IOVS BJO
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

(Investigative Ophthalmology and Visual Science. 2007;48:4725-4732.)
© 2007 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.06-1404
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Morimoto, T.
Right arrow Articles by Tano, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Morimoto, T.
Right arrow Articles by Tano, Y.

Transcorneal Electrical Stimulation Promotes the Survival of Photoreceptors and Preserves Retinal Function in Royal College of Surgeons Rats

Takeshi Morimoto,1 Takashi Fujikado,1 Jun-Sub Choi,2 Hiroyuki Kanda,1 Tomomitsu Miyoshi,3 Yutaka Fukuda,3 and Yasuo Tano2

1From the Departments of Applied Visual Science, 2Ophthalmology, and 3Physiology, Osaka University Graduate School of Medicine, Osaka, Japan.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To determine whether transcorneal electrical stimulation (TES) has neuroprotective effects on photoreceptors and preserves retinal function in Royal College of Surgeons (RCS) rats.

METHODS. Three-week-old RCS rats received TES through a contact lens electrode on the left eye weekly for 2 to 6 weeks. The right eyes received sham stimulation on the same days. Electroretinograms (ERGs) were recorded from the rats at 3 weeks (before TES), and at 5, 7, and 9 weeks of age. After the ERG recordings, the rats were killed for morphologic analyses of the retina.

RESULTS. Morphologic analyses showed that the mean thickness of the outer nuclear layer (ONL) at each time point was significantly thicker in eyes treated with TES of 100 µA than in eyes with sham stimulation (TES 100 µA versus sham: 5, 7, and 9 weeks of age; P < 0.001). ERG studies showed that TES also significantly preserved retinal function up to 7 weeks of age, but did not preserve retinal function at 9 weeks of age.

CONCLUSIONS. TES prolongs the survival of photoreceptors and delays the decrease of retinal function in RCS rats. Although further investigations are necessary before using TES on patients, these findings indicate that TES may be a therapeutic treatment for some patients with diseases of the photoreceptors such as retinitis pigmentosa.

Electrical activity is essential for both the development and survival of neurons. For example, depolarization of neurons exerts some trophic influence on their development,1 2 and depolarization by high KCl concentrations inhibits the death of mature retinal ganglion cells (RGCs) in culture.3 4 5 In the auditory system, chronic electrical stimulation promoted the survival of spiral ganglion cells which otherwise would have degenerated from the administration of an ototoxic drug in vivo.6 7 8 In motor neurons, electrical stimulation activated the cell body and accelerated axonal regeneration and increased the expression of the mRNAs of brain-derived neurotrophic factor (BDNF) and trkB.9 10

In the visual system, we have demonstrated that direct electrical stimulation of the transected optic nerve (ON) stump promotes the survival of axotomized RGCs in adult Wistar rats.11 In addition, we have demonstrated that transcorneal electrical stimulation (TES), which is less invasive than electrical stimulation of the transected ON stump, also promotes the survival of axotomized RGCs in vivo.12 We concluded that electrical stimulation may have a neuroprotective effect on injured RGCs in patients with ON diseases, such as optic neuropathy. In fact, we have applied TES on patients with traumatic optic neuropathy (TON) or with nonarteritic ischemic optic neuropathy (NAION) and have found an improvement of visual function.13

These findings led us to hypothesize that TES would also have a neuroprotective effect on photoreceptors in eyes with photoreceptor degeneration such as in patients with retinitis pigmentosa (RP), one of the leading causes of blindness worldwide. No established treatment is available clinically, although many experimental approaches have been tried to save photoreceptors in various animal models of RP.14 15 16 17 18 19

The purpose of this study was to determine whether TES would have a neuroprotective effect on the photoreceptors and preserve retinal function in RCS rats. RCS rats have been extensively used because they develop photoreceptor degeneration, and they serve as an animal model of RP.20 In morphologic and electrophysiological analyses in the present study, TES had a neuroprotective effect on the photoreceptors and delayed the decrease of visual function in RCS rats.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Animals
All experimental procedures were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and the procedures were approved by the Animal Research Committee, Osaka University Medical School. Tan-hooded, pink-eyed RCS rats (rdy/rdy) were purchased from Clea Japan Inc. (Tokyo, Japan) and inbred at the animal facilities of Osaka University. They were raised on a 12-hour dark–12-hour light cycle with an ambient light intensity of 100 lux.

Transcorneal Electrical Stimulation
The rats were anesthetized intraperitoneally with pentobarbital sodium (60 mg/kg). Only the left eye was electrically stimulated, as described in detail.12 For the stimulation, the cornea was anesthetized with a drop of 0.4% oxybuprocaine HCl, and a contact lens electrode with inner and outer circular concentric electrodes (Kyoto Contact Lens, Kyoto, Japan) was placed on the cornea with a drop of 2.5% methylcellulose to maintain good electrical contact and prevent corneal dehydration.

