Diminished bone perfusion develops in response to disuse and has been proposed as a mechanism underlying bone loss. Bone blood flow (BF) has not been investigated within the unique context of severe contusion spinal cord injury (SCI), a condition that produces neurogenic bone loss that is precipitated by disuse and other physiological consequences of central nervous system injury. Herein, 4-mo-old male Sprague-Dawley rats received T9 laminectomy (SHAM) or laminectomy with severe contusion SCI (n = 20/group). Time course assessments of hindlimb bone microstructure and bone perfusion were performed in vivo at 1- and 2-wk postsurgery via microcomputed tomography (microCT) and intracardiac microsphere infusion, respectively, and bone turnover indices were determined via histomorphometry. Both groups exhibited cancellous bone loss beginning in the initial postsurgical week, with cancellous and cortical bone deficits progressing only in SCI thereafter. Trabecular bone deterioration coincided with uncoupled bone turnover after SCI, as indicated by signs of ongoing osteoclast-mediated bone resorption and a near-complete absence of osteoblasts and cancellous bone formation. Bone BF was not different between groups at 1 wk, when both groups displayed bone loss. In comparison, femur and tibia perfusion was 30%-40% lower in SCI versus SHAM at 2 wk, with the most pronounced regional BF deficits occurring at the distal femur. Significant associations existed between distal femur BF and cancellous and cortical bone loss indices. Our data provide the first direct evidence indicating that bone BF deficits develop in response to SCI and temporally coincide with suppressed bone formation and with cancellous and cortical bone deterioration.NEW & NOTEWORTHY We provide the first direct evidence indicating femur and tibia blood flow (BF) deficits exist in conscious (awake) rats after severe contusion spinal cord injury (SCI), with the distal femur displaying the largest BF deficits. Reduced bone perfusion temporally coincided with unopposed bone resorption, as indicated by ongoing osteoclast-mediated bone resorption and a near absence of surface-level bone formation indices, which resulted in severe cancellous and cortical microstructural deterioration after SCI.
Activity-based physical rehabilitation (e.g., bodyweight supported treadmill training (TM) or passive Cycle training) promotes recovery of voluntary locomotor function after moderate-severity spinal cord injury (SCI). However, little evidence supports efficacy of these treatments following severe SCI. PURPOSE: To determine the effects of TM and passive Cycle training on the recovery of voluntary locomotor function in rodents after severe SCI. METHODS: 16-week old male Sprague-Dawley rats (n=44) received either a T9 laminectomy (SHAM) surgery or T9 laminectomy plus severe (250 kilodyne) contusion SCI using a computer-guided impactor. SCI animals were then stratified into groups that received 1) no training (SCI), 2) TM training (SCI+TM), or 3) Cycle training (SCI+Cycle). TM and Cycle training were initiated 1-week post-surgery and consisted of two 20 min bouts/day, performed 5 days/week for 3 weeks. Hindlimb locomotion was assessed weekly using the BBB Locomotor Rating Scale. RESULTS: One-week post-surgery, all SCI animals exhibited locomotor deficits (BBB score <3 on a 0-21 scale, p<0.01 vs baseline), indicating near-complete hindlimb paralysis. Thereafter, the SCI group spontaneously regained some voluntary hindlimb function (BBB = 6.1 ± 0.993 at week 4, p<0.01 vs week 1). However, SCI animals did not recover the ability to hindlimb weight support in stance or to perform stepping patterns. Similarly, BBB scores improved in the SCI+TM group from week 1 to week 4 (p<0.01), although, hindlimb locomotor recovery was not greater than SCI alone. In contrast, BBB scores did not improve significantly from weeks 1-4 in the SCI+Cycle group. SCI+TM exhibited higher BBB scores than SCI+Cycle at weeks 3-4 (week 4 average: SCI+TM = 8.1 ± 1.025; SCI+Cycle = 4.4 ± 0.561 , p<0.05 ) CONCLUSION: Our findings suggest that neither TM nor Cycle training promoted locomotor recovery after a 3-week time span in male rodents after severe SCI. Additionally, Cycle training may have limited locomotor recovery in our model, given that BBB scores did not improve in SCI+Cycle animals. Future research is needed to determine an alternative treatment that can be used in conjunction with locomotor training to improve ambulatory status after severe SCI.
