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Published online before print March 29, 2005, 10.1148/radiol.2352040003
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(Radiology 2005;235:461-468.)
© RSNA, 2005

Experimental Studies

Quantitative Neuropathologic Correlates of Changes in Ratio of N-acetylaspartate to Creatine in Macaque Brain1

Margaret R. Lentz, PhD, John P. Kim, ScB, Susan V. Westmoreland, VMD, Jane B. Greco, PhD, Robert A. Fuller, BS, Eva M. Ratai, PhD, Julian He, MD, Prabhat K. Sehgal, BVSc, Elkan F. Halpern, PhD, Andrew A. Lackner, DVM, PhD, Eliezer Masliah, MD and R. Gilberto González, MD, PhD

1 From the NMR Center and Neuroradiology Division, Massachusetts General Hospital, 55 Fruit St, GRB 285, Boston, MA 02114-2696 (M.R.L., J.P.K., J.B.G., R.A.F., E.M.R., J.H., E.F.H., R.G.G.); New England Primate Research Center, Southborough, Mass (S.V.W., P.K.S.); Department of Neurosciences, University of California, San Diego, La Jolla, Calif (E.M.); and Tulane National Primate Research Center, Tulane University Health Sciences Center, Covington, La (A.A.L.). Received January 2, 2004; revision requested March 3; final revision received July 8; accepted July 28. Supported by NIH grants RR13213 (R.G.G.), NS34626 (R.G.G.), NS30769 (A.A.L.), MH61192 (A.A.L.), MH45294 (E.M.), RR00168–39 (New England Primate Research Center), and P41RR00995 (Massachusetts Institute of Technology); National Center for Research Resources grant P41RR14075; and the Mental Illness and Neuroscience Discovery Institute. Address correspondence to R.G.G. (e-mail: rggonzalez@partners.org).


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
PURPOSE: To elucidate the neuropathologic basis of transient changes in the ratio of N-acetylaspartate (NAA) to creatine (Cr) in the primate brain by using a simian immunodeficiency virus (SIV)–infected macaque model of the neurologic manifestation of acquired immune deficiency syndrome.

MATERIALS AND METHODS: This study was approved by the Massachusetts General Hospital Subcommittee on Research and Animal Care and the Institutional Animal Care and Use Committee of Harvard University. Rhesus macaques infected with SIV were evaluated during the 1st month of infection. A total of 11 animals were studied, including four control animals, three animals sacrificed 12 days after infection, three animals sacrificed 14 days after infection, and one animal sacrificed 28 days after infection. All animals underwent in vivo proton (1H) magnetic resonance (MR) spectroscopy, and postmortem frontal lobe tissue was investigated by using high-spectral-resolution 1H MR spectroscopy of brain extracts. In addition, quantitative neuropathologic analyses were performed. Stereologic analysis was performed to determine neuronal counts, and immunohistochemical analysis was performed to analyze three neuronal markers: synaptophysin, microtubule-associated protein 2 (MAP2), and calbindin. Analysis of variance (ANOVA) was used to determine substantial changes in neuropathologic and MR spectroscopic markers. Spearman rank correlations were calculated between plasma viral load and neuropathologic and spectroscopic markers.

RESULTS: During acute infection with SIV, the macaque brain exhibited significant changes in NAA/Cr (P < .02, ANOVA) and synaptophysin (P < .013, ANOVA). There was no significant change in the concentration of Cr. No significant changes were found in neuronal counts or other immunohistochemical neuronal markers. With the Spearman rank test, a significant direct correlation was detected between synaptophysin and ex vivo NAA/Cr (rs = 0.72, P < .013). No correlation between NAA/Cr and neuronal counts, calbindin, or MAP2 was found.

CONCLUSION: NAA/Cr is a sensitive marker of neuronal injury, not necessarily neuronal loss, and best correlates with synaptophysin, a marker of synaptodendritic dysfunction.

