Changes in mRNA Levels of the Myoc/Tigr Gene in the Rat Eye after Experimental Elevation of Intraocular Pressure or Optic Nerve Transection

Abstract

purpose. To isolate the rat Myoc/Tigr gene and investigate changes in its expression pattern in normal eyes and in eyes with either pressure-induced optic nerve damage or optic nerve transection.

methods. Expression pattern of the rat Myoc/Tigr gene was investigated by Northern blot hybridization. Optic nerve damage and death of ganglion cells in the retina were induced unilaterally, by injection of hypertonic saline solution, episcleral vein cauterization, or optic nerve transection. The levels of mRNA for Myoc/Tigr were compared between several tissues of the control and surgically altered eyes, by using semiquantitative RT-PCR, real-time PCR, and Northern blot analysis.

results. The rat Myoc/Tigr gene is 10 kb long and contains three exons. Among the eye tissues analyzed, Myoc/Tigr mRNA was detected in the combined tissues of the eye angle, sclera, cornea, retina, and optic nerve head. With pressure-induced optic nerve degeneration, the level of Myoc/Tigr mRNA decreased in the retina and the combined tissues of the eye angle, but increased in the optic nerve head. After optic nerve transection, the level of Myoc/Tigr mRNA increased in the retina, but did not change in the combined tissues of the eye angle.

conclusions. The decreased level of Myoc/Tigr mRNA in the retina after induction of elevated intraocular pressure compared with that in the control retina cannot be explained by ganglion cell death alone. Differences in Myoc/Tigr mRNA levels in eye tissues after elevation of intraocular pressure or optic nerve transection may reflect the activation of different signaling pathways involved in regulation of this gene.

Glaucoma is an optic neuropathy characterized by the death of ganglion cells in the retina accompanied by excavation and degeneration of the optic nerve head. Glaucoma is usually, but not always, associated with elevated intraocular pressure (IOP). Data describing the molecular changes in the eye after elevation of IOP are limited. ^{1 2 3 4} Understanding the molecular changes in the different tissues of the eye after IOP elevation may lead to a better understanding of glaucoma and improved treatment for this disease. In human patients, such studies are difficult to conduct at early stages after IOP elevation, and retina and optic nerve samples can only be obtained after death. Therefore, appropriate animal models may provide valuable information about the molecular events in the retina and the optic nerve during the course of elevated IOP. Although the monkey model may provide the best insight into the processes in the human glaucomatous retina and optic nerve, ^{5 6 7} the cost and limited availability of monkeys make them difficult to use in pilot studies. Several rat models of elevated-pressure–induced optic nerve damage have been developed to study changes in the retina and the optic nerve. ^{8 9 10}  

It has been shown that mutations in the MYOC/TIGR gene are associated with juvenile open-angle glaucoma, often accompanied by high IOP. ^{11 12 13 14} Moreover, between 2.6% and 4.3% cases of sporadic primary-open angle glaucoma are associated with mutations in this gene. ¹⁵ MYOC/TIGR is expressed in the ciliary body, iris, and trabecular meshwork, which is consistent with its proposed role in tissues responsible for aqueous dynamics. ^{12 16 17 18 19} It is also expressed in the retina, sclera, and cornea ^{12 16 20} ; however, it is not known whether these latter tissues are directly affected by mutations in the MYOC/TIGR gene. Although significant variability in the level of MYOC/TIGR protein may occur between different individuals, available data suggest that the level of MYOC/TIGR maybe enhanced in the trabecular meshwork ²¹ and aqueous humor of patients with glaucoma who do not have mutations in the MYOC/TIGR gene. ²²  

Recently, rat Myoc/Tigr cDNA has been isolated, ²³ and expression of the rat Myoc/Tigr gene has been detected in retina, skeletal muscle, and thyroid among tissues analyzed. In this study, we characterized the rat Myoc/Tigr gene and investigated changes in the levels of Myoc/Tigr mRNA in ocular tissues after induction of elevated IOP, by two different methods. These changes were compared with those observed after optic nerve transection. 

