Classic preconditioning decreases the harmful accumulation of nitric oxide during ischemia and reperfusion in rat hearts.

BACKGROUND: The role of NO in the mechanism of preconditioning is not understood. Therefore, we studied the effect of preconditioning and subsequent ischemia/reperfusion on myocardial NO content in the presence of an NO synthase (NOS) inhibitor. METHODS AND RESULTS: Isolated working rat hearts were subjected to preconditioning protocols of 3 intermittent periods of rapid pacing or no-flow ischemia of 5 minutes' duration each followed by a test 30 minutes of global no-flow ischemia and 15 minutes of reperfusion. Test ischemia/reperfusion resulted in a deterioration of myocardial function and a considerable increase in cardiac NO content as assessed by electron spin resonance. Preconditioning improved postischemic myocardial function and markedly decreased test ischemia/reperfusion-induced NO accumulation. In the presence of 4.6 micromol/L N(G)-nitro-L-arginine (LNA), basal cardiac NO content decreased significantly, although test ischemia/reperfusion-induced functional deterioration and NO accumulation were not affected in nonpreconditioned hearts. However, the protective effects of preconditioning on both test ischemia/reperfusion-induced functional depression and NO accumulation were abolished. When 4.6 micromol/L LNA was administered after preconditioning, it failed to block the effect of preconditioning. In the presence of 46 micromol/L LNA, ischemia/reperfusion-induced NO accumulation was significantly decreased and postischemic myocardial function was improved in nonpreconditioned hearts. CONCLUSIONS: Our results show that (1) although NO synthesis by the heart is necessary to trigger classic preconditioning, preconditioning in turn attenuates the accumulation of NO during ischemia/reperfusion, and (2) blockade of ischemia/reperfusion-induced accumulation of cardiac NO by preconditioning or by an appropriate concentration of NOS inhibitor alleviates ischemia/reperfusion injury as demonstrated by enhanced postischemic function.

Measurement of NO in biological samples.

Although the physiological regulatory function of the gasotransmitter NO (a diatomic free radical) was discovered decades ago, NO is still in the frontline research in biomedicine. NO has been implicated in a variety of physiological and pathological processes; therefore, pharmacological modulation of NO levels in various tissues may have significant therapeutic value. NO is generated by NOS in most of cell types and by non-enzymatic reactions. Measurement of NO is technically difficult due to its rapid chemical reactions with a wide range of molecules, such as, for example, free radicals, metals, thiols, etc. Therefore, there are still several contradictory findings on the role of NO in different biological processes. In this review, we briefly discuss the major techniques suitable for measurement of NO (electron paramagnetic resonance, electrochemistry, fluorometry) and its derivatives in biological samples (nitrite/nitrate, NOS, cGMP, nitrosothiols) and discuss the advantages and disadvantages of each method. We conclude that to obtain a meaningful insight into the role of NO and NO modulator compounds in physiological or pathological processes, concomitant assessment of NO synthesis, NO content, as well as molecular targets and reaction products of NO is recommended.

Heme oxygenase and cardiac function in ischemic/reperfused rat hearts.

We investigated whether the expression of heme oxygenase (HO) isozymes was related to the occurrence of ventricular fibrillation (VF) induced by ischemia/reperfusion in nondiabetic and diabetic myocardium. To study the role of HO-1 and HO-2 mRNA expression in VF, isolated hearts obtained from nondiabetic and 8-week diabetic rats were subjected to 30 min of ischemia followed by 2 h of reperfusion. Expression of HO-1 and HO-2 mRNA was studied in fibrillated and nonfibrillated myocardium using Northern blotting and reverse transcription polymerase chain reaction (RT-PCR). The effect of zinc protoporphyrin IX (Zn-PPIX), a potent inhibitor of HO activity, on HO activity was also studied in ischemic/reperfused hearts. Upon reperfusion, an expression of HO-1 was observed in nonfibrillated myocardium. HO-1 mRNA expression was significantly reduced in hearts showed VF. Zn-PPIX (5 microM) treatment reduced HO activity from its control values of 398+/-27 (in nondiabetics) and 370+/-20 pmol bilirubin/h (in diabetics) to 69+/-14 (in nondiabetics, p<.05) and 60+/-11 pmol bilirubin/h (in diabetics, p<.05), respectively, and all hearts, upon reperfusion, showed VF in both nondiabetic and diabetic subjects. HO-2 expression was unchanged in nonfibrillated and fibrillated myocardium. Postischemic function showed no correlation with the expression of these genes. Our data show that the mechanism(s) of ischemia/reperfusion-induced VF involves the downregulation of HO-1 mRNA and a reduction in HO activity. Furthermore, the mechanism(s) of VF at molecular level involving HO isozymes does not show a significant difference between nondiabetics and diabetics.

