Mercurio et al. Journal of Neuroinflammation
https://doi.org/10.1186/s12974-024-03133-4(2024) 21:141

Journal of Neuroinflammation

Open Access

RESEARCH

Inhibition of mannan-binding lectin
associated serine protease (MASP)-2 reduces
the cognitive deficits in a mouse modelof severe traumatic brain injury
Domenico Mercurio1†, Francesca Pischiutta1†, Serena Seminara1, Francesca Tribuzio1, Ilaria Lisi1, Laura Pasetto2,
Valentina Bonetto2, Maria-Grazia De Simoni1, Wilhelm Schwaeble3, Sadam Yaseen4, Thomas Dudler4, Elisa R. Zanier1*
and Stefano Fumagalli1*
Abstract
The lectin pathway (LP) of complement mediates inflammatory processes linked to tissue damage and loss offunction following traumatic brain injury (TBI). LP activation triggers a cascade of proteolytic events initiated by
LP specific enzymes called MASPs (for Mannan-binding lectin Associated Serine Proteases). Elevated serum andbrain levels of MASP-2, the effector enzyme of the LP, were previously reported to be associated with the severityof tissue injury and poor outcomes in patients with TBI. To evaluate the therapeutic potential of LP inhibition in
TBI, we first conducted a pilot study testing the effect of an inhibitory MASP-2 antibody (α-MASP-2), administeredsystemically at 4 and 24 h post-TBI in a mouse model of controlled cortical impact (CCI). Treatment with α-MASP-2
reduced sensorimotor and cognitive deficits for up to 5 weeks post-TBI. As previous studies by others postulateda critical role of MASP-1 in LP activation, we conducted an additional study that also assessed treatment with an
inhibitory MASP-1 antibody (α-MASP-1). A total of 78 mice were treated intraperitoneally with either α-MASP-2, orα-MASP-1, or an isotype control antibody 4 h and 24 h after TBI or sham injury. An amelioration of the cognitive
deficits assessed by Barnes Maze, prespecified as the primary study endpoint, was exclusively observed in the
α-MASP-2-treated group. The behavioral data were paralleled by a reduction of the lesion size when evaluatedhistologically and by reduced systemic LP activity. Our data suggest that inhibition of the LP effector enzyme
MASP-2 is a promising treatment strategy to limit neurological deficits and tissue loss following TBI. Our work has
translational value because a MASP-2 antibody has already completed multiple late-stage clinical trials in other†

Domenico Mercurio and Francesca Pischiutta equal contribution
first-author.
*Correspondence:
Elisa R. Zanier
elisa.zanier@marionegri.it
Stefano Fumagalli
stefano.fumagalli@marionegri.it
Full list of author information is available at the end of the article© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use,sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) andthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in thisarticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not includedin the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you willneed to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The
Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available
in this article, unless otherwise stated in a credit line to the data.

Mercurio et al. Journal of Neuroinflammation(2024) 21:141

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indications and we used a clinically relevant treatment protocol testing the therapeutic mechanism of MASP-2
inhibition in TBI.
Keywords Complement system, Traumatic brain injury, Pharmacology, Mannan-binding lectin-associated serine
proteases

