This work was supported by the Caesarea Edmond &#38; Benjamin de Rothschild Foundations.

The protocol follows the Hadassah Hebrew University Ethics Committee guidelines for studies in human subjects. To avoid the effect of myopia or refractive errors on test results, the protocols should be performed while patients wear their eyeglasses (corrected vision).
Object From Motion (OFM) protocol:
1. Test Initiation and Instructing Subjects
<ol>
	<li>Seat the subject 50 cm in front of the computer screen.</li>
	<li>Open the OFM software.</li>
	<li>Instruct the subject that he will be presented with motion defined objects. Instruct him to respond as correct and as fast as possible by pressing the "A" keyboard and then verbally naming the perceived object.</li>
	<li>Following response, a screen indicating "press the space bar" will appear. Instruct the subject to press the space bar when ready to identify the next stimulus.</li>
	<li>Explain to the subject that stimuli may appear at very hard to-perceive velocities or at some easier to perceive ones.</li>
</ol>
2. Learning Phase
Enter "learning OFM" at the command line. Subject will now be presented with 4 example stimuli. This phase is conducted when subject&#39;s both eyes are open.
3. Testing Phase
In general, each OFM test includes 20 stimuli. All are first presented at the lowest velocity of 4 pixels/sec. Those not recognized will be then presented at the next velocity of 5.5 pixels/sec. Those not recognized, will be then presented at the next velocity of 7.5 pixels/sec and so on, going through 10 pixels/sec, 13.5 pixels/sec, 18 pixels/sec, and till the fastest velocity of 24.5 pixels/sec. Velocities were defined based on the exponent y=3*e0.3. If five consecutive stimuli in a certain velocity were not recognized, the next stimuli will be presented at the next faster velocity to avoid frustration in the patients. This will result in shortening test length which generally is longer as recognition is worse (necessitating the passage through larger number of velocities per stimulus).
<ol>
	<li>Cover subject&#39;s one eye with an eye patch. Every eye patch may be adequate as long as it supplies full coverage.</li>
	<li>Enter "OFM objects" at the command line.</li>
	<li>Choose one stimuli set (software includes 4 stimuli sets. Each set includes 20 different objects. Selection may be random. However, make sure that you apply different stimuli sets for each of the tested eyes; Apply different stimuli sets for subsequent learning time points in case of longitudinal assessment).</li>
	<li>A prompt asking you to enter subject&#39;s name, tested eye and testing date will appear. Complete required information.</li>
	<li>Carefully monitor the subject and respond to the subject while she/he completes the task as follows:
	<ol>
		<li>When a stimulus appears the subject must press the "A" button on the keyboard and name the identity of the presented stimulus.</li>
		<li>Press the left mouse button for a correct answer or the right mouse button for a wrong answer.</li>
		<li>Subject presses the "space bar" on the keyboard for initiation of the next stimulus.</li>
		<li>This procedure (steps 3.5.1-3.1.5.3) continues until all 20 stimuli in the set are either recognized or presented at the fastest velocity.</li>
	</ol>
	</li>
	<li>Repeat the whole procedure (from step 3.1) for the subject&#39;s second eye using a different set of stimuli.</li>
</ol>
Time-constrained Stereo Protocol:
1. Test Initiation and Instructing Subjects
<ol>
	<li>Seat the subject 50 cm in front of the computer screen.</li>
	<li>Open the Stereo software.</li>
	<li>Instruct the subject that he will be presented with 3 dimensional (3D) shapes and will have to name the perceived shape as correct and fast as possible. Shapes will be one of the following: a circle, a square, a triangle, or a star.</li>
	<li>Explain to the subject that stimuli may appear at very hard to perceive conditions or at some easier to perceive ones.</li>
	<li>Instruct the subject to wear the 3D glasses.</li>
	<li>Turn off the room lightening.</li>
</ol>
2. Learning Phase
Enter "learning Stereo" at the command line. Subject will be now presented with 4 repetitions of each shape, presented at the longest stimuli duration (500 msec) and at the easiest disparity (840 sec of arc) conditions. Following the 3D presentation condition, a 2D presentation will follow for each shape. In the latter, a line marking shapes contours will be added, to make sure subject perceived the dimensions of the presented shape. Subjects, who did not succeed at this easy condition, will not be tested at the next phase.
3. Testing Phase: Stereopsis Perception as a Function of Binocular Disparity
The 4 shapes will be presented for 500 msec at 4 different disparity conditions: 120, 300, 540, and 840 sec of arc.
<ol>
	<li>Enter "Stereo Disparity" at the command line.</li>
	<li>A prompt asking you to enter subject&#39;s name and testing date will appear. Complete required information.</li>
	<li>Stimulus appears</li>
	<li>Subject names the presented shape. Responses are coded by the examiner at key buttons 1-4 (i.e. press the 1, 2, 3, or 4 key buttons for subject&#39;s verbal responses of "a circle", "a square", "a triangle", or "a star", respectively). Due to lighting conditions and the fact that the subject wears 3D glasses, he cannot code the responses by himself).</li>
	<li>Subject presses any key to continue to the next stimulus. The order of stimuli presentation is random.</li>
</ol>
4. Testing Phase
Stereopsis perception as a function of stimulus presentation time. The 4 shapes will be presented for disparities of 540 and 840 sec of arc at either 40, 60, or 100 msec durations.
<ol>
	<li>Enter "Stereo Duration" at the command line.</li>
	<li>Repeat steps 3.2-3.5.</li>
</ol>