© RSNA, 2005


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
The N-acetylaspartate (NAA) methyl resonance is typically the largest in the in vivo proton (1H) magnetic resonance (MR) spectrum of the mammalian brain. Results of immunohistochemical examination (1,2) have shown that NAA is localized exclusively to neurons. High-spectral-resolution nuclear MR studies of extracts of brain cells have also demonstrated that NAA is found almost exclusively in neurons, although some NAA was observed in precursor oligodendrocytes, which are generally found only during development (3,4). The role of NAA as a reliable noninvasive neuronal marker at MR spectroscopy has been confirmed with a large number of published studies that have reported decreases in this resonance, most commonly expressed as a ratio with respect to the creatine (Cr) methyl resonance (NAA/Cr), in a variety of central nervous system diseases. We have previouslyreported direct linear correlations between the number of neurons and NAA/Cr in postmortem brains with Alzheimer and Pick disease (5,6). The loss of NAA/Cr, however, is not an absolute marker of neuronal loss because reversal of this ratio has been observed in human studies. A major outstanding question is the neurobiologic basis of transient decreases in NAA/Cr.

In our study of the neurologic manifestation of acquired immune deficiency syndrome (AIDS) (hereafter, neuroAIDS) with a macaque model infected with the simian immunodeficiency virus (SIV), we observed a transient decrease in NAA/Cr, as measured with in vivo 1H MR spectroscopy, in the frontal cortex during acute infection (7). Thus, the purpose of this study was to elucidate the neuropathologic basis of transient changes in primate brain NAA/Cr by using the SIV-infected macaque model of neuroAIDS.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
Animals
All animals were housed according to the standards of the American Association for Accreditation of Laboratory Animal Care. Investigators abided by the Guide for the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council. The study was approved by the Massachusetts General Hospital Subcommittee on Research and Animal Care and the Institutional Animal Care and Use Committee of Harvard University. As previously described (7), we performed in vivo 1H MR spectroscopic studies in 15 rhesus macaques (Macaca mulatta). The macaques were divided into three cohorts of five animals each. The first cohort was imaged before inoculation with SIVmac251 and 11 and 25 days after inoculation, and the second cohort was imaged before inoculation and 13 and 27 days after inoculation. The third cohort was imaged before inoculation and 13 days after inoculation. One animal from the second cohort and three animals from the third cohort were euthanized for postmortem studies (ex vivo MR spectroscopy and quantitative neuropathologic analysis) the day after they underwent their last MR examination.

Also included in the present study were three macaques that were infected with SIVmac251 and euthanized 12 days after inoculation and four uninfected control animals. The three animals inoculated with SIVmac251 were infected as previously described (8,9). Thus, a total of 11 animal brains underwent postmortem evaluation, including four from uninfected control macaques, three from animals euthanized 12 days after inoculation, three from animals euthanized 14 days after inoculation, and one from an animal euthanized 28 days after inoculation.

Viral Load Determinations
Peripheral blood was collected from all animals before inoculation and 11, 12, 14, and 28 days after infection. Virion-associated SIV RNA in plasma was quantified by using a real-time reverse-transcription polymerase chain reaction assay with a sequence detection system (Prism 7700; Applied Biosystems, Foster City, Calif), as described previously (10,11). Results are averages of duplicate determinations.

MR Imaging and MR Spectrocopic Studies
MR images were obtained by one of the authors (M.R.L., J.P.K., J.B.G., R.A.F., E.M.R., or J.H.) by using a clinical 1.5-T unit (Signa; GE Medical Systems, Milwaukee, Wis) with a standard linear extremity coil. The imaging portion of the examination consisted of T1-weighted sagittal imaging, which was followed by dual-echo intermediate- and T2-weighted transverse imaging. Transverse images were used to localize a 1.5 x 1.5 x 1.5-cm spectroscopic voxel in the frontal lobe with the voxel centered on the interhemispheric fissure. In those animals that underwent repeat imaging, the animal head was positioned as in the previous study (7) by using three-plane scout imaging and iterative repositioning. In this manner, voxel placement was highly reproducible. Voxel positioning was performed (J.B.G., M.R.L., and E.M.R.) and reviewed (R.G.G.). MR spectroscopy was performed with the Probe-P (GE Medical Systems) spectroscopy package (12), which consists of a point-resolved spatially localized spectroscopy sequence with chemical shift-selective water suppression (13,14). With the exception of a slightly smaller voxel size, the MR spectroscopic protocol was identical to the one used in multicenter human immunodeficiency virus (HIV) studies (15). That protocol was characterized by high reproducibility. Spectra were collected in the same voxel with identical receiver gains and power settings at echo times of both 35 and 135 msec and a repetition time of 3,000 msec.

