Effects of maternal thiamine deficiency on the lipid composition of rat whole brain, gray matter and white matter

Studies were made of the effects of maternal thiamine deficiency on rat whole brain, gray matter and white matter lipids. Mothers were fed a high protein diet (controls) or thiamine deficient high protein diet (thiamine deficient, TD) from 14th day of gestation through lactation. An additional group (pair fed control, PFC) was pair fed with the thiamine deficient group. The TD pups started showing symptoms of abnormalities in posture, arched back and hind limb paralysis from 16th day of lactation. Significant deficits were found in body weight and brain weight of TD and PFC pups. But the deficits seem to be more in the former group. Significant deficits were observed with regard to the concentration of lipids such as galactolipids, phospholipids and plasmalogens in the whole brain of TD and PFC pups at 21 days of age. Additional deficits were also found in the concentration of cholesterol in PFC pups. Gray matter lipids from TD pups seem to be completely spared. However, deficits were found in galactolipid and ganglioside concentrations in PFC pups. The deficits found in the concentration of different lipids in white matter are similar to those observed in whole brain. These results suggest that the effects of thiamine deficiency may be partly due to resultant growth retardation and partly due to the deficiency of thiamine per se.     

Proteome analysis in cardiovascular pathophysiology using Dahl rat model

Dahl salt-sensitive (DS) and salt-resistant (DR) inbred rat strains represent a well established animal model for cardiovascular research. Upon prolonged administration of high-salt-containing diet, DS rats develop systemic hypertension, and as a consequence they develop left ventricular hypertrophy, followed by heart failure. The aim of this work was to explore whether this animal model is suitable to identify biomarkers that characterize defined stages of cardiac pathophysiological conditions. The work had to be performed in two stages: in the first part proteomic differences that are attributable to the two separate rat lines (DS and DR) had to be established, and in the second part the process of development of heart failure due to feeding the rats with high-salt-containing diet has to be monitored. This work describes the results of the first stage, with the outcome of protein expression profiles of left ventricular tissues of DS and DR rats kept under low salt diet. Substantial extent of quantitative and qualitative expression differences between both strains of Dahl rats in heart tissue was detected. Using Principal Component Analysis, Linear Discriminant Analysis and other statistical means we have established sets of differentially expressed proteins, candidates for further molecular analysis of the heart failure mechanisms.     

Thiamine deficiency in rats affects thiamine metabolism possibly through the formation of oxidized thiamine pyrophosphate

BackgroundThiamine deficiency (TD) has a number of features in common with the neurodegenerative diseases development and close relationship between TD and oxidative stress (OS) has been repeatedly reported in the literature. The aim of this study is to understand how alimentary TD, accompanied by OS, affects the expression and level of two thiamine metabolism proteins in rat brain, namely, thiamine transporter 1 (THTR1) and thiamine pyrophosphokinase (TPK1), and what factors are responsible for the observed changes.MethodsThe effects of OS caused by TD on the THTR1and TPK1 expression in rat cortex, cerebellum and hippocampus were examined. The levels of active and oxidized forms of ThDP (enzymatically measured) in the blood and brain, ROS and SH-groups in the brain were also analyzed.ResultsTD increased the expression of THTR1 and protein level in all studied regions. In contrast, expression of TPK1 was depressed. TD-induced OS led to the accumulation of ThDP oxidized inactive form (ThDP_ox) in the blood and brain. In vitro reduction of ThDP_ox by dithiothreitol regenerates active ThDP suggesting that ThDP_ox is in disulfide form. A single high-dose thiamine administration to TD animals had no effect on THTR1 expression, partly raised TPK1 mRNA and protein levels, but is unable to normalize TPK1 enzyme activity. Brain and blood ThDP levels were increased in these conditions, but ThDP_ox was not decreased.General significanceIt is likely, that the accumulation of ThDP_ox in tissue could be seen as a potential marker of neurocellular dysfunction and thiamine metabolic state.     

