Neuropharmacology 181 (2020) 108357

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Neuropharmacology
journal homepage: www.elsevier.com/locate/neuropharm

Lithium enhances post-stroke blood-brain barrier integrity, activates the

MAPK/ERK1/2 pathway and alters immune cell migration in mice
Matteo Haupt a, Bozena Zechmeister a, Bert Bosche b, c, d, Simone Lieschke a, Xuan Zheng a,
Lin Zhang a, Vivek Venkataramani e, Fengyan Jin f, Katharina Hein a, Martin S. Weber a, g,
Dirk M. Hermann c, Mathias Bähr a, Thorsten R. Doeppner a, *
a
University Medical Center Goettingen, Department of Neurology, Goettingen, Germany
b
Department of Neurocritical Care, First Stage Rehabilitation and Weaning, Reichshof-Eckenhagen, Germany
c
University of Duisburg-Essen Medical School, Department of Neurology, Essen, Germany
d
Institute of Neurophysiology, Medical Faculty, University of Cologne, Cologne, Germany
e
University Medical Center Goettingen, Institute of Pathology, Goettingen, Germany
f
The First Hospital of Jilin University, Department of Hematology, Cancer Center, Changchun, Jilin, China
g
University Medical Center Goettingen, Institute of Neuropathology, Goettingen, Germany

A R T I C L E I N F O A B S T R A C T

Keywords: Lithium induces neuroprotection against cerebral ischemia, although the underlying mechanisms remain elusive.
Blood-brain barrier We have previously suggested a role for lithium in calcium regulation and (extra)cerebral vessel relaxation under
Neurovascular unit non-ischemic conditions. Herein, we aimed to investigate whether or not lithium contributes to post-stroke
Cerebral ischemia
stabilization of the blood-brain barrier (BBB) in mice. Using an oxygen-glucose-deprivation (OGD) model, we
Immune response
first analyzed the impact of lithium treatment on endothelial cells (EC) in vitro. Indeed, such treatment of EC
Lithium
Neuroprotection exposed to OGD resulted in increased cell survival as well as in enhanced expression of tight junction proteins
and P-glycoprotein. Additional in vivo studies demonstrated an increased stabilization of the BBB upon lithium
treatment in stroke mice, as shown by a reduced Evans blue extravasation and an elevation of tight junction
protein expression. Furthermore, stabilization of the BBB as a consequence of lithium treatment was associated
with an inhibition of matrix metalloproteinase-9 activity, independent of calveolin-1 regulation. In line with this,
flow cytometry analysis revealed that lithium treatment led to a decreased neutrophil invasion and an increased
T cell extravasation from the blood compartment towards the brain parenchyma. We finally identified the pro-
survival MAPK/ERK1/2 pathway as the key regulator of the impact of lithium on the BBB. In conclusion, we
demonstrate for the first time that lithium is able to enhance post-stroke BBB integrity. Importantly, our work
delivers novel insights into the exact mechanism of lithium-induced acute neuroprotection, providing critical
information for future clinical trials involving lithium treatment in stroke patients.

1. Introduction outcome in distinct neurobehavioral tests over a period of 56 days after

Lithium has been successfully used for the treatment of bipolar dis­
orders for decades (Manji and Lenox, 1998). Recent evidence in rodents,
moreover, suggests that lithium has protective effects on neurons after
cerebral ischemia. In detail, lithium treatment yielded a reduction of
infarct volume, inhibition of apoptotic cell death and suppression of
neuroinflammation (Chuang et al., 2011; Doeppner et al., 2017; Fan
et al., 2015; Ren et al., 2003; Xu et al., 2003). Furthermore, lithium
treatment enhanced neurological recovery, as observed by an improved
cerebral ischemia (Doeppner et al., 2017). These preclinical studies gave
rise to a clinical trial showing an improved motor recovery after lithium
treatment in patients with cortical ischemic stroke (Abdollahi et al.,
2014). However, none of these studies explained the mechanisms by
which lithium induces its acute neuroprotective effects.
Lithium is a major modulator of the glycogen synthase kinase 3 beta
(GSK3-β), which in turn can regulate a large number of downstream
targets (Li X, Bijur G, 2007). Targets of GSK3-β include NFκB, Bcl-2, and
members of the Wnt signaling pathway, all of which play critical roles in

* Corresponding author. Department of Neurology, University Medical Center Goettingen, Robert-Koch-Str. 40, 37075, Goettingen, Germany.
E-mail address: thorsten.doeppner@med.uni-goettingen.de (T.R. Doeppner).

