Original Paper Ophthalmic Res 2014;51:173–178 DOI: 10.1159/000357736
Received: June 5, 2013 Accepted after revision: December 4, 2013 Published online: March 18, 2014
Factors Influencing the Retrograde Labeling of Retinal Ganglion Cells with Fluorogold in an Animal Optic Nerve Crush Model Tzu-Lun Huang a, b, g Sun-Ping Huang a–c Chung-Hsing Chang e Kung-Hung Lin f Min-Muh Sheu b Rong-Kung Tsai a, b, d a Institute
of Eye Research, Buddhist Tzu Chi General Hospital, and Departments of b Ophthalmology and Visual Science and c Molecular and Human Genetics, d Institute of Medical Sciences, Tzu Chi University, Hualien, e Departments of Dermatology, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, and f Department of Neurology, Taiwan Adventist Hospital, and g Department of Ophthalmology, Far Eastern Memorial Hospital, Banciao District, New Taipei City, Taiwan, ROC
Abstract Purpose: To investigate whether different crush durations or a different fluorogold (FG) injection timing can affect the efficiency of FG retrograde labeling of retinal ganglion cells (RGCs) in the optic nerve (ON) crush model. Methods: We performed the ON crush in rats with a clip at different durations or a jewel forceps to compare the effects of different crush methods with FG staining. RGC density was compared between the FG injection 1 week before the sacrifice of the animals (group A) and the injection before the crush experiment (group B). Double staining with CD11b and FG in the retinal sections was conducted to investigate the relationship between the overcounting of RGCs and microglia. Results: The FG-stained particles were significantly decreased at the distal part of the crush site compared to the proximal site of the ON with a crush duration of over 30 s or when crushed with the jewel forceps. Two weeks after ON crush, the RGC count was higher both in the central and mid-peripheral retinas in group B. The percentage of CD11b-stained cells among the FG-stained cells in the RGC layer of retinas in group B was higher than that of group A (34% in group B
© 2014 S. Karger AG, Basel 0030–3747/14/0514–0173$39.50/0 E-Mail [email protected] www.karger.com/ore
vs. 4% in group A, p = 0.0001). Overcounting of RGC density in group B was due to additional microglia with FG engulfing. Conclusions: Our results suggest that each laboratory should test its setting conditions to avoid factors influencing the RGC density measurement before conducting ON crush experiments. © 2014 S. Karger AG, Basel
Introduction
The optic nerve (ON) crush experiment is one of the commonly used animal models to study the efficacy of various interventions on neuronal survival or damage according to retinal ganglion cell (RGC) counts [1–4]. Retrograde labeling of the fluorescent tracer fluorogold (FG) has been used to study the morphometry of RGCs after insults [5–9]. Two timings of retrograde FG injection have been used in the related literature, either injecting FG before the ON crush [10] or injecting FG 5–7 days before sacrifice of rats [11, 12]. When injecting FG before the ON crush, overcounting the number of RGCs was
Tzu-Lun Huang and Sun-Ping Huang contributed equally to this research project.
Rong-Kung Tsai, MD, PhD Institute of Eye Research, Buddhist Tzu Chi General Hospital, Tzu Chi University No. 707, Section 3, Chung-Yang Road Hualien 97002, Taiwan (ROC) E-Mail rktsai @ tzuchi.com.tw, tsai.rk @ gmail.com
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Key Words Optic nerve crush · Retinal ganglion cell · Fluorogold · Microglia
a
b
c
d
e
f
Fig. 1. a–g Rat ONs 2 weeks after crush injury. a, b There were no differences in FSPs
Proximal part
180
Distal part
160
*
140 FSPs (n)
120
**
100 80 60 40 20
g
0
Proximal
noted by mixing the labeled surviving RGCs with the dyeengulfing macrophages and/or microglia [10, 13–15]. Regarding the method of injecting FG after the crush experiment and 5–7 days before animal sacrifice, the major concern is that the FG will not label RGCs efficiently if the axons of the ON are severely damaged. Several methods of ON injury, including indirect and direct ones, have been used in the literature [1, 2, 7, 16– 22]. The extent of RGC degeneration after ON crush is related to the force exerted by instruments and the applied duration [19]. Using constant force-exerting instruments is frequently chosen to get convincing results [23–26]. The aim of this study is to investigate how the methodology used to crush the ON affects the efficiency of FG 174
Ophthalmic Res 2014;51:173–178 DOI: 10.1159/000357736
Clip/30 s
Clip/60 s
Clip/90 s
**
**
Clip/120 s
Forceps
retrograde labeling and to evaluate whether the different timings of the FG injection influence the accuracy of RGC density measurements and their relationship with the dye-engulfing microglia.
