Project Nr. 1: Objective measures of motor dysfunction after compression spinal cord injury in adult rats: Correlations with locomotor rating scores
Participants: Katharina Wellmann, Janina Ankerne, Gregor Stein, Srebrina K. Angelova, Konrad Mader, Oliver Semler, Maria Grosheva, Sandra Maier, Eckhard Schönau, Andrey Irintchev, Doychin N. Angelov
Precise assessment of motor deficits after traumatic spinal cord injury (SCI) in rodents is crucial for understanding the mechanisms of functional recovery and testing therapeutic approaches. Here we analyzed the applicability to a rat SCI model of an objective approach, the single-frame motion analysis, created and used for functional analysis in mice. Adult female Wistar rats were subjected to graded compression of the spinal cord (Fig. 1). Recovery of locomotion was analyzed using video recordings of beam walking (Fig. 2) and inclined ladder climbing (Fig. 3). Three out of 4 parameters used in mice appeared suitable: the foot-stepping angle (FSA) and the rump-height index (RHI) measured during beam walking and estimating paw placement and body weight support, respectively, and the number of correct ladder steps (CLS) assessing skilled limb movements. These parameters, similar to the Basso, Beattie and Bresnahan (BBB) locomotor rating scores, correlated with lesion volume and showed significant differences between moderately and severely injured rats at 1-9 weeks after SCI (Figs. 4, 5). The beam parameters, but not CLS, correlated well with the BBB scores within ranges of poor and good locomotor abilities (Fig. 6). FSA co-varied with RHI only in the severely impaired rats while RHI and CLS were barely correlated (Fig. 7). Our findings suggest that the numerical parameters estimate, as intended by design, predominantly different aspects of locomotion. The use of these objective measures combined with BBB rating provides a time- and cost-efficient opportunity for versatile and reliable functional evaluations in both severely and moderately impaired rats combining clinical assessment with precise numerical measures.
Project Nr. 2: Functional deficits and morphological changes in the neurogenic bladder match the severity of spinal cord compression
Participants: Ozlem Ozsoy, Umut Ozsoy U, Gregor Stein, Oliver Semler, Emmanouil Skouras, Greta Schempf, Katharina Wellmann, Felicitas Wirth, Srebrina Angelova, Janina Ankerne, Mahak Ashrafi, Eckhard Schönau, Teodora Papamitsou-Sidoropolou, Patrick Jaminet, Levent Sarikcioglu, Andrey Irintchev, Sarah A. Dunlop, Doychin N. Angelov
Following spinal cord injury (SCI), loss of spinal and supraspinal control results in desynchronisation of detrusor vesicae (parasympathicus) and external urethral sphincter (sympathicus) activity. Despite recovery of lower urinary tract function being a high priority in patients with SCI, effective treatment options are unavailable largely because mechanisms are poorly understood (Fig. 8). We used a clinically relevant model of thoracic SCI compression injury in adult female Wistar rats and confirmed that lesion volumes following severe injuries were significantly greater compared to moderate injuries (Figs. 10, 11). Between 1-9 weeks, we assessed recovery of bladder function as well as return of locomotor function using the Basso, Beattie and Bresnahan (BBB) score (Fig. 9). Bladder morphometrics and overall intramural innervation patterns, as assessed with ß-III tubulin immunohistochemistry, were also examined (Figs. 12-14). Despite variability, bladder function was significantly worse following severe compared to moderate compression injury (p<0.05); furthermore, the degree of bladder and locomotor dysfunction were significantly correlated (r=0.59; p<0.05). In addition, at 9 weeks after SCI we saw significantly greater increases in bladder dry weight (p<0.05) and wall thickness following severe compared to moderate injury as well as increases in intramural axon density (moderate: 3x normal values; severe 5x; both p<0.05) that also correlated with injury severity (r=0.89; Fig. 15). The moderate and severe compression models show consistent and correlated deficits in bladder and locomotor function, as well as in gross anatomical and histopathological changes. Increased intramural innervation may contribute to neurogenic detrusor overactivity and suggests the use of therapeutic agents which block visceromotoric efferents.
