Acute and Chronic Exposure to Linagliptin, a Selective Inhibitor of Dipeptidyl Peptidase-4 (DPP-4), Has an Effect on Dopamine, Serotonin and Noradrenaline Level in the Striatum and Hippocampus of Rats.

Linagliptin is a selective dipeptidyl peptidase-4 (DPP-4) inhibitor that indirectly elevates the glucagon-like peptide-1 (GLP-1) level. The aim of the present study was to check whether linagliptin has an influence on neurotransmission in rat brain. Rats were acutely and chronically exposed to linagliptin (10 and 20 mg/kg, intraperitoneally (i.p.)). Twenty-four hours later, the striatum and hippocampus were selected for further studies. In neurochemical experiments, using high-performance liquid chromatography with electrochemical detection (HPLC-ED), the concentrations of three major neurotransmitters-dopamine, serotonin and noradrenaline-and their metabolites were measured. The analysis of mRNA expression of dopamine (D1 and D2), serotonin (5-HT-1 and 5-HT-2) and noradrenaline (α1 and α2a) receptors was also investigated using real-time quantitative reverse transcription polymerase chain reaction (RQ-PCR) in the same brain areas. Linagliptin has the ability to influence the dopaminergic system. In the striatum, the elevation of dopamine and its metabolites was observed after repeated administration of that linagliptin, and in the hippocampus, a reduction in dopamine metabolism was demonstrated. Acute linagliptin exposure increases the serotonin level in both areas, while after chronic linagliptin administration a tendency for the mRNA expression of serotoninergic receptors (5-HT1A and 5-HT2A) to increase was observed. A single instance of exposure to linagliptin significantly modified the noradrenaline level in the striatum and intensified noradrenaline turnover in the hippocampus. The recognition of the interactions in the brain between DPP-4 inhibitors and neurotransmitters and/or receptors is a crucial step for finding novel discoveries in the pharmacology of DPP-4 inhibitors and raises hope for further applications of DPP-4 inhibitors in clinical practices.

Social Interaction in Adolescent Rats with Neonatal Ethanol Exposure: Impact of Sex and CE-123, a Selective Dopamine Reuptake Inhibitor.

Children with fetal alcohol spectrum disorders (FASDs) demonstrate deficits in social functioning that contribute to early withdrawal from school and delinquency, as well as the development of anxiety and depression. Dopamine is involved in reward, motivation, and social behavior. Thus, we evaluated whether neonatal ethanol exposure (in an animal model of FASDs) has an impact on social recognition memory using the three-chamber social novelty discrimination test during early and middle adolescence in male and female rats, and whether the modafinil analog, the novel atypical dopamine reuptake inhibitor CE-123, can modify this effect. Our study shows that male and female rats neonatally exposed to ethanol exhibited sex- and age-dependent deficits in social novelty discrimination in early (male) and middle (female) adolescence. These deficits were specific to the social domain and not simply due to more general deficits in learning and memory because these animals did not exhibit changes in short-term recognition memory in the novel object recognition task. Furthermore, early-adolescent male rats that were neonatally exposed to ethanol did not show changes in the anxiety index but demonstrated an increase in locomotor activity. Chronic treatment with CE-123, however, prevented the appearance of these social deficits. In the hippocampus of adolescent rats, CE-123 increased BDNF and decreased its signal transduction TrkB receptor expression level in ethanol-exposed animals during development, suggesting an increase in neuroplasticity. Thus, selective dopamine reuptake inhibitors, such as CE-123, represent interesting drug candidates for the treatment of deficits in social behavior in adolescent individuals with FASDs.

Comparing the Healing Abilities of Fluorapatite and Hydroxyapatite Ceramics in Regenerating Bone Tissue: An In Vivo Study.

