If you don't remember your password, you can reset it by entering your email address and clicking the Reset Password button. You will then receive an email that contains a secure link for resetting your password
If the address matches a valid account an email will be sent to __email__ with instructions for resetting your password
HIV-associated sensory neuropathy (HIV-SN) is currently the most common neurological complication of chronic HIV infection and continues to substantially affect patient quality of life. Mechanisms underlying the neuronal damage and loss observed in sensory ganglia of HIV-infected individuals have not been sufficiently studied. The present study aimed to develop and characterize a model of HIV-SN using SIV-infected CD8 T-lymphocyte-depleted rhesus macaques (Macaca mulatta). Uninfected controls (n = 5), SIV-infected CD8-depleted (n = 4), and SIV-infected non-CD8-depleted (n = 6) animals were used. Of the six non-CD8-depleted animals, three were conventional progressors (progressing to AIDS >1 year after infection) and three were rapid progressors (AIDS within 6 months). Dorsal root ganglia (DRG) were examined for histological hallmarks of HIV-SN, including satellitosis, presence of Nageotte nodules, and neuronophagia, as well as increased numbers of CD68+ macrophages and abundant viral replication. In contrast to non-CD8-depleted animals, which had mild to moderate DRG pathology, the CD8-depleted SIV-infected animals had moderate to severe DRG damage, with increased numbers of CD68+ satellite cells. Additionally, there was marked active viral replication in the affected DRG. These findings confirm that many features of HIV-SN can be recapitulated in the CD8-depleted SIV-infected rhesus macaque model within a short time frame and illustrate the importance of this model for study of sensory neuropathy.
Since the advent of antiretroviral therapy, many neurological complications resulting from HIV infection, including HIV-associated dementia, have declined.
Abnormalities of the peripheral nervous system (PNS), however, continue to be among the most common neurological complications of HIV-1 infection and are still poorly understood. Existing antiretroviral therapies have marginal effect on the incidence or severity of HIV-associated sensory neuropathy (HIV-SN),
and HIV-SN continues to substantially and negatively affect quality of life for chronically HIV-infected patients. The pathogenesis of non–drug-related toxicities in PNS disease remains to be elucidated. This quest has been hampered in the PNS by a lack of an appropriate model.
Rodents have generally been used as animal models for the study of nerve degeneration.
Although efficient in terms of relative ease of experimental manipulation, rodent models are comparatively poor models for lentiviral-associated sensory neuropathy. Feline immunodeficiency virus (FIV) infection, a lentiviral model of HIV in cats that causes marked immunosuppression, has been used occasionally to study the pathogenesis of sensory neuropathy.
Findings from a study using a model of pigtail macaques that were coinoculated with a neurovirulent cloned SIV virus (SIV/17E-Fr) and an immunosuppressive swarm virus (SIV/DeltaB670) showed development of multifocal trigeminal ganglionitis of varying severity, characterized by multifocal mononuclear infiltrates, neuronophagia, and neuronal loss.
In the present study, we used a CD8 T-lymphocyte-depleted (hereafter, CD8-depleted) SIV-infected rhesus macaque model, which results in the rapid onset of SIV-associated disease; >77% of persistently (>28 days) depleted macaques develop SIV encephalitis, with a progression to terminal AIDS within 12 weeks after infection. This model is highly reproducible and is commonly used to investigate monocyte/macrophage activation and traffic, peripheral immune responses, and central nervous system (CNS) disease.
This model has not previously been examined as a potential model for HIV-SN. Given the similarities between HIV infection and CNS disease and SIV encephalitis, we hypothesized that this rapid model might also be useful for a model of HIV-SN in humans. The present study aimed to develop a model of HIV-SN using SIV-infected CD8-depleted rhesus macaques, to aid in understanding of the pathogenic mechanisms underlying development of HIV-SN.
