Ординатура / Офтальмология / Английские материалы / Ocular Neuroprotection_Levin, Polo _2003

Figure 5 Photomicrographs and graph demonstrating changes in ganglion cell number following optic nerve crush and either no treatment or treatment with different levels of BDNF. Treatment with 30 g of BDNF resulted in the greatest cell survival, primarily by rescuing the medium-sized beta cells. Atrophic ganglion cells in the untreated retina are recognized by their irregular shape and clumped chromatin (arrows). (Adapted from Ref. 13.)

IX. SUMMARY

In this chapter I have attempted to highlight some of the advantages of using the retina as a model for studying neuronal degeneration and neuroprotection. I also have tried, without going into detail, to make the reader aware of some of the more traditional approaches that have been used to study retinal ganglion cell morphology over the years. Although many of these might be considered outdated, there remain areas where such traditional methods could be applied to current studies of retinal degeneration and neuroprotection. Our use of the isolated retina and intracellular staining method, along with the Nissl-stained retinal whole mount, reflects both ends of the spectrum. The highly traditional Nisslstained approach continues to serve as a quick and reliable means for making a

first assessment of neuronal survival based on the evaluation of a large number of cells. The intracellular approach provides a more detailed analysis of the cellular changes that occur at the single cell level. It is hoped that the procedures described here will provide a foundation for others also seeking ways to enhance their understanding of the injured retina and its responses to different neuroprotection treatment strategies.

ACKNOWLEDGMENTS

Supported by NIH/NEI grant EY11159 and grants from the Michigan State University Foundation, The Glaucoma Foundation, Alcon Laboratories, and The American Health Assistance Foundation.

REFERENCES

1.Dowling, JE. The Retina: An Approachable Part of the Brain. Cambridge, MA: Belknap Press of Harvard University Press, 1987.

2.Sherman SM. Functional organization of the W-, Y-, and W-type retinal pathways in the cat: a review and hypothesis. Prog Psychobiol Physiol Psychol 1985; 11: 233–314.

3.Kaplan E, Lee BB, Shapley RM. New views of primate retinal function. In: Osborne N, Chader G, ed. Progress in Retinal Research. Oxford: Pergamon, 1990:273– 336.

4.Dacey DM. Physiology, morphology, and spatial densities of identified ganglion cell types in primate retina. Ciba Foundation Symposium 1994; 412–428.

5.Rodieck RW, The First Steps in Seeing. Sunderland, MA: Sinauer, 1998.

6.Cottee LJ, FitzGibbon T, Westland K, Burke W. Long survival of retinal ganglion cells in the cat after selective crush of the optic nerve. Eur J Neurosci 1991; 3: 1245–1254.

7.Mey J, Thanos S. Intravitreal injections of neurotrophic factors support the survival and regrowth of axotomized retinal ganglion cells. Brain Res 1993; 602:304– 317.

8.Silveira LCL, Russelakis-Carneiro M, Perry VH. The ganglion cell response to optic nerve injury in the cat: differential responses revealed by neurofibrillar staining. J Neurocytol 1994; 23:75–86.

9.Mansour-Robaey S, Clarke DB, Wang Y-C, Bray GM, Aguayo AJ. Effects of ocular injury and administration of brain-derived neurotrophic factor on survival and regrowth of axotomized retinal ganglion cells in adult rats in vivo. Proc Natl Acad Sci USA 1994; 91:1632–1636.

10.Watanabe M, Sawai H, Fukuda Y. Number and dendritic morphology of retinal ganglion cells that survived after axotomy in adult cats. J Neurobiol 1995; 27:189– 203.

11.Peinado-Ramo´n PM, Salvador MP, Villegas-Pe´rez MP, Vidal-Sanz M. Effects of axotomy and intraocular administration of NT-4, NT-3, and brain-derived neurotrophic factor on the survival of adult rat retinal ganglion cells. Invest Ophthalmol Vis Sci 1996; 37:489–500.

12.Sawai H, Clarke DB, Kittelerova P, Bray GM, Aguayo AJ. Brain-derived neurotrophic factor and neurotrophin-4/5 stimulate growth of axonal branches from regenerating retinal ganglion cells. J Neurosci 1996; l6:3887–3894.