Biphasic rectangular (1-ms/phase duration) current pulses were delivered at a frequency of 20 Hz from an electrical stimulation system (Stimulator: SEN-7320, Nihon Kohden, Tokyo, Japan; Isolator: WPI, Sarasota, FL) through the contact lens electrode. The current intensities were 0 (sham stimulation), 50, and 100 µA, and the duration of stimulation was 1 hour.

TES was applied initially on 3-week-old RCS rats (20–23 postnatal days), and was applied once a week for an hour thereafter. The TES-treated RCS rats were divided into three groups. TES was applied between 3 and 5 weeks of age in group 1 (n = 6), between 3 and 7 weeks of age in group 2 (n = 6), and between 3 and 9 weeks of age in group 3 (n = 6). The left eyes received TES (50 or 100 µA), and the right eyes received either sham electrical stimulation or no treatment as the control.

Electroretinography
Electroretinograms (ERGs) were recorded from RCS rats after the end of the TES-treatment (i.e., at 5 weeks of age in group 1, at 7 weeks of age in group 2, and at 9 weeks of age in group 3. For the ERGs, animals were kept in total darkness for at least 12 hours and were prepared for the recordings under dim red light. They were anesthetized intramuscularly with a loading dose of xylazine (13 mg/kg) and ketamine (86 mg/kg). The pupils were dilated with 0.1% atropine, 0.5% tropicamide, and 0.5% phenylephrine HCl. The animals were held steady with a bite bar and nose clamp in a stereotaxic frame. A heating pad maintained the body temperature at approximately 37°C.

ERGs were recorded from both eyes simultaneously with contact lens electrodes with internal and external concentric electrodes embedded in the lens (Kyoto Contact Lens, Kyoto, Japan), and the ground electrode was inserted subcutaneously, near the tail. Responses were amplified 10,000x and bandpass filtered from 0.08 to 1000 Hz with a 60 Hz notch filter. The recordings were digitized at 5 kHz. Ten to 20 responses were averaged with interstimulus intervals from 3 to 30 seconds, depending on the intensity of the stimulus.

ERGs were elicited by white light of 50 ms duration and a maximum luminance of +2.1 log cd/m2. The luminance was attenuated with neural-density filters in 0.5- or 1.0-log-unit steps. The threshold amplitude was set at 10 µV for the b-wave and 5 µV for the scotopic threshold response (STR). During ERG recordings, the shape and amplitude of each response from the first response to the last response were monitored, and we confirmed that the form and amplitude of the first ERG did not differ significantly from the last ERG.

Histologic Analyses
Immediately after the ERG recording, the rats were killed with an overdose of pentobarbital sodium. The eyes were removed and kept overnight at 4°C in 4% glutaraldehyde in 0.1 M phosphate buffer. Eyes were trimmed and postfixed in 1% osmium for 1 hour. The epoxy-embedded tissue was cut into 1-µm sections and stained with toluidine blue for light microscopy. All sections were cut along the vertical meridian of the eye passing through the ON. Three serial sections of each eye were quantified for each experimental animal.

The degree of retinal degeneration was assessed by measuring the thickness of outer nuclear layer (ONL) and inner nuclear layer (INL). In each of the superior and inferior hemispheres, photographs of the retina were taken at nine defined points with a camera attached to a light microscope (model E800; Nikon, Tokyo, Japan). The first photograph was made at approximately 500 µm from the center of the ON head, and subsequent photographs were taken every 400 µm more peripherally. The thickness of ONL and INL were measured on the photographs (Scion Image analyzer; Scion Corp. Frederick, MD). Three measurements were made at defined points separated from the adjacent photograph by 50 µm. The three measurements were averaged for the value plotted at each point. In this way, the 54 measurements in the two hemispheres were measured which represented the thickness over almost the entire retina. Each eye was coded so that the investigator was masked to treatment of the eye.

Statistical Analyses
Data were analyzed by commercial software (SPSS, ver. 10.0J; SPSS Inc, Chicago, IL). The data are expressed as the mean ± SEM. Comparisons between two groups were made by Student’s t-test, when the data were normally distributed, or by the Mann-Whitney rank sum test, when the data were not normally distributed. Comparisons among many groups were made by one-way ANOVA followed by the Tukey test. Statistical significance was set at P < 0.05.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
TES and Survival of Photoreceptors In Vivo
Representative retinal sections from the superior retinas from 7-week-old RCS rats that had 100 µA of TES or had sham stimulation are shown in Figure 1 . The number of rows of nuclei in the ONL layer was four or five in the retina receiving TES (Fig. 1A) and two or three in the retina with sham stimulation (Fig. 1B) .


Figure 1
View larger version (102K):
[in this window]
[in a new window]

  		FIGURE 1. Photomicrographs of TES-treated (A) and sham-stimulated (B) retinas from 7-week-old RCS rats. TES led to better structural preservation of the photoreceptors than sham stimulated. Scale bar, 50 µm.

