MRI can provide anatomical, functional, and physiological images at relatively high spatial resolution and is non-invasive and does not have depth limitation. However, the application of MRI to study the retina is difficult due to the very small size of the retina. This thesis details the development of MRI methods to image blood flow (BF), anatomy, and function of the retina and choroid, and their application to two diseases of the retina: diabetic retinopathy and retinal degeneration. A unique continuous arterial spin labeling technique was developed to image BF in mice and tested by imaging cerebral BF. This method was then applied to image layer-specific BF of the retina and choroid in mice, and to acquire BF functional MRI of the retina and choroid in response to hypoxic challenge. Additionally blood oxygen level dependent functional MRI of the mouse retina and choroid in response to hypoxic challenge was obtained using a balanced steady state free precession sequence which provides fast acquisition, has high signal to noise ratio, and does not have geometric distortion or signal dropout artifacts. In a mouse model of diabetic retinopathy, MRI detected reduced retinal BF in diabetic animals. Visual function in the diabetic mice, as determined by psychophysical tests, was also reduced. Finally, in a mouse model of retinal degeneration, BF and anatomical MRI detected reductions of retinal BF and the thickness of the retina. The studies detailed in this thesis demonstrate the feasibility of layer-specific MRI to study BF, anatomy, and function, in the mouse retina. Further, these methods were shown to provide a novel means of studying animal models of retinal disease in vivo.

[Truncated abstract] Sporadic inclusion body myositis (sIBM) is a chronic inflammatory disease that is the most common myopathy in individuals above the age of 50 in the Caucasian population. sIBM is characterised by cytotoxic immune infiltration of skeletal muscle, consisting primarily of CD8+ T-cells and macrophages, as well as a degenerative process, with muscle fibre vacuolation and intracellular filamentous inclusions. The pathogenesis of sIBM is likely to involve a complex interaction between genetic and environmental factors. Whilst the physiological and pathological characteristics of sIBM have been clearly identified, the exact origin and genetic basis of the disease remains unknown. A number of studies show that sIBM is associated with alleles of the major histocompatibility complex (MHC) on chromosome 6p21.3 and specifically with two ancestral haplotypes (AH) in Caucasians – the 8.1AH, defined by HLA-B*0801, HLA-DRB1*0301 and the 35.2AH, defined by HLA-B*3501, HLA-DRB1*0101. Mapping studies subsequently showed that sIBM susceptibility likely originates from a 389kb region of the MHC, spanning from centromeric of PBX2 to telomeric of HLA-DRB1. The central hypothesis of this thesis was that susceptibility to sIBM is conferred by a single allele found within a region defined using the 8.1AH, which is also carried by other haplotypes associated with sIBM. Three patient cohorts from Australia, the U.S.A and Japan were studied. ... Of the 32 alleles genotyped, none were found in all susceptibility haplotypes and one was common, but not unique, to the 8.1AH, 7.2AH and 52.1AH. Five SNPs were also found in two of the three haplotypes, although none were specific to the sIBM susceptibility haplotypes. These data suggest that the 8.1AH is likely to carry an sIBM susceptibility allele independent of the 35.2AH, 7.2AH and 52.1AH. Based on the possible mechanism of action in cellular differentiation and its location within the 8.1AH-defined sIBM susceptibility region reported in 2004, NOTCH4 was a strong candidate for conferring sIBM susceptibility. NOTCH4 coding region polymorphisms were thus investigated in a Caucasian patient cohort to assess any possible role in sIBM susceptibility. While the frequency of some alleles were increased in sIBM patients, the strong linkage disequilibrium throughout the MHC prevented confirmation of any alleles as playing a direct role in sIBM. The 8.1AH-derived sIBM susceptibility region was further refined using recombination mapping. This approach used markers characterised against multiple haplotypes to genotype patients carrying part of the 8.1AH to locate a common, overlapping susceptibility region. Recombination mapping of patients revealed a common overlapping region of the 8.1AH, extending from BTNL2 to HLA-DRB3. The results of the study indicate that 8.1AH-derived susceptibility for sIBM is likely to originate from a 172kb region encompassing HLA-DRA, HLA-DRB3 and part of BTNL2. These genes warrant further investigation in future studies.

