Cut Optic Nerve Research Articles

Outcomes of Children Diagnosed with Unilateral Retinoblastoma: Retrospective Audit

To evaluate treatment outcomes of children diagnosed with unilateral retinoblastoma. Retrospective study of children diagnosed with unilateral retinoblastoma registered at the Tata Memorial Hospital (TMH), Mumbai from January 2013 to December 2018 and completed the planned curative treatment protocol. For the 98 cases that were analyzed, the median age of presentation was 24 months. The majority of patients had the intraocular disease (n = 72), whereas orbital retinoblastoma was in 26 patients. At the time of presentation, on imaging extra scleral spread was observed in 16 patients whereas 18 patients had optic nerve involvement, 11 patients had both extra scleral invasion and optic nerve involvement. We used the International Classification of Retinoblastoma for grouping. Out of 98 patients, 71 patients were in Group E, 21 were in Group D and 4 were in Group B and 2 were in Group C. For Staging of Retinoblastoma, we used International Retinoblastoma Staging System (IRSS) in our study and 14 patients had Stage 0 disease, 52 patients had Stage I disease, 10 patients had Stage II, 21 patients had Stage III A and 1 patient had Stage III B disease. High-risk features on surgical specimen histopathology were optic nerve cut margin positive in 6 patients, optic nerve involvement in 21 patients, extra scleral spread in 3 patients, Choroidal invasion in 38 and Iris involvement in 10 patients. Primary enucleation was offered in 52 patients whereas 26 patients underwent secondary enucleation. Systemic chemotherapy was received by the patient in the neoadjuvant setting in 17 patients as a form of chemo-reduction and 51 patients received systemic chemotherapy in the adjuvant setting. Intra-arterial chemotherapy was offered to 27 patients as a primary treatment or in conjugation with focal therapy (n = 11). Definitive radiotherapy was offered to only 2 patients and 22 patients received adjuvant radiotherapy. At a median follow-up of 62 months, 2patients had local relapse, which was salvaged by focal therapy in one and enucleation in the other. Ten patients who had leptomeningeal relapse had died. The 5-year local control (LC) is 97.6%, event-free survival (EFS) is 88% and overall survival (OS) is 89.5%. Globe was salvaged in only 16 cases. On univariate analysis, we observed a significant association between overall survival and extraocular and intraocular disease (p-value 0.0), Extra scleral spread (p-value 0.0), optic Nerve involvement (imaging), and an optic nerve cut margin positive (p-value 0.045), ICRB Groups (p-value 0.0) and IRSS stage (p- value 0.024). Retinoblastoma is curable if detected early. Extra ocular disease and high-risk features are associated with inferior outcomes and poor globe salvage rates.

Protective Effect of Curcuma Extract in an Ex Vivo Model of Retinal Degeneration via Antioxidant Activity and Targeting the SUMOylation.

Retinal degeneration is the major and principal cause behind many incurable blindness diseases. Several studies indicated the neuroprotective effect of Curcuma longa in eye pathologies, specifically retinopathy. However, the molecular mechanism behind its effect has not been completely elucidated. Using an ex vivo model of retinal degeneration obtained from an ex vivo optic nerve cut (ONC), we demonstrated that Curcuma extract (Cur) exerted a neuroprotective effect. Importantly, Cur was able to modulate apoptosis and MAPK signaling pathway activation and prevent retinal ganglion cell (RGC) loss. Other well-known neuroprotective pharmacological tools, including memantine (Mem), citicoline (Cit), and ginkgolic acid (GA), were used to compare the potential mechanisms of Cur. The antioxidant activity of retinas treated with Cur following optic nerve cut was significantly higher than control, but Cur failed to change the retina glutamate content. Considering the antioxidant effect of Cur and taking advantage of our recent findings on the crosstalk between oxidative stress and post-translational protein modifiers, in particular, small ubiquitin-related modifier (SUMO), we were interested in exploring the effect of Cur on SUMOylation. We found that Cur significantly prevented the increase of protein SUMOylation, confirming our previous in vitro data indicating the cytoprotective effect of curcumin through modulating the oxidative stress and SUMO-JNK axis. Altogether, these results suggest that Curcuma protects the retina from degeneration via antioxidant activity and targets SUMOylation. Therefore, it might be considered for the combination therapy with other neuroprotective agents with different mechanisms in preclinical studies on retinal degeneration.

