The Expression of Hypoxia-Related Biomarkers: A Significance of HIF-1α C1772T Polymorphism as Predictor of Laryngeal Carcinoma Relapse.

INTRODUCTION: The association between the expression of HIF-1α in the laryngeal carcinoma and the prognosis of disease is quite well documented, but the significance of HIF-1α C1772T polymorphism and its relation to disease phenotype have to be clarified. The aim of this study was to investigate the influence of C1772T polymorphism on the clinical-pathological characteristics and disease-free survival after initial surgical treatment of patients with laryngeal carcinoma. MATERIALS AND METHODS: The prospective cohort study included 65 patients with laryngeal carcinoma. Two representative tumor tissue specimens were taken in each patient during surgery; 1 specimen was used to asses HIF-1α C1772T polymorphism and the other 1 to determine the immunohistochemical expression of HIF-1α, VEGF, as well as CD 34 proteins. The comparison of polymorphism frequency between study and control population was conducted by collecting a 5 mL of peripheral venous blood samples in each subject. RESULTS: Clinicopathological characteristics of laryngeal carcinoma didn't affect the expression of hypoxia-related biomarkers, such as HIF-1α, VEGF or MVD. The statistically significant association between HIF-1α and VEGF expression was found (P = .034), but not between HIF-1α expression and MVD value (P = .696). The expression of HIF-1α was significantly higher among CT heterozygotes (P = .029). We found a significantly more recurrence among CT heterozygotes compared with patients with CC homozygous alleles (57.10% and 24.30%, respectively; P = .007). Patients with C1772T polymorphic variants had significantly worse disease-free survival compared with patients without polymorphism (Log-rank test, P = .007). CONCLUSION: HIF-1α C1772T polymorphism was significantly associated with worse disease-free survival which nominates it as a predictor of laryngeal carcinoma relapse. The preoperative assessment of hypoxia-related biomarkers should be used in everyday practice in order to determine the treatment modalities for laryngeal carcinoma.

Macrophages, the main marker in biocompatibility evaluation of new hydrogels after subcutaneous implantation in rats.

Biocompatibility of materials is one of the most important conditions for their successful application in tissue regeneration and repair. Cell-surface interactions stimulate adhesion and activation of macrophages whose acquaintance can assist in designing novel biomaterials that promote favorable macrophage-biomaterial surface interactions for clinical application. This study is designed to determine the distribution and number of macrophages as a means of biocompatibility evaluation of two newly synthesized materials [silver/poly(vinyl alcohol) (Ag/PVA) and silver/poly(vinyl alcohol)/graphene (Ag/PVA/Gr) nanocomposite hydrogels] in vivo, with approval of the Ethics Committee of the Faculty of Veterinary Medicine, University of Belgrade. Macrophages and giant cells were analyzed in tissue sections stained by routine H&E and immunohistochemical methods (CD68+). Statistical relevance was determined in the statistical software package SPSS 20 (IBM corp). The results of the study in terms of the number of giant cells localized around the implant showed that their number was highest on the seventh postoperative day (p.o.d.) in the group implanted with Ag/PVA hydrogels, and on the 30th p.o.d. in the group implanted with Ag/PVA/Gr. Interestingly, the number of macrophages measured in the capsular and pericapsular space was highest in the group implanted with the commercial Suprasorb© material. The increased macrophage number, registered around the Ag/PVA/Gr implant on 60th p.o.d. indicates that the addition of graphene can, in a specific way, modulate different biological responses of tissues in the process of wound healing, regeneration, and integration.

VHL Dependent Expression of REDD1 and PDK3 Proteins in Clear-cell Renal Cell Carcinoma.

