Ibrahim Tuglu

A histological analysis of the protective effect of ischemic preconditioning in the rat retina.

Purpose. Ischemic preconditioning (IP) protects the retina from the damaging effect of subsequent ischemia in vivo. We aimed to investigate the histological alterations induced by the protective effect of IP to the retina. Methods. The eyes of the rats were rendered ischemic by intra-ocular pressure (IOP) elevation. IP procedure consisted of producing ischemia for 5 minutes. Sham operation was similar to IP procedure except the pressure elevation. The operational eyes of sham and IP group underwent 60 minutes of ischemia 24 hours after the first procedure. The eyes contralateral to the experimental eyes made up the control group. The eyes were histologically analysed one week after the ischemia. Results. The total retinal thickness of the sham group was significantly less than total retinal thickness of the control group (p < 0.001). There was not a significant difference between control and IP group regarding the total retinal thickness (p > 0.05). The thickness of the inner retinal layers of the sham group were significantly less than corresponding retinal layers of the control group (p < 0.001). The inner plexiform layer (IPL) and inner nuclear layer (INL) thickness values of the sham group were significantly less than same layers of the IP group (p < 0.001). Ganglion cell layer (GCL) thickness of the IP group was significantly less than GCL thickness of the control group (p < 0.001). IPL thickness of the IP group was significantly less than that of control groups (p < 0.05). The GCL and total retinal thickness of the IP group were significantly more than thickness of the corresponding layers of the sham group (p < 0.05). Conclusion. IP considerably protects inner retinal layers from subsequent ischemic damage in a high IOP ischemic model. This endogenous process could further be utilized to tailor specific neuroprotective strategies for retinal cells.

The effect of autologous bone marrow stromal cells differentiated on scaffolds for canine tibial bone reconstruction

Bone marrow contains mesenchymal stem cells that form many tissues. Various scaffolds are available for bone reconstruction by tissue engineering. Osteoblastic differentiated bone marrow stromal cells (BMSC) promote osteogenesis on scaffolds and stimulate bone regeneration. We investigated the use of cultured autologous BMSC on different scaffolds for healing defects in tibias of adult male canines. BMSC were isolated from canine humerus bone marrow, differentiated into osteoblasts in culture and loaded onto porous ceramic scaffolds including hydroxyapatite 1, hydroxyapatite gel and calcium phosphate. Osteoblast differentiation was verified by osteonectine and osteocalcine immunocytochemistry. The scaffolds with stromal cells were implanted in the tibial defect. Scaffolds without stromal cells were used as controls. Sections from the defects were processed for histological, ultrastructural, immunohistochemical and histomorphometric analyses to analyze the healing of the defects. BMSC were spread, allowed to proliferate and differentiate to osteoblasts as shown by alizarin red histochemistry, and osteocalcine and osteonectine immunostaining. Scanning electron microscopy showed that BMSC on the scaffolds were more active and adhesive to the calcium phosphate scaffold compared to the others. Macroscopic bone formation was observed in all groups, but scaffolds with stromal cells produced significantly better results. Bone healing occurred earlier and faster with stromal cells on the calcium phosphate scaffold and produced more callus compared to other scaffolds. Tissue healing and osteoblastic marker expression also were better with stromal cells on the scaffolds. Increased trabecula formation, cell density and decreased fibrosis were observed in the calcium phosphate scaffold with stromal cells. Autologous cultured stromal cells on the scaffolds were useful for healing of canine tibial bone defects. The calcium phosphate scaffold was the best for both cell differentiation in vitro and bone regeneration in vivo. It may be possible to improve healing of bone defects in humans using stem cells from bone marrow.

