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Glucose induced IEG expression in the thiamin-deficient rat brain1
Brain Research 892 (2001) 218–227 www.elsevier.com / locate / bres
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Glucose induced IEG expression in the thiamin-deficient rat brain 1 Craig Zimitat 2 , Peter F. Nixon* Department of Biochemistry, The University of Queensland, Brisbane, Queensland, Australia 4072 Accepted 8 December 2000
Abstract Glucose loading of rats made thiamin deficient by dietary deprivation of thiamin and the administration of pyrithiamin (40 mg / 100 g, i.p.) precipitates an acute neuropathy, a model of Wernicke’s encephalopathy in man (Zimitat and Nixon, Metab. Brain Dis. 1999;14:1–20). Immunohistochemical detection of Fos proteins was used as a marker to identify neuronal populations in the thiamin-deficient rat brain affected by glucose loading. As thiamin deficiency progressed, the extent and intensity of Fos-like immunoreactivity (FLI) in brain structures typically affected by thiamin deficiency (the thalamus, mammillary bodies, inferior colliculus, vestibular nucleus and inferior olives) were markedly increased when compared to thiamin-replete controls. Glucose loading for 1–3 days further increased the intensity of FLI in these same regions, consistent with a dependence of Fos expression on carbohydrate metabolism as well as on thiamin deficiency. The timed acute changes that follow a bolus glucose load administered to thiamin-deficient animals may provide a sequential account of events in the pathogenesis of brain damage in this model of Wernicke’s encephalopathy. 2001 Elsevier Science B.V. All rights reserved. Theme: Disorders of the neurons system Topic: Neurotoxicity Keywords: IEG expression; Thiamin deficiency; Wernicke’s encephalography; Brain damage
1. Introduction Induction of thiamin deficiency in the rat by pyrithiamin produces a characteristic sequence of signs of neurological disease and selective pathological lesions in the nuclei of the thalamus and hypothalamus, the collicular plate, vestibular nucleus, inferior olive and mammillary bodies [40]. Glutamate NMDA receptor-mediated excitotoxicity has been proposed as a mechanism for neuronal cell death in pyrithiamin-induced thiamin deficiency (PTD) in rats on the basis of histological studies [45], microdialysis [19,26] and enzymatic studies. Decreased activity of the thiamindependent enzyme 2-oxo-glutarate dehydrogenase (OGDH) is associated with the onset of neurological signs and progress of thiamin deficiency and could lead to the accumulation of glutamate in the brain [4,5,16]. Indeed, 1
Published on the World Wide Web 3 January 2001. Present address: Griffith Institute for Higher Education, Griffith University, Brisbane, Australia, 4111. *Corresponding author. Fax: 161-7-3365-4613. E-mail address: [email protected] (P.F. Nixon). 2
selective increases in extracellular glutamate concentrations have been measured in specific sites of the brain of rats with neurological signs of thiamin deficiency before neuronal death occurred in these sites [19,26,39]. In addition, the characteristic features of neuronal cell death in PTD [2,45] are consistent with those of excitotoxic cell death triggered by glutamate [22]. The strongest evidence in support of a glutamate NMDA -mediated process in PTD is that MK801 (a glutamate NMDA receptor antagonist) when administered to PTD rats blocked the localised increases in extracellular glutamate concentration in the brain [26] and also significantly attenuated neuronal cell death [33]. In thiamin deficiency, the probable source of increased extracellular glutamate is increased intracellular glutamate, accumulated when glucose metabolism is blocked at OGDH. Wernicke’s encephalopathy (WE) is a consequence of thiamin deficiency in humans [42]. WE is not observed in general starvation but a glucose load can precipitate WE in humans with unsuspected thiamin deficiency [42] and exacerbate neurological symptoms in individuals with WE [31,41]. Furthermore, glucose loading of PTD and thiamin-deficient (TD) rats precipitates an
0006-8993 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )03297-2
C. Zimitat, P.F. Nixon / Brain Research 892 (2001) 218 – 227
exacerbation of ataxia [46] preceded by increased blood brain barrier (BBB) permeability and hyper-intensities in vulnerable brain regions detected by magnetic resonance imaging [44,23]. These changes are accompanied by an increase in the total concentration of brain glutamate or glutamate-derivatives (e.g. GABA, glutamine) [34]. Thus glucose metabolism is diverted into glutamate accumulation, at least in part, in thiamin deficiency. Extracellular stimulation of neurons via the glutamate NMDA receptor triggers a series of intracellular events [38]. The basal level of c-fos expression in the brain is low but it is rapidly and transiently increased after some stimulus causes an increase in extracellular calcium, e.g. via the glutamate NMDA receptor or voltage sensitive calcium channels (VSCCs). Stimulated neurons contain c-Fos, hence mapping c-Fos immunoreactivity provides a method for mapping neuronal pathways activated in response to specific stimuli [36]. The expression of c-fos has been associated also with excitotoxic cell death, programmed cell death and delayed neuronal death [37,43]. As a consequence, the expression of c-fos has also been used as a cellular marker of neuronal activity and potentially fatal neuronal damage [11,21]. We report, for the first time, experiments that utilise the expression of c-fos to identify specific neuronal populations in the PTD rat brain that are affected by glucose loading before the onset of neurological signs or of overt histopathology.
2. Materials and methods
2.1. Animals and diets The animals used in these experiments (n538), their housing and the composition of the cereal based control rat chow and the thiamin-deficient diet have been described previously [47,46]. The latter diet contained 30% glucose and 15% starch. All animal procedures were authorised by the Animal Ethics and Experimentation Committee of The University of Queensland.
2.2. Reagents and equipment The following chemicals were purchased from BDH Chemicals Australia Pty Ltd., Kilsyth, NSW: disodium hydrogen phosphate, potassium dihydrogen phosphate and sodium chloride, whilst xylene, hydrogen peroxide and ethanol (AR) were supplied by APS Ajax Finechem, Auburn, NSW. Triton X-100, pyrithiamin, sodium azide, diaminobenzidine, nickel ammonium sulphate and bovine serum albumin were purchased from Sigma–Aldrich Australia Pty Ltd., Castle Hill, NSW. Boehringer Mannheim Australia Pty Ltd., Castle Hill, NSW supplied cobalt chloride. Sectioning was carried out on a freezing microtome (Minotome, Damon / IEC, MA, USA). OCT com-
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pound for embedding was purchased from Miles Inc., IN, USA. Light microscopy and photography were performed using an Axioplan2 microscope (Carl Zeiss Jena GmbH, Germany). Polyclonal sheep anti-Fos antibodies were purchased from Cambridge Research Biochemicals, Cambridge, UK. These antibodies were raised against c-Fos amino acids 127–152. The term Fos-like immunoreactivity (FLI) is used because the antibody precipitates Fos proteins (c-Fos and FosB) and cross-reactive Fos-related antigens (Fra-1 and Fra-2) [9,13,14]. Differences in FLI, detected in the brains of ataxic and opisthotonic PTD rats vs. controls, therefore represent cumulative changes in combined c-Fos, FosB, Fra-1 and Fra-2 immunoreactivity. Increases in FLI 4 h after the saline or glucose load primarily reflect changes in expression of c-fos and the presence of c-Fos protein, because of its brief half life and short induction time [27].
2.3. Effect of glucose loading on c-fos expression in the brains of pyrithiamin-treated thiamin-deficient ( PTD) rats Wistar rats weighing 140–180 g were distributed into six groups for these experiments (Table 1). All animals in groups 2–5 received thiamin-deficient diet and pyrithiamin (PT) injections (40 mg / 100 g, i.p.) for 5 consecutive days out of every 7 days starting from day 1, and additional injections of saline or glucose starting on day 7 as described (Table 1 and hereafter). On the day of the onset of ataxia (days 7–9), rats in groups 2 and 3 received one more dose of PT and glucose or saline and were killed for analysis 4 h later. Groups 1a and 1b control animals were also harvested at this time, 4 h after their saline or glucose injection. On the day after the onset of opisthotonus (11–14 days of PTD treatment), animals in groups 4 and 5 received one more dose of PT and a single glucose or saline load, then they were killed for analysis 4 h later. A group 6 (n55, not shown in Tables) was treated identically to group 5 except that they were killed for analysis 24 h after the single glucose load. Animals that had lost their righting reflex or were observed convulsing were excluded from experiments. The brains were perfused in vivo in preparation for immunohistochemistry [47]. Perfused brain tissue was dissected out and cryoprotected by immersion in 0.3 M sucrose in 0.01 M phosphate buffer, pH 7.4 for 72 h then embedded with OCT compound. Sagittal sections (40 mm) of the right hemispheres were cut in series, from the midline, on a freezing microtome. Brain tissue was sampled for immuno-histochemistry between the midline and 2.4 mm lateral from the midline of the brain, which excludes the lateral vestibular nucleus and the lateral caudal CA1 regions of the hippocampus. Sections were processed for immunohistochemical detection of Fos proteins as freefloating sections in tissue culture plates using the method
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Table 1 Details of treatments for each group in these experiments Group
Treatment
1a
Saline treated control animals (n55) Received standard thiamin-replete rat chow and daily intraperitoneal (i.p.) saline injections commencing on day 7 Harvested at the same time as rats in Group 2.
