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Promotion
of axon regeneration and inhibition of astrocyte activation by alpha
A-crystallin on crushed optic nerve
Wei-Yang Shao1,2,
Xiao Liu1,2, Xian-Liang Gu1,2, Xi Ying1,2, Nan
Wu1,2, Hai-Wei Xu1,2, Yi Wang1,2
1Southwest Hospital/Southwest
Eye Hospital, Third Military Medical University, Chongqing 400038, China
2Key Lab of Visual
Damage and Regeneration & Restoration of Chongqing, Chongqing 400038, China
Correspondence to: Yi Wang. Southwest Hospital/Southwest Eye
Hospital, Third Military Medical University, Chongqing 400038, China. yiwangeye2015@163.com
Received: 2016-03-12 Accepted:
2016-05-25
Abstract
AIM: To explore the effects of αA-crystallin in astrocyte gliosis after optic nerve
crush (ONC) and the mechanism of α-crystallin in neuroprotection and axon
regeneration.
METHODS: ONC was established on the
Sprague-Dawley rat model and αA-crystallin (10-4 g/L, 4 μL) was intravitreously injected into the
rat model. flash-visual evoked
potential (F-VEP) was examined 14d after ONC, and the glial fibrillary acidic
protein (GFAP) levels in the retina and crush site were analyzed 1, 3, 5, 7 and
14d after ONC by immunohistochemistry (IHC) and Western blot respectively. The
levels of beta Tubulin (TUJ1), growth-associated membrane phosphoprotein-43
(GAP-43), chondroitin sulfate proteoglycans (CSPGs) and neurocan were also
determined by IHC 14d after ONC.
RESULTS: GFAP level in the retina and the optic
nerve significantly increased 1d after ONC, and reached the peak level 7d
post-ONC. Injection of αA-crystallin significantly decreased GFAP level in both
the retina and the crush site 3d after ONC, and induced astrocytes architecture
remodeling at the crush site. Quantification of retinal ganglion cell (RGC)
axons indicated αA-crystallin markedly promoted axon regeneration in ONC rats
and enhanced the regenerated axons penetrated into the glial scar. CSPGs and
neurocan expression also decreased 14d after αA-crystallin injection. The
amplitude (N1-P1) and latency (P1) of F-VEP were also restored.
CONCLUSION:
Our results suggest α-crystallin promotes
the axon regeneration of RGCs and suppresses the activation of astrocytes.
KEYWORDS:
αA-crystallin; axonal regeneration;
astrocyte; glial scar; chondroitin sulfate proteoglycans; optic nerve crush
DOI:10.18240/ijo.2016.07.04
Citation: Shao WY, Liu X, Gu XL, Ying X, Wu N, Xu
HW, Wang Y. Promotion of axon regeneration and inhibition of astrocyte
activation by alpha A-crystallin on crushed optic nerve. Int J Ophthalmol 2016;9(7):955-966
INTRODUCTION
Acute optic nerve injury, which is caused
by trauma, ischemia or glaucoma, often leads to retinal ganglion cell (RGC)
damage accompanied by the activation of astrocytes, microglial cells and
oligodendrocytes[1-4].
The cellular response to injury includes migration to the injury site,
proliferation and the secretion of inhibitory molecules and proteins to form an
unfavorable environment for axon regeneration. Astrocytes are a type of cells
that mainly respond to injury to form glial scar at the crush site; they also
secrete inhibitory extracellular matrix (ECM) molecules, such as chondroitin
sulfate proteoglycan (CSPG), which had been thought play an important role in
axon regeneration[5-6].
Some methods were previously reported to rescues the RGC survival and promote
axon regeneration, including enhancing the neurotrophic factor support[7], interfering with the
apoptotic signaling through caspase-3 and RhoA/Rho-kinase (RhoA/Rock) pathway
to reduce the RGC loss[8-9],
promoting the intrinsic capability for axon regeneration, and manipulating the
inhibitory physical and chemical barrier, which was also thought to be an ideal
strategy to enhance neurite outgrowth[4,9-10].
All of the above strategies were limited in improving RGC survival or
short-distance axon regeneration; therefore, it is necessary to identify
multi-target molecules and comprehensive interventions to promote highly
effective axon regeneration.
It is known that the heat shock protein
(HSP) α-crystallin consists of noncovalently associated A and B subunit, and
plays a crucial role in RGC survival and axon regeneration[11-13]. α-crystallin acts as a therapeutic protein
through its anti-apoptotic, anti-inflammatory, anti-aggregation and other
activities[14]. It has
previously been reported that lens injury could stimulate axon regeneration in
the optic nerve cut model and that the growth cone might reach the
retinoreceptive layer of the superior colliculus at 5wk after optic nerve
lesion[15]. α-crystallin
promotes axon outgrowth by regulating the RhoA/Rock signaling pathway[16], α-crystallin also
promotes rat retinal neurite growth on myelin substrates in vitro[17],
promotes RGCs survival and inhibits microglial activation in vivo[3].
