Name: portable thermographic screening for detection of acute wallenberg's syndrome
Uploaded: 2019-09-19T12:30:00
Description: Watch this Scientific Journal Video about Portable Thermographic Screening for Detection of Acute Wallenberg's Syndrome at JoVE.com

Not applicable

All methods described here were approved by the Human Research Ethics Committee Institutional Review Board of Kanto Central Hospital.
NOTE: We used a commercially available portable thermal camera and dedicated software (see the Table of Materials), and have constructed our protocols based on the use of these specific instruments.
1. Preparation for Measurements
<ol>
	<li>Charge the thermal camera before use.</li>
	<li>Turn on the...

<ol>
	<li>Kim, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pure+lateral+medullary+infarction:+Clinical-radiological+correlation+of+130+acute,+consecutive+patients.">Pure lateral medullary infarction: Clinical-radiological correlation of 130 acute, consecutive patients.</a> Brain. 126 (8), 1864-1872 (2003).</li><li>Kameda, W., 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=Lateral+and+medial+medullary+infarction:+a+comparative+analysis+of+214+patients.">Lateral and medial medullary infarction: a comparative analysis of 214 patients.</a> Stroke. 35 (3), 694-699 (2004).</li><li>Nowak, D. A., Topka, H. 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S., Park, S. C., Lee, Y. J., Kang, J. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Clinical Autonomic Disorders: Evaluation and Management. , (1997).</li><li>Takahashi, M., 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=Teaching+NeuroImages:+the+half-split+man.">Teaching NeuroImages: the half-split man.</a> Neurology. 87 (11), (2016).</li><li>Vardasco, R., Ring, F., Plassmann, P., Jones, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermal+symmetry+of+the+upper+and+lower+extremities+in+healthy+subjects.">Thermal symmetry of the upper and lower extremities in healthy subjects.</a> Thermology International. 22 (2), 53-60 (2012).</li><li>Nakazato, Y., Shimazu, K., Tamura, N., Hamaguchi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+study+of+skin+surface+temperature+in+patients+with+unilateral+cerebral+infarction--with+special+reference+to+central+autonomic+regulation+of+skin+vasomotor+response.">A study of skin surface temperature in patients with unilateral cerebral infarction--with special reference to central autonomic regulation of skin vasomotor response.</a> Clinical Neurology. 35 (7), 758-763 (1995).</li><li>Motobe, K., 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=Cut-off+value+of+the+ankle-brachial+pressure+index+at+which+the+accuracy+of+brachial-ankle+pulse+wave+velocity+measurement+is+diminished.">Cut-off value of the ankle-brachial pressure index at which the accuracy of brachial-ankle pulse wave velocity measurement is diminished.</a> Circulation Journal. 69 (1), 55-60 (2005).</li><li>International Academy of Clinical Thermography. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Thermography Guidelines: Standards and protocols in clinical thermographic imaging. , (2002).</li></ol>

Critical steps of this protocol are the establishment the initial suspicion of WS and the decision to acquire thermographic images of the patient. Acquiring thermographic images and assessing the laterality of BST is a simple approach, even for non-neurologists who may examine patients upon initial presentation to the emergency department or a general clinic. If a patient exhibits laterality of BST, especially at multiple locations on the same side, the physician should consider the possibility of WS. Because most WS pat...

