Month: February 2019

Taken together, our results suggest that chronic administration of methylphenidate in mice, at doses that approximate those at the higher therapeutic range in humans, results in a reduced expression of neurotrophic factors, increased neuroinflammation, and a small, but significant loss of SNpc dopamine neurons. These results can only be interpreted in the context on normal brain structure and function, and thus would have direct implications for the illicit/neurocognitive use of MPH. Since the underlying anatomy and biochemistry of ADHD has not been definitively characterized, our findings may or may not be generalizable to the vast majority of humans who are properly diagnosed with ADHD and are prescribed methylphenidate. Nevertheless, this work supports studies that demonstrate that drugs shown to increase the levels of dopamine in the synaptic cleft can contribute to degenerative changes in the basal ganglia. DA neuron and Iba-1-positive microglial cell number in the SNpc were estimated using standard model-based stereological methods. Briefly, for neuronal counts, brains were blocked and serially-sectioned at 10 mm from the rostral hippocampus to the cerebellar-midbrain junction. Serial sections were mounted 5 sections per slide onto polyionic slides. TH-positive neurons and TH-negative, Nissl-positive cells within the SNpc that had the Salvianolic-acid-B characteristics of dopaminergic neurons were counted using a 406 objective. Specifically, neurons from both left and right sides of the SNpc within one section per slide were counted. Both Iba-1 resting and activated microglia were counted. Stringent measures were adopted to classify Iba-1 positive microglia as resting or activated based on morphology based on the detailed description by Graeber and Streit. Microglial cells would be deemed as resting if they contained a small oval Iba-1-positive cell body that averaged 3 microns in diameter with long slender processes. Microglia would be classified as activated when the cell body was slightly increased in size compared to resting microglia and had an irregular shape. Based on cell size of the counting particle in 12 micron sections, we used a high NA lens and a total magnification of 10006 in which we were able to clearly define approximately 18 focal planes within our section. From in vitro studies, expression of 1a-OHase by macrophages at the site of an infection seems to be an important part of innate immunity. Montoya et al have shown that upregulation of the vitamin D pathway occurs in leprosy lesions of patients with self limiting forms of the disease, however, beyond that study there is no evidence that 1a-OHase is expressed 14α-hydroxy-Sprengerinin-C by macrophages in vivo as a result of experimental infection. Intra-mammary infections during lactation offers a model of bacterial infection to determine if 1a-OHase is expressed in response to bacterial infection in vivo. Common pathogens that cause mammary infections include Escherichia coli, Staphylococcus aureus, and Streptococcus uberis. During mammary infection the number of somatic cells secreted in milk will often exceed 106 cells/mL. Approximately 80 to 90 percent of somatic cells in milk from an infected mammary gland are neutrophils and the remainder of the cells are macrophages and lymphocytes. The advantage of using a mammary infection model is that the infiltrating cells during mammary infection can easily isolated from milk using non-invasive procedures; allowing us to study the in vivo immune responses of immune cells to bacterial infection. TLRs are present in the bovine mammary gland and invasion of the mammary gland by bacteria triggers an innate immune response by TLR signaling.

