Immunosuppressant FK506: Focusing on neuroprotective effects following brain and spinal cord injury
Kamila Saganová ⁎, Ján Gálik, Juraj Blaško, Andrea Korimová, Eniko Račeková, Ivo Vanický
Institute of Neurobiology, Center of Excellence, Slovak Academy of Sciences, Košice, Slovak Republic
a r t i c l e i n f o
Received 21 March 2012
Accepted 23 June 2012
Dosing regimen Combined therapy Functional outcome Tissue sparing
a b s t r a c t
The secondary damage that follows central nervous system (CNS) injury is a target for neuroprotective agents aimed at tissue and function sparing. FK506, a clinically used immunosuppressant, acts neuro- protectively in rat models of brain and spinal cord injury and ischemia. Evidence of in vivo experimental studies highlights the neuroprotective role of FK506 by its direct impact on various cell populations within the CNS. The participation of FK506 in modulation of post-traumatic inﬂammatory processes is a further po- tential aspect involved in CNS neuroprotection. In this review we provide an overview of the current labora- tory research focusing on the multiple effects of FK506 on neuroprotection following CNS injury.
© 2012 Elsevier Inc. All rights reserved.
FK506 effects following brain ischemic/traumatic injury 78
FK506 effects following traumatic spinal cord injury 79
FK506 effects following CNS combined therapy 80
Conﬂict of interest statement 81
The immunosuppressive drug FK506 (Tacrolimus) is widely used in transplantation medicine for the reduction of allograft rejection. Immunosuppressant FK506 (Kino et al., 1987; Ochiai et al., 1987) acts by inhibition of calcineurin-mediated T-cell activation via com- plex formation with FK506 binding proteins (Liu et al., 1991). The FK506 inhibits a key step in T‐cell activation, it suppresses the pro- duction of the interleukin-2 (IL-2) by blocking IL-2 gene transcription (Sigal and Dumont, 1992). In addition, FK506 specificity for T-cells is augmented by blocking the activation of the JNK and p38 signaling pathways triggered by antigen recognition (Allison, 2000). The
⁎ Corresponding author at: Institute of Neurobiology, Center of Excellence, Slovak Academy of Sciences, 040 01 Košice, Slovak Republic. Tel.: +421 55 727 6246; fax:
+421 55 727 6202.
E-mail address: [email protected] (K. Saganová).
presence of two distinct targets in T-cell activation makes FK506 a highly potent immunosuppressive drug (Matsuda et al., 2000). FK506 improves graft survival rates, but its use in a number of other pathological conditions, such as central nervous system injury, is expanding. Within the nerve cells, FK506 binds to several intracellu- lar FK506 binding proteins (FKBPs) belonging to the immunophilin family (Sekierka et al., 1989; Sekierka and Sigal, 1992), of which FKBP12 appears to be the most important in the nervous system. Binding of FK506 to FKBP12 leads to formation of a complex which in- hibits calcineurin (Liu et al., 1991; Snyder et al., 1998); this in turn would be expected to increase the amount of the active form of the growth-associated protein GAP-43 in neurons (Bavetta et al., 1999). FKBP and calcineurin are co-localized in most brain and spinal cord regions, and almost identical co-localization has been found within the substantia gelatinosa, dorsal and ventral horn of spinal cord (Dawson et al., 1994). High levels of FKBPs in the brain and spinal cord and their striking calcineurin co-localizations indicate their key regulatory function. Through interactions with different cellular fac- tors, FKBPs play important roles in various physiological processes
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and, more interestingly, in a number of pathological processes. It was found that tacrolimus possesses multiple modes of action such as in- hibition of apoptotic and necrotic cell death, attenuation of microglia activation and attenuation of leukocyte accumulation (Tsujikawa et al., 1998; Wakita et al., 1998; Herr et al., 1999; Wang et al., 1999; Furuichi et al., 2004). Several lines of evidence show that the neuro- protective effect of FK506 is multifactorial, but the individual mecha- nisms of FK506 action are not completely understood (Gold et al., 2004). Detailed evaluation of neuroprotective effects of different im- munosuppressants including immunophilins FK506 and cyclosporin A after traumatic and ischemic CNS injury is a topic of recent reviews (Sosa et al., 2005; Hailer, 2008; Toll et al., 2011).
