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The effect of lesions in the locus coeruleus on the physiological responses of the cerebral blood vessels in cats
Brain Research, 136 (1977) 431--443
431
© Elsevier/North-Holland Biomedical Press
T H E E F F E C T OF LESIONS I N T H E L O C U S C O E R U L E U S O N T H E P H Y S I O L O G I C A L RESPONSES OF T H E C E R E B R A L B L O O D VESSELS I N CATS
DAVID BATES*, RICHARD M. WEINSHILBOUM, R. JEAN CAMPBELL and THORALF M. SUNDT, Jr.** From the Cerebrovascular Clinical Research Center and the Departments of Neurology. Pharmacology, Anatomical Pathology, and Neurologic Surgery, Mayo Clinic and Mayo Foundation, Rochester, Minn. 55901 (U.S.A.)
(Accepted March 3rd, 1977)
SUMMARY The effects on cerebral blood flow (CBF) of lesions placed stereotactically in or near the locus coeruleus were studied in 15 lightly anesthetized cats; 5 control cats in which the electrode was placed but no lesion created, and 10 experimental cats in which a lesion was created. The response of CBF to changes in Paco2 and in mean arterial blood pressure was determined by lSaXe-washout studies 10 days after the stereotactic procedures. The sites of the lesions were studied histologically, and their effects on catecholamine concentrations in the paraventricular hypothalamic nucleus, anterior ventral nucleus of the thalamus, and parietal cortex were determined by radiochemical assay. Control animals and those with lesions near, but not in, the locus coeruleus had normal Paco2-CBF response curves and normal catecholamine concentrations in the areas of biopsy. Bilateral destruction of the locus coeruleus was confirmed in 3 animals on histological examination and in these animals there were decreased levels of catecholamines in the areas of assay, higher resting CBFs at normocapnia, and significantly abnormal CBF-Paco2 response curves. The autoregulatory response to changes in perfusion pressure was preserved. Thus, noradrenergic neurons originating in the locus coeruleus may contribute to the control of intraparenchymal cerebral vessels and disturbance of this control may be important in the pathology of cerebral ischemia.
* David Bates was in receipt of a Harkness Fellowship during the performance of this study. Present address: Department of Neurology, RVI, Newcastle-upon-Tyne, Great Britain. ** To whom reprint requests should be sent: Cerebrovascular Clinical Research Centre, Room 4-437, Alfred Building, St. Mary's Hospital, Rochester, Minn. 55901.
432 INTRODUCTION Current evidence suggests that factors intrinsic to the vessel wall or in its immediate environment control the responses of the cerebral blood vessels ~'~,27. However, a question remains as to the effect of the nerve terminals on the extraparenchymal and intraparenchymal blood vessels1,4,16, 20. The initial belief that the cervical sympathetic and facial nerve parasympathetic nerve fibers control vascular responses has not been universally confirmed7,'~1,28, :~1. Although the sympathetic nervous system may exert some modulating influence on the extracerebral intracranial vessels 26, the evidence that cervical sympathectomy does not result in degeneration of all the catecholaminergic terminals on the intracerebral blood vessels raises the possibility of an alternate route for this perivascular nerve supply H. Molnar and Seylaz 1~ suggested that there may be a brain stem center acting via an intra-axial nerve pathway to control the cerebral blood vessels. The existence of such a pathway is supported by the studies of Edvinsson et al. s and was confirmed in the recent report by Raichle et al. 22. These latter authors suggested that neurons containing norepinephrine and terminating on small cortical and hypothalamic vessels originate from the locus coeruleus, and they demonstrated that the behavior of these vessels may be modified by the instillation of adrenergic-blocking agents intraventricularly and the application of carbachol to the locus coeruleus. The anatomic distribution of the terminals of the norepinephrine-containing neurons originating from the locus coeruleus has been recently defined 13, but their physiologic role is unknown. The present study used an accepted model to study cerebral blood flow (CBF) in order to evaluate the effect of stereotactically produced ablative lesions in the locus coeruleus. The sites of the lesions were verified histologically, and the resultant depletions in concentrations of diencephalic and cortical catecholamines were measured quantitatively. The effects of such lesions on the resting CBF and its response to alterations in blood gas tension and blood pressure were determined. MATERIAL AND METHODS
Experimental animals Six cats were used initially to standardize the techniques and to ascertain the precise stereotactic position of the locus coeruleus. Fifteen cats of either sex and weighing between 2.5 and 4 kg were then used in the investigation. The location of the brain stem structures in cats of similar size was consistent enough to obviate the need for contrast radiologic techniques.
Stereotactic procedure The fifteen cats were placed into an experimental group of 10 and a control group of 5. Experimental group. The cats were lightly anesthetized with pentobarbital intraperitoneaUy (15 mg/kg body weight). Each cat was placed in a stereotactic head
433 holder (Baltimore Instruments). Aseptic techniques were used, and burr holes were fashioned 2 mm apart on either side of the midline in the parietal region at the desired site. The superior sagittal sinus was identified and avoided, and the dura was elevated with a hook and incised with a surgical blade. A 0.5 mm diameter stainless steel electrode coated with an insulating resin to a distance of 0.5 mm from its tip was then introduced to the required stereotactic coordinates. The sites of the lesions were 2 mm posterior to the intervascular plane, 8 mm above the true zero plane, and 1.5 mm to each side of the midline (P2, H - - 2 , R and L 1.5). In some cats, the bony tentorium prevented the perfect insertion of the electrode at the desired site and, in these, the electrode holder was advanced 0.5 mm. When the electrode was correctly positioned, a current of 750/~A was applied for 20 sec. This procedure produced a spherical lesion of approximately 1 mm in diameter (the lesion maker was designed by the Electronics Laboratory of the Mayo Clinic). Postoperatively, the muscle and skin were closed in layers. Each cat was injected intramuscularly with 300,000 units of benzothin penicillin G (Wyeth) and allowed to recover from the anesthetic. Control group. Five cats underwent the identical procedure, with the exception that the electrode was not stimulated.
CBF studies Preparation of cats. On the 10th day after the performance of the stereotactic lesions, the cats were anesthetized with intraperitoneal pentobarbital (20 mg/kg body weight). Arterial and venous catheters (PE 90) were introduced into the right femoral vessels for blood pressure monitoring, arterial blood gas sampling, and administering of drugs. A tracheostomy was performed, and a cannula (PE 50) was inserted into the left lingual artery so that its tip lay in the carotid artery. The skin, subcutaneous tissue, and muscle were excised bilaterally from the skull to the level of the zygoma. Blood loss was minimal. Each cat was paralyzed with tubocurare (5 mg/kg body weight) and ventilated with a mixture of oxygen, nitrogen, and carbon dioxide. Blood pressure was constantly recorded by a strain-gauge transducer in the femoral artery catheter, and core temperature was monitored by a rectal thermometer and maintained at 37 q- 0.5 °C by external warming. Pacoz, Pao2, and pH were determined immediately before each flow measurement. The hematocrit level was measured at the beginning and end of each experiment; no significant change occurred, and no hemolysis was detected. Measurement of CBF. The collimation, calibration, resolution, and positioning of the sodium iodide detector used has been previously described 2, as have the methods of recording the washout curve of lZSXe used in this laboratory 30. Each blood flow measurement was calculated by kinetic analysis 82 of the washout curve of a 0.3 ml bolus of saline containing 300/~Ci of 133Xe injected into the lingual artery catheter. Lambda (2) for whole brain was taken as unity, and the CBF was expressed as ml blood/100 g brain tissue/min. The data were statistically evaluated by Student's t-test for unpaired samples, with P < 0.05 significant.