https://doi.org/10.1016/j.neuropharm.2020.108357
Received 3 March 2020; Received in revised form 10 October 2020; Accepted 12 October 2020
Available online 13 October 2020
0028-3908/© 2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
M. Haupt et al.
the regulation of signal transduction and cell survival (Maurer et al.,
2014). Interestingly, we have previously shown that lithium-regulated
signaling pathways involved in the pathophysiology of cerebral
ischemia are pleiotropic and not solely dependent on GSK3-β (Doeppner
et al., 2017). We found that post-stroke lithium treatment also modu­
lated miR-124 expression, RE1-silencing transcription factor abundance
and protein deubiquitination in mice (Doeppner et al., 2017). Although
these effects contributed to a better neurological recovery in these mice,
the precise downstream targets of lithium during acute stage of the
disease remained elusive at the time.
Among the complex pathophysiology of cerebral ischemia, the
opening of the blood-brain barrier (BBB) during the first hours following
stroke induction is a critical mechanism for cell injury development
(Dirnagl et al., 1999). In addition, the BBB breakdown becomes clini­
cally more relevant for patients during the subacute phase of ischemic
stroke, when vasogenic edema formation and subsequent
space-occupying brain swelling predominantly occurs (Bosche et al.,
2003; Wijdicks et al., 2014; Liebeskind et al., 2019). In the endothelial
cells (EC) of the BBB, energy deficits during hypoxia lead to an increase
of cytosolic Ca2+, contributing to irreversible cell injury (Schäfer et al.,
2001). The latter triggers the dysregulation of the endothelial barrier
function and leads to hyperpermeability (Noll et al., 1995). In vitro ex­
periments suggested that lithium prevents this early Ca2+ overload by
inhibiting the inositol-3-phosphate-sensitive Ca2+ release from the
endoplasmic reticulum of EC (Bosche et al., 2013). Further in vitro ex­
periments under non-hypoxic conditions suggest a complex
lithium-induced stabilization of the endothelium through the decrease
in myosin light chain phosphorylation in vessel grafts from de-nerved
murine aortas and porcine middle cerebral arteries (Bosche et al.,
2016b, 2016a). In addition, Li and colleagues observed a protection of
lithium-treated EC against oxygen-glucose-deprivation (OGD) in vitro
and a preservation of BBB function in vivo following lithium pretreat­
ment in an ischemia–reperfusion model in rats (Li et al., 2014).
Apart from the intracellular processes in EC, activation of matrix
metalloproteinases (MMP) play a key role in the pathophysiology of
cerebral ischemia (Bosche et al., 2006; Gasche et al., 1999; Kurzepa
et al., 2014; Yang et al., 2007). MMPs such as MMP-9 lead to the
degradation of extracellular endothelial tight junction proteins and
hence to a more permeable BBB. MMPs are present as pro-forms, which
become activated through cleavage after stroke (Medsker et al., 2016).
Opening of the BBB does not only directly impair neural cell survival but
also facilitates peripheral immune cell migration into the central ner­
vous system (Chamorro et al., 2012; Dirnagl et al., 1999). Resident
phagocytes (microglia) activation, chemotaxis and expression of cell
adhesion molecules play a pivotal role in this context (Xiong et al., 2016;
Yanamadala and Friedlander, 2010). The importance of these mecha­
nisms is illustrated by the fact that MMP-9 inhibition leads to neuro­
protection under experimental stroke conditions (Chaturvedi and
Kaczmarek, 2014). Indeed, lithium treatment has been shown to reduce
MMP-9 expression, subsequently leading to enhanced BBB integrity in
mice 3 days post-induction of traumatic brain injury (Yu et al., 2011).
Nevertheless, the role of lithium in the context of BBB disruption
after cerebral ischemia and all types of stroke remains largely elusive but
is highly relevant especially for further studies in humans (Bosche and
Macdonald, 2015; Bosche et al., 2020). In our study, we investigated
whether or not lithium elicits its neuroprotective effects through
impacting the BBB in adult stroke mice. More specifically, we focus on
immune cell migration, tight junction protein expression, cell survival
signaling cascades, and the activation of MMP-9 upon in vitro OGD of
cultured EC as well as in stroke mice.
2. Materials and methods
2.1. Cell culture, treatment and lysis
Mouse brain EC (bEnd.3, CRL-2299™; American Type Culture
2
Neuropharmacology 181 (2020) 108357
Collection, Manassas, Virginia, USA) were seeded in TC-plates (Sarstedt,
Nuembrecht, Germany) and cultured under confluent conditions at a
density of 6 × 104 cells/cm2. Cells were incubated with 10% fetal bovine
serum-containing medium (Dulbecco’s Modified Eagle Medium/
Nutrient Mixture F-12; Thermo Fisher Scientific, Waltham, Massachu­
setts, USA) which was enriched with lithium chloride (Sigma-Aldrich,
St. Louis, Missouri, USA; subsequently addressed as lithium only) at a
final concentration of 1.25 mM. Controls were incubated in medium
without lithium. The cells were placed for 14 h under OGD 24 h after
seeding. For OGD, the cells were incubated in deoxygenated glucose-free
salt solution (BSS0) containing 10 x BSS (1.16 M NaCl, 54 mM KCl, 8 mM
MgSO4 and 10 mM NaH2PO4H2O), 1 M NaHCO3, 1 M HEPES, 30 mM
glycine and 1 M CaCl2. For lithium-treated cells, 1.25 mM lithium was
added to the BSS0 solution. OGD atmosphere in the hypoxia chamber
contained 0.2% O2, 5% CO2, and 65% humidity and were confirmed by
the built-in sensor of the chamber (Toepffer Lab Systems, Goeppingen,
Germany). After removing the BSS0 solution, the cells were incubated in
the aforementioned cell culture medium with or without 1.25 mM
lithium for 24 h in the incubator at 37 ◦ C. For Western blot analysis, the
cells were lysed by shock freezing at − 80 ◦ C followed by ultrasound
treatment. After 10 min of centrifugation at 12,000 rpm, the supernatant
was collected and the protein concentration was photometrically
determined (Pierce™ BCA Protein Assay Kit; Thermo Fisher Scientific).
The samples were aliquoted and stored at − 80 ◦ C until the Western blot
analysis were performed.
2.2. Measurement of cell viability
A commercial live dead assay kit (ab115347; Abcam, Cambridge,
UK) was used to analyze cell viability according to the manufacturer’s
protocol. Briefly, cells were incubated after their respective treatments
with the fluorescence dyes for 10 min at room temperature. Subse­
quently, cells labeled either green (live) or red (dead) were viewed and
counted under the Axioplan 2 fluorescence microscope (Carl Zeiss, Jena,
Germany). For quantification, we counted the number of live/dead cells
within two sectors per well (at 10x magnification). The percentage of
live cells in relation to the total number of cells were calculated and
presented in the figures.
MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bro­
mide; Thiazolyl blue) reagent (Sigma-Aldrich) was used in a colori­
metric assay to assess cell metabolism. The MTT assay was performed on
cells 24 h after OGD or without OGD. Therefore, the MTT was directly
added to culture wells and incubated for 3 h at 37 ◦ C. Following removal
of the medium, the dye was solubilized for 5 min in DMSO (dimethyl
sulfoxide; Merck, Darmstadt, Germany) under constant shaking. The
absorbance was photometrically measured at 595 nm using a 96-well
absorbance microplate reader (Tecan, Maennedorf, Switzerland). To
normalize the data, metabolic activity of mouse brain EC without OGD
was measured and set as 100%.
2.3. Induction of focal cerebral ischemia
All animals were kept under circadian rhythm and had free access to
food and water. Experiments were performed according to local au­
thorities (LAVES, Lower Saxony, Germany) following the ARRIVE
guidelines.
The induction of transient focal cerebral ischemia was obtained
using the middle cerebral artery (MCA) occlusion model as previously
described (Weise et al., 2006). In short, under anesthesia with 2% iso­
flurane and 0.8 l/min O2, the right common carotid artery was isolated
and a 6-0 nylon monofilament (Doccol Corporation, Sharon, Massa­
chusetts, USA) inserted. The filament was gently pushed forward to­
wards the MCA where it was placed for 45 min. After filament removal,
the wounds were carefully sutured. The occlusion and the reperfusion
were monitored under constant laser doppler flow. For pain treatment,
0.1 mg/kg buprenorphine was injected subcutaneously (s.c.) before
M. Haupt et al.
surgery and 4 mg/kg carprofen after surgery. This was followed by daily
s.c. injections of 0.1 mg/kg buprenorphine in the morning and 4 mg/kg
carprofen in evening. For the sham group, animals underwent the very
same procedure except for insertion of the nylon filament.
2.4. Animal groups and drug treatment
7-week-old male C57BL6 mice (Charles River, Wilmington, Massa­
chusetts, USA) were randomly divided in groups with survival times of
24 h or 72 h after MCA occlusion. Inclusion criteria include the detection
of stroke lesions within the striatum, whereas exclusion criteria include
huge cortical infarcts, development of seizures, and spinning of animals
when picked up by the tail. The mice received phosphate-buffered saline
(PBS) as solvent control, Lithium was dissolved in PBS whereas the
MEK1/2 inhibitor U0126 (Cell Signaling Technology, Danvers, Massa­
chusetts, USA) was dissolved in DMSO depending on their group allo­
cation. Mice with 24 h survival were further divided into the following
groups: sham, control, lithium, lithium + U0126 and U0126. For sham,
control and U0126 groups, PBS was given at the beginning of reperfu­
sion and at 6 h after MCA occlusion. For lithium and lithium + U0126
groups, lithium was given at the beginning of reperfusion and at 6 h after
MCA occlusion. The lithium dosage for the first injection was 1 mmol/kg
and for the second injection 2 mmol/kg, based on our previous study
(Doeppner et al., 2017). The injection volume for the first injection was
around 50 μl and for the second around 100 μl depending on the indi­
vidual bodyweight. The lithium + U0126 and U0126 groups addition­
ally received intravenous injections of 0.5 mg/kg of U0126 (Cell
Signaling Technology) at the beginning of the reperfusion. Control mice
for the experiments with U0126 received the same volume of DMSO
intravenously. Mice with 72 h survival were further divided into a
control and a lithium group. Both groups received the same treatment as
the control and the lithium groups with 24 h survival and two additional
intraperitoneal injections with 2 mmol/kg of lithium each at 24 h and at
48 h after MCA occlusion. Overview tables showing all treatment groups
and animal numbers are provided in Tables S1 and S2. For each
experiment conducted (i.e., Evans Blue extravasation, Western blot
analysis, immunohistochemical analyses, zymography and flow cytom­
etry), new experimental groups were generated with the
above-described treatment paradigms.
2.5. Evans Blue extravasation
Evans Blue extravasation was performed as previously described
(Doeppner et al., 2011). Briefly, 100 μl of 4% Evans Blue dye (Sig­
ma-Aldrich) was administered using a retrobulbar access route 2 h
before killing. Subsequently, the mice were sedated and cardiac perfu­
sion with PBS was performed. The hemispheres were weighed and lysed
in 50% trichloroacetic acid. After centrifugation for 20 min at 10,000
rpm the supernatant was diluted with ethanol and the absorbance at
620 nm was measured photometrically. The measured Evans Blue con­
centration was calculated based on a standard curve. The standard curve
was plotted by measuring the absorbance versus optical density of
samples with defined concentrations of Evans Blue (0.25–20 μg/ml). The
final concentration was then calculated as (μg) Evans Blue per (g) tissue.
2.6. Tissue preparation for Western blot analysis
The brain tissue was lysed in a buffer containing 50 mM Tris, 1%
Triton-X 100, 131 mM sodium chloride, 1 mM sodium diphosphate, 1
mM sodium fluoride, 1 mM EDTA, protease inhibitors (cOmplete™;
Sigma-Aldrich) and phosphatase inhibitors (PhosSTOP™; Sigma-
Aldrich) using a homogenisator for 10 min followed by centrifugation
at 4 ◦ C with 16,000 rpm for 10 min. The supernatant was collected and
the quantification of the protein concentration photometrically accom­
plished (Pierce™ BCA Protein Assay Kit; Thermo Fisher Scientific). The
samples were aliquoted and stored at − 80 ◦ C until Western blot analysis
3
Neuropharmacology 181 (2020) 108357
performed.
2.7. Western blot analysis
For Western blot analysis, reducing sample buffer (Carl Roth,
Karlsruhe, Germany) was added, and the tissue or cell culture samples
were heated for 5 min at 95 ◦ C. Thereafter, 35 μg of protein were
separated using SDS-PAGE and transferred onto nitrocellulose mem­
branes (Bio-Rad, Hercules, California, USA). Following transfer, the
membranes were blocked using 5% milk in PBS (skim milk powder;
Sigma-Aldrich). The membranes were then incubated with the primary
antibodies occludin (1:1000; ab167161; Abcam), ZO-1 (1:1000;
ab96587; Abcam), caveolin-1 (1:1000; ab2910; Abcam), c-Raf (1:1000;
#9422; Cell Signaling Technology), p-c-Raf (1:1000; #9421; Cell
Signaling Technology), MEK1/2 (1:1000; #9122; Cell Signaling Tech­
nology), p-MEK1/2 (1:1000; ab194754; Abcam), ERK1/2 (1:2000; sc-
93-G; Santa Cruz Biotechnology, Dallas, Texas, USA) p-ERK1/2
(1:1000; #9101; Cell Signaling Technology), Bcl-2 (1:1000; sc-7382;
Santa Cruz Biotechnology), ICAM1 (1:1000; sc-8439; Santa Cruz
Biotechnology), VCAM-1 (1:1000; ab134047; Abcam), P-gp (1:1000;
ab170904; Abcam) and horseradish peroxidase coupled secondary anti-
mouse-antibody (1:10,000; ab97023; Abcam) and anti-rabbit-antibody
(1:10,000; ab6721; Abcam). As loading controls, anti-Tubulin
(1:10,000; sc-8035; Santa Cruz Biotechnology) or anti-GAPDH
(1:10,000; GTX627408; Irvine, GeneTex, California, USA) were used,
after first visualization of the specific protein of interests. All antibodies
used were diluted in 5% milk in PBS. The membranes were bathed in
ECL reagent (Cell Signaling Technology) and developed with the
ChemiDoc™ XRS+ (Bio-Rad) imaging system. For quantification, the
density of the protein of interest and the housekeeping protein tubulin or
GAPDH was measured using the Image Lab software (Bio-Rad). The
results are presented as the ratio between protein of interest and
housekeeping protein in percentage. Separate membranes were used for
each antibody incubation and detection. Representative and the com­
plete, uncropped membranes for each experimental condition are shown
in the supplementary section of the manuscript (Figs. S1–S6).
2.8. Immunohistochemical analyses
For immunohistochemical analyses, the mice were transcardially
perfused with ice cold PBS before killing. The paraffin embedding the
brain sections (diameter: 4 μm) were deparaffined and boiled in citrate
puffer (pH 6.0) and washed in PBS. After blocking with buffer containing
tris buffered saline (pH 7.4), 2% BSA, 10% donkey serum, 0.25% Triton
X-100, the sections were incubated overnight with the following pri­
mary antibodies: Iba-1 (1:1000; 019–19741; Wako Inc., Osaka, Japan),
MMP-9 (1:500; sc-393859; Santa Cruz Biotechnology), claudin-1
(1:1000; sc-137121M; Santa Cruz Biotechnology), ZO-1 (1:500;
ab96587; Abcam), caveolin-1 (1:800; ab2910; Abcam), NeuN (1:1000;
ab104225; Abcam), GFAP (1:1000; AB5541; Merck), CD31 (1:500;
ab7388; Abcam) and 4‘,6-Diamidin-2-phenylindol (1:10,000; A1001;
AppliChem, Darmstadt, Germany). Thereafter, the sections were incu­
bated for 1 h with the following secondary antibodies: AlexaFlour488-
anti-rabbit (1:10,000; 711-545-152; Jackson ImmunoResearch, Ely,
UK), Cy3-anti-mouse (1:10,000; 715-165-150; Jackson ImmunoR­
esearch), Cy3-anti-rabbit (1:10,000; 711-165-152; Jackson ImmunoR­
esearch). For double staining using antibodies from the same host, the
sections were blocked for 4 h with a Fab fragment antibody (1:50; 711-
007-003, Jackson ImmunoResearch) at room temperature before incu­
bation with the second primary antibody. Five pictures per hemisphere
were taken in our regions of interest (ROI) with the Axioplan 2 fluo­
rescence microscope (Carl Zeiss) and the average intensity was calcu­
lated or positive cells were counted. The subventricular zone (SVZ) and
the basal ganglia were defined as our ROIs. Stereotactic coordinates for
the SVZ were 0.14 mm anterior, 2–3 mm ventral and 1–1.25 mm lateral
from bregma and for basal ganglia 0.14 mm anterior, 2.5–3.25 mm
 M. Haupt et al.
ventral and 1.5–2.25 mm lateral from bregma. The software ImageJ
(National Institutes of Health, Bethesda, Maryland, USA) were used for
cell counting and intensity quantification.
2.9. Zymographic measurement of MMP-2 and MMP-9 activity
The gelatin zymography measurement were performed with affinity-
support purification as previously described (Zhang and Gottschall,
1997). Briefly, the mice were transcardially perfused with ice cold PBS
before killing. After collecting the brains, hemispheres were lysed in a
non-reducing lysis buffer containing 50 mmol/l Tris-HCl (pH 7.6), 150
mmol/l NaCl, 5 mmol/l CaCl2, 0.05% BRIJ-35, 0.02% NaN3 and 1%
Triton X-100, and afterwards centrifuged for 5 min at 12,000 rpm. The
supernatant was collected, the protein concentration measured
(Pierce™ BCA Protein Assay Kit; Thermo Fisher Scientific) and incu­
bated with 1:10 volume of sepharose 4B (Sigma-Aldrich) for 60 min.
After incubation and centrifugation, the purified pellet was resuspended
in lysis buffer containing additional 10% DMSO. Equal volumes were
then incubated with non-reducing sample buffer (Carl Roth) and loaded
on a zymogram gel (Novex™) containing 0.1% gelatin. As standards,
0.1 ng pro-MMP-2 (Merck) and pro-MMP9 (Merck) and 0.01 ng of active
MMP-2 (Merck) and active MMP-9 (Merck) were used. After electro­
phoresis, the gel was washed twice with 2.5% Triton X-100 and incu­
bated for 96 h in developing buffer (Novex™). After incubation, the gel
was stained with SimplyBlue staining solution (Novex™) over night and
then destained in deionized water. After destaining, white bands remain
before a dark background. Following this, gels were scanned using a
ChemiDoc™ XRS+ (Bio-Rad) imaging system and densitometrically
analyzed using the software ImageJ (National Institutes of Health).
2.10. Analysis of brain infiltrating leukocyte by flow cytometry
Infiltrating leukocytes and the subset of T cells, B cells, neutrophils,
macrophages and monocytes were determined by flow cytometry with a
fluorescence-activated cell sorter as described (Pösel et al., 2016).
Briefly, the right ischemic hemispheres were broken down with a pestle
by hand in lysis buffer (0.5% BSA, 5% glucose, DNase (10 mg/ml), PBS)
and centrifuged at 1600 rpm for 10 min. Thereafter, the pellet was
solved in 30% Percoll solution (GE Healthcare, Chicago, Illinois, USA)
and loaded on the Percoll gradient containing 45% and 70% Percoll
phases. Following centrifugation, the leukocytes between the phases
were aspirated and dissolved in working solution (3% fetal calf serum in
PBS). After washing, the cells were incubated with Zombie dye (1:500;
BioLegend, San Diego, California, USA) for 10 min as a viability control.
After adding anti-CD16/32 (1:100; BioLegend) for 10 min, the cells were
centrifuged and washed again. Subsequently, the cells were incubated
with anti-CD3 (1:50; Becton Dickinson, Franklin Lakes, New Jersey,
USA), anti-CD45 (1:100; BioLegend), anti-Ly6G (1:50; BioLegend),
anti-CD19 (1:50; BioLegend) and anti-CD11b (1:50; BioLegend) anti­
bodies overnight. Gating and quantification were obtained with the
software FlowJo v. 10.5.3 (Becton Dickinson). The gating pattern was
based on the study by Pösel and colleagues (Pösel et al., 2016) (Fig. 8H).
In detail, leukocytes are defined as CD45high cells and microglia as
CD45int cells out of single cells. For the latter, it is not possible to
distinguish between activated and inactivated cells. The CD45low frac­
tion includes neuronal cells, astroglia, ependymal and endothelial cells
and were not analyzed in the present study. Neutrophils are defined as
Ly6G+CD3− cells and T cells are defined as Ly6G-CD3+ cells out of all
leukocytes. Ly6G-CD3− cells were further divided into B cells and mon­
ocytes/macrophages. Herein, B cells are defined as CD11b-CD19+ cells
out of CD45highLy6G-CD3− cells and monocytes/macrophages are
defined as CD11b+CD19− cells out of CD45highLy6G-CD3− cells. The
three parental gates of single cells, CD45high cells and CD45high
Ly6G-CD3− cells were always set as 100% for further analysis in the
subsequent gates.
4
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Neuropharmacology 181 (2020) 108357
2.11. Statistical analysis
For all experimental analyses, the experimenter was blinded towards
the treatment paradigm, and animals were randomly allocated to either
treatment paradigms. All data were normally distributed as indicated by
the Kolmogorov-Smirnov test. Accordingly, parametric tests were
applied. These tests included the Student’s t-test (comparison between
two groups) and a one-way analysis of variance (ANOVA) followed by
Sidak’s multiple comparisons test (comparison between multiple
groups). Data were presented as means ± standard deviations (SD). p
values < 0.05 were regarded as statistically significant.
3. Results
Lithium increases EC viability, enhances P-gp expression, and in­
creases the abundance of tight junction proteins after OGD injury.
In order to examine a putative impact of lithium on cerebral EC in
vitro, cultured EC were exposed to OGD with subsequent re-cultivation
under standard cell culture conditions. Thereafter, cell viability,
expression of P-glycoprotein (P-gp; synonyms: multidrug resistance
protein 1 (MDR1), ATP-binding cassette sub-family B member 1
(ABCB1), cluster of differentiation 243 (CD243)), and the abundance of
selected tight junction proteins were quantitatively measured 38 h after
induction of OGD. The cell viability assay revealed a significant
enhancement in the ratio of live cells in the lithium-treated group in
comparison to the control group (F(2,26) = 161.1, p = <0.0001)
(Fig. 1A). However, the ratio of live cells in the lithium-treated group
was still significantly lower compared to the non-OGD group (F(2,26) =
161.1, p = <0.0001) (Fig. 1A). Using the MTT assay, we observed a
significant increase (t(35) = 3.5, p = 0.0015) in cellular metabolism of the
lithium-treated group vs the control (Fig. 1B).
Quantitative Western blot analysis also revealed a significantly
higher expression of zonula occludens-1 (ZO-1) (t(26) = 3.786, p =
0.0008) and occludin (t(18) = 2.771, p = 0.0126) in the lithium group
compared to the control group (Fig. 1E–FE). In addition, P-gp abundance
was significantly increased after treatment with lithium when EC were
exposed to OGD (t(16) = 2.875, p = 0.011) (Fig. 1C). These results are in
favor of lithium enhancing both the cell-cell-integrity and modulating P-
gp expression of cultivated ECs after OGD in vitro.
3.1. Lithium reduces post-ischemic brain extravasation of Evans Blue
Next, the Evans Blue extravasation was used to investigate the post-
stroke integrity of the BBB after lithium administration in mice. Evans
Blue concentrations in brain tissue of the right ischemic hemisphere of
both stroke mice and non-ischemic sham mice were analyzed 24 h after
MCA occlusion. Spectrophotometric analysis revealed a significant in­
crease in Evans Blue concentration in the brain parenchyma of controls
compared to the sham group (F(2,21) = 61.51, p = <0.0001) (Fig. 2A).
However, the Evans Blue concentration in the lithium group was
significantly lower compared to the control group (F(2,9) = 37.15, p =
<0.0001) (Fig. 2A), suggesting an enhanced level of BBB stability due to
lithium treatment.
3.2. Lithium minimizes post-ischemic loss of tight junction proteins ZO-1,
occludin and claudin-1 in vivo
Since lithium led to an enhanced expression of tight junction proteins
of EC exposed to OGD, we next wanted to investigate if this also
occurred in vivo. Using immunofluorescence and Western blotting, the
expression of ZO-1, occludin, and claudin-1 was measured in ischemic
hemispheres both 24 h and 72 h after MCA occlusion.
The immunofluorescence staining showed a significantly higher
expression of claudin-1 in the lithium group compared to the control
group 24 h after MCA occlusion (t(15) = 8.654, p = 0.0001) (Fig. 2B).
Further analysis revealed claudin-1 positive cells to co-localize with
M. Haupt et al. Neuropharmacology 181 (2020) 108357

