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Application of focused microwave irradiation for autoradiography: Simultaneous measurements of glucose metabolism, cerebral blood flow and ATP

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APPLICATION OF FOCUSED MICROWAVE IRRADIATION
FOR AUTORADIOGRAPHY:
SIMULTANEOUS MEASUREMENTS OF GLUCOSE METABOLISM,
CEREBRAL BLOOD FLOW AND ATP
A Thesis
Presented
to the Faculty of
California State University Dominguez Hills
In Partial Fulfillment
of the Requirements for the Degree
Master of Science
in
Biology
by
Monica Darlene Wong
Fall 2009
UMI Number: 1481425
All rights reserved
INFORMATION TO ALL USERS
The quality of this reproduction is dependent upon the quality of the copy submitted.
In the unlikely event that the author did not send a complete manuscript
and there are missing pages, these will be noted. Also, if material had to be removed,
a note will indicate the deletion.
UMT
Dissertation Publishing
UMI 1481425
Copyright 2010 by ProQuest LLC.
All rights reserved. This edition of the work is protected against
unauthorized copying under Title 17, United States Code.
ProQuest LLC
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P.O. Box 1346
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ACKNOWLEDGEMENTS
I sincerely thank my sister, Ms. Dara Barrere, for her financial support towards my
educational goals. I am indebted to Dr. Stefan M. Lee for his mentorship and his
continual academic support. I further thank Ms. Sima Ghavim and Mr. John M. Beemer
for their technical contributions and Dr. Richard Sutton for reviewing the text. This study
was supported by NIH/NINDS NS37363 and the UCLA Brain Injury Research Center.
m
TABLE OF CONTENTS
PAGE
APPROVAL PAGE
ii
ACKNOWLEDGEMENTS
iii
TABLE OF CONTENTS
iv
LIST OF TABLES
v
LIST OF FIGURES
vi
ABSTRACT
vii
CHAPTER
1. INTRODUCTION
1
2. MATERIALS AND METHODS
6
Supplies and Experimental Design
Surgical Procedures
Experimental Group 1: [14C]-2-deoxyglucose (2DG) Study
Experimental Group 2: Double Tracer [18F]-fluorodeoxyglucose (FDG) and
[t4C]-iodoantipyrine (IAP) Study with ATP Analysis
Quantitative ATP Bioluminescence Imaging
Autoradiographic Analysis
Statistical Analysis
3. RESULTS
6
7
7
9
10
12
13
14
Physiological and Study Variables
Basal lCMRglc Values - Microwave Versus Fresh Frozen
2DG Autoradiography
Simultaneous lCMRglc, rCBF and ATP Values of Microwave Tissue
14
15
19
4. DISCUSSION
23
REFERENCES
29
iv
LIST OF TABLES
PAGE
1. Absolute lCMRglc for Two Fixation Methods: Fresh Frozen and Microwave
16
2. Absolute lCMRglc, rCBF and ATP Values in Rat Brain
21
V
LIST OF FIGURES
PAGE
1. Scatter Plot of lCMRglc Values Measured from Fresh-Ffozen and
Microwave-Fixed Rat Brains
17
2. Representative [14C]-2-deoxyglucose Autoradiograms of Rat Brain
Preserved by Fresh-Frozen or by Microwave Fixation
18
3. Representative [18F]-FDG and [14C]-IAP Autoradiograms and Resulting
ATP Image of lCMRglc, rCBF and ATP Within the Same Animal
22
VI
ABSTRACT
Multi-tracer autoradiography or bioluminescence imaging has been used to study
the bioenergetic state of normal and injured brains. However, in situ freezing methods
are typically inadequate in maintaining high-energy metabolites sensitive to postmortem
degradation such as ATP. In this study, whole-brain focused microwave fixation was
adapted to multi-tracer autoradiography. Analysis of 25 brain regions indicated that
microwave-based metabolic rates were the same as those obtained from freeze-fixation
method (p < 0.001). ATP content from adjacent cortical and subcortical tissue regions
ranged from 27.3 to 31.3 nmol/mg protein, respectively. The spatial resolution for ATP
bioluminescence images was approximately 900 um . These findings demonstrate that
18
focused microwave fixation can be adapted to measure [ F]-fluoro-deoxyglucose-based
metabolic rates, [14C]-iodoantipyrine-based regional cerebral blood flow and quantitative
ATP content in the same animal, permitting a more precise understanding of the
interaction between glucose utilization and cerebral blood supply in producing energy
under normal and pathophysiologic conditions.
1
CHAPTER 1
INTRODUCTION
The relationship between local cerebral metabolic rates for glucose (lCMRglc),
regional cerebral blood flow (rCBF) and ATP levels is thought to be critical for
maintaining optimum function of the central nervous system. Under physiological states
lCMRglc and rCBF are coupled in supplying glucose for the production of ATP in the
brain (Reivich 1972; Kuschinsky et al. 1981; Sokoloff 1981; Baron et al. 1982; Iadecola
et al. 1983; Tuor et al. 1986; Ginsberg et al. 1997). However, during pathological events
lCMRglc may become uncoupled from rCBF.