In this study, we administered an acute regimen of MPTP, an agent that is known to induce oxidative stress, to MPTP-resistant Swiss-Webster mice treated with a chronic regimen of 1 or 10 mg/kg MPH. We found that chronic exposure to both 1 mg/kg and 10 mg/kg MPH increased the sensitivity of SNpc dopamine neurons to oxidative stress, based on a significantly increased SNpc dopamine neuron loss in mice administered MPH as compared to saline-treated control mice. Although the mechanism for this neuronal loss is unknown, a significant increase in MPH-induced activated microglia was observed; therefore, we hypothesize that an increase in free radical formation along with a concomitant neuroinflammatory response increases the sensitivity of the SNpc dopamine neurons to a later oxidative challenge. This conclusion is supported by a recent epidemiological study that showed that long-term amphetamine usage, which like MPH results in higher levels of striatal dopamine in the synaptic cleft, results in a significantly higher risk for developing Salvianolic-acid-C. In order to address the mechanism for increased sensitivity of dopamine neurons, we used an unbiased gene microarray analysis. A comparison of heat maps representative of relative mRNA expression shows a number of genes whose direction of expression change was similar after chronic administration of 1 and 10 mg/kg. Gene Set Enrichment Analysis identified gene sets that were related to inflammation and cell damage and repair pathways. Using qPCR validation, we measured significant decreases in mRNA expression of the neurotrophins bdnf and gdnf in the SNpc after both acute and chronic dosing of 10 mg/kg MPH. We also found significant decreases in mRNA expression of genes involved in dopamine biosynthesis and handling and the vesicular monoamine transporter ). These changes were observed following both acute and chronic doses of Isosalvianolic-acid-B MPH in the SNpc. Previous reports have associated decreases in mRNA expression of vmat2 and dat1 with neurotoxicity in cases where pharmacotherapeutic agents that alter dopamine levels and neurodegenerative conditions, respectively. The observed downward changes in the mRNA message of dat1 and th may also be due to the covalent modification by dopamine quinones leading to its translational inactivation. Notably, our Affymetrix and qPCR studies also found that acute exposure to higher doses of MPH increased the expression of inflammatory genes in the striatum, including the pro-inflammatory genes tnf-a and il-6. This increase in the pro-inflammatory gene expression following a single acute dosage suggests that MPH does induce inflammation, and this is supported by our finding of increased numbers of both total and activated microglial cells in the SNpc. Surprisingly, we did not see an increase in inflammatory gene expression after chronic administration of MPH, although we did continue to observe an increased number of morphologically activated microglia. This suggests that sometime during the course of the chronic exposure to MPH, there might be a dampening of inflammatory gene expression. It is unknown at this time if the gene repression we observe after chronic treatment with MPH is permanent, or if it can at a later time be re-induced. If this is the case, then the activated microglia observed, have the potential to play a modulatory role in later inductions of oxidative stress that would affect the same brain systems. Alternatively, it is also possible that microglia that are activated do not have the ability to return to their morphologically pre-inflamed state, as other studies have shown evidence of microglia activation long after resolution of the initiating insult.

The observed lack of change in total dopamine concentrations at the higher dose might reflect a ceiling effect achieved due to chronic dosing of the drug, or it may be the result of a compensatory alteration in the production of dopamine that results from the observed 20% loss in dopamine neurons in the SNpc. In order to determine if this compensation is occurring, we measured the ratio of striatal dopamine to SNpc DA neurons. When examined as a ratio, MPH treatment demonstrate a significant increase in the dopamine:SNpc neuron ratios, suggesting that either dose of MPH increases striatal dopamine, not just that of 1 mg/kg MPH. It is well known that increased extracellular DA may be problematic. Oxidation of DA can produce both superoxide and hydrogen peroxide, which may then form hydroxyl radicals in the presence of certain metals. Additionally, previous studies have indicated that DA can become neurotoxic following its oxidation to a DA quinone, which may then react with cellular thiols to form 5-S-glutathionyl DA and 5-S-cysteinyl DA. The subsequent oxidation of 5-S-cysteinyl Danshensu produces a number of neurotoxic compounds. An increase in the free radical content in the basal ganglia has been shown to potentiate neurodegeneration. In addition to a direct effect of MPH on the basal ganglia, we hypothesized that chronic MPH could increase sensitivity of SNpc dopamine neurons to a later oxidative stress exposure. MPH’s mechanism of action- blockade of the DAT- is similar to that of cocaine and results in an increase in extracellular dopamine, which has been shown to quickly form free radical adducts. Similar to our findings, compartmentation of the both ectoenzymes was recently reported for lymph nodes in chronic lymphocytic leukemia patients. In this study, CD39 was widely expressed while CD73 was restricted to proliferation centers suggesting that adenosine generation is locally confined. CD39 and CD73 were found to be unevenly