Fig. 1. In vitro lithium treatment increases endothelial cell (EC) viability and enhances both P-glycoprotein (P-gp) and tight junction protein abundance.
EC underwent 14 h of oxygen glucose deprivation (OGD) followed by subsequent treatment with medium or medium containing 1.25 mM lithium chloride (OGD +
Li) for 24 h. (A) Analysis of live (green) and dead (red) cells using a commercial cell viability kit. n = 10 for non-OGD and OGD, n = 9 for Li + OGD. Scale bars: 50
μm. (B) Analysis of cell viability using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The metabolic activity of non-OGD group
were taken as 100%. n = 16 per group. (C) Quantitative measurement of P-gp receptor protein expression using Western blot analysis normalized with the
housekeeping protein glycerol-3-phosphate dehydrogenase (GAPDH). n = 8 for OGD, n = 10 for Li + OGD. (D) Non-quantitative immunofluorescence staining of ZO-
1, depicting representative staining areas. Scale bars: 20 μm. (E) Quantitative measurement of tight junction protein ZO-1 expression using Western blot analysis
normalized with the housekeeping protein Tubulin. n = 14 per group. (F) Quantitative measurement of tight junction proteins occludin expression using Western blot
analysis normalized with the housekeeping protein GAPDH. n = 6 for OGD, n = 14 for Li + OGD. Data are expressed as mean ± SD. *p < 0.05, **p < 0.01, ***p <
0.001, ****p < 0.0001.f

