We thank Ellen Ytebrouck for her valuable information regarding method application in rat. This work was supported by the Research Council of the KU Leuven (OT09/22) and the Fund for Scientific Research-Flanders (FWO Vlaanderen). Jeroen Aerts is supported by a Ph.D. fellowship of the Agency for Innovation through Science and Technology Flanders (IWT Vlaanderen) and Julie Nys by a Ph.D. grant from FWO Flanders.

All experiments were approved by the ethical research committee of KU Leuven and were in strict accordance with the European Communities Council Directive of 22 September 2010 (2010/63/EU) and with the Belgian legislation (KB of 29 May 2013). Every possible effort was made to minimize animal suffering and to reduce the number of animals.
1. Animal Treatment and Anesthetics
<ol>
	<li>Anesthetize the mouse with an intraperitoneal injection of a mixture of ketamine hydrochloride (75 mg/ml) and medetomidine hydrochloride (1 mg/kg) in saline.</li>
	<li>Check the reflexes by pinching the toes with a forceps to assure the mouse is completely sedated.</li>
	<li>Apply 70% ethanol to disinfect the eyelids and the region surrounding the eye using a cotton tip. Check the eyelid reflex to additionally assess the degree of sedation.</li>
</ol>
2. Removing the Eye
<ol>
	<li>Make sure the animal resides on a flat, dry and smooth surface.</li>
	<li>Sterilize a forceps with a curved, serrated tip (preferred tip size: 0.5 x 0.4 mm).</li>
	<li>Gently press onto the canthus (corner of the eye) with the forceps until the eyeball is displaced from the socket and the optic nerve is reachable.</li>
	<li>Guide the forceps behind the eye. Press and hold the optic nerve firmly,&#160;preferably with the beginning of the curve and not the very tip of the forceps. This will help to lift the globe from the socket and to clamp the complete optic nerve.</li>
	<li>Make circular movements with the hand holding the forceps in the direction with the least resistance while the mouse remains on the flat surface. The mouse will swing along the surface according to the direction of the hand movement.</li>
	<li>Perform this action with gradually increasing speed until the optic nerve is constricted in two (usually between 7 to 15 circular movements, approximately a half to one full turn per second). Hence, the detached eyeball is removed.</li>
</ol>
3. Post-operative Care
<ol>
	<li>In case of bleeding (rare), fill the orbit with a viscous coagulate and hemostatic agent.</li>
	<li>Reverse the anesthesia by injecting 1 mg/kg of atipamezol hydrochloride in saline intraperitoneally.</li>
	<li>Administer 1 mg/kg of Meloxicam intraperitoneally every 24 hr&#160;to relieve pain.</li>
	<li>Apply eye ointment to the remaining eye to prevent dehydration of the cornea.</li>
	<li>Let the animal recover on a heating plate or wrap the animal in insulating material in a separate cage to control body temperature.</li>
	<li>Measure the weight of the mouse each day for at least 2&#160;days. Loss of weight can indicate suffering and in this case, continue Meloxicam treatment until the animal is fully recovered.</li>
</ol>

<ol>
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The authors have nothing to disclose.

