METH-treated Mice Research Articles

The role of the microbiota–gut–brain axis in methamphetamine-induced neurotoxicity: Disruption of microbial composition and short-chain fatty acid metabolism

Methamphetamine (METH) abuse is associated with significant neurotoxicity, high addiction potential, and behavioral abnormalities. Recent studies have identified a connection between the gut microbiota and METH-induced neurotoxicity and behavioral disorders. However, the underlying causal mechanisms linking the gut microbiota to METH pathophysiology remain largely unexplored. In this study, we employed fecal microbiota transplantation (FMT) and antibiotic (Abx) intervention to manipulate the gut microbiota in mice administered METH. Furthermore, we supplemented METH-treated mice with short-chain fatty acids (SCFAs) and pioglitazone (Pio) to determine the protective effects on gut microbiota metabolism. Finally, we assessed the underlying mechanisms of the gut–brain neural circuit in vagotomized mice. Our data provide compelling evidence that modulation of the gut microbiome through FMT or microbiome knockdown by Abx plays a crucial role in METH-induced neurotoxicity, behavioral disorders, gut microbiota disturbances, and intestinal barrier impairment. Furthermore, our findings highlight a novel prevention strategy for mitigating the risks to both the nervous and intestinal systems caused by METH, which involves supplementation with SCFAs or Pio.

Role of MARK4 in methamphetamine-induced acute kidney injury.

Methamphetamine (METH) is an illicit psychoactive substance that can damage various organs, with the urinary system being one of its significant targets. This study aims to explore the role of microtubule affinity-regulating kinase 4 (MARK4) in METH-induced acute kidney injury (AKI). A total of 10 healthy adult male C57BL/6 mice were randomly divided into a control group and a METH group, 5 mice in each group. The METH group was administered METH (20 mg/kg, intraperitoneally, once daily for 3 consecutive days), while the control group received an equal volume of physiological saline. The mice were executed 24 hours after the final injection, and the success of the AKI model was detected by blood serum creatinine, blood urea nitrogen, and renal HE staining. Proteins differentially expressed between kidney tissues with METH-induced AKI and normal kidney tissues were screened by proteomics techniques and subjected to gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and bioinformatics analysis. The accuracy of proteomic data was validated using Western blotting, and the expression levels of MARK4 and cleaved caspase-3 in mouse kidneys were measured. We further explored the role of MARK4 in METH-induced AKI. Firstly, a METH toxicity model was established in BUMPT cells to screen the appropriate concentration and time of METH treatment; the viability of BUMPT cells after METH treatment and the expression of cleaved caspase-3 were detected by interfering with MARK4 expression through inhibitors. The proteomic analysis of kidney tissues from METH and control groups screened for a total of 17 differentially expressed proteins, of which 11 were up-regulated and 6 were down-regulated (all P<0.05). The expression levels of MARK4 and cleaved caspase-3 were elevated in the kidneys of METH-treated mice (both P<0.05). The activity of BUMPT cells gradually decreased with increasing METH treatment concentration (all P<0.05), where the viability of BUMPT cells decreased to about 60% after METH treatment at 4 mmol/L. Compared with the control group, expression levels of MARK4 and cleaved caspase-3 were increased with higher METH concentrations and longer exposure times in a concentration- and time-dependent manner (all P<0.05). Inhibition of MARK4 expression improved METH-induced decrease in BUMPT cell activity, down-regulated the expression of cleaved caspase-3, and decreased the apoptosis of BUMPT cells induced by METH. MARK4 is highly expressed in a mouse model of METH-induced AKI, and MARK4 mediates METH-induced AKI by regulating cell apoptosis.

Single-dose methamphetamine administration impairs ORM retrieval in mice via excessive DA-mediated inhibition of PrLGlu activity.

