The authors recognize Rene Jack Roy for his excellent technical support in the area of MRI. This work was supported by the National Institutes of Health (R01 NS102194 to KSL and R01 CA217953-01 to MW), the Chester Fund (KSL), and the Focused Ultrasound Foundation (KSL and JW).

All methods described here have been approved by the University of Virginia Animal Care and Use Committee.
1. Preparation of reagents
<ol>
	<li>On the day of surgery, prepare 6.0 mL of injectable quinolinic acid (QA). Dissolve 450 mg of QA in 4.0 mL of 1.0 N NaOH. Add 0.6 mL of 10x PBS, pH to 7.4, and bring to a final volume of 6.0 mL with dH2O. Filter through 0.22 &#181;m syringe filter. The solution is stable for 2 weeks at 4 &#176;C.</li>
	<li>Prepare an aqueous dispersion of m...

<ol>
	<li>Wiebe, S., Eliasziw, M., Matijevic, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+quality+of+life+in+epilepsy:+How+large+must+they+be+to+be+real.">Changes in quality of life in epilepsy: How large must they be to be real.</a> Epilepsia. 42, 113-118 (2001).</li><li>McClelland, S., Guo, H., Okuyemi, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Population-based+analysis+of+morbidity+and+mortality+following+surgery+for+intractable+temporal+lobe+epilepsy+in+the+United+States.">Population-based analysis of morbidity and mortality following surgery for intractable temporal lobe epilepsy in the United States.</a> Archives of Neurology. 68, 725-729 (2011).</li><li>Hynynen, K., McDannold, N., Vykhotseva, N., Jolesz, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+MR+imaging-guided+focal+opening+of+the+blood-brain+barrier+in+rabbits.">Noninvasive MR imaging-guided focal opening of the blood-brain barrier in rabbits.</a> Radiology. 220, 640-646 (2001).</li><li>McDannold, N., Vykhodtseva, N., Raymond, S., Jolesz, F. A., Hynynen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MRI-guided+targeted+blood-brain+barrier+disruption+with+focused+ultrasound:+histological+findings+in+rabbits.">MRI-guided targeted blood-brain barrier disruption with focused ultrasound: histological findings in rabbits.</a> Ultrasound in Medicine &amp; Biology. 31, 1527-1537 (2005).</li><li>Park, J., Zhang, Y., Vykhodtseva, N., Jolesz, F. A., McDannold, N. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permeability+assessment+of+the+focused+ultrasound-induced+blood-brain+barrier+opening+using+dynamic+contrast-enhanced+MRI.">Permeability assessment of the focused ultrasound-induced blood-brain barrier opening using dynamic contrast-enhanced MRI.</a> Physics in Medicine and Biology. 55 (18), 5451-5466 (2010).</li><li>Zhang, Y., 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=focal+disconnection+of+brain+circuitry+using+magnetic+resonance-guided+low-intensity+focused+ultrasound+to+deliver+a+Neurotoxin.">focal disconnection of brain circuitry using magnetic resonance-guided low-intensity focused ultrasound to deliver a Neurotoxin.</a> Ultrasound in Medicine &amp; Biology. 42 (9), 2261-2269 (2016).</li><li>Zhang, Y., 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=Testing+different+combinations+of+acoustic+pressure+and+doses+of+quinolinic+acid+to+induce+focal-neuron+loss+in+mice+using+transcranial+low-intensity+focused+ultrasound.">Testing different combinations of acoustic pressure and doses of quinolinic acid to induce focal-neuron loss in mice using transcranial low-intensity focused ultrasound.</a> Ultrasound in Medicine &amp; Biology. 45, 129-136 (2018).</li><li>Foster, A. C., Miller, L. P., Oldendorf, W. H., Schwarcz, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+disposition+of+quinolinic+acid+after+intracerebral+or+systemic+administration+in+the+rat.">Studies on the disposition of quinolinic acid after intracerebral or systemic administration in the rat.</a> Experimental Neurology. 84, 428-440 (1984).</li><li>Beskid, M., R&#243;&#380;ycka, Z., Taraszewska, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quinolinic+acid:+effect+on+the+nucleus+arcuatus+of+the+hypothalamus+in+the+rat+(ultrastructural+evidence).">Quinolinic acid: effect on the nucleus arcuatus of the hypothalamus in the rat (ultrastructural evidence).