Indiana University-Purdue University Indianapolis (IUPUI) / Retinoids play an important role in tissue patterning during development as well as in epithelial formation and health. In the mammalian central nervous system, the meninges are a source of retinoids for brain tissue. Retinoid production has been described in juvenile Axolotl ependymal cells. Retinoid effects may possess a significant role in the regeneration-permissive interaction of the meninges and ependyma of the Axolotl spinal cord after penetrating injury. During spinal cord regeneration in urodele amphibians, the pattern of retinoid production changes as the meninges interact with the injury-reactive ependymal cells reconstructing the injured spinal cord. In order to determine which components of the retinoid metabolism and intracellular signaling pathway act in Urodele spinal cord regeneration, we employed antibody/horseradish peroxidase staining of both intact and regenerating Axolotl spinal cord tissues obtained from adult animals as well as cell culture techniques to determine expression of three retinoid pathway components: Cellular Retinoic Acid Binding Protein II (CRABP 2), Cellular Retinol Binding Protein I (CRBP 1), and Retinaldehyde Dehydrogenase II (RALDH 2). Current results demonstrate the following in the intact cord: 1) CRBP 1 is expressed in the pia and dura mater meningeal layers, in gray matter neurons (including their axonal processes), and the ependymal cell radial processes that produce the glia limitans, 2) CRABP 2 is expressed in the arachnoid and/or dura mater meningeal layers surrounding the spinal cord, and 3) RALDH 2 is expressed in the meninges as well as cytoplasm of grey matter neurons and some ependymal/sub-ependymal cells. In the regenerating cord, CRBP 1 is expressed in ependymal cells that are undergoing epithelial-to-mesenchymal transition (EMT), as is CRABP 2. RALDH 2 staining is very strong in the reactive meninges; in addition, expression is also upregulated in the cytoplasmic and perinuclear regions of reactive grey matter neurons, including motor neurons and in the apical region of ependymal. Preliminary studies culturing reactive meninges and ependymal cells together suggested that the meninges could drive re-epithelialization of the reactive ependymal cells. Experiments to characterize this interaction show an unusual proliferation pattern: Proliferating Cell Nuclear Antigen (PCNA) labeling is present in intact and regenerating cord ependymal cells. However, in culture, the presence of meninges results in no proliferation proximal to the explant, but extensive proliferation in leading cell outgrowth; also, the cultured meninges is positive for RALDH2. In summary, the intact adult cord shows meningeal production of RA, which is upregulated following injury; in addition, during this time, RA production is upregulated in the adult ependymal cells as well. In culture, the reactive meninges appears to modulate the behavior of reactive ependymal cells.

We have developed a highly effective method for in vivo gene silencing in the spinal cord and dorsal root ganglia (DRG) by a cationic lipid facilitated delivery of synthetic, small interfering RNA (siRNA). A siRNA to the delta opioid receptor (DOR), or a mismatch RNA, was mixed with the transfection reagent, i-FectTM (vehicle), and delivered as repeated daily bolus doses (0.5 mug to 4 mug) via implanted intrathecal catheter to the lumbar spinal cord of rats. Twenty-four hours after the last injection, rats were tested for antinociception by the DOR selective agonist, D-Ala2, Glu4]deltorphin II (DELT), or the mu opioid receptor (MOR) selective agonist, D-Ala2, N-Me-Phe4, Gly-ol5]enkephalin (DAMGO). Pretreatment with the siRNA, but not the mismatch RNA or vehicle alone, blocked DELT antinociception dose-dependently. The latter was concomitant with a reduction in the spinal immunoreactivity and receptor density of DOR, and in DOR transcripts in the lumbar DRG and spinal dorsal horn. Neither siRNA nor mismatch RNA pretreatment altered spinal immunoreactivity of MOR or antinociception by spinal DAMGO, and had no effect on the baseline thermal nociceptive threshold. The inhibition of function and expression of DOR by siRNA was reversed by 72 hr after the last RNA injection. The uptake of fluorescence-tagged siRNA was detected in both DRG and spinal cord. The low effective dose of siRNA/i-FectTM complex reflects an efficient delivery of the siRNA to peripheral and spinal neurons, produced no behavioral signs of toxicity. This delivery method may be optimized for other gene targets.

