Changes of endothelial glycocalyx thickness in patients undergoing hip/knee surgery
Astapenko D., Pouska J., Beneš J., Lehmann Ch. a Černý V.
Endothelial glycocalyx (EG) represents a sugar-based gel-like structure coating vascular endothelium. It plays key role in maintaining vascular integrity, however, due to its fragile structure, EG can be affected by various noxious stimuli. Clear evidence of changes of glycocalyx during surgery and/or anaesthesia does not exist. Non- invasive method of evaluating EG thickness by using Perfused Boundary Region (PBR) has been introduced just recently. The aim of the study was to evaluate changes in PBR in patients after hip/knee replacement under general (GA) and regional (RA) anesthesia. We assumed RA influences EG less than GA.
A patient cohort of this multicentric prospective observational study was 60 adults indicated to hip/knee replacement in GA/RA (30 in each group). After obtaining an informed consent selected demographic, clinical and physiological data were recorded. PBR (in µm) was evaluated in sublingual microcirculation area in each patient before and 24 hours after the operation by specialized automated software (GlycoCheck, Maastricht, Netherlands). The normality of the data was tested as well as group analysis (un/paired t-test). A value p=0.05 was considered as statistically significant.
There was no significant difference in PBR (mean ± SEM) between groups before the operation (1.95 ± 0.03 in RA vs. 2.02 ± 0.03 in GA). The PBR value rose significantly in both groups 24 hours after the operation (RA: 2.09 ± 0.02,
p < 0.001; GA: 2.20 ± 0.03, p < 0.001). In addition there was a significant difference between groups (2.09 ± 0.02 vs. 2.20 ± 0.03, p = 0.006).
Hip/knee replacement surgery in GA/RA significantly increased PBR. Patients in GA had significantly higher PBR than those in RA. According to our data a protective influence of RA on PBR and on EG itself can be considered.
ENDOVASCULAR TREATMENT OF ACUTE MIDDLE CEREBRAL ARTERY OCCLUSION – COMPARISON OF TREATMENT METHODS AND
IDENTIFICATION OF OUTCOME PREDICTORS Kateřina Blejchařová, M.D.
blejchak@lfhk.cuni.cz
Department of Neurology, Faculty of Medicine in Hradec Králové, Charles University in Prague
Co-authors: R. Herzig, E. Vítková, M. Roubec, D. Šaňák, A. Tomek, A. Krajina, V.
Procházka, M. Köcher, F. Charvát, D. Krajíčková, M. Kuliha, J. Zapletalová, D. Školoudík, M. Vališ
Tutor: Prof. Roman Herzig, M.D., Ph.D., FESO, FEAN
Introduction
In the treatment of the acute middle cerebral artery occlusion (MCAo), intravenous thrombolysis (IVT) has only limited effectiveness, while endovascular treatment (EVT) represents an alternative treatment method. Recently published studies, such as MR CLEAN [1], EXTEND-IA [2] and ESCAPE [3], and meta-analysis [4] reported the superiority of EVT, used mostly as an additional therapy to IVT (so called bridging therapy) to IVT alone in acute ischemic stroke in the proximal anterior circulation. According to the valid European Stroke Organisation guidelines, intraarterial treatment of acute MCA occlusion within a 6-h time window is recommended as an option. Nevertheless, these recommendations do not specify a preferred treatment method. [5] More recent American Heart Association/American Stroke Association guidelines recommend the use of stent retrievers such as Solitaire FR and Trevo rather than coil retrievers such as Merci, when mechanical thrombectomy is pursued.
According to the same guidelines, the usefulness of emergent intracranial angioplasty and/or stenting is not well established and these procedures should be used in the setting of clinical trials. [6]
Aims
To evaluate safety and efficacy of PTA and MT in the treatment of acute MCAo, including intravenous thrombolysis (IVT) with subsequent EVT, and to identify outcome predictors.
Patients and Methods
In the retrospective study, data from the Czech National Multicenter Registry of Cerebral Mechanical Recanalizations were analyzed. The set consisted of 126 acute ischemic stroke patients (64 males; mean age 68.0±13.3 years). All patients met the following inclusion criteria: neurologic deficit attributable to the MCA circulation; radiologically confirmed MCAo; and the use of PTA or MT (including the combination with IVT). There were no
exclusion criteria. Patient approval was obtained, as necessary. Following data were
collected: baseline characteristics, risk factors, pre-event stroke/transient ischemic attack, pre- event treatment with antithrombotics, treatment with statins, body mass index, glycaemia, systolic and diastolic blood pressure on admission, neurologic deficit on admission and at the time of treatment, presence of brain edema, localization of occlusion, type and estimated time to initiation of therapy, time to therapy, recanalization rate, post-treatment imaging findings, neurologic deficits 24 h and 7 days after the intervention, and clinical outcome on day 90. The following stroke risk factors were noted: arterial hypertension, atrial fibrillation, coronary artery disease, diabetes mellitus, hyperlipidemia, smoking history, alcohol abuse, and pre- event neurologic deficit evaluated using the modified Rankin scale (mRS). Post-treatment imaging was performed using computer tomography or magnetic resonance imaging and was carried out within 24 h after treatment initiation. Hemorrhagic and ischemic changes were systematically recorded. Thrombolysis in Cerebral Infarction (TICI) score was used for the assessment of recanalization.[7] Recanalization in posttreatment angiograms was
dichotomized as complete to partial (successful – TICI 2-3) versus minimal to nil
(unsuccessful – TICI 0-1). Clinical outcome on days 90 was evaluated using the mRS, with a good clinical outcome defined as a score of 0 – 2. Statistical analysis was performed using the software IBM SPSS Statistics version 22.
Results
Good 90-day clinical outcome (mRS 0-2) was achieved more frequently in patients treated with IVT+MT (56.4%) than with IVT+PTA (33.3%) (P=0.04). Other differences found between the particular groups (PTA, MT, IVT+PTA, IVT+MT) were not statistically
significant: successful recanalization in 89.1%, 93.1%, 86.7% and 91.4%, resp., and good 90- day clinical outcome in 41.1%, 51.0%, 33.3% and 56.4%, resp. (P>0.05 in all cases).
Diastolic blood pressure on admission (OR=0.940, 95% CI: 0.902-0.980, P=0.004) and neurologic deficit at the time of treatment (OR=0.820, 95% CI: 0.728-0.922, P=0.001) were identified as independent negative predictors and, achieved recanalization – TICI 2-3 (OR=20.8, 95% CI: 1.400-319.1, P=0.029) as an independent positive predictor of good 90- day clinical outcomes.
Conclusions
Data from this registry showed that both PTA and MT represented safe and effective recanalization methods of acute MCAo.
References
[1] Berkhemer OA, et al.; MR CLEAN Investigators. N Engl J Med 2015; 372: 11 – 20.
[2] Campbell BC, et al.; EXTEND-IA Investigators. N Engl J Med 2015; 372: 1009 – 18.
[3] Goyal M, et al.; ESCAPE Trial Investigators. N Engl J Med 2015; 372: 1019 – 30.
[4] Fargen KM, et al. J Neurointerv Surg 2015; 7: 84 – 9.
[5] The European Stroke Organisation (ESO) Executive Committee and the ESO Writing Committee. Cerebrovasc Dis 2008; 25: 457 – 507.
[6] Jauch EC, et al. Stroke 2013; 44: 870 – 947.
[7] Higashida RT, et al. Stroke 2003; 34: 109 – 37.
ALTERED METHYLATION OF TRANSCRIPTION FACTORS IN OVARIAN CARCINOMA
Ivana Bubancová bubancoi@lfhk.cuni.cz
Institute for Clinical Biochemistry and Diagnostics, Charles University Faculty of Medicine and University Hospital Hradec Kralové
Co-authors: H. Kovaříková, J. Laco, O. Dvořák, V. Palička, M. Chmelařová Tutor: prof. MUDr. Vladimír Palička, CSc., dr. h. c.
