Name: nerve excitability assessment in chemotherapy-induced neurotoxicity
Uploaded: 2012-04-26T12:00:00
Duration: 7 min 42 s
Description: Watch this Scientific Journal Video about Nerve Excitability Assessment in Chemotherapy-induced Neurotoxicity at JoVE.com

1. Patient Preparation
<ol>
	<li>Patients are referred from the Department of Medical Oncology for baseline nerve excitability testing prior to commencement on chemotherapy.</li>
	<li>Patient suitability for excitability testing must be determined. Patients should be excluded if they have a history of or baseline neurophysiological evidence of peripheral neuropathy, have received prior neurotoxic chemotherapy treatment or there are any contra-indications for excitability testing.</li>
</ol>
2. Axonal Excitability Procedures
<ol>
	<li>Undertake sensory and motor excitability protocols on the median nerve, using the semi-automated computerized system QTracS (Institute of Neurology, Queen Square, UK), an isolated linear bipolar constant current stimulator (Digitimer, Welwyn Garden City, UK) and an amplifier (Sapphire IIA, Medelec, UK).</li>
	<li>Prepare the skin surface at the wrist and forearm with an abrasive gel or pad to reduce skin resistance, followed by application of an alcohol wipe.</li>
	<li>Prepare the recording site for motor recordings- with non-polarizable electrodes placed on the muscle belly of abductor pollicis brevis and a reference electrode 4cm distal to record compound motor action potentials (CMAPs).</li>
	<li>Prepare the recording site for sensory recordings - using ring electrodes placed at the proximal and distal interphalangeal joints for recording and reference electrodes respectively, to record compound sensory action potentials (CSAPs).</li>
	<li>Place an electrosurgical neutral earth plate in the palm, with conductive gel.</li>
	<li>Electrical noise in the recording setup should be removed as much as possible, using a Humbug 50/60 Hz Noise eliminator (Quest Scientific Instruments, North Vancouver, Canada).</li>
	<li>Stimulate the median nerve at the wrist. Stimulation site should be selected as the site of lowest threshold, using a repositionable bipolar electrode and subsequently a non-polarizable electrode.</li>
	<li>The anode electrode should be placed 10 cm proximal from the stimulating electrode over bone.</li>
	<li>Monitor temperature at the site of stimulation throughout the test and ensure that the temperature is greater than 32 &deg;C.</li>
</ol>
3. Axonal Excitability Protocols
<ol>
	<li>Record a stimulus-response curve by incrementally increasing the stimulus until the response is maximal and does not augment when stimulus intensity is further increased.</li>
	<li>The target amplitude for threshold tracking is set automatically to 30%-40% of maximal amplitude, corresponding to the area of steepest slope on the stimulus-response curve.</li>
	<li>As per the method of Bostock et al., 1998, changes in threshold current required to achieve the target amplitude are tracked online.</li>
	<li>Record multiple excitability parameters, including threshold electrotonus (TE), recovery cycle (RC) and current-threshold (I/V) relationship as described in Kiernan et al., 2000 and Kiernan et al., 2001.</li>
	<li>Threshold electrotonus is assessed using 100ms subthreshold polarizing currents, with polarizing current set to &plusmn; 40% of control threshold (Fig. 1). The change in threshold current required to maintain target response amplitude following both de- and hyper-polarization is recorded. Threshold electrotonus provides an assessment of internodal conductances and membrane potential, with responses in the hyperpolarizing direction at the end of the polarizing pulse (90-100 ms) strongly associated with membrane potential.</li>
	<li>The recovery cycle is assessed using a paired pulse paradigm (Fig. 2), with an initial supramaximal conditioning stimulus followed at different intervals by a test stimulus (from 2.5 ms to 200 ms). Following the supramaximal stimulus, it is more difficult to generate a subsequent response, termed 'refractoriness', reflecting the inactivation of voltage-gated Na+ channels. Following the refractory period, a period of facilitation known as superexcitability occurs.</li>
