González, R. Gilberto; Cheng, Leo L.a; Westmoreland, Susan V.b; Sakaie, Ken E.; Becerra, Lino R.; Lee, Patricia L.; Masliah, Eliezerc; Lackner, Andrew A.b
HIV infection of the central nervous system (CNS) results in a spectrum of clinical and pathological abnormalities [1–4]. At one extreme, the neurological abnormalities caused by the infection are experienced as only mild cognitive–motor deficits; at the other, they represent a profound neurological impairment, characterized by dementia and/or encephalitis. The disorder known as the AIDS dementia complex (ADC) typically develops late in the course of HIV infection and is characterized by cognitive, motor and behavioral disturbances. These clinical syndromes are attributed to neuronal injury and loss, a view supported by a number of autopsy studies that have demonstrated loss of neurons and damage to the synaptodendritic apparatus in HIV-infected brain [5–12]. Neuronal injury is also suggested by brain magnetic resonance spectroscopy (MRS) studies that have demonstrated loss in the neuronal marker n-acetylaspartate (NAA) [13–21]. The cause of this neuronal injury and when it occurs remain unknown. A preponderance of studies finds no convincing evidence for direct neuronal infection by HIV or SIV (the exception being an ultra-sensitive PCR study ), arguing strongly that indirect mechanisms are involved.
Autopsy evaluation of brain from AIDS patients reveals a correlation between ADC and the presence of HIV-1 in the CNS, dendritic pathology and neuronal loss [5–12]. The pathogenesis and temporal progression of this neuronal loss and damage in relation to HIV neuroinvasion, however, is not understood. Neuroinvasion by HIV has been shown to occur early in infection and can be associated with a mild meningitis and cerebrospinal fluid (CSF) pleocytosis [23,24]. In the SIV–macaque model of AIDS, which allows for careful examination of early events, neuroinvasion is observed as early as 7 days after infection [25,26]. In this model, neuroinvasion is associated with increased numbers of perivascular macrophages, scattered perivascular cuffs, the presence of virus in the brain parenchyma, increased expression of endothelial adhesion molecules on vessels in the brain parenchyma, and elevation of markers of intrathecal immune activation such as quinolinic acid [27–31]. Whether these early events in the neuropathogenesis of AIDS result in cellular injury and how they might be related to later development of cognitive impairment and dementia is not known. We undertook this study to examine whether early neuroinvasion produces cellular injury in the brain.
Materials and methods
Fifteen rhesus macaques (Macaca mulatta) were included for study. Animals were housed in accordance with standards determined by the American Association for Accreditation of Laboratory Animal Care. Investigators adhered to the Guide for the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council. Eleven macaques were infected intravenously with either molecularly cloned SIVmac239 (n = 9) or uncloned SIVmac251 (n = 2). Molecularly cloned SIVmac239 and uncloned SIVmac251 are both highly pathogenic and have a median survival of approximately 1 year [32,33]. The animals infected with SIV were then sacrificed at a range of times after infection (Table 1). Four uninfected macaques were used as controls, and four animals infected with SIVmac239 were sacrificed 14 days after infection, during the peak of viremia (Table 1). Of the remaining seven infected animals, most were sacrificed when moribund with AIDS.
All animals were necropsied immediately after euthanasia, which was effected with an intravenous overdose of pentobarbital. Multiple blocks of brain were collected. Adjacent blocks of tissue were foil wrapped or embedded in O.C.T. compound (Miles Scientific, Elkhart, Indiana, USA), and then snap-frozen by immersion in 2-methylbutane/dry ice and stored at −70 °C. An additional block of tissue, adjacent to those collected for snap freezing, was fixed in 10% neutral buffered formalin. Formalin fixed tissues were embedded in paraffin and sectioned at 6 μm for routine histology. Frozen tissue and paraffin embedded samples of frontal lobe gray matter were analyzed by nuclear magnetic resonance (NMR) spectroscopy and quantitative neuropathology, respectively.
