Aging potentiates the acute and chronic neurological symptoms of pyrithiamine-induced thiamine deficiency in the rodent

Behavioural Brain Research 119 (2001) 167– 177 www.elsevier.com/locate/bbr

Research report

Aging potentiates the acute and chronic neurological symptoms of pyrithiamine-induced thiamine deficiency in the rodent Shane R. Pitkin, Lisa M. Savage * Beha6ioral Neuroscience Program, Department of Psychology, State Uni6ersity of New York at Binghamton, Binghamton, NY 13905, USA Received 19 June 2000; received in revised form 22 September 2000; accepted 26 September 2000

Abstract The present study aimed to assess the role of advanced age in the development and manifestation of thiamine deficiency using an animal model of Wernicke-Korsakoff syndrome (WKS). Interactions between pyrithiamine-induced thiamine deficiency (PTD) and age were examined relative to working memory impairment and neuropathology in Fischer 344 rats. Young (2 – 3 months) and aged (22–23 months) F344 rats were assigned to one of two treatment conditions: PTD or pair-fed control (PF). Rats in the former group were further divided into three groups according to duration of PTD treatment. Working memory was assessed with an operant matching-to-position (MTP) task; after testing, animals were sacrificed and both gross and immunocytochemical measures of brain pathology were obtained. Aged rats exhibited acute neurological disturbances during the PTD treatment regime earlier than did young rats, and also developed more extensive neuropathology with a shorter duration of PTD. Aged rats displayed increased brain shrinkage (smaller frontal cortical and callosal thickness) as well as enhanced astrocytic activity in the thalamus and a decrease in ChAT-positive cell numbers in the medial septum; the latter two measures of neuropathology were potentiated by PTD. In both young and aged rats, and to a greater degree in the latter group, PTD reduced thalamic volume. Behaviorally, aged rats displayed impaired choice accuracy on the delayed MTP task. Regardless of age, rats with lesions centered on the internal medullary lamina of the thalamus also displayed impaired choice accuracy. Moreover, increased PTD treatment duration led to increased response times on the delayed MTP task. These results suggest that aging does indeed potentiate the neuropathology associated with experimental thiamine deficiency, supporting an age coupling hypothesis of alcohol-related neurological disorders. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Rat; Aging; Pyrithiamine-induced thiamine deficiency; Delayed matching-to-position

1. Introduction The mammalian central nervous system undergoes change as a result of normal aging. Evidence suggests that a variety of mechanisms may be compromised in the senescent central nervous system and may increase the vulnerability of the aged brain to neurotoxicity [1]. A number of neurological disorders appear with increased frequency as age increases; among them are the disorders associated with chronic alcohol exposure, alcohol-induced dementia (AID) and Wernicke-Kor* Corresponding author. Tel.: +1-607-7774383; fax: + 1-6077774890. E-mail address: [email protected] (L.M. Savage).

sakoff syndrome (WKS) [2,3]. Theories explaining the development of alcohol-related neurological disorders in humans have invoked interactions between neurotoxic events and aging and include variations on an ‘age coupling’ hypothesis, suggesting that alcohol-related neurotoxicity results in dementia and brain dysmorphology when coupled with aging or, alternately, that alcohol abuse accelerates age-related changes leading to more severe cognitive deficits and neuropathology [4– 15]. Animal models of AID have supported an age coupling hypothesis — aged rats exposed to chronic ethanol have increased neuropathology and greater behavioral dysfunction relative to young rats exposed to the same treatment [16 –18]. However, research using

