Abstract
The frequency of patients diagnosed with cerebral venous sinus thrombosis (CVST) has increased due to the expanded use of noninvasive brain imaging methods. The aim of this study was to assess the correlations between the location and extent of venous sinus impairment, clinical presentation during the acute phase, recanalization, the presence of parenchymal lesions, and clinical outcome after 3 to 4 months in patients with CVST. In a retrospective study, clinical and magnetic resonance imaging data from a cohort of 51 consecutive patients with CVST (mean age 33.1 ± 15.4 years) were collected and analyzed. Good clinical outcome after 3 to 4 months, which was assessed using the modified Rankin scale, significantly negatively correlated with a thrombosis location in the left transverse, left sigmoid, or superior sagittal sinus (P = .022, P = .045, and P = .046, respectively) and positively correlated with recanalization (P = .048). The clinical outcome was significantly more favorable in the females with gender-specific risk factors than in the males (P = .029). In conclusion, successful recanalization substantially helps to achieve good clinical outcome in patients with CVST.
Keywords
Introduction
In recent years, cerebral venous sinus thrombosis (CVST) has been diagnosed substantially more frequently than in the past due to the expanded use of noninvasive brain imaging methods. However, the actual incidence of CVST is most likely even higher. Many cases remain unrecognized due to the high variability of their clinical presentation and the possible absence of any symptoms. In the most recent publication, which was based on an analysis of a cohort of 9270 patients examined between January 2008 and December 2010 in the Netherlands, the reported incidence of CVST was 1.32/100 000 individuals/year; this incidence was the highest in females aged 31 to 50 years (2.78/100 000). 1 This report indirectly confirmed the results of the International Study on Cerebral Vein and Dural Sinus Thrombosis (ISCVT) performed in 2004, which examined the largest cohort of patients with cerebral phlebothrombosis to date and which reported that females consisted of 74.5% of those experiencing CVST. 2
The finding that the ratio of CVST incidence in females and males is 3:1 at young and middle age can only be explained by gender-specific risk factors (GSRFs), which most often include hormonal contraception (HC), pregnancy, puerperium, and hormone replacement therapy (HRT). These factors are likely responsible for the better outcome of CVST in females than in males, despite the same thrombosis severity and a higher frequency of parenchymal lesions. 3 Aside from male gender, impaired consciousness at presentation, deep vein thrombosis, and parenchymal hemorrhage were identified as predictors of unfavorable outcome in the ISCVT study. 4
The aim of our study was to assess the correlations between the location and the extent of venous sinus impairment, clinical presentation during the acute phase, recanalization, the presence of parenchymal lesions, and the clinical outcome in patients with CVST. The identification of (primarily modifiable) outcome predictors and their subsequent control are essential to achieve good clinical outcome in these patients.
Methods
Study Design and Inclusion Criteria
A retrospective single-center study was performed. The medical records (both inpatient and outpatient) of all consecutive patients with CVST who were hospitalized and followed-up at the Comprehensive Stroke Center of the Department of Neurology at University Hospital in Hradec Králové, Czech Republic (including a catchment area of 800 000 inhabitants), between January 2000 and June 2013, were analyzed. The inclusion criteria comprised the availability of all monitored clinical data and at least 2 comparable brain and venous system magnetic resonance imaging (MRI) scans—during the acute phase and after 3 to 4 months. Five patients were excluded due to missing data.
Clinical Data
The observed clinical data included age, gender, risk factors (RFs) including thrombophilic conditions (as listed in Table 1) and GSRFs (HC, pregnancy, puerperium, and HRT), clinical symptoms during the acute phase of the disease, and clinical outcome after 3 to 4 months. Clinical outcome was evaluated during the follow-up visit by a certified neurologist using the modified Rankin scale (mRS [0, 1, or 2 to 3 points]). 5
Patient Age and Occurrence of Risk Factors.
Abbreviations: #, number of patients; GSRF, gender-specific risk factor; HC, hormonal contraception; HRT, hormonal replacement therapy; RF, risk factor; SD, standard deviation.
