Non-invasive Parenchymal, Vascular and Metabolic High-frequency Ultrasound and Photoacoustic Rat Deep Brain Imaging
March 2nd, 2015
The present work describes a new protocol to perform non-invasive high-frequency ultrasound and photoacoustic based imaging on rat brain, to efficiently visualize deep subcortical regions and their vascular patterns by directing signals on skull foramina naturally present on animal cranium.
The overall goal of this procedure is to perform non-invasive high frequency ultrasound and photoacoustic based imaging on rat brain to visualize deep subcortical regions and their vascular patterns. This is accomplished by first preparing the animal for imaging through hair removal and proper positioning of the animal. On an ultrasound and photoacoustic imaging station, worktop, the transducer is placed, aligning it to the virtual axis, connecting the ear to the eye to obtain an optimal beam.
Focalization, the animal is then positioned for image acquisition from the temporal view. Following anatomic image acquisition, vascular images are acquired to visualize internal brain blood vessels as well as determine bloodstream, velocities, and directions. Blood total hemoglobin content and oxygenation degree can also be determined.
The animal is then positioned for image acquisition from the occipital view, followed by ultrasonic and photoacoustic brain anatomic and vascular image acquisition. Ultimately, the photoacoustic method is used to show anatomical brain images and physiological vascular parameters After its development. This technique paved the wave for researcher in neuro imaging to explore physiological, vascular, and anatomical structures.
This method can help answer key question in the brain diseases such as stroke and neurological degeneration, since it can give specific information on the neurological functions. To begin, place the rat inside the appropriate isof fluorine chamber to anesthetize it as described in the text protocol. Once the anesthesia takes effect, remove the rat and weigh it.
Spread a thin layer of water soluble ophthalmic gel on the animal's eyes to protect them and to maintain ocular physiological hydration. Then lay the rat down on an ultrasound and photoacoustic imaging station worktop, and quickly position the nose inside the appropriate mask, providing a constant anesthesia flow to shave the animal. Spread a consistent layer of hair, removing cream on the head surface, covering areas surrounding the ears and neck.
After allowing the cream to act for several minutes, gently take it out with a spatula. Softly remove all cream remnants with a wet sponge to accurately clean the skin. To position the rat, arrange the animal in a spread eagle position.
Lean the pause on the vital parameter sensors on the worktop after applying some drops of electrode cream to monitor the vital signs. Finally fasten the limbs with a hypoallergenic artificial silk patch. Then dispose a consistent layer of hypoallergenic water-soluble ultrasound transmission gel on the animal's head.
Cover the transducer head with a thin layer of the same gel, and put it into contact with the layer on the rat. Raise the animal head and rotate it slightly on one side. Use a cotton roll as a stand keeping the snout well inserted into the anesthesia mask.
Incline the worktop at an angle of about 30 degrees with respect to the horizontal plane. Turn the imaging transducer at an angle of about 30 degrees with respect to the vertical plane. For ultrasonic and photoacoustic anatomic and vascular image acquisition, turn the imaging scan on.
Enter the B mode image acquisition, and properly set all image acquisition parameters to respect possible given requirements of the experiment. Set the transmit center frequency as low as possible in order to have the maximum penetration depth for the transducer. Start image acquisition in B mode and adjust the transducer positioning in real time by identifying anatomical references and by centering the region of interest to the monitor Middle point.
Place the transducer to align it to the virtual axis, connecting the ear to the eye to obtain an optimal beam.Focalization. Acquire different views of the internal brain volume by clockwise or counterclockwise rotation. Ensure that the cerebral region of interest localizes at 10 millimeters of depth with respect to the ultrasound laser transducer source in order to receive an optimal photoacoustic response signal.
Next, enter color Doppler mode to visualize internal brain blood vessels in a highly sensitive way. Then choose the desired acquisition parameter set in color. Doppler mode.
