The Neuronal Regeneration of Adult Zebrafish After Spinal Cord Injury Is Enhanced by Transplanting Optimized Number of Neural Progenitor Cells

Zebrafish Husbandry

Zebrafish wild-type (WT) strain AB/TU and transgenic line huORFZ were cultured indoors ²¹ , and their developmental stages were identified according to standard procedures described by Westerfield ²² . All embryos were cultured in embryo medium (EM) (140 mM NaCl, 5.4 mM KCl, 0.25 mM Na₂HPO₄, 0.44 mM KH₂PO₄, 1.3 mM CaCl₂, 1.0 mM MgSO₄, and 4.2 mM NaHCO₃ at pH 7.2) for 1 day, and the medium was replaced by fresh EM containing 0.003% 1-phenyl-2-thiourea (Sigma, St. Louis, MO, USA).

Fixation and Bone Decalcification of Adult Zebrafish

We followed the procedures described by Kroehne et al. ²³ with some modifications. After we collected the adult zebrafish, they were anesthetized with 100 mg/l Tricaine (the ethylester of 3-aminobenzoic acid; Sigma) and both head and tail were removed. We then fixed trunk samples in a 4% paraformaldehyde (pH 7.2 to 7.4; Sigma) at 25°C for 3 days, followed by treating with 20% of decalcification stock solution (2.5M EDTA, Thermo, Waltham, USA; final concentration of 0.5M EDTA, pH 7.8) at 25°C for 7 days, washing three times with phosphate-buffered saline (PBS) (pH 7.4), keeping samples at 4°C.

Frozen Section of Adult Zebrafish

We followed the procedures described by Zeng et al. ²⁰ , except we used a 25-µm thick slice. The green fluorescence signal shown on each section was detected by primary polyclonal antibody against GFP (Abcam, Cambridge, UK) and observed by an A1 confocal laser-scanning microscope (Nikon, Nagoya, Japan). The green fluorescence signal shown on each frozen section was detected by primary polyclonal antibody against GFP (Abcam, Cambridge, UK) at 1:150 dilution, anti-phospho-histone 3 (PH3) (Millipore, Bedford, MA, USA) at 1:200, and anti-HuCD (Fausett et al., 2006 ) (early neuronal marker; Invitrogen, Carlsbad, CA, USA) at 1:250 ²⁴ . The secondary antibody was either goat anti-rabbit or anti-mouse Cy3-conjugated fluorescence (Millipore) at 1:500.

Confocal Microscopy and Imaging Processes

The fluorescent signal shown on embryos was observed by a Zeiss confocal microscope (LSM 780, Carl Zeiss AG) and an A1 confocal laser-scanning microscope (Nikon). The image processing software with NIS-Elements Confocal and Zeiss LSM 780 (Carl Zeiss AG) Confocal was used for image capture.

Hypoxic Exposure of Zebrafish Embryos from Transgenic Line huORFZ

We followed the procedures described by Zeng et al. ²⁰ to carry out hypoxic treatment of zebrafish embryos. Briefly, 80 ml of EM at pH 5.8 was placed in a 100-ml serum bottle capped with a rubber stopper having a glass tube and kept at 28°C for 5 to 10 min, followed by pumping 99% nitrogen for 5 min to make deoxygenated EM. When 100 zebrafish embryos were developed at 72 hours post-fertilization, the used EM was replaced by fresh deoxygenated EM. The bottle was sealed tightly to avoid bubbles and kept at 28°C for 2 h. After that, the hypoxia-exposed larvae were collected, fresh EM replaced, and kept at 28°C for the following experiment.

SCI of Adult Zebrafish

To prepare mechanical SCI for adult zebrafish, we followed the procedures described by Fang et al. ²⁵ with some modification ²³ . After adult fish were anesthetized in 0.02% Tricaine (Millipore, Bedford, MA, USA), we employed fine tips (Proserv Instruments, New Taipei City, Taiwan) to remove squamae 6 mm in length, starting from the brainstem junction toward the caudal part, using a sterilized surgical blade (Proserv Instruments) to cut off skin and muscle, and a sterilized microsurgical blade to transect the spinal cord. After SCI, the lesion site was treated with vetbond tissue adhesive gel (St. Paul, MN, USA). Finally, the treated zebrafish were kept at 28°C in water with a 0.1% antifungal drug (Universal Medikament, Hamburg, Germany).

