Animals
We used male C57BL/6J mice (Charles River, Italy), 9 weeks of age, weighing 21–26 g for the experiments. Procedures involving animals and their care were conducted in conformity with institutional guidelines, which comply to National (D.L. n.116, G.U. suppl. 40, 18 February 1992) and international laws and policies (EEC Council Directive 86/609, OJ L 358,1; Dec.12,1987; NIH Guide for the Care and Use of Laboratory Animals, U.S. National Research Council 1996). The project was approved by the Italian Ministry of Health with the codes No. 9F5F5.148 (approval number 80/2020-PR) for the first part of the study and No. 9F5F5.206 (approval number 117/2022-PR) for the second part of the study.
Mice were housed 5 per cage and kept at a constant temperature (21° ± 1 °C) and relative humidity (60%) with a regular light/dark schedule (7am-7pm). Food (Altromin pellets for mice) and water were available ad libitum.
Experimental TBI
Mice were anesthetized with isoflurane inhalation (induction 5%; maintenance 2%) in an N2O/O2 (70%/30%) mixture and placed in a stereotaxic frame. Rectal temperature was maintained at 37 °C using a feedback-controlled heating pad. Mice were subjected to craniectomy followed by CCI brain injury as previously described [16]. Briefly, the injury was induced using a 3-mm diameter rigid impactor driven by an electromagnetic piston (Leica, Impact One) mounted at an angle of 20° from the vertical plane and applied vertically to the exposed dura mater, between bregma and lambda, over the left parieto-temporal cortex (antero-posteriority: −2.5 mm, laterality: −2.5 mm), at impactor velocity of 5 m/s and deformation depth 2 mm, resulting in a severe level of injury [9, 17]. The craniotomy was covered with a cranioplasty and the scalp sutured. Sham-operated mice received identical anesthesia and surgery without brain injury.
HG-4, a derivative of the clinically used MASP-2 inhibitor narsoplimab, was used for MASP-2 inhibition in these mouse studies while antibodies 86C3 and motavizumab were used for MASP-1 inhibition and isotype control treatment, respectively [14, 18]. Antibody treatments were administered by intraperitoneal (i.p.) injection at a dose of 10 mg/kg of body weight at 4- and 24-hours post TBI.
Behavioral tests
Sensorimotor deficits
Neuroscore: mice were scored from 4 (normal) to 0 (severely impaired) for each of the following: forelimb function, hindlimb function during walking on a grid, and resistance to lateral left pulse. The best possible score, corresponding to no deficits, was 12 [13].
Simple Neuroassessment of Asymmetric imPairment (SNAP): Mice were evaluated using eight tests measuring: interaction with the handler, grip strength, visual placing, pacing/circling, gait and posture, head tilt, visual field, and coordination and proprioception. Scores ranged from 0 (normal) to 5 (severely impaired) for each test and were summed to give an overall score that ranged from 0 (best) to 40 (worst) [19].
Cognitive deficits
Barnes maze: the test assessed spatial learning and memory. The maze consists of a circular platform with multiple holes around its perimeter, only one of which leads to an escape tunnel or a safe area. The test started with a habituation trial (day 1) during which the mouse was placed in the center of the empty arena under a beaker for 30 s, then guided to the escape tunnel over 10–15 s by slowly moving the beaker and allowing 2 min to enter the escape tunnel spontaneously. The mouse was allowed to stay in the escape for 1 min and then was taken back to its home cage. In the learning phase (days 2–4), mice were placed for 10 s in the center of the arena and then allowed to explore the arena for at least 2 min to find the escape (primary latency) and to enter it (secondary latency). The test phase (day 5) consisted of the removal of the escape tunnel from the maze to assess the primary latency [20]. The escape was sealed (‘false escape’) to prevent the mouse from falling through the hole. The ability of the mouse to find the escape within the 2-minute time was recorded.