 
Quantitative analyses showed that the ONL in the TES-treated eyes was significantly thicker than in the sham-stimulated and control eyes at 7 weeks of age (Fig. 2A) . The mean thickness of ONL in control retinas of 7-week-old RCS rats was 9.8 ± 1.0 µm (mean ± SEM, n = 6) which was not significantly different from that in the sham-stimulated eyes at 10.9 ± 0.6 µm (n = 6). The mean ONL thickness in the retinas treated with TES at a current intensity of 50 µA was 13.7 ± 0.4 µm (n = 6), whereas that with a current intensity of 100 µA was 23.3 ± 1.8 µm (n = 6). The ONL with 50 µA was not significantly thicker than that of sham-stimulated eyes, but with 100 µA, the ONL was significantly thicker than that of the sham-stimulated eyes and that of the 50 µA (P < 0.001 versus sham, P < 0.001 versus 50 µA; Fig. 2A ). Thus, TES at 100 µA was significantly effective on the survival of photoreceptors but TES at 50 µA was not significantly effective in RCS rats at 7 weeks of age; therefore, we used TES at 100 µA in the following experiment.


Figure 2
View larger version (12K):
[in this window]
[in a new window]

  		FIGURE 2. Effect of TES on the thickness of the ONL and INL of RCS rats at 7 weeks of age. Data are the mean ± SEM. (A) Mean thickness of the ONL in the retinas treated with TES (100 µA) is significantly thicker than that of control and sham-stimulated retinas (one-way ANOVA P < 0.001, followed by Tukey test, *P < 0.001 vs. sham). (B) Mean thickness of the ONL along the vertical meridian of the retina of RCS rats. The data are the mean ± SEM. In all cases, the ONL in the eyes receiving 100 µA ({blacksquare}) TES was significantly thicker than that with sham stimulation ({square}) or controls ({circ}) in both superior and inferior retina (one-way ANOVA, P < 0.001). (•) TES at the current intensity of 50 µA. (C) Mean thickness of the INL of the retinas of RCS rats at 7 weeks of age. There was no significant difference among them.

 
To determine whether the differences in the thickness of the ONL was localized or widespread across the retina, the mean thickness of the ONL was determined at 18 points along the superior–inferior plane of the eye in the three groups of RCS rats. The mean ONL thickness at every point in the superior and inferior hemispheres of the retinas treated with 100 µA TES was significantly thicker than that treated with sham stimulation or in the control retinas (one-way ANOVA, P < 0.001; Fig. 2B ). Thus, TES delayed the degeneration of the photoreceptors across the retina.

To determine whether the TES affected other layers of the retina, we measured the thickness of the INL. The mean thickness of the INL was: control = 29.0 ± 0.8 µm; sham = 28.1 ± 1.1 µm; 50 µA TES = 26.9 ± 0.4 µm; and 100 µA TES = 30.1 ± 0.8 µm (mean ± SEM, n = 6 each; Fig. 2C ). None of these differences was significant.

Time Course of Survival of Photoreceptors Treated with TES
Because 100 µA of TES prolonged the survival of photoreceptors, we used this current intensity to follow the time course of the survival of photoreceptors. The ONL of the retina of RCS rats at 3 weeks of age is composed of 10 to 12 rows of nuclei (Fig. 3A) . In the sham-stimulated rats, the ONL of 5-week-old RCS rat was made up of three rows, and at 9 weeks of age, only an occasional nucleus was seen in the ONL, and the photoreceptors were scattered and disorganized (Figs. 3C 3E) . In contrast, the ONL in 5-week-old RCS rats treated with TES had five to six rows, and at 9 weeks, there were one to two rows, were organized in a line (Figs. 3B 3D) .


Figure 3
View larger version (63K):
[in this window]
[in a new window]

  		FIGURE 3. Photomicrographs of the retina from control, TES-treated and sham-stimulated RCS rats. Retinas from control RCS rats at 3 weeks of age (A) and from TES (B) and sham-stimulated (C) eyes at 5 weeks of age, from TES (D) and sham-stimulated (E) eyes at 9 weeks of age. Although the ONL thickness in the sham-stimulated retina decreases with age, the ONL was thicker in the retinas receiving TES. Scale bar, 50 µm.

 
Measurements of the ONL thickness showed that there was a significant difference in the mean ONL thickness between TES-treated and sham-stimulated eyes in 5-week-old RCS rats (TES = 26.5 ± 2.9 µm; sham = 17.1 ± 2.7 µm; mean ± SEM, n = 6 each, t-test; P < 0.001), and at 9 weeks (TES = 8.34 ± 0.5 µm; sham = 4.12 ± 0.5 µm; n = 6 each, t-test; P < 0.001; Fig. 4A ).


