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�:	Title: Neuroprotective Effect of Photoactive Quantum Dots in Progressive Retinal Photoreceptor Degeneration. 
Short Title:  Intravitreal Quantum Dots as neuroprotective agents.  
Authors: 
Jeffrey L. Olson, MD1.    					jeffrey.olson@ucdenver.edu
Raul Velez-Montoya, MD1.  				raul.velez-montoya@ucdenver.edu
Naresh Mandava, MD1.   				naresh.mandava@ucdenver.edu
Conrad R. Stoldt, PhD2.      				conrad.stoldt@ucdenver.edu

Affiliations: 
Department of Ophthalmology, University of Colorado Health and Science Center, Rocky Mountain Lions Eye Institute, University of Colorado School of Medicine, CO, USA. 
Department of Mechanical Engineering, University of Colorado Boulder, Boulder CO, USA.

Corresponding Author: Jeffrey L. Olson, MD. Address: 1675 Aurora Court, Aurora CO 80204. Phone: (720) 848.2500. Fax: (720) 848.5014. Email: jeffrey.olson@ucdenver.edu

A preliminary version of this study has never been presented as a paper or poster or submitted for publication. The authors (JO, NM & CS) have a patent application pending for the disclosed device.  There were no grants or funds allocated to the completion of this study. The authors state that they have full control of all primary data and they agree to allow Journal of Nanomaterials & Molecular Nanotechnology to review their data upon request.


Abstract:
Background: To determine the effect of intraocularly administered photoactive quantum dots in a rodent model of progressive photoreceptor degeneration.
Methods: The Royal College of Surgeons (RCS) rat model of progressive photoreceptor degeneration was used for this study.  Twenty eyes of ten animals were divided into four groups:  active implant, inactive implant, sham surgery, and control group.  The active and inactive implant group received a single intravitreal injection of photoactive quantum dots conjugated with biotin.  The sham surgery group received an intravitreal injection of balanced saline solution, and the control group underwent no surgical procedure.  All procedures were done at week six of life and weekly electroretinograms (ERG) recorded for the following six weeks.
Results: Both the control and sham surgery groups demonstrated a progressive decline in the amplitude of the ERG recordings over the six weeks post-procedure.  In contrast, the eyes receiving intravitreal photoactive quantum dots demonstrated transient, but statistically significant increases in retinal electrical activity post-injection.
Conclusions: The observed increase in retinal electrical activity following intravitreal injection of photoactive quantum dots in the RCS rat model suggests a potential therapeutic role for this technology in progressive retinal degenerations.


Key Words: Nanoparticles, Quantum dots, Electroretinogram, Intravitreal, Photoreceptor degeneration. 




Introduction: 
For millions of Americans suffering from retinal disease, each day brings a further diminution of their vision. Despite recent technological and pharmacologic advances in medicine, the number of people losing sight continues to increase, due in large part to blinding diseases such as macular degeneration, diabetic retinopathy, and hereditary retinal degenerations. Often the vision loss is irreversible � at least by conventional means.

Vision loss is one of the most feared disabilities, and is affecting more Americans than ever before. Alarmingly, the number of people with visual impairment in this country is expected to double over the next thirty year.[1, 2] Age-related macular degeneration (AMD), diabetic retinopathy and retinitis pigmentosa are the leading causes of blindness in the United States. An estimated 27 million Americans are affected by one of these conditions.[3] 

Loss of vision from retinal disorders generally results from degeneration or damage to retinal photoreceptors. Attempts to transplant the retina have met with limited success, largely due to the inherent intricacy of the biological wiring involved. There are over 120 million rods and 6 million cones, which upon exposure to light become hyperpolarized and initiate the process of phototransduction.  This signal then travels through a complex interconnection of bipolar and amacrine cells which process visual information before converging into one million ganglion cells and traveling to the central nervous system via the optic nerve. The retina, like most mammalian nervous tissue, has a limited capacity to regenerate once damaged. It can, however, respond to external or artificial stimulation.[4] 

Reversal of blinding disorders through the use of implantable retinal prosthetics is an area of intense investigation, and numerous groups around the world are actively involved in �artificial vision� projects. There are two basic approaches under investigation: implants which are placed on the surface of the retina (epiretinal) and implants beneath the retina (subretinal).[5, 6] Chip-based retinal prosthetics are a tissue-level therapy best suited to those patients who already have extensive loss of retinal photoreceptors and severe vision loss.  The goal of the chip is to replace the missing phototransduction capabilities of the retinal photoreceptors.

