RESUMO
Extended general anesthesia early in life is neurotoxic in multiple species. However, little is known about the temporal progression of neurodegeneration after general anesthesia. It is also unknown if a reduction in natural cell death, or an increase in cell creation, occurs as a form of compensation after perinatal anesthesia exposure. The goal of this study was to evaluate markers of neurodegeneration and cellular division at 2, 24, or 72 h after sevoflurane (Sevo) exposure (6 h) in fully oxygenated postnatal day (PND) 7 rats. Neurodegeneration was observed in areas throughout the forebrain, while the largest changes (fold increase above vehicle) were observed in areas associated with either the primary olfactory learning pathways or the basal ganglia. These regions included the indusium griseum (IG, 25-fold), the posterior dorso medial hippocampal CA1 (17-fold), bed nucleus of the stria terminalis (Bed Nuclei STM, 5-fold), the shell of the nucleus accumbens (Acb, 5-fold), caudate/putamen (CPu, 5-fold), globus pallidus (GP, 9-fold) and associated thalamic (11-fold) and cortical regions (5-fold). Sevo neurodegeneration was minimal or undetectable in the ventral tegmentum, substantia nigra, and most of the hypothalamus and frontal cortex. In most brain regions where neurodegeneration was increased 2 h post Sevo exposure, the levels returned to <4-fold above control levels by 24 h. However, in the IG, CA1, GP, anterior thalamus, medial preoptic nucleus of the hypothalamus (MPO), anterior hypothalamic area (AHP), and the amygdaloid nuclei, neurodegeneration at 24 h was double or more than that at 2 h post exposure. Anesthesia exposure causes either a prolonged period of neurodegeneration in certain brain regions, or a distinct secondary degenerative event occurs after the initial insult. Moreover, regions most sensitive to Sevo neurodegeneration did not necessarily coincide with areas of new cell birth, and new cell birth was not consistently affected by Sevo. The profile of anesthesia related neurotoxicity changes with time, and multiple mechanisms of toxicity may exist in a time-dependent fashion.
Assuntos
Tonsila do Cerebelo/metabolismo , Gânglios da Base/metabolismo , Hipocampo/metabolismo , Sevoflurano/farmacologia , Animais , Substância Cinzenta/metabolismo , Ratos Sprague-Dawley , Tálamo/metabolismoRESUMO
Bacterial cell wall endotoxins, i.e. lipopolysaccharides (LPS), are some of the original compounds shown to evoke the classic signs of systemic inflammation/innate immune response and neuroinflammation. The term neuroinflammation often is used to infer the elaboration of proinflammatory mediators by microglia elicited by neuronal targeted activity. However, it also is possible that the microglia are responding to vasculature through several signaling mechanisms. Microglial activation relative to the vasculature in the hippocampus and parietal cortex was determined after an acute exposure of a single subcutaneous injection of 2 mg/kg LPS. Antibodies to allograft inflammatory factor (Aif1, a.k.a. Iba1) were used to track and quantify morphological changes in microglia. Immunostaining of platelet/endothelial cell adhesion molecule 1 (Pecam1, a.k.a. Cd31) was used to visualize vasculature in the forebrain and glial acidic fibrillary protein (GFAP) to visualize astrocytes. Neuroinflammation and other aspects of neurotoxicity were evaluated histologically at 3 h, 6 h, 12 h, 24 h, 3 d and 14 d following LPS exposure. LPS did not cause neurodegeneration as determined by Fluoro Jade C labeling. Also, there were no signs of mouse IgG leakage from brain vasculature due to LPS. Some changes in microglia size occurred at 6 h, but by 12 h microglial activation had begun with the combined soma and proximal processes size increasing significantly (1.5-fold). At 24 h, almost all the microglia soma and proximal processes in the hippocampus, parietal cortex, and thalamus were closely associated with the vasculature and had increased almost 2.0-fold in size. In many areas where microglia were juxtaposed to vasculature, astrocytic endfeet appeared to be displaced. The microglial activation had subsided slightly by 3 d with microglial size 1.6-fold that of control. We hypothesize that acute LPS activation can result in vascular mediated microglial responses through several mechanisms: 1) binding to Cd14 and Tlr4 receptors on microglia processes residing on vasculature; 2) damaging vasculature and causing the release of cytokines; and 3) possibly astrocytic endfeet damage resulting in cytokine release. These acute responses may serve as an adaptive mechanism to exposure to circulating LPS where the microglia surround the vasculature. This could further prevent the pathogen(s) circulating in blood from entering the brain. However, diverting microglial interactions away from synaptic remodeling and other types of microglial interactions with neurons may have adverse effects on neuronal function.
