Introduction: The progressive clinical course with delayed neurological damage in carbon monoxide (CO) poisoning may be due to neuron apoptosis. The usefulness of hyperbaric oxygen (HBO) in different time periods after CO exposure in neuronal cell apoptosis reduction has not been evaluated thus far. The aim was to evaluate HBO efficacy in reducing neuronal apoptosis in different time periods after CO exposure.Methods: Wistar rats were exposed to 3000 ppm CO in air for 60 min and 100% oxygen at a pressure of three bar for 30 min 0-12 h after CO exposure. The apoptosis was evaluated by immunohistochemical analysis with antibodies against activated caspase-3 and the percentage of caspase-3 positive hippocampal ganglionic cells was reported.Results: It was shown that CO poisoning results in ganglionic cell apoptosis. The percentage of apoptotic cells in rats exposed to CO was the highest (32%), whereas the percentage of apoptotic cells in rats exposed to HBO 0 and 1 h after CO was similar with a lower percentage than rats exposed to CO. The percentage of apoptotic cells in rats exposed to HBO 3 and 5 h after CO was similar with a lower percentage than rats exposed to HBO 0 and 1 h after CO. The percentage of apoptotic cells in rats exposed to HBO 7-12 h after CO was similar with a higher percentage than rats exposed to HBO 5 h after CO.Conclusion: HBO has a time-dependent protective effect on CO-induced neuron apoptosis with the highest efficiency at 3 and 5 h after CO poisoning.

We compared the quality of debridement of chondral lesions performed by four arthroscopic (SH, shaver; CU, curette; SHCU, shaver and curette; BP, bipolar electrodes) and one open technique (OPEN, scalpel and curette) which are used prior to autologous chondrocyte implantation (ACI). The ex vivo simulation of all five techniques was carried out on six juvenile equine stifle joints. The OPEN, SH and SHCU techniques were tested on knees harvested from six adult human cadavers. The most vertical walls with the least adjacent damage to cartilage were obtained with the OPEN technique. The CU and SHCU methods gave inferior, but still acceptable results whereas the SH technique alone resulted in a crater-like defect and the BP method undermined the cartilage wall. The subchondral bone was severely violated in all the equine samples which might have been peculiar to this model. The predominant depth of the debridement in the adult human samples was at the level of the calcified cartilage. Some minor penetrations of the subchondral end-plate were induced regardless of the instrumentation used. Our study suggests that not all routine arthroscopic instruments are suitable for the preparation of a defect for ACI. We have shown that the preferred debridement technique is either open or arthroscopically-assisted manual curettage. The use of juvenile equine stifles was not appropriate for the study of the cartilage-subchondral bone interface.

BACKGROUND:The high-threshold sensory afferents, which express trkA, are predominantly involved in terminal collateral sprouting in the skin of adult mammals. We explored which sensory axons are capable of sprouting into the end-to-side coapted nerve in the rat.METHOD:The distal stump of the transected peroneal nerve was sutured to the side of the uninjured sural nerve. After 36 weeks, sprouting of sensory axons into the end-to-side coapted nerve was assessed by the electrophysiologic measurements of compound action potential and by counting the myelinated axons. The neurons in the dorsal root ganglia (DRG) L5 whose axons sprouted into the end-to-side coapted nerve were retrogradely labelled by the fluorescent dye Fluorogold. The expression of trkA in sprouting DRG neurons was investigated by immunohistochemistry.FINDINGS:Predominantly thin myelinated axons were found in the end-to-side coapted peroneal nerve. Their mean conduction velocity (CV) was between the average CVs of the Adelta and Abeta fibres in the normal sural nerves. About 90% of the sprouting DRG neurons were small and medium sized, and about 10% were large. About 85% of sprouting DRG neurons was immunoreactive to trkA, but the rest were not.CONCLUSIONS:Mostly the high-threshold sensory afferents sprouted into the end-to-side coapted nerve, which resembles the collateral sprouting of sensory axons in the skin. However, our results suggest that also some low-threshold mechanoreceptors can sprout after the end-to-side nerve repair.

