Evaluating ultrastructural preservation quality in banked brain tissue

Abstract

The ultrastructural analysis of postmortem brain tissue can provide important insights into cellular architecture and disease-related changes. For example, connectomics studies offer a powerful emerging approach for understanding neural circuit organization. However, electron microscopy (EM) data is difficult to interpret when the preservation quality is imperfect, which is common in brain banking and may render it unsuitable for certain research applications. One common issue is that EM images of postmortem brain tissue can have an expansion of regions that appear to be made up of extracellular space and / or degraded cellular material, which we call ambiguous interstitial zones. In this study, we report a method to assess whether EM images have ambiguous interstitial zone artifacts in a cohort of 10 postmortem brains with samples from each of the cortex and thalamus. Next, in matched samples from the contralateral hemisphere of the same brains, we evaluate the structural preservation quality of light microscopy images, including immunostaining for cytoskeletal proteins. Through this analysis, we show that on light microscopy, cell membrane morphology can be largely maintained, and neurite trajectory visualized over micrometer distances, even in specimens for which there are ambiguous interstitial zone artifacts on EM. Additionally, we demonstrate that synaptic structures can be successfully traced across serial EM sections in some postmortem samples, indicating the potential for connectivity studies in banked human brain tissue when appropriate preservation and visualization protocols are employed. Taken together, our analysis may assist in maximizing the usefulness of donated brain tissue by informing tissue selection and preparation protocols for various research goals.

Keywords: Brain banking; Connectomics; Neurofilaments; Perfusion fixation; Postmortem changes; Ultrastructural quality.

Figure 1

Fine-scale annotation of ultrastructural components in one representative EM image from the cortex of donor #7. Color coding identifies distinct cellular structures: Green: membrane-bound structures with electron-dense interior (e.g., organelle-rich neurites); Purple: membrane-bound structures with electron-lucent interior (e.g., swollen astrocytic processes); Blue: myelinated axons; Yellow: cell bodies. Non-colored regions represent ambiguous interstitial zones (AIZs) that lack clearly defined membrane boundaries. Upper scale bar: 2 μm. Lower scale bar: 1 μm.

Figure 2

Overlap of annotations of a sample from the cortex of donor #66 by independent annotators. The top and middle image were done by two independent annotators. The bottom image is a combination of the overlays to compare how alike the two annotations are. Color coding identifies distinct cellular structures: Green: membrane-bound structures with electron-dense interior (e.g., organelle-rich neurites); Purple: membrane-bound structures with electron-lucent interior (e.g., swollen astrocytic processes); Blue: myelinated axons; Yellow: cell bodies. Non-colored regions represent ambiguous interstitial zones (AIZs) that lack clearly defined membrane boundaries. Scale bars: 1 μm.

Figure 3

Fine-scale manual annotation of ambiguous interstitial zones (AIZs) from one EM image across all samples. Samples are sorted by the percentage of ambiguous interstitial zones (AIZs), from the lowest to the highest: 7-C (a), 37-T (b), 34-C (c), 65-C (d), 59-C (e), 55-C (f), 37-C (g), 78-C (h), 57-C (i), 30-C (j), 65-T (k), 57-T (l), 55-T (m), 78-T (n), 30-T (o), 34-T (p), 66-C (q), 7-T (r), 66-T (s), 59-T (t), where the number is the donor ID number, and then “-T” indicates that it is from the thalamus and “-C” from the cortex. Color coding identifies distinct cellular structures: Green: membrane-bound structures with electron-dense interior (e.g., organelle-rich neurites); Purple: membrane-bound structures with electron-lucent interior (e.g., swollen astrocytic processes); Blue: myelinated axons; Yellow: cell bodies. Non-colored regions AIZs that lack clearly defined membrane boundaries. Scale bars: 500 nm.

Figure 4

Representative EM images graded as having or not having ambiguous interstitial zone (AIZ) artifacts. Five examples images are shown that were graded as having AIZ artifacts: 30-T (b), 39-C (d), 59-C (f), 59-T (h), 66-T (j), as well as five examples that were not: 7-C (a), 57-C (c), 65-C (e), 65-T (g), 66-C (i), where the number is the donor ID number, and then “-T” indicates that it is from the thalamus and “-C” from the cortex. Scale bars: 1 μm.

Figure 5

Representative images in the serial section TEM data sets showing that synapses can be traced. Samples from the cortex of donor IDs #7 (a), #35 (b), #65 (c). Synapses are highlighted in color. Scale bars: 1 μm.

Figure 6

Representative serial section image stack with annotations for afferent neurites from one synapse. All images are from a serial section from the cortex sample of donor #65. Scale bars: 1 μm.

Figure 7

Representative H&E-stained images demonstrate that cell membrane morphology is generally intact across a wide range of PMIs. Samples from the thalamus of donor IDs #7 (a, b), #65 (c, d), #57 (e, f), and #59 (g, h) with PMIs of 4.25 h, 1.5 h, 96 h, and 91 h, respectively. Insets on the lower magnification images correspond to the regions shown in the higher magnification images. Scale bars: (a, c, and g) 1 mm; (e) 2 mm; (b, d, f, and h) 50 μm.

Figure 8

Representative H&E-stained images from the thalamus showing variable vascular clearance. a, b: Images from donor #30 show extensive intravascular material. c, d: Images from donor #59 show minimal intravascular material. Scale bars: (a, c) 2 mm; (b, d) 200 μm.

Figure 9

Representative SMI-312-stained images demonstrate that axonal morphology is generally intact across a wide range of PMIs. Samples from the thalamus of donor IDs #65 (a), #78 (b), #7 (c), #37 (d), #59 (e), and #57 (f) with PMIs of 1.5 h, 2.5 h, 4.25 h, 72 h, 91 h, and 96 h, respectively. Scale bars: 50 μm.

Figure 10

Representative SMI-311-stained images demonstrate that dendritosomatic morphology on light microscopy is generally intact across a wide range of PMIs in human brains. Samples from the thalamus of donor IDs #78 (a), #7 (b), #30 (c), #37 (d), #59 (e), and #57 (f) with PMIs of 2.5 h, 4.25 h, 17 h, 72 h, 91 h, and 96 h, respectively. Scale bars: 50 μm.

Figure 11

Representative GFAP-stained images of the cortex show intact subpial astrocyte morphology in images from samples with a wide range of PMIs. Samples from donor IDs #65 (a), #78 (b), #7 (c), #37 (d), #59 (e), and #57 (f) with PMIs of 1.5 h, 2.5 h, 4.25 h, 72 h, 91 h, and 96 h, respectively. Scale bars: 50 μm.