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

then move toward your goal by making and evaluating changes one at a time. Isolate your cell culture activities from other laboratory activities as much as possible. Standardize all procedures. Meticulously document the source, age, lot number, and usage of all media components. Minimize delay in the generation of cultures. Regularly examine cultures after they have become attached (this usually occurs within 8 h). Do not examine too many cultures at one time as they are sensitive to pH and temperature shock. Finally, document your results as completely as possible with each experiment, even when it seems that it did not work. This will often help considerably in identifying problems and developing improved methods for retinal cell cultures.

REFERENCES

1.de Araujo EG, Linden R. Trophic factors produced by retinal cells increase the survival of retinal ganglion cells in vitro. Eur J Neurosci 1993;5:1181–1188.

2.Lindsey JD, Weinreb RN. Survival and differentiation of purified retinal ganglion cells in a chemically defined microenvironment. Invest Ophthalmol Vis Sci 1994; 35:3640–3648.

3.Kashiwagi F, Kashiwagi K, Iizuka Y, Tsukahara S. Effects of brain-derived neurotrophic factor and neurotrophin-4 on isolated cultured retinal ganglion cells: evaluation by flow cytometry. Invest Ophthalmol Vis Sci 2000;41:2373–2377.

4.Shindler KS, Zurakowski D, Dreyer EB. Caspase inhibitors block zinc-chelator induced death of retinal ganglion cells. Neuroreport 2000;11:2299–2302.

5.Yasuyoshi H, Kashii S, Zhang S, et al. Protective effect of bradykinin against glutamate neurotoxicity in cultured rat retinal neurons. Invest Ophthalmol Vis Sci 2000; 41:2273–2278.

6.Morgan J, Caprioli J, Koseki Y. Nitric oxide mediates excitotoxic and anoxic damage in rat retinal ganglion cells cocultured with astroglia. Arch Ophthalmol 1999; 117:1524–1529.

7.Saga T, Scheurer D, Adler R. Development and maintenance of outer segments by isolated chick embryo photoreceptor cells in culture. Invest Ophthalmol Vis Sci 1996;37:561–573.

8.Romano C, Price MT, Olney JW. Delayed excitotoxic neurodegeneration induced by excitatory amino acid agonists in isolated retina. J Neurochem 1995;65:59–67.

9.Sherry DM, St JR, Townes-Anderson E. Morphologic and neurochemical target selectivity of regenerating adult photoreceptors in vitro. J Comp Neurol 1996;376: 476–488.

10.Adler R, Lindsey JD, Elsner CL. Expression of cone-like properties by chick embryo neural retina cells in glial-free monolayer cultures. J Cell Biol 1984;99:1173–1178.

11.Madreperla SA, Adler R. Opposing microtubuleand actin-dependent forces in the development and maintenance of structural polarity in retinal photoreceptors. Dev Biol 1989;131:149–60.

12.Stalmans P, Himpens B. Confocal imaging of Ca signaling in cultured rat retinal

pigment epithelial cells during mechanical and pharmacologic stimulation. Invest Ophthalmol Vis Sci 1997;38:176–187.

13.Marin N, Romero B, Bosch-Morell F, Llansola M, Felipo V, Roma J, Romero FJ. Beta-amyloid-induced activation of caspase-3 in primary cultures of rat neurons. Mech Ageing Dev 2000;119:63–67.

14.Brewer G. Isolation and culture of adult rat hippocampal neurons. J Neurosci Methods 1997;71:143–155.

15.Heidinger V, Hicks D, Sahel J, Dreyfus H. Peptide growth factors but not ganglioside protect against excitotoxicity in rat retinal neurons in vitro. Brain Res 1997; 767:279–288.

16.Meyer-Franke A, Kaplan M, Pfrieger F, Barres B. Characterization of the signaling interactions that promote the survival and growth of developing retinal ganglion cells in culture. Neuron 1995;15:805–819.

17.Rotstein N, Aveldano M, Politi L. Essentiality of docosahexaenoic acid in retina photoreceptor cell development. Lipids (Suppl) 1999;34:S115.

18.Adler R. Regulation of neurite growth in purified retina neuronal cultures: effects of PNPF, a substratum-bound, neurite-promoting factor. J Neurosci Res 1982;8: 165–177.

