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Correlation of intracellular redox states and pH with blood flow in primary and secondary seizure foci.

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ORIGINAL ARTICLES
Correlation of Intracellular
Redox States and pH with Blood Flow
in Primary and Secondary Seizure Foci
Robert T. Tenny, MD, Frank W. Sharbrough, MD, Robert E. Anderson, BAS, and Thoralf M. Sundt, Jr, MD
Epileptogenic foci were created by topical application of penicillin to the cerebral cortex in 40 paralyzed and
artificially ventilated cats receiving halothane anesthesia. The animals were divided into two equal groups to compare primary and secondary foci. The following variables were recorded at normocapnia, hypocapnia, and hypercapnia prior to and during seizure activity: cerebral blood flow (CBF), determined by clearance of xenon 133;
cortical redox states, measured by the fluorescence of reduced pyridine nucleotides (PN);brain pH, measured using
a lipid-soluble, pH-sensitive fluorescent indicator; and electroencephalograms (EEG). Mean arterial blood pressure,
arterial pH, arterial carbon dioxide tension (Pace,), and arterial oxygen tension (Pao,) were monitored in each
animal. All animals had a normal PaCo,-CBF response prior to the creation of a seizure focus, assuring the presence
of autoregulation and normal metabolic function.
CBF increased equally with seizures in the primary and secondary hemispheres. The relative increase was related
to the P q o , but approximated 68% at normocapnia. There was an alteration in the Pwo,-CBF response with seizures,
but the ability of the cerebral vasculature to constrict and dilate with hypocapnia and hypercapnia was retained.
There was no significant difference in the reduced PN signal with variations in Pqo, prior to seizures, but there
was an apparent 10 to 15% fall with seizures. The “equivalent” intracellular pH feil to 6.94 at normocapnia in the
primary focus but remained essentially unchanged from the control value of 7.10 in the secondary focus. These
differences in p H were consistent with the greater degree of seizure activity observed in the primary focus. We
conclude that a nonhypoxic acidosis existed in the primary focus and that changes in CBF were not related to it
because the CBF changed equally in both hemispheres.
Tenny RT, Sharbrough FW, Anderson RE, Sundt TM Jr: Correlation of intracellular redox states and pH
with blood flow in primary and secondary seizure foci. Ann Neurol 8:564-573, 1980
Information correlating the intracellular redox state
and p H of cortical tissue prior to and during a seizure
is required for a fuller understanding of the metabolic and blood flow changes that occur in areas of
seizure activity. Jobsis and co-workers were the first
to explore this area using modifications of the
fluorometric techniques introduced by Chance [ 5,
121. Since that important study, several teams of investigators have directed attention to this subject
using modifications of these methods [16-18, 32,
3 31. Major differences in laboratory seizure preparations and instrumentation have made a comparison of
results difficult. Furthermore, none of the studies
cited correlated cortical redox states and intracellular
p H with cerebral blood flow (CBF) measurements.
This investigation sought to make such a correlation.
on blood flow and intracellular redox states and pH using
vertical illumination techniques. Measurements were made
at varying levels of arterial carbon dioxide tension (Pace,)
prior to and following the creation of a seizure focus. The
electroencephalogram (EEG), reduced pyridine nucleotide
(PN) fluorometric measurements, and mean arterial blood
pressure (MABP) were recorded continuously throughout
the experiment. CBF, PaCO,, arterial oxygen tension (Pao,),
and brain pH were recorded at 15-minuteintervals prior to
and during seizure activity. Differences in the fluorometric
instrumentation used for continuously measuring reduced
PN and the periodic measurement of brain pH prevented
simultaneous recordings. This made it necessary to further
subdivide the 20 primary and 20 secondary seizure preparations into two subgroups of 10 each. Blood flow measurements are therefore based on 40 animals, but reduced
PN and pH determinations are from 20 preparations.
