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>

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</ol>

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><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>Antibiotics (cefazolin, Kefzol)</td><td>Eurocept Pharmaceuticals</td><td>BE 106267</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 (&lt;em&gt;e.g.&lt;/em&gt;, 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="Mouse ocular dissection; eye removal process; optic disc view; brain dissection result." fo:content-width="5in" src="/files/ftp_upload/51936/51936fig1highres.jpg" width="500" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1. 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="Mouse brain schematic showing neural pathways; diagram; connectivity visualization; density scale." fo:content-width="5in" src="/files/ftp_upload/51936/51936fig2highres.jpg" width="500" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2. 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 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 ...

a-highly-reproducible-straightforward-method-to-perform-vivo-ocular

Research

JoVE Journal

Biology

23.3K Views.  KU Leuven - University of Leuven.  Het verwijderen van de ogen, ook wel enucleatie, een bruikbare strategie om aspecten van de visuele, cross-modale en ontwikkelingsplasticiteit bestuderen langs de zoogdieren visuele systeem is omdat het leidt tot onomkeerbare gedeeltelijke (monoculair) of volledige (binoculair) verlies van gezichtsvermogen. Hier beschrijven we een zeer reproduceerbare en duidelijke aanpak om te presteren in vivo enucleatie. 

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

Mouse Eye Enucleation for Remote High-throughput Phenotyping

The mouse eye is an important genetic model for the translational study of human ophthalmic disease. Blinding diseases in humans, such as macular degeneration, photoreceptor degeneration, cataract, glaucoma, retinoblastoma, and diabetic retinopathy have been recapitulated in transgenic mice.1-5 Most transgenic and knockout mice have been generated by laboratories to study non-ophthalmic diseases, but genetic conservation between organ systems suggests that many of the same genes may also play a role in ocular development and disease. Hence, these mice represent an important resource for discovering new genotype-phenotype correlations in the eye. Because these mice are scattered across the globe, it is difficult to acquire, maintain, and phenotype them in an efficient, cost-effective manner. Thus, most high-throughput ophthalmic phenotyping screens are restricted to a few locations that require on-site, ophthalmic expertise to examine eyes in live mice. 6-9 An alternative approach developed by our laboratory is a method for remote tissue-acquisition that can be used in large or small-scale surveys of transgenic mouse eyes. Standardized procedures for video-based surgical skill transfer, tissue fixation, and shipping allow any lab to collect whole eyes from mutant animals and send them for molecular and morphological phenotyping. In this video article, we present techniques to enucleate and transfer both unfixed and perfusion fixed mouse eyes for remote phenotyping analyses.

The dissection technique illustrates enucleation of the mouse eye for tissue fixation to perform phenotyping in high-throughput screens.

The mouse eye is an important genetic model for the translational study of human ophthalmic disease. Blinding diseases in humans, such as macular ...

Medicine

Video-oculography in Mice

Eye movements are very important in order to track an object or to stabilize an image on the retina during movement. Animals without a fovea, such as the mouse, have a limited capacity to lock their eyes onto a target. In contrast to these target directed eye movements, compensatory ocular eye movements are easily elicited in afoveate animals1,2,3,4. Compensatory ocular movements are generated by processing vestibular and optokinetic information into a command signal that will drive the eye muscles. The processing of the vestibular and optokinetic information can be investigated separately and together, allowing the specification of a deficit in the oculomotor system. The oculomotor system can be tested by evoking an optokinetic reflex (OKR), vestibulo-ocular reflex (VOR) or a visually-enhanced vestibulo-ocular reflex (VVOR). The OKR is a reflex movement that compensates for "full-field" image movements on the retina, whereas the VOR is a reflex eye movement that compensates head movements. The VVOR is a reflex eye movement that uses both vestibular as well as optokinetic information to make the appropriate compensation. The cerebellum monitors and is able to adjust these compensatory eye movements. Therefore, oculography is a very powerful tool to investigate brain-behavior relationship under normal as well as under pathological conditions (f.e. of vestibular, ocular and/or cerebellar origin).
Testing the oculomotor system, as a behavioral paradigm, is interesting for several reasons. First, the oculomotor system is a well understood neural system5. Second, the oculomotor system is relative simple6; the amount of possible eye movement is limited by its ball-in-socket architecture ("single joint") and the three pairs of extra-ocular muscles7. Third, the behavioral output and sensory input can easily be measured, which makes this a highly accessible system for quantitative analysis8. Many behavioral tests lack this high level of quantitative power. And finally, both performance as well as plasticity of the oculomotor system can be tested, allowing research on learning and memory processes9.
Genetically modified mice are nowadays widely available and they form an important source for the exploration of brain functions at various levels10. In addition, they can be used as models to mimic human diseases. Applying oculography on normal, pharmacologically-treated or genetically modified mice is a powerful research tool to explore the underlying physiology of motor behaviors under normal and pathological conditions. Here, we describe how to measure video-oculography in mice8.