FK506, as a drug established in clinical practice, is attractive for laboratory investigation of CNS neuroprotection. Tacrolimus has been reported to have potent neuroprotective properties in rodents as well as primate stroke models (Tokime et al., 1996; Yagita et al., 1996; Takamatsu et al., 2001; Furuichi et al., 2003a,b, 2007). Most often, the neuroprotective effect of FK506 has been demonstrated under various experimental conditions using rat models of CNS inju- ry. The documented neuroprotection of FK506 in rat depends on the experimental models and paradigms; however, the achieved results do not sufficiently elucidate the mechanism of FK506 neuroprotective action for the present. Our aim is to describe the neuroprotective ef- fects of FK506 by comparing the available data from the most studied and best understood in vivo adult rat models of traumatic or ischemic CNS injury. One part is directed at studies in which combined therapy including cell transplantation was implemented. In view of the fact that only a limited number of FK506 studies based on the injured CNS has been published, our report might contribute to the interest in intensifying the study of FK506 therapeutic approaches, including combination therapy in animal models of CNS injury, and the transla- tion of such treatment into human clinical trials.
FK506 effects following brain ischemic/traumatic injury
In vivo rat models of ischemic and traumatic brain injury that have been used for investigating the neuroprotective effects of FK506 are included in this part of review (Table 1). The most studied ischemia model was focal ischemia in which FK506 effectiveness was dependent on FK506 dose and time window (Arii et al., 2001; Furuichi et al., 2003a). The most effective neuroprotective dose of FK506 was 1 mg/kg (Furuichi et al., 2003a, 2004; Noto et al., 2004; Zawadska and Kaminska, 2005), which corresponds to the dosage that is used as an immunosupppresive dose in humans (Sharkey and Butcher, 1994). However, other studies have shown that doses in the range 0.1–0.3 mg/kg (Sharkey and Butcher, 1994; Arii et al., 2001; Brecht et al., 2009), which are not sufficient to prevent im- mune rejection of neuronal transplants in rats (Sakai et al., 1991), also had neuroprotective potential. Time window dependency was shown as the other key factor for FK506 neuroprotection. FK506 was demonstrated as a powerful neuroprotective agent when ad- ministered immediately (Furuichi et al., 2004; Noto et al., 2004), up to 60 min (Arii et al., 2001; Sharkey and Butcher, 1994; Zawadska and Kaminska, 2005) or 120 min (Furuichi et al., 2003a) following MCA post-occlusion.
FK506 administration in brain pathology revealed significant effect on diminishing of infarct volume/area following focal ischemia. As a re- sult of immunohistochemical observation, it was shown that reduced infarct volume resulted from reduced number of apoptotic/necrotic neurons, cytochrome c neurons, redistribution of cytosolic cytochrome c, reduced microglia and astrocyte response and T-cell infiltration (Table 1). The effect of FK506 on neurological outcome was investigated only in the study by Zawadska and Kaminska (2005). Recently, neuro- protective effect of FK506 on CA1 neurons of hippocampus was con- firmed following transient global ischemia (Sharifi et al., 2012).
Neuroprotective effects of FK506 following rat brain ischemia/trauma.
