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Toxicology 262 (2009) 192–198

Contents lists available at ScienceDirect

Toxicology

journa l homepage: www.e lsev ier .com/ locate / tox ico l

dverse effects of cyclosporine A on HSP25, alpha B-crystallin and myofibrillarytoskeleton in rat heart

lessandra Stacchiotti a, Francesca Bonominia, Antonio Lavazzab, Luigi Fabrizio Rodellaa, Rita Rezzania,∗

Division of Human Anatomy, Department of Biomedical Sciences and Biotechnology, University of Brescia, V.le Europa 11, I-2513 Brescia, ItalyIstituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia-Romagna, Brescia, Italy

r t i c l e i n f o

rticle history:eceived 29 May 2009eceived in revised form 9 June 2009ccepted 10 June 2009vailable online 18 June 2009

eywords:yclosporine Aytoskeletoneat shock proteins

mmunohistochemistry

a b s t r a c t

Cyclosporine (CsA) is a universally used immunosuppressive drug which induces adverse side effects inseveral organs, but its impact on the heart is still controversial.

Small heat shock proteins (sHSPs), such as HSP25 and alpha B-crystallin, are cytoprotective stress pro-teins exceptionally represented in the heart. They act as myofibrillar chaperones that help actin anddesmin to maintain their optimum configuration and stability, thereby antagonizing oxidative damage.The present study examined: (1) the cardiac distribution and abundance of HSP25 and alpha B-crystallinin rats receiving CsA at a therapeutic dosage (15 mg/kg/day) for 42 days and 63 days; (2) the presenceof myofibrillar proteins, such as actin, alpha-actinin and desmin following the CsA treatments; (3) thesubcellular effects of prolonged CsA exposure on the cardiomyocytes by histopathology and transmissionelectron microscopy. After 63 days CsA intake, sHSPs translocated from a regular sarcomeric pattern to

ransmission electron microscopy peripheral sarcolemma and intercalated discs, together with actin and desmin. In contrast, the sarcomericalpha-actinin pattern did not change in all experimental groups. The abundance of actin and HSP25 wasunchanged in every time point of treatment while after 63 days CsA, alpha B-crystallin and desmin levelssignificantly decreased. Furthermore CsA induced fibrosis, irregular sarcomeric alignment and damageddesmosomes. These findings indicate that following prolonged CsA exposure, the cardiac muscle networkwas affected. In particular, the translocation of sHSPs to intercalated discs merits special consideration

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as a direct compensatory

. Introduction

Cyclosporine A (CsA) is the most frequently used immuno-uppressant in organ transplantation and in the treatment ofutoimmune disorders. However, the main disadvantage of CsAherapy is its side effects in several organs, such as the kidney, liver,essels and the heart as reviewed by Rezzani (2004).

Nevertheless CsA induced cardiotoxicity is still controversialecause there are many confounding effects associated with itsystemic administration (Miller, 2002; Zhang, 2002) and dishomo-eneity in studies with CsA, either in vivo or in vitro, at varyingimes and dosages.

Some studies indicated that CsA is beneficial against myocar-ial ischemia and infarct size (Niemann et al., 2002; Bes et al.,

005). Conversely, other studies reported that CsA causes a cardio-epressive effect at clinically relevant concentrations (Janssen etl., 2000; Zakliczynski et al., 2003).

∗ Corresponding author. Tel.: +39 030 3717483; fax: +39 030 3717486.E-mail address: rezzani@med.unibs.it (R. Rezzani).

300-483X/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.tox.2009.06.007

anism to limit CsA cardiotoxicity.© 2009 Elsevier Ireland Ltd. All rights reserved.

Accumulating evidence demonstrated that heat shock proteins(HSPs), a highly conserved group of proteins, enhanced after expo-sure to a variety of adverse stimuli (Morimoto and Santoro, 1998).Most of HSPs act as “molecular chaperones” that bind to denaturedproteins and assist proper folding or translocation of essential pro-teins and organules affected by redox stress (Papp et al., 2003),and in impaired myocardial performance. Moreover HSPs beneficialfunctions include repairing ion-channels, restoring redox balance,interacting with nitric oxide-induced protection, preventing apop-totic pathway (Latchmann, 2001; Delogu et al., 2002).