After animal imaging, MR spectroscopy was performed with a phantom containing known concentrations of NAA, Cr, choline, and myo-inositol. Phantom MR spectral data were used to confirm instrument stability. All spectra were processed off-line by using a commercial software package (SageIDL; GE Medical Systems), details of which have been previously described (12).

Necropsy and Preparation of Tissue Samples
Animals were sacrificed by means of an intravenous overdose injection of sodium pentobarbital. Necropsy immediately followed euthanasia and was conducted by one of the authors (S.V.W.). Brain tissue was harvested in blocks, which were wrapped in foil, snap frozen by immersion in 2-methylbutane/dry ice, and stored at –70°C. Representative tissues from the frontal cortex, parietal cortex, temporal cortex, occipital cortex, basal nuclei, rostral and caudal thalamus, hippocampus, brainstem, and cerebellum were collected. A complete set of brain tissues was also fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned for histopathologic analysis. For this study, frozen tissue from the frontal cortex was processed and analyzed with proton nuclear MR spectroscopy. Tissues immediately adjacent to those samples were collected, fixed in 10% neutral buffered formalin, embedded in paraffin, and cut in 5-µm-thick slices for routine histologic examination and quantitative neuropathologic examination.

Brain Extracts
Extraction of brain tissue was performed (M.R.L., J.P.K., J.B.G., R.A.F., E.M.R., and J.H.) by using a methanol and chloroform extraction process as previously described (16) and modified for small samples. Special impact-resistant 2-mL screw-cap tubes containing lysing matrix D, also known as the FastRNA Green/Biopulverizing System I (Q-Biogene, Carlsbad, Calif) and specifically designed for use with the FastPrep FP 120 cell disrupter (Thermo-Savant, Holbrook, NY), were filled with 1.2 mL of methanol and a tissue sample of 40–80 mg. The cell disrupter was used to pulverize the sample tissue. The tube was placed in a centrifuge for 3 minutes at 9000 rpm, and 0.8 mL of the solution was placed into a 15-mL centrifuge tube. Water (0.8 mL) was added to the tube, which was then placed into the cell disrupter and subsequently centrifuged. After removal of 1 mL of the solution, the extraction was repeated a third time with 1 mL of chloroform. All extracted solutions and contents of the tube were placed in the 15-mL centrifuge tube. The 2-mL tube was rinsed in succession with 1 mL of chloroform, 0.8 mL of methanol, and 0.2 mL of water. All rinses were combined with the extracted solutions in the 15-mL centrifuge tube. After centrifugation of the mixture for 8 minutes at 3000 rpm, the resulting water-soluble metabolic solution was removed, placed into a dryer (Speedvac Concentrator; Thermo-Savant, Holbrook, NY), and dried at 40°C for 2 hours. The sample was then dried for an additional 8 hours at room temperature. The remaining residue was dissolved in 600 µL of D2O.

High-Spectral-Resolution 1H Nuclear MR Studies
High-spectral-resolution 1H nuclear MR studies were performed by one author (M.R.L.) with a 600-MHz spectrometer (Avance; Bruker Instruments, Billerica, Mass) by using a 5-mm probe. A one-pulse experiment was used with spectral acquisition parameters, including a 90° pulse length of 9 µsec, recycle delays of 20 seconds, a spectral width of 7.2 kHz, 32000 complex points, and 64 scans (which were averaged). All spectra were processed off-line with a spectral analysis software program (Peak Research; PERCH Solutions, Kuopio, Finland) and used to determine the ratio of NAA (2.01 ppm) to Cr compound (3.03 ppm).