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The authors declare no competing financial interests.

Optic neuritis is a demyelinative disease of the optic nerve, causing acute visual loss. Though considered transient when using standard visual testing1, patients continue to perceive difficulties in performing everyday visual tasks. We argue that dynamic visual tests are adequate to identify and quantify these sustained deficits. This is since dynamic but not static visual functions depend on projection rates, and may be more vulnerable to delayed projection following demyelination.
T...

Optic neuritis as a model for tracking tissue degeneration and repair
Multiple sclerosis (MS) is a chronic inflammatory neurodegenerative disease of the central nervous system (CNS) and is the leading cause of nontraumatic neurological disability in young adults in developed countries. Demyelination is considered the most characteristic histopathological feature of MS. Recent studies, however, revealed that MS is also a neurodegenerative disease with early neuroaxonal damage1-3.
Optic neuritis (ON), inflammation of the optic nerve, is the presenting symptom in 20% of MS patients and at least 50% of those suffering from MS experience at least one episode of ON during their lifetime4. Unlike other locations of MS lesions that do not always correlate to the clinical manifestations, demyelinating episode of the optic nerve typically results in distinctive manifestation of acute visual loss. Given its comorbidity with MS and its prominent clinical features, ON offers a unique opportunity for tracking tissue degeneration and repair and their consequences in a single MS lesion.
The need for improved methods for tracking tissue degeneration and repair in vivo
Pathologic studies in MS implicate demyelination as a principal cause of axonal transection and subsequent axonal degeneration. Remyelination may prevent demyelinated axons from degenerating; however, effective remyelination may be limited as a result of repeated attacks. Therefore, current and evolving neuroprotective and regenerative therapeutic strategies in MS are aimed to prevent new attacks and promote remyelination processes in the CNS5.
In order to follow up optic neuritis patients and evaluate the efficacy of their treatment, a fine tool to quantify changes in myelination at the CNS is required. However, the standard measurements, including routine visual tests and MRI scans, are not sensitive enough for this purpose. Routine visual tests (i.e. visual acuity, contrast sensitivity, visual fields, and color perception) may reveal cases of reduced input projection along the visual pathways but are insensitive to identify delayed projection rates, which is the role of the demyelinated fibers6,7. T2 hyperintense lesions, which are the hallmark of the disease, result from residual mixture of edema, inflammation, demyelination, axonal loss and gliosis and thus cannot differentiate between demyelination and other brain pathologies. Furthermore, standard MRI is designed to reveal qualitative tissue contrast. While these are adequate for identifying the location of unusual tissue, they are insufficient to quantitatively assess tissue properties.
Dynamic visual tests may be used as markers of demyelination and remyelination
We argue that dynamic visual functions are more appropriate than static functions to identify and quantify changes in projection latencies along the visual pathways. While accomplishment of both static and dynamic visual functions requires sufficient amount of visual input projection, only dynamic visual functions depend on projection rates. Optic nerve demyelination may thus affect dynamic rather than static visual functions, implicating the need for rapid transmission of visual input in order to perceive motion.
We have developed two behavioral tasks to assess monocular and binocular visual functions which may closely associate with projection latencies along the visual pathways. These include Object From Motion (OFM) extraction and Time-constrained stereo protocols.
In the OFM test, an array of dots compose an object, by moving the dots within the image rightward while moving the dots outside the image leftward or vice versa. The dot pattern generates a camouflaged object that cannot be detected when the dots are stationary or moving as a whole. Importantly, object recognition is dependent on motion perception. Using the OFM protocol, we have demonstrated a sustained deficit in the affected eyes of ON patients, evident even 12 months following the optic neuritis attack, while standard visual functions had recovered8. Furthermore, impaired performance was associated with delayed conductions (delayed P100, reflecting demyelination) and improvement in motion perception was correlated with shortening of conduction rates (reflecting remyelination; linear least squares regression with calculation of the correlation coefficient F=27.3; p=0.0005; r=-0.87)9.
The currently presented OFM protocol was updated in order to fit the test for clinical usage, including test shortening, adjusting the test software to result in an automatic output file, and to result in a motion sensitivity score.
To assess the effect of projection latencies on binocular vision, the Time-constrained Stereo protocol was developed. In this protocol, spatially disparate images are presented for a limited length of time, challenging binocular integration in time. This test was designed to test the hypothesis that due to demyelination at the affected nerve, information from the two eyes will reach the cortex at different time points impairing binocular integration in time. Testing a group of recovered ON patients (1-2.5 years following the attack), we have shown that while most patients had intact performance levels in a standard static stereo task; performance on the time-constrained stereo task was impaired in most cases10.
The OFM and the time-constrained stereo protocols provide a simple, yet powerful, way to identify and quantify processes of demyelination and remyelination along the visual pathways. These protocols may be efficient to diagnose and follow up ON and MS patients in a cost effective manner using an easy to use computer based protocol.

