Name: m&#252;ller glia cell activation in a laser-induced retinal degeneration and regeneration model in zebrafish
Uploaded: 2017-10-27T14:00:00
Description: Watch this Scientific Journal Video about MÃ¼ller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish at JoVE.com

We thank Martin Zinkernagel, MD, PhD and Miriam Reisenhofer, PhD for her scientific input at establishing the model and Federica Bisignani for her excellent technical assistance.

All experiments adhered to the Statement for the Use of Animals in Ophthalmic and Vision Research of the Association for Research in Vision and Ophthalmology (ARVO) and respect the related regulations of the governmental authorities.
1. Animals
<ol>
	<li>Maintain TgBAC (gfap:gfap-GFP) zebrafish 167 (AB) strain aged between 6 - 9 months under standard conditions in water with a temperature of 26.5 &#176;C and a 14/10 h light/dark cycle18.</li>
	<li>Follow the animal ca...

<ol>
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An immunofluorescence study with antibodies to the glial fibrillary acidic (GFA) protein.</a> Exp Eye Res. 28 (1), 63-69 (1979).</li><li>Sherpa, T., Fimbel, S. M., Mallory, D. E., Maaswinkel, H., Spritzer, S. D., Sand, J. A., Li, L., Hyde, D. R., Stenkamp, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ganglion+cell+regeneration+following+whole-retina+destruction+in+zebrafish.">Ganglion cell regeneration following whole-retina destruction in zebrafish.</a> Dev Neurobiol. 68 (2), 166-181 (2008).</li><li>Cameron, D. A., Carney, L. 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Retinal regeneration/degeneration in the zebrafish has been investigated by different approaches such as cytotoxin-mediated cell death22, mechanical injury23, and thermal injury24. We employed a 532 nm diode laser to damage the zebrafish retina. Thereby, our model offers several advantages. For instance, we rapidly created a well-defined area of injury localized in the outer retina, specifically in the PRs layer. Furthermore, this experimental set-up...

