We would like to thank Dr. Kellie Breen Church, Mr. Michael Kreisman, and Ms. Jessica Jang for their contributions to collecting the data shown in the representative results. This work was supported by the National Institutes of Health (NIH) R00 HD104994 (R.B.M.).

All the procedures were approved by the Colorado State University (#3960) and University of California San Diego Institutional Animal Care and Use Committees, where the representative data were collected (S13235, PI Kellie Breen Church). Data from five adult female and two adult male C57/BL6 mice (9-16 weeks old) are depicted in the representative data section. Female mice were ovariectomized 3-4 weeks prior to ICV injection and blood collection as described previously7. Prior to experimentation, ...

Performing Intracerebroventricular Injection in Mice Using a Free-Hand Approach

<ol>
	<li>Roseweir, A. 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=Discovery+of+potent+kisspeptin+antagonists+delineate+physiological+mechanisms+of+gonadotropin+regulation.">Discovery of potent kisspeptin antagonists delineate physiological mechanisms of gonadotropin regulation.</a> Journal of Neuroscience. 29 (12), 3920-3929 (2009).</li><li>Kim, J. Y., Grunke, S. D., Levites, Y., Golde, T. E., Jankowsky, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracerebroventricular+viral+injection+of+the+neonatal+mouse+brain+for+persistent+and+widespread+neuronal+transduction.">Intracerebroventricular viral injection of the neonatal mouse brain for persistent and widespread neuronal transduction.</a> Journal of Visualized Experiments. (91), e51863 (2014).</li><li>Taylor, Z. V., Khand, B., Porgador, A., Monsonego, A., Eremenko, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+intracerebroventricular+injection+of+CD4(+)+T+cells+into+mice.">An optimized intracerebroventricular injection of CD4(+) T cells into mice.</a> STAR Protocols. 2 (3), 100725 (2021).</li><li>Russo, K. 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=Circadian+control+of+the+female+reproductive+axis+through+gated+responsiveness+of+the+RFRP-3+system+to+VIP+signaling.">Circadian control of the female reproductive axis through gated responsiveness of the RFRP-3 system to VIP signaling.</a> Endocrinology. 156 (7), 2608-2618 (2015).</li><li>Laursen, S. E., Belknap, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracerebroventricular+injections+in+mice.+Some+methodological+refinements.">Intracerebroventricular injections in mice. Some methodological refinements.</a> Journal of Pharmacological Methods. 16 (4), 355-357 (1986).</li><li>Haley, T. J., McCormick, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+effects+produced+by+intracerebral+injection+of+drugs+in+the+conscious+mouse.">Pharmacological effects produced by intracerebral injection of drugs in the conscious mouse.</a> British Journal of Pharmacology and Chemotherapy. 12 (1), 12-15 (1957).</li><li>McCosh, R. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin-induced+hypoglycaemia+suppresses+pulsatile+luteinising+hormone+secretion+and+arcuate+Kiss1+cell+activation+in+female+mice.">Insulin-induced hypoglycaemia suppresses pulsatile luteinising hormone secretion and arcuate Kiss1 cell activation in female mice.</a> Journal of Neuroendocrinology. 31 (12), e12813 (2019).</li><li>Wu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcardiac+perfusion+of+the+mouse+for+brain+tissue+dissection+and+fixation.">Transcardiac perfusion of the mouse for brain tissue dissection and fixation.</a> Bio-Protocol. 11 (5), e3988 (2021).</li><li>Comba, 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=Laser+capture+microdissection+of+glioma+subregions+for+spatial+and+molecular+characterization+of+intratumoral+heterogeneity,+oncostreams,+and+invasion.">Laser capture microdissection of glioma subregions for spatial and molecular characterization of intratumoral heterogeneity, oncostreams, and invasion.</a> Journal of Visual Experiments. 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Here, a simple and effective means for making ICV injections in mice is described. Since this technique does not require a stereotaxic frame, this approach for the central delivery of drugs and experimental agents is accessible to more researchers. Additionally, this approach is relatively high throughput since the preparation and injection procedure can be performed quickly.
Since this procedure requires the manipulation of needles and a glass syringe by hand using approximate distances, it i...

