Name: gait analysis of age-dependent motor impairments in mice with neurodegeneration
Uploaded: 2018-06-18T14:00:00
Description: Watch this Scientific Journal Video about Gait Analysis of Age-dependent Motor Impairments in Mice with Neurodegeneration at JoVE.com

We thank animal caretakers at the ENI&#39;s Animal facility for help with breeding, and Dr. Nuno Raimundo for useful comments on the manuscript. I.M. is supported by the grants from the German Research Foundation (DFG) through the collaborative research center SFB-889 (project A8) and SFB-1190 (project P02), and the Emmy Noether Young Investigator Award (1702/1). C.M.R. is supported by the fellowship from the G&#246;ttingen Graduate School for Neurosciences, Biophysics, and Molecular Biosciences (GGNB).

All animal experiments reported here are conducted according to the European Guidelines for animal welfare (2010/63/EU) with approval by the Nieders&#228;chsisches Landesamt f&#252;r Verbraucherschutz und Lebensmittelsicherheit (LAVES), registration number 14/1701.
1. Study Design
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	<li>As animal behavior work requires careful planning, consider the following parameters while designing the experiment.
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		<li>Number of animals needed per group.
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			<li>Use a statistical softwar...

Studying the motor coordination is a useful approach in the characterization of models of neurodegenerative diseases, especially for diseases like PD in which motor coordination is severely affected. With the help of a kinematic gait analysis functional assay, we can identify subtle changes in the gait of animals at the onset of locomotion problems, or in models with weak neurodegeneration and hence relatively modest phenotype. Given the wide range of phenotypes in various models of neurodegenerative diseases that encomp...

