Combinatorial assessments of brain tissue metabolomics and histopathology in rodent models of human immunodeficiency virus infection
- First Online:
Metabolites are biomarkers for a broad range of central nervous system disorders serving as molecular drivers and byproducts of disease pathobiology. However, despite their importance, routine measures of brain tissue metabolomics are not readily available based on the requirements of rapid tissue preservation. They require preservation by microwave irradiation, rapid freezing or other methods designed to reduce post mortem metabolism. Our research on human immunodeficiency virus type one (HIV-1) infection has highlighted immediate needs to better link histology to neural metabolites. To this end, we investigated such needs in well-studied rodent models. First, the dynamics of brain metabolism during ex vivo tissue preparation was shown by proton magnetic resonance spectroscopy in normal mice. Second, tissue preservation methodologies were assessed using liquid chromatography tandem mass spectrometry and immunohistology to measure metabolites and neural antigens. Third, these methods were applied to two animal models. In the first, immunodeficient mice reconstituted with human peripheral blood lymphocytes then acutely infected with HIV-1. In the second, NOD scid IL2 receptor gamma chain knockout mice were humanized with CD34+ human hematopoietic stem cells and chronically infected with HIV-1. Replicate infected animals were treated with nanoformulated antiretroviral therapy (nanoART). Results from chronic infection showed that microgliosis was associated with increased myoinostitol, choline, phosphocholine concentrations and with decreased creatine concentrations. These changes were partially reversed with nanoART. Metabolite responses were contingent on the animal model. Taken together, these studies integrate brain metabolomics with histopathology towards uncovering putative biomarkers for neuroAIDS.
KeywordsFocused beam microwave irradiation Neural antigens Human immunodeficiency virus type one neuroAIDS Antigen preservation Magnetic resonance spectroscopy Metabolomics
Histopathologic assessments of neural integrity are commonly used to study neuropathogenesis following human immunodeficiency virus (HIV) infection. Both liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses of metabolites, proteins applied to cerebrospinal fluids (CSF) (Bonneh-Barkay et al. 2008; Wikoff et al. 2008; Velazquez et al. 2009) and proton magnetic resonance spectroscopy (1H MRS) tests have sought biomarkers for HIV-associated neurocognitive disorders (HAND) in humans (Lentz et al. 2011; Valcour et al. 2012) and in relevant animal models (Boska et al. 2004; Ratai et al. 2011; Dash et al. 2011). Nonetheless, such approaches have fallen short in providing diagnostic information for several reasons. First, LC-MS/MS analysis of CSF only indirectly reflects ongoing brain metabolic activity (Laspiur et al. 2007; Rozek et al. 2007; Angel et al. 2012). Second, while 1H MRS is capable of visualizing regional brain metabolites, its sensitivity is limited (Choi et al. 2007; Holt et al. 2012). There is thus a need for precise investigations into relationships between metabolites and tissue pathologies. We posit that this can be realized by combining targeted LC-MS/MS and tissue immunohistochemistry with efforts that seek optimal metabolite preservation. Such an approach would be most useful when applied to appropriate model systems in HIV-affected brain subregions.
To these ends, we combined investigations of tissue immunohistology with metabolite profiling for the characterization of disease-related events in rodent models of HIV infection and HAND. Focused beam microwave irradiation (FBMI) and rapid tissue freezing techniques were validated for their abilities to preserve brain metabolites. FBMI heat distribution was optimized for murine brain irradiation through the use of phantoms infused with temperature sensitive dyes. Flash tissue freezing was cross-validated against FBMI. We next reasoned that yet another confounder could be the animal system itself and its abilities to reflect human disease. Taking this also into consideration, we used both an acute model of HIV-1 infection using immunodeficient mice reconstituted with human peripheral blood lymphocytes (PBL) (Koyanagi et al. 1997; Gorantla et al. 2010b) and NOD scid IL2 receptor gamma chain knockout humanized mice transplanted with CD34+ hematopoietic stem cells (HSC) then chronically infected (Gorantla et al. 2010a; Dash et al. 2011) to test the genesis of virus-induced brain disease. These models tested the effects of HIV-1 infection on brain immunopathology and metabolite levels. Both models supported HIV-1 infection and showed changes in brain metabolism with concomitant astro- and micro- gliosis associated pathologies. However, only CD34+ humanized animals demonstrated associations between time-dependent losses in CD4+ T lymphocytes, virus infection, metabolite concentrations and glial activation. Importantly, each of these parameters were affected by antiretroviral therapy and supporting a wealth of prior data gathered from our laboratories during past investigation (Dash et al. 2012; Roy et al. 2012). Taken together, these studies demonstrate important technical and methodological considerations needed in assessing metabolic biomarkers of HIV-associated neuropathology.
Materials and methods
Animals, human cell reconstitutions and HIV-1 infection
NOD scid IL2 receptor gamma chain knockout, NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ, (NSG) mice (The Jackson Laboratories, Bar Harbor, Maine, USA; stock number 005557) were obtained from an established breeding colony and housed under pathogen-free conditions in accordance with ethical guidelines for care of laboratory animals at the National Institutes of Health and the University of Nebraska Medical Center. All animal manipulations were performed in laminar flow hoods.
