Changes in heart rate variability in patients with atrial fibrillation after pulmonary vein isolation and ganglionated plexus ablation.

Pulmonary vein isolation (PVI) is the cornerstone in the treatment of patients with paroxysmal atrial fibrillation (PAF). Some research has suggested studies have shown that modification of ganglionated plexuses (GP), performed with PVI, could lead to even better outcomes. The aim of this study was to determine the effect of PVI on the autonomic system. Heart rate variability (HRV) was used as a marker of autonomic system activity. Twenty-six PAF patients underwent PVI (PVI group) and twenty patients underwent PVI plus a GP ablation (GP group). In each group, 5 min long ECG signals obtained before and after the electrophysiology EP study were analyzed. Time and frequency domain parameters were evaluated. Vagal responses during ablation were observed in 15 (58 %) patients in the PVI group and in 12 (60 %) patients in the GP group. The change in normalized power in the low frequency (LF) and in the LF/HF ratio, before and after ablation, was statistically significant in both groups (LF/HF 2.6+/-1.6 before vs. 1.4+/-1.7 after ablation in PVI group and LF/HF 3.3+/-2.6 before vs. 1.8+/-1.9 after ablation in the GP group). Relative to heart rate variability parameters, there were no differences between PVI and PVI + plus GP ablation.

Evaluation of molecular changes of distal organs after small bowel transplantation.

The ischemia and reperfusion of a jejunal graft during transplantation triggers the stress of endoplasmic reticulum thus inducing the synthesis of pro-inflammatory cytokines. Spreading of these signals stimulate immunological reactions in distal tissues, i.e. lung, liver and spleen. The aim of this study was to detect the molecular changes in liver and spleen induced by transplanted jejunal graft with one or six hours of reperfusion (group Tx1 and Tx6). Analysis of gene expression changes of inflammatory mediators (TNF-alpha, IL-10) and specific chaperones (Gadd153, Grp78) derived from endoplasmic reticulum (ER) was done and compared to control group. The qRT-PCR method was used for amplification of the specific genes. The levels of corresponding proteins were detected by Western blot with immunodetection. Protein TNF-alpha was in liver tissue significantly overexpressed in the experimental group Tx1 by 48 % (p<0.001). In the group Tx6 we found decreased levels of the same protein to the level of controls. However, the protein concentrations of TNF-alpha in spleen showed increased levels in group Tx1 by 31 % (p<0.001) but even higher levels in the group Tx6 by 115 % (p<0.001) in comparing to controls. Our data demonstrated that the spleen is more sensitive to post-transplantation inflammation than liver, with consequent stress of ER potentially inducing apoptosis and failure of basic functions of lymphoid tissue.

The immediate response of jejunal mucosa to small bowel heterotopic allotransplatation in rats.

The course of histopathological alterations within jejunal graft architecture during the initial adaptation phase in the host body was investigated. Graft tissues were compared to the intestinal tissues of the recipients. This study demonstrates: (1) renewal of intestinal epithelial lining in the graft biopsies during initial hours after transplantation is more likely caused by migration and extension of remaining epithelial cells than by their increased mitotic division. (2) Distinct decrease in histopathological injury was observed in transplanted grafts after 6h, but the morphometrical parameters, particularly villus height and wall thickness, remained altered. (3) Significant decrease in apoptotic cell death in the epithelial lining within 6h of graft recirculation was accompanied by no effect on apoptosis levels of the cells in lamina propria connective tissue. (4) Although the apoptosis level in the connective tissue cells was not modulated in the grafts within the first hour after transplantation, caspase-3 dependent apoptosis was decreased significantly.

Alterations of epithelial layer after ischemic preconditioning of small intestine in rats.

