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 microbubbles in normal saline by probe sonication from decafluorobutane gas and stabilize with DSPC/PEG stearate monolayer shell12.</li>
	<li>Size microbubbles by flotation at normal gravity. Determine microbubble concentration and size by electrozone sensing using Multisizer III counter. Microbubble concentration and size distribution should be 6 x 108/mL and ~2 &#956;m (mean particle diameter), respectively. The largest bubbles are removed by flotation exclusion/separation.</li>
	<li>Alternatively, purchase commercially available microbubbles.</li>
</ol>
2. Preparation of animals
<ol>
	<li>Acclimatize the animal (rat or mouse) for 3 days after delivery. The experiments described here used Sprague-Dawley rats (5&#8211;6 weeks of age) or telencephalic internal structural heterotopia (tish) rats (local colony).</li>
	<li>House the animals under a 12 hour light: 12 hour dark cycle.</li>
	<li>Record the animals&#8217; weights. This information is important throughout the procedure.</li>
	<li>Obtain T2-weighted MR images the day before the FUS procedure in order to establish preoperative baseline images. Use the following parameters for T2 imaging: repetition time/echo time [TR/TE] =3,000/138 milliseconds, 3 averages, field of view=29 x 45 mm2, matrix size = 125 x 192, slice thickness = 0.23 mm.</li>
	<li>Anesthetize the animal with isoflurane (4% induction, 2% maintenance). Confirm adequate depth of anesthesia using a toe pinch. Apply an ophthalmic ointment to the eyes.</li>
	<li>Shave the animal&#8217;s scalp, and remove the remaining hair with depilatory cream.</li>
	<li>Use a tail vein catheter for the infusion of microbubbles, contrast agent, and QA. The catheters consist of a length of PE10 tubing fitted with a 30 G x &#189; inch needle. Leave a 1 mL syringe with heparinized saline in line, to be removed and reattached when the line is used for infusions.</li>
</ol>
3. MRI and PING procedures
<ol>
	<li>Perform the MRI on a 7 T MR unit with a gradient strength of 600 mT/m/ms (Figure 1 and Figure 2). Perform MRI acquisitions using a surface coil incorporated in the FUS system. 
	NOTE: The FUS system used for the experiments comprises three parts: (i) the sonication system is a MR-compatible pre-focused, 8-element annular array, 1.5 MHz transducer (spherical radius = 20 mm &#177; 2 mm, active diameter = 25 mm (f-number = 0.8), with 80% electric-acoustic efficiency, which is connected to a phased array generator and RF power amplifier; (ii) a MR-compatible motorized positioning stage to move the transducer in the anterior-posterior direction and medio-lateral direction; (iii) a Thermoguide workstation to control the delivery of sonication, including electronic focusing through phase modulation to adjust the focal depth (Figure 2).</li>
	<li>Place the anesthetized animal on the coil sled assembly (Figure 1) of the MR-compatible FUS system in the prone position. Immobilize the animal using the incisor bar and ear bars incorporated in the cradle of the sled.</li>
	<li>Apply acoustic gel to the water-wetted scalp; ensuring that no bubbles exist, place the membrane barrier of the water circulator portion of the transducer assembly above the animal&#8217;s skull, and lower the transducer assembly as far as possible in a parallel planar orientation relative to the skull plate. Place the diaphragm of the transducer firmly against the animal&#8217;s shaven scalp directly over the skull plate.</li>
	<li>Attach a pneumatic sensor to the body with surgical tape, to monitor respiration. Position the pneumatic sensor on the left lower rib cage.</li>
	<li>Move the FUS arm assembly with coil, sled, and animal into the 7T MRI unit (Figure 1).</li>
	<li>Run a T2-scout sequence to determine the general physical position of the transducer assembly relative to the animal&#8217;s head, and make mechanical adjustments as necessary (Figure 2). The parameters for T2 imaging are: repetition time/echo time [TR/TE] = 3,000/138 milliseconds, 3 averages, field of view = 29 x 45 mm2, matrix size = 125 x 192, slice thickness = 0.23 mm. Thermometry is typically not used in this protocol.</li>
	<li>Obtain T2 images to refine the transducer positioning. Precisely, define the transducer location and specify the focal point(s) of sonication using the targeting function of the software. The parameters for T2 imaging are: repetition time/echo time [TR/TE] = 3,000/138 milliseconds, 3 averages, field of view = 29 x 45 mm2, matrix size = 125 x 192, slice thickness = 0.23 mm.</li>
	<li>Just prior to sonication, inject 300 &#956;L/kg of microbubbles14 via the tail vein.</li>
	<li>Use a 1.5 MHz transducer to produce sonications (30 ms wave packet, 3% duty cycle, 1 Hz burst repetition frequency, 240 s duration/sonication.</li>
	<li>Immediately after sonication, inject gadodiamide contrast agent via the tail vein, and then perform T1-weighted plus contrast scans to confirm opening of the BBB and the accuracy of targeting. The parameters for T1-weighted imaging: TR/TE = 900/12 milliseconds, 2 averages, field of view = 24 x 30 mm2, matrix size = 208 x 256, slice thickness = 0.7 mm. Typically, a single T1 scan is performed.</li>
	<li>Remove the FUS arm and sled from the MRI, and place the animal on a heating pad set to 40 &#176;C, while maintaining 2% isoflurane anesthesia.</li>
	<li>Starting 30 min after sonication, use a syringe pump to infuse QA (75 mg/mL stock solution) via the tail vein for 1 h at a rate of 16.8 &#181;L/min to achieve a final dosage of 225 mg/kg (q.s. to 1.0 mL saline).</li>
	<li>When the infusion is complete, discontinue anesthesia, keeping the animal on a heating pad until alert. Place the animal in a cage and make routine checks for its activity every 15 min, for several hours after the procedure.</li>
	<li>Return the animal to the vivarium and check every 6 h for the first day for distress or irregular activity.</li>
	<li>One day post-sonication, perform T2-weighted MR imaging to assess any damages in the area of sonication. The parameters for T2 imaging: repetition time/echo time [TR/TE] = 3,000/138 milliseconds, 3 averages, field of view = 29 x 45 mm2, matrix size = 125 x 192, slice thickness = 0.23 mm. Images are evaluated for areas of hyperintensity to identify possible tissue damage/edema.</li>
</ol>
4. Post-mortem analysis of neuronal loss
<ol>
	<li>Allow a post-sonication, survival period of 4&#8211;5 days for assessing neuronal loss with Fluoro-Jade histochemistry.</li>
	<li>Deeply anesthetize the animal with isoflurane, and euthanize via intracardial perfusion with 0.1 M phosphate buffer (pH 7.4) followed by 4% paraformaldehyde in phosphate buffer.</li>
	<li>Remove the brain from the skull and post-fix for 2 days in 4% paraformaldehyde.</li>
	<li>Immerse the brain in 30% sucrose for cryoprotection, and cut sections at a thickness of 20&#8211;30 &#956;m with a cryostat.</li>
	<li>Mount cryostat sections onto gelatinized slides and air-dry overnight.</li>
	<li>Rehydrate slides in distilled water, and then dehydrate in ascending graded ethanols. After dehydration, rehydrate slides in descending graded ethanols.</li>
	<li>Transfer slides to a solution of 0.06% potassium permanganate for 15 min on an orbital shaker.</li>
	<li>Rinse slides for 1 min in distilled water and transfer to a 0.001% solution of Fluoro-Jade B in 0.1% acetic acid. Incubate under gentle agitation for 30 min at room temperature. Rinse slides three times for 1 min in distilled water.</li>
	<li>Dry the slides on a slide warmer, equilibrate in xylenes for 3 min, and coverslip using DPX mounting media.</li>
</ol>

