2003 Papers - Judd


Evaluation of Rabbit (Oryctolagus cuniculus) Articular Cartilage After Traumatic Injury in the Presence of an Apoptosis Inhibitor N-iminoethyl-L-lysine (L-NIL)
Daniel B. Judd, CPT, MC, USA and William Burtkhalter Jr, LTC, MC, USA
Tripler Army Medical Center

Introduction: Cartilage injury is a significant factor leading to secondary osteoarthritis. Several studies have demonstrated cartilage matrix damage and chondrocyte death in response to mechanical injury. Traumatic injuries may cause cartilage destruction through several mechanisms. Chondrocyte (cartilage cell) death may follow one of two mechanisms chondrocyte necrosis, (cell murder), and apoptosis, (cell suicide). Investigators have begun to reveal the apoptosis pathway in chondrocytes and have identified potential inhibitors of this process. Recent in vitro studies have shown significant decreases in chondrocyte death following traumatic injuries when treated with apoptosis inhibitors. This offers the possibility of a therapeutic window between injury and cell death, in which preservation of these otherwise apoptotic chondrocytes may have a chondroprotective effect by the maintenance of articular cartilage through preservation of chondrocyte viability. With the use of apoptosis inhibitors chondrocyte apoptosis has been inhibited in animal and human in vitro cartilage explants. In vivo animal models have been shown to undergo apoptosis after traumatic injuries. To our knowledge there have been no trials using apoptosis inhibitors to evaluate their in vivo effects after traumatic injury. Nitric oxide (NO) has been shown to induce chondrocyte apoptosis after traumatic injury and in experimentally induced arthritis animal models. The inhibition of inducible nitric oxide synthetase (iNOS, which converts arginine to NO) by L-NIL has been shown to reduce the severity of experimental osteoarthritis in in vivo animal models. NO also activates important proteins involved in the apoptotic phenomenon, i.e. caspase 3 (inducer) and BCL-2 (inhibitor). The inhibition of NO and caspase 3 have been shown to inhibit human chondrocyte apoptosis in in vitro injury models. L-NIL has the advantage of selectively blocking iNOS and thus may inhibit cartilage damage and apoptosis at multiple levels. An anti-apoptotic agent may allow chondrocytes that were destined to die, to survive and more importantly to continue to synthesis and deposit new matrix. Our objective is to investigate, in an existing rabbit impact model, if therapeutic intervention using apoptosis inhibitors can preserve chondrocyte viability in vivo and if normal articular cartilage is maintained after a traumatic injury in the presence of L-NIL.

Materials and Methods: After obtaining Institutional Animal Review committee approval, 10 rabbits were anesthetized and subjected to a unilateral patellofemoral joint impact of 3 Kg from a height of 50cm in a drop tower apparatus. This model was designed by Dr. D’Lima from the Scripps Research Institute, La Jolla CA. They have found this mechanism reliably induces cartilage injury without a concomitant fracture in the bone. The contralateral knee will not be impacted and will serve as an uninjured control. Rabbits were placed into one of two study groups (10 rabbits per group):
1. Control: No treatment; SQ injection of normal saline in the same volume as L-NIL in the experimental group.
2. Treatment: SQ injection of nitric oxide synthase inhibitor N-iminoethyl-L-lysine (L-NIL, Pharmacia) (10 mg/kg) once a day for 10 days.
After 10 days the rabbits were euthanized and the injured and uninjured knees were harvested. Assessment was made of the patellar and femoral trochlear surface of both knees to evaluate chondrocyte apoptosis, cell viability and cartilage health. Evaluators were be blinded as to which group the specimen came from and to the results of the other investigators. Macroscopic evaluation: Each knee was examined for gross morphologic changes and cartilage lesions according to established protocol. (Arth and Rheum 43(6):1290-1299, 2000) Histological Analysis: Sections from injured areas were stained with safranin O-fast green stain and standard H & E staining then examined using light microscopy. A cartilage grading system will be used to assess the health of the articular cartilage.(Sellers R, Peluso D, Morris E, JBJS 79(A) 1997 p 1452) In situ detection of apoptosis: Cartilage tissue sections were examined with TUNEL (TdT-mediated dUTP nick end-labeling) to detect the DNA fragmentation that cells can exhibit when undergoing apoptosis. (Darzynkiewicz A, Li X, Gong J, Methods in Cell Biology 1994;41:15-38) Apoptosis was be measured in situ by detection of nuclear DNA fragmentation using a TUNEL (TdT-mediated X-dUTP nick end labeling) assay. The TUNEL reaction preferentially labels DNA strand breaks generated during apoptosis. This allows discrimination of apoptosis from necrosis and from primary DNA strand breaks induced by DNA-damaging agents.

Results: Gross Morphology revealed 4 of the 10 impacted femoral trochlea with minimal fibrillation of surface cartilage. The remaining trochlea and patella specimens were unremarkable for gross morphologic changes and impacted vs. non-impacted cartilage could not be distinguished. No specimens were fractured. Histology: H & E and Safarin-O/Fast green stains revealed full and partial thickness cartilage fissures in the zone of impact of all impacted specimens. No non-impacted knees revealed cartilage lesions. Safarin-O/Fast green stains revealed a decease in the staining intensity of the impacted cartilage compared to the non-impacted cartilage, consistent with a net loss of proteoglycans. Although the injured knees treated with L-NIL had a greater intensity of Safarin-O/Fast green staining than the control group, this did not reach statistical significance.
In-Situ detection of Apoptosis
Patellar cartilage from impacted knees had 12(+/-3)% apoptotic cells while the contralateral patellar cartilage had 1(+/-)%. Apoptotic rates were reduced to 5% (+/-2) in the L-NIL group. This is statistically significant.