The changes in tissue histomorphology and quality of DNA in healthy human autopsied tissues stored at -20 °C and -150 °C.

The aim of this study was to compare the changes in tissue histomorphology and DNA quality in six different healthy tissues (brain, heart, lung, liver, spleen and kidney) exempted during autopsy of healthy individuals and storage at -20 °C and -150 °C three month. Tissue samples were obtained, divided by tissue and temperature group, and for each sample, tissue histomorphology and DNA (isolated from all tissues in duplicated - 72 samples of DNA) quality were analysed. Morphology of tissue samples was studied using H&E staining. DNA was isolated using the phenol-chloroform-isoamyl alcohol method. To assess the concentration and purity of the DNA samples, we used a spectrophotometer to measure absorbance at wavelengths of 280 nm and 260 nm. The fragments of human telomerase reverse transcriptase (hTERT) gene were amplified from the DNA using PCR reaction and then visualised using the 2 % agarose gel. Samples stored at -150 °C sustained the highest degree of histomorphological damage, while samples stored at -20 °C were less degraded, compared to control. The liver samples stored at -20 °C had a mean DNA concentration (1030.4 ± 51.5 ng/µl) higher than the samples of liver tissue stored at -150 °C (497.4 ± 167.1 ng/µl) (p < 0.001). Other tissues did not have statistically significantly different DNA concentration at both temperatures. Liver samples at -20 °C had degraded DNA, showed as the absence of hTERT gene in most of samples. Other tissue samples in both temperature groups had unfragmented DNA. Storing tissue samples at -20 °C is not inferior in terms of DNA yield and integrity, and possibly superior for tissue histomorphology, comparing with samples stored at -150°C.

DNA isolated from formalin-fixed paraffin-embedded healthy tissue after 30 years of storage can be used for forensic studies.

Tissue formalin fixation and paraffin embedding (FFPE) is a standard method for long-term preservation and morphological and molecular analysis. The aim of this study was to analyze the effect of storage time on the integrity of DNA isolated from three different healthy FFPE tissues. DNA was isolated from FFPE heart, liver and brain tissues obtained from autopsy and archived from 1988 to 2017 using two different methods of DNA isolation: phenol-chloroform-isoamyl alcohol (PCI) and PureLink Genomic DNA Kit. The quantification and purity of DNA was measured spectrophotometrically at 260 nm and 280 nm. The quality of isolated DNA was evaluated by PCR amplification of GPD1 (150 bp), ACTB (262 bp) and RPL4 (407 bp) genes. The histomorphological characteristics of FFPE tissues were not significantly changed during 30 years of storage. Higher yield (272.9 ± 10.3 µg) and purity (A260/280 = 2.05) of DNA was obtained using the PCI method for DNA isolation from FFPE liver tissue. The PCI extraction method showed reproducible and consistent results in PCR amplification of all of three examined genes. The GPD1 gene can be amplified up to 30 years, the ACTB gene in the same samples up to 26 years and the RPL4 gene up to 6 years of storage in FFPE blocks. Although the best yield and purity of isolated DNA (using both isolation methods) was obtained from FFPE liver tissue, the DNA with the most preserved integrity was obtained from brain tissue archived up to 30 years. This is the first report using long-term archived healthy FFPE tissues (up to 30 years) that shows that the DNA isolated from these tissues is of preserved integrity and can be used in molecular autopsy.

The Effects of Thiamine Hydrochloride on Cardiac Function, Redox Status and Morphometric Alterations in Doxorubicin-Treated Rats.

Previous studies have suggested that thiamine has antioxidant activity and could decrease the production of ROS in various disorders. Our study focused on the effect of thiamine hydrochloride in the reversal of DOX-induced cardiotoxicity and compared it with the reversal in the absence of thiamine pre-treatment. Rats were divided into groups as follows: (a) thiamine + doxorubicin (TIA + DOX), (b) doxorubicin (DOX) and c) healthy (CTRL) groups. For 7 days, thiamine hydrochloride was administered at a dose of 25 mg/kg per day intraperitoneally, while a single dose of 15 mg/kg doxorubicin was injected into all groups except the CTRL group. We measured the following parameters: maximum rate of left ventricular development (dp/dt max), minimum rate of left ventricular development (dp/dt min), systolic left ventricular development (SLVP), diastolic left ventricular development (DLVP), heart rate (HR) and coronary flow (CF), pro-oxidative and antioxidative markers, cardiac activity, and histopathological evaluation. In our study, cardiac contractility was significantly altered after DOX treatment and diminished by thiamine pre-treatment. Additionally, pro-oxidant parameters were significantly increased in the DOX group. The levels of O2-, H2O2 and TBARS were significantly increased in the DOX group and decreased in the DOX + T group compared to those in the DOX group. Morphometric analyses showed moderately expressed interstitial fibrosis and degenerately modified cardiac muscle fibres, with signs of interfibrillary congestion, vacuolar degeneration and myocytolysis in the DOX group as visualized by H&E and Masson's Trichrome staining. Pre-treatment of thiamine hydrochloride before doxorubicin administration could decrease oxidative stress production, increase myocardial contractility and enhance the antioxidant defence system.

