DEVELOPMENT AND RESULTS OF MEDICATIONS OF THE BURNS

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RATIONAL BASE OF A COMBINATION GENTAMICIN 0.1 % - UREA 0.2 % IN OINTMENT FORM DURING THE TREATMENT OF PATIENTS WITH SKIN BURNS

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Time Course of Neurobehavioral Disruptions and Regional Brain Metabolism Changes in the Rotenone Mice Model of Parkinson’s Disease

Parkinson’s disease (PD) is characterized by slow progression with a long prodromal stage and the gradual evolution of both neuropsychological symptoms and subtle motor changes, preceding motor dysfunction. Thus, in order for animal models of PD to be valid, they should reproduce these characteristics of the disease. One of such models, in which neuropathology is induced by chronic injections of low doses of mitochondrial toxin rotenone, is well established in rats. However, data on this model adapted to mice remain controversial. We have designed the study to describe the timecourse of motor and non-motor symptoms during chronic subcutaneous administration of rotenone (4 mg/kg daily for 35 days) in C57BL/6 mice. We characterize the underlying neuropathological processes (dopaminergic neuron degeneration, regional brain metabolism, monoamine neurotransmitter and lipid peroxidation changes) at different timepoints: 1 day, 2 weeks and 5 weeks of daily rotenone exposure. Based on the behavioral data, we can describe three stages of pathology: cognitive changes from week 2 of rotenone exposure, subtle motor changes in week 3–4 and motor dysfunction starting roughly from week 4. Neuropathological changes in this model include a general decrease in COX activity in different areas of the brain (acute effect of rotenone) and a more specific decrease in midbrain (chronic effect), followed by significant neurodegeneration in SNpc but not VTA by the 5th week of rotenone exposure. However, we were unable to find changes in the level of monoamine neurotransmitters neither in the striatum nor in the cortex, nor in the level of lipid peroxidation in the brainstem. Thus, the gradual progression of pathology in this model is linked with metabolic changes, rather than with oxidative stress or tonic neurotransmitter release levels. Overall, this study supports the idea that a low-dose rotenone mouse model can also reproduce different stages of PD as well as rats

Striatal Neurons Partially Expressing a Dopaminergic Phenotype: Functional Significance and Regulation

Since the discovery of striatal neurons expressing dopamine-synthesizing enzymes, researchers have attempted to identify their phenotype and functional significance. In this study, it was shown that in transgenic mice expressing green fluorescent protein (GFP) under the tyrosine hydroxylase (TH) gene promoter, (i) there are striatal neurons expressing only TH, only aromatic L-amino acid decarboxylase (AADC), or both enzymes of dopamine synthesis; (ii) striatal neurons expressing dopamine-synthesizing enzymes are not dopaminergic since they lack a dopamine transporter; (iii) monoenzymatic neurons expressing individual complementary dopamine-synthesizing enzymes produce this neurotransmitter in cooperation; (iv) striatal nerve fibers containing only TH, only AADC, or both enzymes project into the lateral ventricles, providing delivery pathways for L-3,4-dihydroxyphenylalanine and dopamine to the cerebrospinal fluid; and (v) striatal GFP neurons express receptor genes for various signaling molecules, i.e., classical neurotransmitters, neuropeptides, and steroids, indicating fine regulation of these neurons. Based on our data, it is assumed that the synthesis of dopamine by striatal neurons is a compensatory response to the death of nigral dopaminergic neurons in Parkinson’s disease, which opens broad prospects for the development of a fundamentally novel antiparkinsonian therapy

The Gene Expression of Proteins Involved in Intercellular Signaling and Neurodegeneration in the Substantia Nigra in a Mouse Subchronic Model of Parkinson’s Disease

