Olfactory ensheathing glia: Repairing injury to the mammalian visual system

Experimental Neurology 229 (2011) 99–108

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Experimental Neurology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y e x n r

Review

Olfactory ensheathing glia: Repairing injury to the mammalian visual system Giles W. Plant a,b,c,⁎, Alan R. Harvey c, Simone G. Leaver c, Seok Voon Lee a,b,c a b c

Stanford Partnership for Spinal Cord Injury and Repair, Department of Neurosurgery, Stanford University School of Medicine, Stanford, CA 94305, USA Eileen Bond Spinal Cord Research Centre, The University of Western Australia, Perth, Western Australia 6009, Australia School of Anatomy and Human Biology, The University of Western Australia, Perth, Western Australia 6009, Australia

a r t i c l e

i n f o

Article history: Received 1 July 2010 Revised 31 August 2010 Accepted 8 September 2010 Available online 17 September 2010 Keywords: Retinal ganglion cells Glia Neurotrauma Myelination Regeneration

a b s t r a c t The visual system is widely used as a model in which to study neurotrauma of the central nervous system and to assess the effects of experimental therapies. Adult mammalian retinal ganglion cell axons do not normally regenerate their axons for long distances following injury. Trauma to the visual system, particularly damage to the optic nerve or central visual tracts, causes loss of electrical communication between the retina and visual processing areas in the brain. After optic nerve crush or transection, axons degenerate and retinal ganglion cells (RGCs) are lost over a period of days. To promote and maintain axonal growth and connectivity, strategies must be developed to limit RGC death and provide regenerating axons with permissive substrates and a sustainable growth milieu that will ultimately provide long term visual function. This review explores the role olfactory glia can play in this repair. We describe the isolation of these cells from the olfactory system, transplantation to the brain, gene therapy and the possible benefits that these cells may have over other cellular therapies to initiate repair, in particular the stimulation of axonal regeneration in visual pathways. This article is part of a Special Issue entitled: Understanding olfactory ensheathing glia and their prospect for nervous system repair. © 2010 Elsevier Inc. All rights reserved.

Contents Introduction . . . . . . . . . . . . . . . . . . OEG population types and transplantation . . OEG transplants into the mammalian brain . OEG transplants into the spinal cord . . . . . Is age a determining factor of OEG potential? Is age a determining factor of OEG potential? OEG transplantation to the visual system . . OEG and RGC regeneration in vitro . . . . . . . OEG and RGC survival and regeneration in vivo . OEG and myelination of RGC axons . . . . . . . Future studies . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . .

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Introduction Over the last two decades, olfactory ensheathing glia (OEG) have been identified as a potential transplantation strategy in central nervous system (CNS) disease and after injury. This is due to their role in the mature olfactory system where OEG guide constantly ⁎ Corresponding author. Department of Neurosurgery, 1201 Welch Road, MSLS P105, Stanford University School of Medicine, Stanford, CA 94305-5487, USA. Fax: + 1 650 736 1949. E-mail address: [email protected] (G.W. Plant). 0014-4886/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2010.09.010

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renewing olfactory axons from the basal cells in the olfactory neuroepithelium through the cribriform plate to reach their glomerular targets deep in the olfactory bulb (Barber and Raisman, 1978; Graziadei et al., 1979). This makes OEG unique as they exist both within peripheral neural tissue and the CNS. As a consequence, OEG can be isolated that are either peripherally or centrally derived (Barnett et al., 1993). Studies on these two types of OEG have shown that they share many similar features and characteristics; however there are also significant differences including the ability to migrate and create a permissive regeneration-promoting environment at a lesion site (Richter et al., 2005).

