Introduction

Non-human primates (NHPs) infected with simian immunodeficiency virus (SIV) exhibit neuropathological symptoms similar to those seen in HIV+ patients and have been employed extensively to study the mechanisms of the disease or therapeutical treatment (Apetrei et al. 2012; Haigwood 2004; Hu 2005; Staprans and Feinberg 2004; Van Rompay 2012; Williams et al. 2008). SIVsmmFGb is a novel strain of SIV, which induces neuropathology (as measured by significant levels of SIV in the brain parenchyma, multi-nucleated giant cells (MNGCs), and SIV encephalitis (SIVE)) in 90−100 % of infected pig-tailed macaques while most SIV strains in 25−40 % of infected macaques (Novembre et al. 1998; O'Neil et al. 2004; Reeve et al. 2009), suggesting that SIVsmmFGb-infected pig-tailed macaques are an excellent model system for studies on neuropathogenesis and therapeutic development.

Neurochemical alterations associated with the inflammatory process and neuronal loss have been commonly observed in previous magnetic resonance spectroscopy (MRS) studies of HIV patients (Chang 1995; Chang et al. 1999; Ernst et al. 2003, 2010; Lentz et al. 2011; Salvan et al. 1997; Tracey et al. 1996; Vion-Dury et al. 1998). Also, rhesus macaques inoculated with a SIV251 swarm (Kodama et al. 1993; Strickland et al. 2011) have been employed to investigate the metabolite alterations of brain injury during SIV infection (Fuller et al. 2004; Gonzalez et al. 2000; Greco et al. 2004; Lentz et al. 2005; Williams et al. 2005; Wu et al. 2013a, b), demonstrating that in vivo MRS is a promising approach to evaluate progressive neurochemical changes in the brain during SIV infection. Our previous studies have demonstrated that SIVsmmFGb is highly neuropathogenic in pig-tailed macaques, but the temporal evolution of brain infection remains not fully understood. In the present study, in vivo MRS was used to investigate temporal cerebral metabolite alterations in pig-tailed macaques infected with SIVsmmFGb.

Materials and methods

Pig-tailed macaques (Macaca nemestrina) (male, 4 years old, n = 5) were infected by intravenous inoculation with 100 TCID50 of the SIVsmmFGb isolate (Novembre et al. 1998; O'Neil et al. 2004).

In vivo proton MRS examination

The single voxel spectra were acquired with the point-resolved spectroscopy (PRESS) sequence with chemical shift selective (CHESS) water suppression and TR/TE = 1,500/30 ms using a circular surface coil (ID = 7 cm) on a Siemens 3 T Trio scanner (Siemens Medical Solutions, PA, USA). A volume of 6 mm × 6 mm × 6 mm (or 0.22 ml) within prefrontal gray matter (PGM) or striatum was chosen as region of interest (ROI) (Fig. 1a, b) and scanned before SIV inoculation (baseline) and at 2, 4, 8, 12, 16, 20, and 24 weeks post inoculation (wpi). For each scan, animals were anesthetized and maintained with 1–1.5 % isoflurane mixed with 100 % oxygen and the animal head was immobilized with a custom-built head holder. Vital physiological parameters including heart rate, breathing rate, rectal temperature, blood pressure, oxygen saturation, inhaled CO2, Et-CO2, were monitored continuously and maintained during scanning. Baseline MRS data were acquired in three separate scanning sessions for each animal, with at least 1 week between sessions. Animals were euthanized after their final scanning session.

Fig. 1
figure 1

Voxels of interest illustrated in a prefrontal gray matter (PGM) and b striatum (caudate and putamen) on the T 2-weighted structural images of a pig-tailed adult macaque monkey. c Representative proton MR spectrum in PGM with fitted by the LCModel software (post SIV inoculation) with the residual shown on the top. Abbreviations: Cr, total creatine; Glx, glutamate/glutamine; mI, myo-inositol; NAA, N-acetylaspartate; Cho, choline

Full size image

CD4+ and CD8+ T cell monitoring

Blood samples were collected from anesthetized animals for quantitation of circulating CD4+ and CD8+ T cell subsets, 1 day before each MRI scan and a complete blood count (CBC) with differential and lymphocyte phenotype analysis was performed. Absolute numbers and percentages of CD3+/CD4+ and CD3+/CD8+ T lymphocytes were measured, in whole blood, using four-color flow cytometry and gating on the lymphocyte population. All monoclonal antibodies (CD3, CD20, CD4, and CD8) used here were purchased from BD company as previously described (Novembre et al. 1998; O'Neil et al. 2004).

