Fish retains a remarkable potential of neuro-regeneration throughout the life, while injury to neuronal system in the central nervous system of mammals results in neural degeneration and loss of function. Thus, understanding of the mechanism of neuro-regeneration in fish could be useful to improve the poor neuro-regenerative capability in mammals. In the present study, we characterized a neuro-regenerative process in the ablated brain of cichlid, tilapia, Oreochromis niloticus by immunohistochemistry for bromodeoxyuridine (BrdU) and TUNEL-assay. Morphological observations showed a complete neuro-regeneration of ablated habenula region by 60 days post-ablation. A lipophilic tracer (DiI) tracing showed a complete recovery of neuronal projection from the habenula to its target, the interpenduncular nucleus by 60-day post-ablation. In the ablated brain, TUNEL assay showed a significant increase of apoptotic cells (~234%, P<0.05) at one day post-ablation, while the number of BrdU-positive cells were significantly increased (~92%, P<0.05) at 7 days post-ablation when it compared with sham-control fish. These observations suggest an important role of apoptosis activity in elimination of degenerated tissues and cell proliferation during neuroregeneration in the fish brain. To confirm this hypothesis, the effect of degenerative neural tissue on cell proliferation was analysed. Implantation of degenerative neural but not non-neural tissue into the brain cavity significantly increased number of BrdU-positive cells in the brain of the intact fish. These results suggest that newborn cells are induced by factors derived from degenerative apoptotic cells, which could be among the earliest signal(s) in the regenerative process in the fish brain.
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In the central nervous system (CNS) in mammals, adult neurogenesis has only been demonstrated in the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the dentate gyrus in the hippocampus where neural stem cells are actively generated (Jin et al., 2003; Leung et al., 2007). However, most of newly generated precursor cells are unable to survive, differentiate and integrate back to their existing neuronal circuitry (Schwartz et al., 1999; Chapouton et al., 2007; Jin, et al., 2006). It has been estimated that approximately 0.2% of the newborn neurons are contributed in the reconstruction of the damaged neural circuitry (Arvidsson et al., 2002, Magavi et al., 2000). In addition, the presence of inhibitory factors and the absence of a permissive environment further restrain neuro-regeneration in the adult mammalian brain (Sirbulescu et al., 2009). Nevertheless, there are several studies showing the capability of newly differentiated neurons successfully being restored in the adult human brain (Arvidssan et al., 2002; Jin et al., 2004; and Jin et al., 2006). In the brain of patients with stroke, new born cells are only seen in the SVZ, but not in ischemic damage area (Arvidsson et al., 2002). These observations suggest that damaged brain regions release some signals which stimulate generation of new born cells to the damaged area. However, the nature of the signaling to attract new born cells to insulted site is still unclear.
Unlike mammals, most non-mammalian vertebrates possess extensive cell proliferative capability in the CNS. Especially in teleost fish, cell proliferative zones are located in several brain regions (Kaslin et al., 2008) and even adult fish has neuroregenerative capability after injury or damage (Zupanc, 2006). Generally in the fish brain, the neural regeneration process starts with a large number of mitotic cells being generated in the cell proliferative zones located at the ventricular surface of the brain (Takeda et al., 2008). The newly generated cells then migrate towards the injured site and participate in restoration process, and then differentiate into neurons and glial cells (Kaslin et al., 2008). The high cell proliferative activity in the CNS of teleost is also counterbalanced by cell apoptosis, especially when brain injury has occurred. Apoptotic process involves cleaning and removing of the damaged or death cells before they are replaced and restored by newly generated cells (Zupanc, 2006). In contrast, in the mammalian CNS, necrosis is the predominant type of cell death after injuries, which leads to preventing the ingrowth of nerve fibers and the migration of cells into the lesion site (Zupanc, 2006). These results suggests that cell apoptosis process might have an essential role in inducing proliferative activity during neuro-regeneration in the brain of teleost (Zupanc et al., 1998)..Therefore, the better understanding of the mechanism of neuro-regeneration in teleost could provide breakthrough to neuro-regeneration in mammalian. However, the mechanism involved in neuroregeneration in the fish brain is still not well understood.