The delivery of experimental agents, such as drugs1, viruses2, or cells3, to the brain is often necessary for neuroendocrine research. If the agent does not readily cross the blood-brain barrier or the experimental objective is to specifically test the central effects of the agent, it is important to have a reliable method for delivering injections into the brain. Moreover, injection into the intracerebroventricular (ICV) space provides the opportunity to distribute the agent widely in the brain and provides a large target area, thus increasing the likelihood of successful injection2. 
A common method for making ICV injections involves the placement of a permanent indwelling cannula. In this approach, a stereotaxic frame is necessary to position the commercially available or custom-made cannula, as the cannula is glued or cemented in place. Often, upon recovery, a supraphysiological dose of angiotensin II is administered through the cannula, and if drinking behavior is immediately observed, then the cannula is considered correctly placed4. This approach has many advantages, including the ability to perform long-term infusion and the ability to inject the same animal multiple times; additionally, if angiotensin II is employed, correct placement can be confirmed prior to the administration of experimental compounds. However, there are some limitations to placing a permanent cannula, including the requirement for expensive equipment (stereotaxic frame), the possibility of damage to the cannula after placement (e.g., mice can chew on the cannula of a cage mate), and the possibility of infections around the permanent cannula. Single ICV injections can be made with the use of a stereotaxic frame3, which, although effective, requires substantial exposure to anesthesia and, thus, may obscure some acute physiological and behavioral effects of the treatment. Additionally, the placement of mice in a stereotaxic frame requires substantial training to achieve stable placement and prevent the rupturing of the ear canals.
Here, an established method for making free-hand injections in mice is described. This method is based on previous reports5,6. The advantages of this technique are that it is simple, rapid, and does not require specialized equipment such as a stereotaxic frame. As described below, this procedure involves manipulating a glass syringe relative to landmarks on the mouse head to make the injections, which can be done rapidly and, thus, requires only a few minutes of gas anesthesia on the experimental day.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>18-gauge blunt needles</td>
			<td>SAI Infusion</td>
			<td>B18-150</td>
			<td></td>
		</tr>
		<tr>
			<td>18-gauge needles</td>
			<td>BD Medical</td>
			<td>305195</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol pads</td>
			<td>Fisher Scientific</td>
			<td>22-363-750</td>
			<td></td>
		</tr>
		<tr>
			<td>Bench pad</td>
			<td>Fisher Scientific</td>
			<td>14-206-62AC22</td>
			<td></td>
		</tr>
		<tr>
			<td>Betadine solution</td>
			<td>Fisher Scientific</td>
			<td>NC1696484</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>Patterson Vet Supply</td>
			<td>07-892-5235</td>
			<td>Controlled substance</td>
		</tr>
		<tr>
			<td>Eyelube</td>
			<td>Fisher Scientific</td>
			<td>50-218-8442</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass syringe</td>
			<td>Hamilton</td>
			<td>7634-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Injection needle</td>
			<td>Hamilton</td>
			<td>7803-01</td>
			<td>27 gauge, Small Hub RN needle, point style: 4, Needle length: 10cm, Angle: 45</td>
		</tr>
		<tr>
			<td>Isoflurane&#160;&#160;</td>
			<td>Patterson Vet Supply</td>
			<td>07-893-8441</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane vaporizer</td>
			<td>Vet Equip</td>
			<td>V-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory Tape</td>
			<td>VWR</td>
			<td>89098-128</td>
			<td></td>
		</tr>
		<tr>
			<td>Medical grade oxygen</td>
			<td>Airgas</td>
			<td>OX USPEA</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Millipore-Sigma</td>
			<td>8.18715.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Fisher Scientific</td>
			<td>J67802.K2</td>
			<td></td>
		</tr>
		<tr>
			<td>PulsaR Software</td>
			<td>Open source, University of Otago</td>
			<td></td>
			<td>See ref 9</td>
		</tr>
		<tr>
			<td>Ruler</td>
			<td>Fisher Scientific</td>
			<td>12-00-152</td>
			<td></td>
		</tr>
		<tr>
			<td>Silastic tubing (0.040&#34; I.D.)</td>
			<td>DOW</td>
			<td>508-005</td>
			<td></td>
		</tr>
		<tr>
			<td>Silastic tubing (0.078&#34; I.D.)</td>
			<td>DOW</td>
			<td>508-009</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile saline</td>
			<td>VWR</td>
			<td>101320-574</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose&#160;</td>
			<td>Fisher Scientific</td>
			<td>S5-500</td>
			<td></td>
		</tr>
	</tbody>
</table>