Vision is probably the most essential sense of the human being and its impairment has a high socio-economic impact. In the industrialized world, retinal degenerative diseases account for the majority of vision loss and blindness among the adult population1. Retinitis pigmentosa (RP) is the most common inherited cause of blindness in people between the ages of 20 and 60, affecting approximately 1.5 million people worldwide2,3. It is a heterogeneous family of inherited retinal disorders characterized by progressive loss of the photoreceptors (PRs) followed by degeneration of retinal pigment epithelium and, subsequently, gliosis and remodeling of inner neurons4. The course of the disease can be explained by the incremental loss of the two PR cell types, usually starting with rods, which are responsible for achromatic vision in dim light, and cones, which are essential for color vision and visual acuity5. A single gene defect is sufficient to cause RP. So far more than 130 mutations in over 45 genes have been associated with the disease6. This leads to varying disease phenotypes and is one reason that gene therapy is non-generalizable and thus an intricate therapeutic approach. Therefore, there is an urgent need to develop new general therapeutic approaches to treat retinal degenerations in blinding diseases.
Retinal degeneration often involves PR loss; therefore, PR cell death is a hallmark of the degenerative processes in the retina7. It has already been demonstrated that PR cell death stimulates M&#252;ller glia cell&#160;(MC) activation and proliferation8. MCs, the major glial cell type in the vertebrate retina, were once considered to be nothing more than a &#34;glue&#34; between retinal neurons. In recent years, many studies have shown that MCs act as more than mere structural support9. Among the different functions, MCs participate also in neurogenesis and repair10. Indeed, in response to diffusible factors from the degenerating retina, MCs significantly increase glial fibrillary acidic protein (GFAP) expression. Therefore, GFAP labelling can be used as a marker for MC activation as a secondary response to retinal injury and degeneration11.
Recently, we developed a novel adaptation of focal injury using a laser to induce retinal degeneration in zebrafish (Danio rerio). Focal injury is advantageous for studying certain biological processes such as the migration of cells into the injured site and the precise timing of events that take place during retinal regeneration12. Furthermore, the zebrafish has become important in visual research because of the similarities between its visual system and that of other vertebrates. Gross morphological and histological features of human and teleost retinae display few differences. Accordingly, human and zebrafish retinae contain the same major cell classes organized in the same layered pattern, where light-sensing photoreceptors occupy the outermost layer, while the retinal projection neurons, the ganglion cells, reside in the innermost neuronal layer, proximal to the lens. The retinal interneurons, the amacrine, bipolar, and horizontal cells, localize in between the photoreceptor and ganglion cell layers13. Furthermore, the zebrafish retina is cone-dominated and therefore closer to the human retina than, for example, the intensively studied rodent retina. A fascinating difference between teleost and mammals is the persistent neurogenesis in fish retina and retinal regeneration after damage. In zebrafish, MCs can dedifferentiate and mediate regeneration in injured retina14,15. In chicken, MCs have some capacity also to re-enter the cell cycle and to dedifferentiate. Following retinal injury in adult fish, MCs adopt certain characteristics of progenitor and stem cells, migrate to the damaged retinal tissue and produce new neurons16. Gene expression profiling of mammalian MCs revealed unexpected similarities to retinal progenitors, and evidence for intrinsic neurogenic potential of MCs in the chicken, rodent, and even human retina is growing17. Nevertheless, why the regenerative response in birds and mammals is lower compared with the robust response in fish is not yet understood. Therefore, understanding the endogenous repair mechanisms in zebrafish may suggest strategies for stimulating retinal regeneration in mammals and humans. Employing the endogenous repair mechanism of MCs as a therapeutic tool for the treatment of patients with retinal degeneration would have an outstanding impact for our society.
Herein, we provide the steps necessary to employ the degeneration/regeneration model in ophthalmic research. We focused first on inducing focal damage in the neurosensory retina, then on the imaging of events developing at the injury site, and finally visualizing involvement of the adjacent MCs. The general protocol is relatively easy to perform and opens a wide variety of possibilities for evaluating the retina afterwards.

