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 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' 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 occurs commonly in the central nervous system (CNS) after traumatic injuries, and following acute and chronic neurodegenerative diseases.1,2,3,4,5 The death of neurons and glial cells as an early consequence of primary pathological events is termed primary degeneration, while secondary degeneration refers to the death of neurons and glial cells, which are not or only partially affected by primary damage. Secondary degeneration of RGCs is also believed to occur in glaucoma.6 Yoles et al.7 confirmed that secondary injury of RGCs occurs in the optic nerve injury model. They showed that neurons whose axons were not injured after an acute damage will eventually degenerate due to degenerative environment produced by the injury surrounding these axons. This secondary degeneration affects the neurons in a progressive fashion related to the severity of the damage inflicted. So far, the mechanisms underlying RGC damage in glaucoma remain unclear, especially those related to secondary injury, which results in insufficient clinical treatment.8,9,10 Therefore, it is necessary to explore the underlying mechanisms of secondary degeneration of RGCs during the development of glaucoma.11 Establishment of animal models of secondary injury that can quantitatively assess the size, distribution, and mechanism of secondary degeneration of RGCs attracts increasing attention from scientists studying secondary injury of RGCs.
To clarify this issue, a rat PONT model was established by Levkovitch-Verbin et al.12 to evaluate axonal injury induced degeneration and death of RGCs. This model is believed to constitute a good tool for exploring the mechanisms of secondary degeneration and identifying the potential neuroprotective agents. The instrument used to generate this model of secondary injury is a diamond knife with a scale to transect quantitatively by setting the depth of abscission through the dial knob, in order to complete a quantitative transection of the optic nerve. The surgical path approaches from the eyeball upward or temporal conjunctiva. During the operative process, the retina and optic nerves might be affected by the force of forceps, which in turn may cause primary injury. More importantly, due to the limited space of the exposed optic nerve, it is difficult to separate the meningeal sheath before incision. Therefore, it is possible to damage the ophthalmic vessels during partial optic nerve transection, which results in retinal ischemia and the failure of the model. In addition, the diamond knife is costly, and each use decreases the tip sharpness. This might in turn affect the depth and effect of modeling.
The model of secondary degeneration of RGCs described in this study was obtained through a new operative approach from the lateral orbital wall of the eyeball. This novel operative approach directly accesses the retrobulbar optic nerve encircled by the orbital muscle cone, avoiding primary injury to the eyeball and the optic nerve when pulling downwards or towards the nasal lateral side of the eyeball. This also increases the space of surgical operation during model establishment, and enables meningeal sheath isolation before partially transecting the optic nerve. It is important to notice that the inadvertent involvement and injury of ophthalmic vessels can lead to the failure of the model. Furthermore, the model enables a follow-up assessment of the transfected cells, drugs and reagents on the stump of the partially transected optic nerve. The self-designed surgical instrument is cheap and can be used multiple times, thereby reducing the cost of modeling. The secondary injury model of RGCs established by this method was shown to have good reproducibility and stability.
Operational Procedure
There are some points worthy of notice in the process of model construction. In step 4.2, the surgical movement should be performed carefully to avoid damaging the vasculature above the subfascial muscle. Especially, when cutting the subcutaneous fascia in the outer lateral canthus, sharp-serrated forceps should be used to pull upward the subcutaneous fascia on the fascia surface vertically; the fascia should be cut with Vannas spring scissors to avoid damage to the orbital vein at the outer canthus, which can result in model failure by excessive bleeding. Step 4.3 has the advantage of potentially preventing bleeding when directly removing from blood vessels. In separating orbital muscles in step 4.5, the reason for picking sharp-serrated forceps but not Vannas spring scissors is to avoid continuous bleeding and hemorrhage. The muscles are bluntly separated on both sides in the perpendicular direction to the skin fascia incision; meanwhile, the deep muscles of the orbit are stretched outward and peripherally. This procedure will reveal deeper portions of the orbital cavity, providing a larger surgical window and allowing unimpeded access to tissues overlaying the optic nerve. In the above procedures, if bleeding occurs, pressure should be applied using sterile surgical or cotton swabs. Minor bleeding will stop after several seconds by this procedure. The purpose of step 4.6 is to facilitate follow-up operations to easily remove some fat and separate muscles in the orbit muscle cone to expose the optic nerve along the direction of the optic nerve in the orbital depth.
