Animals
A total of 40 C57Bl/6 male mice were obtained from the Australian Animal Resource Centre (ARC, Western Australia) for use in this study. Mice were 12 weeks of age at the time of injury, were group housed under a 12-hour (h) light/dark cycle, and were given access to food and water ad libitum for the duration of the experiment. All procedures were approved by the Animal Ethics Committee at La Trobe University (AEC #18–032), were within the guidelines of the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (the Australian National Health and Medical Research Council), and were in compliance with the ARRIVE guidelines on reporting of animal experiments [22].
Experimental Design
Animals received either sham or TBI procedures, and were randomly allocated to either vehicle or hAEC treatment groups. All mice underwent behavioral testing at 24 h post-injury, followed by tissue collection at 48 h for either histological or flow cytometric analysis as detailed in Table 1.
Table 1
| Sham vehicle | TBI vehicle | Sham hAEC | TBI hAEC |
Behavior | 8 | 12 | 8 | 12 |
Histology | 3 | 4 | 2 | 5 |
Flow Cytometry | 5 | 8 | 6 | 7 |
Numbers indicate group n’s. Abbreviations: hAEC, human amnion epithelial cells; TBI, traumatic brain injury.
Controlled Cortical Impact (cci)
The CCI model was used to induce an experimental TBI of moderate severity in mice [23]. In brief, animals were anesthetized with 1.5% isoflurane then secured within a stereotaxic frame. Mice were administered 0.05 mg/kg subcutaneous (s.c.) buprenorphine for analgesia prior to surgery, and isotonic sterile saline (0.9% NaCl s.c.) post-surgery to aid in rehydration. The skull was exposed by midline incision, and a 4 mm craniotomy was performed on the left parietal bone, midway between Bregma and Lambda, to expose the intact dura. For animals randomly assigned to receive a TBI, the impact was delivered via a Leica Impact One device, using a 3 mm convex tip, 4.5 m/s velocity, 1.5 mm depth for 100 ms duration. Following impact, bleeding and swelling were observed then the scalp was sutured closed. Sham animals underwent identical surgical preparation with the exception of the actual impact. Following surgery, mice were individually housed under a heat lamp until a righting reflex was observed and normal activity was resumed, then returned to group housing. Body weights were monitored daily post-surgery.
Haec Isolation And Injection
hAECs were isolated from term placenta donated by healthy volunteers who underwent elective cesarean section delivery as previously described [15] and with approval from Monash Health Human Research Ethics (approval no. 12223B). All volunteers provided informed consent and information were collected in accordance to National Health and Medical Research Council guidelines on Human Research Ethics. Cells were thawed and washed in 2% human serum albumin (HSA) in saline. Cell viability was assessed by trypan blue exclusion and isolates with a minimum of 80% viability were used, with cells were suspended in 2% HSA at 5 × 106 cells/mL. Sixty mins after TBI, mice were randomly assigned to receive either 2% HSA (vehicle) or 1 × 106 hAECs administered via tail vein injection.
Behavioral Testing
Mice underwent behavioral testing in the open field and rotarod at 24 h post-injury, by an experimenter blinded to group assignment.
Open Field
An open field was used to assess locomotion/activity at 24 h post-sham/TBI, similar, to that as described previously [24, 25]. Briefly, this task consisted of a 50 × 50 cm square arena, with 25 cm-high walls to prevent escape. Mice were released into the center of the field and allowed to explore for 5 min before they were returned to their home cage. Total distance traveled was determined. As a measure of anxiety, the arena was divided into a central inner zone (30 × 30 cm) and an outer zone, and the time spent in the inner zone was determined.