Retinal ganglion cell loss in an ex vivo mouse model of optic nerve cut is prevented by curcumin treatment

Retinal ganglion cell (RGC) loss is a pathologic feature common to several retinopathies associated to optic nerve damage, leading to visual loss and blindness. Although several scientific efforts have been spent to understand the molecular and cellular changes occurring in retinal degeneration, an effective therapy to counteract the retinal damage is still not available. Here we show that eyeballs, enucleated with the concomitant optic nerve cut (ONC), when kept in PBS for 24 h showed retinal and optic nerve degeneration. Examining retinas and optic nerves at different time points in a temporal window of 24 h, we found a thinning of some retinal layers especially RGC’s layer, observing a powerful RGC loss after 24 h correlated with an apoptotic, MAPKs and degradative pathways dysfunctions. Specifically, we detected a time-dependent increase of Caspase-3, -9 and pro-apoptotic marker levels, associated with a strong reduction of BRN3A and NeuN levels. Importantly, a powerful activation of JNK, c-Jun, and ERK signaling (MAPKs) were observed, correlated with a significant augmented SUMO-1 and UBC9 protein levels. The degradation signaling pathways was also altered, causing a significant decrease of ubiquitination level and an increased LC3B activation. Notably, it was also detected an augmented Tau protein level. Curcumin, a powerful antioxidant natural compound, prevented the alterations of apoptotic cascade, MAPKs, and SUMO-1 pathways and the degradation system, preserving the RGC survival and the retinal layer thickness. This ex vivo retinal degeneration model could be a useful method to study, in a short time window, the effect of neuroprotective tools like curcumin that could represent a potential treatment to contrast retinal cell death.

Combined association of massive choroidal and optic nerve invasion as a prognostic relevance in primary retinoblastoma: A 10-year study.

To determine the significance of both massive choroidal invasion and optic nerve invasion (retrolaminar [(RL]+cut end [CE]) as a criterion for classifying high metastatic potential retinoblastoma and their relationship with other known histopathological high-risk features. A retrospective review of 650 eyes diagnosed as retinoblastoma over a 10-year period. In our study, there is male predominance and a higher percentage of the poorly differentiated tumors. The age of most of the patients ranges from 1 month to 8years with a median age of 2years. There were 24% of eyes with massive choroidal invasion and 18% of eyes with optic nerve invasion up to the cut end. On performing Cox-proportional hazard analysis, it was found that massive choroidal invasion in association with optic nerve invasion up to the cut end was an independent prognostic parameter. On Kaplan-Meier analysis, overall survival had reduced in patients having both massive choroidal invasion and an optic nerve cut end invasion along with orbital invasion (P<.05). The presence of massive choroidal invasion in association with optic nerve cut end invasion (RL+CE) could be used as a better prognostic predictor in assessing retinoblastoma patients with high metastatic potential and need to be kept for longer follow up.

Ambient Illumination Influence on Photuris Firefly Larval Surface Movements is not Mediated by the Stemmata

Movements of fireflies visible on the surface of soil were measured under controlled laboratory conditions consisting of high and low ambient illumination. High illumination approximating the intensity of light prior to sunset constituted our light condition. Low illumination was consistent with ambient light levels after civil sunset, which we referred to as our dark condition. Surface movements were significantly more frequent during dark conditions compared to light conditions. Stemmata are the larval form of the insect eye and were the only identified visual organs present in Photuris larvae. We investigated whether stemmata provided larvae with the sensory information facilitating the light dependency of surface movements. We disrupted transmission of visual information from the larval eyes to the brain by severing the optic nerves. The amount of surface movement was compared between larvae with intact and severed optic nerves, under light and dark conditions. Light dependency of surface movements was preserved in larvae with cut optic nerves. The presence of the light dependency after cutting the optic nerves indicated that an alternative, extrastemmatal sensory pathway must be providing light intensity information to the animal. Light dependency was abolished upon removal of the head. Thus, these results suggested that the extraocular system providing light intensity information for regulating the frequency of surface movement was located in the head. The precise location of the suggested extraocular receptor and the nature of the associated sensory system remains unknown.