</a> Experimental and Toxicologic Pathology. 49, 477-481 (1997).</li><li>Schwarcz, R., K&#246;hler, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+vulnerability+of+central+neurons+of+the+rat+to+quinolinic+acid.">Differential vulnerability of central neurons of the rat to quinolinic acid.</a> Neuroscience Letters. 38, 85-90 (1983).</li><li>Schwarcz, R., Whetsell, W. O., Mangano, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quinolinic+acid:+an+endogenous+metabolite+that+produces+axon-sparing+lesions+in+rat+brain.">Quinolinic acid: an endogenous metabolite that produces axon-sparing lesions in rat brain.</a> Science. 219, 316-318 (1983).</li><li>Klibanov, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbubble+contrast+agents:+targeted+ultrasound+imaging+and+ultrasound-assisted+drug-delivery+applications.">Microbubble contrast agents: targeted ultrasound imaging and ultrasound-assisted drug-delivery applications.</a> Investigative Radiology. 41 (3), 354-362 (2006).</li><li>Agari, T., 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=Successful+treatment+of+epilepsy+by+resection+of+periventricular+nodular+heterotopia.">Successful treatment of epilepsy by resection of periventricular nodular heterotopia.</a> Acta Medica Okayama. 66 (6), 487-492 (2012).</li><li>Lee, K. S., 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=A+genetic+animal+model+of+human+neocortical+heterotopia+associated+with+seizures.">A genetic animal model of human neocortical heterotopia associated with seizures.</a> The Journal of Neuroscience. 17 (16), 6236-6242 (1997).</li><li>Schottler, F., Couture, D., Rao, A., Kahn, H., Lee, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcortical+connections+of+normotopic+and+heterotopic+neurons+in+sensory+and+motor+cortices+of+the+tish+mutant+rat.">Subcortical connections of normotopic and heterotopic neurons in sensory and motor cortices of the tish mutant rat.</a> The Journal of Comparative Neurology. 395 (1), 29-42 (1998).</li><li>Schottler, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normotopic+and+heterotopic+cortical+representations+of+mystacial+vibrissae+in+rats+with+subcortical+band+heterotopia.">Normotopic and heterotopic cortical representations of mystacial vibrissae in rats with subcortical band heterotopia.</a> Neuroscience. 108 (2), 217-235 (2001).</li><li>Zhang, Y., 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=Effects+of+non-invasive,+targeted,+neuronal+lesions+on+seizures+in+a+mouse+model+of+temporal+lobe+epilepsy.">Effects of non-invasive, targeted, neuronal lesions on seizures in a mouse model of temporal lobe epilepsy.</a> Ultrasound in Medicine and Biology. 46, 1224-1234 (2020).</li><li>Holmes, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+serum+kynurenine+and+hepatic+tryptophan+dioxygenase+activity+by+high-performance+liquid+chromatography.">Determination of serum kynurenine and hepatic tryptophan dioxygenase activity by high-performance liquid chromatography.</a> Analytical Biochemistry. 172, 518-525 (1988).</li><li>Shibata, K., Ohno, T., Sano, M., Fukuwatari, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+urinary+ratio+of+3-hydroxykynurenine/3-hydroxyanthranilic+acid+is+an+index+of+predicting+the+adverse+effects+of+D-trytophan+in+rats.">The urinary ratio of 3-hydroxykynurenine/3-hydroxyanthranilic acid is an index of predicting the adverse effects of D-trytophan in rats.</a> Journal of Nutritional Science and Vitaminology. 60, 261-268 (2014).</li><li>Aubry, J. -. 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The PING method is designed to produce non-invasive, targeted neuronal lesions. The method derives from a strong and growing foundation of research in the field of focused ultrasound3,4,5,6,7. The ability to provide focal access to specific areas of the brain parenchyma via transient opening of the BBB has created an avenue for delivering a wide variety of age...