Cardioprotective effects of Galium verum L. extract against myocardial ischemia-reperfusion injury.

The aim of our study was to determine a chemical composition of methanol extract of Galium verum as well as to assess its effects on functional recovery and redox status of isolated rat heart after ischemia. Rats were divided into control and G. verum group, which included animals treated with 500 mg/kg of methanol extract of G. verum for 28 days. Parameters of heart function and oxidative stress markers were estimated. Cell morphology was evaluated by hematoxylin and eosin (HE) staining. Our results demonstrated for the first time that G. verum extract preserved cardiac contractility, systolic, and diastolic function as wells as structural damage of the heart after ischemia. Furthermore, G. verum extract modulated the activity of antioxidant enzymes and alleviated the production of pro-oxidants.

The influence of folic acid-induced acute kidney injury on cardiac function and redox status in rats.

The aim of this study was to evaluate the influence of acute kidney injury caused by high doses of folic acid on cardiac function and markers of oxidative stress in serum and isolated rat heart. Isolated hearts of Wistar albino rats (divided into two groups: control and folic acid group) were perfused according to Langendorff technique at basal coronary perfusion pressure CPP of 70 cm H2O. After a stabilization period, CPP was lowered to 60 cm H2O and then gradually increased to 80, 100, 120 and finally decreased to 40 cm H2O in order to establish coronary autoregulation. For each perfusion pressure value, the left ventricular function parameters were determined. Samples of coronary venous effluent were collected for determination of coronary flow and biomarkers of oxidative stress. The blood samples were collected in order to examine the values of serum urea, creatinine, Na, K, and parameters of oxidative stress and antioxidant defense system. Heart and kidney tissue samples were collected for histopathological examination. Folic acid group showed reduction of systolic and diastolic left ventricular pressure and increase of coronary flow and minimum left ventricular pressure development rate. In coronary flow, folic acid group showed increased levels of TBARS and reduction of H2O2 and NO2-. Serum ROS concentrations were lower in rats treated with folic acid, particularly levels of TBARS and NO2- in which values were significantly lower. The parameters of systemic antioxidative stress were at significantly high levels especially SOD and GSH. This study is the experimental confirmation of cardio-renal syndrome type 3, which represents the acute kidney injury that causes a damage of a heart function. The data suggest that negative effects of acute kidney injury on myocardium do not necessarily involve oxidative stress, which may lead to future investigations which will be based on inflammation as a one of the important factors in the organ crosstalk.

Clinical significance of neoangiogenesis and index of proliferation in the signet ring type of gastric adenocarcinoma.

PURPOSE: The purpose of this study was to examine whether microvascular density and the level of proliferation in gastric signet ring cell carcinoma (SRCC) are important factors in the locoregional control of the disease. METHODS: Over a period of eight years, gastric resection specimens from 37 patients were examined. The proliferative index (labelled by Ki67) and microvascular density (MVD) index (mvdIDX) (labelled by CD105) were determined for each case of SRCC. RESULTS: Gastric SRCC was diagnosed more often in female than in male patients (21 females, 16 males ; p≤0.05) . The average age of female patients was 63 years, while the male patients were 62 years old on average (p=0.702). Immunohistochemical analysis showed that the median numbers of Ki67 positive cells and CD105 positive blood vessels were higher in tumors compared to surrounding non-tumor tissue. Higher proliferative index and higher mvdIDX were also established relative to tumor stage. Correlation analysis showed a high positive correlation between proliferation index and microvascular density (MVD) index (mvdIDX) (correlation coefficient=0.784). Receiver operating characteristics (ROC) analysis showed progression of both indices examined. CONCLUSION: Our results showed that, although both proliferative and mvdIDXs are reliable, the former had better performance in identifying of disease progression (AUC=0.970).

Effect of formalin fixation on pcr amplification of DNA isolated from healthy autopsy tissues.

The aim of this study is to investigate the effects of formalin fixation on the degradation of DNA molecules in five different healthy tissues exempted during the autopsy, as well as the selection of the method that is most suitable for the DNA isolation. Heart muscle, liver, brain, lung and kidney tissue obtained from the healthy people who suddenly died from a violent death were used. The parts of tissue were fixed in 10% phosphate-buffered formalin as well as in 4% unbuffered formalin at room temperature. Morphology of tissue was studied using H&E staining. The DNA was isolated 6 h, 1-7 days (every 24 h), 10, 14, 28 days and 2 months after fixation using two different methods: extraction with phenol-chloroform-isoamyl alcohol as well as with PureLink Genomic DNA Kit. Yield and purity of the DNA samples were measured spectrophotometrically at 260 nm and 280 nm. The PCR amplifications of the glycerol-3-phosphate dehydrogenase 1 (GPD1, 150 bp), ß actin (ACTB, 262 bp) and ribosomal protein L4 (RPL4, 407 bp) genes were performed to evaluate the degree of DNA fragmentation. The RPL4 gene was amplified up to 72 h, ACTB gene up to 14 days and GPD1 gene up to 28 days from tissue fixed in phosphate-buffered formalin using phenol-chloroform-isoamylalcohol protocol for DNA isolation. Liver and kidney gave better results of PCR amplification, but statistical significance between tissues was not found. Preserving period, fixative and DNA extracting method are important factors for successful PCR amplification. The healthy tissue, fixed in phosphate-formalin up to 28 days, can be useful source in molecular studies. Tissues fixed in unbuffered formalin are suitable for molecular analysis up to 7 days.

