Neurocranial growth in the OIM mouse model of osteogenesis imperfecta

Osteogenesis imperfecta (OI) is a disorder of type I collagen characterized by abnormal bone formation. The OI craniofacial phenotype includes midfacial underdevelopment, as well as neurocranial changes (e.g., macrocephaly and platybasia) that may also affect underlying nervous tissues. This study aims to better understand how OI affects the integrated development of the neurocranium and the brain. Juvenile and adult mice with OI (OIM) and unaffected wild type (WT) littermates were imaged using in vivo micro‐computed tomography (microCT). Virtual endocast models were used to measure brain volume, and 3D landmarks were collected from the cranium and brain endocasts. Geometric morphometric analyses were used to compare brain shape and integration between the genotypes. OIM mice had increased brain volumes (relative to cranial centroid size) only at the juvenile stage. No significant difference was seen in cranial base angle (CBA) between OIM and WT mice. However, CBA was higher in juvenile than in adult OIM mice. Brain shape was significantly different between OIM and WT mice at both stages, with OIM mice having more globular brains than WT mice. Neurocranial and brain morphology were strongly integrated within both genotypes, while adult OIM mice tended to have lower levels of skull‐brain integration than WT mice. These results suggest that neurocranial dysmorphologies in OI may be more severe at earlier stages of postnatal development. Decreased skull‐brain integration in adult mice suggests that compensatory mechanisms may exist during postnatal growth to maintain neurological function despite significant changes in neurocranial morphology.


| INTRODUCTION
Osteogenesis imperfecta (OI) is an inherited disorder of type I collagen, the organic component of the extracellular matrix that provides bone with toughness or resistance to fracture.Low levels and abnormal synthesis of type I collagen results in bones that fracture easily and give the disorder the alternate name of brittle bone disease.Craniofacial phenotypes associated with OI include underdeveloped facial skeletons, progressive hearing loss, dental malocclusions (Arponen, 2012;Cheung et al., 2011;Marini, 1988;O'Connell & Marini, 1999).Abnormalities of the neurocranial skeleton are also present in OI.Cranial base flattening (platybasia) and basilar invagination, or the upward movement of the second cervical vertebrae into the base of the skull, have been documented (Arponen, 2012;Cheung et al., 2011).Cranial vault abnormalities are also seen, including macrocephaly (enlargement of head circumference), an increased incidence of sutural (Wormian) bones, and delayed closure of fontanelles (Cremin et al., 1982;Tsipouras et al., 1986).
These craniocervical abnormalities are widely thought to be related to skeletal dysplasia and force induced deformation in OI (Arponen, 2012).During growth, the weight of the brain may compress the cranial base, causing microfractures resulting in a relative upward migration of the craniocervical structures (Frank et al., 1982;Sillence, 1994).Basilar invagination and basilar impression, which encompass skeletal malformations characterized by translocation of the upper cervical spine and clivus into the foramen magnum, have also been documented in children and adults with OI type types I, III, and IV (Arponen, 2012).
The craniocervical abnormalities seen in OI may also influence cranial vault and brain morphology.Macrocephaly, a condition in which circumference of the human head is abnormally large, may result from shortening of the cranial base in the rostrocaudal direction.This in turn may lead to brain growth in the superior-inferior and medial-lateral directions as a compensatory mechanism (Cheung et al., 2011;Tsipouras et al., 1986).Ventricular dilation and an increase in intracranial pressure are also seen in patients with OI (Tsipouras et al., 1986).
The interrelationship of the brain and skull during growth through genetic, epigenetic, and functional mechanisms is known as integration (Richtsmeier et al., 2006;Smith, 1996).Quantifying integration provides an avenue to better understand the reciprocal influences of brain and skull tissues during development, and may shed light on the etiologies of craniofacial disorders such as craniosynostosis (Motch Perrine et al., 2017;Richtsmeier & Flaherty, 2013) and OI.Here, we use the osteogenesis imperfecta murine (OIM) mouse model to investigate the role of brain-skull integration in the emergence of OI craniofacial phenotypes.
OIM mice are characterized by a spontaneous nucleotide deletion which causes a frameshift in the COL1A2 gene resulting in the absence of functional α2(I) chains of type I collagen (Gentry et al., 2010).Prior studies have demonstrated that adult OIM mice have craniofacial phenotypes similar to those of humans with severe (type III) OI (Menegaz et al., 2020;Steele, 2020).The OIM craniofacial phenotype includes an overall smaller skull, decreased relative facial length, increased relative neurocranial height and width, and decreased relative basicranial length (Menegaz et al., 2020;Steele, 2020).The changes observed in the facial skeleton may be related to structural changes in the neurocranium, as cranial base growth precedes facial growth during early development.This study aims to better understand how OI affects the integrated development of the neurocranium and the brain in the OIM mouse model.
We investigate the neurocranial growth of OIM mice from weaning (postnatal week 4) to skeletal maturity (week 16).We hypothesize that, compared to unaffected mice, OIM mice will have: (1) increased brain volume and macrocephaly (enlargement of the cranial vault due to shortening of the cranial base in the rostrocaudal direction); (2) increased cranial base angles (CBAs), resulting in platybasia; (3) altered brain shape due to differences in skull growth patterns; and (4) different integration patterns between vault shape and brain shape.