Effects of oxidative stress on the expression of antioxidative defense enzymes in spontaneously hypertensive rat hearts.

Little is known concerning the effect of oxidative stress on the expression of antioxidative enzymes in the decompensated cardiac hypertrophy of spontaneously hypertensive rats (SHR), considered as a model of dilative cardiomyopathy in man. Superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx) were characterized in isolated perfused hearts of 18 month old SHR and the age-matched normotensive control Wistar-Kyoto (WKY) rats, before and after 30 min infusion of 25 microM H(2)O(2). After infusion of H(2)O(2), aortic flow decreased in WKY from 26.2 +/- 2.2 to 16.0 +/- 0.8 ml/min (p <.05) but not in SHR (18.2 +/- 1.9 vs. 20.7 +/- 2.2 ml/min). This protection was related to the higher myocardial activities of GPx, MnSOD and CuZnSOD in SHR, compared with those of the WKY group. Although total SOD activity in the SHR fell after H(2)O(2) exposure (to 1.81 +/- 0.13 from 3.56 +/- 0.49 U/mg of protein), catalase activity increased (to 2.46 +/- 0.34 from 1.56 +/- 0.29 k min(-1)mg(-1)protein), compared with the pre-infusion period (p <.05 in each case). In additional studies, hearts were subjected to 30 min of global ischemia followed by 30 min of reperfusion. The results obtained in ischemic/reperfused hearts show the same changes in enzyme activities measured as it was observed in H(2)O(2) perfused hearts, indicating that oxidative stress is independent of the way it was induced. The higher catalase activity derived from elevated mRNA synthesis. The antioxidative system in dilative cardiomyopathic hearts of SHR is induced, probably due to episodes of oxidative stress, during the process of decompensation. This conditioning of the antioxidative potential may help overcome acute stress situations caused by reactive oxygen species in the failing myocardium.

Regulation of ventricular fibrillation by heme oxygenase in ischemic/reperfused hearts.

We have assessed the relationship between reperfusion-induced ventricular fibrillation (VF) and heme oxygenase (HO) mRNA expression using northern blotting, reverse transcription-polymerase chain reaction (RT-PCR), and enzyme activity in isolated working ischemic/reperfused rat hearts. Isolated hearts were subjected to 30 min of global ischemia followed by 120 min of reperfusion. Upon reperfusion with VF, cardiac function was registered (n = 6 in each group), and HO mRNAs and enzyme activities were measured at the end of reperfusion in hearts that showed VF or did not develop VF. The expression of HO-1 mRNA (about fourfold) was observed in ischemic/reperfused nonfibrillated myocardium in comparison with the nonischemic control hearts. In those hearts when VF was developed, the expression of HO-1 mRNA was not observed in comparison with the nonischemic control myocardium. The results measured by RT-PCR and enzyme analysis support the data obtained by northern blotting. In additional studies, we decided to approach the question from a different angle. Thus, the purpose of our work was also to study the role of HO expression and enzyme activity in electrically fibrillated hearts without the ischemic/reperfused protocol. To simulate the period of 10 min of reperfusion-induced VF, hearts were electrically fibrillated, then defibrillated, and perfused for an additional 110 min, and HO-1 mRNA expression and enzyme activities were determined. Thus, electrically induced VF resulted in about 60%, 60%, and 70% reduction in HO-1 mRNA expression, RT-PCR signal intensity, and enzyme activity, respectively, compared with the nonfibrillated ischemic/reperfused group. In conclusion, our data provide evidence that the development of reperfusion-induced VF inhibits HO-1 mRNA expression and enzyme activity in both electrically fibrillated myocardium and ischemic/reperfused fibrillated hearts. The results clearly show that HO-1 mRNA expression and enzyme activity were increased in ischemic/reperfused nonfibrillated myocardium, suggesting that interventions that are able to increase HO-1 mRNA expression and enzyme activity may prevent the development of VF.

Nitroglycerin-induced direct protection of the ischaemic myocardium in isolated working hearts of rats with vascular tolerance to nitroglycerin.

We investigated whether nitroglycerin (NTG) was able to produce an anti-ischaemic effect in isolated working hearts of rats with vascular tolerance to NTG. Hearts isolated from tolerant and non-tolerant rats were subjected to 10 min coronary occlusion in the presence of 10(-7) M NTG and/or its solvent. NTG alleviated ischaemia-induced deterioration of cardiac function and decreased lactate dehydrogenase release whilst having no effect on coronary flow nor the area of the ischaemic zone both in hearts isolated from NTG-tolerant and non-tolerant rats. The magnitude of the effect was similar in the two groups. These results suggest that the anti-ischaemic effect of NTG involves direct myocardial mechanisms independent of its vascular action and that vascular tolerance to NTG does not affect this direct protective action.