This uncoupling has been shown in
patients with traumatic brain injury (TBI) (Bergsneider et al. 1997) and in experimental
TBI models (Back et al. 1995; Ginsberg et al. 1997; Richards et al. 2001). Decreases in
ATP levels have been demonstrated to occur after ischemic insults (Paschen et al. 1983;
Nowicki et al. 1988; Nowak et al. 1985; Mies et al. 1991, Babu and Ramanathan 2009)
and TBI (Lee et al. 1999; Signoretti et al. 2001, 2009; Lifshitz et al. 2003, Aran et al.
2009).
In TBI models that produce little or no histological damage, such as fluid
percussion injury or impact acceleration, ATP concentrations initially decreased by 3050% and either recovered over time after mild-to-moderate TBI (Lee et al. 1999;
Signoretti et al. 2001, 2009) or remained depressed after severe TBI (Signoretti et al.
2001). Delayed cellular degeneration injury models, such as controlled cortical impact or
impact acceleration plus hypoxic hypotension, produced an acute drop in ATP (50-80%)
where concentrations continued to decrease over time after TBI (Lee et al. 1999;
2
Signoretti et al. 2001). It has been suggested (Lee et al. 1999) that this lowered energy
state coupled with metabolic mismatch results in a period of cellular vulnerability where
cells may either recover, i.e., after a mild TBI, or may succumb to secondary insults and
degenerate through apoptosis, i.e., after a severe TBI. Therefore, understanding the
relationship between lCMRglc, rCBF and ATP levels during periods of vulnerability or
secondary insults can help elucidate cellular viability and clarify the mechanisms
involved in neuronal pathology.
Many techniques exist for quantifying lCMRglc, rCBF with or without ATP, but
not all are well suited for combined use in small animals. For longitudinal studies,
positron emission tomography (PET) or phosphorus-31 magnetic resonance spectroscopy
(31P-MRS) were utilized to measure lCMRglc (Moore et al. 2000), rCBF (Ogawa et al.
1996) or the bioenergetic state of high-energy phosphates in brain-injured rats (Vink et al.
1987, 1988, 1994; Headrick et al. 1994, Harris et al. 1996). These techniques, however,
are limited in spatial resolution (Moore et al. 2000) or in the absolute quantification of
ATP (Vink et al. 1988, 1994). On the other hand, traditional autoradiography, with its
higher spatial resolution, has been employed in small animals to measure absolute rates
of lCMRglc or rCBF in vivo by using [14C]-2-deoxy-D-glucose (2DG; Sokoloff et al.
1977) or [14C]-iodoantipyrine (LAP; Sakurada et al. 1978), respectively. Unlike singlelabel autoradiography, double-tracer autoradiography allows for the concurrent
measurements of lCMRglc and rCBF in the same animal by combining two slowdecaying isotope tracers (Furlow et al. 1983; Jones and Greenberg 1985; Ginsberg et al.
1986) or tracers with short and long half-lives (Lear et al. 1981; Mies et al. 1981; Diemer
3
and Rosenorn 1983; Sako et al. 1984; Ackermann and Lear 1989).
Of the two
combinations, the latter is more advantageous because it does not require post-study
processing to remove one of the two labels (Lear et al. 1981; Mies et al. 1981).
With the advent of multi-parametric autoradiography some investigators have also
performed biochemical ATP imaging on tissue adjacent to that used for autoradiography,
although the ATP analyses were either performed qualitatively (Mies et al. 1990a,b;
Paschen et al. 1992) or by indirect biochemical quantification, which was done by
correlating the images with dissected, adjacent tissue blocks (Mies et al. 1991, 1993,
1999). In these prior studies, in situ freezing using liquid N2 was used to preserve
regional ATP. However, as in all freeze fixation methods such as decapitation into liquid
N2 (Mandel and Harth 1961), funnel freezing (Ponten et al. 1973), and freeze-blowing
(Veech et al. 1973), rapid cooling causes uneven freezing across sub-structures (Swaab
1971) and any subsequent tissue thawing will lead to rapid postmortem ATP degradation
by endogenous ATPases, thus making ATP assessment difficult or unreliable (Delaney
and Geiger 1996). Although autoradiography has been used to measure lCMRglc, rCBF
and ATP within the same animal after tissue preservation via funnel freezing (Mies et al.
1990a,b), artifacts from slow freezing and thawing, as well as inadequate ATP
quantification, have limited the use of this multi-parametric technique.
An alternative to the various freezing methods is focused microwave irradiation,
which employs a brief (2-7 s), high-energy microwave beam that is focused directly to
the animal's head, rapidly heating the brain to >70 °C and inactivating thermo-sensitive
enzymes (Schmidt et al. 1972; Stavinoha et al. 1973). This procedure has been used to
4
study glucose kinetics (Cremer et al. 1981; Cunningham et al. 1981), glucose metabolism
(Garriga and Cusso 1992), CBF and the cerebral distribution space of non-diffusible
tracers (Todd et al. 1993) in the rat brain. Since the heat-inactivation is irreversible,
thawing will not degrade intermediate metabolites, and biochemical quantification of
ATP is possible in rat brain after sacrifice by microwave irradiation (Wu and Phillis
1978). Additionally, depending on the microwave's power output, the amount of ATP
preserved has been shown to be better than levels produced by the numerous quick-freeze
methods (Medina et al. 1975; Delaney and Geiger 1996). Despite these advantages,
focused microwave fixation has not been used extensively for autoradiography or ATP
imaging. One potential disadvantage is that the generated heat can cause diffusion of
labile substances (Meyerhoff et al. 1978; Kant et al. 1979) and potentially disrupt cell
membranes (Butcher and Butcher 1976), which may violate one of the principle
assumptions of 2DG autoradiography, e.g., the preservation and compartmentalization of
2DG within cells. To our knowledge, only one autoradiographic study has been reported
previously to use this fixation method. The qualitative glycogenosis study by Swanson
et al. (1992) is the only prior report combining autoradiographic methods with
microwave-preserved brain tissue. The Swanson et al. (1992) study, however, did not
address issues regarding quantitation or the potential degradation of spatial resolution.
