Umbilical Cord Blood-Derived CD34 + Cells Improve Outcomes of Traumatic Brain Injury in Rats by Stimulating Angiogenesis and Neurogenesis

Abstract

Human umbilical cord blood cells (HUCBCs) have been shown to be beneficial in reducing neurological deficits in rats with brain fluid percussion injury (FPI). This study aimed to assess the basic mechanisms underlying the neuroprotective effects of HUCBC-derived cluster of differentiation 34-positive (CD34⁺) cells. Rats were divided into three major groups: (i) sham-operated controls; (ii) FPI rats treated with phosphate-buffered saline (PBS); (iii) FPI rats treated with 0.2%, 50%, or 95% CD34⁺ cells (in 5 × 10⁵ cord blood lymphocytes and monocytes). Intravenous (IV) administration of 0.3 ml of PBS, 0.2% CD34⁺ cells, 50% CD34⁺ cells, or 95% CD34⁺ cells was conducted immediately after FPI. It was found that 4 days post-FPI, CD34⁺ cells could be detected in the ischemic brain tissues for 50% CD34⁺ cell- or 95% CD34⁺ cell-treated FPI rats, but not for the PBS-treated FPI rats or the 0.2% CD34⁺ cell-treated FPI rats. CD34⁺ cell (0.2%)-treated FPI rats or PBS-treated FPI rats displayed neurological and motor deficits, cerebral contusion and apoptosis [e.g., increased numbers of both TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling)-positive cells and caspase-3-positive cells], and activated inflammation (e.g., increased serum levels of tumor necrosis factor-α). FPI-induced neurological motor dysfunction, cerebral contusion and apoptosis, and activated inflammation could be attenuated by 50% CD34⁺ or 95% CD34⁺ cell therapy. In addition 50% or 95% CD34⁺ cell therapy but not PBS or 0.2% CD34⁺ cell therapy significantly promoted angiogenesis (e.g., increased numbers of both vasculoendothelial growth factor-positive cells and 5-bromodeoxyuridine (BrdU)-endothelial double-positive cells), neurogenesis (e.g., increased numbers of both glial cell line-derived neurotrophic factor-positive cells and BrdU/neuronal nuclei double-positive cells) in the ischemic brain after FPI, and migration of endothelial progenitor cells from the bone marrow. Our data suggest that IV administration of HUCBC-derived CD34⁺ cells may improve outcomes of FPI in rats by stimulating both angiogenesis and neurogenesis.

Keywords

Traumatic brain injury Apoptosis Cytokines Angiogenesis Neurogenesis Progenitor cells

Introduction

Traumatic brain injury (TBI) is a leading cause of death and disability in developed countries with limited treatment options (48). The traumatic penumbra is the stage of the reparative process that may develop after hours or days (26,32,57,62). Following fluid percussion injury (FPI) in experimental TBI, secondary cellular and molecular processes including inflammation, excitotoxicity, and oxidative stress may contribute to delayed neuronal loss (43,44), which is typically observed at the cortex or the hippocampus (19,28). Neuronal loss in FPI may be functionally counterbalanced by continual neurogenesis (7,19,55,58) and angiogenesis (7,23,28,63) in the adult brain.

Stem cell therapy has been proposed as an effective regenerative approach for reducing neuronal death in neurodegenerative diseases (35,63). Stem cells can be isolated from various tissues. For example, adult bone marrow contains stem cells that can potentially differentiate into many different cell types (13,54). The activity and number of peripheral blood hematopoietic stem cells under steady-state conditions with no cytokine induction is very low (15). However, human umbilical cord blood cells (HUCBCs) are rich in hematopoietic stem cells (8). Two percent of the HUCBCs are stem cells capable of reconstituting blood lineages. The mononuclear cells derived from HUCBCs can be induced to express class III β-tubulin, glial fibrillary acidic protein, galactosylceramidase (Galc, an oligodendrocyte marker) (18), and microtubule-associated protein 2 (20). When the HUCBCs were administered via the tail vein, surviving HUCBCs were identified in the cortex and striatum of the injured hemisphere (4,68). The neurological deficits produced by stroke (4,64,68), TBI, spinal cord injury (59), and heatstroke injury (8) were significantly improved by intravenous (IV) delivery of HUCBCs.

According to the findings of Lu et al. (37), IV administration of HUCBCs reduced neurological deficits in rats after TBI. However, the basic mechanisms underlying the neuroprotective effects of HUCBCs remain unclear. Stem cells [cluster of differentiation 34-positive (CD34⁺)] derived from umbilical cord blood are of potential use in cell therapy of spinal cord injury (66), stroke (68), and limb ischemia (34). Transplantation of CD34⁺ cells has been proven to enhance neovascularization in the penumbra region of the ischemic brain and subsequently promote prominent neurogenesis (63). In the pathogenesis of TBI, a relationship between angiogenesis and neurogenesis induced by CD34⁺ cells would be consistent with those of the chronic stroke (61).

In this study, to test the hypothesis, we administered HUCB-derived 95% CD34⁺ cells, 50% CD34⁺ cells, 0.2% CD34⁺ cells, or phosphate-buffered saline (PBS) (that contained 0% CD34⁺ cells) intravenously into the tail vein and assessed their effects on neurogenesis [evidenced by increasing the numbers of both glial cell-derived neutrophil growth factor-human nuclear antigen (GDNF/HNA) double-positive cells and 5-bromodeoxyuridine/neuronal nuclei (BrdU/NeuN) double-positive cells], angiogenesis [evidenced by increasing the numbers of both vascular endothelial growth factor (VEGF)-positive cells and BrdU/endothelial-positive cells], cellular levels of ischemia and damage markers in the ischemic brain tissues, and serum levels of systemic inflammatory molecules in a clinically relevant model of TBI such as FPI (71) and ascertained which was more effective in treating TBI.

Materials and Methods

Animals

Adult male Sprague-Dawley rats (weight, 266 ± 12 g) were obtained from the Animal Resource Center of the National Science Council of the Republic of China (Taipei, Taiwan). The animals were housed in groups of four at an ambient temperature of 24°C ± 1°C, with a 12-h light–dark cycle. Pellet rat chow and tap water were available ad libitum. All protocols were approved by the Animal Ethics Committee of the Chi Mei Medical Center (Tainan, Taiwan) in accordance with the National Science Council's guidelines for the care and use of laboratory animals and the Guidelines of the Animal Welfare Act.