free-hand intracerebroventricular injections in mice

The investigation of neuroendocrine systems often requires the delivery of drugs, viruses, or other experimental agents directly into the brains of mice. An intracerebroventricular (ICV) injection allows the widespread delivery of the experimental agent throughout the brain (particularly in the structures near the ventricles). Here, methods for making free-hand ICV injections in adult mice are described. By using visual and tactile landmarks on the heads of mice, injections into the lateral ventricles can be made rapidly and reliably. The injections are made with a glass syringe held in the experimenter&#39;s hand and placed at approximate distances from the landmarks. Thus, this technique does not require a stereotaxic frame. Furthermore, this technique requires only brief isoflurane anesthesia, which permits the subsequent assessment of mouse behavior and/or physiology in awake, freely behaving mice. Free-hand ICV injection is a powerful tool for the efficient delivery of experimental agents into the brains of living mice and can be combined with other techniques such as frequent blood sampling, neural circuit manipulation, or in vivo recording to investigate neuroendocrine processes.

Author Spotlight: Insights, Innovations, and Future Avenues in Reproductive Neurocontrol 

Free-Hand Intracerebroventricular Injections in Mice

We are interested in the neural control of reproductive function, particularly how the brain regulates the release of hormones that control the gonads, both in healthy conditions and in response to stress. The main advantage of this technique is that it is relatively simple and rapid. Delivering drugs into the lateral ventricle is a great way to cast a wide net in testing a signaling pathway to support more detailed follow-up experiments.There's still much to be learned about the neuroendocrine control of reproduction, including the molecular mechanisms for luteinizing hormone pulse generation, mechanisms for modulating pulse frequency during stress, and generation of the preovulatory LH surge. In the future, we are particularly interested in integrating disparate findings into models.

When performed successfully, this technique allows the rapid delivery of an experimental agent into the ventricular system. A luteinizing hormone (LH) pulse profile from an ovariectomized mouse that received an ICV injection of 3 &#181;L of sterile isotonic saline, the vehicle for many pharmacological compounds, is shown in Figure 2A. This example demonstrates that brief exposure to gas anesthesia and the injection of 3 &#181;L of fluid into the ventricular system alone did not alter the pul...

Watch this Scientific Journal Video about Insights, Innovations, and Future Avenues in Reproductive Neurocontrol at JoVE.com

Here, a simple and rapid approach for performing intracerebroventricular injections in mice using a free-hand approach (that is, without a stereotaxic device) is described.

The investigation of neuroendocrine systems often requires the delivery of drugs, viruses, or other experimental agents directly into the brains of mice. ...

free-hand-intracerebroventricular-injections-in-mice

Research

JoVE Journal

Neuroscience

5.6K ビュー.  Colorado State University. 私たちは、生殖機能の神経制御、特に脳が生殖腺を制御するホルモンの放出を、健康な状態とストレスへの応答の両方でどのように調節するかに興味を持っています。この手法の主な利点は、比較的単純で高速であることです。側脳室への薬物送達は、シグナル伝達経路の検査に広く網を張る優れた方法であり、より詳細な追跡実験をサポートします。神経内分泌?...

ビデオ: 著者スポットライト:生殖神経制御における洞察、イノベーション、および将来の道筋 

Here, a simple and rapid approach for performing intracerebroventricular injections in mice using a free-hand approach (that is, without a stereotaxic device) is...

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combining a breath-synchronized olfactometer with brain simulation to study the impact of odors on corticospinal excitability and effective connectivity

Author Spotlight: Exploring Olfactory Influences on Corticospinal Excitability - Insights and Innovations in Neurological Research 

This paper describes using a breath-synchronized olfactometer to trigger single- and dual-coil transcranial magnetic stimulation (TMS) during odorant presentation synchronized to human nasal breathing. This combination allows us to objectively investigate how pleasant and unpleasant odors impact corticospinal excitability and brain-effective connectivity in a given...

Effect of Odor on Brain Excitability Assessed by a Breath-Synchronized Olfactometer

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targeted knockdown of genes in the choroid plexus

Author Spotlight: Insights and Innovations in Gene Expression Manipulation Techniques for Choroid Plexus Research 

Here, we describe a method to selectively alter gene expressions in the choroid plexus while avoiding any impact in other brain...

Selective Gene Silencing in the Choroid Plexus

freezing injury in mouse masseter muscle to establish an orofacial muscle fibrosis model

Author Spotlight: Exploring Orofacial Muscle Regeneration &amp;#8211; Insights and Innovations

The goal of this protocol is to establish an orofacial muscle fibrosis model. Comparison of the histology between mice masseter and tibialis anterior muscle after freezing injury confirmed masseter muscle fibrosis. This model will facilitate further investigation into the mechanism underlying orofacial muscle...

Establishing an Orofacial Muscle Fibrosis Model in Mice via a Freezing Injury

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using motion capture technology in the instrumented timed up and go test to detect the risk of falling in aged adults

Author Spotlight: Innovations in iTUG Test for Enhanced Risk Assessment and Cognitive Insights

The instrumented Timed Up and Go (iTUG) test is gaining increasing attention in body sway and gait analysis with the development of new technologies. We present a protocol to analyze the subcomponents of the iTUG with motion...

Evaluating the Risk of Falling in Elderly Using iTUG Motion Capture Technology

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a modified transcranial middle cerebral artery occlusion model to study stroke outcomes in aged mice

Author Spotlight: Innovative Techniques and Future Directions in Stroke Research 

This protocol demonstrates a unique mouse stroke model with a medium-sized infarct and an excellent survival rate. This model allows preclinical stroke researchers to extend the ischemia duration, use aged mice, and assess long-term functional...

Establishing a Transcranial Middle Cerebral Artery Occlusion Mice Model

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swin-psaxialnet: an efficient multi-organ segmentation technique

Author Spotlight: Advancing Medical AI by Innovations in Deep Learning and Multimodal Algorithms

The present protocol describes an efficient multi-organ segmentation method called Swin-PSAxialNet, which has achieved excellent accuracy compared to previous segmentation methods. The key steps of this procedure include dataset collection, environment configuration, data preprocessing, model training and comparison, and ablation...

Swin-PSAxialNet: A Streamlined Approach for Segmenting Multiple Organs

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a rat dry eye model with lacrimal gland dysfunction induced by scopolamine

Author Spotlight: Challenges in Developing Dry Eye Animal Models and Future Research Directions

Here, we establish a rat model of lacrimal gland dysfunction to provide a basis for the study of aqueous-deficient dry...

Scopolamine-Induced Lacrimal Gland Dysfunction in Rats

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arteriovenous metabolomics to measure in vivo metabolite exchange in brown adipose tissue

Author Spotlight: Illuminating New Avenues for Adipose Tissue Metabolism and Disease Prevention 

In this protocol, methods relevant for BAT-optimized arteriovenous metabolomics using GC-MS in a mouse model are outlined. These methods allow for the acquisition of valuable insights into BAT-mediated metabolite exchange at the organismal...

Exploring Metabolite Exchange in Brown Adipose Tissue

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a mouse model for corneal neovascularization by alkali burn

Author Spotlight: Decoding Corneal Neovascularization with Alkali Burn Model for Future Therapeutic Strategies 

This protocol focuses on alkali burn-induced corneal neovascularization in mice. The method generates a reproducible and controllable corneal disease model to study pathological angiogenesis and the associated molecular mechanisms and to test new pharmacological agents to prevent corneal neovascularization.

Alkali Burn-Induced Corneal Neovascularization in Mice

reproductive techniques for ovarian monitoring and control in amphibians

Reproductive Techniques for Ovarian Monitoring and Control in Amphibians

The study of amphibian biology provides valuable information on the reproductive, physiological, embryological and developmental processes that drive organisms of many taxonomic groups. Here, we present a comprehensive guide on different methodologies that can be used to study ovarian control and monitoring in...

a seamless cloning approach for porcine reproductive and respiratory syndrome virus expression vector construction

Author Spotlight: Exploring Cloning Techniques for Full-Length DNA Fragments

Here, we present a protocol to obtain the pVAX1-PRRSV expression vector by introducing suitable restriction sites at the 3' end of the inserts. We can linearize the vector and join DNA fragments to the vector one by one through homologous recombination technology.

Cloning the Full-Length PRRSV Gene in the pVAX1 Expression Vector

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tracking sugar-elicited local searching behavior in drosophila

Author Spotlight: Exploring Behavioral Pathways Through Cross-Species Insights in Foraging and Communication

This protocol describes a behavioral assay for recording sugar-elicited search behavior using Drosophila melanogaster. The assay can be utilized to study feeding and foraging-related behaviors, as well as the underlying neuronal...

Analysis of Sugar-Induced Foraging Behavior in &lt;i&gt;Drosophila&lt;/i&gt;

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stabilizing a femur osteotomy with a plate fixation in ambystoma mexicanum

Author Spotlight: Exploring Regeneration in Axolotls Through Insights in Cellular and Molecular Mechanisms, Bone Healing, and Implications for Human Therapies

A protocol for femoral osteotomy surgery with the use of internal plate fixation in mature axolotls is presented. The procedure can be used to perform comparative studies on limb regeneration and fracture healing in aquatic...

Using Plate Fixation to Study Bone Healing and Limb Regeneration in Axolotls

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improved hysteroscopic resection of endometrial polyps using 6-fr micro-scissors and forceps

Author Spotlight: A Reproductive Hysteroscopy Approach for Complete Endometrial Polyp Removal and Enhanced Endometrial Receptivity

The novel technique presented here employs a combination of 6-Fr micro-scissors and forceps for hysteroscopic treatment of endometrial polyps, demonstrating encouraging outcomes for infertile patients afflicted with this...

Hysteroscopic Technique with Micro-Scissors and Forceps for Polyp Removal

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ultrasonography in experimental reproductive investigations on rats

Ultrasonography in Experimental Reproductive Investigations on Rats

This manuscript describes the utility of ultrasound performed on female rats to design experimental models for reproductive and gynecological investigation. A step-by-step explanation of how to perform ultrasonographic evaluation is...

high-resolution fluorespirometry to assess dynamic changes in mitochondrial membrane potential in human immune cells

Author Spotlight: New Insights into PBMC Mitochondrial Responses Using Fluorespirometry

Methods for studying mitochondrial bioenergetics under physiologically relevant substrate concentrations in immune cells are limited. We provide a detailed protocol that uses high-resolution fluorespirometry to assess changes in the response of the mitochondrial membrane potential to energy demand in human T-cells, monocytes, and peripheral mononuclear...

High-Resolution Analysis of Mitochondrial Function in Human PBMCs

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mast cells in the microenvironment of hepatocellular carcinoma confer favorable prognosis: a retrospective study using qupath image analysis software

Author Spotlight: Investigating Immune Cell Dynamics in the Tumor Microenvironment &amp;#8212; Challenges and Innovations in Cancer Prognosis

The presence of mast cells in the inner margin and peritumor areas of hepatocellular carcinoma after resection confers a favorable prognosis. This study endorses QuPath image analysis software as a promising platform that could meet the need for reproducibility, consistency, and accuracy in digital pathology.

Prognostic Significance of Immune Cell Infiltration in Hepatocellular Carcinoma

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measurement of healthy and injured triceps surae morphology

Author Spotlight: Exploring the Complexities of Achilles Tendon Injuries &amp;#8212; Research and Future Directions

Ultrasound imaging is becoming more accessible in clinical and research settings, and a consistent protocol will be beneficial for comparison between studies and for clinical interpretations. This protocol for ultrasound evaluation is a valid and reliable method to evaluate Achilles tendon morphology in healthy, tendinopathic, and ruptured tendons.

Ultrasound Evaluation of Achilles Tendon Morphology

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exploring independent effects of follicle-stimulating hormone in vivo in a mouse model

Author Spotlight: Investigating the Relationship Between FSH and Pathophysiological Changes in Perimenopausal Women - Insights from a Mouse Model 

Follicle-stimulating hormone (FSH) in various extragonadal tissues and organs is associated with the pathogenesis of multiple diseases. The ovariectomized and FSH-treated mouse model (OVF) can be used to explore the extragonadal actions of...