-
[show abstract]
[hide abstract]
ABSTRACT: PURPOSE: Retinal laser photocoagulation represents a major treatment strategy for the management of diabetic macular edema (DME). The thermal nature of this procedure, however, defines that collateral tissue injury result, meaning that it cannot be used near the fovea centralis. We studied inflammatory and glial responses resulting from treatment of rats with a conventional laser and with a novel short-duration, non-thermal laser (Retinal Regeneration Therapy; 2RT) at clinically relevant energy levels. METHODS: Pigmented Dark Agouti rats were treated with either a conventional thermal continuous wave (CW; 532nm, 100ms pulse duration) or a short-pulse (2RT; 532nm, Q-switched, 3ns pulse) laser. Settings were at visible threshold for the CW laser (12733mJ/cm2/pulse) and at supra- and sub-visible threshold for the 2RT laser ("High", 2RT-H, 163mJ/cm2/pulse; "Low", 2RT-L, 109mJ/cm2/pulse). Rats were killed at various subsequent time points. Samples were processed for histology, immunohistochemistry, RT-PCR and Western blotting. RESULTS: The CW laser caused outer retinal lesions that were associated with photoreceptor death, astrocyte and Muller cell activation, and infiltration of macrophages and neutrophils. Furthermore, inflammatory cytokines, heat shock proteins, endogenous trophic factors, and matrix metalloproteinases were induced. In comparison, all of these changes were drastically attenuated when the 2RT laser was used, particularly at the sub-threshold setting. CONCLUSIONS: The conventional laser produced marked retinal damage and cellular responses consistent with an inflammatory response to thermal injury. In contrast, the 2RT laser produced negligible retinal damage and cellular responses at clinically relevant settings. These results may have important implications for the treatment of retinal disease.
Investigative ophthalmology & visual science 02/2013; · 3.43 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: PURPOSE: To determine detailed effects to retinal cells, and, in particular, neurons, following laser photocoagulation using a conventional 532nm Nd:YAG continuous wave (CW) laser. Furthermore, to determine whether a novel 3ns pulse laser (retinal regeneration therapy; 2RT) could specifically ablate retinal pigmented epithelium (RPE) cells without causing collateral damage to other retinal cells. METHODS: Adult Dark Agouti (DA) rats were separated into four groups: control, CW laser (12700mJ/cm2/pulse, 100ms pulse duration) or 3ns pulse 2RT laser at one of two energy settings ("High", 2RT-H, 163mJ/cm2/pulse; "Low", 2RT-L, 109mJ/cm2/pulse). Animals were treated and killed after 6h to 7d, and retina/RPE analyzed by histological assessment, Western blot, polymerase chain reaction and immunohistochemistry. RESULTS: Both lasers caused focal loss of RPE cells with no destruction of Bruch's Membrane; RPE cells were present at lesion sites again within 7 days of treatments. CW and 2RT-H treatments caused extensive, and moderate damage to the outer retina, respectively. There were no obvious effects to horizontal, amacrine or ganglion cells, as defined by immunolabeling, but an activation of PKCα within bipolar cells was noted. There was little discernible damage to any cells other than the RPE with the 2RT-L treatment. CONCLUSIONS: Conventional laser photocoagulation caused death of RPE cells with associated widespread damage to the outer retina but little influence to the inner retina. The novel 3ns 2RT laser, however, was able to selectively kill RPE cells without causing collateral damage to photoreceptors. Potential benefits of this laser for clinical treatment of diabetic macular edema are discussed.
Investigative ophthalmology & visual science 02/2013; · 3.43 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: In the 1920s, Otto Warburg and his group at the Kaiser Wilhelm Institute noted the importance of glycolysis to proliferating normal and malignant tissue, formulating the "no growth without glycolysis" dictum.(1,2) Consistent with Warburg's observations, Agathocleous et al(3) have recently shown that embryonic frog (Xenopus laevis) retina tends to divert energy metabolism away from oxidative phosphorylation and produce adenosine triphosphate via the conversion of pyruvate to lactate, despite the presence of oxygen, a phenomenon known as aerobic glycolysis or the Warburg effect. Biosynthetic demands explain the presence of the Warburg effect in neoplastic and normal embryonic tissue: provided that the energy supply is sufficient, proliferating cells divert glycolytic metabolites towards biosynthesis rather than towards oxidative phosphorylation.(4) But the existence of the Warburg effect in the retina has remained mysterious.