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OEG population types and transplantation The purity of OEG obtained for transplantation also affects the capability of the cells in a cell-transplantation regime. There are various methods used to purify OEG including differential adhesion method (Wu et al., 2010), immunopanning (Ramón-Cueto et al., 1993, 1998; Plant et al., 2002), fluorescence activated cell sorting (FACS) and surface marker antibodies (Barnett et al., 1993). Immortalized cell lines have also been studied and provide a useful tool for single cloned sources of human or rodent OEG (Moreno-Flores et al., 2006; García-Escudero et al., 2010). Production of some lines requires the introduction of the catalytic subunit of telomerase (TERT) (Lim et al., 2010; Llamusí et al., 2010) which may complicate interpretation over and above those from purified or FACS sorted cell populations, especially when two viral transductions are required. In experimental studies on repair after spinal cord injury, various OEG populations have been reported to induce axonal regeneration and/or improve functional recovery. Grafted cells were either purified OEG, non-purified cell preparations obtained from the olfactory bulb (Ramón-Cueto et al., 1993, 1998, 2000; Li et al., 1997, 2003a; Plant et al., 2003) or nasal olfactory tissue implanted directly after removal (Lu et al., 2001). Other studies using a mix of meningeal cells and OEG have shown improved remyelination after transplantation into an x-irradiated/ethidium bromide model compared to a purer population of OEG obtained using cytosine arabinoside treatment to reduce the level of fibroblastic contamination (Lakatos et al., 2003). Contrasting results have also been found after dorsal root lesion using OEG grafts as a repair strategy (Ramón-Cueto and Nieto-Sampedro, 1994; Li et al., 2004; Riddell et al., 2004; Ramer et al., 2004) that may be attributed to the method of purification or the age of the animals from which the cell grafts were prepared (Barnett and Riddell, 2004). OEG transplants into the mammalian brain In the brain and spinal cord many neurological diseases such as Alzheimer's, Parkinson's and amyotrophic lateral sclerosis (ALS) are a result of chronic neurodegenerative changes that result in loss of specific neuronal populations. Thus major therapeutic targets include neuroprotection, and replacing cells and restoring compromised circuitries in the brain by enhancing neurogenesis. Neurotrophic factors have been identified as having positive effects in promoting neurogenesis in the brain, particularly in the hippocampal region. These factors include brain derived neurotrophic factor (BDNF; Lee et al., 2002), basic fibroblast growth factor (bFGF, Palmer et al., 1999), and nerve growth factor (NGF, Frielingsdorf et al., 2007). Due to the different neurotrophic factors that they can secrete, OEG could be a key cell in attempting to not only support the survival and extension of remaining axons, but also in promoting neurogenesis. In culture OEG promote the survival and outgrowth of hippocampal neurons that is comparable to the growth of neurons exposed to different growth factors (Pellitteri et al., 2009). Conditioned media obtained from OEG cultures and used to maintain hippocampal neurons also resulted in improved survival and outgrowth of these neurons (Pellitteri et al., 2009) indicating that there is a secretion mechanism underlying the ability of OEG to promote neuronal growth and survival. Transplantation studies of OEG into the brain have utilized different lesion models. Smale et al. (1996) isolated rat embryonic OEG and transplanted the cells in the form of cell/collagen matrix graft into a unilateral lesion of the fimbria–fornix pathway. The studies showed that OEG survived in the graft up to 4 weeks and promoted growth of axons in the septal–hippocampal pathway (Smale et al., 1996). Further evidence showing the integration of OEG in the brain was demonstrated with the transplantation of OEG into the normal thalamus (Pérez-Bouza et al., 1998). The study showed that transplanted OEG can span natural barriers within the brain as well as induce axonal growth between independent CNS structures (Pérez-

Bouza et al., 1998). In addition, transplanted OEG may determine the direction of host axon growth (Pérez-Bouza et al., 1998), a characteristic that has also been described in culture (Sonigra et al., 1999). Other studies also show that transplantation of OEG can be beneficial in neurodegenerative diseases. In rats, a model for studying Parkinson's disease is by performing a (6-OHDA)-lesion in the nigrostriatal dopaminergic pathway. Transplantation of unpurified OEG obtained from the glomerular layers of the adult olfactory bulb with or without fetal ventral mesencephalic cells (VMC) into the model showed an increase in functional recovery (Agrawal et al., 2004). Although OEG transplantation alone showed positive results in the injury model, co-transplantation with VMC showed significant functional restoration compared to individual transplant groups (Agrawal et al., 2004) indicating that a multifunctional approach could be important in the repair of the injured brain. OEG transplants into the spinal cord OEG have been used as a cellular choice in many models of spinal cord injury (for review see Raisman, 2001). In such models, central ascending and descending pathways are subjected to a trauma that leads to axonal injury and often axotomy, in which the axons are completely severed. Olfactory glia have been transplanted into the spinal cord either alone (Li et al., 1997; Ramón-Cueto et al., 2000; Ruitenberg et al., 2002) or in combination with other cell types such as Schwann cells (Ramón-Cueto et al., 1998; Takami et al., 2002; Barakat et al., 2005). These studies have reported varying levels of success including an increase in tissue sparing, axonal sparing/regeneration and functional behavior. Interesting differences do exist between the spinal cord and brain; regenerating axons are able to leave OEG transplant zones within the injured spinal cord and can re-enter the host spinal tracts. However, within the visual system OEG show no evidence of advancing with regenerating RGC axons and co-extending into the astrocytic territory of the distal stump of the injured optic nerve (Li et al., 2003b). Without showing a true correlation of behavior to anatomical outcomes, it is difficult to ascertain the real mechanism of repair; however there is evidence showing the positive uses of olfactory glia in the injured nervous system and particularly the spinal cord. A more in-depth review of spinal cord transplantation studies using olfactory glia can be found in the other reviews within this special edition. Is age a determining factor of OEG potential? We have further explored the differences in OEG cell properties when animals of different ages are used to prepare the primary cultures. Rat age groups for cell isolation can be divided into three broad categories — embryonic, postnatal and adult. Embryonic ages are E18–E19, the postnatal age range is between day 1 (P1) after birth up to day 21 (P21) and the adult group can comprise rats aged anywhere from 3 weeks to 6 months. A number of previous studies have used animals of different ages to obtain primary OEG cultures, but there has been a lack of direct comparison between the cell types. While embryonic and postnatal cells are unlikely to be considered in translational studies due to ethical reasons, it is still important to understand the mechanisms underlying their functions in order to gain a better overall understanding of OEG biology. Furthermore, while the technique used to isolate and prepare OEG may not solely explain contrasting results reported in the literature, it remains an important issue that should be taken into consideration in all OEG studies. Is age a determining factor of OEG potential? Our laboratory has conducted a study on the age-dependent myelination by OEG. Comparison between p75 immunopanned purified embryonic, postnatal and adult OEG showed that only