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In the present study, we analyzed a role of apoptosis in the neuroregeneration in the brain of a cichlid, tilapia (Oreochromis niloticus). To observe neuro-regeneration process, the habenula region, a paired structure located in the diencephalon was chosen to be ablated because it is highly; 1) cell proliferative region in fish (Kaslin et al., 2008), 2) evolutionarily highly conserved region in vertebrate's brain (Bianco and Wilson, 2008), and 3) relatively compact structure and located on brain surface, which allows us to lesion the brain with minimal damage to other brain region. Further, we examined the effects of apoptotic degenerative neural tissue on neurogenesis to identify its potential role in neuroregenerative process.
Materials and Methods
Sexually mature male tilapia, Oreochromis niloticus (standard length, 10-12 cm; body weight, 55 -65 g) were used for analysis. Fish were maintained in freshwater aquaria at 27Â±1 Â°C with a controlled natural photo-regimen (14/10h, light/dark). The fish were anesthetized by immersion in a 0.01% solution of benzocaine (Sigma, St. Louis, MO) before operations and dissection of tissues. Fish were maintained and used in accordance with the Guidelines of the Animal Ethics Committee (AEC) of Monash University (AEC Approval Number: SOBSB/MY/2008/42).
To analyze the neuro-regenerative process in the brain of tilapia, the habenula region was ablated. The anesthetized fish (n=3) was wrapped in moistened tissue paper to keep the body and gills moist, and positioned in the holder of a stereotaxic apparatus (Narishige Co., Tokyo, Japan). Through the landmark (starting from the midline between the eyes) on the fish's head, a hole was made through the skull approximately 5 mm2 with a sterile surgical blade and an 18G disposable needle attached to the stereotaxic apparatus was lowered approximately 2-3 mm from the surface of the skull into the habenula region. The habenula tissue was then ablated by aspiration (~100 ml volume). After the ablation, the opening on the skull was sealed with a water proof instant adhesive (LOCTITE 404, Sunnyvale, CA) and the fish was returned to an isolated individual tank for various post-ablation recovery times: 0, 1, 7, 14, 21, 40 and 60 days. As negative controls, sham operated fish (skull surgically operated without ablation) were prepared and kept for same time periods for recovery.
Apoptotic cells were detected by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) assay using Cell Death Detection Kit (Roche Diagnostics, Mannheim, Germany). Brains of ablated and sham control fish (n=3/group) were dissected and immediately embedded in Tissue-Tek Optimal Cutting Temperature (OCT) compound (Sakura Finetechnical Co. Ltd., Tokyo, Japan), and frozen on dry ice. The brain was sectioned coronally at 14 mm thick on a cryostat and thaw-mounted onto aminopropyltriethoxy silane (APS)-coated glass slides. Subsequently, the sections were fixed in buffered 4% paraformaldehyde (PFA) in 0.1M phosphate buffer (PB) at 4Â°C for 5 min. They were then washed in phosphate buffer saline (PBS, pH 7.4) to remove the fixative solution. The sections were pretreated using microwave irradiation to improve cell permeabilisation (Deng et al., 2001; Dubska et al., 2002). Briefly, the slides were immersed in 0.01M citrate buffer (Citric acid/ Sodium citrate, pH 6.0) and irradiated for 2 min in a microwave at 750W. The slides were immediately immersed in PBS at room temperature for rapid cooling. After pretreatment, the slides were incubated in TUNEL reaction mixture containing TdT enzyme and fluorescein-labeled dUTPs (Roche Diagnostics) for 60 min at 37 Â°C. Positive and negative controls were included and processed in parallel with the test samples. As a negative control, the sections were treated in TUNEL reaction mixture without the TdT enzyme; while for positive control, the sections were treated with 1U/ml of DNase (Promega, Madison, WI) for 5 min at room temperature prior to treatment with TUNEL reaction mixture. The sections were then counterstained with 0.5% cresyl violet (Sigma).
