From positron emission tomography to cell analysis of the 18-kDa Translocator Protein in mild traumatic brain injury | Scientific Reports – Nature.com

Animals

Male Swiss mice were supplied by Janvier labs, weighed 28 to 30g. All care and experiments were in accordance with the ethical approvals stipulated by the Animal Ethics Committee of Paris Descartes University, the French regulations and the European Council Directive of September 22, 2010 (2010/63/EEC) on the protection of animals for experimental use (APAFIS#4765). Animals were housed under temperature (222C) and light (12h per day) controlled conditions with access to food and water ad libitum.

The study was carried out in compliance with the ARRIVE guidelines.

The Controlled Cortical Impact (CCI) model was performed as previously described30. Mice were anesthetized with isoflurane and placed in a stereotaxic frame. Body temperature was monitored throughout surgery by a rectal probe and maintained at 37.00.5C with a homeothermic blanket control unit. A 4-mm craniotomy was performed onto the left temporo-parietal cortex centered between the bregma and lambda (Supplemental Fig. S3A), taking care to leave the dura mater intact. Injury was delivered using a 3mm diameter impactor by a pneumatically controlled device (TBI 0310 Impactor, Precision System Instruments) using the following parameters: diameter 3mm, velocity 3.5m/s, depth of cortical deformation 1.0mm and dwell time 50 ms45. Following the injury, the skullcap was replaced by applying bone wax and the skin sutured. Sham-operated (SO) mice underwent the same surgery without impact. To recover from anesthesia and prevent post-surgery hypothermia, animals were placed in an incubator set at 30C for one hour. Mice were subsequently returned to their home cage. Non-operated (NO) mice had neither surgery nor anesthesia.

Radiosynthesis of [18F]FEPPA was performed at the Unit Claude Kellershohn, in Saint-Louis Hospital, Paris (France), as previously described46. Briefly, the radiosynthesis of [18F]FEPPA was performed using an AllInOne (Trasis) synthesis module and a tosylated precursor for a one-step fluorine nucleophilic aliphatic substitution.

All reagents and solvents were purchased from commercial suppliers (ABX or Sigma-Aldrich) and were used without further purification. Sep-Pak QMA were purchased from ABX. [18F]fluoride ion was produced via the [18O(p,n)18F] nuclear reaction (IBACyclone 18/9 cyclotron). Radioactivity of the final product was measured with a dose calibrator (PET DOSE 5Ci, COMECER).

PET/CT imaging was performed using Inveon micro PET/CT scanner (Siemens Medical Solutions) designed for small laboratory animals. Mice were anesthetized (isoflurane/oxygen, 2.5% for induction at 0.81.5 L/min, and 11.5% at 0.40.8 L/min thereafter) during injection of [18F]FEPPA (9.91.5MBq) in a volume of 0.15mL (0.220.19nmol of FEPPA) through the tail vein, and during PET/CT acquisitions, as previously described46.

The spatial resolution of Inveon PET device was 1.4mm full-width at half-maximum at the center of the field of view. Images were reconstructed using a 3D ordered subset expectation maximization method including corrections for scanner dead time, scatter radiations, and random.

In accordance with metabolism studies46, the [18F]FEPPA concentration decreases rapidly in the plasma, and get stabilized 90min after the injection. Conversely, brain concentration is barely stable through time. The acquisition was then started 75min after injection of [18F]FEPPA, and lasted for 30min. Between the injection and the start of the acquisition, anesthesia was stopped.

PET/CT imaging was performed at 1, 3, and 7days after surgery or TBI. At D1, 8 mice were imaged: 1NO, 3 SO and 4 TBI (Supplemental Fig. S4). At D3 and D7, 16 mice were imaged: 2 NO, 6 SO and 8 TBI. No animal was excluded from this experiment. All NO mice were pooled for SUV comparison. One SO D1 acquisition failed and could not be used for SUV comparison.

PET/CT images were visually assessed. Then quantitative analysis of PET/CT images was performed by PMOD version 3.806 image analysis software (PMOD Technologies). All values of radioactivity concentrations were normalized by the injected dose and the weight of the mouse, and expressed as percentage of the injected dose per g of tissue (% ID/g; Supplemental Fig. S3B) for the visualization and expressed as standard uptake value (SUV) maximum (SUVmax) and mean (SUVmean) for the quantification.

PET images were automatically rigidly matched with corresponding CT and then cropped to keep only the brain images. CT were automatically rigidly matched with a T2 MRI template (M. Mirrione, included in PMOD software), and then the transformation applied to the PET image was cropped. This method allowed us to use the atlas of the brain corresponding to the T2 MRI template. The atlas allowed us to quantify 6 regions (cortex, striatum, hippocampus, amygdala, midbrain and inferior colliculi) on the left (ipsilateral) and right (contralateral) side, and 7 regions non laterally-differentiated (thalamus, cerebellum, basal forebrain septum, hypothalamus, brain stem, central gray and superior colliculi) (Supplemental Fig. S3B).

