Short- and long-term cognitive and metabolic effects of medium-chain triglyceride supplementation in rats.

Medium-chain triglycerides (MCT) possess neuroprotective properties. However, the long-term metabolic consequences of supplementing a regular diet with cognition-enhancing doses of MCT are largely unknown. We studied the effects of chronic (28 days) supplementation of regular diet with different doses of MCT oil (1, 3, or 6 g/kg/day) or water (control) on working memory (Y-maze), behavior in the Open Field, spatial learning (Morris water maze), and weight of internal organs in male Wistar 2.5-m.o. Rats. In a separate experiment, we evaluated acute (single gavage) and chronic (28 days) effects of MCT or lard supplementation (3 g/kg) on blood biochemical parameters. MCT-1 and MCT-3 doses improved working memory in YM. In MWM, MCT-6 treatment improved spatial memory. Chronic MCT-1 or MCT-3 treatment did not affect internal organ weight, while MCT-6 dose increased liver weight and the brown/white adipose tissue ratio. Acutely, MCT administration elevated blood ß-hydroxybutyrate and malondialdehyde levels. Chronic MCT administration (3 g/kg) did not affect the blood levels of glucose, lactate, pyruvate, acetoacetate, ß-hydroxybutyrate, total and HDL cholesterol, triglycerides, malondialdehyde, and aspartate transaminase and alanine transaminase activities. Therefore, daily supplementation of standard feed with MCT resulted in mild intermittent ketosis. It improved working memory at lower concentrations without significant adverse side effects. At higher concentrations, it improved long-term spatial memory but also resulted in organ weight changes and is likely unsafe. These results highlight the importance of monitoring the metabolic effects of MCT supplementation alongside cognitive assessment in future studies of MCT's neuroprotective properties.

Reference gene expression stability within the rat brain under mild intermittent ketosis induced by supplementation with medium-chain triglycerides.

Reverse transcription followed by quantitative (real-time) polymerase chain reaction (RT-qPCR) has become the gold standard in mRNA expression analysis. However, it requires an accurate choice of reference genes for adequate normalization. The aim of this study was to validate the reference genes for qPCR experiments in the brain of rats in the model of mild ketosis established through supplementation with medium-chain triglycerides (MCT) and intermittent fasting. This approach allows to reproduce certain neuroprotective effects of the classical ketogenic diet while avoiding its adverse effects. Ketogenic treatment targets multiple metabolic pathways, which may affect the reference gene expression. The standard chow of adult Wistar rats was supplemented with MCT (2 ml/kg orogastrically, during 6 h of fasting) or water (equivolume) for 1 month. The mRNA expression of 9 housekeeping genes (Actb, B2m, Gapdh, Hprt1, Pgk1, Ppia, Rpl13a, Sdha, Ywhaz) in the medial prefrontal cortex, dorsal and ventral hippocampus was measured by RT-qPCR. Using the RefFinder® online tool, we have found that the reference gene stability ranking strongly depended on the analyzed brain region. The most stably expressed reference genes were found to be Ppia, Actb, and Rpl13a in the medial prefrontal cortex; Rpl13a, Ywhaz, and Pgk1 in the dorsal hippocampus; Ywhaz, Sdha, and Ppia in the ventral hippocampus. The B2m was identified as an invalid reference gene in the ventral hippocampus, while Sdha, Actb, and Gapdh were unstable in the dorsal hippocampus. The stabilities of the examined reference genes were lower in the dorsal hippocampus compared to the ventral hippocampus and the medial prefrontal cortex. When normalized to the three most stably expressed reference genes, the Gapdh mRNA was upregulated, while the Sdha mRNA was downregulated in the medial prefrontal cortex of MCT-fed animals. Thus, the expression stability of reference genes strongly depends on the examined brain regions. The dorsal and ventral hippocampal areas differ in reference genes stability rankings, which should be taken into account in the RT-qPCR experimental design.

Changes in activity of µ- and m-calpains and signs of neuroinflammation in the hippocampus and striatum of rats after single intraperitoneal injection of subseptic dose of endotoxin.

Some mechanisms of neuronal degeneration in endotoxinemia are already well described, but need to be detailed. In this study, we tested the effect of a single intraperitoneal injection of a LPS sub-septic dose (1 mg/kg of animal weight) on calpain activity in the striatum and hippocampus. We showed, that in the hippocampus the day after LPS administration an increase in production of IL-1ß and TNF-α mRNA, followed by elevated mRNA expression and activity of µ- and m-calpains without signs of microglia activation is observed. In striatal cells, the day after LPS injection an increase in expression of IL-1ß, TNF-α, IBA-1, m-calpain and calpastatin mRNA is revealed, which only intensifies over time. The elicited changes are accompanied by a decrease in motor behavior, which can be considered as a sign of sickness behavior. In the hippocampus, 180 days after LPS administration expression of TNF-α, content and activity of µ-calpain are increased. In the striatum, elevation in expression of TNF-α, IBA-1, µ- and m-calpain mRNA, with hyperactivation of only m-calpain, is observed. Significantly reduced motor activity can be a consequence of LPS-induced neuronal death. A long-lasting endotoxin activates microglia that damage neurons via proinflammation cytokines and calpain hyperactivation. The endotoxin hypothesis of neurodegeneration is unproven, but if correct, then neurodegeneration may be reduced by decreasing endotoxin-induced neuroinflammation and m-calpain hyperactivation. Therefore, the drugs, that decrease endotoxin-induced neuroinflammation and differently inhibit µ- or m-calpain, can be used to prevent or reduce the severity of neurodegeneration.

Developmental prefrontal mRNA expression of D2 dopamine receptor splice variants and working memory impairments in rats after early life Interleukin-1ß elevation.

Long (D2L) and Short (D2S) isoforms of D2 dopamine receptor differ in their biochemical and physiological properties, which could affect functioning of prefrontal cortex. Contribution of distinct D2 dopamine receptor isoforms to cognitive dysfunctions and its developmental regulation are currently not fully elucidated. In the present study, we evaluated developmental mRNA expression of D2S/D2L dopamine receptor isoforms within the rat medial prefrontal cortex (mPFC) in the model of neurodevelopmental cognitive dysfunction. Working memory performance (Y-maze spontaneous alternations) and D2S/D2L mRNA expression in the mPFC (by qRT-PCR) were evaluated in juvenile (P27), adolescent (P42-47) and adult (P75-90) rats after chronic early life treatment with proinflammatory cytokine interleukin (IL)-1ß (1 µg/kg i.p. daily P15-21). It was shown that IL-1ß elevation during the 3rd week of life leads to working memory deficit originating in juvenile animals and persisting into adulthood. D2S mRNA expression was strongly downregulated during adolescence, and such downregulation was exaggerated in animals injected with IL-1ß during P15-21. Early life IL-1ß administrations influenced developmental changes in the D2S/D2L mRNA ratio. This measure was found to be decreased in adolescent and adult control (intact and vehicle-treated) rats compared to juvenile control, while in the case of IL-1ß-treated animals, the decrease in D2S/D2L ratio was observed only in adulthood but not in adolescence compared to juvenile rats. During the adolescence, D2S mRNA expression was downregulated and D2S/D2L ratio was upregulated in the mPFC of rats treated with IL-1ß during the 3rd week of life compared to controls. Based on these data we conclude that changes in the developmental expression of D2 dopamine receptor splice variants within mPFC may underlie long-lasting cognitive deficit associated with neonatal pathology.