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CHOLINERGIC MECHANISM IN CO2 REACTIVITY/5crem/« et al.
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Symp. on Neurogenic Control of the Brain Circulation, Stockholm, 1977
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Metabolic Profiles of Canine Cerebrovascular Tree:
A Histochemical Study
B. H. COOK, M.D.,
P H . D . , H. J. GRANGER, P H . D . , D. N . GRANGER,
PH.D.,
A. E. TAYLOR, P H . D . , AND E. E. SMITH, P H . D .
SUMMARY Intriguing questions have recently been raised regarding the applicability of direct observations of the pial microcirculation to the behavior of the total cerebral microcirculation.
Operating under the assumption that arteriolar tone and, thus,
cerebrovascular resistance is, to some extent, directly related to the
intrinsic energy metabolism of the arteriolar wall, a comparative
histochemical analysis of cerebral microvessels, both pial and
parenchymal, was undertaken. Reactions were chosen on the bases of
representation of substrate and of enzymes of glycolysis, the hexose
monophosphate shunt, /3-oxidation of fat, Krebs cycle, cytochrome
system and ATP hydrolysis. Three metabolically distinct segments of
the cerebral microvasculature were delineated with the pial vessels
showing strong capacities for glycolysis, (3-oxidation of fats and
utilization of glucose through the hexose monophosphate shunt.
Microvessels of the gray matter have a qualitatively similar metabolic
profile but the capacities of each pathway are lower when compared
to pial arterioles. Arterioles of the white matter demonstrate the
weakest energy-yielding capacities.
SINCE THE DIRECT observations of the pial circulation
wall which mediate the contractile process through produc-
by Donders 1 in the 19th century and by Forbes2 in the early
tion of adenosine triphosphate. Since previous studies in this
20th century, the responses of the pial vessels have served as
laboratory have indicated that a metabolic heterogeneity
a useful model for the whole brain microcirculatory unit.
between vessels of different calibre within a vascular bed7
Rosenblum and Kontos3 have, however, recently raised the
and between vessels of similar calibre from organ to organ 8
intriguing question of whether the pial vessels do indeed
may exist, the topohistochemica! approach utilized in these
mimic the responses of parenchymal vessels. While pial
studies has been applied to the cerebral microvasculature in
vessels have been shown to respond in a proper manner to
order
those humoral substances (CO 2 , H + ) 4 ' 6 generally accepted to
metabolically similar to parenchymal vessels.
to ascertain
whether
the vessels of the pia
are
be responsible for cerebral autoregulation, other studies6
have indicated that parenchymal vessels behave in a manner
which would tend to compensate for alterations in pial
vascular calibre. In addition to neurogenic and parenchymal
(via vasodilator release) factors, vascular tone is, to some extent, a function of the metabolic properties of the arteriolar
Methods
Ten mongrel dogs were anesthetized with sodium pentobarbital. Skin and scalp muscles overlying the skull were dissected away before a rectangular opening was made in the
fronto-parietal bones for biopsy of underlying cerebral cor-
From the Department of Medical Physiology, College of Medicine, Texas
A & M University, College Station, TX 77843 and Department of Physiology
and Biophysics, University of Mississippi School of Medicine, Jackson, MS
39216.
Supported by grant-in-aid 72-947 from the American Heart Association
and HL 19151 from the National Heart, Lung and Blood Institute.
Reprint requests to Dr. H. J. Granger, Dept. Med. Physiology, Texas A &
M College of Medicine, College Station, TX 77843.
tex. Biopsied samples were immediately frozen at — 170°C
in isopentane precooled in liquid nitrogen. Ten micron sections were cut on an Ames Lab-Tek cryostat and were not
allowed to thaw prior to the application of incubating
medium. Standard histochemical reactions were performed
for representative enzymes of each of the major metabolic
STROKE
166
TABLE 1 Reactions Employed for Hdstochemical Analyses
Reference
A.
B.
