Noninvasive microvascular imaging in newborn rats using high-frequency ultrafast ultrasound

Ultrasound imaging stands as the predominant modality for neonatal health assessment, with recent advancements in ultrafast Doppler ( μ Doppler) technology offering significant promise in fields such as neonatal brain imaging. Combining μ Doppler with high-frequency ultrasound (HF-μ Doppler) presents a potential efficient avenue to enhance in vivo microvascular imaging in small animals, notably newborn rats, a crucial preclinical animal model for neonatal disease and development research. It is necessary to verify the imaging performance of HF-μ Doppler in preclinical trials. This study investigates the microvascular imaging capabilities of HF-μ Doppler using a 30 MHz high-frequency linear array probe in newborn rats. Results demonstrate the clarity of cerebral microvascular imaging in rats aged 1 to 7 postnatal days, extending to whole-body microvascular imaging, encompassing the central nervous system, including the brain and spinal cord. In conclusion, HF-μ Doppler technology emerges as a reliable imaging tool, offering a new perspective for preclinical investigations into neonatal diseases and development.


Introduction
Blood flow monitoring, particularly microvascular monitoring, plays a pivotal role in diagnosing neonatal diseases and investigating neonatal development.Researches have indicated that in cases of neonatal brain injury, early functional abnormalities are often reversible.However, as the injury progresses to structural damage, reversibility diminishes (Baranger et al., 2021).Based on the neurovascular coupling mechanism, cerebral blood flow serves as a reflection of brain function (Hendrikx et al., 2019;Iadecola, 2017).Thus, monitoring neonatal cerebral blood flow offers a valuable means to identify high-risk neonates prone to potential brain injuries before irreversible structural damage ensues.Early intervention strategies can then be promptly devised by clinicians, advancing diagnosis and treatment, thereby improving neurodevelopmental prognosis.Furthermore, studies on neonatal diseases reveal close associations between vascular abnormalities along with hemodynamic changes and common neonatal diseases, such as congenital heart disease, heart failure, pulmonary hemorrhage, and necrotizing enterocolitis (Dijkema et al., 2017;Levy et al., 2018;Wagh and Gill, 2013;Akin et al., 2015).
Most preclinical studies on neonatal disease diagnosis and development employ newborn rats as research subjects (Ahn et al., 2021;Lyu et al., 2021;Charriaut-Marlangue and Baud, 2018).The postnatal days 1 to 7 (P1-P7) in newborn rats correspond to the gestational age of 23 to 36 weeks in humans (Hagberg et al., 2002), constituting a critical period for human brain development (Gilmore et al., 2007;Sidman, 1982).Thus, the study of newborn rats can provide insights for understanding the development and conditions in neonates, creating a pressing need for reliable microvascular imaging methods with high resolution.
In recent years, ultrafast Doppler (μDoppler), an innovative advancement in CDFI technology, has significantly enhanced blood flow imaging quality (Deffieux et al., 2018; Deffieux et al., 2021; Macé et al.,   2011).μDoppler, utilizing multi-plane wave transmission and advanced spatiotemporal clutter filters, enhances blood flow measurement sensitivity by over 10 times without the need for contrast agents, better distinguishing between low blood flow and tissue motion (Deffieux et al., 2018).Compared with traditional CDFI, which is only sensitive to large blood vessels (diameter >200 μm), μDoppler can detect most microvessels (diameter <200 μm) (Liu et al., 2008).Some studies have applied μDoppler to neonatal cerebral blood flow and functional imaging (Baranger et al., 2021;Demené et al., 2014;Demene et al., 2017;Aguet et al., 2023), indicating its potential as a method for bedside cerebral blood flow imaging in neonates.This technology is anticipated to offer valuable insights into neonatal disease diagnosis and development.
