Effect of Paralysis In-ovo on Development of Chick Hind Limb Articular Cartilage:an Evaluation Using Micro Mri Measurement of Delayed Gadolium Uptake

INTRODUCTION: It is known that immobilization causes a reduction of glycosaminoglycan (GAG) in articular cartilage [1, 2]. Further, in-ovo paralysis by decamethonium bromide in developing chick embryos interferes with the formation of normal joint anatomy similar to arthrogryposis [3, 4]. However, the effect of immobilization on the GAG content of articular cartilage in the developing chick embryo is unknown. MRI in conjunction with the gadolinium contrast agent, Gd(DTPA), has been demonstrated to measure local GAG concentration in articular cartilage non-destructively [5]. Since negatively charged Gd(DTPA) distributes in inverse proportion to the concentration of negatively charged GAG molecules in the cartilage matrix, the GAG concentration in the cartilage can be calculated from the concentration of Gd(DTPA) distributed throughout the cartilage. Gd(DTPA) shortens the tissue specific longitudinal T1 relaxation time in a concentration-dependent manner. Thus, a shorter T1 relaxation time implies that a higher Gd(DTPA) concentration is present and that the GAG concentration is decreased in the cartilage. A map of the T1 relaxation times reflects the relative distribution of GAG throughout the cartilage. Using this MRI technique, the effect of decamethonium bromide paralysis in-ovo on the GAG concentration of articular cartilage in the developing knee joint of the embryonic chick was investigated. METHODS: Fertilized Legghorn eggs maintained at 38°C were divided into a paralyzed group and non-paralyzed control group. In the paralyzed group, 0.25 ml decamethonium bromide (DMB)[0.2 mg/ml] was injected into the eggs every other day starting at day 10 after fertilization. Hind limbs were harvested from each group at day 13 (10 paralyzed, 10 control) and day 16 (11 paralyzed, 11 control) after fertilization. The hind limbs were equilibrated in either charged gadolinium aqueous solution (1 mmol Gd/l Gd(DTPA)) for 48 hours paralyzed: 5 at day 13, 6 at day 16; control: 5 at day 13, 6 at day 16 or neutral gadolinium aqueous solution (1 mmol Gd/l Gd(HADO3A)) for 48 hours paralyzed: 5 at day 13, 5 at day 16; control: 5 at day 13, 5 at day 16. The negatively charged Gd(DTPA) distributes through the cartilage in inverse proportion to the negatively charged GAG. On the other hand, Gd(HADO3A) is a neutral molecule, thus its distribution throughout the cartilage is independent of GAG and reflects the diffusion of gadolinium. Therefore differences in the distribution of Gd(DTPA) and Gd(HADO3A) throughout the cartilage reflect the presence GAG [5]. Each hind limb was evaluated using an 8.45 Tesla microimaging system (DRX-350TM, Bruker Bio-spin, Karlsruhe, Germany) to measure the T1 relaxation time at the epiphyses of the knee joint. Initially a T2 weighted fast spin echo sequence was used to identify an appropriate imaging slice that included both the distal femur and proximal tibia epiphyses. T1 measurements were then taken using a conventional spin-echo sequence with six different repetition times (TRs) ranging 0.05 to 3 seconds, 6.6 msec echo time, 0.5 mm slice thickness, 10 mm field of view, 128x128 matrix, and 4 (for day 13) or 2 (for day 16) number of excitation, affording (78 m) in-plane resolution. Five different regions of interest were measured on the processed T1 maps of the femoral and tibial epiphyses. After eliminating the highest and lowest values, the remaining T1 values were averaged to reflect the GAG concentration at each epiphyses. The T1 relaxation times were compared using student’s t-test between the paralysis group and the control group at post fertilization days 13 and 16 for Gd(DTPA) or Gd(HADO3A) respectively. Four of the specimens in each group were fixed in 4% paraformaldehyde in PBS, decalcified with 14% EDTA, embedded in paraffin, cut into 5 μm sections, and stained with Toluidine Blue or Safranin O for histomorphometry. RESULTS: On day 13 post fertilization the hind limbs equilibrated with Gd(DTPA), the femoral T1 relaxation time was shorter in the paralysis group than the control group (p=0.02) whereas no significant difference was found between the groups for the tibia (Fig. 1). However for hind limbs equilibrated with Gd(HPDO3A), the T1 relaxation times were the same for both the tibia and femur for both groups. On day 16 post fertilization the hind limbs equilibrated with Gd(DTPA), the T1 relaxation time was longer for the paralyzed group for both the femur (p<0.01) and tibia (p=0.04). However for hindlimbs equilibrated with Gd(HPDO3A), the T1 relaxation time was shorter for the femur in the paralyzed group (p<0.01). No clear differences were found between control and paralysis groups at day 13 or day 16 evaluated by histology using Toluidine blue and Safranin O.