Name: Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery (Video) | JoVE Journal
Uploaded: 2017-01-31T14:00:00.000+00:00
Duration: 6 min 41 s
Description: Learn a mouse microsurgery infusion technique for targeted substance delivery into the CNS via the internal carotid artery. This method minimizes trauma while achieving high brain concentrations for neuroscience research.

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5 minutes

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%&#160;and oxygen flow at 2 L/min on the commercial machine (See Materials Table). Transfer the animal to the surgery surface, under a stereomicroscope, and sustain anesthesia using a nose cone (use isoflurane setting 1.5 - 2.5 and oxygen flow at 2 L/min).
	<ol>
		<li>Ensure that the mouse respiratory rate is around 1 - 2 respiration/sec without gasping. In addition, ensure that the animal does not exhibit whiskers stimulation reaction and pedal reflex (toe pinch). Monitor respiration rate and effort during the surgery, at least every 5 min.&#160;Follow specific Institutional Animal Care and Use Committee and veterinary guidelines for rodent anesthesia monitoring.</li>
	</ol>
	</li>
	<li>Apply a drop of ophthalmic lubricant on each eye using a sterile swab in order to prevent dryness.&#160;Administration of anti-inflammatory and analgesic to alleviate discomfort is recommended.</li>
	<li>With the animal lying on its back, maintain anesthesia using a nose cone.</li>
	<li>Clean the animal&#39;s neck area by wiping the area three times&#160;with ethanol 70% and chlorhexidine. Shave the animal&#39;s surgery area using a razor (detailed below).</li>
</ol>
2. Dissection of the Common Carotid Artery (CCA)
<ol>
	<li>Perform the entire microsurgery procedure under a stereomicroscope. Using surgical scissors and forceps perform a shallow midline incision in the neck, from above the breastbone to below the jaw (about 3 to 4 cm).</li>
	<li>Using forceps carefully separate fatty and connective tissue to expose the trachea.</li>
	<li>Place a pillow (round object, about 0.5 cm in diameter) on the back of the neck of the mouse to extend the neck, further exposing the area.</li>
	<li>Separate the tissue using either a tissue retractor or hooks.</li>
	<li>On the animal&#39;s left side of the trachea, carefully tweeze apart the connective tissue to expose the left CCA.</li>
	<li>Using forceps carefully remove all connective tissue to expose the CCA bifurcation, and the beginning of both external and internal carotid arteries.</li>
</ol>
3. Preparation of the CCA for Substance Infusion
<ol>
	<li>Insert two segments of nylon suture (about 1 cm each) under external carotid artery (ECA), using forceps.</li>
	<li>Place a permanent knot at the highest point possible of the ECA.</li>
	<li>At the lowest point possible of the ECA, immediately above the CCA bifurcation, place a removable knot. This knot should be loose compared to the upper knot.</li>
	<li>Close the CCA using a vessel clip, at the lowest possible point.</li>
	<li>Close the internal carotid artery (ICA) using a vessel clip.</li>
	<li>Using microdissection spring scissors perform a small cut in the ECA (about 2 mm), between the two knots.</li>
</ol>
4. Substance Infusion via ICA
<ol>
	<li>Assemble an infusion system. Attach 6 inches of capillary tubing (ideal dimensions: 2.5 mm x 1.2 mm) to a tuberculin syringe containing 250 &#181;l of the substance to be infused (drugs, pathogens, extracellular vesicles, among others) and insert the capillary tip gently into the incision performed in step 3.6.</li>
	<li>Continue to slide down the capillary until it reaches a midpoint between the bifurcation and the clip closing the CCA.</li>
	<li>Tie down the lower ECA knot. Verify that the knot is loose enough to allow capillary fluidity and tight enough to prevent leaking.</li>
	<li>Remove clip from the ICA.</li>
	<li>Gently apply pressure to the syringe piston allowing substance infusion at approximately 10 &#181;l per second.</li>
</ol>
5. Post Infusion Procedures
<ol>
	<li>Place clip back on the ICA.</li>
	<li>Gently remove the capillary tubing.</li>
	<li>Tie down the lower ECA knot completely.</li>
	<li>Remove clips from the ICA and the CCA.</li>
</ol>
6. Incision Closing and Post-operative Care
<ol>
	<li>Remove retractor or tissue hooks and pillow.</li>
	<li>Clean suture area using sterile saline.</li>
	<li>Close the incision using nylon suture/needle and forceps.</li>
	<li>Administer anti-inflammatory and analgesic to alleviate post-operative discomfort.</li>
	<li>Place animal in cage placed on heating pad for at least 1 hr and monitor recovery.</li>
</ol>

