Automated detection of squint as a sensitive assay of sex-dependent calcitonin gene-related peptide and amylin-induced pain in mice.

ABSTRACT: We developed an automated squint assay using both black C57BL/6J and white CD1 mice to measure the interpalpebral fissure area between the upper and lower eyelids as an objective quantification of pain. The automated software detected a squint response to the commonly used nociceptive stimulus formalin in C57BL/6J mice. After this validation, we used the automated assay to detect a dose-dependent squint response to a migraine trigger, the neuropeptide calcitonin gene-related peptide, including a response in female mice at a dose below detection by the manual grimace scale. Finally, we found that the calcitonin gene-related peptide amylin induced squinting behavior in female mice, but not males. These data demonstrate that an automated squint assay can be used as an objective, real-time, continuous-scale measure of pain that provides higher precision and real-time analysis compared with manual grimace assessments.

Blast-Mediated Traumatic Brain Injury Exacerbates Retinal Damage and Amyloidosis in the APPswePSENd19e Mouse Model of Alzheimer's Disease.

Purpose: Traumatic brain injury (TBI) is a risk factor for developing chronic neurodegenerative conditions including Alzheimer's disease (AD). The purpose of this study was to examine chronic effects of blast TBI on retinal ganglion cells (RGC), optic nerve, and brain amyloid load in a mouse model of AD amyloidosis. Methods: Transgenic (TG) double-mutant APPswePSENd19e (APP/PS1) mice and nontransgenic (Non-TG) littermates were exposed to a single blast TBI (20 psi) at age 2 to 3 months. RGC cell structure and function was evaluated 2 months later (average age at endpoint = 4.5 months) using pattern electroretinogram (PERG), optical coherence tomography (OCT), and the chromatic pupil light reflex (cPLR), followed by histologic analysis of retina, optic nerve, and brain amyloid pathology. Results: APP/PS1 mice exposed to blast TBI (TG-Blast) had significantly lower PERG and cPLR responses 2 months after injury compared to preblast values and compared to sham groups of APP/PS1 (TG-Sham) and nontransgenic (Non-TG-Sham) mice as well as nontransgenic blast-exposed mice (Non-TG-Blast). The TG-Blast group also had significantly thinner RGC complex and more optic nerve damage compared to all groups. No amyloid-ß (Aß) deposits were detected in retinas of APP/PS1 mice; however, increased amyloid precursor protein (APP)/Aß-immunoreactivity was seen in TG-Blast compared to TG-Sham mice, particularly near blood vessels. TG-Blast and TG-Sham groups exhibited high variability in pathology severity, with a strong, but not statistically significant, trend for greater cerebral cortical Aß plaque load in the TG-Blast compared to TG-Sham group. Conclusions: When combined with a genetic susceptibility for developing amyloidosis of AD, blast TBI exposure leads to earlier RGC and optic nerve damage associated with modest but detectable increase in cerebral cortical Aß pathology. These findings suggest that genetic risk factors for AD may increase the sensitivity of the retina to blast-mediated damage.

Peripherally administered calcitonin gene-related peptide induces spontaneous pain in mice: implications for migraine.

Migraine is the third most common disease in the world (behind dental caries and tension-type headache) with an estimated global prevalence of 15%, yet its etiology remains poorly understood. Recent clinical trials have heralded the potential of therapeutic antibodies that block the actions of the neuropeptide calcitonin gene-related peptide (CGRP) or its receptor to prevent migraine. Calcitonin gene-related peptide is believed to contribute to trigeminal nerve hypersensitivity and photosensitivity in migraine, but a direct role in pain associated with migraine has not been established. In this study, we report that peripherally administered CGRP can act in a light-independent manner to produce spontaneous pain in mice that is manifested as a facial grimace. As an objective validation of the orbital tightening action unit of the grimace response, we developed a squint assay using a video-based measurement of the eyelid fissure, which confirmed a significant squint response after CGRP injection, both in complete darkness and very bright light. These indicators of discomfort were completely blocked by preadministration of a monoclonal anti-CGRP-blocking antibody. However, the nonsteroidal anti-inflammatory drug meloxicam failed to block the effect of CGRP. Interestingly, an apparent sex-specific response to treatment was observed with the antimigraine drug sumatriptan partially blocking the CGRP response in male, but not female mice. These results demonstrate that CGRP can induce spontaneous pain, even in the absence of light, and that the squint response provides an objective biomarker for CGRP-induced pain that is translatable to humans.

