The authors thank Ms Lourdes Carreon G Tan for her assistance in the fish husbandry. This work was funded by NMBU, Grants-in-Aid from Japan Society for the Promotion of Science (JSPS) (Grant number 18H04881 and 18K19323), and grant for Basic Science Research Projects from Sumitomo Foundation to S.K.

All experimentations and animal handling were conducted in accordance with the recommendations on the experimental animal welfare at Norwegian University of Life Sciences. Experiments using gonadectomy were approved by the Norwegian Food Safety Authority (FOTS ID 24305).
NOTE: The experiments were performed using adult male and female (6-7 months old, weight ca. 0.35 g, length ca. 2.7 cm) Japanese medaka. The sex was determined by distinguishing the secondary sexual characteristics, such as the size and shape of dorsal and anal fin, as described in42,47.
1. Instruments and solutions preparation
<ol>
	<li>Prepare anesthetic stock solution (0.6% Tricaine).
	<ol>
		<li>Dilute 0.6 g of Tricaine (MS-222) in 100 mL of 10x Phosphate Buffer Saline (PBS).</li>
		<li>Distribute 1 mL of the Tricaine stock solution into several 1.5 mL plastic tubes and store at -20 &#176;C until use.</li>
	</ol>
	</li>
	<li>Prepare recovery water (0.9% NaCl solution) by adding 18 g of NaCl into 2 L of aquarium water. Store the solution at room temperature until use.</li>
	<li>Prepare the incision tools by breaking a razor diagonally to get a sharp point (Figure 1A).</li>
	<li>Prepare blood anti-coagulant solution (0.05 U/&#181;L of sodium heparin) by diluting 25 &#181;L of sodium heparin into 500 &#181;L of 1x PBS. Store the anti-coagulant solution at 4 &#176;C until use.</li>
	<li>Prepare two glass needles from a 90 mm long glass capillary by pulling a glass capillary with a needle puller (Figure 1B) following the instructions of the manufacturer. 
	NOTE: The outer diameter of the glass needle is 1 mm, while the inner diameter is 0.6 mm.</li>
	<li>Prepare a 1.5 mL plastic tube lid by cutting the lid and make a hole that fits with the needle outer diameter (Figure 1C). To make the hole, heat one end of the 9 mm glass capillary and stab the heated glass capillary through the lid. Alternatively, use a needle to stab through the lid until the diameter of the hole fits with the 9-mm glass capillary.</li>
</ol>
2. Gonadectomy procedure
<ol>
	<li>Prepare 0.02% of anesthetic solution by diluting one tube of Tricaine stock (0.6%) in 30 mL of aquarium&#160;water.</li>
	<li>Prepare dissection tools including one ultra-fine and two fine forceps (one with relatively wide tip), small scissors, nylon thread and razor as described in step 1.3.</li>
	<li>Anesthetize the fish by putting it into the 0.02% anesthetic solution for 30-60 seconds. 
	&#8203;NOTE: The duration of the anesthesia depends on the size and weight of the fish&#160;and must be adapted. To ensure that the fish is fully anesthetized, the fish body can be pinched gently using forceps. If the fish does not react, the gonadectomy can be started.</li>
	<li>Take out the fish from the anesthetic solution and place the fish horizontally on its side, out-of-water under a dissection microscope.</li>
	<li>Ovariectomy (OVX) in females
	<ol>
		<li>Remove oviposited eggs (eggs hanging outside the female body) if any and scrape the scales in the incision area (Figure 2A).</li>
		<li>Gently make an incision about 2-2.5 mm long between the ribs, between the pelvic and anal fins (Figure 2A), using the razor blade. Then, pinch gently the fish abdomen while taking out the ovaries little by little using fine forceps with wide tip.</li>
		<li>Cut the end of the ovaries using fine forceps and place the ovaries aside (Figure 2B). 
		NOTE: Take care not to break the ovarian sac if possible. If the ovarian sac is broken, remove any gonad traces as completely as possible without leaving even any non-ovulated eggs.</li>
	</ol>
	</li>
	<li>Orchidectomy in males
	<ol>
		<li>Gently make an incision between the ribs above the anus (Figure 2A), and open up the incision slowly using fine forceps.</li>