Acquire images in this modality to distinguish bloodstream, velocities and directions until several millimeters of penetration depth. Enter pulsed wave doppler mode and acquire images to detect artery blood pulsation, and to differentiate between arteries and veins. Now, enter power doppler mode and set acquisition parameters to perform a signal quantification on the basis of the number of scattering events caused by the flux movement to evaluate differences in flow rates.
Next, enter Photoacoustic mode and properly refine acquisition parameters to collect data about blood total hemoglobin content or oxygenation degree in a given area By producing laser excitation on an entire wavelength spectrum, the absorption of total hemoglobin present in different chemical states inside a tissue can be quantified to image from the occipital point of view. Keep the animal in a prone position, lower the animal head and use small cotton gauze rolls as lateral stands to correctly arrange the animal's position. Turn the imaging transducer parallel to the transverse plane of the animal head for ultrasonic and photoacoustic anatomic and vascular image acquisition.
Enter the B mode image acquisition and set all image acquisition parameters. As before, spread the necessary ultrasound gel layers on the probe and on the animal nappe. Visualize internal brain blood vessels in power doppler mode by properly setting acquisition parameters.
Localize intensely pulsated arteries by pulsed wave Doppler mode. Collect bloodstream velocities data and directions in color doppler mode by adequately adapting acquisition parameters. After saving all acquired data, turn the laser pulsing off by exiting the photoacoustic acquisition mode and distance the transducer while maintaining the animal under the anesthesia effect.
Start to clean it by gently removing the protective gel from the eyes with the wet cotton swab, use a spatula and several paper towels to completely remove the ultrasound gel from the head and the snout. Then clean them with a wet sponge. Be careful not to damage the delicate shaved skin.
Take out the adhesive patch used to fasten the limbs and disconnect the limbs from the sensors that monitor the physiologic parameters. Rapidly transfer the animal from the acquisition worktop to a different cage aid the animal in recovery as described in the text protocol. This method permits deep imaging of both specific anatomic reference structures and blood vessels at relatively high spatial resolution.
Shown here are resolved images of the middle cerebral artery or MCA that arises from the internal carotid artery or ICA, and further divides into two or more branches that finally surround cortical lobes. Doppler based acoustic imaging reveals small branches while directional information of blood current is available with color Doppler acquisition. MCA artery feature is confirmed by the pulsed wave ultrasonic technique.
Shown here is pulsed wave mode acquisition through the temporal foramen for the individuation of vascular references. Photoacoustic signal of contained hemoglobin into circulating red blood cells can be detected and analyzed to collect data about its molecular oxidative status and to calculate blood oxygen saturation. Hematic oxygen content can be correlated to sonic data in order to confirm the discrimination of arterial blood from venous blood.
The cerebral parenchymal tissue was also recorded with a photoacoustic modality in the occipital projection to show vascular characterization in the spectral plot. With this spectrum, it is possible to distinguish the signal derived from arterial and venous vessels. The main advantage of this technique over existing method is that the brain anatomy and relating vascular behavior can be studied with photo imaging mode without mood skull scar.
After watching this video, you should have a good understanding how to record parenchymal and vascular brain properties and how to record in real time oxygen. The hemoglobin level changes in brain tissue.
Overview
This study presents a protocol for non-invasive high-frequency ultrasound and photoacoustic imaging of the rat brain, enabling visualization of deep subcortical regions and their vascular patterns. The technique utilizes natural skull foramina to direct imaging signals effectively.
Key Study Components
Area of Science
- Neuroscience
- Imaging Techniques
- Vascular Imaging
Background
- Non-invasive imaging methods are crucial for studying brain structures.
- High-frequency ultrasound and photoacoustic imaging provide detailed anatomical and physiological information.
- Understanding vascular patterns is essential for addressing neurological diseases.
- This protocol aims to enhance imaging capabilities in preclinical research.
Purpose of Study
- To develop a method for visualizing deep brain structures in rats.
- To assess vascular patterns and blood flow dynamics in the brain.
- To provide insights into neurological functions and diseases.
