This work was funded by the Austrian Research Promotion Agency (FFG; project number 850681), the University of Applied Sciences Upper Austria Basic Funding initiative (project GlucoSTAR) and the Center for Technological Innovation in Medicine (TIMed Center) that receives basic funding by the province of Upper Austria.

According to the directive 2010/63/EU, experiments with non-hatched avian embryos during the first two-thirds of embryonic development do not need permission by an ethics committee.
1. Storage and Breeding of the Eggs
<ol>
	<li>Obtain fertilized hen eggs (Table of Materials) from a local breeder. 
	NOTE: Eggs may be stored at 14 &#176;C in a humidified atmosphere for up to 10 days after laying before use for experiments.</li>
	<li>Incubate the eggs at 38 &#176;C with an average humidity of 40 - 60% for 10 or 11 days in an incubator that constantly turns the eggs.</li>
</ol>
2. Selection of the Eggs
<ol>
	<li>Check the eggs for fertilization after incubation of up to 11 days. Use a candling light for this step. Dip the fringe of the candling lamp into an inkpad and candle the pointed side of the egg.</li>
	<li>To identify the air bladder, check for differences in brightness on the top of the egg. 
	NOTE: The air bladder appears as a round, more transparent region, while the albumen is darker when the egg is candled.</li>
	<li>After detection of the air bladder, mark the location by pressing the lamp slightly against the egg with the previously applied ink.</li>
	<li>Exclude non-fertilized eggs at this step, which do not show a difference in brightness between the albumen and air bladder.</li>
</ol>
3. Injection of Substances
<ol>
	<li>Prepare the substance of interest (e.g., the herbal extract or insulin) by diluting the substance in the desired concentration in Hank&#39;s balanced salt solution (HBSS) buffer. For example, 3.3 U/mL of a commercially available insulin analog in HBSS.</li>
	<li>For the application of the selected compound, carefully peck the eggshell with a pointed pair of tweezers in the marked area of the air bladder. Do not form a hole larger than the diameter of the needle of the syringe. Inject the buffer solution (300 &#181;L) containing the substance of interest into the pecked area via a syringe. 
	NOTE: Use sterile, 1-ml single-use syringes, pipette tips, and vessels. Wear gloves and a lab coat for the experiments.</li>
	<li>To determine the blood glucose-reducing effect of a substance, place the eggs back into the incubator for 60, 120, and 180 min, respectively. To determine basal blood glucose levels, include at least 10 - 15 non-treated control eggs.</li>
</ol>
4. Toxicity Tests
<ol>
	<li>After application of the selected compound for 24 h, equilibrate the eggshell membrane with phosphate buffered saline (PBS) buffer (enough to cover the whole eggshell membrane) for at least 30 s.</li>
	<li>Remove excess PBS buffer by pouring it away.</li>
	<li>Pull off the eggshell membrane carefully with a pointed pair of tweezers.</li>
	<li>Check blood vessels for lesions and confirm vitality (e.g., movement) of the chick embryo. 
	NOTE: See Figure 1 for comparison of vital versus non-vital embryos after application of a toxic herbal extract.</li>
</ol>
5. Measurement of the Blood Glucose Value
<ol>
	<li>At the respective time point, carefully remove the eggshell above the air bladder and equilibrate the eggshell membrane with PBS buffer (enough to cover the whole eggshell membrane).</li>
	<li>Remove excess PBS buffer by pouring it away.</li>
	<li>Carefully pull off the eggshell membrane with a pointed pair of tweezers.</li>
	<li>Cut and remove the chorioallantoic membrane with a micro-scissor, to enable good access to suitable blood vessels. Cut the membrane at least 2 - 3 cm. Do not cut any large vessels in the membrane itself to avoid unwanted blood loss of the embryo.</li>
	<li>Locate the large vessel originating from the abdomen of the embryo. Lift this vessel out of the albumen with a closed micro-scissor and place it on a plastic pH-strip. Move the pH strip under the vessel, which is held with the micro-scissor, and pull back the micro-scissor away carefully.</li>
	<li>Remove 1 - 2 mL of the liquid beneath the chorioallantoic membrane with a pipette to avoid dilution of the blood in the next step. Dry the vessel and pH-strip with filter paper, before the vessel is cut.</li>
	<li>Cut the blood vessel carefully with a micro-scissor on one side. Do not cut through the vessel completely. 
	NOTE: The vessel is approximately 1-mm thick. Do not cut more than half of the vessel to avoid cutting through the vessel entirely, as it may slip off the strip and no blood collection is possible.</li>
	<li>Collect the leaking blood on the pH-strip using a pipette. Ten microliters of blood are required to determine glucose levels using the blood glucose meter.</li>
</ol>
6. Statistical Evaluation
<ol>
	<li>Calculate the mean value of at least 10 individual eggs per time point and normalize these values to one of the control eggs (non- or buffer-treated eggs).</li>
	<li>Subsequently, determine the percentage of decrease. In addition, use insulin as a positive control. 
	NOTE: Repeat each experiment at least three times.</li>
</ol>