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animal models to study the interactions between thiamine deficiency and aging has been relatively scarce, which is surprising given the evidence implicating thiamine deficiency in the etiology of both AID and WKS [3,19,20]. The most common animal model of thiamine deficiency and WKS is the nonsurgical rodent model termed pyrithiamine-induced thiamine deficiency (PTD; see [21]). In this model, rats are free-fed thiamine-deficient chow and given daily injections of pyrithiamine hydrobromide, whose high rate of absorption into the tissues of the central nervous system leads to a rapid depletetion of thiamine in the brain [22]. This treatment causes a sequence of acute neurological disturbances (loss of the righting reflex, ataxia, convulsions, and tonic –clonic seizure) until thiamine is restored (‘reversal’). The range of chronic brain pathology (neurochemical alterations and lesions) which persists after extended PTD treatment is remarkably consistent with that of WKS [21,23 – 27]. However, there is not an exact correspondence between rodent model and human condition. PTD-induced lesions are symmetrically distributed in the thalamus (midline intralaminar, mediodorsal lateral, and posterior nuclear groups), mammillary bodies, pontine tegmentum, and periaqueductal gray [27 –30]. Particularly prominent are lesions of the internal medullary lamina (IML), with which the most severe behavioral deficits are associated [22,28,31,32]. A number of differences between the model and WKS appear to exist, e.g. a lack of evidence of cholinergic cell loss in the forebrain of PTD-treated rats [21] and contradictory evidence for hippocampal damage [19]. One factor which may contribute to this discrepancy is the age at which the insult occurs. Most studies using the PTD model have been conducted in young male rats (3 months), whereas WKS is most often diagnosed in middle-aged and older adults [3]. Deficits in learning and memory resembling those associated with aging and WKS patients have been documented in the PTD model [29,31,33,34]. In the present study, working memory deficits were assessed using the operant version of matching-to-position (MTP). This procedure has been shown to be sensitive to the effects of aging [35,36] and variations of this task are notably sensitive to PTD [32,37 – 39]. The current study aimed to examine the interactions between age and experimental thiamine deficiency in the Fischer 344 rat with respect to working memory performance as well as gross neuropathology. Analysis of the latter involved neuropathological measures sensitive to both PTD [28,32] and aging [40,41]. It was hypothesized that age would intensify PTD-induced neuropathology and behavioral dysfunction, thus supporting an age coupling hypothesis of alcohol-related neurological disorders.

2. Method

2.1. Subjects A total of 68 naive adult male Fischer 344 rats, both young (2–3 months at start of experiment; N= 29) and aged (22 –23 months at start of experiment; N=39), were used in this study. They were housed two per cage with unlimited access to water and Purina rodent chow (prior to food restriction; see below) in a colony room with a 12:12 h light:dark cycle (onset at 0700 h).

2.2. Treatment Animals in each age group were first randomly assigned to one of the following treatments (see [42]: pair fed control (PF; young, n=5; aged, n=10); (a) PTD, reversal injection prior to seizure activity at indication of loss of righting reflex (LRR; young, n= 8; aged, n= 11); (b) PTD, reversal injection at onset of seizure activity (Early Acute Seizure or EAS; young, n= 8; aged, n= 9); or (c) PTD, reversal injection after 3–5 h of seizure activity (Middle Acute Seizure or MAS; young, n= 8; aged, n= 9). Subjects in the PTD groups were free-fed a thiamine-deficient chow (Teklad Diets, Madison, WI) and given daily injections (0.25 mg/kg, i.p.) of pyrithiamine hydrobromide (Sigma, St. Louis). From days 13 to 16 of treatment, animals in the EAS and MAS groups displayed signs of local tonoclonic movement of the front and hind limbs, and generalized convulsions (seizures). At reversal, animals were given an injection of thiamine (100 mg/kg, i.p.) and returned to regular chow; seizure activity disappeared within 8–12 h and upright posture was regained. The PF subjects were fed an amount of thiamine deficient chow equivalent to the average amount consumed by the PTD subjects on the previous day of treatment (i.e. to equate weight loss) and were given daily injections of thiamine (0.40 mg/kg, i.p.). All animals were allowed 8 weeks to recover. Prior to the onset of behavioral testing, the subjects’ body weights were gradually reduced to  85% of their free-feeding weights by restricted feedings. Their weights were maintained at this level for the duration of behavioral testing by supplemental feedings of rodent chow (15 g) in their home cages at the conclusion of each experimental session.

2.3. Apparatus The training and testing sessions were conducted in commercial small animal operant conditioning chambers (29× 24×30 cm), each enclosed within a soundattenuating chest (Med Associates, St. Albans, VT). The side walls of each chamber were made of Plexiglas, whereas the ceiling and front and back walls were constructed of aluminum. An aperture (5× 5 cm) for

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accessing the food/water dispenser was centrally located on the front wall 1.6 cm above the grid floor. A photobeam sensor was positioned in the food/water aperture to detect head entry. The two response levers (5 cm wide) were located 3 cm to either side of the food hopper and 6.3 cm from the grid floor. A panel light was located 6.5 cm above the center of the aperture containing the food/water dispensers. General illumination was provided by a houselight located in the middle of the back wall opposite the food/water dispenser, 24 cm above the grid floor. Experimental events were controlled and data collected by an IBM-compatible PC (Gateway 486) and Med PC interface and software (Med Associates).