Data analysis

In vivo MRS data were processed using the LC-Model software (Provencher 1993). Total creatine (Cr, plus phosphocreatine) levels were used as an internal reference (Fuller et al. 2004; Greco et al. 2002; Haga et al. 2009; Herndon et al. 1998; Kaiser et al. 2005; Ratai et al. 2009; Wiebenga et al. 2014). Metabolite ratios including NAA/Cr, Cho/Cr, mI/Cr, and glutamate/glutamine (Glx)/Cr were calculated. Representative spectra of the post-inoculation proton MRS in PGM and striatum were shown (Fig. 1c). Also, the preinoculation data of each animal were processed separately, and the metabolite ratios with smallest standard deviation percentage in the LC-model fitting were used as the reference baseline for the following data analysis of that animal.

Statistical analysis

Analysis of variance (ANOVA) with post hoc testing was employed for the statistical analysis of repeated measurements across different time points using SPSS 20.0. Student’s t test was applied to compare the results between the baseline and that in the post-inoculation. Relationships between the percentages of T cells (or CD4/CD8 ratio) and metabolite changes were examined using linear mixed regression analysis (Blackwell et al. 2006). p values less than 0.05 were considered statistically significant. All the p values were adjusted for multiple comparisons by means of the false discovery rate (FDR) procedure (Benjamini and Hochberg 1995).

Results

CD4+ and CD8+ T cell percentage changes post SIV infection

Longitudinal changes in circulating CD4+ and CD8+ T cell percentages and numbers are illustrated in Fig. 2. CD4+ T cell percentage declined progressively as expected, differing significantly from baseline at 12, 16, 20, and 24 wpi. In the acute phase (<1 month), the CD8+ T cell percentages increased significantly and then became steady and declined slightly in the chronic phase after SIV infection (Fig. 2).

Fig. 2
figure 2

CD4+ and CD8+ T cell percentages (a) and absolute numbers (b) of the lymphocyte changes post SIV inoculation. Error bars represent standard error of the mean (SEM). *p < 0.05, compared with the baseline (pre SIV inoculation)

Full size image

Cerebral metabolite ratio changes post SIV inoculation

Compared to the baselines, the mean NAA/Cr and Glx/Cr ratios (averaged cross time post-inoculation) were reduced by 13.6 ± 6.5 % (standard deviation (SD)) and 13.1 ± 9.3 % (SD), respectively, in PGM and 13.2 ± 8.0 % (SD) and 9.6 ± 1.5 % (SD) in striatum, although these declines were not statistically significant (Fig. 3). Also, in comparison with the baselines, the mean Cho/Cr ratios remained almost unchanged in PGM and striatum while the mean mI/Cr ratios showed increasing tendency (Fig. 3).

Fig. 3
figure 3

Cerebral metabolite ratio changes (averaged across time) in prefrontal gray matter (PGM) (a) and striatum (b) after SIV inoculation. Error bars represent standard error of the mean (SEM). Metabolites include the following: N-acetylasparlate (NAA), total creatine (Cr), total choline (Cho), myo-inositol (mI), glutamate/glutamin (Glx). Total creatine (Cr) is used as internal reference

Full size image

In the longitudinal examination, ANOVA analysis indicated that there were no significant changes observed in all interested metabolites in PGM and striatum at most time points after the SIV infection except in the acute stage. Specifically, significant changes of the NAA/Cr ratio in PGM and Glx/Cr and mI/Cr ratios in striatum were observed at 2 wpi (Fig. 4a, b). Significant reduction of Cho/Cr and Glx/Cr ratios in PGM occurred at 4 and 12 wpi (Fig. 4a). The Glx/Cr ratio in PGM continued to decrease over the time, while the Glx/Cr ratio in striatum had a transient drop at 2 wpi. The NAA/Cr ratio in PMG and striatum correlated with the Glx/Cr ratio in striatum (r = 0.36, p = 0.03).