μDoppler holds substantial potential for microvascular imaging in newborn rats during the critical P1-P7 period.Unlike adult rats, the underdeveloped skull and softer spine of newborn rats facilitate the penetration of ultrasound signals.Additionally, the absence of body hair in newborn rats promotes efficient coupling of ultrasound signals.These conditions mirror clinical characteristics observed in neonates, such as the incomplete closure of the anterior fontanel of the skull and minimal body hair.It is worth pointing out that ultrasound imaging necessitates a higher center frequency in neonatal microvascular flow studies with newborn rats due to the shallow imaging depth (Zhang et al., 2021;Tiran et al., 2017).Hence, newborn rats serve as an ideal preclinical animal model of the neonatal condition, and it is useful to know if high-frequency μDoppler (HF-μDoppler) can be used in this model.
Therefore, this study employs a 30 MHz linear array high-frequency probe in conjunction with plane wave μDoppler ultrasound imaging technology with an expected high resolution (~50 μm) (Bhatti et al., 2023;Almairac et al., 2018;Bhatti et al., 2022), aiming to explore the effectiveness of HF-μDoppler in microvascular imaging in newborn rats and verifying its applicability in typical scenarios, including the brain, central nervous system, and injury models.

Ultrasound system
We utilized the Verasonics Vantage 256 high-frequency system (Verasonics Inc, Kirkland, WA) and the L35-16 high-frequency linear array probe (Vermon SA, Tours, France) with a transmission frequency of 30 MHz to conduct HF-μDoppler scanning on newborn rats from the coronal position.For a whole-body scan, a custom-made probe holder was employed to stabilize the high-frequency probe in the coronal position of the newborn rat, performing horizontal movement in the elevation direction from the head to the tail of the rat.The horizontal movement of the probe was precisely controlled by a stepper motor with a step length of 200 μm.The experimental process is shown in Fig. 1.

Animal preparation
In this investigation, Sprague-Dawley newborn rats were utilized as the experimental subjects.To observe the microvascular and structural changes of newborn rats during their development, we conducted observations on four groups of rats at different ages: P1 (5-10 g), P3 (8-14 g), P5 (12-17 g), and P7 (16-25 g), including four males in each group.During the ultrasound scan, each newborn rat was initially anesthetized with isoflurane (3.0 % for induction, 1.0 % for maintenance).Subsequently, the rat was placed in a prone position on the operating table with physiological monitoring and heating function in the Vevo F2 system (VisualSonics Inc, Toronto, Canada) to monitor the electrocardiogram, heart rate, respiration, and core temperature (using a rectal probe), maintaining the temperature at 37 ± 0.5 • C, and fixed using a brain stereotaxic instrument.Meanwhile, the peripheral blood oxygen saturation of newborn rats was monitored using the small animal vital signs monitor (MouseOx, Starr Life Sciences Corp., USA) combined with a foot probe to ensure that the blood oxygen saturation maintained over 95 % when collecting data.The coronal position scanning captured the HF-μDoppler microvascular images of the whole body, encompassing the brain, spinal cord, liver, and kidney.All experiments have followed the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978), and were ethically approved by the Institutional Animal Care and Use Committee of Peking University, Beijing, China.

Data acquisition
The horizontal movement of the probe was precisely controlled by a stepper motor with a step length of 200 μm.In this case, when scanning the whole brain (40 slices), the length in the elevation direction is 8 mm, and the elevation width is 0.7 mm, which makes the field of view in the elevation direction close to the width of the L35-16 probe, making it easier to visualize.
We utilized a multi-angle coherent compounding ultrafast plane wave imaging strategy (Tanter and Fink, 2014).The imaging sequence adopted 7-angle (− 15 • to 15 • , 5 • increment) plane wave emission, with the pulse repetition frequency (PRF) set at 25 kHz.The imaging frame rate was maintained at 500 compounding frames per second, with an imaging depth of approximately 12 mm (about 210 wavelengths).Each slice position took about 5 s, of which 1 s was allocated for raw radio frequency (RF) data collection, and the remaining time for data storage and slice switching.A total of 240 slices (120,000 compounding frames) were collected for the whole-body scan, generating approximately 0.8 TB of corresponding raw RF data.

