Brain Research, 530 (1990) 267-274
267
Elsevier BRES 15942
Cerebral hemodynamics during cortical spreading depression in rabbits M. Shibata, C.W. Leffler and D.W. Busija Department of Physiology and Biophysics, University of Tennessee, Memphis, TN 38163 (U.S.A.) (Accepted 24 April 1990)
Key words: Cortical spreading depression; Pial arteriolar dilation; Cerebral blood flow; Cranial window; Rabbit; Urethane
Effects of a single cortical spreading depression (CSD), elicited by KCI microinjection, on diameter of pial arterioles and venules in the parieto-occipital cortex were examined in urethane-anesthetized adult rabbits using a closed cranial window. The velocity of CSD propagation was 2.7 + 0.1 mm/min (mean + S.E.M.). All arterioles (n = 39) except for those in the retrospleniol region (n = 6) increased their diameter significantly during CSD. The arteriolar dilation lasted for 1.5 + 0.1 min. Location of dilating arteriole and propagating CSD showed that they were always closely associated temporally. As a percentage change, diameters of smaller arterioles significantly increased (from 60 + 1 to 103 + 2/am, 71%, n = 12) more than those of larger ones (from 82 + 2 to 129 + 3/am, 57%, n = 27). While venules with initial diameter of 85 + 4/am (n = 5) did not dilate, those with initial diameter of 49 + 3/am increased to 57 _+ 3/am (16%, n = 8) for 1.4 + 0.2 min during CSD. The majority of the dilated venules started to increase their diameter after nearby arterioles had dilated maximally. Pial arterioles, which dilated during ipsilateral CSD, decreased their diameter significantly from 78 + 2 to 72 + 3/am (8%, n = 11) during contralateral CSD for 13.8 + 3.6 min with similar onset iatencies as those observed for the dilation. Indomethacin pretreatment significantly enhanced arteriolar dilation during CSD (from 73 + 4 to 138 + 6/am, 89%, n = 4). The results indicate that pial arteriolar dilation observed during CSD is an active response, and probably caused by an excitatory rather than inhibitory effect accompanying CSD, and that prostanoids may play an important modulatory role.
INTRODUCTION Cortical spreading depression (CSD) is a wave of n e u r o n a l depolarization which propagates concentrically from its elicited site throughout the whole tissue with the constant velocity of 2 - 3 m m / m i n 4. CSD is elicited by electrical, chemical (e.g. KCI, excitatory amino acid), or mechanical (e.g. puncture) stimuli. A c c o m p a n y i n g CSD is a brief excitation (2-5 s) that is followed by a prolonged and complete inhibition (1-2 min) of cortical n e u r o n s 6'4°. Although it can be induced repeatedly with interwave intervals of 2 - 4 min, which corresponds to the absolute refractory period of the affected tissue, effects of a single CSD on cortical neuronal activity or on metabolic processes could last for as long as 30 min. Since the discovery of CSD in 1944 by Leao 21'22, CSD has been extensively employed in studies involving functions of the cerebral cortex 2-4'25'35-38 because of its unique properties in inducing reversible functional decortication due to the inhibitory effect. Several of the earlier findings reported during CSD include changes in cerebral hemodynamicss" 22,42. Although conflicting results have been reported, it is now generally recognized that the m a j o r hemodynamic
response during CSD is initial arteriolar vasodilation resulting in an increased cerebral blood flow (CBF) 19'26 followed by a prolonged vasoconstriction 16'45. However, there are a n u m b e r of issues that are not addressed. First, how is CSD related temporally and spatially to dilation of specific arterioles? Second, are vascular responses limited to one or both cerebral hemispheres? Third, do pial venules as well as arterioles dilate? Fourth, since indomethacin p r e t r e a t m e n t attenuates vasodilation to certain dilator stimuli8'23, would i n d o m e t h a c i n eliminate cerebrovascular dilation during CSD? The aim of the present study was to answer these questions in order to investigate the mechanism underlying CSD-induced cerebral h e m o d y n a m i c changes. For this purpose, pial arteriolar and venular diameters were examined during CSD in urethane-anesthetized adult rabbits using a closed cranial window. MATERIALS AND METHODS
Animal preparations Adult rabbits (2.1-3.4 kg, either sex, n = 34) were anesthetized initially with pentothal sodium (20 mg/kg, i.v., Abbott Laboratories), and maintained on urethane (1.6-1.8 g/kg, i.p., Sigma Chem.
