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\begin{document}

\title{Hypertension Promotes Neuroinflammation, Brain Injury and Cognitive
Impairment}



\author[1]{Quynh Dinh}%
\author[1]{Antony Vinh}%
\author[1]{Cecilia Lo}%
\author[1]{David Wong Zhang}%
\author[2]{Sharmelee Selvaraji}%
\author[1]{Hyun Ah Kim}%
\author[3]{Sophocles Chrissobolis}%
\author[1]{Thiruma Valavan Arumugam}%
\author[1]{Grant Drummond}%
\author[1]{Christopher Sobey}%
\author[1]{T. Michael De Silva}%
\affil[1]{La Trobe University}%
\affil[2]{National University of Singapore}%
\affil[3]{Ohio Northern University R H Raabe College of Pharmacy}%


\vspace{-1em}



  
  \date{May 26, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Background and purpose: Hypertension increases the risk for cognitive
impairment and promotes vascular and renal inflammation. We tested if
immune cell infiltration occurs in the brain during hypertension and if
it is associated with cognitive impairment. Experimental approach: Male
C57Bl/6 mice were administered vehicle, angiotensin II (0.7 mg/kg/d
S.C.) or aldosterone (0.72 mg/kg/d S.C.) via osmotic minipumps. A subset
of mice also received hydralazine (50 mg/kg) in their drinking water
after minipump implantation. We measured systolic blood pressure,
markers of inflammation, working memory and transcriptomic changes in
the brain. Key results: Administration of angiotensin II or aldosterone
increased blood pressure and promoted blood-brain barrier dysfunction,
leukocyte accumulation and impairment of working memory in mice. When
co-administered with angiotensin II, hydralazine prevented the
development of these changes. In a separate cohort of mice in which
angiotensin II-induced changes were first established, intervention with
hydralazine lowered blood pressure but did not reverse brain
inflammation or cognitive impairment. Finally, angiotensin II infusion
altered the transcriptomic profile of the whole brain, as well as
specifically within the hippocampus, and co-treatment with hydralazine
modulated these changes. Conclusion and implications: Experimental
hypertension leads to brain inflammation and impaired working memory.
Cognitive impairment that develops during hypertension can be inhibited,
but not readily reversed, by anti-hypertensive therapy.%
\end{abstract}%



\sloppy


\textbf{HYPERTENSION PROMOTES NEUROINFLAMMATION, BRAIN INJURY AND
COGNITIVE IMPAIRMENT}

\textbf{Quynh Nhu Dinh\textsuperscript{1}, Antony
Vinh\textsuperscript{1}, Cecilia Lo\textsuperscript{1}, David E. Wong
Zhang\textsuperscript{1}, Sharmelee Selvaraji\textsuperscript{2}, Hyun
Ah Kim\textsuperscript{1}, Sophocles Chrissobolis\textsuperscript{3},
Thiruma V. Arumugam\textsuperscript{1}, Grant R.
Drummond\textsuperscript{1}, Christopher G. Sobey\textsuperscript{1}, T.
Michael De Silva\textsuperscript{1}}

\textsuperscript{1}Centre for Cardiovascular Biology and Disease
Research and Department of Microbiology, Anatomy, Physiology \&
Pharmacology, School of Agriculture, Biomedicine, Environment, La Trobe
University, Victoria, Australia;

\textsuperscript{2}Memory Aging and Cognition Centre, Department of
Pharmacology, National University of Singapore, Singapore;

\textsuperscript{3}Department of Pharmaceutical and Biomedical Sciences,
Raabe College of Pharmacy, Ohio Northern University, Ohio, USA

Short title: Hypertension promotes cognitive impairment

Corresponding Authors:

T. Michael De Silva, PhD Christopher G Sobey, PhD

Dept. of Microbiology, Anatomy, Physiology \& Dept. of Microbiology,
Anatomy, Physiology \& Pharmacology Pharmacology School of Agriculture,
Biomedicine, Environment School of Agriculture, Biomedicine, Environment

