The authors would like to thank the HealthPartners Institutional Animal Care and Use Committee at Regions Hospital.

1. Acclimation to Handling for Intranasal Dosing to Awake Mice
Introduction
Mice must be acclimated to handling for a period of two-four weeks before the onset of intranasal dosing. Acclimation to handling is important, as it helps ensure a correct body position for maximum effectiveness of awake intranasal drug delivery. In addition, mice that have not been properly accustomed to this process can have a severe anxiety reaction after dosing. Mice should spend about 2-3 days on each of nine steps before progressing to the next step, depending upon the animal's comfort to handling. The mouse's stress level should be used as a measure of progress. This means monitoring the mouse's movements, the amount/frequency of urination, defecation, trembling, and biting. If a mouse has a high stress response, stay on that step before progressing to the next until the response is reduced. A sample acclimation schedule can be seen in Table 1. Acclimation of the mice should progress through the following once-a-day steps. Performing these steps more than once per day, in an attempt to accelerate the acclimation, is not recommended.
<ol>
 <li>Place the mouse in the palm of the hand for a period of two to three minutes, no more than one foot above the cage top, as animals frequently jump during this introductory step. If the mouse attempts to crawl out of the hand and up one's arm, simply lift the mouse by the base of the tail and place it back in one's hand.</li>
 <li>Place the mouse in the palm of the hand for three minutes and pet the animal gently. Pet the mouse directionally from the head to the tail, while allowing the animal to move about freely.</li>
 <li>Place the mouse in the palm of the hand for three minutes while massaging behind the ears (lightly pinching together the skin on the back of the neck using the thumb and pointer finger).</li>
 <li>Hold/lift the mouse by the scruff of its neck for 30 sec, letting the mouse rest on the cage top for 30 sec before repeating the hold again.</li>
 <li>Hold the mouse using the intranasal grip, which is described in section 2, but do not invert the animal (complete up to step 2.4). Hold the animal like this for a period of 30 sec and release back to the cage top. Repeat a second time after a one-minute rest period.</li>
 <li>Hold the mouse with the intranasal grip as described in section 2, including inverting the animal so its ventral side is facing up towards the ceiling. Make sure that the animal's neck is parallel to the floor. Hold this position for 30 sec. Repeat this a second time after a one-minute rest period. Some mice will resist the intranasal grip more than others, especially when used for the first time with inversion at this step in the acclimation. If the mouse frees itself from the grip, put the mouse back on the cage top and re-grip. Repeat the re-grip if needed, but monitor the stress level of the mouse and return it to the cage if necessary. Another factor that can play a role in losing the grip at this point in the acclimation process is inexperience of the animal handlers with the intranasal delivery technique.</li>
 <li>Hold the mouse with the intranasal grip, invert, briefly place a pipettor with an empty tip over each nostril, and hold the animals position for 30 sec. Repeat this after a one-minute rest period.</li>
 <li>Hold the mouse with the intranasal grip, invert it, and intranasally administer 6 &mu;l of saline into the left and right nare as described in section 3 (through step 3.7).</li>
 <li>Hold the mouse with the intranasal grip, invert it, and intranasally administer 6 &mu;l of saline into the left and right nare twice placing the animal back on the cage top in between, as described in section 3.</li>
</ol>
2. The Intranasal Grip to Immobilize Awake Mice
<ol>
 <li>Place the mouse on the cage top. </li>
 <li>With the thumb and index finger of your dominant hand, hold the mouse by the tail. Using your middle, ring, and pinky fingers, lightly pin the lower portion of the body to the cage top (Figure 1A). Let the mouse grab onto the bars as you lightly stretch the mouse out and better grab the scruff.</li>
 <li>Using the thumb and middle finger on your non-dominant hand, pinch the mouse firmly at the scruff (excess skin) behind the neck and shoulders. Pull up on the loose skin to make a tent of skin across the shoulder blades (Figure 1B). Take as much skin as possible, but be careful not to pull up muscle as this will cause discomfort to the mouse. Exercise caution so as not to apply too much pressure to the animal's ribcage or spine. Excessive pressure or pulling of the skin may lead to inhibition of respiration in some cases.</li>
 <li>Quickly place the index finger of your non-dominant hand on the mouse's head as close to the nose as possible and pull back on the skin (Figure 1C, 1D). At the same time, slide the thumb and middle fingers toward the head for optimal position. Ideally, the mouse should not be able to move its head vertically or horizontally.</li>