Materials and Methods
Animal Preparation
Cats weighing between 2.5 and 3.5 kg were placed in a cat
restrainer, and anesthesia was induced with 4% inspired
Forty cats were divided into two groups of 20 each to
analyze the effect of primary and secondary seizure activity
~
From the Cerebrovascular Research Center, Mayo Clinic, Rochester, MN.
Received Nov 26, 1979, and in revised form Mar 24 and May 5,
1980. Accepted for publication May 19, 1980.
Address reprint requests to Thoralf M. Sundt, Jr, MD, Cerebrovascular Research Center, Room 4-437, Alfred Bldg, St.
Mary’s
Rochester, MN 55901.
564 0364-5134/80ll20564-10$01.25 @ 1978 by Thoralf M. Sundt, Jr
halothane. They were maintained o n 1.5% halothane for
the surgical preparation; the anesthetic was lightened to
0.8% expired halothane for brain recordings. Variations in
P w o , were achieved by changing the amount of inspired
carbon dioxide. Catheters were inserted into the right
femoral artery and vein for monitoring blood pressure,
sampling arterial blood gases, and administering drugs. A
tracheostomy was performed, with a PE-50 cannula being
inserted into the right lingual artery so that its tip lay in the
carotid artery. The skin, subcutaneous tissue, and muscles
were excised over the right parietal area with the cutting
current of a Bovie electrosurgical unit. Blood loss never
exceeded 5 ml. The bone overlying the parietal area was
removed with a high-speed air drill. Soft bone wax was then
placed in the diplo$ of the bone, and the dura was reflected
over the margins of the craniectomy with the aid of the
operating microscope.
The animals were placed on a Harvard respirator, and
0.15 mg of pancuronium bromide per kilogram of body
weight was given to abolish respiratory effects. Chemical
cerebral vasomotor regulation was evaluated in each animal
prior to the creation of a seizure focus by varying the
amount of inspired carbon dioxide. Measurements were
made at Pwo, levels of40,20,60, and 40 torr (normal Pwo,
in the cat approximates 32 to 36 torr; 40 torr was used to
simplify correlation with other studies).
Following the evaluation of chemical regulation, seizures
were induced by topical application of sodium penicillin G.
In the group in which primary seizures were studied, 0.1
ml of sodium penicillin G (500,000 U/ml) was topically
F i g 1. Typical animal recordings of electroencephalogram;
blood pressure; relative cortical redox tracing, that is, rednced
pyridine nucleotide (PN),fZuorescence;and re,fZectance
monitoring signal in area of primary seizure focus immediately
prior t o and following onset of seizum activity. The seizure
focus wus created 2 minutes prior t o the time of thir tracing by
cortical application of penicillin. Blood pressure (BP) and
redox state did not change in this preparation. (L, = left
frontotemporal; LkP= left frontoparietul; RFP= right frontoparietal; RFT= right frontotemporal.)
applied to the right cerebral cortex. Seizure activity was
usually identified within 3 minutes, and invariably severe
epileptiform activity was present in 10 minutes. In the
group of animals in which the secondary seizure focus was
studied, seizures were induced by a subdural injection of
0.1 ml of sodium penicillin G (500,000 U/ml) over the left
parietal area, and brain determinations were made from the
right cerebral hemisphere.
Ten minutes following the application of cortical penicillin, the P q o , again was varied from 40 to 20 to 60 to 40
tow. The animals were then killed by occlusion of the endotracheal tube, and measurements were continued until
the animals died. Brain measurements of p H , CBF, indicator clearance, PaCO,, and P ~ o ,were made at 15-minute
intervals prior to and after seizure activity. MABP and the
EEG were recorded continuously, as was reduced PN in
animals in which this variable was studied. Figure 1 is a
typical tracing of the EEG and cortical fluorescence at 463
nm adjacent to the area of pencillin application.
M easy remen ts
Arterial blood pressure, as measured by a strain gauge attached to the femoral artery catheter, was displayed on a
polygraph. Core body temperature was monitored with a
rectal digital thermometer, and the animals were kept at
normothermia by the use of a small heating blanket. Arterial bIood samples were taken for measurements of arterial
blood gases and pH measurements as previously described.