La Trobe University La Trobe University

Bundoora, Victoria 3086, Australia Bundoora, Victoria 3086, Australia

e-Mail: t.desilva@latrobe.edu.au e-Mail: c.sobey@latrobe.edu.au

Phone: +61-3-94796876 Phone: +61-3-9479 1316

Quynh Nhu Dinh: q.dinh@latrobe.edu.au

Antony Vinh: a.vinh@latrobe.edu.au

Cecilia Lo: c.desilva@latrobe.edu.au

David E. Wong Zhang: 17141319@students.latrobe.edu.au

Sharmelee Selvaraji: e0358056@u.nus.edu

Hyun Ah Kim: h.kim2@latrobe.edu.au

Sophocles Chrissobolis: s-chrissobolis@onu.edu

Thiruma V. Arumugam: g.arumugam@latrobe.edu.au

Grant R. Drummond: g.drummond@latrobe.edu.au

Christopher G. Sobey: c.sobey@latrobe.edu.au

T. Michael De Silva: t.desilva@latrobe.edu.au

\textbf{ABSTRACT}

\textbf{Background and purpose:} Hypertension increases the risk for
cognitive impairment and promotes vascular and renal inflammation. We
tested if immune cell infiltration occurs in the brain during
hypertension and if it is associated with cognitive impairment.

\textbf{Experimental approach:} Male C57Bl/6 mice were administered
vehicle, angiotensin II (0.7 mg/kg/d S.C.) or aldosterone (0.72 mg/kg/d
S.C.) via osmotic minipumps. A subset of mice also received hydralazine
(50 mg/kg) in their drinking water after minipump implantation. We
measured systolic blood pressure, markers of inflammation, working
memory and transcriptomic changes in the brain.

\textbf{Key results:} Administration of angiotensin II or aldosterone
increased blood pressure and promoted blood-brain barrier dysfunction,
leukocyte accumulation and impairment of working memory in mice. When
co-administered with angiotensin II, hydralazine prevented the
development of these changes. In a separate cohort of mice in which
angiotensin II-induced changes were first established, intervention with
hydralazine lowered blood pressure but did not reverse brain
inflammation or cognitive impairment. Finally, angiotensin II infusion
altered the transcriptomic profile of the whole brain, as well as
specifically within the hippocampus, and co-treatment with hydralazine
modulated these changes.

\textbf{Conclusion and implications:} Experimental hypertension leads to
brain inflammation and impaired working memory. Cognitive impairment
that develops during hypertension can be inhibited, but not readily
reversed, by anti-hypertensive therapy.

Key words: Hypertension, inflammation, brain, cognition, blood-brain
barrier



\textbf{INTRODUCTION}

Hypertension is a prevalent condition affecting at least 30\% of the
global adult population (Mills et al., 2016). The impact of uncontrolled
hypertension is particularly pronounced in the brain and cerebral
circulation, where end-organ disease manifests earlier than in other
parts of the body (Jennings \& Zanstra, 2009). Hypertension is a major
risk factor for two of the most important conditions that impact the
brain: stroke and cognitive impairment (Iadecola et al., 2016). However,
the underlying mechanisms by which hypertension promotes brain disease
are not clear. Importantly, the incidence of cognitive impairment is
rapidly increasing and even mild cognitive decline increases the risk of
developing dementia (Bozoki, Giordani, Heidebrink, Berent, \& Foster,
2001). Patients on anti-hypertensives have a lower risk of cognitive
impairment and dementia (Ou et al., 2020) but it is not conclusive from
clinical trials whether anti-hypertensive therapy reverses established
cognitive dysfunction (Iadecola et al., 2016; Rapp et al., 2020).

Inflammation plays a central role in the pathophysiology of hypertension
(Drummond, Vinh, Guzik, \& Sobey, 2019). For example, mice that lack T
cells have reduced pressor responses (Dinh et al., 2021; Guzik et al.,
2007) and preventing T cell activation attenuates hypertension (Dinh et
al., 2021; Vinh et al., 2010). Hypertension is strongly associated with
inflammation and leukocyte infiltration into the systemic vasculature
and kidneys. While T cells and microglia are upregulated in the brain
during hypertension, it remains unclear whether other leukocyte subtypes
are similarly upregulated. Angiotensin II infusion has been shown to
stimulate tumour necrosis factor (TNF)-\selectlanguage{greek}α \selectlanguage{english}production in the hippocampus
(Iulita et al., 2018), a key brain region for regulating aspects of
cognitive function, particularly memory, and brain inflammation is
associated with cognitive impairment (Faraco et al., 2016).
Interestingly, evidence is accumulating to suggest that immune cells may
directly contribute to cognitive impairment. For example, T cell
infiltration in white matter is associated with cognitive decline in
aged monkeys (Batterman, Cabrera, Moore, \& Rosene, 2021), and T cell
depletion in a mouse model of Alzheimer's disease improves spatial
memory (Laurent et al., 2017). Further, in the setting of hypertension,
depletion of perivascular macrophages inhibits angiotensin II-induced
cognitive impairment (Faraco et al., 2016).