Rotarod
An accelerating rotarod was used to assess sensorimotor ability similar to that to that described previously [26]. Briefly, testing apparatus consisted of 3 cm diameter rotating rod, with 5.7 cm lanes and a 16 cm fall height (47650 Rotarod, Ugo Basile→, Italy). Two days prior to injury, mice firstly completed rotarod training at a constant speed of 16 rpm for 5 min, with mice manually returned to the rod upon falling. On the day prior to injury, mice completed 3 baseline trials. Each trial consisted of the mouse being placed on the rotating barrel, with the speed gradually increased from 5 to 50 rpm over a period of 5 min. The average duration of time the mouse was able to stay on the rotating barrel was recorded for each trial period (maximum time of 5 min). This procedure was repeated at 24 h post-injury. Latency to fall data for each mouse was expressed as a ratio of mean post-injury to mean baseline values.
Tissue Perfusion / Collection
For flow cytometry, mice were euthanized at 48 h post-injury by carbon dioxide asphyxiation, intracardially perfused with phosphate buffered saline (PBS) and then decapitated for brain collection. Brain hemispheres were separated after removal of the cerebellum and olfactory bulbs. Spleen and thymus weights were determined.
For histology, mice were euthanized at 48 h post-injury by carbon dioxide asphyxiation, intracardially perfused with PBS followed by 4% paraformaldehyde (PFA). Brains were collected and post-fixed in 4% PFA for 24 h, cryoprotected in 30% sucrose for 48 h, snap frozen in isopentane and stored at -80 °C until sectioned.
Flow Cytometry
Mice were euthanized at 48 h post-injury by carbon dioxide asphyxiation, intracardially perfused with PBS and then decapitated for brain collection. Brain hemispheres were separated after removal of the cerebellum and olfactory bulbs. The left hemisphere was mechanically dissociated in digestion buffer containing collagenase type XI (125 U/mL), hyaluronidase (60 U/mL), and collagenase type I-S (450 U/mL) in Ca2+/Mg2+-containing PBS, and incubated at 37 °C for 45 min with gentle agitation (100 rpm). The suspension was then passed through a 70 µm nylon cell strainer (Falcon, BR Biosciences) to yield a single-cell suspension. Cells were washed with PBS (350 g, 5 min at 4 °C), the pellet was resuspended in 3 mL of 30% Percoll (GE Healthcare), underlaid with of 2 mL of 70% Percoll, and centrifuged at 1,400 g at room temperature for 20 min without the use of a brake. Mononuclear cells at the interphase of the two Percoll density gradients were collected and washed with PBS.
Cells were stained with the antibodies listed in Table 2. Cells were firstly incubated with LIVE/DEAD™ Fixable Aqua Dead Cell Stain (1:1000 dilution, Invitrogen) for 15 min at 4 °C. Cells were then washed with PBS containing 1% bovine serum albumin (BSA; 350 g, 5 min at 4 °C). An antibody cocktail containing all antibodies except FoxP3-PE-Cy5.5 was then prepared and cells were stained for 25 min at 4 °C. Cells were washed with PBS containing 1% BSA (350 g, 5 min at 4 °C) and incubated with FIX & PERM Cell Fixation & Cell Permeabilization Kit (Invitrogen) for 20 min at 4°C. After incubation, the samples were washed with Permeabilization Wash (Invitrogen, 350 g, 5 min at 4 °C). For intracellular staining of FoxP3, a marker of regulatory T cells, cells were stained with FoxP3 antibody for 15 min at room temperature. The samples were washed with Permeabilization Wash (350 g, 5 min at 4 °C) and resuspended in 1% formalin in PBS containing 1% BSA. Stained cells were quantified on a Cytoflex LX flow cytometer (Beckman Coulter). The total number of brain-infiltrating immune cells and resident microglia were analyzed using FlowJo software (Version 10, Treestar). All data are derived from gating of the live cell population.
Gating strategy
Forward and side scatters were used to identify single cells. Dead cells were excluded with the LIVE/DEAD™ stain. Live cells were gated for CD45+ high and then divided into myeloid cells (CD45+ highCD11b+), and subdivided into monocytes (CD45+ highCD11b+Ly6C+), macrophages (CD45+ highCD11b+F4/80+), neutrophils (CD45+ highCD11b+Ly6C+Ly6G+), and lymphoid cells, which included T cells (CD45+ highCD3+CD4+), helper T cells (CD45+ highCD3+) and regulatory T cells (CD45+ highCD3+ CD4+FoxP3+). All cells are presented as the number of cells per hemisphere.