Surgery

Animals were anesthetized with sodium pentobarbital (25 mg/kg intraperitoneally; Sigma Chemical, St., Louis, MO, USA) and a mixture containing ketamine (4.4 mg/ kg, intramuscularly; Nankuang Pharmaceutical, Tainan, Taiwan), atropine (0.02633 mg/kg, intramuscularly; Sintong Chemical. Ind., Taoyuan, Taiwan), and xylazine (6.77 mg/ kg, intramuscularly; Bayer, Leverkusen, Germany). Both the femoral artery and the vein on the right side were cannulated with PE50 polyethylene tubing (Becton Dickinson & Company, Franklin Lakes, NJ, USA) for monitoring blood pressure and drug injection, respectively. After cannulation, the wound was sutured (Doccol Corporation, Redlands, Sharon, MA, USA) and animals were put into a prone position. The animals were placed in a stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA), and the scalp was incised sagittally. The animals were subjected to a lateral FPI (42). After the scalp was incised, a 4.8-mm circular craniotomy was performed midway between lambda and bregma 3.0 mm to the right of the central suture. A modified Luer Lock connector (trauma cannula; GC Corporation, Tokyo, Japan), 2.6-mm inner diameter, was secured into the craniotomy with cyanoacrylate adhesive and dental acrylic (GC Corporation). A moderate FPI (2.2 atm) was produced by rapidly injecting a small volume of saline into the closed cranial cavity with a fluid percussion device (VCU Biochemical Engineering, Richmond, VA, USA). Immediately after the traumatic event, the animals displayed brief respiratory arrests and limb convulsions lasting for 40 to 70 s. The animals were removed from the device, the acrylic was removed, and the incision was sutured. The animal was then returned to its cage. Each injured and sham-injured animal for the FPI model was closely evaluated immediately after FPI for behavioral recovery.

Physiological Parameters

The right femoral artery of rats was cannulated with polyethylene tubing (PE50) under sodium pentobarbital anesthesia for blood pressure monitoring. For measurement of intracranial pressure (ICP), the animals were positioned in a stereotaxic apparatus (Kopf 1406; Grass Instrument, Quincy, MA, USA) to insert probes for a Statham P23AC transducer (Statham Labs, Inc., Hato Rey, Puerto Rico) via a 20-gauge stainless steel needled probe (diameter 0.90 mm; length 38 mm; Terumo Corporation, Tokyo, Japan), which was introduced into the left cerebral ventricle according to the stereotaxic coordinates of Paxinos and Watson (51): A, interaural, 7.7 mm; L, 2.0 mm from the midline; and H, 3.5 mm from the top of the skull. All recordings were made on a four-channel Gould polygraph (Gould Instruments System Inc, Cleveland, OH, USA). Core temperature (T_co) was monitored continuously by a thermocouple (Gould Instruments System Inc), and mean arterial pressure (MAP) and heart rate (HR) were monitored continuously with a Statham pressure transducer.

Microdialysis Determination of the Hippocampal Glutamate, Lactate-to-Pyruvate Ratio, Glycerol, and Nitric Oxide Metabolites (NO_x)

Each animal was anesthetized with sodium pentobarbital intraperitoneally. The animal's head was mounted in a stereotaxic apparatus (David Kopf Instruments) with the nose bar positioned 3.3 mm below the horizontal line. After a midline incision, the skull was exposed and a burr hole was made in the skull for the insertion of a dialysis probe (4 mm in length, CMA/2; Carnegie Medicine, Stockholm, Sweden). The microdialysis probe was stereotaxically and obliquely (anterior 4.3 mm) implanted into the right hippocampus according to the atlas and coordinates of Paxinos and Watson (51): 8 mm posterior to bregma, 3.0 mm lateral to the midline, and 5 mm to the surface. According to the methods described previously (12), the microdialysis was perfused at 2.0 μl/min and the dialysates were collected every 20 min in a CAM/140 fraction collector (Carnegie Medicine). Aliquots of dialysates (5 μl) were injected onto a CMA600 microdialysis analyzer (Carnegie Medicine) for measurement of lactate, pyruvate, and glutamate. The NO_x^– concentrations in the dialysates were measured with the Eicom ENO-20 NO₂^– analysis system (Eicom, Kyoto, Japan) (10). Only experiments in which the hippocampal localization of the microdialysis probes was confirmed histologically were included in the results. In this series of experiments, no animal was excluded due to incorrect microdialysis probe position. The microdialysis probe was inserted at least 90 min before the start of FPI.

Cell Viability

The trypan blue exclusion method was used to assess the cell viability. The cells were gently harvested and mixed with 0.4% trypan blue solution (Invitrogen, Carlsbad, CA, USA); the resulting cell suspension was counted under a phase contrast inverted microscope (Leica, Solms, Germany). The viable cells with intact cell membranes, which could exclude the dye, were counted using a hemocytometer (Marienfeld Superior, Lauda-Königshofen, Germany) and expressed as percentage of the total cells counted. The percentage of the total cells counted over 95% was used for cell therapy experiments.

Human CD34⁺ Cell Preparation

Human CD34⁺ cells were isolated from the cord blood of 15 females after informed consent from the mother and IRB approval, using a Direct CD34⁺ Progenitor Cell Isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and CD34⁺ Multisort kit (Miltenyi Biotec) according to the manufacturer's protocol. In brief, human cord blood lymphocytes and monocytes were suspended in 300 μl of PBS (Sigma) and 5 mM ethylenediaminetetraacetic acid (EDTA; Sigma). These cells were labeled with a hapten-conjugated monoclonal antibody against CD34 (PharMingen, San Diego, CA, USA), followed by an anti-hapten antibody coupled with microbeads, and were incubated with beads at ratios of 100 μl of beads per 10⁸ cells for 15 min at 4°C. The bead-positive cells (CD34⁺ cells) were enriched on positive-selection columns set in a magnetic field. Fluorescence-activated cell sorting (FACS) analysis using anti-CD34 antibodies (PharMingen) labeled with phycoerythrin (PE) (Becton Dickinson, Mountain View, CA, USA) of MACS-sorted cells showed that 96 ± 3% of the selected cells were positive for CD34. Immediately after FPI, 0.2%, 50%, or 95% human CD34⁺ cells (in 5×10⁵ human cord blood lymphocytes and monocytes) and PBS (that contained 0% CD34⁺ cells) were administered intravenously via the tail vein.

Experimental Groups

Animals were assigned randomly to one of the following five groups. One group of rats was treated with FPI followed immediately by IV infusion of 0.2%, 50%, or 95% CD34⁺ cells or PBS (0.3 ml per rat). The fifth group of animals was used as sham-operated controls. All tests were run with experimenters blinded to the treatment conditions, and the animal codes were revealed only at the end of the behavioral and histologic analysis. The animals used for histologic or behavioral studies were provided food and water ad libitum throughout the study.

Experimental Procedures

In experiment 1, an IV dose of 95% CD34⁺ cells, 0.2% CD34⁺ cells, or PBS (0% CD34⁺ cells) was randomly administered immediately after FPI (n = 16), and their effects on MAP, HR, ICP, cerebral perfusion pressure (CPP), core temperature (T_co) and hippocampal level of glutamate, glycerol, nitric oxide metabolite (NO_x) and lactate-to-pyruvate ratio were assessed immediately during FPI for 120 min.