The occurrence of large acetylcholinesterase-containing neurons in human neostriatum as disclosed in normal and Alzheimer-diseased brains.

The human neostriatum was found to contain large neurons (maximum diameter: 30-40 microns) that stain intensely for acetylcholinesterase (AChE). These neurons are few in number, representing less than 5% of the total striatal neuronal population, and appear uniformly scattered throughout the caudate nucleus and putamen. They are morphologically similar to the AChE-containing neurons disclosed in the striatum of rat, cat and monkey after AChE inhibitor (DFP) pretreatment. In Alzheimer-diseased brains the number, morphological characteristics, and staining intensity of the striatal AChE neurons were found to be unaltered despite a marked loss of AChE cells in the adjoining nucleus basalis. These findings suggest that large intrinsic cholinergic neurons exist in human neostriatum and that these elements, in contrast to those of nucleus basalis, are not affected in Alzheimer's disease.

Capsaicin-sensitive local sensory innervation is involved in pacing-induced preconditioning in rat hearts: role of nitric oxide and CGRP?

UNLABELLED: Among several mediators, nitric oxide (NO) and calcitonin gene-related peptide (CGRP) were suggested to be involved in the mechanism of preconditioning. We examined the possible role of the cardiac capsaicin-sensitive sensory innervation in pacing-induced preconditioning, as well as in the cardiac NO and CGRP content. Wistar rats were treated subcutaneously with capsaicin or its solvent in the sequence of 10, 30, and 50 mg/kg increasing single daily doses for 3 days to deplete neurotransmitters of the sensory innervation. Isolated hearts from both groups were then subjected to either preconditioning induced by three consecutive periods of pacing at 600 beats per minute for 5 min with 5 min interpacing periods, or time-matched non-preconditioning perfusion, followed by a 10-min coronary occlusion. NO content of left ventricular tissue samples was assayed by electron-spin resonance, and CGRP release was determined by radioimmunoassay. CGRP immunohistochemistry was also performed. In the non-preconditioned, solvent-treated group, coronary occlusion decreased cardiac output (CO) from 68.1 to 32.1 mL/min, increased left ventricular end-diastolic pressure (LVEDP) from 0.58 to 1.90 kPa, and resulted in 200 mU/min/g LDH release. Preconditioning significantly increased ischaemic CO to 42.9 mL/min (P < 0.05), decreased ischaemic LVEDP to 1.26 kPa (P < 0.05) and decreased LDH release to 47 mU/min/g (P < 0.05) in the solvent-treated group. Preconditioning did not confer protection in the capsaicin-pretreated group (ischaemic CO: 35.6 mL/min; LVEDP: 1.76 kPa; LDH 156 mU/min/g). Capsaicin-treatment markedly decreased cardiac NO content, CGRP release, and CGRP-immunoreactivity. CONCLUSIONS: (i) The presence of an intact local sensory innervation is a prerequisite to elicit pacing-induced preconditioning in the rat heart. (ii) A significant portion of cardiac basal NO content may be of neural origin. (iii) Release of NO and CGRP from capsaicin-sensitive nerves may be involved in the mechanism of pacing-induced preconditioning.

[Primary adenocarcinoma at a colostomy site]. / Anus praeternaturalisban kialakult primer adenocarcinoma.

The colostomy opening is a very rare site of the large bowel cancer. There is only one case report in the world literature and none in the Hungarian one. The authors report a case of a 51 year old man, who presented a typical malignant growth in his colostomy opening, created 24 years before due to a rectal lesion considered as an inoperable cancer, though not proved histologically. Later the patient was treated by telecobalt irradiation, and the growth perfectly disappeared. Now the second, metachron malignancy arising from the colostomy has been removed and the patient is symptom free four years after the operation. Emphasizing the importance of postoperative follow-up in cases of gastrointestinal malignancies the authors considered this case as an unique but instructive one.

[The role of interval surgery and irradiation in the treatment of primarily non-resectable rectal cancers]. / A helyileg irreszekábilis végbélrákok kezelési lehetósége intervallum-mútéttel és besugárzással.