To perform a successful enucleation according to our method, the most critical steps to consider are: 1) using a forceps with a curved and serrated tip of the appropriate size; 2) performing the enucleation on a smooth and dry surface; and 3) gradually speeding up the circular movements in the direction with the least friction.
For an effective outcome it is essential to use an appropriate forceps characterized by a curved and serrated tip (preferred tip size: mouse: 0.5 x 0.4 mm; rat: 2.15 x 1.3 mm). The curvature allows for an easy access to the optic nerve after eyeball displacement and is necessary for correct hand placement when performing the circular movements. Smooth tips are not recommended since they lack the necessary grip when holding the optic nerve. Failure to hold the optic nerve properly during the circular movements results in rupture of the ophthalmic artery, poor detachment of the eye and hence poor reproducibility.&#160;Therefore it is recommended to first practice this technique on euthanized animals for optimization of the forceps handling in order to guarantee maximal animal welfare once applying the method in vivo. Successful eye enucleation has recently also been performed in the rat in our lab using the same technique except for turning the animal body manually and keeping the forceps stationary.
One limitation of the technique is that it could possibly damage the retina. Therefore this method is less suitable for collecting retinas to perform histology5. Moreover, our method is limited to post eye opening ages since the eyeball needs to be displaced from the socket without removing or cutting the eyelids.
Eye enucleation in different species, including rodents, is routinely performed using alternative methods, which often entail the removal of the eyelids and cutting the optic nerve18,23-25. These methods tend to be more invasive and have a higher learning curve than the technique described here. Without the need for removing or suturing the eyelids, the post surgery recovery time is minimized, resulting in higher animal welfare and more reproducible results.

Removing an eye and thereby irreversibly destructing the sensory receptor surface (retina), imposes a considerable loss of sensory input along the visual pathway. The enucleation model in the juvenile and adult visual system has proven to be valuable in understanding the development, plasticity and function of different visual centers1-4. The molecular, cellular and physiological consequences of this sensory deprivation can provide insights into how normal development is regulated and how established cortical circuits cope and change their structure and function in response to such an extensive alteration in experience.
Different methods of visual deprivation exist and they all have their specific advantages in vision-related research. For example dark rearing specifically eliminates visually driven activity yet it does not affect the spontaneous retinal activity. Similarly, lid sutures or eye patches remove patterned visual input without disturbing spontaneous activity but they allow dispersed light penetration through the closed eyes. Those methods are reversible and have been shown to be valuable in understanding the role of patterned vision and low-level correlation of binocular inputs in sculpting cortical circuits during development6-8. In glaucoma research, the optic nerve crush model in adult animals has been widely used because it establishes a progressive loss of retinal ganglion cell inputs that constitute the optic nerve9,10. On the other hand, enucleation, where the eye and thus the retina is completely and instantly removed, is the appropriate choice of deprivation when the aim is to irreversibly remove both spontaneous and patterned vision at once. It also induces a robust intraocular activity imbalance which can enhance the signal to noise ratio in activity mapping studies11,12. Comparing functional and structural changes in response to enucleation with those after deprivation by less drastic methods such as lid suture for example, might also expose new insights into the role of spontaneous retinal activity in both homeostatic and synaptic types of plasticity.
Enucleation triggers a loss of trophic influences in direct retinal targets. For instance, BDNF levels are significantly downregulated in the lateral geniculate nucleus (LGN) and superior colliculus of adult enucleated rats13. Reactive oxygen species, which function as messenger molecules to mediate structural remodeling, were also detected in subcortical structures of the adult rat visual system14. Furthermore, microglial and astroglial activation across different subcortical visual target structures in the mouse occur in a specific post-enucleation time frame of one week15. Together, optic deafferentation results in different subcortical responses at the glial, structural and molecular level. Despite these subcortical effects, it does not necessarily implicate effects at the cortical level16. Noteworthy, cross-modal cortical plasticity, including modifications in other sensory areas next to the strengthening of non-visual inputs to the deprived visual cortex occur after both monocular (ME)3,4,17,18 and binocular (BE)1,17 enucleation.
Apart from contributing to visual neuroscience, enucleation as a type of deafferentation can be used to study the balance between neuroprotective19 and neurodegenerative20-22 properties of the central nervous system.
Different procedures to perform enucleation are already described in literature. Certain methodologies for in vivo ME in rats and mice are less straightforward due to unnecessary sectioning of orbital muscles and tissue23-25. Other publications such as Mahajan et al. (2011)5 provide a detailed protocol using blunt dissection for a high-throughput collection of eyes to study the genotype-phenotype correlations, likely post-mortem. For their purpose, the method is convenient and fast. However, this method is less suitable for in vivo enucleation when one opts to study the afferent visual pathway following enucleation (in live animals) rather than the eye itself. In such a setting, post-enucleation survival is of high importance. Also, minimal in vivo damage and preservation of the optic nerve and orbital tissue is favorable. Here, we present an alternative enucleation method, more similar to the one described by Faguet et al. (2008)26, that offers certain advantageous properties: it is associated with a swift post-operative recovery and is characterized by a very low learning threshold for researchers. In general, different methods are complementary depending on the focus of subsequent research: eye morphology or visual pathway research.
In sum, enucleation can be applied from vision research towards investigations of homeostatic and cross-modal brain plasticity, glial response properties, and axon stability. In this visualized article, we demonstrate a feasible and reliable method for in vivo eye enucleation in the mouse.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Ketamine hydrochloride (Anesketin)</td>
			<td>Dechra Veterinary Products (Eurovet)</td>
			<td>BE-V136516</td>
			<td></td>
		</tr>
		<tr>
			<td>Medetomidine hydrochloride (Domitor)</td>
			<td>Orion Corporation (Janssen Animal Health)</td>
			<td>BE-V151742</td>
			<td></td>
		</tr>
		<tr>
			<td>Atipamezol hydrochloride (Antisedan)</td>
			<td>Orion Corporation (Elanco Animal Health)</td>
			<td>BE-V153352</td>
			<td></td>
		</tr>
		<tr>
			<td>Meloxicam (Metacam)</td>
			<td>Boehringer Ingelheim Vetmedica</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Eye ointment (Fucithalmic)</td>
			<td>Leo Pharma nv-sa</td>
			<td>BE 144654</td>
			<td></td>
		</tr>
		<tr>
			<td>Moria MC31 Forceps - Serrated Curved</td>
			<td>Fine Science Tools</td>
			<td>11370-31</td>
			<td>For application in the mouse. Any forceps with similar dimensions can be used as long as the tip is curved and serrated.</td>
		</tr>
		<tr>
			<td>Narrow Pattern Forceps - Curved</td>
			<td>Fine Science Tools</td>
			<td>11003-13</td>
			<td>For application in the rat. Any forceps with similar dimensions can be used as long as the tip is curved and serrated.</td>
		</tr>
		<tr>
			<td>Hemostatic cotton wool</td>
			<td>Qualiphar</td>
			<td>N/A</td>
			<td>Other hemostatic agents are equally suitable (e.g. Viscostat, #649, Ultradent Products)</td>
		</tr>
	</tbody>
</table>

