A Single Concussion in Juvenile Mice Leads to Sex Specific Acute Cerebral Vascular Dysfunction and Blood-brain Border Dysfunction

A Single Concussion in Juvenile Mice Leads to Sex Specific Acute Cerebral Vascular Dysfunction and Blood-brain Border Dysfunction | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A Single Concussion in Juvenile Mice Leads to Sex Specific Acute Cerebral Vascular Dysfunction and Blood-brain Border Dysfunction Jiamin Yan, Nathan Nguyen, Terese Garcia, Adam Godzik, Greer Cisneros, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8622019/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Traumatic brain injury (TBI) can induce alterations to the blood–brain border (BBB) that contributes to long-term neurological and behavioral deficits. The temporal progression of post-concussion BBB dysfunction during developmentally sensitive periods remains poorly understood. Therefore, we sought to characterize the temporal evolution of BBB disruption and cerebrovascular alterations acutely after concussion in juvenile mice. Methods Postnatal day 17 (PND17) C57BL/6J male and female mice were subjected to sham or single closed head injury with long-term disorders (CHILD). At 1h, 6h, 1d, 3d, and 7d post-injury, Evans blue (EB) dye was administered intravenously to evaluate BBB permeability, followed by vessel painting to visualize modified cerebrovascular angioarchitecture. MRI-based T2 relaxation mapping at 1dpi has been used for brain tissue properties, including edema. EB and vascular features were modeled to assess ability to discriminate between sham and CHI mice. Results A single early-life concussion induced hyper-acute (hours) structural and functional alterations in brain vasculature. CHILD in PND17 mice resulted in: 1) disruption of physiological functions and developmental trajectories, 2) reduced brain volumes and sex-dependent T2 relaxometry changes (elevated in females, reductions in males), and 3) hyper-acute BBB increases in permeability which correlated with cerebral vascular rarefaction. Notably, males exhibited more robust BBB and vascular perturbations than females, revealing sex-dependent trajectories of vascular response to CHILD. We also highlight differential vulnerability in vessel location with the smaller penetrating cortical vessels displaying greater susceptibility to alterations compared to larger, more resilient pial blood vessels. Modeling demonstrated that vascular features clustered together while trajectory analysis confirmed that female CHI mice were not consistent in their disease trajectory compared to male CHI. Additional analysis suggested that vascular features able to discriminate in a sex- and injury specific manner. Conclusions A single concussion is sufficient to induce hyper-acute BBB and cerebrovascular perturbations in juvenile mice, which may presage long-term deficits during development. Importantly, sex differences in vascular TBI responses evident at PND17 emphasize the need to consider sex as an important variable in future pediatric TBI research. vessels magnetic resonance imaging closed head injury vessel painting Evans Blue Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Traumatic brain injury (TBI) due to external mechanical force, represents a major public health and economic burden, accounting for more than 600,000 emergency department visits each year, with 90% of all pediatric TBIs classified as mild TBI (mTBI) [ 1 ]. Little is known about the unique features of mTBI in children, particularly considering the structural and functional differences as the developing brains transition to adulthood. Thinner and less rigid skulls may provide reduced mechanical protection, increasing susceptibility to fractures and tissue deformation [ 2 , 3 ]. The developing brain is characterized by immature neural networks and active processes of synapse formation and pruning, such that perturbations during these critical epochs can interfere with normative maturation [ 4 ]. Pediatric and juvenile brain injury places children at elevated risk for persistent learning disabilities, psychological disorders, behavioral problems, disruption of academic and social performance [ 1 , 5 , 6 ]. The blood-brain barrier or more recently designated as Blood-Brain Border (BBB) interface [ 7 ], is a very dynamic interface between blood and brain, composed of tight junction proteins linked endothelial cells expressing various transporters finely tuned by pericytes and astrocytic endfeet [ 7 ]. BBB remodeling and vascular dysfunctions have been suggested to contribute to the long-term neurological and behavioral deficits often observed after adult TBI [ 8 , 9 ]. Recent work in patients with moderate to severe TBI exhibited BBB perturbations confined to microvascular regions in pediatric TBI but was predominately in larger vessels in adult patients [ 10 ]. Moreover, increased BBB dysfunctions in juvenile mice evoked an increased microglial response [ 11 ]. Yet, whether similar mechanisms occur following juvenile mTBI, and, if so, the timeline and severity of these alterations in the developing brain are underexplored. Most children appear to recover from mTBI within several weeks but up to one-third experience persistent deficits [ 1 ]. In adults, BBB dysfunction is recognized as a central mechanism contributing to long-term dysfunction after severe TBI in humans [ 12 , 13 ] and in adult rats exposed to severe TBI [ 13 ]. While moderate to severe TBI outcomes have been relatively well documented in both human subjects and in rodent models, far less is known about the sequelae following mTBI/concussion in pediatric brain injury. Indeed, the temporal course of hyperacute BBB changes in pediatric and juvenile mTBI have not been reported. This lack of mechanistic understanding impedes the development of pediatric-specific diagnostic tools, treatments, and strategies to identify children at greatest risk for chronic deficits. To address this gap, we examined BBB integrity and altered angioarchitecture in a juvenile closed head injury with long-term disorders (CHILD) model [ 14 ]. The CHILD model is a robust unrestrained closed head concussion model in postnatal day 17 (PND17) mice and replicates key clinical features of mTBI: a) rotational acceleration and coup–contrecoup injury [ 14 , 15 ], b) behavioral alterations [ 15 – 17 ], c) acute perturbations in tissue oxygenation, neurovascular coupling and long-term cardiac dysfunction [ 16 , 18 ], and d) progressive decrements in white matter [ 19 ] (see Table 1 in reference [ 19 ]). In our study, we utilized the PND17 CHILD model and examined BBB leakage and cerebrovascular perturbations at 1h, 6h, 1-, 3- and 7-days post-injury (dpi) in a sex-specific manner. We report both temporal and sex-specific alterations in BBB and vascular responses to juvenile mTBI. Methods The experimental protocol focused on hyperacute and acute time points after mTBI as outlined in a schematic in Fig. 1 A. Animals Pregnant C57BL/6J female mice (E14) were purchased from Jackson Laboratory (JAX #000664). CHILD or sham procedures were performed on postnatal day 17 (PND17) pups of both sexes. Animals were randomly assigned to one of six groups (Supplemental Table 1): Sham, CHILD 1h (n = 13), CHILD 6h (n = 16), CHILD 1d (n = 21), CHILD 3d (n = 18), and CHILD 7d (n = 19). Pups were excluded if their weight was less than 5.9g on PND 17; all pups were weaned on PND 21. Mice were maintained at 21 ° C with an automated 12-hour light-dark cycle and had ad libitum access to water and standard vivarium chow. All experiments were in accordance with the University of California, Riverside and University of California, Irvine Institutional Animal Care and Use Committees and federal regulations and in accordance with ARRIVE guidelines as well as Animal Welfare Act and Public Health Service policies related to humane care of animals. Closed Head Injury with Long Term Disorders (CHILD) CHILD model details and videos have been published recently [ 14 ]. Briefly, on PND17, animals were weighed and anesthetized with 2.5% isoflurane in 1.5 L/min O 2 for 5 minutes in a chamber heated to 37°C. Each mouse was removed from the isoflurane chamber and quickly placed on a taut and secured aluminum foil (15 x 15 cm) stretched across a stereotactic frame. The mouse position was adjusted so that the impactor tip was directly above the left somatosensory cortex. The impactor tip (3mm diameter rubber tip) was mounted at a 90° angle perpendicular to the stereotactic apparatus. A single impact was then delivered using an electromagnetic impactor (Leica Biosystems, Deer Park, IL, USA) with the following parameters: velocity: 3m/s, dwell time: 0.1s, and depth: 3mm. The resulting injury is equivalent to Grade 2 (G2) level injury, as previously defined [ 15 ]. The presence of apnea and head rotation were recorded. The mouse was then immediately placed on its right side in a warmed (37°C) recovery chamber to assess righting time and time to resume exploratory behaviors. All animals survived the CHILD. The shams underwent identical procedures but without an impact. Evans Blue Injection, Vessel Painting and Tissue Fixation At each time point post-CHILD, a 2% solution of Evan’s Blue (EB) (Acros Organics, Geel, Antwerpen, Belgium) in phosphate buffered saline (PBS) was administered via tail vein injection (3µL/g) while the animals were under light anesthesia (2% isoflurane in 1.5L/min O 2 ). EB was allowed to circulate for 1 hour prior to vessel painting and perfusion. Mice were then anesthetized with 2.5% isoflurane in 1.5L/min oxygen and were given an intraperitoneal (i.p.) injection of Ketamine (200mg/kg) and Xylazine (200mg/kg) to induce deep general anesthesia. Mice were then given an i.p. injection of heparin (1000units/kg) followed by sodium nitroprusside (0.75mg/kg) to dilate vessels. To visualize the cerebrovasculature, we performed an intracardiac injection of 3,3’-dioctadecyloxacarbocyanine (DiO, Biotium, Fremont, CA, USA) (0.75mg/kg) diluted with 4% dextrose in PBS. Mice were then immediately intracardially perfused with 15mL PBS followed by 20mL of 4% paraformaldehyde (PFA). Brain tissues were post-fixed in 4% PFA for 24h, washed with PBS for 3 consecutive days and stored at 4°C in 0.02% sodium azide-PBS solution. Labeling of the vasculature is termed vessel painting (VP) [ 20 ]. IgG Immunohistochemistry (IHC) and Analysis 1hpi mouse brains were used for IgG staining and were incubated in 30% sucrose solution at 4°C for 48hrs. Samples were then frozen in Optimal Cutting Temperature Compound (OCT) on dry ice and stored at -20°C. Brain samples were sectioned coronally into 30µm thick slices and mounted directly onto slides and stored at -80°C. Sections were treated with 1% Sodium Dodecyl Sulfate at room temperature then incubated for 1.5 hours in room temperature with Alexa Fluor™ 594 Goat anti-Mouse IgG (1:1000, Invitrogen, A11005). Slides were then dried and coverslipped with Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA). Wide-field and Confocal Microscopy Fluorescence images from vessel painted brains were acquired with a wide-field fluorescence microscope (Keyence BZ-X810, Keyence Corp, Osaka, Japan). Both axial surface and coronal sections at the level of the dorsal hippocampus (Bregma − 1.82mm) were imaged at 2X using the sectioning and Z-stack functions (step size 25.2 µm, 20 stack). Level correction, black balance, and haze reduction (blur size = 10, brightness = 10, reduction size = 1) were applied to the images using BZ-II Analyzer software (Version: 1.1.30.19). Higher magnification 10X images were taken from regions with EB extravasation and the corresponding region in the contralateral hemisphere. Confocal images at 20X were acquired from 30µm IgG-stained sections using a Zeiss LSM 880 confocal microscope (Carl Zeiss, Oberkochen, Germany). IgG, EB, and VP signals were imaged using excitation wavelengths of 561, 633, and 488 nm, respectively. Single-field images were acquired using the following parameters: 2% laser power; 2.57 Airy units pinhole size; 25 optical sections of z-stack with a step size of 2 µm; and 425.1 µm × 425.1 µm image field. Three-dimensional reconstruction and visualization were performed using Imaris Bitplane software (version 10.2.0; Oxford Instruments, Abingdon, UK). Evans Blue Analysis Quantification of EB extravasation was performed using Fiji (Version: 1.54f) software. First, a known region of EB leakage was outlined in a single CHILD mouse at 1hr post injury. Then the “fire” lookup table was applied and intensity levels > 100 were defined as extravasation. This method was then applied to all mice and regional areas with intensity values > 100 was extracted and summarized in MS Excel. In coronal sections, the integrated density was measured within the identical cortical region of the ipsilateral hemisphere corresponding to the site of the injury in all CHILD and sham mice. Angioarchitecture Analysis Angiotool 0.6 software [ 21 ] was used to quantify classical vessel characteristics (vessel density, length, and junction density in the selected region of interest (ROI). Vessel complexity was assessed using the ImageJ FracLac to derive local fractal dimensions [ 22 ]. Axial regions of interest (ROI) included left and right hemispheres or whole axial brain analyses. Coronal ROIs encompassed cortical regions extending from the mid-line to the ventral-most boundary of the somatosensory cortex and placed ipsi- and contralaterally. Similar analytical methods have been published previously [ 23 ]. Data was extracted and summarized in MS Excel. Magnetic Resonance Imaging (MRI) Acquisition and Analysis T2-weighted (T2WI) and susceptibility-weighted imaging (SWI) were performed on ex vivo , skull-attached samples at 9.4T (Bruker Biospec, Billerica, MA). The following acquisition parameters were used for T2: 4000ms repetition time, 10ms echo time, 10 echoes, 4 averages, field of view 1.25 x 1.25cm, matrix 128 x128, 20 slices, 0.5mm slice thickness, 0.5mm slice interval, acquisition time ~ 25min using Paravision 5.11. SWI was acquired using: 722.9ms repetition time, 10ms echo time, 8 averages, field of view 1.25 x 1.25cm, matrix 128 x128, 20 slices, 0.5mm slice thickness, 0.5mm slice interval, acquisition time ~ 12min. The brain was segmented away from skull and extraneous tissues using ITK_SNAP (Version 3.8.0) software [ 24 ]. The extracted brains were used to generate T2 maps using JIM 7.0 software (Version. 7.0_42 Jan 10 2018, Xinapse Systems, Northants, UK). T2 maps were registered to our modified bilateral Australian Mouse Brain Mapping Consortium Atlas [ 25 ] using Advanced Normalization Tools (ANTS, Version:RRID:SCR_004757, University of Pennsylvania, Philadelphia, USA ). Regional brain volumes and T2 relaxation times were then derived from the registered T2 maps. SWI scans were analyzed using Signal Processing in NMR (SPIN) software (Version: Revision 1872) to identify presence of extra parenchymal bleeds. Modeling Methodology All the data, except MRI, were combined for the analysis to determine if there were potential predictors for CHI BBB disruption. The analysis was performed by the in-house python scripts using SciPy and Scikit-learn libraries. MRI data from a sub-cohort of mice these data were excluded, as they were missing over 70% of samples. Imputation . Up to 17% of data in other feature groups were missing; these were imputed to allow consistent downstream statistical analysis and module construction (see below). Imputation was performed separately for each variable using a hierarchical strategy, as follows. If at least two real observations were available within the same group × sex × timepoint subset, the missing values in that subset were replaced with the mean of the available observations. If a subset contained fewer than two real measurements (i.e., insufficient information for a reliable subgroup mean), the missing values were left unchanged and only replaced with the global mean of that variable if still required for principal component analysis (PCA) or visualization. This approach preserved true biological variability, avoided overfitting sparse subgroups, and prevented downstream analyses (e.g., PCA, clustering) from being dominated by “missingness” patterns rather than biological signal. Heatmap and Clustering . To visualize the correlation structure among measurements and assess relationships between features, pairwise Spearman correlation coefficients were computed for all features across all animals. Correlations were displayed as a heatmap with hierarchical clustering using average linkage and a Euclidean distance metric on the correlation matrix. This unsupervised approach recovered biologically related variables and highlights modules of coordinated change following CHI, supporting intuition that measurements from the same anatomical orientation or imaging modality are correlated with each other. Module Definitions . Clustering results were used to define feature “modules” that both describe related biological processes and are correlated with each other. For example, vascular metrics derived from coronal sections formed a vascular-coronal module, while fractal dimension (LFD) features from axial slices defined as an axial-LFD module. For each module, we used PCA and used the first principal component (PC1) of the standardized module variables to be used as the module’s composite metric. PC1 captures the dominant shared variance of the module and serves as a noise-reduced, direction-consistent representation of the underlying biological process, such as vascular remodeling or vascular complexity. Trajectory Modeling . We took a combined p-values approach for trajectory modeling (averaging over points) to assess changes over time. To quantify sex differences while properly accounting for measurements collected at multiple timepoints after injury, statistical comparisons were performed independently at each timepoint using the Mann–Whitney U test. The resulting per-timepoint p-values (p i ) were then aggregated into a test statistics (𝚿) using Fisher’s combined probability method, as defined: $$\:\psi\:=\:-2\sum\:_{i=1}^{k}\text{l}\text{n}\left({p}_{i}\right)$$ which follows a c² distribution with 2k degrees of freedom (k = number of timepoints) and allows us to calculate the combined p-value. This approach does not assume linear or monotonic changes over time and is robust to heterogeneous variance and missingness across timepoints. For additional robustness, permutation-based combined p-values were computed by shuffling sex labels within each timepoint, recomputing p-values, and comparing the observed Fisher statistic to its permutation distribution. Consistency . Because Fisher’s method combines p-values but not effect directions, we also quantified whether the male–female differences were directionally consistent across timepoints. For each timepoint, the sign of the difference (mean_male − mean_female) was recorded. Directional consistency was defined as the fraction of time points at which the sign matched the majority direction across the trajectory. Values near 1.0 indicate stable directional effects (e.g., males consistently higher than females), whereas values near 0.5 indicate mixed or fluctuating differences. This provides an intuitive measure of biological coherence complementing the combined p-value. t-SNE Embedding of Key Discriminative Features . To visualize multivariate relationships among subjects, we applied t-distributed Stochastic Neighbor Embedding (t-SNE) to a curated feature set consisting of the most biologically discriminative module PC1 scores (e.g., axial LFD, coronal vascular) and key volumetric variables. All features were standardized prior to embedding. Only animals with complete data for the selected features were included, ensuring stable geometry and avoiding distortions driven by missing values. The resulting two-dimensional embedding was plotted with point color indicating sex, point shape indicating Sham or CHI groups, and where small numeric labels marking post-injury timepoints. This provides an intuitive visualization of how injury and sex jointly influence high-dimensional phenotypic space. Statistical Analysis Statistical analysis was performed using GraphPad Prism (Version 9, GraphPad, Boston, MA, USA). We performed one-way analysis of variance (one-way ANOVA) with multiple comparisons for temporal data and group comparisons utilized t-tests. All t-tests were parametric unless specifically stated. Pearson correlations were also performed in GraphPad. All values are presented as mean ± SEM. Statistical significance threshold was defined as p < 0.05 with trending reported in those cases with p < 0.10. Results CHILD induced sex-specific physiological and structural changes. Prior to CHILD induction, PND17 weights between male and female mice were not significantly different, with the average weight of all pups being 7.04 ± 0.67g (n = 117). There were no significant differences (p = 0.724, unpaired t-test) between male average weights (7.06 ± 0.59g, n = 61) and female weights (7.02 ± 0.75, n = 56). No significant weight differences were found at 1dpi between sham and CHILD male or female mice (Fig. 1 B). In contrast, relative to pre-CHILD (baseline), weight gain at the 7dpi period relative was significantly increased in sham compared to CHILD mice (p = 0.0009, unpaired non-parametric t-test) (Fig. 1 D). Male CHILD mice at 7dpi had a significant decrement (p = 0.002, unpaired non-parametric t-test) in weight gain (55.6 ± 0.04% compared to male shams 66.5 ± 0.02%; Supplemental Fig. 1A). Female CHILD mice also had reduced weight gain compared to female sham mice at 7dpi but did not reach significance (p = 0.073, unpaired non-parametric t-test) (Supplemental Fig. 1B). Paired-weight changes between baseline and 1dpi or 7dpi further demonstrate significant increases in weight gain over the 7dpi period (Supplemental Fig. 1C, D). Immediately after CHILD induction we monitored the level of consciousness in all mice by recording the presence and duration of apnea immediately after head impact and the time required to resume a righting position. The prevalence of CHILD mice that exhibited apnea was ~ 20% higher in males (44.44%) than in females (24.32%) (Fig. 1 C), exhibiting a sex-specific immediate physiological response to concussive injury. CHILD mice also exhibited a significantly longer time to resume righting position relative to shams (p < 0.0001, unpaired t test) with no overt sex differences (Fig. 1 D). A randomized subset of sham (n = 11) and CHILD mice (n = 15) at 1dpi underwent ex vivo T2-weighted MRI (Fig. 1 E). Cerebrum