This study was supported by the Ophthalmic Research Institute of Australia (ORIA). We thank Prof. Algis Vingrys and Dr. Bang Bui, University of Melbourne, for initially helping us to develop the VEP recording technique.

Ethics Statement: All procedures involving animals were conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes and the guidelines of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and were approved by the Animal Ethics Committee of Macquarie University.
1. VEP Electrode Implantation
<ol>
	<li>Anesthetize the animal with an intraperitoneal injection of ketamine (75 mg/kg) and medetomidine (0.5 mg/kg). 
	Note: Following induction of anaesthesia, observe the withdrawal reflexes (pinch test, corneal and palpebral reflex, etc.) and their absence as an indication to begin surgery. Continually monitor animals throughout the surgery and administer additional anaesthetic drug (10% of initial ketamine dose per top-up) if the reflexes are present. Adult rats (&#62;12 weeks) are used in the experiments.</li>
	<li>Shave the skin of the surgical area. Place the animal on a warming pad (37 &#176;C) to maintain body temperature during the surgery. Prepare skin through topical application of povidone-iodine. Apply surgical draping. Apply topical ophthalmic ointment to prevent corneal dryness under general anaesthesia. Maintain asepsis by using sterile instruments.</li>
	<li>Make a longitudinal skin incision on the midline of the head skin. Clear the connective tissue to achieve good exposure of the skull.</li>
	<li>Carefully, drill small burr holes manually using a micro hand drill at 7 mm behind the bregma and 3 mm lateral to the midline.</li>
	<li>Implant screw electrodes through the skull into the cortex (area 17), penetrating the cortex to a depth of approximately 0.5 mm. Implant a reference screw electrode on the midline 3 mm rostral to the bregma. Apply dental cement to encase and fix the screws (not always required).</li>
	<li>Suture the skin of the head, administer antibiotic ointment onto the skin and allow the animal to recover from anaesthesia on a warming pad. 
	Note: Alternatively, electrodes can be left exposed, so that skin doesn&#8217;t need to be reopened in each recording. Immediately upon cessation of surgery but prior to anaesthetic recovery, administer a non-steroidal anti-inflammatory drug (NSAID) (if not administered pre-operatively) or an opioid analgesic. Monitor the animals constantly until full recovery from anaesthetic and fully ambulatory.</li>
	<li>Allow at least 1 week for the animals to recover from the surgery prior to VEP recording.</li>
</ol>
2. Optic Nerve Injection
<ol>
	<li>Anesthetize the animal, prepare the skin and apply draping as above (1.1 and 1.2).</li>
	<li>Make a 1- to 1.5-cm incision in the skin above the orbit of a randomly selected eye. Open the subcutaneous tissue to reach the orbital cavity using fine iris scissors. Open the conjunctiva and anterior Tenon&#39;s capsule under the operating microscope.</li>
	<li>Retract the extraocular muscles and intraobital lacrimal glands to expose approximately 3 mm length of the optic nerve. Open the dura and arachnoid matter layers around the optic nerve longitudinally using an ophthalmic blade.</li>
	<li>Insert the glass pipette into the optic nerve at a distance of 2 mm posterior to the globe. The glass micropipette is attached to a Hamilton syringe.</li>
