Application of focused microwave irradiation for autoradiography: Simultaneous measurements of glucose metabolism, cerebral blood flow and ATPкод для вставкиСкачать
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 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 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). 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