Tadeusz Cichocki

Development of sensory innervation in rat tibia: co-localization of CGRP and substance P with growth-associated protein 43 (GAP-43)

The development of sensory innervation in long bones was investigated in rat tibia in fetuses on gestational days (GD) 16–21 and in neonates and juvenile individuals on postnatal days (PD) 1–28. A double immunostaining method was applied to study the co‐localization of the neuronal growth marker growth‐associated protein 43 (GAP‐43) and the pan‐neuronal marker protein gene product 9.5 (PGP 9.5) as well as that of two sensory fibre‐associated neuropeptides, calcitonin gene‐related peptide (CGRP) and substance P (SP). The earliest, not yet chemically coded, nerve fibres were observed on GD17 in the perichondrium of the proximal epiphysis. Further development of the innervation was characterized by the successive appearance of nerve fibres in the perichondrium/periosteum of the shaft (GD19), the bone marrow cavity and intercondylar eminence (GD21), the metaphyses (PD1), the cartilage canals penetrating into the epiphyses (PD7), and finally in the secondary ossification centres (PD10) and epiphyseal bone marrow (PD14). Maturation of the fibres, manifested by their immunoreactivity for CGRP and SP, was visible on GD21 in the epiphyseal perichondrium, the periosteum of the shaft and the bone marrow, on PD1 in the intercondylar eminence and the metaphyses, on PD7 in the cartilage canals, on PD10 in the secondary ossification centres and on PD14 in the epiphyseal bone marrow. The temporal and topographic pattern of nerve fibre appearance corresponds with the development of regions characterized by active mineralization and bone remodelling, suggesting a possible involvement of the sensory innervation in these processes.

Functional effects of eggs, naturally enriched with conjugated linoleic acid, on the blood lipid profile, development of atherosclerosis and composition of atherosclerotic plaque in apolipoprotein E and low-density lipoprotein receptor double-knockout mice (apoE/LDLR-/-).

The objective of this study was to evaluate potential anti-atherogenic properties of hen eggs enriched naturally with conjugated linoleic acid (CLA) isomers (cis-9, trans-11 and trans-10, cis-12). Eighteen apoE and LDL receptor double-knockout mice (apoE/LDLR- / - ), at the age of 4 months with pre-established atherosclerosis, were randomly assigned to three experimental groups (n 6) and fed AIN-93G-based diets for the next 2 months. The experimental diets were: AIN-93G+ CLA-free egg-yolk powder (control); AIN-93G+ CLA-free egg-yolk powder +0.1 % CLA (CLA-supplemented eggs); and AIN-93G+ CLA-enriched egg-yolk powder, providing 0.1 % CLA (CLA-enriched eggs). For assessment of anti-atherogenic properties of CLA-enriched or CLA-supplemented eggs the following criteria were used: (1) serum lipid profile; (2) development of atherosclerosis; and (3) composition of atherosclerotic plaque. CLA-enriched eggs, compared with CLA-supplemented eggs, reduced significantly (P < 0.05) total plasma cholesterol in the mice. At the same time, both CLA-supplemented eggs and CLA-enriched eggs tended to decrease the size of atherosclerotic plaque in aortic roots of mice. Most importantly, atherosclerotic plaques of mice fed CLA-enriched eggs showed significantly (P < 0.05) reduced number of atherogenic macrophages and increased area occupied by smooth muscle cells in atherosclerotic lesions. In conclusion, CLA-enriched eggs exerted an anti-inflammatory effect more effectively than CLA-supplemented eggs. This anti-inflammatory effect can be considered their major functional claim that warrants further exploitation.

