Catecholamine content of cerebral tissue after occlusion or manipulation of middle cerebral artery in cats HAROLD P. COHEN, PH.D. ARTHUR G. WALTZ, M.D., AND RONALD L. JACOBSON, M.S. Cerebrovascular Clinical Research Center, Department of Neurology, University of Minnesota, Minneapolis, Minnesota The authors determined by fluorimetry the norepinephrine-epinephrine content (NE-E) of cerebral tissue from 38 cats, to ascertain whether constriction of hypersensitive arterial vessels by vasoactive agents in ischemic cerebral tissue could cause extension of cerebral infarcts and worsening of neurological deficits. Twenty-three cats had the left middle cerebral artery (MCA) occluded transorbitally, and 10 cats had sham operations. Five cats had only the surgical procedures necessary for obtaining tissue; mean NE-E content was 0.30 ~tg/gm (SD = 0.041). For the other 33 cats, including those with sham operations, values were variable, ranging from 0.07 to 0.60 ~g/gm. Low values usually were obtained for ischemic hemispheres 24 hours and 7 days after MCA occlusion, but at other times values could be high or low on either side. Many factors unrelated to tissue damage, including arterial manipulation, influence the catecholamine content of cerebral tissue. KEY WORDS " a r t e r i a l s p a s m 9 catecholamines 9 cerebral ischemia 9 cerebral infarction 9 cerebral vasomotor responses 9 middle cerebral artery occlusion

HE presence of vasoactive agents Cerebral arterial spasm that occurs after such as catecholamines (largely nor- spontaneous or experimental subarachnoid epinephrine and dopamine), seroto- hemorrhage is probably related to serotonin, or prostaglandins in cerebral tissue nin, 1,2 and can be sufficiently intense to cause damaged by t r a u m a or ischemia may be im- changes of regional cerebral blood flow. ls,19 portant in the pathophysiologieal processes of Recently, regional increases of norepinephcerebral infarction and necrosis, and may in- rine content have been found in association fluence the resulting neuronal dysfunction with spinal cord injury, leading to a speculaand neurological disability. Focal constric- tion that vasomotor responses may influence tion of surface arterial vessels of the brain can development of local necrosis. 1~ occur soon after the onset of ischemia, le C e r t a i n e n z y m e inhibitors and other perhaps because o f hypersensitivity o f agents, such as a-methyl-p-tyrosine, can isehemic arterioles to s e r o t o n i n ? ,~'~7 m o d i f y the c o n t e n t or the release of

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J. Neurosurg. / Volume 43 / July, 1975

Catecholamine content after MCA occlusion in cats norepinephrine in nervous tissue. 1~ Thus, these agents m a y have potential therapeutic value for acute cerebral ischemia, during which vasoconstriction possibly could cause an extension of an area of necrosis and a worsening of neurological deficits. However, the interactions between cerebral ischemia, infarction, and catecholamine content are unknown. In this study we made determinations of the norepinephrine-epinephrine (NE-E) content of samples of brain in an experimental model of acute cerebral ischemia. Methods

For this study we used 33 unselected adult cats. Anesthesia with phencyclidine hydrochloride, 1 m g / k g injected intramuscularly, and sodium pentobarbital, 20 m g / k g injected intraperitoneally, was used for all surgical procedures. The left middle cerebral artery ( M C A ) was exposed transorbitally 9 in all 33 cats; it was occluded by bipolar coagulation in 23, while the other 10 cats had sham operations. In the latter, the left M C A was exposed and grasped with the coagulating forceps but not occluded. Sixteen minutes to 7 days after M C A occlusion or manipulation, the cerebral hemispheres were exposed by bilateral craniectomies. The brain was frozen in s i t u with liquid nitrogen and one core of tissue was removed from the superior portion of the anterior Sylvian gyrus and the subjacent white m a t t e r of each hemisphere. Samples weighed between 0.4 and 1.0 gm. In cats this part of the ipsilateral cerebral hemisphere becomes ischemic, and usually infarcted, after M C A occlusion, s~ Five cats had only those surgical procedures necessary for obtaining the samples of cerebral tissue, without exposure of the left M C A . The frozen samples of brain were either treated immediately or stored for 24 to 72 hours at - 8 5 ~ Each sample was weighed rapidly and fragmented under liquid nitrogen in an insulated porcelain m o r t a r using a chisel and h a m m e r . An extract of the tissue was prepared according to the method of Sharman. 14 Catecholamines were separated from the extract with an ion-exchange resin, contained in columns.* The mean recovery of known amounts of norepinephrine from eight columns was 105%, with a standard deviation *Column Test 5 obtained from Bio-Rad Laboratories, Richmond, Virginia. J. Neurosurg. / V o l u m e 43 / July, 1975