The purpose of this method is to provide a means for producing non-invasive neuronal lesions in a targeted region of the brain. The rationale for developing such an approach is to disconnect neuronal circuitry contributing to neurological disorders. For instance, surgery can be quite effective in treating certain medically intractable neurological disorders, such as drug resistant epilepsy (DRE)1. However, each of the available surgical modalities possess limitations in terms of producing undesirable collateral damage to the brain. Traditional resective surgery can be highly invasive with the risk of bleeding, infection, blood clots, stroke, seizures, swelling of the brain, and nerve damage2. Alternatives to resective surgery that are minimally invasive or non-invasive include laser interstitial thermal therapy and radiosurgery, which have also proved to be effective in suppressing seizures in DRE. More recently, thermal lesions produced by high-intensity focused ultrasound (HIFU) have shown promise in reducing seizures. HIFU is non-invasive; however, its treatment window is currently limited to more central areas of the brain because of the risk of thermal injury to non-target tissue located in the vicinity of the skull. Despite such limitations, the benefits of surgery often outweigh the potential risks. For instance, although surgery for DRE can produce collateral brain damage, its beneficial effects in suppressing seizures and improving quality of life typically prevail over the surgical risks.
The method described herein, Precise Intracerebral Non-invasive Guided surgery (PING), was developed for the purpose of disconnecting neural circuitry, while limiting collateral brain damage. The method utilizes low intensity focused ultrasound combined with intravenous injection of microbubbles to open the BBB, in order to deliver a neurotoxin. This approach does not produce thermal lesions to the brain3,4,5,6,7, and the period of BBB opening can be exploited to deliver BBB-impermeable compounds to the brain parenchyma. The opening of the BBB is transient, and can be produced in a targeted manner using magnetic resonance imaging guidance. In our studies, the period of BBB opening has been utilized to deliver a circulating neurotoxin to a targeted area of the brain parenchyma in rats and mice8,9. Quinolinic acid is a neurotoxin that is well tolerated when administered intravenously10, intraarterially10, or intraperitoneally8,9,11. The lack of QA toxicity is due to its poor BBB permeability, which has been reported to be negligible10. In contrast, direct injection of QA into the brain parenchyma produces neuronal lesions that spare neighboring axons12,13. Thus, when circulating QA gains access to the brain parenchyma in the targeted area of BBB opening, neuronal death is produced8,9. The present method thus produces focal neuronal loss in a precisely targeted and non-invasive manner.

<table>
	<tbody>
		<tr>
			<td>7T-ClinScan MRI System</td>
			<td>Bruker Biospin, Ettinglen, Germany</td>
			<td></td>
			<td>MR Image Acquisition</td>
		</tr>
		<tr>
			<td>Acoustic Gel</td>
			<td>Litho CLEAR</td>
			<td>11-601</td>
			<td>High Viscosity Accoustic Transmission Gel</td>
		</tr>
		<tr>
			<td>DPX Mounting Medium</td>
			<td>Electron Microscopy Sciences</td>
			<td>13512</td>
			<td>Resin Based Cover Glass Mountant</td>
		</tr>
		<tr>
			<td>Fluoro-Jade B</td>
			<td>EDM Millipore</td>
			<td>AG310</td>
			<td>High Affinity Stain For Degenerating Neurons</td>
		</tr>
		<tr>
			<td>Fluovac anesthetic adsorber</td>
			<td>Harvard Apparatus</td>
			<td>34-0388</td>
			<td>Organic Anaesthesia Scavenger</td>
		</tr>
		<tr>
			<td>FUS System</td>
			<td>Image Guided Therapy, Pessac, France</td>
			<td>LabFUS</td>
			<td>MR Compatible Small Animal Focused Ultrasound System</td>
		</tr>
		<tr>
			<td>Gadodiamide</td>
			<td>GE Healthcare AS, Oslo, Norway</td>
			<td>Omniscan</td>
			<td>MR Contrast Agent</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>SAGENT</td>
			<td>NDC2502140010</td>
			<td>Anti-Coagulant</td>
		</tr>
		<tr>
			<td>Hypodermic needle 30G x 1/2</td>
			<td>Becton-Dickinson</td>
			<td>26027</td>
			<td>Tail Vein Catheterization</td>
		</tr>
		<tr>
			<td>Insulin syringe 28G1/2 (1ml)</td>
			<td>EXEL</td>
			<td>26027</td>
			<td>Administration of Injectables to Tail Vein Catheter</td>
		</tr>
		<tr>
			<td>Isofluorane atomizer</td>
			<td>SurgiVet</td>
			<td>VCT302</td>
			<td>Anaesthesia Administration</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Henry Schein</td>
			<td>NDC1169567762</td>
			<td>Anaesthesia</td>
		</tr>
		<tr>
			<td>KMnO4</td>
			<td>Sigma</td>
			<td>223468</td>
			<td>Reagent Used in Fluoro-Jade B Staining</td>
		</tr>
		<tr>
			<td>Microbubbles</td>
			<td>Produced internally: A. Klibanov</td>
			<td>305106</td>
			<td>Blood Brain Barrier Disrupting Agent</td>
		</tr>
		<tr>
			<td>Microbubbles (commercial source)</td>
			<td>Lantheus Medical Imaging, North Billerica, MA</td>
			<td>Definity microbubbles</td>
			<td>Blood Brain Barrier Disrupting Agent</td>
		</tr>
		<tr>
			<td>Monitoring &#38; Gating System</td>
			<td>Small Animal Instruments</td>
			<td>Model 1030</td>
			<td>Respiration Monitoring</td>
		</tr>
		<tr>
			<td>Multisizer 3 Coulter counter</td>
			<td>Beckman-Coulter, Hialeah, FL</td>
			<td>Multisizer 3</td>
			<td>Used to Determine Average Size of Microbubbles</td>
		</tr>
		<tr>
			<td>Optixcare EYE LUBE</td>
			<td>CLC MEDICA, Ontario, Canada</td>
			<td>11611</td>
			<td>Corneal Protectant-Eye Lube</td>
		</tr>
		<tr>
			<td>PE10 tubing</td>
			<td>Becton-Dickinson</td>
			<td>427401</td>
			<td>Tail Vein Catheter Component</td>
		</tr>
		<tr>
			<td>Quinolinic Acid</td>
			<td>Santa Cruz Biotechnology, Dallas, TX</td>
			<td>CAS 89-00-9</td>
			<td>Neurotoxin</td>
		</tr>
		<tr>
			<td>Sprague-Dawley Rats</td>
			<td>Taconic Biosciences</td>
			<td>SD-M</td>
			<td>Rat Model</td>
		</tr>
		<tr>
			<td>Syringe Pump</td>
			<td>Carnegie Medicin</td>
			<td>CMA 100</td>
			<td>Controlled Delivery of Quinolinic Acid</td>
		</tr>
		<tr>
			<td>Thermoguide Software</td>
			<td>Image Guided Therapy, Pessac, France</td>
			<td>Thermoguide</td>
			<td>Drives Lab FUS System</td>
		</tr>
		<tr>
			<td>Tish Rats</td>
			<td>In-house colony</td>
			<td></td>
			<td>Rat Model</td>
		</tr>
		<tr>
			<td>Veet depilatory cream</td>
			<td>Reckitt Benckiser</td>
			<td></td>
			<td>Removal of Scalp Hair</td>
		</tr>
	</tbody>
</table>