CBF was measured by the clearance of a 0.1 ml bolus of
normal saline solution containing 200 pCi of xenon 133
(total volume of each injectate was 0.3 ml and included 0.2
ml of an umbelliferone solution). The bolus was delivered
into a catheter secured in the right lingual artery. (Details
of this technique, the isoresponse curve of the detector,
and collimation factors have been described previously [4,
341.)
For EEG recordings, three screws were placed on each
side of the cat's skull, and scalp muscle was reflected and
retracted to minimize electrical artifacts. Differential EEGs
were recorded in two areas: frontoparietal and frontotemporal. The resultant four-channel EEGs were recorded on a
Grass polygraph (Fig 2).
Tenny et al: Seizures, p H , and Blood Flow
565
k-Jy
A
B
1s
F i g 2. Typical E E G at 25 mmlsec prior t o (A) andfollowing
( B ) a seizure discharge. Note greater degree of activity in rigbt
hemisphere, the side of the primary focus. (FT,= frontotemporul left: FPL = frontoparietal left; FPR = frontopurietal
right; FTR= frontotemporal right.)
The technique for fluorescence and reflectance measurements has been illustrated and described in detail previously [2, 29-31] and is not reviewed here.
A nomogram derived from the ratio of 450 nm fluorescence from 370 nm and 340 nm excitation is illustrated in
Figure 3. The use of this nomogram to measure brain p H ,
as well as the technique for calibration, is illustrated in the
analysis of a typical fluorescence clearance curve in Figure
4. The cortical p H was determined by choosing three
points, 24 seconds apart on the curve, and taking an average of the three ratios. Each p H measurement in all of the
animals was determined from these paired individual
clearance curves [29, 311. Each p H measurement was accompanied by blood gas studies and by a simultaneous CBF
measurement from the clearance of '"Xe.
F i g 3. Excitation of umbelliferone at 370 nm produce.r a variable pH-dependent intensity ofJuoreJcence at 450 nm, but excitation at 340 nm gives an isosbesticfEuoresrentline at 450
nm. T h u ~this
, nomogram can be constructed relating the
changing ratio ofjluorescencefrom these excitation bands to
zjarzations in p H . Nomograms must be reconstructedfor each
group of experimental animals because of filter fatigue 1291.
Statistical Analysis
Student's t test was used for analysis of blood flows and
indicator clearance rates. The analysis of covariance was
used for comparison of brain pH during primary and secondary seizures because it was necessary to consider in that
analysis a secondary variable, the blood P q o , .
'.Ol
0.9
0.8 -
'
.?
*
0.7
-
0.6
-
9.5
-
0.4 -
0.2
-
/x
I
566 Annals of Neurology Vol 8 No 6 December 1980
I
1
I
I
I
I
I
I
I
.-A
fA
Time (seconds)
F i g 4. Dual-channel recording of typical umbelliferone
clearance curves at an arterial PaCO., of 37 torr prior t o
activation of a seizure focus. The formula for the ratio o f
450 nm fluorescence lines at excitations of 3 70 and 340 nm in
the vascular space (point A) and in the tissue {points B, C ,
and D ) is as follows:
where TF,,, is the totalfluorescenceat 450 nm with 370 nm
excitation
TF340is the totalfEuorescence at 450 nm with 340 nm
excitation
BF is the background fluorescence from hydrolyzed
nicotinamide-adenine dinucleotide ( N A D H )N A D H phosphate (NADPH)
This formula represents the 450 nmJuorescence at 370 nm
excitation attributable to the indicator divided by 450 nm
fluorescence at 340 excitation attributable to the indicator.
Thus, at point A (the arterial spike) the formula yields 530 50 + 720 - 54 = 480 + 666 = 0.727 (PH 7.29from
nomogram; simultaneous arterial sample = 7.32).At point B,
theformula equals 310 - 50 + 530 - 54 = 260 + 476 =
0.545 (pH 7.09 from nomogram);similar calculations at
points C and D produce brain pH measurements of 7.09 and
7.10, respectively. Points B, C , and Dare separated from each
other by 24-second intervals. Analysis of points farther along
the clearance came of umbelliferone is less reliable, as a greater
amount of thejuorescence in this portion of the curve is attributable to background fluorescence of NADH-NADPH.