Clinical and Experimental Ophthalmology 12/2012; · 1.98 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: It is increasingly recognised that chronically activated glia contribute to the pathology of various neurodegenerative diseases, including glaucoma. One means by which this can occur is through the release of neurotoxic, proinflammatory factors. In the current study, we therefore investigated the spatio-temporal patterns of expression of three such cytokines, IL-1β, TNFα and IL-6, in a validated rat model of experimental glaucoma. First, only weak evidence was found for increased expression of IL-1β and TNFα following induction of ocular hypertension. Second, and much more striking, was that robust evidence was uncovered showing IL-6 to be synthesised by injured retinal ganglion cells following elevation of intraocular pressure and transported in an orthograde fashion along the nerve, accumulating at sites of axonal disruption in the optic nerve head. Verification that IL-6 represents a novel marker of disrupted axonal transport in this model was obtained by performing double labelling immunofluorescence with recognised markers of fast axonal transport. The stimulus for IL-6 synthesis and axonal transport during experimental glaucoma arose from axonal injury rather than ocular hypertension, as the response was identical after optic nerve crush and bilateral occlusion of the carotid arteries, each of which is independent of elevated intraocular pressure. Moreover, the response of IL-6 was not a generalised feature of the gp130 family of cytokines, as it was not mimicked by another family member, ciliary neurotrophic factor. Finally, further study suggested that IL-6 may be an early part of the endogenous regenerative response as the cytokine colocalised with growth-associated membrane phosphoprotein-43 in some putative regenerating axons, and potently stimulated neuritogenesis in retinal ganglion cells in culture, an effect that was additive to that of ciliary neurotrophic factor. These data comprise clear evidence that IL-6 is actively involved in the attempt of injured retinal ganglion cells to regenerate their axons.
Neurobiology of Disease 08/2012; 48(3):568-81. · 5.40 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Our study aimed to establish a model of energetic and metabolic dysfunction to cultured retinal cells by chemically inhibiting the mitochondrial electron transport chain with sodium azide (NaN(3)), and subsequently investigating toxic mechanisms and potential neuroprotective strategies. Methods. Mixed rat retinal cultures comprising neurons and glia were treated with a range of NaN(3) concentrations for up to 24 hours and toxicity levels were determined by immunologic
Detailed pathologic mechanisms were investigated by assessing apoptosis (TUNEL assay), mitochondrial membrane potential, reactive oxygen species (ROS), and levels of adenosine triphosphate (ATP). Finally, a number of pharmacologic agents were tested to determine whether they could abrogate the effects of NaN(3) to retinal cells.
Neurons and glia were killed by NaN(3) in a concentration- and time-dependent manner, with neurons being relatively more susceptible. Cell loss was via apoptosis for glia but not for neurons. Cell death generally involved a loss of mitochondrial membrane potential, a reduction in cellular ATP, and an increase in intracellular ROS levels. Glucose was partially able to prevent neuron death, as were the antioxidants trolox and pyruvate, calpain inhibitor III, the ryanodine receptor blocker dantrolene, and the nitric oxide synthase inhibitor L-NAME.
Mitochondrial respiratory inhibition via NaN(3) treatment, with delineated mechanisms of toxicity and neuroprotection, represents a valid and reproducible metabolic challenge to cultured retinal cells.
Investigative ophthalmology & visual science 06/2012; 53(8):4897-909. · 3.43 Impact Factor
-
Shiwani Sharma,
Kathryn P Burdon, Glyn Chidlow,
Sonja Klebe,
April Crawford,
David P Dimasi,
Alpana Dave,
Sarah Martin,
Shahrbanou Javadiyan,
John P M Wood,
Robert Casson,
Patrick Danoy,
Kim Griggs,
Alex W Hewitt,
John Landers,
Paul Mitchell,
David A Mackey,
Jamie E Craig
[show abstract]
[hide abstract]
ABSTRACT: Glaucoma is the leading cause of irreversible blindness worldwide. Primary open angle glaucoma (POAG) is the most common subtype. We recently reported association of genetic variants at chromosomal loci, 1q24 and 9p21, with POAG. In this study, we determined association of the most significantly associated single nucleotide polymorphism (SNP) rs4656461, at 1q24 near the TMCO1 gene, with the clinical parameters related to glaucoma risk and diagnosis, and determined ocular expression and subcellular localization of the human TMCO1 protein to understand the mechanism of its involvement in POAG.