targeted neuronal injury for the non-invasive disconnection of brain circuitry

Surgical intervention can be quite effective for treating certain types of medically intractable neurological diseases. This approach is particularly useful for disorders in which identifiable neuronal circuitry plays a key role, such as epilepsy and movement disorders. Currently available surgical modalities, while effective, generally involve an invasive surgical procedure, which can result in surgical injury to non-target tissues. Consequently, it would be of value to expand the range of surgical approaches to include a technique that is both non-invasive and neurotoxic.
Here, a method is presented for producing focal, neuronal lesions in the brain in a non-invasive manner. This approach utilizes low-intensity focused ultrasound together with intravenous microbubbles to transiently and focally open the Blood Brain Barrier (BBB). The period of transient BBB opening is then exploited to focally deliver a systemically administered neurotoxin to a targeted brain area. The neurotoxin quinolinic acid (QA) is normally BBB-impermeable, and is well-tolerated when administered intraperitoneally or intravenously. However, when QA gains direct access to brain tissue, it is toxic to the neurons. This method has been used in rats and mice to target specific brain regions. Immediately after MRgFUS, successful opening of the BBB is confirmed using contrast enhanced T1-weighted imaging. After the procedure, T2 imaging shows injury restricted to the targeted area of the brain and the loss of neurons in the targeted area can be confirmed post-mortem utilizing histological techniques. Notably, animals injected with saline rather than QA do demonstrate opening of the BBB, but dot not exhibit injury or neuronal loss. This method, termed Precise Intracerebral Non-invasive Guided surgery (PING) could provide a non-invasive approach for treating neurological disorders associated with disturbances in neural circuitry.

This section describes the effect of PING on neurons located in a neocortical dysplasia. Tissue dysplasias are a common feature in the brains of patients with drug resistant epilepsy, and surgical removal of seizure-genic dysplasias can provide excellent control of seizures15. Defining the effect of PING on dysplastic brain tissue is therefore an important priority. A rat model of genetic cortical dysplasia, the tish rat, was selected for studying this issue because the tish brain exhibits dysplas...

