Journal of Cerebral Blood Flow and Metabolism
11:66-71 © 1991 Raven Press, Ltd., New York
Effects of Lesioning of the Substantia Innominata on Autoregulation of Local Cerebral Blood Flow in Rats
Shintaro Gomi, Fumio Gotoh, Naoki Ishihara, Kortaro Tanaka, Yoshiki Ishikawa, Shutaro Takashima, and Ban Mihara Department of Neurology, School of Medicine, Keio University, Tokyo, Japan
Summary: Recently, accumulated data have suggested that the nucleus basalis magnocellularis, i. e., the substan tia innominata (S1), may represent the primary source of central cholinergic innervation in the rat cortical vascu lature. We therefore examined the effects of unilateral lesion of the SI on the autoregulation of local CBF (lCBF) during induced hypotension in rats. Male Wistar rats were divided into three groups. The animals of groups 1 and 2 received an injection of 5 fLg of ibotenate into the right SI stereotaxically. At 7 days after the injection, the lCBF was measured by the [14C]iodoantipyrine technique in the awake state. Group 1 was used as the normotensive group (MABP 113.1 ± 12.2 mm Hg). Group 2 formed the hypotensive group, and the lCBF was measured dur ing hypotension (MABP 80.0 ± 5.5 mm Hg) induced by hemorrhage. Group 3, the sham-operated normotensive
group, received vehicle injection into the right SI at 7 days prior to the lCBF measurement. In group 1, lCBF was significantly lower in the frontal, parietal, temporal, and striate cortices on the lesioned side compared to that on the contralateral side. In group 2, lCBF was signifi cantly decreased in the cortices on the lesioned side, but there was no significant difference in magnitude of the ICBF reduction between groups 1 and 2. Group 3 exhib ited no hemispheric asymmetries in lCBF. These findings suggest that the SI exerts an influence on cortical ICBF, but does not play a role in the autoregulation of lCBF during hypotension. Key Words: Cerebral blood flow Autoregulation-Nucleus basalis magnocellularis-Sub stantia innominata-Neurogenic control-Cholinergic in nervation.
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Constancy of CBF despite changes in perfusion
electrical stimulation of the ganglion is reported to
pressure has been designated as autoregulation of
increase CBF in the cerebral cortex (Seylaz et al.,
CBF. Many previous studies have indicated that
1988; Suzuki et al., 1990).
CBF autoregulation is mediated by several mecha
Recently accumulated data have suggested that
nisms, of which the neurogenic mechanism is of
the nucleus basalis magnocellularis, i.e., the sub
greatest importance (Gotoh et al., 1975, 1981).
stantia innominata (SI), represents the primary
However, much effort has been directed towards
source of central cholinergic innervation in the rat
studying the adrenergic system, and little is known
cortex (Wenk et al., 1980; Bigl et al., 1982; McKin
about the influence of the cholinergic system on the
ney et al., 1983; Saper, 1984; Kasa, 1986). Destruc
cerebral circulation (Edvinsson et al., 1977). So far,
tion of this nucleus results in impairment of the per
the extracranial parasympathetic system including
formance of active or passive avoidance tasks (Flick
the sphenopalatine ganglion has been suggested as
er et al., 1983; Tanaka et al., 1987; Mayo et al.,
one of the origins of cerebrovascular cholinergic fi
1988) and in attenuation of cerebral glucose metab olism (Tanaka et al., 1987; Gomi et al., 1988). Fur
bers (Hara et al., 1985; Suzuki et al., 1988) and
thermore, the possible innervation of intracerebral blood vessels by choline acetyltransferase (ChAT) containing neurons from the SI has been reported
Received February 9, 1990; accepted June 26, 1990. Address correspondence and reprint requests to Dr. K.
(Eckenstein and Baughman, 1984; Armstrong, 1986; Luiten et al., 1987). It is possible, therefore,
Tanaka at Department of Neurology, School of Medicine, Keio University, 35 Shinano-machi, Shinjuku-ku, Tokyo 160, Japan. Abbreviations used: AchE, acetylcholinesterase; ChAT, choline acetyltransferase; lAP, iodoantipyrine; Sl, substantia in nominata.
that the cholinergic nerve fibers innervating the ce rebral cortical blood vessels may originate from the SI. Clinical studies in patients with senile dementia
66
67
CBF AUTOREGULATION AND SUBSTANTIA INNOMINATA
of the Alzheimer type, in which loss of cholinergic neurons in the nucleus basalis of Meynert is even tually observed (Whitehouse et al., 1982), have demonstrated a decrease of blood flow in the cere bral cortices. However, reports on the autoregula tion of CBF in such patients are scarce (Simard et al., 1971). The purpose of the present study was to examine the effects of unilateral lesion of the SI on the local cerebral blood flow (lCBF) and its autoregulation during induced hypotension in rats. Part of the present data was presented at the Meeting on Neu rotransmission and Cerebrovascular Function at La Napoule, France, in 1989.
