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Effects of chronic dietary aluminum on local cerebral glucose utilization in rats
Neurobiologyof Aging, Vol. 15, No. 5, pp. 657-661, 1994 Copyright © 1994 ElsevierScience Ltd Printed in the USA. All fights reserved 0197-4580/94 $6.00 + .00
Pergamon 0197-4580(94)00061-1
Effects of Chronic Dietary Aluminum on Local Cerebral Glucose Utilization in Rats M. C L A U B E R G , * C. B E E B E S M I T H , t T. DANG,~" L. SOKOLOFFS" A N D J. G. JOSH1.1
*Department of Biochemistry, University of Tennessee, Knoxville, TN 37996-0840 "~Laboratory of Cerebral Metabolism, National Institute of Mental Health, U.S. Public Health Service, Department of Health and Human Services, Bethesda, MD 20892 R e c e i v e d 24 M a y 1993; R e v i s e d 4 M a r c h 1994; A c c e p t e d 7 April 1994 CLAUBERG, M., C. BEEBE SMITH, T. DANG, L. SOKOLOFF AND J. G. JOSHI. Effects of chronic dietary aluminum on local cerebral glucose utilization in rats. NEUROBIOL AGING 15(5) 657-661, 1994.--Beginning at 4 weeks of age normal, male, Sprague-Dawley rats were reared on Purina Laboratory Chow and drinking water containing 100 IxM AIC13. After 2 years, local rates of cerebral glucose utilization were determined with the autoradiographic [14C] deoxyglucose method in the brain as a whole and in 25 brain regions in 6 treated rats and 4 age-matched controls. The results indicate that any effects of chronic aluminum in the diet on rates of cerebral glucose utilization are small. In the brain as a whole, the mean rate of glucose utilization in the aluminum-treated rats was 6% lower than that of the controls (p = 0.09). In 21 of the 25 brain regions examined mean rates of glucose utilization were generally lower in the aluminum-treated rats but in none of the regions were the effects statistically significant. Aluminum
Aging
[14C]deoxyglucose
THE DEVELOPMENT of a reversible encephalopathy in dialysis patients has been linked to aluminum accumulation (1,2,3), showing that aluminum can be neurotoxic (4). Elevated aluminum concentrations in cerebral cortex from cases of Alzheimer's disease (AD) (12), Down's syndrome (11), and mentally normal elderly patients (37) and in neurofibrillary tangle-bearing neurons in cases of both AD and A L S - P D of Guam (27,28) have been reported. In addition, biochemical studies (8) have shown that aluminum may interfere with the regulation of proteolytic processing of the [3-amyloid precursor protein, a major component of the core of senile plaques. Such findings have led to the implication of aluminum in the pathogenesis of AD. AD, in contrast to dialysis encephalopathy, has a relatively slow onset which suggests that if aluminum is an etiological factor, it may be due to a gradual accumulation of metabolic errors (18). Aluminum administered chronically to rats in the drinking water results in changes in iron homeostasis which could lead to free radical damage (14,18). In addition, chronic aluminum treatment in rats results in decreases in key enzymes in the glycolytic pathway in brain (5). Activities of both hexokinase and glucose6-phosphatedehydrogenase are reduced by as much as 25% after chronic dietary aluminum. In patients, each factor may not be significant, but as suggested (8,18), a time-dependent colocalization of a "critical mass" of metabolic errors may result in disease. Numerous studies have demonstrated that in AD rates of cerebral glucose utilization are reduced (15,21,31). Normally, glucose is the sole source of energy for a mature brain (6). The supply of glucose to the brain by the cerebral circulation and its conversion to glucose-6-phosphate by hexokinase are tightly regulated. Con-
version of glucose-6-phosphate to ribose-5-phosphate by consecutive actions of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase ensures adequate supply of ribose-5phosphate for nucleotide synthesis and of NADPH for lipid synthesis. Hexokinase and glucose-6-phosphate dehydrogenase catalyze the first committed steps in glycolysis and the pentose shunt pathways, respectively. That the activities of these enzymes in brain are reduced after chronic dietary treatment of rats with aluminum suggested that brain glucose utilization may be affected by aluminum treatment. We have investigated the effects of chronic dietary aluminum in rats on local rates of cerebral glucose utilization (lCMRglc). We chose long-term feeding of aluminum-containing drinking water rather than a single intracerebroventricular (ICV) injection, which has been reported to decrease lCMRglc (24), to simulate the chronic process that might be involved in the pathogenesis of AD. METHOD
Animals Twenty male, Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) 4 weeks of age were housed in pairs in standard laboratory cages under standard conditions. Lighting was maintained at a reduced intensity with alternating 12L: 12D cycle. The animals were fed Purina Lab Chow and chlorine-treated water ad lib. The aluminum-treated group was comprised of 10 rats in five randomly selected cages. The drinking water of this group contained 100 ~M aluminum chloride; drinking water solutions were freshly prepared three times per week. The control and aluminum-treated groups were housed in the same room and cage
1 Requests for reprints should be addressed to J. G. Joshi, Department of Biochemistry, University of Tennessee, Knoxville, TN 37996---0840. 657
658
CLAUBERG ET AL.
rack. Rats were maintained in this manner for a full 2 years. Our earlier studies showed that under these conditions the activities of hexokinase and glucose-6-phosphate dehydrogenase in the rat brain are significantly reduced, and such brains have elevated levels of aluminum (5). Prior to the determination of ICMRg~c some of the rats were employed in a behavioral study (7). After 18 months of treatment 2 control and 2 aluminum-treated rats were used in a pilot study of the effects of chronic aluminum treatment on 1CMRg~¢. After 24 months of treatment, 6 of the rats developed "age-associated" malignancies, including fibromas and carcinomas, and chronic glomerulonephropathy, which necessitated euthanasia before the end of the study. No apparent correlation was observed between aluminum treatment and the onset, cause, or rate of mortality. Four control and 6 aluminum-treated rats were transported to the animal facility at NIH and allowed a week to acclimatize before the determination of 1CMRg~. Rats were placed in randomly selected cages and the identity of the rats encrypted. Rats were deprived of food for 15 h prior to surgical preparation. On the day of the experiment animals were lightly anesthetized with halothane and polyethylene catheters were inserted into one femoral artery and vein; the catheters were tunneled under the skin and exited at the nape of the neck. During the 4 h recovery period and throughout the experiment, rats were allowed to move freely in their cages.
