Method Article

Paired Cisterna Magna Nanoinjection and Laser Speckle Contrast Imaging Assay to Study Cerebral Blood Flow Regulation In Vivo

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DOI:

10.3791/67544

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July 8th, 2025

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1Department of Neuroscience, Center for Brain Immunology and Glia (BIG), University of Virginia School of Medicine

In This Article

Summary

To study cerebral blood flow regulation in vivo, traditional pharmacological methods are invasive, often damaging the skull, dura, and leptomeninges. In this work, we developed a minimally invasive technique that combines nanoinjector-guided intracisternal magna (ICM) injection with laser speckle contrast imaging, preserving the integrity of the skull and leptomeninges.

Abstract

Cerebral blood flow (CBF) regulation is crucial for maintaining cognitive functions, but its underlying mechanisms remain poorly understood. Investigating the cellular and molecular processes involved in this regulation requires a pharmacological approach that allows precise drug delivery into cerebral spinal fluid (CSF) while enabling real-time monitoring of cerebral blood flow dynamics. In this paper, we present a method for studying cerebral blood flow regulation in vivo by combining nanoinjector-based intracisternal magna (ICM) injection with laser speckle contrast imaging.

The nanoliter injector-based ICM injection technique allows for precise drug delivery at a dynamic range from nL/min to µL/min. In our experiments, this approach significantly reduced the injection volume from the traditional 5-10 μL to just 1.5 μL, thereby maintaining normal intracranial pressure. When combined with laser speckle contrast imaging, we were able to monitor cerebral blood flow (CBF) dynamics in real time before, during, and after ICM injections.

Using this method, we evaluated CBF changes in response to ICM injection of three different vasodilators: (S)-3,5-Dihydroxyphenylglycine (DHPG), acetylcholine, and adenosine. While all three vasodilators increased basal CBF, they had distinct effects on CBF dynamics. For instance, adenosine's impact was transient, whereas DHPG and acetylcholine produced long-lasting effects. With its unique advantages, this method represents a valuable addition to the toolbox of studying rodent CBF regulation in vivo.

Introduction

Cerebral blood flow (CBF) ensures continuous supply of oxygen and nutrients to the brain while removing metabolic waste, which is essential for maintaining normal cognitive functions1,2. Under homeostatic conditions, CBF remains relatively stable despite fluctuations in perfusion pressure, a process known as autoregulation3,4,5. In addition to autoregulation, local CBF can be dynamically adjusted in response to nearby neuronal activity, a phenomenon referred to as neurovascular coupling6,7. Both autoregulation and neurovascular coupling are regulated by a group of cells-endothelial cells, mural cells, astrocytes, fibroblasts, and neurons-collectively known as neurovascular unit cells4,7. However, the cellular and molecular mechanisms by which these different cell types coordinate to modulate CBF are still largely unknown.

Methods for investigating CBF regulation are generally divided into in vitro and in vivo models. In vitro studies, often using acute brain slices, are typically combined with electrophysiological or pharmacological techniques to examine how neurons and glial cells regulate the dilation or constriction of individual blood vessels8,9,10. The primary limitation of in vitro models is that they do not replicate in vivo conditions, particularly the absence of physiological blood pressure. In vivo models for studying CBF regulation range from macroscale to microscale approaches. Macroscale techniques, such as positron emission tomography (PET) and functional magnetic resonance imaging (fMRI), are noninvasive but suffer low spatial and temporal resolution11,12,13,14.

The primary microscale method for studying CBF in vivo is two-photon imaging, which provides high spatial-temporal resolution9,15,16,17,18. However, this technique requires the removal of the skull and dura mater, and when combined with pharmacological manipulation, it becomes even more invasive, potentially causing hemorrhage, damaging brain tissue, and altering intracranial pressure19. Therefore, it's crucial to have a method for manipulating and monitoring CBF in vivo that meets specific criteria: it should be noninvasive (preserving the integrity of the skull, leptomeninges, and vessels), provide high spatial-temporal resolution and allow precise pharmacological manipulation of CBF. In this paper, we introduce a new method that combines nanoliter injector-based intracisternal magna (ICM) injection and laser speckle contrast imaging, fulfilling all these requirements.

Traditional pharmacological approaches for studying CBF regulation often involve removing the skull and meninges to expose the brain tissue for local drug delivery. In contrast, ICM injection avoids this damage by administering drugs directly into the cerebral spinal fluid (CSF), allowing them to follow the natural flow of CSF and thereby better mimic physiological conditions. One challenge with ICM injection is that it usually requires large volumes of fluid-often 5 µL or more in mice20. Given that the total CSF volume in adult mice is approximately 36 µL, injecting 5 µL can increase intracranial pressure and alter CBF dynamics. Our nanoinjector system, which uses glass pipettes, delivers fluid with precise control over volume and speed without causing CSF leakage at the injection site.