Wallenberg&#39;s syndrome (WS) is a type of brainstem infarction. Acute WS patients are sometimes initially misdiagnosed with non-stroke diseases because of the symptomatic and magnetic resonance imaging (MRI) characteristics of WS. To accurately diagnose acute WS, careful neurological examination is necessary, which may be difficult for non-neurologists to whom affected patients initially present. Here, we present a simple, rapid, noninvasive, and cost-effective method for the detection of acute WS using portable thermography.
WS is caused by the infarction of a wedge of the dorsal lateral medulla oblongata, due to occlusion of the vertebral artery or posterior inferior cerebellar artery1,2. WS may be misdiagnosed as a non-stroke disease because of a combination of unique symptomatic and MRI characteristics that contrast with those typically observed in cerebral infarction. Hemiparesis and tactile sensory disturbance, which tend to be observed in patients with other types of cerebral infarction, are rare in WS patients; however, they exhibit various combinations of clinical symptoms, including hoarseness and dysphagia, dissociated sensory disturbance, vertigo, gaze-induced nystagmus, ataxia, and Horner&#39;s syndrome1,2,3,4,5,6,7,. Another unique characteristic of WS patients is the limited severity of symptoms, which is similar to that in other types of brainstem infarctions7,8,9,10,11. Some patients with brainstem infarctions have arrived at the outpatient clinic on foot and reported only minor complaints7. In some patients with WS, vertigo is the only presenting symptom, and it can therefore be difficult to differentiate between WS and auditory vertigo12. Furthermore, WS can affect young patients, due to its potential etiology of artery dissection2. MRI analysis of brainstem infarction, including WS, is unique in that the high-intensity diffusion-weighted imaging signal may be delayed in some patients7,13,14.
The above characteristics are thought to cause misdiagnosis of WS. Dysphagia may cause aspiration pneumonia or asphyxia, and artery dissection may cause subarachnoid hemorrhage15; therefore, overlooking WS may result in the development of life-threatening conditions for the patient. Although careful neurological examination is necessary to prevent the misdiagnosis of WS, it is likely that a patient will first present to a non-neurologist. Therefore, a rapid and simple method for screening of acute WS may be clinically useful.
Previously, we reported that 89% of acute WS patients exhibit laterality of BST, which is presumed to result from disturbance of the central autonomic nervous tract due to infarction at the lateral medulla7. Because this autonomic nervous tract descends from the lateral brainstem (including the ventro-lateral medulla) and contains the connective pathway of sweating and skin blood flow16, disturbance of sweating and vasoconstriction lead to increased BST on the ipsilateral side of WS. In the prior report, we also showed that the laterality of BST can be easily detected within 2 min using thermographic measurement in most patients with WS7,17. Here, we report a method for the detection of laterality of BST using thermography, which may be useful in preventing misdiagnosis of acute WS.

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			<td>FLIR E5</td>
			<td>FLIR Systems</td>
			<td>P/N: 63905-0501</td>
			<td></td>
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			<td>FLIR Tools</td>
			<td>FLIR Systems</td>
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			<td>RRID:SCR_016330</td>
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portable thermographic screening for detection of acute wallenberg's syndrome

Wallenberg&#39;s syndrome (WS) is a type of brainstem infarction. WS patients often show Horner&#39;s syndrome, dissociated sensory disturbance, truncal ataxia, and hoarseness. However, they rarely show tactile sensory disturbance and paralysis of the extremities. Additionally, acute brainstem infarction is often not apparent in magnetic resonance images. These symptomatic and imaging characteristics sometimes lead to misdiagnosis of WS as a non-stroke disease, including auditory vertigo. Although careful neurological examination is necessary to prevent misdiagnosis of WS, this type of examination may be difficult for non-neurologists to whom affected patients initially present. Lateral differences in body surface temperature (BST) constitute a recognized and widespread symptom of WS. We previously reported that most acute WS patients exhibit lateral differences in BST at multiple locations and that these lateral differences in BST could easily be detected by thermographic measurement. Here, we present the method for use of portable thermography to detect acute WS, using a simple, rapid, noninvasive, and cost-effective approach. To assess lateral differences in BST among patients with suspected WS, BST was measured as soon as possible in the examination room or in the patient&#39;s bedroom. Measurements were performed bilaterally at four locations where images could easily be acquired (face, palm of the hand, abdomen, and dorsum of the foot) using a portable thermal camera. When lateral differences in BST are observed macroscopically, especially in multiple locations on the same side, a diagnosis of WS should be suspected. Macroscopic assessment of BST laterality can be made within 2 min of the acquisition of thermographic images. This method may be useful in preventing misdiagnosis of acute WS as a non-stroke disease, especially when such patients initially present to non-neurologists.

Acquisition of thermographic images and macroscopic assessment to determine whether BST exhibits laterality may be performed within 2 min in most patients. Most acute WS patients exhibit laterality of BST at multiple locations. Some patients exhibit laterality of BST throughout the body (Figure 2A), whereas some exhibit laterality only in a few locations (Figure 2B). The warmer side, as determine...