Methamphetamine (METH), an abused psychostimulant, impairs cognition through prolonged or even single-dose exposure, but animal experiments have shown contradictory effects on memory deficits. In this study we investigated the effects and underlying mechanisms of single-dose METH administration on the retrieval of object recognition memory (ORM) in mice. We showed that single-dose METH administration (2 mg/kg, i.p.) significantly impaired ORM retrieval in mice. Fiber photometry recording in METH-treated mice revealed that the activity of prelimbic cortex glutamatergic neurons (PrLGlu) was significantly reduced during ORM retrieval. Chemogenetic activation of PrLGlu or glutamatergic projections from ventral CA1 to PrL (vCA1Glu-PrL) rescued ORM retrieval impairment. Fiber photometry recording revealed that dopamine (DA) levels in PrL of METH-treated mice were significantly increased, and micro-infusion of the D2 receptor (D2R) antagonist sulpiride (0.25 μg/side) into PrL rescued ORM retrieval impairment. Whole-cell recordings in brain slices containing the PrL revealed that PrLGlu intrinsic excitability and basal glutamatergic synaptic transmission were significantly reduced in METH-treated mice, and the decrease in intrinsic excitability was reversed by micro-infusion of Sulpiride into PrL in METH-treated mice. Thus, the impaired ORM retrieval caused by single-dose METH administration may be attributed to reduced PrLGlu activity, possibly due to excessive DA activity on D2R. Selective activation of PrLGlu or vCA1Glu-PrL may serve as a potential therapeutic strategy for METH-induced cognitive dysfunction.

Blocking Sigmar1 exacerbates methamphetamine-induced hypertension

AimMethamphetamine (METH) chronic exposure is an important risk factor for hypertension development. However, the mechanisms behind METH-induced hypertension remain unclear. Therefore, we aimed to reveal the potential mechanisms underlying METH-induced hypertension. Methods and resultsWe structured the mouse hypertension model by METH, and observed that METH-treated mice have presented vascular remodeling (large-and small-size arteries) with collagen deposit around the vessel and increasing blood pressure (BP) and Sigma1 receptor (Sigmar1) in vascular tissue. We hypothesized that Sigmar1 is crucial in METH-induced hypertension and vascular remodeling. Sigmar1 knockout (KO) mice and antagonist (BD1047) pretreated mice exposed to METH for six-week showed higher BP and more collagen deposited around vessels than wild-type (WT) mice exposed to METH for six-week, in contrast, mice pretreated with Sigmar1 agonist (PRE-084) had unchanged BP and perivascular collagen despite the six-week METH exposure. Furthermore, we found that METH exposure induced vascular smooth muscle cells (VSMCs) and mesenchymal stem cells to differentiate into the myofibroblast-like cell and secrete collagen into surrounding vessels. Mechanically, Sigmar1 can suppress the COL1A1 expression by blocking the classical fibrotic TGF-β/Smad2/3 signaling pathway in METH-exposed VSMCs and mesenchymal stem cells. ConclusionOur results suggest that Sigmar1 is involved in METH-induced hypertension and vascular fibrosis by blocking the activation of the TGF-β/Smad2/3 signaling pathway. Accordingly, Sigmar1 may be a novel therapeutic target for METH-induced hypertension and vascular fibrosis.

The neuroprotective effect of LCZ696 on methamphetamine-induced cognitive impairment in mice

ObjectiveMethamphetamine (METH) exposure commonly causes cognitive impairment. An angiotensin II receptor/neprilysin inhibitor (ARNI), LCZ696 has been demonstrated to inhibit inflammation, oxidative stress and apoptosis. The present study was designed to examine the effect of LCZ696 on METH-induced cognitive impairment and the underlying mechanism. MethodsFollowing daily treatment of either saline or METH (5 mg/kg) for 5 consecutive days, the cognitive function was tested using the Y-maze and the Novel Object Recognition (NOR) in Experiment 1. In Experiment 2, mice were initially treated with saline or LCZ696 (60 mg/kg) for 9 consecutive days, followed by LCZ696, METH or saline for 5 days. Cognitive testing was carried out as Experiment 1. In Experiment 3, SH-SY5Y cells were treated with either METH (2.5 Mm) or ddH2O for 12 h. The apoptosis and reactive oxygen species (ROS) level of SH-SY5Y were examined. In Experiment 4, SH-SY5Y cells were pretreated with either ddH2O or LCZ696 (70um) for 30 min, followed by ddH2O or METH treatment for 12 h. Nrf2 and HO-1 protein expression was examined in the ventral tegemental area (VTA) of all the animals and SH-SY5Y cells. ResultsLCZ696 significantly improved METH-induced cognitive impairment, in conjunction with decreased apoptosis and ROS levels in VTA of METH-treated mice and SH-SY5Y cells. METH significantly decreased Nrf2 and HO-1 protein expression in VTA of mice and SH-SY5Y cells, which was reversed by LCZ696 treatment. ConclusionLCZ696 yields a neuroprotective effect against METH-induced cognitive dysfunction via the Nrf2/HO-1 signaling pathway.

