Neocortical Damage during HIV Infection Clayton A. Wiley, MD, PhD,"? Eliezer Masliah, MD,? Mitchel Morey, MD," Cindy Lemere, MS,t &chard DeTeresa, BS,t Marjorie Grafe, MD, PhD," Lawrence Hansen, MD,*t and Robert Terry, MD"t
Clinical and pathological evidence of subcortical central nervous system (CNS) damage is observed commonly in patients with human immunodeficiency virus (HIV) encephalitis. Whether other CNS regions are also affected has not been well studied. We report neocortical damage in patients with HIV encephalitis. Using quantitative techniques, we demonstrate statistically significant thinning of the neocortex, with a loss of large cortical neurons. Qualitative and quantitative assessments of neocortical neuropil reveal a loss of synaptic density and vacuolation of dendritic processes. Failure to demonstrate an association of these changes with the presence of HIV antigens suggests that neocortical damage may be an indirect effect of HIV infection of the CNS. Wiley CA, Masliah E, Morey M, Lemere C, DeTeresa R, Grafe M, Hansen L, Terry R. Neocortical damage during HIV infection. Ann Neurol 1991;29:651-657
Central nervous system (CNS) damage has commonly been associated with human immunodeficiency virus (HIV) infection 11). In addition to the neurological sequelae of focal opportunistic lesions, a constellation of symptoms ranging from psychomotor slowing with personality changes and impaired memory to severe cognitive and motor disturbances, termed acquired immunodeficiency syndrome (AIDS) dementia complex (ADC), has been attributed to HIV infection of the brain {2]. In an autopsy series of 70 patients with AIDS, Navia and colleagues determined that those patients with the most severe ADC frequently had subcortical damage consisting of macrophage and multinucleated giant-cell infiltration, reactive gliosis, and pallor of white matter { 3 ] . The hypothesis that this damage might be mediated by H I V was supported by the prevalence of H I V nucleic acids within brain tissues [4] and the frequent isolation of HIV from cerebrospinal fluid (CSF) and neural tissues 15, 61. In 1986, we and others directly identified HIV antigens and provirus within damaged subcortical regions 17-91, In addition to this well-documented subcortical pathology, neuroradiological studies commonly have noted cortical atrophy in HIV-infected patients {lo, 111. The importance of the cortical changes may be difficult to appreciate clinically in face of the severe subcortical changes. Little attention has focused on histopathological changes in the neocortex. Artigas and colleagues noted neuronal loss, vacuolation, and gliosis associated with HIV infection in 5 of 5 patients dying with ADC 1121. This finding was recently supported
Materials and Methods A total of 32 autopsies were analyzed. Autopsies were performed between 6 hours and 3 days after death. The brains were removed and fixed in 20% formalin for 7 to 10 days. In addition to the portions of brain sampled for routine neuropathological analysis, blocks of tissue perpendicular to the gyral surfaces from the midfrontal, superior temporal, and inferior parietal regions of the cortex were removed and embedded in paraffin as previously described {14}. Six brains with an average weight of 1,381 & 143 gm were from HIVseropositive patients without clinical neurological involvement, and 12, with an average weight of 1,371 & 143 gm, were from AIDS patients with HIV encephalitis. Fourteen brains, with an average weight of 1,453 ? 141 gm, were from non-HIV, neurologically normal age-matched control subjects. For morphometric analysis of cortical thickness and cell-size distribution, the brains of 8 of the 12 AIDS patients
From the Departments of *Pathology and tNeuosciences, University of California, San Diego, La Jolla, CA.
Address correspondence to Dr Wiley, Department of Pathology, University of California, San Diego, La Job, CA 92093-0612.
by data from Ketzler and colleagues who noted a loss of cortical neurons in the frontal cortex of patients with HIV encephalitis [ 131. We employed quantitative and qualitative techniques to assess histopathological changes in the neocortex. Comparisons of cortical thickness and neuronal density in autopsied brains of patients who had HIV encephalitis with those in age-matched control subjects revealed cortical thinning and neuronal loss. Microdensitometric measurements of the presynaptic terminal marker synaptophysin demonstrated an overall simplification of the cortical neuropil. Review of cortical brain biopsy specimens using electron microscopy identified mild spongiform changes.