What is currently lacking is a method to treat retinal photoreceptors using electrical stimulation before they begin to degenerate.  That is, to prevent vision loss rather than restore it.  To stimulate the retina and upregulate neurotrophic growth factors on a cellular level, rather than a tissue level, is the goal of this study.

The technology described below avoids the problems encountered by other artificial vision projects through the use of photoactive semiconducting nanoparticles deployed on the cellular level, allowing less invasive methods for implantation and minimizing traumatic damage to the retina. These particles, known as quantum dots, are discrete nanometer-scale materials that have photoactive properties similar to bulk semiconductors such that when exposed to electromagnetic energy in the form of visible light, can convert light into an electrical impulse (photovoltaic response) or a different wavelength of light (photoluminescent response).[7] Further, quantum dots can be used in a variety of biological applications.[8-11] Researchers have used these properties to produce a quantum-dot-sensitized solar cell, wherein light activates the quantum dots causing them to release excited electrons.[12-14] Further, quantum dots can be engineered to be sensitive to energy in the infrared region, the visible spectrum, and even ultraviolet range through changes in size and composition.[15] In essence, the quantum dots behave like small solar cells, producing energy in response to light.[13]  

The goal of this study is to harness this photoactive property to deliver electrical stimulation to retinal cells in order to prevent or delay cell death. Using a quantum dot approach, there are no external components. Instead, the stimulus is placed directly at the retinal cells, the origin of the intricate interplay of neural wiring that forms the basis of vision, thereby utilizing several million years of neural evolution, rather than trying to circumvent or duplicate it. Thus, the focus of this study is the potential effect on retinal electrophysiology and anatomy through the surgical implantation of photoactive nanoparticles into the mammalian eye.

Materials and methods: 
The study was reviewed and approved by the University of Colorado Institutional Animal Care and Use Committee. All procedures were performed according to the statement for the use of animals in ophthalmic and visual research from the Association of Research of Vision and Ophthalmology (ARVO). The study was fully conducted at the University of Colorado facilities. There were no funds allocated for the realization of this study. 

Royal College of Surgeons (RCS) rats, a well established model of progressive retinal degeneration, were used for this study.  Originally obtained from Machelle Pardue (Atlanta VA Medical Center), a breeding colony was maintained as a homozygous breeding colony at the Center for Comparative Medicine at the University of Colorado Denver, Anschutz Medical Campus. The RCS rat undergoes a deterioration of the electrical activity in the retina from the third week of life onward, with no recordable activity by week twelve.[16, 17] All animals were housed on a 12 hour light/dark cycle with food and water ad libium. All procedures are done using rats 6 weeks of age with a range of 2 days older or younger to minimize variability and ensure that all rats were at the same chronological stage of degeneration.

A total of 20 eyes from 10 rats were used in the first round of testing.  The rats were divided into four groups to be studied: a control group with no intervention, a sham surgery group receiving intraocular saline injections, an inactive implant group (490 nm quantum dots), and the active implant group (540 nm quantum dots).  Those in the quantum dot group received either particles that absorb at 490 nm or 540nm, both of which were conjugated with biotin (Evident Technologies, Troy, NY).  These two different wavelengths were selected based on the lighting environment the animals would be contained in for the duration of the study.  The animal laboratory was lighted with fluorescent lighting; average luminance during the twelve hour light cycle was around 120 lux and colorimetry ranging from 520 to 660 nm.   As depicted in Figure 1, fluorescent lighting has an initial peak at around 540 nm and negligible intensity below 530 nm. Based on the excitation spectra provided by the manufacturer, the maximum excitatory wavelength for the inactive group (490 nm quantum dot) is around 500 nm, which is below the wavelengths to which the animals were exposed.  In contrast, the maximum excitatory wavelength for the active group (540 nm quantum dot) is about 550, which correlates with an intensity spike for the ambient lighting.  As such, the 490 nm quantum dots are outside the spectrum of light in the facility, and serve as inactive implants.  All animals had baseline electroretinogram (ERG) recordings done prior to any procedures, and then weekly thereafter for six weeks.  Any animals with traumatic crystalline lens damage, vitreous hemorrhage, or retinal detachment were excluded from analysis.

