Download Prolonged Effects of Cholinesterase Inhibition

Journal of Cerebral Blood Flow and Metabolism
13:702-711 © 1993 The International Society of Cerebral Blood Flow and Metabolism
Published by Raven Press, Ltd., New York
Prolonged Effects of Cholinesterase Inhibition with
Eptastigmine on the Cerebral Blood Flow-Metabolism
Ratio of Normal Rats
*tOscar U. Scremin, *:j:A. M. Erika Scremin, *Deborah Heuser, *Raymond Hudgell,
*Elsa Romero, and §Bruno P. Imbimbo
*Veterans Affairs Medical Center and Departments of tPhysiology and tMedicine, UCLA School of Medicine,
Los Angeles, California U.S.A.; §Mediolanum Farmaceutici, Milan, Italy
Summary: The cerebrovascular and metabolic effects of
the novel cholinesterase inhibitor eptastigmine were
tested in conscious rats. The drug was administered by
single intravenous injection, and blood flow or glucose
utilization were assessed in 38 brain regions by quantita­
tive autoradiographic techniques. A dose-dependent in­
crease in regional cerebral blood flow (rCBF) was ob­
tained for i.v. doses ranging from 0.5 to 3 mg kg-I. Forty
minutes after the dose of 1.5 mg kg-I, average rCBF of
the 38 regions studied was (mean ± SD) 2.62 ± 0.62 ml
g- 1 min-I, a value significantly higher than that of saline­
injected controls ( 1. 46 ± 0.26; p < 0.005). In contrast, a
similar dose of eptastigmine did not significantly alter re­
gional cerebral glucose utilization (rCGU) (0.90 ± 0.21
ILmol g- 1 min - I) when compared with saline-injected
controls (0.99 ± 0.08 ILmol g-I min-I). A linear correla­
tion between rCBF and rCGU was observed both in sa­
line (r = 0.871) and eptastigmine (r = 0.873)-injected
animals but the slope of the regression line of rCBF on
rCGU was significantly higher (p < 0.01) in the eptastig­
mine group (2.863 ± 0.266) than in the controls that re­
ceived saline (1.00 ± 0.09 4). The cerebral vasodilatation
induced by eptastigmine peaked at 40 min after drug ad­
ministration. No toxic signs were observed at the doses
used. Mean arterial blood pressure decreased after 0.5 mg
kg-I (control = 109.3 ± 10.56 mm Hg; eptastigmine =
96.6 ± 8.10 mm Hg) but did not differ from control at the
higher doses. It is concluded that eptastigmine induces a
long-lasting increase in rCBF and a significant enhance­
ment of the rCBF:rCGU ratio in most regions. The results
suggest an important role of endogenous acetylcholine in
the control of cerebral perfusion. Key Words: Cerebro­
vascular circulation-Cerebral glucose utilization­
Acetylcholine-Cholinesterase inhibitors-Physo­
stigmine.
Acetylcholine and a number of muscarinic cholin­
ergic agonists are known to enhance cerebral blood
flow or pial vessel diameter when administered lo­
cally to the cerebral cortex (Scremin et aI., 1973;
Kuschinsky, et aI., 1974) or systemically (Rovere et
aI., 1973; Aoyagi et aI., 1975; Scremin, et aI., 1978;
Aubineau et aI., 1977; Heistad and Kontos, 1983;
Triguero et aI., 1988; Molnar, et aI., 1991). This
phenomenon is not associated with metabolic acti­
vation in most brain regions (Scremin et aI., 1982;
Scremin et aI., 1988b) and this has been corroborated
in a number of animal species and humans
(Scremin, 1991). The regional distribution of the
cholinergic effect on cerebral blood flow-metabo­
lism coupling is known for a limited number of cho­
linesterase inhibitors (Scremin et aI., 1988; Sc!emin
and Shih, 1991) and direct cholinergic agonists
(Scremin, et aI., 1988a; Maiese et aI., 1991).
T h e h e p t y l d e r i v a t i v e o f p h y s o s t i gmine
(pyrrolo(2 ,3b )indol-5-01 ,3,3a, 8,8a-hexahydro1,3a,8-trimethylheptylcarbamate [ester, (3aS-cis)]:
eptastigmine (MF201), is a recently characterized
carbamate cholinesterase inhibitor (Brufani et aI.,
1986) with central nervous system actions (Brufani
Received June 22, 1992; final revision received January 5,
1993; accepted January 6, 1993.
Address correspondence and reprint requests to Dr. Oscar U.
Scremin, West Los Angeles VA Medical Center, Bldg 115,
Room 317, Wilshire & Sawtelle Blvds, Los Angeles CA 90073,
U.S.A.
Abbreviations used: ACh, acetylcholine; e4C]2DG, e4C]2deoxyglucose; e4C]lAP, iodo-[14C]antipyrine; GCBF, global
CBF; GCGU, global cerebral glucose uptake; MABP, mean ar­
terial blood pressure; rCBF, regional CBF; rCGU, regional
CGU.
702
CHOLINERGIC EFFECTS ON CBF!CGU COUPLING
et aI., 1987; Marta et aI., 1988; De Sarno et aI.,
1989) and a half-time of enzyme recovery of 2. 9 h
for plasma and 12. 5 h for red blood cells of humans
following a single dose (Unni et aI., 1991). This cho­
linesterase inhibitor reverses behavioral deficits in
rodent and primate models of dementia at doses
within the range used in this study (Dawson et aI.,
1991; Rupniak et aI., 1992).
The present experiments were designed to deter­
mine the potency of eptastigmine as a cerebral va­
sodilator, the time course of this effect, if present,
and the regional distribution of cerebral blood flow
and glucose utilization under the prolonged cholin­
esterase inhibition induced by this drug.
MATERIAL AND METHODS
Wistar male rats, 25�3oo g body weight, were used.
Experiments were carried out at the Albuquerque DVA
Medical Center, altitude 1,600 meters above sea level,
average barometric pressure, 630 mm Hg. The rats were
on a 12-h light/dark full spectrum lighting cycle which was
provided between 0600 and 1800 h. All experiments were
performed according to established guidelines for the care
of experimental animals.
Regional cerebral blood flow (rCBF) was measured
with the iodo-[14C]antipyrine ([14C]lAP) quantitative auto­
radiographic method (Sakurada et aI., 1978). Two arterial
and two venous catheters were implanted in the femoral
vessels under halothane anesthesia (2.5% in air for induc­
tion and 1.5% for maintenance). After surgery, animals
were placed in a Bollman cage and allowed to recover
from anesthesia for 2 h. In these cages the animals rest in
a prone position with their limbs hanging to the sides.