Neuropathologic Analyses
Quantitative neuropathologic analyses were performed by one author (E.M.). The number of cortical neurons was quantified by using stereologic evaluation. Formalin-fixed paraffin sections (20 µm thick) from the frontal cortex were stained with cresyl violet for subsequent computer-aided image analysis. Briefly, as previously described (17), an adaptation of the "selector" was implemented for stereologic estimation of neuronal density per unit volume across the same cortical region as mentioned earlier. This necessitated two steps: estimation of the neuronal volume fraction, occupied by all neurons in the sampled volume, and estimation of the mean number-weighted neuronal volume. The volume-weighted neuronal number density was calculated simply by dividing neuronal volume fraction by the mean number-weighted neuronal volume. These estimations were derived by using an oil immersion objective lens (numerical aperture, 1.25) at x100 magnification to analyze contiguous fields of 65 x 65 µm, with four such fields across and down through full thickness of cortex. Two sides of this frame were forbidden, as in the other method, so that neurons falling on them were not sampled for that field; this ensured that neurons were not counted twice. At x100 magnification, two distinct focal planes, separated by an unknown depth within the section, were identified for each field. Neurons were included only if an in-focus clear nucleolus and Nissl substance could be identified.

{gamma}-Aminobutyrate-ergic neuronal integrity was appraised with monoclonal antibody against the calcium-binding protein calbindin (1:1000) (Sigma Chemical, St Louis, Mo). The integrity of the synapses was evaluated with the monoclonal antibody against synaptophysin (1:10) (Boehringer Mannheim, Indianapolis, Ind). The status of neuronal dendrites was evaluated by using monoclonal antibody against microtubule-associated protein 2 (MAP2) (Boehringer Mannheim). For these purposes, 5-µm-thick paraffin sections from the frontal cortex of both control and SIV-infected macaques were immunolabeled overnight with these monoclonal antibodies, followed by biotinylated horse anti-mouse immunoglobulin G (Avidin-HRP; Vector, Burlingame Calif). The sections were then reacted with diaminobenzadine tetrahydrochloride and H2O2 (0.03%).

Levels of calbindin, synaptophysin, and MAP2 were estimated by means of computer-aided image analysis, as previously described (18). For synaptophysin and MAP2, immunoreactivity was semiquantitatively assessed as corrected optical density by using a microdensitometer (Quantimet 570C; Liaca, Deerfield, Ill). For this purpose, three immunolabeled sections were analyzed from each case. As previously described (18), the system was first calibrated with a set of filters of various densities, and 10 images were obtained for each section at x100 magnification. After the area of interest (layers 2–5) was delineated with the cursor, the optical density within that area was obtained. The optical density in each image was averaged and expressed as the mean per case. The units of all measurements for MAP2 and synaptophysin are in arbitrary optical density units and range from 0 to 500 (ie, 0 indicates all light is allowed to pass through the sample, while 500 indicates no light is allowed to pass through the sample). For calbindin, immunopositive neurons were quantified as the number of positively strained neurons per square millimeter (19). Immunolabeled sections were imaged with the microdensitometer by interactively setting a threshold that covered the calbindin immunoreactive cells. A total of three sections per case and 10 images per section were obtained. For each case, results representing the number of calbindin immunoreactive neurons were averaged and expressed as a mean. All sections were coded while investigators were blinded to the animal cohort, and the code was broken after the results were obtained. All values are expressed as mean ± standard error of the mean.