Association of SNP rs4656461 with five clinical parameters was assessed in 1420 POAG cases using linear regression. The TMCO1 gene was screened for mutations in 95 cases with a strong family history and advanced disease. Ocular expression and subcellular localization of the TMCO1 protein were determined by immunolabeling and as GFP-fusion.
The data suggest that individuals homozygous for the rs4656461 risk allele (GG) are 4 to 5 years younger at diagnosis than noncarriers of this allele. Our data demonstrate expression of the TMCO1 protein in most tissues in the human eye, including the trabecular meshwork and retina. However, the subcellular localization differs from that reported in other studies. We demonstrate that the endogenous protein localizes to the cytoplasm and nucleus in vivo and ex vivo. In the nucleus, the protein localizes to the nucleoli.
This study shows a relationship between genetic variation in and around TMCO1 with age at diagnosis of POAG and provides clues to the potential cellular function/s of this gene.
Investigative ophthalmology & visual science 06/2012; 53(8):4917-25. · 3.43 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The maintenance of vision, through prevention and attenuation of neuronal injury in glaucoma, forms the basis of current clinical practice. Currently, the reduction of intraocular pressure is the only proven method to achieve these goals. Although this strategy enjoys considerable success, some patients progress to blindness; hence, additional management options are highly desirable. Several terms describing treatment modalities of neuronal diseases with potential applicability to glaucoma are used in the literature, including neuroprotection, neurorecovery, neurorescue and neuroregeneration. These phenomena have not been defined within a coherent framework. Here, we suggest a set of definitions, postulates and principles to form a foundation for the successful translation of novel glaucoma therapies from the laboratory to the clinic.
Clinical and Experimental Ophthalmology 05/2012; 40(4):350-7. · 1.98 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We investigated the mechanism of secondary axonal degeneration after perikaryal excitotoxic injury to retinal ganglion cells (RGCs) by comparing pathological responses in wild-type rats and Wld(s) rats, which display delayed Wallerian degeneration. After perikaryal excitotoxic RGC injury, both types of rats exhibited a spatio-temporal pattern of axonal cytoskeletal degeneration consistent with Wallerian degeneration, which was delayed by up to 4 weeks in Wld(s) rats. Furthermore, RGC somal loss was greater in Wld(s) rats. Microglial response in the anterior visual pathway to injury was attenuated in the Wld(s) rats with lymphocytic infiltration that was relatively reduced; however, immunostaining for major histocompatibility complex class II antigens (OX6) was more pronounced in Wld(s) rats. These data indicate that perikaryal excitotoxic RGC injury causes a secondary Wallerian axonal degeneration, and support the notion of a labile, soma-derived axonal survival factor.
Experimental Neurology 04/2012; 236(1):34-45. · 4.70 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Glaucoma is a term describing a group of ocular disorders with multi-factorial etiology united by a clinically characteristic intraocular pressure-associated optic neuropathy. It is not a single entity and is sometimes referred to in the plural as the glaucomas. All forms are potentially progressive and can lead to blindness. The diverse conditions that comprise glaucoma are united by a clinically characteristic optic neuropathy: glaucomatous optic neuropathy (GON). Evidence suggests that the primary site of neurological injury is at the optic nerve head. This fact enables the conditions to be grouped, irrespective of the causal mechanism(s). The term experimental glaucoma implies model resemblance to the human condition. We propose that 'experimental glaucoma' be restricted to animal models with demonstrable features of GON and/or evidence of a primary axonopathy at the optic nerve head. A fundamental inadequacy in this framework is any reference to the pathogenesis of GON, which remains unclear.
Clinical and Experimental Ophthalmology 02/2012; 40(4):341-9. · 1.98 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Full axon counting of optic nerve cross-sections represents the most accurate method to quantify axonal damage, but such analysis is very labour intensive. Recently, a new method has been developed, termed targeted sampling, which combines the salient features of a grading scheme with axon counting. Preliminary findings revealed the method compared favourably with random sampling. The aim of the current study was to advance our understanding of the effect of sampling patterns on axon counts by comparing estimated axon counts from targeted sampling with those obtained from fixed-pattern sampling in a large collection of optic nerves with different severities of axonal injury.