Physiological Measurements Physiological variables were measured 10--20 min before the initiation of the procedure for determination of 1CMR9g~c to evaluate the normalcy of each animal's physiological state. Rectal temperature was monitored with a YSI Model No. 73 TeleThermometer (Yellow Springs Instrument Co., Yellow Springs, OH). Mean arterial blood pressure was measured by means of an air-damped mercury manometer attached to the femoral arterial catheter. Arterial blood pH, pCO 2, and pO 2 were measured with a Coming 158 pH/Blood Gas Analyzer (Coming Ltd.,Halstead, Essex, UK). Arterial blood hematocrit was determined in blood sampies collected in capillary tubes that were subsequently sealed and centrifuged in a Microspin 24S (Sorvall Instruments, DuPont, Wilmington, DE). Arterial plasma glucose concentrations were measured in a Beckman Glucose Analyzer 2 (Beckman Instruments, Fullerton, CA).
mCi/mmol; NEN-DuPont, Wilmington, DE). Timed arterial blood samples (50 p,1) were then collected during the following 45 min for the determination of thetime courses of the plasma concentrations of glucose and [~'*C]deoxyglucose. At the end of the 45-min period the rats were killed with a lethal intravenous dose of sodium pentobarbital. The brains were rapidly removed, frozen in isopentane cooled to -40°C with dry ice, and 20 p,m coronal sections were cut in a cryostat maintained at -20°C. The brain sections were dried on a hot plate at 60°C and autoradiographed together with calibrated [~4C] methylmethacrylate standards (Amersham Corp., Arlington Heights, IL) autoradiographed on OMC-1 film (Eastman Kodak Co., Rochester, NY) as previously described (34). Concentrations of 14C in 25 brain regions were determined by image analysis of the autoradiograms (M1, Imaging Research Inc., St. Catharines, Ontario, Canada). Values for 1CMRg~c were calculated by the operational equation of the deoxyglucose method (34). Each regional value is the average of determinations made in 4 to 15 brain sections, left and fight hemispheric values. The autoradiograms were analyzed for the mass-weighted average CMRglc in the brain as a whole with a Photoscan System P-1000 HS scanning densitometer (50 mm aperture) (Optronics International, Chelmsford, MA) and computerized image processing system (16).
Statistical Analyses Statistical comparisons between groups were carried out by means of Student's t tests. The application of the Bonferroni correction for multiple comparisons (25) requires a p ~< 0.002 for statistical significance with the analysis of 25 brain regions. RESULTS
Physiological Status At the time of the deoxyglucose study there were no statistically significant differences (at p ~< 0.05) in the physiological variables measured between the aluminum-treated and control rats (Table 1).
Average CMRgtc in the Brain as a Whole In comparison with the age-matched control rats, weighted average CMRo¢ was lower in the aluminum-treated rats but the effect was not statistically significant (Table 2).
lCMRgtc Determination of ICMRglc 1CMRglc was determined by means of the quantitative autoradiographic [14C]deoxyglucose method (34). The procedure was initiated by the administration of an IV pulse of 2-deoxy-D[~-~4C]glucose (125 mCi/kg body weight) (specific activity, 50-60
ICMRg~c was determined in 25 brain regions. The results (Table 3) are presented in order of decreasing levels of statistical significance (Student's t tests). By the Bonferoni criterion (i.e., p ~< 0.002) none of the effects of chronic aluminum treatment reached statistical significance. The largest effect,an 11.3% de-
TABLE 1 PHYSIOLOGICALVARIABLESIN CONTROLAND ALUMINUM-TREATEDRATSDURINGMEASUREMENT OF lCMRsl~ PhysiologicalVariable Body weight (g) Mean arterial blood pressure (mm Hg) Hematoerit (%) Arterial plasma glucose concentration(mmoles/ml) Arterial blood pO2 (rrtm Hg) Arterial blood pCO2 (ram Hg) Arterial blood pH
Controls 704 -- 55 107 -5 47 +- 2 7.03 -+ 0.61 86.4 --- 5.8 35.6 -- 1.6 7.45 - 0.01
Aluminum-Treated (4) (4) (4) (4) (2) (2) (2)
Values are the means - SEM for the number of animals indicatedin parentheses.
