Spinal cord from body donors is suitable for multicolor immunofluorescence

Abstract

Immunohistochemistry is a powerful tool for studying neuronal tissue from humans at the molecular level. Obtaining fresh neuronal tissue from human organ donors is difficult and sometimes impossible. In anatomical body donations, neuronal tissue is dedicated to research purposes and because of its easier availability, it may be an alternative source for research. In this study, we harvested spinal cord from a single organ donor 2 h (h) postmortem and spinal cord from body donors 24, 48, and 72 h postmortem and tested how long after death, valid multi-color immunofluorescence or horseradish peroxidase (HRP) immunohistochemistry is possible. We used general and specific neuronal markers and glial markers for immunolabeling experiments. Here we showed that it is possible to visualize molecularly different neuronal elements with high precision in the body donor spinal cord 24 h postmortem and the quality of the image data was comparable to those from the fresh organ donor spinal cord. High-contrast multicolor images of the 24-h spinal cords allowed accurate automated quantification of different neuronal elements in the same sample. Although there was antibody-specific signal reduction over postmortem intervals, the signal quality for most antibodies was acceptable at 48 h but no longer at 72 h postmortem. In conclusion, our study has defined a postmortem time window of more than 24 h during which valid immunohistochemical information can be obtained from the body donor spinal cord. Due to the easier availability, neuronal tissue from body donors is an alternative source for basic and clinical research.

Keywords: Horseradish peroxidase immunohistochemistry; Human spinal cord; Multicolor immunofluorescence; Neuronal markers; Postmortem interval.

Fig. 1

Hematoxylin–eosin staining of human spinal cords after different postmortem intervals. a Cross section of the spinal cord at 24 h postmortem. The structure of the spinal cord appears normal without observable histological changes. Anterior roots (ar), posterior roots (pr). b, c Half of the spinal cord after 48 h (b) and 72 h (c) postmortem interval. At time point 48 h, structural defects including small gaps (inset in b) are present in the spinal cord whereas at 72 h, the gray and white matter exhibit large gaps, indicating advanced autolysis of the tissue (c). Scale bar 1000 μm (a–c)

Fig. 2

Human spinal cord labeled with anti-neurofilament (NF) after 24 h postmortem interval showing lipofuscin autofluorescence. a Scheme with an inset illustrating the part of the spinal cord shown in figure b–b’’’. Posterior root (pr), anterior root (ar). b–b’’’ Spinal cord in neurofilament staining (b), red autofluorescence (b’), green autofluorescence (b’’), and overlay (b’’’). Thethick dashed line demarcates the gray and white matter and thethin dashed lines the surface of the spinal cord and the anterior root. b Grey and white matter and the anterior root (ar) express neurofilament. b’ Motoneurons of the anterior horn exhibit red and b’’ green autofluorescence due to lipofuscin deposits. b’’’ The overlay of b–b’’ shows that the mixture of red and green autofluorescence results in yellow color. c–c’’ High magnification of motoneurons of anterior horn demarcated with a dashed line showing neurofilament signal and red lipofuscin autofluorescence (c), neurofilament and green lipofuscin autofluorescence (c’), and the overlay of c–c’ (c’’). The amount of lipofuscin varies and two cell bodies are fully covered with lipofuscin, one cell partly, and one cell (arrow) is free of lipofuscin. Lipofuscin granules exhibiting red and green autofluorescence are also visible outside the cell bodies in the neuropil.Scale bars 500 µm in b’’’ for b–b’’’ and 50 µm in c’’ for c–c’’