Ischemic-reperfusion (IR) injury of the small intestine makes a serious complications associated with various surgical procedures and is related to changes in motility, secretory activity and structural alterations. Preconditioning can reduce range of this damage. The aim of the experimental study was to determine the influence of ischemic preconditioning (IPC) on IR injury on jejunal epithelial layer. Wistar rats (n = 56) were divided in two experimental groups. IR group was subjected to 60 min ischemia of cranial mesenteric artery and followed by reperfusion periods: 1,4,8,24 h (IR1, IR4, IR8, IR24). Group with ischemic preconditioning (IPC+IR) was subjected to two subsequent ischemic attacks (12 min) with 10 min of reperfusion between them, and after 2nd attack ischemia was induced for 60 min followed by relevant reperfusion period. IPC showed the protective impact on the jejunal tissue architecture after 1 h reperfusion, when in IR1 group the highest and significant damage was observed (p < 0.001) in contrast to IPC+IR1 group. Histopathological damage of the intestine in pretreated groups was postponed to 4 h of reperfusion. Protective effect of IPC together with later accumulation of injury signs were confirmed by weaker impact on goblet cell (p < 0.001) and Paneth cell populations (p < 0.05).The increased cells proliferation in preconditioned groups came later, but stronger after 8 h of reperfusion (p < 0.001) and after 24 h of reperfusion still remained at the high activity level (p < 0.001). Our experimental results on the histopathological changes in the jejunum during ischemic preconditioning proved that IPC may have a positive effect on maintaining intestinal barrier function.

Progression of apoptic signaling from mesenteric ischemia-reperfusion injury to lungs: correlation in the level of ER chaperones expression.

Multiple organ dysfunction syndrome (MODS) is characterized by the development of probably reversible, progressive dysfunction of vital systems in two or more organs, directly undamaged by surgery or other trauma. The organs which have the most common potential dysfunction are lungs, liver, kidneys, heart and gastrointestinal tract. The small intestine is the source of production of proinflammatory mediators leading and contributing to multiorgan failure. The endoplasmic reticulum (ER), after ischemia and post-ischemic reperfusion, is significantly involved in the activation of enterocyte apoptosis. The purpose of this study was to determine the stage of apoptosis in the lungs, initiated through inflammatory response from the small intestine. We analyzed changes in mRNA levels of pro-apoptotic genes Gadd153 (Chop) and anti-apoptotic genes Grp78 (Bip) in the small intestine wall and lung parenchyma. During experimental procedure the rats underwent 60 min of ischemia, caused by complete occlusion of the mesenteric arteria cranialis, with subsequent reperfusion and evaluation after 1 h, 24 h and 30 days (from R1, R24 to R30, respectively, each group n = 8). The gene expression levels were measured using RT-PCR followed by electrophoresis and visualization under UV. In the lungs we detected significantly lower level of expression Grp78 by 45 ± 6.9%. This suggests that ischemic attack and subsequent reperfusion did not promote ER stress in the lungs through induction of Gadd153 expression in the small intestine. There is still no effective approach to the treatment of affected ischemic intestine tissue, to stop the processes with could eventually lead to MODS. Therefore it is necessary to study changes in the damaged tissue at the molecular level and try to suggest possible therapeutic defined routes to the protection of tissue.

NADPH-diaphorase expression in the rat jejunum after intestinal ischemia/reperfusion.

The purpose of this study was to analyze the nicotinamide adenine dinucleotide phosphate - diaphorase (NADPH-d) activity in the rat jejunum after a mesenteric ischemia/reperfusion injury. Nitric oxide, synthetised from L-arginine by the enzyme nitric oxide synthase, is a nonadrenergic noncholinergic relaxant neurotransmitter of the intestinal smooth muscle. It plays an important role in the process of plasticity after the ischemia/reperfusion injury. Experimental animals were divided in two groups: the control group and the ischemic/reperfusion group, with different period of the reperfusion. The NADPH-d histochemical method has been used as a marker for the nitric oxide synthase. NADPH-d activity has been rapidly decreased in the neurons of both enteric nervous systems in plexuses of the jejunum after 1 h mesenteric ischemia and 1 h reperfusion. Differences were predominantly detected in the myenteric plexus; they were seen in change of the neuronal shape, in the arrangement of neurons and in intensity of their staining. The NADPH-d positivity was absent in the intestinal crypts. After 1 h ischemia and 24 h reperfusion, the NADPH-d activity was gradually increased, but it was lower in comparison with the control group. On the 30th day following the ischemia/reperfusion there were no changes in NADPH-d positivity compared with the control animals. These results indicated that the jejunal ischemia/reperfusion has affected the neurons of the enteric nervous system of adult rats and resulted in the early decrease of NADPH-d positivity 1 h of the reperfusion insult. The gradual increasing of NADPH-d activity in 24 h following the reperfusion could be considered as a result of the plasticity process. On the 30(th) day after the ischemia/reperfusion all histochemical changes were returned to the control levels.