Some reports in the literature show the advantages of fluoride-containing apatite ceramics over hydroxyapatite (HAP), at least in some aspects. While HAP has been used extensively in the treatment of bone defects, fluoridated apatite has hardly been tested in vivo. In order to verify the biological properties of fluoride-doped apatite and to assess its therapeutic potential, we synthesized fluorapatite (FAP) and applied it as a filling in bone defects of experimental animals (rabbits). The treatment effects were evaluated on extracted bones after 3 and 6 months from implantation using peripheral quantitative computed tomography (pQCT), dual-energy X-ray absorptiometry (DXA), radiography (X-ray) and histological staining. The study proved the integration between FAP and the bone tissue, thus indicating its stimulating effect on new bone formation and mineralization. The results achieved after 3 months of treatment were difficult to interpret unequivocally and suggested the transient delay in FAP integration of bone in comparison with HAP. The reasons for this phenomenon are unclear. Most likely, these differences between FAP and HAP resulted mainly from the different porosities, densities and ionic reactivity of the ceramics, which in our opinion affected their solubility, integration and degree of bone tissue resorption. However, it was shown that 6 months after implantation, similar level of bone defect regeneration was achieved for both FAP and HAP. In this article, we present our hypothesis concerning the basis of this phenomenon.

Matrix-assisted laser desorption/ionization mass spectrometry imaging sample preparation using wet-interface matrix deposition for lipid analysis.

RATIONALE: Sample preparation is one of the most crucial steps for matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). Scientists beginning their study with this technique may be overwhelmed by the variety of matrices, solvents, and concentrations; the methods of their applications; and the lack of widely available knowledge of the effect of these parameters on the results. Here we present in depth the aspects of matrix deposition, which will be helpful for the scientific community. METHODS: In this study, we tested several MALDI matrices, such as 2,5-dihydroxybenzoic acid (DHB), norharmane, N-(1-naphthyl)ethylenediamine dihydrochloride (NEDC), and 9-aminoacridine (9AA), using the SunCollect system: wet-interface matrix deposition in the context of lipid analysis. We optimized the number of matrix layers and nozzle settings in terms of spectral intensity and the overall quality of the obtained ion maps. RESULTS: Our research presents the effect of the number of matrix layers and nozzle settings on the results and allows for choosing the optimal parameters for the analyses. In positive ionization mode, DHB matrix could be chosen first. In the negative ionization mode, 1,5-diaminonaphthalene matrix produces a higher peak intensity in a lower mass range and seems to provide more information than 9AA. We recommend NEDC for particular processes such as glucose analysis. Compared to the remaining matrices, norharmane shows significant changes in the obtained ion maps. CONCLUSIONS: Such a large amount of data allow us to observe an interesting conclusion: the obtained ion image for a particular ion could differ dramatically with a change in the matrix, the solvent composition, or even the number of matrix layers. This must be considered when interpreting the result, impelling us to compare the results obtained with different matrices with caution.

Impact of Mephedrone on Fear Memory in Adolescent Rats: Involvement of Matrix Metalloproteinase-9 (MMP-9) and N-Methyl-D-aspartate (NMDA) Receptor.

Treatment of Post-Traumatic Stress Disorder (PTSD) is complicated by the presence of drug use disorder comorbidity. Here, we examine whether conditioned fear (PTSD model) modifies the rewarding effect of mephedrone and if repeated mephedrone injections have impact on trauma-related behaviors (fear sensitization, extinction, and recall of the fear reaction). We also analyzed whether these trauma-induced changes were associated with exacerbation in metalloproteinase-9 (MMP-9) and the GluN2A and GluN2B subunits of N-methyl-D-aspartate (NMDA) glutamate receptor expression in such brain structures as the hippocampus and basolateral amygdala. Male adolescent rats underwent trauma exposure (1.5 mA footshock), followed 7 days later by a conditioned place preference training with mephedrone. Next, the post-conditioning test was performed. Fear sensitization, conditioned fear, anxiety-like behavior, extinction acquisition and relapse were then assessed to evaluate behavioral changes. MMP-9, GluN2A and GluN2B were subsequently measured. Trauma-exposed rats subjected to mephedrone treatment acquired a strong place preference and exhibited impairment in fear extinction and reinstatement. Mephedrone had no effect on trauma-induced MMP-9 level in the basolateral amygdala, but decreased it in the hippocampus. GluN2B expression was decreased in the hippocampus, but increased in the basolateral amygdala of mephedrone-treated stressed rats. These data suggest that the modification of the hippocampus and basolateral amygdala due to mephedrone use can induce fear memory impairment and drug seeking behavior in adolescent male rats.