In the second round of testing, 12 eyes of 6 rats were used to determine the rate of quantum dot migration out of the eye after intravitreal injection.  After the injection, animals were euthanized at 1 hour, 6 hours, and days 1, 2, 7, and 14.

The active and inactive quantum dot implants are a colloidal semiconductor nanocrystal composed of a cadmium/selenium (Cd/Se) core covered with a thin outer shell layer of zinc sulfide. The particles are then conjugated with biocompatible proteins or molecules-- in this case with the water soluble B-complex vitamin biotin.  All intraocular injections were done at the fourth week of life, and the animals were monitored for the subsequent six weeks.  Prior to dosing, the animals were anesthetized using ketamine (80mg/kg) and xylazine (5-10 mg/kg) anesthesia and the right eye was prepped with betadine. The animals were monitored every 4-5 minutes while under anesthesia by observing any response to a toe or skin pinch. A colloidal suspension of the quantum dots was injected into the vitreous via pars plana of the study eye of each animal in the active implant group. Sham surgeries consisted of the same surgical procedure, except the eye was injected with sterile saline. The volume of injection for all groups was 5 �L of a 1 �M solution.  Control group animals had no intraocular procedures done.  The animals were monitored daily for the first five days following injection for any signs of infection or alteration in behavior.  All the animals were then evaluated six weeks post-dosing.  

ERGs were performed at baseline prior to any procedures and then weekly for the subsequent six weeks to measure the response of the retina to light.   Both dark- and light-adapted ERGs were recorded.  Prior to recording, the animals were dark-adapted overnight and prepared under dim red illumination. The animals were placed under ketamine (80 mg/kg)/xylazine (5-10 mg/kg) anesthesia and their pupils dilated with topical drops (1% tropicamide, 2% phenylephrine) and cornea anaesthetized (1% tetracaine). 

Using a handheld Ganzfeld stimulator equipped with a Blue Kodak Wratten-Gelatin Filter (Rochester, NY) to block wavelengths below 500 nm, bilateral ERGs were recorded simultaneously with DTL Plus electrodes (Diagnosys, Lowell, MA) in contact with the ocular surface through a coating of 1% methylcellulose.   Monopolar needle electrodes (Advena, Hereford, UK) were placed in the cheek (reference) and the tail (ground).  

The animals were exposed to increasing intensities of light as follows:  1)  Dark adapted -25 dB stimulus (0.01 Scotopic Test),  2) dark adapted 0 dB stimulus (3.0 Scotopic Test), 3) light adatpted 0 dB stimulus (3.0 Photopic Test).    The LKC Epic 4000 electroretinography system (LKC Technologies, Gaithersburg, MD) was used for data filtering, acquisition, and averaging.

For those steps in the testing protocol where an a-wave is recordable (photoreceptor response), most frequently the scotopic 0 dB and photopic 0 dB, the amplitude was measured from baseline to the trough.[18]  These manual measurements were done by an investigator masked to the treatment groups.  

The b-wave amplitude was measured for all light intensities presented (M�ller and bipolar cells function).[18] The amplitude of the b-wave is determined by measuring the difference from the trough of the electronegative a-wave (if present) or from the baseline to the peak of the electropositive b-wave.  The ERG data was acquired at baseline prior to any procedures, and weekly thereafter for six weeks.