Here, we have infused mice with angiotensin II or aldosterone to test if
hypertension can promote brain immune cell infiltration, transcriptomic
changes and cognitive impairment. Using hydralazine hydrochloride, we
also studied whether development of these effects on the brain following
angiotensin II infusion are blood pressure-dependent and reversible.

\textbf{METHODS}

\textbf{Animals}

This study was approved by the La Trobe University Animal Ethics
Committee (AEC16-79; AEC16-93). Male C57BL/6J (WT) mice (n=218; 8-12
weeks old) were obtained from Animal Resource Centre (Canning Vale,
Western Australia, Australia). Standard rodent chow and drinking water
were provided \emph{ad libitum} . Animals were housed in individually
ventilated cages.

\textbf{Administration of pharmacological agents and measurement of
blood pressure}

Mice were randomly assigned to treatment groups. Angiotensin II was
dissolved in 0.9 \% saline and infused S.C. by osmotic minipump (Alzet
model 2002 or 2004) at 0.7 mg/kg/d. Control mice for angiotensin II
infusion received 0.9\% saline. Aldosterone was dissolved in 87 \%
propylene glycol, 9 \% ethanol and 4 \% Milli-Q water and administered
S.C. to some mice by osmotic minipump (Alzet model 2002) at 0.72
mg/kg/d. Control mice for aldosterone treatment were infused with 87 \%
propylene glycol, 9 \% ethanol and 4 \% Milli-Q water. Mice that were
administered vehicle or angiotensin II were maintained on normal
drinking water. Mice that were administered aldosterone had their
drinking water replaced with 0.9 \% saline after minipump implantation.

Mice were anaesthetised with isoflurane (2-4 \% inhaled with oxygen, 0.4
L/min) for \textasciitilde{}20 min. Adequacy of anaesthesia was
monitored by observing respiration and checking for a reflex response to
a toe pinch. An osmotic minipump was implanted S.C. in the mid-scapular
region for infusion for 14 or 28 d. At the time of surgery, mice were
administered bupivacaine (2.5 mg/kg S.C.) at the surgical site and
carprofen (5 mg/kg S.C.). Mice then received carprofen (5 mg/kg S.C.)
daily for 2 days after surgery. A subset of angiotensin II-infused mice
received hydralazine hydrochloride (50 mg/kg) in their drinking water
during the 14-d osmotic minipump infusion, or as an intervention at 2
weeks following the commencement of angiotensin II infusion.

Systolic blood pressure was monitored in conscious mice via tail cuff
plethysmography (MC4000 Multichannel system, Hatteras Instruments).
Prior to surgery, mice were trained for 1 d (i.e. on day -1) to
acclimate to the procedure, and blood pressure was then recorded on days
0 (prior to surgery), 3, 7, 14, 21 and 28 of angiotensin II or
aldosterone infusion.