Methods

Animals

All experiments complied with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Two rat models of pressure-induced optic nerve damage were used. In the first model (method 1), four male Brown Norway rats weighing 300 to 400 g were used. Elevation of IOP in one eye of each animal was induced by injection of 50 μl of a 1.75-M hypertonic saline solution through the episcleral vein, as described previously. ⁸ A tonometer (TonoPen XL; Mentor, Norwell, MA) was used to measure IOP daily in awake animals, as described. ^{8 24} Rats were killed 6 weeks after surgery, and the degree of optic nerve damage was then estimated by several independent observers, as described. ²⁵ A grade scale from 1 (normal) to 5 (total degeneration) was used, based on prior observations of a stereotypic pattern of injury in this model. ⁸  

Sixty-two adult female albino Wistar rats (Charles River Laboratory, Wilmington, MA) weighing 250 to 300 g were used in the second model (method 2). They were kept in standard lighting conditions (14-hour light and 10-hour dark cycle). IOP was elevated in the left eye of the anesthetized animals by cauterizing two or three episcleral veins, as previously described. ⁹ The right eye was subjected to a sham operation, in which the surgery was performed without episcleral vein cauterization, and served as the control. Special care was taken during the surgery not to injure the limbal venous plexus and to minimize the amount of blood loss and damage to the conjunctiva and the underlying sclera. IOP was monitored once a week in the morning with rats under anesthesia (mixture of 45 mg/kg ketamine and 9 mg/kg xylazine). Each IOP value was an average of three consecutive measurements taken with a precalibrated pneumatonometer (Mentor; Bio-Rad, Hercules, CA). In these experiments, the optic nerve damage was estimated for selected animals by fundus observation or fundus photography. Rats were killed in groups of three at days 3 and 5 and at 1, 2, 3, 4, 5, 6, and 8 weeks after the surgery. 

Optic nerve transection was performed in four rats. In brief, the rats were anesthetized and a skin incision was made close to the superior orbital rim. The orbit was opened, leaving the supraorbital vein intact. After subtotal resection of the lacrimal gland, the superior extraocular muscles were spread using a small retractor. The optic nerve was exposed by longitudinal incision of the eye retractor muscle and the perineurium. The optic nerve was cut 3 to 4 mm behind the globe. Special care was taken to avoid damaging the central retinal artery, which passes within the meninges of the nerve. The left optic nerve was cut in each animal, and the right eye served as the sham- operated control, in which the surgery was performed without cutting the optic nerve. Animals were killed 11, 19, and 22 days after the surgery. 

RNA Isolation and Northern Blot Hybridization

Total RNA was isolated from the dissected cornea, retina, lens, sclera, and combined tissues of the iridocorneal angle (trabecular meshwork, iris, and ciliary body) of Wistar or Norway rats using RNA extraction reagent (RNazol; TelTest, Friendswood, TX) or a kit (Total RNA Miniprep; Stratagene, La Jolla, CA). Total RNA (0.5 μg) was separated by electrophoresis on a 1.2% agarose, 2.2 M formaldehyde gel to evaluate the quality of RNA samples. For Northern blot analysis experiments, 2 μg RNA was separated on agarose gel as just described, transferred to a membrane (Nitran; Schleicher & Schuell, Keene, NH), and hybridized with a [³²P]-labeled rat Myoc/Tigr probe (position 17-2004 in AB019393) in hybridization solution (ExpressHyb; Clontech, Palo Alto, CA) at 68°C overnight. Membranes were washed in 2× SSC-0.1% SDS, again in 1× SSC-0.1% SDS, and finally in 0.1× SSC-0.1% SDS solutions at 65^oC. The intensity of the hybridization bands was estimated using a phosphorescence imager (Storm 860; Molecular Dynamics, Inc., Sunnyvale, CA). Filters were stained with 0.02% methylene blue after autoradiography for normalization of the amount of loaded RNA. ²⁶ In some cases, 1 μg ethidium bromide was added to RNA samples before separation, and RNA was visualized after electrophoresis under UV light. 

