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6. Bunt-Milam AH, SaariJC. Immunocytochemical localization of two from bovine and retinal pigment epithelium. / Biol Chem. retinoid-binding proteins in vertebrate retina. / Cell Biol. 1983;97: 1982;257:13329-13333. 703-712. 11. Saari JC, Bredberg D, Noy N. Control of substrate flow at a branch 7. Bok D. The retinal pigment epithelium: a versatile partner in in the visual cycle. Biochemistry. 1994;33:3106-3112. vision./ Cell Sci. 1993;17:189-195. 12. Morimura H, Berson EL, Dryja TP. Recessive mutations in the 8. Bernstein PS, Law WC, Rando RR. Isomerization of all-trans-retin- RLBP1 gene encoding cellular retinaldehyde-binding protein in a oids to 11-czs-retinoids in vitro. Proc Natl Acacl Sci USA. 1987;84: form of punctata albescens. Invest Ophthalmol Vis Sci. 1849-1853. 1999;40:1000-1004. 9. Deigner PS, Law WC, Canada FJ, Rando RR. Membranes as the 13. Broman KW, Murray JC, Sheffield VC, White RL, Weber JL. Compre- energy source in the endergonic transformation of vitamin A to hensive human genetic maps: individual and sex-specific variation in 11-cfe-retinol. Science. 1989;244:968-971. recombination. 1998 http://www.marshmed.org/genetics/. 10. SaariJC, Bredberg L, Garwin GG. Identification of the endogenous 14. Hudson T, Stein L, Gerety S, et al. An STS-based map of the human retinoids associated with three cellular retinoid-binding proteins genome. Science. 1995;270:1945-1954.

Recessive Mutations in the missense: one was a frameshift, and one affected a canon- ical splice donor site. RLBP1 Gene Encoding Cellular CONCLUSIONS. Recessive mutations in the RLBP1 gene are Retinaldehyde-Binding Protein an uncommon cause of retinal degeneration in humans. The phenotype produced by RLBP1 mutations seems to in a Form of Retinitis be a form of retinitis punctata albescens. (Invest Ophthal- Punctata Albescens mol Vis Sci. 1999;40:1000 -1004)

1 2 Hiroyuki Morimura, Eliot L Berson, and he first step in vision occurs when a photon converts an 1 1 Thaddeus P. Dryja ' T 1 l-cis retinal chromophore, which is covalently linked to rod or cone opsin, to the all-trans isomer.1 In mammals, this PURPOSE. TO determine the frequency and spectrum of reaction takes place in the outer segments of the photorecep- mutations in the RLBP1 gene encoding cellular retinalde- tor cells of the retina. The aH-trans chromophore subsequently hyde-binding protein (CRALBP) in patients with heredi- leaves rod or cone opsin and travels as all-fraws-retinol to a 2 tary retinal degeneration. neighboring retinal pigment epithelial cell, where it is con- verted through a series of intermediates back to 11-m-retinal- METHODS. The single-strand conformation polymorphism dehyde. The protein CRALBP seems to play a role in this (SSCP) technique and a direct genomic sequencing tech- pathway. CRALBP is present in the retinal pigment epithelium nique were used to screen the coding exons of this gene and the Miiller cells of the retina.3 The protein forms com- (exons 2-8) for mutations in 324 unrelated patients with plexes with regenerated 11-a's-retinaldehyde and its immediate recessive or isolate , retinitis punctata precursor 11-czs-retinol.4'5 Still uncertain is whether these com- albescens, Leber congenital amaurosis, or a related dis- plexes are essential intermediates in this pathway or whether ease. Variant DNA fragments revealed by SSCP analysis CRALBP has another physiological role. were subsequently sequenced. Selected alleles that altered The requirement for CRALBP in the retina was highlighted the coding region or intron splice sites were evaluated by a recent report of the missense mutation Argl50Gln in the further through segregation analysis in the families of the RLBP1 gene (chromosome 15q26)6 in a family from India with index cases. a recessive retinal degeneration that was termed retinitis pig- mentosa.7 The mutation was homozygous in the affected mem- RESULTS. Four novel mutations were identified in this gene bers but not in the unaffected members of an 11-member among three unrelated patients with recessively inherited sibship that was the product of a first-cousin marriage. Evi- retinitis punctata albescens. Two of the mutations were dence for the pathogenicity of this mutation came from the observations that the mutant protein in vitro was less water soluble than normal and that it did not bind to 11-m-retinal- From the 'Ocular Molecular Genetics Institute and the 2Berman- dehyde.7 In the present report, we provide additional evi- Guncl Laboratory for the Study of Retinal Degenerations, Harvard Med- dence, through the identification of mutations in patients of ical School and the Massachusetts and Ear Infirmary, Boston, Massachusetts. European ancestry, that recessive defects in the human RLBP1 gene are pathogenic. Furthermore, the clinical findings from Supported by Grants EY08683 and EY00169 from the National Eye 8 Institute, National Institutes of Health, Bethesda, Maryland; the Foun- our cases and those in the report by Burstedt et al in this issue dation Fighting Blindness, Hunt Valley, Maryland; and the Massachu- suggest that the phenotype can be distinguished from typical setts Lions Eye Research Fund, Northborough; and by private dona- retinitis pigmentosa. tions to the Taylor Smith Laboratory and the Ocular Molecular Genetics Institute. TPD is a Senior Scientific Investigator of Research to Prevent Blindness, New York, New York. METHODS Submitted for publication October 16, 1998; accepted December 11, 1998. Ascertainment of Patients Proprietary interest category: N. Reprint requests: Thaddeus P. Dryja, MD, Massachusetts Eye and This study, which involved human subjects, conformed to the Ear Infirmary, 243 Charles Street, Boston, MA 02114. tenants of the Declaration of Helsinki. The index patients in