The aim of this study was to quantify the amount of the biologically active wear particles in the periprosthetic tissue around loosened polyacetal isoelastic stems to find the pattern of their distribution in the periprosthetic tissue and determine their origin. Proton microbeam measurements of thin tissue sections enabled us identification and quantified elemental analysis of individual wear particles in the tissue. The Ba concentration measured on the wear particles matches the Ba concentration measured with in-air PIXE on the polyacetal stem directly proving the origin of the wear particles which suppose to play a decisive biological role in the process of premature aseptic loosening of polyacetal stems.

Objectives To find out which form of renal osteodistrophy is predominant in our population of patients with end‐stage renal failure. To elucidate the reasons of abnormal mineralization in mixed uremic osteodystrophy and low‐turnover osteodystrophy.Design and methods Ninety‐three consecutive patients (58 males and 35 females, mean age 50.6 ± 12.2 years) with end‐stage renal failure underwent iliac bone biopsy (after double tetracycline labeling). Histomorphologic as well as histomorphometric analyses were performed in bone tissue sections stained by Goldner, toluidin, Pearls’ blue, tiophlavin, Alumininon, and fluorescence.Results Thirty‐eight (40.9%) of our patients had mixed renal bone disease, predominant hyperparathyroid bone disease was found in 31 (33.3%) patients, and low‐turnover osteodystrophy in 24 patients (25.8%). The deposits of aluminum on mineralization front were found in 10.8%, amyloid in 35.9%, and iron in 6.5%. The defective mineralization in patients with mixed and low‐turnover forms of the disease was present in 22 patients due to amyloid, in 7 patients due to aluminum deposits, in 1 to iron deposits, whereas in 6 patients the reason was the combination of 2 factors: amyloid and iron (3 patients), aluminum and iron (1 patient), amyloid and aluminum (2 patients). In 26 patients, defective mineralization was unknown (the bone sections were free of aluminum, iron and amyloid).Conclusions In the Slovenian population of end‐stage renal failure patients, the predominant form of renal bone disease is mixed renal osteodystrophy. The most important reason for defective mineralization is amyloid deposits on the mineralization front.

Background: Telomerase catalytic subunit ( hTERT) gene re- expression is a rate limiting step for the activity of telomerase, a key enzyme implicated in cellular immortalisation and transformation.Aims: To determine the potential role of hTERT protein in gallbladder carcinogenesis.Material/ Methods: hTERT protein was analysed by means of immunohistochemistry in 89 gallbladder tissue samples: 16 normal epithelia, 14 reactive hyperplasias, 15 low grade dysplasias, 16 high grade dysplasias, and 28 adenocarcinomas. At least 200 nuclei were assessed for each slide and the mean number of positive signals for each nucleus was expressed as the hTERT index.Results: The mean hTERT index increased progressively with the degree of gallbladder epithelial abnormalities: from 0.03 in normal epithelia, 0.04 in hyperplastic epithelia, 0.25 in low grade dysplasia, 0.82 in high grade dysplasia, to 0.93 in adenocarcinoma. Statistical analysis revealed that three different groups of gallbladder epithelial changes can be distinguished according to the number of hTERT signals for each nucleus: ( 1) normal and regenerative gallbladder epithelium, ( 2) low grade dysplasia, and ( 3) high grade dysplasia and adenocarcinoma ( p < 0.001).Conclusions: The occasional presence of hTERT protein in normal and regenerative gallbladder mucosa reflects their regenerative capacity. Nevertheless, significantly higher hTERT indices in low and high grade dysplastic epithelia and in gallbladder adenocarcinomas are probably a consequence of hTERT re-expression - an early event in the multistep process of gallbladder carcinogenesis.