These studies showed that α-crystallin might be a multi-target molecule. However,
it remains unclear why the axon could significantly penetrate the physical and
chemical barrier following α-crystallin treatment. Our hypothesis is that
α-crystallin can directly influence the astrocyte response to injury. We
previously observed that αA-crystallin could suppress the activation and
proliferation of astrocytes in vitro,
and the astrocyte cell scratch assay also showed that a higher concentration,
10 μg/mL, could inhibited astrocyte migration. These results suggested that
αA-crystallin could interact with astrocytes and influence astrocyte
activation.
αA-crystallin shares approximately 55%
sequence identity at the amino acid level with αB-crystallin, which is
abundantly expressed in the ocular system. In this study, we investigated
whether αA-crystallin could enhance axon regeneration by suppressing astrocyte
activation and secretion of inhibitory factors.
MATERIALS
AND METHODS
Adult (150-200 g) female Sprague-Dawley
rats were used for the experiments. All experimental protocols were approved by
the Institutional Animal Care and Use Committee of the Third Military Medical
University, Chongqing, China. All procedures were conducted in accordance with
the Institutional Animal Care and Use Committee of the Third Military Medical
University for the use of animals in ophthalmic and vision research. The
animals were housed with standard chow and water ad libitum, and sustained on a
12h:12h light and dark cycle at a temperature of 21℃-25℃.
Optic Nerve
Crush and Intravitreous Injection The optic nerve crush (ONC) injury model
was performed as previously reported[13,16].
Briefly, the eight-week-old adult rats (150-200 g) were anesthetized by an
intraperitoneal injection of 10% chloral hydrate (0.4 g/kg, it was
allowed to anesthetize rodent animals in China). A 0.5-1 cm incision was made
in the temporal conjunctiva of each eye under a microscope, and 3-5 mm optic
nerve was bluntly exposed. The optic nerve was clamped at 2 mm behind the eyeball
for 10s using an atraumatic artery clamp to cause moderate injury. By avoiding
the injury site, the appearance of the ophthalmic artery and the vascular
integrity of the retina were verified by funduscope examination; the cases in
which the retinal vascular integrity was in question were excluded from the
group. For the sham operation, only 3-5 mm of the optic nerve was exposed.
αA-crystallin (10-4 g/L; 4 μL) [Recombinant Human Crystallin Alpha
A, Cellsciences, USA, Lot no. 3172602, purity: >95% as determined by
reversed phase high-performance liquid chromatography and sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analyses], which was
dissolved in sterile phosphate buffer solution (PBS), was injected into
vitreous cavity using a 10 μL microinjector (made in Ninbo, China) by a
posterior approach to the left eye, taking care not to injure the lens. The
same volume (4 μL) of sterile PBS was injected in the right eye after ONC. A
topical anti-inflammatory ointment was embrocated on the eye surface after
operation. Food and water were provided ad libitum post-injury.
Visual Electrophysiology
Investigation The visual electrophysiology method was
performed according to previously reported protocol[18-19]. Briefly, the rats were maintained with a
12h:12h light dark cycle (7:00 a.m.-7:00 p.m.). The flash-visual evoked
potential (F-VEP) was recorded before the operation, 12h after the operation
and 14d after the operation. Each rat was adapted to a dark room and prepared
under long-wave form red light when the F-VEPs were being recorded. The animals
were anesthetized and immobilized on a special supporter, and the pupils were
dilated with one drop of tropicamide. Three needle electrodes were inserted
subcutaneously, one was inserted at that middle point of the two eyes at a 0.5
cm distance close to the nasal side and served as the reference lead, another
was inserted in the sagittal suture near the visual cortex and served as a
recording lead, and the last one was placed in the tail and served as the
ground lead. The stimulus was a LED flash intensity of 3.93 cd/m2•s with 1 Hz frequency and a band pass width of 1-100 Hz, and
the stable waveforms were superimposed 100 times using the Reti-scan system
(Roland, Germany). When one eye was recorded, the other one was covered. Each
eye was recorded three times with a 5min interval. The recorded F-VEP waveform
was labeled with the latencies N1, P1 and N2 (response time, ms) and amplitudes
N1-P1 (delta between trough N1 and peak P1, μv).
Tissue
Preparation and Fixation The rats were anesthetized by an
intraperitoneal injection of 10% chloral hydrate (0.4 g/kg) and perfused
through the heart with 0.9% normal saline followed by 4% paraformaldehyde
(PFA). The eye cups and optic nerves were carefully isolated and post-fixed for
2h with 4% PFA before being incubated in 30% sucrose/PBS overnight, followed by
freezing in opti-mum cutting temperature compound (O.C.T. Compound)
(Sakura, USA) cryopreservation medium and storage at -80℃. The O.C.T. Compound embedded eyecups
generate 10 μm-thick frozen sections as well as longitudinal optic nerve
sections. The sections were preserved at -20℃ until further use.
