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Projekt POMOST

Application of SIMS and DESI to measure the oxidative stress accompanying morphine administration


Addiction to drugs becomes a severe medical, social and economical problem nowadays. It is recognized as a complex, chronic and relapsing mental disorders, associated with changes in behavior and chemistry of the brain. Apart from the price, which is paid by the abusers themselves (devastated physical and mental health), the whole society bears the costs related to medical treatment, crimes, spreading epidemics of AIDS, hepatitis, tuberculosis etc. Our knowledge on the biological background of drug addiction is constantly increasing, but still many mechanisms remain elusive and need further research. Revealing the biological background of this disease brings the promise to find new medical treatment for the affected people.

Due to those facts, the Work Programme “Health 2011” of the European Union Seventh Framework Programme, focuses on addiction as a life-style related health problem. It indicates that an effort is needed to achieve a better understanding of the underlying causes of this phenomenon, which allows combating it.

 

Addiction and oxidative stress


There is growing number of evidence that heroin, morphine and opiates may be the cause of the oxidative stress in the cell, which may lead to oxidative damage of molecules. Oxidative stress is combined with decreased amount of antioxidants, and with increased production of molecules known as reactive oxygen species. It may lead to oxidative damage of proteins, lipids and DNA and, in consequence to apoptosis, or necrosis of the cell. Oxidative stress is additionally thought to be involved in many neurodegenerative diseases such as Alzheimer or Parkinson disease.

In our previous studies [1,2] we were using 2D gel electrophoresis and mass spectrometry to identify proteins, which expression is changed after morphine administration. As an experimental model, we used neuronal and astrocyte cell cultures. During our studies we found several proteins engaged in the response of the cell to oxidative stress. In the neurons from striatum we found changes in levels of dihydrolipoyl dehydrogenase - NO scavenger [3], aldehyde dehydrogenase - antioxidant enzyme [4], and glyceraldehyde-3-phosphate dehydrogenase, which is known as a sensor for NO stress [5]. Additionally, last protein was found to be affected by morphine administration by Kim et al., which may be considered as an additional validation of our work (finding proteins which were identified by different proteomics studies may be facilitated by the database created by our team www.addiction-proteomics.org) [6].

 

The general aim of the project


Till now oxidative stress combined with morphine administration was examined by using the homogenate from the whole brain (for example: [7]), or by the serum analysis [8]. As an indicator of oxidative stress the level of malondialdehyde, and glutathione peroxidease, as well as DNA damage, and protein carbonyl group were measured.

There is no detailed knowledge about exact places in the brain, where oxidative stress occurs after morphine administration. We do not known the type of the cells responsible for that effect, either. In our project we would like 
to focus on this problem, and examine it by using imaging mass spectrometry techniques such as SIMS and DESI.

 

Addiction model


Various methods may be used to induce morphine dependence. The method of morphine exposure (passive – by experimenter, or active – by animal), the scheme and duration of application as well as used dose may differ. But if only the animals shown acute morphine withdrawal (jumping, wet dog shakes, paw tremor others), after administration of an opiate antagonist, they are considered as morphine dependent. As an addiction model in our study we are planning to use male Wistar rats addicted by subcutaneous implementation of a pellet containing 75 mg of morphine for three days.

To elucidate what types of cells are responsible for oxidative stress we would like to use cortical astrocyte and striatal neuronal cell cultures in monolayers. It is known that opioid receptor stimulation increase reactive oxygen spieces production in cultured microglia [9], thus those cells will be excluded from the study. Considering the cell culture it is difficult to say about the drug dependence, since it is complex physiological, biochemical, behavioral and psychological phenomenon. In this model we may only observe the morphine influence on the biochemistry of the cell, by interaction with μ- opioid receptors. In our model we will treat the cells with morphine at concentration of 10 μM, for five days. It is worth to mention that morphine concentration of 10 μM is a clinically relevant dose, since it is achieved in CSF after epidural or intrathecal administration of opioids [10]. Vlaskowska et. al. considered morphine administration for 5 days as a model of chronic administration [11].

 

How to see oxidative stress in the sample?