434
Assessment of responsiveness All cats were subjected to the same series o f physiologic stimuli. Initially, after stabilization, the C B F was measured at resting mean arterial blood pressure ( M A B P ) (122±6mm H g ) , P a c o z 3 9 r : 2 t o r r , and Pao2 1 1 0 ~ 2 0 t o r r . The Paco2 was then increased to 58 ± 2 torr by altering the inspired gas tensions and, after allowing 10 min for equilibration 24, a further flow was recorded, after which the Paco2 was lowered to 27 ± 2 torr. After 10 min was allowed for the maximal vascular response to occur, a third measurement o f C B F was taken and the Paco2 was returned to 39 :i:: I torr. After a further basal flow was estimated, an infusion o f phenylephrine hydrochloride (100 #g/ml) was adjusted to a rate of between 0.05 and 0.2 ml/min to increase the M A B P approximately 30% above the resting level, and 10 min was allowed for autoregulation to occur and a fifth measurement of C B F was recorded 23. The infusion was stopped, the cat was bled (30-50 ml) into a heparinized reservoir to reduce the M A B P to 70 % of the resting level, and the C B F was recorded. Finally, the blood was reinfused, and a seventh measurement o f C B F was taken at basal conditions. This sequence o f measurements is shown graphically in Fig. 1.
Removal of brain After completion of the blood flow studies, an extensive craniotomy was performed, the cat was sacrificed, and the brain was removed within 2 min. Two slices of "140t-
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Fig. l. Cerebral blood flow in response to changes in Paco~ and MABP. CBF in the groups of control cats, cats with inadequate lesions, and cats with bilateral destruction of locus coeruleus are shown. Lower panel shows MABP and Paco~ during the experiment. CBF measurements were obtained at 30 min intervals (see text for details).
435 cerebrum, each 3 mm thick, were cut immediately caudal and rostral to the optic chiasm. These sections were rapidly frozen on solid carbon dioxide and stored at - - 7 0 °C for catecholamine assay. The portion of the brain that was caudal to these sections, including the brain stem and cerebellum, was fixed in 10 ~ formal-saline for histologic study.
H ist ologic .findings The brain stem was sectioned at the level of the inferior colliculi, and a 3 mm thick slice was embedded in paraffin. Serial sections at 20 # m were cut and stained with Luxol Fast Blue and counterstained with cresyl violet. The sites of the stereotactically induced lesions were identified histologically. Catecholamine assay The sections through the hemispheres were trimmed on a cryostat; the more appropriate one was selected; and a Zeiss binocular operating microscope with a 300 mm focal plane was used to dissect portions of parietal cortex, anterior ventral thalamic nucleus, and paraventricular hypothalamic nucleus from the left hemisphere. The nuclei were identified by comparison to the shape of the brain, ventricles, and major nerve tracts 13. Three areas were chosen to be biopsied: one with a high, a second with a medium, and a third with a low concentration of norepinephrine 1~. Catecholamine levels were measured by the method of Coyle and Henry 5. This procedure utilizes the enzyme catechol-O-methyltransferase to transfer a radioactively labeled methyl group from S-adenosyl-l-methionine to the catechol compound. After the O-methylated radioactive product is separated by organic solvent extraction, periodate cleavage is carried out. This procedure forms [3H]vanillin from the reaction products of both epinephrine and norepinephrine. The radioactivity of the vanillin is then determined in a liquid-scintillation counter after a final organic solvent extraction step. Dopamine is not measured by this procedure. Specifically, tissue samples were weighed, homogenized in 2 ml of 0.4 N perchloric acid at 0 °C, and then were centrifuged at 8000 × g for 10 min. The supernatant was removed, and aliquots of 60 and 300/~1 were used for the catecholamine assays in a final reaction volume of 400/d. Each sample was also assayed with an internal standard of 0.313 ng of norepinephrine. In addition, a standard curve of 0.16-2.50 ng norepinephrine was determined during each assay procedure. Catecholamine concentrations were expressed as ng norepinephrine/mg brain tissue and ng norepinephrine/mg protein. Protein determination The protein pellet obtained after centrifugation of the perchloric acid tissue homogenates was dissolved in 1 N NaOH, and the amount of protein was measured by the method of Lowry et al. la. Bovine serum albumin was used as a standard. RESULTS
Animals Two of the 10 cats in the experimental group died during the 10 day interval.
436
Fig. 2. Midbrain section of lesions in cat L6, showing accurately placed bilateral lesions in locus coeruleus (:~ 16).
TABLE 1
Values of catecholamines (ng norepinephrine/mg protein in each of the three sites biopsied) Cat
Site of lesions
Parietal cortex
Anterior ventral Paraventrieular nucleus of thalamus nucleus of h27~othalamus
L1
Unilateral
0.66
2.63
9.76
L3 L4 L6 M i S.E.
Bilateral Bilateral Bilateral
0.17 0.21 0.25 0.21 ~ 0.02
1.07 0.99 0.99 1.02 ~ 0.03
3.37 3.47 3.36 3.40 ± 0.04
L7 Ls L10 M ± S.E.
Inaccurate Inaccurate Inaccurate
1.17 1.04 1.20 1.14 ~ 0.05
6.80 6.76 4.75 6.10 ± 0.68
17.99 17.78 31.92 22.56 ~ 4.68
C1 Co~ C3 C4 C5 M ± S.E.
Control Control Control Control Control
1.89 1.04 1.08 2.22 1.67 1.58 ± 0.23
8.81 8.61 7.39 6.16 4.18 7.03 -2- 0.86
14.64 14.44 22.83 29.42 14.08 19.08 ± 3.06
437
ICC
NIC
/
MLF a
9 b ~
NIC
MLF
Fig. 3. a: composite diagram through brain stem, showing lesions (areas of cross-hatching) in cats having bilateral destruction of locus coeruleus. ICC, inferior collicula commissure; NIC, nucleus of inferior colliculus; PAG, periaqueductal gray matter; AQ, cerebral aqueduct; LC, locus coeruleus; TDT, dorsal tegmental nucleus; MLF, median longitudinal fasciculus; and VTN, ventral tegmental nucleus, b" composite diagram of lesions in cats having inaccurately placed lesions (areas of crosshatching). Both cats failed to recover from anesthetic, became hypothermic, and developed a profound bradycardia, which was unresponsive to therapy. One other cat in the experimental group was excluded because the brain slices were frozen in liquid nitrogen rather than on solid carbon dioxide and the slices were fragmented, making examination impossible. The remaining 7 cats in the experimental group and all 5 control cats showed no abnormalities in behavior. They ate, drank, and slept normally.