Magnetic Resonance Imaging
Magnetic resonance imaging examinations were performed using 1.5-T Magnetom Avanto and Magnetom Symphony systems (Siemens, Erlangen, Germany). The routine imaging protocol for both scanners included T2 2D Turbo spin echo, 2-dimensional (2D) fluid-attenuated inversion recovery, 2D T2* gradient echo and diffusion-weighted imaging sequences with a b factor of 0, 500 or 1000 and reconstructed apparent diffusion coefficient maps, and a sagittal T1 spin echo sequence. Axial sequences were applied at an identical level using the same slice thickness (5 mm with a 20% gap) and slice number (25) using a rectangular field of view (FOV) of 230 × 190 mm. The bicallosal line was used as an anatomical landmark.
The 2D phase-contrast venography (slice thickness of 30 mm, FOV of 220 mm) at an encoding velocity of 20 cm/s in the sagittal and axial orientations was performed for rapid visualization of the venous sinuses. Then, 2D time-of-flight (TOF) venography in the coronal plane (slice thickness of 3 mm with a 30% overlay, FOV of 200 mm, matrix of 192) was routinely performed in all cases. In inconclusive cases (such as hypoplastic dural sinuses and low flow areas, representing a major difficulty in 2D TOF), 3D contrast magnetic resonance venography was performed (3D volume-interpolated T1w fast low angle shot sequence at an isotropic resolution of 0.9 mm) following the intravenous administration of a standard dose of the contrast agent gadolinium.
The MRI scans were evaluated by a certified neuroradiologist who was blinded to the patient clinical data. The radiological data included the localization and severity of venous sinus impairment during the acute phase, details concerning focal cerebral tissue lesions (edema and/or hemorrhage), and the extent of recanalization after 3 to 4 months. For the identification of the anatomical variant (particularly hypoplastic sinus vs sinus occluded by a thrombus), the morphological results were analyzed in combination with the results of venous magnetic resonance angiography and, in 5 cases, contrast administration.
For the quantification of sinus impairment, a modified scoring system developed by Zubkov et al 6 was utilized, in which 1 point was applied for each individual compromised sinus or cortical vein and 2 points were applied for the impairment of the deep venous system. The changes in the tissue were evaluated using MRI, which was graded as I to IV according to Kawaguchi et al, 7 in which grade I denotes tissue displaying no structural changes, grade II denotes edema, grade III denotes edema and disruption of the blood–brain barrier, which was identified as opacification after contrast administration, and grade IV denotes an apparent hemorrhage.
Statistical Analysis
Statistical analysis was used to calculate the significance of the correlations. We designed contingency tables according to the required classifications, including clinical manifestation during the acute phase (impairment of consciousness, presence of focal lesions, and epileptic symptoms), thrombophilia state, extent of the sinus impairment (score 1-2, 3-5, or 6-9), characteristics of the cerebral tissue lesions (edema and hemorrhage), recanalization quality, and mRS (0, 1, or 2-3) after 3 to 4 months in the entire cohort and separately in the subsets of females with GSRFs (group A) and males (group C). The independence of these classifications was tested using Pearson χ2 test. The analyses were performed at a significance level of P < .05. The group of females without GSRFs (group B) could not be statistically analyzed due to the small number of cases (n = 4). Statistical analysis was performed using NCSS statistical software (NCSS LLC, East Kaysville, Utah).
Ethics
The entire study was conducted in accordance with the Helsinki Declaration of 1975 (as revised in 2004 and 2008). All conscious patients signed informed consent forms to provide the relevant and available diagnostic and treatment data.
Results
Patient Cohort
The study cohort consisted of 51 patients (mean age 33.1 ± 15.4 years, 37 [72.5%] females) who met the inclusion criteria. Five patients were excluded due to missing data. The youngest patients, with an average age of 29.4 (median 26.0) years at the time of definitive diagnosis, were the females in group A. The average age of the males (group C) was 35.9 (median 28.5) years. The females in group B were the oldest, with an average age of 54.8 (median 55.0) years (Table 1).
Risk Factors
Based on the RFs, we separated our cohort into 3 groups (Table 1). Group A consisted of 33 females (89.2%) with GSRFs, among whom 31 (93.9%) were using HC, 1 was in puerperium and 1 was using HRT. The 4 females without GSRFs and the 14 males were assigned to groups B and C, respectively.
Inherited thrombophilia was diagnosed in 15 (29.4%) patients and was more frequent in males (42.9%) than in females (24.3%). All thrombophilic conditions were present in heterozygous form and, in 4 patients, the presence of 2 thrombophilic conditions was detected.