Continuous Ingestion of Lacticaseibacillus rhamnosus JB-1 during Chronic Stress Ensures Neurometabolic and Behavioural Stability in Rats.

The intestinal microbiome composition and dietary supplementation with psychobiotics can result in neurochemical alterations in the brain, which are possible due to the presence of the brain-gut-microbiome axis. In the present study, magnetic resonance spectroscopy (MRS) and behavioural testing were used to evaluate whether treatment with Lacticaseibacillus rhamnosus JB-1 (JB­1) bacteria alters brain metabolites' levels and behaviour during continuous exposure to chronic stress. Twenty Wistar rats were subjected to eight weeks of a chronic unpredictable mild stress protocol. Simultaneously, half of them were fed with JB-1 bacteria, and the second half was given a daily placebo. Animals were examined at three-time points: before starting the stress protocol and after five and eight weeks of stress onset. In the elevated plus maze behavioural test the placebo group displayed increased anxiety expressed by almost complete avoidance of exploration, while the JB-1 dietary supplementation mitigated anxiety which resulted in a longer exploration time. Hippocampal MRS measurements demonstrated a significant decrease in glutamine + glutathione concentration in the placebo group compared to the JB-1 bacteria-supplemented group after five weeks of stress. With the progression of stress the decrease of glutamate, glutathione, taurine, and macromolecular concentrations were observed in the placebo group as compared to baseline. The level of brain metabolites in the JB-1-supplemented rats were stable throughout the experiment, with only the taurine level decreasing between weeks five and eight of stress. These data indicated that the JB-1 bacteria diet might stabilize levels of stress-related neurometabolites in rat brain and could prevent the development of anxiety/depressive-like behaviour.

Linagliptin, a Selective Dipeptidyl Peptidase-4 Inhibitor, Reduces Physical and Behavioral Effects of Morphine Withdrawal.

(1) Background: Recent data indicate that receptors for GLP-1 peptide are involved in the activity of the mesolimbic system. Thus, the purpose of the present study was to examine the effect of the selective dipeptidyl peptidase-4 (DPP-4) inhibitor, linagliptin, on morphine dependence in mice. (2) Methods: Morphine dependence in mice was obtained by administration of increasing doses of morphine for eight consecutive days, twice a day. On the 9th day of the experiment, the naloxone-induced (2 mg/kg, ip) morphine withdrawal signs (jumping) were assessed. Moreover, behavioral effects of short-term (60 h after morphine discontinuation) and long-term (14 days after morphine discontinuation) morphine withdrawal were observed. In terms of behavioral effects, the depressive effect in the forced swim test and anxiety in the elevated plus maze test were investigated. Locomotor activity of mice was also studied. (3) Results: The administration of linagliptin (10 and 20 mg/kg, ip) for 8 consecutive days before morphine injections significantly diminished the number of naloxone-induced morphine withdrawal signs (jumping) in mice. In addition, the cessation of morphine administration induced depressive behavior in mice which were observed during short- and long-term morphine withdrawal. Linagliptin administered during morphine withdrawal significantly reduced the depressive behavior in studied mice. Furthermore, the short-term morphine withdrawal evoked anxiety which also was reduced by linagliptin in mice. (4) Conclusions: The present study reveals that GLP-1 receptors are involved in morphine dependence. What is more, linagliptin might be a valuable drug in attenuating the physical symptoms of morphine dependence. It might be also a useful drug in reducing emotional disturbances which may develop during the morphine withdrawal period.