Chronic ocular hypertension was induced in adult Sprague-Dawley rats for 1-7 weeks by translimbal laser photocoagulation of the trabecular meshwork. Axonal damage on resin-embedded cross-sections was estimated using three different methods: (i) semi-quantitative damage grading; (ii) semi-quantitative, automated axon counting using targeted sampling; and (iii) semi-quantitative, automated axon counting using fixed-pattern sampling.
Estimated axon counts, as generated by targeted sampling and fixed-pattern sampling, correlated equally well with the semi-quantitative grading scheme. Estimated counts obtained with targeted sampling were not statistically different from those yielded by fixed-pattern sampling. Bland-Altman analysis showed a good agreement between the two methods.
The results of our study validate the use of both fixed-pattern sampling and targeted sampling for estimation of axonal damage but do not indicate that the latter method is superior for detection of axon loss in animals with minor damage.
Clinical and Experimental Ophthalmology 12/2011; 40(6):626-33. · 1.98 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: To evaluate the neuroprotective effect of short-term hyperglycemia on the retinal ganglion cell body and axon in a rat model of experimental glaucoma.
Using a well-described limbal laser technique, unilateral ocular hypertension was induced in 2 groups (26 per group) of Sprague-Dawley rats. One group remained normoglycemic; the other was rendered hyperglycemic by means of an intraperitoneal injection of streptozocin. After 2 weeks of elevated intraocular pressure, axonal and retinal damage profiles were compared using several histological techniques. Immunohistochemical changes in the retina and optic nerve were also assessed.
We found convincing evidence of delayed axonal degeneration and retinal ganglion cell death in hyperglycemic rats. Axon loss was reduced by about 50% 2 weeks after induction of ocular hypertension. Survival of retinal ganglion cell perikarya increased to a similar extent in hyperglycemic rats.
The optic nerve and retinal ganglion cells are partially protected by short-term hyperglycemia in this rat model of experimental glaucoma. Energy substrate availability may therefore play a role in glaucomatous optic neuropathy.
Our findings, to some extent, support the claims of the Ocular Hypertension Treatment Study, in which diabetes appeared to protect against the conversion to glaucoma. Targeted manipulation of neuronal energy metabolism may delay optic nerve degeneration and may represent a novel neuroprotective strategy for neurodegenerative diseases of the visual system such as glaucoma.
Archives of ophthalmology 10/2011; 129(10):1337-44. · 3.86 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The preferred fixative for whole eyes is Davidson's solution, which provides optimal tissue preservation while avoiding retinal detachment. Hitherto, the compatibility of Davidson's solution with immunohistochemistry has been largely untested. The goal of the present study was to compare the immunolabeling patterns of a wide-ranging panel of commercially available, previously validated antibodies in formalin- and Davidson's-fixed retinas. Immunohistochemistry was performed in normal pigmented rat eyes and, to facilitate localization of inducible proteins, eyes injected with the bacterial toxin lipopolysaccharide or subjected to laser-induced photoreceptor damage. Specificity of labeling was judged by the morphology and distribution of immunopositive cells, by the absence of signal in appropriate controls, and by comparison with expected staining patterns. Retinas fixed in formalin displayed only adequate morphological integrity but were highly compatible with all 39 antibodies evaluated. Retinas fixed in Davidson's solution displayed morphological integrity superior to those fixed in formalin. Generally, the cellular and subcellular patterns and intensities of immunoreactivities obtained with each fixative were identical; however, Davidson's fixative was less compatible with certain antibodies, such as the neurotransmitter γ-aminobutyric acid, the microglial marker iba1, the macroglial stress protein nestin, and the small heat shock proteins Hsp27 and αB-crystallin, shortfalls that somewhat temper enthusiasm concerning its use.
Journal of Histochemistry and Cytochemistry 08/2011; 59(10):884-98. · 2.72 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Thermal lasers are routinely used to treat certain retinal disorders although they cause collateral damage to photoreceptors. The current study evaluated a confined, non-conductive thermal, 3-nanosecond pulse laser in order to determine how to produce the greatest therapeutic range without causing collateral damage. Data were compared with that obtained from a standard thermal laser.