Acetyl-CoA metabolism in amprolium-evoked thiamine pyrophosphate deficits in cholinergic SN56 neuroblastoma cells

Inhibition of pyruvate (PDHC) and ketoglutarate (KDHC) dehydrogenase complexes induced by thiamine pyrophosphate deficits is known cause of disturbances of cholinergic transmission in the brain, yielding clinical symptoms of cognitive, vegetative and motor deficits. However, particular alterations in distribution of key acetylcholine precursor, acetyl-CoA, in the cholinergic neuron compartment of thiamine pyrophosphate-deficient brain remain unknown. Therefore, the aim of our work was to find out how amprolium-induced thiamine pyrophosphate deficits (TD) affect distribution of acetyl-CoA in the compartment of pure cholinergic neuroblastoma SN56 cells originating from murine septum. Amprolium caused similar concentration-dependent decreases in thiamine pyrophosphate levels in nondifferentiated (NC) and differentiated (DC) cells cultured in low thiamine medium. In such conditions DC displayed significantly greater loss of viability than the NC ones, despite of lesser suppressions of PDHC activities and tetrazolium salt reduction rates in the former. On the other hand, intramitochondrial acetyl-CoA levels in DC were 73% lower than in NC, which explains their greater susceptibility to TD. Choline acetyltransferase activity and acetylcholine content in DC were two times higher than in NC. TD caused 50% decrease of cytoplasmic acetyl-CoA levels that correlated with losses of acetylcholine pool in DC but not in NC. These data indicate that particular sensitivity of DC to TD may result from relative shortage of acetyl-CoA due to its higher utilization in acetylcholine synthesis.     

Activities of thiamine-dependent enzymes in two experimental models of thiamine deficiency encephalopathy: 3. Transketolase

Chronic thiamine deprivation in the rat leads to ataxia, loss of righting reflex and neuropathological damage to lateral vestibular nucleus. Before onset of neurological symptoms, transketolase (TK) activities were found to be selectively reduced by 25% in lateral vestibular nucleus and surrounding pons. Further progression of thiamine deprivation resulted in a generalized reduction in TK activity. Measurement of enzyme activity in the presence of added TPP cofactor in vitro did not lead to normalisation of enzyme activities suggesting loss of apoenzyme. Administration of thiamine to symptomatic thiamine-deprived rats resulted in reversal of neurological symptoms and to normalisation of defective TK activities in less vulnerable structures such as cerebral cortex striatum and hippocampus; reduction of TK activity, however, persisted in brainstem and cerebellar regions. Pyrithiamine treatment results, within 3 weeks, in loss of righting reflex, convulsions and more widespread neuropathological damage compared to that observed following thiamine deprivation. TK activity was found to be significantly decreased before the onset of neurological symptoms in all brain regions and appearance of symptoms was accompanied by more severe reductions of TK. In contrast to chronic thiamine deprivation, TK activities following pyrithiamine treatment were: (i) equally reduced in magnitude in vulnerable and non-vulnerable brain structures, (ii) unchanged following reversal of neurological abnormalities by thiamine administration.     

Vagal control of heart period in alloxan diabetic rats

Autonomic neuropathies are a frequent complication to diabetes in humans. Similar neuropathies have not been well-documented in animal models. To determine if diabetic rats would develop parasympathetic neuropathies, rats were made diabetic by the injection of alloxan into the tail vein and then maintained on daily injections of insulin. At various times subsequent to the induction of diabetes (3–5 weeks, 7–9 weeks, and 14 weeks), the effect of constant frequencies of vagal stimulation on the efferent cardiac chronotropic response was evaluated using analysis of variance techniques. It was found that the vagal parasympathetic effect was accentuated in diabetic rats. That is, at a given frequency of supramaximal vagal stimulation, the heart rate slowed more in diabetic rats than in nondiabetic rats. Whether a similar phenomenon exists in humans is not known.     

Impairment of acetylcholine synthesis in thiamine deficient rats developed by prolonged tea consumption

The synthesis of whole brain acetylcholine is reduced in thiamine deficient rats produced by prolonged administration of tea. In those rats fed a normal diet and given tea (1:50, w/v) instead of drinking water for 20 weeks, the conversion of [14C] pyruvate to [14C]acetylcholine decreased by 35%. However, no neurological symptoms were observed. Administration of tea to rats fed a thiamine half-deficient diet for 7-8 weeks caused not only 60% decrease in acetylcholine synthesis but also neurological symptoms. This decreased synthesis of acetylcholine is related to a decline in pyruvate dehydrogenase activity. The results suggest that prolonged administration of tea to rats cause an impairment of acetyl CoA production resulting in a deficit in acetylcholine synthesizing capacity.     