Analysis of changes in pro (Gadd153) and anti apoptotic (Grp78) gene expression after ischemic-reperfusion injury of the small intestine.

Analysis of changes after ischemia-reperfusion (IR) attack to the small intestine leads to multiple organ dysfunction (multiple organ dysfunction syndrome, MODS) and the subsequent death of patients is a topic for discussion. IR stress affects the endoplasmic reticulum (ER). ER dysfunction induces responses through kinases activation that stimulate anti-apoptotic mechanism, for example Grp78 (Bip) (Yeung et al., 2008) and pro-apoptotic mechanism, for example, activation Gadd153 (Chop) (Allyson et al., 2007). We analyzed the impact of IR damage of epithelium of the small intestine of rats after 1 h ischemia and subsequent 1 h, 24 h and 30 days of reperfusion on the level of apoptotic genes expression (Gadd153) and (Bip). In this study we used RT-PCR for detection of changes in gene expression. Significantly increased levels of mRNA for Gadd153 gene were detected after 1 h ischemia and 1 h reperfusion. The mRNA level of Grp78 gene was increased 24 h after ischemia comparing with the control groups. After 30 days of reperfusion Grp78 was at the level of control groups. Still, it is necessary to analyze the changes in the damaged tissue at the molecular level to define possible pathways leading to the tissue protection.

Adult Respiratory Distress Syndrome and alveolar epithelium apoptosis: an histopathological and immunohistochemical study.

Adult Respiratory Distress Syndrome results from a variety of different initial insults, including trauma, sepsis, pneumonia and aspiration, and represents a severe form of acute lung injury. The lung samples of a 20-year-old man who had suffered a serious motorbike accident were obtained for histological examination. He died on the seventh day as a consequence of respiratory failure. The typical histopathological features of syndrome overlapping the first exudative phase into the second proliferate phase were observed. The apoptotic index of the early apoptotic phase evaluated using M30CytoDEATH was 3.4+/-0.2. The average number of apoptotic cells in the intermediate and late phases measured using the TUNEL method was 9.8+/-0.7. Our findings indicate that alveolar epithelium apoptosis seems to be less important during the early phases of Adult Respiratory Distress Syndrome.

Effects of borneol and thymoquinone on TNBS-induced colitis in mice.

Components of plant essential oils have been reported to have health benefit properties, including antioxidative, anti-tumour, antimicrobial, anti-stress, and immunomodulative activities. We examined the anti-inflammatory effects of thymoquinone, the active ingredient in the volatile oil of Nigella sativa seeds, and borneol, the active component of Salvia officinalis essential oil, on TNBS-induced colitis in mice. Thymoquinone was added to the commercial diet at a concentration of 0.05 % and borneol at two concentrations (0.09% and 0.18%) and fed to ICR mice 5 days before induction of TNBS colitis. Seven days after TNBS administration the mice were killed and macroscopic and histological scores were evaluated. Cytokine mRNA expression in colonic tissue was assessed using quantitative realtime RT-PCR. We did not detect any significant changes in macroscopic and histological scores between experimental and control groups, but we observed a significant decrease in proinflammatory cytokine (IL-1beta and IL-6) mRNA expression in colon tissue in the 0.09% and 0.18% borneol-treated groups of mice in comparison to the control group. Surprisingly, we were not able to confirm anti-inflammatory effects of thymoquinone in TNBS colitis. In conclusion, our data show that borneol is able to significantly suppress proinflammatory cytokine mRNA expression in colonic inflammation, although no significant morphological changes are visible.

Renal activity of Akt kinase in experimental Type 1 diabetes.

