Histologic validation of locus coeruleus MRI contrast in post-mortem tissue

Abstract

The locus coeruleus (LC) noradrenergic system regulates arousal and modulates attention through its extensive projections across the brain. LC dysfunction has been implicated in a broad range of neurodevelopmental, neurodegenerative and psychiatric disorders, as well as in the cognitive changes observed during normal aging. Magnetic resonance imaging (MRI) has been used to characterize the human LC (elevated contrast relative to surrounding structures), but there is limited understanding of the factors underlying putative LC contrast that are critical to successful biomarker development and confidence in localizing nucleus LC. We used ultra-high-field 7 T magnetic resonance imaging (MRI) to acquire T1-weighted microscopy resolution images (78 μm in-plane resolution) of the LC from post-mortem tissue samples. Histological analyses were performed to characterize the distribution of tyrosine hydroxylase (TH) and neuromelanin in the scanned tissue, which allowed for direct comparison with MR microscopy images. Our results indicate that LC-MRI contrast corresponds to the location of neuromelanin cells in LC; these also correspond to norepinephrine neurons. Thus, neuromelanin appears to serve as a natural contrast agent for nucleus LC that can be used to localize nucleus LC and may have the potential to characterize neurodegenerative disease.

Keywords: Brainstem; Histology; Inversion recovery; MRI; Neuromelanin; Norepinephrine; Post-mortem.

Fig. 1

Optimal parameters for ex-vivo LC-MRI. A rapid acquisition with refocused echoes (RARE) sequence was used to obtain axial sections from brainstem tissue embedded in agar. A) A short inversion recovery time (TI) produces distinct periaqueductal gray (PAG) contrast compared to surrounding tissue, but no elevated contrast in putative LC regions (orange triangles). B) Elevated contrast is still observed in the PAG with a long TI, as well as specific increased contrast in putative LC regions (blue triangles). C–E) A short echo time (TE) resulted in diminished PAG contrast relative to increased contrast in LC (blue triangles). Magnified 4th ventricular area (dashed box) is presented in the middle row. Bottom row demonstrates the relative increase in LC-MRI contrast with shorter TE using a linear ROI that was used to collect contrast values across the brainstem for each TE acquisition (y-axis: arbitrary units). An axial slice from the same tissue sample (HB 24) is presented in all images (ROI positioning shown in the inset at bottom right). Image parameters common across scans: number of slices = 8, slice thickness = 2 mm, slice spacing = 0.5 mm, repetition time = 3000 msec, inversion time = 825 ms, number of averages = 3, imaging resonance frequency = 300 MHz, echo train length = 2, flip angle = 180°, matrix size = 128 × 128, field of view = 4.00 × 4.00 cm, in-plane voxel size = 310 μm. Scale bars = 5 mm.

Fig. 2

MRI microscopy of the human LC (HB24). A long (3 h 11 min) scan acquisition was used to obtain microscopy resolution images (panel B) from post-mortem tissue to allow comparative analysis with histology in the same tissue (panel C). To emphasize the benefit of ultra-high resolution imaging for this project, a typical 3 T T1-TSE 11 min scan of the LC is presented for comparison (panel A). A) Axial slice through the human brainstem obtained using a T1-TSE sequence as described in Keren et al. (2009). Increased contrast can be observed in the putative location of the LC (blue arrows). Double arrow indicates ventral (V) and dorsal (D) directions. A magnified view of the 4th ventricle region (dashed box) is displayed in the bottom panel, with additional detail of the observed contrast (inserts). The spatial extent of putative LC-MRI contrast (dashed box in higher power image below) includes only a few voxels due to relatively limited spatial resolution (400 um in plane resolution). B) Microscopy resolution RARE-INV scan obtained at 7 T MRI with postmortem tissue. Elevated contrast is observed in the putative LC region (blue arrows). Lower panels in A and B show magnified regions indicated by dashed boxes in upper panels. Dashed boxes in lower, high power images indicate location of LC-MRI contrast. C) A bright-field photomicrograph of a hematoxylin and eosin (H&E)-stained 40 μm-thick axial section from the same tissue and equivalent rostral–caudal position as the MRI slice displayed in panel B. Magnification of the ventricle and putative LC regions (bottom panels) demonstrates spatial correspondence between microscopy LC-MRI contrast and H&E-stained neurons in the LC region (yellow arrows; compare panels B and C). Scale bars = 5 mm (top panels), and 500 μm (bottom panel inserts).

Fig. 3

7 T MR microscopy (in-plane resolution = 78 μm) through the mid-pons of postmortem brainstem tissue samples across individual cases. For each specimen, an axial section is presented at the level of apparent maximal LC-MRI contrast. The rostral–caudal location of each slice is presented on a mid-sagittal view of the brainstem sample (inserts). Between-sample variation in the putative LC-MRI contrast (blue triangles) can be observed, including focal (e.g. specimen HB13) and diffuse patterns (e.g. specimen HB21) of contrast. The 4th ventricle region of sample HB19 is magnified (top right panel) to demonstrate anatomical detail, varied tissue contrasts, and detailed LC morphology that can be observed utilizing the ex-vivo microscopy-resolution RARE-INV acquisition.

