This study was funded by the Mcihael J. Fox Foundation for Parkinson&#39;s Research 2011 Rapid Response Innovations Awards Program. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Prior to surgery make sure all procedures to be done are approved by IACUC and any additional ethical or legal authorities and use humane animal treatment practices. This includes using sterile surgery conditions, anesthetizing the mouse using IACUC approved method, lubricating mice eyes with vet ointment during surgery, and providing postsurgical care. Do not proceed with surgery if there is any question whether aspects of the procedure are approved. All procedures performed herein were approved by the Boston University Institutional Animal Care and Use Committee.
1. Preparation of Animals and Surgical Supplies
<ol>
	<li>Autoclave all surgical instrument that will be used during the surgery.</li>
	<li>Make sure all techniques that are to be performed are approved by the animal regulatory agencies.</li>
</ol>
2. Harvesting of the Mucosal Graft
<ol>
	<li>Chose a genetically identical mouse of similar age as the experimental mouse and euthanize it in an IACUC approved method (here: isoflurane asphyxiation followed by cervical dislocation).</li>
	<li>Using surgical scissors, remove the skin around the nasal region of mouse head exposing the skull.</li>
	<li>With a pneumatic drill, mark with three lines two of which laterally flank the nasal region and a third in line with the eyes which connects the two lines perpendicularly.</li>
	<li>Drill down ventrally in order to separate the nasal septum from the surrounding tissue. A wider path will prevent damage to the mucosal membrane however it will also make it more difficult to isolate the membrane. A narrow cut closer to midline is recommended.</li>
	<li>Use scissors to cut the septum free from any tissue adhered to it and store it in a sterile saline solution. At this time the graft can be cleaned up to remove any connected tissue. The ideal situation is to have undamaged mucosal membranes exposed on both sides of the cartilage septum. One graft can supply membrane for two mice provided the surface area of the membrane is sufficient to cover the craniotomy sites. It is recommended that the graft is used as quickly as possible and the researcher proceeds to step 3 as soon as the graft is isolated.</li>
</ol>
3. Surgical Implantation of Mucosal Graft
<ol>
	<li>Using standard, aseptic murine surgical procedures, anesthetize and mount a mouse in the stereological frame. Use approximately 2% isoflurane in pure oxygen using a rodent anesthesia machine.</li>
	<li>Immobilize the mouse in a stereotaxic apparatus with ear bars and a nose holder.&#160;Apply ophthalmic ointment to the eyes and scrub the scalp with betadine and 75% ethanol for three rounds.&#160;Using either scissors or a hair trimmer, remove the fur on the head. Expose the skull with a razor blade and level the head. Perform a craniotomy above the location of the brain that will be dosed. For example, when targeting the striatum cut a 1.25 mm diameter circular hole in the skull (centered at AP: 1.00 mm; ML: 0.88 mm) using a pneumatic drill. Wet the drilled area with sterile saline and use a razor blade to remove the skull.</li>
	<li>Carefully remove the dura using the tip of a needle. Additionally, this can be accomplished by applying a minimal amount of tissue adhesive to the moist dural surface. Once this layer has hardened, lateral motion with a razor blade tip can be used to remove the membrane.</li>
	<li>Place the mucosal membrane above the brain surface taking extreme care to keep the epithelial side facing away from the wound. This is best done by transferring the entire septum on to the surface of the skull adjacent to the craniotomy site with tweezers. Using the tip of a pair of surgical scissors, pull the membrane off of the cartilage and onto the skull and brain surface. Do not let the membrane dry out or touch it with any absorbent material. The graft should generously overlap all bony edges of the craniotomy site.</li>
	<li>Cover the graft with a sterile piece of nitrile. This acts to prevent adhesion of the skin to the graft during healing. The nitrile needs to be large enough to cover the entire mucosal membrane. Trim excessive membrane if necessary. Avoid any motion of the nitrile once it has come into contact with the graft.</li>
	<li>Close the skin with a running 5-0 sterile suture and let the mouse recover for 3-7 days before proceeding to the next step. Take care not to perturb the nitrile barrier or the mucosal graft during skin closure.</li>
</ol>
4. Administration of Dosing Solution
<ol>
	<li>After securing the anesthetized mouse in the stereotaxic frame, cut the suture with scissors and remove excess skin around the skull.</li>
	<li>Remove the nitrile barrier and clean the surface of the skull. Use sterile saline and cotton swabs to clean the area until the graft is visible. It may be necessary to cut the graft with a razor if has grown larger than the desired surface area.</li>
	<li>If the experiment will be longer than a few days, it is wise to implant at least two skull screws to reinforce the head implant.</li>
	<li>Place the well above the graft so that the edges are in contact with the skull. Apply cyanoacrylate adhesive at the junction between the well and the skull. Fill the well with sterile saline and check to make sure there are no leaks. Wells are made from cut syringe needles.</li>
	<li>Apply bone cement on the skull to secure the well in place.</li>
	<li>Remove the saline from the well with a pipette. Wash the well several times to verify that adhesive has not leaked in. Add the desired solution; in this case 50 &#956;l of fluorescent dextran is used. It is expected that water soluble compounds of a similar polarity will behave the same as dextran. Delivery of hydrophobic compounds or suspensions has not been explored with this method.</li>
	<li>Cap the top of the well by using a circular piece of nitrile secured to the top using cyanoacrylate adhesive. Make sure the adhesive does not come in contact with the well contents.</li>
</ol>
5. Analysis of Transmucosal Delivery
<ol>
	<li>After the desired amount of time has passed, anesthetize the mouse and exchange the well contents with a solution of Evan&#39;s blue dye. This dye is used to verify that the graft was intact.</li>
	<li>After 30 min, anesthetize the mouse heavily, remove the dye solution, and euthanize via decapitation.</li>
	<li>Manually remove the implant and remove the brain using surgical scissors. Be careful to keep the graft in place.</li>
	<li>Once removed, flash-freeze the brain in a solution of isobutane cooled in a dry ice bath.</li>
	<li>Embed the brain in Optimal Cutting Temperature (OCT) solution and slice at 50 &#956;m.</li>
	<li>Place the desired slices directly on a microscope slide.</li>
	<li>Image the slice using a florescence microscope as soon as the OCT solution has dried.</li>
</ol>