Correspondence: M. Shibata, Department of Physiology and Biophysics, University of Tennessee, 894 Union Avenue, Memphis, TN 38163, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
268 Corp.). A femoral artery and vein were catheterized respectively for monitoring blood gases, pH, and blood pressure (BP), or for administration of fluids or drugs. Following intubation, animals were artificially ventilated. Their heads were fixed to stereotaxic frames, and an opening 20 mm in diameter was made in the skull over the parieto-occipital cortex. The dura was cut and reflected to expose the cortical surface. A cranial window, equipped with inlet and outlet ports, was placed in the opening, and fixed to the skull with acrylic dental cement together with anchor screws to ensure mechanical stability of the window. A small opening of the skull (1.0 mm in diameter) was made over the frontal and the occipital cortex outside the window at coordinates of, respectively, 2.0 and 4.0 mm from the midline. The distance between the two openings ranged from 21.0 to 22.5 mm. Glass semimicropipettes (2.0 mm o.d., 10-15 mm long, tip diameter 100-150 ~m) filled with 0.9% saline solution were implanted 1.0 mm below the cortical surface through the small openings, and fixed to the skull with acrylic dental cement. Open ends of the pipettes were then connected with cotton wicks to recording calomel electrodes (impedance, 3-6 K,Q) for monitoring slow potential changes (SPC, a DC-shift) accompanying CSD. A reference electrode of the same type was placed directly on the exposed skull along the midline over the olfactory bulb. SPC was chosen to monitor CSD because the negative shift of SPC closely follows CSD event4 and the time course of ionic movements across the membrane of neurons and glial cells1°'4°'43. In animals with a pair of glass semimicropipettes in both cortices, a reference calomel electrode was connected to one of the pipettes opposite to CSD induction. An additional small opening was made over the frontal cortex 2-3 mm anterior to the frontal pipette for a microinjection of 5% KCI solution (3-5 MI) 1.0 mm below the cortical surface to elicit CSD. In some animals, a similar opening was made posterior to the occipital pipette for the same purpose. Rectal temperature of animals was maintained at 38.2-38.9 °C with a heating pad. The closed cranial window was filled with warmed artificial cerebrospinal fluid (ACSF) gassed with 6% 02/6.5% CO2 in N 2. The ACSF typically exhibited pO 2, pCO z, and pH, of, respectively, 41-46 mm Hg, 36-42 mm Hg, and 7.39-7.45. The composition of rabbit ACSF was as follows (in mM): KCI 2.9, MgCI 2 0.6, CaCI2 1.5, NaCI 131.9, urea 6.7, dextrose 3.7, NaHCO 3 20.2. Images of the exposed cortical surface were projected onto a video monitor through the microscopic video camera set above the window. Diameter change of pial arterioles and venules was recorded on-line via a dimensional analysis system (VPA 1000, FOR A Corp. Ltd.) on a pen-recorder. SPC accompanying CSD was amplified and recorded on the pen-recorder together with BP in separate channels.
Experimental procedures After an initial washout of the window with ACSF, at least 30 min elapsed before any attempt for the control trial. Freshly prepared ACSF was infused into the window, and SPC, diameter changes of a pial arteriole or venule, and BP were simultaneously recorded for at least 10 min. The window was flushed again with ACSF, and a 5% KCI solution was microinjected into the cortex for CSD trials (see 'KCI' in the inset in Fig. 1). Successful induction of a single CSD was confirmed by a large negative shift (10-15 mV) followed by a small positive shift (20 mM) elicits vasoconstriction 15'33. Since CBF increases during CSD 19"26, it is also unlikely that high extracellular K ÷ concentration by CSD induced constriction of penetrating arterioles but resulted in dilation of pial arterioles. The possibility that leaked KCI from the injection sites diffused into the window and dilated pial arterioles can be ruled out because mechanically induced CSD also elicited arterioiar dilation with similar onset latency, magnitude, and duration as that observed during CSD induced by KCI. A recent study reported an increased release of arachidonic acid (AA) in the cortical tissue during CSD Is. This result raises the possibility that A A metabolites such as prostaglandin E a, or prostacyclin, both