Fluorescence-activated Cell Sorting (FACS) Cell Sorter

First, zebrafish embryos were washed with 1× E3 medium (60× stock preparation: 34.8 g NaCl, 1.6 g KCl, 5.8 g CaCl₂·2H₂O, and 9.78 g MgCl₂·6H₂O, pH 7.2), followed by immersion in Ringer’s solution (116 mM NaCl, 2.6 mM KCl, 5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.0) to gently remove the yolk. Second, embryos underwent cell dissociation using protease solution (0.25% trypsin, 1 mM EDTA, pH 8.0, in PBS) at 28°C for 10 min, followed by homogenization for 5 min at least three times. Third, 200 μl of 1× stop solution (6× stock preparation: 30% fetal bovine serum, 6 mM CaCl₂·2H₂O, in PBS) was added to stop protease reaction. Finally, we saved the cell pellet after centrifugation, added 1 ml of suspension media (1% fetal bovine serum (FBS; Stem Cell Technology, Vancouver, Canada), 0.8 mM CaCl₂, 50 U/ml penicillin, 0.05 mg/ml streptomycin, Dulbecco’s modified Eagle’s medium (GIBCO, InvitrogenTM, Carlsbad, CA, USA)), took 200 μl aliquots to pass through a 40-µm cell strainer and dispensed into a falcon FACS strainer (STEMCELL, Cambridge, USA) for cell sorting. We always kept cell on ice.

Cell Transplantation

All utensils needed to be disinfected with 70% alcohol to avoid infection. We cleaned the lesion site with sterilized swabs and used a stainless-steel pincette to open the wound site ²⁵ . A 1.5-μl PBS solution containing 100, 300, 500, and 1,000 GFP-(+) cells was individually transplanted into the recipient’s lesion site using Fisherbrand aerosol barrier pipette tips (Thermo, Waltham, USA). After transplantation, tissue adhesive gel was used to seal the lesion site to prevent infection. Each cell transplantation process did not exceed 10 s to reduce lethality. Afterwards, DNA-free swabs with cotton tip and wooden handle (Thomas Scientific, Phoenix, AZ, USA) were used to gently remove surface water that covered the lesion site. The treated zebrafish were kept in 28°C water containing a 0.1% antifungal drug, one fish per tank. Water and reagents were replaced daily, and each experiment was carried out on 10 adult zebrafish.

Mortality of SCI-treated Adult Zebrafish After Cell Transplantation

After cell transplantation, the condition of lesion site and the symptom of each SCI-treated adult zebrafish were examined every 24 h for 5 days. Simultaneously, the mortality of recipients in each group was recorded daily and counted in accumulation. Ten adult zebrafish were used in each experimental group. The mortality rate was averaged from three independent experiments, presenting as mean ± SD, using the light-stimulated response approach to examine the effect of transplanting neural progenitor cells on enhancing neuronal regeneration in adult zebrafish after SCI. The swimming pattern of each individual fish was tracked in line for 30 s after light stimulation for 1 s.

Adult Zebrafish Behavioral Assay

The behavioral assay of adult zebrafish followed the protocol described by Shimmura et al. ²⁶ with some modifications. All fish were kept in a test tank with fixed size (15 × 15 × 15 cm³) containing water at 28°C. The locomotion of each fish sample was recorded for 3 min (Dipp-AAM, Ditect, Japan), and two-dimensional data were analyzed by Microsoft Excel.

Quantifications and Statistics

Unless otherwise indicated, each experiment was repeated at least three times or more. Animals were randomly assigned to different experimental groups, but no formal method of randomization was used. We used one-way analysis of variance (ANOVA), followed by Dunn’s multiple comparison test, as well as two-way ANOVA, followed by Student’s t-test (normally distributed data) for comparisons. Error bars indicate the standard error of the mean. A P-value of <0.05 was considered significant.