1b
Glucose treated control animals (n55) Received standard thiamin-replete rat chow and daily glucose injections (10 g / kg; i.p.) commencing on day 7 Harvested at the same time as rats in Group 3
2
PTD animals with serial saline treatments (n55) Thiamin deficiency induced by daily PT injections (5 out of 7 days) and provision of TD diet Received daily saline injections (2 ml, i.p.) every day from day 7 until the onset of ataxia Killed for analysis 4 h after a final dose of saline, which followed the onset of ataxia
3
PTD animals with serial glucose treatments (n55) Thiamin deficiency induced by daily PT injections (5 out of 7 days) and provision of TD diet Received glucose loads (10 g / kg, i.p.) every day from day 7 until the onset of ataxia Killed for analysis 4 h after a final dose of glucose, which followed the onset of ataxia
4
PTD animals with single saline treatment (n55). Thiamin deficiency induced by daily PT injections (5 out of 7 days) and provision of TD diet Received one saline injection (2.5 ml, i.p.) 24 h after the onset of opisthotonus Killed for analysis 4 h after the saline injection
5
PTD animals with single glucose treatment (n58) Thiamin deficiency induced by daily PT injections (5 out of 7 days) and provision of TD diet Received one glucose load only (10 g / kg, i.p.) 24 h after the onset of opisthotonus Killed for analysis 4 h after the glucose load
of [10]. Antibody dilutions were: anti-Fos antibodies 1:1000 dilution in 1% bovine serum albumin (BSA), biotinylated secondary antibodies 1:400 dilution in 1% BSA in phosphate buffered saline. Sections were incubated in avidin peroxidase complex (ABC Elite kit, Vector Laboratories, Burlingame, USA) before visualisation using a solution containing 0.1% diaminobenzidine, 0.01% hydrogen peroxide, 0.01% nickel ammonium sulfate, and 0.01% cobalt chloride. Negative controls omitted the primary antibody. Coverslips were applied to stained sections with DPX mounting solution and the slides left to dry overnight before storage in a slide box at room temperature. Light microscopy was used to identify and count the FLI-positive cells in brain regions identified by correlation with the rat brain atlas of [30]. The number of positively stained cells visible in three high power (340 objective) fields in each different population were counted
and averaged. The extent of staining denotes sparse / disperse staining (5–10 high power field); 11 denotes 11–50 high power field; 111 denotes .50 high power field.
was stratified: 1 positive cells per positive cells per positive cells per
2.4. Statistical analysis Data were subjected to statistical analysis by use of SigmaStat statistical analysis software (Version 1.1, Jandel Scientific, CA, USA). Analysis and comparison of the proportion of treatment groups showing FLI in a particular brain region, as a result of thiamin-deficiency, or saline- or glucose-loading, was undertaken using the Fisher Exact test (FET). The extent and intensity of FLI in each brain region of animals in each treatment group was analysed and compared by summing (the number of affected sites 3 the severity score) and applying the Mann–Whitney rank sum (MWRS) test for non-parametric data. Results were considered statistically significant if P,0.05.
3. Results
3.1. FLI and progression of PTD The effects of PTD on the rat brain were assessed by the induction of FLI in the brains of ataxic and opisthotonic animals 4 h after saline treatment and of controls. Salinetreatment (group 1a) or glucose-treatment (group 1b) of thiamin-replete control rats did not result in significant or consistent FLI in any brain region.
3.1.1. Saline-treated ataxic PTD rats FLI was detected in the pons and brainstem in a significantly (P,0.05; FET) greater proportion of salinetreated ataxic PTD animals (group 2) than of saline-treated controls (group 1a; Table 2). In the brainstem of ataxic PTD rats, the pontine nucleus, inferior olive, and medial vestibular nucleus were most affected, whilst FLI was also seen in the granular layer, the medial nucleus and isolated Purkinje cells in several lobes of the cerebellum of these animals. The extent and intensity of FLI in the cortex, thalamus, hypothalamus and cerebellum of the group of ataxic PTD rats was not significantly different from that seen in the control group, although individual animals showed marked FLI in the medial thalamus and lateral hypothalamus. 3.1.2. Saline-treated opisthotonic PTD rats In the midbrain, pons, brainstem and cerebellum, FLI was detected in a significantly greater (P,0.05; FET) proportion of opisthotonic PTD rats (group 4) than in thiamin-replete controls (group 1a; Table 2). FLI in opisthotonic PTD rat brains was detected generally in the same regions as it was in ataxic PTD rats with an overall
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Table 2 Scored FOS immunoreactivity by treatment groups and affected brain nuclei a ,b Brain region
Control1 daily saline (group 1a; n55)
Control1 daily glucose (group 1b; n55)
Ataxic PTD1 daily saline (group 2: n55)
Ataxic PTD1 daily glucose (group 3: n55)
Opisthot. PTD1 saline once (group 4: n55)
Opisthot. PTD1 glucose once (group 5: n58)
Cerebral cortex
Negative 5 / 5
Negative 5 / 5
Negative 3 / 5 1 CC3V 1 / 5 1 DG 1 / 5
Negative 2 / 5 1 AOL 1 / 5 1 LO 1 / 5 1 VLO 1 / 5
Negative 4 / 5 1 CC3V 1 / 5
Negative 6 / 8 11 CC3V 1 / 8 111 DG 1 / 5
Thalamus [
Negative 5 / 5
Negative 5 / 5
Negative 4 / 5 1 3V 1 / 5 1 AM 1 / 5 1 VM 1 / 5
Negative 0 / 5 1 3V 1 / 5 11 AM 2 / 5 1 VM 1 / 5 11 VM 2 / 5
Negative 2 / 5
Negative 3 / 8 1 3V 1 / 8 1 AM 1 / 8 11 AM 2 / 8 11 VM 2 / 8 111 VM 2 / 8
Negative 4 / 5 11 LH 1 / 5
Negative 0 / 5 1 AH 3 / 5 1 LH 3 / 5 11 LH 1 / 5 11 MM 3 / 5 1 PMV 2 / 5
Negative 2 / 5 1 LH 1 / 5 11 LH 2 / 5 1 LM 3 / 5
Hypothalamus [
Midbrain ‡,
§, [, †
Pons and brainstem F,‡
Cerebellum ‡
Negative 5 / 5
Negative 4 / 5 1 OC 1 / 5
1 AM 1 / 5 1 VM 1 / 5 1 VL 2 / 5
Negative 2 / 8 1 1 1 1
LM 4 / 8 MM 2 / 8 MP 2 / 8 PMV 1 / 8
Negative 5 / 5
Negative 5 / 5
Negative 4 / 5 1 DCIC 1 / 5
Negative 0 / 5 1 DCIC 5 / 5
Negative 1 / 5 1 DCIC 2 / 5 11 DCIC 2 / 5
Negative 4 / 8 1 DCIC 3 / 8 11 DCIC 1 / 8
Negative 5 / 5
Negative 5 / 5
Negative 1 / 5 1 IO 2 / 5 11 IO 1 / 5 1 Pn 1 / 5 11 Pn 1 / 5 1 MVe 1 / 5 11 Mve 1 / 5 1 MVPO 1 / 5
Negative 0 / 5 1 IO 1 / 5 11 IO 2 / 5 1 MVe 1 / 5 11 MVe 3 / 5 1 Pn 2 / 5
Negative 1 / 5 1 GiA / GiV 2 / 5 11 IO 2 / 5 11 Pn 2 / 5 1 MVe 2 / 5 11 MVe 2 / 5 1 MVPO 1 / 5
Negative 3 / 8 1 Cent G 2 / 8 1 Gia / V 2 / 8 1 IO 2 / 8 1 Pn 4 / 8 1 Mve 2 / 8 11 Mve 4 / 8 1 Sol 1 / 8
Negative 2 / 5 1 Isol P 2 / 5 1 gran 2 / 5 1 Med 2 / 5 11 Med 1 / 5
Negative 0 / 5 1 Isol P 4 / 5 1 gran 1 / 5 11 gran 2 / 5 111 gran 1 / 5 1 Med 2 / 5 11 Med 2 / 5
Negative 1 / 5 1 Isol P 3 / 5 1 gran 1 / 5 11 gran 2 / 5 1 Med 3 / 5 11 Med 3 / 5
Negative 1 / 8 1 Isol P 3 / 8 1 gran 3 / 8 11 gran 4 / 8 1 Med 3 / 8 11 Med 2 / 8
Negative 5 / 5
Negative 5 / 5 1 Isol P 1 / 5
