Lack of neuroinflammation in the HIV-1 transgenic rat: an [(18)F]-DPA714 PET imaging study.

BACKGROUND: HIV-associated neuroinflammation is believed to be a major contributing factor in the development of HIV-associated neurocognitive disorders (HAND). In this study, we used micropositron emission tomography (PET) imaging to quantify neuroinflammation in HIV-1 transgenic rat (Tg), a small animal model of HIV, known to develop neurological and behavioral problems. METHODS: Dynamic [(18)F]DPA-714 PET imaging was performed in Tg and age-matched wild-type (WT) rats in three age groups: 3-, 9-, and 16-month-old animals. As a positive control for neuroinflammation, we performed unilateral intrastriatal injection of quinolinic acid (QA) in a separate group of WT rats. To confirm our findings, we performed multiplex immunofluorescent staining for Iba1 and we measured cytokine/chemokine levels in brain lysates of Tg and WT rats at different ages. RESULTS: [(18)F]DPA-714 uptake in HIV-1 Tg rat brains was generally higher than in age-matched WT rats but this was not statistically significant in any age group. [(18)F]DPA-714 uptake in the QA-lesioned rats was significantly higher ipsilateral to the lesion compared to contralateral side indicating neuroinflammatory changes. Iba1 immunofluorescence showed no significant differences in microglial activation between the Tg and WT rats, while the QA-lesioned rats showed significant activation. Finally, cytokine/chemokine levels in brain lysates of the Tg rats and WT rats were not significantly different. CONCLUSION: Microglial activation might not be the primary mechanism for neuropathology in the HIV-1 Tg rats. Although [(18)F]DPA-714 is a good biomarker of neuroinflammation, it cannot be reliably used as an in vivo biomarker of neurodegeneration in the HIV-1 Tg rat.

Decreased norepinephrine transporter availability in obesity: Positron Emission Tomography imaging with (S,S)-[(11)C]O-methylreboxetine.

OBJECTIVES: Noradrenergic dysfunction is implicated in obesity. The norepinephrine transporter (NET) regulates the synaptic availability of norepinephrine. However, NET availability has not been previously characterized in vivo in obese people using Positron Emission Tomography (PET) imaging. Here we report findings evaluating NET availability in individuals with obesity and matched lean (i.e., normal weight) comparison subjects. METHODS: Seventeen obese but otherwise healthy individuals with a mean±SD body mass index (BMI) of 34.7±2.6 and 17 lean individuals with a mean±SD BMI of 23.1±1.4 were studied using a high-resolution research tomograph (HRRT) and (S,S)-[(11)C]O-methylreboxetine ([(11)C]-MRB), a radioligand selective for the NET. The regional brain NET binding potential (BPND) was estimated by the multilinear reference tissue model 2 (MRTM2) with the occipital cortex as a reference region. BPND for regions of interest were obtained with the Automated Anatomic Labeling (AAL) template registered to individual's structural MR scans. RESULTS: Obese individuals had lower NET BPND values in the thalamus (p<0.038, 27% reduction) including within the pulvinar (p<0.083, 30% reduction), but not in the hypothalamus, locus coeruleus or the raphe nuclei, compared to lean individuals. When age was included as a covariate, the difference in NET BPND values remained significant in the thalamus (p<0.025) and pulvinar (p<0.042). CONCLUSIONS: These results indicate that NET availability is decreased in the thalamus, including the pulvinar, in obese individuals. These findings further support data indicating noradrenergic dysfunction in obesity and suggest impaired NE clearance in obesity.

Imaging dopaminergic dysfunction as a surrogate marker of neuropathology in a small-animal model of HIV.