The most critical parts of the current protocol are Steps 5.1-5.6. It is important not to damage the vasculature around the optic nerve head. The optic nerve should be partially transected at least 1.5-2.0 mm from the back of the eye, to avoid any damage to the ophthalmic artery that penetrates the nerve within 1 mm of the eye and supplies blood to the inner retina. The purpose of cutting the lateral rectus is to achieve better exposure of the optic nerve as the lateral rectus is wide and obviously blocks the view of the optic nerve. Meanwhile, to avoid removing the ophthalmic artery that is associated with the meningeal sheath (Figure 5),it is necessary to separate and dissociate the dura around the optic nerve and examine the vascular pattern of the meningeal sheath, using forceps to gently rotate the sheath. In addition, an area devoid of blood vessels should be identified, allowing a longitudinal cut in the meningeal sheath. It is also necessary to maintain a small working distance from the back of the eye, to avoid the portion of the dura that is closely associated with the ophthalmic artery. The retina is normally transparent, and blood vessels can be clearly demarcated. In case of damaged retinal blood supply, the retina is degenerated, leading to a milky-white flocculent appearance. The vitreous chamber of the eye and the lens will typically become cloudy as well, with decreased eye size over time. In this study, preoperative and postoperative images of the fundus confirmed no damage to the fundus blood supply in the model after applying the above steps.
Furthermore, special care is required in several steps of this model. When using sharp-curved-serrated forceps or other surgical instruments to expose the optic nerve, the surgeon should avoid excessive force, as it may damage the optic nerve, eyeball or ophthalmic artery, resulting in primary injury and retinal ischemia. In addition, blood vessels surrounding the eye should not be damaged, to avoid sustained bleeding, which could lead to the failure of modeling. The SSAI used in this experiment requires delicate usage. When the optic nerve is placed within the instrument groove, the optic nerve and grooved surface need to be tightly fitted to ensure good consistency and repeatability of each animal model. With practice, the full surgical procedure can be completed within 15-20 minutes per eye, after the initial entry cuts have been made.
Wang et al.19 published a similar animal model of partial optic nerve transection established using an optic nerve quantitative amputator. The surgical procedures includes: 1) Cutting apart the outer canthus, suspending and fixing the palpebral superior; 2) Exploring the optic nerve, and transecting the superior portion of the optic nerve using the amputator; and 3) Suturing the conjunctiva and skin. Although the surgical procedure was relatively simple, the following problems were encountered during the operation. Although lateral canthus incision could expose certain space for operation, there was an inevitable need to constantly stretch the eyeball in order to expose the retrobulbar optic nerve sheath, especially when the surgeons desired to expose a longer retrobulbar optic nerve sheath to facilitate further sheath isolation; the force for stretching the eyeball was greater, which is likely to cause direct traction injury of the eyeball and optic nerve. No special attention was paid to blood vessels that might be cut together with the optic nerve sheath, and damage to blood vessels is likely to lead to failed model establishment. The principal procedures of secondary injury model described in this paper are: a new operative approach from the lateral orbital wall of the eyeball to directly access the retrobulbar optic nerve surrounded by the orbital muscle cone, avoiding primary injury to the eyeball and optic nerve, when pulling downwards or towards the nasal lateral side of the eyeball. This new operative approach increases the space of surgical operation during modeling, and enables easy isolation of the meningeal sheath, which is closely associated with the ophthalmic artery, before partial transection of the optic nerve. Partial optic nerve transection was performed with a self-designed surgical instrument, which is cost effective and reusable, reducing the overall cost of modeling. The rat's orbital structure is different from those of other mammals, with the orbit closest to the canthus and no bony structure, but covered with muscles. The surgical approach could reach the posterior part of the eyeball without the need of destroying the orbital bone and periosteum. Through strict preoperative disinfection and postoperative antibiotic prophylaxis, infection, inflammation and edema were greatly reduced.
Self-designed Surgical Assistant Instrument
The rat model of partial optic nerve transection was established using the self-designed surgical assistant instrument, whose main features are as follows. It can assist in partial quantitative transection of the optic nerve exposed to the grooved edge, also ensuring transection consistency among different animals. We tested and verified the repeatability of model establishment with SSAI. The maximum coefficient of variation was 1.85%, with an average value 0.67%±0.44%.20 These results indicate that the SSAI could be used to establish partial optic nerve transection models, with satisfactory reproducibility and uniformity.