Thus, while focused microwave irradiation effectively preserves ATP levels postmortem,
the potential heat-induced diffusion of tracers needs to be addressed before this in situ
fixation method can be broadly applied to quantitative autoradiography.
5
The current
studies
were
conducted
to
establish
focused
microwave
autoradiography as a potential tool for simultaneously understanding the relationship
between lCMRglc, rCBF and ATP in the same animal.
We first conducted 2DG
autoradiographic studies to determine the validity, sensitivity and spatial resolution
following focused microwave irradiation. To validate this method, lCMRglc calculated
from microwave 2DG autoradiography were compared to lCMRglc generated from the
traditional 2DG "fresh-frozen" method established by Sokoloff et al. (1977).
The
lCMRglc values for each method were obtained from brain regions of various sizes to
determine the sensitivity and spatial resolution of the microwave autoradiography. In
addition, representative studies were conducted to illustrate that quantitative ATP
imaging in microwave-fixed brain tissue can be combined with double-tracer quantitative
autoradiography in the same animal using [18F]-fluoro-deoxyglucose and [14C]iodoantipyrine. Our study is the first to demonstrate that microwave-irradiated brain
tissue can be used to simultaneously determine quantitative lCMRglc, rCBF and ATP
concentration in the same area within the same animal.
6
CHAPTER 2
MATERIALS AND METHODS
Supplies and Experimental Design
[ 14C]-2-deoxyglucose (2DG) and [14C]-iodoantipyrine (LAP) were purchased
from American Radiolabeled Chemicals Inc. (St. Louis, MO, USA).
The UCLA
Cyclotron Facility supplied the [18F]-fluoro-deoxyglucose (FDG). All chemicals were
obtained from Sigma-Aldrich (St. Louis, MO, USA). Male Sprague-Dawley rats (275325 g) were purchased from Charles River Laboratories (Hollister, CA, USA).
In both experiments, catheterized animals recovered completely from isoflurane
anesthesia 3 h prior to the metabolic studies. Animals in the first experimental group
underwent a 2DG study and were either sacrificed by barbiturate overdose (n = 8) or by a
focused microwave beam (6-6.25 sec, 1.8 kW) to the head (n = 5). Animals in the second
experiment (n = 10) underwent a double-tracer (FDG/IAP) lCMRglc and rCBF study and
were sacrificed by the microwave method.
In all groups, the brains were quickly
removed after decapitation and frozen in -60°C 2-methylbutane. Serial sections of the
frozen brain were prepared for autoradiography (Experimental Groups 1 and 2) and ATP
analysis (Experimental Group 2). The UCLA Chancellor's Animal Research Committee
approved all experimental procedures.
7
Surgical Procedures
General anesthesia was induced with isoflurane (2% in 100% O2,1.5 L/min flow
rate). Body temperature was thermostatically maintained between 37.0 and 38.0 °C with
a rectal probe and heating pad (Harvard Apparatus, Holliston, MA, USA).
Once
surgical-level anesthesia was reached, animals' eyes were covered with an ophthalmic
ointment and the femoral region was shaved and scrubbed repeatedly with betadine
followed by 70 % alcohol. A 1.5 cm incision was applied over the femoral triangle and
the femoral artery and vein were isolated and cannulated with polyethylene tubing (PE50;
Becton, Dickinson and Company, Franklin Lakes, NJ, USA) for blood sampling and drug
administration, respectively.
Bupivicaine (2 mg/kg, s.c; Abbott Laboratories, North
Chicago, IL, USA) was applied to wound margins and openings were carefully sutured
closed around the cannula. Animals were secured to a cardboard plank with adhesive
tape and the anesthesia was slowly withdrawn.
Blood gases (pH, p02, PCO2) were monitored prior to the 2DG, FDG and CBF
portions of the studies with a Chiron 238 blood gas analyzer (Ciba Corning Diagnostics,
East Walpole, MA, USA). If the pC02 value were hypercapnic beyond 50 mmHg, the
study was aborted and animals sacrificed.
Experimental Group 1: [14C]-2-deoxyglucose
(2DG) Study
A 2DG bolus (30 |iCi) was administered intravenously within 30 s and timed
arterial blood samples (120 (J.1) were collected into lithium-heparin coated tubes
8
(Beckman Coulter, Fullerton, CA, USA) and stored on ice for immediate centrifugation
(14,000 rcf, 3 min). Twenty-five minutes following 2DG administration, 1.5 ml warm,
sterile Ringers solution was injected intravenously into the animal, which replenished the
blood volume lost and maintained normal mean arterial blood pressure.