	<li>Inject 1% lysolecithin (0.4 - 1.0 &#181;l, with 0.02% Evan&#8217;s Blue which doesn&#8217;t have effects on myelination) slowly into the nerve approximately over a period of 30 sec.</li>
	<li>Suture the skin incision. Apply antibiotic ointment to prevent infection. Fellow eyes can be served as internal controls for electrophysiology recordings.</li>
	<li>Place the animals on a warming pad to recover from anaesthesia.</li>
</ol>
3. VEP Recording
<ol>
	<li>Anesthetize the animal and prepare the skin as 1.1 and 1.2. 
	Note: Lower dose of anaesthetics can be used for electrophysiological recording (ketamine 40 mg/kg and medetomidine 0.25 mg/kg).</li>
	<li>Place the rat in a dark room and allow it to adapt to darkness for 5 - 30 min. In some cases rats can respectively be dark adapted O/N for scotopic or light adapted for photopic VEP recordings8.</li>
	<li>Maintain the body temperature at 37 &#177; 0.5 &#176;C by the homoeothermic blanket system with a rectal thermometer probe.</li>
	<li>Dilate the pupils with 1.0% tropicamide eye drops. Open the skin over the skull to access the pre-placed in situ screw electrodes.</li>
	<li>Connect the screw over the contralateral visual cortex of the stimulated eye and the reference screw to the amplifier. Insert a needle electrode into the tail as the ground. Measure and maintain the electrode impedance below 5 k&#8486;.</li>
	<li>Place a mini-Ganzfeld stimulator directly on the skin around the eyelids to provide superior eye isolation7. The illumination of the stimulator needs to be calibrated beforehand by a photometer.</li>
	<li>Deliver photic stimulation through light flashes 100 times at a frequency of 1 Hz, with low and high band-pass filter settings of 1 and 100 Hz, respectively. The signal sampling rate is at 5 kHz. 
	Note: Signals should be sampled at least at about 250 - 300 Hz to ensure that more that two samples are collected during each cycle.</li>
	<li>Suture the skin back and keep the animals on a warming pad to recover from anaesthesia. Recording can be repeatedly recorded on individual animals to monitor functional changes over a period of time.</li>
	<li>At the end-point, administer an overdose injection of sodium pentobarbitone (100 mg/kg, i.p.) to euthanize the animal. Confirm euthanasia by cardiac arrest, respiratory arrest and decrease of body temperature.</li>
</ol>
4. Tissue Preparation and Histology
<ol>
	<li>Remove the optic nerves from euthanized animals under the microscope and fix the tissue in 1% paraformaldehyde O/N.</li>
	<li>Wash the tissue thoroughly with saline. Treat the tissue in an automatic tissue processor and embed in paraffin. Cut sections (5 - 10 &#181;m) using a rotary microtome. 
	Note: For immunohistochemistry study, fix tissue in 1% paraformaldehyde, wash with saline and incubate with 30% sucrose O/N. Embed tissue in OCT embedding medium and make cryosections using a cryostat.</li>
	<li>Incubate sections in 0.1% fast blue solution such as Luxol (in 95% ethanol) O/N at 56 &#176;C. Differentiate the sections in 0.05% lithium carbonate for 30 sec and then 70% ethanol for another 30 sec. Finally, counterstain in 0.1% cresyl violet solution for 30 sec before mounting the sections. Use the fast blue staining to identify myelin in the optic nerve5.</li>
</ol>