High resolution magic angle spinning (HRMAS) MRS experiments were performed at 5 °C on an MSL400 NMR spectrometer (proton frequency 400.13 MHz) using a BD-MAS probe (Bruker Instruments, Inc. Billerica, Massachusetts, USA). Sample size was 30–40 mg. Temperature was controlled by a VT-1000 unit in combination with a MAS-DB pneumatic unit (Bruker Instruments, Inc.). The sample spinning rate was stabilized between 2.0 and 2.5 kHz, with fluctuation for a given spinning rate of < 2 Hz. Rotor synchronized and T2-filtered Carr–Purcell–Meiboom–Gill (CPMG) pulse sequences [90-(τ-180-τ)n-acquisition] were used for spectrum acquisition. The inter-pulse delay (t = 2π/ωr) was synchronized to the rotor rotation, where wr/2π is the magic angle spinning (MAS) rate. The value of n was varied between 400 and 500, depending on the sample spinning speed of each sample, in order to achieve a T2-filter of (2nτ = 400 ms). The 90° pulse length was adjusted for each sample individually and varied from 9.9 to 10.5 ms. The number of transients was 512, with an acquisition time of 1 s. A repetition time of 3.0 s and a spectral width of 25 p.p.m. were used. T2 measurements were performed with the same CPMG sequence by varying n from 200 to 900. Before Fourier transformation and phasing, all free induction decays were subjected to 1 Hz apodization. Tetramethylsilane (TMS) at 0.00 p.p.m. was used as an external chemical shift reference, from which the internal reference of the lactate doublet was determined to be 1.32 and 1.34 p.p.m.
Metabolites were quantified in the following manner: specific resonances were corrected for T2 filter effects using the formula Ic(X) = Ir(X) × exp [TE/T2(X)] where Ic is the corrected intensity for resonance X, Ir is the measured intensity for X, and TE is the length of the applied T2 filter. Metabolite levels were expressed as ratios, with creatine (Cr) serving as the denominator. Cr is a reservoir for high-energy phosphate metabolism and supplies phosphate to convert adenosine diphosphate to adenosine triphosphate. Cr levels are often assumed to be stable in the presence of disease, leading to its use as an internal intensity reference for the measurement of other metabolites. Absolute measurements of metabolites in vivo in HIV-infected humans support the validity of this assumption [34–36].
γ-aminobutyrate (GABA)ergic neuronal integrity was appraised with a monoclonal antibody against the calcium-binding protein calbindin (1 : 1000, Sigma Chemical Company, St. Louis, Missouri, USA). The integrity of the synaptic terminals was evaluated with the monoclonal antibody against synaptophysin (1 : 10, Boehringer Mannheim, Indianapolis, Illinois, USA). The degree of reactive astrocytosis was assessed with the monoclonal anti-glial fibrillary acidic protein (GFAP) antibody (1 : 1000, Boehringer Mannheim). For this purpose, 10 μm thick paraffin sections from the frontal cortex of control and SIV-infected macaques were immunolabeled overnight with these monoclonal antibodies, followed by biotinylated horse anti-mouse IgG, Avidin–horse radish peroxidase (ABC Elite kit, Vector, Burlingame, CA) and reacted with diaminobenzidine tetrahydrochloride and H2O2 (0.03%). Levels of calbindin, synaptophysin, and GFAP were estimated by computer aided image analysis, as described previously . For GFAP and synaptophysin, immunoreactivity was assessed semi-quantitatively as corrected optical density by using the Quantimet 570C microdensitometer: from each case three immunolabeled sections were analyzed. Briefly, as described previously , the system was first calibrated with a set of filters of various densities and a total of 10 images at 100 × magnification were obtained for each section. The area of interest (L2–5) was delineated with the cursor and then the optical density within that area was obtained. The optical density in an unstained section was used as a correction factor. The corrected optical density in each image was averaged and expresses as mean per case.
For calbindin, immunopositive neurons were quantified as number/mm2. Immunolabeled sections were imaged with the Quantimet 570C 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 numbers of calbindin immunoreactive neurons were averaged and expressed as a mean. All sections were blind-coded and the code was broken after results were obtained. All values are expressed as mean ± SEM. Comparison among control and SIV-infected groups were performed using either one-factor ANOVA or Student's t test.