Neurodegenerative diseases impose a significant burden on patients, families, and society, and will become of even greater concern as life expectancy increases, and the world population continues to age. One of the most common symptoms of neurodegenerative diseases are balance and mobility problems. Thus, characterization of motor behavior in aging mammalian (e.g., rodent) models, and/or models showing neurodegenerative phenotypes, is a valuable tool to demonstrate the in vivo relevance of the specific animal model(s), or therapeutic treatments that aim to improve the disease symptoms. Almost every approach to treat neurodegenerative diseases ultimately requires testing in an animal model before initiation of a clinical trial in humans. Therefore, it is crucial to have reliable, reproducible behavior tests that can be used to consistently quantify disease-relevant phenotypes along age progression, in order to ensure that a candidate drug, which showed potential in an in vitro model, can effectively ameliorate the phenotype in a living animal.
One aspect of motor behavior assessment in rodents is kinematic gait analysis, which can be performed by VPI (also called ventral plane videography)1,2. This established method capitalizes on continuous recording of the underside of the rodents walking atop a transparent and motorized treadmill belt1,2,3,4. Analysis of the video feed data creates &#34;digital paw prints&#34; of all four limbs that dynamically and reliably recapitulate the rodent&#39;s walking pattern, as originally described by&#160;Kale et al.2 and Amende et al.3.
The principle of imaging-based gait analysis is to measure the paw area in contact with the treadmill belt over time, for each individual paw. Every stance is represented by an increase in paw area (in the braking phase) and a decrease in paw area (in the propulsion phase). This is followed by the swing phase in which no signal is detected. Swing and stance together form a stride. In addition to gait dynamics parameters, posture parameters can also be extracted from the recorded videos. Exemplary parameters and their definition are listed in Table 1 and include stance width (SW; the combined distance from the fore or hind paws to the snout-tail axis), stride length (SL; average distance between two strides of the same paw), or paw placement angle (the angle of the paw to the snout-tail axis). The posture and gait dynamics data allow drawing conclusions on animal balance (by posture parameters and their variability over several steps) and coordination (by gait dynamics parameters). Other parameters, such as ataxia coefficient (the SL variability calculated by [(max. SL&#8722;min. SL)/mean SL]), hind limb shared stance time (time that both hind limbs are in contact with the belt), or paw drag (total area of the paw on the belt from full stance to paw lift-off) can also be extracted, and have been reported to be changed in various neurodegenerative disease models5,6,7,8 (see Table 1).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Parameter</td>
			<td>Unit</td>
			<td>Definition</td>
		</tr>
		<tr>
			<td>swing time</td>
			<td>ms</td>
			<td>duration of time the paw is not in contact with the belt</td>
		</tr>
		<tr>
			<td>stance time</td>
			<td>ms</td>
			<td>duration of time the paw is in contact with the belt</td>
		</tr>
		<tr>
			<td>% brake</td>
			<td>% of stance time</td>
			<td>percentage of stance time the paws are in the brake phase</td>
		</tr>
		<tr>
			<td>% propel</td>
			<td>% of stance time</td>
			<td>percentage of stance time the paws are in the propulsion phase</td>
		</tr>
		<tr>
			<td>stance width</td>
			<td>cm</td>
			<td>combined distance from the fore or hind paws to the snout-tail axis</td>
		</tr>
		<tr>
			<td>stride length</td>
			<td>cm</td>
			<td>average distance between two strides of the same paw</td>
		</tr>
		<tr>
			<td>stride frequency</td>
			<td>strides/s</td>
			<td>number of complete strides per second</td>
		</tr>
		<tr>
			<td>paw placement angle</td>
			<td>deg</td>
			<td>angle of the paw in relation to the snout-tail axis of the animal</td>
		</tr>
		<tr>
			<td>ataxia coefficient</td>
			<td>a.u.</td>
			<td>SL variability calculated by [(max SL-min SL)/mean SL]</td>
		</tr>
		<tr>
			<td>% shared stance</td>
			<td>% of stance</td>
			<td>hind limb shared stance time; time that both hind limbs are in contact with the belt at the same time</td>
		</tr>
		<tr>
			<td>paw drag</td>
			<td>mm2</td>
			<td>total area of the paw on the belt from full stance to paw lift-off</td>
		</tr>
		<tr>
			<td>limb loading</td>
			<td>cm2</td>
			<td>MAX dA/dT; maximal rate of change of paw area in the breaking phase</td>
		</tr>
		<tr>
			<td>step angle variability</td>
			<td>deg</td>
			<td>standard deviation of the angle between the hind paws as a function of SL and SW</td>
		</tr>
	</tbody>
</table>
Table 1. Definition of key gait parameters that can be tested by ventral plane imaging.
Assessing the motor behavior of rodent models for neurodegenerative diseases can be challenging depending on the severity of the phenotype of a specific model at a given age. Several diseases, most prominently PD, show strong motor behavior (locomotion) deficits, both in patients and in animal models. One of the four key symptoms in PD is bradykinesia, which progresses with aging and manifests in severe gait impairments already in early stages of PD9. Studies of the acute PD model, rodents treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridin (MPTP), have already used VPI gait analysis10,11,12. However, given the acute nature of this model, these studies do not address the age-related progression of motor deficits. Several recent studies have conducted gait analysis in aged mice with neurodegenerative changes, for example13,14,15, emphasizing the relevance of understanding the disease progression with advancing age.
In addition to motor deficits, animal models of neurodegenerative diseases often have difficulties focusing on the examination tasks and show prominent cognitive impairments, in particular with advancing age. Such a phenotype can influence the result of motor behavior tests. Namely, one of the most widely used tests to examine motor deficits, the rotarod test16, relies on cognition, attention, and stress17,18. While the willingness to walk on a motorized treadmill also depends on these factors, the recorded read-out is running, which is a more standardized feature and far less influenced by altered cognition. Effects of stress and attention may be visible in specific parameters, like swing/stance time for stress, and SL for attention19,20, but not in overall running ability.
The kinematic gait analysis approach further offers the advantage of having options to adjust the challenge for rodent models. The treadmill with adjustable angle and speed allows walking speeds from 0.1 - 99.9 cm/s, so that rodents with severe walking impairments may still be able to run at a slow speed (~10 cm/s). Non-impaired animals can be measured at faster running speeds (30 - 40 cm/s). The observation of whether or not the tested animals are able to run at a certain speed provides a result by itself. Further, the rodent can be additionally challenged to run up an incline, or down a decline, by tilting the treadmill to a desired angle with the help of a goniometer, or by attaching a weighted sled to mouse or rat hind limbs.
In addition to numerous studies of single proteins that are mutated in patients, there is a recent increasing awareness of the links between defective endocytosis process and neurodegeneration13,21,22,23,24,25,26,27,28. Mouse models with reduced levels of endophilin-A (henceforth endophilin), a key player in both clathrin-mediated endocytosis13,21,29,30,31,32,33,45 and clathrin-independent endocytosis34, were found to show neurodegeneration and age-dependent impairments in locomotor activity13,21. Three genes encode the family of endophilin proteins: endophilin 1, endophilin 2, and endophilin 3. Notably, the phenotype resulting from depletion of endophilin proteins varies greatly depending on the number of missing endophilin genes13,21. While triple knock-out (KO) of all endophilin genes is lethal just a few hours after birth, and mice without both endophilin 1 and 2 fail to thrive and die within 3 weeks after birth, single KO for any of the three endophilins shows no obvious phenotype for tested conditions21. Other endophilin mutant genotypes show reduced lifespan and develop motor impairments with increasing age13. For example, endophilin 1KO-2HT-3KO mice display walking alterations and motor coordination problems (as tested by kinematic gait analysis and rotarod) already at 3 months of age, while their littermates, endophilin 1KO-2WT-3KO animals, display a significant reduction in motor coordination only at 15 months of age13. Due to the vast diversity of phenotypes in these models, it is necessary to identify and apply a test that can integrate a variety of challenges corresponding to the animal&#39;s motor and cognition abilities, as well as the age. Here, we detail the experimental procedures that capitalize on the kinematic gait analysis to assess the onset and progression of motor impairments in a mouse model that shows neurodegenerative changes (i.e., endophilin mutants). This includes measuring gait parameters at various ages and different severities of locomotion impairments.