DNA double-strand break (DSB) repair pathways are implicated in the maintenance of genomic stability. However the alterations of these pathways, as may occur in human tumor cells with strong genomic instability, remain poorly characterized. We analyzed the loss of heterozygosity (LOH) and the presence of mutations for a series of genes implicated in DSB repair by non-homologous end-joining in five radiation-induced sarcomas devoid of both active Tp53 and Rb1. LOH was recurrently observed for 8 of the 9 studied genes (KU70, KU80, XRCC4, LIG4, Artemis, MRE11, RAD50, NBS1) but not for DNA-PKcs. No mutation was found in the remaining allele of the genes with LOH and the mRNA expression did not correlate with the allelic status. Our findings suggest that non-homologous end-joining repair pathway alteration is unlikely to be involved in the high genomic instability observed in these tumors.

Amplification of the epidermal growth factor receptor gene on double minutes is recurrently observed in cells of advanced gliomas, but the structure of these extrachromosomal circular DNA molecules and the mechanisms responsible for their formation are still poorly understood. By using quantitative PCR and chromosome walking, we investigated the genetic content and the organization of the repeats in the double minutes of seven gliomas. It was established that all of the amplicons of a given tumor derive from a single founding extrachromosomal DNA molecule. In each of these gliomas, the founding molecule was generated by a simple event that circularizes a chromosome fragment overlapping the epidermal growth factor receptor gene. In all cases, the fusion of the two ends of this initial amplicon resulted from microhomology-based nonhomologous end-joining. Furthermore, the corresponding chromosomal loci were not rearranged, which strongly suggests that a postreplicative event was responsible for the formation of each of these initial amplicons.

Authors diagnosed the form of renal osteodystrophy as well as the reason for defective mineralization (if it was present) of the bone in 93 consecutive nephrological patients in Slovenia (69 patients with end-stage renal failure and 24 patients after kidney transplantation). The patients passed iliac bone biopsy after double tetracycline labeling. 40.9% of patients had mixed renal bone disease, predominant hyperparathyroid bone disease was found in 33.3%, whereas 25.8% of patients had low-turnover osteodystrophy. The deposits of aluminium on mineralization front were found in 10.8%, amiloid in 35.9%, iron in 6.5% of patients. Mixed renal osteodystrophy is the predominant renal bone disease in Slovenian nephrological patients, but the most important reason for defective mineralization are amiloid deposits on the mineralization front.

AIMS:To investigate survivin expression in laryngeal squamous cell carcinoma (LSCC), its prognostic significance and relation to p53 status. Survivin is an inhibitor of apoptosis protein that is overexpressed in cancer. It has been implicated in both prevention of apoptosis and cell cycle regulation. It has been suggested that wild-type p53 represses survivin expression.METHODS AND RESULTS:Expression of survivin and p53 was analysed in 68 archival biopsy specimens of LSCC by immunohistochemistry. Survivin was detected in 67 of 68 LSCC cases; the proportion of survivin-positive cells varied from 8.2% to 100%. It was localized in the nucleus and/or cytoplasm of tumour cells. Of LSCC cases, 31.8% were p53+. The number of survivin-positive cells was significantly higher in the p53+ group. A high level of survivin expression and a supraglottic site of the tumour were two independent adverse prognostic factors in LSCC.CONCLUSIONS:Survivin is expressed in a varying proportion of cells in virtually all cases of LSCC. A high level of its expression predicts poor survival. Loss of wild-type p53 is a possible mechanism of survivin up-regulation in LSCC.

One difficulty in studying molecular changes of tumours has been the inability to isolate DNA and RNA from a homogeneous cell population. The combination of several new technologies should help overcome these hurdles. Microdissection is a technique for rapid and easy procurement of a pure cellular subpopulation away from its complex tissue milieu. Laser-assisted microdissection has recently been identified as a quick, simple and effective method by which microdissection of complex tissue specimens can be routinely performed for molecular analysis. With the advent of laser microdissection, cDNA libraries can be developed from pure cells obtained directly from stained neoplastic tissue, and microarrays of thousands of genes can now be used to examine gene expression in microdissected tumour tissue samples. This review will concentrate on the application of different microdissection techniques in the area of cancer research.

