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%...

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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.

<table border="0" cellpadding="0" cellspacing="0" width="635">
	<tbody>
		<tr height="21">
			<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Closing suture</td>
			<td style="width:149px;">VWR</td>
			<td style="width:119px;">95057-036</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Isoflurane</td>
			<td style="width:149px;">Piramal</td>
			<td style="width:119px;"></td>
		</tr>
		<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>
		</tr>
	</tbody>
</table>

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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Neuroscience

10.7K 조회수.  University of Miami. 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...