CD31 positive cells (Fig. S7). The quantitative Western blot analysis
revealed higher expression of occludin (t(14) = 3.108, p = 0.0077) and
ZO-1 (t(14) = 3.493, p = 0.0036) in the lithium group compared to the
control group 24 h after MCA occlusion (Fig. 2C). After 72 h of reper­
fusion, the quantitative Western blot analysis showed a higher expres­
sion of ZO-1 (t(10) = 2.468, p = 0.0332) in the lithium group compared to
the control group, with no significant impact of lithium on occludin
expression (t(10) = 0.7897, p = 0.448) at this time point (Fig. 2D). These
results suggest that delivery of lithium minimizes the loss of tight
junction proteins 24 h after MCA occlusion.
3.3. Lithium reduces post-ischemic activity of MMP-9 by caveolin-1
independent pathways
We next evaluated possible mechanisms that might be involved in
the process of stabilization of the BBB. Therefore, the expression and
activity of MMP-9 as well as the expression of caveolin-1 was measured
using immunofluorescence staining and zymography. The immunoflu­
orescence staining showed a significantly higher levels of MMP-9 in the
control group in comparison to the lithium group (F(2,21) = 16.53, p =
0.0057) and sham group (F(2,9) = 10.69, p = <0.0001) (Fig. 3A). This is
in line with our zymography analysis, which revealed a significantly
enhanced pro-MMP-9 activity in control mice when compared to both
sham mice (F(2,27) = 10.12, p = 0.0004) or mice treated with lithium
(F(2,27) = 10.12, p = 0.0196). Moreover, the activity of activated MMP-9
was significantly elevated in control mice compared to sham mice
(F(2,27) = 24.69, p = <0.0001) and lithium treated mice (F(2,27) = 24.69,
p = 0.0007). However, the quantity of activated MMP-9 was still
significantly enhanced in lithium treated mice compared to sham mice
(F(2,27) = 24.69, p = 0.0332) (Fig. 3B). Interestingly, significant
increased pro-MMP-2 activity in control mice compared to sham mice
were observed (F(2,27) = 7.118, p = 0.0024), but no significant difference
between the control and the lithium group was seen (F(2,27) = 7.118, p =
0.2125) (Fig. 3B). Active MMP-2 activity was not detectable in all three
groups. As caveolin-1 has been shown to regulate the activity of MMPs,
we proceeded to investigate the expression of caveolin-1 in our system.
Both our quantitative Western blot analysis (F(2,19) = 8.932, p = 0.9997)
and immunofluorescence staining (F(2,22) = 0.6207, p = 0.8275) showed
no significant difference in caveolin-1 expression between the control
and the lithium group (Fig. 3C–D). However, our Western blot analysis

