Expression of Potato virus Y cytoplasmic inclusion protein in tobacco results in disorganization of parenchyma cells, distortion of epidermal cells, and induces mitochondrial and chloroplast abnormalities, formation of membrane whorls and atypical lipid accumulation

Infection of plant cells by potyviruses induces the formation of cytoplasmic inclusions ranging in size from 200 to 1000 nm. To determine if the ability to form these ordered, insoluble structures is intrinsic to the potyviral cytoplasmic inclusion protein, we have expressed the cytoplasmic inclusion protein from Potato virus Y in tobacco under the control of the chrysanthemum ribulose-1,5-bisphosphate carboxylase small subunit promoter, a highly active, green tissue promoter. No cytoplasmic inclusions were observed in the leaves of transgenic tobacco using transmission electron microscopy, despite being able to clearly visualize these inclusions in Potato virus Y infected tobacco leaves under the same conditions. However, we did observe a wide range of tissue and sub-cellular abnormalities associated with the expression of the Potato virus Y cytoplasmic inclusion protein. These changes included the disruption of normal cell morphology and organization in leaves, mitochondrial and chloroplast internal reorganization, and the formation of atypical lipid accumulations. Despite these significant structural changes, however, transgenic tobacco plants were viable and the results are discussed in the context of potyviral cytoplasmic inclusion protein function.

Myoinositol Abnormalities in Temporal Lobe Epilepsy

Purpose: This study used magnetic resonance spectroscopy (MRS) to examine metabolite abnormalities in the temporal and frontal lobe of patients with temporal lobe epilepsy (TLE) of differing severity. Methods: We investigated myoinositol in TLE by using short-echo MRS in 34 TLE patients [26 late onset (LO-TLE), eight hippocampal sclerosis (HS-TLE)], and 16 controls. Single-voxel short-echo (35 ms) MR spectra of temporal and frontal lobes were acquired at 1.5 T and analyzed by using LCModel. Results: The temporal lobe ipsilateral to seizure origin in HS-TLE, but not LO-TLE, had reduced N-acetylaspartate (NA) and elevated myoinositol (MI; HS-TLE NA, 7.8 ± 1.9 mM, control NA, 9.2 ± 1.3 mM; p < 0.05; HS-TLE MI, 6.1 ± 1.6 mM, control mI 4.9 ± 0.8 mM, p< 0.05). Frontal lobe MI was low in both patient groups (LO-TLE, 4.3 ± 0.8 mM; p < 0.05; HS-TLE, 3.6 ±.05 mM; p < 0.001; controls, 4.8 ± 0.5 mM). Ipsilateral frontal lobes had lower MI (3.8 ± 0.7 mM; p < 0.01) than contralateral frontal lobes (4.3 ± 0.8 mM; p < 0.05). Conclusions: MI changes may distinguish between the seizure focus, where MI is increased, and areas of seizure spread where MI is decreased.

Morphometry and abnormalities of the feet of Kaimanawa feral horses in New Zealand

Objective: The present study investigated the foot health of the Kaimanawa feral horse population and tested the hypotheses that horses would have a large range of foot morphology and that the incidence of foot abnormality would be significantly high. Procedures: Abnormality was defined as a variation from what the two veterinarian assessors considered as optimal morphology and which was considered to impact negatively on the structure and/or function of the foot. Fifteen morphometric variables were measured on four calibrated photographic views of all four feet of 20 adult Kaimanawa feral horses. Four morphometric variables were measured from the lateromedial radiographs of the left forefoot of each horse. In addition, the study identified the incidence of gross abnormality observed on the photographs and radiographs of all 80 feet. Results: There was a large variation between horses in the morphometric dimensions, indicating an inconsistent foot type. Mean hoof variables were outside the normal range recommended by veterinarians and hoof care providers; 35% of all feet had a long toe conformation and 15% had a mediolateral imbalance. Abnormalities included lateral (85% of horses) and dorsal (90% of horses) wall flares, presence of laminar rings (80% of horses) and bull-nose tip of the distal phalanx (75% of horses). Both hypotheses were therefore accepted. Conclusions: The Kaimanawa feral horse population demonstrated a broad range of foot abnormalities and we propose that one reason for the questionable foot health and conformation is lack of abrasive wearing by the environment. In comparison with other feral horse populations in Australia and America there may be less pressure on the natural selection of the foot of the Kaimanawa horses by the forgiving environment of the Kaimanawa Ranges. Contrary to popular belief, the feral horse foot type should not be used as an ideal model for the domestic horse foot.

