The failure of mammalian CNS neurons to regenerate their axons derives from a combination of intrinsic deficits and extrinsic obstacles. Following injury, chondroitin sulfate proteoglycans (CSPGs) accumulate within the glial scar that forms at the lesion site in response to the insult. CSPGs inhibit axonal growth and regeneration, an action mediated by their sulfated glycosaminoglycan (GAG) chains, especially those with 4-sulfated (4S) sugars. Arylsulfatase B (ARSB) selectively cleaves 4S groups from the non-reducing ends of GAG chains without disrupting other, potentially growth-permissive motifs. In this thesis, "Modifying Chondroitin Sulfation Enhances Retinal Ganglion Cell Axon Regeneration," I, Craig Pearson, seek to determine the time course and spatial distribution of CSPG accumulation in the glial scar following acute injury, and then to demonstrate that ARSB is effective in reducing the inhibitory actions of CSPGs. I examine the effects of ARSB in an in vitro model of the glial scar and in vivo, using optic nerve crush (ONC) in adult mice. ARSB is clinically approved for replacement therapy in patients with mucopolysaccharidosis VI and therefore represents an attractive candidate for translation to the human CNS. My findings illustrate the importance of CSPGs as a barrier to axon extension following injury, and show compelling evidence that selective modification of the sulfation pattern on GAG chains results in significant enhancement of RGC axonal regeneration. Finally, I combine ARSB treatment with a host of intrinsic pro-regenerative stimuli and show robust, long-distance regeneration of RGC axons through the optic chiasm and into the optic tract. Taken together, the results of this thesis argue for the therapeutic potential of modifying the extracellular matrix to promote regeneration of axons in the CNS.

Chan, Chung Kit. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 98-110). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract in Chinese --- p.iv / Acknowledgements --- p.v / Table of Abbreviations --- p.vi / Table of Contents --- p.vii / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter Chapter 2 --- Functions of hyaluronan in the development of retinofugal pathway / Introduction --- p.18 / Materials and Methods --- p.19 / Results --- p.23 / Discussion --- p.26 / Figures --- p.32 / Chapter Chapter 3 --- Characterization of Nogo and its receptor in retinofugal pathway using Western blot analysis / Introduction --- p.40 / Materials and Methods --- p.42 / Results --- p.50 / Discussion --- p.52 / Figures --- p.57 / Chapter Chapter 4 --- Expression patterns and functions of Sonic hedgehogin retinofugal pathway / Introduction --- p.62 / Materials and Methods --- p.64 / Results --- p.69 / Discussion --- p.76 / Figures --- p.81 / Chapter Chapter 5 --- General Discussion --- p.91 / Figures --- p.95 / References --- p.98

Spontaneous intracerebral haemorrhage (ICH) is one of the most lethal forms of stroke and has a relatively higher morbidity in Asian people. The high disability rate of 50% in all survivors and lack of clinically effective regeneration medicine make ICH a major unanswered problem worldwide. Considerable preclinical evidence suggests that neuroprotective interventions are urgently needed to lessen the effects of this disease. To date, less preclinical researches with proven positive results have successfully translated to the clinical setting, mainly due to poor modelling, a lack of behavioural testing, inadequate experimental design and failure to consider white matter damage. According to the few previous studies, white matter is a key predictor of ICH outcomes and a potential target of recovery. The long-term axonal degeneration in rodent ICH has been ignored for decades, and almost every preclinical study has focused on mechanisms in the acute phase. Clinically ICH patients may suffer a permanent loss of brain function or long-term deficits that take years to recover from. If the preclinical researches target the long-term axon degeneration/regeneration in the chronic stage, it might help to develop successful clinical interventions for functional reconstruction and rehabilitation. / Thus, to obtain the