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Effect of naloxone on neurologic deficit and cortical blood flow during focal cerebral ischemia in cats
Life Sciences, Vol. 31, pp. 2205-2208 Printed in the U.S.A.
Pergamon Pres~
EFFECT OF NALOXONE ON NEUROLOGIC DEFICIT AND CORTICAL BLOOD FLOW DURING FOCAL CEREBRAL ISCHEMIA IN CATS Robert Levy 1, Paul F e u s t e l 2, John S e v e r i n g h a u s 2 and Yoshio Hosobuchi 1 iDepartment of Neurosurgery and 2Department of Anesthesia, University of California, San Francisco, CA 94143. (Received in final form June 14, 1982) Summary Using middle cerebral artery occlusion in the cat as a model of focal cerebral ischemia, we investigated the effect of naloxone on both neurologic deficit and regional cortical blood flow during cerebral ischemia. In all animals with major strokes, 2 mg/ml naloxone administered intravenously produced a dramatic reversal of neurologic symptoms four hours after the ischemic lesion. Animals were then anesthetized and cortical blood flow was measured by the hydrogen clearance method. Blood flow in the ischemic cortex was noted to be approximately 55% that of the control side. Naloxone produced a significant decrease of approximately 20% in cortical blood flow in the ischemic hemisphere while no effect on blood flow on the control side was noted. Thus, naloxone appears to reverse the neurologic deficits following middle cerebral artery occlusion in the cat. This effect appears to be accompanied by a decrease in local blood flow to the ischemic cortex.
Since the discovery of opiate receptors in the central nervous system (1,2,3), it has become apparent that endogenous opiate peptides subserve important roles in both health and disease. While many studies have elucidated the role of endorphins in the modulation and perception of pain (4,5), these compounds have also recently been implicated in the regulation of heat adaptation (6), pituitary function (7) and mental illness (8) as well as in endotoxic (9), hypovolemic (i0) and spinal shock (II). Hosobuchi and Baskin (12) have observed that the administration of the opiate antagonist naloxone reverses the ischemic neurologic deficit in gerbils following unilateral carotid artery occlusion. These same investigators have also demonstrated that the intravenous administration of naloxone reverses much of the neurologic deficit seen in two patients with symptoms referable to cerebral ischemia (13). Young and his coworkers (14) have demonstrated that the opiate receptor antagonist naloxone can increase blood flow to the ischemic spinal cord following experimental spinal contusion. To determine whether naloxone is effective in reversing the neurologic deficits following stroke in other species, and to determine whether this action is mediated by an alteration in blood flow to the ischemic cortex, we have investigated the role of this opiate antagonist on stroke symptomatology and regional cerebral blood flow using middle cerebral artery occlusion in the cat as a model of acute cerebral ischemia.
0024-3205/82/202205-04503.00/0 Copyright (c) 1982 Pergamon Press Ltd.
2206
Naloxone
in Experimental
Stroke
Vol.
31, No.s
20 & 21, 1982
Methods Nine cats were anesthetized with halothane, intubated, and artificially ventilated. Inspired gas consisted of 70% N 2 and 30%02 with halothane added to achieve an end-tidal fraction of 1.0%. Anesthetic and respiratory gases were monitored with a P e r k i n - E l m e r ii00 mass spectrometer. An added peak detection circuit sampled the m a x i m u m CO 2 as end-tidal CO 2 for m o n i t o r i n g and recording. The transorbital approach to the middle cerebral artery (MCA) was employed. The contents of the right globe were evacuated. Using an operating microscope (Ziess OPMII), the optic foramen was enlarged laterally and the middle cerebral artery was exposed by opening the dura and retracting the frontal lobe superiorly. The MCA was dissected from the surface of the brain as far m e d i a l l y as possible, cauterized with m i c r o b i p o l a r forceps and divided. The craniectomy was closed with dental acrylic, the orbit packed with gelfoam and sutured shut. A venous catheter was placed in