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Lack of neocortical nerve cell loss in Alzheimer's disease: Reality or methodological artifact
Neurobiologyof Aging,Vol. 15, No. 3, pp. 361-362, 1994 Copyright© 1994 ElsevierScienceLtd Printedin the USA. All fightsreserved 0197-4580/94 $6.00 + .00
Pergamon 0197.4580(94)E0003-I
COMMENTARY
Lack of Neocortical Nerve Cell Loss in Alzheimer's Disease: Reality or Methodological Artifact ELLIOTT J. M U F S O N A N D W I L L I A M C. B E N Z I N G
Department of Neurological Sciences, Rush Alzheimer' s Disease Center, Rush Presbyterian-St. Luke's Medical Center, Chicago, IL 60612 tively count neurons and measure cell size (13,14). In fact, Regeur and associates (11) describe the application of a relatively new stereological device termed an optical disector for the evaluation of neuronal numbers in the brain. There are very few studies that have employed this apparatus which makes it difficult to compare its inherent reproducibility to accurately quantify brain cell counts. Previous studies using more standard semiautomatic image analysis apparatus have relied on cell recognition based on cell size or the density of cytoplasmic staining in glial or neuronal perikarya (13,14). Despite the ability of traditional computer-assisted devices to perform automated counts, it is still essential to manually edit contiguous cells, eliminate vessels, and artifacts and fill incompletely stained cell bodies. As noted by Terry and Hansen (14), such editing is essential for accuracy. Although Regeur et al. (11) propose many advantages of the optical disector, no mention is made of editing. More importantly, it is not clear how the optimal disector distinguishes neurons stacked one upon another particularly in a 35 p,m thick Giemsa stained section. Traditionally, paraffin 5-20 ~m thick thionin or cresyl violet stained sections are used in cell counting studies that allow for differentiation of individual neurons either by the observer or the computer. In 35~m thick sections, more neurons in the AD cortex may be counted since they would not be obscured by neighboring and more superfical cells as seen in control brains. Indeed, if the optical disector is not capable of differentiating columns of stacked cortical neurons, it is likely the number of cortical neurons in the aged normal brain were underestimated. Thus, a comparison with AD neocortical neurons may lead to the erroneous conclusion that there is only a minor difference between the diseased brain and age-matched controls. Perhaps use of the optical disector with thinner sections (e.g., 15-20 p,m) may result in different findings. As noted by the Regeur et al. (11) in their Discussion section, the ability to differentiate between glia and neurons is crucial for any accurate morphometric evaluation. They state that after several pilot studies, the Giemsa stain "appeared to provide the best preparations to distinguish nerve cells from glial cells". This is surprising and it would be of interest to delineate how these pilot studies were performed and precisely what characteristics led to the selection of the Giemsa stain. Their final choice is rather odd considering that the Giemsa stain is traditionally used for hemato-
THE WORK OF Regeur and coworkers (11) presents a potentially interesting set of observations demonstrating a nonsignificant loss of nerve cells in frontal, temporal, and parietal neocortex in Alzbeimer's disease (AD) as compared to age-matched controls. Surprisingly, neuronal loss within the occipital lobe, which has previously been demonstrated to be relatively spared in AD (8), approached a statistically significant decrease in neuron number in their study. These findings mark a departure from the long-held belief that there is a significant decrease in the number of large neocortical neurons in the frontal, temporal, and parietal cortex in AD indivduals compared to normal aged individuals (13,14). In fact, previous studies indicate AD patients aged 50-65 lose approximately 50% of the large neocortical neurons, whereas individuals 70-100 years of age lose 28% of these neurons compared with age-matched controls (14). Both of these figures are statistically significant. The age range employed by Regeur and colleagues (10) falls within the latter group, however, they report only a minimal 6% reduction in the total number of cortical neurons in their population of AD patients. These authors do not mention whether this minor loss of neurons was associated with large cortical projection neurons. In fact, their experimental design does not categorize cortical neurons into size classes which also differs from previous studies. Interestingly, it is the large cortical projection neurons, located mainly within layers 3 and 5, that exhibit extensive neurofibrillary tangle (NFTs) formation in AD (9,12). Moreover, reductions in neocortical neuron counts show some correlations with estimates of NFTs and senile plaques (1,10), and it is believed that nerve cells exhibiting these structural abnormalities eventually die. It is this massive loss of cortical projection neurons that has provoked the hypothesis of corticocortical disconnection as the major anatomic substrate for dementia in AD (4,9). Thus, it is perplexing as to why the report by Regeur and associates (11) varies from the classic morphometric investigations which more consistently demonstrate neuronal loss in AD neocortex. If found to be true, the data presented by Regeur et al. would change some of the fundamental tenets of AD pathology. Therefore, it is worthwhile to scrutinize the controversial data presented by Regeur et al. (11). To minimize sampling problems and observer bias, many investigators employ computerized morphometric analyses to objec361