		<li>Gently grab the testes using the fine forceps and slowly take out the testes. Afterwards, cut the end of the testes to completely remove the testes (Figure 2B). For male orchidectomy, all preparations are similar to in females until the incision part. When grabbing the testes, sometimes the fat resembling the testes is obtained. However, after restoring the fat, it is possible to try to find the testes again (Figure 2B). 
		NOTE: For both males and females, it is important to minimize the incision size in the abdomen to prevent excessive damage that can lead to mortality. Sometimes the intestines may also appear through the incision along with the gonads, so make sure they are properly returned inside the incision before closure. Prior knowledge on ovaries and testes location in medaka abdomen is essential.</li>
	</ol>
	</li>
	<li>Suture the incision similarly in males and females (Figure 3).
	<ol>
		<li>Place the nylon thread beside the incision area&#160;and stab the skin from the right side of the incision through inner body cavity using ultra-fine forceps to take the thread in with fine forceps (Figure 3; 1-2).</li>
		<li>Stab the skin from the left side of the incision through outer body cavity to take out the thread ( Figure 3; 3-4).</li>
		<li>Close the incision opening and make two knots and cut the excessive thread (Figure 3; 4-6). 
		NOTE: The suture must be adequately tight, and the remaining thread on the fish must be long enough to prevent loosening of the suture. The whole procedure from anesthesia until suturing commonly takes up to 6 minutes. Longer time&#160;may lead to&#160;mortality.</li>
		<li>Put the fish in the recovery water and leave them for at least 24 hours before transferring them to the aquarium system. 
		&#8203;NOTE: Gonadectomized fish usually show normal behavior after 1-2 hours in the recovery water. Therefore, depending on the experiment purpose, one can sample the fish after this time interval.</li>
	</ol>
	</li>
</ol>
3. Blood sampling procedure
<ol>
	<li>Prepare the tools: a glass needle, a silicone capillary, a plastic tube with a hole, an empty 1.5 mL plastic tube, a minicentrifuge and tape.</li>
	<li>Anesthetize the fish using 0.02% anesthetic solution as described in step 2.1 and place the fish under a dissection microscope in a vertical position (Figure 4A). Place the fish on a bright surface to ease visualization of the caudal puncture vein.</li>
	<li>Install the blood drawer by attaching a glass needle to the silicone capillary (Figure 4B). Break the tip of the needle with wide tip forceps and coat the inside of the needle with anti-coagulant solution by suctioning and blowing. 
	NOTE: The use of a sucker and a silicone capillary with at least 50 cm length are recommended for safety measures to avoid any direct contact of the blood when suctioning. In addition, make sure that the opening of the needle tip is sufficiently large to allow drawing the blood.</li>
	<li>Direct the needle toward the peduncle area of the fish, aim at the caudal peduncle vein (Figure 5A) and draw the blood using mouth until at least one fourth the total volume of the needle is filled (Figure 5B). 
	NOTE: It is important to stop suctioning before removing the needle from fish body.</li>
	<li>Release the needle and put a piece of tape at the proximity of the sharp side of the needle. Place the lid with a hole on a collection tube and put the needle inside the tube through the hole with the needle tip on the outside (Figure 5C).</li>
	<li>Put the fish in the recovery water and leave them for at least 24 hours before transferring them to the aquarium system. 
	NOTE: To perform a second blood sampling from the same fish, sample the blood one week after the first blood sampling.</li>
	<li>Flash spin down the collected blood for 1-2 seconds with&#160;1,000 x g at&#160;room temperature to collect the blood in the tube.</li>
	<li>Proceed directly to downstream applications or store the blood at -20 &#176;C until use. 
	NOTE: Refer to the previous study for sex steroid extraction from the whole blood48.</li>
</ol>

The authors have nothing to disclose.

