Stroke prevents exercise-induced gains in bone microstructure but not composition in mice

Ischemic stroke induces rapid loss in bone mineral density that is up to 13 times greater than during normal aging, leading to a markedly increased risk of fracture. Little is known about skeletal changes following stroke beyond density loss. In this study we use a mild-moderate middle cerebral artery occlusion model to determine the effects of ischemic stroke without bedrest on bone microstructure, dynamic bone formation, and tissue composition. Twenty-seven 12-week-old male C57Bl/6J mice received either a stroke or sham surgery and then either received daily treadmill exercise or remained sedentary for four weeks. All mice were ambulatory immediately following stroke, and limb coordination during treadmill exercise was unaffected by stroke, indicating similar mechanical loading across limbs for both stroke and sham groups. Stroke did not directly detriment microstructure, but exercise only stimulated adaptation in the sham group, not the stroke group, with increased bone volume fraction and trabecular thickness in the sham distal femoral metaphysis. Stroke differentially decreased cortical area in the affected limb relative to the unaffected limb of the distal femoral metaphysis, as well as endosteal bone formation rate in the affected tibial diaphysis. Although exercise failed to improve bone microstructure following stroke, exercise increased mineral-to-matrix content in stroke but not sham. Together, these results show that stroke inhibits exercise-induced changes to femoral microstructure but not tibial composition, even without changes to gait. Similarly, affected-unaffected limb differences in cortical bone structure and bone formation rate in ambulatory mice show that stroke affects bone health even without bedrest.


INTRODUCTION 1
Stroke is the leading cause of long-term disability in the United States -approximately 2 7.0 million Americans have had a stroke, and nearly 4% of the population are projected 3 to have a stroke by 2040 [1]. In addition to cognitive and motor impairments, stroke also 4 severely affects skeletal health, particularly in the paretic limbs, by reducing bone 5 mineral density (BMD) up to 13% per year compared to 1% for healthy aging individuals 6 over 60 years of age [2][3][4][5][6][7]. Reduced BMD and increased susceptibility to falling lead to a 7 15% fracture incidence within 5 years following stroke and a 47% increased risk of 8 fracture compared to age-and sex-matched controls [8,9]. Traditionally, BMD loss 9 following stroke has been attributed to paresis and bedrest. However, in a study 10 examining BMD a year following severe stroke in completely bed-ridden patients, stroke 11 patients still lost more BMD in their paretic limbs, suggesting stroke impacts skeletal 12 health beyond the effects of mechanical unloading [6]. Aside from BMD loss and 13 increased fracture risk, little is known about the effects of stroke on bone health. 14 Understanding more about how stroke affects other bone measures, particularly at the 15 tissue and cellular levels, is a critical first step for identifying mechanisms underlying 16 bone fragility post-stroke and mitigating bone loss in these patients. ischemic stroke in rodents. The MCAo-induced stroke causes ischemia followed by 20 reperfusion damage, mimicking the conditions of the most common type of stroke in 21 human patients [1,10]. In this study, we induced a mild to moderate stroke in mice, 22 ensuring that the animals remained ambulatory after the procedure, to characterize the 23 effect of stroke beyond mechanical disuse effects on bone microstructure, dynamic 24 bone formation, and tissue composition following four weeks of recovery. Since exercise 25 initiated early during stroke recovery is associated with better motor control recovery 26 and BMD maintenance [11][12][13], mice also performed daily treadmill locomotion. The 27 goals of this study were 1) to characterize the effect of stroke without bedrest on bone 28 parameters beyond BMD, and 2) to determine the effect of moderate daily exercise on 29 stroke-induced bone changes.