Urethane and alpha-chloralose are used in our study because they provide a more stable physiological baseline of cerebral blood flow and preserve neurovascular coupling (NVC) better than commonly used alternatives like isoflurane or ketamine/xylazine (KX). Using this system, we tested three differentvasodilators-(S)-3,5-Dihydroxyphenylglycine (DHPG), acetylcholine, and adenosine, and confirmed that it allows precise manipulation and monitoring of CBF dynamics in vivo.

Protocol

This protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Virginia. All experiments adhered to the National Institutes of Health Guide for Care and Use of Laboratory Animals. Six male C57BL/6 mice, aged 10-14 weeks, were used for the experiments. The surgical procedures are outlined in Figure 1A.

1. Surgery preparation

  1. Position a heating pad on the stereotaxic frame and preheat it to 37 °C ~20 min prior to the surgery. Prepare fresh urethane (750 mg/kg) and chloralose (50 mg/kg) in phosphate-buffered saline (PBS) for anesthesia.
    NOTE: For the preparation of the alpha-chloralose solution in PBS, we heated the solution in a 40 °C water bath and vortexed it every 10-15 min until it was fully dissolved. Both the urethane and alpha-chloralose solutions were filtered using a 0.22 µm syringe filter before use.
  2. Before surgery, gather all necessary surgical tools, including scissors, forceps, micro needle holders, hemostats, cotton swabs, wipers, and sutures. Sterilize the instruments by autoclaving (121 °C at 15 psi for 60 min).
  3. Prepare glass capillary tubes (outer diameter 1.14 mm, inner diameter 0.53 mm, 88.9 mm in length). Use a glass puller to create glass pipettes for injection21. The preferred pipette resistance is approximately 80 - 100 MΩ. Sterilize and store the glass pipettes in a clean container.
  4. Prepare a nanoinjector with proper injection volume (25 nL - 4.5 μL) and speed (6 nL/s - 644 nL/s).
  5. Prepare the following vasodilator solutions in artificial cerebrospinal fluid (aCSF): (S)-3,5-dihydroxyphenylglycine (DHPG) at 5 mM, acetylcholine at 0.5 mM, and adenosine at 10 mM.
  6. Prepare meloxicam (4.0 mg/kg) for pain relief during surgery. Administer meloxicam subcutaneously to the mice approximately 30 min before surgery to ensure adequate analgesia.

2. Surgical procedure

  1. Weigh six C57BL/6 male mice (10-14 weeks old) and anesthetize them via intraperitoneal injection of urethane (750 mg/kg) and chloralose (50 mg/kg). Place the mice in an anesthesia chamber with 1% isoflurane to maintain anesthesia. This process takes approximately 40 min to 1 h.
    NOTE: Isoflurane can speed up the induction of anesthetization.
  2. Carefully remove the mice from the anesthesia chamber and assess the depth of anesthesia by confirming the absence of the toe-pinch reflex. Using a cotton swab, apply hair removal cream to the top of the skull and the back of the neck to remove the fur.
  3. Apply ophthalmic ointment to the eyes to prevent dryness and reapply as needed throughout the procedure. Monitor regularly during the experiment and administer additional ointment when necessary.
  4. Position the mouse in a prone orientation within the stereotaxic frame, ensuring adequate ventilation with medical air at a flow rate of 1 to 2 L/min. Secure the mouse with ear bars for proper fixation, then gently tilt the head downward to form a 120° angle with the body, thereby exposing the cisterna magna.
    NOTE: Isoflurane is exclusively used for anesthesia induction and not during maintenance phase.
  5. Disinfect the surgical area using at least three alternating applications of 70% alcohol and a surgical scrub (e.g., chlorhexidine or povidone-iodine) to ensure adequate skin sterilization.
  6. Locate the occipital crest under a surgical microscope. Lift the overlying skin with tweezers and make a 1 cm midline incision. Control any bleeding with cotton swabs.
  7. Expose the neck muscles and carefully incise and separate them along the midline using forceps. Employ two curved forceps, one in each hand, to gently retract the muscles and expose the dura mater, which forms the outer boundary of the cisterna magna.
  8. Adjust the head position by tilting it downward to form a 150° angle with the body for optimal exposure of the cisterna magna.
  9. Make a 5 mm incision along the sagittal suture, covering both lambda and bregma points. Using two curved forceps, gently pull the skin aside, and clean the surface of the skull with saline. Ensure that no hair remains on the exposed skull.
  10. After cleaning, apply a layer of ultrasound gel ~1 mm thick to the exposed skull using cotton swabs to maintain moisture in the area. Since ultrasound gel may contain air bubbles, ensure that all air bubbles are removed prior to imaging.
  11. Position the mouse under the lens of the laser speckle contrast imaging system. Turn on the system and its accompanying software. Ensure the mouse brain is centered within the imaging field, then use the Auto Focus function to achieve the correct focus for imaging. If necessary, gently remove any remaining hairs or air bubbles from the imaging field using tweezers.