Animals
sacrificed as described by Sokoloff et al. (1977) received a lethal pentobarbital dose (100
mg/kg, i.v.; Abbott Laboratories, North Chicago, IL, USA) after the last 45-min blood
collection, followed by quick decapitation and brain removal. After the 40-min time
point, isoflurane anesthesia (1% in 100% CM was re-instated to the microwave group to
maintain proper head positioning of the animal during the microwave procedure. The
animal was removed from its restraints after isoflurane induction, positioned into an
animal holder and secured in a 3.5 kW, 2.4 KHz Metabostat microwave fitted with a WR430 waveguide (Thermex-Thermatron, Louisville, KY, USA).
After the last blood
collection at 45 min, the animal was sacrificed with a focused, microwave beam directed
to the head (6-6.25 s, 1.8 kW), followed by quick decapitation and brain removal. In
both methods, the harvested brain was frozen in cold 2-methylbutane (7-8 sec, - 60°C)
within 150 s of sacrifice.
The plasma aliquots were dissolved in scintillation cocktail (BioSafe II; Research
Products International, Mount Prospect, IL, USA) and the beta decay was measured using
a scintillation counter (Beckman Coulter, Fullerton, CA, USA).
Remaining plasma
samples were frozen and later analyzed for glucose concentration using a hexokinasebased enzymatic diagnostic kit (Sigma Diagnostics, St. Louis, MO, USA).
9
The microwave brains were sectioned in a cryostat (-15°C, Leica Microsystems
Inc., Bannockburn, IL, USA).
Twenty-micron serial, coronal sections were thaw-
mounted on subbed microscope slides and air-dried. The non-microwave "fresh-frozen"
sections (-19°C) were thaw-mounted on glass cover slips and immediately heat dried on
a 60°C slide warmer. The 2DG sections were exposed to Bio-Max MR film (Eastman
Kodak Company, New Haven, CT, USA) together with calibrated [14C]-polymer
standards (Amersham Biosciences Corp, Piscataway, NJ, USA) and developed after a 24
h exposure period.
Experimental Group 2: Double Tracer[18F]-fluorodeoxyglucose (FDG)
and[14C]-iodoantipyrine (IAP) Study with ATP Analysis
The FDG study was similar to the 2DG procedures. Briefly, a 1 mCi FDG bolus
was intravenously administered and the 2DG protocol for blood sampling was followed.
Isoflurane anesthesia was re-administered at 25 min and the animals were positioned into
the microwave. After the 30-min FDG blood and blood gas collections, the CBF study
was initiated.
IAP was constantly infused intravenously by an infusion pump (35
(iCi/min; Harvard Apparatus, Holliston, MA, USA). Single, timed drops of arterial
blood, regulated to 1.5 drops/s, were collected onto pre-weighed filter papers throughout
a 50 s injection period. After the last blood collection, the animal was sacrificed by
microwave irradiation (6-6.25 s, 1.8 kW).
Due to the nature of the [18F] half-life (110 min), all FDG samples required
immediate processing upon termination of study (Moore et al. 2000).
micro waved brains were immediately sectioned in a cryostat.
The frozen,
Additional adjacent
10
sections were thaw-mounted on separate subbed slides, air-dried and stored for
subsequent ATP processing at -70°C. Within 1 h after sacrifice, the FDG sections were
exposed to Bio-Max MR film for 75 min. FDG plasma aliquots were taken for
radiolabeled tracer determination using a gamma radiation counter (COBRA II; Packard
BioScience, Meriden CT, USA).
Remaining plasma samples were frozen for later
glucose analysis. For the CBF blood samples, scintillation cocktail was added to the reweighed filter papers and the samples placed on a shaker, which assisted in maximizing
the [ CI signal by leaching the isotope from the blood to the cocktail. Two days after
development of the FDG autoradiographs (after 26 [18F] half-lives), the [14C] images
were captured on Bio-Max MR film with [14C] standards (24 h exposure) and the [14C]
plasma signal was quantified in a scintillation counter.
Quantitative ATP Bioluminescence Imaging
ATP images were obtained by a modified luciferin-luciferase method of Kogure
and Alonso (1978). The 20-|im coronal brain sections were thawed mounted on subbed
microscope slides and air-dried. A 20-|im section of ATP standard (26 nmol/mg protein)
was thawed mounted adjacent to the tissue sections and also air-dried. The slides were
then cooled in the cryostat and a 100-um section of the frozen enzyme solution was
layered over the frozen tissue and standard sections and quickly placed inside a
chemiluminescence detector (Fluor-S Max Multi-Imager; Bio-Rad Laboratories,
Hercules, CA, USA). The slide was digitally captured and integrated for 30 min. The
sample ATP values were determined from ATP standard images (0.5 to 4 mM), which
11
were generated separately for each enzyme block and data are presented as nmol ATP per
mg protein.
The enzyme block was made in a 0.2 M HEPES/0.1 M Arsenate buffer.
The
buffer was heated to 50 °C and 1 % polyvinylpyrrolidone, 2% gelatin and 1% glycerol
were added. The solution was cooled to 30°C and then 220 mg of powdered firefly
lanterns was added to 10 ml of the buffer. The mixture was centrifuged at 13,000 re/for
2 min and 20 ul of 1M MgCl2 was mixed into the supernatant. The final solution was
poured into small plastic molds (Electron Microscopy Sciences, Hatfield, PA, USA) and
frozen at -70°C.
ATP standards were made using denatured fresh brain homogenates. Fresh brain
tissue was incubated at room temperature for 30 min to rid the tissue of endogenous ATP.
