Jan A. Litwin

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.

Effect of differentiating agents (all-trans retinoic acid and phorbol 12-myristate 13-acetate) on drug sensitivity of HL60 and NB4 cells in vitro.

In vitro studies have shown that human myeloid leukemia cell lines: HL60 and NB4 can be stimulated to differentiation by various agents, for example, all-trans retinoic acid (ATRA) and phorbol 12-myristate 13-acetate (PMA). The purpose of this study was to investigate whether differentiation of HL60 and NB4 leukemia cell lines induced by ATRA and PMA alters their drug sensitivity. The differentiation along the neutrophil lineage (upon stimulation with ATRA) and along the monocyte/macrophage lineage (upon stimulation with PMA) was proved by decreased proliferative potential of cells, changes in their morphology, increased ability for NBT reduction and increased expression of CD11b and CD14 cell surface markers. The effect of drugs: cytosine arabinoside, daunorubicin, mitoxantrone and etoposide was examined by Alamar Blue test (proliferation and survival rates), as well as by evaluation of cell smears stained with Hoechst 33342 (apoptotic index). Differentiation resulted in the change of drug sensitivity in both cell lines: the differentiation along the neutrophil pathway (after stimulation with ATRA) increased sensitivity to cytosine arabinoside and mitoxantrone but decreased sensitivity to etoposide; the differentiation along the monocyte/macrophage pathway (induced by PMA) resulted in the decreased sensitivity of both cell lines to all drugs tested. In conclusion, we have shown that ATRA- and PMA-mediated differentiation of HL60 and NB4 cell lines results in the changes of their drug sensitivity. Our data may provide a contribution to a strategy aimed at a rational combination of differentiating agents and conventional anticancer drugs.

Vascular architecture of human uterine cervix visualized by corrosion casting and scanning electron microscopy

BACKGROUND In contrast to the uterine corpus, the vascular architecture of the human cervix has been the subject of only a few studies, mostly dealing with the ectocervical mucosal vessels. This study presents the vascular system of the cervical wall surrounding the endocervical canal visualized by the best currently available technique, corrosion casting combined with scanning electron microscopy. METHODS Uteri collected at autopsy (n= 20) were perfused via afferent vessels with fixative followed by Mercox resin and corroded after polymerization of the resin. The obtained vascular casts of the cervix visualizing all vessels including capillaries were examined in the scanning electron microscope. RESULTS The vascular system of the cervix was nearly completely replicated in only two (10%) of the samples. In the wall of the cervix, four distinct vascular zones surrounding the endocervical canal were observed: (i) the outer zone containing larger vessels, arteries and veins of 0.3-1 mm diameter; (ii) the zone containing arterioles and venules; (iii) the zone of endocervical mucosal capillaries showing a very high density, parallel arrangement and relatively few interconnections and (iv) the innermost, subepithelial zone containing small veins running along the endocervical canal. CONCLUSIONS Despite the loss of the delicate ectocervical mucosal vessels from the cast during the corrosion step, we have successfully visualized the majority of the cervical vasculature. The vascular pattern of the human cervix, especially that of the endocervical mucosa, may facilitate the adaptation of the cervical vasculature to the extensive remodeling of the cervix during parturition.

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.

Circadian rhythmicity of synapses in mouse somatosensory cortex.

The circadian rhythmicity displayed by motor behavior of mice: activity at night and rest during the day; and the associated changes in the sensory input are reflected by cyclic synaptic plasticity in the whisker representations located in the somatosensory (barrel) cortex. It was not clear whether diurnal rhythmic changes in synapse density previously observed in the barrel cortex resulted from changes in the activity of the animals, from daily light/dark (LD) rhythm or are driven by an endogenous clock. These changes were investigated in the barrel cortex of C57BL/6 mouse strain kept under LD 12 : 12 h conditions and in constant darkness (DD). Stereological analysis of serial electron microscopic sections was used to assess numerical density of synapses. In mice kept under LD conditions, the total density of synapses and the density of excitatory synapses located on dendritic spines was higher during the light period (rest phase). In contrast, the density of inhibitory synapses located on dendritic spines increased during the dark period (activity phase). Under DD conditions, the upregulation of the inhibitory synapses during the activity phase was retained, but the cyclic changes in the density of excitatory synapses were not observed. The results show that the circadian plasticity concerns only synapses located on spines (and not those on dendritic shafts), and that excitatory and inhibitory synapses are differently regulated during the 24 h cycle: the excitatory synapses are influenced by light, whilst the inhibitory synapses are driven by the endogenous circadian clock.

Fear learning increases the number of polyribosomes associated with excitatory and inhibitory synapses in the barrel cortex.

