Pyrithiamine-treated Rats Research Articles

Interaction of Thiamine Deficiency and Voluntary Alcohol Consumption Disrupts Rat Corpus Callosum Ultrastructure

The relative roles of alcohol and thiamine deficiency in causing brain damage remain controversial in alcoholics without the Wernicke-Korsakoff syndrome. Experimental control over alcohol consumption and diet are impossible in humans but can be accomplished in animal models. This experiment was designed to differentiate the separate and combined effects on the macro- and ultrastructure of the corpus callosum of thiamine deficiency and voluntary alcohol consumption. Adult male alcohol-preferring (P) rats (9 chronically alcohol-exposed and 9 water controls) received a thiamine-deficient diet for 2 weeks. There were four groups: five rats previously exposed to alcohol were treated with pyrithiamine (a thiamine phosphorylation inhibitor); five rats never exposed to alcohol were treated with pyrithiamine; four alcohol-exposed rats were treated with thiamine; and four rats never exposed to alcohol were treated with thiamine. On day 14, thiamine was restored in all 18 rats; 2 weeks later the 10 pyrithiamine-treated rats received intraperitoneal thiamine. The rats were perfused 61 days post-pyrithiamine treatment at age 598 days. Brains were dissected and weight and volumes were calculated. Sagittal sections were stained to measure white matter structures. The corpus callosum was examined using transmission electron microscopy to determine density of myelinated fibers, fiber diameter, and myelin thickness. The corpus callosum in the alcohol/pyrithiamine group was significantly thinner, had greater fiber density, higher percentage of small fibers, and myelin thinning than in the alcohol/thiamine and water/thiamine groups. Several measures showed a graded effect, where the alcohol/pyrithiamine group had greater pathology than the water/pyrithiamine group, which had greater pathology than the two thiamine-replete groups. Across all 16 rats, thinner myelin sheaths correlated with higher percentage of small fibers. Myelin thickness and axon diameter together accounted for 71% of the variance associated with percentage of small fibers. Significant abnormalities in the alcohol/pyrithiamine group and lack of abnormality in the alcohol-exposed/thiamine-replete group indicate that thiamine deficiency caused white matter damage. The graded abnormalities across the dually to singly treated animals support a compounding effect of alcohol exposure and thiamine depletion, and indicate the potential for interaction between alcohol and thiamine deficiency in human alcohol-related brain damage.

Loss of [3H]kainate and of NMDA-displaceable [3H]glutamate binding sites in brain in thiamine deficiency: results of a quantitative autoradiographic study.

Previous studies suggest that alterations of brain glutamate synthesis and release occur in experimental thiamine deficiency. In order to assess the integrity of post-synaptic glutamatergic receptors in thiamine deficiency, binding sites for [3H]glutamate (displaced by NMDA), [3H]-kainate, and [3H]quisqualate (AMPA sites) were evaluated using Quantitative Receptor Autoradiography in rat brain following 14 days of treatment with the central thiamine antagonist pyrithiamine. Compared to pair-fed controls, brains of symptomatic thiamine-deficient animals contained significantly fewer NMDA-displaceable binding sites in cerebral cortex, medial septum and hippocampus. It has been suggested that NMDA-receptor mediated glutamate excitotoxicity plays a role in the pathogenesis of neuronal loss in thiamine deficiency. If such is the case, the selective loss of NMDA binding sites in cerebral cortex and hippocampus offers a possible explanation for the relative nonvulnerability of these brain regions to pyrithiamine-induced thiamine deficiency. [3H]quisqualate (AMPA) binding sites were unchanged in all brain regions of pyrithiamine-treated rats whereas [3H]kainate sites were significantly reduced in density in medial and lateral thalamus. The decline in these binding sites may be due to neuronal loss in pyrithiamine-induced thiamine deficiency. Alterations of glutamatergic synaptic function involving both NMDA and kainate receptor subclasses could contribute to the pathogenesis of neurological dysfunction in Wernicke's Encephalopathy in humans.