Within recent literature, internal locus of control (LOC) has been correlated with self-efficacy and improved coping after injury. Service connection (SC) has been linked to well-being after trauma. Level of injury has been associated with physical ability and limitations. This study examines how depression and QOL are associated with LOC and SC for veterans diagnosed with spinal cord injury (SCI). It was hypothesized that internal LOC and SC would correlate with higher QOL and decreased depression. Participants included 58 veterans, 30 diagnosed with paraplegia and 28 with quadriplegia. Measures included a demographics questionnaire, the Rotter's Internal-External Scale of LOC, the Diener's Satisfaction with Life Scale, and the Center for Epidemiological Studies - Depression Scale. Medical records were reviewed for SC. Generalized linear model regressions were run using LOC, SC and level of injury to estimate QOL and depression. Locus of control was a significant predictor of QOL, F=2.961, p=.045, while SC approached significance at F=2.082, p=.077. Locus of control approached significance as a predictor for depression, F=1.977, p=.083 level. Level of injury was not a significant factor for either outcome variable. Follow-up analyses including time since injury (TSI) indicated that the interaction of LOC and TSI was a significant predictor for QOL, F=5.320, p=0.013, and the interaction of SC and TSI was a significant predictor for depression, F=9.800, p=0.002. A significant correlation was found between depression and QOL, r=-.472, p=.000. A significant correlation was found between LOC and TSI, r=-.277, p=.018. Results indicate that internal LOC, SC, and increased TSI are positively associated with improved QOL and lower depression. The negative correlation between LOC and TSI indicates that LOC may be a mutable personality factor that adjusts from being external to internal to improve coping after disability, or those with an internal LOC are more consistent with treatment over time. The negative correlation between depression and QOL indicates that participants endorsing depression perceive having a lower QOL. These findings have important implications for health psychology, as they indicate that increasing internal LOC and access to health care over time can promote lower depression and higher QOL for SCI patients.

In contrast to mammals, adult zebrafish display cellular regeneration of lost motor neurons and achieve functional recovery following a complete spinal cord transection. Using adult zebrafish as a model to study how key developmental pathways can be re-activated to regulate neuroregeneration in cellular recovery, I addressed the following questions: 1) What is the role of Notch signalling during regenerative mechanisms in the lesioned spinal cord of the adult zebrafish? 2) What is the role of Notch overexpression in neurogenesis in the adult zebrafish retina? 3) Which additional signalling pathways are involved in the generation of motor neurons during spinal cord regeneration in adult zebrafish? 1) In the main part of my thesis I have investigated the role of Notch signalling during spinal cord regeneration. The Notch pathway has been shown to regulate neural progenitor maintenance and inhibit neuronal differentiation in the vertebrate nervous system. In the injured mammalian spinal cord, increased Notch signalling is held partly responsible for the low regenerative potential of endogenous progenitors to generate new neurons. However, this is difficult to test in an essentially non-regenerating system. We show that in adult zebrafish, which exhibit lesion-induced neurogenesis, e.g. of motor neurons from endogenous spinal progenitor cells, the Notch pathway is also reactivated. I over-activated the Notch pathway by forced expression of a heat-shock inducible active domain of notch in spinal progenitor cells. I observed that although apparently compatible with functional regeneration in zebrafish, forced activity of the pathway significantly decreased progenitor proliferation and motor neuron generation. Conversely, pharmacological inhibition of the pathway increased proliferation and motor neuron numbers. Thus in summary our work demonstrates that Notch is a negative signal for regenerative neurogenesis in the spinal cord. Importantly, we show for the first time that spinal motor neuron regeneration can be augmented in an adult vertebrate by inhibiting Notch signalling. 2) While in the lesioned spinal cord, over-activation of Notch attenuated neurogenesis, I observed that in the unlesioned retina the same manipulation led to strong proliferation of cells in the inner nuclear layer, presumable Müller glia cells which are the retinal progenitor cells. This coincided with an increase in eye size in adult zebrafish. These preliminary findings provide the first hint that the role of Notch may differ for different adult progenitor cell pools and will lead to future investigations of Notch induced neurogenesis in the retina. 3) We have evidence from previous studies that the dopamine and retinoic acid (RA) signalling pathways may be involved in the generation of motor neurons in the adult lesioned spinal cord. Using in situ hybridisation, I assessed the gene expression patterns a) for all D2-like receptors and b) candidate genes that relate to the RA pathway in the adult lesioned spinal cord to identify the signalling components. a) I found that only the D4a receptor was upregulated in spinal progenitor cells in the ventricular zone rostral to the lesion site, but not caudal to it. This correlates with other results showing that dopamine agonists increase motor neuron regeneration rostral, but not caudal to a spinal lesion site. b) I observed a strong increase in the expression of Cyp26a, a RA catabolising enzyme, in the ventricular progenitor zone caudal to the lesion site, in contrast to the weak expression rostrally. Crabp2a, a cellular retinoic acid binding protein, was also upregulated rostral and in close proximity to the lesion site in a subpopulation of neurons located ventrolaterally in the spinal cord. In summary, we show that the Notch pathway negatively regulates neurogenesis in the spinal cord in contrast to the retina and provide evidence that dopamine from the brain signals via the D4a receptor to promote the generation of motor neurons in addition to RA, which may also play a role in this process. These insights into adult neural progenitor cell activation in zebrafish may ultimately inform therapeutic strategies for spinal cord injury and neurodegenerative diseases such as motor neuron disease.