 <li>While maintaining the grip on the scruff, turn the mouse onto his back. The mouse's back should be in the palm of your hand. Firmly hold the mouse's tail (near the base) against your palm with your last two fingers (ring and pinky) to stabilize the mouse, or you may wrap your fingers across the lower body (Figure 1E, 1F).</li>
 <li>At this point make sure that the mouse's neck and chin are flat and parallel to the floor. The chin should be close to a 180 degree angle with the neck as much as possible to assist with intranasal delivery. Slight changes in angle can cause the drug to end up in the wrong site. Make sure the mouse is able to breathe by watching the abdomen. The mouse should not be able to move, but should be comfortable and not in any pain. If you see the mouse's neck and chin making a "V", then you need to re-grip. </li>
</ol>
3. Intranasal Delivery of Drug to Awake Mice
Introduction
Using the intranasal grip, each mouse is restrained twice and held with their neck parallel to the floor while a volume of 20-30 &mu;l of liquid is administered. This volume is safe and efficacious for small-large mice; we typically use 24 &mu;l, and that is described in this protocol. The awake intranasal method can be used in mice of any age, for example in newborn mice for developmental neuroscience research. In newborn mice, a smaller total volume (8 &mu;l) and droplet size (1 &mu;l) should be used to accommodate the smaller volume of the nasal cavity. Drug is typically delivered to two mice at a time, alternating rest periods to make the most efficient use of time and keep on schedule. Set-up will be a little different depending on if you are dosing group housed mice or individually housed mice. With the individually housed mice, it is helpful to place all of your materials in between the cages to prevent mice from crossing over to the other cage top. With the group-housed mice, dose from that cage only. It is important that each mouse in a group-housed cage receives the same intranasal treatment, as potential for cross contamination of two different treatments exists due to drug expelling into the air or with skin contact. It is highly recommended that a dosing sheet be printed in advance, to remind technicians of treatment assignments, to record any notes of missed drops, to note general health of each animal, etc.
Steps
<ol>
 <li>Set up cages where they should be for dosing, and set up a dosing station (Figure 2).</li>
 <li>Remove mouse from cage and keep on cage top during dosing.</li>
 <li>Pick up the mouse using the intranasal grip described in section 2.</li>
 <li>Using your dominant hand, load the pipettor with 6 &mu;l of drug or vehicle. Place the tip of the filled pipettor near the mouse's left nostril, usually at a 45 degree angle (Figure 1G, 1H). If you have a viscous solution you may need to increase your angle to 90 degrees because the drug may run up the side of the pipette tip instead of making a drop. If very viscous, wiping the pipette tip with a tissue before expelling liquid may help. Slowly eject the drug to form a small droplet with about half of the volume (3 &mu;l).</li>
 <li>The droplet should be placed close enough to the mouse's nostril so that the mouse can inhale the droplet. Immediately after the mouse inhales this small drop, eject the rest of the drug in the pipette tip to form another small droplet for the mouse to inhale through the same nostril about 2-3 sec later. Be sure to watch the mouth. If there is any liquid in the mouth, the drop did not make it fully into the nose and this should be noted.</li>
 <li>Hold the mouse in this position for 15 sec. It is highly recommended that a timer be used to standardize this 15 sec hold.</li>
 <li>Repeat steps 3.3 - 3.6 for the right nostril. If any drops are snorted out, that drop should be replaced and noted.</li>
 <li>Place the mouse back on top of his cage for 2 min. Repeat the entire process starting 2 min. after delivery of the first drop. This gives the mouse four drops of 6 &mu;l each totaling 24 &mu;l of solution. A sample schedule for dosing one or two mice at once can be seen in Table 2.</li>
 <li>After the mouse has received all drops, return the mouse to its cage.</li>
</ol>
4. General Animal Handling Techniques for Acclimation and Intranasal Delivery of Drug to Awake Mice
After each acclimation or intranasal dosing session, mice should be given a treat, such as a Cheerio or peeled sunflower seed as positive reinforcement. Treats are given as a reward and to distract them from scratching their noses, potentially causing injury to themselves. When working with group housed mice, enough treats should be dropped for each mouse. Before returning animals to their cages be sure to restore nests to their original states in order to reduce stress. When dosing group housed animals, gloves must be changed between dosing of cages so as not to transfer scent from cage colony to cage colony, as they will become aggressive towards one another.