Immunofluorescence The sections were immunofluorescently stained as previously
described[20-21]. The
sections were dried in air, washed in PBS three times for 5min each, and
subsequently incubated in 0.3% Triton X-100 (Sigma-Aldrich, USA) for 10min
before incubation in blocking solution (8% goat serum, diluted in PBS) for
60min at room temperature. The sections were then incubated with the following
primary antibodies in 3% goat serum overnight at 4℃: rabbit anti-glial fibrillary acidic
protein (GFAP) (Abcam, Cambrige, MA, USA, 1:500), mouse anti-beta Tubulin
(TUJ1) monoclonal antibody (Earthox, San Francisco, USA, 1:300), mouse
anti-feline CSPG Brain core protein (US Biological, US, 1:400), and mouse
anti-neurocan antibody (EMD Millipore, 1:500). The sections were subsequently
washed four times for 5min each and then incubated with the Alexa
Fluor-568-conjugated (1:500) or -488-conjugated (1:300) IgG secondary
antibodies (Molecular Probes, USA) for 1h in a dim room at 37℃. The nuclei were stained with 40,
6-diamidino-2-phenylindole (DAPI, Beyotime Institute of Biotechnology,
Shanghai, China, and diluted 1:5 in PBS) for 10min. The images were observed
using and Olympus OP70 microscope (Olympus Microscopy, Japan) or a Leica TCS
SP50 confocal microscope (Leica Microsystems, Wetzlar, Germany).
Immunohistochemistry (IHC) was used to
observe the growth-associated membrane phosphoprotein-43 (GAP-43) -positive
regenerating axons. The rabbit anti-GAP43 (Abcam, Cambridge, USA, 1:300)
primary antibody was diluted in 8% goat serum and 0.1% Triton X-100 in PBS.
Subsequently, the sections were incubated with an Alexa Fluor-488-conjugated
secondary antibody IgG (Molecular probes, 1:300) for 2-3h in a dark room at 37℃.
Western Blot
Analysis To examine the protein expression in the
tissues, a Western blot (WB) analysis was performed as previously reported[3,16] .The retinas or optic
nerves were homogenized in ice-cold protein lysis buffer containing protease
and phosphatase inhibitors (150 mmol/L NaCl, 1% Triton X-100, 50 mmol/L of
Tris-HCl buffer, pH 7.4, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate
(SDS), sodium orthovanadate, sodium fluoride, Leupeptin and
Ethylenediaminetetraacetic acid) for approximately 30min. The homogenate was
centrifuged at 15 000 g for 10min at 4℃, and the supernatants were collected and stored at -80℃ until use. A bicinchoninic acid kit
(Beyotime Institute of Biotechnology, Shanghai, China) was used to investigate
the concentration of the samples. The samples (50 μg of protein/lane) were
separated on a 10% SDS-PAGE gel and transferred to an Immobilon membrane
(Millipore) for 60min at 100 V. The membranes were probed with the following
primary antibodies overnight at 4℃: rabbit anti-GFAP (Abcam, Cambridge, USA, 1:2000), mouse
anti- TUJ1 monoclonal antibody (Earthox, San Francisco, USA, 1:1000), rabbit
anti-GAP43 antibody (Abcam, Cambridge, USA, 1:500), mouse anti-feline CSPG
Brain core protein (US Biological, USA, 1:1000), and mouse anti
glyceraldehyde-3-phosphate dehydrogenase monoclonal antibody (GAPDH, CWbio,
China, 1:2000). After washing, the membranes were incubated with different
HRP-conjugated secondary antibodies for 2h at 37℃. The membranes were observed using the Odyssey infrared
imaging system (Bio-Rad Laboratories, Hercules, CA, USA). The relative of
amounts of the proteins were calculated as the ratio of GFAP, TUJ1, GAP-43, or
CSPG to GAPDH, respectively. The experiments were repeated three times for each
protein.
Axon
Regeneration and Quantitation The regenerate axons were quantified by
counting the number of GAP-43-positive axons, as previously described[22]. The number of axons
that had extended 50, 100, 200 and 300 μm from the middle crush site was
counted in six sections per case. The section width of the optic nerve was
measured at the points where the axons were counted and was used to calculate
the number of axons per millimeter of nerve, which was then averaged for the
six sections per case. The total number of extending axons 
from a distance d of the crush site was
calculated by the following formula: 
The radius r was calculated by adding all of the sections with a thickness t (10 μm).
Statistical
Analysis The data were expressed as the means
SD.
The statistical analyses were performed with the SPSS 13.0 software, and the
gray value analyses were performed using the ImageJ software. The statistical
differences between groups were analyzed using Student's t-tests or one-way analysis of variance (ANOVA) and considered to
be significant with P-value
less than 0.05.