	<li>Current-threshold relationship is assessed using polarizing currents of 200 ms which vary in strength from +50% to -100% of threshold.</li>
</ol>
4. Patient Assessment
<ol>
	<li>Using these techniques, patients are assessed both acutely and longitudinally across chemotherapy treatment. To assess acute neurotoxicity, patients return for post-chemotherapy assessment within 48 hours of receiving treatment.</li>
	<li>To examine chronic neurotoxicity, assessments taken prior to chemotherapy infusion are compared longitudinally across treatment cycles.</li>
	<li>In addition to axonal excitability testing, conventional clinical grading scales should be used to assess chemotherapy-induced neurotoxicity, including the National Cancer Institute - Common Criteria for Adverse Events Neuropathy Sensory Subscale, Total Neuropathy Score and patient-reported outcome assessment.</li>
</ol>
5. Analysis and Interpretation
<ol>
	<li>To identify acute neurotoxicity, results are compared pre- and post-chemotherapy treatment. To assess the development of longitudinal changes in nerve function, results are compared across treatment. Key parameters for assessment include refractoriness, superexcitability, extent of threshold change in threshold electrotonus (hyperpolarizing 90-100 ms), in addition to conventional parameters such as peak amplitude and latency.</li>
	<li>To assess overall change in excitability parameters across treatment, a composite excitability score is calculated. The change in three parameters - superexcitability, refractoriness and threshold electrotonus (hyperpolarizing 90-100 ms) - is summed from initial to final treatments to give an overall marker of change.</li>
</ol>
6. Representative Results
Examples of excitability results in a patient treated with oxaliplatin are provided. Immediately post-oxaliplatin infusion, acute changes in both sensory and motor excitability develop, suggestive of the development of a functional Na+ channelopathy10 -13. However, significant change in multiple excitability parameters develops progressively across oxaliplatin treatment only in sensory axons, with motor axons unaffected (Fig. 3), reflecting widespread sensory axonal damage and membrane potential change. This pattern matches the clinical expression of symptoms in chronic oxaliplatin-induced neurotoxicity. Excitability changes in sensory axons precedes reductions in peak amplitude as assessed using conventional nerve conduction techniques, and suggest that axonal excitability techniques may provide a sensitive assessment tool for early oxaliplatin-induced neurotoxicity.
<img alt="Figure 1" src="/files/ftp_upload/3439/3439fig1.jpg" /> 
Figure 1. Threshold electrotonus, depicting waveforms in response to prolonged subthreshold polarizing current (100 ms), with hyperpolarizing direction plotted in the bottom quadrant (blue) and depolarizing direction plotted in the upper quadrant (red). Below is the stimulus waveform applied to generate the threshold electrotonus response.
<img alt="Figure 2" src="/files/ftp_upload/3439/3439fig2.jpg" /> 
Figure 2. Recovery cycle of excitability, demonstrating the characteristic sequence of excitability changes following impulse conduction, with a period of reduced excitability (refractoriness) up to 3 ms following a supramaximal stimulus, followed by a period of increased excitability (superexcitability) peaking at 5-7 ms and subsequently reduced excitability (subexcitability). The paired pulse paradigm stimulus protocol is inset.
<img alt="Figure 3" src="/files/ftp_upload/3439/3439fig3.jpg" /> 
Figure 3. Excitability changes in sensory axons oxaliplatin-treated patients, with baseline recordings shown in black and post-treatment recordings shown in white, following 4-6 months of oxaliplatin treatment. These changes are thought to reflect widespread axonal damage and membrane potential change. A picture of the excitability set-up in the oncology setting is shown at left. <a href="https://www.jove.com/files/ftp_upload/3439/3439fig3large.jpg" target="_blank">Click here to view larger image</a>.