Watch this Scientific Journal Video about Portable Thermographic Screening for Detection of Acute Wallenberg's Syndrome at JoVE.com

Portable Thermographic Screening for Detection of Acute Wallenberg's Syndrome

Acute Wallenberg&#39;s syndrome can be misdiagnosed as a non-stroke disease, such as auditory vertigo. Thus, careful neurological examination, which is sometimes difficult for non-neurologists, is necessary for precise diagnosis. Here, we present a simple, rapid, noninvasive, and cost-effective method for detection of acute Wallenberg&#39;s syndrome using portable thermography.

Wallenberg&#39;s syndrome (WS) is a type of brainstem infarction. WS patients often show Horner&#39;s syndrome, dissociated sensory disturbance, truncal ...

Wallenberg's syndrome is caused by the infarction of the lateral medulla and is sometimes misdiagnosed as non-stroke disease. Using portable thermography may help prevent the misdiagnosis of this neurological condition. This method is a very simple, rapid, non-invasive and cost-effective technique that allows the possibility of Wallenberg's syndrome to be easily determined.Before beginning the measurements, charge and turn on the thermal camera. Press the center button on the camera to display the menu and select the color in iron or rainbow. Then press the center button on the camera to display the menu again and select measurement and center spot measurement to allow measurement of the body surface temperature in real time.For Wallenberg's syndrome screening select patients with dizziness, vertigo, ptosis, hoarseness, dysphagia, and isochoria or dissociated sensory disturbance as all of the symptoms have been associated with Wallenberg's syndrome. It is important to measure the body surface temperature by thermography in any patient in which Wallenberg's syndrome is suspected. To subjectively determine the laterality of the body surface temperature first palpate patients with suspected Wallenberg's syndrome.To acquire thermographic images instruct the patient to remove their socks and shoes and have the patient remove enough clothing to enable examination of the abdominal region as per their consent. As soon as Wallenberg's syndrome is suspected have the patient assume a supine posture, or sitting position, and have a photographer hand 50 to 100 centimeters from the patient. Then have the photographer acquire one image each at the frontal face, bilateral palm, abdomen, and bilateral dorsum of the foot.Consider body surface temperature to exhibit laterality when the body surface temperature between the right and left sides is markedly different macroscopically and the degree of laterality is greater than a half of a degree Celsius. If laterality of the body surface temperature is not obvious or appears to be less than half of a degree Celsius initialize the thermal imaging and analysis software and open the acquired images to be analyzed. Use the ellipse measurement button to indicate ellipses with diameters of greater than five millimeters at that nasolabial fold on the face, the palm of the hand, the thoracic spine levels eight to 10 approximately five centimeters from the umbilicus of the torso, and at the center of the dorsum of the foot.Then measure the average body surface temperature from each location and compare it to the body surface temperature on the contralateral side of the body. Some patients exhibit laterality of the body surface temperature throughout the body, whereas some exhibit laterality in only a few locations. The warmer side, as determined by the body surface temperature, is ipsolateral to the location of Wallenberg's syndromes.Importantly however, a Wallenberg's syndrome patient with a very small infarction may not exhibit body surface temperature laterality. When a patient does not exhibit a central nervous disorder laterality of the body surface temperature is typically not observed. However, when a patient exhibits vascular stenosis the body surface temperature maybe be lower in the extremities with vascular stenosis than in extremities on the contralateral side, and these patients may exhibit vascular stenosis in both the upper and lower limbs.The precise degree of the body surface temperature can be analyzed with dedicated software and may be required when the laterality of the body surface temperature is not apparent macroscopically. It is important to remember that this method is used for the screening of Wallenberg's syndrome, not for confirming the diagnosis of Wallenberg's syndrome. To confirm a Wallenberg's syndrome diagnosis a careful neurological examination and repeated brain stem MRIs are needed.Consider hospitalization when the possibility of Wallenberg's syndrome cannot be excluded.