evidence of axonal degeneration and regeneration in the chronic stage of experimental ICH, this study at first systematically assessed the histological and functional outcomes of axonal regeneration in experimental ICH from day 3 to day 56 and secondly find the effective markers and methods for investigation axons in experimental ICH models in vivo. The long-term gait disturbance of a computer-generated CatWalk system, the electrophysiological changes, axonal loss by PKC gamma (PKCγ) immunohistochemistry and axonal degeneration by Bielschowsky silver staining were examined in unilateral striatum lesioned ICH (ST-ICH) rats. As the ST-ICH model demonstrated a spontaneous functional recovery within one or two month, we further developed a modified internal capsule lesioned ICH (IC-ICH) rat model which mimic clinical conditions, and investigated whether an internal capsule lesion leads to long-term axonal damage and long lasting functional deficits. / The finding demonstrated that haematoma in striatum led to severe axonal degeneration/loss in ipsilateral medullary corticospinal tract (CST) and functional deficits in a long-term duration (1-2 months after ICH). PKCγ level was an effective marker to quantify the axonal loss in experimental ICH and it indicated a significant axonal loss on day 56 after ICH in ipsilateral CST. Bielschowsky silver staining was a useful method to illustrate the morphological changes of axonal degeneration and regeneration in longitudinal sections of CST and it clearly showed the process of axon swelling, disrupted and regenerated in 2 months’ duration after ICH. / Somatosensory evoked potentials (SSEPs) and gait analysis were valuable functional assessments to characterize the long-term behavioral deficits resulting from axonal degeneration/regeneration in experimental ICH. The decrease in electrophysiological parameter SSEP amplitudes was observed in experimental ICH. Multiple gait parameters changed after ICH and some of that such as paw print area, paw pressure, stand index, duty cycle can be used as long-term evaluating markers in chronic stage of experimental ICH. / Compared to ST-ICH, the modified IC-ICH model exhibited a relatively smaller lesion volume with consistent axonal loss/degeneration and long-lasting neurological dysfunction at 2 months after ICH. Functionally, the impairment of the mNSS, ratio of contralateral forelimb usage, four limb stand index, contralateral duty cycle and ipsilateral SSEPs amplitude remained significant in the IC-ICH model at 56 days compared with the sham group, and asymmetries in the hind paw print area of the IC-ICH model exhibited significant differences from the ST-ICH model at 56 days. Structurally, the significant loss of PKCγ in ipsilateral CST of IC-ICH and the consistent axonal degeneration with several axonal retraction bulbs and enlarged tubular space was observed at 56 days after ICH. / In summary, the data from this study systematically characterize the histological and functional outcomes (especially gait parameter and SSEPs changes) in the experimental ICH model. A modified internal capsule lesioned ICH model was developed for rats, and proved to have long lasting neurological deficits. A comprehensive understanding of the dynamic progression after experimental ICH should aid further successful clinic translation in animal ICH studies, and provide new insights into the potential biomarkers and therapeutic targets of ICH. / 原發性腦出血（ICH）是一種致死性較高的卒中類型。在亞洲人群眾發病率相對較高。高致殘率和臨床上缺乏有效的治療手段，使得腦出血成為世界範圍內的健康問題。因此需要大量的臨床前研究尋找有效的治療方法。然而，迄今為止，臨床前研究獲得的陽性結果中，只有少數被成功的轉化到臨床應用。臨床轉化存在的部分失敗，歸結於幾個主要的因素包括動物模型的不足，動物行為學實驗的不恰當使用，實驗設計的缺陷以及對白質損傷機制的忽略。有研究認為，腦白質是卒中後功能恢復的關鍵指標和潛在治療靶點。腦出血慢性期的軸索變性在齧齒類動物模型中的研究被忽視了幾十年，而幾乎所有的臨床前研究都關注於急性期的機制。而臨床上倖存的腦出血病人大多罹患永久性的腦功能損傷，往往需要數年才能恢復或者難以恢復。如果臨床前轉化實驗以腦出血後慢性期的神經軸索損傷/再生作為研究目標，也許可以找到有助於卒中後功能重建和康復的治療手段。 / 為了尋找腦出血慢性期神經軸索損傷的證據，本研究首先從組織學和功能行為學兩個方面對對實驗性腦出血後的軸索再生進行了系統的評價。並建立了有效反應慢性期神經軸索再生的一系列方法和標誌物。本研究將步態分析，電生理評價， Bielschowsky銀染和PKCγ組織學染色結合起來對腦出血後的動物模型的軸索蛻變和再生進行長期觀察。結果顯示傳統的紋狀體損傷模型在1到2個月出現自發的功能恢復。本研究進一步假設內囊出血模型可能會獲得更加持久的功能損傷，也更為接近臨床患者的情況。因此，為了更好地研究腦出血慢性期的白質損傷和類比臨床情況，本研究建立了一種改進的內囊出血大鼠模型，並用組織學和行為學方法對其長期的功能損傷進行評價。 / 研究結果顯示，位於紋狀體的血腫可以引起同側的延髓皮質脊髓束(CST)出現嚴重的慢性期退化和變性，並同時伴有神經功能損傷。