Introduction
Epigenetic alterations, such as DNA methylation, are well-known to be involved in cancer initiation and progression. It has been suggested that epigenetic alterations have a great potential to serve as biomarkers in monitoring response to therapy and for disease screening and detection [1]. Many different genes have been identified as being hypermethylated and silenced in ovarian carcinoma [2]. Transcription factors play an important role in many cellular processes, among others they promote cellular differentiation of numerous tissues and therefore their impaired function or altered expression may contribute to the malignant transformation of affected cells [3]. The purpose of this study was to search for the most important CpG sites for DNA methylation analysis in the genes encoding transcription factors HNF1B and GATA4 and confirm their importance in ovarian carcinogenesis.
Methods
We used next generation sequencing (NGS) platform Illumina for detecting regions with the most altered methylation status of the HNF1B and GATA4 genes in fresh frozen ovarian cancer tissue (n=9) in comparison with normal ovarian tissue (n=6). To confirm discovered alterations in selected regions we further analyzed formalin-fixed, paraffin- embedded ovarian tissue (66 cancer samples, 35 control samples) using high-resolution melting analysis and methylation-specific real-time PCR. We compared detected methylation with clinicopathological characteristics (such as age, stage, histology).
Results
Using NGS we detected statistically significant methylation in analyzed regions in both genes (HNF1B: p = 0.004; GATA4: p < 0.001) in ovarian cancer tissue compared with normal ovarian tissue. In 6 CpGs of HNF1B and 9 CpGs of GATA4 high methylation was present in over than 50 % of tumor samples. Further analysis confirmed detected hypermethylation of selected regions of the HNF1B and GATA4 genes. Methylation-positive pattern in HNF1B was observed in 31.8 % (p < 0.001) and in GATA4 in 45.5 % (p < 0.001) of ovarian cancer tissue samples, whereas there was no methylation present in any of the normal ovarian tissue samples. In the late stage tumors we detected significantly higher methylation of the HNF1B gene than in the early stage tumors (46.2 % vs. 11.1 %; p = 0.003). Borderline significant correlation (p = 0.05) was found between GATA4 methylation in the endometrioid type tumors (69.2 %) and the high-grade serous type ones (35 %). We also observed a decrease of GATA4 methylation in the late stage tumors compared to the early stage ones (from 55.6 % to 38.5 %). No significant correlation between GATA4 or HNF1B methylation and the rest of clinicopathological parameters was observed.
Discussion
NGS approach can be invaluable tool in looking for epigenetic biomarkers of clinical benefit in disease screening, diagnosis and prognosis. While commonly used methods for analysis
of DNA methylation can monitor only few CpGs, NGS can provide a comprehensive view of methylation patterns of more extensive region of selected gene. Since methylation is site specific it is practical to use NGS for preliminary scan and then for further analysis employ another method with focus on the most interesting sites of selected gene. In our study we focused on searching for relevant sites for methylation analysis of the HNF1B and GATA4 genes in ovarian carcinoma using above mentioned approach. Our results are in correlation with previous studies of the HNF1B gene methylation [4, 5] showing a slightly higher percentage that is presumably caused by our choice of more important site for analysis thanks to the initial NGS scan. In addition, we observed statistically significant difference between different stages of ovarian carcinomas. Previous studies of the GATA4 gene methylation [6, 7]
reported higher percentage of methylation in ovarian cancer tissue than our results; however, they detected methylation also in normal ovarian tissue. This difference in results emphasizes the need of selecting the most relevant site for analysis. Our results also showed subtype specific methylation in the GATA4 gene and variances between percentages of methylation in different stages of ovarian cancer. Observed difference in genes methylation between the stages shows that analyzed CpG sites could be a promising targets for early detection of ovarian cancer, especially if detected in plasma.
Conclusions
In our study we employed innovative approach of selecting specific sites of the HNF1B and GATA4 genes for methylation analysis in searching of possible epigenetic biomarkers. We confirmed significance of those genes hypermethylation with emphasis on the need of selecting the most relevant sites for analysis. Epigenetic characteristics observed in our study suggest possible use of the HNF1B and GATA4 genes for ovarian cancer screening, detection and therapeutic strategies.
Funding
This study was supported by Ministry of Health, Czech Republic – conceptual development of research organization (UHHK, 00179906), by the programme PRVOUK P37/11 and by the project BBMRI_CZ LM2015089.
References
1. Graham JS, et al. The promises and pitfalls of epigenetic therapies in solid tumours. European Journal of Cancer. 2009;45(7):1129-36.
2. Koukoura O, et al. DNA methylation profiles in ovarian cancer: implication in diagnosis and therapy (Review). Mol Med Rep. 2014;10(1):3-9.
3. Zheng R, Blobel GA. GATA Transcription Factors and Cancer. Genes & cancer. 2010;1(12):1178-88.
4. Shen H, et al. Epigenetic analysis leads to identification of HNF1B as a subtype-specific susceptibility gene for ovarian cancer. Nat Commun. 2013;4:1628.
5. Terasawa K, et al. Epigenetic inactivation of TCF2 in ovarian cancer and various cancer cell lines.
British journal of cancer. 2006;94(6):914-21.
6. Wakana K, et al. Involvement of GATA-4/-5 transcription factors in ovarian carcinogenesis. Cancer Letters. 2006;241(2):281-8.
7. Montavon C, et al. Prognostic and diagnostic significance of DNA methylation patterns in high grade serous ovarian cancer. Gynecologic Oncology. 2012;124(3):582-8.
USE OF EX-PRESS® IMPLANT IN GLAUCOMA SURGERY TO INCREASE AQUEOUS HUMOUR DRAINAGE – RETROSPECTIVE STUDY
MUDr. Veronika Fialová
veronikahavlickovafialova@gmail.com
1. Department of Ophthalmology, Charles University in Prague, Faculty of Medicine Hradec Kralove, Czech Republic
Head of Ophthalmology clinic: Prof. MUDr. Naďa Jirásková, Ph. D., FEBO 2. Visus spol. s.r.o. – Náchod Eye hospital, Czech republic
Senior consultant: MUDr. Karel Havlíček, MBA Co-author: MUDr. Martin Váša, Ph.D.
Tutor: Prof. MUDr. Naďa Jirásková, Ph. D., FEBO Abstrakt:
Introduction:
Surgical therapy of glaucoma is aimed mainly at decrease or stabilisation of intraocular pressure. Surgery of primary open-angle glaucoma (POAG) is performed in patients with glaucomatous optic neuropathy despite of maximum possible pharmacotherapy. Ex-Press – a miniature drainage implant was developed as an alternative to trabeculectomy, traditional glaucoma surgery to increase aqueous humour drainage.
Purpose:
Assessment of results of POAG surgery using the Ex-Press drainage implant. Decrease of intraocular pressure (IOP), stabilisation of perimetry (T 30-2) or HRT findings and possible reduction of pharmacotherapy were evaluated.
Patients and Methods:
Retrospective analysis of the data of 37 POAG eyes in 25 patients, out of which 14 were women and 11 men, average age 71.76 years. Surgery on all 37 eyes was performed by one surgeon in 2011-2015. The implantation was indicated in POAG with decompensated IOP, failure of maximum conservative therapy or previous antiglaucoma surgery. The set included 13 eyes that had previously undergone antiglaucoma surgery. In all cases, progression was found during the pre-operative period by means of the perimetry, or by HRT. The following parameters were evaluated in all 37 eyes before and after surgery by doctor: IOP (on applanation tonometry), visus, pachymetry, fundus including the state of optic nerve, therapy by antiglaucomatic agents, and regular follow-ups by perimetry and HRT. The average follow-up duration in the set of operated patients was 2,36 years.