Newborn cells (S-phase cells) in the brain were detected by BrdU-labeling. Fish was injected intraperitoneally with a cocktail of 3.0 mg/ml BrdU (Sigma) and 0.3 mg/ml 5-fluoro-2'-deoxyuridine (FdU) (Sigma) in sterile 0.9% w/v NaCl saline with approximately 1.0 ml/ 100g of body weight. FdU was added to enhance incorporation of BrdU into the replicating chromosomal DNA. Twenty-four hours after the injection, the fish was anesthetized and killed by decapitation, and the brain was removed and fixed in buffered 4% PFA for 6 hours at 4 Â°C. Cryoprotection was achieved by transferring the brains into 20% sucrose in PB at 4Â°C overnight. Subsequently, the brain was embedded in OCT compound and quickly frozen on dry ice. The brain was sectioned coronally (for ablation experiment) and sagittally (for implantation experiment) at 14 Âµm thick on a cryostat and thaw-mounted onto APS-coated glass slide. The sections were incubated in 2N HCl at 37Â°C for 1 hour to denature the DNA. The reaction was stopped by a rinse in 0.1M borate buffer (pH 8.5) at room temperature for 10 minutes, followed by three rinses in PBS. The sections were incubated in blocking solution (2% normal horse serum and 0.5% Triton X-100 in PBS) at room temperature for 30 minutes. The sections were then incubated in a mouse anti-BrdU antibody (BD Pharmingen Laboratories, NJ, USA) diluted 1:200 in the blocking solution at 4Â°C overnight and were incubated in Alexa Fluor 594-labeled anti-mouse IgG (Invitrogen, Carlsbad, CA) diluted 1: 200 in blocking solution for 2 hours at room temperature. Sections incubated without antibody were used as negative control (n = 3). Coverslips were applied with Vectashield (Vector Laboratories, Burlingame, CA) for microscopy observation.
Preparation of apoptotic degenerative tissues, cytoplasmic protein extracts and cerebrospinal fluid for implantation studies
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For implantation studies, apoptotic degenerative tissues, protein extracts of apoptotic tissues and cerebrospinal fluid (CSF) were prepared.
To obtain apoptotic tissues, approximately 1 mm2 of neural (habenula and spinal cord) and non-neural (white muscle underneath the skull) tissues were dissected from one same fish and immediately used for the implantation. For protein extracts, the dissected tissues were homogenized in 0.9% NaCl solution and briefly centrifuged 2,000 g for 10 min at 4Â°C to bring down the tissue debris, and supernatant was collected and stored at 4 Â°C until the injection. CSF was isolated from the brain of 1 day-post habenula ablated fish (n=2) following the procedure described elsewhere (Barbieri et al., 1992). Fluid withdrawn was centrifuged 2,000x g for 10 minutes at 4Â°C to separate the CSF from blood and used immediately.
Implantation of apoptotic tissues and protein extracts
Three groups of applications were conducted: (A) implantation of dissected neuronal or non-neuronal tissues; (B) injection of protein extracted from apoptotic tissues; and (C) infusion of CSF extracted from the injured fish.
For (A) tissue implantation, four categories of implantation study were conducted: (i) dissected habenula tissue nearby the habenula region; or (ii) the olfactory bulbular region; (iii) implantation of dissected spinal cord tissue or (iv) white muscle tissue nearby the habenula region (n=3 per each group). A hole was made through the skull approximately 5 mm2 with a sterile surgical blade and the piece of dissected tissues (3 mm2) was implanted into the brain cavity of fish.
For (B) injection study, protein extract of apoptotic neural tissue was injected on the right lobe of telencephalon; while those of apoptotic muscle tissue was injected to the left lobe of telencephalon as control. After making a hole on the skull, an 18G disposable needle attached to the stereotaxic apparatus was lowered approximately 3.0 mm into the telencephalic region and 10Âµl of protein extracts of either neuronal tissue or muscle tissue were injected via microsyringe (n=6/ group).
