This work was supported by grants of the University of Zurich (FK-15-057), the Novartis Foundation for medical-biological Research (16C231) and Swiss Cancer Research (KFS-3852-02-2016, KFS-4146-02-2017) to Johannes vom Berg and BRIDGE Proof of Concept (20B1-1_177300) to Linda Schellhammer.

All methods described here have been approved by the Swiss Cantonal Veterinary Office under license number ZH246/15.
1. Preparation of the Step Catheters
<ol>
	<li>Preparation of a fused silica tubing for the step of the catheter
	<ol>
		<li>Cut the fused silica capillary with inner diameter of 0.1 mm and wall thickness of 0.0325 mm tubing to a length of 30 mm.</li>
		<li>Examine the tubing for cracks and heat polish the ends using a microforge to ensure the tubing openings have a smooth surface.</li>
	</ol>
	</li>
	<li>Fixation of the inner tube in a metal needle
	<ol>
		<li>Mount a 27 G needle on a 10 &#956;L&#160;syringe and place the syringe in a stereotactic robot.</li>
		<li>Using the robot, move the syringe over a hard surface and touch it with the needle tip. This position should be noted or saved in the software because it will serve as a reference surface for setting the length of the catheter step.</li>
		<li>Elevate the needle to enable placing of the fused silica capillary inside of the needle</li>
		<li>Place the fused silica capillary in the needle such that 20 mm of the capillary is protruding from the needle.</li>
		<li>Using a pipette, evenly spread 2 &#956;L of high viscosity cyanoacrylate adhesive over the capillary, starting from the metal needle and finishing 10 mm above the lower end of the capillary, as depicted in Figure 2.</li>
		<li>Using the stereotactic robot, lower the needle until the tip of the metal needle is 1 mm over the reference surface. This way the fused silica capillary will be fixed in the metal needle and will form a 1 mm step from the tip of the metal needle. Remove any excess of glue forming at the end of the metal needle to avoid blunting the step.&#160;</li>
		<li>Wait 15 min for the glue to harden and remove the syringe with the catheter from the stereotactic robot.&#160;Confirm that at the step all excess glue has been removed by checking the tip of the catheter under a microscope.</li>
	</ol>
	</li>
	<li>Testing the step catheter using a block of agarose
	<ol>
		<li>Prepare 0.6% agarose solution in PBS in a conventional gel tray and wait until it polymerizes. Cut the agarose in approximately 20 mm x 20 mm blocks. Until use, keep the blocks immersed in PBS.</li>
		<li>Manually fill the step catheter syringe with 10 &#956;L of 0.4% solution of filtered trypan blue.</li>
		<li>Using the stereotactic robot, dispense 1 &#956;L at 0.2 &#956;L/min in order to assess sealing of the step of the catheter during the fixation procedure. Trypan blue solution should be visible solely on the tip of the catheter. Wipe it off with a paper tissue.</li>
		<li>Place the agarose block in the stereotactic robot and calibrate the robot so the tip of the catheter is referenced against the surface of the agarose block.</li>
		<li>Program the injection parameters for CED.
		<ol>
			<li>For the injection volume of 5 &#181;L, use the following steps: 1 &#181;L at 0.2 &#181;L/min, then 2 &#181;L at 0.5 &#181;L/min and 2 &#181;L at 0.8 &#181;L/min. Adjust the final injection volume according to the specific experimental plan by proportionally changing the duration of each of the steps.</li>
			<li>In order to inject the solution into murine caudate putamen (striatum), perform such injection in a position 1 mm frontal and 1.5&#8211;2 mm lateral from bregma at the depth of 3.5 mm.</li>
			<li>After the injection, leave the catheter in place for 2 min and then retract at 1 mm/min to ensure proper dispersion of the fluid in the brain and sealing of the injection tract during catheter removal. 
			NOTE: Depending on the specific stereotactic robot used, all the parameters can be programmed into a single script. An example script is available as <a href="https://www.jove.com/files/ftp_upload/59675/CED_5_ul_striatum.txt" target="_blank">Supplementary Material.</a>.</li>
