Download Effect of pH on Metabolism and Ultrastructure of Guinea Pig

Effect of pH on Metabolism and Ultrastructure of
Guinea Pig Cerebral Cortex Slices
BY KUSUM K. PATEL, Ph.D., J. FRANCIS HARTMANN, Ph.D., AND MAYNARD M. COHEN, M.D., Ph.D.
Abstract:
Effect of pH on
Metabolism and
Ultrastructure of
Guinea Pig
Cerebral Cortex
Slices
• Lactic acidosis is a prominent biochemical alteration which follows cerebral ischemia
and is of sufficient degree to result in pH decrease in involved and adjacent tissue.
Because of the significance of acidosis in the cerebral ischemic process, we have
investigated ultrastruclural changes and some biochemical parameters under varying pH
conditions using guinea pig cerebral cortex slices.
Metabolic rates decreased markedly during acidic incubations, and increased only
minimally during basic incubations. Acidic pH affected oxygen and glucose utilization
rates and fine structure to a greater degree than did an equal change in pH from 7.4
toward the alkaline. Glucose consumption was affected by pH deviations from 7.4 to a
greater degree than oxygen uptake.
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Additional Key Words
acidosis (in vitro)
O2 uptake
glucose consumption
aerobic incubation
electron microscopy
brain (in vitro)
• Concentrations of lactic acid in both brain and
cerebrospinal fluid rise during and following cerebral
ischemia. In humans, meeting electroencephalographical and clinical criteria of brain death, there is
a marked increase in CSF lactic acid over normal
values.1 Zupping et al.2 noted a significant increase
in the CSF lactate and pyruvate in patients with
cerebral infarcts. Lactic acid increases in tissue and
a rise in the lactate-to-pyruvate ratio is the earliest
observable biochemical alteration following inadequate oxygen supply.3 Hypotensive dogs showed
progressive increase in lactate in the cerebral tissue
correlating with the duration of shock.4 A pH drop
of 0.6 (from 7.1 to 6.5) has been calculated to occur
in tissue as a result of oxygen deprivation which may
be sufficient to produce deleterious effects on neural
metabolism and activity. The acidosis may thus
compound the damaging effect of oxygen deprivation. pH has an additional biological importance
related to determination of net electric charge of the
amino acids, which affects cell membrane permeability.
The advantages of the in vitro experimental
methods are their ease of control, speed, and relative
reproducibility. The results of in vitro studies on
From the Department of Neurological Sciences, RushPresbyterian-St. Luke's Medical Center, Chicago, Illinois,
60612.
This study was aided by Grants NB 07463 91A1 and
NB 05591 from the National Institute of Neurological
Diseases and Stroke, U. S. Public Health Service.
Reprint requests to Dr. Patel.
Stroke, Vol. 4, March-April 1973
alkalosis (in vitro)
brain slices
brain metabolism have generally been confirmed by
results of work in vivo.5 The present work was
undertaken to study the effect of various pH values
on aerobic metabolism of cerebral slices incubating
at 37°C in glucose phosphate medium. Data were
gathered on respiratory and glucose utilization rates
and correlated with changes in the ultrastructure.
Methods
BIOCHEMICAL
Brain slices were prepared from young and mature
guinea pigs and floated into preoxygenated medium as
described by Patel et al.6 After draining a slice gently on
a glass surface it was weighed to the nearest milligram.
Three slices from one hemisphere, incubated at pH 7.4,
served as controls for three slices from the other
hemisphere incubated under the experimental condition
selected. In this manner both control and experimental
tissues were subjected to the same preparative conditions to obviate the effects of swelling, ionic shifts, and
other changes that occur during preparation. Required
basic or acidic pH values were obtained with NaOH and
HCI, respectively.
The main compartment of the Warburg flask
contained 3 ml of oxygenated medium fortified with 10
mM D-glucose and 1 fid (U-14C) glucose when
radioactivity was studied. A filter paper wick was placed
in 0.2 ml of 0.3 N-NaOH in the center well of the flask
to trap the evolved carbon dioxide.
