Haupeltshofer et al. Journal of Neuroinflammation
https://doi.org/10.1186/s12974-024-03149-w

Journal of Neuroinflammation(2024) 21:155

Open Access

RESEARCH

Delayed plasma kallikrein inhibition fosters
post-stroke recovery by reducing thromboinflammation
Steffen Haupeltshofer1*, Stine Mencl1, Rebecca D. Szepanowski1, Christina Hansmann1, Ana I. Casas1,2,
Hanna Abberger3,4,5, Wiebke Hansen3, Alina Blusch6, Cornelius Deuschl7, Michael Forsting7, Dirk M. Hermann1,8,Friederike Langhauser1† and Christoph Kleinschnitz1†
Abstract
Activation of the kallikrein-kinin system promotes vascular leakage, inflammation, and neurodegeneration inischemic stroke. Inhibition of plasma kallikrein (PK) – a key component of the KKS – in the acute phase of ischemic
stroke has been reported to reduce thrombosis, inflammation, and damage to the blood-brain barrier. However,
the role of PK during the recovery phase after cerebral ischemia is unknown. To this end, we evaluated the effectof subacute PK inhibition starting from day 3 on the recovery process after transient middle artery occlusion(tMCAO). Our study demonstrated a protective effect of PK inhibition by reducing infarct volume and improving
functional outcome at day 7 after tMCAO. In addition, we observed reduced thrombus formation in cerebral
microvessels, fewer infiltrated immune cells, and an improvement in blood-brain barrier integrity. This protective
effect was facilitated by promoting tight junction reintegration, reducing detrimental matrix metalloproteinases,
and upregulating regenerative angiogenic markers. Our findings suggest that PK inhibition in the subacute phasemight be a promising approach to accelerate the post-stroke recovery process.
Keywords Thrombo-inflammation, Thrombosis, Ischemic stroke, Recovery, Subacute, Plasma kallikrein, Kallikreinkinin system, Blood-brain barrier, Extravasation, Inflammation†

Friederike Langhauser and Christoph Kleinschnitz contributing
senior authors equally to this work.
*Correspondence:
Steffen Haupeltshofer
steffen.haupeltshofer@uk-essen.de1
Department of Neurology and Center for Translational Neuro- andBehavioral Sciences (C-TNBS), University Hospital Essen, Hufelandstr. 55,
D-45147 Essen, Germany
2
Department of Pharmacology & Personalized Medicine, MeHNS, Facultyof Health, Medicine & Life Science, Maastricht University, Maastricht, The
Netherlands
3
Institute of Medical Microbiology, University Hospital Essen, Virchowstr.
179, D-45147 Essen, Germany

4

Division of Immunology, Walter and Eliza Hall Institute of Medical
Research, Parkville, VIC 3052, Australia
5
Department of Medical Biology, University of Melbourne, Parkville,
VIC 3052, Australia
6
Department of Neurology, Center for Huntington’s Disease NRW, St.
Josef-Hospital, Ruhr-University Bochum, Gudrunstr. 56, D-44791 Bochum,
Germany
7
Institute of Diagnostic and Interventional Radiology and Neuroradiology,
University Hospital Essen, Hufelandstr. 55, D-45147 Essen, Germany
8
Chair of Vascular Neurology, Dementia and Ageing, Departmentof Neurology, Medical Research Centre, University Hospital Essen,
Hufelandstr. 55, D-45147 Essen, Germany© 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.

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Background
Ischemic stroke is a prevalent neurovascular event,impacting more than 60 million patients per year worldwide [1]. Currently, the only available treatments for cerebral ischemia are thrombolysis with tissue plasminogenactivator or mechanical thrombectomy [2, 3]. However,these treatments are restricted to a short time window
in the acute phase after stroke; currently, no treatment
options support the recovery phase after stroke.
Following ischemic stroke, thrombus formation andinflammation are closely connected, a phenomenon
referred to as thrombo-inflammation [4]. The kallikreinkinin system (KKS) represents an interface between
thrombotic and inflammatory circuits and consists ofserially connected serine proteases, initiated by theactivation of factor XII (FXIIa). Specifically, FXIIa activates plasma kallikrein (PK) leading to cleavage of highmolecular weight kininogen and the release of the proinflammatory peptide hormone bradykinin, thus fostering
disruption of the blood-brain barrier (BBB) and cerebral
inflammation [4].
Previous investigations have suggested that inhibition of KKS components improves stroke outcomes anddecreases infarct volumes in acute ischemic stroke [5].
Factor XII inhibition and its genetic depletion mediatedischemic stroke protection with beneficial neurologicaloutcomes, without increasing the intracranial bleedingrisk [6, 7]. Similarly, mice with genetic depletion of kininogen showed less susceptibility to post-ischemic brain
damage by a reduction in the inflammatory response, lessmicrovascular thrombosis, and improved BBB integrity[8]). Acute PK inhibition or genetic depletion of PK also
mediated protection in experimental ischemic stroke by
reducing infarct size and inflammation and enhancing
cerebral blood flow [9]. However, these studies addressed
KKS inhibition in the acute phase of cerebral ischemia
starting at the time of reperfusion or in genetic depletion
models. So far, the impact of delayed KKS inhibition on
stroke recovery has not been evaluated.
To this end, we investigated the effect of PK inhibition in the subacute phase after ischemic stroke in micesubjected to transient middle cerebral artery occlusion(tMCAO). Our aim was to evaluate the effects of delayed
treatment with αPK antibodies (starting from day 3 poststroke) on neurological recovery, thrombosis, inflammation, and BBB integrity.