Human peripheral blood mononuclear cells (PBMC) were separated into a monocyte-and peripheral blood lymphocyte (PBL)-enriched fractions. PBL (30 × 106 cells in 0.5 ml PBS) were injected intraperitoneally (i.p.) into 4 week old NSG mice (hu-PBL) (Gorantla et al. 2010b). HIV-1 infection group were administered HIV-1ADA (a CCR5 strain, (Gendelman et al. 1988)) i.p. at 104 × 50 % tissue culture infective dose (TCID50) on day 7 after PBL engraftment (hu-PBL-NSG HIV-1). Human pan-CD45, CD3, CD4, CD8, CD14 and CD19 markers were assayed as a six-color combination (BD Pharmingen, San Diego, California, USA) using a fluorescence-activated cell sorting (FACS) Diva (BD Immunocytometry Systems, Mountain View, California, USA) system. The percentages of CD4+ and CD8+ cells were obtained from the gate set on human CD3+ cells. Blood collected in EDTA-containing tubes, for immune cell reconstitution profiles, were determined by FACS. All mice demonstrated hu-PBL reconstitution (Roy et al. 2012). Animals were sacrificed at 21 days after virus infection.
CD34+ HSCs were obtained from fetal liver (University of Washington, Laboratory of Developmental Biology supported by NIH Award Number 5R24HD000836) using magnetic beads CD34+ selection kit (Miltenyi Biotec Inc., Auburn, California, USA). Animals were transplanted as previously described (Gorantla et al. 2010a; Dash et al. 2011). Animals were infected with HIV-1ADA i.p. at a dose of 104 TCID50 per mouse at 22 weeks following FACS validation of human immune cell reconstitution. Blood samples were collected at 2-week intervals during HIV infection in all animal groups to monitor human CD4/CD8 and viral load. The levels of viral RNA copies per ml in plasma were monitored by automated COBAS Amplicor System v1.5 (Roche Molecular Diagnostics, Basel, Switzerland). Animals were sacrificed at 18 weeks following HIV infection.
Nanoformulations using the excipient poloxamer-188 (P188; Sigma-Aldrich, St Louis, Missouri, USA), atazanavir (ATV)-sulfate (Gyma Laboratories of America Inc. Westbury, New York, USA) and free-base ritonavir (RTV) (Shengda Pharma-ceutical Co., Zhejiang, China) were prepared by high-pressure homogenization as described (Balkundi et al. 2010; Balkundi et al. 2011). Lyophilized nanoART particles were resuspended in saline and injected subcutaneous in HIV-1-infected animals at 16 weekly doses of 250 mg/kg of ATV and RTV (Dash et al. 2012; Roy et al. 2012). NanoART was initiated following 12 weeks HIV infection.
Spleen and brain were collected after euthanasia and dissected over ice. Brains were initially split with left hemisphere for paraffin embedding and right hemisphere dissected into sub-regions for LC-MS/MS. Sub-regional dissection followed anatomical boundaries to separate hemi-brains into cerebellum, brainstem, cortex, hippocampus, striatum, and midbrain. Cortex was further divided into frontal and middle sections. Control NSG mice non-reconstituted with human cells and not HIV-infected serve for testing heat stabilization of brain tissue processing.
Agar-saline-thermochromic ink mouse (ASTIM) phantoms
A single 20-gram mouse was euthanized, coated in petroleum jelly and covered in plaster to make the primary mold (ArtPlaster™, Activa Products, Inc. Marshall, TX). After drying overnight the animal was removed from the mold and latex replicas were cast from the primary plaster mold (407 Latex Casting Rubber®, EnvironMolds, Summit, NJ). These latex molds were used to make replicate agar phantoms of 2 % agar in water with 0.9 % NaCl brought to boil. When molten agar cooled to 50° concentrated thermochromic ink was added to a dilution of 5 % (Chromax Black NH K60C, LCR Hallcrest, Glenview, IL). Phantoms were made by pouring molten agar mix into the latex molds and cooled over ice.
FBMI euthansia and heat stabilization
Mice were anesthetized by inhalation of 1–2 % isoflurane in oxygen and aligned in water-jacketed animal holder for microwave irradiation in a Muromachi Microwave Fixation System (10 kW model). For phantom testing irradiation times from 400 to 700 ms were varied at 50 ms intervals at constant 4.9 kW for each buffer solution tested in the water jacket (distilled water, 0.5×, 1×, 2×, 3×, 4× PBS). Finally, these irradiation settings were tested on replicate animals followed by 1H MRS validation.