a highly reproducible and straightforward method to perform in vivo ocular enucleation in the mouse after eye opening

Enucleation or the surgical removal of an eye can generally be considered as a model for nerve deafferentation. It provides a valuable tool to study the different aspects of visual, cross-modal and developmental plasticity along the mammalian visual system1-4.
Here, we demonstrate an elegant and straightforward technique for the removal of one or both eyes in the mouse, which is validated in mice of 20 days old up to adults. Briefly, a disinfected curved forceps is used to clamp the optic nerve behind the eye. Subsequently, circular movements are performed to constrict the optic nerve and remove the eyeball. The advantages of this technique are high reproducibility, minimal to no bleeding, rapid post-operative recovery and a very low learning threshold for the experimenter. Hence, a large amount of animals can be manipulated and processed with minimal amount of effort. The nature of the technique may induce slight damage to the retina during the procedure. This side effect makes this method less suitable as compared to Mahajan et al. (2011)5 if the goal is to collect and analyze retinal tissue. Also, our method is limited to post-eye opening ages (mouse: P10 - 13 onwards) since the eyeball needs to be displaced from the socket without removing the eyelids. The in vivo enucleation technique described in this manuscript has recently been successfully applied with minor modifications in rats and appears useful to study the afferent visual pathway of rodents in general.