362
MUFSON AND BENZ1NG
pathological examination and not for differentiation of neurons and glia. Despite this potential caveat, Regeur et al. (11) operationally defined a set of criteria based on Giemsa staining which they " f o u n d useful for differentiating between neurons and g l i a . . . " . These criteria depend on the ability of the Giemsa stain to delineate " a membrane-bound convex nucleus which has a diffuse and uniform chromatin pattern and the presence of a clear nucleolus" as well as " a clearly defined neuronal cytoplasm". However, in a report on the use of the Giemsa stain on nervous tissue, Iniguez and colleagues (7) state that like neurons, the nuclei of astroglia are also colored homogeneously and that a "welldelineated rim, stained red-violet, surrounds the nuclei of these [astroglia] cells" and that nucleoli can sometimes be seen. Perhaps many of the cells exhibiting a homogeneously stained nucleus and containing a clear nucleolus in the AD cases in the present study, were actually astrocytes which are extremely numerous in the AD neocortex (2,5). This would result in an overestimation of neuronal numbers in AD and emphasizes the additional need for cell size criteria. Moreover, Iniguez et al. (7) reported that the nuclear appearance of neurons using the Giemsa stain "depends on the dispersion of the chromatin and in large neurons, the nuclei seem rather empty". Thus, many large neurons may not have been counted in the Regeur study because their nuclei lacked a diffuse chromatin pattern. Thus, large pyramidal neurons would not have fullfilled their operational definiton of a neuron which would result in an underestimation of total neuron number in the normal aged cortex. The type of morphologic differentiation used in the present study is in marked contrast to previous morphometric studies of AD cortex that are based, in part, on cell size. These latter investigations have shown that perikarya smaller than 401xm a were almost always glial, whereas neurons were almost always greater than 40 i~ma (13). Using these criteria many investigators have been able to classify neurons into at least two size groups: small, between 40 and 90 i~ma in crossectional area, and large, more than 90 I~m2. By using these less subjective morphologic standards, it is not only possible to quantify cell numbers but to determine their laminar distribution within the cortex, i.e., supra or infragranular layers. It is these types of detailed analyses that have led to the belief that the large neurons within cortical layers 3 and 5 are significantly reduced in number in AD (13,14). It is important to
keep in mind that precautions also should be taken in interpreting laminar thickness data because the cortical lamination pattern can be disrupted in severe cases of AD. However, it would be intriguing to differentiate neurons and glia based on cell size and laminar location using the optical disector methodology with the present population of brains. Then compare numbers of neurons between age-matched controls and AD individuals with those obtained using more standard semi-automated methods. Obviously the fact that this comparison would be based on Giemsa stained sections would still troublesome. A variety of methods for the calculation of changes in neuron number have been used in attempts to define the effects of AD on neuronal cell number. This methodological diversity has made it extremely difficult to interpret differences between investigations evaluating human tissue neuron counts. In their review of the literature, Coleman and Flood (3) indicated that these sources of confusion range from the clinical and pathologic criteria used for selection of human cases to the microscopic sampling of brain regions and even cells. Moreover, although difficult to document, observer bias may be a major variable in these type of studies and most certainly exists. Therefore, any study that is not "rigorously blind," as Coleman and Flood (3) point out, " m a y be suspect". The finding of a lack of a significant reduction in total cortical neuronal cell numbers in AD, as suggested by Regeur et al. (11) would alter many long-held assumptions regarding the pathologic impact of this disease on neocortical circuitry. Their observations contrast with the current literature dealing with this topic which shows a significant loss of long projection neocortical neurons in both early and late onset cases of AD (14). Moreover, there is also evidence that many small cortical neurons such as those containing somatostatin are decreased in AD (6). The authors of the present article do not discuss how their findings either support or contrast with the existing literature. If as suggested by Regeur et al. (11) that there is a nonsignificant loss of neocortical neurons between age-matched controls and AD individuals, then it would be necessary to rethink current concepts underlying cortical neuronal changes in this neurodegenerative disease. However, before these novel observations are incorporated into the reality of the scientific community it would be important to replicate the present findings (11) using the optical disector methodology with a large series of cases.