Table 2
Antibodies used for flow cytometry analysis.
Antibody | Dilution | Target cells | Host/isotype | Supplier |
CD3-APC | 1:500 | T cells | Hamster IgG | BioLegend |
CD4-BV605 | Helper T cells | Rat IgG2a, κ | BioLegend |
CD11b-BV421 | Myeloid cells | Rat IgG2b, κ | BioLegend |
CD45-A700 | Leukocytes | Rat IgG2b, κ | BioLegend |
F4/80-APC-Cy7 | Microglia/Macrophages | Rat IgG2a, κ | BioLegend |
Ly6C-FITC | Monocytes | Rat IgG2c, κ | BioLegend |
Ly6G-PE-Cy7 | 1:1000 | Neutrophils | Rat IgG2a, κ | BioLegend |
FoxP3-PE-Cy5.5 | 1:500 | Regulatory T cells | Rat IgG2a, κ | eBioscience |
Brain Sections Following Pfa Perfusion:
In a subset of mice (n = 2–5 per group), brain coronal sections spanning the entire injury site (Bregma − 0.9 to -3.5 mm) were collected to determine the extent of tissue damage by histology, as well as immunofluorescence to confirm the presence of hAECs in the injured brain (representative n = 1 per group). Sections were thaw-mounted onto Superfrost™ plus slides (Thermo Scientific, USA) and stored at -80 °C until analysis.
Histology/lesion Volume Measurements
Six equidistant sections per brain (10 µm thickness, 180 µm apart) were stained with 0.25% cresyl violet acetate (15 min) followed by differentiation in descending ethanol concentrations prior to clearance in xylene and coverslip adherence with DPX mountant (Sigma). Brightfield images were captured using a Leica Aperio AT Turbo slide scanner in the Monash Histology Platform, then exported to FIJI (https://imagej.net/Fiji) for analysis using the unbiased Cavalieri method with grid point counting, whereby volume = number of points counted x area represented by each point x distance between sections (taking into account the sampling frequency and section thickness) [27, 28]. Volume measurements were made of the dorsal cortex and hippocampus, both ipsilateral and contralateral to the injury site as previously described in depth [29].
Immunohistochemistry
PFA-fixed coronal brain sections (10 µm) were thaw-mounted onto Superfrost™ plus slides (Thermo Scientific, USA) and immunofluorescently labeled with HLA-G to identify the presence of hAECs in the brain following TBI. Sections were fixed in 4% PFA for 5 min, washed in 0.01 M PBS (2 × 5 min), and then blocked with a Mouse on Mouse (M.O.M.™) Ig blocking reagent (Vector Laboratories, USA) for 1 h. Following wash with 0.01 M PBS (2 × 2 min), sections were incubated in M.O.M.™ diluent (Vector Laboratories, USA) for 5 min, prior to HLA-G antibody incubation for 30 min (1:500; Ab52455; Abcam, UK). Sections were then washed with 0.01 M PBS (2 × 2 min) and incubated with M.O.M Biotinylated Anti-Mouse IgG reagent (Vector Laboratories, USA) for 10 min. Following washes with 0.01 M PBS (2 × 2 min), fluorescein-Avidin DCS (Vector Laboratories, USA) was applied for 5 min. Sections were then washed with 0.01 M PBS (2 × 5 min), cover-slipped with Vectashield DAPI® (Vector Laboratories, USA), and examined with an Olympus fluorescence microscope. Positive HLA-G stains were confirmed upon co-localization with DAPI+ nuclei.
Statistical Analysis
All outcomes were analyzed using GraphPad Prism version 8.02 for macOS (GraphPad Software, CA, USA). All data was analyzed with two-way analysis of variance (ANOVA) and are presented as mean + standard error of the mean (SEM), with Sidak’s multiple comparisons conducted where appropriate. Statistical significance was set at p < 0.05.