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Secretomes of apoptotic mononuclear cells ameliorate neurological damage in rats with focal ischemia.

Altmann P, Mildner M, Haider T, Traxler D, Beer L, Ristl R, Golabi B, Gabriel C, Leutmezer F, Ankersmit HJ - F1000Res (2014)

Bottom Line: Administration of rat as well as human apoptotic MNC-secretomes significantly reduced ischemic lesion volumes by 36% and 37%, respectively.Co-incubation of human astrocytes, Schwann cells and neurons with hMNC (apo sec) resulted in activation of several signaling cascades associated with the regulation of cytoprotective gene products and enhanced neuronal sprouting in vitro.Analysis of neurotrophic factors in hMNC (apo sec) and rat plasma revealed high levels of brain derived neurotrophic factor (BDNF).

View Article: PubMed Central - PubMed

Affiliation: Department of Thoracic Surgery, Medical University of Vienna, Vienna, 1090, Austria ; Christian Doppler Laboratory for Cardiac and Thoracic Diagnosis and Regeneration, Vienna, 1090, Austria.

ABSTRACT
The pursuit of targeting multiple pathways in the ischemic cascade of cerebral stroke is a promising treatment option. We examined the regenerative potential of conditioned medium derived from rat and human apoptotic mononuclear cells (MNC), rMNC (apo sec) and hMNC (apo sec), in experimental stroke. We performed middle cerebral artery occlusion on Wistar rats and administered apoptotic MNC-secretomes intraperitoneally in two experimental settings. Ischemic lesion volumes were determined 48 hours after cerebral ischemia. Neurological evaluations were performed after 6, 24 and 48 hours. Immunoblots were conducted to analyze neuroprotective signal-transduction in human primary glia cells and neurons. Neuronal sprouting assays were performed and neurotrophic factors in both hMNC (apo sec) and rat plasma were quantified using ELISA. Administration of rat as well as human apoptotic MNC-secretomes significantly reduced ischemic lesion volumes by 36% and 37%, respectively. Neurological examinations revealed improvement after stroke in both treatment groups. Co-incubation of human astrocytes, Schwann cells and neurons with hMNC (apo sec) resulted in activation of several signaling cascades associated with the regulation of cytoprotective gene products and enhanced neuronal sprouting in vitro. Analysis of neurotrophic factors in hMNC (apo sec) and rat plasma revealed high levels of brain derived neurotrophic factor (BDNF). Our data indicate that apoptotic MNC-secretomes elicit neuroprotective effects on rats that have undergone ischemic stroke.

No MeSH data available.


Related in: MedlinePlus

Enhanced CREB phosphorylation and neurite length in neurons treated with apoptotic MNC-secretomes.(a) Cell extracts were prepared after stimulation with hMNCapo sec or cell culture medium as control. Western blot analysis for phospho-CREB revealed a dose dependent activation of CREB in astrocytes and in neurons. (b) Neuron cultures treated with hMNCapo sec or control medium for five days were stained with methylene-blue. One representative picture of ten is shown. Bar=10 µm (c) Lengths of neurons treated with hMNCapo sec or cell culture medium as control were calculated using ImageJ software. Bars represent the mean of five different cultures.
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f7: Enhanced CREB phosphorylation and neurite length in neurons treated with apoptotic MNC-secretomes.(a) Cell extracts were prepared after stimulation with hMNCapo sec or cell culture medium as control. Western blot analysis for phospho-CREB revealed a dose dependent activation of CREB in astrocytes and in neurons. (b) Neuron cultures treated with hMNCapo sec or control medium for five days were stained with methylene-blue. One representative picture of ten is shown. Bar=10 µm (c) Lengths of neurons treated with hMNCapo sec or cell culture medium as control were calculated using ImageJ software. Bars represent the mean of five different cultures.

Mentions: To investigate neuronal sprouting of human primary neurons, 1×104 cells (CellSystems) were seeded in 24-well plates (Costar) and allowed to adhere for 24 hours. Cells were further cultivated in neuronal medium (see above) without growth factors for five days together with the secretome of hMNC derived from 2.5×106 cells/mL (hMNCapo sec) or control medium. After five days cells were fixed at room temperature in 100% methanol for 10 minutes and stained with methylene blue (Sigma; 0.5% in methanol). Excess methylene blue was washed out with distilled water, and culture wells were evaluated with an inverted microscope (EvosXL, Life Technologies, Carlsbad, CA, USA). Cell cultures were digitalized using an Olympus Digital Camera E-520 (3648×2736 pixels). A blinded observer set a random representative area of the photographed cell culture (as seen inFigure 7b) using Adobe Photoshop Lightroom Software (Version 5.2, 2013; Adobe, San José, CA, USA). (i) on a photograph of cells treated with human apoptotic MNC-secretomes and (ii) on a photograph showing cells treated with control medium. The areas in each photograph measured 1420×2456 pixels. Subsequently, they picked and marked visible distinct, full-length and non-overlapping 30–35 neurites using ImageJ software (Bethesda, MD). Another blinded investigator measured these marked neurons using ImageJ software.