<table border="0" cellpadding="0" cellspacing="0" width="684">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">DigiGait</td>
			<td style="width:123px;">Mouse Specifics, Inc., Framingham, Massachusetts, USA</td>
			<td style="width:123px;"></td>
			<td style="width:224px;">DigiGait Imager and Analysis Software are included with the hardware</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">non-transparent blanket or dark cloth</td>
			<td style="width:123px;"></td>
			<td style="width:123px;"></td>
			<td style="width:224px;">cover the test chamber to reduce the animal&#39;s feeling of exposure/stress</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">balance</td>
			<td style="width:123px;">e.g. Satorius</td>
			<td style="width:123px;"></td>
			<td style="width:224px;">balance with 0.1 g accuracy and a maximum load of at least 100 g</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">red finger paint</td>
			<td style="width:123px;">e.g. Kreul or Staedtler</td>
			<td style="width:123px;"></td>
			<td>for increasing the contrast between paws and animal&#8217;s body</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">small paint brush</td>
			<td style="width:123px;"></td>
			<td style="width:123px;"></td>
			<td style="width:224px;">soft brush to apply finger paint to the animal paws</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">diluted detergent</td>
			<td style="width:123px;"></td>
			<td style="width:123px;"></td>
			<td style="width:224px;">for cleaning</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">disinfectant, e.g. Meliseptol or 70% ethanol</td>
			<td style="width:123px;">e.g. B.Braun</td>
			<td style="width:123px;"></td>
			<td style="width:224px;">for desinfection</td>
		</tr>
	</tbody>
</table>

gait analysis of age-dependent motor impairments in mice with neurodegeneration

Motor behavior tests are commonly used to determine the functional relevance of a rodent model and to test newly developed treatments in these animals. Specifically, gait analysis allows recapturing disease relevant phenotypes that are observed in human patients, especially in neurodegenerative diseases that affect motor abilities such as Parkinson&#39;s disease (PD), Alzheimer&#39;s disease (AD), amyotrophic lateral sclerosis (ALS), and others. In early studies along this line, the measurement of gait parameters was laborious and depended on factors that were hard to control (e.g., running speed, continuous running). The development of ventral plane imaging (VPI) systems made it feasible to perform gait analysis at a large scale, making this method a useful tool for the assessment of motor behavior in rodents. Here, we present an in-depth protocol of how to use kinematic gait analysis to examine the age-dependent progression of motor deficits in mouse models of neurodegeneration; mouse lines with decreased levels of endophilin, in which neurodegenerative damage progressively increases with age, are used as an example.

To illustrate the use of kinematic gait analysis, we have performed gait analysis on WT C57BL/6J mice with advancing age, as well as several endophilin mutant lines, using commercially available instrumentation and software (please refer to the Table of Materials). In this setup, a high-speed camera under a transparent treadmill records the running of a mouse (Figure 1A). The software then recognizes the contrast between the red colored paws ...