a Symbols legend: Negative, no staining; 1, 5–10 positive cells / hpf; 11, 11–50 positive cells / hpf; 111, .50 positive cells / hpf. F Denotes a significantly (P,0.05, FET) greater proportion of rats with FLI in the designated brain regions of ataxic PTD rats administered saline (group 2) when compared with controls administered saline (group 1a). ‡ Denotes a significantly (P,0.05, FET) greater proportion of rats with FLI in the designated brain regions of opisthotonic PTD rats administered saline (group 4) when compared with controls administered saline (group 1a). § Denotes a significantly (P,0.05, FET) greater proportion of rats with FLI in the designated brain region when opisthotonic PTD rats given saline daily (group 4) are compared with ataxic PTD rats given saline daily (group 2). [ Denotes a significantly (P,0.05, FET) greater proportion of rats with FLI in the designated brain region of glucose-treated ataxic PTD rats (group 3, daily glucose) compared with saline-treated, ataxic PTD rats (group 2, daily saline). † Denotes a significant difference (P,0.05; MWRS) in the extent and intensity of the FLI in the designated brain region of glucose-treated ataxic PTD rats (group 3, daily glucose) compared with saline-treated, ataxic PTD rats (group 2, daily saline). All FLI detected in examined brain tissue is reported, i.e. from the midline to 2.4 mm from the midline. b Brain region legend: 3V, cells lining wall of third ventricle; AH, anterior hypothalamic area; AM, anterior medial thalamic nucleus; AOL, lateral anterior olfactory nucleus; CC3V, corpus callosum near 3rd ventricle; CentG, central gray matter; DCIC, dorsal cortex of inferior colliculus; DG, dentate gyrus; gran, granular layer of cerebellum; GiA / V, gigantocellular reticular nucleus, alpha and ventral; gran, granular layer of cerebellum; IO, inferior olive; Isol P, isolated Purkinje cells; LH, lateral hypothalamus; LM, lateral mammillary nucleus; LO, lateral orbital cortex; Med, medial nucleus of cerebellum; MM, medial mammillary nucleus; MP, posterior medial mammillary nucleus; MVe, medial vestibular nucleus; MVPO, medioventral periolivary nucleus; OC, optic chiasm; PMV, ventral premammillary nucleus; Pn, pontine nuclei; Sol, solitary tract nucleus; SpVe, superior vestibular nucleus; VL, ventrolateral thalamic nucleus; VLO, ventrolateral orbital cortex; VM, ventromedial thalamic nucleus; VMH, ventromedial hypothalamus.
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slight increase in FLI (P50.09; FET) seen in progression from ataxia to opisthotonus. Of affected brain regions, only the midbrain showed FLI in a significantly greater proportion of opisthotonic PTD rats (group 4) than in ataxic PTD rats (group 2; P,0.05; FET) with a tendency (P50.09; MWRS) towards an increase in the extent and intensity of FLI in the hypothalamus and cerebellum of opisthotonic PTD rats (group 4). The medial vestibular nucleus showed FLI in four of five opisthotonic animals. Other structures with FLI included the anteromedial and ventromedial nuclei of the thalamus (Figs. 1 and 2, A and C), the mammillary bodies of the hypothalamus, the inferior colliculus, the central gray matter, the inferior and superior olives and the granular layer, medial nucleus and isolated Purkinje cells of the cerebellum.
3.2. FLI and glucose-loading of PTD rats 3.2.1. Effect of serial glucose loading of ataxic PTD rats A significantly greater proportion of glucose-treated ataxic PTD rats (group 3; P,0.05; FET) showed FLI in the thalamus, hypothalamus and midbrain than did their saline-treated counterparts (group 2; Table 2). In individual, glucose-loaded animals the extent and intensity of
FLI was most marked in the anteromedial and ventromedial nuclei of the thalamus (Figs. 1 and 2, A and B), the mammillary bodies and the anterior and lateral hypothalamus. The medial vestibular nucleus, inferior olives, lateral hypothalamus and medial thalamus showed FLI in the majority of the ataxic animals after glucose loading. When considering specific nuclei, the extent and intensity of FLI was significantly greater (P,0.05; MWRS) in the lateral hypothalamus and inferior colliculus and only marginally increased in the anteromedial and ventromedial nuclei (0.05,P,0.10; MWRS) of glucose-treated ataxic PTD rats (group 3) when compared with the saline-treated animals (group 2).
3.2.2. Effect of a single glucose load in opisthotonic PTD rats The administration of a single glucose load to PTD rats (group 5) with advanced disease did not have a significant effect on the proportion of affected animals or on the degree or extent of FLI detected in any brain region when compared with those of the saline-treated controls (group 4; Table 2). In some regions, such as the thalamus and hypothalamus, there was a tendency (0.05,P,0.10; MWRS) for an increased density of FLI after glucose loading. At the level of specific nuclei, the intensity of FLI
Fig. 1. Changes in FLI in the anteromedial nucleus of the thalamus of PTD rats after saline or glucose loading. FLI staining increased in extent and density after glucose loading when compared with saline treated counterparts. (A) ataxic PTD rats 4 h after saline treatment; (B) ataxic PTD rats 4 h after glucose loading; (C) opisthotonic PTD rats 4 h after saline treatment; and (D) opisthotonic PTD rats 4 h after glucose loading [320].
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Fig. 2. Changes in FLI in the ventromedial nucleus of the thalamus of PTD rats after saline or glucose loading. FLI staining increased in extent and density after glucose loading of ataxic PTD rats when compared with saline treated counterparts. (A) Ataxic PTD rats 4 h after saline treatment; (B) ataxic PTD rats 4 h after glucose loading; (C) opisthotonic PTD rats 4 h after saline treatment; and (D) opisthotonic PTD rats 4 h after glucose loading [320].
in the anteromedial and ventromedial nuclei of the thalamus and the medial vestibular nucleus of the hypothalamus tended (P50.09; FET) to increase following glucose treatment. Generally the structures showing FLI were common to all of the opisthotonic groups regardless of treatment; i.e. positive cells were grouped near the third ventricle and in the medial thalamus, inferior colliculus, inferior olive, medial vestibular nucleus, pontine nucleus and medial nucleus of the cerebellum. FLI was also assessed in group 6 killed for analysis 24 h after a single glucose load (data not shown) and the results were almost identical with those for group 5, killed 4 h after the glucose load. The only difference between these groups was that the extent and intensity of FLI at 24 h was slightly less than at 4 h, consistent with the known peak period of FLI expression in relation to a timed insult [27].
4. Discussion Several significant findings arise from these experiments. FLI was detected in regions of the ataxic and opisthotonic PTD rat brain that show early metabolic changes and sustain neuropathology later in thiamin de-
ficiency. Small increases in FLI occurred in these regions as neurological disease progressed. Glucose treatment of ataxic PTD rats resulted in significant selective increases in FLI within these same brain regions.