<ol>
	<li>Defronzo, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Banting+Lecture.+From+the+triumvirate+to+the+ominous+octet:+a+new+paradigm+for+the+treatment+of+type+2+diabetes+mellitus.">Banting Lecture. From the triumvirate to the ominous octet: a new paradigm for the treatment of type 2 diabetes mellitus.</a> Diabetes. 58 (4), 773-795 (2009).</li><li>American_Diabetes_Association. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+and+classification+of+diabetes+mellitus.">Diagnosis and classification of diabetes mellitus.</a> Diabetes Care. 37, S81-S90 (2014).</li><li>Han Cho, N., Cavan, 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=.">.</a> IDF Diabetes Atlas. 7, (2015).</li><li>Bailey, C. 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=Individualized+glycaemic+targets+and+pharmacotherapy+in+type+2+diabetes.">Individualized glycaemic targets and pharmacotherapy in type 2 diabetes.</a> Diabetes &amp; vascular disease research. 10 (5), 397-409 (2013).</li><li>Maruthur, N. 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=Diabetes+Medications+as+Monotherapy+or+Metformin-Based+Combination+Therapy+for+Type+2+Diabetes:+A+Systematic+Review+and+Meta-analysis.">Diabetes Medications as Monotherapy or Metformin-Based Combination Therapy for Type 2 Diabetes: A Systematic Review and Meta-analysis.</a> Annals of internal medicine. 164 (11), 740-751 (2016).</li><li>Aleman-Gonzalez-Duhart, D., Tamay-Cach, F., Alvarez-Almazan, S., Mendieta-Wejebe, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+Advances+in+the+Biochemical+and+Physiological+Aspects+of+the+Treatment+of+Type+2+Diabetes+Mellitus+with+Thiazolidinediones.">Current Advances in the Biochemical and Physiological Aspects of the Treatment of Type 2 Diabetes Mellitus with Thiazolidinediones.</a> PPAR research. , 7614270 (2016).</li><li>Cohen, F. J., Neslusan, C. A., Conklin, J. E., Song, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+antihyperglycemic+prescribing+trends+for+US+privately+insured+patients+with+type+2+diabetes.">Recent antihyperglycemic prescribing trends for US privately insured patients with type 2 diabetes.</a> Diabetes care. 26 (6), 1847-1851 (2003).</li><li>Desai, N. R., 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=Patterns+of+medication+initiation+in+newly+diagnosed+diabetes+mellitus:+quality+and+cost+implications.">Patterns of medication initiation in newly diagnosed diabetes mellitus: quality and cost implications.</a> The American journal of medicine. 125 (3), e301-e307 (2012).</li><li>Martel, 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=Anti-obesogenic+and+antidiabetic+effects+of+plants+and+mushrooms.">Anti-obesogenic and antidiabetic effects of plants and mushrooms.</a> Nature reviews. Endocrinology. , (2016).