<ol>
	<li>Hausheer, F. H., Schilsky, R. L., Bain, S., Berghorn, E. J., Lieberman, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis,+management,+and+evaluation+of+chemotherapy-induced+peripheral+neuropathy.">Diagnosis, management, and evaluation of chemotherapy-induced peripheral neuropathy.</a> Semin. Oncol. 33, 15-49 (2008).</li><li>Park, S. B., Krishnan, A. V., Lin, C. S., Goldstein, D., Friedlander, M., Kiernan, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+underlying+chemotherapy-induced+neurotoxicity+and+the+potential+for+neuroprotective+strategies.">Mechanisms underlying chemotherapy-induced neurotoxicity and the potential for neuroprotective strategies.</a> Curr. Med. Chem. 15, 3081-3094 (2008).</li><li>Park, S. B., Lin, C. S., Krishnan, A. V., Goldstein, D., Friedlander, M. L., Kiernan, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+Neuropathy+after+Oxaliplatin+Treatment:+Challenging+the+Dictum+of+Reversibility?.">Long-term Neuropathy after Oxaliplatin Treatment: Challenging the Dictum of Reversibility?.</a> The Oncologist. , (2012).</li><li>Postma, T. J., Heimans, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Grading+of+chemotherapy-induced+peripheral+neuropathy.">Grading of chemotherapy-induced peripheral neuropathy.</a> Ann. Oncol. 11, 509-513 (2000).</li><li>Cascinu, S., Catalano, V., Cordella, L., Labianca, R., Giordani, P., Baldelli, A. M., Beretta, G. D., Ubiali, E., Catalano, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotective+effect+of+reduced+gluthathione+on+oxaliplatin-based+chemotherapy+in+advanced+colorectal+cancer:+a+randomized,+double-blind,+placebo-controlled+trial.">Neuroprotective effect of reduced gluthathione on oxaliplatin-based chemotherapy in advanced colorectal cancer: a randomized, double-blind, placebo-controlled trial.</a> J. Clin. Oncol. 20, 3478-3483 (2002).</li><li>Lehky, T. J., Leonard, G. D., Wilson, R. H., Grem, J. L., Floeter, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxaliplatin-induced+neurotoxicity:+acute+hyperexcitability+and+chronic+neuropathy.">Oxaliplatin-induced neurotoxicity: acute hyperexcitability and chronic neuropathy.</a> Muscle Nerve. 29, 387-392 (2004).</li><li>Bostock, H., Cikurel, K., Burke, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Threshold+tracking+techniques+in+the+study+of+human+peripheral+nerve.">Threshold tracking techniques in the study of human peripheral nerve.</a> Muscle Nerve. 21, 137-158 (1998).</li><li>Burke, D., Kiernan, M. C., Bostock, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excitability+of+human+axons.">Excitability of human axons.</a> Clin. Neurophysiol. 112, 1575-1585 (2001).</li><li>Krishnan, A. K., Lin, C. S., Park, S. B., Kiernan, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+ion+channels+from+bench+to+bedside:+a+translational+neuroscience+perspective.">Axonal ion channels from bench to bedside: a translational neuroscience perspective.</a> Prog. Neurobiol. 89, 288-313 (2009).</li><li>Park, S. B., Lin, C. S., Krishnan, A. V., Goldstein, D., Friedlander, M., Kiernan, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxaliplatin-induced+neurotoxicity:+changes+in+axonal+excitability+precede+development+of+neuropathy.">Oxaliplatin-induced neurotoxicity: changes in axonal excitability precede development of neuropathy.</a> Brain. 132, 2712-2723 (2009).</li><li>Park, S. B., Goldstein, D., Lin, C. S., Krishnan, A. V., Friedlander, M. L., Kiernan, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+abnormalities+of+sensory+nerve+function+associated+wtih+oxaliplatin-induced+neurotoxicity.">Acute abnormalities of sensory nerve function associated wtih oxaliplatin-induced neurotoxicity.</a> J. Clin. Oncol. 27, 1243-1249 (2009).</li><li>Krishnan, A. V., Goldstein, D., Friedlander, M., Kiernan, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxaliplatin-induced+neurotoxicity+and+the+development+of+neuropathy.">Oxaliplatin-induced neurotoxicity and the development of neuropathy.</a> Muscle Nerve. 32, 51-60 (2005).</li><li>Krishnan, A. V., Goldstein, D., Friedlander, M., Kiernan, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxaliplatin+and+axonal+Na++channel+function+in+vivo.">Oxaliplatin and axonal Na+ channel function in vivo.</a> Clin. Cancer Res. 12, 4481-4484 (2006).</li></ol>