Porcine ocular explants were used; apposed neuroretina was also in place for actual laser treatment. After treatment, the retina was removed and a calcein-AM assay was used to assess retinal pigmented epithelium (RPE) cell viability in the explants. Histological methods were also employed to examine lased transverse explant sections. Three nanoseconds pulse lasers with either speckle- or gaussian-beam profile were employed in the study. Comparisons were made with a 100 milliseconds continuous wave (CW) 532 nm laser. The therapeutic energy range ratio was defined as the minimum visible effect threshold (VET) versus the minimum detectable RPE kill threshold.
The 3-nanosecond lasers produced markedly lower minimum RPE kill threshold levels than the CW laser (e.g., 36 mJ/cm(2) for speckle-beam and 89 mJ/cm(2) for gaussian-beam profile nanosecond lasers vs. 7,958 mJ/cm(2) for CW laser). VET values were also correspondingly lower for the nanosecond lasers (130 mJ/cm(2) for 3 nanoseconds speckle-beam and 219 mJ/cm(2) for gaussian-beam profile vs. 1,0346 mJ/cm(2) for CW laser). Thus, the therapeutic range ratios obtained with the nanosecond lasers were much more favorable than that obtained by the CW laser: 3.6:1 for the speckle-beam and 2.5:1 for the gaussian-beam profile 3-nanosecond lasers versus 1.3:1 for the CW laser.
Nanosecond lasers, particularly with a speckle-beam profile, provide a much wider therapeutic range of energies over which RPE treatment can be performed, without damage to the apposed retina, as compared with conventional CW lasers. These results may have important implications for the treatment of retinal disease.
Lasers in Surgery and Medicine 08/2011; 43(6):499-510. · 2.75 Impact Factor
-
Kathryn P Burdon,
Stuart Macgregor,
Alex W Hewitt,
Shiwani Sharma, Glyn Chidlow,
Richard A Mills,
Patrick Danoy,
Robert Casson,
Ananth C Viswanathan,
Jimmy Z Liu, [......],
Paul Leo,
Jie Jin Wang,
Elena Rochtchina,
Dale R Nyholt,
Nicholas G Martin,
Grant W Montgomery,
Paul Mitchell,
Matthew A Brown,
David A Mackey,
Jamie E Craig
[show abstract]
[hide abstract]
ABSTRACT: We report a genome-wide association study for open-angle glaucoma (OAG) blindness using a discovery cohort of 590 individuals with severe visual field loss (cases) and 3,956 controls. We identified associated loci at TMCO1 (rs4656461[G] odds ratio (OR) = 1.68, P = 6.1 × 10(-10)) and CDKN2B-AS1 (rs4977756[A] OR = 1.50, P = 4.7 × 10(-9)). We replicated these associations in an independent cohort of cases with advanced OAG (rs4656461 P = 0.010; rs4977756 P = 0.042) and two additional cohorts of less severe OAG (rs4656461 combined discovery and replication P = 6.00 × 10(-14), OR = 1.51, 95% CI 1.35-1.68; rs4977756 combined P = 1.35 × 10(-14), OR = 1.39, 95% CI 1.28-1.51). We show retinal expression of genes at both loci in human ocular tissues. We also show that CDKN2A and CDKN2B are upregulated in the retina of a rat model of glaucoma.
Nature Genetics 06/2011; 43(6):574-8. · 35.53 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The neurodegenerative disease glaucoma is characterised by the progressive death of retinal ganglion cells (RGCs) and structural damage to the optic nerve (ON). New insights have been gained into the pathogenesis of glaucoma through the use of rodent models; however, a coherent picture of the early pathology remains elusive. Here, we use a validated, experimentally induced rat glaucoma model to address fundamental issues relating to the spatio-temporal pattern of RGC injury. The earliest indication of RGC damage was accumulation of proteins, transported by orthograde fast axonal transport within axons in the optic nerve head (ONH), which occurred as soon as 8 h after induction of glaucoma and was maximal by 24 h. Axonal cytoskeletal abnormalities were first observed in the ONH at 24 h. In contrast to the ONH, no axonal cytoskeletal damage was detected in the entire myelinated ON and tract until 3 days, with progressively greater damage at later time points. Likewise, down-regulation of RGC-specific mRNAs, which are sensitive indicators of RGC viability, occurred subsequent to axonal changes at the ONH and later than in retinas subjected to NMDA-induced somatic excitotoxicity. After 1 week, surviving, but injured, RGCs had initiated a regenerative-like response, as delineated by Gap43 immunolabelling, in a response similar to that seen after ON crush. The data presented here provide robust support for the hypothesis that the ONH is the pivotal site of RGC injury following moderate elevation of IOP, with the resulting anterograde degeneration of axons and retrograde injury and death of somas.
