The work performed in this protocol was funded by Hasselblad Foundation (2020-2021, ERF), &#197;ke Wibergs Foundation (M19-0660, ERF), Swedish Research Council (2019-01320, HH; 2021-01872, CM), Public Health Service at the Sahlgrenska University Hospital (ALFGBG-965174, HH ; ALFGBG-966107, CM), The Swedish Brain Foundation (FO2022-0110, CM), &#197;hlen Foundation (223005, CM) and Horizon 2020 Framework Program of European Union (grant agreement no. 87472/PREMSTEM, HH).

All procedures listed in this protocol conformed to the Swedish Board of Agriculture and were approved by the Gothenburg Animal Ethics Committee (825-2017 and 2195-19). C57BL/6 mice and Wistar rats were bred in-house with a 12 h light/dark cycle and free access to food and water. All experimental procedures followed the ARRIVE guidelines10.
1. Workspace setup
<ol>
	<li>For the duration of this procedure, collect the experimental animals from the dam cage and place them in a separate cage on a heated pad (35-37 &#176;C). 
	NOTE: If using a light source (albino animals), a non-heat light source that can be positioned underneath the animal&#39;s head should be used.</li>
</ol>
2. Needle and solution
<ol>
	<li>Use a needle with 29-31 G (around 0.30 mm).</li>
	<li>For accurate volumes, draw up the solution to be injected from a pipetted volume. 
	NOTE: A maximum of 5 &#956;L/ g body weight should be injected in each retroorbital sinus.</li>
</ol>
3. Setup
<ol>
	<li>Place the animals on a flat surface (Figure 1A) in lateral recumbency (Figure 1C).</li>
	<li>Induce full-body isoflurane anesthesia (5% induction, 3% maintenance). 
	NOTE: The animals should be placed under anesthesia using a mouthpiece. The eyelid and tear duct area should not be covered (Figure 1D).</li>
	<li>Check the depth of anesthesia using the paw withdrawal reflex method. 
	NOTE: No pre-operative analgesia is required as this is not considered an invasive procedure11.</li>
</ol>
4. Injection procedure
NOTE: If possible, use a light source under the animal&#39;s head (Figure 1C), to facilitate view of the venous sinus (Figure 1E). No sterilization of the area being injected is required, as the eye lid is still closed.
<ol>
	<li>With its head facing to the right, administer the injection into the right retroorbital sinus (right-handed operator example).</li>
	<li>Insert the needle, bevel down, at the front of the eye socket - the equivalent of the medial canthus, at an angle of approximately 40&#176;. This angle allows the needle to be directed to the back of the eye orbit.</li>
	<li>Advance 1/3 of the needle (around 2 mm) into the area of the retroorbital sinus located behind the eye orbit.</li>
	<li>Inject in a gentle, smooth, and fluid motion.</li>
	<li>Wait for a moment, before withdrawing the needle slowly to avoid backflow. 
	CAUTION: Do not aspirate.</li>
	<li>Use a new sterile syringe for each animal to avoid contamination. 
	NOTE: When injecting a clear solution, the vein should turn momentarily clear.</li>
</ol>
5. Post-injection care
<ol>
	<li>Place the pup in the recovery box, rested on a protected warming device (35-37 &#176;C).</li>
	<li>Wait for the recovery and check for any signs of distress before returning the pup to the dam. 
	NOTE: Dye-injected practice animals should be immediately euthanized as per the IACUC approved protocols. 
	CAUTION: If the eye swells during the injection, it means that the needle is not inserted into the venous plex and is instead in the eye orbit. The neonatal skull is very soft, if the needle perforates it, then the injection will go into the meninges or even the brain parenchyma.</li>
</ol>

