density to [14C] brain tissue content. These data demonstrated that opioids did not change cerebral metabolism. Opioids could contribute to the regulation of cerebral hemodynamics. These peptides are present in the brain TM. Furthermore, opioids have been detected in CSF6'15 and CSF opioid concentrations are in the vasoactive range under control conditions in the newborn piga. Opioid receptor binding has been demonstrated on cerebral microvessels 17 and enkephalin immunoreactivity, indicative of innervation, has been shown in large REFERENCES 1 Abrams, R.M., Ito, M., Frisinger, J.E., Patlak, C.S., Pettigrew,
cerebral arteries of the pig22. Enkephalin and fl-endorphin-containing neurons have been identified throughout the brain through the use of immunohistochemical procedures 9'11. Because CSF opioid concentrations increase during hemorrhagic hypotension3'6, opioids could contribute to pial arteriolar dilation induced by hypotension3. Previous investigations into the ability of opioids to influence the cerebral circulation have resulted in conflicting data. Topical application of enkephalins has been observed to produce modest dilation in feline middle cerebral arteries and minimal dilation in feline pial atteries at very high doses 8'24. Alternatively, topical methionine enkephalin has been shown to decrease feline cortical blood flowTM. In contrast to previous observations, topically applied opioids have prominent effects on pial arteriolar diameter in newborn pigs2'3. Methionine enkephalin and leucine enkephalin elicited prostanoiddependent dilation while dynorphin produced tonedependent responses (prostanoid-dependent dilation during normotension, constriction during hypotension when cerebrovascular tone was reduced2'3). Although synthesic analogues possessing higher affinity for opioid receptor subtypes are available, the present study was designed to evaluate the local cerebrovascular activity of naturally occurring opioids. These results indicate that/~- (methionine enkephalin) 7, 6- (leucine enkephalin) 7 and r (dynorphin)7-receptor activation do not increase the local metabolism of glucose. The concentration of opioids and glutamate used in the present study have previously been shown to produce comparable dilation2-4. Since glutamate increased glucose metabolism, the present method could detect changes if they were to occur. Since these glutamate-induced changes were only observed in the pia and a portion of cerebral cortex layer I directly beneath the cranial window, these results indicate that topically applied agents exert local effects. These results extend previous studies by determining the local effects of naturally occurring opioids on glucose utilization. In conclusion, topical opioids do not appear to produce vascular effects through a change in cerebral metabolism under conditions of anesthesia.
Acknowledgements. The authors thank Drs. R. Ranney Mize and James Reger for imaging and histologicconsultation. The authors also thank Alex Fedinec, John Pirani, Bill Gannawayand Mildred Jackson for excellenttechnicalassistanceduring performanceof the experiments.These studieswere supported by grants from the NIH.
K.D. and Kennedy, C., Local cerebral glucose utilization in fetal and neonatal sheep, Am. Z Physiol., 246 (1984) R608R618.