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this study had a diagnosis of retinal degeneration or malfunc- containing 20 mM Tris-HCl (pH 8.4 or 8.6), 0.25 to 1.5 mM tion made through ophthalmologic examination including MgCl2, 50 mM KC1, 0.02 mM deoxyadenosine triphosphate electroretinography (ERG). Most patients resided in the United (dATP), 0.02 mM deoxythymidine triphosphate (dTTP), 0.02 States or Canada. mM deoxyguanosine triphosphate (dGTP), and 0.002 mM de- Patients with autosomal recessive retinitis pigmentosa had oxycytidine triphosphate (dCTP), 0.6 mCi [a-32P] deoxycyti- unaffected parents and at least one affected sibling or were the dine triphosphate (3000 Ci/millimole), 0.1 mg/ml bovine se- offspring of a consanguineous mating. If all affected siblings rum albumin, 0% or 10% dimethyl sulfoxide, and 0.25 units of with retinitis pigmentosa in a sibship were males, the possibil- Taq polymerase. The pH, Mg2+ concentration, and presence ity of X-linked disease was evaluated through an ophthalmo- or absence of 10% dimethyl sulfoxide were tailored to each logic examination of the mother, including an ERG to search primer pair to yield optimal amplification. After initial denatur- for the carrier state of X-linked retinitis pigmentosa; families ation (94°C for 5 minutes), 35 cycles of PCR amplification were with X-linked retinitis pigmentosa were excluded from this performed. Each cycle consisted of denaturation (94°C for 30 study. Some isolate cases were included. They had no affected seconds), primer annealing (54°C for 30 seconds), and exten- relatives and were not the offspring of a consanguineous mat- sion (71°C for 30 seconds). The final extension was at 71°C for ing. Atypical retinitis pigmentosa was diagnosed in these pa- 5 minutes. The amplified DNA fragments were heat denatured, tients if they had some combination of the following features: and the single-stranded fragments were separated, each reduced but unusually large ERGs for their age, an unusual through two sets of 6% polyacrylamide gels, one set with and pigmentary pattern, or no abnormality of dark adaptation one without 10% glycerol. Gels were run at 8 W to 12 W for 5 threshold after 45 minutes of dark adaptation; all were isolate to 20 hours before drying and autoradiography. cases. Variant bands were evaluated by sequencing of corre- Patients with retinitis punctata albescens had ERG findings sponding PCR-amplified DNA segments using a cycle-sequenc- indicating a generalized retinal degeneration, elevated dark ing protocol in a radiolabeled terminator cycle sequencing kit adaptation threshold after 45 minutes of dark adaptation, and (Thermo Sequenase; Amersham Life Science, Cleveland, OH). fundi that showed attenuated arterioles and subretinal yellow Sequence variations expected to affect protein sequence or or yellow-white deposits as a predominant feature. Some also expression were evaluated further by recruiting the relatives of had intraretinal pigment deposits. the index patient to participate in a study to determine Patients with Leber congenital amaurosis had absent or whether the variant allele cosegregated with the disease. For severely diminished vision within the first year of life and this purpose, leukocyte DNA samples from the relatives were markedly diminished ERGs; all had unaffected parents. Patients analyzed by SSCP or direct genomic sequencing for the pres- with fundus albipunctatus had normal retinal vessels, diffuse ence of the variant sequence. subretinal white deposits, elevated dark adaptation thresholds, and reduced ERGs after 45 minutes of dark adaptation that became normal after 6 to 12 hours of dark adaptation. RESULTS Patients with other forms of stationary night blindness had fundi without the stigmata of retinitis pigmentosa, elevated Based on the previously reported 5' untranslated, 3' untrans- dark-adaptation thresholds, reduced rod ERGs, and cone ERGs lated, and flanking intron sequences for the human RLBPJ 6 that were normal when corrected for age and . gene, we developed assays using the SSCP technique to Control subjects had no known blood relative with hereditary screen for mutations in the entire coding sequence and the retinal degeneration and no symptoms of retinal malfunction. intron sequences immediately flanking exons 2 through 8. After informed consent, we collected between 3 ml and With these assays, we evaluated DNA from 189 patients from 50 ml venous blood from each patient or control subject. separate families with autosomal recessive retinitis pigmen- Leukocyte nuclei were prepared from the blood samples and tosa, 45 unrelated patients with congenital amaurosis, 6 unre- stored at — 70°C before DNA was purified from them. lated patients with retinitis pigmentosa sine pigmento, 16 un- related patients with atypical retinitis pigmentosa, 28 unrelated Screening for RLBP1 Mutations patients with Bardet-Biedl syndrome, 28 unrelated patients Exons 2 through 8 of the RLBP1 gene were individually am- with retinitis punctata albescens, 2 unrelated patients with plified from human genomic DNAs by polymerase chain reac- fundus albipunctatus with night blindness, and 10 unrelated tion (PCR) using the following primer pairs based on the patients with stationary night blindness. published genomic sequence6 (sense, antisense, respectively): Three index patients were found to be homozygotes or exon 2, TGAGATCCACAGTTCTGAGAC, AGGAGAGCCCTG- compound heterozygotes for mutations listed in Table 1. All GAGGACA; exon3, GGCTGATGCGGTTGGCTGTT, CCCCT- reported European ancestors. One patient (003-188) was ho- CATGTTGCCTCCCTA; exon 4, CTCATCACCTGTGTGTCCT- mozygous for a frameshift mutation in codon 278 (Gln278[l-bp GCC, GAGAGCGGATAGCATCCTCATG; exon 5, CTTCTGAG- del]; Fig. 1A). This frameshift changes the amino acid sequence TCCCACTAGGAGG, CCAGTAGAGGCCAGGGTTGA; exon 6, downstream of codon 278 and extends the length of the CCTCAGGACCTCAAGCCTTA, CTGCAAGCACCATGAAAGGA; encoded protein from 316 to 326 residues. This patient's par- exon 7, AATGAGTGGGAGCCTCTGAG, CCCTCTTGTCTCATT- ents were third cousins. Only the mother was alive, and she GTCTGG; and exon 8, CTCCTGCTCAGTTCTGTCTC, AGT- was found to be a heterozygote for this mutation. The patient TCAGCTGGCAGGAGATG. The single-strand conformation had one living sibling. This sister was unaffected and was a polymorphism (SSCP) technique was used to screen for point heterozygote. No other family members were affected. An- mutations and other small-scale sequence changes. PCRs were other patient (097-001) heterozygously carried two sequence performed in the wells of 96-well microtiter plates. In each abnormalities: One was a point mutation involving the canon- well was 50 ng to 100 ng leukocyte DNA in 20 /al of a solution ical splice donor site of intron 3 (TVS3 + 2 T—>C; Fig. IB) and

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TABLE 1. Mutations Found in the RLBPl Gene

Patient Genomic Location Region of Gene or Codon Affected 003-188 9483delC Exon 8, Gln*278(l-bp del) (GAG to -AG) 097-001 Intron 3 splice donor site, IVS3+2 (GT to GC) 097-001 Exon 6, Met°226Lys+ (ATG to AAG) 274-007 Exon 7, Arg+234Trp° (CGG to TGG)

Nucleotide numbers are based on a published RLBPl genomic sequence.6 The codon numbering includes the initiation methionine, a residue that is not present in the final protein product.15 The superscript symbol after each three-letter amino acid abbreviation indicates the type of R group: * polar; 0 nonpolar; + positively charged.

the other a missense change (Met226Lys; Fig. 1C). Each of this were reminiscent of those seen in gyrate atrophy (Fig. 2). patient's parents, who were unaffected, carried one of these Intraretinal pigment deposits were sparse and were sometimes changes heterozygously (the mother had the splice-site muta- seen at the borders of the atrophic patches of retinal pigment tion and the father the missense mutation), indicating that the epithelium. Retinal vessels were attenuated in both patients, mutations were allelic and that the patient was therefore a more so in the older patient. The funduscopic findings of the compound heterozygote (data not shown). There were no Swedish patients with an RLBPl mutation were similar.8 Our other affected members in this family. There was one unaf- patients with RLBPl mutations had reduced rod and cone fected sibling (a brother) who carried the missense mutation ERGs with predominant loss of rod function. heterozygously. A third patient (274-007) was homozygous for the missense mutation Arg234Trp (not shown). This patient was a member of a Swedish kindred that we subsequently DISCUSSION learned was fortuitously and independently being studied by 8 another group (see the accompanying report ). None of the The mutations in the RLBPl gene encountered in this study are four mutations found in these patients was found among 69 or likely to be pathogenic, .because they were not found among 70 normal control subjects. normal control subjects and because affected people, who had Ten other sequence anomalies were encountered during recessively inherited disease, were either homozygotes or com- this study (Table 2). One was a biallelic, single-base polymor- pound heterozygotes. Except for the mutation found in the phism in intron 6 with a minor allele frequency of 035 to 0.39- Swedish patient (see the accompanying report8), the probands Most of the others were interpreted as rare variants, because were from small families and had no affected relatives, so that they were not predicted to alter the sequence or expression of meaningful cosegregation analyses were not possible. Ah1 the the encoded protein. An exception was the missense change mutations could be predicted to have major effects on the Phel82Cys that was found heterozygously in an isolate case of encoded protein. One of the mutations is a frameshift that retinitis pigmentosa sine pigmento (patient identification num- would alter the carboxyl terminus of the protein. Another is a ber 008-005). No defect was found in the homologous allele in splice-site mutation that is likely to interfere with proper pro- this patient, and it was not studied further. cessing of the RNA transcript. Two of the mutations are mis- Clinical examination of patients 097-001 at age 19 years sense and one changes a nonpolar residue to a positively and 003-188 at age 52 years showed small yellow deposits at charged one (Met226Lys), whereas the other changes a posi- the level of the retinal pigment epithelium across the fundus. tively charged residue to a nonpolar one (Arg234Trp). Addi- In the older patient, there were round areas of atrophic retinal tional work will be necessary to determine whether the corre- pigment epithelium in the midperiphery and far periphery that sponding mutant proteins have a reduced affinity for the

