This is the first longitudinal neuroimaging study of human or nonhuman primates that begins before infection by an immunodeficiency virus, and continues through to the development of encephalitis. In a cohort of 8 rhesus macaques, we serially performed in vivo 1H MR spectroscopy and MRI with the goals of demonstrating whether brain injury in the SIV-infected macaque is detectable noninvasively, whether there are metabolic brain markers that predict the development of SIV encephalitis, and whether there are relationships between traditional clinical markers and brain 1H MRS abnormalities. While MRI appeared normal, novel metabolic abnormalities were observed, and they provide new insights into the response of the CNS to SIV infection that are relevant to HIV infection in humans.
H MRS changes seen shortly after infection were expected. Previous work from our laboratory documented similar findings by MRS both in vivo
] and in post mortem
]. Our observations during the subacute and chronic stages of SIV infection are new. Our most interesting finding was an elevation of Cho/Cr in the basal ganglia four weeks post-inoculation in the 2 animals destined to develop SIVE, whereas the other 6 animals had basal ganglia Cho/Cr values below baseline (figure ).
SIV-induced neuropathological changes are variable with SIV encephalitis developing in 25% of infected macaques [18
], similar to the incidence of encephalitis in HIV-infected humans, and the rate of SIVE in the present study. It is not clear why there is a spectrum of individual responses to infection with the same virus. The most likely explanations include viral mutation and the differences in host factors. Throughout the course of infection there is a high rate of mutation [21
]. Our data supports an important role for host factors; at 4 weeks pi, when minimal viral mutation is expected, we found a clear difference in the basal ganglia Cho/Cr ratio between those macaques destined to develop SIVE and the others.
We suggest that the observation of persistent basal ganglia Cho/Cr elevation during the first month after infection is a marker of host susceptibility to SIVE. If confirmed, it may have important implications for design of further research, and potentially in the clinical management of patients. To our knowledge, the only other biomarker with the potential to predict encephalitis is elevated levels of macrophage chemoattractant protein-1 (MCP-1) in the CSF for development of SIVE [25
]. However, the MCP-1 finding was observed in an accelerated SIVE model that employs 2 viruses, results in a high rate of SIVE, and is quite different from the human situation. If persistence of elevated cerebral Cho/Cr is found to also be a predictor of a neurocognitive disorder in HIV-infected patients it could become an important clinical tool. For example, it may be used to identify individuals that may benefit from early, CNS-targeted or more aggressive administration of antiviral, anti-inflammatory, and neuroprotective agents.
Prior studies have demonstrated viral neuroinvasion in the weeks following infection [20
]. Brain abnormalities have been documented by in vivo 1
H MRS [17
], in vitro 1
H MRS [15
], and neuropathology [16
] in post-mortem tissues obtained within 2 weeks of infection. In the present study, we again observed abnormalities in vivo
, specifically, elevation of frontal lobe Cho/Cr at the time of peak viremia (2 weeks pi), and a diminution of this ratio to below baseline at 4 weeks pi, after the immune system responded to the virus. An elevation of Cho/Cr is often observed in CNS inflammatory processes, and is seen in HIV infected patients before evidence of neuronal injury or neurocognitive symptoms [2
]. While a marked astrogliosis was observed in post-mortem tissue [16
], elevations of the choline-containing metabolites in the in vitro
MR spectra were not [15
]. Thus, in vivo
Cho/Cr elevation appears to be related to astrogliosis, but levels of choline-containing compounds measured by MRS in vitro
do not correlate. These findings are curious, and suggest that there is a poorly understood contribution to the Cho resonance measured by 1
H MRS in vivo
that requires further study.
Our observation of a positive linear correlation between plasma viral load and changes in frontal Cho/Cr during both the acute and chronic periods (figure ) is a new finding that may help to illuminate the CNS response to SIV. The brain viral inflammatory process induced by the virus may progress in at least 2 ways [28
]. The virus may enter the CNS shortly after infection, and continue to replicate there, producing a chronic, progressive inflammatory process that ultimately leads to neurodegeneration. Alternatively, virus may be largely cleared from the CNS after the initial viremia is controlled by the immune system, and cerebral inflammation returns only when there is loss of immunological control and recurrence of viremia. Our findings illustrated in figure support a third proposition: brain inflammation tracks, and is directly related to blood levels of the virus which may peak from time to time, even before the onset of AIDS.
We did not detect reduced NAA/Cr as a function of time after infection in this longitudinal study (figure ). We have previously observed declines in NAA/Cr 2 weeks after infection using in vivo
] and in vitro 1
H MRS [15
]. The most likely explanation is that SIVE was infrequent, and CNS disease was mild on average across the 8 animals. Thus NAA changes were too subtle to be detected in this small cohort by in vivo 1
H MRS at 1.5 T. Indeed in our previous in vivo 1
H MRS study [17
], we found changes in NAA at 2 weeks, but a total of 15 animals were studied. This conclusion is also supported by human studies. The imaging protocol we employed in this study is identical to one used in a multicenter study of HIV infected subjects [11
]. That study detected a small difference in NAA/Cr, but required 15 HIV- and 38 neurosymptomatic, HIV+ subjects [11
]. Other in vivo
MRS human studies demonstrating decreased NAA/Cr performed at 1.5 T also employed larger numbers and more advanced disease [1
Variable progression of CNS disease in individual primates may also explain our inability to detect NAA/Cr change as a function of time after infection. If CNS injury in AIDS is characterized by different rates of neuronal injury progression alternating with periods of remission, the averaging of measurements at specific time points within a small cohort would mask evidence of brain injury. We mitigated the effects of temporal averaging by comparing brain metabolite levels with plasma viral load in individual animals, and found evidence of brain injury in the subacute and chronic periods of infection. As illustrated in figures and , SIV viremia significantly correlated with elevated frontal lobe Cho/Cr and decreased basal ganglia NAA/Cr. These correlations between plasma viral load and brain metabolites during the chronic phase of SIV infection suggests that CNS injury is dependent on the presence of the virus, most likely transmitted into the CNS by the trafficking of SIV-infected monocytes [19
The issue of variable response by different brain regions in response to SIV infection merits comment. Such variation in HIV-infected individuals has been reported by many groups [4
]. We also find regional variations in the SIV infected macaque brain. We found that at 2 weeks pi the changes in Cho/Cr ratio appear greater in the frontal cortex than other areas. We do not know the situation in the human condition because such studies have not been reported in the early post infection stage. After the 2 week period, neuronal injury was more evident in basal ganglia than in other brain regions in the macaque, similar to human studies [11
]. It is possible that the tropism of the virus may change with time. That is, it is possible that early metabolic changes are prominent in the cortical regions and only later are they more prominent in the basal ganglia.