102 2 Armstead, W.M., Mirro, R., Busija, D.W. and Leffler, C.W., Prostanoids modulate opioid cerebrovascular responses in newborn pigs, J. Pharm. Exp. Ther., 255 (1990) 1083-1089. 3 Armstead, W.M., Mirro, R., Busija, D.W., Desiderio, D.M. and Leffler, C.W., Opioids in cerebrospinal fluid in hypotensive newborn pigs, Circ. Res., 68 (1991) 922-929. 4 Busija, D.W. and Leffler, C.W., Dilator effects of amino acid transmitters on piglet pial arterioles, Am. J. Physiol., 257 (1989) H1200-H1203. 5 Carlsson, C., Smith, D.S., Keykhoh, M.M., Engleback, I. and Harp, J.R., The effects of high-dose fentanyl on cerebral circulation and metabolism in rats, Anesthesiology, 57 (1982) 375380. 6 Elam, R., Bergmann, F. and Feuerstein, G., Simultaneous changes of catecholamines and of Leu-enkephalin-like immunoreactivity in plasma and cerebrospinal fluid of cats undergoing acute hemorrhage, Brain Research, 303 (1984) 313-317. 7 Feuerstein, G. and Siren, A.L., The opioid peptides, Hypertension, 9 (1987) 561-565. 8 Hanko, J.H. and Hardebo, J.E., Enkephalin-induced dilatation of pial arteries in vitro probably mediated by opiate receptors, Eur. J. Pharmacol., 51 (1978) 295-297. 9 H6kfelt, T., Elde, R., Johannson, O., Terenius, L. and Stein, L., The distribution of enkephalin-immunoreactive cell bodies in the rat central nervous system, Neurosci. Lett., 5 (1977) 25-31. 10 Hughes, J., Kosterlitz, H.W. and Smith, T.W., The distribution of methionine enkephalin and leucine enkephalin in the brain and peripheral tissues, Br. J. Pharmacol., 61 (1977) 639-648. 11 Hughes, J., Smith, T.W., Kosterlitz, H.W., Fothergill, L.A., Morgan, B.A. and Morris, H.R., Identification of two related pentapeptides from the brain with potent opiate agonist activity, Nature, 258 (1975) 577-579. 12 Kirsch, J.R., Hanley, D.F., Wilson, D.A. and Traystman, R.J., Effects of centrally administered encephalinamides on regional cerebral blood flow in the dog, J. Cereb. Blood Flow Metab., 8 (1988) 385-394. 13 Koskinen, L.O. and Bill, A., Regional cerebral, ocular, and peripheral vascular effects of naloxone and morphine in unanesthetized rabbits, Acta Physiol. Scand., 119 (1983) 235-241. 14 Mize, R.R., Holdefer, R.N. and Nabors, L.B., Quantitative immunocytochemistry using an image analyzer. I. Hardware
15 16 17 18
19
20 21 22 23
24 25
evaluation, image processing, and data analysis, J. Neurosci. Methods., 26 (1988) 1-24. Nyberg, E and Terenius, L., Endorphins in human cerebrospinal fluid, Life Sci., 31 (1982) 1737-1740. Pasternak, G.W., Goodman, R. and Snyder, S.H., Endogenous morphine-like factor in mammalian brain, Life Sci., 16 (1975) 1765-1769. Peroutka, S.J., Moskowitz, M.A., Reinhard, J.E and Snyder, S.H., Neurotransmitter receptor binding in bovine cerebral microvessels, Science, 208 (1980) 610-613. Sandor, P., Demchenko, I.T., Morgalyov, Y.N., Moshalenko, Y.E. and Kovach, A.G.B., Cerebral blood flow and tissue pO 2 changes following local met-enkephalin administration in awake, freely moving cats, Acta Physiol. Acad. Sci. Hung., 51 (1983) 155-161. Sokoloff, L., Reivich, M., Kennedy, C., DesRosiers, M.H., Patlak, C.S., Pettigrew, K.D., Sakurada, O. and Shinohara, M., The [14C]deoxyglucose method for the measurement of local cerebral glucose utilization: theory, procedure, and normal values in the conscious and anesthetized albino rat, J. Neurochem., 28 (1977) 897-916. Sokoloff, L., Relation between physiological function and energy metabolism in the central nervous system, J. Neurochem., 29 (1977) 13-26. Takeshita, H., Michenfelder, J.D. and Theye, R.A., The effects of morphine and N-allyl-normorphine on canine cerebral metabolism and circulation, Anesthesiology, 37 (1972) 605-612. Thureson-Klein, A., Kong, J.Y. and Klein, R.L., Enkephalin and neuropeptide Y in large cerebral arteries of the pig after ischemia and reserpine, Blood Vessels, 26 (1989) 177-184. Ukponmwan, O.E., Van der Poel-Heisterkamp, A.L., Haffmans, J. and Dzoljic, M., MAO-B inhibitor deprenyl and fl-phenylethylamine potentiate [~Ala2]-Met-enkephalinamide induced seizures, Naunyn-Schmiedeberg's Arch. Pharmacol., 322 (1983) 38-41. Wahl, M., Effects of enkephalin, morphine, and naloxone on pial arteries during perivascular microapplication, J. Cereb. Blood Flow Metab., 5 (1985) 451-457. Wuster, M., Dirks, B., Krieglstein, J. and Herz, A., The effect of the potent opiate agonist etorphine on local energy metabolisms in the isolated perfused rat brain, Neuropharmacology, 20 (1981) 901-904.