Fig. 3

Double labeling with anti-neurofilament (NF) and anti-ChAT after 24 h postmortem interval. a Scheme with an inset illustrating the part of the spinal cord shown in figure b–b’’. Posterior roots (pr), anterior roots (ar). b The spinal cord in neurofilament staining. The gray and white matter and the anterior (ar) and posterior (pr) roots express neurofilament. Thedashed line demarcates the gray and white matter. Motoneurons of the anterior horn exhibit red autofluorescence due to lipofuscin deposits (arrow). b’ The spinal cord in ChAT staining. Fascicles of the anterior root exhibit ChAT immunoreactivity whereas fascicles of the posterior root lack ChAT immunoreactivity. Thegreen pial contour covering the surface of the posterior root represents an artificial signal. Motoneurons of the anterior horn exhibit green autofluorescence due to lipofuscin (arrow). b’’ Overlay of b and b’, shows that the nerve fascicles of the anterior root are positive for neurofilament and ChAT. c–c’’ The fascicles of the posterior root (inset in the upper part of c’’). Axons of the posterior root are neurofilament positive (c) but lack ChAT (c’). In the overlay (c’’), neurofilament and ChAT staining is merged. d–d’’ Fascicles of the anterior root (inset in the lower part of b’’). Axons of the anterior root express neurofilament (d) and ChAT (d’). In the overlay d’’, the mix of the red neurofilament and green ChAT signal results in yellow color.Scale bars: 1000 µm in b’’ for b–b’’; 100 µm in d’’ for c–c’’ and d–d’’

Fig. 4

Immunolabeling and quantitative analyses of the anterior roots of the organ donor and body donor spinal cord. a–a’’, b–b’’, c–c’’, d–d’’, e–e’’ Labeling with anti-neurofilament (NF) and anti-ChAT of the 2-h organ donor spinal cord (a–a’’), the body donor spinal cord from two different subjects 24 h (b–b’’, c–c’’), and from one subject 48 h (d–d’’), and 72 h postmortem (e–e’’). A reduction in the signal intensity is observed in the neurofilament (a, b, c, d, e) and ChAT staining (a’, b’, c’, d’, e’) although the degree varies. In the neurofilament staining (a, b, c, d, e), the brightness of the signal is similar at time point 2 h (a), 24 h (b and c), and 48 h (d) and appears apparently reduced not until 72 h (e). A stronger signal reduction is observed in the ChAT staining (a’, b’, c’, d’, e’). At time point 2 h (a’) and 24 h (b’ and c’), the ChAT intensity is almost equal, lower at time point 48 h (d’), and lowest at 72 h (e’). Overlays of the neurofilament and ChAT staining are shown in a’’, b’’, c’’, d’’, e’’. The overlay in a’’, b’’, and c’’ appears in yellow color due to the mix of the strong red neurofilament (a, b, c) and green ChAT signal (a’, b’, c’). Overlays in d’’ and e’’ appear inred–orange color due to the stronger decrease of the green ChAT signal (d’, e’) relative to the red neurofilament signal (d, e).Scale bar: 100 µm in e’’ for a–a’’, b–b’’, c–c’’, d–d’’, and e–e’’. f, g Bar chart illustrating the change of the neurofilament and ChAT staining intensity 2, 24, 48, and 72 h postmortem. At time point 2 h, the neurofilament and ChAT staining intensity is set at 100% and values for 24, 48, and 72 h are presented as a percentage of the 2 h reference value (f: neurofilamentred bars, g: ChATgreen bars). At 24 h, the brightness of the neurofilament is at about 90% (f: second bar), at 48 h about 70% (f: third bar), and at 72 h to about 45% (f: fourth bar). In the ChAT staining, the signal reduction is minimal 24 h postmortem and 93% of the 2 h value (g: second bar). Signal reduction is to about 40% at time point 24 h, (g: third bar), and to about 20% at 72 h (g: fourth bar).Black dots (●) indicate significant differences with time point 2 h,asterisks (*) indicate significant differences with time point 24 h, andplus signs (+) mark significant differences with time point 48 h (one-way ANOVA test followed by Holm–Sidak post hoc method, P < 0.05). (h) Percentage of neurofilament and ChAT-positive axons in the anterior root after 24 h and 48 h postmortem interval. At 24 h, the number of neurofilament and ChAT-positive axons is almost the same proving that all neurofilament positive axons in the anterior root express ChAT as well. At 48 h, a higher number of neurofilament than ChAT-positive axons is counted because, in some axons, ChAT signals are too low for being detected by computer-based quantification. (t test; P < 0.05; N = 3 for neurofilament and ChAT)