PKCγ是評價實驗性腦出血後神經軸索損傷程度的有效標誌物，資料表明同側皮質脊髓束PKCγ的表達水準在ICH損傷56天后仍有顯著降低。對延髓椎體CST的Bielschowsky銀染，可以從結構上有效的反應軸索變形和再生的過程，CST縱行切片染色清楚地顯示了腦出血損傷後2個月的時間內軸索水腫、斷裂和再生的過程。 / 體感誘發電位（SSEPs）和步態分析的方法可以從功能上對腦出血後神經軸索損傷進行較為全面的評價和定量分析。單側紋狀體腦出血可以引起同側皮層SSEP波幅的降低。多個步態分析參數在腦出血後也存在明顯的變化，其中前後掌爪印面積（paw print area），爪印壓力（paw pressure），站立指數（stand index），患側肢體站立百分比（duty cycle）都可作為觀察腦出血後慢性期功能損傷和恢復的評價指標。 / 改進後的內囊腦出血模型顯示病灶體積相比較小但神經軸索的損失和神經功能障礙較為持久。從神經功能方面評判，與假手術組相比，神經功能評分（mNSS），對側前肢使用率（cylinder test），四肢站立指數（stand index），患側肢體站立百分比（duty cycle）和患側體感誘發電位波幅（SSEPs amplitude）在出血後2個月仍然顯著降低。後掌的爪印面積（print area）與紋狀體腦出血的動物比較在出血後第56天后仍有顯著差異。從軸索結構評判，內囊出血模型顯示出更為嚴重的神經軸索退變和損傷，表現為在出血後56天PKCγ蛋白表達量的持續降低，軸索斷裂結節和管狀間隙的形成。 / 綜上所述，本研究系統地分析了實驗腦出血後的組織學和功能特點，建立了一個改進的內囊腦出血大鼠模型，並證明該模型存在更為持久的神經功能障礙和神經軸索損傷。 / Liu, Yao. / Thesis Ph.D. Chinese University of Hong Kong 2014. / Includes bibliographical references (leaves 168-200). / Abstracts also in Chinese. / Title from PDF title page (viewed on 18, October, 2016). / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.

During the development of the nervous system, a complex system of chemical and mechanical cues guide nerve cell projections toward appropriate targets by eliciting attractive or repulsive responses from navigating structures called growth cones. Current in vitro models of neural guidance lack the capacity to provide multiple cues within the same substrate. This dissertation presents in vitro models with tunable presentation of multiple chemical and mechanical guidance cues in a single substrate for elucidating a more complete understanding of growth cone responses to the extracellular environment. In the first study, our model utilized a light-sensitive agarose gel as the growth substrate and a polyethylene glycol gel that contained three dimensional neural growth in specific geometries. This model incorporated molecular cues as immobilized proteins and gradients of soluble factors in a quantifiable manner. The agarose gel presented issues with gelation and supporting neural growth, so we sought to improve upon our initial design by changing the growth substrate. The second study employed a novel light-sensitive dextran gel in place of the agarose of the first study. The chemoattractant neurotrophin-3 and chemorepellant semaphorin3A were immobilized within this dextran gel in a spatially defined manner, and the response from growth cones quantified. The growth cones did not respond to the control protein NeutrAvidin, exhibited a moderate response to neurotrophin-3, and showed a strong repulsive response to semaphorin 3A. The third study investigated neural responses to changes in substrate stiffness. Dextran gels with light-degradable crosslinks exhibited different elastic moduli based upon irradiation time. Specified regions of dextran gels were irradiated with light to present growth cones with a choice between two different elastic moduli. Growth cones exhibited preference for a narrow range of elastic moduli spanning less than 200 Pa, an observation unattainable with other in vitro models. The results establish our models as possible platforms for observing and manipulating specific growth cone responses to both chemical and mechanical cues. Understanding how the growth cone interacts with its environment may lead to improved wound healing therapies, and expanding our model into a high-throughput assay could contribute to the development of these new treatments. / acase@tulane.edu

Peripheral nerve damage affects hundreds of thousands of people every year. This study tested the effectiveness of using lysophosphatidylcholine (LPC) in combination with nerve growth factor (NGF) to increase the healing rate of damaged left sciatic nerves in female rats. The rats were randomly divided into eight groups: Sham, Right Sciatic, Crush, LPC, LPC-NGF, Crush- LPC, Crush-NGF, and Crush-LPC-NGF. The healing of the nerves was measured by monitoring gait, electrophysiological parameters (compound muscle action potential amplitudes and nerve conductance velocities) and morphological parameters (total fascicular area, total myelinated fiber counts, fiber densities, fiber diameters, and g-ratio). Gait and electrophysiological parameters were measured three times a week. Morphological