Earliest Mechanisms of Dopaminergic Neurons Sufferance in a Novel Slow Progressing Ex Vivo Model of Parkinson Disease in Rat Organotypic Cultures of Substantia Nigra
Abstract
:1. Introduction
2. Results
2.1. Model Development and Experimental Plan
2.2. Morphometric Analysis of the Temporal Degeneration of Dopaminergic Neurons and Agreement with the Clinical Scenario
2.2.1. Decrease of the Number of the DOPAn’s Neurites
2.2.2. Decrease of the Number of the DOPAn
2.2.3. Decrease of the Length of the DOPAn’s Neurites
2.3. Time-Dependent Comparative Evaluation of the Molecular Events Undergoing DOPAn Sufferance and Demise
2.3.1. Real-Time PCR Analysis of Inflammation, Redox Imbalance, Unfolded Proteins Ubiquitination, Vesicles Transport, and Apoptosis; and Quantification of Glutamate in the Medium
2.3.2. Results at 3 h
2.3.3. Results at 12 h
2.3.4. Results at 24 h, 48 h and 96 h
3. Discussion
4. Materials and Methods
4.1. Organotypic Brain Culture Preparation
4.2. Cultures Medium and Treatment
4.3. LDH Test
4.4. Immunofluorescent Staining of Dopaminergic Neurons
4.5. Counting of Dopaminergic Neurons and Neurites
4.6. Glutamate Quantification in Culture Media
4.7. Quantitative Real-Time PCR of Selected Marker Genes
4.8. Statistical Analysis
5. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
PD | Parkinson disease |
DOPAn | Dopaminergic neurons |
SN | Substantia nigra |
TH+ | Tyrosine hydroxylase |
HY | Hohen & Yhar score |
PET | Positron Emission Tomography |
Hmox1 | Heme-oxygenase 1 |
Srnx1 | Sulfiredoxin 1 |
ROS | Reactive oxygen species |
Tnfα | Tumor necrosis factor alfa α |
Cox2 | Cyclo-oxygenase 2 |
Il6 | Interleukine 6 |
Il1 β | Interleukine 1β |
Cd68 | Cluster of differentiation 68, marker of microglia activation |
Bdnf | Brain-derived neurotrophic factor |
Snca | Alpha-synuclein (also known as Park1,4) |
Uchl1 | Ubiquitin carboxy-terminal hydrolase L1 (also known as Park5) |
Vmat2 | Vesicular monoamine transporter 2 |
Anxa5 | Annexin5 |
Glu | Glutamate |
Gdnf | Glial cell-derived neurotrophic factor |
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Gene | 3 h | 12 h | 24 h | 48 h | 96 h |
---|---|---|---|---|---|
Redox imbalance | |||||
Hmox1 | 1.68 ± 0.40 ** | 1.03 ± 0.16 | 0.64 ± 0.13 | 1.07 ± 0.21 | 1.04 ± 0.46 |
Srnx1 | 1.21 ± 0.35 | 1.71 ± 0.57 * | 1.48 ± 0.38 ** | 1.69 ± 0.80 * | 1.70 ± 0.39 * |
Inflammation | |||||
Tnfa | 1.68 ± 0.54 * | 1.47 ± 0.39 | 1.26 ± 0.19 * | 1.58 ± 0.78 | 0.89 ± 0.29 |
Cox2 | 1.89 ± 0.55 * | 1.40 ± 0.74 | 2.43 ± 0.69 *** | 0.43 ± 0.58 | 0.31 ± 0.21 |
Il6 | 0.82 ± 0.44 | 2.10 ± 0.60 * | 5.52 ± 0.81 *** | 0.92 ± 0.68 | 0.88 ± 0.46 |
Cd68 | 1.35 ± 0.36 | 0.93 ± 0.19 | 0.87 ± 0.15 | 2.16 ± 0.82 | 4.20 ± 1.91 *** |
Il1b | 1.58 ± 0.51 | 1.07 ± 0.37 | 0.73 ± 0.23 | 0.83 ± 0.45 | 0.39 ± 0.12 * |
