(June 22, 2023) Webinar: iNSiGHT & PIXImus: A Tale of Two Systems​

Overview:

In this webinar, Dr. David Maridas from Harvard School of Medicine will shared how the latest Dual X-ray Absorptiometry (DXA) advances helped his research. Dr. Maridas will walked us through comparing different systems used in acquiring and analyzing data.

Dual X-ray absorptiometry allows measurements of fat mass, lean mass, bone mineral density, and bone mineral content in live animals. The short radiation exposure time allows for measurements to be performed in vivo using relatively little anesthesia (e.g., isoflurane), making DXA a powerful tool to frequently measure multiple parameters throughout the life of an animal.

Over the years, multiple DXA systems were implemented with different strengths and weaknesses. During this webinar, we will compared the PIXImus and the iNSiGHT for measurements in adult mice.

Looking at the latest advancements in the field of Dual X-ray Absorptiometry (DXA), Dr. Maridas focused on the comparison of two systems, the iNSiGHT and the PIXImus, which are important tools for the accurate measurement of fat mass, lean mass, bone mineral density, and bone mineral content in live animals.

He critically evaluated and contrast the strengths and weaknesses of these systems in terms of their functionalities, logistics, and the unique challenges they pose, providing guidance for researchers in making the most suitable choice based on their specific requirements and constraints. In conclusion, the selection of a system would depend on the need of scientists and the available space for the machine.

Key Points:

-Evolution of DXA Technology: Watch & learn about the technological advancements in DXA as Dr. Maridas delves into the transformation from legacy to contemporary systems, illustrating how the development of this technology impacts the precision and efficiency of live animals.

-iNSiGHT-ful Comparison: With first-hand experience working with both the iNSiGHT and the PIXImus DXA systems, Dr. Maridas will provided an in-depth, comparative analysis, presenting unique strengths and functionalities.

-Behind-the-Scenes Coordination: Gain exclusive insights into the dynamics and understand how the systems influenced Dr. Maridas’s research outcomes.

1. INSiGHT & PIXImus: A Tale of Two Systems David Maridas, PhD Instructor in Developmental  Biology , Harvard School of Dental Medicine

2. Outline Dual X-ray Absorptiometry (DXA) in skeletal studies. IGFBP4 knockout mice skeletal phenotype. Preliminary results of Alk4-FC treatment in mice. Comparison between two DXA systems. 2

3. Skeletal phenotyping in mice 3 16 wks 8 wks 4 wks 22 wks Female Male 14 wks 52 wks 1mm

4. Skeletal phenotyping in mice 4 16 wks 8 wks 4 wks 22 wks Female Male 14 wks 52 wks 1mm

5. Microcomputed tomography (µCT) limitations 5 Requires sacrifice of mice to isolate bones. Very slow, days worth of work to process a cohort. Measurement thresholds (contouring, segmentation …) set by user. Expensive. In vivo systems available but expensive and time-consuming.

6. Using 2 X-Rays beams with different energy to measure bone mineral density (and other parameters). Strengths: Non-invasive. Minimal dose of radiation. Fast Cheap Weaknesses: No assessment of trabecular bone. No 3D reconstruction and microarchitecture assessment. Dual X-ray Absorptiometry (DXA) 6

7. Dual X-ray Absorptiometry (DXA) 7 DXA scan in clinics DXA ( PIXImus ) for small animals Using 2 X-Rays beams with different energy to measure bone mineral density (and other parameters). Strengths: Non-invasive. Minimal dose of radiation. Fast Cheap Weaknesses: No assessment of trabecular bone. No 3D reconstruction and microarchitecture assessment.

8. Dual X-ray Absorptiometry (DXA) 8 DXA scan in clinics DXA ( PIXImus ) for small animals Using 2 X-Rays beams with different energy to measure bone mineral density (and other parameters). Strengths: Non-invasive. Minimal dose of radiation. Fast Cheap Weaknesses: No assessment of trabecular bone. No 3D reconstruction and microarchitecture assessment.

9. The PIXImus system The PIXImus densitometer is a portable unit. It uses a stationary anode X-ray tube that generates a cone beam X-ray. PIXImus uses low X-ray energies (80/35 kVp at 500 µA). The stationary area detector uses ultrahigh resolution pixels (0.18 x 0.18 mm). A chieves contrast in extremely low-density bone and for tissue comparison analysis. 9 30 x 63 x 33 cm, ~ 60lbs

10. PhD Thesis Growth Hormone Hypothalamus Anterior Pituitary GHRH Somatostatin Liver IGF-I Metabolism Adipose Tissue Bone Skeletal muscle Adapted from Thorner MO, et al. In: Williams Textbook of Endocrinology . 9th ed. 1998:249.* *Clemmons D, Cook D, Kleinberg D

11. Mesenchymal Progenitor IGF-I: mitogenic and differentiating factor IGF-I

12. Mesenchymal Progenitor Adipocyte Chondrocyte IGF-I: mitogenic and differentiating factor Osteoblast IGF-I IGF-I IGF-I IGF-I

13. Mesenchymal Progenitor Adipocyte Chondrocyte IGF-I: mitogenic and differentiating factor Osteoblast IGF-I IGF-I IGF-I IGF-I IGFBP

14. IGFBP4 Growth IGFBP1 Glucose Metabolism IGFBP2 Fat Bone IGFBP3 Growth IGFBP5 Cell Proliferation IGFBP6 Proliferation Migration IGFBPs regulate IGF actions locally

15. IGFBP4 Growth IGFBP1 Glucose Metabolism IGFBP2 Fat Bone IGFBP3 Growth IGFBP5 Cell Proliferation IGFBP6 Proliferation Migration IGFBPs regulate IGF actions locally

16. Actions of IGFBP4 IGFBP4 inhibits IGF actions in almost all in vitro models: Bone cells Vascular smooth muscle cells Colon cancer cells In vivo overexpression of IGFBP4 in bone reduced bone formation. Injections of IGFBP4 in mice induced bone formation. Surprisingly, IGFBP4 KO mice a growth delay but the knockout was generated on a mixed background. IGFBP4 Growth ?

