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1 From the Wilmer Ophthalmological Institute and 2 Wilmer Biostatistics Center, The Johns Hopkins Medical Institutions, Baltimore, Maryland.
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Abstract |
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METHODS. At postnatal day (P)13 C3H/HeJ (rd/rd) retinal degenerate mice received a subretinal transplant, in one eye only, of neural retinal tissue isolated from newborn normal C57/BL6J mice. Between 33 and 35 days after transplantation, local electroretinograms (ERGs) and ganglion cell responses were recorded directly from the retinal surface using a differential bipolar surface electrode. Measurements were performed both with and without light stimulation. Similar recordings were also performed in age-matched eyes subjected to sham transplantation, in control eyes that were not subjected to surgery, and in animals eyes that underwent transplantation at 8 weeks of age. After the recordings, the eyes were processed for light and transmission electron microscopy.
RESULTS. Three of 10 mice showed bursts of ganglion cell action potentials (ON response only) as well as recordable intraocular ERGs over the transplant in response to 1-second and 200-msec light stimuli. Light-driven ganglion cell responses could not be recorded in areas outside the transplant in all transplant-recipient eyes, age-matched control eyes, and sham-transplantation eyes. Light responses also could not be recorded in animal eyes that received transplants at an older age (8 weeks). Electron microscopic examination confirmed the presence of photoreceptor outer segments in the areas affected by transplantation.
CONCLUSIONS. This study demonstrates the presence of light-driven ganglion cell responses after subretinal transplantation in a retinal degenerate model. This finding may reflect functional integration of the transplant with the host, but a rescue effect on remaining host photoreceptors cannot be ruled out. The findings suggest, however, that modification of host parameters, such as host age, may be important approaches for improving the functional success of retinal transplantation.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A key requirement for successful retinal transplantation is that the transplanted retinal tissue integrate functionally with the remaining host retina. For this reason, the functional capabilities and functional integration of neural retinal transplants have been the focus of study for a number of research groups. Adolph et al.13 detected electrophysiological function in retinal transplants that were isolated from the host retina and noted that retinal transplants could respond to light, even when they were organized as rosettes. In addition, in well-developed retinal transplants, Seiler et al.10 observed light-dependent distributional changes of proteins involved in phototransduction. Moreover, a number of histologic studies have suggested the presence of possible synaptic connections between the host retina and transplanted retinal tissue.6 7 8 9 10 11 12 13 14 15 16 17 Despite this morphologic evidence, the presence of functional integration has been difficult to establish. Del Cerro et al.18 19 used the suppressive effect of a warning light flash on the startle reflex response to acoustic stimuli in light-blinded 344 Fisher rats to investigate functional integration after retinal transplantation. They observed partial restoration of this visually mediated behavior after retinal transplantation,18 but no recovery after sham injection or injection of retinal cell homogenates or cerebellar cells.19 In addition, Kwan et al.20 observed a restoration of dark preference behavior at low luminance levels after transplantation in the rd mouse. Interpretation of behavioral changes in animals, however, can be very difficult and imprecise. In the light-blinded Fisher rat, Silverman et al.21 found some recovery of the pupillary reflex and visually evoked potentials (VEPs) after neural retinal transplantation. The pupillary reflex, however, is not a good predictor for retinal recovery, because there is no correlation between the size of the remaining photoreceptor population and the extent of pupillary constriction.9 Although a VEP is a more objective measure, detection of a full-field flash VEP does not establish that the observed response is coming from the precise area of the transplantation.
To enhance the possibility of synaptic integration between the host retina and the transplant, attempts have been made to modify the transplant by using fetal neural retinal tissue with the presumed advantages of greater synaptic plasticity and less immunogenicity.22 23 In addition, some investigators have attempted to remove the inner retinal layers of the transplant with a vibratome,24 in hope of reducing a potential anatomic barrier to integration.
Despite the considerable attention directed to modification of the transplant, host characteristics have not been well studied. Gouras et al.6 were the first to suggest that a glial barrier derived from remaining host Müller cells may hinder the formation of connections between the graft and host retinal cells. Another explanation for the absence of functional integration, however, may be a reduced ability of mature host retinal neurons to form new synapses. The goal of our work was to determine whether the functional success of retinal transplantation can be enhanced by the use of a less mature or younger host retina. In this study, we report the presence of light-driven retinal ganglion cell (RGC) responses after transplantation of normal newborn mouse retina into young (13-day-old) retinal degenerate (rd) mice.
