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From the Department of Experimental Ophthalmology, School of Medicine, University of Münster, Germany.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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METHODS. In adult albino rats, penetrating lens injury was performed by intraocular injection. To test for injury-induced neuroprotective effects in vivo, fluorescence-prelabeled RGCs were axotomized by subsequent crush of the optic nerve (ON) with concomitant lens injury to cause cataract. The numbers of surviving RGCs were determined in retinal wholemounts and compared between the different experimental and control groups. To examine axonal regeneration in vivo, the ON was cut and replaced with an autologous piece of sciatic nerve (SN). Retinal ganglion cells with axons that had regenerated within the SN under lens injury or control conditions were retrogradely labeled with a fluorescent dye and counted on retinal wholemounts. Neurite regeneration was also studied in adult retinal explants obtained either after lens injury or without injury. The numbers of axons were determined after 1 and 2 days in culture. Putative neurotrophins (NTs) were studied within immunohistochemistry and Western blot analysis.
RESULTS. Cataractogenic lens injury performed at the same time as ON crush resulted in highly significant rescue of 746 ± 126 RGCs/mm2 (mean ± SD; approximately 39% of total RGCs) 14 days after injury compared with controls without injury or with injection of buffer into the vitreous body (30 ± 18 RGCs/mm2). When lens injury was performed with a delay of 3 days after ON crush, 49% of RGCs survived, whereas delay of 5 days still rescued 45% of all RGCs. In the grafting paradigm virtually all surviving RGCs after lens injury appeared to have regenerated an axon within the SN graft (763 ± 114 RGCs/mm2 versus 79 ± 17 RGCs/mm2 in controls). This rate of regeneration corresponds to approximately 40% of all RGCs. In the regeneration paradigm in vitro preceding lens injury and ON crush 5 days previous resulted in a maximum of regeneration of 273 ± 39 fibers/explant after 1 day and 574 ± 38 fibers/explant after 2 days in vitro. In comparison, in control retinal pieces without lens injury 28 ± 13 fibers/explant grew out at 1 day, and 97 ± 37 fibers/explant grew out at 2 days in culture. Immunohistochemical and Western blot analysis of potential NTs in the injured lens revealed no expression of ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), NT-4, nerve growth factor (NGF), and basic fibroblast growth factor (bFGF).
CONCLUSIONS. The findings indicate that the lens contains high neuroprotective and neuritogenic activity, which is not caused by NT. Compared with the data available in the literature, this neuroprotection is quantitatively among the highest ever reported within the adult rat visual system.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Other substances reported to influence survival of RGCs after intraocular injection were giant cell line–derived neurotrophic factor (GDNF),15 inhibitors of cpp32-like proteases,16 inhibitors of nitric oxide synthase,17 and inhibitors of microglial cells.13 Some of these NTs are produced by Schwann’s cells11 and may account for survival and regeneration in the peripheral nerve–ON grafting paradigm.1 In addition to targeting of RGCs, bFGF has been intraocularly injected to rescue dystrophic photoreceptors in the rat strain of the Royal College of Surgeons (RCS).18 Finally, several NTs were injected to rescue photoreceptors that undergo degeneration on light illumination.19
Neuroprotective manipulations may have been effective because of complex influences on the retina that involve nonneuronal cells in the retina or even nonretinal compartments. Injections of solvents or merely a heavy penetrating injury to the anterior portion of the eye strengthened the resistance of RGC to ON cut.12 Similarly, experimentally induced intraocular inflammation supported survival of axotomized RGCs, therefore ascribing to immigrating macrophages a supportive role during regeneration.20 Deactivation of resident intraretinal microglial cells13 or increasing macrophage recruitment in the ON enhances the rate of RGC survival, whereas NT-4’s effects on RGCs may be mediated by astrocytes.7 Conceivably, several of the effective substances that are contained in injected media or are represented by the various neurotrophic molecules may prolong the survival of axotomized neurons by modulating the glial responses. Alternatively, they may induce intraocular responses with consecutive local transcription of effective factors other than those originally injected. This interpretation is strengthened by various reports that the rescue effects are limited to a proportion of RGCs, whereas there are almost no reports on additive effects. A mere mechanical injury to the eye induces, for example, transcription of FGF-2 and CNTF.21