We have nothing to disclose.

Chemotherapy-induced neuropathy is a serious side effect of cancer treatment, which may affect treatment course and produce long-lasting patient disability. There is a lack of sensitive and objective assessment measures to specifically measure nerve dysfunction in chemotherapy treated patients. The clinical development of axonal excitability techniques has provided useful and predictive information for the assessment of chemotherapy-induced neurotoxicity. By providing information about ion channel function, resting potential, and axonal membrane function, these techniques enable insight into the pathophysiological processes underlying axonal dysfunction in cancer patients treated with chemotherapy. In addition, axonal excitability techniques have been demonstrated to be feasible in the clinical oncology setting, and a single test may be completed in 5-10 minutes.
In oxaliplatin-treated patients, axonal excitability techniques provide a sensitive biomarker which enables early identification of patients at risk of severe neurotoxicity. In comparison to conventional nerve conduction studies which identify oxaliplatin-induced nerve damage only after axonal loss has already occurred, axonal excitability studies provide predictive markers of nerve dysfunction prior to axonal loss. As such, axonal excitability studies may be utilised to provide assessment of nerve function in clinical trials of potential neuroprotective strategies, to objectively determine neuroprotective efficacy.

<table border="1">
	<tbody>
		<tr>
			<td>Equipment and Materials</td>
			<td>Company</td>
		</tr>
		<tr>
			<td>QTracS program</td>
			<td>Digitimer, Institute of Neurology, Queen Square, UK</td>
		</tr>
		<tr>
			<td>DS5 isolated linear bipolar constant current stimulator</td>
			<td>Digitimer</td>
		</tr>
		<tr>
			<td>Sapphire IIA Amplifier</td>
			<td>Medelec</td>
		</tr>
		<tr>
			<td>Humbug 50/60 Hz Noise eliminator</td>
			<td>Quest Scientific Instruments</td>
		</tr>
		<tr>
			<td>Non-polarizable electrodes</td>
			<td>Unomedical</td>
		</tr>
		<tr>
			<td>Electrosurgical neutral earth plate</td>
			<td>Unomedical</td>
		</tr>
	</tbody>
</table>

nerve excitability assessment in chemotherapy-induced neurotoxicity

Chemotherapy-induced neurotoxicity is a serious consequence of cancer treatment, which occurs with some of the most commonly used chemotherapies1,2. Chemotherapy-induced peripheral neuropathy produces symptoms of numbness and paraesthesia in the limbs and may progress to difficulties with fine motor skills and walking, leading to functional impairment. In addition to producing troubling symptoms, chemotherapy-induced neuropathy may limit treatment success leading to dose reduction or early cessation of treatment. Neuropathic symptoms may persist long-term, leaving permanent nerve damage in patients with an otherwise good prognosis3. As chemotherapy is utilised more often as a preventative measure, and survival rates increase, the importance of long-lasting and significant neurotoxicity will increase.
There are no established neuroprotective or treatment options and a lack of sensitive assessment methods. Appropriate assessment of neurotoxicity will be critical as a prognostic factor and as suitable endpoints for future trials of neuroprotective agents. Current methods to assess the severity of chemotherapy-induced neuropathy utilise clinician-based grading scales which have been demonstrated to lack sensitivity to change and inter-observer objectivity4. Conventional nerve conduction studies provide information about compound action potential amplitude and conduction velocity, which are relatively non-specific measures and do not provide insight into ion channel function or resting membrane potential. Accordingly, prior studies have demonstrated that conventional nerve conduction studies are not sensitive to early change in chemotherapy-induced neurotoxicity4-6. In comparison, nerve excitability studies utilize threshold tracking techniques which have been developed to enable assessment of ion channels, pumps and exchangers in vivo in large myelinated human axons7-9.
Nerve excitability techniques have been established as a tool to examine the development and severity of chemotherapy-induced neurotoxicity10-13. Comprising a number of excitability parameters, nerve excitability studies can be used to assess acute neurotoxicity arising immediately following infusion and the development of chronic, cumulative neurotoxicity. Nerve excitability techniques are feasible in the clinical setting, with each test requiring only 5 -10 minutes to complete. Nerve excitability equipment is readily commercially available, and a portable system has been devised so that patients can be tested in situ in the infusion centre setting. In addition, these techniques can be adapted for use in multiple chemotherapies.
In patients treated with the chemotherapy oxaliplatin, primarily utilised for colorectal cancer, nerve excitability techniques provide a method to identify patients at-risk for neurotoxicity prior to the onset of chronic neuropathy. Nerve excitability studies have revealed the development of an acute Na+ channelopathy in motor and sensory axons10-13. Importantly, patients who demonstrated changes in excitability in early treatment were subsequently more likely to develop moderate to severe neurotoxicity11. However, across treatment, striking longitudinal changes were identified only in sensory axons which were able to predict clinical neurological outcome in 80% of patients10. These changes demonstrated a different pattern to those seen acutely following oxaliplatin infusion, and most likely reflect the development of significant axonal damage and membrane potential change in sensory nerves which develops longitudinally during oxaliplatin treatment10. Significant abnormalities developed during early treatment, prior to any reduction in conventional measures of nerve function, suggesting that excitability parameters may provide a sensitive biomarker.