Fig. 5

High-resolution images of immunolabeled anterior root axons. a–a’’ Axons from the 2-h organ donor spinal cord and b–b’’, c–c’’, d–d’’ from the body donor spinal cord at different postmortem intervals. Axons are shown in anti-neurofilament (NF, a, b, c, d), anti-ChAT staining (a’, b’, c’, d’) and overlays (a’’, b’’, c’’, d’’). At time point 2 and 24 h, all axons exhibit strong neurofilament (a, b) and ChAT (a’, b’) signals and there is a complete overlap of neurofilament and ChAT signals (a’’, b’’). Most axons are of large diameter (putative α-motoneurons) and few are of small diameter (putative γ-motoneurons). In the overlay a’’, b’’, fluorescence and bright-field images are merged showing that all axons are covered with a myelin sheath. At time point 48 h (c–c’’), neurofilament (c) and ChAT signals (c’) decrease but the reduction is stronger in ChAT staining. Some axons (arrow) lack antibody signals. At time 72 h (d–d’’), a further signal reduction is visible, and signals lose sharpness and homogeneity. The number of axons without antibody signal (arrows) has increased. Additionally, axons are more separated from each other.Scale bar: 10 µm in d’’ for a–a’’, b–b’’, c–c’’, and d–d’’

Fig. 6

Immunofluorescence with anti-neurofilament (NF) and anti-ChAT, and HRP immunohistochemistry with anti-ChAT. a–a’’, b–b’’ Immunofluorescence of motoneurons stained with anti-neurofilament (a, b) and anti-ChAT (a’, b’) from the organ donor (a–a’’) and 24 h body donor spinal cord (b–b’’).Dashed lines demarcate the motoneuron cell bodies (a–a’’) including the proximal dendrite (b–b’’). Some motoneurons exhibit red and green autofluorescence due to lipofuscin deposits (LP). A weak ChAT signal is observed 2 h postmortem (a’) but absent 24 h postmortem (b’). Synapses (arrows) contacting motoneuron cell bodies exhibit strong ChAT immunoreactivity (a’, b’). Overlays of the neurofilament (a, b) and ChAT (a’, b’) staining are presented in a’’ and b’’.Arrows in a’’, b’’ indicate ChAT-positive synapses. (c–f) Immunoperoxidase staining with anti-ChAT at lower (c, d) and higher magnification (e, f). At time point 2 h (c, e), the cell bodies exhibit a strong ChAT signal, which is weaker or absent (*, d, f) in cell bodies at time point 24 h (d, f). Lipofuscin is demarcated by adashed line at time point 24 h (LP, f). Synapses (some of them are indicated by arrows) with strong ChAT immunoreactivity cover the cell bodies and proximal dendrites of motoneurons (c, d, f).Scale bars: 20 µm in b’’ for a–a’’, b–b’’, 50 µm in d for c and d, 20 µm in f for e and f

Fig. 7

Double labeling with anti-neurofilament (NF) and anti-synaptophysin (Syn). a Cross section through a spinal cord 24 h postmortem illustrating synaptophysin immunoreactivity throughout the gray matter. Anterior roots (ar) and posterior roots (pr). Dendrites extending into the white matter are indicated byarrows. (b–b’’, c–c’’, d–d’’, e–e’’) Show anterior horn motoneuron cell bodies in neurofilament (b, c, d, e), synaptophysin staining (b’, c’, d’, e’), and overlay (b’’, c’’, d’’, e’’). Some motoneurons (arrows) exhibit red lipofuscin (LP) autofluorescence (c, d, e). At time points 2 h (b–b’’) and 24 h (c–c’’), the cell bodies including proximal dendrites of the motoneurons are fully covered with synaptophysin-positive synapses (b’, b’’, c’, c’’). At time point 48 h (d–d’’), synaptic density appears unchanged but the synaptophysin intensity is decreased (d’, d’’). At time point 72 h (e–e’’), the number of synapses is reduced and the synaptophysin signal (e’, e’’) is further reduced. Additionally, large gaps surround the cell bodies of the motoneuron. f Bar chart showing the reduction of the synaptophysin staining intensity. At the time point 24 h, the signal intensity is about 95% of the 2-h value (f, second bar). At time point 48 h, the signal intensity is around 60% (f, third bar) and at 72 h about 25% (f, third bar) of the initial value (f, first bar).Black dots (●) indicate significant differences with time point 2 h,asterisks (*) indicate significant differences with time point 24 h, andplus signs (+) mark significant differences with time point 48 h. (one-way ANOVA test followed by Holm–Sidak post hoc method, P < 0.05).Scale bar: 1000 µm in a, 25 µm in e’’ for b–b’’, c–c”, d–d’’, and e–e’’