volumes exhibited sex differences with male CHILD mice showing significantly reductions by 8.82% (p = 0.035, unpaired t test) compared to shams (Supplemental Fig. 2A), while female CHILD or sham mice did not report differences. Male CHILD mice exhibited significantly lower cerebrum volumes than female CHILD mice (p = 0.011, unpaired t test) but no differences between male and female shams were reported (Supplemental Fig. 2A). Brain tissue properties were assessed with T2-relaxometry measurements (in ms) from cortical regions (motor, parietal and somatosensory) and white matter structures (corpus callosum, CC) that are at the site of the concussive impact (Fig. 1 F-I). T2 relaxation time was reduced in all four regions in male CHILD mice. There was a significant decrease in T2 relaxation in parietal cortex of male CHILD mice of 12.58% compared to shams (p = 0.014, unpaired t test) (Fig. 1 G). Sex differences were also observed since female CHILD mice had significantly increased T2-values in motor (8.82%), parietal cortices (7.56%), and corpus callosum (10.21%) compared to female shams (p = 0.010, p = 0.021, p = 0.002, respectively, unpaired t test) and a trending significance in the somatosensory cortex (p = 0.074, unpaired t test) (Fig. 1 F-I). Susceptibility-weighted imaging (SWI) was also acquired to assess the presence of extravascular blood (Supplemental Fig. 2B) which was often found at the cortical surface and at the interface between gray and white matter (corpus callosum). Seventy-five percent of male CHILD mice but only 42% of female CHILD mice exhibited visible extravascular bleeding (Supplemental Fig. 2C). Thus, clinically relevant neuroimaging further confirms sex-specific differences in water content and parenchymal bleeds. CHILD induced transient dysfunction of the blood-brain border (BBB) at acute time points followed by recovery. Evans Blue (EB) is a water-soluble fluorescent dye that binds to serum albumin and only permeates into the brain parenchyma when BBB properties are compromised. EB observed within blood vessels confirmed functional perfusion in sham mice, whereas EB accumulation within the brain parenchyma was observed in the ipsilateral cortex in CHILD mice at 1hpi (Fig. 2 A). At higher magnification (Fig. 2 A, right panel), EB extravasation in the parenchyma was observed adjacent to vessels defined by VP at the site of injury in CHILD mice, but not in shams. Integrated intensity of extravasated EB in the parenchyma was quantified at 1hpi, 6hpi, 1dpi, 3dpi and 7dpi in the ipsilateral cortex from coronal tissue sections. Increased EB accumulation peaked at 1hpi and then declined over time in males and females (Fig. 2 B). Female CHILD mice exhibited a significant increased EB extravasation of 71.02% compared to shams at 1hpi (p = 0.047, unpaired t test), while the increase of EB extravasation did not reach significance in male CHILD mice (Fig. 2 B). Higher variability in EB extravasation was observed for the acute timepoints (1hpi and 6hpi) compared to later timepoints in both sexes. Using a two-phase exponential decay model, temporal analysis of BBB dysfunction from coronal images showed a linear decrease in EB extravasation in males, while females exhibited a rapid exponential decrease after injury (Fig. 2 C). We also assessed EB leakage area from the cortical surface (axial) (Fig. 2 A, middle panel) and like the coronal analyses, there was considerable variability in male CHILD mice although less so in the female mice with no significant differences (Fig. 2 D). The number of mice from each sex who had axial EB leakage present were collated as a percent of all the mice (Fig. 2 E). In male CHILD mice there was an increasing proportion that showed BBB leak that peaked at 1dpi (71%) and then precipitously declined by 7dpi (11%) (Fig. 2 E). In contrast, CHILD female mice had 100% of injured mice exhibiting cortical EB leakage at 6hpi that slowly declined by 7dpi (60% of mice, Fig. 2 E). Exemplar micrographs illustrate extravascular EB extravasation from cortical vessels are shown in Supplementary Fig. 3, at 1hpi and 1dpi. These images reveal subtle and vascular localization of EB leakage within cortical regions and those adjacent to the concussive impact site. These findings were in line with sex differences in BBB pathophysiology between concussed male and female mice. CHILD impairs axial cortical angioarchitecture associated with BBB perturbations The inter-relationships between axial cortical vascular features using vessel painting and EB extravasation were examined in sham and CHILD mice across all timepoints (Fig. 3 A). Axial vessel density on the ipsilateral hemisphere was significantly reduced by 41.79% in male CHILD mice compared with shams at 1hpi (p = 0.047, one-way ANOVA), which progressively recovered by 7dpi (Fig. 3 B). Total vessel lengths and number of junctions exhibited no significant changes in CHILD males, but the pattern of changes were like that of vessel density in males (Fig. 3 B, E, H). However, no significant changes were observed in females either in vessel density, total vessel lengths and number of junctions at any of the time points examined (Fig. 3 C). In males the temporal resolution of vessel density was consistent with the peak BBB dysfunction (Fig. 2 B, C). Therefore, the relationships between these outcome measures were calculated (Fig. 3 D, G, J). In male CHILD mice axial surface EB extravasation area were significantly negatively correlated to vessel density (r=-0.578, p = 0.024) (Fig. 3 D) and junction density (r=-0.572, p = 0.026) (Fig. 3 J). CHILD male mice exhibited no significant relationship with total vessel length and surface EB extravasation (Fig. 3 G). This suggests that vessel alterations characterized by a loss in density and number of junctions also demonstrated BBB dysfunction. No significant correlations were observed in CHILD females in any vessel metric compared to EB extravasation area (Fig. 3 D, G, J). These results suggest that impairment of the BBB is strongly associated with morphological vessel alterations in males but not in females, highlighting sex differences in cerebrovascular pathophysiology early post-concussion. Coronal cortical angioarchitecture is decreased after CHILD and is associated with BBB perturbations The ipsilateral coronal cortical vasculature was analyzed at and adjacent to the impact site, examining the vessels penetrating the cortex. We quantified VP angioarchitecture and EB extravasation, similarly to the axial surface findings (Fig. 4 A). Following similar pattern observed on axial analysis (Fig. 3 B), a dramatic and significant reduction by 71.82% cortical vessel density was found in male CHILD mice at 1hpi compared to shams (p < 0.05, one-way ANOVA, Tukey’s post-hoc test), which temporally recovered by 7dpi (Fig. 4 B). Total vessel length was significantly reduced at every time point in CHILD males (p < 0.05 for each, ordinary one-way ANOVA) (Fig. 4 E). Junction density was similarly significantly reduced in CHILD male mice at 1hpi compared to shams (p = 0.05, ordinary one-way ANOVA) (Fig. 4 H). As described for the axial analysis, no significant morphological changes in blood vessel metrics were observed in female CHILD mice compared to shams (Fig. 4 C, F, I), despite an overall trend for vascular reductions at 1hpi. When we examined the relationship between vascular morphological features (vessel density p = 0.040; total length p = 0.037; junctions p = 0.027), we found significant correlation to EB intensity in CHILD male mice independent of time post injury (Fig. 4 D, G, J). Again, no significant correlations between EB extravasation and vessel metrics were found in female CHILD mice(Fig. 4 D, G, J). Thus, in CHILD males but not CHILD females, the severity of the BBB dysfunction was directly related to vasculature morphological changes within the ipsilateral cortex. Acute CHILD elicits a hyper-acute reduction in vessel complexity A key hallmark of vascular damage is a reduction in vascular complexity which can be assessed using fractal measures [ 23 ]. Smaller vessels are potentially more vulnerable to mechanistic forces induced by the head rotation (Rodriguez-Grande, Glia 2018) and thus contribute to pathological progression of BBB breakdown. We assessed vascular complexity in coronal cortical vessels at the lesion site by generating fractal histograms (Fig. 5 A, B; Supplementary Fig. 5). The resultant fractal histograms measures provide quantitative information about complexity (shift in LFD curve) and vessel numbers (area under the curve or AUC) (Fig. 5 B). Early hyper-acute time points (1-6hpi) revealed a significant reduction in AUC at 1hpi in male CHILD mice compared with male shams (p = 0.031, unpaired t test), with no overt differences in female CHILD mice (Fig. 5 C). At 6hpi in male CHILD mice the AUC started to recover with a trending significant reduction (p = 0.115, unpaired t-test) (Fig. 5 C) with no significant differences were observed in either male or female CHILD mice at other time points up to 7dpi (Supplementary Fig. 6A, B). Vessel complexity was assessed using the maximum local fractal dimension (LFD) at the peak frequency (Fig. 5 B). At 1hpi, both CHILD male (p = 0.016, unpaired t test) and CHILD female (p = 0.037, unpaired t test) mice had significant reductions in LFD values consistent with reduced vessel complexity (Fig. 5 B) but no differences in either male or female CHILD mice were observed at any other time points (Supplementary Figs. 4,5). These results further confirm that CHILD in male mice results in reduced brain vasculature and complexity at hyper-acute time points post-injury whereas CHILD in female mice elicited only decrements in complexity but not in vascular density. Presence of Immunoglobulin G (IgG) in CHILD mice signifies BBB breakdown Immunoglobulin G (IgG) extravasation in brain tissue after injury is a marker of BBB dysfunction as we previously described in CHILD at 1dpi (Rodriguez-Grande et al. 2018) and in adult CHI [ 26 ] and in juvenile TBI [ 11 ]. To further confirm BBB dysfunction, we undertook IgG staining at 1hpi when the most robust alterations in cortical vessels and EB extravasation were observed. Low magnification IgG-stained sections (Supplementary Fig. 6) were examined for representative cortical vessels that exhibited EB extravasation in male CHILD mice at 1hpi. Figure 6 illustrates the coincident labeling between vessels and IgG, vessels and EB and vessels, IgG and EB in sham mice (left panel) and CHILD mice (right panel). Sham mice did not exhibit any notable IgG or EB signals outside the vessels although in the merged images both are visible within the vessels themselves. In stark contrast, CHILD mice at 1hpi exhibited IgG extravasation staining protruding from vessels in discrete beads along injuried vessels (Fig. 6 ). Interestingly, extravasation EB staining presented larger coverage along the vessels than IgG staining. However, IgG staining was associated with EB-extravasation (Fig. 6 ), a confirmation of the vascular-BBB dysfunctions. Modeling the Interactions between Vascular Injury and BBB Dysfunction Given the wealth of the data acquired in this study we examined if modeling these vascular and BBB data could provide additional insights into the physiological mechanisms and the potential for predictive capabilities. The first step was to identify the correlation structure between all the variables (data) that were collected. Clustering of the data and its heatmap representation clearly illustrated that vascular measures strongly clustered together (Fig. 7 A). Coronal vascular features, axial vascular features and complexity measures were all strongly aggregated. Physiological features (apnea duration etc.) exhibited a reduced clustering. Based on the clustering and heatmap analysis we undertook a data reduction approach whereby we consolidated groups of related features into modules. The final modules are summarized in Supplemental Table 2 and include Vascular Coronal, Vascular Axial, Leakage Evans Blue and Local Fractal Dimension (coronal and axial combined) and were utilized in subsequent analysis. Modeling of temporal evolution of vascular and BBB disruption following brain injury are of importance, particularly in the context of patient management. Here we undertook trajectory analyses and coupled this to consistency measures to identify which features provide intuitive measures of biological coherence. We found that in the Evans Blue Leakage module sham mice (male or female) exhibited poor consistency values as might be expected as no CHI was induced (Fig. 7 B, top panel). In contrast, the CHI mice showed high consistency at the earlier time points with decreasing consistency at later time points consistent with acute BBB disruption after brain injury (Fig. 7 B, top panel). There were no overt sex differences. When we examined the coronal vascular module, male sham mice exhibited a stable trajectory while female mice had a more variable consistency (Fig. 7 B, bottom panel). In male CHI mice there was a progressive increase in consistency in cortical vascular features that continued over 7dpi (Fig. 7 B, bottom panel). The female CHI mice exhibited no consistency in these coronal vascular features until 1dpi that then precipitously declined by 3dpi with subsequent increased consistency by 7dpi, like that of male CHI mice (Fig. 7 B, bottom panel). These consistency measures suggest that males after injury exhibited a more consistent trajectory of either BBB leakage or coronal vascular features than female CHI mice. We now examined these multivariate relationships by employing t-distributed Stochastic Neighbor Embedding (t-SNE) to visualize these interactions. Specifically, we were interested in how injury and sex may jointly influence our highly dimensional phenotypes particularly in light of the heterogenous nature of concussion. These analyses highlighted sex and injury-specific clustering (Fig. 7 C). While there was some overlap between sham male and female mice (as would be expected) the CHI male and female mice exhibited clear separations. As noted in our vascular and Evans Blue data and in the modeling above, male and female CHI mice clearly have unique features that allow separation. Discussion Pediatric and juvenile mild traumatic brain injury (mTBI, concussions) and their subsequent pathologic sequelae are understudied. Most notably lacking is how blood-brain border (BBB) integrity is impacted by concussive injuries over time in clinic patients, as well as in rodent models. We used our CHILD mouse model utilizing a single impact at postnatal day 17 (PND17) reminiscent of pediatric concussion which includes a rotational component [ 15 ]. We describe here the distinct neurovascular and BBB trajectories in both male and female mice at hyper-acute and acute epochs after impact above the somatosensory cortex. Our key findings are: 1) weight reductions in CHILD mice, particularly males, 2) T2 values in ipsilateral cortical regions were reduced in CHILD males but increased in females, 3) hyper-acute EB extravasation in male and female CHILD mice, 4) hyper-acute decrements in vessel density that correlated with the presence of EB, but only in males but not female CHILD mice, 5) reductions in acute vessel complexity were only apparent in male CHILD mice, and 6) EB and IgG were visible adjacent to intracortical vessels in CHILD mice but not shams. We also demonstrate strong sex- and injury-specific relationships in our modeling approach. Taken together our results herein provide strong evidence for an early hyper-acute vascular and BBB disruption that is more prominent in males compared to female concussed juvenile mice. These early cerebrovascular perturbations may presage the subsequent development of long-term deficits that we have reported previously [ 19 , 27 ]. Changes in physiology and interference with brain development Pediatric and juvenile brains are vulnerable to concussion given the rapid developmental growth of the brain and neuronal connectivity. Early physiological changes after concussion in early childhood have been described (i.e. appetite changes, cognition, mood etc. (see [ 28 ]). Sex differences in either concussion rates [ 29 ], neuroimaging features [ 30 , 31 ] and acute and chronic outcomes [ 32 ] have suggested the existence of potential differences between males and females in both clinical and preclinical studies. In our juvenile model of concussion, male mice displayed a higher prevalence of transient apnea (44.44% of males) than females (24.32%), while both sexes experienced prolonged righting reflex times. The CHILD model is associated with rotational aspects at time of injury [ 15 ] and youth have been modeled to exhibit lower linear and rotational tolerance than adults [ 33 ]. Our rotational CHILD model can result in brain stem injuries, resulting in a transient disruption of the respiratory system function due to disturbance of medullary functions and temporary respiratory arrhythmia [ 34 ]. Righting reflexes in rodents are considered an alternative measurement of consciousness and alertness for determining TBI severity and prognosis [ 35 ] albeit in our study we found no overt sex differences. Thus, transient apnea and delayed righting reflex in the CHILD model represents a concussion landmark of the precipitating event compared to shams. We have previously described for the CHILD model a sex difference in recovery times which were shorter in males compared to female mice, in contrast to our current findings which had a larger number of replicates [ 14 ]. The severity of transient hypoxic events can represent a critical pathological landmark for concussion as the duration of hypoxia has been correlated to future long term cardiac dysfunctions in CHILD [ 18 ]. In the current study, reduced weight gain in CHILD mice was observed by 7dpi. While both sexes had reductions in weight gain, only male CHILD mice reported significant differences. Attenuated weight gain after repeated mild TBI has been described in rodents reflecting problems in pituitary and hypothalamic circuits, impeding normal growth and development [ 36 , 37 ]. In a rat pup model of mild TBI, decrements in weight gain were coincident with growth hormone reductions during the acute period but were elevated chronically [ 38 ]. Such hormonal changes may also underlie progressive decreases in CHILD cerebrum volumes. Therefore, disturbance of hypothalamic function after CHILD may influence a host of physiological changes both acutely and long after the initial event. Early sex-dependent vascular alterations Temporal neurovascular alterations, BBB dysfunction, vessel properties and morphology have been previously investigated in juvenile CHILD males where increased IgG staining in brain parenchyma has been described at 1dpi and resolved by 7dpi [ 15 ]. These alterations in BBB properties were associated with decreased neurovascular reactivity and decreased brain oxygenation peaking at 6hpi which then normalized [ 16 ]. These early functional vascular changes were then assessed 12 months post-concussion and CHILD male mice had increased numbers of capillary vessels [ 17 ]. Lacking from the literature is an assessment of neurovascular properties in male and female acutely after single pediatric concussion. This gap is critical considering known sex differences in youth after concussion injury [ 29 ]. We report significant reductions of pial and intracortical cerebral vessels for male CHILD mice at 1hpi. These decrements in cerebrovascular metrics were not significantly different in female CHILD mice (see Figs. 3 , 4 ). Moreover, these sex-specific differences were also confirmed in our modeling (see Fig. 7 ). The hyper-acute reductions in vessel density, vessel total length and number of junctions normalized at later timepoints. Our current findings relied on the vessel painting method with perfusion of lipophilic dye through the vascular system [ 23 ]. One consideration is that mTBI may result in vascular hypoperfusion [ 39 , 40 ] and reduced neurovascular coupling [ 16 , 41 ], as previously reported. Vasculogenic processes are initiated within acute time windows with angiogenic molecules such as vascular endothelial growth factor (VEGF) being upregulated [ 42 , 43 ]. While VEGF rapidly increases after TBI it is thought to require ~ 2 weeks for the complex molecular cascade to induce endothelial cell proliferation and migration, anastomosis, and glial cell recruitment [ 44 ]. Other vasculogenic pathways are also involved and in adult TBI activation of the Wnt/β-catenin pathway as early as 1dpi with increased Wnt5a levels at 7dpi where related to recovery of vessel density [ 45 ]. Our current study did not directly examine cerebral blood flow (CBF) at these acute time points, but T2-weighted imaging (specifically T2 relaxation) potentially sheds some additional light. We reported T2 relaxation was significantly decreased in male CHILD mice in cortical regions under or adjacent to the impact site but was significantly increased in female CHILD mice (Fig. 1 F-I). Our current interpretation of these opposite sex effects is that in males there is a putative reduction in CBF and in metabolically active tissues results in increased oxygen extraction during transit. In females these changes are less pronounced acutely, and CBF may not be impacted to the same extent as in males. We have described such a mechanism for reduced T2 relaxation previously [ 46 ]. Therefore, multiple systems may contribute to the recovery of decreased vessel density, including recovery of perfusion and vasculogenesis. Intracortical vascular complexity was also only altered in male CHlLD mice at 1hpi with no overt changes at later time points. In male CHILD mice the LFD at peak frequency was always increased (although not significantly) at each time point and normalized by 7dpi, which was not the case in female mice (Supplementary Fig. 4). While vascular complexity has not been extensively reported in TBI, we have noted in a cortical contusion injury model, initial loss of complexity that recovers with resumption of vessel density over time [ 23 ]. It is also important to highlight temporal differences between pial and intracortical vessels in our study, where we observed a longer duration of presumed recovery in intraparenchymal blood vessels (Fig. 4 ). It is well known that pial and intraparenchymal vessels present different anatomical, physiological and functional properties in healthy and diseased brain tissues [ 10 , 47 , 48 ]. These differences could suggest that in response to a mechanical injury, there is a