Received Sep 20, 1990, and in revised form Nov 16. Accepted for publication Dec 16, 1990.
Copyright 0 1991 by the American Neurological Association 651
with HIV encephalitis (as defined by severe white matter and deep gray matter gliosis and the presence of microglial nodules and multinucleated giant cells) were compared to an age-matched control group of 10 brains (part of the original 14) of patients who died from nonneurological diseases.
Laser Conjkal Microscopy To characterize synaptic loss further, we performed laser con-
Morphometry Cortical width and cell-size counts were done on 20-p-thick, paraffin-embedded sections stained with cresyl violet. A Quantimet 970 (Leica Inc, Deerfield, IL) was used for the semiautomated analysis of the tissue sections as described previously 1141. Briefly, automated counting was combined with manual editing to separate contiguous cells, exclude blood vessels, and enhance cell boundaries below the threshold density. Cortical thickness was measured along a 600-p length of pia. The greater optical density of cells was used to differentiate them from the neuropil background. Cells were sorted into nine categories according to diameter, ranging from 5 to 30 p2 to 500 to 800 p’. It was previously determined that in the normal adult human brain almost all cells less than 40 p2 in diameter are glia, and those greater than 40 p2 are neurons [ 147. In addition, immunocytochemical stains for glial fibrillary acidic protein (GFAP) were performed on 20-p-thick sections and then subjected to study with the Quantimet 970 [ 151.
lmmunohistocbemistry Immunocytochemical reactions for GFAP and H I V gp4 1 (mouse monoclonal antibody from Genetic Systems, Seattle, WA) on paraffin- and epoxy resin (Epon)-Araldite-embedded tissues (Electron Microscop Sciences, Philadelphia, PA) were performed as previously described [161. To understand better the nature of the neocortical changes, we analyzed synaptic density using qualitative and quantitative immunohistochemical methods previously validated in studies of Alzheimer’s disease and lesioned rodent brains [17, 181. These quantitative studies are very sensitive to fixation parameters and postmortem time intervals. Only brains fixed in an immersion of 20% formalin within 8 hours of death give valid measurements of synaptophysin content [17, 191. For the present studies we examined the frontal region of the cortex from 6 patients with AIDS who had pathologically confirmed H I V encephalitis and 6 AIDS patients without significant neuropathology (ie., without opportunistic CNS infections or neoplasms). Synaptophysin immunostaining was performed on 5-pthick, paraffin-embedded sections of the midfrontal region. The average postmortem delay in these brains was 5.9 +4.2 hours. Five-micron-thick sections were incubated at 4°C overnight with the monoclonal antibody (SY38) against the presynaptic terminal marker synaptophysin (Boehringer Mannheim Labs, Indianapolis, I N ; 4 pg/ml), followed by incubation in biotinylated horse anti-mouse, avidin D-horseradish peroxidase (Vectastain ABC Elite kit, Vector, Burlingame, CA), and developed in diaminobenzidine (DAB). Sections from all of the brains were processed simultaneously under the same conditions. The optical density of the ancisynaptophysin immunostained sections from the midfrontal cortex was quantified with the Quantimet 970 as previously described [17]. The optical density of the granular reaction product in the neuropil of layers I1 to V was measured and
652 Annals of Neurology
averaged. The optical density of the white matter in each section was subtracted to arrive at a corrected value. A statistical comparison between H I V encephalitis cases and control groups were performed with double-tailed student’s t test.