The activation of intrinsic antiapoptotic genes is an alternative concept to the approach of external substitution with neurotrophic or neuroprotective agents. Members of the bcl-2 family22 have been reported to rescue such neurons in culture with dependence on neurotrophic factors.23 In transgenic mice overexpressing bcl-2, a greater proportion of neonatal RGCs survive and extend axons in culture, although neuritogenesis was assumed to be mediated by a different mechanism than antiapoptosis.24 RGCs also survived better in adult stages of bcl-2 transgenic mice.25 26 27 Bcl-2 seems to regulate apoptosis within the lens epithelium, and lens-specific overexpression of bcl-2 in transgenic mice inhibits lens cell death.28 Also, NTs such as BDNF and NT-3 show a patterned expression within the lens epithelium.29 These molecules are assumed to regulate differentiation and maintenance of ocular tissues including lens epithelia.
The experiments reported here were undertaken to answer the following questions in relation to penetrating injury of the lens: Is injury to the lens and subsequent opacity associated with survival of RGCs? If so, can the activity be attributed to identified NTs or other cell survival promoting factors? Is lens-derived activity due to neuroprotective and/or to neuritogenic activity? To achieve these goals, we performed three major series of experiments. First, we prelabeled RGCs from the superior colliculus (SC) and then axotomized them by crushing the ON and analyzed whether simultaneous or delayed lens injury resulted in greater survival of RGCs. In the second set of experiments, axonal regeneration was studied in a grafting paradigm in vivo. For this, the cut ON was replaced with an autologous piece of SN, and RGCs regenerating after lens injury were retrogradely labeled from the graft. Third, in a series of cell culture experiments, crush of the ON and lens injury were performed to analyze whether the retinal stripes obtained from these eyes showed greater axonal regeneration in culture than control stripes. Finally, immunohistochemical and Western blot analyses were performed to detect candidate molecules involved to prevent neuronal death.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Effects of Lens Injury In Vivo
To search for lens-derived neuroprotection of RGCs, these neurons
were identified by retrograde tracing. To do this, 48 rats received the
fluorescent dye D291,
N-4-[4-didecylaminostryryl]-N-methyl-pyridinium
iodide (4Di-10ASP; Molecular Probes, Eugene, OR) into the SC.
Figure 1 shows the anatomic relationships within the eye and the projection of
ganglion cell axons through the ON to the SC. Eight days later, the ON
contralateral to the injected SC was surgically exposed in its
intraorbital aspect and mechanically crushed at 0.5 mm beyond the ON
head for 10 seconds, as described earlier. The rats were then divided
into six groups. The first group (n = 6) received ON crush,
intraocular penetration and injury of the lens (perforation routes 1
and 2, Fig. 1
) containing the lens proliferative zone of the
epithelium, and injection of 10 µl phosphate-buffered saline (PBS, pH
7.4) according to standard protocols.13
The second group
(n = 6) received ON crush, retrolenticular intraocular
penetration (perforation route 3, Fig. 1
), and injection of 10 µl PBS
without lens damage. A third group of control animals (n =
6) received ON-crush, but no eye injection or lens injury. A fourth
group (n = 18) received ON-crush, with lens injury plus PBS
(perforation route 2, Fig. 1
) by 3, 5, or 7 days (six animals per
subgroup) after ON-crush. The fifth group of control rats (n
= 6) received only retrograde labeling of RGCs but no further
manipulation at the ON or in the eye. This group was used to determine
the numbers of retrogradely filled RGCs, and the rats were killed 8
days after the staining procedure. A group of six rats underwent
labeling only and were killed at 22 days after staining—that is, after
the total study period of the other experimental groups—to assure that
the long-term staining did not affect cell survival by itself. Finally,
a group of five rats were treated the same as the animals of the fourth
group, but without PBS injection to test the effect of the injection
route alone.