The Synergistic Effect of Curcumin and Piperine Nanoparticles on Methamphetamine-induced Neurotoxicity, Oxidative Stress, and Memory Impairments in Mice Brain

Background: Methamphetamine (METH) is a highly addictive neural stimulant that severely affects the CNS and can induce oxidative damage. Piperine and curcumin are active constituents that have numerous properties, including antioxidant, anti-inflammatory, and neuroprotective Objective: In this study, the synergistic effect of piperine and curcumin nanoparticles was investigated on the acute doses of METH-induced neurotoxicity in mice brains. Methods: METH (6 mg/kg, i.p) was administered to 14 groups of mice and piperine-curcumin nanoparticles at different doses (10, 20, 40 mg/kg and 20, 40 and 60 mg/kg, respectively) were administered. Open field test (OFT) and conditioned place preference (CPP) were used to investigate locomotor activity, anxiety-like behavior, and addictive behavior in mice. Oxidative stress biomarkers (reactive oxygen species (ROS), protein carbonyl content, lipid peroxidation, glutathione content, and mitochondrial function were evaluated in isolated brain mitochondria. Results: We found that piperine and curcumin nanoparticles significantly decreased hyperlocomotion and anxiety-like behavior in METH-treated mice. Also, METH enhanced CPP whilst piperine and curcumin nanoparticles suppressed the effect of METH-induced CPP. METH administration significantly increased ROS, protein carbonyl content, and lipid peroxidation and decreased glutathione content and mitochondrial function in the isolated brain mitochondria. Piperine and curcumin nanoparticles (at all doses) showed synergistic effects on reducing oxidative damages in a dosedependent manner compared to the METH group. Conclusion: In conclusion, combined piperine and curcumin nanoparticles showed greater neuroprotective effects against METH-induced neurotoxicity due to their greater permeability and better antioxidant properties than piperine and curcumin alone

Single-nucleus transcriptome profiling of prefrontal cortex induced by chronic methamphetamine treatment

BackgroundMethamphetamine (METH) addiction causes a huge burden on society. The prefrontal cortex (PFC), associated with emotion and cognitive behaviours, is also involved in addiction neurocircuitry. Although bulk RNA sequencing has...

Neuroprotective effect of histamine H3 receptor blockade on methamphetamine-induced cognitive impairment in mice

ObjectiveMethamphetamine (METH) exposure is commonly believed to result in cognitive impairment. Histamine H3 receptor (H3R) antagonists reportedly have potential applications for treating cognitive impairment accompanied by various neuropsychiatric disorders. The present study aimed to investigate the effect of H3R blockade by Thioperamide (THIO) on METH-induced cognitive impairment and the underlying mechanism. MethodsIn Experiment 1, C57BL/6 mice received daily injections of saline or 5 mg/kg METH for 5 consecutive days. The Novel Object Recognition (NOR) and Morris water maze (MWM) tasks were used to assess cognitive functions of mice. H3R protein expression and apoptosis were subsequently measured in the hippocampus. In Experiment 2, HT22 cells were first treated with ddH2O or 3 mM METH. The cell survival rate and H3R protein level were subsequently assessed. In Experiment 3, the animals were first treated with saline or 20 mg/kg THIO for 7 days, followed by co-administration of either saline or 5 mg/kg METH for an additional 5 days. The remaining experiments were carried out in the same manner as Experiment 1. In Experiment 4, HT22 cells were pretreated with either ddH2O or 5 mM THIO for 2 h, followed by ddH2O or 3 mM METH treatment for an additional 12 h. The remaining experiments were carried out in the same manner as Experiment 2. In Experiment 5, the changes in MEK1/2, p-MEK1/2, ERK1/2 and p-ERK1/2 protein levels were examined in the hippocampus of all mice from Experiment 3 and HT22 cells from Experiment 4. ResultsMETH-treated mice showed significantly worsened NOR and MWM performance, along with markably hippocampal apoptosis. A significantly lower cell survival rate was observed in METH-treated HT22 cells. Increased levels of H3R protein were found in both METH-treated mice and HT22 cells. THIO significantly improved METH-induced cognitive impairment in mice and toxicity in HT22 cells. METH significantly increased the level of p-MEK1/2 and p-ERK1/2 proteins in the hippocampus of mice and HT22 cells, which was reversed by THIO pretreatment. ConclusionOur findings reveal that H3R blockade by THIO yields a neuroprotective effect against METH-induced cognitive impairment in mice and toxicity in HT22 cells via the raf-MEK-ERK signaling pathway.

