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Thiamine deficiency-induced neurodegeneration and neurogenesis

ProQuest Dissertations and Theses, 2011
Dissertation
Author: Ryan Peter Vetreno
Abstract:
Wernicke-Korsakoff syndrome (WKS) is a thiamine deficiency-induced neurodegenerative disorder that culminates in a bilateral diencephalic lesion and severe amnesia. The pyrithiamine-induced thiamine deficiency (PTD) animal paradigm models the neuropathology and behavioral impairments observed in WKS. A hallmark feature of WKS/PTD is hippocampal dysfunction in the absence of anatomical lesion. Extensive literature exists demonstrating a vital role for neurogenesis in hippocampus-based learning and memory function. Thus, a reduction of hippocampal neurogenesis may contribute to the amnestic syndrome associated with WKS and PTD. The current series of experiments sought to determine the consequences of PTD treatment on hippocampal neurogenesis. In the first experiment (Chapter 2), stage-dependent alterations of neurogenesis and gliogenesis were assessed in the hippocampal granule cell layer. Experimental thiamine deficiency increased the proliferation, but not survival, of progenitor cells. In contrast, neurogenesis was reduced and astrocytogenesis was increased during the late post-lesion stage of PTD treatment. The altered hippocampal neurogenesis was accompanied by an increase in undifferentiated cell populations during the post-lesion stages. These data demonstrate that long-term changes to neurogenesis might underlie the hippocampal dysfunction in the PTD model. The second experiment (Chapter 3) sought to determine whether voluntary exercise could ameliorate PTD-induced behavioral deficits and rescue hippocampal neurogenesis. Subjects were exposed to a running or stationary wheel for 14 days and were behaviorally tested on two spatial working memory tasks beginning either 24-hours or 2-weeks following the completion of exercise. Wheel running selectively improved PTD-induced behavioral impairments on the plus-maze spontaneous alternation task at the 2-week time point relative to the stationary PTD subjects. At this time point, exercise also increased the survival of progenitor cells, rescued the diminished neurogenesis, and facilitated cellular activation during behavioral testing. Taken together, these data demonstrate that reduced hippocampal neurogenesis (Chapter 2) might underlie some of the spatial memory impairments associated with PTD treatment and that exercise exposure restores behavioral functionality and rescues hippocampal neurogenesis (Chapter 3). Furthermore, diminished hippocampal neurogenesis may contribute to the amnestic symptoms associated with thiamine deficiency and voluntary exercise may serve as a non-invasive therapeutic approach to alleviate the observed long-term memory impairments.

TABLE OF CONTENTS

List of Abbreviations

………………………..……………………………………….

x

Chapter 1 …………………………………………………………………………......

1

Wernicke - Korsakoff syndrome ………………………………………………………..

2

Pyrithiamine - induced Thiamine Deficiency …………………………………………..

5

Chapter 2 …………………………………………………………………………......

12

Introduction –

Experiment 1 ......................................................... .................................

13

Methods –

Experiment 1 ................................................................................................

16

Results –

Experiment 1 ....................................................................... ...........................

26

Discussion –

Experiment 1 ............................................................................................

31

Chapter 3 .......................................................................................... ............................

40

Introduction –

Experiment 2 ..........................................................................................

41

Methods –

Experiment 2 ............................................................................. ...................

45

Results –

Experiment 2 ..................................................................................................

56

Discussion –

Experiment 2 ................................................................................ ............

64

Chapter 4

...................................................................................................................... 72 Stage - Dependent Alterations of Hippocampal Neurogenesis Accompany

PTD Treatment ......................... .....................................................................................

74

The Therapeutic Potential of Voluntary Exercise is Dependent Upon Task Demands

and Time of Assessment Relative to the Conclusion of Exercise ........................ .........

76

Voluntary Exercise Rescued Hippocampal Neurogenesis in a Time - Dependent

Fashion .................................................................................................................... ......

78

Further Issues and Future Direction s ............................................................................

79

ix

Concluding Comments ..................................................................................................

81

References ...................................... ............. .. ............................................................... 83

Table Legends

..............................................................................................................

104

Figure Legends ............................ .................................................................................

105

Tables ............................................................................................................................

107

Figures ........................ ..................................................................................................