As reported in previous literature, gonadectomy and blood sampling have long been used in other model species to investigate questions related to the role of sex steroids in regulation of the BPG axis. However, these techniques seem to be amenable only for bigger animals. Considering the small size of the commonly used teleost model, Japanese medaka, we provide a detailed protocol for gonadectomy and blood sampling that is feasible for this species.
The fact that the survival rate of gonadecto...

In vertebrates, sex steroids, which are mainly produced by the gonads, play important roles in the regulation of the Brain-Pituitary-Gonadal (BPG) axis through various feedback mechanisms1,2,3,4,5. In addition, sex steroids affect the proliferation and activity of neurons in the brain6,7,8 and endocrine cells, including gonadotropes, in the pituitary9,10, and thus serve crucial roles in brain and pituitary plasticity. Despite relatively good knowledge in mammals, the mechanism of BPG axis regulation mediated by sex steroids is far from being understood in non-mammalian species, leading to poor understanding of evolutionary conserved principles11. There is still a limited number of studies documenting the role of sex steroids on brain and pituitary plasticity, thus raising the need for further investigations of the role and effects of sex steroids on diverse vertebrate species.
Among vertebrates, teleosts have become powerful model animals in addressing numerous biological and physiological questions, including stress response12,13, growth14,15, nutritional physiology16,17 and reproduction2. Teleosts, in which sex steroids are mostly represented by estradiol (E2) in females and 11-ketotestosterone (11-KT) in males18,19, have&#160;long been reliable experimental models for investigating the general principle of reproduction across species. Teleosts show uniqueness in their hypothalamic-pituitary connection20,21 and distinct gonadotrope cells22, which are sometimes convenient for the elucidation of regulatory mechanisms. Moreover, due to their amenability to both laboratory and field experiments, teleosts offer many advantages compared to other organisms. They are relatively inexpensive to purchase and maintain23,24. In particular, small teleost models such as zebrafish (Danio rerio) and the Japanese medaka (Oryzias latipes), are species with very high fecundity and a relatively short life cycle enabling rapid analysis of gene function and disease mechanisms23, thus providing even greater advantages in addressing a plethora of biological and physiological questions, considering the numerous well-developed protocols and genetic toolkit available for these&#160;species25.
In numerous studies, the removal of gonads (gonadectomy) along with blood sampling techniques have been used as a method for investigating many physiological questions, including its impact in vertebrate reproductive physiology in mammals26,27,28, birds29 and amphibians30. Although the gonadectomy effect on reproductive physiology can be alternatively mimicked by sex steroid antagonists, such as tamoxifen and clomiphene, the effect of the drugs appears to be inconsistent due to bimodal effects31,32. Chronic exposure to a sex steroid antagonist may lead to ovarian enlargement33,34, which may disable observation of its effects for long-term purposes due to an unhealthy phenotype. In addition, it is impossible to perform a recovery experiment after sex steroid antagonist treatment, to warrant the specific effect of certain sex steroids. Along with those aforementioned points, other trade-offs of sex steroid antagonist use have been extensively reviewed31,32. Therefore, gonadectomy still appears today as a powerful technique to investigate the role of sex steroids.