Fig. 4
figure 4

Longitudinal cerebral metabolite ratio changes in prefrontal gray matter (PGM) (a) and striatum (b) of pig-tailed macaques after SIV inoculation. Error bars represent standard error of the mean (SEM). *p < 0.05, compared with the baseline; “- - - -”, the temporal changes of metabolites/Cr significantly negatively correlated with CD8+ T cell percentage change

Full size image

Relationships of the metabolite alterations with immunologic dysfunction

As seen in Fig. 4 and Table 1, the progressive changes of the NAA/Cr ratios in PGM and striatum and the Glx/Cr ratio in striatum were correlated with the percentage of CD8+ T cells during SIV infection, as indicated by linear mixed effect regression analyses (Table 1). Standardized beta values (slopes) were significantly greater than 0 for the relationship between the NAA/Cr ratio and CD8+ percentage in PGM (F (1,13.87) = 14.55, p = 0.002) and for the relationship between the NAA/Cr ratio and CD8+ percentage in striatum (F (1,15.19) = 9.57, p = 0.007), and for the relationship between the Glx/Cr ratio and CD8+ percentage in striatum (F (1,18.85) = 16.85, p = 0.001). No significant correlation between any metabolite ratios and the CD4+ T cell depletion (or CD4/CD8 ratio) was observed.

Table 1 Standardized β value between CD4+, CD8+ T cell percentages and CD4/CD8 with cerebral metabolites in prefrontal gray matter (PGM) and striatum, *p < 0.05 after FDR correction
Full size table

Discussion

The present in vivo MRS study reveals that the cerebral metabolites in prefrontal gray matter and striatum of pig-tailed macaques are altered progressively during the SIVsmmFGb infection and largely in agreement with those seen in SIVmac251-infected rhesus macaques as well as in HIV-infected patients. Notably, the temporal metabolite changes are associated with the progressive immune dysfunction. In addition, the Glx/Cr ratio changed with different temporal profiles in striatum versus prefrontal gray matter, indicating that the glutamatergic system might be affected differently in these two regions following SIV infection. As SIVsmmFGb enters the brain in over 90 % of the infected animals, we believe that SIVsmmFGb has advantages over SIVmac251 for neuroimaging studies designed to characterize CNS injury following SIV infection of macaque monkeys.

Brain metabolite changes in PGM and striatum

N-acetyl-asparate (NAA)

NAA is a neuronal marker widely used to assess brain injury. Previous studies have demonstrated that no significant changes in NAA/Cr were seen in basal ganglia, frontal cortex, and centrum semiovale at any time point after 2 weeks in rhesus monkeys with SIVmac251 infection (Fuller et al. 2004). In contrast, the present study revealed significant reduction of NAA/Cr in PGM at 2 wpi (Fig. 4a), consistent with the NAA reduction in frontal cortical gray matter reported in prior studies of HIV+ patients in the early stages of infection (Lentz et al. 2009) and the NAA/Cr decrease in frontal cortex at 13 days post-inoculation in SIVmac251-infected macaques (Greco et al. 2004) and those seen at 12 days and 14 days post-inoculation in an ex vivo NMR study (Lentz et al. 2005). Such transient reduction of NAA/Cr correlated with the changes in synaptophysin and might reflect the reversible neuronal injury during the primary SIV infection (Lentz et al. 2005). Also, the NAA/Cr alteration is in agreement with previous finding of whole brain fractional anisotropy (FA) in SIVsmmFGb-infected macaque brains examined with diffusion tensor imaging (DTI) (Li et al. 2011).

It is of use to compare the present macaque experiment done at 3 T with the previous ones at 1.5 T. Even though current 3 T offers higher (nearly 2-fold) sensitivity than 1.5 T in in vivo MRS and a dedicated surface coil was used, the voxel size used in the present study was 6 mm × 6 mm × 6 mm (0.22 ml), much smaller (15-fold) than that (15 mm × 15 mm × 15 mm, or 3.4 ml) used in the previous rhesus macaque studies. In consideration of the spectrum signal to noise ratio (SNR) and the sample size differences, the NAA/Cr reduction at 2 weeks post-inoculation was seen at the present study with n = 5 and previous study with n = 10 (Greco et al. 2004) but not with n = 8 (Fuller et al. 2004), the present MRS results might suggest that SIVsmmFGb in pig-tailed macaques is highly neuropathogenic in comparison with SIVmac251 in rhesus macaques.

