Otakar Krs

Alternative method of rapid drying vascular specimens for scanning electron microscopy.

Purpose: To report the use of hexamethyldisilazane (HMDS) as an alternative to critical point drying for preparing stented canine peripheral vessels for scanning electron microscopy (SEM). Technique: Vascular specimens were fixed in 4% formaldehyde overnight, dehydrated in a graded ethanol series, followed by immersion in 100% hexamethyldisilazane. After air drying, the specimens were mounted on stainless steel stubs, coated with gold, and examined in the SEM. The electron micrographs were of high quality, showing the layers of the vascular wall and the incorporated stent covered by a neointimal layer. The micrographs were comparable to corresponding histological sections, but detailed endothelial patterns were more visible. Conclusions: HMDS treatment and subsequent air drying provides good quality scanning electron micrographs that reveal both endothelial patterns and the layered architecture of stented vessels. The disadvantage of HMDS drying may be a shrinkage and distortion similar to other drying agents. Ease of handling, low cost, and a high rate of success are advantages that favor HMDS desiccation over other drying methods.

Different inhibition of acetylcholinesterase in selected parts of the rat brain following intoxication with VX and Russian VX

Differences between acetylcholinesterase (AChE) inhibition in the brain structures following VX and RVX exposure are not known as well as information on the possible correlation of biochemical and histochemical methods detecting AChE activity. Therefore, inhibition of AChE in different brain parts detected by histochemical and biochemical techniques was compared in rats intoxicated with VX and RVX. AChE activities in defined brain regions 30 min after treating rats with VX and Russian VX intramuscularly (1.0 × LD50) were determined by using biochemical and histochemical methods. AChE inhibition was less expressed for RVX, in comparison with VX. Frontal cortex and pontomedullar areas containing ncl. reticularis has been found as the most sensitive areas for the action of VX. For RVX, these structures were determined to be frontal cortex, dorsal septum, and hippocampus, respectively. Histochemical and biochemical results were in good correlation (Rxy = 0.8337). Determination of AChE activity in defined brain structures was a more sensitive parameter for VX or RVX exposure than the determination of AChE activity in the whole-brain homogenate. This activity represents a “mean” of the activities in different structures. Thus, AChE activity is the main parameter investigated in studies searching for target sites following nerve-agent poisoning contributing to better understanding of toxicodynamics of nerve agents.

Sexual dimorphism of ossified costal cartilage. Radiograph scan study on Caucasian men and women (Czech population)

The aim of our study was to evaluate differences between males and females based on patterns of costal cartilage ossification and also with respect to ageing. We provided diagnosis of ossifications from two files of radiograms. The first group consisted of 1044 chest and abdominal radiograms of patients (537 men and 507 women), ranging in age from 10 to 95 years obtained by using conventional X-ray technique. The second group was a set of 55 radiograms of chest plate fragments of cadavers (29 men and 26 women) aged from 15 to 98, obtained by using soft X-ray imaging in the skiagraphic-skiascopic unit. Ossifications were identified in more than 80% of the cases. They appear in puberta and their occurrence increases with age. The peripheral ossification pattern, typically the male pattern, is characterized by subperichondral deposits which contour the upper and lower margin of cartilage. The female, central lingual ossification pattern, is characterized by the pyramidal (lingual) shape of ossifications with a peak towards the sternum. The existence of another typical female central globular model of ossification was not confirmed in the file of cadavers. Central globular foci were found in both sexes (62% of women and 34% of men) from the 3rd decade. In the sample of Caucasian men and women (Czech population) we detected a frequent occurrence of costal cartilage ossification. Peripheral and central lingual patterns are highly predictive for sex determination. Globular loci of ossifications can be used for age estimation.

Combined approach to demonstrate acetylcholinesterase activity changes in the rat brain following tabun intoxication and its treatment

Reactivation effects of K203 and currently available oximes (obidoxime, HI-6) in combination with atropine on acetylcholinesterase activities in the brain parts of rats poisoned with tabun were studied. The activity was determined by quantitative histochemical and biochemical methods correlating between them very well. The tabun-induced changes in acetylcholinsterase activity as well as in reactivation potency of reactivators used were different in various parts of the brain. Pontomedullar area seems to be important for observed changes following tabun intoxication and its treatment. From the oximes studied, the reactivation effect of K203 was comparable with obidoxime; HI-6 was ineffective. Combination of bio- and histochemical methods allow fine differentiation among the action of different oximes following tabun poisoning.

A comparison of tabun-inhibited rat brain acetylcholinesterase reactivation by three oximes (HI-6, obidoxime, and K048) in vivo detected by biochemical and histochemical techniques.