Brain ischemia FK506 dose FK506 protection Functional outcome FK506 mediated effects References
Transient focal ischemia
Transient focal ischemia
Transient/ 1 mg/kg
0.032 mg/kg Neuroprotective effects
Reduced infarct volume Reduced infarct volume Immunophilins involvement confirmed
Suppressed neuronal damage; time window limitation
Suppressed neuronal damage/death; Sharkey and Butcher, 1994 Arii et al., 2001
Furuichi et al., 2003a
permanent focal ischemia
Transient focal 0.1 mg/kg
1.0 mg/kg 1 mg/kg
Reduced infarct area; suppressed inﬂammation; dose‐dependent effects
Furuichi et al., 2004
ischemia reduced apoptotic/necrotic neurons; reduced cytochrome-c neurons;
reduced microglial response necrotic cell death; suppressed inﬂammation
Permanent focal ischemia
Transient focal 1 mg/kg
1 mg/kg Reduced infarct area; redistribution of cytosolic cytochrome c
Reduced infarct volume;
Improved Modulation of apoptotic and/or necrotic cell death pathway
Modulation of inﬂammation Noto et al., 2004
0.1 mg/kg reduced microglial and
astrocytes response Reduced T-cell infiltration; functional recovery and glial response
Suppressed neuronal damage; Kaminska, 2005
Brecht et al., 2009
ischemia reduced microglial/ suppressed response of resident/
6 mg/kg macrophage cells
Reduced CA1 cell damage peripheral immune cells
Suppressed neuronal damage /death;
Sharifi et al., 2012
ischemia dose‐dependent effect
Traumatic brain injury
Impact-acceleration injury model
Diffuse close head injury
Midline ﬂuid percussion injury
2 mg/kg Improved axonal survival and regeneration
3 mg/kg Reduced traumatic axonal injury
3 mg/kg Reduced axonal injury including unmyelinated axons
Inhibition of calcineurin activity Singleton et al., 2001 Inhibition of calcineurin activity Marmarou and
Suppressed posttraumatic CAP Reeves et al., 2007
In comparison to brain ischemia, a higher FK506 dose was used for axonal neuroprotection in traumatic brain injury (TBI) studies. The dosage was based on the observation that 3 mg/kg of FK506 crosses the blood–brain barrier and establishes therapeutic levels within the brain parenchyma (Singleton et al., 2001). In TBI studies in which FK506 was administered 30 min pre-TBI, it was shown that pre-treatment with FK506 reduced traumatic axonal injury following impact-acceleration injury in rats (Singleton et al., 2001) and severe diffuse closed head TBI (Marmarou and Povlishock, 2006). It was con- cluded that as FK506 attenuates calcineurin, calcineurin-mediated processes would most likely be the target for the protection observed with impaired axonal damage. Using the rat midline ﬂuid precursor model of TBI, it was revealed that FK506 had neuroprotective effects also on unmyelinated axon neuropathology (Reeves et al., 2007). FK506 had strong neuroprotective effect on the function of unmyelin- ated axons of the corpus callosum and provided functional protection in callosal myelinated axons.
FK506 effects following traumatic spinal cord injury
In this part of our review we have included spinal cord injury (SCI) studies in which FK506 administration was used in in vivo rat models of SCI. The rat spinal cords were traumatically injured by different methods; contusion, compression, partial transection or photochem- ical reaction was performed (Table 2). All injuries occurred in the low thoracic spine level within the Th8–Th10 segments. Just as in the brain, the dose and timing of FK506 administration varied signif- icantly in the spinal cord studies. FK506 dosing regimen ranged from single bolus administration (Lopez-Vales et al., 2005) to repeated short-term administration (Madsen et al., 1998; Bavetta et al., 1999; Nottingham et al., 2002) or to daily long-term administration for weeks after injury (Lopez-Vales et al., 2005; Voda et al., 2007; Saganova et al., 2009). Similarly, daily doses of FK506 (Table 2) dif- fered in a wide range from 0.25 mg/kg (Madsen et al., 1998) to 5 mg/kg (Nottingham et al., 2002). Such variations in experimental conditions make it difficult to elucidate FK506 effects in detail. The optimizing of FK506 dosing regimen in SCI conditions needs further investigation.
Functional improvement, especially the neurological outcome, is the most desired effect of SCI neuroprotective strategies. In the ma- jority of FK506 spinal cord studies, behavioral outcome was consid- ered (Table 2). An improvement in neurological function through
FK506 action was found following phototrombotic injury (Madsen et al., 1998), photochemical injury (Lopez-Vales et al., 2005) and spi- nal cord hemisection (Voda et al., 2005). No functional outcome was observed following spinal cord contusion (Voda et al., 2007) or spinal cord compression (Saganova et al., 2009). Histological examination revealed that FK506 had ameliorative effect on the spinal cord paren- chyma (Lopez-Vales et al., 2005) and axonal sparing rostrally to trau- matic lesion (Bavetta et al., 1999; Saganova et al., 2009). Moreover, Voda et al. (2005) demonstrated a significantly greater number of ret- rogradely labeled neurons in the red nucleus. Generally, direct corre- lation between neurological and histopathological outcomes is expected; however, revealed neuropathological improvements due to FK506 treatment following SCI had limited ameliorative effect on neurological outcome. It is apparent that the observed spinal cord pa- renchyma and axonal sparing which was found to be strengthened within the rostral part of the injured spinal cord was not sufficient to improve the final functional outcome following severe SCI.