Silencing the Tlr4 Gene Alleviates Methamphetamine-Induced Hepatotoxicity by Inhibiting Lipopolysaccharide-Mediated Inflammation in Mice.

Methamphetamine (METH) is a stimulant drug. METH abuse induces hepatotoxicity, although the mechanisms are not well understood. METH-induced hepatotoxicity was regulated by TLR4-mediated inflammation in BALB/c mice in our previous study. To further investigate the underlying mechanisms, the wild-type (C57BL/6) and Tlr4−/− mice were treated with METH. Transcriptomics of the mouse liver was performed via RNA-sequencing. Histopathological changes, serum levels of metabolic enzymes and lipopolysaccharide (LPS), and expression of TLR4-mediated proinflammatory cytokines were assessed. Compared to the control, METH treatment induced obvious histopathological changes and significantly increased the levels of metabolic enzymes in wild-type mice. Furthermore, inflammatory pathways were enriched in the liver of METH-treated mice, as demonstrated by expression analysis of RNA-sequencing data. Consistently, the expression of TLR4 pathway members was significantly increased by METH treatment. In addition, increased serum LPS levels in METH-treated mice indicated overproduction of LPS and gut microbiota dysbiosis. However, antibiotic pretreatment or silencing Tlr4 significantly decreased METH-induced hepatic injury, serum LPS levels, and inflammation. In addition, the dampening effects of silencing Tlr4 on inflammatory pathways were verified by the enrichment analysis of RNA-sequencing data in METH-treated Tlr4−/− mice compared to METH-treated wild-type mice. Taken together, these findings implied that Tlr4 silencing, comparable to antibiotic pretreatment, effectively alleviated METH-induced hepatotoxicity by inhibiting LPS-TLR4-mediated inflammation in the liver.

Differentially Expressed Genes in Period 2-Overexpressing Mice Striatum May Underlie Their Lower Sensitivity to Methamphetamine Addiction-Like Behavior.

Previous reports have demonstrated that genetic mechanisms greatly mediate responses to drugs of abuse, including methamphetamine (METH). The circadian gene Period 2 (Per2) has been previously associated with differential responses towards METH in mice. While the behavioral consequences of eliminating Per2 have been illustrated previously, Per2 overexpression has not yet been comprehensively described; although, Per2-overexpressing (Per2 OE) mice previously showed reduced sensitivity towards METH-induced addiction-like behaviors. To further elucidate this distinct behavior of Per2 OE mice to METH, we identified possible candidate biomarkers by determining striatal differentially expressed genes (DEGs) in both drug-naïve and METH-treated Per2 OE mice relative to wild-type (WT), through RNA sequencing. Of the several DEGs in drug naïve Per2 OE mice, we identified six genes that were altered after repeated METH treatment in WT mice, but not in Per2 OE mice. These results, validated by quantitative real-time polymerase chain reaction, could suggest that the identified DEGs might underlie the previously reported weaker METH-induced responses of Per2 OE mice compared to WT. Gene network analysis also revealed that Asic3, Hba-a1, and Rnf17 are possibly associated with Per2 through physical interactions and predicted correlations, and might potentially participate in addiction. Inhibiting the functional protein of Asic3 prior to METH administration resulted in the partial reduction of METH-induced conditioned place preference in WT mice, supporting a possible involvement of Asic3 in METH-induced reward. Although encouraging further investigations, our findings suggest that these DEGs, including Asic3, may play significant roles in the lower sensitivity of Per2 OE mice to METH.

