AXONAL REGENERATION

 

A few days after axonal injury, the associated retinal ganglion cells (RGCs) begin to degenerate. This is especially so if the injury is close to the eye.

The death of RGCs can be prevented almost completely by overexpression of the antiapoptotic Bcl family proteins, such as, Bcl-2 and Bcl-xL.

However, the regeneration and survival of axons is also dependent on numerous intracellular signaling pathways. This is seen when RGCs overexpressing Bcl-2 or Bcl-xL fail to regenerate axons, unless provided with additional growth factors.

A number of trophic factors can slow, but not completely stop, the death of RGCs. These factors include ciliary neurotrophic factor (CNTF), brain derived neurotrophic factor (BDNF), neurotrophin 4/5 nerve growth factor, insulin-like growth factor-1, granulocyte colony-stimulating factor, glial-derived neurotrophic factor, and neurturin. 

The death of axotomized RGCs is also slowed by preventing caspase cleavage, blocking the nuclear enzyme poly (adenosine diphosphate–ribose) polymerase (a substrate for caspases), blocking nitric oxide synthase, introducing reducing agents, or inhibiting cell death via caspase-independent pathways. Long-term prevention of RGC death after axotomy may require the development of long-term delivery systems or a combination of treatments.

Fibroblast growth factor 2 stimulates some axon regeneration through the optic nerve.

Two molecules present in the eye were found to stimulate mature RGCs to regenerate their axons. One is mannose, a simple sugar that is abundant in the vitreous. Mannose stimulates RGCs to extend moderately long axons if cells have sufficiently high levels of intracellular cyclic adenosine monophosphate (cAMP). The second growth factor is oncomodulin (Ocm), a 12-kDa, calcium-binding protein secreted by macrophages. 

The advances during the past few years give hope for the possibility that at least some RGCs will be able to regenerate their axons all the way to their central targets. The future challenges will include finding ways to optimize this regeneration and testing whether they restore functionally meaningful levels of vision.

REFERENCE:

Benowitz LI, Yin Y. Optic Nerve Regeneration. Arch Ophthalmol. 2010;128(8):1059–1064. doi:10.1001/archophthalmol.2010.152

AXONAL RESPONSE TO INJURY

 

The optic nerve is regarded as an extension of the brain. It is usually recognized that once damaged, the optic nerve does not regenerate, leading to visual loss lasting the lifetime of the individual. The degeneration of the optic nerve follows a timeline of events, starting milliseconds to hours after the initiating event, such as trauma or ischemia.

One of the earliest effects is the entry of calcium ions (Ca⁺⁺) into the site, through voltage-gated calcium channels, as well as, possibly from the endoplasmic reticulum. This increased Ca⁺⁺ activates calpains, which are commonly occurring cysteine proteases, mechanistically linking injury-induced calcium signaling to subsequent axonal degeneration by the process of cytoskeletal degradation. Following this, the axons swell and fragment on both sides of the injury. In murine spinal cord, the same fragmentation process can be completely blocked by calpain inhibitors.

A few days later, the distal axon segments fragment through a process called Wallerian degeneration in which the cytoskeleton is degenerated. The axon first forms swellings and then fragments into self-enclosed units, and the myelin disintegrates into elliptical structures. The proximal axonal segment forms a retraction bulb, elliptical in shape and several times the axonal diameter. This bulb grows progressively larger over weeks as the axonal cytoskeleton depolymerizes and the axon dies back towards the soma.  

The first week following the event is critical as the inflammatory response reaches its peak. Infiltrating monocyte-derived macrophages arrive at the optic nerve after the first day.

Astrocytes at the injury site in the optic nerve degenerate within 3 days and begin to repopulate by day 7. Optic nerve head astrocytes become reactive, losing many fine processes and shrinking in total area covered, but thickening both their soma and primary processes. Retinal microglia increase in number, presumably through proliferation.

The retinal ganglion cell soma receives the signal that it has been damaged within the first week, and many stress responses are subsequently activated. Whether the RGC will die or regenerate is determined in that first week after injury, and this fate depends on various intrinsic and extrinsic factors.

REFERENCE:

Fague L, Liu YA, Marsh-Armstrong N. The basic science of optic nerve regeneration. Ann Transl Med. 2021 Aug;9(15):1276. doi: 10.21037/atm-20-5351. PMID: 34532413; PMCID: PMC8421956.

