To our knowledge this is the first neuropathology and neuroimaging study to address whether CD8+ lymphocyte depletion without SIV infection affects brain metabolism and/or results in neuronal injury/astrogliosis. Our results demonstrate that none of the CD8-depleted uninfected macaques (n=4) developed symptoms of AIDS or SIVE, while all infected animals developed AIDS and SIVE (n=4). In the current study, we performed MRS to determine whether metabolic changes detected by MRS could be attributable to CD8 depletion. The noninvasive nature of in vivo
MRS permits data acquisition at multiple time points, eliminating the need for serial sacrifice studies and minimizing the number of animals needed. Nevertheless, there are limitations to the quality of in vivo
spectra, which are usually obtained at low field strengths. To improve the sensitivity in this study, we performed MRS on a high magnetic field (7 Tesla) scanner to ensure we could detect metabolic changes due to CD8 depletion, if they were present. It has been shown that higher field strengths yield better signal-to-noise ratio, higher spectral resolution, and therefore, improved quantification precision [8
]. MRS revealed that neither NAA/Cr nor NAA changed significantly after treatment with anti-CD8 antibody. Similarly, glial markers Cho and MI did not show significant alteration upon CD8 depletion. The lack of observable deviations from baseline in metabolite levels indicates that CD8+ lymphocyte depletion does not significantly impact brain metabolism.
Previously, our group has shown that SIV infection and CD8 depletion result in a rapid decline in NAA/Cr; after eight weeks SIV infection and CD8 depletion, NAA/Cr levels were significantly lower compared to pre-infection values in all brain regions we examined [26
]. In the acute stage of SIV infection without CD8 depletion, we observed transient and reversible NAA/Cr changes in the frontal cortex [10
], which led us to speculate that longitudinal analysis of NAA/Cr could be a marker of the dynamic processes involved in neuronal injury and repair. Additionally, we found that NAA/Cr correlated best with synaptophysin compared to MAP2 and neuronal density during this reversible neuronal injury in the early stages of disease [16
]. Furthermore, NAA/Cr accurately corresponds to degree of CNS inflammation; specifically the magnitude of decrease in NAA/Cr ratio has been found to be associated with severity of SIVE [17
]. Hence, NAA/Cr is believed to be a very sensitive, reliable marker of neuronal injury and dysfunction.
Typically, increases in Cho and MI are considered to reflect increases in glial activity [2
]. In the SIV-infected CD8-depleted macaque model, we found a temporal increase in Cho and Cho/Cr at 2 weeks post infection (wpi), and a subsequent decrease to baseline and below 4 wpi, consistent with the acute phase of SIV infection [10
]. At later stages, 8 wpi, Cho and Cho/Cr increased once again, suggesting a second increase of gliosis. MI level appears to be elevated at 2 and 4 wpi, and subsequently decreases back to baseline levels [26
In addition, we conducted histopathological examination to evaluate the pre-synaptic and dendritic integrity of tissue from the frontal cortex. Decreases in the expression of neuronal markers synaptophysin (SYN), an integral protein in presynaptic terminals, and microtubule-associated protein 2 (MAP2), a marker for neuronal cell bodies and dendrites, have been shown to reflect the severity of neurodegeneration [22
]. Consistent with previous findings [26
], we observed significantly decreased levels of SYN in SIV-infected animals compared to the two uninfected cohorts. Synaptophysin and MAP2 expression in uninfected CD8-depleted animals and uninfected controls showed no statistically significant difference upon comparison, confirming our MRS findings that suggested CD8+ lymphocyte depletion alone does not have measureable effects on neuronal integrity. Glial fibrillary acidic protein (GFAP) is rapidly synthesized by astrocytes in response to neurologic insult, and therefore, serves as a reliable marker of astrogliosis. The GFAP levels measured in the SIV+/CD8- animals were significantly higher than those in the uninfected cohorts, suggesting that astrocytes in the SIV+/CD8- animals suffered an insult as a result of SIV infection and consequently underwent astrogliosis. These results are also consistent with our previous findings [9
]. On the other hand, we observed no significant variation in GFAP levels in the uninfected, CD8-depleted cohort compared to uninfected controls, suggesting that CD8+ lymphocyte depletion alone has no significant effect on astrocytic processes in the macaque brain. In addition, we have demonstrated that the CD8-depleted control animals did not show significant evidence of microgliosis based on ionized calcium binding adaptor molecule 1 (IBA-1) immunohistochemical analysis. Immunohistochemical detection of CD8+ lymphocytes in brain in all three cohorts revealed rare to occasional CD8+ cells.
In conclusion, MR spectroscopy revealed that metabolite concentrations and ratios do not change with anti-CD8 treatment. IHC revealed no changes in neuronal integrity as well as no astroglial response due solely to anti-CD8 treatment. These findings confirm the validity of this accelerated rhesus macaque model of neuroAIDS and also prove that despite viral manipulation of the immune system, brain metabolism is preserved.