The authors review the results of 1181 colorectal operations performed in a 16y period and analyze the last 8y period in which 713 colorectal operations were performed and the irradiation of primarily irresectable rectal cancer was introduced. At the time of the first operation 44% of the rectal cancer cases (69) proved to be irresectable, 14 of them were sent to a proper center as suitable ones for irradiation. 13 patients had got irradiation therapy [in three cases irradiation was combined with chemotherapy (5-fluorouracil)--synchronization chemoradiotherapy) and 11 of them were suitable for resection at the course of the second operation (there were performed 9 abdominoperineal resection and 2 Hartmann's procedures). In this small group (7% of rectal cancer cases) the radiotherapy can change the primarily irresecatable rectal cancer to resectable and for these patients this could be the only chance for cure.

[The role of conservative surgery in Crohn disease: practical results obtained from a case]. / A konzervatív sebészet helye Crohn-betegségben: egy eset gyakorlati tanulsága.

The 42 years old male patient in a 12 year period got over seven laparotomies and repeated bowel resections, due to which there was left only about 50 cm long jejunum, grossly dilatated and connected side to side to the transverse colon after a right hemicolectomy. The patients was admitted in a serious state of malnutrition due to a Y-shaped fistula formation between the too long blind end of the jejunotransversostomy and the first part of the jejunum. The blind end was resected and the 2 holes on the jejunum was closed by one layer wire stitches. The histology demonstrated a typical granulomatosis process. In summary: the Crohn's disease cannot be cured by surgical means. In this panalimentary process the repeated small bowel resections imply a later short bowel syndrome. On the base of the literature and of their modest experience the authors emphasize the need of conservative surgery and for the so called skip-lesions only stricture plasties to be performed instead of resections.

[Routine roentgen examination of the skull in head injuries]. / A fejsérültek rutinszerü koponya röntgenvizsgálatáról.

Authors in their present report are studying the routine skull X-ray examination of head injured patients. They discuss the disadvantages of the routine up to the present. They introduce the classifications found in the literature. With the help of these classifications the injured who do not need skull X-ray examination can be separated. In case of hospitalisation they consider the anamnesis and clinical examination to be primary in contradiction to the X-ray result. They introduce the retrospective results their head injured patients treated between 1977-1986 in their department.

[Pancreatic cystic tumor misdiagnosed as pseudocyst: lessons from an intraoperative error]. / Altömlönek vélt cysticus daganat a hasnyálmirigyben: egy intraoperatív tévedés tanulságai.

A 62-year old woman presented a fistula due to a previously made cystojejunostomy performed because of a small pancreatic lesion considered as a pseudocyst. This intervention was performed in another institute. In the course of the exploration the authors resected the whole mass with the Roux-en-Y loop, and with the spleen. The histological examination proved a cystadenocarcinoma of the pancreas. There are discussed the problems of the intraoperative diagnosis of pancreatic lesions, and the possibilities of the reduction of mistakes. The authors emphasize the experience of the surgeon--which is the most important factor of the cure--and the importance of the fine needle biopsy.

[Biliary fistula, following partial hepatectomy, treated by endoscopic papillotomy (atypical indication for endoscopic sphincterotomy)]. / Májcsonkolást követó epesipoly megszüntetése endoszkópos papillotomiával (az endoszkópos szfinkterotomia atípusos javallata).

The authors present the case of a 33 y old male patient. The young farmer suffered of a large Echinococcus (hydatid) cyst in the right lobe of his liver. The solution of the problem was a practically atypical right lobectomy. On the 5th postoperative day there was manifested a marked external bile fistula through the drain inserted at laparotomy, and the leakage continued until the 38th postoperative day, when after an endoscopic papillotomy the leak nearly immediately has closed. According to the authors, and some literary data published since, in cases of a postoperative external bile fistula the method of choice seems to be the EST.

[Evaluation of 3757 cases of open biliary tract surgery before the introduction of the laparoscopic method (basis for comparison)]. / A laparoscopos módszer elötti 3757 nyitott epemütét értékelése (Alap az összehasonlításra).

3757 elective nonmalignant biliary tract operations are evaluated retrospectively in the 17-year period from January 1, 1974 to December 31, 1990. The operations were divided into four periods according to development and frequency of intraoperative diagnostics: I. period without examinations of common bile duct (511 operations), II. selective period (848 operations), III. routine period (906 operations), IV. restricted routine period (1492 operations). The authors experienced improvement in their results if they carried out intravenous cholangiography routinely. Comparing eight characteristic factors they believe that their results are favourable if they performed intraoperative common bile duct examinations (manometry, cholangiography, flow rate measurement) in 39.6% rate and if they employed praeoperatively EST if necessary. They propose the selective intraoperative cholangiography. In the period of 39.6% intraoperative cholangiography (restricted routine period) they found common bile duct stones in 10.7%, unsuspected stones in 0.4%, retained stones in 1.6%, unnecessary choledochotomies in 2.7% and intraoperative common bile duct injuries in 0.2%. The overall mortality rate was 0.4%. They deal with the intraoperative differential diagnostics of Vater papilla stenosis and spasm.