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embryonic OEG are capable in vitro of myelinating TrkA-dependent DRG neurons under culture conditions containing serum, ascorbate and progesterone (de Mello et al., unpublished data). In vivo studies comparing embryonic to adult OEG myelinating capacity using a lysolecithin-induced demyelination model in rats also showed differing results. While both cell groups possessed a similar proportion of intact myelin compared to a Schwann cell control group, the proportion of axons surrounded by loose uncompacted myelin was significantly less in embryonic OEG groups, and the proportion of axons that were completely unmyelinated was correspondingly higher (de Mello, unpublished data). Our observations indicate a developmental shift in the properties of OEG populations, and suggest that age-related differences may yet be found in the bulb in vivo despite other findings (Magavi et al., 2005). This indicates that comparison studies between age groups could yet be used to gain a better understanding of the function of OEG with the aim of promoting regeneration and functional repair of the damaged CNS. There remains more to be elucidated about OEG biology; it has potential therapeutic uses because of the positive preclinical results obtained thus far in cellular transplantation studies in animal models, and also because OEG are easily obtained for autologous transplantation in the clinical setting. OEG transplantation to the visual system The mammalian visual system is a useful model in which to examine the effects of transplanted OEG on the survival and regrowth of CNS tissue. Injury and degeneration in the visual system can be induced by direct trauma or ischemia, but also occurs as a consequence of more chronic ophthalmic diseases such as glaucoma; all can result in the death of a significant proportion of retinal ganglion cells (RGC). It is well-established that after an optic nerve crush or transection, death of these neurons occurs on a wide scale, in part due to trophic factor deprivation. Promoting RGC survival and then inducing the regeneration of their damaged axons are both required in order to repair the visual system after an injury. Just keeping the neurons alive is, in itself, not sufficient to promote axonal regrowth (Goldberg et al., 2002; Cui et al., 2003; Leaver et al., 2006a); it is necessary to induce RGCs to re-acquire a growth state that seems to be lost or otherwise considerably diminished after the embryonic stage (Fischer et al., 2004; Sun and He, 2010). In many ways, the issues and problems associated with inducing the damaged optic nerve to regenerate to fully functional capability are very similar to those that pertain when attempting to regenerate the injured spinal cord. The inhibitory nature of the lesion site needs to be overcome, and permissive growth substrates need to be available in order to bridge the injury site and induce the regrowth of axons. Should sufficient regrowth be achieved it is also important to ensure that remyelination occurs, thus increasing salutatory conduction speeds and aiding in the return of function. Similar to the rest of the CNS, it has been suggested that appropriate therapeutic manipulation of neurotrophic signaling pathways will promote the survival of injured RGCs (for reviews see Chierzi and Fawcett, 2001; Zhi et al., 2005; Harvey et al., 2006; Johnson et al., 2009). Among the neurotrophic factors known to promote survival of RGCs include BDNF (Rohrer et al., 2001; Leaver et al., 2006b; Peinado-Ramón et al., 1996), neurotrophin 4/5 (Cui et al., 2003; Peinado-Ramón et al., 1996), glial cell-derived neurotrophic factor (GDNF; Jiang et al., 2007) and ciliary neurotrophic factor (CNTF; Cui et al., 2003; Ji et al., 2004; Parrilla-Reverter et al., 2009). Numerous laboratories have tried to improve the efficacy of neurotrophic treatments to the injured optic nerve with varying success (Mey and Thanos, 1993; Chierzi and Fawcett, 2001; Harvey et al., 2006; Berry et al., 2008). OEG are known to secrete multiple neurotrophic factors such as BDNF and CNTF that could prove beneficial in promoting the survival of