Akt kinase regulates numerous cell functions including glucose metabolism, cell growth, survival, protein synthesis, and control of local hemodynamics. mTOR is one of down-stream effectors of Akt involved in the initiation of protein translation. However, renal Akt signaling in Type 1 diabetes (DM) in vivo, in particular under the conditions reflecting differences in metabolic control, has received less attention. Renal cortical activity and expression of Akt and mTOR (kinase assay, western blotting) were determined in streptozotocin-diabetic rats (D) with different levels of glycemic control (blood glucose 22.0+/-1.0, 13.4+/-1.5, 8.1+/-0.4 mmol/l, p<0.05 between the groups), achieved by varying insulin treatment (0, 4 and 12 IU/day), and in control rats with (C4) or without (C) chronic insulin administration. Renal Akt activity was reduced in D rats without insulin treatment and severe hyperglycemia (D-0, -62 %, p<0.01 vs. C), partially restored in moderately hyperglycemic rats (D-4, -30 %, p<0.05 vs. C), and normalized in D rats with intensive insulin and tight metabolic control (D-12). Expression of active mTOR paralleled Akt activity in D-0 (-51 %, p<0.01 vs. C), but not in D-4 and D-12 that demonstrated increases in active mTOR (+55 %, +80 % resp., p<0.05) as compared to C. Moreover, insulin activated renal Akt (+82 %, p<0.01), but not mTOR in C4. In conclusion, glycemic control and intensity of insulin treatment are important modulators of renal Akt and mTOR activity in diabetes. While Akt activity is reversible by tight metabolic control, combination of hyperglycemia and insulin treatment resulted in enhancement of mTOR activity. In addition to Akt, other signaling pathways likely contribute to regulation of renal mTOR activity in diabetes.

Expression of serotonin receptors in mouse oocytes and preimplantation embryos.

Serotonin receptors have been found in several reproductive organs as well as in the central nervous system. Serotonin-binding sites have been demonstrated in duck ovarian follicles and the testis, hamster ovaries, human granulosa cells and mouse placenta. Local production of serotonin by the rat ovary, oviduct, uterus and testis has also been reported. We analyzed the expression of three types of serotonin receptors: 5-HT1B, 5-HT2C and 5-HT1D by reverse transcription-polymerase chain reaction in mouse unfertilized oocytes and preimplantation embryos from zygotes to the blastocyst stage in vivo. Transcripts for 5-HT1B and 5-HT2C serotonin receptors were detected neither in unfertilized oocytes nor at any stages of in vivo developing preimplantation embryos. Serotonin 5-HT1D receptor mRNA was present in unfertilized oocytes, zygotes, 2-cell embryos, compacted morulae and in vivo produced expanded blatocysts. The expression of the mRNA 5-HT1D serotonin receptor was also detected in blastocysts cultured in vitro. When added to the culture medium, specific serotonin 5-HT1D agonist sumatriptan (1 microM) significantly inhibited the development of mouse embryos cultured in vitro. Demonstration of the expression of 5-HT1D serotonin receptor in mouse oocytes and preimplantation embryos supports the idea of a functional serotonin (5-HT1D) receptor in early mammalian development.

[Nitric oxide (NO) in a model of renal ischemia and cyclosporine toxicity]. / Oxid dusnatý (NO) u modelu ischémie ledvin a cyklosporinové toxicity.

BACKGROUND: The role of nitric oxide (NO) after the cadaveric kidney transplantation has not been fully clarified yet. The aim of our study was to examine benefits of the administration of a NO precursor--L-arginine in the model of renal ischaemia and after the subsequent cyclosporine (CsA) treatment, which simulates the state resulting from the kidney transplantation. METHODS AND RESULTS: 60 male rats of the Wistar strain were exposed to ischaemia for 45 minutes. Then they were divided into six groups: 1. Controls, 2. Rats administered by gastric sonde with 300 mg/kg of L-arginine since the first day after ischaemia, 3. Rats administered in a similar way with 10 mg/kg of cyclosporine A, 4. Group of rats receiving both drugs in the same doses, 5. Rats receiving 10 mg/kg of cyclosporine A since the first day after ischaemia and L-arginine in the dose 300 mg/kg since the seventh day, 6. Group of animals administered with L-arginine and cyclosporin A in the same doses with nonselective blocker of NO synthesis--L-NNA in the dose of 5 mg/kg. We examined renal functions (blood and urine levels of creatinine, urea, Na, K, Cl, osmolality, proteinuria), blood and urine levels of NO metabolites (NO2- and NO3-) in the fourth week after ischaemia. We found that L-arginine administration (when groups 1 and 2 were compared) decreased S-creatinine (< 0.05), it increased U-osmolality (p < 0.01), tubular resorption (p < 0.001), and blood levels of NO metabolites (p < 0.05). Changes in urine levels of NO metabolites (U-NOx/U-Cr) and in proteinuria were not found. In animals with renal ischaemia treated with cyclosporine (comparison of groups 3 and 4), L-arginine administration brought about a decrease of blood creatinine levels (p < 0.05), higher creatinine clearance (p < 0.05) and higher blood levels of NO metabolites (S-NOx; p < 0.01). However, differences in tubular functions, proteinuria and U-NOx/U-Cr were not detected. When L-arginine was added 7 days after the beginning of cyclosporine treatment, no significant difference was found between groups 5 and 3 and differences between groups 5 and 4 were similar to those between groups 4 and 3. Animals of the group 6 were not tested due to high mortality indicating high toxicity of the combination ischaemia + cyclosporine + L-arginine + L-NNA. CONCLUSION: Our study shows the benefits of L-arginine treatment in the renal ischaemia and during cyclosporine administration. The treatment should start immediately after the ischaemic period and at the same time as the cyclosporine administration. L-arginine added later has no positive effect.