Figure 4
View larger version (11K):
[in this window]
[in a new window]

  		FIGURE 4. Time course of photoreceptor survival. (A) Mean thickness of ONL in the retina of 3-, 5- and 9-week-old RCS rats treated with TES or sham stimulation. The data are the mean ± SEM. The differences in the mean ONL thickness between TES-treated retinas ({blacksquare}) and sham stimulated retinas (blk12) at 5 and 9 weeks of ages, as well as at 7 weeks of age, is significant (t-test, *P < 0.001). (B) Mean thickness of ONL along the vertical meridian of the eye in RCS rats. The data are the mean ONL thickness ± SEM. In all cases, treatment with TES at a current intensity of 100 µA led to ONL thickness that was significantly thicker than that with sham stimulation in both superior and inferior retina at each time point (one-way ANOVA, P < 0.001). ({circ}) 3-week-old RCS rats; ({blacksquare}) 5-week-old RCS rats treated with TES or ({square}) sham stimulation; and ({blacktriangleup}); 9-week-old RCS rats treated with TES or ({triangleup}) sham stimulation. (C) Measurement of mean INL thickness in the retinas in 5- and 9-week-old RCS rats treated with TES ({blacksquare}) or sham stimulation (blk12). There was no significant difference between them.

 
As shown in Figure 4A , although TES significantly delayed the loss of photoreceptors at each time point, the rapid loss of photoreceptors occurred between 7 and 9 weeks of age. Thus, the degree of neuroprotection changed with an increase in the age of the RCS rats.

Figure 4B shows the time course of the decrease of the mean ONL thickness at 18 points along the superior–inferior plane of the eye in RCS rats at 3, 5, and 9 weeks of age. Although the mean ONL thickness decreased with an increase in the age of the RCS rats, the mean ONL thickness at every point in the superior and inferior hemispheres in the retinas treated with TES was thicker than that in the retinas treated with sham stimulation.

There was no difference in the mean INL thickness between retinas treated with TES and sham stimulation at 5 weeks and 9 weeks of ages (Fig. 4C) .

The eye and fundus were examined at the end of the experiments, and neither retinal detachment nor vitreous hemorrhage was observed. In addition, cataracts or corneal opacities did not develop in all rats.

Retinal Function in RCS Rats Treated with TES
Representative ERGs recorded from RCS rats at 3, 5, and 7 weeks of ages are shown in Figure 5 . In the eye of the RCS rat at 3 weeks of age, the b-wave reached the criterion amplitude at an intensity of –1.9 log cd/m2, and the a-wave was first detected at 1.1 log cd/m2 (Fig. 5A) . In 5-week-old RCS rats with sham stimulation, a negative response dominated the ERG over the whole intensity range, and a b-wave did not appear until nearly the maximum stimulus intensity (Fig. 5C) . In contrast, in the 5-week-old RCS rat treated with 100 µA TES, a b-wave appeared at –0.4 log cd/m2 and the amplitude increased with increasing stimulus intensities. However, the amplitude of the a-wave was reduced (Fig. 5B) .


Figure 5
View larger version (13K):
[in this window]
[in a new window]

  		FIGURE 5. Effect of TES on the ERGs. Typical ERG responses elicited by different stimulus intensities from TES-treated and sham-stimulated eyes of RCS rats at 5 and 7 weeks of age. ERGs were recorded from both eyes simultaneously. ERGs from a 3-week-old RCS control rat (A), a 5-week-old RCS rat after TES (B) and sham stimulation (C), and a 7-week-old RCS rat after TES (D) and sham stimulation (E).

 
Although 50 µA TES also delayed the decrease of retinal function, the effect of 100 µA was more consistent with less variation than that of TES at 50 µA (data not shown).

The b-waves were used to assess retinal function of each RCS animal at 5 weeks of age because of the absence of an a-wave in the sham-stimulated animals. There was a 1.23-log-unit difference in the mean threshold of the b-wave between TES-treated eyes and sham-stimulated eyes (TES: –0.07 ± 0.50 log cd/m2; sham: 1.16 ± 0.58 log cd/m2; mean ± SEM, n = 6, t-test; P < 0.001).

The intensity–response function curve for the b-wave in TES-treated eyes was shifted to the right by approximately 0.9 log units compared with the eyes at 3 weeks of age, whereas the curve from the eyes treated with sham stimulation was shifted to the right by 2.5 log units (Fig. 6A) .


Figure 6
View larger version (9K):
[in this window]
[in a new window]

  		FIGURE 6. Intensity–response curves of the ERGs. (A) Average b-wave amplitudes versus stimulus intensity from TES-treated ({circ}) and sham-stimulated (•) retinas of 5-week-old and untreated 3-week-old ({triangleup}) RCS rats on a log–log scale. (B) Average STR-like negative wave amplitudes versus stimulus intensity from TES-treated ({circ}) and sham-stimulated retinas (•) in 7-week-old RCS rats on a log–log scale. Error bars, SEM. Time course of the decrease of retinal function. (C) Change in mean thresholds for STR with age in retinas with TES ({circ}) or with sham stimulation (•). Error bars, SEM. TES preserved by 7 weeks of age against retinal degeneration (t-test; {dagger}P = 0.027; Mann-Whitney rank sum test; {ddagger}P < 0.001; #unrecordable).