Optimizing the Number of Transplanted Cells

Taking advantage of a zebrafish transgenic line huORFZ in the present study, we first exposed huORFZ embryos to hypoxia to induce GFP-expressing cells (Fig. 1A and B). Second, we used the FACS sorter to isolate GFP-(+) cells, which served as donor cells (Fig. 1C). Meanwhile, we administered mechanical SCI to adult WT zebrafish (Fig. 1D). The GFP-(+) cells were then transplanted into the lesion site of SCI-treated adult zebrafish.

OPEN IN VIEWER

Figure 1.

GFP-(+) cells were transplanted into the spinal cord lesion site of SCI-treated adult zebrafish. (A) Schematic illustration of the procedures for treating and transplanting cells to zebrafish transgenic line huORFZ. After embryos at 72 hpf were exposed to hypoxia for 2 h, the GFP signal was observed after oxygen recovery for 24 h. (B) GFP signal was apparent in brain and spinal cord. (C) Schematic illustration of transplanting isolated GFP-(+) cells into SCI-treated adult zebrafish. After huORFZ embryos were exposed to hypoxia, cells were dissociated, and then GFP-(+) cells were isolated by FACS. Slmultaneously, surgery was performed on adult zebrafish to cut the spinal cord. GFP-(+) cells were then transplanted into the lesion site of SCI-treated adult zebrafish, followed by frozen section and phenotype observation under confocal microscopy. (D) Photos of spinal cord before and after SCI. Arrow indicates the SCI site of adult zebrafish. FACS: fluorescence-activated cell sorting; hpf: hours post-fertilization; hpi: XXX, GFP: green fluorescent protein; SCI: spinal cord injury; WT: wild type.

At two days post-transplantation (dpt), we determined the presence of GFP-(+) cells in the lesion site of adult zebrafish. To accomplish this, we collected the recipients, performed frozen sections, and observed them under the fluorescent microscope. No green fluorescence signals were observed in the nontransplanted control group (Fig. 2A, upper panels). However, GFP-(+) cells were detected at the lesion site of SCI (Fig. 2A, lower panels), suggesting that GFP-(+) cells had been successfully transplanted into recipients. Next, to determine the growth condition of recipients relative to the number of transplanted cells, we divided the experiment into four groups, i.e. those transplanted with 100, 300, 500, and 1,000 cells individually into the SCI lesion site. As shown in Table 1, no obvious effect on morphological appearance was observed in recipients having both 100 and 300 transplanted cells. However, recipients transplanted with 500 cells resulted in inflammation and reddish appearance around the lesion site, and those transplanted with 1,000 cells resulted in seriously swollen and reddish appearance with tissue fluid seepage. Therefore, the recipient adult zebrafish transplanted with 300 cells exhibited better spinal cord regeneration.

OPEN IN VIEWER

Figure 2.

GFP-(+) cells were shown in the lesion site of SCI-treated adult zebrafish and induced the lethality of recipients after transplantation. (A) After the frozen section, the tissue at SCI site was observed under bright-field microscopy and GFP fluorescence microscopy. Upper panels: nontransplantation control; lower panels: transplanted cells. The yellow arrows indicate that the GFP signal originated from the GFP-expressing cells transplanted into the lesion site of SCI-adult zebrafish. Dotted lines indicate the location of the spinal cord. All images were obtained from the cross section. (B) The accumulation of mortality rates of SCI-adult zebrafish. Ten adult zebrafish were used in each experimental group. Each value was averaged from three independent experiments. Mortality rate (%) was accumulated and presented as mean ± SD on every day after cell transplantation. GFP: green fluorescent protein; SCI: spinal cord injury.

OPEN IN VIEWER

Table 1.

Effect of the Number of Transferred Cells on Lesion Site in the Context of Symptoms Observed.