Electrochemical reduction of chloridazon at mercury electrodes, and its analytical application.

The electrochemical behavior of the herbicide chloridazon, I (pyrazon), at different pH is described. The electrode reaction (one wave in acidic media and another in alkaline media), investigated using direct current and pulse voltammetry, controlled-potential coulometry, and HPLC-MS, is a combination of the electroreduction (two-electron in the first step) and a kinetic process as a result of which simple compounds (HCl, NH3) are released and, moreover, a five-membered pyrrole cycle is formed in strongly acid media. Products of the kinetic reaction are further reducible. The dissociation constant of I, pKa = 2.96, was found spectrophotometrically. Fast-scan differential pulse voltammetry (FSDPV) was used for determination of I; the detection limit was 2.7 x 10(-8) mol L(-1) (0.006 microg L(-1)) at pH 2.3. Chloridazon was determined in spiked drinking and river water.

Cellular and subcellular localization of stathmin during oocyte and preimplantation embryo development.

Stathmin is a 19 kDa cytosolic phosphoprotein, proposed to act as a relay integrating diverse intracellular signaling pathways involved in regulation of cell proliferation, differentiation, and function. To gain further information about its significance during early development, we analyzed stathmin expression and subcellular localization in mouse oocytes and preimplantation embryos. RT-PCR analysis revealed a low expression of stathmin mRNA in unfertilized oocytes and a higher expression at the blastocyst stage. A fine cytoplasmic punctuate fluorescent immunoreactive stathmin pattern was detected in the oocyte, while it evolved toward an increasingly speckled pattern in the two-cell and later four- to eight-cell embryo, with even larger speckles at the morula stage. In blastocysts, stathmin immunoreactivity was fine and intense in inner cell mass cells, whereas it was low and variable in trophectodermal cells. Electron microscopic analysis allowed visualization with more detail of two types of stathmin immunolocalization: small clusters in the cytoplasm of oocytes and blastocyst cells, together with loosely arranged clusters around the outer membrane of cytoplasmic vesicles, corresponding to the immunofluorescent speckles in embryos until the morula stage. In conclusion, it appears from our results that maternal stathmin is accumulated in the oocyte and is relocalized within the oocyte and early preimplantation embryonic cell cytoplasm to interact with specific cytoplasmic membrane formations. Probably newly synthesized, embryonic stathmin is expressed in the blastocyst, where it is localized more uniformly in the cytoplasm mostly of inner cell mass (ICM) cells. These expression and localization patterns are probably related to the particular roles of stathmin at the successive steps of oocyte maturation and early embryonic development. They further support the proposed physiologic importance of stathmin in essential biologic regulation.

Nephrotoxicity of cyclosporin A in hereditary hypertriglyceridemic rats.

