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1 From the Department of Ophthalmology, 2 The Dana Center for Preventive Ophthalmology, and 3 Departments of Molecular Biology and Genetics and Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland.
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Abstract |
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METHODS. Eyes were studied in masked fashion by light and electron microscopy and by immunohistochemistry with antibodies directed against the tyrosine kinase receptors (TrkA, B, and C) and against brain-derived neurotrophic factor (BDNF), as well as by autoradiography to identify retrograde axonal transport of 125I-BDNF injected into the superior colliculus.
RESULTS. With acute glaucoma in the rat, RGC axons became abnormally dilated, accumulating vesicles presumed to be moving in axonal transport at the optic nerve head. Label for TrkB, but not TrkA, was relatively increased at and behind the optic nerve head with IOP elevation. Abnormal, focal labeling for TrkB and BDNF was identified in axons of monkey optic nerve heads with chronic glaucoma. With acute IOP elevation in rats, radiolabeled BDNF arrived at cells in the RGC layer at less than half the level of control eyes.
CONCLUSIONS. Interruption of BDNF retrograde transport and accumulation of TrkB at the optic nerve head in acute and chronic glaucoma models suggest a role for neurotrophin deprivation in the pathogenesis of RGC death in glaucoma.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RGC apoptosis has been demonstrated in human eyes with glaucoma, in experimental glaucoma, and after optic nerve transection in monkeys and rats.4 5 6 7 8 Furthermore, evidence from both human eyes and experimental models suggests that glaucoma obstructs anterograde and retrograde axonal transport in RGC axons at the nerve head.9 10 11 12 13 RGC axons undergoing transport obstruction are distended at the nerve head by membrane-bound vesicles known to move bidirectionally between the cell body and axon terminal.12 13 We hypothesize that transport obstruction could prevent the arrival at the cell body of molecules required for the health of RGCs and thereby result in apoptotic cell death.
Neurotrophins such as brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin 3 (NT-3), and neurotrophin 4,5 (NT-4/5) are ideal candidates for factors whose transport interruption could lead to RGC death. Neurotrophins interact with specific receptors, including TrkA, TrkB, and TrkC receptors that are primarily activated by NGF, BDNF, both NT-4/5 and NT-3, respectively. Once activated, neurotrophin/receptor complexes are retrogradely transported to the cell body.14 15 16 17 18 Neurotrophins, particularly BDNF, are known to influence RGC survival in vitro, both during retinal development and after lesioning.19 20 21 22 23 24 25 26 27 28 29 30 31 32 During development, RGCs are produced in excess, and then neurons whose axons fail to contact the correct target cell die, possibly because of lack of appropriate neurotrophin stimulation.33 34 35 36 Although studies of neurotrophins in the retina have focused primarily on their developmental role, adult RGCs also synthesize neurotrophins, exhibit TrkB receptors,23 26 28 and respond to BNDF.20 22 31 32 Continued neurotrophin dependence among adult RGCs was suggested by a study in which cat RGCs died after their target neurons in the dorsal lateral geniculate nucleus were eliminated with kainic acid.37
These findings suggest the hypothesis that RGC death can be initiated by retrograde axonal transport blockade of neurotrophins that normally arrive from central target cells. In this theory, the optic nerve head damage in glaucoma could act as a physiological "transection of the axon." To investigate this possibility, we studied ultrastructural alterations and immunohistochemical localization of BDNF, TrkB, TrkA, and TrkC in optic nerve heads of rats with acute IOP elevation. Furthermore, we localized BDNF and the TrkB receptor in monkey eyes with unilateral, chronic experimental glaucoma and with optic nerve axotomy. Finally, we studied the effect of acute IOP elevation on retrograde transport of radiolabeled BDNF in rats to determine directly if there was disruption of the axonal transport of neurotrophins.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Among the 31 acute IOP model rats, 23 were studied for ultrastructure or immunohistochemistry of Trk receptors and BDNF. Twelve of these rats had unilateral IOP elevation equal to mean BP (PP = 0) and 11 at 25 mm Hg below mean BP (PP = 25). Eyes from half of each perfusion group were fixed overnight by immersion in 2% paraformaldehyde/2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2, for subsequent embedding in epoxy resin and for light and electron microscopy. The eyes of the other 12 animals were fixed for 4 hours in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.2, with paraffin embedding for immunohistochemistry.