Acrylic nontraumatic bars entrap the animal, preventing
locomotion but allowing limb and head movements. The
cage was covered with a cloth in order to prevent cooling
of the animal and to eliminate visual contact with the
environment. Rectal temperature was recorded and main­
tained at 37°C with a BAT-12 thermocouple thermometer
connected to a TCAT-IA (Physitemp, Inc.) temperature
controller and a source of radiant heat. One arterial cath­
eter was connected to a pressure transducer for continu­
ous recording of blood pressure, and the other was used
for sampling arterial blood. One of the venous catheters
was connected to a motor-driven syringe containing the
radioactive tracer solution and the other to a similar sy­
ringe containing the euthanasia solution {n-[2-(m­
methoxyphenyl)-I] }--y-hydroxybutiramide, 200 mg/ml;
4, 4'-methylene-bis(cyclohexyltrimethylammonium io­
dide), 50 mg/ml; tetracaine hydrochloride, 5 mg/ml) (T-61,
American Hoechst Co.). A sample of arterial blood was
obtained for measurement of blood gases and pH in a
Radiometer ABL-30 blood acid-base system and then the
infusion of [14C]lAP was started. Infusate volume was 0.6
ml, dose 100 /LCi/kg and the infusion period was 30 s.
Arterial blood samples (30 /LI) were obtained every 3 s
from a free-flowing catheter. Circulation was arrested by
an injection of 0.5 ml of euthanasia solution delivered
intravenously over the last 5 s of the [14C]lAP infusion.
This was attended by a precipitous fall in blood pressure.
The exact timing of circulatory arrest was determined
from the polygraph record of arterial blood pressure. The
703
brain was then rapidly removed and flash frozen in
methylbutane chilled to -70°C. These tissues were sec­
tioned in a cryostat at - 20°C in 20-/Lm slices, heat dried
and exposed to Kodak AR X-OMAT film in spring-loaded
x-ray cassettes along with eight standards of known ra­
dioactivity to obtain an P4C]lAP autoradiogram.
The regional cerebral glucose utilization (rCGU) was
measured with the autoradiographic quantitative [14C]2_
deoxyglucose ([14C]2DG) technique (Sokoloff et aI.,
1977). Venous and arterial femoral catheters were im­
planted under halothane anesthesia and the animals al­
lowed to recover for 2 h in a Bollman cage as described
above. A sample of arterial blood was obtained for mea­
surement of blood gases and pH in a Radiometer ABL-30
blood microsystem. Then, the procedure was started by
intravenous injection of a bolus of 0.6 ml of saline con­
taining 100 /LCi kg-I of [14C]2DG (Amersham Corp., spe­
cific activity -50 mCi/mmol). Arterial blood samples (60
/Ll) were obtained at times 0, 20, and 40 s and 1, 1.5, 3, 5,
7.5, 10, 15, 25, 35, and 45 min after tracer infusion. After
the last sample, a bolus of euthanasia solution was in­
fused intravenously and the brain rapidly removed and
processed for autoradiography as described above. Auto­
radiographs were digitized with a ChromaPro 45 IAIS
"Dumas" film illumination system and a Phillips CCD
monochrome imaging module coupled to an AT&T Targa
M8 digitizing board on a Tandon PCAl 12 microcomputer.
Optical density of selected brain regions and standards
was measured with JAVA (Jandel Scientific Corp.) soft­
ware and tissue radioactivity calculated by interpolation
from the radioactivity versus optical density relationship
defined by the standards.
Thirty-eight regions were studied, the stereotaxic co­
ordinates (Paxinos and Watson, 1982) of which are given
in Table 1. rCBF and rCGU data were calculated from
film optical density of brain autoradiographs and stan­
dards and arterial blood radioactivity in the case of rCBF,
and arterial plasma radioactivity and glucose concentra­
tion in the case of rCGU by use of the corresponding
operational equations (Sakurada et aI., 1978; Sokoloff et
aI., 1977). For every region, measurements of optical den­
sity were obtained from autoradiographs of two to three
different tissue sections. Global CBF (GCBF) and global
CGU (GCGU) values were calculated by averaging all
regions in every animal. Three groups of experiments
were performed in the following order: (a) Dose-effect
study of rCBF changes induced by eptastigmine: animals
were injected with eptastigmine dissolved in saline at
doses of 0.5, 1, or 3 mg kg-I by rapid i.v. infusion (20 s)
or with an equivalent volume of saline (controls) in
groups of 6, 5, 6, and 7 animals, respectively. rCBF was
measured 20 min after drug administration; (b) time
course of rCBF changes induced by eptastigmine: a dose
of 1.5 mglkg eptastigmine was injected i.v. and rCBF was
measured at 40, 90, 180, and 360 min after in groups in
five animals each; (c) effects of eptastigmine on rCGU:
Five animals were injected with 1.5 mg kg- I eptastigmine
i.v. and [14C]2DG infused 35 min later to initiate the
rCGU measuring procedure. Five additional rats received
saline and served as controls. The methods used for rCBF
and rCGU measurement differ in that the first integrates
blood flow over a period of 30 s whereas the second is
implemented over 45 min. In fact, since [14C]2DG specific
activity of gray matter peaks at 5 min after injection of
this tracer, most of the [14C]2DG phosphorylation takes
place in the initial moments of the procedure (Sokoloff et
J Cereb Blood Flow Me/ab, Vol. 13, No. 4, 1993
O. U. SCREMIN ET AL.
704
TABLE 1. Glucose utilization (ILmo/ g
Controls (saline)
-
J min J)
-
Eptastigmine (1.5 mg/kg i.v.)