A. Gin278(1-bp del) B. IVS3+2T->C C. Met226Lys wild type mutarrt Control 097-001 Control 003-188 Gln228 CTAG CTAG CTAG CTAG Leu227

Met226Lys

Asp225

Val224

Phe27$ Met223

FIGURE 1. Sequence of the mutations found in the RLBPl gene. In each panel, the relevant region of the RLBPl genomic sequence is shown with the sense direction going from the bottom to the top. The corresponding regions from a subject with the wild-type sequence are shown for comparison.

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TABLE 2. RLPB1 Polymorphisms, Rare Silent Variants, and Rare Variants of Uncertain Pathogenicity

Number of Respective Alleles*

Genomic Location Region of Gene or Codon Affected Patients Controls

Exon 2, 5' untranslated region (G versus A) 646:2 140:0 878C-»A Exon 2, 5' untranslated region (C versus A) 647:1 140:0 Exon 2, 5' untranslated region (T versus C) 647:1 140:0 Exon 3, Arg+42Arg+ (CGC versus CGT) 647:1 140:0 IVS3-3 (T versus G) 647:1 138:0 2779delGAGGCC rVS4+3 (GAGGCC versus —) 645:3 134:4 + + 4674G->A Exon 5, Arg 121Arg (CGG versus CGA) 647:1 140:0 Exon 6, Leu°177Leu° (TTG versus CTG) 647:1 138:0 8008T-*G Exon 6, Phe°182Cys* (TTC versus TGC) 647:1 138:0 8l67C-»T IVS6+20 (C versus T) 397:251 (0.39) 90:48 (0.35)

Nucleotide numbering, codon numbering, and symbols are the same as in Table 1. * Minor allele frequency in parentheses.

retinoids that are thought normally to form complexes with Certain clinical characteristics of the patients with RLBP1 CRALBP, an abnormality found in the previously reported mis- mutations are similar to those found in patients with retinitis sense mutation.7 pigmentosa, such as night blindness as an early symptom,

FIGURK 2. (A, B) Fundus photographs of the right eye of patient 097-001 at age 19 show slight retinal arteriolar attenuation with yellow subretinal deposits throughout the retina and areas of atrophic retinal pigment epithelium in the periphery. (A) Temporal midperiphery; (B) posterior pole. (C, D) Photographs of the right eye of patient 003-188 at age 52 show retinal arteriolar attenuation and peripheral and midperipheral atrophy of the retinal pigment epithelium. (C) Posterior pole; (D), nasal midperiphery.

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funduscopic findings of attenuated retinal vessels and intrareti- References nal pigment deposits, and reduced ERGs. Other clinical find- ings shared by patients carrying RLBP1 mutations may be 1. Schoenlein RW, Petenau LA, Mathies RA, Shank CV. The first step sufficiently distinguishing to warrant a separate diagnostic cat- in vision: femtosecond isomerization of rhodopsin. Science. 1991; 254:412-415. egory. All patients had small yellow deposits at the level of the 2. Dowling JE. Chemistry of visual adaptation in the rat. Nature. retinal pigment epithelium. Subretinal yellow deposits were 1960;188:ll4-118. also described in the affected members of the previously re- 7 3- Bunt-Milam AH, Saari JC. Immunocytochemical localization of two ported family with an RLBP1 mutation, suggesting that this retinoid-binding proteins in vertebrate retina. / Cell Biol. 1983;97: feature may be a characteristic of the disease caused by defec- 703-712. tive or absent CRALBP. A second feature is the development of 4. Saari JC, Bredberg DL, Noy N. Control of substrate flow at a branch patches of atrophic retinal pigment epithelium with well-de- in the visual cycle. Biochem. 1994;33:31O6-3112. fined, rounded margins in the retinal periphery that are remi- 5. Crabb JW, Carlson A, Chen Y, et al. Structural and functional niscent of those found in patients with gyrate atrophy. characterization of recombinant human cellular retinaldehyde- binding protein. Protein Sci. 1998;7:746-757. The term "retinitis punctata albescens" is probably the 6. Intres R, Goldflam S, Cook JR, Crabb JW. Molecular cloning and best existing one for the patients with RLBP1 defects, because structural analysis of the human gene encoding cellular retinalde- it is commonly used by ophthalmologists to describe a photo- hyde-binding protein. JBiol Chem. 1994;269:254l 1-25418. receptor degeneration with or without intraretinal pigmenta- 7. Maw MA, Kennedy B, Knight A, et al. Mutation of the gene tion that has minute yellow or yellow-white deposits deep in encoding cellular retinaldehyde-binding protein in autosomal re- the retina or at the level of the retinal pigment epithelium.910 cessive retinitis pigmentosa. Nat Genet. 1997;17:198-200. Areas of chorioretinal atrophy have been described in some 8. Burstedt MSI, Sandgren O, Holmgren G, Forsman-Semb K. Bothnia patients with retinitis punctata albescens.10'11 Similar to retini- dystrophy caused by mutations in the cellular retinaldehyde-bind- tis pigmentosa, retinitis punctata albescens is genetically het- ing protein gene (RLBP1) on chromosome 15q26. Invest Ophthal- mol Vis Sci. 1999;40:995-1000. erogeneous, with reports documenting cases caused by defects 9. Lauber H. Die sogenannte Retinitis punctata albescens. Klin in the RDS gene on chromosome 6p or in the rhodopsin gene 12 13 Monatsbl Augenbeilkd. 1910;48:133-l48. on chromosome 3q. ' 10. Franceschetti A, Francois J, Babel J. Chorioretinal Heredodegen- The RLBP1 gene seems to be a rare cause of recessive erations. Springfield, IL: Charles C. Thomas; 1963. retinal disease, accounting for only 3 of 324 unrelated patients 11. Pillat A. Tapetoretinal degeneration of the central fundus region. with retinitis pigmentosa or an allied retinal degeneration or Am J Ophthalmol. 1930;13:l-12. malfunction in our series. A previous search among an over- 12. Kajiwara K, Sandberg MA, Berson EL, Dryja TP. A null mutation in the human peripherin/RDS gene in a family with autosomal lapping set of patients for mutations in the RLBP1 gene using 14 dominant retinitis punctata albescens. Nat Genet. 1993;3:208- Southern blot techniques was understandably negative, be- 212. cause the types of mutations found here involving single-base 13- Souied E, Soubrane G, Benlian P, et al. Retinitis punctata albescens changes or deletions are beyond the resolution of Southern associated with the Argl35Trp mutation in the rhodopsin gene. blot techniques. With recognition of the clinical features that Am J Ophthalmol. 1996;121:19-25. seem to be characteristic of this disease, it may be possible to 14. Cotran PR, Ringens PJ, Crabb JW, Berson EL, Dryja TP. Analysis identify patients with a similar phenotype and to determine of the DNA of patients with retinitis pigmentosa with a cellular whether they also have RLBP1 gene defects. retinaldehyde binding protein cDNA. Exp Eye Res. 199O;51:15- 19. Acknowledgments 15. Crabb JW, Goldflam S, Harris SE, Saari JC. Cloning of the cDNAs encoding the cellular retinaldehyde binding protein from bovine The authors thank Sten Andreasson for obtaining blood samples from and human retina and comparison of the protein sequences. J Biol some of the patients. Chem. 1988; 263:18688-18702.

