Summary

This study was designed to evaluate potential therapeutic value of intracerebral transplantation of cultured Notch-induced human bone marrow-derived mesenchymal stromal cells (MSCs) (referred to as SB623, supplied by SanBio Inc.) [26,28] in an animal model of TBI. Transplantation was carried out at 7 days after TBI with functional readouts of behavioral and histological deficits conducted during the subsequent three month period after TBI. We characterized locomotor and neurological performance at baseline (prior to TBI), then at 7 days after TBI (prior to transplantation), and monthly thereafter up to three months after TBI. Following completion of behavioral testing at one month or three months after TBI, animals were euthanized by transcardial perfusion and brains harvested to histologically characterize the extent of brain damage. The stem cell engraftment and host tissue endogenous repair mechanisms (e.g., increased host cell survival in peri-TBI lesion area) were examined by immunohistochemical analyses. A total of 40 animals identified at baseline (prior to TBI surgery) as exhibiting normal behaviors (EBST: 50-60% bias swing activity; Rotorod: 60 seconds staying time on rotating rod; Bederson: at most 0-0.5 mean neurologic score), received TBI surgery as described below. Only TBI animals reaching the criterion of behavioral impairment (EBST: at least 75% bias swing activity; Rotorod: 30 seconds or less staying time on rotating rod; Bederson: at least 2.5 mean neurologic score) were randomly assigned to either SB623 transplants (n=20) or vehicle infusion (n=20) on Day 7 post-TBI. All animals were monitored monthly post-grafting for behavioral outcomes. Randomly selected animals were euthanized at one month (n=10 per group) post-TBI, and the remaining animals euthanized at three months post-TBI by transcardial perfusion with 4% paraformaldehyde. For outcome measures, transplant outcome were evaluated using the following parameters: 1) locomotor behavior via elevated body swing test (EBST) and Rotorod; (2) neurological performance via a Bederson-modified neurological examination; 3) lesion volume via hematoxylin and eosin (H&E) histologic stains; 4) graft survival via immunohistochemistry using specific antibody shown to detect human cells, and; 5) mechanism-based immunohistochemial analyses of neuroprotection and/or regeneration using antibodies directed against the grafted human cells and host cells.

Subjects

The University of South Florida Institutional Animal Care and Use Committee approved all procedures used in this study. Animals had free access to food and water, and all were housed under normal conditions (20°C, 50% relative humidity, and a 12 hour light/dark cycle).

TBI surgery

All surgical procedures were conducted under aseptic conditions. Adult male Sprague-Dawley (SD) rats (8-weeks old) were anesthetized with 1.5% isofluorane and checked for pain reflexes. Under deep anesthesia, animals underwent the moderate TBI model. Each animal was placed in a stereotaxic frame (anesthesia maintained via gas mask) with 1-2% isoflurane. After exposing the skull, a 4.0 mm craniectomy was performed over the left frontoparietal cortex (center at −2.0 mm anteroposterior (AP) and +2.0 mm mediolateral (ML) to bregma) [29]. A pneumatically operated metal impactor (diameter = 3.0 mm) impacted the brain at a velocity of 6.0 m/s reaching a depth of 1.0 mm below the dura mater layer and remained in the brain for 150 milliseconds. The impactor rod was angled 15° to the vertical to be perpendicular to the tangential plane of the brain curvature at the impact surface. A linear variable displacement transducer (Macrosensors, Pennsauken, NJ) connected to the impactor measured velocity and duration to verify consistency. After controlled cortical impact injury, the incision was sutured after bleeding ceased. An integrated heating pad and rectal thermometer unit with feedback control allowed maintenance of body temperature at normal limits. All animals were monitored until recovery from anesthesia. In addition, animals were weighed and observed daily for the next three consecutive days following TBI surgery, weighed twice a week thereafter, and monitored daily for health status and any signs that indicate problems or complications throughout the study.