In experiment 2, an IV dose of 95% CD34⁺ cells, 50% CD34⁺ cells, 0.2% CD34⁺ cells, or PBS (0.3 ml) was randomly administered immediately after FPI (n = 8 each), and their effects on the neurological and motor function were assessed 1–7 days after FPI.

In experiment 3, an IV dose of 95% CD34⁺ cells, 50% CD34⁺ cells, 0.2% CD34⁺ cells, or PBS (0.3 ml) was randomly administered immediately after FBI (n = 8 each), and their effects on cerebral infarction zone and serum levels of endothelial progenitor cells (EPCs), tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10), and intercellular adhesion molecule 1 (ICAM-1) were assessed 4 days after FPI.

In experiment 4, an IV dose of 95% CD34⁺ cells, 50% CD34⁺ cells, 0.2% CD34⁺ cells, or PBS (0.3 ml) was randomly administered immediately after FPI (n = 8 each), and their effects on the number of both terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells and caspase-3-positive cells in the injured hippocampus were assessed 4 days after FPI.

In experiment 5, an IV dose of 95% CD34⁺ cells, 0.2% CD34⁺ cells, or PBS (0.3 ml) was randomly administered immediately after FPI (n = 8 each), and their effects on the number of VEGF-positive cells, GDNF-positive cells, BrdU-positive endothelial cells, BrdU/NeuN double-positive cells, and HNA-positive cells in the injured hippocampus were assessed 4 days after FPI.

Neurological Function Evaluation

All rats were evaluated using a neurological severity score (NSS) (60). NSS is a composite of the motor (muscle status, abnormal movement), sensory (visual, tactile, and proprioceptive), and reflex tests. One point was given for failure to perform a task. Thus, the higher the score, the more severe is the injury, with a maximum of 14 points. The NSS was measured on all rats preinjury and on days 1, 4, and 7 after FPI.

Neurological motor function was measured using an inclined plane test (3). The inclined plane was used to measure limb strength. The animals were placed, facing right and then left, perpendicular to the slope of a 20 cm × 20 cm rubber ribbed surface of an inclined plane starting at an angle of 55° (designed in-house). The angle was increased or decreased in 5° increments by a microcontroller (Atmel Corporation, San Jose, CA, USA) to determine the maximal angle an animal could hold to the plane. The data for each day were the mean of left and right side maximal angles defined as the maximal angles attained before the animals fall to the padded surface below. Rats were tested prior to the injury to obtain baseline values. After TBI, the tests were performed on days 1, 4, and 7. All behavioral tests were examined and independently scored by two observers who were unaware of prior treatment. These scores were averaged to arrive at one score for each animal for the behavioral session.

Isolation of EPCs

Bone marrow was harvested by isolating the femurs of rats, which were carefully cleaned of adherent soft tissue. A syringe needle (23 gauge; Terumo Corporation) was inserted into the marrow cavity and flushed with PBS. The marrow cells were harvested by Ficoll (GE Healthcare, Uppsala, Sweden) density gradient centrifugation. Mononuclear cells were washed thrice in PBS. The cells were analyzed for CD133 (ab 19898, Abcam, Cambridge, MA, USA) conjugated with the corresponding fluorescein isothiocyanate (FITC)-labeled secondary antibody (Ab6717, Abcam) and VEGF-2 (ab10972, Abcam) conjugated with the corresponding PE-labeled secondary antibody (ab7004, Abcam). Isotype-matched antibodies (Abcam) served as controls in every experiment.

Cerebral Infarction Assessment

Four days after injury, all the animals were anesthetized with sodium pentobarbital (100 mg/kg intraperitoneally) and then transcardially perfused with 100 ml of saline with 1 U/ml heparin (Sigma) followed by 300 ml of 10% neutral buffered formalin (Sigma). Brains were removed, postfixed in formalin (Sigma), processed, and embedded in paraffin. Coronal sections (6 μm thick) were cut and stained with triphenyltetrazolium chloride (TTC; Alfa Aesar, Ward Hill, MA, USA) for light microscopic analyses of cortical lesion (TTC stain). A 4-day survival was chosen to correspond with the behavioral (neurological and motor function) evaluation. The infarction volume, as revealed by negative TTC stains indicating dehydrogenase-deficient tissue, was measured in each slice and summed using computerized planimetry (PC-based Image Tools Software, Image Research, Brook University, St. Catharines, Ontario, Canada) and calculated as 2 mm (thickness of the slice) × (sum of the infarction area in all brain slices: mm²) (67). For volumetric assessment of this cortical lesion, digitized images of brain sections, taken every millimeter from 2.0 to 7.0 mm posterior to bregma, were captured. All imaging and lesion volume analyses were performed by an independent investigator blinded to postinjury treatment status.

TUNEL Assay

Brain sections for TUNEL assay (Alfa-Aesar) were prepared as described previously (36). The color was developed using 3,3-diamino-benzidine tetrachloride (DAB; Sigma). The sections were sequentially treated with xylene and ethanol for paraffin removal and for dehydration; washed with PBS and incubated in 3% H₂O₂ (Sigma) solution for 20 min; treated with 5 μg/ml proteinase K (Clontech Laboratories Inc., Mountain View, CA, USA) at room temperature for 2 min; rewashed with PBS (0.1 M, pH 7.4); treated with a TUNEL reaction mixture (nucleotide; Roche, Mannheim, Germany) at 37°C for 1 h; washed with distilled water (D/W); reincubated in horseradish peroxidase-conjugated fluorescent antibodies at room temperature for 30 min; rewashed; and visualized using the avidin-biotin- peroxidase complex (ABC) technique and 0.05% DAB as a chromogen. The TUNEL-positive cells were counted by two pathologists in 30 fields per section (×200 magnification). For negative controls, all the procedures were performed in the same manner without the primary antibodies. The tissue was colabeled with 4×6-diamidino-2-phenylindole (DAPI, a nonspecific nuclear marker; Vector Laboratories, Burlingame, CA, USA) and the immunohistochemically positive cells were then quantitatively expressed as a ratio to all DAPI-positive cells. All cell counting was done in a blinded manner to avoid all bias.

BrdU Labeling

BrdU, a thymidine analog that is incorporated into the DNA of dividing cells during S phase, was used for mitotic labeling (50 mg/kg; Roche Diagnostics, Indianapolis, IN, USA). The labeling protocol has been described previously (74). BrdU was administered intraperitoneally daily for 3 consecutive days after TBI. The FPI animals were killed 4 days after TBI for BrdU labeling. BrdU was stained with a specific antibody against BrdU (1:400; Roche Diagnostics) and the quantification of BrdU-immunoreactive cells was performed as has been previously described (74).