<ol>
	<li>Laughon, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+labeling+and+exposure+in+neonates.">Drug labeling and exposure in neonates.</a> JAMA Pediatr. 168 (2), 130-136 (2014).</li><li>Das, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodological+issues+in+the+design+and+analyses+of+neonatal+research+studies:+Experience+of+the+nichd+neonatal+research+network.">Methodological issues in the design and analyses of neonatal research studies: Experience of the nichd neonatal research network.</a> Semin Perinatol. 40 (6), 374-384 (2016).</li><li>Ku, L. C., Smith, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dosing+in+neonates:+Special+considerations+in+physiology+and+trial+design.">Dosing in neonates: Special considerations in physiology and trial design.</a> Pediatr Res. 77 (1-1), 2-9 (2015).</li><li>Linakis, M. W., 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=Challenges+associated+with+route+of+administration+in+neonatal+drug+delivery.">Challenges associated with route of administration in neonatal drug delivery.</a> Clin Pharmacokinet. 55 (2), 185-196 (2016).</li><li>Steel, C. D., Stephens, A. L., Hahto, S. M., Singletary, S. J., Ciavarra, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+lateral+tail+vein+and+the+retro-orbital+venous+sinus+as+routes+of+intravenous+drug+delivery+in+a+transgenic+mouse+model.">Comparison of the lateral tail vein and the retro-orbital venous sinus as routes of intravenous drug delivery in a transgenic mouse model.</a> Lab Anim (NY). 37 (1), 26-32 (2008).</li><li>Gombash Lampe, S. E., Kaspar, B. K., Foust, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravenous+injections+in+neonatal+mice).">Intravenous injections in neonatal mice).</a> J Vis Exp. , e52037 (2014).</li><li>Hamodi, A. S., Martinez Sabino, A., Fitzgerald, N. D., Moschou, D., Crair, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transverse+sinus+injections+drive+robust+whole-brain+expression+of+transgenes.">Transverse sinus injections drive robust whole-brain expression of transgenes.</a> Elife. 9, 53639 (2020).</li><li>Semple, B. D., Blomgren, K., Gimlin, K., Ferriero, D. M., Noble-Haeusslein, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+development+in+rodents+and+humans:+Identifying+benchmarks+of+maturation+and+vulnerability+to+injury+across+species.">Brain development in rodents and humans: Identifying benchmarks of maturation and vulnerability to injury across species.</a> Prog Neurobiol. 106-107, 1-16 (2013).</li><li>Yardeni, T., Eckhaus, M., Morris, H. D., Huizing, M., Hoogstraten-Miller, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retro-orbital+injections+in+mice.">Retro-orbital injections in mice.</a> Lab Anim (NY). 40 (5), 155-160 (2011).</li><li>Kilkenny, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+research:+Reporting+in+vivo+experiments:+The+arrive+guidelines).">Animal research: Reporting in vivo experiments: The arrive guidelines).</a> Br J Pharmacol. 160 (7), 1577-1579 (2010).</li><li>Prabhakar, S., Lule, S., Da Hora, C. C., Breakefield, X. O., Cheah, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aav9+transduction+mediated+by+systemic+delivery+of+vector+via+retro-orbital+injection+in+newborn,+neonatal+and+juvenile+mice.">Aav9 transduction mediated by systemic delivery of vector via retro-orbital injection in newborn, neonatal and juvenile mice.</a> Exp Anim. 70 (4), 450-458 (2021).</li><li>Ek, C. J., Habgood, M. D., Dziegielewska, K. M., Potter, A., Saunders, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permeability+and+route+of+entry+for+lipid-insoluble+molecules+across+brain+barriers+in+developing+monodelphis+domestica.">Permeability and route of entry for lipid-insoluble molecules across brain barriers in developing monodelphis domestica.</a> J Physiol. 536, 841-853 (2001).</li><li>Jinnai, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+of+germinal+matrix+hemorrhage+in+preterm+rat+pups).">A model of germinal matrix hemorrhage in preterm rat pups).</a> Front Cell Neurosci. 14, 535320 (2020).</li><li>Andersson, E. A., Rocha-Ferreira, E., Hagberg, H., Mallard, C., Ek, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+and+biomarkers+of+the+blood-brain+barrier+in+a+neonatal+germinal+matrix+haemorrhage+model.">Function and biomarkers of the blood-brain barrier in a neonatal germinal matrix haemorrhage model.</a> Cells. 10 (7), (2021).</li><li>Ohls, R. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+early+erythropoietin+therapy+on+the+transfusion+requirements+of+preterm+infants+below+1250+grams+birth+weight:+A+multicenter,+randomized,+controlled+trial.">Effects of early erythropoietin therapy on the transfusion requirements of preterm infants below 1250 grams birth weight: A multicenter, randomized, controlled trial.</a> Pediatrics. 108 (4), 934-942 (2001).</li><li>Costa, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+administrate+erythropoietin,+intravenous+or+subcutaneous.">How to administrate erythropoietin, intravenous or subcutaneous.</a> Acta Paediatr. 102 (6), 579-583 (2013).</li><li>Fernandez-Lopez, 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=Blood-brain+barrier+permeability+is+increased+after+acute+adult+stroke+but+not+neonatal+stroke+in+the+rat.">Blood-brain barrier permeability is increased after acute adult stroke but not neonatal stroke in the rat.</a> J Neurosci. 32 (28), 9588-9600 (2012).</li><li>Sugiyama, Y., 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=Intravenous+administration+of+bone+marrow-derived+mesenchymal+stem+cell,+but+not+adipose+tissue-derived+stem+cell,+ameliorated+the+neonatal+hypoxic-ischemic+brain+injury+by+changing+cerebral+inflammatory+state+in+rat.">Intravenous administration of bone marrow-derived mesenchymal stem cell, but not adipose tissue-derived stem cell, ameliorated the neonatal hypoxic-ischemic brain injury by changing cerebral inflammatory state in rat.</a> Front Neurol. 9, 757 (2018).</li><li>Li, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retro-orbital+injection+of+fitc-dextran+is+an+effective+and+economical+method+for+observing+mouse+retinal+vessels.">Retro-orbital injection of fitc-dextran is an effective and economical method for observing mouse retinal vessels.</a> Mol Vis. 17, 3566-3573 (2011).</li><li>Amer, M. H., White, L. J., Shakesheff, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+injection+using+narrow-bore+needles+on+mammalian+cells:+Administration+and+formulation+considerations+for+cell+therapies.">The effect of injection using narrow-bore needles on mammalian cells: Administration and formulation considerations for cell therapies.</a> J Pharm Pharmacol. 67 (5), 640-650 (2015).</li></ol>