Anxiety-like behavior
Elevated plus maze (EPM): the test (set up in-house) measured disinhibition and anxiety-like behaviors. The test consisted of two open and two closed arms (each 35 cm × 5.5 cm) and a central platform (5.5 cm × 5.5 cm) elevated 60 cm above the ground. Mice were acclimatized in the room for 1 h before testing then placed on the central platform facing an open arm, and their movements were recorded for 5 min. Video recording and time spent in the closed and open arms were measured by Ethovision XT, 14 (Noldus Information Technology, Wageningen, The Netherlands).
Health score
To obtain a comparative analysis of the functional outcome, each experimental subject was rated according to a health score calculated on functional outcomes, as shown previously [13]. Briefly, the Barnes maze, EPM, neuroscore, and SNAP performance data sets obtained in motavizumab-treated mice (N = 19) were stratified into four groups according to quartiles. Each quartile was attributed a score ranging from 4 (best) to 1 (worst outcome). Each mouse obtained a final score, which was the sum of the weighted scores of the four parameters, each accounting for 25% of the final score. The effect size (odds ratio) was calculated by a Chi square test using the Woolf logit interval for computing the 95% confidence interval (CI), stratifying mice in terms of good outcome (> 3) versus bad outcome (≤ 3). Odds ratios with 95% CI were reported in the Forest plot to visualize the strength of the association between the treatments and the functional outcome.
Sample collection
Blood for longitudinal assessment was collected from the submandibular vein at 4 days and 6 weeks after TBI. Clotting and complement activation was prevented by collecting samples into BD Microtainer™ Tubes containing K2EDTA as anticoagulant (BD, ref 365,975). Blood was centrifuged at 2000 × g for 15 min at 4 °C and plasma was then stored at − 80 °C before analysis.
Blood biomarker measurements
Single Molecule Array (Simoa): The Simoa assays were performed using the Simoa™ NF-light Advantage Kit (item 103,400), Simoa™ mouse Tau Discovery Kit (item 102,209) and run on the Simoa SR-X platform following the manufacturer instruction (Quanterix Corp, Boston, MA, USA) [21].
AlphaLISA™: The AlphaLISA assay was performed using the Mouse Matrix Metalloproteinase 9 (MMP9) kit (item AL519C, Revvity) for MMP9. AlphaLISA signals were measured using an Ensight Multimode Plate Reader (Revvity) [21].
Lectin pathway activity assay
The LP activity was measured in mouse plasma samples obtained at 4 days after TBI or sham injury. For ex vivo LP assessment, EDTA-plasma samples (2.5% final plasma concentration) were incubated on mannan-coated ELISA plates for 15 min to initiate activation followed by detection of C3c deposition as described elsewhere [22]. Briefly, EDTA-plasma samples were thawed on ice and suspended in barbital buffered saline (BBS; 4 mM barbital, 145 mMNaCl, 2 mM CaCl2, 1mM MgCl2, pH 7.4), to a final plasma concentration of 2.5%. Plasma solutions were incubated on the coated plate at 37 °C for 15 min. The plate was washed and incubated for 1 h 30 min at RT with a polyclonal anti-C3c antibody (Dako, A0062) diluted 1:5000 in washing buffer. After washing, the plate was incubated with an alkaline-phosphatase labelled goat anti-rabbit IgG antibody (Sigma A-3812) diluted 1:5000 in washing buffer for 1 h 30 min at RT. Following washing, the assay was developed by adding 100 µL substrate solution (Sigma Fast p-Nitrophenyl Phosphate tablets, Sigma) and the absorption at 405 nm measured using the Infinite M200 spectrofluorimeter managed by Magellan software (Tecan, CH).
Histological analysis
Sacrifice and tissue collection
Mice were transcardially perfused with chilled paraformaldehyde (4% in PBS). The brains were transferred to 30% sucrose in PBS at 4 °C overnight for cryoprotection. Then they were frozen by immersion in isopentane at − 45 °C for 3 min before being sealed into vials and stored at -80 °C until use. For lesion size determination, twenty-micron coronal brain cryosections were cut serially at 200-µm intervals and stained with cresyl violet (Sigma Aldrich).