Statistical Analysis
Statistical analyses were performed by one author (E.F.H.) with SAS software (version 8; SAS, Cary, NC). Analysis of variance (ANOVA) was used to determine significant changes for each neuropathologic marker, ex vivo NAA/Cr measurements, and plasma viral loads. A normal distribution was assumed. P values may not be precisely correct if there is deviation from normality in the sample. The least squares means t test (one tailed) was used to isolate specific significant changes within groups only if statistical significance was determined with ANOVA (20). Spearman rank correlation coefficients were calculated between NAA/Cr, plasma viral load, and neuropathologic markers. A P value of less than .05 was considered to indicate a statistically significant difference.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
Plasma Viral Load
There was a sharp increase in the plasma levels of SIVmac251 shortly after infection, as illustrated in Figure 1. As the animal mounted an immune response, the plasma viral load decreased, although it was not completely eliminated. Results of ANOVA revealed highly significant changes in the viral load in the blood plasma of the 11 animals that underwent necropsy (P < .001). Least squares means t tests were used to isolate significant differences at specific time points. We found a significant increase in viral load between values obtained before infection and those obtained 11 (P < .001) and 12 (P < .001) days after inoculation (P < .001). We observed significant decreases in viral load between 11 and 12 days after inoculation (P < .001), between 11 and 14 days after inoculation (P < .001), and between 12 and 14 days after inoculation (P < .009).



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  		Figure 1. Graph shows the SIV load in rhesus macaque blood serum during acute infection. The rapid increase in plasma viral load as SIV infection takes hold, and its subsequent rapid decrease due to immunologic control that occurs during the 2nd week of infection, is typical for this model. The data are from the 11 animals used in the postmortem examinations. Plasma viral loads from all 11 animals were used for the "0 Days Post Infection" data point. At the other points, only plasma viral loads from the seven infected animals were used. The error bars represent the standard error of the mean for plasma viral load measured on the specific day after infection.

 
In Vivo and ex Vivo MR Spectroscopy
Fifteen animals underwent in vivo 1H MR spectroscopy before infection with SIVmac251 and 11, 13, 25, and 27 days after inoculation. Spectra were acquired from the frontal cortex of each animal. The results of in vivo 1H MR spectroscopy have been reported elsewhere (7). The effects of SIV infection on NAA/Cr measured in vivo in the frontal cortex during the acute infection period were evaluated. A 7.7% decrease in the neuronal marker NAA/Cr (P = .02) was observed 13 days after inoculation. No statistically significant change was detected at other time points. No significant change was detected in the absolute concentration of Cr.

Extracts from frontal cortex samples of control macaques and those sacrificed 12, 14, and 28 days after inoculation were examined with MR spectroscopy at 600 MHz. Changes in frontal cortex extract NAA/Cr were apparent even with visual inspection, as illustrated in Figure 2, in which spectra from a control animal and an animal sacrificed 14 days after inoculation are shown. Across the entire group, we found significant changes in NAA/Cr (P < .02, ANOVA). We used least squares means t tests to isolate significant changes at specific times after infection. We found a significant decrease in the ex vivo NAA/Cr between control animals and those sacrificed 12 days after inoculation (P = .004) and between control animals and those sacrificed 14 days after inoculation (P < .02). This decrease occurred at approximately the same time as that seen with in vivo NAA/Cr (Fig 3).



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  		Figure 2. Spectra from high-spectral-resolution 1H nuclear MR spectroscopy of frontal cortex extracts from a healthy macaque (left) and one infected with SIV (right). The spectrum on the right was obtained 14 days after intravenous infection with SIV. In the infected animal, a substantial decrease is seen between NAA resonances at 2.01 ppm when both spectra are normalized to the Cr resonance at 3.03 ppm.

 


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  		Figure 3. Graphs show changes in NAA/Cr and synaptophysin level in frontal cortex tissue samples during acute SIV infection. Top graph shows the changes in NAA/Cr obtained with in vivo 1H MR spectroscopy and the synaptophysin levels measured in animals euthanized 1 day after imaging. The bottom graph depicts NAA/Cr measured from frontal cortex extracts and synaptophysin levels from adjacent tissue. After infection, temporal changes in NAA/Cr were similar to changes in synaptophysin level. Error bars indicate the standard error of the mean.