Table 1 summarizes the clinical and pathological data from each animal. Of the 11 animals inoculated with SIV, four had classic SIV encephalitis (SIVE), characterized by perivascular aggregates of macrophages and multinucleated giant cells [28,39]. The remaining seven animals, including the four sacrificed 14 days after infection, did not have significant histopathologic abnormalities of the CNS, although subtle perivascular accumulations of mononuclear cells were noted in the animals 14 days after infection, a phenomenon described previously [30,31]. Systemic pathology was noted in all of the animals that were chronically infected with SIV, most commonly Pneumocystis carinii pneumonia. None of the acutely infected animals had systemic pathology.
In a previous study in our laboratory, proton MRS of macaque brain extracts was used to demonstrate neuronal injury associated with SIV infection. In that preliminary work, we demonstrated that SIV infection results in a significantly reduced concentration of NAA, a neuron specific metabolite . More recently, we developed a high resolution proton MRS methodology, MAS, that may be used to obtain magnetic resonance spectra from unprocessed brain specimens, thereby eliminating extraction artifacts. The quantitative NAA measurements produced by this method correlate well with neuronal counts determined by histology . In the present study, we used HRMAS proton MRS to study intact tissue specimens from SIV-infected macaque brain.
The MRS analysis of NAA and choline resonances of all samples is depicted in Fig. 1. The analysis demonstrates highly statistically significant differences observed in the mean NAA/Cr ratio of brain obtained from the control macaques compared to the SIV-infected macaques. The mean NAA/Cr ratio was approximately 25% lower in the acutely infected macaques, as compared to the controls (P < 0.007). Furthermore, after long-term infection an even greater decrease in this ratio was seen: the NAA/Cr of chronically infected animals was observed to be approximately 35% lower than that of controls (P < 0.0005) and approximately 10% lower than that of the acutely infected animals (P < 0.036). No change was observed in the choline resonance. Linear regression analysis revealed a statistically significant negative relationship between the NAA/Cr ratio and time of infection (r = 0.713;P < 0.014). No significant relationship was found between the presence of SIVE and NAA/Cr (SIVE NAA/Cr = 1.3; no SIVE NAA/Cr = 1.2;P < 0.25).
The results of analysis of neuronal damage are shown in Figs 2 and 3. Calbindin is a neuronal marker that is specific to GABAergic neurons. Previous studies in the brains of individuals infected with HIV have shown that this neuronal population is particularly vulnerable and that injury to these neurons might contribute to the cognitive alterations in AIDS patients . In this context, frontal lobe samples from the same animals that were studied by MRS were used for the calbindin study. Compared to controls (Fig. 2a), SIV-infected animals showed a disruption in morphology of the calbindin immunoreactive neurons, characterized by shrinkage, irregular profiles and fragmentation of the neuritic processes (Fig. 2b). These alterations were more prominent at 2 years (Fig. 2c). Numbers of calbindin immunoreactive neuronal cell bodies were assessed in immunolabeled paraffin sections with the aid of the Quantimet 570C (Fig. 3a). Statistically significant loss of calbindin was seen in animals infected with SIV for 14 days (P < 0.02). A larger decrease was seen in the chronically infected animals (P < 0.004). These data indicate that GABAergic neurons are injured early in the progression of SIV infection.
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.
It is notable that several laboratories using in vivo MRS have reported a decrease in brain NAA in human patients with AIDS corresponding to that observed in the SIV–macaque model [13–21]. This observation has been made both in late stages of dementia and in the early stages of the clinical disease, when patients have few or no cognitive symptoms. The neuropathologic basis of diminished NAA in HIV infection and a variety of other human brain diseases including multiple sclerosis, cerebral infarction and Alzheimer's disease [49–52] has not yet been established. Our findings here suggests that NAA loss may be related to synaptic injury as well as injury to GABAergic neurons. Such injury may ultimately explain the cognitive abnormalities and dementia that accompany HIV infection. Insofar that the SIV–macaque model is similar to human HIV infection, early neuronal injury may also explain studies in which neurobehavioral disorders were observed in patients shortly after 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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