Fig. 4

Axial slices of an ex-vivo brainstem sample (HB24) obtained with 7 T MRI using RARE-INVersion recovery sequence. Slices in panels numbered 1–14 correspond to slices taken through the brainstem in upper left panel. Elevated contrast is observed in regions corresponding to the left substantia nigra, pars compacta (SNpc, white triangle, panel 1), and the locus coeruleus (LC, blue triangles, panels 5–7). Within-specimen morphological asymmetry in putative LC-MRI contrast can be observed for slices 5–7. Anatomical landmarks are indicated to facilitate orientation: 4th ventricle (4 V), cerebral aqueduct (CA), corticospinal tract (CST), inferior cerebellar peduncle (ICP), inferior colliculus (IC), interpeduncular fossa (IPF), medial lemniscus (ML), medial longitudinal fasciculus (MLF), periaqueductal gray (PAG), superior cerebellar peduncle (SCP). Arrows indicates ventral (V), dorsal (D), rostral (R), and caudal (C) directions.

Fig. 5

LC-MRI contrast depends on the consistency of TH+ LC neurons across the thickness of an MRI slice. A) Illustration of a mid-sagittal section through the HB24 brainstem. Lower rectangles and lines extending from the illustration to indicate the histological sections shown in (B). B) TH+ neurons from the left and right LC (left and right column of images) for the 5 equidistant 40 μm-thick section images that span the thickness of the MRI slice. Note the limited TH staining in LC processes (left) in comparison to dense staining for clusters of LC cell bodies on the right. C) The density of staining for the left and right LC was represented by binarizing the TH images and summing to create a TH density map that also demonstrates the greater density of TH+ LC neurons on the right. This also coincides with the more pronounced LC-MRI contrast on the right in (D). Slice 7 can also be seen in Figs. 4 and 6.

Fig. 6

TH+ LC neuron density spatially corresponds to increased RARE-INVersion recovery contrast. The same tissue sample as in Fig. 4 (HB24) underwent MRI scanning and was then sectioned for histological analysis. A) Illustration of a mid-sagittal section through the brainstem. Gray rectangles (MRI slices 5–7) represent the position of three 2-mm-thick consecutive RARE-INV (μMRI) axial sections (see Fig. 4, panels 5–7) in which putative LC-MRI contrast can be observed. The five consecutive, equidistant 40 μm-thick sections (black lines) contained within each of the three MRI slices (indicated at right in panel A) were immunohistochemically stained for TH to visualize NE neurons in LC. Double arrow indicates rostral (R) and caudal (C) directions. B) Magnified view of left and right TH-stained neurons and processes in LC. Both photomicrographs are from histology section TH 74, the most caudal of five sections comprising MRI slice 6 in panel A. Double arrow indicates ventral (V) and dorsal (D) directions. C) LC TH+ density maps and corresponding LC-MRI contrast. In each row a TH+ signal density map (left) is shown next to its corresponding MRI slice (right). The histology sections (panel A) were processed and spatially rigidly aligned to a common stable anatomical landmark to produce TH density maps (see Methods). Note that LC-MRI contrast is most pronounced for slices (white arrows in panels at right) in which TH staining was observed across all 5 sections for that MRI slice (red: TH staining on all 5 sections; blue: TH staining on 1 section).

Fig. 7

TH+ neurons that produce neuromelanin are necessary for LC-MRI contrast. Increased TH+ density in HB24 did not always spatially correspond to increased MRI signal. A) TH+ density map of the 4th ventricle region in the rostral pons (from sections through MRI slice 5 in Figs. 4 and 6C). In addition to the LC TH signal on either side, increased TH+ density can be observed in midline regions flanking the ventral border of the 4th ventricle (white triangles), corresponding to the location of the caudal dorsal raphe nucleus (dRN). B) RARE-INV microscopy (μMRI) of the same tissue as in A. No increase in contrast was observed in the periventricular area corresponding to dRN TH+ signal (white triangles). C) Bright field photomicrograph of the 4th ventricle region at the rostral pons in a 40 μm-thick section stained for TH. The section presented is from the same tissue and from within the slab imaged in (B). As expected, TH+ cells and proximal neurites can be observed (white triangles) in the same region as increased TH+ density in (A), corresponding to the dRN. D) Bright field photomicrograph of the same region in an unstained section, showing neuromelanin (NM+) granule distribution. Although neuromelanin granules can be observed in the same area as the TH+ neurons of the LC (C) near the left and right boundaries of the image, they are absent from the more medial dRN area (white triangles).

Fig. 8

Increased RARE-INV contrast corresponds to the location of TH+ neuromelanin containing neurons of the LC. Photomicrographs and MRI microscopy acquisition from HB24. A) TH-stained tissue showing NE neurons in LC. Magnified panels of left and right LC (lower) show the extent of NE cell bodies and proximal neurites. B) Unstained section showing distribution of neuromelanin (NM) granules in the same tissue section as A. Magnified panels (lower) display depositions of large intracellular granules as well as smaller extracellular neuromelanin in LC. C) RARE-INV (μMRI) scan of the same region with microscopy resolution. Magnified panels in A, B and C demonstrate correspondence of increased MRI contrast with distribution of NM granules in LC, and overlapping with the location of NE cells in LC.