Experimental Induction of ganglion cell death in mice was characterized using crush and intravitreal injections of the glutamate Retinal Ganglion Cell Death in analog ./V-methyl-D-aspartate (NMDA). Adult Mice METHODS. TO expose retinal ganglion cells (RGCs) to ex- citotoxins, adult CB6F1 mice were injected intravitreally Yan Li, Cassandra L Schlamp, and in one eye with NMDA. In an alternative protocol to Robert W. Nickells physically damage the axons in the optic nerve, the nerve was crushed using self-closing fine forceps. Each animal PURPOSE. Retinal ganglion cells die by apoptosis during had one or the other procedure carried out on one eye. development and after trauma such as axonal damage and Loss of RGCs was monitored as a percentage of cells lost exposure to excitotoxins. Apoptosis is associated with relative to the fellow untreated eye. Thyl expression was changes in the expression of genes that regulate this examined using in situ hybridization. DNA fragmentation process. The genes that regulate apoptosis in retinal gan- in dying cells was monitored using terminal transferase- glion cells have not been characterized primarily because dUTP nick-end labeling (TUNEL). previous studies have been limited to animal models in which gene function is not easily manipulated. To over- RESULTS. RGCS comprise 67.5% ± 6.5% (mean ± SD) of come this limitation, the rate and mechanism of retinal cells in the ganglion cell layer (GCL) of control mice based

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on nuclear morphology and the presence of mRNA for the mate toxicity have been implicated in the pathophysiology of ganglion cell marker Thyl. One week after optic nerve .' crush, these cells started to die, progressing to a maximum The genes that regulate the cell death program in RGCs loss of 57.8% ± 8.1% of the cells in the GCL by 3 weeks. have also been studied. These studies have traditionally been Cell loss after NMDA injection was dose dependent, with carried out on rats after axotomy of the optic nerve and entail injections of 10 nanomoles having virtually no effect to a measuring the changes in the expression of select genes such 6 7 maximum loss of 72.5% ± 12.1% of the cells in the GCL as bax and bcl-x. ' These observations are limited, however, in within 6 days after injection of 160 nanomoles NMDA. that they only provide circumstantial evidence of gene func- Cell death exhibited features of apoptosis after both optic tion in RGCs. Ideally, the most rigorous test of function is to nerve crush and NMDA injection, including the formation genetically alter a select gene and test the effect of either the of pyknotic nuclei and TUNEL staining. loss-of-function or the gain-of-function of that gene after exper- imentally activating RGC death. Currently, the ability to alter CONCLUSIONS. Quantitative RGC death can be induced in genes in a mammalian system is most well developed in mice. mice using two distinct signaling pathways, making it Until recently, however, these animals have not been used to possible to test the roles of genes in this process using study RGC death, in part because of the technical difficulties in transgenic animals. (Invest Ophthalmol Vis Set. 1999;40: stimulating the death of these cells. The ability to use mice to 1004-1008) study RGC death would represent an advance in this area of research. Here, we report the characterization of RGC death in adult mice using two protocols that activate apoptosis via etinal ganglion cells (RGCs) are the retinal neurons most different signaling pathways. Raffected in disorders of the optic nerve, of which glaucoma is the most prevalent. Recent studies indicate that RGCs die with characteristics of apoptosis during normal development METHODS of the retina and after injury to the axons of adult RGCs such as axotomy of the optic nerve, experimental glaucoma in Experimental Animals animals, and glaucoma and anterior ischemic The animals used in this study were handled in accordance in humans.' with the ARVO Resolution for the Use of Animals in Ophthal- Various stimuli cause the death of RGCs. During develop- mic and Vision Research. All protocols were reviewed and ment these cells acquire absolute dependence on neurotro- approved by the Research Animal Resource Center of the phins secreted by the brain as part of the mechanism to ensure University of Wisconsin. The experiments shown here were all proper synaptic connection with higher visual centers. Loss of conducted on CB6F1 mice (an Fl hybrid generation of a neurotrophic support stimulates the death of RGCs in culture.2 BALB/c female crossed with a C57BL/6 male) of an average age Similar neurotrophic loss may be encountered after optic nerve of 4 months, weighing 20 g to 25 g. No obvious differences in transection, because RGC death under this condition can be cell loss after treatment were detected in several other strains delayed by injecting exogenous growth factors into the vitre- tested (data not shown). For each surgical procedure the mice ous.3 RGCs are also sensitive to the toxic effects of high were first anesthetized by intraperitoneal injection of 0.2 ml to concentrations of excitatory amino acids such as glutamate. In 0.3 ml of solution containing ketamine (6 mg/mL) and xylazine the retina, RGCs and a subset of cells in the inner nuclear layer (0.4 mg/mL). At select time points after , the mice were express subunits for the 7V-methyl-D-aspartate (NMDA) recep- killed by cervical dislocation. tor. In an excitotoxic response, glutamate hyperactivates the Optic Nerve Crush NMDA receptor and precipitates a Ca2+-dependent chain of events that can lead to apoptosis or necrosis, depending on the Mice were first anesthetized. A lateral canthotomy was made magnitude of the excitatory response. RGCs are exquisitely on one eye to allow access to the posterior pole. The bulbar sensitive to the effects of glutamate and the glutamate analog was cut 90° in die superior temporal region and NMDA, which cause a dose-dependent loss of RGCs both in gently peeled back to the posterior region of the . The vitro and in vivo.4'5 Both neurotrophin deprivation and gluta- optic nerve was then exposed through a small window made between the surrounding muscle bundles by gentle blunt- dissection using a pair of watchmaker forceps. Care was taken not to cut the muscles or the vessels in the surrounding fascia, From the Department of and Visual Sciences, because this leads to excessive bleeding. At a site approxi- University of Wisconsin, Madison. mately 1 mm from the posterior pole, the nerve was clamped Supported by grants from the American Health Assistance Foun- dation, Rockville, Maryland and the Retina Research Foundation, Hous- using a pair of Dumont No. 5 self-closing forceps for 3 to 5 ton, Texas; Grant 1 R29 EY12223 from the National Institutes of seconds. After this procedure, the conjunctiva and lateral can- Health, Bethesda, Maryland; and a Career Development Award to RWN thotomy were closed with sutures and the mice allowed to and an unrestricted gift to the Department of Ophthalmology and recover. These mice exhibited normal eating and drinking Visual Sciences from Research to Prevent Blindness, New York, New York. YL was the recipient of the Shaffer International Fellowship from behaviors and showed no signs of ill effects for a period of at the Glaucoma Research Foundation, San Francisco, California. least 6 weeks. Operated were monitored for signs of Submitted for publication July 16, 1998; revised November 19, ischemia due to damage of the central retinal artery both by 1998; accepted December 21, 1998. direct observation of the retinal blood vessels using a dissect- Proprietary interest category: N. ing microscope and histologically after they had been killed. Reprint requests: Robert W. Nickells, Department of Ophthalmol- ogy and Visual Sciences, K6/458 CSC, University of Wisconsin, 600 Typically, an eye with a severed artery showed signs of acute Highland Avenue, Madison, WT 53792. damage within 2 days after the operation. This occurred in less