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Materials and Methods |
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Recording of In Vivo RGC Responses and ERGs
RGC and electroretinogram (ERG) recordings13
were
performed 33 to 35 days after retinal transplantation. At this age
(P46–P48), in a non–transplant-recipient rd mouse, the rod
photoreceptor degeneration is essentially complete. Animals were dark
adapted for at least 4 hours and then anesthetized with the same
medications and dosages used for transplantation. A 30-gauge needle was
used to make a small incision into the cornea just anterior to the
limbus. Sodium hyaluronate (Healon GV; Pharmacia & Upjohn, Columbus,
OH) was injected into the anterior chamber through the incision site to
maintain intraocular pressure. The cornea was excised, and an
intracapsular lensectomy was performed. The retinal surface was exposed
for electrophysiological testing by dissecting the vitreous with sodium
hyaluronate. All these preparative procedures were performed under dim
red light illumination. Body temperature was maintained at 35°C with
a feedback-controlled heating pad.
For differential RGC recordings, the tips (separated by 200 µm) of two 125-µm diameter paralyene-coated tungsten electrodes (A-M systems, Everett, WA) were positioned on the retinal surface using a microscope and a micromanipulator (MX-100; Newport; Irvine, CA). The ground electrode was placed in the animal’s mouth. A halogen light source was used as the light stimulus, and the eyes were directly illuminated (full-field illumination; intensity: 5 x 105 cd/m2) using a fiber optic (6-mm diameter) positioned at a distance of 1.0 to 1.5 cm from the retina. The onset of the light stimulus was at 0.1 second of the recording. One-second and 200-msec light stimuli were used for the recordings. Ten animals each were used for RGC recordings in the four experimental groups: the transplant host retinal areas (three recording locations) of transplant-recipient eyes posterior from the visible injection site; the areas uninvolved in the transplantation (three recording locations) of these eyes on the side opposite the transplant (at 6 o’clock); the corresponding areas (12 [two locations] and 6 o’clock [one location]) of control and sham-transplantation eyes. In each group, a total of three separate locations within a given experimental host retinal area were sampled.
Ten cycles (1 cycle = 1.6 seconds) of RGC responses were recorded at each location with the light turned either on or off (shutter sound only). Light-driven RGC responses were recorded from a normal B57/Bl6 mouse before each experimental session to confirm that all recording equipment was functioning properly. The examiners and the electrophysiologist were not masked to the transplant location or the treatment group assignment. Transplants were always performed at the same location, and it was usually easy to identify the transplant location during microscopic inspection while positioning the electrodes for the recordings.
The output of the recording electrodes was fed into a differential AC preamplifier (band-pass filter, 300-2000 Hz; gain, 10-fold; EX - 1; Dagan, Minneapolis, MN) that was connected to another AC amplifier (gain, 2000-fold; AM502; Tektronix, Beaverton, OR). Total gain was 20,000-fold. For ERG recordings, an identical setup was used, but with the band-pass filter of the amplifiers set at 0.3 to 300 Hz. A speaker and an oscilloscope were connected to the system to allow both audio and video monitoring of the recording conditions and the RGC responses. The output of the amplifier was fed into a computer (466 MHz; Pentium II microprocessor; Intel, Mountain View, CA). Data were acquired synchronously to the shutter action with a custom-written software program (LabView National Instruments, Austin, TX) and analyzed using another program (MatLab; (MathWorks, Natick, MA). Thresholds were then set manually for each file. For the construction of peristimulus time histograms (PSTHs), spikes were counted only if the amplitude was at least twice the amplitude of baseline. When a light response could be recognized with the speaker and recorded on the computer, it was verified by at least 10 consecutive recordings, either with the shutter action only (light turned off) or light stimulation.
For statistical analysis, the spike counts from 10 consecutive recordings were summed, transformed into logs, and then summed across the three locations for each experimental area (transplant-recipient eyes had six recording locations, three for the transplant area and three for the nontransplant area). Thus, after log transformation and summing, for the purpose of statistical comparisons, 10 animal eyes were used for each experimental group and area. All comparisons of spike counts between groups were made using the two-tailed unpaired t-test, with the exception of the comparison between nontransplant and transplant areas (spontaneous activity) of the same eye, for which the two-tailed paired t-test was used. Using a Bonferroni adjustment for multiple comparisons, P < 0.0083 was considered statistically significant (testing the hypothesis that there was a difference in number of spikes recorded during the stimulus interval among the different experimental groups).