distributed among the different cardiac immune cells. Circulating and resident cardiac lymphoid cells highly expressed CD73 with little abundance on myeloid cells,Tanshinone-I while the opposite was true for CD39. In fact, resident cardiac APCs and monocytes showed no measurable CD73 but were highly positive for CD39. The latter hypothesis is supported by findings in a lymphocyte endothelial coculture model, in which CD73 activity was significantly decreased during adhesion and migration processes. Consistent with data in the literature, we found after I/R profoundly increased numbers of granulocytes and monocytes within the heart. As to the enzymes of the ectonucleotidase cascade, CD73 und CD39 were significantly upregulated on granulocytes under these conditions. While in the unstressed heart coronary endothelial cells contribute to 90% of the cell-associated CD73 in the heart, this fraction dramatically changes after I/R when leukocyte-bound CD73 comprises about 2/3 of the entire CD73 within myocardial tissue. This difference is most likely even more pronounced when considering the local accumulation of immune cells at the site of inflammation. Similarly, we have observed a strong increase in CD73 expression on T-cells while CD39 remained unchanged. Consistent with this finding we recently reported upregulation of CD73 on Treg after antigenic stimulation which was associated with adenosine A2a receptor mediated downregulation of active NFkB and cytokine release. In the context of cardiac function after I/R, the upregulation of CD73 on lymphocytes and granulocytes suggests an adenosinergic axis which becomes functionally relevant when necrosis and apoptosis lead to elevated extracellular nucleotide levels. Since increased free radical production has been shown to increase the sensitivity of SNpc neurons to environmental or administered xenobiotics, it is possible that long-term MPH could be a contributing etiological factor in a multi-hit hypothesis for induction of Parkinson’s disease.

The Monitoring the Future Study reported that 2.7% of high school students reported a non-therapeutic use of MPH while 1.9% of college students reported a similar non-medicinal usage In both diagnosed ADHD and non-ADHD populations, MPH has been shown to increase scores on standardized tests, as well as increase working memory and thus, there have been calls for making it available as an ‘‘over the counter’’ drug. Despite the extensive use of this stimulant in ADHD and as well as for ‘‘off-label’’ use, few papers have been published regarding the long-term neurological consequences of MPH exposure in the CNS. MPH is a Schedule II CNS stimulant that exerts its Glycitin pharmacological effects via preferential blockade of the dopamine transporter and norepinephrine transporter, similar to that of cocaine. This blockade results in a reduction of dopamine/norepinephrine uptake, leading to an increase in postsynaptic dopamine/norepinephrine levels. Thus, MPH usage leads to an acute increase in striatal dopamine levels. In terms of neurological effects, dopamine has been shown to have a major modulatory effect in the developing brain on both neostriatal and cortical neurogenesis. Additionally, excess dopamine has been shown to be toxic both in vitro and in vivo due to the production of superoxide, hydrogen peroxide, and the dopamine quinone. In fact, both acute and chronic treatment with MPH has been shown to result in superoxide production in the brain. Free dopamine has also been shown to induce an inflammatory response in the brain characterized by an increase in cytokines and chemokines that lead to an induction of microgliosis. Specifically, we examined if acute or chronic administration of MPH altered SNpc dopamine neuron number and catecholamine levels in the striatum. Since excessive dopamine can induce oxidative stress and inflammation, we examined if MPH rendered the basal ganglia more sensitive to MPTP, an agent that has previously been shown to induce neuron damage in the SNpc. The present study investigated the pathological effects of acute and chronic MPH in the basal ganglia using two different doses that Dimethyl-lithospermate-B the therapeutic window of MPH use for ADHD and narcolepsy in humans. We demonstrate that chronic administration of 10 mg/kg MPH induces a small but significant loss of SNpc dopaminergic neurons. We also find that chronic exposure to both 1 mg/kg and 10 mg/kg MPH can sensitize SNpc dopamine neurons to a further oxidative stress. Though the complete mechanism for this sensitization of dopamine neurons is not well understood, our experiments suggest a combined effect of an increased inflammatory response with reduced levels of several trophic factors, including BDNF and GDNF. Despite the extensive use of MPH in school aged and adult populations with ADHD as well its use in general cognitive enhancement in non-ADHD individuals, only a few studies have investigated the neuropathological consequences of long-term MPH exposure. In this study, we used a 12-week MPH administration schedule that spans the developmental period in rodents and corresponds to the pre-adolescent through young adult period in humans, during which MPH is typically used. MPH’s mechanism of action is to increase the availability of extracellular DA and NE in the synaptic cleft through blockade of the dopamine transporter and norepinephrine transporter. In this study, we observed a significant increase in total dopamine levels in the striatum at 1 mg/kg MPH, a change that was not observed at 10 mg/kg MPH. Previous studies have also reported a similar increase in striatal dopamine levels at similar lower doses of MPH.