In 3 patients (2 females in group A, 1 male), a myeloproliferative syndrome was diagnosed. Two of these patients, who were also carriers of a JAK2 mutation, exhibited a severe acute phase of CSVT that resulted in parenchymal lesions. In group B, 2 females had an oncological disease (Hodgkin lymphoma and gastric lymphoma), 1 female had Crohn disease, and 1 female exhibited no RFs.
Only 5 (10%) individuals did not exhibit any RFs. More than 1 RF was found in 21 (41%) patients. In females, exhibiting a higher number of RFs was associated with the presence of GSRFs.
Clinical Presentation
The females in group A more frequently presented with severe symptoms during the acute phase of the disease than the other patients (Table 2). Impaired consciousness was present in 5 (15.2%) females in group A but in only 1 (7.1%) male and in no females in group B. Focal neurological deficit, such as hemiparesis, phatic disorders, alexia, and agraphia, was observed more often in group A females (39.4%) than in males (28.6%). Alternatively, the males more often had generalized and focal epileptic episodes than the females in group A (28.6% vs 18.2%). However, these differences were not significant. Five patients (all from group A, 15.2%) were treated via local thrombolysis due to a severe disease course including the progressive impairment of consciousness and, in all cases except one, also due to a focal deficit (hemiparesis, aphasia, or focal epileptic seizures).
Clinical Manifestation and Radiological Parameters of Cerebral Venous Sinus Thrombosis.
Abbreviations: #, number of patients; mRS, modified Rankin scale.
aSubset of females in Group B was not statistically analyzed due to the small number of cases.
Extent and Localization of Thrombosis and Parenchymal Lesions
Regarding the anatomical variants, in 15 patients, hypoplasia of the transverse and/or sigmoid sinus was observed (on the left side in 14 cases and on the right side in 1 case). In 1 patient, hypoplasia of the right occipital sinus was detected.
The sigmoid and transverse sinus were most frequently affected by acute thrombosis, more often on the left side, followed by the superior sagittal sinus. In the majority of patients, multiple sinuses were affected (Tables 2, 3, and 4). The average number of sinuses involved was significantly higher in the males than in the females in group A (4.6 vs 2.8; P = .025). Although the impairment of the deep venous system and cortical veins was more frequent in males than in females, this difference was not significant. The extent of thrombosis significantly correlated with the presence of a thrombophilic condition (P = .031) but not the impairment of consciousness (P = .531) or the occurrence of edema (P = .140) or hemorrhage (P = .426), as presented in Figure 1. The proportion of patients in whom thrombosis did not result in parenchymal lesions was as follows: score 1 to 2, 68%; score 3 to 5, 36%; and score 6 to 9 (most extensive thrombosis), 56%. Alternatively, edema was significantly associated with thrombosis of the deep venous system and cortical veins (P = .048 and P = .005, respectively). Hemorrhage significantly correlated with thrombosis of cortical veins and the right transverse and sigmoid sinus (P = .015 and P = .034, respectively.). No association between impaired consciousness and the impairment of any of the sinus, including the deep venous system and cortical veins, was observed (P > .05 in all cases).
Localization of Thrombosis.a
aThrombosis involved multiple sinuses in the majority of patients.
Significant Correlations With Clinical Outcome in the Whole Cohort.
Abbreviation: mRS, modified Rankin scale.
aFor other sinuses, no statistically significant correlation between thrombosis and brain tissue lesions was found.
bFor other sinuses, no statistically significant correlation between thrombosis and clinical outcome was found.
Extent of the thrombosis and parenchymal lesion.
However, the incidence of impaired consciousness was significantly higher in cases with parenchymal lesions such as edema and hemorrhage (P = .034 and P = .002, respectively). The females in group A more often had edema and hemorrhage than the males. Magnetic resonance imaging grade IV tissue changes were observed in 33.3% of the females in group A, including all 5 patients treated via local thrombolysis but only in 14.3% of the males. Magnetic resonance imaging grade II tissue changes were observed in 18.2% of the females in group A but in only 7.1% of the males. In 48.5% of the females in group A and in 78.0% of the males, no structural changes were detected (grade I). Magnetic resonance imaging grade III tissue changes were not observed in our cohort.
