Ординатура / Офтальмология / Английские материалы / Retinal Degeneration Disease_Hollyfield, Anderson, LaVail_1999
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on the use of either transfer of antiapoptotic genes to the photoreceptors or, more readily, delivery of neuroprotective factors. The endogenous rod-derived cone viability factors (RdCVFs) are some of the most appropriate neuroprotective candidates. In addition, the development of eye delivery methods using genetically engineered and encapsulated cells implanted directly into the vitreous offers a new means of long-term controllable and reversible neuroprotective treatment. Finally, progress in the understanding of endogenous neurogenesisn will perhaps provide new possibilities of treatment in the late stages of RP.
5. AKNOWLEDGEMENTS
This work was supported by the Institut National pour la Santé et la Recherche Médicale (INSERM), Louis Pasteur University (Strasbourg), Pierre et Marie Curie University (Paris VI), the French Ministère des Sciences et des Technologies, The Association Française contre les Myopathies (AFM), the Fondation Bétancourt, the Fédération des Aveugles de France and the European Commission: PROAGERET (# QLK6-2001-00385) and PRORET (# QLK6-2001-00569).
6.REFERENCES
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8.C. Bowes, T. Li, M. Danciger, L. C. Baxter, M. L. Applebury, and D. B. Farber. Retinal degeneration in the rd mouse is caused by a defect in the beta subunit of rod cGMP-phosphodiesterase, Nature. 347(6294):67780 (1990).
9.S. Mohand-Said, D. Hicks, M. Simonutti, D. Tran-Minh, A. Deudon-Combe, H. Dreyfus, M. S. Silverman, J. M. Ogilvie, T. Tenkova, and J. Sahel. Photoreceptor transplants increase host cone survival in the retinal degeneration (rd) mouse, Ophthalmic Res. 29(5):290-7 (1997).
10.S. Mohand-Said, A. Deudon-Combe, D. Hicks, M. Simonutti, V. Forster, A. C. Fintz, T. Leveillard, H. Dreyfus, and J. A. Sahel. Normal retina releases a diffusible factor stimulating cone survival in the retinal degeneration mouse. Proc Natl Acad Sci U S A. 95, 8357-62 (1998).
11.S. Mohand-Said, D. Hicks, H. Dreyfus, and J. A. Sahel. Selective transplantation of rods delays cone loss in a retinitis pigmentosa model. Arch. Ophthalmol. 118:807-811 (2000).
12.J. A. Sahel, S. Mohand-Said, T. Leveillard, D. Hicks, S. Picaud, and H. Dreyfus Rod-cone interdependence: implications for therapy of photoreceptor cell diseases. Prog Brain Res 131:649-61 (2001).
13.A. C. Fintz, I. Audo, D. Hicks, S. Mohand-Said, T. Leveillard, and J. A. Sahel. Partial characterization of retinaderived cone neuroprotection in two culture models of photoreceptor degeneration. Invest Ophthalmol Vis Sci. 44:818-25 (2003).
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14.E. G. Faktorovich, R. H. Steinberg, D. Yasumura, M. T. Matthes, and M. M. LaVail. Photoreceptor degeneration in inherited retinal dystrophy delayed by basic fibroblast growth factor. Nature. 347(6288):83-6 (1990).
15.M. M. LaVail, K. Unoki, D. Yasumura, M. T. Matthes, G. D. Yancopoulos, and R. H. Steinberg. Multiple growth factors, cytokines, and neurotrophins rescue photoreceptors from the damaging effects of constant light. Proc Natl Acad Sci U S A. 89(23):11249-53 (1992).
16.C. K. Chen, M. E. Burns, M. Spencer, G. A. Niemi, J. Chen, J. B. Hurley, D. A. Baylor, and M. I. Simon. Abnormal photoresponses and light-induced apoptosis in rods lacking rhodopsin kinase. Proc Natl Acad Sci U S A. 96(7):3718-22 (1999).
17.M. Frasson, J. A. Sahel, M. Simonutti, H. Dreyfus, and S. Picaud. Retinitis pigmentosa: rod photoreceptor rescue by a Ca2+ channel blocker in the rd mouse. Nature Medicine, 5: 1183-7 (1999).
18.M. Frasson, S. Picaud, T. Léveillard, M. Simonutti, S. Mohand-Said, H. Dreyfus, D. Hicks, and J. A. Sahel. Glial cell line-derived neurotrophic factor induces histologic and functional protection of rod photoreceptors in the rd/rd mouse. Invest Ophthalmol Vis Sci., 40:2724-34 (1999).
