Name: mouse microsurgery infusion technique for targeted substance delivery into the cns via the internal carotid artery
Uploaded: 2017-01-31T14:00:00
Description: Watch this Scientific Journal Video about Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery at JoVE.com

We would like to thank Dr. Lei Chen (Icahn School of Medicine at Mount Sinai, NY) who first established the use of this model in our laboratory, and to Dr. Gretchen Wolff (German Cancer Research Center, Heidelberg, Germany) for disseminating the technique in our laboratory. Supported in part by HL126559, DA039576, MH098891, MH63022, MH072567, DA027569, and NSC 2015/17/B/NZ7/02985.

The procedures involved in the following protocol have been approved by the University of Miami Institutional Animal Care and Use Committee (IACUC). In addition, all procedures are being conducted in facilities approved by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC).
1. Preparation of Mice for Surgery
<ol>
	<li>Anesthetize mouse with isoflurane mixed with oxygen, using a laboratory anesthesia system. Use isoflurane at setting between 4-5%...

<ol>
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The infusion microsurgery described here has been proved to be very successful in delivering exogenous substances of various biological features into the CNS, preventing unwanted dissemination throughout the body 1,12. Disruption of the blood-brain barrier is a pathological characteristic of several CNS-related diseases; therefore assessing the relationship of exogenous substances with the blood-brain barrier is of major importance and interest.
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In vivo models of central nervous system (CNS) diseases require an effective delivery of exogenous substances, such as drugs, pathogens, or exosomes, into the brain. Therefore, an ideal delivery method should cause minimal trauma to the animal, preserve the integrity of the neuronal network, and achieve high substance concentrations in the brain 1.
Several surgical methods of local substance delivery have been described, including intra-sheath, intracerebral, and intraventricular injections or implants 2,3,4,5. These approaches, however, are considered traumatic to the CNS, and allow administration of only low volumes of the substance of interest. Moreover, it has been suggested that exogenous substances can be rapidly removed by the cerebrospinal fluid 6, and a low penetration range to the brain parenchyma has been observed 7 when the above-mentioned techniques are employed. Systemic delivery methods, such as oral, pulmonary, subcutaneous, and intravenous routes, are more commonly used in animal models, though they exhibit low effectiveness in delivering the substances to the CNS, due to uptake by other organs 8,9. Therefore, these routes of delivery require elevated doses of the administrated substances, increasing the risk of side effects and toxicity 10,11.
Here, we describe a mouse infusion microsurgery technique, which effectively delivers substances directly into the brain via the internal carotid artery. In addition to targeting the delivery to the CNS, this technique does not bypass normal physiological barriers and is therefore highly relevant to biological processes involved in the passages of therapeutics or pathogens into the brain.

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			<td height="21" style="height:21px;width:367px;">Anesthesia instrument</td>
			<td style="width:149px;">Vetequip</td>
			<td align="right" style="width:119px;">901806</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Surgical scissors</td>
			<td style="width:149px;">Fine Science Tool</td>
			<td style="width:119px;">14558-09</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Surgical forceps straight tip</td>
			<td style="width:149px;">Fine Science Tool</td>
			<td style="width:119px;">00108-11</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Surgical forceps angled tip</td>
			<td style="width:149px;">Fine Science Tool</td>
			<td style="width:119px;">00109-11</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Spring scissors</td>
			<td style="width:149px;">Fine Science Tool</td>
			<td style="width:119px;">15000-08</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Nylon suture</td>
			<td style="width:149px;">Braintree Scientific</td>
			<td style="width:119px;">SUT-S 104</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Capillary tubing (Micro-Renathane 0.010&#8221; x 0.005&#8221; per ft.)&#160;</td>
			<td style="width:149px;">Braintree Scientific</td>
			<td style="width:119px;">MRE01050</td>
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			<td height="21" style="height:21px;width:367px;">Closing suture</td>
			<td style="width:149px;">VWR</td>
			<td style="width:119px;">95057-036</td>
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			<td height="21" style="height:21px;width:367px;">Isoflurane</td>
			<td style="width:149px;">Piramal</td>
			<td style="width:119px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:367px;">2,3,5-Triphenyltetrazolium chloride</td>
			<td style="width:149px;">FisherScientific</td>
			<td style="width:119px;">50-121-8005</td>
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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.

The mouse infusion microsurgery technique described here is very versatile and has been used to deliver different substances directly into the brain, including the delivery of tumor cells in a representative model of brain metastasis formation 1,12.
This technique is also suitable to assess the pathological aspects of different pathogens in the CNS. In a mouse model of H...

Watch this Scientific Journal Video about Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery at JoVE.com

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.

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 ...