Structure
Coordinates
Mean
SD
Mean
SD
Probability
Motor C
Somatos
Temporal
Area 17
Area 18
Area 18A
Olfactory
Cingulate
Hip CAl
Hip CA2
Hip CA3
Claustrum
N basalis
Caudate-p
Dentate G
Preoptic A
Lat hyp
Dor hyp
D m hyp
M Mam n
Amygdala
Interped n
S sup coli
D sup coli
Infer coli
Pont retic
Cerebellum
Med dor th
Med em
Per gr
M genic
M raphe
Pont n
C call
Int caps
Hip com
Ce med th
S nigra
Mean
SD
-0.3,3,0.7
-0.3,5.5,0.7
-5.8,7,0.7
-5.8,4,0.7
-5.8,2,0.7
-5.8,6,0.7
-1.3,5.5,0.7
0.7,0.5,0.7
-5.3,3.5,3
-5.3,5.5,6
-3.3,3.3,3.8
2.7,2. 5
-1.3,3,8
0.7,2.5,6
-3.3,1,4.2
0.3,1.4,8.3
-1.3,2.2,8.8
-2.3,0.5,8.5
-3.3,0.5,9
-4.8,0,99
-1.8,3.8,8
-5.8,0,9
-6.8,1,3
-6.8,1,4.2
-8.8,1.5,4.4
-7.8,1.3,8.5
-10.3,0,5
-1.8,0.5,5.2
-2.8,0,10
-7.3,0.5,5.5
-5.3,3.5,6
-7.3,0,8.5
-7.3,1,10
0.2,0,3.5
-1.3,2.5,4
1.3,0,4
-2.8,0,6.3
-5.3,2.5,8
1.21
1.34
1.50
l.l5
1.09
1.17
0.94
1.22
0.86
0.80
0.83
1.43
0.81
1.22
0.78
0.88
0.69
0.74
0.75
1.25
0.80
1.30
1.05
1.02
2.29
0.69
0.91
1. 26
0.57
0.82
1.37
l.l1
0.73
0.55
0.56
0.39
1.11
0.80
0.99
0.08
5
0.15
0.11
0.21
0.12
0.14
0.14
0.08
0.14
0.11
0.10
0.10
0.18
0.10
0.12
0.07
0.17
0.06
0.10
0.12
0.13
0.12
0.17
0.10
0.11
0.31
0.06
0.04
0.10
0.10
0.10
0.20
0.09
0.09
0.05
0.09
0.06
0.15
0.09
1.08
l.l4
1.27
0.98
0.87
0.96
0.87
1.08
0.64
0.68
0.70
1.77
0. 64
0. 92
0.67
0.68
0.60
0.64
0. 69
1.11
0.90
l.l9
1.42
1.00
2.04
0.65
0.80
1.16
0.56
0.68
1.24
1.04
0.73
0.44
0.52
0.40
1.03
0.75
0.90
0.21
5
0.34
0.35
0.22
0.19
0.22
0.20
0.20
0.31
O.ll
0.14
0.12
0.34
O.ll
0.23
0.15
0.22
0.12
0.10
0.12
0.35
0.20
0.43
0.36
0.20
0.52
0.16
0.18
0.29
0.12
0.13
0.29
0.24
0.24
0.09
0.13
0.15
0.27
0.16
ns
ns
ns
ns
ns
ns
ns
ns
<0.02
ns
ns
ns
<0.05
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
<0.05
ns
ns
ns
ns
n
Stereotaxic coordinates (mm) of the region's center, listed in column 2 (left to right) as coronal
plane, horizontal coordinate, and distance from pial surface for cortical regions, and as plane, horizontal coordinate, and vertical coordinate for all other regions. Region name (abbreviation): anterior
cingulate cortex (Cingulate); motor cortex (Motor); somatosensory cortex (Somatos); temporal cortex, auditory area (Temporal); occipital cortex, area 17 (Area 17); occipital cortex, area 18 (Area 18);
occipital cortex, area 18a (Area 18a); primary olfactory cortex (Olfactory); field CAl of Ammon's horn
(Hip CAl); field CA2 of Ammon's horn (Hip CA2); field CA3 of Ammon's horn (Hip CA3); dentate
gyrus (Dentate); medial dorsal thalamus (Med d thaI); central medial thalamic nucleus (Ce med thaI);
caudate-putamen (Caudate-p); claustrum (Claustrum); central amygdaloid nucleus (Amygdala); nucleus basalis (N basalis); lateral preoptic area (Preoptic a); lateral hypothalamic area (Lat hypoth);
dorsal hypothalamic area (Dor hypoth); dorsomedial hypothalamic nucleus (D m hypoth); median
eminence (Med em); medial mammillary nucleus (M mam n); interpeduncular nucleus (Interped n);
superficial gray layer of the superior collicullus (S sup coli); deep gray layer of the superior collicullus
(D sup coli); periaqueductal gray (Per gray); medial raphe nucleus (M raphe); pontine reticular nucleus, oral part (Pont ret n); pontine nucleus (pont n); substantia nigra, reticular (S nigra); medial
geniculate body (M genic); central nucleus of the inferior collicullus (Inf coli); cerebellum (Cerebellum); internal capsule (Int caps); ventral hippocampus commissure (Hip commis); corpus callosum (C
callosum).
al., 1977). The 45-min wait period is required to allow for
decline in the nonphosphorylated [14C]2DG in brain tis­
sue. Taking into account these facts, [14C]2DG was in­
jected 35 min after eptastigmine so that the peak rate of
phosphorylation of the tracer would occur at 40 min, the
J Cereb Blood Flow Metab, Vol. 13, No.4, 1993
time after drug injection when a peak effect on rCBF had
been detected in the time course study. In the calculation
of the regression of rCBF on rCGU, the values of rCGU
were paired with those of rCBF measured at 40 min after
eptastigmine. Calculation of a mean GCBF over the 45-
CHOLINERGIC EFFECTS ON CBF/CGU COUPLING
min period of the [14C]2DG procedure, by integrating the
curve representing the time course of GCBF after
eptastigmine within that time period and dividing by the
time interval, yielded a value of 2.39 ml g -I min I, 91 %
of peak value (Fig. 2).
-
Data analysis
The figures and tables represent the primary (untrans­
formed) data in the form of means and standard devia­
tions. As a first step in the statistical analysis, the rCBF
and rCGU values were transformed into the natural log­
arithm to obtain normalization and homoscedasticity
(Bartlett, 19 47).
In the dose-effect study, the significance of the factors,
dose and cerebral region, and their interactions on rCBF
was tested with a two-way analysis of variance with one
repeated measure. In the time course study the signifi­
cance of the factors, time and cerebral region, and their
interaction on rCBF was also tested with a two-way anal­
ysis of variance with one repeated measure. GCBF of
different groups were compared with a one-way analysis
of variance, testing effects of dose or time. All analyses of
variance were handled according to a multivariate least­
squares general linear model. Post hoc tests were con­
ducted with linear contrasts. The number of post hoc
comparisons was confined to the degrees of freedom for
the significant effects. As a result, the nominal ex of the
separate tests is comparable to that of the overall analysis
of variance (Carmer and Swanson, 1973; Bernhatrdson,
1975). Calculations were done with the statistical package
SYSTAT (Wilkinson, 1986).
The relationship between rCBF and rCGU was exam­
ined with a previously described statistical approach that
adopts a model with a linear relationship with unit slope
between the natural logarithms of rCBF and rCGU:
3.5
Y
P
c
=
<
1.45 + 0.37 X
0.0001
01
ex
+ loge (rCGU)
where ex represents the natural logarithm of the constant
of proportionality of rCBF to rCGU (McCulloch et aI.,
1982). For this particular analysis, values of rCBF and
rCGU were expressed as per 100 g of tissue in order to
avoid negative results.
RESULTS
The lowest dose of eptastigmine used (0.5 mg
kg-I ) induced a slowly developing decrease in
mean arterial blood pressure ( M ABP ) that
amounted to a statistically significant decrease of
12% at 20 min after drug administration (Fig. 1).
The effects of eptastigmine on blood gases and
pH, plasma glucose, and body temperature were
tested at the dose of 1. 5 mg kg-I and between 35 to
80 min after drug administration when CBF effects
were maximal. Results are shown in Table 2. The
levels of blood gases are consistent with normal val­
ues at the altitude of the laboratory and were not
affected by the treatment. Plasma glucose level in­
creased significantly following eptastigmine.
