Synthesis of Novel Plant-Derived Encapsulated Radiolabeled Compounds for the Diagnosis of Parkinson's Disease and the Evaluation of Biological Effects with In Vitro/In Vivo Methods.

Parkinson's disease (PD) is a neurodegenerative disorder that affects millions of individuals globally. It is characterized by the loss of dopaminergic neurons in Substantia Nigra pars compacta (SNc) and striatum. Neuroimaging techniques such as single-photon emission computed tomography (SPECT), positron emission tomography (PET), and magnetic resonance imaging (MRI) help diagnosing PD. In this study, the focus was on developing technetium-99 m ([99mTc]Tc) radiolabeled drug delivery systems using plant-derived compounds for the diagnosis of PD. Madecassoside (MA), a plant-derived compound, was conjugated with Levodopa (L-DOPA) to form MA-L-DOPA, which was then encapsulated using Poly Lactic-co-Glycolic Acid (PLGA) to create MA-PLGA and MA-L-DOPA-PLGA nanocapsules. Extensive structural analysis was performed using various methods such as Fourier-transform infrared spectroscopy (FTIR), nuclear magnetic resonance spectroscopy (NMR), liquid chromatography-mass spectrometry (LC-MS), thin layer chromatography (TLC), high performance liquid chromatography (HPLC), dynamic light scattering (DLS), and scanning electron microscopy (SEM) to characterize the synthesized products. Radiochemical yields of radiolabeled compounds were determined using thin layer radio chromatography (TLRC) and high performance liquid radio chromatography (HPLRC) methods. In vitro cell culture studies were conducted on human neuroblastoma (SH-SY5Y) and rat pheochromocytoma (PC-12) cell lines to assess the incorporation of [99mTc]Tc radiolabeled compounds ([99mTc]Tc-MA, [99mTc]Tc-MA-L-DOPA, [99mTc]Tc-MA-PLGA and [99mTc]Tc-MA-L-DOPA-PLGA) and the cytotoxicity of inactive compounds (MA and MA-L-DOPA compounds and encapsulated compounds (MA-PLGA and MA-L-DOPA-PLGA). Additionally, the biodistribution studies were carried out on healthy male Sprague-Dawley rats and a Parkinson's disease experimental model to evaluate the compounds' bioactivity using the radiolabeled compounds. The radiochemical yields of all radiolabeled compounds except [99mTc]Tc-L-DOPA-PLGA were above 95% and had stability over 6 h. The cytotoxic effects of all substances on SH-SY5Y and PC-12 cells increase with increasing concentration values. The uptake values of PLGA-encapsulated compounds are statistically significant in SH-SY5Y and PC-12 cells. The biodistribution studies showed that [99mTc]Tc-MA is predominantly retained in specific organs and brain regions, with notable uptake in the prostate, muscle, and midbrain. PLGA-encapsulation led to higher uptake in certain organs, suggesting its biodegradable nature may enhance tissue retention, and surface modifications might further optimize brain penetration. Overall, the results indicate that radiolabeled plant-derived encapsulated drug delivery systems with [99mTc]Tc hold potential as diagnostic agents for PD symptoms. This study contributes to the advancement of drug delivery agents in the field of brain research.

Evaluating the Glucantime Concentration for the ex vivo Glial Cell Model of Antimony-resistant Leishmania tropica Amastigotes