The evaluation of spinal cord photothrombotic injury showed that FK506 treatment increased the neuronal growth-associated protein GAP-43 immunoreactivity and the number of GAP-43 mRNA-expressing neurons (Madsen et al., 1998). These findings support the direct effect of FK506 on neurons, and by increasing GAP-43 expression also its role in the enhancement of normal sprouting. In addition to the neurons, glial cells within the spinal cord parenchyma also responded to FK506 treatment following SCI. FK506 anti-apoptotic effect led to inhibition of apoptotic marker caspase-3 activation within the oligodendroglia, which promoted oligodendroglial survival (Nottingham et al., 2002). It was also shown that FK506 significantly reduced GFAP reactivity within the astrocytes, although the FK506 effect on reduction of microglia/ macrophage response was less apparent (Lopez-Vales et al., 2005).
Although the mechanisms of FK506 neuroprotection of spinal cord parenchyma and axonal preservation are still not elucidated, ex- cept for the direct effect of FK506 on neurons and glia, indirect FK506 neuroprotective effect can result from its immunosuppressant action. It is known that SCI is associated with an immune response that initiates activation of immune cells and local neuroinﬂammation that can be reduced by immunosuppression. Neuroinﬂammation as a part of the pathogenesis of secondary spinal cord injury (Alexander and Popovich, 2009) is associated with immediate response of resi- dent microglia and astrocytes that by releasing of chemokines and cytokines enhance recruitment of peripheral leukocytes into CNS (Pineau and Lacroix, 2008). FK506 as a potent inhibitor of activation
Neuroprotective effects of FK506 following rat spinal cord injury.
SCI model FK506 FK506 protection Functional outcome FK 506 mediated effects References
Photothrombotic 0.25 mg/kg Increased number of Improved Effects on neurons; Madsen et al., 1998
Partial dorsal column transection
GAP-43 positive neurons
0.5 mg/kg 2.0 mg/kg Increased axonal
Promotion of axonal sparing Bavetta et al., 1999
Contusion 5 mg/kg Reduced caspase-3 expression in oligodendroglia;
increased number of oligodendroglia
Effects on oligodendroglia; effects on apoptosis
Nottingham et al., 2002
2 mg/kg 0.2 mg/kg Increased spinal cord
tissue sparing; reduced microglial response; reduced astroglial response
Improved functional recovery
Neuroprotection based on non-immunosuppressant mechanism(s)
Lopez-Vales et al., 2005
Hemisection 2 mg/kg Increased axonal survival Improved
Neuroprotection based on non-immunosuppressant mechanism(s)
Voda et al., 2005
Contusion 2 mg/kg Reduced SCI-induced pain hypersensivity
No functional improvement
Mediation of distinct mechanisms of motor and sensory dysfunction
Voda et al., 2007
Compression 1 mg/kg Increased axonal sparing (rostrally)
No functional improvement
Multifactorial mechanisms Saganova et al., 2009
of T-cells that also infiltrate the injured spinal cord (Ankeny and Popovich, 2009) can by its immunosuppressive action on immune cells modulate inﬂammation within the spinal cord and ameliorate neuroprotection.
FK506 effects following CNS combined therapy
In this part we have included in vivo rat model studies of CNS injury in which combined therapy with FK506 as one of the neuro- protective agents was used (Table 3). Formerly, the immunosuppres- sive action of FK506 was proved by prevention of graft rejection following attraumatic spinal cord grafting (Kakinohana et al., 2004), spinal cord ischemia and SCI (Marsala et al., 2004; Hayashi et al., 2005; Cizkova et al., 2007). However, the CNS transplantation studies in which FK506 acts as a potent immunosuppressant to stop graft rejection are not covered in this review. In the selected studies, tested drugs or methods that are widely used in CNS experiments were used as second neuroprotective agents. The rationale for combination of therapeutic intervention coupling FK506 with other neuroprotective agents is the expectation that it can extend the therapeutic window and improves the efficacy of CNS neuroprotection.