The authors declare that there is no conflict of interest with respect of the research, authorship, or publication of this protocol.

This protocol provides a clear and precise method for the injection of substances into the retroorbital sinus of neonatal mice and rats. This is important because it shows that retroorbital injections can be performed reliably and reproducibly in rodents older than P2, where the superficial temporal/facial vein is no longer discernible, and in animals younger than P12, where the eye lids have not yet opened, and the eyeball is not exposed. Furthermore, the neonatal retroorbital injection is well tolerated by both the pups and the dams, with minimal risks of side effects once the technique has been mastered.
Injections via iv have an advantage over other routes of administration as they allow for injection of high concentration, as well as both low and high pH, providing that the rate of injection is kept constant and low to avoid rupture of the vessel. Furthermore, iv injections allow for faster distribution of compounds as they enter directly into the systemic circulation thus bypassing potential delays from poor absorption observed in other routes of administration. This allows for immediate access and nearly 100% bioavailability of compounds.
Clinically iv is the preferred route of administration in neonates (&lt; 28 days of age). This is especially true in neonatal intensive care settings as iv cannulation allows for easy access to provide drugs/fluids. Injections via the sc route have been somewhat used in neonates, particularly for the administration of erythropoietin15. However, concerns have been raised, with a study suggesting iv infusion as a superior alternative16. Oral administration is not often a practical option when neonates are in a hospital intensive unit setting. Additionally, compared to adults, neonates have differences in their gastrointestinal tract, including delayed gastric emptying and decreased intestinal motility which can affect drug absorption. Intramuscular injections are difficult to administer as a result of the small muscular mass of neonates3,4.
In rodent research, one of the most widely used methods of iv injections is the tail vein injection. However, this method is inviable when working with neonates. Other iv sites such as the superficial temporal/facial vein6 become invisible at P3. Neonatal transverse sinus has been described in one study and was performed at P0-P1 and, with assistance of a microscope, opening the skin and advancing a capillary needle though the skull into the transverse sinus, allowing 2-4 &#956;l volume injections7. Few studies have documented the use of the external jugular vein at P7 in rats17. However, this is an invasive technique that requires surgical opening of the skin and exposure of the external jugular vein18. In studies in adult rodents, retroorbital administration has been shown to be as effective as tail vein injection5 thus reinforcing the viability and relevancy of the retroorbital route. The retroorbital injection causes minimal distress and once mastered can be performed by a single person with minimal equipment and allows multiple injections (ensuring that eyes are alternated). Previous studies have shown that retroorbital injection has been used to administer adeno-associated virus 9 in mice at P0-P1 or at P14-P2111 or FITC-dextran at P1719 indicating an increasing acceptance of this method.
There are some limitations associated with retroorbital injection in neonates. As with all iv injections, injected volume is limited, and we recommend 5 &#956;L/g for this procedure. Additionally, the retroorbital injection requires full body anesthesia. To minimize complications, it is suggested the use of inhaled anesthesia agents such as isoflurane, as these are quicker in anesthesia induction, have fast metabolism and have a rapid recovery rate. Training is required, preferably using colored dye in terminally anesthetized animals, to avoid potential swelling around the injection site or eye trauma due to incorrect placement of the needle bevel. Due to the small size of these animals, finer needles are required, with small needle gauge. Injection of cells must be performed in single-cell suspension, to avoid blockage of vessels, and to ensure cell viability. Encouragingly, a study by Amer and colleagues has shown that injection of mammalian cells using 30 G syringes still provides reliable cell viability even at high cell density ejection20.
In summary, establishment of a reliable iv route in neonates is of clinical significance, as this is the preferred route of administration in humans. The retroorbital injection can be easily mastered, is reproducible and provides a relevant alternative to other iv injection sites, such as tail and temporal/facial vein which cannot be reliable used throughout the rodent neonatal period. Thus, the neonatal retroorbital injection allows for the delivery of drugs, cells and other compounds at appropriate neonatal ages.