</li><li>Lanzerstorfer, P., 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=Identification+of+novel+insulin+mimetic+drugs+by+quantitative+total+internal+reflection+fluorescence+(TIRF)+microscopy.">Identification of novel insulin mimetic drugs by quantitative total internal reflection fluorescence (TIRF) microscopy.</a> British journal of pharmacology. 171 (23), 5237-5251 (2014).</li><li>Onur, S., St&#246;ckmann, H., Zenthoefer, M., Piker, L., D&#246;ring, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Plant+Extract+Collection+Kiel+in+Schleswig-Holstein+(PECKISH)+Is+an+Open+Access+Screening+Library.">The Plant Extract Collection Kiel in Schleswig-Holstein (PECKISH) Is an Open Access Screening Library.</a> Journal of Food Research. 2 (4), (2013).</li><li>Haselgrubler, R., 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=Gluc-HET,+a+complementary+chick+embryo+model+for+the+characterization+of+antidiabetic+compounds.">Gluc-HET, a complementary chick embryo model for the characterization of antidiabetic compounds.</a> PloS one. 12 (8), e0182788 (2017).</li><li>Stadlbauer, V., 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=Biomolecular+Characterization+of+Putative+Antidiabetic+Herbal+Extracts.">Biomolecular Characterization of Putative Antidiabetic Herbal Extracts.</a> PloS one. 11 (1), e0148109 (2016).</li><li>Greywe, 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=Applicability+and+robustness+of+the+hen's+egg+test+for+analysis+of+micronucleus+induction+(HET-MN):+results+from+an+inter-laboratory+trial.">Applicability and robustness of the hen's egg test for analysis of micronucleus induction (HET-MN): results from an inter-laboratory trial.</a> Mutation research. 747 (1), 118-134 (2012).</li><li>Manakova, E., Hubickova, L., Kostalova, J., Zemanova, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryotoxicity+of+mirtazapine:+a+study+using+Chick+Embryotoxicity+Screening+Test.">Embryotoxicity of mirtazapine: a study using Chick Embryotoxicity Screening Test.</a> Neuro endocrinology letters. 31, 8-10 (2010).</li><li>Ribatti, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chick+embryo+chorioallantoic+membrane+as+a+useful+tool+to+study+angiogenesis.">Chick embryo chorioallantoic membrane as a useful tool to study angiogenesis.</a> International review of cell and molecular biology. 270, 181-224 (2008).</li><li>Yoshiyama, Y., Sugiyama, T., Kanke, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+diabetes+model+in+chick+embryos+treated+with+streptozotocin.">Experimental diabetes model in chick embryos treated with streptozotocin.</a> Biological &amp; pharmaceutical bulletin. 28 (10), 1986-1988 (2005).</li><li>Wolf, T., Niehaus-Rolf, C., Luepke, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+new+methodological+aspects+of+the+hen's+egg+test+for+micronucleus+induction+(HET-MN).">Some new methodological aspects of the hen's egg test for micronucleus induction (HET-MN).</a> Mutation research. 514 (1-2), 59-76 (2002).</li></ol>