The dopaminergic system is especially vulnerable to the effects of human immunodeficiency virus (HIV) infection, rendering dopaminergic deficits early surrogate markers of HIV-associated neuropathology. We quantified dopamine D2/3 receptors in young HIV-1 transgenic (Tg) (n  =  6) and age-matched control rats (n  =  7) and adult Tg (n  =  5) and age-matched control rats (n  =  5) using [18F]fallypride positron emission tomography (PET). Regional uptake was quantified as binding potential (BPND) using the two-tissue reference model with the cerebellum as the reference. Time-activity curves were generated for the ventral striatum, dorsal striatum, thalamus, and cerebellum. Whereas BPND values were significantly lower in the ventral striatum (p < .001) and dorsal striatum (p  =  .001) in the adult Tg rats compared to controls rats, they were significantly lower only in the dorsal striatum (p < .05) in the young rats. Tg rats had smaller striatal volumes on magnetic resonance imaging. We also found lower expression levels of tyrosine hydroxylase on immunohistochemistry in the Tg animals. Our findings suggest that progressive striatal D2/3 receptor deficits occur in Tg rats as they age and can be detected using small-animal PET imaging. The effectiveness of various approaches in preventing or halting this dopaminergic loss in the Tg rat can thus be measured preclinically using [18F]fallypride PET as a molecular imaging biomarker of HIV-associated neuropathology.

Test-retest reproducibility of [11C]-(+)-propyl-hexahydro-naphtho-oxazin positron emission tomography using the bolus plus constant infusion paradigm.

We examined the reproducibility of using the constant infusion paradigm for equilibrium measurement of D2/3 receptors using [11C]-(+)-propyl-hexahydro-naphtho-oxazin (PHNO) positron emission tomography (PET). Six subjects were scanned with a bolus plus constant infusion (Kbol = 80 minutes) of [11C]-(+)-PHNO. Binding potential (BPND) was computed using the equilibrium approach and compared to a simplified reference tissue model (SRTM). The rate of change in the concentration-activity curve from 60 to 90 minutes was -5 ± 13%/h in the caudate, putamen, substantia nigra, thalamus, and cerebellum but was 15 ± 15%/h in the ventral striatum and pallidum. Test-retest variability was lower in striatal compared to extrastriatal regions (4 ± 8% vs -8 ± 22%, respectively) using the equilibrium approach, with comparable results with SRTM. The equilibrium ratio and SRTM yielded reliable BPND estimates (intraclass correlation coefficient = 0.88 and 0.82, respectively). These studies support the reproducibility of the bolus plus constant infusion paradigm with [11C]-(+)-PHNO PET.

P-glycoprotein influence on the brain uptake of a 5-HT(2A) ligand: [(18)F]MH.MZ.

BACKGROUND/AIMS: The serotonergic system, especially the 5-HT(2A) receptor, is involved in various diseases and conditions. We have recently developed a new [(18)F]-5-HT(2A) receptor ligand using an analogue, MDL 100907, as a basis for molecular imaging with positron emission tomography. This tracer, [(18)F]MH.MZ, has been shown to be an adequate tool to visualize the 5-HT(2A) receptors in vivo. However, [(18)F]altanserin, similar in chemical structure, is a substrate of efflux transporters, such as P-glycoprotein (P-gp), of the blood-brain barrier, thus limiting its availability in the central nervous system. The aim of this study was to determine whether transport by P-gp influences the distribution ratio of [(18)F]MH.MZ in the frontal cortex. METHODS: The approach was based on P-gp knockout mice which were compared with wild-type mice under several conditions. In vivo pharmacokinetic and microPET investigations were carried out. RESULTS: All analyses showed that [(18)F]MH.MZ entered the brain and was sensitive to P-gp transport. In P-gp knockout mice, brain concentrations of MH.MZ were about 5-fold higher than in wild-type animals which is reflected by a 2-fold increase in standardized uptake values of [(18)F]MH.MZ in the frontal cortex of P-gp knockout mice. CONCLUSION: Our results give evidence for a functional role of transport mechanisms at the blood-brain barrier, specifically of P-gp, and its subregional distribution. Investigation of these mechanisms will benefit the development of more efficient radioligands and drugs for molecular imaging and pharmacotherapy of the mentally ill.

