Improving Outcomes in NF1 Spine Fusion

NF1 (neurofibromatosis type 1) is a relatively common genetic disease which may be characterized by the presence of scoliosis and altered bone metabolism, amongst its many orthopaedic manifestations. Traditionally, spine fusion procedures have been used to correct and limit the progression of this deformity. NF1 bone healing at the tibia and spine feature impaired bone anabolism, excessive catabolism, and fibrosis. Fibrotic tissue in the tibia and between the vertebrae can lead to pseudarthrosis, which can require substantive clinical intervention. In particular, complications associated with the spine can represent a significant source of morbidity in this population, often presenting with persistent deformity, pain, and hardware failure. Revision procedures, themselves a source of morbidity, are often required when a primary procedure has failed. This thesis explores systematic approaches to modelling deficient NF1 spine healing and treatment to improve outcomes in this patient population. A murine model of posterolateral fusion using rhBMP-2 (bone morphogenetic protein-2) was developed to test a range of pharmacological interventions. This model was first applied to Nf1 heterozygous mice and was reproducible and reliable. Nf1+/- mice exhibited a mild orthopaedic phenotype with increased osteoclasts on histology. Treatment with the bisphosphonate Zoledronic acid (ZA) increased the bone volume of the fusion masses in both control and Nf1+/- mice, though the improvement was larger in controls. Several studies have shown that tibial pseudarthroses can be associated with a localized double inactivation of the Nf1 gene, and we speculated that this could underlie local lesions in the spine. To recapitulate this, we utilized a model where a Cre-expressing adenovirus induced local double inactivation in Nf1flox/flox mice. This was then applied to the established spine fusion model. Consistent with the clinical presentation of spinal pseudarthrosis, a limited amount of rhBMP-2 bone was formed and substantive fibrous tissue was present. Targeted treatments with pharmaceutical agents were next trialled in this model. The MEK inhibitor PD0325901 increased bone volume in all groups while ZA increased bone density. In summary, this model represents a robust platform upon which to test targeted interventions to reduce the fibroproliferative phenotype of NF1. A second goal of this research project was to investigate the cellular contributors to spine fusion in general, which could be used to design new treatments both for NF1 and non-NF1 spine fusion. To accomplish this, a murine genetic model of lineage tracking was employed. This featured Tie2- -creERT2:Col2.3- GFP:Ai9 reporter mice. Spine fusion operations were performed in these mice, and the distribution of lineage-labelled cells were traced using fluorescence. Notably, Tie2 lineage cells co-labelled with TRAP positive cells, suggesting a primary contribution to the osteoclasts but not osteoblasts of the fusion mass. Conversely, lineage cells co-labelled with Col2.3-GFP expressing osteoblasts, suggesting new bone primarily arises from mesenchymal cells with negligible input from endothelial cells undergoing transdifferentiation. In conclusion, treatment of scoliosis remains a challenge in individuals with NF1. The development of a fibroproliferative model of spine fusion in an Nf1 deficient mouse represents a robust platform upon which to test targeted interventions to improve outcomes in NF1. Additionally, advancements in genetic modeling of human disease in animals may provide new models in which to investigate this process

Nutritional influence on oxidative stress in global ischemia

Primary brain injury in stroke is followed by oxidative stress and further neural damage. Glutathione (GSH) is critical in antioxidant defense. Since cysteine is limiting in GSH synthesis, Phase 1 of this study investigated the effect of a dietary sulphur amino acid deficiency (-SAA) on neural damage in global hemispheric hypoxia-ischemia (GHHI). Rats were fed a -SAA or control diet for 6 days, and subjected to GHHI after 3 days. Histologically evaluated neural damage at 7 days post hypoxia-ischemia was greater in -SAA rats. Brain GSH concentration was decreased in -SAA rats 3 days after ischemia. A cysteine precursor, L-2-oxothiazolidine-4-carboxylic acid (OTC) administered to -SAA rats did not ameliorate neural damage. GSH is decreased by protein-energy malnutrition (PEM) in some tissues. Phase 2 investigated the effect of PEM on brain oxidative stress, neural damage and behaviour after global ischemia in adult male gerbils. In a 2x2 factorial design, gerbils were fed an adequate protein (12%; C) or low protein (2%; PEM) diet for 4 weeks, then subjected to transient ischemia (I) or sham surgery (S). After 12 hours of reperfusion, brain from half the gerbils was collected for biochemical analyses. Remaining gerbils were fed pre-surgery diets for 10 more days. To assess functional consequences of ischemia, gerbils were placed in an open field on Days 3, 7 and 10 after surgery. On Day 10, viable hippocampal CA1 neurons were counted. C-I gerbils did not habituate as readily in the open field on day 3 as C-S, but normalized by day 7. PEM-I gerbils failed to habituate by day 10, traveled greater distance than other gerbils and 7 of 12 displayed thigmotaxis, a wall-hugging preference for the outer perimeter of the open field. CA1 neuron loss in I was 61.5% of S, but unaffected by PEM. Four of 12 PEM-I gerbils had marked increases in hippocampal glia. Hippocampus protein thiols were reduced by PEM and by ischemia, consistent with oxidative stress. GSH concentration, glutathione reductase activity and thiobarbituric acid reactive substances were not significantly affected by PEM or ischemia. Findings from these two studies suggest well-nourished but not nutritionally-deficient rodents tolerate a mild brain insult. This is clinically relevant because many elderly stroke victims suffer from PEM at the time of ischemia, which may compromise recovery

Inflammatory Mechanisms of Neurodegeneration in Toxin-Based Models of Parkinson's Disease

