This work was supported by the Korea Ministry of Health &#38; Welfare (HI17C1046) to S.K.L.

All experiments were performed in compliance with guidelines and protocols approved by the Animal Care and Use Committee of the Hanyang University. In this protocol, 6-week-old Balb/c mice weighing 20-25 g were used (n = 3 per group). The animals were housed in a pathogen-free facility with 12 h light/dark cycles at a controlled temperature and humidity level with free access to water and food.
1. Device assembly
<ol>
	<li>Assemble the individual parts of the positioning device as shown in Figure 1A. 
	NOTE: The device is supplied in individual parts that can be easily assembled and disassembled.</li>
</ol>
2. Material setup
NOTE: The IN administration of liquefied drugs requires the following pretreatment steps.
<ol>
	<li>Prepare the required materials, including the positioning device, a 10 &#181;L micropipette, 10 &#181;L micropipette tips, a treatment solution (e.g., RVG9R-siRNA complex), a timer clock, and 70% ethanol (refer to Figure 2A).</li>
	<li>Plug the power code to turn on the device heating system at least 15-20 min prior to the experiment. The optimal temperature for animal experiments is automatically maintained at ~37 &#176;C.</li>
	<li>Prepare the anesthesia ketamine:xylazine:phosphate-buffered saline (PBS) at a ratio of 1:0.5:8.5 and anesthetize each mouse with a single intraperitoneal (i.p.) injection in a final volume of 200 &#181;L per 20 g mouse. 
	NOTE: Please note that any optimized drug for ensuring the anesthesia condition for a period of 30-45 min can be used.</li>
</ol>
3. Mouse positioning
<ol>
	<li>Assess the level of anesthesia by pedal reflex (firm toe pinch) to maintain the surgical plane.</li>
	<li>Place the positioning device at an appropriate distance and height so as to provide for convenient access to all required reagents.</li>
	<li>Once the mice are properly anesthetized, gently lift each mouse by the scruff of their neck with thumb and pointer finger and place it on the designated chair.</li>
	<li>Proper positioning of the mouse on the chair is very important at this stage. Lay the mouse&#39;s back parallel to the back support of the chair, and at 90&#176; to the chair seat. Lift the hands off the mouse and let the animal lie naturally in the head down-and-forward position, without pushing or pressing, as shown in Figure 2B. Make sure the mouse&#39;s forelimbs provide natural support while the animal is in this relaxed position, without any discomfort to the mouse.</li>
	<li>Once the mice are properly positioned, strap the mice in with the chair belt, and immediately start the IN inoculation.</li>
</ol>
4. Intranasal delivery
NOTE: This protocol describes the IN delivery of RVG9R:siRNA complexes using the mouse positioning device. Using this protocol, a maximum of 20-30 &#181;L of solution can be easily administered to each mouse in 2 &#181;L drops. This volume is lower than that of the mouse&#39;s nasal cavity, which is 0.032 cm3, so the procedure does not result in lethal nostril obstruction or asphyxiation. This protocol allows for the simultaneous inoculation of at least four mice and a maximum of eight mice per device, resulting in time optimization and reduced variability.
<ol>
	<li>Take the 10 &#181;L micropipette and adjust it to 2 &#181;L.</li>
	<li>Load the micropipette with 2 &#181;L of RVG9R:siRNA complex solution (e.g., 5.2 &#181;g of siCy5 complexed with 104 &#181;g of RVG9R for the delivery experiment, and 13.5 &#181;g of siSOD1 complexed with 270 &#181;g of RVG9R for the silencing experiment, in a final volume of 25 &#181;L of PBS containing 5% glucose.</li>
	<li>While holding the pipette in the dominant hand, adjust position by placing an elbow on the bench top. Support the hand holding the pipette with the other hand to avoid uncontrolled movements while administering.</li>
	<li>Place a ~2 &#181;L drop very close to one nostril so that the mouse can directly inhale the droplet (refer to Figure 2B). If a tiny drop is not easily formed, then replace the pipette tip with a new one and repeat the operation. Use the stopwatch to verify the time interval between inoculations.</li>
	<li>This device allows for the treatment of at least four mice at a time. If another group of mice is required to repeat the step 4.4, set another bar with four chairs above or below the first bar with four chairs to accommodate up to eight mice at a time on the device.</li>
	<li>Repeat step 4.4 with the other nostril after 3-4 min from the first inoculation. This time interval is sufficient for the mouse to complete the inhalation of the first dose and restore normal breathing. If a drop of the inhaled solution is dislodged due to accidental snorting out from the mouse&#39;s naris, reinoculate an additional 2 &#181;L of solution.</li>
	<li>Repeat the entire process with alternate nostrils until the 20-30 &#181;L dose is finished. It will take ~30-45 min to complete the entire inoculation procedure.</li>
	<li>Put wet ointment on the mice&#39;s eyes before returning the mice back to their designated cages. 
	NOTE: Do not leave the mice unattended until they have regained sufficient consciousness to maintain sternal recumbency.</li>
</ol>
5. Data analysis
<ol>
	<li>To confirm the brain deposition of Fluor-labeled RVG9R, anesthetize the mice (as described in step 2.3) and isolate the brain, as well as other peripheral organs, such as lung, liver, spleen, and kidney. Place each organ on the ImageStation and visualize fluorescence using ImageStation.</li>
	<li>To visualize fluorescence due to the Cy5-label, prepare 20 &#181;m-thick cryosections, costained with Hoechst 33342, and perform full-brain scanning with a confocal microscope.</li>
	<li>To assess gene silencing, extract RNA from various brain regions, such as the olfactory bulb, the cortex, the hippocampus, the thalamus, the hypothalamus, the cerebellum, and the brain stem, with an RNA purification kit, reverse-transcribed into cDNA using a cDNA synthesis kit, and perform qPCR with primer pairs as described previously15.</li>
</ol>

