Quantitative T2 relaxation time and magnetic transfer ratio predict endplate biochemical content of intervertebral disc degeneration in a canine model

Animals and study design

This study was conducted in compliance with the recommendations in the Guide for the
Care and Use of Laboratory Animals written by the National Institutes of Health. The
experimental protocol, using an animal model, was approved by the Institutional Animal
Care and Use Committee of Navy General Hospital (NO. 2013–0724, see Additional file
1 related to this article can be found at the “web link”). Experimental annular incision
of the IVD was performed in sixteen adult domestic dogs (non-chondrodystrophic breeds),
weighing 10 to 12 kg; the surgical procedure avoided naturally occurring spontaneous
disc degeneration. Surgeries were performed under general anesthesia (Ketamine, 10 mg/kg,
and Midazolam, 0.5 mg/kg) 14]. Under sterile surgical conditions, the spine was exposed through a retroperitoneal
approach. After clear identification of the IVD, an 18-gauge needle was inserted through
the AF and into the center of the NP. To avoid interaction with structures from adjacent
discs, anterolateral annular punctures were limited to a depth of about 7 mm, using
an 11-scalpel blade. Three stab incisions were performed, parallel to the endplate,
in the IVD at L3-L4, L4-L5, and L5-L6 (n?=?48). The IVD at L2-L3 and L6-7 were left undisturbed to serve as controls (n?=?32). The wound was closed in layers, and a tattoo marking was made on the back
of the animal at the level of the operated IVDs. Animals were given a course of 3 days
of penicillin by intramuscular injection. For pain control, a fentanyl patch (30 mg/h)
and flunixin meglumine (2.0 mg/kg intramuscularly) were used for 3 days. After the
surgery, animals were kept in separate cages in a temperature-controlled room (23?±?2 °C).
The animals had free access to food and water. Weight, food intake, and sleeping habits
were recorded. No animals died post-surgery.

MR imaging

Repeated MR imaging was performed before the animals were sacrificed. MRI was conducted
using a 3.0 T magnet (GE Signa Echo-Speed; GE Medical Systems, Milwaukee WI), with
all images were obtained under general anesthesia. For imaging, the dogs were placed
in a right lateral decubitus position, wrapped tightly, and supported with pillows.
An HD Cardiac Coil (Medical Systems, Milwaukee, Wisc, USA) was placed over the transverse
process of L4-5. A baseline MR image was obtained for each animal prior to surgery.
Post-surgery, MR images were obtained at 1-week intervals during the first month.
Five dogs were euthanized at 4, 8, and 12 weeks post-surgery immediately after MR
scanning. The lumbar spines of these dogs were harvested and immediately transferred
to the animal laboratory for histological analysis, and subsequently preserved at
?196 °C for biochemical analysis.

Detailed scanning parameters are listed in Table 1. In a first sequence, T2-weighted images (T2WIs) were obtained in the sagittal and
transverse planes for visual analysis. A T2 map was subsequently created (Repetition
Time, TR/ Echo Time, TE: 1500 / 8.5–67.9 milliseconds), using the T2 values from the
mid-sagittal section of the sagittal image centered on the midline of the lumbar spine;
the mapping was optimized using an 8 multispin echo sequence, available in the software
package (ADW 4.3, Functool, GE Medical Systems, Milwaukee WI). The signal intensity
(SI) of the T2 maps was computed on a pixel-by-pixel basis using the formula for each
respective TE: SI?=?e ^{–TE/ T2}15], 16].

Table 1. Scanning parameters

For MTR imaging, scans were centered at the level of the IVDs, with the middle slice
passing through the center of the IVD being imaged. The MTR data were obtained using
a sagittal gradient echo sequence (TR/TE:107/8.0 milliseconds), with dual acquisition,
and collected with and without the application of MT pre-pulses – one with the off-resonance
pulse applied at 1100 Hz down to the free water proton resonance frequency (Ms), and
the other without this off-resonance pulse (Mo) 17]. In all animals, MTR was calculated on a pixel-by-pixel basis using the formula:
MTR?=?(Mo-Ms) / Mo 15], 17].