For histologic investigations, animals were killed with an intraperitoneal injection of pentobarbital (200 mg/kg). The eyes were then enucleated and immersion fixed with 3% glutaraldehyde in 0.1% 1 M phosphate buffer for 24 hours. For light and electron microscopy, specimens were postfixed in 1% veronal acetate-buffered osmium tetroxide, dehydrated in a series of graded ethanols, and embedded in plastic. For transmission electron microscopy, ultrathin sections were stained with uranyl acetate and lead citrate.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PSTHs representing the average spike count per location (Figs. 6A 6B 6C 6D) in the four control groups were calculated as an average of 300 recordings. For each experimental group, recordings were obtained from 10 eyes, with a total of 30 recordings obtained from three retinal locations (10 recordings per location). The same eyes were used for the transplant area and the nontransplant area of a transplant-recipient group. In the transplant locations (Fig. 6A) , the spontaneous activity (recorded in the shutter-only mode with the light source turned off) led to an average RGC spike count of 107 ± 118.9 (log units: 5.17 ± 1.57) spikes per location (spk/loc) ± SD This spontaneous activity was significantly higher (P < 0.004) than the activity observed in the nonsurgical control eyes (Fig. 6C ; 13.9 ± 8 spk/loc [log units: 3.22 ± 0.62]), but was not significantly higher (P = 0.15), although there was a trend, than in the nontransplant locations of the same transplant-recipient eyes (42.9 ± 38.9 spk/loc [log units: 4.35 ± 1.14]; Fig. 6B ). In sham-transplantation eyes (Fig. 6D) , the RGC activity (111.5 ± 119.3 [log units: 5.36 ± 1.32]) did not significantly differ from the spontaneous activity of the transplant areas of the transplant-recipient eyes (P = 0.78). Animal eyes subjected to transplantation at 8 weeks of age showed a similar level of spontaneous activity in the transplant areas as sham-transplantation eyes, but no light-driven responses.26
Local light-driven ERGs were also recorded from the host retinal surface over the light-responsive locations determined by the RGC recordings. A 1-second light stimulus was used. The appearance of a retinal surface ERG is very different from a corneal ERG, with a negative deflection appearing as the predominant component.13 The negative deflection amplitude of the ERG waveform from light-responsive locations was 90 µV (Fig. 7A ), 69% of the amplitude (130 µV) observed in a normal C57/Bl6 mouse (Fig. 7B) recorded at the beginning of the session the same day. Local retinal surface ERGs were not recordable (i.e., were flat) from the transplant-recipient eyes that did not show light-driven RGC activity. Local ERGs also were not recordable from nontransplant areas of all transplant-recipient eyes, nor from any of the other control groups.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Most neuronal cells in the retina, such as the photoreceptors, bipolar cells, and horizontal cells, show graded responses. Only RGCs and some amacrine cells are known to show spike responses (action potentials).29 With our recording technique, the differential bipolar retinal surface electrodes are carefully advanced with a micromanipulator, until the electrodes just contact the retinal surface. Both visual and audio cues are used to monitor the electrode approach. With this method, unintentional penetration of the retinal surface is nearly impossible. Without retinal penetration, we have not been able to record responses from deeper retinal layers. Thus, it is unlikely that the spike responses are from the deeper amacrine cells and are more likely a manifestation of RGC activity.
In this study, we demonstrated the presence of light-driven RGC responses (ON response only) over the graft 5 weeks after transplantation of normal newborn retinal tissue into the subretinal space of 3 of 10 rd mice aged 13 days. A light response could not be elicited in any control eye or nontransplant area. In addition, we could not elicit light responses from rd mice that received transplants at 8 weeks of age or older.26 A reason for this apparent age-dependent effect may be the higher synaptic plasticity of the developing retinal tissue in younger mice. After transplantation of fetal rat retinal tissue over newborn rat superior colliculi, McLoon et al.30 demonstrated survival of the retinal transplants and found histochemical evidence for synaptic connections between graft and host tissue. Furthermore, Lund and Coffey31 performed functional experiments on newborn rats that received, from day 12 to 15 embryos, neural retinal transplants that were placed over their midbrains. Although the retinal transplants were organized as rosettes, the transplanted tissue made preferential axonal innervations into visual centers (i.e., the superior colliculus), and photic stimulation of the transplanted tissue caused reproducible pupillary constriction responses. In addition, learned behaviors were modified by photic inputs received through the transplanted tissue. Thus, by using fetal retinal tissue and a young host, Lund and Coffey demonstrated functional connections between graft and host neural tissue.
Fetal retinal tissue has been suggested as a suitable source of donor tissue because of its presumed greater plasticity and lower antigenicity compared with adult retinal tissue.22 23 Despite these apparent advantages, functional integration of fetal retinal tissue transplanted into the subretinal space has not been demonstrated. In Lund and Coffey31 and in our work, a young host animal was also used. In both studies, the findings suggest that functional connections may have developed between the host and the graft. This observation is consistent with our hypothesis that a younger host retina containing less well-differentiated cells may be more receptive to synaptic contacts, thereby enhancing functional integration with the transplant.
