Animals
Animal experiments were carried out according to ARRIVE guidelines 2.0 [16]; the United Kingdom Animals (Scientific Procedures) Act, 1986; and EU Directive 2010/63/EU for animal experiments. The experiment received ethics approval from the Finnish National Animal Experiment Board (ESAVI/6360/04.10.03/2011). Male Lewis rats (n = 24) were obtained from Charles River Laboratories (Sulzfeld, Germany) and acclimatised for 7 days before the start of the experiments. Animals were housed in accordance with the Treaty of Amsterdam protocol for animal welfare, in pairs in individually ventilated cages with a consistent temperature of 21 (1.2)°C and consistent humidity of 55 (5)% with a 12-h light/dark cycle. Food (CRM(E) Expanded, Special Diet Services, UK) and tap water were provided ad libitum. Values are indicated as mean (SD).
The animals were randomly assigned to the treatment groups (Fig. 1). The fDTH model of MS was induced in the same manner as described previously [13]. Briefly, to induce the fDTH lesion, heat-killed bacillus Calmette–Guérin (BCG, 105 organisms in 2 μL of phosphate-buffered saline) was injected in the right striatum (RC + 1.0 mm, ML + 3.0 mm, DV-4.0 mm from bregma). The injection was performed in four 0.5 µL volumes by using a Hamilton syringe (Sigma-Aldrich) within the dorsal ventral depth − 4.0–2.5 mm over 10 min to avoid back-flow of the thick BCG. Four weeks after the intracranial injection of BCG (i.e. at week 0), the animals were peripherally sensitised by intradermal injection of heat-killed mycobacterium tuberculosis (TB, 1.5 mg, Difco Laboratories, Detroit, MI, USA) in an emulsion of complete Freund’s adjuvant (CFA, 50:50 v/v FA/saline, 100 μL, Sigma Aldrich, Saint Louis, MO, USA) into the flank of the rat, which initiates an fDTH lesion at the site of the intracranial injection of heat-killed BCG. This lesion activation step, at 4 weeks after the intracranial microinjection of BCG, is recorded as experimental day 0. The study animals were divided into three sets of eight rats each (n = 4 treated and n = 4 control): Set A was imaged by PET and euthanised for IHC after the last PET study, Set B was used for IHC after 2 weeks of treatment, and Set C used for IHC after 4 weeks of treatment. These time points were chosen to correspond with the time points of PET imaging in Set A. The time points for PET imaging were chosen for baseline imaging, acute phase of inflammation (at 1–2 weeks) and chronic phase (at 4–8 weeks) and to evaluate the potential rebound effect after halting the treatment for 10 weeks (at 18 weeks). Weight gain was measured throughout the imaging period (Fig. 2).
Starting on day 0, the rats were treated twice daily per os with 15 mg/kg of DMF (Sigma‐Aldrich Chemie GmbH, Steinheim, Germany) diluted in 0.08% aqueous Methocel (200 µL, Sigma‐Aldrich Chemie GmbH, Steinheim, Germany), or vehicle (tap water with 0.08% Methocel) for controls, using an oral gavage. Dosing of DMF was based on previous reports [7, 17]. Methocel was used because of the poor solubility of DMF in water [7]. To aid the dissolution of DMF, the solution was sonicated for 15 min.
Radiochemistry
[18F]GE-180 ((S)-N,N-diethyl-9-(2-[18F]fluoroethyl)-5-methoxy-2,3,4,9-tetrahydro-1H-carbazole-4-carboxamide) was synthesised at the Radiopharmaceutical Chemistry Laboratory of Turku PET Centre as described previously [18]. The molar activity of [18F]GE-180 was 1.6(0.2) TBq/μmol at the end of syntheses (n = 6). The radiochemical purity was ≥ 97.8%.
PET
Animal PET imaging was performed using an Inveon Siemens multimodality PET/computed tomography (CT) scanner (Siemens Medical Solutions USA, Knoxville, TN) designed for small laboratory animals. The rats were anaesthetised with isoflurane/air (4% isoflurane with 700 mL/min air for induction and 2–3.0% isoflurane with 400–500 mL/min air for maintenance on the table, until the animal was placed in the PET scanner) 20 min before the [18F]GE-180 injection. During the PET scan, the rats were under anaesthesia in 2–2.5% isoflurane/700 mL oxygen. Body temperature was maintained during imaging using a heating pad on which the rats slept. Two animals were imaged at once. Following transmission scans for attenuation correction using the CT modality, static PET images (5 × 300 s frames) were obtained 25–50 min after the injection of [18F]GE-180 with an energy window of 350–650 keV. The injected activity for DMF-treated animals was 32.23(1.27) MBq and the injected mass 28.4(13.4) ng/kg. The injected activity for the control animals was 32.56(1.18) MBq and the injected mass 29.2(12.9) ng/kg.
The PET data were reconstructed using the ordered-subsets expectation maximisation algorithm in three dimensions (OSEM3D) twice and MAP iterative reconstruction protocols 18 times in the Inveon™ acquisition software (Siemens Medical Solutions, USA).
Images were analysed using PMOD analysis software (v3.4 PMOD Technologies Ltd., Zürich, Switzerland). Images were divided and aligned to the Schiffer MR template that is inbuilt into the software by first aligning the PET to the CT space, after which the CT was aligned to the MR space. Alignment of the PET image to the MR space was achieved by combining the transformations, applying manual supervision and motion correction whenever needed. The images were summed to standardised uptake value (SUV) maps (25–50 min).