Neuroinflammatory Changes in Relation to Cerebrospinal Fluid Viral Load in Simian Immunodeficiency Virus Encephalitis.

The exact cause of neurocognitive dysfunction in HIV-positive patients despite successful control of the infection in the periphery is not completely understood. One suggested mechanism is a vicious cycle of microglial activation and release of proinflammatory chemokines/cytokines that eventually leads to neuronal loss and dysfunction. However, the exact role of microglial activation in the earliest stages of the infection with high cerebrospinal fluid (CSF) viral loads (VL) is unclear. In this study, we imaged the translocator protein (TSPO), a mitochondrial membrane receptor known to be upregulated in activated microglia and macrophages, in rhesus macaques before and multiple times after inoculation with a neurotropic simian immunodeficiency virus (SIV) strain (SIVsm804E), using 18F-DPA714 positron emission tomography (PET). The whole-brain standardized uptake values of TSPO at equilibrium reflecting total binding (SUVT) and binding potentials (BPND) were calculated and correlated with CSF and serum markers of disease, and a corresponding postmortem immunostaining analysis was also performed. SUVT was found to be inversely correlated with both CSF VL and monocyte chemoattractant protein 1 (MCP-1) levels. In SIV-infected macaques with very high CSF VL at necropsy (>106 copies/ml), we found decreased TSPO binding by PET, and this was supported by immunostaining which showed glial and neuronal apoptosis rather than microglial activation. On the other hand, with only moderately elevated CSF VL (∼104 copies/ml), we found increased TSPO binding as well as focal and diffuse microglial activation on immunostaining. Our results in the SIV-infected macaque model provide insights into the relationship between HIV neuropathology and CSF VL at various stages of the disease.IMPORTANCE Neurological and cognitive problems are a common complication of HIV infection and are prevalent even in treated individuals. Although the molecular processes underlying brain involvement with HIV are not completely understood, inflammation is suspected to play a significant role. Our work presents an in vivo assessment of neuroinflammation in an animal model of HIV, the simian immunodeficiency virus (SIV)-infected rhesus macaque. Using positron emission tomography (PET) imaging, we identified changes in brain inflammation after inoculation with SIV over time. Interestingly, we found decreased binding of the PET ligand in the presence of very high cerebrospinal fluid (CSF) viral loads. These findings were supported by immunostaining which showed marked glial loss instead of inflammation. This study provides insight into glial and neuronal changes associated with very high CSF viral load and could reflect similar changes occurring in HIV-infected patients.

The effects of inhaled acetone on place conditioning in adolescent rats.

INTRODUCTION: Acetone is an ubiquitous ingredient in many household products (e.g., glue solvents, air fresheners, adhesives, nail polish, and paint) that is putatively abused; however, there is little empirical evidence to suggest that acetone alone has any abuse liability. Therefore, we systematically investigated the conditioned response to inhaled acetone in a place conditioning apparatus. METHOD: Three groups of male, Sprague-Dawley rats were exposed to acetone concentrations of 5000, 10,000 or 20,000 ppm for 1 h in a conditioned place preference apparatus alternating with air for 6 pairing sessions. A place preference test ensued in an acetone-free environment. To test the preference of acetone as a function of pairings sessions, the 10,000 ppm group received an additional 6 pairings and an additional group received 3 pairings. The control group received air in both compartments. Locomotor activity was recorded by infrared photocells during each pairing session. RESULTS: We noted a dose response relationship to acetone at levels 5000-20,000 ppm. However, there was no correlation of place preference as a function of pairing sessions at the 10,000 ppm level. Locomotor activity was markedly decreased in animals on acetone-paired days as compared to air-paired days. CONCLUSION: The acetone concentrations we tested for these experiments produced a markedly decreased locomotor activity profile that resemble CNS depressants. Furthermore, a dose response relationship was observed at these pharmacologically active concentrations, however, animals did not exhibit a positive place preference.

Metabolic and behavioral deficits following a routine surgical procedure in rats.

