Early neurogenesis during caudal spinal cord regeneration in adult Gekko japonicus
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- Zhou, Y., Xu, Q., Li, D. et al. J Mol Hist (2013) 44: 291. doi:10.1007/s10735-012-9466-3
Gekko japonicus undergoes dramatic changes in the caudal spinal cord after tail amputation. The amputation induces cell proliferation in the caudal ependymal tube. We performed hematoxylin and eosin staining at different time points in the regeneration process to investigate the morphological characterization of the regenerated appendages. The central canal extended to the blastema post-amputation and the cartilage and muscle tissue appeared 3 weeks after injury. We performed the bromodeoxyuridine (BrdU) incorporation assay to detect proliferating cells during the regeneration process. BrdU positive cells were detected in the peri-central canal. Furthermore, nestin and neuron-specific enolase (NSE) immunocytochemistry were applied to detect neural stem/progenitor cells and neurons. Two weeks after injury, nestin-positive cells undergoing proliferation were located outside of the ependymal tube, and NSE positive cells appeared after 3 weeks of amputation. These data suggest that neurogenesis is an early event during caudal spinal cord regeneration in gecko.
KeywordsNeurogenesisBlastemaGekko japonicusSpinal cordRegeneration
Among amniotes, the most striking example of epimorphic regeneration comes from tail regeneration in lizards. Gekko japonicus, a species belonging to the Gekkonidae family, Sauria (lizard) suborder, lives mainly in China and Japan (Du et al. 2002). Geckos have a remarkable ability to regenerate their tails, and the regeneration mainly includes the skin, muscle, skeleton and nerve system. The most amazing issue is the nascent tail can regenerate the spinal cord and then restore the related function (McLean and Vickaryous 2011). The spinal cord regeneration has been extensively reported in many species, including adult fish, urodele amphibians, and lizard tails (Anderson et al. 1986; Sirbulescu and Zupanc 2011; Dawley et al. 2012; Duffy et al. 1992). Generally, the related respects of the regeneration contain axonal regrowth, neurogenesis and glial responses, while the situation is varied in different species. In the urodele amphibian, regeneration of the spinal cord can occur after tail amputation, and the differentiation of neural cells, axonal growth, and establishment of appropriate connection with their targets is usually complete within a few weeks (Dawley et al. 2012). The neurogenesis in the regenerates was regarded to originate from the radial glia cells lining the ependymal canal, which had been examined using either clonal cell cultures from the adult SC, cell labeling and in situ cell re-implantation, or in situ cell labeling using the biolistic technique (Benraiss et al. 1996, 1997). In addition, some researchers have shown that the nascently differentiated Schwann cells and melanocytes could be observed in regenerated tail which indicated the repair of peripheral nervous system after amputation (Benraiss et al. 1999). In reptile, most reports documented axonal regrowth during trauma-induced tail regeneration. In lizards that undergo regeneration, the regenerating nervous system was reported to be composed of the ependymal tube and axons derived from the proximal portion of the original spinal cord and dorsal root ganglia (Cox 1969; Alibardi 2010; McLean and Vickaryous 2011). However, a few studies also demonstrated the neurogenesis during regeneration in lizards (Molowny et al. 1995). Our question is how about the situation in Gekko japonicas? Whether the axon descending from the proximal spinal cord was occurred in the regenerated tail of gecko? In further, does neurogenesis occur during regeneration? If so, how and when does the neurogenesis occur? In present study, we first investigated the morphological changes of spinal cord during tail regeneration, then detected the location of proliferative cells and the neural stem/progenitor cells in the regenerates, and finally observed axon growth and neurogenesis at different time points after amputation. Our findings support the view that neurogenesis is an early event during caudal spinal cord regeneration in geckos.
Materials and methods
Adult geckos were wild caught and cultured in the laboratory animal center of Nantong University. At the start of the experiment, they were mature individuals and all were 3 ± 0.5 g in weight with an average total body length (snout to vent plus tail) of 520 mm, including males and females. They were freely fed with mealworms and water in homemade cages with hollow bricks, as well as hay, leaves, imitating natural gecko accommodation environment, and housed in an air-conditioned room with controlled temperature (22–25 °C) and saturated humidity. All experimental protocols pertinent to animals were given prior approval by the Laboratory Animal Care and Use Committee of the Nantong University. We minimized the number of animals used and used cooling anesthesia to reduce their suffering. The tail amputation of adult Gekko japonicus was performed according to previously published methods (Jiang et al. 2009). Briefly, the caudatomy was conducted by inserting a nylon slipknot at the site of the sixth tail segment and pulling gently, which mimics the conditions of autotomy in the natural environment of these lizards (Bryant and Bellairs 1970).
Administration of BrdU and tissue collection
In order to label the population of mitotically active cells in tail regenerates, especially in the regenerating caudal SC, animals were injected with BrdU (5-bromo-2′-deoxyuridine, Sigma). BrdU is incorporated into the DNA of dividing cells during S phase and can be visualized using immunohistochemistry. Geckos were anesthetized on ice and injected intraperitoneally with BrdU diluted in physiological saline at a dose of 2 g/kg for 3 days. Three days after the BrdU injections, the geckos were anesthetized with ethyl ether and perfused with 4 % paraformaldehyde and then normal and regenerated tail from perfused geckos were removed with the adjacent stump regions and fixed overnight in 4 % paraformaldehyde (pH 7.0). Tissues were then dehydrated in a 10–30 % sucrose gradient in phosphate-buffered saline (PBS, pH 7.4), and 12 μm thick sections from normal and regenerated tail were cut on a cryostat, thaw mounted onto polylysine-coated slides, and stored at −20 °C until staining was performed.