Assuntos
Encefalite/imunologia , Hipocampo/irrigação sanguínea , Hipocampo/imunologia , Lipopolissacarídeos/toxicidade , Microglia/imunologia , Córtex Pré-Frontal/irrigação sanguínea , Córtex Pré-Frontal/imunologia , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/imunologia , Encefalite/induzido quimicamente , Feminino , Hipocampo/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Microglia/efeitos dos fármacos , Córtex Pré-Frontal/efeitos dos fármacosRESUMO
This work extends the understanding of how toxic exposures to amphetamine (AMPH) adversely affect the immune system and lead to tissue damage. Importantly, it determines which effects of AMPH are and are not due to pronounced hyperthermia. Whole blood messenger RNA (mRNA) and whole blood and serum microRNA (miRNA) transcripts were identified in adult male Sprague-Dawley rats after exposure to toxic AMPH under normothermic conditions, AMPH when it produces pronounced hyperthermia, or environmentally-induced hyperthermia (EIH). mRNA transcripts with large increases in fold-change in treated relative to control rats and very low expression in the control group were a rich source of organ-specific transcripts in blood. When severe hyperthermia was produced by either EIH or AMPH, significant increases in circulating organ-specific transcripts for liver (Alb, Fbg, F2), pancreas (Spink1), bronchi/lungs (F3, Cyp4b1), bone marrow (Np4, RatNP-3b), and kidney (Cesl1, Slc22a8) were observed. Liver damage was suggested also by increased miR-122 levels in the serum. Increases in muscle/heart-enriched transcripts were produced by AMPH even in the absence of hyperthermia. Expression increases in immune-related transcripts, particularly Cd14 and Vcan, indicate that AMPH can activate the innate immune system in the absence of hyperthermia. Most transcripts specific for T-cells decreased 50-70% after AMPH exposure or EIH, with the noted exception of Ccr5 and Chst12. This is probably due to T-cells leaving the circulation and down-regulation of these genes. Transcript changes specific for B-cells or B-lymphoblasts in the AMPH and EIH groups ranged widely from decreasing ≈ 40% (Cd19, Cd180) to increasing 30 to 100% (Tk1, Ahsa1) to increasing ≥500% (Stip1, Ackr3). The marked increases in Ccr2, Ccr5, Pld1, and Ackr3 produced by either AMPH or EIH observed in vivo provide further insight into the initial immune system alterations that result from methamphetamine and AMPH abuse and could modify risk for HIV and other viral infections.
Assuntos
Transtornos Relacionados ao Uso de Anfetaminas/sangue , Anfetamina/administração & dosagem , Febre/sangue , Golpe de Calor/sangue , MicroRNAs/sangue , RNA Mensageiro/sangue , Anfetamina/farmacologia , Animais , Biomarcadores/sangue , Febre/induzido quimicamente , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
The initial goals of these experiments were to determine: 1) if blood-brain barrier (BBB) breakdown was a cause or an effect of METH-induced seizures; 2) all the brain regions where BBB is disrupted as seizures progress; and 3) the correlations between body temperature and vascular leakage and neurodegeneration. A fourth objective was added after initial experimentation to determine if sub-strain differences existed in adult male C57 B6 J (Jackson laboratories, JAX) versus C57 B6N (Charles River, CR) mice involving their susceptibility to BBB breakdown and seizure severity. With the 1st "maximal" intensity myoclonic-tonic seizure (MCT) varying degrees of IgG infiltration across the BBB (≤1 mm2) were prominent in olfactory system (OS) associated regions and in thalamus, hypothalamus and neocortex. IgG infiltration areas in the OS-associated regions of the bed nucleus of the stria terminalis, septum and more medial amygdala nuclei, and the hypothalamus were increased significantly by the time continuous behavioral seizures (CBS) developed. Mice receiving METH that had body temperatures of ≥40 °C had IgG infiltration along with MCT or CBS but peak body temperatures above 40 °C did not significantly increase IgG infiltration. Neurodegeneration seen at ≥6 h was restricted to the OS in both JAX and CR mice and was most prominent in the posteromedial cortical amygdaloid nucleus. Neurodegeneration in the anterior septum (tenia tecta) was seen only in the JAX mice. We hypothesize that METH-induced hypertension and hyperthermia lead to BBB breakdown and other vascular dysfunctions in the OS brain regions resulting in OS hyperexcitation. Excitation of the OS neural network then leads to the development of seizures. These seizures in turn exacerbate the energy depletions and the reactive oxygen stress produced by hyperthermia further damaging the BBB and vascular function. These events form a recurrent cycle that results in ever increasing seizure activity and neurotoxicity.
Assuntos
Barreira Hematoencefálica/metabolismo , Permeabilidade Capilar/fisiologia , Estimulantes do Sistema Nervoso Central/toxicidade , Progressão da Doença , Metanfetamina/toxicidade , Convulsões/sangue , Convulsões/induzido quimicamente , Animais , Permeabilidade Capilar/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Convulsões/diagnóstico , Fatores de TempoRESUMO
Thiamine/vitamin B1 deficiency can lead to behavioral changes and neurotoxicity in humans. This may due in part to vascular damage, neuroinflammation and neuronal degeneration in the diencephalon, which is seen in animal models of pyrithiamine-enhanced thiamine deficiency. However, the time course of the progression of these changes in the animal models has been poorly characterized. Therefore, in this study, the progression of: 1) activated microglial association with vasculature; 2) neurodegeneration; and 3) any vascular leakage in the forebrain during the progress of thiamine deficiency were determined. A thiamine deficient diet along with 0.25â¯mg/kg/d of pyrithiamine was used as the mouse model. Vasculature was identified with Cd31 and microglia with Cd11b and Iba1 immunoreactivity. Neurodegeneration was determined by FJc labeling. The first sign of activated microglia within the thalamic nuclei were detected after 8 d of thiamine deficiency, and by 9 d activated microglia associated primarily with vasculature were clearly present but only in thalamus. At the 8 d time point neurodegeneration was not present in thalamus. However at 9 d, the first signs of neurodegeneration (FJcâ¯+â¯neurons) were seen in most animals. Over 80% of the microglia were activated at 10 d but almost exclusively in the thalamus and the number of degenerating neurons was less than 10% of the activated microglia. At 10 d, there were sporadic minor changes in IgG presence in thalamus indicating minor vascular leakage. Dizocilpine (0.2-0.4â¯mg/kg) or phenobarbital (10-20â¯mg/kg) was administered to groups of mice from day 8 through day 10 to block neurodegeneration but neither did. In summary, activated microglia start to surround vasculature 1-2 d prior to the start of neurodegeneration. This response may be a means of controlling or repairing vascular damage and leakage. Glutamate excitotoxicity and vascular leakage likely only play a minor role in the early neurodegeneration resulting from thiamine deficiency. However, failure of dysfunctional vasculature endothelium to supply sufficient nutrients to neurons could be contributing to the neurodegeneration.