The authors have nothing to disclose.

The HET-CAM assay is a widely used alternative test system to animal testing in the industry12. The in-ovo system described here represents a modified version of the HET-CAM assay. While in its original form, HET-CAM experiments are performed to study the irritating properties of compounds and formulation or the analysis of angiogenesis14,15,16, we have adapted the approach to test compounds with putative insulin-mimetic characteristics12. According to the directive 2010/63/EU, experiments with non-hatched avian embryos during the first two-thirds of embryonic development do not need permission by an ethics committee since these experiments are not considered animal experiments. Recent studies have proven the suitability of the in-ovo system to characterize the efficacy of selected herbal extracts, which have been identified in a GLUT4-translocation-based primary screen10, to reduce blood glucose levels in the absence of insulin12,13. Critical points for a good performance of the in-ovo system include: First, a reliable breeder delivering the fertilized eggs. The Lohmann classic brown chicken is a good choice of breed. Second, the implementation of toxicity tests to exclude toxic effects of the investigated compound needs to be done. In addition, the preparation of a suitable blood vessel for blood collection is important. It is important to avoid the cutting of large vessels in the chorioallantoic membrane itself, as this can lead to a non-preferable loss of blood. Furthermore, before the vessel is cut onto the pH strip, it has to be patted dry using filter paper to prevent dilution of the collected blood. Finally, a sufficient number of experiments appears to be important. We recommend using at least 10 eggs for each time point, and a three-fold repetition of the respective experiment.
Naturally, there are some limitations of this in-ovo approach. Since it takes up to 30 minutes until the substances are absorbed through the eggshell membrane and come into close contact with the chorioallantoic membrane, it is not possible to quantify a rapid response of the embryo to the applied compound. Furthermore, some compounds may be toxic in the applied concentration, which frequently results in lesions of the blood vessels in the chorioallantoic membrane. Thus, cytotoxicity testing based on long-term incubation of the compounds for about 24 hours appears reasonable. Finally, we found that the buffer system used for application of the compounds has a significant effect on the performance of the assay.12 Currently, HBSS buffer is used because it results in the smallest effect on the blood glucose levels of the embryo.
For future applications, additional buffer systems as an alternative to HBSS might be tested. Furthermore, the influence of herbal extracts on additional blood parameters such as cholesterol, triglycerides or lipoproteins could be an attractive question.
Our new in-ovo system is a promising and important tool to test substances with insulin-mimetic characteristics in a living organism without the need of an animal experiment. Therefore, it fills the gap between in-vitro and in-vivo approaches. In comparison to existing in-ovo model systems17 there is no need to induce diabetes by streptozotocin (STZ) treatment, as we use embryos at day 10 or 11 of incubation. At this stage, insulin production has not started, but the embryos are already insulin sensitive. Additionally, permission by an ethics committee might be required if embryos aged 14 - 17 days are used17. Furthermore, compared to alternative strategies18, the compound application as described here is less harmful and laborious, increasing experimental through-put rates.
Taken together, this in-ovo approach is an attractive system if a blood-decreasing effect of an insulin-mimetic substance needs to be tested in a living organism.

Diabetes mellitus is a metabolic disease characterized by hyperglycemia and is caused by defects in insulin secretion or insulin action1. Ninety percent of all diabetes cases are category type 2 diabetes mellitus (T2DM), where individuals demonstrate insulin resistance and mostly insulin deficiency2. Several factors are known to increase the global incidence of T2DM, including changes in lifestyle, particularly those related to overnutrition, aging, and physical inactivity. Approximately 400 million people have diabetes mellitus worldwide according to the International Diabetes Federation (IDF). This number is expected to reach 600 million within the next 20 years3.
Diabetic complications caused by hyperglycemia and insulin-resistance, such as hypertension, dyslipidemia, glucose intolerance, coronary heart disease, and cerebral vascular disease, lead to a significantly reduced life expectancy and quality4. Affected individuals require glucose-lowering pharmacotherapy; however, the usage of drugs, such as metformin5 is often associated with dramatic side-effects6,7,8. Therefore, further antidiabetic approaches that are safe, widely available and inexpensive are necessary.
Different herbal compounds, extracts, plants, and nutraceuticals have been ascribed to feature insulin-mimetic properties by modulating various cellular functions. An important feature is stimulation of the translocation of glucose transporter 4 (GLUT4) to the plasma membrane from intracellular storage compartments, resulting in an increased uptake of glucose in muscle and adipose tissue in the absence of insulin, known as insulin-mimetic properties9. Previously, we implemented a fluorescence microscopy-based approach to quantify the translocation process of GLUT410. Analysis of numerous plant extracts from an extract library11 using this assay resulted in the identification of promising candidates. However, further tests in living organisms are required. The approach described in detail in this paper, a modified chick embryo model, has proven to be a promising model for identifying substances with insulin-mimetic properties. It represents an attractive tool filling the wide gap between in-vitro and in-vivo studies and offers several advantages in comparison to existing systems such as acceptable costs, simple handling procedures, and adequate throughput rates. In addition, permission by an ethics committee is not required.

