A high resolution ex vivo 7T MR imaging protocol is presented, to perform MR-guided histopathological validation of microvascular pathology in post-mortem human brain tissue. Furthermore, guidelines are provided for the assessment of cortical microinfarcts on in vivo 7T as well as 3T MR images.
Cerebral microinfarcts are frequent findings in the post-mortem human brain, and are related to cognitive decline and dementia. Due to their small sizes it is challenging to study them on clinical MRI scans. It was recently demonstrated that cortical microinfarcts can be depicted with MRI scanners using high magnetic field strengths (7T). Based on this experience, a proportion of these lesions is also visible on lower resolution 3T MRI. These findings were corroborated with ex vivo imaging of post-mortem human brain tissue, accompanied by histopathological verification of possible cortical microinfarcts.
Here an ex vivo imaging protocol is presented, for the purpose of validating MR observed cerebral microvascular pathology with histological evaluation. Furthermore, guidelines are provided for the assessment of cortical microinfarcts on both in vivo 7T and 3T MR images. These guidelines provide researchers with a tool to rate cortical microinfarcts on in vivo images of larger patient samples, to further unravel their clinical relevance in cognitive decline and dementia, and establish these lesions as a novel biomarker of cerebral small vessel disease.
The application of ultra-high field 7 Tesla (T) MRI in patient studies is rapidly progressing1. This paper introduces a representative application of 7T MRI in the context of cerebrovascular disease in the aging human brain. Cerebrovascular disease is a major cause of cognitive decline and dementia. This vascular contribution to dementia frequently involves the small vessels of the brain, such as arterioles, small veins, and capillaries. Hence, it is referred to as cerebral small vessel disease (SVD)2. Because the cerebral small vessels are too small to capture with conventional MRI, only the consequences of SVD – i.e., the resulting tissue injury – can be visualized. This includes white matter hyperintensities, cerebral microbleeds, and lacunar infarcts3.
Other important manifestations of SVD are cerebral microinfarcts (CMIs)4. Autopsy studies report high prevalence of CMIs in vascular dementia, and Alzheimer’s disease5. However, due to their small sizes (ranging from 50 µm to a few mm) they escape detection on conventional MRI4,5. 7T MRI provides high resolution images with improved signal-to-noise-ratio and contrast, which enables the detection of certain structures and lesions beyond the detection limit of conventional MRI. This technique was therefore applied to detect CMIs. To identify possible CMIs, many in vivo 7T MRI scans were previously screened for lesions with sizes <5 mm and imaging characteristics consistent with ischemic properties. Such lesions could reliably be identified in the cortex. These focal elongated lesions were hyperintense on 7T FLAIR (0.8 mm isotropic voxels), restricted to the cortex and seemed to extend from the cortical surface, hyperintense on T2 (0.7 mm isotropic voxels), and hypointense on T1 (1.0 mm isotropic voxels). It was confirmed that these lesions were cortical CMIs using an MR-guided histopathology approach in post-mortem human brain tissue6,7.
Here, the ex vivo MRI protocol is presented that was used in previous studies for the histopathological validation of cortical CMIs. Secondly, guidelines are provided for the assessment of cortical CMIs on in vivo 7T MRI. Finally, the assessment of cortical CMIs on 7T has been translated to more widely available 3T MRI, and guidelines are provided how to identify cortical CMIs on 3T MRI.
The use of the autopsy samples and in vivo MR images for this protocol was in accordance with local regulations and approved by the local institutional review board of the University Medical Center Utrecht (UMCU).
1. MR-guided Histopathological Validation of Cortical Microinfarcts
Figure 1. Preparation of formalin-fixed brain slabs for post-mortem scanning at 7T MRI. A purpose-built Perspex container is filled with either 10% formalin or a perfluoropolyether (PFPE) lubricant if MRI signal from the fluid is undesired. Three 10-mm thick formalin-fixed coronal brain slabs are placed in the container. A smaller container is used to keep the slabs in place. Tape the second container to the first one, to prevent movement.
Figure 2. Placement of purpose-built container in 7T head coil. Cover the container with plastic or parafilm to prevent evaporation of the formalin. Place the container, enclosed in a towel or surgical underpad, in the head coil of a 7T MR scanner. Make sure the container cannot move, and that the slabs remain in horizontal position.
2. Assessing Cortical Microinfarcts on In Vivo 7T MRI
Figure 3. Example image viewing platform for the assessment of cortical microinfarcts. An interface is used, integrated in MeVisLab. This program allows to incorporate multiple viewers simultaneously, to switch easily between sagittal / transversal / coronal orientation, and to place and save markers on possible lesion locations. (Different markers can be chosen for different types of lesions).
