We have previously shown that a single application of the growth factors ciliary neurotrophic factor (CNTF) or fibroblast growth factor 2 (FGF-2) to the crushed optic nerve of the frog, Rana pipiens , increases the numbers and elongation rate of regenerating retinal ganglion cell axons. Here we investigate the effects of these factors on the numbers and types of macrophages that invade the regeneration zone. In control PBS-treated nerves, many macrophages are present 100 μm distal to the crush site at 1 week after injury; their numbers halve by 2 weeks. A single application of CNTF at the time of injury triples the numbers of macrophages at 1 week, with this increase compared to control being maintained at 2 weeks. Application of FGF-2 is equally effective at 1 week, but the macrophage numbers have fallen to control levels at 2 weeks. Immunostaining with a pan-macrophage marker, ED1, and a marker for M2-like macrophages, Arg-1, showed that the proportion of the putative M2 phenotype remained at approximately 80% with all treatments. Electron microscopy of the macrophages at 1 week shows strong phagocytic activity with all treatments, with many vacuoles containing axon fragments and membrane debris. At 2 weeks with PBS or FGF-2 treatment the remaining macrophages are less phagocytically active, containing mainly lipid inclusions. With CNTF treatment, at 2 weeks many of the more numerous macrophages are still phagocytosing axonal debris, although they also contain lipid inclusions. We conclude that the increase in macrophage influx seen after growth factor application is beneficial for the regenerating axons, probably due to more extensive removal of degenerating distal axons, but also perhaps to secretion of growth-promoting substances.

Retinoic acid (RA) plays major roles during nervous system development, and during regeneration of the adult nervous system. We have previously shown that components of the RA signaling pathway are upregulated after optic nerve injury, and that exogenous application of all-trans retinoic acid (ATRA) greatly increases the survival of axotomized retinal ganglion cells (RGCs). The objective of the present study is to investigate the effects of ATRA application on the macrophages in the optic nerve after injury, and to determine whether this affects axonal regeneration. The optic nerve was crushed and treated with PBS, ATRA and/or clodronate-loaded liposomes. Nerves were examined at one and two weeks after axotomy with light microscopy, immunocytochemistry and electron microscopy. ATRA application to the optic nerve caused transient increases in the number of macrophages and microglia one week after injury. The macrophages are consistently labeled with M2-type markers, and have considerable phagocytic activity. ATRA increased ultrastructural features of ongoing phagocytic activity in macrophages at one and two weeks. ATRA treatment also significantly increased the numbers of regenerating GAP-43-labeled axons. Clodronate liposome treatment depleted macrophage numbers by 80%, completely eliminated the ATRA-mediated increase in axonal regeneration, and clodronate treatment alone decreased axonal numbers by 30%. These results suggest that the success of axon regeneration is partially dependent on the presence of debris-phagocytosing macrophages, and that the increases in regeneration caused by ATRA are in part due to their increased numbers. Further studies will examine whether macrophage depletion affects RGC survival.

27We have previously shown that a single application of the growth factors ciliary 28 neurotrophic factor (CNTF) or fibroblast growth factor 2 (FGF-2) to the crushed optic nerve of 29 the frog, Rana pipiens, increases the numbers and elongation rate of regenerating retinal 30 ganglion cell axons. Here we investigate the effects of these factors on the numbers and types 31 of macrophages that invade the regeneration zone. In control PBS-treated nerves, many 32 macrophage-like cells are present 100 μm distal to the crush site at 1 week after injury; their 33 numbers halve by 2 weeks. A single application of CNTF at the time of injury triples the 34 numbers of macrophages at 1 week, with this increase compared to control being maintained at 35 2 weeks. Application of FGF-2 is equally effective at 1 week, but the macrophage numbers have 36 fallen to control levels at 2 weeks. Immunostaining with a pan-macrophage marker, ED1, and a 37 marker for M2-like macrophages, Arg-1, showed that the proportion of the putative M2 38 phenotype remained at approximately 80% with all treatments. Electron microscopy of the 39 macrophage-like cells at 1 week shows strong phagocytic activity with all treatments, with many 40 vacuoles containing axon fragments and membrane debris. At 2 weeks with PBS or FGF-2 41 treatment the remaining macrophage-like cells are less phagocytically active, containing mainly 42 lipid inclusions. With CNTF treatment, at 2 weeks many of the more numerous macrophages 43 are still phagocytosing axonal debris, although they also contain lipid inclusions. We conclude 44 that the increase in macrophage influx seen after growth factor application is beneficial for the 45 regenerating axons, probably due to more extensive removal of degenerating distal axons, but 46 also perhaps to secretion of growth-promoting substances. 47 48 49 3 50 Introduction 51 Central nervous system (CNS) neurons react to injury in different ways in different 52 groups of animals -for example, mammalian neurons mostly die and the remainder show poor 53 regrowth [1,2], fish CNS neurons survive and regenerate successfully [3,4], while amphibian 54 neurons show intermediate survival rates and successful regrowth [5]. Neuronal death after55 injury is thought to be due to interruption of the supply of neurotrophic factors from the target 56 region [1,[6][7][8], while poor regrowth is due to an inhibitory environment [9,10], and many studies 57 have been devoted to alleviating these conditions. 59In recent years, it has been proposed that macrophages play a key role in modulating 60 the progression of neurodegenerative diseases [11][12][13][14] and also the response to CNS injury 61 [15][16][17]. Macrophages originate from bone-marrow-derived monocytes, which circulate in the 62 bloodstream [18] and are then capable of infiltrating injured tissues, where they differentiate into 63 macrophages [19,20]. Along with already-resident microglia, these cells phagocytose debris [21] 64 and secrete chemicals that enhance or inhibit the inflammatory ...

Retinoic acid (RA) plays major roles during nervous system development. We have previously shown that RA signaling pathway components are upregulated after optic nerve injury and that exogenous application of RA improves the long‐term survival of axotomized retinal ganglion cells to more than 90%. In addition, there is some evidence that RA may be a factor involved in the induction of phagocytic macrophages, promoting the production of pro‐repair chemokines that could play a role in successful regeneration. The objective of the present study is to determine the effects of RA application on the macrophage populations in the optic nerve after injury. We performed optic nerve crush and applied into the nerve either saline solution or retinoic acid. We examined the optic nerves at 48h, one week, and two weeks after axotomy with immunocytochemistry and electron microscopy. Electron microscope studies of the proximal, injury, and distal sites of the optic nerves show macrophages filled with secondary lysosomes and residual bodies. Immunocytochemistry of various macrophage subtypes was carried out followed by confocal microscopy. Our results indicate that application of RA to the optic nerve causes a significant increase in the number of macrophages and microglia present one week after optic nerve injury. Most cells were CD68‐positive in saline‐ and RA‐treated nerves. We also identified activated microglia (Iba1‐positive) and a sub‐population of anti‐inflammatory M2 macrophages (arginase‐positive) at the injury and distal sites. In conclusion, the application of RA affects the number and the distribution of macrophages after optic nerve injury and it may play a role in the success of optic nerve regeneration. Support or Funding Information REB is supported by NIH grant GM116692, JMB by NIH grant NS081726 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.