Quantitative 1H MRS measurements
Single voxel localized spectra were acquired using point resolved spectroscopy (PRESS) with high bandwidth pulses to optimize sequence performance. Spectra were acquired with a repetition time of 4 s, echo time of 33 ms, 256 averages, using volume coil transmit and surface coil receive on a 7 T/16 cm Bruker Pharmascan (Karlsure, Germany) MRI/MRS system. Single-scan, localized, unsuppressed water signals were acquired as a reference for metabolite quantification. Spectroscopic data were processed by fitting in the time domain using the QUEST algorithm (Ratiney et al. 2005) with spectra (basis set) composed of GAMMA computer models of spectra (Smith et al. 1994) using published values of frequency and coupling constants from 22 abundant metabolites found in the brain by 1H MRS (Govindaraju et al. 2000). These were normalized to water without correction for relaxation. Metabolite concentrations reported were semiquantitative. To preclude concentration corrections for relaxation, water normalized signal amplitudes were presented in institutional units (IU).
Nine Amino acids and myo-inositol (mInos) in mouse brain were quantified by LC-MS/MS conforming to previously published procedures (Bathena et al. 2012). The LC-MS/MS analyses were completed on a Waters ACQUITY ultra-performance liquid chromatography (UPLC) system (Waters, Milford, MA) coupled with a 4500 or 5500 Q TRAP® hybrid quadrupole linear ion trap mass spectrometer (Applied Biosystems, MDS Sciex, Foster City, CA, no). Briefly, brain tissues were homogenized in methanol. Aliquots of homogenate were serially diluted with HPLC grade water, spiked with internal standard (IS) solution (Glu-d5), and extracted by protein precipitation using methanol. Samples were vortexed and centrifuged at 20,000 g for 10 min. The supernatants were aspirated and evaporated under vacuum, the resulting residues were reconstituted in 50 % ACN and 10 μL of 10,000-fold diluted sample extracts were then subjected to LC-MS/MS analyses. The chromatographic separation was carried out with Atlantis® HILIC silica column (150 mm × 2.1 mm ID, 5 μm particles). Mobile phase A consisted of 0.1 % formic acid in water and mobile phase B comprised of acetonitrile. The multiple reaction monitoring (MRM) transitions used for each analyte and IS were: glutamine (Gln) 147.0/129.9, mInos 178.9/160.9, gamma-aminobutyric acid (GABA) 104.0/68.9, glutamate (Glu) 148.0/84.0, N-acetylaspartate (NAA) 176.0/133.9, aspartate (Asp) 133.9/73.9, taurine (Tau) 125.9/107.8, choline (Cho) 104.0/60.0, creatine (Cre) 131.9/87.1, phosphocholine (PCho) 184.0/86.0, and Glu-d5 (IS) 153.0/88.0. Experiments performed on AB Sciex 5500 Q TRAP utilized the following optimized MRM transitions: mInos 178.8/117.1, GABA 104.0/86.9, Glu 146.1/101.9, NAA 173.9/87.9, Asp 132.0/88.1, Cho 104.1/60.0, and Cre 132.1/90.0.
IHC Primary and Secondary Antibodies (Ab)
Mouse monoclonal clone CR3/43, 1:100
DakoCytomation, Carpinteria, CA
HRP-conjugated anti-mouse IgG
HIV-1 core Ag
Mouse monoclonal clone Kal-1, 1:10
DakoCytomation, Carpinteria, CA
HRP-conjugated anti-mouse IgG
Glial fibrillary acidic protein (GFAP)
Rabbit polyclonal, 1:1000
DakoCytomation, Carpinteria, CA
HRP-conjugated anti-rabbit IgG
Ionized calcium binding adaptor molecule 1 (Iba1)
Mouse monoclonal, 1:500
Wako Chemicals USA, Inc., Richmond, VA
HRP-conjugated anti-mouse IgG
Microtubule-associated protein 2 (MAP-2)
Neuronal soma and dendritic microtubulin
Rabbit polyclonal, 1:500
Millipore Corporation, Temecula, CA
Alexa Fluor ® 594 anti-rabbit IgG
200 kDa + 68 kDa neurofilaments (NF)
Neuronal axon intermediate filaments
Mouse monoclonal clone 2 F11, 1:200
DakoCytomation, Carpinteria, CA
Alexa Fluor ® 488 anti-mouse IgG
Vesicular glutamate transporter1 (VGlut1)
Pre-synaptic excitatory neurotranmission
Guinnea Pig polyclonal, 1:1000
Synaptic Systems, Göttingen, Germany
Alexa Fluor ® 488 anti-guinnea pig IgG
Vesicular GABA transporter1 (VGAT)
Pre-synaptic inhibitory neurotransmission
Rabbit polyclonal, 1:500
Synaptic Systems, Göttingen, Germany
Alexa Fluor ® 594 anti-rabbit IgG
synapsin1 conjugated to Oyster© 650 (Snp1)
Mouse monoclonal, 1:200
Synaptic Systems, Göttingen, Germany
Adjacent slide specimens of paraffin-embedded mouse brain were selected for immunofluoresent staining. Brain sections were treated with primary mouse monoclonal antibodies to microtubule-associated protein 2 (MAP-2), vesicular glutamate transporter 1 (VGlut1), synapsin 1 (Snp1), neurofilament (NF), and vesicular GABA transporter (VGAT). Highly cross-adsorbed secondary anti-mouse, anti guinea pig and anti-rabbit antibodies conjugated to the fluorescent probes Alexa Fluor® 488, Alexa Fluor® 488 and Alexa Fluor® 594 respectively (Invitrogen, Carlsbad, CA) were used (Table 1). Cell nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI). Slides were cover slipped with ProLong Gold anti-fade reagent (Invitrogen, Carlsbad, CA), allowed to dry for 24 h at room temp and then stored at −20 °C for future use.