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S., Balyasnikova, I. V. <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+therapeutic+stem+cells+to+glioblastoma+in+a+mouse+model.">Intranasal delivery of therapeutic stem cells to glioblastoma in a mouse model.</a> Journal of Visualized Experiments. (124), e55845 (2017).</li><li>Mittal, D., 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=Insights+into+direct+nose+to+brain+delivery:+current+status+and+future+perspective.">Insights into direct nose to brain delivery: current status and future perspective.</a> Drug Delivery. 21, 75-86 (2014).</li><li>Renner, D. B., Frey, W. H., Hanson, L. 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P.K. and S.K.L. are cofounders of Signet Biotech. P.K. is coinventor of patent PCT/US07/12,152, which partly includes claims relating to RVG9R. I.U., K.C., P.K., and S.K.L. are listed as coinventors of a submitted patent (PCT/KR2016/014220), which includes claims relating to a mouse positioning device.

We have developed a mouse positioning device for optimally positioning mice for the nose-to-brain delivery of therapeutics. The device is equipped with different functionalities, ensuring the easy simultaneous handling of animals. It is also equipped with heating pads for the maintenance of the animals&#39; physiological body temperatures during experimentation. The anesthetized mice can be maintained in the head down-and-forward position by means of specifically designed chairs, with minimal discomfort to the animals. The height of the positioning chairs can be adjusted in such a way as is best to visualize animal nostrils while administering drugs.
IN brain delivery has several intrinsic limitations, including the small surface area of nostrils (allowing maximum volumes of 20-30 &#181;L per administration), nasal irritation, epithelium damage, and limited absorption across the nasal epithelium18. The IN administration of drugs to mice placed in the supine position has been used for brain delivery by dropping liquid drugs into the animals&#39; alternate nares via pipette or polyurethane tubing (24 G x 19 mm) connected to microliter syringes19,20. Although the use of tubing systems to release drugs near the olfactory epithelium is a potentially suitable approach, it causes irritation or nasal inflammation upon repetitive administration. Moreover, the small size of the nasal cavity limits this approach, especially in mice that may be overcome by repeating, as well as by drug formulations that persist in the nasal mucosa. Another option is the IN administration of therapeutics to awake mice10. However, this approach requires skilled procedures for animal handling during IN inoculation. Additionally, it causes animal stress and, therefore, is not ideal for disease models, especially infectious disease models. Furthermore, inconsistent dosing may easily result from drug drainage into the lungs or the stomach due to imperfect animal gripping. The approach presented here allows scientists to overcome these technical barriers. The head down-and-forward position reduces the possibility of drug leakage from the nose to the lungs while inhaling, favoring direct and selective siRNA delivery to the brain. IN delivery using the mouse positioning device does not require any specialized technique for handling or gripping the animals during inoculation. Four animals at a time can be treated for a period of at least 30-45 min. The procedure can be scaled up by including an additional four-chair bar, allowing for the easy management of up to eight mice in the same experimental session. Therefore, following this method, a single operator may induce drug delivery to the brains of large groups of animals over extended periods of time.
The anatomical structure of the nasal cavity strongly influences the nose-to-brain delivery (as reviewed by Merkus et al.11 and Ruigrok and de Lange21). The relative surface area of the nasal cavity in mice is 15 times bigger than that of humans and the relative surface area of the olfactory epithelium is 6 times bigger. Although there are significant differences in the anatomy of the nasal cavity in humans to rodents, there are approximately 45 clinical trials underway using the IN approach to treat several brain disorders (www.clinicaltrials.gov). The study presented here indicates that when using IN delivery of therapeutics, scientists should consider several factors, such as head position, sleeping, and proper delivery agents.
We have shown the efficient and specific deposition of fluorescently labeled siRNA to the mouse brain. Furthermore, the significant decrease in SOD1 gene expression observed after IN siRNA delivery confirmed functional effects. We previously showed consistently that the IN administration of RVG9R-siRNA complexes targeting the West Nile virus (WNV) RNA exerts a strong therapeutic effect on WNV encephalitis15. Notably, nose-to-brain siRNA delivery required a cell-targeting ligand (RVG) and a positively charged molecule (9R) to complex siRNA. In the absence of these elements, molecules were cleared through the systemic circuit and lymphatic vessels 48 h posttreatment (Figure 3A)15. Therefore, in the experimental setup described here, we have examined peptide/siRNA localization 48 h after inoculation to image only the levels retained specifically in the brain. This approach could be easily implemented for the delivery of other molecules, such as proteins, peptides, and nanoparticles, or other therapeutics, for the treatment of a number of brain-related disorders.