After six weeks, the RCS rats in all groups were euthanized using carbon dioxide inhalation. The eyes were enucleated and placed in 2% paraformaldehyde/2.5% glutaraldehyde until ready to be dissected. 

Using a razor blade, the anterior chamber and lens were removed 1mm posterior to the corneal limbus and the posterior eyecups dehydrated then embedded in plastic medium. Then a diamond knife was used to cut sections 0.5 �m thick on an ultramicrotome. The resultant sections were then stained with toludine blue.    Light microscopy using an epiflourescent microscope equipped with a digital camera was used to capture digital images of the retinal sections at a magnification of 20x. Ten representative images were taken from each eye and then analyzed.
Using ImagePro Plus digital image analysis program (MediaCybernetics, Bethesda, MD), the number of cell nuclei in the ganglion cell layer, inner nuclear layer, and outer nuclear layer was quantified by an observer masked to the treatment groups.

Statistical analyses were made using spreadsheet software (Excel 2007, Microsoft Corp, Redmond, WA). A one way ANOVA test was used to identify differences in the variability of the means among groups, using a p value of less than 0.05 for statistical significance.

Results: 
Retina Function: 
The first step of the ERG protocol, the 0.01 scotopic flash stimulus, elicits a predominantly rod photoreceptor response.  As expected in the RCS model, these amplitudes progressively decline with time in the control group, and this trend is mirrored in the sham injection group.  However, in the group receiving the active quantum dots, there is a trend for increased amplitudes in weeks four through six post-operatively, and was statistically significant at post-operative week 5 (ANOVA analysis F (3,13) =3.87, p= 0.038) and week 6 (ANOVA analysis F (3,14) = 6.58, p = 0.005).  This trend was not observed in the group receiving injections of the inactive quantum dots.  These results are depicted in Figure 2.

The second step of the ERG protocol, the 3.0 scotopic stimulus, elicits both rod and cone photoreceptor response and is often referred to as the maximal combined response. The b-wave amplitudes from the scotopic 0 dB flash were measures for all groups. In the control and sham injection groups, there is a progressive decline in amplitude from baseline to week six.  In the active quantum dot injection group, the amplitudes increase over post-operative weeks 1 through 3, and then gradually decline through week 6, although remaining higher than both the sham and control groups post-operatively.  This was statistically significant at week 2 (ANOVA analysis, F (3, 13) = 4.29, p = 0.026).  The average b-wave amplitudes for all three intensities are depicted in Figure 3.

The third step of the ERG protocol was the photopic 0 dB flash after light adaptation, which elicits predominantly cone photoreceptor response.  Overall, the amplitudes tended to be higher in the active quantum dot group compared to all other groups in the later weeks, but this did not reach statistical significance.

Retinal Histology: 
Following the six week post-injection ERG recordings, which were 12 weeks of age, the RCS rats were euthanized, their eyes enucleated, and the retinas examined histologically.  There were no instances of traumatic cataract, vitreous hemorrhage, or retinal detachment in any of the eyes.  The number of nuclei present in the three retinal cell nuclear layers (ganglion cell layer, inner nuclear layer, outer nuclear layer) layers was counted using digital analysis software by a masked observer.

In the RCS rat, by 12 weeks of age there is nearly complete degeneration of the photoreceptor cell nuclei in the outer nuclear layer of the retina, as can be seen in the histological section from an unoperated eye in Figure 4(a).[19]  The outer nuclear layer is merely a cell thick and rests atop a thick layer of photoreceptor debris.  In the sham operated eyes, the outer nuclear layer is better preserved, and is 1 to 2 cell layers thick as seen in Figure 4(b).  In contrast, the eyes injected with 540 nm quantum dots had a pronounced preservation of the photoreceptor nuclei, with an outer nuclear layer 3-4 cells thick as seen in Figure 4(c).  On average, the number nuclei present in the outer nuclear layer in the control group was 72.3, in the 490 nm quantum dot group 135.7, in the sham group 185.8, and 392 in the 540 nm quantum dot group.  Using ANOVA analysis, this was statistically significant:  F (3, 96) = 18.0, p = 0.00025) among all four treatment groups.  Put another way, the number of photoreceptors preserved in the sham group was 2.6 times greater than the control group (Student�s t test, p = 0.0012), and the 540 nm quantum dot group had 2.11 times more photoreceptor nuclei than the sham injection group (Student�s t test, p = 0.0006).  This is graphically represented in Figure 5.