The overall goal of this surgical technique is to deliver exogenous substances into the mouse brain without penetrating the cranium. This method can help answer key questions in neuroscience regarding the action of drugs, pathogens, and exosomes in the brain. The main advantage of this technique is that it causes minimal trauma to the animal, preserves the integrity of the neuronal network, and achieves high concentrations of the substance in the brain.Begin by administering isoflurane mixed with oxygen to anesthetize the mouse in an induction chamber. After induction of anesthesia, transfer the animal to the surgical surface under a stereo microscope, and place the nose cone to maintain a surgical plane of anesthesia. Ensure that the respiratory rate is around one to two respirations per second without gasping.In addition, ensure that the animal does not react to whisker stimulation or a toe pinch. Use a sterile swab to apply a drop of ophthalmic lubricant on each eye to prevent dryness. After shaving the surgical site with a razor, prep the area with 70%ethanol and chlorhexidine.While looking through the stereo microscope, use surgical scissors and forceps to create a midline incision in the neck from above the breastbone to below the jaw. Use forceps to carefully separate fatty and connective tissue to expose the trachea. Then place a soft, round object of about zero point five centimeters in diameter under the back of the neck to extend the neck and further expose the area.Next, separate the tissue around the trachea using hooks or a tissue retractor. On the left side of the trachea, carefully tweeze apart the connective tissue to expose the left common carotid artery, or CCA. Using forceps, carefully remove all connective tissue to expose the CCA bifurcation in the beginning of both the external and internal carotid arteries.Once the ECA and ICA are exposed, use forceps to place a segment of one-centimeter nylon suture under the external carotid artery. Then place a second one-centimeter segment. Then tie a permanent knot at the highest point possible of the ECA.At the lowest point possible of the ECA, immediately above the CCA bifurcation, tie a looser, removable knot. Then use vessel clips to close the CCA at the lowest possible point, and to close the ICA. Properly closing the internal carotid artery before making a small cut in the external carotid artery will prevent bleeding.Now, using microdissection spring scissors, make a two-millimeter cut in the ECA between the two knots. It is important to keep the cuts small, as a large cut will cause leaking of the substance to be infused. Assemble the infusion system by attaching six inches of capillary tubing to a tuberculin syringe containing 250 microliters of the substance to be infused.Then gently insert the tip of the capillary tubing into the incision in the ECA. Keep advancing the tip until it reaches midway between the bifurcation and the clip on the CCA. Tie down the lower ECA knot.Verify that the knot is loose enough to allow capillary fluidity, and tight enough to prevent leaking. Then, remove the clip from the ICA. Gently apply pressure to the syringe piston, allowing substance infusion at approximately 10 microliters per second.Following infusion of the test substance, place the clip back on the ICA. Gently remove the capillary tubing. Then, tie down the lower ECA knot completely.Remove the clip from the ICA and the CCA. Then remove the retractor or tissue hooks and pillow. Clean the area to be sutured with sterile saline, and then close the incision using nylon sutures.After administering an approved anti-inflammatory agent and analgesic to alleviate post-operative discomfort, place the animal in a cage on a heating pad for at least one hour, and monitor recovery. This histogram demonstrates HIV detection by real-time PCRs seven days after viral infusion via the left internal carotid artery. As expected, levels of the virus are higher in the left hemisphere.This immunofluorescence image shows a mouse brain section depicting GFP tag CD63, an exosome marker, after extracellular vesicle infusion via the internal carotid artery. The CD63 expressing extracellular vesicles, shown in green, are associated with the mouse brain microvessel. Once mastered, this technique can be done in 20 minutes if it is performed properly.After its development, this technique paved the way for researchers in the field of neurodegenerative diseases to explore drug, pathogens, and exosome interactions in mice.

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Research

JoVE Journal

Neuroscience

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. However, because the uterine wall is opaque during early embryogenesis, the ability to target specific parts of the embryo for microinjection is greatly limited. Fortunately, high-frequency ultrasound imaging permits the generation of images that can be used in real time to guide a microinjection needle into the embryonic region of interest. Here we describe the use of such imaging to guide the injection of retroviral vectors into the ventricular system of the mouse forebrain at embryonic day (E) 9.5. This method uses a laparotomy to permit access to the uterine horns, and a specially designed plate that permits host embryos to be bathed in saline while they are imaged and injected. Successful surgeries often result in most or all of the injected embryos surviving to any subsequent time point of interest (embryonically or postnatally). The principles described here can be used with slight modifications to perform injections into the amnionic fluid of E8.5 embryos (thereby permitting infection along the anterior posterior extent of the neural tube, which has not yet closed), or into the ventricular system of the brain at E10.5/11.5. Furthermore, at mid-neurogenic ages (~E13.5), ultrasound imaging can be used direct injection into specific brain regions for viral infection or cell transplantation. The use of ultrasound imaging to guide in utero injections in mice is a very powerful technique that permits the molecular and cellular manipulation of mouse embryos in ways that would otherwise be exceptionally difficult if not impossible.

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. Here we describe the use of high-frequency ultrasound imaging to guide the injection of retroviral vectors into the mouse brain at embryonic day (E) 9.5.

In utero survival surgery in mice permits the molecular manipulation of gene expression during development.  However, because the uterine wall is opaque ...