Data processing
Matlab 2021b (The MathWorks, Natick, MA, USA) was used to reconstruct the collected RF data frames and demodulate them through delay and sum (DAS) beam synthesis to obtain the amplitude of the inphase/quadrature (IQ) data.Following the acquisition of the B-mode image, the widely-used spatiotemporal clutter filter (Zhang et al., 2021) based on singular value decomposition (SVD) was applied to eliminate tissue signals and extract blood flow signals.Specifically, for SVD, the first 5 % of the eigenvalues in the SVD-decomposed eigenvalue matrix were discarded for every 500-frame stack.Subsequently, the remaining eigenvalues and decomposition matrix were reconstructed into a new image to derive the blood flow image.The reconstructed HF-μDoppler images from the same slice were overlaid through maximum intensity projection (MIP) using ImageJ (NIH, Bethesda, MD, USA), creating a single-slice blood flow image with a high SNR.To further improve the quality of blood flow images, each single-slice image was processed through 'Subtract Background', 'Square Root', and 'auto Adjust Brightness/Contrast' using ImageJ.After applying the above procedures to all slices, 2.5D (2D + translation)1 microvascular imaging was obtained using the 5D visualization plug-in ClearVolume (Royer et al., 2015).
To quantify the vascular morphological features, vessel regions were extracted from the reconstructed blood flow images using vessel segmentation.Here topology-aware segmentation (TAS) was used to offer higher topological fidelity in the presence of non-uniform signal and background intensities and blurred vessel boundaries (Chen et al., 2023).The essential idea of TAS is to adaptively binarize the images to ensure that the local widths of the extracted vessel regions are consistent with the full widths at half maximum (FWHMs) of the intensity change.Concretely, for each pixel, TAS first estimates the vessel orientation using weighted principal component analysis.Along the normal direction to the estimated orientation, TAS then searches within a radius (10 pixels, in our experiments) to identify the maximum and minimum intensity; the local threshold is thus set at half the sum of the maximum and the minimum.Next, to estimate vessel sizes, the extracted vessel regions, represented by binary masks, are further skeletonized.The vessel size at a skeleton pixel is calculated as the distances between boundaries at both sides along the normal direction.

Whole-body scanning of HF-μDoppler
As illustrated in Fig. 2, HF-μDoppler provides clear microvascular imaging results in newborn rats with a high resolution noninvasively.By calculating the Fourier Ring Correlation (FRC) (Hingot et al., 2021), the spatial resolution of the HF-μDoppler image is 62.09 μm (Sup Fig. 1(a)).
Fig. 2(a-c) displays the 2.5D whole-body microvascular imaging results obtained by ultrasound scanning from the back of newborn rats from various angles, and Fig. 2(d-g) shows the local blood vessels in several specific organs and structures including the brain, neck, liver, and kidneys.Some common physiological structures of blood vessels, such as the superior sagittal sinus (SSS), intercostal artery (IA), thoracic aorta (TA), carotid artery, inferior vena cava and abdominal aorta can be clearly displayed.Moreover, due to 2.5D scanning, how blood vessels are connected and extended can also be well-reflected.
Additionally, we also conducted HF-μDoppler scanning on newborn rats along the central nervous system in the sagittal position, providing the complete vasculature of the whole central nervous system, including the brain and spinal cord in the midsagittal plane, using HF-μDoppler for the first time (Fig. 3).It is evident that, some microvessels, such as the SSS with a diameter of ~250 μm, the posterior spinal artery (PSA) with a diameter of ~220 μm, and the anterior spinal artery (ASA) with a diameter of ~120 μm, are clearly detected.However, due to the attenuation of ultrasound signals caused by the vertebrae, the TA appears discontinuous.The wavefront of the ultrasound signal propagating downward through the vertebral space has changed due to the obstruction of the vertebrae and other reasons, and is no longer suitable for the original plane wave imaging sequence, thus causing certain artifacts below the vertebrae.It is notable that, even for newborn rats on P1, high-frequency ultrasound signals encounter difficulties in penetrating the region at the back of the brain (in the orange box).