438
Histologic findings None of the control cats showed macroscopic or microscopic evidence of brain stem lesions. In two, the needle track could be detected at the level of the inferior colliculi but there was no hemorrhagic reaction. In the experimental group, three cats had satisfactorily placed bilateral lesions (Figs. 2 and 3a). One cat had an adequate lesion on the left, but the lesion on the right was placed dorsal to the locus; and the other three cats had incorrectly placed lesions. A composite diagram of the lesions in these 4 cats is shown as Fig. 3b.
Cateeholamine assay The levels of catecholamines in all 12 cats that survived are shown in Table 1. The values for norepinephrine concentrations in the paraventricular nucleus of the hypothalamus, anterior ventral nucleus of the thalamus, and parietal cortex in the control cats were 19.08 ± 3.06 ng/mg protein, 7.03 ± 0.86 ng/mg protein and 1.58 _~: 0.23 ng/mg protein, respectively. These values are comparable to, although lower than, values reported by Kobayashi et al. 13 in studies of the rat brain. These authors described levels in the paraventricular hypothalamic nucleus, anterior ventral thalamic nucleus, and parietal cortex of 52.7 ± 9.2, 14.3 ± 1.5, and 1.52 ± 0.09, respectively. In the experimental three cats with inadequate lesions, the levels of norepinephrine were not significantly different from those of the control cats with means of 22.56 4.68 ng/mg protein, 6.10 + 0.68 ng/mg protein, and 1.14 ± 0.05 ng/mg protein. The cat with a unilateral lesion had levels of norepinephrine approximately one-half of those found in the control cats; that is 9.76 ng/mg protein, 2.63 ng/mg protein, and 0.66 ng/mg protein. It should be noted that the locus coeruleus lesions were on the side ipsilateral to the site of the brain biopsies. In the cats with accurate bilateral lesions, the catecholamine levels for the three biopsy sites were: paraventricular nucleus, 3.40 ~ 0.04 ng/mg protein; anterior ventral nucleus, 1.02 -3:_0.03 ng/mg protein; parietal cortex, 0.21 ± 0.02 ng/mg protein. These levels are all significantly lower (P < 0.001) than those found in the controls and in cats with misplaced lesions. The levels represent reductions to 18, 15, and 13% of the control values, respectively.
Cerebral blood flow The three cats with bilateral destruction of the locus coeruleus had patterns of response significantly different from the control values, whereas the response patterns of the cats with unilateral lesion and the cats with inappropriately placed lesions were not significantly different from the control values. The results are shown graphically in Fig. 1, together with the mean Paco2 and MABP recordings. The blood flow of the control group at basal conditions was 43 4- 3 ml/100 g/min; in cats with inadequate lesions, 40 4- 3 ml/100 g/min; and in cats with accurately placed lesions 77 -+- 3 ml/100 g/min. The differences were significant (P < 0.001). At hypercapnia, the normal increase in CBF was seen in the control group (111 ± 7 ml/100 g/min) and in the cats with inadequate lesion (117 i 15 ml/100 g/min) - - a difference that was not significantly different. However, the three cats with lesions had a significantly (P < 0.05)
439 lower CBF at hypercapnia (81 i 8 ml/100 g/min) than the control cats. Again at hypocapnia (Paco2 28 4- 1 torr), there was a significant difference (P < 0.01) between the cats with lesions (44 4- 2 ml/100 g/min) and the control cats (30 4- 2 ml/100 g/min), which persisted at the second basal measurement (control 38 4- 3 and lesion 48 4- 3 ml/100 g/min) (P < 0.05). There was no significant difference in the autoregulatory responses to changes in blood pressure between the cats with accurately placed lesions and the control cats. However, the cats with lesions persistently had a slightly greater blood flow than did the control group. Thus, while these studies show a significant difference in the basal CBF between the two groups and in the response to changes in Paco2 they do not show evident changes in the capacity of the cats with lesions to respond to changes in perfusion pressure. DISCUSSION Ten years have passed since the possibility of a brain stem center controlling the cerebral vascular responses was proposed 19. During that time, there have been occasional reports of disturbances of CBF in animals14,25 and in man lz with brain stem lesions. More recently, experimental studies have confirmed these earlier reports and have begun to delineate areas of the brain stem where the control mechanism may lie3,L In addition, the emphasis on neural control of the cerebral vessels has shifted from the acknowledged autonomic pathways of the cervical sympathetic and facial nerve parasympathetic to the possibility of intra-axial nerve pathways11. Although there are still some questions as to the precise relationship of these intra-axial fibers to the cerebral blood vessels17, the recent studies from Washington University22 have raised interesting possibilities as to the role of the locus coeruleus in cerebral vascular control. In the present study, all stereotactic lesions in the brain stem were verified histologically. In three cats, the lesions were accurately bilaterally located in the locus coeruleus. The locus coeruleus in the cat is not pigmented, which makes identification more difficult than in higher mammals, but, as shown in Fig. 2, the lesions when correctly placed lay at the rostral end of this nucleus, caused considerable neuronal loss, and were adequate to ablate the entire A6 efferent tract 6. In the three experimental cats in which lesions were inaccurately placed (Fig. 3b), the most common mistake was inadequate depth of the electrode, and this was seen in the largest cats. Lesions that were placed too far laterally probably resulted from movement of the electrode by the bony tentorium as the electrode was being introduced. The adequacy of the lesion was confirmed in the biochemical assay. The control values (Table I) are comparable to, although somewhat lower than, those reported by Kobayashi et al. 18 in a different species, the rat. The cats with bilaterally inaccurate lesions had levels of norepinephrine comparable to those of the controls. The cat with unilateral ablation of the locus on the left (Table I, L1) had levels of norepinephrine in the ipsilateral paraventricular hypothalamic nucleus that were 51 ~ of control, in the anterior ventral thalamic nucleus 37 ~ of control, and in the parietal cortex 42 ~ of
