EQUINE VETERINARY EDUCATION / AE / july 2022


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Samples of all three sutures were collected for all horses except the 5-day-old foal, where only the Sutura maxillolacrimalis could be evaluated and the 6-year-old horse, where only the specimens of the internasal and nasofrontal (MMA sample only) sutures could be evaluated. The specimens were fixed in a 4% formaldehyde solution.


2 1 3


The specimens were divided into two pieces: one part was completely decalcified with EDTA for 2 months and then embedded in paraffin. The second part was incompletely decalcified and then infiltrated and embedded in methyl methacrylate (MMA). The paraffin sections were regularly cut in a 90° angle to the suture; after five to 10 serial sections of 2-5 µm, the specimens were adapted so that some sections could be cut parallel and within the suture line. The sections were stained with haematoxylin–eosin as a routine staining. The MMA blocks were cut with a diamond-coated band saw in sections (1 mm thick), mounted on polyacrylate slides and then milled with a microtome milling machine (Leica 2600) to sections of 300 µm, which were stained with Giemsa or Giemsa eosin. The Giemsa staining enables differentiation of bone and


Fig 1: Equine skull showing the location of sample collection of the facial suture lines 1: Sutura internasalis. 2: Sutura nasofrontalis. 3: Sutura maxillolacrimalis.


equally distributed. The foals and horses were patients of the University of Zurich and the owners agreed to the collection of post-mortem specimens. The foals and horses were subjected to euthanasia for reasons unrelated to this study and did not present with any clinical indications of suture exostosis. The horses were not exsanguinated, and suture samples were collected betweenone and 24 h after euthanasia. After removal of the skin and subcutaneous tissue over


the expected suture lines, the bone was exposed until the suture was identified. Then, sections of bone of 1.5 9 1.5 cm were removed from the selected sites with a mallet and hammer, with the suture line in the middle of the sample.


a)


connective tissue and cartilage in MMA sections. The MMA sections were cut in the same directions as the paraffin sections. From the serial sections, which showed very similar results, the qualitative best sections were evaluated independently, but unblinded for age and location of the sample by three authors (L.G., L.K. and H.G.). The sections where the suture lines were clearly visible were analysed for width, form, cell density and morphology, vascularisation and morphology of surrounding tissue. The samples were viewed by the authors under a Leica


DM LB2 light microscope, and digital images were obtained using a Leica DC480 camera mounted on the microscope. Measurements of the sutures were made using a magnification of 950 with light microscopy. The sutures were visually examined and then measured at three locations each that appeared to fulfil the following measurement criteria: wide, narrow and most common width. The widest and narrowest measurements were recorded as well as median of the common width. The interdigitation was evaluated subjectively based on the angle of the suture loops visible in the 950 magnification – that is does the suture make a full 180° turn (tight interdigitation) or only 90° (mild) or somewhere in between (moderate). This is illustrated in Fig 2;


b)


CB


1000 µm


1000 µm


Fig 2: a) Sutura internasalis 1-day-old foal, MMA sample, Giemsa stain. The interdigitation is mild–moderate in this sample. b) Sutura nasofrontalis 6-year-old horse,MMAsample, Giemsa–Eosin stain. The suture is visible along the entire length of the sample and is denoted by an arrow. It is surrounded bymature cancellous bone (CB). The interdigitation is tight in this sample. The interdigitationwas evaluated bymeasuring the approximate angles atwhich the suture changed course (lines); a directional change in 90° or lesswas deemed mild interdigitation, greater than 90° but less than 180°moderateand180° tight interdigitation.


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