Preliminary study on the distribution of selected elements in cancerous and non-cancerous kidney tissues

In this study special interest was given to trace elements recognized as to be carcinogenic to humans. The kidney tissue sections were analyzed in order to determine the concentrations of elements present in the sample. The Synchrotron Radiation Induced X-ray Emission (SRIXE) technique was applied using a white photon microbeam. The results from cancerous parts of the kidney tissues were compared to non-cancerous parts and to the control group. In addition the iron concentration level was determined in the serum of those patients. Two-dimensional scans are presented to illustrate the differences between perfused and not-perfused tissues. According to this study there is no significant difference in the Mn concentration between cancerous and non-cancerous parts of the kidney, but the concentrations of Cd, Cr, Ti, V, Cu, Se, and Zn are at a lower concentration level in the cancerous parts than in the non-cancerous parts. A converse observation has been made for Fe. This may be associated with different metabolism and dynamics of the cancer process and both higher vascularization and need of higher blood supply in the cancerous tissue. The two-dimensional scanning of thin kidney sections showed differences in the trace element distributions depending on the analyzed samples: perfused and non-perfused. Perfusion removed blood mostly from the peritubular capillaries while in the glomerulus some capillaries had a relatively high Fe content. A low Fe concentration was observed in nephron tubules while a converse observation has been made for Cd. This may indicate that Cd is localized in the cells but not in the blood.

Development of rat tibia innervation: colocalization of autonomic nerve fiber markers with growth-associated protein 43.

Development of autonomic innervation of the tibia was investigated in rat fetuses on gestational days (GD) 17–21 and in juvenile animals on postnatal days (PD) 1–28. Double immunofluorescence combined with confocal microscopy was applied to study colocalization of neuronal growth- associated protein 43 (GAP-43) and panneuronal marker protein gene product 9.5 (PGP) with markers of the autonomic nervous system: neuropeptide Y (NPY) and dopamine β-hydroxylase (DβH) for adrenergic, as well as vasoactive intestinal polypeptide (VIP) and vesicular acetylcholine transporter (VAChT) for cholinergic fibers. The first GAP-43-immunoreactive (GAP-IR) nerve fibers were seen on GD17 in the perichondrium of the proximal epiphysis. Further GAP- and PGP-IR innervation appeared in the perichondrium/periosteum of the diaphysis and in the distal epiphysis (GD19), then in the bone marrow and in the intercondylar eminence (GD21). On PD1, NPY-IR and DβH-IR fibers appeared within the diaphyseal periosteum and on PD4 within the bone marrow. From PD14, GAP-43 immunoreactivity of NPY-positive fibers decreased. From PD7 on, NPY-IR fibers were observed in cartilage canals of both epiphyses and in the intercondylar eminence. In secondary ossification centers, NPY-IR fibers were seen from PD10, and in the bone marrow of the epiphyses from PD14. First VIP-IR and VAChT-IR fibers were observed on PD4 within the periosteum, bone marrow and patellar ligament. From PD10 on, VIP-positive fibers were seen in the intercondylar eminence, and from PD14 in secondary ossification centers. GAP-43 proved to be superior to PGP 9.5 as marker of growing nerve fibers, mostly due to its earlier appearance. The presence of specific nerve fibers may suggest possible involvement of autonomic innervation in regulation of bone development.

Increased Incidence of Pituitary Microadenomas in Suicide Victims

Background: Current data suggest an influence of the hypothalamic-pituitary-adrenal axis on suicidal behavior. The frequency of pituitary adenomas in suicide victims has not yet been investigated. Objectives: The aim of this study was to assess whether the incidence of pituitary adenomas is correlated with suicide. Methods: Serial sections of 151 human pituitary glands obtained upon autopsy were examined microscopically. The glands were collected from 70 suicide victims and 81 subjects whose deaths were classified as unexpected or accidental (nonsuicidal group). The sections were stained with hematoxylin-eosin and the presence of adenoma was confirmed by immunostaining for collagen III. Results: In the suicidal group, pituitary microadenomas were found in 32 cases (47.7%), while in the nonsuicidal group microadenomas were detected in 15 cases (18.3%). The observed difference was statistically significant (p = 0.0003). The relative risk ratio of suicide in persons with pituitary adenomas was estimated at 1.9. Logistic regression analysis in a model controlled for age and sex showed that microadenomas constituted a unique risk factor in this model. The immunohistochemical phenotyping revealed a higher percentage of immunopositive (secreting) microadenomas in the nonsuicidal group as compared to the suicidal group (80.0 vs. 59.38%) and a predominance of growth hormone-secreting microadenomas in both groups. However, these differences as well as differences in the hormonal profiles of microadenomas between the groups were not significant. Conclusions: These results suggest that pituitary adenomas belong to suicide risk factors.

