All protocols involving the use of animals were in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the University of Texas Health Science Center Institutional Animal Care and Use Committee (AWC-11-120).

Experimental design

Male rats at a starting average weight of 250 g were used in all experiments. Separate group of animals were used for the BBB studies and behavior testing. For BBB, we used four different time points for MAPC treatment. The time points were 2/24, 6/24, 12/36, and 36/72. Similarly, for the behavior testing at 120 days, we had the same MAPC treatment times (2/24, 6/24, 12/36, and 36/72). Animal experiments were performed in cohorts of 15–20 due to logistical consideration, and a positive control difference between sham and cortical contusion injury (CCI) was required in each cohort to be included to ensure the presence of a consistent injury. Animals were randomized to sham, CCI, or a MAPC dose following CCI. Numerous pre-clinical studies have demonstrated the feasibility of MAPC alone; thus, a sham + MAPC was not performed [13].

Controlled cortical injury (CCI)

A controlled cortical impact device (Impact One Stereotaxic Impactor, Leica Microsystems, Buffalo Grove, IL) was used to administer a unilateral brain injury as described previously [30]. Adult male Sprague-Dawley rats weighing 250–300 g were anesthetized with 4% isoflurane and 0 2 , and the head was mounted in a stereotactic frame. The head was held in a horizontal plane, a midline incision was used for exposure, and a 6- to 7-mm craniectomy was performed on the right cranial vault. The center of the craniectomy was placed at the midpoint between bregma and lambda, ~ 3 mm lateral to the midline, overlying the tempoparietal cortex. Animals received a single impact of 2.7 mm depth (cognitive testing) or 3.1 mm depth (BBB) of deformation with an impact velocity of 5.6 m/s and a dwell time of 150 ms (moderate-severe injury) at an angle of 10° from the vertical plane using a 6-mm-diameter impactor tip, making the impact orthogonal to the surface of the cortex. The impact was made to the parietal association cortex. Sham injuries were performed by anesthetizing the animals, making the midline incision, and separating the skin, connective tissue, and aponeurosis from the cranium. The incision was then closed with sterile wound clips. A sham that includes a craniectomy can result in inflammation and anatomical damage that can confound results in a TBI model. Cole et al. demonstrated that a craniectomy resulted in injury that was distinct from the brain injury. In addition, there were changes in pro-inflammatory cytokines, morphological and behavioral damage as well [31]. Therefore, in order to avoid confounding results, we only did a midline incision as a sham.

MAPC preparation and administration

MAPC were obtained from Athersys, Inc. (Cleveland, OH) and stored in liquid nitrogen. Prior to injection, the MAPC were thawed, washed, and suspended in phosphate-buffered saline (PBS) vehicle at a concentration of 10 × 106 cells/kg in 1 ml of PBS (CCl-10). Cells were counted and checked for viability via Trypan blue exclusion. Viability was greater than 90%. Immediately prior to intravenous injection, MAPC (approximately 2.5 million in 1 ml) were titrated gently 8–10 times to ensure a homogeneous mixture of cells and were injected at variable time points after CCI injury via tail vein injection. CCI alone received PBS vehicle injection (1 ml) alone at the same designated time points as the cell-treated animals.

Alexa Fluor 680 dye BBB permeability analysis

Animals that received MAPC at 2/24, 6/24, and 12/36 were euthanized 72 h post injury, and the 36/72 treated group was harvested at 96 h post injury. Prior to euthanasia, rats were anesthetized as described previously and 1 mg/kg of Alexa Fluor 680 dye conjugated to 10 kDa dextran PBS was introduced via tail vein injection [16]. The animals were allowed to recover for 30 min, then euthanized via right cardiac puncture and perfused with ice cold PBS followed by 4% paraformaldehyde. Explanted brains were placed in 4% paraformaldehyde for 1 h and then transferred to PBS. The brains were then sectioned coronally into 1-mm slices using a rat brain matrix slicer (Zivic Instruments, Pittsburgh, PA). Eight anatomically standardized slices encompassing the area of injury were placed on a plastic petri dish using a grid and imaged using an LI-COROdyssey CLx infrared laser scanner (LI-COR, Lincoln, NE) at 700 and 800 nm. Raw images were then stacked, processed, and analyzed in batch using Fiji, the fully open source version of ImageJ 1.48p (http://imagej.nih.gov/ij). Plot profiles were generated for each section along the impactor trajectory to identify the depth and anatomical region corresponding to the maximum Alexa Fluor signal intensity. The one-dimensional plot of Alexa Fluor signal intensity suggested that there may be two- to three-dimensional regions of BBB permeability after TBI corresponding to the injury penumbra. To identify potential regions of BBB permeability, threshold ranges were applied to the stacked raw images to detect various regional patterns of Alexa Fluor signal arising from the brain tissue from the site of cortical impact. Low + narrow signal intensity threshold ranges identified rims surrounding the area of maximal injury (4–5K, 5–7K, and 5–10K) whereas high + wide signal intensity threshold ranges identified the highest foci of signal within the most injured area of the cortex (5–60K). For each rat brain, the area of integrated signal from the Alexa Fluor was plotted against these intensity threshold ranges.