ments, Buffalo, NY, U.S.A.) at -20°C. The slices were thaw-mounted onto glass cover slips, and dried on a hot plate at 70°C. The materials were then exposed to radio graphic films (SB-5, Eastman Kodak, Rochester, NY, U.S.A.) with [14C]methylmethacrylate standards (Amer sham, U.K.) for 7 days. Quantitative densitometric anal ysis of the resultant autoradiograms was carried out with the computerized digital image processing system devel oped in our laboratory, which consists of a charge coupled device (CCD) video camera, video digitizer, frame memory, RGB color monitor, and IBM PC/AT computer (Gomi et aI., 1988; Tanaka et aI., 1988). Values of lCBF were calculated from the 14C radioactivities of the brain tissue and history of the arterial blood samples. The animals were divided into three groups as follows: Group 1 formed the normotensive group (n 9, MABP 113.1 ± 12.2 mm Hg) with an ibotenate lesion. Group 2 (n 8) consisted of animals with the same lesion as group I, but the lCBF was measured during hypotension (MABP 80.0 ± 5.5 mm Hg) induced by hemorrhage: the average volume of blood withdrawn for induction of hypotension was 5.3 ± 0.3 m!. Group 3, the sham operated group (n 7), formed the normotensive group (MABP 120.3 ± 9.0 mm Hg) with vehicle injection. Five coronal brain slices including the SI taken at in tervals of 0.5 mm were stained with hematoxylin and eosin to examine the SI lesions histologically by light mi croscopy. Data were expressed as the mean ± SD. Statistical analysis was performed by Student's paired t test for evaluating side-to-side differences in ICBF and by Bon ferroni's modified t test for examining differences in lCBF and physiological parameters among the groups. The experimental protocol has been approved by the Experimental Animals Committee of Keio University. =
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MATERIALS AND METHODS
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Experiments were performed on 24 male Wistar rats weighing 260-330 g. The animals were allowed access to standard rat pellets and water ad libitum, and were main tained under a 12-h dark/light cycle. The animals were lightly anesthetized with pentobar bital sodium (25 mg/kg, i.p.) and secured in a stereotaxic apparatus (Type SR-6, Narishige Scientific Instrument Lab., Tokyo, Japan). A sagittal scalp incision was made, and 5 f.Lg of ibotenate (Sigma, St. Louis, MO, U.S.A.) in I f.Ll of Krebs-Ringer solution or 1 f.Ll of the vehicle only was injected into the right SI stereotaxically using a Ham ilton microsyringe through a burr hole in the calvarium over a period of 15 min. The needle was left in place for an additional 5 min to prevent diffusion along the needle tract. Employing the stereotaxic atlas of Paxinos and Watson (1982), the infusion coordinates were as follows: 0.8 mm posterior to the bregma, 2.5 mm lateral to the midline, and 8.0 mm ventral to the skull surface. The skin was sutured and the animals were observed until they became alert and returned to their vivarium. At 7 days after the injection, the animals were lightly reanesthetized with pentobarbital sodium (25 mg/kg, i.p.) and the femoral arteries and left femoral vein were can nulated (PE-50) for continuous monitoring of the arterial blood pressure, blood sampling, and tracer administra tion. Following surgery, the animals were immobilized by means of loose-fitting plaster casts. Four hours after the beginning of anesthesia, 0.2 ml of arterial blood was sam pled for Paoz, Pacoz, and pH measurement using a blood gas analyzer (BMS3 Mk2 Blood Micro System, Radiom eter, Copenhagen, Denmark). The ICBF was then deter mined by the [14C]iodoantipyrine ([14C]IAP) autoradio graphic technique (Sakurada et aI., 1978) in the awake state. Briefly, 740 kBq of [14C]IAP (specific activity of 2.18 GBq/mmol; Amersham, Buckinghamshire, U.K.) in 2.5 ml of saline was injected intravenously at a constant rate employing a motor-driven injector, and 35 s later the an imals were decapitated. The brains were immediately re moved and frozen in Freon 22 (Asahi Glass, Tokyo, Ja pan). During the period of injection, 14 arterial blood samples of 20 f.LI were collected, and the 14C radioactivity of each sample was measured with a liquid scintillation counter (LS9800, Beckman Instruments, Irvine, CA, U.S.A.). The brains were cut into 20-f.Lm thick slices in a cryostat (Histostat microtome, AO Scientific Instru-
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RESULTS
The physiological parameters of the three groups are listed in Table I. No significant difference was observed among the three groups except in the mean arterial blood pressure. Other parameters were within the normal ranges in each group. Histological evaluations of the brain slices ob tained from group 1 and group 2 substantiated se lective destruction of the SI. These animals re vealed a sphere of neuronal degeneration and marked infiltration of macrophages in the SI. There was no direct damage within the cerebral cortex. TABLE 1. Physiological parameters MABP (mm Hg)
Paoz (mm Hg)
Pacoz (mm Hg)
pH
113.1 12. 2
102.9 18. 9
37. 9 5. 1
7.36 0.03
80.0 5. 5
105.4 8. 5
33. 5 2. 8
7.41 0.04
120.3 9. 0
90. 8 9. 6
35. 6 3. 1
7.41 0.00
--- ------ "---
Group 1 Mean SD Group 2 Mean SD Group 3 Mean SD
J Cereb Blood Flow
Metab, Vol. 11, No.1, 1991
68
S. GOM! ET AL.
Figure 1 shows representative color-coded [14C]_ iodoantipyrine autoradiograms of groups 1 and 2. In group 1 (normotensive group), the ICBF in the right cerebral cortex was lower than that in the left cor tex at the levels of both the frontal cortex and the thalamus. On the other hand, other deep structures, i.e., the thalamus and hippocampus, exhibited no asymmetry. The decrement of cortical lCBF on the right side in group 2 (hypotensive group) was simi lar to that in group 1 in terms of its magnitude and distribution. In group 2, the ICBF in the deep struc tures also showed no asymmetry and did not differ from that of group 1. The results of ICBF measurements are summa rized in Table 2. In group 1, the lCBF in the cortices on the lesioned side ranged from 116 ml 100 g - 1 min -1 in the frontal cortex to 180 ml 100 g-I min- 1 in the temporal cortex auditory area. The values for ICBF in the frontal, frontoparietal (motor and so matosensory area), temporal, and striate cortices on the lesioned side amounted to 83-95% of those in the corresponding regions on the contralateral side. These differences between the two sides were sta tistically significant. In group 2, the values of lCBF in the cortices on the lesioned side ranged from 116 m] 100 g-I min -I in the frontal cortex to 157 ml 100 g-I min-I in the temporal cortex auditory area, and were 89-95% of those on the contralateral side. The reductions in cortical ICBF on the lesioned side showed a similar pattern to those observed in group 1. These interhemispheric differences in group 2 were also statistically significant in the frontal cor tex, frontoparietal cortex motor area, and somato sensory area. The values of ICBF in the deep struc-
FIG. 1. Representative color coded a u t ora di ograms ob tained from rats of group 1 (nor motensive) (left) and group 2 (hypotensive) (right). The view er's left is the animal's right. In both groups, the leBF in the cortex on the right (Iesioned side) was lower than on the left, and the differences were of sim ilar magnitude. The blood flow in the deep structures did not reveal any asymmetry.