Study Design 34
The protocol for this project was approved by the North Carolina State University 35 Institutional Animal Care and Use Committee. Mice were housed by surgery group (4-5 36 per cage) on a 12-hour diurnal light cycle with access to chow and water ad libitum. 37 Twenty-seven, 12-week-old, male, C57Bl/6J mice (The Jackson Laboratory, Bar Harbor, 38 ME) received either a stroke (n = 15) or sham (n = 12) surgery (Fig. 1). Following surgery, 39 mice were either given daily treadmill exercise (n = 8 stroke-exercise, n = 6 sham-40 exercise) or placed on a stationary treadmill for an equivalent time period (n = 7 stroke-41 sedentary, n = 6 sham-sedentary). For four days following surgery, mice were housed 42 individually in cages with wetted food and hydrogel packs, and their health was 43 monitored at least twice a day. Body mass was measured twice a day during the 4-day 44 Stroke recovery was also assessed using a rotarod, or rotating rod, test [15]. Mice were 109 placed on a plastic rod that rotates at a velocity that accelerates from 4 to 40 rotations 110 per minute over 5 minutes (ENV-576M, Med Associates Inc, St. Albans, VT). The test 111 scores motor coordination by timing how long the mouse can walk on the rod without 112 falling off or hugging the rod for three consecutive rotations without attempting to 113 walk. Mice were acclimated to the rotarod test for two days immediately prior to 114 surgery using a constant velocity of 20 rotations per minute. Mice performed 115 accelerating rotarod tests at 48 hours, 4 days, and then weekly following surgery, on the 116 same day as neuroscore testing. During each acclimation and rotarod test day, each 117 mouse attempted the test three times, and the longest time was recorded, up to a 118 maximum of 300 seconds. Mice were allowed 15 min of rest between each attempt. 119 120

Treadmill Exercise and Gait Pattern Analysis 121
Treadmill exercise was performed using a rodent treadmill (Exer 3/6, Columbus 122 Instruments, Columbus, OH). Mice in the exercise groups were acclimated to the 123 treadmill for two days prior to surgery at 6 m/min (6.56 yds/min) for 10 min. Exercise 124 therapy began at four days after surgery, gradually increasing the protocol for the first 125 two days. On day four, mice were exercised at 5 m/min (5.47 yds/min) for 10 min, 126 followed by 12 m/min (13.12 yds/min) for 10 min. On day five, mice exercised at 5 127 m/min (5.47 yds/min) for 5 min, followed by 12 m/min (13.12 yds/min) for 20 min.  single gait cycle (paw strike to next paw strike), in the affected hindlimb was calculated 143 relative to the duty cycle in the unaffected hindlimb. Phase dispersion, the relative 144 timing of paw strikes between two limbs within the same gait cycle, was calculated 145 between the affected hindlimb and each of the other limbs: unaffected hindlimb 146 (contralateral), unaffected forelimb (diagonal), and affected forelimb (ipsilateral). All 147 parameters were measured in three sets of five consecutive gait cycles for each weekly 148 treadmill session, and the average parameters were calculated for each week. Video 149 was captured at one day prior to surgery, five days after surgery, and weekly thereafter. 150 151

Micro-Computed Tomography 152
Left and right femora were scanned in 70% ethanol with micro-computed tomography 153 (µCT80, SCANCO Medical AG, Brüttisellen, Switzerland) using a 10-µm voxel size, 45 kV 154 peak X-ray tube potential, 177 µA X-ray intensity, and 800-ms integration time. Volumes 155 of interest (VOI) were analyzed in the distal metaphysis and mid-diaphysis. The 156 metaphyseal VOI was defined as 10% of the total femur length, located proximal to the 157 distal growth plate. The cancellous bone and cortical bone were contoured and analyzed 158 separately in the metaphyseal VOI. Standard cancellous and cortical microstructural 159 parameters were analyzed using the scanner's software (SCANCO v.6.6) [24]. The 160 diaphyseal VOI was defined as 15% of the total femur length, centered at the midpoint 161 between the distal growth plate and middle of the third trochanter, and standard 162 cortical bone parameters were analyzed. Following stroke, all neuroscores were above 0 (sham group score) at all post-stroke 251 timepoints, confirming that the MCAo procedure caused neurological impairment (Fig.  252 3A). Based on these neuroscores, the severity of the induced strokes was mild to 253 moderate, and the magnitude of impairment varied between individual mice and 254 tended to improve over time (decreasing neuroscore). Exercise did not affect 255 neuroscore at any timepoint (p = 0.66), suggesting the treadmill exercise protocol used 256 did not affect stroke recovery. The rotarod test was not a useful metric for assessing 257 motor impairments post-stroke, as it did not capture the same differences as the 258 neuroscore, with no significant effects of stroke (p = 0.88) or exercise (p = 0.37) 259 observed at any timepoint (Fig. 3B). Before surgery, sham and stroke groups had similar 260 body mass (p = 0.38), but the stroke group experienced significant mass losses after 261 ischemic stroke and remained smaller than the sham group at all timepoints post-262 surgery (Fig. 3C).