Results:
The average IOP before surgery was 20,14 mmHg, 6 months after surgery was 11,38 mmHg
and currently is 13.54 mmHg. Antiglaucoma therapy before surgery: monotherapy – 1 eye, therapy by 2 agents – 25 eyes, and therapy by 3 agents – 12 eyes. No therapy was needed after surgery in 17 eyes. Monotherapy was required in 5 eyes, therapy by 2 agents in 13 eyes, and therapy by 3 agents in 1 eye. The average number of applied antiglaucomatic agents thus decreased from 2.35 before surgery to 0.94 at the last follow-up visit. Stationary post-surgery perimetry results were found in 36 eyes and mild progression was found in 1 eye only. No severe intraoperative complications were found. During the postoperative period, choroid ablation that was corrected within 6-7 days was found in 10 eyes. Endophthalmitis as a more severe complication was found in 1 eye and was followed by PPV. In the late postoperative period, we have not seen the failure of filtration, or the need of subsequent surgery.
Conclusion:
The above given results show that the use of Ex-Press implants in glaucoma surgery is an effective and safe method with the minimum number of complications.
References :
ROZSÍVAL, Pavel, pořadatel. Trendy soudobé oftalmologie, svazek 9. 1. vydání.
Praha, Galén, 2013
Česká a slovenská oftalmologie, 71. ročník 2015, 6.číslo
DEREGULATION OF SELECTED MICRORNAS IN SINONASAL CARCINOMA AND THEIR VALUE AS PROGNOSTIC BIOMARKERS
Helena Kovaříková kovarikovahe@lfhk.cuni.cz
Institute for Clinical Biochemistry and Diagnostics, Charles University, Faculty of Medicine in Hradec Králové and University Hospital Hradec Králové
Co-authors: I. Bubancová, J. Laco, K. Sieglová, H. Vošmiková, P. Dundr, K. Němejcová, J.
Michálek, M. Vošmik, V. Palička, M. Chmelařová Tutor: prof. MUDr. Vladimír Palička, CSc., dr. h. c.
Introduction
Sinonasal carcinomas (SNCs) are malignant tumors arising from nasal and paranasal sinuses and make up 3 % of all cancers of head and neck area. Tumors occurring in sinonasal area are characterized by unfavorable outcome due to difficult diagnosis, treatment and prognosis of the disease corresponding with the anatomic complexity of the region. Risk factors for developing SNC include cigarette smoking professional exposure to various cancerogenous substances (wood-dust, leather dust) and HPV infection [1]. MicroRNAs (miRNAs) are short (18 – 25 nt) non-coding RNA molecules that are part of gene expression and their primary role is negative regulation of translation as part of the RNA-induced silencing complex (RISC) [2]. The aim of this study was to investigate relative expression levels of selected miRNAs in sinonsal carcinoma samples and to compare the results with recorded
clinicopathological data.
Methods
A total of 87 formalin fixed, paraffin embedded samples of sinonasal carcinoma and normal sinonasal tissue were analyzed (70 sinonasal cancer samples and 17 samples of control tissue). Relative expression of miR-21, miR-9, miR-143 and miR-145 were measured by real-time PCR with specific TaqMan® Advanced miRNA Assays (Applied Biosystems) on Rotor-Gene Q and calculated using the 2-ΔΔCt method [3]. One-way analysis of variance and regression analysis were used to analyze the correlation between relative expression levels of miRNA and recorded clinicopathological characteristics such as gender, age at the time of diagnosis, smoking history, occupation, tumor localization, TNM classification, tumor subtype, invasion, recurrence, metastasis or HPV infection status. The Kaplan-Maier method and Logrank test were used to determine overall survival rate and corresponding statistical significance.
Results
Real-time PCR data show statistically significant upregulation of miR-21 (5.5-fold, p < 0.0001), miR-9 (2.65-fold, p < 0.0001) and miR-143 (1.79-fold, p = 0.044) in SNC samples in comparison to control tissue. On the other hand miR-145 was significantly downregulated (1.92-fold, p = 0.027). We found correlation between shorter overall survival interval of the patients with higher upregulation of miR-21 (p = 0.0030) and downregulation of miR-145 (p = 0.036). After comparing relative expression levels of the miRNAs with clinicopthological data, we found out that smokers and former smokers had significantly higher upregulation of miR-9 than non-smokers (p = 0.021). Additionally, miR-143 was significantly more upregulated in male patients than in female patients (p = 0.036) and the upregulation was also higher in patients with risk occupations (p = 0.026).
Discussion
Although miRNA research is currently on the rise and many authors investigated expression of miRNAs in many cancerous tissues including head and neck cancer subtypes, there are not many information about miRNA expression and regulation in sinonasal cancer. MiR-21 is one of the most well-studied oncomiRs (miRNA functioning as oncogene). We observed not only upregulation of miR-21 in SNC samples, but also correlation between levels of miR-21 overexpression and poor survival rate of patients with sinonasal cancer, which was also reported by Hu et al. [4] in laryngeal carcinoma. Furthermore, our results showing higher upregulation of miR-9 in samples of smokers indicate an influence of nicotine on miR-9 expression which is in agreement with results of Yu et al. [5] in head and neck squamous cell carcinoma. We also observed upregulation of miR-143 and interestingly, it was higher in samples of patients pursuing risk occupations and also in male patients in comparison to female patients. Those results are in agreement with the fact that SNC is more common in men than woman, because there is higher risk of developing SNC due to professional exposure in some occupations usually pursued by men [1]. Finally, our data show not only upregulation of miR-145, but we also found correlation between miR-145 overexpression and reduced survival time of SNC patients which corresponds with study done by Bufalino et al.
[6] in oral squamous cell carcinoma.
Conclusion
Our data show that miR-21, miR-9, mir-143 and miR-145 may represent important regulatory molecules involved in development and progression of the disease and survival time of sinonasal carcinoma patients. On top of that, they could be potentially used as valuable prognostic biomarkers of the disease.
Funding
The study was supported by the program MH CZ - DRO (UHHK, 00179906), by the program PRVOUK P37/11 and by the project BBMRI_CZ LM2015089.
Literature
1. Barnes, L., J.W. Eveson, P. Reichart, et al., eds. Tumours of the Nasal Cavity and
Paranasal Sinuses. World Health Organization Classification of Tumours. Pathology and Genetics of Head and Neck Tumours. 2005, IARC Press: Lyon. 9 - 80.
2. Lee, Y., K. Jeon, J.T. Lee, et al., MicroRNA maturation: stepwise processing and subcellular localization. Embo J, 2002. 21(17): p. 4663-70.
3. Schmittgen, T.D. and K.J. Livak, Analyzing real-time PCR data by the comparative CT method. Nature Protocols, 2008. 3(6): p. 1101-1108.
4. Hu, A., J.J. Huang, W.H. Xu, et al., miR-21 and miR-375 microRNAs as candidate diagnostic biomarkers in squamous cell carcinoma of the larynx: association with patient survival. Am J Transl Res, 2014. 6(5): p. 604-13.
5. Yu, M.A., A. Kiang, J. Wang-Rodriguez, et al., Nicotine promotes acquisition of stem cell and epithelial-to-mesenchymal properties in head and neck squamous cell carcinoma.
PLoS One, 2012. 7(12): p. e51967.
6. Bufalino, A., N.K. Cervigne, C.E. de Oliveira, et al., Low miR-143/miR-145 Cluster Levels Induce Activin A Overexpression in Oral Squamous Cell Carcinomas, Which Contributes to Poor Prognosis. PLoS One, 2015. 10(8): p. e0136599.
EXPERIMENTAL TREATMENT OF THE SPINAL CORD INJURY BY HUMAN MESENCHYMAL STEM CELLS DERIVED FROM WHARTON´S JELLY (WJ-MSC)
MUDr. Petr Krůpa petrkrupa@centrum.cz
Department of Neurosurgery, Faculty of Medicine in Hradec Králové, Charles University in Prague and
Institute of Experimental Medicine v.v.i., The Czech Academy of Sciences Co-authors: Jendelova P., Amemori T., Vackova I., Ruzicka J., Zaviskova K., Dubisova J.
Tutor: Prof. MUDr. Svatupluk Řehák, CSc.