BACKGROUND: Sporadic clear-cell renal cell carcinoma (ccRCC) is associated with mutations in the VHL gene, upregulated mammalian target of rapamycin (mTOR) activity and glycolytic metabolism. Here, we analyze the effect of VHL mutational status on the expression level of mTOR, eIF4E-BP1, AMPK, REDD1, and PDK3 proteins. METHODS: Total proteins were isolated from 21 tumorous samples with biallelic inactivation, 10 with monoallelic inactivation and 6 tumors with a wild-type VHL (wtVHL) gene obtained from patients who underwent total nephrectomy. The expressions of target proteins were assessed using Western blot. RESULTS: Expressions of mTOR, eIF4EBP1 and AMPK were VHL independent. Tumors with monoallelic inactivation of VHL underexpressed REDD1 in comparison to wtVHL tumors (P = 0.042), tumors with biallelic VHL inactivation (P < 0.005) and control tissue (P = 0.004). Additionally, REDD1 expression was higher in tumors with VHL biallelic inactivation than in control tissue (P = 0.008). Only in wt tumor samples PDK3 was overexpressed in comparison to tumors with biallelic inactivation of VHL gene (P = 0.012) and controls (P = 0.016). In wtVHL ccRCC, multivariate linear regression analysis revealed that 97.4% of variability in PDK3 expression can be explained by variations in AMPK amount. CONCLUSION: Expressions of mTOR, eIF4EBP1 and AMPK were VHL independent. We have shown for the first time VHL dependent expression of PDK3 and we provide additional evidence that VHL mutational status affects REDD1 expression in sporadic ccRCC.

Correlation of structural defects in the ascending aortic wall to ultrasound parameters: benefits for decision-making process in aortic valve surgery.

BACKGROUND: Histopathological changes in the ascending aorta wall in patients with severe tricuspid aortic valve (TAV) stenosis were graded and correlated to echocardiographic parameters. Objective was to associate threshold echocardiographic values with structural defects in the ascending aorta providing a tool to improve decision-making process in cases when simultaneous aortic valve replacement (AVR) and ascending aorta replacement is considered. METHODS: Biopsies from 108 TAV stenosis patients subjected to AVR were graded into three grades according to severity of aortic wall changes. Echocardiographic parameters obtained preoperatively and correlated to grade, age, gender and risk factors, were diameters of ventriculo-aortic junction (AA), sinus Valsalva (SV), sinotubular junction (STJ), the largest diameter of the visualized ascending aorta (AscA) as well as indexes: sinus Valsalva (SVI), sinotubular junction (STJI), AscA/AA and STJ/AA. RESULTS: Two echocardiographic parameters portrayed grades with statistical significance: STJ (F = 5.417; p = 0.006 (p < 0.05)) and AscA (F = 3.924; p = 0.023 (p < 0.05)). By using multiple predictors in the setting of Regression analysis, statistically significant differences among grades were reached for AA, SV, STJ, AscA and SVI. With further ROC curves analysis, threshold values for different grades were recognized. Grade 2 is identified in patients with AscA > 3.3 cm, while Grade 3 is identified in patients with values of AscA > 3.5 cm, STJ > 2.9 cm and STJI > 1. CONCLUSIONS: Hemodynamic stress induced by TAV stenosis leads to elastic lamellae disruption in the aortic wall. Those changes could be graded and correlated with echocardiographic parameters of the aortic root and ascending aorta, providing a tool for decision to replace ascending aorta concomitantly with AVR.

Tuberous sclerosis complex protein 1 expression is affected by VHL Gene alterations and HIF-1α production in sporadic clear-cell renal cell carcinoma.

Alterations in von Hippel-Lindau gene (VHL) do not determine deregulation of hypoxia-inducible factors (HIFs) in clear-cell renal carcinoma (ccRCC). Their effects on tuberous sclerosis proteins (TSC1/2) and heat shock protein 90 (Hsp90) expressions in sporadic ccRCC are unknown. Therefore, we analyze the impact of VHL alterations and HIF-α production on the expression of TSC proteins and Hsp90 in these tumors. Alterations in VHL gene region exhibited 37/47 (78.7%) tumors. Monoallelic inactivation (intragenic mutation or LOH) was found in 10 (21.3%) and biallelic inactivation (intragenic mutation plus LOH) in 27 (57.4%) ccRCCs. Tumorous expression of HIF-α mRNAs, HIF-α, Hsp90 and TSC2 were VHL independent; TSC2 was underexpressed in all tumors by immunostaining (P<0.001). Immunoblotting revealed that TSC1 production was lower in tumors with monoallelic VHL inactivation than in control (P=0.01) and tissues with biallelic VHL inactivation (P=0.019), while tumors lacking HIF-1α (16/47) concurrently overexpressed HIF-2α and underexpressed TSC1 in comparison to controls (P=0.01 for both) and HIF-1α positive tumors (P=0.015 and P=0.050). Significant portion of variability (56.4%) in tumor diameter was explained by oscillations in nuclear grade, and TSC1 and HIF-2α expression in VHL altered tumors. In conclusion, while TSC2 is broadly downregulated in sporadic ccRCC, TSC1 expression is reduced in two subsets of these tumors, those with monoallelic VHL gene inactivation and those with concurrent low HIF-1α and high HIF-2α expression. Hence, the involvement of nuclear grade, TSC1 and HIF-2α in the progression of VHL altered tumors, implies the interplay between pVHL and TSC1.