potential greater vulnerability for intracortical compared to pial blood vessels to mechanical deformation. Our observations are strengthened by the importance of mechanical injury-induced cerebrovascular dysfunction that has been described clinically and in mouse models [ 49 , 50 ]. Sex-BBB alterations In this study, BBB properties were assessed using EB injection 1hr prior to VP. Brief and early elevations of EB extravasation were observed for both sexes in CHILD mice, although only significant in females (Fig. 2 B). These acute robust increases in EB leakage also exhibited high consistency in our trajectory modeling of both male and female CHI mice, which then declined with time post injury (Fig. 7 B). Male CHILD mice exhibited an elevated EB extravasation at 1dpi, in concordance with our previous report [ 15 ], that then declined linearly to sham levels by 7dpi (Fig. 2 C). This contrasts with the rapid decline at 1dpi of EB extravasation in female CHILD mice, further demonstrating sex differences in response to concussion and further supported in our trajectory modeling. Our histological demonstration of co-localization of EB and IgG outside of vascular structures in male CHILD mice, clearly demonstrates extravasation during the hyper-acute period after concussion. It is interesting to note that the IgG and EB staining do not consistently overlap and may be due to differences in molecular weights (EB: 0.9kDa, IgG: 150kDa). We and others have reported similar vascular IgG extravasation after concussion [ 51 ] and cortical contusion injury (CCI) [ 9 ]. Importantly, EB extravasation and VP-derived vessel metrics were significantly negatively correlated in CHILD male in both pial and intracortical assessments but not females (Figs. 3 , 4 ). Decreased vessel density strongly correlated with larger EB extravasation. These results further support in male CHILD mice an acute BBB leakage, hypoperfusion and modified vessel angioarchitecture [ 16 ]. Changes in BBB after CHILD have been linked with early increased expression of the water channel aquaporin 4 (APQ4) at 1dpi with late developing astrogliosis (7dpi) (Rodriguez-Grande et al 2018). Even a mild mechanical stress on the brain produces significant vascular changes with decrease in flow and change in BBB properties, similarly to more severe TBI in pediatrics and adults [ 9 , 20 , 45 , 52 , 53 ]. The exact mechanism(s) behind these perturbations are still poorly understood and the pathophysiological vascular response differs over time. Mechanical forces due to linear and rotational acceleration can directly damage endothelial cell structures compromising junctional proteins that transition to BBB dysfunction and subsequent vasogenic edema [ 54 – 56 ]. Local changes in water homeostasis may compress nearby vessels and the presumed loss of perfusion observed in our results. Concurrently, endothelial cells in response to injury also release vasoconstrictors such as endothelin-1 (ET-1) [ 57 , 58 ], further promoting vasoconstriction and altering vascular tone. Together, edema and ET-1-dependent vasoconstrictor activity could reduce CBF and may limit dye perfusion, consistent with the reduction observed in vessel painting and larger EB extravasation. These changes are resolved by 7dpi. The molecular mechanisms involved in resolution of the BBB dysfunction post-injury are very poorly understood and understudied [ 7 ]. There are numerous putative proteins involved in BBB leakage, including caveolin-1 [ 59 ], which is expressed in the neurovascular unit and has been proposed as a key pathway in BBB recovery in juvenile moderate TBI [ 60 ]. Caveolin-1 expression after brain injury follows a similar timeline of the BBB changes and recovery in CHILD [ 60 ]. Other phenomena such as tight junction (TJ) loss after TBI in adult rodents leads to BBB loss of function acutely with subsequent restoration and reduced inflammatory activities [ 61 ]. Compromised BBB function allows entry of peripheral serum proteins (IgG), immune cells, and potential pathogens into the brain parenchyma, activating brain resident immune responses [ 62 ]. The heightened immune response, leading to microgliosis and astrogliosis, can further promote endothelial dysfunction. Thus, despite recovery from transient BBB permeability, the progression of secondary sequalae and the potential for chronic inflammation can lead to a compromised neurovascular unit [ 13 ]. The early neurovascular dysfunctions after CHILD may represent a critical event that leads to long-term neurovascular dysfunction and inflammatory sequelae observed in the CHILD mouse model of pediatric concussion [ 17 – 19 , 63 ]. Sex differences following pediatric concussion A recent literature review found that 44% of animal TBI studies reported that females had better outcomes than males, while only 28% of the studies surveyed showed no sex differences [ 64 ]. Despite increasing studies using both males and females there is still a considerable gap about how sex modulates outcomes in pediatric concussion. It has been hypothesized that estrogen and progesterone play vascular protective roles in females by upregulating vasodilatory factors and reducing vasoconstrictive factors [ 64 , 65 ], thereby improving microcirculation and vessel reactivity. Compared to males, females present with a lower prevalence of cardiovascular disease [ 66 ] and improved CBF after TBI [ 39 , 67 ]. Adult female mice in a CCI model of TBI found acute (1dpi) increased astrogliosis and heme-oxygenase-1 expression (via estrogen) whereas male TBI mice had increased neovascularization via β-catenin [ 9 ]. However, sex hormones alone may not fully account for the differential vascular function, as sex differences in microvascular blood flow have been reported during infancy and adolescence [ 57 , 67 , 68 ]. Another potential contributor to sex differences in prepubertal juveniles is the differential expression of the predominant vasoconstrictor ET-1 in pediatric TBI, which is upregulated in males, but not in females [ 57 ]. The upregulation of ET-1 in males increases their susceptibility to cerebral autoregulatory impairments, leading to vasoconstriction and reduced perfusion. This potentially provides one mechanism for the reduced T2 values and reduction in vessel metrics we report here. Contrary to the males, the reduction of ET-1in females may be protective, thus explaining the lack of vasculature abnormalities despite pronounced BBB disruptions. Indeed, our trajectory and consistency modeling lends additional support related to strong sex differences, even at this early age. Our current study further reveals that sex differences in the vascular response to trauma are present and should incentivize further exploration into mechanisms underlying sex differences in juvenile TBI. Trajectory Modeling of Traumatic Brain Injury (TBI) Nascent studies are now being to address the heterogenous nature of TBI recovery between groups and individuals, primarily focused on physiological, psychological and functional outcomes. Ren and colleagues, using trajectory analyses for emotive symptoms in a relatively small cohort of subjects, were able to discriminate between depression, anxiety and life satisfaction as well as within groups (low vs. high symptomology) [ 69 ]. Similarly functional motor and cognitive scores were able to differentiate recovery patterns in subjects with moderate to severe TBI [ 70 ]. A more comprehensive Track-TBI study with 2100 participants was able to identify seven unique trajectories based on recovery by using Glasgow Outcome Scale Extended (GOSE) [ 71 ]. These trajectories were also strongly associated with initial presentation of the GOS, computed tomography findings and psychiatric comorbidities. Other measures after TBI, such as patterns of sympathetic hyperactivity [ 72 ], executive function [ 73 ] and quality of life measures [ 74 ] also aggregate into distinct trajectories. To our knowledge, there are no clinical or preclinical studies examining trajectories related to vascular impairments and longer-term outcomes. While our study was confined to 7dpi, we demonstrate that trajectory modeling may have enhanced utility in discriminating between sex and injury subtypes bases on vascular and BBB leakage features. We suggest that future longitudinal studies utilize trajectory modeling to enhance our understanding of physiological and psychological outcomes after TBI. Limitations and future directions There are several limitations and future directions related to our findings. Firstly, all the morphological and BBB leakage studies required sacrifice of the mice at discrete epochs. Continuous monitoring from the same animal for neurovascular function and BBB leakage using other techniques, such as MRI, would have provided additional temporal evolution insights. As noted above, the VP method can map the cerebrovascular networks, but only if they are being perfused. Correlation with other histological techniques (ie tomato lectin, etc.) could be applied. We also did not make any effort to disentangle the contribution of arteries and veins in concussion. Finally, there are number proteins responsible for modulating the BBB in health and disease, such as caveolins, claudins and others that likely play an important role not only in the acute injury phase but also during recovery; we did not explicitly assess those in our study. Future studies using imaging approaches such as MRI, in vivo confocal microscopy, miniscopes (as we have done [ 49 ]) or functional ultrasound imaging (fUSI) could and should be used to differentiate direct perfusion changes, particularly as they relate to early in life putative sex differences. Additional replicates or similar data from other published research could strengthen the impact of trajectory modeling. Conclusion The novelty of our research is two-fold: 1) hyper-acute sex differences in a clinically translatable model of juvenile mTBI (CHILD) that exhibits acute, chronic and long-term (12 months) pathophysiology [ 17 , 19 , 63 ], and 2) hyper-acute vessel changes that strongly correlate with BBB leakage in a sex-dependent manner. Our findings demonstrate that CHILD induces an early acute neurovascular change with loss of vascular density and BBB dysfunction transiently, recovering by 7dpi only in males and not females. We posit that these hyper-acute changes within the neurovascular unit primes the injured brain to contribute to the long-term cellular, molecular, neurovascular and behavioral perturbations we, and others have observed at 12–18 months post-concussion [ 17 , 19 , 63 ]. The presence of sex differences during the pre-pubertal period highlights the importance of incorporating biological sex as a determinant of injury response in future mechanistic and therapeutic pediatric mTBI studies. Abbreviations AQP4 : Aquaporin 4 BBB : Blood-brain border CCI: Cortical contusion Injury CHI: Closed head injury CHILD: Closed head injury with long term disorders DiO : 3,3’-dioctadecyloxacarbocyanine dpi : Days post-injury EB: Evans blue ET-1: Endothelin-1 hpi : Hours post-injury IHC: Immunohistochemistry LFD: Local fractal dimension MMP: Matrix metalloproteinase MRI : Magnetic resonance imaging ms: milliseconds mTBI : Mild traumatic brain injury PBS: Phosphate-buffered saline PCA: Principal component analysis PFA: Paraformaldehyde PND: Postnatal day ROI: Region of interest ROS : Reactive oxygen species SWI : Susceptibility-weighted imaging T2: T2-weighted TBI: Traumatic Brain injury TJ: Tight junction t-SNE: t-distributed Stochastic Neighbor Embedding VEGF: Vascular endothelial growth factor Declarations Data Availability : All protocols and data supporting the findings are available upon request to the corresponding authors. Competing Interests : The authors report no competing interests. Funding Sources : The data for this study were funded by NINDS 1R01NS119605 (Andre Obenaus, Jerome Badaut) and NINDS 1RF1NS138032 (Andre Obenaus, Paul Territo) Author Contributions : Animal experiments were performed by JY and AJ. Fluorescent images were acquired by JY and analyzed by JY, NN, JA and RP. MRI analysis was performed by JY and TG. Immunohistochemistry was performed by GC and JY. JY performed data analysis and AO reviewed all data. JY wrote the draft and AO, JB critically revised the manuscript. AO, JY, AG performed all the modeling analysis and related text. JB, AO, JY and AJ contributed to experimental design, data analysis, and manuscript revisions. JY is the leading author. Ethics Approval : All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of University of California, Irvine, and University of California, Riverside. Acknowledgements : The authors would like to acknowledge Dr. Shunshan Li for assistance with MRI acquisition, Brenda Noarbe for advice on MRI analysis, and Tia Ketsan and Safa Hamid for data analysis. Use of Language Models: In the preparation of this manuscript, the authors did not use any generative artificial intelligence tools. At the completion of the final draft, Grammarly (Grammarly Inc., San Francisco, CA, USA) was used to improve the clarity, grammar, and readability of the text. The authors carefully reviewed, revised, and take full responsibility for the content of the manuscript. References Suskauer SJ, Houtrow AJ. Invited Commentary on The Report to Congress on the Management of Traumatic Brain Injury in Children. Arch Phys Med Rehabil. 2018;99(11):2389–91. Araki T, Yokota H, Morita A. Pediatric Traumatic Brain Injury: Characteristic Features, Diagnosis, and Management. Neurol Med Chir (Tokyo). 2017;57(2):82–93. Nwafor DC, Brichacek AL, Foster CH, Lucke-Wold BP, Ali A, Colantonio MA, et al. Pediatric Traumatic Brain Injury: An Update on Preclinical Models, Clinical Biomarkers, and the Implications of Cerebrovascular Dysfunction. J Cent Nerv Syst Dis. 2022;14:11795735221098125. Figaji AA. Anatomical and Physiological Differences between Children and Adults Relevant to Traumatic Brain Injury and the Implications for Clinical Assessment and Care. Front Neurol. 2017;8:685. Lumba-Brown A, Yeates KO, Sarmiento K, Breiding MJ, Haegerich TM, Gioia GA, et al. Centers for Disease Control and Prevention Guideline on the Diagnosis and Management of Mild Traumatic Brain Injury Among Children. JAMA Pediatr. 2018;172(11):e182853. Esterov D, Witkowski J, McCall DM, Weaver AL, Brown AW. Long-Term Risk for Mood and Anxiety Disorders After Pediatric Traumatic Brain Injury: A Population-Based, Birth Cohort Analysis. J Head Trauma Rehabil. 2023;38(3):E212–22. Badaut J, Ghersi-Egea JF, Thorne RG, Konsman JP. Blood-brain borders: a proposal to address limitations of historical blood-brain barrier terminology. Fluids Barriers CNS. 2024;21(1):3. Patabendige A, Janigro D. The role of the blood-brain barrier during neurological disease and infection. Biochem Soc Trans. 2023;51(2):613–26. Jullienne A, Salehi A, Affeldt B, Baghchechi M, Haddad E, Avitua A, et al. Male and Female Mice Exhibit Divergent Responses of the Cortical Vasculature to Traumatic Brain Injury. J Neurotrauma. 2018;35(14):1646–58. Fullerton JL, Hay J, Bryant-Craig C, Atkinson J, Smith DH, Stewart W. Pediatric Traumatic Brain Injury and Microvascular Blood-Brain Barrier Pathology. JAMA Netw Open. 2024;7(11):e2446767. Green TRF, Nguyen T, Dunker V, Ashton D, Ortiz JB, Murphy SM, et al. Blood-Brain Barrier Dysfunction Predicts Microglial Activation After Traumatic Brain Injury in Juvenile Rats. Neurotrauma Rep. 2024;5(1):95–116. Hay JR, Johnson VE, Young AM, Smith DH, Stewart W. Blood-Brain Barrier Disruption Is an Early Event That May Persist for Many Years After Traumatic Brain Injury in Humans. J Neuropathol Exp Neurol. 2015;74(12):1147–57. van Vliet EA, Ndode-Ekane XE, Lehto LJ, Gorter JA, Andrade P, Aronica E, et al. Long-lasting blood-brain barrier dysfunction and neuroinflammation after traumatic brain injury. Neurobiol Dis. 2020;145:105080. Dubois CJ, Yan J, Obenaus A, Badaut J. A Pediatric Concussion Model in Mice: Closed Head Injury with Long-Term Disorders (CHILD). J Vis Exp. 2025(216). Rodriguez-Grande B, Obenaus A, Ichkova A, Aussudre J, Bessy T, Barse E, et al. Gliovascular changes precede white matter damage and long-term disorders in juvenile mild closed head injury. Glia. 2018;66(8):1663–77. Ichkova A, Rodriguez-Grande B, Zub E, Saudi A, Fournier ML, Aussudre J, et al. Early cerebrovascular and long-term neurological modifications ensue following juvenile mild traumatic brain injury in male mice. Neurobiol Dis. 2020;141:104952. Obenaus A, Rodriguez-Grande B, Lee JB, Dubois CJ, Fournier ML, Cador M, et al. A single mild juvenile TBI in male mice leads to regional brain tissue abnormalities at 12 months of age that correlate with cognitive impairment at the middle age. Acta Neuropathol Commun. 2023;11(1):32. Leyba K, Paiyabhroma N, Salvas JP, Damen FW, Janvier A, Zub E, et al. Neurovascular hypoxia after mild traumatic brain injury in juvenile mice correlates with heart-brain dysfunctions in adulthood. Acta Physiol (Oxf). 2023;238(2):e13933. Obenaus A, Noarbe BP, Lee JB, Panchenko PE, Tong F, Noarbe SD, et al. Progressive lifespan modifications in the corpus callosum following a single concussion in juvenile male mice monitored by diffusion MRI. Exp Neurol. 2025;394:115455. Obenaus A, Ng M, Orantes AM, Kinney-Lang E, Rashid F, Hamer M, et al. Traumatic brain injury results in acute rarefication of the vascular network. Sci Rep. 2017;7(1):239. Zudaire E, Gambardella L, Kurcz C, Vermeren S. A computational tool for quantitative analysis of vascular networks. PLoS ONE. 2011;6(11):e27385. Karperien A, Ahammer H, Jelinek HF. Quantitating the subtleties of microglial morphology with fractal analysis. Front Cell Neurosci. 2013;7:3. Salehi A, Jullienne A, Wendel KM, Hamer M, Tang J, Zhang JH et al. A Novel Technique for Visualizing and Analyzing the Cerebral Vasculature in Rodents. Transl Stroke Res. 2018. Yushkevich PA, Piven J, Hazlett HC, Smith RG, Ho S, Gee JC, et al. User-guided 3D active contour segmentation of anatomical structures: significantly improved efficiency and reliability. NeuroImage. 2006;31(3):1116–28. Watson C, Janke AL, Hamalainen C, Bagheri SM, Paxinos G, Reutens DC, et al. An ontologically consistent MRI-based atlas of the mouse diencephalon. NeuroImage. 2017;157:275–87. Nespoli E, Hakani M, Hein TM, May SN, Danzer K, Wirth T, et al. Glial cells react to closed head injury in a distinct and spatiotemporally orchestrated manner. Sci Rep. 2024;14(1):2441. Obenaus A, Rodriguez-Grande B, Lee JB, Dubois CJ, Fournier M-L, Cador M, et al. A single mild juvenile TBI in male mice leads to regional brain tissue abnormalities at 12 months of age that correlate with cognitive impairment at the middle age. Acta Neuropathol Commun. 2023;11(1):32. Beauchamp MH, Anderson V, Ewing-Cobbs L, Haarbauer-Krupa J, McKinlay A, Wade SL et al. Early Child Concussion Pediatr. 2024;154(5). Arambula SE, Reinl EL, El Demerdash N, McCarthy MM, Robertson CL. Sex differences in pediatric traumatic brain injury. Exp Neurol. 2019;317:168–79. Koerte IK, Schultz V, Sydnor VJ, Howell DR, Guenette JP, Dennis E, et al. Sex-Related Differences in the Effects of Sports-Related Concussion: A Review. J Neuroimaging. 2020;30(4):387–409. Sharma B, Nowikow C, DeMatteo C, Noseworthy MD, Timmons BW. Sex-specific differences in resting-state functional brain activity in pediatric concussion. Sci Rep. 2023;13(1):3284. Yeates TM, Taylor HG, Bigler ED, Minich NM, Tang K, Cohen DM, et al. Sex Differences in the Outcomes of Mild Traumatic Brain Injury in Children Presenting to the Emergency Department. J Neurotrauma. 2022;39(1–2):93–101. Campolettano ET, Gellner RA, Smith EP, Bellamkonda S, Tierney CT, Crisco JJ, et al. Development of a Concussion Risk Function for a Youth Population Using Head Linear and Rotational Acceleration. Ann Biomed Eng. 2020;48(1):92–103. Corsellis JA, Bruton CJ, Freeman-Browne D. The aftermath of boxing. Psychol Med. 1973;3(3):270–303. Jain S, Margetis K, Iverson LM. Glasgow Coma Scale. StatPearls. Treasure Island (FL)2025. Greco T, Hovda D, Prins M. The effects of repeat traumatic brain injury on the pituitary in adolescent rats. J Neurotrauma. 2013;30(23):1983–90. Greco T, Hovda DA, Prins ML. Adolescent TBI-induced hypopituitarism causes sexual dysfunction in adult male rats. Dev Neurobiol. 2015;75(2):193–202. Ortiz JB, Tellez S, Rampal G, Mannino GS, Couillard N, Mendez M et al. Diffuse traumatic brain injury substantially alters plasma growth hormone in the juvenile rat. J Endocrinol. 2024;260(1). Bai G, Bai L, Cao J, Sun C, Wang S, Yin B, et al. Sex differences in cerebral perfusion changes after mild traumatic brain injury: Longitudinal investigation and correlation with outcome. Brain Res. 2019;1708:93–9. Hayward NM, Tuunanen PI, Immonen R, Ndode-Ekane XE, Pitkanen A, Grohn O. Magnetic resonance imaging of regional hemodynamic and cerebrovascular recovery after lateral fluid-percussion brain injury in rats. J Cereb Blood Flow Metab. 2011;31(1):166–77. Chen SF, Richards HK, Smielewski P, Johnstrom P, Salvador R, Pickard JD, et al. Relationship between flow-metabolism uncoupling and evolving axonal injury after experimental traumatic brain injury. J Cereb Blood Flow Metab. 2004;24(9):1025–36. Nag S, Takahashi JL, Kilty DW. Role of vascular endothelial growth factor in blood-brain barrier breakdown and angiogenesis in brain trauma. J Neuropathol Exp Neurol. 1997;56(8):912–21. Shore PM, Jackson EK, Wisniewski SR, Clark RS, Adelson PD, Kochanek PM. Vascular endothelial growth factor is increased in cerebrospinal fluid after traumatic brain injury in infants and children. Neurosurgery. 2004;54(3):605–11. discussion 11 – 2. Zhao ZA, Yan L, Wen J, Satyanarayanan SK, Yu F, Lu J, et al. Cellular and molecular mechanisms in vascular repair after traumatic brain injury: a narrative review. Burns Trauma. 2023;11:tkad033. Salehi A, Jullienne A, Baghchechi M, Hamer M, Walsworth M, Donovan V, et al. Up-regulation of Wnt/beta-catenin expression is accompanied with vascular repair after traumatic brain injury. J Cereb Blood Flow Metab. 2018;38(2):274–89. Choy M, Dube CM, Patterson K, Barnes SR, Maras P, Blood AB, et al. A novel, noninvasive, predictive epilepsy biomarker with clinical potential. J neuroscience: official J Soc Neurosci. 2014;34(26):8672–84. Duvernoy HM, Delon S, Vannson JL. Cortical blood vessels of the human brain. Brain Res Bull. 1981;7(5):519–79. Zhang JH, Badaut J, Tang J, Obenaus A, Hartman R, Pearce WJ. The vascular neural network–a new paradigm in stroke pathophysiology. Nat Rev Neurol. 2012;8(12):711–6. Lin X, Chen L, Jullienne A, Zhang H, Salehi A, Hamer M, et al. Longitudinal dynamics of microvascular recovery after acquired cortical injury. Acta Neuropathol Commun. 