Name: A Micro-CT-based Method for Characterizing Lesions and Locating Electrodes in Small Animal Brains (Video) | JoVE Journal
Uploaded: 2018-11-08T14:00:00.000+00:00
Duration: 5 min 12 s
Description: Learn a micro-CT method for lesion characterization and electrode localization in rat brains without sectioning. This technique produces 3D digital volumes for precise neuroscience analysis.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

The authors thank Greg Lin and Arthur McClelland for their expertise with the micro-CT machine, David Richmond and Hunter Elliott at the Image and Data Analysis Core (IDAC) at Harvard Medical School for their image processing advice, and William Liberti at Boston University for graciously providing a zebra finch brain. This work was performed in part at the Center for Nanoscale Systems (CNS), a member of the National Nanotechnology Coordinated Infrastructure Network (NNCI), which is supported by the National Science Foundation under NSF award no. 1541959. CNS is a part of Harvard University. This work was supported by the Richard and Susan Smith Family Foundation and IARPA (contract #D16PC00002). S.B.E.W. was supported by fellowships from the Human Frontier Science Program (HFSP; LT000514/2014) and the European Molecular Biology Organization (EMBO; ALTF1561-2013). G.G. was supported by the National Science Foundation (NSF) Graduate Research Fellowship Program (GRFP).

