Lens injury induced activation of retinal glia, and subsequent release of ciliary neurotrophic factor (CNTF) and leukaemia inhibitory factor (LIF) potently protect axotomised retinal ganglion cells from apoptosis and promotes axon regeneration in the injured optic nerve. The goal of the current study was to investigate if similar effects may also be applicable to rescue photoreceptors from degeneration in a model of retinitis pigmentosa. Lens injury was performed in the Royal College of Surgeons (RCS) rats at the age of one month. The survival of photoreceptors was evaluated histologically, and retinal function was analysed by electroretinography (ERG). Expression of CNTF was also analysed. Lens injury significantly enhanced the survival of photoreceptors 1 month after surgery compared to untreated controls, which was associated with an enhanced ERG response. In addition, lens injury significantly protected photoreceptors from degeneration in the contralateral eye, although to a much lesser extent. We could show that lens injury is sufficient to transiently delay the degeneration of photoreceptors in the RCS rat. The observed neuroprotective effects may be at least partially mediated by an upregulation of CNTF expression seen after lens injury.
It has recently been shown that lens injury or intravitreal applications of lens-derived
The purpose of the current study was to investigate whether lens injury is also sufficient to protect photoreceptors from degeneration. As a model, we chose the Royal College of Surgeons (RCS) rat, which is a commonly used model of retinal degeneration, in particular retinitis pigmentosa. The cells of the retinal pigment epithelium (RPE) are not capable of phagocytosing photoreceptor outer segments due to a mutation in the Mertk gene. This leads to a gradual degeneration of the photoreceptors starting at the time point of eye opening (i.e., approximately at P20) and being completed at the age of 3 months [
The RCS rats were bred in our animal facility. They received food and water
Lens injury was performed using a microscope with illumination in 14 RCS rats at the age of one month, shortly after the onset of retinal degeneration. Eight animals were used for histology, two for immunohistochemistry, and four for Western blot analysis. Animals were anaesthetised by a mixture of ketamine and xylazine (120 mg/kg ketamine, 10 mg/kg xylazine). A small incision was made into the temporal corner of the eyes. The eyeball was rotated into the nasal direction, and the conjunctiva was incised to allow direct access to the sclera. A sharp 25-gauge injection needle was used to puncture the lens by inserting it into the eye through the sclera. In order to assure injury of the lens, the needle was rotated two or three times inside the lens before retracting it. The success of lens penetration was controlled by a funduscopic inspection of the eye. After lens injury, the eyeball was brought back into its normal position, and the eye was covered by antibiotic ointment (Gentamytrex, Dr. Mann Pharma, Berlin). Finally, the animals were kept at 37°C on a heating pad until waking up and then brought back into their cages.
Four RCS rats were killed 1 month after the lens injury and four RCS rats after two months. In addition, four untouched animals were used as controls for the ages of two and three months. Consequently, four eyes were available for histological analysis for each age and each experimental group (control, lens injury, and contralateral to lens injury).
The animals were enucleated, the eyes were fixed in formalin, embedded in paraffin wax, and haematoxylin-stained sections were prepared according to standard procedures. Digital images were taken, and photoreceptor nuclei per 100
Scheme of the rat eye. The regions where evaluation of photoreceptor survival was performed are indicated.
In order to check expression of CNTF after lens injury in RCS rat eyes, paraffin sections of the eyes were deparaffinised using standard procedures. The sections were heated for 3 minutes in Tris buffer/EDTA (pH 9) and blocked with BSA (1% in PBS) for 1 hour. A double staining was performed applying a mixture of primary antibodies against CNTF (goat polyclonal anti-CNTF antibody, Santa Cruz Biotechnology, Santa Cruz, CA, USA, dilution 1 : 100) and the glial fibrillary acidic protein (GFAP, rabbit polyclonal anti-GFAP antibody; DakoCytomation, Glostrup, Denmark, dilution 1 : 4000) at 37°C for 2 hours. The sections were washed three times with TBS and Tween 20. A mixture of secondary antibodies (Alexa Fluor 488-labelled donkey anti-rabbit IgG antibody for GFAP, Invitrogen Corporation, Carlsbad, California, dilution 1 : 1000, and Cy3-labelled AffiniPure donkey anti-goat IgG antibody for CNTF, Jackson ImmunoResearch Laboratories, West Grove, Pennsylvania, dilution 1 : 400) was applied for 45 minutes at room temperature. After washing with distilled water, the samples were embedded in FluorSave (Calbiochem, Darmstadt, Germany), coated with a cover glass slide, and inspected using a fluorescent microscope.
