PROTECTIVE EFFECTS OF CONTROLLED MECHANICAL STRAIN ENVIRONMENTS IN HINDLIMB UNLOADED MALE C57BL/6J MICE

Restricted (Penn State Only)
Author:
Delong, Drew
Graduate Program:
Laboratory Animal Medicine
Degree:
Master of Science
Document Type:
Master Thesis
Date of Defense:
April 01, 2019
Committee Members:
  • Gregory Stephen Lewis, Thesis Advisor
  • Ronald Paul Wilson, Committee Member
  • Henry Donahue, Committee Member
Keywords:
  • Hind-limb suspension
  • in-vivo axial loading
  • tibial compression loading model
  • disuse
  • spaceflight
  • bone
  • countermeasure
  • microCT
  • mouse
Abstract:
Spaceflight and prolonged bed rest can cause significant deleterious effects on bone mass and strength. Disuse osteopenia is a common disorder for bed-ridden patients; furthermore, astronauts can lose up to 1.5% bone volume and 10% muscle mass during one month of space travel. Both of these situations lead to an increased risk for fracture during the recovery phase or during or after spaceflight. Therapeutics, such as pharmacologics, nutrition and exercise, have yielded variable success; therefore, more effective countermeasures are necessary. The hind limb suspension rodent model, which routinely creates consistent and reproducible bone and muscle loss in the tibia and femur, has been largely adopted as the model of choice to simulate spaceflight and disuse. A potential countermeasure to bone loss induced by the hind limb suspension model could be mechanical loading of the limb. The tibial compression loading model is reported to produce osteogenic changes in areas of strain, but has not been previously combined simultaneously with hind limb suspension. Adult male (16 weeks) C57BL/6J mice were randomly assigned into two groups, Hind Limb Suspension (n=15) and Ground Control (n=10). Mice were hind limb suspended, using a modified hind-limb suspension model, originally described by Morey-Holton, for three consecutive weeks and subjected to anesthetized mechanical load (1200 cycles/day at 9N compression at a saw-tooth waveform at 4Hz) four times per week. The right hind limb (loaded) was mechanically loaded in all mice, and the left hind limb was an internal control (unloaded). Micro-Ct analysis of three tibial locations subject to different load-induced strain environments (proximal metaphysis (trabecular), mid-shaft (cortical), distal-shaft (cortical)) was completed at days 0, 11 and 21. Approximately 250 ul blood was collected on day 11 and at the termination of the experiment (day 21) for serum biomarker (CTX and P1NP) analysis. There was an additional control group of HLS animals (n=10) for this assay. At day 21, trabecular bone loss was apparent in three conditions (HLS+load, HLS unloaded, GC+load). Significant differences of bone loss (BV/TV, Trabecular #, Trabecular thickness, Trabecular separation, Bone mineral density) were noted between HLS unloaded and HLS+load limbs, with e.g. -50% loss in BV/TV in unloaded limbs but only -15 % loss in contralateral loaded limbs (p=0.001). In some trabecular parameters (BV/TV, thickness, BMD), the HLS loaded data was not significantly different from GC unloaded. At day 21, cortical bone at mid-shaft displayed significantly less bone loss (Cortical thickness) in HLS loaded compared to unloaded limbs, with e.g. -6% loss in Ct.Th in unloaded limbs but only -2% loss in contralateral loaded limbs (p=0.024). The HLS unloaded group had significant cortical bone loss compared to GC groups in some parameters (Total area, Cortical bone area, Cortical thickness). In some cortical parameters (Total area, Cortical bone area, Cortical Area Fraction, Cortical thickness), the HLS loaded data was not significantly different from GC unloaded Cortical bone at the distal-shaft showed similar changes to values at mid-shaft. At day 21, there was a significant increase of P1NP in the serum in the loaded animals compared to the unloaded controls (p=0.009). There were no differences detected in CTX levels at either time point. Mechanical strain environments, as little as 6 mins/day, 4 days/week can provide protection against trabecular and cortical bone loss induced from an unloaded state. This is most apparent when analyzing the contralateral limbs of experimental animals. Also, of note, many micro-CT parameters of the loaded limbs of suspended animals did not significantly differ from ground control limbs, indicating that a controlled mechanical strain provides protection from bone loss approaching what an animal normally would encounter with age-related bone loss. Furthermore the higher strain cortical region was more protected from bone loss than the lower strain region. The load + suspension group showed significantly higher amounts of P1NP (bone formation marker) than the suspension group that did not receive load, but no differences in CTX, indicating that the protection against bone loss was through increased formation as opposed decreased resorption. This study uniquely isolates the role of bone strain in protecting bone from disuse-related loss, and demonstrates that limited bouts of mechanical loading may be useful during periods of prolonged disuse or bedrest.