All care and experimental manipulation of animals were reviewed and approved by the Harvard Institutional Animal Care and Use Committee. The perfusion described here is specific for rats, but the procedure is applicable to any animals with smaller or similarly sized brains.
1. Perfusion
<ol>
	<li>Prepare 1x phosphate-buffered saline (PBS). For a rat (age: 0.5&#8211;1.5 years old, weight: 250&#8211;600 g), 800&#8211;1,000 mL should be sufficient. Use 400 mL to perfuse the animal and an additional 400 mL to dilute the fixative.</li>
	<li>Prepare the fixative consisting of 2% (w/v) paraformaldehyde (PFA) and 2.5% (w/v) glutaraldehyde (GA) in 1x PBS. For a rat, 400 mL is sufficient. Save 50 mL in a 50 mL conical tube for post-fixation of the brain after perfusion.</li>
	<li>Anesthetize the rat with 4&#8211;5% isoflurane gas (at 0.8 L/min O2 at 1.0 bar at 21 &#176;C) for 15 min.</li>
	<li>Inject a lethal dose of sodium pentobarbital intraperitoneally (180 mg/kg).</li>
	<li>Test for reflex loss in the animal by conducting a toe-pinch reflex exam. Wait to begin the perfusion until the animal has lost its reflex response.</li>
	<li>Follow the previously described intracardial perfusion and brain extraction protocol14 with the following solutions: perfuse the animal with 400 mL of 1x PBS at 125 mm Hg to remove the blood. Once all blood has been removed and replaced with 1x PBS, begin perfusing with 400 mL of a solution of 2% PFA and 2.5% GA dissolved in 1x PBS at 125 mm Hg. 
	NOTE: If the procedure is being conducted in an animal that is not a rat, the only important components of the perfusion procedure are the use of 1x PBS and 2% PFA, 2.5% GA in 1x PBS. The solution volumes and perfusion pressure may be adjusted according to the species.</li>
</ol>
2. Post-fixation
<ol>
	<li>Place the extracted brain in 2% PFA, 2.5% GA in 1x PBS solution (same solution used to perfuse the animal previously). Ensure that the solution volume is at least 10x the volume of the brain. For rats, place the brain in a 50 mL conical tube with 50 mL of solution. Store the sample in post-fixation for 2&#8211;3 days, shaking lightly at 4 &#176;C. 
	NOTE: If the sample is in a 50 mL conical tube, placing it horizontally on an orbital shaker will ensure the best results.</li>
	<li>After the sample has been post-fixed for long enough, wash the sample in double-distilled water (ddH2O), de-ionized water (diH2O), or ultrapure water (see Table of Materials) four times for the following durations: 1, 1, 1, and 15 min. 
	NOTE: For this protocol, ddH2O, diH2O, or ultrapure water should be interchangeable. For simplicity, ddH2O will be used to refer to purified water henceforth.</li>
</ol>
3. Staining
CAUTION: For this step, conduct all solution preparations under a hood while using gloves.
<ol>
	<li>Prepare at least 10x the brain volume of staining solution. For rat brains, prepare 50 mL of 2% (w/v) osmium tetroxide (OsO4) in ddH2O by combining 25 mL of stock 4% OsO4 solution and 25 mL of ddH2O. 
	CAUTION: Osmium tetroxide is volatile and may cause temporary blindness and respiratory problems if not handled appropriately. Discard all materials that contact the osmium tetroxide in an appropriate chemical hazard container within a secondary container.</li>
	<li>Place the brain in a new 50 mL conical tube and add the OsO4 solution. The brain should begin to turn brown as OsO4 reacts with lipids in the tissue.</li>
	<li>Close the tube and seal it thoroughly with paraffin film (see Table of Materials) to ensure that it does not leak during incubation. 
	NOTE: The osmium is volatile and will react mildly with the plastic of the tube, so sealing the tube properly is very important. The tube may be wrapped with aluminum foil for added protection.</li>
	<li>Store the sealed tube at 4 &#176;C, shaking lightly on an orbital shaker at 50 rpm for 2 weeks. Place the tube horizontally to ensure the best mixing. Ensure that the sample is fully submerged in the solution while shaking. 