REFERENCES 1. Ball, M. J. Neuronal loss, neurofibrillary tangles and granulvacuolar degeneration in the hippocampus with aging and dementia. Acta Neuropath. (Bed) 37:111-118; 1977. 2. Beach, T. G.; Walker, R.; McGeer, E. G. Patterns of gliosis in Alzheimer's disease and aging cerebrum. Glia 2:420-436; 1989. 3. Coleman, P. D.; Flood, D. G. Neuron numbers and dendritic extent in normal aging and Alzheimer's disease. Neurobiol. Aging 8:521545; 1987. 4. De Lacoste, M-C.; White, C. L., III. The role of cortical connectivity in Alzheimer's disease pathogenesis: A review and model system. Neurobiol. Aging 14:1-6; 1993. 5. Dixon, D. W.; Mattiace, L. A. Astrocytes and microglia in human brain shoare an epitope recognized by 13-Lymphocyte-specificmonoclonal antibody (LN-1). Am. J. Pathol. 135:135-147; 1989. 6. Gaspar, P.; Duyckaerts, C.; Febvert, A.; Benoit, R.; Beck, B.; Berger, B. Subpopulations of somatostain 28-immunoreactive neurons display different vulnerability in senile dementia of the Alzheimer type. Brain Res. 490:1-13; 1989. 7. Iniguez, C.; Gayoso, M. J.; Carreres, J. A versatile and simple method for staining nervous tissue using Giemsa dye. J. Neurosci. Meth. 13:77-86; 1985. 8. Lewis, D. A.; Campbell, M.; Terry, R. D.: Morrison, J. H. Laminar
9.
10. 11.
12. 13. 14.
and regional distribution of neurofibrillary tangles and neuritic plaques in Alzheimer's disease: A quantitative study of visual and auditory cortices. J. Neurosci. 6:1799-1808; 1987. Pearson, R. C. A.; Esiri, M. M.; Hiorns, R. W.; Wilcock, G. K.; Powell, T. P. S. Anatomical correlates of the distribution of the pathologic changes in the neocortex in Alzheimer disease. Proc. Natl. Acad. Sci. USA 82:4531-4534; 1985. Price, D. L. New perspectives on Alzheimer's disease. Annu. Rev. Neurosci. 9:489-512; 1986. Regeur, L.; Jensen, G. B.; Pakkenberg, H.; Evans, S. M.; Pakkenberg, B. No global neocortical nerve cell loss in brain from patients with senile dementia of Alzheimer's type. Neurobiol. Aging 15:347352; 1994. Rogers, J.; Morrison, J. H. Quantitative morphology and regional and laminar distributions of senile plaques in Alzheimer's disease. J. Neurosci. 5:2801-2808; 1985. Terry, R. D.; Davies, P. Some morphologic and biochemical aspects of Alzheimer's disease. In: Samuel et al., Aging of the brain. New York: Raven Press; 1983: 47-59. Terry, R. D.; Hansen, L. A. Some morphometric aspects of Alzheimer disease and of normal aging. In: R. D. Terry, ed. Aging and the brain. New York: Raven Press; 1988:109-114.
