Domizio Suvà⁽¹⁾, Isabelle Favre⁽²⁾, Rudolf Kraftsik⁽³⁾, Monica Esteban⁽⁴⁾, Alexander Lobrinus⁽⁵⁾, Judit Miklossy^{(6)
(1)(2)(4)(5)(6)}CHUV, Institute of Pathology - Lausanne. Switzerland
⁽³⁾IBCM - Lausanne. Switzerland

	[Neuroscience]	[Pathology]

INTRODUCTION

Since its first description at the beginning of this century ^(1-6), the clinico-pathologic entity of Alzheimer´s disease (AD) ^(7-9) continues to rise many questions, well summarized in a report of Khachaturian published in 1985 ⁽⁸⁾.

In AD, the involvement of the entorhinal cortex, hippocampus, as well as of the frontal and parietal associative cortical areas is well established ^(10-20). It is generally accepted that the primary motor cortex is less involved or even spared in AD ^(21-28). Only few case reports described severe involvement of the primary motor cortex in AD ^(29,30).

As the reported data are contradictory, the aim of this study was to perform a quantitative analysis of senile plaques and neurofibrillary tangles, to answer the question whether or not the primary motor cortex is involved in AD. The severity of the cortical changes in the primary motor cortex was compared to those of the enthorinal cortex, frontal and parietal associative areas known to be severely involved in AD.

MATERIAL & METHODS

The brains of 29 autopsy cases were analyzed. The brains were fixed in 10% formalin for 4 weeks. From all the 29 brains about 3 x 3 x 0.5 cm large samples were taken from the following cortical regions: entorhinal cortex, hippocampus, frontal associative cortex (Brodmann´s area (Br) 8, 9), parietal associative cortex (Br. 39, 40), primary motor cortex (Br. 4) and primary sensory cortex (Br. 3,1,2). In 9 cases an additional sample from the primary visual cortex (Br. 17) was also taken. After embedding in paraffin, from all these blocks 5um thick paraffin sections were cut and stained with haematoxylin and eosin, Thioflavine S, Congo red, Gallyas silver technique ⁽³⁰⁾ and immunostained with monoclonal antibody to beta-amyloid protein (DAKO, M872, dil.1:100). In some cases, sections were also stained with the cresyl-violet technique and immunostained with a rabbit polyclonal anti-myelin basic protein (MBP) (DAKO, A623, dil. 1:100) for the analysis of cyto- and myelo-architectonics, respectively.

For immunostaining the avidin-biotin-peroxidase technique was used. The sections were deparaffinized, rehydrated and incubated in 0.3% mathanolic peroxide for 30 minutes, in order to eliminate endogenous peroxidase activity. The sections were incubated overnight at 4°C with the primary antibody. The biotinylated secondary antibody and the avidin-biotin complex were used following recommendations of the manufacturer (DAKO, ABComplex/HRP-Kit, K0355). For the visualization of the immunoreaction diaminobenzidine was used as chromogen. After immunostaining, the sections were counterstained with haematoxylin. For the detection of beta-amyloid, before immunostaining, the sections were pre-treated with formic acid for 20 minutes.

For the neuropathological diagnosis of AD, in all cases, a semi-quantitative analysis of senile plaques and neurofibrillary tangles was performed on different cortical regions (entorhinal cortex, hippocampus, frontal and parietal associative areas), as described in a previous study ⁽³²⁾. For the diagnosis of AD, criteria proposed by the Consortium to Establish Registery for Alzheimer´s Disease (CERAD) ⁽⁹⁾ and those proposed by Khachaturian were both considered ⁽⁸⁾. The severity of cortical involvement by neurofibrillary tangles was graded following Braak ⁽²⁰⁾. Based on these staging procedures 17 cases with severe AD-type cortical changes fulfilled the criteria of the neuropathological diagnosis of AD (Table 1, cases 1-17). In these AD cases the severity of the cortical AD-type changes corresponded to Braak stages V-VI. The age of these patients varied between and 94 years. Dementia was clinically documented in all cases, therefore these 17 cases also fit the diagnosis of AD following the guidlines of CERAD ⁽⁹⁾. In addition to dementia signs of pyramydal involvement was clinically documented in three cases which included Babinski sign and/or spacticity. Seven patients, aged 76 to 89 years, had discrete to moderate AD-type cortical changes, insufficient for a diagnosis of AD (Table1, cases 18-24). These patients were not demented, except one, in which the neuropathological examination revealed hypertensive encephalopathy. In this group the severity of cortical lesions following Braak corresponded to stages I-II in 5 cases (Table1 cases 18-22) and to III-IV in two cases (Table1, cases 18-22). The remaining 5 non-demented cases, aged 37 to 61 years, without any AD-type cortical changes formed the third group and served as controls (Table1, cases 25-29).