Eight of the 31 rats were first stereotactically injected with 0.75 µCi of 125I-BDNF (specific activity = 162 µCi/µg; NEN Life Science Products, Boston MA) into the superior colliculus on both sides of the brain. Within 15 minutes of collicular injection, IOP was elevated. Four rats had IOP elevations in one eye to PP = 0 and the other four were maintained at PP = 25 for 4 hours. These eyes were fixed overnight by immersion in 2% paraformaldehyde/2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2, for subsequent embedding in epoxy resin for autoradiography.
Chronic Experimental Glaucoma Model
Six male juvenile cynomolgus monkeys (Macaca
fascicularis) were treated with an argon laser one or more times
to scar the trabecular meshwork in one eye, increasing IOP and
producing experimental optic nerve injury similar to that of human
glaucoma.38
IOP was measured weekly throughout the
treatment period with the Tonopen XL (Mentor), and clinical examination
of the fundus was performed monthly to monitor the progression of the
disease. After 4 months to 2 years, animals were euthanatized by
pentobarbital overdose, eyes were rapidly enucleated, and optic nerve
heads were dissected and fixed in 4% paraformaldehyde in 0.1 M
phosphate buffer, pH 7.2, for 4 hours. Optic nerve cross sections were
embedded in epoxy resin to stage the degree of axon loss as mild,
moderate, or severe. This was done by comparison of the remaining
neural area to their normal fellow eyes, as well as to a large number
of additional, control and glaucoma nerves from previously published
experiments in which axon counts were used to validate the three-level
severity grading7
used here. Optic nerve heads were
processed into paraffin for immunohistochemical staining.
Optic Nerve Axotomy Model
Two male juvenile cynomolgus monkeys underwent unilateral
intraorbital optic nerve axotomy to produce specific RGC
death.39
Fibers were severed posterior to the exit of the
central retinal artery and vein to avoid retinal ischemia. Patency of
the retinal circulation was verified within 1 week of the procedure by
fluorescein angiography. Five months later, animals were killed by
pentobarbital overdose, and tissues were prepared as described for
glaucoma monkeys.
All animal procedures were approved and supervised by the Animal Care Committee of the Johns Hopkins University School of Medicine and adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
Transmission Electron Microscopy
Posterior globes of rat eyes (retina, choroid, sclera, optic nerve
head, and optic nerve) were postfixed in 1% buffered osmium tetroxide,
dehydrated in graded ethanol, and embedded in LX112 epoxy resin (Ladd
Research Industries, Burlington VT). The area centered on the optic
nerve head was thin-sectioned and stained by 5% uranyl acetate and
Reynold’s lead citrate before examination in the transmission electron
microscope (JEOL 100CX; JEOL USA, Peabody, MA).
Autoradiography
Plastic-embedded segments of the rat eyes containing 3 to 4 mm of
retina, the optic nerve head, and up to 3 mm of optic nerve were
sectioned at 1 µm and dipped in undiluted Kodak NTB-2 liquid emulsion
(Scientific Imaging Systems/Eastman Kodak Company, Rochester, NY).
After 2 months’ exposure, these were developed, and the number of
grain tracks corresponding to radioemissions were counted in masked
fashion from six sections per eye. The number of tracks per square
micrometer of tissue was counted in six retinal areas, in the optic
nerve head, and in three optic nerve areas on each slide. The mean
values for retina, nerve head, and optic nerve were calculated. We
recognized that there were some grains overlying the outer retina and a
very small number overlying the vitreous humor. Counts were divided
into those from the inner and outer retina, including the inner
plexiform layer with the inner retina and the inner nuclear layer with
the outer retina. Grain counts overlying the outer retina and from
three areas overlying the vitreous on each slide were subtracted from
the inner retinal values to yield data that most likely represented
specifically transported BDNF. To provide additional confirmation that
the observed radioactivity over the inner retina represented axonally
transported BDNF, we performed injections into the colliculus in four 4
additional animals. These were made into only the colliculus on one
side of the brain (unilateral injection). The injection consisted of a
mixture of 125I-BDNF (0.63 µCi) and either
100-fold excess of nonradioactive NGF (2 animals) or 100-fold excess of
nonradioactive BDNF (2 animals). It would be expected that a large
excess of nonradioactive BDNF would decrease axonal transport of the
labeled BDNF, whereas NGF in excess would not compete effectively for
the TrkB receptor and labeled BDNF transport would be unaffected.
The 125I-BDNF was unaffected by labeling in its biological behavior, demonstrating specific binding to HEK293T cells transfected with TrkB, but not to untransfected cells (David D. Ginty, personal communication).