N-Acetyl-l-cysteine enhances ex-vivo amplification of deciduous teeth dental pulp stem cells.

OBJECTIVE: Obtaining high number of stem cells is of interest for cell based therapies. N-Acetyl-l-cysteine (NAC) acts as a source of sulfhydryl groups and an anti-oxidative agent. The aim of this study was to test different NAC concentration on proliferation and differentiation of deciduous teeth dental pulp stem cells (DTSCs) in vitro as well as to define the possible underlining mechanism of its effect. DESIGN: Number of viable, apoptotic and senescent DTSCs was determined after addition of NAC (0.1mM, 1.0mM, 2.0mM). Also, cell cycle analysis, HIF1-α expression, LDH isoenzymes, superoxide-dismutase (SOD) and catalase (CAT) activity, sulfhydryl groups content, the level of lipids' and proteins' oxidative damage and differentiation capacity of NAC treated DTSCs was determined. RESULTS: DTSCs expressed HIF-1α in all conditions. The lowest NAC dose (0.1mM) increased the number of DTSCs by one fifth comparing to the control, most likely stimulating entry of cells into S phase of cell cycle and enhancing the activity of LDH5 isoenzyme. The highest NAC dose (2mM) inhibited DTSCs proliferation. Also, DTSCs had the lowest level of oxidative damage with 0.1mM NAC. All tested NAC concentrations enhanced DTSCs osteo-chondrogenesis. CONCLUSION: The lowest NAC dose exerted significant positive effect on DTSCs proliferation as well as antioxidative protection creating beneficial environment for stem cells in vitro cultivation especially when their clinical use is important for stimulation of osteo-chondrogenesis.

Co-expression of vascular and lymphatic endothelial cell markers on early endothelial cells present in aspirated coronary thrombi from patients with ST-elevation myocardial infarction.

INTRODUCTION: Angiogenesis is the growth of both new vascular and lymphatic blood vessels from the existing vasculature. During this process, blood endothelial cells (BECs) and lymphatic endothelial cells (LECs) express specific markers, which help their discrimination and easier identification. Since the coronary thrombi material aspirated from patients with ST-elevation myocardial infarction (STEMI) proved as good angiogenesis model, we investigated the expression of CD34 and CD31 as BECs markers, and D2-40, LYVE-1 and VEGFR3 as LEC markers in this material. MATERIALS AND METHODS: Aspirated thrombi were stained immunohistochemically for CD34, CD31, D2-40, LYVE-1 and VEGFR3. Organizational patterns of immunopositive cells were graded as single cells, clusters or microvessels. Double immunofluorescence for CD31, D2-40, LYVE-1 and VEGRF3 was done. Thrombi were also graded as fresh (<1day old), lytic (1-5days old) and organized (>5days old). RESULTS: Serial sections of aspirated thrombi showed concordant BEC and LEC markers immunopositivity. Double immunoflorescence proved co-expression of CD31 and LEC markers on the same cells. Cells expressing LEC markers organized in clusters and microvessels were mainly present in lytic and organized thrombi. CONCLUSION: Co-expression of BEC and LEC markers on the same non-tumorous cell during thrombus neovascularization indicates existing in vivo plasticity of endothelial cells under non-tumorous pathological conditions. It also points that CD34 and CD31 on one hand, and D2-40, LYVE-1 and VEGFR3 immunostaining on the other hand, cannot solely be a reliable indicators whether vessel is lymphatic or not.