		</ol>
		</li>
		<li>Start the CED procedure and inject 5 &#956;L of trypan blue solution into the agarose block.</li>
		<li>Assess the shape of cloud of trypan blue in the agarose and potential leakage along the catheter tract. Trypan blue should form an ellipsoid or a round cloud with the center around the catheter tip and a diameter of at least 1 mm. No major backflow over the tip of the metal needle should be visible.</li>
		<li>Place a new agarose block and start a second injection of 1 &#181;L at 0.2 &#181;L/min in order to assess clogging of the catheter with the agarose. Trypan blue should again start forming a cloud from the tip of the catheter immediately after the start of the injection.</li>
		<li>Assess whether the leftover volume in the syringe corresponds to 3 &#181;L. Any variations might point towards a leakage of fluid through the catheter mounting or syringe plunger.</li>
		<li>If all the test injections are successful, the catheter is well sealed, straight and no trypan blue solution is observed from other spots than the catheter tip, wash the catheter with deionized H2O (dH2O) until no traces of trypan blue are visible and then wash ten times as follows: 70% ethanol and 100% ethanol followed by flushing again with 70% ethanol and clean deionized water.</li>
		<li>Store the catheter under dry conditions.</li>
	</ol>
	</li>
</ol>
2. Convection-enhanced Delivery of Antibody Solution into the Murine Brain
NOTE: Depending on local animal welfare regulations, various types of anesthetics, analgesics and antibiotics can be implemented for this procedure. This protocol describes the use of injection anesthesia. Inhalation anesthetics such as isoflurane can also be used by mounting a nose mask on the stereotactic frame. In addition, we recommend adding antibiotics to the drinking water for infection prophylaxis.
<ol>
	<li>Surgical setup
	<ol>
		<li>Prepare anesthetics and antidote solutions. Mice can be safely anesthetized using a three-component anesthesia containing fentanyl (0.05 mg/kg), midazolam (5 mg/kg) and medetomidine (0.5 mg/kg) diluted in sterile dH2O. We perform a two-step wake up procedure using two antidote solutions, one containing flumazenil (0.5 mg/kg) and buprenorphine (0.1 mg/kg) in sterile dH2O (first antidote solution). The second one contains atipamezole (2.5 mg/kg) in sterile dH2O (second antidote solution).</li>
		<li>Prepare analgesia solution containing carprofen (5.667 mg/kg) diluted with sterile dH2O.</li>
		<li>Clean the stereotactic frame, heating pad and elements of the stereotactic robot. Bear in mind that not all the parts of the robot can be cleaned without risk of damage. Refer to the manual of the robot for details on cleaning and preparing for usage.</li>
		<li>Assemble the syringe with the step catheter and flush it multiple times with dH2O, 70% ethanol and 100% ethanol followed by flushing again with 70% ethanol and dH2O. Finally, flush the syringe with PBS or other buffers to be used for preparation of the solution for intracranial injection, e.g. artificial cerebrospinal fluid. The plunger of the syringe should move smoothly and freely during the whole procedure.</li>
		<li>Calibrate the stereotactic robot software with the stereotactic frame.</li>
		<li>Test the stereotactic robot software by ensuring that the robot arms move freely and that the injection pump is properly connected and can perform the CED procedure without any disturbances. This includes testing robot movement, ramping injection, checking the 2 min waiting step and the speed of catheter retraction. All the parameters should fit the preprogrammed CED procedure described in point 1.3.5.</li>
		<li>Insert the drill bit in the drill. It is recommended to sterilize the drill bits before use.</li>
		<li>Prepare antibody solution using PBS or other buffer solutions such as aCSF. 1 to 20 &#181;g of antibody in 5 &#181;L can be injected in a single CED procedure. Other volumes and protein amounts should be tested prior to performing the experiment. Be aware that using high viscosity solutions might lead to catheter clogging.</li>