Immunohistochemistry

Adjacent 50-μm sections corresponding to coronal coordinates 2.0 to 7.0 mm posterior to the bregma were consecutively incubated in 2 mol/L HCl (Sigma) for 30 min and rinsed with 0.1 mol/L boric acid (Sigma) (pH 8.5) at room temperature for 3 min: primary antibodies were incubated in PBS containing 0.5% normal bovine serum (Invitrogen) at 4°C overnight; secondary antibodies were incubated at room temperature for 1 h. The antibodies therein were sequentially rat monoclonal anti-BrdU antibody (1:10; Oxford Biotechnology, Oxfordshire, UK), mouse anti-VEGF (Chemicon, Temecula, CA, USA), mouse anti-GDNF (Chemicon), anti-neuronal-specific nuclear protein (NeuN, Abcam), or anti-HNA (MAB1281, all antibodies at 1:200 dilution unless specified otherwise; Chemicon), followed by FITC-conjugated goat anti-mouse secondary antibody (Alexa-Fluor^® 568; Life Technologies, Grand Island, NY, USA) for detecting the sections. DAPI staining (Molecular Probes, Eugene, OR, USA) was performed to visualize nucleated cells. For endothelial cells, biotinylated Lycopersicon esculentum (tomato) lectin (1:200; Vector Laboratories) was used in the same manner instead of the other primary antibodies and visualized using fluorescein-labeled streptavidin (1:200; Vector Laboratories). For determination of caspase-3-positive cells, cortical sections were serially incubated with the cleaved caspase-3 (ASP/75) antibody (1:200; Cell Signaling Technology, Inc., Beverly, MA, USA), which only recognized the large fragment of activated caspase-3 (17–20 kDa), in 3% bovine serum albumin (BSA; Invitrogen)/PBS/0.1% Triton X-100 (TX-100; Sigma) at 4°C overnight; washed in PBS/0.6% TX-100 and 1:200 FITC-conjugated anti-rabbit IgG incubated in 1% BSA/PBS/0.1% TX-100 for 60 min; and mounted using antifade mounting media (Sigma). The labeled cells were calculated in five coronal sections from each rat and expressed as the mean number of cells per section. For negative control sections, all the procedures were without the primary antibodies.

Images of the fluorescent immunohistochemistry for immune cells were captured at 100× magnification using a fluorescence microscope system (Zeiss Axiovision; Zeiss Gmbh, Göttingen, Germany), and images from bregma levels −2.0, −2.5, −3.0, and −3.5 mm from each animal were evaluated. In each image, immune-positive cells showing staining with a cellular morphology and above background levels were manually and exhaustively counted using the Axiovision image analysis software (Zeiss Gmbh). All cell counts were performed by an investigator (M.T.L.) blinded to the treatment status of each animal. In each image at all evaluated levels, the regions of interests (ROIs) at 100× magnification were evaluated bilaterally in the perilesioned cortex. The ROIs were evaluated in brain-injured animals and compared with the ROIs ipsilateral to the injury in sham-injured animals.

Cytokine Assay

The concentrations of TNF-α, ICAM-1, and IL-10 in the serum were determined using double-antibody sandwich enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions.

Statistical Analysis

All measurements in this study were performed blindly. The data are presented as mean ± standard deviation (SD). Repeated-measures two-way analysis of variance was used for comparison of hemodynamic data of the groups over time (Kolmogorov-Smirnov test). Analyses for all behavioral and histochemical variables used Student's unpaired t tests to compare variables between groups. Bonferroni's analysis was then performed when appropriate to determine post hoc significance at individual time points. A value of p < 0.05 was considered significant in all cases.

Results

CD34⁺ Cells Attenuated Both Intracranial Hypertension and Decreased CPP During FPI

Compared to the sham-operated controls, the FPI rats treated with PBS (that contained 0% CD34⁺ cells) or the FPI rats treated with 0.2% CD34⁺ cells had higher values of ICP but lower values of CCP during the acute stage of FPI (Table 1 and Fig. 1A). However, the FPI rats treated with 95% CD34⁺ cells had significantly lower values of ICP but higher levels of CPP during FPI than did the FPI rats treated with 0.2% CD34⁺ cells or PBS (Table 1 and Fig. 1A).

Figure 1.

Physiologic and biochemical attributes of the FPI rats. Mean ± SD values of (A) mean arterial pressure (MAP), heart rate (HR), intracranial pressure (ICP), and cerebral perfusion pressure (CPP) and (B) body core temperature (T_co) and the hippocampal levels of nitric oxide metabolites (NO_x), glutamate, glycerol, and lactate-pyruvate ratio for the sham-operated rats (●; n = 10), the FPI rats treated with 0.2% CD34⁺ cells (○; n = 10), and the FPI rats treated with 95% CD34⁺ cells (▼; n = 10). *p<0.05, compared with the (●) group; +p <0.05, compared with the FPI + PBS group (Δ, A; ∇, B). FPI, fluid percussion injury. The FPI rats treated with PBS do not have similar physiologic and biochemical responses as the sham controls.

Table 1.

The Behavioral and Histochemical Characteristics for the Sham-Operated Rats, the FPI Rats Treated With PBS (FPI + PBS), the FPI Rats Treated With 0.2% CD34⁺ Cells (FPI + 0.2% CD34⁺), and the FPI Rats Treated With 95% CD34⁺ Cells (FPI + 95% CD34⁺)