Expression of FOXO3a and Correlation With Histopathologic Features in Retinoblastoma.

Forkhead box (FOX) transcription factors are a class of highly conserved proteins, which serve critical cellular functions including cell cycle regulation. The downstream mechanisms of cell cycle regulation involve preservation of retinoblastoma protein function. Its deactivation by phosphorylation and translocation from nucleus to cytoplasm leads to cell proliferation. FOXO3a has been found to be dysregulated in few cancers. However, no study has been reported on role of FOXO3a in retinoblastoma. We assessed the expression of FOXO3a in sections of archived tissue blocks of enucleated/exenterated specimens of retinoblastoma by immunohistochemistry. The histopathologic features were reviewed and correlated with its expression. Effect of FOXO3a expression on survival was assessed. FOXO3a expression was assessed in 100 sections. Six samples did not contain any viable tissue. Retrospective data of 94 patients revealed that median age at presentation was 36 months with male:female ratio of 1.9:1. Fifty-one percent of patients were International Retinoblastoma Staging System stage 1. Of the 94 sections, 68 (72%) showed cytoplasmic expression. Choroidal invasion was associated with cytoplasmic FOXO3a (P=0.04). A trend was also noted in optic nerve cut end involvement (P=0.07). No other histopathologic features were found to be associated with FOXO3a expression. The overall survival and progression-free survival were not found to be affected by FOXO3a expression. Cytoplasmic expression of FOXO3a is frequently found in retinoblastoma and may be involved in pathogenesis. Activation by relocation of FOXO3a to nucleus may activate nonmutated retinoblastoma and may be a potential target of treatment in retinoblastoma.

The involvement of sirtuins during optic nerve injury of rats.

Sirtuins, comprised of seven members, protect cells from injury, possibly through different roles. In this study, we used two young rat optic nerve injury models to analyze the changes in Sirts 1-7 at different time points to better understand the role of sirtuins during optic nerve injury. Twelve-week-old adult male F344 rats (total n=42) were divided randomly into two groups. One group was subjected to optic nerve cut (ON-cut) and the other group was subjected to a peripheral nerve-optic nerve graft (PN-ON graft) on the left eye. At 1 and 3 days and 1, 2, and 4 weeks, rats were euthanized and retinas of both eyes were removed. Total RNA was extracted and first-strand cDNA was synthesized. Sirts 1-7 and housekeeping β-actin quantitative real-time PCR were performed. The quantitative real-time PCR profile showed that sirtuin mRNAs in both groups increased following optic nerve injury with and without peripheral nerve grafting. Sirt1 mRNA increased rapidly, reaching its peak at 3 days after surgery. Sirts 2-7 showed an increasing trend and remained high through 4 weeks after surgery. Sirts 4 and 6 were the only Sirts that increased in number in the PN-graft group at 4 weeks after surgery, where neuronal survival should be higher. Our data indicate that Sirt1 and Sirts 2-7 may play different or complementary roles in optic nerve injury and that Sirts 4 and 6 may play a greater role than the remaining Sirts in axon regeneration.

βB2-Crystallin Promotes Axonal Regeneration in the Injured Optic Nerve in Adult Rats.