	NOTE: If the osmium is not allowed to circulate continuously, it may not fully penetrate the sample, so horizontal placement on the shaker is very important.</li>
</ol>
4. Embedding
<ol>
	<li>After the sample has been incubated in OsO4 for 2 weeks, wash it with ddH2O 5 times at room temperature (RT) for the following durations: 1, 1, 1, 15, and 60 min to remove all the unbound OsO4 in the sample. 
	NOTE: The multiple exchanges, including the last 60 min&#160;exchange, are necessary to allow all the osmium in the circulatory system to diffuse out.</li>
	<li>Wash the sample with ddH2O for 30 min at 4 &#176;C. 
	&#8203;NOTE: Osmium may continue to diffuse out of the sample, but its quantity should be greatly reduced from the previous step.</li>
	<li>Dehydrate the sample with ethanol to eventually infiltrate it with resin. To dehydrate, replace the ddH2O with 10&#8211;20 mL of the following ethanol dilutions (for 30 min each at 4 &#176;C): 20% (v/v) ethanol and 80% (v/v) ddH2O; 50% (v/v) ethanol and 50% (v/v) ddH2O; 70% (v/v) ethanol and 30% (v/v) ddH2O; 90% (v/v) ethanol and 10% (v/v) ddH2O; 100% ethanol.</li>
	<li>Prepare the acetone/resin dilutions as follows.
	<ol>
		<li>Prepare 100 mL of the resin for embedding (see Table of Materials) as per the manufacturer&#39;s instructions.</li>
		<li>To make the 33% (v/v) resin-67% acetone solution, pour 15 mL of resin into a 50 mL conical tube and add 30 mL of 100% glass-distilled acetone.</li>
		<li>To make the 50% (v/v) resin-50% acetone, pour 22.5 mL of resin into a 50 mL conical tube and add 22.5 mL of 100% glass-distilled acetone.</li>
		<li>To make the 67% (v/v) resin-33% acetone solution, pour 30 mL of resin into a 50 mL conical tube and add 15 mL of 100% glass-distilled acetone.</li>
		<li>Use the remaining 32.5 mL of resin as the first solution of 100% resin below.</li>
	</ol>
	</li>
	<li>Begin the resin infiltration process by moving the sample through 10-20 mL of the following acetone and acetone/resin dilutions: 100% acetone for 30 min at 4 &#176;C; 100% acetone for 30 min at 4 &#176;C; and 100% acetone for 30 min at room temperature (RT). 
	NOTE: The rest of the infiltration process will take place at RT.</li>
	<li>Immerse the sample in 33% (v/v) resin 67% acetone for 3 h at RT, then 50% (v/v) resin-50% acetone for 3 h at RT, 67% (v/v) resin 33% acetone for 3 h at RT, and 100% resin for 12 h at RT.</li>
	<li>Make a fresh 50 mL batch of resin following the instructions on the bottle. Transfer the sample to the container in which it will be cured (e.g., the disposable molds described in Table of Materials). Infuse the sample with fresh 100% resin for 4 hours at RT. 
	NOTE: If fresh resin is not used, the resin made the previous day will begin to harden prematurely, and the sample will be hard to manipulate.</li>
	<li>Degas the sample in a vacuum oven for 15 min at 45 &#176;C. 
	NOTE: This step will help remove any trapped air bubbles within the sample, but it is non-essential and will not affect quality of the data.</li>
	<li>Finally, cure the sample in an oven for 48 h at 60 &#176;C.</li>
</ol>
5. micro-CT
<ol>
	<li>Once the sample has been cured, peel off the disposable mold and scan it with the micro-CT machine. 
	NOTE: Depending on the machine used, the settings will be different. For the scanner used by the authors listed in the Table of Materials, the recommended settings are 130 kV, 135 &#181;A with a 0.1 mm copper filter and a molybdenum source, a 1-second exposure, and an average of 4 frames per projection. However, experimenters should calibrate the scanner individually, as many factors will affect optimal settings during a particular session.</li>
	<li>Once the scan is completed, reconstruct the sample with the recommended parameters for the experimenter&#39;s scanner/software combination on a computer with the scanner software.</li>
	<li>Finally, visualize and analyze the reconstructed digital volume using the experimenter&#39;s software of choice (see Table of Materials for examples).</li>
</ol>