Methamphetamine induces intestinal injury by altering gut microbiota and promoting inflammation in mice

Methamphetamine (METH) is a psychostimulant abused worldwide. Its abuse induces intestinal toxicity. Moreover, the gut microbiota is altered by drugs, which induces intestinal injury. Whether gut microbiota mediates METH-induced intestinal toxicity remains to be validated. In the present study, wild-type and TLR4−/− mice were treated with METH. Gut microbiota was determined using 16S rRNA gene sequencing. Transcriptomics of the intestinal mucosa was performed by RNA-Sequencing. Blood levels of pro-inflammatory cytokines and lipopolysaccharide (LPS), the intestinal barrier, and inflammation were also assessed. METH treatment weakened the intestinal barrier and increased pro-inflammatory cytokines and LPS levels in the blood. Moreover, METH treatment significantly decreased the diversity of probiotics but increased the abundance of pathogenic gut microbiota, contributing to the over-production of LPS and disruption of intestinal barrier. Inflammatory pathways were enriched in the intestinal mucosa of METH-treated mice by KEGG analysis. Consistently, activation of the TLR4 pathway was determined in METH-treated mice, which confirmed intestinal inflammation. However, pretreatment with antibiotics or Tlr4 silencing significantly alleviated METH-induced gut microbiota dysbiosis, LPS over-production, intestinal inflammation, and disruption of the intestinal barrier. These findings suggested that the gut microbiota and LPS-mediated inflammation took an important role in METH-induced intestinal injury. Taken together, these findings suggest that METH-induced intestinal injury is mediated by gut microbiota dysbiosis and LPS-associated inflammation.

Methamphetamine Enhances Cryptococcus neoformans Melanization, Antifungal Resistance, and Pathogenesis in a Murine Model of Drug Administration and Systemic Infection.

Methamphetamine (METH) is a major public health and safety problem in the United States. Chronic METH abuse is associated with a 2-fold-higher risk of HIV infection and, possibly, additional infections, particularly those that enter through the respiratory tract or skin. Cryptococcus neoformans is an encapsulated opportunistic yeast-like fungus that is a relatively frequent cause of meningoencephalitis in immunocompromised patients, especially in individuals with AIDS. C. neoformans melanizes during mammalian infection in a process that presumably uses host-supplied compounds such as catecholamines. l-3,4-Dihydroxyphenylalanine (l-Dopa) is a natural catecholamine that is frequently used to induce melanization in C. neoformans. l-Dopa-melanized cryptococci manifest resistance to radiation, phagocytosis, detergents, and heavy metals. Using a systemic mouse model of infection and in vitro assays to critically assess the impact of METH on C. neoformans melanization and pathogenesis, we demonstrated that METH-treated mice infected with melanized yeast cells showed increased fungal burdens in the blood and brain, exacerbating mortality. Interestingly, analyses of cultures of METH-exposed cryptococci supplemented with l-Dopa revealed that METH accelerates fungal melanization, an event of adaptation to external stimuli that can be advantageous to the fungus during pathogenesis. Our findings provide novel evidence of the impact of METH abuse on host homeostasis and increased permissiveness to opportunistic microorganisms.

Retinal hypoxia and angiogenesis with methamphetamine

Central retinal artery occlusion, retinopathy, and retinal neovascularization have been reported in methamphetamine (METH) abusers. In the current study, we investigated whether METH induces retinal neovascularization in a mouse model, and if so, whether the neovascularization is associated with increased hypoxia, hypoxia-inducible factor 1α (HIF-1α), and vascular endothelial growth factor (VEGF). Mice were administrated METH by intraperitoneal injection over a 26-day period, or injected with saline as a vehicle control. The number of retinal arterioles and venules were counted using in vivo live imaging following infusion with fluorescein isothiocyanate-dextran. Excised retinas were stained with griffonia simplicifolia lectin I and flat mounted for a measurement of vascularity (length of vessels per tissue area) with AngioTool. Retinal hypoxia was examined by formation of pimonidazole adducts with an anti-pimonidazole antibody, and HIF-1α and VEGFa protein levels in the retina were detected by immunoblot. METH administration increased vascularity (including the number of arterioles) measured on Day 26. Retinal VEGFa protein level was not changed in METH-treated mice on Day 5, but was increased on Day 12 and Day 26. Hypoxia (pimonidazole adduct formation) was increased in retinas of METH-treated mice on Day 12 and Day 26, as were HIF-1α protein expression levels. These results indicate that METH administration induces hypoxia, HIF-1α, VEGFa, and angiogenesis in the retina.