Introduction:
Spinal cord injury (SCI) remains one of the most physically, psychologically and socially debilitating medicine problems in the world. So far there is no specific or effective primary treatment yet available. Injury to the spinal cord results in motor and sensitive deficit, which severity depends on mechanism of the injury (primary damage) and on endogenous processes following the direct trauma in hours and days (secondary damage). While direct trauma can be hardly affected, modulation of the local inflammatory response, scavenging reaction and production of growth factors can result in better regeneration of the tissue.
In recent decade a variety of stem cells were used to affect the process of cell apoptosis, demyelination and axonal degeneration. Mesenchymal stem cells (MSCs) are a type of multipotent progenitor cells that can be differentiated into several cell types of mesodermal origin including osteocytes or cardiomyocytes. Major sources of MSCs are bone marrow and umbilical blood, but they are also found in adipose tissue or Wharton´s Jelly surrounding the umbilical vessels [1]. Their beneficial effect in spinal cord injury treatment is due to their ability to secrete trophic factors, which can affect secondary processes occurring after SCI, promoting axon regeneration, angiogenesis and reduce inflammatory cell activation and glial scar formation.[2]
Aims:
The aim of the study was to determine, whether the human mesenchymal stem cells derived from Wharton´s Jelly could improve the functional outcome of the rat with ischemic-compression spinal cord injury. Secondary purpose was to evaluate, if the treatment is dose – responsive – e.g. if the repetition or higher doses of transplanted stem cells would result in better recovery.
Methods:
Experiment was performed on 10-weeks-old male Wistar rats with the body weight 300g +-15g.
Ballon-induced spinal cord injury model was utilized as a ischemic-compression model of SCI. Rats were divided into five groups. Group A (single treatment of 0.5mil hWJ-MSC), Group B (single treatment of 1.5mil hWJ-MSC), Group C (repeated treatment of 0.5mil hWJ-MSC), Group D (repeated treatment of 1.5mil hWJ-MSC) and Group E (control group receiving saline). Seven days after SCI,
hWJ-MSC (5 x105 or 1.5 x106/ 50 ml saline) were injected by the lumbar puncture into the subdural space through the L5-L6 intervertebral space. Groups with repeated treatment received another injection 14 and 21 days after SCI. Control rats were injected with 50ml of saline into the L5-L6 interspinous space.
Through the experiment the behavioural assessment was regularly performed. Locomotor function was tested by Basso, Beattie, and Bresnahan (BBB) open field test, flat-beam test and rotarod test every week after lesion.
Eight weeks after spinal cord injury all rats were perfused with 4% paraformaldehyde in PBS and their spinal cords were removed, embedded in paraffin and cut into cross sections. Samples were stained with Cresyl violet Luxol to visualise white and grey matter, with GAP43 to analyse axonal sprouting and with GFAP to recognize the glial scar.
Results:
1. Behavioural testing: Group with single treatment of 1.5 mil hWJ-MSC and groups with repeated treatment of 3x0.5mil or 3x1.5mil hWJ-MSC recovered significantly better then control (two-way RM ANOVA, Treatment p˂0.05). Treatment with single dose of 0.5 mil hWJ- MSC didn’t show any significant difference compared to control. Testing of advance motor function and limb coordination by flat beam test and rotarod test showed no or minimal differences between the treated groups and control.
2. Histological assessment: Grey and white matter sparing was measured as a remained tissue on cross section areas of the treated animals compared to the control group. Significant difference in preserved grey matter achieved only group with 3x1.5 mil hWJ-MSC (two-way RM ANOVA, Treatment p˂0.05). No significant difference in preserving white matter was observed. Glial scar around the main cavity was measured and counted as a percentage of whole cross section tissue. Astrogliosis was significantly lower in higher single dose – 1x1.5mil hWJ-MSC and in both repeated doses – 3x0.5mil and 3x1.5mil hWJ-MSC. Axonal sprouting was counted as a number of fibers in GAP43 staining. There was significant higher number of GAP43+ fibers in all treated groups with exception of 1x0.5mi hWJ-MSC (one-way ANOVA, Treatment p<0.05)
Conclusions/Summary:
In the presented experimental study we concluded, that treatment by single dose of 0.5 mil. hWJ- MSC (group A) had no or minimal benefit on recovery from the spinal cord injury in rat model. We also proved that higher doses and repeated treatment (group B,C,D) of hWJ-MSC significantly improved overall functional outcome. Though there was no difference between the groups B,C and D in behavioural testing, group D had significantly higher number of GAP43+ fibers and had significantly larger area of spared grey matter in the centre of the lesion. These findings can lead to hypothesis that in long term recovery (> 9 weeks) rats in group D would have higher potential to improve than rats in group B and C.
References:
[1] Dasari VR, Veeravalli KK, Dinh DH. Mesenchymal stem cells in the treatment of spinal cord injuries: A review. World J Stem Cells. 2014 Apr 26;6(2):120-33
[2] Serhiy Forostyak, Pavla Jendelova, Eva Sykova. The role of mesenchymal stromal cells in spinal cord injury, regenerative medicine and possible clinical applications, Biochimie. 2013 Dec;95(12):2257-70
"Respiratory movement detection by MS Kinect"
Authors Kuchyňka J. 1, Centzone F. 2,3, Schätz M. 2 ; Procházka A. 2; Vyšata O. 1,4; Cejnar P. 2;
Vališ M 1
1. Neurological clinic of University hospital in Hradec Králové
2. Department of Computing and Control Engineering , Institute of Chemical Technology, Prague 3. Department of Mechanical Engineering, Politecnico di Milano, Milan, Italy
4. The Czech Institute of Informatics, Robotics and Cybernetics (CIIRC) in Prague
Abstract: Laboratory-based polysomnography is considered to be a gold standard to measure sleep objectively, but it is impractical for long term and home utilization. Standards for PSG do not fully reflect actual technology, particulary the fast development of anvanced sensors. Connecting wires and bands hinder the patient's movement and alter normal sleep during study. There is a need for a technician in sleep laboratory or during home sleep study, which increases expenditure. Our clinical study shows posibility to measure respiratory movements with noncontact method using depth camera of MS Kinect system. This is the first step in our plan of development more convenient method for sleep monitoring.
Methods: Initially we tested the MS Kinect depth camera and explored the methods of signal denoising, resampling, and spectral analysis of acquired data as well as feature extraction and their Bayesian classification. Then we applied methodology for analysis of the 8 hours long monitoring of 25 individuals who were observed simultaneously by PSG and MS Kinect in the sleep laboratory.
After time synchronization of polysomnographic and MS Kinect video data, features were extracted from both signals and compared.
Results/Discussion: The average error of the frequency while being evaluated by MS Kinect that was related to that obtained by PSG was 3.75%. The mean accuracy of the Bayesian classification of features into twoclasses(i.e.wake or sleep) was 88.90 and 88.95% for thePSG and MSKinect
measurements, respectively. The strong likeness of features supports the hypothesis that contactless techniques may represent a valid alternative to the present approach of sleep monitoring, thereby allowing data acquisition in the home environment as well.
Keywords: Polysomnography, Breathing analysis, Digital signal processing, Range imaging methods, MS Kinect, Feature extraction, Bayesian classification
DEXMEDETOMIDINE-ISOFLURANE VERSUS FENTANYL-ISOFLURANE ANESTHESIA FOR COLORECTAL SURGERY:
EFFECT ON PERIANASTOMOTIC MICROPERFUSION AND OXYGENATION IN PIGS.
Marian Mynář marian.mynar@gmail.com
Department of Anesthesia and Intensive Care Medicine, University Hospital Hradec Kralove, Charles University, Faculty of Medicine Hradec Kralove
Tutor: MUDr. Zdeněk Turek, Ph.D.