2.4. Procedure 2.4.1. Pretraining Training was initiated with three sessions of habituation to the experimental chamber. During this phase, the subjects were placed in the chambers for 45 min with the house light activated. During sessions four and five the rats were placed on a standard autoshaping procedure, in which either the left or right lever (randomly determined) was extended after the 60 s intertrial interval (ITI) for 8 s and then retracted. Immediately following the retraction of the lever either a 45 mg food pellet (BioServ, Frenchtown, NJ) or 0.1 – 20% sucrose water (randomly determined) was presented. Both lever extension and food presentation were independent of the animal’s behavior. Each session consisted of 48 trials. Next, the rats were trained to lever press. A total of 60 s after the onset of the session, either the left or right lever (randomly determined) was extended. Once the extended lever was pressed, either reinforcer was presented (randomly determined). Each session included 48 trials with a VI-32.5 s ITI. The rats were rewarded on a FR-1 schedule for two sessions. Animals that did not engage in lever pressing under that schedule were returned to the autoshaping procedure for one session, after which the animals were transferred back to the FR-1 schedule. Once the rats had learned to lever press on the FR-1 schedule, they were trained on a FR-2 schedule for two sessions. 2.5. Matching-to-position (MTP) MTP training was comprised of two phases, a sample phase and a choice response phase. During the sample phase either the left or right lever was extended (semirandomly determined), which the animal was required to press. The subject had 10 s to respond to the sample lever (limited hold); if the lever was not pressed within 10 s, the lever was retracted and a VI-30 s ITI was initiated. After the ITI, the same sample lever was

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again inserted into the chamber. Surpassing the limited hold was not counted as an error trial. Once the lever was pressed in the sample phase (register of sample) it was retracted and the panel light was turned on. The panel light remained on until a nose poke was made into the food/water aperture, at which time the panel light was turned off and both levers were extended for the choice response phase. The animal was required to press the lever that had been extended in the sample phase. The trial was recycled if the limited hold was surpassed. Once the rat made a choice, both levers were retracted. If a correct choice was made the panel light was again turned on and remained on during the duration of the reinforcer delivery. In the event of an incorrect choice all lights, including the house light, were turned off for 10 s. All error trials were repeated until the animal made the correct choice. Each trial with an error, including repeated trials, was scored as incorrect; this was done to control for side-biased responding. Each MTP session included 60 trials, and this phase of training was concluded when each animal achieved 90% or greater accuracy for three consecutive sessions. Once the rats had reached criterion on the MTP procedure, delayed matching-to-position (DMTP) was begun. DMTP training was identical to the MTP procedure except that a delay of 0, 4, 8, 16, 32, or 48 s (randomly selected) was introduced between the conclusion of the sample phase and the beginning of the choice response phase, and the number of trials per session was increased to 70. If all trials were not completed within 75 min the session was terminated. After the presentation of and response to the sample lever, the delay interval was initiated. During the delay interval the panel light was turned on and, as during MTP training, the rat was required to make a nose poke into the food hopper. The first nose poke after the completion of the scheduled delay period terminated the delay period, at which time the panel light was turned off and both levers were extended into the chamber (choice response phase). The time from the beginning of ‘set delay’ (0, 4, 8, 16, 32, or 48 s) to the time each subject made a response during the choice phase was recorded as ‘true delay,’ which provided an assessment of vigilance and persistence. As in MTP training, a correct choice resulted in random pellet or sucrose water reinforcement, whereas an incorrect choice resulted in the panel light being turned off for 10 s followed by a repeat of the previous trial. DMTP consisted of 10 sessions with a VI-37 s ITI.

2.6. Histology 2.6.1. Tissue fixation and histological analyses Once behavioral testing was concluded, animals were anesthetized with Nembutal ( 2.0 ml/kg, i.p.) and

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perfused transcardially using 0.9% saline solution and 4% phosphate-buffered paraformaldehyde. Their brains were removed, post fixed in a 10% formalin solution for at least 72 h, then transferred to a 30% sucrose solution. Frozen coronal sections (40 m) were cut from the level of the anterior commissure to the level of the posterior pontine tegmentum. Every fifth section was stained with cresyl violet and the degree of gross pathological damage to thalamic structures was determined. Callosal, frontal cortical, and thalamic measures, known to be altered by PTD [28,32], were also recorded. Measures of medial callosal (CC) thickness and of frontal cortical (FR1) thickness (from the top of the cingulum in the right hemisphere) were taken at interaural 8.60 mm (using as landmarks the pointed cingulum, bulbous septal area, and anterior commissure). Intraventricular distances (from the roof of the ventral third ventricle to the floor of the dorsal third ventricle) were recorded at interaural 7.20 mm (IVD1) and 6.44 mm (IVD2; in both cases using as a landmark the shape of the hippocampal formation). All interaural locations are approximate and judged according to a standard atlas [43]. Pathology was evaluated using video images taken from a Nikon Optophot-2 light microscope using a Polaris CCD camera, digitized by a digital frame grabber card (Scion), and analyzed using an image analyzer program (IMAGE v.1.61, NIH, Bethesda, MD) on a Power Macintosh G3/253 MHz computer. All quantification was performed by two blind raters.

