Neuropathological examination suggests impaired brain iron acquisition in restless legs syndrome

Patient characteristics.

Tissue was analyzed from seven patients with primary RLS.4 The clinical history is summarized in table 1. Five individuals with no neurologic history served as controls. These individuals were a 48-year-old woman, an 84-year-old man, a 67-year-old man, a 74-year-old woman, and a 66-year-old woman.

Evaluation of sections.

A complete histologic and immunohistochemical evaluation was performed on three of the cases at the Hershey Medical Center and the evaluation of the other cases was performed by the Harvard Brain Bank. The tissue was evaluated for AD18 and for dementia with Lewy bodies.19 Vascular disease was evaluated in each case. Arteriosclerosis was graded as mild, moderate, or severe. Congophilic angiopathy was also graded as mild, moderate, or severe.20

Tissue collection.

The brains were harvested at autopsy. One half of each brain was fixed in 10% formalin and processed for routine pathologic evaluation. Paraffin sections were stained with hematoxylin & eosin, Luxol fast blue–hematoxylin and eosin, Bielschowsky silver, and Congo red stains, and evaluated immunochemically using antibodies α-synuclein (1:1000, Chemicon, Temecula, CA, rabbit, polyclonal), ubiquitin (1:800, Dako, Carpinteria, CA, rabbit polyclonal), tau (1:1000, AT8, Innogenetics, Ghent, Belgium, mouse monoclonal), B-amyloid (1:50, βA4, Dako, mouse monoclonal), and tyrosine hydroxylase (1:250, Pel-Freez, Rogers, AR, rabbit polyclonal). For the immunohistochemical studies on the substantia nigra, involving iron management proteins, polyclonal antibodies to divalent metal transporter 1, metal transport protein 1 (also known as IREG1 or ferroprotein), and monoclonal antibodies to the ferritin subunits were used. Iron histochemistry was performed using a modified Perls reaction. The three cases originating at Hershey were examined bilaterally and the others were unilateral examinations.

Immunochemistry was performed on paraffin and cryostat sections ranging in thickness from 10 to 40 μm. A formic acid pretreatment (2 minutes) in concentrated formic acid (Fisher Scientific, Pittsburgh, PA) was routinely carried out on paraffin sections evaluated for immunohistochemistry. Nonspecific staining was blocked using SuperBlock (ScyTech, Logan, UT). The negative staining control slides were incubated with phosphate-buffered saline instead of primary antibody. In all cases, slides were incubated in biotinylated secondary antibody (SensiTec, ScyTek) followed by streptavidin-horseradish peroxidase (ScyTek). The reaction products were visualized by reacting the tissue sections with 3,3′ diaminobenzidine (DAB) and 1% nickel chloride. The addition of nickel chloride turns the brown DAB reaction product blue,21 which enabled us to differentiate the reaction product from neuromelanin.

Histopathology results.

Histochemical and immunohistochemical results.

A semiquantitative assessment was performed independently on all iron stained and immunostained sections by two investigators blinded as to patient condition. Two scores were assigned per section, one for staining intensity within the neuromelanin cells (table 2) and one reflecting the staining within the neuropil and non-neuronal cells of the substantia nigra (table 3). For the assessment of staining in the neuromelanin-containing cells, each section (at least two per patient) was assigned an intensity rating using a score of 0 to indicate no reaction product detectable in the neuromelanin cells and 1 if fewer than 10 cells had any reaction product. Sections were scored a 2 if at least 50% of the cells contained reaction product. A score of 3 meant that most cells were immunoreactive and a 4 was given if all cells were positive. A score of 5+ was assigned only when the immunoreaction was so intense that the neuromelanin pigment was frequently obscured. For the assessment of neuropil and non-neuronal staining in the substantia nigra, the specimens were assigned a score of 0 (no staining) to 5. A median score was tabulated for each investigator and is reported in tables 2 and 3⇓. There was no statistical difference between the scores for the two investigators according to the Mann-Whitney U test.

Tyrosine hydroxylase immunostaining.

Tyrosine hydroxylase immunostaining was used to demonstrate the presence and morphologic integrity of the dopaminergic neurons. No differences between the control and RLS brains were observed in the staining intensity or the appearance of these neurons and associated processes in any of the midbrain structures (figure 1, A and B).