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The authors have nothing to disclose.

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>KMnO&lt;sub&gt;4&lt;/sub&gt;</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 &amp;amp; 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...

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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 ...

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

Research

JoVE Journal

Neuroscience

5.1K Views.  University of Virginia. Chirurgische ingreep kan heel effectief zijn voor de behandeling van bepaalde vormen van medisch hardnekkige neurologische ziekte. Momenteel beschikbaar chirurgische modaliteiten terwijl effectief, over het algemeen betrekken invasieve procedures die kunnen resulteren in chirurgische letsel aan niet-doelweefsels. Bijgevolg zou het een waarde zijn om het bereik van chirurgische benaderingen uit te breiden met een techniek die niet-invasief is en neuronale laesies produceert.Deze video p...

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

Targeting of Deep Brain Structures with Microinjections for Delivery of Drugs, Viral Vectors, or Cell Transplants

Microinjections into the brain parenchyma are important procedures to deliver drugs, viral vectors or cell transplants.  The brain lesion that an injecting needle produces during its trajectory is a major concern especially in the mouse brain for not only the brain is small but also sometimes multiple injections are needed.  We show here a method to produce glass capillary needles with a 50-&mu;m lumen which significantly reduces the brain damage and allows a precise targeting into the rodent brain. This method allows a delivery of small volumes (from 20 to 100 nl), reduces bleeding risks, and minimizes passive diffusion of drugs into the brain parenchyma. By using different size of capillary glass tubes, or changing the needle lumen, several types of substances and cells can be injected. Microinjections with a glass capillary tube represent a significant improvement in injection techniques and deep brain targeting with minimal collateral damage in small rodents.