<table>
	<tbody>
		<tr>
			<td>Acid hematoxylin solution</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>2852</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>A07030</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>5470</td>
			<td></td>
		</tr>
		<tr>
			<td>Dako Pen</td>
			<td>Dako, Glostrup, Danmark</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI mounting medium</td>
			<td>Vector Labs, Burlingame, CA, USA</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin G aqueous solution 0.5%</td>
			<td>Carl Roth, Arlesheim, Switzerland</td>
			<td>X883.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>2860</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylene diamine tetraacetic acid (EDTA)</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>ED</td>
			<td></td>
		</tr>
		<tr>
			<td>Eukitt</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>3989</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG H&#38;L Alexa Fluor&#174; 488</td>
			<td>Life Technologies, Zug, Switzerland</td>
			<td>A11008</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG H&#38;L Alexa Fluor&#174; 594</td>
			<td>Life Technologies, Zug, Switzerland</td>
			<td>A11020</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat normal serum</td>
			<td>Dako, Glostrup, Danmark</td>
			<td>X0907</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogel contact lens</td>
			<td>Johnson &#38; Johnson AG, Zug, Switzerland</td>
			<td>n.a.</td>
			<td>1-Day Acuvue Moist</td>
		</tr>
		<tr>
			<td>Hydroxypropylmethylcellulose 2%</td>
			<td>OmniVision, Neuhausen, Switzerland</td>
			<td>n.a.</td>
			<td>Methocel 2%</td>
		</tr>
		<tr>
			<td>Ethyl 3-aminobenzoate methanesulfonate</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>A5040</td>
			<td>Tricaine, MS-222</td>
		</tr>
		<tr>
			<td>Visulas 532s</td>
			<td>Carl Zeiss Meditec AG, Oberkochen, Germany</td>
			<td>n.a.</td>
			<td>532 nm laser</td>
		</tr>
		<tr>
			<td>Mouse anti-GS monoclonal antibody</td>
			<td>Millipore, Billerica, MA, USA</td>
			<td>MAB302</td>
			<td></td>
		</tr>
		<tr>
			<td>HRA + OCT Imaging System</td>
			<td>Heidelberg Engineering, Heidelberg, Germany</td>
			<td>n.a.</td>
			<td>Spectralis</td>
		</tr>
		<tr>
			<td>Heidelberg Eye Explorer</td>
			<td>Heidelberg Engineering, Heidelberg, Germany</td>
			<td>n.a.</td>
			<td>Version 1.9.10.0</td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>P5368</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>P5368</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-GFAP polyclonal antibody</td>
			<td>Invitrogen, Waltham, MA, USA</td>
			<td>180063</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone pin holder</td>
			<td>Huco Vision AG Switzerland</td>
			<td>n.a.</td>
			<td>Cut by hand from silicone pin mat of the sterilization tray accordingly.</td>
		</tr>
		<tr>
			<td>Slit lamp BM900</td>
			<td>Haag-Streit AG, Koeniz, Switzerland</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Slit lamp adapter</td>
			<td>Iridex Corp., Mountain View, CA, USA</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost Plus glass slides</td>
			<td>Gehard Menzel GmbH, Braunschweig, Germany</td>
			<td>10149870</td>
			<td></td>
		</tr>
		<tr>
			<td>TgBAC (gfap:gfap-GFP) zf167 (AB) strain</td>
			<td>KIT, Karlsruhe, Germany</td>
			<td>15204</td>
			<td>http://zfin.org/ZDB-ALT-100308-3</td>
		</tr>
		<tr>
			<td>Tris buffered saline (TBS)</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>P5912</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr>
			<td>78D non-contact slit lamp lens</td>
			<td>Volk Optical, Mentor, OH, USA</td>
			<td>V78C</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>534056</td>
			<td></td>
		</tr>
		<tr>
			<td>Ocular fundus laser lens</td>
			<td>Ocular Instruments, Bellevue, WA, USA</td>
			<td>OFA2-0</td>
			<td></td>
		</tr>
		<tr>
			<td>2100 Retriever</td>
			<td>Aptum Biologics Ltd., Southampton, United Kingdom</td>
			<td>R2100-EU</td>
			<td>Steamer</td>
		</tr>
	</tbody>
</table>

m&#252;ller glia cell activation in a laser-induced retinal degeneration and regeneration model in zebrafish

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after severe damage. Investigating the regeneration pathways in zebrafish might bring new insights to develop innovative strategies for the treatment of retinal degenerative diseases in mammals. Herein, we focused on the induction of a focal lesion to the outer retina in adult zebrafish by means of a 532 nm diode laser. A localized injury allows investigating biological processes that take place during retinal degeneration and regeneration directly at the area of damage. Using non-invasive optical coherence tomography (OCT), we were able to define the location of the damaged area and monitor subsequent regeneration in vivo. Indeed, OCT imaging produces high-resolution, cross-sectional images of the zebrafish retina, providing information which was previously only available with histological analyses. In order to confirm the data from real-time OCT, histological sections were performed and regenerative response after the induction of the retinal injury was investigated by immunohistochemistry.

Real-time OCT: In order to analyze the role of MCs in retinal repair, we used a laser injury model inducing a well-delineated zone of damage in the zebrafish retina. The site of damage was imaged by means of in vivo OCT for the first time (Day 0) within 60 minutes following the injury (Figure 3). To compensate for the optics of the fish eye, a custom-made contact lens was placed on the cornea. Immediately after laser treatment, a dif...

Watch this Scientific Journal Video about MÃ¼ller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish at JoVE.com

M&amp;#252;ller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish

The zebrafish is a popular animal model to study mechanisms of retinal degeneration/regeneration in vertebrates. This protocol describes a method to induce localized injury disrupting the outer retina with minimal damage to the inner retina. Subsequently, we monitor in vivo the retinal morphology and the M&#252;ller glia response throughout retinal regeneration.