Slices were incubated aerobically in a Warburg
bath at 37°C. Incubation was begun within 25 minutes
of sacrifice of the animal. Oxygen was supplied at the
rate of three liters per minute for five minutes. After an
equilibration for five minutes, manometric readings
were taken at ten-minute intervals and corrected for
221
PATEL, HARTMANN, COHEN
TABLE 1
Rates of Oxygen and Glucose Consumption by Brain Slices in Media at Various pH
pH of
incubation
medium
Glucose consumption
/imol/g wet wt/h
Oxygen consumption
jtmol/g wet wt/h
92.8
70.4
94.7
81.5
106.8
94.5
102.8
107.6
93.8
95.6
7.4
6.2
7.4
6.4
7.4
7.0
7.4
8.4
7.4
9.0
6.4
6.5
4.7
2.0
2.5
2.4
5.0
3.0
2.7
2.5
69.0
29.8
67.6
31.0
72.8
61.1
70.4
87.8
72.0
79.6
4.7
3.0
2.9
4.5
5.5
4.3
2.9
4.8
2.6
4.0
Relative specific
activity of COi
0.72
0.62
0.014
0.016*
0.63 =* 0.026
0.62 ± 0.014
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As the rates of oxygen and glucose consumption were shown statistically not to be affected by the three
slice levels or by the incubation times of 20, 30 or 40 minutes, each value is a mean ± SE of nine observations, except in case of pH 9.0 and its control, where 18 observations were made considering inconsistent
effect of this basic pH.
Composition of incubation medium: NaCl, 98 mM; KC1, 27 raM; MgSO4, 1.2 mM; KH2PO4, 4.0
mM; Na2HPO4, 17.5 mM; and glucose, 10 mM.
*(P = 0.001) significance of difference from preceding figure.
changes in the room pressure or bath temperature as
indicated by thermobarometric readings.
Respiration was terminated at the end of 20, 30, or
40 minutes by immediate removal of flasks from the
manometers, placing them in an ice-filled tray and
quickly adding 0.3 ml 14% perchloric acid to the main
compartment. The contents of the main compartment
and two rinses with 1 % perchloric acid, 1 ml each, were
transferred to a cold homogenizing tube. Homogenization was carried out in the cold, and the homogenizing
tube was rinsed with 1 ml 1% perchloric acid. The
homogenates, including those from the thermobarometers, were centrifuged for 30 minutes at 3,000 g in
the refrigerated Sorvall RC-2. The supematants were
analyzed for glucose on the Technicon Dual Channel
AutoAnalyzer. The difference in values obtained for
glucose concentration in the homogenates from those of
the controls gave glucose consumption by brain slices
during the incubation. The total CO2 evolved and the
amount of glucose carbon incorporated into carbon
dioxide were measured to obtain relative specific activity
(RSA) of CO2. Total carbon dioxide was measured by
titrating the center well contents with HC1 using a
Metrohm automatic titrator. Labeled carbon dioxide
and the initial activity of radioactive glucose were
counted in a Packard Tri-carb Model 3003 scintillation
spectrometer with PPO-POPOP-Toluene mixture. The
RSA of CO., was calculated by dividing the specific
activity of CO2 with the specific activity of glucose and
multiplying the quotient by 6.
TABLE 2
Occurrence of Decline and Increase in the Rates of
Oxygen and Glucose Uptake by Brain Slices, due to
Change in pH of the Incubation Medium from pH 7.4
No. of instances with
uptake change
Increase
Total
Parameter
PH
Decline
Oxygen
Acidic
Basic
Acidic
Basic
27
9
26
6
Glucose
0
18
1
21
27
27
27
27
The number of instances was obtained from individual data
on each slice at each incubation time and at each pH.
222
Effect of pH on rates of oxygen and glucose consumption
by brain slices metabolizing in glucose-phosphate medium
for 20, 30, or 40 minutes. The values are plotted as percent
deviation from the rates at pH 7.4.
Stroke, Vol. 4, March-April 1973
EFFECT OF pH ON METABOLISM AND ULTRASTRUCTURE
ULTRASTRUCTURAL
tion. Animal variation was eliminated by using one
cerebral hemisphere for the experimental pH and the
contralateral hemisphere for control pH 7.4. Statistical analysis indicated no significant difference
attributable to slice levels or to incubation times;
and the mean uptake values using the top two or all
three slices were very comparable. This permitted us
to combine the results at each pH irrespective of
slice level or incubation duration.
Table 1 shows the rates of oxygen uptake and
glucose consumption by brain slices incubated at five
different pH values as compared to control rates at
pH 7.4. Oxygen and glucose consumption rates
decline in acidic incubations and increase in basic
incubations. In figure 1 the effect of three incubation
times is illustrated separately. At pH 9.0, occasional
decline was obtained. The effect of acidic or basic
pH on each slice was therefore examined (table 2 ) .
