Ayhan Bilir

Ultrastructural Changes in Axons Following Exposure to Pulsed Radiofrequency Fields

Pulsed radiofrequency (PRF) fields applied by an electrode to neural structures, such as the peripheral sensory nociceptor axons and dorsal root ganglion, are clinically effective in reducing pain and other neuropathic syndromes. However, a full understanding of the underlying mechanisms by which this occurs has not yet been clarified. In this study, PRF is applied to the afferent axons of the sciatic nerves of rats. A standard radiofrequency (RF) electrode and RF generator is used to apply the RF signal output to the sciatic nerve using standard PRF parameters that have been successfully used in clinical practice. The ultrastructure of the treated axons is observed after 10 days by electron microscopy. A control, sham application is simultaneously applied to the contralateral sciatic nerve to provide a statistical differential comparison. It is found that the internal ultrastructural components of the axons show microscopic damage after PRF exposure, including: abnormal membranes and morphology of mitochondria, and disruption and disorganization of microfilaments and microtubules. The damage appears to be more pronounced for C‐fibers than for A‐delta and A‐beta fibers. The results are discussed in terms of internal electric field strengths and thermodynamic parameters.

Effects of pulsed versus conventional radiofrequency current on rabbit dorsal root ganglion morphology

Lesioning using radiofrequency (RF) current has been increasingly used in clinical practice for the treatment of pain syndromes. Although formation of heat causing “thermocoagulation” of the nervous tissues is thought to be responsible of the clinical outcome, a more recent modality of RF application named pulsed radiofrequency (PRF) delivers the RF current without producing destructive levels of heat. In our study, we compared the effects of conventional RF (CRF) and PRF on rabbit dorsal root ganglion (DRG) morphology, including also control and sham operated groups. The setting of the experiment and the RF parameters used were similar to those used in current clinical practice. The specimens were analyzed both with light microscopy and electron microscopy, two weeks after the procedure. At the light microscopic level, all groups had preserved the normal DRG morphology and no differences were observed between them. In the electron microscopic analysis there were no pathological findings in the control and sham operated groups. But the ganglion cells in the RF groups had enlarged endoplasmic reticulum cisterns and increased number of cytoplasmic vacuoles which were more evident in the CRF group. Some of the ganglion cells in the CRF group had mitochondrial degeneration, nuclear membrane disorders or loss of nuclear membrane and neurolemma integrity. The myelinated and unmyelinated nerve fibers were of normal morphology in all groups. Our results suggest that PRF application is less destructive of cellular morphology than CRF at clinically used “doses”. Before making certain judgements, more experimental and clinical studies should be planned.

The Effects of Melatonin on Angiogenesis and Wound Healing

PurposeThe pineal gland hormone melatonin is a well-known neoroendocrine hormone. In addition to its immunomodulator effect, it also has a positive effect on monocyte, cytokine, and fibroblast proliferations, which also influence angiogenesis. This study investigated the effects of melatonin hormone on angiogenesis in wound healing on 100 Wistar-Albino rats.MethodsThe rats were divided into two groups. Melatonin dissolved in 0.9% NaCl was administered to the study group in a dose of 0.4 mg/kg/rat per day (0.25 cc/rat per day), and 0.9% NaCl to the control group in a dose of 0.25 cc/rat per day. Incisions 5 cm in length were made on the back skin of the rats and the wounds were closed with a skin stapler. Excisional biopsies from healing tissues were taken on the 3rd, 7th, 10th, 14th, and 21st postoperative days. Angiogenesis was evaluated in healing tissues by light and electron microscopy and by hydroxyproline level measurements.ResultsThe commencement of neovascularization and a significant increase (P ≪ 0.05) in the number of vessels were observed at all stages of the study group but not in the control group. The tissue hydroxyproline levels were also higher in the study group than in the control group.ConclusionsMelatonin may have a positive effect on both angiogenesis and wound healing.

The role of cannula diameter in improved adipocyte viability: A quantitative analysis

BACKGROUND Fat injection for soft tissue augmentation is a common procedure in plastic surgery. Because the limitation of fat injection is its resorption, understanding how different handling techniques affect adipocyte survival is crucial to optimizing long-term results. OBJECTIVE In this study; we sought to determine the effect of aspiration and injection cannula diameters on adipocyte viability. METHODS Fat aspiration samples were obtained from 6 female patients undergoing abdominoplasty. Viable adipocytes were counted at fat suspension, which was obtained with 2-, 3-, and 4-mm-diameter aspiration cannulas and injected with 1.6-, 2-, and 2.5-mm-diameter injection cannulas. RESULTS A greater number of viable adipocytes were detected using a 4-mm-diameter aspiration cannula (419 x 10(4) cell/1 mL, P < .05) and a 2.5-mm-diameter injection cannula (410 x 10(4) cell/1 mL, P < .05). CONCLUSIONS The use of wider-diameter cannulas can potentially improve fat graft survival and reduce fat graft resorption.