Background
Traumatic brain injury (TBI) is a leading cause of deathand disability worldwide. The most recent Global Burdenof Disease reported an annual incidence across Europeof at least 4 million [1]. TBI represents a high burden topatients, society, and health-care systems, but there arestill limited treatment options available.
TBI pathophysiology is characterized by secondary events that follow the initial impact and evolve overtime and across brain areas. The initial response to TBI
includes protective endogenous mechanisms as well as
detrimental cascades, with the balance contributing toneurological worsening or recovery [2]. Post-TBI inflammation may contribute to secondary pathophysiologicalmechanisms in opposite manners, because it helps limit
the spread of toxic signals and supports tissue remodeling, but can also aggravate TBI consequences by releasing pro-inflammatory mediators.
Activation of the complement system triggers an
inflammatory cascade that contributes to the exacerbation of TBI damage, leading to tissue damage andloss of neurological functions [3, 4]. Depending on
the initiating event, complement activation may occur
through three different pathways: the classical pathway (CP), the alternative pathway (AP) and the lectin
pathway (LP), each composed of specific initiators andeffector enzymes. Non-self-carbohydrate structuresor acetylated proteins exposed on the surface of damaged cells, including apoptotic or necrotic cells andstressed endothelium [5], represent molecular patterns
recognized by LP recognition subcomponents. Binding of LP recognition subcomponents such as the collectins mannan-binding lectin (MBL) and collectin-10(CL-L1) and collectin-11 (CL-K1), or ficolins, such as
ficolin-1 (M-ficolin), ficolin-2 (L-ficolin) or ficolin-3(H-ficolin) and MBL-A and MBL-C, collectin-10 andcollectin-11 and Ficolin A and Ficolin C in rodents
leads to activation of MASPs and initiates complement
activation via the LP.
Among MASPs, it was established that only MASP-2
can cleave both C2 and C4 to generate a C4b2a type C3
convertase, while MASP-1 cleaves C2, but is unable tocleave C4 and MASP-3 cannot cleave either C2 or C4.
MASP-2 can therefore be considered as the single key
initiator of the LP as it can autoactivate and generate a
C3 convertase by its own, with MASP-1 acting as an
auxiliary enzyme that provides some of the activated C2component for the convertase [6, 7]. As such, MASP-2
and MASP-1 are both potential targets to dampen LP
activation.
Previous studies pointed to activation of the LP as a
critical contributor to TBI pathophysiology [8–10]. LP
recognition molecules are found decorating the vessels
pertinent to the injured areas in models of acute brain
injury such as stroke [11] and TBI [9, 10]. In specimens
obtained from patients with severe TBI undergoing
the surgical removal of the lesion, the LP recognition molecules MBL and ficolin-1, -2, and −3 were all
found to be associated with the affected blood vessels[10]. Importantly, MASP-2 was present at higher levels on TBI vessels when compared to brain specimens
obtained from controls, including gliomas, or autoptic
brains of people who died due to extracranial causes[8]. Clinical observations by Osthoff et al. reported
that elevated blood levels of MASP-2 early after TBI
of all severities were associated with poor 90-day outcomes [12].
In our previous work, we modeled TBI by controlled
cortical impact (CCI) in mice carrying gene deletionsof key LP components to study the effects on the neurological functions and pathology. Using MASP-2(Masp2−/−), ficolin-A (Fcna−/−), CL-11 (Colec11−/−),
MASP-1/3 (Masp1−/−), MBL-C (Mbl2−/−), MBL-A(Mbl1−/−) or MBL (Mbl1−/− / Mbl2−/−)-deficient male
C57BL/6J or WT mice, we reported that the absenceof the essential LP effector enzyme MASP-2 was associated with the highest degree of protection after TBI[13].
Mounting evidence suggests that MASP-2 represents
a superior therapeutic target for ameliorating TBI consequences. Since MASP-2 is a low abundance plasma
protein produced exclusively by the liver, systemic inhibition of the LP using anti-MASP-2 antibody (α-MASP-2)
which blocks MASP-2 functional activity is eminently
feasible. To further evaluate the therapeutic potential of
MASP-2 inhibition for the treatment of TBI, we tested
therapeutic intervention protocols using a recombinant
MASP-2 antibody that was previously shown to ameliorate ischemic stroke deficits [14] and COVID-19-induced
brain inflammation [15] in mouse models, and compared it to an anti-MASP-1 antibody. Our data presented
herein suggest that acute α-MASP-2 treatment is a promising new therapeutic approach to improve recovery following TBI.

Mercurio et al. Journal of Neuroinflammation(2024) 21:141

Methods
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 conductedin conformity with institutional guidelines, which comply to National (D.L. n.116, G.U. suppl. 40, 18 February1992) 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 thestudy and No. 9F5F5.206 (approval number 117/2022PR) for the second part of the study.
Mice were housed 5 per cage and kept at a constanttemperature (21° ± 1 °C) and relative humidity (60%) witha regular light/dark schedule (7am-7pm). Food (Altrominpellets 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 thevertical plane and applied vertically to the exposed duramater, between bregma and lambda, over the left parietotemporal 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 thescalp sutured. Sham-operated mice received identicalanesthesia and surgery without brain injury.
HG-4, a derivative of the clinically used MASP-2 inhibitor narsoplimab, was used for MASP-2 inhibition inthese 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 adose 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].