The high variability induced by the injection and acquisition protocol prevented us from making a straight comparison of SUV between mice. SUV values were then normalized through a ratio with a SUV value of a reference region for each animal. The ratio between left (ipsilateral) and right (contralateral) side of the 6 side-differentiable regions (cortex, striatum, hippocampus, amygdala, midbrain and inferior colliculi), and the addition of all these regions (total brain) were calculated. The comparison of the 7 non side-differentiable brain regions (thalamus, cerebellum, basal forebrain septum, hypothalamus, brain stem, central gray and superior colliculi) was not possible.

The immunohistochemistry and immunocytochemistry protocols are briefly presented in this section. They are fully detailed in supplementary file.

The day after the PET acquisition (Supplemental Error! Reference source not found. S4), mice were transcardially perfused with NaCl followed by a fixative solution. Brains were removed and kept frozen at -80C. Coronal brain Sects.(20m thick) were taken using a cryostat (JUNG CM3000, Leica Microsystems). Sections were fixed in acetone for 5min and then rehydrated in phosphatebuffered saline (PBS) for 15min, before processing to immunostaining.

Transformed mouse brain endothelial cells bEnd.3 (ATCC CRL-2299) were purchased from Sigma-Aldrich and fixed for 12min using 4%paraformaldehyde in PBS prior to immunostaining.

Primary mixed glial cell culture was prepared from cortices of postnatal (day P0 to P3) mice as previously described47,48 and fixed for 12min using 4%paraformaldehyde in PBS.

Slices were incubated overnight at 4C with the primary antibodies and 90min with secondary antibodies. Then, slices were incubated with DAPI (Calbiochem; 268,298; 1:20 000) for 90min at room temperature. For the DAPI/IB4/collagen IV/TSPO staining, a 90-min incubation with a FITC-marked Isolectin B4 from Bandeiraea simplicifolia (Griffonia simplicifolia) (Sigma, L2895, 1:100) was performed. Antibodies references and dilutions are listed in the Supplemental Table S4 and Supplemental Table S5.

The slices were examined under a SP8 laser scanning confocal microscope (Leica Microsystems).

The objective of the flow cytometry experiment was to evaluate the expression of TSPO by microglial cells, in the homeostatic state and the inflammatory context of our TBI model, whether the inflammation originated from the surgery or the TBI. Thus, TSPO expression in SO mice would not have been useful data. Considering also ethical issues, we included and analyzed only two categories of animals (NO and TBI; 10 NO, 8 TBI at 1day and 8 at 3days; Supplemental Fig. S4).

Brain dissociation and immunostaining protocols are briefly presented in this section. They are fully detailed in supplementary file.

At 1 and 3days after TBI, or surgery, mice were anesthetized and transcardially perfused with 0.9% NaCl saline. The brains were removed and each hemisphere represented a sample.

Each sample was then dissociated in a mix from the Miltenyi Biotecs Adult Brain Dissociation Kit (130107-677), following the manufacturers instructions. The cells obtained from the samples were counted and the volume adjusted to obtain 50 to 100.106 cells/mL.

Cells were analyzed with LSRFortessa cell analyzer (BD Biosciences) and data analyzed with FACSDiva (Becton Dickinson) and FlowJo (version 10.5.3; Tree Star) softwares.

After isolating single living cells (Supplemental Fig. S5A):

- monocytes and neutrophils were gated on their Ly-6G and Ly-6C expression profile,

- lymphocytes, macrophages and microglia were gated on their CD45 and CD11b expression profile,

- and endothelial cells were gated on their CD144 expression profile (Supplemental Fig. S5B).

After gating the TSPO+ cells on single live cells, the same gating strategy was applied (Supplemental Fig. S5C). This allowed us to quantify the proportion of the different immune cells and endothelial cells in cells expressing TSPO in the CNS, and its relative expression level.

The number of each immune cell population (monocytes, neutrophils, macrophages, lymphocytes and microglia) was counted and added to determine the total number of immune cells. The proportion of each immune cell was calculated by the ratio of the number of the specific immune cell population over the total number of immune cells.

In each isolated cell population, the proportion of TSPO+ cells was calculated, and in each cellular population, the TSPO expression was quantified using geometric mean intensity.

Data were expressed as meanSEM of n observations, where n represents the number of animals. All figures and statistical analyses were created with GraphPad Prism 5.0 (Graphpad Software). Values of probability lower than 5% (P<0.05) were considered significant.

To proceed to TEP data analysis, SUVmean and SUVmax were extracted from PMOD software (PMOD Technologies) and converted into Microsoft Excel format.

SUVmean and SUVmax of each of the 6 side-differentiable brain regions were compared using one-way ANOVA followed by a Dunnetts test.

Immunostaining quantification values were using a Welsh t test as the variance were significantly different.

The proportion of immune cells measured in flow cytometry, and the geometric mean of the TSPO staining intensity were compared using a one-way ANOVA followed by a Dunnetts test.

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From positron emission tomography to cell analysis of the 18-kDa Translocator Protein in mild traumatic brain injury | Scientific Reports - Nature.com