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.
Projekt 7: The effect of myelotomy following low thoracic spinal cord compression injury in rats
Participants: Carolin Meyer, Habib Bendella, Svenja Rink, Robin Gensch, Robert Seitz, Gregor Stein, Marilena Manthou, Theodora Papamitsou, Makoto Nakamura, Bertil Bouillon, Mary Galea, Peter Batchelor P, Sarah A Dunlop, Doychin N Angelov.
Myelotomy is a surgical procedure allowing removal of extravasated blood and necrotic tissue (Fig. 47) that is thought to attenuate secondary injury as well as promote recovery in experimental spinal cord injury (SCI) models and humans. Here we examined in rat whether myelotomy at 48h after low-thoracic compressive SCI provided any benefit over a 12 week period. Compared to animals receiving SCI alone, myelotomy worsened BBB scores (p<0.05) and also did not improve plantar stepping, ladder climbing, urinary bladder voiding or sensory function (thermal latency) during the 12-week period (Figs. 48, 49). Quantitative analyses of tissue sections at 12 weeks showed that myelotomy also did not reduce lesion volume nor alter immunohistochemical markers of axons in spared white matter bridges, microglia, astrocytes or serotinergic fibres. However, myelotomy reduced synaptophysin expression, a marker of synaptic plasticity (Figs. 50-53). We conclude that further studies are required to evaluate myelotomy after SCI.
Project Nr. 8: Constitutively reduced sensory capacity promotes better recovery after spinal cord-injury (SCI) in blind rats of the dystrophic RCS strain
Participants: SvenjaRink, Habib Bendella, Kurdin Alsolivany, Carolin Meyer, Aliona Woehler, Ramona Jansen, Zeynep Isik, Gregor Stein, Sina Wennmachers, Makoto Nakamura, Doychin N. Angelov
We compared functional, electrophysiological and morphological parameters after SCI in two groups of rats Sprague Dawley (SD) rats with normal vision and blind rats from a SD-substrain "Royal College of Surgeons" (SD/RCS) who lose their photoreceptor cells after birth due to a genetic defect in the retinal pigment epithelium. For these animals skin-, intramuscular-, and tendon receptors are major available means to resolve spatial information. The purpose of this project was to check whether increased sensitivity in SD/RCS rats would promote an improved recovery after SCI. Following conditioning (Fig. 54), all rats were subjected to severe compression of the spinal cord at vertebra Th8, spinal cord segment Th10 (Fig. 55). Recovery of locomotion was analyzed at 1, 3, 6, 9, and 12 weeks after SCI using video recordings of beam walking and inclined ladder climbing. Five functional parameters were studied: foot-stepping angle (FSA), rump-height index (RHI) estimating paw placement and body weight support, respectively, number of correct ladder steps (CLS) assessing skilled hindlimb movements, the BBB-locomotor score and an established urinary bladder score (BS) (Figs. 56 A-D). Sensitivity tests were followed by electrophysiological measurement of M- and H-wave amplitudes from contractions of the plantar musculature after stimulation of the tibial nerve (Fig. 56 E, F). The closing morphological measurements included lesion volume and expression of astro- and microglia below the lesion. Numerical assessments of BBB, FSA, BS, lesion volume and GFAP-expression revealed no significant differences between both strains. However, compared to SD-rats, the blind SD/RCS animals significantly improved RHI and CLS by 6 – 12 weeks after SCI. To our surprise the withdrawal latencies in the blind SD/RCS rats were longer and the amplitudes of M- and H-waves lower (Fig. 57, 58). The expression of IBA1-immunoreactivity in the lumbar enlargement was lower than in the SD-animals (Figs. 59, 60, 61). The longer withdrawal latencies suggest a decreased sensitivity in the blind SD/RCS rats, which promotes better recovery after SCI. In this way our results provide indirect support to earlier work showing, that hypersensitivity and chronic pain after contusive SCI impair the recovery of locomotor function.
Project Nr. 9: Recovery after spinal cord injury by modulation of the proteoglycan receptor PTPσ
SCI is followed by dramatic upregulation of chondroitin sulfate proteoglycans (CSPGs) which limit axonal regeneration, oligodendrocyte replacement and remyelination. The recent discovery of the specific CSPGs signaling receptor protein tyrosine phosphatase sigma (RPTPσ) provided an opportunity to refine the therapeutic approach to overcome CSPGs inhibitory actions. In previously published work, subcutaneous (s.c.) delivery of 44 µg/day of a peptide mimetic of PTPσ called intracellular sigma peptide (ISP), which binds to PTPσ and blocks CSPG-mediated inhibition, facilitated recovery after contusive SCI. Since this result could be of great interest for clinical trials, we independently repeated this study, but modified the method of injury as well as peptide application and the dosage. Following SCI at the Th10-segment, 40 rats were distributed in 3 groups. Animals in group 1 (20 rats) were subjected to SCI, but received no treatment. Rats in group 2 were treated with intraperitoneal (i.p.) injections of 44 µg/day ISP (SCI+ISP44) and animals of group 3 with s.c. injections of 500 µg/day ISP (SCI+ISP500) for 7 weeks after lesioning. Recovery was analyzed at 1, 3, 6, 9 and 12 weeks after SCI by determining (i) BBB-score, (ii) foot-stepping angle, (iii) rump-height index, (iv) number of correct ladder steps, (v) bladder score and (vi) sensitivity (withdrawal latency after thermal stimulus) (Figs. 62, 63). Finally, we determined the amount of serotonergic fibers in the preserved neural tissue bridges (PNTB) around the lesion site (Figs. 64-66). Our results show that, systemic therapy with ISP improved locomotor, sensory and vegetative recovery which correlated with more spared serotonergic fibers in PNTB.