Grafting procedures

All surgical procedures were conducted under aseptic conditions. Animals were anesthetized with 1.5% isofluorane and checked for pain reflexes. Once deep anesthesia was achieved (by checking for pain reflexes), hair was shaved around the area of surgical incision (skull area) with enough border to prevent contaminating the operative site, followed by two surgical germicidal scrubs of site, and draping with sterile drapes. The animal was then fixed to a stereotaxic apparatus (Kopf Instruments). A 26-gauge Hamilton syringe was then lowered into a small burred skull opening (transplant coordinates were adjusted to correspond with the cortical area adjacent to the core injury site: 0.5 mm anterior and 1.0 mm lateral to bregma and 2.0 mm below the dural surface [29]). Within this single needle pass, 3 deposits of the test article (100,000 cells in 3 µl per deposit or a total of 300,000 cells in 9 µl of Plasmalyte A for 3 deposits) were made. The target area was the medial cortex which corresponded to the peri-injured cortical area, based on previously established target sites for similar stereotaxic implants. Each deposit consisted of 100,000 viable cells in 3 µl volume infused over a period of 3 minutes. Following an additional 2-minute absorption time, the needle was retracted and the wound closed stainless steel wound clip. A heating pad and a rectal thermometer allowed maintenance of body temperature at about 37°C throughout surgery and following recovery from anesthesia.

Behavioral and neurological tests

All investigators testing the animals were blinded to the treatment condition. Animals were subjected to elevated body swing test (EBST), neurological exam, and Rotorod. EBST involved handling the animal by its tail and recording the direction of the swings. The test apparatus consisted of a clear Plexiglas box (40 x 40 x 35.5 cm). The animal was gently picked up at the base of the tail, and elevated by the tail until the animal’s nose was at a height of 2 inches (5 cm) above the surface. The direction of the swing, either left or right, was counted once the animals head moved sideways approximately 10 degrees from the midline position of the body. After a single swing, the animal was placed back in the Plexiglas box and allowed to move freely for 30 seconds prior to retesting. These steps were repeated 20 times for each animal. Intact rats display a 50% swing bias, that is, the same number of swings to the left and to the right. A 75% swing bias indicated 15 swings in one direction and 5 in the other during 20 trials. We have previously utilized the EBST, and noted that unilaterally lesioned animals display >75% biased swing activity at one month after a nigrostriatal lesion or unilateral hemispheric injury; asymmetry is stable for up to six months [3,26]. About one hour after the EBST, a modified Bederson-Neurological exam was conducted following the procedures previously described [3,26] with minor modifications. Neurologic score for each rat was obtained using 3 tests which include (1) forelimb retraction, which measured the ability of the animal to replace the forelimb after it was displaced laterally by 2 to 3 cm, graded from 0 (immediate replacement) to 3 (replacement after several seconds or no replacement); (2) beam walking ability, graded 0 for a rat that readily traversed a 2.4-cm-wide, 80-cm-long beam to 3 for a rat unable to stay on the beam for 10 seconds; and (3) bilateral forepaw grasp, which measured the ability to hold onto a 2-mm-diameter steel rod, graded 0 for a rat with normal forepaw grasping behavior to 3 for a rat unable to grasp with the forepaws. The scores from all 3 tests, which were done over a period of about 15 minutes on each assessment day, were added to give a mean neurologic deficit score (maximum possible score, 9 points divided by 3 tests = 3). After an hour of completion of the neurological exam, the animals were then subjected to the Rotorod test. The Rotorod test involved placement of the animal on an accelerating Rotorod (Accuscan, Inc.) that used a rotating treadmill that accelerates from 4 rpm to 40 rpm over a 60-second period. The total number of seconds maintained on the Rotorod was recorded and used as index of motor coordination. We have previously shown that TBI model animals exhibited significantly shorter time staying on the Rotorod compared to sham operated or normal controls. Animals were subjected to this battery of tests at baseline (prior to TBI), then at 7 days after TBI (prior to transplantation) and monthly thereafter up to three months post-TBI.

Histology

Immunohistochemistry

Free floating sections were processed for immunofluorescent microscopy. Briefly, 40 µm cryostat sectioned tissues were examined at 4X magnification and digitized using a PC-based Image Tools computer program. Brain sections were blind-coded and Abercrombie’s formula was used to calculate the total number of immunopositive cells [3,26]. Cell engraftment index for SB623 was assessed using monoclonal human specific antibody (HuNu) that did not cross-react with rodent proteins. Additional brain sections were processed for mechanism-based immunohistochemical analyses of brain tissue samples focusing on cell proliferation (Ki67), migration (doublecortin or DCX) and immature neural marker (nestin).