Images were captured at multiple wavelengths (420–720 nm) capturing the bandwidth of chromagens and the emission spectra of the fluorescent tags. All sections were imaged with 20× objective and synaptic-related stains were acquired using 40× objective. The spectra for each chromogen or fluorophore was determined on control slides and tissue sections were analyzed by multispectral imaging/image analysis, with a brightfield/fluorescence microscope (Nikon Eclipse 55i) and Nuance FX multispectral imaging system (Cambridge Research Instruments, Worburn, MA). A spectral un-mixing algorithm (Nuance system) quantitatively separated the grayscale images representing each spectral component. The grayscale images representing optical density (OD) for brightfield chromagen intensity or fluorescence signal counts per area (mm2) (same exposure times for samples compared) was quantified as mean pixel intensity (12-bit grayscale). Area-weighted average intensity was calculated for all antibodies in triplicate for multiple regions in the brain (cortex, hippocampus, caudate, midbrain) by dividing the sum of the product of area and mean intensity, for each partitioned area, by the sum of the partitioned areas.
Group metabolite data were examined for outliers or unequal variances. Group metabolite means were tested for significant differences using one-way univariate ANOVA with PROC MIXED of SAS. Significance of HIV infection and nanoART treatment variable was evaluated by F-tests; differences among pairs of means were then evaluated with T-Tests (p ≤ 0.05 was assumed). If deemed necessary, an unequal variance ANOVA model was specified. Pairwise comparisons of means were computed and significance and confidence intervals for individual comparisons were adjusted for multiplicity with the Tukey method. All statistical significance tests were two-sided. Statistical analyses were generated with SAS/STAT software, Version 9.3 (© 2002–2010) of the SAS System for Windows (Cary, NC).
FBMI for brain tissue preservation
FBMI and 1H MRS
Employing 1H MRS allows the degree of metabolic stability to be determined by comparing post-mortem metabolite levels to those in live animals. Furthermore, the acquired metabolite profile provides confirmation of FBMI affects following previous works where 1H-MRS scanning of halothane-euthanized rat brain tissue compared to FBMI treatment demonstrated increased lactate, GABA, alanine and reduced NAA levels, representing residual enzymatic activities of energy respiration due to post mortem metabolism (de Graaf et al. 2009). Additionally, MRI also allows visualization of the tissue architecture, providing validation of the integrity of brain tissue after FBMI.
LC-MS/MS measures of brain metabolites and preservation
Comparisons between brain metabolite concentrations from NSG mice with previously reported strains
LC-MS/MS Concentration (mmol/kg)
(Pfeuffer et al. 1999) Concentration (mmol/kg) other species
(Tkac et al. 2004) concentration (mmol/kg) regional variations
(Schwarcz et al. 2003) concentration (mmol/kg) mice
2.3 ± 0.4
Total Choline (Cho + GPC + PCho)
0.3 ± 0.03 (free Cho)
Creatine (including phosphoCre)
14.9 ± 2.0
2.9 ± 0.7
12.2 ± 2.0
3.7 ± 0.7
5.9 ± 0.9
4.9 ± 0.6
0.5 ± 0.06
(included in Cho)
(included in Cho)
13.9 ± 1.6
FBMI facilitates measures of neural antigens and metabolites in hu-PBL mice
Flash freezing preservation of neural antigens and metabolites in humanized HIV-1 infected mice
In the current report, we developed techniques to preclude brain enzymatic activities leading to preservation of both neural antigens and metabolites. Such findings, have notable value for studies of neural metabolism and to develop metabolic biomarkers of tissue injury and disease pathologies. Specifically, the metabolite profiles found in specific brain regions provide early predictive monitoring of HAND as well as to follow therapeutic interventions. As a diagnosis of HAND is currently made by the exclusion of other co-morbid conditions of the CNS, these works are certainly timely and of clinical relevance (Valcour et al. 2011).