In all 29 cases, a quantitative analysis of senile plaques and neurofibrillary tangles was performed on sections derived from the following cortical regions: entorhinal, frontal associative (Br. 8, 9), parietal associative (Br. 39, 40), primary motor (Br. 4) and primary sensory cortex (Br. 3, 1, 2). In order to verify the reproducibility and reliability of the results, in all cases and on all sections the quantitative analysis of plaques and tangles was repeated independently by another investigator.

Morphometric analysis of senile plaques was performed on beta-amyloid stained sections. Immunostained deposits of beta-amyloid of all type of senile plaque were considered ⁽³³⁾. The analyzed cortical areas were always selected from regions free of vessels with beta-amyloid deposits. Using a computer assisted Leitz microscope microscopical images each corresponding to 0.16 mm2 of cortical surface visualized using X10 objective were captured with a video camera and Samba Immuno v.4.05 software (for Microsoft Windows). The morphometric analysis was made on these online images. In selecting the brown color of beta-amyloid deposits we were able to eliminate the blue color of haematoxylin-stained nuclei. After the subtraction of background noise and histology artifacts, the total surface occupied by beta-amyloid plaques was measured and expressed as a percentage of the totalcortical area studied. The analysis was performed in 5 fields selected from the most severely affected parts of each cortical areas. Of the 5 fields a mean was calculated.

The quantitative analysis of neurofibrillary tangles was performed on Gallyas-stained sections using a Leitz microscope. The number of tangles were counted in 0.4mm2 microscopic fields obtained using 10x power eye-piece and 25x power objective lens (250x magnification). Like for senile plaques, the quantitative analysis of neurofibrillary tangles was performed in 5 different fields of the histologically identified most severely affected regions of each cortical area. Tangles localized on the border of the upper half field were counted, those situated on the borderline of the lower half field were ignored. The results were expressed as the mean number of tangles per 0.4mm2 microscopic field (the total cortical area studied).

In order to determine the reliability of the quantitative study, the analysis of variance (ANOVA) was used to compare the results obtained by the two investigators. The same statistical method was used to compare the mean percentage of cortical surface occupied by senile plaques and the mean number of tangles between the 5 different cortical areas.

The Pearson correlation was calculated and tested for significance in order to analyze if an association exists between the severity of cortical changes among different cortical areas.

beta-amyloid and Gallyas-stained sections were used to analyze the laminar distribution of plaques and tangles with the aid of a Zeiss computer microscope system ⁽³⁴⁾. In some cases on Nissl stained sections, the neuronal loss in layer Va and Vb of the primary motor cortex was documented using the same computer microscope and the Neurolucida mapping software from Microbrightfield Inc.

RESULTS

The results of the quantitative analysis of plaques and tangles obtained by both examiners were similar without statistically significant difference, indicating their reproducibility and reliability. We thus used both datasets, giving us a mean value for ten cortical fields for both plaques and tangles. These data are presented in Table1.

In the brain of all 17 AD cases the primary motor cortex was severely affected by senile plaques (fig. 1, A). The percentage of cortical surface occupied by plaques in the primary motor cortex was similar to that found in other cortical areas (Table1, fig. 1 and fig. 2A). In 2 cases the percentage of cortical surface occupied by senile plaques was even higher in the primary motor cortex than in the associative cortical areas (Table1, cases 4, 5), and in two other cases it was higher than in the parietal associative cortex (Table1, cases 6, 16).

The number of neurofibrillary tangles in the primary motor cortex was lower than in the entorhinal cortex, and slightly lower than in the associative cortical areas (Table1, fig. 2B) with the exception of two cases. Case 9 showed higher number of tangles in the primary motor cortex than in the associative cortical areas and in two other cases (cases 12 and 13) the number of tangles was higher than in the entorhinal cortex (Table1).

The majority of cases with discrete to moderate AD-type cortical changes showed the presence of senile plaques in all examined areas including the primary motor cortex, except in one case, in which no plaques were found (case 21) in the primary motor and sensory cortex. Neurofibrillary tangles in these 7 cases were restricted to the entorhinal cortex, except in the two cases where few tangles were counted in the associative cortical areas. In one case (case 24 of Table1) some neurofibrillary tangles were also found in the primary motor cortex. There were no plaques or tangles in any cortical areas of the brain in the control group.

There were some regional differences in the distribution of plaques and tangles with respect to different cortical areas. In 1 case (Table1, case 3) the cortical surface occupied by plaques was more than five times higher in the frontal than in parietal cortex, respectively.