Characterization of deciduous teeth stem cells isolated from crown dental pulp.

BACKGROUND/AIM: The last decade has been profoundly marked by persistent attempts to use ex vivo expanded and manipulated mesenchymal stem cells (MSCs), as a tool in different types of regenerative therapy. In the present study we described immunophenotype and the proliferative and differentiation potential of cells isolated from pulp remnants of exfoliated deciduous teeth in the final phase of root resorption. METHODS: The initial adherent cell population from five donors was obtained by the outgrowth method. Colony forming unit-fibroblast (CFU-F) assay was performed in passage one. Cell expansion was performed until passage three and all tests were done until passage eight. Cells were labeled for early mesenchymal stem cells markers and analysis have been done using flow cytometry. The proliferative potential was assessed by cell counting in defined time points and population doubling time was calculated. Commercial media were used to induce osteoblastic, chondrogenic and adipogenic differentiation. Cytology and histology methods were used for analysis of differentiated cell morphology and extracellular matrix characteristics. RESULTS: According to immunophenotype analyses all undifferentiated cells were positive for the mesenchymal stem cell markers: CD29 and CD73. Some cells expressed CD146 and CD106. The hematopoietic cell marker, CD34, was not detected. In passage one, incidence of CFU-F was 4.7 +/- 0.5/100. Population doubling time did not change significantly during cell subcultivation and was in average 25 h. After induction of differentiation, the multicolony derived cell population had a tri-lineage differentiation potential, since mineralized matrix, cartilage-like tissue and adipocytes were successfully formed after three weeks of incubation. CONCLUSION: Altogether, these data suggest that remnants of deciduous teeth dental pulp contained cell populations with mesenchymal stem cell-like features, with a high proliferation and tri-lineage differentiation potential and that these cultures are suitable for further in vitro evaluation of cell based therapies.

The effect of calorie restriction on acute ethanol-induced oxidative and nitrosative liver injury in rats.

The aim of our study was to examine the effect of calorie restriction (CR) on oxidative and nitrosative liver injury in rats, induced by acute ethanol intoxication. Male Wistar rats were divided into groups: (1) control; (2) calorie-restricted groups with intake of 60-70% (CR60-70) and 40-50% of daily energy needs (CR40-50); (3) ethanol-treated group (E); (4) calorie-restricted, ethanol-treated groups (E+CR60-70 and E+CR40-50). Ethanol was administered in 5 doses of 2g/kg every 12h, and duration of CR was 5 weeks before ethanol treatment. Malondialdehyde and nitrite and nitrate level were significantly lower in E+CR60-70 and higher in E+CR40-50 vs. E group. Liver reduced glutathione content and activity of both superoxide dismutase izoenzymes were significantly higher in E+CR60-70 and lower in E+CR40-50 vs. E group. Oxidative stress may be a potential mechanism of hormetic effects of CR on acute ethanol-induced liver injury.

Glucocorticoid receptor and molecular chaperones in the pathogenesis of adrenal incidentalomas: potential role of reduced sensitivity to glucocorticoids.

Glucocorticoid (GC) sensitivity depends on glucocorticoid receptor (GR) and heat shock proteins (Hsps). We investigated whether common GR genes (ER22/23EK, N363S, Bcl I, and 9ß) and adrenocorticotropin receptor promoter polymorphisms influence susceptibility for unilateral adrenal incidentaloma (AI), plus GR and Hsp expression in tumorous (n = 19), peritumorous (n = 13) and normal adrenocortical (n = 11) tissues. Patients (n = 112), population-matched controls (n = 100) and tumor tissues (n = 32) were genotyped for these polymorphisms. Postdexamethasone serum cortisol was higher in patients (p < 0.001). GR gene variants, larger allele of Bcl I (odds ratio [OR] 2.9; 95% confidence interval [CI] 1.7-5.1; p < 0.001] and minor allele of 9ß (OR 3.0; 95% CI 1.6-5.7; p < 0.001) were independent predictors of AI. In patients, the first allele is linked with larger tumors (p = 0.002) and the latter with higher postdexamethasone cortisol levels (p = 0.025). Both allele carriers had lesser waist circumference (p = 0.02), similar adrenocorticotropin and higher basal (p = 0.024) and postdexamethasone cortisol concentrations (p < 0.001). Tumorous and constitutional genotypes were similar. GR-D is the major receptor isoform in normal adrenal cortex by Western blotting. Loss of other receptor isoforms, decrease in immunostaining for GR (p < 0.0001), underexpression of chaperones (p ≤ 0.01) and the presence of inducible Hsp70 were found in adenomas. In conclusion, GR gene variants, C allele of Bcl I and minor allele of 9ß, are associated with AIs. Their concurrent presence in patients reduces GC sensitivity. Normal adrenal cortex preferentially expresses GR-D. In adenomas, the lack of other GR isoforms and underexpression of heat shock proteins perhaps permanently impair GC signaling, which could promote dysregulated cortisol production and tumor growth. The innate GC sensitivity probably modifies these effects.