Neurotoxic effect of Caulerpa racemosa var. cylindracea by neurite inhibition on the neuroblastoma cell line

In the present study, antiproliferative, apoptotic and especially neurotoxic effects of Caulerpa racemosa var. cylindracea dry and wet extracts on mouse neuroblastoma cell line, NA2B were investigated by neurotoxicity screening test (NST). C. racemosa var. cylindracea wet and dry extracts were obtained by methanol (MT) extraction. The effect of the extracts on viability and proliferation was measured by MTT. NA2B cells were induced to differentiate using 1 μM dcAMP and the amount of inhibition of growing neurites in different dilutions (50, 35, 25, 15, 10 and 5 μl/ml) by extracts was measured. The number of apoptotic cells was computed by TUNEL method using cells in culture. It was found that majority of the cells died with dry extract above the level of 15 μl/ml due to the MT effect. Below this level, on the other hand, presence of cell death and antiproliferative effect was noted due to the toxic effects of C. racemosa var. cylindracea which was independent of MT. In all doses of wet extracts, similar but less prominent dose-dependent effects were observed. Below the level of 15 μl/ml, mild toxic effect presented itself with neurite inhibition. In addition to the toxic, apoptotic and antiproliferative effects of C. racemosa var. cylindracea, its neurotoxic effects possessing property at low concentrations which manifesting itself by neurite inhibition was also showed. This species offers a potential for developing new drugs due to its antiproliferative, toxic and apoptotic effects. Nevertheless, its neurotoxic effect is a factor to be considered as multifunctional agents especially in neuronal metabolism.

The effect of different implant biomaterials on the behavior of canine bone marrow stromal cells during their differentiation into osteoblasts

We investigated the effects of different implant biomaterials on cultured canine bone marrow stromal cells (BMSC) undergoing differentiation into osteoblasts (dBMSC). BMSC were isolated from canine humerus by marrow aspiration, cultured and differentiated on calcium phosphate scaffold (CPS), hydroxyapatite, hydroxyapatite in gel form and titanium mesh. We used the MTT method to determine the effects of osteogenic media on proliferation. The characteristics of dBMSC were assessed using alizarin red (AR), immunocytochemistry and osteoblastic markers including alkaline phosphatase/von Kossa (ALP/VK), osteocalcin (OC) and osteonectin (ON), and ELISA. The morphology of dBMSC on the biomaterials was investigated using inverted phase contrast microscopy and scanning electron microscopy. We detected expression of ALP/VK, AR, OC and ON by day 7 of culture; expression increased from day 14 until day 21. CPS supported the best adhesion, cell spreading, proliferation and differentiation of BMSCs. The effects of the biomaterials depended on their surface properties. Expression of osteoblastic markers showed that canine dBMSCs became functional osteoblasts. Tissue engineered stem cells can be useful clinically for autologous implants for treating bone wounds.

Effects of caffeic acid phenethyl ester on matrix molecules and angiogenetic and anti-angiogenetic factors in gastric cancer cells cultured on different substrates.

Migration, invasion, metastasis and angiogenesis associated with cancer depend on the surrounding microenvironment. Angiogenesis, the growth of new capillaries, is a regulator of cancer growth and a useful target for cancer therapy. We examined matrix protein interactions in a gastric cancer cell culture that was treated with different doses of caffeic acid (3,4-dihydroxycinnamic acid) phenethyl ester (CAPE). We also investigated the relations among the levels of vascular endothelial growth factor (VEGF), matrix metalloproteinase-9 (MMP-9), endostatin (ES) and trombospondin-1 (TSP-1). Cytotoxity of CAPE was measured using the 3-(4,5-dmethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. We examined the behavior of cells on laminin and collagen I coated surfaces in response to the angiogenic effect of these matrix molecules. We examined the protein alterations of these matrix molecules immunohistochemically and measured the levels of VEGF, MMP-9, ES and TSP-1 using the ELISA test. We showed that application of CAPE to the gastric cancer cell line on tissue culture plastic, laminin and collagen I significantly decreased the VEGF and MMP-9 protein levels. We found that TSP-1 levels were increased significantly in the gastric cancer cells after application of CAPE. The protein levels of gastric cancer cells also were increased significantly when tissue was cultured on laminin and collagen I. Application of CAPE to cells on laminin or collagen I coated surfaces significantly increased all of the proteins except ES. ES levels were increased on the collagen I covered surfaces, but the laminin surface decreased the levels of ES significantly. We demonstrated the beneficial effect of CAPE on a gastric cancer cell line including inhibition of proliferation and induction of some proteins that might be related to decreased angiogenesis.

