Alleviation of Constant-Light–Induced Photoreceptor Degeneration by Adaptation of Adult Albino Rat to Bright Cyclic Light

Abstract

purpose. To further test the hypothesis that light-adaptation–mediated photoreceptor protection works through inhibition of apoptosis by activation and/or upregulation of neuroprotective molecules.

methods. Albino rats were born and raised in 5-lux cyclic light (12 hours OFF and ON). At 8 weeks of age, animals were adapted to 400-lux cyclic light for different periods. Light damage was induced by exposure to constant light for 1 day at an illumination of 1700 lux. Animals were killed, and their eyes were removed for morphometric and biochemical analysis. TUNEL assay was used to evaluate photoreceptor cell apoptosis and Western blot analyses were used to determine the levels of basic fibroblast growth factor (bFGF), neuronal nitric oxide synthase (nNOS), and caspase-3.

results. Exposure of dim-reared rats to constant light for 1 day dramatically increased TUNEL-positive cells in the outer nuclear layer. Adaptation to 400-lux bright cyclic light for 4 days significantly reduced TUNEL-positive cells induced by exposure to constant light, which correlated with a significant increase in bFGF expression. Compared with control retinas, caspase-3 levels were not changed by exposure to constant light or after adaptation to 400 lux. There was a significant increase in nNOS level in the constant-light–exposed group, but not in the group adapted to 400-lux bright light before exposure to constant light.

conclusions. The retina of the adult rat can rapidly upregulate neuroprotective mechanisms when switched from dim to bright cyclic light. Identification of the molecules involved in this process may allow rational development of therapeutic approaches to treat retinal degenerative diseases.

Light history has been shown to affect the susceptibility of the retina of the albino rat to light damage. ^{1 2 3} Animals raised in relatively bright environments are protected against light-induced degeneration, compared with rats raised in dim cyclic light or darkness, ^{1 2 4 5 6} suggesting that factors that determine the protection against light-induced apoptosis may be generated in response to light stress. This adaptability encompasses many aspects of retinal cell and molecular biology and physiology, one of which must be the up- or downregulation of retinal photoreceptor cell components. In this regard, several molecules and mechanisms have been found to change in animals raised from birth in bright cyclic light, compared with those raised in dim light, and in animals exposed acutely to damaging levels of light, compared with unexposed control animals. These include upregulation of antioxidant protective mechanisms, ¹ upregulation of cytokines, ⁷ and other neuroprotective proteins, such as heat shock protein ⁸ and heme oxygenase, ⁹ up- or downregulation of visual transduction cascade components, ¹⁰ and upregulation of transcription factor activator protein (AP)-1. ^{11 12} It has been shown recently that hypoxia upregulates erythropoietin, which protects the retina from light damage. ^{13 14} Hypoxia also induces the formation of bFGF and VEGF. ^{15 16 17 18 19 20}  

It is now clear that apoptotic cell death occurs in virtually every cell type and that most pathologic insults can provoke apoptosis if delivered at a dose below that expected to cause acute, necrotic cell death. Apoptosis has been described in a wide variety of hereditary retinal degenerations, ^{21 22 23} in light-induced damage, ²⁴ after retinal detachment, ^{25 26} and in other types of retinal degeneration. ²⁷ Recent observations demonstrating the importance of mitochondrial function and the high metabolic demands of rods make it likely that loss of calcium homeostasis, free radical damage, and any other process leading to mitochondrial failure may also be of significance. ^{28 29 30} We hypothesize that adaptive protection against light-induced apoptosis works through upregulation of neuroprotective molecules. To test this hypothesis, we examined the time course of activation of this adaptation system in adult rats born and raised in dim cyclic light (5 lux) and switched acutely to brighter cyclic light (400 lux). We also determined the levels of expression of bFGF, caspase-3, and neuronal nitric oxide synthase (nNOS) as a function of adaptation. 