773 96 45 6.72 80.8 37.3 7.45
+- 56 + 3 -- 2 - 0.56 + 0.8 -+ 1.4 -+ 0.01
(6) (6) (6) (6) (4) (4) (4)
EFFECTS OF AICI 3 ON LCMRglc
659
their drinking water for most of their adult lives are negative. Behavioral tests of short- and long-term memory in these animals also showed no difference between the control and aluminumtreated groups (7). In parallel studies in our laboratory, aluminum levels on the brains of rats similarly treated for 1 year were twice those of normal, age-matched controls (5). In our small series of rats survival was unaffected by the aluminum treatment, and at the time of the deoxyglucose study physiological variables (Table 1) in both groups of animals were within the normal range for Sprague-Dawley rats (34). CMRgic in the brain as a whole was also unaffected by the aluminum treatment and values in both treated and control groups (Table 2) were similar to those previously reported for aged Sprague-Dawley rats in which CMRg~c was approximately 20% lower than in normal, young adult male rats (32). Twenty-five brain regions (Table 3) were selected for the regional analysis of the effects of chronic aluminum intoxication. We concentrated our analysis on regions such as prefrontal, frontal, and parietal cortex and the hippocampus because of their involvement in associative functions and memory, functions which are impaired in patients with AD. In addition, we examined brain regions involved in vision, audition, movement and olfaction. Many of these regions, particularly the primary sensory areas, exhibit reductions in 1CMRg~c during normal aging in the rat (32). In 21 of the 25 regions examined mean values for 1CMRg~c were lower in the aluminum-treated rats but in none of the regions were the effects statistically significant. Eleven of the 24 gray matter regions analyzed in the present study had also been examined in the previous study of normal aging in rats (32) and in all but two
TABLE 2 EFFECT OF CHRONIC DIETARY ALUMINUMON AVERAGERATE OF GLUCOSE UTILIZATION 1N THE BRAIN AS A WHOLE Rate of Glucose Utilization (~moles/100 g/min) Controls 67.1 ± 0.4 (4)
Aluminum-Treated 63.2 ± 1.8 (6)
Percent Change
p Value
--5.8
0.09
Values are the means ± SEM for the number of animals indicated in parentheses. crease in the aluminum-treated rats, was found in the sensorimotor cortex (p = 0.05). In several other regions, e.g., superior colliculus, ventral pallidum, CA1 molecular layer of the hippocampus, and temporal cortex, 1CMRglc values in the aluminum-treated rats were 6.8%-9.2% lower than controls but these differences did not reach statistical significance (p <~ 0.05). In 21 of the 25 brain regions mean 1CMRg~c tended to be lower in the aluminum-treated rats, while in the remaining four regions mean 1CMRglc was slightly higher in the treated animals. DISCUSSION In spite of many reports of neurotoxic effects of aluminum in both rats (13,20,29,30,35) and in man (2,17,26,33,36), the results of our studies on the effects of aluminum administered to rats in
TABLE 3 EFFECTS OF CHRONIC DIETARY ALUMINUMON 1CMRglcIN AGED RATS
1CMRsic (Ixmoles/10Og/min) Brain Region
Controls (4)
Aluminum-Treated (6)
Percent Change
p Value
Sensorimotor cortex Superior colliculus, superficialis Ventral pallidum Hippocampus, CA1, molecular layer Temporal cortex Parietal cortex Globus pallidus Hippocampus dentate gyrus Piriform cortex Lateral orbital cortex Dorsal lateral geniculate nucleus Agranular insular cortex Caudate-putamen Frontal cortex Infralimbic cortex, dorsal Auditory cortex Anterior cingulate cortex Entorhinal cortex Infralimbic cortex, ventral Caudate-putamen, rostral Corpus callosum Prelimbic cortex Nucleus accumbens Occipital cortex Medial geniculate nucleus
93.1 ± 2.4 87.6 ± 3.2 49.7 ± 0.3 98.5 ± 1.2 100.5 +-- 1.9 98.2 -+ 3.4 55.4 ± 0.6 59.4 ± 1.5 107.3 ± 1.7 104.0 ± 4.4 81.5 ± 2.0 80.9 ± 3.1 96.8 ± 1.8 84.2 ± 2.2 95.4 -+ 2.7 110.1 - 9.7 105.2 --- 1.9 62.6 -+ 3.5 74.7 ± 2.5 92.2 - 2.7 26.0--- 1.1 96.3 ± 1.3 83.2 ± 2.7 97.0 ± 1.1 101.4 ± 2.4
82.6 ± 3.3 79.6 ± 0.9* 45.8 ± 1.9 90.5 ± 4.1 92.4 ± 3.7 91.6 +-- 3.0 53.4 ± 1.2 54.9 ± 2.7 103.6 -- 2.2 110.8 _+ 4.9 78.4 ± 2.6 77.7 ± 2.4 93.0 ± 3.7 80.5 ± 3.8 90.1 ± 4.8 101.1 ± 8.4 102.6 ± 3.7 59.3 ± 5.1" 73.0 ± 1.2 90.4 -+ 3.2 26.4--- 1.2 97.6 ± 5.5 83.9 ± 3.2 96.7 ± 1.6" 101.1 ± 6.0*
-11.3 -9.2 -7.8 - 8.1 - 8.1 -6.8 -3.6 - 7.5 -3.4 6.5 -3.7 -4.0 -3.8 -4.3 - 5.6 -8.2 -2.5 -5.3 -2.3 - 1.9 1.6 1.3 0.9 -0.3 -0.2
0.05 0.09 0.10 0.12 0.13 0.18 0.22 0.24 0.26 0.37 0.43 0.43 0.46 0.49 0.50 0.51 0.61 0.62 0.70 0.71 0.79 0.86 0.87 0.89 0.97
Values are the means ± SEM for the number of animals indicated in parentheses. * Due to technical problems in these regions ICMRglc was determined in only 5 of the rats.
