Name: targeted neuronal injury for the non-invasive disconnection of brain circuitry
Uploaded: 2020-09-27T13:00:00
Description: Watch this Scientific Journal Video about Targeted Neuronal Injury for the Non-Invasive Disconnection of Brain Circuitry at JoVE.com

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>
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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. 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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.

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 ...

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 ...

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

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 ...

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 ...

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 ...

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 ...

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 ...

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, ...

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) ...

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 ...