IBA in life sciences

Quantification and localisation of trace elements in Parkinsonian brain:
A proton beam microscopy study

M. Sc. Nirav Barapatre

The Parkinson's disease is characterised by the severe loss of dopaminergic neurones in the Substantia nigra (SN) of the brain. These neurones also possess a browny-black pigment called neuromelanin (NM), which chelates and accumulates metal ions. The role of neuromelanin in the regulation of trace metal ions has come under scrutiny, since an overload of iron in the SN of Parkinsonian brain has been reported in many studies [1]. In general, these studies do not provide information on the localisation of iron overload within the SN. With the help of proton beam microscopy one can locate and quantify metal ions with trace element sensitivity [2]. The trace element concentration in the SN of three Parkinsonian cases was compared with three controls. Particularly, the trace metal content bound to NM and in the extra-cellular region was analysed. Surprisingly, no significant difference was found in the Fe content either bound to neuromelanin or in the extracellular region when the mean of three Parkinsonian cases was compared against the mean of three controls. The Ca and K content bound to neuromelanin as well as in the extra-cellular region was significantly altered. Also, the Cu content in the extra-cellular region was significantly lower in the Parkinsonian cases.
Figure 1: Left: An optical picture of a neuron containing the browny-black pigment neuromelanin. Right: A three element map of the same neurone showing phophorous, sulphur and iron distribution. Turquoise colour is due to overlap of blue (Fe) and green (S).

This work was supported by the Deutsche Forschungsgemeinschaft in the framework of GRK InterNeuro.

[1] M. Gtz, et al.: Ann. N.Y. Acad. Sci. 1012, 193 (2004)
[2] T. Reinert, et al.: Nucl. Inst. Meth. B249, 734 (2006)


Quantitative Element Microscopy of Human Hippocampi of Opiate Abusers

Dr. Tilo Reinert

Metal-binding metallothioneins (MT) with a strong neuroprotective effect in the mammalian brain were shown to be upregulated in the brains of morphine intoxicated rats. Data from animal models of addiction hint at an e. g. impaired zinc homeostasis. This might particularly affect the large neuroplastic potential of the hippocampus and would reduce capabilities of cognition and memory. However, semi-quantitative analysis of MT protein expression in human hippocampi by means of immunohistochemistry revealed no significant difference of MT expression in drug addicts versus age and gender matched controls. Thus, we assumed that the amount of bound metals such as zinc could be affected, while the antibody-recognized protein expression remains constant.

In order to quantitatively compare the concentrations of zinc and other relevant elements (P, S, Ca, Fe, Ni, Cu) we performed a PIXE study of the hippocampal structures of unstained DePeX embedded brain sections of five opiate abusers and five control individuals. We analysed the hippocampal regions stratum granulare (SG), stratum moleculare (SM), CA1, CA2, CA3, CA4, and alveus (Alv).

Figure 2: Left: Micrograph of the hippocampus with its regions (SG: stratum granulare, SM: stratum moleculare, CA1, CA2, CA3, CA4, Alv: alveus). Right: Zn-content of the regions for opiate  abusers and the control group (error bars: standard error). Significantly lower concentrations are flagged by an asterisk.

Zinc concentrations were found to be significantly decreased in the hippocampi of the addicts in the stratum granulare (p = 0.028), the stratum moleculare (p = 0.047, the CA1 (p = 0.093) and the CA4 (p = 0.050). Except for the alveus where the zinc concentration was higher in the drug addicts all other regions showed a slight trend to lower zinc concentrations in the drug addicts (Fig. 2). The amount of Fe was slightly higher in the hippocampi of the addicts than in the controls in all regions except stratum moleculare and CA4. However, statistical analysis for each region revealed no significant differences.