Development monitoring and evaluation
We assessed the performance of HF-μDoppler in monitoring and evaluating the development of newborn rats.We monitored the whole cerebral vascular development of newborn rats within P7 (Fig. 4(a-d)) and estimated the distribution of whole brain vessel size using the TAS method (Fig. 4(e-h)) (Chen et al., 2023).Since we pay more attention to microvessels, when drawing the distribution map, only blood vessels Fig. 1.Experiment system and data processing steps.Newborn rats are anesthetized using isoflurane and fixed in a prone position using a brain stereotaxic instrument on an operating table with heating function.HF-μDoppler scanning is performed on newborn rats from the coronal position using the Verasonics Vantage 256 high-frequency system and the L35-16 high-frequency linear array probe with a transmission frequency of 30 MHz.The collected radio frequency (RF) data is processed by delay and sum (DAS) and singular value decomposition (SVD) to obtain HF-μDoppler microvascular images, and a 2.5D (2D + translation) whole-body microvascular image of newborn rats is reconstructed.with a diameter <250 μm were distinguished by color, while others were represented by the same color.At the same time, to ensure that the comparison between different postnatal days is not affected by the proportion of the brain in the imaging field of view, we only performed a comparative analysis on the blood vessels in the clear display area of the brain center, which is the elliptical area enclosed by posterior cerebral arteries (PCAs), that is, the yellow region of interest (ROI) in Fig. 4(e-h).Through the histograms of the vessel size distribution (Fig. 4(i-l)), we can find that HF-μDoppler can detect most blood vessels, including microvessels with a diameter <200 μm, and the distribution of blood vessel size approximately conforms to a power law.By comparing the distribution histograms of blood vessels on different postnatal days, we found that as postnatal day increases, the proportion of microvessels continues to increase (Fig. 4(m)).The statistical analysis (4 rats per day) of the mean width of whole brain blood vessels and the proportion of microvessels with a diameter <200 μm also confirms that (Fig. 4(n, o)).
We suspect that this is related to the continuous emergence of new blood vessels during the development process.
We also selected several representative slices to evaluate the effect of HF-μDoppler on development monitoring and evaluation from different dimensions.To help better understand the location of the selected slices and the structures displayed, we compared them with the Allen brain atlas, containing structural information (Wang et al., 2020) and the cerebrovascular atlas obtained by optical methods, containing vascular information (Todorov et al., 2020;Scremin, 2015) respectively (Sup Fig. 2).As shown in Fig. 5, the mean diameter of cortical microvessels (CMs) in the yellow ROI and lenticulostriate arteries (LAs) in the pink ROI, the width of the third ventricle, and the distance between the PCAs on different postnatal days (4 rats per day) were also measured.Subsequently, we made use of these measurements to evaluate and analyze the brain development of newborn rats.Through statistical analysis, we can find that with the increase of postnatal day (P1, P3, P5, P7, respectively), the mean microvessel diameter (108.96± 2.26, 111.22 ± 2.03, 108.08 ± 3.91, 102.35 ± 5.93 μm for CMs, and 108.45 ± 4.73, 111.48 ± 2.60, 104.89 ± 4.76, 104.52 ± 4.16 μm for LAs, respectively) reveals a decreasing trend, which is consistent with the histograms of vessel size distribution.Meanwhile, brain structure size (144.97 ± 17.65, 183.41 ± 20.64, 228.83 ± 19.76, 330.17 ± 21.29 μm, respectively), and blood vessel position distance (3.63 ± 0.11, 3.83 ± 0.08, 4.02 ± 0.10, 4.54 ± 0.09 mm, respectively), reveal an increasing trend with significant differences.This indicates that the imaging strategy can effectively capture the early brain development process of experimental rats.