While gonadectomy and blood sampling techniques are relatively easy to perform in bigger species, such as European sea bass (Dicentrarchus labrax)35, bluehead wrasse (Thalassoma bifasciatum)36, dogfish (Scyliorhinus canicula)37 and catfish (Heteropneustes fossilis and Clarias bathracus)38,39, they raise challenges when applied in small fish as medaka. For instance, the use of Fish Anesthesia Delivery System (FADS)40 is less feasible and appears to be prone to excessive physical damage for small fish. In addition, a gonadectomy procedure that is commonly used for bigger fish40 is not suitable for small fish that requires high precision to avoid excessive damage. Finally, blood sampling is challenging due to the limited access to blood vessels and the small amount of blood in those animals. Therefore, a clear protocol demonstrating every step of gonadectomy and blood sampling in a small teleost is of importance.
This protocol&#160;demonstrates the step-by-step procedures of gonadectomy followed by blood sampling in Japanese medaka, a small freshwater fish native to East Asia. Japanese medaka have a sequenced genome, several molecular and genetic tools available25, and a genetic sex determination system allowing for investigation of sexual differences before secondary sexual characteristics&#160;or gonads are well developed41. Interestingly, Japanese medaka possess fused gonads contrary to many other teleost species42. These two techniques combined take only 8 minutes in total and will complete the list of video protocols already existing for this species that included labeling of blood vessels43, patch-clamp on pituitary sections44 and brain neurons45, and primary cell culture46. These techniques will allow the research community to investigate and better understand the roles of sex steroids in feedback mechanisms as well as brain and pituitary plasticity in the future.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Glass capilary</td>
			<td></td>
			<td>GD1</td>
			<td>Glass Capillary with Filament GD-1; Narishige</td>
		</tr>
		<tr>
			<td>Heparin sodium salt</td>
			<td></td>
			<td>H4784-1G</td>
			<td>Sigma-aldrich</td>
		</tr>
		<tr>
			<td>Needle puller</td>
			<td></td>
			<td>P97</td>
			<td>Flaming/Brown Micropipette puller Model P-97; Sutter Instrument</td>
		</tr>
		<tr>
			<td>Nylon thread</td>
			<td></td>
			<td>N45VL</td>
			<td>Polyamide suture, 0.2 metric; Crownjun</td>
		</tr>
		<tr>
			<td>Plastic tube</td>
			<td></td>
			<td>T9661</td>
			<td>Eppendorf Safe-lock microcentifuge tube 1.5 ml, Sigma-aldrich</td>
		</tr>
		<tr>
			<td>Razor blade</td>
			<td></td>
			<td>-</td>
			<td>Astra Superior Platinum Double Edge Razor Blades Green, salonwholesale.com</td>
		</tr>
		<tr>
			<td>Silicone capillary</td>
			<td></td>
			<td>a16090800ux0403</td>
			<td>Uxcell Silicone Tube 1 mm ID x 2 mm OD, amazon.com&#160;</td>
		</tr>
		<tr>
			<td>Tricaine</td>
			<td></td>
			<td>WXBC9102V</td>
			<td>Aldrich chemistry</td>
		</tr>
	</tbody>
</table>