Connective tissue alterations in women with pelvic organ prolapse and urinary incontinence

Background. Alterations in collagen synthesis and metabolism have previously been reported in patients with pelvic organ prolapse (POP) and/or urodynamic stress incontinence (USI). Since urinary incontinence does not always associate with POP, the objective of this study was to examine connective tissues from patients with USI plus POP, and patients with prolapse only. Methods. Biopsies from the uterosacral ligaments were obtained during the operation from POP patients (n =28), and from continent women (control group, n =12) who underwent surgery for other benign reasons. POP patients were classified following urodynamic tests and symptom questionnaire with respect to the presence (n =14) or absence (n =14) of USI. N‐terminal propeptides of collagen (PINP and PIIINP), TGF‐β and leptin were measured in plasma. Hydroxyproline and glycosaminoglycan (GAGs) concentrations and total hexosaminidase activity were measured in tissue samples. Histological sections were prepared using Massons trichrome technique, and digitised solutions were used for imaging provided by Soft Imaging System GmBh. Statistical evaluations were made by the Kruskal‐Wallis test. Results. A significant decrease in hydroxyproline content was found in USI+POP women in comparison to controls (p<0.05). In contrast, histopathological examination revealed an increased density of collagen in USI+POP patients. Hexosaminidase activity was decreased in both groups with POP, but no change in the amount of GAGs was observed. Markers of collagen synthesis (PINP, PIIINP), and factors related to the collagen synthesis (TGF‐β, leptin) remained unaltered. Conclusion. Our biochemical and morphological findings suggest a different organisation of collagen fibres in tissues of patients with USI+POP, when compared with both the controls and the POP patients.

Olea europaea leaf extract alters microRNA expression in human glioblastoma cells.

PurposeGlioblastoma multiforme (GBM) is the most common and the most lethal form of primary malignant tumors in the central nervous system. There is an increasing need for the development of more efficient therapeutic approaches for the treatment of these patients. One of the most attractive cancer therapy methods to date is the induction of tumor cell death by certain phytochemicals. Interestingly, bioactive compounds have been shown to alter micro RNA (miRNA) expression involved in several biological processes at the posttranscriptional level. The present study aimed to evaluate whether Olea europaea leaf extract (OLE) has an anticancer effect and modulates miRNA expression in GBM.Materials and methodsFirstly, the anti-proliferative activity of OLE and the nature of the interaction with temozolomide (TMZ) of OLE were tested in human glioblastoma cell line T98G cells by trypan blue and WST-1 assays and than realized miRNA PCR array analysis. Potential mRNA targets were analyzed bioinformatically.ResultsOLE exhibited anti-proliferative effects on T98G cell lines. Cells were treated with temozolomide (TMZ) in the presence OLE, and changes to miRNA expression levels were identified by PCR array analysis. miRNA target genes are involved in cell cycle and apoptotic pathways. Specifically, miR-181b, miR-153, miR-145, miR-137, and let-7d were significantly upregulated after treatment with both TMZ and OLE.ConclusionOur results suggest that OLE modulates the expression of some miRNAs related to anticancer activity in GBM and the response to TMZ. Further studies and validations are needed, but we suggest that OLE might be used for in vivo studies and future medical drug studies.

Resveratrol attenuates doxorubicin-induced cellular damage by modulating nitric oxide and apoptosis.

Although doxorubicin (DOX) is a commonly used chemotherapeutic agent its clinical use is restricted due to its organ toxicities. The present investigation relates to reducing DOX induced side effects to the liver, kidney and ileum by usage of the antioxidant, anti-inflammatory agent, resveratrol (RES) and to investigate the role of nitric oxide synthase (NOS) in the process. Wistar rats were divided into four groups: control (saline i.p); DOX (20 mg/kg i.p), RES (20 mg/kg i.p) and DOX (20mg/kg i.p)+RES (20 mg/kg i.p). Immunohistochemical activity of both iNOS and eNOS were evaluated after DOX treatment and ultrastructural changes such as cellular damage and mitochondrial degeneration were evaluated. Degenerative ultrastructural changes were demonstrated especially in the DOX treated group. Variations in biochemical marker levels of oxidative stress on ischemia in tissues were not observed. Our data indicate that RES may prevent cellular damage in the early phase of DOX induced toxicity. RES could be used with its beneficial effects during early cellular damage in organ toxicity after DOX treatment in cancer patients.