Potential for Prebiotic Stabilized Cornus mas L. Lyophilized Extract in the Prophylaxis of Diabetes Mellitus in Streptozotocin Diabetic Rats.

As a systemic disease, diabetes mellitus (DM) is characterized by the disruption of many glucose metabolic pathways. Therefore, it seems critical to study new therapies to support treatment to develop therapeutic systems that can operate across a broad metabolic spectrum. The current state of knowledge indicates an essential role of the gut microbiota in the development and course of the disease. Cornus mas fruits have demonstrated a rich biological activity profile and potential for application in the treatment of DM. As part of a preliminary analysis, the activity of four cultivars of Cornus mas fruits was analyzed. The cultivar Wydubieckij was selected as having the highest activity in in vitro conditions for further prebiotic system preparation. The study aimed to develop a unique therapeutic system based, first of all, on the mechanism of α-glucosidase inhibition and the antioxidant effect resulting from the activity of the plant extract used, combined with the prebiotic effect of inulin. The obtained system was characterized in vitro in terms of antioxidant activity and enzyme inhibition capacity, and was then tested on diabetic rats. The study was coupled with an analysis of changes in the intestinal microflora. The system of prebiotic stabilized Cornus mas L. lyophilized extract with inulin offers valuable support for the prophylaxis and treatment of DM.

Alteration of Ethanol Reward by Prior Mephedrone Exposure: The Role of Age and Matrix Metalloproteinase-9 (MMP-9).

Mephedrone, a synthetic cathinone, is widely abused by adolescents and young adults. The aim of this study was to determine: (i) whether prior mephedrone exposure would alter ethanol reward and (ii) whether age and matrix metalloproteinase-9 (MMP-9) are important in this regard. In our research, male Wistar rats at postnatal day 30 (PND30) received mephedrone at the dose of 10 mg/kg, i.p., 3 times a day for 7 days. To clarify the role of MMP-9 in the mephedrone effects, one mephedrone-treated group received minocycline, as an MMP-9 antagonist. Animals were then assigned to conditioned place preference (CPP) procedure at PND38 (adolescent) or at PND69 (adult). After the CPP test (PND48/79), expression of dopamine D1 receptors (D1R), Cav1.2 (a subtype of L-type calcium channels), and MMP-9 was quantified in the rat ventral striatum (vSTR). The influence of mephedrone administration on the N-methyl-D-aspartate glutamate receptors (NMDAR) subunits (GluN1, GluN2A, and GluN2B) was then assessed in the vSTR of adult rats (only). These results indicate that, in contrast with adolescent rats, adult rats with prior mephedrone administration appear to be more sensitive to the ethanol effect in the CPP test under the drug-free state. The mephedrone effect in adult rats was associated with upregulation of D1R, NMDAR/GluN2B, MMP-9, and Cav1.2 signaling. MMP-9 appears to contribute to these changes in proteins expression because minocycline pretreatment blocked mephedrone-evoked sensitivity to ethanol reward. Thus, our results suggest that prior mephedrone exposure differentially alters ethanol reward in adolescent and adult rats.

Saturation transfer MRI is sensitive to neurochemical changes in the rat brain due to chronic unpredictable mild stress.

Chemical exchange saturation transfer (CEST) MRI was performed for the evaluation of cerebral metabolic changes in a rat model of depressive-like disease induced by chronic unpredictable mild stress (CUMS). CEST Z-spectra were acquired on a 7 T MRI with two saturation B1 amplitudes (0.5 and 0.75 µT) to measure the magnetization transfer ratio (MTR), CEST and relayed nuclear Overhauser effect (rNOE). Cerebral cortex and hippocampus were examined in two groups of animals: healthy control (n = 10) and stressed (n = 14), the latter of which was exposed to eight weeks of the CUMS protocol. The stressed group Z-spectrum parameters, primarily MTRs, were significantly lower than in controls, at all selected frequency offsets (3.5, 3.0, 2.0, - 3.2, - 3.6 ppm) in the cortex (the largest difference of ~ 3.5% at - 3.6 ppm, p = 0.0005) and the hippocampus (MTRs measured with a B1 = 0.5 µT). The hippocampal rNOE contributions decreased significantly in the stressed brains. Glutamate concentration (assessed using ELISA) and MTR at 3 ppm correlated positively in both brain regions. GABA concentration also correlated positively with CEST contributions in both cerebral areas, while such correlation with MTR was positive in hippocampus, and nonsignificant in cortex. Results indicate that CEST is sensitive to neurometabolic changes following chronic stress exposure.