Animal research is an essential step leading to clinical trials, and as such, it is important that animal studies closely mimic procedures and treatments performed in the clinical setting. However, there are several challenges in translating clinical practices to neonatal rodent studies. These include the small size of the neonatal rodent and the gap in neonatal research and knowledge compared to adult research, among others1,2.
Administration of different substances like drugs or cells can be performed via multiple routes, including intraperitoneal (ip), subcutaneous (sc), and intravenous (iv) injections. Injection by iv is the preferential route of administration of compounds in human neonates. In neonates, the iv route of administration is advantageous compared to other routes because it maximizes systemic distribution of drugs and has high bioavailability3,4. A well-maintained iv lines may be used for repeated drug administration. In rodent studies, iv injections must be performed in the tail, facial/temporal veins, or in the retroorbital sinus5. Tail vein injection is routinely used in adult rodents, as it provides two lateral caudal parallel veins to choose from5. However, these veins have a small diameter, which excludes their use in neonates. Most neonatal iv injections have been performed in the superficial facial/temporal vein, as it is visible from postnatal day 0 (P0)-P2 and allows for a relatively large volume administration5. However, this route becomes unreliable around P36 once the animal gains skin coloration, thereby making the superficial facial/temporal vein difficult to see with the unassisted eye. Administration iv via the neonatal transverse sinus has been described in one study7; however, this requires opening the skin above the transverse sinus and injecting AAV9 at P0-P1 with assistance from a microscope.
When investigating a potential treatment or establishing a relevant neonatal injury model, it is important to consider that neonatal rodents can have different organ development timing compared to humans. Our protocol is based on the differences in neonatal central nervous system development between humans and rodents. As an example, the term newborn human brain approximately corresponds to a P7 rat and a P10 mouse brain8. As the distribution of substances injected retroorbitally is similar to that of the other iv sites, with high blood levels rapidly being achieved, we consider it a suitable route. This technique has been well-described by Yardeni and colleagues, who injected compounds into the ophthalmic venous sinus in P1-P2 mice9. In the current protocol, we show a simple and feasible method of performing retroorbital injections in older neonatal rodents which have yet to open their eyes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BD Micro-Fine Demi 0,3 ml 30G (0,30mm)</td>
			<td>BD</td>
			<td>256370</td>
			<td>1 per animal per injection</td>
		</tr>
		<tr>
			<td>Biotin-dextran (BDA) tracer</td>
			<td>ThermoFischer</td>
			<td>D1956</td>
			<td>2.0-2.5 mg per animal</td>
		</tr>
		<tr>
			<td>Fiber optic light source</td>
			<td>Euromex</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HP 062 Heating Plate</td>
			<td>Labotect</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Vetmedic</td>
			<td>Vnr 17 05 79</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptan blue solution (0.4%)</td>
			<td>Sigma</td>
			<td>T8154</td>
			<td>5 &#956;l/ g body weight</td>
		</tr>
	</tbody>
</table>

a neonatal rodent model of retroorbital vein injection

Intravenous (iv) injection is the most used route of drug administration in neonates in the clinical setting. Therefore, retroorbital vein injection is an important method for compound administration in research, where successful proof-of-concept studies can progress into much-needed neonatal clinical trials. Most intravenous studies in neonatal rodents use the superficial temporal/facial vein. However, retroorbital injection becomes unreliable in neonatal rodents older than 2 days after the skin darkens and the vein is no longer visible. In the present protocol, we describe the retroorbital injection of the venous sinus in both the neonatal mouse and rat at ages when the superficial temporal vein is no longer visible, but the eyes have not opened yet. Eye-opening facilitates retro-orbital injection by enabling the researcher to clearly see that they are not perforating the eye when inserting the needle. We demonstrate that this technique can be performed in a reliable and reproducible manner without adverse effects. Additionally, we show that it can be used in many studies, such as administering compounds to study neonatal brain injury.