<ol>
	<li>Dhuria, S. V., Hanson, L. R., Frey, W. H. <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>Thorne, R. G., Pronk, G. J., Padmanabhan, V., Frey, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+of+insulin-like+growth+factor-I+to+the+rat+brain+and+spinal+cord+along+olfactory+and+trigeminal+pathways+following+intranasal+administration.">Delivery of insulin-like growth factor-I to the rat brain and spinal cord along olfactory and trigeminal pathways following intranasal administration.</a> Neuroscience. , 127-481 (2004).</li><li>Benedict, 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=Intranasal+insulin+as+a+therapeutic+option+in+the+treatment+of+cognitive+impairments.">Intranasal insulin as a therapeutic option in the treatment of cognitive impairments.</a> Exp. Gerontol. 46, 112-115 (2011).</li><li>Francis, G. J., 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+insulin+prevents+cognitive+decline,+cerebral+atrophy+and+white+matter+changes+in+murine+type+I+diabetic+encephalopathy.">Intranasal insulin prevents cognitive decline, cerebral atrophy and white matter changes in murine type I diabetic encephalopathy.</a> Brain. 131, 3311-3334 (2008).</li><li>Francis, G., 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+insulin+ameliorates+experimental+diabetic+neuropathy.">Intranasal insulin ameliorates experimental diabetic neuropathy.</a> Diabetes. 58, 934-945 (2009).</li><li>Marks, D. R., Tucker, K., Cavallin, M. A., Mast, T. G., Fadool, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Awake+intranasal+insulin+delivery+modifies+protein+complexes+and+alters+memory,+anxiety,+and+olfactory+behaviors.">Awake intranasal insulin delivery modifies protein complexes and alters memory, anxiety, and olfactory behaviors.</a> J. Neurosci. 29, 6734-6751 (2009).</li><li>Hanson, 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=Intranasal+delivery+of+deferoxamine+reduces+spatial+memory+loss+in+APP/PS1+mice.">Intranasal delivery of deferoxamine reduces spatial memory loss in APP/PS1 mice.</a> Drug Delivery and Translational Research. 2, 160-168 (2012).</li><li>Danielyan, 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=Protective+effects+of+intranasal+losartan+in+the+APP/PS1+transgenic+mouse+model+of+Alzheimer+disease.">Protective effects of intranasal losartan in the APP/PS1 transgenic mouse model of Alzheimer disease.</a> Rejuvenation Research. 13, 195-201 (2010).</li><li>Fine, J. 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=Intranasal+deferoxamine+improves+performance+in+radial+arm+water+maze,+stabilizes+HIF-1alpha,+and+phosphorylates+GSK3beta+in+P301L+tau+transgenic+mice.">Intranasal deferoxamine improves performance in radial arm water maze, stabilizes HIF-1alpha, and phosphorylates GSK3beta in P301L tau transgenic mice.</a> Exp. Brain Res. 219, 381-390 (2012).</li><li>Lochhead, J. J., Thorne, R. G. <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+of+biologics+to+the+central+nervous+system.">Intranasal delivery of biologics to the central nervous system.</a> Adv. Drug Deliv. Rev. 64 (11), 614-628 (2012).</li><li>Hanson, L. R., 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+deferoxamine+provides+increased+brain+exposure+and+significant+protection+in+rat+ischemic+stroke.">Intranasal deferoxamine provides increased brain exposure and significant protection in rat ischemic stroke.</a> J. Pharmacol. Exp Ther. 330, 679-686 (2009).</li><li>Reger, 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=Effects+of+intranasal+insulin+on+cognition+in+memory-impaired+older+adults:+modulation+by+APOE+genotype.">Effects of intranasal insulin on cognition in memory-impaired older adults: modulation by APOE genotype.</a> Neurobiol. Aging. 27, 451-458 (2006).</li><li>Kim, C. H., Song, H. y. u. n., M, E. u. n. A. h. n., Lee, Y., G, J., Yoon, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+hypo-,+iso-+and+hypertonic+saline+irrigation+on+secretory+mucins+and+morphology+of+cultured+human+nasal+epithelial+cells.">Effect of hypo-, iso- and hypertonic saline irrigation on secretory mucins and morphology of cultured human nasal epithelial cells.</a> Acta Otolaryngol. 125, 1296-1300 (2005).</li><li>Greiff, L., Andersson, M., Wollmer, P., Persson, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypertonic+saline+increases+secretory+and+exudative+responsiveness+of+human+nasal+airway+in+vivo.">Hypertonic saline increases secretory and exudative responsiveness of human nasal airway in vivo.</a> Eur. Respir. J. 21, 308-312 (2003).</li></ol>