In this article, we show a method to make glass capillary needles with a 50-&mu;m lumen. This technique significantly reduces the brain damage, minimizes passive diffusion of drugs and allows a precise targeting into the rodent brain.

Microinjections into the brain parenchyma are important procedures to deliver drugs, viral vectors or cell transplants.  The brain lesion that an ...

Investigations on Alterations of Hippocampal Circuit Function Following Mild Traumatic Brain Injury

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological impairments 1. Two pervasive and disabling symptoms experienced by TBI survivors, memory deficits and a reduction in seizure threshold, are thought to be mediated by TBI-induced hippocampal dysfunction 2,3. In order to demonstrate how altered hippocampal circuit function adversely affects behavior after TBI in mice, we employ lateral fluid percussion injury, a commonly used animal model of TBI that recreates many features of human TBI including neuronal cell loss, gliosis, and ionic perturbation 4-6.
	Here we demonstrate a combinatorial method for investigating TBI-induced hippocampal dysfunction. Our approach incorporates multiple ex vivo physiological techniques together with animal behavior and biochemical analysis, in order to analyze post-TBI changes in the hippocampus. We begin with the experimental injury paradigm along with behavioral analysis to assess cognitive disability following TBI. Next, we feature three distinct ex vivo recording techniques: extracellular field potential recording, visualized whole-cell patch-clamping, and voltage sensitive dye recording. Finally, we demonstrate a method for regionally dissecting subregions of the hippocampus that can be useful for detailed analysis of neurochemical and metabolic alterations post-TBI.
	These methods have been used to examine the alterations in hippocampal circuitry following TBI and to probe the opposing changes in network circuit function that occur in the dentate gyrus and CA1 subregions of the hippocampus (see Figure 1). The ability to analyze the post-TBI changes in each subregion is essential to understanding the underlying mechanisms contributing to TBI-induced behavioral and cognitive deficits. 
	The multi-faceted system outlined here allows investigators to push past characterization of phenomenology induced by a disease state (in this case TBI) and determine the mechanisms responsible for the observed pathology associated with TBI.

A multi-faceted approach to investigating functional changes to hippocampal circuitry is explained. Electrophysiological techniques are described along with the injury protocol, behavioral testing and regional dissection method. The combination of these techniques can be applied in similar fashion for other brain regions and scientific questions.

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological ...

Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical insult to secondary injury processes, including apoptosis and inflammation. Animal modeling has been valuable in the search to unravel injury mechanisms and evaluate potential neuroprotective therapies. This protocol describes the controlled cortical impact (CCI) model of focal, open-head TBI. Specifically, parameters for producing a mild unilateral cortical injury are described. Behavioral consequences of CCI are analyzed using the adhesive tape removal test of bilateral sensorimotor integration. Regarding experimental therapy for TBI pathology, this protocol also illustrates a process for transplanting cultured cells into the brain. Neural cell cultures derived from human induced pluripotent stem cells (hiPSCs) were chosen for their potential to show superior functional restoration in human TBI patients. Chronic survival of hiPSCs in the host mouse brain tissue is detected using a modified DAB immunohistochemical process.

This protocol demonstrates methodologies for a mouse model of open-skull traumatic brain injury and transplantation of cultured human induced pluripotent stem cell-derived cells into the injury site. Behavioral and histologic tests of outcomes from these procedures are also described in brief.

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical ...

Developmental Biology

Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no effective treatment for it yet. Limitations of currently used animal models impede understanding of the pathology mechanism. Multiphoton microscopy allows studying cells and tissues within intact animal brains longitudinally under physiological and pathological conditions. Here, we describe two models of acute brain injury studied by means of two-photon imaging of brain cell behavior under posttraumatic conditions. A selected brain region is injured with a sharp needle to produce a trauma of a controlled width and depth in the brain parenchyma. Our method uses stereotaxic prick with a syringe needle, which can be combined with simultaneous drug application. We propose that this method can be used as an advanced tool to study cellular mechanisms of pathophysiological consequences of acute trauma in mammalian brain in vivo. In this video, we combine acute brain injury with two preparations: cranial window and skull thinning. We also discuss advantages and limitations of both preparations for multisession imaging of brain regeneration after trauma.