Substrates
Glycogen
Neutral Fat
Free Fatty Acids
14
15
16
Enzymes
1)
Glycolysis
lactate dehydrogenase
9
2) Hexose Monophosphate Shunt
glucose-6-phosphate dehydrogenase
DPN diaphorase
9
18
3) Lipid Catabolism
lipase
/3-OH butyrate dehydrogenase
13
9
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4) Tricarboxylic Acid Cycle
NAD -linked isocitrate dehydrogenase
succinate dehydrogenase
5) Respiratory Chain
cytochrome oxidase
6)
ATPases
Herman-Padykula ATPase
alkaline phosphatase
9
9
12
10
11
pathways. Three substrates, glycogen, neutral fat, and free
fatty acids were also demonstrated. Samples for glycogen
determination were fixed for 14-18 hours in absolute alcohol
at 0°C and routinely processed through paraffin. Mitochondrial dehydrogenases were demonstrated by the standard methods of Hess, Pearse, and Scarpelli9 utilizing polyvinylpyrrolidone as an osmotic protectant and Nitro BT
(tetrazolium salt) as sole electron acceptor. Soluble enzymes
were demonstrated utilizing viscous gels (gelatin or
polyvinyl alcohol) to minimize enzyme diffusion and
phenazine methosulfate as an intermediate electron acceptor. Myosin ATPase was demonstrated by the calciumcobalt method of Hermann and Padykula10 and alkaline
phosphatase was demonstrated using the naphthol AS
phosphate method of Burstone." Cytochrome oxidase
localization was afforded by Burstone's amino-quinoline
method.12 Lipase was demonstrated utilizing Gomori's
Tween 80 method for "true lipase."13 The dimedone-PAS
method of Bulmer was employed for glycogen
demonstration."
In general, a histochemical profile was constructed for
both the energy producing reactions (glycolysis, hexosemonophosphate shunt, /3-oxidation of fats, and the
respiratory chain) and energy yielding reactions (ATPases).
In addition, cellular equipment for the breakdown of neutral
fat to triglyceride was studied in conjunction with Lillie's Oil
Red O method15 for neutral fat and Holczinger's method for
free fatty acids.16 Table 1 summarizes the reactions performed for delineation of metabolic characteristics of
cerebral vasculature.
Controls for enzyme reaction were performed through
omission of substrate from the incubation medium and
through exposure of the unfixed tissue section to hot formalin vapors for 5 minutes prior to incubation respectively.
"Nothing dehydrogenase" activity was eliminated through
VOL 9, No
2, MARCH-APRIL
1978
rigid pH control. A 30 minute acetone extraction provided
controls for the Oil Red O reaction.
For each histochemical reaction, color photographs of 5
to 6 microvessels were obtained from each brain compartment of each dog under identical magnification and illumination conditions. Hence, 50 to 60 photographs of pial
vessels could be compared with an identical number of
microvessels of grey or white matter. After random pairing
of photographs representing two compartments, two individuals working separately ranked each pair according to
intensity of specific histochemical color products located
within the muscle layer of the microvessels. This process was
repeated until all permutations were ranked (i.e. pia vs white
matter, grey vs white matter, pia vs grey matter). A
difference in reaction intensity was considered highly
probable if 80% of the paired photographs showed a consistent gradient of staining. We assumed that no difference existed if only 40 to 50% of the paired photos for a given reaction yielded positive rankings for an assumed gradient.
Because all tissue sections were identical in thickness, the
variations in intensity cannot be explained by differences in
substrate penetrability unless the physical consistency ol
cytoplasm within microvascular smooth muscle varies from
compartment to compartment, an unlikely possibility.
After the relative rankings of the 3 brain compartments
were established, an attempt was made to provide a subjective index of the magnitude of the gradient existing betweer
2 compartments meeting our criteria for significant
difference. This subjective index provides a crude means ol
determining whether these consistent gradients of histochemical activity are shallow or steep.
Pial vessels larger than 100 n were excluded from this
study due to an apparent heterogeneity which precludec
application of our method of analysis.
Results
Table 2 illustrates the subjectively graded observations foi
vessels of the pial, grey matter, and white matter segment!
of the cerebral vasculature. Pial vessels of 15-75 n diametei
were observed in random distribution within the sulci of th<
cerebral cortex. No significant metabolic differentiatioi
could be distinguished among these pial vessels. Sur
prisingly, however, our results indicate the parenchyma
vessels cannot be compartmentalized as a single entity. Oi
the contrary, we were able to histochemically delineate i
difference between vessels of the grey and white matter. Fo
reasons of coherence the results are categorized witl
reference first to the function of the molecule involved, i.e.
substrate vs enzyme, and enzymes are further subdivide!
with reference to the metabolic pathway which they repre
sent.
I. Substrates
By referring to table 2, the reader will note the gradien
demonstrated for glycogen with the highest levels bein]
found in the pial vasculature. It appears that as the vessel
penetrate the substance of the brain the glycogen o
arteriolar smooth muscle decreases with intermediat
amounts present in vessels of the grey matter and lesse
amounts in those of the white matter.
CEREBRAL MICROVESSEL METABOLISM/Coofc et al.
