Optimal image sample size for corneal nerve morphometry

Purpose Arbitrary numbers of corneal confocal microscopy images have been used for analysis of corneal subbasal nerve parameters under the implicit assumption that these are a representative sample of the central corneal nerve plexus. The purpose of this study is to present a technique for quantifying the number of random central corneal images required to achieve an acceptable level of accuracy in the measurement of corneal nerve fiber length and branch density. Methods Every possible combination of 2 to 16 images (where 16 was deemed the true mean) of the central corneal subbasal nerve plexus, not overlapping by more than 20%, were assessed for nerve fiber length and branch density in 20 subjects with type 2 diabetes and varying degrees of functional nerve deficit. Mean ratios were calculated to allow comparisons between and within subjects. Results In assessing nerve branch density, eight randomly chosen images not overlapping by more than 20% produced an average that was within 30% of the true mean 95% of the time. A similar sampling strategy of five images was 13% within the true mean 80% of the time for corneal nerve fiber length. Conclusions The “sample combination analysis” presented here can be used to determine the sample size required for a desired level of accuracy of quantification of corneal subbasal nerve parameters. This technique may have applications in other biological sampling studies.

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.

A tensor-based morphometry study of genetic influences on brain structure using a new fluid registration method

We incorporated a new Riemannian fluid registration algorithm into a general MRI analysis method called tensor-based morphometry to map the heritability of brain morphology in MR images from 23 monozygotic and 23 dizygotic twin pairs. All 92 3D scans were fluidly registered to a common template. Voxelwise Jacobian determinants were computed from the deformation fields to assess local volumetric differences across subjects. Heritability maps were computed from the intraclass correlations and their significance was assessed using voxelwise permutation tests. Lobar volume heritability was also studied using the ACE genetic model. The performance of this Riemannian algorithm was compared to a more standard fluid registration algorithm: 3D maps from both registration techniques displayed similar heritability patterns throughout the brain. Power improvements were quantified by comparing the cumulative distribution functions of the p-values generated from both competing methods. The Riemannian algorithm outperformed the standard fluid registration.

Mapping the regional influence of genetics on brain structure variability - A Tensor-Based Morphometry study

Genetic and environmental factors influence brain structure and function profoundly. The search for heritable anatomical features and their influencing genes would be accelerated with detailed 3D maps showing the degree to which brain morphometry is genetically determined. As part of an MRI study that will scan 1150 twins, we applied Tensor-Based Morphometry to compute morphometric differences in 23 pairs of identical twins and 23 pairs of same-sex fraternal twins (mean age: 23.8 ± 1.8 SD years). All 92 twins' 3D brain MRI scans were nonlinearly registered to a common space using a Riemannian fluid-based warping approach to compute volumetric differences across subjects. A multi-template method was used to improve volume quantification. Vector fields driving each subject's anatomy onto the common template were analyzed to create maps of local volumetric excesses and deficits relative to the standard template. Using a new structural equation modeling method, we computed the voxelwise proportion of variance in volumes attributable to additive (A) or dominant (D) genetic factors versus shared environmental (C) or unique environmental factors (E). The method was also applied to various anatomical regions of interest (ROIs). As hypothesized, the overall volumes of the brain, basal ganglia, thalamus, and each lobe were under strong genetic control; local white matter volumes were mostly controlled by common environment. After adjusting for individual differences in overall brain scale, genetic influences were still relatively high in the corpus callosum and in early-maturing brain regions such as the occipital lobes, while environmental influences were greater in frontal brain regions that have a more protracted maturational time-course.

Muscle morphometry of the individual hamstring muscles in semi-elite AFL Players: Preliminary findings from a longitudinal study

Hamstring strains in the Australian Football League (AFL) have a high incidence (15%) and recurrence rate (34%) with lateral hamstring injuries most common (83%). Retrospective studies have found significant muscle volume asymmetries ≤23 months post hamstring injury; however examination of the association between hamstring strains and muscle asymmetry has not been investigated prospectively. This study presents baseline data from a longitudinal study focusing on individual hamstring morphometry in uninjured and injured semi-elite AFL players.