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I., Mushin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+visual+evoked+response+in+optic+neuritis.">Delayed visual evoked response in optic neuritis.</a> Lancet. 1, 982-985 (1972).</li><li>You, Y., Klistorner, A., Thie, J., Graham, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Latency+delay+of+visual+evoked+potential+is+a+real+measurement+of+demyelination+in+a+rat+model+of+optic+neuritis.">Latency delay of visual evoked potential is a real measurement of demyelination in a rat model of optic neuritis.</a> Invest Ophthalmol Vis Sci. 52 (9), 6911-6918 (2011).</li><li>You, Y., Klistorner, A., Thie, J., Gupta, V. K., Graham, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+loss+in+a+rat+model+of+optic+neuritis+is+closely+correlated+with+visual+evoked+potential+amplitudes+using+electroencephalogram+based+scaling.">Axonal loss in a rat model of optic neuritis is closely correlated with visual evoked potential amplitudes using electroencephalogram based scaling.</a> Invest Ophthalmol Vis Sci. 53, 3662 (2012).</li><li>You, Y., Klistorner, A., Thie, J., Graham, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+reproducibility+of+VEP+recording+in+rats:+electrodes,+stimulus+source+and+peak+analysis.">Improving reproducibility of VEP recording in rats: electrodes, stimulus source and peak analysis.</a> Doc Ophthalmol. 123 (2), 109-119 (2011).</li><li>Heiduschka, P., Schraermeyer, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+visual+function+in+pigmented+and+albino+rats+by+electroretinography+and+visual+evoked+potentials.">Comparison of visual function in pigmented and albino rats by electroretinography and visual evoked potentials.</a> Graefes Arch Clin Exp Ophthalmol. 246 (11), 1559-1573 (2008).</li><li>You, Y., Thie, J., Klistorner, A., Gupta, V. K., Graham, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normalization+of+visual+evoked+potentials+using+underlying+electroencephalogram+levels+improves+amplitude+reproducibility+in+rats.">Normalization of visual evoked potentials using underlying electroencephalogram levels improves amplitude reproducibility in rats.</a> Invest Ophthalmol Vis Sci. 53 (3), 1473-1478 (2012).</li><li>Levkovitch-Verbin, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+optic+nerve+diseases.">Animal models of optic nerve diseases.</a> Eye (Lond). 18 (11), 1066-1074 (2004).</li><li>Henry, K. R., Rhoades, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relation+of+albinism+and+drugs+to+the+visual+evoked+potential+of+the+mouse).">Relation of albinism and drugs to the visual evoked potential of the mouse).</a> J Comp Physiol Psychol. 92 (2), 271-279 (1978).</li><li>Murrell, J. C., Waters, D., Johnson, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+effects+of+halothane,+isoflurane,+sevoflurane+and+desflurane+on+the+electroencephalogram+of+the+rat.">Comparative effects of halothane, isoflurane, sevoflurane and desflurane on the electroencephalogram of the rat.</a> Lab Anim. 42 (2), 161-170 (2008).</li><li>Makela, K., Hartikainen, K., Rorarius, M., Jantti, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+F-VEP+during+isoflurane-induced+EEG+suppression.">Suppression of F-VEP during isoflurane-induced EEG suppression.</a> Electroencephalogr Clin Neurophysiol. 100 (3), 269-272 (1996).</li><li>Boyes, W. K., Padilla, S., Dyer, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Body+temperature-dependent+and+independent+actions+of+chlordimeform+on+visual+evoked+potentials+and+axonal+transport+in+optic+system+of+rat.">Body temperature-dependent and independent actions of chlordimeform on visual evoked potentials and axonal transport in optic system of rat.</a> Neuropharmacology. 24 (8), 743-749 (1985).</li><li>Hetzler, B. E., Boyes, W. K., Creason, J. P., Dyer, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature-dependent+changes+in+visual+evoked+potentials+of+rats.">Temperature-dependent changes in visual evoked potentials of rats.</a> Electroencephalogr Clin Neurophysiol. 70 (2), 137-154 (1988).</li><li>Mitchell, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+lysolecithin+on+non-myelinated+axons+in+vitro.">The effects of lysolecithin on non-myelinated axons in vitro.</a> Acta Neuropathol. 58 (4), 243-248 (1982).</li><li>Meyer, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+neuronal+apoptosis+in+a+rat+model+of+multiple+sclerosis.">Acute neuronal apoptosis in a rat model of multiple sclerosis.</a> J Neurosci. 21 (16), 6214-6220 (2001).</li><li>Lachapelle, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Failure+of+remyelination+in+the+nonhuman+primate+optic+nerve.">Failure of remyelination in the nonhuman primate optic nerve.</a> Brain Pathol. 15 (3), 198-207 (2005).</li></ol>