Immunohistochemistry
Seven-micrometer longitudinal paraffin sections of rat and monkey
optic nerve heads were collected onto Superfrost Plus slides (Fisher
Scientific; Pittsburgh, PA) before immunolabeling by the
streptavidin-biotin peroxidase technique of Lutty et al.40
The affinity-purified rabbit polyclonal antibodies were directed
against BDNF and against the neurotrophin receptors TrkB, TrkA, and
TrkC (Santa Cruz Biotechnology, Santa Cruz, CA). For the colabeling of
TrkB and GFAP, an affinity-purified goat polyclonal antibody directed
against TrkB was used (Santa Cruz Biotechnology). The antibody
recognizing human BDNF was raised against a peptide corresponding to
amino acids 128 to 147. Both the rabbit and goat polyclonal TrkB
antibodies were directed against an amino acid sequence 794 to 808,
located within the intracellular portion of the full-length murine
gp145TrkB receptor molecule. The TrkA antibody was raised
against a peptide that corresponds to amino acids 763 to 777, adjacent
to the carboxyl-terminus of the precursor form of the porcine
gp140Trk receptor molecule, and the TrkC antibody was
directed against a peptide corresponding to amino acids 798 to 812 of
the precursor form of porcine TrkC, gp140TrkC. TrkB, TrkA,
and TrkC antibodies were noncrossreactive with one another, as
determined by the supplier with immunoprecipitation. Western blot
analysis confirmed the presence of bands at the appropriate molecular
weight (data not shown). Primary antibodies were applied at 0.5 to 1.0
µg/ml. Negative controls included nonimmune serum of the same species
as the primary antibody at the same protein concentration, primary
antibody preincubated with fivefold excess control peptide, and
incubation buffer alone. Labeled sections were mounted in glycerol
jelly and viewed by Nomarski optics (Zeiss Axioskop; Carl Zeiss Inc.,
Thornwood, NY).
In addition, a series of rat and monkey optic nerve head sections were colabeled with goat anti-TrkB and a rabbit polyclonal antibody directed against bovine GFAP (Dako Corporation, Carpinteria, CA) to ascertain if TrkB label was localized in astrocytes. For this experiment, goat anti-TrkB was followed by donkey anti-goat biotinylated secondary antibody (Santa Cruz Biotechnology) and Cy3-labeled streptavidin (Jackson ImmunoResearch Laboratories, West Grove, PA), respectively. After reblocking in normal serum, rabbit anti-GFAP was applied followed by fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit secondary antibody (Santa Cruz Biotechnology). Sections were mounted in Vectashield mounting media (Vector Laboratories, Burlingame, CA) and viewed on a confocal microscope (Zeiss LSM 410; Carl Zeiss Inc.). Images of Cy3-TrkB label were collected using a helium-neon laser at 543-nm excitation, with a 570-nm longpass filter, whereas images of FITC-GFAP were collected using a krypton-argon laser at excitation 488 nm, with a band pass of 505 to 580 nm. Cy3-TrkB images were pseudocolored red and FITC-GFAP green before their superimposition.
Because retinas from the monkey nerve heads had been removed for other studies, additional retinal samples from archival control monkey eyes were labeled with goat anti-trkB as described above. Epifluorescent images of the Cy3-TrkB label were collected with the Zeiss Axioskop using a rhodamine filter (Carl Zeiss Inc.).
Masked Evaluation of BDNF and TrkB Label in Rat and Monkey
Experimental Models
A masked evaluation of control and experimental rat eyes based on
their TrkB and BDNF labeling was performed. The staining intensity of
the nerve fiber layer (NFL), ganglion cell layer (GCL), superficial
optic nerve head, optic nerve head, nonmyelinated zone just behind the
globe, and myelinated optic nerve were noted. In normal rat eyes, TrkB
label was uniformly present from the retina through the optic nerve
head into the optic nerve. For each rat eye, the degree of difference
from normal after IOP elevation was graded on a four-level scale from 0
(normal) to 3 (most abnormal). This score summarized both general
alterations in the distribution of label and focal label accumulation.
In normal rat eyes and in those with acute IOP elevation, BDNF labeling was barely detectable even with the optimal protocol.