<table border="0" cellpadding="0" cellspacing="0" width="638">
	<tbody>
		<tr height="167">
			<td dir="LTR" height="167" style="height:167px;width:231px;">Personal computer, including laptops</td>
			<td style="width:117px;"></td>
			<td style="width:119px;"></td>
			<td dir="LTR" style="width:171px;">The OFM software runs best on Mac or on Windows 7 (or higher) PC. The Stereo software runs on every personal computer.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Monitor&#160; specification</td>
			<td style="width:117px;"></td>
			<td style="width:119px;"></td>
			<td dir="LTR" style="width:171px;">Size: at least 15inch,&#160; Color: at least 16 bit</td>
		</tr>
		<tr height="109">
			<td height="109" style="height:109px;width:231px;">The OFM and the Stereo softwares</td>
			<td dir="LTR" style="width:117px;">These are self-developed softwares</td>
			<td style="width:119px;"></td>
			<td dir="LTR" style="width:171px;">Researchers &#38; physicians who are interested in these softwares may contact us at: fmri-hadassah.org</td>
		</tr>
		<tr height="209">
			<td dir="LTR" height="209" style="height:209px;width:231px;">Red/Cyan 3D glasses (We had tested the Stereo software on the two following 3D glasses):</td>
			<td style="width:117px;">Nvidia &#38; American Paper Optics</td>
			<td dir="LTR" style="width:119px;">3D Vision Ultimate Anaglyph 3D Glasses &#38; Pro X Style Red/Cyan 3D Glasses for Movies and Games on Flat Screens&#160;&#160;</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="63">
			<td dir="LTR" height="63" style="height:63px;width:231px;">Performance on our stereo task was compared to performance on the standard Randot stereo test</td>
			<td dir="LTR" style="width:117px;">Stereo Optical Co.</td>
			<td dir="LTR" style="width:119px;">Randot SO-002&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

dynamic visual tests to identify and quantify visual damage and repair following demyelination in optic neuritis patients

