The authors have declared that no competing interests exist.
Conceived and designed the experiments: EG FL GAL. Performed the experiments: EG RB IL NX GAL. Analyzed the data: EG RB IL NX GAL. Contributed reagents/materials/analysis tools: EG RB GAL PB. Wrote the paper: EG IL FL GAL.
The precise assessment of cerebral saturation changes during an inflammatory injury in the developing brain, such as seen in periventricular leukomalacia, is not well defined. This study investigated the impact of inflammation on locoregional cerebral oxygen saturation in a newborn rodent model using photoacoustic imaging.
1 mg/kg of lipopolysaccharide(LPS) diluted in saline or saline alone was injected under ultrasound guidance directly in the corpus callosum of P3 rat pups. Coronal photoacoustic images were carried out 24 h after LPS exposure. Locoregional oxygen saturation (SO2) and resting state connectivity were assessed in the cortex and the corpus callosum. Microvasculature was then evaluated on cryosection slices by lectin histochemistry.
Significant reduction of SO2 was found in the corpus callosum; reduced SO2 was also found in the cortex ipsilateral to the injection site. Seed-based functional connectivity analysis showed that bilateral connectivity was not affected by LPS exposure. Changes in locoregional oxygen saturation were accompanied by a significant reduction in the average length of microvessels in the left cortex but no differences were observed in the corpus callosum.
Inflammation in the developing brain induces marked reduction of locoregional oxygen saturation, predominantly in the white matter not explained by microvascular degeneration. The ability to examine regional saturation offers a new way to monitor injury and understand physiological disturbance non-invasively.
Periventricular leukomalacia (PVL) stands as a predominant cause of cerebral palsy and significant neurodevelopmental impairment in premature infants
The regional changes seen in white matter injury of the preterm infants have been described using near-infrared spectroscopy (NIRS). This well-established bedside technique is limited by the absence of depth resolved information where for instance the presence of sub-dural hematoma or scalp edema seen in newborns might lead to inaccurate results. Moreover the technique cannot differentiate the cortex from the white matter or saturation extracted from the venous circulation. Although partial depth information can be obtained by using tomographic reconstruction methods
Since the discovery of spatially remote areas connected by spontaneous low frequency in the temporal domain with coherent fluctuations in the BOLD signal (<0.1 Hz)
The objective of this study was to determine the impact of inflammation on the corpus callosum and the cortex both known to be affected by inflammation. Moreover the possibility of assessing resting-state functional connectivity using PAT imaging in immature rat pups was examined.
All surgical procedures performed according to the recommendations of the Canadian Council on Animal Care, were approved by the Animal Research Ethics Committee of the Montreal Heart Institute and by the Institutional Committee for Animal Care in Research of the CHU Sainte-Justine Research Center. All the procedures were performed under isoflurane anesthesia, and all efforts were made to minimize suffering.
Nineteen postnatal day 3 (P3) Sprague-Dawley rat pups from four litters were used in this study (9.8±0.3 g weight, Charles River, Wilmington, MA); the rat pups were given
P3 rat pups (n = 19) were randomized to two experimental groups: LPS (n = 11; nmale = 6, nfemale = 5) and NaCl (sham n = 8; nmale = 7, nfemale = 1). Pups were injected under ultrasound guidance using the Vevo LAZR micro-ultrasound system (FUJIFILM VisualSonics Inc., Toronto, ON, Canada) with LPS (1 mg/kg) suspension (E. Coli, serotype 055:B5, Sigma St Louis, MO) in 0.5 µL of sterile saline or with the same volume of sterile saline alone for the sham group. The injection was placed in the corpus callosum at the level equivalent to P-7, c9
(A) Arrow marks injection site. (B) Seeds placement, overlaid a coronal B-scan, averaged over 11 individual B-scans. Abbreviations: M, motor cortex; S1, primary somatosensory cortex; S1BF, barrel field primary somatosensory cortex; LV, Lateral ventricle; CPu, Caudate putamen. Black dotted line indicates Left Cortex ROI used for locoregional SO2 measurement. White dotted rectangle indicates ROI used for local SO2 measurement in corpus callosum. Scale bar: 1 mm.