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RGCs (Lipson et al., 2003) and the cells can be engineered to secrete biologically active neurotrophins in vivo (Ruitenberg et al., 2003, 2005; Cao et al., 2004). In addition, studies of Schwann cell transplantation into the injured visual system have shown that: 1) there is poor integration of the Schwann cells with host tissue and 2) the number of axons that grow out of the Schwann cell-rich environment into CNS neuropil is limited (Zwimpfer et al., 1992; Harvey et al., 1995; Tan and Harvey, 1999: Symons et al., 2001; Vidal-Sanz et al., 2002; Whiteley et al., 1998; Avilés-Trigueros et al., 2000). This is mainly due to the relative attractiveness of the Schwann cell versus the adult CNS environment, as well as the negative interactions between Schwann cells and CNS glia that can enhance the inhibitory nature of the injury site (Plant et al., 2001; Lakatos et al., 2003) OEG however, are known to be able to overcome this latter obstacle and intermix well with glia in the CNS neuropil (Vukovic et al., 2007). Additionally they do not appear to exacerbate the scar tissue that forms after injury (Ramón-Cueto et al., 1998). In this regard, OEG have been shown to induce less chondroitin sulfate proteoglycan (CSPG) expression compared to Schwann cells (Lakatos et al., 2003). CSPG is found in the scar tissue and contributes to the non-permissive properties of the CNS environment but can be removed or reduced by the use of chondroitinase ABC treatment and if combined with BDNF injections results in significant sprouting of retinal afferents into the denervated superior colliculus (Tropea et al., 2003). One known factor produced from olfactory glia that would reduce the effects of scar inhibition is metalloproteinase 2, as shown by Pastrana et al. (2006) and is thought to be important in the successful regeneration of RGCs when produced in primary OEG. Matrix molecules such as laminin, L1 and N-cadherin are also good candidate molecules known to promote axon outgrowth (Lander et al., 1985; Bixby et al., 1988; Lochter and Schachner, 1993) and interestingly are present in olfactory glia. However, the presence of these molecules alone in RGC regenerative studies was insufficient to induce mature RGC axonal growth (Goldberg et al., 2002). An important property of OEG needs consideration in the context of visual system repair. OEG do not appear to interfere with appropriate axon–target cell interaction and recognition. In recent studies using tissue transplantation in the visual system, Vukovic et al. (2007) showed that Schwann cells but not OEG interfere with retinal target recognition in a fetal co-graft transplant model. Fetal tectal tissue was mixed with either adult Schwann cells or OEG, each purified glial population having been labeled ex vivo with a lentiviral vector that encoded the sequence for green fluorescent protein (GFP) (Ruitenberg et al., 2002). These mixed tissues were transplanted onto the midbrain of neonatal rats where they established neural connections with the underlying host brain. When assessed several weeks later, the majority of host retinal axons were consistently found to innervate appropriate target regions in the tectal grafts in the presence of OEG, but axons were more scattered in grafts containing Schwann cells and only a few retinal fibers found their target sites in the graft neuropil. Therefore OEG have particular potential as a transplant cell within CNS neuropil because they can support axonal growth but also allow correct target innervation. OEG may also have an advantage of being able to ameliorate the inhibitory nature of the damaged visual system while providing suitable growth matrix and growth-promoting factors to assist injured RGCs to survive and regenerate. Given these initial studies highlighting the potential benefits OEG treatment in the injured visual system, we now summarize some more recent studies that have been published in the context of new data from our laboratory. OEG and RGC regeneration in vitro In culture, purified rat P8 RGCs do not extend axons when maintained in the complete absence of neurotrophic signals (Goldberg et al., 2002). However, when RGCs are cultured in the presence of optic nerve glial cell types, there is an increase in RGC axonal growth (Goldberg et al., 2002).