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Figure 1. Representative micrographs from the human substantia nigra comparing control brains (left panels) to restless legs syndrome (RLS) brains (right panels). A magnification bar is placed in H and the magnification listed at the end of the legend for each panel refers to that bar. (A, B) Tyrosine hydroxylase. The number of substantia nigra neuromelanin cells is comparable in both the control (A) and RLS brains (B). The neuromelanin is the brown pigment in the cells. The cell bodies and their process are immunolabeled for tyrosine hydroxylase (chromagen for the reaction product is blue) with no visibly detectable difference between control and RLS brains. The arrows point to typical cells as examples. Bar = 12.5 μm. (C, D) Iron staining. (C) A modified Perls reaction for iron staining is used (see text), and in this figure the neuromelanin is brown and the iron reaction product is blue. In the control brain the neuromelanin containing cells are clearly present and contain no blue reaction product for iron (e.g., the cell at the white arrowhead containing brown neuromelanin pigment). The iron-positive cells (e.g., at arrow) are oligodendrocytes. (D) In the RLS brain, the neuromelanin cells do not stain for iron and there are relatively few iron-positive cells. Occasionally, cells that stain for the Perls reaction are present in the RLS brain. An example of this cell type is indicated in the figure (arrow) and has processes, unlike those iron-positive cells seen in the control brains. Bar = 16 μm. (E, F) H-ferritin. In these figures, the H-ferritin reaction product is blue. The neuromelanin pigment is brown. (E) In the control brain there is blue reaction product in the neuromelanin cells (e.g., at white arrowhead) indicating the presence of H-ferritin. In addition, numerous blue staining processes and cells are present in the parenchyma. Occasionally, an H-ferritin positive oligodendrocyte is visible (arrow). (F) In the RLS brain section there is no detectable H-ferritin in the neuromelanin containing neurons or the processes in the parenchyma. A few positive glial cells are visible (e.g., at arrows). This observation is consistent with an absence of stored iron in the neurons in this region of the RLS brain. Bar = 16 μm. (G, H) L-ferritin. In this figure, L-ferritin immunoreaction product is blue and the neuromelanin is brown. (G) L-ferritin is present in the control brains in small round cells (arrows) but rarely in cell processes. Most of the neuromelanin containing cells do not stain for L-ferritin. (H) In the RLS brain, L-ferritin positive cells are also clearly present, but morphologically distinct from the majority of cells in the control brains. Almost all of the L-ferritin positive cells in the RLS brain are ramified (arrows) and have the morphologic appearance of astrocytes and microglia. L-ferritin positive neuromelanin cells are rare. Bar = 8 μm.

Iron staining.

Iron staining in the substantia nigra is normally found predominantly in oligodendrocytes with some diffuse staining in the neuropil (figure 1C). Neuromelanin cells rarely stain for iron. In the brains from individuals with RLS, there was a dramatic decrease in iron staining in the neuropil and iron-positive oligodendrocytes were rare in 6 of 7 brains examined (figure 1D). In one RLS brain, iron staining in the oligodendrocytes was similar to the control brains, but this individual was still identified as in the low end of normal. This individual was not one in whom infarcts were observed, so infarcts did not influence the staining pattern. There were a few non-neuronal iron-positive cells in the RLS brains that are process bearing and may be astrocytes or microglia or both. Similar to controls, the neuromelanin cells in the RLS brains did not contain reaction product for iron.

Ferritin staining.

In the control brains, the H-chain of ferritin could be detected in some of the neuromelanin cells and the immunoreaction product extended into the primary processes of these cells. Some neuropil staining was also visible. Oligodendrocytes were the cells most strongly stained with H-ferritin (figure 1E). In the RLS brains, there was a dramatic decrease in the H-ferritin staining compared to controls. Staining of the neuromelanin cells in the RLS brains for H-ferritin was rare and the neuropil was unstained. There were a few round, non–process bearing cells scattered in the neuropil that were H-ferritin positive (figure 1F).