4.1. FLI as a marker of neurons at risk of damage in the PTD rat brain At the early stage of PTD characterised by the onset of ataxia, FLI was detected in the brainstem and cerebellum of the majority of rats and in the thalamus, hypothalamus, inferior colliculus and cortex of individual animals. FLI was primarily localised to structures that show corresponding metabolic and functional changes early in thiamin deficiency. Decreases in the activity of the thiamin-dependent enzyme OGDH have been measured in these structures, particularly the vestibular nucleus and inferior olives, before the onset of ataxia [15,4,5]. In rats with advanced PTD characterised by the onset of opisthotonus, FLI was detected in the thalamus, hypothalamus, inferior colliculus, brainstem and cerebellum of the majority of animals. In other studies the onset of opisthotonus typically coincides with the appearance of histopathological changes in the brainstem, inferior col-
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liculus, thalamus and hypothalamus [40]. Metabolic studies have demonstrated continuing regional decreases in OGDH activity at this time [5], whilst progressive increases in ECF glutamate concentrations have been measured by microdialysis in the thalamus as thiamin deficiency progresses [19,26]. FLI was detected in the medial vestibular nucleus and mammillary bodies which most commonly show histopathology in PTD [12,40]. Indeed, the structures in which FLI was detected (Table 2) typically show both light and electron microscopic pathology in advanced PTD in rats [8,40,2]. Fos induction in PTD rats with advanced neurological disease has been examined in two other studies. Hazell et al. [20] examined four brain regions of the PTD rat and detected increased amounts of c-Fos protein in the medial thalamus and inferior colliculus compared with those in the parietal and occipital cortices of non-convulsing animals with opisthotonus and loss of righting reflex. Munujos et al. [29] examined c-fos induction in three areas in the opisthotonic PTD brain and confirmed the presence of c-fos mRNA in the ventromedial hypothalamus and inferior colliculus. There are several differences between the data from this study and those from [29]. First, the latter authors did not detect c-Fos in the vestibular nuclei or mammillary bodies of PTD rats, though it was seen in the majority of our PTD rats. Considering the advanced stage of PTD, the absence of c-Fos from these nuclei in Munujos’ study might reflect extensive neuronal loss occurring after the onset of opisthotonus [40] or limited tissue sampling. Munujos did report significant c-fos mRNA in the paraventricular thalamic nucleus and dentate gyrus and CA1 of the hippocampus of their PTD rats with advanced neurological disease. Induction of c-fos in the paraventricular nucleus is associated with severe food and water deprivation of rats [3]. Since our rats were still capable of feeding, this may explain the difference between our data and those of Munujos for the paraventricular nucleus. Expression of c-fos in the hippocampus is typically associated with seizure activity [28] and has been attributed to convulsions characteristic of end-stage PTD. The hippocampus rarely shows histopathology in PTD rats even 12 h after the onset of convulsions [40,25] although it does exhibit decreased activity of thiamin-dependent enzymes [5] and persistent hyper-intensity on proton magnetic resonance imaging [23]. The lack of FLI in the hippocampus of our rats might be because they were not convulsing; or because its presence was limited to the most lateral and caudal part of the CA1 region and therefore outside our observation. The close correlation between the loci of FLI in the PTD rat brain with the loci of changes in cerebral metabolism, morphology and function reported by other investigators, and with later neuropathology, suggests that FLI does mark neuronal populations at risk of death in the PTD model.
4.2. The effects of glucose loading on FLI in the PTD rat brain This is the first report of the effects of glucose loading on Fos expression in the PTD rat brain. Serial glucose loading of ataxic PTD rats significantly increased FLI in the thalamus, hypothalamus, and midbrain at the onset of ataxia (compare groups 3 and 2, Table 2). Glucose loading may increase the blood glucose concentration to approximately 50 mM [47], whereas the normal blood glucose concentration that results from dietary glucose may be correlated with the smaller amount of FLI in the brains of saline-treated ataxic rats. Indeed, glucose or other carbohydrate from dietary sources may play a role in the pathogenesis of WE. The disease does not occur in general starvation but is precipitated by administration of carbohydrate to starving humans; i.e. carbohydrate administration can unmask covert thiamin deficiency [42]. The particular brain structures containing FLI after glucose loading, such as the inferior olive, the medial vestibular nucleus, the mammillary bodies, the lateral hypothalamus and the medial nuclei of the thalamus, characteristically show decreased activity of OGDH [5], changes in blood brain barrier (BBB) function [6] and light microscopic and electron microscopic pathology in PTD [40,7]. Furthermore, the structures in which FLI was increased after glucose loading included those already affected by PTD in rats whose only source of glucose was their diet. Once PTD rats were opisthotonic, glucose loading did not lead to a further significant increase in brain FLI. There are several possible reasons for these observations. First, it is possible that glucose administration increases the rate of progression of PTD disease up to a ceiling that coincides with the onset of opisthotonus and at which the expression of Fos proteins is already maximal. Second, it is possible that several glucose loads are necessary to produce significant increases of FLI in the brain of opisthotonic PTD rats, but in our study more than one dose is impracticable in animals rapidly approaching death. Third, low energy levels in neurons in advanced PTD [1] might not be sufficient to support biosynthesis of more Fos proteins.
4.3. Glucose loading and brain glucose metabolism Following the administration of a glucose load, similar to that used here, the brain glucose concentration as detected by in vivo NMR spectroscopy rises by 3- to 5-fold in PTD rats but only 1.2- to 1.5-fold in controls [34]. Below we discuss the metabolic fate of this glucose. However, Hakim and Pappius [18] have reported that 2-deoxyglucose uptake into the brains of ataxic PTD rat is impaired by up to 50% and attribute this to a decrease in glycolysis. These data appear to contradict our results, but the clinical observation that WE can be precipitated by a glucose load in sub-clinical thiamin deficiency in humans
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[42] and in rats [47] argues that a glucose load does affect the thiamin-deficient brain, presumably requiring uptake and metabolism of glucose by that brain. Perhaps the impairment of the BBB in thiamin deficiency [6] adversely affects brain retention of the minute tracer dose of 2deoxy-[ 14 C]glucose to yield a result that is unrepresentative of the glucose uptake and metabolism of the thiamindeficient brain under a glucose load. Further, the interpretation [17,18], namely that the 2-deoxy-[ 14 C]glucose data mean that glycolysis is significantly impaired in the thiamin-deficient brain at the pre-symptomatic stage is untenable for several reasons. First, [ 14 C]glucose oxidation by brain slices from PTD rats measured by [ 14 C]CO 2 production is decreased little and only in concert with a decrease in the activity of the TCA cycle enzyme, OGDH [15]. Second, a glucose load causes accumulation of brain glutamate [34], as discussed below, requiring uptake of the glucose into the brain and its metabolism to glutamate.
4.4. Correlation of FLI with neurological effects of glucose loading The increase in neuronal FLI in response to a glucose dose follows within 4 h of the onset of behavioural and neurochemical responses to glucose loading of ataxic PTD rats [34,46]. The neurochemical changes in ataxic rats include increases in cerebral glutamate derivatives that commence within 15 min after glucose loading and persist beyond 120 min [34], consistent with increased brain ECF glutamate concentrations measured by microdialysis in the absence of glucose loading [19,26]. Behavioural changes include episodes of greatly increased ataxia and impairment of the righting reflex commencing within 40 min and persisting beyond 2 h [46]. The temporal relationship of these changes is consistent with the proposition that the increased severity of ataxia and the onset of Fos expression are both due to the earlier increase in brain glutamate concentration.
4.5. A unifying hypothesis for a glutamateNMDA -receptor mediated process in PTD rats The combination of data presented here together with that of other investigations provides considerable circumstantial evidence that allows construction of a plausible sequence of events that are consistent with, and unify, the observations arising from glucose loading of PTD rats. This hypothesis is built upon the evidence for a role of glutamate excitotoxicity in PTD in rats. The trigger for the induction of Fos genes in neurons of the brains of PTD rats could be the selective decrease in the activity of the OGDH enzyme which occurs prior to the onset of ataxia and again before the onset of opisthotonus [15,4,5]. A decrease in OGDH activity is likely to have a greater impact on neurons than glial cells because
225
glial cells have greater metabolic flexibility and also provide neurons with substrates. As a rate-limiting step of the TCA cycle [15], declining OGDH activity could lead to the accumulation of intracellular glutamate and also adversely affect cellular energy levels in the PTD rat brain [1] limiting the function of ATP-dependent pumps of neurons or glial cells. Failure to maintain cellular electrolyte homeostasis could activate swelling-induced anion transporters on glial cell plasma membranes and the release of intracellular glutamate [24]. Changes in glial cell morphology, such as swelling, are the earliest features of thiamin deficiency detected by electron microscopy and occur in the midst of intact neurons [12,32]. Increases in ECF glutamate concentration and disruption of the glutamate / glutamine cycle could also result from failure of glutamate transporters on glial cells [35]. As thiamin deficiency progresses and OGDH activity and cellular energy reserves further decline, ECF glutamate concentrations in affected brain structures could increase, as has been measured in the thalamus of PTD rats [19,26]. Increased ECF concentrations of glutamate would lead to glutamate NMDA -receptor stimulation and thus increased expression of Fos proteins. This postulated sequence of events is consistent with the observations reported here.
4.6. The glucose loading model of Wernicke’ s encephalopathy in the rat Our glucose loading model of Wernicke’s encephalopathy in the rat has been developed and characterised in terms of the neurological, neurochemical and neuromorphological events occurring over an acute timeframe of 15 min to 4 h. Investigators now have the opportunity for continuous collection and correlation of data obtained within a defined short time frame after a specific stimulus. The use of in vivo nuclear magnetic resonance imaging and spectroscopy and of in vivo microdialysis techniques allows the continuous assessment of short-term functional and metabolic changes occurring in the brain after glucose loading of individual animals at different stages of thiamin deficiency. These data can be correlated with the behavioural consequences of glucose loading monitored over the same time frame. Immunohistochemical techniques allow the identification of cellular and molecular events and their timed sequence after glucose loading in thiamin deficiency. Correlation of the sequence of all events occurring after glucose loading in the brains of TD rats should contribute greatly to understanding the pathogenesis of Wernicke’s encephalopathy.