		<li>Manually load the syringe with the diluted antibody.</li>
	</ol>
	</li>
	<li>Antibody injection by CED into striatum
	<ol>
		<li>Weigh the mouse and inject the three-component anesthesia solution into the peritoneum according to the body weight. Note the injection time. Transfer the mouse to a separate cage heated with a heating pad.</li>
		<li>Observe the mouse to determine when the sedation starts. As soon as the mouse stops moving, apply ophthalmic ointment on the eyes to protect the cornea from drying out during the surgery. Full sedation usually starts 10&#8211;15 min from the injection of the three-component anesthesia solution.</li>
		<li>Check pain reactions using the pinch-reflex test to ensure full anesthesia of the animal.</li>
		<li>Shave the head using a hair trimmer.</li>
		<li>Disinfect the skin with cotton swabs soaked in iodine solution.&#160;Scrub the skin three times in circular motion.</li>
		<li>Using a scalpel, make a 10 mm skin incision along the cranial midline finishing on the eye level.</li>
		<li>Fix the mouse in the stereotactic frame using the nose clamp and ear bars. Ensure that the skull surface is horizontal and tightly secured. Apart from correct anatomical navigation this is also crucial to avoid tilting of the skull during the drilling and the CED procedure.</li>
		<li>Place the syringe in the stereotactic robot.</li>
		<li>Synchronize the drill bit with the tip of the catheter on a reference point. It is crucial that the relation between the position of the drill and the syringe is precisely determined in the software, so the injection can be performed in the desired anatomical region of the brain.</li>
		<li>Retract the skin using forceps and localize bregma on the skull surface.</li>
		<li>Reference bregma in the software using the tip of the drill bit.</li>
		<li>Move the drill to a position 1 mm frontal and 2 mm lateral from bregma and drill a burr hole. Be careful not to damage the dura mater.</li>
		<li>Move the syringe over the burr hole.</li>
		<li>Dispense 0.5&#8211;1 &#956;L from the syringe to ensure that no air bubbles are left in the catheter.</li>
		<li>Start the CED program described in point 1.3.5. Observe the skull surface for any traces of fluid backflow from the injection spot. Monitor the breathing rate of the animal.</li>
		<li>Once the CED program is over and the catheter is withdrawn from the brain, start the injection pump at 0.2 &#181;L/min in order to check for catheter clogging during the CED. If no clogging occurred, you should immediately see a droplet of injection mix coming from the catheter tip.</li>
		<li>Prior to reusing or storing the catheter, visually examine the catheter step for any signs of damage or wear under a microscope and clean it as in step 1.3.10.</li>
	</ol>
	</li>
	<li>Waking up procedure
	<ol>
		<li>Gently remove the mouse from the stereotactic frame.</li>
		<li>Wash the surgery site with sterile saline solution.</li>
		<li>Using forceps, fill the burr hole with bone wax.</li>
		<li>Close the skin with thin-tipped forceps and apply surgical glue with a 10 &#181;L pipette over the cut. Wait 15&#8211;30 s for the glue to polymerize.</li>
		<li>Apply analgesia solution by subcutaneous injection. Note the time of injection.</li>
		<li>Apply the first antidote solution. Note the time of injection.</li>
		<li>Transfer the mouse to a separate cage with a heating pad and monitor the animal for startle reflexes.</li>
		<li>If mouse has not gained full consciousness 15 min after administration of the first antidote solution, apply the second antidote solution by subcutaneous injection.</li>
		<li>Monitor animals during recovery phase.</li>
		<li>Check 1&#8211;2 h later as well as the next day for post-operative complications. If necessary, re-apply the analgesia.</li>
		<li>For infection prophylaxis, add sulfadoxin (final concentration 0.08% w/v) and trimethoprim (final concentration 0.016% w/v) to drinking water to which the animals have access ad libitum for 1 week after the surgery.</li>
	</ol>
	</li>
</ol>