Secretomes of apoptotic mononuclear cells ameliorate neurological damage in rats with focal ischemia.

Altmann P, Mildner M, Haider T, Traxler D, Beer L, Ristl R, Golabi B, Gabriel C, Leutmezer F, Ankersmit HJ - F1000Res (2014)

Enhanced CREB phosphorylation and neurite length in neurons treated with apoptotic MNC-secretomes.(a) Cell extracts were prepared after stimulation with hMNCapo sec or cell culture medium as control. Western blot analysis for phospho-CREB revealed a dose dependent activation of CREB in astrocytes and in neurons. (b) Neuron cultures treated with hMNCapo sec or control medium for five days were stained with methylene-blue. One representative picture of ten is shown. Bar=10 µm (c) Lengths of neurons treated with hMNCapo sec or cell culture medium as control were calculated using ImageJ software. Bars represent the mean of five different cultures.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215751&req=5

f7: Enhanced CREB phosphorylation and neurite length in neurons treated with apoptotic MNC-secretomes.(a) Cell extracts were prepared after stimulation with hMNCapo sec or cell culture medium as control. Western blot analysis for phospho-CREB revealed a dose dependent activation of CREB in astrocytes and in neurons. (b) Neuron cultures treated with hMNCapo sec or control medium for five days were stained with methylene-blue. One representative picture of ten is shown. Bar=10 µm (c) Lengths of neurons treated with hMNCapo sec or cell culture medium as control were calculated using ImageJ software. Bars represent the mean of five different cultures.
Mentions: To investigate neuronal sprouting of human primary neurons, 1×104 cells (CellSystems) were seeded in 24-well plates (Costar) and allowed to adhere for 24 hours. Cells were further cultivated in neuronal medium (see above) without growth factors for five days together with the secretome of hMNC derived from 2.5×106 cells/mL (hMNCapo sec) or control medium. After five days cells were fixed at room temperature in 100% methanol for 10 minutes and stained with methylene blue (Sigma; 0.5% in methanol). Excess methylene blue was washed out with distilled water, and culture wells were evaluated with an inverted microscope (EvosXL, Life Technologies, Carlsbad, CA, USA). Cell cultures were digitalized using an Olympus Digital Camera E-520 (3648×2736 pixels). A blinded observer set a random representative area of the photographed cell culture (as seen inFigure 7b) using Adobe Photoshop Lightroom Software (Version 5.2, 2013; Adobe, San José, CA, USA). (i) on a photograph of cells treated with human apoptotic MNC-secretomes and (ii) on a photograph showing cells treated with control medium. The areas in each photograph measured 1420×2456 pixels. Subsequently, they picked and marked visible distinct, full-length and non-overlapping 30–35 neurites using ImageJ software (Bethesda, MD). Another blinded investigator measured these marked neurons using ImageJ software.

Bottom Line: Administration of rat as well as human apoptotic MNC-secretomes significantly reduced ischemic lesion volumes by 36% and 37%, respectively.Co-incubation of human astrocytes, Schwann cells and neurons with hMNC (apo sec) resulted in activation of several signaling cascades associated with the regulation of cytoprotective gene products and enhanced neuronal sprouting in vitro.Analysis of neurotrophic factors in hMNC (apo sec) and rat plasma revealed high levels of brain derived neurotrophic factor (BDNF).

View Article: PubMed Central - PubMed

Affiliation: Department of Thoracic Surgery, Medical University of Vienna, Vienna, 1090, Austria ; Christian Doppler Laboratory for Cardiac and Thoracic Diagnosis and Regeneration, Vienna, 1090, Austria.

ABSTRACT
The pursuit of targeting multiple pathways in the ischemic cascade of cerebral stroke is a promising treatment option. We examined the regenerative potential of conditioned medium derived from rat and human apoptotic mononuclear cells (MNC), rMNC (apo sec) and hMNC (apo sec), in experimental stroke. We performed middle cerebral artery occlusion on Wistar rats and administered apoptotic MNC-secretomes intraperitoneally in two experimental settings. Ischemic lesion volumes were determined 48 hours after cerebral ischemia. Neurological evaluations were performed after 6, 24 and 48 hours. Immunoblots were conducted to analyze neuroprotective signal-transduction in human primary glia cells and neurons. Neuronal sprouting assays were performed and neurotrophic factors in both hMNC (apo sec) and rat plasma were quantified using ELISA. Administration of rat as well as human apoptotic MNC-secretomes significantly reduced ischemic lesion volumes by 36% and 37%, respectively. Neurological examinations revealed improvement after stroke in both treatment groups. Co-incubation of human astrocytes, Schwann cells and neurons with hMNC (apo sec) resulted in activation of several signaling cascades associated with the regulation of cytoprotective gene products and enhanced neuronal sprouting in vitro. Analysis of neurotrophic factors in hMNC (apo sec) and rat plasma revealed high levels of brain derived neurotrophic factor (BDNF). Our data indicate that apoptotic MNC-secretomes elicit neuroprotective effects on rats that have undergone ischemic stroke.

No MeSH data available.


Related in: MedlinePlus