In studies examining combined therapy, the usual dose of FK506 varied between 0.2 and 0.5 mg/kg, which is the drug concentration not associated with its immunosuppressive effect; in some studies higher doses, 1.0-2.0 mg/kg, were used (Table 3). The neuro- protective effect of methylprednisolone (MP) is extensively consid- ered in SCI studies (Rabchevsky et al., 2002; Vanicky et al., 2002; Weaver et al., 2005; Fu and Saporta, 2005); however, its effective- ness varies in different experimental conditions. The advantage of using combined therapy with FK506 and MP was observed following partial spinal cord transection. This combination of drugs was signif- icantly more effective in protecting axons than MP alone, but not
significantly more effective than FK506 alone (Bavetta, et al., 1999). It was concluded that axonal preservation by FK506 is greater than by MP. Studies on brain hypothermia have suggested that mild hypother- mia may increase the benefit of pharmacotherapy or expand the therapeutic window. Following transient focal ischemia, mild hypo- thermia in combination with FK506 significantly reduced infarct and edema volume, while mild hypothermia or FK506 alone failed to improve ischemic brain damage (Nito et al., 2004). Recently (data not included in Table 3) the utility of combined therapy coupling post-traumatic hypothermia and FK506 administration was evaluat- ed following TBI (Fujita et al., 2011; Oda et al., 2011). In accordance with expectations the combined therapy significantly enhanced vas- cular and axonal protection following diffused axonal injury (Oda, et al., 2011). However, the next study by the same authors (Fujita et al., 2011) showed that while separate FK506 therapy proved effica- cious, producing benefits with early as well as delayed administration, it did not increase either vascular or axonal benefit in combination with hypothermia. It was concluded that the different animal models used and/or their associated injury severity might have led to these divergent results. These conﬂicting results affirm the multifactorial
neuroprotective aspects of CNS therapy.
The main strategy for thrombolytic therapy is enhancing resto- ration of blood ﬂow within the ischemic area. Although the recom- binant tissue-plasminogen activator (rt-PA) is an effective thrombolytic agent, it has a limited time window, as decline in its efficacy has been observed when administered beyond 3 h after onset of ischemic insult. It was demonstrated that following permanent focal ischemia the therapeutic time window resulting from combined therapy with rt-PA and FK506 is longer than that resulting from treatment with either drug alone (Maeda et al., 2002). Combined treatment of spontaneously hypertensive rats showed that rt-PA-induced hemorrhagic transformation was suppressed by FK506 following focal ischemia (Maeda et al., 2009). FK506 neuroprotective
Neuroprotective effects of FK506 following combined therapy of rat CNS injury.
FK506 Brain injury
SCI model FK506
FK506 mediated effects
MP 0.5 mg/kg;
Partial dorsal column transection
Increased axonal regeneration
Effects on neurons; effect on glia; immunosuppression
Bavetta et al., 1999
0.3 mg/kg Transient
Reduced infarct volume; reduced edema volume
Improved functional recovery
Inhibition of NO production; suppression of free radical production
Nito et al., 2004
rt-PA 1.0 mg/kg Permanent focal ischemia
Reduced infarct volume
Prolongs/extends therapeutic time window; immunosupression
Maeda et al., 2002
1.0 mg/kg Permanent
Reduced infarct volume; reduced hemorrhagic
vascular permeability; increased integrity of microvascular endothelial cells
against hemorrhagic transformation based on
Maeda et al., 2009
MSCs 0.3 mg/kg Transient focal ischemia
Complete/ partial SCI
Reduced lesion volume;
reduced astrogliosis; axon sparing/ regeneration Reduced infarct volume; reduced
edema index; reduced apoptosis; reduced inﬂammation
Improved functional recovery
Improved functional recovery
No difference in OECs p75 immunoreactivity
Increased survival of transplanted cells
Enhancement of the protective effect
of OECs grafts
Enhanced proliferation/ regeneration of grafted MSCs;
anti-apoptotic and anti-inﬂammatory effects
Lopez-Vales et al., 2006
Suda et al., 2011
MP, methylprednisolon; rt- PA, recombinant tissue plasminogen activator; OECs, olfactory ensheathing cells; MSCs, bone marrow stromal cells.
effects were enhanced by combined treatment, with reduction of infarct volume, hemorrhagic score and vascular permeability being demon- strated, and another benefit was increased integrity of microvascular endothelial cells. Recently, combined therapy with rt-PA and tacrolimus which possessing multiple modes of action for neuroprotection exerted additional protection also in a nonhuman primate stroke model (Furuichi et al., 2007).