Assuntos
Vasos Sanguíneos/patologia , Microglia/metabolismo , Degeneração Neural/patologia , Tálamo/metabolismo , Tálamo/patologia , Deficiência de Tiamina/metabolismo , Deficiência de Tiamina/patologia , Animais , Antígeno CD11b/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Dieta , Maleato de Dizocilpina/farmacologia , Feminino , Camundongos , Proteínas dos Microfilamentos/metabolismo , Degeneração Neural/prevenção & controle , Fenobarbital/farmacologia , Piritiamina , Deficiência de Tiamina/induzido quimicamente , Fatores de TempoRESUMO
Our previous studies have raised the possibility that altered blood glucose levels may influence and/or be predictive of methamphetamine (METH) neurotoxicity. This study evaluated the effects of exogenous glucose and corticosterone (CORT) pretreatment alone or in combination with METH on blood glucose levels and the neural and vascular toxicity produced. METH exposure consisted of four sequential injections of 5, 7.5, 10, and 10 mg/kg (2 h between injections) D-METH. The three groups given METH in combination with saline, glucose (METH+Glucose), or CORT (METH+CORT) had significantly higher glucose levels compared to the corresponding treatment groups without METH except at 3 h after the last injection. At this last time point, the METH and METH+Glucose groups had lower levels than the non-METH groups, while the METH+CORT group did not. CORT alone or glucose alone did not significantly increase blood glucose. Mortality rates for the METH+CORT (40%) and METH+Glucose (44%) groups were substantially higher than the METH (< 10%) group. Additionally, METH+CORT significantly increased neurodegeneration above the other three METH treatment groups (≈ 2.5-fold in the parietal cortex). Thus, maintaining elevated levels of glucose during METH exposure increases lethality and may exacerbate neurodegeneration. Neuroinflammation, specifically microglial activation, was associated with degenerating neurons in the parietal cortex and thalamus after METH exposure. The activated microglia in the parietal cortex were surrounding vasculature in most cases and the extent of microglial activation was exacerbated by CORT pretreatment. Our findings show that acute CORT exposure and elevated blood glucose levels can exacerbate METH-induced vascular damage, neuroinflammation, neurodegeneration and lethality. Cover Image for this issue: doi. 10.1111/jnc.13819.
Assuntos
Glicemia/efeitos dos fármacos , Corticosterona/toxicidade , Glucose/toxicidade , Metanfetamina/toxicidade , Lobo Parietal/efeitos dos fármacos , Tálamo/efeitos dos fármacos , Animais , Glicemia/metabolismo , Corticosterona/administração & dosagem , Combinação de Medicamentos , Glucose/administração & dosagem , Masculino , Metanfetamina/administração & dosagem , Microglia/efeitos dos fármacos , Microglia/metabolismo , Lobo Parietal/irrigação sanguínea , Lobo Parietal/metabolismo , Ratos , Ratos Sprague-Dawley , Tálamo/irrigação sanguínea , Tálamo/metabolismoRESUMO
BACKGROUND: A computational evolution system (CES) is a knowledge discovery engine that can identify subtle, synergistic relationships in large datasets. Pareto optimization allows CESs to balance accuracy with model complexity when evolving classifiers. Using Pareto optimization, a CES is able to identify a very small number of features while maintaining high classification accuracy. A CES can be designed for various types of data, and the user can exploit expert knowledge about the classification problem in order to improve discrimination between classes. These characteristics give CES an advantage over other classification and feature selection algorithms, particularly when the goal is to identify a small number of highly relevant, non-redundant biomarkers. Previously, CESs have been developed only for binary class datasets. In this study, we developed a multi-class CES. RESULTS: The multi-class CES was compared to three common feature selection and classification algorithms: support vector machine (SVM), random k-nearest neighbor (RKNN), and random forest (RF). The algorithms were evaluated on three distinct multi-class RNA sequencing datasets. The comparison criteria were run-time, classification accuracy, number of selected features, and stability of selected feature set (as measured by the Tanimoto distance). The performance of each algorithm was data-dependent. CES performed best on the dataset with the smallest sample size, indicating that CES has a unique advantage since the accuracy of most classification methods suffer when sample size is small. CONCLUSION: The multi-class extension of CES increases the appeal of its application to complex, multi-class datasets in order to identify important biomarkers and features.
