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Keywords:

  • neural stem cells;
  • cerebral ischemia;
  • collagen;
  • tissue engineering

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

Using tissue engineering, a complex of neural stem cells (NSCs) and collagen type I was transplanted for the therapy of cerebral ischemic injury. NSCs from E14 d rats were dissociated and cultured by neurosphere formation in serum-free medium in the presence of basic fibroblast growth factor (bFGF), then seeded onto collagen to measure cell adhesive ability. BrdU was added to the culture medium to label the NSCs. Wistar rats (n=100) were subjected to 2-hour middle cerebral artery occlusion. After 24 hours of reperfusion, rats were assigned randomly to five groups: NSCs-collagen repair group, NSCs repair group, unseeded collagen repair group, MCAO medium group, and sham group. Neurological, immunohistological and electronic microscope assessments were performed to examine the effects of these treatments. Scanning electronic microscopy showed that NSCs assemble in the pores of collagen. At 3, 7, 15, and 30 d after transplantation of the NSC-collagen complex, some of the engrafted NSCs survive, differentiate and form synapses in the brains of rats subjected to cerebral ischemia. Six d after transplantation of the NSC-collagen complex into the brains of ischemic rats, the collagen began to degrade; 30 d after transplantation, the collagen had degraded completely. The implantation of NSCs and type I collagen facilitated the structural and functional recovery of neural tissue following ischemic injury. Anat Rec, 2010. © 2010 Wiley-Liss, Inc.

Neural stem cells (NSCs) have recently aroused a great deal of interest not only because of their importance in basic research of neural development but also for their broad potential for stem cell-based therapy in neurological diseases, such as stroke, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and spinal cord injury (Lee et al., 2007; Sharma et al., 2007; Sugaya et al., 2007; Bürgers et al., 2008; Guzman et al., 2008; O'Keeffe et al., 2008; Wu et al., 2008b). Neural stem cells are capable of self-renewal and can give rise to both neurons and neuroglia (Benzing et al., 2006). This strategy has offered hope for recovery through the ability of the NSCs to differentiate and integrate appropriately into host cytoarchitecture.

Recent work indicates that transplanted human NSCs can survive and differentiate in the brain of rats with stroke and thereby improve behavioral recovery (Darsalia et al., 2007; Guzman et al., 2008). Neural stem cells from the C57BL/6J EGFP transgenic mouse (EGFP mice) could differentiate into the MAP2 positive cells after transplantation into an injured spinal cord (Du et al., 2007). However, it remains unclear whether these cells are capable of acquiring full functionality, which is one of the major prerequisites for NSC-based cell replacement strategies for neurological diseases. The ability to establish and maintain synaptic contacts is one of the basic requirements for intercellular communication and functional integration into existing neuronal networks.

The ideal cerebral reconstruct in stroke is composed of a biodegradable material which is strong enough to resist damage. The material should permit diffusion of nutrients and metabolic waste necessary for cell growth; enable cell adhesion, migration, proliferation, and differentiation; facilitate extracellular matrix formation (Ozawa et al., 2002). Collagen is widely used for biomedical applications and could represent a valid alternative scaffold material for vascular tissue engineering (Boccafoschi et al., 2005). The advantages of collagen matrices are their large surface area for cell seeding, porosity for capillary in growth, stability for mechanical support, biodegradability, and minimal immunogenicity (Kofidis et al., 2003; Gonen-Wadmany et al., 2004). Collagen Type I is the most prevalent collagen, and its relative ease of isolation and low cost have led to numerous studies of this molecule for multiple tissue engineering applications (Stuart and Panitch, 2009).

In this study, we investigated whether the transplantation of NSCs of rats and collagen type I could improve neurological function after middle cerebral artery occlusion (MCAO) in rats. We speculated that combined transplantation of neural stem cells and collagen type I compensates for lost or injured brain tissue.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

Experimental Animals

Adult female Wistar rats weighing 250 to 280 g were purchased from Harbin Veterinary Research Institute (HVRI). The 14-day-old embryos in gestating female Wistar rats were used for NSCs material. All rats were housed in rooms maintained at constant temperature and humidity and subjected to 12-hr light/dark cycle. All animals were housed and experiments conducted according to guidelines established by Harbin Medical University, and the procedures were approved by the Harbin Medical University Animal Supervision Committee. Rats received normal rodent feed and water at random.

Animal Model

Transient middle cerebral artery occlusion (MCAO).