J Cereb Blood Flow Metab. Vol. II. No. I. 1991
TABLE 2. Local cerebral blood flow (ml 100 g-I min-I) Group I Frontal cortex R L
Group 2
Group 3
116± 34* 128±41
116±27** 129±29
122±41 120±40
Motor area R L Somatosensory area R L
119±42** 144± 39
125± 36* 140± 37
151±44 157±43
150±57** 163 ±56
142± 39** 151± 38
174±51 177±51
Caudate-putamen R L
122±46 118± 39
117±27 121± 3 3
135±46 1 3 4±42
Thalamus medial R L
157± 37 156± 37
138± 40 140± 36
142±25 141±25
Thalamus lateral R L
143 ± 38 145± 36
130 ± 39 132 ± 3 3
130±22 1 3 1±24
180±58* 207±64
157±42 168±44
189±43 197±57
149±63* 157±59
124± 31 133 ± 31
137± 34 140± 32
92±24 94±21
90±12 90±II
Temporal cortex R L Striate cortex R L Hippocampus R L
97±19 96±22
Values are expressed as the mean± SO. Asterisks denote a significant difference from the contralateral side by Student's paired t test (*p < 0.05, **p < 0.01). R and L indicate the right (with the SI lesion) and left sides, respectively.
tures, i.e. , the caudate-putamen, thalamus, and hippocampus, did not exhibit any significant differ ences between both sides in the above two groups. In addition, no significant difference was observed in the ICBF of each region between groups 1 and 2.
CBF AUTOREGULATION AND SUBSTANTIA INNOMINATA
The sham-operated animals (group 3) displayed no interhemispheric asymmetry in ICBF, and the val ues of ICBF in each region did not differ from those of group 1 or 2. The ratio of the values of the cor tical lCBF on the right to those on the left for each region did not differ statistically between groups 1 and 2, and the magnitude of the reduction in cortical ICBF on the right side, when compared to that on the left, was similar between the above two groups.
DISCUSSION
The findings of the present study may be summa rized as follows: (a) The values of ICBF in the cor tices on the lesioned side were significantly lower than those on the contralateral side in the nor motensive group. (b) The reduction in cortical lCBF on the lesioned side in the hypotensive group was of similar extent to that in the normotensive group. Accordingly, it seems reasonable to consider that the SI exerts a definite influence on the ICBF in the cortices, but does not play an important role in the autoregulation of ICBF during induced hypoten sIOn. We could not find any statistical differences in ICBF of each region among three groups, although there was some intergroup or intragroup variance. Since respiration of animals was not controlled, Paeo2 also showed slight variability. Variance of ICBF might be ascribable to that of Paeo2. The right/left ratio of ICBF for each region did not differ between groups 1 and 2. We therefore considered that the autoregulation is not impaired after the SI lesioning. It is reported that the cholinergic fibers innervat ing the cerebral vessels exert a vasodilatory effect and play an important role in autoregulation of the CBF (Reis et aI., 1985; MacKenzie and Scatton, 1987), but the origin of these fibers is still a matter of contention. The nucleus basalis magnocellularis represents a major cholinergic input to the cerebral cortex (Wenk et aI., 1980; Bigl et aI., 1982; McKinney et aI., 1983; Saper, 1984; Kasa, 1986). Destruction of this nucleus results in impairment of the perfor mance of active or passive avoidance tasks (Flicker et aI., 1983; Tanaka et aI., 1987; Mayo et aI., 1988) and a decline in the acetylcholine esterase (AchE) and ChAT activities in the cerebral cortex. Arm strong (1986) and Luiten et ai. (1987) suggested the possibility that the intracerebral blood vessels may be innervated by ChAT-containing neurons in the basal forebrain. Eckenstein and Baughman (1984) noted that fibers and varicosities containing vaso-
69
active intestinal polypeptide (VIP) or ChAT immu noreactivity are in intimate association with blood vessels in the cerebral cortex, and that these struc tures might originate partly from the SI. Further more, ladecola et ai. (1983) reported that the in crease in ICBF of the cerebral cortices during se lective electrical stimulation of the cerebellar fastigial nucleus, possibly mediated by a cholinergic mechanism, was abolished by placement of the le sion in the basal forebrain. It may be possible there fore that these neurons in the nucleus basalis mag nocellularis, including those in the SI, contribute to the cholinergic innervation of the cerebral cortical arteries. Recently, Lacombe et ai. (1987) found that elec trical stimulation in the SI could induce significant cortical vasodilation. They speculated that the ob served vasodilation might be mediated directly via vasomotor innervation or indirectly via metabolic stimulation. If the SI exerts a direct influence on the cortical blood vessels, it is considered that both a reduction in blood flow and impairment of its auto regulation in the cerebral cortices should be ob served in animals with SI lesions (Gotoh et aI., 1981; ladecola et aI., 1983; Ishitsuka et aI., 1986; Gotoh and Tanaka, 1988). Lacombe et al. (1989) examined the effects of electrical stimulation of the SI on the cerebral blood flow and tissue P02 and Peo2 in rats. Their results suggested that stimulation of the SI increased the cortical blood flow through a direct vasodilatory ef fect. Itakura et ai. (1989) observed that lesioning of the SI resulted in a decline of cortical blood flow. They attributed the reduction in blood flow to de nervation of vasodilatory fibers probably originat ing in the nucleus basalis magnocellularis. Despite these reports, Araki et al. (1985) failed to find any difference in the autonomic nervous action potential of pial arteries during induced hypoten sion in cats with lesions of the nucleus basalis of Meynert, compared to those without such lesions. They concluded that the nucleus does not play a role in the autoregulation of ICBF in the cerebral cortex. ladecola et ai. (1983) examined the effects of SI lesions on the autoregulation of ICBF by the [14C]IAP method and detected no impairment of the autoregulation. Gotoh et ai. (1975) demonstrated that the change in diameter of pial arteries in response to an alter ation of blood pressure was pronounced in vessels with a diameter greater than 50 J..Lm , while the change occurring during CO2 inhalation or hyper ventilation was apparent in arteries smaller than 50 J..Lm. Accordingly, arteries greater than 50 J..Lm in diameter are thought to be primarily involved in J Cereb Blood Flow
Me/ab. Vol. 11, No.1, 1991
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S. GOMI ET AL.