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Cell densities were determined with the aid of a fluorescence microscope (Axiovert; Carl Zeiss) equipped with an ocular grid of 200 x 275 µm (magnification, x400). The results from the counts, which covered the total retinal surface, were averaged for every retina and were calculated to cell density values per area unit. To estimate the total number of RGCs per retina, the average was multiplied with the retinal surface, which was determined to be 40 mm2 at this age. From the mean densities of all retinas per group, mean values and SDs were calculated. Data of cell survival under the different treatments were compared with an analysis of frequency distribution. The results of the different conditions of injections were compared with the control condition by using Student’s t-test. Data are presented as means ± SD.
The rodent lens has an oval shape that fills most of the posterior eye cavity, thus providing limited space for intravitreal injections of larger amounts of material (Fig. 1) . The approximate sizes of the intraocular cavities and compartments are indicated for an adult rat of approximately 200 g body weight in Figure 1 . The perforation routes 1 and 2 of Figure 1 are commonly reported to be associated with intraocular injury. The only perforation route that avoids no direct injury to lens is route 3 in Figure 1 , which passes through the posterior pole of the eye cup to approach the vitreous body transsclerally and transretinally. It appeared from the injections performed in the present study that the use of a pulled glass capillary of 20 to 30 µm diameter is the most suitable method to obtain consistent results with all the perforation routes. All perforation routes were tested in pilot experiments with injection of 10 µl of PBS. However, the only method that consistently resulted in reproducible cataract was route 2.
To study axonal regeneration in vivo, the exposed ON (n = 7)
was cut and replaced with an autologous piece (
2 cm length) of
sciatic nerve as reported earlier.1
Two groups of animals
were formed. In the first group (n = 4) cataractogenic lens
injury was performed. The animals of the control group (n =
3) received grafting but no lens injury. In both groups rats were
allowed to survive 14 days. In a second control group of three rats, 1
mg bovine serum albumen (BSA; Sigma, Munich, Germany) was injected into
the vitreous body without lens injury. The purpose of this control was
to monitor whether proteins within the vitreous body induce secondary
effects that may result in better regeneration. At this stage the graft
was surgically exposed again, and those RGCs whose axons had
regenerated into the graft were retrogradely labeled, as described
earlier.13
The densities of RGCs were counted 5 days later
in wholemounted retinas by using the same conditions as were used for
the survival studies.
Pretreatment of Retina and Explantation
Lens-derived factors may promote axonal regeneration in culture,
in addition to supporting ganglion cell survival and regeneration in
vivo. The procedure of culturing was identical with that described
previously.4
5
In nine rats, the lens capsule was damaged
by an injection using a microcapillary penetrating through the sclera
(perforation route 2, Fig. 1
). Control animals were rats that received
no lens injury and rats (n = 3) that received an
intravitreal injection of 1 mg BSA through perforation route 3—that
is, without lens injury. The retinas of the different groups were used
to produce explants for axonal regeneration in vitro. The ganglion cell
origin of the growing axons has been studied with various methods
showing that only RGCs are able to extend axons under the conditions
imposed in this study.4
5
The numbers of regrowing axons
were determined after 24 and 48 hours in vitro by means of an inverted
phase-contrast microscope (Axiovert; Carl Zeiss) with a x20 lens.
As a measure of axonal growth, all axons were counted that encountered an imaginary line of between 50 and 100 µm from the explant’s margins. With increasing numbers of axons per explant, fasciculation occurred, in particular in the lens-damaged group. The fasciculation sometimes prevented the clear distinction of individual axons within bundles, even at larger microscope magnifications. The results from the single counts were averaged for each experimental group. To extrapolate over the total retina, the averaged number was multiplied by the number of explants per retina, usually eight. The data of regeneration under the different treatments were evaluated with Student’s t-test. All data of cell densities or cells per retina are presented as mean ± SD.