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than 5% of the cases of partial optic nerve crush, but in 100% of the cases of intraorbital axotomy of the optic nerve (data not shown). Intravitreal Injection of NMDA Mice were first anesthetized. A small incision was made with a 30-gauge needle 0.5 mm to 1,0 mm behind the limbus in the superior region of the globe of one eye, through the conjunc- tiva and . For microinjcctions, a glass micropipet was passed through this incision at a 40° to 50° angle to the equator. In animals with dilated , it was possible to view the needle entering the vitreous. The eyes were routinely injected with 2 /xl of solution. For NMDA injections, we pre- pared balanced saline solution containing 5 mM to 80 mM NMDA (Research Biochemicals International, Natick, MA). For control experiments, eyes were injected with balanced saline solution alone. Only one eye of each mouse was ever injected. Occasionally, this procedure created in mice, possi- bly due to damage to the with the micropipet. Mice that had developed cataracts were killed and not used for analysis. FIGURE 1. DAPi-staLned sections of central regions of mouse Quantification of Cell Loss in the Retinal showing cell loss after optic nerve crush or intravitreal injection of NMDA. (A) Section from a control retina of a mouse 3 weeks after the Ganglion Cell Layer optic nerve of the fellow eye was crushed. ONL, outer nuclear layer; Experimentally induced cell loss in the retinal ganglion cell INL, inner nuclear layer; GCL, ganglion cell layer. (B) Section from the layer (GCL) was measured from histologic sections of the same region of the retina of an experimental eye 3 weeks after optic mouse retina. In each mouse, one eye was operated on leaving nerve crush. (C) Section through the central retina of a control eye 6 the other eye for the unoperated control. At select time points days after intravitreal injection of 160 nanomoles NMDA into the fellow eye. (D) Section through the same region of an experimental eye 6 after surgery, the mice were killed and the eyes were enucle- days after injection. Both treatments cause similar percentage losses of ated and fixed in 100 mM phosphate buffer containing 4% cells in the GCL at the times shown. Scale bar, 20 pm. (wt/vol) paraformaldehyde and 2.5% (wt/vol) glutaraldehyde. After fixation the retinas were removed and embedded in glycolmethacrylate (|B4-Plus; Polysciences, Warrington, PA). hybridization was carried out using a whole-mount procedure Each retina was cut into transverse sections (2-jxm thickness) 7 extending from the peripheral retina to the . Histo- described for the rat retina. Quantification of the number of logic landmarks, such as the ciliary epithelium and the edge of positive cells was determined by comparing photographs of the optic disc, allowed for orientation of the sections from the DAPI-stained image with the Nomarski image of 5 to 8 both eyes of each mouse for counting. Representative sections sections from a minimum of two mice at each time point. (12-16 total for each eye) were cut from both the superior and Analysis of DNA Fragmentation inferior retinas. The sections were then stained with 4,6-dia- midino-2-phenylindole (DAPI; Boehringer Mannheim Bio- Cells with fragmented DNA in histologic sections were identi- chemicals, Indianapolis, IN) to fluorescently label all the nu- fied by TUNEL staining. Harvested retinas were embedded in clei. Stained nuclei in the GCL were then counted in paraffin. Sections of 4-/xm thickness along the horizontal me- photographs of 2 microscopic fields of each section corre- ridian were cut and stained as described previously. To quan- sponding to approximately 400 /u.m of retina extending from tify the TUNEL staining, all the positive cells in each layer of the the ciliary epithelium (peripheral retina) or from the optic disc retina of a section (6 sections per retina) were counted. (central retina). To compensate for slightly oblique sections, the number of cells in the GCL was corrected to the number of cells in the outer nuclear layer in the same field. The amount of RESULTS cell loss was then calculated as a percentage of the cells Cell Loss in the GCL after Optic Nerve Crush and present in the control eye of the same individual. NMDA Injection Optic nerve crush caused a gradual loss of the principle cell In Situ Hybridization type in the GCL, which contained large round nuclei with In situ hybridization to localize Thy-1 mRNA, a DNA sequence prominent nucleoli consistent with the nuclear moq^hology of corresponding to the third exon of murine Thyl was amplified RGCs in the mouse retina (Figs. 1A, IB). A consistent loss of by the polymerase chain reaction using sequence-specific cells was first detected 1 week after crush and maximum loss primers (5'-CTTGCAGGTGTCCCGAGGGC-3'J forward primer; occurred by 3 weeks (Fig. 2A). On average, this maximum loss 5-ATGGGATTCGCGCCCGAGAC-3', reverse primer) and was 57.8% ± 8.1% (mean ± SD, n = 6 mice) of the cells in the mouse genomic DNA as a template. These primers generated a GCL. No further loss was detected in eyes examined up to 6 305-bp DNA that was blunt-end cloned into the Smal site of the weeks after crush (data not shown). plasmid pBK-CMV (Stratagene, La Jolla, CA). Sequence analysis Intravitreal injections of NMDA caused the dose-depen- confirmed both the identity and orientation of this DNA. In situ dent loss of the same principal population of cells in the GCL

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this tissue. In control retinas 67.5% ± 6.5% (n = 8) of the cells in the GCL were Thyl-positive. By 2 weeks after optic nerve crush, no Thy 1 -positive cells were detected in the GCL. Simi- larly, no Thy-1 -positive cells were detected in retinas 4 days after injection of 160 nanomoles NMDA. Time-course experi- ments suggest that loss of Thy-1 mRNA precedes actual cell death in the retina (Li Y, Schlamp CL, and Nickells RW, un- published data). It is also noteworthy that the maximum num- ber of cells lost after NMDA injection (72.5% ± 12.1%) closely 8 10 12 matched the number of Thyl-posixive cells in the control retina (67.5% ± 6.5%). Days Days B Characteristics of Cell Death in the GCL FIGUKE 2. Graphic representation of cell loss in the GCL. All data are Dying cells resulting from optic nerve crush or NMDA injection shown as a percentage of cells in the GCL remaining in the treated eye exhibited identical characteristics, including highly condensed relative to the control fellow eye for each mouse. Each data point (pyknotic) nuclei, fragmentation of the cell body, and a lack of represents the mean of at least four eyes (± SD). (A) Cell loss after residual debris (data not shown). Neither procedure elicited an optic nerve crush. No further loss of cells was detected after 3 weeks (data not shown). (B) Cell loss measured after intravitreal injection of inflammatory response, even though NMDA injection caused 10 nanomoles NMDA (filled circles) or 160 nanomoles NMDA (open the rapid death of the majority of GCL cells. circles'). Injection of 10 nanomoles NMDA caused virtually no cell Dying cells also exhibited DNA fragmentation as as- death, whereas maximal cell loss was measured after injection of 160 sayed by TUNEL staining (Fig. 3). Peak TUNEL staining in the nanomoles. Intermediate doses of NMDA had proportionately interme- GCL occurred 2 days after injection of 160 nanomoles diate effects on the kinetics of cell death (data not shown). Cell NMDA and 1 week after optic nerve crush (Fig. 4). NMDA number was also observed to increase in the GCL after longer periods injection also stimulated cell death in the inner nuclear of exposure to high doses of NMDA. These additional cells did not layer, although the total number of TUNEL-positive cells was resemble ganglion cells and may have appeared in the GCL as a lower than in the GCL. consequence of gliosis.