2.6.2. Immunocytochemical analyses Sections were also stained for choline acetyltransferase (ChAT) at interaural 9.20 mm (using as landmarks the shape of the septal area and position of the anterior commissure relative to the lateral ventricles) and for glial fibrillary acidic protein (GFAP) at interaural 6.20 and 4.70 mm (using as a landmark the shape of the hippocampal formation; all interaural locations approximate; see [43]). Using the multi-well technique, two brain sections from six subjects in each group were initially washed three times for 10 min in either cold phosphate buffer (PB; pH 7.3) for ChAT or cold phosphate-buffered saline (PBS; pH 7.4) for GFAP. This was followed by the tissue being quenched in a hydrogen peroxide solution (0.3%) for 20 min. After the quench, the tissue was blocked in either goat (GFAP) or rabbit (ChAT) serum for 60 min. The free-floating sections were then incubated (at 4°C) in the primary polyclonal antibody (Incstar rabbit anti-GFAP, 1:2000; or Chemicon goat antiChAT; 1:100) for 24 h. After incubation, the tissue was washed in PB/PBS followed by incubation (1 h) in secondary antibody (Vector biotinylated anti-rabbit

[GFAP] or anti-goat [ChAT IgG]). Following additional washes in PB/PBS the tissue was processed for 1 h with avidin-biotin complex (ABC; Vector Labs). Lastly, the tissue underwent a DAB reaction (Sigma, D 7304). The tissue was then mounted and allowed to dry overnight, and coverslipped the next day. Tissue from a positive control (non-treated young rat) was included in each assay to check for consistency. ChAT-labeled cells were counted at 200× magnification in the medial septal nucleus (see [44,45]). Cells were counted bilaterally using multiple non-overlapping fields within a triangular region (1.5× 1.5×1.5 mm) encompassing the medial septal nucleus. Only cells with distinct cytoplasmic nuclei were counted. Density of GFAP staining within the area of the thalamus was subjectively rated at two interaural locations per subject on a scale of 1–6 (least dense to most dense), and the mean density for each subject was recorded. All procedures were performed by two blind raters.

3. Results

3.1. PTD treatment Acute treatment data are summarized in Table 1. The time from first pyrithiamine injection to first sign of seizure activity was recorded for both young and aged EAS and MAS animals and analyzed with a one-factor ANOVA. Aged animals exhibited seizure activity significantly earlier than did young animals [F(1, 39)= 8.19, PB 0.01]. Percentage of weight lost between the first day of treatment and the time of reversal for each subject was analyzed with a two-factor ANOVA. The age × treatment duration interaction was significant [F(3, 71)=6.82, PB 0.01]; aged animals displayed a greater percentage of weight loss than did their young counterparts at every level of PTD treatment (LRR, EAS, and MAS).

Table 1 Acute treatment data Group

Mean time to seizure (h)a

SE

Percent body weight lostb

SE

YLRR YEAS YMAS ALRR AEAS AMAS

– 337.750 331.681 – 315.611 315.188

– 7.535 3.330 – 4.900 7.721

15.085 16.250 18.427 16.734 17.142 18.847

0.954 0.725 1.076 0.476 0.789 0.659

a

Significant effect of age. Significant effects of treatment duration, age×treatment duration. b

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Fig. 1. Examples of normal and IML-lesioned thalami from each age and treatment group, as they appear in cresyl violet photomicrographs.