It was evident from nine observations for each of the
The top two cortical slices were employed for studying
the effect of acidic and basic incubations on the fine
structure. The procedures for incubation, and for
preparation of tissue and micrographs were as described
by Patel et al.u The osmolality of the glucose-phosphate
medium at pH 7.4 was 293 mOsm. It was almost
unchanged in the basic media, pH 8.4 and 9.0, was
increased by 5 units in pH 7.0 medium, and by 11 units
in more acidic media, pH 6.4 and 6.2. The osmolality of
the fixative, 1% OsO4 in glucose-free phosphate
medium, was 310 raOsm. Tonicity of the medium as a
whole can be increased or decreased some 30% without
great effect on respiration or respiratory response.7
Results
BIOCHEMICAL
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Incubation time and pH of the incubation medium
were the two variable factors under study. However,
variations in animal and slice-level were additional
factors introduced by the method of tissue prepara-
••
A
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D
if*1
FIGURE 2
Neuropil from slice incubated under optimum conditions for 30 minutes at pH 7.4. Except for some swelling of an astrocyte
process (A) and a dendritic spine (D), the appearance approaches that of normal well-fixed cerebral cortex, x 46,500.
Stroke, Vol. 4, March-April 1973
223
PATEL, HARTMANN, COHEN
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three acidic pH values corresponding to nine control
pH observations that oxygen consumption declined
in every instance and that glucose consumption
declined in 26 out of 27 instances. Nine observations
for pH 8.4 and 18 observations for pH 9.0 were
gathered. Basic pH caused an increase in the rate of
oxygen consumption in 18 out of 27 instances and in
that of glucose consumption in 21 out of 27
instances. To find if the single rise in the rate of
glucose uptake due to acidic pH and the occasional
declines in the oxygen and glucose consumption
rates due to basic pH were by chance alone, two by
two contingency tests were run. The probability of
occurrence of decline and increase in the consumption rates independent of direction of pH change was
found to be 0.1 X 10~° in case of oxygen consumption and 0.2 x 10~7 in case of glucose consumption.
The fall expressed as percent deviation (fig. 1),
in oxygen and glucose consumption rates from
respective control rates, was greater with greater
acidity, e.g., the rate of oxygen uptake was
diminished by 10% to 15% at pH 7.0 and 21% to
26% at pH 6.2. Secondly, acidosis as well as
alkalosis affected glucose consumption rates more
than oxygen consumption rates. The oxygen uptake
rate at pH 6.2 following 30 minutes of incubation
(fig. 1) was 78.7% of that obtained at pH 7.4, while
the glucose consumption rate under the same
conditions was only 37.3% of that at pH 7.4. This
results in an increased ratio of oxygen uptake to
glucose consumption, a situation occurring in
hypoglycemic conditions.8
The data in table 1 reveal that changes in pH
toward the acidic produced greater metabolic
alterations than those toward the basic. A pH
diminution of 1.0 below pH 7.4 produced deviations
in oxygen and glucose utilization rates greater than
\"&
p
17
*
i
'SS,
FIGURE 3
Forty minutes' incubation at pH 7.0. A swollen astrocyte process (A) is flanked on the left by more or less compressed
cellular processes. The dendrite (D) shows five swollen profiles of endoplasmic reticulum among normal-appearing microtubules. A pseudoextracellular space is particularly prominent at (X). X 37,800.
224
Stroke, Vol. 4, March-April 7973
EFFECT OF pH ON METABOLISM AND ULTRASTRUCTURE
twice that of those resulting from a pH increase of
1.0 above the same value.
Incorporation of radioactive carbon into
evolved carbon dioxide was significantly lower at pH
6.4 and was almost unchanged at pH 8.4 compared
to that at pH 7.4 (table 1).
ULTRASTRUCTURAL
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Figure 2 is a micrograph prepared from a slice
incubated under conditions considered optimal for
biochemical studies. The ultrastructure does not
illustrate ideally fixed normal tissue, but is employed
as a baseline for alterations resulting from experimental incubation. At the end of 30 minutes of
incubation at pH 7.4, the fine structure is well
preserved except for some astrocytic swelling
attributable principally to slicing. When the incubation was extended to 40 minutes, a few swollen
mitochondria and some increase in the volume of
interstitial space were found.