We previously described in rat heart a direct impact of CsA (at15 mg/kg/day) on the expression of different medium and large-sized heat shock proteins such as HSP70 (Rezzani et al., 2006a) andHSP90 (Rezzani et al., 2003), but, at our knowledge, there are nodata regarding the involvement of small sized HSPs.

Small HSPs actually comprise ten known members, weighedfrom 10 to 30 kDa, that are expressed in skeletal and cardiac muscle

(Golenhofen et al., 2004; Macario and Conway de Macario, 2007).So the aim of this study was to analyze in rat heart two funda-mental small HSPs, HSP25 and alpha B-crystallin recently reviewedby Arrigo (2007) and Nakamoto and Vigh (2007). In the heart,they act as myofibrillar chaperones, strictly related to sarcom-

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ric proteins (Lutsch et al., 1997) and their overexpression greatlymproved ventricular function and reduced infarct size (White etl., 2006).

Besides small HSPs, we focused here on the cardiac distribu-ion of actin and its binding protein, alpha-actinin, and desmin,epresentative of the cardiac cytoskeleton (Kumarapeli and Wang,004; Pyle and Solaro, 2004) and strictly linked to mechanical andunctional properties (Kostin et al., 2000).

Actin, the major constituent of thin filaments in the I bandnd its filamentous form, called F-actin, is associated with HSP25Panasenko et al., 2003). Alpha-actinin is located in skeletal andardiac cells in the Z lines, thus outlining the boundary of the sar-omeric unit (Sanger et al., 2000). Desmin is present in costameresnd close to the Z-line (Costa et al., 2004) and in desmin-knockoutouse, the loss of this peculiar association causes a dramatic loss

f cardiac myocytes (Paulin and Li, 2004).In conclusion, in the present study we sought to shed further

ight on the side effects of CsA on the heart by examining the directffect of a prolonged treatment of the immunosuppressant drug onmall HSPs and cardiac cytoskeleton.

Therefore we aimed: (1) to precisely characterize the CsA effectst 63 days on rat ventricular cardiomyocytes by histopathologynd ultrastructural analysis; (2) to analyze the cardiac localizationnd abundance of HSP25 and alpha B-crystallin in rat treated byrolonged CsA treatment at a well established therapeutic dosage15 mg/kg/day) daily for 42 days and 63 days; (3) to determineimilarities and differences with the distribution of cytoskeletalyofibrillar markers such as actin, alpha-actinin and desmin.

. Materials and methods

.1. Animals and experimental treatment

Thirty male adult Sprague Dawley rats (230–250 g body weight) were used.nimals care was made according to National law on the protection of laboratorynimals (D.M.116192) and EC regulations (L358/112/18/1986). All procedures werepproved by Italian Ministry of Health. Rats were housed in a controlled environ-ent at a regular 12 h light–12 h dark cycle at 20 ◦C, relative humidity 50% and fedith a standard diet and water ad libitum. All treatments started after almost 1 week

f stabulation from arrival.Rats were divided into three groups (10 rats each) and treated as follow – group 1:

aily s.c. CsA injection (15 mg/kg/day, Sandimmun; Sandoz); group 2: castor oil, CsAehicle, as control group, s.c. daily; group 3: sterile saline, as an additional controlroup, s.c. daily. All animals were killed by cervical dislocation after 42 days and 63ays.

Rats were perfused by 4% paraformaldehyde (BDH, Milan, Italy) in phosphateuffer 0.1 M, pH 7.4 for 10′ and subsequently heart extracted, hearts dissected, fixed

n the same fixative O.N. at 4 ◦C and embedded in paraffin wax (Paraplast, BDH).o detect cytoskeletal proteins heart after perfusion with 4% paraformaldehyde wasmmediately frozen in liquid nitrogen and stored at −20 ◦C. To perform immunoblot-ing additional groups of five rats were used and, at the end of treatments, heart wasxtracted, immediately frozen in liquid nitrogen and stored at −80 ◦C. For histo-ogic evaluation the sections were stained by Haematoxylin & Eosin and PAS and forbrosis by Masson’s trichrome.