Objective: Because the protocols used in the treatment of leishmaniasis can be toxic and have many limitations, such as the development of resistance against such protocols, new treatment options are needed, especially against resistant patients. Ex vivo models may be a good source for evaluating new drug options for patients with antimony-resistant parasites. This study aimed to evaluate the Glucantime concentration for our ex vivo glial cell amastigote model we had defined in previous work. Methods: We prepared the astroglial cell culture from brains of 2 to 3 day old neonatal Sprague-Dawley rats under sterile conditions by modifying McCarthy's method. Four plates of cells were infected with antimony-resistant Leishmania tropica promastigotes. After 24 h of incubation, we added Glucantime to 3 plates with different concentrations. After 72 h, we removed the supernatant and then dried, fixed, and stained the plates with Giemsa to count the amastigotes in the glial cells. Results: We observed the amastigotes in glial cells in the control flask. Glial cells were ruined in flasks, which include 75 µg/mL and 37.5 µg/mL Glucantime. The number of amastigotes per 100 glial cells was 116 for the flask with 7.5 µg/mL Glucantime concentration, while 487 for the control flask. Conclusion: We found that while high concentrations of Glucantime were toxic for glial cells, 7.5 µg/mL Glucantime concentration managed to reduce the number of Leishmania tropica amastigotes in glial cells.

[Infecting glial cells with antimony resistant Leishmania tropica: A new ex-vivo model]. / Glia hücrelerinin antimona dirençli Leishmania tropica ile enfekte edilmesi: Yeni bir ex-vivo modeli.

Leishmaniasis is a vector-borne zoonotic disease that shows different clinical features like cutaneous, mucocutaneous, visceral and viscerotropic forms. The protocols used in the treatment of leishmaniasis are toxic and have many limitations during administration. One of the limitations of treatment is the resistance against the protocols in practice. There is also a need to define new treatment options especially for resistant patients. Ex-vivo models using primary cell cultures may be a good source for evaluating new drug options in patients with antimony resistance, in addition to in-vitro and in-vivo studies. In this study, it was aimed to define a new ex-vivo culture model to evaluate treatment options in patients with cutaneous leishmaniasis who did not respond to treatment. In our experimental model of ex-vivo infection, Leishmania tropica promastigotes isolated from a case previously diagnosed with cutaneous leishmaniasis were used. The primary astroglial cell culture used for the ex-vivo model was prepared from 2-3 days old neonatal Sprague Dawley rat brains under sterile conditions by the modification McCarthy's method. The astroglia cells, which reached sufficient density, were infected with antimony resistant L.tropica promastigotes. After 24 hours of incubation, the supernatant on the cells were collected, the cell culture plate was dried at room temperature, then fixed with methyl alcohol and stained with Giemsa to search for L.tropica amastigotes. Amastigotes were intensely observed in glia cells in primary cell cultures infected with L.tropica promastigotes. No promastigotes were seen on Giemsa stained preparations of the precipitates prepared from the bottom sediment after the centrifugation of the liquid medium removed from the infected plates. In this study, promastigotes from a cutaneous leishmaniasis patient unable to respond to pentavalent antimony therapy were shown to infect rat glia cells and converted to amastigote form. This amastigote glial cell model, as far as we know, is the first model in the literature produced by L.tropica. The occurrence of L.tropica amastigote forms in glia cells may be indicative of the ability of Leishmania species to infect the central nervous system. The central nervous system may be an area for the Leishmania amastigotes to escape from the immune system in cases of leishmaniasis without a treatment response. Our study is important because it is the first study to show the infection of glia cells with L.tropica amastigotes.

Protective effect of edaravone against manganese-induced toxicity in cultured rat astrocytes.

Manganese (Mn), a trace metal, is essential for maintaining the normal regulation of many biochemical and cellular processes. However, accumulation of Mn due to excessive environmental exposure leads to neurological impairment, referred to as manganism. Edaravone (EDA) is a potent free radical scavenger that has been clinically shown to reduce the neuronal injury after cerebral ischemia. In the present study, we aimed to examine the protective effects of EDA against Mn toxicity in astrocyte cultures. Astrocyte cultures were prepared from cerebral cortices of newborn Sprague-Dawley rats. The experiments were performed between 16 and 18 days of cultures. Astrocytes were treated in DMEM medium containing Mn (1-1000µM) for 24h to test Mn toxicity. In order to assess the effect of EDA, cells were pre-treated with different doses of EDA (10, 100 and 1000µM) 6h before Mn treatment. Cell viability (MTT), apoptotic cell death (Hoechst test) and lipid peroxide levels were evaluated in cultures. Our results showed that Mn significantly and dose-dependently reduced cell viability in astrocyte cultures. The apoptotic cell death and lipid peroxides were significantly higher in Mn treated cultures. Treatment of astrocytes with EDA successfully suppressed oxidative stress and cell death due to Mn exposure. The findings of the present study suggest that Mn cytotoxicity is mainly associated with ROS generation and apoptotic cell death. Besides, EDA may have beneficial effects against Mn toxicity. However, further studies are needed to elucidate the molecular mechanisms underlying protective effect of EDA.