The purpose of the study was to further scrutinize the potential of βB2-crystallin in supporting regeneration of injured retinal ganglion cell axons both in vitro and in vivo. Retinal explants obtained from animals after treatment either with lens injury (LI) alone or with combined LI 5 days or 3 days before or simultaneously with an optic nerve crush (ONC) were cultured for 96 h under regenerative conditions, and the regenerating axons were quantified and compared with untreated controls. These measurements were then repeated with LI replaced by intravitreal injections of γ-crystallin and β-crystallin at 5 days before ONC. Finally, βB2-crystallin-overexpressing transfected neural progenitor cells (βB2-crystallin-NPCs) in the eye were studied after crushing the optic nerve in vivo. Regeneration was monitored with the aid of immunoblotting of the retina and optic nerve both distal and proximal to the lesion site, and this was compared with controls that received injections of phosphate buffer only. LI performed 5 days or 3 days before ONC significantly promoted axonal outgrowth in vitro (p < 0.001), while LI performed alone before explantation did not. Intravitreal injections of β-crystallin and γ-crystallin mimicked the effects of LI and significantly increased axonal regeneration in culture at the same time intervals (p < 0.001). Western blot analysis revealed that crystallins were present in the proximal optic nerve stump at the lesion site in ONC, but were neither expressed in the undamaged distal optic nerve nor in uninjured tissue. βB2-crystallin-NPCs supported the regeneration of cut optic nerve axons within the distal optic nerve stump in vivo. The reported data suggest that βB2-crystallin-producing "cell factories" could be used to provide novel therapeutic drugs for central nervous system injuries.

Inflammatory stimulation preserves physiological properties of retinal ganglion cells after optic nerve injury

Axonal injury in the optic nerve is associated with retinal ganglion cell (RGC) degeneration and irreversible loss of vision. However, inflammatory stimulation (IS) by intravitreal injection of Pam3Cys transforms RGCs into an active regenerative state enabling these neurons to survive injury and to regenerate axons into the injured optic nerve. Although morphological changes have been well studied, the functional correlates of RGCs transformed either into a de- or regenerating state at a sub-cellular level remain unclear. In the current study, we investigated the signal propagation in single intraretinal axons as well as characteristic activity features of RGCs in a naive, a degenerative or a regenerative state in ex vivo retinae 1 week after either optic nerve cut alone (ONC) or additional IS (ONC + IS). Recordings of single RGCs using high-density microelectrode arrays demonstrate that the mean intraretinal axonal conduction velocity significantly decreased within the first week after ONC. In contrast, when ONC was accompanied by regenerative Pam3Cys treatment the mean intraretinal velocity was undistinguishable from control RGCs, indicating a protective effect on the proximal axon. Spontaneous RGC activity decreased for the two most numerous RGC types (ON- and OFF-sustained cells) within one post-operative week, but did not significantly increase in RGCs after IS. The analysis of light-induced activity revealed that RGCs in ONC animals respond on average later and with fewer spikes than control RGCs. IS significantly improved the responsiveness of the two studied RGC types. These results show that the transformation into a regenerative state by IS preserves, at least transiently, the physiological functional properties of injured RGCs.

Remote Ischemic Postconditioning Promotes the Survival of Retinal Ganglion Cells after Optic Nerve Injury

Ischemic conditioning, the application of a mild ischemic stimulus to an ischemia-sensitive structure like the heart or brain either before (preconditioning) or after (postconditioning) its exposure to a lethal ischemic insult, is known to switch on endogenous protective mechanisms. However, most studies of its neuroprotective effect in the central nervous system (CNS) have focused on ischemic damage or related conditions like hypoxia, while its potential in treating other neural diseases remains uncertain. In particular, the recent discovery of remote ischemic postconditioning whereby mild ischemia applied to a region remote from the target after the main ischemic insult also confers protection offers an attractive paradigm to study its potential in other types of neural injury. Retinal ganglion cells damaged by optic nerve transection undergo extensive cell death. However, application of a series of mild ischemic/reperfusion cycles to the hind limb (limb remote ischemic postconditioning) at 10 min or 6 h after optic nerve cut was found to promote ganglion cell survival at 7 days post-injury, with the 10 min postconditioning still exerting protection at 14 days post-injury. Concomitant with the increased ganglion cell survival, 51 % more ganglion cells expressed the small heat shock protein HSP27, when remote ischemic postconditioning was performed at 10 min post-injury, as compared to the sham conditioning group. Our results highlight the potential of using remote ischemic postconditioning as a noninvasive neuroprotective strategy in different CNS disorders like spinal cord and traumatic brain injury.