Hematoxylin and eosin (H&E) staining were used to identify the tissue and cellular compositions of the tails (Fischer et al. 2008). Briefly, sections were rinsed with deionized water (ddH2O; 5 min) and stained in Mayer’s hematoxylin (30 min) and rinsed in running water (5 min), 70 % ethanol for 1.5 min (10 drops of concentrated HCl in 200 ml 70 % ethanol) and bluing solution (10 drops of NH4OH in 200 ml ethanol, 1 min). Slides were dehydrated through three changes of 50 % ethanol (5 min), 70 % ethanol (5 min) and 80 % ethanol (5 min). Sections were stained in eosin (1 min), and then dehydrated through 95 % ethanol (5 min), three changes of absolute ethanol (5 min each), and three changes of xylene (5 min each), prior to coverslipping.
To determine the cell lineage of the proliferating ependymal cells, double-staining experiments were performed using an anti-BrdU antibody with anti-nestin or anti-NSE antibodies. Eight geckos were used/sampled for each of the BrdU, nestin and NSE components, divided into two groups of four, a group was used to detect BrdU and nestin, the other group used to detect BrdU and NSE. We mainly examined sections at the sixth tail segments which is adjacent to the amputation plane, and they demonstrate similar immunostaining pattern. For immunohistochemical detection of BrdU, the tissue sections were quenched with 3 % hydrogen peroxide in ddH20 for 10 min to eliminate endogenous peroxidases, then subsequently incubated in 2 N HCl and 0.5 % Triton X-100 in PBS for 30 min at 37 °C to denature the DNA. The sections were rinsed in 0.1 M sodium tetraborate, pH 8.5. Nonspecific sites were blocked in 10 % bovine serum albumin in PBS at room temperature for at least 2 h. The sections were incubated overnight at 4 °C in a humidified chamber with anti-BrdU antibody at 1:50 dilution in PBS. After washing with PBS, anti-NSE (ProSci Company) or anti-nestin (ProSci Company), antibodies were applied at 1:200 dilution for 16 h at 4 °C. Fluorescein isothiocyanate or tetramethyl rhodamine-conjugated anti-mouse or anti-rabbit IgG, and Texas red conjugated goat anti-rat IgG (Invitrogen) at 1:200 dilution were used as secondary antibodies. Representative sections were observed with epifluorescence on a Leica microscope.
Morphological characterization of the normal spinal cord of adult Gekko japonicus
The normal tail of adult Gekko japonicus is a prominent and complex appendage composed of multiple tissues including spinal cord, muscle, vasculature, adipose tissue, and a bony vertebral column. Like other lizards, the most notable feature of Gekko japonicus is its ability to voluntarily shed or autotomize their tail as a strategy to escape predation (Dawley et al. 2012).
Morphological and histological changes of spinal cord-like tissue of the regenerate tail at different times
Neurogenesis during caudal spinal cord regeneration
Epimorphic regeneration is a post-traumatic morphogenetic event beginning with the formation of a cellular aggregation (reportedly a blastema) at the wound site and developing a replacement appendage that can restore lost tissues and structures (Kierdorf and Kierdorf 2012). Tail regeneration in Gekko japonicus follows a conserved sequence of morphological and histological events comparable with observations made on other lizards (Cristino et al. 2000; Simpson 1968; Rumping and Jayne 1996; Alibardi 2010). Generally, the tail regeneration process can be divided into three stages: wound healing, blastema formation, and growth of the new tail. The tail of the gecko is a prominent and complex appendage composed of multiple tissue types, including striated muscle, vasculature, bony vertebral column, spinal cord, and notochord(Cristino et al. 2000). The spinal cord regeneration after tail amputation was the most amazing issue, and it has been extensively reported in many species. In the fish Apteronotus (Sternarchus) albifrons, ependymal cells in the adult retain the ability to incorporate thymidine and undergo neural differentiation (Anderson and Waxman 1985; Anderson et al. 1994). In amphibians, Benraiss et al. (1997) suggested that new neurons could be generated from some of the ependymal cells within the mature SC. In reptiles, the new spinal cord of the original tail is composed of ependymal tube and descending nerves (Simpson 1968; Rumping and Jayne 1996; Alibardi 2010), and there are also convincing data about neurogenesis occurring in the new tail (Simpson and Duffy 1994). Interestingly, Kehl et al. (1997) also observed cell proliferation and neural differentiation from the postnatal rat SC in primary culture. Another aspect to be concerned is the cell source of neurogenesis. It is now generally accepted that the adult nervous system contains multi-potential precursors for neurons, astrocytes, and oligodendrocytes (McKay 1997). Are these cells actually newly generated cells or do they come from cell de-differentiation, which had been extensively investigated in the regeneration of muscle tissue (Ding and Schultz 2004). Such a similar recycling process could be involved for neural cells in the SC as well. Lin and Matesic (1994) showed that after injury in the adult rodent forebrain, astrocytes may re-express fetal traits of early differentiating glial cells/neurons. We cannot rule out both possibilities for the neurogenesis that occurred in the present study. In conclusion, our results show that the adult gecko SC retains the capacity to have neurogenesis in the regenerating ependymal tube of the nascent tail.
This research supported by the grants from the NSFC (31071874, 31171007, 31171405), the Basic Research Program of Jiangsu Education Department (09KJA180005, 11KJA180004), Natural Science Foundation of Jiangsu Province (BK2010274) and the PAPD of Jiangsu Higher Education Institutions.