Annotation of the results obtained in 2021
The aim of this Project is to develop and assess the effectiveness of methods to alleviate cognitive impairment of different etiologies using medium-chain fatty acid (MCFAs, caprylic and capric) triglycerides and to investigate the mechanisms of the MCFA supplementation effects. The third year of the Project was dedicated to the assessment of the effects of 28 days of medium-chain triglyceride (MCT) administration (3 g/kg/day) on orienting and exploratory behavior, working memory and spatial learning, neuroplasticity in the brain, and possible negative effects on organ systems and inflammation, in male and female adult Wistar rats with a cognitive deficit induced by moderately pyrogenic lipopolysaccharide (LPS) injections during early postnatal development (an animal model of mind cognitive impairment associated with maternal inflammation during perinatal period in humans). The experimental design was as follows. 2-week-old rats received intraperitoneal injections of LPS (25 mcg/kg) or saline as control. At the age of 2.5 months, the animals were subjected to preliminary behavioral testing in the Y-maze test to assess working memory and the Open Field (OF) test to assess orienting and exploratory behavior. Then, the male and female animals were divided into groups that started receiving, orogastrically, either water (control), or lard (3 g/kg/day), or MCT (3 g/kg/day). After 3 weeks of the fat supplementation protocol, the behavioral tests were repeated together with the Morris water maze (MWM) test to assess spatial learning. The animals were decapitated on the next day, the internal organs were weighed, and several brain structures and blood were collected for further analysis. Additionally, in a separate experiment, blood was collected from male and female animals 30, 60, 120, 180 min after single o/g administration of water (control) or MCT (3 g/kg). During the 3rd year of the Project, we also performed biochemical and molecular-genetic analysis of the samples collected from male rats during the 2nd year of the Project realization. In those experiments, male Wistar rats received a single 1 mg/kg dose of LPS. MCT (6 g/kg/day) and lard were then administered for 28 days and were subjected to behavioral tests according to the protocol described above. Additionally, we also analyzed the intestinal microbiome in the fecal samples of the animals from the pilot experiment (where make rats received 28 days of 2 ml/kg MCT). We found that the animals that received 1 mg/kg LPS exhibited a reduced chow consumption and weight gain compared to the animals that received saline. The LPS injection significantly increased the spleen size, while chronic MCT administration decreased the spleen mass to the control values, suggesting an anti-inflammatory effect of MCT. The animals fed with MCT after early postnatal LPS treatment demonstrated reduced locomotor activity during the 2nd round of the OF testing, indicating an improved ability to remember their surrounding environment. In the MWM, MCT-fed animals were faster to learn to find the underwater platform, suggesting improved spatial learning as a result of MCT feeding. LPS injection in moderately pyrogenic dose (25 mcg/kg), led to more weight gain in male but not female rats when the animals were fed lard, but not MCT (3 g/kg). Sex differences were revealed during the preliminary testing in OF. LPS injection led to reduction of the total distance traveled in female rats only, while only male rats demonstrated faster extinction of locomotor activity. During the 2nd round of tests performed after the fat supplementation protocol, MCT-fed male rats no longer showed intergroup differences in extinction of locomotor activity. On the contrary, MCT feeding increased the extinction of locomotor activity in female rats. In MWM, we found significant interaction between LPS x MCT factors and no sex differences. Saline-treated animals that later were fed with MCT traveled longer distance in the target quadrant compared to the control lard-fed group. Notably and unexpectedly, LPS treatment in early postnatal development improved spatial memory compared to the saline treatment, and MCT feeding did not show any additive positive effect. We found that chronically feeding MCT to rats subjected to early postnatal LPS injections had differential effects on the levels of many cytokines and chemokines (interferon-gamma, IL-1beta, IL-10, IL-18, TNF, IP-10, RANTES, MCP-1, EGF, fractalkine), while we found no effects on IL-2, IL-4, IL-5, IL-12p70, IL-17, MIP-1a, MIP-2, VEGF, LIX, and leptin). A single MCT administration (3 g/kg) resulted in a characteristic response pattern in many cytokines (especially IL-1beta, IL-2, IL-10, IL-4) characterized by an acute decrease of their levels 30-60 min post-administration with a gradual increase to pre-administration levels or lower. As expected, acute administration of MCT (3 g/kg) but not lard elevated blood beta-hydroxybutyrate (BHB) level, peaking at 120 min after MCT administration and decreasing 180 min post-administration, indicating that MCT administered to animals fed with regular chow results in intermittent ketosis. Lard administration acutely elevated the blood levels of triglycerides, total cholesterol, HDL-cholesterol, and alanine transaminase (ALT) activity, while MCT administration had no effect on these markers. MCT administration had a slight hypoglycemic effect 30 min post administration. The same markers analyzed in the end of the 28-day protocol in the blood collected 18-22 h after the last fat administration showed no changes compared to control. To assess the effects of MCT administration on the expression of neuroplasticity-related genes, we first analyzed the stability of genetic expression of 9 potential reference genes in medial prefrontal cortex (mPFC), and dorsal and ventral hippocampus (DH, VH) under the condition of intermittent ketosis. Hprt1 and B2m expression was the most stable in all brain structures. Therefore, these genes were selected as references for the following experiments. No changes in the neuroplasticity-related genes (Mmp9, Timp1, Bdnf, Gdnf, Fgf2, Gsk3b, EAAT2, GluN1, GluN2a, GluN2b, GluA1, GluA2) were found in mPFC and VH. In DH, early postnatal LPS exposure resulted in increased levels of GluN1, GluN2a, GluA2 (AMPA), Glut, and the GluN2a/GluN2b ratio, while the MCT administration decreased these levels to control values. A single administration of MCT affected the expressions of Mmp9, Fgf2, Glut3, GluN1 и GluN2a, but not GluN2b in the hippocampus. We also found significant reverse correlation between the total blood ketone body concentrations and the mRNA levels of NMDA receptors GluN1 and GluN2a. The analysis of fecal samples from our pilot experiment revealed that 4 weeks of 2 ml/kg MCT supplementation increased the proportion of Bacteroidota and decreased the proportion of Bacteroidota compared to control. Therefore, daily chronic administration of MCT as a supplement to standard chow resulted in intermittent ketosis and improved cognitive functions in various behavioral tests. Neither acute nor chronic MCT administration (3 g/kg) had significant effects on the markers of metabolic health. MCT administration showed to affect pro- and anti-inflammatory cytokine levels, mRNA levels of genes involved in neuroplasticity, glucose transport, glutamatergic neurotransmission, and gut microbiome composition. The established animal model can be used for further investigation of the effects and mechanisms of MCT administration on cognitive function in normal conditions and in models of cognitive impairment.