Manganese-enhanced MRI to evaluate neurodegenerative changes in a rat model of kainic acid-induced excitotoxicity.

PURPOSE: Manganese-enhanced magnetic resonance imaging (MEMRI) has been used to detect brain activity based on the ability of active neurons to take up manganese ions through calcium channels. Kainic acid (KA), an analog of excitotoxic glutamate, can elicit selective neuronal death in the brains of rodents, of which the pathological changes partially mimic neurodegeneration in the central nervous system. We used in vivo MEMRI to evaluate neurodegenerative changes in an excitotoxicity model induced by KA in rats. MATERIALS AND METHODS: Adult Sprague-Dawley rats (220-250 g) were injected with either KA or saline into the right lateral ventricle. Precontrast and postcontrast MEMRI sessions were obtained. Region of interest (ROI) analyses were performed on both injected (saline and KA) and contralateral (normal) sites in the hippocampal area. All brains were evaluated histologically following MEMRI. RESULTS: Analysis of percentage change in ROI intensities of T1-weighted fluid-attenuated inversion-recovery MR images in the hippocampal area revealed a significant difference between the KA-injected (ipsilateral) and contralateral sites (P = 0.008), whereas no significant difference was observed between the saline-injected and contralateral sites. Furthermore, there was a significant difference between ipsilateral sites of the saline-treated and KA-treated groups (P = 0.026). The histological results supported these findings. CONCLUSION: MEMRI is a simple and useful in vivo method for detecting neurodegenerative changes due to excitotoxicity in the rat brain. The development of a manganese-based contrast agent that can be safely used in humans is warranted to investigate neurological disorders.

Oxytocin inhibits pentylentetrazol-induced seizures in the rat.

We aimed to reveal the anti-convulsant effects of oxytocin (OT) in pentylenetetrazol (PTZ)-induced seizures in rats. Thirty rats were randomly divided into 5 equal groups. Using stereotaxy, we implanted electroencephologram (EEG) electrodes in the left nucleus of the posterior thalamus. After 2 days, the first and second groups were used as the control and PTZ (35 mg/kg) groups, respectively. We administered 40, 80 and 160 nmol/kg OT+35 mg/kg PTZ to the rats, constituting the third, fourth, and fifth groups, respectively, for 5 days. At the end of 5 days, we recorded EEGs via bipolar EEG electrodes. After 12h, all groups except the first received 70 mg/kg PTZ and we determined the dose-response ratio. Racine's Convulsion Scale was used to evaluate seizures. The spike-wave complex percentage in the EEG was determined as 0% for the first group, 38.6%±7.2 for the second group, 36.4%±5.6 for the third group, 4.3%±1.8 for the fifth group and 4.1%±1.1 for the fifth group. The fourth and fifth groups had significantly decreased spike-wave complex percentages compared to the second group (p<0.0001). OT may prevent PTZ-induced epilepsy on an EEG. OT may also be considered for use in the treatment of epilepsy in the future.

Estradiol protects adipose tissue-derived stem cells against H(2)O(2)-induced toxicity.