Results
Redox States
General information regarding the 20 animals in
which the redox state was studied and specific data
relating to fluorescence of reduced PN are summarized in Figure 5 and Table 1, respectively. No
significant difference was found in the levels of reduced P N fluorescence in normal brain during
hypocapnia, hypercapnia, or normocapnia. (Although
the level of fluorescence did appear lower during
hypercapnia, this was not statistically significant.) An
apparently significant fall in the levels of reduced PN
fluorescence occurred with the induction of seizures.
This change was significant at the p < 0.005 level
when the fluorescence of reduced PN during
hypocapnia and normocapnia was compared to preseizure values at similar p = ~ , . During hypercapnia
this difference was significant only at the p < 0.05
level. There was no difference in any of these values
between the primary and secondary foci. A 200%
rise in the fluorescence of reduced P N occurred with
endotracheal tube occlusion, but that finding was not
associated with a change in reflectance.
Intracellular PH
General information regarding the 20 animals in
which the intracellular p H was studied and specific
data relating to p H are summarized in Figure 6 and
Table 2, respectively. A marked intracellular acidosis
was present in the primary seizure focus which was
not identified in the secondary focus. Regression
lines comparing brain p H with arterial P q o , are
drawn in Figure 7.
Cerebral Blood Flow
Data comparing CBF to MABP and Pace, before and
during seizures in both the primary and secondary
foci are illustrated in Figure 5 for the 20 animals in
which fluorescence of reduced PN was recorded and
in Figure 6 for the 20 animals in which brain pH was
recorded. The Pwo,-CBF response curves in normal
brain and in the primary and secondary seizure foci
are plotted in Figure 8. The data points (means) from
which the curves in Figure 8 are constructed are presented in Table 3 (40 animals).
Discussion
Seizure Models
Three general methods have been used to induce
seizure activity in laboratory animals: ( 1 ) systemic
administration of various pharmacological agents
(penicillin [22], pentylenetetrazol [ 131, bicuculline
[25]), ( 2 ) electroconvulsive stimuli [ 11, 16, 241, and
( 3 )topical application to the cortex of irritative drugs
[8, 22, 321. We have used a form of the third type as
it more nearly approximates the clinical setting.
Tenny et al: Seizures, pH, and Blood Flow
567
I
Reflectance
(nW/cm?
X-366
.nrn,srl
500c-
+
“20E+
E-
0
0
P a co?
(torrl
I
-
+-
160
80
MABP
( m m Hgl
Pen G
Pen
G
Tiirie ( m i n u t e s )
A
Table 1. Reduced Pyridine Nacleotide Flaore.icence
Prior t o and Daring Seizure
PacO,
Reduced PN
Paco,
Reduced PN
CONTROL
I
39.3 f 1
20.9 ? 1
61.6 f 2
41.1 t 1
0.98 t 0.02
0.99 i- 0.02
0.87 k 0.02
0.96 t 0.02
39.4
20.5
58.7
41.7
t 1 0.98 t 0.02
f 1 1.05 5 0.02
0.82 ? 0.05
0.82 -t 0.04
0.75 f 0.05
0.79 t 0.05
2.79 f 0.10
40.9
20.6
59.3
43.3
...
t 1 0.86 ? 0.05
f 1 0.94 t 0.03
2
1 1.03 t 0.03
SEIZURE
40.4 ? 1
21.3 t 2
63.5 t 2
42.1 t 1
a
.