Raman Spectroscopy 359
Stroke induced minor changes in bone tissue composition in the diaphyseal bone of 360 affected tibiae, while exercise induced more substantial changes, particularly in the 361 stroke group (Fig. 7). Exercise increased phosphate:amide III mineral-to-matrix ratio in 362 stroke-exercise relative to stroke-sedentary by 27% near the endosteal surface (p = 363 0.019) and by 25% in the mid-cortex (p = 0.0070) but not in sham-exercise relative to 364 sham-sedentary (p = 1.00 endosteal, p = 0.58 mid-cortex) (Fig. 7A). Stroke nearly 365 decreased phosphate:amide III mineral-to-matrix in stroke-sedentary relative to sham-366 sedentary near the endosteal surface (18%, p = 0.10) but not in the midcortex (p = 0.91). 367 Exercise also increased the endosteal phosphate:amide I mineral-to-matrix ratio (23%, p 368 = 0.079, Fig. 7C) and the endosteal carbonate-to-matrix ratio (20%, p = 0.11, Fig. 7D) in 369 stroke-exercise relative to stroke-sedentary, but not in sham-exercise relative to sham-370 sedentary (p = 0.96 and p = 0.80, respectively). Exercise had no effect on the 371 phosphate:(proline+hydroxyproline) mineral-to-matrix ratio (Fig. 7B). 372 373 Stroke decreased carbonate substitution near the periosteal surface by 8% relative to 374 sham (main effect p = 0.019, Fig. 7E) and by 12% in stroke-sedentary relative to sham-375 sedentary (p = 0.029). Stroke also nearly decreased phosphate:(proline+hydroxyproline) 376 mineral-to-matrix ratio near the endosteal surface (14%, p = 0.090, Fig. 7B). For 377 carbonate substitution, carbonate-to-matrix ratio, and the phosphate:amide I and 378 phosphate:amide III mineral-to-matrix ratios, the stroke-sedentary group exhibited the 379 most differences relative to the other groups, while stroke-exercise was similar to sham-380 exercise, suggesting exercise may mitigate the effects of stroke on bone composition. 381 Mineral maturity (crystallinity) was unaffected by stroke or exercise (Fig. 6F). hindlimbs. Therefore, the functional tissue strain was likely similar as well. Gait patterns 411 were not analyzed in the sedentary groups, since high-speed video had to be captured 412 during treadmill exercise, and even a small amount of exercise could confound the 413 sedentary results. The acute recovery period appears to be a critical window during 414 which physical activity could prevent skeletal health decline in human stroke patients 415 [34]. Based on a pilot study, we found that the earliest that mice could perform the full 416 treadmill regimen was beginning at four days after stroke. For earlier intervention, 417 alternative physical therapies or exercise-mimicking pharmacological therapies would 418 need to be explored. Future studies could also implement exercise training prior to 419 MCAo, which has been shown to improve functional recovery following stroke [35]. formation. Similarly, another MCAo study in 11-week-old male rats that also found no 424 changes in femoral microstructure, reported affected-unaffected limb differences in the 425 stroke group [32]. They found that, in the cortical bone surrounding the distal femoral 426 metaphysis, stroke increased total area (reported as total volume) by 2% and BMD by 427 5% in the affected relative to the unaffected side, while we found a 4% reduction in 428 total area and a similar 3% increase in BMD. The difference in our total area could be 429 due to stroke interacting with skeletal growth, since 11-week-old rats are at an earlier 430 point in skeletal development than 12-week-old mice. Skeletal growth in C57Bl/6J mice 431 slows at 12 weeks and reaches skeletal maturity at 16 weeks, while growth in rats slows 432 around 15 weeks and reaches skeletal maturity at 20 weeks [36][37][38]. Since animals were 433 ambulatory following stroke in both experiments, these results suggest that stroke, 434 without bedrest, specifically affects the metaphyseal cortical bone envelope. In the 435 tibial diaphysis, stroke differentially decreased mineral apposition rate and bone 436 formation rate by nearly half relative to the unaffected side. However, a similar 437 unaffected-affected side difference in mineral apposition rate was present in sham, as 438 well. These results are consistent with the MCAo study in rats, which found decreased 439 serum concentration of P1NP, a marker of bone formation, four