Fisetin decreases the duration of ictal-like discharges in mouse hippocampal slices.

There is an increasing interest in the biological and therapeutic effects of fisetin, a natural phenolic compound. Fisetin has affinity on some neuronal targets and may have the potential to modulate neuronal activity. In this study the effects of acute application of fisetin on synchronized events were evaluated electro-physiologically. Besides, interaction of fisetin with closely related channels were investigated in silico. Acute horizontal hippocampal slices were obtained from 32- to 36-day-old C57BL/6 mice. Extracellular field potentials were recorded from CA3 region of the hippocampus. Bath application of 4 aminopyridine (4AP, 100 µM) initiated ictal- and interictal-like synchronized epileptiform discharges in the brain slices. Fifty micromolar fisetin was applied to the recording chamber during the epileptiform activity. The duration and frequencies of both ictal-like and interictal-like activities were calculated from the electrophysiological records. Molecular docking was performed to reveal interaction of fisetin on GABA-A, NMDA, AMPA receptors, and HCN2 channel, which are neuronal structures directly involved in recorded activity. Although fisetin does not affect basal neuronal activity in brain slice, it reduced the duration of ictal-like discharges significantly. Molecular docking results indicated that fisetin has no effect on GABA-A, NMDA, and AMPA receptors. However, fisetin binds to the (5JON) HCN2 channel strongly with the binding energy of -7.66 kcal/mol. Reduction on the duration of 4AP-induced ictal-like discharges can be explained as HCN channels can cause an inhibitory effect via enhancing M-type K + channels which increase K outward currents.

Microencapsulated parathyroid allotransplantation in the omental tissue.

Permanent hypoparathyroidism is a severe clinical condition accompanied by low parathyroid hormone level. Conventional treatment requires lifelong medication, and daily drug usage has some side effects. To avoid this circumstance, transplantation is an alternative and curative option. Microencapsulation may be used as a transplantation approach particularly to evade immune response. In order to define treatment of permanent hypoparathyroidism, a 37-year-old female recipient who has permanent hypoparathyroidism was evaluated for 3 years. Routine tests, viral markers, and T and B lymphocyte cross-match tests were analyzed. In addition intradermal skin test was performed for ultrapure alginate. Microencapsulation of cultured parathyroid cells was performed with ultrapure alginate. Cell suspension was prepared and spheroids were generated with calcium chloride. Afterward, transplantation was performed with a laparoscopic approach in the omental tissue. The recipient was discharged from the hospital without complications. Serum calcium, parathyroid hormone (PTH), and phosphorus levels were observed throughout 1 year. During the follow-up period, no complications were observed. Serum calcium levels were increased significantly on day 10 and PTH levels were increased on day 25 as well. According to our knowledge, this is the first study where ultrapure alginate-based microencapsulated parathyroid cells were transplanted in the omental tissue. A significant increment of PTH levels was detected. Microencapsulated parathyroid cells showed the functionality of this technique for more than 1 year. This study showed that using ultrapure alginate-based microencapsulation without immunosuppression appears to be a promising technique.

Calcium homeostasis in cisplatin resistant epithelial ovarian cancer.

Intracellular calcium concentration ([Ca2+]i) may have an important role in the development of chemoresistance, which is an essential problem in cancer chemotherapy. Cisplatin (DDP), which modulates the intracellular calcium concentration by different mechanisms, is an antineoplastic agent with high success rate in cancer therapies. We investigated the regulatory role of [Ca2+] in cisplatin resistance in epithelial ovarian cancer cell line, in MDAH-2774, and its chemoresistant subclone MDAH-2774/DDP. The measurement of [Ca2+]i using fluorescence microscope, and flow cytometry revealed that the amount of intracellular calcium decreased in cisplatin resistant cells compared to the amounts in parental cells. mRNA expression profiles of calcium homeostasis-associated major genes (IP3R1/2/3, RYR1/2, SERCA1/2/3, NCX1/2/3, PMCA1/2/3, and PMCA4) decreased in cisplatin resistant cell line in comparison to the expression profiles in parental cells. Owing to the changes in the expression of genes involved in calcium regulation, these results show, drug resistance may be prevented by introducing a new perspective on the use of inhibitors and activators of these genes, and thus of cytostatic treatment strategies, due to changes in the expression of genes involved in calcium regulation.