Introduction
Dexmedetomidine, α2 – adrenergic highly selective agonist produce complex clinical effects after binding to α2 – adrenergic receptors. Well documented hypnotic-sedative and analgetics effect of dexmedetomidine is mediated by α2A - receptor in the central nervous system. The influence of selective α2 agonist on peripheral vascular system including hepatosplanchnic circulation is difficult to assess regarding vasodilatatory action via sympatholysis and
vasoconstriction mediated through the receptors in the smooth muscle cells [1]. Last decades, the use of dexmedetomidine especially at intensive care units is more frequent due to its specific effect called conscious-sedation. There is an increasing evidence of dexmedetomidine use during intraoperative period because of its analgetics and anesthetic-sparing effect [2,3].
To our knowledge there are no studies assessing the complex effect of dexmedetomidine on regional splanchnic circulation and oxygenation during major abdominal surgery. This knowledge may be of high importance in colorectal surgery procedures which carry the risk of anastomotic leakage, especially in rectal resection.
Aims
We hypothetized, there is no difference in regional rectal perianastomotic perfusion and oxygenation when using non-opioid dexmedetomidine-isoflurane aneshesia when compared to fentanyl-isoflurane anesthesia. The secondary goal of this study was to evaluate the trends of regional circulation and oxygenation during colorectal surgery in pigs.
Methods
Ten female pigs were randomly divided into two group (Dexmedetomidine, DEX, n = 5, Fentanyl, FNT, n = 5). All pigs were initially anesthetized with ketamine and azaperon for instrumentation. This was followed by either dexmedetomidine (0,7 -1,0 μg/kg/h) or fentanyl ( 6-10 μg/kg/h) intravenous infusion. The model of rectosigmoid resection in pigs was used for the purpose of this study. Two combined laser Doppler flowmetry (LDF) and oxymetry probes were fixed on the antimesenterial site of the rectosigmoid, one orally and second distally to resection zone. LDF and tissue oxymetry measurements were performed at the baseline (T0) and at T1 (after ligation of supply artery), T2 (after anastomosis being performed), T3 (30min. after anastomosis), T4 (60min.) and T5 (90min.). Time course of microhemodynamic data (LDF and oxymetry) are presented as percentage change from baseline. Macrohemodynamic data including cardiac output and preload dynamic parameteres were monitored by using LiDCO plus throughout the study. At the end of the experiment all animals were woken up and extubated. All animals were checked for weight gain and food intake during postoperative period, the anastomotic healing was controlled seven days after
the operation. Data are presented as median (interquartile range) or mean (± standard deviation).
Results
All experimental animals were hemodynamically stable throughout the intraoperative period, no statistically significant difference in baseline values of mean arterial pressure and cardiac output was observed between the groups. All animals survived until the seventh postoperative day. No anastomotic leakage was detected. In both DEX and FNT group statistically
significant decrease (P < 0,001) was found in LDF signal from aboral site of anastomosis at T1 after ligation of supplying artery. When compared to baseline statistically significant decrease in LDF signal from aboral perianastomotic site was observed in both groups throughout the study. In DEX group the median of the LDF signal on aboral site at T5 was 35% (23-49)%, in FNT group the median of the LDF signal was 19% (12-28)%, which was statistically significant lower (P<0,05). When compared to baseline at T5 tissue oxymetry in DEX group (83 ± 14)% did not differ from FNT group (84 ± 19)%.
Discussion
Non-opioid sedation and analgesia with α2 – agonist dexmedetomidine is widely used not only at critical care, but also during perioperative period. There are only limited experimental data describing the effect of dexmedetomidine on splanchnic microperfusion and
oxygenation. This is the first experimental study with dexmedetomidine focusing on
perianastomotic perfusion and oxygenation in model of rectosigmoid resection in pigs when using LDF imaging and VMS oxymetry. In presented study DEX group revealed higher level of LDF signal at the end of intraoperative period when compared to FNT group thus
dexmedetomidine may have potentially positive effect on microperfusion in perianastomotic region. This trend was not observed in tissue oxymetry. Local vasoconstriction may be eliminated by dexmedetomidine induced sympatholysis.
Conclusion-summary
Highly standardized experimental model for anastomotic perfusion assessment in pigs was established. Dexmedetomidine does not impair regional perfusion and oxygenation in experimental model of colorectal surgery. This study has shown some protective effect of dexmedetomidine-isoflurane anesthesia on perianastomotic microcirculation when compared to fentanyl-isoflurane anesthesia.
References
1. Takahiko K., Mervyn M. Clinical Uses of α2 – Adrenergic Agonist, Anesthesiology 2000;
93:1345-9
2. Su Hyun L., Namo K., Chang Yeong L., Min Gi B., Young Jun O. Effects of
dexmedetomidine on oxygenation and lung mechanism in patients with moderate chronic obstructive pulmonary disease undergoing lung cancer surgery, Eur. J. Anaesthesiol. 2016 33:275-282
3. Yu-Chang Y., Wei-Zen S., Wen-Je K., Wing-Sum Ch., Shou-Zen F., Jui-Chang T., Tzu-Yu L. Dexmedetomidine Prevents Alterations of Intestinal Microcirculation That Are Induced by Surgical Stress and Pain a Novel Rat Model, Anesth Analg 2012; 115:46-53
SERUM DRUG LEVELS AS A NEW DIAGNOSTIC TOOL FOR NON-ADHERENCE TO THERAPY IN HEART FAILURE PATIENTS
Radek Pelouch, MD radek.pelouch@fnhk.cz
1st Department of Internal Medicine – Cardioangiology, Charles University - Faculty of Medicine Hradec Kralove, University Hospital Hradec Kralove
Co-authors: J.Ceral, V.Voříšek, V.Furmanová, M.Solař Tutor: Assoc. Prof. Miroslav Solař, MD, PhD Introduction
Heart failure (HF) is a significant clinical problem affecting patient´s prognosis and quality of life. Over the last decades, improvements in treatments have enhanced survival of HF patients and reduced the hospitalization rate for acute decompensated heart failure (ADHF) [1]. However, the non-adherence to recommended therapy is not rare, and may be one of the factors contributing to ADHF [1]. The adherence to prescribed medication can be evaluated by pill counts, rates of prescription refills and electronic medication monitors [2]. These monitoring techniques have high specificity, but sensitivity is reduced, because no technique is able to document if the prescribed drugs were actually ingested [3]. The measurement of serum or urinary drug levels is a novel approach for adherence assessment that provides reliable information on whether the recommended medications were taken [4].
Aims
The aim of the study is to assess the frequency of non-adherence to the recommended therapy in HF patiens.
Methods
Serum levels of prescribed medications were used as an indicator of drug adherence. Serum drug levels (SDLs) were evaluated by liquid chromatography and mass spectrometry.
Two different groups of patients were studied: Group1: patients addmited to hospital for ADHF. Blood sampling was performed at the time of admission. The main cause of ADHF was studied.
Group2: Stable chronic HF outpatients with established medication. They passed three study visits (enrollment (M1), month three (M3) and month nine (M9)) and two-year follow-up.
Blood sampling was performed during all study visits.
The subjects were labeled as non-adherent when the SDL of at least one of the evaluated drugs was below the limit of quantification.
The study was approved by the Local Ethics committee, and the participants gave written informed consent.
Results
Group1: The data of 50 patients were assessed. The ADHF was caused by acute coronary syndrome (5, 10%), infection (5, 10%), arrhythmia (2, 4%), anemia (1, 2%), uncontrolled arterial hypertension (1, 2%), and progression of valvular heart disease (1, 2%). Three (6%) patients conceded omission of recommended medication, and in one individual (2%) the medication was inappropriately changed by the attending physician. In 31 patients (62%) the cause of ADHF remained unclear based on routine clinical exams performed.
All of the evaluated drugs were detected in the sera of 28 (56%) study patients. The non- adherence was diagnosed in the remaining 22 (44%) patients. None of prescribed drugs was found in the sera of 5 (10%) patients.
In patients with unknown precipitating cause of ADHF (31), the non-adherence was diagnosed in 13 (42%), none of prescribed drugs was detected in 2 (7%).