Magnetic resonance spectroscopy and neurocognitive dysfunction in obstructive sleep apnea before and after CPAP treatment

STUDY OBJECTIVES: To determine whether cerebral metabolite changes may underlie abnormalities of neurocognitive function and respiratory control in OSA. DESIGN: Observational, before and after CPAP treatment. SETTING: Two tertiary hospital research institutes. PARTICIPANTS: 30 untreated severe OSA patients, and 25 age-matched healthy controls, all males free of comorbidities, and all having had detailed structural brain analysis using voxel-based morphometry (VBM). MEASUREMENTS AND RESULTS: Single voxel bilateral hippocampal and brainstem, and multivoxel frontal metabolite concentrations were measured using magnetic resonance spectroscopy (MRS) in a high resolution (3T) scanner. Subjects also completed a battery of neurocognitive tests. Patients had repeat testing after 6 months of CPAP. There were significant differences at baseline in frontal N-acetylaspartate/choline (NAA/Cho) ratios (patients [mean (SD)] 4.56 [0.41], controls 4.92 [0.44], P = 0.001), and in hippocampal choline/creatine (Cho/Cr) ratios (0.38 [0.04] vs 0.41 [0.04], P = 0.006), (both ANCOVA, with age and premorbid IQ as covariates). No longitudinal changes were seen with treatment (n = 27, paired t tests), however the hippocampal differences were no longer significant at 6 months, and frontal NAA/Cr ratios were now also significantly different (patients 1.55 [0.13] vs control 1.65 [0.18] P = 0.01). No significant correlations were found between spectroscopy results and neurocognitive test results, but significant negative correlations were seen between arousal index and frontal NAA/Cho (r = -0.39, corrected P = 0.033) and between % total sleep time at SpO(2) < 90% and hippocampal Cho/Cr (r = -0.40, corrected P = 0.01). CONCLUSIONS: OSA patients have brain metabolite changes detected by MRS, suggestive of decreased frontal lobe neuronal viability and integrity, and decreased hippocampal membrane turnover. These regions have previously been shown to have no gross structural lesions using VBM. Little change was seen with treatment with CPAP for 6 months. No correlation of metabolite concentrations was seen with results on neurocognitive tests, but there were significant negative correlations with OSA severity as measured by severity of nocturnal hypoxemia. CITATION: O'Donoghue FJ; Wellard RM; Rochford PD; Dawson A; Barnes M; Ruehland WR; Jackson ML; Howard ME; Pierce RJ; Jackson GD. Magnetic resonance spectroscopy and neurocognitive dysfunction in obstructive sleep apnea before and after CPAP treatment.

Longitudinal assessment of neuropathy in diabetes using novel ophthalmic markers (LANDMark) : baseline findings

Purpose Over the past decade, corneal nerve morphology and corneal sensation threshold have been explored as potential surrogate markers for the evaluation of diabetic neuropathy. We present the baseline findings of a Longitudinal Assessment of Neuropathy in Diabetes using novel ophthalmic Markers (LANDMark). Methods The LANDMark Study is a 5-year, two-site, natural history (observational) study of individuals with Type 1 diabetes stratified into those with (T1W) and without (T1WO) neuropathy according to the Toronto criteria, and control subjects. All study participants undergo detailed annual assessment of neuropathy including corneal nerve parameters measured using corneal confocal microscopy and corneal sensitivity measured using non-contact corneal esthesiometry. Results 396 eligible individuals (208 in Brisbane and 188 in Manchester) were assessed: 76 T1W, 166 T1WO and 154 controls. Corneal sensation threshold (mbars) was significantly higher in T1W (1.0 ± 1.1) than T1WO (0.7 ± 0.7) and controls (0.6 ± 0.4) (P=0.002); post-hoc analysis (PHA) revealed no difference between T1WO and controls (Tukey HSD, P=0.502). Corneal nerve fiber length (mm/mm2) (CNFL) was lower in T1W (13.8 ± 6.4) than T1WO (19.1 ± 5.8) and controls (23.2 ± 6.3) (P<0.001); PHA revealed CNFL to be lower in T1W than T1WO, and lower in both of these groups than controls (P<0.001). Corneal nerve branch density (branches/mm2) (CNBD) was significantly lower in T1W (40 ± 32) than T1WO (62 ± 37) and controls (83 ± 46) (P<0.001); PHA showed CNBD was lower in T1W than T1WO, and lower in both groups than controls (P<0.001). Alcohol and cigarette consumption did not differ between groups, although age, BMI, BP, waist circumference, HbA1c, albumin-creatinine ratio, and cholesterol were slightly greater in T1W than T1WO (p<0.05). Some site differences were observed. Conclusions The LANDMark baseline findings confirm that corneal sensitivity and corneal nerve morphometry can detect differences in neuropathy status in individuals with Type 1 diabetes and healthy controls. Corneal nerve morphology is significantly abnormal even in diabetic patients ‘without neuropathy’ compared to control participants. Results of the longitudinal trial will assess the capability of these tests for monitoring change in these parameters over time as potential surrogate markers for neuropathy.