A quantitative autoradiographic study of muscarinic cholinergic receptor subtypes in the brains of pyrithiamine-treated rats.

Previous studies describe decreased acetylcholine synthesis in brain as well as neurobehavioral evidence for a central muscarinic cholinergic deficit in pyrithiamine-induced thiamine-deficient rats. In order to further evaluate this possibility, quantitative autoradiographic procedures using [3H]quinuclidinyl benzilate (for total muscarinic binding sites), [3H]pirenzepine (for muscarinic M1 sites) and [3H]AF-DX 384 (for muscarinic M2 sites) were performed at early (presymptomatic) and late (symptomatic) stages of thiamine deficiency induced in rats by administration of the central thiamine antagonist, pyrithiamine. No significant alterations in densities of M1, M2 or total muscarinic binding sites were observed in any brain structure evaluated at either early or late stages of thiamine deficiency. These findings do not support a major role for modifications of muscarinic cholinergic function in the pathogenesis of the neurological symptoms of thiamine deficiency.

Regional alterations of thiamine phosphate esters and of thiamine diphosphate-dependent enzymes in relation to function in experimental Wernicke's encephalopathy

Thiamine phosphate esters (thiamine monophosphate-TMP; thiamine diphosphate-TDP and thiamine triphosphate-TTP) were measured as their thiochrome derivatives by High Performance Liquid Chromatography in the brains of pyrithiamine-treated rats at various stages during the development of thiamine deficiency encephalopathy. Severe encephalopathy was accompanied by significant reductions of all three thiamine phosphate esters in brain. Neurological symptoms of thiamine deficiency appeared when brain levels of TMP and TDP fell below 15% of normal values. Activities of the TDP-dependent enzyme alpha-ketoglutarate dehydrogenase were more severely reduced in thalamus compared to cerebral cortex, a less vulnerable brain structure. On the other hand, reductions of TTP, the non-cofactor form of thiamine, occurred to a greater extent in cerebral cortex than thalamus. Early reductions of TDP-dependent enzymes and the ensuing metabolic pertubations such as lactic acidosis impaired brain energy metabolism, and NMDA-receptor mediated excitotoxicity offer rational explanations for the selective vulnerability of brain structures such as thalamus to the deleterious effects of thiamine deficiency.

Pyrithiamine-induced thiamine deficiency results in decreased Ca2+-dependent release of glutamate from rat hippocampal slices

Alterations of excitatory amino acids in brain may be of pathophysiological significance in thiamine-deficiency encephalopathy. The present study was undertaken to evaluate the effects of thiamine deficiency induced by the central thiamine antagonist, pyrithiamine, on the glutamate content of glutamatergic nerve terminals. Electrically-stimulated, Ca(2+)-dependent release of glutamate from hippocampal slices obtained from symptomatic pyrithiamine-treated rats was significantly decreased compared to pair-fed controls. Possible explanations for the decreased "neurotransmitter pool" of glutamate in thiamine-deficient rat brain include decreased synthesis of glutamate as a result of decreased activities of the thiamine-dependent enzyme alpha-ketoglutarate dehydrogenase or increased release of glutamate per se. There is evidence to suggest that the latter mechanism with ensuing excitotoxic neuronal damage could be involved in the pathogenesis of selective neuronal death in thiamine deficiency. Similar mechanisms could be implicated in Wernicke's encephalopathy in humans.

Enzyme-cytochemical study of small ganglion cells in experimental thiamine deficiency: concerning the pain mechanism

In an effort to clarify the mechanism of pain accompanying thiamine deficiency, thiamine monophosphatase (TMPase) activity was demonstrated, by means of electron microscopic cytochemistry, in small ganglion cells in pyrithiamine-treated thiamine-deficient rats. TMPase activity was seen to be present in the reticular part of the Golgi complex and vesicle-like bodies around the trans side of the Golgi complex. By the 10th day after the initial pyrithiamine treatment, a significant increase in the number of TMPase-positive vesicle-like body was observed. These results indicate that thiamine deficiency causes a disturbance in the transportation and/or production of TMPase, which is an integral part of the synaptic transmission of the nociception impulses.