TABLE 2 Comparative Hislochemislry of Cerebral Microvessels
Reaction
Pial
vessels
Grey
matter
vessels
White
matter
vessels
++
+
0
0
++
++
3. Free fatty acids
++++
*
+++
4. Lactate
dehydrogenase
+ + + + +++
1. Glycogen
2. Neutral Fat
5. Glucose-6-phosphate
dehydrogenase
7. Lipase
++
+++
+ + + + +++
0
++
8. /3-OH butyrate
dehydrogenase
++++
9. Isocitrate
dehydrogenase
+++
6. DPN diaphorase
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10. Succinate
dehydrogenase
11. Cytochrome oxidase
12. Myosin ATPase
13. Alkaline phosphatase
0
±
+
-f- +
+++
-f- -+• + +
+ +
+++
0
+
+++
0
+
+++
+
+
+++
+
+
++
+ + + + ++++ ++++
0
+++
+++
= No discernible activity.
= Very weak activity.
= Slight activity,
= Moderate activity.
= Strong activity,
= Very strong activity.
Neutral fat, as demonstrated by Lillie's Oil Red O
technique, was found to be weakly positive in the pial
vasculature and essentially absent from the vessels of grey
and white matter.
Free fatty acids were found to be present in moderate concentrations in the three segments of the vasculature.
II. Enzymes
A. Glycolysis. Lactate dehydrogenase was utilized as an
indicator of glycolytic activity. Zugibe" regards the activity
of this enzyme as the "most useful of all the histochemical
techniques to determine whether the Embden-Meyerhof
pathway is operative." The activity of this enzyme was
found to be greatest in the pial vessels with decreasing activity as the vessels traversed grey and finally white matter.
B. Hexose monophosphate shunt. The relative significance of this pathway was evaluated through the activity
of both the initial enzyme of the pathway, glucosesphosphate dehydrogenase, and of the final mediator of ATP
production from this pathway, DPN diaphorase. Cohen18
regards the latter enzyme as reflecting the activity of transhydrogenase. Maximal glucose-6 phosphate dehydrogenase
activity was found in the superficial (pial) vessels of the brain
with a decrease in the vessels of the grey matter and a further
decrease in the vessels of the white matter. DPN diaphorase
shows a similar pattern with greatest intensity in the surface
vasculature and decreasing through grey and white matter.
C. Tricarboxylic acid cycle. The oxidative capabilities of
the three vascular segments were evaluated through the ac-
167
tivities of NAD-linked isocitrate dehydrogenase and of succinate dehydrogenase. Isocitrate dehydrogenase was quite
intense in all vascular segments studied with no differential
observed. Succinate dehydrogenase, on the other hand,
showed the greatest activity in pial vessels with low intensities in the vasculature of both grey and white matter.
D. Lipid metabolism. As noted before, the level of nonglobular neutral fat, as tested by the Oil Red O technique,
was very slight in the pial vasculature. No detectable nonglobular neutral fat was present in vessels of grey and white
matter. Moderate lipase activity was present in the surface
vasculature of the brain with a total absence in both grey and
white matter. /3-OH butyrate dehydrogenase, utilized as an
indicator of /3-oxidation of fats, was found to be extremely
active in pial vessels with lesser amounts in grey matter and
a further decline in the vasculature of white matter. The
presence of free fatty acids and TCA cycle in the pial vessels
serve to reinforce the concept of a ^-oxidative scheme in
these vessels.
E. The respiratory chain. The activity of this enzyme
reflects the terminus in the cycle of oxygen-dependent ATP
production. Pial vessels demonstrated higher activity of this
enzyme than the vessels of grey and white matter.
F. Enzymes of energy utilization. Myosin ATPase was
noted to be extremely active in all three vascular segments
studied. Alkaline phosphatase, on the other hand, was absent from the pial vasculature but present in high concentration in the arterioles of both grey and white matter.