Injury detection and evaluation
Traumatic brain injury (TBI) is a common clinical disorder in neonates, associated with higher mortality rates and neurodevelopmental defects (Hanlon et al., 2016;Tan et al., 2018).TBI not only causes brain damage such as blood-brain barrier disruption, brain edema, and gliomas, but also leads to neonatal seizures and epilepsy, resulting in functional impairment (Gu et al., 2015).In addition, in the absence of obvious external bleeding, the small amount of blood loss caused by TBI can also lead to neonatal hemorrhagic shock, which cannot be ignored (Araki et al., 2017).Therefore, we designed a corresponding experiment involving puncture-induced bleeding as the TBI model (Kim et al., 2015;Ma et al., 2019) to investigate the feasibility and capability of HF-μDoppler in detecting and assessing brain injury.
Isoflurane (3.0 % induction, 1.0 % maintenance) was administered to anesthetize newborn rats on P3, which were secured in a prone position on an operating table using a brain stereotaxic instrument.A 26G needle was fixed on the brain stereotaxic instrument with the end of the needle located on the right side of the rat's posterior fontanelle.The brain stereotaxic instrument was adjusted to penetrate the brain of the newborn rat slowly with a puncture depth of 3 mm, and then the needle was lifted gradually.
Experimental results indicated the cerebral vasculature of newborn rats was normal before injury (Fig. 6(e)).Upon injury, the signal at the injured site intensified, distinctly differentiating it from the normal site.Notably, the injured site exhibited active acute bleeding compared to the normal site (Fig. 6(f)).For both 1 and 2 h post-injury, the signal at the injured site disappeared, showing clearer distinction from the normal site.At this point, the injured site appeared different from the immediate post-injury state, with observable cessation of active bleeding (Fig. 6(g,  h)).The newborn rats used in the experiment continued to survive for a long time, but their movements slowed, indicating substantial damage to the rats' brain function caused by the puncture.
We also compared the detection ability of plane-wave B mode imaging with HF-μDoppler imaging for brain injury in newborn rats.The ratio of the mean brightness of the injury site to the mean brightness of the surrounding circular site is used to characterize the detection ability of injury.For plane-wave B mode imaging (Fig. 6(a-d)), there is almost no difference in the brightness ratio in the different stages of injury occurrence (Fig. 6(l)), indicating its shortcomings in detecting injury.For HF-μDoppler imaging (Fig. 6(e-h)), there is a significant difference in the brightness ratio in the different stages of injury occurrence (Fig. 6 (m)).Compared with plane-wave B mode imaging, the ability of HF-μDoppler to detect injury is significantly improved at different stages of injury occurrence (Fig. 6(i-k)).

Comparison of using contrast agents
Although HF-μDoppler can achieve reasonable imaging quality without undergoing any invasive surgery, the skull and other bones can cause certain attenuation of ultrasound propagation, making it difficult to distinguish small variations in blood flow and noise (Pinton et al., 2012;Errico et al., 2016).One way to solve this problem is to inject ultrasound contrast agents.Performing HF-μDoppler imaging with contrast agent injection can significantly improve the sensitivity and even completely compensate for signal attenuation caused by the skull (Zhang et al., 2021;Errico et al., 2016).Meanwhile, ultrasound contrast agents are also commonly used in clinical applications such as the kinetics of microbubble flow in cerebral vasculature of neonates, providing important insights for clinical diagnosis and treatment (Knieling et al., 2022;Knieling et al., 2020;Rüffer et al., 2022).Therefore, we investigated and compared the impact of ultrasound contrast agents on microvascular imaging in newborn rats.