112

x

LIST OF ABBREVIATIONS

α7nAChRs: α7 nicotinic ACh receptors

ACh: acetylcholine

AChEIs: acetylcholinesterase inhibitors

BDNF: brain - derived neurotrophic factor

BrdU: 5‟ - bromo - 2‟ - deoxyuridine

ChAT: choline acetyltransferase

GCL: granule cell layer

GFAP: glial fibrillary acidic protein

Iba - 1: ionized calcium - binding adaptor molecule 1

MS/DB: medial septum/diagonal band

NeuN: NEUronal Nuclei

PF: pair - fed

PTD : pyrithiamine - induced thiamine deficiency

SGZ: subgranular zone

WKS: Wernicke - Korsakoff syndrome

1

CHAPTER 1

GENERAL INTRODUCTION

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Wernicke - Korsakoff syndrome

Wernicke - Korsakoff syndrome (WKS) is an insidious neurodegenerative dis order caused by thiamine deficiency (Victor, Adams, & Collins, 1971). Although primarily diagnosed in chronic alcoholics, it is also observed in HIV – AIDS patients, clinical cases of persistent vomiting, and gastric bypass patients as well as in other diso rders of malnutrition (Butterworth, 2003; Harper, Fornes, Duyckaerts, Lecomte, & Hauw, 1995; Victor, Adams, & Collins, 1989). In the general population, the post - mortem prevalence rate of WKS is 1 –

2%, but is increased to 12 –

14% in the chronic alcohol use population (Donnino, Vega, Miller, & Walsh, 2007; Harper, Giles, & Finlay - Jones, 1986; Harper, 1998; 2007; Torvik, Lindboe, & Rogde, 1982).

Wernicke - Korsakoff syndrome consists of an acute Wernicke‟s encephalopathy phase and a chronic Korsakoff‟s synd rome phase (Butters, 1981; Victor et al., 1971; 1989). The acute phase is characterized by ophthalmoplegia, gait abnormalities, and global confusion (Butters, 1981; Victor et al., 1971). Recent neuroimaging studies have revealed that the mammillary bodie s, periaqueductal and periventricular gray matter, collicular bodies, and thalamus are edematous and inflamed during the acute phase of Wernicke‟s encephalopathy, which might contribute to the general confusion state (Sullivan & Pfefferbaum, 2009). If lef t untreated, thiamine deficiency during this phase is fatal in 20% of cases (Harper et al., 1986), whereas thiamine replacement therapy improves a majority of the neurological symptoms that characterize this stage (Butters, 1981).

Although thiamine replen ishment therapy reverses the acute symptoms of Wernicke‟s encephalopathy, the majority of cases progress into the more chronic

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Korsakoff‟s syndrome phase. This is, in part, due to the non - specific nature of the early symptoms of Wernicke‟s encephalopathy (Harper, 2007; Kopelman, Thomson, Guerrini, & Marshall, 2009). Indeed, 80% of patients with Wernicke‟s encephalopathy will go on to develop Korsakoff‟s syndrome following thiamine replacement (Victor et al., 1989). The Korsakoff‟s syndrome phase is chara cterized by amnestic symptoms and the development of confabulation (Butters, 1981; Kopelman et al., 2009; Victor et al., 1989). The amnestic symptoms that characterize WKS involve retrograde and anterograde memory impairments, which are not better account ed for by diminished intellect as these patients exhibit similar intelligence scores to age - matched controls on the Wechsler Adult Intelligence Scale (Butters, 1981; Gold & Squire, 2006; Reed et al., 2003). The retrograde amnesia evidenced by WKS patients

involves compromised recall and recognition memory as evidenced by impaired memory of recent public events. However, the retrograde memory impairments are temporally graded as these patients demonstrate a conservation of earlier memories whereas more rec ent memories, often extending back 25 –

30 years, are liable to the amnesia (Butters, 1981; Fama, Marsh, & Sullivan, 2004; Gold & Squire, 2006; Kopelman, 1989; Mair, Warrington, & Weiskrantz, 1979).