| Experimental model
The osteogenesis imperfecta murine (OIM or B6C3FE a/a-Col1a2 OIM/ J; strain #001815, Jackson Laboratories) is a mouse strain with a nonlethal recessive inherited mutation of the COL1A2 gene first described by Chipman et al. (1993).The homozygous OIM mouse (B6C3FE a/a-Col1a2 OIM/OIM ) used in this study is commonly used as a model for the human presentation of severe type III OI.OIM mice demonstrate high fracture rates, severe osteopenia, small body sizes, and both postcranial and craniofacial phenotypes similar to those seen in humans with OI type III (Chipman et al., 1993;Gentry et al., 2010;Menegaz et al., 2020;Misof et al., 1997;Phillips et al., 2000).Unaffected, wild type (WT) littermates were used as the control in this study.

| Animal husbandry
All procedures and animal care were approved by the Indiana University School of Medicine Institutional Animal Care and Use Committee.WT and homozygous OIM mice were bred in-house at the Indiana University School of Medicine and maintained on a C57BL/6J background (Berman et al., 2020).Mice were group housed in a facility with 12-h light/dark cycles and access to food and water ad libitum.Environmental enrichment objects were of a size and shape (e.g., large spheroids like toy balls) chosen to limit opportunities for non-feedingrelated biting and chewing behaviors.For this experiment, OIM and WT littermates were raised from weaning (postnatal day 21, the beginning of week 4) to skeletal maturity (week 16) (Table 1).This period encompasses the early postnatal brain growth period (birth-6 weeks), the completion of sutural fusion in the neurocranium ($45 days), and the stabilization of adult cranial and brain morphology (after 3 months) in mice (Bradley et al., 1996;Hammelrath et al., 2016).

| MicroCT imaging
All animals were imaged in vivo using micro-computed tomography (microCT) at weeks 4 and 16 through the Indiana University Center for Musculoskeletal Health.MicroCT scans were acquired using a Skyscan 1176 microCT machine (Bruker Corp, MA, USA) operated at 41-65 kV and 385-500 μA.Scans were reconstructed using Skyscan NRecon software (Bruker Corp) into 16-bit TIF stacks using 0.008 or 0.017 mm 3 voxels, depending on cranial length.Image stacks were then cropped to remove excess air space around the specimen using Ima-geJ (Schneider et al., 2012).
During imaging, animals were anesthetized via inhalation anesthesia using an isoflurane nonrebreathing system with a 3%-5% flow rate and maintained at 1.5% for the duration of the scan.Animals were secured to the scanning bed using standard non-porous medical tape to minimize movement during imaging.During scanning, animals were monitored using a camera inside of the microCT machine.Vitals (heart rate, respiration rate) were also monitored from the control computer and isoflurane flow rate adjusted as needed.Following the scan, animals were allowed to fully recover on room air in an empty cage.Once sternal recumbency was achieved, the animal was returned to its home cage.