Adult female animals were anesthetized with 10% chloraldurate. MCAO was induced using a method of intraluminal vascular occlusion modified in our laboratory. Briefly, the right common carotid artery, external carotid artery (ECA), and internal carotid artery (ICA) were exposed. A length of fishing thread (18.5 mm), with a diameter of 0.26 mm, had its tip dipped in 56°C−60°C paraffin, and then the tip was rounded. The fishing thread was inserted though the ECA into the lumen of the ICA until it blocked the origin of the MCA (Chen et al., 2003). In addition, the tip of the fishing thread was removed from the internal carotid artery 2 hr after the onset of occlusion to reperfuse the ischemic area. The animals were returned to their home cage in a room warmed at 26°C to 28°C (Mary et al., 2001). The rats were monitored postoperatively for 4 hrs after surgery. Of the 100 female animals which received the transient middle cerebral artery occlusion surgery, 78 survived. Of the 78 rats, animals were randomly divided into five groups: NSCs-collagen repair group (N = 18), NSCs repair group (N = 18), unseeded collagen repair group (N = 18), MCAO medium group (N = 12), and sham group (N = 12).

Cell culture.

The 14-day-old embryos were isolated and their brains were dissected in cold phosphate-buffered saline (PBS). Briefly, brains were washed twice in 10 mL of ice-cold PBS. The cerebrum was completely dissected with scissors and scalpels. The disrupted brain tissue was filtered using a wire gauze filter. After removal of the supernatant, the cell suspension was transferred to a centrifuge tube in 8 mL of PBS and centrifuged for 5 min at 1,500 rpm. The pellet was resuspended at 1 × 106 cells in 6 mL of Dulbecco's Modified Eagle's Medium (DMEM)-Ham's F12 medium (Gibco) supplemented with 100 units/mL penicillin, 100 units/mL streptomycin, 20 μg/L EGF, 20 μg/L bFGF, and 20 mg/mL neurobasal-B27 at 37°C in 5% CO2-95% air (Pfeifer et al., 2006). Half of the medium was replaced each week, and cells were passaged every 4 days. The neural stem cells were cultured for 1 week until neurospheres began to form. The neural stem cells were cultured for 3 weeks and then purified by continuous passage.

Coculture of NSCs and collagen.

Porous 3-D collagen sponges, insoluble bovine tendon collagen type I, was purchased from Shanghai Qisheng Biological Preparation Company. Collagen sponges were cut into 5 × 5 × 5 mm pieces with scissors and scalpels, and placed into the wells of a 12-well plate. A 1-mL cell suspension containing the neurospheres at 1 × 105 cells/mL was added to each well containing collagen. Thirty minutes after incubation, 2 mL of culture medium were added to the culture plate. NSCs and collagen were cocultured for 6 days to immobilize the neurospheres in three-dimensional collagen.

Measuring the cell adhesive ability.

After the NSCs and collagen had been cocultured for 6 days, the complex of NSCs and collagen was taken out and rinsed with PBS to wash the surface nonadhesive or dead cells. The cells located in the pores of the collagen were digested by 0.25% pancreatin and the number of cells in six plates was counted by a cell counter.

Bromodeoxyuridine Labeling.

Bromodeoxyuridine (BrdU; Sigma), a thymidine analog that can incorporate into the DNA of dividing cells during S-phase, was used to label newly generated cells (Wu et al., 2008a). BrdU (20 mg/mL) was added to the NSCs conditioned culture medium for 6 days.

Transplantation Procedure

NSCs and collagen transplantation.

The left middle cerebral artery (MCA) was exposed via a temporal craniectomy (Tamura et al., 1981). A 1 cm burr hole was made at the junction of the zygomatic arch and squamous bone. The lesion region induced by MCAO was exposed in the left hemisphere, a collagen seeded with NSCs was placed directly into the lesion region without removal of additional brain tissue, subsequently covered by bone wax, and the incision was closed with 4-0 absorbable surgical suture (Lu et al., 2007). We used this same method for the collagen unseeded group.

NSCs transplantation.

Undifferentiated NSCs were resuspended at the concentration of 3,000,000 cell/mL in preparation for cell transplantation. A hole was drilled in the left side of the skull to allow the penetration of a 10-μL Hamilton syringe into the cerebral cortex derived from bregma, with the skull in the flat position: anterior—1.0 mm; lateral—3.0 mm; and ventral—3.0 mm. (Ishibashi et al., 2004). A 5 μL aliquot of suspension (15,000 cells) was infused over 2 min at the site, and the syringe was left in place for an additional 2 min to prevent liquid exudation from the tip.

