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Loss of Neuronal Integrity during Progressive HIV-1 Infection of Humanized Mice: HIV invades CNS within 1-2 weeks after infection, progressive viral infection correlated with loss of neuronal integrity. - pdf attached
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The Journal of Neuroscience, 2 March 2011, 31(9)
Prasanta K. Dash,1 Santhi Gorantla,1 Howard E. Gendelman,1,4 Jaclyn Knibbe,1 George P. Casale,3 Edward Makarov,1 Adrian A. Epstein,1 Harris A. Gelbard,5,6,7,8 Michael D. Boska,2 and Larisa Y. Poluektova1 Departments of 1Pharmacology and Experimental Neuroscience, 2Radiology, 3Surgery, and 4Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska 68198, and 5Center for Neural Disease and Development, and Departments of 6Neurology, 7Pediatrics, and 8Microbiology, University of Rochester Medical Center, Rochester, New York 14642
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"Virus invades the CNS within 1-2 weeks after viral infection coincident with the peak of viremia and seen within perivascular macrophages and lymphocytes......NSG mice were transplanted at birth with CD34+ HSCs (CD34-NSG), and then infected with HIV-1......During progressive viral infection, we investigated when and to what extent neuronal injuries occur after infection onset and how such changes correspond to immune, neuronal, and glial aberrations. The data showed that progressive viral infection correlated with loss of neuronal integrity......HIV-1-associated neuropathology seen during progressive viral infection of humanized CD34-NSG mice was linked to systemic viral infection. The sequence of events consisted of viral invasion of the nervous system, glial neuroinflammatory responses, and neurodegeneration........Such changes reflect substantive neurodegeneration and are also found in the brains of HIV-1-infected individuals........After the initiation of viral infection, development of neuronal abnormalities in cerebral cortex were seen in the animals for up to 15 weeks by spectroscopic and diffusion tensor imaging and confirmed by immunohistological tests......Mouse m983 (with highest VL and lowest CD3+CD4+ cell percentage in spleen) had the most severe brain pathology. The presence of scattered lymphocytes in brain parenchyma, accumulation of human cells in meninges, and formation of perivascular cuffs around brain microvessels were found. In the pons/medulla, activation of microglial cells with nodule formation not directly associated with human cell infiltration was noted."
Abstract
Neuronal damage induced by ongoing human immunodeficiency virus type 1 (HIV-1) infection was investigated in humanized NOD/scid-IL-2Rγcnull 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.
Introduction
Human immunodeficiency virus 1-associated neurocognitive disorders (HAND) remain the most common CNS disease linked to advanced viral infection and present despite the widespread use of antiretroviral therapy (Antinori et al., 2007). Although considerable research is directed towards understanding disease pathobiology, little is known about disease onset and progression. How advanced viral infection elicits neuronal damage is linked to immune, neural cell, and tissue pathobiological events. These are critical in ultimately producing cognitive, behavioral, and motor disorders and causing disabling morbidity in infected people (McArthur et al., 2010).
Virus invades the CNS within 1-2 weeks after viral infection coincident with the peak of viremia and seen within perivascular macrophages and lymphocytes (Davis et al., 1992; Horn et al., 1998; Williams et al., 2001). During the seroconversion reaction, mild aseptic meningitis is observed in up to 20% of infections (Atwood et al., 1993; Newton, 1995; Gray et al., 1996; del Saz et al., 2008). The long-term sequelae of such events to the brain are incompletely understood (Boufassa et al., 1995; Wallace et al., 2001). Although such pathobiological events are best studied in animal models of human disease (Kraft-Terry et al., 2009), gaps in understanding human immunodeficiency virus 1 (HIV-1) neuropathogenesis parallel the limited availability of relevant model systems. As lentiviral infections are host cell specific, the mainstay for studies of human disease have centered on the use of rhesus macaques infected with simian immunodeficiency virus (SIV) (Lackner et al., 1991; Narayan et al., 1995). Although this model has yielded insights into viral pathobiology and can, in part, mimic human disease, existing limitations in terms of cost, conduct, and availability to investigators have hampered research progress. This has galvanized efforts to develop disease-relevant rodent models (Santoro et al., 1994; Toggas et al., 1994; Persidsky et al., 1996; Reid et al., 2001; Kim et al., 2003; Potash et al., 2005). Nonetheless, such models, while demonstrating relevance for studies of human disease diagnosis and therapies, have also met with limitations. These limitations include impaired graft survival and the inability to mimic progressive HIV infection and neuroAIDS, in particular. The recent development of humanized NOD/scid-IL-2Rγcnull (NSG), BALB/c-Rag2-/-γc-/-, and NOD/scid mice by transplanting with human cord blood isolated CD34+ hematopoietic stem cells (HSCs), or with fetal liver and thymus, have overcome many of these limitations. Indeed, humanized mice can facilitate studies of chronic HIV-1 infection in immune grafts that are sustained for the lifetime of the animal (for review, see Van Duyne et al., 2009). Importantly, in the field of neuroAIDS, this model may identify relationships between HIV-1 disease progression and neuropathobiology.