Materials and Methods
Animals, Viral Infection, and CD8 Lymphocyte Depletion
Five cohorts of rhesus macaques (Macaca mulatta) were used, a total of 15 animals. The first cohort (uninfected, CD8-depleted; M1 and M2) consisted of two uninfected control animals that were CD8-depleted by treatment with a human anti-CD8 antibody administered only once (50 mg/kg i.v.). The human anti-CD8 antibody was provided by the NIH Nonhuman Primate Reagent Resource.
The second cohort consisted of three uninfected non-CD8-depleted control animals (uninfected, non-CD8-depleted; M3 to M5). The third cohort (CD8-depleted; M6 to M9) consisted of four animals that were inoculated with a viral swarm SIVmac251 (a generous gift from Ronald Desrosiers, New England Primate Research Center, Southborough, MA) and were treated with human anti-CD8 antibody cM-T807 to achieve rapid disease progression with a high incidence of SIV encephalitis. These animals were administered 10 mg/kg s.c. of anti-CD8 antibody at day 6 after infection and 5 mg/kg i.v. at days 8 and 12 after infection. In the CD8-depleted groups, animals were defined as persistently depleted if the CD8 cells were depleted for >28 days. In the present study, all of the animals were persistently depleted, and CD8 levels were partially recovered at the time of necropsy in all animals except M9. Cohort 4 (non-CD8-depleted, rapid progressors; M10 to M12) consisted of three SIV-infected animals that were not CD8-depleted and that developed AIDS within 6 months after infection. Cohort 5 (non-CD8-depleted, conventional progressors; M13 to M15) consisted of three SIV-infected animals that were not CD8-depleted and that developed AIDS ≥1 year after infection.
To compare only peripheral effects of SIV infection, animals were selected that developed SIV encephalitis, as defined by the presence of multinucleated giant cells and accumulation of parenchymal and perivascular infiltrates of macrophages in the brain.
All animals were anesthetized with ketamine HCl, euthanized by an intravenous pentobarbital overdose, and exsanguinated. Animals from cohorts 1 and 3 were housed at the New England Primate Research Center (Southborough, MA) and animals from cohorts 2, 4, and 5 were housed at Tulane University's National Primate Research Center (Covington, LA) in strict accordance with standards of the American Association for Accreditation of Laboratory Animal Care.
Necropsy and Histopathology
Animals were necropsied immediately after death. Representative sections of all major organs were collected, fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 5 μm, and stained using H&E. Staining was performed as follows. Tissues were deparaffinized in xylene, hydrated in graded alcohols, counterstained with Harris hematoxylin solution (Sigma-Aldrich, St. Louis, MO) for 2 minutes, and rinsed with running water. The slides were then dipped sequentially in acid alcohol (90% methanol, 5% sulfuric acid, 5% acetic acid; Sigma-Aldrich) and ammonia water (15 to 20 drops ammonium hydroxide in 250 mL water; Sigma-Aldrich), with a rinse under running water after each dip, followed by 80% alcohol for 2 minutes and then eosin (Sigma-Aldrich) for 2 minutes. Finally, tissue sections were rinsed in graded alcohols, dehydrated with xylene, and mounted with VectaMount medium (Vector Laboratories, Burlingame, CA).
Histopathological Analysis of Dorsal Root Ganglia Morphology
H&E-stained sections of dorsal root ganglia (DRG) were evaluated blindly for histopathological lesions by a board-certified pathologist (A.D.M.); scoring was based on the presence and severity of infiltrating mononuclear cells, neuronophagia, and neuronal loss/degeneration. Ganglionitis was scored as 1, 2, or 3 according to the following criteria: mild (1), for scattered infiltrating mononuclear cells with rare evidence of neuronophagia and/or neuronal loss; moderate (2), for increased numbers of infiltrating mononuclear cells with occasional neuronophagia and/or neuronal loss; and severe (3), for abundant infiltrating mononuclear cells, frequent neuronophagia, and neuronal loss.