Group2: Forty chronic HF patients were prospectively enrolled. All of the evaluated drugs were detected in the sera of 27patients (67.5%), the non-adherence was diagnosed in 13 patients (32.5%). At the end of two-year follow-up next indicators were compared between adherent and non-adherent group: overall mortality (29.6%vs15.4%, p=0.35), HF mortality (22.2%vs15.4%, p=0.63), need for emergency visit/hospitalization due to ADHF (1event 11.1%vs0%, p=0.23, ≥2 events 3.7%vs0%, p=0,52), gender (men 66.7%vs38.5%, p=0.10), NTpro-BNP (327.8±482.2vs112.1±128.7 pmol/l, p=0.43), LV EF (30.0±10.9%vs31.9±10.8%, p=0.62) and NYHA class (2.4±0.6vs2.4±0.6, p=0.60).
Discussion
The non-adherence rate was not rare in our patients. Most of the previous studies assessing medication adherence in HF patients were based mainly on the self-reporting or the analysis of prescription refills, and the frequency of non-adherence may have been underestimated.
Compared to other techniques, the estimation of serum drug levels allows reliable and easily accessible identification of actual non-adherence, which is a clinically relevant finding that indicates a need for a special attention. These patients require focused counseling and close supervision, because an extensive communication may help to establish good relationships with the patient and to manage the problem successfully.
Study limitations: 1/ Small size of the study population. 2/ We were not able to reliably assess the adherence to angiotensin-converting enzyme inhibitors due to analytical limitations.
3/ In Group1, in a significant number of patients furosemide was excluded from the assessment, because it was administered intravenously as an acute pre-hospital treatment.
Conclusions
Our data suggest that the non-adherence to the recommended therapy may be present in a significant part of chronic HF patients and may be a significant cause of ADHF. We assume, that the estimation of SDLs is an useful diagnostic tool in clinical evaluation of the
medication adherence.
We did not found any difference between groups of adherent and non-adherent chronic HF patients during two-year follow-up, but we are aware of small size of the study population as a principal limitation of the study.
References
[1] Ponikowski P, Voors AA, Anker SD et al.. 2016 ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure: The Task Force for the diagnosis and treatment of acute and chronic heart failure of the European Society of Cardiology (ESC). Eur J Heart Fail.
2016 Aug;18(8):891-975
[2] Osterberg L, Blaschke T. Adherence To Medication. N Engl J Med 2005;353(5):487-97.
[3] Van Onzenoort HA, Verberk WJ, Kessels AG, et al.. Assessing Medication Adherence Simultaneously By Electronic Monitoring And Pill Count In Patients With Mild-To-Moderate Hypertension. Am J Hypertens 010;23(2):149-54.
[4] Ceral J, Habrdova V, Vorisek V et al., Difficult- To-Control Arterial Hypertension Or Uncooperative Patients? TheAssessment Of Serum Antihypertensive Drug Levels To Differentiate Non-Responsiveness From Non-Adherence To Recommended Therapy.
Hypertens Res 2001;34(1):87-90.
Choleresis induced by iron depletion in rats Alena Prasnicka M.Sc.
prasnickaa@lfhk.cuni.cz
Department of Pharmacology, Faculty of Medicine, Hradec Králové, Charles University Co-Authors: Jolana Cermanova; Milos Hroch; Eva Dolezelova; Lucie Rozkydalova; Lucie
Hyrsova; Jaroslav Chladek; Martin Lenicek; Libor Vitek; Petr Pavek; Stanislav Micuda, Tutor: Assoc. Prof. Stanislav Micuda, M.D., Ph.D.
Introduction
Iron depletion is a worldwide nutrition problem affecting 25% of the population. It may result in anemia, metabolic problems and fetal impairment. Reduced iron content is also described in the liver, associated with cholestatic diseases such as primary biliary cholangitis or biliary atresia.
The relationship between bile production and secretion and iron depletion is currently poorly described. Data suggest that iron depletion may induce gene expression of some enzymes mediating bile acid synthesis, and that iron chelators may reduce the gene expression of major transporting proteins in terms of bile secretion in hepatocytes. More complex results do not currently exist.
Aim
This study was designed to describe the relationship between iron depletion and bile production and secretion in rats. Iron depletion was induced by the use of an iron deficient diet.
Methods
Two groups of pair fed female Wistar rats (n = 6) were used: one group was fed with a standard diet (STD) and the second one was fed with an iron deficient diet (IDD - containing 5.155 mg/kg Fe, Altromin Spezialfutter GmBh & CO.KG). Both diets were applied over 4 weeks. Thereafter bile was collected under general anesthesia. Another two groups of rats to which either the STD or the IDD diet was applied also underwent bile collection after 4 weeks of feeding, but with the simultaneous administration of radiolabeled taurocholate. Consequent analysis of radioactivity in the bile enabled an evaluation of transporting protein activity without the contribution of bile acid (BA) synthetic processes. Samples of plasma, bile and liver tissue were analyzed as described previously [Cermanova et al. Toxicol Appl Pharmacol. 2015 May 15;285(1):12-22].
Results
IDD applied over 4 weeks produced a significant reduction in the serum and liver concentrations of iron (p < 0.01). Reduced iron disposition led to a significant increase in cumulative bile flow (p ˂ 0.001) accompanied by an increase in biliary secretion of both major bile osmotic constituents, BAs and glutathione (GSH), as well as cholesterol and phospholipids (p ˂ 0.05).
BA plasma levels were measured at the start and the end of the bile collection study, and showed a significant increase (p ˂ 0.05) in IDD animals. A quantitative RT-PCR analysis of liver mRNA revealed increased gene expression of Abcg5/8 (cholesterol biliary secretion), Abcb4 (biliary phospholipid secretion), bile acid synthesis enzymes Cyp7a1, Cyp8b1, and cholesterol uptake transporter Ldlr, and reduced Abca1 (cholesterol efflux transporter from hepatocyte to blood) in
IDD animals. The detection of proteins through the use of Western blot verified the induction of Abcg5/g8, Cyp7a1, Cyp8b1, and the reduction of Abca1. We also detected a parallel up- regulation of major nuclear receptors regulating the transcription of these molecules, Liver X receptor. In contrast to mRNA, IDD reduced protein levels of Ldlr, Sr-b1, and Mdr2, and induced the expression of Ntcp and Oatp1/2, an uptake of transporters for bile acids and numerous other compounds. The expression of rate-limiting transporting proteins for the biliary secretion of bile acids (Bsep) and glutathione (Mrp2) remained unaffected by IDD. This finding was supported by unchanged biliary secretion of radiolabeled taurocholate. Instead, we detected increased liver concentrations of glutathione in a reduced form. We also analyzed the expression of major BA reabsorbing transporters in ileum, and detected reduced mRNA in the Ost-α/β transporter.
Discussion
Our results demonstrate that iron depletion as a result of IDD increases bile flow. This choleretic effect is the consequence of a simultaneous increase in the biliary secretion of BA and GSH. The rate limiting transporters for these compounds, Bsep and Mrp2, are not directly responsible for such an effect as suggested by their unchanged protein expression, preserved location on apical membrane of hepatocytes, and unaltered biliary excretion of the exogenous Bsep substrate, radio-labeled taurocholate. Instead, we detected increased liver concentrations of GSH and increased plasma concentrations of BA in IDD rats despite the upregulation of uptake transporters Ntcp and Oatp1/2. Together it suggest that choleresis with increased biliary secretion of BA/GSH in IDD animals is the consequence of the increased disposition of these compounds in hepatocytes, and the unaffected function of rate-limiting transporters.
The disposition of BA in organisms is regulated by their synthesis in the liver, and by enterohepatic recycling. Because the overall function of liver transporters for BA was unchanged, and the Ost transporters in ileum, the major site for BA reabsorption, were even reduced by IDD, we believe that the increased synthesis of BA is the major mechanism behind the increased disposition of BA. Increased liver expression of rate limiting enzymes for the synthesis of BA from cholesterol, Cyp7a1 and Cyp8b1, supports this suggestion.