Figure 1 illustrates the successful removal of the eye using the described protocol and is characterized by the absence of bleeding or any apparent physical damage to the orbital tissue or eye socket (Figures 1A, 1B). Furthermore, the removed eye has a smooth cornea, choroid and optical disc, indicative for a completely intact globe (Figure 1C). Since our protocol encompasses the clamping of the optic nerve behind the eye and mechanical turning, the optic nerve of the removed eye is constricted at the base of the retina (Figure 1D). Performing the described procedure results in a clean-cut optic nerve without any damage to the surrounding brain area (Figure 1E).
Monocular enucleation, in combination with activity mapping (Figure 2), allows one to sharply delineate the functional or eye specific input regions in the contralateral visual cortex of the mouse12,27 or even ocular dominance columns in higher-order mammals like monkeys28.
In experiments with mice, the removal of one (ME) or both eyes (BE) combined with targeted visual stimulation and the detection of zif268 mRNA or c-Fos protein expression levels was applied to uncover regional neuronal activation in the visual cortex12,27. In contrast to visually stimulated controls (Figure 2A), BE mice showed basal activity in the visual cortex due to complete lack of visual input (Figure 2B). As such, the borders between visual with non-visual cortex (i.e. somatosensory cortex at more anterior sections and auditory cortex in more posterior sections) were uncovered. Results from ME mice with a one-week survival time visualized the eye specific input regions in the contralateral visual cortex. The two monocularly driven regions were hypoactive and located medial and lateral of the central binocular zone (Figure 2C).
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51936/51936fig1highres.jpg" width="500" /> 
Figure 1.&#160;Qualitative evaluation of the post-enucleation state of the eye socket, the removed eye and the optic nerve. After removal of the eye with a curved forceps (A) no bleeding or damage is observed in the eye socket (B). The removed eye is completely intact as reflected by a normal appearance of the cornea and choroid (C,D). The optic nerve is constricted at the optic disc where it leaves the eye (D). Examination of the ventral part of the brain reveals a clean cut optic nerve (asterisk) and no apparent damage to other structures (E). Scale bars in C, D: 1 mm. Scale bar in E: 5 mm. A: anterior; L: left; P: posterior; R: right.
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51936/51936fig2highres.jpg" width="500" /> 
Figure 2.&#160;Functional eye-input specific subdivisions in the mouse visual cortex as revealed by enucleation. Black and gray lines that connect the eyes and cortex represent the crossing over of retinal afferents and the eye specific input regions. Neural activity is visualized on coronal sections of control (A), BE (B), and ME (C) mice by radioactive in situ hybridization (ISH) for zif268 (gray scale) around Bregma level -3.40 mm. In control animals (A), the visual cortex in both hemispheres expresses high activity following visual stimulation. When one or both eye(s) are extirpated, a clear decrease in the activity signal is visible in corresponding deprived cortical regions. Monocularly enucleated (C) mice show a zone of high activity in the binocular zone of the visual cortex surrounded by a decreased signal in the monocular zones contralateral to the removed eye. Scale bar: 2 mm. Reprinted with permission from Van Brussel&#160;et al12.

Watch this Scientific Journal Video about A Highly Reproducible and Straightforward Method to Perform In Vivo Ocular Enucleation in the Mouse after Eye Opening at JoVE.com

A Highly Reproducible and Straightforward Method to Perform In Vivo Ocular Enucleation in the Mouse after Eye Opening

The removal of eyes, also called enucleation, provides a useful strategy to study aspects of visual, cross-modal, and developmental plasticity along the mammalian visual system since it induces irreversible partial (monocular) or complete (binocular) vision loss. Here we describe a highly reproducible and straightforward approach to perform in vivo enucleation.

A Highly Reproducible and Straightforward Method to Perform In Vivo Ocular Enucleation in the Mouse after Eye Opening

Enucleation or the surgical removal of an eye can generally be considered as a model for nerve deafferentation. It provides a valuable tool to study the ...