Parkinson's disease (PD) has been associated with exposure to a variety of environmental agents, including pesticides, heavy metals, and organic pollutants; and inflammatory processes appear to constitute a common mechanistic link among these insults. Indeed, toxin exposure has been repeatedly demonstrated to induce the release of oxidative and inflammatory factors from immunocompetent microglia, leading to damage and death of midbrain dopamine (DA) neurons. In particular, proinflammatory cytokines such as tumor necrosis factor-α and interferon-γ, which are produced locally within the brain by microglia, have been implicated in the loss of DA neurons in toxin-based models of PD; and mounting evidence suggests a contributory role of the inflammatory enzyme, cyclooxygenase-2. Likewise, immune-activating bacterial and viral agents were reported to have neurodegenerative effects themselves and to augment the deleterious impact of chemical toxins upon DA neurons. The present paper will focus upon the evidence linking microglia and their inflammatory processes to the death of DA neurons following toxin exposure. Particular attention will be devoted to the possibility that environmental toxins can activate microglia, resulting in these cells adopting a “sensitized” state that favors the production of proinflammatory cytokines and damaging oxidative radicals

Use of induced pluripotent stem cell derived neurons engineered to express BDNF for modulation of stressor related pathology

Combined cell and gene-based therapeutic strategies offer potential in the treatment of neurodegenerative and psychiatric conditions that have been associated with structural brain disturbances. In the present investigation, we used a novel virus-free re-programming method to generate induced pluripotent stem cells (iPSCs), and then subsequently transformed these cells into neural cells which over-expressed brain derived neurotrophic factor (BDNF). Importantly, the infusion of iPSC derived neural cells (as a cell replacement and gene delivery tool) and BDNF (as a protective factor) influenced neuronal outcomes Specifically, intracerebroventricular transplantation of iPSC-derived neural progenitors that over-expressed BDNF reversed the impact of immune (lipopolysaccharide) and chronic stressor challenges upon subventricular zone adult neurogenesis and the iPSC-derived neural progenitor cells alone blunted the stressor induced corticosterone response. Moreover, our findings also indicate that mature dopamine producing neurons can also be generated using iPSC procedures and these cells appeared to be viable when infused in vivo. Taken together, these data could have important implications for using gene-plus-cell replacement methods to modulate stressor related pathology

Incubation environment impacts the social cognition of adult lizards

Recent work exploring the relationship between early environmental conditions and cognition has shown that incubation environment can influence both brain anatomy and performance in simple operant tasks in young lizards. It is currently unknown how it impacts other, potentially more sophisticated, cognitive processes. Social-cognitive abilities, such as gaze following and social learning, are thought to be highly adaptive as they provide a short-cut to acquiring new information. Here, we investigated whether egg incubation temperature influenced two aspects of social cognition, gaze following and social learning in adult reptiles (Pogona vitticeps). Incubation temperature did not influence the gaze following ability of the bearded dragons; however, lizards incubated at colder temperatures were quicker at learning a social task and faster at completing that task. These results are the first to show that egg incubation temperature influences the social cognitive abilities of an oviparous reptile species and that it does so differentially depending on the task. Further, the results show that the effect of incubation environment was not ephemeral but lasted long into adulthood. It could thus have potential long-term effects on fitness

Elastic biodegradable starch/ethylene-co-vinyl alcohol fibre-mesh scaffolds for tissue engineering applications

The fabrication of a biomaterial scaffold, with adequate physical and structural properties for tissue engineering applications, is reported. A blend of starch with ethylene-vinyl alcohol (50/50 w/w, SEVA-C) is used to produce 3D fibre-mesh scaffolds by wet-spinning. The scaffolds are characterized in terms of morphology, porosity, interconnectivity, and pore size, using scanning electron microscopy (SEM) and microcomputed tomography (μCT). The degradation behavior, as well as the mechanical properties of the scaffolds, is investigated in presence of alpha-amylase enzyme at physiological concentration. Scaffolds with porosities ranging from 43 to 52%, interconnectivity of ∼70.5% and pore size between 118 and 159 μm, can be fabricated using the proposed methodology. The scaffolds exhibit an elastic behavior in the wet state with a compressive modulus of 7.96±0.32 MPa. Degradation studies show that SEVA-C scaffolds are susceptible to enzymatic degradation by alpha-amylase, confirmed by the increase of weight loss (40% of weight loss after 12 weeks) and presence of degradation products (reducing sugars) in solution. The diameter of SEVA-C scaffolds decreases with degradation time, increasing the overall porosity, interconnectivity and pore size. In vitro cell studies with human osteosarcoma cell line (SaOs-2) showed a nontoxic and cytocompatible behavior of the developed fibre mesh scaffolds. The positive cellular response, together with structural and degradable properties, suggests that 3D SEVA-C fibre-meshes may be good candidates as tissue engineering scaffolds. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014, 131, 40504. Copyright © 2014 Wiley Periodicals, Inc.This work was supported by national funds through the Portuguese Foundation for Science and Technology under the scope of the project PTDC/CTM/67560/2006 and by the European Regional Development Fund (ERDF) through the Operational Competitiveness Programme “COMPETE” (FCOMP-01-0124-FEDER-007148)

Use of a trabecular metal implant in ankle arthrodesis after failed total ankle replacement: A short-term follow-up of 13 patients

Patients and methods 13 patients with a migrated or loose total ankle implant underwent arthrodesis with the use of a retrograde intramedullary nail through a trabecular metal Tibial Cone. The mean follow-up time was 1.4 (0.6-3.4) years. Results At the last examination, 7 patients were pain-free, while 5 had some residual pain but were satisfied with the procedure. 1 patient was dissatisfied and experienced pain and swelling when walking. The implant-bone interfaces showed no radiographic zones or gaps in any patient, indicating union. Interpretation The method is a new way of simplifying and overcoming some of the problems of performing arthrodesis after failed total ankle replacement