Oxidative stress is associated with various pathophysiological processes, including cell survival, adhesion, apoptosis, and cancer. In the present study, we aimed to evaluate the effects of H(2)O(2)-induced toxicity on adipose tissue-derived stem cells (ADSCs) and whether 17ß-estradiol (E(2)) has protective effects on these cells. ADSCs derived from adult Sprague-Dawley rats were pretreated with different doses of E(2) for 24 h and then exposed to 200 µM H(2)O(2) for 4 h. Incubation of ADSCs with H(2)O(2)-decreased cell viability in a concentration-dependent fashion (p < 0.0001), whereas pretreatment of these cells with E(2) significantly reversed toxicity (p < 0.05), inhibited apoptotic changes, and decreased lipid peroxidation (p < 0.0005). Our findings suggest that E(2) protects ADSCs from oxidative-induced cell death, and therefore, it may be used to improve the survival rate and regenerative capacity of stem cells.

Stimulatory effect of 17ß-estradiol on osteogenic differentiation potential of rat adipose tissue-derived stem cells.

Adipose tissue-derived stem cells (ADSCs) are considered as a potential cell source for regenerative medicine and tissue engineering. Although ADSCs have greater proliferation capacity than bone marrow stem cells (BMSCs), lower differentiation ability of these cells limits their utility in experimental and clinical studies. The purpose of this study was to investigate whether 17ß-estradiol (E(2)) has a stimulatory effect on osteogenic differentiation potential of ADSCs in vitro. ADSCs were isolated from visceral adipose tissues of rats and treated with different concentrations of E(2) in osteogenic medium (OM) for 21 days. The differences in osteogenic differentiation potential of the cultures were assessed by von Kossa staining, measurement of alkaline phosphatase (ALP) activity and calcium levels. ADSCs cultured in OM supplemented with E(2) showed greater bone-like nodule formation and mineral deposition in comparing with the cells grown in OM. In addition, ALP activity and calcium levels also were significantly higher in the cultures exposed to E(2) than the cells treated only with OM (p < 0.005, n = 5). Our results suggest that E(2) may stimulate the osteogenic differentiation of ADSCs and therefore, can be used as an inducing agent to improve the efficiency of these cells in in vitro and in vivo studies.

The effect of testosterone treatment on urodynamic findings and histopathomorphology of pelvic floor muscles in female rats with experimentally induced stress urinary incontinence.

OBJECTIVE: In recent studies, it has been observed that androgen receptors are densely located in pelvic floor muscles. We aimed to investigate the effect of testosterone on urodynamic findings and histopathomorphology of pelvic floor muscles in rats with experimentally induced stress urinary incontinence. MATERIALS AND METHODS: Twenty-eight adult female rats were randomized into four groups. Group I: rats in which SUI was induced and single-dose testosterone was administered 30 days later, group II: rats in which SUI was induced and single-dose testosterone was administered within the same session, group III: rats in which SUI was induced and saline was injected intramuscularly 30 days later, and group IV: the sham group. In order to demonstrate objectively the curative and preventive role of testosterone in experimental model of SUI, urodynamic examination and histopathomorphological evaluation of levator ani muscle were performed. RESULTS: Myofiber areas in groups I and II were detected to be significantly larger than those of the control group (P < 0.001). Another parameter was leak point pressure value by urodynamy. Regarding this parameter, LPP values in groups 1, 2 and 4 were observed to be significantly higher than those of group 3 (P < 0.001). The results of the comparison among groups 1, 2 and 4 revealed no significance (P > 0.05), which indicates that testosterone provides continence in a similar way to the group in which sciatic nerve section was not performed. CONCLUSIONS: In the present study, it has been demonstrated that testosterone has both preventive and curative effects on rat models of experimental SUI.

Effects of neuronal and glial restricted precursor cells transplantation on erectile function after experimentally induced spinal cord injury.