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22.M. N. Delyfer, T. Leveillard, S. Mohand-Saïd, D. Hicks, S. Picaud, and J.A. Sahel. Inherited retinal degenerations : therapeutic prospects. Biol. Cell. 96: 261-9 (2004).
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28.N. Wakasugi, Y. Tagaya, H. Wakasugi, A. Mitsui, M. Maeda, J. Yodoi, and T. Tursz. Adult T-cell leukemiaderived factor/thioredoxin, produced by both human T-lymphotropic virus type I- and Epstein-Barr virustransformed lymphocytes, acts as an autocrine growth factor and synergizes with interleukin 1 and interleukin 2. Proc Natl Acad Sci U S A. 87(21):8282-6 (1990).
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CHAPTER 45
NEUROPROTECTION OF PHOTORECEPTORS IN THE RCS RAT AFTER IMPLANTATION OF A SUBRETINAL IMPLANT IN THE SUPERIOR OR INFERIOR RETINA
Machelle T. Pardue1, Michael J. Phillips1, Brett Hanzlicek2, Hang Yin1, Alan Y. Chow3, and Sherry L. Ball2,4
1. INTRODUCTION
The artificial silicon retina (ASRTM) consists of an array of photodiodes on a silicon disk that responds to incident light in a gradient fashion (Peyman et al., 1998; Chow et al., 2001, 2002). This device is designed to be placed in the subretinal space and serve as a replacement for degenerating photoreceptors. Two possible mechanisms for the ASR device to improve visual function include 1) direct activation of the remaining inner retinal neurons and subsequent activation of visual centers in the brain or 2) a delay in photoreceptor loss due to a neurotrophic effect from subretinal electrical stimulation. Initial results of ongoing FDA trials with the ASR device suggest that subretinal electrical stimulation could elicit a neurotrophic effect (Chow et al., 2004). Ten advanced retinitis pigmentosa (RP) patients implanted with the ASR device have increased central visual fields and improved visual acuity and color vision (Chow et al., 2004). These improvements cannot be easily explained by direct activation since the implant was placed 20° from the macula. To determine whether neuroprotection results from subretinal electrical stimulation, the RCS rat model of RP was implanted with an ASR device. Subretinal implantation of an ASR device into the superior retina of the Royal College of Surgeons (RCS) rat resulted in preservation of photoreceptors (Pardue et al., 2004). However, the RCS rat is known to have delayed photoreceptor degeneration in the superior region of the retina (LaVail and Battelle, 1975). To determine whether the superior retina is a “privileged” site in the RCS rat, ASR devices were subretinally implanted in the superior and inferior retina.
1 Rehab R&D, Atlanta VA Medical Center, Decatur, GA 30033, USA and Department of Ophthalmology, Emory University, Atlanta GA 30322, USA; 2 Research Service, Cleveland VA Medical Center, Cleveland, OH 44106, USA; 3 Optobionics, Corp, Naperville, IL 60563; 4 Department of Psychology, Case Western Reserve University, Cleveland, OH 44106.
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2. METHODS
2.1. Experimental and Implant Design
RCS rats were obtained from an in-house breeding colony at the Cleveland VA Medical Center which originated from Dr. Matthew M. LaVail at University of California, San Francisco. Three RCS rats were implanted at 21 days of age such that one eye was implanted superiorly and the other inferiorly. Three other RCS rats served as unoperated controls. All animals were followed until 8 weeks post-implantation or 11 weeks of age. All procedures were carried out in accordance with the Association for Research in Vision and Ophthalmology statement concerning the use of animals in ophthalmic and vision research.
Each implant consisted of a series of 20 mm by 20 mm microphotodiodes on a silicon disk that was 1 mm in diameter and ~30 mm thick. Each implant was backed with a uniform layer of iridium oxide to serve as the electrode. Implants were fabricated by Optobionics Corp.
2.2. Surgical Procedures
As previously described (Ball et al., 2000), each RCS rat was sedated and anaesthetizing drops were applied to the corneal surface. One eye was rotated inferiorly with a suture and an incision made through the eye cup into the vitreous approximately 3 mm from the superior limbus. The implant was then gently placed in the superior subretinal space. The eye was rotated back to the normal position and antibiotic ointment applied. The same surgical procedures were performed in the opposite eye, except the eye was rotated superiorly and the implant was placed 3 mm from the inferior limbus.