Watch this Scientific Journal Video about Targeted Neuronal Injury for the Non-Invasive Disconnection of Brain Circuitry at JoVE.com

Targeted Neuronal Injury for the Non-Invasive Disconnection of Brain Circuitry

The goal of the protocol is to provide a method for producing non-invasive neuronal lesions in the brain. The method utilizes Magnetic Resonance-guided Focused Ultrasound (MRgFUS) to open the Blood Brain Barrier in a transient and focal manner, in order to deliver a circulating neurotoxin to the brain parenchyma.

Surgical intervention can be quite effective for treating certain types of medically intractable neurological diseases. This approach is particularly ...

Surgical intervention can be quite effective for treating certain types of medically intractable neurological disease. Currently available surgical modalities while effective, generally involve invasive procedures that can result in surgical injury to non-target tissues. Consequently, it would be a value to expand the range of surgical approaches to include a technique that is noninvasive and produces neuronal lesions.This video presents a method for producing focal neuronal lesions in the brain in a noninvasive manner. This method is termed precise interest cerebral noninvasive guided surgery and will be referred to here by its acronym Ping. The general approach for this method is to open the blood-brain barrier focally and trenchantly using MR-guided focused ultrasound.Then to administer a blood-brain barrier impermeable neurotoxin systemically. The neurotoxin, then gains access to the brain parenchyma only where the blood-brain barrier has been opened. This producer neuronal loss that is restricted to the target region of the blood-brain barrier opening.The key steps of the method are animal preparation, the ping procedure, and post-mortem cellular analyses. The anesthetized animal is placed on a surgical drape over a heating pad. 2%ISO flooring is delivered through a nose cone for the maintenance phase of anesthesia.A scavenging line is positioned for of the anesthetic. The scalp is shaved in preparation for the later application of acoustic gel. A line is then placed in the tail vein.This line will be used for the administration of micro bubbles and contrast agent during the sonication phase of the ping procedure and will be used for the infusion of quinolinic acid during the post sonication phase. The line is secured with tape. This photograph shows key features of the sled in which the animal is placed for the focused ultrasound procedure.In preparation for placing the animal in the sled. A beaker of water, a syringe containing acoustic gel, and a small screwdriver are required. This view from above the sled shows the procedure for positioning the animal.The transducer is removed and placed to the side of the sled. The animal is then placed in the sled and the head is positioned. An incisor bar and ear bars are used to secure the head.When the ear bars are positioned, they are secured by tightening using a small screwdriver. Water is applied to the surface of the scalp. This is followed by application of acoustic gel with the syringe on the scalp.Additional water is placed on the acoustic gel and on the base of the transducer. The transducer is then lowered onto the acoustic gel and secured. A pneumatic sensor is then taped to the body of the animal for monitoring respiration.The control room for the MRI and focused ultrasound equipment is comprised of two primary stations. The station to the left, where the investigator is sitting, is the planning area for targeting focused ultrasound. The station to the right is the control area for the MRI system.The copper reinforced window B on the MRI station looks into the room housing the seven Tesla magnet. Looking through the window. The magnet can be seen with a sled leading into the aperture of the magnet.Specialized software is used for planning the target of the focused ultrasound. This software controls a combination of electronic and mechanical movement of the targeting system. The trajectory planner defines the target and chose the target area.In this case, the sample target is the striatum and the target site is mapped onto a T2-weighted coronal MRI section. After establishing the targeting, the animal receives an injection of microbubbles via the tail vein line. This process has not visible through the window because it takes place on the far side of the magnet were filming is not possible.Focused ultrasound is delivered 30 seconds after the injection of micro bubbles. Immediately after delivering focused ultrasound, get a dynamite is injected via the tail vein line. And the opening of the blood-brain barrier is confirmed using contrast-enhanced T1-weighted imaging.After receiving sonication, the animal is returned to the surgical hood where 2%ISO fluorine anesthesia is maintained through a nose cone. A syringe filled with quinolinic acid is attached to an infusion pump and the output is connected to the tail vein line for a one-hour infusion into the animal. Cortical malformations are surgical targets in certain neurological disorders, such as drug-resistant epilepsy.The tish rat is a genetic neurological mutant with a cortical malformation characterized by bilateral heterotopia. In this experiment, the heterotopia in a tish rat were targeted on both sides of the brain. Frames A and B showed the same T2-MRI of a tish brain taken one-day post ping.Frame A depicts the location of the normally position neocortex and the underlying heterotopia H the positions of the lateral ventricles are also shown in frame B areas of hyperintensity indicated by arrows correspond to the targets of sonication in the heterotopia. The white arrow indicates a medial target in the heterotopia on the left side of the image, and the yellow arrow indicates a lateral target in the heterotopia and on the right side of the image. Five days post ping the animal was euthanized, and the brain prepared for histological analysis.Fluoro Jade staining was performed to identify degenerating neurons in the brain. The rectangle in the T2 section indicates the fluoro Jaden stained area of the brain shown at the right. The bright yellowish-green stained area contains numerous degenerating neurons.At higher magnification individual degenerating neurons can be seen. The ping method provides a noninvasive approach for destroying neurons in a targeted area of the brain. Trenchant vocal opening of the blood-brain barrier allows a systemically administered neurotoxin to gain access to the brain parenchyma in a precisely targeted manner.This method has been used successfully in rats and mice. Importantly, ping has been used to disconnect neurocircuitry in brain regions that are commonly the target of surgical intervention for treating neurological disorders such as epilepsy.