Recanalization
Complete or partial recanalization after 3 to 4 months was observed in 47 (92.2%) patients (Tables 2 and 4, Figure 2), insignificantly more often in females with GSRFs (94%) than in males (86%). Recanalization was not dependent on thrombophilia (P = .376).
A 21-year-old woman with headache lasting for several days. A, A microcatheter was introduced through the jugular vein and transverse sinus into the superior sagittal sinus for local infusion of thrombolytic. The injection in lateral view revealed thrombi in the superior sagittal sinus and minimal blood flow. B, Blood flow in the sinuses was restored after 24 hours of local thrombolysis followed by neurologic improvement.
Clinical Outcome
Good clinical outcome, which was evaluated using the mRS, after 3 to 4 months significantly negatively correlated with a thrombosis location in the left transverse, left sigmoid, or superior sagittal sinus (P = .022, P = .045, and P = .046, respectively) and positively correlated with recanalization (P = .048). However, good clinical outcome was not significantly associated with the extent of thrombosis (P = .086), the impairment of the deep venous system or cortical veins (P = .324 and P = .1, respectively), impaired consciousness during the acute phase of the disease (P = .56), or the presence of edema or hemorrhage (P = .908 and P = .499, respectively).
None of the patients died during the acute phase or during the follow-up period. The most severe outcome (mRS grade of 3) was detected in 1 patient, an 18-year-old male who had a pronounced worsening of visual acuity resulting from intracranial hypertension (Table 2). The clinical outcome was significantly better in females with GSRFs than in males (P = .029). Almost 80% of the females with GSRFs, including 5 females treated via local thrombolysis due to their severe condition, and 43% of the males completely recovered (mRS grade of 0). Eighteen percent of the females with GSRFs and 36% of the males complained of intermittent headache (mRS grade of 1). Five patients were classified as an mRS grade of 2, included 1 woman with GSRFs, 2 males with an epileptic syndrome, and 2 females in group B with extensive parenchymal lesions during the acute phase (1 who presented with a memory deficit and 1 with alexia). Of 10 patients exhibiting epileptic episodes during the acute phase of CVST (6 females in group A and 4 males), the epileptic syndrome persisted in 3 cases (1 female and 2 males; 5.9%) in the form of focal seizures. No “de novo” epileptic symptoms were detected during the follow-up period.
Discussion
The most valuable source of data concerning CVST remains the ISCVT study published in 2004, which analyzed data collected from 624 patients at 89 centers in 21 countries between 1998 and 2001. 2 Excluding this study, CVST cohorts have been relatively small and have been recruited from individual centers. 6,8,9
The most frequently affected sinuses in our cohort were the left transverse and sigmoid sinus, possibly as a consequence of their morphological inferiority (often being hypoplastic), 4 followed by the superior sagittal sinus. The association of transverse and sigmoid sinus thrombosis with local inflammation in the nasal, tonsillar, and middle ear area, which was commonly observed in the past, has recently been rare. 10 We confirmed this finding, reflecting a globally reduced incidence of inflammatory phlebothrombosis, due to the increased use of antibiotic treatment. In the majority of our patients, multiple sinuses were involved, and more extensive thrombosis was observed in males than in females. Previously, only 2 reports thoroughly analyzing the correlation between the extent of CVST and the risk of parenchymal complications were published. Although Bergui et al in 1999 11 did not detect this relationship, the Mayo Clinic report published 10 years later 9 obtained an opposing conclusion. Of the 56 patients with CVST examined, 34% presented with a parenchymal complication (edema/infarction or hemorrhage), and the risk of complications directly correlated with the extent of thrombosis (in contrast to age, gender, and the presence of thrombophilic conditions). In the present study, no significant correlation was observed between the extent of thrombosis and either edema (detected in 43% of the cases) or brain hemorrhage (present in 29% of the cases). Nevertheless, vasogenic edema was significantly more frequently caused by thrombosis of the deep venous system and cortical veins, whereas hemorrhage was a consequence of the thrombosis of cortical veins or the right transverse or sigmoid sinus. The dominant role of deep and cortical veins in the risk of parenchymal complications is the logical based on their characteristics; these veins are “terminal,” lacking sufficient collaterals that are able to protect the brain from the increased venous pressure. Thrombosis of the right transverse and sigmoid sinus was associated with an elevated risk of hemorrhage, as confirmed in the ISCVT study, 4 likely as a consequence of increased venous congestion due to common hypoplasia of the contralateral sinus. Thrombophilic conditions significantly contributed to more extensive thrombosis but not to the occurrence of parenchymal lesions. Although females with inherited forms of a thrombophilic condition (identified in 24% of the females) exhibited an average thrombosis extent score of 2.8, the average thrombosis extent score in males with identified thrombophilic conditions (present in 43% of the males) was 4.6. Approximately one-third of the patients with CVST had parenchymal hemorrhage, which is typically accompanied by more severe clinical symptoms at disease onset 6 and, occasionally, by worse outcome. 2,8,12 –14 In most cases, small parenchymal hemorrhages surrounded by normal tissue or by a zone of perifocal edema of varying size are observed, whereas subdural hematoma and subarachnoid hemorrhage are rare. In our cohort, 29% of the patients had hemorrhage—in all cases at the region of vasogenic edema. Although both types of brain complications (hemorrhage and edema) were associated with a more severe clinical presentation during the acute phase, accompanied by impaired consciousness in 12% of the cases, their presence did not influence long-term clinical outcome in the present study.