440 control. These data for ipsilateral depletion are similar to those reported by Kobayashi et al. la for the rat. The three cats with adequate lesions histologically had a profound decrease in the norepinephrine level of the hemispheric areas biopsied to approximately 19 % of the control level. This decrease would suggest that a considerable part of the norepinephrine in these sites is in axons with their cell bodies in the locus coeruleus although other nuclei do contribute to this supply ')9. Recently, it has been reported that bilateral destructive lesions in the locus coeruleus of rats, although causing a marked reduction in non-sympathetic perivascular adrenergic nerves, did not result in their complete loss 17. However, these authors used fluorescence techniques to identify the adrenergic nerves and thus could not differentiate among norepinephrine-, epinephrine-, and dopamine-containing neurons. (Dopamine is not measured by the radiochemical technique that we used to measure catecholamines.) The blood flow in the experiments reported herein show a significant increase in resting CBF in the cats with lesions, a statistically significant difference in unresponsiveness to Pac02, but an essentially retained ability to autoregulate to changes in blood pressure. The lack of response to Pat02 in the presence of brain stem lesions agrees with the report by Shalit et al. '~5, but the high initial resting level was not found by these workers, and they did not test the response to changes in perfusion pressure. The more recent reports of Fenske et al. 9, who reported significant loss of vascular response to hypercapnia in cats after a cold lesion had been produced in the brain stem, and by Capon 3, who showed a similar change in Pat02 responsiveness after high pontine transsection in cats, are in keeping with our results. In their preparations, Fenske et al. 9 also noted a retained autoregulation to pressure changes. All three reports have suggested that 'high brain stem' lesions can reduce the response of the cerebral vessels to alteration in Pac02. Our results and the report by Raichle et al. 22 who showed changes in CBF with pharmacologic stimuli to the locus coeruleus, suggest that this area of the brain stem is responsible for these changes. Thus this nucleus probably can alter the caliber of the cerebral vessels and thereby alter the CBF, and of the stimuli tested, this control is most manifest in response to changes in Pac02. Our study indicates that the response of the cerebral vessels to changes in pressure is independent of this control and therefore either is governed by another center or is due to local phenomena in the vascular smooth muscle - - the Bayliss effect~L Alternatively, the response to Paco2 may be due to alteration in the caliber of the smaller, intracerebral vessels, whereas the response to alteration in perfusion pressure is governed by the larger, extracerebral vessels. Thus, whereas lesions of the locus coeruleus may deplete the terminals of the intra-axial noradrenergic neurons, they may spare the fibers that take an alternate route to the pial vessels. Such a suggestion is similar to that raised by Harper et al. TM that the extraparenchymal vessels might be governed by the cervical sympathetic system and that the intraparenchymal vessels might be governed by an alternate system. The difference is that our results suggest that the intracerebral vessels also are subjected to neurogenic control but one with separate route for the brain stem center. In Fig. 1 CBF values are reported as absolute measurements. However, if CBF changes were to be plotted as a percentage change of the basal flow values, which were
441 300
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Fig. 4. Results of Fig. i plotted as percentage of basal flow values. higher in the animals with correctly placed lesions, then the abnormality in the Paco2 response is even more dramatic (Fig. 4). This would show that the response to hypocapnia is remarkably similar in all three groups. This suggests that the basal level and response to hypercapnia are primarily affected by the lesion. In addition, our results raise the possibility that noradrenergic neurons may contribute to an inherent 'resting tone' in the cerebral blood vessels. Release from this tone may result in vasodilatation that would explain the increase in basal CBF in the presence of lesions in the locus coeruleus. If this is true, these mechanisms may have a role in the response to cerebral ischemia and brain stem infarction. ACKNOWLEDGEMENTS This investigation was supported in part by Research Grants NS 6663 and H L 17487-1 from the National Institutes of Health, Public Health Service and by the Minnesota Heart Association Grant M H A 23.
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442 3 Capon, A., Effect of acute sections of the brain stem on hypercapnic vasodilatation of cerebral and spinal vessels. In M. Harper, B. Jennett, D. Miller and J. Rowan (Eds.), Blood Flow and Metabolism in the Brain (Proceedings o f the 7th International Symposium on Cerebral Blood Flow and Metabolism), Churchill Livingstone, Edinburgh, 1975, pp. I. 16-1.17. 4 Cervos-Navarro, J. and Matakas, F., Electron microscopic evidence for innervation of intracerebral arterioles in the cat, Neurology (Minneap.), 24 (1974) 282-286. 5 Coyle, J. T. and Henry, D., Catecholamines in fetal and newborn rat brain, J. Neurochem., 21 (1973) 61-67. 6 Dahlstr6m, A. and Fuxe, K., Evidence for the existence of monoamine-containing neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neurons, Acta physiol, stand., 62, Suppl. 232 (1964) 1-55. 7 D'Alecy, L. G. and Feigl, E. O., Sympathetic control of cerebral blood flow in dogs, Circulat. Res, 31 (1972) 267-283. 8 Edvinsson, L., Lindvall, M., Nielsen, K. C. and Owman, Ch., Are brain vessels innervated also by central (non-sympathetic) adrenergic neurones?, Brain Research, 63 (1973) 496-499. 9 Fenske, A., Hey, O., Theiss, R., Reulen, H. J. and Schfirmann, K., Regional cortical blood flow in the early stage of brain stem oedema. In M. Harper, B. Jennett, D. Miller and R. Rowan (Eds.), Blood Flow and Metabolism in the Brain (Proceedings o f the 7th International Symposium on Cerebral Blood Flow and Metabolism), Churchill Livingstone, Edinburgh, 1975, pp. 1.12-1.15. 10 Harper, A. M., Deshmukh, V. D., Rowan, J. O. and Jennett, W. B., The influence of sympathetic nervous activity on cerebral blood flow, Arch. Neurol. (Chic.), 27 (1972) 1-6. 11 Hartman, B. K., Zide, D. and Udenfriend, S., The use of dopamine /%hydroxylase as a marker for the central noradrenergic nervous system in rat brain, Proc. nat. Aead. Sci. (Wash.), (1972) 69 2722-2726. 12 lngvar, D. H. and Sourander, P., Destruction of the reticular core of the brain stem: a pathoanatomical follow-up of a case of coma of three years' duration, Arch. Neurol. (Chie.), 23 (1970) 1 8.