To test the hypothesis that functional metabolic deficits observed following surgical brain injury are associated with changes in cognitive performance in rodents, we performed serial imaging studies in parallel with behavioral measures in control animals and in animals with surgical implants. Memory function was assessed using the novel object recognition (NOR) test, administered 3 days prior to and 3, 7, 14 and 56 days after surgery. At each time point, general locomotion was also measured. Metabolic imaging with 18F-fluorodeoxyglucose ([18F]FDG) occurred 28 and 58 days after surgery. Animals with surgical implants performed significantly worse on tests of object recognition, while general locomotion was unaffected by the implant. There was a significant decrease in glucose uptake after surgery in most of the hemisphere ipsilateral to the implant relative to the contralateral hemisphere. At both time points, the most significant metabolic deficits occurred in the primary motor cortex (-25%; p<0.001), sensory cortex (-15%, p<0.001) and frontal cortex (-12%; p<0.001). Ipsilateral areas further from the site of insertion became progressively worse, including the sensory cortex, dorsal striatum and thalamus. These data was supported by a voxel-based analysis of the PET data, which revealed again a unilateral decrease in [18F]FDG uptake that extended throughout the ipsilateral cortex and persisted for the duration of the 58-day study. Probe implantation in the striatum results in a widespread and long-lasting decline in cortical glucose metabolism together with a persistent, injury-related deficit in the performance of a cognitive (object recognition) task in rats.

Metabolic correlates of toluene abuse: decline and recovery of function in adolescent animals.

RATIONALE: Children and adolescents will readily abuse household products that contain solvents such as toluene. It is likely that reinforcing exposures to toluene alter brain glucose metabolism. OBJECTIVE: Using an animal model of drug reinforcement, we sought to identify a metabolic signature of toluene abuse in the adolescent rodent brain. Small animal PET (microPET), in combination with the glucose analog radiotracer, (18)FDG, were used to evaluate the metabolic consequences of inhaled toluene. METHODS: The exposure protocol paralleled our previously established method for assessing the conditioned reinforcing effects of toluene (5,000 ppm) using the conditioned place preference (CPP) paradigm. Animals were scanned at baseline and 2 h after the last exposure. Follow-up (18)FDG scans occurred 1 day, 3 weeks, and 2 months later. RESULTS: After six pairings, 38% of the animals preferred the toluene paired chamber and 25% were averse. The immediate metabolic effect in toluene-exposed animals was a 20% decline in whole brain (18)FDG uptake. Twenty-four hours following the last exposure, the whole brain decline was 40%, and 2 months later, the decline was 30% of pretoluene levels. A region-by-region analysis demonstrated significant additional decreases in the pons, cerebellum, striatum, midbrain, temporal cortex, and hippocampus. Two months after toluene cessation, regions of complete metabolic recovery were the thalamus and cerebellum; however, the temporal cortex did not recover. CONCLUSIONS: Brain uptake of (18)FDG appears to be a useful tool for examining the metabolic impact of toluene abuse, which include a profound decline followed by region-specific recovery after cessation.

Concentration-dependent conditioned place preference to inhaled toluene vapors in rats.

OBJECTIVES: Toluene is present in many commercial products and is subject to abuse by inhalation. The goal of this study was to extend previous reports indicating that rats will exhibit a positive conditioned place preference to inhaled toluene vapors and to determine the dose-response relationship for inhaled toluene in terms of exposure concentration and number of exposures. For the conditioned place preference experiments rats were exposed to toluene vapors at concentrations of 800, 2000, 3000 or 5000 ppm in one compartment of a three-compartment box. RESULTS: Following six conditioning sessions with toluene, a significant place preference was obtained at 2000 and 3000 ppm, but not at 800 or 5000 ppm. Extending the number of toluene pairings at the 2000 and 3000 ppm concentration to 12 significantly enhanced the place preference compared to that at six pairings. CONCLUSIONS: These experiments extend our previous finding that rats will show a conditioned place preference to inhaled toluene, and indicate that a reinforcing "dose" of toluene depends on both the concentration and number of pairings.