The anterograde amnestic symptoms that typify WKS invo lve impaired declarative memory as evidenced by deficits on both episodic (recall of words and faces) and semantic (letter and category fluency) tasks (Butters, Granholm, Salmon, Grant, & Wolfe, 1987; Gold & Squire, 2006; Kopelman et al., 2009). In additi on, WKS patients demonstrate increased perseverative responses on the Wisconsin Card Sorting Task relative to age - matched controls (Fama et al., 2004). However, non - declarative memory

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function appears unaffected on measures that assess perceptual priming and perceptuomotor performance in individuals with WKS (d‟Ydewalle & Van Damme, 2007; Fama, Pfefferbaum, & Sullivan, 2006; Gold & Squire, 2006), indicating some conservation of cognitive function. Thus, WKS patients retain difficulty in establishing new e xplicit memories and evidence diminished long - term memory.

Wernicke - Korsakoff syndrome has historically been described as a diencephalic amnesic syndrome because the neuropathology associated with this disorder is localized primarily to the thalamus and ma mmillary bodies (Kopelman, 1995). In support of this classification, neuroimaging and post - mortem studies have consistently revealed sulcal widening and ventricular enlargement that is accompanied by shrinkage of the cortex and diencephalon (i.e., mammill ary bodies and thalamus [Gold & Squire, 2006; Mair et al., 1979; Reed et al., 2003; Sullivan & Pfefferbaum, 2009]). Within the cortex, significant tissue shrinkage has been observed in the temporal, orbitofrontal, frontal, and parietal cortices (Colchester

et al., 2001; Jernigan, Schafer, Butters, & Cermak, 1991; Kril, Halliday, Svoboda, & Cartwright, 1997), whereas the diencephalic insult involves lesion to the anterior, mediodorsal, and midline thalamic nuclei as well as the medial mammillary bodies (Aggl eton & Sahgal, 1993; Harding, Halliday, Caine, & Kril, 2000; Harper, Kril, & Daly, 1987; Harper, Dixon, Sheedy, & Garrick, 2003; Kopelman et al., 2009; Mair et al., 1979; Markowitsch, 1988; Mayes, Meudell, Mann, & Pickering, 1988). Neuroimaging studies ha ve also revealed shrinkage of the medial septum/diagonal band (MS/DB [Sullivan & Pfefferbaum, 2009]) as well as degeneration of the mammillothalamic tract (Markowitsch, 1988; Yoneoka et al., 2004). Furthermore, both in vivo

neuroimaging and post - mortem ex amination has revealed reductions of grey and

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white matter in the anterior superior vermis of the cerebellum as well as a reduction in vermis Purkinje cells (Baker, Harding, Halliday, Kril, & Harper, 1999; Sullivan, Rosenbloom, Lim, & Pfefferbaum, 2000). Thus, the diencephalic and cortical neuropathology appears to contribute to the amnestic symptoms whereas the observed cerebellar neuropathology might underlie the ataxia common in humans and animals following a thiamine deficiency episode.

The declarative

memory deficits characteristic of WKS is indicative of hippocampal neuropathology (Caulo et al., 2005; Fama et al., 2004; Gold & Squire, 2006; Victor et al., 1989). However, there is little evidence of overt lesion or cell loss in the hippocampus (Colche ster et al., 2001; Gold & Squire, 2006; Squire, Amaral, & Press, 1990) despite reports of hippocampal shrinkage (see Sullivan & Pfefferbaum, 2009 for a review). While the physiological mechanisms underlying hippocampal hypofunction in WKS patients are unk nown, Caulo and colleagues (1995) demonstrated that the anterograde amnestic symptoms involve deficits of memory encoding. Furthermore, shrinkage of the MS/DB (see Sullivan & Pfefferbaum, 2009) would culminate in compromised cholinergic function, which co uld contribute to the hippocampal hypofunction observed in WKS. Unfortunately, given the limitations of human research, the utilization of animal models of WKS has provided further insight into the underlying factors contributing to the amnestic symptoms.