<ol>
	<li>Cardoso, F. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Looking+at+the+blood-brain+barrier:+Molecular+anatomy+and+possible+investigation+approaches.">Looking at the blood-brain barrier: Molecular anatomy and possible investigation approaches.</a> Brain Res. Rev. 64, 328-363 (2010).</li><li>Pardridge, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+transport+across+the+blood-brain+barrier.">Drug transport across the blood-brain barrier.</a> J. Cereb. Blood Flow Metab. 32, 1959-1972 (2012).</li><li>Chen, Y., Liu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modern+methods+for+delivery+of+drugs+across+the+blood-brain+barrier.">Modern methods for delivery of drugs across the blood-brain barrier.</a> Adv. Drug Deliv. Rev. 64, 640-665 (2012).</li><li>Cheng, F. -. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+cell+line-derived+neurotrophic+factor+protects+against+1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine+(MPTP)-induced+neurotoxicity+in+C57BL/6+mice.">Glial cell line-derived neurotrophic factor protects against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity in C57BL/6 mice.</a> Neurosci. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+and+gene+targeting+to+the+brain+with+molecular+trojan+horses.">Drug and gene targeting to the brain with molecular trojan horses.</a> Nat. Rev. Drug Discov. 1, 131-139 (2002).</li><li>Pardridge, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-brain+barrier+delivery+of+protein+and+non-viral+gene+therapeutics+with+molecular+Trojan+horses.">Blood-brain barrier delivery of protein and non-viral gene therapeutics with molecular Trojan horses.</a> J. Control. Release. 122, 345-348 (2007).</li><li>Bellavance, M. -. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+blood-brain+barrier+disruption+as+a+CNS+delivery+strategy.">Recent advances in blood-brain barrier disruption as a CNS delivery strategy.</a> AAPS J. 10, 166-177 (2008).</li><li>Merkus, F. H. M., Berg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+nasal+drug+delivery+bypass+the+blood-brain+barrier.">Can nasal drug delivery bypass the blood-brain barrier.</a> Drugs R. D. 8, 133-144 (2007).</li><li>Dhuria, S. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intranasal+delivery+to+the+central+nervous+system:+Mechanisms+and+experimental+considerations.">Intranasal delivery to the central nervous system: Mechanisms and experimental considerations.</a> J. Pharm. Sci. 99, 1654-1673 (2010).</li><li>Illum, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nasal+drug+delivery-possibilities,+problems+and+solutions.">Nasal drug delivery-possibilities, problems and solutions.</a> J. Control. 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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+brain+infusion+of+glial+cell+line-derived+neurotrophic+factor+in+Parkinson+disease.">Direct brain infusion of glial cell line-derived neurotrophic factor in Parkinson disease.</a> Nat. Med. 9, 589-595 (2003).</li><li>Love, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+cell+line-derived+neurotrophic+factor+induces+neuronal+sprouting+in+human+brain.">Glial cell line-derived neurotrophic factor induces neuronal sprouting in human brain.</a> Nat. Med. 11, 703-704 (2005).</li><li>Antunes, M. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+nasal+septa+for+respiratory+epithelial+air-liquid+interface+cultures.">Murine nasal septa for respiratory epithelial air-liquid interface cultures.</a> BioTechniques. 43, 195-204 (2007).</li><li>Bleier, B. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permeabilization+of+the+blood-brain+barrier+via+mucosal+engrafting:+implications+for+drug+delivery+to+the+brain.">Permeabilization of the blood-brain barrier via mucosal engrafting: implications for drug delivery to the brain.</a> PLoS ONE. 8, (2013).</li><li>Bernal-Sprekelsen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Closure+of+cerebrospinal+fluid+leaks+prevents+ascending+bacterial+meningitis.">Closure of cerebrospinal fluid leaks prevents ascending bacterial meningitis.</a> Rhinology. 43, 277-281 (2005).</li><li>Bleier, B. 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Benjamin S. Bleier MD is lead inventor of provisional patent covering methods of drug delivery to the central nervous system.

