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.
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 (2h 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 all other treatments (≈ 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 implicate elevated blood levels of glucose and hyperthermia in METH-induced neurotoxicity, neurovascular damage, and lethality, and that acute elevation of CORT exacerbates both neurotoxicity and neuroinflammation.
BackgroundBrain 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.MethodsMale 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.ResultsDual 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.ConclusionsThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0526-6) contains supplementary material, which is available to authorized users.
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.
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