Joint-specific changes in locomotor complexity in the absence of muscle atrophy following incomplete spinal cord injury

ackground Following incomplete spinal cord injury (iSCI), descending drive is impaired, possibly leading to a decrease in the complexity of gait. To test the hypothesis that iSCI impairs gait coordination and decreases locomotor complexity, we collected 3D joint angle kinematics and muscle parameters of rats with a sham or an incomplete spinal cord injury. Methods 12 adult, female, Long-Evans rats, 6 sham and 6 mild-moderate T8 iSCI, were tested 4 weeks following injury. The Basso Beattie Bresnahan locomotor score was used to verify injury severity. Animals had reflective markers placed on the bony prominences of their limb joints and were filmed in 3D while walking on a treadmill. Joint angles and segment motion were analyzed quantitatively, and complexity of joint angle trajectory and overall gait were calculated using permutation entropy and principal component analysis, respectively. Following treadmill testing, the animals were euthanized and hindlimb muscles removed. Excised muscles were tested for mass, density, fiber length, pennation angle, and relaxed sarcomere length. Results Muscle parameters were similar between groups with no evidence of muscle atrophy. The animals showed overextension of the ankle, which was compensated for by a decreased range of motion at the knee. Left-right coordination was altered, leading to left and right knee movements that are entirely out of phase, with one joint moving while the other is stationary. Movement patterns remained symmetric. Permutation entropy measures indicated changes in complexity on a joint specific basis, with the largest changes at the ankle. No significant difference was seen using principal component analysis. Rats were able to achieve stable weight bearing locomotion at reasonable speeds on the treadmill despite these deficiencies. Conclusions Decrease in supraspinal control following iSCI causes a loss of complexity of ankle kinematics. This loss can be entirely due to loss of supraspinal control in the absence of muscle atrophy and may be quantified using permutation entropy. Joint-specific differences in kinematic complexity may be attributed to different sources of motor control. This work indicates the importance of the ankle for rehabilitation interventions following spinal cord injury.

The construction and characterization of new permeable rho antagonists and their roles after spinal cord injury

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

The construction and characterization of new permeable rho antagonists and their roles after spinal cord injury

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Development of novel therapeutics and in vitro models for spinal cord injury

Spinal cord injury (SCI) is a devastating condition, which results from trauma to the cord, resulting in a primary injury response which leads to a secondary injury cascade, causing damage to both glial and neuronal cells. Following trauma, the central nervous system (CNS) fails to regenerate due to a plethora of both intrinsic and extrinsic factors. Unfortunately, these events lead to loss of both motor and sensory function and lifelong disability and care for sufferers of SCI. There have been tremendous advancements made in our understanding of the mechanisms behind axonal regeneration and remyelination of the damaged cord. These have provided many promising therapeutic targets. However, very few have made it to clinical application, which could potentially be due to inadequate understanding of compound mechanism of action and reliance on poor SCI models. This thesis describes the use of an established neural cell co-culture model of SCI as a medium throughput screen for compounds with potential therapeutic properties. A number of compounds were screened which resulted in a family of compounds, modified heparins, being taken forward for more intense investigation. Modified heparins (mHeps) are made up of the core heparin disaccharide unit with variable sulphation groups on the iduronic acid and glucosamine residues; 2-O-sulphate (C2), 6-O-sulphate (C6) and N-sulphate (N). 2-O-sulphated (mHep6) and N-sulphated (mHep7) heparin isomers were shown to promote both neurite outgrowth and myelination in the SCI model. It was found that both mHeps decreased oligodendrocyte precursor cell (OPC) proliferation and increased oligodendrocyte (OL) number adjacent to the lesion. However, there is a difference in the direct effects on the OL from each of the mHeps; mHep6 increased myelin internode length and mHep7 increased the overall cell size. It was further elucidated that these isoforms interact with and mediate both Wnt and FGF signalling. In OPC monoculture experiments FGF2 treated OPCs displayed increased proliferation but this effect was removed when co-treated with the mHeps. Therefore, suggesting that the mHeps interact with the ligand and inhibit FGF2 signalling. Additionally, it was shown that both mHeps could be partially mediating their effects through the Wnt pathway. mHep effects on both myelination and neurite outgrowth were removed when co-treated with a Wnt signalling inhibitor, suggesting cell signalling mediation by ligand immobilisation and signalling activation as a mechanistic action for the mHeps. However, the initial methods employed in this thesis were not sufficient to provide a more detailed study into the effects the mHeps have on neurite outgrowth. This led to the design and development of a novel microfluidic device (MFD), which provides a platform to study of axonal injury. This novel device is a three chamber device with two chambers converging onto a central open access chamber. This design allows axons from two points of origin to enter a chamber which can be subjected to injury, thus providing a platform in which targeted axonal injury and the regenerative capacity of a compound study can be performed. In conclusion, this thesis contributes to and advances the study of SCI in two ways; 1) identification and investigation of a novel set of compounds with potential therapeutic potential i.e. desulphated modified heparins. These compounds have multiple therapeutic properties and could revolutionise both the understanding of the basic pathological mechanisms underlying SCI but also be a powered therapeutic option. 2) Development of a novel microfluidic device to study in greater detail axonal biology, specifically, targeted axonal injury and treatment, providing a more representative model of SCI than standard in vitro models. Therefore, the MFD could lead to advancements and the identification of factors and compounds relating to axonal regeneration.