Watch this Scientific Journal Video about Nerve Excitability Assessment in Chemotherapy-induced Neurotoxicity at JoVE.com

Nerve Excitability Assessment in Chemotherapy-induced Neurotoxicity

This abstract describes a novel method to assess the development of neurotoxicity in patients receiving chemotherapy treatment. While conventional assessment methods are limited in their ability to detect early changes in nerve function, nerve excitability techniques provide early identification of patients at risk of severe neurotoxicity and insight into pathophysiology.

Chemotherapy-induced neurotoxicity is a serious consequence of cancer treatment, which occurs with some of the most commonly used chemotherapies1,2. ...

nerve-excitability-assessment-in-chemotherapy-induced-neurotoxicity

Research

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Neuroscience

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Assessment of Neuromuscular Function Using Percutaneous Electrical Nerve Stimulation

Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels). The present protocol describes how this method can be used to stimulate the posterior tibial nerve that activates plantar flexor muscles. Percutaneous electrical nerve stimulation consists of inducing an electrical stimulus to a motor nerve to evoke a muscular response. Direct (M-wave) and/or indirect (H-reflex) electrophysiological responses can be recorded at rest using surface electromyography. Mechanical (twitch torque) responses can be quantified with a force/torque ergometer. M-wave and twitch torque reflect neuromuscular transmission and excitation-contraction coupling, whereas H-reflex provides an index of spinal excitability. EMG activity and mechanical (superimposed twitch) responses can also be recorded during maximal voluntary contractions to evaluate voluntary activation level. Percutaneous nerve stimulation provides an assessment of neuromuscular function in humans, and is highly beneficial especially for studies evaluating neuromuscular plasticity following acute (fatigue) or chronic (training/detraining) exercise.

We present a protocol to assess changes in neuromuscular function. Percutaneous electrical nerve stimulation is a non-invasive method that evokes muscular responses. Electrophysiological and mechanical properties of these responses permit the evaluation of neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels).

Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, ...

Microglia as a Surrogate Biosensor to Determine Nanoparticle Neurotoxicity

Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay described here will aid in elucidating the role of microglia in neuroinflammation and neurodegenerative diseases. The use of microglia, resident immune cells of the brain, as a surrogate biosensor provides novel insight into how inflammatory responses mediate neuronal insults. Here, we utilize an immortalized murine microglial cell line, designated BV2, and describe a method for nanoparticle exposure using silver nanoparticles (AgNPs) as a standard. We describe how to expose microglia to nanoparticles, how to remove nanoparticles from supernatant, and how to use supernatant from activated microglia to determine toxicity, using hypothalamic cell survival as a measure. Following AgNP exposure, BV2 microglial activation was validated using a tumor necrosis factor alpha (TNF-&#945;) enzyme linked immunosorbent assay (ELISA). The supernatant was filtered to remove the AgNP and to allow cytokines and other secreted factors to remain in the conditioned media. Hypothalamic cells were then exposed to supernatant from AgNP activated microglia and survival of neurons was determined using a resazurin-based fluorescent assay. This technique is useful for utilizing microglia as a surrogate biomarker of neuroinflammation and determining the effect of neuroinflammation on other cell types.

Microglia (immune cells of the brain), are used as a surrogate biosensor to determine how nanoparticles influence neurotoxicity. We describe a series of experiments designed to assay microglial response to nanoparticles and exposure of hypothalamic neurons to supernatant from activated microglia to determine neurotoxicity.

Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay ...

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, their distinct dendritic morphologies, their specific patterns of axonal connectivity, and their selective firing responses. The molecular and cellular mechanisms responsible for these aspects of differentiation during development are still poorly understood.
Here, we describe combined protocols for labeling and characterizing the structural connectivity and excitability of cortical neurons. Modification of the in utero electroporation (IUE) protocol allows the labeling of a sparse population of neurons. This, in turn, enables the identification and tracking of the dendrites and axons of individual neurons, the precise characterization of the laminar location of axonal projections, and morphometric analysis. IUE can also be used to investigate changes in the excitability of wild-type (WT) or genetically modified neurons by combining it with whole-cell recording from acute slices of electroporated brains. These two techniques contribute to a better understanding of the coupling of structural and functional connectivity and of the molecular mechanisms controlling neuronal diversity during development. These developmental processes have important implications on axonal wiring, the functional diversity of neurons, and the biology of cognitive disorders.

