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Non-Invasive Quantification of Cartilage Using a Novel In Vivo Bioluminescent Reporter Mouse.

Mailhiot SE, Zignego DL, Prigge JR, Wardwell ER, Schmidt EE, June RK - PLoS ONE (2015)

Bottom Line: Ex vivo experiments revealed that collagenase digestion of the femur reduced both luciferase signal intensity and pixel area, demonstrating a link between cartilage degradation and bioluminescence.In an in vivo model of experimental OA, we found decreased bioluminescent signal and pixel area, which correlated with pathological disease.We detected a decrease in both bioluminescent signal intensity and area with natural aging from 2 to 13 months of age.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biosciences Program, Montana State University, Bozeman, MT, United States of America; Department of Mechanical and Industrial Engineering, Montana State University, Bozeman, MT, United States of America.

ABSTRACT
Mouse models are common tools for examining post-traumatic osteoarthritis (OA), which involves cartilage deterioration following injury or stress. One challenge to current mouse models is longitudinal monitoring of the cartilage deterioration in vivo in the same mouse during an experiment. The objective of this study was to assess the feasibility for using a novel transgenic mouse for non-invasive quantification of cartilage. Chondrocytes are defined by expression of the matrix protein aggrecan, and we developed a novel mouse containing a reporter luciferase cassette under the inducible control of the endogenous aggrecan promoter. We generated these mice by crossing a Cre-dependent luciferase reporter allele with an aggrecan creERT2 knockin allele. The advantage of this design is that the targeted knockin retains the intact endogenous aggrecan locus and expresses the tamoxifen-inducible CreERT2 protein from a second IRES-driven open reading frame. These mice display bioluminescence in the joints, tail, and trachea, consistent with patterns of aggrecan expression. To evaluate this mouse as a technology for non-invasive quantification of cartilage loss, we characterized the relationship between loss of bioluminescence and loss of cartilage after induction with (i) ex vivo collagenase digestion, (ii) an in vivo OA model utilizing treadmill running, and (iii) age. Ex vivo experiments revealed that collagenase digestion of the femur reduced both luciferase signal intensity and pixel area, demonstrating a link between cartilage degradation and bioluminescence. In an in vivo model of experimental OA, we found decreased bioluminescent signal and pixel area, which correlated with pathological disease. We detected a decrease in both bioluminescent signal intensity and area with natural aging from 2 to 13 months of age. These results indicate that the bioluminescent signal from this mouse may be used as a non-invasive quantitative measure of cartilage. Future studies may use this reporter mouse to advance basic and preclinical studies of murine experimental OA with applications in synovial joint biology, disease pathogenesis, and drug delivery.

No MeSH data available.


Related in: MedlinePlus

Induction of bioluminescence via tamoxifen.To examine the duration of serial injections of tamoxifen free base on joint-specific bioluminescence, we treated mice for 0–5 days via intraperitoneal injection. (A) There is an increase at 3 days (p = 0.004) and from 3 to 5 days (p = 0.01). No significant bioluminescence was detected after 1 day of Tamoxifen administration. (B) Representative bioluminescence images from 0–5 days of tamoxifen treatment.
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pone.0130564.g002: Induction of bioluminescence via tamoxifen.To examine the duration of serial injections of tamoxifen free base on joint-specific bioluminescence, we treated mice for 0–5 days via intraperitoneal injection. (A) There is an increase at 3 days (p = 0.004) and from 3 to 5 days (p = 0.01). No significant bioluminescence was detected after 1 day of Tamoxifen administration. (B) Representative bioluminescence images from 0–5 days of tamoxifen treatment.

Mentions: Bioluminescence was observed to be under tight control of induction by creERT2 activity in aggrecan expressing cells including cartilage of the ankle, knee, and hip. Scruff injection of tamoxifen to activate cre for genetically encoded expression of luciferase in chondrocytes resulted in genetic induction of detectable luminescence after 3 daily injections (Fig 2, n = 4 mice). Pilot studies found substantial tracheal bioluminescence (not shown) in addition to bioluminescence within all of the joints of the lower limbs (i.e. ankle, knee, and hip). One day of tamoxifen treatment did not result in detectable bioluminescence, but three and five days of tamoxifen injections did produce detectable bioluminescence as compared to the single injection (both p < 0.01, Fig 2). Furthermore, bioluminescence was greater with five as compared to three tamoxifen injections (p = 0.01, Fig 2) indicating that increased numbers of cells activated creERT2 due to administering additional tamoxifen, which also highlights the challenges to articular cartilage drug delivery [2].