3. Nanoinjection setup

  1. Secure the pump to the stereotaxic manipulator arm and connect the cable to the controller. Turn on the controller and select the target pump. In the control panel, set DIRECTION to INFUSE. Set the speed to 2μl per minute and volume to 4.5 μl.
    NOTE: The nanoinjection system consists of two main components: the injection pump and the controller. Each controller can operate two pumps simultaneously. For ICM injection, we only use one pump.
  2. After the INFUSE is complete, take one glass pipette out from the container and fill it with colorless, odorless mineral oil using a 34 G MicroFil flexible needle or a similar type of needle. Make sure that the entire glass pipette is filled with oil.
  3. Unscrew the collet of the pump, insert the glass pipette, and then tighten the collet. Make sure there are no air bubbles present inside the glass pipette.
  4. Change the DIRECTION in the control panel for WITHDRAW. Set the speed to 2 μ L/min and the volume to 4.5 μ L.
  5. Move the stereotaxic arm to gently introduce the pipette into the prepared solution and withdraw the desired volume.

4. Injection of vasodilators into the cisterna magna and monitoring CBF

  1. Ensure the pump is securely fixed to the stereotaxic manipulator arm. Adjust the angle of the stereotaxic manipulator arm to 45° in the anterior-posterior plane.
  2. Using curved tweezers under the dissection microscope, carefully expose the cisterna magna. Clean the area with saline to remove any blood. Utilize the stereotaxic micromanipulator to position the glass pipette near the dura mater of the cisterna magna.
  3. Once the glass pipette touches the dura, gently maneuver it to pass into the center of the cisterna magna, avoiding any penetration into the cerebellum or medulla. Ensure the tip of the glass pipette is inserted to a depth of 1-2 mm before releasing the tweezers.
    NOTE: A digital stereotaxic frame is essential for monitoring the depth of glass pipette penetration into cisterna magna.
  4. Change the DIRECTION in the control panel to INFUSE. Set the injection speed to 300 nL/min and the volume to 1.5 µL.
  5. Launch the software for laser speckle contrast imaging (LSCI) and adjust the focus to achieve the clearest image of the blood vessels. Ensure there are no hair or air bubbles in the field of view.
  6. Set the imaging interval to 5 s in the LSCI control panel. Record the baseline CBF for approximately 10 min, ensuring that the baseline remains stable. If there are fluctuations in the baseline, continue recording for a longer duration until the stability is achieved.
  7. After 10 min of stable baseline recording, initiate the nanoinjection and note the injection start time in the LSCI recording system. Once the injection is complete, mark the finish time and continue recording for an additional 20 min or longer. After completing the experiment, transcardiac perfusion the mice with PBS and collect brain tissues for histological analysis.

Results

After completing the surgery, we secured the mouse in a stereotaxic frame with a digital readout (Figure 1A,B). For cerebral blood flow (CBF) measurement, we carefully pulled apart the skin over the skull following the incision and applied a transparent ultrasound gel to maintain moisture on the skull's surface (Figure 1C). To facilitate intracisternal magna (ICM) injection, we dissected the neck muscles around the occipital crest to expose the cisterna magna. After that, we withdraw target solution with glass pipette, and used the micromanipulator to guide the glass pipette to penetrate the membranes of cisterna magna to a depth of 1-2 mm (Figure 1D,E). The stereotaxic arm was tilted at a 45o angle to assist with the insertion.