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M. Haupt et al. Neuropharmacology 181 (2020) 108357

Fig. 2. Lithium preserves blood-brain

barrier (BBB) integrity and expres­
sion of tight junction proteins
claudin-1, zonula occludens-1 (ZO-1)
and occludin after cerebral ischemia
in mice. Mice were exposed to 45 min of
middle cerebral artery (MCA) occlusion
followed by 24 h or 72 h survival with
reperfusion. Sham group mice under­
went the surgery procedure without
MCA occlusion. Mice were treated with
phosphate buffered saline (PBS, control)
or intraperitoneally treated at the
beginning of reperfusion with 1 mmol/
kg lithium chloride and 6 h later with
another 2 mmol/kg of lithium chloride.
Mice with 72 h survival got two addi­
tional injections of PBS (control) or 2
mmol/kg lithium chloride 24 h and 48 h
after MCA occlusion. (A) Analysis of the
BBB integrity using the Evans Blue
extravasation assay of the right ischemic
hemisphere. n = 8 per group. (B)
Quantitative analysis of claudin-1
expression by immunofluorescence
staining within the right ischemic stria­
tum. Scale bars: 20 μm. n = 9 for control,
n = 8 for lithium. (C) Quantitative
analysis of ZO-1 and occludin expression
24 h after MCA occlusion using Western
blot analysis of the right ischemic
hemisphere. Normalized with house­
keeping proteins tubulin or glycerol-3-
phosphate dehydrogenase (GAPDH). n
= 6 for control, n = 10 for lithium. (D)
Quantitative analysis of ZO-1 and
occludin expression 72 h after MCA oc­
clusion using Western blot analysis of
the right ischemic hemisphere. Normal­
ized with housekeeping proteins tubulin
or GAPDH. n = 6 per group. Data are
expressed as mean ± SD. *p < 0.05, **p
< 0.01, ***p < 0.001, ****p < 0.0001.

revealed a significant induction of caveolin-1 expression in the control
(F(2,19) = 8.932, p = 0.0064) and in the lithium group (F(2,19) = 8.932, p
= 0.003) in comparison to the sham group. Immunohistochemical
analysis revealed caveolin-1 to be expressed in ECs, astroglial cells, and
neurons (Fig. S8). Taken together, lithium significantly reduces MMP-9
activity through a caveolin-1 independent pathway.
3.4. Lithium activates the MAPK/ERK1/2 pathway by elevated c-Raf,
MEK1/2, and ERK1/2 phosphorylation
the ischemic hemisphere (Fig. 4A,C,E). However, we observed through
Western blot analysis that the phosphorylation of c-Raf (p-c-Raf) (F(2,19)
= 5.823, p = 0.0282, p = 0.0165), MEK1/2 (p-MEK1/2) (F(2,19) = 13.18,
p = 0.0002, p = 0.0129) and ERK1/2 (p-ERK1/2) (F(2,19) = 13.19, p =
0.0003, p = 0.0048), which marks these kinases as active were signifi­
cantly increased in the lithium group compared to the sham (first p
value) and control (second p value) (Fig. 4B,D,F). In addition, Western
blot analysis revealed significantly higher levels of Bcl-2 in the sham
(F(2,19) = 6.041, p = 0.0409) and lithium group (F(2,19) = 6.041, p =
0.0466) both compared to control (Fig. 4G).

Evidence suggests that ERK1/2 affects the BBB integrity by regu­

lating ZO-1 and occludin expression (Liu et al., 2014). In view of these 3.5. Inhibition of the MAPK/ERK1/2 pathway reverses lithium-induced
findings, we analyzed the MAPK/ERK1/2 signaling pathway. Western effects on MMP-9 and BBB integrity
blot analysis of absolute c-Raf, MEK1/2 and ERK1/2 expression revealed
no difference between the sham, control and lithium group after 24 h in To further analyze whether the MAPK/ERK1/2 pathway is a key

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M. Haupt et al. Neuropharmacology 181 (2020) 108357

Fig. 3. Lithium decreases matrix metallopeptidase- 9 (MMP-9) activity independent of caveolin-1 expression. Mice were exposed to 45 min of middle cerebral
artery (MCA) occlusion followed by 24 h survival. Sham group mice underwent the surgery procedure without MCA occlusion. Mice were treated with phosphate
buffered saline (PBS, control and sham) or intraperitoneally treated at the beginning of reperfusion with 1 mmol/kg lithium chloride and 6 h later with another 2
mmol/kg of lithium chloride. (A) Determination of MMP-9 quantity by immunofluorescence staining and measurement of fluorescence intensity of the ischemic and
sham striatum normalized with negative control. n = 8 per group. (B) Analysis of MMP-9 and MMP-2 activity using gelatine zymography of the right ischemic
hemisphere or right sham hemisphere. Commercially available pro-MPP-2/MMP-9 and active MMP-2/MMP-9 were used as standards (Std). n = 10 per group. (C)
Analysis of caveolin-1 expression using immunofluorescence staining and measurement of fluorescence intensity in the ischemic and sham striatum. Scale bars: 20
μm. n = 8 for sham and lithium, n = 9 for control. (D) Quantitative analysis of caveolin-1 expression by Western blot analysis of the right ischemic hemispheres and
sham hemispheres. Western blot was normalized with the housekeeping protein tubulin. n = 6 for sham and control, n = 10 for lithium. Data are expressed as mean
± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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M. Haupt et al. Neuropharmacology 181 (2020) 108357

