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.

[Experimental use of the peritoneal graft as a vascular prosthesis]. / Vyuzitie peritoneálneho stepu ako cievnej náhrady v experimente.

INTRODUCTION: The parietal peritoneum appears to be a suitable material for the vascular system reconstructions. AIM: The aim was to assess and compare thrombogenicity and ability for endothelization of the mesothelial and submesothelial side of the parietal peritoneum in the canine venous system. EXPERIMENTAL ANIMALS: canis familiaris (n = 13), half-breeds of both sexes, aged between 1 and 2 years, weighting 15-25kgs, underwent authological transplantations of the peritoneal grafts with the mesothelial side in the lumen- the group M (n = 5) and with the submesothelial side in the lumen the group S (n = 5). In the control group K (n = 3) a part of the venous wall was used as a graft and was affixed back to its original place. The bioptic samples collected on the 10th, 20th, 30th and 40th postoperative day (POD) were stained using the HE staining, NADPH-d and imunohistochemically on the intermedial filaments. The endothelization rate of the peritoneal graft was measured using morphometry and the trombogenicity was assessed peroperatively. RESULTS: In none of the trial groups a presence of thrombi was detected peroperatively. In the first trial group (group M), the onset of the peritoneal graft epithelization (reaching 20%) was recorded on the 10th POD. The endothelization process was completed on the 30th POD in this trial group. In the second trial group (group S), the peritoneal graft epithelization reaching 10% was recorded on the 10th POD. The process was completed on the 40th POD. In the third trial group K, no endothelial changes were recorded during the experiment. CONCLUSION: Both sides of the peritoneum do not show signs of thrombogenicity and possess ability for endothelization.

Preservation injury of jejunal grafts and its modulation by custodiol and university of wisconsin perfusion solutions in wistar rats.

BACKGROUND: The two preservation solutions most commonly used in human transplantation surgery are University of Wisconsin (UW) and Custodiol (histidine-tryptophan-ketoglutarate; HTK). The aim of our study was to compare the protective effect of UW and HTK solutions on preservation-induced injury of jejunal grafts, as evaluated by the histological changes (semiquantitative method) and small bowel mucosal serotonin levels (as a possible new quantitative method). METHODS: Male Wistar rats (n = 50) weighing 316 +/- 52 g were divided into two main groups according to which preservation solution was used, i.e. UW (n = 25) or HTK (n = 25), and each of these groups was divided into five subgroups according to cold ischemic time (0, 1, 6, 9 and 12 h). Jejunal mucosa biopsy specimens were obtained to determine the serotonin concentration in mucosa and for standard light histology. To grade histological changes in mucosa, Park's small bowel injury grading system was used. RESULTS: Histological examination revealed that injury increased with cold ischemic time in the UW as well as in the HTK group, and there were no significant differences in injury between the two groups, except for the 6-hour cold ischemic period (p < 0.05), when HTK-preserved grafts showed a lower degree of injury (0.97 +/- 0.41) compared with UW-preserved grafts (1.25 +/- 0.39). The mucosal serotonin concentration decreased with cold ischemic time in both groups, and there were significant differences (p < 0.05) in concentrations between the groups after 9 and 12 h of cold ischemia. A significantly higher concentration was measured in grafts preserved in UW solution at these time points. CONCLUSION: The concentration of mucosal serotonin in rat small bowel grafts preserved for 9 and 12 h in UW preservation solution was significantly higher than that in HTK solution. These findings indicate a better protective effect of UW solution on small bowel injury after 9 h of cold ischemia.

Morphological changes of small bowel graft in Wistar rats after preservation injury.

