Title Page:



Title Page:

CHANGES DURING TIME IN MR IMAGES OF EQUINE ISOLATED LIMBS REFRIGERATED AT 4°C.

Géraldine Bolen, Dimitri Haye, Robert Dondelinger, Valeria Busoni

Running head: MR CHANGES OF REFRIGERATED EQUINE LIMBS.

Abstract

When ex-vivo magnetic resonance (MR) imaging studies are undertaken, conservation of the specimen may be critical and should be taken into account when interpreting MR imaging results. The purpose of this study was to assess changes of MR signal during time in the anatomical structures of the equine digit on cadaver limbs stored at 4°C. Four feet were scanned immediately after the horse death (T0), 24 hours (T24h), 48 hours (T48h), 1 week (T1w) and 2 weeks (T2w) after death. Sequences used were Turbo Spin Echo (TSE) T1, TSE T2, Short Tau Inversion Recovery (STIR) and Double Echo Steady State (DESS). Images obtained at different times after death were subjectively compared side by side and a quantitative analysis was realized by drawing regions of interest (ROI) to assess signal changes between examinations. A mild size reduction of the synovial recesses was detected subjectively. No signal change was subjectively seen between examinations except for bone marrow that appeared slightly hyperintense in STIR and slightly hypointense in TSE T2 sequence at T24h compared to T0. Quantitative analysis demonstrated no changes in the TSE T1 sequence while a significant change was observed in bone marrow signal between T0 and T24h in TSE T2 sequence. No change in bone marrow signal was statistically demonstrated in the STIR sequence.

Introduction

Pain located in the digit is a common cause of forelimb lameness in the horse.1 The equine digit is composed by several anatomical structures which are partially contained within the horny hoof. The presence of the hoof complicates the routine clinical and imaging examination of these anatomical structures by palpation, radiography and ultrasonography.1 For this reason, because of its cross sectional ability and its high contrast resolution, the use of magnetic resonance (MR) imaging to evaluate the equine digit is increasing.2-25 A large number of MR imaging examinations are undertaken under general anesthesia, which represents a risk for the horses.26 In some equine clinics, standing MR units are used on the sedated equine patients.12,13,20,24,25,27,28

Because of the costs of in vivo MR imaging examination, because of the need to compare MR findings to histopathology or because of late evening availability of the MR units for research purposes, a lot of experimental studies on MR imaging have been undertaken on cadaver limbs.2,3,7,10,15,16,29-39 Some of these studies had been realized on frozen limbs.7,15,16,22,29,37,38 Because frozen tissue does not contain sufficient mobile protons to generate a radiofrequency signal, specimens must be thawed prior to imaging.31 No differences have been detected between ante-mortem and post-mortem examinations on the same feet when it was possible to compare.15 When in an ex-vivo study several diagnostic techniques should be applied and realized at different geographic sites or at different times, preservation of the specimen is critical.40 A technique for preserving equine cadaver specimens has been described.31 Preservation of the cadaver specimens was obtained by sealing the limbs with a paraffin-polymer combination.31 Although this technique allows multiple freeze-defrost cycles to be performed on the same specimen without degrading image quality, the method is relatively time-consuming.31

Because the equine distal limbs contains no muscles and the distal extremity is embedded in the hoof, we hypothesized that simple refrigeration at 4°C could be used to preserve equine limbs without major changes in MR signal of the digital anatomical structures. This technique will permit a delay examination without freezing-thawing process and without using paraffin.

The purpose of this study was then to assess changes of MR signal during time in the anatomical structures of the equine digit on cadaver limbs stored at 4°C and imaged just after death and 24 hours, 48 hours, 1 week and 2 weeks after the horse’s death.

Materials and Methods

Collection and preservation of the limbs

Four fresh equine cadaver forelimbs, considered normal because of their normal appearance at inspection, palpation and radiographic examination, were selected from a series of equine limbs collected at an abattoir. These forelimbs were sectioned at the level of the carpo-metacarpal joint to prevent air introduction around the tendons and in the digital tendon sheath. The proximal end of each specimen was covered by absorbing material and latex glove to prevent blood loss during handling. The shoes were removed if the feet were shoed. The feet were cleaned and excess frog trimmed. The digits were preserved at 4°C in a cold room between the MR imaging examinations.

MR imaging examination

The feet were not warmed at room temperature before each MR imaging examination in order to decrease the degradation of the tissues. MR images of the specimens were acquired with a human knee radiofrequency coil in a 1.5-T magnetic field (Siemens Symphony 1.5T, Siemens S.A., Bruxelles, Belgium). Sequences used were: Turbo Spin Echo (TSE) T1-weighted in a transverse plane, TSE T2-weighted in a sagittal plane, Short Tau Inversion Recovery (STIR) in a sagittal plane and water-excited 3D Double-Echo Steady State (DESS) in a dorsal plane. Parameters used are reported in Table 1. The transverse plane was oriented perpendicular to the proximodistal axis of the distal sesamoid bone (DSB), the dorsal plane was oriented parallel to the proximodistal axis of the DSB. Imaging series were obtained by the same technologist together with the first author by manually selecting the section prescription on the basis of a three-plane localizer series. Anatomic features visible on the localizer images were used as reference points. The digits were positioned with the dorsal aspect on the table to avoid the magic angle effect.33 The feet were scanned immediately after the horse death (T0) and then 24 hours (T24h), 48 hours (T48h), 1 week (T1w) and 2 weeks (T2w) after death.

MR images analysis

Images obtained at different times after death were subjectively compared by one operator side by side to assess visual differences. The reader was asked to define the signal of each anatomical structure (trabecular bone, synovial recesses, digital cushion, deep digital flexor tendon – DDFT) at T24h, T48h, T1w and T2w, as being isointense, hypointense or hyperintense to the signal of the same structure at T0 and to see if there was any change in size of the synovial recesses with time.

A quantitative analysis was realized to compare the signal change between examinations and by drawing regions of interest (ROI) in each sequence. ROI were drawn in trabecular bone of the distal phalanx (P3), the middle phalanx (P2) and the DSB, in the palmar proximal recesses of the distal interphalangeal joint (DIPJ), in the digital cushion and in the DDFT. Size of each ROI was decided subjectively in relation to the size of the anatomical structure to be evaluated. A ROI of 1 cm2 was drawn in the distal half of P2, just proximal to the distal subchondral bone plate, in the sagittal area. A ROI of 0.5 cm2 was drawn in the proximal half of P3, just distal to the proximal subchondral bone plate, in the sagittal area. For the DSB, a ROI of 0.2 cm2 was drawn in the middle of the trabecular bone. A ROI of 0.1 cm2 was used to assess the palmar proximal recesses of the DIPJ in the sagittal area. The digital cushion was assessed palmar to the collateral sesamoidean ligaments (CSL) in the sagittal area by drawing a ROI of 2 cm2. A ROI of 0.1cm2 was drawn in the DDFT proximal to the CSL in the sagittal area.

A linear model with a mixed procedure was realized using a SAS software (SAS Institute, Inc., Gary, North Carolina, USA) to test statistical significance of signal changes (P ................
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