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Research

JoVE Journal

Neuroscience

Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. Since then, the technique was developed further and currently there are many different ways to prepare organotypic cultures. The method presented here was adapted from the one described by Stoppini et al. for the preparation of the slices and from Gogolla et al. for the staining procedure 3,4.
A unique feature of this technique is that it allows you to study different parts of the brain such as hippocampus or cerebellum in their original structure, providing a big advantage over dissociated cultures in which all the cellular organization and neuronal networks are disrupted. In the case of the cerebellum it is even more advantageous because it allows the study of Purkinje cells, extremely difficult to obtain as dissociated primary culture. This method can be used to study certain developmental features of the cerebellum in vitro, as well as for electrophysiological and pharmacological experiments in both wild type and mutant mice.
The method described here was designed to study the effect of apoptotic stimuli such as Fas ligand in the developing cerebellum, using TUNEL staining to measure apoptotic cell death. If TUNEL staining is combined with cell type specific markers, such as Calbindin for Purkinje cells, it is possible to evaluate cell death in a cell population specific manner. The Calbindin staining also serves the purpose of evaluating the quality of the cerebellar cultures.

This method describes the generation of organotypic cerebellar cultures and the effect of certain apoptotic stimuli on the viability of different cerebellar cell types.

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. ...

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells.
Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis.
Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to neuroblasts able to move along the rostral migratory stream (RMS) towards the olfactory bulb (OB). This long-distance migration is required for the subsequent maturation of newborn neurons in the OB, but the molecular mechanisms regulating this process are still unclear. Investigating the signaling pathways controlling neuroblast motility may not only help understand a fundamental step in neurogenesis, but also have therapeutic regenerative potential, given the ability of these neuroblasts to target brain sites affected by injury, stroke, or degeneration.
In this manuscript we describe a detailed protocol for in vivo postnatal electroporation and subsequent time-lapse imaging of neuroblast migration in the mouse RMS. Postnatal electroporation can efficiently transfect SVZ progenitor cells, which in turn generate neuroblasts migrating along the RMS. Using confocal spinning disk time-lapse microscopy on acute brain slice cultures, neuroblast migration can be monitored in an environment closely resembling the in vivo condition. Moreover, neuroblast motility can be tracked and quantitatively analyzed. As an example, we describe how to use in vivo postnatal electroporation of a GFP-expressing plasmid to label and visualize neuroblasts migrating along the RMS. Electroporation of shRNA or CRE recombinase-expressing plasmids in conditional knockout mice employing the LoxP system can also be used to target genes of interest. Pharmacological manipulation of acute brain slice cultures can be performed to investigate the role of different signaling molecules in neuroblast migration. By coupling in vivo electroporation with time-lapse imaging, we hope to understand the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair.

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video tracking.

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to ...

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the experimental time, and increases the number of animals that are utilized per study. This limitation specifically impacts protocols such as calcium imaging that require prolonged pre-incubation with bath-applied dyes. Exponential bacterial growth within 3 - 4 h after slicing is tightly correlated with a decrease in tissue health. This study describes a method for&#160;limiting the proliferation of bacteria in acute preparations to maintain viable neuronal tissue for prolonged periods of time (&#62;24 h) without the need for antibiotics, sterile procedures, or tissue culture media containing growth factors. By cycling the extracellular fluid through UV irradiation and keeping the tissue in a custom holding chamber at 15 - 16 &#176;C, the tissue shows no difference in electrophysiological properties, or calcium signaling through intracellular calcium dyes at &#62;24 h postdissection. These methods will not only extend experimental time for those using acute neuronal tissue, but will reduce the number of animals required to complete experimental goals, and will set a gold standard for acute neuronal tissue incubation.

Once removed from the body, neuronal tissue is greatly affected by environmental conditions, leading to eventual degradation of the tissue after 6 - 8 h. Using a unique incubation method, which closely monitors and regulates the extracellular environment of the tissue, tissue viability can be significantly extended for &#62;24 h.

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...