In monkey tissues, a masked examination for BDNF and TrkB labeling of the glaucoma and transection eyes and fellow eye controls was performed. In the normal monkey eye, TrkB labeling was somewhat heavier in the NFL and nerve head than in the myelinated optic nerve. Previous demonstrations of axonal transport interruption in monkeys with radiolabeled materials showed that IOP-induced blockade is associated with an increase in the labeling in the nerve head compared to the retina and the optic nerve.10 This pattern was present with the immunohistochemical techniques used here. Each monkey nerve head was placed into one of three categories: (1) normal pattern; (2) borderline (some relative increase in nerve head label); or (3) abnormal (definitely increased label in nerve head relative to retina and optic nerve). We also noted the presence of focal accumulations of TrkB and BDNF in the nerve head.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Trk and BDNF Labeling in Normal Rat Retina and Optic Nerve Head
In the normal rat, TrkB staining was found in the NFL, GCL, and
inner plexiform layer, with the most intense labeling in the NFL (Fig. 2A
). (Findings in the outer retina will be reported separately by
McKinnon et al.) TrkB label was uniformly distributed in the neural
bundles of the optic nerve head and optic nerve (Fig. 2D)
. Glial cells
of the nerve head also were labeled, but at an intensity less than that
of the axonal fibers. There was no labeling in slides with primary
antibody blocked with excess control peptide (Figs. 2C
2F)
, nor in
slides stained with nonimmune IgG or incubation buffer (data not
shown).
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Trk Localization with Acute IOP Elevation in Rats
In five of six PP = 0 eyes, TrkB labeling was more intense
than in normal retinas in the NFL and GCL (Fig. 2B)
. In the optic nerve
head, focal accumulations of antibody label were identified in neural
bundles passing through the nerve head (Fig. 2E)
. Similar accumulations
also were found in the unmyelinated optic nerve and occasionally among
myelinated axons (Figs. 2G
2H)
. In areas with focal labeling, both
dilated and intact axons were densely labeled for TrkB. Eyes at PP = 25 had less intense accumulation of TrkB label than that of eyes at
PP = 0 at the same antibody concentration. The PP = 0 eyes
had a statistically higher mean severity score (2.2) than the PP =
25 eyes (mean = 2.0) or the normal eyes (mean = 0.8). The IOP
ranges were 51 to 81 mm Hg for the PP = 0 group and 19 to 58 mm Hg
for the PP = 25 group (Table 1)
. In two eyes noted to have some leakage at the corneal needle,
the IOP appeared to be above the normal range during the experiment. We
did monitor the IOP levels by Tonopen XL tonometry and did not confirm
that these eyes failed to achieve the desired IOP. However, it was
interesting to us that their TrkB results were closer to eyes with
normal IOP than to the other eyes with elevated IOP.
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Two interesting results occurred as byproducts of the experimental system. First, some animals had relatively low BP during the 4-hour experiment, causing the actual PP in control eyes with lower BP to be close to that in PP = 25 rats with normal BP. To assess the effect of this factor, the actual PP of the experimental and control eyes was compared to their severity score (Table 1) . Abnormality in TrkB labeling closely followed actual PP, with the majority of eyes less than 30 mm Hg having severity scores of 2 or 3, and those with higher PP usually scoring 0 or 1. Another link between PP and transport block was the fact that four of the five controls that were graded as abnormal in TrkB labeling had low BP throughout the experiment. With controls divided into those whose PP was between 25 and 50 mm Hg and those higher than 50 mm Hg, the mean severity scores of the groups were 1.6 and 0.2, respectively (Table 1) . In a few eyes, there was detectable leakage around the corneal cannula, leading to a failure to achieve the desired IOP for some portion of the experiment. Consistent with this interpretation, two of four experimental eyes with a normal TrkB label pattern by masked grading were noted during the time of surgery to have leakage of fluid at the corneal cannula (Table 1) .
TrkA and BDNF localization in the retina, nerve head, and optic nerve was not detectably altered by acute IOP elevation in areas occupied by RGC and their axons (data not shown).
Autoradiography of 125I-BDNF
The eight eyes with IOP elevation had a 60% reduction in
transported radioactivity overlying the inner retina (48.8 ± 41.9
grain tracks/µm2) compared to the mean of
control eyes that also had bilateral colliculus injections of
125I-BDNF (121.8 ± 22.1 grain
tracks/µm2, P = 0.002) (Fig. 3)
. The values for the PP = 25 and the PP = 0 eyes were both
depressed to a similar degree. In the two animals in which competitive
inhibition was tested by unilaterally injecting a mixture of 100-fold
excess unlabeled BDNF with 125I-BDNF into the
superior colliculus, there was a 37% decrease in radioactivity
arriving at the retina (60.8 ± 15.8 compared to 96.8 ± 46.2
grain tracks/µm2 for matched control animals
that had unilateral collicular injection of
125I-BDNF). Coinjection of cold NGF with
125I-BDNF in two animals did not cause any
decrease in transport (94.2 ± 19.4 compared to 96.8 ± 46.2
grain tracks/µm2 for the control eyes).