The distribution of the cases with respect to the increasing percentage of cortical surface (by 2% intervals) occupied by plaques and the increasing number of neurofibrillary tangles (by intervals of 3 tangles) in the primary motor cortex is illustrated on fig. 3 A and B, respectively. In the majority of AD cases (11 of the 17) the percentage of cortical surface occupied by senile plaques in the primary motor cortex was high (14-20%). In the 7 cases with discrete to moderate cortical changes it was equal or lower than 12%.

In the majority of AD cases the number of tangles in the motor cortex varied between 3 and 18. In all the seven cases with discrete to moderate cortical changes the number of tangles was less than 3 and in the 5 control cases it was zero.

The primary sensory cortex was also severely involved by AD-type changes by both plaques and tangles, in proportions similar to that of the primary motor cortex (Table1).

In the group of the 17 AD cases the statistical analysis did not reveal significant difference between cortical areas regarding the percentage of cortical surface occupied by senile plaques in the primary motor cortex and in the other cortical areas. The number of tangles was significantly higher in the entorhinal cortex when compared to the other cortical areas (p<0.001). Their number was somewhat lower in the primary motor and primary sensory cortex than in the associative cortical areas, with a difference of borderline significance (p=0.04) There was no significant difference between the number of tangles in frontal and parietal associative cortical areas, or between the primary motor and sensory cortex.

The severity of the primary motor cortex involvement by senile plaques strongly correlated with that of primary sensory (R=0.8; p<0.001), enthorhinal (R= 0.7; p=0.004) and frontal associative cortex (R= 0.6; p=0.01) (fig. 4). Concerning the severity of the cortical involvement by neurofibrillary tangles a high correlation was found between the primary motor cortex and the sensory (R=0.9; p<0.001), parietal (R=0.7; p=0.009) and frontal associative cortex (R=0.6; p=0.007).

Due to the severe degenerative process, the cyto- and myeloarchitectonic hallmarks to delineate cortical layers are less distinct in the 17 AD cases than in the control cases. We did not observed laminar distribution of senile plaques in the primary motor cortex (fig. 5). In a few cases a slight tendency for a laminar distribution with a higher density of plaques in layers III and V was noticed. A tendency for laminar distribution of neurofibrillary tangles was observed in the primary motor cortex, where tangles were more numerous in the superior part of layer III and in layer V (fig. 5). In the primary sensory cortex, a laminar distribution, with high density of plaques in the inferior part of layer III and in layer IV was observed. We did not observe laminar distribution of neurofibrillary tangles in the primary sensory cortex. In the 17 AD cases on haematoxylin-eosin and cresyl-violet-stained sections neuronal loss was observed in all cortical layers of the primary motor cortex including layers Va and Vb. The loss of neurons in layer V, including those of the Betz cells in layer V/b in a familial AD case is illustrated in fig. 1E and F. The severity of neuronal loss, including those of Betz cells varied in different cases. We did not observe neuronal loss in the 5 control cases.

In the 9 severe AD cases (group AD, cases 1, 7, 8, 9, 10, 11, 12, 14, 16) where the primary visual cortex was available for analysis, a high number of senile plaques and a high to moderate number of neurofibrillary tangles was observed in this area. Only in one case (case 10) we did not find tangles in the primary visual cortex.

DISCUSSION

It is generally accepted that the entorhinal cortex, hippocampus but also the frontal and parietal associative areas are severely involved in AD. The primary motor cortex is known to be less involved or even spared in AD ^{(11,13-16,18,20,23,24,28)}. Based on these data, several authors have proposed that the distribution of plaques and tangles through the cerebral cortex may follow neuronal connections ^{(12,17,19,22,26)}, and that the involvement of the associative cortical areas is correlated with their connections to the limbic areas.

Senile plaques are generally reported as being the most abundant in the associative neocortical areas, less numerous in entorhinal cortex and hippocampus, with the lowest density in the primary cortical areas ^{(12,16,20,21)}. Neurofibrillary tangles are consistently reported as being very numerous in the entorhinal cortex, followed by hippocampus, the neocortical associative areas and finally the primary projection areas.

Severe involvement of the primary motor cortex in AD was only occasionally reported ^(29,30,35). Perretti ⁽³⁶⁾ suggested, after obtaining abnormal electrophysiologic responses in abductor pollicis brevis and tibialis anterior muscles in AD patients using transcranial magnetic stimulation of the motor cortex, that sub-clinical dysfunction of the motor cortex neurons is present in AD before the clinical signs become apparent. Specific signs of pyramidal involvement, including Babinski sign, increased deep tendon reflexes and spasticity have been reported to occur in rare AD cases ^(29,30,37). Moreover, the occurrence of AD-type cortical changes in the primary motor cortex has been described in progressive supra-nuclear palsy and in amyotrophic lateral sclerosis ^(38,39).