\textbf{Flow cytometric analysis of leukocytes in brain}

Mice were killed by carbon dioxide inhalation and perfused through the
left cardiac ventricle with 0.2 \% clexane (400 IU, Sanofi Aventis,
Australia) in 0.01 M phosphate-buffered saline (PBS). The left brain
hemisphere was harvested for flow cytometry. Brain samples were minced
with scissors and digested in PBS (with MgCl\textsubscript{2} and
CaCl\textsubscript{2}) containing a mixture of collagenase type XI (125
U/ml), collagenase type I-S (460 U/ml) and hyaluronidase (60 U/ml)
(Sigma-Aldrich, USA) for 30 min at 37 \selectlanguage{ngerman}°C. Brain samples were then passed
through a 70 \selectlanguage{greek}μ\selectlanguage{english}m filter and subjected to a
Percoll\textsuperscript{TM}gradient (70 \% and 30 \% isotonic Percoll)
centrifugation from which the layer of mononuclear cells was collected
from the interface of the Percoll solutions. Brain cells were stained
with an antibody cocktail of anti-CD45 APC-Cy7 (30-F11, Biolegend, USA),
anti-CD11b Pacific Blue (M1-70, eBioscience, USA), anti-Ly6G PE-Cy7
(1A8, Biolegend, USA), anti-Ly6C FITC (HK1.4, Biolegend, USA), anti-CD3\selectlanguage{greek}ε
\selectlanguage{english}APC (145-2C11, eBioscience, USA), anti-CD4 Alexa Fluor 700 (GK1.5,
eBioscience, USA), anti-F4/80 BV711 (BM8, Biolegend, USA) and anti-CD19
PE-Cy5 (6D5, Biolegend, USA), and diluted in PBS containing 0.5\% bovine
serum albumin. Samples were analysed via flow cytometry using a CytoFLEX
LX flow cytometer (Beckman Coulter, USA) and FlowJo Software (version
10.1, Tree Star Inc, USA, Supplementary Figure 1). Cell numbers were
expressed as total cells per brain hemisphere.

\textbf{Assessment of blood-brain barrier permeability}

Blood-brain barrier permeability was assessed by staining brain sections
for endogenous immunoglobulin type G (IgG). Whole brains were
snap-frozen in liquid nitrogen and stored at -80 \selectlanguage{ngerman}°C. Ten \selectlanguage{greek}µ\selectlanguage{english}m coronal
sections were cut and thaw-mounted onto Superfrost Ultra Plus slides
(Thermo Fisher Scientific, USA). Frozen brain sections were air-dried (5
min), fixed with 100 \% acetone at 4 @\selectlanguage{english}C (10 min) and washed 3 x 5 min
with 0.01 M PBS. Sections were incubated with goat anti-mouse IgG (goat
anti-mouse IgG (ab150118), Alexa 555, 1:200 dilution) (Abcam, Cambridge,
UK) in antibody diluent (3 h), then washed 3 x 5 min with 0.01 M PBS in
a dark room. Sections were mounted with
VECTASHIELD\selectlanguage{ngerman}\textsuperscript{®}mounting medium containing the nuclear
stain DAPI (Vector Laboratories, Inc. Burlingame, USA) and cover
slipped. Edges were sealed with nail polish and sections were stored at
4 \selectlanguage{english}@C until imaging. The hippocampus and cortex (2 images per animal)
were imaged with an Olympus DP73 Camera (Olympus Corporation, Tokyo,
Japan) connected to an Olympus BX53 microscope (Olympus Corporation,
Tokyo, Japan) at 100x and 200x magnification running CellSens Standard
Software (version 1.17, Olympus Corporation). Exposure settings, ISO and
black balance were kept consistent across all images. Percentage area of
staining was analysed using FIJI (National Institute of Health, USA) and
the threshold settings were kept consistent across all images.

\textbf{Gene expression in brain}

Messenger RNA (mRNA) expression of inflammatory markers in the brain was
determined using TaqMan\selectlanguage{ngerman}® real-time PCR. The right brain hemisphere was
harvested and snap frozen in liquid nitrogen. Brain hemispheres were
sonicated in TRIzol\textsuperscript{TM} (Life Technologies, USA), mixed
with chloroform, and centrifuged at 824 x \emph{g} for 15 min at 4 °C.
The aqueous phase was collected and RNA was extracted using the
RNeasy\textsuperscript{®} Mini Kit (Qiagen, USA). RNA was quantified
using a NanoDrop One spectrophotometer (Thermo Scientific, USA) and
converted to 1\textsuperscript{st} strand cDNA using a High Capacity
cDNA RT Kit (Applied Biosystems, USA). Commercially available primers
(Applied Biosystems, USA) were used to measure mRNA expression of
inflammatory markers, and a house-keeping gene, GAPDH, on a CFX96 Touch
Real-Time PCR Detection machine (Bio-Rad, USA). Changes in gene
expression were assessed using the delta-delta C\textsubscript{T} method
(Schmittgen \& Livak, 2008).

\textbf{Assessment of working memory}

Working memory was assessed by the novel object recognition test
(Lueptow, 2017). Mice were acclimated for 2 d by placement in the empty
testing box for 10 min. On the day of testing, the mouse was placed in
the same box with two identical objects for 10 min. One h later, the
mouse was placed back in the same box for 5 min with one of the familiar
objects replaced by a novel object. The mouse's interactions with the
objects (nose point entering a zone 2 cm wide around the object) were
tracked using automated software (Ethovision XT, Noldus Information
Technology BV, Wageningen, The Netherlands).