The present technique was performed on a flat surface, with a mouthpiece for global anesthesia (Figure 1A). The mouthpiece should not block access to the medial canthus (Figure 1B). In albino animals, a fiber optic light source was placed below the animal, to assist with the visualization of the veins (Figure 1B). The needle was placed at an angle of approximately 40&#176;, and advanced around 2 mm into the medial canthus (Figure 1C). Injection of trypan blue dye in a P5 albino rat allowed clear visualization of the dye in the retroorbital sinus (Figure 1C).
The retroorbital injection technique described in this protocol was successfully used to administer the tracer biotin-dextran (BDA, 10,000 Da)12. The use of visible tracers in vascular research can, for example, provide an alternative to using radioactive sucrose extravasation from the blood vessels, enabling the use of the same brains for other histological measurements12.
Recently, we have established a neonatal rat model of germinal matrix hemorrhage (GMH)13. In brief, P5 Wistar rats received a single intracranial injection of 0.3 U of collagenase VII into the medial striatum. GMH results in rupture of the vessels in the germinal matrix and is one of the prevalent causes of preterm brain injury and mortality14. To further characterize the GMH model, we used a retroorbital injection of the BDA tracer (Figure 2) to investigate the effects of GMH in blood-brain barrier function and integrity14.
When compared to saline-injected controls (Figure 2A), successful retroorbital injection of the BDA tracer14 allowed assessment of the tracer presence in the brain vasculature 10 min after BDA injection (Figure 2B). This technique was then used to detect penumbra vascular leakage of BDA at the individual blood vessel level in GMH-injured animals (Figure 2C, red arrows) which can then be quantified10.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65386/65386fig01.jpg" /> 
Figure 1: Experimental setup with diagram view of the blood vessels after trypan blue dye administration.&#160;(A) Anesthesia setup (B) without and (C) with fiber optic light source. (D) Retroorbital injection of trypan blue dye in the P10 C5BL/6 mouse. (E) Diagram view of the blood vessels in the P5 Wistar rat following trypan blue dye injection. <a href="https://www.jove.com/files/ftp_upload/65386/65386fig01large.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/65386/65386fig02.jpg" /> 
Figure 2: Representative brain micrographs showing the distribution of the BDA tracer. (A) No positive stain in saline-injected control animals. (B) BDA tracer dissolved in saline, at a dose concentration of 2.0 - 2.5 mg per animal was visible within blood vessels of the brain (cortex). (C) BDA tracer leaking into the brain parenchyma following GMH (red arrows). Scale bar = 200 &#956;m. Adapted from Andersson et al., 202114. <a href="https://www.jove.com/files/ftp_upload/65386/65386fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Neonatal Rodent Model of Retroorbital Vein Injection at JoVE.com

A Neonatal Rodent Model of Retroorbital Vein Injection

This protocol aims to demonstrate a reproducible venous administration route that can be used in rats and mice throughout the neonatal period. This procedure is important for preclinical rodent studies that wish to mirror drug administration in neonatal care units primarily using intravenous administration.

Intravenous (iv) injection is the most used route of drug administration in neonates in the clinical setting. Therefore, retroorbital vein injection is an ...

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4.8K Views.  University of Gothenburg. This protocol aims to demonstrate a reproducible venous administration route that can be used in rats and mice throughout the neonatal period. This procedure is important for preclinical rodent studies that wish to mirror drug administration in neonatal care units primarily using intravenous administration.

Video: A Neonatal Rodent Model of Retroorbital Vein Injection

Neonatal Subventricular Zone Electroporation

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal cells1. Understanding the molecular pathways responsible for NSC self-renewal, commitment, and differentiation is critical for harnessing their unique potential to repair the brain and better understand central nervous system disorders. Previous methods for the manipulation of mammalian systems required the time consuming and expensive endeavor of genetic engineering at the whole animal level2. Thus, the vast majority of studies have explored the functions of NSC molecules in vitro or in invertebrates.
Here, we demonstrate the simple and rapid technique to manipulate neonatal NPCs that is referred to as neonatal subventricular zone (SVZ) electroporation. Similar techniques were developed a decade ago to study embryonic NSCs and have aided studies on cortical development3,4 . More recently this was applied to study the postnatal rodent forebrain5-7. This technique results in robust labeling of SVZ NSCs and their progeny. Thus, postnatal SVZ electroporation provides a cost and time effective alternative for mammalian NSC genetic engineering.

We demonstrate a minimally invasive technique referred to as neonatal subventricular zone electroporation. The technique consists of injecting plasmid DNA into the lateral ventricles of neonatal pups and applying electrical current to deliver and genetically manipulate neural stem cells

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal ...