Comparison of cytological categories atypical (C3) and suspected (C4) with histopathological diagnoses of breast lesions.

PURPOSE: Fine needle aspiration cytology (FNAC) is a diagnostic method characterized by high sensitivity, specificity and predictive value. In order to obtain uniformed results of FNAC breast changes, the following categories are introduced: C1 (non-representative), C2 (benign), C3 (atypical), C4 (suspected) and C5 (malignant). The purpose of this study was to establish which pathological processes are most frequently diagnosed as C3 and C4 categories, which carry a malignant tumor risk. METHODS: The frequency of all cytological categories was determined in a retrospective analysis which included 1605 patients, all of whom had undergone FNAC of breast lesions, over a period of 5 years (2012-2016). Furthermore, histopathological diagnoses of 212 patients with cytological categories C3 (77) or C4 (135) were compared. RESULTS: In the sample of 1605 patients, 212 belonged to C3 or C4 cytological category ( frequency for C3 4.8%, for C4 8.4%). Also, in the group of patients with cytological categories C3 and C4 there were 208 women. The patients with C3 were younger than C4 patients. There was a statistically significant difference between the number of benign and malignant diagnoses in patients diagnosed with C3 or C4 cytological category (p<0.001). In C3 category, in 57.1% of the cases a benign condition was histopathologically diagnosed, while in C4 category, in 90.4% of the cases malignant tumor was histopathologically diagnosed. CONCLUSIONS: After histopathological analysis, C3 category in FNAC breast lesions is most commonly diagnosed as a fibrocystic breast disease or fibroadenoma, while C4 category is diagnosed as well-differentiated malignant tumor.

Vitamin B complex mitigates cardiac dysfunction in high-methionine diet-induced hyperhomocysteinemia.

This research is designed to test the hypothesis that elevated homocysteine (Hcy) levels in vivo, caused by a deficit in vitamin B complex, promote changes in cardiac function and redox status that lead to heart failure. In order to conduct the study, we used adult male Wistar albino rats (n = 30; 4 weeks old; 100 ± 15 g body weight). Hyperhomocysteinaemia (HHcy) in these animals was achieved by dietary manipulation. For 4 weeks, the animals were fed with a standard rodent chow (control, CF), a diet enriched in methionine with no deficiency in B vitamins (i.e., folic acid, B6 and B12) (HMNV) or a diet enriched in methionine and deficient in B vitamins (HMLV). After 28 days of dietary manipulation, all animals were killed. The rat hearts were isolated and retrogradely perfused according to the Langendorff technique at a gradually increasing perfusion pressure. We found a negative correlation between elevated serum Hcy and total body and heart weight. The maximum rate of left ventricular pressure development was significantly increased in the HMNV group compared with in the other groups. Systolic left ventricular pressure was significantly changed in all groups. HHcy induces remodelling of the cardiac tissues, as moderate HHcy is associated with more prominent interstitial and perivascular fibrosis. Our results suggest that a high methionine diet without vitamin B complex causes profound negative effects associated with HHcy.

Clinical significance of microvessel density and proliferation in prostate cancer core biopsy.

PURPOSE: To investigate the microvessel density (MVD) and proliferation in prostate cancer (PC) core biopsies. METHODS: Core biopsy samples of PC tissue from 45 patients were routinely processed and embedded in paraffin. The samples of PC formed the investigated group (n=25), while samples of benign prostatic hyperplasia (BPH) served as controls (n=20). From paraffin blocks, 3-5 µm-thick sections were made and routine hematoxylin-eosin method and immunohistochemical ABC method with Ki67 and CD34 antibodies were applied. Immunohistochemical expression of Ki67 and CD34 was stereometrically quantified. RESULTS: The median number of Ki67 and CD34 positive cells per mm2 in PC were significantly higher in comparison to the median of these cells in BHP. The average age and Gleason score in patients with high proliferation index (proIDX) and MVD index (mvdIDX) was significantly greater in comparison to those with low proIDX and low mvdIDX. The absolute values of Ki67 expression were in highly positive and significant correlation with the absolute values of CD34 expression. Highly significant correlation was found between Gleason score and proIDX and mvdIDX. CONCLUSION: This study showed that PC expressed significantly higher values of Ki67 and CD34 in comparison to BPH. The values of proIDX and mvdIDX obtained by core biopsy could clearly show the level of cancer progression expressed through highly correlated Gleason score. In this way it is possible to identify the patients at high risk for disease progression.