To these ends, NSG mice were transplanted at birth with CD34+ HSCs (CD34-NSG), and then infected with HIV-1. During progressive viral infection, we investigated when and to what extent neuronal injuries occur after infection onset and how such changes correspond to immune, neuronal, and glial aberrations. The data showed that progressive viral infection correlated with loss of neuronal integrity. As such, this model can uncover relationships between ongoing viral replication and its associated neural dysfunction, and is useful for studies of HIV-1 neuropathogenesis and therapeutic development.
Discussion
HIV-1-associated neuropathology seen during progressive viral infection of humanized CD34-NSG mice was linked to systemic viral infection. The sequence of events consisted of viral invasion of the nervous system, glial neuroinflammatory responses, and neurodegeneration. Early development of aseptic meningitis and CD4+ T-lymphocyte depletion was also observed, as reported (Gorantla et al., 2007, 2010a,b). After the initiation of viral infection, development of neuronal abnormalities in cerebral cortex were seen in the animals for up to 15 weeks by spectroscopic and diffusion tensor imaging and confirmed by immunohistological tests. These revealed decreased expression of SYN, an integral protein in presynaptic terminals, and MAP2, a marker for neuronal cell bodies and dendrites, and axonal NF in cerebral cortex. Such changes reflect substantive neurodegeneration and are also found in the brains of HIV-1-infected individuals.
Human neuropathology was previously studied in some depth during the later stages of viral infection in the absence of combination antiretroviral therapy. This is where encephalitis was commonly seen and was a result of virus-infected mononuclear phagocytes (blood-derived perivascular macrophages and microglia) and formation of multinucleated giant cells, astrogliosis, myelin pallor, and neuronal morphological changes. The latter was seen as decreased synaptic density and neuronal dropout (Price et al., 1988; Budka, 1991; Gelman, 1993; Wiley, 1995; Kraft-Terry et al., 2009). These morphological features of HIV-1-induced neurodegeneration have diminished in severity as a consequence of antiretroviral medicines (Masliah et al., 1996; Everall et al., 1999; Zheng et al., 2001; McCarthy et al., 2006).
