Glia influence neuronal development and aging. Few translational studies have examined how age at injury affects the glial response to traumatic brain injury (TBI). We hypothesized that rats injured before sexual maturity would exhibit a greater glial response, that persists into early adulthood, compared to rats injured near the onset of sexual maturity.

Postnatal day (PND)17 and PND35 rats received midline fluid percussion injury or sham surgery. In three cortical regions (peri-injury, S1BF, perirhinal), we investigated the glial response relative to age at injury, time post-injury (2H, 1D 7D, 25D, and 43D), and post-natal age, such that rats injured at PND17 or PND35 were compared at the same post-natal-age (e.g., PND17+25d post-injury=PND42; PND35+7d post-injury=PND42). We measured Iba1+ microglia cells and quantified their activation status. GFAP expression was examined using immunohistochemistry. Data were analyzed using Bayesian multivariate multi-level models.

Independent of age at injury, TBI activated microglia (shorter branches, fewer endpoints) in the cortex with more microglia in all regions compared to shams. TBI-induced microglial activation was sustained in the S1BF into early adulthood (PND60). PND17 injured rats had more microglial activation in the perirhinal cortex than PND35 injured rats. Activation was not confounded by age-dependent cell size changes, and microglial cell body sizes were similar between ages.

Increased microglial activation in PND17 injured rats suggests that TBI upregulates the glial response at discrete stages of development. Age at injury and aging with an injury are translationally important because experiencing a TBI during early childhood may trigger an exaggerated glial response.

Study design

We investigated the glial response relative to age at injury (PND17/PND35), time post-injury (tissue harvest: 2H, 1D, 7D, 25D, and 43D), and post-natal age, such that rats injured at PND17 or PND35 were compared at the same post-natal age (e.g., PND17 + 25d post-injury = PND42; PND35 + 7d post-injury = PND42).

Animals

Male Sprague Dawley rats (Envigo, Indianapolis, IN) were used for all experiments. Rats were housed in a 12h light:12h dark cycle at a constant temperature (23°C ± 2° C) with food and water available ad libitum according to the Association for Assessment and Accreditation of Laboratory Animal Care International guidelines. PND17 rats were received from shipment at PND10 with the dam and acclimated to their environment for one week prior to experiments to control for the stress associated with shipping. Each dam arrived with 10 pups. After surgery, post-operative care via physical examination took place to monitor each animal’s condition. PND17 rats were returned to the dam following surgery and midline fluid percussion (mFPI), until tissue collection at 2H, 1D or 7D post-injury. Rats were weaned at PND21 or were allowed to stay with the dam until their 7D tissue collection time point (PND24) to avoid stress before tissue collection. Weaned animals (for PND42 and PND60 tissue collection) were housed in groups of 3-4 littermates until PND42, when they were pair housed. Average PND17 pre-surgical weight was 28.2g ± 3.2g. PND35 rats arrived at PND28 and were housed in groups of 3-4 until they reached PND42, when they were separated and pair housed. Average PND35 pre-surgical weight was 135.5g ± 14.3g. Weights and health conditions were monitored and documented throughout the experiment. Animal care and experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Arizona (protocol 13-460).

Midline fluid percussion injury (mFPI)

For surgery, all rats were administered 5% isoflurane in 100% oxygen for 5 minutes and then secured in a stereotaxic frame. Anesthetization was maintained with continuous isoflurane delivery at 2.5% via nosecone. A midline incision was made and a craniectomy (outer diameter 3mm in PND17 rats and 4mm in PND35 rats) was trephined midway between bregma and lambda (Rowe, Harrison, Ellis, Adelson, & Lifshitz, 2018). The skull flap was then removed with care not to disrupt the dura or superior sagittal sinus underlying the craniectomy site. An injury hub (prepared from the female portion of a Luer-Loc needle hub) was fixed over the craniectomy using cyanoacrylate gel and methyl-methacrylate (Hygenic Corp., Akron, OH). Post-surgery, animals were placed on a heating pad and monitored until ambulatory.

Approximately 60-120 minutes after surgery, rats were subjected to mFPI with methods previously described (Rowe et al., 2018; Rowe et al., 2016b). Rats were re-anesthetized with 5% isoflurane in 100% oxygen delivered for 3 minutes. The hub assembly on the skull was filled with saline and attached to the male end of the FPI device (custom design and fabrication, Virginia Commonwealth University, Richmond, VA). PND35 rats were connected directly to the FPI device. PND17 rats were connected to the FPI device using an extension tube (Baxter #2C5643). When a toe pinch withdrawal response was detected, the pendulum was released causing a fluid pulse directly onto the dura resulting in a diffuse brain injury. Sham rats were connected to the device but the pendulum was not released. This injury model is routinely used on adult rats. Here, we used our previously optimized methods for mFPI in juveniles to give a consistently mild to moderate injury in all animals (PND17 = 1.5 atmospheres pressure (atm), PND35 = 1.9 atm) (Rowe et al., 2018; Rowe et al., 2016b). Hubs were removed immediately after injury or sham injury and rats were monitored for apnea, seizure-like behavior, righting reflex time (time from the initial impact until the rat spontaneously righted itself from a supine position), and a fencing response. The fencing response is a tonic posturing characterized by extension and flexion of the arms that has been validated as a physical indicator of TBI (Hosseini & Lifshitz, 2009). After rats spontaneously righted, their brains were inspected for herniation, hematomas, and integrity of the dura. Rats included in this study had a righting reflex time between 3 and 10 minutes, indicative of a mild to moderate injury, and had no dura damage. Rats were placed in a heated recovery cage and monitored until ambulatory after the incision was cleaned with sterile saline and closed. PND17 rats were rubbed with bedding from their home cage to ensure the dam accepted them after surgery/injury. Rat welfare was evaluated and documented daily during post-operative care via physical examination.