RESUMO
BACKGROUND: Atrial Fibrillation (AF) is a common condition associated with impaired quality of life (QOL) and recurrent hospitalisation. Catheter ablation for AF is a well-established treatment for symptomatic patients despite medical therapy. We sought to examine the effect of point specific nurse-led education on QOL, AF symptomatology and readmission rate post AF ablation. METHODS: Forty-one patients undergoing AF ablation were randomised to Nurse Intervention (NI) versus Control (C), n=22 vs. 19. Both groups were well matched with respect to age, sex and AF subtype. All patients completed SF36 and AF Symptom Checklist, Frequency and Severity Scale questionnaires at baseline and six months post ablation. The NI group underwent nurse education on admission, prior to discharge, and with telephone contact. RESULTS: Baseline SF-36 and AF Symptom Checklist, Frequency and Severity scores were similar. The NI group showed significant differences compared to Control with respect to higher QOL on the SF-36 score of Physical Functioning and Vitality at six months. There were significant improvements in seven components of the AF Symptom Checklist, Frequency and Severity at six months in the NI group with a trend in a further seven. There was no difference in AF related hospital readmissions at six months between C and NI groups (10.5% vs. 13.6%, p=ns). CONCLUSION: Nurse-led education at time of AF ablation is associated with improved QOL and reduced symptom frequency and severity compared to usual care.
Assuntos
Fibrilação Atrial/cirurgia , Ablação por Cateter , Educação em Enfermagem , Enfermagem/métodos , Readmissão do Paciente , Qualidade de Vida , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de TempoRESUMO
Prolonged vitamin B1 (thiamine) deficiency can lead to neurological disorders such as Wernicke's encephalopathy and Wernicke-Korsakoff Syndrome (WKS) in humans. These thiamine deficiency disorders have been attributed to vascular leakage, blood-brain barrier breakdown and neuronal loss in the diencephalon and brain stem. However, endothelial dysfunction following thiamine deficiency and its relationship to the phenomenon of neurodegeneration has not been clearly elucidated. The present study sought to begin to address this issue by evaluating vascular morphology and integrity in a pyrithiamine (PT)-induced rat model of thiamine deficiency. Adjacent brain sections were used to either assess vascular integrity through immunohistochemical localization of rat endothelial cell antigen (RECA-1) and endothelial brain barrier antigen (EBA-1) or neurodegeneration using the de Olmos cupric silver method. GFAP and CD11b immunolabeling was used to evaluate astrocytic and microglial/macrophagic changes. Extensive neurodegeneration occurred concomitant with both vascular damage (thinning and breakage) and microglial activation in the inferior olive, medial thalamic area, and medial geniculate nuclei of pyrithiamine treated rats. Likewise, glucose transporter-1 (Glut-1), which is mostly expressed in endothelial cells, was also severely decreased in this pyrithiamine induced thiamine deficient rat model. MRI scans of these animals prior to sacrifice show that the pyrithiamine induced thiamine deficient animals have abnormal T2 relaxation values, which are commensurate with, and possibly predictive of, the neurodegeneration and/or endothelial dysfunction subsequently observed histologically in these same animals.
Assuntos
Antimetabólitos/toxicidade , Encéfalo/patologia , Células Endoteliais/efeitos dos fármacos , Piritiamina/toxicidade , Deficiência de Tiamina/induzido quimicamente , Deficiência de Tiamina/patologia , Animais , Antígenos de Superfície/metabolismo , Astrócitos/patologia , Astrócitos/ultraestrutura , Encéfalo/diagnóstico por imagem , Antígeno CD11b/metabolismo , Modelos Animais de Doenças , Células Endoteliais/ultraestrutura , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Transportador de Glucose Tipo 1/metabolismo , Imageamento por Ressonância Magnética , Masculino , Doenças Neurodegenerativas/diagnóstico por imagem , Doenças Neurodegenerativas/etiologia , Ratos , Ratos Sprague-Dawley , Coloração pela Prata , Deficiência de Tiamina/complicações , Deficiência de Tiamina/diagnóstico por imagemRESUMO
BACKGROUND: Brain microglial activations and damage responses are most commonly associated with neurodegeneration or systemic innate immune system activation. Here, we used histological methods to focus on microglial responses that are directed towards brain vasculature, previously undescribed, after a neurotoxic exposure to methamphetamine. METHODS: Male rats were given doses of methamphetamine that produce pronounced hyperthermia, hypertension, and toxicity. Identification of microglia and microglia-like cells (pericytes and possibly perivascular cells) was done using immunoreactivity to allograft inflammatory factor 1 (Aif1 a.k.a Iba1) and alpha M integrin (Itgam a.k.a. Cd11b) while vasculature endothelium was identified using rat endothelial cell antigen 1 (RECA-1). Regions of neuronal, axonal, and nerve terminal degeneration were determined using Fluoro-Jade C. RESULTS: Dual labeling of vasculature (RECA-1) and microglia (Iba1) showed a strong association of hypertrophied cells surrounding and juxtaposed to vasculature in the septum, medial dorsal hippocampus, piriform cortex, and thalamus. The Iba1 labeling was more pronounced in the cell body while Cd11b more so in the processes of activated microglia. These regions have been previously identified to have vascular leakage after neurotoxic methamphetamine exposure. Dual labeling with Fluoro-Jade C and Iba1 indicated that there was minimal or no evidence of neuronal damage in the septum and hippocampus where many hypertrophied Iba1-labeled cells were found to be associated with vasculature. Although microglial activation around the prominent neurodegeneration was found in the thalamus, there were also many examples of activated microglia associated with vasculature. CONCLUSIONS: The data implicate microglia, and possibly related cell types, in playing a major role in responding to methamphetamine-induced vascular damage, and possibly repair, in the absence of neurodegeneration. Identifying brain regions with hypertrophied/activated microglial-like cells associated with vasculature has the potential for identifying regions of more subtle examples of vascular damage and BBB compromise.