Number of transplanted cells	SCI only	100	300	500	1,000
Symptoms of spinal cord injuries	No effect	No effect	No effect	Inflammation and swelling	Inflammation, swelling, and tissue fluid exudation

SCI: spinal cord injury; No effect: inflammation was not observed at the lesion site.

We also calculated the mortality rate of adult SCI-treated zebrafish transplanted with different numbers of cells. As shown in Fig. 2B, zebrafish transplanted with 100 cells had only 6.67% ± 5.77% mortality on the fourth day after transplantation. The 300-cell transplantation group had a 13.33% ± 5.77% mortality rate on the third day and 23.33% ± 5.77% mortality rate on the fourth day. The 500-cell transplantation group had a 20.00% ± 5.77% mortality rate on the first day and up to 75.00% ± 5.77% mortality rate on the fourth day. The 1,000-cell transplantation group had a 53.33% ± 5.77% mortality rate on the first day and 96.66% ± 5.77% mortality rate on the fourth day. These data suggested that the transplantation of different numbers of cells in SCI-treated adult zebrafish had different consequences. Most importantly, disputing “the higher the number the better the results” hypothesis, herein the highest number of transplanted cells did not result in the best physiological results for the recipients.

Furthermore, to determine whether the 300 donor GFP-(+) cells were correctly transplanted to the lesion site in zebrafish after SCI, we performed nine series cross sections to detect donor GFP-(+) cells (Fig. 3A). The results shown on each section were 8.67 ± 2.52, 11.67 ± 3.79, 33.33 ± 2.52, 36.67 ± 4.04, 46 ± 20, 28 ± 2.65, 17.67 ± 3.06, 13.33 ± 2.08, and 1.00 ± 1.00 GFP-(+) cells counted on sections B, C, D, E, F, G, H, I, and J, respectively, at 1 dpt (Fig. 3B–J). In total, there were 196.33 ± 5.03 GFP-(+) cells counted from nine sections (Fig. 3K). We noticed that, since there was a 0.3 cm distance between each section and each section was only 25 µm thick, the number of transplanted cells counted from total frozen sections, such as 196.33 ± 5.03 cells shown here, should be underestimated. Additionally, not all GFP-(+) cells transplanted into host could entirely survive after 1 dpt.

OPEN IN VIEWER

Figure 3.

The number of survival cells transplanted into SCI-treated adult zebrafish. (A) Schematic diagram illustrates how we prepared samples for performing frozen sections to count the survival cells in adult zebrafish after cell transplantation. In total, 300 donor GFP-expressing cells obtained from the embryos from transgenic line huORFZ under the hypoxia exposure were transplanted into the recipient wild-type adult fish treated with SCI. At 1 dpt, head and tail parts of recipients were removed, followed by performing fixation and decalcification. The frozen section of fish was cut at a 0.3-cm distance away from the injury site, and there was a 0.3-cm distance between each cross section (B–J lines). (B–J) The GFP-expressing cells were apparent on each frozen section corresponding to B–J lines shown in Panel A. (K) The number distribution of GFP-expressing cells counted from section B through J is presented. The number of GFP-expressing cells shown in each section was averaged from three fish samples. Error bars indicate mean ± SD. dpt: day post-transplantation; GFP: green fluorescent protein; SCI: spinal cord injury.

GFP-expressing Donor Cells Could Improve Neuronal Regeneration in Adult Zebrafish After SCI

