convection-enhanced delivery of antibodies into a mouse brain

Convection-Enhanced Delivery of Antibodies into a Mouse Brain

For antibody injection into the murine striatum, confirm a lack of response to skin pinch in an anesthetized mouse, and shave the head with a hair trimmer. Disinfect the skin with cotton swabs soaked in iodine solution.
Use a scalpel to make a 10-millimeter skin incision along the cranial midline finishing at the eye level. Fix the mouse in the stereotactic frame using the nose clamp and ear bars, taking care that the skull surface is horizontal and tightly secured.
Place the syringe in the stereotactic robot, and synchronize the drill bit with the tip of the catheter on a reference point. Use forceps to retract the skin, and localize bregma on the skull surface. Reference bregma in the software using the tip of the drill bit, and move the drill to a 1-millimeter frontal and 2-millimeter lateral from bregma position.
Drill a burr hole, taking care not to damage the dura mater, and move the syringe over the burr hole. Dispense 0.5 to 1 microliter from the syringe to ensure that no air bubbles are left in the catheter. Start the convection-enhanced delivery as demonstrated, observing the skull surface for any traces of fluid backflow from the injection spot.
At the end of the delivery and catheter removal, start the injection pump at 0.2 microliters per minute to check for evidence of catheter clogging during the injection. If no clogging occurred, a droplet of injection mix should immediately be observed coming from the catheter tip.

convection-enhanced-delivery-of-antibodies-into-a-mouse-brain

Universidad San Sebastian

Research

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Antibody-Based Imaging and Detection Methods

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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 an 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 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 the fused silica capillary to be placed 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 1.</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, forming a 1 mm step from the tip. Remove any excess glue forming at the end of the metal needle to avoid blunting the step.</li>
		<li>Wait 15 min for the glue to harden, and remove the syringe with the catheter from the stereotactic robot. Confirm that all excess glue has been removed at the step 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 phosphate-buffered saline (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 to assess the sealing of the catheter step 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>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 a 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 fluid dispersion in the brain and seal the injection tract during catheter removal. 
			NOTE: All parameters can be programmed into a single script depending on the specific stereotactic robot.</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 a 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 to assess the catheter&#39;s clogging 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 indicate fluid leakage through the catheter mounting or syringe plunger.</li>
		<li>If all the test injections are successful, the catheter is well sealed and 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 distilled water (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 has atipamezole (2.5 mg/kg) in sterile dH2O (second antidote solution).</li>
		<li>Prepare an 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 robot parts can be cleaned without risk of damage. Refer to the robot manual 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 prepare the solution for intracranial injection, e.g., artificial cerebrospinal fluid. The syringe plunger should move smoothly and freely during the 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, that the injection pump is appropriately connected, and that it can perform the CED procedure without any disturbances. This includes testing robot movement, ramping injection, checking the 2-minute waiting step, and checking 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. Sterilizing the drill bits before use is recommended.</li>
		<li>Prepare antibody solution using PBS or other buffer solutions such as artificial cerebrospinal fluid (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 before 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 the 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. When 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 after the injection of the three-component anesthesia solution.</li>
		<li>Check pain reactions using the pinch-reflex test to ensure the animal&#39;s complete anesthesia.</li>
		<li>Shave the head using a hair trimmer.</li>
		<li>Disinfect the skin with cotton swabs soaked in iodine solution. Scrub the skin three times in a circular motion.</li>
		<li>Make a 10 mm skin incision along the cranial midline, finishing on the eye level using a scalpel.</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. Besides correct anatomical navigation, it is also crucial to avoid tilting 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. The relation between the position of the drill and the syringe must be 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 the bregma on the skull&#39;s surface.</li>
		<li>Reference Bregma in the software using the drill bit tip.</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 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 to check for catheter clogging during the CED. If no clogging occurred, you should immediately see a droplet of injection mix from the catheter tip.</li>
		<li>Before reusing or storing the catheter, visually examine the catheter step under a microscope for any signs of damage or wear and clean it as in step 1.3.10.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>Sucrose</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>

Source: Beffinger, M., et al. <a href="https://app.jove.com/v/59675">Delivery of Antibodies into the Murine Brain via Convection-enhanced Delivery.</a> J. Vis. Exp. (2019).
This video demonstrates a convection-enhanced delivery (CED) technique for antibody delivery into the mouse brain. The skull of an anesthetized mouse is exposed, and a burr hole is created at the target area for antibody delivery. A step catheter containing antibodies is inserted at the target site, and via an infusion pump, a pressure gradient is created to deliver antibodies at a controlled rate.