Immunohistochemistry
Eyes obtained from damaged lens samples (n = 3) and
those from control (n = 3) rats were examined for the
appearance of molecules that are known to exert neuroprotective
effects. The eyes were frozen in liquid nitrogen and cryosectioned for
immunohistochemistry. The following monoclonal antibodies and antisera
were used: antibodies to CNTF (dilution 1:100; Promocell, Heidelberg,
Germany), NT-4 (dilution 1:200; Chemicon, Temecula, CA), NGF
(dilution 1:100; Sigma), and bFGF (dilution 1:100; Promocell). To
examine immigrating immunocompetent cells as result of the lens injury,
CD45 antigen staining was performed on all rat samples (leukocyte
common antigen [LCA] clone CD45; Serotec, Oxford, UK; dilution
1:200). In addition to the antibodies against NTs or leukocytes, glial
markers were used to examine whether lens injury is associated with
glial cell activation. Sections from the same samples were therefore
processed to stain either astrocytes and Müller cells (glial
fibrillary acidic protein, [GFAP] dilution 1:200–1:500;
Boehringer–Mannheim, Mannheim, Germany) or intraretinal microglial
cells (OX-42 antibody that recognizes the complement factor 3 receptor,
dilution 1:500; Dako, Zug, Switzerland). Immunohistochemistry protocols
were performed according to instructions of the suppliers. The
secondary antibodies were coupled with fluorescein-isothiocyanate, and
evaluation was performed with the aid of a fluorescence microscope
equipped with photographic camera and computer-assisted image analysis.
Biochemistry and Western Blot Analysis
The anterior halves of five lens capsules carrying the epithelium
were dissected at the level of the equator, removed from the lens
cortex with jeweller’s forceps, and collected in PBS. Histologic
examination of wholemount samples with hematoxylin stain revealed that
with this method most of the lens epithelium remained attached to the
capsule. Epithelium-containing capsules were homogenized with an
ultrasound homogenizer (10 minutes; 20 W) in 200 µl PBS. In addition
to epithelium, five whole lenses were dissected into small pieces
followed by ultrasound homogenization (5–10 minutes). After
centrifugation at 7,500 rpm for 5 minutes, the supernatant was
collected, and protein contents were determined photometrically with
protein dye binding of Coomassie brilliant blue. Separation of proteins
was performed with 14% sodium-dodecyl-sulfate–polyacryamide gel
electrophoresis (SDS-PAGE) according to standardized protocols (Slab
gels; Bio-Rad, Hercules, CA). After SDS-PAGE, the gels were fixed and
stained with the silver method, or used for Western blot analysis.
To immunologically identify neurotrophic factors, we separated five total lens homogenates including epithelium with SDS-PAGE in an electrophoresis chamber (Bio-Rad) using 1-mm-thick acrylamide gels. After SDS-PAGE the proteins were transferred to nitrocellulose membranes (0.45-µm thick). The blots were blocked with 5% dried milk in Tris-buffered saline-Tween 20 (TBS-T) and processed for immunostaining with primary antibodies to the NTs (CNTF, NT4, bFGF, and NGF, dilution 1:2000 each). The monoclonal antibody to human BDNF (gift from Ilse Bartke, Roche Diagnostics, Panzberg, Germany) was diluted 1:2000 to 1:5000. Antigen antibody complexes were detected by enhanced chemiluminescence (ECL; Amersham, Amersham, UK).