Analysis of the images

The structure of the IVD in dogs is different from the structure in humans 18], 19]. Based on previous reports of the anatomical characteristics of canine IVDs 19], 20], we used the CEP zone (CEPZ) in our study. On the T2 Fast sequence echo, the region
near the CEP exhibited low and heterogeneous SI which was indistinct from those of
some parts of bone marrow, namely, the bony endplate which is analogous to the secondary
ossification center. The double low SI regions adjacent to the bony endplate were
identified as the growth plate and CEP, respectively. Therefore, we defined the CEPZ
as including the cartilaginous surface, the bony endplate, and the growth plate (Fig. 1, Fig. 2a–e). A calibrated phantom model (i.e., continuous small rectangle drawing) was used
before imaging data was collected and SI measurements calculated.

Fig. 1. The components of the cartilaginous plate zone. a: Division based on anatomical characteristics; the cartilaginous plate zone (CEPZ),
which includes the growth plate, the cartilaginous surface, and the bony endplate,
shown at low magnification; b: The arrow shows the anatomical structures at high magnification

Fig. 2. Regions of interest (ROIs) were drawn on the imaged IVD. a–d: The first echo and colored T2 map and the MTR are shown. e: The CEPZ is visualized in T2WI. f–g: The ROIs for the CEPZ were drawn from cephalic to caudal vertebrae. To ensure the
best possible anatomical alignment, all ROIs were selected from the morphological
images (first echo image of the T2 map or MTR mapping sequence) and transferred by
“copy and paste” into the T2 and MTR maps) to cover the anterior, middle, and posterior
part of IVD (f, Low magnification). Arrows show the specific drawing (g, high magnification). The area of ROIs and signal intensity can be automatically
calculated using the segmentation software (h). The lower edge of the ROI is covered with the following upper edge of the ROI of
the adjacent vertebra to avoid omitting tissue information. ?indicates CEPZ

To obtain images for analysis, a suitable ellipse was drawn on the T2 map or MTR to
demarcate one IVD, avoiding including other tissues within the regions of interest
(ROIs). Two liners were placed at points of inflection, based on the anatomical characteristics
of the canine IVD previously described 18]. A non-parallel anatomical structure was observed in the ventral and dorsal CEPZ,
with both parts being angled with the cross-section of the IVD but being relatively
parallel to the center of the CEPZ. Anterior and posterior rectangular ROIs were localized
parallel to the corresponding CEPZ (Fig. 2f, g). Measurements were obtained from the images by manual segmentation of the ROIs using
a workstation (GE Medical Systems, Milwaukee, WI). As a means of reducing the partial
volume effect, a suitable rectangular ROI was selected for the IVD from the cephalic
to caudal CEPZ including the vertebra, NP and AF, and the lower edge of the ROI was
covered with the upper edge of the ROI at the subsequent level (Fig. 2f, g). To ensure the best possible anatomical alignment, ROIs were selected from the morphological
images (first echo image of the T2 mapping sequence 11] or MTR mapping sequence) and transferred using “copy and paste” function into the
T2 and MTR maps. All ROIs were selected manually by an experienced senior musculoskeletal
radiologist (R.A.J., 20 years of experience). The areas of the ROI were calculated
automatically by the software, with areas of 1.00?±?0.13 mm
²
, 2.35?±?0.12 mm
²
and 0.70?±?0.14 mm
²
used to cover the anterior, middle, and posterior portions of the IVD, respectively
(Fig. 2h). In order to distinguish boundaries between adjacent single pixels, the smallest
possible ROIs were drawn by software. The analyzed SI data were transferred to Excel
(Microsoft, Excel 2003) for curve analysis; and both peak and trough values were included
to distinguish different tissues. The mean and 2 standard deviations (SDs) were calculated
for all measured values within a slice, and the 2 SD value used to set a subtraction
threshold for all pixels in that slice. Pixels with T2 or MTR values lower than the
calculated threshold were subtracted out, a method which has been shown to be effective
for image-based histological assessments 21]. Measurements were performed 3 times for each slice, and average values used for
analysis.

Disc tissue dissection

To obtain the samples of the CEP and growth plate, all cartilage was carefully cleaned
from the endplate surface, and parallel incisions were made to the depth of the growth
plate, followed by an incision parallel to the growth plate 22]. To obtain the AF, a full thickness rectangular strip, approximately 20 mm in width,
was cut from the anterior to the posterior part of the disc, and sliced into sections
of approximately 1 mm in width 23]. Sections were then cut along their sagittal plane into 2 roughly unequal pieces,
with the small section used to measure water content and the larger section to quantify
PG and collagen content.