L-chain ferritin subunit was found in occasional neuromelanin cells in control brains, but most of the L-ferritin positive cells were small and round (figure 1G). In the RLS brains, L-ferritin positive neuromelanin cells were rare. There is a considerable number of L-ferritin stained cells in RLS brains (figure 1H) but these cells differ morphologically from those seen in control brains. The L-ferritin positive cells in RLS brains are mostly process bearing and appear to be a mixture of astrocytes and microglial cells.

Metal transporter 1 (ferroprotein).

In control brains (figure 2A), neuromelanin cells contained MTP and numerous processes in the neuropil were also visible. In contrast, in RLS brains (figure 2B), there was minimally detectable staining in the soma of the neuromelanin cells. There was also a decrease in the number of neuromelanin cells with MTP positive processes. The immunostaining in the neuropil was similar in intensity between the RLS and control brains.

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Figure 2. Micrographs from the human substantia nigra of proteins involved in iron transport. Micrographs from control brains are in the panels on the left and micrographs from restless legs syndrome (RLS) brains are in the panels on the right. A magnification bar is placed in H and the magnification listed at the end of each panel legend refers to that bar. (A, B) Metal transport protein 1 (MTP1). The control brain (A) shows the presence of MTP1 (blue reaction product) in the neuromelanin cells. The MTP1 reaction product is confined mostly to the soma (e.g., at arrows), but a few primary processes are visible. The melanin pigment appears brown in the micrograph. There is a considerable amount of process staining in the neuropil. (B) In the RLS brain, the neuromelanin pigment is brown. Most of the neuromelanin cells are devoid of reaction product (e.g., cell at arrow) but immunostained processes in the neuropil are prominent. These immunostained processes have a similar appearance to those in the control brains. Bar = 8 μm. (C, D) Divalent metal transporter 1 (DMT1). In C, the staining in the control brains (blue reaction product) shows a strong reaction in the brown neuromelanin containing cells extending into the primary process (e.g., cell at arrow) whereas the staining in the RLS brains (D) is rare with only an occasional small cell (e.g., at the arrow) present. Bar = 30 μm. (E, F) Transferrin receptor. (E) Transferrin receptor immunoreaction product (blue) is present on most of the brown neuromelanin containing cells in the control brains. The immunoreaction product is found on the soma and extends into a primary process in all cases (e.g., cell at arrow). (F) In the RLS brains, the immunoreaction product for Tf receptor is minimal and immunostained cell processes are rare on the brown neuromelanin cells (e.g., cell at arrow). Bar = 16 μm. (G, H) Transferrin. (G) Transferrin (blue reaction product) is visible in the neuromelanin cells (e.g., at arrow) in the control brain and in oligodendrocytes, some of which are near the neuromelanin containing neurons (e.g., arrowhead). There are also processes that are Tf positive in the neuropil. (H) By comparison, the RLS brain has considerably more Tf reaction product in the neuromelanin cells (e.g., at arrow) and in the processes of these cells than the control brain. The Tf positive processes in the neuropil are also much more striking in the RLS brain than in the control brain. Occasional small, round cells are present (e.g., arrowhead) as in the control brain. Bar = 8 μm.

Divalent metal transporter 1.

Divalent metal transport protein 1 (DMT1) was expressed in the neuromelanin cells including their proximal processes in normal brains (figure 2C) but the reaction product was rarely found in the neuromelanin cells in RLS brains (figure 2D). In the neuropil, DMT1 staining was light and cells were rarely found.

Transferrin receptor.

In the control brains, transferrin receptor staining on neuromelanin cells was found on both the soma and processes (figure 2E). However, in the RLS brains, transferrin receptor staining was minimal (figure 2F). The neuropil was unstained and no non-neuronal cells were immunostained in either RLS or control brains.

Transferrin.

Immunostaining for transferrin was found in the neuromelanin cells in the control brains, but the staining was relatively light and confined to the soma (figure 2G). In addition to the staining in the neuromelanin cells in the control brains, transferrin immunopositive oligodendrocytes were also visible. In RLS, the staining intensity for transferrin was much more robust in the neuromelanin cells than in the control brains (figure 2H) and the immunoreaction product extended into the primary processes. In addition, numerous darkly stained Tf-positive processes were seen in the neuropil.