Influence of selenium (antioxidant) on gliclazide induced hypoglycaemia/anti hyperglycaemia in normal/alloxan-induced diabetic rats

Oxidative stress is involved in diabetes mellitus and its complications. Since diabetes is a stress-related disorder, supplementation with antioxidants may improve the condition. The purpose of this study is to know the effect of oral administration of selenium on blood glucose and its influence on gliclazide induced hypoglycaemia/antihyperglycaemia in normal and alloxan-induced diabetic rats. Albino rats of either sex were divided into three groups of six each. Group-I/II/III were treated with selenium 1/2 TD (0.9 μg/200 g rat)/TD (1.8 μg/200 g rat)/2TD (3.6 μg/200 g rat), respectively. Later group II was treated with gliclazide TD (1.44 mg/200 g rat)/selenium TD + gliclazide TD with a washout period of 1 week between the treatments. Diabetes was induced by alloxan monohydrate 100 mg/kg body weight i.p. A group of six rats showing fasting blood glucose levels ranging from 175–250 mg/dl were selected for the study. Rats were treated with selenium TD, gliclazide TD and selenium TD + gliclazide TD with a washout period of 1 week between the treatments. Selenium 1/2 TD and TD produced hypoglycaemia while 2TD produced hyperglycaemia. The combination of selenium TD with gliclazide TD, significantly enhanced the glucose lowering effect of gliclazide in normal and diabetic rats.     

Effect of cervical sympathetic trunk transection on renal sympathetic nerve activity in rats

Stellate ganglion blockade (SGB) with a local anesthetic increases muscle sympathetic nerve activity in the tibial nerve in humans. However, whether this sympathetic excitation in the tibial nerve is due to a sympathetic blockade in the neck itself, or due to infiltration of a local anesthetic to adjacent nerves including the vagus nerve remains unknown. To rule out one mechanism, we examined the effects of cervical sympathetic trunk transection on renal sympathetic nerve activity (RSNA) in anesthetized rats. Seven rats were anesthetized with intraperitoneal urethane. RSNA together with arterial blood pressure and heart rate were recorded for 15 min before and 30 min after left cervical sympathetic trunk transection. The baroreceptor unloading RSNA obtained by decreasing arterial blood pressure with administration of sodium nitroprusside was also measured. Left cervical sympathetic trunk transection did not have any significant effects on RSNA, baroreceptor unloading RSNA, arterial blood pressure, and heart rate. These data suggest that there was no compensatory increase in RSNA when cervical sympathetic trunk was transected and that the increase in sympathetic nerve activity in the tibial nerve during SGB in humans may result from infiltration of a local anesthetic to adjacent nerves rather than a sympathetic blockade in the neck itself.     

In vivo microdialysis in an animal model of neurological disease: thiamine deficiency (Wernicke) encephalopathy

In vivo microdialysis allows for the constant monitoring of brain neurotransmitters in the extracellular fluid of awake and freely moving animals. Considerations including factors affecting probe recoveries, the blood-brain barrier, and tissue reactions to probe implantation are discussed in this paper. Details of the application of in vivo microdialysis to an animal model of encephalopathy are then presented. Thiamine deficiency encephalopathy is an animal model of Wernicke encephalopathy, a neurological disorder observed in alcoholics and in patients with severely compromised nutrition. Regionally selective neuronal cell death is observed in both patients and animals with thiamine deficiency (TD). Various thalamic nuclei suffer significant TD-induced cell death, and NMDA receptor-mediated glutamate excitotoxicity has been proposed as an underlying causative factor. A detailed methodology for the examination of the role of glutamate excitotoxicity using in vivo microdialysis in the neuronal cell death due to thiamine deficiency is presented.     