Given the limited access to clinical material for studying the pathogenesis of Parkinson’s disease (PD), these studies should be carried out on experimental models. We have recently developed a subchronic model of the progressive development of PD with a gradual transition from the preclinical (asymptomatic) stage to the clinical (symptomatic) one. The aim of this study was to evaluate changes in the expression of a wide range of genes in the substantia nigra (SN), the central link in the regulation of motor function, in mice in our subchronic model of PD. We have found changes in the expression of a number of genes encoding enzymes involved in the synthesis and degradation of dopamine as well as proteins involved in the vesicular cycle, axonal transport, protein degradation in the proteasome system, neuroinflammation, and cell death in the SN of our mouse model of the clinical stage of PD. Similar changes in gene expression were previously demonstrated in patients (postmortem), indicating good reproducibility of PD in our model. Further analysis of the gene expression in the SN of mice has shown that the expression of some genes also changes in the model of the preclinical stage, when dopaminergic neurons have not yet died. Thus, this study opens up broad prospects for further evaluation of the molecular mechanisms of PD pathogenesis and the development of a test system for drug screening

Image_6_Isolation of living dopaminergic neurons labeled with a fluorescent ligand of the dopamine transporter from mouse substantia nigra as a new tool for basic and applied research.JPEG

Dopaminergic neurons (DNs) of the nigrostriatal system control the motor function, and their degeneration leads to the development of Parkinson’s disease (PD). A stumbling block in the study of DNs in the whole substantia nigra (SN) is the lack of tools to analyze the expression of most of the genes involved in neurotransmission, neurodegeneration, and neuroplasticity, since they are also expressed in other cells of the SN. Therefore, this study aimed to develop a fluorescence-activated cell sorting method for isolating living DNs from the SN of wild-type mice using two fluorescent dyes, DRAQ5 (nuclear stain) and a dopamine uptake inhibitor GBR 12909 coupled to a fluorophore (DN stain). We have developed a method for selecting a population of DNs from the SN of mice, as evidenced by: (i) immunopositivity of 95% of the sorted cells for tyrosine hydroxylase, the first enzyme of dopamine synthesis; (ii) the sorted cells expressing the genes for specific proteins of the dopaminergic phenotype, tyrosine hydroxylase, the dopamine transporter, and vesicular monoamine transporter 2 and non-specific proteins, such as aromatic L-amino acid decarboxylase, non-specific enzyme of dopamine synthesis. We then compared the changes in gene expression found in the sorted DNs and in the SN homogenate in a PD model we developed, reproduced in mice by treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Using quantitative PCR, we obtained evidence of the same changes in the expression of specific genes in the sorted DNs of SN and in the SN homogenate of a MPTP mouse model of PD, compared with the control. The undoubted advantage of our approach is the possibility of obtaining a large amount of readily available and relatively cheap primary material (SN) from wild-type mice, which can be used to solve both research and applied problems. In addition, this method can be easily adapted to the isolation of DNs from the SN in other animal species, including non-human primates.</p

Image_2_Isolation of living dopaminergic neurons labeled with a fluorescent ligand of the dopamine transporter from mouse substantia nigra as a new tool for basic and applied research.JPEG

Dopaminergic neurons (DNs) of the nigrostriatal system control the motor function, and their degeneration leads to the development of Parkinson’s disease (PD). A stumbling block in the study of DNs in the whole substantia nigra (SN) is the lack of tools to analyze the expression of most of the genes involved in neurotransmission, neurodegeneration, and neuroplasticity, since they are also expressed in other cells of the SN. Therefore, this study aimed to develop a fluorescence-activated cell sorting method for isolating living DNs from the SN of wild-type mice using two fluorescent dyes, DRAQ5 (nuclear stain) and a dopamine uptake inhibitor GBR 12909 coupled to a fluorophore (DN stain). We have developed a method for selecting a population of DNs from the SN of mice, as evidenced by: (i) immunopositivity of 95% of the sorted cells for tyrosine hydroxylase, the first enzyme of dopamine synthesis; (ii) the sorted cells expressing the genes for specific proteins of the dopaminergic phenotype, tyrosine hydroxylase, the dopamine transporter, and vesicular monoamine transporter 2 and non-specific proteins, such as aromatic L-amino acid decarboxylase, non-specific enzyme of dopamine synthesis. We then compared the changes in gene expression found in the sorted DNs and in the SN homogenate in a PD model we developed, reproduced in mice by treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Using quantitative PCR, we obtained evidence of the same changes in the expression of specific genes in the sorted DNs of SN and in the SN homogenate of a MPTP mouse model of PD, compared with the control. The undoubted advantage of our approach is the possibility of obtaining a large amount of readily available and relatively cheap primary material (SN) from wild-type mice, which can be used to solve both research and applied problems. In addition, this method can be easily adapted to the isolation of DNs from the SN in other animal species, including non-human primates.</p