13 Kobayashi, R. M., Palkovits, M., Jacobowitz, D. M. and Kopin, i. J., Biochemical mapping of the noradrenergic projection from the locus coeruleus: a model for studies of brain neuronal pathways, Neurology (Minneap.), 25 (1975) 223-233. 14 Langfitt, T. W. and Kassell, N. F., Cerebral vasodilatation produced by brain-stem stimulation: neurogenic control vs. autoregulation, Amer. J. Physiol., 215 (1968) 90-97. 15 Lassen, N. A., Brain extracellular pH: the main factor controlling cerebral blood flow (editorial), Seand. J. Clin. lab. Invest., 22 (1968) 247-251. 16 Licata, R. H., Olson, D. R. and Mack, E. W., Cholinergic and adrenergic innervation of cerebral vessels. In T. W. Langfitt, L. C. McHenry, M. Reivich and H. Woltman (Eds.), Cerebral Circulation and MetaboRsm (6th International CBF Symposium), Springer, New York, 1975, pp. 466-469. t7 Lindvall, M., Cervos-Navarro, J., Edvinsson, L., Owman, Ch. and Stenevi, V., Nonsympathetic perivascular nerves in the brain. In M. Harper, B. Jennett, D. Miller and J. Rowan (Eds.), Blood Flow and Metabolism in the Brain (Proceedings o f the 7th International Symposium on Cerebral Blood Fow and Metabolism), Churchill Livingstone, Edinburgh, 1975, pp. 1.7-1.9. 18 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 19 Molnar, L. et Seylaz, J., Mise en evidence et interpretation des effects de la decerebration et des sinus carotidiens sur la circulation cerebrale, C.R. Aead. Sci. (Paris), 260 (1965) 3164-3167. 20 Neilsen, K. C., Edvinsson, L. and Owman, Ch., Cholinergic innervation and vasomotor response of brain vessels. In T. W. Langfitt, L. C. McHenry, M. Reivich and H. Wollman (Eds.), Cerebral Circulation and Metabolism (6th International CBF Symposium), Springer, New York, 1975, pp. 473-475. 21 Ponte, J. and Purves, M. J., The role of the carotid body chemoreceptors and carotid sinus baroreceptors in the control of cerebral blood vessels, J. Physiol. (Lond.), 237 (1974) 315-340. 22 Raichle, M. E., Hartman, B. K., Eichling, J. O. and Sharpe, L. G., Central noradrenergic regulation of cerebral blood flow and vascular permeability, Proe. nat. Aead. Sei. (Wash.), 72 (1975) 37263730. 23 Rapela, E. E. and Green, H. D., Autoregulation of canine cerebral blood flow, Circulat. Res., 15, Suppl. I (1964) 205-21 I. 24 Raper, A. J., Kontos, H. A. and Patterson, J. L., Jr., Response of pial precapillary vessels to changes in arterial carbon dioxide tension, Circulat. Res., 28 (1971) 518-523.
443 25 Shalit, M. N., Rcinmuth, O. M., Shimojyo, S. and Scheinberg, P., Carbon dioxide and cerebral circulatory control. IlL The effects of brain stem lesions, Arch. NeuroL (Chic.), 17 (1967) 342-353. 26 Sundt, T. M., Jr., Subject review: the cerebral autonomic nervous system; a proposed physiologic functionand pathophysiologic response in subarachnoid hemorrhage and in focal cerebral ischemia, Mayo Clin. Proc., 48 (1973) 127-137. 27 Symon, L., Held, K. and Dorsch, N. W. C., A study of regional autoregulation in the cerebral circulation to increased pcrfusion pressure in normocapnia and hypercapnia, Stroke, 4 (1973) 139-147. 28 Traystman, R. J. and Rapcla, C. E., Effect of sympathetic nerve stimulation on cerebral and cephalic blood flow in dogs, Circulat. Res., 36 (1975) 620-630. 29 Ungerstedt, U., Stcreotaxic mapping of the monoamine pathways in the rat brain, Acta physioL scand. Suppl. 367 (1971) 1-48. 30 Waltz, A. G., Wanek, A. R. and Anderson, R. E., Comparison of analytic methods for calculation of cerebral blood flow after intracarotid injection of 133Xe,Jr. nucl. Med., 13 (1972) 66-72. 31 Waltz, A. G., Yamaguchi, T. and Regli, F., Regulatory responses of cerebral vasculature after sympathetic denervation, Amer. J. Physiol., 221 (1971) 298-302. 32 Zierler, K. L., Equations for measuring blood flow by external monitoring of radioisotopes, Circular. Res., 16 (1965) 309-321.
431
© Elsevier/North-Holland Biomedical Press
T H E E F F E C T OF LESIONS I N T H E L O C U S C O E R U L E U S O N T H E P H Y S I O L O G I C A L RESPONSES OF T H E C E R E B R A L B L O O D VESSELS I N CATS
DAVID BATES*, RICHARD M. WEINSHILBOUM, R. JEAN CAMPBELL and THORALF M. SUNDT, Jr.** From the Cerebrovascular Clinical Research Center and the Departments of Neurology. Pharmacology, Anatomical Pathology, and Neurologic Surgery, Mayo Clinic and Mayo Foundation, Rochester, Minn. 55901 (U.S.A.)
(Accepted March 3rd, 1977)
SUMMARY The effects on cerebral blood flow (CBF) of lesions placed stereotactically in or near the locus coeruleus were studied in 15 lightly anesthetized cats; 5 control cats in which the electrode was placed but no lesion created, and 10 experimental cats in which a lesion was created. The response of CBF to changes in Paco2 and in mean arterial blood pressure was determined by lSaXe-washout studies 10 days after the stereotactic procedures. The sites of the lesions were studied histologically, and their effects on catecholamine concentrations in the paraventricular hypothalamic nucleus, anterior ventral nucleus of the thalamus, and parietal cortex were determined by radiochemical assay. Control animals and those with lesions near, but not in, the locus coeruleus had normal Paco2-CBF response curves and normal catecholamine concentrations in the areas of biopsy. Bilateral destruction of the locus coeruleus was confirmed in 3 animals on histological examination and in these animals there were decreased levels of catecholamines in the areas of assay, higher resting CBFs at normocapnia, and significantly abnormal CBF-Paco2 response curves. The autoregulatory response to changes in perfusion pressure was preserved. Thus, noradrenergic neurons originating in the locus coeruleus may contribute to the control of intraparenchymal cerebral vessels and disturbance of this control may be important in the pathology of cerebral ischemia.
* David Bates was in receipt of a Harkness Fellowship during the performance of this study. Present address: Department of Neurology, RVI, Newcastle-upon-Tyne, Great Britain. ** To whom reprint requests should be sent: Cerebrovascular Clinical Research Centre, Room 4-437, Alfred Building, St. Mary's Hospital, Rochester, Minn. 55901.
432 INTRODUCTION Current evidence suggests that factors intrinsic to the vessel wall or in its immediate environment control the responses of the cerebral blood vessels ~'~,27. However, a question remains as to the effect of the nerve terminals on the extraparenchymal and intraparenchymal blood vessels1,4,16, 20. The initial belief that the cervical sympathetic and facial nerve parasympathetic nerve fibers control vascular responses has not been universally confirmed7,'~1,28, :~1. Although the sympathetic nervous system may exert some modulating influence on the extracerebral intracranial vessels 26, the evidence that cervical sympathectomy does not result in degeneration of all the catecholaminergic terminals on the intracerebral blood vessels raises the possibility of an alternate route for this perivascular nerve supply H. Molnar and Seylaz 1~ suggested that there may be a brain stem center acting via an intra-axial nerve pathway to control the cerebral blood vessels. The existence of such a pathway is supported by the studies of Edvinsson et al. s and was confirmed in the recent report by Raichle et al. 22. These latter authors suggested that neurons containing norepinephrine and terminating on small cortical and hypothalamic vessels originate from the locus coeruleus, and they demonstrated that the behavior of these vessels may be modified by the instillation of adrenergic-blocking agents intraventricularly and the application of carbachol to the locus coeruleus. The anatomic distribution of the terminals of the norepinephrine-containing neurons originating from the locus coeruleus has been recently defined 13, but their physiologic role is unknown. The present study used an accepted model to study cerebral blood flow (CBF) in order to evaluate the effect of stereotactically produced ablative lesions in the locus coeruleus. The sites of the lesions were verified histologically, and the resultant depletions in concentrations of diencephalic and cortical catecholamines were measured quantitatively. The effects of such lesions on the resting CBF and its response to alterations in blood gas tension and blood pressure were determined. MATERIAL AND METHODS
Experimental animals Six cats were used initially to standardize the techniques and to ascertain the precise stereotactic position of the locus coeruleus. Fifteen cats of either sex and weighing between 2.5 and 4 kg were then used in the investigation. The location of the brain stem structures in cats of similar size was consistent enough to obviate the need for contrast radiologic techniques.