Projekt Nr. 3: Whole-body vibration improves functional recovery in spinal cord-injured rats
Participants: Felicitas Wirth, Greta Schempf, Gregor Stein, Katharina Wellmann, Marilena Manthou, Maina Sidorenko, Oliver Semler, Leonie Eisel, Rachida Harrach, Srebrina Angelova, Patrick Jaminet, Janina Ankerne, Mahak Ashrafi, Ozlem Ozsoy, Umut Ozsoy U, Harald Schubert, Diana Abdulla, Sarah A. Dunlop, Andrey Irintchev, Eckhard Schönau, Doychin N. Angelov
Whole-body vibration (WBV) is a relatively novel form of exercise used to improve neuromuscular performance in healthy individuals. Its usefulness as a therapy for patients with neurological disorders, in particular spinal cord injury (SCI), has received little attention in clinical settings and, surprisingly, even less in animal SCI models. We performed severe compression SCI at a low-thoracic level in Wistar rats followed by daily WBV (Fig. 16) starting 7 (10 rats) or 14 (10 rats) days after injury (WBV7 and WBV14, respectively) and continued over a 12-week post-injury period. Rats with SCI but no WBV training (sham, 10 rats) and intact animals (10 rats) served as controls. Compared to sham treated rats, WBV did not improve BBB score, plantar stepping or ladder stepping during the 12-week period (Fig. 17). Accordingly, WBV did not significantly alter plantar H-reflex (Fig. 18), lesion volume (Fig. 19), serotonergic input to the lumbar spinal cord (Fig. 20) nor cholinergic (Fig. 21) or glutamatergic (Fig. 22) inputs to lumbar motoneurons at 12 weeks after SCI. However, compared to sham, WBV14, but not WBV7, significantly improved body weight support (rump-height index) during ground locomotion and overall recovery between 6 - 12 weeks and also restored the density of synaptic terminals in the lumbar spinal cord at 12 week (Fig. 23). Most remarkably, WBV14 lead to a significant improvement of bladder function at 6 - 12 weeks after injury (Fig. 24). These findings provide the first evidence for functional benefits of WBV in an animal SCI model and warrant further preclinical investigations to determine mechanisms underpinning this non-invasive, inexpensive and easily delivered potential rehabilitation therapy for SCI.
Project Nr. 4: Functional recovery after experimental spinal cord compression and whole body vibration therapy requires a balanced revascularization of the injured site
Participants: Marilena Manthou, Klaus Nohroudi, Samantha Moscarino, Felix Rehberg, Gregor Stein, Ramona Jansen, Diana Abdulla, Patrick Jaminet, Oliver Semler, Eckhard Schoenau, Doychin N. Angelov
Based on several positive effects of whole-body-vibration (WBV) therapy on recovery after SCI, we looked for correlations between functional (analysis of locomotion), electrophysiological (H-reflex) and morphological (density of functioning capillaries) measurements after SCI and WBV-treatment. Severe compression SCI at low-thoracic level (T8) in adult female Wistar rats was followed by WBV twice a day (2 x WBV) over a 12-week post-injury period (Fig. 25). Intact rats and rats with SCI but no WBV-therapy ("No-WBV") served as controls. Recovery of locomotion was determined by BBB-locomotor rating, foot stepping angle (FSA), rump-height index (RHI), correct ladder steps (CLS) and H-reflex at 1, 3, 6, 9, and 12 weeks after SCI (Figs. 26, 27). Animals were sacrificed by an overdose of Isoflurane. One hour later their spinal cords were fixed in 4% PFA for 24 h. Samples from the thoracic cord containing the lesion site and from the lumbar intumescence were cut into 10 µm thick longitudinal frozen sections. All functioning capillaries were unequivocally identified because the endogenous peroxidase of the erythrocytes was clearly visualized with 0.05% diaminobenzidine (DAB; Fig. 28). A determination of their absolute (in µm2) and proportional areas (percent of photographed tissue) revealed a significantly denser capillary network in the WBV-treated rats: 1,66 ± 0,41% in the "vibrated" rats versus 0,79 ± 0,19% in the "No-WBV" animals. The portion of the capillary network in intact rats was 1,51 ± 0,69% (Fig. 29). Surprisingly, even though the vascularization in the treated animals was significantly increased, this had no beneficial influence on the recovery of functions after SCI. The results of this study provide for the first time evidence that intensive WBV-therapy leads to a significantly denser capillary network in the lesioned spinal cord. However, since this higher capillary density is not associated with improved functional recovery (possibly because it exceeded the balance necessary for functional improvements), optional treatments with lower intensity or less time of WBV-therapy should be tested.