Materials and Methods

Animals and Constant Light Exposure

Sprague-Dawley albino rats were born and raised in 5-lux cyclic light (12 hours ON and OFF). At 8 weeks of age, animals were adapted to 400-lux cyclic light for 1, 2, 3, 4, 6, or 9 days. Light damage was then induced by exposure to constant light for 24 hours at an illumination of 1700 lux. Except as noted, after exposure to 400-lux cyclic light, control animals were returned to 5-lux cyclic light for an additional 5 days before any measurements were made. After constant exposure to light, animals were placed in the dark for 24 hours and then returned to 5-lux cyclic light for an additional 4 days, except as noted. All experimental procedures adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the University of Oklahoma Faculty of Medicine Guidelines for Animals in Research. All research protocols were reviewed and approved by the Institutional Animal Care and Utilization Committees of the University of Oklahoma Health Sciences Center and the Dean A. McGee Eye Institute. 

Functional Evaluation by Electroretinography

Animals were kept in total darkness overnight before electroretinograms (ERG) were recorded. Animals were anesthetized with ketamine (120 mg/kg body weight intramuscularly [IM]) and xylazine (6 mg/kg body weight IM). Pupils were dilated with 1.0% tropicamide and 2.5% phenylephrine HCl, and ERG responses were recorded with a gold electrode placed in the cornea with 1% tetracaine topical anesthesia. A reference electrode was positioned at the mouth and a ground electrode on one foot. The duration of white-light stimulation was 10 ms with a 60-second delay between flashes at seven light intensities presented in ascending order, beginning below threshold, to record the b-wave sensitivity curves and allow calculation of the saturated b-wave amplitude (B_max). 

Morphologic Evaluation by Quantitative Histology

After ERG testing, animals were killed by an overdose of carbon dioxide. The eyes were enucleated, fixed, and embedded in paraffin, and 5-μm-thick sections were taken along the vertical meridian, to allow comparison of all regions of the retina in the superior and inferior hemispheres. We used the method established by Rapp et al. ³¹ and LaVail et al. ³² to evaluate the morphologic changes quantitatively. In each of the hemispheres, outer nuclear layer (ONL) thickness was measured in nine defined areas, starting at the optic nerve head and extending along the vertical meridian toward the superior and inferior ora serrata. Measurements were made at 450-μm intervals. Mean ONL thickness was then calculated for the entire retinal section, ³² as was ONL thickness of the superior region of retina, which is most sensitive to the damaging effects of light. In each experiment, a single section from the retinas of at least six eyes was measured. 

Photoreceptor Cell Apoptosis Evaluation by TUNEL Assay

The TUNEL assay was used to study the effect of environmental light change on photoreceptor cell apoptosis and on the ability of the retina to adapt to 400-lux cyclic light. An apoptosis detection kit (ApopTag Peroxidase In Situ Apoptosis Detection; Intergen Co., Purchase, NY) was used in histochemical staining of the paraffin-embedded tissues to examine photoreceptor cell apoptosis through DNA fragmentation. 

SDS-PAGE and Western Blot Analysis

Protein samples containing 50 to 100 μg of retinal protein were resolved by 7.5%, 10%, or 15% SDS-PAGE and transferred to nitrocellulose membranes. The blots were washed two times for 5 minutes each with TTBS (100 mM NaCl, 20 mM Tris-HCl [pH 7.4], and 0.1% Tween-20) and blocked with 10% nonfat dry milk in TTBS overnight at 4°C. The blots were then incubated with bFGF (1:500; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), caspase-3 (1:500; Santa Cruz Biotechnology, Inc.), nNOS (1:2500; BD Biosciences, San Diego, CA), or rhodopsin (R1D4, 1:10,000) antibodies for 2 hours at room temperature. After primary antibody incubation, the blots were incubated with horseradish peroxidase (HRP)–linked secondary antibodies (anti-rabbit, anti-mouse, or anti-goat IgG) and developed by enhanced chemiluminescence (ECL), according to the manufacturer’s instructions. 

Statistical Analysis

Results are expressed as the mean ± SD. Differences were assessed by ANOVA or t-test, depending on experimental design. P < 0.05 was considered significant. 