potent vasodilator stimuli 3~, may have increased in CSF and resulted in arteriolar dilation. This is, however, unlikely since, in the present study, indomethacin enhanced arteriolar dilation during CSD rather than abolishing it. Our result, therefore, implicates the possibility that an increased level of prostanoids by CSD may play a modulatory constrictor role but not causative dilator role. Further study is required to clarify the role of CSF prostanoids in CSD-induced arteriolar dilation. During CSD, lactic acid increases, and creatine phosphate, glucose, and glycogen decrease in the cortical tissue TM 14,39. Cortical pH shifts toward acidity probably due to the increased lactic acid 28'32. It is not known to what extent any of these might influence changes of pial arteriolar tone or CBF observed during and after CSD. Neurogenic factors may be considered as a cause for CSD-induced arteriolar dilation. Two cortical regions which were not invaded by CSD because of their morphological differences have been reported. These include regions close to the midline (i.e., retrosplenial area), and the lateral boundaries (entorhinal area) 9"41. The retrosplenial region lies between the parasagittal sulcus and the midline in the parietal and occipital cortex. The present finding that pial arterioles in the retrosplenial region failed to dilate during CSD favors a neurogenic factor. If CSD-induced arteriolar dilation is primarily caused by humoral factors released into CSF, arterioles in the retrosplenial region should have exhibited some degree of diameter change due to diffusion of that factor when CSD traveled nearby. Second, though circumstantial, it seems unlikely that the diffusing humoral factors could increase the diameter of pial arterioles as much as 57% to 71% over the baseline level and allow a decrease in arteriolar diameter to the baseline level within 1.5 min (Table I). It is worth to note that Leao predicted neurogenic factors for this same reason some 46 years ago 22. The failure of retrosplenial arterioles to dilate during CSD may also suggest that the system involved in CSD-induced arteriolar dilation may operate exclusively within the cerebral cortex. This view is supported by another finding that there was a close relationship between the onset of arteriolar dilation and the position of extrapolated CSD. Of particular interest is a possibility that cortical gtial cells might play an important role in this system since their foot processes surround the brain capillaries 1'12. In this context, it may be noted that cortical glial cells are also depolarized during CSD 4°. Another possibility to explain pial arteriolar dilation is that the dilator stimuli act originally on parenchymal arterioles and the dilator 'signal' is transmitted to arterioles with larger diameters. Recently, propagation of arteriolar dilation was reported in the hamster cheek pouch 34. The propagating dilation was
273 induced by acetyicholine or norepinephrine and stopped only by gap junction blockers. It was hypothetized that signals spread electrically along arterioles through gap junctions coupling smooth muscle and/or endothelial cells on the cerebral circulation because of the close relationship between glial cells and blood vessels, the former may be mediators of signal propagation. It is unknown whether such mechanism operates in arterioles of the cerebral cortex. The present finding that venular dilation started after arteriolar diameter had reached its maximum, and that only smaller venules showed detectable diameter increases during CSD suggest that the arteriolar dilation is an active response, and that dilation of venules is secondary to the increased blood flow. The mechanism underlying arteriolar vasoconstriction observed during contralateral CSD is not clear. One possibility would be that the vasoconstriction may have been induced by effects of CSD in the contralateral cortex via the corpus callosum since its onset latency closely followed that of vasodilation observed during ipsilateral CSD. This assumption is strengthened by histological and electrophysiological evidence that cortical regions are homotopically interconnected, in part, through the corpus callosum 22"27'29. It is, however, unlikely that the vasoconstriction was induced by autoregulation or elevated systemic BP since no changes in
arterial BP were observed during CSD in the present study. It is also not likely that interhemispheric redistribution of blood due to increased blood flow in the depressed cortex caused the vasoconstriction in the unaffected hemisphere since either induced increase 11 or decrease 7 in blood flow in one hemisphere did not affect blood flow in the other hemisphere. Observations made during vasoconstriction after ipsilateral CSD and those during human migraine headache led some researchers to conclude that CSD-like p h e n o m e n a may be involved in migraine headache 16'17'19'3°. The hypothesis is based on the observations that both CSD in animals and migraine headache in humans share some similarities, such as propagation of cortical area with the reduced blood flow, decreased vasoreactivity to pH, potassium ions, adenosine, bradykinin and CO216'17'20'45, as well as manifestation of paresthesia 2°'ss. In summary, the present study demonstrated that dilation of pial arterioles observed during CSD is an active response, and probably caused by an excitatory effect accompanying CSD involving neurons, glial cells, and perivascular nerves, and that CSF prostanoids may play an important modulatory role.
Acknowledgements. The present study was supported in part by NIH Grant HL 30260 to D.W.B., HL 34059 to C.W.L., and a grant from the University of Tennessee Center of Excellence to M.S.