M&#252;ller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after ...

The overall goal of this video is to show how to monitor cellular changes in vivo following a focal, laser-induced retinal damage in Zebrafish. This model can help you answer key questions of retinal regeneration, such as morphological changes, kinetics, as well as cell types involved. The main advantage of this technique is that by inducing a focal injury, biological processes can be investigated directly at the site of injury.Though this method can provide insight into regeneration of Zebrafish retina, it can also be applied to study the repair mechanism in different animal models. Prepare a stock solution of anesthetic by dissolving 400 milligrams of tricaine powder in 97.9 millimeters of tank water, and 2.1 millimeters of one molar TBS. Adjust to pH 7.0 with one molar tris at pH 9.To make the working solution, dilute the tricaine stock solution 1 to 25 in tank water and transfer 50 milliliters to a Petri dish. Then, place the Zebrafish into the anesthetic solution for two to five minutes until they become immobile and do not respond to external stimuli. Transfer each fish by hand to a custom made silicone pin holder for laser treatment.Appropriate anesthesia is critical for the well-being of the animal and the success of procedure. Therefore, freshly prepared tricaine solution is pivotal, and the time out of the water should not far exceed 10 minutes. Set up the output power of the 532 nanometer dialed laser to 70 milliwatts, the pulse duration to 100 milliseconds, and the aerial diameter to 50 microns.Then, apply one to two drops of 2%hydroxypropyl methylcellulose to the eye. When apply methylcell to the eye, ensure that the viscous solution doesn't go in the gills. Next, use a 2 millimeter fundus laser lens to focus the laser aiming beam on the retina.Place four laser spots around the optic nerve on the left eye, and use the right, untreated eye as internal control. Immediately after laser induction, place the still anesthetized Zebrafish on a custom made silicone pin holder in the imaging area. To obtain optimal images, cut a commercially available hydrogel contact lens to fit the Zebrafish eye by means of a hole punch.Fill the concave surface of the lens with methylcellulose, then place it over the cornea. Equip the optical coherence tomography system with a 78D non-contact slit lamp lens. Focus the infrared image in IR plus OCT mode to visualize the fundus of the eye.Then, take the IR pictures by clicking the acquire button to localize the laser spots on the retina. Then, visualize a three dimensional section of the retinal layers in IR plus OCT mode, and take the pictures by clicking the acquire button. Observe the severity of injury in the outer nuclear layer in these images.To reverse anesthesia after treatment and imaging, place the zebrafish in a container containing tank water. To support recovery, create a flow of fresh tank water over the gills by moving the zebrafish back and forth in the water. Immediately after laser treatment, a diffuse hyper-reflective signal was localized to the outer retina.It extended from the retinal pigmented epithelium to the outer plexiform layer. The diffuse hyper-reflective signal is absent in the retina from the control un-lesioned eye. A similar diffuse hyper-reflective signal detected on day one after injury.After day three, this diffuse signal became more organized and dense. It was consistently seen in the outer nuclear layer, extending into the photoreceptor layer. Following the first week, there was a significant decrease in average lesion size, and only a small hyper-reflective signal was detected.Starting from day 14 until the latest time point investigated, the laser spots were no longer visible in IR and OCT images, and the morphology of the retina is comparable to the control side, seen here. H&E staining was employed to investigate the extent and kinetics of retinal degeneration and regeneration. This image shows the control section.This image shows H&E staining three days after injury, when maximum photoreceptor loss is apparent. Immunohistochemistry to visualize the glial cell markers, glutamine synthetase, in red, and glial fibrillary acidic protein, in green, was performed. This image shows a control retina where there is very little green fluorescence, which is indicative of GFAP.The GFAP signal was up-regulated at day three post injury, whereas the red glutamine synthetase signal remained unchanged. Following this procedure, other methods like two photon microscopy as well as cellular and molecular analysis can be performed in order to study the involvement of molecular cells in endogenous repair mechanisms. After watching this video, you should have a good understanding how to induce focal damage to the zebrafish retina and to monitor in vivo the following degenerative and regenerative processes.