The most difficult step of the procedure described herein is the successful transfer of an adequately sized mucosal membrane onto the brain surface. This step is made significantly easier if the harvested nasal septum is large enough and cleaned well. If the ventral portion of the septum is truncated, a new graft should be obtained. The drilling angle should be perpendicular to the mouse head to ensure that the mucosal membrane is not damaged by the drill. If a wider than recommended drilling path is taken it will be muc...

The treatment of neurological and psychiatric disease is severely hindered by the presence of the blood-brain barrier (BBB) which prevents over 95% of all potential pharmaceutical agents from reaching the central nervous system1-3. For example, Glial Derived Neurotrophic Factor (GDNF) has been shown to be effective in treating Parkinson&#39;s Disease when injected directly into the brain, however is ineffective when delivered systemically because it cannot penetrate the BBB4-6.
Numerous approached have been developed to try to circumvent this problem. Improvement in systemic delivery of neurotheraputics has been demonstrated by using drug conjugates containing antibodies selective for transport proteins located on the brain capillary endothelium; however this method has not been shown to be applicable for a broad range of pharmaceuticals7,8. Additionally, osmotic opening of the BBB has been used clinically, however this method suffers from systemic drug dosing as opposed to a more direct delivery to the brain region of interest9. Substantial effort has been put into optimizing transnasal delivery in the hopes of directly targeting the brain10-12. Although some success has been achieved, conclusive results have only been obtained for drugs that possess endogenous receptors, such as insulin13,14. Furthermore the mechanism of transnasal delivery has been controversial with evidence suggesting indirect entry into the brain via olfactory neuron uptake or through the bloodstream11. Direct, transcranial delivery using implantable catheters has been achieved, however this procedure is highly invasive and associated with numerous complications15,16. To date, there is no general, minimally invasive method to deliver high molecular weight compounds into the brain.
Presented herein is a murine surgical procedure that creates a semipermeable interface with the brain. This is accomplished by engrafting a mucosal membrane explant17 over a surgical craniotomy defect in a mouse. Using this procedure it has been shown that soluble compounds up to 500 kDa can be delivered into the central nervous system (directly into brain parenchyma as well as into cerebrospinal fluid) in both a time and molecular weight dependent fashion18. This method of bypassing the BBB is a model for skull base defect repairs in humans which uses vascularized mucosal grafts to repair holes in the skull following transnasal endoscopic surgery19,20.