The brain tissue was then denatured in a laboratory grade microwave. The tissue was
weighed and homogenized with adenosine-5'-triphosphate dissolved in 5% sterile gelatin
to the consistency of ~3.2% protein/weight.
The brain paste was packed into an
embedding capsule (Electron Microscopy Sciences, Hatfield, PA, USA) and frozen at
-70°C. To calibrate each standard, the frozen standards were released from the capsule
and portions were analyzed for ATP using a luciferin-luciferase assay (FLAA kit; Sigma
Aldrich, St. Louis, MO, USA) and for protein content (DC Protein Assay; Bio-Rad
Laboratories, Hercules, CA, USA). Previous work (not reported) determined that
microwaved brain tissue dehydrated 22% compared to fresh brain tissue.
This
dehydration effect was also factored into the standards' values. The remaining columns
were mounted onto labeled cryostat chucks and stored at -70°C. Twenty-micron sections
12
of each standard block were thawed mounted on a slide, air-dried and imaged using the
protocol described above. Five standard curves were generated per enzyme block and the
mean value for each standard was used to generate a unique standard curve for each
enzyme block. One reference standard was used as an internal reference for the samples
to account for variability in enzyme activity on a given day. Twenty-micron sections of
the normalizing standard were included on each sample slide.
Images were read for luminescence using computer-based optical densitometry
(Discovery Series Quantity One 1-D Analysis Software version 4.1.1; Bio-Rad
Laboratories, Hercules, CA, USA). The internal reference was compared to the standard
curve generated for the respective enzyme block used. The imaging process and analysis
was repeated per case until six sets of slides with normalizing standard values were used
to calculate the mean ATP value (± SEM) and reported as nmol/mg protein.
Autoradiographic Analysis
Autoradiographic films were scanned and read for optical density using a
densitometry program (NIH Image 1.61 program; (http://rsb.info.nih.gov/nih-image/,
National Institutes of Health, Bethesda, MD, USA). The lCMRglc and rCBF were
determined by the operational equations of Sokoloff et al. (1977) and Sakurada et al.
(1978), respectively. The following constants were used for 2DG (Sokoloff et al. 1977)
and FDG (Ackermann and Lear 1989) studies, respectively: (gray matter) kl = 0.19,
0.30; k2 = 0.25, 0.40; k3 = 0.052, 0.068; and LC = 0.46, 0.60. The lambda value for CBF
studies was 0.8.
13
For the first experiment, 25 regions of interest (ROIs) were selected to represent
different structures and sub-structures throughout the brain. ROIs were manually drawn
and read from each hemisphere for the two fixation methods to determine the validity and
sensitivity of the microwave method on 2DG autoradiography.
For the second
experiment, six ROIs were chosen to demonstrate the application of FDG microwave
autoradiography.
Statistical Analysis
Linear regression analysis was performed to determine similarity of lCMRglc
values calculated from 2DG autoradiography resulting from fresh-frozen and microwave
methods. Student's two-tailed t-tests were performed on lateral ROI values. Significance
was set at p < 0.05.
14
CHAPTER 3
RESULTS
Physiological and Study Variables
In Experimental Group 1, mean (± SEM) blood gas levels for fresh frozen (n = 8)
and microwave (n = 5) cases, respectively were similar and within normal physiological
range (pH = 7.47 ± 0.01, 7.45 ± 0.02; pC02 = 36.9 ± 1.5, 35.8 ± 1.6 mmHg; p02 84.9 ±
2.6, 98.0 ± 6.9 mmHg, respectively). Animals in the microwave group were under
isoflurane anesthesia for 6.5 ± 0.4 min prior to termination of the 2DG study. Mean (±
SEM) plasma glucose values were 1.86 ±0.1 and 1.88 ±0.1 mg/ml for fresh-frozen and
microwave cases, respectively. No animals from this study were excluded from further
analysis.
In Experimental Group 2 (n = 11), the first readings for blood pH, pCC>2 and p02
levels were within normal physiological range (7.43 ± 0.01; 39.5 ± 1.0 mmHg; 112.0 ±
23.8 mmHg, respectively). The second blood gas readings were taken after isoflurane
induction just prior to the CBF study and were 7.41 ± 0.01 pH, 40.0 ±1.3 mmHgpC0 2
and 249.9 ± 43.3 mmHg p02- The animals were under anesthesia for 13.0 ± 1.2 min
prior to termination after the CBF study. Mean plasma glucose level was 1.95 ± 0.06
mg/ml. One animal's lCMRglc for all six ROIs were consistently 58% lower than the
other cases and was statistically an outlier (Q = 0.59, p < 0.05). Therefore, its FDG and
corresponding CBF and ATP data were removed from further analysis.
15
Basal lCMRglc Values - Microwave Versus
Fresh Frozen 2DG Autoradiography
Mean lCMRglc values (± SEM) calculated from fresh frozen and microwave 2DG
autoradiographic cases are summarized in Table 1. There was no hemispheric, metabolic
asymmetry in any structure examined (p > 0.05). Twenty-one of 25 regions read from
microwave cases were found to be within range of the corresponding fresh-frozen
structures.
Linear regression analysis of all structures indicates that the microwave
technique on 2DG autoradiography is significantly similar to the traditional freezing
method (pi = 1.06, r2 = 0.85, p < 0.001, Table 1, Figure 1).
