Imaging Vital and Non-vital Brain Pericytes in Brain Slices following Subarachnoid Hemorrhage
Summary
The preliminary inquiry confirms that subarachnoid hemorrhage (SAH) causes brain pericyte demise. Evaluating pericyte contractility post-SAH requires differentiation between viable and non-viable brain pericytes. Hence, a procedure has been developed to label viable and non-viable brain pericytes concurrently in brain sections, facilitating observation using a high-resolution confocal microscope.
Abstract
Pericytes are crucial mural cells situated within cerebral microcirculation, pivotal in actively modulating cerebral blood flow via contractility adjustments. Conventionally, their contractility is gauged by observing morphological shifts and nearby capillary diameter changes under specific circumstances. Yet, post-tissue fixation, evaluating vitality and ensuing pericyte contractility of imaged brain pericytes becomes compromised. Similarly, genetically labeling brain pericytes falls short in distinguishing between viable and non-viable pericytes, particularly in neurologic conditions like subarachnoid hemorrhage (SAH), where our preliminary investigation validates brain pericyte demise. A reliable protocol has been devised to surmount these constraints, enabling simultaneous fluorescent tagging of both functional and non-functional brain pericytes in brain sections. This labeling method allows high-resolution confocal microscope visualization, concurrently marking the brain slice microvasculature. This innovative protocol offers a means to appraise brain pericyte contractility, its impact on capillary diameter, and pericyte structure. Investigating brain pericyte contractility within the SAH context yields insightful comprehension of its effects on cerebral microcirculation.
Introduction
Brain pericytes, distinguished by their slender protuberances and protruding cell bodies, encircle the microcirculation1,2. While cerebral blood flow augmentation is predominantly driven by capillary dilation, smaller arteries exhibit slower rates of dilation3. Pericyte contractility exerts influence over capillary diameter and pericyte morphology, impacting vascular dynamics4. Contraction of brain pericytes leads to capillary constriction, and in pathological scenarios, excessive contraction may impede erythrocyte flow5. Various factors, including norepinephrine released from the locus coeruleus, can induce brain pericyte contraction within capillaries6. With a regulatory role in cerebral blood flow, pericytes exhibit 20-HETE synthesis, serving as an oxygen sensor during hyperoxia7. Oxidative-nitrative stress-triggered contraction of brain pericytes detrimentally affects capillaries5. Despite both in vivo and ex vivo investigations into brain pericyte contraction8, limited knowledge persists regarding the imaging of viable and non-viable brain pericytes within brain slices.
Crucially, post-tissue fixation imaging of brain pericytes compromises their vitality and subsequent contractility assessment. Moreover, in scenarios such as neurological disorders (e.g., subarachnoid hemorrhage – SAH), transgenic labeling of brain pericytes fails to differentiate between viable and non-viable pericytes, as confirmed by our preliminary SAH-induced brain pericyte death study9.
To surmount these challenges, we employed TO-PRO-3 to label live pericytes, while deceased ones were stained with propidium iodide (PI). We used high-resolution confocal imaging technologies to visualize viable and non-viable brain pericytes in brain slices while preserving slice activity during imaging. This article aims to present a reproducible method for imaging viable and non-viable brain pericytes in brain slices, serving as a valuable tool to probe the impact of brain pericytes on cerebral microcirculation post SAH.
Protocol
Representative Results
Discussion
Developed are high-resolution confocal imaging techniques for visualizing vital brain pericytes, non-vital brain pericytes, and the microvasculature in brain slices. In acute rat brain slices, the process entails initial labeling of pericytes with TO-PRO-311, followed by microvascular endothelial cells with IB412; subsequently, identification of deceased pericytes is conducted using PI. This protocol is straightforward, reproducible, and highly applicable for functional res…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
The study was supported by grants from the National Natural Science Foundation of China (81960226,81760223); the Natural Science Foundation of Yunnan Province (202001AS070045,202301AY070001-011)
Materials
| 6-well plate | ABC biochemistry | ABC703006 | RT |
| Adobe Photoshop | Adobe | Adobe Illustrator CS6 16.0.0 | RT |
| Aluminium foil | MIAOJIE | 225 mm x 273 mm | RT |
| CaCl2·2H2O | Sigma-Aldrich | C3881 | RT |
| Confocal imaging software | Nikon | NIS-Elements 4.10.00 | RT |
| Confocal Laser Scanning Microscope | Nikon | N-SIM/C2si | RT |
| Gas tank (5% CO2, 95% O2) | PENGYIDA | 40L | RT |
| Glass Bottom Confocal Dishes | Beyotime | FCFC020-10pcs | RT |
| Glucose | Sigma-Aldrich | G5767 | RT |
| Glue | EVOBOND | KH-502 | RT |
| Ice machine | XUEKE | IMS-20 | RT |
| Image analysis software | National Institutes of Health | Image J | RT |
| Inhalation anesthesia system | SCIENCE | QAF700 | RT |
| Isolectin B 4-FITC | SIGMA | L2895–2MG | Store aliquots at –20 °C |
| KCl | Sigma-Aldrich | 7447–40–7 | RT |
| KH2PO4 | Sigma-Aldrich | P0662 | RT |
| MgSO4 | Sigma-Aldrich | M7506 | RT |
| NaCl | Sigma-Aldrich | 7647–14–5 | RT |
| NaH2PO4·H2O | Sigma-Aldrich | 10049–21–5 | RT |
| NaHCO3 | Sigma-Aldrich | S5761 | RT |
| Pasteur pipette | NEST Biotechnology | 318314 | RT |
| Peristaltic Pump | Scientific Industries Inc | Model 203 | RT |
| Propidium (Iodide) | Med Chem Express | HY-D0815/CS-7538 | Store aliquots at –20 °C |
| Stereotaxic apparatus | SCIENCE | QA | RT |
| Syringe pump | Harvard PUMP | PUMP 11 ELITE Nanomite | RT |
| Thermostatic water bath | OLABO | HH-2 | RT |
| Vibrating microtome | Leica | VT1200 | RT |
Referencias
- Dalkara, T., Gursoy-Ozdemir, Y., Yemisci, M. Brain microvascular pericytes in health and disease. Acta Neuropathologica. 122 (1), 1-9 (2011).