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This indicates that there are growth-promoting factors provided by the supporting cells that assist in the survival and growth potential of RGCs after injury. By this reasoning it is also possible that OEG, which normally assist and guide olfactory axons to their target sites, can aid in promoting the survival and axonal growth of RGCs. An in vitro study by Sonigra et al. (1999) showed that adult OEG can indeed promote neurite outgrowth of RGCs. This has been further confirmed by other studies. Immortalized OEG clonal lines established by transfection of adult olfactory bulb OEG, as well as primary cultures of OEG and Schwann cells were co-cultured as monolayers with P21 RGCs (Moreno-Flores et al., 2003). When the RGCs were co-cultured with either glia type there was an increase in the frequency of longer neurites compared to controls (Moreno-Flores et al., 2003). Quantification of axonal markers (MAP1B and MAP2C) and synaptic markers (synaptophysin) showed that OEG were better at promoting regeneration when compared to Schwann cells (Moreno-Flores et al., 2003). Experiments undertaken in our laboratories have also shown that RGCs exhibit greater regrowth when cultured in the presence of OEG, as compared to Schwann cells. Analysis of outgrowth from adult retinal explants co-cultured with purified adult OEG obtained from the olfactory bulb revealed a substantial number of regenerating RGC neurites, the number significantly greater compared to outgrowth from explants co-cultured with adult Schwann cells (Leaver et al., 2006c). As mentioned earlier, a further advantage that OEG may have over Schwann cells for use in the visual system is their ability to nondisruptively integrate among endogenous astrocytes, similar to conditions found in the injured spinal cord. Co-culture of OEG and Schwann cells together with RGCs did not further improve RGC outgrowth compared to OEG alone indicating that there is a specific OEG/RGC contact-mediated mechanism responsible for the regrowth induced by OEG (Leaver et al., 2006c), rather than just a secretion based response as seen with hippocampal neurons (Pellitteri et al., 2009). In new experiments in our laboratory we have found that adult rat RGC explants plus the addition of OEG to Schwann cell/astrocyte/ RGC co-cultures provided a growth-promoting environment that resulted in significant increases in the number and length of RGC neurites (Fig. 1). Retinal neurites (per mm from the explant perimeter) were significantly increased from the control value of 0.87 ± 0.26 to 4.31 ± 0.30 (p b 0.05) when OEG were co-cultured with astrocytes in the growth substrate. This was not the case when Schwann cells were added to astrocytes (0.87 ± 0.26 compared to 0.76 ± 0.01). The average neurite length was also significantly increased when OEG were added to co-cultures. When astrocytes alone were added to RGC explants the average length of adult RGC growth was 779 ± 148 μm. The addition of Schwann cells had no significant effect giving axon lengths of 801 ± 122 μm. OEG in contrast when combined with astrocytes increased the length to 1461 ± 85 μm and when combined with Schwann cells was 1578 ± 242. The longest retinal neurite was measured at 8886 μm for OEG co-cultured with astrocytes. These data support the hypothesis that OEG could be an ideal cell candidate for transplantation into the injured visual system to promote survival and axonal regrowth. However, detailed in vivo studies need to be undertaken to determine the true potential of these cells. OEG and RGC survival and regeneration in vivo In the first study of its kind, Li et al. (2003b) obtained unpurified GFP-positive OEG, consisting of around 50% p75-positive OEG and 50% fibronectin-positive olfactory nerve fibroblasts (ONF), embedded in a matrix of their own production and transplanted it into a complete transection of the adult rat optic nerve about 2 mm from the optic disk. It was reported that the transplanted OEG survived within the complete transection of the adult optic nerve and were able to stimulate and guide regeneration of cut RGC axons for up to 10 mm into the distal stump (Li et al., 2003b). The results seem to show that

OEG have a permissive role in limiting the loss of RGCs and also can support axon growth across the injury gap. However, other factors would be necessary in order to achieve functional repair because the regenerating RGC only advanced in co-extension with OEG and ONF and their processes (Li et al., 2003b). It seems that the fibroblast-like ONFs played a key role in the neurite outgrowth observed by acting as a conduit for the OEG to interact with the host axons (Li et al., 2003b). Two further studies have indicated the synergistic role fibroblasts may have with OEG when used in a CNS injury. Ramón-Cueto et al. (1998) showed OEG interact with host spinal cord fibroblasts when transplanted with Schwann cell/matrigel grafts to repair the complete transacted spinal cord. Long tract serotonergic axon regeneration was seen primarily, in areas where OEG and fibroblasts co-existed. One further observation in a spinal cord demyelination model was after OEG were transplanted with meningeal cells there was an increase in the number of myelinated axons by OEG (Lakatos et al., 2003) when compared to OEG transplants alone. So does a pure population of OEG transplanted into the injured visual system elicit RGC survival and regrowth? OEG isolated from P13 transgenic rats expressing green fluorescent protein (GFP) and purified using the differential adhesion method was transplanted into the ocular stump of rats immediately after having their left optic nerve completely transected 1.5 mm from the optic disk (Wu et al., 2010). The study found that OEG-transplanted retinas had a significantly higher survival rate at day 7 as compared to the control group, suggesting neuroprotection of RGCs after OEG transplantation. Deprivation of target-derived neurotrophic factors is a known cause of RGC apoptosis after optic nerve transection (Batistatou and Greene, 1991; Harvey et al., 2006) and it was found that there was an elevated amount of BDNF in OEG-treated animals at 7 days post transplantation (Wu et al., 2010). Importantly however, the study found that transplanted OEG only had a transient neuroprotective effect in the damage optic nerve; while grafted OEG were seen at 7 days, no surviving OEG could be detected in the optic nerve stump at 14 days (Wu et al., 2010). We have also found that OEG survive poorly after transplantation into the lesioned rat optic tract (Ooi and Harvey, unpublished observations). We carried out a series of experiments to investigate the ability of OEG to promote RGC survival and regrowth in vivo. (Leaver et al., unpublished observations). We used purified adult OEG prepared as previously described by Plant et al. (2003), including the p75 immunopanning technique of purifying the cells. We feel it is critical to obtain purified populations of OEG in order to better assess if any improvement is due to the OEG themselves, rather than supporting/ contaminating cells. In our experiments, all OEG cultures were at least 98% pure. In addition, we transduced the OEG cultures used for transplantation with either LV-GFP or LV-CNTF at a multiplicity of infection (MOI) of 50 (Ruitenberg et al., 2002) to produce OEG-GFP or OEG-CNTF respectively. Immunostaining of the transfected cells showed that around 90% and 40% of the OEG were transduced with LV-GFP and LV-CNTF respectively, and the cells still displayed characteristics analogous to those previously described (Fig. 2; Plant et al., 2002, 2003). The bioactivity of the LV-CNTF used for these experiments has also been previously described (Hu et al., 2005). We wanted to examine if OEG together with an over-expression of a secretable form of CNTF will assist in the survival and regrowth of RGC compared to OEG alone. The model of visual system impairment used was an optic nerve crush rather than an optic nerve transection. Briefly, anesthetized adult female Fischer 344 rats had the left optic nerve crushed 1.5 mm behind the nerve head for 5 s, avoiding injury to the ophthalmic artery. Five days later, following the reduction in inflammation caused by the optic nerve crush surgery, the site of the crush was reopened and exposed to allow the transplantation of 0.75 μl (50,000 cells) of OEG-GFP, OEG-CNTF or media containing no cells. Transplantation of cells or media only was done via injection through a glass micropipette into the injury site at