gonadectomy and blood sampling procedures in the small size teleost model japanese medaka (oryzias latipes)

Sex steroids, produced by the gonads, play an essential role in brain and pituitary tissue plasticity and in the neuroendocrine control of reproduction in all vertebrates by providing feedback to the brain and pituitary. Teleost fishes possess a higher degree of tissue plasticity and variation in reproductive&#160;strategies compared to mammals and appear to be useful models to investigate the role of sex steroids and the mechanisms by which they act. The removal of the main source of sex steroid production using gonadectomy together with blood sampling to measure steroid levels has been well-established and fairly feasible in bigger fish and is a powerful technique to investigate the role and effects of sex steroids. However, these techniques raise challenges when implemented in small size teleost models. Here, we describe the step-by-step procedures of gonadectomy in both males and female Japanese medaka followed by blood sampling. These protocols are shown to be highly feasible in medaka indicated by a high survival rate, safety for the life span and phenotype of the fish, and reproducibility in terms of sex steroid clearance. The use of these procedures combined with the other advantages of using this small teleost model will greatly improve the understanding of feedback mechanisms in the neuroendocrine control of reproduction and tissue plasticity provided by sex steroids in vertebrates.

This protocol describes every step for performing gonadectomy and blood sampling in a small size model teleost, the Japanese medaka. The survival rate of the fish after ovariectomy (OVX) in females is 100% (10 out of 10 fish) while 94% (17 out of 18 fish) of the males survived after orchidectomy. Meanwhile, after the blood sampling procedure was performed, all (38 fish) fish survived.
Sham-operated females show oviposition (<str...

Watch this Scientific Journal Video about Gonadectomy and Blood Sampling Procedures in the Small Size Teleost Model Japanese Medaka Oryzias latipes at JoVE.com

Gonadectomy and Blood Sampling Procedures in the Small Size Teleost Model Japanese Medaka Oryzias latipes

The article describes a quick protocol to gonadectomize and sample blood from the small teleost fish, using Japanese medaka (Oryzias latipes) as a model, to investigate the role of sex steroids in animal physiology.

Gonadectomy and Blood Sampling Procedures in the Small Size Teleost Model Japanese Medaka (Oryzias latipes)

Sex steroids, produced by the gonads, play an essential role in brain and pituitary tissue plasticity and in the neuroendocrine control of reproduction in ...

gonadectomy-blood-sampling-procedures-small-size-teleost-model

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4.3K Views.  Norwegian University of Life Sciences. The article describes a quick protocol to gonadectomize and sample blood from the small teleost fish, using Japanese medaka (Oryzias latipes) as a model, to investigate the role of sex steroids in animal physiology. 

Video: Gonadectomy and Blood Sampling Procedures in the Small Size Teleost Model Japanese Medaka Oryzias latipes

Seawater Sampling and Collection

This video documents methods for collecting coastal marine water samples and processing them for various downstream applications including biomass concentration, nucleic acid purification, cell abundance, nutrient and trace gas analyses. For today's demonstration samples were collected from the deck of the HMS John Strickland operating in Saanich Inlet. An A-frame derrick, with a multi-purpose winch and cable system, is used in combination with Niskin or Go-Flo water sampling bottles. Conductivity, Temperature, and Depth (CTD) sensors are also used to sample the underlying water mass. To minimize outgassing, trace gas samples are collected first.  Then, nutrients, water chemistry, and cell counts are determined. Finally, waters are collected for biomass filtration. The set-up and collection time for a single cast is ~1.5 hours at a maximum depth of 215 meters. Therefore, a total of 6 hours is generally needed to complete the collection series described here.

This video documents methods for collecting coastal marine water samples and processing them for various downstream applications including biomass concentration, nucleic acid purification, cell abundance, nutrient and trace gas analyses.

This video documents methods for collecting coastal marine water samples and processing them for various downstream applications including biomass ...

Noninvasive In Vivo Small Animal MRI and MRS: Basic Experimental Procedures

Small animal Magnetic Resonance (MR) research has emerged as an important element of modern biomedical research due to its non-invasive nature and the richness of biological information it provides. MR does not require any ionizing radiation and can noninvasively provide higher resolution and better signal-to-noise ratio in comparison to other tomographic or spectroscopic modalities. In this protocol, we will focus on small animal MR imaging and MR spectroscopy (MRI/MRS) to noninvasively acquire relaxation weighted 1H images of mouse and to obtain 31P spectra of mouse muscle. This work does not attempt to cover every aspect of small animal MRI/MRS but rather introduces basic procedures of mouse MRI/MRS experiments. The main goal of this work is to inform researchers of the basic procedures for in vivo MR experiments on small animals. The goal is to provide a better understanding of basic experimental procedures to allow researchers new to the MR field to better plan for non-MR components of their studies so that both MR and non-MR procedures are seamlessly integrated.

Noninvasive In Vivo Small Animal MRI and MRS: Basic Experimental Procedures

This work describes basic procedures of noninvasive small animal MRI and MRS in vivo.

Small animal Magnetic Resonance (MR) research has emerged as an important element of modern biomedical research due to its non-invasive nature and the ...