RAD51-associated Protein 1 (RAD51AP1) Interacts with the Meiotic Recombinase DMC1 through a Conserved Motif

Homologous recombination (HR) reactions mediated by the RAD51 recombinase are essential for DNA and replication fork repair, genome stability, and tumor suppression. RAD51-associated protein 1 (RAD51AP1) is an important HR factor that associates with and stimulates the recombinase activity of RAD51. We have recently shown that RAD51AP1 also partners with the meiotic recombinase DMC1, displaying isoform-specific interactions with DMC1. Here, we have characterized the DMC1 interaction site in RAD51AP1 by a series of truncations and point mutations to uncover a highly conserved WVPP motif critical for DMC1 interaction but dispensable for RAD51 association. This RAD51AP1 motif is reminiscent of the FVPP motif in the tumor suppressor protein BRCA2 that mediates DMC1 interaction. These results further implicate RAD51AP1 in meiotic HR via RAD51 and DMC1.

Molecular basis for enhancement of the meiotic DMC1 recombinase by RAD51 associated protein 1 (RAD51AP1)

Homologous recombination is needed for meiotic chromosome segregation, genome maintenance, and tumor suppression. RAD51AP1 (RAD51 associated protein 1) has been shown to interact with and enhance the recombinase activity of RAD51. Accordingly, genetic ablation of RAD51AP1 leads to enhanced sensitivity to and also chromosome aberrations upon DNA damage, demonstrating a role for RAD51AP1 in mitotic homologous recombination. Here we show physical association of RAD51AP1 with the meiosis-specific recombinase DMC1 and a stimulatory effect of RAD51AP1 on the DMC1-mediated D-loop reaction. Mechanistic studies have revealed that RAD51AP1 enhances the ability of the DMC1 presynaptic filament to capture the duplex-DNA partner and to assemble the synaptic complex, in which the recombining DNA strands are homologously aligned. We also provide evidence that functional cooperation is dependent on complex formation between DMC1 and RAD51AP1 and that distinct epitopes in RAD51AP1 mediate interactions with RAD51 and DMC1. Finally, we show that RAD51AP1 is expressed in mouse testes, and that RAD51AP1 foci colocalize with a subset of DMC1 foci in spermatocytes. These results suggest that RAD51AP1 also serves an important role in meiotic homologous recombination.

Senataxin plays an essential role with DNA damage response proteins in meiotic recombination and gene silencing

Senataxin, mutated in the human genetic disorder ataxia with oculomotor apraxia type 2 (AOA2), plays an important role in maintaining genome integrity by coordination of transcription, DNA replication, and the DNA damage response. We demonstrate that senataxin is essential for spermatogenesis and that it functions at two stages in meiosis during crossing-over in homologous recombination and in meiotic sex chromosome inactivation (MSCI). Disruption of the Setx gene caused persistence of DNA double-strand breaks, a defect in disassembly of Rad51 filaments, accumulation of DNA:RNA hybrids (R-loops), and ultimately a failure of crossing-over. Senataxin localised to the XY body in a Brca1-dependent manner, and in its absence there was incomplete localisation of DNA damage response proteins to the XY chromosomes and ATR was retained on the axial elements of these chromosomes, failing to diffuse out into chromatin. Furthermore persistence of RNA polymerase II activity, altered ubH2A distribution, and abnormal XY-linked gene expression in Setx⁻/⁻ revealed an essential role for senataxin in MSCI. These data support key roles for senataxin in coordinating meiotic crossing-over with transcription and in gene silencing to protect the integrity of the genome.

Senataxin controls meiotic silencing through ATR activation and chromatin remodeling

Senataxin, defective in ataxia oculomotor apraxia type 2, protects the genome by facilitating the resolution of RNA–DNA hybrids (R-loops) and other aspects of RNA processing. Disruption of this gene in mice causes failure of meiotic recombination and defective meiotic sex chromosome inactivation, leading to male infertility. Here we provide evidence that the disruption of Setx leads to reduced SUMOylation and disruption of protein localization across the XY body during meiosis. We demonstrate that senataxin and other DNA damage repair proteins, including ataxia telangiectasia and Rad3-related protein-interacting partner, are SUMOylated, and a marked downregulation of both ataxia telangiectasia and Rad3-related protein-interacting partner and TopBP1 leading to defective activation and signaling through ataxia telangiectasia and Rad3-related protein occurs in the absence of senataxin. Furthermore, chromodomain helicase DNA-binding protein 4, a component of the nucleosome remodeling and deacetylase chromatin remodeler that interacts with both ataxia telangiectasia and Rad3-related protein and senataxin was not recruited efficiently to the XY body, triggering altered histone acetylation and chromatin conformation in Setx−/− pachytene-staged spermatocytes. These results demonstrate that senataxin has a critical role in ataxia telangiectasia and Rad3-related protein- and chromodomain helicase DNA-binding protein 4-mediated transcriptional silencing and chromatin remodeling during meiosis providing greater insight into its critical role in gene regulation to protect against neurodegeneration.