autoregulation of the CBF. If smaller blood vessels in the cerebral cortex were mainly innervated by fibers originating in the ChAT-containing neurons of the SI, there would be a possibility that lesioning or stimulation of the SI might exert an influence only on the baseline ICBF, and not on the autoreg ulation of the ICBF. We reported previously that the cortical glucose metabolic rate as measured by the e4C]deoxyglu cose autoradiographic method was decreased on the lesioned side in rats with unilateral lesion of the SI (Gomi et ai., 1988). The extent of reduction in the glucose metabolism was identical with that of the cortical blood flow observed in the normotensive animals with SI lesions in the present study. It is well known that CBF has a close relationship with glucose metabolism under physiological con ditions and provides useful information concerning the level of functional activity in the brain (Soko loff, 1981). It could be considered, therefore, that the observed decrease of cortical blood flow in nor motensive animals with SI lesions is partly due to metabolic suppression. Acknowledgment: This study was supported in part by a Grant from the Research Committee on Dementia, the Ministry of Health and Welfare, Japan.
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Cereb Blood Flow Metab 5(suppl 1):S517-S518 Armstrong DM (1986) Ultrastructural characterization of choline acetyltransferase-containing neurons in the basal forebrain of rat: Evidence for a cholinergic innervation of intracere bral blood vessels. J Comp Neurol 250:81-92 Bigl V, Woolf NJ, Butcher LL (1982) Cholinergic projections from the basal forebrain to frontal, parietal, temporal, oc cipital, and cingulate cortices: A combined fluorescent tracer and acetylcholinesterase analysis. Brain Res Bull 8:727-749 Eckenstein F, Baughman RW (1984) Two types of cholinergic innervation in cortex, one co-localized with vasoactive in testinal polypeptide. Nature (Lond) 309:153-155 Edvinsson L, Falck B, Owman C (1977) Possibilities for a cholin ergic action on smooth musculature and on sympathetic ax ons in brain vessels mediated by muscarinic and nicotinic receptors. J Pharmacol Exp Ther 200:117-126 Flicker C, Dean RL, Watkins DL, Fisher SK, Bartus R (1983) Behavioral and neurochemical effects following neurotoxic lesions of a major cholinergic input to the cerebral cortex in the rat. Pharmacol Bie)('hem Behav 18:973-981 Gomi S, Gotoh F, Ishihara N, Tanaka K, Ishikawa Y, Taka shima S, Mihara B (1988) Effects of lesioning of the nucleus basalis magnocellularis on local cerebral glucose utilization in the conscious rat. Autonom Nerv Syst (Tokyo) 25:527-534 Gotoh F, Muramatsu F, Fukuuchi Y, Amano T (1975) Dual con trol of cerebral circulation: Separate sites of action in vas cular tree in autoregulation and chemical control. In: Cere bral Circulation and Metaholism (Langfitt TW, McHenry LC Jr, Reivich M, Wollman H, eds), Berlin, Springer Verlag, pp 43�5 Gotoh F, Fukuuchi Y, Shimazu K, Amano T, Tanaka K, Ko-
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ogy. Neural Control Mechanisms (Kovach AGB, Sandor P, Kollai M, eds), Budapest, Akademiai Kiad6, pp 127-136 Gotoh F, Tanaka K (1988) Regulation of cerebral blood flow. In: Handhook of Clinical Neurology, Vol. 53. Vascular Dis eases, part I (Vinken PJ, Bruyn GW, Klawans HL, TooIJF, eds), Amsterdam, Elsevier, pp 47-77 Hara H, Hamill GS, Jacobowitz DM (1985) Origin of cholinergic nerves to the rat major cerebral arteries: Coexistence with vasoactive intestinal polypeptide. Brain Res Bull 14:179-188 Iadecola C, Mraovitch S, Meeley MP, Reis DJ (1983) Lesions of basal forebrain in rat selectively impair the cortical vasodi lation elicited from cerebellar fastigial nucleus. Brain Res 279:41-52 Ishitsuka T, Iadecola C, Underwood MD, Reis DJ (1986) Le sions of nucleus tractus solitarii globally impair cerebrovas cular autoregulation. Am J Physiol 251:H269-H281 Itakura T, Yokote H, Yukawa S, Imai H, Kamei I, Komai N (1989) Transplantation of cholinergic neurons into Alz heimer model rat brain: Behavioral change and local cere bral blood flow. J Cereb Blood Flow Metah 9(suppl I):S527 Kasa P (1986) The cholinergic systems in brain and spinal cord. Prog Neurobiol 26:211-272 Lacombe P, Sercombe R, Correze JL, Philipson V, MacKenzie ET, Seylaz J (1987) Cortical vasodilation induced by elec trical stimulation in the substantia innominata in anesthe tized rats. J Cereb Blood Flow Metab 7(suppl I):S393 Lacombe P, Sercombe R, Verrecchia C, Philipson V, MacKen zie ET, Seylaz J (1989) Cortical blood flow increases in duced by stimulation of the substantia innominata in the unanesthetized rat. Brain Res 491:1-14 Luiten PGM, Gaykema RPA, Traber J, Spencer DG Jr (1987) Cortical projection patterns of magnocellular basal nucleus subdivisions as revealed by anterogradely transported Phaseolus vulgaris leucoagglutinin. Brain Res 