Zymography

A separate cohort of animals consisting of TBI plus SB623 cells, TBI plus vehicle, and control-sham operated age-matched adult SD rats (n=3 per group) was subjected to the same experimental paradigm as above, but tissues were processed for zymography, a process involving electrophoretic separation of proteins for assessment of proteolytic activity [30,31]. The tissue corresponding to the biobridge formed by the migrating cells from the SVZ to the impacted cortex was laser captured. After extraction, the tissue was placed in cryotubes and flash frozen in liquid nitrogen. The tubes were stored in a -80°C freezer until homogenization. The samples were homogenized in 450 µL of cold working buffer containing 50 mM Tris-HCl (pH 7.5), 75 mM NaCl, and 1 mM PMSF. The tissue was processed with a homogenizer for 10 minutes and centrifuged at 4°C for 20 minutes at 13000 rpm. The supernatants were separated, frozen and kept at -80°C until use. The total protein concentration was assessed by the Bradford method. On the day of the zymography, the volume equivalent to 50 µg of total protein was loaded into fresh made gelatin zymography gels. The gels were then electrophoretically separated under non-reducing conditions and 100 V. After electrophoresis the zymogram gels were washed in 125 ml 2.5% Triton twice for 20 minutes. The gels were then incubated in activation buffer (Zymogram Development Buffer, Bio-Rad, Hercules, CA) for 20 hours at 37°C. The next day, the gels were stained with Coomassie Blue R-250 Staining Solution (Bio-Rad) for 3 hours and destained for 25 minutes with Destain Solution (Bio-Rad). The gelatinolytic activity of the samples was assessed by densitometric analysis (Gel-Pro v 3.1, Media Cybernetics, Carlsbad, CA) of the bands as a relative comparison to a standard band of recombinant enzyme. To minimize inter-gel variability, all gels had a control lane loaded with 0.5 ng recombinant enzyme, which was used as a standard optical density and enzyme amount (in ng). The lytic bands identified in the zymogram gels were subjected to molecular weight identification with the use of pre-stained standard protein marker (Bio-Rad). Membranes were blocked with blotting grade blocker non-fat dry milk (Bio-Rad). After washing with 0.1% tween 20- tris-buffered saline (TTBS), the membranes were incubated with anti-matrix metalloproteinase (MMP)-9 monoclonal mouse antibody overnight at 4°C. Membranes were washed again in TTBS, incubated with secondary antibody (goat anti-mouse IgG, horseradish peroxidase conjugated antibody, Calbiochem) for one hour and finally developed with horseradish peroxidase development solution (ECL advance detection kit, Amersham). The membranes were exposed to autoradiography films (Hyblot CL, Denville Scientific Inc.). The density of the sample bands for the zymograms was expressed as maximal optical density relative to the standard band.

Cell migration assay

Using a transwell assay, primary rat neuronal cells, PRNCs, (embryos at Day 18; BrainBits) (1×10⁵ cells/well) seeded onto the upper chamber of a Boyden chamber (Costar Transwell assay, Corning, NY, USA) supplemented with NbActive4 (BrainBits) in the absence of antibiotics. The chamber was placed in a 24-well plate containing confluent SB623 cells (1×10⁵ cells/well) and starved with serum-free DMEM/F-12 medium in the presence or absence of Cyclosporine-A (a known MMP-9 inhibitor; 10⁴ ng/mL in dimethyl sulfoxide; Sigma-Aldrich Inc., St Louis, MO, USA) for 24 h in the cell incubator. Next, the upper chamber was removed and wiped clean, then the lower side of the filter was washed and fixed in 4% paraformaldehyde. For quantification, migratory cells that reached the lower chamber and attached to the lower side of the filter were counted from five randomly captured microscopic fields (X400) and averaged for each treatment condition. This migratory assay was performed in triplicates.