Evaluation of Neurological Function

Behavioral tests were performed on all rats at 1, 2, 3, 4, 5, and 6 days after transplantation by a blinded investigator, using the neurological severity scores modified by Chen et al. (2001). Neurological function was graded on a scale of 0 to 18 (0 = normal score; 18 = maximal deficit score). The functional evaluation included motor, sensory, reflex and balance tests. According to the severity scores of brain injury, one point was awarded for the inability to perform a task or for the lack of a test reflex (Hanabusa et al., 2005). The higher the score, the more severe the injury. Rats with a score of 15 or higher usually died 24 hrs after MCAO. The complex of NSCs and collagen was transplanted under anesthesia 24 hrs post-MCAO (Nystedt et al., 2006).

Immunostaining of Brain Sections

Tissue preparation.

Rats were perfused through the left ventricle with 100 mL of ice-cold NaCl followed by 50 mL of 4% paraformaldehyde in PBS. Brains were removed quickly, and postfixed with 4% paraformaldehyde for 2–4 days at 4°C.

The brains were quickly harvested. Coronal sections of 4 μm were cut using a microtome and processed as free-floating sections. Sections were incubated in 3% H2O2 for 10 min followed by three washes in 0.01 M PBS (Kelly et al., 2004). Sections were blocked in 5% rabbit serum and then incubated with either anti-Tau (Tau, Bioss, 1:100, for detection of neurons) or anti-BrdU (BrdU, Sigma, 1:400, for detection of newly generated cells) primary antibodies. Sections were subsequently incubated overnight with the primary antibodies at 4°C. Sections were washed and incubated with biotinylated anti-rabbit or anti-mouse IgG secondary antibodies (ZSGB-BIO) for 30 min at 37°C. For visualization, sections were colorized with diaminobenzidine (DAB). Slides were counterstained slightly with hematoxylin before dehydration and mounting.

Synapse formation.

Rat brain sections were fixed with 4% paraformaldehyde, and then incubated in 30 g/L (3%) glutaraldehyde for 2 hrs, and subsequently postfixed in 1% osmium tetroxide (in 0.1 M phosphate buffer, pH 7.4) for 2 hrs at 4°C. Then, the samples were dehydrated in a graded ethanol series with acetone, permeated, and embedded in epoxide resin. Semi-thin sections of about 75 nm were prepared, stained with uranyl acetate and lead citrate, and then observed with an H-300 transmission electron microscope (Hitachi Electronic Instruments, Japan).

Statistical Analyses

All data are expressed as means and standard errors and were analyzed using Student's t tests by SPSS 13.0 software to compare NSCs differentiation, new synapse formation and evaluation of neurological function between groups. All values are presented as the mean ± SD. P < 0.05 was taken as statistically significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

Cocultures of NSCs and Collagen

Light microscopy showed that the neurospheres purify by continuous passage after 3 weeks in serum free medium containing EGF, bFGF, and neurobasal-B27 (Fig. 1). After NSCs and collagen were cocultured for 6 days in vitro, Scanning electronic microscopy showed that the neurospheres grew in the pore of the three-dimensional collagen sponge (Fig. 2).

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Figure 1. The neural stem cells were cultured for three weeks in vitro in the conditional medium containing EGF, bFGF, and neurobasal-B27. There are neurospheres forming under a inverted phase contrast microscope.

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Figure 2. After NSCs and collagen were cocultured for 6 days in vitro. Scanning electronic microscopy showed that the neurospheres grew in the pore of the three-dimensional collagen sponge.

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Measuring the Cell Adhesive Ability

The cell adhesive ability was 31.47% (53.90 ± 0.48 × 104 in the NSCs and collagen group; 56.98 ± 0.31 × 104 in the control group) (P = 4.84, P > 0.05). Thus, the collagen sponge did not influence cell growth and had good cell compatibility (Table 1).

Table 1. The cell adhesive ability
GroupAverage cell number of 6 well (104)The cell adhesive ability (%)P
NSCs and collagenAdhesive cell number16.96 ± 0.3231.474.84
Free cell number36.94 ± 0.26
Total cell number53.90 ± 0.48
Control groupTotal cell number56.98 ± 0.31  

Immunostaining of Brain Sections

In the transplantation area, the nuclei of BrdU-positive cells appeared brown (Fig. 3). The nucleolus was obvious. The cytoplasms of the Tau-positive cells were stained brown (Fig. 4). The Tau-positive cells were 91.90% ± 1.46 in the NSCs-collagen repair group; 81.50% ± 1.60 in the NSCs repair group. (P = 0.000, P < 0.01). The vast majority of transplanted NSCs in the cerebral ischemia adopted a neuronal fate. The NSCs-collagen repair group has more Tau-positive cells. Statistically, significant differences between the groups were determined by analysis of variance.