660
CLAUBERG E'[ A L
of the regions there is very good agreement between the values in the present study and those reported previously in normal aged rats. The two exceptions are the eaudate-putamen and the nucleus accumbens, regions rich in dopamine receptors and involved in motor function; in these regions, the measured values for ICMRglc in the present series of control rats were 10% and 14% higher, respectively, than values reported previously (32). A possible explanation for these differences is that in the present study rats were freely moving during the determination of 1CMRglc whereas in the study of aging per se (32) rats were partially immobilized. A study of the effects of partial restraint in young adult rats (10) on 1CMRglc, however, did not show decreases in 1CMRg~c in the caudateputamen and nucleus accumbens. Our results appear to be at variance with those of Lipman and Tolchard (24), who reported that aluminum treatment in rats resuits in a reduction in 1CMRs~c in selective brain regions as well as profound behavioral alterations (23). In those studies, however, the aluminum was administered either as a single ICV or IP injection of aluminum tartrate, the 1CMRg~c measurements were only relative rates that depend on the stability of the whole brain values
for their validity, and the rats were tested seven and 14 days after a single treatment. Other behavioral studies (19) of the effects of a single intracerebral injection of aluminum show that an initial acquisition deficit seen immediately following treatment is reversible. That aluminum inhibits brain hexokinase has been shown in vitro (22). Similarly, long term in vivo aluminum treatment results in a 27% inhibition of brain hexokinase activity as measured in vitro (5). Our results suggest, however, that this inhibition, if it in fact exists in the brain in vivo,has little or no importance in vivo where there is a vast excess of hexokinase (6). Our results fail, therefore, to support a neurotoxic effect of long-term treatment with aluminum in rats. ACKNOWLEDGEMENTS We thank Jane Jehle for her expert assistance in preparing the brain sections for autoradiography. This article was presented in part at the 30th Meeting of the American Society for Cell Biology, San Diego, CA, November, 1990. The research was supported in part by The Council for Tobacco Research.
REFERENCES 1. Ackrill, P.; Ralston, A. J.; Day, J. P.; Hodge, K. C. Successful removal of aluminum from patient with dialysis encephalopathy. Lancet 2:692-693; 1980. 2. Alfrey, A. C.; LeGendre, G. R.; Kaehny, W. D. The dialysis encephalopathy syndrome. New Eng. J. Med. 294:184-188; 1976. 3. Altmann, P.; Dhanesha, U.; Hamon, C.; Cunningham, J.; Blair, J.; Marsh, F. Disturbance of cerebral function by aluminum in hemodialysis patients without overt aluminum toxicity. Lancet 2:7-12; 1989. 4. Boeganm, R. J.; Bates, L. A. Neurotoxicity of aluminum. Can. J. Physiol. Pharmacol. 62:1010-1014; 1984. 5. Cho, S.-W.; Joshi, J. G. Effects of long-term feeding of aluminum chloride on hexokinase and glucose-6-phosphate dehydrogenase in the brain. Toxicology 48:61-69; 1988. 6. Clarke, D. C.; Sokoloff, L. Circulation and energy metabolism of the brain. In: Siegel, G. J.; Agranoff, B. W.; Albers, R. W.; Molinoff, P. B., eds. Basic neurochemistry, 5th ed. New York: Raven Press; 1993:645--680. 7. Clauberg, M.; Joshi, J. G. Effects of prolonged chronic dietary treatment with AIC13 on selected metabolisms and memory in rats. Ann. NY Acad. Sci. 648:289-290; 1992. 8. Clanberg, M.; Joshi, J. G. Regulation of serine protease activity by aluminum: Implications for Alzheimer's disease. Proc. Natl. Acad. Sci. USA 90:1009-1012; 1993. 9. Clanberg, M.; Smith, C. B.; Dang, T.; Sokoloff, L.; Joshi, J. G. Effects of chronic dietary aluminum on local cerebral glucose utilization in rats. J. Cell Biol. 111(5):496a; 1990. 10. Crane, A. M.; Porrino, L. J. Adaptation of the quantitative 2-[t4C]deoxyglucose method for use in freely moving rats. Brain Res. 499:87-92; 1989. 11. Crapper, D. R.; Dalton, A. J.; Skopitz, M.; Scott, J. W.; Hachinski, V. C. Alzheimer degeneration in Down syndrome. Etectrophysiologic alterations and histopathologic findings. Arch. Neurol. 32:618623; 1975. 12. Crapper, D. R.; Krishnan, S. S.; Quittkat, S. Aluminum, neurofibrillary degeneration and Alzheimer's disease. Brain 99:67-80; 1976. 13. D611ken, A. fiber die Wirkmag des Aluminum mater besorderer Berucksichtigung der dureh das Aluminum Rersursachten Lasionenin Zentrai Nervensystem. Naunyn-Schmiedebergs Arch. Exp.Pathol. Pharmakol. 40:58-120; 1897. 14. Fleming, J.; Joshi, J. G. Ferritin: Isolation of aluminum-ferritin complex from brain. Proc. Natl. Acad. Sci. USA 84:7866-7870; 1987. 15. Friedland, R. P.; Jugust, W. J.; Huesman, R. H.; Koss, E.; Knittel, B.; Mathis, C. A.; Ober, B. A.; Mazoyer; B. M.; Budinger, T. F. Regional cerebral glucose transport and utilization in Alzheimer's disease. Neurology 39:1427-1434; 1989.