Generalized and focal epileptic symptoms during the acute phase occur in up to 40% of patients (in 20% of the patients in the present study), and residual epileptic syndrome persists in 10% to 30% of these patients (in only 6% of these patients in the present study). 2 The prophylactic use of anticonvulsants during the acute phase of CVST is controversial and has yet to be supported by any evidence. The observation that the probability of the development of a secondary epileptic syndrome is higher in patients with a supratentorial lesion, as observed in our patients, supports their use in this subgroup of patients. 15
Partial or complete recanalization after 3 to 4 months was detected in 92% of our patients. In the majority of reported patients, partial or complete recanalization is achieved within the first 3 months, 16,17 after which the recanalization rate does not further increase. 18 Therefore, in routine clinical practice, it may not be necessary to perform MRI imaging at long-term follow-up if a noncomplicated course of CVST is observed clinically. Successful recanalization in our cohort did not depend on the presence of a thrombophilic condition but significantly influenced clinical outcome.
The prognosis of CVST is markedly better than brain infarction due to arterial occlusion. The predictors of poor outcome (mRS > 3) observed in 13% of the patients in the ISCVT study (with a 30-day mortality rate of 3.4%) and in the National Inpatient Sample database from the 2000 to 2007 (with a mortality rate of 4.4%) include older age, brain hemorrhage, and concomitant diseases, for example, hematological pathologies, systemic malignancy, and central nervous system infection. 2,19 However, other studies reported even lower mortality—approximately 1%. 1 In the present study, as previously stated, good clinical outcome after 3 to 4 months significantly negatively correlated with a thrombosis location in the left transverse, left sigmoid, or superior sagittal sinus and positively correlated with the achievement of recanalization. However, good clinical outcome did not correlate with other factors, such as the extent of thrombosis, impairment of the deep venous system and cortical veins, impairment of consciousness, edema, or hemorrhage. Regarding the thrombosis location, our finding did not confirm those from the ISCVT study, in which poor clinical outcome among the patients having CVST with intracerebral hemorrhage was associated with thrombosis of the right lateral sinus. This discrepancy might be attributable to the larger size of this sinus, explaining that the drainage cannot be compensated by the contralateral hypoplastic side. 4
None of the patients in our cohort died. The worst outcome (mRS grade 3) was observed in an 18-year-old male who had a pronounced worsening of visual acuity. An mRS grade of 2 was observed in 10% of the patients; this clinical outcome was due to a residual epileptic syndrome in 3 patients, memory impairment in 1 patient, and persisting alexia in 1 patient.