Inflammatory pain is a debilitating condition that can occur following tissue injury or inflammation and results in touch evoked pain (allodynia), exaggerated pain (hyperalgesia) and spontaneous pain, yet the neural plasticity underlying these symptoms is not fully understood. However, it is known that lamina I neurokinin 1 receptor expressing (NK1R+) spinal cord output neurons are crucial for the manifestation of inflammatory pain. There is also evidence that the afferent input to and the postsynaptic response of these neurons may be altered in inflammatory pain, which could be relevant for inflammatory pain hypersensitivity. Therefore, the aim of this thesis was to study inflammatory pain spinal plasticity mechanisms by investigating the synaptic input to lamina I NK1R+ neurons. In ex vivo spinal cord and dorsal root preparations from the rat, electrophysiological techniques were used to assess inflammation-induced changes in and pharmacological manipulation of the primary afferent drive to lamina I NK1R+ neurons. The excitatory input to lamina I NK1R+ neurons was examined and it was found that inflammation did not alter the relative distribution of the type of primary afferent input received and did not potentiate monosynaptic A δ or monosynaptic C-fibre input, the predominant input to these neurons. Spontaneous excitatory input was significantly elevated in the subset of neurons that received monosynaptic A δ-fibre input only, regardless of inflammation. It has recently been shown that the chemerin receptor 23 (ChemR23) represents a novel inflammatory pain target, whereby ChemR23 agonists can decrease inflammatory pain hypersensitivity, by a mechanism that involves the attenuation of potentiated spinal cord responses. This study has found that the ChemR23 agonist, chemerin, attenuated capsaicin potentiation of excitatory input to lamina I NK1R+ neurons and significantly reduced monosynaptic C-fibre input to a subset of these neurons in inflammatory pain. However, chemerin was without effect in non-potentiated conditions. In exploring potential inflammatory pain spinal plasticity mechanisms, I have investigated a phenomenon called activity-dependent slowing (ADS), whereby repetitive stimulation of C-fibres at frequencies of 1Hz or above results in a progressive slowing of action potential conduction velocity, which manifests as a progressive increase in response latency. This is proposed to limit nociceptive input to the spinal cord, thus regulating plasticity. Results demonstrate that inflammation significantly attenuated C-fibre ADS in isolated dorsal roots. Furthermore, ADS in monosynaptic C-fibre input to lamina I NK1R+ neurons was significantly reduced in inflammatory pain, which could facilitate nociceptive drive to these key spinal cord output neurons and promote inflammatory pain spinal cord plasticity. In conclusion, the major novel findings of this thesis are firstly, that chemerin can attenuate primary afferent input to lamina I NK1R+ neurons in potentiated conditions, which supports recent studies that suggest ChemR23 is a potential target for the development of new analgesics. Secondly, it was discovered that ADS in monosynaptic C-fibre inputs to lamina I NK1R+ neurons is altered in inflammatory pain, which could be relevant for inflammatory pain spinal plasticity. The findings presented in this thesis could contribute to the development of novel inflammatory pain treatments.