Image_3_Isolation of living dopaminergic neurons labeled with a fluorescent ligand of the dopamine transporter from mouse substantia nigra as a new tool for basic and applied research.JPEG

Dopaminergic neurons (DNs) of the nigrostriatal system control the motor function, and their degeneration leads to the development of Parkinson’s disease (PD). A stumbling block in the study of DNs in the whole substantia nigra (SN) is the lack of tools to analyze the expression of most of the genes involved in neurotransmission, neurodegeneration, and neuroplasticity, since they are also expressed in other cells of the SN. Therefore, this study aimed to develop a fluorescence-activated cell sorting method for isolating living DNs from the SN of wild-type mice using two fluorescent dyes, DRAQ5 (nuclear stain) and a dopamine uptake inhibitor GBR 12909 coupled to a fluorophore (DN stain). We have developed a method for selecting a population of DNs from the SN of mice, as evidenced by: (i) immunopositivity of 95% of the sorted cells for tyrosine hydroxylase, the first enzyme of dopamine synthesis; (ii) the sorted cells expressing the genes for specific proteins of the dopaminergic phenotype, tyrosine hydroxylase, the dopamine transporter, and vesicular monoamine transporter 2 and non-specific proteins, such as aromatic L-amino acid decarboxylase, non-specific enzyme of dopamine synthesis. We then compared the changes in gene expression found in the sorted DNs and in the SN homogenate in a PD model we developed, reproduced in mice by treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Using quantitative PCR, we obtained evidence of the same changes in the expression of specific genes in the sorted DNs of SN and in the SN homogenate of a MPTP mouse model of PD, compared with the control. The undoubted advantage of our approach is the possibility of obtaining a large amount of readily available and relatively cheap primary material (SN) from wild-type mice, which can be used to solve both research and applied problems. In addition, this method can be easily adapted to the isolation of DNs from the SN in other animal species, including non-human primates.</p

Time Course of Neurobehavioral Disruptions and Regional Brain Metabolism Changes in the Rotenone Mice Model of Parkinson’s Disease

Parkinson’s disease (PD) is characterized by slow progression with a long prodromal stage and the gradual evolution of both neuropsychological symptoms and subtle motor changes, preceding motor dysfunction. Thus, in order for animal models of PD to be valid, they should reproduce these characteristics of the disease. One of such models, in which neuropathology is induced by chronic injections of low doses of mitochondrial toxin rotenone, is well established in rats. However, data on this model adapted to mice remain controversial. We have designed the study to describe the timecourse of motor and non-motor symptoms during chronic subcutaneous administration of rotenone (4 mg/kg daily for 35 days) in C57BL/6 mice. We characterize the underlying neuropathological processes (dopaminergic neuron degeneration, regional brain metabolism, monoamine neurotransmitter and lipid peroxidation changes) at different timepoints: 1 day, 2 weeks and 5 weeks of daily rotenone exposure. Based on the behavioral data, we can describe three stages of pathology: cognitive changes from week 2 of rotenone exposure, subtle motor changes in week 3–4 and motor dysfunction starting roughly from week 4. Neuropathological changes in this model include a general decrease in COX activity in different areas of the brain (acute effect of rotenone) and a more specific decrease in midbrain (chronic effect), followed by significant neurodegeneration in SNpc but not VTA by the 5th week of rotenone exposure. However, we were unable to find changes in the level of monoamine neurotransmitters neither in the striatum nor in the cortex, nor in the level of lipid peroxidation in the brainstem. Thus, the gradual progression of pathology in this model is linked with metabolic changes, rather than with oxidative stress or tonic neurotransmitter release levels. Overall, this study supports the idea that a low-dose rotenone mouse model can also reproduce different stages of PD as well as rats