Regional cerebral metabolic rate for glucose in beagle dogs of different ages

Neurobiology of Aging, Vol. 4, pp. 121-126, 1983. © A n k h o International Inc. Printed in the U.S.A.

Regional Cerebral Metabolic Rate for Glucose in Beagle Dogs of Different Ages E D Y T H E D. L O N D O N , M A S A H I R O O H A T A , H I D E N O R I T A K E I , A. W A Y N E F R E N C H A N D S T A N L E Y I. R A P O P O R T

Laboratory o f Neurosciences, Gerontology Research Center, National Institute on Aging Baltimore City Hospitals, Baltimore, MD 21224 R e c e i v e d 27 July 1983 LONDON, E. D., M. OHATA, H. TAKEI, A. W. FRENCH AND S. I. RAPOPORT. Regionalcerebralmetabolicratefor glucose in Beagledogs ofd(fiferentages. NEUROBIOL AGING 4(2) 121-126, 1983.--Regional cerebral metabolic rates for glucose (rCMl~k.) were studied in unanesthetized Beagle dogs in five age groups. Significant age-related differences did not occur in the cingulate, pyriform or visual cortices, cerebellar flocculus, corpus callosum, or cerebellar white matter. However, age-related decrements were apparent in 15 of the 22 brain regions examined. The apparent time course of age effect on rCMl~c varied among the brain regions. Most regions had significantly lower rCMRt~c at 6 years than at 1 year. Decrements of more than 25% were seen in the mammillary bodies, pons, hippocampus, superior colliculus, basis of the midbrain, temporal cortex, geniculate bodies, caudate nucleus, and superior frontal gyrus. There were no age differences in rCMRslc at 10-12 years compared with 6 years. Senescence-associated decrements (after 6 years) were noted in only 5 regions: the frontal and temporal cortices, mammillary bodies, and areas involved in sensory functions. The results indicate that rCMR~jc in the adult Beagle brain declines by midlffe, and continues to decline in some brain regions through senescence. Beagle

Cerebral metabolism

Canine brain

Aging

2-Deoxy-D-glucose

Glucose utilization

10, 17, 23, 35, 38, 39, 41], may reflect compensatory changes. We hypothesized that with minimal age-related brain pathology, compensation could sustain the adult level of rCMRslc throughout senescence. In the aged rat brain, axonal terminal degeneration [39], deterioration of pyramidal cell dendrites [9,38] and dendritic spine loss [10] have been observed. However, pathological changes such as senile (neuritic) plaques, amyloidosis, neurofibrillary tangles and granulovacuolar degeneration, some of which occur in the senescent human brain [5,43], have not been observed in the rat brain. In contrast, the aged canine brain exhibits cerebral amyloidosis [22,42] and occasional structures which resemble human neuritic (senile) plaques [21, 40, 42], as well as histopathology which is comparable to that observed in the senescent human cortex [18, 26, 27, 28]. The presence of these neuropathologic findings in the senescent canine brain suggests that rCMR~tc, a measure of functional activity, may not be sustained throughout senescence, as it is in the relatively pathology-free rat brain. We therefore thought it of interest to measure rCMR,tc in Beagle dogs of different ages. An abstract o f this paper has been published [12].