FRTL-5 cell line is a cloned epithelial follicular cell line from Fischer rat thyroids. This cell line expresses many of the thyroid differentiated markers in vitro. Their growth and function depend on thyrotropin (TSH) as the main regulatory hormone. In this astereological analysis, the authors concentrate on FRTL-5 nuclei as the most vulnerable part of the cell. Using morphometrical variables, they wished to discover the morphologically identifiable sign of transformation of FRTL-5 cells after irradiation and to study the effect of different TSH concentrations. FRTL-5 cells were grown in a medium of 4 different concentrations of TSH (0, 0.1, 1, 10 mU/ml) and irradiated with 0 Gy, 2 Gy, and 4 Gy. The results showed that the nuclear-cytoplasmic ratio decreases after irradiation with doses of 4 Gy or if TSH was included in the medium. The nuclear maximum diameter of FRTL-5 cells increased with higher concentrations of TSH more obviously after irradiation with 4 Gy than with 2 Gy. On the basis of astereological analysis, it was concluded that different concentrations of TSH and irradiation exert an effect especially upon FRTL-5 cell nuclei. The possible transformation of FRTL-5 cells after culturing in TSH medium and after irradiation could be confirmed by injection into an animal of the Fischer strain.

We analysed revised Mathys isoelastic polyacetal femoral stems with stainless-steel heads and polyethylene acetabular cups from eight patients in order to differentiate various types of particle of wear debris. Loosening of isoelastic femoral stems is associated with the formation of polyacetal wear particles as well as those of polyethylene and metal. All three types of particle were isolated simultaneously by tissue digestion followed by sucrose gradient centrifugation. Polyacetal particles were either elongated, ranging from 10 to 150 microm in size, or shred-like and up to 100 microm in size. Polyethylene particles were elongated or granules, and were typically submicron or micron-sized. Polyacetal and polyethylene polymer particles were differentiated by the presence of BaSO4, which is added as a radiopaque agent to polyacetal but not to polyethylene. This was easily detectable by back-scattered SEM analysis and verified by energy dispersive x-ray analysis. Two types of foreign-body giant cell (FBGC) were recognised in the histological specimens. Extremely large FBGCs with irregular polygonal particles showing an uneven, spotty birefringence in polarised light were ascribed to polyacetal debris. Smaller FBGCs with slender elongated particles shining uniformly brightly in polarisation were related to polyethylene. Mononucleated histiocytes containing both types of particle were also present. Our findings offer a better understanding of the processes involved in the loosening of polyacetal stems and indicate why the idea of 'isoelasticity' proved to be unsuccessful in clinical practice.

For the first time, 3, 7 and 10 days growth of normal thyroid follicular FRTL-5 cell colonies in a semi-solid medium of 0.6% methocel over 1% agar base was morphometrically analyzed. It was found that the areas of FRTL-5 colonies as well as their perimeters and maximum diameters increased, while according to their form factors the FRTL-5 colonies were regular in shape. After 10 days in a semi-solid medium, 83% of the FRTL-5 colonies had maximum diameters greater than 50 gMm. It is obvious that after long culturing of FRTL-5 cells under the influence of the pituitary thyroid-stimulating hormone (TSH) these cells are not uniform anymore and that they grow in a semi-solid medium.

The morphological changes of in vitro irradiated FRTL-5 cells and their ability to grow in semi-solid medium were studied morphometrically. FRTL-5 cells were grown in medium with 4 different concentrations of TSH (0, 0.1, 1, 10 mU/ml). After irradiation with 0 Gy, 2 Gy and 4 Gy, the cells were seeded on glass cover-slips and in methocel. Fourteen days after irradiation, the morphometric analysis of FRTL5 cells and their nuclei was performed. The results showed that irradiation and different doses of TSH have influence on FRTL-5 cell size, more on their nuclei than on the cells as a whole. Growing of FRTL-5 cells in the methocel indicates the possible transformation of these cells after long-culturing in the TSH medium and after irradiation.