Eptastigmine induced a dose-dependent increase
in GCBF (Fig. 1) with a 74% increase over controls
(p = 0.001) at 3 mg kg-I . Regression analysis dem­
onstrated a highly significant dependence of GCBF
on dose (Intercept = 1. 45, slope = 0. 37, p <
0. 0001). rCBF also increased dose dependently in
most regions (Table 3). Analysis of variance
125
'E
'i
=
150
::-- 3.0
I
loge (rCBF)
705
2.5
E
u...
�
2.0
-;
.c
o
a
75
1.5
50 �----r---'----�--r-�
o
O�
1�
1�
2�
DOSE (mg kg-I)
2�
3
o
0.5
1.0
1.5
2.0
2.5
3
DOSE (mg kg-I)
FIG. 1. Effects of eptastigmine on mean arterial blood pressure (MABP) and global cerebral blood flow (GCBF). MABP was
measured at the time of CBF determination, 20 min after drug or saline administration, from a catheter in the iliac artery. GCBF
represents the average of 38 regions in which rCBF was assessed with the quantitative autoradiographic [14C]IAP technique. A
significant (p < 0.0001) linear dependence of GCBF on dose was found. Values represent the mean and SO of 5-7 animals per
dose. The decrease in MABP with 0.5 mg kg -1 eptastigmine was statistically different from control (ANOVA F(3.23) 3.756, P
0.0249; protected "t" test, p < 0.05).
=
J Cereb Blood Flow Metab, Vol. 13, No.4, 1993
O. U. SCREMIN ET AL.
706
TABLE 2. Effects of eptastigmine on physiological variables
PaC02
(mm Hg)
pH
Condition
Saline i.v.
Eptastigmine i. v., \.5 mg/kg
7.409
7.411
±
±
31. 9
30.2
0.007
0.006
±
±
Pao2
(mm Hg)
\.89
1.29
7\,1
68. 6
±
±
Rectal temp.
(OC)
4.75
6.45
37.3
37.2
±
±
Plasma glucose
(mM)
0.72
0.49
6.37
8.14
±
±
0.31
0.31a
Data expressed as mean ± SD. Variables were measured between 35 and 80 min after eptastigmine or saline administration. Values
were averaged in each animal (two blood gases and five plasma glucose values per animal).
a Significantly different from saline (p < 0.001); n = 5 animals/group.
showed a significant effect of the factors dose (p
0.011) and cerebral region (p
0.001) as well as a
significant effect of the interaction between the two
factors (p < 0.05).
In the time course study, 1. 5 mg kg 1 eptastigmine increased GCBF by 81% at 40 min after injection (p
0. 004) and by 35% at 90 min (p
0.040)
=
=
-
=
=
compared with controls. At 180 min, the drug was
still able to enhance GCBF ( + 35%) but the significance of the difference against controls had a borderline value (p
0.062). GCBF returned to control values at 360 min (Fig. 2).
The study of effects of eptastigmine on GCGU
(1. 5 mg kg-1 eptastigmine i.v., 35 min before mea=
TABLE 3. reBF (ml g-I min-I).
Dose
Control
Time
0.5 mg/kg
I mg/kg
3 mg/kg
20 min
20 min
20 min
Structure
Mean
SD
Mean
SD
Mean
SD
Mean
SD
Motor C
Somatos
Temporal
Area 17
Area 18
Area 18A
Olfactory
Cingulate
Hip CAl
Hip CA2
Hip CA3
Claustrum
N basalis
Caudate-p
Dentate gy.
Preoptic a
Lat hyp
Dor hyp
D m hyp
M mam n
Amygdala
Interped n
S sup coli
D sup coli
Infer coli
Pont retic
Cerebellum
Med dor th
Med em
Per gr
M genic
M raphe
Pont n
C call
Int caps
Hip com
Ce med th
S nigra
1.53
1.95
2.09
1.36
1.19
1.53
1.34
1.53
1.06
1.18
1.02
1.59
1.09
1.43
1.07
1.33
1.28
1.35
1.46
1.84
1.30
1.79
1.78
1.58
2.74
1.31
1.41
1.75
1.19
1.43
1.95
2.00
1.55
0.76
0.79
0.74
1.71
1.35
7
1.46
0.334
0.520
0.405
0.227
0.201
0.333
0.261
0.346
0.277
0.242
0.206
0.254
0.198
0.375
0.236
0.285
0.249
0.299
0.247
0.543
0.295
0.533
0.464
0.328
0.892
0.233
0.327
0.302
0.228
0.339
0.625
0.464
0.423
0.122
0.156
0.113
0.304
0.324
\.92
2.09
2.95
\.60
1.39
1.78
1.43
1.84
1.35
1.58
1.05
2.21
1.31
1.56
1.24
1.41
1.23
1.40
1.38
1.72
1.32
2.21
1.62
1.50
3.17
1.20
1.52
1. 15
1.20
1.26
2.39
1.82
1.30
0.93
0.83
0.64
\. 65
1.36
6
1.62
0.610
0.694
1.384
0.569
0.529
0.551
0.363
0.511
0.444
0.629
0.319
0.729
0.540
0.470
0.315
0.309
0.335
0.313
0.392
0.589
0.277
0.610
0.278
0.260
1.230
0.239
0.467
0.339
0.397
0.296
1.127
0.566
0.300
0.225
0.194
0.220
0.482
0.518
\.97
2.22
3.30
1.85
1.58
1.90
1.50
1.94
1.46
1.61
1.04
2.29
1.55
1.34
1.36
1.54
1.47
1.53
1.59
2.28
1.32
2.51
2.61
2.11
3.25
1.68
2.04
1.47
1.30
1.56
2.52
2.49
2.13
0.94
0.79
0.76
1.86
1.47
5
1.86
0.218
0.462
0.951
0.299
0.234
0.198
0.241
0.454
0.147
0.227
0.212
0.446
0.210
0.223
0.349
0.254
0.184
0.265
0.311
0.529
0.160
0.569
0.652
0.440
1.153
0.354
0.484
0.278
0.222
0.259
0.571
0.648
0.757
0.242
0.099
0.148
0.576
0.421
3.77
3.35
4.96
2.45
2.14
2.90
2.25
3.04
1.57
1.94
1.58
4.04
2.21
1.84
1.87
1.89
2.09
1.93
1.96
2.93
1.93
3.52
3.59
2.75
4.44
2.19
2.13
2.30
1.59
1.59
1.89
3. 90
2.98
2.43
1.13
1.09
1.87
2.80
6
2.61
1.233
\.149
0.756
0.837
0.661
0.723
1.281
1.152
0.423
0.376
0.373
1.123
1.575
0.523
0.369
0.736
1.199
0.468
0.509
1.078
0.496
1.311
0.748
0.570
1.150
1.314
0.714
0.977
0.401
0.401
0.355
0.725
1.406
1.240
0.275
0.193
0.499
1.010
n
GCBF
0.26
0.41
0.26
0.76
Eptastigmine was administered intravenously at the dose indicated. Time represents the interval between drug administration and
rCBF measurement.