Spatiotemporal neuromodulation therapies engaging muscle synergies improve motor control after spinal cord injury.

Electrical neuromodulation of lumbar segments improves motor control after spinal cord injury in animal models and humans. However, the physiological principles underlying the effect of this intervention remain poorly understood, which has limited the therapeutic approach to continuous stimulation applied to restricted spinal cord locations. Here we developed stimulation protocols that reproduce the natural dynamics of motoneuron activation during locomotion. For this, we computed the spatiotemporal activation pattern of muscle synergies during locomotion in healthy rats. Computer simulations identified optimal electrode locations to target each synergy through the recruitment of proprioceptive feedback circuits. This framework steered the design of spatially selective spinal implants and real-time control software that modulate extensor and flexor synergies with precise temporal resolution. Spatiotemporal neuromodulation therapies improved gait quality, weight-bearing capacity, endurance and skilled locomotion in several rodent models of spinal cord injury. These new concepts are directly translatable to strategies to improve motor control in humans.

Dipsacus asperoides (Xue Duan) inhibits spinal cord injury-induced inflammatory responses in rats

Purpose: To investigate the effect of Dipsacus asperoides (Xue Duan), a traditional Chinese medicine, on rats with spinal cord injury (SCI). Methods: In this study a total of 40 adult rats were used after inducing SCI where Xue Duan was applied on experimental group and phosphate-buffered saline (PBS) was administered in corresponding control groups. Intraperitoneal administration of both compounds for a period of four weeks (28 days) was carried out at a dose of 10 mg/kg/day. Bright field microscopy was performed on the tissues. Results: Bright Field microscopy of tissue sections showed significant reduction in cavity area that resulted from injury, that is from 0.19 ± 0.05 mm2 to 0.09 ± 0.03 mm2 (p < 0.01) in untreated and treated groups respectively. Similarly western blotting results showed a decrease in the expression of NF-kB p65 and I-kBα (p < 0.01). These two compounds are important in increasing secondary pathophysiology in SCI. The results for MPO activity also revealed significantly reduced infiltration of leukocytes to the injury site (p < 0.01). Conclusion: This study reveals the positive effect of the plant material in reducing inflammation in rats with traumatic SCI.

Dose-dependent protection of reseveratrol against spinal cord ischemic-reperfusion injury in rats

Purpose: To examine the protective effects of resveratrol (RESV) against spinal cord ischemic reperfusion (SCIR) injury. Methods: Forty-eight male rats were divided into six groups: sham-operated (control-I), SCIR-treated (SCIR-II), rats receiving 20 mg/kg of RESV with SCIR (RESV 20+SCIR-III), rats receiving 40 mg/kg of RESV with SCIR (RESV 40+SCIR-IV), rats receiving 60 mg/kg of RESV with SCIR (RESV 60+SCIR-V), and rats receiving 50 mg/kg of methylprednisolone (MP) with SCIR (MP + SCIR-VI), for 7 days prior to IR (pre-treatment) and 7 days after IR (post-treatment). Results: The levels of oxidative markers (TBARS, MPO) and inflammatory markers (IL-1β, IL-6, TNF-α, and NF-p65) were concomitantly suppressed in RESV-treated rats, which showed improved locomotor function. A pronounced increase in the activities of antioxidant enzymes (SOD, CAT and GSH) was noted in the RESV group compared with the MP and SCIR groups. RESV and MP supplementation increased neuronal count with decreased nuclear degeneration. RESV (40 mg) exhibited greater protective effect than 20 mg and 60 mg of RESV and 50 mg of MP. Conclusion: The results show the neurotherapeutic potential of RESV (40 mg) to attenuate oxidative stress and the inflammatory response to SCIR injury.