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Research

JoVE Journal

Neuroscience

Optogenetic Activation of Zebrafish Somatosensory Neurons using ChEF-tdTomato

Larval zebrafish are emerging as a model for describing the development and function of simple neural circuits. Due to their external fertilization, rapid development, and translucency, zebrafish are particularly well suited for optogenetic approaches to investigate neural circuit function. In this approach, light-sensitive ion channels are expressed in specific neurons, enabling the experimenter to activate or inhibit them at will and thus assess their contribution to specific behaviors. Applying these methods in larval zebrafish is conceptually simple but requires the optimization of technical details. Here we demonstrate a procedure for expressing a channelrhodopsin variant in larval zebrafish somatosensory neurons, photo-activating single cells, and recording the resulting behaviors. By introducing a few modifications to previously established methods, this approach could be used to elicit behavioral responses from single neurons activated up to at least 4 days post-fertilization (dpf). Specifically, we created a transgene using a somatosensory neuron enhancer, CREST3, to drive the expression of the tagged channelrhodopsin variant, ChEF-tdTomato. Injecting this transgene into 1-cell stage embryos results in mosaic expression in somatosensory neurons, which can be imaged with confocal microscopy. Illuminating identified cells in these animals with light from a 473 nm DPSS laser, guided through a fiber optic cable, elicits behaviors that can be recorded with a high-speed video camera and analyzed quantitatively. This technique could be adapted to study behaviors elicited by activating any zebrafish neuron. Combining this approach with genetic or pharmacological perturbations will be a powerful way to investigate circuit formation and function.

Optogenetic techniques have made it possible to study the contribution of specific neurons to behavior. We describe a method in larval zebrafish for activating single somatosensory neurons expressing a channelrhodopsin variant (ChEF) with a diode-pumped solid state (DPSS) laser and recording the elicited behaviors with a high-speed video camera.

Larval  zebrafish are emerging as a model for describing the development and function  of simple neural circuits. Due to their  external fertilization, ...

A Novel Light Damage Paradigm for Use in Retinal Regeneration Studies in Adult Zebrafish

Light-induced retinal degeneration (LIRD) is commonly used in both rodents and zebrafish to damage rod and cone photoreceptors. In adult zebrafish, photoreceptor degeneration triggers M&#252;ller glial cells to re-enter the cell cycle and produce transient-amplifying progenitors. These progenitors continue to proliferate as they migrate to the damaged area, where they ultimately give rise to new photoreceptors. Currently, there are two widely-used LIRD paradigms, each of which results in varying degrees of photoreceptor loss and corresponding differences in the regeneration response. As more genetic and pharmacological tools are available to test the role of individual genes of interest during regeneration, there is a need to develop a robust LIRD paradigm. Here we describe a LIRD protocol that results in widespread and consistent loss of both rod and cone photoreceptors in which we have combined the use of two previously established LIRD techniques. Furthermore, this protocol can be extended for use in pigmented animals, which eliminates the need to maintain transgenic lines of interest on the albino background for LIRD studies.

Multiple light damage protocols have been described to damage photoreceptors and consequently induce a retinal regeneration response in adult zebrafish. This protocol describes an improved method that can be used in pigmented animals and that damages the vast majority of rod and cone photoreceptors across the entire retina.