DISCUSSION (Figs. 1C, ID). Low doses of NMDA (10 nanomoles) had virtu- ally no effect on cell number, whereas higher doses (160 We adapted two protocols that stimulate RGC death in rats to nanomoles injected) rapidly stimulated cell loss (Fig. 2B). Max- study RGC death in mice. The first method, optic nerve crush, imum loss in the GCL occurred 6 days after injection of 160 causes ganglion cell death possibly by blocking retrograde nanomoles NMDA and averaged 72.5% ± 12.1% (n = 6) of the transport of neurotrophic factors from the brain to the RGC cells. soma. In mice, this procedure causes a gradual loss of cells in the GCL over a 3-week period, which is similar to the rate of Depletion of Cells in the GCL Correlates with the cell loss observed in rats after optic nerve transection.8 The Loss of Thyl mRNA major difficulties in adapting this protocol for mice were ac- To confirm that optic nerve crush and NMDA injection caused cessing the optic nerve in the without causing excessive the death of RGCs, we also examined the loss of expression of bleeding from damaged vessels in the fascia surrounding the Thyl by in situ hybridization. Thyl is predominantly expressed ocular muscles and damaging the RGC axons without severing by RGCs in the retina, so it serves as a marker for these cells in the central retinal artery. Bleeding can be avoided by using

GCL A — B ft, FIGURE 3- TUNEL-stained mouse retinas after various treatments. (A) Control retina from a mouse 2 days after intravitreal injection with 2 /xl balanced saline solution. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. (B) Control retina treated with DNase I to nick the DNA in all the nuclei as a positive control for TUNEL. (C) Retina harvested from an eye 1 week after optic nerve crush. A single TUNEL-positive cell is present in the GCL. (D) Retina harvested from an eye 2 days after intravitreal injection of 160 nanomoles NMDA. At least 7 TUNEL-positive cells are visible in the GCL. Scale bar, 20 /urn.

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c do not survive this procedure long enough to measure an effect o on RGC survival (data not shown). 5, In mice, the injection of NMDA causes a dose-dependent loss of cells in the GCL, which occurs over a period of 6 days at the 4- I highest concentration tested. As with optic nerve crush, similar 0) kinetics of RGC loss have been reported in rats injected with a> comparable doses of NMDA.4 NMDA injection also stimulated the death of a small number of cells in the inner nuclear layer. These '55 o cells were situated in the proximal region of this cell layer, which a. • 1. is generally populated by amacrine cells. Several different cell UJ types in this layer express NMDA receptor subunits, although z only amacrine cells appear to express the same subunits detected 12 3 4 in RGCs. In addition, biochemical and histologic evidence also suggest that amacrine cells are adversely affected by NMDA.4 It is Weeks also possible that some of the affected cells were displaced RGCs, although the number of TUNEL-positive cells in this layer ex- ceeded the number of 7frj//-positive cells identified in control retinas (data not shown). The characteristics of cell death stimulated by both optic nerve crush and NMDA injection included features of apopto- sis. Dying cells contained pyknotic condensed nuclei, cellular fragmentation, and DNA fragmentation. In addition, neither treatment resulted in the accumulation of cellular debris and inflammation normally associated with necrosis. The observa- tion that cells in the GCL of mice die by apoptosis is consistent with several other studies in rats, rabbits, and primates.1

Acknowledgments

The authors thank Paul Kaufman and Leonard Levin of the University of Wisconsin, Madison, for helpful discussions and critical review of the manuscript and Soesiawati Darjatmoko for assistance with the 8 10 12 handling of the mice. B Days References FIGURE 4. Graphs showing the peak periods of cell death in sections 1. Nickells RW. Retinal ganglion cell death in glaucoma: the how, the of the entire retina as a function of TUNEL staining. Each data point why, and the maybe./ Glaucoma. 1996;5:345-356. represents the mean number of TUNEL-positive cells/section obtained 2. Johnson JE, Barde Y-A, Schwab M, Thoenen H. Brain-derived from 4 mice (6 sections each) (± SD). (A) Optic nerve crush showing neurotrophic factor supports the survival of cultured rat retinal ganglion cells. / Neurosci. 1986;6:3031-3038. the results for the GCL only. No TUNEL-positive cells were detected in other retinal layers after optic nerve crush. (B) NMDA (160 nanomoles) 3. Mey J, Thanos S. Intravitreal injections of neurotrophic factors support the survival of axotomized retinal ganglion cells in adult injection showing the counts for both the ganglion cell layer (GCL) and rats in vivo. Brain Res. 1993;602:304-317. the inner nuclear layer (INL). For each treatment, maximum TUNEL 4. Siliprandi R, Canella R, Carmignoto G, et al. N-methyl-D-aspartate- staining preceded the point of maximum cell loss. induced neurotoxicity in the adult rat retina. Vis Neurosci. 1992; 8:567-573. 5. Vorwerk CK, Lipton SA, Zurakowski D, Hyman BT, Sabel BA, blunt dissection to create a window in the fascia to access the Dreyer EB. Chronic low-dose glutamate is toxic to retinal ganglion nerve. Damage to the central retinal artery can be avoided by cells: toxicity blocked by memantine. Invest Ophthalmol Vis Set. clamping the nerve with a pair of self-closing Dumont forceps. 1996;37:l6l8-l624. 6. Isenmann S, Wahl C, Krajewski S, Reed JC, Bahr M. Up-regulation This clamp is sufficient to damage axons without permanently of Bax protein in degenerating retinal ganglion cells precedes damaging the major blood vessels. The alternative to crushing apoptotic cell death after optic nerve lesion in the rat. Eur JNeu- the optic nerve is axotomy. A complete transection of the optic rosci. 1997:9:1763-1772. nerve in the eye orbit is impossible because the artery is still 7. Levin LA, Schlamp CL, Spieldoch RL, Geszvain KM, Nickells RW. closely apposed to the nerve at this point. Others have circum- Identification of bcl-2 family genes in the rat retina. Invest Oph- thalmol Vis Sci. 1997;38:2545-2553. vented this problem by accessing the nerves of newborn mice 8. Berkelaar M, Clarke DB, Wang Y-C, Aguayo AJ. Axotomy results in at a more posterior site using an intracranial approach. This delayed death and apoptosis of retinal ganglion cells in adult rats. method proved to be unsuitable for adult mice, many of which J Neurosci. 1994;l4:4368-4374.