 
Neuropathologic Analysis
The frontal cortex from each animal was fixed in formalin, and quantitative neurohistopathologic analysis was performed. Synaptophysin measurements obtained at different time points after SIV infection are shown in Figure 3. Across the different time points, we found significant changes in synaptophysin (P < .013, ANOVA). By using the least squares means t test to isolate differences between specific time points, we found significant differences in synaptophysin between control macaques and those sacrificed 12 (P < .016) and 14 (P < .003) days after inoculation.

Heretofore, the only well-established neuropathologic correlate of a decrease in NAA/Cr has been a decrease in neuronal number (5,6). With stereologic analysis, we quantified the number of neurons from the samples and found essentially the same number of neurons in the frontal cortex of control animals and those infected with SIV (P > .77, ANOVA). Investigating other commonly used neuronal markers, we found no significant changes in the levels of calbindin (P > .18, ANOVA) and MAP2 (P > .58, ANOVA). These observations are depicted in Figure 4.



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  		Figure 4. Chart shows the neuronal counts, MAP2, and calbindin in the frontal cortex during acute SIV infection. Neuronal density was determined with stereology, whereas MAP2 and calbindin were quantified by using immunohistochemical examination. No changes in any of these measures were detected with acute SIV infection. Error bars represent the standard error of the mean. dpi = days after infection.

 
Direct visual inspection of micrographs demonstrates the contrast between changes in synaptophysin and no change in neuronal number. As can be appreciated in Figure 5, A and B, decreases in synaptophysin are visible 14 days after inoculation when compared with control values. Photomicrographs stained with cresyl violet that clearly depict neurons are shown in Figure 5, C (control animal) and D (animal sacrificed 14 days after inoculation). No difference in neuronal number is observed.



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  		Figure 5. Photomicrographs of frontal cortex tissue from one healthy rhesus macaque and one infected with SIV. Photomicrographs obtained with antisynaptophysin immunocytochemical stain in, A, a control macaque and, B, a macaque euthanized 14 days after infection with SIV. A decrease in synaptophysin (stained brown) with SIV infection is easily visible. Photomicrographs stained with cresyl violet show no alterations in the numbers of neurons (stained purple) found between C (frontal cortex tissue from the same macaque as in A) and D (the same macaque as in B). The bar located at the bottom of D represents 25 µm.

 
We performed correlation analyses between the extract NAA/Cr and the neuropathologic markers. By using the Spearman rank test, we found a significant direct correlation between synaptophysin and NAA/Cr (rs = 0.72, P < .013) (Fig 6). In addition, we found significant correlation between viral load and synaptophysin (rs = –0.67, P < .034). We found no significant correlation between NAA/Cr and neuronal counts, calbindin, or MAP2.



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  		Figure 6. Graph shows the correlation between NAA/Cr and synaptophysin in frontal lobe tissue extracts. A significant positive linear correlation was found between NAA/Cr measurements and synaptophysin from the same location in the same animals (rs = 0.72, P = .013).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
 
An outstanding question in clinical brain 1H MR spectroscopy is the pathobiologic basis of the decrease in NAA/Cr that is commonly observed in a variety of neurologic diseases. Although there is ample evidence to indicate that neuronal loss will result in a decrease in NAA/Cr, transient decreases in this ratio are not understood. By using the SIV-infected macaque model of neuroAIDS during the acute and transient phase of infection, we found that decreases in NAA/Cr correlate best with changes in synaptophysin levels, a marker of synaptic integrity. Neuronal number, MAP2 (a dendritic cytoskeletal marker), and calbindin (a {gamma}-aminobutyrate-ergic–specific neuronal protein) were unchanged in this acute period and did not show correlation with changes in NAA/Cr.