Electrospun biomaterial scaffolds can be engineered to support the neural differentiation of induced pluripotent stem cells. As electrospinning produces scaffolds consisting of nano or microfibers, these topographical features can be used as cues to direct stem cell differentiation. These nano and microscale scaffolds can also be used to deliver chemical cues, such as small molecules and growth factors, to direct the differentiation of induced pluripotent stem cells into neural phenotypes. Induced pluripotent stem cells can become any cell type found in the body, making them a powerful tool for engineering tissues. Therefore, a combination of an engineered biomaterial scaffold with induced pluripotent stem cells is a promising approach for neural tissue engineering applications. As detailed in this thesis, electrospun scaffolds support the neuronal differentiation of induced pluripotent stem cells through delivering the appropriate chemical cues and also presenting physical cues, specifically topography to enhance neuronal regeneration. This thesis seeks to evaluate the following topics: multifunctional electrospun scaffolds for promoting neuronal differentiation of induced pluripotent stem cells, neuronal differentiation of human induced pluripotent stem cells seeded on electrospun scaffolds with varied topographies, and controlled release of glial cell-derived neurotrophic factor from random and aligned electrospun nanofibers. / Graduate / nkhadem@uvic.ca

Spinal cord injury (SCI) is a debilitating condition accompanied by motor and sensory deficits and a reduced quality of life. Current treatment options are limited and are associated with variable efficacy and a risk of adverse effects. The pathophysiology of SCI is initiated by a primary mechanical insult to the spinal cord, followed by a complex series of deleterious events known as secondary injury. Secondary injury processes include free radical formation, glutamate excitotoxicity, inflammation and cell death. Apoptotic cell death in particular plays a key role in the secondary injury processes and exacerbates tissue degradation and loss of function. The role of Fas-mediated apoptosis in SCI pathophysiology is poorly defined in the literature to date. Correlative evidence suggests that this form of cell death is delayed and occurs in white matter adjacent to sites of primary damage. The cellular and temporal mechanisms of Fas-mediated apoptosis following experimental SCI were evaluated using a clinically relevant clip compression SCI model in the rat. Furthermore, therapeutic manipulation of Fas activation using a soluble form of the Fas receptor (sFasR) was carried out to establish the efficacy and clinical relevance of targeting this aspect of secondary injury. This work shows that Fas-mediated apoptosis is an important contributor to secondary SCI pathology. Oligodendrocytes are targeted by this form of cell death in a delayed fashion post-injury, providing an opportunity for therapeutic intervention. Intrathecal administration of sFasR following SCI reduced post-traumatic apoptosis, improved cell survival, enhanced tissue preservation and resulted in an improved motor recovery. Administration of sFasR was effectively delayed by up to 24 hours post-injury, however a shorter delay of 8 hours post-injury was most efficacious. A surprising result emerged from this work. Delayed intrathecal administration of IgG following SCI showed significant efficacy in both cellular and tissue level outcomes, as well as at the functional level. Fas-mediated apoptosis is an important aspect of secondary SCI pathophysiology and is an attractive therapeutic target. The beneficial outcomes of manipulating Fas activation using sFasR provide further evidence for this. Future work will refine this treatment strategy, bringing it into the SCI patient population.