No conflicts of interest declared.

This method shows how mice can be given regular doses of intranasal drug to target the brain without the use of anesthesia. Intranasal drug can be delivered to large groups of mice for extended periods of time with minimal effort. A single group of up to 20 mice can be intranasally treated by a single person in less than one hour. A vast array of molecules, ranging from small iron chelators to growth factors to mesenchymal stem cells, have been demonstrated to rapidly target the CNS and have shown efficacy in rodents mod...

<table border="1">
 <tbody>
 <tr>
 <td>Materials</td>
 <td>Company</td>
 <td>Cat #</td>
 <td>Comment</td>
 </tr>
 <tr>
 <td>P20 pipettor set at 6 &mu;l</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Gel loading pipette tips</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Timer</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Waste receptacle for tips</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Dosing sheet for notes</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Pen</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Drug and vehicle</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Container to hold drug vials</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Animals to be dosed</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Spare gloves</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Paper towels to wipe urine</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Table 3. Materials Needed</td>
 </tr>
 </tbody>
</table>

intranasal administration of cns therapeutics to awake mice

Intranasal administration is a method of delivering therapeutic agents to the central nervous system (CNS). It is non-invasive and allows large molecules that do not cross the blood-brain barrier access to the CNS. Drugs are directly targeted to the CNS with intranasal delivery, reducing systemic exposure and thus unwanted systemic side effects1. Delivery from the nose to the CNS occurs within minutes along both the olfactory and trigeminal neural pathways via an extracellular route and does not require drug to bind to any receptor or axonal transport2. Intranasal delivery is a widely publicized method and is currently being used in human clinical trials3.
Intranasal delivery of drugs in animal models allows for initial evaluation of pharmacokinetic distribution and efficacy. With mice, it is possible to administer drugs to awake (non-anesthetized) animals on a regular basis using a specialized intranasal grip. Awake delivery is beneficial because it allows for long-term chronic dosing without anesthesia, it takes less time than with anesthesia, and can be learned and done by many people so that teams of technicians can dose large numbers of mice in short periods. Efficacy of therapeutics administered intranasally in this way to mice has been demonstrated in a number of studies including insulin in diabetic mouse models 4-6 and deferoxamine in Alzheimer's mouse models. 7,8
The intranasal grip for mice can be learned, but is not easy and requires practice, skill, and a precise grip to effectively deliver drug to the brain and avoid drainage to the lung and stomach. Mice are restrained by hand using a modified scruff in the non-dominant hand with the neck held parallel to the floor, while drug is delivered with a pipettor using the dominant hand. It usually takes 3-4 weeks of acclimating to handling before mice can be held with this grip without a stress response. We have prepared this JoVE video to make this intranasal delivery technique more accessible.