We describe three prostheses with cemented titanium-alloy stems and Al(2)O(3) ceramic femoral heads which had to be revised after a mean period of implantation of 78 months. In each case, the neck of the prosthesis had been so severely worn that the profile was elliptical rather than circular, There was severe metallosis of the periprosthetic tissues. Metal particles isolated from the tissues were approximately one nanometre in size and the ratios of titanium, aluminium and vanadium in the particles were the same as in the original alloy. Histologically, the high concentration of metal particles masked the presence of high-density polyethylene (HDP) debris, but again particles about one nanometre in size were isolated from the tissues. The severe necrobiosis and necrosis noted were consistent with other reports of the presence of extensive wear particles in periprosthetic tissues. Wear is presumed to have occurred as a result of mismatch between the shape or size of the taper cone and the femoral head, or to changes in the geometry of loading due to migration of the cup. To facilitate early intervention, patients with this design of prosthesis should be monitored radiologically.

Purpose: To visualize directly a sequence of genetic changes underlying the entire spectrum of epithelial hyperplastic laryngeal lesions (EHLL) and laryngeal cancer by the use of non-isotopic in situ hybridization (ISH) for chromosomes 7 and 17 in correlation with overexpression of p53 protein and epidermal growth factor receptor (EGFR). The specific aim was to compare the results and prognostic significance between the two types of EHLL: isolated, mainly atypical hyperplasia or risky epithelium, and EHLL associated with squamous cell carcinoma (SCC). Patients and Methods: 59 tissue specimens of EHLL obtained from 34 patients, graded according to the Ljubljana classification into simple (SH), abnormal (AbH) and atypical hyperplasia (AtH), and carcinoma in situ (CIS) were included in the study. Non-fluorescent ISH for chromosomes 7 and 17 was performed by biotinylated α-satellite DNA probes. Immunohistochemical staining for EGFR and p53 protein was analyzed on the same tissue samples. Results: Polysomy for both chromosomes increased in correlation with progressive grades of EHLL. The most important finding was the statistically significant difference in chromosome copy numbers between the isolated AtH and AtH associated with SCC. Overexpression of EGFR and p53 protein was found in 61 (36/59) and 52% (31/59) of cases, respectively. The immunoreactivity for both markers increased with the grade of lesions, but the staining pattern was not so uniform in isolated EHLL. On the other hand, the immunoreactivity was expressed more constantly in EHLL adjacent to SCC. Conclusions: Numerical changes in chromosomes 7 and 17 might be associated with an upregulation of EGFR and p53 genes, and could contribute to critical events in laryngeal carcinogenesis. For daily practice, the cytogenetic and immunohistochemical analyses could be of assistance in distinguishing between low- and high-risk groups of AtH. However, the isolated forms of atypical hyperplasia need considerable further study by evaluating genetic changes with the described methods regarding their ultimate transformation to carcinoma.

To improve our understanding of the basic biological mechanisms of Hürthle cell tumours (HCT), proliferative activity in 20 HCT was determined by Ag-NOR (nucleolar organiser regions) counting and by immunohistochemical markers proliferating cell nuclear antigen (PCNA) and MIB-1. Ten surgically removed Hürthle cell adenomas (HCA) and 10 Hürthle cell carcinomas (HCC) were used for the study. For comparison, a group of 10 thyroid adenomas without Hürthle cells (SA) was also evaluated. There were significant differences in the percentages of PCNA- and MIB-1-positive nuclear areas between HCA and HCC as well as between SA and HCA. The mean number of Ag-NORs per nucleus showed no significant differences between our groups. However, determination of percentages with 5 or more Ag-NORs per nucleus within one focal plane of sections was more useful for the differentiation between thyroid tumours included in our study. The combination of three types of cell kinetic markers may provide insight into the still poorly understood relationships among karyological abnormalities, enhanced proliferation speed, clonal expansion and invasiveness of HCT.

The morphological changes and proliferative activity of FRTL-5 cells irradiated in vitro with one dose of 0, 8, 12, 16, 24 or 32 Gy were studied morphometrically 7, 14 and 21 days after irradiation. The number of cells irradiated with 24 Gy and 32 Gy decreased during the 3 weeks of the experiment. Morphometrical analysis confirmed that the cells repaired their injuries up to 16 Gy of irradiation, while doses of 24 and 32 Gy damaged them irreversibly. Thyroglobulin was detected in cells after all doses of irradiation.