19.Carri NG, Perris R, Johansson S, Ebendal T. Differential outgrowth of retinal neurites on purified extracellular matrix molecules. J Neurosci Res 1988;19:428–439.

20.Hewitt AT, Lindsey JD, Carbott D, Adler R. Photoreceptor survival-promoting activity in interphotoreceptor matrix preparations: characterization and partial purification. Exp Eye Res 1990;50:79–88.

21.Tezel GM, Seigel GM, Wax MB. Density-dependent resistance to apoptosis in retinal cells. Curr Eye Res 1999;19:377–388.

22.Rowe-Rendleman C, Mitchell CK, Habrecht M, Redburn DA. Expression and downregulation of the GABAergic phenotype in explants of cultured rabbit retina. Invest Ophthalmol Vis Sci 1996;37:1074–1083.

23.Germer A, Jahnke C, Mack A, Enzmann V, Reichenbach A. Modification of glutamine synthetase expression by mammalian Muller (glial) cells in retinal organ cultures. Neuroreport 1997;8:3067–3072.

24.Feigenspan A, Bormann J, Wassle H. Organotypic slice culture of the mammalian retina. Vis Neurosci 1993;10:203–217.

25.Hoff A, Hammerle H, Schlosshauer B. Organotypic culture system of chicken retina. Brain Res Protocols 1999;4:237–248.

26.Vollmer G, Layer PG, Gierer A. Reaggregation of embryonic chick retina cells: pigment epithelial cells induce a high order of stratification. Neurosci Lett 1984; 48:191–196.

27.Pearlman EM, Seigel GM, Notter MF. Induction of c-fos by excitatory amino acids in developing chick retina is affected by changes in cellular interactions. J Neurosci Res 1993;36:252–259.

28.Watanabe T, Voyvodic JT, Chan-Ling T, et al. Differentiation and morphogenesis in pellet cultures of developing rat retinal cells. J Comp Neurol 1997;377:341–350.

29.Politi LE, Lehar M, Adler R. Development of neonatal mouse retinal neurons and photoreceptors in low density cell culture. Invest Ophthalmol Vis Sci 1988;29:534– 543.

30.Abrams L, Politi LE, Adler R. Differential susceptibility of isolated mouse retinal neurons and photoreceptors to kainic acid toxicity. In vitro studies. Invest Ophthalmol Vis Sci 1989;30:2300–2308.

31.Politi LE, Adler R. Generation of enriched populations of cultured photoreceptor cells. Invest Ophthalmol Vis Sci 1986;27:656–665.

32.Fontaine V, Kinkl N, Sahel J, Dreyfus H, Hicks D. Survival of purified rat photoreceptors in vitro is stimulated directly by fibroblast growth factor-2. J Neurosci 1998; 18:9662–9672.

33.Barres BA, Silverstein BE, Corey DP, Chun LL. Immunological, morphological, and electrophysiological variation among retinal ganglion cells purified by panning. Neuron 1988;1:791–803.

34.Freshney RI. Culture of Animal Cells: A Manual of Basic Technique. New York: Wiley-Liss, 2000.

35.Mather J. Introduction to cell and tissue culture: theory and technique. In: Introductory Cell and Molecular Biology Techniques. New York: Plenum Press, 1998.

36.Barnes D, Sirbasku D, Sato G. Methods for preparation of media, supplements, and substrata for serum-free animal cell culture. In: Cell Culture Methods for Molecular and Cell Biology. New York: A.R. Liss, 1984.

37.Finlay D, Wilkinson G, Kypta R, de Curtis I, Reichardt L. Retinal cultures. Methods Cell Biol 1996;51:265–283.

38.Helmrich A, Barnes D. Animal cell culture equipment and techniques. Methods Cell Biol 1998;57:3–17.

39.Lincoln CK, Gabridge MG. Cell culture contamination: sources, consequences, prevention, and elimination. Methods Cell Biol 1998;57:49–65.

40.Loo DT, Rillema JR. Measurement of cell death. Methods Cell Biol 1998;57:251– 264.

41.Malicki J. Development of the retina. Methods Cell Biol 1999;59:273–299.

42.Mather JP. Making informed choices: medium, serum, and serum-free medium. How to choose the appropriate medium and culture system for the model you wish to create. Methods Cell Biol 1998;57:19–30.

43.Mattson MP, Barger SW, Begley JG, Mark RJ. Calcium, free radicals, and excitotoxic neuronal death in primary cell culture. Methods Cell Biol 1995;46:187–216.