Visual Fixation Instability in Multiple Sclerosis Measured Using SLO-OCT.

Purpose: Precise measurements of visual fixation and its instability were recorded during optical coherence tomography (OCT) as a marker of neural network dysfunction in multiple sclerosis (MS), which could be used to monitor disease progression or response to treatment. Methods: A total of 16 MS patients and 26 normal subjects underwent 30 seconds of scanning laser ophthalmoscope (SLO)-based eye tracking during OCT scanning of retinal layer thickness. Study groups consisted of normal eyes, MS eyes without prior optic neuritis (MS wo ON), and MS eyes with prior optic neuritis (MS + ON). Kernel density estimation quantified fixation instability from the distribution of fixation points on the retina. In MS wo ON eyes, fixation instability was compared to other measures of visual and neurologic function. Results: Fixation instability was increased in MS wo ON eyes (0.062 deg2) compared to normal eyes (0.030 deg2, P = 0.015). A further increase was seen for MS + ON eyes (0.11 deg2) compared to MS wo ON (P = 0.04) and normal (P = 0.006) eyes. Fixation instability correlated weakly with ganglion cell layer (GCL) volume and showed no correlation with low-contrast letter acuity, EDSS score, or SDMT score. Conclusions: Fixation instability reflects the integrity of a widespread neural network germane to visual processing and ocular motor control, and is disturbed in MS. Further study of visual fixation, including the contribution of microsaccades to fixation instability, may provide insight into the localization of fixation abnormalities in MS and introduce innovative and easily measured outcomes for monitoring progression and treatment response.

Pupillary response abnormalities in depressive disorders.

Depressive disorders lack objective physiological measurements to characterize the affected population and facilitate study of relevant mechanisms. The melanopsin-mediated light signaling pathway may contribute to seasonal variation and can be measured non-invasively by pupillometry. We prospectively studied changes in melanopsin-mediated pupillary constriction in 19 participants with major depressive disorder (MDD) and 10 control across the summer and winter solstices. The melanopsin-mediated response, as measured by the pupil's sustained constriction six s after a high intensity blue light stimulus, was marginally attenuated in those with MDD relative to controls (p=0.071). The participants with MDD unexpectedly showed a significantly reduced transient pupillary response to low intensity red (p=0.011) and blue light (p=0.013), but not high intensity red and blue light. Sustained pupillary constriction in response to high intensity blue light was more pronounced with increasing daylight hours (p=0.037) and was more strongly related to objectively measured versus estimated light exposure. Melanopsin-mediated impairments in pupil response may serve as a biological marker for vulnerability to depression in low light conditions. Assessment of these and other responses to light stimuli, such as response to low intensity light, may be useful for the study of the neurobiology of MDD and related mood disorders.

The Pattern of Visual Fixation Eccentricity and Instability in Optic Neuropathy and Its Spatial Relationship to Retinal Ganglion Cell Layer Thickness.

PURPOSE: The purpose of this study was to assess whether clinically useful measures of fixation instability and eccentricity can be derived from retinal tracking data obtained during optical coherence tomography (OCT) in patients with optic neuropathy (ON) and to develop a method for relating fixation to the retinal ganglion cell complex (GCC) thickness. METHODS: Twenty-nine patients with ON underwent macular volume OCT with 30 seconds of confocal scanning laser ophthalmoscope (cSLO)-based eye tracking during fixation. Kernel density estimation quantified fixation instability and fixation eccentricity from the distribution of fixation points on the retina. Preferred ganglion cell layer loci (PGCL) and their relationship to the GCC thickness map were derived, accounting for radial displacement of retinal ganglion cell soma from their corresponding cones. RESULTS: Fixation instability was increased in ON eyes (0.21 deg2) compared with normal eyes (0.06982 deg2; P < 0.001), and fixation eccentricity was increased in ON eyes (0.48°) compared with normal eyes (0.24°; P = 0.03). Fixation instability and eccentricity each correlated moderately with logMAR acuity and were highly predictive of central visual field loss. Twenty-six of 35 ON eyes had PGCL skewed toward local maxima of the GCC thickness map. Patients with bilateral dense central scotomas had PGCL in homonymous retinal locations with respect to the fovea. CONCLUSIONS: Fixation instability and eccentricity measures obtained during cSLO-OCT assess the function of perifoveal retinal elements and predict central visual field loss in patients with ON. A model relating fixation to the GCC thickness map offers a method to assess the structure-function relationship between fixation and areas of preserved GCC in patients with ON.