	Animal Groups
Parameters	Sham	FPI + PBS	FPI + 0.2% CD34⁺	FPI + 95% CD34⁺
1. ICP (mmHg)	6 ± 1 (10)	24 ± 4 (5)	22 ± 5 (10)^*	6 ± 1 (10)^†
2. Cerebral perfusion pressure (mmHg)	72 ± 4 (10)	51 ± 7 (5)	56 ± 8 (10)^*	79 ± 4 (10)^†
3. Glutamate (μmol/L)	6 ± 2 (10)	16 ± 2 (5)^*	15 ± 1 (10)^*	6 ± 2 (10)^†
4. Nitric oxide (μmol/L)	2 ± 1 (10)	20 ± 2 (5)^*	18 ± 2 (10)^*	2 ± 1 (10)^†
5. Glycerol (μmol/L)	10 ± 1 (10)	30 ± 2 (5)^*	29 ± 1 (10)^*	10 ± 1 (10)^†
6. Lactate/pyruvate	18 ± 1 (10)	58 ± 2 (5)^*	60 ± 1 (10)^*	18 ± 1 (10)^†
7. NSS	0 ± 0 (10)	9.4 ± 0.8 (5)^*	9.3 ± 0.7 (10)^*	7.0 ± 0.6 (10)^†
8. Maximal angle (degrees)	61 ± 1 (10)	30 ± 2 (5)^*	34 ± 3 (10)^*	43 ± 3 (10)^†
9. Infarction volume (mm³): TTC	0 ± 0 (10)	180 ± 31 (5)^*	175 ± 26 (10)^*	22 ± 4 (10)^†
10. Numbers of TUNEL-positive cells (per section)	0 ± 0 (10)	379 ± 25 (5)^*	385 ± 26 (10)^*	76 ± 17 (10)^†
11. Numbers of caspase-3-positive cells (per section)	5 ± 2 (10)	184 ± 22 (5)^*	175 ± 21 (10)^*	74 ± 7 (10)^†
12. Numbers of VEGF-positive cells (per section)	2 ± 1 (10)	2 ± 1 (5)	2 ± 1 (10)	130 ± 18 (10)^†
13. Numbers of BrdU/endothelial cells (per section)	1 ± 1 (10)	1 ± 1 (5)	1 ± 1 (10)	8 ± 2 (10)^†
14. Numbers of BrdU/NeuN double-positive cells (per section)	2 ± 1 (10)	2 ± 1 (5)	2 ± 1 (10)	47 ± 1 (10)^†
15. Numbers of HNA/GDNF-positive cells (per section)	2 ± 1 (10)	2 ± 1 (5)	2 ± 1 (10)	118 ± 23 (10)^†
16. HNA-positive cells/mm²	0 ± 0 (10)	0 ± 0 (10)	0 ± 0 (10)	15 ± 5 (10)^†
17. TNF-α (pg/ml)	11 ± 2 (10)	290 ± 32 (5)^*	275 ± 29 (10)^*	64 ± 6 (10)^†
18. ICAM-1 (pg/ml)	9 ± 1 (10)	378 ± 37 (5)^*	366 ± 34 (10)^*	23 ± 4 (10)^†
19. IL-10 (pg/ml)	12 ± 1 (10)	17 ± 2 (5)^*	16 ± 2 (10)^*	338 ± 8 (10)^†
20. EPC expression	0 ± 0 (10)	7 ± 2 (5)^*	8 ± 2 (10)^*	45 ± 3 (10)^†

Data are means ± SD followed by the numbers of animals in parentheses. Data 7–19 were obtained 1–7 days after FPI; data 1–6 were obtained within 120 min after FPI. TTC, triphenyltetrazolium chloride; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; VEGF, vascular endothelial growth factor; BrdU, 5-bromodeoxyuridine; NeuN, nuclear antigen; HNA, human nuclear antigen; GDNF, glial cell-derived growth factor; TNF, tumor necrosis factor; ICAM-1, intercellular adhesion molecule-1; IL-10, interleukin 10; EPC, endothelial precursor cell.

p < 0.05 in comparison with sham controls;

†

p < 0.05 in comparison with FPI+0.2% CD34⁺ group.

CD34⁺ Cells Attenuated Cerebral Markers of Both Ischemia and Tissue Damage During FPI

Compared to the sham-operated controls, the FPI rats treated with PBS or 0.2% CD34⁺ cells had significantly higher cellular levels of both ischemia markers (such as glutamate and lactate-to-pyruvate ratio) and damage markers (such as glycerol and NO_x) in the brain tissue (Table 1 and Fig. 1B). The FPI rats treated with 95% CD34⁺ cells had significantly lower cellular levels of both ischemia and damage markers than did the FPI rats treated with 0.2% CD34⁺ cells or PBS (Table 1 and Fig. 1B).

CD34⁺ Cells Reduced Neurological and Motor Dysfunction During FPI

Behavioral tests revealed that the 0.2% CD34⁺ cell-treated or PBS-treated FPI animals performed significantly less well in both neurological and motor (Fig. 2 and Table 1) function tests than did the sham-operated controls evaluated at 1–7 days after FPI injury. However, the FPI rats treated with 95% CD34⁺ cells performed significantly better in both neurological and motor function tests than did the FPI rats treated with 0.2% CD34⁺ cells or PBS (Table 1 and Fig. 2).

Figure 2.

Behavioral testing of rats after FPI. Means ± SD values of (A) neurological severity scores (NSS) and means ± standard error value of the (B) maximal angle animals could cling to an inclined plane from sham controls (□; n = 10), FPI rats treated with PBS (; n = 5), FPI rats treated with 0.2% CD34⁺ cells (; n = 10), FPI rats treated with 50% CD34⁺ cells (; n = 10), and FPI rats treated with 95% CD34⁺ cells (; n = 10). The NSS and the inclined plane tests were measured on all rats preinjury and on days 1, 4, and 7 after FPI. *p<0.05 compared with the sham group; #p<0.05 compared with the FPI + 0.2% CD34⁺ cells group.

CD34⁺ Cells Reduced Infarction Volume During FPI

The TTC staining data revealed that 4 days after FPI, the PBS-treated or the 0.2% CD34⁺ cell-treated FPI animals had significantly higher TTC staining volume than did the sham-operated controls (Fig. 3 and Table 1). However, the FPI rats treated with 95% CD34⁺ cells had significantly lower infarction volume during FPI than did the FPI rats treated with 0.2% CD34⁺ cells or PBS (Table 1 and Fig. 3).

Figure 3.

Brain tissue infarct volumes 4 days post-FPI. Representative pictures of triphenyltetrazolium chloride (TTC) staining from a sham FPI rat (A), an FPI rat treated with PBS (B), an FPI rat treated with 0.2% CD34⁺ cells (C), and an FPI rat treated with 95% CD34⁺ cells (D) are shown. (E) Infarction volume of brain tissue from sham controls (□; n = 10), the 4 days post-FPI rats treated with PBS (; n = 5), the 4 days post-FPI rats treated with 0.2% CD34⁺ cells (; n = 10), the 4 days post-FPI rats treated with 50% CD34⁺ cells (; n = 10), and the 4 days post-FPI rats treated with 95% CD34⁺ cells (; n = 10). *p<0.05 compared with the sham controls; #p<0.05 compared with the FPI + PBS group or the FPI + 0.2% CD34⁺ group.

CD34⁺ Cell Therapy Reduced Cerebral Neuronal Apoptosis During FPI

Numbers of both the TUNEL-positive cells (Table 1 and Fig. 4) and the caspase-3-positive cells (Table 1 and Fig. 5) in the brain tissue of the FPI rats treated with 0.2% CD34⁺ cells or PBS were significantly higher than those of the sham-operated controls. Compared to those of the FPI rats treated with 0.2% CD34⁺ cells or PBS, the FPI rats treated with 95% CD34⁺ cells had significantly lower numbers of both the TUNEL-positive cells and the caspase-3-positive cells in the brain tissues (Figs. 4 and 5).

Figure 4.