Glutamate and glutamine (Glx)

Neurons and glial cells are believed to be the primary sources of glutamate and glutamine in which glutamate is considered as the principal excitatory neurotransmitter in the brain. Disturbance of these substances could reflect dysfunction in one or both of these cell types (Lentz et al. 2009). Significant Glx reduction has been observed in the frontal cortical gray matter of HIV patients at early and chronic stages (Lentz et al. 2009) and in basal ganglia at chronic stage (Lentz et al. 2011). Also, no changes in Glx or Glx/Cr were reported in the frontal cortex and centrum semiovale, but the reduction was nearly significant in basal ganglia in previous macaque study (Ratai et al. 2009). In contrast, Glx/Cr in PGM demonstrated continuous decline, and significant reduction at 12 wpi, consistent with the changes seen in HIV patients with early and chronic infection (Lentz et al. 2009). Also, significant Glx/Cr reduction in striatum was seen in 2 wpi in the present study while such change was also observed in basal ganglia in HIV patients at chronic stage (Lentz et al. 2011), consistent with that seen in the SIVmac251-infected macaques with CD8 depletion (Ratai et al. 2009). In addition, the difference of Glx/Cr evolutions in PGM and striatum of the SIVsmmFGb-infected macaques suggests that the glutamatergic system may be affected differently in these regions probably due to the higher viral load in PGM than in striatum in the unique SIV model of neuroAIDS (O'Neil et al. 2004).

Choline (Cho)

As a marker for inflammation and membrane disruption, increased Cho levels, seen in patients with chronic HIV infection, are typically thought to represent increased lipid membrane turnover due to glial activation in response to the HIV infection (Lentz et al. 2011). This interpretation is supported by the observation of pathologic changes during early HIV and SIV infections (Gray et al. 1996; Kim et al. 2005).

Elevation in Cho has been observed in the frontal white matter of chronic HIV infection (Chang et al. 1999; Meyerhoff et al. 1999) and in Cho/Cr of the frontal cortex and white matter of HIV patients in early and chronic stage (Lentz et al. 2011). Ratai and colleagues reported that Cho and Cho/Cr increased at 2 wpi and decrease at 4 wpi in the frontal cortex and basal ganglia during SIV infection (Ratai et al. 2009). In contrast, reduction of Cho/Cr in PGM was only seen at 4 wpi in the present study.

Myo-inositol (mI)

Because mI exists primarily in glia, its elevation is considered to reflect increased glial cell activity (Brand et al. 1993). Higher mI has been commonly observed in the white matter of asymptomatic HIV+ patients (Chang et al. 2004). Also, such mI increase was seen in the frontal cortex in SIVmac251-infected macaques during acute infection (Ratai et al. 2009). In contrast, no changes in mI/Cr were reported in the frontal cortex and basal ganglia of SIVmac251-infected macaques in the early and chronic stages (Fuller et al. 2004). In the present study, mI/Cr was stable in PGM in acute stage but significantly elevated in striatum at 2 wpi and then reduced at 4 wpi, different from that seen in the SIVmac251-infected macaques (Ratai et al. 2009).

Correlation between temporal changes of brain metabolite ratios in PGM and striatum

Region-specific metabolic responses have been reported previously in SIV-infected macaques (Ratai et al. 2009). The temporal changes in NAA/Cr and Glx/Cr ratios of striatum and NAA/Cr in PGM were correlated with each other, suggesting that the temporal association of neuronal injury in the frontal cortex and basal ganglia during SIVsmmFGb infection. In contrast, significant correlation was observed in Glx but not NAA in frontal cortex and basal ganglia in the study of Ratai et al.