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Johannes vom Berg is mentioned as an inventor on patent application (PCT/EP2012/070088) of the University of Zurich. Michal Beffinger, Linda Schellhammer and Johannes vom Berg are mentioned as inventors on a patent application (EP19166231) of the University of Zurich. The authors have no additional financial interests.

Convection-enhanced delivery, or pressure-mediated drug infusion into the brain, was first proposed in the early 19903. This approach promises perfusion of large brain volumes behind the blood brain barrier in a controlled manner2. However, so far, only a few clinical trials have been performed using this approach, partially because CED in a clinical setup has shown to be technically demanding24,25. Recent developme...

The blood brain barrier (BBB) forms a semipermeable border separating the central nervous system (CNS) from the blood circulation. Reaching the CNS with therapeutics is however necessary in context of various diseases, like brain tumors, Alzheimer&#8217;s disease (AD) or Parkinson&#8217;s disease (PD) among others1. This becomes important in the development of new therapies, especially if the tested drug exhibits poor BBB permeability or its systemic exposure can lead to dangerous toxicity1,2. Some of the clinically used antibodies display both of these features. A solution to this problem would be to deliver the therapeutics directly behind the BBB.
Convection-enhanced delivery (CED) is a neurosurgical technique enabling effective perfusion of large brain volumes. This is achieved by surgically installing one or more catheters in the target area. During the drug application, a pressure gradient is formed at the opening of the catheter, which becomes the driving force of the infusate dispersion in the tissue3,4. It is thus the duration of infusion and not the diffusion coefficients that determine the perfusion range2,4,5. This provides uniform delivery of the infusate over a much larger brain volume compared to conventional, diffusion based intracerebral injection methods2,6. At the same time, this delivery modality has a lower risk of tissue damage2. Accordingly, CED can enable safe and efficacious administration of conventional chemotherapeutics for treatment of CNS tumors, as well as delivery of immunomodulatory agents or agonistic and antagonistic antibodies in a multitude of other CNS disorders2,7,8,9. CED is currently tested in therapies of Parkinson&#8217;s disease, Alzheimer&#8217;s disease, as well as high-grade glioma2,7,8,10,11.
Catheter design and the injection regimen are among the most important factors influencing the outcome of CED 10,12,13,14,15,16. Furthermore, it requires specific physicochemical properties of the infusate, including moderate size of the particles, an anionic charge, and low tissue affinity 10,17. Each of these parameters has to be potentially adjusted according to the histological features of the brain region to be targeted2,10,17.
Here we describe methodology for performing CED of an antibody solution into the caudate putamen (striatum) of mice. Furthermore, the protocol includes preparation of step catheters in a laboratory setup, testing them in vitro and performing the CED.
There are multiple catheter designs available in the literature, differing by the shape of the cannula, the materials used and the number of catheter openings12,15,18,19,20,21,22. We are using a step catheter made of a fused silica capillary protruding 1 mm from a blunt end metal needle. This catheter design can be easily manufactured in a research laboratory and reproducibly gives good CED results when tested in vitro with agarose blocks with physical parameters resembling brain parenchyma in vivo23.
Moreover, we implement a ramping regimen for delivering 5 &#181;L of infusate in vivo. In such a protocol the injection rate is increased from 0.2 &#181;L/min to a maximum of 0.8 &#181;L/min, thus minimizing chances of infusate reflux along the catheter as well as risk of tissue damage16. Using this protocol, we have successfully administered mice with up to 20 &#181;g of antibody in 5 &#181;L of PBS over the course of 11 min 30 s.
The protocol can be readily adjusted for other infusion volumes or for injecting various other substances, e.g. chemotherapeutics, cytokines, viral particles or liposomes2,10,14,18,22. In case of using infusate with drastically different physicochemical properties compared to a phosphate buffered saline (PBS) or artificial cerebrospinal fluid (aCSF) solution of antibodies, additional validation steps are recommended. For catheter assembly, validation and CED, we describe all steps using a stereotactic robot with a drill and injection unit mounted onto a regular stereotactic frame. This procedure can also be performed with a manual stereotactic frame connected to programmable microinfusion pump that can drive the described glass microsyringes.