Melatonin ameliorates methamphetamine-induced cognitive impairments by inhibiting neuroinflammation via suppression of the TLR4/MyD88/NFκB signaling pathway in the mouse hippocampus

Methamphetamine (METH) is a highly addictive psychostimulant that causes significant health issues due to high prevalence of its illegal use. Chronic use of METH is associated with cognitive impairments in both human and animal studies, but the underlying mechanism remains unclear. METH-induced neuroinflammation is, potentially, one of the factors that causes cognitive impairments. Therefore, the present study aimed to assess whether melatonin could provide protection against inflammation, in a manner comparable to the anti-inflammatory agent, minocycline, with consequent improvements of METH-induced cognitive impairments and associated abnormalities in the mouse hippocampus. Results from the Morris water maze (MWM) test and the novel object recognition test (NORT) showed that melatonin given after METH injections could ameliorate both METH-induced spatial and recognition memory impairments. These memory impairments are associated with changes in the neuroinflammatory profiles, including IL-6, IL-1β, and TNF-α, both in the blood serum and hippocampus of adult mice. METH-treated mice also exhibited reactive astrocytes and activated microglia in the hippocampus. METH-induced activation of glial cells is associated with the activation of the TLR4/MyD88/NFκB signaling pathway. Moreover, melatonin administration led to recovery of these METH-induced markers to control levels. Thus, we conclude that melatonin could potentially be used as a cognitive enhancer and anti-inflammatory agent in the treatment of METH use disorder in humans.

Methamphetamine‐induced retinal hyperpermeability, hypoxia, and vascularization

PurposeMethamphetamine (METH), a highly addictive stimulant of neurotransmitters induces central retinal artery occlusion with retinal atrophy and neovascularization. We previously have identified METH‐induced loss of endothelial surface molecules including platelet and endothelial cell adhesion molecule‐1 (PECAM‐1) and glycocalyx in mice. However, the pathophysiologic pathway of METH‐induced retinal vascular dysfunction has been insufficiently investigated. The purpose of this study is to investigate the METH‐induced alteration of the retinal vascular circulation.Materials and methodsC57BL/6J male and female mice were administrated progressively increasing doses of METH (0 to 6 mg/kg) by repetitive intraperitoneal injections (4 times per day) for 4 weeks. Norepinephrine levels in plasma were measured by ELISA. Cross‐sections of the retina were stained with hematoxylin and eosin. Levels of hypoxia in the retina were determined by immunostaining of pimonidazole adducts with Hypoxyprobe. Retinal blood vessels were visualized with fluorescent plasma tracers injected via femoral vein injection for live imaging, and immunostained with griffonia simplicifolia lectin 1 (GSL‐1) for retinal flatmounts. Expression of various proteins was determined by immunoblot.ResultsThe level of pimonidazole adduct was increased in METH‐treated retinas, compared with saline‐treated retinas, indicating METH‐induced retinal hypoxia. Plasma norepinephrine levels, which can promote hypoxia‐inducible factor 1a (HIF‐1a) at the transcriptional level, were elevated by METH, compared with saline treatment at 1 week (2.2±0.9 vs 7.8±1.6 ng/mL, P&lt;0.01), 2 weeks (0.8±0.3 vs 5.5±1.8 ng/mL, P&lt;0.05), and 4 weeks (0.9±0.2 vs 2.0±0.4 ng/mL, P&lt;0.05). Retinal protein expression levels of HIF‐1a and angiogenic proteins, such as vascular endothelial growth factor a (VEGFa), erythroblast transformation‐specific (ETS)‐related gene (ERG), and Protein C‐ets‐1 (ETS‐1) were increased in the METH‐treated retinas. Counts of retinal arterioles and capillaries were higher in METH‐treated mice than in saline‐treated mice. Moreover, protein concentration in the vitreous humor and immunoreactivity of immunoglobulin in retina, which are measures of retinal permeability, were increased by METH treatment.ConclusionOur present data suggest that METH is associated with retinal neovascularization and hyperpermeability with increases in hypoxia, plasma norepinephrine, and HIF‐1a activation.Support or Funding InformationThis work was supported by funding from the National Institute of Health (NIH) EY025632.