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

This manuscript provides protocols that use in utero electroporation (IUE) to describe the structural connectivity of neurons at the single-cell level and the excitability of fluorescently labeled neurons. Histology is used to characterize dendritic and axonal projections. Whole-cell recording in acute slices is used to investigate excitability.

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...

In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing

Electrophysiology enables the objective assessment of peripheral nerve function in vivo. Traditional nerve conduction measures such as amplitude and latency detect chronic axon loss and demyelination, respectively. Axonal excitability techniques &#34;by threshold tracking&#34; expand upon these measures by providing information regarding the activity of ion channels, pumps and exchangers that relate to acute function and may precede degenerative events. As such, the use of axonal excitability in animal models of neurological disorders may provide a useful in vivo measure to assess novel therapeutic interventions. Here we describe an experimental setup for multiple measures of motor axonal excitability techniques in the rat ulnar nerve.
The animals are anesthetized with isoflurane and carefully monitored to ensure constant and adequate depth of anesthesia. Body temperature, respiration rate, heart rate and saturation of oxygen in the blood are continuously monitored. Axonal excitability studies are performed using percutaneous stimulation of the ulnar nerve and recording from the hypothenar muscles of the forelimb paw. With correct electrode placement, a clear compound muscle action potential that increases in amplitude with increasing stimulus intensity is recorded. An automated program is then utilized to deliver a series of electrical pulses which generate 5 specific excitability measures in the following sequence: stimulus response behavior, strength duration time constant, threshold electrotonus, current-threshold relationship and the recovery cycle.
Data presented here indicate that these measures are repeatable and show similarity between left and right ulnar nerves when assessed on the same day. A limitation of these techniques in this setting is the effect of dose and time under anesthesia. Careful monitoring and recording of these variables should be undertaken for consideration at the time of analysis.

In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing

Axonal excitability techniques provide a powerful tool to examine pathophysiology and biophysical changes that precede irreversible degenerative events. This manuscript demonstrates the use of these techniques on the ulnar nerve of anesthetized rats.

Electrophysiology enables the objective assessment of peripheral nerve function in vivo. Traditional nerve conduction measures such as amplitude and ...

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 ...

Purification of Fibroblasts and Schwann Cells from Sensory and Motor Nerves in Vitro

The principal cells in the peripheral nervous system are the Schwann cells (SCs) and the fibroblasts. Both these cells distinctly express the sensory and motor phenotypes involved in different patterns of neurotrophic factor gene expression and other biological processes, affecting nerve regeneration. The present study has established a protocol to obtain highly purified rat sensory and motor SCs and fibroblasts more rapidly. The ventral root (motor nerve) and the dorsal root (sensory nerve) of neonatal rats (7-days-old) were dissociated and the cells were cultured for 4-5 days, followed by isolation of sensory and motor fibroblasts and SCs by combining differential digestion and differential adherence methods sequentially. The results of immunocytochemistry and flow cytometry analyses showed that the purity of the sensory and motor SCs and fibroblasts were &#62;90%. This protocol can be used to obtain a large number of sensory and motor fibroblasts/SCs more rapidly, contributing to the exploration of sensory and motor nerve regeneration.

Here, we present a method to purify fibroblasts and Schwann cells from sensory and motor nerves in vitro.

The principal cells in the peripheral nervous system are the Schwann cells (SCs) and the fibroblasts. Both these cells distinctly express the sensory and ...

Multifactorial Assessment of Motor Behavior in Rats after Unilateral Sciatic Nerve Crush Injury

The induction of a peripheral nerve injury is a widely used method in neuroscience for the assessment of repair and pain mechanisms among others. In addition, in the research field of movement disorders, sciatic crush injury has been employed to trigger a dystonia-like phenotype in genetically predisposed DYT-TOR1A rodent models of dystonia. To achieve consistent, reproducible and comparable results after a sciatic nerve crush injury, a standardized method for inducing the nerve crush is essential, in addition to a standardized phenotypical characterization. Attention must be paid not only to the specific assortment of behavioral tests, but also to the technical requirements, the correct execution and consecutive data analysis. This protocol describes in detail how to perform a sciatic nerve crush injury and provides a behavioral test battery for the assessment of motor deficits in rats that includes the open field test, the CatWalk XT gait analysis, the beam walking task, and the ladder rung walking task.