Stereotactic procedure The fifteen cats were placed into an experimental group of 10 and a control group of 5. Experimental group. The cats were lightly anesthetized with pentobarbital intraperitoneaUy (15 mg/kg body weight). Each cat was placed in a stereotactic head
433 holder (Baltimore Instruments). Aseptic techniques were used, and burr holes were fashioned 2 mm apart on either side of the midline in the parietal region at the desired site. The superior sagittal sinus was identified and avoided, and the dura was elevated with a hook and incised with a surgical blade. A 0.5 mm diameter stainless steel electrode coated with an insulating resin to a distance of 0.5 mm from its tip was then introduced to the required stereotactic coordinates. The sites of the lesions were 2 mm posterior to the intervascular plane, 8 mm above the true zero plane, and 1.5 mm to each side of the midline (P2, H - - 2 , R and L 1.5). In some cats, the bony tentorium prevented the perfect insertion of the electrode at the desired site and, in these, the electrode holder was advanced 0.5 mm. When the electrode was correctly positioned, a current of 750/~A was applied for 20 sec. This procedure produced a spherical lesion of approximately 1 mm in diameter (the lesion maker was designed by the Electronics Laboratory of the Mayo Clinic). Postoperatively, the muscle and skin were closed in layers. Each cat was injected intramuscularly with 300,000 units of benzothin penicillin G (Wyeth) and allowed to recover from the anesthetic. Control group. Five cats underwent the identical procedure, with the exception that the electrode was not stimulated.
CBF studies Preparation of cats. On the 10th day after the performance of the stereotactic lesions, the cats were anesthetized with intraperitoneal pentobarbital (20 mg/kg body weight). Arterial and venous catheters (PE 90) were introduced into the right femoral vessels for blood pressure monitoring, arterial blood gas sampling, and administering of drugs. A tracheostomy was performed, and a cannula (PE 50) was inserted into the left lingual artery so that its tip lay in the carotid artery. The skin, subcutaneous tissue, and muscle were excised bilaterally from the skull to the level of the zygoma. Blood loss was minimal. Each cat was paralyzed with tubocurare (5 mg/kg body weight) and ventilated with a mixture of oxygen, nitrogen, and carbon dioxide. Blood pressure was constantly recorded by a strain-gauge transducer in the femoral artery catheter, and core temperature was monitored by a rectal thermometer and maintained at 37 q- 0.5 °C by external warming. Pacoz, Pao2, and pH were determined immediately before each flow measurement. The hematocrit level was measured at the beginning and end of each experiment; no significant change occurred, and no hemolysis was detected. Measurement of CBF. The collimation, calibration, resolution, and positioning of the sodium iodide detector used has been previously described 2, as have the methods of recording the washout curve of lZSXe used in this laboratory 30. Each blood flow measurement was calculated by kinetic analysis 82 of the washout curve of a 0.3 ml bolus of saline containing 300/~Ci of 133Xe injected into the lingual artery catheter. Lambda (2) for whole brain was taken as unity, and the CBF was expressed as ml blood/100 g brain tissue/min. The data were statistically evaluated by Student's t-test for unpaired samples, with P < 0.05 significant.
434
Assessment of responsiveness All cats were subjected to the same series o f physiologic stimuli. Initially, after stabilization, the C B F was measured at resting mean arterial blood pressure ( M A B P ) (122±6mm H g ) , P a c o z 3 9 r : 2 t o r r , and Pao2 1 1 0 ~ 2 0 t o r r . The Paco2 was then increased to 58 ± 2 torr by altering the inspired gas tensions and, after allowing 10 min for equilibration 24, a further flow was recorded, after which the Paco2 was lowered to 27 ± 2 torr. After 10 min was allowed for the maximal vascular response to occur, a third measurement o f C B F was taken and the Paco2 was returned to 39 :i:: I torr. After a further basal flow was estimated, an infusion o f phenylephrine hydrochloride (100 #g/ml) was adjusted to a rate of between 0.05 and 0.2 ml/min to increase the M A B P approximately 30% above the resting level, and 10 min was allowed for autoregulation to occur and a fifth measurement of C B F was recorded 23. The infusion was stopped, the cat was bled (30-50 ml) into a heparinized reservoir to reduce the M A B P to 70 % of the resting level, and the C B F was recorded. Finally, the blood was reinfused, and a seventh measurement o f C B F was taken at basal conditions. This sequence o f measurements is shown graphically in Fig. 1.
Removal of brain After completion of the blood flow studies, an extensive craniotomy was performed, the cat was sacrificed, and the brain was removed within 2 min. Two slices of "140t-
~2ol-
•
Lesion Non-lesion .... o . - Control
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90 120 M~nufes
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Fig. l. Cerebral blood flow in response to changes in Paco~ and MABP. CBF in the groups of control cats, cats with inadequate lesions, and cats with bilateral destruction of locus coeruleus are shown. Lower panel shows MABP and Paco~ during the experiment. CBF measurements were obtained at 30 min intervals (see text for details).
435 cerebrum, each 3 mm thick, were cut immediately caudal and rostral to the optic chiasm. These sections were rapidly frozen on solid carbon dioxide and stored at - - 7 0 °C for catecholamine assay. The portion of the brain that was caudal to these sections, including the brain stem and cerebellum, was fixed in 10 ~ formal-saline for histologic study.