2.3. Electroretinography (ERG)
ERGs were recorded every 1 or 2 weeks until 8 weeks post-op using the LKC ERG system (Gaithersburg, MD). Rats were dark-adapted overnight, anesthetized, and their pupils dilated. ERGs were recorded from both eyes simultaneously with silver wire electrode loops contacting the corneal surface through a layer of 1% methylcellulose. Platinum needle electrodes placed in the cheek and tail served as reference and ground, respectively.
To record a dark-adapted response series, stimuli were presented in order of increasing luminance from 0.001 to 137 cd sec/m2; interstimulus intervals increased from 15 sec to 1 min with increasing flash intensity. Cone-mediated responses were isolated by superimposing stimuli upon a rod-desensitizing adapting field (30 cd/m2), and presenting the stimulus at 2.1 Hz after a 10 min period of light adaptation.
2.4. Histology
After eight weeks of implantation or at 11 weeks of age, rats were sacrificed by anesthetic overdose, and their eyes enucleated. Each eye was marked for orientation and immersed in 2% paraformaldehyde/ 2.5% glutaraldehyde overnight. Eyes were then dehydrated through a graded alcohol series and embedded in Embed 812/Der736. Blocks were cut at 0.5 mm on a Reichert Ultramicrotome and stained with toluidine blue.
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Retinal morphometry was analyzed from sections taken from both operated and unoperated groups. From each retinal section, six 0.5 mm regions were analyzed as indicated in Figure 45.3A. Digital images were taken of each retinal region and photoreceptor cells counted with samplings from five sections averaged for each region.
3. RESULTS
3.1. Electroretinography
Figure 45.1A shows the dark-adapted ERG responses recorded from RCS rat eyes in the three treatment groups: superior-implantation, inferior-implantation, and unoperated. Each waveform is the maximal dark-adapted response recorded at each post-implantation time indicated for a single eye. At 2 weeks after implantation, retinal responses from all eyes are similar. However, by 4 weeks, eyes implanted with a subretinal device in either the superior or inferior retina have larger amplitude b-waves than unoperated eyes. At 5 weeks after implantation, the unoperated eyes have begun to develop the negative scotopic threshold response (STR; arrow) while the implanted eyes do not develop the STR until 7 or 8 weeks post-implantation. At 8 weeks post-op, the unoperated eyes have a larger amplitude STR response than the implanted eyes.
Figure 45.1B summaries the average maximal dark-adapted b-wave or STR amplitude response at each post-operative timepoint. Eyes with a superiorly or inferiorly placed subretinal implant have very similar responses and have significant functional preservation compared to unoperated control eyes. Responses of implanted eyes were significantly greater
Figure 45.1. Dark-adapted ERG responses from RCS rats implanted with a subretinal implant in the superior or inferior retina versus unoperated rats. A) Maximal dark-adapted response from a single eye in each treatment group elicited by a 137 cd sec/m2 flash at each post-operative week indicated. The arrow indicates the appearance of the STR in the unoperated eye. B) Average maximal dark-adapted b-wave across time (± standard error). The horizontal line marks the division between the positive b-wave and negative STR that are plotted on a continuum.
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than unoperated controls at 4 to 6 weeks post-op (Student’s t-test, p < 0.005). While response amplitudes decrease over the 8 weeks of follow-up, only three of six implanted eyes develop the STR response at 7 and 8 weeks post-op. In contrast, the unoperated eyes have a faster rate of retinal function loss with only the STR visible in the majority of animals by 4 weeks post-op. Light-adapted b-wave responses from implanted eyes also had significantly higher amplitudes than unoperated eyes from 2 to 6 weeks post-op (Student’s t-test, p < 0.05; data not shown).
3.2. Histology
Figure 45.2 presents photomicrographs of the RCS retina implanted superiorly (A) or inferiorly (B) or age-matched unoperated RCS rats (C). In eyes implanted with an ASR device, 5-7 layers of photoreceptor nuclei are still present at 8 weeks after implantation. In the unoperated retina, a sparse single row of photoreceptor nuclei is visible along with a large layer of photoreceptor segment debris.
These results are summarized in Figure 45.3 which plots the number of photoreceptor cells in each retinal region across the retina for each treatment group. While unoperated eyes have very few photoreceptors with a small increase in the superior retina, both the superiorand inferior-implanted eyes have significantly more photoreceptors across all the regions of the retina examined (Repeated measures ANOVA for main effect of treatment; superior vs unoperated F(1,4) = 91.5, p = 0.001; inferior vs unoperated F(1,4) = 51.0, p = 0.002). Photoreceptor preservation peaks in regions directly overlying the implant (regions 2 and 7). A direct comparison of the number of photoreceptors in the inferior and superior regions of the eye (i.e. overlaying the curves) reveals no significant differences (Repeated measures ANOVA, F(5,20) = 0.34, p = 0.88).
