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1

Organotypic retinal explants model for evaluation of neurotoxicity and neuroprotection - the impact of metallothionein treatment on HuR protein content

100%
Pietrucha-Dutczak M.
Acta Neurobiologiae Experimentalis
|
2019
|
tom 79
|
nr Suppl.1
Retina and optic nerve diseases are one of the major causes of irreversible blindness worldwide, with increasing prevalence associated with aging of the population. Since there is no fully reliable and successful method for culturing retinal neurons (i.e., retinal ganglion cells, RGC) and furthermore, in vivo studies are relatively costly and time consuming, the ex vivo organotypic retinal culture could be a competitive and highly efficient method for initial drug toxicity screening. Neuroprotection and neuroregeneration are topics of a growing number of studies regarding eye diseases. Ocular pathologies result in neurodegeneration primary or secondary involving RGCs. Metallothioneins (MTs) are low molecular weight cysteine rich proteins. It is suggested that MTs, especially the isoform 2, are important neuroprotective substances for cerebral ischemia and retinal diseases. MT2 as a secondary antioxidant cooperates with reduced glutathione in the cellular protective system against oxidative stress. Additionally, it has been demonstrated that MT treatment can alleviate neurodegeneration of RGC in retinal explants exposed to toxic concentration of gentamycin. This protective effect, linked most probably to antioxidant activity, is associated with delayed increase of HuR protein content in retinal explants, which could be considered as a marker of delayed cells stress response.
2

Schwann cells induced neuroprotection and regeneration of retinal ganglion cells

51%
Lewin-Kowalik J., Smedowski A., Pietrucha-Dutczak M.
Acta Neurobiologiae Experimentalis
|
2015
|
tom 75
|
nr Supl.
To investigate neuroprotective effect of intravitreally applied Schwann cells therapy towards Retinal Ganglion Cells (RGCs) in rat experimental glaucoma. Twenty male Wistar rats were included to this study. Experimental glaucoma was induced in the left eye of each rat by intraocular pressure (IOP) elevation using intracameral injection of polystyrene microbeads. The right eye served as a healthy control. Ten animals received intravitreal injection of 5 μl Schwann cells suspension (about 106 cells), another 10 received injection of equivallent volume of PBS. Animals were breaded for 6 weeks and IOP was monitored using laboratory tonometer once a week. After 6 weeks animals were sacrificed, eyes with optic nerves were enucleated and processed for histology and immunohistochemistry. RGCs survival was compared by counting RGCs bodies and optic nerve axons from control eyes (healthy and PBS) and Schwann cells treated. Mean 6 weeks IOP in ocular hypertension eyes was significantly higher in comparison to healthy contralateral eyes (31.02±5.5 mmHg and 10.32±0.54 mmHg, mean±SD, Wilcoxon paired test, P<0.05). There were significant differences between RGCs bodies and optic nerve axons numbers in Schwann cell-treated vs. PBS-treated vs. healthy control eyes (P<0.05, Kruskall-Wallis test). Mean 6-weeks loss of RGCs bodies was 21.7% in glaucoma eyes treated with Schwann cells and 45% in glaucoma eyes treated with PBS. Immunofluorescent staining with GAP43 showed neurites outgrowth within optic nerves from eyes treated with Schwann cells. Applied cellular therapy using predegenerated Schwann cells showed neuroprotective and regenerative effect towards RGCs in rat glaucoma model.
3