Representative
autoradiograms of fresh-frozen and microwave tissue are presented in Figure 2. Of the
25 regions of interest, four structures, ventromedial hypothalamus, CA dorsal
hippocampus, red nucleus and corpus callosum-splenium, were significantly different
between the two methods (p < 0.05, Student's t-test, two-tailed with unequal variance).
The areas measured for these four regions along with two additional structures, medialis
dorsalis and anterior corpus callosum, were all below 500 Jim2 (Table 1).
16
Table 1. Absolute lCMRglc for Two Fixation Methods: Fresh Frozen and
Microwave.
Region of interest
lCMRglc (umol/lOOg/min)
Area
(mm 2 )
fresh frozen
(n=8)
Mean
SEM
microwave
(n=5)
Mean
SEM
parietal cortex
6.47
93.0
4.8
94.6
2.5
dorsal hippocampus
5.95
59.2
2.3
67.0
3.1
frontal cortex
4.89
91.8
3.6
83.9
4.8
occipital cortex
4.86
93.4
3.9
100.0
3.7
temporal cortex
3.85
105.4
3.4
110.4
4.1
thalamus
3.63
95.0
3.7
98.3
3.6
globus pallidus
2.22
44.0
2.9
51.0
2.3
medial geniculate nucleus
1.67
74.0
3.4
81.1
2.8
lateral geniculate nucleus
1.56
82.5
3.5
90.5
3.3
ventral nucleus thalamus
1.55
85.7
4.0
90.6
3.4
substantia nigra reticulate
1.45
69.1
2.6
76.4
2.8
caudate/putamen
1.36
78.9
3.4
78.4
2.9
superior colliculus (superficial layer)
1.18
79.2
4.0
87.3
3.4
medial geniculate
0.94
93.4
4.3
88.8
2.9
anterior thalamus
0.93
86.6
6.2
96.0
4.6
septum
0.68
53.9
4.3
55.6
1.8
amygdaloid nucleus
0.63
85.8
5.0
78.6
3.6
CA3 dorsal hippocampus
0.61
61.3
2.4
69.3
4.8
medial dorsal nucleus of the thalamus
0.54
90.4
1.9
92.5
3.8
ventromedial hypothalamus
0.51
46.9
2.2
*59.8
3.0
medialis dorsali
0.48
92.5
6.8
100.6
6.3
CA1 dorsal hippocampus
0.46
50.8
2.0
*62.7
3.7
anterior corpus callosum
0.41
24.1
4.4
34.2
6.1
red nucleus
0.40
62.0
3.7
*83.4
2.4
corpus callosum - splenium
0.23
32.3
2.8
*45.8
3.7
* p <0.05; Student's t-test, two-tailed with unequal variance
17
120.0
y = 1.06x
R2 = 0.85
"5, 100.0
o
o
o
E
80.0 A
3
B
60.0
«
40.0
>
o
o
E
20.0 J
0.0
0.0
20.0
40.0
60.0
80.0
100.0
120.0
fresh frozen ICMRglc (umol/100g/min)
Figure 1.
Scatter plot of ICMRglc values measured from fresh-frozen and microwave-
fixed rat brains. Linear regression analysis demonstrated significant relationship between
the two fixation methods (r = 0.85).
18
Figure. 2
Representative [ C]-2-deoxyglucose autoradiograms of rat brain preserved
by (A) fresh-frozen method or by (B) microwave fixation. Twenty-micron sections were
thawed mounted onto coverslips (A) or subbed slides (B) and exposed to Kodak BioMax
MR film for 24 h.
19
Simultaneous lCMRglc, rCBF and
ATP Values of Microwave Tissue
Summarized lCMRglc, rCBF and the resulting ATP value for normal rats (n = 10)
are presented in Table 2. There was no hemispheric asymmetry in any of the structures
examined for all three metabolic studies (p > 0.05). Representative colorized lCMRglc,
rCBF and ATP densitometry images are presented in Figure 3.
r18Fl-FDG-based lCMRglc
The mean lCMRglc (± SEM) calculated from the double-label studies were 90.9 ± 5.3,
92.9 ± 8.7 and 105.4 ± 9.5 (imol/lOOg/min for parietal, occipital and temporal cortices,
respectively and 64.6 ± 2.7, 101.9 ± 3.1 and 74.4 ± 2.3 umol/lOOg/min for the subcortical structures, dorsal hippocampus, thalamus and caudate-putamen, respectively.
These values were similar to 2DG-based lCMRglc (p > 0.1) values from Experimental
Group 1.
r14C1-IAP-based rCBF
The mean rCBF (± SEM) portion of the double-label studies were 116.4 ± 9.1, 115.0 ±
9.1 and 133.5 ± 10.4 ml/lOOg/min for parietal, occipital and temporal cortices,
respectively. Dorsal hippocampus, thalamus and caudate-putamen were calculated to be
92.6 ± 6.9, 132.9 ± 9.2 and 99.7 ± 7.2 ml/lOOg/min, respectively. There were greater
variations in the rCBF portion of the study (Table 2) than for the lCMRglc.
ATP Standard curve
One set of standard blocks and 5 enzyme blocks were used during the study. The mean
slope (± SEM) was 4.4 x 106 ± 6.3 x 105 (r2 = 0.92, Figure 3D). The normalizing
20
standards used in the study had a mean variability (± SEM) of 8.0 ± 1.5% to the standard
curve.