H ist ologic .findings The brain stem was sectioned at the level of the inferior colliculi, and a 3 mm thick slice was embedded in paraffin. Serial sections at 20 # m were cut and stained with Luxol Fast Blue and counterstained with cresyl violet. The sites of the stereotactically induced lesions were identified histologically. Catecholamine assay The sections through the hemispheres were trimmed on a cryostat; the more appropriate one was selected; and a Zeiss binocular operating microscope with a 300 mm focal plane was used to dissect portions of parietal cortex, anterior ventral thalamic nucleus, and paraventricular hypothalamic nucleus from the left hemisphere. The nuclei were identified by comparison to the shape of the brain, ventricles, and major nerve tracts 13. Three areas were chosen to be biopsied: one with a high, a second with a medium, and a third with a low concentration of norepinephrine 1~. Catecholamine levels were measured by the method of Coyle and Henry 5. This procedure utilizes the enzyme catechol-O-methyltransferase to transfer a radioactively labeled methyl group from S-adenosyl-l-methionine to the catechol compound. After the O-methylated radioactive product is separated by organic solvent extraction, periodate cleavage is carried out. This procedure forms [3H]vanillin from the reaction products of both epinephrine and norepinephrine. The radioactivity of the vanillin is then determined in a liquid-scintillation counter after a final organic solvent extraction step. Dopamine is not measured by this procedure. Specifically, tissue samples were weighed, homogenized in 2 ml of 0.4 N perchloric acid at 0 °C, and then were centrifuged at 8000 × g for 10 min. The supernatant was removed, and aliquots of 60 and 300/~1 were used for the catecholamine assays in a final reaction volume of 400/d. Each sample was also assayed with an internal standard of 0.313 ng of norepinephrine. In addition, a standard curve of 0.16-2.50 ng norepinephrine was determined during each assay procedure. Catecholamine concentrations were expressed as ng norepinephrine/mg brain tissue and ng norepinephrine/mg protein. Protein determination The protein pellet obtained after centrifugation of the perchloric acid tissue homogenates was dissolved in 1 N NaOH, and the amount of protein was measured by the method of Lowry et al. la. Bovine serum albumin was used as a standard. RESULTS
Animals Two of the 10 cats in the experimental group died during the 10 day interval.
436
Fig. 2. Midbrain section of lesions in cat L6, showing accurately placed bilateral lesions in locus coeruleus (:~ 16).
TABLE 1
Values of catecholamines (ng norepinephrine/mg protein in each of the three sites biopsied) Cat
Site of lesions
Parietal cortex
Anterior ventral Paraventrieular nucleus of thalamus nucleus of h27~othalamus
L1
Unilateral
0.66
2.63
9.76
L3 L4 L6 M i S.E.
Bilateral Bilateral Bilateral
0.17 0.21 0.25 0.21 ~ 0.02
1.07 0.99 0.99 1.02 ~ 0.03
3.37 3.47 3.36 3.40 ± 0.04
L7 Ls L10 M ± S.E.
Inaccurate Inaccurate Inaccurate
1.17 1.04 1.20 1.14 ~ 0.05
6.80 6.76 4.75 6.10 ± 0.68
17.99 17.78 31.92 22.56 ~ 4.68
C1 Co~ C3 C4 C5 M ± S.E.
Control Control Control Control Control
1.89 1.04 1.08 2.22 1.67 1.58 ± 0.23
8.81 8.61 7.39 6.16 4.18 7.03 -2- 0.86
14.64 14.44 22.83 29.42 14.08 19.08 ± 3.06
437
ICC
NIC
/
MLF a
9 b ~
NIC
MLF
Fig. 3. a: composite diagram through brain stem, showing lesions (areas of cross-hatching) in cats having bilateral destruction of locus coeruleus. ICC, inferior collicula commissure; NIC, nucleus of inferior colliculus; PAG, periaqueductal gray matter; AQ, cerebral aqueduct; LC, locus coeruleus; TDT, dorsal tegmental nucleus; MLF, median longitudinal fasciculus; and VTN, ventral tegmental nucleus, b" composite diagram of lesions in cats having inaccurately placed lesions (areas of crosshatching). Both cats failed to recover from anesthetic, became hypothermic, and developed a profound bradycardia, which was unresponsive to therapy. One other cat in the experimental group was excluded because the brain slices were frozen in liquid nitrogen rather than on solid carbon dioxide and the slices were fragmented, making examination impossible. The remaining 7 cats in the experimental group and all 5 control cats showed no abnormalities in behavior. They ate, drank, and slept normally.
438
Histologic findings None of the control cats showed macroscopic or microscopic evidence of brain stem lesions. In two, the needle track could be detected at the level of the inferior colliculi but there was no hemorrhagic reaction. In the experimental group, three cats had satisfactorily placed bilateral lesions (Figs. 2 and 3a). One cat had an adequate lesion on the left, but the lesion on the right was placed dorsal to the locus; and the other three cats had incorrectly placed lesions. A composite diagram of the lesions in these 4 cats is shown as Fig. 3b.
Cateeholamine assay The levels of catecholamines in all 12 cats that survived are shown in Table 1. The values for norepinephrine concentrations in the paraventricular nucleus of the hypothalamus, anterior ventral nucleus of the thalamus, and parietal cortex in the control cats were 19.08 ± 3.06 ng/mg protein, 7.03 ± 0.86 ng/mg protein and 1.58 _~: 0.23 ng/mg protein, respectively. These values are comparable to, although lower than, values reported by Kobayashi et al. 13 in studies of the rat brain. These authors described levels in the paraventricular hypothalamic nucleus, anterior ventral thalamic nucleus, and parietal cortex of 52.7 ± 9.2, 14.3 ± 1.5, and 1.52 ± 0.09, respectively. In the experimental three cats with inadequate lesions, the levels of norepinephrine were not significantly different from those of the control cats with means of 22.56 4.68 ng/mg protein, 6.10 + 0.68 ng/mg protein, and 1.14 ± 0.05 ng/mg protein. The cat with a unilateral lesion had levels of norepinephrine approximately one-half of those found in the control cats; that is 9.76 ng/mg protein, 2.63 ng/mg protein, and 0.66 ng/mg protein. It should be noted that the locus coeruleus lesions were on the side ipsilateral to the site of the brain biopsies. In the cats with accurate bilateral lesions, the catecholamine levels for the three biopsy sites were: paraventricular nucleus, 3.40 ~ 0.04 ng/mg protein; anterior ventral nucleus, 1.02 -3:_0.03 ng/mg protein; parietal cortex, 0.21 ± 0.02 ng/mg protein. These levels are all significantly lower (P < 0.001) than those found in the controls and in cats with misplaced lesions. The levels represent reductions to 18, 15, and 13% of the control values, respectively.