Insulin: does it induce follicular arrest in the rat ovary?

The goal of this study was to investigate histological changes of the rat ovary treated with either insulin or insulin plus human chorionic gonadotropin (hCG). The study was conducted in Celal Bayar University, School of Medicine, Animal Research Laboratory. Eighteen adult female Wistar rats were divided into three groups to receive saline, or insulin, or insulin plus hCG for 4 weeks. At the end of treatment the rats were sacrificed and the ovaries were evaluated with hematoxylin and eosin. There was no abnormal change in rats treated with saline. A thickened capsule, stromal hypertrophy and stromal cell hyperplasia, and no developing follicles, were observed in the insulin-only group. A thin capsule, developing follicles and corpora lutea, and normal theca cells and stroma were observed in the insulin-plus-hCG group. We conclude that insulin may lead to histological changes similar to stromal hyperthecosis and polycystic ovary syndrome, and may be one of the factors causing follicular arrest.

Attachment and growth of dental pulp stem cells on dentin in presence of extra calcium.

OBJECTIVE We aimed to differentiate dental pulp stem cells (DPSC) to odontoblast-like cells (ODPSC) and to investigate their attachment and growth on dentin in the presence of extra calcium by colorimetric assay and scanning electron microscopy (SEM). METHODS After isolation of DPSC, they were differentiated to ODPSC. Standard dentin discs from human molar teeth were prepared. While the dentin discs in Group 1 did not receive any extra treatment, the discs in Group 2 were treated with acidic calcium phosphate precipitation (CPP) solution. In Group 3, the discs were suspended in phosphate buffered saline containing calcium. DPSC or ODPSC (3×10(4) cells/mL) were seeded on all discs and incubated for 7, 14 or 21 days. Attachment and growth of 7-day cell cultures on extra dentin samples were examined by SEM. MTT assay showed that number of cells on dentin surfaces was increased by time periods regardless of type of treatment and cells (p<0.05). RESULTS While DPSC and ODPSC showed similar proliferation rates at 7 and 14days (p>0.05), the number of ODPSC was higher than DPSC in 21-day samples (p=0.039). MTT assay showed that number of cells on dentin surfaces was increased by time periods regardless of type of treatment and cells (p<0.05). Calcium-treated dentin surfaces always had lower number of cells; being significant for only CPP-treated surfaces (p<0.01). Both types of cells demonstrated good attachment and proliferation on dentin surfaces regardless of type of dentin treatment. CONCLUSIONS Because the nature of dentin surface itself showed good adhesive characteristics with ODPSC and DPSC, additional calcium treatment of dentin surfaces may not be necessary.

THE effect of tibolone on endometrial IGF-1 and IGFBP-1 levels in ovariectomized rats

OBJECTIVE The goal of this study was to search the effects of two different doses of tibolone on endometrial IGF-1 and IGFBP-1 levels in ovariectomized rats. METHODS Eighteen adult, female, 80-90-days-old, Wistar rats with an average weight of 250 g underwent bilateral ovariectomy under general anesthesia. After waiting for 4 weeks, they were randomized into three groups to receive either oral tibolone in two different doses or placebo. The treatment was continued for 5 weeks, and then the rats were sacrificed and the endometria were analyzed. RESULTS Low columnar epithelium of the endometrial surface, longer epithelium and stratified squamous epithelium were seen in the control, low-dose and high-dose groups, respectively. The staining intensity of IGF-1 was mild in control, and moderate in both treatment groups, the difference between control the treatment groups was significant (P=0.015 for group L, and P=0.03 for group H). The staining intensity of IGFBP-1 was moderate in control, and strong in groups L and H. Again the difference was significant between control and both treatment groups (P=0.039 for grup L, and P=0.03 for group H). No significant difference was noted between each treatment group for both IGF-1 and IGFBP-1. CONCLUSION Tibolone caused histological changes in endometrium and stimulated IGF-1 and IGFBP-1 staining. Both low and high dose treatments led to moderate and strong staining intensities for IGF-1 and IGFBP-1, respectively. The strong staining intensity of IGFBP-1 is likely due to the progestagenic effect of tibolone.