Isolation and Characterization of the Rat Myoc/Tigr Gene

Primers 6955 and 6957 (see Table 1 ), located in exon 1 of the rat Myoc/Tigr gene, were used to screen a rat P1 genomic library. The screening was performed as a service by Genome System, Inc. (St. Louis, MO). Two P1 clones, P21991 and P21992, were identified and used in all subsequent experiments. DNA was isolated from P1 clones, with a kit (Qiagen, Chatsworth, CA). P1 DNA was digested with the EcoRI restriction enzyme, and the 5.2-kb restriction fragment, containing the 5′ end of the rat Myoc/Tigr cDNA, as determined by Southern hybridization, was cloned into a vector (BlueScript SK; Stratagene). The fragment containing a complete intron 2 sequence was obtained by PCR using DNA of P1 clone 21991 as a template and primers 6921 and 6922 (see Table 1 ). This fragment was cloned into the vector (BlueScript SK; Stratagene). The complete nucleotide sequences of the rat Myoc/Tigr gene were obtained by direct sequencing of P1 clones and by sequencing of plasmid DNAs containing the 5.2-kb EcoRI restriction fragment or the intron 2 sequence. P1 and plasmid DNAs were sequenced with fluorescent dideoxynucleotides on an automated sequencer (model 310; PE Biosystems, Foster City, CA). The complete nucleotide sequence of the rat Myoc/Tigr gene was deposited into GenBank (accession numbers AF289235; GenBank is provided in the public domain by the National Center for Biotechnology Information, Bethesda, MD, and is available at http://www.ncbi.nlm.nih.gov/genbank). 

Semiquantitative RT-PCR and Real-Time PCR

Total RNA (1 μg) was used for cDNA synthesis using reverse transcriptase (SuperScript; Gibco, Grand Island, NY) and oligo(dT)-primer. The amount of synthesized cDNA was evaluated by PCR using primers specific for opsin, cyclophilin, hypoxanthine-guanine phosphoribosyltransferase (HPRT), and ribosomal protein L19, depending on the source of RNA (Table 1) . PCR reactions were performed in a thermocycler (PTC-200; MJ Research, Watertown, MA), using polymerase (AmpliTaq; PE Biosystems). Each PCR reaction was repeated at least twice. The thermal cycling parameters were as follows: 1 minute 30 seconds at 94°C followed by 30 cycles of 30 seconds at 94°C, 1 minute 30 seconds at 59°C, and 1 minute at 72°C, and final incubation for 5 minutes at 72°C. PCR reaction products were analyzed by agarose gel-electrophoresis. After adjustment of cDNA concentration for each pair of samples from the control and experimental eyes of the same animal, relative abundance of mRNAs for Myoc/Tigr, Thy-1, and GFAP were estimated. Primers for each gene were located in different exons. Different dilutions of cDNA samples were used for different genes to provide a linear range of PCR reactions. The intensity of DNA bands was estimated by computer (Chemilimager 4000 software; Alpha Innotech Inc., San Leandro, CA). Correlation coefficient analysis was also performed (Sigmaplot 2000; SPSS Science, San Raphael, CA; and Excel; Microsoft, Redmond, WA.) The correlation coefficients obtained for each set of data were very similar for both programs. Real-time PCR was performed in a sequence-detection system (GenAmp 5700; PE Biosystems) using a green fluorescence PCR kit under conditions recommended by the manufacturer (SYBR Green; PE Biosystems). 