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Fig. 1. Graph of βIII-tubulin positive retinal ganglion explants (Ret) axons extending from the explants perimeter when co-cultured with astrocytes (As), olfactory ensheathing glia (OEG) and/or Schwann cells (SC). The number of axons exiting the retinal ganglion cell (RGC) explants was per millimeter of the explants perimeter. There were significantly higher (*) numbers of axons when green fluorescent protein (GFP) positive OEG were present in the co-cultures (A). Mean length of axons extending from the explants (mm) (B). Mean length of axons extending from the explants perimeter was significantly greater (*) when OEG were present in the co-cultures. Photographs C to F show appearance of axons under different co-culture conditions. Scale bar in C (100 μm) also serves D–F. Antibodies: glial fibrillary acidic protein (GFAP), TUJ-1 (clone-βIII tubulin).

0.5 μl/min using a microinjector (Harvard Apparatus Syringe Pump Model 22 #55-2222). Animals were sacrificed at either 24 h following transplantation or 7 weeks following transplantation for the remaining animals. OEG were transplanted on day 5 following axotomy, because phagocytic cells responsible for the removal of oligodendrocytes and myelin debris would have already infiltrated the injury site (Frank and Wolburg, 1996). From this study it was determined that at 7 weeks post-axotomy, the transplantation of OEG did not improve RGC survival although some RGC axons regenerate long distances along the optic nerve (Fig. 3) this number was greater when OEG were modified to produce the cytokine CNTF (see Fig. 4). However, no OEG could be identified in the optic nerve 7 weeks following transplantation. It may be more beneficial to consider alternative transplantation strategies such as the use of a matrix (Li et al., 2003a) or peripheral nerve sheaths (Cui et al., 2003), although OEG were again reported to exhibit poor viability when incorporated into the nerve sheaths (Cui et al., 2003) and the method of directly grafting OEG into CNS tissue allows the cells to migrate within the CNS environment and potentially accompany axons they induce to regenerate through the degenerating optic nerve (Vukovic et al., 2007). In our study only one time-point (5 days post injury) was studied and thus it is possible that the presence of OEG, or the additional availability of CNTF, had a transient effect on RGC viability (see also Wu et al., 2010). Another consideration is that the time window for optimal RGC survivability had passed for there to be any beneficial effect on long term RGC viability when using OEG transplants This seems unlikely because when either following a intraorbital nerve transection (0.5 mm behind the eye) or intraorbital nerve crush (3 mm) cell loss is only statistically different to controls after day 7 (Parrilla-Reverter et al., 2009) and maximal RGC cell loss is between 5 and 7 days post lesion (Kanamori et al., 2010). Earlier OEG transplant time points prior to 5 days may increase RGC numbers and axonal regeneration this certainly is worth investigating. Nonetheless, these preliminary transplant results in vivo provide the basis for future work aimed at determining the usefulness of OEG in promoting the survival and regeneration of RGCs after trauma.