the femoral vein for the later a d m i n i s t r a t i o n of naloxone or saline. The halothane was removed and the cat was allowed to ~ecover from anesthesia. Four hours after middle cerebral artery occlusion, animals were evaluated for neurologic deficit in an open field paradigm. Naloxone was then tested in a crossover double blind fashion against saline. Animals were injected with either saline or 2 mg /kg naloxone via an indwelling venous catheter and observed for 30 minutes following injection. Three observers were then asked to identify the naloxone injection on the basis of neurologic improvement over the testing period. Animals were then placed under light halothane anesthesia, intubated and artificially ventilated. A tracheostomy was performed and the animals paralyzed as needed with gallamine triethiodide (2.5 mg/kg). A femoral arterial cannula was inserted for sampling arterial blood and monitoring of blood pressure. The femoral venous line was used for administration of bicarbonate to correct any metabolic acidosis. Temperature was monitored with a rectal probe and controlled with a heating pad. The head was fixed and a 20mm by 3mm opening was made on the right side of the skull parallel and approxim a t e l y lOmm lateral to the midline along the suprasylvian and ectosylvian gyri from the rostral suprasylvian sulcus to the caudal suprasylvian sulcus in the occipital cortex. A smaller 3mm by 3mm hole was made on the left, corresponding in position to the anterior portion of the contralateral opening. Electrodes were cut from 25 m i c r o n diameter teflon coated platinum (Pt) wire. The dura was then opened and the electrodes were inserted into the cortex a distance of 1 to 2 mm. Pial vessel were avoided. Three electrodes were placed into the left cortex. Seven electrodes were placed on the right side over an area extending from the suprasylvian gyrus to the occipital cortex. Monitoring of end tidal CO 2 and periodic blood gas determinations (Pco2, Po2, and pH) prevented CO 2 changes and systemic acidosis throughout the experiment. The output from all ten electrodes together with the blood pressure and end-tidal Pco 2 signals were digitized and stored by the PDP 11/34 computer. Local cerebral blood flow was measured by the H 2 clearance technique (15). The Pt electrodes were maintained at 0.3 volts positive with respect to a silver/silver chloride reference electrode. The H 2 o x i d a t i o n reaction at the platinum surface generated a current proportional to tissue H 2 concentration. After waiting 20 m i n u t e s for stabilization, N 2 delivery was shut off and H 2 substituted for a short period of time. H 2 o x i d a t i o n current was sampled at one hertz. The H 2 clearance from the tissue was then measured. This procedure was repeated three times and the flows calculated from the H 2 clearance averaged. Following three baseline clearance measurements, naloxone (2 mg/kg) was administered via the indwelling venous catheter. Repeated infusions of 1 mg/kg naloxone every 15 minutes were a d m i n i s t e r e d over the time course of three clearance m e a s u r e m e n t s as described above. One hour following the final
Vol. 31, No.s 20 & 21, 1982
Naloxone in Experimental Stroke
2207
dose of naloxone, a series of three post-naloxone H 2 clearances were obtained. To calculate regional cortical blood flow, computer analysis with a single compartment model was employed. The first 20% was eliminated to permit the arterial concentration to fall to a negligible level before beginning analysis. If a curve was multiexponential, as described by some investigators using larger electrodes (16,17,18) then this technique yields a flow closer to the higher component, both because of the weighting and the elimination of the later points (unless, of course, the faster component has a very small contribution to the total flow, e.g. a contribution of less than 20% will not be seen). The natural logarithm of the difference of current over the interval of time for all points beginning between 80% and 30% of the peak H 2 current was determined. The slope of the linear regression with time of these values is the inverse time constant.