DRG sections were deparaffinized with xylene, hydrated in a series of graded alcohols, and stained with a pan-macrophage marker anti-CD68 (Dako, Carpinteria, CA), anti-SIV protein p28 (Bartels; Trinity Biotech USA, Jamestown, NY), a pan-T-cell marker anti-CD3 (Dako) or anti-CD8 (Vector Laboratories). Sections were counterstained with hematoxylin, dehydrated, and mounted using VectaMount permanent mounting medium (Vector Laboratories). Sections were visualized and photographs taken using a Zeiss Axio Imager M1 microscope (Carl Zeiss MicroImaging, Thornwood, NY) with Plan-Apochromat 20×/0.8 and 40×/0.95 Korr objectives. The number of CD68+, viral p28+, CD3+, and CD8+ cells were counted manually. Data are expressed as a percentage of total satellite cells, calculated by dividing the number of positively stained cells by the total number of cells stained with hematoxylin and multiplying by 100. The absolute number of cells (positive cells/mm2) was calculated by dividing the number of positive cells in the field by the size of the field examined (each 20× field was 0.147 mm2). Cell counts were performed on eight nonoverlapping fields per DRG tissue section per animal at ×200 magnification.
Bromodeoxyuridine (5-bromo-2′-deoxyuridine; BrdU) was administered 24 hours before necropsy as a slow bolus injection (60 mg BrdU/kg body weight, i.v.).
Two uninfected CD8-depleted (cohort 1), three uninfected non-CD8-depleted (cohort 2), four SIV-infected CD8-depleted (cohort 3), and six SIV-infected non-CD8-depleted rhesus macaques (cohorts 4 and 5) were used in the present study (Table 1). Of the six SIV-infected non-CD8-depleted animals, three were rapid progressors (AIDS progression within 6 months) (non-CD8 depleted: rapid progressors) and three were conventional progressors (progressed to AIDS >1 year after infection) (non-CD8 depleted: conventional progressors) (Table 1). DRG tissue sections from uninfected controls had healthy neurons surrounded by a thin layer of supportive satellite cells (Figure 1A), regardless of CD8 T-cell depletion status. DRG from all CD8-depleted SIV-infected animals revealed variable satellitosis, neuronophagia, and development of Nageotte nodules, which are compact areas of satellite cells that accompany DRG neuronal loss (Figure 1B). Morphological assessment of DRG from non-CD8-depleted infected macaques demonstrated less severe peripheral neuropathy than that found in CD8-depleted macaques, with three animals having mild lesions and three animals having mild to moderate DRG pathology (Table 1). The DRG pathology was similar in nondepleted SIV-infected animals regardless of whether they were rapid or conventional progressors (Figure 1, C and D).
Table 1Animals Used in Developing an Rhesus Macaque Model of HIV-Induced Sensory Neuropathy
Data are expressed as means ± SEM. The percentage of positive cells was calculated as the number of positively stained cells divided by the total number of satellite cells (total hematoxylin-positive cells) surrounding the DRGs, multiplied by 100. Eight 20× fields were used for each measurement. The absolute number of cells was calculated as the cells/mm2 in eight separate 0.147-mm2 fields by dividing the number of positive cells in the field by the size of the field examined (eg, for a 20× field, it was 0.147 mm2).