<table border="0" cellpadding="0" cellspacing="0" style="width:848px;" width="849">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Name of Material/ Equipment</td>
			<td style="width:220px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:263px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">mice</td>
			<td style="width:220px;">Taconic</td>
			<td>C57BL/6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Isoflurane</td>
			<td style="width:220px;">Piramal Healthcare</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Student Fine Scissors</td>
			<td style="width:220px;">Fine Science Tools</td>
			<td>91461-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">pneumatic drill</td>
			<td style="width:220px;">MTI Dental</td>
			<td style="width:119px;">333-CB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">drill bit</td>
			<td style="width:220px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">forcepts</td>
			<td style="width:220px;">Fine Science Tools</td>
			<td>91106-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">0.9% Sodium Chloride Injection USP</td>
			<td style="width:220px;">Abbott Laboratories</td>
			<td>4925</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Polystyrene Detri Dish</td>
			<td style="width:220px;">Fisher</td>
			<td>08-757-12</td>
			<td>for temporarily storing graft</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bead Sterilizer</td>
			<td style="width:220px;">Fine Science Tools</td>
			<td>18000-45</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Oxygen/Isoflurane System</td>
			<td style="width:220px;">SurgiVet</td>
			<td>V720100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Temperature Control System</td>
			<td style="width:220px;">Physitemp</td>
			<td style="width:119px;">TCAT-2LV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Small Animal Stereotaxic Instrument</td>
			<td style="width:220px;">KOPF</td>
			<td style="width:119px;">Model 940</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eye Ointment</td>
			<td style="width:220px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Electric shaver</td>
			<td style="width:220px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Cotton-Tipped Applicators</td>
			<td style="width:220px;">Fisher</td>
			<td>23-400-106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">7.5% Provadone Iodine</td>
			<td style="width:220px;"></td>
			<td style="width:119px;"></td>
			<td>Betadine surgical scrub</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70% Ethanol</td>
			<td style="width:220px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Surgical Blade Stainless</td>
			<td style="width:220px;">Feather</td>
			<td style="width:119px;">2976#10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Scalpel Handle - #3</td>
			<td style="width:220px;">Fine Science Tools</td>
			<td>10003-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3% Hydogen Peroxide</td>
			<td style="width:220px;"></td>
			<td style="width:119px;"></td>
			<td>for cleaning the skull</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Vetbond Tissue Adhesive</td>
			<td style="width:220px;">3M</td>
			<td>1469SB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">needles</td>
			<td>Becton, Dickinson and Company</td>
			<td>305176</td>
			<td>needle tip cut off and used as well</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">syringes</td>
			<td>Becton, Dickinson and Company</td>
			<td>309597</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">nitrile gloves</td>
			<td style="width:220px;">Denville Scientific Inc</td>
			<td>G4162</td>
			<td>for well closure and protection of graft</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5-0 Nylon Suture Thread</td>
			<td style="width:220px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Student Halsey Needle Holder</td>
			<td style="width:220px;">Fine Science Tools</td>
			<td>91201-13</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">cyanoacrylate adhesive</td>
			<td style="width:220px;"></td>
			<td style="width:119px;"></td>
			<td>commecially available super glue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dental Cement Kit, 1 lb, Pink Opaque</td>
			<td style="width:220px;">Stoelting</td>
			<td>51458</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">isobutane (2-methylbutane)</td>
			<td style="width:220px;">Aldrich</td>
			<td>M32631</td>
			<td>for dry ice bath</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">dry ice</td>
			<td style="width:220px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

heterotopic mucosal engrafting procedure for direct drug delivery to the brain in mice

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high molecular weight compounds from the bloodstream and into brain tissue. Some direct delivery success in humans has been achieved via implantation of transcranial catheters; however this method is highly invasive and associated with numerous complications. A less invasive alternative would be to dose the brain through a surgically implanted, semipermeable membrane such as the nasal mucosa that is used to repair skull base defects following endoscopic transnasal tumor removal surgery in humans. Drug transfer though this membrane would effectively bypass the BBB and diffuse directly into the brain and cerebrospinal fluid. Inspired by this approach, a surgical approach in mice was developed that uses a donor septal mucosal membrane engrafted over an extracranial surgical BBB defect. This model has been shown to effectively allow the passage of high molecular weight compounds into the brain. Since numerous drug candidates are incapable of crossing the BBB, this model is valuable for performing preclinical testing of novel therapies for neurological and psychiatric diseases.

Obtaining a large enough nasal septum explant is crucial for the subsequent steps. This can be accomplished by drilling at the location on the donor mouse&#39;s skull shown in Figure 1a. Cutting along this path will produce an explant of sufficient the size as shown in Figure 1b. If the drilling depth is not deep enough, the graft will be truncated and it will be hard to obtain a large enough membrane to cover the brain surface. Drilling more laterally than the suggested path is not advi...