A 1H-MRS investigation of the medial temporal lobe in antipsychotic-naïve and early-treated first episode psychosis

Schizophrenia is associated with significant brain abnormalities, including changes in brain metabolites as measured by proton magnetic resonance spectroscopy (MRS). What remains unclear is the extent to which these changes are a consequence of the emergence of psychotic disorders or the result of treatment with antipsychotic medication. We assessed 34 patients with first episode psychosis (15 antipsychotic naïve) and 19 age- and gender-matched controls using short-echo MRS in the medial temporal lobe bilaterally. Overall, there were no differences in any metabolite, regardless of treatment status. However, when the analysis was limited to patients with a diagnosis of schizophrenia, schizophreniform or schizoaffective disorder, significant elevations of creatine/phosphocreatine (Cr/PCr) and myo-inositol (mI) were found in the treated group. These data indicate a relative absence of temporal lobe metabolic abnormalities in first episode psychosis, but suggest that some treatment-related changes in mI might be apparent in patients with schizophrenia-spectrum diagnoses. Seemingly illness-related Cr/PCr elevations were also specific to the diagnosis of schizophrenia-spectrum disorder and seem worthy of future study.

The manipulation of apoptosis-related genes to generate resistance to Fusarium wilt and water stress in banana

Bananas are susceptible to a diverse range of biotic and abiotic stresses, many of which cause serious production constraints worldwide. One of the most destructive banana diseases is Fusarium wilt caused by the soil-borne fungus, Fusarium oxysporum f. sp. cubense (Foc). No effective control strategy currently exists for this disease which threatens global banana production. Although disease resistance exists in some wild bananas, attempts to introduce resistance into commercially acceptable bananas by conventional breeding have been hampered by low fertility, long generation times and association of poor agronomical traits with resistance genes. With the advent of reliable banana transformation protocols, molecular breeding is now regarded as a viable alternative strategy to generate disease-resistant banana plants. Recently, a novel strategy involving the expression of anti-apoptosis genes in plants was shown to result in resistance against several necrotrophic fungi. Further, the transgenic plants showed increased resistance to a range of abiotic stresses. In this thesis, the use of anti-apoptosis genes to generate transgenic banana plants with resistance to Fusarium wilt was investigated. Since water stress is an important abiotic constraint to banana production, the resistance of the transgenic plants to water stress was also examined. Embryogenic cell suspensions (ECS) of two commercially important banana cultivars, Grand Naine (GN) and Lady Finger (LF), were transformed using Agrobacterium with the anti-apoptosis genes, Bcl-xL, Bcl-xL G138A, Ced-9 and Bcl- 2 3’ UTR. An interesting, and potentially important, outcome was that the use of anti-apoptosis genes resulted in up to a 50-fold increase in Agrobacterium-mediated transformation efficiency of both LF and GN cells over vector controls. Regenerated plants were subjected to a complete molecular characterisation in order to detect the presence of the transgene (PCR), transcript (RT-PCR) and gene product (Western blot) and to determine the gene copy number (Southern blot). A total of 36 independently-transformed GN lines (8 x Bcl-xL, 5 x Bcl-xL G138A, 15 x Ced-9 and 8 x Bcl-2 3’ UTR) and 41 independently-transformed LF lines (8 x Bcl-xL, 7 x BclxL G138A, 13 x Ced-9 and 13 x Bcl-2 3’ UTR) were identified. The 41 transgenic LF lines were multiplied and clones from each line were acclimatised and grown under glasshouse conditions for 8 weeks to allow monitoring for phenotypic abnormalities. Plants derived from 3 x Bcl-xL, 2 x Ced-9 and 5 x Bcl-2 3’ UTR lines displayed a variety of aberrant phenotypes. However, all but one of these abnormalities were off-types commonly observed in tissue-cultured, non-transgenic banana plants and were therefore unlikely to be transgene-related. Prior to determining the resistance of the transgenic plants to Foc race 1, the apoptotic effects of the fungus on both wild-type and Bcl-2 3’ UTR-transgenic LF banana cells were investigated using rapid in vitro root assays. The results from these assays showed that apoptotic-like cell death was elicited in wild-type banana root cells as early as 6 hours post-exposure to fungal spores. In contrast, these effects were attenuated in the root cells of Bcl-2 3’ UTR-transgenic lines that were exposed to fungal spores. Thirty eight of the 41 transgenic