While not as pronounced as the photoreceptor nuclear counts, there were differences noted in the two other retinal nuclear cells layers between treatment groups as well.  The average cell count in the ganglion cell layer for the control group was 30.4, and approximately twice this number was found for both quantum dot groups as well as the sham group.  This was statistically significant by ANOVA analysis F(3, 87) = 3.08, p = 0.032, see Figure 5.  Further, there was a statistically significant difference in the number of cells in the inner nuclear layer as well, with an average of 300 in the control group, 347 in the 540 nm quantum dot group, 433 in the 490 nm quantum dot group, and 464 in the sham group (ANOVA, F(3,96) = 2.90, p = 0.039).

Quantum Dots Histological migration:
In the initial groups of animals tested, no quantum dots were retained within the eye as evidenced by histological examination after six weeks had elapsed from the initial injection, and no animals received secondary injections.  Based on the pharmacokinetics of similarly sized proteins, such as bevacizumab, which tend to have a half-life of 10 days in the non-vitrectomized eye, this was not unexpected.[20]

A second set of animals received intravitreal injections of quantum dots, and were than euthanized at 1 hour, 6 hours, 1 day, 2 days, 7 days, and 14 days to determine the rate of quantum dot migration.  One hour after injection, the quantum dots were contained within the vitreous and adherent to the internal limiting membrane of the retina.  Six hours after injection, the quantum dots can be seen migrating across the retina (Figure 6).  By 2 days the majorities of the quantum dots has crossed the retina and are taken up by the retinal pigment epithelium (RPE), where they may remain for up to four months.[21] 

Discussion: 
In this study, we found a beneficial, although transient, effect on retinal electrophysiology from injecting intraocular photoactive quantum dots conjugated with biotin in a rodent model of progressive retinal degeneration.  Eyes injected with the active 540 nm quantum dots showed a statistically significant increase in ERG activity for the 0.01 scotopic ERG recording at week 2 when compared with na�ve controls, saline injections, and inactive 490 nm quantum dots.  There was also a statistically significant increase in the ERG amplitudes of the active quantum dot group for weeks 5 and 6 of the 0.01 scotopic ERGs when compared with these groups.  Further, for both the 3.0 scotopic and 3.0 photopic flash intensities, there was a trend for the ERG amplitudes to increase over time in the active quantum dot group, while the other groups experienced a progressive decline in amplitude.  While there was a trend for the overall average amplitudes to be greatest in the active treated group, this difference did not always reach statistical significance.

Additionally, there appears to be protective effects on a cellular level from intraocular quantum dot injection as well.  In comparing the active quantum dot injection group with na�ve controls, saline injections, and inactive quantum dots, there were statistically significant differences in histological cells counts for all three nuclear layers within the retina.  In brief, intraocularly administered photoactive quantum dots appear to preserve both the function and the anatomy of the retina in this model of progressive blindness.
Four factors need to be considered in determining the observed effects of intraocular photoactive quantum dots in a rodent model of progressive retinal deterioration:  mechanical trauma, electrical stimulation, unique photoelectric and biotoxic properties of quantum dots, and the conjugating element biotin.  

Mechanical trauma to the eye, as noted earlier, is a known stimulus for the production of growth hormones which may ameliorate the progressive retinal deterioration in this model.  The experimental design of this study accounted for this factor through the use of a na�ve control group, a saline injection group, and an inactive quantum dot injection group.  At one week post-injection, there was a trend for an increase in the ERG amplitude in three groups receiving a needle injection, regardless of the material injected.  Otherwise, there was a trend for progressive decline in ERG amplitudes in both the saline and inactive quantum dot injection groups for the duration of the study.