Fig. 4. Lithium modulates the MAPK/ERK1/2 pathway. Mice were exposed to 45 min of middle cerebral artery (MCA) occlusion followed by 24 h survival. Sham
group mice underwent the surgery procedure without MCA occlusion. Mice were treated with phosphate buffered saline (PBS, control) or intraperitoneally treated at
the beginning of reperfusion with 1 mmol/kg lithium chloride and 6 h later with another 2 mmol/kg of lithium chloride. (A–G) Quantitative analysis of c-Raf, p-c-
Raf, MEK1/2, p-MEK1/2, ERK1/2, p-ERK1/2, and Bcl-2 expression by Western blot analysis of the right ischemic hemisphere and sham hemispheres. Normalized
with the housekeeping proteins tubulin or glycerol-3-phosphate dehydrogenase (GAPDH). n = 6 for sham and control, n = 10 for lithium. Data are expressed as mean
± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

player in lithium-induced enhancement of post-stroke BBB integrity, the
MEK1/2 inhibitor U0126 was applied in order to inhibit the pathway.
Expression levels of MEK1/2, p-MEK1/2, ERK1/2, and p-ERK1/2 after
treatment with U0126 were determined using Western blot analysis at
24 h after MCA occlusion. Whereas absolute expression levels of MEK1/
2 did not differ between the treatment groups (F(2,20) = 0.04393, p =
0.9571), the levels of p-MEK1/2 were significantly decreased in the
U0126 group in comparison to the control group (F(2,20) = 3.842, p =
0.0387) (Fig. 5A–B). Likewise, total levels of ERK1/2 were not affected
by the various treatment schemes (F(2,20) = 0.2932, p = 0.7490), while
the levels of p-ERK1/2 were significantly decreased in the U0126 group
(F(2,20) = 6.577, p = 0.0037) compared to control (Fig. 5C–D), further
confirming inhibition of the MAPK/ERK1/2 pathway by U0126. The
subsequent zymography analysis showed no difference in pro-MMP-9
(F(2,21) = 0.7083, p = 0.5039), active-MMP-9 (F(2,21) = 0.001703, p =
0.9983) and pro-MMP-2 (F(2,21) = 0.3667, p = 0.6974) activity between
the controls and mice treated with either lithium + U0126 or U0126
alone (Fig. 5E). In addition, spectrophotometric analysis revealed no
significant difference in Evans Blue concentrations between controls and
mice treated with either lithium + U0126 or U0126 alone (F(2,20) = 1.16,
p = 0.3337) (Fig. 5F). These data show that activation of the MAPK/
ERK1/2 pathway and subsequent inhibition of MMP-9 activity is critical
in lithium-induced stability of the post-stroke BBB.
3.6. Lithium reduces post-ischemic activation of phagocytes
Neuroinflammation and BBB opening after hypoxia are closely
linked (Jiang et al., 2018). To evaluate whether lithium modulates
phagocyte activation 24 h after MCA occlusion, immunofluorescence
phagocytes and thereby a modulated neuroinflammatory response by
lithium.
3.7. Lithium modulates the expression of the endothelial surface proteins
ICAM-1 and P-gp after stroke in vivo
Besides BBB opening, the expression of the intercellular adhesion
molecule 1 (ICAM-1) and vascular cell adhesion protein 1 (VCAM-1) also
play important roles in leukocyte infiltration into the brain parenchyma
(Yanamadala and Friedlander, 2010). Indeed, Western blot analysis of
the ischemic hemispheres showed a significantly lower expression of
ICAM-1 in lithium-treated mice when compared to controls (t(14) =
3.659, p = 0.0026) (Fig. 7A). However, no difference in VCAM-1
expression was observed (Fig. 7B). Since lithium showed an effect on
P-gp expression in ECs, we analyzed P-gp expression levels in mice
exposed to cerebral ischemia. Likewise, P-gp expression levels were
significantly increased in lithium-treated mice when compared to con­
trols (t(14) = 2.371, p = 0.0326) (Fig. 7C).
3.8. Lithium modulates the early migration of immune cells into the
infarct area
Next, we analyzed the impact of lithium on very early infiltrating
leukocytes. Herein, we focused on neutrophil, T cell, B cell, monocyte
and macrophage markers. The brain tissue of the ischemic hemisphere
was analyzed by flow cytometry 24 h after MCA occlusion. The subsets
of leukocytes (CD45high), resident microglia (CD45int), T cells
(CD45highCD3+), neutrophils (CD45highLy6G+), B cells
high -
(CD45 CD3 Ly6G CD19 ) and macrophages and monocytes
− +

staining for the calcium-binding adapter molecule 1 (Iba-1) of the right
ischemic hemispheres was performed. The immunofluorescence staining
showed a significant decrease in morphologically activated phagocytes
in the lithium group compared to the control group (t(14) = 5.702, p =
0.0001) (Fig. 6). These data suggest a very early effect on activation of
(CD45highCD3-Ly6G− CD11b+) were analyzed (Fig. 8H). The analysis
showed a significantly higher proportion of leukocytes in the control
group vs both the lithium group (F(2,12) = 28.65, p = <0.0001) and sham
group (F(2,12) = 28.65, p = 0.0002) (Fig. 8A). No difference in the subset
of resident microglia was observed (Fig. 8B). Since extracranial

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M. Haupt et al. Neuropharmacology 181 (2020) 108357

Fig. 5. The MEK1/2 inhibitor U0126 reverses lithium-induced effects on MMP-9 and post-stroke blood-brain barrier (BBB) integrity. Mice were exposed to
45 min of middle cerebral artery (MCA) occlusion followed by 24 h of survival. Animals were treated with phosphate buffered saline (PBS, control) or with 0.5 mg/kg
U0126 at the beginning of the reperfusion. The combined treatment approach consisted of an intravenous injection of 0.5 mg/kg U0126 plus an intraperitoneal
injection of 1 mmol/kg lithium chloride at the beginning of the reperfusion followed by another intraperitoneal injection of 2 mmol/kg lithium chloride 6 h later.
(A–D) Quantitative analysis of MEK1/2, p-MEK1/2, ERK1/2 and p-ERK1/2 expression patterns by Western blot analysis of the right ischemic hemisphere. Western
blots were normalized with the housekeeping protein glycerol-3-phosphate dehydrogenase (GAPDH). n = 8 for control and lithium + U0126, n = 7 for U0126. (E)
Analysis of MMP-9 and MMP-2 activity using gelatine zymography of the right ischemic hemisphere. Commercially available pro-MPP-2/MMP-9 and active MMP-2/
MMP-9 were used as standards (Std). n = 8 per group. (F) Analysis of the BBB integrity using the Evans Blue extravasation assay of the right ischemic hemisphere. n
= 8 for control and U0126, n = 7 for lithium + U0126. Data are expressed as mean ± SD. *p < 0.05, **p < 0.01.