To characterize the degree of satellitosis and neuronophagia in the CD8-depleted SIV-infected animals, sections of DRG tissues were immunostained with an anti-CD68 monoclonal antibody (Figure 2). In uninfected CD8-depleted macaques, CD68 immunostaining identified a resident population of CD68+ macrophages diffusely scattered around the DRG neurons (Figure 2A). In the two CD8-depleted controls, 13.8% and 11.6% of the total satellite cells were CD68+ (491.5 and 683.2 CD68+ satellite cells/mm2 of tissue, respectively) (Table 1 and Figure 2A). Immunostaining for CD68 in DRG of the CD8-depleted SIV-infected macaques revealed a significant 1.5-fold to 2.0-fold increase in CD68+ cells, compared with uninfected controls (P < 0.05, unpaired t-test) (Table 1). The quantity of CD68+ cells in DRG tissue from CD8-depleted SIV-infected animals ranged from 814.5 to 1342.0 cells/mm2. Activated CD68+ macrophages (based on IHC and morphology) in the CD8-depleted SIV-infected animals had more prominent cell processes, compared with control DRG tissues, consistent with increased reactivity (Figure 2B). In the non-CD8-depleted uninfected controls, the proportion of CD68+ satellite cells ranged from 11.8% to 15.4% of total satellite cells; absolute numbers ranged from 420.6 to 569.7 CD68+ satellite cells/mm2 of DRG tissue (Table 1). The CD68 immunostaining in the non-CD8-depleted SIV-infected groups was more variable between animals, but both the overall percentage of CD68+ satellite cells and the absolute number of CD68+ cells in the DRG tissue were elevated, compared with uninfected animals (Table 1 and Figure 2, C and D).
DRG tissue was stained with an anti-SIV p28 antibody to analyze the number of productively SIV-infected satellite cells and to determine whether productive infection correlated with DRG pathology (Figure 3). Within the CD8-depleted SIV-infected animals SIV p28+ cells ranged from 1.2% to 8.2%; absolute numbers of SIV p28+ satellite cells ranged from 60.1 to 317.4 cells/mm2 of DRG tissue (Table 1). In these animals, productively infected cells were consistently localized to ganglionic lesions of infiltrating cells and prominent neuronophagia (Figure 3A). In non-CD8-depleted SIV-infected animals with rapid disease progression, cells staining positively for SIV p28 ranged from 4.8% to 45.6%; absolute numbers of SIV p28+ satellite cells ranged from 143.5 to 1921.8 cells/mm2 of DRG tissue (Table 1 and Figure 3B). In contrast, there were few productively infected satellite cells in the non-CD8-depleted SIV-infected animals that were conventional progressors (2.0% to 2.6% of satellite cells and the absolute number of cells ranging from 70.0 to 127.1 cells/mm2 of DRG tissue staining positively for SIV p28) (Table 1 and Figure 3C).
To examine the presence of T cells in DRG tissue and their possible role in DRG inflammation or in controlling SIV replication, sections of DRG tissue were immunostained with an anti-CD3 antibody. In the two CD8-depleted uninfected animals, the basal level of CD3+ T cells in the DRG tissue was 101.1 and 191.2 cells/mm2, with 2.6% to 6.4% of the satellite cells expressing CD3 (Table 1). In the SIV-infected CD8-depleted animals, the number of CD3+ T cells in the DRG tissues was not elevated, compared with uninfected controls. The percentage of CD3+ T cells of total satellite cells ranged from 1.7% to 7.4% and the absolute number ranged from 96.3 to 252.6 CD3+ cells/mm2 of DRG tissue (Table 1). In the non-CD8-depleted uninfected animals, the basal number of CD3+ T cells in the DRG tissue was slightly higher than the CD8-depleted animals, ranging from 293.5 to 597.6 cells/mm2, with 7.0% to 11.2% of the satellite cells expressing CD3 (Table 1). In the SIV-infected CD8-depleted animals, the quantity of CD3+ T cells in the DRG tissues was not significantly elevated in the rapid progressors, compared with non-CD8-depleted uninfected animals. The percentage of CD3+ T cells in the total satellite cells in the conventional progressors was higher than in the uninfected animals, ranging from 5.3% to 19.3%; the absolute number ranged from 243.2 to 778.0 CD3+ cells/mm2 of DRG tissue (Table 1).
To assess the possible role or roles that CD8+ T cells may have in either controlling SIV replication or in promoting inflammatory response in DRG tissue during SIV peripheral neuropathy, we examined the number of CD8+ T cells present in the DRG tissue. In the CD8-depleted animals, both uninfected and infected, there was a similar range (0.0% to 6.2%) of total satellite cells that were CD8+ (Table 1). There were more CD8+ cells in DRG of the non-CD8-depleted animals (5.1% to 17.8% of total satellite cells), with the greatest number in the conventional progressors (Table 1).