44.Mills JC, Wang S, Erecinska M, Pittman RN. Use of cultured neurons and neuronal cell lines to study morphological, biochemical, and molecular changes occurring in cell death. Methods Cell Biol 1995;46:217–242.

45.Moore A, Donahue CJ, Bauer KD, Mather JP. Simultaneous measurement of cell cycle and apoptotic cell death. Methods Cell Biol 1998;57:265–278.

Figure 1 Anatomy of the rat visual system.

II. ANATOMY OF THE RAT VISUAL SYSTEM

The axons of RGCs in the rat and mice, after exiting the retina through the optic nerve head, form the optic nerve. At the optic chiasm most of the fibers cross to the contralateral optic tract to reach the optic tectum (superior colliculus). Fewer than 10% of the fibers beyond the optic chiasm are ipsilateral [15]. Nearly all of the RGCs in each tract project to the superior colliculus on that side, and fewer than 40% of them have collateral projections to the dorsal lateral geniculate nucleus (LGN) [15–17].

III.PARTIAL CRUSH INJURY OF THE RAT OPTIC NERVE

A.Surgical Exposure of the Optic Nerve Intraorbitally

The intraorbital part of the optic nerve is longer in rodents than in other species, making it relatively easy to carry out experimental manipulations without impinging on adjacent tissues or harming the nerve itself. All surgical procedures are done under general anesthesia. We use a binocular operating microscope; the conjuctiva is incised lateral to the cornea, the retractor bulbi muscles are separated using curved blunt forceps, and the optic nerve is identified and exposed near

Figure 2 Cross-action forceps.

the eyeball by blunt dissection for 2.5–3 mm, and care is taken not to stretch the nerve.

B. Calibrated Crush Injury

A reproducible crush injury of graded severity is inflicted on the optic nerve by the use of pre-calibrated cross-acting (self-closing) forceps, which open when the handles are pressed and close when the handles are released [4]. The force exerted by the grasping jaws (and thus the severity of the crush lesion inflicted) is adjusted by varying the number of revolutions of the screw attached to the handle.

Using the forceps, a moderate, mild, or very mild crush injury is inflicted on the exposed optic nerve about 1 mm distal to the eye, for a period of 30 seconds.

IV. SEVERE CRUSH INJURY OF THE MOUSE

OPTIC NERVE

To identify and characterize the molecules participating in the process of RGC death, it is necessary to devise an animal model that allows molecular manipulation. Establishment of the mouse model makes it possible to study the effects of severe optic nerve injury in genetically manipulated mice. For this purpose, all RGCs must be labeled 72 h before optic nerve crush. With the aid of a binocular operating microscope, the conjunctiva over the posterior pole of the eye of the anesthetized mouse is incised. The optic nerve is exposed by gentle blunt dissection between the surrounding muscle and the retrobulbar region, as described above for the rat. Using cross-action forceps and taking care not to interfere with the blood supply, we then crush the nerve for 2 s.

V.RETROGRADE LABELING OF RETINAL GANGLION CELLS

Because of the anatomical construction of the visual system, retinal ganglion cell (RGC) survival at any time after axonal injury can be quantified by the use of retrograde neuronal tracers. Properties of the tracers selected for this purpose should include (1) lack of any effect on neuronal viability and activity; (2) intense fluorescence; (3) resistance to fading; (4) absence of diffusion from labeled cells; and (5) relatively prolonged survival time.

The RGCs that survive an optic nerve crush injury, and are potentially capable of being rescued by neuroprotective therapy, are the cell bodies of damaged fibers and of intact fibers that escaped the injury. To determine the total number of surviving RGCs, the protocol of choice is of the labeling prior to the injury. To assess the number of surviving RGCs with still-intact fibers the protocol of choice is the post-injury labeling. These two protocols are done by employing the retrograde labeling procedures, as described in the following section.

A.Labeling of All Retinal Ganglion Cells Prior to Injury

The total number of RGCs in the retina is determined after stereotactic injection of a fluorescent dye to the superior colliculus of both hemispheres, where almost all of the optic axons form synapses.