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Results |
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Injury-Induced Cataractogenesis
On fundoscopic examination at the day of injection using a cornea
applanation coverslip and a surgical microscope, perforation the of
lens could be visualized easily and documented. Re-examination of the
same animals was performed at the day of retinal explantation to
prepare cultures or on the day when retinal flatmounts were prepared to
count RGCs. In all cases, the degree of lens injury–induced swelling,
nuclear opacity, and capsular rupture and the progression of cataract
were documented and related to the method of eye penetration.
It appeared that perforation route 1 through the cornea and the pupil to damage the anterior lens capsule did not induce cataract during the 14 days of analysis. Nor did perforation route 3 induce any cataract, (Fig. 2A ), and the extracted lenses were found to be translucent and free of opacity (Fig. 2B) . Such transparency was independent of whether buffer was injected into the vitreous. In contrast, most lenses that received injury through perforation route 2 showed a fast reduction or loss of transparency (within 1 day), swelling of lens cortex, and a progressive cataractous opacity in the lens nucleus (Figs. 2C 2D) . Cataractogenesis continued during the 14 days of observation and reached the typical shapes and morphologies shown in Figures 2C and 2D and in Figures 2E and 2F after lens extraction. The anterior part of the cortex of such lenses could be described as crystalline and transparent, although they also showed some signs of swelling and moderate opacity (Figs. 2E 2F) . In control animals (n = 3), perforation route 2 was used to damage only the ciliary body without affecting the lens capsule.
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In the experimental series used to analyze and quantify effects of lens injury, the lens was perforated according to perforation route 2 (Fig. 1) , because pilot experiments using the noncataractogenic perforation routes 1 and 3 did not show a marked effect on survival of RGCs. Also the pilot experiments using perforation route 2 to damage the ciliary body and pars plana did not reveal higher incidence of cell survival in the retina (data not shown). It appeared that ON crush and concomitant or delayed cataractogenic lens injury resulted in massive protection of retrogradely labeled RGCs (Figs. 3C 3D) . Morphologically intact RGCs (Fig. 4C) were uniformly distributed over the whole retinal surface and over its total eccentricity (Figs. 3C 3D) . Quantitatively, 746 ± 126 RGCs/mm2 or 29,840 ± 5,040 RGCS/retina were present (that is, approximately 39% of the total population; n = 6, Fig. 5 ). In the same retinas, labeled microglial cells were reduced to 288 ± 74/mm2 or 11,520 ± 2,960 RGCs/retina (Fig. 5) . The number of rescued RGCs could even be increased, when the cataractogenic lens injury was performed with a delay of 3 or 5 days after ON crush. In these cases 49% (3 days’ delay) and 45% (5 days’ delay) of all RGCs survived (Fig. 5) . The 7 days’ delayed lens injury rescued fewer RGCs (Fig. 5) . In the cases with delayed injuries (5 and 7 days), the peripheral retina contained as many viable cells (Fig. 3F) as the control retina (Fig. 3A) . These cells are morphologically shown in Figures 3E and 3F and enlarged in Figure 4D . The difference between the procedures with concomitant injuries was the significantly higher density of ganglion and microglial cells (467 ± 294 RGCs/mm2; Fig. 5 ), which indicated that some phagocytosis was performed at days 5 and 7 after crush.
Regeneration of Retinal Ganglion Cell Axons
To assess whether lens injury promoted survival of ganglion cells
with regenerative properties (proof of viability), axonal regrowth was
studied in vivo (grafting example) and in culture by using a chemically
defined medium containing neither serum nor neurotrophic
substitutes.4
5
In the grafting paradigm in vivo, lens injury resulted in cataract that was visible in all animals of this group (n = 4). Deposition of the fluorescent dye 4Di-10ASP into the graft 14 days after grafting and examination of the flatmounted retinas 5 days later revealed that fluorescent, and thus regenerating, cells were distributed across the retina (Figs. 6A 6B 6C 6D) . All regions of the retina contributed to regeneration, whereas a slight central-to-peripheral decrease in cell density was observed (Figs. 6A 6C) . Quantitatively, an average of 763 ± 114 RGCs/mm2 (n = 4) were retrogradely labeled across the retinal surface (Fig. 6E) . Extrapolated over the total retinal surface, this number corresponds to 30,520 ± 4,560 RGCs/retina, which is approximately 40% of the total population. This number is 10 times higher, and thus significantly higher (P < 0.001), than that in the control group (79 ± 17 RGCs/mm2 or 3,160 ± 680 RGCs/retina (n = 3; Fig. 6E ). The comparison of this number of regenerating RGCs with the numbers of surviving RGCs under conditions of cataractogenic lens injury (compare with Fig. 5 ) shows that it is likely that all surviving neurons were able to regenerate their axons into the SN graft.