TABLE 1 NE-E content o f cerebral tissue from five cats without M C A exposure *

Cat No. 1

2 3 4 5

Hemisphere Left Right (ug/gm) (ug/gm) 0.26 0.28 0.30 0.32 0.30

Ratio Left :Right

0.27 0.26 0.33 0.39 0.29

0.96 1.08 0.91 0.82 1.03

* Mean=0.30 ug/gm; standard deviation=0.041. NE-E= norepinephrine-epinephrine; MCA = middle cerebral artery. TABLE 2 N E - E content o f cerebral tissue .from 10 cats with the left M C A manipulated but not occluded *

Cat No. 1 2 3 4 5 6 7 8 9 10

Hemisphere Ratio Time after Left Right Left :Right Manipulation (~g/gm) (ug/gm) 16 min 24 min 4 hrs 4 hrs 24 hrs 24 hrs 24 hrs 7 days 7 days 7 days

0.17 0.18 0.40 0.34 0.30 0.33 0.28 0.33 0.23 0.27

0.13 0.14 0.44 0.48 0.31 0.24 0.42 0.27 0.24 0.27

1.31 1.29 0.91 0.71 0.97 1.38 0.67 1.22 0.96 1.00

* NE-E= norepinephrine-epinephrine; MCA = middle cerebral artery. of 5.6%. D o p a m i n e added to the column was found not to interfere with the results, and metanephrines were not eluted. Norepinephrine and e p i n e p h r i n e were d e t e r m i n e d together by a modification of the method of Anton and S a y r e ? M e a s u r e m e n t s were m a d e with a Farrand spectrofluorometert (excitation: 400 mu; emission: 502 m~, uncorrected). Results

The N E - E content of the 10 samples of brain taken from the five cats without M C A exposure, occlusion, or manipulation was relatively uniform (Table 1). However, the tFarrand spectrofluorometer manufactured by Farrand Optical Company, Inc., Commercial Products Division, 117 Wall Street, Valhalla, New York 10595. 33

H. P. Cohen, A. G. Waltz and R. L. Jacobson values for samples from animals with a sham operation (Table 2) or an occluded M C A (Table 3) were highly variable. The unilateral sham operation appeared to have a transient effect on the N E - E content of cerebral tissue bilaterally: values were low at 16 and 24 minutes and high for three of four samples 4 hours after the surgical procedure. Unilateral M C A occlusion likewise appeared to p r o d u c e changes of N E - E content bilaterally. Values for either hemisphere could be low or high at any of the times after occlusion at which d e t e r m i n a t i o n s were made; however, at 24 hours and 7 days values were low bilaterally in six of eight cats. There were no consistent side-to-side differences of N E - E content after M C A occlusion, although at 6 hours, 24 hours, and 7 days values for the side of occlusion were lower than those for the opposite side in nine of 1 1 cats.

Discussion The great variability of the values for the N E - E content of samples of brain obtained from cats with M C A manipulation or occlusion was surprising; it was not related to the methods used for analysis, since there was much less variability of the values for the cats w i t h o u t M C A exposure, for which the samples of brain were obtained and analyzed in the same way as for the cats with M C A manipulation or occlusion. One reason for the variability could have been the unavoidable differences in weights and a m o u n t s of ischemic, infarcted, edematous, and hyperemic tissue in different samples; while in cats the region of brain we sampled becomes ischemic after M C A occlusion, 2~the effects of ischemia are not uniform? '15'2~ The undoubted heterogeneity of the samples of ischemic brain could not have caused much variability; the values obtained for samples from the nonischemic cerebral hemispheres opposite an exposed M C A were also variable. Furthermore, the directions of the changes of N E - E content caused either by M C A manipulation or by occlusion were not consistent, as would be expected if different a m o u n t s of affected tissue were solely responsible for the variability. T h e variability of the results was most p r o b a b l y related to multiple factors second a r y to M C A exposure and manipulation or occlusion, such as changes of synthesis, uptake, utilization, or storage of NE-E; release 34