Downloaded from iovs.arvojournals.org on 09/25/2021 JOVS, April 1999, Vol. 40, No. 5 Reports 1009 Effect of Staurosporine on muscle contraction, we determined the effect of staurosporine Outflow Facility in Monkeys on the outflow facility in living monkey eyes. Baohe Tian, B'Ann T. Gabelt, and Paul L Kaufman METHODS Fourteen adult cynomolgus monkeys (Macaca fascicularis) of PURPOSE. TO determine the effect of the serine-threonine both sexes, weighing 2.5 kg to 6.0 kg, were anesthetized with kinase inhibitor staurosporine on outflow facility in living intramuscular ketamine (10 mg/kg), followed by intramuscular monkeys. (35 mg/kg) or intravenous (15 mg/kg) pentobarbital-Na. Total METHODS. Total outflow facility was determined by two- outflow facility was determined by two-level constant pressure level constant pressure perfusion of the anterior chamber perfusion of the anterior chamber (AC) with Bariiny's solution, bilaterally before and after intracameral infusion of stau- using one double-branched and one single-branched needle 9 rosporine or vehicle in opposite eyes. and correcting for internal apparatus resistance. Most mon- keys were used in more than one protocol, had undergone RESULTS. Intracameral staurosporine dose-dependently more than one prior perfusion, or both, but none within the doubled outflow facility, with 0.1 \xM, 1 /LIM, and 10 juM preceding 5 to 6 weeks; all were free of AC cells and flare. After being subthreshold, effective, and maximal doses, respec- 35 minutes of baseline facility measurement, the AC was ex- tively. At 50 /xM, intracameral staurosporine was less ef- changed with 2 ml of 0.1 /nM to 50 /xM staurosporine (Sigma, fective than 10 /xM on facility and induced corneal tox- St. Louis, MO) solution in one eye, vehicle (0.01% to 5% icity. dimethyl sulfoxide [DMSO] in Barany's solution) in the other, CONCLUSIONS. The broad-spectrum protein kinase inhibitor for 10 to 15 minutes. The reservoirs were then immediately staurosporine increases outflow facility in living monkeys, filled with the corresponding solutions, closed for 45 minutes, perhaps by affecting the trabecular meshwork cytoskele- and reopened for 45 minutes of post-drug facility measure- ton. (Invest Ophthalmol Vis Sci. 1999;40:1009 -1011) ment. In some experiments, the monkey received two differ- ent doses on one occasion, with a higher dose (1.0 JLLM or 10 /xM) administered to the same eye following post-lower dose he serine-threonine kinase inhibitor H-7 (l-(5-isoquinolinyl- (0.1 JLLM or 1.0 /xM) facility measurements. All investigations Tsulfonyl)-2-methylpiperazine) inhibits acto-myosin contrac- were in accordance with University of Wisconsin and NIH tility and thereby leads to deterioration of the actin microfila- guidelines and with the ARVO Statement on the Use of Animals ment system and perturbation of its membrane anchorage, in in Ophthalmic and Vision Research. turn reducing tension at cell- cell and cell- extracellular matrix adherens junctions in several types of cultured cells, including human trabecular meshwork cells.1"4 H-7 also inhibits pilo- RESULTS carpine-induced contraction of isolated monkey ciliary muscle strips in vitro and pupillary constriction in vivo, probably by AC exchange with 0.1 /LtM to 50 /xM staurosporine produced a disrupting the actin filament network in the ciliary and dose-dependent facility increase; 0.1 /xM was ineffective, whereas sphincter smooth muscle cells.5 In living monkeys, topical or 1 /nM, 10 /LIM, and 50 /uM staurosporine increased facility by 69% intracameral H-7 increases outflow facility and reduces intraoc- ± 20% (n = 8, P < 0.02), 168% ± 48% (n = 8, P < 0.01), and ular pressure,3 perhaps due to similar effects on the actin 117% ± 39% (n = 4, P < 0.1), respectively, relative to baseline cytoskeleton of the trabecular meshwork. and adjusted for contralateral control eye changes (Fig. 1, Table The serine-threonine kinase inhibitor staurosporine de- 1). The initial facility values on restarting the perfusion after die pletes actin microfilament bundles in cultured rat astrocytes,6 closed reservoir waiting period were only slightly increased rela- inhibits contraction of guinea pig airway smooth muscle cells tive to the control eyes, but the increase was dose-dependent (Fig. induced by the protein kinase C activator phorbol myristate 1). At 50 ju,M, corneal cloudiness and edema were present the day acetate,7 and inhibits intracellular Ca2+-dependent contrac- after perfusion and persisted for at least several months. No tions induced by various agonists in rabbit aortic strips.8 In apparent corneal toxicity was observed by casual examination view of the similarity of the effects of H-7 and staurosoprine on under normal room light 1 day after or by slit-lamp 5 to 8 weeks the actin filament network in cultured cells and on smooth after 0.1 jixM, 1 /LtM, or 10 /xM intracamerally.

From the Department of Ophthalmology and Visual Sciences, DISCUSSION University of Wisconsin, Madison. Supported by grants from the (EY02698), The serine-threonine kinase inhibitor staurosporine, which has National Institutes of Health, Bethesda, Maryland; Glaucoma Research cytoskeletal effects similar to those of H-7 in cultured cells,6 also Foundation, San Francisco, California; Research to Prevent Blindness, New York, New York; and the Ocular Research & Educa- has a similar facility-increasing effect in the living monkey eye at tion Foundation, Madison, Wisconsin. concentrations (^1 jtxM) comparable to that (100 nM) reported Submitted for publication May 7, 1998; revised November 3, 1998; for cytoskeletal effects in rat astrocytes.6 Similar to H-7, the initial accepted December 17, 1998. facility values were only slightly elevated compared with the Proprietary interest category: P, C3, C5, Ccl, Cc3, Cc4, Cc6. ipsilateral baselines after intracameral exchange infusion of 1 /xM Reprint requests: Paul L. Kaufman, Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, F4/328 CSC, or 10 |LtM staurosporine, with substantial facility elevation pro- 3 600 Highland Avenue, Madison, WI 53792-3220. duced only after continued perfusion. Most likely, drug-induced

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1.3 Staurosporine n=4 n=8 1.1 Vehicle

0.9 0.1 p.M Staurosporine LOjiM Staurosporine

0.7- X 0.5- T 0.3" 1 - Res Closed Res Closed Hi- ! I I I I 0.1- I- 0.1 \iM Staurosporine l.OuM Staurosporine a 1.2- n=8 n=4

O 1^ lOuM Staurosporine 50nM Staurosporine 9 O 0.8 H

0.6-

0.4- X^

j.-1- Res Closed 0.2- Res Closed

10(iM Staurosporine 50)iM Staurosporine

10 30 50 70 90 110 130 10 30 50 70 90 110 130 I i i 1 BL BL Time (min)

FIGURE 1. Effect of intracameral exchange infusion with 0.1 JUM to 50 JU-M staurosporine on outflow facility in monkeys. BL, Baseline; Res, Reservoir. Data are means ± SEM /xl/min per mm Hg for n animals, each contributing one staurosporine- and one vehicle-treated eye. Difference between eyes =£0.0 by the two-tailed paired f-test: *P < 0.1, }P < 0.05.

architectural changes within the meshwork produce only slight are more substantial.3 At 50 /xM, staurosporine produced a sub- resistance washout at physiological flow rates, whereas at the stantial initial facility increase, perhaps suggesting that at higher higher flow rates and pressure heads during perfusion, disruption doses the drug may render the meshwork architecture unstable of resistance-relevant structures and reduction of flow resistance even at normal flow rates.

TABLE 1. Effect of Intracameral Staurosporine on Outflow Facility Outflow Facility (fil/min/mm Hg)

Dose Stau Veh Stau/Veh 0.1 pM in 0.01% DMSO (n = 4) BL 0.30 ± 0.09 0.29 ± 0.09 1.04 ± 0.14 Rx 0.27 ± 0.07 0.28 ± 0.08 1.03 ± 0.17 Rx/BL 0.97 ± 0.05 0.99 ± 0.04 0.98 ± 0.04 1.0 jaM in 0.1% DMSO (n = 8) BL 0.28 ± 0.05 0.30 ± 0.05 0.96 ±0.11 Rx 0.50 ± 0.08 0.33 ± 0.04 1.57 ± 0.25 Rx/BL 1.96 ± 0.25 1.19 ± 0.12 1.69 ± 0.20t 10 JU-M in 1% DMSO (n = 8) BL 0.32 ± 0.05 0.44 ± 0.08 0.81 ± 0.12 Rx 0.74 ± 0.18 0.39 ± 0.08 1.99 ± 0.43 Rx/BL 2.26 ± 0.33 1.00 ± 0.20 2.68 ± 0.48* 50 jaM in 5% DMSO (n = 4) BL 0.33 ± 0.06 0.32 ± 0.07 1.05 ± 0.09 Rx 0.51 ± 0.10 0.24 ± 0.04 2.17 ± 0.28f Rx/BL 1.55 ± 0.14 0.79 ± 0.15 2.17 ± 0.39*

Values are means ± SEM for n animals. Staurosporine administered by exchange perfusion; BL, baseline; Rx, post-drug facility; Stau, staurosporine-treated eye; Veh, vehicle-treated eye; post-drug data encompasses 45 min, beginning 45 min after drug administration. i * P < 0.1, t P < 0.05, t P < 0.01 for ratios different from 1.0 by the two-tailed paired f-test.