Acute changes in the retina and central retinal artery with methamphetamine

Methamphetamine (METH), an addictive stimulant of neurotransmitters, is associated with cardiovascular and neurological diseases. METH-induced ophthalmic complications are also present but have been insufficiently investigated. The purpose of this study is to investigate the retinal effects of METH. C57BL/6 mice were administrated progressively increasing doses of METH (0–6 mg/kg) by repetitive intraperitoneal injections for 5 days (4 times per day). Retinal degeneration was examined by morphological changes and terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling (TUNEL) assay. Norepinephrine levels were measured by ELISA, protein expression levels were determined by immunoblot and immunostaining, and gelatinase activity was examined by zymography. The thickness of the retina and the number of nuclei in the inner and outer nuclear layers were decreased by METH. Retinal cell death and astrocyte activation by METH treatment were confirmed by TUNEL assay and glial fibrillary acidic protein expression, respectively. Increased tumor necrosis factor-α protein in the retina and elevated norepinephrine levels in plasma were found in METH-treated mice. Platelet endothelial cell adhesion molecule-1 (PECAM-1) protein expression level was decreased in the retina and central retinal artery (CRA) by METH treatment, along with the endothelial proteoglycans glypican-1 and syndecan-1. Moreover, a regulator of the extracellular matrix, matrix metalloproteinase-14 (MMP-14) in the retina, and MMP-2 and MMP-9 in plasma, were increased by METH treatment. In conclusion, METH administration is involved in retinal degeneration with a vascular loss of PECAM-1 and the glycocalyx in the CRA and retina, and an increase of MMPs.

Chronic methamphetamine interacts with BDNF Val66Met to remodel psychosis pathways in the mesocorticolimbic proteome.

Methamphetamine (Meth) abuse has reached epidemic proportions in many countries and can induce psychotic episodes mimicking the clinical profile of schizophrenia. Brain-derived neurotrophic factor (BDNF) is implicated in both Meth effects and schizophrenia. We therefore studied the long-term effects of chronic Meth exposure in transgenic mice engineered to harbor the human BDNFVal66Met polymorphism expressed via endogenous mouse promoters. These mice were chronically treated with an escalating Meth regime during late adolescence. At least 4 weeks later, all hBDNFVal66Met Meth-treated mice exhibited sensitization confirming persistent behavioral effects of Meth. We used high-resolution quantitative mass spectrometry-based proteomics to biochemically map the long-term effects of Meth within the brain, resulting in the unbiased detection of 4808 proteins across the mesocorticolimbic circuitry. Meth differentially altered dopamine signaling markers (e.g., Dat, Comt, and Th) between hBDNFVal/Val and hBDNFMet/Met mice, implicating involvement of BDNF in Meth-induced reprogramming of the mesolimbic proteome. Targeted analysis of 336 schizophrenia-risk genes, as well as 82 growth factor cascade markers, similarly revealed that hBDNFVal66Met genotype gated the recruitment of these factors by Meth in a region-specific manner. Cumulatively, these data represent the first comprehensive analysis of the long-term effects of chronic Meth exposure within the mesocorticolimbic circuitry. In addition, these data reveal that long-term Meth-induced brain changes are strongly dependent upon BDNF genetic variation, illustrating how drug-induced psychosis may be modulated at the molecular level by a single genetic locus.

In vivo long-lasting alterations of central serotonin transporter activity and associated dopamine synthesis after acute repeated administration of methamphetamine

BackgroundMethamphetamine (METH)-associated alterations in the striatal dopamine (DA) system or dopamine transport (DAT) have been identified in clinical and preclinical studies with positron emission tomography (PET) imaging but have not been well correlated with in vivo serotonin transporter (SERT) availability due to the lack of appropriate imaging agents to assess SERTs. N,N-dimethyl-2-(2-amino-4-[18F]-fluorophenylthio) benzylamine (4-[18F]-ADAM) has been developed by our group and validated for its high affinity and selectivity for SERTs, allowing the in vivo examination of SERT density, location, and binding function. The aims of this study were to investigate the potential of SERT imaging using 4-[18F]-ADAM PET to estimate the long-lasting effects of METH-induced serotonergic neurotoxicity, and further determine whether a correlative relationship exists between SERT availability/activity and tyrosine hydroxylase (TH) activity in various brain regions due to the long-lasting consequences of METH treatment.ResultsMale rats received four administrations of METH (5 or 10 mg/kg, s.c.) or saline (1 ml/kg, s.c.) at 1-h intervals. At 30 days post-administration, in vivo SERT availability and activity were measured by 4-[18F]ADAM PET imaging. In contrast to the controls, the uptake of 4-[18F]ADAM in METH-treated mice was significantly reduced in a dose-dependent manner in the midbrain, followed by the hypothalamus, thalamus, striatum, hippocampus, and frontal cortex. The regional effects of METH on TH activity were assessed by quantitative immunohistochemistry and presented as integrated optical density (IOD). A significant decrease in TH immunostaining and IOD ratios was seen in the caudate, putamen, nucleus accumbens, substantia nigra pars compacta, and substantia nigra pars reticulata in the METH-treated rats compared to controls.ConclusionThe present results suggested that the long-lasting response to METH decreased the uptake of 4-[18F]-ADAM and varied regionally along with TH immunoreactivity. In addition, 4-[18F]ADAM PET could be used to detect serotonergic neuron loss and to evaluate the severity of serotonergic neurotoxicity of METH.