Fresh tissue parathyroid allotransplantation with short-term immunosuppression: 1-year follow-up.

BACKGROUND: Permanent hypoparathyroidism is a serious problem and requires medications indefinitely. Parathyroid allotransplantation (PA) with short-term immunosuppression is definitive choice but long-term results are not clear. METHOD: We performed PA from two donors to two recipients. Both recipients were 39-year-old females. Donors were a 32-year-old female and a 36-year-old male, who both have chronic kidney disease. Routine tests, viral markers, and cross-matches were analyzed individually. The parathyroid glands were resected from the living donors, fragmented quickly in the operation room and injected into the left deltoid muscles of the two recipients. RESULTS: Methylprednisolone was administered on post-PA day one and two. Recipients were discharged from the hospital without complications. Calcium and PTH levels were observed throughout 1 year. We did not observe any complications during the follow-up period. Medications ceased in post-transplantation week 1 for Case 1 and after 1 month for Case 2. CONCLUSION: Fresh tissue PA with short-term immunosuppression appears to be a promising technique that is easy to perform, is cost-effective, has low risk of side effects and minimal complications with compatibility for HLA conditions. A longer follow-up period and more case studies are needed to determine the risks and benefits of this procedure for future cases.

Effects of cilostazol on oxidative stress, systemic cytokine release, and spinal cord injury in a rat model of transient aortic occlusion.

BACKGROUND: Cilostazol is a phosphodiesterase inhibitor that has anti-inflammatory potential in addition to vasodilator and antiplatelet effects. The aim of this study was to determine the influence of cilostazol on biochemical markers of oxidative damage, proinflammatory cytokine release, and spinal cord injury after transient aortic occlusion in rats. METHODS: Animals were randomized into 3 groups. Sham group rats were subjected to laparotomy without aortic occlusion. Control group rats were pretreated with intraperitoneal dimethyl sulfoxide, and cilostazol group rats received intraperitoneal cilostazol (20 mg/kg/day) for 3 days before the induction of ischemia. Ischemia was induced by clamping of the infrarenal aorta, and 48 hours after reperfusion, Tarlov grades were assessed and spinal cord conduction velocities (SCCVs) were measured using epidural electrical stimulation. Erythrocyte superoxide dismutase (SOD) and catalase activities and plasma malondialdehyde, serum tumor necrosis factor-α, interleukin-1ß, and interleukin-6 levels were analyzed. Spinal cord histopathology was examined to determine neuronal damage and tissue inflammation. RESULTS: Aortic occlusion caused significant increases in SOD, catalase activities, and malondialdehyde and cytokine levels accompanied by spinal cord injury. Cilostazol significantly reduced malondialdehyde levels but did not significantly alter the activations of antioxidant enzymes, levels of proinflammatory cytokines, or histologic severity of inflammation. The differences regarding the results of Tarlov grading, SCCVs, and neuronal viability between the ischemic and cilostazol pretreated groups were statistically nonsignificant. CONCLUSION: The present experimental study indicated that cilostazol pretreatment used in this study before aortic occlusion decreased lipid peroxidation, which may be related to the reduction of reactive oxygen species. Cilostazol did not significantly suppress systemic cytokine release and prevent spinal cord inflammation and injury; however, it did show some benefit. Additional investigations might be needed to determine the critical dose of cilostazol for clarifying the protective role of this drug in spinal cord ischemia/reperfusion injury.

Alarin potentiates ongoing epileptiform activity in rat brain slices: an in vitro electrophysiological study.

