Astrocytes are responsible for a wide variety of essential functions throughout the central nervous system. The protein markers glial fibrillary acidic protein (GFAP), glutamate aspartate transporter (GLAST), glutamate transporter‐1 (GLT‐1), glutamine synthetase (GS), 10‐formyltetrahydrofolate dehydrogenase (ALDH1L1), and the transcription factor SOX9 are routinely used to label astrocytes in primary rodent cultures. However, GLAST, GLT‐1, GS, and SOX9 are also produced by microglia and oligodendrocytes and GFAP, GLAST, GLT‐1, and GS production levels are affected by astrocyte phenotypic changes associated with reactive astrogliosis. No group has performed a comprehensive immunocytochemical evaluation to quantify the percentage of cells labeled by these markers
This content will become publicly available on January 10, 2025
Multiple therapies have been studied to ameliorate the neuroinhibitory cues present after traumatic injury to the central nervous system. Two previous in vitro studies have demonstrated the efficacy of the Food and Drug Administration‐approved cardiovascular therapeutic, protamine (PRM), to overcome neuroinhibitory cues presented by chondroitin sulfates; however, the effect of a wide range of PRM concentrations on neuronal and glial cells has not been evaluated. In this study, we investigate the therapeutic efficacy of PRM with primary cortical neurons, hippocampal neurons, mixed glial cultures, and astrocytes. The threshold for PRM toxicity is shown to be at or above 10 µg mL−1depending on the cell population, that 10 µg mL−1PRM enables neurons to overcome the inhibitory cues presented by chondroitin sulfate type A, and that soluble PRM allows neurons to more effectively overcome inhibition compared to a PRM coating. Changes in gene expression of reactive astrocytes is also assessed with soluble PRM and it is determined that PRM does not increase their neurotoxic phenotype and that PRM may reduce brevican and serpin transcription in cortical and spinal cord astrocytes. This is the first study to thoroughly assess the toxicity threshold of PRM with neural cells and study astrocyte response after acute exposure to PRM in vitro.
more » « less- NSF-PAR ID:
- 10487227
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Advanced Therapeutics
- ISSN:
- 2366-3987
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract in vitro , nor compared changes in staining between cortex‐ and spinal cord‐derived cells in naïve and stimulated cultures. Here, we quantified the percentage of cells positively stained for these six markers in astrocyte, microglia, and oligodendrocyte cultures isolated from neonatal rat cortices and spinal cords. Additionally, we incubated the astrocytes with transforming growth factor (TGF)‐β1 or TGF‐β3 to determine if the labeling of these markers is altered by these stimuli. We found that only SOX9 in cortical cultures and ALDH1L1 in spinal cord cultures labeled more than 75% of the cells in naïve and stimulated astrocyte cultures and stained less than 5% of the cells in microglia and oligodendrocyte cultures. Furthermore, significantly more cortical than spinal cord astrocytes stained for GFAP, GLAST, and ALDH1L1 in naïve cultures, whereas significantly more spinal cord than cortical astrocytes stained for GLAST and GS in TGF‐β1‐treated cultures. These findings are important as variability in marker staining may lead to misinterpretation of the astrocyte response in cocultures, migration assays, or engineered disease models. -
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