The changes supporting increased BA synthesis as the major mechanism of their disposition in IDD are reduced systemic concentrations of cholesterol. We have, for the first time, demonstrated the increased biliary secretion of cholesterol through the up-regulated transporter, Abcg5/g8. Liver concentrations of cholesterol were unchanged due its increased uptake from blood by induced Ldlr and Sr-b1 and by the reduced export by Abca1. The up-regulation of the Liver X receptor, a nuclear receptor regulating the transcription of Abcg5/g8 transporters, and also Cyp7a1, seem to be major mechanism for the modulation of cholesterol metabolism in IDD rats.
Conclusion
This study demonstrate that iron depletion has a stimulating effect on bile production by enhancing the BA dependent and independent bile flow. Our results also indicate the cholesterol lowering effect of iron depletion by the increased conversion of cholesterol to bile acids, and also by its enhanced biliary secretion. The regulating role of the Liver X receptor with regard to this effect of IDD has been suggested.
Acknowledgement
The study was supported by the Prvouk P37/05, and SVV-2016-260287 Research Projects.
MITOCHONDRIAL FUNCTIONS IN STEATOTIC RAT LIVER Ondřej Sobotka
sobotkao@lfhk.cuni.cz
Department of Physiology, Faculty of Medicine in Hradec Kralove, Charles University in Prague
Co-authors: O. Kučera, P. Staňková, R. Endlicher, K. Nožičková, Z. Červinková Tutor: Prof. MUDr. Zuzana Červinková, CSc.
Introduction
Non-alcoholic fatty liver disease (NAFLD) is a frequent cause of chronic liver disease in western population. This hepatic disorder is characterized by increased accumulation of lipids in hepatocytes and by potential progression to inflammation called steatohepatitis. This process can advance to the liver cirrhosis an eventual end-stage liver failure with a need of liver transplantation. Cirrhosis can be also complicated by the development of hepatocellular carcinoma. The prevalence of NAFLD is more than one-third of all people in western countries with more than 80% prevalence in obese population. NAFLD is mostly linked to lack of physical activity and unhealthy western type of diet (high cholesterol, high saturated fatty acids content). Liver steatosis is often accompanied by other pathological conditions and disorders such as atherosclerosis, metabolic syndrome and type II diabetes. The pathophysiology of NAFLD is associated with insulin resistance, impaired lipid metabolism and increase in reactive oxygen species (ROS) production. However, the inflammation is also associated with lipid accumulation and the question of cause and consequence of NAFLD has not been sufficiently answered. The role of the liver mitochondria in NAFLD development and progression has not been fully elucidated yet and studies which would satisfactorily describe the changes in mitochondrial functions over the NAFLD progression are lacking.
The vast majority of studies describe some kind of mitochondrial alterations, but facing the critical differences in study designs (type of animal, type and duration of high fat diet, assessment mitochondrial functions) we have to be careful with mitochondrial data interpretation.
Aim
The aim of this study was to investigate the liver mitochondrial respiration and oxidative stress during high-fat and high-cholesterol diet for 1 to 3 weeks, which mimics NAFLD in humans.
Methods
Experiments were performed on male Wistar rats fed with a commercially prepared diet (Altromin) with high cholesterol and high fat content (HFD, 70 % of energy from lard enriched by 1.25 % cholesterol) for 1 and 3 weeks. Histological changes of liver tissue were evaluated by Hematoxylin eosin, Masson’s trichrome and Oil red O staining. Liver functions were estimated by measurement of biochemical markers (ALT, AST, ALP, bilirubin, urea) in
serum. Triglycerides (TG) and cholesterol were assessed both in serum and in liver homogenates using commercial kits. Mitochondria were isolated from liver by differential centrifugation. Mitochondrial respiration was assessed using OROBOROS Oxygraph 2k with using comparative reference protocols. Tricarboxylic acid cycle, fatty acid oxidation (FAO) and glycerophosphate dehydrogenase were evaluated by titrations of respective substrates and inhibitors.
Results
High fat diet did not influence the body weight or body weight gain even after 3 weeks in comparison to low fat diet control. Serum TG, HDL and LDL cholesterol as well as glycaemia were not different between both groups, but we detected progression of liver steatosis already after one week of HFD. Relative liver weight was significantly higher after 3 weeks of HFD feeding. Increased accumulation of lipids and micro vesicular steatosis were present in HFD group after staining by Oil red O and Hematoxylin eosin respectively. We also detected higher content of TG and cholesterol in liver homogenates. Mitochondrial respiration demonstrated significant increase in FAO capacity and relative inhibition of succinate stimulated respiration in HFD group. Maximal mitochondrial respiratory capacity was significantly increased after 3 weeks and mitochondria from HFD group exhibited more efficient oxidative phosphorylation.
Discussion
We described the development of early phase of NAFLD in rats fed by HFD. After the first three weeks of experiment we observed no significant biometrical changes even though the energy intake of HFD group was higher by more than 30% of controls. Already after one week of HFD there was a mild grade of fat accumulation in hepatocytes, but we did not observe any signs of impaired lipid metabolism or liver functional damage in serum biochemical markers. Liver mitochondria revealed higher capacity for FAO from the beginning of our study with more pronounced effect after three weeks of HFD. We may thus conclude that mitochondria started an adaptation process to reflect the diet with high fat and high cholesterol content by enhancing their capacity for FAO. To this day we were able to gain the results only from first and third week of the study, but the planned duration is 12 weeks.
Summary
We succeeded to simulate liver steatosis by HFD in rats already after one week of feeding.
We did not detect and biometric changes in rats but we observed higher TG accumulation in hepatocytes and mitochondrial modifications demonstrating increase in FAO capacity.
Acknowledgements
This work was funded from grants PRVOUK P37/02 and SVV-2016-260287
EXECUTIVE FUNCTIONS IN ADOLESCENTS WITH TYPE 1 DIABETES MELLITUS
Tereza VITVAROVA, M.D.1, David NEUMANN, M.D., Ph.D.1, Jan KREMLACEK, Ing., Ph.D.2, Radka SIMAKOVA, Mgr.3
1Dpt. of Paediatrics, Faculty of Medicine in Hradec Kralove, Charles University in Prague, and University Hospital Hradec Kralove, Czech Republic
2Department of Pathological Physiology, Faculty of Medicine in Hradec Kralove, Charles University in Prague, Czech Republic
3Philosophy faculty, Palacky University Olomouc, Czech Republic
Background: Unfavorable metabolic control of type 1 diabetes mellitus (T1D) has a negative impact on the developing brain. Hyperglycemia and glycemic fluctuations disrupt mainly executive functions, which control the adaptive human behavior, activation of human motivation and persistence or change in the direction of acting. These functions coordinate various cognitive, emotional and behavioral happening with respect to achieving the target. While they are disrupted, patients may have difficulties to maintain the diet and the daily program.
Methods: Visual evoked potentials were examined in a cohort of twenty-two 13-19 years old T1D patients at euglycaemia and in nineteen 10-19 years old healthy controls.
Responses were recorded using monocular stimulation. "Oddball" stimulus pattern reflects the executive functions in the wave P300. Further reactions of the primary visual and the extrastriate area were evaluated.
Results: The results of T1D adolescents were physiological if compared to the general reference. When compared with the reuslts of the control group, T1D patients had a statistically significant prolongation of latency in the primary visual area, but the P300 wave did not differ.
Conclusions: T1D adolescents differ from the control group with prolonged latency of Reversal 20 in selected visual reactions. The study confirms the influence of the long-term metabolic control in T1D children and adolescents on executive functions, especially on inputs of primary visual area.
Stroke after cardiac surgery in extracorporeal circulation – a pilot study
E. Vítková1, R. Herzig1, O. Vyšata1, D. Krajíčková1, J. Harrer2, M. Vališ1
1Department of Neurology, Comprehensive Stroke Center, Charles University Faculty of Medicine and University Hospital, Hradec Králové
2Department of Cardiosurgery, Charles University Faculty of Medicine and University Hospital, Hradec Králové
Introduction: Stroke after cardiac surgery represents a devastating complication leading to the excess of mortality and prolongation of hospitalization. The aim was to assess risk factors associated with the occurrence of ischemic stroke in patients undergoing cardiac surgery procedure in extracorporeal circulation.