<table>
	<tbody>
		<tr>
			<td>10 &#956;L syringe</td>
			<td>Hamilton</td>
			<td>7635-01</td>
			<td></td>
		</tr>
		<tr>
			<td>27 G blunt end needle</td>
			<td>Hamilton</td>
			<td>7762-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Promega</td>
			<td>V3121</td>
			<td></td>
		</tr>
		<tr>
			<td>Atipamezol</td>
			<td>Janssen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bone wax</td>
			<td>Braun</td>
			<td>1029754</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>Indivior Schweiz AG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Carprofen</td>
			<td>Pfizer AG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dental drill bits, steel, size ISO 009</td>
			<td>Hager &#38; Meisinger</td>
			<td>1RF009</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 100%</td>
			<td>Reuss-Chemie AG</td>
			<td>179-VL03K-/1</td>
			<td></td>
		</tr>
		<tr>
			<td>Fentanyl</td>
			<td>Helvepharm AG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FITC-Dextran, 2000 kDa</td>
			<td>Sigma Aldrich</td>
			<td>FD2000S</td>
			<td></td>
		</tr>
		<tr>
			<td>Flumazenil</td>
			<td>Labatec Pharma AG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Sigma Aldrich</td>
			<td>F8775-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>High viscosity cyanoacrylate glue</td>
			<td>Migros</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Iodine solution</td>
			<td>Mundipharma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Medetomidin</td>
			<td>Orion Pharma AG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microforge</td>
			<td>Narishige</td>
			<td>MF-900</td>
			<td></td>
		</tr>
		<tr>
			<td>Midazolam</td>
			<td>Roche Pharma AG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ophthalmic ointment</td>
			<td>Bausch + Lomb</td>
			<td>Vitamin A Blache</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>ThermoFischer Scientific</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyclonal goat anti-rat IgG (H+L) antibody coupled with Alexa Fluor 647</td>
			<td>Jackson Immuno</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpels</td>
			<td>Braun</td>
			<td>BB518</td>
			<td></td>
		</tr>
		<tr>
			<td>Silica tubing internal diameter 0.1 mm, wall thickness of 0.0325 mm</td>
			<td>Postnova</td>
			<td>Z-FSS-100165</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotactic frame for mice</td>
			<td>Stoelting</td>
			<td>51615</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotactic robot</td>
			<td>Neurostar</td>
			<td>Drill and Injection Robot</td>
			<td></td>
		</tr>
		<tr>
			<td>Succrose</td>
			<td>Sigma Aldrich</td>
			<td>S0389-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Topical tissue adhesive</td>
			<td>Zoetis</td>
			<td>GLUture</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>ThermoFischer Scientific</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>Water</td>
			<td>Bichsel</td>
			<td>1000004</td>
			<td></td>
		</tr>
	</tbody>
</table>

delivery of antibodies into the murine brain via convection-enhanced delivery

Convection-enhanced delivery (CED) is a neurosurgical technique enabling effective perfusion of large brain volumes using a catheter system. Such an approach provides a safe delivery method by-passing the blood brain barrier (BBB), thus allowing treatment with therapeutics with poor BBB-permeability or those for which systemic exposure is not desired, e.g., due to toxicity. CED requires optimization of the catheter design, injection protocol, and properties of the infusate. With this protocol we describe how to perform CED of a solution containing up to 20 &#181;g of an antibody into the caudate putamen of mice. It describes preparation of step catheters, testing them in vitro and performing the CED in mice using a ramping injection program. The protocol can be readily adjusted for other infusion volumes and can be used for injecting various tracers or pharmacologically active or inactive substances, including chemotherapeutics, cytokines, viral particles, and liposomes.

This protocol enables preparation of step catheters (Figure 1) for use in the CED procedure in a laboratory environment. In order to control the catheters for leakage, reflux along the needle tract and clogging, we recommend performing injections of a dye, e.g., trypan blue solution, into an agarose block. Figure 3 depicts a cloud of trypan blue forming after injection of 1 &#181;L at 0.5 &#181;L/minute using a CED catheter (Figure 3A</stron...