ORAL CANNABINOID (PEA) FOR GLAUCOMA TREATMENT

 

A cannabinoid system composed of several receptors and endogenous cannabinoids, including anandamide (AEA) and 2-arachidonylglycerol (2-AG), has been identified in the brain, the peripheral tissue, and the eye. Two principal cannabinoid receptors have been described (CB1, which is predominant in neurons, and CB2, which is localized in immune cells and peripheral tissue cells).

In the human eye, CB1 receptors have been reported from the trabecular meshwork (TM) and Schlemm canal cells. Also, CB2 receptors have been demonstrated in porcine TM cells in culture.

AEA and the CB1- and CB2-selective agonists enhance aqueous humor outflow through the conventional pathway and significantly decrease intra-ocular pressure (IOP) in rabbits, primates, and humans after topical application. Conversely, CB1 antagonists elevate IOP.

AEA, the most investigated endo-cannabinoid, acts as a partial CB1 agonist and a weak CB2 agonist and activates the vanilloid type 1 receptor.

Palmitoyl-ethanol-amide (PEA) is an endogenous congener of AEA that is co-synthesized with AEA by most cell types.

PEA does not bind to CB1 or CB2 receptors, but it is a competing substrate with AEA for the enzyme fatty acid amide hydrolase (FAAH) active site, and it has been hypothesized to increase or prolong the effect of AEA (entourage effect) without the systemic side effects of cannabinoids.

A study was reported by Gagliano et al., to assess the effect of oral PEA administration on IOP in primary open angle glaucoma (POAG) and ocular hypertension (OH). In the study, 42 patients with POAG or OH who were treated with timolol 0.5% and whose IOP was between 19- and 24-mm Hg received oral PEA (300-mg tablets twice a day) or placebo (PEA vehicle tablets twice a day) for 2 months (period 1), and, after a 2-month washout, received the other treatment for 1 month (period 2). [1]

After PEA treatment (mean baseline IOP, 21.6 ± 1.7 mm Hg), IOP was reduced by 3.2 ± 1.3 mm Hg at 1 month and by 3.5 ± 1.2 mm Hg (15.9% ± 5.1%) at 2 months (ANOVA, P < 0.001; both Tukey-Kramer, P < 0.01 vs. baseline).

Conversely, after placebo (mean baseline IOP, 21.5 ± 1.5 mm Hg), IOP was reduced by 0.4 ± 1.2 mm Hg at 1 month and by 0.3 ± 1.3 mm Hg at 2 months (t-test at both time points, P < 0.001 vs. PEA). No statistically significant vital signs, visual field, visual acuity changes, or adverse events were detected in either group.

No specific side effects were reported with PEA, and no contraindications have been defined.

The study found no significant changes in systemic blood pressure. Although, cannabinoids reduce blood pressure, with an effect particularly involving CB1 receptors.

The decrease in IOP was 16% of the baseline level, which was comparable to the levels achieved with other currently used anti-hypertensive medications, such as topical Carbonic Anhydrase Inhibitors and alpha agonists.

Kumar et al., have also performed a study to analyze the effect of PEA on aqueous humor outflow facility. According to them PEA caused a concentration-dependent enhancement of outflow facility through the trabecular meshwork. These effects are mediated by GPR55 and PPARα receptors through activation of p42/44 MAPK. [2]

Another novel and significant effect of PEA is neuroprotection. We know that glaucoma is a neuropathy affecting the nervous tissue of the retina and the optic nerve. It is presumed that this neuropathy has an inflammatory component, especially in the glia (neuroinflammation). It is known that PEA has significant anti-inflammatory effects and this property could contribute to neuroprotection by reducing the inflammatory component of glaucoma. [3]

Strobbe et al., have found that PEA improves the endothelium-dependent flow-mediated vasodilation (FMD) characteristics in OH patients. These patients have elevated plasma and aqueous levels of endothelin-1 (ET-1), causing chronic impairment of ocular blood flow, especially of the optic nerve head. [4]

Therefore, PEA, a drug of a class that has been proposed for the treatment of glaucoma but that is not used because of concerns about side effects, could be a valuable tool in the treatment of such diseases.