Methods Used
- Preparation of the rat includes anesthesia and hair removal.
- Ultrasound and photoacoustic imaging are performed using a specialized imaging station.
- Multiple imaging modes are utilized to capture anatomical and vascular data.
- Parameters are adjusted for optimal image acquisition and analysis.
Main Results
- Successful visualization of internal brain structures and blood vessels.
- Quantification of blood total hemoglobin content and oxygenation levels.
- Detailed imaging of blood flow velocities and directions.
- Demonstration of the technique's potential for studying brain diseases.
Conclusions
- The developed protocol enhances the ability to study brain anatomy and physiology.
- This imaging technique can contribute to understanding neurological disorders.
- Future applications may include research on stroke and neurodegeneration.
Frequently Asked Questions
Executive Industry Relevance
Non-invasive high-frequency ultrasound and photoacoustic imaging of the rat brain enables deep visualization of parenchymal and vascular structures without the need for skull removal, addressing a critical bottleneck in preclinical neurovascular research. This protocol enhances predictive confidence in disease modeling and mechanistic de-risking for CNS-targeted drug discovery. The approach supports translational continuity by providing quantitative, real-time data on brain hemodynamics and oxygenation in intact animal models.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables interrogation of neurovascular pathways and functional target validation in vivo.
- Supports mechanistic de-risking by providing real-time hemodynamic and oxygenation data.
- Facilitates portfolio triage by distinguishing disease-relevant vascular phenotypes non-invasively.
Screening & Assay Development
- Prepares validated animal models for downstream compound evaluation in neurovascular contexts.
- Delivers standardized, reproducible imaging outputs for quantitative assessment of vascular and metabolic endpoints.
- Enables scalable, high-resolution imaging workflows for screening contrast agents or vascular modulators.
Translational & Preclinical Research
- Aligns with disease-relevant models for stroke and neurodegeneration by capturing deep brain vascular features.
- Provides continuity from discovery through preclinical validation with non-invasive, longitudinal imaging capability.
- Supports risk-adjusted advancement decisions by quantifying blood flow, vessel integrity, and oxygenation states.
Pipeline & Workflow Integration
This imaging protocol integrates into the discovery-to-preclinical continuum, enabling hypothesis testing, target validation, and translational biomarker development in CNS research.
- Discovery Biology: Supports hypothesis-driven interrogation of neurovascular mechanisms and pathway mapping.
- Screening: Provides reproducible, quantitative imaging endpoints for compound or biomarker evaluation.
- Analytics: Delivers real-time measurements of blood flow, vessel architecture, and hemoglobin oxygenation for comparative analysis.
- Translational Research: Bridges preclinical and disease-relevant models by enabling non-invasive, longitudinal studies.
- Enterprise Reuse: Establishes a reusable imaging platform for diverse CNS and vascular research programs.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in neurovascular target validation.
- Operational Value: Standardizes non-invasive imaging workflows and enhances reproducibility across studies.
- Strategic Value: Improves go/no-go decision-making and capital efficiency by enabling early, quantitative assessment of CNS drug candidates.
- Portfolio Impact: Supports risk-adjusted prioritization and advancement of neurovascular assets.
Implementation Considerations
- Requires expertise in high-frequency ultrasound and photoacoustic imaging modalities.
- Demands access to specialized imaging stations and compatible transducers.
- Necessitates cross-team standardization of animal preparation and imaging parameters.
- May require adaptation for different animal models or cranial anatomies.
- Imaging depth and resolution are influenced by skull thickness and anatomical variability.
Biopharma FAQs
Chapters in this video
0:05
Title
2:08
Preparation of Animal
7:13
Imaging from the Occipital Point of View
8:23
End of Acquisition and Animal Removal
9:27
Non-invasive Parenchymal, Ultrasound Vascular, and Metabolic Images by Ultrasound and Photo-acoustic Tools
11:07
Conclusion
3:46
Image Acquisition from Temporal Point of View