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Interactive report
Glucose induced IEG expression in the thiamin-deficient rat brain 1 Craig Zimitat 2 , Peter F. Nixon* Department of Biochemistry, The University of Queensland, Brisbane, Queensland, Australia 4072 Accepted 8 December 2000
Abstract Glucose loading of rats made thiamin deficient by dietary deprivation of thiamin and the administration of pyrithiamin (40 mg / 100 g, i.p.) precipitates an acute neuropathy, a model of Wernicke’s encephalopathy in man (Zimitat and Nixon, Metab. Brain Dis. 1999;14:1–20). Immunohistochemical detection of Fos proteins was used as a marker to identify neuronal populations in the thiamin-deficient rat brain affected by glucose loading. As thiamin deficiency progressed, the extent and intensity of Fos-like immunoreactivity (FLI) in brain structures typically affected by thiamin deficiency (the thalamus, mammillary bodies, inferior colliculus, vestibular nucleus and inferior olives) were markedly increased when compared to thiamin-replete controls. Glucose loading for 1–3 days further increased the intensity of FLI in these same regions, consistent with a dependence of Fos expression on carbohydrate metabolism as well as on thiamin deficiency. The timed acute changes that follow a bolus glucose load administered to thiamin-deficient animals may provide a sequential account of events in the pathogenesis of brain damage in this model of Wernicke’s encephalopathy. 2001 Elsevier Science B.V. All rights reserved. Theme: Disorders of the neurons system Topic: Neurotoxicity Keywords: IEG expression; Thiamin deficiency; Wernicke’s encephalography; Brain damage
1. Introduction Induction of thiamin deficiency in the rat by pyrithiamin produces a characteristic sequence of signs of neurological disease and selective pathological lesions in the nuclei of the thalamus and hypothalamus, the collicular plate, vestibular nucleus, inferior olive and mammillary bodies [40]. Glutamate NMDA receptor-mediated excitotoxicity has been proposed as a mechanism for neuronal cell death in pyrithiamin-induced thiamin deficiency (PTD) in rats on the basis of histological studies [45], microdialysis [19,26] and enzymatic studies. Decreased activity of the thiamindependent enzyme 2-oxo-glutarate dehydrogenase (OGDH) is associated with the onset of neurological signs and progress of thiamin deficiency and could lead to the accumulation of glutamate in the brain [4,5,16]. Indeed, 1
Published on the World Wide Web 3 January 2001. Present address: Griffith Institute for Higher Education, Griffith University, Brisbane, Australia, 4111. *Corresponding author. Fax: 161-7-3365-4613. E-mail address: [email protected] (P.F. Nixon). 2
selective increases in extracellular glutamate concentrations have been measured in specific sites of the brain of rats with neurological signs of thiamin deficiency before neuronal death occurred in these sites [19,26,39]. In addition, the characteristic features of neuronal cell death in PTD [2,45] are consistent with those of excitotoxic cell death triggered by glutamate [22]. The strongest evidence in support of a glutamate NMDA -mediated process in PTD is that MK801 (a glutamate NMDA receptor antagonist) when administered to PTD rats blocked the localised increases in extracellular glutamate concentration in the brain [26] and also significantly attenuated neuronal cell death [33]. In thiamin deficiency, the probable source of increased extracellular glutamate is increased intracellular glutamate, accumulated when glucose metabolism is blocked at OGDH. Wernicke’s encephalopathy (WE) is a consequence of thiamin deficiency in humans [42]. WE is not observed in general starvation but a glucose load can precipitate WE in humans with unsuspected thiamin deficiency [42] and exacerbate neurological symptoms in individuals with WE [31,41]. Furthermore, glucose loading of PTD and thiamin-deficient (TD) rats precipitates an
0006-8993 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )03297-2
C. Zimitat, P.F. Nixon / Brain Research 892 (2001) 218 – 227
exacerbation of ataxia [46] preceded by increased blood brain barrier (BBB) permeability and hyper-intensities in vulnerable brain regions detected by magnetic resonance imaging [44,23]. These changes are accompanied by an increase in the total concentration of brain glutamate or glutamate-derivatives (e.g. GABA, glutamine) [34]. Thus glucose metabolism is diverted into glutamate accumulation, at least in part, in thiamin deficiency. Extracellular stimulation of neurons via the glutamate NMDA receptor triggers a series of intracellular events [38]. The basal level of c-fos expression in the brain is low but it is rapidly and transiently increased after some stimulus causes an increase in extracellular calcium, e.g. via the glutamate NMDA receptor or voltage sensitive calcium channels (VSCCs). Stimulated neurons contain c-Fos, hence mapping c-Fos immunoreactivity provides a method for mapping neuronal pathways activated in response to specific stimuli [36]. The expression of c-fos has been associated also with excitotoxic cell death, programmed cell death and delayed neuronal death [37,43]. As a consequence, the expression of c-fos has also been used as a cellular marker of neuronal activity and potentially fatal neuronal damage [11,21]. We report, for the first time, experiments that utilise the expression of c-fos to identify specific neuronal populations in the PTD rat brain that are affected by glucose loading before the onset of neurological signs or of overt histopathology.
2. Materials and methods
2.1. Animals and diets The animals used in these experiments (n538), their housing and the composition of the cereal based control rat chow and the thiamin-deficient diet have been described previously [47,46]. The latter diet contained 30% glucose and 15% starch. All animal procedures were authorised by the Animal Ethics and Experimentation Committee of The University of Queensland.
2.2. Reagents and equipment The following chemicals were purchased from BDH Chemicals Australia Pty Ltd., Kilsyth, NSW: disodium hydrogen phosphate, potassium dihydrogen phosphate and sodium chloride, whilst xylene, hydrogen peroxide and ethanol (AR) were supplied by APS Ajax Finechem, Auburn, NSW. Triton X-100, pyrithiamin, sodium azide, diaminobenzidine, nickel ammonium sulphate and bovine serum albumin were purchased from Sigma–Aldrich Australia Pty Ltd., Castle Hill, NSW. Boehringer Mannheim Australia Pty Ltd., Castle Hill, NSW supplied cobalt chloride. Sectioning was carried out on a freezing microtome (Minotome, Damon / IEC, MA, USA). OCT com-
219
pound for embedding was purchased from Miles Inc., IN, USA. Light microscopy and photography were performed using an Axioplan2 microscope (Carl Zeiss Jena GmbH, Germany). Polyclonal sheep anti-Fos antibodies were purchased from Cambridge Research Biochemicals, Cambridge, UK. These antibodies were raised against c-Fos amino acids 127–152. The term Fos-like immunoreactivity (FLI) is used because the antibody precipitates Fos proteins (c-Fos and FosB) and cross-reactive Fos-related antigens (Fra-1 and Fra-2) [9,13,14]. Differences in FLI, detected in the brains of ataxic and opisthotonic PTD rats vs. controls, therefore represent cumulative changes in combined c-Fos, FosB, Fra-1 and Fra-2 immunoreactivity. Increases in FLI 4 h after the saline or glucose load primarily reflect changes in expression of c-fos and the presence of c-Fos protein, because of its brief half life and short induction time [27].
2.3. Effect of glucose loading on c-fos expression in the brains of pyrithiamin-treated thiamin-deficient ( PTD) rats Wistar rats weighing 140–180 g were distributed into six groups for these experiments (Table 1). All animals in groups 2–5 received thiamin-deficient diet and pyrithiamin (PT) injections (40 mg / 100 g, i.p.) for 5 consecutive days out of every 7 days starting from day 1, and additional injections of saline or glucose starting on day 7 as described (Table 1 and hereafter). On the day of the onset of ataxia (days 7–9), rats in groups 2 and 3 received one more dose of PT and glucose or saline and were killed for analysis 4 h later. Groups 1a and 1b control animals were also harvested at this time, 4 h after their saline or glucose injection. On the day after the onset of opisthotonus (11–14 days of PTD treatment), animals in groups 4 and 5 received one more dose of PT and a single glucose or saline load, then they were killed for analysis 4 h later. A group 6 (n55, not shown in Tables) was treated identically to group 5 except that they were killed for analysis 24 h after the single glucose load. Animals that had lost their righting reflex or were observed convulsing were excluded from experiments. The brains were perfused in vivo in preparation for immunohistochemistry [47]. Perfused brain tissue was dissected out and cryoprotected by immersion in 0.3 M sucrose in 0.01 M phosphate buffer, pH 7.4 for 72 h then embedded with OCT compound. Sagittal sections (40 mm) of the right hemispheres were cut in series, from the midline, on a freezing microtome. Brain tissue was sampled for immuno-histochemistry between the midline and 2.4 mm lateral from the midline of the brain, which excludes the lateral vestibular nucleus and the lateral caudal CA1 regions of the hippocampus. Sections were processed for immunohistochemical detection of Fos proteins as freefloating sections in tissue culture plates using the method
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Table 1 Details of treatments for each group in these experiments Group
Treatment
1a
Saline treated control animals (n55) Received standard thiamin-replete rat chow and daily intraperitoneal (i.p.) saline injections commencing on day 7 Harvested at the same time as rats in Group 2.