 
We also compared the mean amplitude of the b-waves elicited by the maximum intensity (2.1 log cd/m2) from the TES-treated eyes with that from the sham-stimulated eyes at 5 weeks of age. There was a significant difference between them (TES: 47.1 ± 9.90 µV; sham: 19.2 ± 6.71 µV, mean ± SEM; n = 6 each, Mann-Whitney rank sum test; P = 0.038).

At 7 weeks of age, the retinal function of RCS rat was more depressed, the b-wave was not present, and a negative response called the "STR-like negative response" dominated the ERG over the whole intensity range. Therefore, the STR-like negative response was used to determine the neuroprotective effect of TES on the eye of 7-week-old RCS rats. In the eyes treated with sham stimulation, the STR reached the criterion amplitude at 0.1 log cd/m2 (Fig. 5E) . In the eye treated with TES, the STR appeared at –0.9 log cd/m2, and the amplitude of the STR-like negative response increased with increasing stimulus intensities (Fig. 5D) . There was a 0.88-log-unit difference in the mean threshold for the STR between the TES-treated eyes and sham-stimulated eyes (TES: –0.45 ± 0.46 log cd/m2; sham: 0.43 ± 0.50 log cd/m2; mean ± SEM; n = 6 each; Mann-Whitney rank sum test; P = 0.027).

In the eyes with sham stimulation, the intensity–response curve for the negative wave was shifted to the right by approximately 0.8 log units compared with the eyes with TES (Fig. 6B) . The mean amplitude of the negative response at the maximum intensity in the TES-treated eyes was significantly larger than that in the sham-stimulated eyes (TES: 25.1 ± 2.79 µV; sham: 11.9 ± 3.00 µV, mean ± SEM; t-test; P < 0.001).

We also recorded ERGs from 9-week-old RCS rats after TES or sham stimulation, but the responses from these animals were too weak to be assessed (data not shown).

Because STRs were recordable from 3- to 7-week-old RCS rats, we used the thresholds of the STR to follow the time course of loss of retinal function. The threshold for the STR as a function of age is plotted in Figure 6C . In RCS rats at 3 weeks of age, the mean threshold of STR was –2.21 ± 0.13 log cd/m2 (mean ± SEM). Although the threshold of STR increased with age, the mean threshold in the retina at 5 weeks of age with TES was significantly lower than that in the retina with sham stimulation (TES: –1.48 ± 0.23 log cd/m2; sham: –0.65 ± 0.21 log cd/m2, mean ± SEM; t-test; P = 0.027). However, as shown in Figure 6C , TES delayed the decrease of retinal function by 7 weeks of age, but could not preserve the retinal function at 9 weeks.


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Our morphologic and electrophysiological analyses showed that 100 µA of TES prolonged the survival of photoreceptors and retinal function against the inherited photoreceptor degeneration of RCS rats. Our present study is, as best as we know, the first report to show that noninvasive electrical stimulation which only electrically stimulates the retina by using contact lens electrode, has a neuroprotective effect on the photoreceptors both functionally and anatomically, although Pardue et al.21 had reported that electrical stimulation by using subretinally implanted electrodes had a neuroprotective effect on photoreceptor degeneration in RCS rat.

Preservation of Retinal Morphology by TES
It has been reported that intravitreal injection of neurotrophic factors,14 15 22 23 24 neuroprotective genes,16 17 25 or transplantation of cells such as RPE cells,18 26 27 have a strong neuroprotective effects on photoreceptors in animal models of RP. However, the survival of the photoreceptors was limited to the area of the injected site. Limited and localized protection of photoreceptors in the retina of RCS rat has also been demonstrated with mechanical injury alone22 28 and by laser burns.29

For TES, the neuroprotective effect on the photoreceptors extended over the entire retina (Figs. 2 4) . This suggests that with our stimulating protocol, the electrical current may spread over the entire retina to exert neuroprotection on the entire retina. This neuroprotective effect was similar to the neuroprotective effect induced by light stress, which also has a neuroprotective effect on the photoreceptors over the whole retina.19 30

We also measured the thickness of the INL to determine whether TES also has a neuroprotective effect on the inner retinal cells. Unlike the significant effects on the photoreceptors, the inner retina appeared less affected by TES, although we have demonstrated that TES enhances the survival of axotomized RGCs in vivo.12 In this study, the mean thickness of INL in the retinas of 5-, 7-, and 9-week-old RCS rats with TES or without TES were not significantly different, but they were thinner than that at 3 weeks. Although our data on mean INL thickness were similar to those presented by Pardue et al.,21 there are no reports on the time course of thickness of the INL with age in pink-eyed RCS rats, and we did not determine why the INL was thinner.