leukocytes usually do not invade the brain parenchyma under physio­
logical conditions, we only observed some leukocytes in the sham group.
Consequently, a further analysis of different cellular subpopulations in
sham animals was not performed.
Further analyzing of the cell subtypes in control and lithium group
revealed a significant increase in the subset of T cells (t(8) = 3.44, p =
0.0088), and a significant decrease in the subset of neutrophils (t(8) =
2.34, p = 0.0474) in the lithium group compared to the control group
(Fig. 8C–D). Lithium did not affect the population of B cells (t(8) =
0.1148, p = 0.9114), monocytes and macrophages (t(8) = 0.8556, p =
0.4171) (Fig. 8E–F). Taken together, these results indicate that lithium
treatment decreases the infiltration of leukocytes and especially
neutrophil invasion into the brain parenchyma at 24 h post-stroke. On
the contrary, the ratio of T cells is elevated at this early stage upon
lithium treatment.
4. Discussion
Lithium has been recently recognized to exert neuroprotection upon
experimental induction of stroke. Although the precise mechanism of
action still remains elusive, results from a clinical study seemed prom­
ising, demonstrating a trend towards better neurological recovery in
stroke patients treated with lithium (Abdollahi et al., 2014). While
additional clinical trials should be conducted to verify the significance of
lithium treatment in stroke patients, understanding of the downstream
mechanisms by which lithium induces its neuroprotective effects upon
stroke is necessary to identify stroke patients who will benefit from such
a lithium treatment. The present study therefore aimed to analyze, if
lithium plays a role in the BBB opening, a key mechanism in the acute
stage of stroke. For the first time, we were able to show that lithium
significantly increases the stability of the BBB upon experimental stroke
in mice. Lithium acts through impairing the activity of MMP-9 and
enhancing phosphorylation as well as activation of the pro-survival
MAPK/ERK1/2 pathway, all of which resulting in reduced BBB
leakage. Conversely, inhibition of the MAPK/ERK1/2 pathway reverses
the lithium-induced reduction of MMP-9 activity and abolished the ef­
fect on BBB permeability. In addition, lithium decreases ICAM-1
expression which reduces neutrophil invasion and increases T cell
migration into the brain.
Previous studies have already reported a modulation of Ca2+ ho­
meostasis and the survival-promoting effects by lithium treatment in
vitro under both ischemic and non-ischemic conditions (Bosche et al.,
2013, 2016a; 2016b; Cimarosti et al., 2001; Silachev et al., 2016).
However, the role of tight junction protein expression has not been
explored in such studies. Opening of the BBB is a key event during the
acute phase of cerebral ischemia, where the degradation of tight junc­
tion proteins such as claudin-1, ZO-1 and occludin appears to be critical
(Jiang et al., 2018; Jiao et al., 2011; Yadav and Shin, 2015). Through an
in vitro approach, we showed that lithium enhances the resistance of
cultivated EC against OGD, inhibiting the OGD-induced downregulation
of tight junction proteins. In this context, Krueger and colleagues iden­
tified four different stages of BBB breakdown after cerebral ischemia,
with the ultimate loss of endothelial cells occurring during the last stage,
indicating the importance of the present findings (Krueger et al., 2015).
Furthermore, our in vivo studies support this observation where we
found lithium treatment to increase expressions of claudin-1, ZO-1 and
occludin 24 h post-MCA occlusion. Interestingly, 72 h after MCA oc­
clusion resulted in only a significant difference in ZO-1 expression

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M. Haupt et al.
Fig. 6. Lithium reduces post ischemic phagocyte activation. Mice were
exposed to 45 min of middle cerebral artery (MCA) occlusion followed by 24 h
survival. Mice were treated with phosphate buffered saline (PBS, control) or
intraperitoneally treated at the beginning of reperfusion with 1 mmol/kg
lithium chloride and 6 h later with another 2 mmol/kg of lithium chloride.
Analysis of morphologically activated IBA-1 positive phagocytes using immu­
nofluorescence staining of the right ischemic striatum. Representative immu­
nofluorescence stainings are shown with scale bars: 20 μm. n = 8 per group,
data are expressed as mean ± SD. ****p < 0.0001.
between the control and the lithium-treated group. This can be
explained by the fact that development of BBB leakage upon induction of
cerebral ischemia occurs with temporal resolution. Indeed, Jiao and
colleagues have previously analyzed the time-dependent dynamics of
tight junction protein degradation and restoration following cerebral
ischemia in mice (Jiao et al., 2011). Whereas Jiao et al. suggest a
restoration of tight junction protein expression at 120 h post-stroke (Jiao
et al., 2011), our results are in favor of a partial restoration of the BBB at
72 h, indicated by the increased expression of occludin in the control
group. However, this could be context-dependent and likely to be a
consequence of the short duration of cerebral ischemia chosen for our
study. In our study, we focused mainly on EC and tight junction
expression as the predominant factor for BBB integrity. Although there is
no doubt about the role of EC in maintaining BBB integrity, the inter­
action of EC with mural cells, immune cells, glial cells, and neural cells is
also critical (Daneman and Prat, 2015). Indeed, Sliachev and colleagues
demonstrated that lithium treatment protects astrocytes against OGD in
vitro, indicating that the protective effect of lithium is not limited to EC
(Silachev et al., 2016). Therefore, lithium appears to modulate the entire
neurovascular unit.
Post-ischemic degradation of tight junction proteins is mainly
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Neuropharmacology 181 (2020) 108357
Fig. 7. Lithium modulates expression of the endothelial surface protein
intercellular adhesion molecule 1 (ICAM-1) and the P-glycoprotein (P-gp)
Mice were exposed to 45 min of middle cerebral artery (MCA) occlusion fol­
lowed by 24 h survival. Mice were treated with phosphate buffered saline (PBS,
control) or intraperitoneally treated at the beginning of reperfusion with 1
mmol/kg lithium chloride and 6 h later with another 2 mmol/kg of lithium
chloride. (A) Measurement of ICAM-1 expression using Western blot analysis of
the right ischemic hemisphere normalized with the housekeeping protein
tubulin. (B) Quantification of vascular cell adhesion protein 1 (VCAM-1)
expression by Western blot analysis of the right ischemic hemisphere normal­
ized with the housekeeping protein glycerol-3-phosphate dehydrogenase
(GAPDH). (C) Measurement of P-gp expression using Western blot analysis of
the right ischemic hemisphere normalized with the housekeeping protein
tubulin. n = 6 for control and n = 10 for lithium. Data are expressed as mean ±
SD. *p < 0.05, **p < 0.01.
induced by an upregulated expression and increased activity of MMPs
such as MMP-2 and MMP-9 (Kurzepa et al., 2014). Based on this
observation, different studies have proposed an antagonization of
MMP-9 activity to be a potential therapeutic strategy against cerebral
ischemia and subsequent BBB leakage (Medsker et al., 2016). Indeed,
some MMP-9 inhibitors have shown promising neuroprotective effects
(Jiang et al., 2001; Murozono et al., 2009). In line with former reports,
we were able to observe a stroke-induced increase of pro-MMP-9,
pro-MMP-2 and activated MMP-9 (Park et al., 2009). Here, we showed
that lithium inhibits the post-ischemic increase of pro-MMP-9 expres­
sion together with a reduced quantity of activated MMP-9. Apart from its
expression, MMP-9 activity can also be regulated by other means such as
the MAPK pathway, which has been shown to negatively regulate the
activity of MMP-9 (Curry et al., 2010). In fact, we demonstrated that
lithium treatment led to an activation of the MAPK pathway as seen by
the induction of phosphorylated cRaf, MEK1/2 and ERK1/2.
M. Haupt et al. Neuropharmacology 181 (2020) 108357

Fig. 8. Lithium decreases post ischemic infiltration of neutrophils but increases T cell invasion into the ischemic hemisphere. Mice were exposed to 45 min
of middle cerebral artery (MCA) occlusion followed by 24 h survival. Sham group mice underwent the surgery procedure without MCA occlusion. Mice were treated
with phosphate buffered saline (PBS, control) or intraperitoneally treated at the beginning of reperfusion with 1 mmol/kg lithium chloride and 6 h later with another
2 mmol/kg lithium chloride. (A–F) Flow cytometry of brain tissue from sham, control and lithium group analysing leukocytes (CD45high), resident microglia
(CD45int), neutrophils (CD45highLy6G+), T cells (CD45highCD3+), macrophages/monocytes (CD45highCD3-Ly6G-CD11b+) and B cells (CD45highCD3-Ly6G-CD19+). (G)
Representative flow cytometry measurements of brain tissue from sham, control and lithium treated mice with three parent gates: single cells, CD45high and
CD45highCD3-Ly6G-. (H) Scheme of gating strategy for flow cytometry analysis. n = 5 per group, data are expressed as mean ± SD. *p < 0.05, ***p < 0.001, ****p
< 0.0001.