RESULTS
αA-crystallin suppressed the activation of gliosis
in the retinas of the ONC rats. To study the effect of αA-crystallin on
the retinal gliosis response in vivo
after ONC, we examined the expression of GFAP at different time points after
ONC by IHC and WB. GFAP expression was restricted to the ganglion cell layer in
the normal retinas (Figure 1A, a, g, m, and s), and the sham operation did not
influence the GFAP expression and distribution in the retinas (Figure 1A. b-f).
ONC injury led to obvious GFAP-positive processes that were distributed in the
inner plexiform layer (IPL) and inner nuclear layer (INL) at only 1d after ONC,
and more intense GFAP-positive processes were observed in all retinal layers
after 3, 5, 7 and 14d (Figure 1A, g-l). However, the αA-crystallin (10-4
g/L, 4 μL) treatment effectively attenuated the expression of GFAP. The time
period for the processes to extend across the entire width of the retina was
delayed to 5d after ONC, and fewer processes appeared (Figure 1A, s-x). The
intravitreous injection of PBS (4 μL) did not influence the ONC-induced
increase in the GFAP-positive processes. We observed that the GFAP-positive
processes were also distributed in the outer layers at 3d after ONC injury
(Figure 1A, m-r).
Immunoblot analysis confirmed that the
ONC injury induced the activation of gliosis in the retinas (Figure 1B, 1C).
The retinal GFAP levels were increased approximately 0.7-fold at 3d after ONC
injury compared to the sham operation group, and the GFAP levels increased
approximately 0.8-fold at 14d after ONC (Figure 1C). A similar result was
observed in the PBS-treated group. However, the GFAP expression level was
significantly attenuated by αA-crystallin treatment at 3 (P<0.01), 5 (P<0.05),
7 (P<0.01) and 14d (P<0.01) compared to the injury only
group (Figure 1C). Notably, the activation of gliosis in the retinas after ONC
injury has been described previously[1,21,23-24].
We observed that the GFAP levels reached a peak at 2wk in the injury only
group, which is consistent with the results described by Rong et al[24]. There was no significant difference in the GFAP
levels after an additional two weeks after optic nerve injury (at 4wk, data not
shown). It has also been reported that α-crystallin pretreatment effectively
diminished the systemic inflammation-induced GFAP levels[11] and inhibited the retinal microglial activation
induced by ONC[3,25], but
there was no report that described the influence of αA-crystallin in glial cell
activation in the total retinas following traumatic optic nerve injury. Our
results indicate that alpha-crystallin could suppress the ONC-induced
activation of gliosis in the retinas.
Figure 1 Intravitreous
injection of αA-crystallin suppressed the activation of gliosis in the retinas
of the ONC rats A:
Immunofluorescence staining showed the level of GFAP (green) in the retinas of
the optic nerve sham injury group (sham) (a-f), ONC injury group (injury only)
(g-l), PBS-treated ONC injury group (PBS injection) (m-r) and
αA-crystallin-treated ONC injury group (α-A injection) (s-x). B, C: WB analysis
showing that the GFAP levels were significantly attenuated by αA-crystallin
treatment at 3 (P<0.01), 5 (P<0.05), 7 (P<0.01) and 14d (P<0.01)
after ONC. GCL: Ganglion cell layer; IPL: Inner plexiform layer; INL: Inner
nuclear layer; OPL: Outer plexiform cell; ONL: Outer nuclear cell. The
quantitative data represent the means±SD (n=3).
aP<0.05, bP<0.01. Scale bar=100 μm.
Intravitreous
injection of αA-crystallin suppressed the activation and
morphological remodeling of astrocytes at the ONC site. We
next observed whether αA-crystallin treatment could also affect astrocyte
activation after ONC. Frozen sections of the optic nerve were stained with GFAP
antibody (Figure 2A). After the optic nerve injury, the cellular architecture
at the crush site was disrupted, and the astrocytes were degenerated and formed
a GFAP-immunofluorescence reactivity (GFAP-IR)-free zone at 3d after the ONC
injury (Figure 2A, g-l). At approximately 7d after injury, the astrocytes at
the distal side of the optic nerve migrated into this zone and formed a glial
scar. The arrangement of the astrocyte processes at the marginal crush site was
more arbitrary (Figure 2A, k, Z2). By 14d, the GFAP-IR-free zone was partially
filled by GFAP-positive processes (Figure 3A, a-d), which is consistent with
previous reports[26-27]
and suggested that a glial scar had formed. The PBS treatment did not influence
the ONC-induced changes in the astrocytes (Figure 2A, m-r). Fortunately, the
intravitreous αA-crystallin injection effectively suppressed the astrocyte
migration and changed the processes (Figure 2A, s-x, Z4); only a few
GFAP-positive processes were present in the GFAP-IR-free gap.
WB analysis was used to determine the
levels of the GFAP protein to further characterize the influence of astrocyte
activation by αA-crystallin treatment. The result showed that GFAP was
significantly inhibited in the αA-crystallin-treated group compared to the
injury only and PBS-treated groups (Figure 2B, 2C). The GFAP levels increased
approximately 0.6-fold (n=3) at only
1d after ONC. Compared to the sham operation, the ONC group reached a 1.23-fold
peak increase at 7d and was maintained at approximately 1.2-fold at 14d; PBS
treatment did not significantly influence the ONC-induced GFAP expression. A
previous report demonstrated that GFAP expression began in the distal part of
the ONC site at 1d after crush and was maintained for at least 2wk[28], similar to changes in
GFAP expression we observed following the ONC. However, GFAP was suppressed by the
αA-crystallin
treatment compared to the ONC and PBS treatment groups, respectively, after 5 (P<0.05), 7 (P<0.01) and 14d (P<0.01).
Our results indicated that αA-crystallin can suppress the activation and
morphological remodeling of astrocytes.
Figure 2 Intravitreous injection αA-crystallin
suppressed the astrocyte activation at the ONC site A: Immunofluorescence staining showed GFAP (green) activation
at the crush site in optic nerve sham injury group (sham) (a-f, and e zoomed
image Z1), ONC injury group (injury only) (g-l, and k zoomed image Z2),
PBS-treated ONC injury group (PBS injection) (m-r, and q zoomed image Z3) and
αA-crystallin-treated ONC injury group (α-A injection) (s-x, and w zoomed image
Z4). B, C: The WB analysis of GFAP showed that the GFAP levels were
significantly inhibited in the αA-crystallin-treated group compared to the ONC
and PBS-treated groups after 5 (P<0.05),
7 (P<0.01) and 14 (P<0.01), respectively. cr: the proximal side of the crush site.
The quantitative data represent the means±SD (n=3). aP<0.05, bP<0.01.
Scale bars=100 or 50 μm.
αA-crystallin increased the
number of TUJ1-positive process at 14d after ONC. TUJ1 is a marker for microtubules in neuronal cells, and it
is a specific marker for RGCs. The number of TUJ1-positive processes indicates
the number of surviving axons. After ONC injury, the GFAP-expressing astrocytes
progressively invaded the perilesional zone at 14d after ONC (Figure 3A, 3B),
and a few TUJ1-positive axons were observed across the crush site (Figure 3A,
3C). Conversely, the αA-crystallin (10-4 g/L, 4 μL) treatment
inhibited astrocyte activation and the GFAP-negative region was obviously
larger than that in the PBS-treated group. The number of TUJ1-postive processes
was obviously increased and some had extended across the crush site (Figure 3A,
j, k). The intravitreous PBS injection (4 μL) did not suppress the ONC-induced
astrocyte invasion or improve axon survival (Figure 3A, f, g).
Western blotting confirmed the levels of
GFAP and TUJ1 in the optic nerve (Figure 3B, 3C). Compared to the sham
operation, GFAP expression increased 1.1-fold (n=5) in the injury only group, while αA-crystallin treatment
increased the GFAP levels 0.3-fold (n=5)
at only 14d after ONC. The GFAP levels were significantly decreased in the
αA-crystallin-treated group compared to the injury only (P<0.01) and PBS-treated (P<0.01)
groups. The TUJ1 expression level decreased by 30 percent (n=5) in the αA-crystallin-treated group, but was decreased by 50
percent (n=5) in the injury only
group. Our results suggested that the αA-crystallin treatment rescued the
number of TUJ1 positive processes at 14d after ONC.
Figure 3 Intravitreous
injection of αA-crystallin increased the number of TUJ1-positive processes at
14d after ONC A: Immunofluorescence
staining showed the GFAP-positive fibers (green) and TUJ1-positive processes
(red) in the crush site following ONC. The sections were obtained from the ONC
injury group (injury only) (a-d), PBS-treated ONC injury group (PBS injection)
(e-h) and αA-crystallin-treated ONC injury group (α-A injection) (i-l). B, C:
WB analysis showed that the GFAP levels in the optic nerve were significantly
decreased in the αA-crystallin-treated group compared to the injury only (P<0.01) and PBS-treated groups (P<0.01). However, the TUJ1 levels
were higher in the αA-crystallin-treated group compared to the injury only
group (P<0.05). cr: The middle of
crush site; ONC: Optic nerve crush. The quantitative data represent the
means±SD (n=5). aP<0.05, bP<0.01. Scale bar=100 μm.
Intravitreous
injection of αA-crystallin enhanced axon regeneration at 14d after ONC. GAP-43 expression was observed to
confirm the hypothesis that αA-crystallin treatment can enhance axon
regeneration. Because RGCs express the GAP-43 protein during axon regeneration,
probing for the protein could indicate that the RGCs are growing[29-31]. Fourteen days after
ONC, there were a few regenerating axons in the crush site of the ONC injury
only and PBS-treated groups (Figure 4A), and very few axons had extended beyond
the crush site. However, in the αA-crystallin-treated group, the axons had
regenerated extensively and passed the crush site (Figure 4A, i-l). The quantification
of the axons revealed that more axonal fibers had extended to 300 μm past the
crush site in the αA-crystallin-treated group than in
the other groups (Figure 4B); the number of axonal fibers was significantly
higher in the αA-crystallin treated group (mean number of
axons per optic nerves±SD: 50 μm, 536±44; 100 μm, 316±61; 200 μm, 206±55; 300
μm, 95±34; n=6) than in the
PBS-treated (mean number of axons per optic nerves±SD: 50 μm, 269±104; 100 μm,
151±42; 200 μm, 38±41; 300 μm, 11±17; n=6)
and injury only groups (mean number of axons per optic nerves±SD: 50 μm,
286±105; 100 μm, 180±51; 200 μm, 40±34; 300 μm, 12±20; n=6). We also analyzed the levels of the GAP-43 protein by Western
blotting (Figure 4C). We found that the level of GAP-43 in the
αA-crystallin-treated group increased 1.8-fold compared to the injury only
group (n=4, P<0.01); the levels in the PBS-treated group increased 0.5-fold
compared to the injury only group, and there was no significant difference. The
result indicated that αA-crystallin might play an important role in axon
outgrowth after traumatic optic nerve injury.
Figure 4 Intravitreous
injection of αA-crystallin enhanced axon regeneration at 14d after ONC A: Longitudinal sections through the
adult mouse optic nerve showing the regenerating GAP-43-positive axons in ONC
injury (injury only) (a-c, and c zoomed image d), PBS-treated ONC injury (PBS
injection) (e-g, and g zoomed image h) and αA-crystallin treated ONC injury
groups (α-A injection) (i-k, and k zoomed image l). B: The quantification of the
axons revealed that more axonal fibers extended to 300 μm past the crush site in
the αA-crystallin-treated group than in the other groups (n=6, one-way ANOVA, bP<0.01).
C: WB analysis showed that the level of GAP-43 in αA-crystallin-treated group
was significantly improved compared to the injury only and PBS treated groups (n=4, bP<0.01). cr: The middle of crush site; n.s: No significance. The
quantitative data represent the means±SD. Scale bar=100 μm.
Intravitreous
injection of αA-crystallin restored the F-VEP of rats at 14d after ONC. To functionally evaluate the
αA-crystallin influence on the optic nerve electrophysiology, flash visual
evoked potential investigations were performed using the Roland reimport visual
electrophysiology system. The waveforms recorded from the control and treated
rats are shown in Figure 5A. At 12h after the ONC injury, the F-VEP waveforms
appeared silent and the peaks were dramatically decreased. Two weeks after
injury, the latency and peaks were not obviously restored in the injury only
and PBS-treated groups, while the waveforms were restored to approximately the
pre-ONC level by αA-crystallin treatment. The statistical analysis of the
amplitudes (N1-P1) and latency (P1) indicated the changes were specific; in the
normal rats, the average amplitudes for N1-P1 were approximately 22.97±5.94 μV (n=27). At
12h after ONC, the amplitudes for N1-P1 were 4.86±2.25 μV (n=8) and the latency of P1 was 117±9ms. At 14d after ONC, the
amplitudes for N1-P1 were 5.24±1.67 μV, and PBS treatment did not restore the
amplitude. However, the amplitudes for N1-P1 were obviously restored
(12.02±1.64 μV) (P<0.01) by
αA-crystallin treatment at 2wk after injury (Figure 5B, a). Meanwhile, the
latency of P1 was approximately 78±4ms (n=27)
in the normal rats, and it was delayed to 126±9ms 14d after ONC; intravitreous
injection of αA-crystallin restored the latency to 100±9ms (P<0.01) at 14d after ONC. Our findings
indicated that Alpha-crystallin
can also improve the electrical functions of F-VEPs in the ONC injury model.
Figure 5 Intravitreous
injection of αA-crystallin restored the F-VEP of rats at 14d after ONC A: Representative waveforms of the ONC
injury group (injury only), PBS-treated ONC injury group (PBS injection) and
αA-crystallin-treated ONC injury group (α-A injection). B: The amplitudes
(N1-P1) and latency (P1) were altered by αA-crystallin treatment after ONC. a:
The amplitude (N1-P1) diagram showed a significant increase in the amplitude at
14d after ONC in the αA-crystallin-treated group compared to the ONC injury (P<0.01) and PBS-treated groups (P<0.01). b: The latency (P1) diagram
showed that P1 was obviously delayed after the ONC and was significantly
restored by αA-crystallin treatment at 2wk after ONC (P<0.01). The data were shown as mean±SD (n=9). aP<0.05,
bP<0.01.
Intravitreous injection of αA-crystallin decreased the expression of
ECM of the crush site at 14d after ONC. The ECM macromolecules that are deposited
by reactive astrocytes in response to injury are one of the main barriers that
prevent axon outgrowth. The ECM molecules CSPGs include NG2,
neurocan, collagens, fibronectin, tenascins and other classes of proteoglycans.
Notably, the CSPGs inhibit neurite outgrowth in vitro[32-33].
Two remarkable CSPG proteins, the NG2 proteoglycan and neurocan, were observed
to investigate whether αA- crystallin can reduce the deposition of ECM (Figures
6, 7). Two weeks after ONC, substantial CSPG expression was observed around the
injury site (Figure 6A, a-d). In particular, the CSPG expression at the distal
crush site was much higher than that at the proximal site. PBS treatment did
not influence the obvious increase in CSPG expression following ONC injury
(Figure 6A, e-h). However, in the αA-crystallin-treated group, CSPG expression
was reduced (Figure 6A, i-l). Western blotting confirmed that the expression of
CSPGs was obviously increased in the injury only (increased 0.72-fold
compared to the sham operation) and PBS-treated (increased 0.77-fold compared
to the sham operation) groups. However, in the αA-crystallin-treated group, the
CSPGs were significantly suppressed compared to the injury only (P<0.05) and PBS-treated (P<0.05) groups.
Figure 6 Intravitreous injection of αA-crystallin
decreased the expression of CSPGs in the crush site at 14d after ONC A: Longitudinal sections through the
adult mouse optic nerve showing the CSPG levels (green) in the ONC injury
(injury only) (a-c, and c zoomed image d), PBS-treated ONC injury group (PBS injection) (e-g, and g zoomed image h) and
αA-crystallin-treated ONC injury groups (α-A injection) (i-k, and k zoomed image l). B, C: Western blots
showing that the expression of CSPGs was obviously increased in the injury only
and PBS-treated groups. However, in the αA-crystallin-treated group, the CSPGs
were significantly decreased compared to the injury and PBS-treated (P<0.05) groups. cr: The middle of crush site. The arrows indicate
CSPG-positive labeling. The data were shown as means±SD (n=5). aP<0.05.
Neurocan is one of
the major CSPGs in the nervous tissue, and this axonal extension inhibitor was
upregulated in the scar region after stroke (Figure 7). Extensive neurocan
expression was observed around the crush site at 14d after ONC (Figure 7A-7D).
αA-crystallin treatment obviously decreased the expression of neurocan (Figure
7I-7L). Our findings indicated that the αA-crystallin-enhanced axon
regeneration might occur by suppressing astrocyte activation and influencing
astrocyte secretions.
Figure
7 Intravitreous injection of αA-crystallin
decreased the expression of neurocan in the crush site at 14d after ONC Immunofluorescence staining showed the
neurocan (green) expression in the crush site following ONC in the ONC injury
(injury only) (A-C, and C zoomed image D), PBS-treated ONC injury (PBS injection) (E-G, and G zoomed
image H) and αA-crystallin-treated ONC injury groups (α-A injection) (I-K, and K zoomed
image L). The arrows indicate neurocan-positive labeling. cr: The middle of crush site. The arrows indicate
neurocan-positive labeling.
DISCUSSION
In this study, we found that the
intravitreous injection of αA-crystallin can suppress the activation of gliosis
in retinas and influence the cellular architecture of astrocyte remodeling
around the crush site in the rats with ONC injury.
Many of the retinal gliosis responses
observed in vivo after ONC injury and
treatment have been studied using the intravitreous injection method[1,2,16,21]. In traumatic
brain and optic nerve injury, astrocytes play an important role in the injury
response. Quiescent cells are activated only a few minutes after optic nerve
injury[34], and may be
sustained for up to three month to form a mature glial scar at the injury site[27]. The activated
astrocyte exhibited a hypertrophic soma, the number of processes increased and
extended, GFAP expression increased[35],
and a glial barrier formed through the retinas and nerves. We found that GFAP
expression increased in the retinas at only 1d after ONC and reached a peak
level at 14d (increased approximately 0.8-fold). Similarly, the GFAP levels
increased significantly after the operation. No significant difference in the
GFAP levels was found after an additional two weeks following the optic nerve
injury (at 4wk, data not shown).
We further observed that the
αA-crystallin (10-4 g/L, 4 μL) treatment decreased the GFAP levels
in the retinas and optic nerves and which concentration was used in previous
studies[16-17,25].
Furthermore, the arrangement of astrocytes around the crush site was less arbitrary,
and the astrocyte migration into the GFAP-IR-free zone was inhibited. It has
been reported that HSPs could influence astrocyte activation and proliferation.
βA3/A1-crystallin plays an important role in mediating STAT3 signaling to
promote GFAP expression and VEGF secretion from optic nerve astrocytes[36]. αB-crystallin takes
part in suppressing neuroinflammation by the astrocyte dopamine D2 receptor[37]. GFAP toxicity and
aggregation was suppressed by αB-crystallin in an Alexander disease mouse[38]. Our findings suggested
that crystallin can also affect the activation and remodeling of astrocytes in
the ONC model. However, we cannot maintain the concentration of crystallin in
the retinas and nerves at an optimal level for 14d or longer, and it is not
clear how αA-crystallin was delivered to the crush site. One explanation was
transported via the optic nerve stump to reach the lesion site[12].