NAA is one of the most abundant free amino acids in the brain and has been shown to be produced and localized exclusively to neurons at immunohistochemical examination (1,2) and to be localized almost exclusively to neurons at high-spectral-resolution nuclear MR studies of brain cell extracts (3,4). The function of this abundant amino acid is obscure. It may act as an osmolyte, which regulates water gradients, or it may be a precursor to aspartate (21). NAA has also been implicated in the proper myelination of neurons (21). Baslow (22), in a review of the topic, has hypothesized that NAA and its derivative N-acetylaspartylglutamate serve in the vertebrate brain in cell signaling and that it is important in the regulation of interactions of brain cells and in the establishment and maintenance of the nervous system.

The level of NAA/Cr in the human brain is low at birth but increases rapidly as the brain matures. The NAA/Cr in the rhesus macaque brain displays a similar progression (23). In the adult, NAA/Cr remains stable until old age, when it decreases at a slow rate in neurologically healthy people. NAA/Cr decreases in a wide variety of central nervous system diseases that are known to result in neuronal loss. For example, a decrease in NAA/Cr has been reported in people with Alzheimer disease (24–28), multiple sclerosis (29–31), traumatic brain injury (32,33), and HIV (9,34–39).

Results of research in animals and humans support the inference that the decreases in NAA/Cr reported in clinical studies reflect neuronal injury and loss. Direct infusion of the excitotoxin N-methyl-D-aspartate into rat putamen resulted in a decrease in NAA/Cr in vivo and in brain extracts; the loss was shown to correlate with decreases in neuron-specific enzymes (40). Furthermore, a reduction in NAA/Cr has been shown to directly correlate with neuronal cell loss in Pick disease (5) and Alzheimer disease (6). With use of quantitative neuropathologic methods, correlations between NAA/Cr and neuronal counts were found to be direct and linear.

Although results of tissue investigations confirm that decreases in NAA/Cr occur with neuronal loss, they do not explain several reports of reversal of NAA/Cr, including spontaneous reversal in patients with multiple sclerosis and reversal following treatment in patients with multiple sclerosis, epilepsy, moyamoya disease, and neuroAIDS (41–44). In addition, there is a class of diseases that have not been clearly identified with neuronal loss but that also display decreases in NAA/Cr (eg, schizophrenia [45–47] and bipolar disease [48]).

The findings we report herein help illuminate the issue of NAA/Cr decrease that is reversible and not associated with neuronal loss. The SIV macaque model is ideal for this purpose in several respects. The macaque is a primate with a brain that is biochemically and functionally similar to that of humans in gross and microscopic structure. Moreover, the macaque brain 1H MR spectrum is similar to that of the human (23). SIV infection is highly analogous to HIV infection (49). SIV is a lentivirus with extensive sequence homology to HIV (50,51); in rhesus macaques, SIV produces a clinical syndrome similar to that seen in patients with AIDS (52). These animals develop AIDS and encephalitis that is virtually identical to HIV encephalitis. Decreases in NAA/Cr during HIV infection have been well documented (9,34–39), and we have reported similar changes in macaques with SIV encephalitis (16,53). Interestingly, we recently observed, during acute SIV infection, strikingly stereotypical changes in metabolites detectable with in vivo 1H MR spectroscopy, including a decrease in NAA/Cr (7). Because it is possible to directly evaluate brain tissue at the time of MR spectroscopic changes, it is ideal to help elucidate the pathobiologic basis of potentially reversible changes in NAA/Cr.

In evaluating brain tissue samples from macaques with acute SIV infection, we found statistically significant changes in NAA/Cr, with both in vivo and brain extract high-spectral-resolution 1H MR spectroscopy, and immunohistochemical examination of synaptophysin. In addition, we observed a significant correlation between brain extract NAA/Cr and synaptophysin. Conversely, we found no significant changes in neuronal counts, calbindin, or MAP2 and no significant correlations between NAA/Cr and any of these markers. Taken together, these data indicate that there is early mild neuronal injury with acute SIV infection that is reflected by decreases in NAA/Cr and synaptophysin.