<img alt="Figure 1" src="/files/ftp_upload/22092/22092_Figure1.jpg" /> 
Figure 1: A schematic drawing depicting the application area of the adhesive. The upper 10 mm of the fused silica tubing is inserted into the metal needle. Apply the glue on the 10 mm tubing starting from the tip of the metal needle.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Secure an anesthetized mouse that has a cranial midline incision to a stereotactic frame. 
Detect the bregma, a reference point on the skull, and determine the injection location.
Create a burr hole at the location, avoiding damage to the dura mater covering the brain.
Take a step catheter loaded with antibodies, mounted on a stereotactic robot, and connected to an infusion pump.
The catheter comprises a fused silica capillary protruding from a metal needle, creating a stepped outer diameter at the fusion point.
Insert the catheter into the brain parenchyma. Start the infusion pump to create a pressure gradient at the catheter opening and deliver the antibodies at a controlled rate, a technique known as convection-enhanced delivery.
The stepped diameter compresses the tissue, creating a seal.
The seal prevents fluid reflux and enables the distribution of large antibody volumes while lowering the risk of tissue damage.

52 Views. Somministrazione di anticorpi potenziata dalla convezione nel cervello di un topo

Video: Somministrazione di anticorpi potenziata dalla convezione nel cervello di un topo - Experiment

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.

Convezione Consegna migliorata del vettore virale optogenetico adeno-associato alla Corteccia di Rhesus Macaque Sotto la guida di immagini MRI online

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

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Delivery of Antibodies into the Brain Using Focused Scanning Ultrasound

Only a small fraction of therapeutic antibodies targeting brain diseases are taken up by the brain. Focused ultrasound offers a possibility to increase uptake of antibodies and engagement through transient opening of the blood-brain barrier (BBB). In our laboratory, we are developing therapeutic approaches for neurodegenerative diseases in which an antibody in various formats is delivered across the BBB using microbubbles, concomitant with focused ultrasound application through the skull targeting multiple spots, an approach we refer to as scanning ultrasound (SUS). The mechanical effects of microbubbles and ultrasound on blood vessels increases paracellular transport across the BBB by transiently separating tight junctions and enhances vesicle- mediated transcytosis, allowing antibodies and therapeutic agents to effectively cross. Moreover, ultrasound also facilitates the uptake of antibodies from the interstitial brain into brain cells such as neurons where the antibody distributes throughout the cell body and even into neuritic processes. In our studies, fluorescently labeled antibodies are prepared, mixed with in-house prepared lipid-based microbubbles and injected into mice immediately before SUS is applied to the brain. The increased antibody concentration in the brain is then quantified. To account for alterations in normal brain homeostasis, microglial phagocytosis can be used as a cellular marker. The generated data suggest that ultrasound delivery of antibodies is an attractive approach to treat neurodegenerative diseases.

Presented here is a protocol to transiently open the blood-brain barrier (BBB) either focally or throughout a mouse brain to deliver fluorescently-labeled antibodies and activate microglia. Also presented is a method to detect the delivery of antibodies and microglia activation by histology.

Consegna di anticorpi nel cervello utilizzando ultrasuoni a scansione focalizzata

Only a small fraction of therapeutic antibodies targeting brain diseases are taken up by the brain. Focused ultrasound offers a possibility to increase ...

Magnetic Resonance-Guided Stereotaxy for Infusions to the Pig Brain

The overall goal of this procedure is to perform stereotaxy in the pig brain with real-time magnetic resonance (MR) visualization guidance to provide precise infusions. The subject was positioned prone in the MR bore for optimal access to the top of the skull with the torso raised, the neck flexed, and the head inclined downward. Two anchor pins anchored on the bilateral zygoma held the head steady using the head holder. A magnetic resonance imaging (MRI) flex-coil was placed rostrally across the head holder so that the skull was accessible for the intervention procedure. A planning grid placed on the scalp was used to determine the appropriate entry point of the cannula. The stereotactic frame was secured and aligned iteratively through software projection until the projected radial error was less than 0.5 mm. A hand drill was used to create a burr hole for insertion of the cannula. A gadolinium-enhanced co-infusion was used to visualize the infusion of a cell suspension. Repeated T1-weighted MRI scans were registered in real time during the agent delivery process to visualize the volume of gadolinium distribution. MRI-guided stereotaxy allows for precise and controlled infusion into the pig brain, with concurrent monitoring of cannula insertion accuracy and determination of the agent volume of distribution.

The protocol presented here demonstrates stereotaxy to the pig brain using convection-enhanced infusions, with real-time magnetic resonance imaging (MRI) visualization guidance and real-time infusion distribution visualization.

Stereotassia guidata da risonanza magnetica per infusioni al cervello di maiale

The overall goal of this procedure is to perform stereotaxy in the pig brain with real-time magnetic resonance (MR) visualization guidance to provide ...

Convection-Enhanced Delivery of Antibodies into a Mouse Brain

Trascrizione

Convection-Enhanced Delivery of Antibodies into a Mouse Brain