Assessment of contusion volume
Eight coronal sections from bregma + 1.2 mm to − 4 mm were acquired from each mouse and visualized at 2x magnification with an Olympus BX-61 Virtual Stage microscope with a pixel size of 3.49 mm. The injured area was calculated by subtracting the contralateral hemisphere minus the ipsilateral hemisphere as previously described [13].
Neuronal loss
Three 20-µm coronal sections at 0.4, 1.6, and 2.8 mm posterior to bregma and stained with Cresyl violet (Sigma-Aldrich, St. Louis, MO) were selected from each mouse brain to quantify neuronal cell loss. The entire sections were acquired with an Olympus BX-61 Virtual Stage microscope using a 20x objective lens, with a pixel size of 0.346 μm. Acquisition was done over 10-µm thick stacks with a step size of 2 μm. The different focal planes were merged into a single stack by mean intensity projection to ensure consistent focus throughout the sample. Neuronal count was performed by segmenting the cells over a cortical region located within 350 μm from the contusion edge and in the corresponding contralateral hemisphere. The segmented cells with a round-shaped signal sized < 25 mm2, corresponding to glial cells, were excluded from the analysis. Quantification was performed by Fiji software. Neuronal loss was calculated as follow: 1- (#neurons in ipsilateral cortex / #neurons in contralateral cortex). Value = 0 indicates no neuronal loss.
Study design and randomization
The study was designed in two parts, a pilot study to compare α-MASP-2 to the control Ab and a follow-up study to compare α-MASP-2 to α-MASP-1 and control Ab. In both studies we produced a randomization list using randomize.org. Mice were randomly allocated to surgery (sham or TBI) and to the treatment in a balanced manner through the different experimental days. For the pilot study, we dedicated two days for surgeries, each with a total of 24 operated mice. The allocation ratio in each day was 1:1 for surgery (sham: TBI) and treatment (α-MASP-2:control Ab). For the follow-up study, we dedicated four days for surgery, three with a total of 20 and one with a total of 18 operated mice. The allocation ratio in each day was 1:3 for surgery (sham: TBI) and 1:1:1 for treatment (α-MASP-2: α-MASP-1:control Ab). A detailed report of mice allocation to experimental days and groups of the follow-up study is accessible in the online data repository linked to this paper (see ‘Availability of data and materials’).
Sample size and statistics
The first part of the study, in its explorative nature, relied on the usual numerosity applied for this type of study, i.e. a n = 12 per experimental group and for both sham and TBI mice. When we designed the comparative study between α-MASP-2 and α-MASP-1, we pre-defined a primary study endpoint – the decrease in cognitive deficits calculated by Barnes maze test – which was used to calculate the sample size. We considered a 50% cognitive deficit reduction, corresponding to actual animals’ better performance (e.g., half of the time spent to reach the escape tunnel compared to the expected time of 40 s for a TBI mouse). Group size was 19 defined by the formula: n = 2σ2f(α,β)/Δ2 (SD in groups = σ, type 1 error α = 0.02, type II error β = 0.2, the percentage difference between groups Δ = 50). The standard deviation used in the formula for each assessment was calculated based on previous experiments with the same outcome measure (e.g., latency seconds to reach the escape tunnel), resulting in σ = 49 and n = 19. To avoid an excessive total number of mice and being mainly interested in the comparisons among TBI mice, we decided to limit the number of sham mice at n = 7. A further reason of doing so was that the pilot study indicated a limited variability in the sham group assessing the latency time to escape in the Barnes maze. The data distribution was assessed using tests for normality (D’Agostino & Pearson, Shapiro-Wilk, or Kolmogorov-Smirnov tests). Group comparisons were conducted using t-tests or Mann-Whitney tests, or relevant two-way analysis of variance (ANOVA) followed by the appropriate post hoc test. Equal variances were checked by Bartlett’s test and, if not equal, a Welch’s correction was applied to the test. The identification of outliers was performed by ROUT method setting Q to 0.5%. Any removed outlier is detailed in the figure legends. Statistical analyses were performed using standard software package GraphPad Prism (GraphPad Software Inc., San Diego, CA, USA, version 9.0). All data were presented as mean and standard deviation (SD). P-values lower than 0.05 were considered statistically significant.
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