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 microcirculation^1,2. 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

The experimental protocol was approved by the Animal Ethics and Use Committee of Kunming Medical University (kmmu20220945). Sprague-Dawley (SD) rats of both sexes, 300-350 g, were used for the present study.

1. Inducing the SAH model

1. Anesthetize the rats using 2% isoflurane and 100% oxygen. Maintain anesthesia by supplying continuous inhalation anesthesia with isoflurane (1%-3%). Secure the rat's head using a stereotaxic apparatus (see Table of Materials).
2. Create the SAH model following the steps below.
   1. Insert a microinjection needle into the suprasellar cistern. Then, inject autologous blood from the rat's tail artery (without anticoagulant) into the suprasellar cistern using a syringe pump (see Table of Materials).
   2. Implant a microinjection needle into the right lateral cerebral ventricle using the mentioned coordinates: bregma, −0.8 mm; lateral, 1.4 mm; depth, 4 mm⁹. For SAH groups, inject 0.2 mL of rat tail artery blood. For sham groups, administer the same volume of isotonic saline (Figure 1A).

2. Brain slice preparation and stabilization

1. Deeply anesthetize the rats using 2% isoflurane inhalation. After decapitation, extract the entire brains and immerse them in ice-cold artificial cerebrospinal fluid (ACSF).
   NOTE: Use freshly prepared ACSF solutions with the following composition: 134 mM NaCl, 2.8 mM KCl, 29 mM NaHCO₃, 1.1 mM NaH₂PO₄, 1.5 mM MgSO₄, 2.5 mM CaCl₂, and 10.11 mM D-Glucose (see Table of Materials). Maintain a pH between 7.3 and 7.4.
2. Use a vibratome (see Table of Materials) to prepare acute brain slices with a thickness of 200 µm from the SD rats in ice-cold ACSF that's aerated with 5% CO₂ and 95% O₂ (Figure 2).
3. Place the 200 µm-thick acute brain slices in a storage chamber on a nylon mesh submerged in ACSF. Equilibrate the ACSF solution with 95% O₂ and 5% CO₂, maintaining a temperature range of 35-37 °C. Allow brain slices to stabilize for 2 h, giving them time to adjust (Figure 3).

3. Labeling pericytes in acute brain slices with TO-PRO-3

NOTE: Pericytes from acute brain slices were fluorescently labeled using the tracer TO-PRO-3¹⁰.

1. Add the fluorescent dye TO-PRO-3 to the ACSF, achieving a final concentration of 1 µM. Incubate the acute brain slices at room temperature in a dark environment with the TO-PRO-3-containing ACSF for 20 min.
   NOTE: Remember to wear latex gloves while handling TO-PRO-3 for protection.
2. Transfer the nylon mesh strainer, carrying the brain slices, from the loading chamber to a 6-well plate rinsing chamber (see Table of Materials). Allow the brain slices to stay in the rinsing chamber for 10 min (Figure 4D).
   NOTE: This rinsing step terminates the staining process, decreases background labeling, and prevents nonspecific staining.
3. Rinse the preparations for a total of 15 min using ACSF solution that's equilibrated with a mixture of 5% CO₂ and 95% O₂. This rinsing step halts dye uptake and minimizes background labeling (Figure 4C).

4. Staining non-vital pericytes of cerebral microcirculation in acute brain slices

1. Incubate brain slices preloaded with TO-PRO-3 at 37 °C with isolectin B4 conjugated to Alexa Fluor 488 (FITC-ISOB4; 5 µg/mL, see Table of Materials) for 30 min in a dark environment. After incubation, rinse the brain slices for 15 min in ACSF (Figure 4E).
2. Incubate the brain slices in ACSF gassed with 5% CO₂ and 95% O₂ as usual. Add propidium iodide (PI) at a concentration of 37 µM to both solutions at 37 °C. This will label non-vital brain pericytes. Incubate the preparations in this ACSF solution for 60 min in the dark (Figure 4F).
3. Next, rinse the preparations for 15 min with ACSF solutions to halt dye uptake and minimize background labeling.