<ol><li>Scoville, W., Milner, B. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/10678523">Loss of recent memory after bilateral hippocampal lesions.</a> Journal of Neuropsychiatry and Clinical Neuroscience. 12, 103-113 (2000).</li>
<li>Damasio, H., Grabowski, T., Frank, R., Galaburda, A. M., Damasio, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+return+of+Phineas+Gage%3A+clues+about+the+brain+from+the+skull+of+a+famous+patient%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">The return of Phineas Gage: clues about the brain from the skull of a famous patient.</a> Science. 264 (5162), 1102-1105 (1994).</li>
<li>Kawai, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Motor+cortex+is+required+for+learning+but+not+for+executing+a+motor+skill%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Motor cortex is required for learning but not for executing a motor skill.</a> Neuron. 86, 800-812 (2015).</li>
<li>Otchy, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Acute+off-target+effects+of+neural+circuit+manipulations%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Acute off-target effects of neural circuit manipulations.</a> Nature. 528, 358-363 (2015).</li>
<li>Wright, N., Vann, S., Aggleton, J., Nelson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+critical+role+for+the+anterior+thalamus+in+directing+attention+to+task-relevant+stimuli%5BTitle%5D)+AND+%22Journal+of+Neuroscience%22%5BJournal%5D)">A critical role for the anterior thalamus in directing attention to task-relevant stimuli.</a> Journal of Neuroscience. 35, 5480-5488 (2015).</li>
<li>Kapgal, V., Prem, N., Hegde, P., Laxmi, T., Kutty, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long+term+exposure+to+combination+paradigm+of+environmental+enrichment%2C+physical+exercise+and+diet+reverses+the+spatial+memory+deficits+and+restores+hippocampal+neurogenesis+in+ventral+subicular+lesioned+rats%5BTitle%5D)+AND+%22Neurobiology+of+Learning+and+Memory%22%5BJournal%5D)">Long term exposure to combination paradigm of environmental enrichment, physical exercise and diet reverses the spatial memory deficits and restores hippocampal neurogenesis in ventral subicular lesioned rats.</a> Neurobiology of Learning and Memory. 130, 61-70 (2016).</li>
<li>Hosseini, N., Alaei, H., Reisi, P., Radahmadi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+effects+of+NBM-+lesion+on+synaptic+plasticity+in+rats%5BTitle%5D)+AND+%22Brain+Research%22%5BJournal%5D)">The effects of NBM- lesion on synaptic plasticity in rats.</a> Brain Research. 1655, 122-127 (2017).</li>
<li>Palagina, G., Meyer, J., Smirnakis, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Complex+visual+motion+representation+in+mouse+area+V1%5BTitle%5D)+AND+%22Journal+of+Neuroscience%22%5BJournal%5D)">Complex visual motion representation in mouse area V1.</a> Journal of Neuroscience. 37, 164-183 (2017).</li>
<li>Ranjbar, H., Radahmadi, M., Reisi, P., Alaei, H. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/28063155">Effects of electrical lesion of basolateral amygdala nucleus on rat anxiety-like behavior under acute, sub-chronic, and chronic stresses.</a> Clinical and Experimental Pharmacology and Physiology. , (2017).</li>
<li>Wood, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+honeycomb+maze+provides+a+novel+test+to+study+hippocampal-dependent+spatial+navigation%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">The honeycomb maze provides a novel test to study hippocampal-dependent spatial navigation.</a> Nature. , (2018).</li>
<li>Vermaercke, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+specialization+in+rat+occipital+and+temporal+visual+cortex%5BTitle%5D)+AND+%22Journal+of+Neurophysiology%22%5BJournal%5D)">Functional specialization in rat occipital and temporal visual cortex.</a> Journal of Neurophysiology. 112, 1963-1983 (2014).</li>
<li>Jun, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fully+integrated+silicon+probes+for+high-density+recording+of+neural+activity%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Fully integrated silicon probes for high-density recording of neural activity.</a> Nature. 551, 232-236 (2017).</li>
<li>Masís, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((micro-CT-based+method+for+quantitative+brain+lesion+characterization+and+electrode+localization%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">micro-CT-based method for quantitative brain lesion characterization and electrode localization.</a> Scientific Reports. 8, 5184(2018).</li>
<li>Gage, G., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Whole+Animal+Perfusion+Fixation+for+Rodents%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Whole Animal Perfusion Fixation for Rodents.</a> Journal of Visualized Experiments. 65, 3564(2012).</li>
<li>Helander, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Kinetic+studies+of+formaldehyde+binding+in+tissue%5BTitle%5D)+AND+%22Biotechnic+%26+Histochemistry%22%5BJournal%5D)">Kinetic studies of formaldehyde binding in tissue.</a> Biotechnic & Histochemistry. , (1994).</li>
<li>Paljärvi, L., Garcia, J., Kalimo, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+efficiency+of+aldehyde+fixation+for+electron+microscopy%3A+stabilization+of+rat+brain+tissue+to+withstand+osmotic+stress%5BTitle%5D)+AND+%22Histochemical+Journal%22%5BJournal%5D)">The efficiency of aldehyde fixation for electron microscopy: stabilization of rat brain tissue to withstand osmotic stress.</a> Histochemical Journal. , (1979).</li>
<li>Okuda, K., Urabe, I., Yamada, Y., Okada, H. <a target="_blank" href="https://www.sciencedirect.com/science/article/pii/0922338X91902315">Reaction of glutaraldehyde with amino and thiol compounds.</a> Journal of Fermentation and Bioengineering. 71, (1991).</li>