Incubation of brain slices at pH values above or
below 7.4 results in ultrastructural changes that are
much more severe in acidic than in basic media. This
is in conformity with the report on rabbit brain slices
incubated in glycylglycine medium at pH 6.4 and in
bicarbonate medium at pH 8.4.° The changes
principally involve swelling of astrocytic and
neuronal cell bodies and processes, with the
exception of the smallest neuronal processes near
their terminations, where they appear compressed
and exhibit increased electron density that is
accentuated by the staining technique used (figs. 2
and 3 ) . Mitochondria also exhibit swelling which is
of two types not correlated with the conditions of
incubation or limited to any particular cell type. In
some mitochondria, only the space within the cristae
is enlarged with little increase in overall size, while
the mitochondrial matrix shows nearly normal
The mitochondrion in the swollen dendrite (D) exhibits swelling that involves primarily the intracristal spaces while the matrix
shows nearly normal density. Incubation at pH 7.0 for 20 minutes. X 48,500.
Stroke, Vol. 4, March-April 1973
225
PATEL, HARTMANN, COHEN
T
V? YvU'
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FIGURE 5
Same conditions of incubation as for figure 4, but a different type of swelling is seen in the dendritic mitochondrion (M) in
which the cristae have nearly normal width but the initochond rial matrix is greatly decreased in density. X 49,800.
density (fig. 4). Other mitochondria show generalized swelling up to two or three times normal size
with a greatly decreased density of the matrix (fig.
5). From pH 7.0 to pH 6.2, swelling of cells and
processes is progressively more severe, and at the
latter value the tissue is so disrupted as to be all but
unrecognizable (figs. 6 and 7).
Throughout the pH range studied it was noted
that the extracellular space was enlarged and
resembled the "pseudoextracellular space" found by
Long et al.10 in the gray matter in cerebral edema.
For a given deviation from the baseline value of
pH 7.4 the changes were much more severe on the
acid side. For instance, while the tissue incubated at
pH 6.4 for 20 minutes is severely damaged (fig. 6),
that incubated at pH 8.4 appears much better
preserved (figs. 8 and 9). It seems worth noting that
while the cellular swelling at low pH is accompanied
by a general loss of cytoplasmic density, that
226
occurring in basic media involves mostly mitochondria and cisterns of endoplasmic reticulum without
dissolution of the intervening cytoplasmic matrix
(fig-9).
Discussion
Studies simulating alkalosis and acidosis in patients
have been conducted on various isolated tissues
incubated in media at pH values above or below 7.4.
Earlier investigations on cerebral respiratory rate
under unfavorable pH conditions"- u~18 concluded
that both acidosis and alkalosis alter the chemistry
of the tissue. Our in vitro experiments demonstrated
that distinct changes in both metabolism and
ultrastructure of the tissue occurred upon incubating
the brain slices in acidic or basic media.
Both oxygen and glucose consumption in vitro
are augmented at the alkaline pH values but are
diminished when the pH is acidic. Table 1 and figure
Sfroke, Vol. 4, March-April 1973
EFFECT OF pH ON METABOLISM AND ULTRASTRUCTURE
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FIGURE 6
Incubation for 20 minutes at pH 6.4. Severe swelling with general loss of background density is apparent throughout. The
dendrite (D) is identifiable only by the presence of a few randomly oriented microtubules, while the remaining cellular
processes are not identifiable with certainty. X 46,500.
1 reveal that this effect is more pronounced at acidic
than basic pH. In clinical studies, patients with
alveolar hypoventilation showed no significant
abnormalities in mental state if the arterial CO2
tension was less than 90 mm Hg and the pH above
7.25, while a semicomatose or comatose state was
observed when the arterial CO 2 was above 130 mm
Hg and the pH below 7.14. 19
Swanson18 demonstrated significantly lower
concentrations of creatine phosphate and adenosine
triphosphate in acidotic brain slices (pH 6.5). He
interpreted this change to be due to decreased
synthesis rather than increased utilization of these
compounds.
The generally better-preserved appearance of
slices incubated at high pH is contrasted with those
incubated in acid media. Palade 20 showed that with
fixation in unbuffered OsO 4 , the penetration of the
Stroke, Vol. 4. March-April 1973
fixative was preceded by a wave of tissue acidification that resulted in loss of cytoplasmic densities in
the electron micrographs, as well as to coarse
precipitation of nucleoplasm. Since the present
experiments involved buffered fixatives throughout,
it seems likely that the slices incubated below pH 7.4
were damaged by H + concentrations before the start
of fixation.