413:229-250 MacKenzie ET, Scatton B (1987) Cerebral circulatory and met abolic effects of perivascular neurotransmitters. CRC Crit Rev Clin Neurohiol 2:357�19 Mayo W, Kharouby M, Le Moal M, Simon H (1988) Memory disturbances following ibotenic acid injections in the nucleus basalis magnocellularis of the rat. Brain Res 455:213-222 McKinney M, Coyle JT, Hedreen JC (1983) Topographic analy sis of the innervation of the rat neocortex and hippocampus by the basal forebrain cholinergic system. J Comp Neurol 217:103-121 Paxinos G, Watson C (1982) The Rat Brain in Stereotaxic Coor dinates. Sydney, Academic Press Reis DJ, Iadecola C, Nakai M (1985) Control of cerebral blood flow and metabolism by intrinsic neural system in brain. In: Cerehrovascular Diseases (Plum F, Pulsinelli W, eds), New York, Raven Press, pp 1-25 Sakurada 0, Kennedy C, Jehle J, Brown JD, Sokoloff L (1978) Measurement of local cerebral blood flow with iodo [,4C]antipyrine. Am J Physiol 234:H59-H66 Saper CB (1984) Organization of cerebral cortical afferent sys tems in the rat. II. Magnocellular basal nucleus. J Comp NeuroI222:313-342 Seylaz J, Ham H, Pinard E, Mraovitch S, MacKenzie ET, Ed vinsson L (1988) Effect of stimulation of the sphenopalatine ganglion on cortical blood flow in the rat. J Cereb Blood Flow Metab 8:875-878 Simard D, Olesen J, Paulson OB, Lassen NA, Skinhoj E (1971) Regional cerebral blood flow and its regulation in dementia. Brain 94:273-288 Sokoloff L (1981) Relationships among local functional activity, energy metabolism, and blood flow in the central nervous system. Fed Proc 40:2311-2316 Suzuki N, Hardebo JE, Owman C (1988) Origins and pathways of cerebrovascular vasoactive intestinal polypeptide positive nerves in rat. J Cereb Blood Flow Metab 8:697-712 Suzuki N, Hardebo JE, Kahrstrom J, Owman C (1990) Selective
CBF AUTOREGULATION AND SUBSTANTIA INNOMINATA
electrical stimulation of postganglionic cerebrovascular parasympathetic nerve fibers originating from the spheno palatine ganglion enhances cortical blood flow in the rat. J Cereb Blood Flow Metab 10:383-391 Tanaka K, Gotoh F, Ishihara N, Ishikawa Y, Gomi S, Takash ima S, Mihara B (1987) Behavior and local cerebral glucose metabolism in rats with lesions of nucleus basalis magnocel lularis. J Cereb Blood Flow Metab 7(suppl 1):S380 Tanaka K, Gotoh F, Ishihara N, Gomi S, Takashima S, Mihara
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B (1988) Autoradiographic analysis of second-messenger systems in the gerbil brain. Brain Res Bull 21 :693-700 Wenk H, Bigl V, Meyer U (I980) Cholinergic proj ections from magnocellular nuclei of the basal forebrain to cortical areas in rats. Brain Res Rev 2:295-316 Whitehouse PJ, Price DL, Struble RG, Clark AW, Coyle JT, DeLong MR (1982) Alzheimer's disease and senile demen tia: Loss of neurons in the basal forebrain. Science 215:1237-1239
J
Cereb Blood Flow Metab, Vol. 11, No.1, 1991
11:66-71 © 1991 Raven Press, Ltd., New York
Effects of Lesioning of the Substantia Innominata on Autoregulation of Local Cerebral Blood Flow in Rats
Shintaro Gomi, Fumio Gotoh, Naoki Ishihara, Kortaro Tanaka, Yoshiki Ishikawa, Shutaro Takashima, and Ban Mihara Department of Neurology, School of Medicine, Keio University, Tokyo, Japan
Summary: Recently, accumulated data have suggested that the nucleus basalis magnocellularis, i. e., the substan tia innominata (S1), may represent the primary source of central cholinergic innervation in the rat cortical vascu lature. We therefore examined the effects of unilateral lesion of the SI on the autoregulation of local CBF (lCBF) during induced hypotension in rats. Male Wistar rats were divided into three groups. The animals of groups 1 and 2 received an injection of 5 fLg of ibotenate into the right SI stereotaxically. At 7 days after the injection, the lCBF was measured by the [14C]iodoantipyrine technique in the awake state. Group 1 was used as the normotensive group (MABP 113.1 ± 12.2 mm Hg). Group 2 formed the hypotensive group, and the lCBF was measured dur ing hypotension (MABP 80.0 ± 5.5 mm Hg) induced by hemorrhage. Group 3, the sham-operated normotensive
group, received vehicle injection into the right SI at 7 days prior to the lCBF measurement. In group 1, lCBF was significantly lower in the frontal, parietal, temporal, and striate cortices on the lesioned side compared to that on the contralateral side. In group 2, lCBF was signifi cantly decreased in the cortices on the lesioned side, but there was no significant difference in magnitude of the ICBF reduction between groups 1 and 2. Group 3 exhib ited no hemispheric asymmetries in lCBF. These findings suggest that the SI exerts an influence on cortical ICBF, but does not play a role in the autoregulation of lCBF during hypotension. Key Words: Cerebral blood flow Autoregulation-Nucleus basalis magnocellularis-Sub stantia innominata-Neurogenic control-Cholinergic in nervation.