Pergamon 0197.4580(94)E0003-I
COMMENTARY
Lack of Neocortical Nerve Cell Loss in Alzheimer's Disease: Reality or Methodological Artifact ELLIOTT J. M U F S O N A N D W I L L I A M C. B E N Z I N G
Department of Neurological Sciences, Rush Alzheimer' s Disease Center, Rush Presbyterian-St. Luke's Medical Center, Chicago, IL 60612 tively count neurons and measure cell size (13,14). In fact, Regeur and associates (11) describe the application of a relatively new stereological device termed an optical disector for the evaluation of neuronal numbers in the brain. There are very few studies that have employed this apparatus which makes it difficult to compare its inherent reproducibility to accurately quantify brain cell counts. Previous studies using more standard semiautomatic image analysis apparatus have relied on cell recognition based on cell size or the density of cytoplasmic staining in glial or neuronal perikarya (13,14). Despite the ability of traditional computer-assisted devices to perform automated counts, it is still essential to manually edit contiguous cells, eliminate vessels, and artifacts and fill incompletely stained cell bodies. As noted by Terry and Hansen (14), such editing is essential for accuracy. Although Regeur et al. (11) propose many advantages of the optical disector, no mention is made of editing. More importantly, it is not clear how the optimal disector distinguishes neurons stacked one upon another particularly in a 35 p,m thick Giemsa stained section. Traditionally, paraffin 5-20 ~m thick thionin or cresyl violet stained sections are used in cell counting studies that allow for differentiation of individual neurons either by the observer or the computer. In 35~m thick sections, more neurons in the AD cortex may be counted since they would not be obscured by neighboring and more superfical cells as seen in control brains. Indeed, if the optical disector is not capable of differentiating columns of stacked cortical neurons, it is likely the number of cortical neurons in the aged normal brain were underestimated. Thus, a comparison with AD neocortical neurons may lead to the erroneous conclusion that there is only a minor difference between the diseased brain and age-matched controls. Perhaps use of the optical disector with thinner sections (e.g., 15-20 p,m) may result in different findings. As noted by the Regeur et al. (11) in their Discussion section, the ability to differentiate between glia and neurons is crucial for any accurate morphometric evaluation. They state that after several pilot studies, the Giemsa stain "appeared to provide the best preparations to distinguish nerve cells from glial cells". This is surprising and it would be of interest to delineate how these pilot studies were performed and precisely what characteristics led to the selection of the Giemsa stain. Their final choice is rather odd considering that the Giemsa stain is traditionally used for hemato-
THE WORK OF Regeur and coworkers (11) presents a potentially interesting set of observations demonstrating a nonsignificant loss of nerve cells in frontal, temporal, and parietal neocortex in Alzbeimer's disease (AD) as compared to age-matched controls. Surprisingly, neuronal loss within the occipital lobe, which has previously been demonstrated to be relatively spared in AD (8), approached a statistically significant decrease in neuron number in their study. These findings mark a departure from the long-held belief that there is a significant decrease in the number of large neocortical neurons in the frontal, temporal, and parietal cortex in AD indivduals compared to normal aged individuals (13,14). In fact, previous studies indicate AD patients aged 50-65 lose approximately 50% of the large neocortical neurons, whereas individuals 70-100 years of age lose 28% of these neurons compared with age-matched controls (14). Both of these figures are statistically significant. The age range employed by Regeur and colleagues (10) falls within the latter group, however, they report only a minimal 6% reduction in the total number of cortical neurons in their population of AD patients. These authors do not mention whether this minor loss of neurons was associated with large cortical projection neurons. In fact, their experimental design does not categorize cortical neurons into size classes which also differs from previous studies. Interestingly, it is the large cortical projection neurons, located mainly within layers 3 and 5, that exhibit extensive neurofibrillary tangle (NFTs) formation in AD (9,12). Moreover, reductions in neocortical neuron counts show some correlations with estimates of NFTs and senile plaques (1,10), and it is believed that nerve cells exhibiting these structural abnormalities eventually die. It is this massive loss of cortical projection neurons that has provoked the hypothesis of corticocortical disconnection as the major anatomic substrate for dementia in AD (4,9). Thus, it is perplexing as to why the report by Regeur and associates (11) varies from the classic morphometric investigations which more consistently demonstrate neuronal loss in AD neocortex. If found to be true, the data presented by Regeur et al. would change some of the fundamental tenets of AD pathology. Therefore, it is worthwhile to scrutinize the controversial data presented by Regeur et al. (11). To minimize sampling problems and observer bias, many investigators employ computerized morphometric analyses to objec361
