Previous studies have shown that neurodegeneration in HIV infection is accompanied by damage to synaptic terminals  and that synaptic injury might be an early event which may precede neuronal cell loss. For this purpose synaptophysin immunoreactivity was evaluated in the frontal lobes of the same control and infected macaques using paraffin embedded material and computer aided microdensitometry. The results are shown in Fig 3b. A clear trend (P < 0.08) of decreased (approximately 20%) synaptophysin immunoreactivity was observed at 14 days. Statistically significant differences in synaptophysin immunoreactivity were observed in chronically infected macaques with an approximate 30% decrease (P < 0.005). These data demonstrate that, in the SIV–macaque model, injury to the synaptic terminals might be an early pathogenic event.
Finally, to determine if the neuronal damage in the brains of SIV-infected macaques was associated with reactive astrogliosis, patterns and levels of GFAP immunoreactivity were investigated in paraffin embedded tissue using optical densitometry. Compared to controls (Fig. 2d), at 14 days post-SIV infection the frontal cortex of the macaque showed the presence of abundant hypertrophied protoplasmic GFAP immunoreactive astroglial cells (Fig. 2e). At 2 years post-infection abundant fibrillary astroglial cells were observed (Fig. 2f). The results of the densitometric analysis are shown in Fig. 3c. A twofold increase in GFAP immunoreactivity with respect to controls was demonstrated in both the 14 day post-infection animals (P < 0.00005), and those animals chronically infected with SIV (P < 0.001). A previous study also found increased GFAP immunoreactivity in the cortex of SIV-infected macaques suffering from cognitive and/or motor impairment – a condition with onset long after infection . In contrast, this study finds a large increase in GFAP immunoreactivity almost immediately after infection. We conclude that SIV infection causes activation of astrocytes very shortly after systemic infection with SIV, and that this astrogliosis persists in chronically infected animals.
The SIV-infected macaque is an excellent animal model for studying the pathogenesis of HIV infection in general and the neuropathogenesis of HIV infection in particular. Although neuronal injury has not been studied extensively in the SIV–macaque model, neuronal loss in the hippocampus has been reported in one limited study . SIV is the closest known relative of HIV and, like HIV, it infects CD4 macrophages, lymphocytes and microglia. Most importantly, systemic infection with HIV and SIV result in similar neuropathology [39,45,46]. Notably, neuroinvasion occurs early in infection for both HIV [23,24] and in the SIV–macaque model of AIDS [25,26]. However, the relationship between acute neuroinvasion and cellular injury in the brain has not been established.
The decreases in NAA, calbindin, and synaptophysin immunoreactivity observed 2 weeks after SIV infection corresponds to a period of peak viremia, neuroinvasion, increased numbers of perivascular macrophages, and elevation of quinolinic acid [25–31]. The changes we have observed probably reflect a response to these events. Subsequently, as the host mounts an immune response, there is a decrease in viral load and a temporary reversal of these abnormalities. It is possible that changes in NAA, calbindin and synaptophysin may follow a similar course, and thus, may represent reversible neuronal injury.
Our finding here of a progression in the loss of neuronal and synaptic markers in the SIV-infected macaque at an average of 2 years after systemic SIV infection attests to the significant neuronal injury sustained in this model. Of note, we further observed that NAA and calbindin were reduced whether or not the brain exhibited evidence of classic SIVE. This finding corroborates reports of neuronal loss in human brain in the absence of HIV encephalitis [5–12] and points to indirect mechanisms as the cause of brain injury after infection with HIV or SIV. It is important to point out, however, that this does not imply that the virus is not necessary as in the SIV-infected macaque the virus can be detected in almost all infected animals using highly sensitive methods such as in situ hybridization and PCR.
The striking elevation of GFAP that was observed in the macaque 14 days after SIV infection indicates a substantive reaction by the brain to the early neuroinvasion by the virus. Increased expression of GFAP is the hallmark of reactive gliosis, which occurs in response to virtually any damage or disturbance to the CNS. Gliosis is the most frequent reaction to brain insult occurring in a variety of diseases including viral infections, infarction, trauma, spongiform encephalopathies, and neurodegenerative diseases. GFAP elevation and gliosis is commonly observed in the late stages of HIV infection. Notably, early GFAP elevation has been suggested in the rare autopsy studies in acutely HIV-infected people who died of other causes [47,48]. The role of astrogliosis in the pathogenesis HIV related cognitive disorders is unknown but may be important, and may be explored further with the SIV–macaque model.
Neuroinvasion has been found as early as 14 days after infection in the SIV–macaque model and in rare cases among HIV-infected humans. CNS damage occurs subsequently, but before the onset of cognitive deficits. These studies show that damage can be found as early as 14 days after infection and appear to be progressive.
A relationship between acute and chronic brain injury is likely, although this requires further investigation. Even if the early brain injury represents reversible injury, it is plausible that the underlying neurotoxic mechanisms are similar for both early and late injury. If this can be demonstrated, then the acutely infected macaque may be ideal for the study of the pathogenesis of the late motor and cognitive manifestations of HIV infection.
Our findings are especially relevant to recent physiological studies of the SIV–macaque model. It has been reported that macaques demonstrated disruptions in circadian rhythm, reductions of motor activity and changes in auditory-evoked potential latencies in the weeks following infection with SIV . Most of these physiological abnormalities were reversible. The injury to neurons that we report here may explain these abnormalities.
The magnitude of changes in NAA that we have measured suggests that MRS may be used to observe this phenomenon in vivo. If our observations of an early decrease in NAA in unprocessed macaque brain at 2 weeks post-SIV infection are confirmed in vivo, the macaque model may be used to test hypotheses related to the various proposed mechanisms of neuronal injury directly. In vivo studies are currently underway in our laboratory. Furthermore, this model may be used with MRS to test pharmacotherapies to prevent or reverse neuronal injury that may produce the neurocognitive abnormalities resulting from HIV infection in humans, especially if it can be demonstrated that the early neuronal effects we have observed are related to late neuronal injury.
The authors wish to thank Ms. Joane Fordham for help in the preparation of this manuscript.
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