Acute brain trauma is a severe injury that has no adequate treatment to date. Multiphoton microscopy allows studying longitudinally the process of acute brain trauma development and probing therapeutical strategies in rodents. Two models of acute brain trauma studied with in vivo two-photon imaging of brain are demonstrated in this protocol.

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no ...

Medicine

Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing system was developed that allows for the injection of a drug while recording electrophysiological signals from the affected neuronal population. This method provides a simple and economical alternative to commercial solutions. A glass pipette was modified by combining it with a hypodermic needle and a silver filament. The injectrode is attached to commercial microsyringe pump for drug delivery. This results in a technique that provides real-time pharmacodynamics feedback through multi-unit extracellular signals originating from the site of drug delivery. As a proof of concept, we recorded neuronal activity from the superior colliculus elicited by flashes of light in rats, concomitantly with delivery of drugs through the injectrode. The injectrode recording capacity permits the functional characterization of the injection site favoring precise control over the localization of drug delivery. Application of this method also extends far beyond what is demonstrated here, as the choice of chemical substance loaded into the injectrode is vast, including tracing markers for anatomic experiments.

Here, we craft a glass pipette with dual functions: inhibition of deep brain structures by microinjections of drugs and real-time monitoring of their effects through simultaneous electrophysiological recordings.

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing ...

Targeting Neuronal Fiber Tracts for Deep Brain Stimulation Therapy Using Interactive, Patient-Specific Models

Deep brain stimulation (DBS), which involves insertion of an electrode to deliver stimulation to a localized brain region, is an established therapy for movement disorders and is being applied to a growing number of disorders. Computational modeling has been successfully used to predict the clinical effects of DBS; however, there is a need for novel modeling techniques to keep pace with the growing complexity of DBS devices. These models also need to generate predictions quickly and accurately. The goal of this project is to develop an image processing pipeline to incorporate structural magnetic resonance imaging (MRI) and diffusion weighted imaging (DWI) into an interactive, patient specific model to simulate the effects of DBS. A virtual DBS lead can be placed inside of the patient model, along with active contacts and stimulation settings, where changes in lead position or orientation generate a new finite element mesh and solution of the bioelectric field problem in near real-time, a timespan of approximately 10 seconds. This system also enables the simulation of multiple leads in close proximity to allow for current steering by varying anodes and cathodes on different leads. The techniques presented in this paper reduce the burden of generating and using computational models while providing meaningful feedback about the effects of electrode position, electrode design, and stimulation configurations to researchers or clinicians who may not be modeling experts.

The goal of this project is to develop an interactive, patient-specific modeling pipeline to simulate the effects of deep brain stimulation in near real-time and provide meaningful feedback as to how these devices influence neural activity in the brain.

Deep brain stimulation (DBS), which involves insertion of an electrode to deliver stimulation to a localized brain region, is an established therapy for ...

Bioengineering

Anatomically Inspired Three-dimensional Micro-tissue Engineered Neural Networks for Nervous System Reconstruction, Modulation, and Modeling

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory environment and the limited capacity for neurogenesis. We are developing&#160;a strategy to simultaneously address&#160;neuronal and axonal pathway loss within the damaged CNS. This manuscript presents the fabrication protocol for micro-tissue engineered neural networks (micro-TENNs), implantable constructs consisting of neurons and aligned axonal tracts spanning the extracellular matrix (ECM) lumen of a preformed hydrogel cylinder hundreds of microns in diameter that may extend centimeters in length. Neuronal aggregates are delimited to the extremes of the three-dimensional encasement and are spanned by axonal projections. Micro-TENNs are uniquely poised as a strategy for CNS reconstruction, emulating aspects of brain connectome cytoarchitecture and potentially providing means for network replacement. The neuronal aggregates may synapse with host tissue to form new functional relays to restore and/or modulate missing or damaged circuitry. These constructs may also act as pro-regenerative&#160;&#34;living scaffolds&#34; capable of exploiting developmental mechanisms for cell migration and axonal pathfinding, providing synergistic structural and soluble cues based on the state of regeneration. Micro-TENNs are fabricated by pouring liquid hydrogel into a cylindrical mold containing a longitudinally centered needle. Once the hydrogel has gelled, the needle is removed, leaving a hollow micro-column. An ECM solution is added to the lumen to provide an environment suitable for neuronal adhesion and axonal outgrowth. Dissociated neurons are mechanically aggregated for precise seeding within one or both ends of the micro-column. This methodology reliably produces self-contained miniature constructs with long-projecting axonal tracts that may recapitulate features of brain neuroanatomy. Synaptic immunolabeling and genetically encoded calcium indicators suggest that micro-TENNs possess extensive synaptic distribution and intrinsic electrical activity. Consequently, micro-TENNs represent a promising strategy for targeted neurosurgical reconstruction of brain pathways and may also be applied as biofidelic models to study neurobiological phenomena in vitro.