1b
Glucose treated control animals (n55) Received standard thiamin-replete rat chow and daily glucose injections (10 g / kg; i.p.) commencing on day 7 Harvested at the same time as rats in Group 3
2
PTD animals with serial saline treatments (n55) Thiamin deficiency induced by daily PT injections (5 out of 7 days) and provision of TD diet Received daily saline injections (2 ml, i.p.) every day from day 7 until the onset of ataxia Killed for analysis 4 h after a final dose of saline, which followed the onset of ataxia
3
PTD animals with serial glucose treatments (n55) Thiamin deficiency induced by daily PT injections (5 out of 7 days) and provision of TD diet Received glucose loads (10 g / kg, i.p.) every day from day 7 until the onset of ataxia Killed for analysis 4 h after a final dose of glucose, which followed the onset of ataxia
4
PTD animals with single saline treatment (n55). Thiamin deficiency induced by daily PT injections (5 out of 7 days) and provision of TD diet Received one saline injection (2.5 ml, i.p.) 24 h after the onset of opisthotonus Killed for analysis 4 h after the saline injection
5
PTD animals with single glucose treatment (n58) Thiamin deficiency induced by daily PT injections (5 out of 7 days) and provision of TD diet Received one glucose load only (10 g / kg, i.p.) 24 h after the onset of opisthotonus Killed for analysis 4 h after the glucose load
of [10]. Antibody dilutions were: anti-Fos antibodies 1:1000 dilution in 1% bovine serum albumin (BSA), biotinylated secondary antibodies 1:400 dilution in 1% BSA in phosphate buffered saline. Sections were incubated in avidin peroxidase complex (ABC Elite kit, Vector Laboratories, Burlingame, USA) before visualisation using a solution containing 0.1% diaminobenzidine, 0.01% hydrogen peroxide, 0.01% nickel ammonium sulfate, and 0.01% cobalt chloride. Negative controls omitted the primary antibody. Coverslips were applied to stained sections with DPX mounting solution and the slides left to dry overnight before storage in a slide box at room temperature. Light microscopy was used to identify and count the FLI-positive cells in brain regions identified by correlation with the rat brain atlas of [30]. The number of positively stained cells visible in three high power (340 objective) fields in each different population were counted
and averaged. The extent of staining denotes sparse / disperse staining (5–10 high power field); 11 denotes 11–50 high power field; 111 denotes .50 high power field.
was stratified: 1 positive cells per positive cells per positive cells per
2.4. Statistical analysis Data were subjected to statistical analysis by use of SigmaStat statistical analysis software (Version 1.1, Jandel Scientific, CA, USA). Analysis and comparison of the proportion of treatment groups showing FLI in a particular brain region, as a result of thiamin-deficiency, or saline- or glucose-loading, was undertaken using the Fisher Exact test (FET). The extent and intensity of FLI in each brain region of animals in each treatment group was analysed and compared by summing (the number of affected sites 3 the severity score) and applying the Mann–Whitney rank sum (MWRS) test for non-parametric data. Results were considered statistically significant if P,0.05.
3. Results
3.1. FLI and progression of PTD The effects of PTD on the rat brain were assessed by the induction of FLI in the brains of ataxic and opisthotonic animals 4 h after saline treatment and of controls. Salinetreatment (group 1a) or glucose-treatment (group 1b) of thiamin-replete control rats did not result in significant or consistent FLI in any brain region.
3.1.1. Saline-treated ataxic PTD rats FLI was detected in the pons and brainstem in a significantly (P,0.05; FET) greater proportion of salinetreated ataxic PTD animals (group 2) than of saline-treated controls (group 1a; Table 2). In the brainstem of ataxic PTD rats, the pontine nucleus, inferior olive, and medial vestibular nucleus were most affected, whilst FLI was also seen in the granular layer, the medial nucleus and isolated Purkinje cells in several lobes of the cerebellum of these animals. The extent and intensity of FLI in the cortex, thalamus, hypothalamus and cerebellum of the group of ataxic PTD rats was not significantly different from that seen in the control group, although individual animals showed marked FLI in the medial thalamus and lateral hypothalamus. 3.1.2. Saline-treated opisthotonic PTD rats In the midbrain, pons, brainstem and cerebellum, FLI was detected in a significantly greater (P,0.05; FET) proportion of opisthotonic PTD rats (group 4) than in thiamin-replete controls (group 1a; Table 2). FLI in opisthotonic PTD rat brains was detected generally in the same regions as it was in ataxic PTD rats with an overall
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Table 2 Scored FOS immunoreactivity by treatment groups and affected brain nuclei a ,b Brain region
Control1 daily saline (group 1a; n55)
Control1 daily glucose (group 1b; n55)
Ataxic PTD1 daily saline (group 2: n55)
Ataxic PTD1 daily glucose (group 3: n55)
Opisthot. PTD1 saline once (group 4: n55)
Opisthot. PTD1 glucose once (group 5: n58)
Cerebral cortex
Negative 5 / 5
Negative 5 / 5
Negative 3 / 5 1 CC3V 1 / 5 1 DG 1 / 5
Negative 2 / 5 1 AOL 1 / 5 1 LO 1 / 5 1 VLO 1 / 5
Negative 4 / 5 1 CC3V 1 / 5
Negative 6 / 8 11 CC3V 1 / 8 111 DG 1 / 5
Thalamus [
Negative 5 / 5
Negative 5 / 5
Negative 4 / 5 1 3V 1 / 5 1 AM 1 / 5 1 VM 1 / 5
Negative 0 / 5 1 3V 1 / 5 11 AM 2 / 5 1 VM 1 / 5 11 VM 2 / 5
Negative 2 / 5
Negative 3 / 8 1 3V 1 / 8 1 AM 1 / 8 11 AM 2 / 8 11 VM 2 / 8 111 VM 2 / 8
Negative 4 / 5 11 LH 1 / 5
Negative 0 / 5 1 AH 3 / 5 1 LH 3 / 5 11 LH 1 / 5 11 MM 3 / 5 1 PMV 2 / 5
Negative 2 / 5 1 LH 1 / 5 11 LH 2 / 5 1 LM 3 / 5
Hypothalamus [
Midbrain ‡,
§, [, †
Pons and brainstem F,‡
Cerebellum ‡
Negative 5 / 5
Negative 4 / 5 1 OC 1 / 5
1 AM 1 / 5 1 VM 1 / 5 1 VL 2 / 5
Negative 2 / 8 1 1 1 1
LM 4 / 8 MM 2 / 8 MP 2 / 8 PMV 1 / 8
Negative 5 / 5
Negative 5 / 5
Negative 4 / 5 1 DCIC 1 / 5
Negative 0 / 5 1 DCIC 5 / 5
Negative 1 / 5 1 DCIC 2 / 5 11 DCIC 2 / 5
Negative 4 / 8 1 DCIC 3 / 8 11 DCIC 1 / 8
Negative 5 / 5
Negative 5 / 5
Negative 1 / 5 1 IO 2 / 5 11 IO 1 / 5 1 Pn 1 / 5 11 Pn 1 / 5 1 MVe 1 / 5 11 Mve 1 / 5 1 MVPO 1 / 5
Negative 0 / 5 1 IO 1 / 5 11 IO 2 / 5 1 MVe 1 / 5 11 MVe 3 / 5 1 Pn 2 / 5
Negative 1 / 5 1 GiA / GiV 2 / 5 11 IO 2 / 5 11 Pn 2 / 5 1 MVe 2 / 5 11 MVe 2 / 5 1 MVPO 1 / 5
Negative 3 / 8 1 Cent G 2 / 8 1 Gia / V 2 / 8 1 IO 2 / 8 1 Pn 4 / 8 1 Mve 2 / 8 11 Mve 4 / 8 1 Sol 1 / 8
Negative 2 / 5 1 Isol P 2 / 5 1 gran 2 / 5 1 Med 2 / 5 11 Med 1 / 5
Negative 0 / 5 1 Isol P 4 / 5 1 gran 1 / 5 11 gran 2 / 5 111 gran 1 / 5 1 Med 2 / 5 11 Med 2 / 5
Negative 1 / 5 1 Isol P 3 / 5 1 gran 1 / 5 11 gran 2 / 5 1 Med 3 / 5 11 Med 3 / 5
Negative 1 / 8 1 Isol P 3 / 8 1 gran 3 / 8 11 gran 4 / 8 1 Med 3 / 8 11 Med 2 / 8
Negative 5 / 5
Negative 5 / 5 1 Isol P 1 / 5