Synaptophysin is a neuronal protein localized to synaptic vesicles (54). Synaptophysin has been implicated in synaptic formation or stabilization (55). Knockout mice lacking both synaptophysin and a protein thought to be redundant to synaptophysin have shown defects in neuronal potentiation (56). Traditionally, synaptophysin has been used in immunohistochemistry of neuronal sections as a simple direct measure of neuronal health (18).

Our results have shown that NAA/Cr and synaptophysin display the same pattern of decreases temporally. NAA/Cr does not correlate with the other pathologic neuronal markers in the 1st weeks of acute SIV infection. Neuronal counts, MAP2, and calbindin did not undergo significant changes. MAP2 localizes to the dendritic compartment of neurons and is involved in microtubule assembly. In culture, knockout cerebellar neurons lacking MAP2 are unable to initiate the dendrite outgrowth normally seen (57). Neurons studied in vivo in knockout mice display shorter dendrites than those in wild-type counterparts (58). Although calbindin is a calcium-binding protein found in many organs, in the central nervous system it is localized to {gamma}-aminobutyrate-ergic neurons. Calbindin may have a neuroprotective effect by inhibiting the onset of calcium-mediated excitotoxicity. Calbindin gene expression is decreased in particular areas of the brain with aging and in certain neurodegenerative diseases (59). Overexpression of calbindin in vivo decreased the amount of hippocampal damage after the administration of neurotoxins (60). The dissimilarity between the functions of calbindin and synaptophysin may explain their different behaviors in acute SIV infection. If calbindin were indeed a neuroprotective protein, then it would be overexpressed in damaged neurons. Thus, calbindin levels would not be seen to decrease.

In conclusion, in the macaque with acute SIV infection, we observed transient decreases in NAA/Cr that followed peak viremia and its subsequent control. During this period, quantitative neuropathologic evaluations revealed decreases in synaptophysin but not in neuronal counts, calbindin, or MAP2. Moreover, we found a significant correlation between NAA/Cr in brain extracts and synaptophysin. We conclude that a decrease in NAA/Cr observed at 1H MR spectroscopy may be a sensitive indicator of early neuronal injury, not necessarily neuronal loss, and that this injury may be related to synaptic dysfunction.

Practical applications: The clinical use of brain 1H MR spectroscopy is widespread and continues to expand. A decrease in NAA/Cr is commonly observed and is interpreted as evidence of neuronal injury. Although decreases in NAA/Cr that occur with diseases associated with neuronal loss are well documented, the observation of spontaneous NAA/Cr recovery, such as in patients with multiple sclerosis, and reversal after treatment in several diseases remained unexplained. The investigations reported herein help answer this outstanding question. In this primate model of neuroAIDS, in which transient decreases in NAA/Cr were observed in the 1st month after viral infection, we found that changes in this widely used clinical measure best correlate with dysfunction of neurons that are also reflected in changes in synaptophysin.


   FOOTNOTES
 
Abbreviations: AIDS = acquired immunodeficiency syndrome, ANOVA = analysis of variance, Cr = creatine, HIV = human immunodeficiency virus, MAP2 = microtubule-associated protein 2, NAA = N-acetylaspartate, SIV = simian immunodeficiency virus

Authors stated no financial relationship to disclose.

Author contributions: Guarantor of integrity of entire study, R.G.G.; study concepts, R.G.G., A.A.L., E.M.; study design, R.G.G., A.A.L., E.M., M.R.L., J.P.K., J.B.G.; literature research, M.R.L., J.P.K., R.A.F., E.M.R., J.H.; experimental studies, all authors; data acquisition, M.R.L., J.P.K., S.V.W., J.B.G., R.A.F., E.M.R., J.H., P.K.S.; data analysis/interpretation, M.R.L., J.P.K., S.V.W., J.B.G., E.M.R., J.H., P.K.S., E.F.H., E.M., R.G.G.; statistical analysis, E.F.H.; manuscript preparation, M.R.L., J.P.K.; manuscript definition of intellectual content, editing, revision/review, and final version approval, all authors

M.R.L. and J.P.K. contributed equally to this work.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
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