Watch this Scientific Journal Video about Gait Analysis of Age-dependent Motor Impairments in Mice with Neurodegeneration at JoVE.com

Gait Analysis of Age-dependent Motor Impairments in Mice with Neurodegeneration

In this study, we demonstrate the use of kinematic gait analysis based on ventral plane imaging to monitor the subtle changes in motor coordination as well as the progression of neurodegeneration with advancing age in mouse models (e.g., endophilin mutant mouse lines).

Motor behavior tests are commonly used to determine the functional relevance of a rodent model and to test newly developed treatments in these animals. ...

This method can help answering key questions in the Aging and Neurodegeneration Research such as the onset and progression of gait and motor abnormalities with age and in particular, in mouse models of neurodegenerative diseases. The result of such status can be translated to a much better understanding of motor impairments in patients with neurodegenerative diseases for example, Parkinson's Disease or ataxias. The main advantage of using video based gait analysis is that it allows studying both subtle and severe motor impairments in mouse models of neurodegeneration.Begin by determining the health status of each animal by examining it closely and weighting it on a balance. Gently apply red finger paint to the animal's paws with a brush. Place the animal in a spare, clean cage while the paint dries.Next, set the speed of the treadmill on the top right panel of the apparatus. Then, place the animal in the test chamber and cover it with a dark cloth to allow each animal to adjust for one to two minutes. Turn on the light in the test chamber by turning the treadmill light rotary switch to the on position.Open the cover a little bit so that you can observe the animal during the task. Start the treadmill for about half a second to warn the animal that the task is about to begin. Turn the treadmill rotary switch to forward to start the treadmill.Then, click the Record button in the imager software. Once the animal runs in a stable manner, record for at least five seconds before stopping the treadmill. Stop the recording by clicking Stop on the imager software and turn the treadmill rotary switch back to the off position.Next, click the Edit button in the imager software to open a menu in which the start and end point of the video section can be set. Navigate through the video using the slider on the bottom of the screen. To select the current time point as the start or end point, click From Frame Number and To, respectively.Make sure the section contains at least seven steps per paw or fourteen steps in total of the animal running stably at a constant speed. Finally, enter the animal identification, birthdate, weight, and sex. Save the data on a desired location on the computer or server.The principle of video based gait analysis is to measure the paw area in contact with the surface over time for each individual paw. Many gait dynamics and posture parameters can be extracted from the recorded videos, for example, the swing time, stance time and width, stride length, and frequency. Other gait features such as ataxia coefficient or step angle variability can then be calculated from the primary measurements.Begin the video analysis by starting the analysis software and clicking Select Study Folder to select the folder with the recorded videos. Select one video and click Go.Use the re-draw function to select the area where the mouse is running. Note, this section should only contain the mouse and white background.Next, enter values in the length and width boxes to change the mask that excludes the red area around the snout of the animal for analysis. Adjust the sliders filter noise and filter fur and dark patches to optimize the black and white paw print. Set the filter noise slider to 800 to 950 for black animals and to 700 to 800 for brown or white animals, depending on the exact fur color of the animal, and select Okay.Then select a video that passed the first adjustment and select the Go function to start the analysis of the video. Next, select an analyzed video and click Go.Note that the paw print area in contact with the belt over time for each separate paw, can now be seen. To compare the original video and the calculated paw print for the selected area, select the Play Video function.To correct small mistakes, use either the Correct, Connect, or Delete option. Use the Correct option to delete a wrong signal. Click once to zoom into the relevant area and mark the left border of the object to remove with a second click and the right border with the third click.Use the Connect option to combine two signals. Click once to zoom into the relevant area and double click in the middle of the two objects to combine. Use the Delete option to remove time points from the analysis completely.Click once to zoom into the relevant area and mark the left border of the area to remove with the second click and the right border with the third click. Then select Next Limb to proceed through the four limbs. Note, after Next Limb is clicked after the last paw, the software completes the analysis and shows the results for the animal on four screens.Finally, after all videos from one experiment are analyzed, select all videos and click Reorganize Results to export the results. About 50 gait parameters are calculated and listed in the exported file. Sort the result by group, age, and running speed, and analyze all these conditions independently.In the gait analysis in wild type mice with advancing age, results indicated that the stance with normalized animal width does not change with advancing age. However, hind limb double support and limb loading do increase with age in wild type animals. Furthermore, while all tested animals can run at 30 meters per second at one month, the endophilin mutants develop running deficits as they age.Additionally, gait rhythm is impaired in endophilin mutants. For example, the propel time decreases in the endophilin mutants as the mice age. When attempting the procedure, it's important to remember that the most critical step is the recording of the original video.All further analysis depends on good quality videos. Gait analysis is a useful method to categorize gait and motor behavior in mouse models of neurodegeneration. However, some aspects of motor behavior, for example, muscle strength, are not adequately assessed by this method.But therefore a command complimenting video based gait analysis with a battery of other motor behavior tests, like grip strength, and beam walking. After watching this video, you should have a good understanding of how to perform gait analysis in mouse models that develop motor impairment with aging.