| Endocranial modeling
The imaging software 3D Slicer 4.11 (Kikinis et al., 2014) was used to create digital models of the skull from microCT scans (Figure 1; Figure S1).A single user (ATS) created all skull models based on a qualitative analysis of the thresholds at which cortical bone was selected and the resulting models.Next, a single user (TSH) created models of the endocranial cavity (endocasts) following Kerney (2022) (Figure 1; Figure S1) and endocast volumes were generated using the Segmentation Statistics module.Because the microCT scans used in the study were optimized for the visualization of bone and not soft tissue, endocast volumes were used as a proxy for brain volume.We recognize that total endocranial volume may also include other soft tissues such as meninges and vasculature (Watanabe et al., 2019).
In order to investigate the effects of scaling on brain size, endocast volumes were scaled to cranial centroid size.Centroid size was calculated as the square root of the sum of squared differences of 72 neurocranial and face landmarks (Table S1).Because a Shapiro-Wilk test revealed that the data were non-normally distributed, Mann-Whitney U-tests (α = 0.05) were used to compare both unscaled and scaled (to centroid size) endocast volumes in OIM versus WT littermates.

| Cranial base angle data collection
CBA was calculated by using the angle tool in 3D slicer using three established CBA landmarks: foramen cecum (FC), mid-sphenoidal synchondrosis (MSS), and basion (bas, midline point on the anterior margin of the foramen magnum) (Lieberman et al., 2000) (Figure 2).To ensure precision of the CBA measurement, the mean of three replicated measures was used for each individual.Because a Shapiro-Wilk test revealed that the data were non-normally distributed, Mann-Whitney U-tests (α = 0.05) were used to compare CBAs in OIM versus WT littermates.

| Brain shape & skull-brain integration data collection
Brain shape was assessed using coordinate data from 28 homologous landmarks on the virtual endocasts (Figure 3; Table 2) (see Weisbecker et al., 2021).Prior to landmarking the entire sample, we first conducted a repeatability study using three specimens and three replicates per specimen to ensure correct placement of landmarks.A Procrustes ANOVA was then used to evaluate the role of inter-replicate variation to total variation.An r 2 value for brain landmark placement was 0.95665, with 95.66% of the variation being intra-individual and the remaining 4.34% of variation due to inter-replicate variation.
In order to investigate differences in brain shape between the OIM and WT mice, General Procrustes analysis (GPA) and principal component analyses (PCA) were performed in the Geomorph package in R Studio (Adams et al., 2021).A Procrustes ANOVA was used to test for significant differences in brain shape between the genotypes (α = 0.05).To visualize differences in brain shape between the genotypes, two methods were used.First, the plotRefToTarget function in the Geomorph package was used to generate PC extreme warps (e.g., minimum/maximum scores of PC1) at a 3Â magnification.Second, the coordinates.differenceand procrustes.var.plotfunctions in the landvR package (Guillerme & Weisbecker, 2019) were used to visualize landmark variation in 3D space at a 1Â magnification.
Developmental integration of the brain and the neurocranium was quantified using the "integration.test"function in the Geomorph package in R Studio.This function quantified the strength of integration between the brain (n = 28) and neurocranial (n = 24) landmark datasets (Figure 3; Table 2 and TableS1) using a twoblock PLS analysis (or singular-warp analysis in the case of this analysis) (Bookstein et al., 2003).The average pairwise r-PLS correlation functioned as the test statistic which measured integration within genotypes.Significant integration was determined when this test statistic was larger than the permuted null distribution (Evans et al., 2017).Effect size correlation functioned as the test statistic which compared integration between genotypes.The "compare.pls"function to statistically compare brain-skull integration between the genotypes (α = 0.05).3 | RESULTS

| Brain volume
Juvenile OIM mice were found to have significantly smaller brains than their WT littermates (p = 0.008) (Figure 4a; Table 3).Adult OIM mice were also found to have smaller brains than their WT littermates, however this trend was not significant (p = 0.063).Similarly, juvenile (p < 0.001) and adult (p = 0.003) OIM mice also have significantly smaller crania than their WT littermates (Figure 4b; Table 3).When brain size was scaled to cranial centroid size, juvenile OIM mice had relatively larger (macrocephalic) brains compared to their WT littermates (p = 0.016) (Figure 4c; Table 3).No significant difference was found between scaled adult OIM and WT mice brain size (p = 0.342) (Figure 4c; Table 3).Thus, relative brain sizes are significantly different between OIM and WT mice only in juveniles.In adults, brain volumes (relative to cranial size) are similar between OIM and WT mice.