In order to follow optic neuritis patients and evaluate the effectiveness of their treatment, a handy, accurate and quantifiable tool is required to assess changes in myelination at the central nervous system (CNS). However, standard measurements, including routine visual tests and MRI scans, are not sensitive enough for this purpose. We present two visual tests addressing dynamic monocular and binocular functions which may closely associate with the extent of myelination along visual pathways. These include Object From Motion (OFM) extraction and Time-constrained stereo protocols. In the OFM test, an array of dots compose an object, by moving the dots within the image rightward while moving the dots outside the image leftward or vice versa. The dot pattern generates a camouflaged object that cannot be detected when the dots are stationary or moving as a whole. Importantly, object recognition is critically dependent on motion perception. In the Time-constrained Stereo protocol, spatially disparate images are presented for a limited length of time, challenging binocular 3-dimensional integration in time. Both tests are appropriate for clinical usage and provide a simple, yet powerful, way to identify and quantify processes of demyelination and remyelination along visual pathways. These protocols may be efficient to diagnose and follow optic neuritis and multiple sclerosis patients.
In the diagnostic process, these protocols may reveal visual deficits that cannot be identified via current standard visual measurements. Moreover, these protocols sensitively identify the basis of the currently unexplained continued visual complaints of patients following recovery of visual acuity. In the longitudinal follow up course, the protocols can be used as a sensitive marker of demyelinating and remyelinating processes along time. These protocols may therefore be used to evaluate the efficacy of current and evolving therapeutic strategies, targeting myelination of the CNS.

OFM protocol
The protocol results in a text file, automatically summarizing subject's responses. Outcome can be analyzed in two ways:
Total score: Each stimulus presented is assigned with a stimulus weight, and the sum of weights of all identified stimuli is set as the subject's response score. The weight of a particular stimulus is set based on the speed of the stimulus, with higher weights to lower speeds. Unidentified stimuli are assigned with a zero weight. General...

Watch this Scientific Journal Video about Dynamic Visual Tests to Identify and Quantify Visual Damage and Repair Following Demyelination in Optic Neuritis Patients at JoVE.com

Dynamic Visual Tests to Identify and Quantify Visual Damage and Repair Following Demyelination in Optic Neuritis Patients

Object From Motion (OFM) and Time-constrained stereo protocols are sensitive tools to identify monocular and binocular dynamic visual function deficits, which are uniquely affected in optic neuritis patients. Furthermore, these tests may be used as quantitative noninvasive tools to assess the extent of myelination along visual pathways.

In order to follow optic neuritis patients and evaluate the effectiveness of their treatment, a handy, accurate and quantifiable tool is required to ...

dynamic-visual-tests-to-identify-quantify-visual-damage-repair

Research

JoVE Journal

Medicine

14.0K vistas.  Hadassah Hebrew-University Medical Center.  Objeto De Movimiento (OFM) y protocolos estéreo de duración limitada son instrumentos sensibles para identificar monocular y déficit de la función visual binocular dinámicas, las cuales se ven afectadas de forma exclusiva en pacientes con neuritis óptica. Además, estas pruebas pueden ser utilizados como herramientas no invasivas cuantitativos para evaluar el grado de mielinización largo de las vías visuales. 

Video: Dynamic Visual Tests to Identify and Quantify Visual Damage and Repair Following Demyelination in Optic Neuritis Patients