- Dore-Duffy, P., Cleary, K. Morphology and properties of pericytes. Methods in Molecular Biology (Clifton, N.J). 686, 49-68 (2011).
- Peppiatt, C. M., Howarth, C., Mobbs, P., Attwell, D. Bidirectional control of CNS capillary diameter by pericytes. Nature. 443 (7112), 700-704 (2006).
- Attwell, D., Mishra, A., Hall, C. N., O’Farrell, F. M., Dalkara, T. What is a pericyte. Journal of Cerebral Blood Flow and Metabolism: Official Journal of the International Society of Cerebral Blood Flow and Metabolism. 36 (2), 451-455 (2016).
- Yemisci, M., Gursoy-Ozdemir, Y., Vural, A., Can, A., Topalkara, K., Dalkara, T. Pericyte contraction induced by oxidative-nitrative stress impairs capillary reflow despite successful opening of an occluded cerebral artery. Nature Medicine. 15 (9), 1031-1037 (2009).
- Korte, N., et al. Noradrenaline released from locus coeruleus axons contracts cerebral capillary pericytes via α2 adrenergic receptors. Journal of Cerebral Blood Flow and Metabolism: Official Journal of the International Society of Cerebral Blood Flow and Metabolism. , (2023).
- Hirunpattarasilp, C., Barkaway, A., Davis, H., Pfeiffer, T., Sethi, H., Attwell, D. Hyperoxia evokes pericyte-mediated capillary constriction. Journal of Cerebral Blood Flow and Metabolism: Official Journal of the International Society of Cerebral Blood Flow and Metabolism. 42 (11), 2032-2047 (2022).
- Neuhaus, A. A., Couch, Y., Sutherland, B. A., Buchan, A. M. Novel method to study pericyte contractility and responses to ischaemia in vitro using electrical impedance. Journal of Cerebral Blood Flow and Metabolism: Official Journal of the International Society of Cerebral Blood Flow and Metabolism. 37 (6), 2013-2024 (2017).
- Gong, Y., et al. Increased TRPM4 Activity in cerebral artery myocytes contributes to cerebral blood flow reduction after subarachnoid hemorrhage in rats. Neurotherapeutics: The Journal of the American Society for Experimental NeuroTherapeutics. 16 (3), 901-911 (2019).
- Mai-Morente, S. P., et al. Pericyte mapping in cerebral slices with the far-red fluorophore TO-PRO-3. Bio-protocol. 11 (22), e4222 (2021).
- Mai-Morente, S. P., Marset, V. M., Blanco, F., Isasi, E. E., Abudara, V. A nuclear fluorescent dye identifies pericytes at the neurovascular unit. Journal of Neurochemistry. 157 (4), 1377-1391 (2021).
- Zhao, H., et al. Rationale for the real-time and dynamic cell death assays using propidium iodide. Cytometry. Part A: The Journal of the International Society for Analytical Cytology. 77 (4), 399-405 (2010).
- Van Hooijdonk, C. A., Glade, C. P., Van Erp, P. E. TO-PRO-3 iodide: A novel HeNe laser-excitable DNA stain as an alternative for propidium iodide in multiparameter flow cytometry. Cytometry. 17 (2), 185-189 (1994).
- Lacar, B., Herman, P., Platel, J. C., Kubera, C., Hyder, F., Bordey, A. Neural progenitor cells regulate capillary blood flow in the postnatal subventricular zone. The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 32 (46), 16435-16448 (2012).
- Mai-Morente, S. P., Marset, V. M., Blanco, F., Isasi, E. E., Abudara, V. A nuclear fluorescent dye identifies pericytes at the neurovascular unit. Journal of Neurochemistry. 157 (4), 1377-1391 (2021).
- Hezel, M., Ebrahimi, F., Koch, M., Dehghani, F. Propidium iodide staining: a new application in fluorescence microscopy for analysis of cytoarchitecture in adult and developing rodent brain. Micron (Oxford, England). 43 (10), 1031-1038 (2012).
- Mathiisen, T. M., Lehre, K. P., Danbolt, N. C., Ottersen, O. P. The perivascular astroglial sheath provides a complete covering of the brain microvessels: An electron microscopic 3D reconstruction. Glia. 58 (9), 1094-1103 (2010).