<li>Bahr, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Osmium+tetroxide+and+ruthenium+tetroxide+and+their+reactions+with+biologically+important+substances%3A+electron+stains+III%5BTitle%5D)+AND+%22Experimental+Cell+Research%22%5BJournal%5D)">Osmium tetroxide and ruthenium tetroxide and their reactions with biologically important substances: electron stains III.</a> Experimental Cell Research. , (1954).</li>
<li>Khan, A. A., Riemersma, J. C., Booij, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+reactions+of+osmium+tetroxide+with+lipids+and+other+compounds%5BTitle%5D)+AND+%22Journal+of+Histochemistry+%26+Cytochemistry%22%5BJournal%5D)">The reactions of osmium tetroxide with lipids and other compounds.</a> Journal of Histochemistry & Cytochemistry. 9, 560-563 (1961).</li>
<li>Riemersma, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Osmium+tetroxide+fixation+of+lipids+for+electron+microscopy+a+possible+reaction+mechanism%5BTitle%5D)+AND+%22Biochimica+et+Biophysica+Acta%22%5BJournal%5D)">Osmium tetroxide fixation of lipids for electron microscopy a possible reaction mechanism.</a> Biochimica et Biophysica Acta. 152, (1968).</li>
<li>Mikula, S., Binding, J., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Staining+and+embedding+the+whole+mouse+brain+for+electron+microscopy%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">Staining and embedding the whole mouse brain for electron microscopy.</a> Nature Methods. 9, 1198-1201 (2012).</li>
<li>Mikula, S., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-resolution+whole-brain+staining+for+electron+microscopic+circuit+reconstruction%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">High-resolution whole-brain staining for electron microscopic circuit reconstruction.</a> Nature Methods. 12, 541-546 (2015).</li>
<li>Crespigny, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((3D+micro-CT+imaging+of+the+postmortem+brain%5BTitle%5D)+AND+%22Journal+of+Neuroscience+Methods%22%5BJournal%5D)">3D micro-CT imaging of the postmortem brain.</a> Journal of Neuroscience Methods. 171, 207-213 (2008).</li>
<li>Anderson, R., Maga, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+procedure+for+rapid+imaging+of+adult+mouse+brains+with+MicroCT+using+Iodine-Based+contrast%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">A novel procedure for rapid imaging of adult mouse brains with MicroCT using Iodine-Based contrast.</a> PLoS One. 10, 0142974(2015).</li>
<li>Zhou, Z., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/27027284">Cerebral cavernous malformations arise from endothelial gain of MEKK3-KLF2/4 signalling.</a> Nature. 532, 122-126 (2016).</li>
<li>Choi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Micro-CT+imaging+reveals+mekk3+heterozygosity+prevents+cerebral+cavernous+malformations+in+Ccm2-Deficient+mice%5BTitle%5D)+AND+%22PloS+One%22%5BJournal%5D)">Micro-CT imaging reveals mekk3 heterozygosity prevents cerebral cavernous malformations in Ccm2-Deficient mice.</a> PloS One. 11, 0160833(2016).</li>
<li>Choi, J., Yang, X., Foley, M., Wang, X., Zheng, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induction+and+Micro-CT+imaging+of+cerebral+cavernous+malformations+in+mouse+model%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Induction and Micro-CT imaging of cerebral cavernous malformations in mouse model.</a> Journal of Visualized Experiments. , (2017).</li>
<li>Benveniste, H., Kim, K., Zhang, L., Johnson, G. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/10860789">Magnetic resonance microscopy of the C57BL mouse brain.</a> Neuroimage. 11, 601-611 (2000).</li>
<li>Weninger, W. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-resolution+episcopic+microscopy%3A+a+rapid+technique+for+high+detailed+3D+analysis+of+gene+activity+in+the+context+of+tissue+architecture+and+morphology%5BTitle%5D)+AND+%22Anat+Embryol%22%5BJournal%5D)">High-resolution episcopic microscopy: a rapid technique for high detailed 3D analysis of gene activity in the context of tissue architecture and morphology.</a> Anat Embryol. 211, 213-221 (2006).</li>
<li>Schneider, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((high-throughput+magnetic+paragraph+sign+resonance+imaging+of+mouse+embryonic+paragraph+sign+anatomy+using+a+fast+gradient-echo+sequence%5BTitle%5D)+AND+%22MAGMA%22%5BJournal%5D)">high-throughput magnetic paragraph sign resonance imaging of mouse embryonic paragraph sign anatomy using a fast gradient-echo sequence.</a> MAGMA. 16, 43-51 (2003).</li>
<li>Sharpe, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optical+projection+tomography%5BTitle%5D)+AND+%22Annual+Review+of+Biomedical+Engineering%22%5BJournal%5D)">Optical projection tomography.</a> Annual Review of Biomedical Engineering. 6, 209-228 (2004).</li>
<li>Cox, D. D., Papanastassiou, A., Oreper, D., Andken, B., James, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-Resolution+Three-Dimensional+microelectrode+brain+mapping+using+stereo+microfocal+x-ray+imaging%5BTitle%5D)+AND+%22Journal+of+Neurophysiology%22%5BJournal%5D)">High-Resolution Three-Dimensional microelectrode brain mapping using stereo microfocal x-ray imaging.</a> Journal of Neurophysiology. 100, 2966-2976 (2008).</li>
<li>Borg, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Localization+of+metal+electrodes+in+the+intact+rat+brain+using+registration+of+3D+microcomputed+tomography+images+to+a+magnetic+resonance+histology+atlas%5BTitle%5D)+AND+%22eNeuro%22%5BJournal%5D)">Localization of metal electrodes in the intact rat brain using registration of 3D microcomputed tomography images to a magnetic resonance histology atlas.</a> eNeuro. 2, (2015).</li>
<li>Fu, T. -M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stable+long-term+chronic+brain+mapping+at+the+single-neuron+level%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">Stable long-term chronic brain mapping at the single-neuron level.</a> Nature Methods. 13, 875-882 (2016).</li>
</ol>