targeted-neuronal-injury-for-non-invasive-disconnection-brain

Research

JoVE Journal

Neuroscience

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of the hippocampus is relatively preserved 1. The organotypic culture system allows for the examination of neuronal, astrocytic and microglial effects, but as an ex vivo preparation, does not address effects of blood flow, or recruitment of peripheral inflammatory cells. To that end, this culture method is frequently used to examine excitotoxic and hypoxic injury to pyramidal neurons of the hippocampus, but has also been used to examine the inflammatory response. Herein we describe the methods for generating hippocampal slice cultures from postnatal rodent brain, administering toxic stimuli to induce neuronal injury, and assaying and quantifying hippocampal neuronal death.

The organoptypic hippocampal slice culture model is an in vitro model used to examine neuronal injury in a variety of paradigms. In this article, we describe the methods for generating slice cultures and quantifying neuronal injury.

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of ...

Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises

The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that influence a cell's membrane potential and which we address in these experiments are: (1) Ion concentration of K+ on the outside of the membrane, and (2) the permeability of the membrane to specific ions. The crayfish abdominal extensor muscles are in groupings with some being tonic (slow) and others phasic (fast) in their biochemical and physiological phenotypes, as well as in their structure; the motor neurons that innervate these muscles are correspondingly different in functional characteristics. We use these muscles as well as the superficial, tonic abdominal flexor muscle to demonstrate properties in synaptic transmission. In addition, we introduce a sensory-CNS-motor neuron-muscle circuit to demonstrate the effect of cuticular sensory stimulation as well as the influence of neuromodulators on certain aspects of the circuit. With the techniques obtained in this exercise, one can begin to answer many questions remaining in other experimental preparations as well as in physiological applications related to medicine and health. We have demonstrated the usefulness of model invertebrate preparations to address fundamental questions pertinent to all animals.

The experiments demonstrate an easy approach for students to gain experience in examining muscle structure, synaptic responses, the effects of ion gradients and permeability on membrane potentials. Also, a sensory-CNS-motor-muscle circuit is presented to show a means to test effects of compounds on a neuronal circuit.

The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Non-invasive Parenchymal, Vascular and Metabolic High-frequency Ultrasound and Photoacoustic Rat Deep Brain Imaging

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central nervous system of small animals. However, transdermal and transcranial neuroimaging is frequently affected by low sensitivity, image aberrations and loss of space resolution, requiring scalp or even skull removal before imaging. To overcome this challenge, a new protocol is presented to gain significant insights in brain hemodynamics by photoacoustic and high-frequency ultrasounds imaging with the animal skin and skull intact. The procedure relies on the passage of ultrasound (US) waves and laser directly through the fissures that are naturally present on the animal cranium. By juxtaposing the imaging transducer device exactly in correspondence to these selected areas where the skull has a reduced thickness or is totally absent, one can acquire high quality deep images and explore internal brain regions that are usually difficult to anatomically or functionally describe without an invasive approach. By applying this experimental procedure, significant data can be collected in both sonic and optoacoustic modalities, enabling to image the parenchymal and the vascular anatomy far below the head surface. Deep brain features such as parenchymal convolutions and fissures separating the lobes were clearly visible. Moreover, the configuration of large and small blood vessels was imaged at several millimeters of depth, and precise information were collected about blood fluxes, vascular stream velocities and the hemoglobin chemical state. This repertoire of data could be crucial in several research contests, ranging from brain vascular disease studies to experimental techniques involving the systemic administration of exogenous chemicals or other objects endowed with imaging contrast enhancement properties. In conclusion, thanks to the presented protocol, the US and PA techniques become an attractive noninvasive performance-competitive means for cortical and internal brain imaging, retaining a significant potential in many neurologic fields.