Associative fear learning, resulting from whisker stimulation paired with application of a mild electric shock to the tail in a classical conditioning paradigm, changes the motor behavior of mice and modifies the cortical functional representation of sensory receptors involved in the conditioning. It also induces the formation of new inhibitory synapses on double-synapse spines of the cognate barrel hollows. We studied density and distribution of polyribosomes, the putative structural markers of enhanced synaptic activation, following conditioning. By analyzing serial sections of the barrel cortex by electron microscopy and stereology, we found that the density of polyribosomes was significantly increased in dendrites of the barrel activated during conditioning. The results revealed fear learning-induced increase in the density of polyribosomes associated with both excitatory and inhibitory synapses located on dendritic spines (in both single- and double-synapse spines) and only with the inhibitory synapses located on dendritic shafts. This effect was accompanied by a significant increase in the postsynaptic density area of the excitatory synapses on single-synapse spines and of the inhibitory synapses on double-synapse spines containing polyribosomes. The present results show that associative fear learning not only induces inhibitory synaptogenesis, as demonstrated in the previous studies, but also stimulates local protein synthesis and produces modifications of the synapses that indicate their potentiation.

Microvessel Density and Area in Pituitary Microadenomas

Microvessel density (MVD) and area (MVA) were assessed in 53 clinically silent pituitary microadenomas and in surrounding normal pituitary tissue using CD34 immunostaining and digital image analysis system. Twenty-one microadenomas (40%) were avascular; in the others, both MVD and MVA were significantly lower than in the normal pituitary tissue. No significant differences in MVD and MVA were found between hormonally immunonegative and immunopositive tumors or between different hormonal subtypes of immunopositive microadenomas. In microadenomas and in normal pituitary tissue, MVD and MVA were not significantly influenced by age or sex. These results suggest that pituitary adenomas at early developmental stage are either avascular or significantly less vascular than normal pituitary tissue and that differences in vascularity of pituitary adenomas associated with the variables of sex, age, clinical presentation and immunohistochemical phenotype reported in the literature can evolve with tumor growth and possibly acquire statistical significance in large macroadenomas.

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.

Vascular Effects of Advanced Glycation End-Products: Content of Immunohistochemically Detected AGEs in Radial Artery Samples as a Predictor for Arterial Calcification and Cardiovascular Risk in Asymptomatic Patients with Chronic Kidney Disease

Objectives. Our aim was to determine whether vascular deposition of advanced glycation end-products (AGEs) is associated with arterial calcification and cardiovascular mortality in chronic kidney disease (CKD) patients and to assess the relationships between vascular content of AGEs and selected clinical and biochemical parameters. Materials and Methods. The study comprised 54 CKD patients (33 hemodialyzed, 21 predialyzed). Examined parameters included BMI, incidence of diabetes, plasma fasting glucose, AGEs, soluble receptor for AGEs and 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging, serum C-reactive protein (hsCRP), plasminogen activator inhibitor-1 (PAI-1), and fetuin-A. Fragments of radial artery obtained during creation of hemodialysis access were stained for calcifications using alizarin red. AGEs deposits were identified immunohistochemically and their relative content was quantified. Results. Vascular content of AGEs was positively correlated with BMI, hsCRP, fetuin-A, PAI-1, and DPPH scavenging in simple regression; only fetuin-A was an independent predictor in multiple regression. There was a significant positive trend in the intensity of AGEs immunostaining among patients with grades 1, 2, and 3 calcifications. AGEs immunostaining intensity predicted 3-year cardiovascular mortality irrespective of patients age. Conclusions. The present study demonstrates an involvement of AGEs in the development of medial arterial calcification and the impact of arterial AGE deposition on cardiovascular mortality in CKD patients.

Impaired Fasting Glucose and Diabetes as Predictors for Radial Artery Calcification in End Stage Renal Disease Patients

Objective. The objective of the study was to assess the relationship between selected clinical and biochemical parameters of end stage renal disease (ESRD) patients and arterial calcification. Materials and Methods. The study comprised 59 stage 5 chronic kidney disease patients (36 hemodialyzed and 23 predialysis). The examined parameters included common carotid artery intima-media thickness (CCA-IMT), BMI, incidence of diabetes and impaired fasting glucose (IFG), dyslipidemia, hypertension, and 3-year mortality. Plasma levels asymmetric dimethylarginine (ADMA), osteopontin (OPN), osteoprotegerin (OPG), and osteocalcin (OC) were also measured. Fragments of radial artery obtained during creation of hemodialysis access were stained for calcifications using von Kossa method and alizarin red. Results. Calcification of radial artery was significantly associated with higher prevalence of IFG and diabetes (P = 0.0004) and older age (P = 0.003), as well as higher OPG (P = 0.014) and ADMA concentrations (P = 0.022). Fasting glucose >5.6 mmol/l (IFG and diabetes) significantly predicted vascular calcification in multiple logistic regression. The calcification was also associated with higher CCA-IMT (P = 0.006) and mortality (P = 0.004; OR for death 5.39 [1.20–24.1] after adjustment for dialysis status and age). Conclusion. Combination of renal insufficiency and hyperglycemic conditions exerts a synergistic effect on vascular calcification and increases the risk of death.