None of the authors have competing interests or conflicting interests.

The optic nerve is very susceptible to mechanical damage. Optic nerve crush injury over a duration of 1 s can lead to about 75% loss of RGC over a period of 2 weeks10. Therefore, extreme care is required while performing the surgical procedures. According to the authors&#8217; experience, it is much better to adapt a blunt dissection approach to expose and make way through the tissues around the optic nerve along the orientation of the nerve, rather than penetrating in a perpendicular orientation to the optic ...

Optic neuritis is one of the most common form of optic neuropathy, causing complete or partial loss of vision1. Histologically, it is featured by inflammatory demyelination, retinal ganglion cell axonal loss and varying degrees of remyelination in the optic nerve2. Optic neuritis is usually the manifest onset of multiple sclerosis. The visual evoked potential (VEP) is a non-invasive tool for investigating the function of the visual system. It reflects the post-retinal function from the retina to the primary visual cortex and is affected in many optic nerve disease conditions3. The VEP has been predominantly used in optic neuritis patients to assess the integrity of the visual pathway4. 
The latency of VEP, which reflects the velocity of signal conduction along the visual pathway, is considered to be an accurate measurement of the level of myelin associated changes in the optic nerve5; while the amplitude of VEP is believed to be closely correlated with axonal damage of the retinal ganglion cells (RGC)6. This hypothesis has been fairly well established using the rat model of lysolecithin-induced optic nerve demyelination5. 
Here, we explicate a comprehensive protocol of optic nerve microinjection technique in rodents, which can minimise the surgical manipulation-related damage to the nerve per se as well as to the adjacent tissues such as extraocular muscles and blood vessels. Also, the skull electrode implantation surgery has been described for VEP recording in animals7. The VEP recordings can be repeatedly carried out on animals over a period of time to assess demyelination/remyelination related changes as well as impact on axonal integrity in the optic nerve.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Ketamine 100 mg/ml (Ketamil)</td>
			<td>Troy Laboratories</td>
			<td>AC 116</td>
			<td></td>
		</tr>
		<tr>
			<td>Medetomidine 1 mg/ml (Domitor)</td>
			<td>Pfizer</td>
			<td>sc-204073</td>
			<td></td>
		</tr>
		<tr>
			<td>Tropicamide 1.0% (Mydriacyl)</td>
			<td>Alcon</td>
			<td>sc-202371</td>
			<td></td>
		</tr>
		<tr>
			<td>Homoeothermic blanket system</td>
			<td>Harvard Apparatus</td>
			<td>NC9203819</td>
			<td></td>
		</tr>
		<tr>
			<td>Impedance meter&#160;</td>
			<td>Grass</td>
			<td>F-EZM5</td>
			<td></td>
		</tr>
		<tr>
			<td>Screw electrodes&#160;</td>
			<td>Micro Fasteners</td>
			<td>M1.0&#215;3mm Csk Slot M/T 304 S/S</td>
			<td></td>
		</tr>
		<tr>
			<td>Subdermal needle electrodes&#160;</td>
			<td>Grass</td>
			<td>F-E3M-72</td>
			<td></td>
		</tr>
		<tr>
			<td>Rapid Repair&#160;</td>
			<td>DeguDent GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Light-emitting diode&#160;</td>
			<td>Nichia</td>
			<td>NSPG300A</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioamplifier</td>
			<td>CWE, Inc.</td>
			<td>BMA-400</td>
			<td></td>
		</tr>
		<tr>
			<td>CED system</td>
			<td>Cambridge Electronic Design, Ltd.</td>
			<td>Power1401</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe&#160;</td>
			<td>Hamilton</td>
			<td>87930</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysolecithin</td>
			<td>Sigma</td>
			<td>L4129</td>
			<td></td>
		</tr>
		<tr>
			<td>Evan&#8217;s blue&#160;</td>
			<td>Sigma</td>
			<td>E2129</td>
			<td></td>
		</tr>
	</tbody>
</table>

visual evoked potential recording in a rat model of experimental optic nerve demyelination

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to monitor the disease course in the follow-up period. Changes in the VEP parameters closely correlate with pathological damage in the optic nerve. This protocol provides a detailed description about the rodent model of optic nerve microinjection, in which a partial demyelination lesion is produced in the optic nerve. VEP recording techniques are also discussed. Using skull implanted electrodes, we are able to acquire reproducible intra-session and between-session VEP traces. VEPs can be recorded on individual animals over a period of time to assess the functional changes in the optic nerve longitudinally. The optic nerve demyelination model, in conjunction with the VEP recording protocol, provides a tool to investigate the disease processes associated with demyelination and remyelination, and can potentially be employed to evaluate the effects of new remyelinating drugs or neuroprotective therapies.

Reproducible intra-sessional VEP traces are shown in Figure 1 and a significant delay in N1 latency can be seen after the optic nerve injection. Partial optic nerve lesions of demyelination can be observed on histological sections using Luxol fast blue staining5. Figure 2 shows a representative section with a small focal demyelinated lesion in the centre of the optic nerve. Note that cross section does not represent total volume of lesion. The demyelinated area can be measured...

Watch this Scientific Journal Video about Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination at JoVE.com

Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination

Focal demyelination is induced in the optic nerve using lysolecithin microinjection. Visual evoked potentials are recorded via skull electrodes implanted over the visual cortex to examine the signal conduction along the visual pathway in vivo. This protocol details the surgical procedures underlying electrode implantation and optic nerve microinjection.