The authors have nothing to disclose.

The following are critical steps to the protocol: first, the use of a combination of PFA and GA to perfuse the animal and subsequently post-fix the brain was paramount to achieving consistent full osmium penetration of the tissue. Although we did not test this explicitly, a plausible explanation is that PFA fixation is reversible15, whereas GA fixation is not reversible16,17. Because a two-week incubation in osmium tetroxide is required fo...

Neuroscientists have relied on lesions for a long time in order to understand the relationship between function and location in the brain. For example, our understanding of the hippocampus as being indispensable for learning and memory and of the prefrontal cortex as being key for impulse control were both products of serendipitous lesions in humans1,2. The use of animal models, however, has allowed neuroscientists to harness the power of lesions by going beyond serendipity, and the function of countless brain areas has been elucidated through systematic studies of structure-function relationships through lesions3,4.
To correctly assign function to a structure, however, lesion studies require precise quantification procedures, which is an area that has been lacking. The current gold standard for quantifying lesions is to section, mount, and image brains with a light microscope. The imaged slices are then matched to the closest sections on an atlas, and the approximate coordinates of the lesions across subjects are indirectly reported, often through the use of camera lucida images or example histological slices3,4,5,6,7,8,9,10.
Beyond the imprecision of current lesion quantification procedures, these techniques are time-consuming and prone to failure. Small changes in brain stiffness, blade sharpness, and temperature can lead to botched, warped, or torn sections. Sections can also stain unevenly and be improperly imaged because of bubbles in the mounting medium. Importantly, upon sectioning, the three-dimensional context of the lesion&#39;s location in the brain is lost, making precise 3D reconstruction of the lesion in the brain challenging.
Another common application for lesions has been to determine the location of single and multiple electrode recordings in the brain. At the end of the final recording session, researchers induce small electrolytic lesions at the electrode tip and process the brain histologically as done in a conventional lesion experiment11. This technique suffers from the same drawbacks described above, with additional problems being that the electrolytic lesions are usually larger than the electrodes used to make them but are usually small enough that they are challenging to find histologically. When multiple electrodes are inserted, as in the case of a tetrode array, verification through electrolytic lesions is even more challenging. An alternative to electrolytic lesions is the use of a dye on the electrode to later verify histologically12, but this technique suffers from the same drawbacks that come with conventional histology.
Here, we describe in-depth a recently described method13 based on staining techniques in electron microscopy (EM) and X-ray computed tomography (micro-CT) that quantifies lesions and locates electrodes in small animal brains better than current methods. Micro-CT is an imaging technique in which X-rays are shot at a sample that is rotated 360&#176; while a scintillator collects the X-rays not deflected by the sample. The result is a high-resolution digital 3D reconstruction of the sample that can be visualized in any orientation and quantified precisely. Many academic institutions have micro-CT scanners, which are also available commercially.