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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 andwere 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 witha habituation trial (day 1) during which the mouse wasplaced in the center of the empty arena under a beakerfor 30 s, then guided to the escape tunnel over 10–15 sby 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 backto its home cage. In the learning phase (days 2–4), mice
were placed for 10 s in the center of the arena and thenallowed to explore the arena for at least 2 min to findthe escape (primary latency) and to enter it (secondary
latency). The test phase (day 5) consisted of the removalof the escape tunnel from the maze to assess the primarylatency [20]. The escape was sealed (‘false escape’) to prevent the mouse from falling through the hole. The abilityof 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 (each35 cm × 5.5 cm) and a central platform (5.5 cm × 5.5 cm)
elevated 60 cm above the ground. Mice were acclimatizedin 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 spentin 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 obtainedin 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Mercurio et al. Journal of Neuroinflammation(2024) 21:141

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 strengthof the association between the treatments and the functional outcome.
Sample collection

Blood for longitudinal assessment was collected fromthe submandibular vein at 4 days and 6 weeks after TBI.
Clotting and complement activation was prevented bycollecting 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 (item102,209) 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 witha polyclonal anti-C3c antibody (Dako, A0062) diluted
1:5000 in washing buffer. After washing, the plate
was incubated with an alkaline-phosphatase labelledgoat 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 100µL substrate solution (Sigma Fast p-Nitrophenyl Phosphate tablets, Sigma) and the absorption at 405 nm

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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, twentymicron 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 Stagemicroscope 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 eachmouse brain to quantify neuronal cell loss. The entire
sections were acquired with an Olympus BX-61 VirtualStage microscope using a 20x objective lens, with a pixel
size of 0.346 μm. Acquisition was done over 10-µm thickstacks 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 thecontusion edge and in the corresponding contralateralhemisphere. 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 asfollow: 1- (#neurons in ipsilateral cortex / #neurons incontralateral 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Mercurio et al. Journal of Neuroinflammation(2024) 21:141

total of 24 operated mice. The allocation ratio in each day
was 1:1 for surgery (sham: TBI) and treatment (α-MASP2:control Ab). For the follow-up study, we dedicated fourdays 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 ofmice allocation to experimental days and groups of thefollow-up study is accessible in the online data repository linked to this paper (see ‘Availability of data andmaterials’).
Sample size and statistics

The first part of the study, in its explorative nature,relied on the usual numerosity applied for this typeof study, i.e. a n = 12 per experimental group and forboth 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 decreasein 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 thetime 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 ingroups = 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 experimentswith the same outcome measure (e.g., latency seconds
to reach the escape tunnel), resulting in σ = 49 andn = 19. To avoid an excessive total number of mice andbeing 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 groupassessing the latency time to escape in the Barnes
maze. The data distribution was assessed using tests
for normality (D’Agostino & Pearson, Shapiro-Wilk, orKolmogorov-Smirnov tests). Group comparisons wereconducted using t-tests or Mann-Whitney tests, or relevant two-way analysis of variance (ANOVA) followedby the appropriate post hoc test. Equal variances werechecked by Bartlett’s test and, if not equal, a Welch’s
correction was applied to the test. The identificationof 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). Alldata were presented as mean and standard deviation(SD). P-values lower than 0.05 were considered statistically significant.

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Results
Our previous work demonstrated that, compared to othercomplement targets, ablation of MASP-2 was associatedwith the highest degree of protection in the mouse CCI
model of TBI [13]. To evaluate the therapeutic potential of MASP-2 inhibition using clinically relevant treatment approaches, a pilot study was conducted according
to the plan depicted in Fig. 1A. The treatment consistedof α-MASP-2 administration at 4 and 24 h post-TBI andmice were followed-up for 6 weeks.
TBI mice receiving α-MASP-2 experienced fasterrecovery of sensorimotor function, which was evident 2
weeks after TBI, and a notable amelioration of the cognitive deficits per the Barnes maze test at 5 weeks after TBI(Fig. 1B and C). Importantly, the α-MASP-2 treated TBI
mice took 24 s less than control mAb treated TBI mice
to reach the escape hole during a 2-minute test, which
is a large reduction of the cognitive deficits. The analysis of the circulating biomarkers NfL and tau, performed
at 6 weeks after TBI, showed no difference between thegroups (Fig. 1D).
To confirm the findings of the pilot study, we evaluatedthe efficacy of α-MASP-2 treatment in a follow-on study
using a larger cohort of mice. This study also included an
additional group of TBI mice treated with α-MASP-1.
This was done because previous reports suggested that
MASP-1 may be needed for efficient MASP-2 and LP
activation [6], whereas our previous work showed that
MASP-1/3-deficient mice (which lack MASP-1 but also
MASP-3 required for activation of the alternative pathway of complement) were not protected in the TBI model[13]. By comparing the inhibition of MASP-1 and MASP2, we aimed to dissect the roles of LP activation enzymes
in the context of TBI. We followed the experimental plan
depicted in Fig. 2A and prespecified cognitive deficits
evaluated by the Barnes maze at 5 weeks after TBI as theprimary outcome.
While improving gradually over time, TBI-induced
sensorimotor deficits persisted till the 4th week ofobservation, and neither antibody treatment improved
recovery of sensorimotor function compared to isotype control (Fig. 2B and C). Cognitive assessmentby the Barnes maze showed increased latency to false
escape after TBI in control antibody-treated mice.
The treatment with α-MASP-2 significantly improved
cognitive deficits compared to isotype control or
α-MASP-1 treatment (latency of 22.06 ± 20.28 s compared to 51.74 ± 43.89 s [p < 0.01] and 59.68 ± 41.42[p < 0.05], respectively) 5 weeks after TBI (Fig. 2D). In
the elevated plus maze, control mAb-treated TBI mice
showed higher anxiety as indicated by less time spentin the closed than open arms compared to sham mice.
While α-MASP-2 treated mice showed a trend toward
improvement, the effect was not statistically significant,Mercurio et al. Journal of Neuroinflammation(2024) 21:141