Retinas were isolated 5 days or 1 month after a lens injury and frozen in liquid nitrogen before further processing. In addition, eyes from untreated animals and the eyes contralateral to the lens injury were analysed. Retinal tissue of two eyes was pooled in each group. Isolated retinas were transferred into lysis buffer (20 mM Tris/HCl pH 7.5, 10 mM KCl, 250 mM sucrose, 10 mM NaF, 1 mM DTT, 0.1 mM Na3VO4, 1% TritonX-100, and 0.1% SDS) with 1/100 protease inhibitor (Calbiochem, CA, USA). Retinas were homogenised and centrifuged at 5,000 rpm for 10 min at 4°C. The supernatants were analysed by Western blot assay. Separation of proteins was performed by 10% sodium-dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), according to standard protocols (Bio-Rad, Hercules, USA). After SDS-PAGE, proteins were transferred to nitrocellulose membranes (Amersham, UK). The blots were blocked either in 5% dried milk or in 2% ECL Advance blocking agent in Tris-buffered saline-Tween 20 (TBS-T). They were then processed for immunostaining with either a polyclonal antibody against rat CNTF (Serotec, 1 : 5000) or an antibody against tubulin (Babco, Richmond, CA, 1 : 2000) at 4°C overnight. Bound antibodies were visualised with anti-rabbit or anti-mouse immunoglobulin G (IgG) secondary antibodies conjugated with horseradish peroxidase diluted at 1 : 80,000 (all Sigma, St. Louis, USA). The antigen-antibody complexes were detected by enhanced chemiluminescence (ECL, Amersham, Buckinghamshire, UK).
ERG analysis was performed in all RCS rats listed in the histology paragraph at the ages of two and three months. Animals were dark adapted over a period of at least 24 hours. They were anaesthetised by an intraperitoneal injection of a mixture of ketamine and xylazine (120 mg/kg ketamine, 10 mg/kg xylazine). The corneas of the eyes of the anaesthetised animals were desensitised with Novesine (Novartis Ophthalmics). The animals were placed on a heated platform (37°C) to keep their body temperature constant during the measurements. Gold wire ring electrodes placed on the corneas of both eyes served as working electrodes. A gold wire ring electrode was placed in the mouth to serve as a reference electrode. The pupils were dilated with Tropicamide (Novartis Ophthalmics). Standard electroretinographic measurements were performed using the commercial RetiPort32 device from Roland Consult Systems (Brandenburg, Germany), with scotopic flash ERG at light intensities of 0.0003 and 100 cd·s/m², an additional run for scotopic oscillatory potentials, photopic flash ERG after 10 minutes of light adaptation, and photopic oscillatory potentials. The light intensity used for the flashes in the photopic ERG measurements was 100 cd·s/m². The analogue filters of the ERG device were set to the frequency ranges of 0.5 to 200 Hz for both scotopic and photopic flash ERG and 50 to 500 Hz for oscillatory potentials.
ERG measurements were performed simultaneously on both eyes to compare lens injury treated and contralateral eyes, and the performing person did not know which eyes had been lens-injured.
The ERG waveforms obtained at the age of 2 months, that is, one month after lens injury, are shown in Figure
Typical electroretinographic waveforms recorded in 2-month-old RCS rats. The waveforms obtained in the control rat without treatment are shown in the left column, and the waveforms of the lens-injured animal are shown in the right column. The solid lines represent the waveforms recorded in the lens-injured eye, whereas dotted lines represent waveforms recorded in the contralateral eye. For the scotopic flash ERGs, only two traces are shown to be obtained at light intensities as indicated, one for a pure rod ERG and one for maximal mixed rod-cone response. Please note the different scales.
No ERG response was found in lens-injured eyes 2 months after lens injury (not shown).