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23.1K Views.  KU Leuven - University of Leuven. The removal of eyes, also called enucleation, provides a useful strategy to study aspects of visual, cross-modal, and developmental plasticity along the mammalian visual system since it induces irreversible partial (monocular) or complete (binocular) vision loss. Here we describe a highly reproducible and straightforward approach to perform in vivo enucleation.

Video: A Highly Reproducible and Straightforward Method to Perform In Vivo Ocular Enucleation in the Mouse after Eye Opening

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.

Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

A Method for Islet Transplantation to the Omentum in Mouse

Islet transplantation has been proposed to be a potential treatment for type 1 diabetes. Recent compelling evidence indicates that intravascular islet infusion is far from ideal and therefore, the omentum is re-emerging as a potentially valuable site for islet transplantation. This experiment requires the isolation of high quality islets and the implantation of the islets to the diabetic recipients. Transplantation to the omentum requires surgical steps that can be better demonstrated visually. Here, the detailed steps for this procedure are presented. Two methods of mixing the isolated islets with hydrogel before placing the mixture into the omental pouch of diabetic mice are described here. Different hydrogels are used for the different conditions. Blood glucose levels of diabetic mouse recipients of syngeneic islets in the omentum were monitored for up to 35 days. Some animals were sacrificed after 14 days to perform immuno-histochemical analysis. This pre-clinical transplantation approach can be used as preliminary data leading up to translation to clinical transplantation.

A method for the omental transplantation of islets in a mouse is introduced. The isolated islets are mixed with hydrogel and the mixture is placed into the omental pouch of a diabetic mouse. Then, the blood glucose is monitored, and immuno-histochemical analysis is performed.

Islet transplantation has been proposed to be a potential treatment for type 1 diabetes. Recent compelling evidence indicates that intravascular islet ...

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to ...

A Highly Scalable Approach to Perform Ecological Surveys of Selfing Caenorhabditis Nematodes

Caenorhabditis elegans is one of the major model organisms in biology, but only recently have researchers focused on its natural ecology. The relative sparsity of information about C. elegans in its natural context comes from the challenges involved in the identification of the small nematode in nature. Despite these challenges, an increasing focus on the ecology of C. elegans has caused a wealth of new information regarding its life outside of the laboratory. The intensified search for C. elegans in nature has contributed to the discovery of many new Caenorhabditis species and revealed that congeneric nematodes frequently cohabitate in the wild, where they feed on microbial blooms associated with rotting plant material. The identification of new species has also revealed that the androdioecious mating system of males and self-fertilizing hermaphrodites has evolved three times independently within Caenorhabditis. The other two selfing species, C. briggsae and C. tropicalis, share the experimental advantages of C. elegans and have enabled comparative studies into the mechanistic basis of important traits, including self-fertilization. Despite these advances, much remains to be learned about the ecology and natural diversity of these important species. For example, we still lack functional information for many of their genes, which might only be attained through an understanding of their natural ecology. To facilitate ecological research of selfing Caenorhabditis nematodes, we developed a highly scalable method to collect nematodes from the wild. Our method makes use of mobile data collection platforms, cloud-based databases, and the R software environment to enhance researchers&#39; ability to collect nematodes from the wild, record associated ecological data, and identify wild nematodes using molecular barcodes.

A Highly Scalable Approach to Perform Ecological Surveys of Selfing Caenorhabditis Nematodes

This protocol can be used to perform large-scale ecological surveys of selfing Caenorhabditis nematodes. The primary advantage of this method is the efficient organization and analysis of ecological and molecular data associated with the nematodes collected from nature.

Caenorhabditis elegans is one of the major model organisms in biology, but only recently have researchers focused on its natural ecology. The relative ...