 

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Annotation of the results obtained in 2019
The Project seeks to develop effective methods to treat mild cognitive impairments of different genesis using saturated medium-chain fatty acids (capric and caprylic). The first year of the Project realization was dedicated to selecting the most optimal doses and procedures for administering the triglycerides of the saturated medium-chain fatty acids (MCT), based on the maximal beneficial effects on memory with minimal adverse impact on the integral parameters and molecular markers of the whole organism and its organ system functions. To achieve these goals, we conducted preliminary behavioral testing of male Wistar rats in the Y-Maze test to assess working memory and in the Open Field test to assess exploratory behavior. The animals were then divided into groups, which were administered MCT in the dose of 1, 3, or 6 g/kg daily via orogastric route, or had unlimited access to feed supplemented with MCT. The animals in the control groups were receiving orogastric administration of either water or lard (which is rich in saturated long-chain fatty acids), or had unlimited access to feed supplemented with lard. After 3 weeks of the administration, the behavioral tests were repeated. Additionally, the animals were tested in the Morris water maze to assess spatial memory. To investigate the mechanisms of MCT action, we aimed to assess the blood markers of the energy metabolism, oxidative stress, and inflammation, as well as the markers of the anabolic and catabolic processes in the liver the neuroplasticity markers in the brain. Due to the enforcement of the quarantine measures to prevent the spread of COVID-19 infection, we were unable to finish one series of behavioral tests, and therefore, the analysis was conducted based on incomplete samples and is considered preliminary. We also were not able to conduct the molecular analysis of the collected experimental animal samples. However, we did complete the measurements of the metabolic blood markers, as well as the analysis of the expression in the medial prefrontal cortex of the genes involved in neuroplasticity, in the samples collected from the animals in the pilot experiment, where, in behavioral tests, we have previously demonstrated the beneficial effects of MCT administration on memory (this behavioral data had become the basis for this grant’s application). The preliminary analysis of the effects on memory of the continuous administration of the triglycerides of the saturated medium-chain fatty acids has shown a decrease in learning during the second trial in both the Y-maze and Open-field tests in the control groups, including those receiving lard. MCT supplementation of the feed did not lead to any measurable improvements in any of the behavioral tests. However, the administration of MCT in fixed dosage demonstrated a dose-dependent improvement of memory of the animals in the selected tests. A direct comparison of the control group and the group receiving 6 g/kg of MCT daily demonstrated effects similar to those discovered in our pilot study, where MCT was administered daily in the dose of 3 g/kg after 6 hours of fasting. These results suggest that a short period of fasting prior to the administration of MCT may enhance the beneficial effect of MCT, possibly, by allowing a higher blood concentration of the ketone bodies. The biochemical analysis of blood obtained from the animals in the pilot experiment showed an increase of blood concentration of β-hydroxybutyrate (one of the ketone bodies) after administering MCT in the dose of 3 g/kg daily after 6 hours of not having access to the feed. The blood glucose level was the same as in the control group. However, the blood concentration of pyruvate was lower in animals receiving MCT compared to control animals, which may suggest a higher rate of the Krebs cycle due to using ketone bodies instead of pyruvate for the acetyl-CoA synthesis, which in turn would allow increasing the capacity of the Krebs cycle by redirecting pyruvate towards oxaloacetate synthesis. The analysis of gene expression of the ionotropic glutamate NMDA- and AMPA-receptor subunit genes in the medial prefrontal cortex revealed an increase of the mRNA level of the GluN2a- and GluN2b-subunits of the NMDA receptors and a decrease of the GluN2a/GluN2b ratio, as well as an increase of gene expression of the GluA1- и GluA2-subunits of the AMPA receptors, in animals receiving MCT compared to the control animals. Such changes in the subunit ratios of the NMDA receptors and the increase of the AMPA receptor genes in the cortex are characteristic of the development of stronger synaptic connections between neurons, which may reflect one of the molecular mechanisms of the beneficial effects of the MCT administration on learning and memory. Overall, the current research suggests a cumulative positive effect of MCT and intermittent fasting. Based on this preliminary research, for the further investigation of the effects of MCT on the cognitive functions in the model of induced cognitive impairment, we are going to administer MCT via orogastric route either in the dose of 6 g/kg daily or in the dose of 3 kg/d daily after a short period of limiting the access to feed. The optimal dose and administration method will be finally determined after completing the behavioral testing of the 3rd cohort of animals, a full statistical analysis based on larger samples, and a molecular analysis of the beneficial effects of MCT and possible side effects on the functions of various organs and organ systems.