17. Hypothesis: IGFBP4 regulates growth by affecting skeletal postnatal acquisition

18. Hypothesis: IGFBP4 regulates growth by affecting skeletal postnatal acquisition Investigate postnatal growth of IGFBP4 knockout mice on C54BL6/J background.

19. Hypothesis: IGFBP4 regulates growth by affecting skeletal postnatal acquisition Investigate postnatal growth of IGFBP4 knockout mice on C54BL6/J background.

20. Hypothesis: IGFBP4 regulates growth by affecting skeletal postnatal acquisition Investigate postnatal growth of IGFBP4 knockout mice on C54BL6/J background.

21. Hypothesis: IGFBP4 regulates growth by affecting skeletal postnatal acquisition Investigate postnatal growth of IGFBP4 knockout mice on C54BL6/J background.

22. Igfbp4 +/+ Igfbp4 -/- Females Males

23. DXA revealed that female Igfbp4 -/- mice had decreased BMD at 16wks

24. MicroCT results confirmed our DXA data

25. Igfbp4 -/- mice are protected against ovariectomy-induced bone loss

26. Model: Igfbp4 is regulated by estrogen and enhances IGFI availability

27. Not enough to graduate!

28. Igfbp4 -/- mice have decreased adiposity

29. Igfbp4 deletion inhibits adipogenesis in vitro.

30. Igfbp4 deletion inhibits adipogenesis in vitro.

31. Igfbp4 deletion inhibits adipogenesis in vitro.

32. I could graduate!

33. I could graduate! HSDM And move on

34. Audience Poll

35. Alk2 Acvr1 Alk3 Bmpr1a Alk6 Bmpr1b Acvr2a Acvr2b Bmpr2 BMP signaling Type I receptors Type II receptors Smad1/5/8 Acvr2a Acvr2b Activin signaling Alk4 Acvr1b Smad2/3 TGFβr2 TGF β signaling Alk5 TGFβr1 Smad2/3 BMPs, activins and TGFβ receptors in skeletal progenitors

36. Alk2 Acvr1 Alk3 Bmpr1a Alk6 Bmpr1b Acvr2a Acvr2b Bmpr2 BMP signaling Type I receptors Type II receptors Smad1/5/8 Acvr2a Acvr2b Activin signaling Alk4 Acvr1b Smad2/3 TGFβr2 TGF β signaling Alk5 TGFβr1 Smad2/3 BMPs, activins and TGFβ receptors in skeletal progenitors

37. Activin signaling Alk4 Acvr1b Smad2/3

38. Activin signaling Alk4 Acvr1b Smad2/3

39. Activin signaling Alk4 Acvr1b Smad2/3 Female Male Alk4 fl/fl Alk4 fl/fl , Prx1-Cre Age (months)

40. Systemic approach! : Alk4 binding ligands Alk4 Alk4-FC Smad2/3 : Alk4 binding ligands Alk4 Smad2/3 Smad2/3 Overarching hypothesis Circulating Alk4-binding ligands regulate bone homeostasis in mice

41. Systemic approach! : Alk4 binding ligands Alk4 Alk4-FC Smad2/3 : Alk4 binding ligands Alk4 Smad2/3 Smad2/3 Overarching hypothesis Circulating Alk4-binding ligands regulate bone homeostasis in mice Grant deadline: needed pilot data FAST!

42. Inject 12-week-old C57BL6J (n=3) with 3mg/kg BW of Alk4-FC for 2 weeks

43. Inject 12-week-old C57BL6J (n=3) with 3mg/kg BW of Alk4-FC for 2 weeks 660 x 581 x 1127 mm, ~ 570lbs

44. Inject 12-week-old C57BL6J (n=3) with 3mg/kg BW of Alk4-FC for 2 weeks 660 x 581 x 1127 mm, ~ 570lbs

45. Inject 12-week-old C57BL6J (n=3) with 3mg/kg BW of Alk4-FC for 2 weeks Secured funding! Project ongoing... 660 x 581 x 1127 mm, ~ 570lbs

46. Comparison of DXA systems PIXImus Insight Inalyzer

47. Comparison of DXA systems * Significantly different from MRI

48. Comparison of DXA systems * Significantly different from MRI

49. Comparison of DXA systems * Significantly different from MRI Scanning time was different too!

50. Acknowledgements Rosen Lab Vicki Rosen , PhD Emily Van Doren, DMD Laura Gamer, PhD Emily Moore, PhD Bouxsein Lab Jennifer Coulombe, PhD Daniel Brooks, PhD Mary Bouxsein , PhD

51. Q&A Session WWW.SCINTICA.COM [email protected] Please enter your questions in the Q&A section. Thank You!