Preservation of Retinal Function by TES
We used the b-wave and the STR-like negative response, which are not direct indicators of functioning photoreceptors, to evaluate retinal function. In RCS rats, the a-wave was detected only at relatively high intensities and became unrecordable in the early stage of retinal degeneration. Therefore the a-wave was not suitable for evaluating the efficacy of the TES because of the advanced photoreceptor degeneration.31 Sugawara et al.32 have demonstrated that the b-wave threshold and amplitude can be reliably used to track photoreceptors cell loss due to the damaging effects of constant light. Because STR has been shown to be a prominent component of the ERG in the RCS rat retina with advanced photoreceptor degeneration, STR is more useful than the b-wave in detecting loss of light sensitivity in advanced degenerated RCS rats.31 We therefore used the STR-like negative responses to evaluate the retinal function in RCS rats at 7 weeks of age. The STR threshold was also used to track the time course of the loss of retinal function in the advanced degenerated retina.

The amplitudes of the b-waves (5-week-old) or STR-like negative responses (7-week-old) were significantly larger in TES-treated eyes than in sham-stimulated eyes of RCS rats (Fig. 5) . These functional results were consistent with the histologic results obtained by measuring the thickness of the ONL (Fig. 4) .

However, at the age of 9 weeks, there was no significant difference in the amplitudes of the b-waves of TES-treated and sham-stimulated animals. These results are in agreement with the rapid decrease of ONL thickness from 7 to 9 weeks of age, although the mean thickness of ONL was still significantly thicker in the TES-treated eyes at 9 weeks. These findings demonstrated the limitation of TES for the treatment of the retina with a rapid course of photoreceptor degeneration. In humans with RP, the degeneration is relatively slow, and so other RP models such as RPE65-deficient mouse33 may be more suitable to investigate the effects of TES for slowly progressing photoreceptor degeneration.

Possible Mechanism of TES-Induced Neuroprotection of Photoreceptors
We have demonstrated earlier that TES induces a significant upregulation of endogenous IGF-1, which is produced by Müller cells.12 We also analyzed TES-induced upregulation of the mRNAs of various neurotrophic factors (e.g., IGF-1, bFGF, CNTF, NT-3, NT-4/5, GDNF, and BDNF) in the RCS rat retinas treated with TES, using real-time PCR. We also found that the mRNA of IGF-1 was also upregulated in RCS rats (Morimoto et al., unpublished data, February 2005). However, IGF-1 was reported to have a weak neuroprotective effect on the survival of photoreceptors in light-damaged retinas,15 and thus, IGF-1 may not be the key molecule that is neuroprotective of photoreceptors.

The neuroprotective effect of TES on the photoreceptors was dependent on the intensity of the electrical current (Fig. 2A) . This suggests that the increase of electrical activity of the photoreceptors exerts neuroprotective effect on photoreceptors. We did observe that TES of 100 µA at 1 ms/phase evoked electrical responses in the superior colliculus (data not shown). We could not determine whether TES activated photoreceptors, inner retinal neurons, or Müller cells to induce the rescue effect.

A subretinal implant of an artificial retina has been shown to stimulate the retina electrically and has a neuroprotective effect on the photoreceptors of the RCS rats21 and the retinal function in patients.34 The subretinal implantation of an retinal prosthesis has two possible mechanisms for its neuroprotective effects: one is due to the foreign body effect of the subretinal implant, and the second is the effect of subretinal electrical stimulation (SES).21 Pardue et al.21 showed that SES provided better preservation of retinal function than that of an inactive subretinal implant. They implied that SES had a neuroprotective effect on photoreceptors. Although the strength and stimuli of SES by the artificial retina are different from those of TES and the extent of invasion of the retina is different, both SES and TES have neuroprotective effects on photoreceptors.

All evidence considered, electrical stimulation may directly or indirectly alter the electrical activity or electrical charge balance of photoreceptors and exert a neuroprotective effect on the photoreceptors. Additional experiments are needed to determine the mechanism of TES-induced neuroprotection for photoreceptors.

TES as a Potential Clinical Technique
Our findings indicate that electrical stimulation can delay the photoreceptor loss in eyes with inherited photoreceptor degeneration. We applied TES once a week for 6 weeks to delay the retinal degeneration, and ocular side effects, such as cataracts, were not observed. In RCS rats, a mutation of the gene coding for the receptor tyrosine kinase gene Mertk has been identified.35 The same gene mutation has been identified in patients with autosomal recessive retinitis pigmentosa,36 indicating that the RCS rat is a counterpart of one type of human RP. Although photoreceptor loss was not eventually prevented against the rapid photoreceptor degeneration, the protective effect of TES on photoreceptor degeneration in RCS rats suggests that TES could delay the progression of some forms of RP. Additional studies are needed to determine the optimal electrical stimulus parameters to obtain photoreceptor survival. We must also apply TES to other animals with different genetic mutations to evaluate the neuroprotective effects before consideration of TES for clinical use.

In summary, TES prolonged the survival of photoreceptors and delayed the loss of retinal function in RCS rats. Although the neuroprotective effect of TES in treatment of the retina with photoreceptor degeneration was limited, the present results open the possibility that TES could be used to delay the photoreceptor degeneration in patients with inherited retinal degeneration such as RP.