I
.
t 1 0.86 5 0.06
f 2 0.71 t 0.05
t 2 0.81 5 0.04
2.77 2 0.10
“Died
The creation of seizures from parenteral medications introduces a number of variables that confuse
and obscure variations in cerebral metabolic function
which are primarily the result of cerebral epileptic
activity [2 11. These systemic changes include: hypertension, hypoxemia, hypercapnia, systemic acidosis,
and a direct pharmacological effect on the cerebral metabolic rate from the drug administered. Although some of these effects can be minimized by
B
Fig 5 . Data szimmary for the 20 animals in which redox state
waJ studied. Reduced pyridine nucleotide (PN), reflectance,
and mean arterial blood pressare (MABP) were recorded continuously; means of these tracings at times of cerebral bloodflow
(CBF) measurements and arterial samplings are illnstrated
here. There are no signijkant diffwences between the data from
the primary (Aj and secondaiy o r pr~jected(3)seizure f w i .
There was a signzjkant increase in CBF with the onset of
seizures and an apfiarent decrease in redaced P N .
paralyzing and ventilating the animal artificially, they
cannot be eliminated [251.
Cerebrul Redox Stute
Artifacts relating to photodecomposition, light scattering, vascular blood volume in the field, and transition of the hemoglobin redox state were reduced, but
not eliminated, by the following: monochromators
for narrow bandwidth excitation, emission, and
reflected light; low-intensity vertical excitation energy and high-sensitivity recording instrumentation;
and a small (106 p m in diameter), avascular field of
measurement [2]. However, the meaning o r importance of the recorded change is subject to question
[151.
There was no dramatic change (fall) in t h e fluorescence signal at the time seizures were induced, as was
noted, for example, in the monkey with middle cerebral artery occlusion, in which a very significant and
easily reproducible rise occurs [30].The levels shown
in Figure 5 and Table 1 for fluorescence of reduced
568 Annals of Neurology Vol 8 No 6 December 1980
P H Brain
[-
7.40
7.00
6.60
Tissue indicator 100
clearance
40
(m1/100 g / m i n l
t
t
-
€4
v-
80
(torr)
20
T i m e lminutesl
A
B
Fig 6. Data summary for 20 animals rn which p H was
studied. Brain p H became szgnifcantly acidic i n the primar3,
(A)seizure focus but not in the secondavy ( B )form. It continued t o uary with alterations in Pa,-O, in both primary and
secovdary f o c i after the onset of epileptjfrm activity.
PN were, for comparative purposes, measured at the
time arterial blood gases were drawn, and the means
and standard errors were computed from these
values. However, these were continuous tracings,
and the change in the fluorescence signal was indeed
gradual. It was not a change that occurred in the first
few minutes of seizure activity and then persisted,
thus implying a new metabolic steady state. It did not
approximate or resemble the dramatic change noted
with endotracheal occlusion (a rise that confirms the
sensitivity of our methodology and instrumentation).
Although no statistically significant variation was
Table 2. Intracellular Brain p H Prior t o and During Seizure
~
~~
Secondary Focus
Primary Focus
pH (mean i SEM, N
Pacoz
(torr)
=
10)
pH (mean
PWO,
* SEM, N = 10)
Brain
Arterial
(torr)
Brain
Arterial
7.139 i 0.022
7.230 5 0.044
7.019 0.030
7.148 i 0.039
*
7.337 t 0.019
7.600 f 0.024
7.109 +- 0.024
7.283 i 0.009
38.9 i 1.2
17.9 i 1.8
60.4 1.7
40.5 0.8
*
*
7.107 i 0.033
7.195 i 0.021
7.000 0.030
7.061 f 0.027
*
7.350 i 0 022
7.632 t 0.019
7.148 & 0.011
7.286 t 0.014
6.937 f 0.058
7.003 i 0.078
6.809 2 0.050
6.976 2 0.046
7.294 & 0.009
7.531 f 0.020
7.106 i 0.011
7.271 i 0.023
40.5 i 1.3
19.7 f 1.7
60.4 f 1.5
41.6 2 1.2
7.143 i 0.028
7.176 f 0.065
6.955 2 0.035
7.053 f 0.059
7.299 t 0.019
7.546 t 0.026
7.1 16 -C 0.019
7.251 f 0.024
~
40.1 k
19.0 2
59.1 ?