Methods for Experimental Manipulations after Optic Nerve Transection in the Mammalian CNS

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals through the vitreous fluid ensures that they act upon the entire RGC population. Viral vectors, plasmids or short interfering RNAs (siRNAs) can also be delivered to the vitreous chamber in order to infect or transfect retinal cells 5-12. The high tropism of Adeno-Associated Virus (AAV) vectors is beneficial to target RGCs, with an infection rate approaching 90% of cells near the injection site 6, 7, 13-15. Moreover, RGCs can be selectively transfected by applying siRNAs, plasmids, or viral vectors to the cut end of the optic nerve 16-19 or injecting vectors into their target the superior colliculus 10. This allows researchers to study apoptotic mechanisms in the injured neuronal population without confounding effects on other bystander neurons or surrounding glia. RGC apoptosis has a characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a window for experimental manipulations directed against pathways involved in apoptosis. Manipulations that directly target RGCs from the transected optic nerve stump are performed at the time of axotomy, immediately after cutting the nerve. In contrast, when substances are delivered via an intraocular route, they can be injected prior to surgery or within the first 3 days after surgery, preceding the initiation of apoptosis in axotomized RGCs. In the present article, we demonstrate several methods for experimental manipulations after optic nerve transection.

Optic Nerve transection is a widely used model of adult CNS injury. This model is ideal for performing a number of experimental manipulations that target the retina globally or directly target the injured neuronal population of retinal ganglion cells.

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be ...

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 ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). It is increasingly being recognized that several neurodegenerative diseases such as Alzheimer&#8217;s, multiple sclerosis, and amyotrophic lateral sclerosis present elements of vascular compromise. In addition, the most prominent causes of blindness in pediatric and working age populations (retinopathy of prematurity and diabetic retinopathy, respectively) are characterized by vascular degeneration and failure of physiological vascular regrowth. The aim of this technical paper is to provide a detailed protocol to study CNS vascular regeneration in the retina. The method can be employed to elucidate molecular mechanisms that lead to failure of vascular growth after ischemic injury. In addition, potential therapeutic modalities to accelerate and restore healthy vascular plexuses can be explored. Findings obtained using the described approach may provide therapeutic avenues for ischemic retinopathies such as that of diabetes or prematurity and possibly benefit other vascular disorders of the CNS.

The rodent retina has long been recognized as an accessible window to the brain. In this technical paper we provide a protocol that employs the mouse model of oxygen-induced retinopathy to study the mechanisms that lead to failure of vascular regeneration within the central nervous system after ischemic injury. The described system can also be harnessed to explore strategies to promote regrowth of functional blood vessels within the retina and CNS.

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

Autonomous and Rechargeable Microneurostimulator Endoscopically Implantable into the Submucosa

Gastric dysmotility can be a sign of common diseases such as longstanding diabetes mellitus. It is known that the application of high-frequency low-energetic stimulation can help to effectively moderate and alleviate the symptoms of gastric dysmotility. The goal of the research was the development of a miniature, endoscopically implantable device to a submucosal pocket. The implantable device is a fully customized electronic package which was specifically designed for the purpose of experiments in the submucosa. The device is equipped with a lithium-ion battery which can be recharged wirelessly by receiving an incident magnetic field from the charging/transmitting coil. The uplink communication is achieved in a MedRadio band at 432 MHz. The device was endoscopically inserted into the submucosal pocket of a live domestic pig used as an in vivo model, specifically in the stomach antrum. The experiment confirmed that the designed device can be implanted into the submucosa and is capable of bidirectional communication. The device can perform bipolar stimulation of muscle tissue.

The application of high-frequency low-energetic stimulation can alleviate the symptoms of gastric dysmotility. In this research, a miniature, endoscopically implantable and wirelessly rechargeable device which is implanted into a submucosal pocket is presented. Successful both-way communication and stimulation control were achieved during an experiment on live pig.

Gastric dysmotility can be a sign of common diseases such as longstanding diabetes mellitus. It is known that the application of high-frequency ...

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is a complicated task, which is particularly difficult in regards to the development of &#947;-aminobutyric acid (GABA)ergic cortical interneurons (CIs). CIs are the main inhibitory neurons in the cerebral cortex, and they play key roles in neuronal networks, by regulating both the activity of individual pyramidal neurons, as well as the oscillatory behavior of neuronal ensembles. They are generated in transient embryonic structures (medial and caudal ganglionic eminences -&#160;MGE and CGE) that are very difficult to efficiently target using in utero electroporation approaches. Interneuron progenitors migrate long distances during normal embryonic development, before they integrate in the cortical circuit. This remarkable ability to disperse and integrate into a developing network can be hijacked by transplanting embryonic interneuron precursors into early post-natal host cortices. Here, we present a protocol that allows genetic modification of embryonic interneuron progenitors using focal ex vivo electroporation. These engineered interneuron precursors are then transplanted into early post-natal host cortices, where they will mature into easily identifiable CIs. This protocol allows the use of multiple genetically encoded tools, or the ability to regulate the expression of specific genes in interneuron progenitors, in order to investigate the impact of either genetic or environmental variables on the maturation and integration of CIs.

Here we present a protocol, designed to use chemogenetic tools to manipulate the activity of cortical interneuron progenitors transplanted into the cortex of early postnatal mice.

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is ...

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.

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 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.

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