In a previous study, we demonstrated that layer II islands are visible in the entorhinal cortex using ex vivo
] and that these islands could be used to localize entorhinal cortex in vivo
]. Here we built on this model, and created entorhinal surface reconstructions to quantify spatial and morphometric properties of the verrucae. Utilizing ex vivo
MRI, we confirmed that entorhinal islands colocalize with entorhinal verrucae, that is, that each island is beneath a verruca, as originally detailed previous neuroanatomical studies [30
]. We validated our model with qualitative assessment in the gross specimen and showed that verrucae measurements accurately reflect their true size. The validated models were then used to demonstrate that entorhinal surfaces in control cases differed significantly from other cortical regions. Finally, with this morphometric model, we determined that entorhinal verrucae in control cases were significantly larger than mild AD cases, and that verrucae size significantly and negatively correlated to Braak and Braak stage determined by NFTs in all stages.
The novelties of our study include unique application of imaging methods on entorhinal surface reconstructions (i.e. new model development), quantitative demonstration of individual verruca size (height, width, surface area and volume), establishment of verrucae ratings or grades (i.e. qualitative assessment of verrucae in control and Alzheimer’s disease cases) and finally correlation of verrucae rating to Braak & Braak neurofibrillary tangle stages. No prior study has described individual verruca size quantitatively, or its correlation to AD severity and with high resolution imaging methods.
Visible to the unaided eye, neuroanatomists described entorhinal verrucae a century ago [30
]. The human eye (20/20 vision) can resolve objects about 0.1mm long which is consistent with our verrucae measurements of approximately 0.13 mm in height (mean) and 1.26 mm in width (mean) for control cases. In AD, however, verrucae become atrophic and shrink, and are not observed in severe cases [44
]. Our results confirm these findings, and demonstrate that a semi-automated model derived from neuroimaging data can provide a quantitative assessment of individual verrucae sensitive enough to detect and quantify AD-related atrophy even in its mildest stage.
We compared our findings from entorhinal cortex to neocortical and other limbic surfaces to demonstrate that the method utilized is specific to regions of the brain containing verrucae. The lack of height in neocortical areas demonstrates that our quantitative measures correspond to what can be observed visually. It is noteworthy that since our voxel size represents isotropic data at 100μm3 and since verrucae means were 0.13 mm and 1.26 mm for control cases, each verrucae measurement symbolizes several (~13) voxels in width and at minimum one voxel in height. In our mild AD cases, verrucae width (mean) was 1.14 mm and height was 0.11 mm and contained several (~11) voxels in width and at minimum one voxel in height. Given this relationship and high resolution, we avoid partial voluming issues even in mild AD cases as quantitative measurements were not performed on severe cases.
Prior work has examined entorhinal verrucae. Our data demonstrate a decrease in verrucae size with a disease process (Alzheimer’s disease) whereas the Simic and colleagues examined normal aging [43
]. These two studies examined different matters. The extensive study by Simic which used molded casts reported the total number of verrucae on the surface, overall entorhinal surface area and counted neurons in non-demented aging. Our study quantified the size of individual verrucae (height, width, volume and surface area) and compared the size to Alzheimer diagnosis and severity (Braak and Braak stage). Additionally, the prior study examined a sample of tissue spanning a wide range of adult life from age 23 to age 85 and had 60 cases without cognitive impairment (i.e. normal controls and/or normal aging) nor neurological incident. In the current study, NFTs were correlated with verrucae morphology. It could be postulated that verrucae increase in total number during aging from cross-sectional data [43
] but once the Alzheimer’s cascade begins, then the verrucae decrease in size and eventually disappear due to neuronal loss that gives way to neurofibrillary tangles. Alternatively, Simic and colleagues suggested that verrucae may split into smaller verrucae and that may account for the increase in total number of verrucae during aging. With regard to normal aging, the Simic study remains the most significant report to date cataloging the number of verrucae as a function of age, and has provided the best examination of verrucae in younger persons and healthy older adults with most of their samples gifted from individuals without neurodegenerative diseases. Our study further characterized the verrucae trajectory in Alzheimer’s disease and showed differences in verrucae size, suggesting that verrucae height and volume were most affected in our AD samples and related to disease severity. We further showed that all verrucae measures were significantly different in mild AD compared to normal controls. Taken together, these results begin to establish the normal architecture and magnitude of the verrucae in healthy aging and AD.