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to ...

visual-evoked-potential-recording-rat-model-experimental-optic-nerve

Research

JoVE Journal

Neuroscience

12.1K Views.  Macquarie University. Focal demyelination is induced in the optic nerve using lysolecithin microinjection. Visual evoked potentials are recorded via skull electrodes implanted over the visual cortex to examine the signal conduction along the visual pathway in vivo. This protocol details the surgical procedures underlying electrode implantation and optic nerve microinjection. 

Video: Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and postsynaptic projections to the visual cortex. Based on its distinct anatomical structure and convenient accessibility, it has become the favored structure for studies on neuronal survival, axonal regeneration, and synaptic plasticity. Recent advancements in MR imaging have enabled the in vivo visualization of the retino-tectal part of this projection using manganese mediated contrast enhancement (MEMRI). Here, we present a MEMRI protocol for illustration of the visual projection in mice, by which resolutions of (200 &#181;m)3 can be achieved using common 3 Tesla scanners. We demonstrate how intravitreal injection of a single dosage of 15 nmol MnCl2 leads to a saturated enhancement of the intact projection within 24 hr. With exception of the retina, changes in signal intensity are independent of coincided visual stimulation or physiological aging. We further apply this technique to longitudinally monitor axonal degeneration in response to acute optic nerve injury, a paradigm by which Mn2+ transport completely arrests at the lesion site. Conversely, active Mn2+ transport is quantitatively proportionate to the viability, number, and electrical activity of axon fibers. For such an analysis, we exemplify Mn2+ transport kinetics along the visual path in a transgenic mouse model (NF-&#954;B p50KO) displaying spontaneous atrophy of sensory, including visual, projections. In these mice, MEMRI indicates reduced but not delayed Mn2+ transport as compared to wild type mice, thus revealing signs of structural and/or functional impairments by NF-&#954;B mutations.
In summary, MEMRI conveniently bridges in vivo assays and post mortem histology for the characterization of nerve fiber integrity and activity. It is highly useful for longitudinal studies on axonal degeneration and regeneration, and investigations of mutant mice for genuine or inducible phenotypes.

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

This video illustrates a method, using a clinical 3 T scanner, for contrast-enhanced MR imaging of the na&#239;ve mouse visual projection and for repetitive and longitudinal in vivo studies of optic nerve degeneration associated with acute optic nerve crush injury and chronic optic nerve degeneration in knock-out mice (p50KO).

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and ...

Minimally-invasive Technique for Injection into Rat Optic Nerve

The rat optic nerve is a useful model for stem cell regeneration research. Direct injection into the rat optic nerve allows delivery into the central nervous system in a minimally-invasive surgery without bone removal. This technique describes an approach to visualization and direct injection of the optic nerve following minor fascial dissection from the orbital ridge, using a conjunctival traction suture to gently pull the eye down and out. Representative examples of an injected optic nerve show successful injection of dyed beads.

Direct injection into the rat optic nerve is useful for regenerative research. We demonstrate a minimally-invasive technique for direct injection into a rat optic nerve that does not involve opening the skull. Using this method, surgical complications are minimized and recovery is more rapid.

The rat optic nerve is a useful model for stem cell regeneration research. Direct injection into the rat optic nerve allows delivery into the central ...

Simultaneous Recording of Electroretinography and Visual Evoked Potentials in Anesthetized Rats

The electroretinogram (ERG) and visual evoked potential (VEP) are commonly used to assess the integrity of the visual pathway. The ERG measures the electrical responses of the retina to light stimulation, while the VEP measures the corresponding functional integrity of the visual pathways from the retina to the primary visual cortex following the same light event. The ERG waveform can be broken down into components that reflect responses from different retinal neuronal and glial cell classes. The early components of the VEP waveform represent the integrity of the optic nerve and higher cortical centers. These recordings can be conducted in isolation or together, depending on the application. The methodology described in this paper allows simultaneous assessment of retinal and cortical visual evoked electrophysiology from both eyes and both hemispheres. This is a useful way to more comprehensively assess retinal function and the upstream effects that changes in retinal function can have on visual evoked cortical function.

This protocol describes simultaneous measurement of electroretinogram and visual evoked potentials in anesthetized rats.

