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<0.05).
Results:
A minimal difference in section planes was sometimes seen between examinations of the same limb at different times.
Visibility and margination of the anatomical structures of the digits and overall image quality were considered unchanged.
No signal change was subjectively seen except for bone marrow in STIR and in TSE T2 sequences. Subjectively, at T24h, trabecular bone appeared slightly hypointense compared to T0 in the TSE T2 sequence and slightly hyperintense compared to T0 in the STIR sequence (Fig. 1 and Fig. 2). No changes were subjectively seen in bone marrow signal between T24h, T48h, T1w and T2w (Fig. 1 and Fig. 2).
Quantitative analysis demonstrated no statistical change in signal during time in TSE T1 images in any of the anatomical structures evaluated, while a significant change was observed in signal values of the trabecular bone (of P3, P2 and DSB) between T0 and T24h, T0 and T48h, T0 and T1w, T0 and T2w in TSE T2 sequence. No change was statistically demonstrated in trabecular bone in the STIR sequence. The signal in the palmaro-proximal DIPJ synovial recess showed statistically significant changes in TSE T2 sequence between T0 and T24h and between T0 and T2w, and in the STIR and DESS sequences between T2w and T0, T2w and T24h, T2w and T48h, T2w and T1w.
For all synovial recesses assessed, a mild reduction in size of the synovial recesses was found subjectively.
Discussion:
This study presents the MR changes during time in a group of limbs that have been preserved by refrigeration at 4°C before MR imaging. 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, it is often difficult to image the foot just after the horse’s death.40 Some studies had been previously made on frozen limbs to solve this problem.7,15,16,22,37,38 and a technique for preserving equine cadaver specimens using a freezing/thawing process has been described.31 However, the thawing process take time and the MR examination has to be scheduled in order to permit the foot to be completely thawed. The simple method presented in this study has the advantage to be less time consuming than the method previously presented31 and to allow MR examination to be done at any time with no need of limb preparation for the thawing. Freezing and thawing have been reported to be responsible for cell membrane damage and may consequently reduce the quality of histo- and histopathology samples.41 Simple refrigeration at 4°C as in this study potentially induces a lower cellular damage compared to the freezing and thawing process.
There was a minimal difference in section planes obtained between some examinations. Although attention was paid to orient the section planes as established, positioning of the foot in the magnet may have been slightly different between examinations and have indirectly produced slightly different planes of section. Imaging series were obtained by the same technologist 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. Methods for automatic section prescription have been described and demonstrated to give more reliable section prescription.42 Although differences in section plane were considered minimal, they may have potentially produced different values in the selected ROI. However as significant differences in signal were seen in all ROIs where trabecular bone was selected, this is very unlikely.
Overall image quality was considered good and this suggest that refrigeration only is sufficient to correctly preserve equine cadaver limbs prior to MR examination and to prevent putrefaction during the first 2 weeks after death. Most of the bacterial contamination at slaughter arises from hide and intestinal tract.43 The foot is not in contact with the gastro-intestinal tract at the abattoir which reduces the contamination by bacteria. The distal extremity is a part of the body with only few and mainly poor water contained structures (bones, tendons, ligaments, fat, skin and horn), without muscles and viscera. These anatomical characteristics are probably a major reason why refrigeration at 4°C is enough to preserve the distal limbs for a relative long time as two weeks. Long-term storage at 4°C has been previously used in several studies to preserve allograft.44-49 In these studies, the graft was stored in different preservation solutions.44-49 In one article, a comparison of different methods of preservation at 4°C is presented.45 One method consisted in wrapping the amputated rat limb in gauze moistened with cold saline solution for 6 to 72h.45 Even if the survival rates of the grafted limbs were decreased with this method, some limbs had a successful preservation after 48h of storage before grafting.45 A preservation solution or a preservation by wrapping in moistened tissue was not used in our study to try keeping the limbs externally dry and facilitate handling. Preservation by soaking in a solution or by wrapping is supposed to reduce dehydration of the tissues. Because the isolated equine limbs were kept with the skin and because of the presence of the hoof capsule, we supposed dehydration to be relatively slow and we decided to avoid external humidification to limit external tissues deterioration and decrease bacterial proliferation on the skin.
No change in signal in the distal limbs used in this study suggests increase in water or air content during time. In a study made on food, internal necrosis in over-ripe melons was seen as areas of decrease in signal intensity.50 The reason for that decrease signal is the presence of more air in the damaged tissues, which locally increases magnetic susceptibility of those tissues. The same study shows that gradient echo (GE) sequence is superior to SE sequence to demonstrate the effects of internal necrosis in over-ripe melons.50 In our study, we did not find any of these decreased signal intensity areas but we did not use a true GE sequence. However a DESS sequence was realized and this sequence is more sensitive to magnetic susceptibility artifact than SE sequence.51