Glutamate neurotoxicity | |||||
Glu | 1.12 ± 0.2 | 1.34 ± 0.12 | 1.38 ± 0.21 * | 1.53 ± 0.37 ** | 1.52 ± 0.54 ** |
Neurotrophic growth factor | |||||
Bdnf | 1.16 ± 0.29 | 0.84 ± 0.48 | 2.01 ± 0.68 *** | 0.19 ± 0.04 ** | 0.17 ± 0.17 ** |
Sinucleopathy | |||||
Snca | 0.80 ± 0.21 | 1.38 ± 0.14 | 1.06 ± 0.27 | 1.09 ± 0.48 | 0.90 ± 0.60 |
Ubiquitination | |||||
Uchl1 | 0.88 ± 0.17 | 1.08 ± 0.14 | 1.13 ± 0.24 | 0.63 ± 0.19 | 0.46 ± 0.22 ** |
Vesicular transport | |||||
Vmat2 | 1.11 ± 0.21 | 1.16 ± 0.56 | 0.46 ± 0.28 | 1.07 ± 0.38 | 0.47 ± 0.11 |
Apoptosis | |||||
Anxa5 | 1.20 ± 0.14 | 1.20 ± 0.31 | 1.00 ± 0.23 | 1.57 ± 0.37 * | 1.79 ± 0.58 *** |
Gene | Accession Number | Forward 5′-3′ | Reverse 3′–5′ |
---|---|---|---|
Gapdh | NM_017008.2 | CTCTCTGCTCCTCCCTGTTC | CACCGACCTTCACCATCTTG |
Tbp | NM_001004198.1 | CAATGACTCCTATGACCCCT | TTTACAGCCAAGATTCACGG |
Bdnf | NM_012513.4 | GGACATATCCATGACCAGAA | GGCAACAAACCACAACAT |
Uchl1 | NM_017237.3 | GGAACTGAAGGGACAAGAAG | ATCCATCCTCAAATTCCAGC |
Vmat2 | NM_013031.1 | AACGTCGCCAAATGTTTAAC | CAATGGATGGTGGGACTAAG |
Snca | NM_019169.2 | ACCCCTCTTGCATTGAAATT | CATGAACACATCCATGGCTA |
Hmox1 | NM_012580.2 | GGTGATGGCCTCCTTGTA | ATAGACTGGGTTCTGCTTGT |
Srxn1 | NM_001047858.3 | AAGGCGGTGACTACTACT | TTGGCAGGAATGGTCTCT |
Tnfα | NM_012675.2 | CAACTACGATGCTCAGAAACAC | AGACAGCCTGATCCACTCC |
Il6 | NM_012589.1 | GCCCACCAGGAACGAAAGTC | ATCCTCTGTGAAGTCTCCTCTCC |
IL1β | NM_031512.2 | AACAAGATAGAAGTCAAGA | ATGGTGAAGTCAACTATG |
Cox2 | NM_017232.3 | CTTTCAATGTGCAAGACC | TACTGTAGGGTTAATGTCATC |
CD68 | NM_001031638.1 | ACTTGGCTCTCTCATTCC | GACTGTACTGTGGCTCTG |
Anxa5 | NM_013132.1 | TAATGACCAAAGCTGTCTCG | TTGATTGACAGCACTTCCAA |
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Dal Ben, M.; Bongiovanni, R.; Tuniz, S.; Fioriti, E.; Tiribelli, C.; Moretti, R.; Gazzin, S. Earliest Mechanisms of Dopaminergic Neurons Sufferance in a Novel Slow Progressing Ex Vivo Model of Parkinson Disease in Rat Organotypic Cultures of Substantia Nigra. Int. J. Mol. Sci. 2019, 20, 2224. https://doi.org/10.3390/ijms20092224
Dal Ben M, Bongiovanni R, Tuniz S, Fioriti E, Tiribelli C, Moretti R, Gazzin S. Earliest Mechanisms of Dopaminergic Neurons Sufferance in a Novel Slow Progressing Ex Vivo Model of Parkinson Disease in Rat Organotypic Cultures of Substantia Nigra. International Journal of Molecular Sciences. 2019; 20(9):2224. https://doi.org/10.3390/ijms20092224
Chicago/Turabian StyleDal Ben, Matteo, Rosario Bongiovanni, Simone Tuniz, Emanuela Fioriti, Claudio Tiribelli, Rita Moretti, and Silvia Gazzin. 2019. "Earliest Mechanisms of Dopaminergic Neurons Sufferance in a Novel Slow Progressing Ex Vivo Model of Parkinson Disease in Rat Organotypic Cultures of Substantia Nigra" International Journal of Molecular Sciences 20, no. 9: 2224. https://doi.org/10.3390/ijms20092224