Cerebral blood flow The three cats with bilateral destruction of the locus coeruleus had patterns of response significantly different from the control values, whereas the response patterns of the cats with unilateral lesion and the cats with inappropriately placed lesions were not significantly different from the control values. The results are shown graphically in Fig. 1, together with the mean Paco2 and MABP recordings. The blood flow of the control group at basal conditions was 43 4- 3 ml/100 g/min; in cats with inadequate lesions, 40 4- 3 ml/100 g/min; and in cats with accurately placed lesions 77 -+- 3 ml/100 g/min. The differences were significant (P < 0.001). At hypercapnia, the normal increase in CBF was seen in the control group (111 ± 7 ml/100 g/min) and in the cats with inadequate lesion (117 i 15 ml/100 g/min) - - a difference that was not significantly different. However, the three cats with lesions had a significantly (P < 0.05)
439 lower CBF at hypercapnia (81 i 8 ml/100 g/min) than the control cats. Again at hypocapnia (Paco2 28 4- 1 torr), there was a significant difference (P < 0.01) between the cats with lesions (44 4- 2 ml/100 g/min) and the control cats (30 4- 2 ml/100 g/min), which persisted at the second basal measurement (control 38 4- 3 and lesion 48 4- 3 ml/100 g/min) (P < 0.05). There was no significant difference in the autoregulatory responses to changes in blood pressure between the cats with accurately placed lesions and the control cats. However, the cats with lesions persistently had a slightly greater blood flow than did the control group. Thus, while these studies show a significant difference in the basal CBF between the two groups and in the response to changes in Paco2 they do not show evident changes in the capacity of the cats with lesions to respond to changes in perfusion pressure. DISCUSSION Ten years have passed since the possibility of a brain stem center controlling the cerebral vascular responses was proposed 19. During that time, there have been occasional reports of disturbances of CBF in animals14,25 and in man lz with brain stem lesions. More recently, experimental studies have confirmed these earlier reports and have begun to delineate areas of the brain stem where the control mechanism may lie3,L In addition, the emphasis on neural control of the cerebral vessels has shifted from the acknowledged autonomic pathways of the cervical sympathetic and facial nerve parasympathetic to the possibility of intra-axial nerve pathways11. Although there are still some questions as to the precise relationship of these intra-axial fibers to the cerebral blood vessels17, the recent studies from Washington University22 have raised interesting possibilities as to the role of the locus coeruleus in cerebral vascular control. In the present study, all stereotactic lesions in the brain stem were verified histologically. In three cats, the lesions were accurately bilaterally located in the locus coeruleus. The locus coeruleus in the cat is not pigmented, which makes identification more difficult than in higher mammals, but, as shown in Fig. 2, the lesions when correctly placed lay at the rostral end of this nucleus, caused considerable neuronal loss, and were adequate to ablate the entire A6 efferent tract 6. In the three experimental cats in which lesions were inaccurately placed (Fig. 3b), the most common mistake was inadequate depth of the electrode, and this was seen in the largest cats. Lesions that were placed too far laterally probably resulted from movement of the electrode by the bony tentorium as the electrode was being introduced. The adequacy of the lesion was confirmed in the biochemical assay. The control values (Table I) are comparable to, although somewhat lower than, those reported by Kobayashi et al. 18 in a different species, the rat. The cats with bilaterally inaccurate lesions had levels of norepinephrine comparable to those of the controls. The cat with unilateral ablation of the locus on the left (Table I, L1) had levels of norepinephrine in the ipsilateral paraventricular hypothalamic nucleus that were 51 ~ of control, in the anterior ventral thalamic nucleus 37 ~ of control, and in the parietal cortex 42 ~ of
440 control. These data for ipsilateral depletion are similar to those reported by Kobayashi et al. la for the rat. The three cats with adequate lesions histologically had a profound decrease in the norepinephrine level of the hemispheric areas biopsied to approximately 19 % of the control level. This decrease would suggest that a considerable part of the norepinephrine in these sites is in axons with their cell bodies in the locus coeruleus although other nuclei do contribute to this supply ')9. Recently, it has been reported that bilateral destructive lesions in the locus coeruleus of rats, although causing a marked reduction in non-sympathetic perivascular adrenergic nerves, did not result in their complete loss 17. However, these authors used fluorescence techniques to identify the adrenergic nerves and thus could not differentiate among norepinephrine-, epinephrine-, and dopamine-containing neurons. (Dopamine is not measured by the radiochemical technique that we used to measure catecholamines.) The blood flow in the experiments reported herein show a significant increase in resting CBF in the cats with lesions, a statistically significant difference in unresponsiveness to Pac02, but an essentially retained ability to autoregulate to changes in blood pressure. The lack of response to Pat02 in the presence of brain stem lesions agrees with the report by Shalit et al. '~5, but the high initial resting level was not found by these workers, and they did not test the response to changes in perfusion pressure. The more recent reports of Fenske et al. 9, who reported significant loss of vascular response to hypercapnia in cats after a cold lesion had been produced in the brain stem, and by Capon 3, who showed a similar change in Pat02 responsiveness after high pontine transsection in cats, are in keeping with our results. In their preparations, Fenske et al. 9 also noted a retained autoregulation to pressure changes. All three reports have suggested that 'high brain stem' lesions can reduce the response of the cerebral vessels to alteration in Pac02. Our results and the report by Raichle et al. 22 who showed changes in CBF with pharmacologic stimuli to the locus coeruleus, suggest that this area of the brain stem is responsible for these changes. Thus this nucleus probably can alter the caliber of the cerebral vessels and thereby alter the CBF, and of the stimuli tested, this control is most manifest in response to changes in Pac02. Our study indicates that the response of the cerebral vessels to changes in pressure is independent of this control and therefore either is governed by another center or is due to local phenomena in the vascular smooth muscle - - the Bayliss effect~L Alternatively, the response to Paco2 may be due to alteration in the caliber of the smaller, intracerebral vessels, whereas the response to alteration in perfusion pressure is governed by the larger, extracerebral vessels. Thus, whereas lesions of the locus coeruleus may deplete the terminals of the intra-axial noradrenergic neurons, they may spare the fibers that take an alternate route to the pial vessels. Such a suggestion is similar to that raised by Harper et al. TM that the extraparenchymal vessels might be governed by the cervical sympathetic system and that the intraparenchymal vessels might be governed by an alternate system. The difference is that our results suggest that the intracerebral vessels also are subjected to neurogenic control but one with separate route for the brain stem center. In Fig. 1 CBF values are reported as absolute measurements. However, if CBF changes were to be plotted as a percentage change of the basal flow values, which were
441 300
• • Lesion - - 4 - - Non*lesion ..... • ..... Control
= i~~1/ I/"l
,~'
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~ 200
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160
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60
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480
Fig. 4. Results of Fig. i plotted as percentage of basal flow values. higher in the animals with correctly placed lesions, then the abnormality in the Paco2 response is even more dramatic (Fig. 4). This would show that the response to hypocapnia is remarkably similar in all three groups. This suggests that the basal level and response to hypercapnia are primarily affected by the lesion. In addition, our results raise the possibility that noradrenergic neurons may contribute to an inherent 'resting tone' in the cerebral blood vessels. Release from this tone may result in vasodilatation that would explain the increase in basal CBF in the presence of lesions in the locus coeruleus. If this is true, these mechanisms may have a role in the response to cerebral ischemia and brain stem infarction. ACKNOWLEDGEMENTS This investigation was supported in part by Research Grants NS 6663 and H L 17487-1 from the National Institutes of Health, Public Health Service and by the Minnesota Heart Association Grant M H A 23.
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