Decrease of RGCs number in glaucoma model vs. optic nerve transaction in rats

51%
Pietrucha-Dutczak M., Smedowski A., Wylegala E., Lewin-Kowalik J.
Acta Neurobiologiae Experimentalis
|
2013
|
tom 73
|
nr Suppl.1
Introduction: Transection of the optic nerve and glaucoma causes both structural and functional damage to retinal ganglion cells with subsequent vision defect or loss. This study was undertaken to compare the lost of RGCs after optic nerve transection and glaucoma model. Materials and methods: Wistar rats were divided into two groups. The first group underwent bilateral stereotactic injection of fluorescent tracer – Fluorogold (FG) into the superior colliculus to label RGCs. After one week the right optic nerve was transected. Left eye without optic nerve axotomy was established as control. In the second group intraocular pressure (right eye) was elevated by injection of polystyrene microbeads into anterior chamber (Bead model) and measured by Icare TonoLab. RGCs were labeled by FG before euthanasia. Fourteenth days following optic nerve transection and intraocular pressure elevation the total number of FG-positive RGCs was counted in seven radial sections through the optic disk. Results and conclusions: After axotomy the number of surviving cells was reduced to 20.2 % (from 2249.5 ± 127.2 – in control group to 454.7 ± 96.5 – in group after axotomy), in glaucoma model to 79.9% (from 2249.5 ± 127.2 – in control group to 1798.3 ± 118.96 – in glaucoma model).
4

Matrix metalloproteinases activity in distal stumps of rat sciatic nerves 1-7 days following transection

39%
Pietrucha-Dutczak M., Lewin-Kowalik J., Gorka D., Korczynska I., Matuszek I., Wolwender K., Larysz-Brysz M.
Acta Neurobiologiae Experimentalis
|
2001
|
tom 61
|
nr 3
5

Elevated intraocular pressure impairs distribution of RNA-binding protein HuR in glaucomatous rat retina

33%
Lewin-Kowalik J., Smedowski A., Liu X., Podracka L., Trzeciecka A., Pietrucha-Dutczak M., Varjosalo M., Kaarniranta K., Amadio M.
Acta Neurobiologiae Experimentalis
|
2017
|
tom 77
|
nr Suppl.1
INTRODUCTION: Glaucoma is a serious social problem as it may result in blindness. Most often, it is related to increased intraocular pressure, but the exact biologic mechanism is not known yet. RNA-binding proteins may be one of the pathogenetic factors for this disease. AIM(S): To evaluate impact of increased intraocular pressure (IOP) on HuR protein expression in retina and optic nerve in rat glaucoma model. METHOD(S): IOP was increased unilaterally using modified rat bead model. Fellow eye was used as a healthy control. Animals were sacrificed 1 day, 1‑, 4‑, 6‑ or 8‑weeks after beads injection. Retinas and optic nerves were collected and processed for Western blot (WB) analysis, mass spectrometry (MS), PCR and immunostainings. RESULTS: The loss of retinal ganglion cells (RGCs) was at the level of 36% after 8‑weeks of IOP elevation. The presence of HuR protein and its transcipt was confirmed in retinas and optic nerves using WB, MS and PCR analysis. Additionally, Gene Ontology enrichment analysis revealed that the most significant alterations in glaucoma retinas were linked to the molecular function of binding proteins. In fractionated WB of retinal homogenates, the level of cytoplasmic fraction of HuR was decreased approximately 3-times when compared with healthy tissue (p<0.05). This decrease was accompanied by alteration of the cytoplasmic level of HuR-regulated proteins, i.e. Hsp70 decrease in retina and p53 decrease in optic nerve. Stereological analysis of retinas revealed that some RGCs have lost visible HuR expression. Immunostaining of retinal and optic nerves cross sections showed decreased staining for HuR within RGCs and increased within optic nerve glia, with nuclear polarisation of HuR expression in glaucoma samples. CONCLUSIONS: Increased intraocular pressure results in alteration of RNA-binding protein HuR within retina and, subsequently, decreased expression of HuR-dependent stress-response regulatory proteins. This alterations might contribute to the development of glaucomatous degeneration.
6

Regeneration of sciatic nerves of adults rats induced by extracts from distal stumps of peripheral nerves

33%
Marcol W., Lewin-Kowalik J., Larysz-Brysz M., Swiech-Sabuda E., Gorka D., Golka B., Wolwender K., Pietrucha-Dutczak M.
Acta Neurobiologiae Experimentalis
|
2001
|
tom 61
|
nr 3
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