Influence of age, irradiation and humanization on NSG mouse phenotypes.

Humanized mice are frequently utilized in bench to bedside therapeutic tests to combat human infectious, cancerous and degenerative diseases. For the fields of hematology-oncology, regenerative medicine, and infectious diseases, the immune deficient mice have been used commonly in basic research efforts. Obstacles in true translational efforts abound, as the relationship between mouse and human cells in disease pathogenesis and therapeutic studies requires lengthy investigations. The interplay between human immunity and mouse biology proves ever more complicated when aging, irradiation, and human immune reconstitution are considered. All can affect a range of biochemical and behavioral functions. To such ends, we show age- and irradiation-dependent influences for the development of macrocytic hyper chromic anemia, myelodysplasia, blood protein reductions and body composition changes. Humanization contributes to hematologic abnormalities. Home cage behavior revealed day and dark cycle locomotion also influenced by human cell reconstitutions. Significant age-related day-to-day variability in movement, feeding and drinking behaviors were observed. We posit that this data serves to enable researchers to better design translational studies in this rapidly emerging field of mouse humanization.

Associations between brain microstructures, metabolites, and cognitive deficits during chronic HIV-1 infection of humanized mice.

BACKGROUND: Host-species specificity of the human immunodeficiency virus (HIV) limits pathobiologic, diagnostic and therapeutic research investigations to humans and non-human primates. The emergence of humanized mice as a model for viral infection of the nervous system has overcome such restrictions enabling research for HIV-associated end organ disease including behavioral, cognitive and neuropathologic deficits reflective of neuroAIDS. Chronic HIV-1 infection of NOD/scid-IL-2Rgcnull mice transplanted with human CD34+ hematopoietic stem cells (CD34-NSG) leads to persistent viremia, profound CD4+ T lymphocyte loss and infection of human monocyte-macrophages in the meninges and perivascular spaces. Murine cells are not infected with virus. METHODS: Changes in mouse behavior were measured, starting at 8 weeks after viral infection. These were recorded coordinate with magnetic resonance spectroscopy metabolites including N-acetylaspartate (NAA), creatine and choline. Diffusion tensor magnetic resonance imaging (DTI) was recorded against multispectral immunohistochemical staining for neuronal markers that included microtubule associated protein-2 (MAP2), neurofilament (NF) and synaptophysin (SYN); for astrocyte glial fibrillary acidic protein (GFAP); and for microglial ionized calcium binding adaptor molecule 1 (Iba-1). Oligodendrocyte numbers and integrity were measured for myelin associated glycoprotein (MAG) and myelin oligodendrocyte glycoprotein (MOG) antigens. RESULTS: Behavioral abnormalities were readily observed in HIV-1 infected mice. Longitudinal open field activity tests demonstrated lack of habituation indicating potential for memory loss and persistent anxiety in HIV-1 infected mice compared to uninfected controls. End-point NAA and creatine in the cerebral cortex increased with decreased MAG. NAA and glutamate decreased with decreased SYN and MAG. Robust inflammation reflected GFAP and Iba-1 staining intensities. DTI metrics were coordinate with deregulation of NF, Iba-1, MOG and MAG levels in the whisker barrel and MAP2, NF, MAG, MOG and SYN in the corpus callosum. CONCLUSIONS: The findings are consistent with some of the clinical, biochemical and pathobiologic features of human HIV-1 nervous system infections. This model will prove useful towards investigating the mechanisms of HIV-1 induced neuropathology and in developing novel biomarkers and therapeutic strategies for disease.

Brain region mapping using global metabolomics.

Historically, studies of brain metabolism have been based on targeted analyses of a limited number of metabolites. Here we present an untargeted mass spectrometry-based metabolomic strategy that has successfully uncovered differences in a broad array of metabolites across anatomical regions of the mouse brain. The NSG immunodeficient mouse model was chosen because of its ability to undergo humanization leading to numerous applications in oncology and infectious disease research. Metabolic phenotyping by hydrophilic interaction liquid chromatography and nanostructure imaging mass spectrometry revealed both water-soluble and lipid metabolite patterns across brain regions. Neurochemical differences in metabolic phenotypes were mainly defined by various phospholipids and several intriguing metabolites including carnosine, cholesterol sulfate, lipoamino acids, uric acid, and sialic acid, whose physiological roles in brain metabolism are poorly understood. This study helps define regional homeostasis for the normal mouse brain to give context to the reaction to pathological events.

Improved visualization of neuronal injury following glial activation by manganese enhanced MRI.

Research directed at anatomical, integrative and functional activities of the central nervous system (CNS) can be realized through bioimaging. A wealth of data now demonstrates the utility of magnetic resonance imaging (MRI) towards unraveling complex neural connectivity operative in health and disease. A means to improve MRI sensitivity is through contrast agents and notably manganese (Mn²âº). The Mn²âº ions enter neurons through voltage-gated calcium channels and unlike other contrast agents such as gadolinium, iron oxide, iron platinum and imaging proteins, provide unique insights into brain physiology. Nonetheless, a critical question that remains is the brain target cells serving as sources for the signal of Mn²âº enhanced MRI (MEMRI). To this end, we investigated MEMRI's abilities to detect glial (astrocyte and microglia) and neuronal activation signals following treatment with known inflammatory inducing agents. The idea is to distinguish between gliosis (glial activation) and neuronal injury for the MEMRI signal and as such use the agent as a marker for neural activity in inflammatory and degenerative disease. We now demonstrate that glial inflammation facilitates Mn²âº neuronal ion uptake. Glial Mn²âº content was not linked to its activation. MEMRI performed on mice injected intracranially with lipopolysaccharide was associated with increased neuronal activity. These results support the notion that MEMRI reflects neuronal excitotoxicity and impairment that can occur through a range of insults including neuroinflammation. We conclude that the MEMRI signal enhancement is induced by inflammation stimulating neuronal Mn²âº uptake.

Combinatorial assessments of brain tissue metabolomics and histopathology in rodent models of human immunodeficiency virus infection.

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.

Loss of neuronal integrity during progressive HIV-1 infection of humanized mice.

Neuronal damage induced by ongoing human immunodeficiency virus type 1 (HIV-1) infection was investigated in humanized NOD/scid-IL-2Rγ(c)(null) mice transplanted at birth with human CD34-positive hematopoietic stem cells. Mice infected at 5 months of age and followed for up to 15 weeks maintained significant plasma viral loads and showed reduced numbers of CD4(+) T-cells. Prospective serial proton magnetic resonance spectroscopy tests showed selective reductions in cortical N-acetyl aspartate in infected animals. Diffusion tensor imaging revealed structural changes in cortical gray matter. Postmortem immunofluorescence brain tissue examinations for neuronal and glial markers, captured by multispectral imaging microscopy and quantified by morphometric and fluorescence emission, showed regional reduction of neuronal soma and synaptic architectures. This was evidenced by loss of microtubule-associated protein 2, synaptophysin, and neurofilament antigens. This study is the first, to our knowledge, demonstrating lost neuronal integrity after HIV-1 infection in humanized mice. As such, the model permits studies of the relationships between ongoing viral replication and virus-associated neurodegeneration.