16. Goochee, C.; Rasband, W.; Sokoloff, L. Computerized densitometry and color-coding of [14C] deoxyglucose autoradiographs. Ann. Neurol. 7:359-370; 1980. 17. Griswold, W. R.; Reznik, V.; Mendoza, A.; Alfrey, A. C. Accumulation of aluminum in a nondialyzed uremic child receiving aluminum hydroxide. Pediatrics 71:56-58; 1983. 18. Joshi, J. G. Aluminum, a neurotoxin which affects diverse metabolic reactions. BioFactors 2:163-169; 1990. 19. King, G. A.; DeBoni, U.; Crapper, D. R. Effect of aluminum upon conditioned avoidance response acquisition in the absence of neurofibrlllary degeneration. Pharm. Biochem. Behav. 3:1003-1009; 1975. 20. Kopelhoff, L. M.; Barrera, S. E.; Kopeloff, N. Recurrent convulsive seizures in animals produced by immunologic and chemical means. Am. J. Psychiat. 98:881-902; 1942. 21. Kuhl, D. E.; Metter, E. J.; Riege, W. H. Patterns of glucose utilization in depression, multiple infarct dementia, and Alzheimer's disease. In: Sokoloff, L., ed. Brain imaging and brain function. New York: Raven Press; 1985:211-226. 22. Lai, J. C.; Blass, J. P. Inhibition of brain g]ycolysis by aluminum. J. Neurochem. 42:438--446; 1984. 23. Lipman, J. J.; Colowick, S. P.; Lawrence, P. L.; Abumrad, N. N. Aluminum induced encephaiopathy in the rat. Life Sci. 42:863-875: 1985. 24. Lipman, J. J.; Tolchard, S. Comparison of the effects of central and peripheral aluminum administration on regional 2-deoxy-D-glucose incorporation in the rat brain. Life Sci. 45:1977-1987; 1989. 25. Miller, R. G. Simultaneous statistical inference. New York: McGrawHill; 1966. 26. Nathan, E.; Pedersen, S. Dialysis encephalopathy in anon-dialyzed uraemic boy treated with aluminum hydroxide oraUy. Acta Pediatr. Scand. 69:793-796; 1980. 27. Perl, D. P.; Brody, A R. Alzheimer's disease: X-rayspectrophotometric evidence of aluminum accumulation in neurofibrillary tanglebearing neurons. Science 208:297-299; 1980. 28. Per, D. P.; Gadjusek, D. C.; Garruto, R. M.; Yanaghihara, R. T.: Gibbs, C. J. Intraneuronal aluminum accumulation in amyotrophic lateral sclerosis and parkinsonism-dementiai of Guam. Science 217: 1053-1055; 1982. 29. Seibert, B.; Wells, H. The effects of aluminum on mammalian blood and tissues. Arch. Pathol. 8:230-262; 1929. 30. Seil, P. J.; Lambert, P. W.; Klatzo, I. Neurofibrillary spheroids induced by aluminum phosphate in dorsal root ganglianeurons in vitro. J. Neuropathol. Exp. Neurol. 28:74-85; 1969. 31. Small, G. W.; Kuhl, D. E.; Riege, W. H.; Fujikawa, J. W.; Ashford, J. W.; Metter, E. J.; Mazziotta, J. C. Ceregral glucose meta-
EFFECTS OF AIC13 ON LCMRglc
bolic patterns in Alzheimer's disease: Effect of gender and age at dementia onset. Arch. Gen. Psych. 46:527-532; 1989. 32. Smith, C. B.; Goochee, C.; Rapoport, S. I.; Sokoloff, L. Effects of ageing on local rates of cerebral glucose utilization in the rat. Brain 103:351-365; 1980. 33. Smith, D. B.; Lewis, J. A.; Burks, J. S.; Alfrey, A. C. Dialysisencephalopathy in peritoneal dialysis. JAMA 244:365-366; 1980. 34. Sokoloff, L.; Reivich, M.; Kennedy, C.; DesRosiers, M. H.; Patlak, C. S.; Pettigrew, K. D.; Sakurada, O.; Shinohara, M. The [14C]deoxyglucose method for the measurement of local cerebral glucose utili-
661 zation: Theory, procedure, and normal values in the conscious and anesthetized albino rat. J. Neurochem. 28:897-916;1977. 35. Troncosco, J. C.; Price, D. L.; Griffin, J. W.; Parhad, I. M. Neurofibrillary axonal pathology in aluminum intoxication. Ann. Neurol. 12:278-283; 1982. 36. Wills, M. R.; Savory, J. Aluminum poisoning: Dialysisencepahalopathy, osteomalacia, and anaemia. Lancet ii:29-34; 1983. 37. Wisniewski, H. M.; Iqbal, K. Aluminum-induced neurofibrillary changes its relationship to senile dementia of the Alzheimer'stype. Neurotoxicology 1:121-124; 1980.