Characterization and immunosuppressive properties of mesenchymal stem cells from periapical lesions.

AIM: Mesenchymal stem cells (MSCs) isolated from healthy dental tissues are being investigated as an alternative source of MSCs for the treatment of damaged tissues and inflammatory diseases. Here we investigated whether MSCs from periapical lesions (PL-MSCs) also possess multi-lineage differentiation capacity and immunomodulatory properties. MATERIAL & METHODS: PL-MSCs, isolated by collagenase/DNAse digestion from surgically extracted PLs, were compared with MSCs from non-inflamed dental pulp (DP-MSCs) and dental follicle (DF-MSCs) for their phenotype and multi-potent differentiation potential. The anti-inflammatory and immunomodulatory effects of PL-MSCs were studied in co-culture with peripheral blood mononuclear cells (PB-MNCs) and PL-inflammatory cells (PL-ICs). RESULTS: PL-MSCs were characterized by typical MSCs phenotype, lower clonogenicity and self-renewal rate, compared to DF-MSCs and DP-MSCs. These cells possess the potential to differentiate into adipocyte-, osteoblast- and chondrocyte-like cells in vitro, which differs from that of DP-MSCs and DF-MSCs. PL-MSCs inhibited phytohemaglutinine-induced proliferation of PB-MNCs and production of IL-2, IFNγ and IL-5 in the co-culture, probably via TGF-ß-dependent mechanisms. These cells also suppressed the production of IL-1ß, IL-6, and TNF-α by PL-ICs via soluble mediators, whereas the suppression of IL-8 production required a direct cell-to-cell contact. CONCLUSION: The differentiation potential of PL-MSCs and their immunosuppressive/anti-inflammatory properties could be beneficial for the treatment of chronic periodontal diseases.

[Significance of pathohistological findings and the expression of Bcl-2 in diagnosis and treatment of oral planocellular carcinoma].

BACKGROUND/AIM: Numerous studies were aimed to detect and characterize various tumor markers in patients with oral planocellular carcinoma in order to reduce moratlity and mobidity rates of these patients, as well as to establish the correlation between the expression of specific tumor marker and prognostic outcome. The aim of this study was to determine patohistological characteristics of tumor and peritumor tissue in patients with oral planocellular carcinoma, with special regard to the expression of Bcl-2, as well as to point out the significance of clinicomorphological correlations for clinical use. METHODS: Sixty-two patients with oral planocellular carcinoma, stage II and III, were examined. The patients were surgically treated for this condition at the Clinic for Maxillofacial Surgery, Military Medical Academy, Belgrade. Surgical specimens were obtained from both tumor and peritumoral tissues. Patohistologic degree of tumor differentiation and the immunohistochemical expression of Bcl-2 were determinated for each specimens. RESULTS: Twenty-four (39%) patients had tumor dimension T1, while six (9%) and thirty-two (52%) patients had tumor dimension T2 and T3, respectively. Patohistologic analysis of peritumor connective, fat, muscle and bone tissue samples confirmed the presence of tumor infiltration. The expression of Bcl-2 in peritumor tissue samples correlated significantly with tumor's histologic grade (rho = 0.468; p < 0.001), nuclear grade (rho = 0.430; p < 0.001) and nucleocytoplasmic ratio (rho = 0.410; p = 0.001). CONCLUSION: This results suggest that the expression of Bcl-2 in combination with patohistologic findings could have a prognostic value in patients with oral planocellular carcinoma.