Here, using a quantitative analysis, the primary motor cortex of 17 AD cases was analyzed for the occurrence of cortical AD-type changes. The great variation in size and shape of senile plaques for a same patient and from a patient to another ^(20,33) makes counting of plaques poorly reproducible. We therefore, we have chose to measure the percentage of cortical surface occupied by senile plaques. The results of the morphometric analysis showed that in all the 17 AD cases the primary motor cortex was severely affected. The percentage of cortical surface occupied by senile plaques was as high as in the other cortical areas, including the associative frontal and parietal areas. In a few AD cases the number of plaques and/or tangles was even higher in the primary motor cortex than in the entorhinal or associative cortical areas.

In agreement with the generally accepted view our quantitative analysis of neurofibrillary tangles showed that the most involved cortical region is the entorhinal cortex. The number of tangles was significantly lower in the associative cortical areas followed by the primary motor and primary sensory cortex. The difference between the number of tangles in associative areas and primary motor cortex showed only borderline significance.

In the majority of cases with discrete to moderate AD-type cortical changes, senile plaques were found in reduced number in all cortical areas including the primary motor cortex, suggesting an early appearance of plaques in the primary motor cortex. In one case with Braak stage IV a few neurofibrillary tangles were also present in the primary motor cortex.

In two AD cases the accumulation of plaques in the frontal cortex was much more severe than in the parietal cortex, indicating that regional variation of the severity of cortical changes may occur in AD.

The severity of the primary motor cortex involvement by senile plaques was strongly correlated with that of primary sensory, enthorhinal and frontal associative cortex. The correlation was also high between the number of neurofibrillary tangles the primary motor cortex and the sensory, parietal and frontal associative cortex.

The other primary cortical areas, the primary sensory and visual cortex were also involved in AD. There was a high correlation between the involvement of the primary motor and sensory cortex.

If we consider the histopathological criteria of AD following Khachaturian ⁽⁸⁾ or CERAD ⁽⁹⁾, where the diagnosis of AD depends particularly on the number of senile plaques, our results indicate that the involvement of the primary motor cortex is as severe as those of the frontal and parietal associative areas. However, if we consider the number of neurofibrillary tangles, which is even more significantly correlated with dementia severity ⁽⁴¹⁾, in spite of an important number of tangles in the primary motor cortex in all AD cases, their number is somewhat lower than in the associative areas. The distribution of neurofibrillary tangles suggest a progressive involvement of the cerebral cortex from the entorhinal cortex, hippocampus, through the associative cortical areas, to the primary motor and sensory cortical areas. Further analysis of the distribution of tangles in a high number of cases with discrete, moderate and severe AD-type cortical changes would be of interest to address this point more accurately.

In addition to the accumulation of plaques and tangles we have observed neuronal loss in all layers of the primary motor cortex, including those of the Betz cells in layer V/b. A quantitative analysis of neuronal loss in the primary motor cortex would add further information concerning the severity of the primary motor cortex involvement in AD.

Our results indicate the primary motor cortex is affected in AD and may well lead to severe motor dysfunctions in the late stages of the disease. In a prospective study, the neurological examination of motor functions followed by the neuropathological analysis of the primary motor cortex in AD patients would permit to define more accurately the clinical significance of the involvement of the primary motor cortex observed in this study.

Our findings suggest that AD affects not only the phylogenetically new and vulnerable associative brain regions and their connections ⁽²⁴⁾. With the progression of the disease the primary motor cortex became also severely involved, suggesting that motor dysfunctions will appear in late and terminal stages of the disease.

ACKNOWLEDGEMENTS

We are grateful to S. Burki and J. Maillardet for the photographic support, to P. Darekar, S. Testuz, S. Gros and S. Trepey for technical assistance in histology, to F. T. Bosman for interesting advices, and finally to T. Suvà for editorial advice.

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Discussion Board

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[ABSTRACT] [INTRODUCTION] [MATERIAL & METHODS] [RESULTS] [IMAGES] [DISCUSSION] [ACKNOWLEDGEMENTS] [BIBLIOGRAPHY] [Discussion Board]

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	[Neuroscience]	[Pathology]

Domizio Suvà, Isabelle Favre, Rudolf Kraftsik, Monica Esteban, Alexander Lobrinus, Judit Miklossy
Copyright © 1999-2000. All rights reserved.

Last update: 14/01/00