=
=
Constancy of CBF despite changes in perfusion
electrical stimulation of the ganglion is reported to
pressure has been designated as autoregulation of
increase CBF in the cerebral cortex (Seylaz et al.,
CBF. Many previous studies have indicated that
1988; Suzuki et al., 1990).
CBF autoregulation is mediated by several mecha
Recently accumulated data have suggested that
nisms, of which the neurogenic mechanism is of
the nucleus basalis magnocellularis, i.e., the sub
greatest importance (Gotoh et al., 1975, 1981).
stantia innominata (SI), represents the primary
However, much effort has been directed towards
source of central cholinergic innervation in the rat
studying the adrenergic system, and little is known
cortex (Wenk et al., 1980; Bigl et al., 1982; McKin
about the influence of the cholinergic system on the
ney et al., 1983; Saper, 1984; Kasa, 1986). Destruc
cerebral circulation (Edvinsson et al., 1977). So far,
tion of this nucleus results in impairment of the per
the extracranial parasympathetic system including
formance of active or passive avoidance tasks (Flick
the sphenopalatine ganglion has been suggested as
er et al., 1983; Tanaka et al., 1987; Mayo et al.,
one of the origins of cerebrovascular cholinergic fi
1988) and in attenuation of cerebral glucose metab olism (Tanaka et al., 1987; Gomi et al., 1988). Fur
bers (Hara et al., 1985; Suzuki et al., 1988) and
thermore, the possible innervation of intracerebral blood vessels by choline acetyltransferase (ChAT) containing neurons from the SI has been reported
Received February 9, 1990; accepted June 26, 1990. Address correspondence and reprint requests to Dr. K.
(Eckenstein and Baughman, 1984; Armstrong, 1986; Luiten et al., 1987). It is possible, therefore,
Tanaka at Department of Neurology, School of Medicine, Keio University, 35 Shinano-machi, Shinjuku-ku, Tokyo 160, Japan. Abbreviations used: AchE, acetylcholinesterase; ChAT, choline acetyltransferase; lAP, iodoantipyrine; Sl, substantia in nominata.
that the cholinergic nerve fibers innervating the ce rebral cortical blood vessels may originate from the SI. Clinical studies in patients with senile dementia
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CBF AUTOREGULATION AND SUBSTANTIA INNOMINATA
of the Alzheimer type, in which loss of cholinergic neurons in the nucleus basalis of Meynert is even tually observed (Whitehouse et al., 1982), have demonstrated a decrease of blood flow in the cere bral cortices. However, reports on the autoregula tion of CBF in such patients are scarce (Simard et al., 1971). The purpose of the present study was to examine the effects of unilateral lesion of the SI on the local cerebral blood flow (lCBF) and its autoregulation during induced hypotension in rats. Part of the present data was presented at the Meeting on Neu rotransmission and Cerebrovascular Function at La Napoule, France, in 1989.
ments, Buffalo, NY, U.S.A.) at -20°C. The slices were thaw-mounted onto glass cover slips, and dried on a hot plate at 70°C. The materials were then exposed to radio graphic films (SB-5, Eastman Kodak, Rochester, NY, U.S.A.) with [14C]methylmethacrylate standards (Amer sham, U.K.) for 7 days. Quantitative densitometric anal ysis of the resultant autoradiograms was carried out with the computerized digital image processing system devel oped in our laboratory, which consists of a charge coupled device (CCD) video camera, video digitizer, frame memory, RGB color monitor, and IBM PC/AT computer (Gomi et aI., 1988; Tanaka et aI., 1988). Values of lCBF were calculated from the 14C radioactivities of the brain tissue and history of the arterial blood samples. The animals were divided into three groups as follows: Group 1 formed the normotensive group (n 9, MABP 113.1 ± 12.2 mm Hg) with an ibotenate lesion. Group 2 (n 8) consisted of animals with the same lesion as group I, but the lCBF was measured during hypotension (MABP 80.0 ± 5.5 mm Hg) induced by hemorrhage: the average volume of blood withdrawn for induction of hypotension was 5.3 ± 0.3 m!. Group 3, the sham operated group (n 7), formed the normotensive group (MABP 120.3 ± 9.0 mm Hg) with vehicle injection. Five coronal brain slices including the SI taken at in tervals of 0.5 mm were stained with hematoxylin and eosin to examine the SI lesions histologically by light mi croscopy. Data were expressed as the mean ± SD. Statistical analysis was performed by Student's paired t test for evaluating side-to-side differences in ICBF and by Bon ferroni's modified t test for examining differences in lCBF and physiological parameters among the groups. The experimental protocol has been approved by the Experimental Animals Committee of Keio University. =
=
=
MATERIALS AND METHODS
=
Experiments were performed on 24 male Wistar rats weighing 260-330 g. The animals were allowed access to standard rat pellets and water ad libitum, and were main tained under a 12-h dark/light cycle. The animals were lightly anesthetized with pentobar bital sodium (25 mg/kg, i.p.) and secured in a stereotaxic apparatus (Type SR-6, Narishige Scientific Instrument Lab., Tokyo, Japan). A sagittal scalp incision was made, and 5 f.Lg of ibotenate (Sigma, St. Louis, MO, U.S.A.) in I f.Ll of Krebs-Ringer solution or 1 f.Ll of the vehicle only was injected into the right SI stereotaxically using a Ham ilton microsyringe through a burr hole in the calvarium over a period of 15 min. The needle was left in place for an additional 5 min to prevent diffusion along the needle tract. Employing the stereotaxic atlas of Paxinos and Watson (1982), the infusion coordinates were as follows: 0.8 mm posterior to the bregma, 2.5 mm lateral to the midline, and 8.0 mm ventral