This manuscript details the fabrication of micro-tissue engineered neural networks: three-dimensional micron-sized constructs comprised of long aligned axonal tracts spanning aggregated neuronal population(s) encased in a tubular hydrogel. These living scaffolds can serve as functional relays to reconstruct or modulate neural circuitry or as biofidelic test-beds mimicking gray-white matter neuroanatomy.

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory ...

Stereotactic Atlas-Guided Laser Capture Microdissection of Brain Regions Affected by Traumatic Injury

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial distribution. Such techniques also potentially skew gene expression analysis because the process itself can alter expression patterns in individual cells. Here we describe a laser capture microdissection (LCM) method to selectively collect specific brain regions affected by traumatic brain injury (TBI) by using a modified Nissl (cresyl violet) staining protocol and the guidance of a rat brain atlas. LCM provides access to brain regions in their native positions and the ability to use anatomical landmarks for identification of each specific region. To this end, LCM has been used previously to examine brain region specific gene expression in TBI. This protocol allows examination of TBI-induced alterations in gene and microRNA expression in distinct brain areas within the same animal. The principles of this protocol can be amended and applied to a wide range of studies examining genomic expression in other disease and/or animal models.

We describe the use of laser capture microdissection to obtain samples of distinct cell populations from different brain regions for gene and microRNA analysis. This technique allows the study of differential effects of traumatic brain injury in specific regions of the rat brain.

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial ...

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various intrinsic and extrinsic inhibitory factors involved in the restriction of axon regeneration have been identified. However, simply removing these inhibitory cues is insufficient for successful regeneration, indicating the existence of additional regulatory machinery. Drosophila melanogaster, the fruit fly, shares evolutionarily conserved genes and signaling pathways with vertebrates, including humans. Combining the powerful genetic toolbox of flies with two-photon laser axotomy/dendriotomy, we describe here the Drosophila sensory neuron &#8211; dendritic arborization (da) neuron injury model as a platform for systematically screening for novel regeneration regulators. Briefly, this paradigm includes a) the preparation of larvae, b) lesion induction to dendrite(s) or axon(s) using a two-photon laser, c) live confocal imaging post-injury and d) data analysis. Our model enables highly reproducible injury of single labeled neurons, axons, and dendrites of well-defined neuronal subtypes, in both the peripheral and central nervous system.

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

Here, we present a protocol using the Drosophila sensory neuron - dendritic arborization (da) neuron injury model, which combines in vivo live imaging, two-photon laser axotomy/dendriotomy, and the powerful fly genetic toolbox, as a platform for screening potential promoters and inhibitors of neuroregeneration.

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various ...

In Vivo Intracerebral Stereotaxic Injections for Optogenetic Stimulation of Long-Range Inputs in Mouse Brain Slices

Knowledge of cell-type specific synaptic connectivity is a crucial prerequisite for understanding brain-wide neuronal circuits. The functional investigation of long-range connections requires targeted recordings of single neurons combined with the specific stimulation of identified distant inputs. This is often difficult to achieve with conventional and electrical stimulation techniques, because axons from converging upstream brain areas may intermingle in the target region. The stereotaxic targeting of a specific brain region for virus-mediated expression of light-sensitive ion channels allows selective stimulation of axons originating from that region with light. Intracerebral stereotaxic injections can be used in well-delimited structures, such as the anterior thalamic nuclei, in addition to other subcortical or cortical areas throughout the brain.
Described here is a set of techniques for precise stereotaxic injection of viral vectors expressing channelrhodopsin in the mouse brain, followed by photostimulation of axon terminals in the brain slice preparation. These protocols are simple and widely applicable. In combination with whole-cell patch clamp recording from a postsynaptically connected neuron, photostimulation of axons allows the detection of functional synaptic connections, pharmacological characterization, and evaluation of their strength. In addition, biocytin filling of the recorded neuron can be used for post-hoc morphological identification of the postsynaptic neuron.

This protocol describes a set of methods to identify the cell-type specific functional connectivity of long-range inputs from distant brain regions using optogenetic stimulations in ex vivo brain slices.

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

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