J Cereb Blood Flow Metab, Vol. 13, No.4,
1993
CHOLINERGIC EFFECTS ON CBF/CGU COUPLING
surements) revealed no significant change in this
variable (control: 0. 99 ± 0. 08 flmol g-I min-I ; n =
5; eptastigmine: 0. 89 ± 0. 21 flmol g-I min-I ; n =
5). When differences were tested for individual re­
gions, hippocampus (CAl), nucleus basalis, and
corpus callosum showed significant decreases in
rCGU (Table l). In contrast, a similar analysis ap­
plied to GCBF in a group of animals injected with
the same dose of eptastigmine and the same time
interval between injection and measurement
showed a marked increase (control: 1. 46 ± 0. 26 ml
g-I min-I ; n = 7; eptastigmine: 2. 62 ± 0. 62 ml g-I
min-I ; n = 5; p < 0. 005). The majority of regions
sampled showed significant increases of rCBF over
controls in eptastigmine-injected animals (Table 3).
The relationship between rCBF and rCGU values of
707
every region in these four groups of animals was
evaluated with linear regression analysis. It was
found that these variables were well correlated in
animals that received saline (r = 0. 87) or eptastig­
mine i.v. (r = 0. 87). However, the slope of the
regression line of rCBF on rCGU was significantly
higher in the eptastigmine group (2. 86 ± 0. 26) than
in the controls that received saline (1. 00 ± 0. 93).
These slopes were statistically different from each
other at a level of p < 0. 001 (Fig. 3). This analysis,
although in keeping with numerous previous reports
on the relationship between rCBF and rCGU, may
overestimate the true probability due to lack of in­
dependence of observations on different regions of
the same animal. Use of the alternate approach,
described in Methods (McCulloch et aI. , 1982),
Dose-effect and time course studies
1.5 mg/kg
1.5 mg/kg
1.5 mg/kg
1.5 mg/kg
40 min
90 min
180 min
360 min
Mean
SD
Probaba
Mean
SD
Mean
SD
Mean
SD
3.60
3.65
4.76
2.13
1.74
2.50
2.03
3.21
1.55
1.74
1.50
3.79
1.64
1.97
1.92
2.27
1.99
2.09
1.84
3.09
1.90
4.15
3.91
3.21
5.90
2.39
2.62
3.16
1.60
2.36
3.98
3.92
3.52
0.95
0.95
0.80
2.80
2.16
5
2.62
1.291
1.197
1.437
0.517
0.299
0.822
0.564
1.229
0.512
0.493
0.309
1.484
0.296
0.347
0.590
0.613
0.559
0.576
0.370
0.848
0.397
1.220
1.146
0.883
1.409
0.729
0.812
0.708
0.441
0.414
1.224
1.144
1.523
0.260
0.118
0.211
0.689
0.699
<0.01
<0.02
<0.01
<0.01
<0.01
<0.05
<0.05
<0.02
2.26
2.60
4.03
1.83
1.43
2.05
1.53
2.31
1.54
1.51
1.03
2.25
1.65
1.55
1.43
1.63
1.53
1.36
1.39
2.78
1.33
2.97
2.51
2.27
4.49
1.43
2.06
2.47
1.17
1.86
3.32
3.36
2.28
0.70
0.67
0.76
1.86
1.46
5
1.964
0.277
0.136
0.533
0.183
0.154
0.\31
0.168
0.315
0.269
0.165
0.239
0.269
0.326
0.153
0.469
0.406
0.230
0.538
0.272
0.227
0.407
1.558
0.379
0.251
1.201
0.267
0.826
0.789
0.437
0.452
0.662
0.969
0.522
0.096
0.301
0.201
0.476
0.355
2.43
2.89
4.74
1.90
1.40
2.05
1.55
2.31
1.54
1.67
1.17
2.61
1.69
1.53
1.48
1.43
1.50
1.38
1.51
2.60
1.41
2.38
2.76
2.29
4.20
1.40
1.88
2.38
1.06
1.66
3.62
2.55
1.99
0.65
0.79
0.80
1.97
1.58
5
1.95
0.600
0.943
1.176
0.482
0.405
0.483
0.473
0.773
0.355
0.355
0.343
0.358
0.504
0.414
0.376
0.538
0.599
0.506
0.357
0.785
0.472
0.450
0.743
0.494
0.955
0.351
0.447
0.786
0.358
0.381
1.171
0.993
0.513
0.106
0.288
0.169
0.483
0.411
2.01
2.07
3.24
1.33
1.19
1.61
1.22
1.89
1.10
1.33
0.82
2.52
1.17
1.30
0.93
1.31
1.17
1.06
1.10
1.81
1.01
2.04
1.77
1.46
3.66
1.05
1.37
1.66
0.79
1.21
2.16
1.89
1.37
0.38
0.54
0.61
1.40
1.11
5
1.45
0.562
0.647
1.368
0.261
0.299
0.457
0.230
0.544
0.213
0.298
0.224
0.574
0.204
0.369
0.227
0.303
0.256
0.128
0.179
0.405
0.267
0.172
0.584
0.380
1.261
0.260
0.572
0.399
0.056
0.133
0.535
0.345
0.263
0.127
0.141
0.158
0.313
0.281
0.62
ns
<0.05
<0.05
<0.01
<0.01
<0.05
<0.02
<0.01
<0.05
<0.05
<0.01
<0.01
<0.01
<0.005
<0.005
<0.05
<0.05
<0.01
<0.01
<0.005
ns
<0.005
<0.01
<0.01
<0.02
ns
ns
ns
<0.01
<0.05
0.22
0.477
0.29
aProbability of the difference between 1.5 mg/kg, 40 min, and controls. For details on statistical procedures see Methods section.
Stereotaxic coordinates and definition of abbreviations for structures studied are listed in the legend of Table I.
J Cereb Blood Flow Metab, Vol. 13, No.4, 1993
O. U. SCREMIN ET AL.
708
yielded the following: saline group, a = 0.39, r =
0.77, p < 0.001; eptastigmine group, a = 1.03, r =
0.88, p < 0. 001. The intercepts ( a) representing the
natural logarithm of the constant of proportionality
between rCBF and rCGU in the two groups, were
significantly different (p < 0.01).
No overt signs of eptastigmine on behavior, pe­
ripheral cholinergic effects, or any other signs of
toxicity were observed.
DISCUSSION
Physostigmine and its heptyl derivative, eptastig­
mine are carbamate cholinesterase inhibitors.