3.2. Gross pathology and brain measurements Fig. 1 provides examples of cresyl violet-stained sections displaying both IML-spared and IML-lesioned thalami. Animals were rated IML-lesioned when significant tissue damage specific to the IML region was observed in multiple adjacent sections. Among the young animals, no IML lesions were found in either the LRR group (0/8) or the EAS group (0/8), but 37.5% (3/8) of the animals in the MAS group had IML lesions. Among the aged animals, no IML lesions were found in the LRR group, but 50.0% (4/8) of the rats in the EAS group and 71.4% (5/7) of rats in the MAS group exhibited IML lesions. A chi square test indicated that aged PTD-treated rats had a higher incidence of IML lesions than young PTD-treated rats [ 2(1)= 5.55, PB 0.05]. Note that tissue integrity in one of the aged EAS animals and two of the aged MAS animals was not sufficient to conclusively determine IML lesion status; these subjects were therefore excluded from the analysis. Table 2 displays mean callosal, frontal cortical, and thalamic measures. Corpus callosum (CC) measures were analyzed with a two-factor ANOVA, with aged animals displaying significantly smaller measures of callosal thickness than young animals [F(1, 51)= 9.36, PB 0.01]. A two-factor ANOVA analyzing frontal cortical (FR1) measures also revealed a main effect of Age [F(1, 51)=7.13, PB0.05], again with smaller measures in the aged animals than in the young. Although there was a trend for aged animals to exhibit progressively smaller measures with duration of treatment, Age× Treatment Duration interactions were not significant for either callosal [F(3, 51)= 0.39, P =0.76] or frontal cortical [F(3, 51)=0.98, P =0.41] measures. The two separate measures of intraventricular distance (IVD) were each analyzed with a two-factor ANOVA. IVD measures decreased with Treatment Du-

ration at both interaural 7.20 mm (IVD1) [F(3, 41)= 6.16, PB 0.01] and interaural 6.44 mm (IVD2) [F(3, 47)=7.91, PB 0.01]. Additionally, at 6.44 mm, an Age ×Treatment Duration interaction was found, with aged animals exhibiting significantly greater decreases with duration of treatment than young [F(3, 47)= 5.11, PB0.01].

Table 2 Histological measures: corpus callosum, frontal cortex, intraventricular distance (see text) Mean measure (mm) 9 SE Group

CCa

FR1a

IVD1b

IVD2c

Young PF

0.395 90.014

1.936 90.024

2.384 9 0.075

2.840 90.074

Young 0.381 PTD-LRR 90.012

1.876 9 0.019

2.340 90.077

2.765 9 0.060

Young 0.401 PTD-EAS 9 0.014

1.901 90.038

2.303 9 0.075

2.889 90.046

Young 0.375 PTD-MAS 90.011

1.930 90.034

2.139 90.141

2.680 9 0.117

Aged PF

0.368 9 0.020

1.877 90.035

2.552 9 0.088

2.905 90.057

Aged 0.364 PTD-LRR 90.015

1.874 90.041

2.732 90.084

2.939 9 0.049

Aged 0.355 PTD-EAS 90.008

1.822 9 0.041

2.233 90.149

2.548 9 0.102

Aged 0.332 PTD-MAS 90.018

1.810 9 0.038

2.118 90.123

2.321 9 0.131

a

Significant effect of age. Significant effect of treatment duration. c Significant age×treatment duration interaction. b

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3.4. Beha6ior 3.4.1. Acquisition of matching-to-position Trials to criterion for the MTP task were analyzed with a two-factor ANOVA. Neither advanced Age [F(1, 46)= 2.39, P= 0.13] nor Treatment Duration [F(3, 46)= 1.42, P= 0.25] affected the number of trials to reach criterion; likewise, there was no significant Age× Treatment Duration interaction [F(3, 46)=0.89, P= 0.46]. The mean number of trials to reach criterion was 599.17 (SE9 67.78) for the young animals and 757.12 (SE963.62) for the aged animals.

Fig. 2. Above, shaded region represents area in which medial septal ChAT cell counts were taken (approximately interaural 9.20 mm); below, mean counts for each age and treatment group. Significant effects of Age [F(1, 40) =5.00, PB 0.05], Treatment Duration [F(3, 40)=4.52, P B0.01].

3.3. Immunocytochemistry 3.3.1. ChAT ChAT-positive cells were counted in the medial septal area, and analysis was accomplished using a two-factor (Age, Treatment Duration) ANOVA. Significant effects of Age [F(1, 40)=5.00, P B 0.05] and Treatment Duration [F(3, 40)=4.52, P B0.01] were found, with cell counts decreasing from young to old and from PF to MAS progressively. Furthermore, planned comparisons revealed no effect of Treatment Duration among the young animals [F(3, 20)= 0.84, P = 0.49] but a significant Treatment Duration effect among the aged animals [F(3, 20)=6.88, P B 0.01]. This suggests that the Treatment Duration effect was driven primarily by the latter group. This trend is illustrated in Fig. 2. 3.3.2. GFAP A two-factor ANOVA was used to analyze the averaged ratings. A main effect of Age indicated that aged animals displayed a significantly greater density of GFAP in the thalamus than did young animals [F(1, 87) =21.80, P B0.01]. A main effect of Treatment Duration revealed that the expected trend of increased GFAP density from PF to LRR to EAS to MAS groups was indeed significant [F(3, 87)= 7.81, PB 0.01]. Planned comparisons revealed no effect of Treatment Duration among the young animals [F(3, 43)= 1.64, P= 0.19] but a significant Treatment Duration effect among the aged animals [F(3, 44) = 7.43, P B 0.01]. Fig. 3 displays mean thalamic GFAP ratings and illustrates this trend.