PYRITHIAMINE - INDUCED THIAMINE DEFICIENCY

The pyrithiamine - induced thiamine deficiency (PTD) animal paradigm models both the acute neurological symptoms that characterize Wernicke‟s encephalopathy and

6

the chronic neuropathological and cognitive deficits t hat define WKS (Langlais, Zhang, & Savage, 1996; Troncoso, Johnston, Hess, Griffin, & Price, 1981). During the acute stages of PTD treatment, anatomical damage is primarily localized to the gelatinosus and anteroventral thalamic nuclei (Zhang et al., 1995 ). As thiamine deficiency continues unabated, cell death extends throughout several nuclei within the thalamus (Langlais & Mair, 1990; Zhang et al., 1995). Following thiamine replenishment, the neuropathological insult extends to encompass the medial mam millary bodies (Langlais & Savage, 1995; Langlais et al., 1996). In addition, key fiber tracts that interconnect the limbic structures are atrophied (i.e., fimbria/fornix and mammillothalamic tract) (Langlais & Zhang, 1997; Markowitsch, 1988), which likel y results in a „disconnect‟ between the hippocampus and diencephalon (Jenkins, Dias, Amin, Brown, & Aggleton, 2002; Markowitsch, 1988; Markowitsch & Pritzel, 1985; Reed et al., 2003; Vann & Aggleton, 2003).

Diminished cholinergic input to the hippocampus is another factor that likely contributes to the „disconnect‟ associated with the PTD model. Although there is a modest population of cholinergic neurons in the hippocampus (Frotscher, Schlander, & Levanth, 1986), it receives the majority of acetylcholine

(ACh) innervation from the MS/DB (Mesulam, Mufson, Wainer, & Levey, 1983). Dysfunction of the MS/DB cholinergic system has been implicated the amnestic symptoms associated with both WKS and PTD treatment. Indeed, pharmacotherapy aimed at enhancing hippo campal cholinergic bioavailability has been demonstrated to reverse thiamine deficiency - induced memory impairment. Treatment with acetylcholinesterase inhibitors (AChEIs) reverses the cognitive impairments associated with WKS (Angunawela & Barker, 2001;

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C ochrane, Cochrane, Jauhar, & Ashton, 2005). Similarly, during the acute stages of thiamine deficiency, memory deficits in rats are reversed by administration of AChEIs (Nakagawasai, 2005). Furthermore, systemic or intrahippocampal administration of AChEI s (Roland, Mark, Vetreno, & Savage, 2008; Roland, Levinson, Vetreno, & Savage, 2010), or intraseptal administration of bicuculline (a GABAergic antagonist [Roland & Savage, 2009b]) following thiamine replacement therapy temporally reverses the cognitive im pairments associated with PTD treatment.

The cholinergic dysfunction in WKS and the PTD model arises from a loss of ACh producing cells. Our laboratory has consistently observed an estimated 20 –

30% reduction of choline acetyltransferase (ChAT) - positiv e neurons in the MS/DB following thiamine recovery in the PTD model (Pitkin & Savage, 2001, 2004; Roland & Savage, 2009a; Savage, Roland, & Klintsova, 2007), an effect that is correlated with impairment on spatial memory tasks (e.g, Roland & Savage, 2009a) . The observation of reduced ChAT - positive cells in the PTD model has also been demonstrated in human WKS (Arendt, Bruckner, Bigl, & Marcova, 1995). However, the thiamine deficiency - induced degeneration of neurons within the MS/DB is specific to ChAT - pos itive cells as populations of GABAergic cells within this region are unaltered in the PTD brain (see Roland & Savage, 2009a). The apparent reduction of ChAT - positive cells appears to be the consequence of a vulnerability to thiamine deficiency, as thiamin e is a co - factor for the development of several enzymes necessary for proper cellular function, including transketolase, pyruvate dehydrogenase , and α - ketoglutarate dehydrogenase (Butterworth, 2003). Indeed, pyruvate dehydrogenase is critically involved in the production of ACh as it coverts pyruvate to acetyl - CoA during glycolysis (Todd & Butterworth, 1999). The

8

loss of ChAT - positive cells in the MS/DB, in turn, results in both diminished hippocampal ACh fiber densities (Anzalone, Vetreno, Ramos, & Savage, 2010) and hippocampal cholinergic efflux during behavioral testing (Pires, Pereira, Oliveira - Silva, Franco, & Ribeiro, 2005; Roland & Savage , 2007; Roland et al., 2008; Savage, Chang, & Gold, 2003; Savage et al., 2007; Vetreno, Anzalone, & Savage, 2008), which further supports the “disconnection syndrome” hypothesis. Thus, the extended hippocampal system atrophy, coupled with reduced hippocam pal cholinergic innervation, culminates in severe and long - lasting learning and memory impairments.