Similar developmental patterns of ghrelin- and glucagon-expressing cells in the human pancreas.

The pancreas appears to be a major source of ghrelin during fetal development, but the ontogeny of ghrelin cells in the human pancreas and their developmental relationship with α- and ß-cells remain largely unknown. In the present study, we examined the dynamics of ghrelin cell growth, colocalization of ghrelin with major pancreatic hormones and defined the similarities and differences among developmental patterns of ghrelin-, glucagon- and insulin-expressing cells in the human pancreas. To this end, paraffin-embedded pancreatic tissue sections from human embryos and fetuses were assessed by immunohistochemistry. Ghrelin-positive cells were first detected in the pancreas of 11-week-old fetuses. With advancing gestational age, both ghrelin- and glucagon-expressing cells were increasingly observed at the periphery of the developing islets, whereas insulin-containing cells were typically found in the islet core. Double immunohistochemistry showed that ghrelin-expressing cells were clearly separate from insulin-, somatostatin- and pancreatic polypeptide-containing cells. In contrast, cells coexpressing ghrelin and glucagon were sporadically detected during both the early and late fetal periods. Furthermore, morphometric analysis revealed a similar trend in the volume density of ghrelin- and glucagon-positive cells, and a contrasting pattern in ß-cell density at specific time points during the development of the human pancreas. This study demonstrates that the developmental pattern of ghrelin cells, although clearly distinct, is quite similar to that of glucagon-expressing cells. The obtained findings indicate a close lineage relationship between these cell populations, a functional relationship between their secretory products and an auto/paracrine mode of ghrelin-glucagon interaction in pancreatic development.

Increased angiogenesis-associated poor outcome in acute lymphoblastic leukemia: a single center study.

Angiogenesis in solid tumors is important for tumor growth, invasion, and metastasis. However, angiogenesis plays also an important role in hematological malignancies. We have analyzed the expression of vascular endothelial growth factor (VEGF) in the leukemic blast cells and microvessel density (MVD) in the bone marrow biopsy samples of the patients with acute lymphoblastic leukemia (ALL). Bone marrow MVD of the patients with ALL was significantly higher compared with normal controls and complete remission (P<0.001), but slightly lower than in patients with relapsed ALL (P>0.05). The bone marrow blast VEGF expression was significantly higher in newly diagnosed ALL patients, with predominant strong VEGF expression as compared with complete remission patients (who had negative or weak VEGF expression) (P<0.05), whereas initial values were slightly lower than in relapsed patients. There was a strong positive correlation between VEGF expression and MVD at presentation of ALL. Stronger expression of VEGF on blast cells indicates shorter overall survival in ALL. Furthermore, initial values of MVD had positive correlation with overall survival and leukemia-free survival (P=0.024 and P=0.017, respectively). Our data suggest that increased angiogenesis (confirmed by immunohistochemical expression of VEGF in leukemic blasts), and MVD may play an important role in the pathophysiology of ALL with prognostic implications. Thus, targeting VEGF pathway may bring the new approach for ALL treatment-using antiangiogenic drugs and tyrosine kinase inhibitors in combination with standard chemotherapy regimens.

Immunomodulatory properties of mesenchymal stem cells derived from dental pulp and dental follicle are susceptible to activation by toll-like receptor agonists.

