Hypoxic ischemic brain injury

The modified Vannucci model was used and evaluated with morphology and behavioral tests. There was no mortality, severe illness or need for early euthanasia. WT C57/bl6 specific pathogen free (SPF) mice (Charles River Laboratories) of either sex were used, with free access to pelleted food and housed in open cages with standard enrichment with daily monitoring of the animals in accordance with local institutional guidelines. Pups were kept with their mother at all times except during surgery and hypoxia. Randomly allocated (https://www.random.org), single-dose caffeine 5 mg/kg or PBS was administered i.p. directly after HI (n = 29 and n = 13 respectively). Evaluations were performed by investigators blinded to treatment. Animals were sacrificed by injection of 240 mg/kg sodium pentobarbiturate i.p. and cardiac perfusion with 12 mL phosphate buffered saline (PBS) to remove intravascular blood cells.

HI was performed at postnatal day 10. Unilateral electrocoagulation at 8 Watt of the right carotid artery was carried out via midline neck incision under isoflurane sedation and local bupivacaine infiltration to minimize pain and distress. Pups rested one hour with the dam after surgery, prior to one hour of 10% O₂ in 90% N₂ at 36˚C skin temperature. During sham operation the carotid artery was visualized as described above but not electrocoagulated.

Immunohistochemistry

Brains were collected after behavioral evaluation in the Caffeine or PBS treatment groups (n = 29 and n = 13 respectively) at postnatal day 24, two weeks after HI. In accordance with previous studies [5], three levels of each brain were collected, corresponding to bregma 1.3 mm, -1.64 mm and -2.92 mm in the adult mouse brain using cryostat 10–12 μm sections. Tissue sections were fixed for 10 min in 4% paraformaldehyde. Endogenous peroxidase was blocked by 0.3% H₂O₂ in 3% Normal Horse Serum for 10 min. Microtubule associated protein-2 (MAP-2) staining VECTOR® M.O.M.™ Immunodetection Kit and VECTASTAIN® Elite ABC-Peroxidase Kit were used according to the manufacturer’s specifications. The enzymatic coloration of immunoreactivity was performed by simultaneous full immersion in 3,3´-diaminobenzidine, DAB for all slides. Imaging was performed with a Nikon eclipse E800 microscope with an Olympus DP70 camera with DP controller, and version 3.1.1.267 acquisition software. Percent MAP-2 stained area and infarction size were manually delineated using Adobe Photoshop version 12.0.4 in brain slices with investigators blinded to genotype and treatment. Atrophy was calculated as the remaining tissue loss not described by the infarction area delineated by loss of MAP-2 compared to the uninjured hemisphere using the formula 1-(Damaged hemisphere area including infarction area)/(Undamaged hemisphere area) [6].

Behavioral tests

Behavioral assessments were done at 24 days age, two weeks after HI in caffeine or PBS treated animals (n = 29 and n = 13 respectively). Open Field test (Kungsbacka Mät- och Reglerteknik AB, Fjärås, Sweden) was used to investigate explorative behavior during 30 min. The animals were taken directly from their home litters and put in the open field box without further handling. In addition, the beam walking test, where mice walk three times back and forth on a beam (10 mm wide, 600 mm long), was performed directly after the open field test. Mice were handled with care to assure a calm explorative state of mind in the animal, and the number of slips with the hind limbs recorded by investigator blinded to treatment group.

2.6 FACS analysis

Randomization to caffeine or PBS treatment and sham or HI operation was performed at postnatal day 10 and samples were analyzed at 24h, 72h and 2 weeks after HI (total n = 52, treatment allocation is discernible in Fig 2).

Damaged and undamaged brain hemispheres were gently homogenized using a loose fit glass homogenizer, and cell suspension passed through a 100um cell strainer prior to staining with fluorochrome conjugated antibodies. Splenocyte cell suspensions were prepared by gentle homogenization through a 100um cell strainer followed by red blood cell lysis with ACK buffer for 5 min at room temperature. For antibodies used see S2 Table. Flow cytometric data was collected on a FACS Aria (BD Biosciences). 2.5x10⁶ events were collected from each brain hemisphere.

A data driven gating strategy was used with the FlowCore package for R [7] as previously described in detail [8]. Debris was manually removed using a polygon gate in forward and side scatter. A curvfilter was used to filter out immune cell populations. Dead cells were excluded using DAPI stain. Negative population was selected as reference and cells with higher expression than that were regarded as positive cells. Median fluorescent intensities of the selected populations were used to discriminate between positive and negative populations. The same filter settings were used for each marker in all brain samples. After automated batching, all gate settings were visually confirmed and no manual adjustments were required.

Statistical analysis

Mann-Whitney U test was used to analyze data on morphology and behavior data. Statistical calculations were made in StatSoft, Inc. (2011) STATISTICA, version 10. Heatmaps were made in R version 2.15.