INTRODUCTION: Erectile dysfunction is common among patients with spinal cord injury (SCI). AIM: This study aims to investigate the recovery of penile erectile functions of the rats with spinal cord injury (SCI) following transplantation of endogenous neuronal precursors cell (neuronal restricted precursors [NRP]/glial restricted precursors [GRP]) into the injured area of spinal cord. METHODS: Twenty-two rats were experimented in three groups. Group 1 (N = 6): Sham; Group 2 (N = 10): SCI + NRP/GRP transplanted in day 9 after operation; Group 3 (N = 6): SCI + culture medium transplanted in day 9 after operation. Analysis of penile reflexes and cavernosal nerve stimulation studies were performed in day 28 after transplantation for each group. All rats in three groups were then sacrificed and the injured regions of spinal cords underwent histological investigation. MAIN OUTCOME MEASURES: These results show improvements to some extent in locomotor and erectile functions although these improvements are far from full functional recovery. RESULTS: Cavernosal nerve stimulation resulted in significantly higher intracavernosal pressure in Group 3 (SCI) although there was no difference between Group 1 (sham) and Group 2 (SCI + NRP/GRP). Number of clusters was similar between groups. Number of erections was higher in Group 3 (SCI) than Groups 1 and 2, and number of cups was higher in Group 2 (SCI + NRP/GRP) than the other two groups. Number of flips was similar in Groups 1 and 2 but lower in Group 3. Number of long flips was highest in Group 1 and lowest in Group 3. The differences between groups were significant. CONCLUSION: This study emphasized the healing potential of NRP/GRP transplantation following experimental SCI. However, further experimental and clinical studies are required to advance this treatment modality.

Bladder function recovery in rats with traumatic spinal cord injury after transplantation of neuronal-glial restricted precursors or bone marrow stromal cells.

PURPOSE: We investigated functional recovery of the lower urinary system in rats with spinal cord injury after transplanting neuronal restricted precursors/glial restricted precursors or neural cells derived from bone marrow stromal cells into the injured area of the spinal cord. MATERIALS AND METHODS: A total of 30 rats underwent experimentation in 4 groups, including group 1--sham operation, group 2--spinal cord injury plus neuronal restricted precursor/glial restricted precursor transplantation, group 3--spinal cord injury plus bone marrow stromal cell transplantation and group 4--spinal cord injury control. All rats in the 4 groups were investigated urodynamically and sacrificed on day 28 after transplantation. The cells transplanted into the injured spinal cord underwent histological investigation. RESULTS: Transplanted cells (neuronal and glial restricted precursors, and bone marrow stromal cells) were found to maintain a presence in the injured spinal cord area. Baseline pressure, maximum capacity, mean uninhibited contraction amplitude, mean voiding pressure, voided volume and post-void residual volume were significantly better in groups 2 and 3 than in group 4, while baseline pressure in group 2 was better than that in group 3. We found no significant difference among the groups according to mean uninhibited contraction frequency. CONCLUSIONS: Although neuronal/glial restricted precursor transplanted rats seemed to have more improvement, all rats in groups 2 and 3 showed some significant improvement in lower urinary system function. On the other hand, the level of this improvement was far from complete functional recovery.

The effects of dexmedetomidine on spontaneous contractions of isolated gravid rat myometrium.

The direct effects of dexmedetomidine on isolated gravid rat myometrium were investigated in this in vitro study; such effects may have clinical repercussions in the administration of anesthesia to obstetric patients. Samples of myometrium were taken from 12 gravid rats. Myometrial strips were dissected microscopically and mounted on the myograph at a resting tension of 1 g in bath that contained Krebs solution. After spontaneous contractions of the myometrium had been steadily established, increasing concentrations of dexmedetomidine were added to baths via micropipette, and the effects of these additions were recorded via myograph. Dexmedetomidine in vitro caused a significant increase in the amplitude, frequency, and area under the curve of myometrial contractions in a dose-dependent manner. Results of this study demonstrate that dexmedetomidine increases spontaneous contractions in rat myometrium; however, further investigation is needed to clarify the usefulness of dexmedetomidine in the administration of obstetric anesthesia.