Adult mesenchymal stem cells (MSCs) have recently become a potent tool in regenerative medicine. Due to certain shortcomings of obtaining bone marrow MSCs, alternate sources of MSCs have been sought. In this work, we studied MSCs from dental pulp (DP-MSCs) and dental follicle (DF-MSCs), isolated from the same tooth/donor, to define differences in their phenotypic properties, differentiation potential, and immunomodulatory activities. Both cell types showed colony-forming ability and expressed typical MSCs markers, but differed in the levels of their expression. DF-MSCs proliferated faster, contained cells larger in diameter, exhibited a higher potential to form adipocytes and a lower potential to form chondrocytes and osteoblasts, compared with DP-MSCs. In contrast to DF-MSCs, DP-MSCs produced the transforming growth factor (TGF)-ß and suppressed proliferation of peripheral blood mononuclear cells, which could be neutralized with anti-TGF-ß antibody. The treatment with toll-like receptor 3 (TLR3) agonist augmented the suppressive potential of both cell types and potentiated TGF-ß and interleukin-6 secretions by these cells. TLR4 agonist augmented the suppressive potential of DF-MSCs and increased TGF-ß production, but abrogated the immunosuppressive activity of DP-MSCs by inhibiting TGF-ß production and the expression of indolamine-2,3-dioxygenase-1. Some of these effects correlated with the higher expression of TLR3 and TLR4 by DP-MSCs compared with DF-MSCs. When transplanted in imunocompetent xenogenic host, both cell types induced formation of granulomatous tissue. In conclusion, our results suggest that dental MSCs are functionally different and each of these functions should be further explored in vivo before their specific biomedical applications.

Vasculature and neurovascular relationships of the trigeminal nerve root.

BACKGROUND: Specific microanatomical characteristics of the trigeminal nerve root (TNR) blood supply and close neurovascular relationships with surrounding vessels as well as their possible clinical significance were the main reasons for this study. METHOD: The vasculature of 25 adult and four fetal TNRs were microdissected and examined under the stereoscopic microscope, after injecting their arteries with India ink. RESULTS: The trigeminal vessels, which varied between two and five in number, arose from two or three of the following arteries: the superolateral pontine (92%), anterior inferior cerebellar (AICA) (88%), inferolateral pontine (72%), and superior cerebellar (SCA) (12%). The trigeminal vascular twigs had a mean diameter of 0.215 mm. A single vessel may supply either the motor portion of the nerve root or the sensory portion or both. The trigeminal vasculature formed the proximal and distal rings. The proximal ring was located at the trigeminal root entry zone. Its central branches extended along the TNR to the principal sensory and motor trigeminal nuclei while its peripheral longitudinal twigs followed the TNR fascicles. The incomplete distal arterial ring embraced the middle portion of the TNR before the level of its entrance into the arachnoid sleeve. The most frequent contact of the TNR was noticed with the SCA (20%), the petrosal or Dandy's vein (24%), and the AICA (12%). CONCLUSIONS: The observed characteristics of the TNR vasculature could be the anatomical basis for decompressive neurovascular surgery.

The internal thoracic artery as a transitional type of artery: a morphological and morphometric study.

Coronary artery by-pass grafting (CABG) with arterial grafts is widely accepted as the procedure of choice in the treatment of coronary ischemic disease. It brings back focus on morphological studies of arteries used as conduits in this procedure. One of the most frequently used CABG grafts is the internal thoracic artery with an excellent graft prognosis and patency rate. The aim of the study was a detailed morphological and morphometric description of the internal thoracic artery with an emphasis on its basic histological structure and its changes in aging and atherosclerosis. Therefore, 42 full-length arteries were obtained during forensic autopsies from 27 persons, aged between 20 and 81 years, who had died from non-vascular causes. The arteries were classified into three different age groups. Analysis of the serial arterial segments has shown that the internal thoracic artery is an artery of the transitional type whose media is organized into two layers: the internal, muscular layer and the external layer with spirally oriented elastic lamellae and smooth muscle cells in between. The number of elastic lamellae progressively decreases throughout the length of the examined arteries. As opposed to previous assumptions, we have proven that the grade of atherosclerosis is independent of the number of elastic lamellae in the external media. Perfectly formed elastic lamellae are not a persistent feature of the internal thoracic artery, as previously claimed. We have confirmed that the thickness of elastic lamellae decreases, while the number and the size of their fenestrations steadily increase with aging.

Immunohistochemistry of displaced sensory neurons in the trigeminal nerve root.