For (C) infusion study, an 18G disposable needle attached to the stereotaxic apparatus was lowered approximately 2-3 mm from the surface of the skull into the brain cavity through the opening on the skull and approximately 100Âµl of the extracted CSF was infused into the brain cavity of fish (n = 3) without causing any injury.
After the each application, the opening on the skull was sealed with a water proof instant adhesive. The fish was returned to an isolated individual tank and allowed to survive for 3 days before decapitation. As controls, the sham operated fish were kept for same time periods for recovery (n=3). The fish were injected with 5-bromo-2-deoxyuridine (BrdU) 24 hrs before sampling for proliferative cell study.
Cell counts and statistical analysis
The sections were examined on an inverted fluorescence microscope (TE2000, Nikon Instruments)and images were captured with digital camera (DXM1200, Nikon Instruments, Tokyo, Japan) with a B-2A filter (Nikon Instruments) to reveal TUNEL-labeled cells and with a G-2A filter (Nikon Instruments) to reveal BrdU-positive cells with 4X and 10X objective lens. Numbers of TUNEL-labeled and BrdU-positive cells were determined by image analysis using Image-Pro Plus 6.0 software (Media Cybernetics Inc, USA). In the ablation experiment, number of TUNEL-labeled and BrdU-positive cells were counted in the habenula region on approximately 15 sections (n = 3). For apoptotic tissue implantation studies, 200 mm2 sampling grids were made for systematic random cell counting (on medial sagittal sections with habenula) to remove bias in selection. All values are expressed as mean Â± SEM. Data were compared by using an analysis of variance (ANOVA) for multiple comparison with the Tukey-Kramer post-hoc test.
Carbocyanine dye (DiI) tracing
To examine regeneration of neural pathway, a neural tracer was applied in the brain of fish at 60-day post ablation. Brains of ablated and sham control fish (n=2/group) were dissected and fixed in buffered 4% PFA at 4Â°C for overnight. The fixed brains were cut along the longitudinal axis to expose the habenula, and small crystal of 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI, Molecular Probes, Eugene, OR) was inserted into the habenula region under a stereoscopic microscope. After DiI application, the brains were then embedded in 3% agarose to seal the tracer and stored in 4% PFA supplemented with 0.1% EDTA for 1 week in darkness at 37Â°C. The brain was then removed from agarose and re-embedded into 7% gelatin to be sectioned sagittally on a vibratome (Vibratome 3000, Vibratome Co. Inc., MO) at 200 Âµm thickness. The sections were mounted with Vectashield (Vector Laboratories), observed and photographed under a laser confocal microscope (C1si, Nikon Instruments).
Observation of neuroregenerative process in the tilapia brain
The habenula region was successfully ablated by aspiration with syringe (Fig. 1). In the habenula-ablated fish, a significant reduction of their appetite and aggressive behaviours was observed for three days to few weeks during the recovery (Data not shown). Morphological observations in the ablated brain showed gradual restoration of the tissue at the ablation site after various survival times (Fig. 2A-D). The ablated habenula was observed starting to reform from 7 days post-ablation and it regenerated to an "oval-shaped"-original structure of the habenula by 60 days post-ablation (Fig. 2D).
TUNEL assay revealed the patterns of apoptotic activity during the neuro-regeneration (Fig. 2E-L). In the ablated habenula region, a small number of TUNEL-labeled cells were seen on day 0 post-ablation (~71 cells/ cm2) (Figs. 2I, 3A). After one day of recovery, the number of TUNEL-labeled cells was increased dramatically (~500 cells/ cm2, P<0.05) in the ablated habenula region (Figs. 2F, J and 3A), and it gradually declined (~76% decrease, P<0.05) by one week post ablation (Fig. 3A). There was no change in the number of TUNEL-labeled cells in the sham-operated over the period of survival time after the operation. The detached or destroyed tissue at the ablation site was disappeared by one week post-ablation (data not shown).