Hippocampal TUNEL staining of FPI rats. TUNEL staining of hippocampal tissue from sham controls (□; n = 10), the 4 days post-FPI rats treated with PBS (FPI + PBS, ; n = 5), post-FPI rats treated with 0.2% CD34⁺ cells (FPI + 0.2% CD34⁺ cells, ; n = 10), and the 4 days post-FPI rats treated with 95% CD34⁺ cells (FPI + 95% CD34⁺) (; n = 10). *p<0.05 compared with the sham group; #p<0.05 compared with the FPI + 0.2% CD34⁺ group. Top: representative brain TUNEL staining picture from a sham rat, an FPI + PBS rat, an FPI + 0.2% CD34⁺ rat, and an FPI + 95% CD34⁺ rat.

Figure 5.

Caspase-3 staining of the FPI rat hippocampus. Immunohistochemical staining of caspase-3 cells of hippocampal tissue from sham controls (□; n = 10), the 4 days post-FPI rats treated with PBS (FPI + PBS; ; n = 5), the 4 days post-FPI rats treated with 0.2% CD34⁺ cells (FPI + 0.2% CD34⁺; ; n = 10), and the 4 days post-FPI rats treated with 95% CD34⁺ cells (FPI + 95% CD34⁺; ; n = 10). *p<0.05 compared with the sham group; #p<0.05 compared with the FPI + PBS group or the FPI + 0.2% CD34⁺ group. Top: representative caspase-3 staining picture from a sham rat, an FPI + PBS rat, an FPI + 0.2% CD34⁺ rat, and an FPI + 95% CD34⁺ rat.

CD34⁺ Cell Therapy Improved Angiogenesis During FPI

Numbers of both BrdU-endothelial double-positive cells (Table 1 and Fig. 6) and VEGF-positive cells (Table 1 and Fig. 7) in brain tissue of the 0.2% CD34⁺ cell-treated or PBS-treated FPI rats were insignificantly different from those of the sham-operated controls. However, the numbers of both the BrdU-positive endothelial cells and VEGF-positive cells in the brain tissue of the FPI rats treated with 95% CD34⁺ cells were significantly higher than those of the FPI rats treated with 0.2% CD34⁺ cells or PBS (Table 1 and Figs. 6 and 7).

Figure 6.

BrdU staining of endothelial cells in FPI rat hippocampus. Immunohistochemical staining of 5-bromodeoxyuridine (BrdU)-positive endothelial cells of hippocampal tissue from sham controls (□; n = 10), the 4 days post-FPI rats treated with PBS (FPI + PBS; ; n = 5), the 4 days post-FPI rats treated with 0.2% CD34⁺ cells (FPI + 0.2% CD34⁺; ; n = 10), and the 4 days post-FPI rats treated with 95% CD34⁺ cells (FPI + 95% CD34⁺; ; n = 10). #p<0.05 compared with the FPI + PBS group or the FPI + 0.2% CD34⁺ group. Top: representative BrdU-endothelial cell staining from a FPI + 95% CD34⁺ rat. Green, endothelium; red, BrdU; orange, merged. Biotinylated L. esculentum (tomato) lectin was used to label the endothelium.

Figure 7.

VEGF staining of FPI rat hippocampus. Immunohistochemical staining of VEGF-positive cells of hippocampal tissue from sham controls (□; n = 10), the 4 days post-FPI rats treated with PBS (FPI + PBS; ; n = 5), the 4 days post-FPI rats treated with 0.2% CD34⁺ cells (FPI + 0.2% CD34⁺; ; n = 10), and the 4 days post-FPI rats treated with 95% CD34⁺ cells (FPI + 95% CD34⁺; ; n = 10). #p<0.05 compared with the FPI + PBS group or the FPI + 0.2% CD34⁺ group. Top: representative pictures from a sham rat, an FPI + 95% CD34⁺ rat.

CD34⁺ Cell Therapy Improved Neurogenesis During FPI

Numbers of both the BrdU/NeuN double-labeled cells (Table 1 and Fig. 8) and the HNA/GDNF double-labeled cells (Table 1 and Fig. 9) in the brain tissue of the 0.2% CD34⁺ cell-treated or PBS-treated FPI rats were insignificantly different from those of the sham-operated controls. However, numbers of both the BrdU/NeuN double-labeled cells and the HNA/GDNF double-positive cells in the brain tissue of the FPI rats treated with 95% CD34⁺ cells were significantly higher than those of the FPI rats treated with 0.2% CD34⁺ cells or PBS (Table 1).

Figure 8.

BrdU/NeuN double staining of FPI rat hippocampus. Immunohistochemical staining of BrdU/neuronal nuclei (NeuN) double-positive cells of hippocampal tissue from sham controls (□; n = 10), the 4 days post-FPI rats treated with PBS (FPI + PBS; ; n = 5), the 4 days post-FPI rats treated with 0.2% CD34⁺ cells (FPI + 0.2% CD34⁺; ; n = 10), and the 4 days post-FPI rats treated with 95% CD34⁺ cells (FPI + 95% CD34⁺; ; n = 10). #p<0.05, compared with the FPI rats treated with PBS or the FPI rats treated with 0.2% CD34⁺ cells. Top: representative pictures from a sham rat, an FPI + PBS rat, an FPI + 0.2% CD34⁺ rat, and an FPI + 95% CD34⁺ rat.

Delivered CD34⁺ Cells Were Localized to FPI-Injured Area as Determined by Using Anti-Human Nuclei Antibody

HNA-positive cells were detected in the FPI-injured brain area by HNA/DAPI/GDNF triple staining (Fig. 9) of the FPI rats treated with 95% CD34⁺ cells only. This indicated that CD34⁺ cells could reach the injured brain region 4 days post-FPI after transplantation of 95% CD34⁺ cells.

Figure 9.

HNA/GDNF double staining of FPI rat hippocampus. Immunohistochemical staining of human nuclear antigen/glial cell-derived neurotrophic factor (HNA/GDNF) double-positive cells of hippocampal tissue from sham controls (□; n = 10), the 4 days post-FPI rats treated with PBS (FPI + PBS; ; n = 5), the 4 days post-FPI rats treated with 0.2% CD34⁺ cells (FPI + 0.2% CD34⁺; ; n = 10), and the 4 days post-FPI rats treated with 95% CD34⁺ cells (FPI + 95% CD34⁺; ; n = 10). #p<0.05 compared with the FPI + 0.2% CD34⁺ cells group or the FPI + PBS group. Top: representative pictures from a sham rat, an FPI + PBS rat, an FPI + 0.2% CD34⁺ rat, and an FPI + 95% CD34⁺ rat.