The BBB prevents systemically administered molecules of &#62;400-600 Da from entering the brain, posing a significant challenge to the delivery of therapeutic biomolecules for diseases affecting the CNS and brain1. Direct drug delivery to the brain can be achieved by stereotactic injection; however, this requires surgical expertise and is highly restricted in delivery to areas proximal to the injection site, making it unsuitable for routine clinical use2. IN delivery to the brain can also result in direct brain delivery by bypassing the BBB, allowing for the direct and rapid transfer of a variety of substances to the brain3,4. This transfer is thought to occur by transport mechanisms via the olfactory and trigeminal nerves that connect the nasal passage to the brain, the cerebrospinal fluid, and the lymphatic systems5. As the direct nose-to-brain route does not involve peripheral organs and tissues, it substantially reduces systemic side effects and improves potency. IN administration is a promising noninvasive alternative to both local and systemic routes for brain delivery of therapeutic agents and may represent a powerful approach to combat neurological disorders, including Alzheimer&#39;s disease, Parkinson&#39;s disease, and brain cancer, and is being explored in several clinical trials6,7,8.
Several experimental factors, such as volume and method of inoculation, as well as formulation pH, strongly influence drug delivery to the CNS via the nose-to-brain pathway9. In studies with mice, the success of IN drug delivery strongly depends on proper head positioning, which is critical for efficient brain deposition and to avoid drug drainage into the external environment or the airways. Notably, the majority of rodent studies employ a head-back position (supine) with a 70&#176;-90&#176; tilt for drug delivery to the olfactory epithelium, even though head positioning at 0&#176; may favor drainage into the trachea9. IN delivery of drugs in mice that are awake results in reduced brain deposition compared to any application in the supine position, mostly due to scientists&#39; inability to hold mice in the desired position for longer periods of time. Moreover, the upside-down position required by the skin grip method employed for awake mice results in drug deposition predominantly in the trigeminal nerve and the olfactory bulb, as well as peripheral organs, such as kidneys and lungs, within 30 min postinoculation10. The most appropriate body position for the delivery of therapeutics through the olfactory or trigeminal nerves in larger animals such as nonhuman primates in clinical studies appears to be the head down-and-forward position (i.e., the so-called &#34;praying to Mecca position&#34;)11. However, this position has not been well-studied in the mouse model, and the supine position is more widely used in rodent studies.
Previously, we have shown that the RVG9R, a peptide designed based on the minimal receptor binding domain of the Rabies virus, displays tropism to cells expressing nicotinic acetylcholine receptor subunits like neurons and macrophages and mediates the intracellular delivery of siRNA by a mechanism involving receptor engagement and temporary plasma membrane delocalization at the site of receptor aggregation12,13. Importantly, systemic intravenous administration of RVG9R-siRNA complexes enables the transvascular delivery of siRNA into the CNS14. However, the systemic route dilutes the amount of siRNA delivered to the CNS, and recent data demonstrate that the IN administration of RVG9R:siRNA complexes to mice positioned in the head down-and-forward position elicits wide-spread target gene knockdown in multiple regions of the brain15. Importantly, this level of knockdown was achieved with as little as 13.5 &#181;g of siRNA administered over a four-dose, 2 day regimen while the IV route requires a ~5 times higher dose per injection to achieve comparable knockdown. The only shortcoming of the IN approach is that it is an arduous procedure, requiring the use of both hands during the administration of the solution while continuously gripping the mice alternatively in the head down-and-forward and in relaxed positions between each inhalation for a considerable long treatment period (a 30-45 min procedure for the effective uptake of a ~20-30 &#181;L volume per mouse). Using the mouse positioning device presented here enables the proper placement of mice with little physical duress to the animals and the personnel performing the protocol, as well as the treatment of multiple cohorts of mice within a reasonable period of time, enabling an in-depth study on using siRNAs as a therapeutic for West Nile encephalitis in mice at late stages of the disease15.