Primers were designed with a melting temperature (T_m) of 60^o to amplify short fragments within the target sequences (Table 1) . Each PCR reaction contained 5 μl of the 10× fluorescent green buffer, 6 μl of 25 mM MgCl₂, 4 μl dNTP mix (2.5 mM dCTP, 2.5 mM dGTP, 2.5 mM dATP, and 5 mM dUTP), 0.25 μl polymerase (5 U/μl; AmpliTaq Gold; PE Biosystems), 0.5 μl uracil-N-glycosylase (1 U/μl UNG; AmpErase; PE Biosystems), 1 μl of the forward and reverse primers (10-μM concentration), 5 μl cDNA, and water to a final volume of 50 μl. Each reaction was repeated four times in optical tubes (MicroAmp; PE Biosystems). The thermal cycling parameters were as follows: 2 minutes at 50°C to activate the UNG, and then 10 minutes at 95°C to activate the polymerase (AmpliTaq Gold; PE Biosystems), followed by 40 cycles of 15 seconds at 95°C and 1 minute at 60°C. The amounts of PCR products were estimated, using software provided by the manufacturer (PE Biosystems). After completion of PCR cycles, the reactions were heat denatured over a 35°C temperature gradient from 60°C to 95°C. The primer pairs used gave a single peak of dissociation as judged by the data analysis software (PE Biosystems). The amplification of a single band with the expected size was also confirmed for the Myoc/Tigr and cyclophilin primer pairs by normal RT-PCR with subsequent analysis of the reaction products by 5% polyacrylamide gel electrophoresis. The relative abundance of Myoc/Tigr mRNA in each sample was calculated by comparison of threshold cycle (C _T) values for Myoc/Tigr and cyclophilin, as suggested by the manufacturer (PE Biosystems). 

Results

Organization of the Rat Myoc/Tigr Gene and Its Expression in Ocular Tissues

The cDNA structure of the rat Myoc/Tigr cDNA has been recently described. ²³ This sequence (GenBank accession no. AB019393) was used to design primers 6955 and 6957 that produced a single band in PCR reactions with rat genomic DNA as a template. This combination of primers was used to screen a rat genomic P1 library. Two overlapping P1 clones, 21991 and 21992, were isolated from the rat genomic library. Sequencing of these clones, as described in the Materials and Methods section, gave the complete sequence of the rat Myoc/Tigr gene. The rat gene is 10 kb long (Fig. 1) and contains three exons. The length of the rat Myoc/Tigr gene is similar to that of the mouse gene ²⁷ but is approximately 6.5 kb shorter than the human gene. This difference in length is due to shorter introns in the rodent Myoc/Tigr genes than in the human gene. The exon–intron boundaries of the rat Myoc/Tigr gene are shown in Table 2 , together with the corresponding data for the human gene. 

The expression pattern of the Myoc/Tigr gene in several rat eye tissues was investigated by Northern blot hybridization (Fig. 2) . Among tissues analyzed, sclera contained the highest levels of Myoc/Tigr mRNA with the expected length of 2000 nucleotides (nt). A 3500-nt band observed in the sclera sample could correspond to partially spliced Myoc/Tigr mRNA containing intron 2. The length of intron 2 in the rat Myoc/Tigr gene was 1343 nt (Table 2) . 

Variations in the Level of Myoc/Tigr mRNA in Rat Eye Tissues after Experimental IOP Elevation

To gain insight into mechanisms involved in the regulation of the Myoc/Tigr gene, levels of Myoc/Tigr mRNA were estimated in tissues of the eye expressing Myoc/Tigr after experimental induction of elevated IOP or after optic nerve transection. Because of significant (up to two three times in the retina) variability in the level of Myoc/Tigr gene expression between different animals, changes in the level of Myoc/Tigr message after the experimental treatment were always estimated separately for each animal. One eye served as the control and the other eye was experimentally treated. Tables 3 and 4 provide data on average IOP in the eyes after saline injection (method 1) and cauterization (method 2), respectively. IOP values obtained for anesthetized Wistar rats (method 2) were lower than those measured in awake Norway rats (method 1; Tables 3 4 ). The main reason for these differences is that general anesthetics cause substantial decreases in IOP. ²⁸ Changes in mRNA levels were first estimated by semiquantitative RT-PCR. Opsin, ribosomal protein L19, and cyclophilin mRNA levels were used to normalize the amount of retinal cDNA synthesized. 