3.4.2. Delayed matching-to-position True delays (i.e. time from the beginning of ‘set delay’ to subject’s response) and choice accuracy means were calculated and averaged for sessions 1 and 2 (Block 1), sessions 3 and 4 (Block 2), sessions 5 and 6 (Block 3), sessions 7 and 8 (Block 4), and sessions 9 and 10 (Block 5) and analyzed with two-between subjects (Age, Treatment Duration), two-within subjects (Block, Delay) repeated measures ANOVAs. True-delay means and standard errors are provided by Table 3. True delays of the aged animals were significantly greater than those of the young animals [F(1, 55)=39.20, PB0.01]. An effect of Treatment Duration [F(3, 55)= 4.06, PB 0.05], with PF animals’ true delays shortest and those of MAS animals longest, was also found, in addition to an Age× Treatment Duration interaction [F(3, 55)=3.04, PB 0.05]. Finally, there was an expected decrease in true delays across Blocks [F(4, 220)= 15.84, P B0.01] and a Block× Age interaction [F(4, 220)=9.52, PB 0.01]. Fig. 4 provides mean choice accuracy for each age and treatment group. There was a main effect of Age [F(1, 55)= 9.59, PB 0.01], with young rats having

Fig. 3. Mean GFAP ratings (on a scale of 1 – 6) for each age and treatment group, averaged across brain levels (approximately interaural 6.20 and 4.70 mm). Significant effects of Age [F(1, 87) = 21.80, P B0.01], Treatment Duration [F(3, 87) = 7.81, PB 0.01].

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Table 3 True delay means 9 standard errors (s) for DMTP task Group

YPF YLRR YEAS YMAS APF ALRR AEAS AMAS

Set delay (s) 0

4

8

16

32

48

0.325 90.034 0.637 90.073 0.508 90.084 0.249 90.047 0.362 90.042 0.405 90.083 0.661 90.125 0.633 90.085

4.98490.179 5.03890.155 5.32790.265 6.305 90.553 6.72190.642 6.29190.520 15.600 94.051 7.38791.280

9.325 9 0.216 9.679 9 0.227 10.208 9 0.430 10.850 9 0.676 12.223 9 0.723 13.809 9 1.365 17.391 9 2.033 40.407 9 20.326

17.830 9 0.264 18.078 90.245 18.525 90.449 19.551 90.870 22.022 90.881 29.377 9 4.027 23.900 91.225 41.559 915.196

34.207 9 0.233 34.985 90.354 35.342 90.295 35.247 90.435 47.449 93.706 46.508 9 2.830 51.483 93.601 56.177 95.827

50.586 9 0.291 51.392 90.333 51.214 9 0.353 52.213 90.403 65.621 9 2.910 74.311 9 4.843 64.120 92.754 99.455 917.972

higher choice accuracy than aged rats. Both young and aged animals showed significant improvement across Blocks [F(4, 220)=54.21, P B0.01], though to a lesser extent in the aged group as illustrated by a Block× Age interaction [F(4, 220)=3.60, P B0.01]. Additionally, a Block× Delay interaction [F(20, 1100)= 13.09, PB 0.01] indicated that the improvement across sessions was delay-dependent. As expected, performance consistently decreased with increasing Delay interval [F(5, 275) = 240.29, PB 0.01], again to a significantly greater degree in the aged group as revealed by a Delay× Age interaction [F(5, 275)=11.56, P B0.01]. However, no significant effect of Treatment Duration on DMTP choice accuracy was observed. Although callosal, frontal cortical, and thalamic measures did not correlate with either true-delay response times or choice accuracy in the DMTP task (all P \ 0.11), a one-between subjects (Lesion Status, i.e. IML-lesioned versus IML-spared versus control), twowithin subjects (Block, Delay) repeated measures ANOVA revealed a main effect of Lesion Status on choice accuracy [F(2, 59)= 3.22, PB 0.05]. This result emphasizes the importance of IML lesion status (in lieu of duration of treatment) on behavioral impairment among PTD animals.