Memantine Prevents the WIN 55,212-2 Evoked Cross-Priming of Ethanol-Induced Conditioned Place Preference (CPP).

The activation of the endocannabinoid system controls the release of many neurotransmitters involved in the brain reward pathways, including glutamate. Both endocannabinoid and glutamate systems are crucial for alcohol relapse. In the present study, we hypothesize that N-methyl-D-aspartate (NMDA) glutamate receptors regulate the ability of a priming dose of WIN 55,212-2 to cross-reinstate ethanol-induced conditioned place preference (CPP). To test this hypothesis, ethanol-induced (1.0 g/kg, 10% w/v, i.p.) CPP (unbiased method) was established using male adult Wistar rats. After CPP extinction, one group of animals received WIN 55,212-2 (1.0 and 2.0 mg/kg, i.p.), the cannabinoid receptor 1 (CB1) agonist, or ethanol, and the other group received memantine (3.0 or 10 mg/kg, i.p.), the NMDA antagonist and WIN 55,212-2 on the reinstatement day. Our results showed that a priming injection of WIN 55,212-2 (2.0 mg/kg, i.p.) reinstated (cross-reinstated) ethanol-induced CPP with similar efficacy to ethanol. Memantine (3.0 or 10 mg/kg, i.p.) pretreatment blocked this WIN 55,212-2 effect. Furthermore, our experiments indicated that ethanol withdrawal (7 days withdrawal after 10 days ethanol administration) down-regulated the CNR1 (encoding CB1), GRIN1/2A (encoding GluN1 and GluN2A subunit of the NMDA receptor) genes expression in the prefrontal cortex and dorsal striatum, but up-regulated these in the hippocampus, confirming the involvement of these receptors in ethanol rewarding effects. Thus, our results show that the endocannabinoid system is involved in the motivational properties of ethanol, and glutamate may control cannabinoid induced relapse into ethanol seeking behavior.

The Study of Derivatization Prior MALDI MSI Analysis-Charge Tagging Based on the Cholesterol and Betaine Aldehyde.

Mass spectrometry imaging is a powerful tool for analyzing the different kinds of molecules in tissue sections, but some substances cannot be measured easily, due to their physicochemical properties. In such cases, chemical derivatization could be applied to introduce the charge into the molecule and facilitate its detection. Here, we study cholesterol derivatization with betaine aldehyde from tissue slices and evaluate how different sample preparation methods influence the signal from the derivatization product. In this study, we have tested different solutions for betaine aldehyde, different approaches to betaine aldehyde deposition (number of layers, deposition nozzle height), and different MALDI matrices for its analysis. As a result, we proved that the proposed approach could be used for the analysis of cholesterol in different tissues.

Insight into Glutamatergic Involvement in Rewarding Effects of Mephedrone in Rats: In Vivo and Ex Vivo Study.