13.Chen H, Weber AJ. BDNF enhances retinal ganglion cell survival in cats with optic nerve damage. Invest Ophthalmol Vis Sci 2001; 45:966–974.

14.Pearson HE, Labar DL, Payne BR, Cornwell P, Aggarwal N. Transneuronal retrograde degeneration in the cat retina following neonatal ablation of visual cortex. Brain Res 1981; 212:470–475.

15.Sherman SM, Spear PD. Organization of visual pathways in normal and visually deprived cats. Physiol Rev 1982; 62:738–855.

16.Kalil RE. Removal of visual cortex in the cat: effects on the morphological development of the retino-geniculo-cortical pathway. In: Stone J, Dreher B, Rapaport DH, eds. Development of Visual Pathways in Mammals. New York: Alan R. Liss, 1984: 257–274.

17.Payne BR, Pearson HE, Cornwell P. Transneuronal degeneration of beta retinal ganglion cells in the cat. Proc R Soc 1984; 222:15–32.

18.Kalil RE. Reorganization of retinogeniculate connections in the cat following damage to the visual cortex. In: Bjorkland A, Aguayo AJ, Ottoson D, eds. Brain Repair. London: MacMillan Press, 1990; 285–307.

19.Weber AJ, Kalil RE, Stanford LR. Dendritic field development of retinal ganglion cells in the cat following neonatal damage to visual cortex: evidence for cell class specific interactions. J Comp Neurol 1998; 390:470–480.

20.Watanabe M, Rodieck RW. Parasol and midget ganglion cell of the primate retina. J Comp Neurol 1989; 289:434–454.

21.Watanabe M, Sawai H, Fukuda Y. Number, distribution, and morphology of retinal ganglion cells with axons regenerated into peripheral nerve graft in adult cats. J Neurosci 1993; 13:2105–2117.

22.Dacey D, Lee BB. The “blue-on” opponent pathway in primate retina originates from a distinct bistratified ganglion cell type. Nature 1994; 367:731–735.

23.Izumi Y, Benz AM, Kirby CO, Labruyere J, Zorumski CF, Price MT, Olney JW. An ex vivo rat retinal preparation for excitoxicity studies. J Neurosci Meth 1995; 60:219–225.

24.Romano C, Chen Q, Olney JW. The intact isolated (ex vivo) retina as a model system for the study of excitotoxicity. Progr Retinal Eye Res 1998; 17:465– 483.

25.Weber AJ, Kaufman PL, Hubbard WC. Morphology of single ganglion cells in the glaucomatous primate retina. Invest Ophthalmol Vis Sci 1998; 39:2304–2320.

26.Boycott BB, Wa¨ssle H. The morphological types of ganglion cells of the domestic cat’s retina. J Physiol (Lond) 1974; 240:397–419.

27.Kolb H, Nelson R, Mariani A. Amacrine cells, bipolar cells and ganglion cells of the cat retina: a Golgi study. Vision Res 1981; 21:1081–1114.

28.Isayama T, Berson DM, Pu M. Theta ganglion cell type of cat retina. J Comp Neurol 2000; 417:32–48.

29.Saito H. Morphology and physiologically identified X-, Y-, and W-type retinal ganglion cells of the cat. J Comp Neurol 1983; 221:279–288.

30.Fukuda Y, Hsiao C-F, Watanabe M, Ito H. Morphological correlates of physiologically identified Y-, X-, and W-cells in cat retina. J Neurophysiol 1984; 52:999– 1013.

31.Stanford LR, Sherman SM. Structure/function relationships of retinal ganglion cells in the cat. Brain Res 1984; 297:381–386.

32.Rodieck RW, Binmoeller KF, Dineen J. Parasol and midget ganglion cells of the human retina. J Comp Neurol 1985; 233:115–132.

33.Kolb H, Linberg KA, Fisher SK. Neurons of the human retina: a Golgi study. J Comp Neurol 1992; 318:147–187.