ATP values
Mean ATP values (± SEM) for normal rat brain (n = 10) were 26.9 ± 0.7, 27.4 ± 0.8 and
27.6 ± 0.9 nmol/mg protein for parietal, occipital and temporal cortical regions,
respectively.
The sub-cortical regions (dorsal hippocampus, thalamus and caudate-
putamen) exhibited slightly higher ATP values: 30.0 ± 0.8, 31.2 ± 0.7 and 32.7 ± 0.7
nmol/mg protein, respectively.
21
Table 2. Absolute lCMRglc, rCBF and ATP Values in Rat Brain (n = 10).
Region of interest
parietal cortex
dorsal hippocampus
Thalamus
caudate/putamen
occipital cortex
temporal cortex
lCMRglc
(umol/lOOg/min)
Mean (SEM)
90.9
64.6
101.8
74.4
92.9
105.4
1.6
2.7
3.1
2.3
2.8
3.0
rCBF
(ml/lOOg/min);
Mean (SEM)
116.4
92.6
132.9
99.7
115.0
133.4
9.1
6.9
9.2
7.2
9.1
10.4
ATP
(nmol/mg protein);
Mean (SEM)
26.9
0.73
30.0
31.2
32.7
27.4
27.6
0.79
0.71
0.68
0.81
0.86
22
A
B
18
F-FDG
lCMRglc
H,
C-IAP
rCBF
200
140
t
•* *f t • i *
|imol/100g/min
u
ml/lOOg/min
D
ATP
16000000
14000000
12000000
10000000
8000000
6000000
4000000
2000000
0
nmol/mg protein
Figure 3.
10
15
20
ATP (nmol/mg protein)
Representative (A) 18F-FDG and (B) 14C-IAP autoradiograms and resulting
(C) ATP image of lCMRglc, rCBF and ATP within the same animal. (D) ATP calibration
curves were generated throughout the study and the mean value for each standard was
used to demonstrate a linear relationship between ATP concentration and optical density
(r2 = 0.92). Color scales represent the metabolic rates of lCMRglc and rCBF. Gray scale
represents ATP concentration.
23
CHAPTER 4
DISCUSSION
Previously, high-resolution autoradiography has been used in conjunction with
bioluminescent imaging to provide regional and spatial information of lCMRglc, rCBF,
and cerebral protein synthesis with tissue contents of ATP, glucose, lactate or pH and
NADH in the same animal. Various combinations of these imaging techniques have been
applied to characterize stroke prone rats (Mies et al. 1999), brain tumors (Hossmann et al.
1986), cortical spreading depression (Mies and Paschen 1984), monitor extracorporeal
circulation after cardiac arrest (Iijima et al. 1993) or determine the effects of
hypoglycemia in brain tissue (Kiesling et al. 1988). Moreover, multi-parametric imaging
has afforded insight into the pathophysiology of ischemic processes by examining interrelationships between rCBF, lCMRglc, energy state failure and protein synthesis
inhibition in the ischemic penumbra (Mies et al. 1990a, 1991, 1993; Paschen et al. 1992).
The key advantage of multi-parametric imaging is that it permits the simultaneous study
of multiple physiological parameters within the same tissue.
The high-resolution
combination of autoradiography and bioluminescence imaging not only provides more
accurate and sensitive evaluations, but further information can be garnered from pixelbased analysis. For example, Mies et al. (1990b) analyzed regional CBF/CMRglc pixel
ratios and concluded that although relative hypoperfusion persisted after global ischemia,
neither this nor decreased glucose utilization were the direct cause of selective
vulnerability within CA1 hippocampus. In fact, pixel-based analyses revealed that the
24
extent of delayed brain injury from focal cerebral ischemia was determined by the
ischemic threshold of cerebral protein synthesis rather than by the ischemic threshold of
acute energy state failure (Mies et al. 1991). Despite these advantages, high-resolution
multi-parametric imaging has not become widely used or accepted in research using
small animals. This may be due, in part, to the traditional use of in situ liquid nitrogen as
a preservation method.
Since brain metabolites are highly susceptible to enzymatic activity, rapid fixation
methods are necessary to prevent postmortem-induced changes in substrate levels (Swaab
1971; Delaney and Geiger 1996). Freeze fixation methods involve freezing the whole
animal or the decapitated head in liquid nitrogen (Mandel and Harth 1961) or pouring
liquid nitrogen directly onto the brain (funnel freezing; Ponten et al. 1973). These
techniques preserve substrates better than the decapitation-extraction-freezing method but
were found to freeze the brain's sub-cortical structures slowly (Swaab 1971), which can
falsely provide lower values.
A more efficient freezing method is the freeze-blow
technique (Veech et al. 1973), which instantaneously blows the tissue onto a freezing
plate. The freeze-blow technique does not preserve regional information however, and
thus it cannot be applied to autoradiography. As with other freezing methods, the freezeblow technique does not completely deactivate enzymes and any thawing will further
deplete metabolite pools.
In contrast, focused microwave fixation permanently inactivates enzymes (Nelson
1973; Medina et al. 1975; Hampson et al. 1982) and, depending on the efficiency of
microwave fixation, can be more effective than quick-freeze methods.