Fig. 8

Double labeling with anti-neurofilament (NF) and anti-CGRP. a A segment of the human spinal cord at 24-h postmortem interval. Intense CGRP immunoreactivity is seen in the superficial layer of the posterior horn. Thedashed line demarcates the gray and white matter of the spinal cord. Anterior roots (ar), posterior roots (pr). Motoneuron cell bodies exhibiting lipofuscin autofluorescence are indicated by an arrow. b–e The CGRP-immunoreactivity in the dorsal horn of the organ donor (b) and body donors (c–e) at different postmortem intervals. At time points 2 h (b) and 24 h (c), a high level of CGRP is seen in the superficial layer of the dorsal horn. There is a downward trend in the staining intensity at 48 h (d) and at 72 h (e). Several tissue gaps are present 72 h postmortem (e). Scale bars: 1000 µm in a, 100 µm in b, and 100 µm in e for c, d, and e. f Bar chart showing the reduction of the CGRP signal over the postmortem interval. At time point 24 h, the CGRP intensity is 85% of the 2-h value (f, second bar). At time point 48 h, the CGRP signal intensity is 55% (f third bar) and at 72 h at about 30% (f, third bar) of the initial value (f, first bar).Black dots (●) indicate significant differences with time point 2 h,asterisks (*) indicate significant differences with time point 24 h, andplus signs (+) mark significant differences with time point 48 h. (one-way ANOVA test followed by Holm–Sidak post hoc method, P < 0.05)

Fig. 9

Double fluorescent labeling with anti-neurofilament (NF) and anti-CGRP and HRP immunohistochemistry with anti-CGRP. a–a’’, b–b’’ Immunofluorescence of motoneurons stained with anti-neurofilament and anti-CGRP 2 h (a–a’’) and 24 h postmortem (b–b’’).Dashed lines demarcate motoneuron cell bodies. At time point 2 h (a’), motoneurons exhibit weak CGRP signals but CGRP signals are not visible 24 h postmortem (b’). Lipofuscin (LP) exhibiting red and green autofluorescence fills a part of the cell body (b, b’). Overlays of the neurofilament (a, b) and CGRP (a’, b’) staining are presented in a’’, b’’. The overlay shows lipofuscin autofluorescence in orange color due to the mix of the strong red and green autofluorescence signal (b”).Scale bar: 20 µm in b’’ for a–a’’ and b–b’’. c, d HRP immunohistochemistry with anti-CGRP showing motoneuron cell bodies 2 h (c) and 24 h postmortem (d). Clod-like CGRP signals appear inbrown color and are visible at both postmortem intervals in cell bodies. In c, a cell body without a CGRP signal (arrow) is present. Lipofuscin (LP).Scale bar: 20 µm in d for c and d