Results Of the nine operated animals, all developed neurologic symptoms. Four of the nine were able to walk following surgery (minor strokes) while the remaining five had neurologic deficits so severe that they were unable to ambulate (major strokes). Of the animals with minor strokes, two were unaffected by either saline or naloxone in double blind testing, and as such, were not included in subsequent measures of cerebral blood flow. The two other cats with minor symptoms were effected equally by both naloxone and saline, with a small decrease in their neurologic deficits. In the severely stroked animals, on the other hand, naloxone alone produced a dramatic reversal of neurologic symptoms. This effect began within two minutes of intravenous administration of naloxone and in all but one case was gone within 20 minutes of injection. One cat in this group of major strokes developed cerebral edema of such severity that cortical blood flow was essentially absent, and subsequent blood flow measurements in this animal were impossible. Thus, regional cerebral blood flow was measured in six animals. Cortical blood flow in the control hemisphere, as determined by the average of 15 electrodes in six animals, was 39.34 ~ 4.16 ml/lO0 gm/mln, while blood flow on the occluded hemisphere, with a sample size of 28 electrodes, was 21.61 + 2.22 ml/lOOgm/min. This represents a 45.1% decrease in flow six hours follow~ng middle cerebral artery occlusion. Administration of naloxone as described above was not accompanied by any changes in systemic blood pressure. Naloxone had no significant effect on the cortical blood flow in the control hemisphere (40.84 + 5.40 ml/lO0 gm/min). In the ischemic hemisphere, however, naloxone produced a significant decrease in cortical blood flow (17.58 ~ 2.10 ml/lO0 gm/min; t=3.52, p
2208
Naloxone in Experimental
Stroke
Vol. 31, No.s 20 & 21, 1982
TABLE I EFFECT OF NALOXONE ON CORTICAL BLOOD FLOW AFTER MIDDLE CEREBRAL ARTERY OCCLUSION IN CATS. CONTROL HEMISPHERE CONDITION
FLOW (ml/lOOgm/min)
STROKED HEMISPHERE FLOW (ml/lO0gm/min)
% CONTROL
% CONTROL
Control
39.34 + 4.16
Naloxone
40.84 + 5.40
102.9 + 7.3
17.58 + 2.10
82.4 + 5.0
Post-Naloxone
44.50 + 5.93
112.3 + 7.6
17.29 + 2.57
78.7 + 7.8
* n=25,
t=2.73,
p
21.61 + 2.22
** n=2 , t=3.58,
p<0.005
increasing blood flow to those subcortlcal centers subserving movement, this increase in flow might draw from the already diminished cortical flow. Unlike the healthy cortical vessel, the vessels in the ischemlc cortex are unable to compensate for this decrease in pressure and blood flow is decreased. This hypothesis gains support from the observation that the movements initiated by naloxone administration in baboons with ischemlc lesions appear to be of the gross subcortical, rather than the fine cortical variety (19). We have thus demonstrated the reversal of neurologic deficits following middle cerebral artery occlusion in the cat. This effect appears to he accompanied by a decrease in local blood flow in the ischemic cortex. This work suggests that not only may .endogenous opioid peptldes be involved in the pathophysiology of the neurologic deficit associated with cerebral ischemia, but that the severe deficits seen in patients with stroke may be at least partially reversible. References I. C.B. PERT and S.H. SNYDER, Science 179, 1011-1014 (1973). 2. L. TERENIUS, Acta Pharmacol. Toxicol. 33, 377-384 (1973). 3. E.J. SIMON, J.M. HILLER and I. EDELMAN, Proc. Nat. Acad. Sci. 70, 19471949 (1973). 4. H. AKIL, et al., Science 201, 463-465 (1978). 5. Y. HOSOBUCHI, et al., Science 201 279-281 (1979). 6. A. COWAN, P.W. DETTMAR and G. METCALF, in: Endogenous and Exogenous Opiate Agonists and Antagonists (Ed: E.L. WAY), 475-478, Pergamon Press: New York (1979). 7. D.A. VAN VUGT and J. MEITES, Fed. Proc. 39, 2533-2538 (1980). 8. L.H. LINDSTROM, et al., Acta Psychiat. Scand. 57, 153-164 (1978). 9. D.G. REYNOLDS, et al., Circulatory Shock ~, 39-48 (1980). I0. A.I. FADEN and J.W. HOLADAY, Science 205, 317-318 (1979). Ii. A.I. FADEN, T.P. JACOBS and J.W. HOLADAY, Science 211, 493-494 (1981). 12. Y. HOSOBUCHI, D.S. BASKIN and S.K. WOO, Science 215, 69-71 (1981). 13. D.S. BASKIN and Y. HOSOBUCHI, Lancet 11-1981, 272-275 (1981). 14. W. YOUNG, et al., J. Neurosurg. 55, 209-219 (1981). 15. K. AUKLAND, B.F. BOWER and R.W. BERLINER, Circ. Res. 14, 164-187 (1964). 16. E. PAZSTOR, et al., Stroke ~, 556-567 (1973). 17. L. SYMON, N.M. BRANSTON and O. CHIKOVANI, Stroke iO, 184-191 (1979). 18. A.D. JAMIESON and J.H. HALSEY, Stroke 4, 904-911 (1973). 19. D.S. BASKIN and Y. HOSOBUCHI, personal communication (1982).