Histology

The IVD allografts were immersed into cold 10 % neutral-buffered formalin. Each sample
was subsequently immersed into a decalcifying liquid composed of 12 mL of 30 % chromic
acid solution, 12 mL of 30 % absolute ethyl alcohol, and 12 mL of 40 % hydrochloric
acid. Following decalcifying, macrosections were embedded into paraffin and 4-mm thin
sections prepared. Prepared sections were stained using hematoxylin and eosin (HE),
safranin-O and Picrosirius Red stains for histological analysis. A semi-quantitative
total score of endplate degeneration was used, which included sclerosis, fibrosis
and cellularity of the endplates 24], 25].

Biochemical analysis

All the samples were dissected from the discs and cut into three sections of 1 mm
²
each, using a scalpel. To determine the percent water content and dry tissue weight,
one portion of the tissue was dried at 110 °C for 4 days, until constant weight was
obtained. The percent water content was calculated as the ratio of the wet weight
to the dry weight 15]. The same set of samples was used for the analysis of hydroxyproline and uronic acid
contents. The biochemical composition was assessed using an enzyme-linked immunosorbent
assay (ELISA) 26]–28]. For the ELISA, all the samples (AF, NP, and CEPZ) were carefully separated and stored
at ?80 °C. For each sample, care was taken to separate the AF, EP, and CEPZ, avoiding
contamination from surrounding tissue. Frozen NP, AF, and CEZP tissues were weighed
before extraction of the PG and collagen. These tissues were homogenized in a buffer
solution (100 mg of tissue per mL of RIPA lysis buffer), with the homogenizer immersed
in an ice bath. The solution was centrifuged (3000 r/min) at 4 °C for 10 min. The
supernate and the non-solubilized material (the pellet) were separated. The supernate
was dialyzed against 20 volumes of sterile deionized water overnight and lyophilized
to dryness. The supernatants were collected to measure the PG content using ELISA
kits (Boyao, Shanghai, China) and standard methods. The color of the samples was quantified
by measuring the difference in absorption of a 450-nm wave, using an El
_x
800-microplate reader (Bio-Tek Instruments, Winooski, VT, USA). The total protein
concentration was determined using previously described methods 29].

Another set of samples was used for measurement of the hydroxyproline contents in
the enzyme-digested fractions 30]–32]. Considering the hydroxyproline content to be equivalent to 10 % of the weight of
each collagen alpha chain, the total collagen content per dry weight was estimated
from the proteinase K-digested fraction of the dried tissue 31], 32]. Collagen was extracted from the pellets using 10 mL of a solution consisting of
0.2 mol/L NaCl, 0.5 mol/L acetic acid, and 1 mg/mL of pepsin (Sigma Chemical, St Louis,
MO, USA). The suspension was stirred (4 °C, 24 h), followed be centrifugation and
separation of the supernatant. Three days later, the pH of the solution was adjusted
to 8.0 with a solution of 5 mol/L NaOH. The suspension was then centrifuged (3000 r/min)
at 4 °C for 10 min. The supernate was removed, dialyzed against sterile water, and
lyophilized to measure the amount of total collagen using ELISA kits (Boyao, Shanghai,
China) and standard methods. The remaining assay was carried out as described for
the proteoglycan ELISA 27].

Statistical analysis

Statistical analyses were conducted and graphs were generated using SPSS 19.0 (SPSS
Inc., Chicago, IL, USA). Pearson or Spearman correlation analysis was performed, as
appropriate for the distribution of the data, to compare biochemical content, histological
score with T2 relaxation time values and MTR. The strength of the correlation was
evaluated from the absolute value of significant correlation coefficients, ‘r’, as
follows: a very strong correlation (r?=?0.80 to 1.00), a strong correlation (r?=?0.60 to 0.79), a moderate correlation (r?=?0.40 to 0.59), a weak correlation (r?=?0.20 to 0.39), or no correlation (r??0.20). The significance of changes pre- and post-surgery was evaluated using one-way
analysis of variance (ANOVA). The statistical significance of the matrix components
(water, glycosaminoglycan, and collagen), and the MR parameters (T2 relaxation time
values and Ms/Mo ratio) as a function of time post-surgery was determined by one-way
ANOVA. For, all statistical analysis, a p-value less than 0.05 was considered to be
significant.