362
MUFSON AND BENZ1NG
pathological examination and not for differentiation of neurons and glia. Despite this potential caveat, Regeur et al. (11) operationally defined a set of criteria based on Giemsa staining which they " f o u n d useful for differentiating between neurons and g l i a . . . " . These criteria depend on the ability of the Giemsa stain to delineate " a membrane-bound convex nucleus which has a diffuse and uniform chromatin pattern and the presence of a clear nucleolus" as well as " a clearly defined neuronal cytoplasm". However, in a report on the use of the Giemsa stain on nervous tissue, Iniguez and colleagues (7) state that like neurons, the nuclei of astroglia are also colored homogeneously and that a "welldelineated rim, stained red-violet, surrounds the nuclei of these [astroglia] cells" and that nucleoli can sometimes be seen. Perhaps many of the cells exhibiting a homogeneously stained nucleus and containing a clear nucleolus in the AD cases in the present study, were actually astrocytes which are extremely numerous in the AD neocortex (2,5). This would result in an overestimation of neuronal numbers in AD and emphasizes the additional need for cell size criteria. Moreover, Iniguez et al. (7) reported that the nuclear appearance of neurons using the Giemsa stain "depends on the dispersion of the chromatin and in large neurons, the nuclei seem rather empty". Thus, many large neurons may not have been counted in the Regeur study because their nuclei lacked a diffuse chromatin pattern. Thus, large pyramidal neurons would not have fullfilled their operational definiton of a neuron which would result in an underestimation of total neuron number in the normal aged cortex. The type of morphologic differentiation used in the present study is in marked contrast to previous morphometric studies of AD cortex that are based, in part, on cell size. These latter investigations have shown that perikarya smaller than 401xm a were almost always glial, whereas neurons were almost always greater than 40 i~ma (13). Using these criteria many investigators have been able to classify neurons into at least two size groups: small, between 40 and 90 i~ma in crossectional area, and large, more than 90 I~m2. By using these less subjective morphologic standards, it is not only possible to quantify cell numbers but to determine their laminar distribution within the cortex, i.e., supra or infragranular layers. It is these types of detailed analyses that have led to the belief that the large neurons within cortical layers 3 and 5 are significantly reduced in number in AD (13,14). It is important to
keep in mind that precautions also should be taken in interpreting laminar thickness data because the cortical lamination pattern can be disrupted in severe cases of AD. However, it would be intriguing to differentiate neurons and glia based on cell size and laminar location using the optical disector methodology with the present population of brains. Then compare numbers of neurons between age-matched controls and AD individuals with those obtained using more standard semi-automated methods. Obviously the fact that this comparison would be based on Giemsa stained sections would still troublesome. A variety of methods for the calculation of changes in neuron number have been used in attempts to define the effects of AD on neuronal cell number. This methodological diversity has made it extremely difficult to interpret differences between investigations evaluating human tissue neuron counts. In their review of the literature, Coleman and Flood (3) indicated that these sources of confusion range from the clinical and pathologic criteria used for selection of human cases to the microscopic sampling of brain regions and even cells. Moreover, although difficult to document, observer bias may be a major variable in these type of studies and most certainly exists. Therefore, any study that is not "rigorously blind," as Coleman and Flood (3) point out, " m a y be suspect". The finding of a lack of a significant reduction in total cortical neuronal cell numbers in AD, as suggested by Regeur et al. (11) would alter many long-held assumptions regarding the pathologic impact of this disease on neocortical circuitry. Their observations contrast with the current literature dealing with this topic which shows a significant loss of long projection neocortical neurons in both early and late onset cases of AD (14). Moreover, there is also evidence that many small cortical neurons such as those containing somatostatin are decreased in AD (6). The authors of the present article do not discuss how their findings either support or contrast with the existing literature. If as suggested by Regeur et al. (11) that there is a nonsignificant loss of neocortical neurons between age-matched controls and AD individuals, then it would be necessary to rethink current concepts underlying cortical neuronal changes in this neurodegenerative disease. However, before these novel observations are incorporated into the reality of the scientific community it would be important to replicate the present findings (11) using the optical disector methodology with a large series of cases.