The electroretinogram (ERG) and visual evoked potential (VEP) are commonly used to assess the integrity of the visual pathway. The ERG measures the ...

In Vitro Recording of Mesenteric Afferent Nerve Activity in Mouse Jejunal and Colonic Segments

Afferent nerves not only convey information concerning normal physiology, but also signal disturbed homeostasis and pathophysiological processes of the different organ systems from the periphery towards the central nervous system. As such, the increased activity or &#39;sensitization&#39; of mesenteric afferent nerves has been allocated an important role in the pathophysiology of visceral hypersensitivity and abdominal pain syndromes.
Mesenteric afferent nerve activity can be measured in vitro in an isolated intestinal segment that is mounted in a purpose-built organ bath and from which the splanchnic nerve is isolated, allowing researchers to directly assess nerve activity adjacent to the gastrointestinal segment. Activity can be recorded at baseline in standardized conditions, during distension of the segment or following the addition of pharmacological compounds delivered intraluminally or serosally. This technique allows the researcher to easily study the effect of drugs targeting the peripheral nervous system in control specimens; besides, it provides crucial information on how neuronal activity is altered during disease. It should be noted however that measuring afferent neuronal firing activity only constitutes one relay station in the complex neuronal signaling cascade, and researchers should bear in mind not to overlook neuronal activity at other levels (e.g., dorsal root ganglia, spinal cord or central nervous system) in order to fully elucidate the complex neuronal physiology in health and disease.
Commonly used applications include the study of neuronal activity in response to the administration of lipopolysaccharide, and the study of afferent nerve activity in animal models of irritable bowel syndrome. In a more translational approach, the isolated mouse intestinal segment can be exposed to colonic supernatants from IBS patients. Furthermore, a modification of this technique has been recently shown to be applicable in human colonic specimens.

In Vitro Recording of Mesenteric Afferent Nerve Activity in Mouse Jejunal and Colonic Segments

Mesenteric afferent nerves convey information from the gastrointestinal tract towards the brain regarding normal homeostasis as well as pathophysiology. Gastrointestinal afferent nerve activity can be assessed by mounting isolated intestinal segments with attached afferent nerves into an organ bath, isolating the nerve, and assessing basal as well as stimulated activity.

Afferent nerves not only convey information concerning normal physiology, but also signal disturbed homeostasis and pathophysiological processes of the ...

Partial Optic Nerve Transection in Rats: A Model Established with a New Operative Approach to Assess Secondary Degeneration of Retinal Ganglion Cells

Previous studies have shown that the secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. Partial optic nerve transection is considered a useful and reproducible model. Compared with other optic nerve injury models used commonly for assessing secondary degeneration, e.g. complete optic nerve transection and optic nerve crush models, the partial optic nerve transection model is superior as it distinguishes primary from secondary degeneration in situ. Therefore, it serves as an excellent tool for evaluating secondary degeneration. This study describes a novel operative approach of partial optic nerve transection by directly accessing the area of the retrobulbar optic nerve through the orbital lateral wall of the eyeball. Moreover, we present a newly designed, low cost surgical instrument to assist with transection. As demonstrated by the representative results in distinguishing the boundary of primary and secondary injury areas, the new approach and instrument ensures high efficiency and stability of the model by providing adequate space for surgical operation. This in turn makes it easy to separate the meningeal sheath and ophthalmic vessels from the optic nerve before transection. An additional benefit is that this space-saving operative approach improves the investigators&#39; ability to administer drugs, carriers, or selective RGC tracers to the stump of the partially transected optic nerve, allowing the exploration of mechanisms behind secondary injury in RGCs, in a new way.

Secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. This study describes an innovative operative approach for partial optic nerve transection. The use of this space-saving operative approach extends the model's application range, and allows exploration of secondary injury mechanisms in RGCs in a new way.

Previous studies have shown that the secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. Partial optic nerve transection ...