 

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Annotation of the results obtained in 2020
The aim of this Project is to develop and assess the effectiveness of methods to alleviate cognitive impairment of different etiologies using medium-chain fatty acid (MCFAs, caprylic and capric) triglycerides. The second year of the Project was dedicated to the assessment of the effects of 28 days of medium-chain triglyceride (MCT) administration (6 g/kg/day) on orienting and exploratory behavior, working memory and spatial learning in animals with a cognitive deficit induced by a single lipopolysaccharide (LPS) injection. In order to achieve this, male Wistar rats were subjected to a preliminary behavioral testing in the Y maze test to assess working memory and the Open Field test to assess orienting and exploratory behavior. Then, the animals were divided into groups that received a single intraperitoneal injection of either LPS (1 mg/kg) or saline. Immediately after that, the animals started receiving, orogastrically, either water (control), or lard, or MCT. After 3 weeks of the treatments, the behavioral tests were repeated together with the Morris water maze test to assess spatial learning. To investigate the mechanisms of the MCT-induced effects we thought to measure the energy metabolism, oxidative stress, and pro inflammatory markers in the blood, catabolic and anabolic biomarkers in the liver, and the neuroplasticity markers in the brain. We found that the animals that received LPS exhibited a reduced feeding motivation compared to the animals that received saline. These LPS-treated animals ate less chow and weighed less during the 1st week after the injection. Among the LPS-treated animals, the reduction of the feeding motivation was less pronounced in the animals that received MCT immediately after the LPS injection compared to the animals that received water or lard. We also found that the spleens of the LPS-treated animals that did not receive MCT were enlarged 28 days after the endotoxin injection compared to the animals that received saline. On the other hand, the spleens of the LPS-treated animals that were receiving MCT did not show any changes of the spleen mass, which may be a sign of the anti-inflammatory effect of the MCT treatment. In the Open Field and Morris water maze tests, MCT treatment resulted in a small but significant improvement of cognitive functions. These animals tended to travel a shorter distance during the second Open Field test trial compared to the animals that were treated with water or lard. In the Morris water maze test, the animals that received MCT were able to find the platform hidden under the water faster compared to the animals in other groups. This effect was observed both in the LPS-treated and the saline-treated animals. There were no changes found in the time spent in the target sector during the probe trial with no platform. The obtained integral and behavioral data require a more detailed analysis. The expression levels of the neuroplasticity related genes in several brain structures will be measured in the near future. We have already designed original sequences for primers and TaqMan probes, which have demonstrated a high fidelity in multiplex reactions. One of the goals this year was to assess the acute and chronic effects of the MCT administration, administered in various doses, on the energy metabolism and metabolic health blood markers. The acute effects were assessed in a separate experiment, in which the animals were given 3 g/kg MCT, lard, or water, and where then decapitated 30 or 120 minutes later. Blood was collected for biochemical analysis. We measured the blood levels of glucose, lactate, pyruvate, acetoacetate, β-hydroxybutyrate (BHB), total and HDL cholesterol, triglycerides, malondialdehyde, and the activities of aspartate transaminase (AST) and alanine transaminase (ALT). The effects of chronic MCT administration were assessed using the blood plasma samples collected during the first year of the Project. The chronic administration of neither fat had any effect on the blood levels of glucose, triglycerides, total and HDL cholesterol. It also did not affect the organ masses of the heart, lungs, thymus, liver, spleen, white and brown adipose tissues. It also had no effect on the levels of serotonin, dopamine and its metabolites measured in the striatum. The acute MCT administration increased the blood BHB levels for 2 hours after the administration, whereas the lard or water administration showed no effect. Lard administration increased the blood levels of triglycerides, cholesterol and ALT activity 2 hours after the administration, while MCT administration did not. Therefore, daily MCT administration as a supplement to standard feed allowed to establish a state of mild ketosis and improved cognitive functions in multiple behavioral tests. Neither acute nor chronic administration of MCT had any adverse effects on the metabolic health markers. This animal model may be further used to investigate the mechanisms of the effects of MCFAs on cognitive functions in healthy animals and in animal models of various cognitive impairments.

 

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