<table border="0" cellpadding="0" cellspacing="0" width="621">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:279px;">Phosphate buffered saline (PBS)</td>
			<td style="width:140px;">Biochrom</td>
			<td style="width:123px;">L1825</td>
			<td style="width:80px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hank&#8217;s balanced salt solution (HBSS)</td>
			<td>Thermo Scientific</td>
			<td>SH30268.02</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NovoRapid</td>
			<td>Novo Nordisk</td>
			<td>8-0905-82-304-6</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hens eggs (Lohmann classic brown chicken)</td>
			<td>Local Breeder</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Accu-check performa</td>
			<td>Roche</td>
			<td>6454011</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Accu-check Inform II Teststreifen</td>
			<td>Roche&#160;</td>
			<td>5942861</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Incubator&#160; HEKA- Turbo 288</td>
			<td>HEKA Brutger&#228;te</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe Omnican F 1 mL</td>
			<td>Braun</td>
			<td>09161502S&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Extracts</td>
			<td>PEKISH extract library</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lamp Tempo Nr. 119</td>
			<td>ORBAN</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

an in ovo model for testing insulin-mimetic compounds

Elevated blood glucose levels in type 2 diabetes mellitus (T2DM), a complex and multifactorial metabolic disease, are caused by insulin resistance and &#946;-cell failure. Various strategies, including the injection of insulin or the usage of insulin-sensitizing drugs, were pursued to treat T2DM or at least reduce the symptoms. In addition, the application of herbal compounds has attracted increasing attention. Thus, it is necessary to find efficient test systems to identify and characterize insulin-mimetic compounds. Here we developed a modified chick embryo model, which enables testing of synthetic compounds and herbal extracts with insulin-mimetic properties. Using a fluorescence microscopy-based primary screen, which quantifies the translocation of Glucose transporter 4 (Glut4) to the plasma membrane, we were able to identify compounds, mainly herbal extracts, which lead to an increase of intracellular glucose concentrations in adipocytes. However, the efficacy of these substances requires further verification in a living organism. Thus, we used an in-ovo approach to identify their blood glucose-reducing properties. The approval by an ethics committee is not needed since the use of chicken embryos during the first two-thirds of embryonic development is not considered an animal experiment. Here, the application of this model is described in detail.

