calcein-am and Disease-Models--Animal

Parkinson's disease (PD) is characterized by the degeneration of the dopaminergic neurons in the substantia nigra pars compacta (SNpc). Clinical and experimental evidence suggest that the activation of the nicotinic acetylcholine receptor (nAChR) could be protective for PD. In this study, we investigated the neuroprotective capacity of nicotine in a rat PD model. Considering that iron metabolism has been implicated in PD pathophysiology and nicotine has been described to chelate this metal, we also studied the effect of nicotine on the cellular labile iron pool (LIP) levels. Rotenone (1 μg) was unilaterally injected into the median forebrain bundle to induce the degeneration of the nigrostriatal pathway. Nicotine administration (1 mg/K, s.c. daily injection, starting 5 days before rotenone and continuing for 30 days) attenuated the dopaminergic cell loss in the SNpc and the degeneration of the dopaminergic terminals provoked by rotenone, as assessed by immunohistochemistry. Furthermore, nicotine partially prevented the reduction on dopamine levels in the striatum and improved the motor deficits, as determined by HPLC-ED and the forelimb use asymmetry test, respectively. Studies in primary mesencephalic cultures showed that pretreatment with nicotine (50 μM) improved the survival of tyrosine hydroxylase-positive neurons after rotenone (20 nM) exposure. Besides, nicotine induced a reduction in the LIP levels assessed by the calcein dequenching method only at the neuroprotective dose. These effects were prevented by addition of the nAChRs antagonist mecamylamine (100 μM). Overall, we demonstrate a neuroprotective effect of nicotine in a model of PD in rats and that a reduction in iron availability could be an underlying mechanism.

Topics: Analysis of Variance; Animals; Cell Count; Disease Models, Animal; Dopamine; Dopaminergic Neurons; Dose-Response Relationship, Drug; Embryo, Mammalian; Exploratory Behavior; Fluoresceins; Forelimb; Insecticides; Iron; Male; Medial Forebrain Bundle; Mesencephalon; Motor Activity; Nicotine; Nicotinic Agonists; Parkinson Disease; Pars Compacta; Rats; Rats, Sprague-Dawley; Rotenone; Tubulin; Tyrosine 3-Monooxygenase

Glaucoma is a progressive neurodegenerative disease, characterized by retinal ganglion cells (RGCs) and axon degeneration. The development of neuroprotective drug is required for improving the efficiency of glaucoma treatment. Eucommia ulmoides Oliv. has been used as a source of traditional medicine and as a beneficial health food. Lignans is one of the main bioactive components of Eucommia ulmoides. Here, we show that lignans protects RGCs against oxidative stress-induced injury in vitro. Moreover, lignans exerts neuroprotective effect on glaucoma-associated optic neuropathy in glaucomatous rats. Lignans treatment could improve oxidative stress response in RGCs and retinas of glaucomatous rats. Lignans plays an anti-oxidative stress role via the activation of AMPK signaling. This study provides evidence that lignans possesses protective effect on glaucoma-associated optic neuropathy. Lignans might be an alternative for the prevention and treatment of glaucomatous neurodegeneration.

Topics: Animals; Cells, Cultured; Disease Models, Animal; Dose-Response Relationship, Drug; Eucommiaceae; Fluoresceins; Gene Expression Regulation; Glaucoma; Hydrogen Peroxide; Lignans; Male; Neuroprotective Agents; Optic Nerve Diseases; Phosphopyruvate Hydratase; Rats; Rats, Wistar; Retinal Ganglion Cells; RNA, Long Noncoding; Signal Transduction; Sincalide; Tubulin

Iron-overload condition can be found in β-thalassemic patients with regular blood transfusion, leading to iron deposition in various organs including the heart. Elevated cardiac iron causes iron-overload cardiomyopathy, a condition that provokes mortality because of heart failure in patients with thalassemia. Previous studies demonstrated that myocardial iron uptake may occur via L-type calcium channels (LTCCs). However, direct evidence regarding the claimed pathway in thalassemic cardiomyocytes has never been investigated.. Hearts from genetic-altered β-thalassemic mice and adult wild-type mice were used for cultured ventricular cardiomyocytes. Blockers for LTCC, T-type calcium channel (TTCC), transferrin receptor1 (TfR1), and divalent metal transporter1 (DMT1) were used, and quantification of cellular iron uptake under various iron loading conditions was performed by Calcein-AM fluorescence assay. Microarray analysis was performed to investigate gene expressions in the hearts of these mice.. This study demonstrated that iron uptake under iron-overload conditions in the cultured ventricular myocytes of thalassemic mice was greater than that of wild-type cells (P <0.01). TTCC blocker, efonidipine, and an iron chelator, deferoxamine, could prevent iron uptake into cultured cardiomyocytes, whereas blockers of TfR1, DMT1, and LTCC could not. Microarray analysis from thalassemic hearts demonstrated highly up-regulated genes of TTCC, zinc transporter, and transferrin receptor2.. Our findings indicated that iron uptake mechanisms in cultured thalassemic cardiomyocytes are mainly mediated by TTCC, suggesting that TTCC is the important pathway for iron uptake in this cultured thalassemic cardiomyocyte model.