The rat pups were secured in a custom-designed stereotaxic neonatal frame and placed on an imaging station; monitoring of the respiration and body temperature were done using the 1025L monitoring system (SA Instruments Inc., Stony Brook, NY). The Vevo LAZR photoacoustic imaging system was used to acquire all images with the LZ250 ultrasound probe at a central frequency of 21 MHz. The chosen coronal B-scan was located at the injection site. The acquisition time was 1.4 s per B-scan, with a field of view (FOV) of 13 mm width ×18 mm depth (512 A-lines, 416 samples) using a 256-element array transducer that provides 75 μm axial resolution. A time-series of B-scans were acquired in resting-state conditions for a 5-minute period. Although skin removal has been shown to improve image quality
First, all functional images (PAT) within each scan were realigned to the first image to reduce the impact of motion artifacts. Images of each contrast were spatially smoothed with a Gaussian kernel of FWHM = 0.2 mm. Imaging of the anatomy was performed by an ultrasonic B-scan simultaneously recorded, from which the region corresponding to brain was manually selected using a closed spline curve to create a brain mask. All further processing was performed only on those pixels belonging to the brain mask. The image time series were temporally band-pass filtered (zero phase-shift fourth-order Butterworth filter) at 0.009–0.08 Hz, according to previous functional connectivity studies
All seeds were placed
Movement during scanning influences the quality of PAT data. Head motion causes spurious changes in photoacoustic signal intensity. After frame realignment and motion parameters regression, a technique called “scrubbing” was applied to remove measures distorted by unwanted movement
FD represents instantaneous head motion and is an empirical measurement computed from the motion parameters; in
DVARS was computed as the RMS of the derivative of the global brain signal; this measure represents how much the intensity of a brain image changed in comparison to the previous frame.
In
With the unilateral LPS injection we investigated bilateral differences between brain regions so the metric used to evaluate functional connectivity was a regional bilateral functional correlation, defined as the correlation between each seed time-course and its contralateral homologue, yielding six values for each rat pup. The Pearson’s coefficient r-values were converted to Fisher Z measures using
LPS and NaCl-injected brains were fixed after PAT imaging by transcardiac saline perfusion followed by 4% paraformaldehyde. The whole brains were kept at in paraformaldehyde 4°C and transferred to 30% sucrose after twenty-four hours. Cryosections (50 μm) were obtained and kept in cryoprotectant solution at −20°C.
Four consecutive brain slices were chosen from each brain, spanning the injection site for staining and analysis with tetramethylrhodamine isothiocyanate (TRITC)-conjugated lectin to characterise microvasculature
Statistical significance in imaging and in microvasculature morphometry measures was evaluated using Wilcoxon-Mann-Whitney test. Since histology and locoregional SO2 measures were performed over only a few
After removal of four animals due to excessive motion (these animals were not included in the population description above) average SO2 was measured both on the left cortex (unilateral to LPS injection), the corpus callosum (cc) and on the right cortex. Group-averaged SO2 weighted images of sham controls and LPS are respectively depicted in
(A) Group averaged SO2 weighted PAT image for sham controls (NaCl), (B) Group averaged SO2 weighted PAT image for LPS group, (C) SO2 Comparison performed between LPS group (N = 11), and NaCl (sham) group (N = 8). SO2 showed significant decrease in the LPS group compared to NaCl group in left cortex (L) and corpus callosum (cc). The hemisphere contralateral (R) to injection showed no difference. (D) HbT comparison between LPS and NaCl groups, no significant differences were found in any of the explored ROIs. Scale bar: 1 mm; *P<0.05, ***P<0.001.
We analyzed changes in vessel lengths in rat pup brains post LPS injection using lectin fluorescent staining due to its sensitivity in detecting microvessels
(A) left cortex; sham, (B) left cortex; LPS, (C) right cortex; sham, (D) right cortex; LPS, (E) corpus callosum; sham, (F) corpus callosum; LPS. The average microvessel length in the left cortex of brains injected with LPS (B) is reduced with vessels appearing fragmented compared to the brain injected with saline (A). Bar graphs of total microvessel length (G) and average microvessel length (H) in rat brain sections over the cortex and corpus callosum using the skeleton technique with AngioTool. Note the significant decrease in average microvessel length on the left cortex in brains injected with LPS (* p = 0.0093). Scale bar = 100 μm.
After scrubbing, a total of four scans were removed from the study due to excessive motion; (the same animals removed for computing locoregional SO2). The results displayed in
Comparison performed between LPS group (N = 11), and NaCl (sham) group (N = 8); analysis done for every seed time-trace and its contralateral part. Contrasts shown: (A) SO2, (B) HbT (C) HbO2 and (D) HbR. Abbreviations: (M, motor cortex; S1, primary somatosensory cortex; S1BF, barrel field primary somatosensory cortex).
Locoregional SO2 in the corpus callosum and on the cortex ipsilateral to the injection site were lower in the injured group, as compared to the sham group. This result coincides with lower regional cerebral tissue oxygen saturation found in preterm infants with transient PVL
Furthermore, these findings support the use of PAT as a non-invasive tool to study PVL animal models; due to its non-invasive and contrast agent-free nature, PAT could be applied to longitudinal studies either in adult humans
When measuring bi-lateral seed connectivity, there was a strong correlation between the motor ROIs and less so in the S1 and S1BF ROIs and measured correlation values were similar that published in previous studies
The results of this exploratory work reveal lower locoregional SO2 values in LPS group, showing decreased values in the corpus callosum and in the left cortex, ipsilateral to the injection site. Our findings support the use of PAT as a completely non-invasive tool to assess average oxygenation values
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The authors would like to express their gratitude to the laboratory of Dr. Sylvain Chemtob for fruitful discussions and advice regarding microvascular quantification. The authors also wish to thank Andrew Needles from Visual Sonics for his technical assistance.