Disc degenerative changes are directly or indirectly associated with spinal pain and disability. Literature revealed a high prevalence of disc degeneration in the thoracic region, however thoracic MRI degeneration trends and information on disc biochemical matrix constituents are limited for thoracic discs compared to lumbar and cervical discs. The objective of this thesis was to use MRI to investigate the prevalence of disc degenerative changes affecting the human thoracic spine, and to determine the factors affecting spinal disc biochemical matrix. A 3-point subjective MRI grading scale was used to grade the films. The feasibility of using archived formalin-fixed cadaver material was investigated to analyse collagen and elastin crosslinks. The prevalence of degenerative changes in human thoracic discs and vertebrae (T1 to T12) was determined retrospectively from an audit of 216 MRI cases, using sagittal T1- and T2-weighted MR images. In a subsequent series of ex-vivo studies, human thoracic discs and LF from 26 formalin-fixed and two fresh spines, involving all thoracic levels, were examined macroscopically to determine the degeneration status. Subsequently, disc and ligament tissues were analysed biochemically for collagen (pyridinoline and deoxypyridinoline) and elastin (desmosine and isodesmosine) crosslinks. These crosslinks were extracted from hydrolysed samples by cellulose partition chromatography, and analysed by reverse-phase HPLC. Collagen content was determined using its hydroxyproline content, and proteoglycan content was assayed using a modified DMB assay for chondroitin sulphate. Finally the MRI and macroscopic assessments of thoracic discs, were compared with the biochemical data from two fresh cadaver thoracic spines. The 3-point MRI grading scale had a high inter- (k = 0.57 to 0.78) and intra-rater (k = 0.71 to 0.87) reliability. There were no significant differences in the collagen and elastin content and extent of collagen crosslinks between formalin fixed and unfixed ligament and disc tissues, after 25 weeks of formalin fixation. From the in-vivo MRI series of investigations (n = 216 MRI films), the prevalence of thoracic disc degenerative and vertebral morphological changes revealed significant age, gender and spinal level trends (p < 0.05).Generally, males had a higher propensity for disc degeneration in contrast to females, especially older females, where the trend showed a higher prevalence of osteophytes and vertebral body changes. In particular, the mid and lower thoracic levels have a higher prevalence of degenerative changes, except for osteophytes and anterior vertebral wedging. With increased age, there was a concomitant increase in anterior wedging and bi-concavity and disc degenerative changes except for end-plates. The biochemical investigations on the ex-vivo series of formalin-fixed thoracic discs (n = 303) also revealed significant changes in the disc matrix due to degeneration status, age, gender and spinal regional factors. With increased age, normal disc matrices have significantly lower collagen content and extent of pyridinoline (p < 0.001). In contrast, the degenerated disc matrix revealed significantly higher collagen content and extent of deoxypyridinoline (p < 0.05). These findings suggest that an altered matrix existed in normal ageing discs, which render the disc prone to injury and degeneration over the life span. The higher collagen and deoxypyridinoline in degenerated disc matrices reflects an increase in chondrocyte synthesis, and is also a novel finding, suggesting that they may be used as markers of ageing and degeneration processes. The biochemical investigations on another series of ex-vivo spinal LF tissues (n = 364), revealed that this had a lower collagen and pyridinoline, but significantly higher elastin and deoxypyridinoline compared to spinal discs (p < 0.05). Elastin crosslinks however were difficult to detect in spinal discs, being present in negligible amounts in a few lumbar discs. The elastin crosslinks in the LF were not significantly affected by age, but were significantly higher in calcified, and female ligamentum tissues, and also in the lumbar region (p < 0.05). These MRI prevalence findings enhanced our knowledge of vertebral body and disc degeneration trends in the thoracic region and contributed to the interpretation of MR images for pathology in the human thoracic spine. Information on the associated collagenous and elastic changes in the disc and ligamentum matrices provide original data and insight on the pathogenesis of degeneration in the disc matrix from a biochemical perspective, highlighting gender, age and spinal level influences on the matrix tensile strength and cellular synthetic activities.

