Dissection and tomographic imaging of the brain specimen

The ventral aspect of the brain, after the removal of the leptomeninges, clearly showed the scars of the operation on both hemispheres (Fig. 1). On the surface, the right and left lesions appeared roughly symmetrical in length and in respect to anatomical landmarks; however, the full extent of the injury could only be determined upon dissection. Serial sectioning and direct tomographic imaging was performed in the coronal plane in alignment with the anterior and posterior commissures (AC-PC); these interhemispheric fibre bundles run perpendicular to the major axis of the brain and form the reference points for a widely-used standard radiologic stereotaxic system23. AC-PC alignment ensured that histological sections could be compared with previous scans of H.M.’s brain19,20, those of other patients24,25,26,27, and neuroimaging data from population-based studies28. Rigorous stereotaxic orientation also guaranteed that any interhemispheric differences in the length of the lesion or in the morphology of the spared hippocampus noted in the histological images reflected actual anatomical asymmetry rather than unconventional planes of section.

Figure 1: Ventral surface of H.M.’s brain. The fixed specimen was photographed after removal of the leptomeninges. Evidence of the surgical lesions in the temporal lobes is highlighted by white geometric contours (a, b). A mark produced by the oxidation of one of the surgical clips inserted by Scoville is visible on the parahippocampal gyrus of the right hemisphere (black arrow). (c) encloses a lesion in the orbitofrontal gyrus that affects the cortex and WM. Marked cerebellar atrophy is consistent with H.M.’s long-term treatment with phenytoin. Scale bar, 1 cm. Full size image

The results of our examination are based on 2,401 digital anatomical images and selected corresponding histological sections that were collected at an interval of 70 μm over the course of an uninterrupted 53-hour procedure. The series of digital images of the block’s surface was obtained using a digital camera mounted directly above the microtome stage. Volumetric reconstruction from these images was the basis for subsequent visualization and 3D measurements along arbitrary planes. The dissection of the brain was video-recorded and streamed live on the web to permit scientific scrutiny and to foster public engagement in the study29.

3D anatomical measurements in the MTL

Using the 3D measurement tools in AMIRA (FEI Visualization Science Group, Burlington, MA, USA) we calculated the distance in each hemisphere from the anterior tip of the temporal lobe to the posterior boundary of the surgical lesion on each side. This limit was marked by the most anterior coronal anatomical images that did not show any sign of disruption in the normal anatomy, which was confirmed in corresponding stained histological slices. The lesion followed a straight, but slightly oblique path relative to the long axis of each hemisphere; measured along this axis, its length was 54.5 mm and 44.0 mm in the left and right hemispheres, respectively. The value for the left hemisphere was consistent with earlier measurements made on H.M.’s MRI scans acquired in 1992–1993 (ref. 19) and 2002–2004 (ref. 20). The lesion in the right hemisphere as measured in our postmortem data was 7 mm shorter than in the above-mentioned reports that were based on in vivo imaging.

The borders of the surgical resection were clearly demarcated in the anatomical and histological images; the latter clearly showed that the WM underlying the excised medial temporal cortex was also damaged (Fig. 2). We identified a small portion of the superior-most region of the EC in both hemispheres based on its distinctive cytoarchitecture; specifically, 0.03 cm3 and 0.11 cm3 for the left and right hemispheres, respectively (these values correspond approximately to 1.7% and 6.5% of the normal volume, based on published MRI estimates30). Portions of the centromedial nucleus of the amygdala were also preserved (Fig. 2h,i).