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Heterotopic Mucosal Engrafting Procedure for Direct Drug Delivery to the Brain in Mice

A mouse model of human endoscopic skull base reconstruction has been developed that creates a semipermeable interface between the brain and nose using nasal mucosal grafts. This method allows researchers to study delivery to the central nervous system of high molecular weight therapeutics which are otherwise excluded by the blood-brain barrier when administered systemically.

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high ...

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Research

JoVE Journal

Medicine

10.8K Views.  Boston University.  Модель мыши эндоскопической основания черепа реконструкции человеческого была разработана, что создает полупроницаемую интерфейс между мозгом и носа с использованием слизистой оболочки носа трансплантатов. Этот метод позволяет исследователей изучать до?...

Video: Heterotopic Mucosal Engrafting Procedure for Direct Drug Delivery to the Brain in Mice

Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to stimulate microcirculation. The choice of delivery method for these agents is important because the route of administration profoundly affects the bioactivity and efficacy of these agents1,2. In this article, we demonstrate how to locally administer a substance in ischemic hindlimbs by using a catheterized osmotic pump. This pump can deliver a fixed volume of aqueous solution continuously for an allotted period of time. We also present our mouse model of unilateral hindlimb ischemia induced by ligation of the common femoral artery proximal to the origin of profunda femoris and epigastrica arteries in the left hindlimb. Lastly, we describe the in vivo cannulation and ligation of the infrarenal abdominal aorta and perfusion of the hindlimb vasculature with Microfil, a silicone radiopaque casting agent. Microfil can perfuse and fill the entire vascular bed (arterial and venous), and because we have ligated the major vascular conduit for exit, the agent can be retained in the vasculature for future ex vivo imaging with the use of small specimen micro-CT3.

We show here the in vivo insertion of an osmotic pump for constant local drug delivery and the creation of hindlimb ischemia in a mouse model. Moreover, the hindlimb vasculature is perfused with Microfil, a silicone radiopaque agent, to prepare for micro-computed tomography (micro-CT) imaging.

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to ...

Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these diseases. Chronic retinal diseases such as retinal oedema or neovascularization usually require multiple intraocular injections to effectively treat the condition. However, the risks associated with these injections increase with repeated delivery of the drug. Therefore, alternative delivery methods need to be established in order to minimize the risks of reinjection. Several other investigations have developed methods to deliver drugs over extended time, through materials capable of releasing chemicals slowly into the eye. In this investigation, we outline the use of Elvax 40W, a copolymer resin, to act as a vehicle for drug delivery to the adult rat retina. The resin is made and loaded with the drug. The drug-resin complex is then implanted into the vitreous cavity, where it will slowly release the drug over time. This method was tested using 2-amino-4-phosphonobutyrate (APB), a glutamate analogue that blocks the light response of the retina. It was demonstrated that the APB was slowly released from the resin, and was able to block the retinal response by 7 days after implantation. This indicates that slow-release drug delivery using this copolymer resin is effective for treating the retina, and could be used therapeutically with further testing.

This paper details how Elvax 40W can be used as a slow-release method for drug delivery to the adult rat retina. The protocol for preparing, loading, and delivering the drug-resin complex to the eye is described.

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these ...

Direct Mouse Trauma/Burn Model of Heterotopic Ossification

Heterotopic ossification (HO) is the formation of bone outside of the skeleton which forms following major trauma, burn injuries, and orthopaedic surgical procedures. The majority of animal models used to study HO rely on the application of exogenous substances, such as bone morphogenetic protein (BMP), exogenous cell constructs, or genetic mutations in BMP signaling. While these models are useful they do not accurately reproduce the inflammatory states that cause the majority of cases of HO. Here we describe a burn/tenotomy model in mice that reliably produces focused HO. This protocol involves creating a 30% total body surface area partial thickness contact burn on the dorsal skin as well as division of the Achilles tendon at its midpoint. Relying solely on traumatic injury to induce HO at a predictable location allows for time-course study of endochondral heterotopic bone formation from intrinsic physiologic processes and environment only. This method could prove instrumental in understanding the inflammatory and osteogenic pathways involved in trauma-induced HO. Furthermore, because HO develops in a predictable location and time-course in this model, it allows for research to improve early imaging strategies and treatment modalities to prevent HO formation.