5. Imaging of vital and non-vital brain pericytes in acute brain slices

1. Gently transfer brain slices to glass bottom confocal dishes (see Table of Materials) using plastic Pasteur pipettes. Fill the dishes with ACSF solution previously equilibrated with 5% CO₂ and 95% O₂. Use an iron mesh to secure rat brain slices in place.
2. Transfer the glass bottom confocal dishes to the stage of a confocal microscope. Position the acute brain slice at the bottom of the dish and perfuse⁹ it with ACSF solution equilibrated with a mixture of 95% O₂ and 5% CO₂ during confocal microscopy imaging (Figure 5A and Figure 5Ab').
3. Visualize the wall cells of the cerebral cortical microvasculature using a 40x objective. Capture image stacks using compatible software (see Table of Materials). Use appropriate filters for IB4 (excitation/emission 460 nm/520 nm), PI (excitation/emission 545 nm/595 nm), and TO-PRO-3 (excitation/emission 606 nm/666 nm) (Figure 5D).
   NOTE: Capture images with the following details: 40× DIC N1 objective lens, 3 × 12 bit: 512 × 512 pixels (0.22 × 0.22 mm), calibration: 0.42 µm/px.
4. Identify cerebral microvasculature and pericytes based on their network and morphology. Locate a specific region containing a cerebral microvasculature network on each brain slice and capture images.
5. Carefully note the imaging location to ensure consistency in subsequent captures (Figure 5B,C). For further processing and analysis, use image analysis software (ImageJ) and photo editing software (see Table of Materials).

Representative Results

Under normal physiological conditions, brain pericytes generally do not undergo cell death. Figure 6 illustrates this phenomenon, with yellow indicating the presence of vital brain pericytes; brain pericytes show no staining with PI, indicating their viability. To further investigate whether pericytes remain attached to the microvasculature following cell death, methods were employed in a SAH rat model, and subsequent imaging was conducted.

Methods for imaging both vital and non-vital brain pericytes in brain slices after SAH have been developed. As depicted in Figure 7, vital brain pericytes (blue arrows) are located within the microvasculature, while non-vital brain pericytes are represented by white arrows. This simultaneous visualization allows for the identification of both vital and non-vital brain pericytes within brain slices. Furthermore, it was observed that PI-labeled non-vital brain pericytes remained attached to the entire microvasculature.

Figure 1: SAH model. (A) The rat's head was firmly secured to the stereotaxic apparatus to ensure stability. The SAH model was induced by carefully inserting a stereotaxic needle into the suprasellar cistern. (B,C) In the SAH model, blood enters the subarachnoid space within the skull, affecting the brain. The cerebral hemispheres fill with blood approximately 24 h after SAH. Please click here to view a larger version of this figure.

Figure 2: Acute whole brain slice preparation. (A) The bottom chamber was coated with agarose glue for fixation. (B) The glue was meticulously applied to the bottom chamber to adhere firmly to the brain. (C) The surrounding tank was filled with ice-cold ACSF to maintain temperature. (D) The desired section thickness was carefully defined. (E,F). The brain slices were meticulously transferred to a 6-well plate. Please click here to view a larger version of this figure.

Figure 3: Transfer pipettes and dye-loading in a 6-well plate. (A) Acute brain slices were transferred using plastic Pasteur pipettes (3 mL). The length of the pipette used in (a) was 18.2 cm. For (b) and (c), the fine tip of the plastic Pasteur pipette was carefully trimmed to prevent any potential damage to the brain slices during transfer. The 6-well plates were efficiently transitioned from (B) to (C) for fluorescence staining in a 37 °C water bath. (B) One well of the 6-well plate accommodated a small nylon mesh strainer, with fine tubing positioned for gas delivery. Please click here to view a larger version of this figure.

Figure 4: Incubation of acute brain slices. A systematic procedure was meticulously followed to label pericytes with TO-PRO-3 in the acute brain slices. (A) Initially, one well of a six-well plate (12 × 8 cm) was filled with 10 mL of ACSF, ensuring proper aeration by bubbling the solution using fine tubing connected to a mixture of 95% O₂ and 5% CO₂. (B) Subsequently, the brain slices were meticulously transferred to the six-well plate. (C) 10 µL of the TO-PRO-3 stock solution was introduced into the dye-loading chamber, gently agitating to facilitate dye dissolution (D). (E,F) To further characterize the brain slices, staining with IB4 (1 µM in ACSF) and PI (1 µM in ACSF) was conducted. These sequential steps enabled the successful labeling of pericytes with TO-PRO-3 in acute brain slices, thus facilitating subsequent analysis and examination of the labeled brain pericytes. Please click here to view a larger version of this figure.