Results

Morphologic Evaluation of Photoreceptor Cell Protection

Morphologic evaluation of photoreceptor cell protection by adaptation to bright cyclic light was determined by quantitative histology. As mentioned earlier, after exposure to constant light for 24 hours or to 400-lux cyclic light, all animals were returned to 5-lux cyclic light for an additional 5 days before any measurement. Figure 1 shows the ONL thickness along the vertical meridian of the eye of rats maintained either in dim cyclic light (5 lux) or adapted to bright cyclic light (400 lux) for different periods ranging from 1 to 9 days, with or without exposure to constant light (1700 lux for 24 hours). Before adaptation, exposure to constant light caused a dramatic loss of photoreceptor cells, as evidenced by thinning of the ONL in both the superior and inferior hemispheres (Fig. 1A) . The average ONL thickness of constant-light–stressed rats was 15% of that in the nonstressed control animals (Fig. 1H) . Adaptation to 400-lux cyclic light had two measurable effects. First, there was a modest but significant time-dependent loss of photoreceptor cells due to the adaptive process, such that after 9 days of cyclic 400-lux light, the ONL thickness was reduced to 65% of control thickness (Fig. 1H) . The greatest loss occurred in the superior retina and was evident from the second day of exposure. Second, there was a significant, time-dependent increase in survival of photoreceptors after adaptation to 400-lux cyclic light. After 9 days, 78% of the photoreceptor cells survived the constant-light challenge. 

Functional Evaluation of Photoreceptor Cell Protection

The effect of adaptation on retinal function was determined by electroretinography 5 days after exposure to constant light. Figure 2 shows the b-wave amplitudes of rats adapted to 1, 2, 3, 4, 6, or 9 days of cyclic 400-lux light, before and after constant-light challenge. Histograms of B_max values are presented in Figure 2H . As expected, exposure of nonadapted animals to constant light for 24 hours almost completely abolished the ERG response. Adaptation to 400-lux bright cyclic light gradually reduced the b-wave amplitudes in nonexposed animals, evidenced by the decline of the sensitivity curves (Figs. 2B 2C 2D 2E 2F 2G) and in B_max values (Fig. 2H) . B_max was significantly reduced to 68% or 60% of the time 0 control after 6 (P < 0.03) or 9 (P < 0.009) days of adaptation, respectively (Fig. 2H) . Significant protection of function was first noted at 3 days of light adaptation (P < 0.05). B_max increased from 0 μV in nonadapted rats to 285 μV in animals adapted for 3 days and increased to 434 μV and 493 μV after 6 and 9 days of adaptation, respectively. Thus, although there was some loss of function as a result of the adaptation, exposure to 400-lux cyclic light significantly preserved function when the animals were challenged by 24 hours of constant illumination. 

Protection through Inhibition of Apoptosis

We used the TUNEL assay to examine the possibility that the protection provided by retinal adaptation to bright cyclic light works through inhibition of apoptosis. In these studies, animals were killed immediately after bright-light adaptation and/or exposure to constant light, and sections were taken between 0.5 and 1 mm from the optic nerve head along the superior meridian. Control retinas from animals born and raised in 5-lux cyclic light environment have no TUNEL-positive cells (Fig. 3A) . However, constant exposure to light for 24 hours induced extensive photoreceptor cell apoptosis, as indicated by TUNEL-positive staining in the ONL (Fig. 3B) . In comparison with control retinas (Fig. 3A) , there was a significant increase in TUNEL-positive cells in the ONL after adaptation to 400-lux bright cyclic light for 4 days (Fig. 3C) . However, when challenged with constant light, retinas from these animals showed a significant reduction in TUNEL-positive cells (Fig. 3D) compared with control animals (Fig. 3B) . 

Increased bFGF Expression after Adaptation to Bright Cyclic Light

Because bFGF has been shown to protect photoreceptor cells and to inhibit photoreceptor apoptosis induced by damage from constant light, ^{32 33 34 35} we investigated whether adaptation to bright cyclic light could increase bFGF expression in the retina in a time frame contemporaneous with adaptive neuroprotection. Animals born and raised in 5-lux cyclic light environment were adapted to bright cyclic light (400 lux) for 1, 2, 3, 4, 6, or 9 days before retinas were collected. Animals not adapted to bright cyclic light served as the control. Western blot analysis of bFGF showed an increase after 2 days of adaptation (Fig. 4A) . Scanning of the blots of four independent experiments showed a significant increase in bFGF expression after 4 days of adaptation (Fig. 4B) . This increase was maintained after 6 and 9 days of adaptation, the longest examined time point in this study. As a loading control, we also ran a parallel blot to detect rhodopsin expression. Although a slight but not statistically significant decrease in rhodopsin expression was seen after 4 or 6 days of adaptation, a significant decrease in rhodopsin expression was seen after 9 days’ adaptation to bright cyclic light (Fig. 4A) . This probably reflects the loss of photoreceptor cells during adaptation, as well as the shortening of outer segments ^{1 2 3} and reduction in rhodopsin packing density ^{6 36} in animals raised in bright cyclic light. 