Electrical stimulation to the retina has been demonstrated by many different researchers, using many different techniques, to provide beneficial electrophysiologic and anatomic changes in various animal models of retinal degeneration.[22-24]  The experimental design of this study, using both active 540 nm and inactive 490 nm quantum dots, tested for the effect of delivering small, localized electric charge on a cellular scale.  As noted above, there was a brief, but statistically significant increase in the ERG amplitudes of the active versus the inactive quantum dot groups at week 2 for the 3.0 scotopic testing, and at weeks 5 and 6 for the 0.01 scotopic testing.  This suggests that the transient electrical stimulation provided by the active quantum dots can have an ameliorating effect on the course of progressive retinal photoreceptor degeneration.

There are two factors that may have decreased the efficacy of the active quantum dots used in this study.  First, the quantum dots used in this study are primarily photoluminescent.  That is, they are engineered to respond to certain wavelengths of light in turn with the emission of a different wavelength of light, similar to fluorescent dyes.[14]  However, photoluminescent quantum dots typically have a degree of photovoltaic effect, from the generation of excited electrons that occurs during light exposure.  These electrons in turn create a local electric field, which can be transferred to adherent retinal cells.  For a single quantum dot, this effect would be neglible � but when injected in aggregate, hundreds of thousands of particles can stimulate retinal cells and produce a titratable effect.  Second, the quantum dots used in this study contain the heavy metals cadmium and selenium, which are employed as semiconductor material, but have been shown to be biotoxic in many animal models and humans as well.[25-28] While cadmium/selenium quantum dots with a outer shell of zinc sulfide, such as the ones used in this study, have been shown to be biocompatible in cells at very high concentrations and for long-term exposure, the potential leakage of heavy metals from the materials may have dampened the beneficial effects seen in this study.[14, 29] 

The quantum dots used in this study were conjugated with biotin, a water soluble B-complex vitamin, which is often used in cell labeling techniques due to the high binding affinity for avidin and streptavidin.[30, 31]  Biotin acts as a co-factor for several enzymatic processes, and plays a critical role in fatty acid synthesis and gluconeogenesis.  Biotin also appears to play a role in retinal cell development and apoptosis.[32] The effect of biotin in this study was  controlled for in the experimental design, in that both the active 540 nm quantum dots and the inactive 490 nm quantum dots were both conjugated to biotin, but the possibility that it had some effect, whether beneficial or adverse, cannot be ruled out.  The ability to conjugate nanoparticles to proteins allows for precise targeting of therapeutics on a cellular level, and has been used extensively in the areas of sustained drug delivery and cancer treatment.[33] 

In summary, the use of photoactive quantum dots as an agent to deliver localized electrical stimulation in retinal degeneration provides a means to overcome several large therapeutic hurdles which currently exist in the treatment of blinding retinal diseases.  First, the treatment can be directed at a cellular rather than tissue level, meaning that more precise delivery is possible with less collateral tissue damage.  Second, therapy can be delivered with a needle injection rather than major eye surgery.  Third, and perhaps most importantly, the treatment can begin before pronounced photoreceptor degeneration occurs, with the goal of preventing rather than reversing vision loss.  Fourth, the ability to bioconjugate the quantum dots allows for targeting different cells within the retina.  Lastly, injecting quantum dots into the vitreous is a potentially repeatable, titratable procedure which can be customized to individual patient needs.

The main challenge in using quantum dots as a means of delivering electrical stimulation to retinal photoreceptors is the high rate of migration out of the eye after injection into the vitreous cavity.  In this sense, the quantum dots behave more like a drug, like ranibizumab � the drug used to treat exudative macular degeneration � which requires frequent re-dosing every 4-6 weeks.[34] There is evidence that nanoparticles injected into or around the eye are cleared by the reticulo-endothelial system, and may be detected in the spleen and liver.[35] This pilot study demonstrates a potential benefit in the use of intraocular photoactive quantum dots in a rodent model of progressive retinal degeneration, and further studies are underway.