A Lateralized Odor Learning Model in Neonatal Rats for Dissecting Neural Circuitry Underpinning Memory Formation

Rat pups during a critical postnatal period (&#8804; 10 days) readily form a preference for an odor that is associated with stimuli mimicking maternal care. Such a preference memory can last from hours, to days, even life-long, depending on training parameters. Early odor preference learning provides us with a model in which the critical changes for a natural form of learning occur in the olfactory circuitry. An additional feature that makes it a powerful tool for the analysis of memory processes is that early odor preference learning can be lateralized via single naris occlusion within the critical period. This is due to the lack of mature anterior commissural connections of the olfactory hemispheres at this early age. This work outlines behavioral protocols for lateralized odor learning using nose plugs. Acute, reversible naris occlusion minimizes tissue and neuronal damages associated with long-term occlusion and more aggressive methods such as cauterization. The lateralized odor learning model permits within-animal comparison, therefore greatly reducing variance compared to between-animal designs. This method has been used successfully to probe the circuit changes in the olfactory system produced by training. Future directions include exploring molecular underpinnings of odor memory using this lateralized learning model; and correlating physiological change with memory strength and durations.

This protocol introduces lateralized early odor preference learning in rats using acute single naris occlusion. Lateralized learning permits the examination of behavioral outcomes and underpinning biological mechanisms within the same animals, reducing variance induced by between-animal designs. This protocol can be used to investigate molecular mechanisms underpinning early odor learning.

Rat pups during a critical postnatal period (&#8804; 10 days) readily form a preference for an odor that is associated with stimuli mimicking maternal ...

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.

Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

Intracerebroventricular and Intravascular Injection of Viral Particles and Fluorescent Microbeads into the Neonatal Brain

In the study on the pathogenesis of viral encephalitis, the infection method is critical. The first of the two main infectious routes to the brain is the hematogenous route, which involves infection of the endothelial cells and pericytes of the brain. The second is the intracerebroventricular (ICV) route. Once within the central nervous system (CNS), viruses may spread to the subarachnoid space, meninges, and choroid plexus via the cerebrospinal fluid. In experimental models, the earliest stages of CNS viral distribution are not well characterized, and it is unclear whether only certain cells are initially infected. Here, we have analyzed the distribution of cytomegalovirus (CMV) particles during the acute phase of infection, termed primary viremia, following ICV or intravascular (IV) injection into the neonatal mouse brain. In the ICV injection model, 5 &#181;l of murine CMV (MCMV) or fluorescent microbeads were injected into the lateral ventricle at the midpoint between the ear and eye using a 10-&#181;l syringe with a 27 G needle. In the IV injection model, a 1-ml syringe with a 35 G needle was used. A transilluminator was used to visualize the superficial temporal (facial) vein of the neonatal mouse. We infused 50 &#181;l of MCMV or fluorescent microbeads into the superficial temporal vein. Brains were harvested at different time points post-injection. MCMV genomes were detected using the in situ hybridization method. Fluorescent microbeads or green fluorescent protein expressing recombinant MCMV particles were observed by fluorescent microscopy. These techniques can be applied to many other pathogens to investigate the pathogenesis of encephalitis.

Here, we describe a simple method of intracerebroventricular and intravascular injection of viral particles or fluorescent microbeads into the neonatal mouse brain. The localization pattern of the virus and nanoparticles could be detected by microscopic evaluation or by in situ hybridization.

In the study on the pathogenesis of viral encephalitis, the infection method is critical. The first of the two main infectious routes to the brain is the ...

Neurobehavioral Assessments in a Mouse Model of Neonatal Hypoxic-ischemic Brain Injury

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI brain injury by studying neurobehavioral functions in these mice compared to non-operated (i.e., normal) mice. During the study, Rice-Vannucci&#39;s method was used to induce neonatal HI brain damage in postnatal day 7-10 (P7-10) mice. The HI operation was performed on the pups by unilateral carotid artery ligation and exposure to hypoxia (8% O2 and 92% N2 for 90 min). One week after the operation, the damaged brains were evaluated with the naked eye through the semi-transparent skull and were categorized into subgroups based on the absence (&#34;no cortical injury&#34; group) or presence (&#34;cortical injury&#34; group) of cortical injury, such as a lesion in the right hemisphere. On week 6, the following neurobehavioral tests were performed to evaluate the cognitive and motor functions: passive avoidance task (PAT), ladder walking test, and grip strength test. These behavioral tests are helpful in determining the effects of neonatal HI brain injury and are used in other mouse models of neurodegenerative diseases. In this study, neonatal HI brain injury mice showed motor deficits that corresponded to right hemisphere damage. The behavioral test results are relevant to the deficits observed in human neonatal HI patients, such as cerebral palsy or neonatal stroke patients. In this study, a mouse model of neonatal HI brain injury was established and showed different degrees of motor deficits and cognitive impairment compared to non-operated mice. This work provides basic information on the HI mouse model. MRI images demonstrate the different phenotypes, separated according to the severity of brain damage by motor and cognitive tests.