40.0 5
1.2
1.1
2.1
1.1
SEIZURE
37.9 f 1.5
19.7 % 1.3
61.0 i 2.2
41.7 2 1.5
Tenny et al: Seizures, pH, and Blood Flow
569
20
40
60
80
P a c o l [torrl
F i g 7 . Brain p H plotted against arterial PaCO,. Straight line
represents the period prior to seizure; dashed line, the primary
seizure focus; dotted line, the secondary seizure focus.
Control n = 16
Primary n 8
0.-Q Secondary n - 8
0-4
A--A
l60C
.,
.s
E
z
O
140
I
120-
100-
L
E
L
5
60-
2ot
L
,
,
,
_
,
,
,
,
10
20
30 4 0 5 0 60 7 0
PaC02 (torr)
F i g 8. Response curves of CBF plotted against P a c o pi n the
primary and secondary (mirror)foci prior to and during seizure.s. There were no signzficant differences between bloodjlou?
values in the primary hemispheres and those i n secondary
hemispheres. Each graph marking i~the mean of1 0 CBF
measurements; thus the control curve is constructed from 160
measurements, the primary focus from 80, and the secondary or
mirror focus from 80. The CBF-Pace, response curve.r were
altered but not abolished by seizure activity, indicating some
preservation of autoregulation. In the control determinations,
the second normocapnic CBF measurement immediately prior to
the creation of a .seizurefocus was invariably lower than the
initial or baseline urormocapnic CBF. The reason for this is
unexplained, but it is ob.iwved more commonly i n cats than i n
monkeys [ I , 4: 93. I t may be related t o adaptive autoregulatorjf mechanisms.
noted in the reduced PN signal in normal brain with
fluctuations in Par-, prior to seizure activity, variation did occur which we have not noted in the rat
or monkey-animals that seemingly experience fewer
changes in brain volume and coloration with variations in Pwo, 121. This change was not identified
by altered reflectance, but the monitoring technique
used must be evaluated very cautiously 1151. It is
quite clear that there was not a rise; however, a hemodynamic component cannot be excluded from
these or any other in vivo microfluorometric studies,
and therefore minimal changes should be interpreted
cautiously [ 13, 181.
Fluorescent lndicator Analysis
Umbelliferone is a fat-soluble, pH-sensitive fluorescent indicator that is freely diffusible across the
blood-brain barrier and is nontoxic 1311. Because both
the neutral and ionic forms are fluorophors 1311, a
nomogram can be constructed from the fluorescence
emitted at 450 nm from 370 and 340 nm excitation of
this molecule. It is then possible to determine brain
p H from the ratio of the emission intensity at these
two wavelengths of the indicator’s washout curve,
using appropriate microspectrofluorometric instrumentation and a custom-built filter selector wheel
that permits synchronization of excitation and emission light. The in vivo calibration techniques for
these types of studies along with possible artifacts are
related to alteration of the pK of the indicator in a
fat-soluble environment, concentration of the indicator, solvent effect with differential quenching, and
changes in the indicator redox potential. These have
been considered previously [29] and are not repeated
here. The calibration data for our fluorescence curves
cannot be extrapolated to other spectrofluorometers
unless those fluorometers have the same relative intensities of excitation at the wavelengths studied.
Also, because of filter fatigue, it is necessary to recalibrate the instrumentation frequently. Furthermore, the instrument must be focused on a small
avascular area to minimize hemodynamic artifacts
[ 3 11. Measurements using this method are uniquely
free of the tissue damage associated with any type of
microelectrode. No diffusion barriers, such as the
arachnoidal membrane, are present to interfere with
measurements.