Optimized Management of Endovascular Treatment for Acute Ischemic Stroke

This manuscript describes a streamlined protocol for the management of patients with acute ischemic stroke, which aims at the minimization of time from hospital admission to reperfusion. Rapid restoration of cerebral blood flow is essential for the outcomes of patients with acute ischemic stroke. Endovascular treatment (EVT) has become the standard of care to accomplish this in patients with acute stroke due to large vessel occlusion (LVO). To achieve reperfusion of ischemic brain regions as fast as possible, all in-hospital time delays have to be carefully avoided. Therefore, management of patients with acute ischemic stroke was optimized with an interdisciplinary standard operating procedure (SOP). Stroke neurologists, diagnostic as well as interventional neuroradiologists, and anesthesiologists streamlined all necessary processes from patient admission and diagnosis to EVT of eligible patients. Target times for every step were established. Actually achieved times were prospectively recorded along with clinical data and imaging scores for all endovascularly treated stroke patients. These data were regularly analyzed and discussed in interdisciplinary team meetings. Potential issues were evaluated and all staff involved was trained to adhere to the SOP. This streamlined patient management approach and enhanced interdisciplinary collaboration reduced time from patient admission to reperfusion significantly and was accompanied by a beneficial effect on clinical outcomes.

The outcome of patients with acute ischemic stroke depends on swift restoration of cerebral blood flow. This protocol aims at optimizing the management of such patients by minimizing peri-procedural timings and rendering the time from hospital admission to reperfusion as short as possible.

This manuscript describes a streamlined protocol for the management of patients with acute ischemic stroke, which aims at the minimization of time from ...

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc superoxide dismutase 1 (SOD1). The structural instability of SOD1 and the detection of SOD1-positive inclusions in familial-ALS patients supports a potential causal role for misfolded and/or aggregated SOD1 in ALS pathology. In this study, we describe the development of a cell-based assay designed to quantify the dynamics of SOD1 aggregation in living cells by high content screening approaches. Using lentiviral vectors, we generated stable cell lines expressing wild-type and mutant A4V SOD1 tagged with yellow fluorescent protein and found that both proteins were expressed in the cytosol without any sign of aggregation. Interestingly, only SOD1 A4V stably expressed in HEK-293, but not in U2OS or SH-SY5Y cell lines, formed aggregates upon proteasome inhibitor treatment. We show that it is possible to quantify aggregation based on dose-response analysis of various proteasome inhibitors, and to track aggregate-formation kinetics by time-lapse microscopy. Our approach introduces the possibility of quantifying the effect of ALS mutations on the role of SOD1 in aggregate formation as well as screening for small molecules that prevent SOD1 A4V aggregation.

We describe a method to quantify the aggregation of misfolded proteins. Our protocol details lentiviral induced stable cell line generation, automated confocal imaging, and image analysis of protein aggregates. As an illustrative application, we studied the effect of small molecules in promoting SOD1 aggregation in a time- and dose-dependent manner.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...

Screening of Axonal Degeneration in Carpal Tunnel Syndrome Using Ultrasonography and Nerve Conduction Studies

Axonal degeneration, indicative of surgical decompression, may coexist in carpal tunnel syndrome (CTS) as the disease progresses. However, the current diagnostic and severity gradation system cannot clearly indicate its coexistence, resulting in&#160;confusion of appropriate treatment prescription. There are also constraints in conventional methods for&#160;differentiation as well. This study aims at introducing an innovative, efficient, and quick screening protocol to differentiate axonal degeneration associated with CTS, using ultrasound and nerve conduction studies (NCS). It starts by using NCS to perform orthodromic stimulation at the wrist, to obtain the sensory conduction of the median and the ulnar nerves respectively. Meanwhile, the motor conduction&#160;of the median nerve is collected by stimulating the palm, wrist, and elbow, followed by the stimulation of the ulnar nerve at the wrist,&#160;below and above the elbow. Then, an ultrasound assessment is performed, using a linear array transducer, with cross-sectional area (CSA) and perimeter (P) at the wrist and at the one-third distal forearm calipered. Ratios (R-CSA, R-P) and changes from wrist to one-third distal forearm (&#916;CSA and &#916;P) are calculated according to a standard format. Potential axonal degeneration coexisting in CTS will be screened according to the criteria of NCS and cut-off values of ultrasound measurements established in a previous study. In terms of its noninvasiveness, low cost, convenience, and efficiency, it is easy to apply ultrasound complimentarily in clinical practice to prescreen patients with potential coexisting axonal degeneration. Nevertheless, the ultrasonographic imaging cannot directly reflect axonal degeneration. It still relies on conventional but invasive methods&#160;such as electromyography (EMG) and biopsy&#160;for confirmation if needed.