to the skull surface. The skin was sutured and the animals were observed until they became alert and returned to their vivarium. At 7 days after the injection, the animals were lightly reanesthetized with pentobarbital sodium (25 mg/kg, i.p.) and the femoral arteries and left femoral vein were can nulated (PE-50) for continuous monitoring of the arterial blood pressure, blood sampling, and tracer administra tion. Following surgery, the animals were immobilized by means of loose-fitting plaster casts. Four hours after the beginning of anesthesia, 0.2 ml of arterial blood was sam pled for Paoz, Pacoz, and pH measurement using a blood gas analyzer (BMS3 Mk2 Blood Micro System, Radiom eter, Copenhagen, Denmark). The ICBF was then deter mined by the [14C]iodoantipyrine ([14C]IAP) autoradio graphic technique (Sakurada et aI., 1978) in the awake state. Briefly, 740 kBq of [14C]IAP (specific activity of 2.18 GBq/mmol; Amersham, Buckinghamshire, U.K.) in 2.5 ml of saline was injected intravenously at a constant rate employing a motor-driven injector, and 35 s later the an imals were decapitated. The brains were immediately re moved and frozen in Freon 22 (Asahi Glass, Tokyo, Ja pan). During the period of injection, 14 arterial blood samples of 20 f.LI were collected, and the 14C radioactivity of each sample was measured with a liquid scintillation counter (LS9800, Beckman Instruments, Irvine, CA, U.S.A.). The brains were cut into 20-f.Lm thick slices in a cryostat (Histostat microtome, AO Scientific Instru-
=
=
RESULTS
The physiological parameters of the three groups are listed in Table I. No significant difference was observed among the three groups except in the mean arterial blood pressure. Other parameters were within the normal ranges in each group. Histological evaluations of the brain slices ob tained from group 1 and group 2 substantiated se lective destruction of the SI. These animals re vealed a sphere of neuronal degeneration and marked infiltration of macrophages in the SI. There was no direct damage within the cerebral cortex. TABLE 1. Physiological parameters MABP (mm Hg)
Paoz (mm Hg)
Pacoz (mm Hg)
pH
113.1 12. 2
102.9 18. 9
37. 9 5. 1
7.36 0.03
80.0 5. 5
105.4 8. 5
33. 5 2. 8
7.41 0.04
120.3 9. 0
90. 8 9. 6
35. 6 3. 1
7.41 0.00
--- ------ "---
Group 1 Mean SD Group 2 Mean SD Group 3 Mean SD
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Figure 1 shows representative color-coded [14C]_ iodoantipyrine autoradiograms of groups 1 and 2. In group 1 (normotensive group), the ICBF in the right cerebral cortex was lower than that in the left cor tex at the levels of both the frontal cortex and the thalamus. On the other hand, other deep structures, i.e., the thalamus and hippocampus, exhibited no asymmetry. The decrement of cortical lCBF on the right side in group 2 (hypotensive group) was simi lar to that in group 1 in terms of its magnitude and distribution. In group 2, the ICBF in the deep struc tures also showed no asymmetry and did not differ from that of group 1. The results of ICBF measurements are summa rized in Table 2. In group 1, the lCBF in the cortices on the lesioned side ranged from 116 ml 100 g - 1 min -1 in the frontal cortex to 180 ml 100 g-I min- 1 in the temporal cortex auditory area. The values for ICBF in the frontal, frontoparietal (motor and so matosensory area), temporal, and striate cortices on the lesioned side amounted to 83-95% of those in the corresponding regions on the contralateral side. These differences between the two sides were sta tistically significant. In group 2, the values of lCBF in the cortices on the lesioned side ranged from 116 m] 100 g-I min -I in the frontal cortex to 157 ml 100 g-I min-I in the temporal cortex auditory area, and were 89-95% of those on the contralateral side. The reductions in cortical ICBF on the lesioned side showed a similar pattern to those observed in group 1. These interhemispheric differences in group 2 were also statistically significant in the frontal cor tex, frontoparietal cortex motor area, and somato sensory area. The values of ICBF in the deep struc-
FIG. 1. Representative color coded a u t ora di ograms ob tained from rats of group 1 (nor motensive) (left) and group 2 (hypotensive) (right). The view er's left is the animal's right. In both groups, the leBF in the cortex on the right (Iesioned side) was lower than on the left, and the differences were of sim ilar magnitude. The blood flow in the deep structures did not reveal any asymmetry.
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TABLE 2. Local cerebral blood flow (ml 100 g-I min-I) Group I Frontal cortex R L
Group 2
Group 3
116± 34* 128±41
116±27** 129±29
122±41 120±40
Motor area R L Somatosensory area R L
119±42** 144± 39
125± 36* 140± 37
151±44 157±43
150±57** 163 ±56
142± 39** 151± 38
174±51 177±51
Caudate-putamen R L
122±46 118± 39
117±27 121± 3 3
135±46 1 3 4±42
Thalamus medial R L
157± 37 156± 37
138± 40 140± 36
142±25 141±25
Thalamus lateral R L
143 ± 38 145± 36
130 ± 39 132 ± 3 3
130±22 1 3 1±24
180±58* 207±64
157±42 168±44
189±43 197±57
149±63* 157±59
124± 31 133 ± 31
137± 34 140± 32
92±24 94±21
90±12 90±II
Temporal cortex R L Striate cortex R L Hippocampus R L
97±19 96±22
Values are expressed as the mean± SO. Asterisks denote a significant difference from the contralateral side by Student's paired t test (*p < 0.05, **p < 0.01). R and L indicate the right (with the SI lesion) and left sides, respectively.
tures, i.e. , the caudate-putamen, thalamus, and hippocampus, did not exhibit any significant differ ences between both sides in the above two groups. In addition, no significant difference was observed in the ICBF of each region between groups 1 and 2.