Fig. 10

Triple labeling with anti-neurofilament (NF), anti-NeuN, and Hoechst 33342. a Schematic drawing with an inset illustrating the part of the spinal cord shown in figures b–b’’, and d–d’’. Posterior roots (pr), anterior roots (ar). b–b’’, d–d’’ Overview images of the anterior horn 2 h (b–b’’) and 24 h postmortem (d–d’’) are shown in neurofilament and NeuN staining (b, d), neurofilament and Hoechst staining (b’, d’), and overlays (b’’, d’’). The gray and white matter are separated by adashed line. Motoneuron nuclei of the anterior horn express NeuN immunoreactivity (arrowheads in b, d). The inset in d shows small nuclei positive for NeuN (arrowheads). c–c’’, e–e’’ High magnification images of motoneurons cell bodies (surrounded by adashed line) 2 h (d–d’’) and 24 h postmortem (e–e’’) in neurofilament and NeuN staining (c, e), neurofilament and Hoechst staining (c’, e’) and overlay (c’’, e’’). The motoneuron nucleus expresses NeuN (c, e) and is visualized by Hoechst stain in c’ and e’. The overlays of c and c’ and e and e’ show co-localization of NeuN and the Hoechst signal. Small nuclei expressing NeuN (arrowheads in c, e) and Hoechst signals (c’, e’) are also visible.Scale bar: 200 µm in b’’ for b–b’’; 15 µm in c’’ for c–c’’; 200 µm in d’’ for d–d’’; 15 µm in e’ for e–e’’

Fig. 11

Triple labeling of a body donor spinal cord 24 h postmortem with anti-neurofilament (NF), anti-synaptophysin (syn), and anti-CGRP. a Cross section of a body donor spinal cord at 24 h postmortem interval. The whole gray matter exhibits synaptophysin immunoreactivity whereas CGRP is expressed in the superficial layer of the posterior horn. Anterior roots (ar), posterior roots (pr). b–b’’ High-resolution images of the posterior horn (square box in a) in neurofilament/synaptophysin staining (b) neurofilament/CGRP staining (b’) and overlay b’’. The colocalization of synaptophysin (red) and CGRP (green) results in yellow color (b’’).Scale bars: 1000 µm in a, 100 µm in b’’ for b–b’’

Fig. 12

Triple labeling of a body donor spinal cord 24 h postmortem. (a–a’’, b–b’’) Labeling with anti-neurofilament (NF), anti-synaptophysin (Syn), and anti-VAChT. Cross section of the anterior horn (a–a’’) showing motoneurons (arrows in a) covered with synaptophysin (a) and VAChT (a’) positive synapses. The dashed line demarcates the border between the white and gray matter. The overlay (a’’) shows the mix of neurofilament, synaptophysin, and VAChT staining. (b–b’’) High-resolution image of a motoneuron cell body (*) covered with synapses expressing synaptophysin (b) and VAChT (b’). The overlay (b’’) shows colocalization of synaptophysin/VAChT in some synapses which appear in yellow color. (c–c’’) Labeling of the posterior root with anti-neurofilament, anti-MBP, and anti-CGRP. Most axons are covered with a myelin sheath (c) and only a few axons (arrows in c) are without myelin. Some thin axons express CGRP (c’) and are with myelin (arrowheads in c’’) or without a myelin sheath (arrows in c’’). Scale bars: 100 µm in a’’ for a–a’’, 25 µm in b’’ for b–b’’, 10 µm in c’’ for c–c’’

Fig. 13

Schematic diagram summarizing the staining pattern (a) and decay (b). a Anti-neurofilament (red) is present in the white and grey matter (outlined in black color) of the spinal cord as well as in axons of the anterior (ar) and posterior roots (pr). Axons of the anterior roots express ChAT as well (red/purple) Synaptophysin (blue) is expressed throughout the gray matter and CGRP (yellow) in the superficial layer of the dorsal horn. Lower inset of a shows anterior horn motoneurons covered with synapses that express synaptophysin/VAChT (blue andblack) and other synapses expressing synaptophysin (blue) only. Motoneuron cell bodies express CGRP and ChAT (purple yellow lines) and the nucleus NeuN (brown). Upper inset of a showing a detail of the posterior root with myelinated axons (red/green) and unmyelinated axons (red) some of which express CGRP (red/yellow). (b) Histogram showing the mean decay of the staining intensity in different antibodies over postmortem intervals