Microinjection of Medaka Embryos for use as a Model Genetic Organism

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1, as in zebrafish and the mouse. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species 2, thus medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions.
To take advantage of medaka fish whose embryos are transparent and develop externally, microinjection is an essential technique to inject cell-tracers for labeling cells, mRNAs or anti-sense oligonucleotides for over-expressing and knocking-down genes of interest, and DNAs for making transgenic lines.

Medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions. This protocol highlights the key points for successful microinjection into medaka embryos, an important technique for embryological and genetic analysis using medaka and zebrafish in a laboratory.

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows ...

Dechorionation of Medaka Embryos and Cell Transplantation for the Generation of Chimeras

Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the three vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species 2, thus medaka and zebrafish are complementary for genetic dissection of the vertebrate genome functions. Manipulation of medaka embryos, such as dechorionation, mounting embryos for imaging and cell transplantation, are key procedures to work on both medaka and zebrafish in a laboratory. Cell transplantation examines cell autonomy of medaka mutations. Chimeras are generated by transplanting labeled cells from donor embryos into unlabeled recipient embryos. Donor cells can be transplanted to specific areas of the recipient embryos based on the fate maps 3 so that clones from transplanted cells can be integrated in the tissue of interest during development. Due to the hard chorion and soft embryos, manipulation of medaka embryos is more involved than in zebrafish. In this video, we show detailed procedures to manipulate medaka embryos.

Due to the hard chorion and soft embryos, manipulation of medaka embryos is more involved than in zebrafish. This video shows step-by-step procedures for how to manipulate medaka embryos, including dechorionation, mounting in agarose for imaging and cell transplantation for the production of chimeras. These procedures are essential to use medaka and zebrafish in a laboratory to take full advantage of their complementary features for the genetic dissection of vertebrate genome functions.

Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the three vertebrate model organisms in ...

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using noninvasive sampling techniques to investigate population genetics and demography of wild populations1. This approach has proven to be especially useful when dealing with rare or elusive species2. While a number of these methods have been developed to sample hair, feces and other biological material from carnivores and medium-sized mammals, they have largely remained untested in elusive small mammals. In this video, we present a novel, inexpensive and noninvasive hair snare targeted at an elusive small mammal, the American pika (Ochotona princeps). We describe the general set-up of the hair snare, which consists of strips of packing tape arranged in a web-like fashion and placed along travelling routes in the pikas&#x2019; habitat. We illustrate the efficiency of the snare at collecting a large quantity of hair that can then be collected and brought back to the lab. We then demonstrate the use of the DNA IQ system (Promega) to isolate DNA and showcase the utility of this method to amplify commonly used molecular markers including nuclear microsatellites, amplified fragment length polymorphisms (AFLPs), mitochondrial sequences (800bp) as well as a molecular sexing marker. Overall, we demonstrate the utility of this novel noninvasive hair snare as a sampling technique for wildlife population biologists. We anticipate that this approach will be applicable to a variety of small mammals, opening up areas of investigation within natural populations, while minimizing impact to study organisms.

We present a noninvasive sampling approach to efficiently collect hair samples from elusive small mammals, as shown for the American pika. We demonstrate the utility of this method by extracting DNA from sampled hair and amplifying several types of molecular markers commonly used in studies of wildlife ecology and conservation.

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using ...