Differentiation in Retinoblastoma and Histopathological Risk Factors in Mexico

To describe the relationship between retinoblastoma differentiation, histopathological risk factors, age at enucleation, laterality, and genetic type (hereditary or sporadic) in a series of enucleated eyes in a referral hospital. The criteria used in other studies to classify retinoblastoma differentiation are discussed. The authors retrospectively studied histopathological preparations of eyes with retinoblastoma treated with primary enucleation. Tumors were classified as well differentiated, undifferentiated, and moderately differentiated. Patient age at enucleation, laterality of disease, genetic form (hereditary or sporadic), and presence of histopathological risk factors (massive choroidal infiltration, postlaminar optic nerve invasion, tumor in optic nerve cut, scleral invasion, and involvement of orbital soft tissues) were analyzed. The chi-square test was used for categorical variables and analysis of variance for test mean differences. Histopathological risk factors were present in 23 (36%) of 63 eyes. Moderately differentiated tumors occurred at advanced ages and were more frequently associated with histopathological risk factors. A consensus is needed to establish the histopathological criteria of retinoblastoma differentiation. The value of rosettes as a marker of cell differentiation should be reviewed.[J Pediatr Ophthalmol Strabismus 2013;50(3):174-177.].

Changes in fibroblast growth factor-2 and FGF receptors in the frog visual system during optic nerve regeneration

We have previously shown that application of fibroblast growth factor-2 (FGF-2) to cut optic nerve axons enhances retinal ganglion cell (RGC) survival in the adult frog visual system. These actions are mediated via activation of its high affinity receptor FGFR1, enhanced BDNF and TrkB expression, increased CREB phosphorylation, and by promoting MAPK and PKA signaling pathways. The role of endogenous FGF-2 in this system is less well understood. In this study, we determine the distribution of FGF-2 and its receptors in normal animals and in animals at different times after optic nerve cut. Immunohistochemistry and Western blot analysis were conducted using specific antibodies against FGF-2 and its receptors in control retinas and optic tecta, and after one, three, and six weeks post nerve injury. FGF-2 was transiently increased in the retina while it was reduced in the optic tectum just one week after optic nerve transection. Axotomy induced a prolonged upregulation of FGFR1 and FGFR3 in both retina and tectum. FGFR4 levels decreased in the retina shortly after axotomy, whereas a significant increase was detected in the optic tectum. FGFR2 distribution was not affected by the optic nerve lesion. Changes in the presence of these proteins after axotomy suggest a potential role during regeneration.

Outcome, Pathologic Findings, and Compliance in Orbital Retinoblastoma (International Retinoblastoma Staging System Stage III) Treated with Neoadjuvant Chemotherapy: A Prospective Study

The present study prospectively evaluated outcome, pathologic findings, and compliance in orbital retinoblastoma patients (International Retinoblastoma Staging System [IRSS] stage III). Prospective study and case series. Twenty-eight consecutive IRSS stage III retinoblastoma patients were enrolled prospectively in the study after ethics committee approval and written informed consent. Planned therapy for patients included treatment with a uniform protocol consisting of neoadjuvant chemotherapy followed by enucleation, adjuvant radiotherapy, and chemotherapy. Prospective computerized data entry and telephone contact were undertaken to improve compliance, a major issue in treatment of orbital retinoblastoma. Event-free survival (EFS) and overall survival (OS) were calculated using Kaplan-Meier survival analysis. The difference between groups was analyzed using the log-rank test. Median follow-up was 14.75 months (range, 2.23-26.3 months). The OS for all patients was 40.4% and the EFS was 33.33% at a follow-up of 26.3 months. The median EFS and OS were 10.37 months and 15.73 months, respectively. At baseline, bilateral retinoblastoma predicted inferior EFS (P = 0.0006) and OS (P = 0.0081). Twenty-two of 28 patients underwent enucleation, and viable tumor was present in 21 of 22 enucleated specimens (95%). Neoadjuvant chemotherapy prevented orbital exenterations. Presence of tumor in the cut end of the optic nerve significantly correlated with inferior EFS (P = 0.012) and OS (P = 0.0098). Central nervous system metastasis was the most common site of relapse and death. The overall rate of compliance with treatment was 67.8% (19/28), which was twice the compliance rate at the authors' center before initiation of the present study. There is a paucity of information in the literature on outcomes and pathologic findings after neoadjuvant chemotherapy in IRSS stage III retinoblastoma treated with a uniform treatment protocol, and as far as the authors are aware, there has been no prospective study. Neoadjuvant chemotherapy was able to avoid exenteration in all operated patients; however, residual viable tumor was present in 95% of enucleated specimens. Bilaterality and tumor in the optic nerve cut end after neoadjuvant chemotherapy were associated with inferior outcome. Compliance could be improved with computerized data entry and regular telephone contact.