Acknowledgments: 
The authors (JO, NM & CS) have a patent application pending for the disclosed device.  There were no grants or funds allocated to the completion of this study.

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Figure Legends and Footnotes: 

Figure 1:  Wavelengths and intensity of fluorescent lighting.  The dark line represents the relative intensities of wavelengths of light present in the ambient fluorescent lighting that the animals were exposed to during the entire course of the study.  The excitation spectra of the quantum dots used in the studied are overlaid in green (active 540 nm quantum dots) and red (inactive 490 nm quantum dots).  The active quantum dots excitation spectrum overlaps with that of the fluorescent light emission, indicating that the particles would be stimulated by the ambient light.  Conversely, the excitation spectrum of the inactive 490 nm quantum dots does not overlap with the fluorescent light emission, indicating that no stimulation of these particles would occur.

Figure 2: Electroretinogram waveforms to bright flash stimuli (0.01 Scotopic, 0 dB flash) in dark adapted rats.  One waveform from a representative subject is depicted from each group at four different time points:  1, 2, 5, and 6 weeks post-operatively.  The arrowheads indicate the trough of the a-wave, (photoreceptor response), and the arrows indicate the peak of the b-wave (inner retinal response).  (A) At one week post-operatively, there was no difference between the groups.  (B)  At two week post-operatively, there was an increase in the amplitude of the b-wave in the active implant group which was statistically significant (in A and B all the waves are superimposed in the same graph, therefore the measure in the y-axis is the same for all of them).  (C, D)  By weeks 5 and 6, the amplitude of the b-wave had decreased in all groups, although there was a trend for higher amplitudes in the active implant group which did not reach statistical significance (in C and D the waves were separated for a better appreciation of the morphology, therefore the y-axis do not depict the actual value in amplitude of each wave). However, in the active implant group, there was preservation of the a-wave as well as oscillatory potentials (which are seen as the undulations on the rising portion of the b-wave), which were not evident in the other groups.

Figure 3:  Graphical depiction of the average b-wave response over the range of flash intensities presented for all four groups, at four time points post-operatively:  1, 2, 5, and 6 weeks.  Error bars represent +/- SEM.  (A)  At one week, there was no difference between the groups for any of the light intensities.  (B)  Two weeks post-operatively, there was a statistically significant rise in the amplitude of the b-wave for the 3.0 scotopic testing in the active implant group.  Although not statistically significant, there was a trend for higher amplitudes in the active implant group in the 3.0 photopic testing as well.  (C, D)  At five and six weeks post-operatively, there was a trend for higher amplitudes in the active implant group across all three intensities, which reached statistical significance in the 0.01 scotopic testing at both time points.

Figure 4: Histologic appearance of retina 6 weeks post-operatively.  (A)  The control group has a marked loss of photoreceptor cell nuclei as seen in the ONL which is a single cell thick.  (B)  The eyes receiving sham injections had better preservation of the ONL, which is 2 to 3 cell layers thick.  The appearance was similar in the inactive implant group (not depicted).  (C)  In the eyes receiving the active implant, there was a marked preservation of the ONL layer, which is 4 cell nuclei thick.  Notably, all groups had a loss of outer segments at six weeks, although this was attenuated in the active implant group.

Figure 5:  Graph depicting the cell counts for each of the four groups (+/- SEM).  Ten sections were taken from each eye and scanned by a masked observer using digital analysis software.  There was a statistically significant increase in the number of ganglion cell nuclei in all eyes receiving an injection (sham, active, inactive) compared to control.  There was no difference among the groups in the number of cells in the inner nuclear layer.  In the photoreceptor cell counts, there was a statistically significant increase in the number of nuclei present in the active implant group compared to all others.

Figure 6:   A) One hour after injection, the quantum dots (arrowheads) were contained within the vitreous and adherent to the internal limiting membrane of thenz������
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