There are other possible explanations, however, for the apparent preservation of RGC responses in some of the transplant-recipient rd mice in our study. Mohand–Said et al.32 demonstrated an apparent remote protective effect of the transplant on the host retina that leads to survival of remaining photoreceptor cells. They found increased survival of cones in the central quadrants after transplantation in the mid- and far periphery. In our experiments there were still a few cone nuclei remaining in both age-matched control animals and in the 7-week-old transplant recipients. There was no difference, however, in the number of cone nuclei between control eyes and the nontransplant areas of the transplant-recipient eyes. Because the donor tissue was not labeled, it was not possible to reliably distinguish between host and transplant cones. Consequently, it was not possible to compare the number of host cones within and outside the transplant area. Nonetheless, no photoreceptor outer segments could be identified in the control eyes or the nontransplant areas of the transplant-recipient eyes. Indeed, photoreceptor outer segments have never been reported in rd mice of this age.33 34 In the light-responsive transplant areas, however, we found photoreceptor outer segments that showed a well-developed stacked-disc configuration (Fig. 2C) . No outer segments were found outside the transplant area, and no light response was elicited from any nontransplant region. Thus, if photoreceptor rescue was responsible for preservation of light responsiveness in these transplant-recipient eyes, the effect was highly localized and regional, in contrast to the remote effect reported by Mohand–Said et al.32
It is also possible that residual host cone cells without outer segments contributed to the light response that was observed. The rd mouse harbors a mutation in the gene (rd/rd) that encodes the rod cGMP phosphodiesterase.35 The retinal degeneration starts at P10, with near total loss of the rods by P21. For unknown reasons, the cones are also severely affected as well and degenerate subsequently.36 Corneal ERGs cannot be recorded after 4 weeks of age.35 Although the remaining cones do not have outer segments after P21, Dräger37 was able to record a light-driven decrease in superior colliculus activity for several weeks after birth. These responses were only detectable after stimulation of a peripheral ring of retina, and a dense scotoma was noted centrally. The physiologic mechanism or origin of these responses, however, is unclear. One hypothesis is that because the outer retinal degeneration progresses centrifugally (i.e., from the center to the periphery), some rods or cones may remain near the ora serrata. We have never found, however, any histologic evidence for photoreceptor outer segments, even in the retinal periphery. In addition, the strong bursts of light-driven RGC activity could only be recorded over the transplant areas, which were located posterior to the equator.
Another explanation for the preservation of ganglion cell responses may be an effect of the surgical procedure itself. An increase in spontaneous RGC activity was observed in both transplant-recipient and sham-transplantation animals. The cause for the increased spontaneous activity is uncertain, but it may have been due to some increased release of neurochemically active mediators as a result of the surgical manipulation. Only transplant-recipient eyes, however, exhibited light-dependent activity, and the magnitude (spike count) of the light response was markedly higher than the spontaneous activity. This finding suggests that the effect was due to the transplanted tissue itself.
Another possible explanation for the light-driven RGC responses is that they are derived from ganglion cells originating in the transplant. Although we have never histologically observed a ganglion cell layer in our transplants, it is possible that some ganglion cells develop. Our recording technique, however, appears to sample only highly localized responses. We are not able to record the graded responses of bipolar cells unless we penetrate deep into the retina. Thus, because we are recording from the retinal surface, we would not expect to record from the transplant ganglion cells unless they have migrated into the host ganglion cell layer. Future investigations using labeled transplanted tissue may help explore this possibility.
In summary, we report the preservation of light-driven RGC responses in 3 of 10 eyes of rd mice that received subretinal newborn retinal transplants at only 13 days of age. This effect was not observed in control animals or animals that received transplants at an older age. Although the effect may be due to factors other than functional integration, the finding suggests that modification of host parameters (such as host age in this case) may be important in improving the functional success of retinal transplantation. Further study of host parameters, including efforts designed to improve the efficacy of transplantation into older animals, appears warranted.
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Footnotes |
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Submitted for publication April 19, 2000; revised July 31 and November 21, 2000; accepted December 15, 2000.
Commercial relationships policy: N.
Corresponding author: Eugene de Juan, Jr, Department of Ophthalmology, Wilmer Ophthalmological Institute, Johns Hopkins Hospital, 721 Maumenee, 600 North Wolfe Street, Baltimore, MD 21287. edejuan{at}jhmi.edu
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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