Assuntos
Vasos Sanguíneos/patologia , Estimulantes do Sistema Nervoso Central/toxicidade , Metanfetamina/toxicidade , Microglia/efeitos dos fármacos , Síndromes Neurotóxicas/patologia , Animais , Antígenos de Superfície/metabolismo , Antígeno CD11b/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Degeneração Neural/induzido quimicamente , Degeneração Neural/patologia , Ratos , Ratos Sprague-DawleyRESUMO
Methods were developed to evaluate the stability of rat whole blood expression obtained from RNA sequencing (RNA-seq) and assess changes in whole blood transcriptome profiles in experiments replicated over time. Expression was measured in globin-depleted RNA extracted from the whole blood of Sprague-Dawley rats, given either saline (control) or neurotoxic doses of amphetamine (AMPH). The experiment was repeated four times (paired control and AMPH groups) over a 2-year span. The transcriptome of the control and AMPH-treated groups was evaluated on: 1) transcript levels for ribosomal protein subunits; 2) relative expression of immune-related genes; 3) stability of the control transcriptome over 2 years; and 4) stability of the effects of AMPH on immune-related genes over 2 years. All, except one, of the 70 genes that encode the 80s ribosome had levels that ranked in the top 5% of all mean expression levels. Deviations in sequencing performance led to significant changes in the ribosomal transcripts. The overall expression profile of immune-related genes and genes specific to monocytes, T-cells or B-cells were well represented and consistent within treatment groups. There were no differences between the levels of ribosomal transcripts in time-matched control and AMPH groups but significant differences in the expression of immune-related genes between control and AMPH groups. AMPH significantly increased expression of some genes related to monocytes but down-regulated those specific to T-cells. These changes were partially due to changes in the two types of leukocytes present in blood, which indicate an activation of the innate immune system by AMPH. Thus, the stability of RNA-seq whole blood transcriptome can be verified by assessing ribosomal protein subunits and immune-related gene expression. Such stability enables the pooling of samples from replicate experiments to carry out differential expression analysis with acceptable power.
Assuntos
Anfetamina/farmacologia , Sangue/metabolismo , Perfilação da Expressão Gênica , Imunidade/genética , Estabilidade de RNA/efeitos dos fármacos , Análise de Sequência de RNA , Transcriptoma/genética , Animais , Sangue/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Imunidade/efeitos dos fármacos , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Masculino , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Subunidades Ribossômicas/genética , Subunidades Ribossômicas/metabolismo , Transcriptoma/efeitos dos fármacosRESUMO
The discrete data structure and large sequencing depth of RNA sequencing (RNA-seq) experiments can often generate outlier read counts in one or more RNA samples within a homogeneous group. Thus, how to identify and manage outlier observations in RNA-seq data is an emerging topic of interest. One of the main objectives in these research efforts is to develop statistical methodology that effectively balances the impact of outlier observations and achieves maximal power for statistical testing. To reach that goal, strengthening the accuracy of outlier detection is an important precursor. Current outlier detection algorithms for RNA-seq data are executed within a testing framework and may be sensitive to sparse data and heavy-tailed distributions. Therefore, we propose a univariate algorithm that utilizes a probabilistic approach to measure the deviation between an observation and the distribution generating the remaining data and implement it within in an iterative leave-one-out design strategy. Analyses of real and simulated RNA-seq data show that the proposed methodology has higher outlier detection rates for both non-normalized and normalized negative binomial distributed data.
Assuntos
DNA/genética , Bases de Dados de Ácidos Nucleicos , RNA/genética , Análise de Sequência de DNA/métodos , Análise de Sequência de RNA/métodosRESUMO
Parkinson's disease (PD) is a progressive motor disease of unknown etiology in the majority of cases. The clinical features of PD emerge due to selective degeneration of dopamine (DA) neurons in the substantia nigra pars compacta (SNc), which project to the caudate putamen (CPu) where they release DA. In the current in vivo mouse model study, we tested trehalose for its ability to protect against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced damage to DA neurons. Trehalose is a naturally occurring disaccharide present in plants and animals and appears capable of protecting cells against various environmental stresses. The effect of trehalose is likely due to its action as a pharmacological chaperone which promotes protein stability. In the present study, there were four treatment groups: saline only (control); probenecid only; MPTP+probenecid; and trehalose+MPTP+probenecid. MPTP-induced losses in tyrosine hydroxylase and DA transporter immunoreactivity in the ventral midbrain SNc and CPu were significantly reduced by trehalose. Decreases in CPu dopamine levels produced by MPTP were also blocked by trehalose. Microglial activation and astrocytic hypertrophy induced by MPTP were greatly reduced by trehalose, indicating protection against neuroinflammation. These effects are commensurate with the observed trehalose sparing of motor deficits produced by MPTP in this mouse model. Two tight junctional proteins, ZO-1 and occludin, are downregulated following MPTP treatment and trehalose blocks this effect. Likewise, the glucose transporter-1 that is expressed in brain endothelial cells is also protected by trehalose from MPTP-induced down-regulation. This study is the first to demonstrate using fluoro-turoquoise FT gel perfusion techniques, the protection afforded by trehalose from MPTP-induced damage to microvessels and endothelial and suggests that trehalose therapy may have the potential to slow or ameliorate PD pathology.