Quantitative LC-MS/MS evaluation of amino acids and mInos in brain tissues was made possible by both FBMI-treatment and flash freezing tissue preservation techniques. This provides a metabolite signature comparable to 1H MRS metabolite stability measurements (de Graaf et al. 2009) and validated by high resolution magic angle spinning NMR (Detour et al. 2011). Importantly, the targeted metabolite profiling assays performed show the role of tissue preservation dependent on processing techniques. Furthermore, comparisons between histochemical assessments of neuroinflammation and metabolite profiling provided confirmation of disease processes, yet specific correlations between IHC and LC-MS/MS data did not yield specific correlations (data not shown). Indeed, such findings indicate the sensitivity of metabolomic research to neuroinflammatory processes where cell processes are disrupted before structural alterations are evident. Moreover, the metabolite concentrations are representative of entire brain regions while IHC is limited to individual slices. Future studies would explore other methods to link molecular biochemical processes during neuroinflammation utilizing methods such as in situ hybridization, laser capture microdissection and/or cytokine profiling for measurements of cell phenotype across many brain sections. Additionally, we anticipate that future studies will even better validate metabolite quantitation between these methodologies to refine the quantitative potential of 1H MRS and provide even more exact measurements of a broad range of brain metabolites with post-mortem LC-MS/MS validation at the experimental endpoint in rodent models of disease.
FBMI was pursued as a lead technique to preserve brain metabolites as it has been proven effective in preventing the degradation of compounds after animal sacrifice. Indeed, studies linking behavior changes with energy metabolism and neurotransmitter deficits in animal models of Huntington Disease (Lucas et al. 2012; Mochel et al. 2012) and Alzheimer’s (Francis et al. 2012) have relied on FBMI euthanasia to identify early disease events. Moreover, it has proven successful in stabilizing one of the most labile compounds in the brain, adenosine triphosphate (ATP). Indeed, efforts to obtain accurate ATP levels from brain tissue have led to the conclusion that ATP level stability can be seen in vivo only by high power (10 kW) FBMI and through freeze-blow procedures (Delaney and Geiger 1996). However the freeze-blow technique, which involves blowing the brain tissue onto a very cold plate that prevents enzyme degradation, does not allow subsequent histological analyses. Apropos of the FBMI procedures and as performed in rats heated to 85 °C, high power were believed needed to keep total heating time ≤1 s (Delaney and Geiger 1996). In the current study and as performed in mice, irradiation times of 0.6 s were below this threshold. Comparisons of the quantitative values obtained by LC-MS/MS showed that they were within the range of what was previously reported (Table 2).
What was clear is that metabolomic studies require attention to post mortem metabolism. Specifically, neural enzymatic activities seen as a consequence of hypoxic injury clearly affect metabolomics results. Studies using any disease animal model system must thus be evaluated for confounding effects due to tissue preparation (Nomura et al. 2011). Prior metabolomic studies while relying on rapid tissue preparations controlled for post mortem metabolism but did not report effects of tissue preparations (Cho et al. 2012; Fujieda et al. 2012; Patti et al. 2012). This study confirmed post-mortem metabolite dynamics and links between brain histology and metabolite profiles. This was done in relevant animal models of HIV-1 infection of the nervous system. Two model systems were used. In the first, HIV infection of hu-PBL-NSG mice was chosen for initial investigations. The model readily generates high levels of viral infection but is limited in the fact that mice survive for periods of several weeks due to lethal graft-versus-host disease. Even utilizing FBMI and ex vivo 1H MRS we failed to show substantive group metabolomic differences. Indeed, histological analysis of these brains revealed significant variation in regional neuroimmune responses within reconstituted and in HIV infected animals. These variations may be explained by the differential severities of graft versus host disease with human PBL reconstitution (King et al. 2009). While the model proved helpful for developing the techniques employed, we also realized that if any disease biomarker signature would be obtained from such metabolomics approaches a more robust animal model would need be developed and employed in study. This was found by HIV infection of humanized mice. In this model system human CD34+ cells are engrafted in newborn mice and reconstitute both innate and adaptive arms of the human immune system (Gorantla et al. 2012). Most importantly, humanized mice maintain human cell engraftment for over a year with minimal graft versus host disease. Furthermore, HIV infection leads to neuroimmune response (Gorantla et al. 2010a) and has been shown in our past works to provide a model system for developing new antiretroviral therapies (Dash et al. 2012; Nischang et al. 2012). Employing the model we were able to demonstrate, for the first time, CNS metabolite alterations in brain regions of infected mice. Specific metabolite changes following chronic (18 weeks) infection paralleled microglial profiles in the hippocampus and were, in part, resolved by nanoART-induced suppression of viral loads (Fig. 7). Similar increases of mInos/Cre and Cho/Cre were reported in brain subregions of HIV infected humans (Lentz et al. 2011) and SIV infected macaques (Greco et al. 2004). Additionally, hippocampal neuroinflammation was reported in rats peripherally administered IL-2 in an anxiety model showing concordance between myoinositol and microgliosis (Schneider et al. 2012).