Muscle atrophy is a major sequela occurring after spinal cord injury (SCI) that results from disuse. Additionally, other secondary complications of SCI (e.g., alterations in muscle blood flow) may contribute to muscle loss. PURPOSE: To determine the time course of muscle blood flow changes in relation to muscle atrophy in a rodent severe contusion SCI model. METHODS: Sixty-three 4-months-old (skeletally-mature) male Sprague-Dawley rats received SHAM surgery (T9 laminectomy) or severe (250 kdyne) contusion SCI using a computer-guided impactor. At 1-, 2-, and 4-weeks (wk) post-surgery, an intravenous catheter was implanted into the tail vein of SHAM and SCI animals. Colored microspheres (15μm diameter) were then infused into the circulation, allowing for the measurement of regional blood flow (ml/min/g tissue mass). Subsequently, the animals were euthanized and the mass of the dissected right and left soleus, gastrocnemius, and plantaris were taken. Concentrations of the colored microspheres within each muscle were determined via spectrophotometry, following chemical digestion of the muscle. Muscle blood flow calculations were then averaged across the contralateral hindlimbs for the aforementioned muscles. SCI vs SHAM comparisons were made at each time point using independent samples t-tests and Pearson's correlation coefficients. RESULTS: SCI animals exhibited 23-41% lower soleus mass, 17-27% lower gastrocnemius mass, and 16-29% lower plantaris mass vs SHAM, at all time points (p<0.001). Soleus and gastrocnemius blood flow (corrected for tissue mass, ml/min/g) was 51% lower (p<0.001) and 25% lower (p<0.05) after SCI, respectfully, at 1-wk only. Additionally, a positive relationship between soleus mass and blood flow (corrected for mass) was identified at 1-wk (r= 0.687, p<0.01). No significant alterations in plantaris blood flow were identified at any time point. CONCLUSION: Hindlimb muscle atrophy and reduced muscle blood flow occurred within 1-wk of severe contusion SCI. Thereafter, muscle blood renormalized in comparison with SHAM animals. Further research is needed to determine whether the reductions in muscle blood flow occurring after SCI contribute to muscle loss and/or whether prevention of blood flow deficits preserves muscle mass.
It is unknown whether activity-based physical therapy (ABPT) modalities that mobilize the paralyzed limbs improve bone integrity at the highly fracture-prone epiphyseal regions of the distal femur and proximal tibia following severe spinal cord injury (SCI). In this study, 4-mo-old skeletally mature littermate-matched male Sprague-Dawley rats received either SHAM surgery or severe contusion SCI. At 1 wk postsurgery, SCI rats were stratified to undergo no-ABPT, two 20-min bouts/day of quadrupedal bodyweight-supported treadmill training (qBWSTT), or hindlimb passive isokinetic bicycle (cycle) training, 5 days/wk for another 3 wk. We assessed locomotor recovery and plantar flexor muscle mass, tracked cancellous and cortical bone microstructure at the distal femoral and proximal tibial epiphyses using in vivo microcomputed tomography (microCT), and evaluated bone turnover at the tibial epiphysis with histomorphometry. All SCI animals displayed persistent hindlimb paralysis and pervasive muscle atrophy. Over the initial 2 wk, which included 1 wk of no exercise and 1 wk of ABPT acclimation, a similar magnitude of bone loss developed in all SCI groups. Thereafter, cancellous bone loss and cortical bone decrements increased in the SCI no-ABPT group. qBWSTT attenuated this trabecular bone loss but did not prevent the ongoing cortical bone deficits. In comparison, twice-daily cycle training increased the number and activity of osteoblasts versus other SCI groups and restored all bone microstructural parameters to SHAM levels at both epiphyseal sites. These data indicate that a novel passive isokinetic cycle training regimen reversed cancellous and cortical bone deterioration at key epiphyseal sites after experimental SCI via osteoblast-mediated bone anabolic mechanisms, independent of locomotor recovery or increased muscle mass.
