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.

De betrokken bij de volgende protocol procedures zijn goedgekeurd door de Universiteit van Miami Institutional Animal Care en gebruik Comite (IACUC). Daarnaast worden alle procedures worden uitgevoerd in voorzieningen die door de Vereniging voor evaluatie en accreditatie van Laboratory Animal Care International (AAALAC) goedgekeurd. 1. Voorbereiding van de muizen voor Heelkunde <ol><li> Verdoven muis met isofluraan gemengd met zuurstof met een klinisch anesthesiesysteem. Gebruik isofluraan...

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De infusie microchirurgie beschreven is zeer succesvol gebleken bij het leveren exogene stoffen van verschillende biologische functies in het centraal zenuwstelsel en voorkomt verspreiding door het lichaam 1, 12 te zijn. Verstoring van de bloed-hersenbarrière is een pathologisch kenmerk van verschillende CNS-gerelateerde aandoeningen; dus de beoordeling van de relatie van exogene stoffen met de bloed-hersenbarrière is van groot belang en het belang. <p cla...

In vivo modellering van het centraal zenuwstelsel (CNS) aandoeningen vereisen een effectieve aflevering van exogene stoffen, zoals geneesmiddelen, pathogenen of exosomes, in de hersenen. Daartoe moet een ideale levermethode minimaal trauma veroorzaken aan het dier, de integriteit van het neurale netwerk en het bereiken van hoge concentraties van stoffen in de hersenen 1. Verschillende chirurgische methoden plaatselijke afgifte van een substantie zijn beschreven, inclusief interne schede, intracerebrale, intraventriculaire en injecties of implantaten 2, 3, 4, 5. Deze benaderingen zijn echter traumatische beschouwd als de CNS, en laat het beheer van slechts kleine hoeveelheden van de stof van belang. Bovendien is gesuggereerd dat exogene stoffen snel kunnen worden verwijderd door de cerebrospinale vloeistof 6 </sup&gt; en een lage penetratie bereik om de hersenparenchym waargenomen bij 7 van de bovengenoemde technieken worden gebruikt. Systemische afgifte werkwijzen, zoals orale, pulmonaire, subcutane, en intraveneuze routes worden vaker gebruikt in diermodellen, hoewel ze lage werkzaamheid vertonen in het leveren van de stoffen aan het CNS, door opname van andere organen 8, 9. Daarom zijn deze toedieningsroutes vereisen verhoogde doses voor toediening van stoffen, waardoor het risico op bijwerkingen en toxiciteit 10, 11. Hier beschrijven we een muis infusie microchirurgie technieken, die effectief levert stoffen rechtstreeks in de hersenen via de inwendige halsslagader. Naast richt de aflevering aan het centrale zenuwstelsel, is deze techniek niet voorbij normale fysiologische barrières en reden belangrijk biological-processen voor passages therapeutica of pathogenen in de hersenen.

<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.

De muis infusie microchirurgische techniek beschreven is zeer veelzijdig en is gebruikt om verschillende stoffen te leveren rechtstreeks in de hersenen, inclusief de levering van tumorcellen in een representatief model van hersenmetastasen vorming 1, 12. Deze techniek is ook geschikt voor de pathologische aspecten van verschillende pathogenen in het CZS te beoordelen. ...

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 Microchirurgie Infusion Techniek voor gerichte afgifte van een substantie in het centraal zenuwstelsel<i&gt; via</i&gt; De interne halsslagader

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.

Research

JoVE Journal

Neuroscience

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

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