OBJECTIVE: In our study we decided to define the harvesting and preservation injury of the graft using Park's scheme for the quantification of morphological changes. ANIMALS: Male Wistar rats (n=25) weighting 322+/-18 g. Harvesting preservation technique: Proximal jejunum (5-7 cm) was flushed with 5 ml of HTK solution and preserved for 0, 1, 6, 9 and 12 hours at 4 degrees C in the same solution. Biopsies for light microscopic evaluations were obtained after the preservation period. Park's scheme was used for the quantitative assessment of histological changes. t-test for two independent samples was used to evaluate statistical significance. HISTOLOGY: The extent of the preservation injury in the samples obtained at 0 hours was of grade 0 and increased to 1.84 after 12 hours of preservation time. At 0 hours of preservation a degree of 0 (s=0: no changes) was observed, after 1 hour, a degree of 0.50 (s=0.47: slight changes, similar to 0 time), after 6 hours a degree of 1 (0.97, s=0.41: discrete subepitelial oedemas in villi apexes), after 9 hours a degree of 2 (1.74, s=0.64: extension of subeppitelial spaces in villi apexes), and after 12 hours a degree of 2 (1.83, s=0.64: more extension of subepitelial spaces) was observed, except for these findings, there were also changes of grade 3 (massive subepitelial edema and sequestration of the mucosa from lamina propria) in the 12-hour group. A statistically significant (p=0.05) difference was found between all groups except for 9 and 12-hour groups. CONCLUSIONS: HISTOLOGY revealed increasing preservation injury with a increasing duration of preservation and its dynamic. (Tab. 1, Fig. 2, Ref: 6.)

[Histologic changes in the jejunal wall after autotransplantation in a dog]. / Histologické zmeny v stene jejuna po jeho autotransplantácii u psa.

INTRODUCTION: Damages to the small intestinal wall resulting from ischemic-reperfusion changes, represent common complications of the clinical transplantation of the small intestine. AIM: Evaluation and quantification of the histological changes in the jejunal wall following its autotransplantation in a dog using the scale according to Park. MATERIAL AND METHODS: In dogs (n = 8), mongrel of both sexes, aged from 6 months to 2 years, weighting from 10 to 25 kgs, a resection of the jejunum followed by its autotransplantation was performed. At the time of the jejunal harvest, then after one-hour-long cold ischemia, 20 minutes after its reperfusion and then the 10th and the 20th day after the transplantation, bioptic samples of the whole jejunal wall were taken to be examined histologically. After being stained with hematoxyllin-eosine, the samples were evaluated according to the Park grading system. STATISTICS: The severity of the jejunal wall damage at the respective biopsy samples collection times was evaluated using the t-test for two dependent samples. RESULTS: After an hour-long cold ischemia, signs of increasing damage to the intestinal wall were observed, when compared to the peroperative sample (0 +/- 0) up to the degree 0.68 +/- 0.5 of the Park grading schema (p < 0.05). This damage degree increased 20 minutes after the reperfusion up to the value of 4 +/- 0 (p < 0.05). On the 10th and the 20th day a practically normal histological picture of the jejunal wall was observed. The histological changes in both cases were graded 0.38 +/- 0.5 (p < 0.05). CONCLUSION: Maximum histological changes following the autotransplantation of the small intestine with an hour-long cold ischemia were observed 20 minutes after the reperfusion. After 10 postoperative days, a practically normal histological picture of the small intestinal wall structure was observed. It remained unchanged even on the 20th postoperative day.

Temporal kinetics of calcium in neocortical neuronal nuclei after global cerebral ischemia and reperfusion. An ultrastructural study.

New facts about control of nuclear milieu were recently described. Because calcium is known as an important cellular regulator we designed this study to describe temporal calcium kinetics in neuronal nuclei in canine neocortex after ischemia-reperfusion injury. Seven-minute global cerebral ischemia was followed by reperfusion phase lasting from 0 to 24 hours. Ultrastructural histopathology of brain tissue was assessed both qualitatively and semi quantitatively using scoring system. For electron microscopic calcium detection pyroantimonate histochemistry was used. Significant increases in calcium-pyroantimonate deposits were noted in reperfusion time 0, 8 and 24 hours. Reperfusion time 30 minutes showed lower load of calcium deposits than control, this, however, did not reach statistical significance. In conclusion we have found biphasic pattern of nuclear sequestration of calcium in neocortical neurons, which is in agreement with phasic changes of calcium content in other neuronal compartments.

Apoptosis and cell death (mechanisms, pharmacology and promise for the future).

Rapidly growing body of evidence on cell death mechanisms and its disorders during last five years has replaced old paradigms and opened new horizons in medicine. Identification of different morphological and signaling aspects, as well as variances in requirement for energy enabled us to construct a theory of three main types of cell death: necrosis, apoptosis, and lysosomal cell death. Mitochondria, certain oncoproteins such as Bcl-2 family, and special catabolic enzymes participating in cellular demise might serve as targets for pharmacological manipulation. Upregulation or downregulation of programmed cell death has been implicated in ischemic, neurodegenerative, and autoimmune disorders, as well as in oncology and chronic inflammation. This minireview brings a short overview of genesis and development of theories on programmed cell death and apoptosis, summarizes basic relevant facts on apoptotic mechanisms and draws a new hypothesis on possible implication in medicine and surgery.