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REFERENCES:

1.  Gagliano C, Ortisi E, Pulvirenti L, Reibaldi M, Scollo D, Amato R, Avitabile T, Longo A. Ocular hypotensive effect of oral palmitoyl-ethanolamide: a clinical trial. Invest Ophthalmol Vis Sci. 2011 Aug 3;52(9):6096-100. doi: 10.1167/iovs.10-7057. PMID: 21705689.
2. Kumar A, Qiao Z, Kumar P, Song ZH. Effects of palmitoylethanolamide on aqueous humor outflow. Invest Ophthalmol Vis Sci. 2012 Jul 3;53(8):4416-25. doi: 10.1167/iovs.11-9294. PMID: 22589443; PMCID: PMC4625824.
3. https://areaoftalmologica.com/en/blog/glaucoma/palmitoylethanolamide-pea-a-new-focus-on-the-treatment-of-glaucoma/
4. Strobbe E, Cellini M, Campos EC. Effectiveness of palmitoylethanolamide on endothelial dysfunction in ocular hypertensive patients: a randomized, placebo-controlled cross-over study. Invest Ophthalmol Vis Sci. 2013 Feb 1;54(2):968-73. doi: 10.1167/iovs.12-10899. PMID: 23307959.

COMPASS PERIMETER

 

iCare COMPASS is an automatic perimeter which also combines an active retinal tracker and a scanning ophthalmoscope. This technology provides retinal threshold sensitivity as well as TrueColor confocal images of the retina and fixation analysis, which is eye movement artefact-free.

The COMPASS is compatible with SAP and offers full range of 24-2, 30-2 and 10-2 visual field-testing programs, containing age-matched databases of retinal sensitivity in normal subjects.

The COMPASS supra-threshold testing is used to perform fast screening for visual field (VF) loss.

The iCare COMPASS measures sensitivity at specific retinal locations with high topographic accuracy due to the retinal tracking technology which compensates for any eye movements during VF examination, providing higher accuracy of the test results.

Continuous, automated, tracking of eye movements yields to active compensation of fixation losses, with perimetric stimuli being automatically re-positioned prior to and during projection based on the current eye position.

This mechanism is critical to ensure accurate correlation between function (i.e. retinal threshold values) and structure (fundus image). In the absence of this mechanism, any shift in eye position occurring at the time of stimuli projection would easily produce artifacts in VF results, with an inaccurate sensitivity being reported.

The COMPASS superimposes the threshold map over a 60° image of the fundus. This allows simultaneous assessment of retinal function and structure.

The COMPASS is equipped with an automatic refractive correction system, thus, eliminating the need for trial lenses.

The unique 3D Stereo View technology of COMPASS captures automatically two separate photos of the nasal field, at different angles and different focal planes (bifocal), creating outstanding 3D perception of the disc.

The COMPASS provides 60° confocal images of the retina in different modalities: TrueColor, Infrared and Red-free, enhancing diagnostic and prognostic capabilities in glaucoma management. The true color confocal imaging system, allows the visualization of the retina in real color, with outstanding details of the Optic Nerve Head (ONH).

The COMPASS offers embedded capabilities for network connectivity, for both remote data review and data backup. The COMPASS Remote Viewer is a browser-based software that allows for reviewing from any network computer on the same local area network (LAN), with password protection. The Remote Viewer provides image comparison tools, anatomic measurements, post-processing tools and more. Images taken at different times can be registered and displayed as rapidly alternating to facilitate detection of morphologic changes over time. Cup to disc calculation – ratios can be measured and stored.

The iCare COMPASS is marketed as relatively more patient friendly because the test is straight forward, relatively fast, and can be discontinued at any time and continued again without data loss.

EXOSOME TREATMENT OF RGCs

 

Mesenchymal stem cells (MSC) are self-replicating multipotent stromal cells isolated from mesenchymal tissues such as bone marrow (BMSC), adipose tissues, dental pulp and umbilical cord blood as well as other tissues.

MSCs promote the protection and regeneration of central nervous system (CNS) neurons, which lack the capacity to regenerate, or be replaced following their loss. The retina is an outgrowth of the brain and is thus part of the CNS and subject to the same regenerative limitations.

MSCs secrete exosomes, which are endocytic-derived structures composed of proteins, lipids, and mRNA surrounded by a phospholipid bi-layer that are secreted into the extracellular space. Their size ranges from 30 to 100 nm. Exosomes contain mRNA and miRNA, which are both functional and, when delivered to another cell via fusion with the cell membrane, lead to the translation of new proteins.

They can be easily stored and do not proliferate, making the application of specific doses easier. Due to their smaller size, they are also capable of migrating into the ganglion cell layer (GCL) from the vitreous (unlike transplanted cells) and delivering their content to the retinal ganglion cells (RGC). The surrounding phospholipid bilayer of exosomes protects the contents against degradation and makes them immunologically inert, qualities important for a therapeutic delivery system.

Treatment of primary retinal cultures with BMSC-exosomes demonstrated significant neuroprotective and neuritogenic effects. BMSC-derived exosomes promoted statistically significant survival of RGCs and regeneration of their axons while partially preventing RGC axonal loss and RGC dysfunction.