NRW, LANUV) and guided by the German Welfare Act(German Ministry of Agriculture, Health, and Economic
Cooperation). Male C57BL/6J mice were purchased fromCharles River (Sulzfeld, Germany) and housed in a temperature- and humidity-controlled, specific pathogenfree animal facility with a 12-h light-dark cycle, as well
as food and water available ad libitum. Mice were randomly assigned to experimental groups by investigators
not involved in the data acquisition or analysis. All efforts
were made to minimize animal suffering and the numberof animals used. Surgeries, treatments, and data analyses
were performed by investigators blinded to the experimental animals. Experimental analyses were mainly
limited to male mice for reasons of consistency with previous studies, in which we evaluated the effects of PK
inhibition on ischemic injury and neurological outcomein the acute stroke phase in male mice [9].

Methods
Experimental animals

All experiments were conducted in accordance with the
IMPROVE guidelines for animal experimentation [10]and reported based on ARRIVE guidelines [11]. The study
was approved by the regional government authorities(Landesamt für Natur, Umwelt und Verbraucherschutz

Ischemia model and neurological assessment

In 10-12-week-old mice, focal cerebral ischemia was
induced by tMCAO for 60 min occlusion time [12]. Mice
were anesthetized with 4% isoflurane (Piramal) in 100%
oxygen for 3–5 min (World Precision Instruments, Small
Animal Anesthesia System, EZ-7000). Anesthesia wasmaintained with ∼ 2% isoflurane, and body temperatures
were kept at 37 °C during surgery, using a feedback-controlled warming device (World Precision Instruments,
Small Animal Anesthesia System, EZ-7000). Occlusion of the middle cerebral artery (MCA) was induced
as described [12]. In brief, the filament (#602112PK5Re,
Doccol) was introduced to the MCA via the external
carotid artery with a reopening of transient ligation at thecommon carotid artery to ensure full reperfusion. Cerebral blood flow was measured for successful occlusion
and reperfusion using Laser Doppler flowmetry (PeriFlux
6000, Perimed, Järfälla, Sweden). Neurological deficits
following tMCAO were measured utilizing a modified
version of the neuroscore [13]. This score is composed ofa global and focal deficit subscore. Points covered by the
global score include spontaneous activity and fur abnormalities, while focal deficits encompass body symmetry,gait circling behavior, symmetry of paws and body, andwhisker response. In this study, we used the combined
score. Motor strength, sensorimotor coordination, andendurance were measured using the rotarod (RR) test
(Rotarod Advanced for Mice, TSE Systems, Germany)[14], adhesive removal (AR) test [14], and the tightrope (TR) test [15], which were performed as recently
described by our group [16]. Better behavioral results
were reflected by higher point or time scores in the RR
and TR tests. In the AR test and the modified neuroscore,less time or lower point scores were indicative of a better
behavioral performance. All analyses were carried out ina day 3 and day 7 time frame after tMCAO.

Haupeltshofer et al. Journal of Neuroinflammation(2024) 21:155

MRI scanning and analyses

To analyze infarct volume and identify intracerebral
bleeding, we performed serial stroke assessment using
magnetic resonance imaging (MRI) with a 3 Tesla MRI
unit (Biograph mMR, Siemens Healthineers, Erlangen/Germany) on days 3 and 7 after tMCAO. We used dual
channel surface coils designed for animal experimentation (Rapid Biomedical GmbH, Rimpar/Germany). The
imaging protocol included a coronal T2-weighted, turbo
spin-echo (TSE) sequence (resolution 0.2 × 0.2 × 0.8 mm³,
15 slices, TE = 105 ms, TR = 1660 ms, TA = 3:47 min) to
identify infarct volume, and a susceptibility-weighted,
coronal T2-weighted gradient echo sequence (SWI,
0.1 × 0.1 × 0.2 mm³, 40 slices, TE = 20.6 ms, TR = 38 ms,
TA = 6:01 min) to detect hemorrhage. Refined localization was done with the help of short T1-weighted TSE
sequences along each axis (0.2 × 0.2 × 1 mm³, seven slices,
TA = 17 s). Brain edema and infarct were measured from
MRI coronal T2 images. Percentage brain edema wascalculated based on the following equation: ([ipsilateralhemisphere volume – contralateral hemisphere volume]/[contralateral hemisphere volume]) × 100. Edema wascalculated as follows: ([contralateral hemisphere volume]– [ipsilateral hemisphere volume]) x100. By multiplying
the area of tissue loss by the distances between selected
brain sections, brain infarct volume and volume of tissue loss was determined. Only mice without bleeding
and comparable infarct volume between 50 and 70 mm3
on day 3 were included in the experiment. Infarct sizes
were additionally visualized using triphenyl tetrazolium
chloride (TTC) and hematoxylin and eosin (H&E) staining. Of 63 mice with tMCAO, 54 were included in the
final analysis (n = 18 each group). 9 mice were excluded
due to either hemorrhagic transformation or inconsistentinfarct sizes.