REFERENCES 1. Ball, M. J. Neuronal loss, neurofibrillary tangles and granulvacuolar degeneration in the hippocampus with aging and dementia. Acta Neuropath. (Bed) 37:111-118; 1977. 2. Beach, T. G.; Walker, R.; McGeer, E. G. Patterns of gliosis in Alzheimer's disease and aging cerebrum. Glia 2:420-436; 1989. 3. Coleman, P. D.; Flood, D. G. Neuron numbers and dendritic extent in normal aging and Alzheimer's disease. Neurobiol. Aging 8:521545; 1987. 4. De Lacoste, M-C.; White, C. L., III. The role of cortical connectivity in Alzheimer's disease pathogenesis: A review and model system. Neurobiol. Aging 14:1-6; 1993. 5. Dixon, D. W.; Mattiace, L. A. Astrocytes and microglia in human brain shoare an epitope recognized by 13-Lymphocyte-specificmonoclonal antibody (LN-1). Am. J. Pathol. 135:135-147; 1989. 6. Gaspar, P.; Duyckaerts, C.; Febvert, A.; Benoit, R.; Beck, B.; Berger, B. Subpopulations of somatostain 28-immunoreactive neurons display different vulnerability in senile dementia of the Alzheimer type. Brain Res. 490:1-13; 1989. 7. Iniguez, C.; Gayoso, M. J.; Carreres, J. A versatile and simple method for staining nervous tissue using Giemsa dye. J. Neurosci. Meth. 13:77-86; 1985. 8. Lewis, D. A.; Campbell, M.; Terry, R. D.: Morrison, J. H. Laminar
9.
10. 11.
12. 13. 14.
and regional distribution of neurofibrillary tangles and neuritic plaques in Alzheimer's disease: A quantitative study of visual and auditory cortices. J. Neurosci. 6:1799-1808; 1987. Pearson, R. C. A.; Esiri, M. M.; Hiorns, R. W.; Wilcock, G. K.; Powell, T. P. S. Anatomical correlates of the distribution of the pathologic changes in the neocortex in Alzheimer disease. Proc. Natl. Acad. Sci. USA 82:4531-4534; 1985. Price, D. L. New perspectives on Alzheimer's disease. Annu. Rev. Neurosci. 9:489-512; 1986. Regeur, L.; Jensen, G. B.; Pakkenberg, H.; Evans, S. M.; Pakkenberg, B. No global neocortical nerve cell loss in brain from patients with senile dementia of Alzheimer's type. Neurobiol. Aging 15:347352; 1994. Rogers, J.; Morrison, J. H. Quantitative morphology and regional and laminar distributions of senile plaques in Alzheimer's disease. J. Neurosci. 5:2801-2808; 1985. Terry, R. D.; Davies, P. Some morphologic and biochemical aspects of Alzheimer's disease. In: Samuel et al., Aging of the brain. New York: Raven Press; 1983: 47-59. Terry, R. D.; Hansen, L. A. Some morphometric aspects of Alzheimer disease and of normal aging. In: R. D. Terry, ed. Aging and the brain. New York: Raven Press; 1988:109-114.