Using Visual and Narrative Methods to Achieve Fair Process in Clinical Care

The Institute of Medicine has targeted patient-centeredness as an important area of quality improvement. A major dimension of patient-centeredness is respect for patient's values, preferences, and expressed needs. Yet specific approaches to gaining this understanding and translating it to quality care in the clinical setting are lacking. From a patient perspective quality is not a simple concept but is best understood in terms of five dimensions: technical outcomes; decision-making efficiency; amenities and convenience; information and emotional support; and overall patient satisfaction. Failure to consider quality from this five-pronged perspective results in a focus on medical outcomes, without considering the processes central to quality from the patient's perspective and vital to achieving good outcomes. In this paper, we argue for applying the concept of fair process in clinical settings. Fair process involves using a collaborative approach to exploring diagnostic issues and treatments with patients, explaining the rationale for decisions, setting expectations about roles and responsibilities, and implementing a core plan and ongoing evaluation. Fair process opens the door to bringing patient expertise into the clinical setting and the work of developing health care goals and strategies. This paper provides a step by step illustration of an innovative visual approach, called photovoice or photo-elicitation, to achieve fair process in clinical work with acquired brain injury survivors and others living with chronic health conditions. Applying this visual tool and methodology in the clinical setting will enhance patient-provider communication; engage patients as partners in identifying challenges, strengths, goals, and strategies; and support evaluation of progress over time. Asking patients to bring visuals of their lives into the clinical interaction can help to illuminate gaps in clinical knowledge, forge better therapeutic relationships with patients living with chronic conditions such as brain injury, and identify patient-centered goals and possibilities for healing. The process illustrated here can be used by clinicians, (primary care physicians, rehabilitation therapists, neurologists, neuropsychologists, psychologists, and others) working with people living with chronic conditions such as acquired brain injury, mental illness, physical disabilities, HIV/AIDS, substance abuse, or post-traumatic stress, and by leaders of support groups for the types of patients described above and their family members or caregivers.

This paper illustrates an innovative visual approach (photovoice or photo-elicitation) to achieve fair process in clinical care for patients living with chronic health conditions, illuminate gaps in clinical knowledge, forge better therapeutic relationships, and identify patient-centered goals and possibilities for healing.

The Institute of Medicine has targeted patient-centeredness as an important area of quality improvement. A major dimension of patient-centeredness is ...

Dissection of Human Vitreous Body Elements for Proteomic Analysis

The vitreous is an optically clear, collagenous extracellular matrix that fills the inside of the eye and overlies the retina. 1,2 Abnormal interactions between vitreous substructures and the retina underlie several vitreoretinal diseases, including retinal tear and detachment, macular pucker, macular hole, age-related macular degeneration, vitreomacular traction, proliferative vitreoretinopathy, proliferative diabetic retinopathy, and inherited vitreoretinopathies. 1,2 The molecular composition of the vitreous substructures is not known. Since the vitreous body is transparent with limited surgical access, it has been difficult to study its substructures at the molecular level. We developed a method to separate and preserve these tissues for proteomic and biochemical analysis. The dissection technique in this experimental video shows how to isolate vitreous base, anterior hyaloid, vitreous core, and vitreous cortex from postmortem human eyes. One-dimensional SDS-PAGE analyses of each vitreous component showed that our dissection technique resulted in four unique protein profiles corresponding to each substructure of the human vitreous body. Identification of differentially compartmentalized proteins will reveal candidate molecules underlying various vitreoretinal diseases.

This video shows an effective technique for differentiating and dissecting the various semi-transparent structures of the human vitreous body in post mortem eyes.

The vitreous is an optically clear, collagenous extracellular matrix that fills the inside of the eye and overlies the retina. 1,2 Abnormal interactions ...

Driving Simulation in the Clinic: Testing Visual Exploratory Behavior in Daily Life Activities in Patients with Visual Field Defects