Patients and Methods: In a retrospective, hospital-based study, we analyzed the set of 457 patients (299 males; mean age 70.0 ± 11.4 years), who underwent cardiac surgery in extracorporeal
circulation during a 9-month period (August 2012 – April 2013). The following factors were analyzed:
patient gender and age; history of transient ischemic attack or stroke, myocardial infarction, angina pectoris, atrial fibrillation, coronary artery disease, arterial hypertension, diabetes mellitus;
occurrence of atrial fibrillation after surgery; occurrence of ischemic stroke after surgery. Fisher exact test was used for statistical analysis.
Results: Ischemic stroke occurred in 22 (4.8 %) patients. Its incidence varied according to the procedure localization – ascending aorta 50.0 %, isolated valve 22.7 %, aortic dissection 18.1 %, coronary artery bypass graft 4.5 %, atrial septal defect 4.5 %. None of the observed parameters was identified as a statistically significant risk factor of ischemic stroke occurrence. However, occurrence of atrial fibrillation after surgery was identified as a risk factor with the highest difference between patients with complicating ischemic stroke versus those without ischemic stroke after surgery (63.6
% vs. 33.8 %; P = 0.07).
Conclusion: We demonstrated a relatively high incidence of ischemic stroke (4.8 %) in the set of patients undergoing cardiac surgery procedure in extracorporeal circulation. Cardioembolism, mostly due to atrial fibrillation after surgery, was identified as the most common etiology of complicating ischemic stroke.
IRON DEFICIENCY IN PATIENTS INDICATED FOR PRIMARY PREVENTIVE CARDIOVERTER DEFIBRILLATOR
MUDr. Petr Zdráhal petr.zdrahal@fnhk.cz
1st Department of Internal Medicine - Cardioangiology, Charles University - Faculty of Medicine Hradec Kralove, University Hospital Hradec Kralove
Co-authors: M. Tauchman, M. Měšťan, Z. Tušl, L. Haman, L. Chalupníková, P. Pařízek Tutor: Assoc. Prof. Petr Pařízek, MD., PhD.
Inroduction
Iron deficiency (ID) is recently intensively studied comorbidity of heart failure (HF).
According to European Society of Cardiology guidelines [1], ID should be routinely screened in chronic heart failure (CHHF) patients. The substitution therapy of ID together with conventional therapy can bring addictional profit for the patients. Iron plays an important role in redox reactions, including electron transport chain. So, iron is not only a transporter for oxygen (heamoglobin), it also plays irreplaceable role in cell's energy metabolism. The negative aspect of iron characteristics is its potential to cause oxidative stress by the production of reactive oxygen species. It is also potent bacterial grow factor.
As human organism has only limited capabilities how to dispose of redundant iron, intestinal absorption must be well regulated. Main regulator is Hepcidine, protein produced in liver.
Its production increases in response to iron levels and inflammation and decreases in response to erythropoiesis. HF is accompanied by systemic inflammatory reaction. Mechanisms described above explains the cause of ID and ineffectiveness of oral iron substitution therapy in HF patients [2]. ID in these patients is associated with decreased excercise capacity, more severe HF symptoms, poorer quality of life and it seems to be a strong, independent predictor of death and heart transplantation. Studies following effect of intravenous iron substitution therapy (CONFIRM-HF, FAIR HF) confirmed a significant improvement in excercise capacity, measured as increased walk distance in six-minute walk test and quality of life assessed by the questionnaire. This improvement was observed independently on occurrence of anemia [3,4]. There are no data about ID in primary preventive cardioverter defibrillator candidates published before.
Aims
ID and anaemia screening in HF patients indicated for primary preventive (ICD).
Methods
Serum levels of Ferritin, Transferrin, blood count and Transferrin saturation were investigated in 108 consecutive patients (88 men, 20 women, average age 69.1 ± 9.4 years, EF 10-37.5%, 54 NYHA ≤ II, 54 NYHA > II, 60 with ischemic etiology of HF, 48 with non-ischemic etiology of HF) who underwent primary preventive ICD implantation on our Department. A control group consisted of 27 consecutive patients (23 men, 4 women, average age 59.5±15.2) without history of HF who underwent secondary preventive ICD implantation.ID in CHHF patients is defined as serum Ferritin levels < 100 ug/l (absolute deficit) or serum Ferritin 100- 299 ug/l together with Transferrin saturation (TSAT) < 20% (functional deficit). In control group ID was defined as Ferritin < 21.8 ug/l. Anaemia was defined as Haemoglobin level
<125 g/l in men and < 115 g/l in women. Statistical analysis was performed by Fisher's exact test.
Results
ID was proved in 43 pts. ( 38.9%) in our primary preventive ICD group. The occurrance of ID in this group was statistically significant in comparison with conrol group (p < 0,0001).
We did not prove any relationship between ID incidence and sex (female vs. male, p = 0,621), CHHF etiology (ischemic vs. non-ischemic, p = 0,696), New York Heart Association class (NYHA ≤ II vs. NYHA ˃ II, p = 0,694) or severity of left ventricular (LV) systolic
dysfunction (EF ≤ 25 vs. EF > 25, p = 1). 13 pts. (12%) in studied group were anaemic.
Normochromic normocytic anaemia was observed in 9 pts. and 4 pts. had microcytic hypochromic anaemia. There was a higher incidence of ID among anaemic patients in
comparison to non-anaemic patients (p ˂ 0,03). ID was not associated with anaemia in 79% of ID the patients. 5 pts. died (4 pts. due to progress of HF and 1 due to sepsis soon after ICD implantation), 3 of them were diagnosed with ID.
Discussion
Incidence of ID in our population is comparable with data previously published by other authors in heart failure patients. In comparison with data published by Jankowska and co- workers [5] we did not prove a higher incidence of ID in women and in patients with
advanced NYHA class. We also did not prove the association between ID and severity of LV systolic dysfunction. We gues that it is caused by quite narrow spectrum of CHHF patients in our group, selected according to primary preventive ICD indication criteria. Thus NYHA IV patients were excluded, only 4 NYHA I patients were included and only 1 patient in our group had LV ejection fraction over 35%. In agreement with Anker, von Haeling, Jankovska et.al.
[5,6] we have found that most of the patients with ID (79%) are not anaemic. According to these authors even these patients can profit from intravenous iron substitution therapy. We do not have enough data for mortality analysis yet.
Conclusions/Summary
We are presenting first data from our study. We proved, that ID is common co-morbidity in ICD candidates. ID should not be diagnosed according to blood cout as almost 80% of ID patients do not have any changes in haemoglobin leves. Potential of iron substution therapy in these patients (quality of life, excercise tolerance, possible efect on occurrence arrhythmias) requires further investigation.
References
1. Ponikowski P, Voors AA, Anker SD et al. 2016 ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure. 2016
2. Gulec S, Anderson GJ, Collins JF. Mechanistic and regulatory aspects of intestinal iron absorbtion. Am J Physiol Gastrointest Liver Physiol 2014,307: G397-G409, 3. Anker SD, Comin-Colet J, Filippatos G et al. Rationale and design of Ferinject
assessment in patients with iron deficiency and chronic heart failure (FAIR-HF) study: a randomized, placebo-controlled study of intravenous iron supplementation in 4. Comín-Colet J,Enjuanes C, González G et al. Iron deficiency is key determinant of
health related quality of life quality of life in patients with chronic heart failure regardless of anemia status. European Heart Journal 2013, 15:1164-1172 5. Jankowska EA,von Haeling S,Anker SD et al. Iron deficiency and heart failure:
diagnostic dilammas and therapeutic perspectives. European Heart Journal 2013, 34:
816-826
6. Ebner N, von Haeling S. Iron deficiency in Heart Failure: A practical guide. Nutrients 2013, 5:3730-3739.