In direct
25
comparisons to quick-freeze methods, focused microwave fixation has been shown to be
far more effective in preserving in vivo levels of free fatty acids (Cenedella et al. 1975),
neuropeptides (Theodorsson et al. 1990), inositol isomers (Meek 1986) prostaglandins
(Anton et al. 1983) and, in particular, high energy phosphates (Medina et al. 1975;
Delaney and Geiger 1996) and adenosine (Clark and Dar 1988; Delaney and Geiger
1996).
As further validation of this method, Medina and colleagues (1975, 1977)
compared the application of a 6kW microwave to the freeze-blown brains of mice or
small rats (75-100 g) and demonstrated comparable ATP values (2.8 vs. 2.5 jimol/g tissue
weight, respectively).
Thermo-sensitive enzymes have been demonstrated to denature at different
temperatures (Nelson 1973; Hampson et al. 1982).
For ATP-degrading enzymes,
temperatures ranging from 70°C to 95°C have been used to preserve ATP content
(Schmidt et al. 1972; Stavinoha et al. 1973; Medina et al. 1975; Medina and Stavinoha
1977; Delaney and Geiger 1996). The ATP values presented in this study represent brain
tissue fixed with a microwave power output of 1.8 kW and an irradiation time of 6 s,
which resulted in cortical brain temperatures between 90 and 95 °C.
Under these
conditions the averaged, overall ATP content was 29.3 nmol/mg protein, which is
comparable to those reported previously by Chan et al. (1991) and Delaney and Geiger
(1996) (25 - 26 and 21 - 29 nmol/mg, respectively).
In the current study ATP images were captured using a digital camera in a light
proof box (Bio-Rad Fluoro-Max Imager), providing an image resolution of 4.5
pixels/mm. However, several factors will limit the overall spatial resolution of ATP
26
images. Firefly lanterns were used in this study to make the enzyme blocks, which may
contain other active enzymes such as myokinase. These "contaminants" can convert
ADP to ATP (Strehler and Totter 1952) leading to abnormally high ATP values. Future
studies on ATP imaging may be improved by the use of purer forms of luciferase. In
addition, a slight halo of luminescence can be detected surrounding the borders of the
imaged tissue (image not shown), which may be due to elution of ATP by the thawing
enzyme solution (Kogure and Alonso 1978). To minimize this artifact, Paschen et al.
(1981) freeze-dried their brain sections prior to imaging, thereby eliminating the need to
immerse the sections in a fixative cocktail. As an alternative, Kobayashi et al. (1989)
immobilized the reagent layer on a cover glass to inhibit the movement of enzymes. In
our own technique, ATP elution was minimized by the fact that microwaved brain tissue
was much more dehydrated than traditional quick-frozen brains.
An alternative
explanation for the halo effect may be related to our use of CCD technology. In addition
to the image resolution limitation, the use of a digital CCD camera can produce a
blooming effect, which is typified by a decaying exponential curve that is non-linearly
dependent on the light intensity. These imaging artifacts reduced the overall spatial
resolution of our ATP images to a minimum of 900 |im2 (by comparing the
bioluminescent image to the video image). Thus, future studies may improve on the
current reported techniques by transitioning to a more sensitive imaging media such as
film and using a purer form of luciferase.
Although microwave fixation is advantageous for the measurement of ATP, one
must consider the limitations of the technique for its application in autoradiography. The
27
heat caused by microwave irradiation not only dehydrates the tissue and denatures
proteins but can also disrupt cellular membranes. Meyerhoff and colleagues (1978; Kant
et al. 1979) confirmed the disruption of cellular membranes caused by microwave
irradiation when they measured dopamine in rats using quick-freeze and microwave
techniques. Microwave irradiation led to significant increases in dopamine found in
some structures and lower dopamine levels in other regions not normally seen after liquid
nitrogen fixation, despite whole brain values being the same between the two groups.
Histological examination confirmed that the microwave tissue contained ruptured and
fragmented axons and loss of fibrils. Dopamine diffusion occurred by membrane rupture
and the subsequent movement down its concentration gradient (Meyerhoff et al. 1978).
Hampson et al (1982) also verified that focused microwave irradiation ruptured
mitochondria membranes when assessing whether sub-cellular fractionation separation
can be performed post-microwave fixation.
Although mitochondrial enzymes were
irreversibly inactivated, these authors demonstrated that fixation resulted in significant
ATP and NAD+ leakage into intact tissue. Based on these prior reports, in order to apply
microwave fixation to 2DG autoradiography we had to first determine the potential loss
in resolution since the phosphorylated form of 2DG is compartmentalized in the cell.
Twenty-five ROIs of varying sizes were compared to the equivalent ROIs in fresh frozen
autoradiographs. The data does support the idea that focused microwave irradiation
affects spatial resolution. Typically high regions of 2DG uptake such as layer 4 in the
cortex and the molecular layer in the hippocampus were not as robust in the microwave
autoradiograms as they were in the fresh frozen autoradiograms (Figure 2). In general,
28
the microwave regions where the lCMRglc values differed significantly from the fresh
frozen regions were those regions with areas under -500 urn2 (Table 1), suggesting the
resolution of microwave autoradiography will be limited to regions larger than 500-900
um2. Despite these caveats, the multi-tracer techniques described in the study present a
new, powerful tool for understanding the inter-relationship between glucose metabolism,
cerebral blood flow and energy production in normal and pathophysiological states.
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30
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