Figure 45.2. Photomicrograph of RCS retina implanted with a subretinal implant in the superior (A), inferior (B) retina or an unoperated control (C). The larger number of photoreceptors in the implanted eyes is indicated by the arrows. The black material on the RPE side of the retina is the remnants of the implant after sectioning. The separation of the retina from the implant is an artifact of tissue processing. The black arrowhead indicates the inner nuclear layer and the white arrowhead indicates the ganglion cell layer.
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Figure 45.3. A) Location of the six regions examined across the retina in each implanted retina. In eyes implanted inferiorly, the region nearest to the inferior limbus was indicated as region 1. In eyes implanted superiorly, the region nearest to the superior limbus was indicated as region 8. The arrow indicates the implant. B) Average photoreceptor counts (± standard error) for each retinal location. A greater number of photoreceptors was measured directly over the implant in both the inferior and superior retina. Note that the regions only overlap in areas 3-6 in the two implanted eyes.
4. DISCUSSION
Implantation of an ASR device significantly preserved retinal function and photoreceptor cells in the RCS rat implanted at 21 days of age, as previously reported (Pardue et al., 2004). The amount of functional or anatomical preservation was not regionally dependent. Eyes implanted in either the superior or inferior region both had significant preservation of retinal function. In addition, both the superior and inferior regions of the retina had similar photoreceptor counts. As reported previously (LaVail & Battelle, 1975), more photoreceptor nuclei were counted in the superior retina of unoperated rats. However, this delay in photoreceptor degeneration in the superior region of the RCS rat did not influence the amount of neuropreservation caused by the subretinal implant. These data provide additional evidence that subretinal implantation of an ASR device in the RCS rat results in significant preservation of retinal function and morphology.
5. REFERENCES
Ball, S.L., Pardue, M.T., Chow, A.Y., Chow, V.Y., and Peachey, N.S., 2001, Subretinal implantation of photodiodes in rodent models of photoreceptor degeneration, in: New Insights into Retinal Degenerative Diseases, R.E. Anderson RE, M.M. LaVail MM, J.G. Hollyfield, eds., Kluwer/Plenum, New York, pp.175-182.
Chow, A.Y., Pardue, M.T., Chow, V.Y., Peyman, G.A., Liang, C., Perlman, J.I., and Peachey, N.S., 2001, Implantation of silicon chip microphotodiode arrays into the cat subretinal space. IEEE Trans Neural Syst Rehabil Eng. 9:86-95.
Chow, A.Y., Pardue, M.T., Perlman, J.I., Ball, S.L., Chow, V.Y., Hetling, J.R., Peyman G.A., Liang, C., Stubbs, E.B, Jr., and Peachey, N.S., 2002, Subretinal implantation of semiconductor-based photodiodes: durability of novel implant designs. J Rehabil Res Dev. 39:313-321.
Chow, A.Y., Chow, V.Y., Peyman, G.A., Packo, K.H., Pollack, J.S., and Schuchard, R., 2004, The artificial silicon retinaTM (ASRTM) chip for the treatment of vision loss from retinitis pigmentosa. Arch Ophthalmol. 122:460469.
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LaVail, M.M., and Battelle, B.A., 1975, Influence of eye pigmentation and light deprivation on inherited retinal dystrophy in the rat. Exp. Eye Res. 21:167-192.
Pardue, M.T., Phillips, M.J., Yin, H., Sippy, B.D., Webb-wood, S., Chow, A.Y., Ball, S.L, in press, Neuroprotective effect of subretinal implants in the RCS rat. Invest Ophthalmol Vis Sci.
Peyman, G., Chow, A.Y., Liang, C., Chow, V.Y., Perlman, J.I., and Peachey, N.S., 1998, Subretinal semiconductor microphotodiode array. Ophthalmic. Surg. Lasers 29:234-241.