Watch this Scientific Journal Video about Delivery of Antibodies into the Murine Brain via Convection-enhanced Delivery at JoVE.com

Delivery of Antibodies into the Murine Brain via Convection-enhanced Delivery

Convection-enhanced delivery (CED) is a method enabling effective delivery of therapeutics into the brain by direct perfusion of large tissue volumes. The procedure requires the use of catheters and an optimized injection procedure. This protocol describes a methodology for CED of an antibody into a mouse brain.

Convection-enhanced delivery (CED) is a neurosurgical technique enabling effective perfusion of large brain volumes using a catheter system. Such an ...

delivery-antibodies-into-murine-brain-via-convection-enhanced

Research

JoVE Journal

Neuroscience

8.2K Views.  University of Zurich. Convection-enhanced delivery (CED) is a method enabling effective delivery of therapeutics into the brain by direct perfusion of large tissue volumes. The procedure requires the use of catheters and an optimized injection procedure. This protocol describes a methodology for CED of an antibody into a mouse brain.

Video: Delivery of Antibodies into the Murine Brain via Convection-enhanced Delivery

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 ...

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals are not viable in those cases where the modified gene is expressed or deleted in the whole organism. Moreover, a variety of compensatory mechanisms often make it difficult to interpret the results. The compensatory effects can be alleviated by either timing the gene expression or limiting the amount of transfected cells.
The method of postnatal non-ventricular microinjection and in vivo electroporation allows targeted delivery of genes, siRNA or dye molecules directly to a small region of interest in the newborn rodent brain. In contrast to conventional ventricular injection technique, this method allows transfection of non-migratory cell types. Animals transfected by means of the method described here can be used, for example, for two-photon in vivo imaging or in electrophysiological experiments on acute brain slices.

This protocol describes a non-viral method of delivery of genetic constructs to a certain area of living rodent brain. The method consists of plasmid preparation, micropipette fabrication, neonatal rat pup surgery, microinjection of the construct, and in vivo electroporation.

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals ...

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental pathways. Several powerful and well established transgenic technologies are available to manipulate gene expression levels in mouse, allowing for the generation of both loss- and gain-of-function models. However, the generation of mouse transgenics is both costly and time consuming. Alternative methods of gene manipulation have therefore been widely sought. In utero electroporation is a method of gene delivery into live mouse embryos1,2 that we have successfully adapted3,4. It is largely based on the success of in ovo electroporation technologies that are commonly used in chick5. Briefly, DNA is injected into the open ventricles of the developing brain and the application of an electrical current causes the formation of transient pores in cell membranes, allowing for the uptake of DNA into the cell. In our hands, embryos can be efficiently electroporated as early as embryonic day (E) 11.5, while the targeting of younger embryos would require an ultrasound-guided microinjection protocol, as previously described6. Conversely, E15.5 is the latest stage we can easily electroporate, due to the onset of parietal and frontal bone differentiation, which hampers microinjection into the brain. In contrast, the retina is accessible through the end of embryogenesis. Embryos can be collected at any time point throughout the embryonic or early postnatal period. Injection of a reporter construct facilitates the identification of transfected cells.
To date, in utero electroporation has been most widely used for the analysis of neocortical development1,2,3,4. More recent studies have targeted the embryonic retina7,8,9 and thalamus10,11,12. Here, we present a modified in utero electroporation protocol that can be easily adapted to target different domains of the embryonic CNS. We provide evidence that by using this technique, we can target the embryonic telencephalon, diencephalon and retina. Representative results are presented, first showing the use of this technique to introduce DNA expression constructs into the lateral ventricles, allowing us to monitor progenitor maturation, differentiation and migration in the embryonic telencephalon. We also show that this technique can be used to target DNA to the diencephalic territories surrounding the 3rd ventricle, allowing the migratory routes of differentiating neurons into diencephalic nuclei to be monitored. Finally, we show that the use of micromanipulators allows us to accurately introduce DNA constructs into small target areas, including the subretinal space, allowing us to analyse the effects of manipulating gene expression on retinal development.