.2. Immunohistochemistry

4 �m sections were collected on polyLlysine (Sigma, St. Louis, USA) coatedlides and dried overnight at 37 ◦C. Immunocytochemical localization of differ-nt markers was performed using ABC-peroxidase method. We tested for theetection of stress proteins polyclonal anti-HSP25 and anti-alpha B-crystallin anti-odies (StressGene, USA), diluted 1:200 and 1:300, for cytoskeleton monoclonalnti-alpha-actinin (sarcomeric) and anti-desmin (Sigma Chemicals), diluted 1:800,:20 and an anti-actin antibody (Chemicon International) diluted 1:250 after aicrowave oven treatment in citrate buffer at pH 6.0. All incubations of primary

ntibodies were made overnight at 4 ◦C. Both secondary biotinylated antibodiesnd ABC reaction were applied sequentially and performed according to commer-ial kit instructions (Vectastain-Elite, Vector Laboratories, Burlingame, CA, USA).

he localization was visualised with DAB and counterstained in Mayer’s haema-oxylin, dehydrated and mounted in DPEX (BDH, Italy). To ensure specificity ofhe immunostaining, adjacent control sections were subjected to all above proce-ure with the exception that primary antibodies were replaced by non-immuneoat or horse serum or preabsorbed with their relative natural or recombinantroteins.

y 262 (2009) 192–198 193

2.3. Immunoblotting

The cardiac tissue was homogenised in TEN buffer (50 mM Tris–HCl pH 7.6,150 mM NaCl, 5 mM EDTA) containing protease inhibitors as previously reported(Stacchiotti et al., 2002) and centrifuged at 10,000 × g for 15 min at 4 ◦C. Protein con-centrations were determined with bovine serum albumin (BSA) serving as standardprotein by absorption spectroscopy. Supernatants were analyzed by 12% SDS-PAGEreducing gels and electro-transferred to a nitro-cellulose membrane (pore size0.45 �m; BioRad) by Western blotting at 100 V for 1 h. The membranes were blockedwith 5% non-fat dry milk in Tris-buffered saline pH 7.4 Tween-20 (TTBS) at 4 ◦C toinhibit non-specific binding sites. After three washing with TTBS, the blots wereincubated overnight at 4 ◦C, with constant shaking, with relative primary antibodiesanti-HSP25, anti-alpha B-crystallin, anti-actin, anti-desmin, anti-actinin all diluted1:1000. Then membranes were washed, reblocked, incubated with a biotinylatedsecondary antibody (Dakopatts) as appropriate; finally with an avidin–peroxidasecomplex according to the manufacturer‘s instructions (ABC kit, Dakopatts), witha solution of 0.05% DAB (3.3-diamino-benzedine tetrahydrochloride) and 0.03%hydrogen peroxide. The developing was made using the ECL-system (Amersham).Densitometric analysis was performed after normalization to tubulin.

2.4. Transmission electron microscopy

Small pieces of ventricules were fixed in 2.5% glutaraldehyde for 3 h at 4 ◦C, post-fixed for 1 h in 1% osmium tetroxide, dehydrated in ethanol and propylene oxide andembedded in Araldite epoxy resin (Serva) for electron microscopic analysis. Bothsemithin (1 �m thick) and ultrathin sections (80 nm thick) were obtained by anultramicrotome (Ultracut E, Reichert-Jung, Germany) using glass or diamond blades(Microstar, USA). For microscopic analysis, semithin sections were stained by tolui-dine blue and ultrathins, collected on cupper formvar-coated grids, double-stainedwith uranyl acetate and lead citrate solutions. Electron micrographs were taken bya Philips CM10 (FEI, Eindhoven, The Netherlands) transmission electron microscopyset at 80 kV.

2.5. Statistical analysis

A quantitative analysis of immunohistochemical and immunoblotting data wasperformed by different investigators unaware of the treatment examined. For quan-titative immunostaining data were collected on sections observed at 100× from 25randomly chosen fields for each experimental group using light microscope (Olym-pus, Germany) equipped with an Image analyzer (Image Pro Plus Milan, Italy).

All data are expressed as mean ± S.D. and statistical analysis between differ-ent experimental groups was made by analysis of variance (ANOVA) corrected byBonferroni test (significant at P < 0.05).

3. Results

Considering that CsA vehicle is castor oil, if not specifically indi-cated, we referred here to castor oil-injected animals as to “controlgroup”.