The altered capacity to utilize oxygen and
glucose at an unfavorable pH would be followed by
change in the capacity to maintain the respiratory
chain and hence the high energy phosphate
concentrations. Alterations in the mitochondrial fine
structure under unfavorable pH conditions (figs. 4
and 5) are conceivable since cerebral mitochondria
carry carbohydrate utilizing enzyme systems21 and
play an active role in oxidative phosphorylation.22
Chordikian et al.- 3 reported rat brain mitochondrial
227
PATEL, HARTMANN, COHEN
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FIGURE 7
The fine structure in this slice after 40 minutes' incubation at pH 6.2 shows gross deterioration. Pseudoextracellular space
(X) is large. X 38,100.
fraction in isotonic phosphate buffer in the pH range
5 to 9 to show progressively increased swelling and
to give a straight line relationship upon plotting pH
against AO.D.
At an acidic pH the oxygen uptake is reduced
from values obtained at pH 7.4, to a lesser extent
than is glucose consumption. The relative specific
activity of CO., at pH 7.4 was significantly different
(P = 0.001) from that at pH 6.4 (table 1),
indicating increased endogenous substrate utilization
at pH 6.4. Since brain has little freely available
endogenous substrate (small amounts of glycogen
and glucose and amino acids related to glutamate),
some ultrastructural changes observed following the
incubations in acidic media may be secondary to
combustion of endogenous substrates essential to the
integrity of cells. The alkaline pH 8.4 did not
produce a significant effect on the relative specific
activity of CO* (table 1).
228
Enerson and Berman24 noted cellular swelling
under hypoxic conditions, but the fall in respiration
secondary to acidosis was greater than that which
resulted from swelling alone. The ionic and fluid
movements as controlled by internal and/or external
milieu appear to determine the volume of the
interstitial and astrocytic spaces. The swelling and
resulting increased volume of astrocytic processes
may be reducing the interstitial space volume by the
compressing effect on the adjoining areas or may be
found with accompanying enlargement of interstitial
space volume. Both combinations were encountered
in the micrographs prepared from our pH treatments. An enlarged interstitial space in the presence
of astrocytic swelling suggests a phenomenon
comparable to formation of pseudoextracellular
space in intact animals with cerebral edema.10
In vitro studies of the effect of graded hypoxia
showed that a smaller degree of hypoxia produced
Stroke, Vol. 4, March-April 1973
EFFECT OF pH ON METABOLISM AND ULTRASTRUCTURE
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FIGURE 8
In contrast to figure 6 this tissue incubated at pH 8.4 for 20 minutes shows much better retention of background densities
except in the swollen astrocyte processes. X 38,500.
more severe alterations in rat kidney cortical slices
in comparison to brain cortex and liver slices.23 With
90% N 2 -10% O 2 the kidney slices respired at onethird the rate and the brain and liver slices at onehalf the rate of that with 100% O,.
It is evident that pH exerts an effect different
from anoxia. Whereas oxygen uptake falls and
glucose consumption rises in anoxia in order to meet
the increased energy demands, both glucose and
oxygen utilization rates decline during acidosis and
increase during alkalosis. Under anaerobic conditions, the utilization of glucose was diminished with
a rise in pH. 16 At alkaline pH values, no more
carbohydrate appeared to be oxidized aerobically
than that at pH 7.4 since the increase in glucose
utilized could be accounted for by an increase in
lactate produced. The acceleration of glycolysis
under alkaline conditions has been reported for
brain 10 ' 17 and other tissues.20 Our work demonStroke, Vol. 4, March-April
1973
strates that acidic pH causes derangement in the
glycolytic pathway in the sense that glucose
degradation was suppressed. In unreported current
work decreased formation of pyruvate and lactate
from glucose occurred under acidosis. Investigations
are in progress to determine whether inhibition of
oxidative metabolism by acidic pH also contributes
to the observed reduction in oxygen consumption.
Acknowledgments
The authors wish to thank Miss Elaine Grandt for technical
assistance and Miss Ruth Becker and Mrs. Diane M. Harms
for help with the electron micrographs.
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229
PATEL, HARTMANN, COHEN
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FIGURE 9
Part of a neuron from a slice incubated at pfi 8.4 for 20 minutes. Many swollen profiles of endoplasmic reticulum, the
largest of which is labeled ER, are present in the cytoplasm which contains clusters of RNP particles which stain darkly
against a moderately dense background. X 34,800.
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Stroke, Vol. 4, March-April
1973
231
Effect of pH on Metabolism and Ultrastructure of Guinea Pig Cerebral Cortex Slices
KUSUM K. PATEL, J. FRANCIS HARTMANN and MAYNARD M. COHEN
Stroke. 1973;4:221-231
doi: 10.1161/01.STR.4.2.221
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