34.Dacey DM, Petersen MR. Dendritic field size and morphology of midget and parasol ganglion cells in the human retina. Proc Natl Acad Sci USA 1992; 89:9666–9670.

35.Rodieck RW, Watanabe M. Survey of the morphology of macaque retinal ganglion cells that project to the pretectum superior colliculus, and parvicellular laminae of the lateral geniculate nucleus. J Comp Neurol 1993; 338:289–303.

36.Dacey DM. Morphology of a small-field bistratified ganglion cell type in the macaque and human retina. Vis Neurosci 1993; 10:1081–1098.

37.DeMonasterio FM, Gouras P. Functional properties of ganglion cells of the rhesus monkey retina. J Physiol (Lond) 1975; 251:167–195.

38.Kaplan E, Shapley RM. The primate retina contains two types of ganglion cells, with high and low contrasts sensitivity. Proc Natl Acad Sci USA 1986; 83:2755– 2757.

39.Merigan WH. Chromatic and achromatic vision of macques: role of the P pathway. J Neurosci 1989; 9:776–783.

40.Lee BB. Receptive field structure in the primate retina (Minireview). Vision Res 1996; 36:631–644.

41.Cajal SRY. The structure of the retina. In: Thorpe SA, Glickstein M, eds. Springfield, IL: Charles C Thomas, 1972.

42.Wa¨ssle H, Peichl L, Boycott BB. Morphology and topography of onand off-alpha cells in the cat retina. Proc R Soc Lond B 1981; 212:157–175.

43.Villegas-Pe´rez MP, Vidal Sanz M, Bray GM, Aguayo AJ. Influences of peripheral nerve grafts on the survival and regrowth of axotomized retinal ganglion cells in adult rats. J Neurosci 1998; 8:265–280.

44.Watanabe M, Sawai H, Fukuda Y. Axonal regeneration of retinal ganglion cells in the cat geniculocortical pathway. Brain Res 1991; 560:330–333.

45.Mey J, Thanos S. Intravitreal injections of neurotrophic support the survival of axotomized retinal ganglion cells in adult rats in vivo. Brain Res 1993; 602:304–317.

46.Zhan XJ, Troy JB. An, efficient method that reveals both the dendrites and the soma mosaics of retinal ganglion cells. J Neurosci Meth 1997; 72:109–116.

47.Leventhal AG, Rodieck RW, Dreher B. Retinal ganglion cell classes in the old world monkey: morphology and central projections. Science 1981; 213:1139–1142.

48.Perry VH, Oehler R, Cowey A. Retinal ganglion cells that project to the dorsal

lateral geniculate nucleus in the macaque monkey. Neuroscience 1984; 12:1101– 1123.

49.Martin PR. The projection of different retinal ganglion cell classes to the dorsal lateral geniculate nucleus in the hooded rat. Exp Brain Res 1986; 62:77–88.

50.Stone J. The number and distribution of ganglion cells in the cat’s retina. J Comp Neurol 1978; 180:753–772.

51.Vidal-Sanz M, Villegas-Pe´rez MP, Bray GM, Aguayo AJ. Persistent retrograde labeling of adult rat retinal ganglion cells with the carbocyanine dye Dil. Exp Neurol 1988; 102:92–101.

52.Hughes A. Population magnitudes and distribution of the major modal classes of cat retinal ganglion cell as estimated from HRP filling and a systematic survey of the soma diameter spectra for classical neurons. J Comp Neurol 1981; 197:303– 339.

53.Wong ROL, Hughes A. The morphology, number, and distribution of a large population of confirmed displaced amacrine cells in the adult cat retina. J Comp Neurol 1987; 255:159–177.

54.Vaney DI. The mosaic of amacrine cells in the mammalian retina. In: Osborne N, Chader G, eds. Progress in Retinal Research Oxford: Pergamon, 1999:49–99.

Figure 1 Schematic illustrating the three phases of apoptosis: the initiation phase, decisional phase, and degradative phase. Four hypothetical initiating insults are shown: three that involve a mitochondrial decision phase and are mitochondrially dependent and a fourth that is mitochondrially independent. The multiple initiation and decision phase signaling pathways converge onto common events in the degradation phase. The diagram is meant to emphasize the role of mitochondrial decisional processes in some forms of apoptosis, including the role of BAD, BAX, and BCL-2-like proteins in the mitochondrial decisional process. (From Ref. 39.) The diagram is not meant to fully illustrate all apoptosis signaling pathways.