gait-analysis-age-dependent-motor-impairments-mice-with

Research

JoVE Journal

Developmental Biology

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple cellular and developmental processes principally via activation of ERK, the terminal kinase of the pathway. Tight regulation of ERK activity is essential for normal development and homeostasis; overly active ERK results in excessive cellular proliferation, while underactive ERK causes cell death. C. elegans is a powerful model system that has helped characterize the function and regulation of RTK-RAS-ERK signaling pathway during development. In particular, the RTK-RAS-ERK pathway is essential for C. elegans germline development, which is the focus of this method. Using antibodies specific to the active, diphosphorylated form of ERK (dpERK), the stereotypical localization pattern can be visualized within the germline. Because this pattern is both spatially and temporally controlled, the ability to reproducibly assay dpERK is useful to identify regulators of the pathway that affect dpERK signal duration and amplitude and thus germline development. Here we demonstrate how to successfully dissect, stain, and image dpERK within the C. elegans gonad. This method can be adapted for spatial localization of any signaling or structural protein in the C. elegans gonad, provided an antibody compatible with immunofluorescence is available.

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

We present an immunofluorescence&#160;imaging-based method for spatial and temporal localization of active ERK in the dissected C. elegans gonad. The protocol described here can be adapted for visualization of any signaling or structural protein in the C. elegans gonad, provided a suitable antibody reagent is available.

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple ...

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the cells are located. Specifically, they have been developed to analyze the electrical activity of excitable cells, such as spinal neurons. In such a scenario, it is crucial to preserve the integrity of the spinal cell, but it is also important to preserve the anatomy and physiological shape of the systems involved. Indeed, the comprehension of the manner in which the nervous system-and other complex systems-works must be based on a systemic approach. For this reason, the live zebrafish embryo was chosen as a model system, and the spinal neuron membrane voltage changes were evaluated without interfering with the physiological conditions of the embryos. 
Here, an approach combining the employment of zebrafish embryos with a FRET-based biosensor is described. Zebrafish embryos are characterized by a very simplified nervous system and are particularly suited for imaging applications thanks to their transparency, allowing for the employment of fluorescence-based voltage indicators at the plasma membrane during zebrafish development. The synergy between these two components makes it possible to analyze the electrical activity of the cells in intact living organisms, without perturbing the physiological state. Finally, this non-invasive approach can co-exist with other analyses (e.g., spontaneous movement recordings, as shown here).

Protocols described here allow for the study of the electrical properties of excitable cells in the most non-invasive physiological conditions by employing zebrafish embryos in an in vivo system together with a fluorescence resonance energy transfer (FRET)-based genetically encoded voltage indicator (GEVI) selectively expressed in the cell type of interest.

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

Correlative Super-resolution and Electron Microscopy to Resolve Protein Localization in Zebrafish Retina

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and scanning electron microscopy. The sub-diffraction limit resolution capabilities of super-resolution light microscopes allow improving the accuracy of the correlated data. Briefly, 110 nanometer thick cryo-sections are transferred to a silicon wafer and, after immunofluorescence staining, are imaged by super-resolution light microscopy. Subsequently, the sections are preserved in methylcellulose and platinum shadowed prior to imaging in a scanning electron microscope (SEM). The images from these two microscopy modalities are easily merged using tissue landmarks with open source software. Here we describe the adapted method for the larval zebrafish retina. However, this method is also applicable to other types of tissues and organisms. We demonstrate that the complementary information obtained by this correlation is able to resolve the expression of mitochondrial proteins in relation with the membranes and cristae of mitochondria as well as to other compartments of the cell.

This protocol describes the necessary steps to obtain subcellular protein localization results on zebrafish retina by correlating super-resolution light microscopy and scanning electron microscopy images.

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and ...