Video-oculography is a very quantitative method to investigate ocular motor performance as well as motor learning. Here, we describe how to measure video-oculography in mice. Applying this technique on normal, pharmacologically-treated or genetically modified mice is a powerful research tool to explore the underlying physiology of motor behaviors.

Eye movements are very important in order to track an object or to stabilize an image on the retina during movement. Animals without a fovea, such as the ...

Neuroscience

In vivo-like Organotypic Murine Retinal Wholemount Culture

Targeted ablations of genes and analysis of animal models is the classical strategy for enrolling specific retinal gene function. However, transgenic, retina-specific or conditional knockout mouse models often display early lethality or suffer from severe malformations, preventing an analysis beyond embryonic or early postnatal stages.
Primary cell culture is an alternative to investigate the effects of exogenously applied recombinant factors, overexpression of genes or siRNA-mediated gene knockdown in a controlled environment. Dissociated cell culture has the advantage that the endogenous signals reaching the target cells are reduced, thereby facilitating the identification of exogenously triggered effects after pharmacological manipulation. However, important cell-cell interactions are initially destroyed by enzymatic digestion or mechanical dissociation, even if re-aggregated retinospheroid cultures1 are used.
By contrast, organotypic retinal wholemount cultures provide a system close to the physiological in vivo situation with neuronal interactions and connections still preserved2-5.
In this video article we provide a step by step demonstration of (1) the establishment of in vivo-like organotypic retinal wholemount cultures including dissection peculiarities of embryonic, postnatal and adult murine eyes and (2) a dissociation and cytospin procedure for analysis of neuronal apoptosis and retinal cell proliferation in organotypic wholemounts, e.g. after culture in the presence of exogenously applied recombinant factors.

In vivo-like Organotypic Murine Retinal Wholemount Culture

This video article demonstrates the establishment of organotypic retinal wholemount cultures and a cytospin procedure for analysis of exogenously induced effects. Organotypic retinal wholemount cultures mimic the in vivo situation and significantly facilitate the accessibility of murine retinas for experimental manipulations while circumventing the disadvantages of classical murine animal models.

Targeted ablations of genes and analysis of animal models is the classical strategy for enrolling specific retinal gene function. However, transgenic, ...

Monocular Visual Deprivation and Ocular Dominance Plasticity Measurement in the Mouse Primary Visual Cortex

Monocular visual deprivation is an excellent experimental paradigm to induce primary visual cortical response plasticity. In general, the response of the cortex to the contralateral eye to a stimulus is much stronger than the response of the ipsilateral eye in the binocular segment of the mouse primary visual cortex (V1). During the mammalian critical period, suturing the contralateral eye will result in a rapid loss of responsiveness of V1 cells to contralateral eye stimulation. With the continuing development of transgenic technologies, more and more studies are using transgenic mice as experimental models to examine the effects of specific genes on ocular dominance (OD) plasticity. In this study, we introduce detailed protocols for monocular visual deprivation and calculate the change in OD plasticity in mouse V1. After monocular deprivation (MD) for 4 days during the critical period, the orientation tuning curves of each neuron are measured, and the tuning curves of layer four neurons in V1 are compared between stimulation of the ipsilateral and contralateral eyes. The contralateral bias index (CBI) can be calculated using each cell&#39;s ocular OD score to indicate the degree of OD plasticity. This experimental technique is important for studying the neural mechanisms of OD plasticity during the critical period and for surveying the roles of specific genes in neural development. The major limitation is that the acute study cannot investigate the change in neural plasticity of the same mouse at a different time.

Here, we present detailed protocols for monocular visual deprivation and ocular dominance plasticity analysis, which are important methods for studying the neural mechanisms of visual plasticity during the critical period and the effects of specific genes on visual development.

Monocular visual deprivation is an excellent experimental paradigm to induce primary visual cortical response plasticity. In general, the response of the ...

Developmental Biology

Puncture-Induced Iris Neovascularization as a Mouse Model of Rubeosis Iridis

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant for targeting neovascular glaucoma, a sight-threatening complication of diabetic retinopathy. This method is based on the induction of iris vascular response by a series of self-sealing uveal punctures on BALB/c mice and takes advantage of the postpartum maturation of mouse ocular vasculature. Mouse pups undergo uveal punctures from postnatal day 12.5, when the pups naturally open their eyes, until postnatal day 24.5. Due to the transparency of the cornea, iris vasculature can be analyzed easily through time by noninvasive in vivo methods. Furthermore, the semitransparent iris of BALB/c mice can be flatmounted for detailed immunohistologic analysis with minimal non-specific background staining. In this model, angiogenesis is mainly driven by the inflammatory and plasminogen activating systems. The puncture-induced model is the first to induce iris neovascularization in small rodents, and has the advantage of allowing direct noninvasive in vivo analysis of the angiogenic process. Moreover, the model can be combined with angiogenic modulating substances, which highlights its potential in the study of angiogenesis with an in vivo perspective.