BACKGROUND 3D image registration is a technique where in‐vivo microCT scans are collected at different timepoints and regions of interest (ROI) are constructed and aligned to improve the precision of determining bone microstructure. In the rodent spinal cord injury (SCI) model, the rapid bone loss occurring at the distal femur precludes the use of standard 3D registration strategies. PURPOSE To (1) adapt a microCT‐based 3D registration protocol to our rodent SCI model, (2) determine the degree of cancellous bone loss at the distal femoral epiphysis after SCI, and (3) assess the effects of bodyweight‐supported treadmill training (TM) or passive bicycle training (PBT) on bone loss after SCI. METHODS 16‐wk old male Sprague‐Dawley rats were stratified to receive: 1) T9 laminectomy (SHAM) (n=9), 2) severe T9 contusion (SCI) (n=10), 3) SCI+TM (n=10), or 4) SCI+PBT (n=14). TM and PBT began 1‐wk post‐surgery (post‐sx, two 20‐min bouts/day, 5‐d/wk for 3‐wks). In‐vivo microCT scans were performed pre‐sx and 2‐ and 4‐wks post‐sx. Images were aligned with a 3D registration protocol. ROIs were developed to assess cancellous bone microstructure at the distal femoral epiphysis using two separate protocols that either included or excluded new bone formed by periosteal bone expansion over the 4‐wk experiment. RESULTS Differences were noted between the ROI protocols, with the ROI that included periosteal bone growth underestimating SCI‐induced bone loss. As such, the results reported hereafter were derived from the ROI that excluded new bone resulting from periosteal bone expansion. No differences in bone outcomes were present in SHAMs at any timepoint except for a slightly higher trabecular separation (Tb.Sp) at 4‐wks (p<.05). At 2‐wks, SCI displayed 14% lower cancellous bone volume (BV/TV) than pre‐sx (p<.01), characterized by 13% lower trabecular number (Tb.N) (p<.05) and 7% higher Tb.Sp (p<.05). Bone loss was more pronounced at 4‐wks after SCI, evidenced by lower trabecular thickness (Tb.Th) and higher Tb.Sp vs 2‐wks (both p<.01). SCI+TM and SCI+PBT displayed a similar magnitude of bone loss to SCI at 2‐wks (1‐wk after starting exercise). Thereafter, SCI+TM displayed no further bone loss, resulting in 9% less BV/TV loss than SCI (p<.01). In comparison, PBT increased BV/TV 15% from 2‐ to 4‐wks (p<.01), due to 5% higher Tb.Th (p<.01) and 12% higher Tb.N (p<.05), ultimately restoring BV/TV to pre‐sx levels. Structural model index (SMI) and trabecular pattern factor (Tb.Pf) increased in SCI (p<0.05) and SCI+TM at 4‐wks (p<.05 to <.01), signifying transition from rod‐like to weaker plate‐like trabeculae and a less connected trabecular network, respectively. In comparison, SMI (p<.05) and Tb.Pf (p<.01) increased in SCI+PBT at 2‐wks before returning to pre‐sx levels by 4‐wks. CONCLUSION Using our 3D registration protocol, we determined that SCI causes severe cancellous bone loss and changes indicative of an overall weakening of bone architecture at the distal femoral epiphysis. TM attenuated bone loss at this skeletal site, while PBT promoted new bone formation and restored BV/TV to pre‐sx levels. Support or Funding Information This work was supported by the APS UGSRF.
Spinal cord injury (SCI) produces diminished bone perfusion and bone loss in the paralyzed limbs. Activity-based physical therapy (ABPT) modalities that mobilize and/or reload the paralyzed limbs (e.g., bodyweight-supported treadmill training (BWSTT) and passive-isokinetic bicycle training) transiently promote lower-extremity blood flow (BF). However, it remains unknown whether ABPT alter resting-state bone BF or improve skeletal integrity after SCI.Four-month-old male Sprague-Dawley rats received T 9 laminectomy alone (SHAM; n = 13) or T 9 laminectomy with severe contusion SCI ( n = 48). On postsurgery day 7, SCI rats were stratified to undergo 3 wk of no ABPT, quadrupedal (q)BWSTT, or passive-isokinetic hindlimb bicycle training. Both ABPT regimens involved two 20-min bouts per day, performed 5 d·wk -1 . We assessed locomotor recovery, bone turnover with serum assays and histomorphometry, distal femur bone microstructure using in vivo microcomputed tomography, and femur and tibia resting-state bone BF after in vivo microsphere infusion.All SCI animals displayed immediate hindlimb paralysis. SCI without ABPT exhibited uncoupled bone turnover and progressive cancellous and cortical bone loss. qBWSTT did not prevent these deficits. In comparison, hindlimb bicycle training suppressed surface-level bone resorption indices without suppressing bone formation indices and produced robust cancellous and cortical bone recovery at the distal femur. No bone BF deficits existed 4 wk after SCI, and neither qBWSTT nor bicycle altered resting-state bone perfusion or locomotor recovery. However, proximal tibia BF correlated with several histomorphometry-derived bone formation and resorption indices at this skeletal site across SCI groups.These data indicate that passive-isokinetic bicycle training reversed cancellous and cortical bone loss after severe SCI through antiresorptive and/or bone anabolic actions, independent of locomotor recovery or changes in resting-state bone perfusion.