Animals exposed to PTD treatment evidence spatial working memory deficits on both non - matching - to - sample and - position tasks as well as matching - to - sample a nd - position tasks (Langlais & Savage, 1995; Mumby, Cameli, & Glenn, 1999). These behavioral measures require the subject to remember, for varying latencies, the order of stimulus presentation and use this information to guide behavior on future trials. Similar spatial working memory impairments have been observed on the spontaneous alternation task following PTD treatment (Roland et al., 2008; Roland & Savage, 2009b; Savage et al., 2003; 2007; Vetreno et al., 2008), which also provides a good measure of hippocampal - dependent spatial memory (McIntyre, Marriott, & Gold, 2002; McIntyre, Pal, Marriott, & Gold, 2003). In addition, PTD - treated animals evidence diminished long - term learning and memory function on the Morris water maze and radial arm maze. On t he Morris water maze, animals exposed to PTD treatment exhibit longer latencies to locate the hidden platform as well as increased thigmotaxic behavior (Langlais et al., 1992; Pires et al., 2005). Similarly, PTD - treated subjects evidence impaired working memory and moderate reference memory impairments on the radial arm maze (Robinson

9

& Mair, 1992). Thus, thiamine recovered PTD - treated rats demonstrate persistent impairments in hippocampal - dependent tasks, similar to humans, despite anatomical conservatio n of the hippocampus.

The declarative memory deficits characteristic of WKS and the PTD model are indicative of hippocampal neuropathology (Caulo et al., 2005; Fama et al., 2004; Gold & Squire, 2006; Savage et al., 2003; Vetreno et al., 2008; Victor et al. , 1989). However, there is little evidence of overt lesion or cell loss in the hippocampus (Colchester et al., 2001; Gold & Squire, 2006; Langlais et al., 1992; Squire et al., 1990), although there are reports of hippocampal shrinkage in both humans and t he PTD animal model (Pfefferbaum et al., 2006; Pfefferbaum, Adalsteinsson, Bell, & Sullivan, 2007; Sullivan & Pfefferbaum, 2009). Recent work from Zhao, Zhong, and colleagues (2008) suggest that the hippocampal dysfunction associated with thiamine deficie ncy may stem from a reduction of hippocampal neurogenesis. Indeed, they demonstrated reduced hippocampal neurogenesis during an acute episode of dietary - induced thiamine deficiency that was correlated with deficits on a spontaneous alternation task. Furt hermore, reversal of thiamine deficiency ameliorated the observed reductions in neurogenesis as well as the hippocampal behavioral deficits. However, this model does not recapitulate the full spectrum of neuropathology associated with WKS. Thus, the curr ent series of experiments were designed to evaluate the effects of PTD treatment, a reliable model of WKS and post - thiamine deficiency consequences, on hippocampal neurogenesis.

The first experiment (Chapter 2) sought to determine whether neurogenesis an d/or gliogenesis were altered as thiamine deficiency progressed from the early acute Wernicke encephalopathy phase to the more chronic WKS (as defined by Zhang et al., 2005).

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Levels of neurogenesis (BrdU/NeuN co - localization) and gliogenesis (astrocytes [ BrdU/GFAP], microglia [BrdU/Iba - 1], oligodendrocytes [BrdU/O4]) were assessed in separate groups of PTD and pair - fed (PF) control rats that were administered the S - phase mitotic marker 5‟ - bromo - 2‟ - deoxyuridine (BrdU) at one of 4 cytopathological stages of thiamine deficiency: (1) the early pre - lesion stage (8 days after treatment onset), (2) the late pre - lesion stage (15 days after treatment onset), (3) the early post - lesion stage (24 - hours after thiamine replacement therapy), or (4) the late post - lesion st age (28 days after thiamine replacement therapy). It was hypothesized that PTD treatment would: (1) not affect neurogenesis/gliogenesis during the early pre - lesion stage, (2) increase neural stem cell activity/neurogenesis/gliogenesis during the late pre - lesion and early post - lesion stages, and (3) decrease neural stem cell activity/neurogenesis/gliogenesis during the late post - lesion stage.