Zeng et al. ²⁰ found that the GFP-expressing cells are hypoxia-responsive and -resistant, but play a role in neuronal recovery. Specifically, only GFP-expressing-NSPC and GFP-expressing-RG subtypes can proliferate, migrate, and differentiate into functionally mature neurons during recovery. Finally, most mature neurons are differentiating unipolar neurons. In this study, the transplanted cells we used were those GFP-expressing cells able to differentiate into functional and mature neurons in hypoxia-exposed huORFZ larvae. Therefore, it is reasonable for us to hypothesize that the transplanted GFP-expressing cells at the lesion site of SCI-treated adult zebrafish are not only able to undergo proliferation but also able to differentiate into functional neurons. To confirm this hypothesis, we collected SCI-treated WT adult zebrafish after transplantation for 5 and 7 days and performed frozen cross sections to detect the proliferation and differentiation of cells transplanted into recipients using proliferation marker PH3 and early neuron marker HuCD, respectively. Thereafter, we examined whether the transplanted GFP-expressing donor cells could also express PH3 and HuCD markers labeled with red fluorescent protein (RFP) at 5 and 7 dpt. The results demonstrated that the GFP-expressing cells transplanted into recipients did exhibit yellow fluorescence signal, which was due to the colocalization of two signals, GFP and RFP (Fig. 4). Besides, 53.33% ± 8.50% GFP-expressing cells could express HuCD at 7 dpt. The line of evidence suggested that the cells transplanted in SCI-treated recipients were, to some extent, able to undergo proliferation and differentiation into neurons.

OPEN IN VIEWER

Figure 4.

The cells transplanted into the lesion site of SCI-treated adult fish are able to undergo proliferation and differentiation. (A) Schematic diagram illustrates the experimental flowchart. In total, 300 donor GFP-expressing cells were obtained from the embryos from transgenic line huORFZ exposure to hypoxia. The wild-type adult fish treated with SCI served as recipients. The GFP-(+) donor cells were then transplanted into the lesion site. Several days after transplantation, head and tail parts were removed from recipients. The remainder trunk underwent fixation and decalcification, and was then subjected to perform freezing of sections. The section was cut at 0.3-cm distance from injury site and each section was 25 µm thick. (B) The proliferation marker PH3 labeled with RFP was used to determine the proliferation of GFP-expressing cells transplanted into recipients at 5 dpt, while (C) the early neuron marker HuCD labeled with RFP was used to determine the differentiation of GFP-expressing cells transplanted into recipients at 7 dpt. Arrows indicate the cells exhibiting yellow fluorescence signal due to the colocalization of GFP-(+) and RFP-(+) signals. dpt: day post-transplantation; HuCD: neuron-specific RNA-binding protein; GFP: green fluorescent protein; PH3: phospho-histone 3; RFP: red fluorescent protein; SCI: spinal cord injury.

Finally, we determined if swimming distance would improve even more in fish transplanted with GFP-expressing cells. We performed behavioral analysis in which we compared the swimming distance between the transplanted and nontransplanted adult zebrafish after SCI. Compared to the nontransplanted zebrafish, results showed that the surging distance of transplanted zebrafish with GFP-expressing cells increased 1.54-fold (Fig. 5), suggesting that GFP-expressing donor cells transplanted into host fish did help recipients to improve neuronal regeneration in adult zebrafish after SCI.

OPEN IN VIEWER

Figure 5.

Transplantation of GFP-(+) cells into adult zebrafish treated with SCI enhances its neuronal regeneration. (A) Schematic diagram illustrates the outline of the experimental flowchart regarding how we performed cell transplantation in adult SCI-treated zebrafish. Zebrafish embryos from transgenic line huORFZ at 72 hpf were exposed to hypoxic stress. After these embryos developed 96 hpf were sacrificed, tissues were dissociated, and then employed on FACS to sort out the GFP-(+) cells, followed by transplanting GFP-(+) cells into SCI-treated adult zebrafish. After transplantation, fish samples were cultured in a tank sized 15 × 15 × 15 cm³ for behavior analysis. The swimming distance from X-Y axis (in cm) of each fish was recorded every 2 days during recovery using the touch-evoked response approach. (B) The locomtion pattern of each examined fish from three experimental groups, as indicated, was tracked in line. (C) The swimming distance was averaged from nine fish samples obtained from the SCI-treated zebrafish (served as a control) and SCI-treated zebrafish with transplantation. The t-test was used to perform statistical analysis (***P < 0.001; error bars indicate mean ± SD). dpt: day post-transplantation; FACS: fluorescence-activated cell sorting; GFP: green fluorescent protein; hpi: hours post-injury; hpf: hours post-fertilization; SCI: spinal cord injury; WT: wild type.