Long-Term Gene Therapy Causes Transgene-Specific Changes in the Morphology of Regenerating Retinal Ganglion Cells

Recombinant adeno-associated viral (rAAV) vectors can be used to introduce neurotrophic genes into injured CNS neurons, promoting survival and axonal regeneration. Gene therapy holds much promise for the treatment of neurotrauma and neurodegenerative diseases; however, neurotrophic factors are known to alter dendritic architecture, and thus we set out to determine whether such transgenes also change the morphology of transduced neurons. We compared changes in dendritic morphology of regenerating adult rat retinal ganglion cells (RGCs) after long-term transduction with rAAV2 encoding: (i) green fluorescent protein (GFP), or (ii) bi-cistronic vectors encoding GFP and ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF) or growth-associated protein-43 (GAP43). To enhance regeneration, rats received an autologous peripheral nerve graft onto the cut optic nerve of each rAAV2 injected eye. After 5–8 months, RGCs with regenerated axons were retrogradely labeled with fluorogold (FG). Live retinal wholemounts were prepared and GFP positive (transduced) or GFP negative (non-transduced) RGCs injected iontophoretically with 2% lucifer yellow. Dendritic morphology was analyzed using Neurolucida software. Significant changes in dendritic architecture were found, in both transduced and non-transduced populations. Multivariate analysis revealed that transgenic BDNF increased dendritic field area whereas GAP43 increased dendritic complexity. CNTF decreased complexity but only in a subset of RGCs. Sholl analysis showed changes in dendritic branching in rAAV2-BDNF-GFP and rAAV2-CNTF-GFP groups and the proportion of FG positive RGCs with aberrant morphology tripled in these groups compared to controls. RGCs in all transgene groups displayed abnormal stratification. Thus in addition to promoting cell survival and axonal regeneration, vector-mediated expression of neurotrophic factors has measurable, gene-specific effects on the morphology of injured adult neurons. Such changes will likely alter the functional properties of neurons and may need to be considered when designing vector-based protocols for the treatment of neurotrauma and neurodegeneration.

A Study of Pathologic Risk Factors in Postchemoreduced, Enucleated Specimens of Advanced Retinoblastomas in a Developing Country

Advanced cases of retinoblastoma are treated with chemoreduction followed by enucleation. Further adjuvant therapy is recommended in patients with known pathologic risk factors (PRFs). To determine the PRFs in enucleated specimens after chemoreduction and their association for adverse events of recurrence, metastasis, or death. This was a retrospective study of 77 enucleation specimens from patients treated between January 2000 and September 2008 with prior chemoreduction that were accessioned in the pathology department of a tertiary referral cancer center with an average follow-up of 24 months. Various PRFs were noted and their association with the development of an adverse event was recorded. Of 77 patients, (male to female ratio, 51∶26), the incidence of overall PRF was 51.9%, and retrolaminar optic nerve invasion (32.5%), optic nerve cut margin (12.9%), massive choroidal invasion (26%), scleral invasion (23.4%), vitreous seedings (44.2%), and anterior segment invasion (20.8%). Undifferentiated tumor (>60%) was seen in 60.3% of cases (41 of 68 patients with differentiation available). Adverse event occurred in 18 of 72 patients with available follow-up (25%). Retrolaminar optic nerve invasion, optic nerve cut margin involvement, and scleral invasion were independent prognostic factors predicting the occurrence of an adverse event. Undifferentiated tumor (>60%) was a significant risk factor in univariate analysis, which is the unique feature in this study. Classic PRF with the addition of a predominant presence from the undifferentiated component were associated with adverse outcomes in retinoblastoma treated with anterior chemotherapy. The latter may represent chemoresistant clones and more intensive adjuvant chemotherapy may be warranted in these patients.