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

In utero electroporation allows for rapid gene delivery in a spatially- and temporally-controlled manner in the developing central nervous system (CNS). Here we describe a highly adaptable in utero electroporation protocol that can be used to deliver expression constructs into multiple embryonic CNS domains, including the telencephalon, diencephalon and retina.

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

Designing, Packaging, and Delivery of High Titer CRISPR Retro and Lentiviruses via Stereotaxic Injection

Replication defective lentiviruses or retroviruses are capable of stably integrating transgenes into the genome of an infected host cell. This technique has been widely used to encode fluorescent proteins, opto- or chemo-genetic controllers of cell activity, or heterologous expression of human genes in model organisms. These viruses have also successfully been used to deliver recombinases to relevant target sites in transgenic animals, or even deliver small hairpin or micro RNAs in order to manipulate gene expression. While these techniques have been fruitful, they rely on transgenic animals (recombinases) or frequently lack high efficacy and specificity (shRNA/miRNA). In contrast, the CRISPR/Cas system uses an exogenous Cas nuclease which targets specific sites in an organism's genome via an exogenous guide RNA in order to induce double stranded breaks in DNA. These breaks are then repaired by non-homologous end joining (NHEJ), producing insertion and deletion (indel) mutations that can result in deleterious missense or nonsense mutations. This manuscript provides detailed methods for the design, production, injection, and validation of single lenti/retro virus particles that can stably transduce neurons to express a fluorescent reporter, Cas9, and sgRNAs to knockout genes in a model organism.

The CRISPR/Cas9 system offers the potential to make targeted genome editing accessible and affordable to the scientific community. This protocol is intended to demonstrate how to create viruses that will knockout a gene of interest using the CRISPR/Cas9 system, and then inject them stereotaxically into the adult mouse brain.

Replication defective lentiviruses or retroviruses are capable of stably integrating transgenes into the genome of an infected host cell. This technique ...

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous substances into the brain. These exogenous substances may induce injurious impact or constitute therapeutic strategy. The most common delivery methods of exogenous substances into the brain are based on systemic deliveries, such as subcutaneous or intravenous routes. Although commonly used, these approaches have several limitations, including low delivery efficacy into the brain. In contrast, surgical methods that locally deliver substances into the CNS are more specific and prevent the uptake of the exogenous substances by other organs. Several surgical methods for CNS delivery are available; however, they tend to be very traumatic. Here, we describe a mouse infusion microsurgery technique, which effectively delivers substances into the brain via the internal carotid artery, with minimal trauma and no interference with normal CNS functionality.

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

The present protocol describes a mouse microsurgery infusion technique, which effectively delivers substances directly into the brain via the internal carotid artery.

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous ...

Convection Enhanced Delivery of Optogenetic Adeno-associated Viral Vector to the Cortex of Rhesus Macaque Under Guidance of Online MRI Images

In non-human primate (NHP) optogenetics, infecting large cortical areas with viral vectors is often a difficult and time-consuming task. Here, we demonstrate the use of magnetic resonance (MR)-guided convection enhanced delivery (CED) of optogenetic viral vectors into primary somatosensory (S1) and motor (M1) cortices of macaques to obtain efficient, widespread cortical expression of light-sensitive ion channels. Adeno-associated viral (AAV) vectors encoding the red-shifted opsin C1V1 fused to yellow fluorescent protein (EYFP) were injected into the cortex of rhesus macaques under MR-guided CED. Three months post-infusion, epifluorescent imaging confirmed large regions of optogenetic expression (&#62;130 mm2) in M1 and S1 in two macaques. Furthermore, we were able to record reliable light-evoked electrophysiology responses from the expressing areas using micro-electrocorticographic arrays. Later histological analysis and immunostaining against the reporter revealed widespread and dense optogenetic expression in M1 and S1 corresponding to the distribution indicated by epifluorescent imaging. This technique enables us to obtain expression across large areas of the cortex within a shorter period of time with minimal damage compared to the traditional techniques and can be an optimal approach for optogenetic viral delivery in large animals such as NHPs. This approach demonstrates great potential for network-level manipulation of neural circuits with cell-type specificity in animal models evolutionarily close to humans.&#160;

Here, we demonstrate magnetic resonance (MR)-guided convection enhanced delivery (CED) of viral vectors into the cortex as an efficient and simplified approach for achieving optogenetic expression across large cortical areas in the macaque brain.