In previous studies using this method we have
found the following characteristics of brain pH: (1) it
is relatively more acid at normocapnia under light
halothane anesthesia than under light barbiturate
anesthesia [ 11; (2) brain pH varies with the depth of
anesthesia [ 11; (3) it changes with Pacoz but not with
acute changes in arterial p H [29]; and ( 4 ) the p H of
the brain agrees quite closely with the calculated
570 Annals of Neurology Vol 8 No 6 December 1980
Tuble 3. Relation between CBF and Pace, during Control and Primary and Secondary Seizure Activitj
PH Group
Reduced PN Group
(mean 2 SEM)
Primary
PacO,
(torr)
CBF (mli
39.320.7
20.9 1.4
61.6 I 2.5
41.121.3
82.2%
34.3 2
151.0 %
60.8-c
40
40.4 -c 1.2
20
21.3 t 2.5
63.5 2 2.3
42.1 ? 1.0
98.2 f 4.7"
78.4 2 9.1
132.2 + 9.1
95.8 2 10.7
Protocol
Paco,
Primary
Secondary
(N = 10)
(N = 10)
100 gm/min)
PaCo,
(rorr)
Secondary
(N = 10)
(N = 10)
CBF (mli
100 gmimin)
Pace,
(torr)
Paco,
(rorr)
CBF (mli
100 gmimin)
61.526.7
26.3 t 1.3
146.2 z 7.9
56.2t4.7
38.921.2
1 7 . 9 t 1.8
60.4 5 1.7
40.5t0.8
76.1?
34.5 2
142.2 2
57.72
94.9 % '8.7"
63.1 f 8.9
119.1 2 6.4
110.4 2 6.1
40.5 f 1.2
19.7 2 1.7
60.4 f 1.5
41.6 2 1.2
97.5 t
82.7 t
135.6 &
116.8 2
CBF (mli
100 gmimin)
CONTROL
40
20
60
40
*
9.8
3.9
7.3
6.8
39.4+1.3
20.5 f 1.0
58.7 t 1.4
41.7k0.9
85.3%
37.3 2
140.6"
62.0t
40.9
106.8 t
76.9 2
123.82
92.6 2
8.7
3.8
11.2
7.9
40.121.2
19.0 t 1.1
59.1 2 2.1
40.0%1.1
7.5
3.7
13.7
6.5
SEIZURE
60
40
1.0
20.6 2 1.0
59.3 t 1.5
43.3 .+ 1.6
11.2b 37.9 t 1.5
15.6
19.7 2 1.3
10.6 61.0 2 2.2
10.6 41.7 t 1.5
14.3d
13.6
7.3
13.0
There was a statistically significant increase in CBF with seizure activity (" = 62%; = 74%; = 68%; = 70%). with no significant
difference among
- the RrouDs.
- - N = number of animals. The average increase in CBF at normocapnia with seizure activity among groups was
68%.
"equivalent intracellular pH" reported by Siesjo [25]
and Davidian et a1 [6].
Important information can also be acquired from
analysis of the indicator's clearance curves. Immediately on leaving the intravascular space, umbelliferone enters a compartment too acid to be the extracellular compartment [28]. The meaning of this
observation has been discussed previously. It is
strong evidence for an intracellular transport route
across the blood-brain barrier (that is, capillary to
glial cell to neuron). Deep halothane anesthesia slows
the clearance of indicator, but deep levels of barbiturate anesthesia do not [l]. In the present study,
clearance of indicator was faster in the primary focus
than in the secondary focus, which could be explained by p H differences in the two foci. The more
acid environment in the primary focus results in
less ionization of the umbelliferone (pK, = 7.5).
Because only the nonionized o r molecular form
of the indicator should cross the cell membrane,
clearance should be increased by this change in relative concentrations.
Relation between Measwements of CBF,
MetaboIiJm, and P H
Measurements of CBF in this setting are free of regions of focal ischemia; as a result, artifacts related to
"look through" and Compton scatter are not important [9]. The overlying scalp muscles were removed
in these animals, and the isoresponse curves of the
probes ensured that extracerebral contamination was
minimal. For these reasons, the findings of a pre-
served but altered Paco,-CBF response is meaningful [19].