Tabun belongs to the most toxic nerve agents. Its mechanism of action is based on acetylcholinesterase (AChE) inhibition at the peripheral and central nervous systems. Therapeutic countermeasures comprise administration of atropine with cholinesterase reactivators able to reactivate the inhibited enzyme. Reactivation of AChE is determined mostly biochemically without specification of different brain structures. Histochemical determination allows a fine search for different structures but is performed mostly without quantitative evaluation. In rats intoxicated with tabun and treated with a combination of atropine and HI-6, obidoxime, or new oxime K048, AChE activities in different brain structures were determined using biochemical and quantitative histochemical methods. Inhibition of AChE following untreated tabun intoxication was different in the various brain structures, having the highest degree in the frontal cortex and reticular formation and lowest in the basal ganglia and substantia nigra. Treatment resulted in an increase of AChE activity detected by both methods. The highest increase was observed in the frontal cortex. This reactivation was increased in the order HI-6 < K048 < obidoxime; however, this order was not uniform for all brain parts studied. A correlation between AChE activity detected by histochemical and biochemical methods was demonstrated. The results suggest that for the mechanism of action of the nerve agent tabun, reactivation in various parts of the brain is not of the same physiological importance. AChE activity in the pontomedullar area and frontal cortex seems to be the most important for the therapeutic effect of the reactivators. HI-6 was not a good reactivator for the treatment of tabun intoxication.

Structural changes arising from different thawing protocols on cryopreserved human allograft’s aortic valve leaflets

BACKGROUND The aim of our experimental work was to assess the impact and morphological changes that arise during different thawing protocols on human aortic valve (AV) leaflets resected from cryopreserved aortic root allografts (CARAs). OBJECTIVES Two thawing protocols were tested: 1. CARAs were thawed at a room temperature (23°C); 2. CARAs were placed directly into a water bath at a temperature of 37°C. After all the samples were thawed, non-coronary AV leaflets were sampled from each specimen and fixed in a 4% formaldehyde solution before they were sent for morphological analysis. MATERIAL AND METHODS All the samples were washed in distilled water for 5 min and dehydrated in a graded ethanol series (70%, 85%, 95%, and 100%) for 5 min at each level. The tissue samples were then immersed in 100% hexamethyldisilazane (HMDS) for 10 min, and then air-dried in an exhaust hood at room temperature. Processed samples were mounted on stainless steel stubs and coated with gold. Histological analysis was performed with the use of an electron microscope on a scanning mode operating at 25 kV - BS 301. RESULTS Thawing protocol 1 (room temperature at 23°C): 6 (100%) samples showed loss of the endothelial covering of the basal membrane with no damage to the basal lamina. Thawing protocol 2 (water bath at 37°C): 5 (83%) samples showed loss of the endothelial covering of the basal membrane with no damage to the basal lamina. One (17%) sample showed loss of the endothelial covering the basal membrane with significant damage to the basal membrane. CONCLUSIONS Based on our experimental work, we can clearly conclude that cryopreserved AV leaflet allografts show identical structural changes at different rates of thawing.

Cryopreserved human aortic root allografts arterial wall: Structural changes occurring during thawing

Background The aim of our experimental work was to assess morphological changes of arterial wall that arise during different thawing protocols of a cryopreserved human aortic root allograft (CHARA) arterial wall. Methods The experiment was performed on CHARAs. Two thawing protocols were tested: 1, CHARAs were thawed at a room temperature at +23°C; 2, CHARAs were placed directly into a water bath at +37°C. Microscopic samples preparation After fixation, all samples were washed in distilled water for 5 min, and dehydrated in a graded ethanol series (70, 85, 95, and 100%) for 5 min at each level. The tissue samples were then immersed in 100% hexamethyldisilazane for 10 minutes and air dried in an exhaust hood at room temperature. Processed samples were mounted on stainless steel stubs, coated with gold. Results Thawing protocol 1: All 6 (100%) samples showed loss of the endothelium and damage to the subendothelial layers with randomly dispersed circular defects and micro-fractures without smooth muscle cells contractions in the tunica media. Thawing protocol 2: All 6 (100%) samples showed loss of endothelium from the luminal surface, longitudinal corrugations in the direction of blood flow caused by smooth muscle cells contractions in the tunica media with frequent fractures in the subendothelial layer Conclusion All the samples thawed at the room temperature showed smaller structural damage to the CHARA arterial wall with no smooth muscle cell contraction in tunica media when compared to the samples thawed in a water bath.