3.1. Morphology of ventricular cardiomyocytes following vehicleor cyclosporine A

In control group, heart tissue stained with Masson’s trichromeshowed minimal fibrosis but after 63 days CsA treatment, myocar-dial tissue showed disorganization of muscle fibers, connectivetissue was clearly increased and fibrosis was evident (Fig. 1A and B).After 42 days of treatment an intermediate degree of fibrosis wasobserved (data not shown).

By high resolution light microscopy on semithin sections, weobserved a regular distribution of myofibrils and intercalated discsin the control group (Fig. 1C) that became irregular after prolongedCsA treatment where many misaligned fibrillar units appearedoften associated to heterogeneous nuclear morphology (Fig. 1D).

Through ultrastructural analysis in the control group, weshowed several cross-bridges necessary to maintain neighbour-ing cardiomyocytes in a syncytium and well-aligned intercalated

discs components such as desmosomes (Fig. 1E). Remarkably weobserved that bridges between neighbouring cardiomyocytes weredisorganized after 63 days CsA administration, intercalated discsmisaligned and sometimes projected on the extracellular space(Fig. 1F).

194 A. Stacchiotti et al. / Toxicology 262 (2009) 192–198

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ig. 1. Morphology of heart in control (A, C, E) and CsA treated (63 days) (B, D, Fltrastructure (E and F) (×39,000) Note fibrosis and sarcomeric network derangemeparate experiments were performed and in all of them similar results were obser

.2. Small heat shock protein distribution in rat heart afteryclosporine A treatments

In control group, HSP25 signal was intense and located inside theytoplasm according to a regular myofibrillar pattern in ventricu-ar cardiomyocytes (Fig. 2A). Nuclei were devoid of signals. Afterrolonged CsA treatment, HSP25 immunostaining become irreg-lar and distributed along intercalated discs that joint differentyofibrils even if their intensity grade did not change (Fig. 2B).

Regarding to alpha B-crystallin, we observed a moderate peri-dic pattern along cardiac ventricles in control rats (Fig. 2C) but,fter 63 days CsA treatment, the staining translocated to peripheralarcoplasm and intercalated discs, lacking any periodism (Fig. 2D).uclei were always devoid of brown staining.

.3. Cardiac myofibrillar proteins distribution after cyclosporine Areatments

In controls, alpha-actinin immunostaining was faint and asso-iated to Z lines in I bands (Fig. 3A), after 42 days and 63 days CsAreatment the periodic signal was similar even if sometimes was

etected in external borders and extracellular spaces (Fig. 3B).

However, desmin staining was similarly associated to I bands inmyofibrillar pattern and moderately expressed in the sarcolemma

n ventricular cardiomyocytes of control groups (Fig. 3C). By con-rast, after 63 days CsA exposure, desmin pattern became restricted

Masson staining (A and B) (×400), toluidine blue staining (C and D) (×400) andduced by CsA, that affected the tight junctions, (→) indicates desmosomes. Three

to sarcolemma membrane and its myofibrillar periodism was com-pletely lost (Fig. 3D).

Finally, actin immunostaining was intense and detected as aregular sarcomeric pattern in control groups (Fig. 3E), but, afterprolonged CsA treatment, it became irregular and associated to dis-rupted intercalated discs (Fig. 3F). The quantification of myofibrillarproteins distribution in different experimental groups is reportedin Fig. 4.

3.4. Western blotting of HSP25, alpha B-crystallin andcytoskeletal proteins

The overall amount of tested proteins was consistent withimmunohistochemical trend.

Due to low abundance the relevant Western data on alpha-actinin showed thin bands, similar during all treatments at 42 daysand 63 days (data not shown). Considering the functional asso-ciation between cardiac chaperones and cytoskeletal proteins wedecided to present data of HSP25 and actin together and separatelyfrom alpha B-crystallin and desmin.

After treatment with CsA at both time periods, HSP25 and

actin abundance was consistent and similar to control levels (datanot shown). In contrast, alpha B-crystallin and desmin contentdecreased after both CsA treatments with respect to relative con-trols (data not shown). Quantitative densitometric data are givenbelow the respective bands in Fig. 5.