Next, we examined CBF dynamic changes in response to three different vasodilators: (S)-3,5-dihydroxyphenylglycine (DHPG), acetylcholine, and adenosine22,23,24,25. We initially recorded a 10-minute baseline before injecting 1.5 µL of each vasodilator at the speed of 300 nL/min over 5 min. After the injection, we recorded an additional 20 min of data. Our results showed that all three vasodilators increased CBF, though with distinct dynamics. DHPG induced a sharp increase in CBF, followed by a gradual decline after injection (Figure 2A,B). In contrast, acetylcholine elicited a gradual increase in CBF throughout the recording period (Figure 2C,D). Adenosine also produced a transient increase in CBF, which returned to baseline level approximately 15-20 min after injection (Figure 2E,F).

Nano-injection diagram and setup: cranial surgery, glass pipette, LSCI monitoring, cisterna magna injection.
Figure 1: Summary of surgery procedures. (A) Step-by-step main surgical procedures for nanoinjection + LSCI model. (B) Schematics showing the combined nanoinjection + LSCI system. The nanoinjector is fixed stably on the stereotaxic arm and the arm is tilted by 45o to facilitate penetration of the atlanto-occipital membrane in the cisterna magna. (C) Top-down view of the mouse skull under the LSCI system. The skull is covered with ultrasound gel. (D) Back view showing the glass pipette penetrating the cisternal magna. The glass pipette was filled with Evans blue to facilitate visualization. Use the digital stereotaxic frame for this experiment so that the penetration distance can be precisely quantified. (E) Schematics showing the glass pipette tip location in cisternal magna. The mouse's head was tilted by 30o to facilitate the cisternal magna injection. Abbreviation: LCSI = laser speckle contrast imaging. Please click here to view a larger version of this figure.

Brain blood flow change by DHPG, acetylcholine, adenosine; heat maps, graphs, CBF analysis.
Figure 2: Increased cerebral blood flow after nanoinjection of vasodilators. (A) Representative images showing cerebral blood flow changes before and after DHPG injection. Blood speed is color-coded. Blue represents low speed and red represents high speed. (B) Real-time blood flow in the region marked with a red line, which indicates the time window for the DHPG injection. (C) Representative images showing cerebral blood flow changes before and after ACh injection. Blood speed is color-coded. Blue and red indicate low and high speeds, respectively. (D) Real-time blood flow in the region marked by the red line, which indicates the time window for ACh injection. (E) Representative images showing cerebral blood flow changes before and after adenosine injection. Blood speed is color-coded. Blue and red indicate low and high speeds, respectively. (F) Real-time blood flow in the region marked by the red line, which indicates the time window for adenosine injection. Abbreviations: DHPG = (S)-3,5-Dihydroxyphenylglycine; ACh = acetylcholine. Please click here to view a larger version of this figure.

Discussion

We present a novel method for investigating cerebral blood flow (CBF) regulation in vivo by combining precise cisternal magna nanoinjection with laser speckle contrast imaging (LSCI). This approach allows for accurate drug delivery into the cerebral spinal fluid (CSF) while enabling real-time monitoring of CBF, facilitating the evaluation of cerebral vascular reactivity.

Pharmacological manipulation and drug delivery techniques are critical for studying CBF and cerebral vascular reactivity in vivo. Traditional methods for drug application, such as the use of vasodilators or constrictors, often necessitate the removal of the skull, which can damage the leptomeninges and even the brain parenchyma9,10,18. These invasive techniques can disrupt the brain microenvironment, particularly by causing vascular damage and hemorrhage, thereby failing to replicate the real physiological conditions. In contrast, our method offers four significant advantages.

First, our approach for drug application is noninvasive and preserves physiological conditions. Second, classical ICM injection methods typically rely on syringes or cannulas, which can lead to leakage of drugs or traces and CSF due to the large size of the tips. These traditional methods often require large amounts of drug or trace -- usually 5 µL or more, which can alter the intracranial pressure. In contrast, our nanoinjector-guided ICM injection requires only a small volume of as little as 1.5 µL or less. Additionally, the injection speed can be precisely controlled across a wide range, from 1 nL/s to 100 nL/s. We typically use a speed of 5 nL/s (300 nL/min), as this rate does not affect intracranial pressure. Third, unlike pipette-guided drug injections into brain parenchyma or drug perfusion onto the cortical surface, our cisternal magna injection-based drug delivery follows the natural flow of CSF, closely mimicking physiological conditions. Finally, since our method preserves the integrity of the skull and meninges, we can conduct measurements on the same group of mice at different time points, allowing for longitudinal comparisons -- something that is not feasible with other drug application methods. For survival studies and longitudinal comparisons, strict aseptic techniques must be followed, including proper skin preparation, use of sterile instruments and materials, and compliance with institutional animal care guidelines. A two-week recovery period should be allowed for wound healing.