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Pharmaceuticals, EK-009-01) and proceeded as describedin the manufacturer’s protocol. Inflammatory cytokines (IL-1β, IL-6, RANTES, MCP-1, MCP-2, GM-CSF,
TNFα), degradation markers (MMP-2, MMP-9) andangiogenic markers (VEGF, PDGFb) were measured
using R&D Systems customized LUMINEX plates. Samples were processed according to the manufacturer’s protocol, measured with a MAGPIX Luminex device and thedata analyzed with the Luminex xPONENT software.
Flow cytometry

Animals were euthanized and perfused with cold saline.
Brains were dissected into the ipsilateral (left) and contralateral (right) hemisphere. Immune cells were isolatedusing the Neural Tissue Dissociation Kit™ (Miltenyi Biotec) and a gentleMACS dissociator with heaters (Miltenyi Biotec), following the manufacturer’s instructions.
Fluorochrome compensation was performed with BD
Diva integrated compensation matrix and single-stained,
mixed-tissue controls. Flow cytometric analysis was performed on the FACSAria III flow cytometer (BD Bioscience). Data analyses were performed using FlowJo
software. Antibodies were used as following: anti-CD3
PacBlue (100,214, Biolegend, 1:400), anti-CD45-HorizonV500 (561,487, Biolegend, 1:400), eFluor 780 Viabilitydye (103,210, Thermo Fisher, 1:400), anti-CD11b-APC(101,212, Biolegend, 1:400), and anti-Ly6G-PerCP-Cy5.5(127,615, Biolegend, 1:400).
Histological and western blot analyses

Based on the MRI infarct volume calculation on day
3, animals with comparable infarct sizes (50–70 mm3)
were included for further experimentation. Animals
were randomly assigned to treatment groups from outside assistance not involved in data acquisition or analysis. Antibodies to block PK (αPK, Ab1006, Abcam) or its
corresponding isotype control (immunoglobulin G (IgG);
BioLegend) were administered intravenously (tail vein) as
follows: 400 µg/kg on days 3 and 4 and 200 µg/kg at days
5 to 7 after stroke induction. The antibody concentration
was based on our previous study of PK inhibition in theacute phase of ischemic stroke [9].

Coronal brain sections were subjected to immunofluorescence staining as described previously [16]. The following primary antibodies were used for histologicalstainings: anti-PK (αPK; ab1006, Abcam, 1:200), antizonula occludens (ZO)-1 (ab216880, Abcam, 1:200),
anti-Claudin-5 (ab131259, Abcam, 1:200), anti-occludin(ab216327, Abcam, 1:200), anti-CD31 (mca2388, BioRad,
1:100), anti-GPIX (M052-0, Emfret, 1:100), anti-albumin(ab207327, Abcam, 1:200), anti-bradykinin-1 receptor (B1R; ABR-011, Alomone Labs, 1:200), anti-CD11b(MCA74G, Biorad, 1:200, unfixed tissue) and anti-NG2(ab5320, Millipore, 1:200). All secondary antibodies werediluted 1:1000. Fluorescence stainings were visualizedusing a Leica DMi8 microscope, Hamamatsu C1144022 CU camera, and Leica Application Software X (LasX
3.0.2.16120). Images were processed using Image J(National Institutes of Health). Western blot analyses
using anti-GP1bα (M043-0, Emfret, 1:200) was performed as previously described [9].

Enzyme-linked immunosorbent assay (ELISA)

Quantitative real-time PCR analyses

To investigate activation of the KKS after tMCAO in
respective treatment conditions, we measured serum
levels of PK (Arigo ARG82751) and bradykinin (Phoenix

To analyze transcriptional changes in the brain or purified endothelial cells from tMCAO animals, real-timepolymerase chain reaction (PCR) was performed. The