A. Stacchiotti et al. / Toxicology 262 (2009) 192–198 195

Fig. 2. HSP25 immunolocalization in rat heart. Control group (A) moderate sarcomeric pattern; 63 days CsA treated group (B) disrupted sarcomeric pattern (×1000). AlphaB-crystallin immunostaining in rat heart. Castor-oil group (C) sarcomeric brown signal in the control group; 63 days CsA-treated group (D) abnormal signal associated toi n. Thro

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. Discussion

For prevention of graft rejection and for treatment of autoim-une disorders, CsA is the most widely used immunosuppressive

rug. Unfortunately, it exerts side effects on the cardiovascularystem which have been related to altered levels of stress pro-eins expression (HSP70 and HSP90) and to morphological damageRezzani et al., 2003, 2009). Small heat shock proteins (sHSPs) inhe heart stabilize the sarcomeric organization and protect otherundamental cytoskeletal components from toxic and ischemiconditions (Sun and Mac Rae, 2005; Macario and Conway deacario, 2007).Herein, using the rat model, we focused on the effects of pro-

onged CsA treatment on the cardiac localization and abundancef two fundamental sHSPs, HSP25 and alpha B-crystallin, and onyofibrillar cytoskeletal proteins, such as desmin, actin and alpha-

ctinin.Considering that in the heart, sHSPs and myofibrillar cytoskele-

on are essential for the correct function of cardiomyocytes, theireciprocal balance may greatly influence both mechanical and func-ional properties of this fundamental organ (Hein et al., 2000;atchmann, 2001).

Our main findings were that CsA administration resulted in:1) elevated fibrosis in rat heart and focal changes in the regularyncytial organization and intercalated discs components such asesmosomes; (2) translocation to the peripheral sarcolemma and

ntercalated discs of HSP25 and alpha B-crystallin from a cross-triated pattern; (3) similar distribution of desmin pattern whichecame superimposable with alpha B-crystallin; (4) significantnd time-dependent reduced abundance of alpha B-crystallin andesmin versus controls.

We previously reported that CsA, at therapeutical doses, affectsxtracellular matrix and induces oxidative damage and apoptosisn rat cardiomyocytes, partly attenuated by the use of antioxidantompounds (Rezzani et al., 2005a, 2006a, 2009). Moreover CsAhanged sHSPs expression in other fundamental rat organs such

ee separate experiments were performed and in all of them similar results were

as kidney (Stacchiotti et al., 2002), aorta (Rezzani et al., 2005b) andliver (Rezzani et al., 2005a).

An important new finding reported here, was that HSP25, themain actin chaperone, and alpha B-crystallin, strictly associatedto desmin, lost regular myofibrillar periodism after prolonged CsAexposure. It is known that alpha B-crystallin strictly binds actinand desmin in the heart and this association is necessary to form afunctional network (Perng et al., 2004; Singh et al., 2007). Moreoveralpha B-crystallin is associated to desmin in the cardiac conductingfibers, mainly in His branches (Leach et al., 1994).

However, desmin mutations or disruption of the desmin net-work, together with mutant crystallin, damaged cardiac function(Kumarapeli and Wang, 2004). Remarkably our data are in agree-ment with those obtained by Heling et al. (2000) and Wanget al. (2001) where desmin location, but not its overexpression,was strongly related with a cardiac deficit. Furthermore, desminhelped maintain lateral alignment of myofibrils and anchored theZ disc to the sarcolemma, so reduced desmin content might con-tribute to sarcomeric disorganization observed after CsA. Paslaruet al. (2007) reported that CsA affected cardiomyocytes differen-tiation and properties and other in vitro studies described CsAinduced damaged to tonofibrils meshwork in human keratinocytes(Prignano et al., 1996), chick cardiomyocytes (Kolcz et al., 1999),and to cytoskeletal proteins, such as desmin and vimentin, in thethymus (Rezzani et al., 2000).

In contrast with alpha B-crystallin and desmin that werereduced, we demonstrated that HSP25 abundance and phospho-rylation were unchanged in all experimental groups. This could bedue to proteasomal inhibition that seemed independent from phos-phorylation (Verschuure et al., 2002). The prevalence in the heart ofunphosphorylated HSP25 might be linked with fibrosis and upregu-

lation of matrix metalloprotease 2 (MMP2) induced by CsA (Bianchiet al., 2003). Remarkably sHSPs were inversely related to apoptosis,ROS production and manganese superoxide dismutase activity (Leeet al., 2004) that enhanced in this experimental model (Rezzani etal., 2009). In particular, alpha B-crystallin prevented the release of