LF lines were subsequently assessed for resistance to Foc race 1 in small-plant glasshouse bioassays. To overcome inconsistencies in rating the internal (vascular discolouration) disease symptoms, a MatLab-based computer program was developed to accurately and reliably assess the level of vascular discolouration in banana corms. Of the transgenic LF banana lines challenged with Foc race 1, 2 x Bcl-xL, 3 x Ced-9, 2 x Bcl-2 3’ UTR and 1 x Bcl-xL G138A-transgenic line were found to show significantly less external and internal symptoms than wild-type LF banana plants used as susceptible controls at 12 weeks post-inoculation. Of these lines, Bcl-2 3’ UTR-transgenic line #6 appeared most resistant, displaying very mild symptoms similar to the wild-type Cavendish banana plants that were included as resistant controls. This line remained resistant for up to 23 weeks post-inoculation. Since anti-apoptosis genes have been shown to confer resistance to various abiotic stresses in other crops, the ability of these genes to confer resistance against water stress in banana was also investigated. Clonal plants derived from each of the 38 transgenic LF banana plants were subjected to water stress for a total of 32 days. Several different lines of transgenic plants transformed with either Bcl-xL, Bcl-xL G138A, Ced-9 or Bcl-2 3’ UTR showed a delay in visual water stress symptoms compared with the wild-type control plants. These plants all began producing new growth from the pseudostem following daily rewatering for one month. In an attempt to determine whether the protective effect of anti-apoptosis genes in transgenic banana plants was linked with reactive oxygen species (ROS)-associated programmed cell death (PCD), the effect of the chloroplast-targeting, ROS-inducing herbicide, Paraquat, on wild-type and transgenic LF was investigated. When leaf discs from wild-type LF banana plants were exposed to 10 ìM Paraquat, complete decolourisation occurred after 48 hours which was confirmed to be associated with cell death and ROS production by trypan blue and 3,3-diaminobenzidine (DAB) staining, respectively. When leaf discs from the transgenic lines were exposed to Paraquat, those derived from some lines showed a delay in decolourisation, suggesting only a weak protective effect from the transgenes. Finally, the protective effect of anti-apoptosis genes against juglone, a ROS-inducing phytotoxin produced by the causal agent of black Sigatoka, Mycosphaerella fijiensis, was investigated. When leaf discs from wild-type LF banana plants were exposed to 25 ppm juglone, complete decolourisation occurred after 48 hours which was again confirmed to be associated with cell death and ROS production by trypan blue and DAB staining, respectively. Further, TdT-mediated dUTP nick-end labelling (TUNEL) assays on these discs suggested that the cell death was apoptotic. When leaf discs from the transgenic lines were exposed to juglone, discs from some lines showed a clear delay in decolourisation, suggesting a protective effect. Whether these plants are resistant to black Sigatoka is unknown and will require future glasshouse and field trials. The work presented in this thesis provides the first report of the use of anti-apoptosis genes as a strategy to confer resistance to Fusarium wilt and water stress in a nongraminaceous monocot, banana. Such a strategy may be exploited to generate resistance to necrotrophic pathogens and abiotic stresses in other economically important crop plants.

Early objective detection of lymphoedema

The aetiology of secondary lymphoedema seems to be multifactorial, with acquired abnormalities as well as pre-existing conditions being contributory factors. Many characteristics bear inconsistent relationships to lymphoedema risk, and the few that are consistently associated with an increased risk of developing the condition, do not alone distinguish the at-risk population. Further, our current prevention and management recommendations are not backed by strong evidence. Consequently, there remains much to be learned about who gets it, how can it be prevented and how can we best treat it. Nonetheless, it is clear that lymphoedema is associated with adverse side effects, which have a profound impact on daily life, and that preliminary evidence suggests that early detection may lead to more effective treatment and lack of treatment may lead to progression. These represent important reasons as to why lymphoedema deserves clinical attention. However, several pragmatic issues must be considered when discussing whether a routine objective measure of lymphoedema could be integrated among the standard clinical care of those undertaking treatment for cancers known to be associated with the development of lymphoedema.