Acute and Chronic Pretreatment With Atenolol Attenuates Intestinal Ischemia and Reperfusion Injury in Hypercholesterolemic Rats

OBJECTIVE To evaluate the protective effects of preinjury atenolol (acute v chronic) on apoptosis, contractility, oxidative stress, and inflammatory markers in hypercholesterolemic rats undergoing intestinal ischemia-reperfusion (I/R) injury. DESIGN Prospective, experimental animal study. SETTING University laboratory. PARTICIPANTS Male Wistar rats (n = 32). INTERVENTIONS Rats were divided into the following 4 groups: 1 group was fed a normal diet (ND) (group ND+NoAT [no atenolol]), and the other 3 groups were fed a high-cholesterol diet (HCD)-group HCD+NoAT, group HCD+ChAT (chronic atenolol, 3 mg/kg/day for 8 weeks), and group HCD+AcAT (acute atenolol, 1.5 mg/kg, given 5 minutes before intestinal clamping). All rats underwent I/R injury. The superior mesenteric artery was clamped for 60 minutes, then opened for 120 minutes (reperfusion). Apoptotic cells and stimulated contractions of ileal segments were examined. Tissue markers of intestinal I/R injury were examined. Intestinal malondialdehyde, superoxide dismutase, and nitrate/nitrite levels were measured. MEASUREMENTS AND MAIN RESULTS The chronic atenolol group had fewer apoptotic cells and higher superoxide dismutase activity compared with the other groups. Intestinal contraction was higher in both atenolol pretreatment groups compared with the NoAT groups. Chronic and acute atenolol resulted in lower ileal levels of malondialdehyde and immunolabeling-positive cells (intestinal inducible nitric oxide synthase, endothelial nitric oxide synthase, interleukin-1, and interleukin-8) after I/R injury compared with the no atenolol groups. CONCLUSIONS Both chronic and acute pre-I/R injury treatment with atenolol attenuated I/R injury in this hypercholesterolemic rat model. These findings should encourage future studies of atenolol in hypercholesterolemic patients undergoing procedures with a high risk of intestinal ischemia.

In vitro investigation of the effect of matrix molecules on the behavior of colon cancer cells under the effect of geldanamycin derivative

The chaperone-binding drug, 17-allylamino-17-demethoxygeldanamycin, has recently come into clinical use. It is a derivative of geldanamycin, an ansamycin benzoquinone antibiotic with anti-carcinogenic effect. Understanding the effect of this drug on the cancer cells and their niche is important for treatment. We applied 17-allylamino-17-demethoxygeldanamycin to colon cancer cell line (Colo 205) on matrix molecules to investigate the relationship of apoptosis with terminal deoxynucleotidyl transferase dUTP nick end labeling immunocytochemistry and related gene expression. We used laminin and collagen I for matrix molecules and vascular endothelial growth factor for angiogenic structure. We also examined apoptosis-related signaling pathway including mitochondrial proteins, cytochrome c, Bcl-2, caspase-9, Apaf-1 expression using real-time polymerase chain reaction. There was clear effect of 17-allylamino-17-demethoxygeldanamycin that killed more cells on tissue culture plastic compared to matrix molecules. The IC50 value was 0.58 µg/mL for tissue culture plastic compared with 0.64 µg/mL for laminin and 0.75 µg/mL for collagen I. The analyses showed that more cells on matrix molecules underwent apoptosis compared to that on tissue culture plastic. Apoptosis-related gene expression was similar in which Bcl-2 expression decreased and proapoptotic gene expression of the cells on matrix molecules increased compared to that on tissue culture plastic. However, the application of 17-allylamino-17-demethoxygeldanamycin was more effective for the cells on collagen I compared to the cells on laminin. There was also a decrease in angiogenesis as shown by the vascular endothelial growth factor staining. This was more pronounced by coating of the tissue culture plastic with matrix molecules. Our results supported the anti-cancer effect of 17-allylamino-17-demethoxygeldanamycin, and this effect depended on matrix molecules. This effect occurs through apoptosis, and related genes were also altered. All these genes may serve for novel target under the effect of matrix substrate. However, correct interpretation of the results requires further studies.