We found a positive correlation between the degree of DRG histopathology and the number of activated CD68+ macrophages. Of the four CD8-depleted SIV-infected animals, M6 had the most severe pathology and the greatest percentage of CD68+ satellite cells (severe DRG pathology with 28.1% CD68+ satellite cells). M7 and M8 had moderate to severe DRG damage, with 26.1% and 24.4% CD68+ satellite cells, respectively. M9 had moderate DRG pathology, with 19.1% CD68+ satellite cells. Of the six animals in the non-CD8-depleted SIV-infected groups, those with mild DRG damage (M12, M14, and M15) had lower percentages of CD68+ satellite cells (16.1% ± 0.8, 19.6% ± 1.4, and 10.6% ± 0.3, respectively) than those with moderate DRG pathology (M10, M11, and M13; 38.0% ± 1.6, 27.3% ± 3.2, and 24.5% ± 1.2, respectively) (Table 1). This trend did not hold true across all animals. Overall, CD8-depleted SIV-infected animals had the most severe pathology, but not the highest percentage of CD68+ satellite cells.
To determine whether there is an accumulation of satellite cells around DRG neurons, total satellite cells were counted in H&E-stained sections; this total was divided by the number of neurons per section of tissue. In the uninfected control animals, the number of satellite cells ranged from 10.3 to 13.8 cells per DRG neuronal body (Table 1). Only two animals, both of them CD8-depleted SIV-infected, had a significantly increased number of satellite cells, compared with uninfected animals, with 26.0 ± 1.2 and 23.1 ± 1.5 cells per DRG neuronal body. BrdU labeling showed that <1% of the satellite cells in CD8-depleted uninfected animals were BrdU+, whereas in CD8-depleted SIV-infected animals the percentage of BrdU+ satellite cells ranged from 1.5% to 5% 24 hours after the BrdU bolus (data not shown). These data demonstrate that, in this model, macrophages traffic from bone marrow and accumulate around the DRG neurons within 24 hours after the BrdU bolus, which is increased during SIV-SN.
With the present study, we have developed and characterized a CD8-depleted SIV-infected rhesus macaque model of SIV-SN. Using this model, we found that satellite cell activation and recruitment are critical components of DRG pathology. This rapid-progression model accurately resembles the peripheral nerve pathology evidenced in non-CD8-depleted SIV-infected animals with AIDS, but results in a higher incidence of SIV encephalitis in the CNS and more severe DRG pathogenesis. It is therefore an accurate and valuable model for elucidating the pathogenic mechanism or mechanisms of HIV-SN and consequent future treatment modalities.
In HIV infection, DRG are a site of neuronal damage, which is associated with reactive mononuclear phagocytes and HIV-infected macrophages. Increasingly, reports from human studies highlight the possible contribution of satellite cell activation and dorsal root inflammation to the persistence of pathological pain in peripheral neuropathy via activation of the sensory pathways that propagate peripheral signals back into the spinal cord and brain.
Our observations of morphological and IHC alterations in satellite cells within the DRG of SIV-infected CD8-depleted macaques recapitulates lesions of sensory neuropathy in a shorter time frame of AIDS progression: 3 to 4 months in CD8-depleted macaques, compared with >10 years in humans and >1 year in conventional progressor nondepleted macaques. Moderate to severe DRG pathology was found in the SIV-infected CD8-depleted macaques, which was characterized by abundant infiltration of mononuclear cells and subsequent formation of Nageotte nodules, thus substantiating the observation that neuronal loss occurs in the DRG of SIV-infected macaques. Consistent with these data, another report described trigeminal ganglionitis with neuronal loss and neuronophagia in SIV-infected pigtail macaques.