Effect of Pyrithiamine Treatment and Subsequent Thiamine Rehabilitation on Regional Cerebral Amino Acids and Thiamine‐Dependent Enzymes

Pyrithiamine-induced thiamine-deficiency encephalopathy in the rat shows many neuropathological and biochemical similarities to Wernicke's encephalopathy in humans. Treatment of rats with pyrithiamine resulted in moderate reductions of glutamate in thalamus and pons and in generalized severe reductions of aspartate in pons (by 89%, p less than 0.01), thalamus (by 83%, p less than 0.01), cerebellum (by 53%, p less than 0.01), and cerebral cortex (by 33%, p less than 0.05). Alanine concentrations were concomitantly increased. Activities of the thiamine-dependent enzyme alpha-ketoglutarate dehydrogenase (alpha KGDH) were decreased in parallel with the aspartate decreases; pyruvate dehydrogenase complex activities were unchanged in all brain regions. Following thiamine administration to symptomatic pyrithiamine-treated rats, neurological symptoms were reversed and concentrations of glutamate, aspartate, and alanine, as well as alpha KGDH activities, were restored to normal in cerebral cortex and pons. Aspartate levels and alpha KGDH activities remained below normal values, however, in thalamus. Thus, pyrithiamine treatment leads to reductions of cerebral alpha KGDH and (1) decreased glucose (pyruvate) oxidation resulting in accumulation of alanine and (2) decreased brain content of glutamate and aspartate. Such changes may be of key significance in the pathophysiology of the reversible and irreversible signs of Wernicke's encephalopathy in humans.

Metabolic studies of the edematous cerebral cortex of the pyrithiamine-treated thiamine-deficient rat

The pyrithiamine-treated rats produced by our method histopathologically exhibited the edematous lesion not only around the third ventricle and at the tegmentum of pons, as expected, but also in the cerebral cortex. These animals were, therefore, used in a biochemical study of the cerebral cortex, in particular, glucose metabolism. The results indicate an accumulation of lactate and severe impairment of the lipid metabolism in the edematous lesion of the cerebral cortex among the pyrithiamine-treated rats. The fact that oxygen consumption was hardly diminished, however, strongly suggested that amino acids were selectively supplied to the tricarboxylic acid cycle from the free amino acid pool.

Activities of thiamine-dependent enzymes in two experimental models of thiamine-deficiency encephalopathy: 1. The pyruvate dehydrogenase complex.

Chronic thiamine deprivation in the rat leads to selective neuropathological damage in brainstem structures whereas treatment with the central thiamine antagonist, pyrithiamine, results in more widespread damage. In order to further elucidate the neurochemical mechanisms responsible for this selective damage, the thiamine-dependent enzyme complex pyruvate dehydrogenase (PDHC) was measured in 10 brain structures in the rat during progression of thiamine deficiency produced by chronic deprivation or by pyrithiamine treatment. Feeding of a thiamine-deficient diet to adult rats resulted in 5-7 weeks in ataxia and loss of righting reflex accompanied by decreased blood transketolase activities. PDHC activities were selectively decreased by 15-30% in midbrain and pons (lateral vestibular nucleus). Thiamine treatment of symptomatic rats led to reversal of neurological signs and to concomitant reductions of the cerebral PDHC abnormalities. Daily pyrithiamine treatment led within 3 weeks to loss of righting reflex and convulsions and to decreased blood transketolase of a comparable magnitude to that observed in chronic thiamine-deprived rats. No significant regional alterations of PDHC, however, were observed in pyrithiamine-treated rats.