Description of the Gluc-HET approach:
After incubation with the substances of interest, the fertilized eggs are opened and prepared for blood collection by removal of the eggshell (Figure 2). Further preparation includes equilibration and removal of the eggshell membrane to gain access to the chorioallantoic membrane. This membrane is then cut carefully to provide access to a suitable blood vessel for blood collection. Preferably, the large vessel originating from the abdomen of the embryo is placed on a plastic pH-strip. To avoid dilution of the blood during collection, the vessel and pH-strip is patted dry with filter paper. The blood vessel is then cut and blood leaking onto the pH-strip is collected for analysis using a pipette.
Effects of selected herbal extracts on blood glucose levels:
With a recently established GLUT4-translocation quantitation-based primary screen, we are able to identify substances with an insulin-mimetic characteristic10. Screening of hundreds of water-soluble herbal extracts resulted in the identification of several hits. These extracts were tested in our in-ovo model. Extracts prepared from Combretum indicum (Rangoon creeper) and a non-disclosed extract (termed extract 0845) were used for these experiments. We dissolved the extracts in HBSS buffer at 300 mg/L, a concentration that turned out to be appropriate in previous studies12,13. Substances were applied to the embryos incubated for 11 days. As indicated in Figure 3, the extract from Rangoon creeper did not result in a significant blood glucose reduction in-ovo. However, the blood glucose concentration was successfully reduced with extract 0845, with a minor decrease after 60 min, which is comparable to the effect observed using a commercially available insulin analog. A significant effect was observed when the incubation time of the eggs was prolonged.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57237/57237fig1.jpg" /> 
Figure 1: Influence of toxic compounds on embryo vitality. Eggs were incubated for 11 days (A and C) or treated with a toxic herbal extract on day 10 for another 24 h (B and D). Application of the extract led to severe lesions of blood vessels in the chorioallantoic membrane (D) and the embryo (B).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57237/57237fig2.jpg" /> 
Figure 2: Description of the in-ovo model. From left to right: Opening and removal of the eggshell (1-2); equilibration and removal of the eggshell membrane with chorioallantoic membrane exposed (3); prepared vessel on a pH-strip (4); the collection of blood and measurement of glucose concentration using a glucose meter (5). Adapted from Haselgruebler et al., 201712. <a href="https://cloudflare.jove.com/files/ftp_upload/57237/57237fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57237/57237fig3.jpg" /> 
Figure 3: Effect of herbal extracts on blood glucose levels compared to untreated or insulin-treated eggs. Eggs were incubated for 11 days and treated with the indicated substances (commercially available insulin analog: 3.3 U/mL; extracts: 300 mg/L) dissolved in HBSS buffer (300 &#181;L volume) for up to 3 h. Blood glucose levels were determined using a blood glucose meter. Results are shown without the effect of the buffer. Error bars are based on the standard error of the mean. *P &#60;0.05 and ****P &#60;0.0001, significant decrease with respect to HBSS-treated eggs of the same incubation time. Adapted from Haselgruebler et al., 201712. <a href="https://cloudflare.jove.com/files/ftp_upload/57237/57237fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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An In Ovo Model for Testing Insulin-mimetic Compounds

In this study, we describe an in-ovo system as a promising tool to test insulin-mimetic compounds in a living organism. The main advantages include adequate throughput rates and acceptable costs, which enable the identification of phytochemicals with insulin-mimetic properties.

An In Ovo Model for Testing Insulin-mimetic Compounds

Elevated blood glucose levels in type 2 diabetes mellitus (T2DM), a complex and multifactorial metabolic disease, are caused by insulin resistance and ...

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9.6K Views.  University of Applied Sciences Upper Austria. In this study, we describe an in-ovo system as a promising tool to test insulin-mimetic compounds in a living organism. The main advantages include adequate throughput rates and acceptable costs, which enable the identification of phytochemicals with insulin-mimetic properties.

Video: An In Ovo Model for Testing Insulin-mimetic Compounds

Flash Photolysis of Caged Compounds in the Cilia of Olfactory Sensory  Neurons

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. Caged compounds are molecules made physiologically inactive by a chemical cage that can be broken by a flash of ultraviolet light. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged compounds for the study of olfactory transduction in dissociated mouse olfactory sensory neurons. The process of olfactory transduction (Figure 1) takes place in the cilia of olfactory sensory neurons, where odorant binding to receptors leads to the increase of cAMP that opens cyclic nucleotide-gated (CNG) channels2. Ca entry through CNG channels activates Ca-activated Cl channels. We show how to dissociate neurons from the mouse olfactory epithelium3 and how to activate CNG channels or Ca-activated Cl channels by photolysis of caged cAMP4 or caged Ca5. We use a flash lamp6,7 to apply ultraviolet flashes to the ciliary region to uncage cAMP or Ca while patch-clamp recordings are taken to measure the current in the whole-cell voltage-clamp configuration8-11.

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged cAMP or caged Ca for the study of olfactory transduction in dissociated mouse olfactory sensory neurons.

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. ...