Iris neovascularization, a common complication of ischemic retinal disease, may lead to sight-threatening neovascular glaucoma. Here, we describe a murine protocol for inducing experimental iris neovascularization that may be used for noninvasive evaluation of angiogenesis-modulating substances.

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant ...

Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

To fully understand the cellular physiology of neurons and glia in behaving animals, it is necessary to visualize their morphology and record their activity in vivo in behaving mice. This paper describes a method for the implantation of a chronic cranial window to allow for the longitudinal imaging of brain cells in awake, head-restrained mice. In combination with genetic strategies and viral injections, it is possible to label specific cells and regions of interest with structural or physiological markers. This protocol demonstrates how to combine viral injections to label neurons in the vicinity of GCaMP6-expressing astrocytes in the cortex for simultaneous imaging of both cells through a cranial window. Multiphoton imaging of the same cells can be performed for days, weeks, or months in awake, behaving animals. This approach provides researchers with a method for viewing cellular dynamics in real time and can be applied to answer a number of questions in neuroscience.

Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

Presented here is a protocol for the implantation of a chronic cranial window for the longitudinal imaging of brain cells in awake, head-restrained mice.

To fully understand the cellular physiology of neurons and glia in behaving animals, it is necessary to visualize their morphology and record their ...

Corneal Sensory Fiber Damage Protocols for Axon Regeneration Studies

Three Strategies to Induce Neurotrophic Keratitis and Nerve Regeneration in Murine Cornea

The cornea is a transparent tissue that covers the eye and is crucial for clear vision. It is the most innervated tissue in the body. This innervation provides sensation and trophic function to the eye and contributes to preserving corneal integrity. The pathological disruption of this innervation is termed neurotrophic keratitis. This can be triggered by injury to the eye, surgery, or disease. In this study, we propose three different protocols for inflicting damage on the innervation in ways that recapitulate the three types of cases generally encountered in the clinic.
The first method consists in making an abrasion of the epithelium with an ophthalmic burr. This involves the removal of the epithelial layer, the free nerve endings, and the subbasal plexus in a manner similar to the photorefractive keratectomy surgery performed in the clinic. The second method&#160;only targets the innervation by sectioning it at the periphery with a biopsy punch, maintaining the integrity of the epithelium. This method is similar to the first steps of lamellar keratoplasty and leads to a degeneration of the innervation followed by regrowth of the axons in the central cornea. The last method damages the innervation of a transgenic mouse model using a multiphoton microscope, which specifically localizes the site of cauterization of the fluorescent nerve fibers. This method inflicts the same damage as photokeratitis, an overexposure to UV light.
This study describes different options for investigating the physiopathology of corneal innervation, particularly the degeneration and regeneration of the axons. Promoting regeneration is crucial for avoiding such complications as epithelium defects or even perforation of the cornea. The proposed models can help test new pharmacological molecules or gene therapy that enhance nerve regeneration and limit disease progression.

Author Spotlight: Advancing Corneal Innervation Research Through Innovative Models 

Here, we propose three different methods of damaging the sensory fibres innervating the cornea. These methods facilitate the study of axon regeneration in mice. These three methods, which are adaptable to other animal models, are ideal for the study of corneal innervation physiology and regeneration.

The cornea is a transparent tissue that covers the eye and is crucial for clear vision. It is the most innervated tissue in the body. This innervation ...

Microdissection of the Rodent Eye

The ocular micro-dissection of the rodent eye involves the segmentation of the enucleated eyeball with the attached nictitating membrane, or third eyelid, to obtain the anterior and posterior eyecups. With this technique, the sub-parts of the eye, including the corneal tissue, neural tissue, retinal pigment epithelial (RPE) tissue, and lens, can be obtained for wholemounts, cryo-sectioning, and/or single-cell suspensions of a specific ocular tissue. The presence of the third eyelid presents unique and significant advantages, as it benefits the maintenance of the orientation of the eye, which is important for understanding eye physiology following any localized intervention or in studies involving ocular analysis relating to the eye&#39;s spatial topography.
In this method, we enucleated the eyeball at the socket along with the third eyelid by carefully and slowly cutting through the extraocular muscles and severing the optic nerve. The eyeball was pierced through the corneal limbus using a microblade.&#160;The incision was used as the point of entry, allowing for cutting along the corneal-scleral junction by inserting micro-scissors through the incision point. Small and continuous cuts along the circumference were made until the cups separated. These could be further dissected by gently peeling the translucent layer of the neural retina using Colibri suturing forceps to obtain the neural retina and RPE layers. Further, three/four equidistant cuts were made from the periphery perpendicularly to the optic center until the optic nerve was reached. This opened the hemispherical cups into a floret shape so that they fell flat and could be easily mounted. This technique has been used in our lab for corneal wholemounts and retinal sections. The presence of the third eyelid delineates the nasal-temporal orientation, which allows for the study of various cell therapy interventions post-transplantation and, thus, the targeted physiological validation vital for visualization and accurate representation in such studies.