<table><tbody><tr><td>&lt;strong&gt;Comercial assays&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>iScript cDNA synthesis kit</td><td>BioRad</td><td>Cat# 1708891</td><td></td></tr><tr><td>RNAiso plus</td><td>TaKaRa Bio</td><td>Cat# 9108</td><td></td></tr><tr><td>SYBR Premix ExTaq</td><td>TaKaRa Bio</td><td>Cat# RR420A</td><td></td></tr><tr><td>&lt;strong&gt;Dyes&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Alexa fluor 488</td><td>ThermoFisher</td><td>Cat# A30052</td><td></td></tr><tr><td>&lt;strong&gt;Mouse strain&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Balb/c</td><td>Orient Bio</td><td>N/A</td><td>6-8 week old, 20-30g</td></tr><tr><td>&lt;strong&gt;Oligonucleotides and primers&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Human CD4</td><td>ST Pharm</td><td>N/A</td><td>Sense: 5&amp;rsquo;-GAUCAAGAGACUCCUCAGU-3&amp;rsquo;</td></tr><tr><td>siSOD1</td><td>ST Pharm</td><td>N/A</td><td>Sense: 5&amp;rsquo;-GGUGGAAAUGAAGAAAGUA-3&amp;rsquo;</td></tr><tr><td>GAPDH primers</td><td>ST Pharm</td><td>N/A</td><td>F: 5&amp;rsquo;-AACTTTGGCATTGTGGAAGG-3&amp;rsquo; R: 5&amp;rsquo;-GGAGACAACCTGGTCCTCAG-3&amp;rsquo;</td></tr><tr><td>SOD1 primers</td><td>ST Pharm</td><td>N/A</td><td>F: 5&amp;rsquo;-CCAGTGCAGGACCTCATTTT-3&amp;rsquo; R: 5&amp;rsquo;-CACCTTTGCCCAAGTCATCT-3&amp;rsquo;</td></tr><tr><td>&lt;strong&gt;Peptides&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>RVG9R</td><td>Peptron</td><td>N/A</td><td>YTIWMPENPRPGTPCDIFTNSR&lt;br/&gt; GKRASNGGGGRRRRRRRRR</td></tr><tr><td>RVM9R</td><td>Peptron</td><td>N/A</td><td>MNLLRKIVKNRRDEDTQKSS&lt;br/&gt; PASAPLDGGGGRRRRRRRRR</td></tr><tr><td>&lt;strong&gt;Software, algorithms and devices&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>FlowJo software 4.3</td><td>FlowJO, LLC</td><td>N/A</td><td>http://docs.flowjo.com/vx/</td></tr><tr><td>Mouse positioning device</td><td>Signet Biotech</td><td>N/A</td><td></td></tr><tr><td>Prism software</td><td>Graphpad</td><td>N/A</td><td>https://www.graphpad.com/scientificsoftware/prism/</td></tr><tr><td>Prism software</td><td>Graphpad</td><td>N/A</td><td>https://www.graphpad.com/scientificsoftware/prism/</td></tr></tbody></table>

a positioning device for the placement of mice during intranasal sirna delivery to the central nervous system

Intranasal (IN) drug delivery to the brain has emerged as a promising method to bypass the blood-brain barrier (BBB) for the delivery of drugs into the central nervous system (CNS). Recent studies demonstrate the use of a peptide, RVG9R, incorporating the minimal receptor-binding domain of the Rabies virus glycoprotein, in eliciting the delivery of siRNA into neurons in the brain. In this protocol, the peptide-siRNA formulation is delivered intranasally with a pipette in the dominant hand, while the anesthetized mouse is restrained by the scruff with the nondominant hand in a "head down-and-forward position" to avoid drainage into the lung and stomach upon inhalation. This precise gripping of mice can be learned but is not easy and requires practice and skill to result in effective CNS uptake. Furthermore, the process is long-drawn, requiring about 45 min for the administration of a total volume of ~20-30 &#181;L of solution in 1-2 &#181;L droplet volumes per inhalation, with 3-4 min rest periods between each inhalation. The objective of this study is to disclose a mouse positioning device that enables the appropriate placement of mice for efficient IN administration of the peptide-siRNA formulation. Multiple features are incorporated into the design of the device, such as four or eight positioning chairs with adjustable height and tilt to restrain anesthetized mice in the head down-and-forward position, enabling easy visualization of the mice's nares and a built-in heating pad to maintain the mice's body temperatures during the procedure. Importantly, the ability to treat four or eight mice simultaneously with RVG9R-siRNA complexes in this manner enables studies on a much quicker time scale, for the testing of an IN therapeutic siRNA approach. In conclusion, this device allows for appropriate and controlled mouse head positioning for IN application of RVG9R-siRNA and other therapeutic molecules, such as nanoparticles or antibodies, for CNS delivery.

Head position during IN administration is a major influence on the efficiency of drug delivery to the brain. Here we described the head down-and-forward position using a mouse positioning device for the IN administration of a brain-targeting peptide-siRNA formulation for the delivery of the mixture to the mouse&#39;s CNS. To verify delivery through the IN route, we used the RVG9R peptide, previously shown to efficiently bind to neuronal cells both in vitro and in vivo14,16.
We first tested nose-to-brain delivery of the RVG9R peptide labeled with Alexa Fluor 488 (RVG9R-A488) by using the mouse positioning device described here. At 48 h postinoculation, various organs, including the brain, the sinus, lungs, the liver, the spleen, and kidneys were excised to measure tissue-associated A488 fluorescence. A488 alone, or a control peptide (RVM9R-A488)14 that does not bind nAchR, were used as negative controls. As expected, neither A488 nor RVM9R-A488 were detected in any of the organs at 48 h postinoculation (Figure 3A, left). On the other hand, a strong fluorescent A488 signal was detected exclusively in the brains of the RVG9R-inoculated group. In addition, we compared this mouse placement position to the supine position method that has been used previously17, as well as to the awake method, for efficacy. We inoculated a fixed amount of RVG9R-A488 (100 &#181;g) and assayed biodistribution at 48 h postinoculation. The results indicated that positioning the mice head down-and-forward improved the penetrance and deposition of RVG9R-A488 throughout the brain tissue (Figure 3A, right). In contrast, the animals inoculated in a supine position resulted in the delivery of RVG9R-A488 to the brain but not as strong as seen with the positioning device method. To further confirm siRNA delivery to the brain, we performed full-brain scanning after a single IN administration of RVG9R complexed to 400 pmol (5.2 &#181;g) of Cy5-labeled siRNA. In contrast to PBS or RVM9R, complexing with RVG9R resulted in a strong accumulation of siRNA in major brain regions, including the olfactory bulb, the cortex, the hippocampus, the thalamus, the hypothalamus, the midbrain, and the cerebellum (Figure 3B).
Finally, we employed RT-qPCR analysis to verify the intracellular delivery of functional siRNA targeting the superoxide dismutase-1 (SOD1) gene. Mice were inoculated intranasally for 3 times on consecutive days with RVG9R:siSOD1 (13.2 &#181;g of siRNA), and the SOD1 mRNA expression was analyzed 24 h after the last inoculation. RVG9R:siSOD1 administration resulted in a substantial reduction of the SOD1 expression in the olfactory bulb (63%), the cortex (47%), the hippocampus (61%), the thalamus (53%), the hypothalamus (55%), the midbrain (39%), and the cerebellum (30%) (Figure 4). In conclusion, the use of a positioning device enables an easy IN inoculation of RVG9R-complexed siRNA in mice, resulting in brain-specific siRNA delivery inducing functional silencing of the target gene.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59201/59201fig1.jpg" /> 
Figure 1: Photographic presentation of the device assembly. The positioning chairs are assembled on the stand at the appropriate height with screws. After the assembly, the device is connected to a power supply for heating to the physiological temperature during inoculation. <a href="https://www.jove.com/files/ftp_upload/59201/59201fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59201/59201fig2.jpg" /> 
Figure 2: Photographic presentation of the IN inoculation procedure using the positioning device. (A) Experimental equipment required for the IN inoculation. (B) Animals in the head down-and-forward position seated on the device (left) and the deposition of a 2 &#181;L droplet for IN inoculation (right). <a href="https://www.jove.com/files/ftp_upload/59201/59201fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59201/59201fig3.jpg" /> 
Figure 3: Distribution of fluorescently labeled peptide and siRNA after IN administration. (A) Biodistribution of intranasally inoculated RVG9R peptide at 48 h after a single IN inoculation. The brain and other peripheral organs were evaluated for the presence of fluorescence in mice inoculated with saline (PBS), A488, RVM-A488, and RVG-A488 (left). Imaging of the mice treated as mentioned above while they are awake, in the supine position, and in the mouse positioning devise in the head down-and-forward position (right). (B) Biodistribution of the RVG9R:siRNA complex in the brain (n = 3 per group). The distribution of Cy5-labeled siRNA was visualized in full brain cryosections with a confocal laser microscope in animals inoculated with saline (PBS), siRNA complexed with control peptide (RVM9R-siCy5), or the brain-targeting peptide RVG9R (RVG9R-siCy5). The scale bar represents 1 mm. Abbreviations: o.b = olfactory bulb; ctx = cortex; hippo = hippocampus; hypo = hypothalamus; tha = thalamus; m.b = midbrain; cer = cerebellum. <a href="https://www.jove.com/files/ftp_upload/59201/59201fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59201/59201fig4.jpg" /> 
Figure 4: IN application of RVG9R:siSOD1 induces target gene silencing in multiple regions of the brain. qPCR analysis for murine SOD1 mRNA in indicated brain regions 24 h after the last IN inoculation of saline (PBS), RVG9R:siCD4 (siCD4), RVM9R:siSOD1 (RVM9R), and RVG9R:siSOD1 (RVG9R). The relative SOD1 silencing in the olfactory bulb, the cortex, the hippocampus (upper), the thalamus, the hypothalamus, the cerebellum (middle), and the midbrain (lower) is shown. Data represent the mean &#177; SD relative to GAPDH after normalizing with the corresponding data of saline-treated mice (n = 3 per group). The cartoon depicting the percentage of SOD1 mRNA in the indicated brain region after the IN inoculation is shown (lower right). **P &#60; 0.01, ***P &#60; 0.001. Abbreviations: o.b = olfactory bulb; ctx = cortex; hippo = hippocampus; hypo = hippocampus; tha = thalamus; m.b = midbrain; cer = cerebellum. <a href="https://www.jove.com/files/ftp_upload/59201/59201fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Positioning Device for the Placement of Mice During Intranasal siRNA Delivery to the Central Nervous System at JoVE.com

A Positioning Device for the Placement of Mice During Intranasal siRNA Delivery to the Central Nervous System

Here, we present a protocol using a mouse positioning device that enables the appropriate placement of mice for the intranasal administration of a brain-targeting peptide-siRNA formulation enabling effective gene silencing in the central nervous system.

Intranasal (IN) drug delivery to the brain has emerged as a promising method to bypass the blood-brain barrier (BBB) for the delivery of drugs into the ...

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23.0K Views.  Hanyang University. Here, we present a protocol using a mouse positioning device that enables the appropriate placement of mice for the intranasal administration of a brain-targeting peptide-siRNA formulation enabling effective gene silencing in the central nervous system.

Video: A Positioning Device for the Placement of Mice During Intranasal siRNA Delivery to the Central Nervous System

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.

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 ...

Medicine

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

Neuroscience

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 ...

Long-term Silencing of Intersectin-1s in Mouse Lungs by Repeated Delivery of a Specific siRNA via Cationic Liposomes. Evaluation of Knockdown Effects by Electron Microscopy

Previous studies showed that knockdown of ITSN-1s (KDITSN), an endocytic protein involved in regulating lung vascular permeability and endothelial cells (ECs) survival, induced apoptotic cell death, a major obstacle in developing a cell culture system with prolonged ITSN-1s inhibition1. Using cationic liposomes as carriers, we explored the silencing of ITSN-1s gene in mouse lungs by systemic administration of siRNA targeting ITSN-1 gene (siRNAITSN). Cationic liposomes offer several advantages for siRNA delivery: safe with repeated dosing, nonimmunogenic, nontoxic, and easy to produce2. Liposomes performance and biological activity depend on their size, charge, lipid composition, stability, dose and route of administration3Here, efficient and specific KDITSN in mouse lungs has been obtained using a cholesterol and dimethyl dioctadecyl ammonium bromide combination. Intravenous delivery of siRNAITSN/cationic liposome complexes transiently knocked down ITSN-1s protein and mRNA in mouse lungs at day 3, which recovered after additional 3 days. Taking advantage of the cationic liposomes as a repeatable safe carrier, the study extended for 24 days. Thus, retro-orbital treatment with freshly generated complexes was administered every 3rd day, inducing sustained KDITSN throughout the study4. Mouse tissues collected at several time points post-siRNAITSN were subjected to electron microscopy (EM) analyses to evaluate the effects of chronic KDITSN, in lung endothelium. High-resolution EM imaging allowed us to evaluate the morphological changes caused by KDITSN in the lung vascular bed (i.e. disruption of the endothelial barrier, decreased number of caveolae and upregulation of alternative transport pathways), characteristics non-detectable by light microscopy. Overall these findings established an important role of ITSN-1s in the ECs function and lung homeostasis, while illustrating the effectiveness of siRNA-liposomes delivery in vivo.

Repeated retro-orbital injections of cationic liposomes/siRNA/ITSN-1s complexes in mice, every 72 hours for 24 days, efficiently deliver the siRNA duplex to the mouse lung microvasculature reducing ITSN-1s mRNA and protein expression by 75%. This technique is highly reproducible in an animal model, has no adverse effects and avoids fatalities.

Previous  studies showed that knockdown of ITSN-1s (KDITSN), an endocytic  protein involved in regulating lung vascular permeability and endothelial cells ...

Biology

Intrathecal Delivery of Antisense Oligonucleotides in the Rat Central Nervous System

The blood brain barrier (BBB) is an important defense against the entrance of potentially toxic or pathogenic agents from the blood into the central nervous system (CNS). However, its existence also dramatically lowers the accessibility of systemically administered therapeutic agents to the CNS. One method to overcome this, is to inject those agents directly into the cerebrospinal fluid (CSF), thus bypassing the BBB. This can be done via implantation of a catheter for either continuous infusion using an osmotic pump, or for single bolus delivery. In this article, we describe a surgical protocol for delivery of CNS-targeting antisense oligonucleotides (ASOs) via a catheter implanted directly into the cauda equina space of the adult rat spine. As representative results, we show the efficacy of a single bolus ASO intrathecal (IT) injection via this catheterization system in knocking down the target RNA in different regions of the rat CNS. The procedure is safe, effective and does not require expensive equipment or surgical tools. The technique described here can be adapted to deliver drugs in other modalities as well.

Here, we describe a method for delivering drugs to the rat central nervous system by implanting a catheter into the lumbar intrathecal space of the spine. We focus on the delivery of antisense oligonucleotides, though this method is suitable for delivery of other therapeutic modalities as well.

The blood brain barrier (BBB) is an important defense against the entrance of potentially toxic or pathogenic agents from the blood into the central ...

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 ...

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

This report describes a step-by-step guide to the technique of acute intrathecal needle injections in a noninvasive manner, i.e. independent of catheter implantation. The technical limitation of this surgical technique lies in the finesse of the hands. The injection is rapid, especially for a trained experimenter, and since tissue disruption with this technique is minimal, repeated injections are possible; moreover immune reaction to foreign tools (e.g. catheter) does not occur, thereby giving a better and more specific read out of spinal cord modulation. Since the application of the substance is largely limited to the target region of the spinal cord, drugs do not need to be applied in large dosages, and more importantly unwanted effects on other tissue, as observed with a systemic delivery, could be circumvented1,2. Moreover, we combine this technique with in vivo transfection of nucleic acid with the help of polyethylenimine (PEI) reagent3, which provides tremendous versatility for studying spinal functions via delivery of pharmacological agents as well as gene, RNA, and protein modulators.

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

This report describes a simple and rapid technique of intrathecal needle puncture for a localized transfection of siRNA in the lumbar spinal cord in mouse under short lasting light anesthesia.

This report describes a step-by-step guide to the technique of acute intrathecal needle injections in a noninvasive manner, i.e. independent of catheter ...

Tracking Superparamagnetic Iron Oxide-labeled Mesenchymal Stem Cells using MRI after Intranasal Delivery in a Traumatic Brain Injury Murine Model

Stem cell-based therapies for brain injuries, such as traumatic brain injury (TBI), are a promising approach for clinical trials. However, technical hurdles such as invasive cell delivery and tracking with low transplantation efficiency remain challenges in translational stem-based therapy. This article describes an emerging technique for stem cell labeling and tracking based on the labeling of the mesenchymal stem cells (MSCs) with superparamagnetic iron oxide (SPIO)&#160;nanoparticles, as well as intranasal delivery of the labeled MSCs. These nanoparticles are fluorescein isothiocyanate (FITC)-embedded and safe to label the MSCs, which are subsequently delivered to the brains of TBI-induced mice by the intranasal route. They are then tracked non-invasively in vivo by real-time magnetic resonance imaging (MRI). Important advantages of this technique that combines SPIO for cell labeling and intranasal delivery include (1) non-invasive, in vivo MSC tracking after delivery for long tracking periods, (2) the possibility of multiple dosing regimens due to the non-invasive route of MSC delivery, and (3) possible applications to humans, owing to the safety of SPIO, non-invasive nature of the cell-tracking method by MRI, and route of administration.

Presented here is a protocol for non-invasive mesenchymal stem cell (MSC) delivery and tracking in a mouse model of traumatic brain injury. Superparamagnetic iron oxide&#160;nanoparticles are employed as a magnetic resonance imaging (MRI) probe for MSC labeling and non-invasive in vivo tracking following intranasal delivery using real-time MRI.

Stem cell-based therapies for brain injuries, such as traumatic brain injury (TBI), are a promising approach for clinical trials. However, technical ...

Compound Administration III

Source: Kay Stewart, RVT, RLATG, CMAR; Valerie A. Schroeder, RVT, RLATG. University of Notre Dame, IN
There are many commonly used routes for compound administration in laboratory mice and rats. However, certain protocols may require the use of less commonly used routes, including intradermal, intranasal, and intracranial injections. Specialized training is essential for these procedures to be performed successfully. Justification for these routes may need to be provided to gain Institutional Animal Care and Use Committee (IACUC) approval.

Compound Administration in Rodents- Intradermal, Intranasal &amp; Intracranial Routes

Source: Kay Stewart, RVT, RLATG, CMAR; Valerie A. Schroeder, RVT, RLATG. University of Notre Dame, IN
There are many commonly used routes for compound ...

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Intranasal Delivery of Therapeutic Neural Stem Cells (NSCs) in Murine Model: A Procedure for Tumor-targeted Delivery of Oncolytic Virus Loaded NSCs into Brain Tumor Bearing Mouse Model

To load human neural stem cells with oncolytic virus, infect the stem cells with 50 conditional replicate of adenovirus viral particles per cell according to standard adenovirus transfection protocols for 2 hours at room temperature with periodic tapping. Then, wash the cells three times with fresh medium to remove any unbound viral particles and suspend the cells in sterile saline for intranasal delivery.
When the cells have been modified as experimentally appropriate, place the anesthetized tumor-bearing animals in the supine position on a clean drape over a heat pad and place a padded pillow under the heads to maintain the nostrils at an upright angle. The pillow material should be either alcohol-wipeable plastic or disposable rolled-up paper towels to avoid cross animal contamination.
When the mice are in position, use a micropipette to dispense 2 microliters of the experimental human neural stem cell population of interest into each nostril of the first mouse, visually confirming aspiration of the droplet before applying the next volume of cells. Repeat the delivery to each mouse in turn. Then, wait 5 minutes and apply the next round of cells. When all four rounds of cells have been administered, place the animals in a recovery chamber with monitoring until full recovery.

A Positioning Device for the Placement of Mice During Intranasal siRNA Delivery to the Central Nervous System

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Chapters in this video

Intranasal Administration of CNS Therapeutics to Awake Mice

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

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

Long-term Silencing of Intersectin-1s in Mouse Lungs by Repeated Delivery of a Specific siRNA via Cationic Liposomes. Evaluation of Knockdown Effects by Electron Microscopy

Intrathecal Delivery of Antisense Oligonucleotides in the Rat Central Nervous System

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

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

Tracking Superparamagnetic Iron Oxide-labeled Mesenchymal Stem Cells using MRI after Intranasal Delivery in a Traumatic Brain Injury Murine Model

Compound Administration III

Intranasal Delivery of Therapeutic Neural Stem Cells (NSCs) in Murine Model: A Procedure for Tumor-targeted Delivery of Oncolytic Virus Loaded NSCs into Brain Tumor Bearing Mouse Model