Here we present a protocol using nerve conduction studies and ultrasound to screen potential axonal degeneration associated with carpal tunnel syndrome. The criteria for differentiation are established. Compared to conventional approaches, this method is noninvasive, convenient, and efficient, with an overall satisfactory accuracy, sensitivity, and specificity.

Axonal degeneration, indicative of surgical decompression, may coexist in carpal tunnel syndrome (CTS) as the disease progresses. However, the current ...

Preparation of Acute Human Hippocampal Slices for Electrophysiological Recordings

Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for sudden death. Despite an abundance of available treatment options, about 30% of patients are drug-resistant. Several novel therapeutics have been developed using animal models, though the rate of drug-resistant patients remains unaltered. One of probable reasons is the lack of translation between rodent models and humans, such as a weak representation of human pharmacoresistance in animal models. Resected human brain tissue as a preclinical evaluation tool has the advantage to bridge this translational gap. Described here is a method for high quality preparation of human hippocampal brain slices and subsequent stable induction of epileptiform activity. The protocol describes the induction of burst activity during application of 8 mM KCl and 4-aminopyridin. This activity is sensitive to established AED lacosamide or novel antiepileptic candidates, such as dimethylethanolamine (DMEA). In addition, the method describes induction of seizure-like events in CA1 of human hippocampal brain slices by reduction of extracellular Mg2+ and application of bicuculline, a GABAA receptor blocker. The experimental set-up can be used to screen potential antiepileptic substances for their effects on epileptiform activity. Furthermore, mechanisms of action postulated for specific compounds can be validated using this approach in human tissue (e.g., using patch-clamp recordings). To conclude, investigation of vital human brain tissue ex vivo (here, resected hippocampus from patients suffering from temporal lobe epilepsy) will improve the current knowledge of physiological and pathological mechanisms in the human brain.

The presented protocol describes the transport and preparation of resected human hippocampal tissue with the ultimate goal to use vital brain slices as a preclinical evaluation tool for potential antiepileptic substances.

Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for ...

Detection of Abnormal Prion Protein by Immunohistochemistry

Abnormal prion proteins (PrPSc) are the disease-associated isoform of cellular prion protein and diagnostic markers of transmissible spongiform encephalopathies (TSEs). These neurodegenerative diseases affect humans and several animal species and include scrapie, zoonotic bovine spongiform encephalopathy (BSE), chronic wasting disease of cervids (CWD), and the newly identified camel prion disease (CPD). Diagnosis of TSEs relies on immunodetection of PrPSc by application of both immunohistochemistry (IHC) and western immunoblot methods (WB) on encephalon tissues, namely, the brainstem (obex level). IHC is a widely used method that uses&#160;primary antibodies (monoclonal or polyclonal) against antigens of interest in cells of a tissue section. The antibody-antigen binding can be visualized by a color reaction that remains localized in the area of the tissue or cell where the antibody was targeted. As such, in prion diseases, as in other fields of research, the immunohistochemistry techniques are not solely used for diagnostic purposes but also in pathogenesis studies. Such studies involve detecting the PrPSc patterns and types from those previously described to identify the new prion strains. As BSE can infect humans, it is recommended that biosafety laboratory level-3 (BSL-3) facilities and/or practices are used to handle cattle, small ruminants, and cervid samples included in the TSE surveillance. Additionally, containment and prion-dedicated equipment are recommended, whenever possible, to limit contamination. The PrPSc IHC procedure consists of a formic acid epitope-demasking step also acting as a prion inactivation measure, as formalin-fixed and paraffin-embedded tissues used in this technique remain infectious. When interpreting the results, care must be taken to distinguish non-specific immunolabeling from target labeling. For this purpose, it is important to recognize artifacts of immunolabeling obtained in known TSE-negative control animals to differentiate those from specific PrPSc immunolabeling types, which can vary between TSE strains, host species, and prnp genotype, further described herein.

Immunolabeling abnormal prion protein using immunohistochemistry protocols requires specific sample and anti-PrP antibody preparation methodologies. The present protocol describes the key steps in epitope demasking to ensure proper PrP immunolabeling and to minimize non-specific background staining. Also, this approach considers biosafety measures when conducting immunohistochemistry studies with the prion-infected tissues.