\textbf{RNA sequencing}

RNA sequencing was performed as previously described (Baik et al.,
2021). Brain hemispheres were snap frozen in liquid nitrogen. In a
separate cohort of mice, the hippocampus was dissected from the brain
and snap frozen in liquid nitrogen. Brain hemispheres and hippocampi
were sonicated in TRIzol\textsuperscript{TM} (Life Technologies, USA),
mixed with chloroform, and centrifuged at 824 x \emph{g} for 15 min at 4
°C. The aqueous phase was collected and RNA was extracted using the
RNeasy\textsuperscript{®} Micro Kit (Qiagen, USA). RNA was quantified
using a NanoDrop One spectrophotometer (Thermo Scientific, USA) and then
stored at -80°C. The RNA samples were shipped to NovogeneAIT Genomics
(Singapore) for cDNA library preparation and RNA sequencing. mRNA was
purified from total RNA using poly-T oligo-attached magnetics. mRNA was
converted to cDNA and purified using AMPure XP Beads (Beckman Coulter
Life Sciences, USA). cDNA libraries were acquired by PCR amplification.
High-throughput sequencing was conducted using the HiSeqTM2500 platform
(Illumina, USA). The results were mapped to the Ensembl-released mouse
genome sequence and annotation. Differential expression analysis was
conducted using the DESeq R Package V.1.10.1 and P-values were adjusted
using the Banjamini and Hochberg's approach for controlling the false
discovery rate. Genes were considered differentially expressed if the
adjusted P-value was less than 0.05. R package heatmap3 and
log2Fold-Change output from EdgeR V.3.2.4 were used to create heatmaps
for differentially expressed genes.

\textbf{Statistical analysis}

Results are expressed as mean ± S.E.M. Power calculations were performed
using G*Power. Statistical analyses between groups were performed using
Student's unpaired t-test, one-sample t-test, or a one- or two-way ANOVA
followed by a Tukey's or Sidak's post-hoc test, as appropriate.
P\textless{}0.05 was considered to be significant. RNA sequencing data
were analysed using R Studio. GraphPad Prism software (version 8.0,
GraphPad Software Inc., USA) was used to perform all other statistical
analyses.



\textbf{RESULTS}

\textbf{Infusion of angiotensin II or aldosterone promotes immune cell
infiltration in the brain}

Angiotensin II infusion caused a marked increase in systolic blood
pressure (by \textasciitilde{}40 mmHg, Figure 1A). Elevation of blood
pressure had no effect on the number of microglia (Figure 1C), but there
was accumulation of circulating CD45\textsuperscript{+} leukocytes
(Figures 1B and 1D; gating strategy for other leukocytes are shown in
the Supplementary Figure 1), specifically T cells (Figures 1E and 1F),
myeloid cells (Figure 1G), neutrophils (Figure 1H), monocytes (Figure 1I
and 1J) and B cells (Figure 1L) in the brain. There was also a tendency
for more macrophages (Figure 1K; \emph{P} =0.15). Similarly, aldosterone
infusion caused elevation of systolic blood pressure (by
\textasciitilde{}20 mmHg, Figure 2A), without affecting the number of
microglia (Figure 2C). Furthermore, aldosterone also promoted
accumulation of leukocytes (Figures 2B and 2D), including T cells
(Figures 2E and 2F), myeloid cells (Figure 2G) and macrophages (Figure
2H) in the brain.

\textbf{Hydralazine prevents angiotensin II-induced hypertension,
cognitive dysfunction and blood brain barrier breakdown}

Co-administration of hydralazine blunted the pressor response to
angiotensin II and reduced systolic blood pressure in vehicle-infused
mice at days 3 and 7 (Figure 3A). In the novel object recognition test
to assess working memory, vehicle-infused mice spent a majority of time
interacting with the novel object (Figure 3B and 3C) whereas angiotensin
II-infused mice did not discriminate between novel and familiar objects
(Figure 3B and 3C). Importantly, no such cognitive dysfunction was
evident in angiotensin II-infused mice co-treated with hydralazine
(Figure 3B and 3C). There was also evidence of memory impairment in a
minority of the vehicle-infused mice treated with hydralazine (Figure
3C).