Abstract 120: Methamphetamine-induced Cardiomyopathy Associated With Mitochondrial Dysfunction, Cardiac Fibrosis and Hypertrophy

Introduction: Methamphetamine (METH) is one of the most commonly abused illicit drugs in the United States, exerting a range of adverse effects upon multiple organ systems. Cardiovascular complications are among the major causes of death in METH users. METH-induced cardiomyopathy is a poorly characterized disease entity as METH-induced molecular perturbations, and histopathological changes in the heart remain under-explored. Objectives: We studied histopathology in the hearts of human METH users. We also observed the histological alteration and changes in mitochondrial function in mice that received ‘binge’ administration of METH. Methods and Results: We obtained 32 autopsy heart samples from humans with positive toxicology for chronic METH use and performed Sirius Red and Masson’s Trichrome (MT) staining on left ventricular (LV) sections. Notably, chronic METH user hearts showed intense perivascular and interstitial fibrosis in LVs. ‘Binge’ METH administration in mice for 4 weeks showed an increase in heart weight-to-tibia length and increase in myocyte cross-sectional area in WGA stained LVs compared to saline-treated mice. Sirius red and MT staining also showed an increase in perivascular and interstitial fibrosis in METH mice heart. Isolated mitochondria from METH-treated mice heart showed suppressed mitochondrial bioenergetics measured by Seahorse Analyzer. Immunoblotting in heart lysates and mitochondrial fractions showed altered mitochondrial dynamics regulatory proteins expression in METH mice compared to control saline group. METH-treated cultured neonatal rat ventricular cardiomyocytes also showed suppression of mitochondrial respiration and mitochondrial network disorganization indicating a direct effect of METH on cardiomyocytes. Conclusions: We report that maladaptive cardiac fibrotic remodeling is typical in a human and pre-clinical mouse model of METH abuse. ‘Binge’ METH exposure in mice induces cardiac hypertrophy, cardiac fibrosis, and suppression of mitochondrial respiration. Thus, chronic METH use induces maladaptive cardiac remodeling associated with mitochondrial dysfunction.

Diffusion Kurtosis Imaging Detects Microstructural Changes in a Methamphetamine-Induced Mouse Model of Parkinson's Disease.

Methamphetamine (METH) abuse is known to increase the risk of Parkinson's disease (PD) due to its dopaminergic neurotoxicity. This is the rationale for the METH model of PD developed by toxic METH dosing (10mg/kg four times every 2h) which features robust neurodegeneration and typical motor impairment in mice. In this study, we used diffusion kurtosis imaging to reveal microstructural brain changes caused by METH-induced neurodegeneration. The METH-treated mice and saline-treated controls underwent diffusion kurtosis imaging scanning using the Bruker Avance 9.4 Tesla MRI system at two time-points: 5days and 1month to capture both early and late changes induced by METH. At 5days, we found a decrease in kurtosis in substantia nigra, striatum and sensorimotor cortex, which is likely to indicate loss of DAergic neurons. At 1month, we found an increase of kurtosis in striatum and sensorimotor cortex and hippocampus, which may reflect certain recovery processes. Furthermore, we performed tract-based spatial statistics analysis in the white matter and at 1month, we observed increased kurtosis in ventral nucleus of the lateral lemniscus and some of the lateral thalamic nuclei. No changes were present at the early stage. This study confirms the ability of diffusion kurtosis imaging to detect microstructural pathological processes in both grey and white matter in the METH model of PD. The exact mechanisms underlying the kurtosis changes remain to be elucidated but kurtosis seems to be a valuable biomarker for tracking microstructural brain changes in PD and potentially other neurodegenerative disorders.