Species differences in histomorphometry
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1 Species differences in histomorphometry Reinhold G. Erben Department of Biomedical Sciences Institute of Physiology and Pathophysiology University of Veterinary Medicine Vienna
2 Purpose of histomorphometry Histomorphometry is always related to mechanistic questions or safety issues Bone structure Bone formation Bone resorption Bone mineralization Bone modeling and remodeling
3 Important issues Strain-, gender-, age-related and site specific differences in bone structure and bone mass Differences in osteoclast morphology Differences in osteoblast morphology and assessment of bone formation Differences in cancellous and cortical bone remodeling Differences in the physiologic response to bone-active drugs
4 Gender-related differences in mice Female Female Male Male Estrogen is a bone anabolic hormone in mice, unlike other mammalian species. Schmidt et al. Am J Pathol 155:557, 1999
5 Age-related changes in mice 3 mo 3 mo 1 y
6 Age-related changes in rats 4 mo 2 2 y yorx
7 Vertebrae in mice and rats 3 mo 10 mo 22-mo-old rats SHAM ORX
8 Example: Efficacy of denosumab in GIO model Murine RANKL genomic structure Exon non-coding region RANKL targeting vector Human Ex 5 Neo Short arm Long arm LoxP LoxP TK TK Mu 3 non-coding region hurankl Knock-in allele Human Ex 5 Neo LoxP LoxP Exon Mu 3 non-coding region (Kostenuik et al., JBMR 24:182, 2009)
9 OPG/RANKL system RANK: Receptor activator of NFκB OPG: Osteoprotegerin RANKL: RANK ligand Stromal cell/t cell Oc precursor srankl RANK Denosumab Osteoclast Differentiation Fusion RANKL Denosumab OPG Osteoblasts/Stromal cells
10 Example: Efficacy of denosumab in GIO model 0.3 Bone formation rate L1 125 Serum osteocalcin (µm²/µm/day) (ng/ml) C Pred WT PBS Ab Control PBS Pred hrankl KI Ab Two-way ANOVA Effect of Pred: NS Effect of Denosumab: p< C WT Pred PBS Ab Control PBS Pred hrankl KI Ab Two-way ANOVA Effect of Pred: NS Effect of Denosumab: p=0.012 Hofbauer et al. Arthritis Rheum 60:1427, 2009
11 Example: Efficacy of denosumab in GIO model Bone formation rate distal femur (µm²/µm/day) PBS Ab PBS Ab C Pred Control WT Pred hrankl KI Two-way ANOVA Effect of Pred: NS Effect of Denosumab: p<0.001 In aged mice or severely osteopenic rats, always take out the verts! Hofbauer et al. Arthritis Rheum 60:1427, 2009
12 Differences in osteoclast morphology
13 Differences in osteoclast morphology Reliable detection of osteoclasts in mice should be based on TRAPstained sections! Therefore, it is necessary to use a methylmethacrylate mixture suitable for histochemistry (e.g., Erben, J Histochem Cytochem 45:307,1997), or to decalcify bone specimens.
14 Differences in osteoblast morphology Marker interval (cancellous bone formation!) 1 day in 3 4-wk-old mice 2 days in 8 12-wk-old mice 2-3 days in aged mice >5 mo 4-5 days in rats
15 Do mice and rats have bone remodeling? Rabbit Rat Mouse Gefäßkanal
16 Do rats and mice have bone remodeling?
17 Age dependent changes in modeling and remodeling Proximal tibial cancellous bone % of all formation sites months 6 months 9 months 12 months Age %Remodeling %Modeling %Uncertain (Erben 1996) Erben Anat Rec 246:39, 1996
18 Age dependent changes in modeling and remodeling Lumbar vertebral cancellous bone % of all formation sites months 6 months 12 months Age %Remodeling %Modeling %Uncertain (Erben, 1996) Erben Anat Rec 246:39, 1996
19 Age dependent changes in modeling and remodeling Longitudinal bone growth in the proximal tibia and L1 of female Fischer rats µm/day Proximal tibia L1, cranial growth plate L1, caudal growth plate months 6 months 9 months 12 months Age Erben Anat Rec 246:39, 1996
20 Labeling escape error MI1 MI2 To minimize the labeling escape error, the marker interval should be less than about 1/5 of the formation period. Time MI3 More information: Erben RG (2003) Bone Labeling Techniques. In: Handbook of Histology Methods for Bone and Cartilage. An YH, Martin KL (eds) Humana Press Inc., Totowa, NJ, USA, pp
21 Bone remodeling in rats and mice Rat vertebral cancellous bone FP RsP RmP (days) months 6 months 9 months 12 months Age
22 Bone remodeling in rats and mice Murine vertebral cancellous bone FP RsP RmP (days) months 6 months 9 months 12 months Age
23 Differences in physiological response to drugs (mg/cm³) Total BMD L4 * P<0.05 vs. Veh * Veh PTH Veh PTH Male Female Gender-related skeletal effect of cyclosporin A (ng/ml) Erben et al. Endocrinology 144:40, CsA blood concentration 800 Males Females SHAM GX GX+HRT Female rats have a 7-fold higher hepatic CsA excretion rate relative to males in steady state.
24 Betacellulin transgenic mice Male Female 3 wk 6 wk 9 wk 3 wk 6 wk 9 wk wt tg Schneider et al. JBMR 24:455, 2009
25 Take home messages Always take out vertebrae in aged mice and severely osteopenic rats. For the quantification of osteoclasts in mice always use TRAP staining. For the assessment of bone formation fluorochrome labeling with an appropriate marker interval is essential. Rats and mice lack true Haversian cortical bone remodeling, but not cancellous bone remodeling activity. Consider species specific effects of certain drugs in rats and mice (e.g., CsA in rats, estrogen and PTH in mice).
26 Acknowledgements Institute of Physiology & Pathophysiology University of Veterinary Medicine Vienna Ute Zeitz Claudia Bergow Christiane Schüler Kerstin Klien Institute of Animal Physiology LMU Munich Karin Begsteiger Sieglinde Hirmer Technical University Dresden Lorenz Hofbauer Gene Center LMU Munich Marlon Schneider Eckhard Wolf
27 ISBM Meeting, Zell am See, Austria, May 28 30, 2009 Special feature: One-day hands-on practical training course on May 28
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