a Symbols legend: Negative, no staining; 1, 5–10 positive cells / hpf; 11, 11–50 positive cells / hpf; 111, .50 positive cells / hpf. F Denotes a significantly (P,0.05, FET) greater proportion of rats with FLI in the designated brain regions of ataxic PTD rats administered saline (group 2) when compared with controls administered saline (group 1a). ‡ Denotes a significantly (P,0.05, FET) greater proportion of rats with FLI in the designated brain regions of opisthotonic PTD rats administered saline (group 4) when compared with controls administered saline (group 1a). § Denotes a significantly (P,0.05, FET) greater proportion of rats with FLI in the designated brain region when opisthotonic PTD rats given saline daily (group 4) are compared with ataxic PTD rats given saline daily (group 2). [ Denotes a significantly (P,0.05, FET) greater proportion of rats with FLI in the designated brain region of glucose-treated ataxic PTD rats (group 3, daily glucose) compared with saline-treated, ataxic PTD rats (group 2, daily saline). † Denotes a significant difference (P,0.05; MWRS) in the extent and intensity of the FLI in the designated brain region of glucose-treated ataxic PTD rats (group 3, daily glucose) compared with saline-treated, ataxic PTD rats (group 2, daily saline). All FLI detected in examined brain tissue is reported, i.e. from the midline to 2.4 mm from the midline. b Brain region legend: 3V, cells lining wall of third ventricle; AH, anterior hypothalamic area; AM, anterior medial thalamic nucleus; AOL, lateral anterior olfactory nucleus; CC3V, corpus callosum near 3rd ventricle; CentG, central gray matter; DCIC, dorsal cortex of inferior colliculus; DG, dentate gyrus; gran, granular layer of cerebellum; GiA / V, gigantocellular reticular nucleus, alpha and ventral; gran, granular layer of cerebellum; IO, inferior olive; Isol P, isolated Purkinje cells; LH, lateral hypothalamus; LM, lateral mammillary nucleus; LO, lateral orbital cortex; Med, medial nucleus of cerebellum; MM, medial mammillary nucleus; MP, posterior medial mammillary nucleus; MVe, medial vestibular nucleus; MVPO, medioventral periolivary nucleus; OC, optic chiasm; PMV, ventral premammillary nucleus; Pn, pontine nuclei; Sol, solitary tract nucleus; SpVe, superior vestibular nucleus; VL, ventrolateral thalamic nucleus; VLO, ventrolateral orbital cortex; VM, ventromedial thalamic nucleus; VMH, ventromedial hypothalamus.
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slight increase in FLI (P50.09; FET) seen in progression from ataxia to opisthotonus. Of affected brain regions, only the midbrain showed FLI in a significantly greater proportion of opisthotonic PTD rats (group 4) than in ataxic PTD rats (group 2; P,0.05; FET) with a tendency (P50.09; MWRS) towards an increase in the extent and intensity of FLI in the hypothalamus and cerebellum of opisthotonic PTD rats (group 4). The medial vestibular nucleus showed FLI in four of five opisthotonic animals. Other structures with FLI included the anteromedial and ventromedial nuclei of the thalamus (Figs. 1 and 2, A and C), the mammillary bodies of the hypothalamus, the inferior colliculus, the central gray matter, the inferior and superior olives and the granular layer, medial nucleus and isolated Purkinje cells of the cerebellum.
3.2. FLI and glucose-loading of PTD rats 3.2.1. Effect of serial glucose loading of ataxic PTD rats A significantly greater proportion of glucose-treated ataxic PTD rats (group 3; P,0.05; FET) showed FLI in the thalamus, hypothalamus and midbrain than did their saline-treated counterparts (group 2; Table 2). In individual, glucose-loaded animals the extent and intensity of
FLI was most marked in the anteromedial and ventromedial nuclei of the thalamus (Figs. 1 and 2, A and B), the mammillary bodies and the anterior and lateral hypothalamus. The medial vestibular nucleus, inferior olives, lateral hypothalamus and medial thalamus showed FLI in the majority of the ataxic animals after glucose loading. When considering specific nuclei, the extent and intensity of FLI was significantly greater (P,0.05; MWRS) in the lateral hypothalamus and inferior colliculus and only marginally increased in the anteromedial and ventromedial nuclei (0.05,P,0.10; MWRS) of glucose-treated ataxic PTD rats (group 3) when compared with the saline-treated animals (group 2).
3.2.2. Effect of a single glucose load in opisthotonic PTD rats The administration of a single glucose load to PTD rats (group 5) with advanced disease did not have a significant effect on the proportion of affected animals or on the degree or extent of FLI detected in any brain region when compared with those of the saline-treated controls (group 4; Table 2). In some regions, such as the thalamus and hypothalamus, there was a tendency (0.05,P,0.10; MWRS) for an increased density of FLI after glucose loading. At the level of specific nuclei, the intensity of FLI
Fig. 1. Changes in FLI in the anteromedial nucleus of the thalamus of PTD rats after saline or glucose loading. FLI staining increased in extent and density after glucose loading when compared with saline treated counterparts. (A) ataxic PTD rats 4 h after saline treatment; (B) ataxic PTD rats 4 h after glucose loading; (C) opisthotonic PTD rats 4 h after saline treatment; and (D) opisthotonic PTD rats 4 h after glucose loading [320].
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Fig. 2. Changes in FLI in the ventromedial nucleus of the thalamus of PTD rats after saline or glucose loading. FLI staining increased in extent and density after glucose loading of ataxic PTD rats when compared with saline treated counterparts. (A) Ataxic PTD rats 4 h after saline treatment; (B) ataxic PTD rats 4 h after glucose loading; (C) opisthotonic PTD rats 4 h after saline treatment; and (D) opisthotonic PTD rats 4 h after glucose loading [320].
in the anteromedial and ventromedial nuclei of the thalamus and the medial vestibular nucleus of the hypothalamus tended (P50.09; FET) to increase following glucose treatment. Generally the structures showing FLI were common to all of the opisthotonic groups regardless of treatment; i.e. positive cells were grouped near the third ventricle and in the medial thalamus, inferior colliculus, inferior olive, medial vestibular nucleus, pontine nucleus and medial nucleus of the cerebellum. FLI was also assessed in group 6 killed for analysis 24 h after a single glucose load (data not shown) and the results were almost identical with those for group 5, killed 4 h after the glucose load. The only difference between these groups was that the extent and intensity of FLI at 24 h was slightly less than at 4 h, consistent with the known peak period of FLI expression in relation to a timed insult [27].
4. Discussion Several significant findings arise from these experiments. FLI was detected in regions of the ataxic and opisthotonic PTD rat brain that show early metabolic changes and sustain neuropathology later in thiamin de-
ficiency. Small increases in FLI occurred in these regions as neurological disease progressed. Glucose treatment of ataxic PTD rats resulted in significant selective increases in FLI within these same brain regions.