In the second experiment (Chapter 3), the therapeutic potential of physical activity to alleviate hippocampal dysfun ction was assessed in PTD -

and PF - treated rats during the chronic WKS phase of PTD treatment. Following thiamine replacement therapy, all animals were allotted a 2 week convalescence period to recover from their respective treatments. Afterwards, the two

treatment groups (PTD/PF) were divided so that half were exposed to a running wheel and the remaining animals were exposed to a locked running wheel. All animals were provided 2 week access (24 hr/day) to their respective exercise conditions during which

BrdU was administered to label dividing cells within the hippocampus. At the conclusion of exercise exposure, subjects were assessed on two versions of the spontaneous alternation task (Y - maze and plus - maze) beginning either 24 - hours or 2 - weeks later. T issue was collected and rates of neurogenesis (BrdU/NeuN co -

11

localization) and cellular activation of newly generated cells (BrdU/NeuN/c - Fos co - localization) were assessed. It was hypothesized that voluntary exercise would recover spontaneous alternation b ehavior and perseveration rates in PTD - treated animals in a task -

(complex task vs. simple task) and time - dependent manner (24 - hours vs. 2 - weeks [see Snyder et al., 2009]). Furthermore, it was hypothesized that exposure to voluntary exercise would facilit ate the maturation (co - expression of BrdU/NeuN) and functional integration (co - expression of BrdU/c - Fos) of neurons at both time points relative to the stationary control subjects (see Zhao, Teng, Summers, Ming, & Gage, 2006).

The aforementioned studies ar e an important step in identifying potential mechanisms underlying amnesia - associated learning and memory impairments. Furthermore, assessment of the ability of voluntary exercise to recovery behavioral performance and rescue neurogenesis would provide in sight into the therapeutic potential of physical activity to serve as a non - invasive long - term therapy.

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CHAPTER 2

STAGE - DEPENDENT ALTERNATIONS OF PROGENITOR CELL PROLIFERATION AND NEUROGENESIS IN AN ANIMAL MODEL OF WERNICKE - KORSAKOFF SYNDROME

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INTRODUCTION

Wernicke - Korsakoff syndrome (WKS) is an insidious neurodegenerative disorder caused by thiamine deficiency. It involves an acute Wernicke‟s encephalopathy phase and a chronic Korsakoff‟s syndrome phase (Butters, 1981; Kopelman et al. , 2009; Victor et al., 1971; 1989). Although WKS is observed in disorders associated with malnutrition, it is most commonly diagnosed in chronic alcoholics (Harper & Kril, 1990; Kopelman, 1995; Kril, 1996; Langlais et al., 1996; Victor et al., 1989). The

anatomical consequence of prolonged thiamine deficiency is a bilateral lesion of multiple thalamic nuclei (anterior n., midline n., mediodoral n., and posterior n.) and the mammillary bodies as well as degeneration of the fimbria/fornix and mammillothalam ic tract that interconnect the limbic structures (Caulo et al., 2005; Gold & Squire, 2006; Kopelman et al., 2009; Kril, 1996; Langlais et al., 1996; Sullivan & Pfefferbaum, 2009; Victor et al., 1989). This neuropathological constellation culminates in tem porally graded retrograde amnesia and severe anterograde amnesia characterized by impaired explicit memory, indicative of hippocampal dysfunction (Caulo et al., 2005; Fama et al., 2004; Victor et al., 1989). However, there is limited evidence of overt hip pocampal pathology in the WKS brain (Caulo et al., 2005; Fama et al., 2004; Gold & Squire, 2006).

The pyrithiamine - induced thiamine deficiency (PTD) animal paradigm models both the diencephalic neuropathology and behavioral impairments observed in human WK S (Langlais & Savage, 1995; Langlais et al., 1996; Langlais & Zhang, 1997; Markowits ch, 1988; Troncoso et al., 1981;

Witt, 1985). Similar to WKS, there is little evidence of gross hippocampal pathology in the PTD model (Langlais et al., 1992; Mair, 1994) despite functional impairment on several spatial memory tasks, including the

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delayed non - matching - to - position task (Langlais & Savage, 1995; Roland & Savage, 2007), Morris water maze (Langlais et al., 1992), and the spontaneous alternation task (Savage et al., 2003; Vetreno et al., 2008). The deficits on hippocampal - dependent spatial memory tasks are paralleled by blunted hippocampal cholinergic efflux assessed during maze learning (Roland & Savage, 2007; Vetreno et al., 2008) .