2022;10(1):59. Griffin AD, Turtzo LC, Parikh GY, Tolpygo A, Lodato Z, Moses AD, et al. Traumatic microbleeds suggest vascular injury and predict disability in traumatic brain injury. Brain. 2019;142(11):3550–64. Hiles-Murison B, Lavender AP, Hackett MJ, Armstrong JJ, Nesbit M, Rawlings S, et al. Blood-brain barrier disruption and ventricular enlargement are the earliest neuropathological changes in rats with repeated sub-concussive impacts over 2 weeks. Sci Rep. 2021;11(1):9261. Fukuda AM, Adami A, Pop V, Bellone JA, Coats JS, Hartman RE, et al. Posttraumatic reduction of edema with aquaporin-4 RNA interference improves acute and chronic functional recovery. J Cereb Blood Flow Metab. 2013;33(10):1621–32. Iliff JJ, Chen MJ, Plog BA, Zeppenfeld DM, Soltero M, Yang L, et al. Impairment of glymphatic pathway function promotes tau pathology after traumatic brain injury. J Neurosci. 2014;34(49):16180–93. Salehi A, Zhang JH, Obenaus A. Response of the cerebral vasculature following traumatic brain injury. J Cereb Blood Flow Metab. 2017;37(7):2320–39. Ichkova A, Rodriguez-Grande B, Bar C, Villega F, Konsman JP, Badaut J. Vascular impairment as a pathological mechanism underlying long-lasting cognitive dysfunction after pediatric traumatic brain injury. Neurochem Int. 2017;111:93–102. Rodriguez-Grande B, Ichkova A, Lemarchant S, Badaut J. Early to Long-Term Alterations of CNS Barriers After Traumatic Brain Injury: Considerations for Drug Development. AAPS J. 2017;19(6):1615–25. Armstead WM, Riley J, Vavilala MS. TBI sex dependently upregulates ET-1 to impair autoregulation, which is aggravated by phenylephrine in males but is abrogated in females. J Neurotrauma. 2012;29(7):1483–90. Salonia R, Empey PE, Poloyac SM, Wisniewski SR, Klamerus M, Ozawa H, et al. Endothelin-1 is increased in cerebrospinal fluid and associated with unfavorable outcomes in children after severe traumatic brain injury. J Neurotrauma. 2010;27(10):1819–25. Badaut J, Blochet C, Obenaus A, Hirt L. Physiological and pathological roles of caveolins in the central nervous system. Trends Neurosci. 2024;47(8):651–64. Badaut J, Ajao DO, Sorensen DW, Fukuda AM, Pellerin L. Caveolin expression changes in the neurovascular unit after juvenile traumatic brain injury: signs of blood-brain barrier healing? Neuroscience. 2015;285:215–26. Tang J, Kang Y, Zhou Y, Shang N, Li X, Wang H et al. TIMP2 ameliorates blood-brain barrier disruption in traumatic brain injury by inhibiting Src-dependent VE-cadherin internalization. J Clin Invest. 2023;134(3). Corrigan F, Mander KA, Leonard AV, Vink R. Neurogenic inflammation after traumatic brain injury and its potentiation of classical inflammation. J Neuroinflammation. 2016;13(1):264. Badaut J, Hippauf L, Malinconi M, Noarbe BP, Obenaus A, Dubois CJ. Endocannabinoid-mediated rescue of somatosensory cortex activity, plasticity and related behaviors following an early in life concussion. bioRxiv. 2024. Gupte R, Brooks W, Vukas R, Pierce J, Harris J. Sex Differences in Traumatic Brain Injury: What We Know and What We Should Know. J Neurotrauma. 2019;36(22):3063–91. Xing D, Nozell S, Chen YF, Hage F, Oparil S. Estrogen and mechanisms of vascular protection. Arterioscler Thromb Vasc Biol. 2009;29(3):289–95. Betai D, Ahmed AS, Saxena P, Rashid H, Patel H, Shahzadi A, et al. Gender Disparities in Cardiovascular Disease and Their Management: A Review. Cureus. 2024;16(5):e59663. Armstead WM, Kiessling JW, Riley J, Kofke WA, Vavilala MS. Phenylephrine infusion prevents impairment of ATP- and calcium-sensitive potassium channel-mediated cerebrovasodilation after brain injury in female, but aggravates impairment in male, piglets through modulation of ERK MAPK upregulation. J Neurotrauma. 2011;28(1):105–11. Stark MJ, Clifton VL, Wright IM. Sex-specific differences in peripheral microvascular blood flow in preterm infants. Pediatr Res. 2008;63(4):415–9. Ren D, Fan J, Puccio AM, Okonkwo DO, Beers SR, Conley Y. Group-Based Trajectory Analysis of Emotional Symptoms Among Survivors After Severe Traumatic Brain Injury. J Head Trauma Rehabil. 2017;32(6):E29–37. Lu J, Roe C, Sigurdardottir S, Andelic N, Forslund M. Trajectory of Functional Independent Measurements during First Five Years after Moderate and Severe Traumatic Brain Injury. J Neurotrauma. 2018;35(14):1596–603. Curpen PP, To XV, Lu M, Winter C, Bellapart J, Newcombe VF et al. Trajectories of Glasgow Outcome Scale-Extended after traumatic brain injury: an analysis of the TRACK-TBI cohort. J Neurol Neurosurg Psychiatry. 2025. Chowdhury SH, Chen LK, Hu P, Badjatia N, Podell JE. Group-Based Trajectory Modeling Identifies Distinct Patterns of Sympathetic Hyperactivity Following Traumatic Brain Injury. Neurocrit Care. 2025;42(3):985–95. Keenan HT, Clark AE, Holubkov R, Cox CS Jr., Ewing-Cobbs L. Trajectories of Children's Executive Function After Traumatic Brain Injury. JAMA Netw Open. 2021;4(3):e212624. Cairns K, Beaulieu-Bonneau S, Jomphe V, Lamontagne ME, de Guise E, Moore L, et al. Four-Year Trajectories of Symptoms and Quality of Life in Individuals Hospitalized After Mild Traumatic Brain Injury. Arch Phys Med Rehabil. 2025;106(3):358–65. Additional Declarations No competing interests reported. Supplementary Files Cav1CHIBBBSupplementalFigures011926ao.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8622019","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":585053943,"identity":"85a02868-bb74-4739-a7de-5e78252edd64","order_by":0,"name":"Jiamin Yan","email":"","orcid":"","institution":"University of California, Riverside","correspondingAuthor":false,"prefix":"","firstName":"Jiamin","middleName":"","lastName":"Yan","suffix":""},{"id":585053944,"identity":"8ca37323-23a8-4184-bdbd-d7d997ab8130","order_by":1,"name":"Nathan Nguyen","email":"","orcid":"","institution":"University of California, Riverside","correspondingAuthor":false,"prefix":"","firstName":"Nathan","middleName":"","lastName":"Nguyen","suffix":""},{"id":585053945,"identity":"f895cbe7-9f15-4a7c-b7fb-c0f7bf4f8a36","order_by":2,"name":"Terese Garcia","email":"","orcid":"","institution":"University of California, Riverside","correspondingAuthor":false,"prefix":"","firstName":"Terese","middleName":"","lastName":"Garcia","suffix":""},{"id":585053946,"identity":"da0226c0-d00c-40c8-bdc5-84811ce1392e","order_by":3,"name":"Adam Godzik","email":"","orcid":"","institution":"University of California, Riverside","correspondingAuthor":false,"prefix":"","firstName":"Adam","middleName":"","lastName":"Godzik","suffix":""},{"id":585053947,"identity":"ef0a45a9-4b90-4458-b2a3-410bf01402ae","order_by":4,"name":"Greer Cisneros","email":"","orcid":"","institution":"University of California, Riverside","correspondingAuthor":false,"prefix":"","firstName":"Greer","middleName":"","lastName":"Cisneros","suffix":""},{"id":585053948,"identity":"34ca01fb-b416-43cf-aeea-94669721fb54","order_by":5,"name":"Amandine Jullienne","email":"","orcid":"","institution":"University of California, Riverside","correspondingAuthor":false,"prefix":"","firstName":"Amandine","middleName":"","lastName":"Jullienne","suffix":""},{"id":585053949,"identity":"b4db2aea-9e18-4929-814b-38028087baa3","order_by":6,"name":"Junuen Alvarado","email":"","orcid":"","institution":"University of California, Riverside","correspondingAuthor":false,"prefix":"","firstName":"Junuen","middleName":"","lastName":"Alvarado","suffix":""},{"id":585053950,"identity":"f68b750f-964c-4796-908a-d6c69d5e8429","order_by":7,"name":"Rojina Pad","email":"","orcid":"","institution":"University of California, Riverside","correspondingAuthor":false,"prefix":"","firstName":"Rojina","middleName":"","lastName":"Pad","suffix":""},{"id":585053951,"identity":"86752be4-d754-4303-9e62-089618bf8cee","order_by":8,"name":"Jerome Badaut","email":"","orcid":"","institution":"Centre d’Études Biologiques de Chizé","correspondingAuthor":false,"prefix":"","firstName":"Jerome","middleName":"","lastName":"Badaut","suffix":""},{"id":585053952,"identity":"8ecd4dc1-7292-4cfb-9cf0-c565f6303c81","order_by":9,"name":"Andre Obenaus","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7ElEQVRIiWNgGAWjYFACHgjFD8QSDAwHoKJsRGiRbCBZi8EBYrXwt/cefFxQc9je+Nrhgzd/1NxhMDh+9gHDh7LDOLVInDmXbDzj2OHEbbfTkq15jj1jMDiTbsA44xxuLQw3csykedgOJ5jdBjIYGw4zGNxgY2DmbcOtRf5Gjvlvnn9Ah83O/yb5E6blLx4tBkBbQGYybpDOYZPghWlhxKPFEOgXad6+9MQZt9OMgX45zCN5Jo3hYM+5dJxa5I73HvzM883ann928kNgiB2W4zt+jPHBjzJr3N5HB+BoOkC8+lEwCkbBKBgF2AAAxylXOhZMUKoAAAAASUVORK5CYII=","orcid":"","institution":"University of California, Riverside","correspondingAuthor":true,"prefix":"","firstName":"Andre","middleName":"","lastName":"Obenaus","suffix":""}],"badges":[],"createdAt":"2026-01-16 20:23:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8622019/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8622019/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102438328,"identity":"918cb5bb-cbf6-45fd-8da4-125dc61789af","added_by":"auto","created_at":"2026-02-11 16:32:07","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":86499,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSex differences emerge in temporal characterization of CHILD. \u0026nbsp;(A) \u003c/strong\u003eSchematic of experimental design and timeline. \u003cstrong\u003e(B)\u003c/strong\u003e Body weight relative to pre-CHI weights were not different at 1dpi but were significantly reduced at 7dpi compared to shams. \u0026nbsp;(*p ≤0.05, unpaired t-test).\u003cstrong\u003e(C)\u003c/strong\u003e Pie charts report that 44.44% of males had apnea compared to 24.32% of females after CHILD induction. \u003cstrong\u003e(D)\u003c/strong\u003e Time to right was significantly increased after CHILD compared to shams (****p≤0.0001, unpaired t-test). \u003cstrong\u003e(E)\u003c/strong\u003eRepresentative MRI T2-weighted images (T2WI) and T2 maps of sham and CHILD brains at 1dpi (*denotes the injury). \u003cstrong\u003e(F-I)\u003c/strong\u003e Ipsilateral T2 relaxation times (ms) were used to assess edema. \u003cstrong\u003e(F)\u003c/strong\u003e T2 relaxation times in the motor cortex were significantly increased in females (**p≤0.01, unpaired t-test) but not in males relative to shams. \u003cstrong\u003e(G)\u003c/strong\u003e T2 values in parietal cortex were significantly increased in both males and females (*p≤0.05, unpaired t-test). \u003cstrong\u003e(H)\u003c/strong\u003eT2 relaxation (ms) did not differ significantly but were increased compared to shams in the ipsilateral somatosensory cortex from males and females. (\u003cstrong\u003eI)\u003c/strong\u003eT2 in the ipsilateral corpus callosum (CC) was significantly increased in females (**p≤0.01, unpaired t-test) but not in males \u003cstrong\u003e(J)\u003c/strong\u003e. \u0026nbsp;All Bar graphs were represented as mean ±SD.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8622019/v1/02bb1255d338a1cc9dc1ea0a.jpg"},{"id":102438319,"identity":"751e262b-fde3-47af-a10c-4155ae73e698","added_by":"auto","created_at":"2026-02-11 16:32:06","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":80434,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBBB disruption is most evident at hyper-acute periods following CHILD. (A)\u003c/strong\u003e Representative 2X images of Evans Blue (EB) fluorescence from axial and coronal aspects of sham and CHILD brains (left) at 1hpi. White dotted outlines indicate the quantification regions.10X images of representative blood vessels from the axial surface of sham mice which exhibit no vascular leakage compared to CHI mice whose vessels exhibit EB extravasation. \u003cstrong\u003e(B)\u003c/strong\u003eCoronal EB integrated intensity from the lesion site on the ipsilateral cortex in males and females were elevated at 1hpi (significance in females p\u0026lt;0.05, one-way ANOVA) that slowly declined over the 7dpi timepoints. \u003cstrong\u003e(C)\u003c/strong\u003e Integrated density values across the experimental period were curve-fitted using a two-phase exponential decay model and revealed more linear leakage over time in males, while females exhibited a rapid exponential decrease in EB extravasation after a single CHI. \u003cstrong\u003e(D)\u003c/strong\u003e EB extravasation area was measured from the brain surface but while elevated no significant differences were observed between time points nor sex. \u003cstrong\u003e(E)\u003c/strong\u003e The percentage of male CHILD mice that exhibited BBB at each time point was elevated over the first 24hrs but then rapidly declined. Female mice showed a higher percentage of mice with EB leakage that was relatively sustained over the 7dpi experimental period. All bar graphs were represented as mean ±SD\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8622019/v1/f5864199f7c3c5b972471f1a.jpg"},{"id":102438321,"identity":"f2028e62-ee64-4588-a30a-d482e9fa4596","added_by":"auto","created_at":"2026-02-11 16:32:07","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":145354,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAcute vessel perturbations correlate with severity of Blood-Brain-Border (BBB) disruptions. (A)\u003c/strong\u003e Representative axial and EB extravasation (magenta), cerebrovasculature (green), merged EB and VP, and vessel analysis maps illustrating junctions (blue dots) and vessels (red lines) in representative sham and CHILD mice. White dotted outlines denote regions of EB extravasation and axial surface analyzed. \u003cstrong\u003e(B)\u003c/strong\u003e Vessel density in CHILD males was significantly reduced at 1hpi compared to shams (*p≤0.05, one-way ANOVA). \u003cstrong\u003e(C) \u003c/strong\u003eNo significant differences in vessel density were observed in females. \u003cstrong\u003e(D)\u003c/strong\u003e In males, EB extravasation area was significantly correlated with vessel density across all time points (Pearson’s r= -0.5781, *p≤0.05) but not in CHILD females. \u003cstrong\u003e(E-F) \u003c/strong\u003eTotal vessel length in both CHILD males \u003cstrong\u003e(E) \u003c/strong\u003eand females \u003cstrong\u003e(F) \u003c/strong\u003edid not differ significantly from shams and yielded no significant correlations EB extravasation area and total vessel length \u003cstrong\u003e(G)\u003c/strong\u003e. \u003cstrong\u003e(H)\u003c/strong\u003e Junction density at 1hpi exhibited a trending reduction in CHILD males compared to shams (p=0.1009, one-way ANOVA). \u003cstrong\u003e\u0026nbsp;(I)\u003c/strong\u003e No significant alterations in junctions were reported in females. \u003cstrong\u003e(J)\u003c/strong\u003e In CHILD males, EB extravasation area was significantly correlated with junction density (Pearson’s r= -0.5723, *p≤0.05) but not in CHILD female mice. All bar graphs were represented as mean ±SD\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8622019/v1/33249b559cd6b579c9cdf6bd.jpg"},{"id":102438323,"identity":"9d1fce09-fa6d-4c95-aa24-3cfecc3c239f","added_by":"auto","created_at":"2026-02-11 16:32:07","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":159084,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCortical cerebrovascular alterations correlate with severity of BBB disruptions. (A)\u003c/strong\u003e Representative coronal images of EB extravasation (magenta), vessel painting (green), merged EB and VP images, and derived cortical vessel analysis from sham and CHILD brains. White dotted outlines denoted regions of EB extravasation and coronal VP included in the analysis. \u003cstrong\u003e(B)\u003c/strong\u003e in CHILD males exhibited significant reductions in vessel density at 1hpi compared to shams (*p≤0.05, one-way ANOVA) with a trending reduction at 6hpi (p=0.0664, one-way ANOVA). \u003cstrong\u003e(C)\u003c/strong\u003e No significant vessel density differences were observed in females. \u003cstrong\u003e(D)\u003c/strong\u003e In CHILD males, EB extravasation (integrated density) in the injured cortex (* denotes CHI site) significantly correlated with vessel density (Pearson’s r= -0.3701, *p≤0.05) but not in females. \u003cstrong\u003e(E) \u003c/strong\u003eTotal vessel length in CHILD males exhibited significant reductions at all time points (*p≤0.05, **p≤0.01, *** p≤0.001, one-way ANOVA). \u003cstrong\u003e(F)\u003c/strong\u003e No significant differences were observed in CHILD females. \u003cstrong\u003e(G)\u003c/strong\u003e In CHILD males EB extravasation at the lesion site significantly correlated with total vessel length (Pearson’s r= -0.3765, *p≤0.05) but not in females. \u003cstrong\u003e(H)\u003c/strong\u003e CHILD males reported a significant reduction in junction density at 1hpi compared to shams (*p≤0.05, one-way ANOVA). \u003cstrong\u003e(I)\u003c/strong\u003e No changes in junction density were found in females (p\u0026gt;0.05, one-way ANOVA). \u003cstrong\u003e(J)\u003c/strong\u003e EB extravasation in CHILD males significantly correlated with junction density (Pearson’s r= -0.3981 *p≤0.05) but not in females. All bar graphs were represented as mean ±SD.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8622019/v1/3084e18954e21ddbe2c70b10.jpg"},{"id":102438325,"identity":"32aeb147-b0a8-401c-b509-9d36d3c925e9","added_by":"auto","created_at":"2026-02-11 16:32:07","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":110727,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCortical vascular complexity was reduced at acute epochs post-injury.\u003c/strong\u003e \u003cstrong\u003e(A) \u003c/strong\u003eRepresentative vessel painted (VP) coronal sections from sham and CHILD mice with their corresponding fractal analysis images. White dotted outlines denote analysis regions. \u003cstrong\u003e(B)\u003c/strong\u003e Exemplar data from sham and CHILD mice for area under the curve and local fractal dimension (LFD) values at peak frequencies calculations derived from LFD histograms. CHILD resulted in a leftward shift in the average LFD histogram relative to shams, consistent with decreased vascular complexity. \u003cstrong\u003e(C) \u003c/strong\u003eArea under the LFD curve (AUC) measures for males and females at acute (1, 6hpi) reported a significant decreased AUC in CHILD males (*p≤0.05, unpaired t-test) but not in CHILD females. \u003cstrong\u003e(D)\u003c/strong\u003e LFD values at peak frequency were significantly reduced 1hpi (*p≤0.05, unpaired t-test) in males and females but not at 6hpi. (see Supplementary Figure 4 for additional time points). All bar graphs were represented as mean ±SD\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8622019/v1/44f442c7863f24ba41cdc5d5.jpg"},{"id":102438320,"identity":"a2ae451f-8e09-45bf-ad89-bbee6790ab6e","added_by":"auto","created_at":"2026-02-11 16:32:06","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":64507,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExtensive BBB disruptions are apparent in vascular reconstructions at 1hpi CHI.\u003c/strong\u003e Representative 20X confocal images of ipsilateral cortex sections stained for IgG (red), vessel painting (VP, green), and Evans Blue (EB, purple), along with the corresponding 3D reconstructions. In sham mice (left panel), both IgG and EB remain confined within the vasculature. In contrast, in CHILD mice at 1hpi (right panel), there is extensive extravasation of IgG and EB from vessels in the ipsilateral cortex, confirming perivascular leakage.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8622019/v1/1923fb034018636f5df4bd91.jpg"},{"id":102438322,"identity":"3d1c2434-bac5-4584-9e5c-7dca38ada46d","added_by":"auto","created_at":"2026-02-11 16:32:07","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":121972,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVascular features strongly cluster in CHI mice.\u003c/strong\u003e A) Clustering and heatmap representation illustrate the strong concordance between vascular features in CHI mice. B) Data reduction into feature related modules (see Supplemental Table 2) to model the temporal relationship. Consistency trajectories revealed that Evans Blue Leakage module exhibited poor consistency in sham mice while CHI mice showed high consistency at early time points. The Coronal Vascular Module sham male mice showed a stable trajectory while female sham mice were more variable. In contrast, CHI female mice showed no trajectory consistency whereas males had a relatively stable trajectory. C) To visualize the high dimensional phenotypes and their interactions in CHI and sham mice we employed t-distributed Stochastic Neighbor Embedding (t-SNE) from a set of the first principal component scores (PC1). As shown irrespective to time points, our vascular features were able to discriminate in a sex- and injury specific manner.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8622019/v1/c7cedfb883a9ebbe234f2363.jpg"},{"id":106725733,"identity":"30d16b5a-b982-4b00-b9ad-e564f02589eb","added_by":"auto","created_at":"2026-04-12 18:33:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2262044,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8622019/v1/f410d3a2-3ea6-4a21-bb55-7cae20863256.pdf"},{"id":102438324,"identity":"f45a73ce-eafe-4e0b-b1b4-8fb713825cbc","added_by":"auto","created_at":"2026-02-11 16:32:07","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":7443104,"visible":true,"origin":"","legend":"","description":"","filename":"Cav1CHIBBBSupplementalFigures011926ao.docx","url":"https://assets-eu.researchsquare.com/files/rs-8622019/v1/38603fd8175cb2f813afc9c3.