1. The Rat Model

The anaesthetized rat is placed in a stereotactic device, the skull is exposed and kept dry and clean, and the bregma is identified and marked (see diagram below). The designated point of injection is 6 mm rostral to the bregma and 1.2 mm lateral to the midline. A window is drilled in the scalp above the designated coordinates in both hemispheres. Using a Hamilton syringe, 2 L of the neurotracer dye FluoroGold [18] (5% solution in saline; Fluorochrome, Denver, CO), which meets all of the criteria mentioned above, is injected into the superior colliculus 3.8 mm, 4 mm, and 4.2 mm below the bony surface, at a rate of 1 L/min at each of the three depths. The needle is then slowly withdrawn and the skin is sutured.

2. The Mouse Model

Anesthetized mice are placed in a stereotactic device, the skull is exposed and kept dry and clean, and the bregma is identified and marked. The designated point of injection is at a depth of 2 mm from the brain surface, 2.92 mm posterior to the bregma and 0.5 mm lateral to the midline. A window is drilled in the scalp above the designated coordinates in both hemispheres. With a Hamilton syringe,

Figure 5 Whole-mounted flattened retina.

VI. COUNTING OF LABELED RGCs

At the end of the experimental period, the rats or mice are killed and their eyes are excised into petri dishes containing phosphate-buffered saline (PBS). The retina is detached from the eye without the vitreous body and fixed in freshly prepared 4% paraformaldehyde. Four cuts are made in the fixed retina to allow flattening of the retina onto a nitrocellulose filter.

Labeled RGCs are counted using the fluorescent microscope. It should be noted that RGC density across the rat retina ranges from about 1000 cells/mm2 at the periphery to 6000 cells/mm2 in the center. However, over most of the retina, except at the outer periphery, the average density is about 3000 cells/mm2 [5,15]. Nevertheless, after optic nerve injury the rate of RGC death is higher at the periphery than at the center of the retina [5]. Accordingly, labeled RGCs are counted in four to six fields at the same distance from the center of the retina, at a magnification of 250. The numbers of labeled RGCs per field are averaged, and the mean number of RGCs per square millimeter is calculated.

VII. ELECTROPHYSIOLOGICAL MEASUREMENTS

The visual evoked potential (VEP) response to light indicates the integrity of an animal’s visual system and can be used to assess the effects of injury and treatment on the system’s functional integrity. Only those axons that escaped the primary lesion and remain intact, with or without protection from secondary de-

generative processes, are capable of conducting action potentials. The pattern of field potentials in response to light stimulation is recorded from the primary visual cortex. The potential evoked by the light originates in the retina and is propagated along the surviving axons to reach the superior colliculus, their target in the brain. Electrodes are implanted above the primary visual cortex as follows: The anesthetized rat is placed in a small stereotactic instrument and two holes are drilled in the skull. Through each hole an electrode is implanted epidurally, with the dura kept intact to minimize cortical damage. The electrodes are gold contact pins (Wire-Pro, Salem, NJ) soldered to stainless steel screws, which are screwed into the holes and cemented to the skull with acrylic cement. An electrode inserted through a hole drilled in the nasal bone is used as a reference point. The second hole is in area V1 (primary visual cortex), with coordinates bregma 8 mm and lateral 3 mm.

Field potentials, before and after injury, are recorded from the visual cortex in response to stroboscopic light stimulation (xenon flash tube 4 W/s, 1–2 ms duration, 0.3 Hz). The signal evoked in the cortex is amplified 1000 times with a microelectrode AC amplifier, model 1800 (AM Systems) and digitized (12 bits, 5000 samples/s) with an MIO16–9 board and the LabView 2.2.1 data acquisition and management system (National Instruments). Potentials should be presented as the means of six recordings, three with and three without light, each involving 60 light flashes.

VIII. SOME PRACTICAL TIPS

1.When incising the conjunctiva, make sure that you do it as far as possible from the limbal area, which has abundant vasculature. An incision at this site might cause massive bleeding, obscuring the area close to the optic nerve.

2.While exposing the optic nerve, separate it as much as possible from the adjacent fat and fascia.

3.While labeling the RGCs with a lipophilic fluorescent dye such as 4- Di-10-Asp, make sure that the dye is completely immersed in the hole you have made in the dura sheath by injecting a drop of incomplete Freund’s adjuvant.

4.If the view of the retina under the microscope is too blurred to count the cells, the problem might be caused by one or more of the following:

The vitreous body is still attached to the retina.

The paraformaldehyde solution in which the retina is soaked is not fresh.

Dye application process went wrong.