97
Elsevier BRES 17379
The influence of opioids on local cerebral glucose utilization in the newborn pig William M. Armstead, Robert Mirro, Samuel Zuckerman, David W. Busija and Charles W. Leffler Laboratory for Research in Neonatal Physiology, Departments of Physiology~Biophysics, Pediatrics and Obstetrics~Gynecology, University of Tennessee, Memphis, TN 38163 (U.S.A.) (Accepted 10 September 1991)
Key words: Opioid; Newborn; Cerebral circulation; Cerebral metabolic rate
Topical methionine enkephalin, leucine enkephalin, and dynorphin (10-6 M) previously have been observed to produce prominent p i a l arteriolar dilation. Dilation to these opioids could be caused directly by opioids acting on vascular receptors, or indirectly, as a consequence of increased metabolism. Therefore, we examined this possibility by determining the influence of opioids on cerebral glucose utilization in piglets with closed cranial windows using the [14C]deoxyglucose method. Qualitatively, the autoradiographic images expressed as a change in relative optical density from vehicle were unchanged by these opioids. Quantitatively, the opioids similarly had no effect on cerebral glucose utilization (53 + 5, 70 + 8, 63 + 5, and 52 + 3,/zmol.100 g-l.min-1 for vehicle, methionine enkephalin, leueine enkephalin, and dynorphin, respectively). In contrast, topical glutamate (10-3 M) produced similar dilation but increased cerebral glucose utilization (41 + 3 vs 89 + 8 /~mol.100 g-Lmin-1 for vehicle and glutamate, respectively). Therefore, these opioids do not appear to produce vascular effects through a change in cerebral metabolic utilization of glucose. INTRODUCTION
O p i o i d peptides were discovered in the brain in 197511 and recent observations indicate that these peptides could contribute to the regulation of cerebral hemodynamics, R e c e p t o r binding for opioids has b e e n d e m o n s t r a t e d on cerebral microvessels 17, and e n k e p h a l i n immunoreactivity, indicative o f innervation, has b e e n o b s e r v e d in large cerebral arteries of the pig 2~. Opioids have b e e n d e t e c t e d in the CSF, and C S F opioid concentrations are in the vasoactive range u n d e r control conditions in the n e w b o r n pig 2,a. H o w e v e r , opioids have been o b s e r v e d to either have p r o m i n e n t dilator, constrictor, o r minimal cerebrovascular activity~'a's'24. The p o t e n t dilation o b s e r v e d after the topical administration of methionine e n k e p h a -
lin, leucine enkephafin and dynorphin in the newborn pig 2'3 could result from either direct effects on vascular smooth muscle o r be due to increased cerebral m e t a b o lism 24. The [14C]deoxyglucose m e t h o d provides a means to measure the rates of cerebral glucose utilization 19. Because functional activity and energy metabolism a p p e a r to be closely c o r r e l a t e d in the brain, local alterations in glucose utilization reflect local changes in functional brain activities 19'2°.