We compared and studied the microvascular imaging quality of the same newborn rats on P3 with and without ultrasound contrast agent injection.We used isoflurane (3.0 % induction, 1.0 % maintenance) to anesthetize newborn rats and injected 0.05 ml of long-lasting microbubbles (Zhang et al., 2021) solution with a concentration of 1 × 10 8 /ml into their bodies through the caudal vein with the aid of an intravenous visible mouse tail injection fixator.Ultrasound imaging was performed immediately after injection, and the imaging settings were the same with or without contrast agents.Finally, we used FRC and SNR to compare and evaluate the imaging quality with and without contrast agents.When calculating SNR, we used the background area without blood flow above the scalp (yellow box in Fig. 7(a, b, e, f)) as noise, selected representative ROIs including large blood vessels, small blood     Fig. 6.Single-slice HF-μDoppler microvascular images and form comparison at different stages of brain injury in newborn rats on P3.Pre-injury, immediate postinjury, 1 and 2 h after injury in the case of (a-d) B mode imaging, and (e-h) HF-μDoppler imaging.The arrows indicate the injury site.Statistical differences between B mode imaging and HF-μDoppler imaging conditions in the brightness ratio of the injury site to its surrounding site in the case of (i) immediate post-injury, (j) 1 and (k) 2 h after injury.Statistical differences among pre-injury, immediate post-injury, 1 and 2 h after injury conditions in the brightness ratio of the injury site to its surrounding site in the case of (l) B mode imaging and (m) HF-μDoppler imaging.vessels, and background in the near field (blue box in Fig. 7(a, b, e, f)) and far field (green box in Fig. 7(a, b, e, f)) respectively, and used the difference between these ROIs and the background as the signal to calculate SNR (Sui et al., 2022).
We selected two representative single-slice images (Fig. 7(a, b, e, f)) and the 2.5D whole-brain images obtained by MIP (Fig. 7(i, j)) to compare the imaging quality.Compared with images without contrast, the FRC of images with contrast is significantly reduced (Fig. 7(k, l)), which means that the spatial resolution is improved.The SNR of the contrast images also improved in the 4 ROIs (Fig. 7(m-p)).As shown in Fig. 7, the cerebral blood flow signal of newborn rats was improved after the injection.It is particularly noteworthy that the reconstructed cerebral blood flow structure from the HF-μDoppler data collected within 1 s, without contrast agent injection (Fig. 7(a, e, i)), was highly consistent with the HF-μDoppler contrast enhanced ultrasound results obtained through long-term acquisition and accumulation (Fig. 7(b, f, j)).This suggests that HF-μDoppler remains a valuable method of cerebral blood flow imaging even in the absence of contrast agents, while meeting the clinical safety standards for neonatal imaging.
In addition, we perform the color map and super-resolution ultrasound (SRUS) of representative slices.Color map (Fig. 7(c, g)) used the same data as Fig. 7(a, e), and added blood flow direction and velocity information to the original image by autocorrelation phase estimation (Kasai et al., 1985), where red represents upward flow (toward the probe), blue represents downward flow (away from the probe) and the different shades of color represent the blood flow velocity.SRUS (Fig. 7  (d, h)) used the standard Ultrasound Localization Microscopy (ULM) framework (Heiles et al., 2022), collected 20 s contrast data at the corresponding slice and reconstructed it.To improve the accuracy of detection, the super-resolution image interpolation factor of brain imaging was set to 4, the maximum link distance of microbubbles between two frames was set to two original pixel sizes, and microbubble trajectories with a continuous length of less than 10 frames were discarded.
Compared to HF-μDoppler (62.09 μm), The FRC of SRUS is 24.09 μm (Sup Fig. 1(b)), while the half-wavelength of the high-frequency ultrasound we used is 27.5 μm, which shows that the SRUS we used has broken through the diffraction limit and achieved super-resolution effects.In future clinical and preclinical applications, these two imaging modes will provide additional structural and functional information on blood flow and become a good complement to the HF-μDoppler imaging mode.

Discussion
In this study, we utilized HF-μDoppler technology to conduct noninvasive whole-body microvascular imaging, aiming to explore the effectiveness of HF-μDoppler imaging in observing newborn rat brain development and detecting injuries.The results of whole-body microvascular imaging of newborn rats reveal that HF-μDoppler imaging adeptly distinguishes typical organs and structures, such as the brain, kidneys, and spinal cord.It also vividly displays their microvascular structures, especially the microscale blood vessels that were previously challenging to other imaging methods.The findings suggest that HF-μDoppler microvascular imaging holds promise as a direct imaging method in future preclinical research.It can contribute to exploring the development of most neonatal organs and structures and diagnosing relevant diseases, such as intraventricular hemorrhage (a leading cause of cognitive impairment and neurodevelopmental delay in premature infants Gilard et al., 2020), pulmonary hemorrhage (considered life-threatening for extremely premature infants Wagh and Gill, 2013), and necrotizing enterocolitis (the most common gastrointestinal emergency in neonates Akin et al., 2015).