   Acknowledgements
 
The authors thank Yozo Miyake and Mineo Kondo for helpful advice.


   Footnotes
 
Supported by Health Sciences Research Grant from the Ministry of Health, Labor and Welfare, Japan (YT, YF). TMo was supported by the JSPS Research Fellowship for Young Scientists.

Submitted for publication November 23, 2006; revised March 22 and May 21, 2007; accepted August 17, 2007.

Disclosure: T. Morimoto, None; T. Fujikado, None; J.-S. Choi, None; H. Kanda, None; T. Miyoshi, None; Y. Fukuda, None; Y. Tano, 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.

Corresponding author: Takashi Fujikado, Department of Applied Visual Science, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita City, Osaka 565-0871, Japan; fujikado{at}ophthal.med.osaka-u.ac.jp.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Galli-Resta L, Ensini M, Fusco E, Gravina A, Margheritti B. Afferent spontaneous electrical activity promotes the survival of target cells in the developing retinotectal system of the rat. J Neurosci. 1993;13:243–250.[Abstract]
  2. Linden R. The survival of developing neurons: a review of afferent control. Neuroscience. 1994;58:671–682.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  3. Meyer-Franke A, Kaplan MR, Pfrieger FW, Barres BA. Characterization of the signaling interactions that promote the survival and growth of developing retinal ganglion cells in culture. Neuron. 1995;15:805–819.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  4. Meyer-Franke A, Wilkinson GA, Kruttgen A, et al. Depolarization and cAMP elevation rapidly recruit TrkB to the plasma membrane of CNS neurons. Neuron. 1998;21:681–693.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  5. Shen S, Wiemelt AP, McMorris FA, Barres BA. Retinal ganglion cells lose trophic responsiveness after axotomy. Neuron. 1999;23:285–295.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  6. Lousteau RJ. Increased spiral ganglion cell survival in electrically stimulated, deafened guinea pig cochleae. Laryngoscope. 1987;97:836–842.[Web of Science][Medline][Order article via Infotrieve]
  7. Leake PA, Hradek GT, Snyder RL. Chronic electrical stimulation by a cochlear implant promotes survival of spiral ganglion neurons after neonatal deafness. J Comp Neurol. 1999;412:543–562.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  8. Miller JM, Altschuler RA. Effectiveness of different electrical stimulation conditions in preservation of spiral ganglion cells following deafness. Ann Otol Rhinol Laryngol Suppl. 1995;166:57–60.[Medline][Order article via Infotrieve]
  9. Al-Majed AA, Brushart TM, Gordon T. Electrical stimulation accelerates and increases expression of BDNF and trkB mRNA in regenerating rat femoral motoneurons. Eur J Neurosci. 2000;12:4381–4390.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  10. Al-Majed AA, Neumann CM, Brushart TM, Gordon T. Brief electrical stimulation promotes the speed and accuracy of motor axonal regeneration. J Neurosci. 2000;20:2602–2608.[Abstract/Free Full Text]
  11. Morimoto T, Miyoshi T, Fujikado T, Tano Y, Fukuda Y. Electrical stimulation enhances the survival of axotomized retinal ganglion cells in vivo. Neuroreport. 2002;13:227–230.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  12. Morimoto T, Miyoshi T, Matsuda S, Tano Y, Fujikado T, Fukuda Y. Transcorneal electrical stimulation rescues axotomized retinal ganglion cells by activating endogenous retinal IGF-1 system. Invest Ophthalmol Vis Sci. 2005;46:2147–2155.[Abstract/Free Full Text]
  13. Fujikado T, Morimoto T, Matsushita K, Shimojo H, Okawa Y, Tano Y. Effect of transcorneal electrical stimulation in patients with nonarteritic ischemic optic neuropathy or traumatic optic neuropathy. Jpn J Ophthalmol. 2006;50:266–273.[CrossRef][Medline][Order article via Infotrieve]
  14. Faktorovich EG, Steinberg RH, Yasumura D, et al. Photoreceptor degeneration in inherited retinal dystrophy delayed by basic fibroblast growth factor. Nature. 1990;347:83–86.[CrossRef][Medline][Order article via Infotrieve]
  15. LaVail MM, Unoki K, Yasumura D, et al. Multiple growth factors, cytokines, and neurotrophins rescue photoreceptors from the damaging effects of constant light. Proc Natl Acad Sci USA. 1992;89:11249–11253.[Abstract/Free Full Text]
  16. Cayouette M, Behn D, Sendtner M, et al. Intraocular gene transfer of ciliary neurotrophic factor prevents death and increases responsiveness of rod photoreceptors in the retinal degeneration slow mouse. J Neurosci. 1998;18:9282–9293.[Abstract/Free Full Text]