An endogenous resident population of scattered CD68+ macrophages is located within tissue of uninfected animals and is unchanged by CD8 T cell depletion itself in the absence of SIV infection. In infected animals, CD68+ macrophages increased in number, surrounded neuronal bodies, and accumulated in distinct foci (rather than being dispersed evenly throughout the tissue), suggesting a role for activated satellite cells in perhaps initiating neuronophagia. These activated cells exhibited more prominent cell processes and extensive contact with the DRG, indicating increased reactivity and enhanced phagocytic function. The phagocytic activation of macrophages presumably occurs in an attempt to combat the underlying viral infection and to clear degenerate neuronal residue.
The DRG pathology observed in the CD8-depleted animals was more severe than in the non-CD8-depleted SIV-infected animals, as measured by satellitosis and frequency of neuronophagia, whether the animals were conventional or rapid progressors, suggesting that CD8+ T cells may play a role in protecting DRG neurons from damage. However, CD8+ T cell numbers were elevated in the DRG only of the conventional progressors, and numbers in the rapid progressors were similar to those in the CD8-depleted animals, indicating that the CD8+ T cells are not likely to be solely responsible for protecting the DRG neurons from damage. In addition, for examining the number of CD3+ T cells and subtracting the number of CD8+ T cells, we predict that the few remaining cells are likely to be CD4+ T cells in the DRG. (CD4 IHC is not reliable in rhesus macaque paraffin tissue sections, because of low antigenicity, so we used a pan-T-cell marker against CD3.) The non-CD8-depleted animals with mild to moderate peripheral neuropathy generally had more productively infected satellite cells, compared with the CD8-depleted SIV-infected animals, including M6 (with severe DRG pathology), supporting the idea that productive infection of satellite cells is not a prerequisite nor is directly related to DRG damage. Of note, both of the non-CD8-depleted animals with the highest numbers of productively infected satellite cells were rapid progressors, so increased productive infection cannot be explained by a longer time progression to AIDS but may be explained in terms of the quantity of CD8 cells. The greatest numbers of CD8+ T cells were found in the animals with the lowest numbers of productively infected cells.
To compare only peripheral effects of SIV infection with known CNS pathology, we selected animals that developed SIV encephalitis. As common as multinucleated giant cells are to CNS pathology in SIV encephalitis, we found no multinucleated giant cells present in the sensory ganglia examined from the same animals. Although similarities exist between the CNS and PNS with regard to SIV-associated pathologies, there are likely fundamental differences between the pathogenic mechanisms in the two affected areas, due in part to differences between the blood-brain barrier of the CNS and the blood-neural barrier of the PNS, as well as to the capacity of PNS neurons (but not CNS neurons) for extensive regeneration.
We conclude that with a more rapid progression to AIDS and typically a higher severity of peripheral neuropathy, the CD8-depleted SIV-infected rhesus macaque can serve as a valuable model for studying the pathogenesis of HIV-induced PNS disease. In this model, activation of endogenous CD68+ macrophages plays substantial roles in perpetuating DRG damage and neuronal loss, as characterized by frequent neuronophagia and formation of Nageotte nodules.
We thank Drs. Andrew A. Lackner, Ronald S. Veazey, and Patricia E. Molina for the use of non-CD8-depleted SIV-infected DRG tissues, Dr. Peter J. Didier for collection of retrospective DRG tissues from the non-CD8-depleted SIV-infected animals, Cecily Conerly Midkiff and Dr. Xavier Alvarez for assistance in obtaining uninfected non-CD8-depleted tissues, the veterinary staff at both the New England Primate Research Center and the Tulane National Primate Research Center for animal care, and pathology residents and staff for assisting with necropsies and tissue collection.
Positive effects of combined antiretroviral therapy on CD4+ T cell homeostasis and function in advanced HIV disease.
Supported by a pilot grant from the Tulane National Primate Research Center ( NIH P51-RR00164 to T.B.) and by NIH grants R01-NS40237 and R01-NS37654 (K.W.). The in vivo CD8-depletion antibodies used in these studies were provided by the NIH Nonhuman Primate Reagent Resource under grants RR016001 and AI040101 .