Figure 1.
Serum inflammatory markers in normal, untreated and PPS treated MPS VI rats.
Tumor necrosis factor-α (TNF-α (A), tumor necrosis factor receptor-1 (TNFR1) (B), interleukin-8 (IL-8) (C), protein S100A8/A9 (D), and C-reactive protein (CRP) (E), were quantified by ELISA assays as described in the Materials and Methods. White columns, normal rats; black columns, untreated MPS VI rats; light gray columns, 4 mg/kg sc PPS treated MPS VI rats; dark gray columns, 2 mg/kg sc PPS treated MPS VI rats; grey hatched columns, 1 mg/kg sc PPS treated MPS VI rats and white hatched columns, 4 mg/kg oral PPS treated MPS VI rats. N = 6/group. All doses are HED [22]. The vertical lines in each column indicate the ranges. * P<0.05 comparing treated to untreated MPS VI rats. **P<0.05 comparing sc PPS administration to oral.
Figure 2.
Histological analysis of MPS VI rat femoral growth plates and articular cartilage.
Representative images are shown for untreated MPS VI rats and each of the sc treatment groups (H & E staining, 20× magnification). (A) Growth plate analyses. Seven-month-old MPS VI rats revealed a complete loss of columnar arrangement in the knee growth plates as compared to normal animals. Overall, PPS sc treatment resulted in moderately improved chondrocyte orientation and growth plate organization, although significant vacuolated chondrocytes were still observed. *Indicates the growth plate. (B) Articular cartilage analyses. Micrographs revealed that sc PPS treatment reduced vacuole formation, suggesting reduced GAG storage in MPS VI articular chondrocytes, in a dose dependent manner. Weekly sc injections of 1 mg/kg had little to no effect, while improvements can be seen at 2 and 4 mg/kg.
Figure 3.
GAG reduction in PPS-treated MPS VI rats.
(A) Total urine GAGs were significantly reduced in all PPS-treated MPS VI rat groups compared to untreated MPS control animals, regardless of the mode of administration. Subcutaneous treatment also significantly reduced urine GAGs compared to oral treatment. Tissue GAGs were significantly reduced only in the MPS VI rats treated with sc PPS. (B) kidney, (C) liver and (D) spleen. A dose responsive reduction was observed in the kidney and liver. *P<0.05 comparing treated to untreated MPS VI rats. ** P<0.05 comparing sc to oral treatment.
Figure 4.
Femoral trabecular analysis of PPS-treated MPS VI rats.
MicroCT analysis of the femurs was performed as described in the Materials and Methods. Representative images are shown for 7-month-old (A) normal, (B) untreated MPS VI, (C) 1 mg/kg, (D) 2 mg/kg, and (E) 4 mg/kg sc PPS-treated animals. Black arrows indicate the trabecular regions. MicroCT quantification of the trabeculae is shown below the images. Dose responsive improvements in several criteria were observed, including % bone volume/tissue volume (BV/TV), bone surface/volume ratio (BS/BV), trabecular number (Tb.N), trabecular separation (Tb.Sp), total porosity Po(tot), and bone mineral density (BMD). *indicates statistical differences between untreated and treatment groups (p<0.05).
Figure 5.
Flexural mechanics of normal, untreated MPS VI, and PPS-treated MPS VI femurs.
The whole bone stiffness (A) and flexural modulus (B) were decreased in MPS VI compared to normal animals, and no changes were observed in the PPS treatment groups. However, force at break (C) and flexural strength (D) did reveal increasing improvements in a dose dependent manner, indicating that sc PPS treatment moderately enhanced the fracture strength of the femurs.
Figure 6.
Spinal trabecular analysis of PPS-treated MPS VI rats.
3-D microCT reconstructions of (A) normal, (B) untreated MPS VI, (C) 1 mg/kg, (D) 2 mg/kg, and (E) 4 mg/kg sc treated T1 thoracic vertebrae. All images were taken at the point where the pedicle connected with the spinal body. As seen in the untreated MPS VI image (B), a significant amount of dense cortical tissue (indicated by a whiter color intensity) is present compared to the normal animal (A). The cortical tissue also appeared to grow into the trabecular area in MPS VI animals (indicated by arrows and arrowheads). The 2 and 4 mg/kg PPS doses reduced the cortical in-growth and promoted the formation of better-oriented trabeculae in the MPS VI rat vertebrae. P values to represent statistical differences are presented as * for p<0.05 and ** for p<0.01.
Figure 7.
Motor activity in PPS treated MPS VI rats.
Two weeks after the last PPS dose, treated MPS VI animals were subjected to rotarod analysis at five different speeds (range: 20–40 revolutions per min (RPM)), and their performance was compared to normal and untreated, age and gender-matched animals. Black columns represent untreated MPS VI rats; white hatched columns, oral 4 mg/kg PPS-treated MPS VI rats; light gray columns, 4 mg/kg sc PPS-treated MPS VI rats; dark gray columns, 2 mg/kg sc PPS-treated MPS VI rats; grey hatched columns, and 1 mg/kg sc PPS-treated MPS VI rats. All groups of treated MPS VI rats (oral and sc) remained on the rotating rod significantly longer than untreated animals (p<0.005). At the higher speeds (30, 35 and 40 RPM) MPS VI animals receiving all sc PPS doses had significantly better endurance and remained on the apparatus longer than those receiving oral PPS (p<0.05).