We speculate that the mechanisms that account for the reduction of verrucae are neuronal loss, neuronal shrinkage due to neurofibrillary tangles and/or decreased synaptic connections in entorhinal cortex layer II. Neurofibrillary tangles are typically smaller in size than a functioning healthy neuron [28
] so that decrease could account for the decrease in verrucae size we observed in AD. Neuronal diameter measurements have been reported in AD where decreased neuronal diameter was demonstrated in AD compared to controls and that neuronal size was 30% smaller in neurons with neurofibrillary tangles [34
]. Decreased neuronal measurements have also been observed in the aging brain in neocortical areas, especially frontal and temporal areas [48
]. It is important to note that that the presence of a neurofibrillary tangle means that a neuron has died [56
] and that neurofibrillary tangles indicate neuronal loss [5
]. Age related and Alzheimer’s related neuronal loss has been reported [20
], but future studies will have to determine whether or not verrucae size relates to stereologically defined neuronal loss. Since Braak and Braak staging is based on the presence of neurofibrillary tangles in the medial temporal lobe [10
] and given the fact that neurofibrillary tangles have been strongly correlated to clinical dementia by several groups [2
] and in large sample studies [41
], we consider using Braak and Braak staging to assess the severity of cases the closest pathological metric to cognitive behavior. There is little doubt that the presence of neurofibrillary tangles, more than a few isolated tangles, signifies the person has memory impairment [36
]. Synaptic degeneration is also a variable in AD and several studies have demonstrated decreases in the number of synapses [1
], and decreases in synaptic proteins (i.e. synaptophysin) [11
] in hippocampus and entorhinal cortex in AD, notably early in disease progression. Some evidence suggests that decreases in synaptic density occur after age 65 [38
] and others have demonstrated a correlation between dystrophic neurites and severity of dementia [6
]. Even so, neuronal cell death is likely the principal basis for the reduction in verrucae size with a smaller contribution due to other mechanisms such as synaptic degeneration. It is possible that collectively these mechanisms may be responsible for the reduction in verrucae size in AD.
Given that cases with large verrucae were less likely to have AD, our results beg the question; do large verrucae represent cognitive resilience and healthy aging? It is unquestionable that cognitive resilience, or cognitively normal cases with AD pathological changes, is an existing, albeit small, category in neuropathology [8
]. Even in cognitively normal cases, most of our cases contained a few NFTs and this was consistent with existing studies that have described NFTs in aging [3
]. The density of NFTs and decreased neuronal numbers remain reliable correlates that predict dementia [2
]. Our results suggest that verrucae height measures below 0.12 mm predict a mild AD diagnosis. Given that we observed the largest height in an individual verrucae was 0.25mm and that verrucae in mild AD cases were typically below 0.12 mm and that flat cortex is less than 0.10 mm, a quantitative range exists that may correspond to cognitively healthy aging and mild cognitive impairment. Variability in neurofibrillary tangles, early in the disease, has been reported in Alzheimer’s disease [22
]. Currently it is unknown what accounts for this variability in verrucae size. This may be explained by cognitive resilience where variation in verrucae size across cases may account for categorical groups: AD (Alzheimer’s dementia), frail (dementia without neuropathological markers), cognitive resilience (no clinical dementia but with neuropathological markers), and normal controls (cognitively normal and no pathological markers). Future studies will have to establish these relationships with cognitive and behavioral data. Nonetheless, the characterization of verrucae not only contributes to understanding a biological phenomenon but also establishes verrucae absence as a metric for earlier AD diagnosis and may provide initial evidence for future cognitive resilience hypotheses.
While the resolution used here isn’t achievable using current clinical in vivo
technology, further characterization of verrucae with ex vivo
methods allows us to define the neural correlates of this unique structure, and examine the variability between individuals. Verrucae detection will require isotropic voxels that will challenge neuroimaging technologies as structures require 3D assessment. Nevertheless, neuroimaging continually improves resolution and acquisition techniques such as motion correction or phase imaging that may advance avenues of early detection [16
]. Most importantly, the correlation of histology and ex vivo
MR imaging provides a tool for validation with many proteins, neurons and pathological states and these results offer a bridge to applying these tools to in vivo
imaging when available.
In summary, our data indicate that verrucae changes, particularly in height and volume, may reflect the pathological presence of NFTs (i.e. neuronal loss) and provide a morphological marker to diagnose AD. Entorhinal verrucae represent a highly specific structural marker for detecting AD early in the course of the disease, and provide a potentially sensitive marker for future neuroimaging. The capacity to determine localized changes in relation to pathology may have considerable repercussions in terms of clinical diagnosis of AD and future applications of this technique could identify changes in other disorders as well.