<ol><li>Chen, L., Swartz, K. R., Toborek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vessel+microport+technique+for+applications+in+cerebrovascular+research%5BTitle%5D)+AND+%22J+Neurosci+Res%22%5BJournal%5D)">Vessel microport technique for applications in cerebrovascular research.</a> J Neurosci Res. 87 (7), 1718-1727 (2009).</li>
<li>Frisella, W. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intracranial+injection+of+recombinant+adeno-associated+virus+improves+cognitive+function+in+a+murine+model+of+mucopolysaccharidosis+type+VII%5BTitle%5D)+AND+%22Mol+Ther%22%5BJournal%5D)">Intracranial injection of recombinant adeno-associated virus improves cognitive function in a murine model of mucopolysaccharidosis type VII.</a> Mol Ther. 3 (3), 351-358 (2001).</li>
<li>Wei, L., Erinjeri, J. P., Rovainen, C. M., Woolsey, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Collateral+growth+and+angiogenesis+around+cortical+stroke%5BTitle%5D)+AND+%22Stroke%22%5BJournal%5D)">Collateral growth and angiogenesis around cortical stroke.</a> Stroke. 32 (9), 2179-2184 (2001).</li>
<li>Wu, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeted+delivery+of+methotrexate+to+epidermal+growth+factor+receptor-positive+brain+tumors+by+means+of+cetuximab+%28IMC-C225%29+dendrimer+bioconjugates%5BTitle%5D)+AND+%22Mol+Cancer+Ther%22%5BJournal%5D)">Targeted delivery of methotrexate to epidermal growth factor receptor-positive brain tumors by means of cetuximab (IMC-C225) dendrimer bioconjugates.</a> Mol Cancer Ther. 5 (1), 52-59 (2006).</li>
<li>Pignataro, G., Studer, F. E., Wilz, A., Simon, R. P., Boison, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neuroprotection+in+ischemic+mouse+brain+induced+by+stem+cell-derived+brain+implants%5BTitle%5D)+AND+%22J+Cereb+Blood+Flow+Metab%22%5BJournal%5D)">Neuroprotection in ischemic mouse brain induced by stem cell-derived brain implants.</a> J Cereb Blood Flow Metab. 27 (5), 919-927 (2007).</li>
<li>Sugiyama, Y., Kusuhara, H., Suzuki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Kinetic+and+biochemical+analysis+of+carrier-mediated+efflux+of+drugs+through+the+blood-brain+and+blood-cerebrospinal+fluid+barriers%3A+importance+in+the+drug+delivery+to+the+brain%5BTitle%5D)+AND+%22J+Control+Release%22%5BJournal%5D)">Kinetic and biochemical analysis of carrier-mediated efflux of drugs through the blood-brain and blood-cerebrospinal fluid barriers: importance in the drug delivery to the brain.</a> J Control Release. 62 (1-2), 179-186 (1999).</li>
<li>Pardridge, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Drug+and+gene+delivery+to+the+brain%3A+the+vascular+route%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Drug and gene delivery to the brain: the vascular route.</a> Neuron. 36 (4), 555-558 (2002).</li>
<li>Vantyghem, S. A., Postenka, C. O., Chambers, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Estrous+cycle+influences+organ-specific+metastasis+of+B16F10+melanoma+cells%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">Estrous cycle influences organ-specific metastasis of B16F10 melanoma cells.</a> Cancer Res. 63 (16), 4763-4765 (2003).</li>
<li>Huang, R. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+gene+delivery+targeted+to+the+brain+using+a+transferrin-conjugated+polyethyleneglycol-modified+polyamidoamine+dendrimer%5BTitle%5D)+AND+%22FASEB+J%22%5BJournal%5D)">Efficient gene delivery targeted to the brain using a transferrin-conjugated polyethyleneglycol-modified polyamidoamine dendrimer.</a> FASEB J. 21 (4), 1117-1125 (2007).</li>
<li>Liu, R., Martuza, R. L., Rabkin, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intracarotid+delivery+of+oncolytic+HSV+vector+G47Delta+to+metastatic+breast+cancer+in+the+brain%5BTitle%5D)+AND+%22Gene+Ther%22%5BJournal%5D)">Intracarotid delivery of oncolytic HSV vector G47Delta to metastatic breast cancer in the brain.</a> Gene Ther. 12 (8), 647-654 (2005).</li>
<li>Kumar, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transvascular+delivery+of+small+interfering+RNA+to+the+central+nervous+system%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Transvascular delivery of small interfering RNA to the central nervous system.</a> Nature. 448 (7149), 39-43 (2007).</li>
<li>Wrobel, J. K., Wolff, G., Xiao, R., Power, R. F., Toborek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dietary+Selenium+Supplementation+Modulates+Growth+of+Brain+Metastatic+Tumors+and+Changes+the+Expression+of+Adhesion+Molecules+in+Brain+Microvessels%5BTitle%5D)+AND+%22Biol+Trace+Elem+Res%22%5BJournal%5D)">Dietary Selenium Supplementation Modulates Growth of Brain Metastatic Tumors and Changes the Expression of Adhesion Molecules in Brain Microvessels.</a> Biol Trace Elem Res. , (2015).</li>
</ol>

The authors have nothing to disclose.

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.