The present study was designed to address the hypothesis that o p i o i d - m e d i a t e d cerebrovascular dilation is associated with changes in cerebral glucose utilization. In o r d e r to investigate this hypothesis, the effects of topical methionine enkephalin, leucine e n k e p h a l i n and dynorphin on pial arteriolar d i a m e t e r and cerebral glucose utilization were d e t e r m i n e d . MATERIALS AND METHODS The animal protocols used were reviewed and approved by the Animal Care and Use Committee of the University of Tennessee, Memphis. Newborn pigs (1-7 days old, 0.9-2.9 kg) of either sex were used in these experiments. They were anesthetized with ketamine hydrochloride (33 mg/kg, i.v.) and acepromazine (3.3 mg/kg, i.v.). Anesthesia was maintained with a-chloralose (30-50 mg/kg initially, supplemented with 5 mg/kg per h i.v.). The trachea was cannulated and the animals were ventilated with room air. Body temperature was maintained at 37-38"C with an overhead radiant heater. Catheters were placed in both femoral arteries and both femoral veins. One set of catheters was used to monitor blood pressure, sample arterial blood, and provide venous access as necessary. The other femoral artery and vein were connected together with a 5-era length of silicon tubing to form an arteriovenous shunt. This shunt allowed for rapid, sequential sampling of arterial blood. Just prior to shunt sampling, heparin (50 U/kg) was given intravenousiy to maintain shunt patency. For insertion of the cranial windows, the scalp was removed and openings were made in the skull over both the left and right parietal cortex. The dura was cut and retracted over the cut bone edge. The cranial windows were placed in the holes and cemented in place
Correspondence: W.M. Armstead, Department of Physiology & Biophysics, University of Tennessee, Memphis, TN 38163, U.S.A.
98 with dental acrylic. The space under the windows was filled with artificial CSF of the following composition: 220 mg KC1, 123 mg MgC12, 221 mg CaC12, 7710 mg NaC1, 402 mg urea, 665 mg dextrose and 2066 mg NaHCO3/I, pH 7.33, pCO 2, 46 mmHg and pO 2 43 mmHg. We have previously shown that this artificial CSF does not affect resting pial arteriolar diameter in piglets2. Pial arteriolar diameter was determined through the use of a microscope and an optical image shearing device, Protocol
Experiments were designed to obtain parallel control and experimental data from a single animal using the two cranial windows, CSF or CSF containing a drug was injected under the window at the start of the experiment and replenished every 10 rain for the duration of the protocol. Responses to topical vehicle (0.9% saline), methionine enkephalin, leucine enkephalin, dynorphin, and glutam a t e (10-3 M Glut, 10-6 M for all others; Sigma Chemical Co., St. Louis, MO) in CSF were obtained. Although several dynorphin peptides are known to exist, we have chosen to investigate the dynorphin peptide containing 13 amino acids ('13jr-Gly-Gly-PheLeu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys). Throughout the text, this form is referred to simply as dynorphin. The effect of only one opioid and its vehicle were observed per each animal. Each animal had two closed cranial windows. One contained the test substance, the other contained the vehicle control. The effect of the test substance was determined by comparing the [14C]deoxyglucose uptake by the two windows (see below). Animals were divided into 5 groups according to which substance was placed under each of the two cranial windows labeled as a and b in each of the following groups: (1) (a) artificial CSF (aCSF), (b) vehicle in aCSF, (2) (a) vehicle in aCSF, (b) methionine enkephalin, (3) (a) vehicle in aCSF, (b) leucine enkephalin, (4) (a) vehicle in aCSF, (b) dynorphin and (5) (a) vehicle in aCSF, (b) glutamate (n = 6 for each group), Therefore, autoradiographic measurements were made for each agent and its corresponding vehicle in each animal. Data for the calculation of cerebral glucose utilization in vehicle animals were obtained from group 1. The design of the experiments allowed for a comparison to this baseline autoradiographic data to be made in each group. All drug solutions were made fresh on the day of use.
The stock opioid solution (10-3 M) was diluted appropriately and aliquoted for experimentation. These aliquots were stored at -20°C until the day of use when small aliquots were then added to CSF for topical application. For measurement of cerebral glucose metabolism 19, piglets were administered a bolus injection of 40/tCi/kg of 2 [1-14C]deoxy-Dglucose (NEN, Boston, MA) in saline via the femoral vein. Blood samples (300 #1) were taken from the arteriovenous shunt at 0, 10, 15, 30, 45, 60, 120, 180, 300, 450, 600, 900, 1500, 2100 and 3600 s and the plasma separated by centrifugation. 10-#1 Aliquots of plasma were used for glucose analysis and 50-/A aliquots were used for scintillation counting. Corrections for quench were made from quenched standards and data handled as dissociations per min per ml of blood. Animals were sacrificed at 60 min by an intravenous dose of KCI. Brain sections beneath the cranial window were stained with a 5% Evans blue solution for orientation, rapidly removed, frozen in isopentane at -38°C, and stored at -60°C until further processing. The tissue was equilibrated to -20°C before sectioning in a cryostat. Coronal sections (20-/zm thick) were collected onto glass coverslips, dried at 50°C, and mounted on Bainbridge board. Sequential sections were collected on glass slides and stained with hemotoxilin and eosin for histological identification of brain structures. The mounted tissue sections were placed in x-ray cassettes with [14C]methylmethacrylate standards (American Radiolabeled Chemicals, St. Louis, MO) and OMC-1 film (Kodak, Rochester, NY) for 12 days. Local tissue concentrations of [14C] were determined using the standard curve and a computer-based image analyzer. The image analyzer used was a Joyce-Loebl Magiscan 2A, installed in the Neuroscience Imaging Center at the University of Tennessee, Memphis. The components and use of this system have been previously described TM. Briefly, this system digitizes 64 gray levels (63 the lightest, 0 the darkest) and automatically subtracts the background for image analysis. Typical autoradiographs with relative optical density determined at particular points were obtained. We were careful to choose points that appeared to be representative of a particular area. Since an agent could have variable effects on relative optical density depending on where the determination was made, we averaged the optical density for the whole surface area.
Fig. 1. Typical autoradiograph with relative optical density indication (0 dark, 63 light). Left panel: influence of topical CSF o n [14C] brain content. Right panel: influence of topical 0.9% saline (vehicle) on brain [14C] content.
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Fig. 2. Typical autoradiograph with relative optical density indication (0 dark, 63 light). Left panel: influence of topical vehicle on [14C] brain content. Right panel: influence of topical methionine enkephalin (10-6 M) on [~4C] brain content.
This area was somewhat variable but the average size was 180 mm 2. Therefore, data were normalized to account for this fact. Local rates of glucose metabolism were computed from the autoradiographic and plasma concentrations of [14C] according to the operational equation of the methods TM. The lumped constant of 0.401 previously determined for the neonatal sheep 1 was used in the present study.
Statistical analysis Data were analyzed by t-test. An a level of P < 0.05 was considered significant in all statistical tests. Values are represented as mean + S.E.M. of absolute value or as percentage of changes from control values.
Fig. 3. Typical autoradiograph with relative optical density indication (0 dark, 63 light). Left panel: influence of topical vehicle on [14C] brain content. Right panel: influence of topical glutamate (10-3 M) on [14C] brain content.
100 100
...................
X
]
80 t
oJ
, CSF V
V ME V LEU V DYN V GLU
Fig. 5. Influence of opioids and glutamate on glucose metabohsm. V, vehicle; MF., methionine enkeph~n (I0 -~ M); LEU, Leucine enkephalin (10-6 M), DYN, dynorphin (10- 6 M); GLU, glutamate (10-3 M); glucose metabolism expressed as/zmol/100g/min, n = 6; *P < 0.05 compared to corresponding vehicle.
, Fig. 4. Typical hemotoxilin- and eosin-stained coronal brain section.