Pergamon 0197-4580(94)00061-1
Effects of Chronic Dietary Aluminum on Local Cerebral Glucose Utilization in Rats M. C L A U B E R G , * C. B E E B E S M I T H , t T. DANG,~" L. SOKOLOFFS" A N D J. G. JOSH1.1
*Department of Biochemistry, University of Tennessee, Knoxville, TN 37996-0840 "~Laboratory of Cerebral Metabolism, National Institute of Mental Health, U.S. Public Health Service, Department of Health and Human Services, Bethesda, MD 20892 R e c e i v e d 24 M a y 1993; R e v i s e d 4 M a r c h 1994; A c c e p t e d 7 April 1994 CLAUBERG, M., C. BEEBE SMITH, T. DANG, L. SOKOLOFF AND J. G. JOSHI. Effects of chronic dietary aluminum on local cerebral glucose utilization in rats. NEUROBIOL AGING 15(5) 657-661, 1994.--Beginning at 4 weeks of age normal, male, Sprague-Dawley rats were reared on Purina Laboratory Chow and drinking water containing 100 IxM AIC13. After 2 years, local rates of cerebral glucose utilization were determined with the autoradiographic [14C] deoxyglucose method in the brain as a whole and in 25 brain regions in 6 treated rats and 4 age-matched controls. The results indicate that any effects of chronic aluminum in the diet on rates of cerebral glucose utilization are small. In the brain as a whole, the mean rate of glucose utilization in the aluminum-treated rats was 6% lower than that of the controls (p = 0.09). In 21 of the 25 brain regions examined mean rates of glucose utilization were generally lower in the aluminum-treated rats but in none of the regions were the effects statistically significant. Aluminum
Aging
[14C]deoxyglucose
THE DEVELOPMENT of a reversible encephalopathy in dialysis patients has been linked to aluminum accumulation (1,2,3), showing that aluminum can be neurotoxic (4). Elevated aluminum concentrations in cerebral cortex from cases of Alzheimer's disease (AD) (12), Down's syndrome (11), and mentally normal elderly patients (37) and in neurofibrillary tangle-bearing neurons in cases of both AD and A L S - P D of Guam (27,28) have been reported. In addition, biochemical studies (8) have shown that aluminum may interfere with the regulation of proteolytic processing of the [3-amyloid precursor protein, a major component of the core of senile plaques. Such findings have led to the implication of aluminum in the pathogenesis of AD. AD, in contrast to dialysis encephalopathy, has a relatively slow onset which suggests that if aluminum is an etiological factor, it may be due to a gradual accumulation of metabolic errors (18). Aluminum administered chronically to rats in the drinking water results in changes in iron homeostasis which could lead to free radical damage (14,18). In addition, chronic aluminum treatment in rats results in decreases in key enzymes in the glycolytic pathway in brain (5). Activities of both hexokinase and glucose6-phosphatedehydrogenase are reduced by as much as 25% after chronic dietary aluminum. In patients, each factor may not be significant, but as suggested (8,18), a time-dependent colocalization of a "critical mass" of metabolic errors may result in disease. Numerous studies have demonstrated that in AD rates of cerebral glucose utilization are reduced (15,21,31). Normally, glucose is the sole source of energy for a mature brain (6). The supply of glucose to the brain by the cerebral circulation and its conversion to glucose-6-phosphate by hexokinase are tightly regulated. Con-
version of glucose-6-phosphate to ribose-5-phosphate by consecutive actions of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase ensures adequate supply of ribose-5phosphate for nucleotide synthesis and of NADPH for lipid synthesis. Hexokinase and glucose-6-phosphate dehydrogenase catalyze the first committed steps in glycolysis and the pentose shunt pathways, respectively. That the activities of these enzymes in brain are reduced after chronic dietary treatment of rats with aluminum suggested that brain glucose utilization may be affected by aluminum treatment. We have investigated the effects of chronic dietary aluminum in rats on local rates of cerebral glucose utilization (lCMRglc). We chose long-term feeding of aluminum-containing drinking water rather than a single intracerebroventricular (ICV) injection, which has been reported to decrease lCMRglc (24), to simulate the chronic process that might be involved in the pathogenesis of AD. METHOD
Animals Twenty male, Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) 4 weeks of age were housed in pairs in standard laboratory cages under standard conditions. Lighting was maintained at a reduced intensity with alternating 12L: 12D cycle. The animals were fed Purina Lab Chow and chlorine-treated water ad lib. The aluminum-treated group was comprised of 10 rats in five randomly selected cages. The drinking water of this group contained 100 ~M aluminum chloride; drinking water solutions were freshly prepared three times per week. The control and aluminum-treated groups were housed in the same room and cage
1 Requests for reprints should be addressed to J. G. Joshi, Department of Biochemistry, University of Tennessee, Knoxville, TN 37996---0840. 657
658
CLAUBERG ET AL.
rack. Rats were maintained in this manner for a full 2 years. Our earlier studies showed that under these conditions the activities of hexokinase and glucose-6-phosphate dehydrogenase in the rat brain are significantly reduced, and such brains have elevated levels of aluminum (5). Prior to the determination of ICMRg~c some of the rats were employed in a behavioral study (7). After 18 months of treatment 2 control and 2 aluminum-treated rats were used in a pilot study of the effects of chronic aluminum treatment on 1CMRg~¢. After 24 months of treatment, 6 of the rats developed "age-associated" malignancies, including fibromas and carcinomas, and chronic glomerulonephropathy, which necessitated euthanasia before the end of the study. No apparent correlation was observed between aluminum treatment and the onset, cause, or rate of mortality. Four control and 6 aluminum-treated rats were transported to the animal facility at NIH and allowed a week to acclimatize before the determination of 1CMRg~. Rats were placed in randomly selected cages and the identity of the rats encrypted. Rats were deprived of food for 15 h prior to surgical preparation. On the day of the experiment animals were lightly anesthetized with halothane and polyethylene catheters were inserted into one femoral artery and vein; the catheters were tunneled under the skin and exited at the nape of the neck. During the 4 h recovery period and throughout the experiment, rats were allowed to move freely in their cages.