Western Blot Analysis of Caspase-3 and nNOS

Because it has been reported that the expression of caspase-3 is increased in the retina of transgenic rats with the S334ter rhodopsin mutation, ³⁷ we determined whether there was an effect of adaptation for 4 days to bright cyclic light on retinal caspase-3 levels. Western blot analysis of retinal extracts of four independent experiments (Fig. 5A) probed with anti-caspase-3 antibodies did not show any effect of adaptation or of exposure to 24 hours of constant illumination. However, nNOS, which has been shown to be increased in mice exposed to 6 hours of very bright light (6000 lux), ³⁸ was significantly increased in the retinas of both groups of rats exposed to constant light, although there was no difference in nNOS expression in control animals and rats adapted to bright cyclic light for 4 days (Fig. 5) . 

Discussion

It is now well known that rats raised in dim cyclic light or darkness are more susceptible to light damage than animals raised in bright cyclic light. ^{1 2 10 39} A number of biochemical and morphologic differences have been described in rats raised under these two conditions. Morphologically, bright cyclic light rearing leads to shortened and more disorganized rod outer segments than dim cyclic light rearing. ¹ Biochemically, three types of changes occur: (1) a change in components of the visual cascade pathway to reduce the efficiency of photon capture and transduction, which includes reduced rhodopsin-packing density in ROS, ^{6 36} reduced rhodopsin protein and opsin mRNA, ^{10 40} reduced transducin protein and mRNA, ^{10 40} and increased arrestin protein and mRNA ^{10 40} ; (2) an enhancement of mechanisms that protect against lipid peroxidation, including increases in enzymatic activities of glutathione enzymes, ¹ decreased levels of polyunsaturated fatty acid substrates of lipid peroxidation, ³⁶ and increased levels of the antioxidants vitamin E and ascorbic acid ¹ ; (3) an upregulation of neuroprotective cytokines such as bFGF and CNTF. ⁴¹ Many of these changes and others occur during acute light damage, in which case it is difficult to differentiate between the neuroprotective effects and a response to an acute insult. 

In an earlier study, ³⁹ adult rats switched from a cyclic light environment of less than 250 to 800 lux for 1 week were protected against constant light damage to photoreceptor cells. In the present study, the time course of adaptation was determined for rats born and raised in 5-lux cyclic light and moved at age 8 weeks into a cyclic light environment of 400 lux. This intensity has been shown to be sufficient to upregulate neuroprotection against light damage. ¹ We used this level in an attempt to reduce light damage, which occurs when adult rats are brought from a dim to bright cyclic light environment. ³⁹ We confirmed our earlier work showing that the adult albino rat retina has a remarkable ability for rapidly upregulation of neuroprotective mechanisms that prevent retinal damage due to intense constant illumination. ³⁹ Only one 12-hour exposure to 400-lux light was sufficient to provide some protection of retinal function (Figs. 1 2) . Significant protection of retinal structure was evident after two 12-hour exposures to 400-lux light. Under our experimental conditions, there was a 65% loss of photoreceptor cells in rats adapted for 9 days to 400-lux cyclic light, indicating a competition between the adaptive and degenerative processes. However, of the cells that adapted to the acute 400-lux cyclic light challenge, 78% survived an acute 1700-lux exposure for 24 hours, compared with only 15% in nonadapted control cells (Fig. 1) . 

An interesting finding in this study was that the reduction in function (B_max) over the 9-day adaptive period was greater than the reduction in ONL thickness. This could reflect an adaptive change in the components of the visual cascade, mentioned earlier. However, the ERG measurements of animals that were not exposed to 1700 lux for 24 hours were made 5 days after the animals were returned to 5-lux cyclic light, which should have been sufficient time for some recovery of function. Also, after acute exposure, the relative loss of B_max in adapted animals at all time points was greater than the relative loss of ONL thickness. Thus, it may be possible that adaptation preserves retinal structure to a greater extent than it preserves function. This is not a new idea, as it has been reported that retinal structure in transgenic rats containing rhodopsin mutations is better preserved than function in animals treated with neuroprotective cytokines ^{42 43} (Steinberg RH, et al. IOVS 1997;38:ARVO Abstract 1069). 

Apoptosis is the mode of photoreceptor cell death in inherited and light-induced retinal degeneration. ^{21 22 23 24} In this study, we demonstrated that the protection provided by retinal adaptation to bright cyclic light works through inhibition of apoptosis, although the molecular basis for this protection has yet to be elucidated. Nevertheless, a diverse range of agents has been used to prevent photoreceptor apoptosis in this model. ^{35 44 45 46} These include survival factors such as bFGF, ^{33 47 48} implicating a role for growth-promoting factors in the inhibition of photoreceptor apoptosis. It has also been demonstrated that bFGF promotes photoreceptor survival in RCS rats, ⁴⁹ which have a defect in phagocytosis of rod outer segments by RPE cells due to a mutation in the receptor tyrosine kinase gene Mertk. ⁵⁰ In albino rats, bFGF also protects photoreceptors from the damaging effects of constant light. ^{33 35} We have shown in this study that adaptation to bright cyclic light increases bFGF expression in the retina in a time frame contemporaneous with that of development of neuroprotection, suggesting that upregulation of bFGF expression may play a role in photoreceptor cell protection by adaptation to bright cyclic light. ⁴¹ It has been demonstrated in differentiated pheochromocytoma (PC12) cells that bFGF rapidly inhibits whole-cell sodium channels, which are fundamental for the regulation of electrical excitability in neuronal cells. This inhibition is coincident with a hyperpolarizing shift in the voltage dependence of inactivation. ⁵¹ However, the relation between such inhibition and bFGF induced by light adaptation in the photoreceptor protection in this study remains unknown. We have also studied the FGFR-1 protein level in the retina in response to light adaptation from 1 to 9 days. No difference between control and light-adapted groups was observed at any time point. Caspase-3 is recognized as one of the key executioners in apoptosis. The role of caspase-3 in photoreceptor degeneration has been examined in a line of transgenic rats that carry a rhodopsin mutation S334ter, and an increase in caspase-3–like activity was noted, ³⁷ suggesting the involvement of caspase-3 in hereditary photoreceptor cell death. Donovan et al. ³⁸ examined the activation status of caspase-3 during photoreceptor apoptosis in a mouse model and found that light-induced photoreceptor degeneration occurs independent of caspase-3. In three independent experiments, we found no obvious changes in retinal caspase-3 expression after adaptation, constant light exposure, or adaptation plus constant light exposure. The slight increase in caspase-3 level in the retinas of rats treated for 4 days with bright light shown in Figure 5A is probably due to the slight relative decrease in rhodopsin in total retinal proteins that occurs when photoreceptor cells are lost. Our Western blot analyses are similar to those presented by Donovan et al. ³⁸ in mice and quite different from the Western blots and enzyme activities reported by Liu et al. ⁵² in rats with a mutant rhodopsin (S334ter). This suggests that the mechanisms involved in photoreceptor cell death by light damage, which are likely to involve oxidative damage, are different from those that occur in cell death due to mutations in photoreceptor-specific genes. 

Because the free radical NO appears to play a role in light-induced retinal degeneration, ^{38 53 54 55} we examined the effect of 400-lux cyclic light adaptation on the expression of nNOS. Our results showed that nNOS expression was increased in the retinas exposed to constant light, but that adaptation to 400-lux cyclic light did not change nNOS expression. Inhibition of nNOS by N ^G-nitro-l-arginine methyl ester (l-NAME) is sufficient to prevent light-induced photoreceptor degeneration in mice ³⁸ and rats. ^{53 54 55} Therefore, the protection by adaptation to 400-lux cyclic light observed in this study is not through reduction in nNOS expression, but probably involves some other pathways. 

The greater sensitivity of the superior retina to light-induced apoptosis is a well-known phenomenon, ⁵⁶ and selective loss of rod photoreceptor cells in this region of the retina has been reported in numerous studies in which the entire retinal expanse was examined. ^{46 57} In our present study, the regional difference is not due to regional differences in light exposure, because our light box was designed to provide uniform exposure. In the studies of Vaughan et al., ⁵⁷ S334ter and P23H rhodopsin mutant rats in green Plexiglas chambers ⁴ were placed inside a circular fluorescent light to guarantee uniform exposure, and there was a selective loss of superior photoreceptor cells in both mutant groups. Early studies suggested a diurnal susceptibility to light damage. ^{58 59} Organisciak et al. ⁶⁰ used short-term light exposures at different times of the light cycle and found that albino rats exposed at 1 AM were four times more susceptible to light damage than those exposed at 5 PM. They suggested, “The expression profile of an intrinsic retinal factor(s) at the onset of light exposure appears to be important in determining light damage susceptibility”. Because studies have shown a genetic predisposition to light damage among different albino rat ⁶¹ and mice ⁶² strains, and because we ^{1 2} and others ^{7 8 10 60} have reported the capacity of the retina to alter protein expression in response to environmental light changes, we suggest that the susceptibility of the superior region of the rat retina is due to differential expression of neuroprotective factors. 

 Supported by Grants EY00871, EY04149, EY06973, EY12190, from the National Eye Institute; and Grant RR17703 from the Division of Research Resources, National Institutes of Health; Research to Prevent Blindness, Inc., New York, New York; The Foundation Fighting Blindness; and Presbyterian Health Foundation, Oklahoma City, Oklahoma.

 Submitted for publication February 7, 2003; revised March 26, 2003; accepted April 2, 2003.

 Disclosure: F. Li, None; W. Cao, None; R.E. Anderson, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Quantification of morphologic changes. Measurements of ONL thickness along the vertical meridian of the eye in albino rats maintained in dim cyclic light (5 lux) or adapted to bright cyclic light (400 lux) for 0 to 9 days, either with (CL) or without (control) exposure for 1 day to constant light (1700 lux). (A–G) Average of six retinas of animals adapted for 0, 1, 2, 3, 4, 6, and 9 days. (H) Histogram of the average ONL thickness with or without constant light exposure. ¶P < 0.05 vs. day 0 non-light-damage groups. *P < 0.05 vs. day 0 light damage groups.

Figure 2.

 

Functional protection by retinal adaptation to bright cyclic light. Measurements of electroretinogram (ERG) b-wave amplitudes in albino rats maintained in dim cyclic light (5 lux) or adapted to bright cyclic light (400 lux) for 0 to 9 days, either with (CL) or without (control) exposure for 1 day to constant light (1700 lux). (A–G) are the average b-wave amplitudes of six retinas of animals adapted for 0, 1, 2, 3, 4, 6, and 9 days. (H) Average B_max values with or without constant light exposure. ¶P < 0.05 vs. day 0 non-light-damage groups. *P < 0.05 vs. day 0 light damage groups.

Figure 3.

 

Inhibition of light-induced apoptosis by retinal adaptation to bright cyclic light. The ability of retinal adaptation in the inhibition of photoreceptor cell apoptosis was measured by TUNEL assay. (A) Control rat born and raised in 5-lux cyclic light. (B) Rat born and raised in 5-lux cyclic light and exposed to 1700-lux constant light for 1 day. (C) Rat born and raised in 5-lux cyclic light and adapted to 400-lux cyclic light for 4 days. (D). Rat born and raised in 5-lux cyclic light, adapted to 400-lux cyclic light for 4 days, and exposed to 1700-lux constant light for 1 day. These are representative micrographs from four animals per experimental group.

Figure 4.

 

Western blot analysis of bFGF and rhodopsin as a function of time of adaptation to 400-lux cyclic light. (A) bFGF was detected as a 17-kDa protein with anti-bFGF antibody. Rhodopsin, a 40-kDa protein, was detected with anti-rhodopsin antibody in a parallel blot. (B) Quantitation of relative bFGF levels. Results from four independent experiments were averaged and presented relative to control levels (no adaptation). Each gel contained 50 μg of retinal protein.

Figure 5.

 

Western blot analysis of caspase-3 and nNOS expression after retinal adaptation to 400-lux cyclic light for 4 days. (A) Caspase-3 was detected as a 32-kDa protein with anti-caspase-3 antibody. nNOS is a 155-kDa protein detected with anti-nNOS antibody in a parallel blot. (B) Quantitation of relative nNOS levels. Data from three independent experiments were averaged and presented relative to control levels.

The authors thank Isabelle Ranchon for helpful advice and Mark Dittmar, Mark McClellan, Kathleen Alvarez, and Sherry Chen for technical support.