RESULTS Qualitatively, topical vehicle (0.9% saline) and methionine enkephalin (10 -6 M) (right panels) had no effect on the amount of [14C] in brain tissue as compared to control (left panels) when the data were expressed as a change in relative optical density from vehicle at specific points in the autoradiograph (Figs. 1, 2). In contrast, glutamate (10 -3 M) produced a large change in relative optical density point determination (right panel) from the corresponding vehicle treated autoradiograph (left panel) that extended approximately 500/~m into the brain parenchyma (Fig. 3). When comparable sections were stained with hemotoxilin and eosin, the area associated with a change in gray level was anatomically determined to constitute the pia and a portion of layer I in the cerebrum (Fig. 4).
TABLE I Influence o[ methionine enkephalin, leucine enkephalin, dynorphin, and glutamate on pial arteriolar diameter in newborn pigs
DISCUSSION
n = 3. Values are given aS mean + S.E.M. Diameter (l~m)
Control Agent
Methionine enkephalin (10-6 M) 151 + 7 186 + 5* Leucine enkephalin (10 -6 M) 146 + 8 182 + 8* Dynorphin (104 M) 158 + 8 190 +_ 5* Glutamate (10- 3 M) 147 _+ 6 181 +__5* Vehicle 145 + 9 146 + 9 * P < 0.05 compared to corresponding control,
Quantitatively, the vehicle, methionine enkephalin, leucine enkephalin, and dynorphin had no effect on the amount of [14C] in brain tissue. The values were 119 _+ 9, 139 ___ 13, 121 + 5, and 109 + 9/~Ci/g for vehicle, methionine enkephalin, leucine enkephalin, and dynorphin, respectively. In contrast, glutamate increased the [14C] brain content (119 + 9 vs 223 + 16/~Ci/g for vehicle and glutamate, respectively). Rates of glucose metabolism were unchanged by the vehicle, methionine enkephalin, leucine enkephalin, and dynorphin (Fig. 5). In contrast, glutamate significantly increased glucose metabolism (Fig. 5). Topical methionine enkephalin, leucine enkephalin, dynorphin, and glutamate all increased pial arteriolar diameter (Table I). When compared on a percentage basis, all of the above agents increased pial arteriolar diameter by the same percent (Table I). The vehicle had no effect on pial arteriolar diameter. Blood gases and p H were measured at the beginning and at the end of all experiments. These values were 7.46 + 0.01 vs 7.48 + 0.01; 31 + 1 vs 32 + 1 and 85 + 2 vs 86 + 1 for pH, pCO2, and pO2, respectively (n = 30). Topical administration of all agents had no effect on systemic arterial blood pressure (65 + 1 vs 64 + 1, n = 30).
Percent Change
23 + 3 25 + 2 20 _+ 3 24 + 2 1+ 1
Results of the present study show that opioid-mediated cerebrovascular dilation does not result from a secondary change in metabolism. The topical administration of methionine enkephalin, leucine enkephalin and dynorphin did not increase the amount of [I4c] in the cerebral cortex. Using the autoradiographic and plasma concentrations of [14C] according to the operational equation of the method 19, topical opioids had no effect on cerebral
101
glucose metabolism. However, topical glutamate increased glucose metabolism. Therefore, this method could detect changes if they were to occur and glutamate served as a positive control in these experiments, The previously observed ability of topical methionine enkephalin, leucine enkephalin and dynorphin to produce potent pial arteriolar dilation in the piglet2'3 could result from secondary changes in neuronal discharge and metabolism24. Topical methionine enkephalin has been observed to decrease cerebral blood flow and tissue pO2 TM while the synthetic opioids morphine and fentanyl decreased global cerebral metabolic rate and cerebral blood flow5'21. Alternatively, the synthetic opioid agonist etorphine increased local energy metabolism in the rat parietal cortex25 while [D-Ala2]met-enkephalinamide increased neuronal discharge23. Depending on the presence or absence, as well as the level of anesthesia, opioids have been observed to increase, decrease, or have no effect on cerebral blood flow 12'13'21. Therefore, baseline metabolic rate may also affect vascular reactivity to opioids. The [14C]2-deoxyglucose method has been used in a variety of experimental conditions to measure local