Alarin is a newly discovered neuropeptide that belongs to the galanin peptide family with a wide range of bioactivity in the nervous system. Its function in the brain's autonomic areas has been studied, and it has been reported that alarin is involved in the regulation of excitability in hypothalamic neurons. Its role in the regulation of excitability in the hippocampus, however, is unknown. In this study, we investigated if alarin induced any synchronous discharges or epileptiform activity, and if it had any effect on already initiated epileptiform discharges. We used thick acute horizontal hippocampal slices obtained from 30­ to 35­day­old rats. Extracellular field potential recordings were evaluated in the CA1 region of the hippocampus. Our data demonstrated that, alarin application did not result in any epileptiform activity or abnormal discharges. 4­aminopyridine was applied to induce epileptiform activity in the slices. We found that alarin increased the frequency of interictal­like events and the mean power of local field potentials in the CA1 region of the hippocampus, which was induced by 4­aminopyridine. These results demonstrated for the first time that alarin has a modulatory effect on synchronized neuronal discharges and showed the contribution of the neuropeptide alarin to epilepsy­like conditions.

Effects of housing conditions on stress, depressive like behavior and sensory-motor performances of C57BL/6 mice.

BACKGROUND: The effects of housing conditions on animal physiology, behavior or stress are still debated. The aim of this study was to investigate the effects of three different housing systems, individually ventilated cages (IVC), classical small cages with floor surface area of 500 cm2 (CC500) and classical large cages with floor surface area of 800 cm2 (CC800) on body weight, sensory-motor performances, depression-like behavior, plasma corticosterone and brain oxidative stress parameters in C57BL/6 mice. The mice housed in one of the cages from birth to 6 months of age. Hang wire and adhesive removal tests were performed to evaluate somatosensory and motor performances. The extent of depression was determined by the forced swim test. Blood corticosterone levels were measured. In addition, brain malondialdehyde (MDA), total antioxidant status (TAS) and total oxidant status (TOS) levels were analyzed. RESULTS: The depression-like behavior of the groups was similar. Although there were no significant differences in hang wire test among groups, CC500 group required longer durations in adhesive removal test. The body weight and plasma corticosterone levels of CC800 group were significantly higher than other groups. The oxidative stress parameters were highest in CC500 cage. CONCLUSIONS: Our study showed that the least stressful housing condition was IVC cage systems. Interestingly, the number of mice in the classical cages had a significant effect on stress levels and sensory-motor performance.

Synthesis and photodynamic efficacy of water-soluble protoporphyrin IX homologue with mPEG550.

Protoporphyrin IX (PpIX), which is an efficient photosensitive agent, cannot be used directly in photodynamic therapy due to its aggregation in physiological environment. If PpIX is made water-soluble without losing its photosensitive properties, it can be used in many medical fields, including cancer treatment. Here we report synthesis of PpIX homologue with mPEG550 (Porfipeg) and its photodynamic effects on both in-vitro and in-vivo environment. Porfipeg is synthesized to give PpIX the ability to dissolve in water. Spectrometric (FT-IR, NMR, MS, UV-vis and Fluorescence) measurements were performed. Porfipeg can penetrate into the cells and indicates no cytotoxicity in the dark whereas cell viability significantly reduced with light irradiation. The cells can be visualized by fluorescence microscope. In-vivo experiment revealed that intravenous injection of Porfipeg is more efficient than intraperitoneal injection for the acute photodynamic effects within 30 min. Moreover it is excreted by the kidneys. In conclusion, Porfipeg has remarkable potentials to be used in both fluorescence guidance in surgeries and photodynamic therapy for cancer treatment.

In silicoinvestigation of Alliin as potential activator for AMPA receptor.