The present work describes a new protocol to perform non-invasive high-frequency ultrasound and photoacoustic based imaging on rat brain, to efficiently visualize deep subcortical regions and their vascular patterns by directing signals on skull foramina naturally present on animal cranium.

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central ...

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral vascular effects. Impact (i.e., non-blast) traumatic brain injury (TBI) is known to decrease pressure autoregulation in the cerebral vasculature in both humans and experimental animals. The hypothesis that blast-induced traumatic brain injury (bTBI), like impact TBI, results in impaired cerebral vascular reactivity was tested by measuring myogenic dilatory responses to reduced intravascular pressure in rodent middle cerebral arterial (MCA) segments from rats subjected to mild bTBI using an Advanced Blast Simulator (ABS) shock tube. Adult, male Sprague-Dawley rats were anesthetized, intubated, ventilated and prepared for Sham bTBI (identical manipulation and anesthesia except for blast injury) or mild bTBI. Rats were randomly assigned to receive Sham bTBI or mild bTBI followed by sacrifice 30 or 60 min post-injury. Immediately after bTBI, righting reflex (RR) suppression times were assessed, euthanasia at the time points post-injury was completed, the brain was harvested and the individual MCA segments were collected, mounted and pressurized. As the intraluminal pressure perfused through the arterial segments was reduced in 20 mmHg increments from 100 to 20 mmHg, MCA diameters were measured and recorded. With decreasing intraluminal pressure, MCA diameters steadily increased significantly above baseline in the Sham bTBI groups while MCA dilator responses were significantly reduced (p &#60; 0.05) in both bTBI groups as evidenced by the impaired, smaller MCA diameters recorded for the bTBI groups. In addition, RR suppression in the bTBI groups was significantly (p &#60; 0.05) higher than in the Sham bTBI groups. MCA&#39;s collected from the Sham bTBI groups exhibited typical vasodilatory properties to decreases in intraluminal pressure while MCA&#39;s collected following bTBI exhibited significantly impaired myogenic vasodilatory responses to reduced pressure that persisted for at least 60 min after bTBI.

Here, we present a protocol to describe methods for ex vivo vascular reactivity determination following a primary blast traumatic brain injury (bTBI) using isolated, pressurized, rodent middle cerebral arterial (MCA) segments. bTBI induction is accomplished using a shock tube, also known as an Advanced Blast Simulator (ABS) device.

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral ...

Non-invasive Strategies for Chronic Manipulation of DREADD-controlled Neuronal Activity

Chemogenetic strategies have emerged as reliable tools for remote control of neuronal activity. Among these, designer receptors exclusively activated by designer drugs (DREADDs) have become the most popular chemogenetic approach used in modern neuroscience. Most studies deliver the ligand clozapine-N-oxide (CNO) using a single intraperitoneal injection, which is suitable for the acute activation/inhibition of the targeted neuronal population. There are, however, only a few examples of strategies for chronic modulation of DREADD-controlled neurons, the majority of which rely on the use of delivery systems that require surgical intervention. Here, we expand on two non-invasive strategies for delivering the ligand CNO to chronically manipulate neural population in mice. CNO was administered either by using repetitive (daily) eye-drops, or chronically through the animal&#39;s drinking water. These non-invasive paradigms result in robust activation of the designer receptors that persisted throughout the CNO treatments. The methods described here offer alternatives for the chronic DREADD-mediated control of neuronal activity and may be useful for experiments designed to evaluate behavior in freely moving animals, focusing on less-invasive CNO delivery methods.

Here we describe two non-invasive methods to chronically control neuronal activity using chemogenetics in mice. Eye-drops were used to deliver clozapine-N-oxide (CNO) daily. We also describe two methods for prolonged administration of CNO in drinking water. These strategies for chronic neuronal control require minimal intervention reducing animals&#8217; stress.

Chemogenetic strategies have emerged as reliable tools for remote control of neuronal activity. Among these, designer receptors exclusively activated by ...