[Adsorption of human serum proteins to titanium dioxide]. / Humán szérumfehérjék adszorpciója titán-dioxidhoz.

In order to find an in vitro biochemical model for investigation of osseointegration in terms of dental implantology, the aim of the present study was to analyse the adsorption of human serum proteins to titanium dioxide. Titanium dioxide powder was suspended in human serum. After incubation and centrifugation the TiO2 with the adsorbed proteins were washed with distilled water, ethylendiaminetetraacetic acid (EDTA) supplemented with ammonium hydrogencarbonate (NH4HCO3) solution, and sodium dodecylsulphate (SDS), after centrifugation the supernatant fluid was collected and SDS polyacrylamide gel and native (Biomidi) gel electrophoresis were conducted to determine the type of the adsorbed proteins. Our results show, that albumin was adsorbed to TiO2, but it could be easily removed. The adsorption of a 94 kDa protein was much stronger than the other proteins. The method seems to be useful in the investigation of the protein adsorbing ability of differently treated titanium implant surfaces.

Matrix metalloproteinase activity assays: Importance of zymography.

INTRODUCTION: Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases capable of degrading extracellular matrix, including the basement membrane. MMPs are associated with various physiological processes such as morphogenesis, angiogenesis, and tissue repair. Moreover, due to the novel non-matrix related intra- and extracellular targets of MMPs, dysregulation of MMP activity has been implicated in a number of acute and chronic pathological processes, such as arthritis, acute myocardial infarction, chronic heart failure, chronic obstructive pulmonary disease, inflammation, and cancer metastasis. MMPs are considered as viable drug targets in the therapy of the above diseases. METHODS: For the development of selective MMP inhibitor molecules, reliable methods are necessary for target validation and lead development. Here, we discuss the major methods used for MMP assays, focusing on substrate zymography. We highlight some problems frequently encountered during sample preparations, electrophoresis, and data analysis of zymograms. RESULTS AND DISCUSSION: Zymography is a widely used technique to study extracellular matrix-degrading enzymes, such as MMPs, from tissue extracts, cell cultures, serum or urine. This simple and sensitive technique identifies MMPs by the degradation of their substrate and by their molecular weight and therefore helps to understand the widespread role of MMPs in different pathologies and cellular pathways.

Protective effect of ischaemic preconditioning on ischaemia/reperfusion-induced microvascular obstruction determined by on-line measurements of coronary pressure and blood flow in pigs.

We investigated the protective effect of ischaemic preconditioning (IP) on the maintenance of coronary patency using on-line measurements of coronary pressures and blood flow in a closed-chest reperfused acute myocardial infarction (MI) model in pigs. Catheter-based 90-min occlusion followed by 60-min reperfusion of the left anterior descending coronary artery (LAD) was performed in anesthetised pigs (MI group). IP was applied (IP group) through two cycles of 5-min occlusion and 5-min reperfusion of the LAD before MI induction. Coronary patency was determined by measurements of coronary wedge pressure, collateral fractional flow reserve (FFRcoll), collateral pressure index (CPI) and absolute coronary blood flow (CBF). Inducible and constitutive nitric oxide synthase (iNOS/cNOS) activities and expressions were determined in the myocardium. Plasma levels of myeloperoxidase (MPO, index of activated leukocytes) and mean platelet volume (MPV, index of activated platelets) were measured. IP resulted in significantly lower levels of MPO (0.52 +/- 0.19 vs. 1.05 +/- 0.24 U/l, p<0.001) and MPV (9.1 +/- 0.6 vs. 9.6 +/- 1.0 fl, p=0.04), higher FFRcoll (0.17 +/- 0.05 vs. 0.04 +/- 0.05, p<0.001), CPI (0.13 +/- 0.05 vs. 0.02 +/- 0.05, p<0.001) and CBF (70.7 +/- 4.2 vs. 50.8 +/- 4.8 m/min, p<0.001) post-reperfusion as compared with the MI group. IP resulted in significantly higher cNOS activity and eNOS expression. Significant negative correlation was found between MPO and measures of coronary patency (FFRcoll, CPI and CBF) and cNOS activity. Moreover, cNOS activity correlated significantly with FFRcoll, CPI and CBF. In conclusion, IP attenuates the release of MPO and platelet activation, thereby contributing to the maintenance of vessel patency at microvascular level after reperfusion of the infarct-related artery.