The Lived Experience of Families Living with Spinal Cord Disability in Northeast Thailand

The experience of disability in the global South remains relatively underreported in spite of the greater focus on disability as both an impediment to development and frequently as a result of development. This article reports a qualitative study using ethnographic techniques undertaken in the province of Khon Kaen in Northeast Thailand. The primary participants were men who had experienced a severe spinal cord injury at a time when they were breadwinners, a role which is significant in the context of a modernising state that is an active participant in a global economy. The experiences, constructions and beliefs of these men, their family carers, and other informants illustrate the complex ways in which social and cultural factors interact with the opportunities, challenges and constraints of the transition modernity. The findings, interpreted according to the ‘three bodies’ approach, illustrate the intersection of colonising effects, governmentality and resistance, and embodied experience in a cultural context.

How useful are x-rays in severe paediatric cervical spinal injuries?

INTRODUCTION Radiological evaluation of the paediatric cervical spine can be a challenge due to the normal anatomic variants and injuries that are unique to children. We aimed to identify the usefulness of plain X-rays in comparison with CT and MRI in diagnosing Paediatric cervical spinal injuries. METHODS Retrospective review of imaging studies of children diagnosed with paediatric cervical spine injuries who had presented to two tertiary hospitals in Queensland. RESULTS There were 38 patients with 18 male and 20 female .The mean age was 8.6 years. Plain Cervical Spine X-rays (3views, AP lateral and open mouth views) were done in 34 patients. The remaining 8 children had a suspected head injury and hence had CT scans of their neck done at the time of CT head scan. Of these images taken, X-rays were diagnostic in 28 (82%) patients. CONCLUSION X- Rays still have a role to play in the diagnosis of pediatric cervical spinal injuries and should be considered as the first line in fully conscious patients and their usefulness should not be overlooked in light of the newer imaging modalities.

CNS injury, γ1 laminin, and its KDI peptide

Nisäkkäillä keskushermoston uudistuminen on rajallista. Keskushermostovamman jälkeen aktivoituu monien paranemista edistävien tekijöiden lisäksi myös estäviä tekijöitä. Monella molekyylillä, kuten laminiinilla, on keskushermoston paranemista tehostava vaikutus. Laminiinit ovat myös kehon tyvikalvojen oleellisia rakennuskomponentteja. Keskushermoston laminiinit ovat tärkeitä sikiökehityksen aikana, esimerkiksi hermosäikeiden ohjauksessa. Myöhemmin ne osallistuvat veriaivoesteen ylläpitoon sekä vammojen jälkeiseen kudosreaktioon. Väitöskirjatutkimuksessani olen selvittänyt lamiiniinien, erityisesti γ1 laminiinin ja sen KDI peptidin, ekspressiota keskushermoston vammatilanteissa. Kokeellisessa soluviljelmäasetelmassa, joka simuloi vammautunutta keskushermostoympäristöä, osoitimme että KDI peptidi voimistaa sekä hermosolujen selviytymistä että hermosäikeiden kasvua. Kainihappo on glutamaattianalogi, ja glutamaattitoksisuudella uskotaan olevan tärkeä merkitys keskushermoston eri vamma- ja sairaustilanteissa tapahtuvassa hermosolukuolemassa. Toisessa väitöskirjani osatyössä osoitimme eläinmallissa KDI peptidin suojaavan rotan aivojen hippokampuksen hermosoluja kainihapon aiheuttamalta solutuholta. Elektrofysiologisilla mittauksilla osoitimme kolmannessa osatyössäni, että KDI peptidi estää glutamaattireseptorivirtoja ja suojaa siten glutamaattitoksisuudelta. Aivoveritulpan aiheuttama aivovaurio on yleinen syy aivohalvaukseen. Viimeisessä osatyössäni tutkimme eläinmallissa laminiinien ekspressiota iskemian vaurioittamassa aivokudoksessa. Laminiiniekspression todettiin voimistuvan vaurion jälkeen sekä tyvikalvo- että soluväliainerakenteissa. Vaurion ympärillä havaittiin astrosyyttejä, jotka jo melko aikaisessa vaiheessa vamman jälkeen ekspressoivat γ1 laminiinia ja KDI peptidiä. Tästä voidaan päätellä laminiinien osallistuvan aivoiskeemisen vaurion patofysiologiaan. Yleisesti väitöskirjatyöni kartoitti laminiinien ekspressiota sekä terveessä että vammautuneessa keskushermostossa. Väitöskirjatyöni tukee hypoteesia, jonka mukaan KDI peptidi suojaa keskushermostoa vaurioilta.