cerebral metabolic utilization of glucose 19'20. Glucose has been shown to be the prime substrate for cerebral energy metabolism in the fetus and newborn 1 and it is thought that all the oxygen consumed by the brain can be accounted for by the oxidation of glucose 19'20. The method employs [14C]2-deoxyglucose as a tracer for glucose metabolism and uses a quantitative autoradiographic technique to measure local [14C] concentrations. The operational equation of the method derives from a model based on the biochemical properties of 2-deoxyglucose in the brain and the kinetics of the exchange of glucose and 2-deoxyglucose between plasma and brain tissue and their phosphorylation by hexokinase 19'2°. In the present study, quantitative changes in cerebral glucose utilization were obtained through the use of a standard curve relating optical density to [14C] brain tissue content. These data demonstrated that opioids did not change cerebral metabolism. Opioids could contribute to the regulation of cerebral hemodynamics. These peptides are present in the brain TM. Furthermore, opioids have been detected in CSF6'15 and CSF opioid concentrations are in the vasoactive range under control conditions in the newborn piga. Opioid receptor binding has been demonstrated on cerebral microvessels 17 and enkephalin immunoreactivity, indicative of innervation, has been shown in large REFERENCES 1 Abrams, R.M., Ito, M., Frisinger, J.E., Patlak, C.S., Pettigrew,
cerebral arteries of the pig22. Enkephalin and fl-endorphin-containing neurons have been identified throughout the brain through the use of immunohistochemical procedures 9'11. Because CSF opioid concentrations increase during hemorrhagic hypotension3'6, opioids could contribute to pial arteriolar dilation induced by hypotension3. Previous investigations into the ability of opioids to influence the cerebral circulation have resulted in conflicting data. Topical application of enkephalins has been observed to produce modest dilation in feline middle cerebral arteries and minimal dilation in feline pial atteries at very high doses 8'24. Alternatively, topical methionine enkephalin has been shown to decrease feline cortical blood flowTM. In contrast to previous observations, topically applied opioids have prominent effects on pial arteriolar diameter in newborn pigs2'3. Methionine enkephalin and leucine enkephalin elicited prostanoiddependent dilation while dynorphin produced tonedependent responses (prostanoid-dependent dilation during normotension, constriction during hypotension when cerebrovascular tone was reduced2'3). Although synthesic analogues possessing higher affinity for opioid receptor subtypes are available, the present study was designed to evaluate the local cerebrovascular activity of naturally occurring opioids. These results indicate that/~- (methionine enkephalin) 7, 6- (leucine enkephalin) 7 and r (dynorphin)7-receptor activation do not increase the local metabolism of glucose. The concentration of opioids and glutamate used in the present study have previously been shown to produce comparable dilation2-4. Since glutamate increased glucose metabolism, the present method could detect changes if they were to occur. Since these glutamate-induced changes were only observed in the pia and a portion of cerebral cortex layer I directly beneath the cranial window, these results indicate that topically applied agents exert local effects. These results extend previous studies by determining the local effects of naturally occurring opioids on glucose utilization. In conclusion, topical opioids do not appear to produce vascular effects through a change in cerebral metabolism under conditions of anesthesia.
Acknowledgements. The authors thank Drs. R. Ranney Mize and James Reger for imaging and histologicconsultation. The authors also thank Alex Fedinec, John Pirani, Bill Gannawayand Mildred Jackson for excellenttechnicalassistanceduring performanceof the experiments.These studieswere supported by grants from the NIH.
K.D. and Kennedy, C., Local cerebral glucose utilization in fetal and neonatal sheep, Am. Z Physiol., 246 (1984) R608R618.
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