Imaging mass spectrometry (MS imaging) is a new, constantly developing, and innovative technology, which allows for the investigation of spatial distribution of molecules at complex surfaces, such as monolayers of the cells in culture, or tissue sections. An important feature of this technique is that it does not demand any additional labeling of the investigated substances (such as immunostaining). The molecular weight and specific structure of the substance (for example amino acid sequence) might be deciphered from the MS spectrum to unambiguously identify it.

Surface analysis has several advantages in comparison to “classical” approach involving tissue dissection and analysis. First, in the case of tissue sections, by using brain atlases, which exactly describe position of particular structure, we may be sure about the proper localization of the observed changes. Secondly, this method allows for analyzing brain structures that are too small to be undoubtedly removed from the brain. In the case of monolayer cell culture analysis, there is no need to do any special sample preparation, apart from removing cell culture media, by rinsing the cells with buffered ammonium acetate and drying the surface [13].

 

DESI (desorption by electrospray) is an example of MS imaging approach [14]. This technique is capable of localization and identification of wide range of substances in biological tissues (mass range 100 – 60 000 Da). DESI uses electrospray source to produce charged droplets of organic solvent, which are focused on analyzed material. Those droplets impact the surface and produce a second generation of charged droplets containing surface molecules, which become ionized. Gaseous ions are introduced to the mass spectrometer inlet and are analyzed according to their mass to charge ratio. Such construction is especially suitable for rapid and nondestructive surface analysis. The entire procedure is very fast and does not demand special sample preparation. Thus, arduous and prone to human mistake steps sample preparation characteristic for example for classical proteomics approach may be omitted. The resolution of such analysis is about 0.01mm2.

 

SIMS [15]. (Secondary Ion Mass Spectrometry) uses high-energy primary ions (mainly Ga+, Cs+, Aun+, O2+, O-, Ar+, Xe+ or electrons) to strike the sample surface and released secondary ions of substances, which are present there. Due to extensive surface fragmentation, its mass range is limited to small molecules (< 1000 Da). This is compensating by much higher spatial resolution than DESI (submicrometer), high chemical specificity and surface sensitivity. High resolution of this method allows for subcellular observation. On the contrary to DESI, SIMS needs ultra high vaccum for analysis, but like DESI, it does not demand any special sample preparation.

Vitamin E functions as a chain breaking antioxidant that prevents the propagation of free radical reactions. It is considered as the main, lipid soluble antioxidant in the body [16]. Localization of vitamin E in the certain area of the tissue may be the indicator of oxidative stress. TOF-SIMS technology was used by Touboul et al. confirm the presence of this phenomena in mouse model of Duchenne dystrophy  [17]. Apart from SIMS analysis of vitamin E distribution in tissue section, this compound was analyzed in single neuronal cell [18], which confirm usefulness of this method for our purposes.

DESI, apart from its ability to measure the distribution of vitamin E, which was shown in the study of Ozmen et aL. [19], allows for the analysis, called reactive DESI. Here, a suitable chemical reagent may be dissolved in the spray solvent, which allows for characteristic, rapid reaction occurs at a spot being sampled, concurrently with acquisition of mass spectra to improve sensitivity and selectivity of detection of target molecules [20]. We would like to use this attempt to confirm the presence of oxidative stress by measuring the marker of peroxitation processes - malondialdehyde (MDA). This aldehyde is produced by the radical breakdown of hydroperoxides resulting from lipids and fatty acids peroxidation, containing at least two double bonds. Its presence may be measured by derivatisation with dinitrophenylhydrazine (DNPH).

In our experiments addicted and control rat brains will be cut into slices. The surface of each slice will be examined, by SIMS and DESI to find the distribution of vitamin E in each case. Comparison of intensities of the peaks characteristics for vitamin E, between addicted and control animals, will allow us to indicate the area of the brain affected by oxidative stress. Additionally, level of MDA will be measured by DESI, as a confirmation of previous results, and validation of using vitamin E as an indicator of oxidative stress. Using the same methodology, we would like to examine the monolayer cell culture of neuron and astrocytes.

Experiments leading to obtain addicted and nonaddicted rat brains will be conducted in Medical Academy in Lublin in cooperation with professor Jolanta Kotlińska. For this project we would like to buy microtome, which would facilitate brain tissue sections. Existing data about vitamin E distribution in normal state may be used as a reference for our results.