Partial colocalization of NADPH-diaphorase and acetylcholinesterase positivity in spinal cord neurons.

The freely diffusible radical, nitric oxide (NO), has been assumed to act as a retrograde signaling molecule that modulates transmitter release. Acetylcholine (ACh) is known to function as a typical neurotransmitter. In the present work we have examined the presence of both transmitters (NO and ACh) and their possible relations in the rabbit spinal cord. In our experiments we have used histochemical methods for the visualization of acetylcholinesterase (AChE) and NADPH diaphorase (NADPH-d) which label neurons that express nitric oxide synthase (NOS). Both histochemical methods were performed separately or together on the same sections of the thoracic spinal cord. NADPH-d positive dark blue stained neurons were seen mostly in superficial and deep layers of the dorsal horn, preganglionic autonomic neurons and pericentral area. The presence of AChE positive amber yellow neurons was confirmed mostly in motoneurons located in the ventral horns and in neurons of the pericentral and intermediate zone. Besides the above mentioned neurons, also double-labeled neurons were found which contained both the yellow and dark blue histochemical product. Their presence was confirmed in the intermediate zone and in the pericentral area. Thus, the co-existence of NADPH-d and AChE occurred in the location of interneurons. Our observations suggest that NO may play a role in the control of cholinergic neuronal activity and that NO can be involved in the modulation of synaptic transmission.

Stobadine protects against ischemia-reperfusion induced morphological alterations of cerebral microcirculation in dogs.

Vascular diseases of the CNS are a major medical, social and economic problem. From the number of causes leading to nervous malfunction and damage, ischemia is most prominent. Thus, neuronal protection from ischemic damage may provide significant preventive and treatment potential. This study was designed to test possible protective effects of stobadine in a canine model of global cerebral ischemia. Seven minute ischemia was induced by four vessel ligation and maintained using a controlled systemic hypotension. Stobadine pretreated animals were infused with 2 mg/kg stobadine 30 minutes prior to ischemia, while control animals received vehicle. After a 24 hour reperfusion phase, animals were perfusion-fixed and evaluated using electron microscopy. Stobadine pretreated dogs showed much less damage to both endothelial lining and pericapillary structures of the blood-brain barrier. This included preservation of cellular shape of the endothelium, patency of microvessels, lack of intraluminal blebs material, near normal cytoplasmic osmiophilia, decreased thickness of endothelial basement membrane, significantly less edema of astrocyte end-feet, and preservation of fine mitochondrial structure compared to the control group. Ischemic neuronal changes were observed less frequently in the stobadine pretreated group. In summary, we conclude that stobadine protects both cerebral microcirculation and neurons from injury induced by global cerebral ischemia and reperfusion.

Validation of a four-vessel occlusion model for transient global cerebral ischemia in dogs.

A new model of transient global cerebral ischemia in dogs with minimal measures of intervention is described together with a simple scale for evaluation of functional outcome. During pentobarbital anesthesia, a global cerebral ischemia lasting seven minutes was induced by a four-vessel occlusion and a controlled systemic hypotension. The reperfusion phase begun after removal of arterial clamps, and the animals were sacrificed by perfusion fixation 24 hours latter. The efficiency of controlled systemic hypotension in diminishing collateral blood flow was validated in two experimental groups with different cerebral filling pressure (CFP). Severe ischemia group (CFP 1.0-1.5 kPa) underwent near-complete ischemia as indicated by rCBF, electroencephalography, and histologically documented ischemic neuronal changes. Mild ischemia group (CFP 2.5-3 kPa) animals experienced reduction in cerebral blood flow well above the ischemic threshold, had better functional outcome as well as no ischemic neuronal changes on light microscopy. This model consistently produces global cerebral ischemia in dogs with minimal surgical intervention and pharmacological support, and without intracranial hypertension, cardiac arrest or asphyxia. We recommend this model for outcome-oriented studies of complete forebrain ischemia in dogs.