Methods Animals Sixty adult male Wistar rats weighing 150–180 g (7–8 weeks old) were used in this study. The rats were obtained from BioLASCO Co., Taiwan. Animal care and experimental procedures were performed in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. The Institutional Animal Care
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between the proximal site and the distal site of the crush area in the sham group and the 30-second group (p > 0.05). c–f There were significantly fewer FSPs at the distal part of the ON than at the proximal part in the groups with crush durations of 60 s (* p = 0.03). Compared with the mean number of FSPs of the proximal side, the mean number of FSPs of the distal area after crush durations of 90 and 120 s, and in the group using jewel forceps was also significantly different (all ** p = 0.0001). Bar =100 μm (n = 6 in each group).
200
90 80 FSPs (n/HPF)
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60 50 40 30 20 10
d b
0
P
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c
Fig. 2. ON sections 2 weeks after ON crush with a microvascular clip for 30 s. a Representation of the ON demonstrated the FSP in the proximal (P) and distal (D) parts to the injury site. Bar = 100 μm. b Representation of zoom-in on the proximal part to the insult site. c Representation of zoom-in on the distal part to the insult site. Bar = 20 μm. d There was no significant difference in the number of FSPs between the proximal and distal parts
to the crush site (p = 0.063, n = 6 in each group).
ON Crush Experiments ON crush injuries were induced as described in our previous reports [12, 24, 26]. Briefly, a vascular clip (60-gram microvascular clip, item No. 14121, World Precision Instruments, Fla., USA) was applied to the ON at a distance of 2 mm posterior to the globe for durations of 30, 60, 90 and 120 s. We also performed ON crushing with jewel forceps at the same site for 15 s. A sham operation entailed the ON exposed without the crush. Efficiency of Retrograde Flow of FG in ON among Different Crush Settings We compared the FG-stained particles (FSPs) in 10 randomly selected high-power fields (HPFs) between the proximal and distal parts to the crush site of the ON under different crush settings (n = 6 in each group). All rats were euthanized 2 weeks after crush injury.
Two weeks after crush injury, the rats were sacrificed, and the flatmounted retinas were examined with a ×400 epifluorescence microscope (Axioskop; Carl Zeiss Meditech Inc., Thornwood, N.Y., USA). Immunohistochemistry: Double Staining of CD11b and FG in the Ganglion Cells of Retinas We conducted double immunohistochemical staining of retinal sections with a microglial marker (CD11b) and FG as described previously [12, 24]. CD11b primary antibodies (1:20, mouse antirat monoclonal antibody, AbD Serotec, Oxford, UK) and Cy3conjugated affinipure goat anti-mouse secondary antibody (1:100, Jackson Immunoreseach Laboratories, West Grove, Pa., USA) were used. The images were observed with a fluorescent microscope (Axioplan 2; Carl Zeiss Inc., Germany). Statistical Analysis Analysis of statistical significance was determined using 2-tailed Student’s t tests and 1-way ANOVA followed by Bonferroni’s multiple comparison test. Data are presented as means ± standard error of the mean. In all cases, p < 0.05 denotes statistical significance.
Results
Retrograde Labeling of RGCs with FG and Morphometry of RGCs before Crush or before Sacrifice The retrograde labeling of RGCs with FG (Fluorochrome, LLC, Denver, Colo., USA) before sacrificing animals has been described in our previous reports [12, 24, 26]. We defined group A by the timing of FG labeling ‘1 week after the crushing and 1 week before the sacrifice’ and group B by the timing of FG labeling ‘5 days before crushing and 19 days before the sacrifice’. The surgical procedures were identical in both groups. There were at least 12 rats in each group.
FSPs Adjacent to the Lesion Site of the ON in Different Settings of ON Crush The flow of FSPs was not blocked by the vascular clip/30-second group, as compared with the sham, but significantly blocked in the clip/60-second, clip/90-second, clip/120-second and jewel forceps/15-second groups
Retrograde Labeling of RGCs by Fluorogold in Optic Nerve Crush
Ophthalmic Res 2014;51:173–178 DOI: 10.1159/000357736
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and Use Committee of the Tzu Chi Medical Center approved all animal experiments. All manipulations were performed with animals under general anesthesia by intramuscular injection of a mixture of ketamine (40 mg/kg body weight) and xylazine (4 mg/kg body weight; Sigma, St. Louis, Mo., USA), and using topical 0.5% Alcaine eyedrops (Alcon, Puurs, Belgium). The rats were maintained in an environmentally controlled room.
a
b
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g
h
i
j 5 days before crush
2,500
*
p = 0.028
a
b c 1 week
**
p = 0.0001
d e 2 weeks
2,000
*
p = 0.017
1,500
**
p = 0.0001
1,000 500 0
f
l
g h 1 week
i j 2 weeks
Fig. 3. Whole mount preparation of retinas and morphometry of RGCs in central and mid-peripheral retinas 1 and 2 weeks after ON crush. Bar = 20 μm. a, e Sham operation. b, d, g, i Retrograde FG labeling 1 week before sacrifice (group A). c, e, h, j Retrograde FG labeling 5 days before ON crush experiments (group B). k, l In both central and mid-peripheral retinas, RGC densities were higher in group B than in group A 1 and 2 weeks after ON crush. b, d One week after crush injury, in group A, the survival rate of FG+ RGCs
was 59.8 and 48.2% in the central and mid-peripheral retinas, respectively. c, e In group B, the survival rate of FG+ RGCs was 68.7 and 68.2% in the central and mid-peripheral retinas, respectively. g, i Two weeks after crush injury, in group A, the survival rate of FG+ RGCs was 36.4 and 27.7% in the central and mid-peripheral retinas, respectively. h, j In group B, the survival rate of FG+ RGCs was 59.2 and 56.2% in the central and mid-peripheral retinas, respectively. * p < 0.05, ** p < 0.01 (n = 8 in each group, 32 rats in total).
(fig. 1).The mean number of FSPs in the proximal site of the crush area is 155 ± 24.4 (n = 6). The mean number of FSPs at the distal part to the crush site in the groups crushed for 30 s (clip/30 s), 60 s (clip/60 s), 90 s (clip/ 90 s), 120 s (clip/120 s) and 15 s (jewel forceps/15 s) were 135.7 ± 24.2/HPF, 126.8 ± 13.1/HPF, 84.1 ± 26.1/HPF, 1.3 ± 1.5/HPF and 1.8 ± 2.4/HPF, respectively. Moreover, the mean FSP number between the proximal and distal parts of the ON to the crush site in the setting of clip/30 s showed no significant difference (p = 0.063; fig. 2).
and 1,700 ± 470/mm2, respectively (fig. 3a, f). Both 1 and 2 weeks after ON crush, the densities of RGCs in group B were significantly higher than those of group A in both the central and mid-peripheral retinas (fig. 3).
Retrograde Labeling of RGCs with FG and the Morphometry of RGCs before Crush or before Sacrifice The densities of RGCs in the central and mid-peripheral retinas in the sham-operated eyes were 2,390 ± 630 176
Ophthalmic Res 2014;51:173–178 DOI: 10.1159/000357736
Dual Staining of CD11b and FG in the Ganglion Cell Layer of Retinas Two weeks after ON crush, the ratio of CD11b+ and FG+ cells in the RGC layer of the retinas in group B was higher than that of group A (34 ± 11 and 4 ± 3%/HPF, respectively, p = 0.0001; fig. 4). There was overcounting of RGCs in group B as evidenced by merged stained cells, which were dye-engulfing microglia instead of surviving RGCs.
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k
1 week before sacrifice
RGCs (n/mm2)
RGCs (n/mm2)
Sham 3,500 3,000 2,500 2,000 1,500 1,000 500 0
Color version available online
b
50 45 40 35 30 25 20 15 10 5 0
**
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Before crush (group B)
In experimental animal studies, ON crush provides a reliable method for studying quantitative anatomical effects on novel neuroprotection or neurodegeneration after insults [23, 27–30]. Our results demonstrate that a complete block of axoplasmal flow was caused by the jewel forceps for 15 s. The jewel forceps has been used in other experiments of ON crush [21, 22]. Qu and Jakobs [21] used jewel forceps for 10 s and found severe axon degeneration of the ON at 1 week, and nearly all the axons were lost after 3 weeks. In the 60-gram microvascular clip groups with crush durations over 30 s, the axons were damaged and showed attenuation of FG flow beyond the area of the lesion site. With crush duration shorter than 30 s with a microvascular clip, the FG flow was well preserved. Other reports also demonstrated that various pressures exerted by a clip and different crush durations could result in a different optic nerve damage [18, 23, 31]. In sum, our results indicate that different instruments to induce crush injuries with variable
durations may influence the efficiency of retrograde labeling. A higher RGC survival rate was noted in both central and mid-peripheral retinas in the group with FG injection before ON crush. These results indicate that the ON crush injuries may result in a reactivation of microglial cells engulfing FG instead of true RGCs, particularly when the labeled RGCs were degenerated [14, 32–34]. Our double immunohistochemical staining with CD11b and FG provided alternative evidence of microglial phagocytosis in group B. This implies that earlier injection of FG before crush experiments may result in a higher RGC survival rate due to FG+ microglia increasing in the retinas. Consequently, injecting FG 1 week before the sacrifice of the animals will result in a more accurate count of RGC density. In conclusion, our results suggest that different instruments and durations of crush injury have different effects on the efficiency of FG retrograde labeling in the ON crush models. Retrograde FG labeling should be conducted 5–7 days before euthanizing the animals to avoid the overcounting of RGCs.
Retrograde Labeling of RGCs by Fluorogold in Optic Nerve Crush
Ophthalmic Res 2014;51:173–178 DOI: 10.1159/000357736
Discussion
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Fig. 4. Immunohistochemical staining of CD11b (microglial marker) and FG in the ganglion cell layer (GCL) of the retinas. INL = Inner nuclear layer. a FG-stained cells presented as green particles (arrowheads) and CD11b+ cells as red particles (arrows). b Two weeks after ON crush, the proportion of CD11b+/FG+ in the RGC layer of the retinas in group B was higher than that of group A (34 ± 11 and 4 ± 3%, respectively, ** p = 0.0001; n = 6 in each group). There was overcounting of RGCs in group B as evidenced by merged staining cells proving that FG+ cells are microglia instead of vital RGCs.
CD11b + cells/ FG + cells (%)
a
Acknowledgements
Disclosure Statement
The authors wish to thank Mr. Malcolm Higgins for his assistance in the English editing and Ms. Su-Zen Chen for figure and data assistance.
Proprietary interest and grant support: none.
References
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23 Feng DF, Chen ET, Li XY, Liu Y, Wang Y: Standardizing optic nerve crushes with an aneurysm clip. Neurol Res 2010;32:476–481. 24 Huang TL, Chang CH, Lin KH, Sheu MM, Tsai RK: Lack of protective effect of local administration of triamcinolone or systemic treatment with methylprednisolone against damages caused by optic nerve crush in rats. Exp Eye Res 2011;92:112–119. 25 Sarikcioglu L, Demir N, Demirtop A: A standardized method to create optic nerve crush: Yasargil aneurysm clip. Exp Eye Res 2007;84: 373–377. 26 Tsai RK, Chang CH, Sheu MM, Huang ZL: Anti-apoptotic effects of human granulocyte colony-stimulating factor (G-CSF) on retinal ganglion cells after optic nerve crush are PI3K/AKT-dependent. Exp Eye Res 2010; 90: 537–545. 27 Kanamori A, Catrinescu MM, Kanamori N, Mears KA, Beaubien R, Levin LA: Superoxide is an associated signal for apoptosis in axonal injury. Brain 2010;133:2612–2625. 28 Li Y, Schlamp CL, Nickells RW: Experimental induction of retinal ganglion cell death in adult mice. Invest Ophthalmol Vis Sci 1999; 40:1004–1008. 29 Lieven CJ, Levin LA: Tools for studying early events in optic neuropathies. Eye (Lond) 2007;21(suppl 1):S21–S24. 30 Okada T, Ichikawa M, Tokita Y, Horie H, Saito K, Yoshida J, Watanabe M: Intravitreal macrophage activation enables cat retinal ganglion cells to regenerate injured axons into the mature optic nerve. Exp Neurol 2005;196: 153–163. 31 Sugita K, Hirota T, Iguchi I, Mizutani T: Comparative study of the pressure of various aneurysm clips. J Neurosurg 1976; 44: 723– 727. 32 Allcutt D, Berry M, Sievers J: A quantitative comparison of the reactions of retinal ganglion cells to optic nerve crush in neonatal and adult mice. Brain Res 1984;318:219–230. 33 Bodeutsch N, Thanos S: Migration of phagocytotic cells and development of the murine intraretinal microglial network: an in vivo study using fluorescent dyes. Glia 2000; 32: 91–101. 34 Thanos S, Kacza J, Seeger J, Mey J: Old dyes for new scopes: the phagocytosis-dependent long-term fluorescence labelling of microglial cells in vivo. Trends Neurosci 1994;17:177.
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1 Misantone LJ, Gershenbaum M, Murray M: Viability of retinal ganglion cells after optic nerve crush in adult rats. J Neurocytol 1984; 13:449–465. 2 Frank M, Wolburg H: Cellular reactions at the lesion site after crushing of the rat optic nerve. Glia 1996;16:227–240. 3 Levkovitch-Verbin H, Harris-Cerruti C, Groner Y, Wheeler LA, Schwartz M, Yoles E: RGC death in mice after optic nerve crush injury: oxidative stress and neuroprotection. Invest Ophthalmol Vis Sci 2000;41:4169–4174. 4 Isenmann S, Wahl C, Krajewski S, Reed JC, Bahr M: Up-regulation of Bax protein in degenerating retinal ganglion cells precedes apoptotic cell death after optic nerve lesion in the rat. Eur J Neurosci 1997;9:1763–1772. 5 Lund RD, Land PW, Boles J: Normal and abnormal uncrossed retinotectal pathways in rats: an HRP study in adults. J Comp Neurol 1980;189:711–720. 6 Jeffery G, Perry VH: Evidence for ganglion cell death during development of the ipsilateral retinal projection in the rat. Brain Res 1981;254:176–180. 7 Jehle T, Wingert K, Dimitriu C, Meschede W, Lasseck J, Bach M, Lagreze WA: Quantification of ischemic damage in the rat retina: a comparative study using evoked potentials, electroretinography, and histology. Invest Ophthalmol Vis Sci 2008;49:1056–1064. 8 Vidal-Sanz M, Lafuente MP, Mayor S, de Imperial JM, Villegas-Perez MP: Retinal ganglion cell death induced by retinal ischemia: neuroprotective effects of two alpha-2 agonists. Surv Ophthalmol 2001; 45(suppl 3):S261–S267, discussion S273–S276. 9 Selles-Navarro I, Villegas-Perez MP, Salvador-Silva M, Ruiz-Gomez JM, Vidal-Sanz M: Retinal ganglion cell death after different transient periods of pressure-induced ischemia and survival intervals. A quantitative in vivo study. Invest Ophthalmol Vis Sci 1996; 37:2002–2014. 10 Levkovitch-Verbin H, Quigley HA, Martin KR, Zack DJ, Pease ME, Valenta DF: A model to study differences between primary and secondary degeneration of retinal ganglion cells in rats by partial optic nerve transection. Invest Ophthalmol Vis Sci 2003;44:3388–3393.
Received: June 5, 2013 Accepted after revision: December 4, 2013 Published online: March 18, 2014
Factors Influencing the Retrograde Labeling of Retinal Ganglion Cells with Fluorogold in an Animal Optic Nerve Crush Model Tzu-Lun Huang a, b, g Sun-Ping Huang a–c Chung-Hsing Chang e Kung-Hung Lin f Min-Muh Sheu b Rong-Kung Tsai a, b, d a Institute
of Eye Research, Buddhist Tzu Chi General Hospital, and Departments of b Ophthalmology and Visual Science and c Molecular and Human Genetics, d Institute of Medical Sciences, Tzu Chi University, Hualien, e Departments of Dermatology, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, and f Department of Neurology, Taiwan Adventist Hospital, and g Department of Ophthalmology, Far Eastern Memorial Hospital, Banciao District, New Taipei City, Taiwan, ROC
Abstract Purpose: To investigate whether different crush durations or a different fluorogold (FG) injection timing can affect the efficiency of FG retrograde labeling of retinal ganglion cells (RGCs) in the optic nerve (ON) crush model. Methods: We performed the ON crush in rats with a clip at different durations or a jewel forceps to compare the effects of different crush methods with FG staining. RGC density was compared between the FG injection 1 week before the sacrifice of the animals (group A) and the injection before the crush experiment (group B). Double staining with CD11b and FG in the retinal sections was conducted to investigate the relationship between the overcounting of RGCs and microglia. Results: The FG-stained particles were significantly decreased at the distal part of the crush site compared to the proximal site of the ON with a crush duration of over 30 s or when crushed with the jewel forceps. Two weeks after ON crush, the RGC count was higher both in the central and mid-peripheral retinas in group B. The percentage of CD11b-stained cells among the FG-stained cells in the RGC layer of retinas in group B was higher than that of group A (34% in group B
© 2014 S. Karger AG, Basel 0030–3747/14/0514–0173$39.50/0 E-Mail [email protected] www.karger.com/ore
vs. 4% in group A, p = 0.0001). Overcounting of RGC density in group B was due to additional microglia with FG engulfing. Conclusions: Our results suggest that each laboratory should test its setting conditions to avoid factors influencing the RGC density measurement before conducting ON crush experiments. © 2014 S. Karger AG, Basel
Introduction
The optic nerve (ON) crush experiment is one of the commonly used animal models to study the efficacy of various interventions on neuronal survival or damage according to retinal ganglion cell (RGC) counts [1–4]. Retrograde labeling of the fluorescent tracer fluorogold (FG) has been used to study the morphometry of RGCs after insults [5–9]. Two timings of retrograde FG injection have been used in the related literature, either injecting FG before the ON crush [10] or injecting FG 5–7 days before sacrifice of rats [11, 12]. When injecting FG before the ON crush, overcounting the number of RGCs was
Tzu-Lun Huang and Sun-Ping Huang contributed equally to this research project.
Rong-Kung Tsai, MD, PhD Institute of Eye Research, Buddhist Tzu Chi General Hospital, Tzu Chi University No. 707, Section 3, Chung-Yang Road Hualien 97002, Taiwan (ROC) E-Mail rktsai @ tzuchi.com.tw, tsai.rk @ gmail.com
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Key Words Optic nerve crush · Retinal ganglion cell · Fluorogold · Microglia
a
b
c
d
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f
Fig. 1. a–g Rat ONs 2 weeks after crush injury. a, b There were no differences in FSPs
Proximal part
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*
140 FSPs (n)
120
**
100 80 60 40 20
g
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noted by mixing the labeled surviving RGCs with the dyeengulfing macrophages and/or microglia [10, 13–15]. Regarding the method of injecting FG after the crush experiment and 5–7 days before animal sacrifice, the major concern is that the FG will not label RGCs efficiently if the axons of the ON are severely damaged. Several methods of ON injury, including indirect and direct ones, have been used in the literature [1, 2, 7, 16– 22]. The extent of RGC degeneration after ON crush is related to the force exerted by instruments and the applied duration [19]. Using constant force-exerting instruments is frequently chosen to get convincing results [23–26]. The aim of this study is to investigate how the methodology used to crush the ON affects the efficiency of FG 174
Ophthalmic Res 2014;51:173–178 DOI: 10.1159/000357736
Clip/30 s
Clip/60 s
Clip/90 s
**
**
Clip/120 s
Forceps
retrograde labeling and to evaluate whether the different timings of the FG injection influence the accuracy of RGC density measurements and their relationship with the dye-engulfing microglia.
Methods Animals Sixty adult male Wistar rats weighing 150–180 g (7–8 weeks old) were used in this study. The rats were obtained from BioLASCO Co., Taiwan. Animal care and experimental procedures were performed in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. The Institutional Animal Care
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between the proximal site and the distal site of the crush area in the sham group and the 30-second group (p > 0.05). c–f There were significantly fewer FSPs at the distal part of the ON than at the proximal part in the groups with crush durations of 60 s (* p = 0.03). Compared with the mean number of FSPs of the proximal side, the mean number of FSPs of the distal area after crush durations of 90 and 120 s, and in the group using jewel forceps was also significantly different (all ** p = 0.0001). Bar =100 μm (n = 6 in each group).
200
90 80 FSPs (n/HPF)
70
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Fig. 2. ON sections 2 weeks after ON crush with a microvascular clip for 30 s. a Representation of the ON demonstrated the FSP in the proximal (P) and distal (D) parts to the injury site. Bar = 100 μm. b Representation of zoom-in on the proximal part to the insult site. c Representation of zoom-in on the distal part to the insult site. Bar = 20 μm. d There was no significant difference in the number of FSPs between the proximal and distal parts
to the crush site (p = 0.063, n = 6 in each group).
ON Crush Experiments ON crush injuries were induced as described in our previous reports [12, 24, 26]. Briefly, a vascular clip (60-gram microvascular clip, item No. 14121, World Precision Instruments, Fla., USA) was applied to the ON at a distance of 2 mm posterior to the globe for durations of 30, 60, 90 and 120 s. We also performed ON crushing with jewel forceps at the same site for 15 s. A sham operation entailed the ON exposed without the crush. Efficiency of Retrograde Flow of FG in ON among Different Crush Settings We compared the FG-stained particles (FSPs) in 10 randomly selected high-power fields (HPFs) between the proximal and distal parts to the crush site of the ON under different crush settings (n = 6 in each group). All rats were euthanized 2 weeks after crush injury.
Two weeks after crush injury, the rats were sacrificed, and the flatmounted retinas were examined with a ×400 epifluorescence microscope (Axioskop; Carl Zeiss Meditech Inc., Thornwood, N.Y., USA). Immunohistochemistry: Double Staining of CD11b and FG in the Ganglion Cells of Retinas We conducted double immunohistochemical staining of retinal sections with a microglial marker (CD11b) and FG as described previously [12, 24]. CD11b primary antibodies (1:20, mouse antirat monoclonal antibody, AbD Serotec, Oxford, UK) and Cy3conjugated affinipure goat anti-mouse secondary antibody (1:100, Jackson Immunoreseach Laboratories, West Grove, Pa., USA) were used. The images were observed with a fluorescent microscope (Axioplan 2; Carl Zeiss Inc., Germany). Statistical Analysis Analysis of statistical significance was determined using 2-tailed Student’s t tests and 1-way ANOVA followed by Bonferroni’s multiple comparison test. Data are presented as means ± standard error of the mean. In all cases, p < 0.05 denotes statistical significance.
Results
Retrograde Labeling of RGCs with FG and Morphometry of RGCs before Crush or before Sacrifice The retrograde labeling of RGCs with FG (Fluorochrome, LLC, Denver, Colo., USA) before sacrificing animals has been described in our previous reports [12, 24, 26]. We defined group A by the timing of FG labeling ‘1 week after the crushing and 1 week before the sacrifice’ and group B by the timing of FG labeling ‘5 days before crushing and 19 days before the sacrifice’. The surgical procedures were identical in both groups. There were at least 12 rats in each group.
FSPs Adjacent to the Lesion Site of the ON in Different Settings of ON Crush The flow of FSPs was not blocked by the vascular clip/30-second group, as compared with the sham, but significantly blocked in the clip/60-second, clip/90-second, clip/120-second and jewel forceps/15-second groups
Retrograde Labeling of RGCs by Fluorogold in Optic Nerve Crush
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and Use Committee of the Tzu Chi Medical Center approved all animal experiments. All manipulations were performed with animals under general anesthesia by intramuscular injection of a mixture of ketamine (40 mg/kg body weight) and xylazine (4 mg/kg body weight; Sigma, St. Louis, Mo., USA), and using topical 0.5% Alcaine eyedrops (Alcon, Puurs, Belgium). The rats were maintained in an environmentally controlled room.
a
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c
d
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2,500
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**
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2,000
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p = 0.017
1,500
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p = 0.0001
1,000 500 0
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i j 2 weeks
Fig. 3. Whole mount preparation of retinas and morphometry of RGCs in central and mid-peripheral retinas 1 and 2 weeks after ON crush. Bar = 20 μm. a, e Sham operation. b, d, g, i Retrograde FG labeling 1 week before sacrifice (group A). c, e, h, j Retrograde FG labeling 5 days before ON crush experiments (group B). k, l In both central and mid-peripheral retinas, RGC densities were higher in group B than in group A 1 and 2 weeks after ON crush. b, d One week after crush injury, in group A, the survival rate of FG+ RGCs
was 59.8 and 48.2% in the central and mid-peripheral retinas, respectively. c, e In group B, the survival rate of FG+ RGCs was 68.7 and 68.2% in the central and mid-peripheral retinas, respectively. g, i Two weeks after crush injury, in group A, the survival rate of FG+ RGCs was 36.4 and 27.7% in the central and mid-peripheral retinas, respectively. h, j In group B, the survival rate of FG+ RGCs was 59.2 and 56.2% in the central and mid-peripheral retinas, respectively. * p < 0.05, ** p < 0.01 (n = 8 in each group, 32 rats in total).