Mephedrone is a widely used drug of abuse, exerting its effects by interacting with monoamine transporters. Although this mechanism has been widely studied heretofore, little is known about the involvement of glutamatergic transmission in mephedrone effects. In this study, we comprehensively evaluated glutamatergic involvement in rewarding effects of mephedrone using an interdisciplinary approach including (1) behavioural study on effects of memantine (non-selective NMDA antagonist) on expression of mephedrone-induced conditioned place preference (CPP) in rats; (2) evaluation of glutamate concentrations in the hippocampus of rats following 6 days of mephedrone administration, using in vivo magnetic resonance spectroscopy (MRS); and (3) determination of glutamate levels in the hippocampus of rats treated with mephedrone and subjected to MRS, using ion-exchange chromatography. In the presented research, we confirmed priorly reported mephedrone-induced rewarding effects in the CPP paradigm and showed that memantine (5 mg/kg) was able to reverse the expression of this effect. MRS study showed that subchronic mephedrone administration increased glutamate level in the hippocampus when measured in vivo 24 h (5 mg/kg, 10 mg/kg and 20 mg/kg) and 2 weeks (5 mg/kg and 20 mg/kg) after last injection. Ex vivo chromatographic analysis did not show significant changes in hippocampal glutamate concentrations; however, it showed similar results as obtained in the MRS study proving its validity. Taken together, the presented study provides new insight into glutamatergic involvement in rewarding properties of mephedrone.

Dietary supplementation with Lactobacillus rhamnosus JB-1 restores brain neurochemical balance and mitigates the progression of mood disorder in a rat model of chronic unpredictable mild stress.

Major depressive disorder is a stress-related disease associated with brain metabolic dysregulation in the glutamine-glutamate/γ-aminobutyric acid (Gln-Glu/GABA) cycle. Recent studies have demonstrated that microbiome-gut-brain interactions have the potential to influence mental health. The hypothesis of this study was that Lactobacillus rhamnosus JB-1 (LR-JB1™) dietary supplementation has a positive impact on neuro-metabolism which can be quantified in vivo using magnetic resonance spectroscopy (MRS). A rat model of depressive-like disorder, chronic unpredictable mild stress (CUMS), was used. Baseline comparisons of MRS and behavior were obtained in a control group and in a stressed group subjected to CUMS. Of the 22 metabolites measured using MRS, stressed rats had significantly lower concentrations of GABA, glutamate, glutamine + glutathione, glutamate + glutamine, total creatine, and total N-acetylaspartate (tNAA). Stressed rats were then separated into 2 groups and supplemented with either LR-JB1™ or placebo and re-evaluated after 4 weeks of continued CUMS. The LR-JB1™ microbiotic diet restored these metabolites to levels previously observed in controls, while the placebo diet resulted in further significant decrease of glutamate, total choline, and tNAA. LR-JB1™ treated animals also exhibited calmer and more relaxed behavior, as compared with placebo treated animals. In summary, significant cerebral biochemical downregulation of major brain metabolites following prolonged stress were measured in vivo using MRS, and these decreases were reversed using a microbiotic dietary supplement of LR-JB1™, even in the presence of continued stress, which also resulted in a reduction of stress-induced behavior in a rat model of depressive-like disorder.

Effect of a carbonated HAP/ß-glucan composite bone substitute on healing of drilled bone voids in the proximal tibial metaphysis of rabbits.

A novel elastic hydroxyapatite-based composite of high surgical handiness has been developed. Its potential application in orthopedics as a filler of bone defects has been studied. The biomaterial was composed of carbonated hydroxyapatite (CHAP) granules and polysaccharide polymer (ß-1,3-glucan). Cylinders of 4mm in diameter and 6mm in length were implanted into bone cavities created in the proximal metaphysis of tibiae of 24 New Zealand white rabbits. 18 sham-operated animals were used as controls. After 1, 3 or 6 months, the rabbits were euthanized, the bones were harvested and subjected to analysis. Radiological images and histological sections revealed integration of implants with bone tissue with no signs of graft rejection. Peripheral quantitative computed tomography (pQCT) indicated the stimulating effect of the biomaterial on bone formation and mineralization. Densitometry (DXA) analysis suggested that biomineralization of bones was preceded by bioresorption and gradual disappearance of porous ceramic granules. The findings suggest that the CHAP-glucan composite material enables regeneration of bone tissue and could serve as a bone defect filler.