[NADPH-diaphorase activity in autonomic preganglionic neurons in the rabbit]. / Aktivita NADPH-diaforázy v autonómnych pregangliových neurónoch králika.

BACKGROUND: In order to provide a morphological basis for the understanding of the role of nitric oxide (NO) in autonomic preganglionic neurons, the present study was designed to clarify the localization and distribution of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) activity, a marker of NO synthase, in the rabbit spinal cord. METHODS: 11 Chinchilla rabbits of both sexes were used in this study. Animals were kept under conditions of controlled light and heat regimens. NADPH-d activity was examined by histochemical methods. RESULTS: The presence of NADPH-d activity in the spinal cord was detected in the dorsal horn, around the central canal (lamina X) and in autonomic preganglionic neurons. Focused on the latter, there was a prominent NADPH-d activity between T1 and L5 segments in the intermediate zone and less intensive NADPH-d staining between S1 and S3 segments. Differences between the two parts of the autonomic system were seen in the arrangement (periodical in sympathetic, and continuous in parasympathetic), in the length of neuronal processes length (shorter in sacral preganglionic neurons) and in their localization (both are seen in the intermediolateral nucleus, but neurons of the sympathetic autonomic system can be found also medially towards the central canal, whilst those of the parasympathetic system were not). CONCLUSIONS: The present results suggest that NO can be used as a transmitter in preganglionic neurons and can be involved in autonomic and sensory processes. (Fig. 10, Ref. 37.)

How do we teach anatomy?

Two approaches to anatomy teaching have been studied and compared. We have concentrated on the teaching time spent for all parts of anatomy and we have compared external recommendations with the teaching time actually given to the subject at our Faculty of Medicine. In addition to that, various opinions on informational and educational aims in medical anatomy teaching are presented. The comparison mentioned above can be used to select the most useful way in which anatomy may be taught, in the light of current curricular emphases and pressures, and in the context of sound educational principles.

[The effect of global cerebral ischemia on erythrocyte deformability and cerebral microcirculation in dogs]. / Vplyv globálnej ischémie mozgu na erytrocytárnu deformabilitu a cerebrálnu mikrocirkuláciu u psa.

The state of cerebral ischemia and the following recirculation affect also the blood rheology. The erythrocyte microrheology was studied less than the blood plasma changes. The presented paper is focused on the erythrocyte microrheology changes after global brain ischemia. Both mild and serious global ischemia were induced by the exclusion of cerebral blood circulation for seven min. Thereafter followed the period of recirculation. The changes of erythrocyte microrheology were studied using the method of colloid-osmotic hemolysis. After 180 minutes of recirculation, a significant increase of colloid-osmotic hemolysis was observed. After this until the 240th a significant decrease of colloid-osmotic hemolysis followed. In the group in which mild cerebral ischemia was induced the above mentioned changes were only slightly presented. Important is the fact that the changes in erythrocyte microrheology after serious brain ischemia are in correlation with the changes in cerebral microcirculation. After serious brain ischemia the trombotisation as well as the blood stasis occurred (no-reflow phenomenon).

Effect of stobadine on gastric mucosal injury after ischaemia/reperfusion.

The ability of stobadine to prevent gastric mucosal injury was tested in rat gastric ischaemia induced by 30 min clamping of the coeliac artery with subsequent 30 min reperfusion. Serious injury of gastric mucosa (macroscopic and microscopic) and the increase of microvascular permeability was found after ischaemia/reperfusion in rats without stobadine. After oral pretreatment with stobadine (5 mg.kg-1, 30 min before surgery), the development of gastric mucosal lesions and changes of vascular permeability were significantly decreased.

Experimental studies of some microrheological factors in serious brain ischemia after stobadine pretreatment.

Serious brain ischemia was induced by occlusion of cerebral arteries in dogs. The occlusion time was 7 min. The blood was collected at various intervals of reperfusion (5, 60, 180, 240 min and 24 h). Thirty minutes before ischemization, stobadine was given (1, 2, or 5 mg/kg). The changes of erythrocyte membrane fluidity were evaluated using colloid-osmotic hemolysis induced by brilliant cresyl blue. In the control group (without stobadine) the colloid-osmotic hemolysis was significantly increased immediately after ischemization and after 5 and 60 min. However, after 240 min of reperfusion, a significant decrease of hemolysis was observed. The increase of colloid-osmotic hemolysis after ischemization in the control group was prevented after stobadine pretreatment. The thrombotization of microcirculation that was observed in the control group was not present after stobadine pretreatment.