The grooved surface's width and the design of the semi-circle of the groove's inner surface can have a more fixed effect on the optic nerve and make the grooved surface and optic nerve attach more tightly, also decreasing experimental errors and adverse reactions. The grooved edge allows a better protection of the optic nerve in the groove during the operation, which will not damage the optic nerve in the groove, regardless of the cutter's sharpness. Another advantage of the grooved edge is crush injury prevention during optic nerve transection.
It is appropriate for operating in deep and narrow space. Although the new operative approach has been expanded, the pathway remains deep, and the hand-held pole and joint section can be used to place the grooved head easily under the optic nerve sheath to perform follow-up operations. When the instrument is used for operation, a wide range of cutters can be used for transection, e.g. a 26 G needle tip. Even a sapphire surgical probe knife can be selected to avoid contusion and crush injuries caused by scissors. Groove surfaces can be made in different vertical depths to complete varying degrees of optic nerve cutting.
Compared with the amputator of Wang et al. the SSAI has a simpler structure. In addition, the cutting step is more convenient using the SSAI, with improved consistency and repeatability of the animal model. Finally, the range of tools applicable for cutting with the SSAI is also wider. In conclusion, SSAI, which makes quantitative and uniform incisions of the nerve, may serve as an effective instrument for establishing rat models for assessing optic nerve transection.
Characteristics of the Rat Partial Optic Nerve Transection Model
The partial optic nerve transection model is useful to assess secondary degeneration in RGCs. The potential merit of this model is the ability to separate primary from secondary degeneration accurately in situ, both in the optic nerve and retina. The central and ventral optic nerves were more susceptible to secondary injury after partial transection (about 1/3 to 1/2) of the dorsal optic nerve (Figure 6). In the retina, the regional location of primary and secondary injuries of RGCs should be based on the topography of the optic nerve corresponding to retinal RGCs after partial transection. If the entire retina of the rat is divided into dorsal (superior) and ventral (inferior) parts, secondary and primary injuries are present in both parts. However, based on the relationship between RGCs on the retina and the optic nerve axon, RGC death in the ventral retina should be mainly attributed to secondary injury (Figure 3).12,22,23 The advantages of this model include: simple and easy-to-operate instrument with standard procedures; no effect on ophthalmic vessels; good reproducibility and stability. This technique might be used to transfect RGCs from this space-saving operative approach by applying short interfering RNAs (siRNAs), plasmids, and viral vectors to the partial optic nerve stump; in addition, reagents could be placed on the partial optic nerve stump for the selective treatment or labeling of RGCs.
Overall, primary and secondary injuries of RGCs coexisted after partial optic nerve transection in this animal model, with a clear boundary in the retina between the two injury types. Although the association of optic nerve axons and RGC location on the retina needs further investigation for a more precise distinction, this space-saving operative approach extends the application range of the model and allows researchers to explore the mechanisms of secondary injury in RGCs in a new way.
The authors have nothing to disclose.
This work was supported by the Beijing Natural Science Foundation (7152038), the Fundamental Research Funds for the Central Universities of Central South University (2016zzts162) and the Science Research Foundation of Aier Eye Hospital Group (Grant No. AF156D11). Finally, Fancheng Yan thanks Yiping Xu for the invaluable support over the years.
Animal Aneathesia Ventilator System | MIDMARK | Matrx VMR | |
Isoflurane | RWD Life Science Co. | R510-22 | |
Surgical Microscope | Leica AG, Heerbrugg, Switzerland | M620 F20 | |
Tobramycin Eye ointment | Alcon | H20110312 | |
Fluorogold | Biotium | 80014 | |
Iris scissors | 66vision Co. | 54026 | |
Vannas spring scissor | 66vision Co. | 54137B | |
Sharp-serrated forceps/0.12mm toothed forceps | 66vision Co. | 53329A | |
Sharp-curved forceps | 66vision Co. | 53324A | |
Sapphire surgical probe | 66vision Co. | 50205TA | |
26G needle tip | Shandong Weigao Group Medical Polymer Co. | 3151474 | |
10 μl Hamilton Syringe | Hamilton Co. | 80030 | |
5-0 non-absorbable suture | Johnson & Johnson International Co. | W580 | |
Chlorhexidine | Sigma-Aldrich | 282227 | |
Stereotaxie apparatus | RWD Life Science Co. | 68026 | |
Retinal Imaging System | OptoProbe Ltd. | OPTO-RIS | |
RetCamII wide field imaging system | Clarity Medical Systems,Inc. | RetCamII | |
Fluorescence microscope | Leica Microsystems Inc. | DM6000 |