Tom Weaver (Presenter)
Teledyne CETAC Technologies
Authorship: Christina Koeppen (1), Olga Reifschneider (1), Indra Castanheira (1), Michael Sperling (1,2), Uwe Karst (1), Guiliano Ciarimboli (1), Dhinesh Asogan (3)
(1) University of Muenster, Muenster, Germany (2) European Virtual Institute for Speciation Analysis (EVISA), Muenster, Germany (3) Teledyne CETAC Technologies, Omaha, NE
| Short Abstract This work presents a quantitative bioimaging method for platinum based on laser ablation-inductively coupled plasma-mass spectrometry and its application for a biomedical study concerning toxic side effects of cisplatin. To trace the histopathology back to cisplatin, platinum was localized and quantified in major functional units of testicle, cochlea, kidney, nerve and brain sections from cisplatin treated mice. The direct consideration of the histology enables precise interpretation of the Pt images and the novel quantitative evaluation approach allows significantly more precise investigations than the pure image. |
Long Abstract
Introduction
Cisplatin is a widely used chemotherapy drug with high cure rates for several types of cancer but its application is limited due to toxic side effects.[1,2] In particular, the induced kidney injury limits further usage of cisplatin and application of effective doses.[1,2] Beside nephrotoxicity, ototoxicity, infertility and neurotoxicity were reported to be adverse effects of cisplatin.[1,2] Several histological studies examined the changes of the injured organs to identify affected cells or functional areas. In most studies, organ sections were investigated by light microscopy to obtain more information about the pathogenesis of the respective toxicity.[1,3–5] Histopathological evaluation of the injured kidneys has identified the renal tubule including proximal tubule as major injured structure in kidneys.[4,6,7] To trace the histopathology back to cisplatin, elemental imaging methods such as X-ray microanalysis[8–10] or radiography[7,11] (combine with radiolabeling or neutron activation) were used to detect the platinum distribution in the affected organs. These methods allow the detection of platinum at sub-micrometer scale with mostly sufficient sensitivity. For kidneys of cisplatin-treated pigs, Makita et al. observed high platinum signals in the injured proximal tubule cells.[8,9] However, the application of these methods in medical studies requires special sample preparations such as ultrathin organ sections (40 nm) on beryllium grids[9] or enormous experimental effort such as nuclear reactors.[11]
Methods
For LA-ICP-MS measurements, a laser ablation (LA) system LSX-213 (Teledyne CETAC Technologies, Omaha, USA) with a frequency quintupled, Q-switched Nd:YAG laser (213 nm) was coupled to a quadrupole-based ICP-MS iCAP Qc (Thermo Fisher Scientific, Bremen, Germany). The LA system was equipped for improved washout behaviour with a home-built laminar flow cell with an effective volume of 7.3 ml (washout time of 500 ms for platinum in polymer sections).
Tissue sections of 3 um for cochlea or 5 um for the other organs (kidneys, testicles, nerves, brains) were obtained and mounted onto slides. To visualize the structure of the tissue, cochlea sections were stained with HE (haematoxylin and eosin, Carl Roth, Karlsruhe, Germany) according to manufacturer’s instructions and optical images
were recorded with the inverted microscope in bright field mode. The structure of the other organs was sufficiently visible in autofluorescence (excitation wavelength of 450 nm) or phase contrast mode. Micrographs of kidneys, testicles and nerves were taken in fluorescence mode, while brain sections were recorded in phase contrast mode.
For imaging, organ sections were ablated by subsequent line scans with averaged output energies of 3 mJ per shot (complete ablation of 3 or 5 mm thick polymer sections), laser spot sizes between 10 mm and 50 mm and a shot repetition rate of 20 Hz. To obtain the desired spatial resolution (Rs), the distance between the lines was set to Rs and the laser scan speed to Rs per duty cycle of ICP-MS. In this way, the sampling setup was allowed to 'oversample' the tissue sections, achieving a spatial resolution better than the chosen laser spot size.
Results
To use LA-ICP-MS for biomedical studies, the method has to be adjusted for the respective biomedical question and for the requirements of the particular samples. The objects of this study are different organs with soft and hard tissues. In LA-ICP-MS, the sample matrix influences the ablation behaviour. To unify the sample matrix and thus the ablation behaviour, a recently published method was adapted and all organs were embedded in the resin Technovit 7100.[12] The major advantage of this embedding for LA-ICP-MS is the polymerization in every compartment of the tissue, so that the whole tissue and all types of tissue have the same density and hardness as the resin. This allowed both direct comparison with traditional micrographs to identify migration and aggregation within various structures of the tissue sections at a spatial resolution of 14 um and accurate determination of the Pt concentration within.
Conclusion
In summary, our results clearly show the great performance of
LA-ICP-MS for biomedical imaging of platinum and contribute to the understanding of the mechanism of the investigated toxic side effects of cisplatin. For the first time, platinum was detected and quantified in all major injured structures of testicle, cochlea, kidney and nerve. Especially its localization in organ of Corti of cochlea and in the seminiferous tubule of testicle offers new insight into pathogenesis of cisplatininduced side effects. It illustrates the high sensitivity and precision of the developed method, as existing methods failed for these important structures for hearing and fertility.
References & Acknowledgements:
1 C. A. Rabik and M. E. Dolan, Cancer Treat. Rev., 2007, 33, 9–23.
2 A.-M. Florea and D. Bu¨sselberg, Cancers, 2011, 3, 1351–1371.
3 R. P. Miller, R. K. Tadagavadi, G. Ramesh and W. B. Reeves, Toxins, 2010, 2, 2490–2518.
4 N. Pabla and Z. Dong, Kidney Int., 2008, 73, 994–1007.
5 L. A. B. Peres and A. D. da Cunha, J. Bras. Nefrol., 2013, 35, 332–340.
6 N. E. Madias and J. T. Harrington, Am. J. Med., 1978, 65, 307–314.
7 J. P. Fillastre and G. Raguenez-Viotte, Toxicol. Lett., 1989, 46, 163–175.
8 T. Makita, K. Hakoi and T. Ohokawa, Cell Biol. Int. Rep., 1986, 10, 447–454.
9 T. Makita, S. Itagaki and T. Ohokawa, Jpn. J. Cancer Res., 1985, 76, 895–901.
10 T. Saito and J. M. Aran, ORL, 1994, 56, 310–314.
11 P. S. Tjioe, K. J. Volkers, J. J. Kroon, J. J. M. de Goeij and S. K. The, Int. J. Environ. Anal. Chem., 1984, 17, 13–24.
12 O. Reifschneider, C. A. Wehe, I. Raj, J. Ehmcke, G. Ciarimboli, M. Sperling and U. Karst, Metallomics, 2013, 5, 1440–1447.
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | Teledyne CETAC Technologies |
| Board Member | no | |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | yes |