CHAPTER 46
GLUTAMATE TRANSPORT MODULATION: A POSSIBLE ROLE IN RETINAL NEUROPROTECTION
Nigel L. Barnett, Kei Takamoto, and Natalie D. Bull*
1. INTRODUCTION
The regulation of extracellular glutamate levels in the retina, under physiological and pathophysiological conditions, is essential for the prevention of excitotoxic neurodegeneration. Glial and neuronal high-affinity glutamate transporters (excitatory amino acid transporters, EAATs) facilitate the rapid removal of glutamate from the extracellular space, thereby terminating the excitatory signal and reducing the possibility of excitotoxic neuronal damage. Failure or reversal of these transport systems leads to raised levels of extracellular glutamate and contributes to the development of excitotoxic retinal degeneration.
Five distinct human EAATs and their rodent homologues (i.e. GLAST, GLT-1, EAAC- 1, EAAT4 and EAAT5) have been cloned and localised by immunohistochemistry in the rodent retina.1-3 GLAST and EAAT4 are associated with the Müller and astroglial cells respectively.1,4 Uptake studies indicate that Müller cells dominate normal retinal glutamate transport, utilising GLAST.5,6 GLT-1 is associated with cones and cone bipolar cells,7 EAAC- 1 is localised to amacrine cells plus horizontal cells and ganglion cells8 while EAAT5 is associated with photoreceptors.3 We have shown that GLAST activity is more susceptible to an ischaemic attack than the other classes of retinal glutamate transporters,6 with its failure contributing to the dangerous ischaemia-induced elevation of extracellular glutamate and consequent neurodegeneration. Thus, posing the question, can the activity of retinal glutamate transporters be enhanced during an ischaemic episode to provide neuroprotection? In this study we modulated the activity of rat retinal glutamate transporters pharmacologically during an acute ischaemic attack in an attempt to protect the retina from degeneration and dysfunction.
* Vision, Touch & Hearing Research Centre, School of Biomedical Sciences, The University of Queensland, Brisbane, 4072, Australia. Email: n.barnett@uq.edu.au.
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2. METHODS
2.1. Pharmacological Modulation of Ischaemic Glutamate Transport
Glutamate transport activity was assessed immunohistochemically, by tracking the accumulation of the non-endogenous, non-metabolisable glutamate transporter substrate, D- aspartate, as previously described.9,10 D-Aspartate antiserum was generously provided by Prof. D. Pow (University of Newcastle, Australia). We have previously shown that inhibitors of protein kinase C (PKC)9 and activators of protein kinase A (PKA)11 can inhibit retinal glutamate transport. In an attempt to stimulate retinal glutamate transport we investigated the effect of the PKCd/e activator, ingenol (300 mM) and the protein kinase A inhibitor, KT5720 (1 mM).
2.2. Acute Ischaemic Insult
All procedures were approved by the University of Queensland Animal Experimentation Committee and were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Reseach. Adult Dark Agouti rats (200-250 g) were anaesthetised with ketamine (100 mg/kg) and xylazine (12 mg/kg). Twenty minutes after a 2 ml intravitreal injection of a pharmacological agent (or saline control), retinal ischaemia was induced by elevating intraocular pressure to 110 mmHg for 60 minutes as previously described.12 Following the ischaemic insult, reperfusion was permitted for 7 days.
2.3. Histological Analysis
Semi-thin (500 mm) sections obtained from either the nasal, temporal, superior or inferior regions of the retina approximately 2 mm from the optic disk were stained with toluidine blue, viewed on a Zeiss Axioskop microscope and digitally imaged. Morphometric analysis of the inner plexiform layer (IPL), the inner nuclear layer (INL), the outer nuclear layer (ONL) and the total retinal thickness was performed using Adobe Photoshop software to quantify retinal damage following the ischaemic insult. Cell count analysis was also performed on the same sections. To do this, 100 mm-length sections of the INL and ONL were cropped out of each image and the number of cell bodies in the cropped area was manually counted. The retinas of four rats for each experimental condition were analysed and a oneway ANOVA with Tukey post hoc test was used to compare values obtained from protein kinase modulator-treated eyes with those from control eyes.
2.4. Electroretinography
Following overnight dark adaptation, full field flash electroretinograms were recorded over a 4.8 log unit intensity range (-3 to 1.8 log cd.s.m-2) to assess retinal function as previously described.13 The a- and b-wave amplitudes were measured and expressed as the mean wave amplitude ± SEM. Two-way repeated measures ANOVA was performed on log transformed data to compare the responses from the control and protein kinase treated retinas. A post-hoc Bonferroni test was used to isolate significant differences ( p < 0.05) between control and experimental responses at each stimulus intensity. All rats were subjected to the same conditions for ERG measurements.