These compounds serve as alternate substrates and
are hydrolyzed by acetylcholinesterase at a consid­
erably slower rate than acetylcholine (ACh) (Wilson
et aI., 1960). The alcohol moiety of physostigmine
or its heptyl derivative are cleaved, giving rise to
methyl or heptyl carbamylated enzymes, respec­
tively, that are more stable than the acetylated en­
zyme. The value of the enzyme reactivation con­
stant is considerably smaller in the case of the hep­
tyl derivative, resulting in a reactivation time of
�20 min for physostigmine and
140 min for
eptastigmine (Marta et aI., 1988). The carbamylated
enzyme is thus effectively sequestered and the en­
zymatic hydrolysis of ACh is prevented for ex­
tended periods of time. This results in elevated lev­
els of ACh at synaptic sites with consequent pro­
longed activation of cholinergic receptors. In
addition, these cholinesterase inhibitors can also
bind directly to cholinergic receptors but, in the
case of physostigmine and its heptyl analogue, at
greater concentration than that required to inhibit
the enzyme (De Sarno et aI., 1989). The substitution
of the methylcarbamyl group of physostigmine by a
heptyl group alters the rate of decarbamylation of
the enzyme but does not confer new pharmacolog­
ical properties other than those demonstrated for
physostigmine and derived from inhibition of ace­
tylcholinesterase (Brufani et aI., 1987; Marta et aI.,
1988; Brufani et aI., 1986; De Sarno et aI., 1989). In
the case of physostigmine, its cerebrovascular ef­
fect is completely blocked by atropine (Scremin et
aI., 1982). When ACh release is inhibited pharma­
cologically with pentobarbital, the cerebrovascular
effect of physostigmine also disappears, implying a
lack of direct cholinergic cerebrovascular effect of
this drug (Scremin et aI., 1983). In the case of
eptastigmine, a lack of direct cerebrovascular ef­
fects has also been demonstrated in anesthetized
animals, a phenomenon that can be reversed by
stimulation of the nucleus basalis, which is known
to release ACh at cortical levels (Linville et aI.,
�
J Cereb Blood Flow Metab, Vol.
13, No.4, 1993
1992). Physostigmine can also bind to peripheral
nicotinic receptors (Shaw et aI., 1985), but the ce­
rebrovascular effects of this drug have been shown
to be blocked by antimuscarinic but not by antini­
cotinic agents (Scremin et aI., 1982). At lethal or
highly toxic dose levels, some actions of certain
cholinesterase inhibitors cannot be reversed with
atropine (Shih et aI., 1991; Somani and Dube, 1989).
However, at nontoxic dosages, all actions of these
compounds derive from activation of cholinergic re­
ceptors by endogenous ACh (Taylor, 1990). En­
hancement of a process by action of a carbamate
cholinesterase inhibitor is considered a specific test
for intervention of a cholinergic step. In view of the
clearly demonstrated ability of eptastigmine to in­
hibit cerebral acetylcholinesterase (Brufani et aI.,
1986; Brufani et aI., 1987; Marta et aI., 1988), en­
hance ACh concentration in brain tissue (De Sarno
et aI., 1989), and antagonize central cholinergic
blockade induced by scopolamine (Dawson et aI.,
1991), it is assumed that its cerebrovascular effects
have been produced by enhancement of cholinergic
transmission.
The cerebrovascular effects of central cholines­
terase inhibition are well known (Scremin, 1991).
Physostigmine induces a large increase in rCBF
which can be blocked by atropine, and is not ac­
companied by metabolic activation (Scremin et aI.,
1982) with exception of the superior colliculus
(Scremin et aI., 1988b). The increase in rCBF in­
duced by this cholinesterase inhibitor, however, is
restricted to the neocortex, claustrum, and superior
colliculus, sparing most subcortical regions. This
particular regional distribution of the cerebral va­
sodilatation induced by physostigmine has been dif­
ficult to interpret since it does not follow the pattern
of distribution of cholinergic markers or drug avail­
ability (Scremin et aI., 1990). Interestingly enough,
eptastigmine induced, in the present experiments, a
generalized increase in rCBF, a result fundamen­
tally different from that observed with physostig­
mine. The difference in duration of action of both
drugs cannot explain the difference in the regional
distribution of their effects because, as shown in a
previous study, continuous infusion of physostig­
mine also failed to induce vasodilatation in most
subcortical structures (Scremin et aI., 1990). It is
conceivable that the kinetics of association of
eptastigmine with acetylcholinesterase, which dif­
fers from that of physostigmine (Unni et aI., 1991),
or a greater affinity of eptastigmine for acetylcho­
linesterase associated with blood vessels might un­
derly the differences observed. There is still the
possibility that eptastigmine could exert a direct
cholinergic agonist action on blood vessels that
CHOLINERGIC EFFECTS ON CBF/CGU COUPLING
709
3.5
I
FIG. 2. Time course of changes in
GCBF (mean ± SD) after Lv. injection
of 1.5 mg kg -1 eptastigmine. The drug
was administered at time
0 min. The
open symbol represents a value interpolated from the dose-effect study
performed at 20 min after drug injection. Projection of the dashed line to
the Y axis represents average GCBF
during the [14C]2DG procedure (2.39
ml g-1 min-1) and beginning and end
of line represent the time period of
such procedure with regard to drug injection (3�0 min). Statistical significance of differences from control was
40 min
0.004; 90 min
0.040; and
180 min
0.062.
3.0
C
E
2.5
=
=
I
C>
2.0
E
1.5
LL
III
a
"
1.0
=
=
.5
0
100
200
TIME
would not require the presence of endogenous ace­
tylcholine. Unfortunately there are no data avail­
able on the affinity of eptastigmine for vascular
cholinergic receptors in vivo. The affinity of this
drug for parenchymal cholinergic muscarinic and
nicotinic receptors in vitro has been found to be
greater than that of physostigmine (De Sarno et aI.,
1989).
On the basis of the effects of acetylcholine, other
direct cholinergic agonists and physostigmine, we
300
400
(min)
earlier proposed a role of endogenous acetylcholine
in the regulation of cerebral cortical blood flow
(Scremin et aI., 1973). The study of rCBF-rCGU
coupling under the action of physostigmine con­
firmed this phenomenon in the neocortex but failed
to provide evidence of its existence elsewhere in the
brain. However, the present results with eptastig­
mine, an apparently more efficient and less toxic
central cholinesterase inhibitor, demonstrate that
this drug, presumably by increasing the availability
7.0
.I
c::::
E
.I
6.0
•
Eptastigmine
y = 0.012 + 2.86 x
r
0.87
5.0
=
•
4.0
•
0)
E
u..
ID
3.0
•
• •
• •
•
•
•
•
•
•
FIG. 3. Regression analysis of rCBF
on rCGU for 38 pairs of values corre­
sponding to the regions enumerated
in the legend of Table 1. Two groups
of animals were injected with 1.5 mg
kg-1 eptastigmine and CBF (n
5) or
CGU (n
5) measured. In two addi­
tional groups CBF (n
7) or CGU (n
5) were measured after saline injec­
tion.