Pharmacological intervention

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procedure of using the SYBR green system (Promega,
A6001) was as previously described [17] and processing took place on a QuantStudio 3 RT-PCR machine(Life Technologies). In brief, endothelial cells were isolated from the ipsilateral hemisphere of tMCAO miceusing Kit1 for dissociation of inflamed neural tissue(Miltenyi Biotec, #130-110-201). Endothelial cells werethen purified using the CD31 MicroBeads isolation kit(Miltenyi Biotec, #130-097-418). For both the wholebrain sections and the purified cells, mRNA was isolated (RNeasy MicroKit, Qiagen, 74,004) and converted
into cDNA (QuantiTect Reverse Transcription Kit, Qiagen, 205,311), as per the manufacturer’s protocol. The
following sequence-specific sense (sen) and antisense(ase) primers were designed to measure mRNA expression levels: arginase 1 (Arg-1; Fwd CAT TCT TCG CTG
CCA TTC TG; Rev GCA CAT TGC CCA TGT TGAATC), Ras-related C3 botulinum toxin substrate 1 (Rac1;Fwd GGG GAT CCC AGG CCA TCA AGT GTG TGG
TGG; Rev GGA ATT CTT ACA ACA GCA GCA GGC
ATT TTC TCT TCC), vascular endothelial growth factor (VEGF; Fwd GGA GAT CCT TCG AGG AGC ACT
T; Rev GGC GAT TTA GCA GCA GAT ATA AGA A),
Rho-associated kinases (ROCK; Fwd AAC ATG CTG
CTG GAT AAA TCT GG; Rev TGT ATC ACA TCG
TAC CAT CCT), alpha-5 integrin (α5; Fwd TGT CAC
CGT CCT TAA TGG; Rev CAT TGT AGC CGT CTT
GGT), β-actin (Fwd CCA AGG CCA ACC GCG AGA
AGA TGA C; Rev AGG GTA CAT GGT GGT GCC GCCAGA C), and glyceraldehyde-3-phosphate dehydrogenase(GAPDH; Fwd AGG TCG GTG TGA ACG GAT TTG;
Rev TGT AGA CCA TGT AGT TGA GGT C). Relative
target mRNA expression was normalized to the geometric expression average of the housekeeping genes β-actin
and GAPDH. We applied each sample in two technicalreplicates for each data point. The mean cycle threshold(Ct) was used in the equation for the housekeeping genes
and Ct for the genes of interest.

was used for normally distributed data if applicable;
otherwise, we used the Mann-Whitney U test for nonnormally distributed datasets. The differences among
multiple groups were analyzed using 1-way or 2-way
ANOVA or non-parametric Kruskal-Wallis tests asappropriate. Differences between groups were assessedwith post hoc Bonferroni (comparisons between all conditions), Dunnett (all conditions compared with a control
group), or Dunn’s (following Kruskal-Wallis) tests. In all
analyses, a P-value ≤ 0.05 was considered statisticallysignificant.

Statistical analysis

Sample sizes for animal studies were determined using
power analyses, allowing us to detect differences with
80% power at the expected effect sizes and with the
expected variance observed in that assay. The expected
effect size was 0.5. Power analyses were primarily basedon our previous work in preclinical stroke [6–9]. The typeI error rate was limited using an α level of 5%, with Bonferroni’s adjustment for pairwise comparisons. Results
are presented as mean ± standard deviation. Of note,
explanatory schematics were created with biorender.
GraphPad Prism software (v9.0.0) was used for statistical analyses and visualization of the results. To evaluate
the normal distribution of the data sets, we performed
the D’Agostino-Pearson test. The 2-tailed Student’s t-test

Results
Inhibition of PK in the subacute phase reduced infarct
volume and improved neurological recovery after ischemic
strokeInhibition of PK in the acute ischemic stroke has beenshown to reduce infarct volumes and improve neurological outcome [9]. Therefore, we investigated whether
inhibition of PK in the subacute phase is also protective.
C57BL/6J mice were subjected to 60-min tMCAO andafter complete infarct maturation on day 3, magnetic
resonance imaging (MRI) was performed to evaluate
infarct volume. Additionally, successful occlusion of the
MCA and reperfusion was monitored using Laser Doppler flowmetry (Suppl. Figure 1). To ensure comparable
baseline conditions, only animals with similar infarct
sizes were randomly assigned for treatment with either
αPK (at doses of 400 µg/kg on days 3 and 4, followed by
200 µg/kg on days 5–7) or IgG as control (Fig. 1A + B;
Suppl. Figure 2). Quantitative analysis of infarct volumes
and edema formation via MRI demonstrated a significant reduction in animals treated with αPK compared tocontrols at day 7 post-stroke (Fig. 1C + D). The reduction
in infarct size was associated with markedly enhanced
functional outcomes, as evidenced by modified neuroscore assessments (Fig. 1E). Detailed functional evaluations encompassing sensorimotor abilities, including AR,
RR, and TR, revealed substantial improvement in αPKtreated mice relative to controls at day 7 after tMCAO(Fig. 1F–H). Notably, αPK-treated mice exhibited consistent weight gain from day 5 onwards, indicative of
progressive functional recovery (Fig. 1I). Correlation
analyses further underscored a significant relationshipbetween reduced neurological deficits and improved
functional outcomes in αPK-treated mice versus controls
at both day 3 and day 7 post-stroke (Fig. J–L).
Delayed PK inhibition reduced thrombo-inflammation
after brain ischemia