This paper presents a protocol for ocular micro-dissection in rodents. The process involves the enucleation of the eyeball along with the nictitating membrane (i.e., the third eyelid). This is then followed by the separation of the posterior and anterior eye cups.

The ocular micro-dissection of the rodent eye involves the segmentation of the enucleated eyeball with the attached nictitating membrane, or third eyelid, ...

A Modified Biopsy Punch Method for Precise Alkali Burns in Mice

Corneal and Limbal Alkali Injury Induction Using a Punch-Trephine Technique in a Mouse Model

The cornea is critical for vision, and corneal healing after trauma is fundamental in maintaining its transparency and function. Through the study of corneal injury models, researchers aim to enhance their understanding of how the cornea heals and develop strategies to prevent and manage corneal opacities. Chemical injury is one of the most popular injury models that has extensively been studied on mice. Most previous investigators have used a flat paper soaked in sodium hydroxide to induce corneal injury. However, inducing corneal and limbal injury using flat filter paper is unreliable, since the mouse cornea is highly curved. Here, we present a new instrument, a modified biopsy punch, that enables the researchers to create a well-circumscribed, localized, and evenly distributed alkali injury to the murine cornea and limbus. This punch-trephine method enables researchers to induce an accurate and reproducible chemical burn to the entire murine cornea and limbus while leaving other structures, such as the eyelids, unaffected by the chemical. Moreover, this study introduces an enucleation technique that preserves the medial caruncle as a landmark for identifying the nasal side of the globe. The bulbar and palpebral conjunctiva, and lacrimal gland are also kept intact using this technique. Ophthalmologic examinations were performed via slit lamp biomicroscope and fluorescein staining on days 0, 1, 2, 6, 8, and 14 post-injury. Clinical, histological, and immunohistochemical findings confirmed limbal stem cell deficiency and ocular surface regeneration failure in all experimental mice. The presented alkali corneal injury model is ideal for studying limbal stem cell deficiency, corneal inflammation, and fibrosis. This method is also suitable for investigating pre-clinical and clinical efficacies of topical ophthalmologic medications on the murine corneal surface.

Author Spotlight: Advancing Research in Corneal Opacity Treatment and Regeneration 

This protocol describes a method to induce an accurate and reproducible corneal and limbal alkali injury in a mouse model. The protocol is advantageous as it allows for an evenly distributed injury to the highly curved mouse cornea and limbus.

The cornea is critical for vision, and corneal healing after trauma is fundamental in maintaining its transparency and function. Through the study of ...

Replicating Cataract Surgical Techniques in a Mouse Model Through LEC Extraction

Modeling Cataract Surgery in Mice

Cataract surgery (CS) is an effective treatment for cataracts, a major cause of visual disability worldwide. However, CS leads to ocular inflammation, and in the long term, it can result in posterior capsular opacification (PCO) and/or lens dislocation driven by the post-surgical overgrowth of lens epithelial cells (LECs) and their conversion to myofibroblasts and/or aberrant fiber cells. However, the molecular mechanisms by which CS results in inflammation and PCO are still obscure because most in vitro models do not recapitulate the wound healing response of LECs seen in vivo, while traditional animal models of cataract surgery, such as rabbits, do not allow the genetic manipulation of gene expression to test mechanisms. Recently, our laboratory and others have successfully used genetically modified mice to study the molecular mechanisms that drive the induction of proinflammatory signaling and LEC epithelial to mesenchymal transition, leading to new insight into PCO pathogenesis. Here, we report the established protocol for modeling cataract surgery in mice, which allows for robust transcriptional profiling of the response of LECs to lens fiber cell removal via RNAseq, the evaluation of protein expression by semi-quantitative immunofluorescence, and the use of modern mouse genetics tools to test the function of genes that are hypothesized to participate in the pathogenesis of acute sequelae like inflammation as well as the later conversion of LECs to myofibroblasts and/or aberrant lens fiber cells.

Author Spotlight: Unraveling the Molecular Mechanisms in PCO and Fibrosis Following Cataract Surgery

This method models cataract surgery in vivo by removing lens fiber cells from adult mice and leaving behind the capsular bag with attached lens epithelial cells (LECs). The injury response is then assessed at various times post-surgery using molecular and morphological criteria.

Cataract surgery (CS) is an effective treatment for cataracts, a major cause of visual disability worldwide. However, CS leads to ocular inflammation, and ...

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

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