<table><tbody><tr><td>Paraformaldehyde (PFA)</td><td>Electron Microscopy Sciences (EMS)</td><td>15710</td><td>2% (w/v/) in 1X PBS</td></tr><tr><td>Glutaraldehyde (GA)</td><td>EMS</td><td>16220</td><td>2.5% (w/v) GA in 1x PBS</td></tr><tr><td>OsO&lt;sub&gt;4&lt;/sub&gt;</td><td>EMS</td><td>19190</td><td>Work in fume hood</td></tr><tr><td>Ethanol</td><td>Decon Labs</td><td>Koptec</td><td>140, 190, 200 proof</td></tr><tr><td>Acetone</td><td>EMS</td><td>10015</td><td>Glass-distilled</td></tr><tr><td>Durcupan ACM resin</td><td>Sigma-Aldrich</td><td>44610</td><td>A, B, C and D components, resin for embedding</td></tr><tr><td>Disposable molds</td><td>Ted Pella</td><td>27114</td><td>Suggested</td></tr><tr><td>milliQ water (ultrapure water)</td><td>Millipore Sigma</td><td>QGARD00R1 (or related purifier)</td><td>Suggested</td></tr><tr><td>Parafilm (paraffin film)</td><td>Millipore Sigma</td><td>P7793</td><td>Suggested paraffin film</td></tr><tr><td>Micro-CT scanner</td><td>Nikon Metrology Ltd., Tring, UK</td><td>X-Tek HMS ST 225</td><td>Used by authors</td></tr><tr><td>&lt;strong&gt;Software for visualizing and analyzing micro-CT scans:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td></td><td>Volume Graphics</td><td>VG Studio Max</td><td>Used by authors</td></tr><tr><td></td><td>FEI / Thermo Scientific</td><td>Avizo</td><td>Used by authors</td></tr><tr><td></td><td>FEI / Thermo Scientific</td><td>Amira</td><td>Similar to Avizo</td></tr><tr><td></td><td>Mark Sutton &amp;amp; Russell Garwood</td><td>Spiers</td><td>Free, http://spiers-software.org/</td></tr><tr><td></td><td>Pixmeo Sarl</td><td>Osirix Lite</td><td>Free, https://www.osirix-viewer.com/</td></tr><tr><td></td><td>Open Source</td><td>FIJI</td><td>Free, https://fiji.sc/</td></tr><tr><td></td><td>Adobe</td><td>Photoshop</td><td>Good for analyzing one slice at a time</td></tr></tbody></table>

a micro-ct-based method for characterizing lesions and locating electrodes in small animal brains

A Micro-CT-based Method for Characterizing Lesions and Locating Electrodes in Small Animal Brains

Lesion and electrode location verification are traditionally done via histological examination of stained brain slices, a time-consuming procedure that requires manual estimation. Here, we describe a simple, straightforward method for quantifying lesions and locating electrodes in the brain that is less laborious and yields more detailed results. Whole brains are stained with osmium tetroxide, embedded in resin, and imaged with a micro-CT scanner. The scans result in 3D digital volumes of the brains with resolutions and virtual section thicknesses dependent on the sample size (12&#8211;15 and 5&#8211;6 &#181;m&#160;per voxel for rat and zebra finch brains, respectively). Surface and deep lesions can be characterized, and single tetrodes, tetrode arrays, electrolytic lesions, and silicon probes can also be localized. Free and proprietary software allows experimenters to examine the sample volume from any plane and segment the volume manually or automatically. Because this method generates whole brain volume, lesions and electrodes can be quantified to a much higher degree than in current methods, which will help standardize comparisons within and across studies.

Traditionally, brains are sectioned and stained in order to quantify lesions and locate electrodes, but this method is error-prone, labor-intensive, and typically requires estimation of the results. By preparing whole brains for micro-CT imaging, the probability of damaging the samples is greatly reduced, features of interest may be analyzed in the context of the entire brain, and the method lends itself to parallel processing of many samples, considerably speeding up sample preparation.<...

This article describes a straightforward method to prepare small animal brains for micro-CT imaging, in which lesions can be quantified and electrodes located with high precision in the context of the whole brain.

Lesion and electrode location verification are traditionally done via histological examination of stained brain slices, a time-consuming procedure that ...

a-micro-ct-based-method-for-characterizing-lesions-locating

Research

JoVE Journal

Neuroscience

The video demonstrates a procedure for implanting a wireless bidirectional multichannel electrode unit into a rat brain to record and stimulate multichannel neural networks.