Assuntos
Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Fármacos Neuroprotetores/uso terapêutico , Transtornos Parkinsonianos/tratamento farmacológico , Trealose/uso terapêutico , Animais , Corpo Estriado/irrigação sanguínea , Corpo Estriado/química , Modelos Animais de Doenças , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Encefalite/metabolismo , Encefalite/prevenção & controle , Proteína Glial Fibrilar Ácida , Transportador de Glucose Tipo 1/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Microglia/metabolismo , Chaperonas Moleculares/farmacologia , Chaperonas Moleculares/uso terapêutico , Proteínas do Tecido Nervoso/metabolismo , Fármacos Neuroprotetores/farmacologia , Trealose/farmacologia , Tirosina 3-Mono-Oxigenase/metabolismo , Proteína da Zônula de Oclusão-1/metabolismoRESUMO
The adverse effects of amphetamine- (AMPH) and methamphetamine- (METH) induced hyperthermia on vasculature, peripheral organs and peripheral immune system are discussed. Hyperthermia alone does not produce amphetamine-like neurotoxicity but AMPH and METH exposures that do not produce hyperthermia (≥40°C) are minimally neurotoxic. Hyperthermia likely enhances AMPH and METH neurotoxicity directly through disruption of protein function, ion channels and enhanced ROS production. Forebrain neurotoxicity can also be indirectly influenced through the effects of AMPH- and METH- induced hyperthermia on vasculature. The hyperthermia and the hypertension produced by high doses amphetamines are a primary cause of transient breakdowns in the blood-brain barrier (BBB) resulting in concomitant regional neurodegeneration and neuroinflammation in laboratory animals. This BBB breakdown can occur in the amygdala, thalamus, striatum, sensory and motor cortex and hippocampus. Under these conditions, repetitive seizures greatly enhance neurodegeneration in hippocampus, thalamus and amygdala. Even when the BBB is less disrupted, AMPH- or METH- induced hyperthermia effects on brain vasculature may play a role in neurotoxicity. In this case, striatal and cortical vascular function are adversely affected, and even greater ROS, immune and damage responses are seen in the meninges and cortical surface vasculature. Finally, muscle and liver damage and elevated cytokines in blood can result when amphetamines produce hyperthermia. Proteins, from damaged muscle may activate the peripheral immune system and exacerbate liver damage. Liver damage can further increase cytokine levels, immune system activation and increase ammonia levels. These effects could potentially enhance vascular damage and neurotoxicity.
RESUMO
Fluoro-Gold (F-G) has been used extensively as a fluorescent retrograde neuronal-track tracer in the past. We now report that intraperitoneal administration of 10 to 30 mg/ kg of F-G from 30 min to 7 days prior to sacrifice labels vascular endothelial cells of the brain, choroid plexus and meninges and can be used to assess vascular integrity and damage. F-G vascular labeling co-localized with rat endothelial cell antigen (RECA-1) in the membrane. F-G also intensely labeled the nuclei of the endothelial cells, and co-localized with propidium iodide staining of these nuclei. As well, the administration of F-G during neurotoxic insults produced by amphetamine, kainic acid or "penetrating" wound to the brain can detect where vascular leakage/ hemorrhage has occurred. Histological methods to detect F-G labeled brain vasculature were performed in the same manner as that used for fluorescent visualization of neuronal elements labeled with F-G after perfusion fixation and coronal sectioning (15 to 40 µm) of the brain. This in vivo F-G labeling of endothelial cells and their nuclei yields a clear picture of the integrity of the vasculature and can be used to detect changes in structure. Vascular leaks after "penetrating" wounds through the cortex and striatum, hyperthermic amphetamine exposure or excitotoxic kainate exposure were detected by F-G in the extracellular space and via parenchymal F-G subsequently labeling the terminals and neurons adjacent to the lesioned or damaged vasculature. Further studies are necessary to determine the extent of the leakage necessary to detect vasculature damage. Visualization of the F-G labeling of vasculature structure and leakage is compatible with standard fluorescent immuno-labeling methods used to detect the presence and distribution of a protein in histological sections. This method should be directly applicable to studying brain vascular damage that occurs in the progression of Alzheimer's disease, diabetes and for monitoring the brain vascular changes during development.