Patients suffering from homonymous hemianopia after infarction of the posterior cerebral artery (PCA) report different degrees of constraint in daily life, despite similar visual deficits. We assume this could be due to variable development of compensatory strategies such as altered visual scanning behavior. Scanning compensatory therapy (SCT) is studied as part of the visual training after infarction next to vision restoration therapy. SCT consists of learning to make larger eye movements into the blind field enlarging the visual field of search, which has been proven to be the most useful strategy1, not only in natural search tasks but also in mastering daily life activities2. Nevertheless, in clinical routine it is difficult to identify individual levels and training effects of compensatory behavior, since it requires measurement of eye movements in a head unrestrained condition. Studies demonstrated that unrestrained head movements alter the visual exploratory behavior compared to a head-restrained laboratory condition3. Martin et al.4 and Hayhoe et al.5 showed that behavior demonstrated in a laboratory setting cannot be assigned easily to a natural condition. Hence, our goal was to develop a study set-up which uncovers different compensatory oculomotor strategies quickly in a realistic testing situation: Patients are tested in the clinical environment in a driving simulator. SILAB software (Wuerzburg Institute for Traffic Sciences GmbH (WIVW)) was used to program driving scenarios of varying complexity and recording the driver's performance. The software was combined with a head mounted infrared video pupil tracker, recording head- and eye-movements (EyeSeeCam, University of Munich Hospital, Clinical Neurosciences).
The positioning of the patient in the driving simulator and the positioning, adjustment and calibration of the camera is demonstrated. Typical performances of a patient with and without compensatory strategy and a healthy control are illustrated in this pilot study. Different oculomotor behaviors (frequency and amplitude of eye- and head-movements) are evaluated very quickly during the drive itself by dynamic overlay pictures indicating where the subjects gaze is located on the screen, and by analyzing the data. Compensatory gaze behavior in a patient leads to a driving performance comparable to a healthy control, while the performance of a patient without compensatory behavior is significantly worse. The data of eye- and head-movement-behavior as well as driving performance are discussed with respect to different oculomotor strategies and in a broader context with respect to possible training effects throughout the testing session and implications on rehabilitation potential.

Patients with visual deficits after stroke report about different constraints in daily life most likely due to variable compensatory strategies, which are difficult to differentiate in clinical routine. We present a clinical set-up which allows measurement of different compensatory head- and eye-movement-strategies and evaluating their effects on driving performance.

Patients suffering from homonymous hemianopia after infarction of the posterior cerebral artery (PCA) report different degrees of constraint in daily ...

Development of an Audio-based Virtual Gaming Environment to Assist with Navigation Skills in the Blind

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind. Using only audio based cues and set within the context of a video game metaphor, users gather relevant spatial information regarding a building's layout. This allows the user to develop an accurate spatial cognitive map of a large-scale three-dimensional space that can be manipulated for the purposes of a real indoor navigation task. After game play, participants are then assessed on their ability to navigate within the target physical building represented in the game. Preliminary results suggest that early blind users were able to acquire relevant information regarding the spatial layout of a previously unfamiliar building as indexed by their performance on a series of navigation tasks. These tasks included path finding through the virtual and physical building, as well as a series of drop off tasks. We find that the immersive and highly interactive nature of the AbES software appears to greatly engage the blind user to actively explore the virtual environment. Applications of this approach may extend to larger populations of visually impaired individuals.

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind.

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind. Using only audio ...

DTI of the Visual Pathway - White Matter Tracts and Cerebral Lesions

DTI is a technique that identifies white matter tracts (WMT) non-invasively in healthy and non-healthy patients using diffusion measurements. Similar to visual pathways (VP), WMT are not visible with classical MRI or intra-operatively with microscope. DTI will help neurosurgeons to prevent destruction of the VP while removing lesions adjacent to this WMT. We have performed DTI on fifty patients before and after surgery between March 2012 to January 2014. To navigate we used a 3DT1-weighted sequence. Additionally, we performed a T2-weighted and DTI-sequences. The parameters used were, FOV: 200 x 200 mm, slice thickness: 2 mm, and acquisition matrix: 96 x 96 yielding nearly isotropic voxels of 2 x 2 x 2 mm. Axial MRI was carried out using a 32 gradient direction and one b0-image. We used Echo-Planar-Imaging (EPI) and ASSET parallel imaging with an acceleration factor of 2 and b-value of 800 s/mm&#178;. The scanning time was less than 9 min.
The DTI-data obtained were processed using a FDA approved surgical navigation system program which uses a straightforward fiber-tracking approach known as fiber assignment by continuous tracking (FACT). This is based on the propagation of lines between regions of interest (ROI) which is defined by a physician. A maximum angle of 50, FA start value of 0.10 and ADC stop value of 0.20 mm&#178;/s were the parameters used for tractography.
There are some limitations to this technique. The limited acquisition time frame enforces trade-offs in the image quality. Another important point not to be neglected is the brain shift during surgery. As for the latter intra-operative MRI might be helpful. Furthermore the risk of false positive or false negative tracts needs to be taken into account which might compromise the final results.