Discussed approach could not only indicate the areas of the brain subjected to oxidative stress after morphine administration, but also allows for comparison of the two methods of MS imaging: SIMS and DESI. Confirmation that those two methods are compatible, offers a great opportunity to extend the range of SIMS analysis by DESI. Additionally DESI offers an opportunity to perform MS/MS or even multi step fragmentation. Schematically, during this procedure, ionized substance is broken into ionized pieces, and its MS spectrum is acquired. MS spectrum of obtained fragments is characteristic for the substance and may be used for its identification. Usually special bioinformatics’ algorithms and databases facilitate this process. SIMS cannot identify unknown species in this way due to it construction, thus if we show compatibility of those methods, in future research, we may try to use DESI to identify unknown molecules discovered by SIMS.

All SIMS experiments will be performed in cooperation with professor Roman Pędrys group from Department of Medical Physics at Institute of Physic, Jagiellonian Uniwersity. All the equipment necessary for DESI analysis will be purchased by our group from grant of Ministry of Science and Education. Since optimisation of DESI ion source for biological sample analysis will demand extensive work we planned to engage two students to help us in this work and to optimise the way of measurements for the cell cultures and tissue sections. This should be a main subject of their master thesis. In all the experiments concern DESI and cell cultures dr. Piotr Suder will be engaged.

 

Expected results.

To sum up, as a result of this project we would like:

  • To indicate structures of the brain subjected to oxidative stress by morphine administration, by using elevated level of vitamin E  (SIMS, DESI), and MDA measurements (DESI) as an indicator of this state.
  • To specify the type of the cells, which are the sources of oxidative stress.
  • To create a methodology which may be applied to examine oxidative stress in different states, or diseases.
  • To check the compatibility between DESI and SIMS which could be beneficial for further analysis.

In the future perspective, creating a laboratory unit which will be able to perform spatial analysis will be beneficial not only for our group, but also for different scientific departments. In our research we are focused on biochemical application of discussed methods, but DESI may be use in analysis of spatial distribution of great variety of different substances. Additionally, since at the AGH University there is dynamic developing unit working which biomaterials we hope that this new method of analysis will be also beneficial for their research. Furthermore, possibility of using this technique in analysis of clinical samples such as tumour tissues may make this technique interested for clinicians, which may benefit in cooperation with Medical College UJ, or Analytical Department of UJ.

 

 

 

Conferences:


Participant

Date

Conference

Subject

 

Anna Bodzoń-Kułakowska

29.IV.2012

30th Informal Meeting on Mass Spectrometry

Cholesterol and vitamin E analysis in prefrontal cortex
of morphine-addicted rat brain using TOF-SIMS mass spectrometry

 

Poster

Magdalena Skalska

29.IV.2012

30th Informal Meeting on Mass Spectrometry

Preparation of astrocyte primary cell culture
for TOF-SIMS analysis

 

Poster

Anna Bodzoń-Kułakowska

15.IX.2012

19th International Mass Spectrometry Conference

The potential use of reactive DESI to assess oxidative stress in the cell culture

 

Poster

Piotr Suder

15.IX.2012

19th International Mass Spectrometry Conference

DESI-MS imaging of differences between physiological and morphine-treated rat brains

Poster

Anna Bodzoń-Kułakowska

5.V.2013

31th Informal Meeting on Mass Spectrometry

Cell culture preparation for DESI analysis

 

Poster

Anna Bodzoń-Kułakowska

11.V.2014

32th Informal Meeting on Mass Spectrometry

DESI MS in the cel cultire analysis

Oral presentation

 

 

Articles:


Desorption electrospray ionisation (DESI) for beginners--how to adjust settings for tissue imaging.
Bodzon-Kulakowska A, Drabik A, Ner J, Kotlinska JH, Suder P, Rapid Commun Mass Spectrom. 2014 Jan 15;28(1):1-9. doi: 10.1002/rcm.6755.

 

DESI-MS as a tool for direct lipid analysis in cultured cells.
Bodzon-Kulakowska A, Cichon T, Golec A, Drabik A, Ner J, Suder P Cytotechnology. 2014 May 7.

 

DESI analysis of mammalian cell cultures – sample preparation and method optimisation,
Journal of Mass Spectrometry