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Quantification of Masked Evaluation of TrkB Labeling in Monkey
Models
Eight control, six glaucoma, and two transection nerve heads were
graded by a masked observer. Seven of eight controls were graded
normal, and the eighth was graded as borderline (Tables 3 4)
. Five of the six experimental glaucoma nerve heads had increased nerve
head accumulation of TrkB. The sixth glaucoma nerve head had severe
atrophy, eliminating any RGC label. Focal TrkB accumulation was seen in
five of six glaucoma eyes, but in only one of eight controls. The
differences between control and glaucoma eyes were significant
(P = 0.026, Fisher’s exact test; Table 4
). Focal TrkB
accumulation observed in transection eyes was associated with glia and
axons were uniformly absent (Fig. 4F)
. Because of the limited number of
monkey sections available, quantification of the findings from the
masked evaluation of BDNF labeling was not performed.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our data suggest that TrkB receptors moving in axonal transport within optic nerve fibers are blocked at the optic nerve head in rat and monkey eyes in glaucoma models. Acute IOP elevation causes disruption of axonal transport in RGCs, indicated by accumulation of smooth-surfaced vesicles within axons, a finding that is similar to observations after chronic IOP elevations in rat eyes.42 Such vesicle collections colocalize with blocked radioactive protein moving in anterograde axonal transport after acute IOP elevation in the monkey.9 10 12 13 Retrograde transport of horseradish peroxidase was found to be obstructed at the site of such vesicular accumulation with short-term IOP elevation in monkey eyes.11 Acute IOP elevation has been previously shown to obstruct retrograde axonal transport in rat eyes.43 The dramatic swelling of axons just behind the sclera in the rat optic nerve has not been described after acute IOP increase in monkey eyes. In monkeys, axons are myelinated immediately behind the nerve head, perhaps preventing the swelling observed in these nonmyelinated segments of rat axons. The frequent association of these clear areas with accumulations of intracellular vesicles and mitochondria suggests transport obstruction.
The immunohistochemical findings provide further evidence for transport obstruction that affected the distribution of BDNF and TrkB in experimental glaucoma. The normal pattern of TrkB labeling was altered in four important ways in both rat (acute model) and monkey eyes (chronic model). These were (1) alteration of normal TrkB axonal distribution, (2) focal accumulations of TrkB and BDNF, (3) increased label in GCL neurons for TrkB, and (4) increased TrkB label in glia.
In seven of nine rat eyes at PP 
25 mm Hg, TrkB label was more
apparent in the optic nerve than in the retina. Because the degree of
labeling varies from slide to slide, we depended on masked comparison
within each slide of intraretinal, nerve head, and optic nerve label to
make reproducible, valid judgments. Relative increase in axonal
labeling behind the nerve head compared to labeling within the eye
indicates obstruction of the retrograde TrkB receptor movement. In
monkeys with chronic glaucoma, TrkB labeling increased in the nerve
head compared to the retina or optic nerve, and BDNF and TrkB
accumulated focally in the nerve head. In addition, acute IOP elevation
in the rat decreased retrograde transport of radioactive BDNF. In eyes
with PP = 0, the IOP level clearly impaired retinal blood flow,
but in PP = 25 eyes, blood flow was normal, yet IOP elevation
blocked BDNF retrograde movement.
We did not demonstrate labeling for BDNF either normally or with acute IOP elevation in rats, though there was focal BDNF accumulation by immunohistochemistry in chronic monkey glaucoma eyes. This may be due to species differences in the specificity of the antibody to human BDNF used here.
TrkB receptors have been shown to be internalized after ligand binding and move to the cell body by retrograde transport in activated form within smooth-surfaced vesicles.14 15 16 17 44 We observed the accumulation of such vesicles in both the chronic glaucoma model in monkeys12 and in acute rat model eyes by electron microscopy. The intensely stained, focal collections of TrkB and BDNF label probably represent activated TrkB receptor/BDNF complexes at the nerve head level. It would be logical that significant IOP elevation would lead to a generalized breakdown in transport of many molecules. Yet, although we found TrkA to be present in axons, we found no change in distribution of this receptor. Either our methods did not produce sufficient labeling to observe transport obstruction, or the blockade affects TrkB more than TrkA.