Importantly, the inhibition of the MAPK pathway using a MEK1/2 in­
hibitor, abolished the effect of lithium on MMP-9 activity and inhibited
the maintenance of BBB integrity, suggesting the significance of MAP­
K/ERK1/2 activation in this context. It is important to note that the
MAPK/ERK1/2 pathway can play a dual role in the pathophysiology of
cerebral ischemia, where it can lead to both neuroprotective or detri­
mental effects (Sawe et al., 2008). However, our study showed that
lithium protects BBB integrity following stroke through activating the
MAPK pathway and subsequently inhibiting MMP-9 activity. In addi­
tion, the level of Bcl-2, an anti-apoptotic protein downstream of the
MAPK pathway, was increased after lithium treatment. This is in line
with previous studies reporting reduced apoptosis upon lithium treat­
ment after ischemic stroke in mice (Doeppner et al., 2017).
Apart from the MAPK pathway, the activity of MMP-9 can be regu­
lated by multiple different signaling molecules, and caveolin-1 has also
been shown to play a role in impairing MMP activity (Gu et al., 2011). In
fact, expression of caveolin-1 is downregulated after cerebral ischemia
which leads to a post-ischemic increase of MMP-9 activity (Gu et al.,
2012). In this study, we found caveolin-1 to be ubiquitously expressed in
the ischemic brain parenchyma, but we did not observe any change of
caveolin-1 expression patterns in the lithium group. Taken together, we
conclude that lithium modulates BBB integrity and MMP-9 activity in­
dependent of caveolin-1.
Besides these effects on cell survival and tight junction protein
expression, we also observed a lithium-induced increase in P-gp
expression in EC in vitro and in the ischemic hemisphere of mice 24 h
after cerebral ischemia in vivo. Physiologically, P-gp is expressed on the
luminal side of EC of the BBB and it is an important gate-keeper of the
BBB (Schinkel, 1999). As a gate-keeper, P-gp plays a role in controlling
the efflux of both small endogenous molecules and a large fraction of
prescribed drugs from the brain side to the blood side (Abbott et al.,
2002; Schinkel, 1999). A previous study has shown that P-gp expression
is upregulated in the capillary endothelium at 24 h after focal cerebral
ischemia (Spudich et al., 2006). Additionally, Ramos and colleagues
demonstrated that ischemic stress in rats induce the expression of P-gp
in normally non-expressive cells such as astrocytes and neurons (Ramos
et al., 2004). However, the role of P-gp after cerebral ischemia is not
clear. A study in P-gp knockout mice suggests that the reduction of P-gp
expression is neuroprotective (Murozono et al., 2009). On the other
hand, P-gp has been shown to protect cells from intracellular accumu­
lation of toxic components and was found to be elevated in
hypoxia-induced apoptosis in cells (Johnstone et al., 1999; Lazarowski
et al., 2007; Robinson et al., 1997). Moreover, Kraya et al. suggest that
P-gp activity positively affects tight junctional protein expression and
BBB resistance (Kraya et al., 2016). Therefore, further studies on the role
of P-gp in the context of cerebral ischemia are important to determine if
the elevated P-gp expression observed upon lithium treatment in our
system contributes to its neuroprotective properties.
The post-stroke immune cell response is associated with opening of
the BBB, and the immune system is known to be a key player in the
pathophysiology of stroke (Dirnagl et al., 1999). Consistent with the
observed stabilization of the BBB by lithium, we found a decrease in
early infiltration of leukocytes into the ischemic hemisphere following
lithium treatment. Of note, the proportion of neutrophils within the
infiltrated leukocytes was significantly reduced. Neutrophils are among
the first hematogenous immune cells found in the brain after cerebral
ischemia (Anrather and Iadecola, 2016). Their extravasation is regu­
lated by the activation of EC, release of chemokines and by the expres­
sion of adhesion molecules such as ICAM1 and VCAM-1 (Dirnagl et al.,
1999). Previous studies suggest that neutrophils play an important role

11
M. Haupt et al.
in the exacerbation of ischemic brain injury (Garcia-Bonilla et al., 2014;
Herz et al., 2015). In fact, neutrophils can contribute to the destabili­
zation of the BBB by releasing active MMP-9 (Ludewig et al., 2013;
Rosell et al., 2008). Moreover, inhibition of ICAM1 is associated with
decreased brain damage and improved outcome of experimental stroke
(Kitagawa et al., 1998; Vemuganti et al., 2004). In contrast, inhibition of
VCAM-1 expression showed no effect on neutrophil invasion and infarct
size (Justicia et al., 2006). Interestingly, we found lithium to reduce the
expression of ICAM1, but not VCAM-1 expression. Taken together, the
reduced ICAM-1 expression and decreased neutrophil invasion might be
another important aspect of the lithium-induced decrease in MMP-9
activity and subsequent maintenance of BBB integrity.
Apart from the reduction in neutrophil invasion, we found an
increased proportion of T cells within the infiltrated leukocytes in
ischemic hemispheres of the lithium-treated group. This could indicate
that lithium selectively decreases neutrophil invasion, which leads to an
increase in the proportion of T cells. It is important to note that the role
of T cells however, remains controversial with some studies having re­
ported a detrimental effect of early T cell infiltration after cerebral
ischemia (Hurn et al., 2007; Kleinschnitz et al., 2010). On the other
hand, Li and colleagues found that post-ischemic administration of
regulatory T cells markedly reduced brain infarct size by stabilizing the
BBB through suppression of MMP-9 (Li et al., 2013). Notably, we have
not established, if lithium directly increases T cells invasion or if it
indirectly enhances the proportion of T cells within the infiltrated
leukocyte population by decreasing the population of neutrophils.
Therefore, further studies on the effect of lithium on immune cell
migration are warranted, including a more detailed analysis of the
subpopulation of infiltrated T cells involved after stroke induction.
Finally, it is noteworthy that lithium is a substance with pleiotropic
effects, which can modulate many different pathways after cerebral
ischemia. Our previous study indicated that lithium induced an increase
in miR-124 expression that is involved in neuroprotection (Doeppner
et al., 2017). This suggests that lithium does not exclusively affect the
MAPK/ERK1/2-pathway followed by stabilization of the BBB. We pro­
pose that lithium affects different signaling pathways, depending on the
temporal resolution of the disease.
Taken together, we showed for the first time that lithium stabilizes
the BBB after experimental stroke. Furthermore, our results provide
novel mechanistic insights of lithium-induced neuroprotection in the
context of post-ischemic BBB opening in mice. Importantly, with only a
single study conducted using lithium as a treatment for stroke patients,
both our in vitro and in vivo work provide substantial translational and
clinical relevance, opening up novel stroke treatment possibilities
through targeting BBB stabilization.
Sources of funding
The study was supported by grants of the Deutsche For­
schungsgemeinschaft (DFG) to BB (BO 4229/1-1 and BO 4229/2-1).
Declarations of interest
None.
CRediT authorship contribution statement
Matteo Haupt: Conceptualization, Investigation, Formal analysis,
Visualization, Writing - original draft, Writing - review & editing.
Bozena Zechmeister: Methodology, Writing - review & editing. Bert
Bosche: Investigation, Writing - review & editing. Simone Lieschke:
Investigation, Writing - original draft, Writing - review & editing. Xuan
Zheng: Investigation. Lin Zhang: Investigation, Methodology. Vivek
Venkataramani: Investigation, Writing - review & editing. Fengyan
Jin: Conceptualization, Writing - original draft. Katharina Hein:
Visualization, Resources. Martin S. Weber: Investigation,
12
Neuropharmacology 181 (2020) 108357
Methodology. Dirk M. Hermann: Conceptualization, Writing - original
draft. Mathias Bähr: Conceptualization, Writing - original draft.
Thorsten R. Doeppner: Conceptualization, Formal analysis, Writing -
original draft, Writing - review & editing, Supervision.
Acknowledgment
We thank both Irina Graf and Regine Kruse for excellent technical
assistance.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.neuropharm.2020.108357.
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