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"A Single Concussion in Juvenile Mice Leads to Sex Specific Acute Cerebral Vascular Dysfunction and Blood-brain Border Dysfunction","fulltext":[{"header":"Background","content":"\u003cp\u003eTraumatic brain injury (TBI) due to external mechanical force, represents a major public health and economic burden, accounting for more than 600,000 emergency department visits each year, with 90% of all pediatric TBIs classified as mild TBI (mTBI) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Little is known about the unique features of mTBI in children, particularly considering the structural and functional differences as the developing brains transition to adulthood. Thinner and less rigid skulls may provide reduced mechanical protection, increasing susceptibility to fractures and tissue deformation [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The developing brain is characterized by immature neural networks and active processes of synapse formation and pruning, such that perturbations during these critical epochs can interfere with normative maturation [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Pediatric and juvenile brain injury places children at elevated risk for persistent learning disabilities, psychological disorders, behavioral problems, disruption of academic and social performance [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe blood-brain barrier or more recently designated as Blood-Brain Border (BBB) interface [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], is a very dynamic interface between blood and brain, composed of tight junction proteins linked endothelial cells expressing various transporters finely tuned by pericytes and astrocytic endfeet [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. BBB remodeling and vascular dysfunctions have been suggested to contribute to the long-term neurological and behavioral deficits often observed after adult TBI [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Recent work in patients with moderate to severe TBI exhibited BBB perturbations confined to microvascular regions in pediatric TBI but was predominately in larger vessels in adult patients [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Moreover, increased BBB dysfunctions in juvenile mice evoked an increased microglial response [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Yet, whether similar mechanisms occur following juvenile mTBI, and, if so, the timeline and severity of these alterations in the developing brain are underexplored.\u003c/p\u003e \u003cp\u003eMost children appear to recover from mTBI within several weeks but up to one-third experience persistent deficits [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In adults, BBB dysfunction is recognized as a central mechanism contributing to long-term dysfunction after severe TBI in humans [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] and in adult rats exposed to severe TBI [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. While moderate to severe TBI outcomes have been relatively well documented in both human subjects and in rodent models, far less is known about the sequelae following mTBI/concussion in pediatric brain injury. Indeed, the temporal course of hyperacute BBB changes in pediatric and juvenile mTBI have not been reported. This lack of mechanistic understanding impedes the development of pediatric-specific diagnostic tools, treatments, and strategies to identify children at greatest risk for chronic deficits.\u003c/p\u003e \u003cp\u003eTo address this gap, we examined BBB integrity and altered angioarchitecture in a juvenile closed head injury with long-term disorders (CHILD) model [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The CHILD model is a robust unrestrained closed head concussion model in postnatal day 17 (PND17) mice and replicates key clinical features of mTBI: a) rotational acceleration and coup\u0026ndash;contrecoup injury [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], b) behavioral alterations [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], c) acute perturbations in tissue oxygenation, neurovascular coupling and long-term cardiac dysfunction [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], and d) progressive decrements in white matter [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] (see Table\u0026nbsp;1 in reference [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]). In our study, we utilized the PND17 CHILD model and examined BBB leakage and cerebrovascular perturbations at 1h, 6h, 1-, 3- and 7-days post-injury (dpi) in a sex-specific manner. We report both temporal and sex-specific alterations in BBB and vascular responses to juvenile mTBI.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eThe experimental protocol focused on hyperacute and acute time points after mTBI as outlined in a schematic in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003ePregnant C57BL/6J female mice (E14) were purchased from Jackson Laboratory (JAX #000664). CHILD or sham procedures were performed on postnatal day 17 (PND17) pups of both sexes. Animals were randomly assigned to one of six groups (Supplemental Table\u0026nbsp;1): Sham, CHILD 1h (n\u0026thinsp;=\u0026thinsp;13), CHILD 6h (n\u0026thinsp;=\u0026thinsp;16), CHILD 1d (n\u0026thinsp;=\u0026thinsp;21), CHILD 3d (n\u0026thinsp;=\u0026thinsp;18), and CHILD 7d (n\u0026thinsp;=\u0026thinsp;19). Pups were excluded if their weight was less than 5.9g on PND 17; all pups were weaned on PND 21. Mice were maintained at 21\u003cb\u003e\u0026deg;\u003c/b\u003eC with an automated 12-hour light-dark cycle and had \u003cem\u003ead libitum\u003c/em\u003e access to water and standard vivarium chow. All experiments were in accordance with the University of California, Riverside and University of California, Irvine Institutional Animal Care and Use Committees and federal regulations and in accordance with ARRIVE guidelines as well as Animal Welfare Act and Public Health Service policies related to humane care of animals.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eClosed Head Injury with Long Term Disorders (CHILD)\u003c/h3\u003e\n\u003cp\u003eCHILD model details and videos have been published recently [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Briefly, on PND17, animals were weighed and anesthetized with 2.5% isoflurane in 1.5 L/min O\u003csub\u003e2\u003c/sub\u003e for 5 minutes in a chamber heated to 37\u0026deg;C. Each mouse was removed from the isoflurane chamber and quickly placed on a taut and secured aluminum foil (15 x 15 cm) stretched across a stereotactic frame. The mouse position was adjusted so that the impactor tip was directly above the left somatosensory cortex. The impactor tip (3mm diameter rubber tip) was mounted at a 90\u0026deg; angle perpendicular to the stereotactic apparatus. A single impact was then delivered using an electromagnetic impactor (Leica Biosystems, Deer Park, IL, USA) with the following parameters: velocity: 3m/s, dwell time: 0.1s, and depth: 3mm. The resulting injury is equivalent to Grade 2 (G2) level injury, as previously defined [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The presence of apnea and head rotation were recorded. The mouse was then immediately placed on its right side in a warmed (37\u0026deg;C) recovery chamber to assess righting time and time to resume exploratory behaviors. All animals survived the CHILD. The shams underwent identical procedures but without an impact.\u003c/p\u003e\n\u003ch3\u003eEvans Blue Injection, Vessel Painting and Tissue Fixation\u003c/h3\u003e\n\u003cp\u003eAt each time point post-CHILD, a 2% solution of Evan\u0026rsquo;s Blue (EB) (Acros Organics, Geel, Antwerpen, Belgium) in phosphate buffered saline (PBS) was administered via tail vein injection (3\u0026micro;L/g) while the animals were under light anesthesia (2% isoflurane in 1.5L/min O\u003csub\u003e2\u003c/sub\u003e). EB was allowed to circulate for 1 hour prior to vessel painting and perfusion. Mice were then anesthetized with 2.5% isoflurane in 1.5L/min oxygen and were given an intraperitoneal (i.p.) injection of Ketamine (200mg/kg) and Xylazine (200mg/kg) to induce deep general anesthesia. Mice were then given an i.p. injection of heparin (1000units/kg) followed by sodium nitroprusside (0.75mg/kg) to dilate vessels. To visualize the cerebrovasculature, we performed an intracardiac injection of 3,3\u0026rsquo;-dioctadecyloxacarbocyanine (DiO, Biotium, Fremont, CA, USA) (0.75mg/kg) diluted with 4% dextrose in PBS. Mice were then immediately intracardially perfused with 15mL PBS followed by 20mL of 4% paraformaldehyde (PFA). Brain tissues were post-fixed in 4% PFA for 24h, washed with PBS for 3 consecutive days and stored at 4\u0026deg;C in 0.02% sodium azide-PBS solution. Labeling of the vasculature is termed vessel painting (VP) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eIgG Immunohistochemistry (IHC) and Analysis\u003c/h3\u003e\n\u003cp\u003e1hpi mouse brains were used for IgG staining and were incubated in 30% sucrose solution at 4\u0026deg;C for 48hrs. Samples were then frozen in Optimal Cutting Temperature Compound (OCT) on dry ice and stored at -20\u0026deg;C. Brain samples were sectioned coronally into 30\u0026micro;m thick slices and mounted directly onto slides and stored at -80\u0026deg;C. Sections were treated with 1% Sodium Dodecyl Sulfate at room temperature then incubated for 1.5 hours in room temperature with Alexa Fluor\u0026trade; 594 Goat anti-Mouse IgG (1:1000, Invitrogen, A11005). Slides were then dried and coverslipped with Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA).\u003c/p\u003e\n\u003ch3\u003eWide-field and Confocal Microscopy\u003c/h3\u003e\n\u003cp\u003eFluorescence images from vessel painted brains were acquired with a wide-field fluorescence microscope (Keyence BZ-X810, Keyence Corp, Osaka, Japan). Both axial surface and coronal sections at the level of the dorsal hippocampus (Bregma \u0026minus;\u0026thinsp;1.82mm) were imaged at 2X using the sectioning and Z-stack functions (step size 25.2 \u0026micro;m, 20 stack). Level correction, black balance, and haze reduction (blur size\u0026thinsp;=\u0026thinsp;10, brightness\u0026thinsp;=\u0026thinsp;10, reduction size\u0026thinsp;=\u0026thinsp;1) were applied to the images using BZ-II Analyzer software (Version: 1.1.30.19). Higher magnification 10X images were taken from regions with EB extravasation and the corresponding region in the contralateral hemisphere.\u003c/p\u003e \u003cp\u003eConfocal images at 20X were acquired from 30\u0026micro;m IgG-stained sections using a Zeiss LSM 880 confocal microscope (Carl Zeiss, Oberkochen, Germany). IgG, EB, and VP signals were imaged using excitation wavelengths of 561, 633, and 488 nm, respectively. Single-field images were acquired using the following parameters: 2% laser power; 2.57 Airy units pinhole size; 25 optical sections of z-stack with a step size of 2 \u0026micro;m; and 425.1 \u0026micro;m \u0026times; 425.1 \u0026micro;m image field. Three-dimensional reconstruction and visualization were performed using Imaris Bitplane software (version 10.2.0; Oxford Instruments, Abingdon, UK).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEvans Blue Analysis\u003c/h2\u003e \u003cp\u003eQuantification of EB extravasation was performed using Fiji (Version: 1.54f) software. First, a known region of EB leakage was outlined in a single CHILD mouse at 1hr post injury. Then the \u0026ldquo;fire\u0026rdquo; lookup table was applied and intensity levels\u0026thinsp;\u0026gt;\u0026thinsp;100 were defined as extravasation. This method was then applied to all mice and regional areas with intensity values\u0026thinsp;\u0026gt;\u0026thinsp;100 was extracted and summarized in MS Excel. In coronal sections, the integrated density was measured within the identical cortical region of the ipsilateral hemisphere corresponding to the site of the injury in all CHILD and sham mice.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAngioarchitecture Analysis\u003c/h3\u003e\n\u003cp\u003eAngiotool 0.6 software [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] was used to quantify classical vessel characteristics (vessel density, length, and junction density in the selected region of interest (ROI). Vessel complexity was assessed using the ImageJ FracLac to derive local fractal dimensions [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Axial regions of interest (ROI) included left and right hemispheres or whole axial brain analyses. Coronal ROIs encompassed cortical regions extending from the mid-line to the ventral-most boundary of the somatosensory cortex and placed ipsi- and contralaterally. Similar analytical methods have been published previously [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Data was extracted and summarized in MS Excel.\u003c/p\u003e\n\u003ch3\u003eMagnetic Resonance Imaging (MRI) Acquisition and Analysis\u003c/h3\u003e\n\u003cp\u003eT2-weighted (T2WI) and susceptibility-weighted imaging (SWI) were performed on \u003cem\u003eex vivo\u003c/em\u003e, skull-attached samples at 9.4T (Bruker Biospec, Billerica, MA). The following acquisition parameters were used for T2: 4000ms repetition time, 10ms echo time, 10 echoes, 4 averages, field of view 1.25 x 1.25cm, matrix 128 x128, 20 slices, 0.5mm slice thickness, 0.5mm slice interval, acquisition time\u0026thinsp;~\u0026thinsp;25min using Paravision 5.11. SWI was acquired using: 722.9ms repetition time, 10ms echo time, 8 averages, field of view 1.25 x 1.25cm, matrix 128 x128, 20 slices, 0.5mm slice thickness, 0.5mm slice interval, acquisition time\u0026thinsp;~\u0026thinsp;12min.\u003c/p\u003e \u003cp\u003eThe brain was segmented away from skull and extraneous tissues using ITK_SNAP (Version 3.8.0) software [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The extracted brains were used to generate T2 maps using JIM 7.0 software (Version. 7.0_42 Jan 10 2018, Xinapse Systems, Northants, UK). T2 maps were registered to our modified bilateral Australian Mouse Brain Mapping Consortium Atlas [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] using Advanced Normalization Tools (ANTS, Version:RRID:SCR_004757, University of Pennsylvania, Philadelphia, USA ). Regional brain volumes and T2 relaxation times were then derived from the registered T2 maps. SWI scans were analyzed using Signal Processing in NMR (SPIN) software (Version: Revision 1872) to identify presence of extra parenchymal bleeds.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eModeling Methodology\u003c/h2\u003e \u003cp\u003eAll the data, except MRI, were combined for the analysis to determine if there were potential predictors for CHI BBB disruption. The analysis was performed by the in-house python scripts using SciPy and Scikit-learn libraries. MRI data from a sub-cohort of mice these data were excluded, as they were missing over 70% of samples. \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eImputation\u003c/span\u003e. Up to 17% of data in other feature groups were missing; these were imputed to allow consistent downstream statistical analysis and module construction (see below). Imputation was performed separately for each variable using a hierarchical strategy, as follows. If at least two real observations were available within the same group \u0026times; sex \u0026times; timepoint subset, the missing values in that subset were replaced with the mean of the available observations. If a subset contained fewer than two real measurements (i.e., insufficient information for a reliable subgroup mean), the missing values were left unchanged and only replaced with the global mean of that variable if still required for principal component analysis (PCA) or visualization. This approach preserved true biological variability, avoided overfitting sparse subgroups, and prevented downstream analyses (e.g., PCA, clustering) from being dominated by \u0026ldquo;missingness\u0026rdquo; patterns rather than biological signal.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eHeatmap and Clustering\u003c/span\u003e. To visualize the correlation structure among measurements and assess relationships between features, pairwise Spearman correlation coefficients were computed for all features across all animals. Correlations were displayed as a heatmap with hierarchical clustering using average linkage and a Euclidean distance metric on the correlation matrix. This unsupervised approach recovered biologically related variables and highlights modules of coordinated change following CHI, supporting intuition that measurements from the same anatomical orientation or imaging modality are correlated with each other. \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eModule Definitions\u003c/span\u003e. Clustering results were used to define feature \u0026ldquo;modules\u0026rdquo; that both describe related biological processes and are correlated with each other. For example, vascular metrics derived from coronal sections formed a vascular-coronal module, while fractal dimension (LFD) features from axial slices defined as an axial-LFD module. For each module, we used PCA and used the first principal component (PC1) of the standardized module variables to be used as the module\u0026rsquo;s composite metric. PC1 captures the dominant shared variance of the module and serves as a noise-reduced, direction-consistent representation of the underlying biological process, such as vascular remodeling or vascular complexity.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eTrajectory Modeling\u003c/span\u003e. We took a combined p-values approach for trajectory modeling (averaging over points) to assess changes over time. To quantify sex differences while properly accounting for measurements collected at multiple timepoints after injury, statistical comparisons were performed independently at each timepoint using the Mann\u0026ndash;Whitney U test. The resulting per-timepoint p-values (p\u003csub\u003ei\u003c/sub\u003e) were then aggregated into a test statistics (\u0026#120511;) using Fisher\u0026rsquo;s combined probability method, as defined:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\psi\\:=\\:-2\\sum\\:_{i=1}^{k}\\text{l}\\text{n}\\left({p}_{i}\\right)$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhich follows a c\u0026sup2; distribution with 2k degrees of freedom (k\u0026thinsp;=\u0026thinsp;number of timepoints) and allows us to calculate the combined p-value. This approach does not assume linear or monotonic changes over time and is robust to heterogeneous variance and missingness across timepoints. For additional robustness, permutation-based combined p-values were computed by shuffling sex labels within each timepoint, recomputing p-values, and comparing the observed Fisher statistic to its permutation distribution. \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eConsistency\u003c/span\u003e. Because Fisher\u0026rsquo;s method combines p-values but not effect directions, we also quantified whether the male\u0026ndash;female differences were directionally consistent across timepoints. For each timepoint, the sign of the difference (mean_male\u0026thinsp;\u0026minus;\u0026thinsp;mean_female) was recorded. Directional consistency was defined as the fraction of time points at which the sign matched the majority direction across the trajectory. Values near 1.0 indicate stable directional effects (e.g., males consistently higher than females), whereas values near 0.5 indicate mixed or fluctuating differences. This provides an intuitive measure of biological coherence complementing the combined p-value.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003et-SNE Embedding of Key Discriminative Features\u003c/span\u003e. To visualize multivariate relationships among subjects, we applied t-distributed Stochastic Neighbor Embedding (t-SNE) to a curated feature set consisting of the most biologically discriminative module PC1 scores (e.g., axial LFD, coronal vascular) and key volumetric variables. All features were standardized prior to embedding. Only animals with complete data for the selected features were included, ensuring stable geometry and avoiding distortions driven by missing values. The resulting two-dimensional embedding was plotted with point color indicating sex, point shape indicating Sham or CHI groups, and where small numeric labels marking post-injury timepoints. This provides an intuitive visualization of how injury and sex jointly influence high-dimensional phenotypic space.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using GraphPad Prism (Version 9, GraphPad, Boston, MA, USA). We performed one-way analysis of variance (one-way ANOVA) with multiple comparisons for temporal data and group comparisons utilized t-tests. All t-tests were parametric unless specifically stated. Pearson correlations were also performed in GraphPad. All values are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Statistical significance threshold was defined as p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 with trending reported in those cases with p\u0026thinsp;\u0026lt;\u0026thinsp;0.10.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eCHILD induced sex-specific physiological and structural changes.\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePrior to CHILD induction, PND17 weights between male and female mice were not significantly different, with the average weight of all pups being 7.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67g (n\u0026thinsp;=\u0026thinsp;117). There were no significant differences (p\u0026thinsp;=\u0026thinsp;0.724, unpaired t-test) between male average weights (7.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.59g, n\u0026thinsp;=\u0026thinsp;61) and female weights (7.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.75, n\u0026thinsp;=\u0026thinsp;56). No significant weight differences were found at 1dpi between sham and CHILD male or female mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). In contrast, relative to pre-CHILD (baseline), weight gain at the 7dpi period relative was significantly increased in sham compared to CHILD mice (p\u0026thinsp;=\u0026thinsp;0.0009, unpaired non-parametric t-test) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Male CHILD mice at 7dpi had a significant decrement (p\u0026thinsp;=\u0026thinsp;0.002, unpaired non-parametric t-test) in weight gain (55.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04% compared to male shams 66.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02%; Supplemental Fig.\u0026nbsp;1A). Female CHILD mice also had reduced weight gain compared to female sham mice at 7dpi but did not reach significance (p\u0026thinsp;=\u0026thinsp;0.073, unpaired non-parametric t-test) (Supplemental Fig.\u0026nbsp;1B). Paired-weight changes between baseline and 1dpi or 7dpi further demonstrate significant increases in weight gain over the 7dpi period (Supplemental Fig.\u0026nbsp;1C, D).\u003c/p\u003e \u003cp\u003eImmediately after CHILD induction we monitored the level of consciousness in all mice by recording the presence and duration of apnea immediately after head impact and the time required to resume a righting position. The prevalence of CHILD mice that exhibited apnea was ~\u0026thinsp;20% higher in males (44.44%) than in females (24.32%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), exhibiting a sex-specific immediate physiological response to concussive injury. CHILD mice also exhibited a significantly longer time to resume righting position relative to shams (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, unpaired t test) with no overt sex differences (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eA randomized subset of sham (n\u0026thinsp;=\u0026thinsp;11) and CHILD mice (n\u0026thinsp;=\u0026thinsp;15) at 1dpi underwent \u003cem\u003eex vivo\u003c/em\u003e T2-weighted MRI (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Cerebrum volumes exhibited sex differences with male CHILD mice showing significantly reductions by 8.82% (p\u0026thinsp;=\u0026thinsp;0.035, unpaired t test) compared to shams (Supplemental Fig.