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Potential Neuroprotective Substances within the Lens
To analyze whether some known neurotrophic factors are produced
and released from the injured lens, immunohistochemical analysis was
performed on cryostat sections, and Western blot analysis in SDS gels.
It appeared that immunohistochemistry was negative when antibodies were
used against CNTF, NT 4/5, bFGF, NGF, and BDNF. Western blot analysis
confirmed these data (Fig. 8)
and supported the view that lens injury did not release
immunodetectable amounts of either of the factors investigated.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Survival and Regeneration of Axotomized RGCs In Vivo
Perforation of the ocular tissue through the cornea, or through
the pars plana–ciliary body, or through the posterior sclera without
lens injury did not increase the numbers of surviving RGC, thus
indicating that opening of the lens capsule is essential to release the
intralens trophic agents. The findings do not completely exclude the
appearance and the possible release of additional factors from such
nonlens tissues as iris, cornea, ciliary body, vitreous body, uvea, and
retina. The different routes of penetration and the combinations of
lesions are best explained by the selective release of lens factors
when injury was followed by onset of swelling and cataractogenesis.
Immunologic responses cannot be completely excluded, although the
injection of BSA into the vitreous body did not cause
regeneration-relevant responses. In addition, the failure to
immunohistochemically detect CD45 antigen on leukocytes in the retina
is a further line of evidence against a massive immigration of these
cells in this experimental paradigm.
The greater survival of RGCs after targeted lens injury and cataractogenesis is consistent with previous observations that show that penetrating lesions causes release of neurotrophic activities. Moreover, it has been reported12 that eye injury through an anterior approach has a more beneficial effect than that performed through a posterior approach. Although we cannot conclude from their description whether they used perforation route 1 or 2, these investigators may have used a combination of perforation routes 1 and 2. To localize the source of lens-derived trophic activities within a certain intraocular compartment, we used a fine glass capillary instead of a 26-gauge needle and injured the lens without affecting the iris or the ciliary body. In addition, we compared three perforation routes that allowed an exact localization of the lens injury, ranging from noncataractogenic retrolenticular injection or anterior capsule rupture to induction of cataractogenic changes of varying degrees. It clearly appeared that induced lens swelling and cataractogenesis enhanced RGC survival, but not intravitreal injection or puncture of lens capsule without traumatic cataract.
That delayed lens injury preferentially affected survival of the RGC in the peripheral half of the retina can be explained with the central-to-peripheral gradient in the progression of death after ON crush. Indeed, more peripheral neurons were rescued with delayed lens injury. In the same context, more microglial cells compared with those found after simultaneous injury may indicate onset of phagocytosis before the fifth day. The most likely interpretation is that the event of lens swelling initiates the release of neuroprotective factors. Most likely, because RGCs enter the apoptosis at days 4 to 6 after ON transection,2 delayed lens injury can retard this apoptotic cascade even when performed 5 days later than nerve lesion. The results do not exclude, however, that other intraocular structures such as the iris or ciliary body may contribute secondarily to the production of neurotrophic agents in response to the phacolytic events. This is of particular relevance, because NT-4 and BDNF mRNAs were detected in the iris.12 BDNF and NT-3 mRNAs are thought to play a regulatory role in the developing mouse eye including the lens epithelium.29 However, the Western blot analysis performed in the present work did not reveal detectable protein levels of BDNF in the lens homogenates. This is explicable by either a downregulation of protein in the adult lens or by low-level protein production not detectable by the monoclonal antibodies. Cellular candidates for release of such factors may be Schwann cells in the iris,30 iris muscle cells,31 or glial cells that are activated after lens injury, or even immigrating immune cells. Regardless of which cells produce these additional neurotrophic or protective factors, it appears from the present work that the lens injury and not the ocular injection itself initiates their production, and thus causes the marked survival effects on RGCs.