Diffusion tensor imaging (DTI) was performed to try to depict the major parts of the visual pathway. The goal was to use an FDA approved standard commercial workstation which could be used for everyday routine in order to try to reduce postoperative damage of the visual pathway in the patients.

DTI is a technique that identifies white matter tracts (WMT) non-invasively in healthy and non-healthy patients using diffusion measurements. Similar to ...

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with distinct functions and gene expression profiles. The roles of microglial activation in health, injury and disease remain incompletely understood due to their dynamic and complex regulation in response to changes in their microenvironment. Thus, it is critical to non-invasively monitor and analyze changes in microglial activation over time in the intact organism. In vivo studies of microglial activation have been delayed by technical limitations to tracking microglial behavior without altering the CNS environment. This has been particularly challenging during chronic neurodegeneration, where long-term changes must be tracked. The retina, a CNS organ amenable to non-invasive live imaging, offers a powerful system to visualize and characterize the dynamics of microglia activation during chronic disorders.
This protocol outlines methods for long-term, in vivo imaging of retinal microglia, using confocal ophthalmoscopy (cSLO) and CX3CR1GFP/+ reporter mice, to visualize microglia with cellular resolution. Also, we describe methods to quantify monthly changes in cell activation and density in large cell subsets (200-300 cells per retina). We confirm the use of somal area as a useful metric for live tracking of microglial activation in the retina by applying automated threshold-based morphometric analysis of in vivo images. We use these live image acquisition and analyses strategies to monitor the dynamic changes in microglial activation and microgliosis during early stages of retinal neurodegeneration in a mouse model of chronic glaucoma. This approach should be useful to investigate the contributions of microglia to neuronal and axonal decline in chronic CNS disorders that affect the retina and optic nerve.

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia activation and microgliosis are key responses to chronic neurodegeneration. Here, we present methods for in vivo, long-term visualization of retinal CX3CR1-GFP+ microglial cells by confocal ophthalmoscopy, and for threshold and morphometric analyses to identify and quantify their activation. We monitor microglial changes during early stages of age-related glaucoma.

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with ...

The Rodent Model of Nonarteritic Anterior Ischemic Optic Neuropathy (rNAION)

Nonarteritic anterior ischemic optic neuropathy (NAION) is a focal ischemic lesion of the optic nerve that affects 1/700 individuals throughout their lifetime. NAION results in optic nerve edema, selective loss of the retinal ganglion cell neurons (RGCs) and atrophy of the optic nerve. A rodent model of NAION that expresses most NAION features and sequelae has been developed, which is applicable to both rats and mice. This model utilizes a focal laser application of 532 nm wavelength to illuminate a photoactive dye, Rose Bengal (RB), to cause capillary damage and leakage at the targeted anterior optic nerve (the laminar region). After rNAION induction, there is an early optic nerve ischemia, optic nerve edema, and intraneural inflammation, followed by selective RGC and axonal loss. Since the optic nerve is a CNS white matter tract, the rNAION model is applicable to mechanistic studies of selective white matter ischemia, as well as neuroprotective analyses and short and long-term mechanisms of glial and neuronal response to ischemia.

The Rodent Model of Nonarteritic Anterior Ischemic Optic Neuropathy rNAION

The following report describes how to replicate the rodent model of nonarteritic anterior ischemic optic neuropathy (rNAION), using the appropriate dye, contact lens and laser parameters. We also reveal the appropriate steps for evaluating the rNAION lesion in vivo.

Nonarteritic anterior ischemic optic neuropathy (NAION) is a focal ischemic lesion of the optic nerve that affects 1/700 individuals throughout their ...