Vol 29 No 6 June 1991
focal imaging in double immunolabeled sections in order to evaluate the changes in three dimensions. Tissue blocks from the midfrontal cortex of 3 patients with H I V encephalitis and 3 HIV-seropositive patients without encephalitis were sectioned at 40 p with a Microslicer 3000 vibratome (Ted Pella, Redding, CA). The sections were double immunolabeied as previously described 1201, with mouse monoclonal anti-synaptophysin and rabbit anti-microtubule-associated protein (a specific dendritic marker {21], diluted 1: 100; Sigma, St. Louis, MO). The sections were then incubated with a combination of secondary antibodies tagged with fluorescein isothiocyanate (FITC) or Texas red. The double immunolabeled sections were transferred to gelatin-coated slides and mounted with antifading media (4% n-propyl gallate; Sigma). The double immunolabeled sections were studied with the Bio-Rad MRC-600 laser confocal microscope (Watford, UK), as previously described 1191. This preparation permits simultaneous recording of presynaptic terminals and dendrites in the same optical plane of less than 1-c~ thickness.
Ultrastrxtural Methods There are severe limitations in the use of paraffin-embedded autopsy material for quantitative analysis of CNS neuropil. While the techniques described above have been systematically validated for animal and human studies, we sought to appreciate better the nature of the neuropil changes in the brains affected by H I V encephalitis using electron microscopy. Given the nature of autopsy artifacts, electron microscopic studies of ultrastructural perturbations require perfusion-fixed or biopsy material. Obviously only the latter is available for human studies. Over a 2-year period we examined brain needle biopsy specimens from 11 AIDS patients. These specimens have been compared to numerous brain specimens from patients without AIDS. At the time of biopsy, one portion was fixed in 10% buffered formalin for paraffin embedding, while a separate portion was diced in buffered 2.5% paraformaldehyde and 1% glutaraldehyde for electron microscopy. Primary aldehyde fixation was continued for 2 to 18 hours; then the specimens were rinsed in buffer and postfixed in 1% buffered osmium tetroxide for 1 hour at 4°C. After rinsing and dehydration, the tissue was embedded in Epon-Araldite.
Results Measurements of neocortical width in brains affected by HIV encephalitis showed an average of 20% reduction compared to HIV-seropositive controls (Fig 1A). The atrophy of the cortical ribbon was more evident in the parietal and temporal regions of neocortex. These changes were consistent with the cell counts, which showed a significant 30 to 50% decrease in the number of large neurons (200 to 500 k 2 )in all three neocortical
" 1 7 -
Mid Frontal
P
Clinical and pathological evidence of subcortical central nervous system (CNS) damage is observed commonly in patients with human immunodeficiency virus (HIV) encephalitis. Whether other CNS regions are also affected has not been well studied. We report neocortical damage in patients with HIV encephalitis. Using quantitative techniques, we demonstrate statistically significant thinning of the neocortex, with a loss of large cortical neurons. Qualitative and quantitative assessments of neocortical neuropil reveal a loss of synaptic density and vacuolation of dendritic processes. Failure to demonstrate an association of these changes with the presence of HIV antigens suggests that neocortical damage may be an indirect effect of HIV infection of the CNS. Wiley CA, Masliah E, Morey M, Lemere C, DeTeresa R, Grafe M, Hansen L, Terry R. Neocortical damage during HIV infection. Ann Neurol 1991;29:651-657
Central nervous system (CNS) damage has commonly been associated with human immunodeficiency virus (HIV) infection 11). In addition to the neurological sequelae of focal opportunistic lesions, a constellation of symptoms ranging from psychomotor slowing with personality changes and impaired memory to severe cognitive and motor disturbances, termed acquired immunodeficiency syndrome (AIDS) dementia complex (ADC), has been attributed to HIV infection of the brain {2]. In an autopsy series of 70 patients with AIDS, Navia and colleagues determined that those patients with the most severe ADC frequently had subcortical damage consisting of macrophage and multinucleated giant-cell