\u0026nbsp;2A), while female CHILD or sham mice did not report differences. Male CHILD mice exhibited significantly lower cerebrum volumes than female CHILD mice (p\u0026thinsp;=\u0026thinsp;0.011, unpaired t test) but no differences between male and female shams were reported (Supplemental Fig.\u0026nbsp;2A). Brain tissue properties were assessed with T2-relaxometry measurements (in ms) from cortical regions (motor, parietal and somatosensory) and white matter structures (corpus callosum, CC) that are at the site of the concussive impact (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF-I). T2 relaxation time was reduced in all four regions in male CHILD mice. There was a significant decrease in T2 relaxation in parietal cortex of male CHILD mice of 12.58% compared to shams (p\u0026thinsp;=\u0026thinsp;0.014, unpaired t test) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Sex differences were also observed since female CHILD mice had significantly increased T2-values in motor (8.82%), parietal cortices (7.56%), and corpus callosum (10.21%) compared to female shams (p\u0026thinsp;=\u0026thinsp;0.010, p\u0026thinsp;=\u0026thinsp;0.021, p\u0026thinsp;=\u0026thinsp;0.002, respectively, unpaired t test) and a trending significance in the somatosensory cortex (p\u0026thinsp;=\u0026thinsp;0.074, unpaired t test) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF-I).\u003c/p\u003e \u003cp\u003eSusceptibility-weighted imaging (SWI) was also acquired to assess the presence of extravascular blood (Supplemental Fig.\u0026nbsp;2B) which was often found at the cortical surface and at the interface between gray and white matter (corpus callosum). Seventy-five percent of male CHILD mice but only 42% of female CHILD mice exhibited visible extravascular bleeding (Supplemental Fig.\u0026nbsp;2C). Thus, clinically relevant neuroimaging further confirms sex-specific differences in water content and parenchymal bleeds.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCHILD induced transient dysfunction of the blood-brain border (BBB) at acute time points followed by recovery.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eEvans Blue (EB) is a water-soluble fluorescent dye that binds to serum albumin and only permeates into the brain parenchyma when BBB properties are compromised. EB observed within blood vessels confirmed functional perfusion in sham mice, whereas EB accumulation within the brain parenchyma was observed in the ipsilateral cortex in CHILD mice at 1hpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). At higher magnification (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, right panel), EB extravasation in the parenchyma was observed adjacent to vessels defined by VP at the site of injury in CHILD mice, but not in shams. Integrated intensity of extravasated EB in the parenchyma was quantified at 1hpi, 6hpi, 1dpi, 3dpi and 7dpi in the ipsilateral cortex from coronal tissue sections. Increased EB accumulation peaked at 1hpi and then declined over time in males and females (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Female CHILD mice exhibited a significant increased EB extravasation of 71.02% compared to shams at 1hpi (p\u0026thinsp;=\u0026thinsp;0.047, unpaired t test), while the increase of EB extravasation did not reach significance in male CHILD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Higher variability in EB extravasation was observed for the acute timepoints (1hpi and 6hpi) compared to later timepoints in both sexes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUsing a two-phase exponential decay model, temporal analysis of BBB dysfunction from coronal images showed a linear decrease in EB extravasation in males, while females exhibited a rapid exponential decrease after injury (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). We also assessed EB leakage area from the cortical surface (axial) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, middle panel) and like the coronal analyses, there was considerable variability in male CHILD mice although less so in the female mice with no significant differences (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). The number of mice from each sex who had axial EB leakage present were collated as a percent of all the mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). In male CHILD mice there was an increasing proportion that showed BBB leak that peaked at 1dpi (71%) and then precipitously declined by 7dpi (11%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). In contrast, CHILD female mice had 100% of injured mice exhibiting cortical EB leakage at 6hpi that slowly declined by 7dpi (60% of mice, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Exemplar micrographs illustrate extravascular EB extravasation from cortical vessels are shown in Supplementary Fig.\u0026nbsp;3, at 1hpi and 1dpi. These images reveal subtle and vascular localization of EB leakage within cortical regions and those adjacent to the concussive impact site. These findings were in line with sex differences in BBB pathophysiology between concussed male and female mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eCHILD impairs axial cortical angioarchitecture associated with BBB perturbations\u003c/h2\u003e \u003cp\u003eThe inter-relationships between axial cortical vascular features using vessel painting and EB extravasation were examined in sham and CHILD mice across all timepoints (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Axial vessel density on the ipsilateral hemisphere was significantly reduced by 41.79% in male CHILD mice compared with shams at 1hpi (p\u0026thinsp;=\u0026thinsp;0.047, one-way ANOVA), which progressively recovered by 7dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Total vessel lengths and number of junctions exhibited no significant changes in CHILD males, but the pattern of changes were like that of vessel density in males (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, E, H). However, no significant changes were observed in females either in vessel density, total vessel lengths and number of junctions at any of the time points examined (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eIn males the temporal resolution of vessel density was consistent with the peak BBB dysfunction (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, C). Therefore, the relationships between these outcome measures were calculated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, G, J). In male CHILD mice axial surface EB extravasation area were significantly negatively correlated to vessel density (r=-0.578, p\u0026thinsp;=\u0026thinsp;0.024) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD) and junction density (r=-0.572, p\u0026thinsp;=\u0026thinsp;0.026) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ). CHILD male mice exhibited no significant relationship with total vessel length and surface EB extravasation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). This suggests that vessel alterations characterized by a loss in density and number of junctions also demonstrated BBB dysfunction. No significant correlations were observed in CHILD females in any vessel metric compared to EB extravasation area (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, G, J). These results suggest that impairment of the BBB is strongly associated with morphological vessel alterations in males but not in females, highlighting sex differences in cerebrovascular pathophysiology early post-concussion.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCoronal cortical angioarchitecture is decreased after CHILD and is associated with BBB perturbations\u003c/h2\u003e \u003cp\u003eThe ipsilateral coronal cortical vasculature was analyzed at and adjacent to the impact site, examining the vessels penetrating the cortex. We quantified VP angioarchitecture and EB extravasation, similarly to the axial surface findings (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Following similar pattern observed on axial analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), a dramatic and significant reduction by 71.82% cortical vessel density was found in male CHILD mice at 1hpi compared to shams (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, one-way ANOVA, Tukey\u0026rsquo;s post-hoc test), which temporally recovered by 7dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Total vessel length was significantly reduced at every time point in CHILD males (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 for each, ordinary one-way ANOVA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Junction density was similarly significantly reduced in CHILD male mice at 1hpi compared to shams (p\u0026thinsp;=\u0026thinsp;0.05, ordinary one-way ANOVA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). As described for the axial analysis, no significant morphological changes in blood vessel metrics were observed in female CHILD mice compared to shams (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, F, I), despite an overall trend for vascular reductions at 1hpi.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWhen we examined the relationship between vascular morphological features (vessel density p\u0026thinsp;=\u0026thinsp;0.040; total length p\u0026thinsp;=\u0026thinsp;0.037; junctions p\u0026thinsp;=\u0026thinsp;0.027), we found significant correlation to EB intensity in CHILD male mice independent of time post injury (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, G, J). Again, no significant correlations between EB extravasation and vessel metrics were found in female CHILD mice(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, G, J). Thus, in CHILD males but not CHILD females, the severity of the BBB dysfunction was directly related to vasculature morphological changes within the ipsilateral cortex.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eAcute CHILD elicits a hyper-acute reduction in vessel complexity\u003c/h2\u003e \u003cp\u003eA key hallmark of vascular damage is a reduction in vascular complexity which can be assessed using fractal measures [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Smaller vessels are potentially more vulnerable to mechanistic forces induced by the head rotation (Rodriguez-Grande, Glia 2018) and thus contribute to pathological progression of BBB breakdown. We assessed vascular complexity in coronal cortical vessels at the lesion site by generating fractal histograms (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B; Supplementary Fig.\u0026nbsp;5). The resultant fractal histograms measures provide quantitative information about complexity (shift in LFD curve) and vessel numbers (area under the curve or AUC) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Early hyper-acute time points (1-6hpi) revealed a significant reduction in AUC at 1hpi in male CHILD mice compared with male shams (p\u0026thinsp;=\u0026thinsp;0.031, unpaired t test), with no overt differences in female CHILD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). At 6hpi in male CHILD mice the AUC started to recover with a trending significant reduction (p\u0026thinsp;=\u0026thinsp;0.115, unpaired t-test) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) with no significant differences were observed in either male or female CHILD mice at other time points up to 7dpi (Supplementary Fig.\u0026nbsp;6A, B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eVessel complexity was assessed using the maximum local fractal dimension (LFD) at the peak frequency (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). At 1hpi, both CHILD male (p\u0026thinsp;=\u0026thinsp;0.016, unpaired t test) and CHILD female (p\u0026thinsp;=\u0026thinsp;0.037, unpaired t test) mice had significant reductions in LFD values consistent with reduced vessel complexity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) but no differences in either male or female CHILD mice were observed at any other time points (Supplementary Figs.\u0026nbsp;4,5). These results further confirm that CHILD in male mice results in reduced brain vasculature and complexity at hyper-acute time points post-injury whereas CHILD in female mice elicited only decrements in complexity but not in vascular density.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003ePresence of Immunoglobulin G (IgG) in CHILD mice signifies BBB breakdown\u003c/h2\u003e \u003cp\u003eImmunoglobulin G (IgG) extravasation in brain tissue after injury is a marker of BBB dysfunction as we previously described in CHILD at 1dpi (Rodriguez-Grande et al. 2018) and in adult CHI [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] and in juvenile TBI [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. To further confirm BBB dysfunction, we undertook IgG staining at 1hpi when the most robust alterations in cortical vessels and EB extravasation were observed. Low magnification IgG-stained sections (Supplementary Fig.\u0026nbsp;6) were examined for representative cortical vessels that exhibited EB extravasation in male CHILD mice at 1hpi. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e illustrates the coincident labeling between vessels and IgG, vessels and EB and vessels, IgG and EB in sham mice (left panel) and CHILD mice (right panel). Sham mice did not exhibit any notable IgG or EB signals outside the vessels although in the merged images both are visible within the vessels themselves. In stark contrast, CHILD mice at 1hpi exhibited IgG extravasation staining protruding from vessels in discrete beads along injuried vessels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Interestingly, extravasation EB staining presented larger coverage along the vessels than IgG staining. However, IgG staining was associated with EB-extravasation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), a confirmation of the vascular-BBB dysfunctions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eModeling the Interactions between Vascular Injury and BBB Dysfunction\u003c/h2\u003e \u003cp\u003eGiven the wealth of the data acquired in this study we examined if modeling these vascular and BBB data could provide additional insights into the physiological mechanisms and the potential for predictive capabilities. The first step was to identify the correlation structure between all the variables (data) that were collected. Clustering of the data and its heatmap representation clearly illustrated that vascular measures strongly clustered together (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Coronal vascular features, axial vascular features and complexity measures were all strongly aggregated. Physiological features (apnea duration etc.) exhibited a reduced clustering. Based on the clustering and heatmap analysis we undertook a data reduction approach whereby we consolidated groups of related features into modules. The final modules are summarized in Supplemental Table\u0026nbsp;2 and include Vascular Coronal, Vascular Axial, Leakage Evans Blue and Local Fractal Dimension (coronal and axial combined) and were utilized in subsequent analysis.\u003c/p\u003e \u003cp\u003eModeling of temporal evolution of vascular and BBB disruption following brain injury are of importance, particularly in the context of patient management. Here we undertook trajectory analyses and coupled this to consistency measures to identify which features provide intuitive measures of biological coherence. We found that in the Evans Blue Leakage module sham mice (male or female) exhibited poor consistency values as might be expected as no CHI was induced (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, top panel). In contrast, the CHI mice showed high consistency at the earlier time points with decreasing consistency at later time points consistent with acute BBB disruption after brain injury (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, top panel). There were no overt sex differences. When we examined the coronal vascular module, male sham mice exhibited a stable trajectory while female mice had a more variable consistency (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, bottom panel). In male CHI mice there was a progressive increase in consistency in cortical vascular features that continued over 7dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, bottom panel). The female CHI mice exhibited no consistency in these coronal vascular features until 1dpi that then precipitously declined by 3dpi with subsequent increased consistency by 7dpi, like that of male CHI mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, bottom panel). These consistency measures suggest that males after injury exhibited a more consistent trajectory of either BBB leakage or coronal vascular features than female CHI mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe now examined these multivariate relationships by employing t-distributed Stochastic Neighbor Embedding (t-SNE) to visualize these interactions. Specifically, we were interested in how injury and sex may jointly influence our highly dimensional phenotypes particularly in light of the heterogenous nature of concussion. These analyses highlighted sex and injury-specific clustering (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). While there was some overlap between sham male and female mice (as would be expected) the CHI male and female mice exhibited clear separations. As noted in our vascular and Evans Blue data and in the modeling above, male and female CHI mice clearly have unique features that allow separation.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePediatric and juvenile mild traumatic brain injury (mTBI, concussions) and their subsequent pathologic sequelae are understudied. Most notably lacking is how blood-brain border (BBB) integrity is impacted by concussive injuries over time in clinic patients, as well as in rodent models. We used our CHILD mouse model utilizing a single impact at postnatal day 17 (PND17) reminiscent of pediatric concussion which includes a rotational component [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. We describe here the distinct neurovascular and BBB trajectories in both male and female mice at hyper-acute and acute epochs after impact above the somatosensory cortex. Our key findings are: 1) weight reductions in CHILD mice, particularly males, 2) T2 values in ipsilateral cortical regions were reduced in CHILD males but increased in females, 3) hyper-acute EB extravasation in male and female CHILD mice, 4) hyper-acute decrements in vessel density that correlated with the presence of EB, but only in males but not female CHILD mice, 5) reductions in acute vessel complexity were only apparent in male CHILD mice, and 6) EB and IgG were visible adjacent to intracortical vessels in CHILD mice but not shams. We also demonstrate strong sex- and injury-specific relationships in our modeling approach. Taken together our results herein provide strong evidence for an early hyper-acute vascular and BBB disruption that is more prominent in males compared to female concussed juvenile mice. These early cerebrovascular perturbations may presage the subsequent development of long-term deficits that we have reported previously [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eChanges in physiology and interference with brain development\u003c/h2\u003e \u003cp\u003ePediatric and juvenile brains are vulnerable to concussion given the rapid developmental growth of the brain and neuronal connectivity. Early physiological changes after concussion in early childhood have been described (i.e. appetite changes, cognition, mood etc. (see [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]). Sex differences in either concussion rates [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], neuroimaging features [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] and acute and chronic outcomes [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] have suggested the existence of potential differences between males and females in both clinical and preclinical studies. In our juvenile model of concussion, male mice displayed a higher prevalence of transient apnea (44.44% of males) than females (24.32%), while both sexes experienced prolonged righting reflex times. The CHILD model is associated with rotational aspects at time of injury [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] and youth have been modeled to exhibit lower linear and rotational tolerance than adults [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Our rotational CHILD model can result in brain stem injuries, resulting in a transient disruption of the respiratory system function due to disturbance of medullary functions and temporary respiratory arrhythmia [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Righting reflexes in rodents are considered an alternative measurement of consciousness and alertness for determining TBI severity and prognosis [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] albeit in our study we found no overt sex differences. Thus, transient apnea and delayed righting reflex in the CHILD model represents a concussion landmark of the precipitating event compared to shams. We have previously described for the CHILD model a sex difference in recovery times which were shorter in males compared to female mice, in contrast to our current findings which had a larger number of replicates [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The severity of transient hypoxic events can represent a critical pathological landmark for concussion as the duration of hypoxia has been correlated to future long term cardiac dysfunctions in CHILD [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the current study, reduced weight gain in CHILD mice was observed by 7dpi. While both sexes had reductions in weight gain, only male CHILD mice reported significant differences. Attenuated weight gain after repeated mild TBI has been described in rodents reflecting problems in pituitary and hypothalamic circuits, impeding normal growth and development [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In a rat pup model of mild TBI, decrements in weight gain were coincident with growth hormone reductions during the acute period but were elevated chronically [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Such hormonal changes may also underlie progressive decreases in CHILD cerebrum volumes. Therefore, disturbance of hypothalamic function after CHILD may influence a host of physiological changes both acutely and long after the initial event.