II.APOPTOTIC DEGRADATION FACTORS RELEASED BY DECREASED MITOCHONDRIAL MEMBRANE PERMEABILITY

At least two factors that signal for apoptotic degradation and that are released from mitochondria have been identified : cytochrome c (CytC) and apoptosis initiation factor (AIF), a 50 kD flavoprotein. Released CytC interacts with apoptosis protease activating factor-1 (Apaf-1) [2], dATP/ATP and procaspase 9 to form a complex known as the apoptosome [3], in which procaspase 9 is converted to caspase 9 (see Fig. 1). Caspase 9 then converts procaspase 3 to activated caspase 3 along with activating caspase 7. The apoptosome seems to function as a multicaspase activating complex (see Ref. 4). Caspase 3 in turn activates DNA frag-

mentation factor [5], an endonuclease activator which enables DNA cleavage and acinus which enables chromatin condensation [6]. The second factor released from mitochondria, AIF, induces DNA loss, peripheral chromatin condensation, and digestion of chromatin into 50 kbp-fragments [7]. The caspase-3 inhibitor ACE-DVAD-FMC inhibits chromatin condensation induced by AIF. The proteases that occur in the early stages of the apoptotic caspase-activation cascade, pro-caspase 2 and 9, have been found to be concentrated in the intermembranous space of mitochondria [8].

III.METHODS OF DETECTING SINGLE-CELL APOPTOSIS

We have developed a number of protocols to examine apoptosis in situ, with particular interest in human postmortem tissue. These include a fluorescent dou- ble-labeling method to simultaneously visualize DNA fragmentation and apoptotic chromatin condensation [9] and several microwave-based antigen retrieval methods for immunocytochemistry of apoptosis-related proteins on paraffin sections. We have also modified these protocols for use on fixed, frozen sections and on cultured cells when examining apoptotic changes in different model systems. Gavrieli first published a method of in situ end labeling [10] that allowed the visualization of DNA fragmentation in tissue sections. Popularly known as the TUNEL method, or ISEL (in situ end labeling), this was a breakthrough in the examination of apoptotic cell death in tissue sections and in cultured cells.

Unfortunately, ISEL when used alone, cannot provide unequivocal identification of an apoptotic nucleus. Endonucleases that digest DNA can create single- and/or double-strand breaks reflected as high molecular weight (50–300 kbp) DNA fragments on gel electrophoresis [11]. Often the DNA digestion continues, with the ultimate production of low molecular weight oligonucleosome-sized fragments [12,13]. Single-strand DNA breaks likely accumulate in the linker regions between oligonucleosomes prior to the double-strand DNA breaks which create a “ladder” pattern when observed by gel electrophoresis [14]. The ISEL method was considered to be selective in detecting these double-stranded breaks between oligonucleosomes.

However, the terminal transferase enzyme (TdT) commonly used for ISEL is generally supplied with a protocol that requires increased cobalt chloride (2.5 mM) in the reaction mix, allowing TdT to label double-strand breaks (protruding, blunt, and recessed ends) as well as single-strand DNA breaks [15,16]. It is important to note that cells dying by necrosis have also been reported to contain singleand/or double-strand breaks, and thus the ISEL method may not always distinguish between the two modes of cell death [17–19]. A second, independent method is therefore required to confirm apoptosis where there is demonstrable

DNA fragmentation. The cyanine dye YOYO-1 (Molecular Probes) binds to DNA (and RNA) and allows the visualization of apoptotic chromatin condensa- tion—an event that is independent of DNA fragmentation. Use of confocal laser microscopy has offered us the additional advantage of high resolution of these nuclear labels (see Refs. 9,20,21).

IV. ISEL/YOYO FOR HUMAN BRAIN POSTMORTEM PARAFFIN SECTIONS

Standard, 5- m-thick paraffin sections are stored at 4°C after sectioning. Slides are run through a standard series of baths to remove all paraffin and rehydrate the sections.