infiltration, reactive gliosis, and pallor of white matter { 3 ] . The hypothesis that this damage might be mediated by H I V was supported by the prevalence of H I V nucleic acids within brain tissues [4] and the frequent isolation of HIV from cerebrospinal fluid (CSF) and neural tissues 15, 61. In 1986, we and others directly identified HIV antigens and provirus within damaged subcortical regions 17-91, In addition to this well-documented subcortical pathology, neuroradiological studies commonly have noted cortical atrophy in HIV-infected patients {lo, 111. The importance of the cortical changes may be difficult to appreciate clinically in face of the severe subcortical changes. Little attention has focused on histopathological changes in the neocortex. Artigas and colleagues noted neuronal loss, vacuolation, and gliosis associated with HIV infection in 5 of 5 patients dying with ADC 1121. This finding was recently supported
Materials and Methods A total of 32 autopsies were analyzed. Autopsies were performed between 6 hours and 3 days after death. The brains were removed and fixed in 20% formalin for 7 to 10 days. In addition to the portions of brain sampled for routine neuropathological analysis, blocks of tissue perpendicular to the gyral surfaces from the midfrontal, superior temporal, and inferior parietal regions of the cortex were removed and embedded in paraffin as previously described {14}. Six brains with an average weight of 1,381 & 143 gm were from HIVseropositive patients without clinical neurological involvement, and 12, with an average weight of 1,371 & 143 gm, were from AIDS patients with HIV encephalitis. Fourteen brains, with an average weight of 1,453 ? 141 gm, were from non-HIV, neurologically normal age-matched control subjects. For morphometric analysis of cortical thickness and cell-size distribution, the brains of 8 of the 12 AIDS patients
From the Departments of *Pathology and tNeuosciences, University of California, San Diego, La Jolla, CA.
Address correspondence to Dr Wiley, Department of Pathology, University of California, San Diego, La Job, CA 92093-0612.
by data from Ketzler and colleagues who noted a loss of cortical neurons in the frontal cortex of patients with HIV encephalitis [ 131. We employed quantitative and qualitative techniques to assess histopathological changes in the neocortex. Comparisons of cortical thickness and neuronal density in autopsied brains of patients who had HIV encephalitis with those in age-matched control subjects revealed cortical thinning and neuronal loss. Microdensitometric measurements of the presynaptic terminal marker synaptophysin demonstrated an overall simplification of the cortical neuropil. Review of cortical brain biopsy specimens using electron microscopy identified mild spongiform changes.
Received Sep 20, 1990, and in revised form Nov 16. Accepted for publication Dec 16, 1990.
Copyright 0 1991 by the American Neurological Association 651
with HIV encephalitis (as defined by severe white matter and deep gray matter gliosis and the presence of microglial nodules and multinucleated giant cells) were compared to an age-matched control group of 10 brains (part of the original 14) of patients who died from nonneurological diseases.
Laser Conjkal Microscopy To characterize synaptic loss further, we performed laser con-
Morphometry Cortical width and cell-size counts were done on 20-p-thick, paraffin-embedded sections stained with cresyl violet. A Quantimet 970 (Leica Inc, Deerfield, IL) was used for the semiautomated analysis of the tissue sections as described previously 1141. Briefly, automated counting was combined with manual editing to separate contiguous cells, exclude blood vessels, and enhance cell boundaries below the threshold density. Cortical thickness was measured along a 600-p length of pia. The greater optical density of cells was used to differentiate them from the neuropil background. Cells were sorted into nine categories according to diameter, ranging from 5 to 30 p2 to 500 to 800 p’. It was previously determined that in the normal adult human brain almost all cells less than 40 p2 in diameter are glia, and those greater than 40 p2 are neurons [ 147. In addition, immunocytochemical stains for glial fibrillary acidic protein (GFAP) were performed on 20-p-thick sections and then subjected to study with the Quantimet 970 [ 151.