The aim of this study was to examine the morphology and the immunohistochemical features of displaced ganglion cells in the trigeminal nerve root (TNR). Forty human TNRs of 20 persons, obtained during routine autopsy in accordance with the Declaration of Helsinki, were examined following Klüver-Barrera and azan trichrome histological staining, and immunohistochemical reactions against certain neuronal markers, neuropeptides and neurotransmitters. A total number of 61 displaced neurons were investigated, which were present in 80% of individuals studied. Displaced neurons were found in 55.0% of the TNRs, either in the sensory portion (22.5%), motor portion (22.5%) or both (10.0%). Neuronal diameter varied from 12.5 x 25.0 to 45.0 x 63.7 (mean 27.6 x 41.6) microm, and in area between 245 and 2,065 (mean 927) microm(2). Each neuron was surrounded by 2-17 elongated satellite cells per slice. The immune reaction was positive in all the neurons studied for neuron-specific enolase, protein gene product 9.5, neurofilament protein and synaptophysin, and in some neurons for calcitonin gene-related peptide (CGRP; 24.4%), cholecystokinin (CCK; 13.3%), somatostatin (SST; 17.8%), substance P (SP; 15.6%), vasoactive intestinal polypeptide (4.4%), neuropeptide Y (8.9%), and serotonin (11.1%). The immune reactions were most frequent against the CGRP, SP, CCK and SST. We concluded that displaced neurons in the TNR morphologically and immunohistochemically resembled the sensory neurons in the trigeminal ganglion.

Ultrastructure and immunohistochemistry of the trigeminal peripheral myelinated axons in patients with neuralgia.

OBJECTIVE: Detailed ultrastructural and immunohistochemical examination of the trigeminal axons surrounded by the peripheral type of the myelin could add new information about the extent of the trigeminal nerve lesion in neuralgia. PATIENTS, MATERIALS AND METHODS: The examination comprised, firstly, the 10 trigeminal nerve roots (TNRs) in which the neurovascular contact was found in 20% of the cases, and the 2 additional control TNRs. Secondly, the biopsy specimens were taken from 6 patients with trigeminal neuralgia and 2 patients with trigeminal neuropathy following a partial TNR rhizotomy. The specimens were examined under the electron microscope (EM) and/or using the immunohistochemical (IHC) methods. RESULTS: In addition to the central zone of demyelination, the EM examination of the TNR also revealed alterations of the peripheral myelin, i.e. deformation, thickening, demyelination and remyelination, as well as changes of the peripheral axons, that is, atrophy or hypertrophy, neurofilaments increase, loss of the myelin and sprouting occasionally. Some Schwann cells were also damaged. The IHC examination usually showed a moderate immune reaction against neuron-specific enolase (NSE) and protein gene product 9.5 (PGP9.5), but sporadically weaker reaction against the S-100 protein, synaptophysin (SY), neurofilament protein (NFP) and glial fibrillary acidic protein (GFAP). The substance P (SP) and calcitonin gene-related peptide (CGRP) immunoreactivity was weak at some sites, but strong at some other places. CONCLUSIONS: The pathological changes affect not only the central nerve fibers of the TNR, but also some of the peripheral axons, their myelin sheath and Schwann cells. These are signs of the retrograde ultrastructural and biochemical alterations, which could participate in the pathophysiological mechanism underlying the trigeminal neuralgia.

Insulin-like growth factor-I in wound healing of rat skin.

Growth factors play an important role in orchestrating and enabling the cellular responses required for successful wound healing. In the present study, rat surgical incision was used to investigate insulin-like growth factor-I (IGF-I) expression in skin cells as well as its systemic and cutaneous tissue concentrations during acute phase of wound healing. Thirty two animals were sacrificed at days 2, 3, 5 and 9 after surgery. Eight animals were used as control. Tissue expression of IGF-I in both incisional and periincisional skin areas, as well as in skin of control unwounded animals was determined by immunohistochemistry. Serum and tissue concentrations of IGF-I were measured using RIA. Immunohistochemical analysis revealed enhanced IGF-I immunostaining in the incisional area at day 2 post-wounding. Presence of IGF-I immunoreactivity in the epidermis, as well as in dermal fibroblasts and monocytes within perivascular inflammatory infiltrate suggests its local synthesis. Although serum levels of IGF-I were not altered during wound healing, their tissue contents in the incisional area were significantly increased compared with periincisional area at days 2 and 3 after injury, as well as compared with skin content of unwounded control rats in all examined time points. Obtained results support a paracrine role of IGF-I during the acute phase of wound healing by primary intention in the rat.