3. Assessing Cortical Microinfarcts on In Vivo 3T MRI
An impression of the high resolution and high image quality of an ex vivo sequence acquired at 7T is provided here (Figure 4). This is a 3D T2* weighted ex vivo scan, with an isotropic resolution of 0.18 mm. Tissue was derived from an 84-year old demented female with pathologically proven Alzheimer’s disease and severe cerebral amyloid angiopathy (CAA). The detail of the image allows the identification of cortical microvascular pathology. T2* is susceptible for iron, as well as air. This tissue contains a high burden of microvascular pathology within the cortex. The hypointensities in the sulci of these slabs are the results of air bubbles, which can interfere with rating cortical microvascular pathology. For the identification of cortical CMIs, a T2 weighted sequence is required.
Figure 5 represents a cortical CMI identified on ex vivo images at 7T. This cortical CMI was found in the post-mortem brain tissue of an 86-year old female with moderate Alzheimer pathology (Braak & Braak stage IV). The corresponding H&E section verified that this lesion is a chronic gliotic CMI with cavitation7.
Figure 6 is a representative probable cortical microinfarct, detected on in vivo 7T MRI.
Figure 7 is a representative probable cortical microinfarct, detected on in vivo 3T MRI.
Figure 4. Representative post-mortem images acquired at 7T. Please click here to view this video.
This is a 3D movie of a 0.18 mm isotropic T2* weighted image of a case with severe amyloid angiopathy. These brain slabs have been generously provided by Dr. Annemieke Rozemuller, VUMC, Amsterdam.
Figure 5. MR-guided histopathology of cortical microinfarct.
Depicted are a FLAIR, T2, T1, wet tissue, and H&E staining, showing a cortical chronic gliotic microinfarct with cavitation. This figure has been modified from7. Please click here to view a larger version of this figure.
Figure 6. Representative probable cortical microinfarct on 7T MRI.
A cortical microinfarct on 7T is hyperintense on FLAIR and T2, and hypointense on T1. This case is a 45-year old female who suffered from a lobar intracerebral hemorrhage. MR images are a courtesy of Dr. Karin Klijn, UMCU, Utrecht. Please click here to view a larger version of this figure.
Figure 7. Representative probable cortical microinfarct on 3T MRI.
A cortical microinfarct on 3T can best be identified as a hypointense lesion on a 3D T1. The corresponding location on FLAIR and T2 should be hyperintense (in this case) or isointense. This case is a 76-year old female with a clinical diagnosis of Alzheimer’s disease. MR images are a courtesy of Dr. Christopher Chen, NUS, Singapore. Please click here to view a larger version of this figure.
TI | TR / TE | Flip / refocusing angle | Acquired resolution | Matrix size | Slices | Averages | Scan duration | |
(ms) | (ms) | (°) | (µm3) | (h:min:sec) | ||||
T2 | – | 3,500 / 164 | 90 / 40 | 400x400x400 | 500×280 | 100 | 4 | 1:52:03 |
FLAIR | 1,600# | 8,000 / 164 | 90 / 40 | 400x400x400 | 500×280 | 100 | 4 | 4:16:08 |
T1 | 280 | 7.7 / 3.5 | 6 / – | 400x400x400 | 348×348 | 80 | 3 | 0:55:38 |
T2* | – | 75 / 20 | 25 / – | 180x180x180 | 832×834 | 278 | 1 | 4:59:31 |
No sensitivity encoding (SENSE) acceleration was applied. # The TI was determined based on 10% formalin. |
Table 1. Post-mortem scan parameters.
CMIs have attracted increasing attention over the last few years. A growing body of evidence derived from autopsy studies has identified CMIs as important contributors to age-related cognitive decline and dementia4,5. CMIs are now detectable on 7T and also 3T MRI. Optimization and standardization of assessment protocols for these lesions will support rapid implementation of robust and valid CMI detection in cohort studies throughout the world. This will enable a widespread evaluation of the clinical relevance of CMIs in the context of aging, cerebrovascular disease, and dementia in future clinical studies, both cross-sectional as well as longitudinal9,10.
The described method in this work is actually a ´meta-method’ in the sense that it can be regarded as a procedure to develop (and successively improve) methods for in vivo detection of CMIs, which could thus far only be assessed post-mortem by a neuropathologist. The most critical step in developing improved in vivo CMI detection methods by new MRI protocols and image processing techniques is to validate it with histology, which is currently the gold standard. An important limitation of the in vivo detection of CMIs compared to histology is resolution. However, despite the fact that in vivo MRI will not be able to detect the smaller CMIs, it does provide whole-brain coverage, which might prove to be as effective as looking for microscopic CMIs on just a few histological sections.
An important step to establish the in vivo guidance for CMI rating was the histopathological validation of CMIs, guided by ex vivo high resolution MRI of post-mortem human brain tissue. The ex vivo scan protocol presented here is optimized for the validation of cortical microvascular pathology, but can be applied in a broader research context, to support in vivo rating of other novel brain imaging markers. Scanning post-mortem human brain tissue has its challenges, which should be acknowledged here. Prolonged storage of formalin-fixed tissue can cause artifacts8. Other challenges are MRI artifacts caused by air bubbles, because air bubbles can interfere with the MR signal, especially at fluid-tissue boundaries. Therefore, the removal of the air bubbles is an important step. Air easily accumulates in empty blood vessels, between gyri, and between slabs. To overcome the latter, one would ideally scan a whole block of un-cut tissue. However, part of standardized autopsy procedures is cutting the formalin-fixed tissue in 10-mm thick slabs. Scanning post-mortem tissue requires extra attention to ensure sufficient B0 shimming and correct RF power optimization (step 1.1.10). This may need specific attention from a local MRI physicist. These steps are dependent on the vendor of the 7T MRI scanner and may be optimized according to the desires of the individual research group. During scanning, slabs are either submerged in 10% formalin or in a PFPE lubricant, which is a proton-free fluid without MR signal. The advantage of using a proton-free fluid is that it minimizes the required field-of-view, it enables better B0 shimming, and it doesn’t penetrate the tissue as it is highly hydrophobic. Disadvantages are that it is expensive, and can be impractical in use (being an oily substance). Formalin is much easier in use, is cheap, and doesn’t interfere with the tissue when the tissue is already formalin-fixed. The disadvantage of formalin is that it may cause RF inhomogeneity at 7T, when scanning large volumes (e.g., whole brains), and that the substance is toxic. Another frequently applied embedding substance for ex vivo MRI is agar gel. Agar is ideal for scanning single slabs or individual pathological specimens, and the major advantage is the reduction of potential movement. Also, it allows the placement of fiducials to use as artificial landmarks11.
In the current in vivo imaging example of cerebral SVD at 7T, the following sequences were used: a 3D FLAIR (0.8 mm isotropic voxels), 3D T2 (0.7 mm isotropic voxels), 3D T1 (1.0 mm isotropic voxels), and T2* (0.5×0.5×0.7 mm3 voxels). The applied 7T FLAIR is heavily T2 weighted, and therefore highly suitable for visualizing minute ischemic lesions12. The T2* sequence has been included for the detection of cerebral microbleeds13, but may also be used to check CMI locations in the absence of a T2. It should be noted that the current 7T FLAIR sequence does not allow a reliable assessment of the temporal lobes, due to a low signal-to-noise ratio in these areas. Other research groups inevitably want to use their own FLAIR and T2 weighted protocols, but this may lead to different sensitivity regarding CMI detection.
The translation of the 7T rating criteria to the assessment of cortical CMIs on in vivo 3T MR images is important to allow the investigation of CMIs in larger patient samples. However, there are a few challenges to take into account. First, ensure to include at least one 3D sequence in the 3T MRI scan protocol, which might not be the standard procedure in most clinically applied protocols. Secondly, a FLAIR sequence at 3T is usually less heavily T2 weighted than on 7T. That is the reason it is recommended to assess cortical CMIs on 3D T1 weighted images, with confirmation on FLAIR and T2 (if available), and in case of doubt T2*. For the purpose of the current CMI rating guidelines described in this protocol, a high resolution 3D T1 (1.0 mm isotropic voxels), 2D FLAIR (1.0×1.0x3.0 mm3 voxels), and 2D T2 (1.0×1.0x3.0 mm3 voxels)9 were used. These images were acquired on a 3T MRI system, with a 32-channel receive head coil.
A few limitations of in vivo CMI rating are in place. Assessing CMIs visually on in vivo MR images remains challenging and is clearly rater dependent. It requires training, but even with the proper experience these small lesions easily escape detection by the human eye. Furthermore, rating CMIs is rather labor intensive and time-consuming, especially when applied to larger samples. Therefore, it is of importance to develop (semi-)automatic detection methods for the identification of CMIs that aid the visual rating14. It should be acknowledged that 3T MRI only detects the larger CMIs. The same applies for 7T, albeit to a lesser extent. Nevertheless, the CMIs that are detected on MRI do have important and specific clinical correlates9.
The authors have nothing to disclose.
The research leading to these results has received funding from the European Research Council under the European Union’s Seventh Framework Programme [FP7/2007-2013] / ERC grant agreement [337333]. The research of SvV and GJB is supported by a VIDI grant [91711384] from ZonMw, the Netherlands Organization for Health Research and Development.
Fomblin / Galden PFPE | Solvay Solexis, Bollate, Italy | ||
7T MR system | Philips Healthcare, Cleveland, OH, USA | ||
32-channel receive head coil | Nova Medical, Wilmington, MA, USA | ||
MeVisLab | MeVis Medical Solutions AG, Bremen, Germany |