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the Notch receptor with ligands from neighboring cells induces activation of the signaling pathway (trans-activation), while interaction with ligands from the same cell inhibits signaling (cis-inhibition). Proper balance between trans-activation and cis-inhibition helps establish optimal levels of Notch signaling in some contexts during animal development. Because of the overlapping expression domains of Notch and its ligands in many cell types and the existence of feedback mechanisms, studying the effects of a given post-translational modification on trans- versus cis-interactions of Notch and its ligands in vivo is difficult. Here, we describe a protocol for using Drosophila S2 cells in cell-aggregation assays to assess the effects of knocking down a Notch pathway modifier on the binding of Notch to each ligand in trans and in cis. S2 cells stably or transiently transfected with a Notch-expressing vector are mixed with cells expressing each Notch ligand (S2-Delta or S2-Serrate). Trans-binding between the receptor and ligands results in the formation of heterotypic cell aggregates and is measured in terms of the number of aggregates per mL composed of &#62;6 cells. To examine the inhibitory effect of cis-ligands, S2 cells co-expressing Notch and each ligand are mixed with S2-Delta or S2-Serrate cells and the number of aggregates is quantified as described above. The relative decrease in the number of aggregates due to the presence of cis-ligands provides a measure of cis-ligand-mediated inhibition of trans-binding. These straightforward assays can provide semi-quantitative data on the effects of genetic or pharmacological manipulations on the binding of Notch to its ligands, and can help deciphering the molecular mechanisms underlying the in vivo effects of such manipulations on Notch signaling.

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Complexity of in vivo systems makes it difficult to distinguish between the activation and inhibition of Notch receptor by trans- and cis-ligands, respectively. Here, we present a protocol based on in vitro cell-aggregation assays for qualitative and semi-quantitative evaluation of the binding of Drosophila Notch to trans-ligands vs cis-ligands.

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...

Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that makes up much of the parenchyma of the human placenta. The distal villous capillary network is the terminus of the fetal blood supply after several generations of branching of vessels extending out from the umbilical cord. This network has a contiguous cellular sheath, the syncytial trophoblast barrier layer, which prevents mixing of fetal blood and the maternal blood in which it is continuously bathed. Insults to the integrity of the placental capillary network, occurring in disorders such as maternal diabetes, hypertension and obesity, have consequences that present serious health risks for the fetus, infant, and adult. To better define the structural effects of these insults, a protocol was developed for this study that captures capillary network structure on the order of 1 - 2 mm3 wherein one can investigate its topological features in its full complexity. To accomplish this, clusters of terminal villi from placenta are dissected, and the trophoblast layer and the capillary endothelia are immunolabeled. These samples are then clarified with a new tissue clearing process which makes it possible to acquire confocal image stacks to z- depths of ~1 mm. The three-dimensional renderings of these stacks are then processed and analyzed to generate basic capillary network measures such as volume, number of capillary branches, and capillary branch end points, as validation of the suitability of this approach for capillary network characterization.

This study presents a protocol for the reversible tissue clearing, immunostaining, 3D-rendering and analysis of vascular networks in human placenta villi samples on the order of 1 - 2 mm3.

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that ...

Cell Cycle Analysis in the C. elegans Germline with the Thymidine Analog EdU

Cell cycle analysis in eukaryotes frequently utilizes chromosome morphology, expression and/or localization of gene products required for various phases of the cell cycle, or the incorporation of nucleoside analogs. During S-phase, DNA polymerases incorporate thymidine analogs such as EdU or BrdU into chromosomal DNA, marking the cells for analysis. For C. elegans, the nucleoside analog EdU is fed to the worms during regular culture and is compatible with immunofluorescent techniques. The germline of C. elegans is a powerful model system for the studies of signaling pathways, stem cells, meiosis, and cell cycle because it is transparent, genetically facile, and meiotic prophase and cellular differentiation/gametogenesis occur in a linear assembly-like fashion. These features make EdU a great tool to study dynamic aspects of mitotically cycling cells and germline development. This protocol describes how to successfully prepare EdU bacteria, feed them to wild-type C. elegans hermaphrodites, dissect the hermaphrodite gonad, stain for EdU incorporation into DNA, stain with antibodies to detect various cell cycle and developmental markers, image the gonad and analyze the results. The protocol describes the variations in the method and analysis for the measurement of S-phase index, M-phase index, G2 duration, cell cycle duration, rate of meiotic entry, and rate of meiotic prophase progression. This method can be adapted to study the cell cycle or cell history in other tissues, stages, genetic backgrounds, and physiological conditions.