In recent years, clinical investigators have used MRS and DTI to assess the underlying basis for cognitive dysfunction during progressive HIV-1 infection in its human host (Pomara et al., 2001; Lim and Helpern, 2002; Schifitto et al., 2009; MŸller-Oehring et al., 2010). In the present study, we used both 1H-MRS and DTI, to monitor biochemical and structural alterations, respectively, in brain regions during the progression of HIV infection in humanized mice. Our 1H-MRS tests showed significant decreases in NAA concentration in cerebral cortex. NAA, the most abundant neuron-specific metabolite reflects the degree of neuronal injury (Chong et al., 1994; Meyerhoff et al., 1994; Laubenberger et al., 1996; Simone et al., 1998; Moffett et al., 2007). Moreover, NAA concentration is often used as a marker of neuronal viability in a range of neurodegenerative disorders that includes HAND, Alzheimer's disease, epilepsy, multiple sclerosis, and spinal cord injury (Meyerhoff et al., 1993, 1994; Marcus et al., 1998; Suwanwelaa et al., 2000; Taras—w et al., 2003; Chang et al., 2004; Soares and Law, 2009; Mohamed et al., 2010). Reductions in NAA were also seen in 1H-MRS studies of SIV-infected monkeys (Lentz et al., 2005, 2008a,b; Ratai et al., 2009). Although previous studies showed changes in NAA concentrations as reflective of human cognitive impairment, their use has been hampered by variations in levels over time, comorbid conditions, and the inability to coregister histopathological correlates of affected brain regions. The decrease in NAA concentration in the cerebral cortex occurred in all four HIV-1-infected humanized mice and correlated with diminution of MAP2, SYN expression. However, in two mice with the highest levels of viral infection (m983 and m985), NF expression was also reduced. In contrast, astrogliosis, as measured by GFAP expression, was not uniformly altered. The diminished NAA concentration, seen in the cortex of the HIV-1-infected humanized mice, may not be directly related to neuronal apoptosis but rather may be an indicator of neuronal abnormalities and is supported by the findings that the biosynthetic enzyme for NAA, N-acetyl transferase, is localized in neuronal mitochondria and synaptosomal cell fractions (Moffett et al., 2007). More work certainly remains to be done to quantify the extent of neuronal damage and validate biomarkers of virus-associated neurodegenerative disease in this model system.
In addition to biochemical changes, we found structural brain tissue alterations by DTI. We observed an increase of FA and a decrease of diffusivity in several gray matter regions starting 4 wpi in animals with higher viral load and established immunopathology. DTI can assess displacement of water on the micrometer scale, yielding information about white matter fiber integrity (Takahashi et al., 2000; Assaf and Pasternak, 2008; Y. Chen et al., 2009; Gongvatana et al., 2009) as well as gray matter during CNS development (McKinstry et al., 2002; Mukherjee et al., 2002; Mukherjee and McKinstry, 2006; Neil and Inder, 2006; Yassa et al., 2010) and neurodegeneration such as Alzheimer's disease (Head et al., 2004; Rose et al., 2008; T. F. Chen et al., 2009; Cherubini et al., 2010; Kantarci et al., 2010; Sexton et al., 2010). FA is highly sensitive to microstructural changes but is not very specific to the type of changes. If in the developing brain a decrease in FA represents ongoing establishment of cortical neuronal interconnectivity, then in earlier stages of HIV-1 infection in humanized mice the changes observed could be associated with structural alteration of synaptic and dendritic processes. The reduction in expression of MAP2 and SYN and the abnormal appearance of SYN-positive structures in the cortex support our findings of FA and diffusivity values.
We also systematically studied, and in replicate manner to the bioimaging and histopathological tests, cognitive and sensorimotor functions studying NSG mice. Others' and our previous experiences in short-term rodent models of HIV-1 encephalitis were founded on Morris water maze testing (Avgeropoulos et al., 1998; Zink et al., 2002) and a water escape radial arm maze test series (Sas et al., 2007). In these works, cognitive deficits (spatial memory development and retention) were shown as was seen in aged HIV-1 gp120 transgenic mice (D'Hooge et al., 1999). However, water maze could not be easily used for our current model as NSG animals are substantively immunologically impaired and any exposure to cold water significantly increases the risk of infection and subsequent death of the animal. To overcome such impediments and to test learning and memory, we selected the Barnes circular platform task (Markowska et al., 1989). This system was used to perform several basic testing. We found that NSG mice had gender-dependent differences as males had significantly increased exploratory behavior on the platform compared with females. We also observed age-dependent decline in learning and memory in 12-month-old compared with 6-month-old males. We then compared learning and memory in unmanipulated versus animals irradiated at birth. Here, females were reconstituted at 6 months of age. The unmanipulated and irradiated/reconstituted mice did not show statistically significant differences in cognition (our unpublished observations) and suggested that such an approach would not readily yield changes in learning and memory after HIV infection. Currently, we are in the process of standardizing tests for behavioral testing in this unique model.