Project Nr. 10: Anatomic conditions for bypass surgery between rostral (T7-T9) and caudal (L2, L4, S1) ventral roots to treat paralysis after spinal cord injury
Participants: HabibBendella, Svenja Rink, Aliona Wöhler, Janna Feiser, Andre Wilden, Maria Grosheva, Hans-Jürgen Stoffels, Carolin Meyer, Marilena Manthou, Makoto Nakamura, Doychin N. Angelov
Severe spinal cord injuries cause permanent neurological deficits and are still considered as inaccessible to efficient therapy. Injured spinal cord axons are unable to spontaneously regenerate. Re-establishing functional activity especially in the lower limbs by reinnervation of the caudal infra-lesional territories might represent an effective therapeutic strategy. Numerous surgical neurotizations have been developed to bridge the spinal cord lesion site and connect the intact supra-lesional portions of the spinal cord to peripheral nerves (spinal nerves, intercostal nerves) and muscles. The major disadvantage of these techniques is the increased hypersensitivity, spasticity and pathologic pain in the spinal cord injured patients, which occur due to the vigorous sprouting of injured afferent sensory fibers after reconstructive surgery. Using micro-surgical instruments and an operation microscope we performed detailed anatomical preparation of the vertebral canal and its content in five human cadavers. Our observations allow us to put forward the possibility to develop a more precise surgical approach, the so called "ventral root bypass" that avoids lesion of the dorsal roots and eliminates sensitivity complications (Fig. 67). The proposed kind of neurotization has been neither used, nor put forward. The general opinion is that radix ventralis and radix dorsalis unite to form the spinal nerve inside the dural sac. This assumption is not accurate, because both radices leave the dural sac separately (Fig. 68). This neglected anatomical feature allows a reliable intravertebral exposure of the dura-mater ensheathed ventral roots and their damage-preventing end-to-side neurorrhaphy by interpositional nerve grafts (Figs. 69, 70).
Project Nr. 11: Amount of axons in spared neural tissue bridges, but not their width or area, correlates with functional recovery in spinal cord-injured rats.
Participants: Svenja Rink, Stoyan Pavlov, Aliona Wöhler, Habib Bendella, Marilena Manthou, Theodora Papamitsou, Sarah A. Dunlop, Doychin N. Angelov
After spinal cord injury (SCI) the relationship between various parameters of tissue damage and subsequent functional recovery is not well understood: patients may regain micturition control and walking despite large post-injury medullar cavities. The objective of the present study was to establish possible correlations between morphological findings and degree of functional recovery after spinal cord compression at vertebra Th8 in rats. Recovery of motor (BBB, foot-stepping angle, rump-height index, ladder climbing), sensory (withdrawal latency) and bladder functions was analysed at 1, 3, 6, 9 and 12 weeks post SCI (Figs. 71, 22). Following perfusion f7xation, spinal cord tissue encompassing the injury site was cut in longitudinal frontal sections. Lesion length, lesion volume and area of the perilesional neural tissue bridges were determined after staining with cresyl violet. The amount of axons in these bridges was quantified after staining for class III b-tubulin (Fig. 73, 74). We found that it was not the area of the spared tissue bridges (that is routinely determined during magnetic resonance imaging, MRI), but the amount of axons in them which correlated with functional recovery Spearman's ρ > 0.8; p < 0.001 after SCI (Fig. 75) and conclude that prognostic statements based only on MRI-measurements should be considered with caution
Project Nr. 12: Motor, sensitive, and vegetative recovery in rats with compressive spinal-cord injury after combined treatment with erythropoietin and whole-body vibration
Participants: Svenja Rink, Maria Eleni Manthou, Julia Arnold, Merle Grigo, Paulina Dicken, Diana Abdulla, Habib Bendella, Klaus Nohroudi, Doychin N. Angelov
Physical therapy with whole body vibration (WBV) following compressive spinal cord injury (SCI) in rats restores density of perisomatic synapses, improves body weight support and leads to a better bladder function. The purpose of the study was to determine whether the combined treatment with WBV plus erythropoietin (EPO) would further improve motor, sensory and vegetative functions after SCI in rats. Severe compressive SCI at low thoracic level was followed by a single i.p. injection of 2,5 µg (250 IU) human recombinant EPO. Physical therapy with WBV started on 14th day after injury and continued over a 12-week post injury period. Locomotor recovery, sensitivity tests and urinary bladder scores were analysed at 1, 3, 6, 9, and 12 weeks after SCI. The closing morphological measurements included lesion volume and numbers of axons in the preserved perilesional neural tissue bridges (PNTB). Assessment of motor performance sensitivity and bladder function revealed no significant effects of EPO when compared to the control treatments (Figs. 76-79). EPO treatment neither reduced the lesion volume, nor increased the number of axons in PNTB (Figs. 80-81).
The combination of WBV+EPO exerts no positive effects on hind limbs motor performance and bladder function after compressive SCI in rats.