A PacOz of 40 torr was used for normocapnia in
this investigation, although there is some evidence
that normocapnia in the cat approximates 34 to 36
torr. Seizure activity produced an increase in CBF
from 59 to 99 m1/100 gm/min (68%) at normocapnia
(see Table 3). This major rise in CBF did not (in this
model) prevent a further elevation with hypercapnia,
which suggests that the neural tissue was not
exhausted and that energy reserves were still available for further demands on the metabolic-CBF response. This is consistent with data from the laboratory of Plum and associates [20] in a different seizure
model; in their model, the animals were ventilated
and paralyzed-as were the preparations reported
here-and they found that seizures produced only a
IOql fall in adenosine triphosphate.
It has now been directly demonstrated by Sokoloff, Reivich, and their colleagues 123, 26, 271
that functional activity in cerebral tissues is closely
coupled to local energy metabolism and that regional
CBF normally varies in direct proportion to the rate
of glucose metabolism. Using a seizure model similar
to that reported here, other workers [141 have found
increases in glucose consumption in areas of motor
cortex adjacent to the penicillin locus. However,
hypercapnia produces desynchronization of the ratio
of glucose consumption to CUF. Thus, although increased flow with hypercapnia does not imply
heightened glucose consumption, it does imply that
preservation of metabolic activity as a cerebral au-
Tenny et al: Seizures, pH, and Blood Flow
571
toregulatory response is dependent on functional
tissue and is lost in anoxia and ischemia. (The best
definition of cerebral autoregulation is Siesjo’s: “the
capability of the cerebral circulation to adjust to the
nutritional needs of the tissue” [ 2 5 ] . If cerebral autoregulation is intact, CBF should vary with changes
in Paco, and not with physiological changes in
MABP.)
Although the umbelliferone technique measures
the p H of the intracellular space, it is probable that
extracellular p H closely follows the changes observed in the intracellular compartment. There was
n o difference between CBF in the two hemispheres,
but brain p H did vary. This suggests that extracellular
p H may not be the primary regulator or coupler between rnctabolism and blood flow. This inference is
consistent with recent conclusions by Astrup et a1 [ 3 ] .
However, it is equally possible that CBF is normally
coupled to hydrogen ion production and that the increased acidosis in the primary focus merely reflects
the inability of the blood flow to keep up with the
metabolic demand of the tissue. The preservation of
some further reactivity to changes in Pace, supports
this possibility.
Metabolism of Epilepsy
When studying the biochemical changes that result
from epileptiform activity, one must distinguish between those that result from the increased metabolic
rate of neural tissue and those that occur from the
systemic effects of convulsive motor activity [20].
The latter effects were eliminated in these preparations inasmuch as the animal was intubated,
paralyzed, and artificially ventilated throughout the
experiment. No animals became hypotensive, and
blood gases and arterial p H were maintained and
controlled carefully.
It is not our purpose to review in detail the literature on energy changes during seizures; such information can be found in recent reviews [21, 251.
However, it is appropriate to correlate our results
with major studies on the subject. Plum, Howse, and
Duffy [lo, 201 found increased levels of lactic acid in
regions of seizure activity without significant changes
in brain energy reserves, and proposed the term
nonbypoxic lactacidosis. The findings of our investigation support this hypothesis. It has been proposed
that this cytoplasmic acidosis results from an increase in reduced nicotinamide-adenine dinucleotide
(NADH) in the cytoplasm, which is in turn the result
of an overloading of the carrier system necessary for
intramitochondrial oxidation of the N A D H formed
in the cytoplasm. Siesjo has noted that seizures represent a case in which cytoplasmic and mitochondria1
redox changes may occur in opposite directions [ 2 5 ] .
The results of the in vivo fluorometric measurements
of reduced P N in which no increase was identified do
not conflict with this theory, because .these types of
measurements are thought to reflect primarily mitochondrial NADH.
The difference in “equivalent intracellular pH”
between the primary and secondary foci correlates
quite closely with differences in EEG activity. The
greater degree of activity in the primary focus suggests that this higher level of metabolic activity was
sufficient to produce a “nonhypoxic lactacidosis” in
the primary, but not the secondary focus.
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