Assuntos
Vasos Sanguíneos/patologia , Encéfalo/patologia , Plexo Corióideo/patologia , Estado Epiléptico/patologia , Estilbamidinas , Ferimentos Penetrantes/diagnóstico , Animais , Encéfalo/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Moléculas de Adesão Celular/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Agonistas de Aminoácidos Excitatórios/toxicidade , Ácido Caínico/toxicidade , Masculino , Proteínas dos Microfilamentos/metabolismo , Propídio , Ratos , Ratos Sprague-Dawley , Estado Epiléptico/induzido quimicamente , Estilbamidinas/administração & dosagem , Fatores de TempoRESUMO
Although selective neurodegeneration of nigro-striatal dopaminergic neurons is widely accepted as a cause of Parkinson's disease (PD), the role of vascular components in the brain in PD pathology is not well understood. However, the neurodegeneration seen in PD is known to be associated with neuroinflammatory-like changes that can affect or be associated with brain vascular function. Thus, dysfunction of the capillary endothelial cell component of neurovascular units present in the brain may contribute to the damage to dopaminergic neurons that occurs in PD. An animal model of PD employing acute, sub-acute and chronic exposures of mice to methyl-phenyl-tetrahydropyridine (MPTP) was used to determine the extent to which brain vasculature may be damaged in PD. Fluoro-Turquoise gelatin labeling of microvessels and endothelial cells was used to determine the extent of vascular damage produced by MPTP. In addition, tyrosine hydroxylase (TH) and NeuN were employed to detect and quantify dopaminergic neuron damage in the striatum (CPu) and substantia nigra (SNc). Gliosis was evaluated through GFAP immunohistochemistry. MPTP treatment drastically reduced TH immunoreactive neurons in the SNc (20.68 ± 2.83 in acute; 22.98 ± 2.14 in sub-acute; 10.20 ± 2.24 in chronic vs 34.88 ± 2.91 in controls; p<0.001). Similarly, TH immunoreactive terminals were dramatically reduced in the CPu of MPTP treated mice. Additionally, all three MPTP exposures resulted in a decrease in the intensity, length, and number of vessels in both CPu and SNc. Degenerative vascular changes such as endothelial cell 'clusters' were also observed after MPTP suggesting that vasculature damage may be modifying the availability of nutrients and exposing blood cells and/or toxic substances to neurons and glia. In summary, vascular damage and degeneration could be an additional exacerbating factor in the progression of PD, and therapeutics that protect and insure vascular integrity may be novel treatments for PD.
Assuntos
Encéfalo/patologia , Ventrículos Cerebrais/patologia , Transtornos Parkinsonianos/patologia , Análise de Variância , Animais , Modelos Animais de Doenças , Proteína Glial Fibrilar Ácida/metabolismo , Imageamento por Ressonância Magnética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transtornos Parkinsonianos/induzido quimicamente , Fosfopiruvato Hidratase/metabolismo , Estilbamidinas , Fatores de Tempo , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Determinants of amphetamine (AMPH)-induced neurotoxicity are poorly understood. The role of lipopolysaccharides (LPS) and organ injury in AMPH-induced neurotoxicity was examined in adult male Sprague-Dawley rats that were give AMPH and became hyperthermic during the exposure. Environmentally-induced hyperthermia (EIH) in the rat was compared to AMPH to determine whether AMPH-induced increases in LPS and peripheral toxicities were solely attributable to hyperthermia. Muscle, liver, and kidney function were determined biochemically at 3h or 1 day after AMPH or EIH exposure and histopathology at 1 day after treatment. Circulating levels of LPS were monitored (via limulus amoebocyte coagulation assay) during AMPH or EIH exposure. Blood LPS levels were detected in 40-50% of the AMPH and EIH rats, but the presence of LPS in the serum had no effect on organ damage or striatal dopamine depletions (neurotoxicity). In both CR and NCTR rats, serum bound urea nitrogen and creatinine levels increased at 3h after EIH or AMPH (2- to 3-fold above control) but subsided by 1 day. Alanine transaminase was increased (indicating liver dysfunction) by both AMPH and EIH at 3 h (2- to 10-fold above control) in CR rats, but the levels were not significantly different between the control and AMPH groups in NCTR animals. Mild liver necrosis was detected in 1 of 7 rats examined in the AMPH group and in 1 of 5 rats examined in the EIH group (only NCTR rats were examined). Serum myoglobin increased (indicating muscle damage) in both CR and NCTR rats at 3h and was more pronounced with AMPH (≈5-fold above control) than EIH. Our results indicate that: (1) "free" blood borne LPS often increases with EIH and AMPH but may not be necessary for striatal neurotoxicity and CNS immune responses; (2) liver or kidney dysfunction may result from muscle damage; however, it is not sufficient nor necessary to produce, but may exacerbate, neurotoxicity; (3) AMPH-induced serum myoglobin release is a potential biomarker and possibly a factor in AMPH-induced toxicity processes.