Project Nr. 5: Changes in the musculoskeletal system after experimental compressive spinal cord injury and whole body vibration in rats
Participants: Alisa Schwarz, Carina Pick, Rachida Harrach, Gregor Stein, Habib Bendella, Ozlem Ozsoy, Umut Ozsoy, Eckhard Schoenau, Patrick Jaminet, Levent Sarikcioglu, Sarah A. Dunlop, Doychin N. Angelov
Traumatic spinal cord injury (SCI) causes a loss of locomotor function with associated compromise of the musculo-skeletal system. Whole body vibration (WBV) is a potential therapy following SCI, but little is known about its effects on the musculo-skeletal system. Following conditioning (Fig. 30 A, B), we examined locomotor recovery and the muskulo-skeletal system after thoracic (T7-9) compression SCI in adult rats. Daily WBV (Fig. 30 C) was started at 1, 7, 14 and 28 days after injury (WBV1-WBV28 respectively) and continued over a 12-week post-injury period. Intact rats, rats with SCI but no WBV (sham-treated) and a group that received passive flexion and extension (PFE) of their hind limbs served as controls (Fig. 30 D, E). Compared to sham-treated rats, neither WBV nor PFE improved motor function. Only WBV14 and PFE improved bdy support (Fig. 31). In line with earlier studies we failed to detect signs of soleus muscle atrophy (weight, cross sectional diameter, total amount of fibers, mean fiber diameter) (Fig. 32, 33) or bone loss in the femur (length, weight, bone mineral density) (Fig. 34, 35). One possible explanation is that, despite of injury extent, the preservation of some axons in the white matter, in combination with quadripedal locomotion, may provide sufficient trophic and neuronal support for the musculoskeletal system.
Project Nr. 6: Whole body vibration (WBV) following spinal cord injury (SCI) in rats: timing of intervention
Participants: Marilena Manthou, Diana Abdulla, Stoyan Pavlov, Ramona Jansen, Habib Bendella, Klaus Nohroudi, Gregor Stein, Carolin Meyer, Ozlem Ozsoy, Umut Ozsoy, Jasemin Behram Kandemir, Levent Sarikcioglu, Oliver Semler, Eckhard Schoenau, Sarah A Dunlop, Doychin N Angelov
Following spinal cord injury (SCI), exercise training provides a wide range of benefits and promotes activity-dependent synaptic plasticity. Whole body vibration (WBV) in SCI patients improves walking and spasticity as well as bone and muscle mass. However, little is known about the effects of timing or frequency of intervention. The aim of this project is to determine which WBV-onset improves locomotor and bladder functions and influences synaptic plasticity beneficially. SCI was followed by WBV starting 1, 7, 14, 28 days after injury (WBV1, WBV7, etc.) and continued for 12 weeks. Intact animals and those receiving SCI but no WBV (No WBV), SCI plus WBV twice daily (2 x WBV) and SCI followed by passive hindlimb flexion-extension (PFE) served as controls. Locomotor [BBB rating, foot stepping angle (FSA) and rump-height index (RHI)] as well as bladder function were determined at 1, 3, 6, 9, and 12 weeks. Following perfusion fixation at 12 weeks, lesion volume (Fig. 36) and immunofluorescence for astrogliosis (GFAP), microglia (IBA1) and synaptic vesicles (synaptophysin, SYN) were determined. Compared to the No WBV group, the WB7 and WBV14 groups showed significantly faster speeds of BBB score recovery though this effect was temporary (Fig. 37, 38). Treatment type did not significantly affect the changes in the FSA: the observed changes in the different treatment groups were similar and the estimated differences were not significantly different from 0 (Fig. 39, 40). Considering RHI we detected a sustained improvement in the WBV14 and PFE groups (Fig. 41, 42). Bladder function was better in the WBV14, WBV28, 2 x WBV and PFE groups (Fig. 43). Synaptophysin levels improved in response to WBV7 and WBV14, but worsened after WBV28 in parallel to an increased IBA1 and GFAP expression (Fig. 44, 45, 46). Correlation- and principal components analysis revealed complex relationships between behavioural (BBB, FSA, RHI) and morphological (GFAP, SYN) measurements. We conclude that WBV started 14 days after SCI provides the most benefit (RHI, bladder); starting at 1day after SCI provides no benefit and starting at 28 days may be detrimental. Increasing the intensity of WBV to twice daily did not provide additional benefit.