Corneal Sensitivity to Hyperosmolar Eye Drops: A Novel Behavioral Assay to Assess Diabetic Peripheral Neuropathy.

PURPOSE: Diagnosis of peripheral neuropathy (PN), which affects approximately 50% of the diabetic population, is subjective, with many patients seeking a diagnosis only after presenting with symptoms. Recently, in vivo confocal microscopy of subepithelial corneal nerve density has been promoted as a surrogate marker for early detection of PN, but imaging of corneal nerves requires sophisticated instrumentation, expertise in confocal imaging, cooperative patients, and automated analysis tools to derive corneal nerve density. As an alternative, we developed a simple screening method that is based on the sensitivity of corneal nerves to cause reflex eyelid squinting in response to hyperosmolar eye drops. METHODS: Eyes of control and type 2 diabetic rats were given an eye drop of a 290- to 900-mOsm solution, and the ocular response was video recorded. Other neuropathic end points including nerve conduction velocity and subepithelial cornea nerve density were determined. RESULTS: Motor and sensory nerve conduction velocity and total nerve fiber length of corneal nerves in the subepithelial layer were significantly decreased in diabetic rats. Applying the hyperosmotic solutions to the ocular surface caused an osmolarity-dependent increase in squinting of the treated eye in control rats. Squinting was almost totally blocked by preapplication of proparacaine or N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-1(2H)-carbox-amide, a transient receptor potential melastatin-8 channel blocker. Squinting in response to the 900-mOsm solution was significantly reduced in diabetic rats. CONCLUSIONS: Preclinical studies show that evaluation of corneal sensitivity may be an alternative method for the early detection of PN and has potential for translation to clinical studies.

A single-trial analytic framework for EEG analysis and its application to target detection and classification.

Modern neuroimaging technologies afford a non-invasive view into the functions of the human brain with great spatial (fMRI) and temporal resolution (EEG). However, common signal analytic methods require averaging over many trials, which limits the potential for practical application of these technologies. In this paper we advance a novel single-trial analysis method for EEG and demonstrate this approach with a target detection task. The method utilizes a framework consisting of multiple processing modules that can be applied in whole or in part, including noise mitigation, source-space transformation, discriminant analysis, and performance evaluation. The framework introduces an enhanced noise mitigation technology based on Directed Components Analysis (DCA) that improves upon existing spatial filtering techniques. Source-space transformation, utilizing a finite difference model (FDM) of the human head, estimates activity measures of the cortical sources involved in task performance. Such a source-space discrimination provides measurement invariance between training and testing sessions and holds the promise of providing a degree of classification not possible with scalp-recorded EEG. The framework's discrimination modules interface with performance evaluation modules to generate classification performance statistics. When applied to EEG acquired during performance of a target detection task, this method demonstrated that neural signatures of target recognition correctly classified up to 87% of targets in a rapid serial visual presentation (RSVP) of target/non-target images. On average, the single-trial classification method resulted in greater than 60% improvement over behavioral performance for target detection.

Geodesic photogrammetry for localizing sensor positions in dense-array EEG.

OBJECTIVE: An important goal for functional brain studies using EEG technology is to estimate the location of brain sources that produce the scalp-recorded signals. The accuracy of source estimates is dependent upon many variables, one of which is the accurate description of the scalp positions of the EEG sensors. The objective of the present research was to develop a photogrammatic method for sensor localization that is fast, accurate, and easy to use. METHODS: With the novel photogrammetric method, multiple cameras were arranged in a geodesic array, and images of the sensors on the subject's head were acquired allowing for the reconstruction of the 3D sensor positions. RESULTS: Data from the photogrammetric method were compared with data acquired with the conventional electromagnetic method. The accuracy of the photogrammatic method, quantified as RMS of the measured positions and the actual known positions, was similar (mean error = 1.27 mm) to the electromagnetic method (mean error = 1.02 mm), and both approximated the localization error of the calibration object (mean error = 0.56 mm). CONCLUSIONS: Accurate determination of 3D sensor positions can be accomplished with minimal demands on the time of the subject and the experimenter using the photogrammetric method.