PK has been described as a key mediator of thrombus
formation and inflammation after ischemic stroke [9, 18].
To understand the impact of PK on the recovery phase,
we determined thrombo-inflammatory consequences ofHaupeltshofer et al. Journal of Neuroinflammation(2024) 21:155

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Fig. 1 Delayed anti-plasma kallikrein (αPK) treatment transiently improved focal deficits and reduced edema formation. (A) Mice were treated with isotype immunoglobulin G (IgG) or αPK at days 3 and 4 (400 µg/kg) and days 5 to 7 (200 µg/kg) after 60-min transient middle artery occlusion (tMCAO). Cerebral damage was visualized using magnetic resonance imaging (MRI) at days 3 and 7. (B) Representative images of T2 MRI scans 3 and 7 days after tMCAO.
The yellow dotted lines depict infarct areas. Quantification of (C) brain infarct volume and (D) brain edema (n = 24/12/12). (E) Assessment of general andfocal deficits at 3 and 7 days of tMCAO mice treated with IgG or αPK. Evaluation of sensorimotor function of tMCAO with (F) adhesive removal test (AR),(G) rotarod test (RR), and (H) tightrope test (TR) (n = 20/10/10). (I) Quantification of body weight at days 0 to 7 after tMCAO (TR) (n = 10/10). (J–L) Spearman
correlation of sensorimotor deficits with neurological score evaluation. Two-way ANOVA and post hoc Sídák test. *P < 0.05, **P < 0.01, ***P < 0.001 for αPK
vs. IgG; grey lines represent the baseline values measured in healthy micedelayed PK inhibition. Administration of αPK (400 µg/
kg at day 3 + 4, 200 µg/kg at day 5–7) reduced activatedPK and its downstream protein bradykinin in the bloodat day 5 and day 7 after cerebral ischemia compared to
IgG-treated control mice (Fig. 2A + B). Histological analysis revealed significantly reduced B1R on CD31+ vessels

in αPK-treated mice at day 7 after tMCAO compared tocontrols (Fig. 2C). In line with reduced KKS activation,
inhibition of PK reduced microvascular thrombus formation at day 7 after tMCAO compared to control mice
(Fig. 2D + E).

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Fig. 2 Subacute inhibition of plasma kallikrein (PK) protected against persistent thrombosis in microvasculature transient middle artery occlusion(tMCAO). (A) Quantification of activated PK and (B) its respective downstream target bradykinin in the blood at day − 1 (baseline) and days 1, 3, 5, and 7
after tMCAO of mice treated with immunoglobulin G (IgG) or anti-PK (αPK) (n = 8). (C) Histological staining of CD31+ blood vessels and the bradykinin-1
receptor (B1R+) at days 3 and 7 (white arrows) (n = 5). (D) Histological staining of CD31+ blood vessels and GPIX+ platelet adhesion molecules showing
thrombi at days 3 and 7 after tMCAO (white arrows) (n = 9/5/5). (E) Protein density analysis of platelet adhesion molecule GP1b at days 3 and 7 aftertMCAO (n = 10/8/8). One-way ANOVA and post hoc Dunn’s test. *P < 0.05, **P < 0.01, ***P < 0.001 for αPK vs. IgG

PK inhibition reduced BBB breakdown after brain ischemia

After ischemia, the BBB opens within hours after infarction, permitting the influx of blood-borne components
and immune cells into the brain [19, 20]. Therefore, we
determined whether PK affects the BBB permeability inthe subacute phase. Inhibition of PK led to significantly
reduced extravasated albumin and PK in the brain parenchyma at day 7 after cerebral ischemia in αPK-treatedmice (Fig. 3A–C). As a next step, we determined theunderlying mechanisms that stabilized the BBB. Cerebral
ischemia induces tight junction (TJ) protein degradation
via mechanisms involving activation of the B1R/ROCK/
Rac1 pathway [21] (Fig. 3D). Reverse transcription PCR(RT-PCR) analysis revealed significantly increased Rac1
expression (Rac1 stabilized the BBB) and decreased
ROCK expression (ROCK-promoted BBB degradation)in purified endothelial cells derived from αPK-treatedmice compared to control endothelial cells at day 7 after
ischemia (Fig. 3E + F). αPK-treated mice exhibited significantly increased numbers of CD31+ blood vessels in
the peri-infarct area (sensory and motor cortex), but notin the infarct core (basal ganglia) at day 7 after tMCAO
compared to controls (Fig. 3G + H). In addition, αPK
treatment significantly increased claudin-5, occludin, andZO-1 co-localization with vessels in the peri-infarct area,
while the infarct core was unaffected at day 7 after stroke
compared to control mice (Fig. 3F–H), suggesting stabilization of the BBB by PK blockade in the subacute stroke
phase.
Subacute PK inhibition reduced BBB-permeabilizing
proteases and increased angiogenic markers