Discussion
Controversy has existed for some time regarding the
validity of direct observation of pial vasculature and extrapolation of this data to the microvasculature of the brain as a
whole. While our results do not negate this approach, we
have found intrinsic metabolic dissimilarities between not
only pial and parenchymal microvessels but have, in addition, observed a further differentiation of microvascular
metabolic capabilities between vessels of grey and those of
white matter. In referring to table 2, it will be noted that pial
vessels possess strong LDH activity, suggesting an active
role of the Embden-Meyerhof pathway in the metabolism of
these arteriolar circuits. The strong activity of hexose
monophosphate shunt enzymes in conjunction with high
glycogen levels suggests, on the other hand, that this
pathway may play a role in the maintenance of arteriolar
wall tone. Again, examination of this table with reference to
the metabolic paraphenalia required for fat catabolism
reveals a great potential for this conduit for ATP production. A slight reaction for nonglobular neutral fat probably
reflects small amounts of either constitutive or
metabolizable liquid. In conjunction with this we find a
moderate lipase activity and an intense reaction for free fatty acids. Quite striking is the presence, in quantity, of reaction products for the enzymes necessary to catabolize free
fatty acid to CO 2 and H 2 O. This is reflected in the reactions
for (8-hydroxybutyrate dehydrogenase, N A D + linked
isocitrate dehydrogenase, succinate dehydrogenase, and
cytochrome oxidase. In addition, the intense activities of
Hermann-Padykula ("myosin") ATPase suggests a high
capacity for utilization of generated adenosine triphosphate.
168
STROKE
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Microvessels of grey matter seem quite capable of utilizing the Embden-Meyerhof pathway as suggested by
moderate lactate dehydrogenase activity. These vessels,
although seemingly capable of utilization of oxidative
pathways for both lipid and carbohydrate, seem somewhat
less endowed than those of the pia. The absence of neutral
fat and lipase together with the moderate reaction for free
fatty acids suggests little intramural lipid storage with fatty
acids of the arteriolar wall provided exclusively via delivery
by the circulatory system. The importance of the hexosemonophosphate shunt, as judged by the intensity of its enzymes, would seem comparatively less than in pial vessels.
"Myosin" ATPase was demonstrated to have an activity
equal to that of the pial vessels. Interestingly, alkaline
phosphatase, another of the ATPases, was found to be reactive in the arterioles of grey matter while totally absent in
the pial vasculature.
Finally, the microvessels of white matter demonstrated, in
general, markedly reduced levels for most of the reactions
performed. In contrast to pial vasculature, this bed seems to
lack the capacity for anaerobic glycolysis, as indicated by
the absence of detectable levels of lactate dehydrogenase activity. Like the vessels of grey matter there seemed to be no
endogenous mechanism for storage and initial
(triglyceride -»free fatty acid) catabolism of neutral fat.
There seemed, in addition, to be a definite, yet once again
greatly reduced, capacity for aerobic lipid metabolism. This
may, however, be misleading in view of the intense activity
of NAD+ linked isocitrate dehydrogenase, a rate limiting
step in the tricarboxylic acid cycle. It is therefore plausible
that the rate of oxidative metabolism in these vessels is
greater than indicated by the relatively weak reactions for
succinate dehydrogenase and cytochrome oxidase. The total
carbohydrate metabolism of these vessels seems to be
operative through the hexose monophosphate shunt and the
aerobic component of the Embden-Meyerhof pathway.
"Myosin" ATPase and alkaline phosphatase, as has been
shown in the vessels of grey matter, were noted to be quite
intense in white matter arterioles.
In summary, the superficial vessels of the canine cerebral
cortex seem metabolically to be the most active vessels of
the cerebral microcirculation with high capacities for
glycolysis, Krebs cycle, lipid catabolism and the hexose
monophosphate shunt. Arterioles of the grey matter have
metabolic capabilities qualitatively similar but quantitatively reduced when compared to the pial microvasculature. Vessels of the white matter were demonstrated
to possess, in the smallest quantities noted, the enzymes of
glycolysis, /3-oxidation, the tricarboxylic acid cycle, the hexose monophosphate shunt, and the respiratory chain. All
vessels studied were found to be rich in myosin ATPase with
VOL 9, No 2, MARCH-APRIL
1978
only the arterioles of grey and white matter demonstrating
alkaline phosphatase activity.
Thus, insofar as the vascular wall of the pial, grey matter
and white matter circulation contribute directly to cerebrovascular resistances, these enzyme and substrate patterns
assume some degree of importance. It is quite conceivable
that changes in cerebrovascular resistance in response to
altered pH, Pco2, etc., could well be mediated through
effects on enzyme systems of the arteriolar wall. If this
assumption is tenable, then it is well to note the existence of
the three metabolically distinct vascular segments described
herein and to analyze their behavior in view of the metabolic
pathways which they employ.
Acknowledgment
The authors are indebted to Teresa Stoddard and Sandy McCormick for
secretarial assistance.
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Metabolic profiles of canine cerebrovascular tree: a histochemical study.
B H Cook, H J Granger, D N Granger, A E Taylor and E E Smith
Stroke. 1978;9:165-168
doi: 10.1161/01.STR.9.2.165
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