Loading and testosterone may influence musculoskeletal recovery after spinal cord injury (SCI). Our objectives were to determine (a) the acute effects of bodyweight-supported treadmill training (TM) on hindlimb cancellous bone microstructure and muscle mass in adult rats after severe contusion SCI and (b) whether longer-term TM with adjuvant testosterone enanthate (TE) delivers musculoskeletal benefit. In Study 1, TM (40 min/day, 5 days/week, beginning 1 week postsurgery) did not prevent SCI-induced hindlimb cancellous bone loss after 3 weeks. In Study 2, TM did not attenuate SCI-induced plantar flexor muscles atrophy nor improve locomotor recovery after 4 weeks. In our main study, SCI produced extensive distal femur and proximal tibia cancellous bone deficits, a deleterious slow-to-fast fiber-type transition in soleus, lower muscle fiber cross-sectional area (fCSA), impaired muscle force production, and levator ani/bulbocavernosus (LABC) muscle atrophy after 8 weeks. TE alone (7.0 mg/week) suppressed bone resorption, attenuated cancellous bone loss, constrained the soleus fiber-type transition, and prevented LABC atrophy. In comparison, TE+TM concomitantly suppressed bone resorption and stimulated bone formation after SCI, produced near-complete cancellous bone preservation, prevented the soleus fiber-type transition, attenuated soleus fCSA atrophy, maintained soleus force production, and increased LABC mass. 75% of SCI+TE+TM animals recovered voluntary over-ground hindlimb stepping, while no SCI and only 20% of SCI+TE animals regained stepping ability. Positive associations between testosterone and locomotor function suggest that TE influenced locomotor recovery. In conclusion, short-term TM alone did not improve bone, muscle, or locomotor recovery in adult rats after severe SCI, while longer-term TE+TM provided more comprehensive musculoskeletal benefit than TE alone.
Severe cancellous bone loss occurs after spinal cord injury (SCI), which increases fracture risk. Bodyweight-supported treadmill training (TM) and passive Cycle training are activity-based rehabilitation therapies that improve neuromuscular plasticity after SCI. However, the skeletal adaptations to these therapies remain unknown. PURPOSE: Determine whether TM or Cycle training alter the rate of cancellous bone loss in a rodent severe contusion SCI model. METHODS: 16-wk old male Sprague-Dawley rats received: 1) SHAM surgery (T9 laminectomy) (n=9), 2) T9 laminectomy plus severe contusion SCI (n=8), 3) SCI+TM (n=14), or 4) SCI+Cycle (n=7). TM and Cycle were initiated 1-wk post-SCI and consisted of two 20 min bouts/day for 3 wks. For TM, 40% bodyweight support was provided and the paralyzed hindlimbs were manually positioned into plantar stepping (3.5 m/min, increasing 0.1 m/min/day). For Cycle, the paralyzed hindlimbs were secured to pedals on a motor-driven bike and moved passively through a cycling motion that mimicked normal gait patterning (12 rotations/min). Distal femur cancellous bone was quantified before surgery (baseline), and at 2- and 4-wk post-surgery via in vivo microCT. Outcomes are reported as percent change from baseline. RESULTS: Across all groups, cancellous bone volume (cBV/TV) was reduced 52-75% at 2-wk and 54-84% at 4-wk, compared with baseline (p<0.01). cBV/TV loss was 22% greater in SCI at 2-wk and 29% greater at 4-wk vs SHAM (p<0.01), characterized by 28% lower trabecular number (Tb.N) and 90% higher trabecular separation (Tb.Sp) (p<0.01) and a higher trabecular pattern factor (Tb.Pf) (p<0.05) that indicates a less connected trabecular network. At 2-wk, neither TM nor Cycle prevented SCI-induced bone deficits. However, at 4-wk SCI+Cycle displayed 25-30% higher cBV/TV, 23-24% higher trabecular thickness (Tb.Th), 17-22% higher Tb.N, and lower Tb.Pf vs SCI and SCI+TM (p<0.01). Ultimately, no differences in cancellous bone outcomes were present between SCI+Cycle and SHAM at 4-wk, except for 16% higher Tb.Th in SCI+Cycle (p<0.01). CONCLUSION: Our data indicate Cycle better attenuated cancellous bone loss in rodents after severe SCI. The higher cBV/TV and Tb.Th in SCI+Cycle at 4-wk also suggests that this modality stimulated bone formation; although, further investigation is needed.