Pergamon Pres~
EFFECT OF NALOXONE ON NEUROLOGIC DEFICIT AND CORTICAL BLOOD FLOW DURING FOCAL CEREBRAL ISCHEMIA IN CATS Robert Levy 1, Paul F e u s t e l 2, John S e v e r i n g h a u s 2 and Yoshio Hosobuchi 1 iDepartment of Neurosurgery and 2Department of Anesthesia, University of California, San Francisco, CA 94143. (Received in final form June 14, 1982) Summary Using middle cerebral artery occlusion in the cat as a model of focal cerebral ischemia, we investigated the effect of naloxone on both neurologic deficit and regional cortical blood flow during cerebral ischemia. In all animals with major strokes, 2 mg/ml naloxone administered intravenously produced a dramatic reversal of neurologic symptoms four hours after the ischemic lesion. Animals were then anesthetized and cortical blood flow was measured by the hydrogen clearance method. Blood flow in the ischemic cortex was noted to be approximately 55% that of the control side. Naloxone produced a significant decrease of approximately 20% in cortical blood flow in the ischemic hemisphere while no effect on blood flow on the control side was noted. Thus, naloxone appears to reverse the neurologic deficits following middle cerebral artery occlusion in the cat. This effect appears to be accompanied by a decrease in local blood flow to the ischemic cortex.
Since the discovery of opiate receptors in the central nervous system (1,2,3), it has become apparent that endogenous opiate peptides subserve important roles in both health and disease. While many studies have elucidated the role of endorphins in the modulation and perception of pain (4,5), these compounds have also recently been implicated in the regulation of heat adaptation (6), pituitary function (7) and mental illness (8) as well as in endotoxic (9), hypovolemic (i0) and spinal shock (II). Hosobuchi and Baskin (12) have observed that the administration of the opiate antagonist naloxone reverses the ischemic neurologic deficit in gerbils following unilateral carotid artery occlusion. These same investigators have also demonstrated that the intravenous administration of naloxone reverses much of the neurologic deficit seen in two patients with symptoms referable to cerebral ischemia (13). Young and his coworkers (14) have demonstrated that the opiate receptor antagonist naloxone can increase blood flow to the ischemic spinal cord following experimental spinal contusion. To determine whether naloxone is effective in reversing the neurologic deficits following stroke in other species, and to determine whether this action is mediated by an alteration in blood flow to the ischemic cortex, we have investigated the role of this opiate antagonist on stroke symptomatology and regional cerebral blood flow using middle cerebral artery occlusion in the cat as a model of acute cerebral ischemia.
0024-3205/82/202205-04503.00/0 Copyright (c) 1982 Pergamon Press Ltd.
2206
Naloxone
in Experimental
Stroke
Vol.
31, No.s
20 & 21, 1982
Methods Nine cats were anesthetized with halothane, intubated, and artificially ventilated. Inspired gas consisted of 70% N 2 and 30%02 with halothane added to achieve an end-tidal fraction of 1.0%. Anesthetic and respiratory gases were monitored with a P e r k i n - E l m e r ii00 mass spectrometer. An added peak detection circuit sampled the m a x i m u m CO 2 as end-tidal CO 2 for m o n i t o r i n g and recording. The transorbital approach to the middle cerebral artery (MCA) was employed. The contents of the right globe were evacuated. Using an operating microscope (Ziess OPMII), the optic foramen was enlarged laterally and the middle cerebral artery was exposed by opening the dura and retracting the frontal lobe superiorly. The MCA was dissected from the surface of the brain as far m e d i a l l y as possible, cauterized with m i c r o b i p o l a r forceps and divided. The craniectomy was closed with dental acrylic, the orbit packed with gelfoam and sutured shut. A venous catheter was placed in the femoral vein for the later a d m i n i s t r a t i o n of naloxone or saline. The halothane was removed