Clinical predictors of high risk histopathology in retinoblastoma

Previous studies show that clinical features at presentation, in retinoblastoma patients, like glaucoma and neovascularization of iris are associated with a higher incidence of high risk histopathology findings (HRF) in enucleated eyes. Herein, we analyze association between clinical features at time of enucleation and occurrence of HRF including invasion of anterior chamber, iris, ciliary body, choroid (massive), sclera, extrascleral tissue, optic nerve beyond lamina cribrosa, and optic nerve cut end, in a large series of eyes enucleated for retinoblastoma. We retrospectively studied demographic, clinical, and histopathology findings in all retinoblastoma patients who underwent primary enucleation at our center, over a 5 years duration. Statistical analysis was done to find any association between clinical features at presentation and the presence of HRF. Three hundred twenty-six eyes were studied. Median age of presentation was 2 years. Glaucoma was the most common clinical finding at presentation apart from leucocoria. Out of 326 enucleated eyes, 28 (8.6%) had extrascleral and/or optic nerve transection invasion. Among remaining 298 eyes, with completely resected tumor, 115 (38.6%) had massive choroidal invasion, 54 (17%) had retrolaminar optic nerve invasion, and 24 (7%), 29 (9%), and 23(7%) had anterior chamber, iris, and ciliary body invasion, respectively. Age more than 2 years, lag period more than 3 months, hyphema, pseudohypopyon, staphyloma, and orbital cellulitis were associated with occurrence of three or more HRF on univariate analysis. Clinical variables including older age, longer lag period, hyphema, pseudohypopyon, staphyloma, and orbital cellulitis were strongly associated with occurrence of HRF in this study.

Long-term morphometric stabilization of regenerating retinal ganglion cells in the adult rat

Adult retinal ganglion cells (RGCs) can regenerate their cut axons within peripheral nerve grafts used to replace the distal optic nerve stump. We examined the long-term stabilization of RGCs by guiding their regenerating axons into different termination areas. The optic nerve (ON) of adult rats was completely cut intraorbitally and its ocular stump was connected with different visual target areas (cortex, midbrain) or with non-visual areas (e.g. muscle). Control groups consisted of blind ending graft and ON cut without graft. The function of the retina was regularly examined by electroretinography. At one, six and nine months postsurgery RGCs were retrogradely labelled with 4-(4-(didecylamino)styryl)-N-methylpyridinium iodide and examined morphometrically. Regenerating RGCs were categorized into three major classes representing the morphological types I, II and III. Our data show that regenerating RGCs remain stable up to nine months after grafting at the ON, although the numbers of axons are low, that is less than 1%, and this number is not significantly effected by reconnection with targets. However, there are significant quantitatively and morphometrically assessable differences between the experimental groups depending on the tissue the RGCs are connected with visual targets. Regenerating RGCs show the highest stability in morphology if reconnected with visual target tissue. Adult RGCs of the rat can be reconnected with visual centers using a peripheral nerve graft. This reconnection stabilizes the cells at morphological and the retina at functional levels for a long period of time, although it does not significantly increase cell survival.

Negative impact of rAAV2 mediated expression of SOCS3 on the regeneration of adult retinal ganglion cell axons

Intravitreal injections of recombinant ciliary neurotrophic factor (rCNTF) protect adult rat retinal ganglion cells (RGCs) after injury and stimulate regeneration, an effect enhanced by co-injection with a cAMP analogue (CPT-cAMP). This effect is partly mediated by PKA and associated signaling pathways, but CPT-cAMP also moderates upregulation of suppressor of cytokine signaling (SOCS) pathways after rCNTF injection, which will also enhance the responsiveness of RGCs to this and perhaps other cytokines. We now report that intravitreal injections of CPT-cAMP do not potentiate RGC axonal regeneration when CNTF is expressed via an adeno-associated viral vector (rAAV2), and concomitantly we show that increases in retinal SOCS mRNA expression are less when CNTF is delivered using the vector. We also directly tested the impact of elevated SOCS3 expression on the survival and regeneration of injured adult RGCs by injecting a bicistronic rAAV2-SOCS3-GFP vector into the vitreous of eyes in rats with a peripheral nerve graft sutured onto the cut optic nerve. Overexpression of SOCS3 resulted in an overall reduction in axonal regrowth and almost complete regeneration failure of RGCs transduced with the rAAV2-SOCS3-GFP vector. Furthermore, rAAV2-mediated expression of SOCS3 abolished the normally neurotrophic effects elicited by intravitreal rCNTF injections. In summary, CNTF delivery to the retina using viral vectors may be more effective than bolus rCNTF injections because the gene therapy approach has a less pronounced effect on neuron-intrinsic SOCS repressor pathways. Our new gain of function data using rAAV2-SOCS3-GFP demonstrate the negative impact of enhanced SOCS3 expression on the regenerative potential of mature CNS neurons.