parameters were measured at three weeks and at six weeks. The LPC and LPC-NGF groups were not statistically different from the controls (Sham and Right Sciatic) at either of the morphological time points but were statistically different from the controls for the first three weeks for the electrophysiological parameters and gait. The LPC-NGF group did not differ from the LPC group at any time point for any of the parameters. Crush, Crush-LPC, Crush-NGF, and Crush-LPC-NGF groups statistically differed from the controls at week 3 for all parameters and only in the electrophysiological parameters at week 6. Crush-LPC, Crush-NGF, and Crush-LPC-NGF did not differ from each other or from the Crush group. The combination of LPC and NGF did not prove to be an effective treatment for peripheral nerve damage. Future work is recommended to test multiple injections of LPC and NGF.

The primary olfactory pathway in adult mammals has retained a remarkable potential for self-repair. Olfactory ensheathing cells (OECs), specialized glial cells within the olfactory nerve, are thought to play an important role in the ongoing growth and replenishment of sensory connections in this system. To gain insight into novel molecules that could mediate OEC-supported growth of axons within the olfactory nerve, gene expression profiling experiments revealed very high expression of the fibulin-3 glycoprotein in OECs. To date, research on fibulin-3 has been limited and mainly focused on its involvement in Doyne honeycomb retinal dystrophy, vasculogenesis and tumor formation. As the extracellular matrix associated with OECs is thought to be an important contributor to a growth-permissive environment, the main aim of this thesis was to define a putative role for fibulin-3 during olfactory receptor neuron replacement and regeneration. This hypothesis was investigated in a series of in vitro and in vivo experiments that involved lentiviral vectors to manipulate fibulin-3 gene expression in OECs as well as the use of knock-out mice. Using genetically-modified OECs, experimental data showed that increased levels of fibulin-3 induced morphological changes in OECs and also impeded their migration. Lentiviral vector-mediated expression of fibulin-3 in OECs also had an inhibitory effect on neurite outgrowth from dorsal root ganglion explants. On the other hand, knock-down of fibulin-3 levels via siRNA technology resulted in reduced proliferation. Comparative lesioning experiments in fibulin-3 knock-out and wild-type mice allowed for further assessment of a role for fibulin-3 in olfactory nerve repair in vivo. Two experimental injury models, i.e. epithelial (Triton-X) lesioning and olfactory bulbectomy, were employed. The results obtained were in line with in vitro observations. A lack of fibulin-3 in knock-out mice resulted in a seemingly augmented regeneration of the olfactory epithelium at 10 days post-injury. However, at the latest recovery time point of 42 days post-injury, an impaired recovery of the olfactory epithelium from the experimental insults was observed. Although the precise mechanism for the latter phenomenon is not yet fully understood, our data point towards several factors which include vascular abnormalities and altered cell proliferation within the olfactory epithelium. Additionally, the precise protein distribution of another wide-spread family of extracellular matrix molecules, the laminins, was investigated in this thesis. It was of interest to investigate the spatiotemporal expression of laminin isoforms during iii olfactory nerve development and regeneration as these molecules may have distinct roles in promoting olfactory sensory neuron growth and patterning. In situ hybridization and immunohistochemical studies concluded that laminin-211 and laminin-411 were the most likely candidates to play such a role. In summary, this thesis provides new insights into the role of the extracellular matrix, fibulin-3 in particular, in regulating cell migration, division and axonal growth in the primary olfactory pathway. Such knowledge also gives a greater understanding of the molecular mechanisms by which OEC transplants may enhance axonal regeneration elsewhere in the CNS.