REFERENCES 1 Abbott, N.J., Bundgaard, M. and Hughes, C.C.M., Morphology of brain microvesseis: a comparative approach. In E Hammersen and K. Messmer (Eds.), Cerebral Microcirculation, Karger, Basel, 1989, pp. 1-19. 2 Bures, J. and Buresova, O., The use of Leao's spreading depression in the study of interhemispheric transfer of memory traces, J. Comp. Physiol. Psychol., 53 (1960) 558-563. 3 Bures, J., Buresova, O., Fifkova, E., Olds, J., Olds, M.E. and Travis, R.P., Spreading depression and subcortical drive centers, Physiol. Bohemoslov., 10 (1961) 321-331. 4 Bures, J., Buresova, O. and Krivanek, J., The Mechanism and Applications of Leao's Spreading Depression of Electroencephalographic Activity, Academia, Prague, 1974. 5 Buresova, O., Changes in cerebral circulation in rats during EEG depression, Physiol. Bohemoslov., 6 (1957) 159-166. 6 Buresova, O., Shima, I., Bures, J. and Fifkova, E., Unit activity in regions affected by the spreading depression, Physiol. Bohemoslov., 12 (1963) 488-494. 7 Busija, D.W., Unilateral and bilateral sympathetic effects on cerebral blood flow during normocapnia, Am. J. Physiol., 250 (1986) H498-H502. 8 Busija, D.W. and Leffler, C.W., Role of prostanoids in cerebrovascular responses during seizures in piglets, Am. J. Physiol., 256 (1989) H120-H125. 9 Fifkova, E., Spreading depression in the neo- paleo- and archicortical structures of the brain of the rat, Physiol. Bohemoslov., 13 (1964) 1-15. 10 Hansen, A.J. and Zeuthen, T., Extracellular ion concentrations during spreading depression and ischemia in the rat brain cortex, Acta Physiol. Scand., 113 (1981) 437-445. 11 Heistad, D.D., Marcus, M.L., Gourley, J.K. and Busija, D.W.,
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Effect of adenosine and dipyridamole on cerebral blood flow, Am. J. Physiol., 240 (1981) H775-H780. Kimelberg, H.K. and Norenberg, M.D., Astrocytes, Sci. Am., April (1989) 66-81. Krivanek, J., Some metabolic changes accompanying Leao's spreading cortical depression, J. Neurochem., 6 (1961) 183-189. Krivanek, J., Concerning the dynamics of the metabolic changes accompanying cortical spreading depression, Physiol. Bohemoslov., 11 (1962) 383-391. Kuschinsky, W. and Wahl, M., Local chemical and neurogenic regulation of cerebral vascular resistance, Physiol. Rev., 58 (1978) 656-689. Lauritzen, M., Long-lasting reduction of cortical blood flow of the rat brain after spreading depression with preserved autoregulation and impaired CO 2 response, J. Cereb. Blood Flow Metabol., 4 (1984) 546-554. Lauritzen, M., Regional cerebral blood flow during cortical spreading depression in rat brain: Increased reactive hyperfusion in low-flow states, Acta Neurol. Scand., 75 (1987) 1-8. Lauritzen, M., Hansen, A.J., Kronborg, D. and Wieloch, T., Cortical spreading depression is associated with arachidonic acid accumulation and preservation of energy charge, J. Cereb. Blood Flow Metabol., 10 (1990) 115-122. Lauritzen, M., Joergensen, M.B., Diemer, N.H., Gjedde, A. and Hansen, A.J., Persistent oligemia of rat cerebral cortex in the wake of spreading depression, Ann. Neurol., 12 (1982) 469-474. Lauritzen, M. and Olesen, J., Regional cerebral blood flow during migraine attacks by Xenon-133 inhalation and emission tomography, Brain, 107 (1984) 447-461. Leao, A.A.P., Spreading depression of activity in the cerebral cortex, J. Neurophysiol., 7 (1944) 359-390. Leao, A.A.P., Pial circulation and spreading depression of