Non-Invasive Modulation and Robotic Mapping of Motor Cortex in the Developing Brain

Mapping the motor cortex with transcranial magnetic stimulation (TMS) has potential to interrogate motor cortex physiology and plasticity but carries unique challenges in children. Similarly, transcranial direct current stimulation (tDCS) can improve motor learning in adults but has only recently been applied to children. The use of tDCS and emerging techniques like high&#8211;definition tDCS (HD-tDCS) require special methodological considerations in the developing brain. Robotic TMS motor mapping may confer unique advantages for mapping, particularly in the developing brain. Here, we aim to provide a practical, standardized approach for two integrated methods capable of simultaneously exploring motor cortex modulation and motor maps in children. First, we describe a protocol for robotic TMS motor mapping. Individualized, MRI-navigated 12x12 grids centered on the motor cortex guide a robot to administer single-pulse TMS. Mean motor evoked potential (MEP) amplitudes per grid point are used to generate 3D motor maps of individual hand muscles with outcomes including map area, volume, and center of gravity. Tools to measure safety and tolerability of both methods are also included. Second, we describe the application of both tDCS and HD-tDCS to modulate the motor cortex and motor learning. An experimental training paradigm and sample results are described. These methods will advance the application of non-invasive brain stimulation in children.

We demonstrate protocols for the modulation (tDCS, HD-tDCS) and mapping (robotic TMS) of the motor cortex in children.

Mapping the motor cortex with transcranial magnetic stimulation (TMS) has potential to interrogate motor cortex physiology and plasticity but carries ...

Statistical Modelling of Cortical Connectivity Using Non-invasive Electroencephalograms

Non-invasive electrophysiological recordings are useful for the evaluation of nervous system function. These techniques are inexpensive, fast, replicable, and less resource-intensive than imaging. Further, the functional data produced have excellent temporal resolution, which is not achievable with structural imaging.
Current applications of electroencephalograms (EEG) are limited by data processing methods. Standard analysis techniques using raw time series data at individual channels are very limited methods of interrogating nervous system activity. More detailed information about cortical function can be achieved by examining relationships between channels and deriving statistical models of how areas are interacting, allowing visualization of connectivity between networks.
This manuscript describes a method for deriving statistical models of cortical network activity by recording EEG in a standard manner, then examining the interelectrode coherence measures to assess relationships between the recorded areas. Higher order interactions can be further examined by assessing the covariance between the coherence pairs, producing high-dimensional &#34;maps&#34; of network interactions. These data constructs can be examined to assess cortical network function and its relationship to pathology in ways not achievable with traditional techniques.
This approach offers greater sensitivity to network level interactions than is achievable with raw time series analysis. It is, however, limited by the complexity of drawing specific mechanistic conclusions about the underlying neural populations and the high volumes of data generated, requiring more advanced statistical techniques for evaluation, including dimensionality reduction and classifier-based approaches.

Standard EEG analysis techniques offer limited insight into nervous system function. Deriving statistical models of cortical connectivity offers far greater ability to investigate underlying network dynamics. Improved functional assessment opens new possibilities for diagnosis, prognostication, and outcome prediction in nervous system diseases.

Non-invasive electrophysiological recordings are useful for the evaluation of nervous system function. These techniques are inexpensive, fast, replicable, ...

Laser-Induced Brain Injury in the Motor Cortex of Rats

A common technique for inducing stroke in experimental rodent models involves the transient (often denoted as MCAO-t) or permanent (designated as MCAO-p) occlusion of the middle cerebral artery (MCA) using a catheter. This generally accepted technique, however, has some limitations, thereby limiting its extensive use. Stroke induction by this method is often characterized by high variability in the localization and size of the ischemic area, periodical occurrences of hemorrhage, and high death rates. Also, the successful completion of any of the transient or permanent procedures requires expertise and often lasts for about 30 minutes. In this protocol, a laser irradiation technique is presented that can serve as an alternative method for inducing and studying brain injury in rodent models.
When compared to rats in the control and MCAO groups, the brain injury by laser induction showed reduced variability in body temperature, infarct volume, brain edema, intracranial hemorrhage, and mortality. Furthermore, the use of a laser-induced injury caused damage to the brain tissues only in the motor cortex unlike in the MCAO experiments where destruction of both the motor cortex and striatal tissues is observed.
Findings from this investigation suggest that laser irradiation could serve as an alternative and effective technique for inducing brain injury in the motor cortex. The method also shortens the time for completing the procedure and does not require expert handlers.

The protocol presented here shows a technique to create a rodent model of brain injury. The method described here uses laser irradiation and targets motor cortex.

A common technique for inducing stroke in experimental rodent models involves the transient (often denoted as MCAO-t) or permanent (designated as MCAO-p) ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...