Visual Evoked Potential Recordings in Mice Using a Dry Non-invasive Multi-channel Scalp EEG Sensor

For scalp EEG research environments with laboratory mice, we designed a dry-type 16 channel EEG sensor which is non-invasive, deformable, and re-usable because of the plunger-spring-barrel structural facet and mechanical strengths resulting from metal materials. The whole process for acquiring the VEP responses in vivo from a mouse consists of four steps: (1) sensor assembly, (2) animal preparation, (3) VEP measurement, and (4) signal processing. This paper presents representative measurements of VEP responses from multiple mice with a submicro-voltage signal resolution and sub-hundred millisecond temporal resolution. Although the proposed method is safer and more convenient compared to other previously reported animal EEG acquiring methods, there are remaining issues including how to enhance the signal-to-noise ratio and how to apply this technique with freely moving animals. The proposed method utilizes easily available resources and shows a repetitive VEP response with a satisfactory signal quality. Therefore, this method could be utilized for longitudinal experimental studies and reliable translational research exploiting non-invasive paradigms.

We designed a dry-type 16 channel EEG sensor which is non-invasive, deformable, and re-usable. This paper describes the whole process from manufacturing the proposed EEG electrode to signal processing of visual evoked potential (VEP) signals measured on a mouse scalp using a dry non-invasive multi-channel EEG sensor.

For scalp EEG research environments with laboratory mice, we designed a dry-type 16 channel EEG sensor which is non-invasive, deformable, and re-usable ...

Rat Model of Widespread Cerebral Cortical Demyelination Induced by an Intracerebral Injection of Pro-Inflammatory Cytokines

Multiple sclerosis (MS) is the most common immune-mediated disease of the central nervous system (CNS) and progressively leads to physical disability and death, caused by white matter lesions in the spinal cord and cerebellum, as well as by demyelination in grey matter. Whilst conventional models of experimental allergic encephalomyelitis are suitable for the investigation of the cell-mediated inflammation in the spinal and cerebellar white matter, they fail to address grey matter pathologies. Here, we present the experimental protocol for a novel rat model of cortical demyelination allowing the investigation of the pathological and molecular mechanisms leading to cortical lesions. The demyelination is induced by an immunization with low-dose myelin oligodendrocyte glycoprotein (MOG) in an incomplete Freund&#39;s adjuvant followed by a catheter-mediated intracerebral delivery of pro-inflammatory cytokines. The catheter, moreover, enables multiple rounds of demyelination without causing injection-induced trauma, as well as the intracerebral delivery of potential therapeutic drugs undergoing a preclinical investigation. The method is also ethically favorable as animal pain and distress or disability are controlled and relatively minimal. The expected timeframe for the implementation of the entire protocol is around 8 - 10 weeks.

The protocol presented here allows the reproduction of a widespread grey matter demyelination of both cortical hemispheres in adult male Dark Agouti rats. The method comprises of intracerebral implantation of a catheter, subclinical immunization against myelin oligodendrocyte glycoprotein, and intracerebral injection of a pro-inflammatory cytokine mixture through the implanted catheter.

Multiple sclerosis (MS) is the most common immune-mediated disease of the central nervous system (CNS) and progressively leads to physical disability and ...

Stimulus-specific Cortical Visual Evoked Potential Morphological Patterns

This paper presents a methodology for the recording and analysis of cortical visual evoked potentials (CVEPs) in response to various visual stimuli using 128-channel high-density electroencephalography (EEG). The specific aim of the described stimuli and analyses is to examine whether it is feasible to replicate previously reported CVEP morphological patterns elicited by an apparent motion stimulus, designed to simultaneously stimulate both ventral and dorsal central visual networks, using object and motion stimuli designed to separately stimulate ventral and dorsal visual cortical networks.&#160; Four visual paradigms are presented: 1. Randomized visual objects with consistent temporal presentation. 2. Randomized visual objects with inconsistent temporal presentation (or jitter).&#160; 3. Visual motion via a radial field of coherent central dot motion without jitter.&#160; 4. Visual motion via a radial field of coherent central dot motion with jitter.&#160; These four paradigms are presented in a pseudo-randomized order for each participant.&#160; Jitter is introduced in order to view how possible anticipatory-related effects may affect the morphology of the object-onset and motion-onset CVEP response.&#160; EEG data analyses are described in detail, including steps of data exportation from and importation to signal processing platforms, bad channel identification&#160;and removal, artifact rejection, averaging, and categorization of average CVEP morphological pattern type based upon latency ranges of component peaks. Representative data show that the methodological approach is indeed sensitive in eliciting differential object-onset and motion-onset CVEP morphological patterns and may, therefore, be useful in addressing the larger research aim. Given the high temporal resolution of EEG and the possible application of high-density EEG in source localization analyses, this protocol is ideal for the investigation of distinct CVEP morphological patterns and the underlying neural mechanisms which generate these differential responses.&#160; &#160; &#160;&#160;