Injury severs the axons in the spinal cord causing permanent functional loss. After injury, a series of events occur around the lesion site, including the deposition of growth cone inhibitory astroglial scar tissue containing chondroitin sulfate proteoglycan (CSPG)- rich regions. It is important to encourage axons to extend through these inhibitory regions for regeneration to occur. The work presented in this dissertation investigates the effect of three proteins, constitutively active (CA)-Cdc42, CA-Rac1, and brain-derived neurotrophic factor (BDNF) on axonal outgrowth through CSPGs-rich inhibitory regions after spinal cord injury (SCI). Cdc42 and Rac1 are members of the Rho GTPase family and BDNF is a member of the neurotrophin sub-family. These three proteins affect the actin cytoskeleton dynamics. Therefore, Cdc42, Rac1, and BDNF promote axonal outgrowth. The effect of CA-Cdc42 and CA-Rac1 on neurite extension through CSPG regions was determined in an in vitro model. Rac1 and Cdc42 s ability to modulate CSPG-dependent inhibition has yet to be explored. In this study, a stripe assay was utilized to examine the effects of modulating all three Rho GTPases on neurite extension across inhibitory CSPG lanes. Alternating laminin (LN) and CSPG lanes were created and NG108-15 cells and E9 chick dorsal root ganglions (DRGs), were cultured on the lanes. Using the protein delivery agent Chariot, the neuronal response to exposure of CA and dominant negative (DN) Rho GTPases, along with the bacterial toxin C3, was determined by quantifying the percent ratio of neurites crossing the CSPG lanes. CA-Cdc42, CA-Rac1, and C3 transferase significantly increased the number of neurites crossing into the CSPG lanes compared to the negative controls for both the NG108-15 cells and the E9 chick DRGs. We also show that these mutant proteins require the delivery vehicle, Chariot, to enter the neurons and affect neurite extension. Therefore, activation of Cdc42 and Rac helps overcome the CSPG-dependent inhibition of neurite extension. In an in vivo study, CA-Cdc42 and CA-Rac1 were locally delivered into a spinal cord cavity. Additionally, BDNF was delivered to the lesion site, either individually or in combination with either CA-Cdc42 or CA-Rac1. The dorsal over-hemisection model was utilized, creating a ~2mm defect that was filled with an in situ gelling hydrogel scaffold containing lipid microtubules loaded with the protein(s) to encourage axons. The lipid microtubules enable slow release of proteins while the hydrogel serves to localize them to the lesion site and permit axonal growth. The results from this study demonstrate that groups treated with BDNF, CA-Cdc42, CA-Rac1, BDNF/CA-Cdc42, and BDNF/CA-Rac1 had significantly higher percentage of axons from the corticospinal tract (CST) that traversed the CSPG-inhibitory regions, as well as penetrate the glial scar compared to the untreated and agarose controls. Although axons from the CST tract did not infiltrate the scaffold-filled lesion, NF-160+ axons were observed in the scaffold. Treatment with BDNF, CA-Cdc42, and CA-Rac1 also reduced the inflammatory response, quantified by analyzing GFAP and CS-56 intensity for reactive astrocytes and CSPGs, respectively, at the interface of the scaffold and host tissue. Therefore, the local delivery of CA-Cdc42, CA-Rac1 and BDNF, individual and combination demonstrated the ability of axons to extend through CSPG inhibitory regions, as well as reduce the glial scar components.

Transplantation of olfactory ensheathing cells (OECs) into injured spinal cord results in improved functional outcome through axonal regeneration, remyelination, and neuroprotection. However, because little is known of the fate of OECs transplanted into injured peripheral nerve, their myelin forming potential requires investigation. To study these issues OECs were isolated from the olfactory bulbs of adult green fluorescent protein (GFP)-expressing transgenic rats and transplanted into a sciatic nerve crush lesions. Five weeks to six months after transplantation the nerves were studied histologically and it was determined that GFP-expressing OECs survived in the lesion and distributed longitudinally across the lesion zone. Immunostaining revealed a high density of isoform Nav1.6 at the newly formed nodes of Ranvier which were flanked by paranodal Caspr staining. Immuno-electron microscopy for GFP revealed transplanted OECs form peripheral type myelin. These results indicate that transplanted OECs extensively integrate into transected peripheral nerve, form myelin on regenerated peripheral nerve fibers, and reconstruct nodes of Ranvier with proper sodium channel structure.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2009.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2009. / Härtill 4 uppsatser.