两种心衰模型大鼠心功能的比较

目的　比较两种心衰模型大鼠心功能的特点。方法　用腹主动脉、下腔静脉穿刺造瘘法及冠脉结扎法建立不同的心衰模型 ,用Doppler超声心动图及心脏称重的方法比较其心功能的各项参数。结果　两组大鼠的相对心脏重量均有所增高。造瘘组射血分数有所下降 ,但心输出量、血压维持正常 ,而冠脉结扎组术后 3周射血分散、心输出量和平均动脉压均明显下降 ,等容舒张期延长。结论　腹主动脉、下腔静脉穿刺造瘘所造成的是高输出量心衰 ,而冠脉结扎法所造成的是低输出量心衰 ,其心衰程度更为严重。Doppler超声心动图为大鼠心功能的检测提供了一种简单、可靠、可随访的无创伤性检查方法。     

Hazards of urethane (ethyl carbamate): a review of the literature

Urethane (ethyl carbamate) is used alone or in combination with other drugs to produce anaesthesia in laboratory animals. Although originally studied as a potential phytocide, urethane demonstrated antineoplastic properties when administered to rats with the Walker rat carcinoma 256. Subsequent trials in humans led to its use as a chemotherapeutic agent for various leukaemias. Mice develop pulmonary adenomas earlier in life and at a higher incidence following urethane administration. Urethane's carcinogenic influence is greater in neonatal mice; it also has a transplacental influence in mice. In rats, urethane increases the incidence of pulmonary adenomas, Zymbal Gland tumours, and a variety of other neoplasms. Urethane is absorbed sufficiently from the skin of laboratory animals to produce a transient narcosis. The carcinogenic effect appears to be due to an undefined oncogenic intermediate formed in the blood. Considering the properties urethane demonstrates in animals, the safety of its use by laboratory personnel is in question. However, if appropriate guidelines are followed, urethane should continue to be a useful anaesthetic agent for laboratory animals.     

A model of chronic heart failure in spontaneous hypertensive rats (SHR)

Common models of chronic heart failure (CHF) do not always result in parameters and symptoms that can be extrapolated to the clinical situation of patients with end-stage heart failure. The aim of this study was to establish and validate a new model of CHF in the rat. CHF was induced in Wistar Kyoto (WKY/NHsd) and spontaneously hypertensive (SHR/NHsd) rats by creating a permanent (8-week) occlusion of the left coronary artery 2 mm distal to the origin from the aorta by a modified technique. This resulted in a large infarction of the free left ventricular wall. The focus of attention was the validation of the geometric properties of the left ventricle and its contractility. The validation of the geometric properties of the left ventricle was done by a non-invasive magnetic resonance imaging (MRI) technique and by planimetry (stereology). Cardiodynamics (e.g. contractility) were evaluated in the isolated 'working heart' model. We were able to establish a new and predictive model of heart failure in the spontaneously hypertensive rat 8 weeks after coronary artery ligation. At this time point, the WKY rat did not show any symptoms of CHF. The model represents characteristic parameters and symptoms that can be extrapolated to the clinical situation of patients with end-stage heart failure (NYHA III-IV). Upon inspection, severe clinical symptoms of congestive heart failure were prominent, such as dyspnoea, subcutaneous oedema, pale-bluish limbs and impaired motion. Non-invasive sequential measurements by NMR techniques showed lung oedema, hydrothorax, large dilated left and right ventricular chambers and hypertrophy of the septum. The infarcted animals showed a reduced heart power, diminished contractility and enhanced heart work, much more so in the SHR/NHsd rat than in the WKY/NHsd rat. Furthermore the infarcted animals showed enhanced levels of hydroxyproline/proline ratios, again much more so in the SHR/NHsd rat than in the WKY/NHsd rat.     

Poster Sessions BP01: Energy Metabolism. NMR investigation of metabolic alterations in the brain of thiamine‐deficient rats

Thiamine deficiency (TD) results in region‐selective impairment of brain metabolism. Since thiamine is a cofactor for enzymes involved in glucose metabolism, ¹H and ¹³C‐NMR was used to investigate metabolic fluxes through the major pathways of glucose metabolism in vulnerable (medial thalamus, MT; inferior colliculus, IC) and nonvulnerable brain structures of rats made thiamine deficient following treatment with the central thiamine antagonist pyrithiamine vs. pair‐fed controls. Symptomatic stages of TD resulted in decreased glutamate and GABA in MT an IC confirming previous biochemical studies. ¹³C‐isotopomer analysis revealed decreased de novo synthesis of [4–¹³C]glutamate (30%p < 0.02) and [2–¹³C]GABA (60%p < 0.01) in MT and IC consistent with decreased activities of pyruvate‐ and α‐ketoglutarate dehydrogenases. These changes were accompanied by decreased consumption of glucose and increased synthesis of lactate from [1–¹³C]glucose confirming decreased mitochondrial metabolism. Accumulation of glyceraldehyde‐3‐phosphate suggested inhibition of glucose flux through the thiamine‐deficient enzyme transketolase. Onset of symptoms of TD and significant cell death was accompanied by decreased neuronal marker molecules NAA and NAAG in MT. Focal lactate accumulation resulting from decreased activities of mitochondrial thiamine‐dependent enzymes appears to play a key role in the pathogenesis of selective neuronal cell death in TD. [funded by CIHR Canada].     