•
•
=
=
=
2.0
=
()
....
o
y
1.0
r
Control
0.45
=
=
+
1.00 x
0.87
0.0
0.0
0.5
1.5
1.0
rCGU
(j.lmol
9
2.0
2.5
-1
J Cereb Blood Flow Metab, Vol. 13, No.4, 1993
O. U. SCREMIN ET AL.
710
of endogenous acetylcholine,can enhance the ratio
of rCBF to rCGU in practically all regions of the rat
brain. The fact that the correlation between levels
of cerebral blood flow and metabolism is preserved
indicates that the action of endogenous acetylcho­
line is not a nonspecific vasodilatation but rather an
increase in the gain of the rCBF-rCGU coupling
mechanism,which was enhanced in the present ex­
periments by a factor of three under the action of
eptastigmine. Because this phenomenon occurs
throughout the brain, it is probably related to ace­
tylcholine sources that are not restricted to the well­
known central cholinergic pathways (Woolf et aI.,
1983,1984; Woolf and Butcher,1985, 1986,1989) or
to cholinergic fibers from peripheral nerves (Ed­
vinsson et aI., 1977; Florence and Bevan, 1979;
Saito et aI., 1985; Suzuki et aI., 1990), because the
distribution of these specific pathways is much nar­
rower than the presently reported distribution of
cholinergic vasodilatation induced by eptastigmine.
The nature of such sources remains elusive at the
moment.
Eptastigmine induced a moderate increase in
plasma glucose levels (averaging between 35 and 80
min of administration),of considerably lesser mag­
nitude than that observed with physostigmine infu­
sion during a comparable time (Scremin et aI. ,
1988b). This change in glucose level is known not to
affect the measurement of CGU (Scremin et aI.,
1988b; Schuier et aI., 1990).
Eptastigmine induced a small decrease in MABP
at the lowest dose used with no significant changes
in this variable at higher doses. This is also at vari­
ance with previous observations in which physo­
stigmine induced a moderate hypertension at the
doses at which it enhanced CBF in rabbits,rats,and
humans (Scremin et aI. , 1983; Scremin and
Scremin, 1986; Scremin et aI., 1992). Although the
cerebrovascular and pressor effects can be sepa­
rated pharmacologically (Scremin and Scremin,
1986),hypertension is an undesirable effect of phy­
sostigmine for potential therapeutic applications in
cerebral ischemia.
Physostigmine has been shown to improve CBF
in areas of ischemia in rats and humans (Scremin
and Scremin, 1986; Scremin et aI., 1992) although
its therapeutic use is limited by its tendency to in­
crease arterial blood pressure and to induce toxic
effects as well as by its short half-life. The present
results demonstrate that eptastigmine can enhance
CBF at doses devoid of toxicity and without in­
creasing arterial blood pressure. The effect is long
lasting. These characteristics make this drug a
likely candidate for the objective of enhancing CBF
in cerebral ischemia.
J Cereb Blood Flow Metab, Vol. 13, No.4,
1993
REFERENCES
Aoyagi M, Meyer JS, Deshmukh VD, Ott EO, Tagashira Y,
Kawamura Y, Matsuda M, Amrit A, Chee ANC (1975) Cen­
tral cholinergic control of cerebral blood flow in the baboon:
effect of cholinesterase inhibition with neostigmine on auto­
regulation and CO2 responsiveness.] Neurosurg 43:689-705
Aubineau P, Sercombe R, Edvinsson L, Owman C (1977)
Cholinergic influence on the cerebrovascular bed mediated
through actions on the vascular smooth musculature and on
the perivascular sympathetic nerves. In: Neurogenic Con­
trol of the Brain Circulation (Owman C, Edvinsson L, eds)
New York, Pergamon Press, pp 331-342
Bartlett MS (1947) The use of transformations. Biometrics 3:3952
Bernhatrdson CS (1975) Type I error rates when mUltiple com­
parison procedures follow a significant F test of ANOV A.
Biometrics 31:229-232
Brufani M, Marta M, Pomponi M (1986) Anticholinesterase ac­
tivity of a new carbamate, hepty1physostigmine, in view of
its use in patients with Alzheimer-type dementia. Eur J Bio­
chem 157:115-120
Brufani M, Castellano C, Marta M, Oliverio A, Pagella PO, Pa­
vone F, Pomponi M, Rugarli PL (1987) A long-lasting cho­
linesterase inhibitor affecting neural and behavioral pro­
cesses. Pharmacol Biochem Behav 26:625-629
Carmer SO, Swanson MR (1973) An evaluation of ten pairwise
mUltiple comparison procedures by Monte Carlo methods. J
Am Stat Assoc 68:6&-74
Dawson OR, Bentley 0, Draper F, Rycroft W, Iversen SD, Pa­
gella PO (1991) The behavioral effects of hepty1physostig­
mine, a new cholinesterase inhibitor, in tests of long term
and working memory in rodents (Abstract). Pharmacol Bio­
chem Behav 39:865-871
De Sarno P, Pomponi M, Oiacobini E, Tang XC, Williams E
(1989) The effect of heptyl-physostigmine, a new cholines­
terase inhibitor, on the central cholinergic system of the rat.