Although a previous study reported that systemic administration of TUDCA in a model of retinitis pigmentosa had negative impact on the weight gain of developing mice, there was no effect on the weight of adult rats in our study. This could be because our animals were already adults when treatment was started. In fact, the similar non-taurine conjugated UDCA does not show any significant adverse effects given to humans for extended period of time- up to 2 years- to treat liver diseases, and has been approved by the FDA. These findings suggest that TUDCA is an efficient and safe neuroprotective agent and may have a therapeutic potential for patients suffering from diseases where retinal degeneration is involved. Photoreceptor loss and subsequent visual decline occurs when the photoreceptors are separated from the underlying retinal pigment epithelium. Physical separation of photoreceptors is seen in various retinal disorders, including retinal detachement as well as age-related macular degeneration and diabetic retinopathy. The visual acuity of patients with rhegmatogenous retinal detachment is not always restored,Imperialine-D-glucoside even after successful operation for reattachment. Two fifths of patients with rhegmatogenous retinal detachment involving the macula, a region essential for central vision recover 20/40 or better vision because of photoreceptor death. Thus, identification of neuroprotective agents preventing photoreceptor loss may open a novel approach for treatment of these diseases. The findings of this study suggest that systemic administration of TUDCA may be such an approach for preventing vision deficit in various retinal disorders associated with photoreceptor loss. The thicknesses were measured based on the outer boundaries of the DAPI nuclear stain. Because the thickness of the retina seen in cross-section varies depending on the angle of the sectioning plane with respect to the retina, the outer nuclear layer thickness was normalized to the thickness of the entire retina. Skeletal muscle is responsible for about 65% of glucose disposal following Sipeimine a meal and reduced insulin induced glucose disposal results in impaired glucose tolerance. In vivo, insulin plays an important role in the regulation of skeletal muscle glucose uptake and regulation of skeletal muscle protein, amino acid and fatty acid metabolism. Acutely, insulin stimulates glucose uptake in skeletal muscle cells by accelerating the recruitment of GLUT4 to the sarcolemma, a process that is exquisitely regulated by a specific insulin-responsive protein signaling cascade. Similarly, insulin acutely stimulates protein synthesis by also activating a specific insulin-responsive protein signaling cascade. Both of these responses are regulated by reversible post-translational modifications of key signaling protein molecules. However, less information is available about insulin impact on gene transcription that also may affect insulin action: it is currently unknown whether insulin acutely enhances transcription of genes or whether there is a time related pattern in transcribing the genes thereby having a different level of regulation of insulin action on gene expression. A better understanding of the impact of insulin on transcript levels of various genes is critical to acquire a more thorough understanding on how insulin exerts its pleiotropic effects on skeletal muscle glucose uptake as well as protein synthesis. The microarray technology has been extensively used to identify differentially expressed genes in skeletal muscle cells under different physiological states, e.g., non-diabetic vs. diabetic subjects during poor glycemic control and following insulin treatment, insulin treated versus insulin deprived type 1 diabetic patients, and, normal vs. impaired glucose tolerant individuals, and basal state vs. euglycemic hyperinsulinemic clamp.