An Achilles tenotomy and burn injury model of heterotopic ossification allows for the reliable study of trauma induced ectopic bone formation without the application of exogenous factors.

Heterotopic ossification (HO) is the formation of bone outside of the skeleton which forms following major trauma, burn injuries, and orthopaedic surgical ...

Heterotopic Cervical Heart Transplantation in Mice

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies this model by avoiding challenging suture anastomoses of small vessels thereby reducing warm ischemia time. In comparison to abdominal graft implantation the cervical model is less invasive and the implanted graft is easily accessible for further follow-up examinations. Anastomoses are performed by pulling the ascending aorta of the graft over the cuff with the recipient&#8217;s common carotid artery and by pulling the main pulmonary artery over the cuff with the external jugular vein. Selection of appropriate cuff size and complete mobilization of the vessels are important for successful revascularization. Ischemia-reperfusion (I/R) injury can be minimized by perfusing the graft with a cardioplegic solution and by hypothermia. In this article, we provide technical details for a simplified and improved cuff technique, which should allow surgeons with basic microsurgical skills to perform the procedure with a high success rate.

The cuff technique shortens and facilitates the model of heterotopic cervical heart transplantation in mice by avoiding technically challenging suture anastomoses of small vessels. The technical details shown in this video paper should allow researches to establish this model in their laboratories.

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies ...

Intraluminal Drug Delivery to the Mouse Arteriovenous Fistula Endothelium

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The simple murine AVF model was combined with intraluminal administration of drug solution to the venous endothelium at the same time as fistula creation. Technical aspects of this model are discussed. Under general anesthesia, an abdominal incision is made and the aorta and inferior vena cava (IVC) are exposed. The infra-renal aorta and IVC are dissected for clamping. After proximal and distal clamping, the puncture site is exposed and a 25 G needle is used to puncture both walls of the aorta and into the IVC. Immediately after the puncture, a reporter gene-expressing viral vector was infused in the IVC via the same needle, followed by 15 min of incubation. The intraluminal administration method enabled more robust viral gene delivery to the venous endothelium compared to administration by the external route. This novel method of delivery will facilitate studies that explore the role of the endothelium in AVF maturation and enable intraluminal drug delivery at the time of surgical operation.

After puncturing the aorta through the inferior vena cava (IVC) to create an aorto-caval fistula in the mouse, solution containing a drug is infused into the IVC via the same needle, followed by incubation. This method enables more robust drug delivery to the venous endothelium compared to the external route.

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The ...

Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cystic Fibrosis Patients

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening pulmonary manifestations. Ivacaftor (IVA) and ivacaftor-lumacaftor (LUMA) combination are two new breakthrough CF drugs that directly modulate the activity and trafficking of the defective CFTR-protein. However, there is still a dearth of understanding on pharmacokinetic/pharmacodynamic parameters and the pharmacology of ivacaftor and lumacaftor. The HPLC-MS technique for the simultaneous analysis of the concentrations of ivacaftor, hydroxymethyl-ivacaftor, ivacaftor-carboxylate, and lumacaftor in biological fluids in patients receiving standard ivacaftor or ivacaftor-lumacaftor combination therapy has previously been developed by our group and partially validated to FDA standards. However, to allow the high-throughput analysis of a larger number of patient samples, our group has optimized the reported method through the use of a smaller pore size reverse-phase chromatography column (2.6 &#181;m, C8 100 &#197;; 50 x 2.1 mm) and a gradient solvent system (0-1 min: 40% B; 1-2 min: 40-70% B; 2-2.7 min: held at 70% B; 2.7-2.8 min: 70-90% B; 2.8-4.0 min: 90% B washing; 4.0-4.1 min: 90-40% B; 4.1-6.0 min: held at 40% B) instead of an isocratic elution. The goal of this study was to reduce the HPLC-MS analysis time per sample dramatically from ~15 min to only 6 min per sample, which is essential for the analysis of a large amount of patient samples. This expedient method will be of considerable utility for studies into the exposure-response relationships of these breakthrough CF drugs.

Ivacaftor and ivacaftor-lumacaftor combination are two new CF drugs. However, there is still a dearth of understanding on their PK/PD and pharmacology. We present an optimized HPLC-MS technique for the simultaneous analysis of ivacaftor and its major metabolites, and lumacaftor.