CD34⁺ Cell Therapy Improved Inflammation During FPI

In the FPI rats treated with 0.2% CD34⁺ cells or PBS, the concentrations of both TNF-α and ICAM-1 (Table 1) in the serum were significantly higher than those of the sham-operated controls. However, the serum levels of TNF-α and ICAM-1 in the FPI rats treated with 95% CD34⁺ cells were significantly lower than those of the FPI rats treated with 0.2% CD34⁺ cells or PBS (Table 1). Table 1 also shows that the concentrations of IL-10 in the serum of the 95% CD34⁺ cell-treated FPI rats were significantly higher than those of the sham-operated controls, the PBS-treated FPI rats, or the 0.2% CD34⁺ cell-treated FPI rats.

CD34⁺ Cell Therapy Increased Circulating Levels of EPCs During FPI

Compared to the sham-operated controls, the FPI rats treated with 0.2% CD34⁺ cells or PBS (0% CD34⁺ cells) had significantly higher serum levels of EPCs (Table 1). Additionally, the serum levels of EPCs of the 95% CD34⁺ cell-treated FPI rats were further significantly higher than those of the FPI rats treated with 0.2% CD34⁺ cells or PBS (Table 1).

Discussion

The FPI model of TBI in the rodent is currently the most commonly used and well-characterized preclinical model of TBI (30). Long-term investigations have detected persistent neurological motor function impairment up to 1 year after severe lateral FPI (52). Indeed, as shown in the present study, 4 days post-FPI, rats displayed neurological motor dysfunction, cerebral contusion and neuronal apoptosis (e.g., increased numbers of both the TUNEL-positive cells and the caspase-3-positive cells), and activated inflammation (e.g., increased serum levels of both TNF-α and ICAM-1). In the present study, we demonstrated that HNA-positive cells could be detected in the ischemic brain tissues of FPI rats treated with the 95% CD34⁺ cells, but not the 0.2% CD34⁺ cells or the PBS (that contained 0% CD34⁺ cells). This strongly indicates that systemic delivery of appropriate dosages of CD34⁺ cells are able to pass through the blood–brain barrier and to migrate into the perilesioned parenchyma of the brain tissue.

Although CD34⁺ cells were localized to the FPI-injured brain area, it still cannot prove CD34⁺ cell function in its recovery. Mesenchymal stem cells (MSCs) are adult multipotent cells found in bone marrow and other adult tissues. MSCs derived from donor rats (37,40) or humans (27,39,41) have been shown to improve outcome in the rat model of TBI. As only a small proportion of transplanted MSCs appear to differentiate into neurons and astrocytes, tissue repair by MSCs may not be the main source of functional recovery (39,41). It has been proposed that in addition to cellular replacement, an increased production of neurotrophic growth factors following administration of MSCs may be among the possible mechanisms of MSC-induced functional recovery (27). Therefore, it is possible that an increased production of GDNF, VEGF, and IL-10 following administration of CD34⁺ cells in the ischemic brain may protect neurons from injury and promote endogenous repair. In in vitro studies, several groups detected the synthesis of various growth factors, including nerve growth factor, brain- derived neurotrophic factor, GDNF, and VEGF (2,6,11). More recently, we demonstrated that systemically delivering normoxia or hypoxia preconditioned human MSC secretomes effectively and potently inhibited brain damage and functional impairment in TBI rats (2,11). The paracrine mechanisms mediated by factors secreted from HUCB-derived CD34⁺ cells may be different from those of MSCs. Additionally, MSCs were cultured with TBI extracts of rat brain in vitro and quantitative sandwich enzyme-linked immunosorbent assays (ELISAs) were performed (9). TBI-conditioned MSC cultures demonstrated a time-dependent increase of various growth factors, indicating a responsive production of these growth factors by the MSCs. In studies in mice, after intraventricular injection of MSCs, nerve growth factors were increased significantly in cerebrospinal fluid by ELISA, confirming their cell cultures (9). Putting these observations together, it can be inferred that transplanted CD34⁺ cells may provide therapeutic benefit in TBI rats via a responsible secretion of an array of growth factors that can foster neuroprotection and angiogenesis.

In addition, we showed that FPI-induced neurological motor dysfunction, cerebral contusion and neuronal apoptosis, and activated inflammation were significantly attenuated by 50% or 95% CD34⁺ cell therapy, but not by 0.2% CD34⁺ cells or PBS therapy. Furthermore, CD34⁺ (95%) cell therapy may improve outcome of TBI by promoting neurogenesis (e.g., increased numbers of both GDNF-positive cells and BrdU/NeuN double-positive cells), angiogenesis (e.g., increased numbers of both VEGF-positive cells and BrdU/endothelial double-positive cells), and migration of EPCs from the bone marrow. Both previous (38) and present results suggest that human umbilical cord blood-derived CD34⁺ cells can be potentially a therapeutic agent for treating TBI.

It was found that FPI caused increased levels of the cellular ischemia markers (e.g., glutamate and lactate/ pyruvate ratio) (1,21,22), the injury marker (e.g., glycerol) (22), and the toxic oxidizing radical like nitric oxide metabolites (NO_x) (69) as well as cerebral contusion. Although the posttraumatic rise in cellular ischemia and injury markers were presented during the short period of 120 min in our study, intracerebral microdialysis studies performed on human head-injured patients at the bedside demonstrated that glutamate concentrations were elevated for an extended period of time (days) following clinical head injury (73). Furthermore, we showed that the FPI-induced intracranial hypertension, cerebral hypoperfusion, cerebral infarction, and motor dysfunctions were all suppressed by CD34⁺ cell (95%) therapy adopted immediately after FPI.

In this study, the amounts of both BrdU/endothelial double-positive cells and VEGF-positive cells in the perilesioned brain regions were significantly increased following CD34⁺ cell (95%) therapy, suggesting that CD34⁺ cell therapy might promote an environment assisting to revascularize lesioned brain regions to proceed to neuronal regeneration (23,63). A rich vascular environment along with VEGF generation might enhance subsequent angiogenesis and neurogenesis. In addition, both the BrdU-labeled cells that expressed NeuN (a post-mitotic neuron-expressed nuclear protein) and the GDNF-positive cells increased significantly 4 days post-FPI in the 95% CD34⁺ cell-treated FPI rats, indicating that CD34⁺ cell therapy might stimulate the neuronal proliferation in the perilesioned brain regions. However, it is not known whether the CD34⁺ cells prevented neuronal loss during FPI via the proliferated cells generated from the hippocampus and subventricular zone (17,55) that migrated and differentiated in the perilesioned brain regions or whether the activation of the latent endogenous neural progenitor cells occurred at the sites of injury. Our present results show that the number of the HNA/GDNF/DAPI-positive cells in the ischemic brain tissue during FPI is significantly increased following 95% CD34⁺ cell therapy. This indicates that CD34⁺ cells of exogenous origin may improve the outcome of FPI by stimulating both endogenous angiogenesis and neurogenesis in the injured brain. The contention is supported by several investigations. For example, HUCBCs can integrate into brain parenchyma and serve as a source of trophic factors (16,33,72). HUCBCs integrating into the vascular structure might increase angiogenesis after FPI and subsequently promote improvement of the neurological function (37). Postnatal vasculogenesis was considered to be involved in neovascularization of adult tissues because bone marrow-derived EPCs isolated from circulating mononuclear cells in peripheral blood were shown to incorporate into sites of physiological and pathological neovascularization and to differentiate into mature endothelial cells (46). Indeed, our present study for the treatment of FPI-induced brain ischemia with CD34⁺ cells showed that CD34⁺ cells increased the endogenous EPC expression in the blood. These bone marrow-derived EPCs might incorporate into the sites of neovascularization in the lesioned brain.