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

Non-Terminal Blood Sampling Techniques in Guinea Pigs

Guinea pigs possess several biological similarities to humans and are validated experimental animal models1-3. However, the use of guinea pigs currently represents a relatively narrow area of research and descriptive data on specific methodology is correspondingly scarce. The anatomical features of guinea pigs are slightly different from other rodent models, hence modulation of sampling techniques to accommodate for species-specific differences, e.g., compared to mice and rats, are necessary to obtain sufficient and high quality samples. As both long and short term in vivo studies often require repeated blood sampling the choice of technique should be well considered in order to reduce stress and discomfort in the animals but also to ensure survival as well as compliance with requirements of sample size and accessibility. Venous blood samples can be obtained at a number of sites in guinea pigs e.g., the saphenous and jugular veins, each technique containing both advantages and disadvantages4,5. Here, we present four different blood sampling techniques for either conscious or anaesthetized guinea pigs. The procedures are all non-terminal procedures provided that sample volumes and number of samples do not exceed guidelines for blood collection in laboratory animals6. All the described methods have been thoroughly tested and applied for repeated in vivo blood sampling in studies within our research facility.

Though a known model, the guinea pig currently represents a niche in experimental animal sciences and limited data is available on the execution of most procedures. Here we present four different approaches to non-terminal in vivo blood sampling techniques in either conscious or anaesthetized guinea pigs.

Guinea pigs possess several biological similarities to humans and are validated experimental animal models1-3. However, the use of guinea pigs currently ...

Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, proteins, and glucose, yet the composition of the blood is dynamically regulated and easily perturbed. One factor that can change the blood composition is the stress response triggered by the sampling procedure, which can contribute to variability in the measures of interest. Here we describe a procedure for blood sampling from the lateral tail vein in the rat. This procedure offers significant advantages over other more commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, without anesthesia or analgesia. Additionally, it can be used to obtain large volume samples (upwards of 1 ml in some rats), and it may be used repeatedly across experimental days. By minimizing the stress response and pain resulting from blood sampling, measures can more accurately reflect the true basal state of the animal, with minimal influence from the sampling procedure itself.

Blood samples are useful for assessing biomarkers of physiological states or disease in vivo. Here we describe the methodology to sample blood from the lateral tail vein in the rat. This method provides rapid samples with minimal pain and invasiveness.

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, ...

Noninvasive Monitoring of Lesion Size in a Heterologous Mouse Model of Endometriosis

Here, we describe a protocol for the implementation of a heterologous mouse model in which progression of endometriosis can be assessed in real time through noninvasive monitoring of fluorescence emitted by implanted ectopic human endometrial tissue. For this purpose, biopsies of human endometrium are obtained from donor women ongoing oocyte donation. Human endometrial fragments are cultured in the presence of adenoviruses engineered to express cDNA for the reporter fluorescent protein mCherry. Upon visualization, labeled tissues with an optimal rate of fluorescence after infection are subsequently chosen for the implantation in recipient mice. One week prior to the implantation surgery, recipient mice are oophorectomized, and estradiol pellets are placed subcutaneously to sustain the survival and growth of lesions. On the day of surgery mice are anesthetized, and peritoneal cavity accessed through a small (1.5 cm) incision by the linea-alba. Fluorescently labeled implants are tweezed, briefly soaked in glue and attached to the peritoneal layer. Incisions are sutured, and animals left to recover for a couple of days. Fluorescence emitted by endometriotic implants is usually non-invasively monitored every 3 days for 4 weeks with an in vivo imaging system. Variations in the size of endometriotic implants can be estimated in real time by quantification of the mCherry signal and normalization against the initial time-point showing maximal fluorescence intensity.
Traditional preclinical rodents of models of endometriosis do not allow non-invasive monitoring of lesion in real time but rather allow evaluation of the effects of drugs assayed at the end point. This protocol allows one to track lesions in real time and is more useful to explore the therapeutic potential of drugs in preclinical models of endometriosis. The main limitation of the model thus generated is that non-invasive monitoring is not possible over long periods of time due to the episomal expression of Ad-virus.

Here, we present a protocol for live-imaging of fluorescently labeled human endometrial fragments grafted in mice. The method allows studying the effects of drugs of choice on endometriotic lesion size through monitoring and quantification of fluorescence emitted by the fluorescent reporter on real time

Here, we describe a protocol for the implementation of a heterologous mouse model in which progression of endometriosis can be assessed in real time ...