Following the in vivo study described above, an additional in vitro experiment was carried out using engineered OEG and retinal explants. Neurite extension was measured at distances from the explant edge (250 μm, 500 μm, 1000 μm and 2000 μm). The groups analyzed were: no cells, OEG-GFP, OEG-CNTF, OEG-GFP conditioned medium and OEGCNTF conditioned medium. Results indicated that the number of regenerating axons was higher in the OEG-GFP and OEG-CNTF groups compared to control (no cell) RGC explant cultures (Fig. 5). Collagen coated dishes with adult RGC explants showed 146 ± 30.43 neurites. Retinal explants co-cultured with OEG-GFP (499± 42.4) or OEG-CNTF (421± 45.1) was higher than controls, but no significant increase in numbers was noted with the addition of CNTF. Conditioned medium from OEG-GFP and OEG-CNTF cells also showed no difference in neurite numbers (198 ± 23.25) and (190± 72.76) (see Fig. 5). Analysis of the cultures and immunostaining with βIII-tubulin shows OEG-GFP and OEG-CNTF positive cells closely associated with growing neurites and interacting with host astrocytes emanating from the retinal explants (Fig. 6). In conclusion, engineered OEG secreting CNTF provided no increased potential for regeneration compared to OEG alone. It is our belief in vitro that the impact of OEG on retinal growth is primarily a contact-mediated effect, rather than a secreted factor effect. OEG and myelination of RGC axons In order to achieve effective neuronal recovery after an injury RGCs that survive, regrow their axons and reconnect to targets also need to be remyelinated. Studies utilizing OEG to promote remyelination of axons have shown conflicting results (Devon and Doucette, 1992; Franklin et al., 1996; Plant et al., 2002; Boyd et al., 2004; Li et al., 2007). In the visual system however, there has been only one study to date on the effects of remyelination by OEG on RGC axons. Li et al. (2007) reported that primary OEG transduced with GFP and transplanted into the optic nerve showed no myelination ability. In fact the authors suggested the myelination came from Schwann cells. One explanation as to why myelination is not observed in some coculture and in vivo studies relates to the axon diameter, which is

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Fig. 2. Photomicrographs of the phenotype of OEG used for transplantation. OEG were transduced using a non-replicative lentiviral vector encoding a green fluorescent reporter gene (LV-GFP) (A, C, E) or a lentivirus encoding the ciliary neurotrophic gene (LV-CNTF) (B, D, F). These transduced cells were immunostained with S100 (A, C) glial fibrillary acidic protein (GFAP) (C, D), green fluorescent protein (GFP) (E) or ciliary neurotrophic factor (CNTF) (F). Immunocytochemically stained cells were mounted in media containing Hoechst 33342 (blue). Scale bar 50 μm.

known to affect the ability of peripherally-derived Schwann cells to myelinate axons. Another explanation is the neuronal origin and their extrinsic and intrinsic differences. Neurons from the PNS or CNS are more appropriate for OEG myelination studies. RGC are centrallyderived axons which are myelinated by host oligodendrocytes and can be successfully myelinated by Schwann cells contained in peripheral nerve grafts and also cultured quite successfully. To test if OEG can successfully myelinate RGCs, we also conducted an in vitro study using purified adult OEG (as previously described by Plant et al., 2002) and compared their potential to adult Schwann cells (purified as described by Morrissey et al., 1991), as well as postnatal oligodendrocyte precursor cells (OPC; purified as described by Milner and Ffrench-Constant, 1994). Retinal explants were obtained from adult rats and placed on collagen coated coverslips and maintained in Neurobasal-A media supplemented with 2% (v/v) B27, 100 μl/ml gentamicin and 1% (w/v) glutamine (NLA-A to provide optimal neuronal growth and survival (Leaver et al., 2006c). Two types of in vitro study were carried out (Doslo et al., unpublished). For the first study, RGC explants were co-cultured with either 250,000 Schwann cells or OEG in the presence of different myelination media: NLA-A containing serum, NLA-A containing 50 μg/ ml ascorbic acid or NLA-A containing serum and 50 μg/ml ascorbic acid, for 14 days before being fixed and processed for immunocytochemistry.

In the second study, RGC explants were placed on OEG which were in turn were cultured on top of collagen coated coverslips, shown previously to facilitate extensive outgrowth of RGC neurites (Leaver et al., 2006c). Following this either OEG, Schwann cells or OPC were placed on top of the explants and OEG monolayer to initiate any myelination process. These cultures were then fed with the different myelinating media, supplemented with either NGF or BDNF and maintained for 12 days before being fixed and processed for immunocytochemistry. The first study yielded poor cell and neurite attachment to the collagen substrate and no myelination was observed in any of the groups. The second method, in which neurite outgrowth was promoted by culturing retinal explants on a feeder layer of OEG (Leaver et al., 2006c) yielded better results. Using the OEG feeder layer, we found more than double the amount of neurite outgrowth compared to explants without the feeder layer. Importantly, in the positive controls using OPC co-cultured with RGC we found MBP positive cells and compact myelin structures, indicative of mature myelin sheaths (data not shown). The Schwann cell co-culture group also contained MBP positive cells and compact structures, but in lower numbers compared to the OPC co-culture group. In the OEG co-culture group, we also saw MBP positive cells but there were no MBP tubular structures identified in any of the culture conditions. In addition,