Acta Neuropathologica 02/2011; 121(6):737-51. · 9.32 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Glia are the main cellular CNS elements initiating defense mechanisms against destructive influences and promoting regenerative processes. The aim of the current work was to characterize the microglial response within the visual pathway in a rat model of experimental glaucoma and to correlate the microglial response with the severity of axonal degeneration.
Experimental glaucoma was induced in each right eye of adult Sprague-Dawley rats by translimbal laser photocoagulation of the trabecular meshwork. Rats were subsequently killed at various times from 3 days to 6 weeks. Tissue sections were obtained from globes, optic nerves, chiasmata, and optic tracts for immunohistochemistry and toluidine blue staining.
This model of experimental glaucoma led to a marked activation of microglia in the retina, optic nerve, and tract. Indeed, microglial activity remained elevated, even after intraocular pressure returned to basal levels. It is postulated that this process accompanies ongoing axonal degeneration. The degree of activation in the optic nerve correlated with axonal damage. Activation was characterized by increased density and morphologic changes. Both major histocompatibility complex (MHC) class I and MHC class II surface proteins were persistently upregulated in optic nerves and localized to microglial cells; however, this did not correlate with any significant T-cell infiltration. Interestingly, MHC class II expression was not detected in the retina.
The present data may have implications for the study of the pathology associated with the visual pathway in diseases such as glaucoma.
Investigative ophthalmology & visual science 12/2010; 51(12):6448-60. · 3.43 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Selective laser trabeculoplasty (SLT) is becoming increasingly employed to reduce elevated intraocular pressure in glaucoma patients. SLT is known to target the ocular trabecular meshwork (TM), but the exact response mechanisms to this treatment have not been clearly delineated. The aim of the present study, therefore, was to investigate the modes of death of cultured bovine TM cells subjected to SLT in vitro.
Bovine TM cell cultures were established, pigmented with exogenous melanin and irradiated with a Q-switched, frequency doubled, Nd:YAG laser, at different energy settings (0.05-1.0 mJ). Influences on cells were determined for up to 10 days post-treatment by trypan blue exclusion, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and by morphological assessment. Furthermore, homogeneous mixtures of pigmented and non-pigmented TM cells were irradiated to ascertain selectivity of laser effects.
At higher energy levels (1.0, 0.75 mJ), immediate loss of cells was detected at the irradiated site. Trypan blue exclusion analysis showed that necrotic cell death subsequently occurred up to 8 hours following irradiation, peaking at 60 minutes. This was followed by delayed cell death peripheral to the irradiated area which was characteristic of apoptosis and which peaked at 2-3 days post-treatment. When mixed cultures were tested, laser treatment selectively killed pigmented cells at an energy level equivalent to the lower cell killing threshold in the initial studies (0.2 mJ) but at the higher laser energy of 0.35 mJ, all cells were non-selectively killed.
SLT treatment killed pigmented TM cells in culture by a variety of processes (instant vaporization, rapid necrosis, delayed apoptosis), depending on the magnitude of the energy used and the distance from the center of the irradiated zone. These data may assist in the elucidation of the mechanism of action of the SLT procedure on TM cells in situ.
Lasers in Surgery and Medicine 04/2010; 42(4):326-37. · 2.75 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: PURPOSE. Although the RGC-5 cell line is widely used in retinal ganglion cell (RGC) research, recent data have raised questions about the nature of these cells. The authors performed a systematic analysis of RGC-5 cells to determine which RGC or neuronal markers are expressed after treatment with known differentiating agents, thus providing further insight into the nature of these cells and assisting in defining their future use. METHODS. RGC-5 cells were treated for 5 days with staurosporine (STSN; 316 nM), trichostatin A (TSA; 500 nM), or succinyl-concanavalin A (sConA; 50 microg/mL), after which they were assayed for specific marker antigen/mRNA expression. Treated cells were also assayed for excitotoxic responsiveness. RESULTS. Neither treated nor untreated RGC-5 cells expressed any specific RGC marker mRNAs or proteins (Brn-3, neurofilaments, Thy-1) or calbindin, calretinin, synaptophysin, PKCalpha, or glial fibrillary acidic protein. However, control RGC-5 cells did express the neuronal markers tau, betaIII-tubulin, microtubule-associated protein (MAP)-1b, MAP2, and PGP9.5. Although treatment with sConA had no effect on the expression of these markers, STSN and (dose dependently) TSA increased their expression and induced excitotoxic responsiveness. All cells, treated or not, expressed high levels of nestin but no other progenitor cell markers. All cells also expressed cone-specific, but not rod-specific, opsin indicative of cone photoreceptor lineage. CONCLUSIONS. RGC-5 cells expressed neuronal, but not RGC-specific, markers that were dose dependently upregulated by TSA. Hence, TSA provided the best tested means to terminally differentiate the cells to a neuronal phenotype from a precursor-like lineage.