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The overall facility increase, relative to both ipsilateral 2. Volberg T, Geiger B, Citi S, Bershadsky AD. Effect of protein kinase baseline values and contralateral vehicle-treated control eyes, inhibitor H-7 on the contractility, integrity, and membrane anchor- induced by 50 p,M staurosporine was no greater, and may have age of the microfilament system. Cell Motil Cytoskel, 1994;29:321- been less, than that induced by 10 yM staurosporine. A similar 338. 3. Tian B, Kaufman PL, Volberg T, Gabelt BT, Geiger B. H-7 disrupts phenomenon was observed with cytochalasin D,l0 where a 5X the actin cytoskeleton and increases outflow facility. Arch Oph- maximal intracameral dose for increasing facility actually thalmol. 1998; 116:633-643. caused a facility decrease in —40% of eyes. Also, because a 4. Liu X, Glasser A, Croft MA, Polansky JR, Fauss DJ, Kaufman PL. concentration of DMSO in the AC of 4% or greater tends to Effect of H-7 on cultured human trabecular meshwork (HTM) cells reduce outflow facility and blunt resistance washout,'' the 5% [ARVO Abstract]. Invest Ophthalmol Vis Set. 1998;39(4):S705. concentration likely caused the trend toward reduction of Abstract nr 3235. facility in the control eyes, and presumably in the drug-treated 5. Tian B, Millar C, Kaufman PL, Bershadsky A, Becker E, Geiger B. eyes as well, for 50 /AM staurosporine. Staurosporine at 50 JLLM H-7 effects on the iris and ciliary muscle in monkeys. Arch Oph- produced corneal cloudness and edema, perhaps suggesting thalmol. 1998;116:1070-1077. 6. Mobley PL, Hedberg K, Bonin L, Chen B, Griffith OH. Decreased separation of corneal endothelial cells and elevation of corneal phosphorylation of four 20-kDa proteins precedes staurosporine- endothelium permeability. However, 1 JXM and 10 ju,M stauro- induced disruption of the actin/myosin cytoskeleton in nit astro- sporine significantly increased facility without corneal toxicity cytes. Exp Cell Res. 1994;2l4:55-66. apparent to gross observation. Studies involving multiple 7. Souhrada M, Souhrada JF. Inhibitor)' effect of staurosporine on doses, more sensitive measurements of corneal structure and protein kinase C stimulation of airway smooth muscle cells. Am function, and older animals are needed. Rev Respir Dis. 1993; 148:425- 430. Preliminary ultrastructural studies indicate that administra- 8. Sasaki Y, Seto M, Komatsu K, Omura S. Staurosporine, a protein kinase inhibitor, attenuates intracellular Ca (2+)-dependent con- tion of H-7 into monkey eyes induces generalized relaxation and tractions of strips of rabbit aorta. EurJPharmacol. 1991 ;202:367- apparent expansion of the trabecular meshwork and Schlemm's 372. canal (Sabanai I, Gabelt B, Tian B, Kaufman P, and Geiger B, 9. Baniny EH. Simultaneous measurement of changing intraocular unpublished data, August 1998). This, in conjunction with data pressure and outflow facility in the vervet monkey by constant showing that H-7 relaxes human trabecular meshwork cells in pressure infusion. Invest Ophthalmol, 1964;3:135-l43. culture,12 suggests that H-7 may affect facility by inhibiting cell 10. Kaufman PL, Erickson KA. Cytochalasin B and D dose-outflow contractility, leading to "relaxation" of the trabecular outflow facility response relationships in the cynomolgus monkey. Invest pathway and expansion of the draining surface, and thus allowing Ophthalmol Vis Sci. 1982;23:646-650. 11. Kiland JA, Peterson JA, Gabelt BT, Kaufman PL. Effect of DMSO more extensive flow through the meshwork. Although stauro- 13 14 and exchange volume on outflow resistance washout and re- sporine has somewhat different specificities than H-7, ' both sponse to pilocarpine during anterior chamber perfusion in mon- are relatively broad serine-threonine protein kinase inhibitors and keys. Curr Eye Res. 1997;l6:1215-1221. have similar cytoskeletal and physiological effects in cultured cells 12. Gills JP, Roberts BC, Epstein DL. Microtubule disruption leads to and smooth muscles, and on outflow facility in living monkeys. cellular contraction in human trabecular meshwork cells. Invest Nonetheless, further physiological and morphologic studies are Ophthalmol Vis Sci, 1998;39:653-658. needed to clarify the mechanisms by which these and related 13- Takagi A, Yamada Mizutani M, Tomioka N, Itai A. Inhibition mech- anisms of staurosporine and H7 to cAMP-dependent protein kinase compounds affect outflow facility. through docking study. Chem Pharm Bull. 1996;44:6l8-620. References 14. Cagnoli CM, Kharlamov E, Atabay C, Uz T, Manev H. Apoptosis induced in neuronal cultures by either the phosphatase inhibitor 1. Birrell GB, Hedberg KK, Habliston DL, Griffith OH. Protein kinase okadaic acid or the kinase inhibitor staurosporine is attenuated by C inhibitor H-7 alters the actin cytoskeleton of cultured cells.y Cell isoquinolinesulfonamides H-7, H-8, and H-9. /Mol Neurosci. 1996; Physiol. 1989;l4l:74-84. 7:65-76.

Mechanism of Exercise-Induced mOsM). In each experiment, venous blood taken before and immediately after exercise was analyzed for hemato- Ocular Hypotension crit, plasma protein concentration, total plasma osmolal- ity, and plasma COP. 2 1 Bruce Martin? Alon Harris, Ted Hammel, and RESULTS. Exercise in both experiments significantly re- 5 Vic Malinovsky duced IOP and elevated COP (each P < 0.01). Dehydra- tion, compared with hydration, also significantly reduced PURPOSE. Although acute dynamic exercise reduces intraoc- IOP and elevated COP, when measured before and after ular pressure (IOP), the factors that provoke this response exercise (P < 0.05). The correlation of mean IOP with remain ill-defined. To determine whether changes in colloid mean COP, over the entire range created by varying ex- osmotic pressure (COP) cause the IOP changes during exer- ercise and hydration statuses, was statistically significant (r = -0.99; P < 0.001). In contrast, other indexes of cise, standardized exercise was performed after dehydration hydration status, including hematocrit, total plasma osmo- and hydration with isosmotic fluid. lality, and plasma protein concentration, failed to change METHODS. Progressive cycle ergometer exercise to voli- as IOP changed and failed to correlate with IOP, on either tional exhaustion was performed after 4 hours' dehydra- a group or individual basis, in conditions of varying levels tion, and after hydration with 946 ml isosmotic liquid (345 of exercise and hydration.

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