Thiamine deficiency in vivo produces fiber cell degeneration in mouse lenses.

Thiamine (Vitamin B1) is a co-factor for enzymes key in bridging aerobic and anaerobic metabolism. One such enzyme, transketolase, catalyzes two of three reactions for entry into the pentose-phosphate pathway, a major source of chemical reducing power. Thus, thiamine deprivation (TD) is considered a classic model of systemic oxidative stress and is linked with degenerative diseases. TD in mice and rats produces neurodegeneration with Alzheimer's disease characteristics. Age-related disease of the lens, commonly cataract, is also linked with thiamine and oxidative stress. To test the effects of TD on mice, we used a previously defined protocol involving a thiamine free diet and a thiamine antagonist. After 12 days, lens fiber cell degeneration was observed primarily along the lens posterior beneath the intact capsule. These regions exhibited a localized increased expression of Alzheimer precursor protein, Abeta peptides, and presenilin 1. These data indicate that TD in mice produces fiber cell degeneration and suggest common mechanisms for TD-induced lens fiber and neuronal cell degeneration.     

The isolated working heart model in infarcted rat hearts

Congestive heart failure (CHF) is one of the most common causes of death in western countries. The aim of this study was to establish and validate the working heart model in rat hearts with CHF. In the rat model the animals show parameters and symptoms that can be extrapolated to the clinical situation of patients with end-stage heart failure. The focus of attention was the evaluation of cardiodynamics (e.g.contractility) in the isolated 'working heart' model. The geometric properties of the left ventricle were measured by planimetry (stereology). Formulae available in the past for determining certain parameters in the working heart model (e.g.external heart work) have to be fitted to the circumstances of the infarcted rat hearts with its different organ properties.CHF was induced in Wistar Kyoto (WKY/NHsd) and spontaneously hypertensive rats (SHR/NHsd) by creating a permanent (8 week) occlusion of the left coronary artery, 2 mm distal to the origin from the aorta, by a modified technique (Itter et al. 2004). This resulted in a large infarction of the free left ventricular wall.We were able to establish and adapt a new and predictive working heart model in spontaneously hypertensive rat hearts with myocardial infarction (MI) 8-12 weeks after coronary artery ligation. At this stage the WKY rat did not show any symptoms of CHF. The SHR rat represented characteristic parameters and symptoms that could be extrapolated to the clinical situation of patients with end-stage heart failure (NYHA III-IV). Upon inspection, severe clinical symptoms of CHF such as dyspnoea, subcutaneous oedema, palebluish limbs and impaired motion were prominent. On necropsy the SHR showed lung oedema, hydrothorax, large dilated left and right ventricular chambers and hypertrophy of the septum. In the working heart model the infarcted animals showed reduced heart power, diminished contractility and enhanced heart work, much more so in the SHR/NHsd than in the Wistar Kyoto rat (WKY/NHsd).The aim for the future is to find a causal therapy of heart failure treatment. At present, only palliative therapy is possible for patients with heart failure. For this reason the working heart model in CHF rat hearts should provide a valuable method for early testing of new therapeutic approaches for patients with CHF.     

Higher mortality in heterozygous neuropilin-1 mice after cardiac pressure overload

We previously identified that neuropilin-1 (NP-1) was a co-receptor of vascular endothelial growth factor receptor 2 (VEGFR2) and confirmed that NP-1 knockout mice were embryonic lethal due to impairment of vascular development, while VEGF was reported to be involved in the progression of heart failure. However, it is unknown whether NP-1 has any influence on cardiac function, and it also remains poor understood concerning cardiac expression of NP-1 and its interaction with other VEGF receptors in the heart. Here, we first showed that NP-1 heterozygous mice had significantly higher mortality due to either acute or chronic heart failure in response to left ventricular pressure overload. We also observed that NP-1 mRNA and protein were expressed in both neonatal rat cardiomyocytes and adult murine heart. Furthermore, we found that NP-1 formed complexes with VEGFR1 and VEGFR2, respectively, in cardiomyocytes. These findings suggest that NP-1 should play beneficial role in heart failure.     