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain dopaminergic neurons is implicated in Parkinson's disease, Schizophrenia, depression, and dementia1-5. Thus, studying the regulation of midbrain dopaminergic neuron differentiation could not only provide important insight into mechanisms regulating midbrain development and neural progenitor fate specification, but also help develop new therapeutic strategies for treating a variety of human neurological disorders.
Dopaminergic neurons differentiate from neural progenitors lining the ventricular zone of embryonic ventral midbrain. The development of neural progenitors is controlled by gene expression programs6,7. Here we report techniques utilizing electroporation to express genes specifically in the midbrain of Hamburger Hamilton (HH) stage 11 (thirteen somites, 42 hours) chick embryos8,9. The external development of chick embryos allows for convenient experimental manipulations at specific embryonic stages, with the effects determined at later developmental time points10-13. Chick embryonic neural tubes earlier than HH stage 13 (nineteen somites, 48 hours) consist of multipotent neural progenitors that are capable of differentiating into distinct cell types of the nervous system. The pCAG vector, which contains both a CMV promoter and a chick &beta;-actin enhancer, allows for robust expression of Flag or other epitope-tagged constructs in embryonic chick neural tubes14. In this report, we emphasize special measures to achieve regionally restricted gene expression in embryonic midbrain dopaminergic neuron progenitors, including how to inject DNA constructs specifically into the embryonic midbrain region and how to pinpoint electroporation with small custom-made electrodes. Analyzing chick midbrain at later stages provides an excellent in vivo system for plasmid vector-mediated gain-of-function and loss-of-function studies of midbrain development. Modification of the experimental system may extend the assay to other parts of the nervous system for performing fate mapping analysis and for investigating the regulation of gene expression.

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

To assess the function and the regulation of genes during the development of midbrain dopaminergic neurons, we describe a method that involves in ovo electroporation of plasmid DNA constructs into embryonic chick ventral midbrain dopaminergic neuron progenitors. This technique can be used to achieve efficient expression of genes of interest to study different aspects of midbrain development and dopaminergic neuron differentiation.

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain ...

Design and Assembly of an Ultra-light Motorized Microdrive for Chronic Neural Recordings in Small Animals

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of neural circuits underlying a variety of natural behaviors, including navigation1 , decision making 2,3, and the generation of complex motor sequences4,5,6. Advances in precision machining has allowed for the fabrication of light-weight devices suitable for chronic recordings in small animals, such as mice and songbirds. The ability to adjust the electrode position with small remotely controlled motors has further increased the recording yield in various behavioral contexts by reducing animal handling.6,7
 Here we describe a protocol to build an ultra-light motorized microdrive for long-term chronic recordings in small animals. Our design evolved from an earlier published version7, and has been adapted for ease-of use and cost-effectiveness to be more practical and accessible to a wide array of researchers. This proven design 8,9,10,11 allows for fine, remote positioning of electrodes over a range of ~ 5 mm and weighs less than 750 mg when fully assembled. We present the complete protocol for how to build and assemble these drives, including 3D CAD drawings for all custom microdrive components.

The design, fabrication and assembly of an ultra-light motorized microdrive is described. The device provides a cost-effective and easy-to-use solution for chronic recordings of single units in small behaving animals.

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of ...

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial and temporal control of ectopic microRNA expression. However, ventral brain structures like ventral midbrain are rather difficult to reach for any manipulations. Here, we demonstrate an efficient way to electroporate miRNA into ventral midbrain using thin platinum electrodes. This method offers a reliable way to transfect specific areas of the midbrain and a useful tool for in vivo studies.

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Ectopic expression is one technique to elucidate the microRNAs role in brain development. However, targeting specific areas using in ovo electroporation is challenging. Here, we show an efficient way to selectively electroporate ventral and dorsal midbrain regions.

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial ...