Rare chromosome disorders and their developmental consequences

Professionals working in disability services often encounter clients who have chromosome disorders such as Williams, Angelman or Down syndromes. As chromosome testing becomes increasingly sophisticated, however, more people are being diagnosed with very rare chromosome disorders that are identified not by a syndrome name, but rather by a description of the number, size and shape of their chromosomes (called the karyotype) or by a report of chromosome losses and gains detected through an advanced process known as microarray-based comparative genomic hybridisation (array CGH). For practitioners who work with individuals with rare chromosome disorders and their families, a basic level of knowledge about the evolving field of genetics, as well as specific knowledge about chromosome abnormalities, is essential since they must be able to demonstrate their knowledge and skills to clients (Simic & Turk, 2004). In addition, knowledge about the developmental consequences of various rare chromosome disorders is important for guiding prognoses, expectations, decisions and interventions. The current article provides information that aims to help practitioners work more effectively with this population. It begins by presenting essential information about chromosomes and their numerical and structural abnormalities and then considers the developmental consequences of rare chromosome disorders through a critical review of relevant literature.

Role of exercise in the prevention and management of lymphedema after breast cancer

Swelling or lymphedema of the limb, trunk, or breast is considered the most problematic and dreaded concern after treatment for breast cancer and has significant physical, psychological, and social ramifications. Conservative incidence estimates suggest that 20%-30% of breast cancer survivors will experience lymphedema, with the majority of cases (up to 80%) occurring within the first year after surgery. The etiology of secondary lymphedema seems to be multifactorial, with acquired abnormalities as well as preexisting conditions being contributory factors.

Magnetic resonance spectroscopy in seizure disorders

Key points • The clinical aims of MR spectroscopy (MRS) in seizure disorders are to help identify, localize and characterize epileptogenic foci. • Lateralizing MRS abnormalities in temporal lobe epilepsy (TLE) may be used clinically in combination with structural and T2 MRI measurements together with other techniques such as EEG, PET and SPECT. • Characteristic metabolite abnormalities are decreased N-acetylaspartate (NAA) with increased choline (Cho) and myoinositol (mI) (short-echo time). • Contralateral metabolite abnormalities are frequently seen in TLE, but are of uncertain significance. • In extra-temporal epilepsy, metabolite abnormalities may be seen where MR imaging (MRI) is normal; but may not be sufficiently localized to be useful clinically. • MRS may help to characterize epileptogenic lesions visible on MRI (aggressive vs. indolent neoplastic, dysplasia). • Spectral editing techniques are required to evaluate specific epilepsy-relevant metabolites (e.g. -aminobutyric acid (GABA)), which may be useful in drug development and evaluation. • MRS with phosphorus (31P) and other nuclei probe metabolism of epilepsy, but are less useful clinically. • There is potential for assessing the of drug mode of action and efficacy through 13C carbon metabolite measurements, while changes in sodium homeostasis resulting from seizure activity may be detected with 23Na MRS.

Swept frequency biompedance analysis for the determination of body water compartments