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eEarly sex-dependent vascular alterations\u003c/h2\u003e \u003cp\u003eTemporal neurovascular alterations, BBB dysfunction, vessel properties and morphology have been previously investigated in juvenile CHILD males where increased IgG staining in brain parenchyma has been described at 1dpi and resolved by 7dpi [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. These alterations in BBB properties were associated with decreased neurovascular reactivity and decreased brain oxygenation peaking at 6hpi which then normalized [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. These early functional vascular changes were then assessed 12 months post-concussion and CHILD male mice had increased numbers of capillary vessels [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Lacking from the literature is an assessment of neurovascular properties in male and female acutely after single pediatric concussion. This gap is critical considering known sex differences in youth after concussion injury [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe report significant reductions of pial and intracortical cerebral vessels for male CHILD mice at 1hpi. These decrements in cerebrovascular metrics were not significantly different in female CHILD mice (see Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Moreover, these sex-specific differences were also confirmed in our modeling (see Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The hyper-acute reductions in vessel density, vessel total length and number of junctions normalized at later timepoints. Our current findings relied on the vessel painting method with perfusion of lipophilic dye through the vascular system [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. One consideration is that mTBI may result in vascular hypoperfusion [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] and reduced neurovascular coupling [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], as previously reported. Vasculogenic processes are initiated within acute time windows with angiogenic molecules such as vascular endothelial growth factor (VEGF) being upregulated [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. While VEGF rapidly increases after TBI it is thought to require\u0026thinsp;~\u0026thinsp;2 weeks for the complex molecular cascade to induce endothelial cell proliferation and migration, anastomosis, and glial cell recruitment [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Other vasculogenic pathways are also involved and in adult TBI activation of the Wnt/β-catenin pathway as early as 1dpi with increased Wnt5a levels at 7dpi where related to recovery of vessel density [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Our current study did not directly examine cerebral blood flow (CBF) at these acute time points, but T2-weighted imaging (specifically T2 relaxation) potentially sheds some additional light. We reported T2 relaxation was significantly decreased in male CHILD mice in cortical regions under or adjacent to the impact site but was significantly increased in female CHILD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF-I). Our current interpretation of these opposite sex effects is that in males there is a putative reduction in CBF and in metabolically active tissues results in increased oxygen extraction during transit. In females these changes are less pronounced acutely, and CBF may not be impacted to the same extent as in males. We have described such a mechanism for reduced T2 relaxation previously [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Therefore, multiple systems may contribute to the recovery of decreased vessel density, including recovery of perfusion and vasculogenesis.\u003c/p\u003e \u003cp\u003eIntracortical vascular complexity was also only altered in male CHlLD mice at 1hpi with no overt changes at later time points. In male CHILD mice the LFD at peak frequency was always increased (although not significantly) at each time point and normalized by 7dpi, which was not the case in female mice (Supplementary Fig.\u0026nbsp;4). While vascular complexity has not been extensively reported in TBI, we have noted in a cortical contusion injury model, initial loss of complexity that recovers with resumption of vessel density over time [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. It is also important to highlight temporal differences between pial and intracortical vessels in our study, where we observed a longer duration of presumed recovery in intraparenchymal blood vessels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). It is well known that pial and intraparenchymal vessels present different anatomical, physiological and functional properties in healthy and diseased brain tissues [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. These differences could suggest that in response to a mechanical injury, there is a potential greater vulnerability for intracortical compared to pial blood vessels to mechanical deformation. Our observations are strengthened by the importance of mechanical injury-induced cerebrovascular dysfunction that has been described clinically and in mouse models [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eSex-BBB alterations\u003c/h2\u003e \u003cp\u003eIn this study, BBB properties were assessed using EB injection 1hr prior to VP. Brief and early elevations of EB extravasation were observed for both sexes in CHILD mice, although only significant in females (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). These acute robust increases in EB leakage also exhibited high consistency in our trajectory modeling of both male and female CHI mice, which then declined with time post injury (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Male CHILD mice exhibited an elevated EB extravasation at 1dpi, in concordance with our previous report [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], that then declined linearly to sham levels by 7dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). This contrasts with the rapid decline at 1dpi of EB extravasation in female CHILD mice, further demonstrating sex differences in response to concussion and further supported in our trajectory modeling. Our histological demonstration of co-localization of EB and IgG outside of vascular structures in male CHILD mice, clearly demonstrates extravasation during the hyper-acute period after concussion. It is interesting to note that the IgG and EB staining do not consistently overlap and may be due to differences in molecular weights (EB: 0.9kDa, IgG: 150kDa). We and others have reported similar vascular IgG extravasation after concussion [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e] and cortical contusion injury (CCI) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Importantly, EB extravasation and VP-derived vessel metrics were significantly negatively correlated in CHILD male in both pial and intracortical assessments but not females (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Decreased vessel density strongly correlated with larger EB extravasation. These results further support in male CHILD mice an acute BBB leakage, hypoperfusion and modified vessel angioarchitecture [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eChanges in BBB after CHILD have been linked with early increased expression of the water channel aquaporin 4 (APQ4) at 1dpi with late developing astrogliosis (7dpi) (Rodriguez-Grande et al 2018). Even a mild mechanical stress on the brain produces significant vascular changes with decrease in flow and change in BBB properties, similarly to more severe TBI in pediatrics and adults [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. The exact mechanism(s) behind these perturbations are still poorly understood and the pathophysiological vascular response differs over time. Mechanical forces due to linear and rotational acceleration can directly damage endothelial cell structures compromising junctional proteins that transition to BBB dysfunction and subsequent vasogenic edema [\u003cspan additionalcitationids=\"CR55\" citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Local changes in water homeostasis may compress nearby vessels and the presumed loss of perfusion observed in our results. Concurrently, endothelial cells in response to injury also release vasoconstrictors such as endothelin-1 (ET-1) [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e], further promoting vasoconstriction and altering vascular tone. Together, edema and ET-1-dependent vasoconstrictor activity could reduce CBF and may limit dye perfusion, consistent with the reduction observed in vessel painting and larger EB extravasation. These changes are resolved by 7dpi.\u003c/p\u003e \u003cp\u003eThe molecular mechanisms involved in resolution of the BBB dysfunction post-injury are very poorly understood and understudied [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. There are numerous putative proteins involved in BBB leakage, including caveolin-1 [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e], which is expressed in the neurovascular unit and has been proposed as a key pathway in BBB recovery in juvenile moderate TBI [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Caveolin-1 expression after brain injury follows a similar timeline of the BBB changes and recovery in CHILD [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Other phenomena such as tight junction (TJ) loss after TBI in adult rodents leads to BBB loss of function acutely with subsequent restoration and reduced inflammatory activities [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Compromised BBB function allows entry of peripheral serum proteins (IgG), immune cells, and potential pathogens into the brain parenchyma, activating brain resident immune responses [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. The heightened immune response, leading to microgliosis and astrogliosis, can further promote endothelial dysfunction. Thus, despite recovery from transient BBB permeability, the progression of secondary sequalae and the potential for chronic inflammation can lead to a compromised neurovascular unit [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The early neurovascular dysfunctions after CHILD may represent a critical event that leads to long-term neurovascular dysfunction and inflammatory sequelae observed in the CHILD mouse model of pediatric concussion [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eSex differences following pediatric concussion\u003c/h2\u003e \u003cp\u003eA recent literature review found that 44% of animal TBI studies reported that females had better outcomes than males, while only 28% of the studies surveyed showed no sex differences [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Despite increasing studies using both males and females there is still a considerable gap about how sex modulates outcomes in pediatric concussion. It has been hypothesized that estrogen and progesterone play vascular protective roles in females by upregulating vasodilatory factors and reducing vasoconstrictive factors [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e], thereby improving microcirculation and vessel reactivity. Compared to males, females present with a lower prevalence of cardiovascular disease [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e] and improved CBF after TBI [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. Adult female mice in a CCI model of TBI found acute (1dpi) increased astrogliosis and heme-oxygenase-1 expression (via estrogen) whereas male TBI mice had increased neovascularization via β-catenin [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, sex hormones alone may not fully account for the differential vascular function, as sex differences in microvascular blood flow have been reported during infancy and adolescence [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAnother potential contributor to sex differences in prepubertal juveniles is the differential expression of the predominant vasoconstrictor ET-1 in pediatric TBI, which is upregulated in males, but not in females [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. The upregulation of ET-1 in males increases their susceptibility to cerebral autoregulatory impairments, leading to vasoconstriction and reduced perfusion. This potentially provides one mechanism for the reduced T2 values and reduction in vessel metrics we report here. Contrary to the males, the reduction of ET-1in females may be protective, thus explaining the lack of vasculature abnormalities despite pronounced BBB disruptions. Indeed, our trajectory and consistency modeling lends additional support related to strong sex differences, even at this early age. Our current study further reveals that sex differences in the vascular response to trauma are present and should incentivize further exploration into mechanisms underlying sex differences in juvenile TBI.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eTrajectory Modeling of Traumatic Brain Injury (TBI)\u003c/h2\u003e \u003cp\u003eNascent studies are now being to address the heterogenous nature of TBI recovery between groups and individuals, primarily focused on physiological, psychological and functional outcomes. Ren and colleagues, using trajectory analyses for emotive symptoms in a relatively small cohort of subjects, were able to discriminate between depression, anxiety and life satisfaction as well as within groups (low vs. high symptomology) [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. Similarly functional motor and cognitive scores were able to differentiate recovery patterns in subjects with moderate to severe TBI [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. A more comprehensive Track-TBI study with 2100 participants was able to identify seven unique trajectories based on recovery by using Glasgow Outcome Scale Extended (GOSE) [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. These trajectories were also strongly associated with initial presentation of the GOS, computed tomography findings and psychiatric comorbidities. Other measures after TBI, such as patterns of sympathetic hyperactivity [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e], executive function [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e] and quality of life measures [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e] also aggregate into distinct trajectories. To our knowledge, there are no clinical or preclinical studies examining trajectories related to vascular impairments and longer-term outcomes. While our study was confined to 7dpi, we demonstrate that trajectory modeling may have enhanced utility in discriminating between sex and injury subtypes bases on vascular and BBB leakage features. We suggest that future longitudinal studies utilize trajectory modeling to enhance our understanding of physiological and psychological outcomes after TBI.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eLimitations and future directions\u003c/h2\u003e \u003cp\u003eThere are several limitations and future directions related to our findings. Firstly, all the morphological and BBB leakage studies required sacrifice of the mice at discrete epochs. Continuous monitoring from the same animal for neurovascular function and BBB leakage using other techniques, such as MRI, would have provided additional temporal evolution insights. As noted above, the VP method can map the cerebrovascular networks, but only if they are being perfused. Correlation with other histological techniques (ie tomato lectin, etc.) could be applied. We also did not make any effort to disentangle the contribution of arteries and veins in concussion. Finally, there are number proteins responsible for modulating the BBB in health and disease, such as caveolins, claudins and others that likely play an important role not only in the acute injury phase but also during recovery; we did not explicitly assess those in our study. Future studies using imaging approaches such as MRI, in vivo confocal microscopy, miniscopes (as we have done [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]) or functional ultrasound imaging (fUSI) could and should be used to differentiate direct perfusion changes, particularly as they relate to early in life putative sex differences. Additional replicates or similar data from other published research could strengthen the impact of trajectory modeling.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe novelty of our research is two-fold: 1) hyper-acute sex differences in a clinically translatable model of juvenile mTBI (CHILD) that exhibits acute, chronic and long-term (12 months) pathophysiology [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e], and 2) hyper-acute vessel changes that strongly correlate with BBB leakage in a sex-dependent manner. Our findings demonstrate that CHILD induces an early acute neurovascular change with loss of vascular density and BBB dysfunction transiently, recovering by 7dpi only in males and not females. We posit that these hyper-acute changes within the neurovascular unit primes the injured brain to contribute to the long-term cellular, molecular, neurovascular and behavioral perturbations we, and others have observed at 12\u0026ndash;18 months post-concussion [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. The presence of sex differences during the pre-pubertal period highlights the importance of incorporating biological sex as a determinant of injury response in future mechanistic and therapeutic pediatric mTBI studies.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003eAQP4\u003c/strong\u003e: Aquaporin 4\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBBB\u003c/strong\u003e: Blood-brain border\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCCI:\u0026nbsp;\u003c/strong\u003eCortical contusion Injury\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCHI:\u0026nbsp;\u003c/strong\u003eClosed head injury\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCHILD:\u003c/strong\u003e Closed head injury with long term disorders\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDiO\u003c/strong\u003e:\u0026nbsp;3,3\u0026rsquo;-dioctadecyloxacarbocyanine\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003edpi\u003c/strong\u003e: Days post-injury\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEB:\u0026nbsp;\u003c/strong\u003eEvans blue\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eET-1:\u0026nbsp;\u003c/strong\u003eEndothelin-1\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ehpi\u003c/strong\u003e: Hours post-injury\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIHC:\u003c/strong\u003e Immunohistochemistry\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLFD:\u0026nbsp;\u003c/strong\u003eLocal fractal dimension\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMMP:\u0026nbsp;\u003c/strong\u003eMatrix metalloproteinase\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMRI\u003c/strong\u003e: Magnetic resonance imaging\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ems:\u0026nbsp;\u003c/strong\u003emilliseconds\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003emTBI\u003c/strong\u003e: Mild traumatic brain injury\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePBS:\u003c/strong\u003e Phosphate-buffered saline\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePCA:\u003c/strong\u003e Principal component analysis\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePFA:\u0026nbsp;\u003c/strong\u003eParaformaldehyde \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePND:\u0026nbsp;\u003c/strong\u003ePostnatal day\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eROI:\u003c/strong\u003e Region of interest\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eROS\u003c/strong\u003e: Reactive oxygen species\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSWI\u003c/strong\u003e: Susceptibility-weighted imaging\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eT2:\u0026nbsp;\u003c/strong\u003eT2-weighted\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTBI:\u003c/strong\u003e Traumatic Brain injury\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTJ:\u0026nbsp;\u003c/strong\u003eTight junction\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003et-SNE:\u003c/strong\u003e t-distributed Stochastic Neighbor Embedding\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVEGF:\u0026nbsp;\u003c/strong\u003eVascular endothelial growth factor\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e: All protocols and data supporting the findings are available upon request to the corresponding authors.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e: The authors report no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Sources\u003c/strong\u003e: The data for this study were funded by NINDS 1R01NS119605 (Andre Obenaus, Jerome Badaut) and NINDS 1RF1NS138032 (Andre Obenaus, Paul Territo)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e: Animal experiments were performed by JY and AJ. Fluorescent images were acquired by JY and analyzed by JY, NN, JA and RP. MRI analysis was performed by JY and TG. Immunohistochemistry was performed by GC and JY. JY performed data analysis and AO reviewed all data. JY wrote the draft and AO, JB critically revised the manuscript. AO, JY, AG performed all the modeling analysis and related text. JB, AO, JY and AJ contributed to experimental design, data analysis, and manuscript revisions. JY is the leading author.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval\u003c/strong\u003e: All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of University of California, Irvine, and University of California, Riverside.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e: The authors would like to acknowledge Dr. Shunshan Li for assistance with MRI acquisition, Brenda Noarbe for advice on MRI analysis, and Tia Ketsan and Safa Hamid for data analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eUse of Language Models:\u0026nbsp;\u003c/strong\u003eIn the preparation of this manuscript, the authors did not use any generative artificial intelligence tools. At the completion of the final draft, Grammarly (Grammarly Inc., San Francisco, CA, USA) was used to improve the clarity, grammar, and readability of the text. The authors carefully reviewed, revised, and take full responsibility for the content of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSuskauer SJ, Houtrow AJ. Invited Commentary on The Report to Congress on the Management of Traumatic Brain Injury in Children. Arch Phys Med Rehabil. 