Topics: Animals; beta-Thalassemia; Calcium Channels, T-Type; Cell Survival; Cells, Cultured; Deferoxamine; Disease Models, Animal; Fluoresceins; Heterozygote; Immunohistochemistry; Iron; Mice; Mice, Inbred C57BL; Myocytes, Cardiac; Verapamil

To determine whether oxidative adduct formation or heme oxygenase-1 (HO-1) expression are altered in retinal ganglion cell (RGC) cultures exposed to elevated hydrostatic pressure and in a mouse model of glaucoma.. Cultured RGC-5 cells were subjected to 0, 30, 60, or 100 mm Hg hydrostatic pressure for 2 hours, and the cells were harvested. Parallel experiments examined the recovery from this stress, the effect of direct 4-hydroxy-2-nonenal (HNE) treatment, and the effect of pretreatment with resveratrol or quercetin. Mice were anesthetized and intraocular pressure was increased to 30, 60, or 100 mm Hg for 1 hour; then the retinas were harvested. HNE adduct formation and HO-1 expression were assessed by immunocytochemistry and immunoblotting.. Increases of HNE-protein adducts (up to 5-fold) and HO-1 expression (up to 2.5 fold) in pressure-treated RGC-5 cells were dose dependent. During recovery experiments, HNE-protein adducts continued to increase for up to 10 hours; in contrast, HO-1 expression decreased immediately. HNE, at a concentration as low as 5 muM, led to neurotoxicity in RGC-5 cells. HNE adducts and HO-1 expression increased in the mouse retina and optic nerve after acute IOP elevation up to 5.5-fold and 2-fold, respectively. Antioxidant treatment reduced the oxidative stress level in pressure-treated RGC-5 cells.. This study demonstrates that oxidative stress is an early event in hydrostatic pressure/IOP-induced neuronal damage. These findings support the view that oxidative damage contributes early to glaucomatous optic neuropathy.

Topics: Aldehydes; Animals; Apoptosis; Blotting, Western; Cell Line, Transformed; Cell Survival; Cells, Cultured; Disease Models, Animal; Fluoresceins; Fluorescent Antibody Technique, Indirect; Glaucoma; Heme Oxygenase-1; Hydrostatic Pressure; Intraocular Pressure; Mice; Mice, Inbred C57BL; Microscopy, Fluorescence; Nuclear Proteins; Optic Nerve Diseases; Oxidative Stress; Rats; Retinal Diseases; Retinal Ganglion Cells

For the reconstruction of 3-dimensional (3D) tissues, we exploited an original method of tissue engineering that layers individual cell sheets harvested from temperature-responsive culture dishes. Stacked cardiomyocyte sheets demonstrated electrical and morphologic communication, resulting in synchronously beating myocardial tissue. When these bioengineered 3D tissue grafts are transplanted onto damaged hearts, gap junction communication between graft and host is likely critical for synchronized beating and functional improvement. In this study, these graft-to-heart morphologic communications were examined.. Neonatal rat cardiomyocyte sheets were harvested from temperature-responsive culture dishes and layered to create 3D tissues. These constructs were then transplanted onto infarcted rat hearts. Histologic analyses and transmission electron microscopy (TEM) were performed to examine morphologic communications. The passage of small molecules through functional gap junctions was also detected using a dye-transfer assay.. Transplanted cardiomyocytes bridged between the grafts and hearts in intact areas. Connexin-43 staining and TEM revealed the existence of gap junctions and intercalated disks between the bridging cardiomyocytes. Furthermore, it was confirmed that a low-molecule fluorescent dye, calcein, was transferred from the grafts to the hearts via the bridging cardiomyocytes. Immunohistochemistry with anti-intercellular adhesion molecule-1 antibodies revealed that mesothelial cells in the epicardium scattered and transdifferentiated into mesenchymal cells between the graft and host.. The direct attachment of layered cardiomyocyte sheets on the heart surface promotes mesothelial cell transdifferentiation and cardiomyocyte bridging, leading to functional communication via gap junctions. These results indicate that these bioengineered myocardial tissues may improve damaged heart function via synchronized beating.

Topics: Animals; Animals, Newborn; Cell Communication; Disease Models, Animal; Epithelial Cells; Extracellular Matrix; Fibroblasts; Fluoresceins; Fluorescent Dyes; Gap Junctions; Heart Conduction System; Intercellular Adhesion Molecule-1; Mesoderm; Microscopy, Electron, Transmission; Myocardial Infarction; Myocardium; Rats; Rats, Sprague-Dawley; Tissue Engineering; Tissue Transplantation