Longitudinal Assessment of Neuropathy in Diabetes using novel ophthalmic MARKers (LANDMark): Baseline findings

Purpose:Over the past decade, corneal nerve morphology and corneal sensation threshold have been explored as potential surrogate markers for the evaluation of diabetic neuropathy. We present the baseline findings of a Longitudinal Assessment of Neuropathy in Diabetes using novel ophthalmic Markers (LANDMark). Methods:The LANDMark Study is a 5-year, two-site, natural history (observational) study of individuals with Type 1 diabetes stratified into those with (T1W) and without (T1WO) neuropathy according to the Toronto criteria, and control subjects. All study participants undergo detailed annual assessment of neuropathy including corneal nerve parameters measured using corneal confocal microscopy and corneal sensitivity measured using non-contact corneal esthesiometry. Results:396 eligible individuals (208 in Brisbane and 188 in Manchester) were assessed: 76 T1W, 166 T1WO and 154 controls. Corneal sensation threshold (mbars) was significantly higher in T1W (1.0 ± 1.1) than T1WO (0.7 ± 0.7) and controls (0.6 ± 0.4) (P=0.002); post-hoc analysis (PHA) revealed no difference between T1WO and controls (Tukey HSD, P=0.502). Corneal nerve fiber length (mm/mm2) (CNFL) was lower in T1W (13.8 ± 6.4) than T1WO (19.1 ± 5.8) and controls (23.2 ± 6.3) (P<0.001); PHA revealed CNFL to be lower in T1W than T1WO, and lower in both of these groups than controls (P<0.001). Corneal nerve branch density (branches/mm2) (CNBD) was significantly lower in T1W (40 ± 32) than T1WO (62 ± 37) and controls (83 ± 46) (P<0.001); PHA showed CNBD was lower in T1W than T1WO, and lower in both groups than controls (P<0.001). Alcohol and cigarette consumption did not differ between groups, although age, BMI, BP, waist circumference, HbA1c, albumin-creatinine ratio, and cholesterol were slightly greater in T1W than T1WO (p<0.05). Some site differences were observed. Conclusions:The LANDMark baseline findings confirm that corneal sensitivity and corneal nerve morphometry can detect differences in neuropathy status in individuals with Type 1 diabetes and healthy controls. Corneal nerve morphology is significantly abnormal even in diabetic patients ‘without neuropathy’ compared to control participants. Results of the longitudinal trial will assess the capability of these tests for monitoring change in these parameters over time as potential surrogate markers for neuropathy.

Propagating excitatory potentials within the fear conditioning circuit of the lateral amygdala

In the Hebbian postulate, transiently reverberating cellular ensembles can sustain activity to facilitate temporal coincidence detection. Auditory fear conditioning is believed to be formed in the lateral amygdala (LA), by way of plasticity at auditory input synapses on principal neurons. To evaluate the contribution of LA cellular ensembles in the formation of conditioned fear memories, we investigated the LA micro-circuitry by electrophysiological and anatomical approaches. Polysynaptic field potentials evoked in the LA by stimulation of auditory thalamus(MGm/PIN) or auditory cortical (TE3) afferents were analyzed in vitro and in vivo. In vivo, two potentials were identified following stimulation of either pathway. In vitro, these multiple potentials were revealed by adding 75uM Picrotoxin or 30uM Bicuculine, with the first potential peaking at 15-20 ms, followed by two additional potentials at 20 – 25 and 30 – 35 ms, respectively. These data show single stimulation events can result in multiple synchronized excitatory events within the lateral amygdala. In order to determine underlying mechanisms of auditory signal propagation, LA principal neuron axon collateral trajectory patterns and morphology were analyzed. Neurons were found to have local axon collaterals that are topographically organized. Each axon collateral within the LA totaled 14.1 ± 2.73mm, had 29.8 ± 9.1 branch points and 1870.8 ± 1035 boutons (n=9). Electrophysiological and anatomical data show that a network of extensive axon collaterals within the LA may facilitate preservation of auditory afferent signals.

Quantification of the total neuronal structure of the fear conditioning circuit of the lateral amygdala of the rat

During Pavlovian auditory fear conditioning a previously neutral auditory stimulus (CS) gains emotional significance through pairing with a noxious unconditioned stimulus (US). These associations are believed to be formed by way of plasticity at auditory input synapses on principal neurons in the lateral nucleus of the amygdala (LA). In order to begin to understand how fear memories are stored and processed by synaptic changes in the LA, we have quantified both the entire neural number and the sub-cellular structure of LA principal neurons.We first used stereological cell counting methods on Gimsa or GABA immunostained rat brain. We identified 60,322+/-1408 neurons in the LA unilaterally (n=7). Of these 16,917+/-471 were GABA positive. The intercalated nuclei were excluded from the counts and thus GABA cells are believed to represent GABAergic interneurons. The sub-nuclei of the LA were also independently counted. We then quantified the morphometric properties of in vitro electrophysiologically identified principal neurons of the LA, corrected for shrinkage in xyz planes. The total dendritic length was 9.97+/-2.57mm, with 21+/-4 nodes (n=6). Dendritic spine density was 0.19+/-0.03 spines/um (n=6). Intra-LA axon collaterals had a bouton density of 0.1+/-0.02 boutons/um (n=5). These data begin to reveal the finite cellular and sub-cellular processing capacity of the lateral amygdala, and should facilitate efforts to understand mechanisms of plasticity in LA.