Light-induced retinal degeneration (LIRD) is commonly used in both rodents and zebrafish to damage rod and cone photoreceptors. In adult zebrafish, ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this protocol, we aim to guide the reader not simply through the technical considerations of generating laser-induced lesions to trigger neovascular processes, but rather focus on the powerful information that can be obtained from multimodal longitudinal in vivo imaging throughout the follow-up period.
The laser-induced mouse CNV model was generated by a diode laser administration. Multimodal in vivo imaging techniques were used to monitor CNV induction, progression and regression. First, spectral domain optical coherence tomography (SD-OCT) was performed immediately after the lasering to verify a break of Bruch&#39;s membrane. Subsequent in vivo imaging using fluorescein angiography (FA) confirmed successful damage of Bruch&#39;s membrane from serial images acquired at the choroidal level. Longitudinal follow-up of CNV proliferation and regression on days 5, 10, and 14 after the lasering was performed using both SD-OCT and FA. Simple and reliable grading of leaky CNV leasions from FA images is&#160;presented. Automated segmentation for measurement of total retinal thickness, combined with manual caliber application for measurement of retinal thickness at CNV sites, allow unbiased evaluation of the presence of edema. Finally, histological verification of CNV is performed using isolectin GS-IB4 staining on choroidal flatmounts. The staining is thresholded, and the isolectin-positive area is calculated with ImageJ.
This protocol is especially useful in therapeutics studies requiring high-throughput-like screening of CNV pathology as it allows fast, multimodal, and reliable classification of CNV pathology and retinal edema. In addition, high resolution SD-OCT enables the recording of other pathological hallmarks, such as the accumulation of subretinal or intraretinal fluid. However, this method does not provide a possibility to automate CNV volume analysis from SD-OCT images, which has to be performed manually.

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Here, we present the usefulness of longitudinal in vivo imaging in the follow-up of morphological changes of laser-induced choroidal neovascularization in mice.

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this ...

Partial Optic Nerve Transection in Rats: A Model Established with a New Operative Approach to Assess Secondary Degeneration of Retinal Ganglion Cells

Previous studies have shown that the secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. Partial optic nerve transection is considered a useful and reproducible model. Compared with other optic nerve injury models used commonly for assessing secondary degeneration, e.g. complete optic nerve transection and optic nerve crush models, the partial optic nerve transection model is superior as it distinguishes primary from secondary degeneration in situ. Therefore, it serves as an excellent tool for evaluating secondary degeneration. This study describes a novel operative approach of partial optic nerve transection by directly accessing the area of the retrobulbar optic nerve through the orbital lateral wall of the eyeball. Moreover, we present a newly designed, low cost surgical instrument to assist with transection. As demonstrated by the representative results in distinguishing the boundary of primary and secondary injury areas, the new approach and instrument ensures high efficiency and stability of the model by providing adequate space for surgical operation. This in turn makes it easy to separate the meningeal sheath and ophthalmic vessels from the optic nerve before transection. An additional benefit is that this space-saving operative approach improves the investigators&#39; ability to administer drugs, carriers, or selective RGC tracers to the stump of the partially transected optic nerve, allowing the exploration of mechanisms behind secondary injury in RGCs, in a new way.

Secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. This study describes an innovative operative approach for partial optic nerve transection. The use of this space-saving operative approach extends the model's application range, and allows exploration of secondary injury mechanisms in RGCs in a new way.

Previous studies have shown that the secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. Partial optic nerve transection ...

A Neurosphere Assay to Evaluate Endogenous Neural Stem Cell Activation in a Mouse Model of Minimal Spinal Cord Injury

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied in vitro using the neurosphere assay. This colony-forming assay is a powerful tool to study the response of NSCs to exogenous factors in a dish; however, this can also be used to study the effect of in vivo manipulations with the proper understanding of the strengths and limitations of the assay. One manipulation of the clinical interest is the effect of injury on endogenous NSC activation. Current models of spinal cord injury provide a challenge to study this as the severity of common contusion, compression, and transection models cause the destruction of the NSC niche at the site of the injury where the stem cells reside. Here, we describe a minimal injury model that causes localized damage at the superficial dorsolateral surface of the lower thoracic level (T7/8) of the adult mouse spinal cord. This injury model spares the central canal at the level of injury and permits analysis of the NSCs that reside at the level of the lesion at various time points following injury. Here, we show how the neurosphere assay can be utilized to study the activation of the two distinct, lineally-related, populations of NSCs that reside in the spinal cord periventricular region - primitive and definitive NSCs (pNSCs and dNSCs, respectively). We demonstrate how to isolate and culture these NSCs from the periventricular region at the level of injury and the white matter injury site. Our post-surgical spinal cord dissections show increased numbers of pNSC and dNSC-derived neurospheres from the periventricular region of injured cords compared to controls, speaking to their activation via injury. Furthermore, following injury, dNSC-derived neurospheres can be isolated from the injury site &#8212; demonstrating the ability of NSCs to migrate from their periventricular niche to sites of injury.