There was a relative increase in TrkB labeling of GCL cell bodies in both rats with acute IOP elevation and in the chronic monkey model. In normal rat eyes, there was minimal label overlying cells in the GCL at antibody concentrations that intensely label axons. With IOP elevation, the neuronal cytoplasm was more darkly labeled than axons by TrkB antibodies. This could result from decreased receptor proteolysis, a failure of synthesized receptor to leave the cell body, increased production of receptor, or increased arrival of receptors from the axon-terminal. Increased arrival is made unlikely by the evidence for blockade of retrograde transport. Immunohistochemical methods cannot measure increased receptor synthesis. Such a response might be an interesting feedback response to lower levels of activated TrkB caused by the blockade of retrograde transport from experimental glaucoma.
The increased TrkB label over glial cells in the nerve head and optic nerve with experimental glaucoma and transection appeared to be associated with astrocytes, as shown by cells double-labeled for TrkB and GFAP. In the retrobulbar nerve, increased glial labeling might involve oligodendrocytes, astrocytes, or microglia, perhaps by increased production of TrkB by glia, which normally synthesize both full-length and truncated TrkB.45 46 47 48 The antibodies used here recognize the full-length TrkB. Alternatively, glia could have acquired more receptor by phagocytosing axonal debris as neurodegeneration occurred.12 49
Two technical issues in the acute rat model are worthy of comment. Leakage around the anterior chamber cannula was detected in 4 of 11 rats. Three of these four failed to show alteration in TrkB labeling. In subsequent eyes without visible leakage, abnormal TrkB labeling was present on masked evaluation in each case. Secondly, 5 rat eyes at normal IOP had increased labeling in the retina compared to the optic nerve, instead of the normal, uniform TrkB label seen in 6 other controls. These 5 animals had mean BP of 54 ± 8.8 mm Hg compared to 76 ± 5.0 mm Hg in the other controls (t-test, P = 0.000). Excessive pentobarbital anesthesia may have lowered BP in these animals, and this was corrected by lower doses in later animals. The control eyes with lower BP therefore had PP intermediate between eyes with high IOP and the other controls (mean actual PP = 38.3 ± 8.9 mm Hg, compared to mean of other 6 controls = 60.0 ± 6.3 mm Hg; t-test, P = 0.001). This is probably explains their intermediate levels of abnormality in TrkB labeling.
Although a masked grader evaluated rat and monkey tissues, severe chronic glaucoma injury and axotomy produce alterations that are clearly visible to the experienced observer. However, in two monkey eyes with mild optic nerve fiber loss and no histologically visible atrophy, there was relative increase and focal accumulation of TrkB in the nerve head.
The additional experiments with acute IOP elevation in rats after 125I-BNDF injection into the superior colliculus show that movement of axonally transported neurotrophin is substantially inhibited by increased IOP. Furthermore, the data with coinjection of nonradioactive NGF and BDNF show that only the latter affects the arrival of 125I-BNDF in the retina. This strongly supports the concept that the movement of BDNF is being mediated by the TrkB receptor. Hence, our studies of movement of the TrkB receptor are made more relevant to delivery of BDNF under normal conditions in the adult rat eye and to inhibition of neurotrophin delivery in experimental glaucoma.
Our interest in TrkB receptors derived from the fact that its ligand BDNF is known to be an important trophic factor for RGCs. BDNF supports the survival of retinal explants and cultures, inhibits apoptosis, and increases the rate of axonal elongation.19 20 24 50 51 Rat RGCs have been reported to demonstrate a modestly extended life after axotomy when BDNF is injected intravitreally.22 24 27 31 32 52 Our experiments suggest that signaling from one endogenous source of BDNF, target cells in the superior colliculus (rat), and lateral geniculate (monkey) may be interrupted in glaucoma, thereby affecting the influence this pathway has on RGC survival. These results support the hypothesis that trophic actions of tyrosine kinase receptor-mediated mechanisms on RGCs may be an important component of the pathway from axonal injury to RGC death.
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Acknowledgements |
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Footnotes |
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Submitted for publication May 14, 1999; revised September 15, 1999; accepted October 26, 1999.
Commercial relationships policy: N.
Corresponding author: Harry A. Quigley, Wilmer 120, 600 North Wolfe Street, Johns Hopkins Hospital, Baltimore, MD 21287. hquigley{at}jhmi.edu
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