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

Trans-Tympanic Drug Delivery for the Treatment of Ototoxicity

The systemic administration of protective agents to treat drug-induced ototoxicity is limited by the possibility that these protective agents could interfere with the chemotherapeutic efficacy of the primary drugs. This is especially true for the drug cisplatin, whose anticancer actions are attenuated by antioxidants which provide adequate protection against hearing loss. Other current or potential otoprotective agents could pose a similar problem, if administered systemically. The application of various biologicals or protective agents directly to the cochlea would allow for high levels of these agents locally with limited systemic side effects. In this report, we demonstrate a trans-tympanic method of delivery of various drugs or biological reagents to the cochlea, which should enhance basic science research on the cochlea and provide a simple way of directing the use of otoprotective agents in the clinics. This report details a method of trans-tympanic drug delivery and provides examples of how this technique has been used successfully in experimental animals to treat cisplatin ototoxicity.

We present a technique for localized administration of drugs through the trans-tympanic route into the cochlea. Drug delivery through this route would not interfere with the anti-cancer efficacy of the chemotherapeutic drugs such as cisplatin.

The systemic administration of protective agents to treat drug-induced ototoxicity is limited by the possibility that these protective agents could ...

In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice

Enteric bacterial communities are established early in life and influence immune cell development and function. The neonatal microbiota is susceptible to numerous external influences including antibiotics use and diet, which impacts susceptibility to autoimmune and inflammatory diseases. Disorders such as Inflammatory Bowel Disease (IBD) are characterized by a massive influx of immune cells to the intestines. However, immune cells conditioned by the microbiota may additionally emigrate out of the intestines to influence immune responses at extra-intestinal sites. Thus, there is a need to identify and characterize cells that may carry microbial messages from the intestines to distal sites. Here, we describe a method to label cells in the colon of newborn mice in vivo that enables their identification at extra-intestinal sites after migration.

In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice

The protocol described here utilizes a photolabeling approach in newborn mice to specifically identify immune cells that emigrate from the colon to extra-intestinal sites. This strategy will be useful to study host-microbiome interactions in early life.

Enteric bacterial communities are established early in life and influence immune cell development and function. The neonatal microbiota is susceptible to ...

Novel Methods for Intranasal Administration Under Inhalation Anesthesia to Evaluate Nose-to-Brain Drug Delivery

Intranasal administration has been reported to be a potential pathway for nose-to-brain delivery of therapeutic agents that circumvents the blood-brain barrier. However, there have been few reports regarding not only the quantitative analysis but also optimal administration conditions and dosing regimens for investigations of nose-to-brain delivery. The limited progress in research on nose-to-brain pathway mechanisms using rodents represents a significant impediment in terms of designing nose-to-brain delivery systems for candidate drugs.
To gain some headway in this regard, we developed and evaluated two novel methods of stable intranasal administration under inhalation anesthesia for experimental animals. We also describe a method for the evaluation of drug distribution levels in the brain via the nose-to-brain pathway using radio-labeled [14C]-inulin (molecular weight: 5,000) as a model substrate of water-soluble macromolecules.
Initially, we developed a pipette-based intranasal administration protocol using temporarily openable masks, which enabled us to perform reliable administration to animals under stable anesthesia. Using this system, [14C]-inulin could be delivered to the brain with little experimental error.
We subsequently developed an intranasal administration protocol entailing reverse cannulation from the airway side through the esophagus, which was developed to minimize the effects of mucociliary clearance (MC). This technique led to significantly higher levels of [14C]-inulin, which was quantitatively detected in the olfactory bulb, cerebrum, and medulla oblongata, than the pipette method. This appears to be because retention of the drug solution in the nasal cavity was substantially increased by active administration using a syringe pump in a direction opposite to the MC into the nasal cavity.
In conclusion, the two methods of intranasal administration developed in this study can be expected to be extremely useful techniques for evaluating pharmacokinetics in rodents. The reverse cannulation method, in particular, could be useful for evaluating the full potential of nose-to-brain delivery of drug candidates.

Here, we describe two novel methods of stable intranasal administration under inhalation anesthesia with minimal physical stress for experimental animals. We also describe a method for quantitative evaluation of drug distribution levels in the brain via the nose-to-brain pathway using radiolabeled [14C]-inulin as a model substrate of water-soluble macromolecules.

Intranasal administration has been reported to be a potential pathway for nose-to-brain delivery of therapeutic agents that circumvents the blood-brain ...