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Fig. 1 MASP-2 inhibition protects from sensorimotor and cognitive deficits after TBI. (A) The experimental plan. (B) Sensorimotor deficits were assessedusing neuroscore from 1 to 4 weeks following TBI or sham surgery. The neuroscore showed slight, but significant better recovery in the α-MASP-2 treated
TBI mice. Two-way ANOVA for repeated measures (p of surgery < 0.0001) followed by Tukey’s post-hoc comparison test, *p < 0.05. (C) Cognitive deficits
were assessed by Barnes maze test. The α-MASP-2 treated mice showed significantly reduced latency to false escape in the probe trial compared to control mAb treated mice. Two-way ANOVA, p of surgery: <0.01. Tukey’s post-hoc test, *p-value < 0.05, **p-value < 0.01. (D) Quantification of NfL and tau levels
in mouse plasma obtained 6 weeks after TBI. No differences were observed among the treatments in sham or TBI mice. Data in C and D are presented as
scattered dot plot, line at mean ± SD

and no treatment effect was apparent for α-MASP-1
treatment (Fig. 2E).
To obtain a comparative evaluation across treatmentgroups, a health score based on the four behavioral tests(neuroscore, SNAP, Barnes, and EPM) was calculated.
Mice were rated from 1 (bad outcome) to 4 (good outcome; Fig. 2F) based on quartile distribution calculated
for the isotype control-treated mice. The α-MASP-2
treatment was the most protective, showing a positive
association with a good outcome (odds ratio 8.5 [95% CI
1.5–49.5], p = 0.01). The α-MASP-1 treatment showed aweaker, non-significant association with a good outcome(odds ratio 2.3 [95% CI 0.4–14.2], p = 0.37, Fig. 2F).
MMP9 levels did not differ between sham and TBI
mice at 4 days or 6 weeks after TBI, nor were the levels
affected by treatment (Fig. 3A, B). As for NfL, while TBI
mice showed higher plasma levels compared to sham animals 4 days after TBI as expected, no treatment effects
were detected (sham: control Ab: 98.5 ± 41.24 pg/ml ± SD;
α-MASP-2: 90.51 ± 22.89; α-MASP-1: 180.0 ± 87.36;
TBI: control Ab: 1411 ± 293.6; α-MASP-2: 1467 ± 290.3;
α-MASP-1: 1559 ± 494.8, Fig. 3C).