| Cranial base angle
CBA in juvenile OIM mice tended to be larger than in WT mice, however this difference was not statistically significant (p = 0.065).No significant differences were seen in CBA at the adult (p = 0.171) stages.As OIM mice aged, CBA decreased until the angles were normalized relative to the WT control by adulthood (Figure 5; Table 4).
The cumulative effect of these shape changes (increased width and height, decreased length) resulted in juvenile OIM mice having more globular brains compared to WT mice.Additionally, adult OIM and WT mice had significantly different brain shapes ( p = 0.001) (Figure 6b).The genotypes were separated by the first principal component (25.26% variance).PC1 described variance in the following dimensions of brain shape: bitemporal brain width (landmarks 9-10, 12-13), midsagittal brain height (landmarks 1-3), and rostrocaudal brain length (landmarks 4-5, 8, 11, 20-23, 25-28) especially at the olfactory bulb (4-5) (Figure S2).As in juvenile mice, the cumulative effect of these shape changes (increased width and height, decreased length) resulted in adult OIM mice having more globular brains compared to WT mice.While the specific shape dimensions identified by the PCA differed between juvenile and adult analyses, the overall result of OIM having more globular brains was similar between the age groups.

| Skull-brain integration
Juvenile brain shape and neurocranial shape were strongly and significantly integrated within each genotype, signified by r-PLS values close to 1 (Table 5).Zscores indicated that juvenile OIM (z-score = 3.093) and WT (z-score = 2.768) mice had similar levels of brainskull integration ( p = 0.270).
Adult brain shape and neurocranial shape were also strongly and significantly integrated within each genotype signified by r-PLS values near or equivalent to 1 (Table 5).However, adult OIM mice (z-score = 1.671) tended to have lower levels of brain-neurocranial integration than adult WT mice (z-score = 2.540) although these differences were not significant (p = 0.522).Effect size, defined as the strength of integration between the brain and neurocranial landmark datasets, was compared between the genotypes, with genotypic differences in integration only observed in the adult mice (Figure 7).

| DISCUSSION
The aim of this study was to better understand how OI affects the integrated development of the neurocranium and the brain.First, we hypothesized that mice with OI would have increased brain volumes related to macrocephaly, or the enlargement of the cranial vault.Our results partially supported this hypothesis, with OIM mice having relatively larger endocranial volumes than their WT littermates at the juvenile stage only.Previous work in humans proposed that the basicranial shortening seen in patients with type III OI resulted in compensatory vertical and horizontal growth, resulting in the macrocephaly seen in these patients (Cheung et al., 2011;Tsipouras et al., 1986).This hypothesis has been supported by studies demonstrating that OIM mice display increased neurocranial dimensions and basicranial shortening at both the juvenile and adult stages (Menegaz et al., 2020;Steele, 2020).While we found that the brains of OIM mice are rostrocaudally shorter, dorsoventrally taller, and laterally wider than those of WT mice (see shape changes discussed below), this is accompanied by endocranial volumetric changes only in juvenile mice with no differences in volume seen for adults.This discrepancy could potentially result from variation in methodologies.Here, we examined macrocephaly by measuring endocranial volumes while previous studies of mice (Menegaz et al., 2020) and humans (Arponen, 2012;Tsipouras et al., 1986) examined macrocephaly via external measurements of the neurocranial skeleton.Alternatively, compensatory growth patterns in postnatal mice could result in macrocephaly while preserving relative brain volume by the adult stage.
Second, we hypothesized that mice with OI would have increased CBA compared to unaffected mice, suggesting that OIM mice experience platybasia (skull flattening) similar to human patients with OI.Our results only partially supported this hypothesis.Juvenile OIM mice tended to have an increased CBA relative to WT mice, however, this difference disappeared by the adult stage.Previous studies in humans have suggested a positive correlation between endocranial volume and basicranial flexion (Jeffery, 2003;Jeffery & Spoor, 2002).However, this hypothesis has yet to be fully examined in mice, in part due to their quadrupedal stature.As we found no difference in relative endocranial volume or CBA between adult OIM and WT mice, our data are unable to address this hypothesis.OIM mice are potentially a poor model for basicranial flattening in adult humans with OI due to the differences between quadrupedal and bipedal postures.Third, we hypothesized that mice with OI would have altered brain shape related to differences in skull growth patterns compared to unaffected mice.Previous studies have assessed skull growth patterns in OIM mice compared to their WT littermates and documented  basicranial shortening, cranial vault expansion, and significant skull shape changes (Menegaz et al., 2020;Steele, 2020).We expected that these differences in skull growth would also be reflected in brain shape.Our results supported our hypothesis as they indicated that both adult and juvenile OIM mouse brains were rostrocaudally shorter particularly in the olfactory region, dorsoventrally taller, and slightly wider.Increases in height and width may reflect compensatory growth in response to shortening of the brain in the rostrocaudal dimension.
Localized changes in the olfactory region suggest that more modular, isolated, changes (sensu Klingenberg, 2013) may contribute to this area of the brain being differentially affected during growth.However, due to the use of endocasts, dividing the landmarks into regional subunits (modules) proved difficult and we were not able to examine the effects of modularity in this mouse model.Future studies should focus on methodological improvements (e.g., the use of contrast-enhanced CT scanning) in order to improve morphometric analyses of soft tissue structures including the brain.Finally, we predicted that compared to unaffected mice, mice with OI would have different patterns of integration between neurocranial and brain shape.Previous studies found significant morphometric differences between OIM and WT mouse skulls (Menegaz et al., 2020;Steele, 2020), with some localized changes along the coronal and sagittal sutures (unpublished data).These regions of skeletal change corresponded to the regions we observed changing with brain shape, leading us to investigate the potential role of integration in brainskull interactions during growth.We found that brain  shape and skull shape were strongly and significantly integrated within each genotype (r-PLS 0.9-1.0).While there were no significant differences in the effect size of integration between juvenile OIM and WT mice, adult OIM mice tended to have lower levels of brain-skull integration in comparison to their WT littermates.Lower integration levels in adult OIM mice may reflect a decoupling between external and internal cranial morphology in this genotype, for example, in the differences between macrocephaly as determined by external (Menegaz et al., 2020) versus internal cranial morphology (this study).These results suggest that overall brain shape may be more independent from skull shape in adult OIM mice compared to WT mice, allowing for compensatory growth mechanisms to maintain neurological function despite significant changes in neurocranial morphology.