Cryoprotection and tissue sectioning

At pre-determined time points post-injury (2H, 1D, 7D, 25D, and 43D), a lethal dose of Euthasol® was administered. Animals underwent transcardial perfusion with 4% paraformaldehyde (PFA) after flushing vasculature with phosphate buffered saline (1× PBS). From the time of tissue harvest, tissue samples were treated identically throughout the experiment to reduce variation. Brains were harvested from the skull and drop fixed in 4% PFA for 24 hours. Brains were cryoprotected by successive incubation in 15% and 30% sucrose, each for 24 hours. Brains were then removed from sucrose and one hemisphere from each animal was frozen in groups of 6-9 using the Megabrain technique as previously described by our lab (Green, Ortiz, Harrison, Lifshitz, & Rowe, 2018). Megabrains were cryosectioned in the coronal plane at 40 µm and mounted on superfrost slides and stored at -80 ºC. Sections were removed from the freezer and baked at 56ºC for 3 hours prior to undergoing immunohistochemistry.

Immunohistochemistry and analysis

Each immunohistochemistry stain was performed on 4 brain slices from each animal, and 3 images per slice were analyzed.

Iba1: To analyze microglia morphology, brains were stained for ionized calcium binding protein adapter molecule 1 (Iba1). To improve scientific rigor, Iba1 staining was performed in a single round of staining to minimize variance and allow comparisons. Slides were rehydrated in 1× PBS after baking (3 hours). Antigen retrieval was performed using sodium citrate buffer (pH 6.0). Slides were then washed in 1× PBS. Hydrophobic barrier pen was applied to the perimeter of the slide and slides were placed in a humidity chamber. Blocking solution was immediately applied (4% normal horse serum [NHS], 0.1% Triton-100 in 1× PBS) with an incubation time of 60 minutes. Following blocking, primary antibody solution (rabbit anti-Iba1; WAKO cat #019919741; RRID: AB_839504; at 1:1000 concentration in 1% NHS, 0.1% triton-100 in 1× PBS) was applied and left to bind overnight at 4 ºC. Slides were then washed in 1× PBS + 0.1% tween-20. Secondary antibody solution (biotinylated horse anti-rabbit IgG (H +L); vector BA-1100; RRID: AB_2336201; at 1:250 concentration in 4% NHS and 0.4% triton-100 in 1× PBS) was applied and incubated for 60 minutes. Slides were washed in 1× PBS + 0.1% tween-20. Endogenous peroxidases were blocked in 200ml 1× PBS + 8 ml H₂O₂ for 30 minutes. After washing in 1× PBS + 0.1% tween-20, Avidin-Biotin Complex (ABC) solution (Vectastain ABC kit PK-6100) was applied and incubated for 30 minutes. Slides were washed in PBS + 0.1% tween-20 and then 3,3′-diam-inobenzidine (DAB) solution (from Vector DAB peroxidase substrate kit SK-4100) was applied and incubated for 10 minutes and, following this, slides were immediately placed in water. Tissue was dehydrated in ethanol (70%, 90% and 100%) and cleared with citrosolve. Coverslips, matching microscope specifications, were applied using dibutylphthalate polystyrene xylene mounting medium.

GFAP: To analyze astrocytes, brains were stained for GFAP. An identical protocol was followed to that described for Iba1, using solutions; blocking = 5% NHS, 0.1% triton X-100 in 1× PBS; primary antibody solution = polyclonal rabbit anti-glial fibrillary acidic protein #Z0334; RRID: AB_10013382; at a 1:1000 concentration in 2% NHS and 1× PBS solution; secondary solution = biotinylated horse anti-rabbit IgG (H +L); vector BA-1100 at 1:250 concentration in 4% NHS and 0.4% triton-100 in 1X PBS. The DAB incubation time was 5 minutes.

Imaging and analysis: Z-stack images of stained tissue were taken at 400× (40× objective lens, 10× ocular lens) using Zeiss Imager A2 microscope via AxioCam MRc5 digital camera and Neurolucida 360 software, with consistent brightness, numerical aperture, and Z-stack height (Figure 2). Nyquist theorem was followed to ensure the signal adequately represented our biological samples. Iba1 staining was analyzed using the skeletal analysis plugin following the protocol previously published (Morrison, Young, Qureshi, Rowe, & Lifshitz, 2017; Young & Morrison, 2018). Cell somas were counted manually to obtain total microglial count. Branch length and endpoints were recorded and divided by the number of cells in each region of interest. Microglial cell bodies were measured using the multipoint area selection tool to calculate cell body area and perimeter. For GFAP staining, images were analyzed for number of GFAP+ cells and average number of pixels per cell using ImageJ software. Cell somas were counted manually. The average number of pixels per cell (referred to as cell coverage henceforth) was recorded to assess gross morphological changes in GFAP⁺ astrocytes following mFPI. No alternations/settings changes were made to any images prior to analysis. All imaging analyses were performed on Z-stack images, which is the most appropriate stereological sampling method to capture our sample.