To elucidate how BBB stabilization was supported by thebrain environment, we determined matrix metalloproteinases (MMPs) and angiogenic markers by ELISA. Following PK inhibition, a significant reduction in MMP-2
and MMP-9 was found in brain tissue extracts at day 7
after tMCAO compared to control mice (Fig. 4A), suggesting less extracellular matrix degradation which
supports BBB integration. We further elucidated theabundance of angiogenic molecules in the ischemic
hemisphere and measured no alterations in VEGF levels

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Fig. 3 Blockade of plasma kallikrein (PK) was essential for blood-brain barrier (BBB) stabilization and attenuation of extravasation after transient middle
artery occlusion (tMCAO). (A) Exemplary staining of albumin and PK (white arrows) in the ischemic lesion. Extravasation of (B) albumin and (C) bloodresident PK into the brain parenchyma of immunoglobulin G (IgG)- and anti-PK (αPK)-treated mice (n = 6). (D) Schematic pathway of tight junction (TJ)
protein degradation in endothelial cells bradykinin-1 receptor (B1R) activation under pathophysiological conditions. (E) Ras-related C3 botulinum toxin
substrate 1 (Rac1)-axis activation led to stabilization of the BBB (n = 8). (F) Intracellular Rho-associated kinases (ROCK)-axis activation mediated degradation of the BBB after ischemic stroke (n = 8). (G) Representative image of anti-CD31 histological staining and determination of region of interest (ROI) in
the basal ganglia and sensory and motor cortex. (H) Quantitative analysis of histological CD31+ vessel density from respective brain regions at days 3 and7 (IgG vs. αPK) (n = 6–8). (I–K) Representative images of TJ protein staining and analysis of histological staining for CD31 vessel density and co-expressionof claudin-5, occludin, and ZO-1 at days 3 and 7 after tMCAO in the respective IgG and αPK groups (n = 5–6). One-way ANOVA and post hoc Dunn’s test.
*P < 0.05, **P < 0.01, ***P < 0.001 for αPK vs. IgG

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Fig. 4 Delayed plasma kallikrein (PK) inhibition blocked destabilization of the blood-brain barrier (BBB), leading to enhanced angiogenic marker expression after transient middle artery occlusion (tMCAO). Secretion patterns on the ipsilateral side of ischemic brains at day 3 and day 7. Amount of disruptive (A) matrix metalloproteinases (MMP)-2 and − 9, (B) vascular endothelial growth factor (VEGF), and platelet-derived growth factor subunit B (PDGFb)
angiogenic markers (n = 6). Expression analysis of angiogenic-supporting factors (C) VEGF, (D) angiopoietin-1 (Ang-1), and (E) integrin-5 (α5) subunit fromthe basal ganglia (BG; infarct core) and cortex (CTX) from ischemic mouse brains (n = 5). One-way ANOVA and post hoc Dunn’s test. *P < 0.05, **P < 0.01,***P < 0.001 for αPK vs. IgG

and platelet-derived growth factor subunit B (PDGFb)
levels at day 7 (Fig. 3B). However, RT-PCR analysis of
VEGF (Fig. 4C), angiopoietin-1 (Ang-1) (Fig. 4D), andalpha-5 integrin subunit (Fig. 4E) revealed increased
expression of the angiogenic markers in the peri-infarctarea, indicating prominent microvascular remodeling in
the peri-infarct brain tissue.
Delayed PK inhibition decreased inflammation during
recovery after cerebral ischemia

Investigating the influx of immune cells did not yieldalterations in overall CD45high leukocytes between treatment groups at day 7 (Fig. 5A + B). Deeper analysis of
immune cell composition, however, revealed reduced
numbers of CD45highCD3+ T cells and CD45highCD11b+
macrophages/microglia in the brains of αPK-treated micecompared to controls at day 7 after ischemia (Fig. 5C + D,
Suppl. Figure 3). The number of CD45highCD11b+Ly6G+
neutrophils was unaffected by PK blockade (Fig. 5E).

Cytokine analysis of the ischemic hemisphere revealed
a pro-inflammatory milieu at day 3 (Fig. 5F). Treatment
with ⍺PK reduced the level of interleukin (IL)-1β, IL-6,
and RANTES (i.e., regulated and normal T cell expressedand secreted) in tMCAO mice. Granulocyte-macrophagecolony-stimulating factor (GM-CSF), tumor necrosis
factor alpha (TNFα), and monocyte chemoattractant
protein (MCP)-1 and − 2 were highly abundant at day 3compared to concentrations found at day 7 after tMCAO.
However, GM-CSF and TNFα were no longer detectableat day 7, while MCP-1 and − 2 were comparably reduced
and not influenced by αPK treatment at this time point(Fig. 5F–L).

Discussion
We studied the benefits of subacute PK inhibition in amurine model of ischemic stroke. Prior research hassuggested that blockade of KKS, in particular PK, canimprove acute phase outcomes and decrease infarctHaupeltshofer et al. Journal of Neuroinflammation(2024) 21:155

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Fig. 5 Plasma kallikrein (PK) inhibition induced alteration of immune cell influx and ameliorated inflammatory cytokines after transient middle artery
occlusion (tMCAO). (A + B) Representative dot plot of brain-isolated immune cells and quantitative analysis of infiltrated immune cells into the ischemic
brain at days 3 and 7 after immunoglobulin G (IgG) and anti-PK (αPK) in tMCAO mice (n = 5). Analysis of (C) CD3+ lymphocytes (adaptive immune system),(D) macrophages/microglia, and (E) neutrophil granulocytes (innate immune system) from IgG- and αPK-treated tMCAO mice (n = 5). Inflammatory cytokines (F) interleukin (IL)-1β, (G) IL-6, (H) tumor necrosis factor alpha (TNFα), (I) monocyte chemoattractant protein (MCP)-1, (J) regulated and normal T
cell expressed and secreted (RANTES), (K) MCP-2, and (L) granulocyte-macrophage colony-stimulating factor (GM-CSF) in the brain parenchyma (n = 4).
One-way ANOVA and post hoc Dunn’s test. *P < 0.05 for αPK vs. IgG

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volumes after tMCAO [4]. We have shown that a delayedand sustained blockade of PK starting from day 3
improved stroke recovery in tMCAO mice. Administration of αPK reduced inflammation, improved functional
outcomes, and ameliorated neurological deficits, accompanied by increased early markers for vessel recovery andvascularization.
Currently, only a few studies have elaborated the longterm role of KKS components in the chronic phase of
cerebral ischemia. Longitudinal brain proteome analysis
revealed enhanced abundance of kininogen 1 and bradykinin up to 7 days in the ischemic mouse brain [22].
This is in line with our results, where elevated PK and
bradykinin concentrations were measured in the bloodfrom day 1 onwards. Other studies have shown significant upregulation of B1R expression during the first48 h after ischemic stroke in mice and focal brain injury,
respectively [23, 24]. We observed increased expressionof B1R on vessels at day 7, which was ameliorated by ⍺PKtreatment. Among KKS components, PK inhabits a dual
mode of action as a proinflammatory and prothrombotic
enzyme [25]. In this manner, activation of the KKS has
been shown to interact with adhesion molecules andfibrinogen, potentially affecting thrombus formation [26,27]. Thrombotic activity has been reported to persist
for up to 24 h after reperfusion in 60-min tMCAO mice
[28, 29]. Furthermore, thrombus formation has been
described until day 28 in a photothrombosis model ofischemic stroke [28, 29], although this may not be ideal
to mimic the microvascular consequences of humanstroke. In addition, thrombi have been found to persist
in the microvasculature until day 28 in a 30-min tMCAO
model, but subacute depletion of platelets was unable
to protect mice from ischemic stroke consequences andfailed to improve functional outcome [16]. In contrast,
our study demonstrated that administration of αPK
antibodies in the subacute phase after tMCAO reduced
KKS activation, diminished persistent microvascularthrombosis, and improved functional outcomes at day 7.
In light of these results, it is likely that the regenerative
effect of PK inhibition is probably not due to the thrombogenic properties of PK but rather mediated throughcell-modulatory effects, presumably at the neurovascularunit.
One of the major complications after ischemic strokeis the breakdown of the BBB. Increased permeability follows a multiphasic pattern of an open-close-open
phenomenon (∼ 6 h, ∼ 72/96 h, ∼ 1 week) in rodent andhuman stroke [30]. By blocking PK in our study, theextravasation of albumin and PK into the brain parenchyma was mitigated, indicating stabilization of the BBB
after the start of treatment on day 3. Endothelial-stabilizing mechanisms are predominantly mediated by intracellular Rac1 in conjunction with activating RhoA, leading

to cytoskeletal rearrangement [21]. Under inflammatory conditions, activation of B1R leads to the formationof myosin light chains and actin stress fibers and phosphorylation of belt-like adherent junctions, causing dissociation of TJ, e.g., ZO and occludins at the endothelial
barrier [31]. This process was significantly ameliorated
in purified endothelial cells of αPK antibody-treated animals in our study, as indicated by increased Rac1 anddecreased ROCK expression, accompanied by increased
TJ coverage of vessels in peri-infarct areas. Additionally,ischemic stroke triggers MMP activity, notably MMP-2
and MMP-9, implicated in BBB disruption and extracellular matrix degradation. MMP-9 induces complete basal
lamina and TJ breakdown, exacerbating barrier impairment and vasogenic edema post-stroke [32–34]. In ourstudy, subacute administration of αPK reduced MMP-2
and MMP-9 levels and attenuated BBB breakdown andedema formation, supporting recovery after cerebral
ischemia. Ang-1 and α5 integrin have been described as
subacute angiogenic markers after tMCAO, expressed at
the ischemic core border and penumbra [35], and bothmarkers were increased after PK inhibition at day 7. In
contrast, VEGF expression increases early after stroke[36] and was not changed by our treatment.
Another consequence of BBB breakdown during cerebral ischemia is an increase in inflammatory processes
and leukocyte infiltration. The KKS influences activation of immune cells, their growth, movement, and functions [37]. Moreover, it plays a role in recruiting immune
cells from the bloodstream, particularly monocytes andT lymphocytes, as shown in a mouse model of infection [38]. Reduced numbers of infiltrated CD11b+ macrophages/microglia and Ly6B.2+ neutrophils, as well as
reduced expression of inflammatory cytokines, have been
observed in PK−/− mice at 24 h after 60-min tMCAO[9]. Interestingly, in experimental encephalomyelitis (ananimal model of multiple sclerosis), deficiency or pharmacologic blockade of PK significantly reduced immune
cell migration into the brain by modulation of the BBB[39]. We observed a significant reduction in infiltrated
CD11b+ macrophages/microglia and CD3+ T cells in
αPK-treated mice. Additionally, cytokines IL-1β, IL-6,
and RANTES were significantly reduced in the ischemic
brain in αPK-treated mice in our study, underlying the
reduction in T cells and macrophages described as major
peripheral sources of these cytokines [40].
From a clinical perspective, drugs supporting poststroke recovery have distinct therapeutic targets that are
related to plasticity and neuronal growths after stroke;
improvements in behavioral outcomes are not necessarily
connected to a reduction in infarct volume [41]. Amongthe drugs that enhance motor recovery in humans, serotonergic and dopaminergic agents currently represent
promising targets. Restorative stroke therapies have

Haupeltshofer et al. Journal of Neuroinflammation(2024) 21:155

generally been targeted at the brain, rather than arterioles, clots, or platelets [41]. However, in our study wedescribe an intervention window that connects the vascular pathology and the recovery process and both can betargeted by PK inhibition. Therapeutic success and safety
for PK inhibition has already been demonstrated in the
disease of hereditary angioedema (HAE), which harbors
deficiency or dysfunction in the C1-esterase inhibitorthat normally inhibits PK and activates FXII [42]. Consequently, PK activity increases blood vessel permeability, allowing fluid to pass through the blood vessel walls
and causing edema formation – a process that we also
observed in our study during cerebral ischemia. In a permanent model of MCA occlusion in mice, treatment withthe human recombinant PK inhibitor DX-88 (approvedfor HAE) in the acute phase of ischemic stroke reduced
infarct volume and brain swelling, lowered the amountof dying neurons, and drastically diminished neurological damage for up to 7 days post-ischemia [43]. Thus,
although successful PK inhibition has been approved inother diseases, it has not yet been translated to ischemic
stroke therapy.
In summary, we found that inhibition of PK administered 3 days post-stroke reduced infarct volume
and improved neurological outcomes, with sustained
effects until day 7. Mechanisms supporting the recovery
included stabilization of the BBB, a reduction in thrombosis and the inflammatory response, and increased
angiogenic signaling.

Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s12974-024-03149-w.
Supplementary Material 1
Acknowledgements
We kindly thank Stefanie Hezel, Kristina Wagner and Sina Luppus for their
excellent technical support as well as Dr. Marcel Gratz for his MRI-related
support. Schematic overviews were created with biorender.com.
Author contributions
S.H., A.I.C., F.L., C.K. designed research; S.H., S.M., C.H., H.A., A.B. performed
research and data analysis S.H., S.M., C.H., R.S., H.A., W.H. A.B., C.D., M.F.,
contributed to new reagents/analytic tools; and S.H., A.I.C., D.M.H., F.L, C.K.
wrote the paper. All authors reviewed the manuscript.
Funding
Open Access funding enabled and organized by Projekt DEAL. This
study was funded by the German Research Foundation (DFG, Deutsche
Forschungsgemeinschaft) - FOR2879: ImmunoStroke - Project number:
405358801. AIC was supported by the Förderprogramm der Corona-Stiftung
im Stifterverband and BMBF FerroPath consortium.
Open Access funding enabled and organized by Projekt DEAL.
Data availability
Data used for this manuscript are available upon reasonable request.

Page 11 of 12

Declarations
Ethics approval
The study was approved by the regional government authorities (Landesamt
für Natur, Umwelt und Verbraucherschutz NRW, LANUV) and guided by theGerman Welfare Act (German Ministry of Agriculture, Health, and Economic
Cooperation).
Consent for publication
All authors have seen and agree with the contents of the submitted
manuscript.
Competing interests
The authors declare no competing interests.
Received: 4 March 2024 / Accepted: 6 June 2024

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