We have shown that reductions of viral load readily observed by nanoART paralleled similar reductions of hippocampal microgliosis and myoinositol. However, the levels of Cho, PCho and Cre were reduced with HIV infection but did not return to baseline as was present for uninfected control animals. We suspect that such findings represent more permanent neurological damage seen as a consequence of the sustained high viral loads and acquired before treatment. This is also reflective of patients where initial neurocognitive deficits, although in part reversed by ART, can affect later cognitive function (Gendelman et al. 1998; Cysique et al. 2009). Additionally, the blood brain barrier limits ATV and RTV efficacy for the brain and the maintenance of the viral reservoir may limit neural recovery. This has previously been shown in studies comparing ART regimens according to a CNS penetrance index where neurocognitive recovery is linked to the drug regimen (Cysique et al. 2011). Though circulating levels of HIV RNA are significantly reduced with 6 weekly injections of nanoART (Dash et al. 2012) other inflammatory factors may perpetuate the reactive phenotype of neural cells (Kraft-Terry et al. 2009; Yadav and Collman 2009). Finally, such limitations in metabolite recovery may reflect the hu-CD34-NSG HIV-1 mouse model itself. Differences in donor human cells are one source of variability and levels of reconstitution are yet another. A third are immune effects seen by the genetic non-obese diabetic background, scid mutation, common cytokine gamma chain knockout, or within the chimeric immune system itself (Gong et al. 2011).
This study, in part, employed rapid flash freezing while controlling for tissue preparation dissection time. Future studies including FBMI euthanasia and profiling metabolite levels will be approached by targeted bioanalytical sample analyses reported here to validate sample preparation before untargeted metabolomic analyses (Maher et al. 2011; Zgoda-Pols et al. 2011) in conjunction with tissue morphological changes. Such studies will identify the biochemical neuroinflammatory pathways that influence synaptic dynamics. Moreover, such multidisciplinary studies including early and late time points of infection, behavioral studies and combinations of nanoART with neuroprotective adjuvants are certain to yield further insights into neuroimmune processes and the biochemical pathways mediating neurodegeneration. All together, the combinations of rapid tissue preparation for careful histological analyses that employ 1H MRS and LC-MS/MS conjointly permit the acquisition of data not previously possible for biomarker discoveries. Such approaches also go a long way in substantiating the humanized mouse model system for studies of HIV neuropathogenesis as well as providing new predictive insights for disease.
We thank Shantanu Balkundi for providing the nanoART materials and Edward Makarov, Jacklyn Knibbe, Tanuja Gutti, Sidra P Akhter, Melissa Mellon, Lindsay Rice, and Nagsen Gautam for technical assistance.
- Angel TE, Jacobs JM, Spudich SS, Gritsenko MA, Fuchs D, Liegler T, Zetterberg H, Camp DG 2nd, Price RW, Smith RD (2012) The cerebrospinal fluid proteome in HIV infection: change associated with disease severity. Clinical proteomics 9(1):3. doi:10.1186/1559-0275-9-3 PubMedCrossRefPubMedCentralGoogle Scholar
- Balkundi S, Nowacek AS, Roy U, Martinez-Skinner A, McMillan J, Gendelman HE (2010) Methods development for blood borne macrophage carriage of nanoformulated antiretroviral drugs. J Visual Exp (46). doi:10.3791/2460
- Balkundi S, Nowacek AS, Veerubhotla RS, Chen H, Martinez-Skinner A, Roy U, Mosley RL, Kanmogne G, Liu X, Kabanov AV, Bronich T, McMillan J, Gendelman HE (2011) Comparative manufacture and cell-based delivery of antiretroviral nanoformulations. Int J Nanomedicine 6:3393–3404. doi:10.2147/IJN.S27830 PubMedPubMedCentralGoogle Scholar
- Bathena SP, Huang J, Epstein AA, Gendelman HE, Boska MD, Alnouti Y (2012) Rapid and reliable quantitation of amino acids and myo-inositol in mouse brain by high performance liquid chromatography and tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 893–894:15–20. doi:10.1016/j.jchromb.2012.01.035 PubMedCrossRefPubMedCentralGoogle Scholar
- Bonneh-Barkay D, Bissel SJ, Wang G, Fish KN, Nicholl GC, Darko SW, Medina-Flores R, Murphey-Corb M, Rajakumar PA, Nyaundi J, Mellors JW, Bowser R, Wiley CA (2008) YKL-40, a marker of simian immunodeficiency virus encephalitis, modulates the biological activity of basic fibroblast growth factor. Am J Pathol 173(1):130–143. doi:10.2353/ajpath.2008.080045 PubMedCrossRefPubMedCentralGoogle Scholar
- Boska MD, Mosley RL, Nawab M, Nelson JA, Zelivyanskaya M, Poluektova L, Uberti M, Dou H, Lewis TB, Gendelman HE (2004) Advances in neuroimaging for HIV-1 associated neurological dysfunction: Clues to the diagnosis, pathogenesis and therapeutic monitoring. Curr HIV Res 2(1):61–78PubMedCrossRefGoogle Scholar
- Cho KI, Searle K, Webb M, Yi H, Ferreira PA (2012) Ranbp2 haploinsufficiency mediates distinct cellular and biochemical phenotypes in brain and retinal dopaminergic and glia cells elicited by the Parkinsonian neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Cell Mol Life Sci 69(20):3511–3527. doi:10.1007/s00018-012-1071-9 PubMedCrossRefPubMedCentralGoogle Scholar
- Cysique LA, Vaida F, Letendre S, Gibson S, Cherner M, Woods SP, McCutchan JA, Heaton RK, Ellis RJ (2009) Dynamics of cognitive change in impaired HIV-positive patients initiating antiretroviral therapy. Neurology 73(5):342–348. doi:10.1212/WNL.0b013e3181ab2b3b PubMedCrossRefPubMedCentralGoogle Scholar
- Dash PK, Gendelman HE, Roy U, Balkundi S, Alnouti Y, Mosley RL, Gelbard HA, McMillan J, Gorantla S, Poluektova LY (2012) Long-acting nanoformulated antiretroviral therapy elicits potent antiretroviral and neuroprotective responses in HIV-1-infected humanized mice. AIDS 26(17):2135–2144. doi:10.1097/QAD.0b013e328357f5ad PubMedCrossRefGoogle Scholar
- Dash PK, Gorantla S, Gendelman HE, Knibbe J, Casale GP, Makarov E, Epstein AA, Gelbard HA, Boska MD, Poluektova LY (2011) Loss of neuronal integrity during progressive HIV-1 infection of humanized mice. J Neurosci 31(9):3148–3157. doi:10.1523/JNEUROSCI.5473-10.2011 PubMedCrossRefPubMedCentralGoogle Scholar
- Francis BM, Yang J, Hajderi E, Brown ME, Michalski B, McLaurin J, Fahnestock M, Mount HT (2012) Reduced tissue levels of noradrenaline are associated with behavioral phenotypes of the TgCRND8 mouse model of Alzheimer’s disease. Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology 37(8):1934–1944. doi:10.1038/npp.2012.40 CrossRefGoogle Scholar
- Gendelman HE, Orenstein JM, Martin MA, Ferrua C, Mitra R, Phipps T, Wahl LA, Lane HC, Fauci AS, Burke DS et al (1988) Efficient isolation and propagation of human immunodeficiency virus on recombinant colony-stimulating factor 1-treated monocytes. J Exp Med 167(4):1428–1441PubMedCrossRefGoogle Scholar
- Gendelman HE, Zheng J, Coulter CL, Ghorpade A, Che M, Thylin M, Rubocki R, Persidsky Y, Hahn F, Reinhard J Jr, Swindells S (1998) Suppression of inflammatory neurotoxins by highly active antiretroviral therapy in human immunodeficiency virus-associated dementia. J Infect Dis 178(4):1000–1007PubMedCrossRefGoogle Scholar
- Gorantla S, Makarov E, Finke-Dwyer J, Castanedo A, Holguin A, Gebhart CL, Gendelman HE, Poluektova L (2010a) Links between progressive HIV-1 infection of humanized mice and viral neuropathogenesis. Am J Pathol 177(6):2938–2949. doi:10.2353/ajpath.2010.100536 PubMedCrossRefPubMedCentralGoogle Scholar
- Gorantla S, Makarov E, Roy D, Finke-Dwyer J, Murrin LC, Gendelman HE, Poluektova L (2010b) Immunoregulation of a CB2 receptor agonist in a murine model of neuroAIDS. Journal of neuroimmune pharmacology: The official journal of the Society on NeuroImmune Pharmacology 5(3):456–468. doi:10.1007/s11481-010-9225-8 CrossRefGoogle Scholar
- Greco JB, Westmoreland SV, Ratai EM, Lentz MR, Sakaie K, He J, Sehgal PK, Masliah E, Lackner AA, Gonzalez RG (2004) In vivo 1H MRS of brain injury and repair during acute SIV infection in the macaque model of neuroAIDS. Magnetic resonance in medicine: Official journal of the Society of Magnetic Resonance in Medicine/Society of Magnetic Resonance in Medicine 51(6):1108–1114. doi:10.1002/mrm.20073 CrossRefGoogle Scholar
- King MA, Covassin L, Brehm MA, Racki W, Pearson T, Leif J, Laning J, Fodor W, Foreman O, Burzenski L, Chase TH, Gott B, Rossini AA, Bortell R, Shultz LD, Greiner DL (2009) Human peripheral blood leucocyte non-obese diabetic-severe combined immunodeficiency interleukin-2 receptor gamma chain gene mouse model of xenogeneic graft-versus-host-like disease and the role of host major histocompatibility complex. Clin Exp Immunol 157(1):104–118. doi:10.1111/j.1365-2249.2009.03933.x PubMedCrossRefPubMedCentralGoogle Scholar
- Koyanagi Y, Tanaka Y, Kira J, Ito M, Hioki K, Misawa N, Kawano Y, Yamasaki K, Tanaka R, Suzuki Y, Ueyama Y, Terada E, Tanaka T, Miyasaka M, Kobayashi T, Kumazawa Y, Yamamoto N (1997) Primary human immunodeficiency virus type 1 viremia and central nervous system invasion in a novel hu-PBL-immunodeficient mouse strain. J Virol 71(3):2417–2424PubMedPubMedCentralGoogle Scholar
- Laspiur JP, Anderson ER, Ciborowski P, Wojna V, Rozek W, Duan F, Mayo R, Rodriguez E, Plaud-Valentin M, Rodriguez-Orengo J, Gendelman HE, Melendez LM (2007) CSF proteomic fingerprints for HIV-associated cognitive impairment. J Neuroimmunol 192(1–2):157–170. doi:10.1016/j.jneuroim.2007.08.004 PubMedCrossRefPubMedCentralGoogle Scholar
- Maher AD, Cysique LA, Brew BJ, Rae CD (2011) Statistical integration of 1H NMR and MRS data from different biofluids and tissues enhances recovery of biological information from individuals with HIV-1 infection. Journal of proteome research 10(4):1737–1745. doi:10.1021/pr1010263 PubMedCrossRefGoogle Scholar
- Nischang M, Sutmuller R, Gers-Huber G, Audige A, Li D, Rochat MA, Baenziger S, Hofer U, Schlaepfer E, Regenass S, Amssoms K, Stoops B, Van Cauwenberge A, Boden D, Kraus G, Speck RF (2012) Humanized mice recapitulate key features of HIV-1 infection: a novel concept using long-acting anti-retroviral drugs for treating HIV-1. PLoS One 7(6):e38853. doi:10.1371/journal.pone.0038853 PubMedCrossRefPubMedCentralGoogle Scholar
- Nomura DK, Morrison BE, Blankman JL, Long JZ, Kinsey SG, Marcondes MC, Ward AM, Hahn YK, Lichtman AH, Conti B, Cravatt BF (2011) Endocannabinoid hydrolysis generates brain prostaglandins that promote neuroinflammation. Science 334(6057):809–813. doi:10.1126/science.1209200 PubMedCrossRefPubMedCentralGoogle Scholar
- Ratai EM, Annamalai L, Burdo T, Joo CG, Bombardier JP, Fell R, Hakimelahi R, He J, Lentz MR, Campbell J, Curran E, Halpern EF, Masliah E, Westmoreland SV, Williams KC, Gonzalez RG (2011) Brain creatine elevation and N-Acetylaspartate reduction indicates neuronal dysfunction in the setting of enhanced glial energy metabolism in a macaque model of neuroAIDS. Magnetic resonance in medicine: Official journal of the Society of Magnetic Resonance in Medicine/Society of Magnetic Resonance in Medicine 66(3):625–634. doi:10.1002/mrm.22821 CrossRefGoogle Scholar
- Roy U, McMillan J, Alnouti Y, Gautum N, Smith N, Balkundi S, Dash P, Gorantla S, Martinez-Skinner A, Meza J, Kanmogne G, Swindells S, Cohen SM, Mosley RL, Poluektova L, Gendelman HE (2012) Pharmacodynamic and antiretroviral activities of combination nanoformulated antiretrovirals in HIV-1-infected human peripheral blood lymphocyte-reconstituted mice. J Infect Dis 206(10):1577–1588. doi:10.1093/infdis/jis395 PubMedCrossRefPubMedCentralGoogle Scholar
- Schwarcz A, Natt O, Watanabe T, Boretius S, Frahm J, Michaelis T (2003) Localized proton MRS of cerebral metabolite profiles in different mouse strains. Magnetic resonance in medicine: Official journal of the Society of Magnetic Resonance in Medicine/Society of Magnetic Resonance in Medicine 49(5):822–827. doi:10.1002/mrm.10445 CrossRefGoogle Scholar
- Tkac I, Henry PG, Andersen P, Keene CD, Low WC, Gruetter R (2004) Highly resolved in vivo 1H NMR spectroscopy of the mouse brain at 9.4 T. Magnetic resonance in medicine: Official journal of the Society of Magnetic Resonance in Medicine/Society of Magnetic Resonance in Medicine 52(3):478–484. doi:10.1002/mrm.20184 CrossRefGoogle Scholar
- Valcour V, Chalermchai T, Sailasuta N, Marovich M, Lerdlum S, Suttichom D, Suwanwela NC, Jagodzinski L, Michael N, Spudich S, van Griensven F, de Souza M, Kim J, Ananworanich J (2012) Central nervous system viral invasion and inflammation during acute HIV infection. J Infect Dis 206(2):275–282. doi:10.1093/infdis/jis326 PubMedCrossRefPubMedCentralGoogle Scholar
- Zgoda-Pols JR, Chowdhury S, Wirth M, Milburn MV, Alexander DC, Alton KB (2011) Metabolomics analysis reveals elevation of 3-indoxyl sulfate in plasma and brain during chemically-induced acute kidney injury in mice: Investigation of nicotinic acid receptor agonists. Toxicol Appl Pharmacol 255(1):48–56. doi:10.1016/j.taap.2011.05.015 PubMedCrossRefGoogle Scholar
Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.