The life, death and regenerative ability of immature and mature rat retinal ganglion cells are influenced by their birthdate

The extensive period of retinal ganglion cell (RGC) neurogenesis in the rat is associated with a protracted sequence of arrival of their axons into central targets such as the superior colliculus (SC) (Dallimore et al., 2002). Using in utero 5-bromo-2'-deoxyrudine (BrdU) injections to label early (embryonic day (E) 15) or late (E18 or E19) born RGCs, we now show that E15 RGCs with axons that enter the SC prenatally undergo programmed cell death earlier than late-born RGCs whose axons only reach the SC late in the first postnatal week. These late-born RGCs do not begin to die until postnatal day (P) 5/6. Removal of retrograde trophic support by P1 SC ablation initially only affects E15 RGCs; however by P5 death of late-born RGCs is increased, confirming that a switch to target dependency is delayed in this cohort. In a further experiment it was found that, following complete rostral SC transection at P2, the proportion of post-lesion axons originating from E19 RGCs was significantly greater than the proportion that normally makes up the retinotectal projection. Thus, even in neonatal brain, uninjured late-arriving axons are more likely to grow across a lesion site than injured axons undergoing regeneration. To study if birth date also affects regenerative potential in adulthood, autologous peripheral nerve (PN) was grafted onto the cut optic nerve in mature BrdU labelled rats. We found that, compared to E15 RGCs, a significantly greater proportion of late-born RGCs survived axotomy, but comparatively fewer of these surviving E19 RGCs regrew an axon into a graft. In summary, this research shows that the birthdate of RGCs significantly impacts on their subsequent life history and response to injury. Understanding how developing central nervous system (CNS) neurons acquire dependency on target-derived trophic support may lead to new strategies for enhancing survival and regeneration in adult CNS.

Dendritic differentiation in the periphery of the growing zebrafish retina

In the retina of teleost fish, new ganglion cells are generated from a circumferential peripheral growth zone at the edge of the eye throughout life. Addressing the question how new cells are fitted into the existing retina, we investigated newly formed ganglion cells in the zebrafish retina morphologically, by tracing them from the cut optic nerve with rhodamine dextran. We identified proliferating cells by antibody detection against proliferating cell nuclear antigen. In addition, newly formed bipolar cell and amacrine cell dendrites were investigated by antibodies against protein kinase C (PKC) and choline-acetyl-transferase (ChaT), respectively, and analyzed in sections or wholemount preparations using confocal microscopy. In retinal sections we observed that ganglion cell dendritic branches in the inner plexiform layer were in close apposition to dividing cells. In the periphery of retinal wholemounts, we detected rhodamine traced ganglion cells adjacent to the growth zone, extending dendrites in proximity to the growth zone, typically branching off in opposite directions running parallel to the retinal rim over more then 100 μm. Ganglion cells with similar dendritic branching patterns were not found in more central retinal areas. Similarly, the dendrites of ChaT-positive amacrine cells showed a preference for running parallel to the circumference in the periphery. Dendritic branches of PKC-positive bipolar cells did not show similar preferred orientation. The change in shape of the dendritic tree with distance from the periphery was studied for the Ma type ganglion cell. The data are consistent with the idea that existing ganglion cells might control differentiation of new ganglion cells. Moreover, ganglion cells with specific branching patterns towards the retinal periphery undergo a restructuring of their dendritic trees.