The volume of interest (VOI) was drawn with the Automatic Isocontour Detection tool for each animal individually by choosing the stage of the lesion at its largest (varied between individuals from week 1 to week 2) and applying this VOI to all other time points within one individual. In addition, a contralateral, spherical VOI was drawn on the contralateral striatum. The non-displaceable binding potential (BPND) was calculated as: (ipsilateral uptake − contralateral uptake) / contralateral uptake.
Immunohistochemistry
Animals were perfuse-fixed with periodate–lysine–paraformaldehyde fixative with 0.1 vol% glutaraldehyde and cryoprotected with sucrose. The method has been described in more detail previously [13, 19]. Staining was performed in a semiautomatic LabVision autostainer (Thermo-Fisher Scientific). Sections were pre-heated in citrate buffer (pH 6, Genmed), blocked with hydrogen peroxide and pre-protein block (Draco antibody diluent; WellMed), and incubated with either Iba1-Ab (Wako, 1:2000 dilution, RT), anti-CD4 (Abcam, ab33775, dilution 1:50), or anti-CD8 (Abcam, ab33786, dilution 1:200) for 60 min at room temperature (RT). For the anti-Iba1 staining, the Orion 1 step detection system (Goat anti-rabbit HRP; WellMed) was used as a secondary antibody (Ab) for 30 min at RT. For anti-CD4 and anti-CD8 staining, the Bright vision 1 step detection system (Goat anti-mouse HRP) was applied. The sections were stained with DAB (Taurus; WellMed) and Mayer’s haematoxylin.
Anti-Iba1 staining was performed to detect activated microglia within and around the lesion core. Ten-micrometre-thick brain sections were obtained with a gap of a 100 µm through the fDTH lesion area of 750 μm from the lesion centre on either side. The Iba1-immunopositive area of the perilesional area or the hypercellular core (depicted in Fig. 4d) was assessed using CaseViewer 2.1 software (3DHISTECH Ltd., Budapest, Hungary). The semi-quantitative volume calculation was performed by drawing the area of activated microglia for each brain section and extrapolating the area in between using the trapezoidal rule. The perilesional volume (Fig. 4a) and the lesion core volume (Fig. 4b) were calculated for both the control and the DMF-treated animals.
To detect T lymphocytes, brain sections (n = 3) adjacent to the lesion core with anti-Iba1 staining were selected. Because individual CD4+ and CD8+ T cells (Fig. 5) could not be calculated, quantitation was performed by measuring the optical density (OD) of a region of interest (ROI) at the lesion core, in the perilesional area, and at the contralateral site corresponding to the lesion core on the ipsilateral side of the coronal brain section. The image was deconvolved to an 8-bit image, which corresponds to the DAB staining, using Fiji software (ImageJ v1.52p). It is, on this image, that the ROIs were drawn. ODROI was calculated using the formula log(maximum intensity/mean intensityROI), where maximum intensity is 255 that corresponds to white in an 8-bit image, and mean intensityROI is the mean intensity from the lesional or perilesional area obtained from three brain sections within one individual [20, 21]. The function is logarithmic since the signal from a microscopy image is nonlinear. The final OD count was obtained by reducing the contralateral ODROI from the lesion or perilesional ODROI, i.e. OD = ODROIlesion or perilesion − ODROIcontra.
Statistical analyses
The statistical analysis for the weight gain in Set A animals (Fig. 2) at all studied time points (weeks—4, 0, 1, 2, 4, 8, 11, 14, 15, and 18) was performed using a linear mixed model with a compound symmetry covariance structure. The model included a time factor, a group factor, and their interaction. The interaction term was used to assess whether the change over time was significantly different in the DMF-treated group compared to the control group. In case the interaction term was significant, the differences in changes at all time points were checked in a post hoc analysis. The logarithm of the response was used instead of the original values for the model to fulfil the normality assumption. With the original values, the assumptions were violated due to non-normal distribution of the values.
Statistical analysis of the change in BPND compared to the baseline was analysed using a two-tailed unpaired Student’s t-test for each time point separately (GraphPad Prism 9, GraphPad Software, San Diego, CA, USA), and additionally, in the same manner as the weight gain analysis, by using a linear mixed model with a compound symmetry covariance structure. However, the logarithm of the response was used instead of the original values for the model to fulfil the normality assumption.
Anti-CD4 and anti-CD8 staining data, with OD as the response, were analysed separately using linear mixed models with compound symmetry covariance structures. The models included an ODROI factor (lesion or perilesional area), a group factor (DMF-treated or control), week as a group factor with a separate set of subjects for weeks 2 and 4, and the interaction term between ODROI and group, with possible post hoc analysis in case of significance. The square root of the response was used instead of the original values for the model to fulfil the normality assumption in both models.
In post hoc analyses, p-values were adjusted using the Tukey–Kramer method. The normality assumption was checked using the studentised residuals. The linear mixed model analyses were performed using SAS version 9.4 for Windows (SAS Institute Inc., Cary, NC, USA). All statistical tests were two-sided with the significance level set at 0.05. Values for animal weight data, injected radiochemical masses, BPND and OD are reported as means (SDs).