Background: Axonal degeneration caused by damage to the optic nerve can result in a gradual death of retinal ganglion cells (RGC), leading to irreversible vision loss. An example of such diseases in humans includes optic nerve degeneration in glaucoma. Glaucoma is characterized by the progressive degeneration of the optic nerve and the loss of RGCs that can lead to loss of vision. The different animal models developed to mimic glaucomatous neurodegeneration, all result in RGC loss consequent optic nerve damage.
Methods: The present article summarizes experimental procedures and analytical methodologies related to one experimental model of glaucoma induced by optic nerve crush (ONC). Point-by-point protocol is reported with a particular focus on the critical point for the realization of the model. Moreover, information on the electroretinogram procedure and the immunohistochemical detection of RGCs are described to evaluate the morpho-functional consequences of ONC.
Discussion: Although the model of ONC is improperly assimilated to glaucoma, then the ONC model simulates most of the signaling responses consequent to RGC apoptosis as observed in models of experimental glaucoma. In this respect, the ONC model may be essential to elucidate the cellular and molecular mechanisms of glaucomatous diseases and may help to develop novel neuroprotective therapies.
Neurodegenerative disorders of the retina involve optic nerve (ON) head injury and the progressive death of retinal ganglion cells (RGCs). The axons of the RGCs form the ON and transmit visual information to the visual cortex. The death of RGC is one of the main causes of irreversible blindness in the world (1).
Glaucoma is a collection of diseases that lead to an irreversible vision loss due to RGC damage. Although the causes leading to RGC loss are not fully understood, the onset and progression of glaucomatous damage and RGC death may share similar mechanisms.
Despite several models have been developed to mimic glaucomatous neurodegeneration, all of them result in RGC loss consequent to optic nerve damage, probably as the initial insult in glaucomatous diseases. In this respect, the optic nerve crush (ONC) model although not directly mimicking the pathophysiological mechanisms of the human glaucoma has allowed us to acquire information on RGC degeneration.
In particular, the ONC model has been used to investigate mechanisms involved in RGC degeneration in order to identify molecules with therapeutic potential to be eventually used as pharmacological treatments against glaucomatous neurodegeneration.
The ONC model is not novel, having been first described in detail in 1999 (2) and has been highly refined to demonstrate a clear association between RGC loss and nerve crush force-impulse and duration (3,4). In addition, both RGCs themselves and the mouse strain used for the ONC protocol may differ in their resilience to the mechanical injury (5,6). Nevertheless, its quick and easy procedure makes the ONC model attractive to replicate RGCs loss that characterizes optic neuropathies including glaucoma.
The purpose of the present manuscript is to review the protocol to induce ONC in mice. Although the protocol is well consolidated, here we emphasize those peculiar strategies that are often neglected in detail or not even discussed about the pros and cons of different strategies in order to provide readers with specific information, which may be of great help to determine the success of a given methodology. In the surgical procedure, for instance, the methodology used to access the ON, the force and the duration of the crush are critical to determine the extent of the ON damage. In addition, detailed information on the Pattern ERG procedure to evidentiate RGC function and the immunohistochemical detection of RGCs can make the difference when evaluating the morpho-functional consequences of ONC.
The survival time between ONC and electrophysiological detection of RGC function has a dramatic bearing on monitoring RGC pathology and has been established in several studies. After mechanical optic nerve damage, RGC degeneration begins rather quickly. RGCs die in a proportion of approximately 50% and 70% one or two weeks after injury, respectively. However, different outcomes are likely to result from substantial differences in the extent of crush thus confirming the necessity to check for the severity (strength and duration) of the crush. In fact, injury to RGCs after ONC can vary between studies and depends on how much force is applied at the injury site, the distance from the globe where the injury is applied and the species or strain of the experimental animal (7-9).
Let the mice to survive for 7 days until to proceed with further investigations including recording of the pattern-induced electroretinogram (PERG) that is an accurate measure of RGC function and the RGC immunohistochemical detection to determine RGC loss in comparison to control retinas.
To evaluate RGCs function, analyze the amplitude and implied time of the components of the PERG responses. In particular measure the amplitude of the N35-P50 wave from the depression of the negative peak, N35, to the peak of the positive peak, P50, and the amplitude of the P50-N95 wave from the peak of the positive peak, P50, to the depression. respectively of the negative peak, N95. In addition, measure the implicit time from the onset of the stimulus to the P50 and N95 peaks. After 7 days from ONC, both components N35-P50 and P50-N95 are significantly reduced compared to controls, while implicit times are increased (Figure 4).
To demonstrate the efficacy of ONC, count the RBPMS immunopositive cells/mm2 in a masked manner. As shown in Figure 5, in the ONC model the number of RBPMS-positive cells is significantly reduced compared to the control retina, indicating a consistent loss of RGCs as successfully induced by ONC.
While the ONC is used as a model for glaucoma in the research setting, the causes of glaucoma cannot be attributed to axonal injury alone. Intraocular pressure (IOP) may also play an important role and there are rodent models that simulate glaucoma by increasing the IOP. In fact, IOP elevation, caused by an imbalance in the production and drainage of aqueous humor in the anterior chamber of the eye, is considered one of the main risk factors in glaucoma (11). Although mouse models of ocular hypertension can be considered as good models of glaucoma, minimum acceptable standards need to be evaluated to validate these models. Each model has its strengths and weaknesses, for example, models using injection of viscous agent or laser photocoagulation are useful to study the effect of IOP elevation on RGC degeneration. In addition, the DBA/2J mouse is used as a model for congenital experimental glaucoma. However, when using this model, it can lead to difficulties in assessing disease progression due to its variability (12,13). In the ONC model discussed here, the contribution of axonal injury to the glaucomatous pathology is independent on the effects of ocular hypertension on RGC dysfunction thus mimicking more precisely traumatic optic neuropathies and representing an excellent model for studying to which extent axonal injury plays a role in glaucomatous pathology.
The advantages of this model are the ease of carrying out the procedure and the rapid onset of pathology. Furthermore, this model involves some features similar to human glaucoma. In fact, a significant portion of RGC injury in glaucoma is believed to occur through mechanical stress at the optic nerve head. Therefore, the ONC model represents a good model for studying the role of axonal injury in glaucomatous pathology (14).
Overall, each of the glaucomatous models has inherent advantages and disadvantages. In general, experimental designs attempt to maximize the information available for each research task. An optimal experiment allows the inferred models with the highest degree of confidence expected. However, in the situation where some models are incomplete, the role of the experimental design must be carefully examined to avoid a complex interpretation of the results.