Investigative ophthalmology & visual science 02/2010; 51(7):3774-83. · 3.43 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Permanent, bilateral occlusion of the common carotid arteries (2VO) is an established model of chronic hypoperfusion. Previous studies have noted the vulnerability of the optic nerve (ON) to 2VO; however, little information is available regarding the spatiotemporal pattern of axonal degeneration and the accompanying glial cell responses. The present study was conducted to investigate these topics.
At various times after surgery, ONs were removed for mRNA or Western blot analysis or to be processed for histology and immunohistochemistry.
2VO precipitated an infarct within the proximal ON, whereas the retinal ganglion cells, unmyelinated axons of the optic nerve head, and the distal portion of the ON were initially preserved. The onset of degeneration was rapid, with disturbances in fast axonal transport occurring by 6 hours, damage to the axonal cytoskeleton in the proximal ON detectable by 24 hours, and complete axonal loss within the infarcted area manifest within 3 days. Wallerian degeneration of the distal segment of the ON proceeded thereafter, with almost complete loss of the ON axonal cytoskeleton evident by 30 days. Degradation of the axonal cytoskeleton was accompanied by increasing microglial activation and proliferation and a delayed infiltration of macrophages into the lesion site. Robust and persistent upregulation of stress proteins by astrocytes and oligodendrocytes, which correlated with axonal damage, was found throughout the ON after 2VO. Extracellular matrix remodeling was evident in the optic nerve head and proximal ON.
2VO causes rapid degeneration of the ON, with some similarity to rodent ischemic optic neuropathy.
Investigative ophthalmology & visual science 10/2009; 51(3):1483-97. · 3.43 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Purpose. Because of differences in energy metabolism between the brain and retina, the hypothesis for the study was that, in a model of ocular and cerebral hypoperfusion, the retina would be protected by short-term hyperglycemia, whereas brain injury would be exacerbated. Methods. Hyperglycemia was induced by intraperitoneal streptozotocin. An initial experiment determined the effect of hyperglycemia alone in sham-surgery rats. Simultaneous retinal and cerebral hypoperfusion was achieved by two-vessel occlusion (2VO; permanent ligation of both common carotid arteries). Hyperglycemia was induced 3 days before 2VO by streptozotocin injection. The rats were killed 7 days after 2VO or sham surgery. The retina of one eye was collected for histology/immunohistochemistry and the fellow retina was collected for real-time RT-PCR. The retinas were analyzed for neuronal and glial markers and heat shock protein-27. The brains were processed for histology and immunohistochemistry. Results. Short-term (approximately 10 days) hyperglycemia alone caused no discernible injury to the retina. The retinas of the normoglycemic 2VO animals showed a marked loss of retinal ganglion cells and horizontal cells, thinning of the inner retina, glial cell activation, and infiltration of macrophages. The hyperglycemic 2VO rats displayed remarkable protection of the retinal structure and reduced glial cell activation compared with the normoglycemic 2VO animals. There was a significantly greater number of heat shock protein-27-positive retinal ganglion cells in the normoglycemic animals than in the hyperglycemic ones. Brains of both the normoglycemic and hyperglycemic 2VO animals displayed scattered ischemic infarcts and mild white matter injury. Conclusions. Short-term hyperglycemia affords robust protection against retinal hypoperfusion injury, but in the same animals, brain injury is not ameliorated. The mechanism of this retinal hyperglycemia-induced neuroprotection requires further study.
Investigative ophthalmology & visual science 10/2009; 51(4):2197-207. · 3.43 Impact Factor