After optic nerve crush injury, MSCs transplanted into the vitreous are able to promote significant neuroprotection of RGCs and moderate regeneration of their axons. In animal models of glaucoma, MSCs promote the survival of RGCs and their axons and preserve their function.

The mechanism of exosome-derived neuroprotection is apparently through a paracrine-mediated effect with secreted factors being necessary.

In culture, MSC are efficacious when cocultured (yet physically separated) from the injured retinal cells. The assumption that neurotrophic growth factors (NTF) are important is corroborated both by the expansive NTF rich secretome of MSC and by the attenuated neuroprotective and neuritogenic effects when particular NTF receptors are inhibited. Secreted NTF such as platelet-derived growth factor and brain-derived neurotrophic factor have been shown to be important to the neuroprotection of RGCs whereas MSC mediated-neuritogenesis depended more on nerve growth factor. Other secreted factors, such as Wnt3a have been implicated in the neuroprotective effect of MSC on CNS neurons.

Transplantation into the vitreous of healthy and diseased eyes yields no evidence of differentiation or migration/integration into retinal tissue, strongly implicating paracrine over cell replacement as the dominant mechanism.

Exosomes offer a cell-free alternative to BMSC therapy, which can be easily isolated, purified and stored. They lack the risk of complications associated with transplanting live cells into the vitreous (immune rejection, unwanted proliferation/differentiation).

A limitation of exosome-related treatment is that regeneration is only significant at short distances from the lesion site (<1 mm) limiting its potential at promoting functional reconnection of the visual pathway. It is currently unknown what the ideal timeframe for treatment is, whether a single injection of exosomes is sufficient or weekly/bi-weekly/monthly injections are required.

PRESERFLO MICROSHUNT DEVICE

 

The PRESERFLO MicroShunt is an ab externo glaucoma drainage device made of an extremely flexible SIBS [poly (Styrene-block-IsoButylene-block-Styrene)] polymer with a tube of 350 μm outer diameter and a lumen of 70 μm. 

It has triangular fins that prevent migration of the tube into the AC. The device is designed to be implanted under the subconjunctival/Tenon space.

SIBS (Poly(styrene-block-isobutylene-block-styrene)) is a uniquely biocompatible, degradation-resistant material proven to minimize inflammation, scarring, and encapsulation.

Contraindications to implantation of the PRESERFLO Microshunt may include cases of shallow anterior chamber, inability of the patient to adhere to postoperative visits and/or medications, and/or intolerance or allergy to Mitomycin-C (MMC).

SURGICAL TECHNIQUE:

A 3 to 4 clock-hour fornix-based conjunctival peritomy is created – typically in the superonasal or superotemporal quadrants. Mitomycin-C soaked sponges are then applied to the scleral bed.

Mitomycin-C concentration and duration of applications are left to the surgeon’s discretion based on judgment of risk for postoperative fibrosis.

After removal of the sponges, the scleral bed is rinsed with balanced salt solution.

The sclera is then marked 3 mm posterior the limbus.

A scleral tunnel is initiated at this point along the curvature of the globe, followed by change trajectory at the level of the trabecular meshwork before entering the anterior chamber parallel to the iris plane.

The device is then inserted through this tract using non-toothed forceps, with care to maintain the proximal tip in a bevel-up position.

The device is advanced until the fins are just tucked within the distal end of the scleral tunnel.

In this position, 2 to 3 mm of the proximal end of the device should extend into the mid-anterior chamber and parallel to iris plane.

When properly positioned and unobstructed, aqueous flow should be visible at the distal end of the device. If no flow is visible and an obvious proximal obstruction is not visualized, the distal end may be “primed” using an anterior chamber cannula and balanced salt solution. A corneal paracentesis may be created at surgeon discretion for additional control of the anterior chamber or to test device patency via injection of balanced salt solution.

Once the device has been placed and flow has been verified, conjunctiva and Tenon capsule are repositioned over the device, taking care to avoid distal obstruction of implant.

The distal end of the implant should lie flush with the sclera and a second instrument may be used to direct it in this manner.

The conjunctiva and Tenon capsule are then closed using the surgeon’s preferred technique.

A fluorescein strip is used to confirm adequate wound closure and the eye is protected according to the surgeon’s usual protocol.

RESULTS:

Studies have largely shown positive outcomes with the PRESERFLO Microshunt device. The success rates with this device are comparable to trabeculectomy success rates. However, higher doses of MMC are associated with better results.