4. Discussion This study represents the first attempt to examine the age coupling hypothesis in relation to thiamine deficiency and the resulting neuropathology and behavioral dysfunction. Advanced age in the rat altered the effects of thiamine deficiency in the following respects: (a) accelerated onset of acute neurological symptoms that occur during the thiamine-deficient episode; (b) increased prevalence of thalamic lesions and gliosis after extended thiamine deficiency; and (c) increased ChATlabeled cell loss in the medial septal region after extended thiamine deficiency. Behaviorally, while we

found no interaction between PTD treatment duration and age in the analysis of choice accuracy in the DMTP task, such an interaction was observed for response time. Moreover, when PTD animals were categorized as IML-lesioned versus IML-spared, those in the former group displayed choice accuracy deficits. The next sections will examine these findings in greater detail.

4.1. Acute PTD treatment measures The consequences of acute PTD treatment include progressive weight loss and a series of neurological symptoms (splayed legs, loss of the righting reflex, ipsilateral circling, retropulsion, ataxia) followed by tonic-clonic seizure. Our data imply a greater susceptibility of the aged rodent to the acute effects of thiamine deficiency. It was found that aged PTD rats entered the seizure phase earlier than young PTD rats; moreover, the percentage of body weight lost during treatment revealed two expected patterns: percent weight lost increased with duration of treatment, and aged rats exhibited a higher percentage of weight loss than did young rats at every level of treatment. Among the most likely possibilities for this increased vulnerability are PTD-induced (and age-augmented) decreases in the activity of three thiamine-dependent enzyme systems involved in cerebral glucose metabolism: a-ketoglutarate dehydrogenase, the pyruvate dehydrogenase complex, and transketolase [42,46,47]. Declines in activity and concentration of a-ketoglutarate dehydrogenase in particular have been documented both with advanced age [47] and in the acute phase of PTD (persisting to some degree following thiamine reversal; [46]). Thiamine’s essential role in glucose metabolism implies decreased intracellular energy levels with thiamine deficiency, which may in turn lead to an increased susceptibility to glutamate excitotoxicity via NMDA receptors [48,49], a potential factor in explaining increased thiamine-deficiency-induced thalamic pathology (see section 3.2 and [50]).

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4.2. Brain pathology Lesions of the medial thalamic region, in particular the internal medullary lamina, as a result of PTD treatment were found in a small subset of the young animals at only the most severe treatment level (MAS); among the aged animals, a significantly larger percentage of animals exhibited IML lesions, from both the EAS and MAS groups. Thus, thiamine deficiency needed not be of an extended duration (as was the case for the young brain) to bring about severe neuropathology in the aged brain. This age-related vulnerability to the neuropathological effects of PTD may involve multiple mechanisms, perhaps chief among them the age-related susceptibility to excitatory amino acid neurotoxicity mentioned above [48,51,52]. Observations of increased extracellular glutamate in the medial thalamus in the later stages of acute thiamine deficiency in PTD rats [32,53,54], in addition to the protective effect of the noncompetitive NMDA receptor antagonist MK-801 [32,42,55,54], have strongly implicated glutamate-mediated excitotoxicity in PTD neuropathology. Aged animals also displayed indications of brain shrinkage, including smaller callosal and frontal cortical measures than their young counterparts; furthermore, for each measure, there was a nonsignificant trend of decreasing thickness from PF to LRR to EAS to MAS among the aged animals, but not among the young. Intraventricular distances decreased with duration of PTD treatment, as anticipated [28,32], and this decrease was significantly more precipitous in the aged animals. The fact that only the more posterior of the two intraventricular distance measures demonstrated a significant Age× Treatment Duration interaction (with

only nonsignificant trends toward interactions in callosal, frontal cortical, and immunocytochemical measures) emphasizes the importance of the integrity of this region in the response to thiamine deficiency [22,28,29,32]. These data, in conjunction with the increased prevalence of IML lesions in the aged PTD animals and the increased thalamic GFAP density with age, suggest that a compromised medial/posterior diencephalic region may be in part responsible for age-related vulnerability to thiamine deficiency. Basal forebrain cholinergic cell groups have been found to exhibit age-related degeneration in both human and rat ([40,56 –60]; but see [61]). The present study replicated those findings — there was a significant decrease in number of ChAT-labeled cells in the medial septal area of aged rats as compared to young rats. Furthermore, a decrease in the total number of basal forebrain ChAT-immunoreactive cells with duration of PTD treatment was observed, due to the fact that the aged PTD rats exhibited decreased ChAT cell numbers with increasing duration of PTD treatment (Fig. 2). Such an effect has not been reported in young rats exposed to PTD in the past [21]. However, patients with WKS do reveal a loss of cholinergic neurons in the basal forebrain [3,62,63]. Age may be one of the factors responsible for this discrepancy between WKS and this experimental model of the disorder. Aged rats exhibited significantly higher GFAP immunoreactivity in thalamus than did young rats, indicating enhanced astrocytic activity and, by association, greater neuropathology. This finding is in accordance with previous studies indicating general gliosis in the aged rat brain [41]. As anticipated, GFAP concentration in the thalamus was also found to increase with