We provide a protocol for the assessment of motor behavior via a behavioral test battery in rats after sciatic nerve crush injury.

The induction of a peripheral nerve injury is a widely used method in neuroscience for the assessment of repair and pain mechanisms among others. In ...

Brain Ventricular Microinjections of Lipopolysaccharide into Larval Zebrafish to Assess Neuroinflammation and Neurotoxicity

Neuroinflammation is a key player in various neurological disorders, including neurodegenerative diseases. Therefore, it is of great interest to research and develop alternative in vivo neuroinflammation models to understand the role of neuroinflammation in neurodegeneration. In this study, a larval zebrafish model of neuroinflammation mediated by ventricular microinjection of lipopolysaccharide (LPS) to induce an immune response and neurotoxicity was developed and validated. The transgenic zebrafish lines elavl3:mCherry, ETvmat2:GFP, and mpo:EGFP were used for real-time quantification of brain neuron viability by fluorescence live imaging integrated with fluorescence intensity analysis. The locomotor behavior of zebrafish larvae was recorded automatically using a video-tracking recorder. The content of nitric oxide (NO), and the mRNA expression levels of inflammatory cytokines including interleukin-6 (IL-6), interleukin-1&#946; (IL-1&#946;), and human tumor necrosis factor &#945; (TNF-&#945;) were investigated to assess the LPS-induced immune response in the larval zebrafish head. At 24 h after the brain ventricular injection of LPS, loss of neurons and locomotion deficiency were observed in zebrafish larvae. In addition, LPS-induced neuroinflammation increased NO release and the mRNA expression of IL-6, IL-1&#946;, and TNF-&#945; in the head of 6 days post fertilization (dpf) zebrafish larvae, and resulted in the recruitment of neutrophils in the zebrafish brain. In this study, injection of zebrafish with LPS at a concentration of 2.5-5 mg/mL at 5 dpf was determined as the optimum condition for this pharmacological neuroinflammation assay. This protocol presents a new, quick, and efficient methodology for brain ventricle microinjection of LPS to induce LPS-mediated neuroinflammation and neurotoxicity in a zebrafish larva, which is useful for studying neuroinflammation and could also be used as a high-throughput in vivo drug screening assay.

This protocol demonstrates the microinjection of lipopolysaccharide into the brain ventricular region in a zebrafish larval model to study the resulting neuroinflammatory response and neurotoxicity.

Neuroinflammation is a key player in various neurological disorders, including neurodegenerative diseases. Therefore, it is of great interest to research ...

Assessing Corticospinal Excitability During Goal-Directed Reaching Behavior

Reaching is a widely studied behavior in motor physiology and neuroscience research. While reaching has been examined using a variety of behavioral manipulations, there remain significant gaps in the understanding of the neural processes involved in reach planning, execution, and control. The novel approach described here combines a two-dimensional reaching task with transcranial magnetic stimulation (TMS) and concurrent electromyography (EMG) recording from multiple muscles. This method allows for the noninvasive detection of corticospinal activity at precise time points during the unfolding of reaching movements. The example task code includes a delayed response reaching task with two possible targets displayed &#177; 45&#176; off the midline. Single pulse TMS is delivered on the majority of task trials, either at the onset of the preparatory cue (baseline) or 100 ms prior to the imperative cue (delay). This sample design is suitable for investigating changes in corticospinal excitability during reach preparation. The sample code also includes a visuomotor perturbation (i.e., cursor rotation of &#177; 20&#176;) to investigate the effects of adaptation on corticospinal excitability during reach preparation. The task parameters and TMS delivery can be adjusted to address specific hypotheses about the state of the motor system during reaching behavior. In the initial implementation, motor evoked potentials (MEPs) were successfully elicited on 83% of TMS trials, and reach trajectories were recorded on all trials.

Reaching is a fundamental skill that allows humans to interact with the environment. Several studies have aimed to characterize reaching behavior using a variety of methodologies. This paper offers an open-source application of transcranial magnetic stimulation to assess the state of corticospinal excitability in humans during reaching task performance.

Reaching is a widely studied behavior in motor physiology and neuroscience research. While reaching has been examined using a variety of behavioral ...