We performed unilateral carotid artery occlusion on postnatal day 7-10 CD-1 mouse pups to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of HI brain injury. We studied neurobehavioral functions in these mice compared to non-operated normal mice.

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI ...

Establishing Mouse Models for Zika Virus-induced Neurological Disorders Using Intracerebral Injection Strategies: Embryonic, Neonatal, and Adult

The Zika virus (ZIKV) is a flavivirus currently endemic in North, Central, and South America. It is now established that the ZIKV can cause microcephaly and additional brain abnormalities. However, the mechanism underlying the pathogenesis of ZIKV in the developing brain remains unclear. Intracerebral surgical methods are frequently used in neuroscience research to address questions about both normal and abnormal brain development and brain function. This protocol utilizes classical surgical techniques and describes methods that allow one to model ZIKV-associated human neurological disease in the mouse nervous system. While direct brain inoculation does not model the normal mode of virus transmission, the method allows investigators to ask targeted questions concerning the consequence after ZIKV infection of the developing brain. This protocol describes embryonic, neonatal, and adult stages of intraventricular inoculation of ZIKV. Once mastered, this method can become a straightforward and reproducible technique that only takes a few hours to perform.

Here we describe a method for establishing a model of Zika virus-induced microcephaly in mouse. This protocol includes methods for embryonic, neonatal, and adult-stage intracerebral inoculation of the Zika virus.

The Zika virus (ZIKV) is a flavivirus currently endemic in North, Central, and South America. It is now established that the ZIKV can cause microcephaly ...

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral vascular effects. Impact (i.e., non-blast) traumatic brain injury (TBI) is known to decrease pressure autoregulation in the cerebral vasculature in both humans and experimental animals. The hypothesis that blast-induced traumatic brain injury (bTBI), like impact TBI, results in impaired cerebral vascular reactivity was tested by measuring myogenic dilatory responses to reduced intravascular pressure in rodent middle cerebral arterial (MCA) segments from rats subjected to mild bTBI using an Advanced Blast Simulator (ABS) shock tube. Adult, male Sprague-Dawley rats were anesthetized, intubated, ventilated and prepared for Sham bTBI (identical manipulation and anesthesia except for blast injury) or mild bTBI. Rats were randomly assigned to receive Sham bTBI or mild bTBI followed by sacrifice 30 or 60 min post-injury. Immediately after bTBI, righting reflex (RR) suppression times were assessed, euthanasia at the time points post-injury was completed, the brain was harvested and the individual MCA segments were collected, mounted and pressurized. As the intraluminal pressure perfused through the arterial segments was reduced in 20 mmHg increments from 100 to 20 mmHg, MCA diameters were measured and recorded. With decreasing intraluminal pressure, MCA diameters steadily increased significantly above baseline in the Sham bTBI groups while MCA dilator responses were significantly reduced (p &#60; 0.05) in both bTBI groups as evidenced by the impaired, smaller MCA diameters recorded for the bTBI groups. In addition, RR suppression in the bTBI groups was significantly (p &#60; 0.05) higher than in the Sham bTBI groups. MCA&#39;s collected from the Sham bTBI groups exhibited typical vasodilatory properties to decreases in intraluminal pressure while MCA&#39;s collected following bTBI exhibited significantly impaired myogenic vasodilatory responses to reduced pressure that persisted for at least 60 min after bTBI.

Here, we present a protocol to describe methods for ex vivo vascular reactivity determination following a primary blast traumatic brain injury (bTBI) using isolated, pressurized, rodent middle cerebral arterial (MCA) segments. bTBI induction is accomplished using a shock tube, also known as an Advanced Blast Simulator (ABS) device.

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral ...