Assuntos
Anfetamina , Gânglios da Base/metabolismo , Lipopolissacarídeos/sangue , Mioglobina/sangue , Síndromes Neurotóxicas/sangue , Animais , Gânglios da Base/patologia , Biomarcadores/sangue , Regulação da Temperatura Corporal , Modelos Animais de Doenças , Dopamina/metabolismo , Febre/sangue , Febre/etiologia , Febre/fisiopatologia , Hipertermia Induzida , Rim/metabolismo , Rim/patologia , Fígado/metabolismo , Fígado/patologia , Masculino , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Necrose , Síndromes Neurotóxicas/etiologia , Síndromes Neurotóxicas/patologia , Síndromes Neurotóxicas/fisiopatologia , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Regulação para CimaRESUMO
BACKGROUND: The meninges (arachnoid and pial membranes) and associated vasculature (MAV) and choroid plexus are important in maintaining cerebrospinal fluid (CSF) generation and flow. MAV vasculature was previously observed to be adversely affected by environmentally-induced hyperthermia (EIH) and more so by a neurotoxic amphetamine (AMPH) exposure. Herein, microarray and RT-PCR analysis was used to compare the gene expression profiles between choroid plexus and MAV under control conditions and at 3 hours and 1 day after EIH or AMPH exposure. Since AMPH and EIH are so disruptive to vasculature, genes related to vasculature integrity and function were of interest. RESULTS: Our data shows that, under control conditions, many of the genes with relatively high expression in both the MAV and choroid plexus are also abundant in many epithelial tissues. These genes function in transport of water, ions, and solutes, and likely play a role in CSF regulation. Most genes that help form the blood-brain barrier (BBB) and tight junctions were also highly expressed in MAV but not in choroid plexus. In MAV, exposure to EIH and more so to AMPH decreased the expression of BBB-related genes such as Sox18, Ocln, and Cldn5, but they were much less affected in the choroid plexus. There was a correlation between the genes related to reactive oxidative stress and damage that were significantly altered in the MAV and choroid plexus after either EIH or AMPH. However, AMPH (at 3 hr) significantly affected about 5 times as many genes as EIH in the MAV, while in the choroid plexus EIH affected more genes than AMPH. Several unique genes that are not specifically related to vascular damage increased to a much greater extent after AMPH compared to EIH in the MAV (Lbp, Reg3a, Reg3b, Slc15a1, Sct and Fst) and choroid plexus (Bmp4, Dio2 and Lbp). CONCLUSIONS: Our study indicates that the disruption of choroid plexus function and damage produced by AMPH and EIH is significant, but the changes may not be as pronounced as they are in the MAV, particularly for AMPH. Expression profiles in the MAV and choroid plexus differed to some extent and differences were not restricted to vascular related genes.
Assuntos
Encéfalo/metabolismo , Líquido Cefalorraquidiano/metabolismo , Plexo Corióideo/metabolismo , Meninges/metabolismo , Anfetamina/toxicidade , Aracnoide-Máter/irrigação sanguínea , Aracnoide-Máter/metabolismo , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Encéfalo/irrigação sanguínea , Plexo Corióideo/irrigação sanguínea , Plexo Corióideo/efeitos dos fármacos , Meio Ambiente , Febre , Humanos , Meninges/irrigação sanguínea , Meninges/efeitos dos fármacos , Proteínas Associadas a Pancreatite , TranscriptomaRESUMO
This video presentation was created to show a method of harvesting the two most important highly vascular structures, not residing within the brain proper, that support forebrain function. They are the cerebral surface (superficial) vasculature along with associated meninges (MAV) and the choroid plexus which are necessary for cerebral blood flow and cerebrospinal fluid (CSF) homeostasis. The tissue harvested is suitable for biochemical and physiological analysis, and the MAV has been shown to be sensitive to damage produced by amphetamine and hyperthermia. As well, the major and minor cerebral vasculatures harvested in MAV are of potentially high interest when investigating concussive types of head trauma. The MAV dissected in this presentation consists of the pial and some of the arachnoid membrane (less dura) of the meninges and the major and minor cerebral surface vasculature. The choroid plexus dissected is the structure that resides in the lateral ventricles as described by Oldfield and McKinley. The methods used for harvesting these two tissues also facilitate the harvesting of regional cortical tissue devoid of meninges and larger cerebral surface vasculature, and is compatible with harvesting other brain tissues such as striatum, hypothalamus, hippocampus, etc. The dissection of the two tissues takes from 5 to 10 min total. The gene expression levels for the dissected MAV and choroid plexus, as shown and described in this presentation can be found at GSE23093 (MAV) and GSE29733 (choroid plexus) at the NCBI GEO repository. This data has been, and is being, used to help further understand the functioning of the MAV and choroid plexus and how neurotoxic events such as severe hyperthermia and AMPH adversely affect their function.
Assuntos
Encéfalo/irrigação sanguínea , Encéfalo/cirurgia , Plexo Corióideo/cirurgia , Meninges/cirurgia , Animais , Encéfalo/fisiologia , Circulação Cerebrovascular , Plexo Corióideo/irrigação sanguínea , Plexo Corióideo/fisiologia , Dissecação/métodos , Expressão Gênica , Meninges/irrigação sanguínea , RatosRESUMO
Up-regulation of proinflammatory cytokines and chemokines in brain ("neuroinflammation") accompanies neurological disease and neurotoxicity. Previously, we documented a striatal neuroinflammatory response to acute administration of a neurotoxic dose of methamphetamine (METH), i.e. one associated with evidence of dopaminergic terminal damage and activation of microglia and astroglia. When we used minocycline to suppress METH-induced neuroinflammation, indices of dopaminergic neurotoxicity were not affected, but suppression of neuroinflammation was incomplete. Here, we administered the classic anti-inflammatory glucocorticoid, corticosterone (CORT), in an attempt to completely suppress METH-related neuroinflammation. METH alone caused large increases in striatal proinflammatory cytokine/chemokine mRNA and subsequent astrocytic hypertrophy, microglial activation, and dopaminergic nerve terminal damage. Pre-treatment of mice with acute CORT failed to prevent neuroinflammatory responses to METH. Surprisingly, when mice were pre-treated with chronic CORT in the drinking water, an enhanced striatal neuroinflammatory response to METH was observed, an effect that was accompanied by enhanced METH-induced astrogliosis and dopaminergic neurotoxicity. Chronic CORT pre-treatment also sensitized frontal cortex and hippocampus to mount a neuroinflammatory response to METH. Because the levels of chronic CORT used are associated with high physiological stress, our data suggest that chronic CORT therapy or sustained physiological stress may sensitize the neuroinflammatory and neurotoxicity responses to METH.