Effect of cultured autologous oral keratinocyte suspension in fibrin glue on oral wound healing in rabbits

The effect of cultured autologous oral keratinocyte suspension in fibrin glue on the healing of surgically produced oral mucosal wounds was assessed in the rabbit model. Using the light microscope and a digital image analysis system, the epithelization parameters (marginal epithelization and percentage of wound re-epithelization) were measured in haematoxylin-eosin stained sections of the wound area and compared with those of wounds treated with fibrin glue alone and untreated ones. The epithelization was significantly higher in keratinocytes plus fibrin glue-treated wounds on postoperative days 3 and 7. No significant differences were observed on postoperative day 1, when the healing process had just begun, and on postoperative day 14, when re-epithelization was completed or nearly completed in all groups. The inflammatory infiltration of the wounded mucosa was weakest in keratinocyte-treated wounds and strongest in untreated wounds. In conclusion, suspension of cultured autologous oral keratinocytes in fibrin glue significantly accelerates oral wound healing in the rabbit model and could be beneficial in the treatment of oral wounds in patients.

Development of Galanin‐Containing Nerve Fibres in Rat Tibia

Galanin exerts tonic inhibition of nociceptive input to the central nervous system. Recently, this peptide was demonstrated in several neuronal and non‐neuronal structures in bones and joints. In this study, the time of appearance and topographic localization of galanin‐containing nerve fibres in bone were studied in rats from gestational day 16 (GD16) to postnatal day 21 (PD21). The tibia was chosen as a model of developing long bone and indirect immunofluorescence combined with confocal laser scanning microscopy was used to identify galanin‐immunoreactive (GAL‐IR) nerve fibres. The earliest, sparse GAL‐IR fibres were observed on GD21 in the perichondrium of both epiphyses and in the periosteum of the diaphysis. From PD1 onwards, GAL‐IR fibres were also seen in the bone marrow cavity and in the region of the inter‐condylar eminence of the knee joint. Intramedullary GAL‐IR fibres in proximal and distal metaphyses appeared around PD1. Some of them accompanied blood vessels, although free fibres were also seen. GAL‐IR fibres located in the cartilage canals of both epiphyses were observed from PD7, in the secondary ossification centres from PD10 and in the bone marrow of both epiphyses from PD14. The time course and localization of galanin‐containing nerve fibres resemble the development of substance P‐ and CGRP‐expressing nerve fibres, thus suggesting their sensory origin.

Expression of basal cell marker revealed by RAM11 antibody during epithelial regeneration in rabbits.

RAM11 is a mouse monoclonal anti-rabbit macrophage antibody recognizing connective tissue and vascular macrophages. Our previous report showed that RAM11 reacted with basal cells of stratified squamous epithelia of rabbit skin, oral mucosa and esophagus. The aim of the present study was to follow the appearance of RAM11 immunoreactivity in basal cells of regenerating oral epithelium in rabbits. No RAM11 immunostaining was observed in the regenerating epithelium examined on days 1 and 3 of wound healing. A weak immunofluorescence first appeared on day 7 in single basal cells and 32% of RAM11- positive basal cells were observed on day 14. These findings indicate that expression of the antigen recognized by RAM11 antibody is a transient event in the differentiation of oral keratinocytes which not always occurs during epithelial repair, although it is a constant feature of epithelial turnover in mature epithelium. Therefore this antigen can be regarded as basal cell marker only in mature stratified squamous epithelia.

Histochemical observations on the pulmonary macrophages in cystic fibrosis

The pulmonary macrophages collected with bronchial mucus from patients with cystic fibrosis and other chronic lung diseases were investigated histochemically for acid phosphatase, beta-glucuronidase and beta-glucosaminidase activity, and also for mucopolysaccharide content. In macrophages from patients with cystic fibrosis decreased beta-glucuronidase activity was observed, together with large cytoplasmic vacuoles suggesting storage of partially digested material. It thus seems very likely that apart from the increased synthesis of mucopolysaccharides described in cystic fibrosis by several authors, the degradation of mucosubstances produced can be impaired.