no / Retinal dystrophies are an overlapping group of genetically heterogeneous conditions resulting from mutations in more than 250 genes. Here we describe five families affected by an adult-onset retinal dystrophy with early macular involvement and associated central visual loss in the third or fourth decade of life. Affected individuals were found to harbor disease-causing variants in DRAM2 (DNA-damage regulated autophagy modulator protein 2). Homozygosity mapping and exome sequencing in a large, consanguineous British family of Pakistani origin revealed a homozygous frameshift variant (c.140delG [p.Gly47Valfs∗3]) in nine affected family members. Sanger sequencing of DRAM2 in 322 unrelated probands with retinal dystrophy revealed one European subject with compound heterozygous DRAM2 changes (c.494G>A [p.Trp165∗] and c.131G>A [p.Ser44Asn]). Inspection of previously generated exome sequencing data in unsolved retinal dystrophy cases identified a homozygous variant in an individual of Indian origin (c.64_66del [p.Ala22del]). Independently, a gene-based case-control association study was conducted via an exome sequencing dataset of 18 phenotypically similar case subjects and 1,917 control subjects. Using a recessive model and a binomial test for rare, presumed biallelic, variants, we found DRAM2 to be the most statistically enriched gene; one subject was a homozygote (c.362A>T [p.His121Leu]) and another a compound heterozygote (c.79T>C [p.Tyr27His] and c.217_225del [p.Val73_Tyr75del]). DRAM2 encodes a transmembrane lysosomal protein thought to play a role in the initiation of autophagy. Immunohistochemical analysis showed DRAM2 localization to photoreceptor inner segments and to the apical surface of retinal pigment epithelial cells where it might be involved in the process of photoreceptor renewal and recycling to preserve visual function.

Indiana University-Purdue University Indianapolis (IUPUI) / Tendinopathy is a common degenerative pathology that is characterized by activity related pain, focal tendon tenderness, intratendinous imaging changes, and typically results in changes in the histological, mechanical, and molecular properties of the tendon. Tendinopathy is difficult to study in humans, which has contributed to limited knowledge of the pathology, and thus a lack of appropriate treatment options. However, most believe that the pathology is degenerative as a result of a combination of both extrinsic and intrinsic factors. In order to gain understanding of this pathology, animal models are required. Because each tendon is naturally exposed to different conditions, a universal model is not feasible; therefore, an appropriate animal model must be established for each tendon susceptible to degenerative changes. While acceptable models have been developed for several tendons, a reliable model for the Achilles tendon remains elusive. The purpose of this dissertation was to develop an animal model of Achilles tendinopathy by investigating the individual and combined effects of an intrinsic and extrinsic factor on the rodent Achilles tendon. Rats selectively bred for high capacity running and Sprague Dawley rats underwent uphill treadmill running (an extrinsic factor) to mechanically overload the Achilles tendon or served as cage controls. Collagenase (intrinsic factor) was injected into one Achilles tendon in each animal to intrinsically break down the tendon. There were no interactions between uphill running and collagenase injection, indicating that the influence of the two factors was independent. Uphill treadmill running alone failed to produce any pathological changes in the histological or mechanical characteristics of the Achilles tendon, but did modify molecular activity. Intratendinous collagenase injection had negative effects on the histological, mechanical, and molecular properties of the tendon. The results of this dissertation demonstrated that the combined introduction of uphill treadmill running and collagenase injection did not lead to degenerative changes consistent with human Achilles tendinopathy. Intratendiouns collagenase injection negatively influenced the tendon; however, these changes were generally transient and not influenced by mechanical overload. Future studies should consider combinations of other intrinsic and extrinsic factors in an effort to develop an animal model that replicates human Achilles tendinopathy.

The alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor (AMPAR) subunit GluR2, which regulates excitotoxicity and the inflammatory cytokine tumour necrosis factor alpha (TNFalpha) have both been implicated in motor neurone vulnerability in amyotrophic lateral sclerosis/motor neurone disease. TNFalpha has been reported to increase cell surface expression of AMPAR subunits to increase synaptic strength and enhance excitotoxicity, but whether this mechanism occurs in motor neurones is unknown. We used primary cultures of mouse motor neurones and cortical neurones to examine the interaction between TNFalpha receptor activation, GluR2 availability, AMPAR-mediated calcium entry and susceptibility to excitotoxicity. Short exposure to a physiologically relevant concentration of TNFalpha (10 ng/mL, 15 min) caused a marked redistribution of both GluR1 and GluR2 to the cell surface as determined by cell surface biotinylation and immunofluorescence. Using fura-2-acetoxymethyl ester microfluorimetry, we showed that exposure to TNFalpha caused a rapid reduction in the peak amplitude of AMPA-mediated calcium entry in a PI3-kinase and p38 kinase-dependent manner, consistent with increased insertion of GluR2-containing AMPAR into the plasma membrane. This resulted in a protection of motor neurones against kainate-induced cell death. Our data therefore, suggest that TNFalpha acts primarily as a physiological regulator of synaptic activity in motor neurones rather than a pathological drive in amyotrophic lateral sclerosis.