<table><tbody><tr><td>Anesthesia instrument</td><td>Vetequip</td><td>901806</td><td></td></tr><tr><td>Surgical scissors</td><td>Fine Science Tool</td><td>14558-09</td><td></td></tr><tr><td>Surgical forceps straight tip</td><td>Fine Science Tool</td><td>00108-11</td><td></td></tr><tr><td>Surgical forceps angled tip</td><td>Fine Science Tool</td><td>00109-11</td><td></td></tr><tr><td>Spring scissors</td><td>Fine Science Tool</td><td>15000-08</td><td></td></tr><tr><td>Nylon suture</td><td>Braintree Scientific</td><td>SUT-S 104</td><td></td></tr><tr><td>Capillary tubing (Micro-Renathane 0.010&amp;rdquo; x 0.005&amp;rdquo; per ft.)&amp;nbsp;</td><td>Braintree Scientific</td><td>MRE01050</td><td></td></tr><tr><td>Closing suture</td><td>VWR</td><td>95057-036</td><td></td></tr><tr><td>Isoflurane</td><td>Piramal</td><td></td><td></td></tr><tr><td>2,3,5-Triphenyltetrazolium chloride</td><td>FisherScientific</td><td>50-121-8005</td><td></td></tr></tbody></table>

mouse microsurgery infusion technique for targeted substance delivery into the cns 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 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...

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 ...

mouse-microsurgery-infusion-technique-for-targeted-substance-delivery

Research

JoVE Journal

Neuroscience

As neuroscience inquiry becomes more sophisticated, investigation of brain structures and circuitry requires improved levels of accuracy and higher resolution. We have developed a method for the preparation and implantation of a chronic infusion system within the brain utilizing a borosilicate microcannula with a tip diameter of 50 microns.

Construction and Implantation of a Microinfusion System for Sustained Delivery of Neuroactive Agents.

Source: Zamboni, M., et al., Induction of Leptomeningeal Cells Modification Via Intracisternal Injection. J. Vis. Exp. (2020)This video demonstrates the procedure of delivering an inducer drug into the cisterna magna of an anesthetized mouse. The injection selectively induces fluorescent marker expression in brain surface cells accessible via cerebrospinal fluid, confirmed by fluorescence imaging.

Targeted Drug Delivery in a Mouse Model via Cisterna Magna Injection

Source: Lim, M., et al. Modeling Brain Metastasis by Internal Carotid Artery Injection of Cancer Cells. J. Vis. Exp. (2022)In this video, an anesthetized mouse undergoes a surgical procedure in which cancer cells are injected into the common carotid artery (CCA) to facilitate targeted delivery to the brain. The procedure involves precise suturing and manipulation of the carotid arteries to direct the injected cells toward the internal carotid artery (ICA), promoting tumor formation in the brain...

Modeling Brain Tumor Metastasis via Internal Carotid Artery Injection of Cancer Cells

Source: Leda, A. R., et al. Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery. J. Vis. Exp. (2017)This video demonstrates a microsurgical procedure designed to facilitate the targeted infusion of a test substance into the brain via the internal carotid artery (ICA). The procedure involves precise suturing and vascular manipulation to temporarily restrict blood flow, ensuring controlled delivery. A capillary tube is inserted into...

Targeted Substance Delivery to the Mouse Brain via Infusion into the Internal Carotid Artery

Blood-brain barrier disruption aids the delivery of certain drugs to the brain. Mannitol delivered intra-arterially shrinks cells surrounding blood vessels in order to physically disrupt the barrier.

Rat Model of Blood-brain Barrier Disruption to Allow Targeted Neurovascular Therapeutics

Brain metastasis has become an urgent unmet medical need as its incidence has increased while therapeutic options have remained palliative. Creating experimental animal models of brain metastasis via intracarotid arterial injection of cancer cells facilitates mechanistic studies of the disease biology and evaluation of novel intervention regimens.

Intracarotid Cancer Cell Injection to Produce Mouse Models of Brain Metastasis

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.

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Most brain metastasis mouse models are complicated by systemic metastases confounding analysis of mortality and therapeutic intervention outcomes. Presented here is a protocol for internal carotid injection of cancer cells that produces consistent intracranial tumors with minimal systemic tumors.

Modeling Brain Metastasis by Internal Carotid Artery Injection of Cancer Cells

Repair of the intracarotid artery in a mouse model after injection returns blood flow to the artery without negatively impacting the distribution of the injected material. Injection site repair facilitates subsequent injections through the same artery and prevents cerebral ischemia in mouse strains that lack a complete Circle of Willis.

Facilitating Repeat Intracarotid Injections in Mouse Models by a Novel Injection Site Repair Technique

Here, we present a protocol to assess immune cell function following ischemic stroke using flow cytometric analysis in a mini-stroke model. This method enables detailed examination of neuroimmune interactions and can be adapted for studying other neurodegenerative diseases, enhancing our understanding of immune responses in stroke pathology.

Isolation and Flow Cytometric Assessment of Neuroimmune Interactions in a Mini-Stroke Murine Model

Learn a mouse microsurgery infusion technique for targeted substance delivery into the CNS via the internal carotid artery. This method minimizes trauma while achieving high brain concentrations for neuroscience research.

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

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