Mercurio et al. Journal of Neuroinflammation(2024) 21:141

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Fig. 2 MASP-2, but not MASP-1 inhibition ameliorates the outcome of TBI mice. A) The experimental plan. B, C) Sensorimotor deficits were assessed using
neuroscore (B) and SNAP (C) from 1 to 4 weeks following TBI or sham surgery. Neither the neuroscore nor the SNAP tests showed significant differences
between the treatments. Two-way ANOVA for repeated measures (p of surgery < 0.0001) followed by Tukey’s post-hoc test. Data are presented as themean ± SD. D) Cognitive deficits were assessed by Barnes maze test. The α-MASP-2 treated mice showed significantly reduced latency to false escapein the probe trial compared to α-MASP-1 or control mAb treated mice. Two-way ANOVA, p of surgery: <0.01. Tukey’s post-hoc test, *p-value < 0.05, **pvalue < 0.01, ***p-value < 0.001. Outlier identification per Rout, Q 0.5%:1 outlier sham control mAb, 1 outlier sham α-MASP-1, 3 outliers TBI α-MASP-2. Data
are presented as scattered dot plot, line at mean ± SD. E) Anxious behavior was assessed by the elevated plus maze and expressed as seconds spent in theclosed arms minus those in the open arm. Two-way ANOVA, p of surgery < 0.001. Data are presented as scattered dot plot, mean ± SD. F, F’) Associationof the treatments with the functional outcome of TBI mice over 5 weeks. The health score was obtained by rating mice from 4 (good outcome) to 1 (badoutcome) based on quartiles (Q) of the Barnes, EPM, neuroscore and SNAP values over the 5 weeks of observation. The final score was the sum of theweighted scores of the four parameters each accounting for 25% of the final score (F). The odds ratio was calculated by a Chi square test using the Woolf
logit interval for calculating the 95% confidence interval (CI 95%), stratifying mice in terms of good outcome (defined as a score > 3) versus bad outcome(score ≤ 3). Forest plot showing that the α-MASP-2 treatment was significantly associated with a good outcome (F’)

To verify LP suppression in α-MASP-1 and α-MASP2-treated mice, we evaluated LP functional activity inplasma samples collected during the treatment phaseusing ex vivo functional assays [15, 23]. As expected, bothα-MASP-1 and α-MASP-2 treatment resulted in a significant reduction of LP-induced C3b deposition compared
to isotype control treatment in both sham and TBI mice
(Fig. 3D).

Mercurio et al. Journal of Neuroinflammation(2024) 21:141

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Fig. 3 MASP-2 or MASP-1 inhibition reduce LP activation, but not the levels of circulating biomarkers of neuronal or vascular damage. A) Quantification of MMP9 levels in mouse plasma obtained 4 days (A) and 6 weeks (B) after TBI or sham injury. No differences were observed among the treatments
in sham or TBI mice. C) Measurement of NfL levels in mouse plasma obtained 4 days after TBI or sham injury. NfL levels increased significantly after TBI
without any difference among the treatments. Two-way ANOVA, p of surgery < 0.0001. Data are presented as scattered dot plots, mean ± SD. D) Quantification of C3b deposition on mannan-coated plates exposed to mouse plasma collected 4 days after TBI/sham surgery. Mice treated with α-MASP-2 andα-MASP-1 showed a significant reduction in C3b levels, indicating a reduced activation of the lectin pathway. Two-way ANOVA, Tukey’s post-hoc test,**p-value < 0.01, *** p-value < 0.001. Data are presented as scattered dot plot, line at mean ± SD

In our final analysis, we gathered the data from all micetreated with α-MASP-2 in the two studies and included
a histological analysis of the lesion. We observed that
TBI mice treated with the α-MASP-2 had a cognitive
improvement (52.78 ± 41.51 vs. 20.56 ± 17.23 latency seconds ± SD; Cohen’s d = 1.01, Fig. 4A), which was associated with a decreased lesion volume (18.24 ± 4.91 versus
21.03 ± 3.10; Cohen’s d = 0.68, Fig. 4B) compared to micereceiving isotype control treatment. In contrast, no differences in neuronal loss were observed (Fig. 4C).

Discussion
In this series of studies, we aimed to evaluate an interventional treatment schedule targeting MASP-2, the effector
enzyme of the LP, whose detrimental roles in TBI werepreviously documented in experimental TBI models [13].
The data reported herein show that treating TBI micewith α-MASP-2 for a short time post-TBI (α-MASP-2
administered at 4- and 24-hours post-TBI) reduced thecognitive deficits at 5 weeks post-TBI.
A key role for MASP-2 in TBI pathophysiology waspreviously suggested by Osthoff et al. based on an observational clinical study reporting that high circulating
MASP-2 levels early after injury were associated withpoor outcomes at 90 days post-TBI [12]. These data
were paralleled by our own observations in patients withsevere TBI, which documented increased intracerebral
levels of MASP-2 at the contusion site compared to controls [8]. Elevated MASP-2 brain levels were associatedwith increased TBI severity, as identified by altered pupillary reactivity and the presence of subarachnoid hemorrhage [8]. Collectively, these data support the evaluationof α-MASP-2 as a potential treatment to counteract andmitigate brain damage and improve outcomes after TBI.
The α-MASP-2 used in this study is a derivative of narsoplimab, an experimental drug that has already been
evaluated in clinical trials for stem cell transplant-associated thrombotic microangiopathy, COVID-19 infection, and glomerular diseases. As such, the molecule has
already shown clinical safety and effective targeting of
MASP-2 in humans, holding the promise for its future
use in TBI patients. Young adults represent the largest demographic among severe TBI patients, with men
facing more than double the risk compared to women[24] thus we first tested our intervention in male mice.
However it is important to note that women tend to
experience poorer outcomes, particularly in terms of
post-injury depression and anxiety [25], highlighting theMercurio et al. Journal of Neuroinflammation(2024) 21:141

Page 9 of 11

Fig. 4 Aggregated data from the two studies relative to the α-MASP-2 treatment. (A) Barnes maze test showing a strong amelioration of cognitive deficits
in the α-MASP-2 treated TBI mice at 5 weeks after injury. Two-way ANOVA, p of surgery: <0.0001. Tukey’s post-hoc test, **** p-value < 0.0001. (B) Lesion
volume. The microphotographs (scale bar 1 mm) indicate the lesion induced by the CCI model (colored surface). Visual inspection and volume quantification showed a decreased size of the lesion in the α-MASP-2 treated TBI mice at 6 weeks after injury. Unpaired t-test, * p-value < 0.05. (C) Neuronal loss. The
microphotographs (scale bar 1 mm) show brain slices at the maximal burden of the lesion, with inserts (scale bar 100 μm) highlighting the Cresyl violet
staining in the proximity of the contusion edge, where the quantification of viable neurons was done. The neuronal loss did not differ between groups at
6 weeks after injury. Unpaired t-test, p-value = 0.49. All data are presented as scattered dot plot, line at mean ± SD

importance to investigate sex-dependent pathologicalmechanisms. Furthermore, TBI epidemiology is shifting
to the old-age individuals, where sex differences in TBI
prevalence are less prominent and comorbidities may
play a major role in modifying disease trajectories. Therefore, future studies should be conducted to extend our
research to experimental models incorporating biologicalvariables such as sex, age, and comorbidities to accurately
assess the efficacy of α-MASP-2.
The treatment schedule in the present study was
designed according to a time-to-treatment which is feasible in the clinical setting, i.e. the first α-MASP-2 dosing was administered 4 h after the injury, and tested in
two independent cohorts of mice. The second cohort also
included a group treated with α-MASP-1 for comparisonand prespecified improvement in the long-term cognitive
performances (Barnes maze test 5 weeks after TBI) as theprimary endpoint. In aggregate, the study demonstrated
that inhibition of MASP-2, but not of MASP-1, improved

the cognitive deficits in the TBI mice with a modest protection of the sensorimotor functions. We also observed
smaller lesion volumes at sacrifice (6 weeks after TBI)
with α-MASP-2 treatment, but no preservation of thenumbers of neurons at the contusion edge. This latter
observation aligns with the observation that circulating
NfL levels were not affected by treatment.
A previous report showed that complement activation via LP promotes blood-brain barrier breakdown
and exacerbates axonal damage after TBI [26]. We canhypothesize that at least part of the beneficial effects
associated with MASP-2 inhibition may be due to reduction of the vasogenic edema, which was mirrored by the
reduced volume of the lesion. To explore this further, weanalyzed the blood biomarker of vascular injury MMP9
as early as 4 days after TBI. However, MMP9 levels were
unchanged after TBI thus limiting its utility as a proxy for
vascular damage in our study. By contrast, NfL blood levels were increased in TBI vs. sham mice. Given the role

Mercurio et al. Journal of Neuroinflammation(2024) 21:141

of the LP in exacerbating the axonal damage, it is unclear
why we were unable to observe a reduction of circulating
NfL. This observation suggests that a further optimization of the treatment schedule may be needed for optimal
efficacy of α-MASP-2 treatment.
Of note, while both α-MASP-2 and α-MASP-1 treatment suppressed systemic LP activity as demonstrated by
reduced ex vivo LP activity in plasma samples collected
4 days after sham or TBI surgery, only the inhibition of
MASP-2 resulted in appreciable protection from TBI.
While we have no clear explanation for the apparent lackof protection for α-MASP-1 treatment, the results presented here are consistent with our previous work comparing experimental TBI outcomes in MASP-2-deficientand MASP-1/3-deficient mice, suggesting that commonlyused LP activity assays, such as the ex vivo assay used inthis study, are too blunt to assess the relative effectiveness of different LP blocking agents. Briefly, in the assay
we incubated plasma on mannan-coated plates andmeasured the formation of C3b active fragments, which
requires C4bC2 convertases downstream to recognition of danger signals by MBL. MASP-2 was reported toinduce direct cleavage of C3 into its active fragments inabsence of C2 and C4 in specific circumstances, including ischemia/reperfusion injury of the gut or the brain[27]. If our in vitro LP activation assay failed to reproduce
this direct mechanism of C3 cleavage by MASP-2, whichmight occur in vivo, it is explained why, although the
inhibition of both MASPs counteracted the LP activationin vitro, only that of MASP-2 was protective after TBI.
A different interpretation of our results relies on theobservation by Degn et al., who have reported MASP-2
activation downstream to a rapid MASP-1 activation[28]. While the hypothesis is not fully supported by ourprevious observations in MASP-1/3-deficient TBI mice,
if this is the case for our TBI model, the first intervention
at 4 h might have been too late for inhibiting MASP-1,
but well timed for MASP-2, which was already activatedat the point of intervention. Thus, only MASP-2 inhibition would be effective, supporting the therapeutic
approach proposed by our study. As we analyzed theLP activation at 4 days after TBI, it is possible that weselected a too late time-point to display relevance for theacute injury, when activated MASPs are relatively quickly
inhibited by circulating enzyme inhibitors such as C1inh
and alpha-2-macroglobulin. Hence the LP activation at
day 4 could rely on renewed MASP activation, which was
efficiently inhibited by α-MASP-1 or α-MASP-2.

Conclusions
Our data point to inhibition of the essential LP effector
enzyme MASP-2 as a promising therapeutic intervention for the treatment of TBI. Among the key aspects ofPage 10 of 11

our study are: (1) we applied a clinically relevant protocol with a post-TBI treatment, (2) the study was done in
two independent cohorts, demonstrating a similar amelioration of cognitive deficits, (3) we used an inhibitory
antibody derived from an antibody that has already completed multiple late-stage clinical trials in other indications, and (4) we targeted a hepatically expressed plasma
protein, thereby assuring target blockade in the brain
without the antibody needing to cross the blood-brain
barrier.
Abbreviations
CCI	Controlled cortical impact
CI	Confidence interval
EPM	Elevated plus mazei.p.	IntraperitonealLP	Lectin pathway
MASPs	Mannan binding lectin associated serine proteases
MBL	Mannan binding lectin
MMP9	Matrix matalloproteinase-9
NfL	Neurofilament light chain
SD	Standard deviation
SNAP	Simple Neuroassessment of Asymmetric imPairment
TBI	Traumatic brain injury
Acknowledgements
None.
Author contributions
DM, FP executed the experiments, analyzed the data and drafted the msSS, FT,
IL, LP, VB executed the experiments, analyzed the dataMGDS, WS, SY, TD, ERZ,SF designed the work, analyzed the data, drafted the ms.
Funding
The study received support from Omeros Corporation, Seattle, WA.
Data availability
All data will be publicly available at this online repository on Figshare: 10.6084/
m9.figshare.25508812.

Declarations
Ethics approval and consent to participate
The project was approved by the Italian Ministry of Health with the codes
No. 9F5F5.148 (approval number 80/2020-PR) and No. 9F5F5.206 (approval
number 117/2022-PR).
Consent for publication
All authors agreed on the content of this manuscript before its submission.
Competing interests
SY and TD are employees of Omeros Corporation.
Author details
1
Department of Acute Brain and Cardiovascular Injury, Istituto di Ricerche
Farmacologiche Mario Negri IRCCS, Milan, Italy2
Department of Neuroscience, Istituto di Ricerche Farmacologiche Mario
Negri IRCCS, Milan, Italy
3
Department of Veterinary Medicine, School of Biological Sciences,
University of Cambridge, Cambridge, UK
4
Omeros Corporation, Seattle, WA, USA
Received: 30 March 2024 / Accepted: 15 May 2024

Mercurio et al. Journal of Neuroinflammation(2024) 21:141

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