Original Contributions
Iron accumulation in the substantia nigra in rats visualized by ultrasound

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Abstract

In recent studies, we have found a marked increase in substantia nigra (SN) echogenicity in patients with Parkinson’s disease (PD) using transcranial ultrasound. Because a substantial body of evidence has accumulated indicating a selective elevation of iron in the SN from patients with PD, we set out to test the hypothesis that trace metals like iron could lead to the observed increase of SN echogenicity in PD. Rat brains were scanned after stereotactic injection of iron in different concentrations into the SN and after injecting ferritin, zinc and 6-OHDA alone, and after the addition of desferrioxamine. The amount of iron in the SN was measured spectroscopically. For iron, and partly for 6-OHDA, in different concentrations, a dose–dependent increase of SN echogenicity could be visualized, corresponding to an increase of iron measured by spectroscopy. No increase of echogenicity was visualized after the injection of ferritin and the addition of desferrioxamine to 6-OHDA, though an increase of iron was measured by spectroscopy. Therefore, we conclude that iron not bound to these proteins may lead to an increase of echogenicity of the SN.

Introduction

In recent studies, we were able to show different patterns of substantia nigra (SN) echogenicity in human beings using trasncranial ultrasound (for details see Becker and Bogdahn 1993, Bogdahn et al 1998). In patients with Parkinson’s disease (PD), we have found a marked increase of SN echogenicity (Becker et al. 1995). This hyperechogenicity was attributed to either an increase of iron Dexter et al 1989, Dexter et al 1993, Gerlach et al 1994, Hirsch et al 1991, Riederer et al 1989, Sofic et al 1988, Sofic et al 1991, Wang et al 1995 or a change of the cytoarchitecture and glial-cell composition of the SN due to the degenerative changes that are known to occur in PD Barron 1995, Montine et al 1995. However, gliosis in the SN in PD is only mild, and histopathological findings disclosed more a cell loss than a profound increase in cell density. Interestingly, a hyperechogenicity of the SN was also detected in about 9% of healthy subjects in a group of 420 volunteers (unpublished data). This observation once more raised the question about the etiology of the change of SN echogenicity. Keeping in mind that animal models can only give limited insights into the pathology of PD, we tested if trace metals like iron or iron-binding proteins could lead to a change in SN echogenicity by performing ultrasound examinations in a rat model, followed by a spectroscopic measurement of iron in the SN of both sides.

Section snippets

Animal preparation

Wistar rats, 3–4 months old, were housed under controlled temperature and light conditions. They were anesthetized with chloral hydrate. Rat brains were injected stereotactically into the right SN with:

  • 1.

    iron in the concentration of 0.3, 1.5 and 7.5 μg of FeCl3 (in 2 μl tris-citrate buffer);

  • 2.

    ferritin (3.2 μg ferritin in 2 μl tris-citrate buffer), to mimic possible alterations of protein-bound iron;

  • 3.

    6-OH-dopamine (6-OHDA) (1μg and 8μg 6-OHDA in 2 μl saline 0.9% and 0.02% ascorbate), which is known

Ultrasound findings

Echogenicity of the right SN and adjacent areas increased with the concentration of iron injected (Fig. 1). After the injection of low concentrations of iron (0.3 μg), a slight increase of echogenicity was realized; after injection of 1.5 and 7.5 μg of iron, echogenicity was marked. Additionally, a wider spread of the dose–dependent inreasingly echogenic signal could be visualized. At the lowest concentration, the signal was restricted to the area of the SN, at 1.5 μg, close adjacent brain

Discussion

This pilot study demonstrates a dose–dependent increase of SN echogenicity for iron in different concentrations and, partly, for 6-OHDA in different concentrations that corresponded to a dose–dependent increase of iron in the injected SN measured by spectroscopy. This is in accordance with the hypothesis of an increase of SN echogenicity due to iron accumulation. Because the amount of iron measured by spectroscopy was only slightly higher after the injection of 8 μg compared with the injection

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