It has been suggested that cyclosporin A (CsA) nephrotoxicity can be reduced by the concomitant administration of omega-3 fatty acids or vitamin E. The present study was designed to establish whether the effect of the above substances can also be demonstrated in rats with hereditary hypertriglyceridemia (HTG) whose sensitivity to the nephrotoxic effect is greater than in control AVN rats. CsA administration at a dose of 10 mg/kg/day to HTG rats resulted in a significant rise (p<0.001) in serum levels of creatinine (from 66.0+/-7.6 to 108.4+/-11.6 micromol/l) and urea (from 8.3+/-0.7 to 22.3+/-18 mmol/l) which was not found in AVN rats. The baseline values of systolic blood pressure (SBP) were significantly higher in HTG rats. However, in both strains CsA administration was associated with a similar SBP increase which was not prevented by omega-3 fatty acids (EPAX) or vitamin E administration. Concomitant administration of CsA with EPAX at a dose of 600 mg/kg b.w./day in HTG rats prevented the rise in the serum levels of creatinine (65.4+/-14.7 micromol/l) and reduced the increase in the serum urea levels (11.9+/-7.6 mmol/l). Concomitant administration of CsA and vitamin E (at a dose of 25 mg/kg/day) also reduced the increase (p<0.05) in the serum levels of creatinine (70.7+/-14.3 micromol/l) and urea (9.8+/-3.4 mmol/l) compared to the effects elicited by the administration of CsA alone (p<0.05). Administration of CsA alone or in combination with EPAX or vitamin E did not have a marked effect on diuresis, proteinuria, urinary osmolality, urinary excretion of urea, creatinine and potassium. Under all experimental conditions, the rate of urinary excretion of sodium in HTG rats was significantly lower (p<0.01) than in AVN rats. The results obtained support the assumption that omega-3 fatty acids and vitamin E at the doses used reduce CsA nephrotoxicity in rats with hereditary hypertriglyceridemia whose sensitivity to the nephrotoxic effect of CsA is significantly higher than in AVN rats.

A modified method of intracytoplasmic sperm injection without the use of polyvinylpyrrolidone.

In a controlled study we compared the outcome of intracytoplasmic sperm injection (ICSI) performed by two different methods. The oocytes from 20 patients were equally divided into two groups and injected either by conventional ICSI using polyvinylpyrrolidone (PVP) or by a modified PVP-free ICSI procedure. While in the conventional ICSI method the spermatozoon is aspirated into the injection pipette, in the modified ICSI procedure the spermatozoon is attached to the end of the narrow micropipette by aspirating its tail. The sperm head is never drawn into the pipette. Accordingly, even a fast-moving spermatozoon can be 'caught' easily. As a result of such an aspiration the spermatozoon loses its motility. Therefore, PVP is required neither to slow down the movement of the spermatozoon nor to facilitate the movement of the solution in the injection pipette. A total of 230 mature oocytes were injected by both methods and the results were analysed. No differences were observed in survival rate between the two ICSI procedures (89% and 91%, respectively). However, the proportion of normally fertilized oocytes was significantly higher after microfertilization by modified ICSI (74%) when compared with the outcome of the conventional ICSI method (62%). The frequency of abnormal fertilization was not influenced by the method of ICSI used. The cleavage rate and quality of resulting embryos were also comparable. In conclusion, we have demonstrated a modified ICSI method which does not require the use of PVP. When compared with the conventional ICSI procedure, even better fertilization rates can be achieved. The proposed ICSI modification may provide an alternative procedure for elimination of the potentially harmful effects which may be associated with conventional ICSI.

Preimplantation embryo development in ICR mice after streptozotocin treatment.

To investigate the significance of impaired insulin secretion on preimplantation embryo development, outbred ICR female mice received a single injection of streptozotocin 130 mg (low) and 160 mg (subdiabetic) kg(-1), 14-17 days before fertilization. Preimplantation embryos were collected on day 3 of pregnancy, four to eight-cell embryos were cultured in vitro 48 h (day 5) and their cell number was estimated. After spontaneous ovulation, the significantly different distribution pattern in comparison with the controls was detected only in preimplantation embryos isolated from subdiabetic (160 mg x kg(-1) streptozotocin) mice. Furthermore, the incidence of degenerated embryos was significantly increased after 48 h in vitro cultivation. The analysis of cell number distribution in embryos after cultivation in vitro indicated a significant delay in cell proliferation in both experimental groups (130 and 160 mg x kg(-1) streptozotocin) in comparison with control mice. After superovulation, the only significant difference was found in the distribution pattern of embryos isolated on day 3 of pregnancy from subdiabetic (160 mg x kg(-1) streptozotocin) mice. No significant differences were found after embryo cultivation in vitro. It could be concluded that, in outbred ICR mice, lower streptozotocin treatment (130 mg x kg(-1)) influenced only cell distribution of in vitro cultured embryos after spontaneous ovulation. In ICR mice, marked changes in preimplantation embryo development were detected only after subdiabetic (160 mg x kg(-1)) streptozotocin treatment. During in vitro cultivation delayed effects of impaired insulin secretion resulted in an increase of embryo degeneration at the time after the third mitotic cleavage. Our results indicate that the effects of impaired maternal insulin secretion on preimplantation embryo development in mice are marked and consistent after spontaneous ovulation. Superovulation apparently disguises subtle changes in preimplantation embryo development after low and subdiabetic streptozotocin treatment.