Neurobehavioral Abnormalities in the HIV-1 Transgenic Rat Do Not Correspond to Neuronal Hypometabolism on 18F-FDG-PET.

Motor and behavioral abnormalities are common presentations among individuals with HIV-1 associated neurocognitive disorders (HAND). We investigated whether longitudinal motor and behavioral performance in the HIV-1 transgenic rat (Tg), a commonly used neuro-HIV model, corresponded to in vivo neuronal death/dysfunction, by using rotarod and open field testing in parallel to [18F] 2-fluoro-2-deoxy-D-glucose (FDG) positron emission tomography (PET). We demonstrated that age-matched non-Tg wild type (WT) rats outperformed the HIV-1 Tg rats at most time points on rotarod testing. Habituation to rotarod occurred at 8 weeks of age (fifth weekly testing session) in the WT rats but it never occurred in the Tg rats, suggesting deficits in motor learning. Similarly, in open field testing, WT rats outperformed the Tg rats at most time points, suggesting defective exploratory/motor behavior and increased emotionality in the Tg rat. Despite the neurobehavioral abnormalities, there were no concomitant deficits in 18F-FDG uptake in Tg rats on PET compared to age-matched WT rats and no significant longitudinal loss of FDG uptake in either group. The negative PET findings were confirmed using 14C- Deoxy-D-glucose autoradiography in 32 week-old Tg and WT rats. We believe that the neuropathology in the HIV-1 Tg rat is more likely a consequence of neuronal dysfunction rather than overt neurodegeneration/neuronal cell death, similar to what is seen in HIV-positive patients in the post-ART era.

Characterization of neuropathology in the HIV-1 transgenic rat at different ages.

The transgenic HIV-1 rat (Tg) is a commonly used neuroHIV model with documented neurologic/behavioral deficits. Using immunofluorescent staining of the Tg brain, we found astrocytic dysfunction/damage, as well as dopaminergic neuronal loss/dysfunction, both of which worsening significantly in the striatum with age. We saw mild microglial activation in young Tg brains, but this decreased with age. There were no differences in neurogenesis potential suggesting a neurodegenerative rather than a neurodevelopmental process. Gp120 CSF levels exceeded serum gp120 levels in some animals, suggesting local viral protein production in the brain. Further probing of the pathophysiology underlying astrocytic injury in this model is warranted.

Imaging addiction with PET: is insight in sight?

Neurochemical imaging studies can identify molecular targets of abused drugs and link them to the underlying pathology associated with behaviors such as drug dependence, addiction and withdrawal. positron emission tomography (PET) is opening new avenues for the investigation of the neurochemical disturbances underlying drug abuse and addiction and the in vivo mechanisms by which medications might ameliorate these conditions. PET can identify vulnerable human populations, treatment strategies and monitor treatment efficacy. Thus, with this tool and the knowledge it provides, the potential for developing novel drugs and treatment strategies for drug addiction is now close at hand.

Diffusion tensor and volumetric magnetic resonance measures as biomarkers of brain damage in a small animal model of HIV.

BACKGROUND: There are currently no widely accepted neuro-HIV small animal models. We wanted to validate the HIV-1 Transgenic rat (Tg) as an appropriate neuro-HIV model and then establish in vivo imaging biomarkers of neuropathology, within this model, using MR structural and diffusion tensor imaging (DTI). METHODS: Young and middle-aged Tg and control rats were imaged using MRI. A subset of middle-aged animals underwent longitudinal repeat imaging six months later. Total brain volume (TBV), ventricular volume (VV) and parenchymal volume (PV = TBV-VV) were measured. Fractional anisotropy (FA) and mean diffusivity (MD) values of the corpus callosum (CC) were calculated from DTI data. RESULTS: TBV and PV were smaller in Tg compared to control rats in young and middle-aged cohorts (p<0.0001). VV increased significantly (p = 0.005) over time in the longitudinal Tg cohort. There were lower FA (p<0.002) and higher MD (p<0.003) values in the CC of middle-aged Tg rats compared to age-matched controls. Longitudinally, MD significantly decreased over time in Tg rats (p<0.03) while it did not change significantly in the control cohort over the same period of time (p>0.05). CONCLUSIONS: We detected brain volume loss in the Tg rat, probably due to astrocytic dysfunction/loss, loss of structural/axonal matrix and striatal neuronal loss as suggested by immunofluorescence. Increased MD and decreased FA in the CC probably reflect microstructural differences between the Tg and Control rats which could include increased extracellular space between white matter tracts, demyelination and axonal degeneration, among other pathologies. We believe that the Tg rat is an adequate model of neuropathology in HIV and that volumetric MR and DTI measures can be potentially used as biomarkers of disease progression.