It is worth mentioning that our study pioneered HF-μDoppler microvascular imaging of the whole central nervous system in newborn rats.The central nervous system, the core of the human nervous system, undergoes the fastest and the most sensitive development during the neonatal period (Gilmore et al., 2007).This period is crucial.Changes in the internal and external environment, along with other high-risk factors, can consequently impact subsequent learning ability, social cognition, as well as motor and sensory functions.Therefore, evaluating the development of the central nervous system of newborn rats through HF-μDoppler microvascular imaging provides essential insights for preclinical research on developmental abnormalities of the neonatal central nervous system and clinical prognosis assessment.
Meanwhile, the imaging strategy of HF-μDoppler easily enables continuously monitoring of the cerebral blood vessel diameter, brain structure, and the positional relationships between different blood vessels in newborn rats.This includes tracking their changes across postnatal days to reflect the process and trends of development.Through this strategy, our study identified changes of development among newborn rats of various postnatal days.It implies that HF-μDoppler microvascular imaging can sensitively monitor the development status of newborn rats and promptly detect differences in various development states.
Moreover, HF-μDoppler proves effective not only in detecting brain injury, but also in distinguishing different stages of brain injury in newborn rats.Newborn rats aged P1-P7 correspond to humans of gestational age 23 to 36 weeks (Hagberg et al., 2002).Considering that most current preclinical research on neonatal development and diseases employs newborn rats as research subjects (Ahn et al., 2021;Lyu et al., 2021;Charriaut-Marlangue and Baud, 2018), HF-μDoppler microvascular imaging stands to provide valuable quantitative insights for understanding the development and disease diagnosis of premature and full-term infants across various gestational ages.
In addition, the introduction of color map and SRUS further improves the image quality of HF-μDoppler, while supplementing more hemodynamic information.Considering that the occurrence of some diseases is closely related to hemodynamic changes (Nixon et al., 2010), the direction and magnitude of blood flow velocity contained in the color map combining HF-μDoppler can help diagnose and prognosis diseases more sensitively.In response to the fact that the development of most vascular diseases starts from the end region of blood vessels (Faure et al., 2024), SRUS can monitor smaller vessels with higher resolution, which may help to obtain information with better prognostic value.
Despite the general belief in a trade-off between sound wave penetration depth and frequency (Tiran et al., 2017), our choice of a relatively high-frequency probe (30 MHz) proves beneficial.As red blood cells within the blood flow are easily penetrated by low-frequency sound waves (Zhang et al., 2021), the resulting reflected signal tends to be notably weak.Consequently, the extraction of this blood flow signal from the low-frequency backscattered signal becomes difficult, thereby causing a diminished sensitivity in microvascular imaging.By the strategic enhancement of the frequency, the sound wave signal reflected by red blood cells are strengthened.This amplification, in turn, augments the blood flow signal in some areas (Moran and Thomson, 2020), i.e., high-frequency signals are more sensitive to blood flow.
In our research, owing to the diminutive size of newborn rats, the 30 MHz ultrasound exhibits sufficient penetration depth, enabling a comprehensive whole-body microvascular imaging.Considering the imperative to minimize invasive procedures in neonatal clinical settings and the softness of the skulls and spines of newborn rats within P7, we abstained from interventions such as craniotomy and vertebrae removal.
It is noteworthy that throughout the experiment, ultrasound signals faced difficulties to penetrate a specific region at the posterior of the brain in all newborn rats, including those on P1, rendering HF-μDoppler imaging impracticable.This difficulty suggests a potential hardening of the skull in this particular region, which was confirmed by dissection, revealing increased hardness compared to other skull regions.The region is anatomically associated with the brainstem, responsible for vital functions such as breathing and voluntary heart rate control.We posit that this structural resilience serves an important role in safeguarding the brainstem from damage and maintaining overall life.