lmmunohistocbemistry Immunocytochemical reactions for GFAP and H I V gp4 1 (mouse monoclonal antibody from Genetic Systems, Seattle, WA) on paraffin- and epoxy resin (Epon)-Araldite-embedded tissues (Electron Microscop Sciences, Philadelphia, PA) were performed as previously described [161. To understand better the nature of the neocortical changes, we analyzed synaptic density using qualitative and quantitative immunohistochemical methods previously validated in studies of Alzheimer’s disease and lesioned rodent brains [17, 181. These quantitative studies are very sensitive to fixation parameters and postmortem time intervals. Only brains fixed in an immersion of 20% formalin within 8 hours of death give valid measurements of synaptophysin content [17, 191. For the present studies we examined the frontal region of the cortex from 6 patients with AIDS who had pathologically confirmed H I V encephalitis and 6 AIDS patients without significant neuropathology (ie., without opportunistic CNS infections or neoplasms). Synaptophysin immunostaining was performed on 5-pthick, paraffin-embedded sections of the midfrontal region. The average postmortem delay in these brains was 5.9 +4.2 hours. Five-micron-thick sections were incubated at 4°C overnight with the monoclonal antibody (SY38) against the presynaptic terminal marker synaptophysin (Boehringer Mannheim Labs, Indianapolis, I N ; 4 pg/ml), followed by incubation in biotinylated horse anti-mouse, avidin D-horseradish peroxidase (Vectastain ABC Elite kit, Vector, Burlingame, CA), and developed in diaminobenzidine (DAB). Sections from all of the brains were processed simultaneously under the same conditions. The optical density of the ancisynaptophysin immunostained sections from the midfrontal cortex was quantified with the Quantimet 970 as previously described [17]. The optical density of the granular reaction product in the neuropil of layers I1 to V was measured and
652 Annals of Neurology
averaged. The optical density of the white matter in each section was subtracted to arrive at a corrected value. A statistical comparison between H I V encephalitis cases and control groups were performed with double-tailed student’s t test.
Vol 29 No 6 June 1991
focal imaging in double immunolabeled sections in order to evaluate the changes in three dimensions. Tissue blocks from the midfrontal cortex of 3 patients with H I V encephalitis and 3 HIV-seropositive patients without encephalitis were sectioned at 40 p with a Microslicer 3000 vibratome (Ted Pella, Redding, CA). The sections were double immunolabeied as previously described 1201, with mouse monoclonal anti-synaptophysin and rabbit anti-microtubule-associated protein (a specific dendritic marker {21], diluted 1: 100; Sigma, St. Louis, MO). The sections were then incubated with a combination of secondary antibodies tagged with fluorescein isothiocyanate (FITC) or Texas red. The double immunolabeled sections were transferred to gelatin-coated slides and mounted with antifading media (4% n-propyl gallate; Sigma). The double immunolabeled sections were studied with the Bio-Rad MRC-600 laser confocal microscope (Watford, UK), as previously described 1191. This preparation permits simultaneous recording of presynaptic terminals and dendrites in the same optical plane of less than 1-c~ thickness.
Ultrastrxtural Methods There are severe limitations in the use of paraffin-embedded autopsy material for quantitative analysis of CNS neuropil. While the techniques described above have been systematically validated for animal and human studies, we sought to appreciate better the nature of the neuropil changes in the brains affected by H I V encephalitis using electron microscopy. Given the nature of autopsy artifacts, electron microscopic studies of ultrastructural perturbations require perfusion-fixed or biopsy material. Obviously only the latter is available for human studies. Over a 2-year period we examined brain needle biopsy specimens from 11 AIDS patients. These specimens have been compared to numerous brain specimens from patients without AIDS. At the time of biopsy, one portion was fixed in 10% buffered formalin for paraffin embedding, while a separate portion was diced in buffered 2.5% paraformaldehyde and 1% glutaraldehyde for electron microscopy. Primary aldehyde fixation was continued for 2 to 18 hours; then the specimens were rinsed in buffer and postfixed in 1% buffered osmium tetroxide for 1 hour at 4°C. After rinsing and dehydration, the tissue was embedded in Epon-Araldite.
Results Measurements of neocortical width in brains affected by HIV encephalitis showed an average of 20% reduction compared to HIV-seropositive controls (Fig 1A). The atrophy of the cortical ribbon was more evident in the parietal and temporal regions of neocortex. These changes were consistent with the cell counts, which showed a significant 30 to 50% decrease in the number of large neurons (200 to 500 k 2 )in all three neocortical
" 1 7 -
Mid Frontal
P