Dopamine D3 receptor alterations in cocaine-dependent humans imaged with [¹¹C](+)PHNO.

BACKGROUND: Evidence from animal models and postmortem human studies points to the importance of the dopamine D3 receptor (D3R) in cocaine dependence (CD). The objective of this pilot study was to use the D3R-preferring radioligand [(11)C](+)PHNO to compare receptor availability in groups with and without CD. METHODS: Ten medically healthy, non-treatment seeking CD subjects (mean age 41 ± 8) in early abstinence were compared to 10 healthy control (HC) subjects (mean age 41 ± 6) with no history of cocaine or illicit substance abuse. Binding potential (BPND), a measure of available receptors, was determined with parametric images, computed using the simplified reference tissue model (SRTM2) with the cerebellum as the reference region. RESULTS: BPND in CD subjects was higher in D3R-rich areas including the substantia nigra ((SN) 29%; P=0.03), hypothalamus (28%; P=0.02) and amygdala (35%; P=0.03). No between-group differences were observed in the striatum or pallidum. BPND values in the SN (r=+0.83; P=0.008) and pallidum (r=+0.67; P=0.03) correlated with years of cocaine use. CONCLUSIONS: Between-group differences suggest an important role for dopaminergic transmission in the SN, hypothalamus and amygdala in CD. Such findings also highlight the potential relevance of D3R as a medication development target in CD.

Imaging dopamine release with Positron Emission Tomography (PET) and (11)C-raclopride in freely moving animals.

We investigated an imaging strategy that provides simultaneous measurements of radiotracer binding and behavior in awake, freely moving animals. In this strategy, animals are injected intravenously (i.v.) through a catheterized line and permitted to move freely for 30 min during uptake of the imaging agent, in this case 11C-raclopride. After this Awake Uptake period, animals are anesthetized and scanned for 25 min. We tested the utility of this strategy for measuring changes in striatal 11C-raclopride binding under control conditions (awake and freely moving in the home cage) and with several drug challenges: a loading dose of unlabeled raclopride, pretreatment with methamphetamine (METH) or pretreatment with gamma-vinyl-GABA [S+-GVG] followed by METH. An additional group of animals underwent a stress paradigm that we have previously shown increases brain dopamine. For drug challenge experiments, the change in 11C-raclopride binding was compared to data from animals that were anesthetized for the uptake period ("Anesthetized Uptake") and full time activity curves were used to calculate 11C-raclopride binding. Regardless of the drug treatment protocol, there was no difference in 11C-raclopride striatum to cerebellum ratio between the Awake versus the Anesthetized Uptake conditions. Awake and Anesthetized groups demonstrated over 90% occupancy of dopamine receptors with a loading dose of cold raclopride, both groups demonstrated approximately 30% reduction in 11C-raclopride binding from METH pretreatment and this effect was modulated to the same degree by GVG under both uptake conditions. Restraint during Awake Uptake decreased 11C-raclopride binding by 29%. These studies support a unique molecular imaging strategy in which radiotracer uptake occurs in freely moving animals, after which they are anesthetized and scanned. This imaging strategy extends the applicability of small animal PET to include functional neurotransmitter imaging and the neurochemical correlates of behavioral tasks.