Cell Cycle Analysis in the C. elegans Germline with the Thymidine Analog EdU

An imaging-based method is described that can be used to identify S-phase and analyze cell cycle dynamics in the C. elegans hermaphrodite germline using the thymidine analog EdU. This method requires no transgenes and is compatible with immunofluorescent staining.

Cell cycle analysis in eukaryotes frequently utilizes chromosome morphology, expression and/or localization of gene products required for various phases ...

Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous system can be observed, with single cell resolution, in vivo. Here, we present an integrated protocol for the examination of neurodevelopment in C. elegans embryos. Our protocol combines imaging, lineaging and neuroanatomical tracing of single cells in developing embryos. We achieve long-term, four-dimensional (4D) imaging of living C. elegans&#160;embryos with nearly isotropic spatial resolution through the use of Dual-view Inverted Selective Plane Illumination Microscopy (diSPIM). Nuclei and neuronal structures in the nematode embryos are imaged and isotropically fused to yield images with resolution of ~330 nm in all three dimensions. These minute-by-minute high-resolution 4D data sets are then analyzed to correlate definitive cell-lineage identities with gene expression and morphological dynamics at single-cell and subcellular levels of detail. Our protocol is structured to enable modular implementation of each of the described steps and enhance studies on embryogenesis, gene expression, or neurodevelopment.

Here, we present a combinatorial approach using high-resolution microscopy, computational tools, and single-cell labeling in living C. elegans embryos to understand single cell dynamics during neurodevelopment.

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous ...

Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical insult to secondary injury processes, including apoptosis and inflammation. Animal modeling has been valuable in the search to unravel injury mechanisms and evaluate potential neuroprotective therapies. This protocol describes the controlled cortical impact (CCI) model of focal, open-head TBI. Specifically, parameters for producing a mild unilateral cortical injury are described. Behavioral consequences of CCI are analyzed using the adhesive tape removal test of bilateral sensorimotor integration. Regarding experimental therapy for TBI pathology, this protocol also illustrates a process for transplanting cultured cells into the brain. Neural cell cultures derived from human induced pluripotent stem cells (hiPSCs) were chosen for their potential to show superior functional restoration in human TBI patients. Chronic survival of hiPSCs in the host mouse brain tissue is detected using a modified DAB immunohistochemical process.

This protocol demonstrates methodologies for a mouse model of open-skull traumatic brain injury and transplantation of cultured human induced pluripotent stem cell-derived cells into the injury site. Behavioral and histologic tests of outcomes from these procedures are also described in brief.

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical ...

Generation of Induced Neural Stem Cells from Peripheral Mononuclear Cells and Differentiation Toward Dopaminergic Neuron Precursors for Transplantation Studies

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral mesencephalon (VM). Cell replacement therapy holds great promise for treatment of PD. Recently, induced neural stem cells (iNSCs) have emerged as a potential candidate for cell replacement therapy due to the reduced risk of tumor formation and the plasticity to give rise to region-specific neurons and glia. iNSCs can be reprogrammed from autologous somatic cellular sources, such as fibroblasts, peripheral blood mononuclear cells (PBMNCs) and various other types of cells. Compared with other types of somatic cells, PBMNCs are an appealing starter cell type because of the ease to access and expand in culture. Sendai virus (SeV), an RNA non-integrative virus, encoding reprogramming factors including human OCT3/4, SOX2, KLF4 and c-MYC, has a negative-sense, single-stranded, non-segmented genome that does not integrate into host genome, but only replicates in the cytoplasm of infected cells, offering an efficient and safe vehicle for reprogramming. In this study, we describe a protocol in which iNSCs are obtained by reprogramming PBMNCs, and differentiated into specialized VM DA neurons by a two-stage method. Then DA precursors are transplanted into unilaterally 6-hyroxydopamine (6-OHDA)-lesioned PD mouse models to evaluate the safety and efficacy for treatment of PD. This method provides a platform to investigate the functions and therapeutic effects of patient-specific DA neural cells in vitro and in vivo.

The protocol presents the reprogramming of peripheral blood mononuclear cells to induce neural stem cells by Sendai virus infection, differentiation of iNSCs into dopaminergic neurons, transplantation of DA precursors into the unilaterally-lesioned Parkinson&#39;s disease mouse models, and evaluation of the safety and efficacy of iNSC-derived DA precursors for PD treatment.