Figure 2: Anatomical and histological views of the lesion and residual hippocampus. (a,d,g,j) Cross-sectional anatomy of patient H.M.’s MTL shown at four different levels. The values below the tissue indicate the section number and in parenthesis, the distance from the origin of the standard coordinate system23 (positive if the level is anterior to the anterior commissure, negative if posterior). Scale bar, 1 cm. (b,c), (e,f), (h,i), (k,l): close-up images acquired from thionin-stained tissue slices; these panels illustrate histological detail for the selection boxes in a, d, g, j, respectively. Scale bar, 5 mm. (m) Horizontal cross-sectional view reconstructed orthogonally from the original coronal images showing the correct alignment of the anterior and posterior commissures. (n–q): normal anatomy and histology of the MTL at the levels shown for the brain of patient H.M. The images were derived from brain slices belonging to a neurologically normal, age-matched individual. Scale bar, 5 mm. PPo, planum polare; MTG, middle temporal gyrus; ITG, inferior temporal gyrus; ITP, inferior temporopolar cortex; PRC, perirhinal cortex; CMA, centromedial amygdala; opt, optic tract; LV, lateral ventricle; PHG, parahippocampal gyrus; Ent, entorhinal cortex; LGN, lateral geniculate nucleus; Hp, hippocampus; FuG, fusiform gyrus; fi, fimbria; SN, substantia nigra; FL, frontal lobe; Cd, caudate; Pu, putamen; I, insula; AC, anterior commissure; Cl, claustrum; TL, temporal lobe; PC, posterior commissure; Ce, cerebellum; OL, occipital lobe; OrG, orbital gyrus; Pir, pirifom cortex; Amg, amygdala. The asterisks delimit the entorhinal cortex. Full size image

The extent of the spared hippocampus measured along the horizontal axis of the brain (which in our study coincided with the AC–PC line) was 23.6 mm in the left hemisphere and 24.3 mm in the right. Our measurements included the alveus and the thin band of WM that encapsulates the posterior end of the hippocampus; the fimbria was excluded from our delineations. The discrepancy between these new values and those obtained from earlier MRI data (19 and 22 mm, respectively) is small and can be explained by differences in the quality of the image data19. These linear measurements, however, do not fully account for the geometry of H.M.’s spared hippocampus. The high-resolution anatomical volume that we created from microtome images allowed us to inspect the MTL from multiple angles, revealing that the posterior hippocampus was bent steeply in the dorso-medial direction (Fig. 3). This curvature is a normal feature of the periventricular portion of the hippocampus in the human brain, while the most anterior portion, which is immediately posterior to the amygdala and adjacent to the EC, is aligned to the horizontal plane (or major axis) of the hemisphere.

Figure 3: Sagittal views of the right (a) and left (b) hemispheres reconstructed from the original series of coronal microtome images. The grey lines intersect at the origin of the origins of the standard coordinate system23 used for the orientation of the specimen. The blue line indicates the most anterior level of the temporal lobes (the temporal poles, which are damaged are not shown in this image). The orange rectangle represents the bounding box that contains the posterior spared hippocampus (outlined in green), the widest extent of which is at a more medial level than this image shows. The black arrows identify the level at which a surgical clip was positioned on a blood vessel. The red arrows indicate the presence of lesions in the subcortical WM. AC, plane of the anterior commissure; PC, plane of the posterior commissure; AC–PC, ideal plane at the level of both the AC and PC; Ce, cerebellum; LG, lateral geniculate nucleus; Pu, putamen; TP, temporal pole; RH, right hemisphere; LH, left hemisphere. Scale bar, 1 cm. Full size image

Measuring the dorso-ventral oblique length of the posterior segment of H.M.’s hippocampus rather than along the AC–PC line produced higher values, specifically, 36.0 mm for the left hemisphere and 40.0 mm for the right. The values were greater when our calculations took curvature into account: in this case, the actual ‘geodesic’ length of the preserved hippocampus amounted to 45.4 mm in the left hemisphere and 47.2 mm in the right (Fig. 4).

Figure 4: Triangulated 3D surface reconstruction of H.M.’s spared left hippocampus. The orange bounding box delimits the dimensions of the structure; different segments indicate different measurements; a: anterior-to-posterior extent in the coronal plane, b (dotted): major diagonal axis, c: anatomical length. The compass cube (A: anterior, L: lateral, S: superior) also functions as scale bar: 5 mm. The insert at the bottom right shows a similarly constructed model from the brain of a neurologically normal subject (78-year-old female donor; volume of the right hippocampus=3.04 mm3; total brain weight 1,278). Ce, cerebellum; FuGR, fusiform gyrus of right hemisphere; IT, inferior temporal gyrus; SN, substantia nigra; Amg, amygdala. Full size image

The fact that multiple results can be obtained based on different measuring criteria makes it necessary to establish a clear terminology to describe the anatomy of H.M.’s lesion and the remaining hippocampus in relation to previous reports. In the context of this communication, we refer to the extent of H.M.’s hippocampus and related structures as their linear span in the rostro-caudal direction. This measure can be obtained simply by multiplying the number of tomographic images by the interval between them (that is, slice thickness). The anatomical length of the hippocampus is different and depends on its 3D shape and orientation in the brain. With this distinction in mind, our findings can be more easily reconciled with previous reports that were based on low-resolution scans, where only the extent of the hippocampus was actually measured19.

The level of sampling and image quality afforded by the current study represents a significant advance over the first clinical MRI that was performed with H.M.19. At that time, the two-dimensional (2D) MRI sequences produced 4–5-mm thick slabs with a 1-mm gap between each slice and an in-plane resolution slightly better than 1 mm per voxel; a T1-weighted 3D MRI scan was also acquired and produced non-isotropic voxels (1 × 1 × 3.2 mm). In these early scans, the boundaries of the posterior hippocampus were blurred by partial volume effects (the presence of multiple tissue types or structures in single large MRI voxels).

In a more recent MRI study with H.M., Salat et al.20 calculated the volume of tissue ascribed to the posterior hippocampus (voxel size: 1 mm × 1 mm × 1.3 mm) and obtained values of 0.65 cm3 for the left hemisphere and 0.88 cm3 for the right. We repeated these measurements based on manual delineations made on three orthogonal views of the digital anatomical reconstruction, and determined that 2.02 cm3 of hippocampal tissue (including the cornu ammonis, the dentate gyrus and the subiculum) was spared in the left hemisphere and 1.96 cm3 in the right. The comparison should be interpreted with caution in view of the fact that sufficient normative data on hippocampal volume based on our new postmortem methodology is not yet available. The labelled fields representing the posterior hippocampus in each hemisphere were used to compute triangulated surface models for visualization and shape analyses (Fig. 4).

Histology

Regularly spaced tissue sections through the brain were selected at an interval of 1.26 mm; these were mounted on large-format glass slides (5 × 7 in) and stained using thionin. Nissl staining with thionin showed preservation of neuronal cell bodies in CA1, CA2, CA3, CA4 and the subiculum of the hippocampal formation, posterior to the surgical lesion (Fig. 5).

Figure 5: Thionin-stained histological section at the level of the lateral geniculate nuclei. (a) Whole section. Scale bar, 1 cm. (b,c) Higher-magnification cross-sectional image of the dentate gyrus of the spared hippocampus. The fimbria is visible below the lateral geniculate nucleus. Scale bar, 1 mm. (d,e) × 20 magnification image of neurons in the CA4 region of the hippocampus (in the location of the box). Scale bar, 50 μm. PrG, precentral gyrus; PL, parietal lobe; CG, cingulate gyrus; LV, lateral ventricle; cc, corpus callosum; fx, fornix; Th, thalamus; TL, temporal lobe; LG, lateral geniculate nucleus; RN, red nucleus; SN, substantia nigra; DG, dentate gyrus; fi, fimbria. Full size image

Given that H.M. required surgery because of epilepsy and that there was partial response to the surgical intervention, it is of interest that the typical neuropathologic hallmarks associated with idiopathic temporal lobe epilepsy (granule cell dispersion in the dentate gyrus, neuronal loss from the pyramidal cell layer particularly in area CA4) were not present in the residual hippocampus.

Pathologic anatomy beyond the MTL

While the general size and cortical folding of the cerebral hemispheres appeared normal for an individual of H.M.’s age, multiple WM lesions consistent with lacunar infarctions were present. Cerebellar atrophy was also evident, likely a consequence of long-term exposure to Dilantin (phenytoin sodium), which was part of H.M.’s seizure management pre- and postoperatively31,32.

Review of the MRI scans and histological sections demonstrated a spectrum of additional lesions in the WM (Fig. 6). We also discovered a small focal lesion in the left lateral orbital gyrus, which was visible on the surface (Fig. 1c) and involved both cortex and underlying WM (Fig. 7).

Figure 6: Deep WM pathology. (a,b) Lesions in the deep WM were visible in postmortem T1-weighted MRI images acquired ex situ (scale bar, 1 cm) and were confirmed by myelin silver-impregnation40,46 (c) and haematoxylin and eosin (H&E) staining (d). Scale bar, 5 mm. Full size image