Background: Hemophilia A results from a congenital deficiency of clotting factor VIII. Patients with hemophila are unable to generate adequate thrombin during blood coagulation and therefore have a bleeding diathesis. Degenerative joint disease is the largest source of morbidity for hemophilic patients. The pathophysiology of this process is poorly understood but clinically, repeated bleeding into a joint leads to changes in the synovium, articular cartilage and underlying bone. It is not well known how quickly cartilage changes occur or if animal age has any impact on susceptibility to blood induced cartilage damage.

Materials and Methods: The E16 fVIII knockout mouse is a well-established model for hemophilia research. These mice contain a targeted disruption of the factor VIII gene at exon 16 (E16) in a C57BL/6 background. Despite having no detectable circulating fVIII activity they rarely suffer from spontaneous hemarthroses. Therefore hemarthroses was mimicked by injecting 5 μL of autologous whole blood into the hind knee. Six male mice per group in each of three ages; 12, 24 and 52 weeks were utilized. The animals were sacrificed 48 hours after injection. The injected and control contralateral knee joint from each animal was fixed in 10% formalin and then mounted in paraffin. After decalcification, five micrometer sections through the joint were obtained. Joint architecture was examined after hematoxylin and eosin staining. Proteoglycan content of articular cartilage was evaluated after alcian blue staining as part of the modified Mankin score. The pathologic specimens were also scored utilizing Valentino’s visual bleeding score. The Student’s t-test was utilized for significance testing.

Results: All mice showed evidence of blood remaining in the injected joint at 48 hours. The mean visual bleeding score was significantly higher for the injected knees versus the control knees of all age groups p<0.001. The mean visual bleeding score was significantly between the 12 and 24 week animals p=0.0112 and the 12 and 52 week animals p=0.0379, but not between the 24 and 52 week animals p=0.5423. The modified Mankin score was significantly higher between the injected and control knees of all age groups as seen in Figure 1 where the mean and standard deviation is shown for 6 animals in each group. Interestingly, one control knee in a 24 week old animal demonstrated synovial hyperplasia and decreased proteoglycan staining but had no evidence of blood in the joint. This likely represented a previous spontaneous hemorrhage. The modified Mankin score was highest for the 12 week old animals and lowest for the 52 week animals but did not achieve statistical significance p=0.5746.

Conclusions: Injection of autologous blood mimicked clinical hemarthrosis. Evidence of decreased proteoglycan staining of articular cartilage was evident within 48 hours of blood exposure. Additionally, there was a trend toward more prominent proteoglycan loss in the youngest animals.

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Figure

Topics:
cartilage, hemophilia a, mice, hemorrhage, proteoglycan, hemarthrosis, alcian blue, bleeding diathesis, blood coagulation factors, cartilage damage

Author notes

Disclosure:Research Funding: Hemophilia of Georgia Clinical Scientist Development Grant. Membership Information: ZLB Behring advisory board; Bayer advisory board.

2007, The American Society of Hematology
2007
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