The time course of blood brain barrier leakage and its implications on the progression of methamphetamine-induced seizures.

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.

Microglial activation and vascular responses that are associated with early thalamic neurodegeneration resulting from thiamine deficiency.

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 0.25 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 (FJc + 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 (0.2-0.4 mg/kg) or phenobarbital (10-20 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.

Corticosterone and exogenous glucose alter blood glucose levels, neurotoxicity, and vascular toxicity produced by methamphetamine.

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.

Vascular-directed responses of microglia produced by methamphetamine exposure: indirect evidence that microglia are involved in vascular repair?

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.

Evaluating the Stability of RNA-Seq Transcriptome Profiles and Drug-Induced Immune-Related Expression Changes in Whole Blood.

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.

Systemic administration of fluoro-gold for the histological assessment of vascular structure, integrity and damage.

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.

Serum myoglobin, but not lipopolysaccharides, is predictive of AMPH-induced striatal neurotoxicity.

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.