After the ERG measurements, the animals were enucleated, and paraffin sections of the eyes were prepared. We compared paraffin sections of RCS rat eyes after lens injury, corresponding contralateral eyes, and eyes of untreated age-matched control animals. Representative histological sections are shown in Figure
(a) Histological sections of retinas of 2-month-old RCS rat eyes. From left to right, the retina of an untouched animal (nontreated control), a retina where the lens was injured, and a retina of an eye contralateral to lens injury are shown. The black bars indicate the thickness of the outer nuclear layer. Scale bar: 50
Compared to untreated control groups, significantly more photoreceptors survived in the lens-injured eyes in all three regions of the retina that were separately evaluated (Figure
We analysed the eyes immunohistochemically for CNTF expression (Figure
Immunohistochemical staining against CNTF of retinal paraffin sections of a 2-month-old lens-injured RCS rat. In the lens-injured eye, CNTF-positive cells are visible mainly in the ganglion layer. Some photoreceptor nuclei with CNTF immune reactivity were also found. Scale bar: 50
Finally, the lens-injury induced upregulation of retinal CNTF expression was confirmed by Western blot analysis for CNTF, showing enhanced CNTF levels in lens-injured eyes, and, to a lesser extent, also in the contralateral eyes (Figure
Result of the Western blot analysis for CNTF in retinal samples obtained from lens-injured eyes and contralateral eyes five days and one month after lens injury. As a measure for the applied sample size,
Since the discovery of the significant neuroprotective and neuroregenerative effects of lens injury on damaged retinal ganglion cells, this experimental paradigm has been successfully applied by several groups [
As shown, a significantly higher portion of photoreceptors survived in lens-injured eyes in 2-month-old rats, whereas no neuroprotection was found in 3-month-old rats, suggesting that the lens injury effects are only effective for a limited time period. This is consistent with the idea that crystallins are released from the injured lens and subsequently stimulate retinal glial cells. After stimulation, the endogenous CNTF levels return to basal levels and, therefore, can no longer protect the photoreceptors [
Consistently, we found CNTF immunoreactivity mainly in the nerve fibre and ganglion cell layers and additionally in deeper layers of the retina. Comparison with the GFAP immunoreactivity suggests that CNTF is produced by astrocytes and Müller cells (not shown). Moreover, we have also seen occasional CNTF immunoreactivity in the outer nuclear layer indicating that even a few photoreceptors themselves could probably produce or bind CNTF upon stimulation by lens injury. A major role of CNTF in mediating the neuroprotective effects of lens injury on photoreceptors is also supported by previous reports, demonstrating that CNTF is neuroprotective to photoreceptors in different animal models of retinal degeneration [
Another interesting finding of the current study was that moderate neuroprotective effects were also found in the eyes contralateral to the lens-injured eye. This observation was in line with a noticeable CNTF immunoreactivity in the contralateral eyes. Mutual influences between the two eyes have been known for a number of years [
We performed electroretinographic measurements in the RCS rats to check if lens injury resulted in an improved retinal function. In the eyes contralateral to lens injury, no retinal response to light stimulation could be measured. The scotopic and photopic ERG waveforms showed a negative slope, which is typical for RCS rats with progressed photoreceptor degeneration [
By contrast, an ERG response could be measured in three out of four lens-injured eyes in 2-month-old rats. Obviously, the extent of protection in these eyes was sufficient to maintain a residual retinal activity. Whether a function of photoreceptors is preserved or not seems to depend on the amount of surviving photoreceptors. The one lens-injured eye where we could not detect any improvement of the ERG response was also the eye with the smallest morphological effect, that is, with the smallest number of surviving photoreceptors. We speculate that there could be a kind of “bystander” effect between the photoreceptors, enabling them to respond considerably to light as long as there is a sufficient number of them.
Lens injury causes cataract and, consequently, opacity of the lens that hinders access of light to the retina. It is thus all the more noteworthy that we could record an enhanced ERG response at all one month after lens injury in three out of four lens-injured animals compared to the contralateral eyes with their nonopaque lenses.
Two months after lens injury, the protective effects were almost completely lost. Corresponding to the poor, that is, no longer existing morphological effects of lens injury, we could not detect any differences in the ERG response between lens-injured and contralateral eyes.
In summary, we conclude that lens injury exerts protective effects on photoreceptors in the RCS rat as a model of retinal degeneration. As this treatment does not address the actual defect in the RCS rat, the protective effect is only moderate and transient. Nevertheless, we propose that lens injury, or injection of lens crystallins as an alternative, could be a useful option for an accompanying treatment during gene therapy or other therapeutical approaches.