The only other animal system that shows the changes observed herein in humanized mice is the SIV-infected rhesus macaque (Bissel et al., 2002; Lentz et al., 2005; Scheller et al., 2005; Thompson et al., 2009). Importantly, and also operative in SIV-infected monkeys, few numbers of virus-infected and immune-competent perivascular mononuclear phagocytes can affect widespread neuronal dysfunction (Williams et al., 2001, 2005; Marcario et al., 2004; Crews et al., 2008). Nonetheless, expense and species specificity preclude the widespread use of SIV models for the study of the early stages of infection. Thus, relevant rodent models that mimic human disease are sorely needed. To our knowledge, this is the first report to show associations between 1H-MRS and DTI evidence of deficits in neuronal integrity. These deficits were confirmed by immunohistochemical evaluation. This study highlights the relevance of humanized mouse models for research on the pathobiology of human disease and underlies the future applications of noninvasively monitoring the systemic effects of antiretrovirals and adjunctive therapeutics.
Results
Human cell reconstitution and HIV-1 infection of mice
Four humanized animals were monitored prospectively for numbers of CD4+ and CD8+ cells in circulation and, at the end point of observation, in spleen. At 22 weeks of age, mice were infected with HIV-1ADA and viral load (VL) was assayed at 2, 4, 8, 12, and 15 weeks postinfection (wpi) (Fig. 1A-D); this was performed at the same time as MRS/DTI imaging. VL peaked at 4 weeks after viral infection and all infected animals had a sustained VL (1.7 x 104 to 7.6 x 105 RNA copies/ml). The decline of CD4+ cells progressed rapidly because of disease between 8 and 15 weeks (Fig. 1B) but did not change in control noninfected mice observed over the same time period (n = 30 weeks) (Fig. 1C). The rapid decrease in the CD4 count during the course of infection, forced us to terminate the study at 15 wpi. The human cell profiles in the spleens of infected animals at the termination point were also analyzed (Fig. 1E). Animals were stratified according to their immune status and peripheral VL. Animals m969 and m973 had 21.1 and 36.4% of CD3+CD4+ cells in splenic lymphoid tissue, CD4:CD8 cell ratios of 1.0 and 0.8 (lower compared with 2:1-3:1 in uninfected animals), and peripheral VLs of 1.71 x 105 and 0.65 x 105 viral RNA copies/ml, respectively. They were considered less affected by HIV-1 infection compared with two other mice m983 and m985, which had higher VLs (7.6 x 105 and 4.81 x 105 viral RNA copies/ml) and inverted CD4:CD8 ratios (0.1 and 0.4), and m983 had the lowest number (3.4%) CD3+CD4+ cells in spleen.
Brain immunopathobiology
Brain pathologies, which included influx of activated HLA-DR+ human cells, activation of microglial cells (Iba-1), and astrogliosis (GFAP) were determined by immunohistology of paraffin-embedded 5-μm-thick sections (Fig. 2). Brain regions including frontal cortex, whisker barrel (WB) somatosensory cortex (data not shown), cerebellum, corpus callosum, hippocampus, and brainstem were investigated. Mouse m983 (with highest VL and lowest CD3+CD4+ cell percentage in spleen) had the most severe brain pathology. The presence of scattered lymphocytes in brain parenchyma, accumulation of human cells in meninges, and formation of perivascular cuffs around brain microvessels were found (Fig. 2). In the pons/medulla, activation of microglial cells with nodule formation not directly associated with human cell infiltration was noted. The same markers in control uninfected humanized mice of the same age group were analyzed, and the data of one representative mouse (m975) are shown in Figure 2. Based on the CD4 cell profiles, viral load profiles, and brain pathologies observed, the animals were grouped into two categories as severe brain pathology (m983 and m985) and moderate brain pathology (m969 and m973).