There are several ways to determine the efficacy of drug delivery. The first is to intranasally administer a dye, such as India ink or methylene blue and to visualize the dye after euthanasia of the animal within the olfactory epithelium and after the brain is removed, entering the CNS through the cribriform plate. The second is to measure the drug delivered either by using a radio-labeled tag or using ELISA detection of the unlabeled molecule. A good example of drug distribution in the CNS after administration t...

Watch this Scientific Journal Video about Intranasal Administration of CNS Therapeutics to Awake Mice at JoVE.com

Intranasal Administration of CNS Therapeutics to Awake Mice

A method to intranasally administer drugs to awake mice for the purpose of targeting the brain is described. This method allows for repeat dosing over long periods using intranasal administration of drug without anesthesia, and nose-to-brain delivery with minimal systemic exposure.

Intranasal administration is a method of delivering  therapeutic agents to the central nervous system (CNS). It is  non-invasive and allows large ...

intranasal-administration-of-cns-therapeutics-to-awake-mice

Research

JoVE Journal

Medicine

58.5K Views.  HealthPartners Institute for Education and Research. A method to intranasally administer drugs to awake mice for the purpose of targeting the brain is described. This method allows for repeat dosing over long periods using intranasal administration of drug without anesthesia, and nose-to-brain delivery with minimal systemic exposure.

Video: Intranasal Administration of CNS Therapeutics to Awake Mice

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

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain barrier1. The barrier selectively excludes molecules from crossing between the blood and the brain based upon their size and charge. This function can impede the delivery of therapeutics for neurological disorders. A number of chemotherapeutic drugs, for example, will not effectively cross the blood-brain barrier to reach tumor cells2. Thus, improving the delivery of drugs across the blood-brain barrier is an area of interest.
The most prevalent methods for enhancing the delivery of drugs to the brain are direct cerebral infusion and blood-brain barrier disruption3. Direct intracerebral infusion guarantees that therapies reach the brain; however, this method has a limited ability to disperse the drug4. Blood-brain barrier disruption (BBBD) allows drugs to flow directly from the circulatory system into the brain and thus more effectively reach dispersed tumor cells. Three methods of barrier disruption include osmotic barrier disruption, pharmacological barrier disruption, and focused ultrasound with microbubbles. Osmotic disruption, pioneered by Neuwelt, uses a hypertonic solution of 25% mannitol that dehydrates the cells of the blood-brain barrier causing them to shrink and disrupt their tight junctions. Barrier disruption can also be accomplished pharmacologically with vasoactive compounds such as histamine5 and bradykinin6. This method, however, is selective primarily for the brain-tumor barrier7. Additionally, RMP-7, an analog of the peptide bradykinin, was found to be inferior when compared head-to-head with osmotic BBBD with 25% mannitol8. Another method, focused ultrasound (FUS) in conjunction with microbubble ultrasound contrast agents, has also been shown to reversibly open the blood-brain barrier9. In comparison to FUS, though, 25% mannitol has a longer history of safety in human patients that makes it a proven tool for translational research10-12.
In order to accomplish BBBD, mannitol must be delivered at a high rate directly into the brain's arterial circulation. In humans, an endovascular catheter is guided to the brain where rapid, direct flow can be accomplished. This protocol models human BBBD as closely as possible. Following a cut-down to the bifurcation of the common carotid artery, a catheter is inserted retrograde into the ECA and used to deliver mannitol directly into the internal carotid artery (ICA) circulation. Propofol and N2O anesthesia are used for their ability to maximize the effectiveness of barrier disruption13. If executed properly, this procedure has the ability to safely, effectively, and reversibly open the blood-brain barrier and improve the delivery of drugs that do not ordinarily reach the brain 8,13,14.

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.