Neurochem Res 14:971-977
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
Florence VM, Bevan JA (1979) Biochemical determination of
cholinergic innervation in cerebral arteries. Circ Res 45:212218
Heistad DD, Kontos HA (1983) Cerebral circulation. In: The
Cardiovascular System, Vol 3, Peripheral Circulation and
Organ Blood Flow (Shepherd JT, Abboud FM, eds) Be­
thesda, American Physiological Society, pp 137-182
Kuschinsky W, Wahl M, Neiss A (1974) Evidence for cholinergic
dilatatory receptors in pial arteries of cats. Pfliigers Arch
347:199-208
Linville DO, Oiacobini E, Arneric SP ( 1 9 9 2 ) Heptyl­
physostigmine enhances basal forebrain control of cortical
cerebral blood flow. J Neurosci Res 31:573-577
Maiese K, Holloway HW, Larson DM, Reis DJ, Soncrant TT
(1991) Coupling of regional cerebral metabolism and blood
flow in the conscious rat is maintained after treatment with
the muscarinic agonist arecoline. Soc Neurosci Abstr 17:26
Marta M, Castellano C, Oliverio A, Pavone F, Pagella PO,
Brufani M, Pomponi M (1988) New analogs of physostig­
mine: alternative drugs for Alzheimer's disease? Life Sci
43:1 ,921-1 ,928
McCulloch J, Kelly PAT, Ford I (1982) Effect of apomorphine on
the relationship between local cerebral glucose utilization
and local cerebral blood flow (with an appendix on its sta­
tistical analysis). J Cereb Blood Flow Metab 2:487-499
Molnar L, Hegedus K, Fekete I (1991) Difference between the
cerebrovascular effect of purinergic Co-ATP and that of the
cholinesterase inhibitor, physostigmine, in vivo. Eur J Phar­
macoI209:81-86
Paxinos 0, Watson C (1982) The Rat Brain in Stereotaxic Coor­
dinates. Sidney, Academic Press
CHOLINERGIC EFFECTS ON CBFICGU COUPLING
Rovere AC, Scremin OU, Beresi MR, Raynald AC, Giardini A
(1973) Cholinergic mechanism in the cerebrovascular action
of carbon dioxide. Stroke 4:969-972
Rupniak NMJ, Tye SJ, Brazell C, Heald A, Iversen SD, Pagella
PG (1992) Reversal of cognitive impairment by heptylphy­
sostigmine, a long lasting cholinesterase inhibitor, in pri­
mates. J Neurol Sci 107:246-249
Saito GA, Wu JY, Lee TJF (1985) Evidence for the presence of
cholinergic nerves in cerebral arteries: an immunohisto­
chemical demonstration of choline acetyltransferase. J
Cereb Blood Flow Metab 5:327-334
Sakurada 0, Kennedy C, Jehle J, Brown JD, Carbin GL,
Sokoloff L (1978) Measurement of local cerebral blood flow
with iodo[14C]antipyrine. Am J Physiol 234:H59-H66
Schuier F, Orzi F, Suda S, Lucignani G, Kennedy D, Sokoloff L
(1990) Influence of plasma glucose concentration on lumped
constant of the deoxyglucose method: effects of hypergly­
cemia in the rat. J Cereb Blood Flow Metab 10:765-773
Scremin OU (1991) Pharmacological control of the cerebral cir­
culation. Annu Rev Pharmacol Toxicol 31:229-251
Scremin OU, Scremin AME (1986) Physostigmine induced re­
versal of ischemia following middle cerebral artery occlusion
in the rat. Stroke 17:1,004-1,009
Scremin OU, Shih T (1991) Cerebral blood flow-metabolism cou­
pling after administration of soman at nontoxic levels. Brain
Res Bull 26:353-356
Scremin OU, Rovere AA, Raynald AC, Giardini A (1973)
Cholinergic control of blood flow in the cerebral cortex of
the rat. Stroke 4:232-239
Scremin 0U, Rubinstein EH, Sonnenschein RR (1978) Cerebro­
vascular CO2 reactivity: role of a cholinergic mechanism
modulated by anesthesia. Stroke 9:160-165
Scremin OU, Sonnenschein RR, Rubinstein EH (1982) Cholin­
ergic cerebral vasodilatation in the rabbit: absence of con­
comitant metabolic activation. J Cereb Blood Flow Metab
2:241-247
Scremin OU, Sonnenschein RR, Rubinstein EH (1983) Cholin­
ergic cerebral vasodilatation: lack of involvement of cranial
parasympathetic nerves. J Cereb Blood Flow Metab 3:362368
Scremin AME, Allen K, Scremin OU (1988a) Regional distribu­
tion of the cerebral vasodilatation induced by a cholinergic
agonist (Abstract). FASEB J 2:A722
Scremin OU, Allen K, Torres CD, Scremin AME (1988b) Phy­
sostigmine enhances blood flow metabolism ratio in neo­
cortex. Neuropsychopharmacology 1:297-303
Scremin OU, Scremin AME, Somani SM, Giacobini E (1990)
Brain regional distribution of physostigmine and its relation
to cerebral blood flow following intravenous administration
in rats. J Neurosci Res 26:188-195
Scremin AME, Kunkel CF, Eisenberg B, Elliot T, Scremin OU
(1992) Physostigmine improves cerebral blood flow in hu­
man focal cerebral ischemia. J Stroke Cerebrovasc Dis 2:
87-91
711
Shaw KP, Aracava Y, Akaike A, Daly JW, Rickett DL, Albu­
querque EX (1985) The reversible cholinesterase inhibitor
physostigmine has channel-blocking and agonist effects on
the acetylcholine receptor-ion channel complex. Mol Phar­
macol 28:527-538
Shih T-M, Koviak TA, Capacio BR (1991) Anticonvulsants for
poisoning by the organophosphorus compound soman: phar­
macological mechanisms. Neurosci Biobehav Rev 15:349362
Sokoloff L, Reivich M, Kennedy C, Des Rosiers MH, Patlak CS,
Pettigrew KD, Sakurada 0, Shinohara M (1977) The [C14]_
deoxyglucose method for the measurement of local cerebral
glucose utilization: theory, procedure and normal values in
the conscious and anesthetized albino rat. J Neurochem 28:
897-916
Somani SM, Dube SN (1989) Physostigmine: an overview as
pretreatment drug for organophosphate intoxication. Int J
Clin Pharmacol Ther Toxicol 27:367-387
Suzuki N, Hardebo JE, Owman C (1990) Origin and pathways of
choline acetyltransferase-positive parasympathetic nerve fi­
bers to cerebral vessels in rats. J Cereb Blood Flow Metab
10:399-408
Taylor P (1990) Anticholinesterase agents. In: Goodman and Gil­
man's The Pharmacological Basis of Therapeutics (Good­
man Gilman A, Rall TW, Nies AS, Taylor P, eds) New
York, Pergamon Press, pp 131-149
Triguero D, L6pez de Pablo AL, G6mez B, Estrada C (1988)
Regional differences in cerebrovascular cholinergic innerva­
tion in goats. Stroke 19:736-740
Unni LK, Hutt V, Imbimbo BP, Becker RE (1991) Kinetics of
cholinesterase inhibition by eptastigmine in man. Eur J Clin
Pharmacol 41:83-84
Wilkinson L (1986) SYSTAT: the system for statistics. Evan­
ston, IL, SYSTAT Inc.
Wilson IB, Hatch MA, Ginsburg S (1960) Carbamylation of ace­
tylcholinesterase. J BioI Chem 235:2,312-2,315
Woolf NJ, Butcher LL (1985) Cholinergic systems in the rat
brain. II. Projections to the interpeduncular nucleus. Brain
Res Bull 14:63-83
Woolf NJ, Butcher LL (1986) Cholinergic systems in the rat
brain. III. Projections from the pontomesencephalic tegmen­
tum to the thalamus, tectum, basal ganglia and basal fore­
brain. Brain Res Bull 16:603--637
Woolf NJ, Butcher LL (1989) Cholinergic systems in the rat
brain. IV. Descending projections of the pontomesencepha­
lic tegmentum. Brain Res Bull 23:519-540
Woolf NJ, Eckenstein F, Butcher LL (1983) Cholinergic projec­
tions from the basal forebrain to the frontal cortex: a com­
bined fluorescent tracer and immunohistochemical analysis
in the rat. Neurosci Lett 40:93-98
Woolf NJ, Eckenstein F, Butcher LL (1984) Cholinergic systems
in the rat brain. I. Projections to the limbic telencephalon.
Brain Res Bull 13:751-784
J Cereb Blood Flow Metab, Vol. 13, No.4, 1993