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response is a vision-based, reflexive behavior that has been observed in all vertebrates tested. Tadpole eyes are small so the tail flip response was used as alternative measure, which requires a trained technician to record the subtle response. We developed an alternative behavior assay based on the fact that tadpoles prefer to swim on the white side of a tank when placed in a tank with both black and white sides. The assay presented here is an inexpensive, simple alternative that creates a response that is easily measured. The setup consists of a tripod, webcam and nested testing tanks, readily available in most Xenopus laboratories. This article includes a movie showing the behavior of tadpoles, before and after severing the optic nerve. In order to test the function of one eye, we also include representative results of a tadpole in which each eye underwent retinal axotomy on consecutive days. Future studies could develop an automated version of this assay for testing the vision of many tadpoles at once.

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Xenopus laevis tadpoles prefer swimming on the white side of a black/white tank. This behavior is guided by their vision. Based on this behavior, we present a simple assay to test the visual function of tadpoles.

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response ...

An In Vitro Model for the Study of Cellular Pathophysiology in Globoid Cell Leukodystrophy

The precise function of multi-nucleated microglia, called globoid cells, that are uniquely abundant in the central nervous system of globoid cell leukodystrophy (GLD) is unclear. This gap in knowledge has been hindered by the lack of an appropriate in vitro model for study. Herein, we describe a primary murine glial culture system in which treatment with psychosine results in multinucleation of microglia resembling the characteristic globoid cells found in GLD. Using this novel system, we defined the conditions and modes of analysis for study of globoid cells. The potential use of this model system was validated in our previous study, which identified a potential role for matrix metalloproteinase (MMP)-3 in GLD. This novel in vitro system may be a useful model in which to study the formation and function, but also the potential therapeutic manipulation, of these unique cells.

An In Vitro Model for the Study of Cellular Pathophysiology in Globoid Cell Leukodystrophy

Globoid cells are a defining pathological feature of Krabbe disease, a leukodystrophy currently lacking an effective long-term therapy. We have developed a cell culture model to study the innate biology and pathogenic potential of activated microglia and their transformation into globoid cells.

The precise function of multi-nucleated microglia, called globoid cells, that are uniquely abundant in the central nervous system of globoid cell ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier (BBB). Rats with induced ischemic stroke were established and used as in vivo models to investigate the functional integrity of the BBB. Spectrophotometric detection of Evans blue (EB) in the brain samples with ischemic injury could provide reliable justification for the research and development of novel therapeutic modalities. This method generates reproducible results, and is applicable in any laboratory without a need for special equipment. Here, we present a visualized and technical guideline on the detection of the extravasation of EB following induction of ischemic stroke in rats.

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

The overall goal of this procedure is to provide a highly reproducible technique for in vivo assessment of the blood-brain barrier disruption in rat models of ischemic stroke.

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier ...

A Preclinical Model to Assess Brain Recovery After Acute Stroke in Rats

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery occlusion/reperfusion (MCAO/R) model in male Sprague-Dawley rats was chosen. By changing the rat&#39;s weight (260&#8722;330 g), the thread bolt type (2636/2838/3040/3043) and the brain infarct time (2-3 h), a higher Longa&#39;s score, a larger infarct volume and a greater model success ratio were screened using the Longa&#39;s score and TTC staining. The optimum model condition (300 g, 3040 thread bolt, 3 h brain infarct time) was acquired and used in a 1-90 day observation period after reperfusion via assessment of sensorimotor functions and infarct volume. At these conditions, the bilateral asymmetry test had a significant difference from 1 to 90 days, and the grid-walking test had a significant difference from 1 to 60 days; both differences could be a suitable sensorimotor functional test. Thus, the most appropriate condition of a novel rat model in the recovery and sequela stages of brain ischemia was found: 300 g rats that underwent MCAO with a 3040 thread bolt for a 3 h brain infarct and then reperfused. The appropriate sensorimotor functional tests were a bilateral asymmetry test and a grid-walking test.

The&#160;purpose of this study is to establish and validate an animal model for research in the recovery and sequela stages of brain ischemia by testing brain infarction and sensorimotor function after middle cerebral artery occlusion/reperfusion (MCAO/R) after 1-90 days in rats.

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery ...