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Sprouting of axon-like processes from axotomized retinal ganglion cells induced by normal and preinjured intravitreal optic nerve grafts
Brain Research 991 (2003) 150 – 162 www.elsevier.com/locate/brainres
Research report
Sprouting of axon-like processes from axotomized retinal ganglion cells induced by normal and preinjured intravitreal optic nerve grafts H.X. Su, E.Y.P. Cho * Department of Anatomy, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China Accepted 13 August 2003
Abstract The failure of axonal regeneration in the mammalian central nervous system (CNS) is currently attributed to the glial environment of the lesion site which elaborates a multitude of inhibitory factors. Less attention has been paid to the potential of trophic support associated with the CNS, especially in relation to the status of the damaged CNS after an injury has been evoked. Using a grafting paradigm to implant an optic nerve (ON) segment into the vitreous, we have addressed how a prior damage of the ON before grafting influences its ability to stimulate retinal ganglion cells (RGCs) to sprout axon-like processes. Our results showed that a normal noninjured ON implanted intravitreally stimulated sprouting of RGCs, as revealed by sliver staining of the sprouting cells, as well as increasing the number of RGCs which express GAP-43. A prior crush injury of the ON 7 days before its implantation into the vitreous resulted in a significant decrease in its ability to stimulate RGC sprouting when the crush lesion segment was used as the graft, whereas grafts taken from segments proximal and distal to the lesion segment had potencies similar to that of the noninjured graft. Both astrocytes and oligodendrocytes were drastically reduced in number in the lesion segment graft, suggesting their involvement in the secretion of soluble trophic factors that may play a role in the sprouting and regeneration of damaged neurons. D 2003 Elsevier B.V. All rights reserved. Theme: Development and regeneration Topic: Regeneration Keywords: Retinal ganglion cell; Regeneration; Sprouting; Optic nerve; Hamster; Astrocyte; Oligodendrocyte
1. Introduction Retinal ganglion cells (RGCs) and their associated projections to the brain have recently become an attractive experimental model to study the issue of axonal regeneration in the central nervous system (CNS) (e.g., see reviews in Refs. [13,21,23,53]. A number of experimental manipulations have been found to enhance the axonal regeneration of rodent RGCs after injury of the optic nerve (ON), including apposition of a peripheral nerve (PN) segment to the cut ON [49] or implantation of a PN into the vitreous [3], intraocular administration of trophic factors [12], inactivation of the Rho signaling pathway [30], injury of the lens [15,16,32], and introduction of
* Corresponding author. Tel.: +852-26096842; fax: +852-26035031. E-mail address: [email protected] (E.Y.P. Cho). 0006-8993/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2003.08.010
activated macrophages into the ON [28] or intraocularly [52]. Our work in the hamster have also demonstrated that axotomized RGCs exhibit an alternative form of regeneration, namely, the sprouting of axon-like processes from the somatodendritic compartment under the influence of an intravitreally implanted PN [8,9]. One of the advantages of this intravitreal grafting paradigm as compared to grafting directly to the ON is that interactions between putative diffusible factors emanating from the graft and the somata of the RGCs can be observed without the complications of substrate interactions, since the graft and retina remain separate from each other by a small distance [8]. Thus, previous studies with an intravitreally transplanted PN have demonstrated that diffusible factors secreted from the graft stimulate sprouting of axon-like processes, upregulation of the growth associated protein GAP-43, as well as enhancement of axonal regeneration in the crushed ON in situ [3,8,37]. Utilizing the same paradigm, we have recently shown that the isolated epineu-
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rium of a PN also stimulates the sprouting of axotomized RGCs [27]. The glial environment of the damaged CNS has been implicated as a major factor in impeding regeneration [14,47], especially with regard to some of the well-characterized inhibitory factors associated with oligodendrocytes and astrocytes [1,20,22,34,36,50]. In contrast, the issue of the existence of factors associated with the damaged CNS, especially soluble ones, which can stimulate regeneration, has not been adequately addressed. One example is the series of studies done by Cotman et al. in the 1980s which show that the wound adjacent to a damaged brain area contains soluble factors with neuronotrophic activities [38,39]. However, most of these early studies utilized in vitro assays to quantify the injury-associated neurotrophic activities, so that their effects in vivo remain obscure. Recently, So and colleagues reported that the concurrent intravitreal grafting of a normal ON segment together with either an intravitreal PN or a PN grafted to the cut ON potentiate the sprouting response of RGCs by the PN, but that the normal ON when grafted alone does not stimulate sprouting [10,11]. In these studies, although the normal ON by itself seems unable to elaborate significant soluble trophic factors, we reasoned that it may behave differently after injury with regard to trophic factor production, based on the findings with the injured brain as mentioned above. Thus, in this study, we utilize the intravitreal grafting paradigm to assess whether a preinjured white matter fibre tract, the ON, possesses diffusible factors to elicit sprouting of RGCs. This was performed by damaging the ON with a crush lesion followed by intravitreal transplantation of the lesioned segment at 7 days post-crush. The sprout-inducing potential of this lesion segment was compared to that of segments taken both proximal and distal to the lesion segment, as well as to that of a normal intravitreal ON graft which had not been preinjured (noninjured ON graft). Our results indicate that both the noninjured ON as well as the segments proximal and distal to the crush lesion stimulated sprouting of axon-like processes from RGCs. In contrast, the segment encompassing the lesion did not induce sprouting. The results suggest that while an intact CNS white matter like the ON possesses diffusible sprout-inducing activity, this could become abolished after injury, an observation which may have some bearing associated with the failure of axonal regeneration in the CNS.
2. Materials and methods A total of 64 adult female hamsters (Mesocricetus auratus) 6 –8 weeks old were used in this study. Animals were randomly assigned to various experimental groups to receive different grafting procedures. All operations were performed under general anesthesia induced by intraperitoneal injection of a mixture of ketamine (200 mg/kg) and xylazine (20 mg/kg).
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2.1. Intraorbital ON crush to axotomize RGCs All animals were inflicted with an intraorbital ON crush to serve as the experimental model of damaging the RGC axons. The right ON was exposed through a superior intraorbital approach and crushed with fine forceps for 10 s about 2 mm behind the globe. Under the operating microscope, the nerve was seen to turn translucent at the crush lesion site but the dural sheath remained intact. 2.2. Intravitreal graft preparation The experimental layout of the different grafting paradigms was as illustrated in Fig. 1. The grafts in this study consisted of one of the following groups prepared from either normal noninjured ONs or from ONs which had been preinjured 1week before intravitreal grafting: (A) rinoninjured viable ON (vON) allografts (B) noninjured viable ON (vaON) autografts (C) noninjured nonviable ON (nvON) allografts –graft rendered nonviable before implantation into the vitreous (see below) (D) lesion segment of ON (LON) – the region that has been crushed 1 week previously (E) proximal segment (PON) –the region between the eyeball and the crush lesion (F) distal segment (DON) –the region beyond the lesion towards the optic chiasm (G) nonviable distal segment (nvDON) Most experiments in this study were performed by transplanting a segment of ON isolated from 1 hamster (allograft) to the vitreous body of the eye of another hamster which had been inflicted with an experimental intraorbital ON crush. This procedure avoided simultaneous damage of both ONs in the same animal and caused less trauma to the animal. Moreover, it has been reported that damage of one ON would result in injury-associated cellular changes of RGCs and glia of the contralateral eye [5], thus potentially complicating the interpretation of the results if both eyes were damaged in one animal. However, in order to address whether potential allograft-induced immune reactions might influence the responses of the axotomized RGCs, autologous grafting of a noninjured ON into the vitreous was performed in another group of animals to look for any possible differences from the allografted animals in terms of the stimulation of RGC sprouting. 2.2.1. Normal noninjured ON grafts 2.2.1.1. Viable allografts (vON). After dividing the superior rectus muscle, the eyeball was protracted slightly outwards by sutures anchored to the fascia around the
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Fig. 1. Schematic diagram showing the intravitreal transplantation of (A) a normal noninjured ON, or (B) either the proximal (P), lesion (L), or distal (D) segment of an ON which had been crushed 7 days ago. The crushed site is denoted by crosshatching.
eyeball in order to visualize the dorsal part of the ON. A cut was made on the dorsal part of the dura using a pair of fine iris scissors at approximately 1 mm behind the globe to expose the ON proper which was then transected completely. The whole length of ON distal to the cut was pulled out gently using fine forceps. After removing the meninges, the ON was transferred to cold tissue culture medium (DMEM/F12) and cut into 2-mm-long segments to be used for allografting. Each ON from a donor could provide grafts for four animals. The donor was killed by a lethal dose of anesthetic immediately after removal of the ON. 2.2.1.2. Viable autografts (vaON). In another group of animals, autologous grafting of a viable noninjured ON was performed by removing 2 mm of the left ON and implanted into the vitreous of the right eye whose ON has been concurrently crushed. 2.2.1.3. Nonviable allografts (nvON). In order to assess the influence of cellular components of the ON graft and any potential nonspecific influence of surgical procedures on the axotomized RGCs, the ON graft was rendered nonviable (destruction of all cells in the graft) by alternately freezing the graft in liquid nitrogen for 5 min followed by thawing at room temperature for another 5 min for a total of 5 cycles. After the freezing/thawing procedure was com-
pleted, the ON graft was transferred to cold culture medium and cut into 2-mm-long segments to be used as nonviable allografts. 2.2.2. Preinjured ON grafts A crush lesion spanning 2.5 mm was made at about 2 mm behind the eyeball. At 1 week post-crush, the ON was exposed again and the crush site identified by its distinctly narrower caliber compared to the regions proximal and distal to it. A 2-mm-long segment consisting exclusively of either the crush lesion (LON), proximal (PON) or distal segment (DON) was removed and transferred to cold culture medium before transplantation. A control group using the distal segment and rendering it nonviable (nvDON) was also examined and compared with the viable grafts. 2.3. Implantation of an ON segment into the vitreous body Intravitreal ON transplantation was carried out immediately after intraorbital ON crush (Fig. 1), following the procedures as described in Cho and So [8]. Briefly, a small penetrating lesion was made at a point slightly behind the limbus of the superior aspect of the eyeball with a 27-G needle. The ON graft was gently introduced into the vitreous body through the lesion with the aid of a finetipped glass micropipette, after which the lesion was closed by a single 10/0 suture.
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2.4. Intravitreal PN grafting In order to compare the propensity of an intravitreal ON to stimulate RGC sprouting to that of a PN graft, transplantation of a 2-mm desheathed viable PN segment (vPN) into the vitreous was performed in a separate group of animals, as described in Cho and So [8]. The retinas from the grafted eyes were processed in the same manner as that from animals with ON grafts (see below). 2.5. Examination of the intravitreal ON graft at 2 weeks post-grafting To assess the cellular changes and viability status of the ON graft, it was removed from the eyeball at 2 weeks postgrafting, the time when the animal was sacrificed for examination of RGC regenerative sprouting in the retina. After the animal was sacrificed with a lethal dose of 20% chloral hydrate, it was perfused transcardially with phosphate-buffered saline (PBS). The eyeball was removed and placed in 4% paraformaldehyde (in 0.1 M phosphate buffer [PB] pH 7.4). The cornea and lens were dissected away rapidly to expose the ON graft. After separation from the eyecup, the graft was fixed for 1 h and then washed in PBS repeatedly for 1 – 2 h before being immersed in 20% sucrose/0.1 M PB overnight at 4 jC. The following day,
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the graft was embedded in OCT, cut into 10-Am sections with a cryostat and mounted onto 3% gelatin-coated slides. The sections were immunostained with either anti-GFAP antibody (Dako, rabbit polyclonal, 1:1000) to label astrocytes, or anti-Rip antibody (Chemicon, mouse monoclonal, 1:2000) to label oligodendrocytes [18]. The primary antibodies were visualized with either anti-rabbit or anti-mouse IgG conjugated to Cy3 (Jackson ImmunoResearch Labs, 1:400, 2 h at room temperature). Both the primary and secondary antibodies were diluted in PBS containing 0.1% Triton-X/0.5% bovine serum albumin/0.9% NaCl. Sections were coverslipped with glycerol and observed under an epifluorescence microscope. In control experiments to assess antibody specificity, some sections were processed without incubation of the primary antibody (anti-GFAP or Rip) to ascertain for the absence of labelling under these circumstances. 2.6. Silver staining of the retina At 2 weeks post-ON crush, the animals were sacrificed with a lethal dose of 20% chloral hydrate. The operated eyeballs were removed immediately without perfusion. Retinas were dissected in 2% paraformaldehyde/0.1 M PB and fixed for 1 h. They were then silver stained by a modified protocol of Leicester and Stone [31] which had
Fig. 2. Photomicrographs showing the cellular components of the ON graft after 2 weeks in the vitreous body. (A) anti-GFAP staining showed that astrocytes were still present in the viable allograft of normal ON (vON). (B) No GFAP-positive staining was detected in the nonviable allograft (nvON), indicating the astrocytes in the graft had been killed before grafting. (C) Rip-positive staining showed oligodendrocytes were still present in the vON. (D) No Rip staining was detected in the nvON, indicating that the oligodendrocytes in the graft had been killed before grafting. Scale bar = 100 Am (A – D).
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been found to reveal the detailed morphology of RGCs exhibiting regenerative sprouting of axon-like processes [8,27]. After silver staining, the retinas were mounted onto 3% gelatin-coated slides, dehydrated in ethanol, cleared in xylene, and coverslipped with Permount. 2.7. Anti-GAP-43 labeling of the retina At 2 weeks post-ON crush, the animals were sacrificed with a lethal dose of 20% chloral hydrate and perfused with PBS transcardially. The retinas were dissected and fixed in 4% paraformaldehyde/0.1 M PB. After 1 h fixation, the
retinas were washed in PBS for about 2 h and then transferred to PBS/1% Triton-X for another hour. This was followed by incubation in 20% fetal bovine serum for 1 h at room temperature to block nonspecific staining. Subsequently, the retinas were incubated in anti-GAP-43 antibody (mouse monoclonal from Sigma) at a dilution of 1:1000 (in PBS containing 1% Triton-X/0.5% bovine serum albumin/ 0.9% NaCl) overnight at 4 jC. After several washes in the same buffer used as the antibody diluent, the retinas were incubated overnight with an anti-mouse IgG secondary antibody conjugated to biotin (Jackson ImmunoResearch Labs, 1:500 in the same diluent of the primary antibody) at
Fig. 3. Photomicrographs showing silver-stained RGCs in the central part of the superior temporal retinal quadrant in eyes with normal noninjured ON (A – C) and PN (D) grafts. (A) In the eye implanted with a vON, a small population of axotomized RGCs with large and irregular cell sizes (examples indicated by arrows) were revealed by the silver staining. (B) In the eye implanted with a vaON, sprouting cells similar to that in (A) were also present (shown by the arrows). (C) In the eye implanted with a nvON, sprouting cells were rarely seen. (D) In the eye implanted with a PN, more sprouting cells were observed compared to that seen in viable ON grafts. (E) Detailed morphology of a sprouting RGC under high magnification. Arrows indicate the haphazard course taken by a sprout. Asterisk indicates a branching point, and the curved arrow a loop. Scale bar = 500 Am (A – D); 100 Am (E).
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4 jC. On the following day, retinas were incubated with Streptavidin-HRP (Jackson ImmunoResearch Labs, 1:1000 in PBS/1% Triton-X) overnight at 4 jC. The HRP label was developed by the diaminobenzidine(DAB) and glucose oxidase reaction [45]. After DAB development, the retinas were mounted onto 3% gelatin-coated slides, dehydrated, cleared, and coverslipped with Permount. The specificity of the immunostaining was assessed by omitting the primary anti-GAP-43 during staining of the grafted retinas whereupon no labelling of RGCs could be observed. 2.8. Quantification of the number of silver-stained RGCs and GAP-43-positive RGCs The morphometric analysis program Stereo Investigator (MicroBrightfield Inc.) was used to compute the total number of silver-stained sprouting RGCs and GAP-43positive RGCs by scanning the whole retinal area under 400 magnification via the motorized control of the microscope stage. All silver-stained RGCs which exhibited sprouting (as judged by the criteria listed in the Results) and all GAP-43-positive RGCs in each retina were counted. 2.9. Statistical analysis of the data
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further since it has been shown that the survival and regeneration of RGCs could be enhanced by injury to the lens or other intraocular structures [16,32,33]. Immunohistological examination of the intravitreal graft showed that astrocytes and oligodendrocytes were still present in the vON graft as revealed by the strong staining of anti-GFAP and anti-Rip (Fig. 2A and C), whereas no positive anti-GFAP or anti-Rip staining was detected in the nvON graft (Fig. 2B and D), indicating that cells initially present in the graft were killed by the repeated freezing and thawing procedures. 3.1.2. Silver staining of RGCs In eyes implanted with either a vON or vaON (Fig. 3A and B), silver staining of the retina revealed the presence of a population of RGCs with large and irregular cell sizes. Most of these cells were located in the superior quadrants close to the intravitreal graft. On detailed examination under high magnification, some of these cells exhibited sprouting of newly formed processes from their somatodendritic compartment. The de novo growth nature of these processes was suggested by their morphology (Fig. 3E): They were uniform in thickness, sparsely branched, and extended far beyond the maximum dendritic domain of normal RGCs. Many of them ran haphazardly and unconfined to a partic-
The variations in the mean number of silver-stained sprouting RGCs and GAP-43-positive RGCs in the different experimental groups were analyzed by one-way ANOVA followed by a post hoc pairwise comparison with the level of significance set at P = 0.05.
3. Results In the following descriptions of the results, the abbreviations used for the different groups of grafts were as listed in Materials and methods. The results obtained from the transplantation of normal noninjured ON grafts would be presented first and followed by that associated with the preinjured ON grafts. 3.1. Normal noninjured ON grafts 3.1.1. Appearances of intravitreal ON graft When the animals were sacrificed at 2 weeks postgrafting, the ON graft was usually found to be located in one of three sites in the eye: attached to the ora serrata adjacent to the limbal lesion site (most common), freely suspended in the vitreous body, or rarely attached to the posterior surface of the lens. There was no direct correlation between the location of the graft and the magnitude of the sprouting response of the RGCs. Great attention was being paid to see whether there were abnormalities in appearance of intraocular structures, such as damage to the lens or distortion of the retina. Although such damages were uncommon, eyes with such abnormalities were not analyzed
Fig. 4. A comparison of the mean number of (A) silver-stained sprouting RGCs and (B) GAP-43-positive RGCs, among the viable and nonviable noninjured ON and the PN graft groups, at 2 weeks post-ON crush and grafting.
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ular retinal layer, forming turns and even complete loops along their course. Thus, they could be distinguished from the dendrites of RGCs and were termed axon-like processes [8]. RGCs which exhibited formation of these axon-like processes were called sprouting RGCs in this study. However, not all the large and irregular-sized RGCs which were revealed by silver staining have identifiable sprouts as judged by the above criteria. Therefore, in this study, every silver-stained RGC in each retina was examined in detail under high magnification, and the number of silver-stained cells with identifiable sprouts was quantified according to the above criteria. When compared to the viable ON graft, RGC sprouting induced by the nonviable graft was minimal (Fig. 3C). The mean number of sprouting RGCs in the vON group (41.2 F 28.8, n = 5) was significantly higher than the control nvON group (2.2 F 1.9, n = 5) ( p < 0.05; Fig. 4A). Eyes grafted with a vaON exhibited comparable RGC sprouting (mean number = 35.3 F 2.2, n = 4) to the vON group, and there was no significant difference between these two viable groups ( p>0.1; Fig. 3B and 4A). Thus, a normal noninjured ON grafted intravitreally induced RGC sprouting irrespective of it being an allo- or autograft. Since allografts did not differ from autografts in their potency to stimulate RGC sprouting, other experiments in this study were performed with ON allografts as explained in Materials and methods.
Compared to an intravitreal PN graft, both allo- and auto-intravitreal ONs were less potent in stimulating RGCs to sprout (compare Fig. 3D to A and B). The mean number of sprouting RGCs in the PN-grafted eyes (186 F 52.9, n = 4) was about 4.5 times that seen in ON-grafted eyes (Fig. 4A). 3.1.3. Anti-GAP-43 staining of RGCs Similar to the silver staining experiments, anti-GAP-43 labeled a number of large and irregular-sized RGCs which were also mainly located in the superior retinal quadrants of the vON-grafted eye (Fig. 5A). Unlike the silver-stained cells, however, RGCs with smaller cell bodies were also marked by anti-GAP-43 (Fig. 5A). The large and irregular cells were also different from the sliver-stained RGCs in a number of aspects. For example, GAP-43-positive cells possessed short filamentous processes, mostly on their cell body surface, but sometimes also on the dendrites and axon (Fig. 5B). Sprouts tipped by an expanded growth cone-like structure emerging from either the cell body or axon at a site close to the cell body was also sometimes observed in GAP43-positive but not silver-stained RGCs (Fig. 5B), whereas the long sprouts arising from dendrites in the silver-stained cells were not as easy to spot in GAP-43-labeled cells. Thus, although both silver staining and anti-GAP-43 labeled the large and irregular sprouting cells, different aspects of the
Fig. 5. Photomicrographs showing GAP-43-positive RGCs in the central superior retinal quadrant in the vON (A), nvON (C), and vPN (D) groups. More GAP43-positive cells were induced by the vON than the nvON, but the vPN is the most potent. Large and irregular-sized RGCs (black arrows in A and C) were observed in both the vON and nvON groups, but they were higher in number in the vON group. In addition, cells with small somata (white arrows) were present in all groups as well, but they tend to predominate in the nvON group. (B) shows a GAP-43 labeled large irregular-sized RGC with short filamentous processes (arrow), and a sprout tipped by a growth cone-like profile (*) which is shown at a higher magnification in the inset; a = axon of the cell. Scale bar in (D) also applies to (A) and (C).
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detailed morphology of these cells were selectively discernable by one of the two methods. The mean number of GAP-43-positive RGCs in the vON group was significantly higher than that in the nvON control group: 345.8 F 122.3 (n = 4) versus 140.5 F 39.2 (n = 4; p < 0.05; Fig. 4B). Moreover, most of the labeled cells in
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the nvON group were of smaller soma sizes (compare Fig. 5C to A). Anti-GAP-43 staining labeled more RGCs than silver staining did because GAP-43 expression was more related to an attempt to regenerate, whereas silver staining revealed only those RGCs which have actually sprouted axon-like processes. The predominance of smaller soma-
Fig. 6. Photomicrographs illustrating the appearances of preinjured ON grafts after 2 weeks post-grafting into the vitreous. The staining of GFAP (A – D) in the proximal segment (PON) and distal segment (DON) grafts were equally strong after 2 weeks in the vitreous. However, the GFAP staining was greatly reduced in the graft originating from the lesion site (LON). In the nonviable distal segment graft (nvDON), GFAP staining was totally absent. Rip staining (E – H) in the PON and DON grafts were strong after 2 weeks in the vitreous. However, the Rip staining in the LON graft was confined to scattered profiles. In the nvDON, Rip staining appeared as granules which may represent debris of dead oligodendrocytes. Scale bar = 100 Am (A – H).
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sized GAP-43-positive cells in the nvON control group was due to the absence of the large sprouting RGCs and possibly more shrinkage of the somata of the surviving cells in the absence of a viable ON. When compared to an intravitreal PN graft, the ON graft was less effective in inducing GAP-43 expression from axotomized RGCs (Fig. 5D). The mean number of RGCs expressing GAP-43 in PN-grafted eyes (1228 F 318, n = 3)
was four times that found in the ON-grafted eyes ( p < 0.05; Fig. 4B). 3.2. Preinjured ON grafts 3.2.1. Appearances of the preinjured intravitreal ON grafts At 2 weeks post-grafting, the staining of GFAP in the PON and DON grafts were equally strong (Fig. 6A and C)
Fig. 7. Photomicrographs comparing the appearances of silver-stained sprouting RGCs (A – D) and GAP-43-positive RGCs (E – H) among the different preinjured ON graft groups. The PON and DON grafts induced sprouting from some RGCs (arrows in A), while sprouting induced by the LON and nvON grafts were minimal. More GAP-43-positive RGCs (arrows in E) were induced by the PON and DON grafts compared to the LON and nvDON grafts. Scale bar = 500 Am (A – D); 100 Am (E – H).
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and similar to that of intravitreal ON grafts which were uninjured at the time of transplantation into the vitreous (cf. Fig. 2A). There was also strong staining of oligodendrocytes in the PON and DON grafts (Fig. 6E and G). However, both GFAP and Rip staining were greatly reduced in the LON graft (Fig. 6B and F), with the labeling being confined to scattered profiles. In the nvDON graft, GFAP staining was totally absent, while fine granules of Rip labeling was seen dispersed throughout the graft (Fig. 6D and H). The granular-like labeling of Rip in the nonviable distal segment graft may be due to the persistence of degenerating debris of oligodendrocytes which had not been totally cleared away. 3.2.2. Silver staining of RGCs Some large and irregular sized RGCs were stained in the PON and DON groups, their numbers (57 F 28.4, n = 4 and 67.2 F 22.1, n = 5 respectively) not appreciably different from each other (Fig. 7A and C; Fig. 8A), nor to the vON group (Fig. 8A). The nvDON elicited almost no sprouting cells (1.4 F 0.9, n = 5; Figs. 7D and 8A), indicating that any nonspecific stimuli arising from surgical disturbance of the intraocular milieu was probably minimal, and that the sprouting induced by the PON or DON necessitated the presence of viable cells in the graft.
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In contrast, the LON graft stimulated significantly fewer RGCs (6.2 F 4.9, n = 5) to sprout (Figs. 7B and 8A) than the PON, DON or vON groups. In fact, there was no significant difference in the number of sprouting RGCs induced by the LON versus the nvDON graft (Fig. 8A). 3.2.3. Anti-GAP-43 staining of RGCs Both the PON and DON grafts stimulated GAP-43 expression in a comparable number of RGCs (Fig. 7E and G; Fig. 8B), whereas the LON graft was significantly less potent in this aspect: the number of GAP- 43-positive RGCs induced by the LON (139 F 67.6, n = 4) was less than onehalf of that in the DON group (312 F 108, n = 4), as well as being significantly lower than in the PON (265 F 54, n = 4) or vON groups (Figs. 7F and 8B). The control nvDON also induced significantly smaller numbers of RGCs (139 F 20, n = 4) to express GAP-43 compared to the DON group (Figs. 7H and 8B). Again, there was no significant difference between the LON and nvDON graft in terms of their induction of GAP-43 expression in RGCs (Fig. 8B).
4. Discussion 4.1. A normal noninjured intravitreal ON graft induces sprouting of axon-like processes from RGCs and enhances GAP-43 expression
Fig. 8. A comparison of the mean number of (A) silver-stained sprouting RGCs and (B) GAP-43-positive RGCs among the different preinjured ON groups. The data from the vON group were included for comparison with that of the preinjured grafts.
In this study, a viable intravitreal ON is found to induce a small population of axotomized RGCs to sprout axon-like processes. Although the ON graft is not as potent as an intravitreal PN in eliciting RGC sprouting, it exerts a clearcut stimulus on the axotomized RGCs, as indicated by the 20-fold difference in the number of sprouting RGCs induced by the viable versus the nonviable ON grafts, as well as the upregulation of GAP-43 in more axotomized RGCs after axotomy. These results are at variance with a previous study by Cho et al. [10] which stated that an intravitreal ON only augmented the effects of a concurrently implanted PN but did not induce sprouting by itself. Differences in experimental design and procedures may be responsible for the discrepancies between the two studies. Cho et al.’s study [10] employed transection of the ON as the axotomy paradigm, while ON crush was used in this study. It is known that a crush lesion leads to a smaller degree of cell death compared to a cut [2], so more surviving RGCs may be stimulated by an intravitreal ON. Secondly, a shorter (1-mm) segment of ON was used for the transplant in Cho et al.’s study which would theoretically produce less stimulus, compared to the 2-mm graft in our experiments. Thus, the ON cut axotomy paradigm coupled with a shorter intravitreal ON graft in Cho et al.’s study may explain why they were unable to see a positive effect. In contrast, our present results clearly show that an intravitreal ON by itself has regeneration-promoting effects on the axotomized RGCs, as assessed by two
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parameters: sprouting of axon-like processes and increased GAP-43 expression. 4.2. Possible mechanisms associated with the action of the intravitreal ON The viable intravitreal ON graft contains an abundance of astrocytes and oligodendrocytes as revealed by anti-GFAP and anti-Rip staining, indicating that they have survived throughout the 2 weeks grafting period. Astrocytes are known to produce an impressive list of molecules which enhance neuronal survival and growth (reviewed in Refs. [6,40,44]). CNTF, which is known to stimulate sprouting and regeneration of RGCs [12,24] and is present in the normal ON [26], is one of the possible candidates. Basic FGF is also constitutively present in the ON and enhances the survival of RGCs when applied to the cut end of ON [46]. Oligodendrocytes have been reported to secrete IGF-1 in culture to promote neuronal survival [51]. The differential contribution of astrocytes and oligodendrocytes to RGC sprouting can be addressed by selectively ablating the astrocytes in the ON with aminoadipate [25] before grafting so that the graft becomes predominated by oligodendrocytes. The status of microglia in the graft have not been examined in this study. Macrophages and activated microglia release certain types of cytokines or proimflammogens [19,29] that in turn may regulate the function of astrocytes in the ON graft. Thus, different cell types in the ON may interact in a complex manner to result in the positive effects observed in this study. 4.3. Both proximal (PON) and distal (DON) segments of the preinjured graft are effective in stimulating RGCs to sprout Examination of the intravitreal PON and DON at 2 weeks post-grafting reveals that the cellular morphology of the astrocytes and oligodendrocytes are not appreciably different from those in a normal intravitreal ON graft. In fact, their appearances are similar to that of the proximal and distal segments of the ON injured in situ, in which the staining intensity of GFAP [4,17] and oligodendrocytes [7,41] are also not appreciably different from that of uninjured nerves. However, it should be remembered that there would be comparatively fewer axons in the proximal segment since over 70 –80% of RGCs have degenerated by 2 weeks post-injury [2], while the disconnected axons in the distal segment are undergoing Wallerian degeneration. Such profound changes in axon to glia relationships would be expected to alter the functional status of the latter. Thus, oligodendrocytes exhibit a decrease in expression of certain myelin protein genes after ON transection [35], while the production of CNTF mRNA by astrocytes also suffers a small drop in both segments after ON crush [26]. In addition, there is a change in phagocytic activity of microglia in the two segments (especially at the distal segment) by virtue of their positive staining with the ED1 antibody
[17,41]. However, it seems that such changes of glial cells in the proximal and distal segments of the injured ON have only limited effects on their sprout-inducing propensity, as reflected by the present results that the PON and DON grafts stimulate RGC sprouting to magnitudes similar to that of the normal ON. Thus, a basal level of stimulus may exist in the normal ON and which is maintained in the segments proximal and distal to the lesion after injury. The exact cellular/molecular identity responsible for this ‘‘basal level’’ can only be speculated at present, but CNTF may be one of the possibilities. The amount of CNTF in the normal ON is very high for a CNS tissue [48], and it remains at 60% of the normal level in the distal segment of a crushed ON [26]. Such relatively small drop in CNTF level in the distal segment of the preinjured ON may explain the constancy of the sprouting response of RGCs induced by it. 4.4. The lesion site graft (LON) is significantly less potent in stimulating RGCs Grafting of an intravitreal LON results in dramatically fewer RGCs to form sprouts and express GAP-43, the values of which approach that induced by the nvDON graft, indicating that the source of stimulus is almost eliminated in the LON. Apart from damaging the RGC axons, a crush injury of the ON leads to extensive cell death within the lesion area [17,41]. From 2 days post-crush onwards, astrocytes which initially express only S-100 but not GFAP begin to repopulate the lesion site at slow rate [17], hence, the number of GFAP-positive cells at 7 days post-crush is very small. The same is true for oligodendrocytes which is almost totally absent at the crush site during the first week post-injury [7,41]. In contrast, there is a massive influx of macrophages from the circulatory system into the lesion area, so that by 1 week, the lesion is already filled with ED1-positive macrophages which only extend a small distance into the proximal and distal segments [17,41]. Thus, at the time of transplantation of the LON into the vitreous, astrocytes and oligodendrocytes should be scarce, while macrophages would be plentiful in the graft. When the LON graft is examined 2 weeks after being implanted into the vitreous, astrocytes and oligodendrocytes are still scarce. Although we have not examined the status of the macrophage population in the intravitreal graft, it is highly likely they are still present in the LON graft, if they resemble the in situ pattern of distribution [3,41]. Thus, some of the more prominent differences between the LON and other viable intravitreal grafts (vON, PON, and DON) is the scarcity of astrocytes and oligodendrocytes, together with an abundance of macrophages, in the former. Dynamic changes in the different glial cells may be associated with the observed changes in RGC sprouting. For example, the amount of CNTF mRNA at the ON crush site is dramatically decreased compared to both the proximal and distal segments, in association with a marked reduction of CNTF-
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positive astrocytes at the lesion site [26]. Taken together, the scarcity of astrocytes and the concomitant drop in CNTF (and possibly other growth factors) in the LON graft may lead to the decrease in RGC sprouting. On the other hand, the role of macrophages in the LON graft remains to be determined. Although macrophages/microglia have been demonstrated to promote axonal regrowth in a number of studies [42,43,52], future experiments designed to address differences in macrophage numbers, subpopulations, and activities (e.g., in terms of phagocytosis) in the intravitreal ON grafts and correlating them to the induced sprouting response may shed light on this issue.
[8]
[9]
[10]
[11]
5. Conclusions [12]
This study highlights the existence of the ability of the intact CNS in stimulating neural regeneration. We have demonstrated that a normal CNS white matter region, the ON, produces soluble factors which promote regenerative sprouting of RGCs upon intravitreal grafting. However, prior damage of the same region leads to a greatly attenuated effect on the RGCs. Such changes in trophic support of the CNS after injury may partly explain the usually meager response of the damaged neurons to regenerate in a CNS environment. It would be interesting to see whether such changes occur universally in other CNS regions and their consequences to the recovery of the damaged neurons.
[13]
[14] [15]
[16]
[17] [18]
Acknowledgements The work reported in this paper was fully supported by a grant from the Research Grants Council of Hong Kong (CUHK4265/98M).
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Research report
Sprouting of axon-like processes from axotomized retinal ganglion cells induced by normal and preinjured intravitreal optic nerve grafts H.X. Su, E.Y.P. Cho * Department of Anatomy, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China Accepted 13 August 2003
Abstract The failure of axonal regeneration in the mammalian central nervous system (CNS) is currently attributed to the glial environment of the lesion site which elaborates a multitude of inhibitory factors. Less attention has been paid to the potential of trophic support associated with the CNS, especially in relation to the status of the damaged CNS after an injury has been evoked. Using a grafting paradigm to implant an optic nerve (ON) segment into the vitreous, we have addressed how a prior damage of the ON before grafting influences its ability to stimulate retinal ganglion cells (RGCs) to sprout axon-like processes. Our results showed that a normal noninjured ON implanted intravitreally stimulated sprouting of RGCs, as revealed by sliver staining of the sprouting cells, as well as increasing the number of RGCs which express GAP-43. A prior crush injury of the ON 7 days before its implantation into the vitreous resulted in a significant decrease in its ability to stimulate RGC sprouting when the crush lesion segment was used as the graft, whereas grafts taken from segments proximal and distal to the lesion segment had potencies similar to that of the noninjured graft. Both astrocytes and oligodendrocytes were drastically reduced in number in the lesion segment graft, suggesting their involvement in the secretion of soluble trophic factors that may play a role in the sprouting and regeneration of damaged neurons. D 2003 Elsevier B.V. All rights reserved. Theme: Development and regeneration Topic: Regeneration Keywords: Retinal ganglion cell; Regeneration; Sprouting; Optic nerve; Hamster; Astrocyte; Oligodendrocyte
1. Introduction Retinal ganglion cells (RGCs) and their associated projections to the brain have recently become an attractive experimental model to study the issue of axonal regeneration in the central nervous system (CNS) (e.g., see reviews in Refs. [13,21,23,53]. A number of experimental manipulations have been found to enhance the axonal regeneration of rodent RGCs after injury of the optic nerve (ON), including apposition of a peripheral nerve (PN) segment to the cut ON [49] or implantation of a PN into the vitreous [3], intraocular administration of trophic factors [12], inactivation of the Rho signaling pathway [30], injury of the lens [15,16,32], and introduction of
* Corresponding author. Tel.: +852-26096842; fax: +852-26035031. E-mail address: [email protected] (E.Y.P. Cho). 0006-8993/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2003.08.010
activated macrophages into the ON [28] or intraocularly [52]. Our work in the hamster have also demonstrated that axotomized RGCs exhibit an alternative form of regeneration, namely, the sprouting of axon-like processes from the somatodendritic compartment under the influence of an intravitreally implanted PN [8,9]. One of the advantages of this intravitreal grafting paradigm as compared to grafting directly to the ON is that interactions between putative diffusible factors emanating from the graft and the somata of the RGCs can be observed without the complications of substrate interactions, since the graft and retina remain separate from each other by a small distance [8]. Thus, previous studies with an intravitreally transplanted PN have demonstrated that diffusible factors secreted from the graft stimulate sprouting of axon-like processes, upregulation of the growth associated protein GAP-43, as well as enhancement of axonal regeneration in the crushed ON in situ [3,8,37]. Utilizing the same paradigm, we have recently shown that the isolated epineu-
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rium of a PN also stimulates the sprouting of axotomized RGCs [27]. The glial environment of the damaged CNS has been implicated as a major factor in impeding regeneration [14,47], especially with regard to some of the well-characterized inhibitory factors associated with oligodendrocytes and astrocytes [1,20,22,34,36,50]. In contrast, the issue of the existence of factors associated with the damaged CNS, especially soluble ones, which can stimulate regeneration, has not been adequately addressed. One example is the series of studies done by Cotman et al. in the 1980s which show that the wound adjacent to a damaged brain area contains soluble factors with neuronotrophic activities [38,39]. However, most of these early studies utilized in vitro assays to quantify the injury-associated neurotrophic activities, so that their effects in vivo remain obscure. Recently, So and colleagues reported that the concurrent intravitreal grafting of a normal ON segment together with either an intravitreal PN or a PN grafted to the cut ON potentiate the sprouting response of RGCs by the PN, but that the normal ON when grafted alone does not stimulate sprouting [10,11]. In these studies, although the normal ON by itself seems unable to elaborate significant soluble trophic factors, we reasoned that it may behave differently after injury with regard to trophic factor production, based on the findings with the injured brain as mentioned above. Thus, in this study, we utilize the intravitreal grafting paradigm to assess whether a preinjured white matter fibre tract, the ON, possesses diffusible factors to elicit sprouting of RGCs. This was performed by damaging the ON with a crush lesion followed by intravitreal transplantation of the lesioned segment at 7 days post-crush. The sprout-inducing potential of this lesion segment was compared to that of segments taken both proximal and distal to the lesion segment, as well as to that of a normal intravitreal ON graft which had not been preinjured (noninjured ON graft). Our results indicate that both the noninjured ON as well as the segments proximal and distal to the crush lesion stimulated sprouting of axon-like processes from RGCs. In contrast, the segment encompassing the lesion did not induce sprouting. The results suggest that while an intact CNS white matter like the ON possesses diffusible sprout-inducing activity, this could become abolished after injury, an observation which may have some bearing associated with the failure of axonal regeneration in the CNS.
2. Materials and methods A total of 64 adult female hamsters (Mesocricetus auratus) 6 –8 weeks old were used in this study. Animals were randomly assigned to various experimental groups to receive different grafting procedures. All operations were performed under general anesthesia induced by intraperitoneal injection of a mixture of ketamine (200 mg/kg) and xylazine (20 mg/kg).
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2.1. Intraorbital ON crush to axotomize RGCs All animals were inflicted with an intraorbital ON crush to serve as the experimental model of damaging the RGC axons. The right ON was exposed through a superior intraorbital approach and crushed with fine forceps for 10 s about 2 mm behind the globe. Under the operating microscope, the nerve was seen to turn translucent at the crush lesion site but the dural sheath remained intact. 2.2. Intravitreal graft preparation The experimental layout of the different grafting paradigms was as illustrated in Fig. 1. The grafts in this study consisted of one of the following groups prepared from either normal noninjured ONs or from ONs which had been preinjured 1week before intravitreal grafting: (A) rinoninjured viable ON (vON) allografts (B) noninjured viable ON (vaON) autografts (C) noninjured nonviable ON (nvON) allografts –graft rendered nonviable before implantation into the vitreous (see below) (D) lesion segment of ON (LON) – the region that has been crushed 1 week previously (E) proximal segment (PON) –the region between the eyeball and the crush lesion (F) distal segment (DON) –the region beyond the lesion towards the optic chiasm (G) nonviable distal segment (nvDON) Most experiments in this study were performed by transplanting a segment of ON isolated from 1 hamster (allograft) to the vitreous body of the eye of another hamster which had been inflicted with an experimental intraorbital ON crush. This procedure avoided simultaneous damage of both ONs in the same animal and caused less trauma to the animal. Moreover, it has been reported that damage of one ON would result in injury-associated cellular changes of RGCs and glia of the contralateral eye [5], thus potentially complicating the interpretation of the results if both eyes were damaged in one animal. However, in order to address whether potential allograft-induced immune reactions might influence the responses of the axotomized RGCs, autologous grafting of a noninjured ON into the vitreous was performed in another group of animals to look for any possible differences from the allografted animals in terms of the stimulation of RGC sprouting. 2.2.1. Normal noninjured ON grafts 2.2.1.1. Viable allografts (vON). After dividing the superior rectus muscle, the eyeball was protracted slightly outwards by sutures anchored to the fascia around the
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Fig. 1. Schematic diagram showing the intravitreal transplantation of (A) a normal noninjured ON, or (B) either the proximal (P), lesion (L), or distal (D) segment of an ON which had been crushed 7 days ago. The crushed site is denoted by crosshatching.
eyeball in order to visualize the dorsal part of the ON. A cut was made on the dorsal part of the dura using a pair of fine iris scissors at approximately 1 mm behind the globe to expose the ON proper which was then transected completely. The whole length of ON distal to the cut was pulled out gently using fine forceps. After removing the meninges, the ON was transferred to cold tissue culture medium (DMEM/F12) and cut into 2-mm-long segments to be used for allografting. Each ON from a donor could provide grafts for four animals. The donor was killed by a lethal dose of anesthetic immediately after removal of the ON. 2.2.1.2. Viable autografts (vaON). In another group of animals, autologous grafting of a viable noninjured ON was performed by removing 2 mm of the left ON and implanted into the vitreous of the right eye whose ON has been concurrently crushed. 2.2.1.3. Nonviable allografts (nvON). In order to assess the influence of cellular components of the ON graft and any potential nonspecific influence of surgical procedures on the axotomized RGCs, the ON graft was rendered nonviable (destruction of all cells in the graft) by alternately freezing the graft in liquid nitrogen for 5 min followed by thawing at room temperature for another 5 min for a total of 5 cycles. After the freezing/thawing procedure was com-
pleted, the ON graft was transferred to cold culture medium and cut into 2-mm-long segments to be used as nonviable allografts. 2.2.2. Preinjured ON grafts A crush lesion spanning 2.5 mm was made at about 2 mm behind the eyeball. At 1 week post-crush, the ON was exposed again and the crush site identified by its distinctly narrower caliber compared to the regions proximal and distal to it. A 2-mm-long segment consisting exclusively of either the crush lesion (LON), proximal (PON) or distal segment (DON) was removed and transferred to cold culture medium before transplantation. A control group using the distal segment and rendering it nonviable (nvDON) was also examined and compared with the viable grafts. 2.3. Implantation of an ON segment into the vitreous body Intravitreal ON transplantation was carried out immediately after intraorbital ON crush (Fig. 1), following the procedures as described in Cho and So [8]. Briefly, a small penetrating lesion was made at a point slightly behind the limbus of the superior aspect of the eyeball with a 27-G needle. The ON graft was gently introduced into the vitreous body through the lesion with the aid of a finetipped glass micropipette, after which the lesion was closed by a single 10/0 suture.
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2.4. Intravitreal PN grafting In order to compare the propensity of an intravitreal ON to stimulate RGC sprouting to that of a PN graft, transplantation of a 2-mm desheathed viable PN segment (vPN) into the vitreous was performed in a separate group of animals, as described in Cho and So [8]. The retinas from the grafted eyes were processed in the same manner as that from animals with ON grafts (see below). 2.5. Examination of the intravitreal ON graft at 2 weeks post-grafting To assess the cellular changes and viability status of the ON graft, it was removed from the eyeball at 2 weeks postgrafting, the time when the animal was sacrificed for examination of RGC regenerative sprouting in the retina. After the animal was sacrificed with a lethal dose of 20% chloral hydrate, it was perfused transcardially with phosphate-buffered saline (PBS). The eyeball was removed and placed in 4% paraformaldehyde (in 0.1 M phosphate buffer [PB] pH 7.4). The cornea and lens were dissected away rapidly to expose the ON graft. After separation from the eyecup, the graft was fixed for 1 h and then washed in PBS repeatedly for 1 – 2 h before being immersed in 20% sucrose/0.1 M PB overnight at 4 jC. The following day,
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the graft was embedded in OCT, cut into 10-Am sections with a cryostat and mounted onto 3% gelatin-coated slides. The sections were immunostained with either anti-GFAP antibody (Dako, rabbit polyclonal, 1:1000) to label astrocytes, or anti-Rip antibody (Chemicon, mouse monoclonal, 1:2000) to label oligodendrocytes [18]. The primary antibodies were visualized with either anti-rabbit or anti-mouse IgG conjugated to Cy3 (Jackson ImmunoResearch Labs, 1:400, 2 h at room temperature). Both the primary and secondary antibodies were diluted in PBS containing 0.1% Triton-X/0.5% bovine serum albumin/0.9% NaCl. Sections were coverslipped with glycerol and observed under an epifluorescence microscope. In control experiments to assess antibody specificity, some sections were processed without incubation of the primary antibody (anti-GFAP or Rip) to ascertain for the absence of labelling under these circumstances. 2.6. Silver staining of the retina At 2 weeks post-ON crush, the animals were sacrificed with a lethal dose of 20% chloral hydrate. The operated eyeballs were removed immediately without perfusion. Retinas were dissected in 2% paraformaldehyde/0.1 M PB and fixed for 1 h. They were then silver stained by a modified protocol of Leicester and Stone [31] which had
Fig. 2. Photomicrographs showing the cellular components of the ON graft after 2 weeks in the vitreous body. (A) anti-GFAP staining showed that astrocytes were still present in the viable allograft of normal ON (vON). (B) No GFAP-positive staining was detected in the nonviable allograft (nvON), indicating the astrocytes in the graft had been killed before grafting. (C) Rip-positive staining showed oligodendrocytes were still present in the vON. (D) No Rip staining was detected in the nvON, indicating that the oligodendrocytes in the graft had been killed before grafting. Scale bar = 100 Am (A – D).
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been found to reveal the detailed morphology of RGCs exhibiting regenerative sprouting of axon-like processes [8,27]. After silver staining, the retinas were mounted onto 3% gelatin-coated slides, dehydrated in ethanol, cleared in xylene, and coverslipped with Permount. 2.7. Anti-GAP-43 labeling of the retina At 2 weeks post-ON crush, the animals were sacrificed with a lethal dose of 20% chloral hydrate and perfused with PBS transcardially. The retinas were dissected and fixed in 4% paraformaldehyde/0.1 M PB. After 1 h fixation, the
retinas were washed in PBS for about 2 h and then transferred to PBS/1% Triton-X for another hour. This was followed by incubation in 20% fetal bovine serum for 1 h at room temperature to block nonspecific staining. Subsequently, the retinas were incubated in anti-GAP-43 antibody (mouse monoclonal from Sigma) at a dilution of 1:1000 (in PBS containing 1% Triton-X/0.5% bovine serum albumin/ 0.9% NaCl) overnight at 4 jC. After several washes in the same buffer used as the antibody diluent, the retinas were incubated overnight with an anti-mouse IgG secondary antibody conjugated to biotin (Jackson ImmunoResearch Labs, 1:500 in the same diluent of the primary antibody) at
Fig. 3. Photomicrographs showing silver-stained RGCs in the central part of the superior temporal retinal quadrant in eyes with normal noninjured ON (A – C) and PN (D) grafts. (A) In the eye implanted with a vON, a small population of axotomized RGCs with large and irregular cell sizes (examples indicated by arrows) were revealed by the silver staining. (B) In the eye implanted with a vaON, sprouting cells similar to that in (A) were also present (shown by the arrows). (C) In the eye implanted with a nvON, sprouting cells were rarely seen. (D) In the eye implanted with a PN, more sprouting cells were observed compared to that seen in viable ON grafts. (E) Detailed morphology of a sprouting RGC under high magnification. Arrows indicate the haphazard course taken by a sprout. Asterisk indicates a branching point, and the curved arrow a loop. Scale bar = 500 Am (A – D); 100 Am (E).
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4 jC. On the following day, retinas were incubated with Streptavidin-HRP (Jackson ImmunoResearch Labs, 1:1000 in PBS/1% Triton-X) overnight at 4 jC. The HRP label was developed by the diaminobenzidine(DAB) and glucose oxidase reaction [45]. After DAB development, the retinas were mounted onto 3% gelatin-coated slides, dehydrated, cleared, and coverslipped with Permount. The specificity of the immunostaining was assessed by omitting the primary anti-GAP-43 during staining of the grafted retinas whereupon no labelling of RGCs could be observed. 2.8. Quantification of the number of silver-stained RGCs and GAP-43-positive RGCs The morphometric analysis program Stereo Investigator (MicroBrightfield Inc.) was used to compute the total number of silver-stained sprouting RGCs and GAP-43positive RGCs by scanning the whole retinal area under 400 magnification via the motorized control of the microscope stage. All silver-stained RGCs which exhibited sprouting (as judged by the criteria listed in the Results) and all GAP-43-positive RGCs in each retina were counted. 2.9. Statistical analysis of the data
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further since it has been shown that the survival and regeneration of RGCs could be enhanced by injury to the lens or other intraocular structures [16,32,33]. Immunohistological examination of the intravitreal graft showed that astrocytes and oligodendrocytes were still present in the vON graft as revealed by the strong staining of anti-GFAP and anti-Rip (Fig. 2A and C), whereas no positive anti-GFAP or anti-Rip staining was detected in the nvON graft (Fig. 2B and D), indicating that cells initially present in the graft were killed by the repeated freezing and thawing procedures. 3.1.2. Silver staining of RGCs In eyes implanted with either a vON or vaON (Fig. 3A and B), silver staining of the retina revealed the presence of a population of RGCs with large and irregular cell sizes. Most of these cells were located in the superior quadrants close to the intravitreal graft. On detailed examination under high magnification, some of these cells exhibited sprouting of newly formed processes from their somatodendritic compartment. The de novo growth nature of these processes was suggested by their morphology (Fig. 3E): They were uniform in thickness, sparsely branched, and extended far beyond the maximum dendritic domain of normal RGCs. Many of them ran haphazardly and unconfined to a partic-
The variations in the mean number of silver-stained sprouting RGCs and GAP-43-positive RGCs in the different experimental groups were analyzed by one-way ANOVA followed by a post hoc pairwise comparison with the level of significance set at P = 0.05.
3. Results In the following descriptions of the results, the abbreviations used for the different groups of grafts were as listed in Materials and methods. The results obtained from the transplantation of normal noninjured ON grafts would be presented first and followed by that associated with the preinjured ON grafts. 3.1. Normal noninjured ON grafts 3.1.1. Appearances of intravitreal ON graft When the animals were sacrificed at 2 weeks postgrafting, the ON graft was usually found to be located in one of three sites in the eye: attached to the ora serrata adjacent to the limbal lesion site (most common), freely suspended in the vitreous body, or rarely attached to the posterior surface of the lens. There was no direct correlation between the location of the graft and the magnitude of the sprouting response of the RGCs. Great attention was being paid to see whether there were abnormalities in appearance of intraocular structures, such as damage to the lens or distortion of the retina. Although such damages were uncommon, eyes with such abnormalities were not analyzed
Fig. 4. A comparison of the mean number of (A) silver-stained sprouting RGCs and (B) GAP-43-positive RGCs, among the viable and nonviable noninjured ON and the PN graft groups, at 2 weeks post-ON crush and grafting.
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ular retinal layer, forming turns and even complete loops along their course. Thus, they could be distinguished from the dendrites of RGCs and were termed axon-like processes [8]. RGCs which exhibited formation of these axon-like processes were called sprouting RGCs in this study. However, not all the large and irregular-sized RGCs which were revealed by silver staining have identifiable sprouts as judged by the above criteria. Therefore, in this study, every silver-stained RGC in each retina was examined in detail under high magnification, and the number of silver-stained cells with identifiable sprouts was quantified according to the above criteria. When compared to the viable ON graft, RGC sprouting induced by the nonviable graft was minimal (Fig. 3C). The mean number of sprouting RGCs in the vON group (41.2 F 28.8, n = 5) was significantly higher than the control nvON group (2.2 F 1.9, n = 5) ( p < 0.05; Fig. 4A). Eyes grafted with a vaON exhibited comparable RGC sprouting (mean number = 35.3 F 2.2, n = 4) to the vON group, and there was no significant difference between these two viable groups ( p>0.1; Fig. 3B and 4A). Thus, a normal noninjured ON grafted intravitreally induced RGC sprouting irrespective of it being an allo- or autograft. Since allografts did not differ from autografts in their potency to stimulate RGC sprouting, other experiments in this study were performed with ON allografts as explained in Materials and methods.
Compared to an intravitreal PN graft, both allo- and auto-intravitreal ONs were less potent in stimulating RGCs to sprout (compare Fig. 3D to A and B). The mean number of sprouting RGCs in the PN-grafted eyes (186 F 52.9, n = 4) was about 4.5 times that seen in ON-grafted eyes (Fig. 4A). 3.1.3. Anti-GAP-43 staining of RGCs Similar to the silver staining experiments, anti-GAP-43 labeled a number of large and irregular-sized RGCs which were also mainly located in the superior retinal quadrants of the vON-grafted eye (Fig. 5A). Unlike the silver-stained cells, however, RGCs with smaller cell bodies were also marked by anti-GAP-43 (Fig. 5A). The large and irregular cells were also different from the sliver-stained RGCs in a number of aspects. For example, GAP-43-positive cells possessed short filamentous processes, mostly on their cell body surface, but sometimes also on the dendrites and axon (Fig. 5B). Sprouts tipped by an expanded growth cone-like structure emerging from either the cell body or axon at a site close to the cell body was also sometimes observed in GAP43-positive but not silver-stained RGCs (Fig. 5B), whereas the long sprouts arising from dendrites in the silver-stained cells were not as easy to spot in GAP-43-labeled cells. Thus, although both silver staining and anti-GAP-43 labeled the large and irregular sprouting cells, different aspects of the
Fig. 5. Photomicrographs showing GAP-43-positive RGCs in the central superior retinal quadrant in the vON (A), nvON (C), and vPN (D) groups. More GAP43-positive cells were induced by the vON than the nvON, but the vPN is the most potent. Large and irregular-sized RGCs (black arrows in A and C) were observed in both the vON and nvON groups, but they were higher in number in the vON group. In addition, cells with small somata (white arrows) were present in all groups as well, but they tend to predominate in the nvON group. (B) shows a GAP-43 labeled large irregular-sized RGC with short filamentous processes (arrow), and a sprout tipped by a growth cone-like profile (*) which is shown at a higher magnification in the inset; a = axon of the cell. Scale bar in (D) also applies to (A) and (C).
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detailed morphology of these cells were selectively discernable by one of the two methods. The mean number of GAP-43-positive RGCs in the vON group was significantly higher than that in the nvON control group: 345.8 F 122.3 (n = 4) versus 140.5 F 39.2 (n = 4; p < 0.05; Fig. 4B). Moreover, most of the labeled cells in
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the nvON group were of smaller soma sizes (compare Fig. 5C to A). Anti-GAP-43 staining labeled more RGCs than silver staining did because GAP-43 expression was more related to an attempt to regenerate, whereas silver staining revealed only those RGCs which have actually sprouted axon-like processes. The predominance of smaller soma-
Fig. 6. Photomicrographs illustrating the appearances of preinjured ON grafts after 2 weeks post-grafting into the vitreous. The staining of GFAP (A – D) in the proximal segment (PON) and distal segment (DON) grafts were equally strong after 2 weeks in the vitreous. However, the GFAP staining was greatly reduced in the graft originating from the lesion site (LON). In the nonviable distal segment graft (nvDON), GFAP staining was totally absent. Rip staining (E – H) in the PON and DON grafts were strong after 2 weeks in the vitreous. However, the Rip staining in the LON graft was confined to scattered profiles. In the nvDON, Rip staining appeared as granules which may represent debris of dead oligodendrocytes. Scale bar = 100 Am (A – H).
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sized GAP-43-positive cells in the nvON control group was due to the absence of the large sprouting RGCs and possibly more shrinkage of the somata of the surviving cells in the absence of a viable ON. When compared to an intravitreal PN graft, the ON graft was less effective in inducing GAP-43 expression from axotomized RGCs (Fig. 5D). The mean number of RGCs expressing GAP-43 in PN-grafted eyes (1228 F 318, n = 3)
was four times that found in the ON-grafted eyes ( p < 0.05; Fig. 4B). 3.2. Preinjured ON grafts 3.2.1. Appearances of the preinjured intravitreal ON grafts At 2 weeks post-grafting, the staining of GFAP in the PON and DON grafts were equally strong (Fig. 6A and C)
Fig. 7. Photomicrographs comparing the appearances of silver-stained sprouting RGCs (A – D) and GAP-43-positive RGCs (E – H) among the different preinjured ON graft groups. The PON and DON grafts induced sprouting from some RGCs (arrows in A), while sprouting induced by the LON and nvON grafts were minimal. More GAP-43-positive RGCs (arrows in E) were induced by the PON and DON grafts compared to the LON and nvDON grafts. Scale bar = 500 Am (A – D); 100 Am (E – H).
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and similar to that of intravitreal ON grafts which were uninjured at the time of transplantation into the vitreous (cf. Fig. 2A). There was also strong staining of oligodendrocytes in the PON and DON grafts (Fig. 6E and G). However, both GFAP and Rip staining were greatly reduced in the LON graft (Fig. 6B and F), with the labeling being confined to scattered profiles. In the nvDON graft, GFAP staining was totally absent, while fine granules of Rip labeling was seen dispersed throughout the graft (Fig. 6D and H). The granular-like labeling of Rip in the nonviable distal segment graft may be due to the persistence of degenerating debris of oligodendrocytes which had not been totally cleared away. 3.2.2. Silver staining of RGCs Some large and irregular sized RGCs were stained in the PON and DON groups, their numbers (57 F 28.4, n = 4 and 67.2 F 22.1, n = 5 respectively) not appreciably different from each other (Fig. 7A and C; Fig. 8A), nor to the vON group (Fig. 8A). The nvDON elicited almost no sprouting cells (1.4 F 0.9, n = 5; Figs. 7D and 8A), indicating that any nonspecific stimuli arising from surgical disturbance of the intraocular milieu was probably minimal, and that the sprouting induced by the PON or DON necessitated the presence of viable cells in the graft.
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In contrast, the LON graft stimulated significantly fewer RGCs (6.2 F 4.9, n = 5) to sprout (Figs. 7B and 8A) than the PON, DON or vON groups. In fact, there was no significant difference in the number of sprouting RGCs induced by the LON versus the nvDON graft (Fig. 8A). 3.2.3. Anti-GAP-43 staining of RGCs Both the PON and DON grafts stimulated GAP-43 expression in a comparable number of RGCs (Fig. 7E and G; Fig. 8B), whereas the LON graft was significantly less potent in this aspect: the number of GAP- 43-positive RGCs induced by the LON (139 F 67.6, n = 4) was less than onehalf of that in the DON group (312 F 108, n = 4), as well as being significantly lower than in the PON (265 F 54, n = 4) or vON groups (Figs. 7F and 8B). The control nvDON also induced significantly smaller numbers of RGCs (139 F 20, n = 4) to express GAP-43 compared to the DON group (Figs. 7H and 8B). Again, there was no significant difference between the LON and nvDON graft in terms of their induction of GAP-43 expression in RGCs (Fig. 8B).
4. Discussion 4.1. A normal noninjured intravitreal ON graft induces sprouting of axon-like processes from RGCs and enhances GAP-43 expression
Fig. 8. A comparison of the mean number of (A) silver-stained sprouting RGCs and (B) GAP-43-positive RGCs among the different preinjured ON groups. The data from the vON group were included for comparison with that of the preinjured grafts.
In this study, a viable intravitreal ON is found to induce a small population of axotomized RGCs to sprout axon-like processes. Although the ON graft is not as potent as an intravitreal PN in eliciting RGC sprouting, it exerts a clearcut stimulus on the axotomized RGCs, as indicated by the 20-fold difference in the number of sprouting RGCs induced by the viable versus the nonviable ON grafts, as well as the upregulation of GAP-43 in more axotomized RGCs after axotomy. These results are at variance with a previous study by Cho et al. [10] which stated that an intravitreal ON only augmented the effects of a concurrently implanted PN but did not induce sprouting by itself. Differences in experimental design and procedures may be responsible for the discrepancies between the two studies. Cho et al.’s study [10] employed transection of the ON as the axotomy paradigm, while ON crush was used in this study. It is known that a crush lesion leads to a smaller degree of cell death compared to a cut [2], so more surviving RGCs may be stimulated by an intravitreal ON. Secondly, a shorter (1-mm) segment of ON was used for the transplant in Cho et al.’s study which would theoretically produce less stimulus, compared to the 2-mm graft in our experiments. Thus, the ON cut axotomy paradigm coupled with a shorter intravitreal ON graft in Cho et al.’s study may explain why they were unable to see a positive effect. In contrast, our present results clearly show that an intravitreal ON by itself has regeneration-promoting effects on the axotomized RGCs, as assessed by two
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parameters: sprouting of axon-like processes and increased GAP-43 expression. 4.2. Possible mechanisms associated with the action of the intravitreal ON The viable intravitreal ON graft contains an abundance of astrocytes and oligodendrocytes as revealed by anti-GFAP and anti-Rip staining, indicating that they have survived throughout the 2 weeks grafting period. Astrocytes are known to produce an impressive list of molecules which enhance neuronal survival and growth (reviewed in Refs. [6,40,44]). CNTF, which is known to stimulate sprouting and regeneration of RGCs [12,24] and is present in the normal ON [26], is one of the possible candidates. Basic FGF is also constitutively present in the ON and enhances the survival of RGCs when applied to the cut end of ON [46]. Oligodendrocytes have been reported to secrete IGF-1 in culture to promote neuronal survival [51]. The differential contribution of astrocytes and oligodendrocytes to RGC sprouting can be addressed by selectively ablating the astrocytes in the ON with aminoadipate [25] before grafting so that the graft becomes predominated by oligodendrocytes. The status of microglia in the graft have not been examined in this study. Macrophages and activated microglia release certain types of cytokines or proimflammogens [19,29] that in turn may regulate the function of astrocytes in the ON graft. Thus, different cell types in the ON may interact in a complex manner to result in the positive effects observed in this study. 4.3. Both proximal (PON) and distal (DON) segments of the preinjured graft are effective in stimulating RGCs to sprout Examination of the intravitreal PON and DON at 2 weeks post-grafting reveals that the cellular morphology of the astrocytes and oligodendrocytes are not appreciably different from those in a normal intravitreal ON graft. In fact, their appearances are similar to that of the proximal and distal segments of the ON injured in situ, in which the staining intensity of GFAP [4,17] and oligodendrocytes [7,41] are also not appreciably different from that of uninjured nerves. However, it should be remembered that there would be comparatively fewer axons in the proximal segment since over 70 –80% of RGCs have degenerated by 2 weeks post-injury [2], while the disconnected axons in the distal segment are undergoing Wallerian degeneration. Such profound changes in axon to glia relationships would be expected to alter the functional status of the latter. Thus, oligodendrocytes exhibit a decrease in expression of certain myelin protein genes after ON transection [35], while the production of CNTF mRNA by astrocytes also suffers a small drop in both segments after ON crush [26]. In addition, there is a change in phagocytic activity of microglia in the two segments (especially at the distal segment) by virtue of their positive staining with the ED1 antibody
[17,41]. However, it seems that such changes of glial cells in the proximal and distal segments of the injured ON have only limited effects on their sprout-inducing propensity, as reflected by the present results that the PON and DON grafts stimulate RGC sprouting to magnitudes similar to that of the normal ON. Thus, a basal level of stimulus may exist in the normal ON and which is maintained in the segments proximal and distal to the lesion after injury. The exact cellular/molecular identity responsible for this ‘‘basal level’’ can only be speculated at present, but CNTF may be one of the possibilities. The amount of CNTF in the normal ON is very high for a CNS tissue [48], and it remains at 60% of the normal level in the distal segment of a crushed ON [26]. Such relatively small drop in CNTF level in the distal segment of the preinjured ON may explain the constancy of the sprouting response of RGCs induced by it. 4.4. The lesion site graft (LON) is significantly less potent in stimulating RGCs Grafting of an intravitreal LON results in dramatically fewer RGCs to form sprouts and express GAP-43, the values of which approach that induced by the nvDON graft, indicating that the source of stimulus is almost eliminated in the LON. Apart from damaging the RGC axons, a crush injury of the ON leads to extensive cell death within the lesion area [17,41]. From 2 days post-crush onwards, astrocytes which initially express only S-100 but not GFAP begin to repopulate the lesion site at slow rate [17], hence, the number of GFAP-positive cells at 7 days post-crush is very small. The same is true for oligodendrocytes which is almost totally absent at the crush site during the first week post-injury [7,41]. In contrast, there is a massive influx of macrophages from the circulatory system into the lesion area, so that by 1 week, the lesion is already filled with ED1-positive macrophages which only extend a small distance into the proximal and distal segments [17,41]. Thus, at the time of transplantation of the LON into the vitreous, astrocytes and oligodendrocytes should be scarce, while macrophages would be plentiful in the graft. When the LON graft is examined 2 weeks after being implanted into the vitreous, astrocytes and oligodendrocytes are still scarce. Although we have not examined the status of the macrophage population in the intravitreal graft, it is highly likely they are still present in the LON graft, if they resemble the in situ pattern of distribution [3,41]. Thus, some of the more prominent differences between the LON and other viable intravitreal grafts (vON, PON, and DON) is the scarcity of astrocytes and oligodendrocytes, together with an abundance of macrophages, in the former. Dynamic changes in the different glial cells may be associated with the observed changes in RGC sprouting. For example, the amount of CNTF mRNA at the ON crush site is dramatically decreased compared to both the proximal and distal segments, in association with a marked reduction of CNTF-
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positive astrocytes at the lesion site [26]. Taken together, the scarcity of astrocytes and the concomitant drop in CNTF (and possibly other growth factors) in the LON graft may lead to the decrease in RGC sprouting. On the other hand, the role of macrophages in the LON graft remains to be determined. Although macrophages/microglia have been demonstrated to promote axonal regrowth in a number of studies [42,43,52], future experiments designed to address differences in macrophage numbers, subpopulations, and activities (e.g., in terms of phagocytosis) in the intravitreal ON grafts and correlating them to the induced sprouting response may shed light on this issue.
[8]
[9]
[10]
[11]
5. Conclusions [12]
This study highlights the existence of the ability of the intact CNS in stimulating neural regeneration. We have demonstrated that a normal CNS white matter region, the ON, produces soluble factors which promote regenerative sprouting of RGCs upon intravitreal grafting. However, prior damage of the same region leads to a greatly attenuated effect on the RGCs. Such changes in trophic support of the CNS after injury may partly explain the usually meager response of the damaged neurons to regenerate in a CNS environment. It would be interesting to see whether such changes occur universally in other CNS regions and their consequences to the recovery of the damaged neurons.
[13]
[14] [15]
[16]
[17] [18]
Acknowledgements The work reported in this paper was fully supported by a grant from the Research Grants Council of Hong Kong (CUHK4265/98M).
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