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.
1
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.
2
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 paraffinpolymer 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
3
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 carpometacarpal 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.
4
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 cm 2 was used to
assess the palmar proximal recesses of the DIPJ in the sagittal area. The digital cushion was
5
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.
6
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
7
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 gastrointestinal 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.
8
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
The reduction of the synovial content, probably due to fluid loss, may be responsible for
subjective impression of size reduction of the synovial recesses and for statistical change in
signal in the DIPJ recess especially between T2w and other times. However, as the synovial
recesses are small structures with amorphous shape, drawing a ROI of 0.1cm 2 is hard to
realize without taking peri-recesses tissues. With a reduction of size even if less than 1mm,
the ROI may easily include peri-recess tissue giving a high change in the signal measurement
in this area. The impression of size reduction of the synovial recesses could not be confirmed
by a statistical analysis as a reliable quantitative evaluation of the size of the synovial recesses
was considered unfeasible mainly because of the variable shape of the recesses.
In the present study, the feet were not warmed before the examination to avoid bacterial
proliferation and to limit tissues deterioration. Signal change with temperature has been
described in a study on excised atherosclerotic carotid tissue.52 In this study, plaque lipid T2weighted image intensity was initially relatively hyperintense in some samples and decreased
with time on cooling, reflecting a decrease in the lipid transverse (T2) relaxation time. In
another study on isolated tissue samples bone marrow T1 and T2 relaxation times decreased
slightly to moderately, with temperature reduction to about -5°C at which temperature a
9
sudden marked decrease occurred.53 In our study, we found a decrease in signal intensity in
bone marrow in T2 weighted images after 24h. As the feet were not warmed at room
temperature and as the signal did not change between the first examination (T24h) and the
following ones (T48h, T1w and T2w), the most probable explanation for this initial signal
change is a different temperature of the limbs between before and after refrigeration. Any
change because of tissue autolysis is in fact supposed to increase with time and no signal
change increasing with time was visible in the present study.
No MR examination before death of the animal was made. Therefore, no comparison between
ante- and post-mortem images was made.
In our study, the STIR images of bone marrow were slightly hyperintense at T24h in
comparison to images at T0 in the qualitative subjective study. However these changes were
not significant in the quantitative study. Optimal inversion time (TI) may vary between
individuals or different body part.54,55 It has been hypothesized that this may be due to
variations in the T1 of fat between patients or between different parts of the body.54,55 It has
also been shown that T1 and T2 of bone marrow isolated tissue decreases slightly to
moderately with decreasing temperature.53 This may explain incomplete suppression of fat in
the STIR sequences of refrigerated limbs. A technique is described to select the best TI.55
Even though slight hyperintensity was only subjectively visible after refrigeration, it may be
interesting to vary the TI in cadaver limbs examined after refrigeration without warming at
room temperature in order to produce lowest fat signal intensity and improve lesions
detection.
The feet examined in this study were normal feet. Studies on pathological feet are necessary
to assess if the results of the present study can be applied to feet with lesions and if signal
differences in bone marrow in refrigerated limbs alter the lesion signal and/or the detection of
lesions with time.
10
Conclusion
Mild changes in signal can be only seen in trabecular bone in STIR and TSE T2 sequences
between the first examination just after death and the examination after refrigeration at 4°C
24h after death. No significant changes seem to occur in refrigerated limbs between 24h and 2
weeks. Within 2 weeks, simple storage at 4°C seems therefore sufficient to preserve feet from
putrefaction between MR examinations.
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Table 1. MR Imaging Protocols
Type
Plane
TR
TE
(msec) (msec)
FOV
(cm)
Matrix Thickn Gap Flip TA NEX
(mm) (mm) (°) (min)
TSE T1 Transverse
512
12
180-180 307x512
3
1
3:14
3
TSE T2
Sagittal
3800
84
160-160 288x384
3
0,3
3:31
1
STIR
Sagittal
3140
46
160-160 256x320
3
0,3
4
2
DESS
Dorsal
20,7
6,34
180-180 256x256
1,5
0,3
3:38
2
40
TI
150
TR: repetition time; TE: echo time; FOV: field of view; Thickn: thickness; TA: acquisition
time; NEX: number of acquisitions; TI: inversion time; TSE: turbo spin echo; STIR: short tau
inversion recovery; DESS: double echo steady state
20
ACKNOWLEDGEMENTS: The authors thank Laurent Massart for his help in the statistical
analysis
21
Figure Legends:
Fig. 1: T2 turbo spin echo sequence in sagittal plane at T0 (A), at T24h (B), at T48h (C), at
T1w (D), at T2w (E): Subjectively, at T24h (B), bones appeared slightly hypointense
compared to T0 (A). No changes were subjectively seen in bones between T24h (B), T48h
(C), T1w (D) and T2w (E).
Fig. 2: STIR sequence in sagittal plane at T0 (A), at T24h (B), at T48h (C), at T1w (D), at
T2w (E): Subjectively, at T24h (B), bones appeared slightly hyperintense compared to T0
(A). No changes were subjectively seen in bones between T24h (B), T48h (C), T1w (D) and
T2w (E).
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