S O K O L O F F and associates have developed the 2deoxy-D-glucose method to measure metabolic rates for glucose by individual brain regions in anesthetized and awake animals [33]. In this method, radioactive 2-deoxy-D-glucose, a glucose analogue, is used to trace glucose phosphorylation by hexokinase in the brain. When cerebral glucose consumption is in a steady state, the net rate o f flux of glucose through the hexokinase-catalyzed step is assumed to equal the rate of glucose utilization. The 2-deoxy-D-glueose method has been used to demonstrate that the regional cerebral metabolic rate for glucose (rCMR,~c) is a measure of local cerebral functional activity [32], in agreement with the coupling between neuronal firing and glucose metabolism, which has been demonstrated in brain slices [16]. In awake Sprague-Dawley rats, rCMRj]~ is lower at 14-16 months than at 4-6 months. Statistically significant differences occur in the sensorimotor and parietal cortices, components of the visual, auditory and extrapyramidal motor systems, the inferior olivary and gracile nuclei, and the supragranular zone of the dentate gyrus [30]. In general, no differences are seen in rats 26-36 months of age compared with 14-16 month old rats [30]. In 12 month old Fischer-344 rats, rCMI~t~ is lower than at 3 months, but not different than at 24 or 34 months [15]. Because the median survival is 23-25 months in male Sprague-Dawley rats [ 13,14] and 27.5 months in male Fischer-344 rats [24], it appears that decrements in rCMP~c occur by midlife in rats, but not during senescence (second and third years of life). The lack of a decrement in rCMR,~ during senescence of the rat brain, despite various neurochernical and morphological sequelae of aging that occur after the first year of life [9,

METHOD A total o f 24 pedigreed breeder female Beagle dogs, aged 1, 3, 6, 10-12 or 14-16 years, were used in this study. Ten dogs from the different age groups were purchased from White Eagle Laboratories (Doylestown, PA), 13 were purchased from Hazleton Research Animals, Inc. (Cumberland, VA), and one was purchased from Laboratory Research Enterprises, Inc. (Kalamazxm, MI). The animals were main-

121

LONDON ET AL.

122 tained in the kennel at the Gerontology Research Center for at least one week before preparation for experiments. They were fed Purina dog chow and water ad lib.

Preparation of Animals During the three days prior to the experimental procedure, each dog was placed in a restraining harness for five training sessions so as to adapt it to the experimental procedure. Heart rate was monitored as an index of stress. The duration of the first session was 30 min, and subsequent sessions continued for 1 hr each. The dogs were fasted for approximately 15 hr prior to surgery. On the morning of the experiment, each dog received atropine sulfate, 0.25 rag, SC (A. J. Buck, Cockeysville, MD). Twenty minutes later, anesthesia was induced with sodium thiamylal, 11 mg/kg, IV (Surital, Parke-Davis and Co., Detroit, MI), and was maintained with methoxyfluorane (Metofane, Pitman-Moore, Inc., Washington Crossing, NJ) for approximately 20 min, while polyethylene catheters were inserted into the left femoral artery and vein. The wound was apposed with sutures, and the surrounding tissues were infiltrated with xylocaine HCI, 1% (Lidocaine, Astra Pharmaceutical Products, Worcester, MA).

Physiological Measurements Four hr after the withdrawal of anesthesia, each dog was placed in the restraining harness for 20 min prior to the experimental procedure. Blood pressures and pulse rates were recorded by connecting the arterial catheter to a strain gauge transducer (Statham Instruments Co., Hatorey, PR) which led into a paper chart recorder (Gould Recorder 2200, Gould Inc., Cleveland, OH). Immediately prior to the experimental procedure, arterial blood samples were withdrawn for determination of pH, Pao2 and Pacoo (pH-Blood Gas Analyzer, No. 213, Instrumentation Labora'tories, Lexington, M A).

Determination of the Regional Cerebral Metabolic Rates for Glucose Regional cerebral metabolic rates for glucose were measured as described by Sokoloff et al. [33]. [14C]2-deoxy-Dglucose (['4C]DG) (specific activity=55-57 mCi/mmol, New England Nuclear, Boston, MA), dissolved in 0.9% NaC1 (100 /~Ci/ml) was administered as an IV bolus. The dogs received 50 ttCi/kg body weight in an injection volume of 0.5 ml/kg body weight. Arterial blood samples were withdrawn at 14 specified times over 45 rain. They were centrifuged for assessment of plasma glucose and [~4C]DG concentration with a Glucose Analyzer II (Beckman Instruments, Irvine, CA) and a liquid scintillation counter (Model LS9000 Liquid Scintillation Spectrometer, Beckman), respectively. Counting efficiency was determined and quench correction was performed by the H-number method. This method, developed by Dr. D. L. Horrocks of Beckman Instruments, Inc., is based on comparing the Compton edge of a sample relative to the Compton edge in an unquenched standard. At the end of the experimental period, each animal was killed by a rapid injection of 0.32 ml/kg of Euthanasia solution containing 200 mg N-[2(m-methoxy-phenyl)-2-ethyibutyl-(l)]-gamma-hydroxybutyramide, 50 mg 4,4'-methylenebis (cyclohexyl-trimethyl-ammonium iodide, 5 mg tetracaine hydrochloride, 0.6 ml dimethylformamide in double distilled water, National Laboratories Corporation, Toledo, OH). The brain was removed and dissected into 22 regions,

each of which was weighed and solubilized in 1-2 ml of hyamine hydroxide (Soluene 350, Packard, Downers Grove, IL). Radioactivity in brain regions was assessed by liquid scintillation counting with Dimilume 30 (Packard) as the cocktail. An operational equation was used to calcul~tte rCMRs,~ [25]. This equation states that the rate of glucose utilization is equal to the quotient obtained when the amount of labeled product (['4C]2-deoxy-D-glucose-6-phosphate) formed during the time between ['4C]DG injection and the death of the animal, is divided by the product of the integrated specific activity in the brain and a factor termed the "lumped constant." The measured variables entered into this equation are the concentrations of "C in the dissected brain region, and the concentrations of'4C and glucose in the plasma samples taken during the experimental period. Also entered are the rate constants (k*,, k*~ and k'a) for the transport and phosphorylation of ['4C]DG, and the lumped constant. The rate constants used in these experiments were those which have been determined in the rat [33]. Although the rate constants may vary among brain regions, extending the experimental period to 45 minutes attenuates the effects of errors in these terms on calculated rCMP~,c [33]. A lumped constant of 0.558, derived for the Beagle puppy [7], was used in these calculations because the lumped constant has not been determined in adult dogs. The lumped constant is thought to be characteristic of the species of animal, but it is possible that it changes in the Beagle with development of the blood-brain barrier. Values of rCMP~tc in the present study are to be taken as relative rather than absolute measures of rCMR~,c, but they could be corrected if the lumped constant in adult dogs did not equal 0.558. An assumption of the present work was that after maturity is reached, the transfer and lumped constants in the Beagle are sufficiently invariate with age as to not affect the results. No data are available regarding this assumption.

Statistical Analyses Mean values of physiological parameters and rCMRs,c at different ages were compared by one-way analysis of variance [34]. In each case, when a significant F was obtained (p~0.05), the significance of differences between ind/vidual means at the p<~0.05 level was assessed by Duncan's new multiple range test [8]. RESULTS

Table 1 presents mean values for physiological parameters in Beagle dogs of 5 different age groups. There were no significant age differences among the groups in mean arterial blood pressure, plasma glucose concentration, and arterial blood pH, Pao2 or Paco2. Body weight was significantly higher in the 10-12 yr old dogs than in the one year old dogs (p~0.05). At the time of the experiment, heart rate was significantly higher by 24% at 10-12 years than at 6 years and 35% higher at 14--16years than at 10-12 years, F(4,19)=5.95, p ~<0.005. Heart rate fell during successive sessions, and in each session, reached a stable value after 20 min in the harness. In the 1-, 3- and 6 year old animals, heart rate on the day of the experiment was within the range observed for each dog during the last training session. In the older dogs, however, heart rate generally was higher 4 hr after surgery (Table 1) than during the last training session (87_+7.2 beats/min for 10-12 year old dogs, 106-'&-14beats/min for 14-16 year old dogs).

BRAIN GLUCOSE METABOLISM, AGING, BEAGLES

123

TABLE 1 PHYSIOLOGICALPARAMETERSIN BEAGLEDOGS OF DIFFERENT AGES Age

Body Weight (kg) Heart Rate (beats/min) Mean Arterial Blood Pressure (mm Hg) Plasma Glucose (mg/100 ml) Arterial Blood pH Arterial Blood Pao, (mm Hg) Arterial Blood Paco2 (mm Hg)

I Year (6)

3 Years (5)

6 Years (4)

10-12 Years (6)

14-16 Years (3)

8.5 + I.I 108 ± II

9.7 _+ 0.4 91 +_ 5§

10.4 ± 1.0 99 ± 7§

11.5 ± 0.5* 123 -+ 127:[:

!1.5 _+ 2.4 166 _+ 13"?~§

125 - 10

121 _+ II

118 ± 3

127 - 5

121 +_ 10

105 -+ 5

102 _+ 3

103 ±

101 -+ 4

101 _+ 7

3

7.41 _+ 0.02 85 ± 5

7.44 + 0.02 86 ± 2

7.40 +_ 0.01 84 ± 2

7.43 ± 0.02 83 -4- 2

7.39 ± 0.01 77 _ 6

32 ± 2

29 ± 3

29 ± 2

29 ± 2

28 _+ 2

Each value is the mean - S.E.M. for the number of animals indicated in parentheses. Mean values were compared by one-way analysis of variance [36]. In each case where a significant F was obtained (p~<0.05), the significance of differences between individual means at the p~0.05 level was assessed by Duncan's new multiple range test [8]. *Significant difference from i year. ?Significant difference from 3 years. ~tSignificant difference from 6 years. §Significant differences from 10-12 years.

Mean values o f rCMR~,~ for 22 brain regions are presented in Table 2. Significant age-related differences did not occur in the cingulate, pyriform or visual cortices, cerebellar flocculus, corpus callosum, or cerebellar white matter, but most brain regions showed lower rCMP~,~ at later ages. Significant age-related differences were observed in the olfactory bulb, superior frontal gyrus, temporal cortex, caudate nucleus, amygdala, hippocampus, hypothalamus, geniculate bodies, thalamus less the geniculate bodies, mammillary bodies, superior and inferior colliculi, basis o f the midbrain, pons, and medulla. (See Table 2 for details regarding the significance o f these age differences.) The apparent time course of age effects on rCMR~,c varied among the brain regions (see Fig. l); however 14 of the 15 regions that showed significant age differences had lower rCMRs~ ~ at 6 years than at I year. The most striking decrements were noted in the mammillary body (42% of r C M P ~ at l year), ports (33%), hippocampus (32%), superior colliculus (32%), basis o f the midbrain (28%), temporal cortex (28%), geniculate bodies (27%), caudate nucleus (27%), and superior frontal gyrus (26%). Lesser decrements were noted in the thalamus less the geniculate bodies (25%), inferior colliculus (24%), hypothalamus (23%) and amygdala (17%). A fall in rCMR,Ic apparently occurred in the temporal cortex and pons between 1- and 3-years with no further reduction between 3- and 6-years. In other regions, where rCMl~a c was lower at 3 years than at l year, further decrements occurred between 3 years and 6 years; examples o f this were the caudate nucleus, geniculate bodies, and the superior colliculus. In contrast to the many age differences in rCMP~oc among l- , 3- and 6-year old dogs, no age differences were seen comparing values in 10--12 y e a r old dogs with those in 6 year old dogs. In fact, only five brain regions showed significant differences when 6 year, 10-12 year and 14-16 year old groups were compared. Values of rCMl~lc were significantly

lower at 14-16 year as compared with 12 years in the superior frontal gyrus (24%), temporal cortex (23%), geniculate bodies (21%), mammillary bodies (55%), and superior colliculus (22%). In the inferior colliculus, where rCMRB]¢ was not different at 14-16 years as compared with 10-12 years, it was lower (21%) at 14--16 years as compared with 6 year. DISCUSSION Five age groups of Beagle dogs subjected to the [I*C]DG procedure did not differ significantly from one another in various physiological parameters, except for body weight and heart rate. The elevation in heart rate on the day of the experiment in 10-12 year old and 14-16 year old dogs as compared with younger dogs may reflect a greater stress response to surgery in the old dogs. However, preliminary findings in this laboratory suggest that such stress would not affect rCMI~j~. Rats subjected to immobilization stress with associated hyperglycemia, did not have substantially altered rCMRs,~ except for slight decreases in some cortical areas (E. D. London, H. Holloway, S. Nespor, and S . I . Rapoport, unpublished observations). Values of cortical rCMR~c in 1- and 3 year old dogs in the present study are similar to values obtained in 0.5-1.5 year old dogs (46_+4.44/~mol/100 g/rain) and 1.5-3 year old dogs (38+_2/~mol/100 g/min) o f unspecified species, as the product of the cerebral arteriovenous difference for glucose and cerebral blood flow [20]. However, values o f rCMRmc in the present study are considerably higher than those obtained by the [14C]DG method in 2--6 day old Beagle puppies (9.2-26 ~mol/100 g/rain in various cortical regions) [7]. This difference may reflect an increase in rCMRs~ with functional development during the first postnatal year; in this regard, a developmental increase in rCMR~a~ has been observed in

L O N D O N E T AL.

124

TABLE 2 REGIONAL CEREBRAL METABOLIC RATE FOR GLUCOSE IN BEAGLE DOGS OF DIFFERENT AGES Age 1 Year (6)

3 Years (5)

6 Years (4)

10-12 Years (6)

14--16 Years (3)

Regional Cerebral Metabolic Rate for Glucose (/zmol/100 g/min) Brain Region Olfactory bulb Superior Frontal Gyrus Cingulate Cortex Temporal Cortex Pyriform Cortex Visual Cortex Caudate Nucleus, head Amygdala Hippocampus Hypothalamus Geniculate Bodies Thalamus, less Geniculate bodies Mammillary Body Superior Colliculus Inferior Colliculus Basis of Midbrain Pons Medulla Cerebellar Flocculus Corpus Callosum, genu Corpus Callosum, remainder Cerebellar White

F(4,19), p 3.38,<~0.05 8.39,~<0.005

27 +- 2 4 6 ± 2¢

24 +- 1 44+- 3¢

23 +- 2 34 +- l*t

24 +- 1 37 ± 2*t

19 ± I* 28 +- 3*t§

2.74,>0.05 7.48,<~0.005 1.38,>0.05 1.51,>0.05 13.9,<~0.005

43 35 31 42 44

± 3 -+ 1#¢§ -4 +- 3 +- 2t¢§

36+- 6 30 +- 2* 28+- 1 38 +- 3 37 +- l*~t§

33 +- 3 26 +- 1" 26-+ 2 32 +- 1 32 +- l*t

34+- 2 29 +- 1" 32 +- 3 35 +- 4 29 +- l*t

26± 1 23 +- 2*t§ 24-+ 0.1 31 +- 5 28 +- 2*t

4.20,~0.025 12.2,~<0.005 4.17,~<0.025 19.4,~<0.005

24 22 22 37

+++±

22 20 20 32

+- 1 +- 1¢§ +- 1 +- 2*t§

20 15 17 27

19 17 18 24

18 +- 0.5** 17 +- l*t 16 ± l*t 19 +- 3*t~§

7.30,<~0.005 4.47,~<0.025 10.4,<~0.005 22.6,<~0.005 5.75,~<0.005 8.51,~<0.005 5.17,<~0.01 1.62,>0.05 1.58,>0.05

32 +- 2~:§ 43 +- 5¢ 41 -+ 2t¢§ 57 ± 3~§ 21 +- It§ 18 +- ITS;§ 17 +- 1~§ 13 +- 1 12+-2

28 +- 2 35 +- 4 34 +- 2*~t 57 +- l~t§ 18 +- 1 15 +- 1" 16 +- 1§ 23 +- 1 10+-3

24 25 28 43 15 12 14

It§ I¢§ 1~§ lt~§

± 1" +- l*t +- 1" +- 0.4*t

+- 1" +- 6* +- l*t +- 0.5*t +- 1" +- 0.4 +-- 1" 13 ± 10 7+-0.5

0.43,>0.05

7+-1

6+-I

6+-1

0.98, >0.05

19-+ 3

18+2

16 +_ I

+± ++-

1" l*t 1" l*t

24 -4- 0.5* 38 +- 3 32 ± !* 39 +- 2"* 15 +- 2* 13 +- 1" 14 -+ 0.5*t 24- 2 8+- 1 6+-0.4 18+- 1

20 ± 3*'t 17 +- l*'t§ 25 +- 2*t§ 34 +- 3*t:~ 13 ± l*t 11 = l*t 13 +- l*t 23 +- 2 7 + - 1 7±1 13+- 2

All values are the means +_ SEM for the number of animals indiciated in parentheses. Mean values were compared by one-way analysis of variance [36]. In each case where a significant F was obtained (p~<0.05),the significance of differences between individual means at the p~<0.05 level was assessed by Duncan's new multiple range test [8]. *Significant difference from one year. t'Significant difference from 3 years. $SignificanI difference from 6 years. §Significant difference from 10-12 years.

F i s c h e r - 3 4 4 r a t s [15]. A l t e r n a t i v e l y , it m a y b e related to a c h a n g e in t h e l u m p e d c o n s t a n t with d e v e l o p m e n t . I n a p r e v i o u s s t u d y o f t h e r e l a t i o n b e t w e e n age a n d c a n i n e c e r e b r a l m e t a b o l i c rate, t h e m e a n c e r e b r a l m e t a b o l i c rate for o x y g e n w a s significantly l o w e r in dogs t h a t w e r e o l d e r t h a n 3 y e a r s as c o m p a r e d w i t h g r o u p s t h a t w e r e 0.5-1 y e a r old a n d 1.5-3 y e a r s o l d [20]. A l t h o u g h c e r e b r a l b l o o d flow a n d t h e c e r e b r a l m e t a b o l i c r a t e f o r g l u c o s e did n o t differ significantly a m o n g t h e t h r e e g r o u p s o f 5 dogs e a c h , t h e v a l u e s o f t h e s e p a r a m e t e r s t e n d e d to b e l o w e r in t h e o l d e r animals. In t h a t s t u d y , t h e c e r e b r a l m e t a b o l i c r a t e s for o x y g e n a n d glucose w e r e c a l c u l a t e d as t h e p r o d u c t s o f t h e c e r e b r a l a r t e r i o v e n o u s d i f f e r e n c e s f o r o x y g e n a n d glucose, r e s p e c t i v e l y , a n d cerebral b l o o d flow as m e a s u r e d directly f r o m t h e isolated s u p e r i o r sagittal sinus. W i t h t h e t e c h n i q u e e m p l o y e d , t h e c a n n u l a t e d sagittal s i n u s d r a i n s 43% o f t h e b r a i n , primarily t h e c e r e b r a l h e m i s p h e r e s [19]. T h e s e findings, w h i c h suggest

a n age-related decline in e n e r g y m e t a b o l i s m in the c e r e b r a l c o r t e x , are in a c c o r d with the p r e s e n t o b s e r v a t i o n s o f l o w e r v a l u e s o f r C M I ~ e in c o n i c a l regions o f o l d e r Beagle dogs as c o m p a r e d with y o u n g e r dogs. T h e results o f this s t u d y indicate t h a t rCMRs, c in the f e m a l e Beagle declines b e t w e e n the ages o f 1 y e a r a n d 6 y e a r s in 12 o u t o f 19 grey m a t t e r regions. By 1 y e a r o f age, t h e d i s t r i b u t i o n o f blood flow values in the dog brain h a s a c h i e v e d its m a t u r e p a t t e r n [31]. F u r t h e r m o r e , various structural, b i o c h e m i c a l a n d e l e c t r o p h y s i o l o g i c a l p a r a m e t e r s h a v e r e a c h e d adult levels b y this time [11]. T h u s , d e c r e m e n t s in rCMR~ac t h a t o c c u r after 1 y e a r o f a g e c a n be c o n s i d e r e d to o c c u r d u r i n g a d u l t h o o d . B e c a u s e t h e m e d i a n survival age for f e m a l e Beagles in captivity is 12.5 y e a r s [1], it is e v i d e n t t h a t , as in t h e rat, d e c r e m e n t s in rCMP-~c o c c u r b y midlife. H o w e v e r , unlike t h e situation in t h e rat, w h e r e rCMl~l~ is cons t a n t in t h e last o n e - h a l f to t w o - t h i r d s o f the lifespan,

BRAIN G L U C O S E M E T A B O L I S M , A G I N G , B E A G L E S I

5O

!