In this paper, we present a protocol to investigate differential cortical visual evoked potential morphological patterns through stimulation of ventral and dorsal networks using high-density EEG. Visual object and motion stimulus paradigms, with and without temporal jitter, are described. Visual evoked potential morphological analyses are also outlined.&#160;&#160;

This paper presents a methodology for the recording and analysis of cortical visual evoked potentials (CVEPs) in response to various visual stimuli using ...

Facial Nerve Surgery in the Rat Model to Study Axonal Inhibition and Regeneration

This protocol describes consistent and reproducible methods to study axonal regeneration and inhibition in a rat facial nerve injury model. The facial nerve can be manipulated along its entire length, from its intracranial segment to its extratemporal course. There are three primary types of nerve injury used for the experimental study of regenerative properties: nerve crush, transection, and nerve gap. The range of possible interventions is vast, including surgical manipulation of the nerve, delivery of neuroactive reagents or cells, and either central or end-organ manipulations. Advantages of this model for studying nerve regeneration include simplicity, reproducibility, interspecies consistency, reliable survival rates of the rat, and an increased anatomic size relative to murine models. Its limitations involve a more limited genetic manipulation versus the mouse model and the superlative regenerative capability of the rat, such that the facial nerve scientist must carefully assess time points for recovery and whether to translate results to higher animals and human studies. The rat model for facial nerve injury allows for functional, electrophysiological, and histomorphometric parameters for the interpretation and comparison of nerve regeneration. It thereby boasts tremendous potential toward furthering the understanding and treatment of the devastating consequences of facial nerve injury in human patients.

This protocol describes a reproducible approach to facial nerve surgery in the rat model, including descriptions of various inducible patterns of injury.

This protocol describes consistent and reproducible methods to study axonal regeneration and inhibition in a rat facial nerve injury model. The facial ...

Assessing Early Stage Open-Angle Glaucoma in Patients by Isolated-Check Visual Evoked Potential

Recently, the isolated-check visual evoked potential (icVEP) technique was designed and has been reported to detect glaucomatous damage earlier and faster. It creates low spatial frequency/high temporal frequency bright stimuli and records cortical activity initiated primarily by afferents in the magnocellular ON pathway. This pathway contains neurons with larger volumes and axonal diameters, and it is preferentially damaged in early glaucoma, which can result in visual field loss. The study presented here uses standard operative procedures (SOP) of icVEP to obtain reliable results. It can detect visual function loss using a signal-to-noise ratio (SNR) corresponding to the defects of retinal nerve fiber layer (RNFL) in early stage open-angle glaucoma (OAG). A setting of 10 Hz and condition of 15% positive-contrast (bright) are selected to differentiate OAG patients and control subjects, with each check containing eight runs. Each run persists for 2 s (for 20 total cycles). A flowchart is constructed, which consists of pupil size and intraocular pressure over a 30 min rest period before each examination. Additionally, the testing order of eyes is performed to obtain reliable electroencephalographic signals. VEPs are recorded and analyzed automatically by software, and SNRs are derived based on a multivariate statistic. An SNR of &#8804; 1 is considered abnormal. A receiver-operating-characteristic (ROC) curve is applied to analyze the accuracy of group classification. Then, the SOP is applied in a cross-sectional study, showing that icVEP can detect glaucomatous visual function abnormality in the central visual field in the form of SNR. This value also correlates with the thickness thinning of RNFL and produces high classification accuracy for early stage OAG. Thus, it serves as a useful and objective diagnostic technology for the early detection of glaucoma.

The isolated-check visual evoked potential (icVEP) method is implemented here to assess the magnocellular ON pathway that is initially damaged in glaucoma. The study shows standard operative procedures using icVEP to obtain reliable results. It is proved to serve as a useful objective diagnosing technology for the early detection of glaucoma.

Recently, the isolated-check visual evoked potential (icVEP) technique was designed and has been reported to detect glaucomatous damage earlier and ...