Fig. 4. Mean choice accuracy (across sessions) in the DMTP task at each delay for all treatment groups. At left, young animals; at right, aged animals. Significant effects of note: Age [F(1, 55) = 9.59, PB0.01], Block × Age [F(4, 220) = 3.60, P B 0.01], Delay ×Age [F(5, 275) = 11.56, P B0.01].

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duration of PTD treatment; this increase appeared to be greater in the aged animals than in the young. Recall that a higher percentage of aged PTD than young PTD rats exhibited severe IML lesions, i.e. gliotic scars — which may account in part for an overall greater level of GFAP immunoreactivity in the aged group.

4.3. Beha6ior Although young rats, as anticipated, averaged fewer trials to reach criterion on the MTP task than did aged rats, there were no significant differences between young and aged animals, or among levels of PTD treatment, on this measure. However, the performance of aged rats was significantly impaired on the DMTP task. Both measures of performance were affected by age — true-delay response times were found to increase with age, and accuracy decreased with increasing delay intervals to a greater degree in the aged group. In other words, performance of both young and aged subjects was roughly equivalent at the shortest delays, but that of the latter group showed a progressively greater decrease at longer delays. This type of working memory impairment has been seen in aged humans [64], in WKS patients [65,66], and in previous studies involving aged rats [35,36,38,39]. The duration of PTD treatment influenced true-delay response times, a measure of response readiness, during the DMTP sessions. This suggests that a diminished vigilance or attentiveness may be associated with the pathology resulting from extended PTD treatment [22,38,39]. Despite this response deficit, a significant effect of PTD treatment duration on DMTP choice accuracy was not observed. Although previous studies have shown a working memory impairment in thiamine-deficient rats [22,29,31,34,37], the degree of impairment is related to the extent of thalamic pathology (IML lesion) rather than the duration of PTD treatment [22,28]; the latter does not guarantee the latter. In accordance with these findings, an effect of IML lesion status on DMTP choice accuracy was observed. Thus, the lack of an effect of PTD treatment duration on memory impairment in the present study may be explained by the relatively limited number of subjects with neuropathology in the young treatment groups. Furthermore, the lack of a treatment duration effect in the aged rats may be due to the fact that advanced age produces such a severe deficit in working memory by itself that additional brain insult fails to potentiate this deficit.

5. Conclusions Aging does appear to accelerate acute neurological symptoms and potentiate neuropathology associated

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with experimental thiamine deficiency. With the caveat that many factors not exclusive to the nervous system (metabolism, nutrition, and so on) may interact to influence the response to PTD treatment with age, the data presented here indicate a decreased capability of the senescent animal to cope with general neurological insult, and effectively expand the coupling hypothesis of alcohol-associated dementia [10] to consider thiamine deficiency (generally implicated in alcohol-related disorders) and the magnitude of its associated pathology in relation to age. Our data support an age coupling hypothesis whereby advanced age increases the susceptibility of the brain to the neurological insult associated with PTD, resulting in more rapid onset of acute symptomatology, increased thalamic pathology, and an apparently more compromised medial septal cholinergic system in the aged rat as compared to the young. Inadequate dietary intake may predispose the elderly human population to thiamine deficiency, with estimated prevalence rates among the elderly ranging from 8 to 20% of non-institutionalized persons to \40% of those institutionalized [67,68]. The pathology associated with thiamine deficiency would appear to be particularly severe when the system is already compromised by age. This study suggests, significantly, that aged individuals may develop neuropathology to thiamine deficiency before or at the time when severe acute neurological symptoms appear. The clinical implications of these findings — the importance of early detection of thiamine deficiency, and more importantly of prevention — are evident.

Acknowledgements The authors wish to thank David Tuttle for his photographic assistance. This research was funded by NIH grant No. MH55001 to L.M.S.

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