In Vitro Method to Study Sex-Based Differences in Conjunctival Goblet Cells

Dry eye is a multi-factorial disease affecting ocular surface health, with a profoundly higher prevalence in women. Disruption of the gel-forming mucin that is secreted by conjunctival goblet cells (CGCs) onto the ocular surface contributes to multiple ocular surface diseases. The elimination of exogenous sex hormones is essential to obtain consistent results during in vitro study of sex-based differences in CGCs. This paper describes a method to minimize the presence of exogenous hormones in the study of sex-based differences in CGCs while maintaining their physiological function. CGCs from postmortem human donors of both sexes were cultured from pieces of the conjunctiva in RPMI medium with 10% fetal bovine serum (FBS) (referred to as the complete medium) until confluency. Nearly 48 h before the start of the experiments, CGCs were transferred to RPMI medium without phenol red or FBS but with 1% BSA (referred to as phenol-red-free medium). The normal cellular function was studied by measuring the increase in intracellular [Ca2+] ([Ca2+]i) after carbachol (Cch, 1 x 10-4 M) stimulation using fura 2/acetoxymethyl (AM) microscopy. The result shows that CGCs maintained normal function in the phenol-red-free media after 48 h. No significant difference in [Ca2+]i response was observed between phenol red-free RPMI medium and complete medium upon Cch stimulation. Therefore, we recommend using the phenol-red free RPMI medium with 1% BSA to eliminate exogenous hormones without altering the normal function of CGCs in the study of sex-based differences.

In Vitro Method to Study Sex-Based Differences in Conjunctival Goblet Cells

Phenol red-free/fetal bovine serum-free medium is a better option than advanced RPMI to eliminate exogenous hormones without altering the normal function of conjunctival goblet cells in the study of sex-based differences.

Dry eye is a multi-factorial disease affecting ocular surface health, with a profoundly higher prevalence in women. Disruption of the gel-forming mucin ...

Modeling and Analyzing Retinal Degeneration

LipidUNet-Machine Learning-Based Method of Characterization and Quantification of Lipid Deposits Using iPSC-Derived Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a monolayer of hexagonal cells located at the back of the eye. It provides nourishment and support to photoreceptors and choroidal capillaries, performs phagocytosis of photoreceptor outer segments (POS), and secretes cytokines in a polarized manner for maintaining the homeostasis of the outer retina. Dysfunctional RPE, caused by mutations, aging, and environmental factors, results in the degeneration of other retinal layers and causes vision loss. A hallmark phenotypic feature of degenerating RPE is intra and sub-cellular lipid-rich deposits. These deposits are a common phenotype across different retinal degenerative diseases. To reproduce the lipid deposit phenotype of monogenic retinal degenerations in vitro, induced pluripotent stem cell-derived RPE (iRPE) was generated from patients&#39; fibroblasts. Cell lines generated from patients with Stargardt and Late-onset retinal degeneration (L-ORD) disease were fed with POS for 7 days to replicate RPE physiological function, which caused POS phagocytosis-induced pathology in these diseases. To generate a model for age-related macular degeneration (AMD), a polygenic disease associated with alternate complement activation, iRPE was challenged with alternate complement anaphylatoxins. The intra and sub-cellular lipid deposits were characterized using Nile Red, boron-dipyrromethene (BODIPY), and apolipoprotein E (APOE). To quantify the density of lipid deposits, a machine learning-based software, LipidUNet, was developed. The software was trained on maximum-intensity projection images of iRPE on culture surfaces. In the future, it will be trained to analyze three-dimensional (3D) images and quantify the volume of lipid droplets. The LipidUNet software will be a valuable resource for discovering drugs that decrease lipid accumulation in disease models.

Author Spotlight: Unraveling the Pathogenesis of Age-Related Macular Degeneration and Discovering Potential Therapies 

Degenerative eye diseases that affect the retinal pigment epithelium layer of the eye have monogenic and polygenic origins. Several disease models and a software application, LipidUNet, have been developed to study mechanisms of disease, as well as potential therapeutic interventions.