  17. Akimoto M, Miyatake S, Kogishi J, et al. Adenovirally expressed basic fibroblast growth factor rescues photoreceptor cells in RCS rats. Invest Ophthalmol Vis Sci. 1999;40:273–279.[Abstract/Free Full Text]
  18. Litchfield TM, Whiteley SJ, Lund RD. Transplantation of retinal pigment epithelial, photoreceptor and other cells as treatment for retinal degeneration. Exp Eye Res. 1997;64:655–666.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  19. Liu C, Peng M, Laties AM, Wen R. Preconditioning with bright light evokes a protective response against light damage in the rat retina. J Neurosci. 1998;18:1337–1344.[Abstract/Free Full Text]
  20. LaVail MM. Legacy of the RCS rat: impact of a seminal study on retinal cell biology and retinal degenerative diseases. Prog Brain Res. 2001;131:617–627.[Medline][Order article via Infotrieve]
  21. Pardue MT, Phillips MJ, Yin H, et al. Neuroprotective effect of subretinal implants in the RCS rat. Invest Ophthalmol Vis Sci. 2005;46:674–682.[Abstract/Free Full Text]
  22. Faktorovich EG, Steinberg RH, Yasumura D, et al. Basic fibroblast growth factor and local injury protect photoreceptors from light damage in the rat. J Neurosci. 1992;12:3554–3567.[Abstract]
  23. Machida S, Chaudhry P, Shinohara T, et al. Lens epithelium-derived growth factor promotes photoreceptor survival in light-damaged and RCS rats. Invest Ophthalmol Vis Sci. 2001;42:1087–1095.[Abstract/Free Full Text]
  24. Machida S, Tanaka M, Ishii T, et al. Neuroprotective effect of hepatocyte growth factor against photoreceptor degeneration in rats. Invest Ophthalmol Vis Sci. 2004;45:4174–4182.[Abstract/Free Full Text]
  25. Gauthier R, Joly S, Pernet V, et al. Brain-derived neurotrophic factor gene delivery to Müller glia preserves structure and function of light-damaged photoreceptors. Invest Ophthalmol Vis Sci. 2005;46:3383–3392.[Abstract/Free Full Text]
  26. Jiang LQ, Hamasaki D. Corneal electroretinographic function rescued by normal retinal pigment epithelial grafts in retinal degenerative Royal College of Surgeons rats. Invest Ophthalmol Vis Sci. 1994;35:4300–4309.[Abstract/Free Full Text]
  27. Lawrence JM, Sauve Y, Keegan DJ, et al. Schwann cell grafting into the retina of the dystrophic RCS rat limits functional deterioration: Royal College of Surgeons. Invest Ophthalmol Vis Sci. 2000;41:518–528.[Abstract/Free Full Text]
  28. Wen R, Song Y, Cheng T, et al. Injury-induced upregulation of bFGF and CNTF mRNAs in the rat retina. J Neurosci. 1995;15:7377–7385.[Abstract]
  29. Xiao M, Sastry SM, Li ZY, et al. Effects of retinal laser photocoagulation on photoreceptor basic fibroblast growth factor and survival. Invest Ophthalmol Vis Sci. 1998;39:618–630.[Abstract/Free Full Text]
  30. Nir I, Liu C, Wen R. Light treatment enhances photoreceptor survival in dystrophic retinas of Royal College of Surgeons rats. Invest Ophthalmol Vis Sci. 1999;40:2383–2390.[Abstract/Free Full Text]
  31. Bush RA, Hawks KW, Sieving PA. Preservation of inner retinal responses in the aged Royal College of Surgeons rat: evidence against glutamate excitotoxicity in photoreceptor degeneration. Invest Ophthalmol Vis Sci. 1995;36:2054–2062.[Abstract/Free Full Text]
  32. Sugawara T, Sieving PA, Bush RA. Quantitative relationship of the scotopic and photopic ERG to photoreceptor cell loss in light damaged rats. Exp Eye Res. 2000;70:693–705.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  33. Redmond TM, Yu S, Lee E, et al. Rpe65 is necessary for production of 11-cis-vitamin A in the retinal visual cycle. Nat Genet. 1998;20:344–351.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  34. Chow AY, Chow VY, Packo KH, Pollack JS, Peyman GA, Schuchard R. The artificial silicon retina microchip for the treatment of vision loss from retinitis pigmentosa. Arch Ophthalmol. 2004;122:460–469.[Abstract/Free Full Text]
  35. D’Cruz PM, Yasumura D, Weir J, et al. Mutation of the receptor tyrosine kinase gene Mertk in the retinal dystrophic RCS rat. Hum Mol Genet. 2000;9:645–651.[Abstract/Free Full Text]
  36. Gal A, Li Y, Thompson DA, et al. Mutations in MERTK, the human orthologue of the RCS rat retinal dystrophy gene, cause retinitis pigmentosa. Nat Genet. 2000;26:270–271.[CrossRef][Web of Science][Medline][Order article via Infotrieve]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Morimoto, T.
Right arrow Articles by Tano, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Morimoto, T.
Right arrow Articles by Tano, Y.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS