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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W., Zhai, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+brain+targeting+drug+delivery+system+by+nasal+route.">Progress in brain targeting drug delivery system by nasal route.</a> Journal of Controlled Release. 268, 364-389 (2017).</li><li>Meredith, M. E., Salameh, T. S., Banks, W. A. <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+proteins+and+peptides+in+the+treatment+of+neurodegenerative+diseases.">Intranasal delivery of proteins and peptides in the treatment of neurodegenerative diseases.</a> The AAPS Journal. 17, 780-787 (2015).</li><li>Pardeshi, C. V., Belgamwar, V. 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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 border="0" cellpadding="0" cellspacing="0" style="width:1107px;" width="1107">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:1107px;">Comercial assays</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:287px;">iScript cDNA synthesis kit</td>
			<td style="width:173px;">BioRad</td>
			<td style="width:143px;">Cat# 1708891</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:287px;">RNAiso plus</td>
			<td style="width:173px;">TaKaRa Bio</td>
			<td style="width:143px;">Cat# 9108</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:287px;">SYBR Premix ExTaq</td>
			<td style="width:173px;">TaKaRa Bio</td>
			<td style="width:143px;">Cat# RR420A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dyes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:287px;">Alexa fluor 488</td>
			<td style="width:173px;">ThermoFisher</td>
			<td style="width:143px;">Cat# A30052</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse strain</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:287px;">Balb/c</td>
			<td style="width:173px;">Orient Bio</td>
			<td style="width:143px;">N/A</td>
			<td>6-8 week old, 20-30g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Oligonucleotides and primers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:287px;">Human CD4</td>
			<td style="width:173px;">ST Pharm</td>
			<td style="width:143px;">N/A</td>
			<td>Sense: 5&#8217;-GAUCAAGAGACUCCUCAGU-3&#8217;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:287px;">siSOD1</td>
			<td style="width:173px;">ST Pharm</td>
			<td style="width:143px;">N/A</td>
			<td>Sense: 5&#8217;-GGUGGAAAUGAAGAAAGUA-3&#8217;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:287px;">GAPDH primers</td>
			<td style="width:173px;">ST Pharm</td>
			<td style="width:143px;">N/A</td>
			<td>F: 5&#8217;-AACTTTGGCATTGTGGAAGG-3&#8217; R: 5&#8217;-GGAGACAACCTGGTCCTCAG-3&#8217;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:287px;">SOD1 primers</td>
			<td style="width:173px;">ST Pharm</td>
			<td style="width:143px;">N/A</td>
			<td>F: 5&#8217;-CCAGTGCAGGACCTCATTTT-3&#8217; R: 5&#8217;-CACCTTTGCCCAAGTCATCT-3&#8217;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:1107px;">Peptides</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:287px;">RVG9R</td>
			<td style="width:173px;">Peptron</td>
			<td style="width:143px;">N/A</td>
			<td>YTIWMPENPRPGTPCDIFTNSR 
			GKRASNGGGGRRRRRRRRR</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:287px;">RVM9R</td>
			<td style="width:173px;">Peptron</td>
			<td style="width:143px;">N/A</td>
			<td>MNLLRKIVKNRRDEDTQKSS 
			PASAPLDGGGGRRRRRRRRR</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Software, algorithms and devices</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:287px;">FlowJo software 4.3</td>
			<td style="width:173px;">FlowJO, LLC</td>
			<td style="width:143px;">N/A</td>
			<td>http://docs.flowjo.com/vx/</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:287px;">Mouse positioning device</td>
			<td style="width:173px;">Signet Biotech</td>
			<td style="width:143px;">N/A</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:287px;">Prism software</td>
			<td style="width:173px;">Graphpad</td>
			<td style="width:143px;">N/A</td>
			<td style="width:504px;">https://www.graphpad.com/scientificsoftware/prism/</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:287px;">Prism software</td>
			<td style="width:173px;">Graphpad</td>
			<td style="width:143px;">N/A</td>
			<td style="width:504px;">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 ...

a-positioning-device-for-placement-mice-during-intranasal-sirna

Research

JoVE Journal

Immunology and Infection

22.8K 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

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse models allow us to study how diseases unfold, often providing a good replica of the same processes occurring in humans. For some autoimmune diseases, like type 1A diabetes, there are models (the NOD mouse) that spontaneously develop a disease similar to the human counterpart. For many other autoimmune diseases, however, the model needs to be induced experimentally. A common approach in this regard is to inject the mouse with a dominant antigen derived from the organ being studied. For example, investigators interested in autoimmune thyroiditis inject mice with thyroglobulin2, and those interested in myasthenia gravis inject them with the acetylcholine receptor3. If the autoantigen for a particular autoimmune disease is not known, investigators inject a crude protein extract from the organ targeted by the autoimmune reaction. For autoimmune hypophysitis, the pathogenic autoantigen(s) remain to be identified4, and thus a crude pituitary protein preparation is used. In this video article we demonstrate how to induce experimental autoimmune hypophysitis in SJL mice.

This video shows how to induce autoimmune hypophysitis in SJL mice and how to assess its severity by histopathology.

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse ...

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering (si)RNAs to inhibit the expression of complementary RNA transcripts is being exploited as a new class of therapeutics for a variety of diseases including HIV. Many previous reports have shown that novel RNAi-based anti-HIV/AIDS therapeutic strategies have considerable promise; however, a key obstacle to the successful therapeutic application and clinical translation of siRNAs is efficient delivery. Particularly, considering the safety and efficacy of RNAi-based therapeutics, it is highly desirable to develop a targeted intracellular siRNA delivery approach to specific cell populations or tissues. The HIV-1 gp120 protein, a glycoprotein envelope on the surface of HIV-1, plays an important role in viral entry into CD4 cells. The interaction of gp120 and CD4 that triggers HIV-1 entry and initiates cell fusion has been validated as a clinically relevant anti-viral strategy for drug discovery. 
Herein, we firstly discuss the selection and identification of 2'-F modified anti-HIV gp120 RNA aptamers. Using a conventional nitrocellulose filter SELEX method, several new aptamers with nanomolar affinity were isolated from a 50 random nt RNA library. In order to successfully obtain bound species with higher affinity, the selection stringency is carefully controlled by adjusting the conditions. The selected aptamers can specifically bind and be rapidly internalized into cells expressing the HIV-1 envelope protein. Additionally, the aptamers alone can neutralize HIV-1 infectivity. Based upon the best aptamer A-1, we also create a novel dual inhibitory function anti-gp120 aptamer-siRNA chimera in which both the aptamer and the siRNA portions have potent anti-HIV activities. Further, we utilize the gp120 aptamer-siRNA chimeras for cell-type specific delivery of the siRNA into HIV-1 infected cells. This dual function chimera shows considerable potential for combining various nucleic acid therapeutic agents (aptamer and siRNA) in suppressing HIV-1 infection, making the aptamer-siRNA chimeras attractive therapeutic candidates for patients failing highly active antiretroviral therapy (HAART).

Several 2&#x2019;-Fluoro RNA aptamers against HIV-1Ba-L gp120 with nanomole affinity are isolated from a RNA library by in vitro SELEX procedure. A new dual inhibitory function anti-gp120 aptamer-siRNA chimera is created and shows considerable promise for systemic anti-HIV therapy.

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering ...

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

The Mesenteric Lymph Duct Cannulated Rat Model: Application to the Assessment of Intestinal Lymphatic Drug Transport

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine via a series of lymphatic vessels and nodes that converge at the superior mesenteric lymph duct. Cannulation of the mesenteric lymph duct thus enables the collection of mesenteric lymph flowing from the intestine. Mesenteric lymph consists of a cellular fraction of immune cells (99% lymphocytes), aqueous fraction (fluid, peptides and proteins such as cytokines and gut hormones) and lipoprotein fraction (lipids, lipophilic molecules and apo-proteins). The mesenteric lymph duct cannulation model can therefore be used to measure the concentration and rate of transport of a range of factors from the intestine via the lymphatic system. Changes to these factors in response to different challenges (e.g., diets, antigens, drugs) and in disease (e.g., inflammatory bowel disease, HIV, diabetes) can also be determined. An area of expanding interest is the role of lymphatic transport in the absorption of orally administered lipophilic drugs and prodrugs that associate with intestinal lipid absorption pathways. Here we describe, in detail, a mesenteric lymph duct cannulated rat model which enables evaluation of the rate and extent of lipid and drug transport via the lymphatic system for several hours following intestinal delivery. The method is easily adaptable to the measurement of other parameters in lymph. We provide detailed descriptions of the difficulties that may be encountered when establishing this complex surgical method, as well as representative data from failed and successful experiments to provide instruction on how to confirm experimental success and interpret the data obtained.

Here we describe a technique to cannulate the mesenteric lymph duct in rats which enables quantification of lipid and drug transport via the lymphatic system following intestinal delivery. The technique can be adapted to assess mesenteric lymph concentrations and/or transport of fluid, immune cells, peptides, proteins and lipophilic molecules.

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine ...

Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a replicative niche. A cohort of effectors are translocated through this system into the host cell to manipulate host processes and allow the establishment of a unique lysosome-derived vacuole for replication. The method presented here involves the combination of two well-established techniques: specific gene silencing using siRNA and measurement of effector translocation using a FRET-based substrate that relies on &#946;-lactamase activity. Applying these two approaches, we can begin to understand the role of host factors in bacterial secretion system function and effector translocation. In this study we examined the role of Rab5A and Rab7A, both important regulators of the endocytic trafficking pathway. We demonstrate that silencing the expression of either protein results in a decrease in effector translocation efficiency. These methods can be easily modified to examine other intracellular and extracellular pathogens that also utilize secretion systems. In this way, a global picture of host factors involved in bacterial effector translocation may be revealed.

Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Investigating the interactions between bacterial pathogens and their hosts is an important area of biological research. Here, we describe the necessary techniques to measure effector translocation by Coxiella burnetii during siRNA gene silencing using BlaM substrate.

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main macrophage populations in the CNS: (i) the microglia, which are the resident macrophages of the CNS and are derived from yolk sac progenitors during embryogenesis, and (ii) the monocyte-derived macrophages (MDM), which can infiltrate the CNS during disease and are derived from bone marrow progenitors. The roles of each macrophage subpopulation differ depending on the pathology being studied. Furthermore, there is no consensus on the histological markers or the distinguishing criteria used for these macrophage subpopulations. However, the analysis of the expression profiles of the CD11b and CD45 markers by flow cytometry allows us to distinguish the microglia (CD11b+CD45med) from the MDM (CD11b+CD45high). In this protocol, we show that the density gradient centrifugation and the flow cytometry analysis can be used to characterize these CNS macrophage subpopulations, and to study several markers of interest expressed by these cells as we recently published. Thus, this technique can further our understanding of the role of macrophages in mouse models of neurological diseases and can also be used to evaluate drug effects on these cells.

This protocol provides an analysis of the macrophage subpopulations in the adult mouse central nervous system by flow cytometry and is helpful for the study of multiple markers expressed by these cells.

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...