274 activity in cerebral cortex, J. Neurophysiol., 7 (1944) 391-396. 23 Leffler, C.W., Busija, D.W., Fletcher, A.M., Beasley, D.G., Hessler, J.R. and Green, R.S., Effects of indomethacin upon cerebral hemodynamics of newborn pigs, Pediatr. Res., 19 (1985) 1160-1164. 24 Lepore, E, Ptito, M., Richter, L. and Guillemot, J.P., Corticocortical callosal connectivity: evidence derived from electrophysiological studies. In T.P. Hicks and G. Benedek (Ed.), Progress in Brain Research, Vol. 75, Elsevier, Amsterdam, 1988, pp. 187-195. 25 Megirian, D. and Bures, J., Unilateral cortical spreading depression and conditioned eyblink responses in the rabbit, Exp. Neurol., 27 (1970) 34-45. 26 Mies, G. and Hossman, K.A., Double tracer autoradiographic investigation of regional blood flow and glucose metabolism during spreading depression, J. Cereb. Blood Flow Metabol., 1 (1981) $94-$95. 27 Miller, M.W. and Vogt, B.A., Heterotopic and homotopic callosal connections in rat visual cortex, Brain Research, 297 (1984) 75-89. 28 Mutch, W.A.C. and Hansen, A.J., Extracellular pH changes during spreading depression and cerebral ischemia: mechanisms of brain pH regulation, J. Cereb. Blood Flow Metabol., 4 (1984) 17-27. 29 Olavarria, J. and Van Sluyters, R., Organization and postnatal development of callosal connections in the visual cortex of the rat, J. Comp. Neurol., 239 (1985) 1-26. 30 Oiesen, J., Larsen, B. and Lauritzen, M., Focal hyperemia followed by spreading oligemia and impaired activation of rCBF in classic migraine, Ann. Neurol., 9 (1981) 344-352. 31 Pourcyrous, M., Leffler, C.W. and Busija, D.W., Post-asphyxial increases in prostaglandins in cerebrospinal fluid in piglets, Ped. Res., 24 (1988) 229-232. 32 Rapoport, J.L. and Marshall, W.H., Measurement of cortical pH in spreading cortical depression, Am. J. Physiol., 206 (1964) 1177-1180. 33 Ryman, T., Brandt, L., Andersson, K.-E. and Mellergard, P., Regional and species differences in vascular reactivity to extracellular potassium, Acta Physiol. Scand., 136 (1989) 151159.
34 Segal, S.S. and Duling, B.R., Conduction of vasomotor responses in arterioles: a role for cell-to-cell coupling? Am. J. Physiol., 256 (1989) H838-H845. 35 Shibita, M., Unilateral cortical spreading depression in the rat: effects on feeding, drinking and other behaviors, Brain Res. Bull., 3 (1978) 395-400. 36 Shibita, M., Hori, T., Kiyohara, T. and Nakashima, T., Impairment of thermoregulatory cooling behavior by single cortical spreading depression in the rat, Physiol. Behav., 30 (1983) 599-605. 37 Shibata, M., Hori, T., Kiyohara, T. and Nakashima, T., Facilitation of thermoregulatory heating behavior by single cortical spreading depression in the rat, Physiol Behav., 31 (1983) 651-656. 38 Shibata, M., Hori, T., Kiyohara, T. and Nakashima, T., Activity of hypothalamic thermosensitive neurons during cortical spreading depression in the rat, Brain Research, 308 (1984) 255-262. 39 Shinohara, M., DoUinger, B., Brown, G., Rapoport, S. and Sokoloff, L., Cerebral glucose utilization: local changes during and after recovery from spreading cortical depression, Science, 203 (1979) 188-190. 40 Sugaya, E., Takato, M. and Noda, Y., Neuronal and glial activity during spreading depression in cerebral cortex of cat, J. Neurophysiol., 38 (1975) 822-841. 41 Van Harreveld, A. and Bogen, J.E., Regional differences in propagation of spreading cortical depression in the rabbit, Proc. Soc. Exp. Biol. Med., 91 (1956)297-302. 42 Van Harreveld, A. and Ochs, S., Electrical and vascular concomitants of spreading depression, Am. J. Physiol., 189 (1957) 159-166. 43 Vyskocil, F., Kriz, N. and Bures, J., Potassium-selective microelectrodes used for measuring the extracellular brain potassium during spreading depression and anoxic depolarization in rats, Brain Research, 39 (1972) 255-259. 44 Wahl, M. and Kuschinsky, W., Influence of H ÷ or K ÷ on adenosine-induced dilation at pial arterioles of cats, Blood Vessels, 14 (1977) 285-293. 45 Wahl, M., Lauritzen, M. and Schilling, L., Change of cerebrovascular reactivity after cortical spreading depression in cats and rats, Brain Research, 411 (1987) 72-80.