196 A. Stacchiotti et al. / Toxicology 262 (2009) 192–198

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ig. 3. Myofibrillar markers immunostaining in controls (A, C, E) and CsA treated (63arcomeric in controls (C) but moved peripherally in CsA treated (D); actin associatedith Mayer’s haematoxylin. Three separate experiments were performed and in all

ytochrome c and directly suppressed caspase 3 activation (Mao etl., 2004), so its limited expression detected in the heart could be aonsequence of CsA apoptosis. Moreover also in a model of diabeticeart, less HSP25 phosphorylation and apoptosis were associated toontractile dysfunctions (Williamson et al., 2007). Another impor-

ant finding of this study was that we did not observe the nuclearranslocation of sHSPS or their assembly with Z-line as reportedfter acute ischemia (Golenhofen et al., 2006) even if CsA inducedxidative damage (Rezzani et al., 2003). This discrepancy could be

ig. 4. Quantitative analysis (IOD) of HSP25, alpha B-crystallin, alpha-actinin,esmin and actin immunostaining in heart of control and CsA treated (63 days)ats. Values are expressed as mean ± S.D. *Statistically significant at P < 0.05 whenompared with control.

rat heart (B, D, F) alpha-actinin periodism was maintained in all groups; desmin wasstameres (E) but loss of periodism after CsA (F) (×1000). Nuclei were counterstainedm similar results were observed.

due to a different duration of stressful stimuli, i.e. a rapid ischemiaversus our prolonged immunosuppressive schedule.

Curiously, alpha-actinin, a Z-line marker, maintains its intactperiodism and abundance in both control and CsA treated groups.On the external sarcomeric side, extramyofibrillar actin complexes

with cytoskeletal proteins, such as alpha-actinin, by connectors thatco-localize to the fascia adherens of intercalated discs (Vasioukhinet al., 2000). So the maintenance of a regular alpha-actinin dis-tribution, detected here in control and CsA treated groups, might

Fig. 5. Quantitative analysis (IOD) of HSP25, alpha B-crystallin, alpha-actinin,desmin and actin Western blotting in heart of controls, CsA treated rats for 42days and for 63 days. Values are expressed as mean ± S.D. *Statistically significant atP < 0.05 when compared with control.

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ndicate that the drug did not affect directly the intrinsic myofibril-ar design of the heart but probably the overall sarcomeric texture.his finding is corroborated by our previous study on non-muscleyosin localization in the rat heart receiving a similar CsA regimen

Rezzani et al., 2006b)It is well known that the heart is a functional syncytium where

he transmission of the stimulus from cell to another dependsn the integrity of intercalated discs (Kostin and Schaper, 2001).orphologically each intercalated disc is composed by three con-

tituents: gap junction, fascia adherens and desmosome (Borrmannt al., 2006). Therefore, the disruption of one of these componentsompromises the whole structure. Remarkably here, by ultrastruc-ural observations, we showed a well preserved Z-line but the

isalignment of intercalated discs and destroyed desmosomesn the tight junctions. Similar ultrastructural findings have beenescribed in cardiac hypertrophy and in dilated human cardiomy-pathy (Eigenthaler et al., 2003). In a previous ultrastructural study,ith lower CsA dosage (5 mg/kg daily) but for a major time (up to

0 days) after heart transplantation, Jurado et al. (1998) reporteditochondrial damage, hypertrophy of sarcoplasmic reticulum and

vident fibrosis in the rat.Taken together, our in vivo data indicate that prolonged CsA

ntake, at therapeutic dosage, directly affects the distribution andolubility of HSP25 and alpha B-crystallin in the rat heart. This phe-omenon was more evident after 63 days and might represent anndogenous compensatory reaction to CsA damage. In addition, weemonstrated a concomitant myofibrillar disorganization of spe-ific cytoskeletal markers that might be considered a morphologicalallmark of CsA cardiotoxicity.

onflict of interest

The authors declare that there are no conflicts of interest.

cknowledgements

Authors would like to thank Dr Francesca Filippini, Ms Stefaniaastrezzati, Mr Giuseppe Bertocchi and Mr Giovanni Bozzoni for thekilful assistance in electron microscopy and digital microscopy.his study was supported by local institutional grants (ex 60%008–2009).

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