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Figure 3. After the transplantation of the NSCs-collagen complex 7 days. The nuclei of BrdU-positive cells in the transplantation area are brown under a light microscope.

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Figure 4. After the transplantation of the NSCs-collagen complex 7 days. The cytoplasms of Tau-positive cells are stained brown in the transplantation area under a light microscope, indicating that the vast majority of transplanted NSCs in the cerebral ischemia adopted a neuronal fate.

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Evaluation of Neurological Function

Neurological severity scores were evaluated 1, 2, 3, 4, 5, and 6 days after ischemia-reperfusion injury. Compared with the functional improvements made by the media injected control group, the improvement in neurological function was significantly after cell transplantation in both cell transplant groups (in the NSCs-collagen repair group P = 0.037, P < 0.05; in the NSCs repair group P = 0.036, P < 0.05). NSCs-collagen repair group and the NSCs repair group scored lower than those in the media control group (Media), indicating that the animals were transplanted NSCs had better neural functioning (Fig. 5). Statistically, significant differences between the groups were determined by analysis of variance.

The Degradation of the Collagen

Three days after transplantation of NSC-collagen complexes, there was a space between the NSCs-collagen complex and the adult brain. Seven days after transplantation, the space disappeared and the glia limitans formed. At the same time, collagen began to degrade. Thirty days after transplantation, the collagen had degraded completely.

Synapse Formation

Light microscopy showed that some engrafted NSCs survive and differentiate in the brains of rats subjected to cerebral ischemia. Thirty days after transplantation of NSC-collagen complexes, TEM showed new synapses forming between the NSCs, Synaptic vesicles were obvious (Fig. 6). The new synapse were 18.4 ± 1.40 in the NSCs and collagen group; 8.86 ± 1.35 in the control group (P = 0.000, P < 0.01). The data showed statistically significant differences between the groups by analysis the new synapse formation.

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Figure 6. After the transplantation of the NSCs-collagen complex 30 days. New synapses form between the NSCs under the transmission electron microscopy. The synaptic vesicles were obvious.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

Neural stem cells (NSCs) are widely endorsed as a cell source for replacement strategies in neurodegenerative disease. NSCs retain the ability to self-renew and produce the major cell types of the brain. This opens new avenues for restorative therapy for neurodegenerative disorders (Kim et al., 2006; Walton et al., 2006; Hsu et al., 2007; Waldau and Shetty, 2008). Isolated from the fetal central nervous system, these cells can also be maintained in vitro and retain the potential to differentiate into nervous tissue. The vast majority of NSCs transplanted into the cerebral ischemic brain adopted a neuronal fate. Most migrating cells had a neuronal phenotype. Therefore, treatment with neurosphere-collagen complexes may offer an additional avenue for stroke therapy. We show here that transplantation of NSC-collagen complexes into the brain improved neurological function evaluation.

The typical therapeutic effect of NSCs is that differentiated cells integrated into host neural circuits and thereby replace the damage host neurons. Here, we demonstrate that intracerebral grafts of purified, unmodified, NSC-derived neurosphere-collagen complexes survive in the ischemic brains of adult rats. The cells differentiate and infused the cerebral tissue of the host.

Although many studies have focused on stroke therapy, the capacity of stem cells to adapt their fate and function to a changing pathological environment after ischemia is the basis for transplantation (Kiss and Muller, 2001; Viguié et al., 2001). Cell-mediated strategies seek to partially restore the neural controls for complex cognitive, sensory, or motor functions (Dobkin, 2007). This study shows that new synapses form between NSCs within 30 days after transplantation of NSC-collagen complexes; under the TEM, synaptic vesicles were obvious. The beneficial effect of the complexes is mediated by a neuroprotective rather than a regenerative mechanism.

This study shows that collagen has good cell and tissue compatibility (Boccafoschi et al., 2005). Its long-term safety, stability and efficacy in vivo have been adequately established in humans (Nunes et al., 2003; Candrian et al., 2008; Mimura et al., 2008). We used a biodegradable porous collagen type I, which is effective in facilitating cell migration and the delivery of oxygen and nutrients to the migrating cells. Collagen can be used as a vehicle to transplant NSCs into the cerebral cortex of adult rats. It does not influence cell growth or proliferation. This study shows that porous collagen sponge provides a good microenvironment to activate endogenous restorative mechanisms in ischemic brains. Thirty days after transplantation of NSC-collagen complexes, the collagen had degraded completely.

In conclusion, this study demonstrates NSCs can survive, differentiate and form new synapses in the lesion of the brain after MACO. The implantation of NSCs and type I collagen facilitated the structural and functional recovery of neural tissue following ischemic injury.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED
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