Effects of stobadine on survival, histopathologic outcome and acid-base status after global brain ischemia in dogs.

Present study is designed to examine an effect of Stobadine, a new cell-protective agent with antiarrhythmic properties, on survival, electron microscopic changes in microvasculatory bed of selected brain areas and acid-base parameters of arterial blood after global brain ischemia and reperfusion. Forty dogs (weighting 6 to 15 kg) were anesthetized using pentobarbital i.v. (5%, 35 mg/kg). An intubation and controlled ventilation was performed. One catheter was placed into v. femoralis (for drug administration), another to a. femoralis (for blood samples) and third one into the left common carotid artery (continuous brain blood feeding pressure measurement). Each dog underwent an surgical obstruction of principal brain-supplying arteries and immediate administration of hypotensive agent (Arfonad, 0.062%) resulting in 7 minutes lasting global brain ischemia (brain feeding pressure 1.0-1.5 kPa). If survived, animals were killed at one (perfusion-fixed for electron microscopy) or three days after ischemia. Ultrastructural changes were evaluated at 24 hour of recirculation (control and S2 groups only). Vehicle or 1, 2 or 5 mg/kg of Stobadine (group S1, S2, S5 resp.) i.v. was given 30 minutes prior to ischemia. Significantly longer survival was observed in group S2 (8 of 11 until 72 hour) as compared to control group (none of 7, p < 0.005 by Student's t-test). The ultrastructural changes of blood-brain barrier structures were none or minimal in S2 (single damage type), but in control group three major types of capillary damages has appeared at 24 hour after insult. They include intravascular coagulopathy (type I), no-reflow (type II) phenomenon with astrocyte edema, and capillary necrosis (type III) finally. Stobadine pretreated animals experienced hypercapnia, elevated arterial O2 and slight deeper acidemia (depending on dosage) as compared to control group. Respiratory compensation of metabolic acidosis was present in control group, but lacking in all stobadine pretreated animals. Stobadine at 2 mg/kg improves survival (Student p < 0.005, Mantel-Cox p < 0.05, Fischer p = 0.004). Stobadine has a protective effect on neurons and structures of blood-brain barrier (endothel, astrocytes, basement membrane) seen in electron microscope.

The state of the microcirculatory bed after total ischaemia of the brain. An experimental ultrastructural study.

The study was focused on the changes in morphological structure of cerebral microcirculation after two stages of 7 min dog's total brain ischaemia followed by 24 recirculation. In the first experimental group (brain blood pressure 2.5-3.0 kPa), there was observed ultrastructural picture of damaged microvessels, including dilated and irregularly shaped lumens, thick finger-like endothelial processes, dark osmiophilic cytoplasm of the endothelial cells with light spaces, clusters of ribosomal structures, impairment of endoplasmic reticulum and mitochondria, dilated tight junctions, irregular and thickened basement membranes. The alteration of astrocytes consisted of accumulation of beta-glycogen particles and lipofuscin and altered lysosomal structures. On the other hand, in the second experimental group (brain blood pressure 1.0-1.5 kPa) the signs of impaired microcirculation were found. Type A pattern of vessel damage was delineated by dilated capillary lumen, despite the presence of marked perivascular oedema. Type B represented the no-reflow phenomenon. Type C was defined by a conspicuous lobular nucleus of endothelial cells obstructing the lumen of the capillary. Type D was characterized by ischemically damaged erythrocytes, despite the adequate perfusion fixation. Finally, type E included necrotic endothelial cells and endothelial blebs.

[Pathomorphology of damaged cerebral microcirculation after global ischemia in the dog. Study of the cortical ectosylvian rostral gyrus]. / Patologicko-morfologický popis poskození mozkové mikrocirkulace po globální ischémii u psa. Studie na kúre gyrus ectosylvius rostralis.

Pathological-morphological changes in the brain microcirculatory blood vessel in dog after global brain ischemia have been evaluated qualitatively. In series of cut sections thick 200 microns dyed with benzidine, a marked microthrombotization of the brain capillaries without vasostasis as well as cylindrical microthrombi and microthrombi with vasostasis were observed. The described light microscopic angioarchitectonic findings account for the phenomenon of vasomotoric paralysis.