Isolation of mineralizing Nestin+ Nkx6.1+ vascular muscular cells from the adult human spinal cord

Background: The adult central nervous system (CNS) contains different populations of immature cells that could possibly be used to repair brain and spinal cord lesions. The diversity and the properties of these cells in the human adult CNS remain to be fully explored. We previously isolated Nestin(+) Sox2(+) neural multipotential cells from the adult human spinal cord using the neurosphere method (i.e. non adherent conditions and defined medium). -- Results: Here we report the isolation and long term propagation of another population of Nestin(+) cells from this tissue using adherent culture conditions and serum. QPCR and immunofluorescence indicated that these cells had mesenchymal features as evidenced by the expression of Snai2 and Twist1 and lack of expression of neural markers such as Sox2, Olig2 or GFAP. Indeed, these cells expressed markers typical of smooth muscle vascular cells such as Calponin, Caldesmone and Acta2 (Smooth muscle actin). These cells could not differentiate into chondrocytes, adipocytes, neuronal and glial cells, however they readily mineralized when placed in osteogenic conditions. Further characterization allowed us to identify the Nkx6.1 transcription factor as a marker for these cells. Nkx6.1 was expressed in vivo by CNS vascular muscular cells located in the parenchyma and the meninges. -- Conclusion: Smooth muscle cells expressing Nestin and Nkx6.1 is the main cell population derived from culturing human spinal cord cells in adherent conditions with serum. Mineralization of these cells in vitro could represent a valuable model for studying calcifications of CNS vessels which are observed in pathological situations or as part of the normal aging. In addition, long term propagation of these cells will allow the study of their interaction with other CNS cells and their implication in scar formation during spinal cord injury.

Microelectrode Implants for Spinal Cord Stimulation in Rats

Paralysis is a debilitating condition afflicting millions of people across the globe, and is particularly deleterious to quality of life when motor function of the legs is severely impaired or completely absent. Fortunately, spinal cord stimulation has shown great potential for improving motor function after spinal cord injury and other pathological conditions. Many animal studies have shown stimulation of the neural networks in the spinal cord can improve motor ability so dramatically that the animals can even stand and step after a complete spinal cord transaction.

This thesis presents work to successfully provide a chronically implantable device for rats that greatly enhances the ability to control the site of spinal cord stimulation. This is achieved through the use of a parylene-C based microelectrode array, which enables a density of stimulation sites unattainable with conventional wire electrodes. While many microelectrode devices have been proposed in the past, the spinal cord is a particularly challenging environment due to the bending and movement it undergoes in a live animal. The developed microelectrode array is the first to have been implanted in vivo while retaining functionality for over a month. In doing so, different neural pathways can be selectively activated to facilitate standing and stepping in spinalized rats using various electrode combinations, and important differences in responses are observed.

An engineering challenge for the usability of any high density electrode array is connecting the numerous electrodes to a stimulation source. This thesis develops several technologies to address this challenge, beginning with a fully passive implant that uses one wire per electrode to connect to an external stimulation source. The number of wires passing through the body and the skin proved to be a hazard for the health of the animal, so a multiplexed implant was devised in which active electronics reduce the number of wires. Finally, a fully wireless implant was developed. As these implants are tested in vivo, encapsulation is of critical importance to retain functionality in a chronic experiment, especially for the active implants, and it was achieved without the use of costly ceramic or metallic hermetic packaging. Active implants were built that retained functionality 8 weeks after implantation, and achieved stepping in spinalized rats after just 8-10 days, which is far sooner than wire-based electrical stimulation has achieved in prior work.

Pretreatment with Resveratrol Prevents Neuronal Injury and Cognitive Deficits Induced by Perinatal Hypoxia-Ischemia in Rats

Despite advances in neonatal care, hypoxic-ischemic brain injury is still a serious clinical problem, which is responsible for many cases of perinatal mortality, cerebral palsy, motor impairment and cognitive deficits. Resveratrol, a natural polyphenol with important anti-oxidant and anti-inflammatory properties, is present in grapevines, peanuts and pomegranates. The aim of the present work was to evaluate the possible neuroprotective effect of resveratrol when administered before or immediately after a hypoxic-ischemic brain event in neonatal rats by analyzing brain damage, the mitochondrial status and long-term cognitive impairment. Our results indicate that pretreatment with resveratrol protects against brain damage, reducing infarct volume, preserving myelination and minimizing the astroglial reactive response. Moreover its neuroprotective effect was found to be long lasting, as behavioral outcomes were significantly improved at adulthood. We speculate that one of the mechanisms for this neuroprotection may be related to the maintenance of the mitochondrial inner membrane integrity and potential, and to the reduction of reactive oxygen species. Curiously, none of these protective features was observed when resveratrol was administered immediately after hypoxia-ischemia.