Several studies demonstrate that systemic administration of human CD34⁺ cells to mice subjected to stroke induces neovascularization in the ischemic zone and provides a favorable environment for neuronal regeneration (63). Such a rich vascular environment, along with generation of other nurturing neuronal mediators by CD34⁺ cells (such as VEGF) enhances subsequent neuronal regeneration (14,47). In contrast, in the presence of an antiangiogenic agent, the beneficial effects of CD34⁺ cells were lost (63).

Proapoptotic effects and apoptosis caused by inflammatory cytokines is the important pathway of secondary damage after FPI (25,50,56,65). Neurophils may contribute to secondary neuronal death by releasing proinflammatory cytokines and free oxygen radicals (31,45). As shown in the present results, increased serum production of systemic inflammatory response molecules like TNF-α and ICAM-1 and toxic oxidizing indicators like NO_x^– occurred during FPI. The serum TNF-α and ICAM-1 levels can be considered as markers for the systemic inflammatory response because they indirectly reflect the whole-body production of TNF-α and ICAM-1 in various organs (5,29,49,70). NO_x is believed to be a toxic oxidizing radical (22). Increased serum levels of TNF-α, ICAM-1, and NO_x may contribute to the neuronal apoptosis that occurred during FPI. In the rat model of FPI, our present study demonstrated that CD34⁺ cell therapy decreased proinflammatory cytokine production (such as TNF-α and ICAM-1), decreased toxic oxidizing radical production (such as NO_x^–), increased anti- inflammatory cytokine production (such as IL-10), reduced brain apoptosis, and improved motor dysfunction. It should be stressed that inflammatory responses in the initial phase of FPI might have a role in aggravating brain damage, whereas in the later stages, these inflammatory mediators might contribute to recovery or repair (24).

After experimental TBI in mice, tumor protein p53 (p53) rapidly accumulated in the injured brain tissue and was translocated to the nucleus of damaged neurons, whereas NF-kB (nuclear factor k-light-chain enhancer of activated B cells) transcriptional activity simultaneously declined (53). Posttraumatic neurodegeneration correlated with the increase in p53 levels and was significantly reduced by the selective p53 inhibitor pifithrin-α. Inhibition of p53 activity resulted in the concomitant increase in NF-kB transcriptional activity and upregulation of NF-kB target proteins. In future studies, we should investigate whether TUNEL-positive and caspase-3-positive cells are p53 dependent and/or p53 independent in 95% CD34⁺ treatment cells.

In summary, our data show that the systemic delivery of human umbilical cord blood-derived CD34⁺ cells reduced functional deficits after severe FPI in the rat. The injected CD34⁺ cells entered the brain, migrated into the injured area, and reduced cerebral infarction and apoptosis by stimulating both angiogenesis and neurogenesis, inhibiting production of systemic inflammatory response molecules, and promoting migration of EPCs from the bone marrow. Thus, our results revealed a potential for human cord blood-derived CD34⁺ cells used as a therapeutic agent for brain FPI.

Footnotes

Acknowledgments

This study was funded in part by the National Science Council of the Republic of China (Grant Nos. NSC100-2314-B-218-001, NSC 102-2314-B-218-001-MY2, and NSC99-2314-B-218-006-MY2) and the Department of Health of the Republic of China (DOH99-TD-B-111-003, Center of Excellence for Clinical Trial and Research in Neuroscience). The authors declare no conflict of interest.

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Umbilical Cord Blood-Derived CD34 + Cells Improve Outcomes of Traumatic Brain Injury in Rats by Stimulating Angiogenesis and Neurogenesis

Abstract

Keywords

Introduction

Materials and Methods

Animals

Surgery

Physiological Parameters

Microdialysis Determination of the Hippocampal Glutamate, Lactate-to-Pyruvate Ratio, Glycerol, and Nitric Oxide Metabolites (NOx)

Cell Viability

Human CD34+ Cell Preparation

Experimental Groups

Experimental Procedures

Neurological Function Evaluation

Isolation of EPCs

Cerebral Infarction Assessment

TUNEL Assay

BrdU Labeling

Immunohistochemistry

Cytokine Assay

Statistical Analysis

Results

CD34+ Cells Attenuated Both Intracranial Hypertension and Decreased CPP During FPI

CD34+ Cells Attenuated Cerebral Markers of Both Ischemia and Tissue Damage During FPI

CD34+ Cells Reduced Neurological and Motor Dysfunction During FPI

CD34+ Cells Reduced Infarction Volume During FPI

CD34+ Cell Therapy Reduced Cerebral Neuronal Apoptosis During FPI

CD34+ Cell Therapy Improved Angiogenesis During FPI

CD34+ Cell Therapy Improved Neurogenesis During FPI

Delivered CD34+ Cells Were Localized to FPI-Injured Area as Determined by Using Anti-Human Nuclei Antibody

CD34+ Cell Therapy Improved Inflammation During FPI

CD34+ Cell Therapy Increased Circulating Levels of EPCs During FPI

Discussion

Footnotes

Acknowledgments

References

Microdialysis Determination of the Hippocampal Glutamate, Lactate-to-Pyruvate Ratio, Glycerol, and Nitric Oxide Metabolites (NO_x)

Human CD34⁺ Cell Preparation

CD34⁺ Cells Attenuated Both Intracranial Hypertension and Decreased CPP During FPI

CD34⁺ Cells Attenuated Cerebral Markers of Both Ischemia and Tissue Damage During FPI

CD34⁺ Cells Reduced Neurological and Motor Dysfunction During FPI

CD34⁺ Cells Reduced Infarction Volume During FPI

CD34⁺ Cell Therapy Reduced Cerebral Neuronal Apoptosis During FPI

CD34⁺ Cell Therapy Improved Angiogenesis During FPI

CD34⁺ Cell Therapy Improved Neurogenesis During FPI

Delivered CD34⁺ Cells Were Localized to FPI-Injured Area as Determined by Using Anti-Human Nuclei Antibody

CD34⁺ Cell Therapy Improved Inflammation During FPI

CD34⁺ Cell Therapy Increased Circulating Levels of EPCs During FPI