267
Elsevier BRES 15942
Cerebral hemodynamics during cortical spreading depression in rabbits M. Shibata, C.W. Leffler and D.W. Busija Department of Physiology and Biophysics, University of Tennessee, Memphis, TN 38163 (U.S.A.) (Accepted 24 April 1990)
Key words: Cortical spreading depression; Pial arteriolar dilation; Cerebral blood flow; Cranial window; Rabbit; Urethane
Effects of a single cortical spreading depression (CSD), elicited by KCI microinjection, on diameter of pial arterioles and venules in the parieto-occipital cortex were examined in urethane-anesthetized adult rabbits using a closed cranial window. The velocity of CSD propagation was 2.7 + 0.1 mm/min (mean + S.E.M.). All arterioles (n = 39) except for those in the retrospleniol region (n = 6) increased their diameter significantly during CSD. The arteriolar dilation lasted for 1.5 + 0.1 min. Location of dilating arteriole and propagating CSD showed that they were always closely associated temporally. As a percentage change, diameters of smaller arterioles significantly increased (from 60 + 1 to 103 + 2/am, 71%, n = 12) more than those of larger ones (from 82 + 2 to 129 + 3/am, 57%, n = 27). While venules with initial diameter of 85 + 4/am (n = 5) did not dilate, those with initial diameter of 49 + 3/am increased to 57 _+ 3/am (16%, n = 8) for 1.4 + 0.2 min during CSD. The majority of the dilated venules started to increase their diameter after nearby arterioles had dilated maximally. Pial arterioles, which dilated during ipsilateral CSD, decreased their diameter significantly from 78 + 2 to 72 + 3/am (8%, n = 11) during contralateral CSD for 13.8 + 3.6 min with similar onset iatencies as those observed for the dilation. Indomethacin pretreatment significantly enhanced arteriolar dilation during CSD (from 73 + 4 to 138 + 6/am, 89%, n = 4). The results indicate that pial arteriolar dilation observed during CSD is an active response, and probably caused by an excitatory rather than inhibitory effect accompanying CSD, and that prostanoids may play an important modulatory role.
INTRODUCTION Cortical spreading depression (CSD) is a wave of n e u r o n a l depolarization which propagates concentrically from its elicited site throughout the whole tissue with the constant velocity of 2 - 3 m m / m i n 4. CSD is elicited by electrical, chemical (e.g. KCI, excitatory amino acid), or mechanical (e.g. puncture) stimuli. A c c o m p a n y i n g CSD is a brief excitation (2-5 s) that is followed by a prolonged and complete inhibition (1-2 min) of cortical n e u r o n s 6'4°. Although it can be induced repeatedly with interwave intervals of 2 - 4 min, which corresponds to the absolute refractory period of the affected tissue, effects of a single CSD on cortical neuronal activity or on metabolic processes could last for as long as 30 min. Since the discovery of CSD in 1944 by Leao 21'22, CSD has been extensively employed in studies involving functions of the cerebral cortex 2-4'25'35-38 because of its unique properties in inducing reversible functional decortication due to the inhibitory effect. Several of the earlier findings reported during CSD include changes in cerebral hemodynamicss" 22,42. Although conflicting results have been reported, it is now generally recognized that the m a j o r hemodynamic
response during CSD is initial arteriolar vasodilation resulting in an increased cerebral blood flow (CBF) 19'26 followed by a prolonged vasoconstriction 16'45. However, there are a n u m b e r of issues that are not addressed. First, how is CSD related temporally and spatially to dilation of specific arterioles? Second, are vascular responses limited to one or both cerebral hemispheres? Third, do pial venules as well as arterioles dilate? Fourth, since indomethacin p r e t r e a t m e n t attenuates vasodilation to certain dilator stimuli8'23, would i n d o m e t h a c i n eliminate cerebrovascular dilation during CSD? The aim of the present study was to answer these questions in order to investigate the mechanism underlying CSD-induced cerebral h e m o d y n a m i c changes. For this purpose, pial arteriolar and venular diameters were examined during CSD in urethane-anesthetized adult rabbits using a closed cranial window. MATERIALS AND METHODS
Animal preparations Adult rabbits (2.1-3.4 kg, either sex, n = 34) were anesthetized initially with pentothal sodium (20 mg/kg, i.v., Abbott Laboratories), and maintained on urethane (1.6-1.8 g/kg, i.p., Sigma Chem.