Natural products from plants, such as flavonoids, arouse immense interest in medicine because of the therapeutic and many other bioactive properties. The molecular docking is a very useful method to screen the molecules based on their free binding energies and give important structural suggestions about how molecules might activate or inhibit the target receptor by comparing reference molecules. Alliin and Allicin differ from many other flavonoids because of containing no benzene rings and having nitrogen and sulfur atoms in their structure. In this study Alliin and Allicin affinity on AMPA, NMDA and GABA-A receptors were evaluated in the central nervous system by using the molecular docking method. Both Alliin and Allicin indicated no inhibitory effects. However Alliin showed significant selectivity to human AMPA receptor (3RN8) as an excitatory. The binding energy of glutamate to 3RN8 was -6.61 kcal mol-1, while the binding energy of Allin was -8.08 kcal mol-1. Furthermore Alliin's affinity to the other AMPA and NMDA receptors is quite satisfactory compared to the reference molecule glutamate. In conclusion based on the molecular docking study, Alliin can be useful for synaptic plasticity studies whereas might be enhance seizure activity because of the increased permeability to cations. It also can be beneficial to improve learning and memory and can be used as a supportive product to the hypofunction of NMDA associated problems.

Xenotransplantation of Microencapsulated Parathyroid Cells as a Potential Treatment for Autoimmune-Related Hypoparathyroidism.

OBJECTIVES: Hypoparathyroidism occurs as a part of a complex autoimmune syndrome or iatrogenically after neck surgery. The disease presents many challenges, such as hypocalcemia, hyperphosphatemia, and low/undetectable parathormone levels. Allotrans - plantation of parathyroid tissue or cells has been reported as a promising option to overcome these effects. Transplantation of microencapsulated parathy - roid tissue or cells offers an immune escape, which particularly restores the parathyroid function for autoimmune-related hypoparathyroidism. So far, clinical and in vivo studies have demonstrated limited graft survival and instability for the available biocompatible materials. In addition, the transplant site, proper local isolation, and biocompatibility of materials are directly related to survival rate. MATERIALS AND METHODS: A microencapsulated parathy - roid xenotransplant model by using high guluronic acid-containing ultrapure alginate transplanted into rat omentum was tested in vivo for 1 year. RESULTS: After stability of empty microcapsules was ensured, parathyroid cells were microencapsulated and transplanted in rats, with results compared versus rats with naked (nonencapsulated) parathyroid cells (both groups followed for 64 weeks). Rats remained normocalcemic, and preinflammatory cytokine levels showed dramatic changes. Despite a delay posttransplant, parathormone levels increased significantly. All retrieved microencapsules elicited pericapsular fibrotic overgrowth; however, the fibrosis area was shown to be well tolerated. CONCLUSIONS: The possible role of accumulation/cell infiltration of immune response remains to be elucidated. In conjunction with this, the use of nonencapsulated parathyroid cells was also positively correlated with survival rates. A similar evaluation using ultrapure alginate materials and omental transplantation may enable the future determination for the long-term effects of correction of parathor - mone insufficiency in patients with severe hypocalcemic responses and other endocrine diseases.

Magnetic nanoparticle-mediated gene therapy to induce Fas apoptosis pathway in breast cancer.

CD95 (Fas) is a complex integral protein that can be expressed in many cells. It induces apoptosis when interacted with its ligand CD95L (FasL). However, cancer cells are resistant to CD95-induced apoptosis because of the changes in death domain (DD) of CD95 (procaspase-8 and c-Flip). In this study, magnetic nanoparticles and lipid-based gene transfection methods were performed to provide active Fas expression in breast cancer cells. Plasmid DNA (pDNA), which can express both human Fas and GFP, was transfected to MCF-7 breast cancer cells. Expression of c-FLIP and caspase-8 and effect of monoclonal antibody FasL for apoptosis stimulation were investigated. Also transfection success of methods and effects on surface protein were compared. Western blot results indicated that MCF-7 cells do not express caspase-8 but express large amount of c-FLIPL. Both lipid-based and magnetic nanoparticle-mediated gene transfection methods successfully applied. Caspase-8 apoptosis pathway was activated on transfected cells. Magnetic nanoparticle-mediated gene transfer is a successful non-viral method for transfection, and it does not affect the expression of other cell proteins, such as beta actin and lamin-B1. The raised c-FLIPL concentration in cytosol inhibits apoptosis. However, transfection of CD95-GFP-tagged pDNA significantly increases apoptosis by activating caspase-8 pathway. FasL interaction indicated a slight increase of apoptosis in the transfected cells. The method and pDNA applied in this study have potentials to be used in gene therapy for breast cancer.