4.1. FLI as a marker of neurons at risk of damage in the PTD rat brain At the early stage of PTD characterised by the onset of ataxia, FLI was detected in the brainstem and cerebellum of the majority of rats and in the thalamus, hypothalamus, inferior colliculus and cortex of individual animals. FLI was primarily localised to structures that show corresponding metabolic and functional changes early in thiamin deficiency. Decreases in the activity of the thiamin-dependent enzyme OGDH have been measured in these structures, particularly the vestibular nucleus and inferior olives, before the onset of ataxia [15,4,5]. In rats with advanced PTD characterised by the onset of opisthotonus, FLI was detected in the thalamus, hypothalamus, inferior colliculus, brainstem and cerebellum of the majority of animals. In other studies the onset of opisthotonus typically coincides with the appearance of histopathological changes in the brainstem, inferior col-
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liculus, thalamus and hypothalamus [40]. Metabolic studies have demonstrated continuing regional decreases in OGDH activity at this time [5], whilst progressive increases in ECF glutamate concentrations have been measured by microdialysis in the thalamus as thiamin deficiency progresses [19,26]. FLI was detected in the medial vestibular nucleus and mammillary bodies which most commonly show histopathology in PTD [12,40]. Indeed, the structures in which FLI was detected (Table 2) typically show both light and electron microscopic pathology in advanced PTD in rats [8,40,2]. Fos induction in PTD rats with advanced neurological disease has been examined in two other studies. Hazell et al. [20] examined four brain regions of the PTD rat and detected increased amounts of c-Fos protein in the medial thalamus and inferior colliculus compared with those in the parietal and occipital cortices of non-convulsing animals with opisthotonus and loss of righting reflex. Munujos et al. [29] examined c-fos induction in three areas in the opisthotonic PTD brain and confirmed the presence of c-fos mRNA in the ventromedial hypothalamus and inferior colliculus. There are several differences between the data from this study and those from [29]. First, the latter authors did not detect c-Fos in the vestibular nuclei or mammillary bodies of PTD rats, though it was seen in the majority of our PTD rats. Considering the advanced stage of PTD, the absence of c-Fos from these nuclei in Munujos’ study might reflect extensive neuronal loss occurring after the onset of opisthotonus [40] or limited tissue sampling. Munujos did report significant c-fos mRNA in the paraventricular thalamic nucleus and dentate gyrus and CA1 of the hippocampus of their PTD rats with advanced neurological disease. Induction of c-fos in the paraventricular nucleus is associated with severe food and water deprivation of rats [3]. Since our rats were still capable of feeding, this may explain the difference between our data and those of Munujos for the paraventricular nucleus. Expression of c-fos in the hippocampus is typically associated with seizure activity [28] and has been attributed to convulsions characteristic of end-stage PTD. The hippocampus rarely shows histopathology in PTD rats even 12 h after the onset of convulsions [40,25] although it does exhibit decreased activity of thiamin-dependent enzymes [5] and persistent hyper-intensity on proton magnetic resonance imaging [23]. The lack of FLI in the hippocampus of our rats might be because they were not convulsing; or because its presence was limited to the most lateral and caudal part of the CA1 region and therefore outside our observation. The close correlation between the loci of FLI in the PTD rat brain with the loci of changes in cerebral metabolism, morphology and function reported by other investigators, and with later neuropathology, suggests that FLI does mark neuronal populations at risk of death in the PTD model.
4.2. The effects of glucose loading on FLI in the PTD rat brain This is the first report of the effects of glucose loading on Fos expression in the PTD rat brain. Serial glucose loading of ataxic PTD rats significantly increased FLI in the thalamus, hypothalamus, and midbrain at the onset of ataxia (compare groups 3 and 2, Table 2). Glucose loading may increase the blood glucose concentration to approximately 50 mM [47], whereas the normal blood glucose concentration that results from dietary glucose may be correlated with the smaller amount of FLI in the brains of saline-treated ataxic rats. Indeed, glucose or other carbohydrate from dietary sources may play a role in the pathogenesis of WE. The disease does not occur in general starvation but is precipitated by administration of carbohydrate to starving humans; i.e. carbohydrate administration can unmask covert thiamin deficiency [42]. The particular brain structures containing FLI after glucose loading, such as the inferior olive, the medial vestibular nucleus, the mammillary bodies, the lateral hypothalamus and the medial nuclei of the thalamus, characteristically show decreased activity of OGDH [5], changes in blood brain barrier (BBB) function [6] and light microscopic and electron microscopic pathology in PTD [40,7]. Furthermore, the structures in which FLI was increased after glucose loading included those already affected by PTD in rats whose only source of glucose was their diet. Once PTD rats were opisthotonic, glucose loading did not lead to a further significant increase in brain FLI. There are several possible reasons for these observations. First, it is possible that glucose administration increases the rate of progression of PTD disease up to a ceiling that coincides with the onset of opisthotonus and at which the expression of Fos proteins is already maximal. Second, it is possible that several glucose loads are necessary to produce significant increases of FLI in the brain of opisthotonic PTD rats, but in our study more than one dose is impracticable in animals rapidly approaching death. Third, low energy levels in neurons in advanced PTD [1] might not be sufficient to support biosynthesis of more Fos proteins.
4.3. Glucose loading and brain glucose metabolism Following the administration of a glucose load, similar to that used here, the brain glucose concentration as detected by in vivo NMR spectroscopy rises by 3- to 5-fold in PTD rats but only 1.2- to 1.5-fold in controls [34]. Below we discuss the metabolic fate of this glucose. However, Hakim and Pappius [18] have reported that 2-deoxyglucose uptake into the brains of ataxic PTD rat is impaired by up to 50% and attribute this to a decrease in glycolysis. These data appear to contradict our results, but the clinical observation that WE can be precipitated by a glucose load in sub-clinical thiamin deficiency in humans
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[42] and in rats [47] argues that a glucose load does affect the thiamin-deficient brain, presumably requiring uptake and metabolism of glucose by that brain. Perhaps the impairment of the BBB in thiamin deficiency [6] adversely affects brain retention of the minute tracer dose of 2deoxy-[ 14 C]glucose to yield a result that is unrepresentative of the glucose uptake and metabolism of the thiamindeficient brain under a glucose load. Further, the interpretation [17,18], namely that the 2-deoxy-[ 14 C]glucose data mean that glycolysis is significantly impaired in the thiamin-deficient brain at the pre-symptomatic stage is untenable for several reasons. First, [ 14 C]glucose oxidation by brain slices from PTD rats measured by [ 14 C]CO 2 production is decreased little and only in concert with a decrease in the activity of the TCA cycle enzyme, OGDH [15]. Second, a glucose load causes accumulation of brain glutamate [34], as discussed below, requiring uptake of the glucose into the brain and its metabolism to glutamate.
4.4. Correlation of FLI with neurological effects of glucose loading The increase in neuronal FLI in response to a glucose dose follows within 4 h of the onset of behavioural and neurochemical responses to glucose loading of ataxic PTD rats [34,46]. The neurochemical changes in ataxic rats include increases in cerebral glutamate derivatives that commence within 15 min after glucose loading and persist beyond 120 min [34], consistent with increased brain ECF glutamate concentrations measured by microdialysis in the absence of glucose loading [19,26]. Behavioural changes include episodes of greatly increased ataxia and impairment of the righting reflex commencing within 40 min and persisting beyond 2 h [46]. The temporal relationship of these changes is consistent with the proposition that the increased severity of ataxia and the onset of Fos expression are both due to the earlier increase in brain glutamate concentration.
4.5. A unifying hypothesis for a glutamateNMDA -receptor mediated process in PTD rats The combination of data presented here together with that of other investigations provides considerable circumstantial evidence that allows construction of a plausible sequence of events that are consistent with, and unify, the observations arising from glucose loading of PTD rats. This hypothesis is built upon the evidence for a role of glutamate excitotoxicity in PTD in rats. The trigger for the induction of Fos genes in neurons of the brains of PTD rats could be the selective decrease in the activity of the OGDH enzyme which occurs prior to the onset of ataxia and again before the onset of opisthotonus [15,4,5]. A decrease in OGDH activity is likely to have a greater impact on neurons than glial cells because
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glial cells have greater metabolic flexibility and also provide neurons with substrates. As a rate-limiting step of the TCA cycle [15], declining OGDH activity could lead to the accumulation of intracellular glutamate and also adversely affect cellular energy levels in the PTD rat brain [1] limiting the function of ATP-dependent pumps of neurons or glial cells. Failure to maintain cellular electrolyte homeostasis could activate swelling-induced anion transporters on glial cell plasma membranes and the release of intracellular glutamate [24]. Changes in glial cell morphology, such as swelling, are the earliest features of thiamin deficiency detected by electron microscopy and occur in the midst of intact neurons [12,32]. Increases in ECF glutamate concentration and disruption of the glutamate / glutamine cycle could also result from failure of glutamate transporters on glial cells [35]. As thiamin deficiency progresses and OGDH activity and cellular energy reserves further decline, ECF glutamate concentrations in affected brain structures could increase, as has been measured in the thalamus of PTD rats [19,26]. Increased ECF concentrations of glutamate would lead to glutamate NMDA -receptor stimulation and thus increased expression of Fos proteins. This postulated sequence of events is consistent with the observations reported here.
4.6. The glucose loading model of Wernicke’ s encephalopathy in the rat Our glucose loading model of Wernicke’s encephalopathy in the rat has been developed and characterised in terms of the neurological, neurochemical and neuromorphological events occurring over an acute timeframe of 15 min to 4 h. Investigators now have the opportunity for continuous collection and correlation of data obtained within a defined short time frame after a specific stimulus. The use of in vivo nuclear magnetic resonance imaging and spectroscopy and of in vivo microdialysis techniques allows the continuous assessment of short-term functional and metabolic changes occurring in the brain after glucose loading of individual animals at different stages of thiamin deficiency. These data can be correlated with the behavioural consequences of glucose loading monitored over the same time frame. Immunohistochemical techniques allow the identification of cellular and molecular events and their timed sequence after glucose loading in thiamin deficiency. Correlation of the sequence of all events occurring after glucose loading in the brains of TD rats should contribute greatly to understanding the pathogenesis of Wernicke’s encephalopathy.
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