Correspondence: M. Shibata, Department of Physiology and Biophysics, University of Tennessee, 894 Union Avenue, Memphis, TN 38163, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
268 Corp.). A femoral artery and vein were catheterized respectively for monitoring blood gases, pH, and blood pressure (BP), or for administration of fluids or drugs. Following intubation, animals were artificially ventilated. Their heads were fixed to stereotaxic frames, and an opening 20 mm in diameter was made in the skull over the parieto-occipital cortex. The dura was cut and reflected to expose the cortical surface. A cranial window, equipped with inlet and outlet ports, was placed in the opening, and fixed to the skull with acrylic dental cement together with anchor screws to ensure mechanical stability of the window. A small opening of the skull (1.0 mm in diameter) was made over the frontal and the occipital cortex outside the window at coordinates of, respectively, 2.0 and 4.0 mm from the midline. The distance between the two openings ranged from 21.0 to 22.5 mm. Glass semimicropipettes (2.0 mm o.d., 10-15 mm long, tip diameter 100-150 ~m) filled with 0.9% saline solution were implanted 1.0 mm below the cortical surface through the small openings, and fixed to the skull with acrylic dental cement. Open ends of the pipettes were then connected with cotton wicks to recording calomel electrodes (impedance, 3-6 K,Q) for monitoring slow potential changes (SPC, a DC-shift) accompanying CSD. A reference electrode of the same type was placed directly on the exposed skull along the midline over the olfactory bulb. SPC was chosen to monitor CSD because the negative shift of SPC closely follows CSD event4 and the time course of ionic movements across the membrane of neurons and glial cells1°'4°'43. In animals with a pair of glass semimicropipettes in both cortices, a reference calomel electrode was connected to one of the pipettes opposite to CSD induction. An additional small opening was made over the frontal cortex 2-3 mm anterior to the frontal pipette for a microinjection of 5% KCI solution (3-5 MI) 1.0 mm below the cortical surface to elicit CSD. In some animals, a similar opening was made posterior to the occipital pipette for the same purpose. Rectal temperature of animals was maintained at 38.2-38.9 °C with a heating pad. The closed cranial window was filled with warmed artificial cerebrospinal fluid (ACSF) gassed with 6% 02/6.5% CO2 in N 2. The ACSF typically exhibited pO 2, pCO z, and pH, of, respectively, 41-46 mm Hg, 36-42 mm Hg, and 7.39-7.45. The composition of rabbit ACSF was as follows (in mM): KCI 2.9, MgCI 2 0.6, CaCI2 1.5, NaCI 131.9, urea 6.7, dextrose 3.7, NaHCO 3 20.2. Images of the exposed cortical surface were projected onto a video monitor through the microscopic video camera set above the window. Diameter change of pial arterioles and venules was recorded on-line via a dimensional analysis system (VPA 1000, FOR A Corp. Ltd.) on a pen-recorder. SPC accompanying CSD was amplified and recorded on the pen-recorder together with BP in separate channels.
Experimental procedures After an initial washout of the window with ACSF, at least 30 min elapsed before any attempt for the control trial. Freshly prepared ACSF was infused into the window, and SPC, diameter changes of a pial arteriole or venule, and BP were simultaneously recorded for at least 10 min. The window was flushed again with ACSF, and a 5% KCI solution was microinjected into the cortex for CSD trials (see 'KCI' in the inset in Fig. 1). Successful induction of a single CSD was confirmed by a large negative shift (10-15 mV) followed by a small positive shift (20 mM) elicits vasoconstriction 15'33. Since CBF increases during CSD 19"26, it is also unlikely that high extracellular K ÷ concentration by CSD induced constriction of penetrating arterioles but resulted in dilation of pial arterioles. The possibility that leaked KCI from the injection sites diffused into the window and dilated pial arterioles can be ruled out because mechanically induced CSD also elicited arterioiar dilation with similar onset latency, magnitude, and duration as that observed during CSD induced by KCI. A recent study reported an increased release of arachidonic acid (AA) in the cortical tissue during CSD Is. This result raises the possibility that A A metabolites such as prostaglandin E a, or prostacyclin, both