1.Xylene 1—15 min.

2.Xylene 2—10 min.

3.100% Ethanol 1—10 min.

4.100% Ethanol 2—10 min.

5.50% Ethanol—10 min.

6.dH2O—10 min.

7.Antigen Unmasking Solution (Vector Labs). Dilute 3.77 mL of this stock solution in 400 mL distilled H2O in a glass container (a Pyrex measuring cup is good). Add eight slides in a plastic rack. Microwave

slides for 3.5 – 4 min, power setting 7—final temperature reached is approximately 60°C. Temperature should not be above 62°C in order

to avoid producing DNA degeneration due to excessive heating (i.e., 65°C or more) and nonspecific labeling. Let slides cool, submerged in the buffer for 30 min on the bench.

Important: Only eight slides should be done at one time. We have found that if there are more slides in the rack, the heating is uneven across all slides, resulting in unreliable labeling. Power settings and

time vary with each microwave to achieve a final temperature of 60°C (we use an 850 W Kenmore with a turntable).

8.3% H2O2/Methanol, room temperature, l0 min. PBS rinse—3 min.

9.RNAseA digest (100 g/mL in 2 SSC) at 37°C for 25 min). Digest time should be decreased for retinal sections and for embryonic tissue. The RNAse digest decreases background since terminal transferase (TdT) can tail from single-strand RNA.

10.2 SSC rinse, 5 min.

11.Proteinase K digest (20 g/mL in T.E. pH 8) at 37°C for 3–5 min. Too long a digest time will increase nonspecific labeling. We do a very brief digest for embryonic tissue (or retina), always less than 3 min.

12.0.1 M glycine/0.1 M Tris pH 7.2, ice-cold, 10–15 min. Stops the enzyme digest and also helps reduce nonspecific background.

13.Equilibration buffer (Intergen), room temperature, 20–30 min. Blot off excess before adding reaction mix.

14.TdT/BODIPY-dUTP reaction mix, at 37°C for 60 min. Thirty minutes provides inadequate and unreliable labeling; 90 min does not provide any appreciable increase in signal to noise ratio. Cover each

tissue section with a parafilm “coverslip” (allows the spread of a small volume of labeling reaction mix evenly over section). Can use 12 L on a mouse brain cross-section, 20 L on most human SNc sections (unless it is really big, then 25 L).

15.2 SSC stop bath, 3 10 min, prewarmed to 37°C. Slides are washed in 400 mL volumes of 2 SSC to effectively remove nonspecifically

bound BODIPY-dUTP. It is important to use a large volume to remove excess label and that the temperature be 37°C.

16.PBS, 2 5 min. Important to rinse all 2 SSC away to get good YOYO staining.

17.YOYO (1 : 500 in PBS), 30 min, dark, RT.

18.PBS rinses, 5 .

19.Use Aquamount (Gurr) or GelMount (Biomeda) for coverslips—both are water soluble and dry quickly. Let slides dry in fume hood overnight, then store in fridge. We have found that the best color/brightness will be in the first 72 h (apoptotic nuclei appear orange if you have a filter that lets you do simultaneous red/green imaging). After 72 h, the fluorescence is a little less bright, but still good for at least another 3–4 months.

In order to get consistent labeling it is important that all concentrations of enzymes and their buffers are accurate and are applied at the stated temperature. This is also essential for the 2 SSC stop baths that follow the labeling reaction. We prewarm all digestion enzymes before applying to the slides. The TdT reaction mix is made up in the sequence given below just before use. The TdT enzyme is stored at 20°C and should be kept in a freezer block or on ice when in use. We use less enzyme and dUTP in our labeling reaction than is given in the product spec sheet. We have tested a broad range and found that 1.4 L of dUTP and 1.4 L of TdT per 100 L reaction volume gave as good or better labeling on tissue sections than the volumes recommended in the manufacturer’s spec sheet.

TdT Reaction Mix

20 L 5 TdT buffer

10 L CoCl2

67.2 L dH2O