The method of anesthesia can significantly impact the CBF. For instance, ketamine-xylazine anesthesia can elevate CBF by over 50% above the baseline levels, while isoflurane can double the basal CBF level. In our experiments, we opted urethane and chloralose due to their minimal effects on cardiovascular system, which helps maintain physiological levels of CBF, as commonly used in the field26,27. In addition, we skipped intraarterial cannulation for repeated blood gas measurement to be non-invasive, although such procedures can be combined with our method26.

Our approach integrates two systems: the nanoinjection-based ICM injection system28 and the laser speckle contrast imaging (LSCI) system29. Experimenters must be trained in both systems to perform the procedure successfully. Since the LSCI system occupies all the space above the mouse's head, tilting the arm by 45o not only facilitates ICM injection but also ensures compatibility between the two systems. This setup is critical for the experiment's success.

For beginners using the nanoinjector-guided ICM injection, extensive practice is required to master the coordination of separating the muscles with tweezers in one hand while maneuvering the micromanipulator to guide the glass pipette with the other. A good laser light and an articulating arm stereo microscope are essential for optimal visualization during the procedure.

Using this method, we assessed how classical vasodilators such as DHPG, acetylcholine, and adenosine affect CBF. As anticipated, all three vasodilators increased CBF; however, they induced different dynamics. DHPG caused a sharp increase in CBF, followed by a slow decay, while acetylcholine elicited a gradual increase. In contrast, adenosine produced only a transient rise in CBF.

This method should also be applicable to rat models. However, given that the rat skull thickness is approximately 0.5-1 mm, it will need to be thinned for laser speckle contrast imaging. Additionally, while the current method can be adapted for head-fixed awake mice, it necessitates extensive training to minimize the animals' movement during experiments. We used a limited number of mice to demonstrate our method; therefore, our study is statistically constrained, and caution should be exercised when drawing broad conclusions from our findings.

For consistent and reliable CBF measurements, it is important for experimenters to recognize that other physiological parameters, such as body temperature, blood pressure, and CO2 levels, play a critical role in comprehensive physiological assessment and should be carefully considered in future studies. In summary, we present a new in vivo method for studying CBF regulation in mice. This approach is precise and minimally invasive, making it well-suited for investigating drug-induced CBF dynamics.

Disclosures

The authors have no conflicts of interest to disclose.

Acknowledgements

This work was supported by the NIH grants (NS108763, NS127392, NS125788, NS125677).

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
(RS)-3,5-DihydroxyphenylglycineTocrisCat. No. 0342
Acetylcholine chlorideSigmaA6625-25G
AdenosineSigmaA9251-25G
Alcohol Swab 70% Isopropyl AlcoholVitrex Medical A/S520213
AmScope SM-8T Series Articulating Arm Zoom Stereo MicroscopeAmScopeSM-8TZ-144S-10M
Aquasonic Clear Ultrasound Transmission GelAquasonicGelPLI 03-50
Digital Mouse Stereotaxic InstrumentsStoelting51730D
Ethyl alcoholSigma459836-2L
Fiber-Lite DC950 IlluminatorDolan-Jenner industriesDC950H
Glass replacement 1.14 MMWorld Precision Instruments504949
Kimtech Science Precision Wipes Tissue WipersKimberly Clark Professional5511
Laser Speckle Contrast Imagermoor instrumentsMOORFLPI-2
Lasik Eye Spear (100 Per Case) hospeqBV40815
MICROFIL FLEXIBLE NEEDLEWorld Precision InstrumentsMF34G-5
Mineral oilSigmaM5904
Mouse heating padFisher scientific50-195-4000
Nair Cocoa Butter Hair Remover LotionAmazonASIN ‏ : ‎ B000GCW5CS
NANOLITER 2020 INJECTORWorld Precision InstrumentsNANOLITER2020
P-2000 micropipette pullerSutter InstrumentP-2000
Puritan® Swabs, Cotton Head, Wood Shaft,Puritan Medical806-WC
Sterile Ocular LubricantMedi-Vet11897
UrethaneSigmaU2500
Vetbond tissue glueWorld Precision InstrumentsVETBOND
α-ChloraloseSigma23120

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Cisterna Magna InjectionLaser Speckle ImagingNeurovascular CouplingMicroglial RegulationVasodilator InjectionIn Vivo AssayStereotaxic SurgeryRodent Brain ImagingIntracisternal Injection
Paired Cisterna Magna Nanoinjection and Laser Speckle Contrast Imaging Assay to Study Cerebral Blood Flow Regulation In Vivo
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