TABLE 3 NE-E content of cerebral tissue from 23 cats with the left MCA occluded *

Time after Cat No. Occlusion 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

38 min 30 min 30 min 1 hr 1 hr 1 hr 2 hrs 2 hrs 2 hrs 4 hrs 4 hrs 4 hrs 6 hrs 6 hrs 6 hrs 24 hrs 24 hrs 24 hrs 24 hrs 24 hrs 7 days 7 days 7 days

Hemisphere Ratio Left Right Left:Right (~g/gm) (~g/gm) 0.25 0.15 0.30 0.17 0.21 0.28 0.26 0.38 0.32 0.29 0.19 0.30 0.27 0.25 0.25 0.08 0.33 0.15 0.12 0.12 0.07 0.15 0.60

0.26 0.11 0.29 0.12 0.29 0.40 0.26 0.33 0.26 0.20 0.15 0.24 0.30 0.38 0.32 0.17 0.29 0.20 0.20 0.20 0.17 0.22 0.40

0.96 1.36 1.03 1.42 0.72 0.70 1.00 1.15 1.23 1.45 1.27 1.25 0.90 0.66 0.78 0.47 1.14 0.75 0.60 0.60 0.41 0.68 1.50

* NE-E = norepinephrine-epinephrine; MCA = middle cerebral artery.

of N E - E f r o m cells; or removal of released N E - E by the circulatory system. The N E - E content of a region of brain could vary greatly at different times with changes in dynamic processes such as these. A single value for the N E - E content of a sample of brain obtained at a given time after M C A occlusion or manipulation cannot provide adequate information about possible changes in dynamic processes. I n f o r m a t i o n about directions and rates of change or turnover rates would be valuable but could be derived in practice only with r a d i o t r a c e r - l a b e l i n g techniques. A c h r o m a t o g r a p h i c procedure under development in our l a b o r a t o r y will m a k e such determinations feasible, and will permit measurem e n t of c h a n g e s o f the relative specific activity of the bioamines present in small amounts of cerebral tissue. At least three treatment groups should be included in the design of studies of experimental cerebral ischemia and infarction. Results must be obtained for animals that have had as few surgical procedures as possiJ. Neurosurg. / Volume 43 / July, 1975

Catecholamine

content

a f t e r M C A o c c l u s i o n in c a t s

ble, for animals that have had all the necessary surgical procedures except the crucial one (in the present study, MCA occlusion), and for animals that have had the crucial procedure. In addition, both nonischemic and ischemic cerebral tissue should be obtained from animals with induced ischemia. If no sham operations are done, for example, and only side-to-side differences or differences from normal values are considered, results may be ascribed to ischemia that might be caused by other factors such as anesthesia, manipulation of blood vessels, exposure of the brain, or the nonspecific stress of surgical procedures. Conversely, if no results are obtained for animals with few or no surgical procedures, changes caused by stress or arterial manipulation might not be appreciated. Unfortunately, several studies of catecholamines and experimental cerebral ischemia reported by others have not included all the necessary groups. 7,s,xs'21 Because of the variability and inconsistency of the results in the present study, no comment can be made about the significance of catecholamines for the pathophysiological processes involved in ischemic cerebral infarction, or about the potential therapeutic usefulness of enzyme inhibitors and other agents that influence catecholamine metabolism. Results of catecholamine determinations made by others are similar to those reported here. 7,s,ls,~ It is possible that catecholamines may have importance for the processes of cerebral infarction, because of vasoactive effects or effects on necrosis and edema, but at present the evidence is inconclusive. Acknowledgments

Technical assistance was provided by Chester Yee and Carlos Verdeja. References

1. Allen GS, Gold LHA, Chou SN, et al: Cerebral arterial spasm. Part 3: In vivo intracisternal production of spasm by serotonin and blood and its reversal by phenoxybenzamine. J Neurosurg 40:451-458, 1974 2. Allen GS, Henderson LM, Chou SN, et al: Cerebral arterial spasm. Part 1: In vitro contractile activity of vasoactive agents on canine basilar and middle cerebral arteries. J Neurosurg 40:433-441, 1974 J. Neurosurg. / Volume 43 / J u l y , 1975