Physiological Measurements Physiological variables were measured 10--20 min before the initiation of the procedure for determination of 1CMR9g~c to evaluate the normalcy of each animal's physiological state. Rectal temperature was monitored with a YSI Model No. 73 TeleThermometer (Yellow Springs Instrument Co., Yellow Springs, OH). Mean arterial blood pressure was measured by means of an air-damped mercury manometer attached to the femoral arterial catheter. Arterial blood pH, pCO 2, and pO 2 were measured with a Coming 158 pH/Blood Gas Analyzer (Coming Ltd.,Halstead, Essex, UK). Arterial blood hematocrit was determined in blood sampies collected in capillary tubes that were subsequently sealed and centrifuged in a Microspin 24S (Sorvall Instruments, DuPont, Wilmington, DE). Arterial plasma glucose concentrations were measured in a Beckman Glucose Analyzer 2 (Beckman Instruments, Fullerton, CA).
mCi/mmol; NEN-DuPont, Wilmington, DE). Timed arterial blood samples (50 p,1) were then collected during the following 45 min for the determination of thetime courses of the plasma concentrations of glucose and [~'*C]deoxyglucose. At the end of the 45-min period the rats were killed with a lethal intravenous dose of sodium pentobarbital. The brains were rapidly removed, frozen in isopentane cooled to -40°C with dry ice, and 20 p,m coronal sections were cut in a cryostat maintained at -20°C. The brain sections were dried on a hot plate at 60°C and autoradiographed together with calibrated [~4C] methylmethacrylate standards (Amersham Corp., Arlington Heights, IL) autoradiographed on OMC-1 film (Eastman Kodak Co., Rochester, NY) as previously described (34). Concentrations of 14C in 25 brain regions were determined by image analysis of the autoradiograms (M1, Imaging Research Inc., St. Catharines, Ontario, Canada). Values for 1CMRg~c were calculated by the operational equation of the deoxyglucose method (34). Each regional value is the average of determinations made in 4 to 15 brain sections, left and fight hemispheric values. The autoradiograms were analyzed for the mass-weighted average CMRglc in the brain as a whole with a Photoscan System P-1000 HS scanning densitometer (50 mm aperture) (Optronics International, Chelmsford, MA) and computerized image processing system (16).
Statistical Analyses Statistical comparisons between groups were carried out by means of Student's t tests. The application of the Bonferroni correction for multiple comparisons (25) requires a p ~< 0.002 for statistical significance with the analysis of 25 brain regions. RESULTS
Physiological Status At the time of the deoxyglucose study there were no statistically significant differences (at p ~< 0.05) in the physiological variables measured between the aluminum-treated and control rats (Table 1).
Average CMRgtc in the Brain as a Whole In comparison with the age-matched control rats, weighted average CMRo¢ was lower in the aluminum-treated rats but the effect was not statistically significant (Table 2).
lCMRgtc Determination of ICMRglc 1CMRglc was determined by means of the quantitative autoradiographic [14C]deoxyglucose method (34). The procedure was initiated by the administration of an IV pulse of 2-deoxy-D[~-~4C]glucose (125 mCi/kg body weight) (specific activity, 50-60
ICMRg~c was determined in 25 brain regions. The results (Table 3) are presented in order of decreasing levels of statistical significance (Student's t tests). By the Bonferoni criterion (i.e., p ~< 0.002) none of the effects of chronic aluminum treatment reached statistical significance. The largest effect,an 11.3% de-
TABLE 1 PHYSIOLOGICALVARIABLESIN CONTROLAND ALUMINUM-TREATEDRATSDURINGMEASUREMENT OF lCMRsl~ PhysiologicalVariable Body weight (g) Mean arterial blood pressure (mm Hg) Hematoerit (%) Arterial plasma glucose concentration(mmoles/ml) Arterial blood pO2 (rrtm Hg) Arterial blood pCO2 (ram Hg) Arterial blood pH
Controls 704 -- 55 107 -5 47 +- 2 7.03 -+ 0.61 86.4 --- 5.8 35.6 -- 1.6 7.45 - 0.01
Aluminum-Treated (4) (4) (4) (4) (2) (2) (2)
Values are the means - SEM for the number of animals indicatedin parentheses.
773 96 45 6.72 80.8 37.3 7.45
+- 56 + 3 -- 2 - 0.56 + 0.8 -+ 1.4 -+ 0.01
(6) (6) (6) (6) (4) (4) (4)
EFFECTS OF AICI 3 ON LCMRglc
659
their drinking water for most of their adult lives are negative. Behavioral tests of short- and long-term memory in these animals also showed no difference between the control and aluminumtreated groups (7). In parallel studies in our laboratory, aluminum levels on the brains of rats similarly treated for 1 year were twice those of normal, age-matched controls (5). In our small series of rats survival was unaffected by the aluminum treatment, and at the time of the deoxyglucose study physiological variables (Table 1) in both groups of animals were within the normal range for Sprague-Dawley rats (34). CMRgic in the brain as a whole was also unaffected by the aluminum treatment and values in both treated and control groups (Table 2) were similar to those previously reported for aged Sprague-Dawley rats in which CMRg~c was approximately 20% lower than in normal, young adult male rats (32). Twenty-five brain regions (Table 3) were selected for the regional analysis of the effects of chronic aluminum intoxication. We concentrated our analysis on regions such as prefrontal, frontal, and parietal cortex and the hippocampus because of their involvement in associative functions and memory, functions which are impaired in patients with AD. In addition, we examined brain regions involved in vision, audition, movement and olfaction. Many of these regions, particularly the primary sensory areas, exhibit reductions in 1CMRg~c during normal aging in the rat (32). In 21 of the 25 regions examined mean values for 1CMRg~c were lower in the aluminum-treated rats but in none of the regions were the effects statistically significant. Eleven of the 24 gray matter regions analyzed in the present study had also been examined in the previous study of normal aging in rats (32) and in all but two