potent vasodilator stimuli 3~, may have increased in CSF and resulted in arteriolar dilation. This is, however, unlikely since, in the present study, indomethacin enhanced arteriolar dilation during CSD rather than abolishing it. Our result, therefore, implicates the possibility that an increased level of prostanoids by CSD may play a modulatory constrictor role but not causative dilator role. Further study is required to clarify the role of CSF prostanoids in CSD-induced arteriolar dilation. During CSD, lactic acid increases, and creatine phosphate, glucose, and glycogen decrease in the cortical tissue TM 14,39. Cortical pH shifts toward acidity probably due to the increased lactic acid 28'32. It is not known to what extent any of these might influence changes of pial arteriolar tone or CBF observed during and after CSD. Neurogenic factors may be considered as a cause for CSD-induced arteriolar dilation. Two cortical regions which were not invaded by CSD because of their morphological differences have been reported. These include regions close to the midline (i.e., retrosplenial area), and the lateral boundaries (entorhinal area) 9"41. The retrosplenial region lies between the parasagittal sulcus and the midline in the parietal and occipital cortex. The present finding that pial arterioles in the retrosplenial region failed to dilate during CSD favors a neurogenic factor. If CSD-induced arteriolar dilation is primarily caused by humoral factors released into CSF, arterioles in the retrosplenial region should have exhibited some degree of diameter change due to diffusion of that factor when CSD traveled nearby. Second, though circumstantial, it seems unlikely that the diffusing humoral factors could increase the diameter of pial arterioles as much as 57% to 71% over the baseline level and allow a decrease in arteriolar diameter to the baseline level within 1.5 min (Table I). It is worth to note that Leao predicted neurogenic factors for this same reason some 46 years ago 22. The failure of retrosplenial arterioles to dilate during CSD may also suggest that the system involved in CSD-induced arteriolar dilation may operate exclusively within the cerebral cortex. This view is supported by another finding that there was a close relationship between the onset of arteriolar dilation and the position of extrapolated CSD. Of particular interest is a possibility that cortical gtial cells might play an important role in this system since their foot processes surround the brain capillaries 1'12. In this context, it may be noted that cortical glial cells are also depolarized during CSD 4°. Another possibility to explain pial arteriolar dilation is that the dilator stimuli act originally on parenchymal arterioles and the dilator 'signal' is transmitted to arterioles with larger diameters. Recently, propagation of arteriolar dilation was reported in the hamster cheek pouch 34. The propagating dilation was
273 induced by acetyicholine or norepinephrine and stopped only by gap junction blockers. It was hypothetized that signals spread electrically along arterioles through gap junctions coupling smooth muscle and/or endothelial cells on the cerebral circulation because of the close relationship between glial cells and blood vessels, the former may be mediators of signal propagation. It is unknown whether such mechanism operates in arterioles of the cerebral cortex. The present finding that venular dilation started after arteriolar diameter had reached its maximum, and that only smaller venules showed detectable diameter increases during CSD suggest that the arteriolar dilation is an active response, and that dilation of venules is secondary to the increased blood flow. The mechanism underlying arteriolar vasoconstriction observed during contralateral CSD is not clear. One possibility would be that the vasoconstriction may have been induced by effects of CSD in the contralateral cortex via the corpus callosum since its onset latency closely followed that of vasodilation observed during ipsilateral CSD. This assumption is strengthened by histological and electrophysiological evidence that cortical regions are homotopically interconnected, in part, through the corpus callosum 22"27'29. It is, however, unlikely that the vasoconstriction was induced by autoregulation or elevated systemic BP since no changes in
arterial BP were observed during CSD in the present study. It is also not likely that interhemispheric redistribution of blood due to increased blood flow in the depressed cortex caused the vasoconstriction in the unaffected hemisphere since either induced increase 11 or decrease 7 in blood flow in one hemisphere did not affect blood flow in the other hemisphere. Observations made during vasoconstriction after ipsilateral CSD and those during human migraine headache led some researchers to conclude that CSD-like p h e n o m e n a may be involved in migraine headache 16'17'19'3°. The hypothesis is based on the observations that both CSD in animals and migraine headache in humans share some similarities, such as propagation of cortical area with the reduced blood flow, decreased vasoreactivity to pH, potassium ions, adenosine, bradykinin and CO216'17'20'45, as well as manifestation of paresthesia 2°'ss. In summary, the present study demonstrated that dilation of pial arterioles observed during CSD is an active response, and probably caused by an excitatory effect accompanying CSD involving neurons, glial cells, and perivascular nerves, and that CSF prostanoids may play an important modulatory role.
Acknowledgements. The present study was supported in part by NIH Grant HL 30260 to D.W.B., HL 34059 to C.W.L., and a grant from the University of Tennessee Center of Excellence to M.S.
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