weeks after stroke [32]. to-matrix ratios and carbonate-to-matrix ratio compared to stroke-sedentary. Since 449 minimal affected-to-unaffected limb differences were observed in other metrics, only 450 affected side tibiae were analyzed. Increased mineral-to-matrix and carbonate-to-matrix 451 ratios indicate stiffer, but potentially more brittle, bone that has not been recently 452 remodeled [29,31]. However, increased mineralization may also be a mechanism by 453 which bone adapts to mechanical load without changing tibial morphology [20,31,40]. A 454 study with the same daily treadmill regimen in male C57Bl/6J mice, initiated at 16 455 weeks-of-age for three weeks, found no changes to tibial morphology but a 15% higher 456 phosphate:(proline+hydroxyproline) mineral-to-matrix ratio and approximately 10% 457 higher ultimate tensile strain in the tibia with exercise relative to sedentary [40]. 458 Similarly, a study in young and old rats found that the phosphate:amide I mineral-to-459 matrix ratio was positively correlated with stiffness but negatively correlated with (e.g., no change in bone formation rate and reduced mineralizing surface per surface 473 relative to sham endosteally), but more samples from the sham-exercise group are 474 needed to confirm this observation. However, two recent studies using a similar 475 treadmill regimen in male C57Bl/6J of similar age also found that exercise had no effect 476 on morphology of the tibial diaphysis or on endosteal bone formation rate, but did 477 increase periosteal bone formation rate in the tibial diaphysis and bone volume fraction From the American Heart Association," Circulation, 139 (10)   Experimental design: Twenty-seven C57Bl/6J mice received either a sham or stroke surgery at 12 weeks of age. Mice were further divided into either a moving treadmill exercise group or a stationary treadmill sedentary group. After four weeks of recovery, mice were sacrificed, and their femora and tibiae were collected for analysis. Figure 2. A) In cortical sections of the affected (left) tibial diaphysis, Raman spectra were collected at 6 equallyspaced regions, along a 10-µm long line on the anterior and posterior sides at three locations: endosteal edge, mid-cortex, and periosteal edge. B) Raman spectra were normalized to the phosphate phosphate ν1 band intensity, and band intensity ratios were calculated: phosphate v1/(proline+hydroxyproline), phosphate v1/carbonate v1 (carbonate substitution), phosphate v1/amide I, phosphate v1/amide III, and carbonate v1/amide I. Crystallinity was measured as the inverse of the full width at half-maximum (FWHM) of the phosphate v1 band.  . Femoral microstructure was assessed for group differences in the distal metaphysis (A-C) and mid-diaphysis (D-F) and for affected-unaffected limb differences in the distal metaphysis (G-I). In the metaphysis, exercise in sham, but not stroke, caused increased A) cortical area, B) trabecular thickness, and C) bone volume fraction. In the diaphysis, D) Ct.Ar also increased with sham-exercise relative to sham-sedentary and stroke-exercise; E) cortical thickness and F) cortical area per total area were unaffected by stroke or exercise. In the metaphysis, affected-to-unaffected limb differences were observed in stroke but not sham groups for G) Ct.Ar/Tt.Ar and H) Ct.Ar but not I) Tt.Ar. a: p < 0.05 sham-exercise vs. stroke exercise. a': p < 0.1 sham-exercise vs. stroke exercise. b: p < 0.05 sham-exercise vs. sham-sedentary. b': p < 0.1 sham-exercise vs. sham-sedentary. c: p < 0.05 vs. unaffected side within surgery group. Figure 6. Dynamic cortical bone formation was assessed in the tibial diaphysis with dynamic histomorphometry. No differences were found on the periosteal surface. On the endosteal surface, exercise decreased (relative to sedentary) A) mineralizing surface per bone surface (MS/BS) but not B) mineral apposition rate (MAR) or C) bone formation rate (BFR). For affected (Aff) vs. unaffected (Un) limb differences, neither sham nor stroke group showed D) differences for MS/BS, but E) both sham and stroke had decreased MAR in the affected limbs, and F) stroke decreased BFR in the affected limb. (A-C) Data presented as estimated least-squares mean ± 95% confidence interval. c: p < 0.05 vs. unaffected side within surgery group. d: p < 0.05 exercise vs. sedentary (main effect).