Here, we demonstrate the performance of a minimal spinal cord injury model in an adult mouse that spares the central canal niche housing endogenous neural stem cells (NSCs). We show how the neurosphere assay can be used to quantify activation and migration of definitive and primitive NSCs following injury.

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...

A Cell Culture Model for Studying the Role of Neuron-Glia Interactions in Ischemia

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent neuronal loss. Numerous evidence suggests that the interaction between glial and neuronal cells exert beneficial effects after an ischemic event. Therefore, to explore potential protective mechanisms, it is important to develop models that allow studying neuron-glia interactions in an ischemic environment. Herein we present a simple approach to isolate astrocytes and neurons from the rat embryonic cortex, and that by using specific culture media, allows the establishment of neuron- or astrocyte-enriched cultures or neuron-glia cultures with high yield and reproducibility.
To study the crosstalk between astrocytes and neurons, we propose an approach based on a co-culture system in which neurons cultured in coverslips are maintained in contact with a monolayer of astrocytes plated in multiwell plates. The two cultures are maintained apart by small paraffin spheres. This approach allows the independent manipulation and the application of specific treatments to each cell type, which represents an advantage in many studies.
To simulate what occurs during an ischemic stroke, the cultures are subjected to an oxygen and glucose deprivation protocol. This protocol represents a useful tool to study the role of neuron-glia interactions in ischemic stroke.

Here, we present a simple approach using specific culture media that allows the establishment of neuron- and astrocyte-enriched cultures, or neuron-glia cultures from the embryonic cortex, with high yield and reproducibility.

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent ...

A Fibrin-Enriched and tPA-Sensitive Photothrombotic Stroke Model

An ideal thromboembolic stroke model requires certain properties, including relatively simple surgical procedures with low mortality, a consistent infarction size and location, precipitation of platelet:fibrin intermixed blood clots similar to those in patients, and an adequate sensitivity to fibrinolytic treatment. The rose bengal (RB) dye-based photothrombotic stroke model meets the first two requirements but is highly refractory to tPA-mediated lytic treatment, presumably due to its platelet-rich, but fibrin-poor clot composition. We reason that combination of RB dye (50 mg/kg) and a sub-thrombotic dose of thrombin (80 U/kg) for photoactivation aimed at the proximal branch of middle cerebral artery (MCA) may produce fibrin-enriched and tPA-sensitive clots. Indeed, the thrombin and RB (T+RB)-combined photothrombosis model triggered mixed platelet:fibrin blood clots, as shown by immunostaining and immunoblots, and maintained consistent infarct sizes and locations plus low mortality. Moreover, intravenous injection of tPA (Alteplase, 10 mg/kg) within 2 h post-photoactivation significantly decreased the infarct size in T+RB photothrombosis. Thus, the thrombin-enhanced photothrombotic stroke model may be a useful experimental model to test novel thrombolytic therapies.

Traditional photothrombotic stroke (PTS) models mainly induce dense platelet aggregates of a high resistance to tissue plasminogen activator (tPA)-lytic treatment. Here a modified murine PTS model is introduced by co-injecting thrombin and photosensitive dye for photoactivation. The thrombin-enhanced PTS model produces mixed platelet:fibrin clots and is highly sensitive to tPA-thrombolysis.