In both human WKS and the P TD animal model, assessment of neural activity during memory testing revealed that the hippocampus is functionally impaired and likely contributes to the amnestic state (see Caulo et al., 2005; Savage et al., 2003). However, the precise physiological mech anisms underlying the hippocampal dysfunction remain to be elucidated. Recent work suggests that diminished neuroplasticity within the hippocampal network might be a contributing factor.

Neurogenesis involves the generation, differentiation, migration, a nd functional integration of newly generated neurons into mature brain tissue (Altman & Das, 1965; Zhao, Deng et al., 2008; Zhao et al., 2006), and occurs across mammalian species (Gould, Tanapat et al., 1999; Kuhn et al., 1996), including humans (Eriksson

et al., 1998). The subventricular zone of the lateral ventricles and the subgranular zone (SGZ) of the hippocampal dentate gyrus represent two distinct mitotically active microenvironments wherein neurogenesis continues to occur throughout life (Abrous e t al., 2005). Neurogenesis within the hippocampus has been heavily implicated in hippocampal - mediated cognitive function (Ambrogini et al., 2000; Gould, Beylin et al., 1999; Kee et al., 2007; Kempermann et al., 2004; Shors et al., 2001). Experimental fac ilitation or inhibition of neurogenesis enhances and diminishes performance on spatial tasks, respectively (Fabel et al., 2009; Gould, Beylin et al., 1999; Madsen et al., 2003;

15

Shors et al., 2001; van Praag, Kempermann et al., 1999; Winocur et al., 2006).

Furthermore, it is a highly dynamic process affected by many intrinsic and extrinsic factors, including neurotransmitters (e.g., acetylcholine [Cooper - Kuhn et al., 2004]), drug abuse (e.g., alcoholism [Crews et al., 2004; He et al., 2009; Klintsova et al. , 2007]), environmental enrichment (Cotman & Berchtold, 2002; Fabel et al., 2009), and pathological insults (Richardson et al., 2007; Rola et al., 2006; Zhao, Zhong et al., 2008).

There is also evidence indicating that altered neurogenesis plays a role in

behavioral abnormalities observed during, and potentially after, an episode of thiamine deficiency. Indeed, work assessing the effects of thiamine deficiency on neurogenesis in mice demonstrated reductions beginning on the 9 th

day of exposure to a thiami ne deficient diet, which were paralleled by behavioral impairments on a Y - maze avoidance task (Zhao, Zhong et al., 2008). However, the animals in this experiment were only exposed to dietary restriction of thiamine, a paradigm that does not completely mod el the brain pathology associated with the chronic condition of WKS (see Langlais et al., 1996).

Thus, the present study sought to determine whether neurogenesis was altered in a model (PTD treatment) that reliable reproduces the pathology and behavioral

impairments associated with both Wernicke‟s encephalopathy and WKS. Both neurogenesis and gliogenesis were assessed at different stages of PTD treatment in the granule cell layer (GCL) and SGZ of the hippocampal dentate gyrus. Subjects were assessed at one of 4 stages during PTD treatment: early pre - lesion stage, late pre - lesion stage, early post - lesion stage, or the late post - lesion stage. Tissue was collected and processed either 24 - hours or 28 - days following BrdU administration to assess cell prolife ration, or survival and phenotype of these cells. The goal of this study was to

16

document how progenitor cell proliferation and survival as well as neurogenesis and gliogenesis change as thiamine deficiency progresses to the culmination of neuropathology a nd the chronic post - lesion state that defines WKS.

METHODS

SUBJECTS

One hundred and twenty - eight male Sprague - Dawley rats (275 –

325 g, 2 ½ months old, Harlan Corp., IN) served as subjects. The animals were pair - housed and maintained on a 12 hr light /dark cycle (onset at 06:00 hr; offset at 18:00 hr) in a temperature (20  C) controlled vivarium. All experiments were conducted according to the National Institute of Health guide for the care and use of laboratory animals. The Institutional Animal Care and Use Committee of the State University of New York at Binghamton approved the experimental procedures used in this study. Furthermore, every effort was made to minimize animal suffering and the number of animals used.

PYRITHIAMINE - INDUCED THIAMINE DEF ICIENCY AND PAIR - FED TREATMENT

Animals were randomly assigned to one of the following treatment conditions: (i) pyrithiamine - induced thiamine deficiency (PTD, n = 64) or (ii) pair - fed control (PF, n = 64). An additional 12 rats were included as a non - man ipulated control group (CON) to determine whether the PF subjects would serve as an adequate control. Within each individual treatment condition (PTD/PF), subjects were further randomly divided into 4 stages based on previous research (see Langlais & Mair , 1990; Langlais & Savage, 1995;

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Langlais & Zhang, 1995; Pfefferbaum et al., 2007; Zhang et al., 1995 [n = 16 per stage (early pre - lesion, late pre - lesion, early post - lesion, late post - lesion [see below])]), each with two separate time points (n = 8 per ti me point [24 - hour, 28 day]). The control subjects were also divided into 2 separate time points (n = 6 per time point) similar to the PTD and PF animals.

Subjects in the PTD treatment group were free - fed thiamine - deficient chow (Teklad Diets, Madison, WI)

and received daily injections of pyrithiamine hydrobromide (0.25 mg/kg, i.p. [Sigma - Aldrich, St. Louis, MO]). Pyrithiamine is an inhibitor of thiamine pyrophosphokinase, an enzyme that converts thiamine to thiamine pyrophosphate (Butterworth & Heroux, 19 89). Subjects in the PF treatment group were fed an amount of thiamine deficient chow equivalent to the amount consumed by the PTD animals the previous day (to replicate the anorexic effects) and received daily injections of thiamine hydrochloride (0.4 mg /kg, i.p. [Sigma - Aldrich, St. Louis, MO]). Animals that receive PF treatment do not demonstrate any changes in brain levels of thiamine - dependent enzymes (Butterworth & Heroux, 1989).

Full document contains 133 pages
Abstract: Wernicke-Korsakoff syndrome (WKS) is a thiamine deficiency-induced neurodegenerative disorder that culminates in a bilateral diencephalic lesion and severe amnesia. The pyrithiamine-induced thiamine deficiency (PTD) animal paradigm models the neuropathology and behavioral impairments observed in WKS. A hallmark feature of WKS/PTD is hippocampal dysfunction in the absence of anatomical lesion. Extensive literature exists demonstrating a vital role for neurogenesis in hippocampus-based learning and memory function. Thus, a reduction of hippocampal neurogenesis may contribute to the amnestic syndrome associated with WKS and PTD. The current series of experiments sought to determine the consequences of PTD treatment on hippocampal neurogenesis. In the first experiment (Chapter 2), stage-dependent alterations of neurogenesis and gliogenesis were assessed in the hippocampal granule cell layer. Experimental thiamine deficiency increased the proliferation, but not survival, of progenitor cells. In contrast, neurogenesis was reduced and astrocytogenesis was increased during the late post-lesion stage of PTD treatment. The altered hippocampal neurogenesis was accompanied by an increase in undifferentiated cell populations during the post-lesion stages. These data demonstrate that long-term changes to neurogenesis might underlie the hippocampal dysfunction in the PTD model. The second experiment (Chapter 3) sought to determine whether voluntary exercise could ameliorate PTD-induced behavioral deficits and rescue hippocampal neurogenesis. Subjects were exposed to a running or stationary wheel for 14 days and were behaviorally tested on two spatial working memory tasks beginning either 24-hours or 2-weeks following the completion of exercise. Wheel running selectively improved PTD-induced behavioral impairments on the plus-maze spontaneous alternation task at the 2-week time point relative to the stationary PTD subjects. At this time point, exercise also increased the survival of progenitor cells, rescued the diminished neurogenesis, and facilitated cellular activation during behavioral testing. Taken together, these data demonstrate that reduced hippocampal neurogenesis (Chapter 2) might underlie some of the spatial memory impairments associated with PTD treatment and that exercise exposure restores behavioral functionality and rescues hippocampal neurogenesis (Chapter 3). Furthermore, diminished hippocampal neurogenesis may contribute to the amnestic symptoms associated with thiamine deficiency and voluntary exercise may serve as a non-invasive therapeutic approach to alleviate the observed long-term memory impairments.