Preparation of Rhythmically-active In Vitro Neonatal Rodent Brainstem-spinal Cord and Thin Slice

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a signal driving the rhythmic contraction of inspiratory muscles. Rhythmic neural activity generated in the pBC and carried to other neuronal pools to drive the musculature of breathing may be studied using various approaches, including en bloc nerve recordings and transverse slice recordings. However, previously published methods have not extensively described the brainstem-spinal cord dissection process in a transparent and reproducible manner for future studies. Here, we present a comprehensive overview of a method used to reproducibly cut rhythmically-active brainstem slices containing the necessary and sufficient neuronal circuitry for generating and transmitting inspiratory drive. This work builds upon previous brainstem-spinal cord electrophysiology protocols to enhance the likelihood of reliably obtaining viable and rhythmically-active slices for recording neuronal output from the pBC, hypoglossal premotor neurons (XII pMN), and hypoglossal motor neurons (XII MN). The work presented expands upon previous published methods by providing detailed, step-by-step illustrations of the dissection, from whole rat pup, to in vitro slice containing the XII rootlets.

This protocol both visually communicates the brainstem-spinal cord preparation and clarifies the preparation of brainstem transverse slices in a comprehensive step-by-step manner. It was designed to increase reproducibility and enhance the likelihood of obtaining viable, long lasting, rhythmically-active slices for recording neural output from the respiratory regions of the brainstem.

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a ...

Double Direct Injection of Blood into the Cisterna Magna as a Model of Subarachnoid Hemorrhage

Among strokes, subarachnoid hemorrhage (SAH) consecutive to the rupture of a cerebral arterial aneurysm represents 5-9% but is responsible for about 30% of the total stroke-related mortality with an important morbidity in terms of neurological outcome. A delayed cerebral vasospasm (CVS) may occur most often in association with a delayed cerebral ischemia. Different animal models of SAH are now being used including endovascular perforation and direct injection of blood into the cisterna magna or even the prechiasmatic cistern, each exhibiting distinct advantages and disadvantages. In this article, a standardized mouse model of SAH by double direct injection of determined volumes of autologous whole blood into the cisterna magna is presented. Briefly, mice were weighed and then anesthetized by isoflurane inhalation. Then, the animal was placed in a reclining position on a heated blanket maintaining a rectal temperature of 37 &#176;C and positioned in a stereotactic frame with a cervical bend of about 30&#176;. Once in place, the tip of an elongated glass micropipette filled with the homologous arterial blood taken from carotid artery of another mouse of the same age and gender (C57Bl/6J) was positioned at a right angle in contact with the atlanto-occipital membrane by means of a micromanipulator. Then 60 &#181;L of blood was injected in the cisterna magna followed by a 30&#176; downward tilt of the animal for 2 minutes. The second infusion of 30 &#181;L of blood into the cisterna magna was performed 24 h after the first one. The individual follow-up of each animal is carried out daily (careful evaluation of weight and well-being). This procedure allows a predictable and highly reproducible distribution of blood, likely accompanied by intracranial pressure elevation that can be mimicked by an equivalent injection of an artificial cerebral spinal fluid (CSF), and represents an acute to mild-model of SAH inducing low mortality.

We described in this protocol a standardized subarachnoid hemorrhage (SAH) mouse model by a double injection of autologous whole-blood into the cisterna magna. The high degree of standardization of the double-injection procedure represents a middle-to-acute model of SAH with relative safety regarding mortality.

Among strokes, subarachnoid hemorrhage (SAH) consecutive to the rupture of a cerebral arterial aneurysm represents 5-9% but is responsible for about 30% ...

Optimization of the Retinal Vein Occlusion Mouse Model to Limit Variability

Mouse models of retinal vein occlusion (RVO) are often used in ophthalmology to study hypoxic-ischemic injury in the neural retina. In this report, a detailed method pointing out critical steps is provided with recommendations for optimization to achieve consistently successful occlusion rates across different genetically modified mouse strains. The RVO mouse model consists primarily of the intravenous administration of a photosensitizer dye followed by laser photocoagulation using a retinal imaging microscope attached to an ophthalmic guided laser. Three variables were identified as determinants of occlusion consistency. By adjusting the wait time after rose bengal administration and balancing the baseline and experimental laser output, the variability across experiments can be limited and a higher success rate of occlusions achieved. This method can be used to study retinal diseases that are characterized by retinal edema and hypoxic-ischemic injury. Additionally, as this model induces vascular injury, it can also be applied to study the neurovasculature, neuronal death, and inflammation.

Here, we describe an optimized protocol for retinal vein occlusion using rose bengal and a laser-guided retinal imaging microscope system with recommendations to maximize its reproducibility in genetically modified strains.

Mouse models of retinal vein occlusion (RVO) are often used in ophthalmology to study hypoxic-ischemic injury in the neural retina. In this report, a ...