TABLE 2 EFFECT OF CHRONIC DIETARY ALUMINUMON AVERAGERATE OF GLUCOSE UTILIZATION 1N THE BRAIN AS A WHOLE Rate of Glucose Utilization (~moles/100 g/min) Controls 67.1 ± 0.4 (4)
Aluminum-Treated 63.2 ± 1.8 (6)
Percent Change
p Value
--5.8
0.09
Values are the means ± SEM for the number of animals indicated in parentheses. crease in the aluminum-treated rats, was found in the sensorimotor cortex (p = 0.05). In several other regions, e.g., superior colliculus, ventral pallidum, CA1 molecular layer of the hippocampus, and temporal cortex, 1CMRglc values in the aluminum-treated rats were 6.8%-9.2% lower than controls but these differences did not reach statistical significance (p <~ 0.05). In 21 of the 25 brain regions mean 1CMRg~c tended to be lower in the aluminum-treated rats, while in the remaining four regions mean 1CMRglc was slightly higher in the treated animals. DISCUSSION In spite of many reports of neurotoxic effects of aluminum in both rats (13,20,29,30,35) and in man (2,17,26,33,36), the results of our studies on the effects of aluminum administered to rats in
TABLE 3 EFFECTS OF CHRONIC DIETARY ALUMINUMON 1CMRglcIN AGED RATS
1CMRsic (Ixmoles/10Og/min) Brain Region
Controls (4)
Aluminum-Treated (6)
Percent Change
p Value
Sensorimotor cortex Superior colliculus, superficialis Ventral pallidum Hippocampus, CA1, molecular layer Temporal cortex Parietal cortex Globus pallidus Hippocampus dentate gyrus Piriform cortex Lateral orbital cortex Dorsal lateral geniculate nucleus Agranular insular cortex Caudate-putamen Frontal cortex Infralimbic cortex, dorsal Auditory cortex Anterior cingulate cortex Entorhinal cortex Infralimbic cortex, ventral Caudate-putamen, rostral Corpus callosum Prelimbic cortex Nucleus accumbens Occipital cortex Medial geniculate nucleus
93.1 ± 2.4 87.6 ± 3.2 49.7 ± 0.3 98.5 ± 1.2 100.5 +-- 1.9 98.2 -+ 3.4 55.4 ± 0.6 59.4 ± 1.5 107.3 ± 1.7 104.0 ± 4.4 81.5 ± 2.0 80.9 ± 3.1 96.8 ± 1.8 84.2 ± 2.2 95.4 -+ 2.7 110.1 - 9.7 105.2 --- 1.9 62.6 -+ 3.5 74.7 ± 2.5 92.2 - 2.7 26.0--- 1.1 96.3 ± 1.3 83.2 ± 2.7 97.0 ± 1.1 101.4 ± 2.4
82.6 ± 3.3 79.6 ± 0.9* 45.8 ± 1.9 90.5 ± 4.1 92.4 ± 3.7 91.6 +-- 3.0 53.4 ± 1.2 54.9 ± 2.7 103.6 -- 2.2 110.8 _+ 4.9 78.4 ± 2.6 77.7 ± 2.4 93.0 ± 3.7 80.5 ± 3.8 90.1 ± 4.8 101.1 ± 8.4 102.6 ± 3.7 59.3 ± 5.1" 73.0 ± 1.2 90.4 -+ 3.2 26.4--- 1.2 97.6 ± 5.5 83.9 ± 3.2 96.7 ± 1.6" 101.1 ± 6.0*
-11.3 -9.2 -7.8 - 8.1 - 8.1 -6.8 -3.6 - 7.5 -3.4 6.5 -3.7 -4.0 -3.8 -4.3 - 5.6 -8.2 -2.5 -5.3 -2.3 - 1.9 1.6 1.3 0.9 -0.3 -0.2
0.05 0.09 0.10 0.12 0.13 0.18 0.22 0.24 0.26 0.37 0.43 0.43 0.46 0.49 0.50 0.51 0.61 0.62 0.70 0.71 0.79 0.86 0.87 0.89 0.97
Values are the means ± SEM for the number of animals indicated in parentheses. * Due to technical problems in these regions ICMRglc was determined in only 5 of the rats.
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CLAUBERG E'[ A L
of the regions there is very good agreement between the values in the present study and those reported previously in normal aged rats. The two exceptions are the eaudate-putamen and the nucleus accumbens, regions rich in dopamine receptors and involved in motor function; in these regions, the measured values for ICMRglc in the present series of control rats were 10% and 14% higher, respectively, than values reported previously (32). A possible explanation for these differences is that in the present study rats were freely moving during the determination of 1CMRglc whereas in the study of aging per se (32) rats were partially immobilized. A study of the effects of partial restraint in young adult rats (10) on 1CMRglc, however, did not show decreases in 1CMRg~c in the caudateputamen and nucleus accumbens. Our results appear to be at variance with those of Lipman and Tolchard (24), who reported that aluminum treatment in rats resuits in a reduction in 1CMRs~c in selective brain regions as well as profound behavioral alterations (23). In those studies, however, the aluminum was administered either as a single ICV or IP injection of aluminum tartrate, the 1CMRg~c measurements were only relative rates that depend on the stability of the whole brain values
for their validity, and the rats were tested seven and 14 days after a single treatment. Other behavioral studies (19) of the effects of a single intracerebral injection of aluminum show that an initial acquisition deficit seen immediately following treatment is reversible. That aluminum inhibits brain hexokinase has been shown in vitro (22). Similarly, long term in vivo aluminum treatment results in a 27% inhibition of brain hexokinase activity as measured in vitro (5). Our results suggest, however, that this inhibition, if it in fact exists in the brain in vivo,has little or no importance in vivo where there is a vast excess of hexokinase (6). Our results fail, therefore, to support a neurotoxic effect of long-term treatment with aluminum in rats. ACKNOWLEDGEMENTS We thank Jane Jehle for her expert assistance in preparing the brain sections for autoradiography. This article was presented in part at the 30th Meeting of the American Society for Cell Biology, San Diego, CA, November, 1990. The research was supported in part by The Council for Tobacco Research.
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