In non-human primate (NHP) optogenetics, infecting large cortical areas with viral vectors is often a difficult and time-consuming task. Here, we ...

Electrophoretic Delivery of &#x3B3;-aminobutyric Acid (GABA) into Epileptic Focus Prevents Seizures in Mice

Epilepsy is a group of neurological disorders which affects millions of people worldwide. Although treatment with medication is helpful in 70% of the cases, serious side effects affect the quality of life of patients. Moreover, a high percentage of epileptic patients are drug resistant; in their case, neurosurgery or neurostimulation are necessary. Therefore, the major goal of epilepsy research is to discover new therapies which are either capable of curing epilepsy without side effects or preventing recurrent seizures in drug-resistant patients. Neuroengineering provides new approaches by using novel strategies and technologies to find better solutions to cure epileptic patients at risk.
As a demonstration of a novel experimental protocol in an acute mouse model of epilepsy, a direct in situ electrophoretic drug delivery system is used. Namely, a neural probe incorporating a microfluidic ion pump (&#181;FIP) for on-demand drug delivery and simultaneous recording of local neural activity is implanted and demonstrated to be capable of controlling 4-aminopyridine-induced (4AP-induced) seizure-like event (SLE) activity. The &#947;-aminobutyric acid (GABA) concentration is kept in the physiological range by the precise control of GABA delivery to reach an antiepileptic effect in the seizure focus but not to cause overinhibition-induced rebound bursts. The method allows both the detection of pathological activity and intervention to stop seizures by delivering inhibitory neurotransmitters directly to the epileptic focus with precise spatiotemporal control.
As a result of the developments to the experimental method, SLEs can be induced in a highly localized manner that allows seizure control by the precisely tuned GABA delivery at the seizure onset.

Electrophoretic Delivery of &#947;-aminobutyric Acid GABA into Epileptic Focus Prevents Seizures in Mice

The challenge of epilepsy research is to develop novel treatments for patients where classical therapy is inadequate. Using a new protocol&#8212;with the help of an implantable drug delivery system&#8212;we are able to control seizures in anesthetized mice by the electrophoretic delivery of GABA into the epileptic focus.

Epilepsy is a group of neurological disorders which affects millions of people worldwide. Although treatment with medication is helpful in 70% of the ...

Intrathecal Delivery of Antisense Oligonucleotides in the Rat Central Nervous System

The blood brain barrier (BBB) is an important defense against the entrance of potentially toxic or pathogenic agents from the blood into the central nervous system (CNS). However, its existence also dramatically lowers the accessibility of systemically administered therapeutic agents to the CNS. One method to overcome this, is to inject those agents directly into the cerebrospinal fluid (CSF), thus bypassing the BBB. This can be done via implantation of a catheter for either continuous infusion using an osmotic pump, or for single bolus delivery. In this article, we describe a surgical protocol for delivery of CNS-targeting antisense oligonucleotides (ASOs) via a catheter implanted directly into the cauda equina space of the adult rat spine. As representative results, we show the efficacy of a single bolus ASO intrathecal (IT) injection via this catheterization system in knocking down the target RNA in different regions of the rat CNS. The procedure is safe, effective and does not require expensive equipment or surgical tools. The technique described here can be adapted to deliver drugs in other modalities as well.

Here, we describe a method for delivering drugs to the rat central nervous system by implanting a catheter into the lumbar intrathecal space of the spine. We focus on the delivery of antisense oligonucleotides, though this method is suitable for delivery of other therapeutic modalities as well.

The blood brain barrier (BBB) is an important defense against the entrance of potentially toxic or pathogenic agents from the blood into the central ...

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 ...