Cell proliferative activity during neuro-regeneration in the ablated brain was analyzed by BrdU-immunohistochemistry (Fig. 2Q-T). A small number of BrdU-positive cells was started to appear 7 days post-ablation (~270 cells/ cm2)(Figs. 2 and 3B) and it was significantly (P< 0.05) increased (~620 cells/ cm2, 6-fold higher compared to day 0) by 21 days post-ablation (Fig. 3B).
DiI tracing of habenula projection
The regeneration of afferent and efferent connections of the habenula in the ablated fish was analyzed using a fluorescent lipophylic tracer, DiI. On 60 days post-ablation, DiI-labeled neural projections from the regenerated habenula were seen innervation toward the telencephalon (data not shown) and the interpeduncular nucleus (IPN) as those seen in the brain of intact fish (Fig. 5B and C).
Newborn cells induced by ablation and apoptotic neuronal tissue implantation
To analyze the stimulatory effect of apoptotic neural tissues on cell proliferative activity, the dissected habenula tissue was implanted in the brain cavity of intact fish. On 3 days post-ablation, a significantly higher (P<0.05, 75-150% increase) number of BrdU-positive cells was seen in the telencephalon, optic tectum, and dorsal and ventral hypothalamus (Fig. 6D-G) in comparison to sham-operated fish. Similarly, in the brain of fish implanted with apoptotic neural tissue, a significantly higher (P<0.05, 52-57% increase) number of BrdU-positive cells was seen in the telencephalon, optic tectum, and dorsal and ventral hypothalamus (Fig. 6D-G) in comparison to sham-operated fish. There was a significant difference in the number of BrdU-positive cells in the telencephalic area between implanted fish and ablated fish (Fig. 6D).
Newborn cells induced by apoptotic neural versus non-neural tissue types
Neural tissue (spinal cord and habenula) and non-neural tissue (white muscle) were infused into the brain of intact fish to confirm tissue-type specificity of their cell proliferation inducible effects (Fig. 7 A-D). There was no significant difference in the number of BrdU-positive cells in the habenula and optic tectum regions between fish implanted with white muscle and sham-operated fish (Fig. 7E and F). In the fish implanted with apoptotic neural tissues (habenula and spinal cord), a significantly higher (P<0.05) number of BrdU-positive cells was seen in their habenula region (56-67% increase) and optic tectum region (53-61% increase) in comparison to sham-operated fish (Fig. 7E and F).
Site specificity of newborn cells induction
Apoptotic neuronal tissues were infused into different sites in the brain cavity to investigate their site specificity on cell proliferative activity (Fig. 9A-C). A significant higher (P<0.05, 52-63% increase) number of BrdU-positive cells was seen in the telencephalon (63% increase), habenula, optic tectum and hypothalamus but not in the olfactory bulb (Fig. 9D-I) when the apoptotic neural tissue was implanted nearby the habenula. On the other hand, a significantly higher (P<0.05, 64-79% increase) number of BrdU-positive cells was only seen in the olfactory bulb and telencephalon (Fig. 9D and E), when the apoptotic neural tissues was implanted nearby the olfactory bulb.
Effect of protein extracts and CSF on cell proliferation
To study cell proliferation inducing effects of the apoptotic tissues, the protein extracts of apoptotic neural and non-neural tissues were injected into the brain of intact fish. A significantly (P< 0.01) higher number of BrdU-positive cells was seen in right lobe of telencephalon injected with protein extract compared to those seen in the left lobe injected with muscle tissue extracts (Fig 8).
In the brain of fish infused with CSF extracted from the injured fish brain, a significantly higher number of BrdU-positive cells in the telencephalon region (75% increase, P< 0.01) and the ventral part of hypothalamus (41.5% increase, P< 0.05), but not in other regions (Fig. 10).