Autophagy and Nuclear Changes in FM3A Breast Tumor Cells after Epirubicin, Medroxyprogesterone and Tamoxifen Treatment in vitro

Objective: Autophagy is a form of physiological programmed cell death which is observable after hormonal withdrawal. In this study, the FM3A murine breast tumor cell line was treated with epirubicin alone and with medroxyprogesterone acetate (MPA) or tamoxifen, to determine if structural and kinetic signs of autophagy may also occur in an enhanced manner during epirubicin sensitization via hormonal agents. Methods: One-week soft agar colony growth, 96-hour values of plating and pulse thymidine labeling and electron microscopical examinations were performed following treatment with MPA and tamoxifen alone or with epirubicin. Results: Tamoxifen induced signs of autophagy, which was enhanced when it was combined with MPA. Epirubicin also induced autophagy of secretory granules, which coalesced to form an intracytoplasmic lumen. Combining MPA with epirubicin enhanced the formation of apoptotic blebs and chromatin fragmentation. When epirubicin was combined with tamoxifen, peculiar nuclear structures were formed. Conclusions: Hormonal agents may modulate anthracycline activity towards specific patterns in eliciting cell death, via autophagy and/or as yet unknown nucleolus-specific interactions.

The flavonoid apigenin reduces prostate cancer CD44(+) stem cell survival and migration through PI3K/Akt/NF-κB signaling.

AIMS Cancer stem cells (CSCs) are involved in drug resistance, metastasis and recurrence of cancers. The efficacy of apigenin on cell survival, apoptosis, migration and stemness properties were analyzed in CSCs. MAIN METHODS Prostate CSCs (CD44(+)) were isolated from human prostate cancer (PCa) PC3 cells using a magnetic-activated cell sorting system. PC3 and CSCs were treated with various concentrations of apigenin, docetaxel and their combinations for 48h. KEY FINDINGS Apigenin dose dependently inhibited CSCs and PC3 cell survival, and this was accompanied with a significant increase of p21 and p27. Apigenin induced apoptosis via an extrinsic caspase-dependent pathway by upregulating the mRNA expressions of caspases-8, -3 and TNF-α, but failed to regulate the intrinsic pathway as determined by the Bax, cytochrome c (Cyt-c) and APAF-1 in CSCs. In contrary to CSCs, apigenin induced intrinsic apoptosis pathway as evidenced by the induction of Bax, Cyt-c and caspase-3 while caspase-8, TNF-α and Bcl-2 levels remained unchanged in PC3 cells. The flavonoid strongly suppressed the migration rate of CSCs compared to untreated cells. Significant downregulation of matrix metallopeptidases-2, -9, Snail and Slug exhibits the ability of apigenin treatment to suppress invasion. The expressions of NF-κB p105/p50, PI3K, Akt and the phosphorylation of pAkt were decreased after apigenin treatment. Moreover, apigenin treatment significantly reduced pluripotency marker Oct3/4 protein expression which might be associated with the down-regulation of PI3K/Akt/NF-κB signaling. SIGNIFICANCE Our data indicated that, apigenin could be a useful compound to prevent proliferation and migration of cancer cells as well as CSCs.

Expression profiling of stem cell signaling alters with spheroid formation in CD133high/CD44high prostate cancer stem cells

Cancer stem cells (CSC) isolated from multiple tumor types differentiate in vivo and in vitro when cultured in serum; however, the factors responsible for their differentiation have not yet been identified. The first aim of the present study was to identify CD133high/CD44high DU145 prostate CSCs and compare their profiles with non-CSCs as bulk counterparts of the population. Subsequently, the two populations continued to be three-dimensional multicellular spheroids. Differentiation was then investigated with stem cell-related genomic characteristics. Polymerase chain reaction array analyses of cell cycle regulation, embryonic and mesenchymal cell lineage-related markers, and telomerase reverse transcriptase (TERT) and Notch signaling were performed. Immunohistochemistry of CD117, Notch1, Jagged1, Delta1, Sox2, c-Myc, Oct4, KLF4, CD90 and SSEA1 were determined in CSC and non-CSC monolayer and spheroid subcultures. Significant gene alterations were observed in the CD133high/CD44high population when cultured as a monolayer and continued as spheroid. In this group, marked gene upregulation was determined in collagen type 9 α1, Islet1 and cyclin D2. Jagged1, Delta-like 3 and Notch1 were respectively upregulated genes in the Notch signaling pathway. According to immunoreactivity, the staining density of Jagged1, Sox2, Oct4 and Klf-4 increased significantly in CSC spheroids. Isolated CSCs alter their cellular characterization over the course of time and exhibit a differentiation profile while maintaining their former surface antigens at a level of transcription or translation. The current study suggested that this differentiation process may be a mechanism responsible for the malignant process and tumor growth.