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Non-invasive Assessment of the Efficacy of New Therapeutics for Intestinal Pathologies Using Serial Endoscopic Imaging of Live Mice

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic mechanisms that contribute to the onset and progression of intestinal diseases. The identification of new molecules that promote these pathologies has led to a flurry of activity focused on the development of potential new therapies to inhibit their function. As a result, various pre-clinical mouse models with an intact immune system and stromal microenvironment are now heavily used. Here we describe three experimental protocols to test the efficacy of new therapeutics in pre-clinical models of (1) acute mucosal damage, (2) chronic colitis and/or colitis-associated colon cancer, and (3) sporadic colorectal cancer. We also outline procedures for serial endoscopic examination that can be used to document the therapeutic response of an individual tumor and to monitor the health of individual mice. These protocols provide complementary experimental platforms to test the effectiveness of therapeutic compounds shown to be well tolerated by mice.

We describe methods for longitudinal monitoring of the efficacy of therapeutics for the treatment of colonic pathologies in mice using a rigid endoscope. This protocol can be readily used for the characterization of the therapeutic response of an individual tumor in live mice and also for monitoring potential disease relapse.

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic ...

A Hydrogel Construct and Fibrin-based Glue Approach to Deliver Therapeutics in a Murine Myocardial Infarction Model.

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of treatments in vivo1. The traditional, established protocol has been further fine-tuned to minimize the damage to the animal. Notably, the pectoral muscle layers are teased away rather than simply cut, and the thoracotomy is approached intercostally as opposed to breaking the ribs in a sternotomy, preserving the integrity of the ribcage. With these changes, the overall stress on the animal is decreased. 
Stem cell therapies aimed to alleviate the damage caused by MIs have shown promise over the years for their pro-angiogenic and anti-apoptotic benefits. Current approaches of delivering cells to the heart surface typically involve the injection of the cells either near the damaged site, within a coronary artery, or into the peripheral blood stream2-4. While the cells have proven to home to the damaged myocardium, functionality is limited by their poor engraftment at the site of injury, resulting in diffusion into the blood stream5. This manuscript highlights a procedure that overcomes this obstacle with the use of a cell-encapsulated hydrogel patch. The patch is fabricated prior to the surgical procedure and is placed on the injured myocardium immediately following the occlusion of the left coronary artery. To adhere the patch in place, biocompatible external fibrin glue is placed directly on top of the patch, allowing for it to dry to both the patch and the heart surface. This approach provides a novel adhesion method for the application of a delicate cell-encapsulating therapeutic construct.

This protocol aims to alleviate the limitation of poor cell engraftment for stem cell treatment of myocardial infarctions through the use of a hydrogel system and a fibrin-based glue. With this approach, cell-to-tissue contact post-infarction can be maintained, increasing the therapeutic potential of beneficial agents at the site of injury.

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of ...

A Rat Carotid Balloon Injury Model to Test Anti-vascular Remodeling Therapeutics

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is also a valuable model system to test, and to evaluate therapeutics and drugs that negate maladaptive remodeling in the vessel. The injury, or barotrauma, in the vessel lumen caused by an inflated balloon via an inserted catheter induces subsequent neointimal growth, often leading to hyperplasia or thickening of the vessel wall that narrows, or obstructs the lumen. The method described here is sufficiently sensitive, and the results can be obtained in relatively short time (2 weeks after the surgery). The efficacy of the drug or therapeutic against the induced-remodeling can be evaluated either by the post-mortem pathological and histomorphological analysis, or by ultrasound sonography in live animals. In addition, this model system has also been used to determine the therapeutic window or the time course of the administered drug. These studies can leadto the development of a better administrative strategy and a better therapeutic outcome. The procedure described here provides a tool for translational studies that bring drug and therapeutic candidates from bench research to clinical applications.

The rat carotid balloon injury model described below allows researchers to evaluate drugs or therapeutics that negate injury-induced arterial hyperplasia. Detailed pre-surgical preparation, surgical procedure, and post-surgical cares of the animal are described.

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is ...

Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing factor in the overall outcome. Immunoblotting, flow cytometry and fixed cell experiments are often used to study how cells respond to anti-cancer drugs. These methods are important, but they have several shortcomings. Variability in drug responses between cancer and normal cells, and between cells of different cancer origin, and transient and rare responses are difficult to understand using population averaging assays and without being able to directly track and analyze them longitudinally. The microscope is particularly well suited to image live cells. Advancements in technology enable us to routinely image cells at a resolution that enables not only cell tracking, but also the observation of a variety of cellular responses. We describe an approach in detail that allows for the continuous time-lapse imaging of cells during the drug response for essentially as long as desired, typically up to 96 hr. Using variations of the approach, cells can be monitored for weeks. With the employment of genetically encoded fluorescent biosensors numerous processes, pathways and responses can be followed. We show examples that include tracking and quantification of cell growth and cell cycle progression, chromosome dynamics, DNA damage, and cell death. We also discuss variations of the technique and its flexibility, and highlight some common pitfalls.

Here, we describe a method of long-term time-lapse microscopy to longitudinally track single cells in response to anti-cancer therapeutics.

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing ...

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular plasticity or damage occurring overtime and in relation to neuronal activity or blood flow. In vivo two-photon microscopy allows the study of the structural and functional plasticity of large cellular units in the living brain. In particular, the thinned-skull window preparation allows the visualization of cortical regions of interest (ROI) without inducing significant brain inflammation. Repetitive imaging sessions of cortical ROI are feasible, providing the characterization of disease hallmarks over time during the progression of numerous CNS diseases. This technique accessing the pial structures within 250 &#956;m of the brain relies on the detection of fluorescent probes encoded by genetic cellular markers and/or vital dyes. The latter (e.g., fluorescent dextrans) are used to map the luminal compartment of cerebrovascular structures. Germane to the protocol described herein is the use of an in vivo marker of amyloid deposits, Methoxy-O4, to assess Alzheimer&#39;s disease (AD) progression. We also describe the post-acquisition image processing used to track vascular changes and amyloid depositions. While focusing presently on a model of AD, the described protocol is relevant to other CNS disorders where pathological cerebrovascular changes occur.

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

This manuscript describes a procedure to track the remodeling of the cerebrovasculature during amyloid plaque accumulation in vivo using longitudinal two-photon microscopy. A thinned-skull preparation enables the visualization of fluorescent dyes to assess the progression of cerebrovascular damage in a mouse model of Alzheimer&#39;s disease.

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular ...

Intranasal Delivery of Therapeutic Stem Cells to Glioblastoma in a Mouse Model

The intrinsic tropism towards brain malignancies renders stem cells as promising carriers of therapeutic agents against malignant tumors. The delivery of therapeutic stem cells via the intranasal route is a recently discovered alternative strategy, with strong potential for clinical translation, due to its non-invasive nature compared to intracranial implantation or delivery via systemic routes. The lack of blood brain barrier further strengthens the therapeutic potential of stem cells undergoing intranasal brain entry. This article summarizes the essential techniques utilized in our studies and outlines the basic principles of intranasal strategy for stem cell delivery using a mouse model of intracranial glioma xenografts. We demonstrate the optimized procedures that generate consistent and reproducible results with specific predetermined experimental parameters and offer guidelines for streamlined work flow that ensure efficient execution and reliable experimental outcome. The article is designed to serve as a baseline for further experimental customization based on hypothesis, stem cell types, or tumor specifics.

Stem cells are promising therapeutic carriers to treat brain tumors due to their intrinsic tumor tropism. Non-invasive intranasal stem cell delivery bypasses the blood brain barrier and demonstrates strong potential for clinical translation. This article summarizes the basic principles of intranasal stem cell delivery in a mouse model of glioma.

The intrinsic tropism towards brain malignancies renders stem cells as promising carriers of therapeutic agents against malignant tumors. The delivery of ...

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 ...

Intranasal Administration of CNS Therapeutics to Awake Mice

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

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

Non-invasive Assessment of the Efficacy of New Therapeutics for Intestinal Pathologies Using Serial Endoscopic Imaging of Live Mice

A Hydrogel Construct and Fibrin-based Glue Approach to Deliver Therapeutics in a Murine Myocardial Infarction Model.

A Rat Carotid Balloon Injury Model to Test Anti-vascular Remodeling Therapeutics

Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

Intranasal Delivery of Therapeutic Stem Cells to Glioblastoma in a Mouse Model

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