Effect of pyrithiamine treatment of potassium ion fluxes in rat cortical slices

The effect of thiamine deficiency on energy-requiring processes in brain tissue was studied by comparing cortical slices prepared from control and pyrithiamine-treated rats. Veratridine was used to stimulate energy metabolism by opening voltage-sensitive sodium channels resulting in enchanced Na +/K + pumping; subsequent tetrodotoxin addition closed the sodium channels. Pyrithiamine-treated slices showed both lower basal and veratridine-stimulated respiration rates compared to control slices. K + was released from the tissue upon addition of veratridine and was taken up again upon addition of tetrodotoxin. The movement of K + was monitored directly with a K +-sensitive electrode as well as by measuring the rubidium diffusion potential. There was no difference between control and pyrithiamine-treated slices in K + fluxes in response to veratridine and tetrodotoxin. The extent of reuptake of K + upon tetrodotoxin addition was inversely related to the extracellular Ca 2+ concentration and to the incubation temperature. Veratridine resulted in a marked decrease in tissue levels of ATP and creatine phosphate; these levels remained quite low upon tetrodotoxin addition. Despite the different respiration rates, control and pyrithiamine-treated slices showed the same ATP and creatine phosphate levels in response to veratridine and tetrodotoxin.

Effects of Forced-feeding on Blood Levels of Pyruvate, Glucocorticoids and Glucose and on Adrenal Weight in Thiamine-deprived and Thiamine Antagonist-treated Rats

To study the factors involved in the anorexia and hyperpyruvicemia of thiamine deficiency, rats were force-fed a liquefied thiamine-deficient diet by stomach tube. Force-feeding decreased the life expectancy of thiamine-deprived and oxythiamine-treated (OTh) rats by 0.33 to 0.50%, compared with similar rats fed ad libitum, but had no effect on life expectancy of pyrithiamine-treated (PTh) rats. Bloating was observed in all force-fed deficient groups and appeared to correlate well with the development of anorexia in rats fed ad libitum. It was so severe in force-fed OTh-treated rats that the daily food ration had to be cut by one-half. Thiamine-deprived and OTh-treated rats still showed significant increases in blood pyruvate and plasma corticosterone levels, and in gut and adrenal weights despite force-feeding to reduce inanition. Longer treatment with OTh appeared to exhaust the ability of the adrenals to put out corticosterone. PTh treatment resulted in increases in plasma corticosterone and adrenal weight in the ataxic but preconvulsive state, which increased further after convulsions had started, along with a significant increase in blood pyruvate. Because the animals are unable to use the food these results suggest that the anorexia is the result of some local biochemical disturbance in the gut, and that elevations of blood pyruvate in thiamine-deficient states are possibly due to two factors: a hyperactivity of the hypophyseal-adrenal system, and specific disturbances in pyruvate metabolism.

Studies on the physiological functions of thiamine: V. Effects of thiamine deprivation and thiamine antagonists on blood pyruvate and lactate levels and activity of lactate dehydrogenase and its isozymes in blood and tissues

Rats were made thiamine deficient by thiamine deprivation and treatment with the thiamine antagonists, oxythiamine and pyrithiamine. Thiamine deprivation and oxythiamine treatment caused significant elevations of blood pyruvate and lactate, the changes being greatest after oxythiamine. Neither had any effect on plasma lactate dehydrogenase activity. Pyrithiamine-treated rats showed no elevation in blood lactate nor pyruvate if tested before convulsions started, but showed marked elevations in both, and also in plasma lactate dehydrogenase activity after 24–36 h in convulsions. No significant changes were observed in lactate dehydrogenase activity in brain and kidney in thiamine-deprived and pyrithiamine-treated rats, but an increase was observed in oxythiamine-treated rats. A decrease in lactate dehydrogenase activity was found in the heart in thiamine-deprived rats and in the liver of rats exhibiting all three types of experimental deficiencies. A decrease in the relative abundance of LDH 3 and LDH 4 isozymes and an increase in LDH 1 was observed in the hearts of thiamine-deprived, oxythiamine-treated and pyrithiamine-treated rats.