Pain is a remarkably complex and a multifaceted process, involving the interaction between physiological and psychological factors in unique ways. Among many other factors, the size of the affected surface area contributes to the pain experience, altering one’s pain perception. Spatial summation is the term used to describe this phenomenon, and is characterized by an increase in pain perception, or a decrease in pain threshold, when the affected surface area is increased. This project investigated the neuronal processes underlying spatial summation of heat sensations in healthy female volunteers, by means of functional magnetic resonance imaging (fMRI) of the central nervous system. The first study of this project involved increasing the surface area of skin stimulated by manipulating the number of thermal probes delivering thermal stimulation, which was delivered just below participants’ measured pain threshold. Surface area was increased on one hand and across two hands to determine the extent of spatial summation, and furthermore, to determine the effect on neural activity in the spinal cord and brainstem. The second study of this project involved increasing the surface area of skin stimulated by a noxious heat stimulus and its effect on pain perception and corresponding neural activity in the spinal cord, brainstem, and brain. Results from this project suggest that the central mechanisms contributing to the spatial summation of heat sensations involve many of many of the brainstem and brain regions involved in processing the emotional, motivational, and cognitive aspects of pain. Therefore, increasing the surface area of stimulation may alter pain perception by influencing the affective dimension of the sensation, rather than the sensory/discriminatory component. The combination of such structures may interact in a unique way to protect the body from potential, or further damage, by increasing the perception of pain through emotional, motivational and cognitive mechanisms. / Thesis (Master, Neuroscience Studies) -- Queen's University, 2013-07-15 19:01:37.694

Class of 2012 Abstract / Specific Aims: The goal of this project was to evaluate the use of a preparation of the brainstem and spinal cord of neonatal rats that has been widely used for observing and quantifying central nervous activity, as well as the response to pharmacological manipulation. To achieve this, we specifically aimed to remove the intact brainstem and spinal cord of newborn rats, and develop a preparation that would maintain physiological function and allow for recording of electrical activity. Methods: Multiple dissections were performed on neonatal rats. Conditions during the dissections were controlled to maintain physiological function. Once removed, the intact brainstem and spinal cord was placed in a preparation that allowed for manipulation and access to nerve rootlets. Finally, glass suction electrodes were used to record electrical activity directly from the nerve rootlets. Once recorded, the data were stored on a hard drive for further analysis. Main Results: We were successful in isolating the intact brainstem and spinal cord in neonatal rats while maintaining physiological conditions and nervous activity. The preparation allowed for easy access to nerve roots as well as customization for different experiments. We were also successful in recording nerve activity in the preparation and collection of data for use in future experiments Conclusions: We conclude that the brainstem spinal cord preparation described in this study is a valuable tool that allows for recording and analysis of nerve activity, and specifically for measurement of respiratory motor output. This is a preparation that can be used in a variety of experiments that attempt to observe or quantify the activity of central nerve cells and allows for pharmacological interventions that could be applied in various experiments.

Injury to the spinal cord results in the disruption of signal transmission between the brain and distal targets. It often presents with the loss of motor function and sensory perception below the level of injury. There are many obstacles following injury that must be overcome in order to encourage axon regeneration and improve functional recovery. A combinatorial approach is necessary to combat physical and chemical barriers to recovery. The fluid filled cyst that forms in the majority of spinal cord injuries presents a physical barrier that we treat with our electrospun bridges. We implanted our bridges into female Long Evans Hooded rats following a complete transection. Using a molecular fusogen, polyethylene glycol, known to seal damaged membranes in conjunction with our bridges, we were able to increase functional recovery compared to animals treated with a bridge and saline. In Chapter 1, we introduce spinal cord anatomy, the pathological classifications, axon pathology, and our therapeutic strategy. Chapter 2 details the materials and methods. Chapter 3 examines previous uses of polyethylene glycol as a molecular fusogen, previous studies utilizing it in spinal cord injury, and our strategy of fusing damaged axons to improve functional recovery. Finally in Chapter 4, I discuss our behavioral results, compare histology, and detail the future of our research including protocol improvements and future combination therapies that include PEG to improve outcome.