Latency to platform

All behavior testing were done under blinded conditions, with unblinding after all the behavior testing was done. Cognitive function was tested using the Morris water maze (MWM) 120 days after injury to assess spatial memory and spatial learning. Animals were tested using four trials per day, over 6 consecutive days (during each week of testing). Each trial consisted of placing the animal in one of four starting locations (south, east, west, and north) chosen at random. The animal was gently placed in the tank facing the wall and allowed to search for the platform (located in north quadrant) for up to 60 s. If the animal failed to find the platform, it was placed upon the platform and allowed to remain for 30 s. Animal movement within the maze was monitored by a video camera linked to tracking software (Ethovision 3.1). Latency to platform was measured.

Probe

To test memory retention, probe trials were administered 24 h after completion of the platform testing. Probe testing (60 s) involved removal of the platform and, using the tracking software, monitoring the animal movement. Calculations were then completed to determine the time the animal spent in the same quadrant as the platform (north quadrant) and time spent in the area three times the size of the platform (platform proximity duration). In addition, animal velocity was automatically calculated via the tracking software to evaluate for significant motor deficits as a result of injury.

Tissue harvest

Following completion of all behavior testing, the animals were taken to the surgical suite where they were again anesthetized using isoflurane anesthesia. Under anesthesia, the animals’ chests were opened. Using a right ventrical puncture technique, the animals were simultaneously exsanguinated and perfused with 60 ml of ice cold PBS followed by 60 ml of ice cold 4% paraformaldehyde (PFA) at a rate of 20 cm3/min using a syringe pump. Following tissue fixation, the brains were removed and placed in 4% PFA and stored at 4° C.

Immunohistochemistry

After harvest, the brains were transferred to a 30% sucrose solution, where they were maintained for at least 72 h. The brains were then put in a 3% agar mold and sectioned at 30 μm using a vibrating blade microtome (Leica Microsystems, Bannockburn, IL, USA). The sections were stained using a standard free-floating staining protocol. They were washed twice in PBS with 0.01% Triton X-100 [(PBST) T-8787, (Sigma Aldrich, St. Louis, MO, USA)] for 1 min and then incubated for 20–30 min in PBS with 0.2% Triton X-100. The sections were then blocked for 1 h at room temperature (RT) in 3% goat serum (# 005-000-121, Jackson Immune Research, PA) in PBST. A primary antibody was used to identify microglia/macrophages [Anti IBA1 Rabbit (1:100, Wake, Richmond, VA, USA)]. The antibody was prepared in PBTB [PBS with 0.01% Triton X-100, 2% bovine serum albumin (A9647, Sigma Aldrich, St. Louis, MO, USA)] and 1% goat serum and incubated at 4 °C overnight. The next day, the sections were rinsed briefly then washed with PBST and incubated with a secondary antibody [1:500; Goat anti-Rabbit IgG (H+L) Cross-Adsorbed, Alexa Fluor® 568:A 11011, lnvitrogen) in PBTB for 2 h at RT. The sections were again rinsed briefly and mounted, and cover-slipped with Fluoromount-G (Southern Biotechnology Associates, Birmingham, AL). Similar procedures were utilized to identify neurogenic cells [Anti Doublecortin Rabbit (DCX), AB55253, 1:1000 EMD Millipore, Billerica, MA, secondary antibody: 1:500; Goat anti-Rabbit IgG (H+L) Cross-Adsorbed, Alexa Fluor® 568:A 11011, lnvitrogen)].

Quantification of immunohistochemistry

Immunohistochemistry was done after harvesting the brains post behavioral testing. Photomicrographs were taken of the hippocampus at × 200 using a Leica fluorescent microscope (Dm4000B LED). To maintain unbiased stereology [16], all image acquisition was performed by an investigator blinded to treatment of the individual histological sections. A single histological section per animal from mid-injury (interaural 5.70 mm, bregma − 3.30 mm) was examined. The dentate gyrus (DG) of the ipsilateral (right) and contralateral (left) hippocampus were photomicrographed (three random sections per slice) and quantified. Labeled cells were then classified based on morphology: small, static cell body with dynamic and branched processes was classified as inactivated, and cells that had an amoeboid morphology were classified as activated. A separate investigator, who was also blind to the group of each photomicrograph counted and characterized the phenotype of IBA1-positive cells. Images were unblinded after all the photomicrographs were analyzed. For the DCX-positive cells, the entire hippocampus was photomicrographed and counted. We photomicrographed and counted all groups.

Statistical analysis

Unless otherwise indicated, all values are represented as mean ± SEM. Between group comparisons were analyzed using analysis of variance (ANOVA), and if found significant, they were further analyzed using Sidak’s multiple comparison test. A priori, we chose to use a built-in program in PRISM (GraphPad software) called ROUT (robust regression and outlier removal) and Grubbs to eliminate outliers for BBB and behavior testing. Additionally, animals were eliminated from analysis due to either death or procedural errors. Procedural errors included training in incorrect order in comparison to the rest of the animals in that group. p value of ≤ 0.05 was used to denote statistical significance. Statistical significance is indicated with “*” for p ≤ 0.05, “**” indicates statistical significance for p ≤ 0.01, “***” indicates statistical significance p ≤ 0.001, and “****” indicates statistical significance p ≤ 0.0001.