MR microscopy of the developing upper extremity of the chicken in ovo using 7 Tesla MRI

MR microscopy (MRM) is known as ultra‐high‐field (UHF) magnetic resonance imaging with an in‐plane spatial resolution of <100 μm, yields highly resolved non‐invasive anatomical imaging and allows longitudinal assessment of embryonic avian development. The aim of the present study was to evaluate the feasibility of in vivo anatomical MRI assessment of the developing upper extremity of the chicken. Thirty‐eight fertilized chicken eggs were examined at 7 Tesla acquiring high‐resolution T2‐weighted images with an in‐plane resolution of 74 × 74 μm. To reduce motion artefacts, the eggs were moderately cooled before and during MRI. Development of the upper extremity was anatomically and quantitatively assessed. Chondrification and ossification on MRI were correlated with histological examination. MRM allowed the identification of the embryo from stage D5 onwards. First chondrification of the upper extremity was visible at stage D7, and the differentiation of the forearm was possible from stage D9 throughout the developmental period with excellent correlation to histology. MRM also allowed the differentiation between cortical and medullary bone as well as the detection of chondrified areas. UHF MRM allows the in vivo and in ovo evaluation of the upper limb during embryonic development and provides non‐invasive longitudinal anatomical information. This technique allows longitudinal studies of the same embryo during the developmental period and may therefore provide further insights into the development of the upper extremity. With improved coil technique and increasing availability of UHF MR systems, there is great potential regarding several research topics in experimental musculoskeletal radiology.

. However, in the latter case, for these studies, embryological development had to be terminated. Therefore, repeated examination of the same embryo for longitudinal studies is not possible.
The developing chicken is a well-established model in the field of embryological research Li et al., 2007;Stern, 2004). Due to the accessibility and economic availability of fertilized chicken eggs, the in ovo embryo has become a widely used animal model also regarding limb development (Smith et al., 2013).
The developmental stages and morphology of chick limbs match the typical plan of tetrapode appendicular skeleton with the exception that there are only three digits in the wing and four in the leg (Davey et al., 2018). Chick limbs arise from populations of mesenchymal cells that are surrounded by ectoderm in the limb buds; these buds elongate and differentiate into limb-like structures that further elongate (Tickle, 2004). The long bones develop via enchondral ossification as mesenchymal cells differentiate into chondrocytes, which are later replaced by osteoblasts (Daumer et al., 2004).
Magnetic resonance imaging (MRI) is a non-invasive imaging modality, which allows multiplanar imaging with excellent soft tissue contrast. With increasing field strength of the MR system, spatial resolution can be improved. Ultra-high-field MRI (UHF MRI) at 7 Tesla (T) with an in-plane spatial resolution of <100 μm is known as MR microscopy (MRM) Niendorf et al., 2014).
In recent studies, we demonstrated that MRM is capable of evaluating the development of the chicken in ovo through the entire development period without affecting normal development Streckenbach et al., 2018). Our recent studies focussed especially on the development of the eyes Lindner et al., 2017;Streckenbach et al., 2018). Therefore, we want to use the established model of chicken embryos to investigate to what extent other organ systems can be assessed using MRM. Taking the upper extremity of the chicken as an example, this study explores the potential of MRM for the in vivo assessment of the skeletal development of the chicken in ovo and discusses its correlation with histopathological findings.

| Animal model
Thirty-eight fertilized White Leghorn chicken eggs (Valo BioMedia) were simultaneously incubated at optimal conditions (37.8°C, 60% relative humidity) using an incubator (Heka-Turbo 168; HEKA). The eggs were divided into two groups: Two eggs were scanned every day (Group A) and 36 eggs were scanned only at one time point between day 1 (D1) and day 20 with two eggs at each time point (Group B). In the latter group incubation of the eggs was terminated immediately after imaging by cooling the embryos on crushed ice for 60 min with following decapitation. Afterwards, embryos underwent histological workup.
In the former group, incubation was terminated on day 20 and the fetal chicken was euthanized before hatching by cooling the embryos on crushed ice for 60 min with following decapitation. The number of eggs was chosen to minimize the number of embryos needed for the study and to ensure, that in each group is at least one embryo that could be evaluated in case fetal development was terminated by other causes.
The experiments were compliant with national animal welfare legislation.

| MR imaging
MR imaging was performed using a 7T MRI Scanner (ClinScan; Bruker Biospin) with a bore size of 13 cm. During the first 10 days, a 4-channel volume coil (rat body coil) was used for signal detection. Motion artefacts of the embryos increased from D10 significantly and impaired the image quality. Therefore, after this embryonic stage, the eggs were placed on crushed ice starting 10 min prior to MR imaging. Temperature was monitored during the entire scanning time using a fibre-optical thermometer (1025T, Monitoring & Gating system, Small Animal Instruments) as described previously Streckenbach et al., 2018).