Primarily, FK506 is a potent immunosuppressant serving for preventing graft rejection. In rare rat CNS studies the neuroprotective effect of FK506 was studied in combination with cell transplantation. The reason for using a combination of two experimental approaches, which have been previously reported as having neuroprotective ef- fects after CNS injury, was to evaluate if it can promote synergic re- storative effects. The concentration 0.2–0.3 mg/kg of FK506 selected for combined therapy neuroprotection has no immunosuppressive effects (Udina et al., 2003). It has been documented that the combina- tion of olfactory ensheathing cell (OEC) graft with the administration of FK506 following SCI promoted additional repair to that exerted by single treatment, and the effect was more remarkable following partial SCI (Lopez-Vales et al., 2006). The achieved improvement in neuroprotection, axonal regeneration and functional recovery sug- gest an additive effect of FK506 administration when combined with OEC transplants. Although the synergic restorative mechanisms underlying the combination of OEC graft and FK506 are unknown, one of the relevant common events of combined treatment is reduc- tion of astrogliosis.
In addition to the ability of bone marrow stromal cells (MSCs) to differentiate into lineage, MSCs secrete various cytokines and growth factors. This production is considered key to the benefits provided by MSC transplantation. Suda et al. (2011), taking into account the extra neurogenic effect of MSCs, examined whether FK506 may act addi- tively or synergistically with MSC transplantation following brain focal ischemia. They found that combined therapy with FK506 and MSCs led to reduction of infarct volume, edema index and neurologi- cal score, and moreover, the number of engrafted MSCs was signifi- cantly higher than with MSCs alone. It was shown that FK506 enhanced the anti-apoptotic and anti-inﬂammatory the survival of transplanted cells and expanded the therapeutic time window for MSCs.
There is no question that the immunosuppressant FK506 acts as a neuroprotective agent in various CNS pathological conditions. Our knowledge of the FK506 neuroprotective effects within injured and ischemic CNS and mechanisms by which FK506 can inﬂuence the pathophysiology and severity of CNS injury is far from clear. The non-immunosuppressive action of FK506 through its effects on FKBP-12 and inhibition of calcineurin action has direct impact on neurons and glial cells. The indirect mechanism for the FK506 protec- tive effect results from immunosuppressant action since CNS injury is associated with immune response that can be reduced by immuno- suppression. Consistent results from the rat injury CNS studies dem- onstrate the role of FK506 as a potent neuroprotectant that can serve for FK506 monotherapy or combined therapy, FK506 potential for clinical use should be considered in the future.
In the large number of rat studies on FK506 effects is the common effort to improve outcomes by preventing progression of cascade of secondary injury following trauma and delayed injury following is- chemia. Although FK506 was described to have powerful neuro- protective properties within CNS recent data are not sufficient for an assessment if FK506 treatment should be more helpful for trau- matic or ischemic CNS injury. Critical should be the therapeutic dose regimen that differs significantly in the studied models of CNS inju- ry/ischemia. Generally, the used effective dose of FK506 was higher in traumatic injury than those following ischemia. One of the critical
factors considering in the treatment of CNS damage is the possible side effect of drug, in view of that the high doses of FK506 should be problematic for long-term studies. The known limitation of FK506 administration is its toxic effects which sometimes led to seri- ous complication such is renal dysfunction, hypertension, neurologi- cal toxicity, insulin‐dependent diabetes, and several gastrointestinal disturbances (Sigal and Dumont, 1992). In our study, using FK506 long-term administration we observed significant decrease in body weight that was most likely caused by several side effects of FK506 (Saganova et al., 2009). The safe dose used for FK506 neuroprotection following CNS injury or ischemia is the potential limitation of FK506 administration. The therapeutic dose regimen seems to be a key fac- tor that can affect the efficacy of FK506 as a neuroprotectant, howev- er, the published data show significant variation in the FK506 regimen used. This makes it difficult to evaluate and elucidate FK506 effects in detail, which is important for the explanation of its participation in amelioration of outcomes following CNS injury. In the future, it will be critical to determine the optimal dosing regimen of FK506 for its maximal neuroprotective benefit within individual parts of CNS. This can help enable us to develop new FK506 protection strategies that can have good prospects for CNS neuroprotection.
Conﬂict of interest statement
The authors declare no conﬂict of interest.
The study was supported by VEGA Grants 2/0182/11, 2/0202/10 and 2/0114/12 from the Slovak Academy of Sciences.
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