and the cat was allowed to ~ecover from anesthesia. Four hours after middle cerebral artery occlusion, animals were evaluated for neurologic deficit in an open field paradigm. Naloxone was then tested in a crossover double blind fashion against saline. Animals were injected with either saline or 2 mg /kg naloxone via an indwelling venous catheter and observed for 30 minutes following injection. Three observers were then asked to identify the naloxone injection on the basis of neurologic improvement over the testing period. Animals were then placed under light halothane anesthesia, intubated and artificially ventilated. A tracheostomy was performed and the animals paralyzed as needed with gallamine triethiodide (2.5 mg/kg). A femoral arterial cannula was inserted for sampling arterial blood and monitoring of blood pressure. The femoral venous line was used for administration of bicarbonate to correct any metabolic acidosis. Temperature was monitored with a rectal probe and controlled with a heating pad. The head was fixed and a 20mm by 3mm opening was made on the right side of the skull parallel and approxim a t e l y lOmm lateral to the midline along the suprasylvian and ectosylvian gyri from the rostral suprasylvian sulcus to the caudal suprasylvian sulcus in the occipital cortex. A smaller 3mm by 3mm hole was made on the left, corresponding in position to the anterior portion of the contralateral opening. Electrodes were cut from 25 m i c r o n diameter teflon coated platinum (Pt) wire. The dura was then opened and the electrodes were inserted into the cortex a distance of 1 to 2 mm. Pial vessel were avoided. Three electrodes were placed into the left cortex. Seven electrodes were placed on the right side over an area extending from the suprasylvian gyrus to the occipital cortex. Monitoring of end tidal CO 2 and periodic blood gas determinations (Pco2, Po2, and pH) prevented CO 2 changes and systemic acidosis throughout the experiment. The output from all ten electrodes together with the blood pressure and end-tidal Pco 2 signals were digitized and stored by the PDP 11/34 computer. Local cerebral blood flow was measured by the H 2 clearance technique (15). The Pt electrodes were maintained at 0.3 volts positive with respect to a silver/silver chloride reference electrode. The H 2 o x i d a t i o n reaction at the platinum surface generated a current proportional to tissue H 2 concentration. After waiting 20 m i n u t e s for stabilization, N 2 delivery was shut off and H 2 substituted for a short period of time. H 2 o x i d a t i o n current was sampled at one hertz. The H 2 clearance from the tissue was then measured. This procedure was repeated three times and the flows calculated from the H 2 clearance averaged. Following three baseline clearance measurements, naloxone (2 mg/kg) was administered via the indwelling venous catheter. Repeated infusions of 1 mg/kg naloxone every 15 minutes were a d m i n i s t e r e d over the time course of three clearance m e a s u r e m e n t s as described above. One hour following the final
Vol. 31, No.s 20 & 21, 1982
Naloxone in Experimental Stroke
2207
dose of naloxone, a series of three post-naloxone H 2 clearances were obtained. To calculate regional cortical blood flow, computer analysis with a single compartment model was employed. The first 20% was eliminated to permit the arterial concentration to fall to a negligible level before beginning analysis. If a curve was multiexponential, as described by some investigators using larger electrodes (16,17,18) then this technique yields a flow closer to the higher component, both because of the weighting and the elimination of the later points (unless, of course, the faster component has a very small contribution to the total flow, e.g. a contribution of less than 20% will not be seen). The natural logarithm of the difference of current over the interval of time for all points beginning between 80% and 30% of the peak H 2 current was determined. The slope of the linear regression with time of these values is the inverse time constant.