Increased cell death in rat blastocysts exposed to maternal diabetes in utero and to high glucose or tumor necrosis factor-alpha in vitro.

The morphogenetic function of the transient phase of cell death that occurs during blastocyst maturation is not known but it is thought that its regulation results from a delicate balance between survival and lethal signals in the uterine milieu. In this paper, we show that blastocysts from diabetic rats have a higher incidence of dead cells than control embryos. Differential lineage staining indicated that increased nuclear fragmentation occurred mainly in the inner cell mass. In addition, terminal transferase-mediated dUTP nick end labeling (TUNEL) demonstrated an increase in the incidence of non-fragmented DNA-damaged nuclei in these blastocysts. Analysis of the expression of clusterin, a gene associated with apoptosis, by quantitative reverse transcription-polymerase chain reaction detected an increase in the steady-state level of its transcripts in blastocysts from diabetic rats. In situ hybridization revealed that about half the cells identified as expressing clusterin mRNA exhibited signs of nuclear fragmentation. In vitro experiments demonstrated that high D-glucose increased nuclear fragmentation, TUNEL labeling and clusterin transcription. Tumor necrosis factor-alpha (TNF-alpha), a cytokine whose synthesis is up-regulated in the diabetic uterus, did not induce nuclear fragmentation nor clusterin expression but increased the incidence of TUNEL-positive nuclei. The data suggest that excessive cell death in the blastocyst, most probably resulting from the overstimulation of a basal suicidal program by such inducers as glucose and TNF-alpha, may be a contributing factor of the early embryopathy associated with maternal diabetes.

Prevalence of psychopathology in patients suffering from breast and gastrointestinal cancer.

Prevalence of psychopathology in 107 in- and outpatients suffering from cancer was assessed by means of self-rating inventory (Symptom Check List (SCL-90)) and an interview. There were 86 women with breast cancer and 21 patients with gastrointestinal cancer (9 women and 12 men). Inventory was administered once after surgery. Psychopathology exceeding so called mean borderline values derived from control samples ranged from 2% to 33% of the patients in particular dimensions of the inventory. Maximal mean values of the psychopathology were found in the dimensions of somatisation, depression, anxiety, phobic anxiety, hostility and of items not included, reflecting mostly sleep and eating distortions. Mean values of the psychopathology were insignificantly lower in patients after mastectomy, compared with gastrointestinal cancer patients. No significant differences between initial (I and II) and advanced (III and IV) stages of the illness were achieved in the patients from both investigated samples. Screened patients are suitable for psychiatric or psychologic therapeutic intervention.

Effects of impaired maternal insulin secretion on preimplantation embryo development in ICR mice.

To investigate the significance of impaired insulin secretion on preimplantation embryo development, outbred ICR female mice received an injection of a single dose of streptozotocin 200 mg.kg-1 14-17 days before fertilization. Oocytes were collected 24-26 h after hCG injection. Morphological evaluation revealed a lower percentage of oocytes with second polar bodies from streptozotocin-treated females in comparison with controls. Furthermore, in this group the incidence of degenerated embryos significantly increased after 120 h in vitro cultivation. Insulin (5 U per 100 g b.w.) administered twice daily to streptozotocin-treated mice significantly improved the Embryonic development. Morphological analysis of oocyte maturation in streptozotocin-treated mice showed no significant differences in comparison with control mice. It could be concluded that marked changes in preimplantation embryo development were detected in outbred ICR mice after streptozotocin administration and this process was partly reversible by insulin treatment. Furthermore, it was shown that the process of fertilization was negatively influenced and that during in vitro cultivation the delayed effects of impaired insulin secretion resulted in an increase of embryo degeneration at the time following the third mitotic cleavage.