| CONCLUSION
Neurocranial dysmorphologies such as macrocephaly may be more severe at earlier stages of postnatal development in OIM mice.The decreased integration of skull and brain shape seen in adult OIM mice may explain the lack of differences in relative brain volume and CBAs between genotypes following skeletal maturity.Due to low integration levels in adult mice, brain shape and skull shape are less interdependent in OIM mice compared to WT mice.This may allow for the normalization of CBAs and brain volumes during growth, as these variables appear to "catch up" to WT norms by the adult stage.These findings provide a better understanding of how OI affects the integrated development of the neurocranium and the brain in the OIM mouse model.This study focused on the development of neurocranial abnormalities in OI between weaning and adulthood.At weaning (postnatal day 21), juvenile OIM mice already exhibit significantly difference brain morphology and endocranial volumes relative to WT mice.Future studies could investigate earlier growth patterns, as significant neurocranial growth occurs before weaning (Jin et al., 2016;Maga, 2016).Currently, treatment options for cranial base and vault abnormalities in OI are limited to shunting for patients with co-occurrent hydrocephaly, or traction and surgical fixation for patients with basilar invagination (Arponen, 2012).A better understanding of the patterns and timing of skull and brain growth in OI will aid the future development and implementation of interventions to improve craniofacial outcomes in these patients.F I G U R E 7 Effect size, defined as the strength of integration between the brain and neurocranial landmark datasets between osteogenesis imperfecta murine (OIM) and wild type (WT) mice at week 4 and week 16.
Cranial segmentation and model of a 16-week old mouse in (a) transverse view, (b) sagittal view, (c) coronal view, and (d) rendered 3D model.Cranial base angle as measured in a 16-week-old mouse based on three landmarks: foramen cecum (FC), midsphenoidal synchondrosis (MSS), and the basion (bas).

F
I G U R E 3 Superior views of endocast (left) and neurocranial (right) landmarks.
U R E 4 (a) Brain volumes, (b) cranial centroid sizes (CCS), and (c) relative brain volumes scaled to CCS in osteogenesis imperfecta murine (OIM) and wild type (WT) mice at week 4 and week 16.*p ≤ 0.05.
Sample size.
T A B L E 1Abbreviations: OIM, osteogenesis imperfecta murine; WT, wild type.HUSAIN ET AL.
Brain volume in OIM and WT mice.
Cranial base angles in OIM and WT mice.