To obtain independent confirmation of the success of the surgery, we analyzed changes in mRNA levels of two genes that are known to alter their expression in glaucoma or after optic nerve transection. It has been reported that the level of GFAP message goes up in the retina after optic nerve transection or in glaucoma, ²⁹ whereas the level of Thy-1 gene message goes down after optic nerve transection. ³⁰ When IOP was induced by hypertonic saline injection, GFAP message levels increased 2.1 to 3.3-fold compared with the control eyes, whereas the levels of mRNA for Thy-1 were reduced 2.1 to 5.9-fold in the samples with a high degree of optic nerve damage (Fig. 3) . In the sample with a low degree of optic nerve damage (pair E598-C599), the mRNA level for GFAP increased 1.75-fold in the experimental eye compared with the normal eye, whereas the mRNA level for Thy-1 did not change significantly (Fig. 3) . When IOP was induced by cauterization, the results were similar. The highest increase (two- to threefold) in GFAP mRNA levels (not shown) and the highest decrease (three- to fourfold) in the Thy-1 message levels (Fig. 4) were observed in retinas exposed to 5 to 6 weeks of elevated IOP. Previous experiments demonstrated that long exposures (5–6 weeks) of the retina to elevated IOP lead to more severe retinal damage than short exposures (2–3 weeks). ³¹ Similarly, more dramatic increases in GFAP and decreases in Thy-1 mRNA levels were observed 22 days after optic nerve transection than in those levels only 11 days after transection (Fig. 5) . 

The Myoc/Tigr gene responded differently to elevation of IOP and optic nerve transection. In contrast to our results with GFAP and Thy-1, Myoc/Tigr mRNA levels decreased in retinas from eyes with elevated IOP but increased after optic nerve transection. As was the case for GFAP and Thy-1, these changes were more pronounced in retinas with more severe damage. In three samples from animals with a high degree of optic nerve damage obtained by method 1, the levels of Myoc/Tigr message were reduced 3.6- to 33-fold, whereas in the sample from the animal with less damage, there was no discernible difference (Fig. 3) . In the samples obtained in rats with IOP elevated by method 2, an average two- to threefold decrease was observed 5 to 6 weeks after IOP elevation. Changes were less pronounced during the first 2 to 3 weeks after surgery. A summary of changes in mRNA levels for Myoc/Tigr and Thy-1 observed in the retina after elevation of IOP by method 2 is shown in Figure 4 . The level of Myoc/Tigr message was increased after optic nerve transection (1.5–1.9-fold) with a slightly higher increase 22 days after surgery compared with 11 days after surgery (Fig. 5) . 

To be sure that differences in the Myoc/Tigr levels obtained by semiquantitative RT-PCR could be reproduced using other methods, two other techniques, real-time PCR and Northern blot hybridization, were used for several RNA samples from control and experimental retinas. Figure 6 shows a typical result of real-time PCR estimation of differences in Myoc/Tigr mRNA levels after surgery. Although the exact numbers were different for the two techniques, they were very similar. For example, the Myoc/Tigr mRNA levels were reduced after surgery 14.3- and 2.6-fold in pairs C592-E593 and C596-E597, as estimated by real-time PCR and 6.7- and 3.6-fold as estimated by semiquantitative RT-PCR. The same was true of Northern blot experiments (Fig. 7) . In the samples presented in Figure 7 , the hybridization intensity was reduced 2.1- and 3.3-fold in experimental retinas in the 6- and 8-week samples, respectively. By semiquantitative RT-PCR the corresponding numbers were 2.9- and 3.1-fold. We concluded that under conditions used in our experiments, semiquantitative RT-PCR provided reliable estimates of the changes in the level of analyzed mRNA. Therefore, in most cases, changes in the Myoc/Tigr mRNA levels were evaluated by semiquantitative RT-PCR only, because this required less RNA and was less expensive. 

Changes in Myoc/Tigr mRNA levels were also estimated in the tissues of the eye angle and in the optic nerve head. Only method 2 was used to prepare samples for these experiments. As was the case in the retina, the levels of Myoc/Tigr mRNA in the combined tissues of the iridocorneal angle after surgery were reduced relative to those in control eyes. However, the kinetics of downregulation differed between angle and retina. In the tissues of the angle, a significant reduction of the Myoc/Tigr mRNA levels (2–2.5 times) was detected as early as 3 to 7 days after surgery (Fig. 8) and returned to practically normal levels 6 to 7 weeks after surgery. Myoc/Tigr mRNA levels did not change significantly in the tissues of the eye angle after optic nerve transection (data not shown). In contrast to the situation in the retina and tissues of the eye angle, Myoc/Tigr mRNA levels increased in the optic nerve head after IOP elevation (Fig. 9) . This upregulation was detected within 3 days after the surgery and was sustained throughout the period of observation (5–6 weeks after the surgery). No changes in Myoc/Tigr levels were detected in the sclera and cornea (not shown). 

Discussion

Although it is well established that mutations in the human MYOC/TIGR gene are associated with glaucoma, the function of this gene is not clear. Animal models of glaucoma may provide helpful hints concerning its role in the tissues of the eye. Because several rat models of glaucoma have been developed, we isolated and characterized the rat Myoc/Tigr gene to study its regulation in normal eyes and in eyes with experimentally induced ganglion cell damage. We investigated the expression pattern of the rat Myoc/Tigr gene in several ocular tissues, by using Northern blot hybridization. In general, the distribution of Myoc/Tigr mRNA in the rat eye was similar to those observed in human and mouse eyes. Expression of the Myoc/Tigr gene in the same ocular tissues in rats and humans has provided an additional argument in favor of using rat models of pressure-induced optic nerve damage to study changes in the expression pattern of this gene after IOP elevation. We demonstrated that levels of Myoc/Tigr mRNA decreased in the retina and in the combined tissues of the eye angle and increased in the optic nerve head after IOP elevation. The decreased levels of Myoc/Tigr mRNA in the rat retina were observed after IOP elevation by two different methods. Because Myoc/Tigr is expressed in ganglion cells ³² and nerve fiber layer, ³³ these results may be explained by a reduced number of ganglion cells in the treated retinas. According to the published estimates, 3% to 4% and 5% to 6% of ganglion cells die each week during the first 2 months after two- and three-vein cauterization, respectively. ^{9 31} Therefore, at least 50% of ganglion cells should survive even in the case of maximum retinal damage (8 weeks, three-vein cauterization). Optic nerve transection produces a more dramatic decrease in ganglion cell number. More than 50% of ganglion cells would be expected to die 2 to 3 weeks after optic nerve transection 3 to 4 mm behind the bulbar exit. ³⁴  

Nevertheless, in retinas from eyes with optic nerve transection, there was an increased level of Myoc/Tigr mRNA. Similar increased levels of Myoc/Tigr mRNA have been observed in preliminary experiments in retinas of transgenic rats expressing γ-interferon under the control of the α-crystallin promoter (Ahmed F, Egwaugu C, Tomarev SI, unpublished data, 2000). As in glaucoma, ganglion cells die by apoptosis in these transgenic rats. ³⁵ On the basis of these results, we concluded that ganglion cell death alone cannot explain changes in the level of Myoc/Tigr mRNA in the retina after experimental treatments. Decreased levels of Myoc/Tigr mRNA were also observed in the combined tissues of the angle of eyes with induced elevated IOP, compared with the fellow control eyes. Although it took several weeks to produce significant changes in the Myoc/Tigr levels in the retina, it took only a few days to produce similar changes in the tissues of the angle. Episcleral vein cauterization leads to nearly instantaneous increases in IOP. Thus, if the Myoc/Tigr promoter contains pressure-sensitive elements, increased IOP may quickly suppress its activity in the tissues of the angle. The level of Myoc/Tigr mRNA was unexpectedly increased in the optic nerve head after induction of high IOP. Such an increase may be connected to a remodeling of the optic nerve head and deposition of extracellular matrix proteins in response to elevated IOP. ^{36 37 38} These results indicate that mechanisms involved in the regulation of the Myoc/Tigr gene may vary in different ocular tissues and are consistent with the suggestion that cellular specificity and differentiation factors should be considered in the understanding MYOC/TIGR gene regulation in different tissues in humans. ³⁹  

We have tested mRNA levels for more than 25 different genes in the eye tissues, by RT-PCR after induction of elevated IOP (Tomarev SI, Ahmed F, Torrado M, Zinovieva RD, unpublished data, 2000). These include stress-response genes, transcription factors, and genes encoding members of signal transduction pathways, cytoskeletal proteins, and enzymes. We have detected changes in mRNA levels of only a few of these genes. Therefore, we believe the observed variations in Myoc/Tigr mRNA to be specific rather than the consequence of general activation or inhibition of transcriptional activity in the eye tissues after experimental treatment. 

It is possible that similar changes may happen in the tissues of the human eye after elevation of IOP. We suggest that decreases in the levels of wild-type MYOC/TIGR mRNA in different ocular tissues are not sufficient to produce glaucoma. It has been demonstrated that decreased levels of MYOC/TIGR protein, due to an Arg46Stop mutation, do not necessarily lead to glaucoma, even when this mutation is present in the homozygous state. ⁴⁰ Knocking out the mouse Myoc/Tigr gene also does not lead to any profound phenotype and does not change IOP. ⁴¹ The decreased levels of Myoc/Tigr mRNA in the retina and the trabecular meshwork after induction of high IOP and increased levels of the same message after optic nerve transection probably reflect the activation of different signaling pathways involved in regulation of the gene. 

 Submitted for publication March 29, 2001; revised July 12, 2001; accepted September 5, 2001.

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Table 1.

 

Oligonucleotides Used in the Study

Gene	Number	Sequence	Product Size (bp)
Myoc/Tigr	6921	5′-agaactaacagaggttcctgctt-3′	1613
Myoc/Tigr	6922	5′-tcgaactggcttatctgactgtact-3′
Myoc/Tigr	6955	5′-tccagacaccttgcagcagag-3′	278
Myoc/Tigr	6957	5′-ctgaaggtcctggatggctga-3′
Myoc/Tigr	8626	5′-aactaacagaggttcctgctt-3′	92
Myoc/Tigr	8627	5′-attgtctcagctgtcctcag-3′
Opsin	7770	5′-gatcgtcatcttcttctgctac-3′	345
Opsin	7771	5′-tcatctcccagtggattcttac-3′
GFAP	7962	5′-aggaacatcgtggtaaagacgg-3′	387
GFAP	7963	5′-tctggcaacggtttccataaca-3′
Thy-1	7964	5′-cgctttatcaaggtccttactc-3′	343
Thy-1	7965	5′-gcgttttgagatatttgaaggtc-3′
RPL19	7975	5′-ggtactgccaacgctcggat-3′	325
RPL19	7976	5′-ccttggacagagtcttgatgat-3′
Cyclophilin	8538	5′-tcctcctttcacagaattattcc-3′	345
Cyclophilin	8539	5′-aattagagttgtccacagtcgg-3′
Cyclophilin	8630	5′-atggcaagaccagcaagaag-3′	59
Cyclophilin	8631	5′-agctacagaaggaatggtttg-3′
HPRT	8540	5′-gtgaaaaggacctctcgaagtg-3′	445
HPRT	8541	5′-cagatacaatctcagtattattattt-3′

Figure 1.

 

Exon–intron structure of the rat Myoc/Tigr gene. Localization of the overlapping P1 clones used in this work is shown. Arrows: boundaries of the sequenced areas. Vertical line in P21992 corresponds to the end of the genomic insert in this clone.

Table 2.

 

Exon-Intron Boundaries in the Rat and Human Myoc/Tigr Genes

Exon No.	Exon Size (bp)	5′ Splice Donor	3′ Splice Acceptor	Intron Size (bp)
I R	676	TCCAGGGAAG/gtaagaatgc	tcgaatacag/TCTCTCAGTG	6987
I H	681	TCCAGAGAAG/gtaagaatgc	tttaatgcag/TTTCTACGTG	13285
II R	123	GGAGACAAAG/gtatcaagtt	aattcatcag/GATGTGGAGT	1343
II H	126	GGAGACACCG/gtatgaagtt	aatttaccag/GATGTGGAGA	1887
III R	1256
III H	1291

 Data for the human MYOC/TIGR gene are from the sequence in GenBank with accession number Z98750.

Figure 2.

 

Northern blot hybridization analysis of Myoc/Tigr expression in rat eye tissues. Total RNA (2 μg per lane) was hybridized with ³²P-labeled rat Myoc/Tigr cDNA (A). Loaded RNA was visualized by staining with ethidium bromide (B). Lane 1: cornea; lane 2: combined trabecular meshwork, iris, and ciliary body; lane 3: sclera; lane 4: retina; and lane 5: lens.

Table 3.

 

Average IOP and Estimated Degree of Optic Nerve Damage in the Experimental Eyes after Injection of Hypertonic Saline Solution (Method 1)

Eye No.	Average IOP (mm Hg)	Injury Grade
E591	37.6	5
E593	33.9	5
E597	37.4	5
E598	31.7	1

 Average IOP is the mean of daily IOP determinations during the 35-day experimental period. The mean IOP for the control eyes was between 28 and 29 mm Hg.

Table 4.

 

Average IOP in Experimental Eyes after Two or Three Episcleral Vein Cauterizations (Method 2)

Surgery	Experimental Eye IOP (mm Hg)
2 weeks, 2 veins	23.5 ± 0.6
3 weeks, 2 veins	22.9 ± 0.8
4 weeks, 2 veins	22.5 ± 0.7
5 weeks, 2 veins	22.9 ± 0.5
6 weeks, 2 veins	21.9 ± 0.6
5 weeks, 3 veins	30.5 ± 0.9
6 weeks, 3 veins	28.9 ± 0.7
8 weeks, 3 veins	28.5 ± 0.6

 IOP data are expressed as mean mm Hg ± SEM.

 Average of three consecutive measurements on a weekly basis. Each group consisted of three animals. Average IOP in the control eyes was between 13 and 14 mm Hg.

Figure 3.

 

Estimation of mRNA level for several genes in total retina after elevation of IOP by method 1, using semiquantitative RT-PCR. Numbers below each panel show the calculated difference between control (C) and experimental (E) samples.

Figure 4.

 

Correlation coefficient analysis of changes in mRNA levels for Myoc/Tigr and Thy-1 in the retina after elevation of IOP by method 2. Changes in mRNA levels after the surgery were measured relative to the control fellow eye. Each point corresponds to a separate pair of eyes. Nonlinear regression curves were then obtained.

Figure 5.

 

Estimation of mRNA level for several genes in total retina after optic nerve transection using semiquantitative RT-PCR. Numbers below each panel show the calculated difference between control (C) and experimental (E) samples. Numbers after the letters correspond to days after transection.

Figure 6.

 

Estimation of mRNA level for Myoc/Tigr by real-time PCR. In this experiment, normalization was obtained by using cyclophilin mRNA. (A, B) Real-time PCR curves for Myoc/Tigr (A) and cyclophilin (B) messages for samples C592 and E593 (see Fig. 3 for comparison). (C, D) Melting curves of the PCR products for Myoc/Tigr (C) and cyclophilin (D). Fluorescence indicates the magnitude of the signal generated by the given set of PCR conditions. The threshold cycle (C _T) value is the cycle at which a statistically significant increase in fluorescence is first detected.

Figure 7.

 

Northern blot hybridization of Myoc/Tigr expression in rat eye tissues. Total RNA (2 μg per lane) was hybridized with ³²P-labeled rat Myoc/Tigr cDNA (bottom). The amount of loaded RNA was evaluated by staining of 18S RNA with methylene blue (top), and intensity of the hybridization bands was estimated. W, weeks; 3v, three-vein cauterization; E, experimental; C, control.

Figure 8.

 

Correlation coefficientanalysis of changes in mRNA levels for Myoc/Tigr in the combined tissues of the eye angle after elevation of IOP by method 2. Analysis was performed as described in the legend to Figure 4 .

Figure 9.

 

Estimation of mRNA level for Myoc/Tigr in the optic nerve head by semiquantitative RT-PCR. Numbers below each panel show the calculated differences between control (C) and experimental (E) samples. W, weeks; 2v and 3v, two- and three-vein cauterization, respectively.

The authors thank Marianna Mertts for purification of the recombinant Myoc/Tigr protein used for the antiserum production and Carl Kupfer, Joram Piatigorsky, and Robert Wheelock for critical reading of the manuscript and for advice.