A new registration method based on Log-Euclidean tensor metrics and its application to genetic studies

In structural brain MRI, group differences or changes in brain structures can be detected using Tensor-Based Morphometry (TBM). This method consists of two steps: (1) a non-linear registration step, that aligns all of the images to a common template, and (2) a subsequent statistical analysis. The numerous registration methods that have recently been developed differ in their detection sensitivity when used for TBM, and detection power is paramount in epidemological studies or drug trials. We therefore developed a new fluid registration method that computes the mappings and performs statistics on them in a consistent way, providing a bridge between TBM registration and statistics. We used the Log-Euclidean framework to define a new regularizer that is a fluid extension of the Riemannian elasticity, which assures diffeomorphic transformations. This regularizer constrains the symmetrized Jacobian matrix, also called the deformation tensor. We applied our method to an MRI dataset from 40 fraternal and identical twins, to revealed voxelwise measures of average volumetric differences in brain structure for subjects with different degrees of genetic resemblance.

Statistically assisted fluid registration algorithm - SAFIRA

In this paper, we develop and validate a new Statistically Assisted Fluid Registration Algorithm (SAFIRA) for brain images. A non-statistical version of this algorithm was first implemented in [2] and re-formulated using Lagrangian mechanics in [3]. Here we extend this algorithm to 3D: given 3D brain images from a population, vector fields and their corresponding deformation matrices are computed in a first round of registrations using the non-statistical implementation. Covariance matrices for both the deformation matrices and the vector fields are then obtained and incorporated (separately or jointly) in the regularizing (i.e., the non-conservative Lagrangian) terms, creating four versions of the algorithm. We evaluated the accuracy of each algorithm variant using the manually labeled LPBA40 dataset, which provides us with ground truth anatomical segmentations. We also compared the power of the different algorithms using tensor-based morphometry -a technique to analyze local volumetric differences in brain structure- applied to 46 3D brain scans from healthy monozygotic twins.

A nonconservative Lagrangian framework for statistical fluid registration - SAFIRA

In this paper, we used a nonconservative Lagrangian mechanics approach to formulate a new statistical algorithm for fluid registration of 3-D brain images. This algorithm is named SAFIRA, acronym for statistically-assisted fluid image registration algorithm. A nonstatistical version of this algorithm was implemented, where the deformation was regularized by penalizing deviations from a zero rate of strain. In, the terms regularizing the deformation included the covariance of the deformation matrices Σ and the vector fields (q). Here, we used a Lagrangian framework to reformulate this algorithm, showing that the regularizing terms essentially allow nonconservative work to occur during the flow. Given 3-D brain images from a group of subjects, vector fields and their corresponding deformation matrices are computed in a first round of registrations using the nonstatistical implementation. Covariance matrices for both the deformation matrices and the vector fields are then obtained and incorporated (separately or jointly) in the nonconservative terms, creating four versions of SAFIRA. We evaluated and compared our algorithms' performance on 92 3-D brain scans from healthy monozygotic and dizygotic twins; 2-D validations are also shown for corpus callosum shapes delineated at midline in the same subjects. After preliminary tests to demonstrate each method, we compared their detection power using tensor-based morphometry (TBM), a technique to analyze local volumetric differences in brain structure. We compared the accuracy of each algorithm variant using various statistical metrics derived from the images and deformation fields. All these tests were also run with a traditional fluid method, which has been quite widely used in TBM studies. The versions incorporating vector-based empirical statistics on brain variation were consistently more accurate than their counterparts, when used for automated volumetric quantification in new brain images. This suggests the advantages of this approach for large-scale neuroimaging studies.

Improving fluid registration through white matter segmentation in a twin study design

Robust and automatic non-rigid registration depends on many parameters that have not yet been systematically explored. Here we determined how tissue classification influences non-linear fluid registration of brain MRI. Twin data is ideal for studying this question, as volumetric correlations between corresponding brain regions that are under genetic control should be higher in monozygotic twins (MZ) who share 100% of their genes when compared to dizygotic twins (DZ) who share half their genes on average. When these substructure volumes are quantified using tensor-based morphometry, improved registration can be defined based on which method gives higher MZ twin correlations when compared to DZs, as registration errors tend to deplete these correlations. In a study of 92 subjects, higher effect sizes were found in cumulative distribution functions derived from statistical maps when performing tissue classification before fluid registration, versus fluidly registering the raw images. This gives empirical evidence in favor of pre-segmenting images for tensor-based morphometry.