2018;99(11):2389\u0026ndash;91.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAraki T, Yokota H, Morita A. Pediatric Traumatic Brain Injury: Characteristic Features, Diagnosis, and Management. Neurol Med Chir (Tokyo). 2017;57(2):82\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNwafor DC, Brichacek AL, Foster CH, Lucke-Wold BP, Ali A, Colantonio MA, et al. Pediatric Traumatic Brain Injury: An Update on Preclinical Models, Clinical Biomarkers, and the Implications of Cerebrovascular Dysfunction. J Cent Nerv Syst Dis. 2022;14:11795735221098125.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFigaji AA. Anatomical and Physiological Differences between Children and Adults Relevant to Traumatic Brain Injury and the Implications for Clinical Assessment and Care. Front Neurol. 2017;8:685.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLumba-Brown A, Yeates KO, Sarmiento K, Breiding MJ, Haegerich TM, Gioia GA, et al. Centers for Disease Control and Prevention Guideline on the Diagnosis and Management of Mild Traumatic Brain Injury Among Children. JAMA Pediatr. 2018;172(11):e182853.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEsterov D, Witkowski J, McCall DM, Weaver AL, Brown AW. Long-Term Risk for Mood and Anxiety Disorders After Pediatric Traumatic Brain Injury: A Population-Based, Birth Cohort Analysis. J Head Trauma Rehabil. 2023;38(3):E212\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBadaut J, Ghersi-Egea JF, Thorne RG, Konsman JP. Blood-brain borders: a proposal to address limitations of historical blood-brain barrier terminology. Fluids Barriers CNS. 2024;21(1):3.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePatabendige A, Janigro D. The role of the blood-brain barrier during neurological disease and infection. Biochem Soc Trans. 2023;51(2):613\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJullienne A, Salehi A, Affeldt B, Baghchechi M, Haddad E, Avitua A, et al. Male and Female Mice Exhibit Divergent Responses of the Cortical Vasculature to Traumatic Brain Injury. J Neurotrauma. 2018;35(14):1646\u0026ndash;58.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFullerton JL, Hay J, Bryant-Craig C, Atkinson J, Smith DH, Stewart W. Pediatric Traumatic Brain Injury and Microvascular Blood-Brain Barrier Pathology. JAMA Netw Open. 2024;7(11):e2446767.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGreen TRF, Nguyen T, Dunker V, Ashton D, Ortiz JB, Murphy SM, et al. Blood-Brain Barrier Dysfunction Predicts Microglial Activation After Traumatic Brain Injury in Juvenile Rats. Neurotrauma Rep. 2024;5(1):95\u0026ndash;116.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHay JR, Johnson VE, Young AM, Smith DH, Stewart W. Blood-Brain Barrier Disruption Is an Early Event That May Persist for Many Years After Traumatic Brain Injury in Humans. J Neuropathol Exp Neurol. 2015;74(12):1147\u0026ndash;57.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003evan Vliet EA, Ndode-Ekane XE, Lehto LJ, Gorter JA, Andrade P, Aronica E, et al. Long-lasting blood-brain barrier dysfunction and neuroinflammation after traumatic brain injury. Neurobiol Dis. 2020;145:105080.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDubois CJ, Yan J, Obenaus A, Badaut J. A Pediatric Concussion Model in Mice: Closed Head Injury with Long-Term Disorders (CHILD). J Vis Exp. 2025(216).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRodriguez-Grande B, Obenaus A, Ichkova A, Aussudre J, Bessy T, Barse E, et al. Gliovascular changes precede white matter damage and long-term disorders in juvenile mild closed head injury. Glia. 2018;66(8):1663\u0026ndash;77.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIchkova A, Rodriguez-Grande B, Zub E, Saudi A, Fournier ML, Aussudre J, et al. Early cerebrovascular and long-term neurological modifications ensue following juvenile mild traumatic brain injury in male mice. Neurobiol Dis. 2020;141:104952.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eObenaus A, Rodriguez-Grande B, Lee JB, Dubois CJ, Fournier ML, Cador M, et al. A single mild juvenile TBI in male mice leads to regional brain tissue abnormalities at 12 months of age that correlate with cognitive impairment at the middle age. Acta Neuropathol Commun. 2023;11(1):32.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeyba K, Paiyabhroma N, Salvas JP, Damen FW, Janvier A, Zub E, et al. Neurovascular hypoxia after mild traumatic brain injury in juvenile mice correlates with heart-brain dysfunctions in adulthood. Acta Physiol (Oxf). 2023;238(2):e13933.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eObenaus A, Noarbe BP, Lee JB, Panchenko PE, Tong F, Noarbe SD, et al. Progressive lifespan modifications in the corpus callosum following a single concussion in juvenile male mice monitored by diffusion MRI. Exp Neurol. 2025;394:115455.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eObenaus A, Ng M, Orantes AM, Kinney-Lang E, Rashid F, Hamer M, et al. Traumatic brain injury results in acute rarefication of the vascular network. Sci Rep. 2017;7(1):239.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZudaire E, Gambardella L, Kurcz C, Vermeren S. A computational tool for quantitative analysis of vascular networks. PLoS ONE. 2011;6(11):e27385.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKarperien A, Ahammer H, Jelinek HF. Quantitating the subtleties of microglial morphology with fractal analysis. Front Cell Neurosci. 2013;7:3.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSalehi A, Jullienne A, Wendel KM, Hamer M, Tang J, Zhang JH et al. A Novel Technique for Visualizing and Analyzing the Cerebral Vasculature in Rodents. Transl Stroke Res. 2018.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYushkevich PA, Piven J, Hazlett HC, Smith RG, Ho S, Gee JC, et al. User-guided 3D active contour segmentation of anatomical structures: significantly improved efficiency and reliability. NeuroImage. 2006;31(3):1116\u0026ndash;28.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWatson C, Janke AL, Hamalainen C, Bagheri SM, Paxinos G, Reutens DC, et al. An ontologically consistent MRI-based atlas of the mouse diencephalon. NeuroImage. 2017;157:275\u0026ndash;87.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNespoli E, Hakani M, Hein TM, May SN, Danzer K, Wirth T, et al. Glial cells react to closed head injury in a distinct and spatiotemporally orchestrated manner. Sci Rep. 2024;14(1):2441.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eObenaus A, Rodriguez-Grande B, Lee JB, Dubois CJ, Fournier M-L, Cador M, et al. A single mild juvenile TBI in male mice leads to regional brain tissue abnormalities at 12 months of age that correlate with cognitive impairment at the middle age. Acta Neuropathol Commun. 2023;11(1):32.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBeauchamp MH, Anderson V, Ewing-Cobbs L, Haarbauer-Krupa J, McKinlay A, Wade SL et al. Early Child Concussion Pediatr. 2024;154(5).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArambula SE, Reinl EL, El Demerdash N, McCarthy MM, Robertson CL. Sex differences in pediatric traumatic brain injury. Exp Neurol. 2019;317:168\u0026ndash;79.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKoerte IK, Schultz V, Sydnor VJ, Howell DR, Guenette JP, Dennis E, et al. Sex-Related Differences in the Effects of Sports-Related Concussion: A Review. J Neuroimaging. 2020;30(4):387\u0026ndash;409.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSharma B, Nowikow C, DeMatteo C, Noseworthy MD, Timmons BW. Sex-specific differences in resting-state functional brain activity in pediatric concussion. Sci Rep. 2023;13(1):3284.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYeates TM, Taylor HG, Bigler ED, Minich NM, Tang K, Cohen DM, et al. Sex Differences in the Outcomes of Mild Traumatic Brain Injury in Children Presenting to the Emergency Department. J Neurotrauma. 2022;39(1\u0026ndash;2):93\u0026ndash;101.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCampolettano ET, Gellner RA, Smith EP, Bellamkonda S, Tierney CT, Crisco JJ, et al. Development of a Concussion Risk Function for a Youth Population Using Head Linear and Rotational Acceleration. Ann Biomed Eng. 2020;48(1):92\u0026ndash;103.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCorsellis JA, Bruton CJ, Freeman-Browne D. The aftermath of boxing. Psychol Med. 1973;3(3):270\u0026ndash;303.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJain S, Margetis K, Iverson LM. Glasgow Coma Scale. StatPearls. Treasure Island (FL)2025.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGreco T, Hovda D, Prins M. The effects of repeat traumatic brain injury on the pituitary in adolescent rats. J Neurotrauma. 2013;30(23):1983\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGreco T, Hovda DA, Prins ML. Adolescent TBI-induced hypopituitarism causes sexual dysfunction in adult male rats. Dev Neurobiol. 2015;75(2):193\u0026ndash;202.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOrtiz JB, Tellez S, Rampal G, Mannino GS, Couillard N, Mendez M et al. Diffuse traumatic brain injury substantially alters plasma growth hormone in the juvenile rat. J Endocrinol. 2024;260(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBai G, Bai L, Cao J, Sun C, Wang S, Yin B, et al. Sex differences in cerebral perfusion changes after mild traumatic brain injury: Longitudinal investigation and correlation with outcome. Brain Res. 2019;1708:93\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHayward NM, Tuunanen PI, Immonen R, Ndode-Ekane XE, Pitkanen A, Grohn O. Magnetic resonance imaging of regional hemodynamic and cerebrovascular recovery after lateral fluid-percussion brain injury in rats. J Cereb Blood Flow Metab. 2011;31(1):166\u0026ndash;77.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen SF, Richards HK, Smielewski P, Johnstrom P, Salvador R, Pickard JD, et al. Relationship between flow-metabolism uncoupling and evolving axonal injury after experimental traumatic brain injury. J Cereb Blood Flow Metab. 2004;24(9):1025\u0026ndash;36.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNag S, Takahashi JL, Kilty DW. Role of vascular endothelial growth factor in blood-brain barrier breakdown and angiogenesis in brain trauma. J Neuropathol Exp Neurol. 1997;56(8):912\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShore PM, Jackson EK, Wisniewski SR, Clark RS, Adelson PD, Kochanek PM. Vascular endothelial growth factor is increased in cerebrospinal fluid after traumatic brain injury in infants and children. Neurosurgery. 2004;54(3):605\u0026ndash;11. discussion 11\u0026thinsp;\u0026ndash;\u0026thinsp;2.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao ZA, Yan L, Wen J, Satyanarayanan SK, Yu F, Lu J, et al. Cellular and molecular mechanisms in vascular repair after traumatic brain injury: a narrative review. Burns Trauma. 2023;11:tkad033.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSalehi A, Jullienne A, Baghchechi M, Hamer M, Walsworth M, Donovan V, et al. Up-regulation of Wnt/beta-catenin expression is accompanied with vascular repair after traumatic brain injury. J Cereb Blood Flow Metab. 2018;38(2):274\u0026ndash;89.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChoy M, Dube CM, Patterson K, Barnes SR, Maras P, Blood AB, et al. A novel, noninvasive, predictive epilepsy biomarker with clinical potential. J neuroscience: official J Soc Neurosci. 2014;34(26):8672\u0026ndash;84.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDuvernoy HM, Delon S, Vannson JL. Cortical blood vessels of the human brain. Brain Res Bull. 1981;7(5):519\u0026ndash;79.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang JH, Badaut J, Tang J, Obenaus A, Hartman R, Pearce WJ. The vascular neural network\u0026ndash;a new paradigm in stroke pathophysiology. Nat Rev Neurol. 2012;8(12):711\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin X, Chen L, Jullienne A, Zhang H, Salehi A, Hamer M, et al. Longitudinal dynamics of microvascular recovery after acquired cortical injury. Acta Neuropathol Commun. 2022;10(1):59.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGriffin AD, Turtzo LC, Parikh GY, Tolpygo A, Lodato Z, Moses AD, et al. Traumatic microbleeds suggest vascular injury and predict disability in traumatic brain injury. Brain. 2019;142(11):3550\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHiles-Murison B, Lavender AP, Hackett MJ, Armstrong JJ, Nesbit M, Rawlings S, et al. Blood-brain barrier disruption and ventricular enlargement are the earliest neuropathological changes in rats with repeated sub-concussive impacts over 2 weeks. Sci Rep. 2021;11(1):9261.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFukuda AM, Adami A, Pop V, Bellone JA, Coats JS, Hartman RE, et al. Posttraumatic reduction of edema with aquaporin-4 RNA interference improves acute and chronic functional recovery. J Cereb Blood Flow Metab. 2013;33(10):1621\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIliff JJ, Chen MJ, Plog BA, Zeppenfeld DM, Soltero M, Yang L, et al. Impairment of glymphatic pathway function promotes tau pathology after traumatic brain injury. J Neurosci. 2014;34(49):16180\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSalehi A, Zhang JH, Obenaus A. Response of the cerebral vasculature following traumatic brain injury. J Cereb Blood Flow Metab. 2017;37(7):2320\u0026ndash;39.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIchkova A, Rodriguez-Grande B, Bar C, Villega F, Konsman JP, Badaut J. Vascular impairment as a pathological mechanism underlying long-lasting cognitive dysfunction after pediatric traumatic brain injury. Neurochem Int. 2017;111:93\u0026ndash;102.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRodriguez-Grande B, Ichkova A, Lemarchant S, Badaut J. Early to Long-Term Alterations of CNS Barriers After Traumatic Brain Injury: Considerations for Drug Development. AAPS J. 2017;19(6):1615\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArmstead WM, Riley J, Vavilala MS. TBI sex dependently upregulates ET-1 to impair autoregulation, which is aggravated by phenylephrine in males but is abrogated in females. J Neurotrauma. 2012;29(7):1483\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSalonia R, Empey PE, Poloyac SM, Wisniewski SR, Klamerus M, Ozawa H, et al. Endothelin-1 is increased in cerebrospinal fluid and associated with unfavorable outcomes in children after severe traumatic brain injury. J Neurotrauma. 2010;27(10):1819\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBadaut J, Blochet C, Obenaus A, Hirt L. Physiological and pathological roles of caveolins in the central nervous system. Trends Neurosci. 2024;47(8):651\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBadaut J, Ajao DO, Sorensen DW, Fukuda AM, Pellerin L. Caveolin expression changes in the neurovascular unit after juvenile traumatic brain injury: signs of blood-brain barrier healing? Neuroscience. 2015;285:215\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTang J, Kang Y, Zhou Y, Shang N, Li X, Wang H et al. TIMP2 ameliorates blood-brain barrier disruption in traumatic brain injury by inhibiting Src-dependent VE-cadherin internalization. J Clin Invest. 2023;134(3).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCorrigan F, Mander KA, Leonard AV, Vink R. Neurogenic inflammation after traumatic brain injury and its potentiation of classical inflammation. J Neuroinflammation. 2016;13(1):264.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBadaut J, Hippauf L, Malinconi M, Noarbe BP, Obenaus A, Dubois CJ. Endocannabinoid-mediated rescue of somatosensory cortex activity, plasticity and related behaviors following an early in life concussion. bioRxiv. 2024.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGupte R, Brooks W, Vukas R, Pierce J, Harris J. Sex Differences in Traumatic Brain Injury: What We Know and What We Should Know. J Neurotrauma. 2019;36(22):3063\u0026ndash;91.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXing D, Nozell S, Chen YF, Hage F, Oparil S. Estrogen and mechanisms of vascular protection. Arterioscler Thromb Vasc Biol. 2009;29(3):289\u0026ndash;95.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBetai D, Ahmed AS, Saxena P, Rashid H, Patel H, Shahzadi A, et al. Gender Disparities in Cardiovascular Disease and Their Management: A Review. Cureus. 2024;16(5):e59663.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArmstead WM, Kiessling JW, Riley J, Kofke WA, Vavilala MS. Phenylephrine infusion prevents impairment of ATP- and calcium-sensitive potassium channel-mediated cerebrovasodilation after brain injury in female, but aggravates impairment in male, piglets through modulation of ERK MAPK upregulation. J Neurotrauma. 2011;28(1):105\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStark MJ, Clifton VL, Wright IM. Sex-specific differences in peripheral microvascular blood flow in preterm infants. Pediatr Res. 2008;63(4):415\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRen D, Fan J, Puccio AM, Okonkwo DO, Beers SR, Conley Y. Group-Based Trajectory Analysis of Emotional Symptoms Among Survivors After Severe Traumatic Brain Injury. J Head Trauma Rehabil. 2017;32(6):E29\u0026ndash;37.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLu J, Roe C, Sigurdardottir S, Andelic N, Forslund M. Trajectory of Functional Independent Measurements during First Five Years after Moderate and Severe Traumatic Brain Injury. J Neurotrauma. 2018;35(14):1596\u0026ndash;603.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCurpen PP, To XV, Lu M, Winter C, Bellapart J, Newcombe VF et al. Trajectories of Glasgow Outcome Scale-Extended after traumatic brain injury: an analysis of the TRACK-TBI cohort. J Neurol Neurosurg Psychiatry. 2025.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChowdhury SH, Chen LK, Hu P, Badjatia N, Podell JE. Group-Based Trajectory Modeling Identifies Distinct Patterns of Sympathetic Hyperactivity Following Traumatic Brain Injury. Neurocrit Care. 2025;42(3):985\u0026ndash;95.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKeenan HT, Clark AE, Holubkov R, Cox CS Jr., Ewing-Cobbs L. Trajectories of Children's Executive Function After Traumatic Brain Injury. JAMA Netw Open. 2021;4(3):e212624.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCairns K, Beaulieu-Bonneau S, Jomphe V, Lamontagne ME, de Guise E, Moore L, et al. Four-Year Trajectories of Symptoms and Quality of Life in Individuals Hospitalized After Mild Traumatic Brain Injury. Arch Phys Med Rehabil. 2025;106(3):358\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"vessels, magnetic resonance imaging, closed head injury, vessel painting, Evans Blue ","lastPublishedDoi":"10.21203/rs.3.rs-8622019/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8622019/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eBackground\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTraumatic brain injury (TBI) can induce alterations to the blood\u0026ndash;brain border (BBB) that contributes to long-term neurological and behavioral deficits. The temporal progression of post-concussion BBB dysfunction during developmentally sensitive periods remains poorly understood. Therefore, we sought to characterize the temporal evolution of BBB disruption and cerebrovascular alterations acutely after concussion in juvenile mice.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePostnatal day 17 (PND17) C57BL/6J male and female mice were subjected to sham or single closed head injury with long-term disorders (CHILD). At 1h, 6h, 1d, 3d, and 7d post-injury, Evans blue (EB) dye was administered intravenously to evaluate BBB permeability, followed by vessel painting to visualize modified cerebrovascular angioarchitecture. MRI-based T2 relaxation mapping at 1dpi has been used for brain tissue properties, including edema. EB and vascular features were modeled to assess ability to discriminate between sham and CHI mice.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA single early-life concussion induced hyper-acute (hours) structural and functional alterations in brain vasculature. CHILD in PND17 mice resulted in: 1) disruption of physiological functions and developmental trajectories, 2) reduced brain volumes and sex-dependent T2 relaxometry changes (elevated in females, reductions in males), and 3) hyper-acute BBB increases in permeability which correlated with cerebral vascular rarefaction. Notably, males exhibited more robust BBB and vascular perturbations than females, revealing sex-dependent trajectories of vascular response to CHILD. We also highlight differential vulnerability in vessel location with the smaller penetrating cortical vessels displaying greater susceptibility to alterations compared to larger, more resilient pial blood vessels. Modeling demonstrated that vascular features clustered together while trajectory analysis confirmed that female CHI mice were not consistent in their disease trajectory compared to male CHI. Additional analysis suggested that vascular features able to discriminate in a sex- and injury specific manner.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusions\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA single concussion is sufficient to induce hyper-acute BBB and cerebrovascular perturbations in juvenile mice, which may presage long-term deficits during development. Importantly, sex differences in vascular TBI responses evident at PND17 emphasize the need to consider sex as an important variable in future pediatric TBI research.\u003c/p\u003e","manuscriptTitle":"A Single Concussion in Juvenile Mice Leads to Sex Specific Acute Cerebral Vascular Dysfunction and Blood-brain Border Dysfunction","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-11 16:32:02","doi":"10.21203/rs.3.rs-8622019/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"35e2fd2d-aad2-42ee-ba70-eac13995b38a","owner":[],"postedDate":"February 11th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-09T15:56:03+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-11 16:32:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8622019","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8622019","identity":"rs-8622019","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}