3. Anton AH, Sayre DF: A study of the factors affecting the aluminum oxide - - trihydroxyindole procedure for the analysis of catecholamines. J Pharmacol Exp Ther 138: 360-375, 1962 4. Bell WH 3rd, Sundt TM Jr, Nofzinger JD: The response of cortical vessels to serotonin in experimental cerebral infarction. J Neurosurg 26:203-212, 1967 5. Bio-Rad Laboratories Clinical Division: Catecholamines by Column Test, Technical Bulletin 4002. Richmond, Bio-Rad Laboratories, Inc., 1972 6. Garcia JH: Morphological changes associated with metabolic abnormalities, in Sandok BA, Whisnant JP (eds): Cerebral Vascular Diseases, Ninth Conference. New York: Grune and Stratton, in press 7. Held K, Jacobsen O, Kraft K, et al: Regional cerebral metabolism in experimental brain infarction. Stroke 4:331, 1974 (Abstract) 8. Kogure K, Busto R, Reinmuth O, et al: Energy metabolites, water content and catecholamine changes in a model of cerebral embolic infarction. Stroke 4:331, 1974 (Abstract) 9. O'Brien MD, Waltz AG: Transorbital approach for occluding the middle cerebral artery without craniectomy. Stroke 4:201-206, 1973 10. Osterholm JL: The pathophysiological response to spinal cord injury: the current status of related research. J Neurosurg 40:5-33, 1974 11. Osterholm JL, Mathews G J: Altered norepinephrine metabolism following experimental spinal cord injury. Part 1: Relationship to hemorrhagic necrosis and post-wounding neurological deficits. J Neurosurg 36:386-394, 1972 12. Osterholm JL, Mathews GH: Altered norepinephrine metabolism following experimental spinal cord injury. Part 2: Protection against traumatic spinal cord hemorrhagic necrosis by norepinephrine synthesis blockade with alpha methyl tyrosine. J Neurosurg 36:395-401, 1972 13. Petruk KC, Weir BKA, Marriott MR, et al: Clinical grade, regional cerebral blood flow and angiographical spasm in the monkey after subarachnoid and subdural hemorrhage. Stroke 4:431-445, 1973 14. Sharman DF: Methods of determination of catecholamines and their metabolites, in Fried R (ed): Methods of Neurochemistry, Volume 1. New York, M Dekker, 1971, pp 83-127 15. Siesj~ BK, EklSf B, MacMillan V: Energy metabolism in the brain in ischemia, in McDowell FH, Brennan RW (eds): Cerebral Vascular Diseases, Eighth Conference. New York, Grune and Stratton, 1973, pp 99-114 35

H. P. Cohen, A. G. Waltz and R. L. Jaeobson 16. Waltz AG, Sundt TM Jr: The microvasculature and microcirculation of the cerebral cortex after arterial occlusion. Brain 90:681-696, 1967 17. Welch KMA, Hashi K, Meyer JS: Cerebrovascular response to intraearotid injection of serotonin before and after middle cerebral artery occlusion. J Neurol Neurosurg Psychiatry 36:724-735, 1973 18. Wurtman R J, Zervas NT: Monoamine neurotransmitters and the pathophysiology of stroke and central nervous system trauma. J Neurosurg 40:34-36, 1974 19. Yamaguchi T, Waltz AG: Effects of subarachnoid hemorrhage from puncture of the middle cerebral artery on blood flow and vasculature of the cerebral cortex in the cat. J Neurosurg 35:664--671, 1971

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20. Yamaguchi T, Waltz AG, Okazaki H: Hyperemia and ischemia in experimental cerebral infarction: correlation of histopathology and regional blood flow. Neurology 21:565-578, 1971 21. Zervas NT, Hori H: Effect of alpha methyl tyrosine on cerebral infarction. Stroke 4:331, 1974 (Abstract)

This investigation was supported in part by Research Grant NS-3364 from the National Insitutes of Health, Public Health Service. Address reprint requests to: Harold P. Cohen, M.D., Cerebrovascular Clinical Research Center, Department of Neurology, University of Minnesota, Minneapolis, Minnesota 55455.

J. Neurosurg. / Volume 43 / July, 1975

Catecholamine content of cerebral tissue after occlusion or manipulation of middle cerebral artery in cats.

The authors determined by fluorimetry the norepinephrine-epinephrine content (NE-E) of cerebral tissue from 38 cats, to ascertain whether constriction...
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