In a number of ganglion cell rescue studies, it has been reported that intraocular injection of either of several molecules results in prolonged survival of ganglion cells. These studies include injection of NGF,9 bFGF,10 BDNF, CNTF,11 32 and inhibitors of caspases.16 17 In addition, intraocular injection of CNTF results in better axonal regeneration within a peripheral nerve graft.14 However, the exact methodologies of injection and in particular whether injection was accompanied with lens injury is not described in any of these studies. The only description of performing such injury in connection with survival was in a study by Mansour–Robaey et al.,12 whereas further experiments applying intravitreal injections were performed without lens injury and using transscleral perforation route 3.11 13 Injections of neurotrophic factors to rescue retinal photoreceptors in dystrophy models were performed with bFGF and CNTF.18 19 Although these investigators also reported higher survival with injection of PBS, there is no clear determination of whether this effect was due to lens injury or not. The present immunohistochemical and Western blot studies did not show abundant production of any of the aforementioned factors in the normal or the injured lens. However, these factors may be produced at lower concentrations not detectable with the methods used in the present study. Although transfer of peripheral nerve pieces into the vitreous body resulted in regeneration of ganglion cell axons within the severed ON,33 this study does not report whether the lens was injured.
The lens is an evolutionary highly conserved tissue that subserves refractive functions and therefore contains very high amounts of soluble structural proteins called crystallins.34 35 The complete encapsulation of the tissue keeps it protected from vascularization, whereas the subcapsular epithelial layer serves as a stem cell population to recruit equatorial addition of new fibers (Fig. 1) . A remarkable feature of this process of cell proliferation and transformation of cells into fibers occurs by incomplete autophagy and subsequent elongation of the crystallin-containing fibers.
The transformation occurs throughout life with the crystallins being the structural proteins in vertebrates with the longest turnover half-life. In addition to functioning as source of transparent lens fibers, the lens epithelial cells contain high amounts of factors that are essential to the protection of the tissue from light-induced damage—for instance solar UVA and UVB irradiation.36 37 38 Such cell survival factors may account for the neuroprotection of ganglion cells as well.22 23 24 25 The importance of lens factors in rescuing retinal cells has also been demonstrated by showing that lensectomy and vitrectomy but not vitrectomy alone decrease the rate of photoreceptor loss in transgenic pigs.39 It seems therefore reasonable to be aware of accidental lens injury and avoid it, if the scope of a study is targeted to intraocular injection of neuroprotective or neurotrophic agents to rescue retinal cells in the various retinodegenerative diseases.
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Acknowledgements |
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Footnotes |
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Submitted for publication March 20, 2000; revised June 13, 2000; accepted July 11, 2000.
Commercial relationships policy: N.
Corresponding author: Solon Thanos, Department of Experimental Ophthalmology, School of Medicine, University of Münster, Domagkstraße 15, D-48149 Münster, Germany. solon{at}uni.muenster.de
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Y. Yin, Q. Cui, Y. Li, N. Irwin, D. Fischer, A. R. Harvey, and L. I. Benowitz Macrophage-Derived Factors Stimulate Optic Nerve Regeneration J. Neurosci., March 15, 2003; 23(6): 2284 - 2293. [Abstract] [Full Text] [PDF]
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