Figure 5: Imaging of vital and non-vital brain pericytes in brain slices. (A) The solution is equilibrated with a mixture of 95% O₂ and 5% CO₂ delivered through the tubing. (B-E) Setup for high-resolution imaging of TO-PRO-3-labeled, IB4-labeled endothelial, and PI-labeled cells in acute brain slices. (E) Confocal microscopy. Please click here to view a larger version of this figure.

Figure 6: Image of vital brain pericytes in brain slices. (A) Cerebral microvasculature was labeled with IB4 (green). (B) Non-vital cells were labeled with PI. (C) Vital pericytes were labeled with TO-PRO-3 (yellow). (D) The merged image is displayed. Please click here to view a larger version of this figure.

Figure 7: Representative image of vital and non-vital brain pericytes in a brain slice after SAH. (A) Cerebral microvasculature was labeled with IB4 (green). (B) Non-vital cells were labeled with PI (red). (C) Vital pericytes were labeled with TO-PRO-3 (yellow). (D) The merged image is displayed. Blue arrows indicate examples of vital brain pericytes, while white arrows indicate examples of non-vital brain pericytes. Notably, the nuclei of pericytes do not protrude above the microvascular surface. Please click here to view a larger version of this figure.

Figure 8: Positive correlation between the number of non-vital pericytes and time after SAH. High-resolution confocal imaging was employed to capture brain pericytes at various time points post SAH. Noticeable cell death of brain pericytes commenced at 6 h after SAH, as indicated by white arrows in (A). Subsequently, there was a substantial increase in pericyte death from the 6 h mark onward. The count of non-vital pericytes displayed a positive correlation with the time elapsed after SAH, as depicted in (B). Please click here to view a larger version of this figure.

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 research.

To specifically trace brain pericytes within the nervous system, the far-red fluorophore TO-PRO-3 is employed. While TO-PRO-3 predominantly stains the nuclei of fixed tissue¹³, it selectively incorporates into living pericytes ex vivo when applied in physiological saline¹⁴. Prior studies have affirmed the unequivocal identification of murine brain pericytes using TO-PRO-3¹⁵. This fluorophore effectively labels vital brain pericytes¹¹. In contrast, PI, a commonly utilized charged fluorochrome for real-time cell viability assessment¹², serves as a marker for deteriorating cells when applied before fixation (pre-fixation PI staining)¹⁶. However, since the morphological characteristics of pericytes, particularly their nuclei, do not extend above the microvascular surface, distinguishing between endothelial and pericyte nuclei using PI staining can be challenging, as indicated by the white arrow in Figure 8A. Studies involving electron microscopy for three-dimensional reconstruction of the CA1 region of the rat hippocampus have suggested that approximately one-third of the endothelium is covered by the somas and processes of pericytes¹⁷.

In Figure 6, brain pericytes labeled with TO-PRO-3 are observed within the microvasculature. Confocal images of the cerebral microvasculature labeled with IB4 and PI reveal that only a few cells undergo cell death during the sectioning process, as evidenced by the absence of PI-positive dead cells in Figure 6. Consequently, the SAH model is utilized to investigate the presence of non-vital pericytes. Figure 7 illustrates that, within brain slices, cell death is more prominent in pericytes compared to endothelial cells. Additionally, in Figure 8A, a significant increase in pericyte death is observed starting 6 h after SAH. Figure 8B demonstrates a positive correlation between the number of non-vital pericytes and the time elapsed after SAH. Nevertheless, a comprehensive study imaging both vital and non-vital brain pericytes have not been documented to the best of the current knowledge.

Currently, it remains uncertain whether pericytes from other species or systems (e.g., heart, small intestine) also manifest vital and non-vital states in acute slices after SAH. In conclusion, the provided protocol offers a replicable approach for imaging both vital and non-vital brain pericytes in brain slices post-SAH. Due to its simplicity and reliability, this technique can be employed to unveil the functional and structural diversity of pericytes in brain slices and facilitate detailed cellular investigations in various disease models related to pericyte pathophysiology.

Materials

Name	Company	Catalog Number	Comments
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