| Histopathological evaluation
Both embryos from group B underwent histological workup. The right upper extremity of each of the embryos was examined on days 9-20 and preserved in 4% buffered formalin, respectively.
Subsequently, one of the wings was stained in toto with Alizarin red to visualize ossification areas within the upper extremity. Images were taken under a microscope with 10-fold magnification.
The wing of the second embryo of group B was embedded in paraffin and cut with a microtome at a thickness of 5 μm in accordance with the imaging plane of MRM. The slices were then stained with Alcian blue for the evaluation of the chondrified segments, as well as with haematoxylin/eosin (HE) and with Azan on alternating sections to evaluate the differentiation of nuclear and cytoplasmatic structures and the distribution of collagenous fibres. Afterwards, the wings were photographed under a microscopic view with 20fold magnification.
Normal development of the avian embryo was evaluated as described elsewhere (Streckenbach et al., 2018) using Groups A and B of the same cohort.

| MR image evaluation and estimation of bone length
For further workup, the MR datasets were transferred to a horos workstation (Ver. 3.3.6, Horos Project), and the length of the entire bone was measured manually for radius and ulna in Group B from D9 onwards.
Bone length of radius and ulna was also digitally measured on the specimens that were stained using Alizarin red on days 9-20. All measurements were correlated for corresponding development days 9, 13, 17, 18 and 20.

| DISCUSS ION
Current knowledge of the development of the human upper extremity is based on early descriptive studies of human embryos or experimental studies in animal models with the termination of the developmental period and subsequent histological evaluation (Czerwiński et al., 1996;Fritsch, 2003;Hita-Contreras et al., 2012;Rodríguez-Niedenführ et al., 2001).
The present study demonstrated the feasibility of MRM for the evaluation of the development of the upper limb of the chicken throughout the entire developmental period in ovo and in vivo. Li et al. (2007) evaluated the upper extremity of the chicken using a 7.1 T wide bore nuclear magnetic resonance spectrometer with an additional image gradient system. However, due to the diameter of the bore, only the upper extremity could be imaged and unlike in the present study, the developmental period also had to be terminated.
Furthermore, using MRM we were able to achieve an in-plane spatial resolution of 74 × 74 μm compared with 160 × 160 μm as reported by Li et al. (2007).
The developing upper extremity was first visible on developmental stage D5. Chondrogenesis of the bones is described from developmental stage D6 onwards with ossification starting at stage D8 (Holder, 1978)/stage 32 according to Hamburger and Hamilton (1951).
In the present study, it was possible to assess the entire right upper extremity on the following days: 9, 13, 17, 18 and 20. Due to the fact that the main aim of this chicken embryo cohort was the examination of the eyes, the right upper extremity was just partially displayed on the remaining days from day 5 to 20. This could be a reason why we were not able to identify the ossification centres until day 9. This difference in comparison with Hamburger and Hamilton (1951) might also be due to partial volume effects if the imaging plane was not exactly parallel to the course of the bone.
MRM has demonstrated good correlations with conventional histology for the ex vivo evaluation of the human skeletal development (Inga Langner et al., 2016) and the anatomy of the human finger . These reports are in concordance with the present study. There was a good correlation between the length and location of the ossification centres between MRM and conventional histology. MRM also allowed the differentiation between cortical and medullary bone, as well as the detection of chondrified areas. These imaging findings also correlated well with histological staining.
It is known that the acquisition time of MRM is long. While this is not an issue in ex vivo imaging, especially in advanced developmental stages image quality may be altered by motion artefacts. Therefore, we used mild cooling prior to and during MR imaging to reduce motion artefacts as described elsewhere (Streckenbach et al., 2018). Streckenbach et al. (2018) reported no effect of repeated cooling and exposure to ultra-high-field magnetic field on the normal development of the chicken embryo. This is supported by the findings of our study as the appearance of ossification centres occurred at the same developmental stages as described previously (Hamburger & Hamilton, 1951;Holder, 1978). Correlation of conventional histology with the imaging plane of MRM may be difficult. This limitation is shared by other MRM studies Li et al., 2007;Streckenbach et al., 2018) and could be overcome by the acquisition of a 3D dataset as described elsewhere (Inga Langner et al., 2016). However, this will increase imaging time and subsequently the risk of motion artefacts. Therefore, optimization of imaging acquisition towards faster sequences will be necessary (Setsompop et al., 2016).
In the present study, we acquired only anatomical T2w datasets. MRM is also capable of acquiring functional MR images.
These data may provide further quantitative information on the ultrastructure of the tissue comparable to immune stains or in situ hybridization techniques. The major advantage of MRM is its non-invasiveness. Spatial resolution, however, is still inferior to conventional histology. With further developments in coil and MR technology (Budinger & Bird, 2018;Thylur et al., 2017) this limitation will be overcome. OS, SL, IL and AW involved in the provision of study materials. OS, SL and IL involved in responsibility for the integrity of the work as a whole. All authors read and approved the final manuscript.

ACK N OWLED G M ENTS
All authors meet the criteria for authorship. There was no external financial and material support for this study. Open Access funding enabled and organized by Projekt DEAL.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.