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Fig. 3. Photomicrographs of adult rat optic nerves 7 weeks following transplantation of OEG-CNTF to the crush zone. A and B show immunofluorescent stained sections for βIII tubulin which identifies retinal ganglion cell (RGC) axons at the site of injury (A) and regrowing toward the optic chiasm (B). Images shown in A and B are sections from the same nerve. Asterisk indicates site of crush injury, arrowheads (A) point to axons with unusual trajectories, arrows (B) indicate axons projecting towards to optic chiasm. Scale bars 100 μm.

when neurotrophic factors were added to the myelinating media, there was a positive effect on SC and OPC myelination but such an effect was not observed in the OEG group. This study showed that adult OEG failed to induce myelination of RGC under conditions containing serum and ascorbate, similar to previously reported work using DRG explants (Plant et al., 2002). In addition, adult OEG also failed to myelinate in the presence of specific neurotrophic factors (NGF and BDNF). Despite these observations, the studies still do not provide definitive proof that adult OEG are unable to myelinate RGC axons. It could be argued that the appropriate culture conditions were not achieved; for example recent evidence has suggested that progesterone has promyelinating effects (De Nicola et al., 2006).

Thus it remains to be seen if other extrinsic factors or intrinsic axonal factors may induce myelination of RGC by purified, adult OEG. Future studies Over a number of years, results obtained with OEG have indicated their successful role in supporting axonal regeneration in the CNS. This review has concentrated on the capability of OEG to stimulate RGC axon regeneration either in vivo or in vitro. Adult OEG are capable of inducing regeneration of RGC neurites for significant distances (Leaver et al., 2006c). Future studies should look at the molecules or matrices involved in the induction and promotion of RGC regeneration and the

Fig. 4. Graph showing the total number of βIII tubulin axons observed in the distal segment of the crushed optic nerve at distances of 0.5 mm, 1.0 mm and 1.5 mm from the injury site 7 weeks post-axotomy following transplantation of OEG-CNTF or OEG-GFP.

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Fig. 5. In vitro analysis of retinal ganglion cell axonal growth. The graph illustrates the total number and length of RGC axonal growth at distances from the retinal explant edge (250, 500, 1000 and 2000 μm) following different treatment conditions including control explants (RGC only), n = 5, LV-GFP transduced OEG conditioned media, n = 5, LV-CNTF transduced OEG conditioned media, n = 5, LV-GFP transduced OEG, n = 6 and LV-CNTF transduced OEG, n = 6. Asterisks indicate significant difference in axonal growth (pb 0.05 Bonferroni post-hoc test) when compared to the control group at the same distance.

Fig. 6. Axonal regeneration from adult rat retinal explants in (A, B) collagen alone control (βIII tubulin (red) and glial fibrillary acidic protein (GFAP) (green); (C) OEG-CNTF conditioned media treatment (βIII tubulin (red) and GFAP (green)). The presence of (D) OEG-GFP (βIII tubulin (red) and GFP (green)) or (E, F) OEG-CNTF (βIII tubulin (red) and S100 (green)) stimulated increased amounts of axonal regeneration. βIII tubulin axons (red) show high affinity for OEG (green), shown under higher magnification in F. Scale bars (A, E, F, shown on A) 100 μm and (B–D, shown on B) 200 μm.

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use of gene therapy to increase or decrease key molecules of interest. Some groups, including ours, have explored this by the over-secretion of neurotrophic factors such as CNTF. Other important key molecules including inflammatory cytokines and chemokines secreted by OEG may provide better results or provide a better starting platform for combinatorial approaches. New insights into the use of optogenetics (Boyden et al., 2005) may also allow the visual system to be manipulated without invasive techniques and a concomitant reduction in inflammatory responses. To date, most in vivo studies using OEG in the visual system have been acute because of the lack of survival of RGC neurons after a short period of time after injury. One intriguing but as yet unanswered question is the capability of OEG to protect and stimulate regeneration of chronically injured RGC neurons. In conclusion, OEG have a role to play in the CNS regenerative field and particularly it seems in the spinal cord. Further studies are also needed to discover if they potentially have a role in the injured visual system that will lead to measurable functional improvements in vivo.

Acknowledgments This work was supported by the Neurotrauma Research Program of Western Australia to GWP and ARH. ARH was supported by the National Health and Medical Research Council (NHMRC Grant No. 254507). GWP was supported by an NHMRC RD Wright Fellow (Grant No. 303265). The authors would like to thank present and previous laboratory staff members involved in the collection of data involved in this review in particular Dr. Thalles De Mello, Dr. Helen Barbour, Marisa Gibbs and Sanja Doslo. We are also grateful to Christine Plant for critical reading and valuable comments on the manuscript.

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