CBF AUTOREGULATION AND SUBSTANTIA INNOMINATA
The sham-operated animals (group 3) displayed no interhemispheric asymmetry in ICBF, and the val ues of ICBF in each region did not differ from those of group 1 or 2. The ratio of the values of the cor tical lCBF on the right to those on the left for each region did not differ statistically between groups 1 and 2, and the magnitude of the reduction in cortical ICBF on the right side, when compared to that on the left, was similar between the above two groups.
DISCUSSION
The findings of the present study may be summa rized as follows: (a) The values of ICBF in the cor tices on the lesioned side were significantly lower than those on the contralateral side in the nor motensive group. (b) The reduction in cortical lCBF on the lesioned side in the hypotensive group was of similar extent to that in the normotensive group. Accordingly, it seems reasonable to consider that the SI exerts a definite influence on the ICBF in the cortices, but does not play an important role in the autoregulation of ICBF during induced hypoten sIOn. We could not find any statistical differences in ICBF of each region among three groups, although there was some intergroup or intragroup variance. Since respiration of animals was not controlled, Paeo2 also showed slight variability. Variance of ICBF might be ascribable to that of Paeo2. The right/left ratio of ICBF for each region did not differ between groups 1 and 2. We therefore considered that the autoregulation is not impaired after the SI lesioning. It is reported that the cholinergic fibers innervat ing the cerebral vessels exert a vasodilatory effect and play an important role in autoregulation of the CBF (Reis et aI., 1985; MacKenzie and Scatton, 1987), but the origin of these fibers is still a matter of contention. The nucleus basalis magnocellularis represents a major cholinergic input to the cerebral cortex (Wenk et aI., 1980; Bigl et aI., 1982; McKinney et aI., 1983; Saper, 1984; Kasa, 1986). Destruction of this nucleus results in impairment of the perfor mance of active or passive avoidance tasks (Flicker et aI., 1983; Tanaka et aI., 1987; Mayo et aI., 1988) and a decline in the acetylcholine esterase (AchE) and ChAT activities in the cerebral cortex. Arm strong (1986) and Luiten et ai. (1987) suggested the possibility that the intracerebral blood vessels may be innervated by ChAT-containing neurons in the basal forebrain. Eckenstein and Baughman (1984) noted that fibers and varicosities containing vaso-
69
active intestinal polypeptide (VIP) or ChAT immu noreactivity are in intimate association with blood vessels in the cerebral cortex, and that these struc tures might originate partly from the SI. Further more, ladecola et ai. (1983) reported that the in crease in ICBF of the cerebral cortices during se lective electrical stimulation of the cerebellar fastigial nucleus, possibly mediated by a cholinergic mechanism, was abolished by placement of the le sion in the basal forebrain. It may be possible there fore that these neurons in the nucleus basalis mag nocellularis, including those in the SI, contribute to the cholinergic innervation of the cerebral cortical arteries. Recently, Lacombe et ai. (1987) found that elec trical stimulation in the SI could induce significant cortical vasodilation. They speculated that the ob served vasodilation might be mediated directly via vasomotor innervation or indirectly via metabolic stimulation. If the SI exerts a direct influence on the cortical blood vessels, it is considered that both a reduction in blood flow and impairment of its auto regulation in the cerebral cortices should be ob served in animals with SI lesions (Gotoh et aI., 1981; ladecola et aI., 1983; Ishitsuka et aI., 1986; Gotoh and Tanaka, 1988). Lacombe et al. (1989) examined the effects of electrical stimulation of the SI on the cerebral blood flow and tissue P02 and Peo2 in rats. Their results suggested that stimulation of the SI increased the cortical blood flow through a direct vasodilatory ef fect. Itakura et ai. (1989) observed that lesioning of the SI resulted in a decline of cortical blood flow. They attributed the reduction in blood flow to de nervation of vasodilatory fibers probably originat ing in the nucleus basalis magnocellularis. Despite these reports, Araki et al. (1985) failed to find any difference in the autonomic nervous action potential of pial arteries during induced hypoten sion in cats with lesions of the nucleus basalis of Meynert, compared to those without such lesions. They concluded that the nucleus does not play a role in the autoregulation of ICBF in the cerebral cortex. ladecola et ai. (1983) examined the effects of SI lesions on the autoregulation of ICBF by the [14C]IAP method and detected no impairment of the autoregulation. Gotoh et ai. (1975) demonstrated that the change in diameter of pial arteries in response to an alter ation of blood pressure was pronounced in vessels with a diameter greater than 50 J..Lm , while the change occurring during CO2 inhalation or hyper ventilation was apparent in arteries smaller than 50 J..Lm. Accordingly, arteries greater than 50 J..Lm in diameter are thought to be primarily involved in J Cereb Blood Flow
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S. GOMI ET AL.
autoregulation of the CBF. If smaller blood vessels in the cerebral cortex were mainly innervated by fibers originating in the ChAT-containing neurons of the SI, there would be a possibility that lesioning or stimulation of the SI might exert an influence only on the baseline ICBF, and not on the autoreg ulation of the ICBF. We reported previously that the cortical glucose metabolic rate as measured by the e4C]deoxyglu cose autoradiographic method was decreased on the lesioned side in rats with unilateral lesion of the SI (Gomi et ai., 1988). The extent of reduction in the glucose metabolism was identical with that of the cortical blood flow observed in the normotensive animals with SI lesions in the present study. It is well known that CBF has a close relationship with glucose metabolism under physiological con ditions and provides useful information concerning the level of functional activity in the brain (Soko loff, 1981). It could be considered, therefore, that the observed decrease of cortical blood flow in nor motensive animals with SI lesions is partly due to metabolic suppression. Acknowledgment: This study was supported in part by a Grant from the Research Committee on Dementia, the Ministry of Health and Welfare, Japan.
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