Bioelectrical impedance analysis, (BIA), is a method of body composition analysis first investigated in 1962 which has recently received much attention by a number of research groups. The reasons for this recent interest are its advantages, (viz: inexpensive, non-invasive and portable) and also the increasing interest in the diagnostic value of body composition analysis. The concept utilised by BIA to predict body water volumes is the proportional relationship for a simple cylindrical conductor, (volume oc length2/resistance), which allows the volume to be predicted from the measured resistance and length. Most of the research to date has measured the body's resistance to the passage of a 50· kHz AC current to predict total body water, (TBW). Several research groups have investigated the application of AC currents at lower frequencies, (eg 5 kHz), to predict extracellular water, (ECW). However all research to date using BIA to predict body water volumes has used the impedance measured at a discrete frequency or frequencies. This thesis investigates the variation of impedance and phase of biological systems over a range of frequencies and describes the development of a swept frequency bioimpedance meter which measures impedance and phase at 496 frequencies ranging from 4 kHz to 1 MHz. The impedance of any biological system varies with the frequency of the applied current. The graph of reactance vs resistance yields a circular arc with the resistance decreasing with increasing frequency and reactance increasing from zero to a maximum then decreasing to zero. Computer programs were written to analyse the measured impedance spectrum and determine the impedance, Zc, at the characteristic frequency, (the frequency at which the reactance is a maximum). The fitted locus of the measured data was extrapolated to determine the resistance, Ro, at zero frequency; a value that cannot be measured directly using surface electrodes. The explanation of the theoretical basis for selecting these impedance values (Zc and Ro), to predict TBW and ECW is presented. Studies were conducted on a group of normal healthy animals, (n=42), in which TBW and ECW were determined by the gold standard of isotope dilution. The prediction quotients L2/Zc and L2/Ro, (L=length), yielded standard errors of 4.2% and 3.2% respectively, and were found to be significantly better than previously reported, empirically determined prediction quotients derived from measurements at a single frequency. The prediction equations established in this group of normal healthy animals were applied to a group of animals with abnormally low fluid levels, (n=20), and also to a group with an abnormal balance of extra-cellular to intracellular fluids, (n=20). In both cases the equations using L2/Zc and L2/Ro accurately and precisely predicted TBW and ECW. This demonstrated that the technique developed using multiple frequency bioelectrical impedance analysis, (MFBIA), can accurately predict both TBW and ECW in both normal and abnormal animals, (with standard errors of the estimate of 6% and 3% for TBW and ECW respectively). Isotope dilution techniques were used to determine TBW and ECW in a group of 60 healthy human subjects, (male. and female, aged between 18 and 45). Whole body impedance measurements were recorded on each subject using the MFBIA technique and the correlations between body water volumes, (TBW and ECW), and heighe/impedance, (for all measured frequencies), were compared. The prediction quotients H2/Zc and H2/Ro, (H=height), again yielded the highest correlation with TBW and ECW respectively with corresponding standard errors of 5.2% and 10%. The values of the correlation coefficients obtained in this study were very similar to those recently reported by others. It was also observed that in healthy human subjects the impedance measured at virtually any frequency yielded correlations not significantly different from those obtained from the MFBIA quotients. This phenomenon has been reported by other research groups and emphasises the need to validate the technique by investigating its application in one or more groups with abnormalities in fluid levels. The clinical application of MFBIA was trialled and its capability of detecting lymphoedema, (an excess of extracellular fluid), was investigated. The MFBIA technique was demonstrated to be significantly more sensitive, (P<.05), in detecting lymphoedema than the current technique of circumferential measurements. MFBIA was also shown to provide valuable information describing the changes in the quantity of muscle mass of the patient during the course of the treatment. The determination of body composition, (viz TBW and ECW), by MFBIA has been shown to be a significant improvement on previous bioelectrical impedance techniques. The merit of the MFBIA technique is evidenced in its accurate, precise and valid application in animal groups with a wide variation in body fluid volumes and balances. The multiple frequency bioelectrical impedance analysis technique developed in this study provides accurate and precise estimates of body composition, (viz TBW and ECW), regardless of the individual's state of health.

Corneal confocal microscopy : a novel means to detect nerve fibre damage in idiopathic small fibre neuropathy

Patients with idiopathic small fibre neuropathy (ISFN) have been shown to have significant intraepidermal nerve fibre loss and an increased prevalence of impaired glucose tolerance (IGT). It has been suggested that the dysglycemia of IGT and additional metabolic risk factors may contribute to small nerve fibre damage in these patients. Twenty-five patients with ISFN and 12 aged-matched control subjects underwent a detailed evaluation of neuropathic symptoms, neurological deficits (Neuropathy deficit score (NDS); Nerve Conduction Studies (NCS); Quantitative Sensory Testing (QST) and Corneal Confocal Microscopy (CCM)) to quantify small nerve fibre pathology. Eight (32%) patients had IGT. Whilst all patients with ISFN had significant neuropathic symptoms, NDS, NCS and QST except for warm thresholds were normal. Corneal sensitivity was reduced and CCM demonstrated a significant reduction in corneal nerve fibre density (NFD) (Pb0.0001), nerve branch density (NBD) (Pb0.0001), nerve fibre length (NFL) (Pb0.0001) and an increase in nerve fibre tortuosity (NFT) (Pb0.0001). However these parameters did not differ between ISFN patients with and without IGT, nor did they correlate with BMI, lipids and blood pressure. Corneal confocal microscopy provides a sensitive non-invasive means to detect small nerve fibre damage in patients with ISFN and metabolic abnormalities do not relate to nerve damage.