I

~" ~COWCULUS

==2o tllPR)C~Pt~ ~

z~

=

_

11]

,'v'.

AGEWEARS} FIG. I. Regional cerebral metabolic rates for glucose (rCMR~c) in Beagle dogs of different ages. Each point represents the mean-+SEM for 3-6 animals per age group. The numbers of dogs in each group were as follows: I year, n=6; 3 years, n=5; 6 years, n=4; 10-12 years, n=6; 14-16 years, n=3. Each of the four brain regions in the figure showed significant age differences in rCMl~tc (p~0.05). See Table 2 for details of the statistical significance of differences between mean values for rCMR~c.

rCMRs~c in the Beagle continues to decline in some brain regions throughout the period o f senescence. Age-related differences in rCMRBtc after midlife (6 years) occur in the geniculate bodies and the inferior and superior colliculi. These brain regions are involved in visual and auditory functions, and these decrements in rCMRs~c may be secondary to decreased sensory input by way o f the sense organs. In this regard, retinal degeneration is very common in Beagles over 8 years of age [2]. Furthermore, aging Fischer-344 rats reared in a 12-hr light/dark cycle, show a progressive retinal degeneration that is associated with a decrease in rCMP~jc in the superior colliculus [29]. Alternatively, decrements in rCMRs~c in brain regions associated with vision and audition in aging Beagles may reflect specific pathology, such as Lafora-like inclusion

125 bodies. In dogs o f unspecified breeds between the ages of 1 month and 16 years, Suzuki et al. [37] noted an agedependent appearance o f abnormal intraneuronal inclusion bodies, similar to those described in Lafora-body disease [3], presenile dementia [36] and human senescence [4]. Laforalike inclusion bodies were found in all dogs older than 8 years, but not in dogs younger than 2 years. Although the inclusion bodies were disseminated throughout the brain, the nucleus o f the medial geniculate body and the molecular layer o f the superior colliculus were among the most severely affected brain regions [39]. Senescence-associated decrements in rCMP~c also occur in the mammillary bodies, superior frontal gyrus, and the temporal cortex of the dog brain. We originally believed that these decrements might reflect selective degeneration in the frontal and temporal areas o f the neocortex. In this regard, a decrease in the number of neurons, especially in layers III and IV o f the temporal and occipital lobes, had been noted in the cerebral cortex o f the aged dog [42]. Similarly, agerelated loss o f human neuronal cell bodies was greater in the frontal and temporal cortices than elsewhere [6]. Nonetheless, examination o f neuronal densities in brain regions from the same dogs used in the "~resent study revealed no relation o f density with age except in the superior colliculus and cingulate cortex [5a]. It therefore appears that age differences in r C M R ~ do not occur only when cell number decrease, but may reflect more subtle structural or functional alterations. The cerebral cortices o f aged dogs of mixed breeds exhibit neuritic plaques, the ultrastructure of which resembles human plaques, except for the absence o f paired helical filaments [42]. Examination of the brains from Beagle dogs in the present study, however, did not reveal neuritic plaques in the cerebral cortex or elsewhere [5a]. Thus, although agerelated decrements in rCMR~c m a y reflect brain pathology, the neuritic plaque does not appear to be a correlate of decreased rCMR~c in the Beagle. The present findings demonstrate senescence-associated decrements in rCMR~c o f the Beagle that generally are not related to regional neuronal densities or the presence of neuritic (senile) plaques. These rCMR~c deficiencies may not entirely reflect primary brain pathology, but possibly reflect an age-related structural abnormality, such as a loss o f dendritic spines, that could influence cerebral function.

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