Non-Invasive Quantification of Cartilage Using a Novel In Vivo Bioluminescent Reporter Mouse.

Mailhiot SE, Zignego DL, Prigge JR, Wardwell ER, Schmidt EE, June RK - PLoS ONE (2015)

Induction of bioluminescence via tamoxifen.To examine the duration of serial injections of tamoxifen free base on joint-specific bioluminescence, we treated mice for 0–5 days via intraperitoneal injection. (A) There is an increase at 3 days (p = 0.004) and from 3 to 5 days (p = 0.01). No significant bioluminescence was detected after 1 day of Tamoxifen administration. (B) Representative bioluminescence images from 0–5 days of tamoxifen treatment.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4495059&req=5

pone.0130564.g002: Induction of bioluminescence via tamoxifen.To examine the duration of serial injections of tamoxifen free base on joint-specific bioluminescence, we treated mice for 0–5 days via intraperitoneal injection. (A) There is an increase at 3 days (p = 0.004) and from 3 to 5 days (p = 0.01). No significant bioluminescence was detected after 1 day of Tamoxifen administration. (B) Representative bioluminescence images from 0–5 days of tamoxifen treatment.
Mentions: Bioluminescence was observed to be under tight control of induction by creERT2 activity in aggrecan expressing cells including cartilage of the ankle, knee, and hip. Scruff injection of tamoxifen to activate cre for genetically encoded expression of luciferase in chondrocytes resulted in genetic induction of detectable luminescence after 3 daily injections (Fig 2, n = 4 mice). Pilot studies found substantial tracheal bioluminescence (not shown) in addition to bioluminescence within all of the joints of the lower limbs (i.e. ankle, knee, and hip). One day of tamoxifen treatment did not result in detectable bioluminescence, but three and five days of tamoxifen injections did produce detectable bioluminescence as compared to the single injection (both p < 0.01, Fig 2). Furthermore, bioluminescence was greater with five as compared to three tamoxifen injections (p = 0.01, Fig 2) indicating that increased numbers of cells activated creERT2 due to administering additional tamoxifen, which also highlights the challenges to articular cartilage drug delivery [2].

Bottom Line: Ex vivo experiments revealed that collagenase digestion of the femur reduced both luciferase signal intensity and pixel area, demonstrating a link between cartilage degradation and bioluminescence.In an in vivo model of experimental OA, we found decreased bioluminescent signal and pixel area, which correlated with pathological disease.We detected a decrease in both bioluminescent signal intensity and area with natural aging from 2 to 13 months of age.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biosciences Program, Montana State University, Bozeman, MT, United States of America; Department of Mechanical and Industrial Engineering, Montana State University, Bozeman, MT, United States of America.

ABSTRACT
Mouse models are common tools for examining post-traumatic osteoarthritis (OA), which involves cartilage deterioration following injury or stress. One challenge to current mouse models is longitudinal monitoring of the cartilage deterioration in vivo in the same mouse during an experiment. The objective of this study was to assess the feasibility for using a novel transgenic mouse for non-invasive quantification of cartilage. Chondrocytes are defined by expression of the matrix protein aggrecan, and we developed a novel mouse containing a reporter luciferase cassette under the inducible control of the endogenous aggrecan promoter. We generated these mice by crossing a Cre-dependent luciferase reporter allele with an aggrecan creERT2 knockin allele. The advantage of this design is that the targeted knockin retains the intact endogenous aggrecan locus and expresses the tamoxifen-inducible CreERT2 protein from a second IRES-driven open reading frame. These mice display bioluminescence in the joints, tail, and trachea, consistent with patterns of aggrecan expression. To evaluate this mouse as a technology for non-invasive quantification of cartilage loss, we characterized the relationship between loss of bioluminescence and loss of cartilage after induction with (i) ex vivo collagenase digestion, (ii) an in vivo OA model utilizing treadmill running, and (iii) age. Ex vivo experiments revealed that collagenase digestion of the femur reduced both luciferase signal intensity and pixel area, demonstrating a link between cartilage degradation and bioluminescence. In an in vivo model of experimental OA, we found decreased bioluminescent signal and pixel area, which correlated with pathological disease. We detected a decrease in both bioluminescent signal intensity and area with natural aging from 2 to 13 months of age. These results indicate that the bioluminescent signal from this mouse may be used as a non-invasive quantitative measure of cartilage. Future studies may use this reporter mouse to advance basic and preclinical studies of murine experimental OA with applications in synovial joint biology, disease pathogenesis, and drug delivery.

No MeSH data available.


Related in: MedlinePlus