The individualized prediction of the disease course is extremely difficult. Nevertheless, in contrast to arterial brain infarction, the achievement of a very good recovery is possible even in severe cases. The prognosis of CVST is better during pregnancy and puerperium, as its common spontaneous regression and high recanalization rate are much more favorable than those of other conditions. In these cases, the probability of recurrence is very low (≤ 1%) 18,20 ; therefore, it is not necessary to discourage these patients from further pregnancies. 21
In 2009, a subgroup analysis of the data from the ISCVT study dedicated to gender differences in CSVT was published. 3 Females accounted for 75% of the cases and were significantly younger, and those (65%) with at least 1 GSRF exhibited a more favorable prognosis. Similar results were observed in the present study. Although the females with GSRFs in our cohort insignificantly more frequently exhibited a severe clinical presentation during the acute phase of the disease and experienced edema and hemorrhage than the males, they exhibited a significantly better clinical outcome than the males. Complete functional recovery (mRS 0-1) was achieved in 97% of the females with GSRFs but only in 79% of the males. Compared with females, males exhibited a higher incidence of an inherited thrombophilic condition (43% vs 24%), significantly more extensive thrombosis, and insignificantly more frequent impairment of the deep and cortical veins.
The better outcome observed in the females with GSRFs could be explained by their younger age (which is typically associated with reduced comorbidities). However, multifactorial analysis performed on the females in the ISCVT study did not identify age as a significant outcome predictor. Alternatively, the absence of GSRFs was a strong independent predictor of unfavorable outcome in females. Another explanation could be the generally better physical condition of females using HC because they are medically supervised, as HC would not be prescribed to females known to exhibit RFs for a thrombotic complication. Similarly, pregnancy is less likely or is postponed in females with health problems. In accordance with this explanation is the finding of the ISCVT study that females without GSRFs share the same risk profile as males. 22 However, one may argue that less frequent systemic illness among younger females could also play a role in their more favorable outcome.
In recent years, several studies have revealed possible mechanisms by which the estrogens contained in HC increase the risk of thrombosis. 23 –27 In response to the use of HC, the levels of procoagulant factors such as II, VII, VIII, X, and vWf are increased, the concentrations of protein S and antithrombin are decreased, and a resistance to activated protein C develops. The progestin component of HC also enhances the risk of venous thrombosis. However, the thrombogenic potential of HC does not exclusively depend on its composition. For the initiation of cerebral venous and sinus thrombosis, the presence of a multiple causative factors is typically required. The highest risk represents a combination of RFs at several levels—at the HC level (impact of different generations of progestins, the form of the drug, and the length of exposure), at the patient level (age, smoking status, arterial hypertension, obesity, immobility, etc), and at the inherited thrombophilic condition level. 27 A meta-analysis published in 2006 reported an odds ratio of 5.59 for CVST risk of HC users relative to those who do not use an HC (95% confidence interval [CI]: 3.95-7.91); in combination with a factor V Leiden or prothrombin mutation, the odds ratio increased to 30 and 79.3, respectively. 28 One may assume that to prevent the synergic effect of RFs, it would be beneficial, in addition to assessing for inherited thrombophilic conditions, to avoid prescribing an HC to females with other identified RFs. Although the long-term outcome of CVST was highly favorable in the vast majority of our females, it is unclear whether this result would be the same if 14% (5 of 37) of them had not undergone invasive and potentially dangerous recanalization treatment.
Several limitations of the present study should be mentioned. Its retrospective observational nature represents the first limitation. Second, a relatively small sample size and the very limited comparison of females without GSRFs make it difficult to draw conclusions regarding the impact that gender on CVST. Third, this sample size did not enable the use of logistic regression analysis for the statistical evaluation of the results. Fourth, the clinical outcomes were confounded by a lack of uniform therapy.
In conclusion, CVST represents a relatively benign disease that primarily affects young individuals. However, CVST may result in unfavorable outcome, particularly in females with GSRFs, specifically HC users, in whom CVST develops more frequently and exhibits a more severe course during the acute phase. Nevertheless, in the present study, the clinical outcome after 3 to 4 months was significantly better in the females with GSRFs than in the males. Moreover, good clinical outcome significantly negatively correlated with a thrombosis location in the left transverse, left sigmoid, or superior sagittal sinus and positively correlated with successful recanalization.
Footnotes
Authors’ Note
This article has been presented in part as a lecture at the Neurovascular Congress 2014—42nd Czech and Slovak Cerebrovascular Congress in Mikulov, Czech Republic in September, 2014.
Acknowledgment
The authors thank Josef Bukač, MSc, PhD, Charles University Faculty of Medicine in Hradec Králové, for testing the statistical significance of the results of this study.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: Study was partially supported by the grant projects number FNHK 00179906 and PRVOUK: P37/08.