Results Of the nine operated animals, all developed neurologic symptoms. Four of the nine were able to walk following surgery (minor strokes) while the remaining five had neurologic deficits so severe that they were unable to ambulate (major strokes). Of the animals with minor strokes, two were unaffected by either saline or naloxone in double blind testing, and as such, were not included in subsequent measures of cerebral blood flow. The two other cats with minor symptoms were effected equally by both naloxone and saline, with a small decrease in their neurologic deficits. In the severely stroked animals, on the other hand, naloxone alone produced a dramatic reversal of neurologic symptoms. This effect began within two minutes of intravenous administration of naloxone and in all but one case was gone within 20 minutes of injection. One cat in this group of major strokes developed cerebral edema of such severity that cortical blood flow was essentially absent, and subsequent blood flow measurements in this animal were impossible. Thus, regional cerebral blood flow was measured in six animals. Cortical blood flow in the control hemisphere, as determined by the average of 15 electrodes in six animals, was 39.34 ~ 4.16 ml/lO0 gm/mln, while blood flow on the occluded hemisphere, with a sample size of 28 electrodes, was 21.61 + 2.22 ml/lOOgm/min. This represents a 45.1% decrease in flow six hours follow~ng middle cerebral artery occlusion. Administration of naloxone as described above was not accompanied by any changes in systemic blood pressure. Naloxone had no significant effect on the cortical blood flow in the control hemisphere (40.84 + 5.40 ml/lO0 gm/min). In the ischemic hemisphere, however, naloxone produced a significant decrease in cortical blood flow (17.58 ~ 2.10 ml/lO0 gm/min; t=3.52, p
2208
Naloxone in Experimental
Stroke
Vol. 31, No.s 20 & 21, 1982
TABLE I EFFECT OF NALOXONE ON CORTICAL BLOOD FLOW AFTER MIDDLE CEREBRAL ARTERY OCCLUSION IN CATS. CONTROL HEMISPHERE CONDITION
FLOW (ml/lOOgm/min)
STROKED HEMISPHERE FLOW (ml/lO0gm/min)
% CONTROL
% CONTROL
Control
39.34 + 4.16
Naloxone
40.84 + 5.40
102.9 + 7.3
17.58 + 2.10
82.4 + 5.0
Post-Naloxone
44.50 + 5.93
112.3 + 7.6
17.29 + 2.57
78.7 + 7.8
* n=25,
t=2.73,
p
21.61 + 2.22
** n=2 , t=3.58,
p<0.005
increasing blood flow to those subcortlcal centers subserving movement, this increase in flow might draw from the already diminished cortical flow. Unlike the healthy cortical vessel, the vessels in the ischemlc cortex are unable to compensate for this decrease in pressure and blood flow is decreased. This hypothesis gains support from the observation that the movements initiated by naloxone administration in baboons with ischemlc lesions appear to be of the gross subcortical, rather than the fine cortical variety (19). We have thus demonstrated the reversal of neurologic deficits following middle cerebral artery occlusion in the cat. This effect appears to he accompanied by a decrease in local blood flow in the ischemic cortex. This work suggests that not only may .endogenous opioid peptldes be involved in the pathophysiology of the neurologic deficit associated with cerebral ischemia, but that the severe deficits seen in patients with stroke may be at least partially reversible. References I. C.B. PERT and S.H. SNYDER, Science 179, 1011-1014 (1973). 2. L. TERENIUS, Acta Pharmacol. Toxicol. 33, 377-384 (1973). 3. E.J. SIMON, J.M. HILLER and I. EDELMAN, Proc. Nat. Acad. Sci. 70, 19471949 (1973). 4. H. AKIL, et al., Science 201, 463-465 (1978). 5. Y. HOSOBUCHI, et al., Science 201 279-281 (1979). 6. A. COWAN, P.W. DETTMAR and G. METCALF, in: Endogenous and Exogenous Opiate Agonists and Antagonists (Ed: E.L. WAY), 475-478, Pergamon Press: New York (1979). 7. D.A. VAN VUGT and J. MEITES, Fed. Proc. 39, 2533-2538 (1980). 8. L.H. LINDSTROM, et al., Acta Psychiat. Scand. 57, 153-164 (1978). 9. D.G. REYNOLDS, et al., Circulatory Shock ~, 39-48 (1980). I0. A.I. FADEN and J.W. HOLADAY, Science 205, 317-318 (1979). Ii. A.I. FADEN, T.P. JACOBS and J.W. HOLADAY, Science 211, 493-494 (1981). 12. Y. HOSOBUCHI, D.S. BASKIN and S.K. WOO, Science 215, 69-71 (1981). 13. D.S. BASKIN and Y. HOSOBUCHI, Lancet 11-1981, 272-275 (1981). 14. W. YOUNG, et al., J. Neurosurg. 55, 209-219 (1981). 15. K. AUKLAND, B.F. BOWER and R.W. BERLINER, Circ. Res. 14, 164-187 (1964). 16. E. PAZSTOR, et al., Stroke ~, 556-567 (1973). 17. L. SYMON, N.M. BRANSTON and O. CHIKOVANI, Stroke iO, 184-191 (1979). 18. A.D. JAMIESON and J.H. HALSEY, Stroke 4, 904-911 (1973). 19. D.S. BASKIN and Y. HOSOBUCHI, personal communication (1982).