ONC injury induces dramatic apoptosis of
RGCs, leading to severe axon loss and the failure to regenerate[39]. We found that
αA-crystallin treatment improved TUJ1-positive process survival and neurite
outgrowth. TUJ1 is known as neuronal βⅢ tubulin and is widely used as
a RGCs marker, the number of TUJ1-positive processes reflected RGC survival. Crystallin promoted RGC survival in the
retinas, as previously described[3,12],
although the authors have not further investigated whether the RGC survival
were functional. We next found that the amplitudes (N1-P1) and latency (P1) of
the F-VEP waveforms were restored by the αA-crystallin treatment. The
amplitudes (N1-P1), which reflected the number of synaptic contacts between the
intact axons and their targets [40],
was
increased to 12.02±1.64 μV compared to the ONC only group (4.86±2.25 μV).
Furthermore, the latency was restored to 100±9ms compared
to the ONC only group (126±9ms), which reflected the conduction and axon myelin
sheath integrity. We described the protective effect of α-crystallin in
electrophysiology, which suggested that crystallin protected the RGCs, the
axons’ synaptic contacts and the myelin sheath integrity. Previous studies also
indicated that Alpha-crystallin treatment could improve optic nerve function, Pangratz-Fuehrer et
al[41] found that an intravenous α-crystallin injection led to the acceleration of visual-evoked
potential latency over 3wk in ischemic optic neuropathy. While we lacked direct
evidence that the F-VEPs were promoted by the rescued axons, it was possible
that crystallin protected the moderately damaged axons through the “shock period”,
which then recovered their electrical function. Our future studies may uncover
more specific evidence to explain the crystallin-mediated rescue of optic nerve
electrical potentials.
We used GAP-43 to identify the new growth
cones and axons that passed through the crush site, and found that the GAP-43
positive processes had obviously penetrated the crush site, which was
accompanied by reduced glial scar formation at 14d after αA-crystallin
treatment. The astrocyte architecture was also less arbitrary. The more robust
axons that may be regenerated in the Bcl-2tg GFAP-/-Vim-/- mice after ONC indicated the
critical role of the glial scar in forbidden axon regeneration[42]. By dismissing the idea
that the glial barrier promoted axon regeneration, Robitta et al[43] observed that the neuronal density in co-cultures
with GFAP-/-Vim-/-
astrocytes was different from those cultured with wild type astrocytes, which
was related to attenuated scar formation. Rodriguez et al[44]
found that the abrogation of β-catenin signaling could reduce glial scarring
and improve axon regeneration. Our hypothesis is αA-crystallin enhances axon
regeneration by suppressing astrocyte activation and proliferation. While we
cannot exclude the possibility that crystallin intrinsically enhances a
neurite-promoting activity, one option is that crystallin acts as a
neurite-promoting factor through autocrine and paracrine mechanisms[45].
To study the mechanism by which
αA-crystallin enhanced the axon regeneration by suppressing astrocyte activity,
we next observed the secretion of CSPG molecules from astrocyte, including NG2
and neurocan. We found that the CSPGs and neurocan were significantly decreased
by the αA-crystallin treatment. In response to injury, astrocytes release the
inhibitory CSPG molecules, creating a chemical barrier to regeneration[46]. CSPGs’ inhibitory
activity depends on the glycosaminoglycan components[47-48]. Other studies demonstrated that eliminating the
inhibition of CSPGs promoted axon regeneration; matrix metalloproteinases and a
related protease called ADAMTS-4 degrade CSPGs to promote
astrocyte remodeling for axon regeneration[6].
In spinal cord injury, chondroitinase ABC had been used as a strategy to
promote axon regeneration and repair plasticity and function[5]. Previous reports also
demonstrated that retinal neuron cultures containing α-crystallin could
significantly promote increased neurite density and length on myelin-coated
dishes via α-crystallin binding to myelin-associated inhibitory molecules or
their receptors[17]. Our
findings suggest that αA-crystallin could promote neurite outgrowth by
suppressing the expression and secretion of CSPGs from astrocytes.
Our findings suggest that α-crystallin is
a multi-target molecule that enhances axon regeneration. This molecule might
rescue the RGCs following optic nerve injury and also suppress astrocyte
activation and the secretion of inhibitory molecules. However, the mechanism is
still unclear, and future studies are needed. Our studies provide a strategy to
cure optic nerve injury diseases.
ACKNOWLEDGEMENTS
Authors’
Contributions: Shao WY, Xu
HW, and Wang Y contributed to data analysis, manuscripts preparation and
revised version. Shao WY, Gu XL, and Liu X contributed the experiments Western
blots and immunofluorescences, other experiments were finished by Shao WY. Wang
Y, Xu HW, Wu N, and Ying X given some good advices in project design.
Foundation: Supported by the National Nature Science Foundation of China
(No.81270996).
Conflicts
of Interest: Shao WY, None; Liu X, None; Gu XL, None; Ying X, None; Wu N, None; Xu HW, None; Wang Y, None.
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