Energy and glucose pathways in thiamine deficient primary rat brain microvascular endothelial cells

Thiamine deficiency (TD) results in lactate acidosis, which is associated with neurodegeneration. The aim of this study was to investigate this alteration in primary rat brain endothelia. Spectrophotometric analysis of culture media revealed that only a higher concentration of pyrithiamine, which accelerates the intracellular blocking of thiamine, significantly elevated the lactate level and lactate dehydrogenase activity within 7 days. The medium without pyrithiamine and with a thiamine concentration comparable to pathophysiological plasma levels mildly reduced only the activity of transketolase. This suggests that significant metabolic changes may not occur at the early phase of TD in cerebral capillary cells, while anaerobic glycolysis in capillaries may be mediated during late stage/chronic TD.     

Chronic alcoholism in rats induces a compensatory response, preserving brain thiamine diphosphate, but the brain 2-oxo acid dehydrogenases are inactivated despite unchanged coenzyme levels

Thiamine-dependent changes in alcoholic brain were studied using a rat model. Brain thiamine and its mono- and diphosphates were not reduced after 20 weeks of alcohol exposure. However, alcoholism increased both synaptosomal thiamine uptake and thiamine diphosphate synthesis in brain, pointing to mechanisms preserving thiamine diphosphate in the alcoholic brain. In spite of the unchanged level of the coenzyme thiamine diphosphate, activities of the mitochondrial 2-oxoglutarate and pyruvate dehydrogenase complexes decreased in alcoholic brain. The inactivation of pyruvate dehydrogenase complex was caused by its increased phosphorylation. The inactivation of 2-oxoglutarate dehydrogenase complex (OGDHC) correlated with a decrease in free thiols resulting from an elevation of reactive oxygen species. Abstinence from alcohol following exposure to alcohol reactivated OGDHC along with restoration of the free thiol content. However, restoration of enzyme activity occurred before normalization of reactive oxygen species levels. Hence, the redox status of cellular thiols mediates the action of oxidative stress on OGDHC in alcoholic brain. As a result, upon chronic alcohol consumption, physiological mechanisms to counteract the thiamine deficiency and silence pyruvate dehydrogenase are activated in rat brain, whereas OGDHC is inactivated due to impaired antioxidant ability.     

Vitamins and brain development.

Effects of deficiency of vitamins on early development of brain have been reviewed. Unusual developmental problems in neurogenesis specific for the brain and impairment of its functional capacities due to vitamin deficiency have been discussed. The species-specific "critical periods" in development of various systems have been mentioned. Indices such as reflex activity, locomotion, special senses, cognition and adaptive behavior were used for assessing brain maturation in experimental models and humans. Significant examples include brain anomalies in humans and other mammals caused by retinoid excess or deficit; increase in calbindin D28K, a vitamin D dependent calcium-binding protein during postnatal period in rat; hydrocephalus and exencephaly in prenatal rats and subarachnoidal or intracerebral hemorrhage in infants caused by vitamin E deficiency. Peripheral neuropathic lesions leading to infantile beriberi is caused by thiamine deficiency. Impaired growth in retinal layers leading to delay in maturation of electroretinogram and depth-perception in postnatal rats occur due to pyridoxine deficiency. Infants of severely vitamin B12 deficient mothers show abnormalities in behavior involving basal ganglia and pyramidal tract. Folic acid deficiency results in delayed maturation of the basic electroencepalographic patterns. In addition, vitamin-interactions leading to developmental errors have been pointed out. Vitamin B6 deficiency impairs vitamin B12 absorption and biotin deficiency may be aggravated by pantothenic acid deficiency. Vitamin C deficiency resulting in impaired metabolism may produce symptoms of deficiency of folic acid. Another characteristic examples is that iron absorption from dietary sources is dependent on ascorbic acid.