Implanting a Wireless Bidirectional Microelectrode System in a Rat Brain

Source: Choi, J. P. et al. Induction and Micro-CT Imaging of Cerebral Cavernous Malformations in Mouse Model. J. Vis. Exp. (2017)This video demonstrates the use of micro-CT imaging to visualize cerebral cavernous malformation (CCM) lesions in a mouse brain. It details the steps for sample preparation, imaging, and 3D image reconstruction to evaluate the density and location of CCM lesions in brain tissue.

Contrast-Enhanced Micro-CT Imaging of Cerebral Cavernous Malformation Lesions in a Mouse Brain

Here we present a histological method for capturing, labeling, optically clearing, and imaging the intact brain tissue interface around chronically implanted microdevices in rodent brain tissue.  Results from the techniques comprising this method are useful for understanding the impact of various penetrating brain-implants on their surrounding tissue.

Intact Histological Characterization of Brain-implanted Microdevices and Surrounding Tissue

This protocol demonstrates the induction of cerebral cavernous malformation disease in a mouse model and uses contrast enhanced micro computed tomography to measure lesion burden. This method enhances the value of established mouse models to study the molecular basis and potential therapies for cerebral cavernous malformation and other cerebrovascular diseases.

Induction and Micro-CT Imaging of Cerebral Cavernous Malformations in Mouse Model

Whereas multiphoton imaging is only effective at limited depths from the tissue’s surface, it is possible to achieve 3 µm resolution imaging at any depth via pCLE. Here, we present a protocol to conduct pCLE imaging to measure microvascular dynamics in the hippocampus of ictal and wild-type mice.

In Vivo Fiber-Coupled Pre-Clinical Confocal Laser-scanning Endomicroscopy (pCLE) of Hippocampal Capillaries in Awake Mice

Here we describe a procedure for tissue clearing, fluorescent labeling, and large-scale imaging of mouse brain tissue which, thereby, enables visualization of the three-dimensional organization of cell types in the neocortex.

Large-scale Three-dimensional Imaging of Cellular Organization in the Mouse Neocortex

Here, we present a method for the long-term performance and safety assessment of soft subdural electrode arrays in a minipig model, describing surgical method and tools, postoperative magnetic resonance imaging, electrophysiology of the auditory cortex, electrochemical properties of the implant, and postmortem immunochemistry.

Subdural Soft Electrocorticography (ECoG) Array Implantation and Long-Term Cortical Recording in Minipigs

This study describes the steps for obtaining high-resolution images of neonatal mouse brains by combining micro-computed tomography (micro-CT) and a contrast agent in ex vivo samples. We describe basic morphometric analyses to quantify brain size and shape in these images.

Micro-CT Imaging and Morphometric Analysis of Mouse Neonatal Brains

We describe a protocol for assessing dose-response curves for extracranial stimulation in terms of brain electrical field measurements and a relevant biomarker-cerebral blood flow. Since this protocol involves invasive electrode placement into the brain, general anesthesia is needed, with spontaneous breathing preferred rather than controlled respirations.

Placement of Extracranial Stimulating Electrodes and Measurement of Cerebral Blood Flow and Intracranial Electrical Fields in Anesthetized Mice

A low-cost electroencephalographic recording system combined with a millimeter-sized coil is proposed to drive transcranial magnetic stimulation of the mouse brain in vivo. Using conventional screw electrodes with a custom-made, flexible, multielectrode array substrate, multi-site recording can be carried out from the mouse brain in response to transcranial magnetic stimulation.

Low-Cost Electroencephalographic Recording System Combined with a Millimeter-Sized Coil to Transcranially Stimulate the Mouse Brain In Vivo

Here, we present two protocols for high-density micro-electrocorticography (µEcoG) recording in rats and mice, including surgical, implantation, and recording methods. µECoG recordings are performed in combination with either laminar polytrode recording in the rat auditory cortex or with optogenetic manipulation of neural activity in the mouse somatosensory cortex.

Multiscale Investigations of Cortical Processing by Integrating Laminar Polytrodes and Optogenetics with Micro Electrocorticography in Rodents

Learn a micro-CT method for lesion characterization and electrode localization in rat brains without sectioning. This technique produces 3D digital volumes for precise neuroscience analysis.

A Micro-CT-based Method for Characterizing Lesions and Locating Electrodes in Small Animal Brains

Explore More Videos