A New Transport Solution for Parathyroid Allotransplantation: Effects on Cell Viability and Calcium-Sensing Receptors.

BACKGROUND: Cold ischemia protects organs and tissues by slowing their metabolism, but it also causes ischemic injury. Minimizing cold ischemia has been an important goal in parathyroid auto- and allotransplantation, as well as the transplantation of other major organs. Parathyroid glands are responsible for calcium homeostasis by releasing parathormone (PTH) into the blood circulation. Functionality of a new parathyroid transport solution (NPTS) and effects on cell viability, PTH secretion, and calcium-sensing receptor (CaSR) levels during cold ischemia were evaluated. MATERIALS AND METHODS: A NPTS was prepared, and the pH was adjusted to a range of 7.2-7.4 and kept at +4°C until use. Seven patients with parathyroid hyperplasia secondary to chronic renal failure who were scheduled to undergo subtotal parathyroidectomy were enrolled in the study. Glands were cold-preserved in NPTS with different time intervals (0, 6, 12, 18, and 24 hours), and then parathyroid cell viability before and after cryopreservation, PTH secretion, and CaSR levels were determined. RESULTS: The mean cell viability before cryopreservation was 92.7% (range 89.2%-97.2%). There were no significant differences in cell viability rates before and after cryopreservation (p = 0.1168 and p = 0.4085, respectively), and CaSR levels (p = 0.5446) were not significant. CONCLUSIONS: NPTS is a solution designed specifically for parathyroid tissue transplantation. This patent pending product can support cellular viability and PTH release, as well as protect CaSR functionality for up to 24 hours of cold ischemia.

Protoporphyrin IX-loaded magnetoliposomes as a potential drug delivery system for photodynamic therapy: Fabrication, characterization and in vitro study.

BACKGROUND: Protoporphyrin IX (PpIX) is a well-known photosensitizer that has great potential for use in photodynamic therapy (PDT). However, aggregation behavior of PpIX in neutral water makes it inappropriate for physiological studies. PpIX-loaded magnetoliposomes (MLs) were fabricated to increase PpIX biocompatibility. PpIX-loaded ML physical properties were characterized, and PpIX-loaded ML drug release behavior was investigated under the influence of an external magnetic field and heat. Toxicity and photodynamic effects of the complex were also examined using in vitro experiments with MCF-7 human breast cancer cells. METHODS: The magnetoliposomes were prepared with DPPC, DSPE-PEG2000 lipids and Fe3O4 nanoparticles. The toxicity and in vitro photodynamic effects of the PpIX-loaded MLs at various concentrations were studied using the MCF-7 cell line. RESULTS: The produced PpIX-loaded MLs exhibited an average hydrodynamic diameter of 221nm; however, TEM measurements indicated that the diameter of the PpIX-loaded MLs varied between 166 and 720nm. The iron content of the MLs affected cell viability less than the content of the iron free liposomes. Cell viability was reduced to 66% when the concentration of the PpIX-loaded MLs was 350nM, but when white light was applied for 5min, all of the cells that were exposed to concentrations of 250nM and higher PpIX died within 24h. CONCLUSION: The results of this study demonstrated the effective application of PpIX-loaded MLs for in vitro photodynamic therapy at nanomolar concentrations. The results also indicated that an LED light source provided sufficient energy to stimulate the PpIX molecules.