1H-MRS measures
1H-MRS tests were used to determine whether changes in neuronal integrity could be present. These results were validated by immunocytochemical tests for neuronal integrity. The regions selected for spectroscopic examination using the PRESS volume selective spectroscopic acquisitions included cerebral cortex, hippocampus, cerebellum, and brainstem/pons (Fig. 3A). 1H-MRS was collected from four humanized NSG mice at different time points (0, 4, 8, 12, and 15 weeks after infection with HIV). Preinfection reconstituted (time, 0 weeks), unmanipulated humanized NSG mice aged 3 months (n = 5), and NSG mice aged 6 months (n = 3), showed no significant differences in N-acetyl aspartate (NAA) concentrations among the different brain regions studied (data not shown). The NAA concentration of uninfected mice of equivalent age (36 weeks) and time (equal to 15 weeks after infection) was 10-12 IU as measured in the cerebral cortex. The nervous system-specific metabolite NAA, which is synthesized in neurons and appears to be a key link in distinct biochemical features of CNS metabolism, was measured at the time points described above. The means ± SEMs of NAA concentration normalized to water for quantitative assessments are presented in Figure 3B. It can be clearly seen that NAA is significantly reduced over the course of infection in the cerebral cortex (Fig. 3B) but not in the cerebellum, hippocampus, or brainstem/pons regions. Moreover, one mouse (m983) showed a substantial reduction in NAA in the cerebellum (NAA, 6.85 IU) at the 15 wpi. As NAA is commonly presented as a ratio to creatine, we compared NAA levels normalized to creatine or to water. Both measures were nearly identical. Creatine remains constant over time in the animals (data not shown). Hence, presentation of NAA as either concentrations or metabolite ratios to creatine provides equivalent results in the animals.
DTI tests
The DTI measures for FA reflect changes of the tissue organization at a microscopic level by measuring water diffusion in different directions, which changes as a consequence of structural neuronal geometry. DTI examinations were performed over the same time period as 1H-MRS measurements. DTI results from the selected regions measured bilaterally from the four animals were divided based on peripheral immune status, VL, and brain immunopathology analyzed independently. Results from the severely affected mice (m983 and m985) are presented in Figure 4. Notable and consistent changes include increased FA in the frontal cortex and hippocampus as well as decreased Dav, λ||, and λ⊥ in the WB along with other cortical areas and hippocampus. Multiple gray matter regions show statistically significant changes over the course of disease development, more than were found from moderately affected mice m969 and m973 (data not shown). In the WB, there was a strong linear correlation between VL and FA, specifically when correlated magnitude of FA increase (∇) from preinfection to 15 wpi (R2 = 0.9987). The same strong trends were found for decline of Dav diffusivity (Fig. 4E,F). Similar correlations of FA and VL were observed in the pons/medulla (R2 = -0.9811) (data not shown).
Quantitative multispectral fluorescence microscopy
To determine whether in vivo reduction of NAA concentration detected by 1H-MRS and changes in DTI values correspond to changes of brain architecture, at termination of the experiment, immunofluorescence neuronal and glial antigens were analyzed by multispectral fluorescence microscopy of replicate brain regions. The images captured with the Nuance multispectral imaging system (20x objective) were converted to 12 bit grayscale images and quantified using ImagePro Plus software. Representative images of the labeled neuronal markers captured (40x objective) with the Nuance multispectral imaging system are presented in Figure 5. The cortical neurons of control animals showed normal MAP2-immunolabeled dendrites, whereas the HIV-1-infected humanized mice showed decreased expression of MAP2-immunolabeled dendrites with irregular and wavy contours. SYN labeling in cortical areas of control animals was observed as diffusely punctate, whereas SYN expression was irregularly shaped in all infected animals. Reduction of NF-positive fibers was observed in the cortex of animals with significant immune and brain pathology (m983 and m985) compared with less affected (m969 and m973) and control animals. In WB of all infected animals, the amount of NF was significantly reduced (Fig. 5). However, in the pons/medulla, these changes were not as significant as seen in cortex and WB (Fig. 5, bottom right panel).
For MAP2 and SYN, the quantitative data were expressed as total positive area/total area analyzed in square micrometers and their corresponding mean pixel density intensity (Fig. 6). For NF and GFAP, data were expressed as total area labeled. There was a diminution in the cerebral cortex and WB of MAP2 and SYN levels compared with uninfected animal but not in the pons/medulla. The most dramatic reduction of NF labeling was found in WB (Fig. 6). Observed changes allowed us to suggest that in addition to dendritic/synaptic protein aberrations, the damage represented by NF also contributed to the changes of [NAA] and MRI/DTI metrics.
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