T cell phenotypes and their association with brain metabolite alterations

Immune system responses to HIV or SIV infection have been reported previously in both SIV and HIV immunology literature (Marcondes et al. 2001; von Geldern et al. 2007). Depletion of CD4+ T cell and accumulation of CD8+ T lymphocytes are the most significant predictor of the clinical disease severity (Chang et al. 2000; Grassi et al. 1999; Langford et al. 2007; Gray et al. 1998). Downregulation of viral replication in the brain coincides with increased CD8+ T cell infiltration, and reduced glial and macrophage activation has been observed in SIV-infected macaques (Clements et al. 2002). The results of the temporal correlations between NAA/Cr in PGM and striatum and Glx/Cr in striatum and the percentage of CD8+ T cells during the SIV infection are consistent with the findings in patients with early HIV+ infection (Lentz et al. 2009), suggesting that the recruited CD8+ T cells may damage bystander neurons during downregulated virus expression, as indicated by reduced NAA/Cr and Glx/Cr probably due to neuronal loss or dysfunction (Crews et al. 2008; De Stefano et al. 1995; Guimaraes et al. 1995; Urenjak et al. 1993).

Previous MRS studies of HIV patients showed that such relationship was driven only by the early infection cohort, and no correlation between any CD8+ T cell population and the NAA or Glx levels in chronic infection cohort were reported (Lentz et al. 2009). However, in the present study of SIV-infected monkeys, it is seen that NAA/Cr and Glx/Cr correlated with the percentage of CD8+ T cells but not with the percentage of CD4+ T cells. The difference in MRS findings between the SIV monkeys and HIV patients may be attributed to the unknown or variable factors including the mode and time course of infection and the medication in HIV patients.

Macaque models are widely used in HIV/AIDS studies and therapeutic development due to the difficulty of studying the disease in humans. The neurochemical alteration during SIV infection has previously been investigated extensively in macaques infected with SIVmac251. Here, we report similar findings in SIVsmmFGb-infected macaques. However, as demonstrated in our previous studies (Reeve et al. 2009, 2010), SIVsmmFGb induces neuropathology in over 90 % of the infected pig-tailed macaques, a much higher infection rate than seen in macaques infected with SIVmac251 and other strains (Westmoreland et al. 1998). Although the sample size (n = 5) in the present study was smaller than those used in previous MRS studies of SIVmac251-infected macaques (n = 8–18) (Fuller et al. 2004; Greco et al. 2004; Ratai et al. 2009), we were still able to detect significant metabolite changes, as described above. Further, these changes were detected in a shorter time course than reported in other models (6-month vs 24-month end point) (Fuller et al. 2004). Therefore, our results suggest that SIVsmmFGb-infected macaques are a more sensitive and responsive model than others for characterizing neuronal injury during SIV infection.

HIV infection leads to immune deficiency and a range of neurological disorders due to the infection of central nervous system and associated dementia. MRI holds great potential to evaluate the physiological, microstructural, neurochemical, and functional changes in HIV patients (Chang 1995; Chang et al. 2004; Offiah and Turnbull 2006; Shyam babu et al. 2013; Tucker et al. 2004; Zhang and Li 2013). However, to date, MRI is still not a sensitive approach in screening HIV patients without neurological symptoms (Nishijima et al. 2014). Our previous study has demonstrated the sensitivity of DTI and perfusion techniques in SIVsmmFGb-infected brains using high-field MRI (Li et al. 2011). Currently, high-field (3 T) and ultra-high field (7 T and above) MRI techniques exhibit improved image quality and expanded range of applications in preclinical and clinical studies (Duyn 2012; Oz et al. 2013). In particular, some novel diffusion, perfusion, and multi-parameter MRI methods may be used to facilitate better detection of brain injury in SIV-infected macaques (Li et al. 2014; Meng and Zhang 2014; Zhang et al. 2014). Taken together with the present MRS results at 3 T, our findings suggest that SIVsmmFGb-infected macaques could serve as an excellent platform for exploring advanced imaging techniques in HIV/AIDS studies.

Conclusions

The temporal cerebral metabolite changes during the SIVsmmFGb infection are evaluated with in vivo MRS at 3 T. The MRS results demonstrate that the metabolite alterations are largely consistent with the previous findings of neuronal injury in SIVmac251-infected rhesus monkeys and HIV patients and, in particular, associated with progressive immune dysfunction. The findings suggest that the SIVsmmFGb-infected pig-tailed macaques are an excellent model for characterizing CNS infection using novel neuroimaging approaches and exploring advanced neuroimaging techniques as well.