Neuro-regeneration of habenula in tilapia
This study for the first time showed neuro-regenerative ability in the tilapia brain after brain-ablation. Morphological characterization revealed the complete regeneration of the ablated brain tissues by 60 days post-ablation. Furthermore, neural tracer application in the ablated habenula confirmed a complete recovery of ascending (from the telencephalon) and descending (to the IPN) fiber connections (Yañez and Anadón, 1996), which indicates the complete regeneration of innervations and the reconnecting of neuronal circuit from the newly generated habenula tissue.
One day after the ablation, the detached and destroyed tissues were highly stained with TUNEL signals, which indicates occurrence of apoptotic activity in the ablated region. One week after the ablation, those apoptotic cells observed around the ablated region were greatly reduced to the background level. At the same time, a small number of BrdU-positive proliferative cells were observed around the ablated site. This distinct difference in the pattern of proliferative and apoptotic activities at the ablation suggests that apoptosis plays a important role in the removal and elimination of degenerated tissues before replacement of degenerated neuronal tissues, which is uniquely observed in non-mammalian brain (Zupanc and Zupanc, 2006). In mammals, brain injuries result predominantly or exclusively, in necrosis, which caused by inflammatory response at the site of injury (Zupanc and Zupanc, 2006). This necrotic event eventually acts as mechanical and biochemical barriers preventing the ingrowth of nerve fibers and the migration of cells into the site of lesion (Zupanc and Zupanc, 2006). By contrast, in teleost, the predominant type of cell death used for removal of damaged cells after brain injuries appears to be apoptosis. Moreover, the negative side effects accompanied with necrosis, such as inflammation of the surrounding tissue, are typically absent in apoptosis (Zupanc and Zupanc, 2006). Therefore, the mechanism taken place on damaged cell removal in fish brain could be one of the crucial factors on limited regenerative capability in mammalian brain.
Stimulatory effects of apoptosis degenerative neural tissue on neurogenesis
In fish brain, there is a continuous provision of newborn cells or undifferentiated cells under intact condition, whereas during injury, newborn cells are recruited more rapidly to compensate the cell lost (Zupanc and Ott, 1999), which is similar to those seen in the injury- or diseased- human brain (Jin et al., 2004; Jin et al., 2006). It is known that adult mammalian brain has their own self-renewal mechanism where neurogenic regions are stimulated to generate more neural stem cell to replace the dead neural tissues (Biebl et al., 2000). It is also hypothesized that signals could be transmitted from apoptotic neurons to call for replacement of new neuron (Arvidsson et al., 2002). However, the mechanism of self-stimulation of progenitor cells the damaged CNS is still unknown. The present study showed that apoptotic neural tissue as well as its protein extract stimulates the cell proliferation activity and accumulation of newborn cells at the implanted site and its surrounding areas, which indicates that apoptotic neural tissues could release some stimulant(s) which able to induce and activate the production of newborn cells from the proliferative zones. Further, the significant changes of newborn cells were not only seen in the ablated brain region, but also in other non-damaged brain regions. These results suggest that the stimulant(s) released from the apoptotic tissues could be delivered and stimulate cell proliferation in these brain regions possibly via CSF.
The present study rises the question of what kind of factors are released from apoptotic neural tissues to stimulate neuro-regeneration in the adult fish brain. In mammals, several cellular elements are known to release from the degenerated neural tissues with cell proliferation stimulative effects, which include protein, peptide, trace element and/ or phagocytic effect (Zhao et al., 2008; Wiessner et al., 1993; Gould and Tanapat, 1997; Ivanoc et al., 1998; Burke et al., 1981; Liedtke et al., 2007).
In summary, apoptosis activity in elimination of degenerated neural tissues after injury will release stimulant to call for cell proliferation for restoration. They could be the earliest signal(s) in the regenerative process in the fish brain with cerebrospinal fluid as the carrier of the signal(s). However, their role in the neuroregenerative process and the molecular mechanism is still unknown, which remains to be investigated. Identification of these signals could provide a breakthrough in adult mammalian neurogenesis, especially in areas other than subventricular zone and dentate gyrus areas (the only neurogenic regions in mammalian brain).