A Simple Mechanical Procedure to Create Limbal Stem Cell Deficiency in Mouse

Limbal stem cell deficiency (LSCD) is a state of malfunction or loss of limbal epithelial stem cells, after which the corneal epithelium is replaced with conjunctiva. Patients suffer from recurrent corneal defects, pain, inflammation, and loss of vision.
Previously, a murine model of LSCD was described and compared to two other models. The goal was to produce a consistent mouse model of LSCD that both mimics the phenotype in humans and lasts long enough to make it possible to study the disease pathophysiology and to evaluate new treatments. Here, the technique is described in more detail.
A motorized tool with a rotating burr has been designed to remove the rust rings from the corneal surface or to smooth the pterygium bed in patients. It is a suitable device to create the desired LSCD model. It is a readily available, easy-to-use tool with a fine tip that makes it appropriate for working on small eyes, as in mice. Its application prevents unnecessary trauma to the eye and it does not result in unwanted injuries, as often is the case with chemical injury models. As opposed to a blunt scraper, it removes the epithelium with the basement membrane. In this protocol, the limbal area was abraded two times, and then the whole corneal epithelium was shaved from limbus to limbus. To avoid stroma injury, care was taken not to brush the corneal surface once the epithelium was already removed.

The following article provides an easy, reproducible technique to effectively create a sustainable mouse model of limbal stem cell deficiency (LSCD). This animal model is useful in testing and comparing the efficacy of treatments for limbal stem cell diseases.

Limbal stem cell deficiency (LSCD) is a state of malfunction or loss of limbal epithelial stem cells, after which the corneal epithelium is replaced with ...

Bioluminescence and Near-infrared Imaging of Optic Neuritis and Brain Inflammation in the EAE Model of Multiple Sclerosis in Mice

Experimental autoimmune encephalomyelitis (EAE) in SJL/J mice is a model for relapsing-remitting multiple sclerosis (RRMS). Clinical EAE scores describing motor function deficits are basic readouts of the immune-mediated inflammation of the spinal cord. However, scores and body weight do not allow for an in vivo assessment of brain inflammation and optic neuritis. The latter is an early and frequent manifestation in about 2/3 of MS patients. Here, we show methods for bioluminescence and near-infrared live imaging to assess EAE evoked optic neuritis, brain inflammation, and blood-brain barrier (BBB) disruption in living mice using an in vivo imaging system. A bioluminescent substrate activated by oxidases primarily showed optic neuritis. The signal was specific and allowed the visualization of medication effects and disease time courses, which paralleled the clinical scores. Pegylated fluorescent nanoparticles that remained within the vasculature for extended periods of time were used to assess the BBB integrity. Near-infrared imaging revealed a BBB leak at the peak of the disease. The signal was the strongest around the eyes. A near-infrared substrate for matrix metalloproteinases was used to assess EAE-evoked inflammation. Auto-fluorescence interfered with the signal, requiring spectral unmixing for quantification. Overall, bioluminescence imaging was a reliable method to assess EAE-associated optic neuritis and medication effects and was superior to the near-infrared techniques in terms of signal specificity, robustness, ease of quantification, and cost.

We show a technique for in vivo live bioluminescence and near-infrared imaging of optic neuritis and encephalitis in the experimental autoimmune encephalomyelitis (EAE) model for multiple sclerosis in SJL/J mice.

Experimental autoimmune encephalomyelitis (EAE) in SJL/J mice is a model for relapsing-remitting multiple sclerosis (RRMS). Clinical EAE scores describing ...

Smartphone Fundus Photography

Smartphone fundus photography is a simple technique to obtain ocular fundus pictures using a smartphone camera and a conventional handheld indirect ophthalmoscopy lens. This technique is indispensable when picture documentation of optic nerve, retina, and retinal vessels is necessary but a fundus camera is not available. The main advantage of this technique is the widespread&#160;availability of smartphones that allows documentation of macula and optic nerve changes in many settings that was not previously possible. Following the well-defined steps detailed here, such as proper alignment of the phone camera, handheld lens, and the patient&#39;s pupil, is the key for obtaining a clear retina picture with no interfering light reflections and aberrations. In this paper, the optical principles of indirect ophthalmoscopy and fundus photography will be reviewed first. Then, the step-by-step method to record a good quality retinal image using a smartphone will be explained.

Fundus photography normally requires specialized fundus cameras that are not always available in all clinical settings.&#160;Here, a simple method to record ocular fundus images using a smartphone camera and a conventional high-plus handheld indirect ophthalmoscopy lens is described.

Smartphone fundus photography is a simple technique to obtain ocular fundus pictures using a smartphone camera and a conventional handheld indirect ...