(fig. 1).The mean number of FSPs in the proximal site of the crush area is 155 ± 24.4 (n = 6). The mean number of FSPs at the distal part to the crush site in the groups crushed for 30 s (clip/30 s), 60 s (clip/60 s), 90 s (clip/ 90 s), 120 s (clip/120 s) and 15 s (jewel forceps/15 s) were 135.7 ± 24.2/HPF, 126.8 ± 13.1/HPF, 84.1 ± 26.1/HPF, 1.3 ± 1.5/HPF and 1.8 ± 2.4/HPF, respectively. Moreover, the mean FSP number between the proximal and distal parts of the ON to the crush site in the setting of clip/30 s showed no significant difference (p = 0.063; fig. 2).
and 1,700 ± 470/mm2, respectively (fig. 3a, f). Both 1 and 2 weeks after ON crush, the densities of RGCs in group B were significantly higher than those of group A in both the central and mid-peripheral retinas (fig. 3).
Retrograde Labeling of RGCs with FG and the Morphometry of RGCs before Crush or before Sacrifice The densities of RGCs in the central and mid-peripheral retinas in the sham-operated eyes were 2,390 ± 630 176
Ophthalmic Res 2014;51:173–178 DOI: 10.1159/000357736
Dual Staining of CD11b and FG in the Ganglion Cell Layer of Retinas Two weeks after ON crush, the ratio of CD11b+ and FG+ cells in the RGC layer of the retinas in group B was higher than that of group A (34 ± 11 and 4 ± 3%/HPF, respectively, p = 0.0001; fig. 4). There was overcounting of RGCs in group B as evidenced by merged stained cells, which were dye-engulfing microglia instead of surviving RGCs.
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k
1 week before sacrifice
RGCs (n/mm2)
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b
50 45 40 35 30 25 20 15 10 5 0
**
Before sacrifice (group A)
Before crush (group B)
In experimental animal studies, ON crush provides a reliable method for studying quantitative anatomical effects on novel neuroprotection or neurodegeneration after insults [23, 27–30]. Our results demonstrate that a complete block of axoplasmal flow was caused by the jewel forceps for 15 s. The jewel forceps has been used in other experiments of ON crush [21, 22]. Qu and Jakobs [21] used jewel forceps for 10 s and found severe axon degeneration of the ON at 1 week, and nearly all the axons were lost after 3 weeks. In the 60-gram microvascular clip groups with crush durations over 30 s, the axons were damaged and showed attenuation of FG flow beyond the area of the lesion site. With crush duration shorter than 30 s with a microvascular clip, the FG flow was well preserved. Other reports also demonstrated that various pressures exerted by a clip and different crush durations could result in a different optic nerve damage [18, 23, 31]. In sum, our results indicate that different instruments to induce crush injuries with variable
durations may influence the efficiency of retrograde labeling. A higher RGC survival rate was noted in both central and mid-peripheral retinas in the group with FG injection before ON crush. These results indicate that the ON crush injuries may result in a reactivation of microglial cells engulfing FG instead of true RGCs, particularly when the labeled RGCs were degenerated [14, 32–34]. Our double immunohistochemical staining with CD11b and FG provided alternative evidence of microglial phagocytosis in group B. This implies that earlier injection of FG before crush experiments may result in a higher RGC survival rate due to FG+ microglia increasing in the retinas. Consequently, injecting FG 1 week before the sacrifice of the animals will result in a more accurate count of RGC density. In conclusion, our results suggest that different instruments and durations of crush injury have different effects on the efficiency of FG retrograde labeling in the ON crush models. Retrograde FG labeling should be conducted 5–7 days before euthanizing the animals to avoid the overcounting of RGCs.
Retrograde Labeling of RGCs by Fluorogold in Optic Nerve Crush
Ophthalmic Res 2014;51:173–178 DOI: 10.1159/000357736
Discussion
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Fig. 4. Immunohistochemical staining of CD11b (microglial marker) and FG in the ganglion cell layer (GCL) of the retinas. INL = Inner nuclear layer. a FG-stained cells presented as green particles (arrowheads) and CD11b+ cells as red particles (arrows). b Two weeks after ON crush, the proportion of CD11b+/FG+ in the RGC layer of the retinas in group B was higher than that of group A (34 ± 11 and 4 ± 3%, respectively, ** p = 0.0001; n = 6 in each group). There was overcounting of RGCs in group B as evidenced by merged staining cells proving that FG+ cells are microglia instead of vital RGCs.
CD11b + cells/ FG + cells (%)
a
Acknowledgements
Disclosure Statement
The authors wish to thank Mr. Malcolm Higgins for his assistance in the English editing and Ms. Su-Zen Chen for figure and data assistance.
Proprietary interest and grant support: none.
References
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