This study is not exempt from some limitations.Despite effectively Y. Zhao et al. monitoring and evaluating newborn rat development, this study faces limitations in achieving continuous monitoring across different postnatal days due to constraints in animal housing conditions.Future efforts should focus on enhanced follow-up evaluations for individual subjects, particularly in investing the development and prognosis of certain disease models.In addition, in terms of injury detection in newborn rats, our exploration has focused solely on the feasibility of utilizing HF-μDoppler for detecting and assessing brain injuries.Subsequent advancements should expand this research to encompass diverse neonatal disease models such as intraventricular hemorrhage and necrotizing enterocolitis.

Conclusions
This study unprecedentedly achieves noninvasive whole-body HF-μDoppler microvascular imaging in newborn rats and demonstrates its excellent microvessel detection capabilities.The research outcomes highlight the versatility of HF-μDoppler, demonstrating its ability to assess the neonatal development and brain injuries of newborn rats.This positions it as a distinctive and promising microvascular imaging method, which is particularly suitable for real-time and continuous bedside monitoring.The technology holds the potential to serve as a valuable preclinical research tool for the diagnosis of neonatal diseases and the assessment of neonatal development.

Fig. 3 .
Fig. 3. HF-μDoppler microvascular images of the whole central nervous system of newborn rat on P1.(a) HF-μDoppler scan of the central nervous system of a newborn rat from the sagittal position.(b-i) Different single-slices of the central nervous system from head to tail, with the brain mainly in (b) and the spinal cord mainly in (c-i).(j) HF-μDoppler microvascular imaging of the whole central nervous system after registering the 8 single-slices (b-i), highlighting the SSS (diameter ~250 μm) used the yellow arrow, the PSA used the green arrow (diameter ~220 μm), the ASA used the purple arrow (diameter ~120 μm), and the TA appearing discontinuous used the blue arrow.The region where ultrasound signals struggle to penetrate is indicated with the orange box.

Fig. 4 .
Fig. 4. Distribution of whole brain blood vessel size in newborn rats in HF-μDoppler microvascular images and a comparative quantitative assessment on different postnatal days (n = 4).(a-d) Representative 2.5D whole-brain HF-μDoppler microvascular images, (e-h) illustration of vessel size, and (i-l) histogram of vessel size distribution of newborn rats on P1, P3, P5, and P7.(m) The changes in the histogram of vessel size distribution.Statistical differences in (n) the mean width of whole brain blood vessels, and (o) the proportion of microvessels <200 μm on different postnatal days.All the above quantitative statistics were performed in the yellow ROIs in (e-h).
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Fig. 5 .
Fig. 5.Comparative developmental quantitative assessment on different postnatal days (n = 4).Changes of (a-h) the size of CMs (highlighted in yellow boxes) and LAs (highlighted in pink boxes), (i-l) the width of the third ventricle, and (m-p) the distance between the PCAs of newborn rats on P1, P3, P5, and P7 in sequence in single-slices.Statistical differences in (q) the diameter of the CMs, (r) the diameter of the LAs, (s) the width of the third ventricle, and (t) the distances between PCAs on different postnatal days.
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Fig. 7 .
Fig. 7. HF-μDoppler cerebral blood flow images of newborn rats on P3 with and without ultrasound contrast agents.(a) Single-slice HF-μDoppler blood flow image without contrast agents, and (b) with contrast agents, (c) color map image characterizing blood flow direction and velocity, and (d) SRUS image of representative slice 1. (e) Single-slice HF-μDoppler blood flow image without contrast agents, and (f) with contrast agents, (g) color map image, and (h) SRUS image of representative slice 2. HF-μDoppler blood flow MIP image (i) without contrast agents, and (j) with contrast agents.Statistical differences in the FRC of (k) slice 1 and (l) slice 2 between with and without contrast agents.Statistical differences in the SNR of (m) ROI 1(blue box) and (n) ROI 2(green box) in slice 1, and (o) ROI 1(blue box) and (p) ROI 2(green box) in slice 2 between with and without contrast agents.