Angiotensin II increased IgG deposition in the hippocampus (Figures
3D-F) and cortex (Figures 3G-I), and this was prevented by co-treatment
with hydralazine. Across all mice, IgG deposition was positively
correlated in hippocampus (R\textsuperscript{2}=0.13, P\textless{}0.05,
Figure 3F) and cortex (R\textsuperscript{2}=0.26, P\textless{}0.05,
Figure 3I) with systolic blood pressure.

\textbf{Hydralazine prevents angiotensin II-induced immune cell
infiltration and neuroinflammation}

Co-treatment with hydralazine prevented angiotensin II-induced increases
in CD45+ leukocytes in the brain (Figure 4A and 4B), including T cells
(Figures 4C and 4D), myeloid cells (Figure 4E), neutrophils (Figure 4F),
monocytes (Figures 4G and 4H) and B cells (Figure 4J). Numbers of
macrophages were not different among all groups (Figure 4I).

Angiotensin II promoted gene expression of chemokine (C-C motif)
receptor 2 (CCR2) (Figure 5A), chemokine (C-C motif) ligand 2 (CCL 2)
(Figure 5B), CCL8 (Figure 5D) and TNF-\selectlanguage{greek}α \selectlanguage{english}(Figure 5E) but had no effect on
CCL7 (Figure 5C) and interleukin-1\selectlanguage{greek}β \selectlanguage{english}(Figure 5F). Co-treatment with
hydralazine prevented the angiotensin II-induced increases in gene
expression of CCR2 (Figure 5A), CCL2 (Figure 5B), CCL8 (Figure 5D) and
TNF- \selectlanguage{greek}α \selectlanguage{english}(Figure 5E).

\textbf{Hydralazine modulates expression of genes in the brain}

Bulk RNA sequencing was performed on the brains of mice infused with
vehicle, angiotensin II or angiotensin II + hydralazine. When comparing
mice infused with angiotensin II alone versus vehicle alone, there were
1193 differentially expressed genes in the whole brain (687 upregulated
and 506 downregulated; Figure 6A). There were 751 differentially
expressed genes (327 upregulated and 424 downregulated) when comparing
mice infused with angiotensin II alone versus angiotensin II +
hydralazine in the whole brain (Figure 6B).

We next performed further analysis focussing on genes related to
inflammation. In this analysis, the genes most upregulated by
angiotensin II included cd163 molecule like 1 (\emph{Cd163l1} ), growth
differentiation factor 7 (\emph{Gdf7} ), e74 like ETS transcription
factor 3 (\emph{Elf3} ), cd3d delta subunit of t cell receptor complex
(\emph{Cd3d} ), cd209 molecule (\emph{Cd209} ), fibroblast factor growth
factor 23 (\emph{Fgf23} ), interleukin 6 (\emph{Il6} ), t cell receptor
beta joining 2-3 (\emph{Trbjj2-3)} , histocompatibility 2, Q region
locus 6 (\emph{H2-q6} ), z-dna binding protein 1 (\emph{Zbp1} ),
coagulation factor XII (\emph{F12} ), CC motif chemokine ligand 8
(\emph{Ccl8} ), hematopoietic SH2 domain containing (\emph{Hsh2d} ) and
CC motif chemokine receptor 1 (\emph{Ccr1} ) (Figure 6C). The top
downregulated genes following infusion with angiotensin II compared to
vehicle included early growth response 2 (\emph{Egr2} ), fos
proto-oncogene, AP-1 transcription factor subunit (\emph{Fos} ) activity
regulated cytoskeleton associated protein (\emph{Arc} ), complement c7
(\emph{C7} ), BTG anti-proliferation factor 2 (\emph{Btg2} )\emph{,} jun
B proto-oncogene (\emph{Junb} ), nuclear receptor subfamily 4 group A
member (\emph{Nr4a1} ), dual specificity phosphatase 1 (\emph{Dusp1} )
and complement receptor 2 (\emph{Cr2} ) (Figure 6C).

The genes most upregulated by angiotensin + hydralazine compared to
angiotensin II in the brain included endothelin 2 (\emph{Edn2),} T cell
receptor delta variable 1 (\emph{Trdv1} ), ubiquitin D
\emph{(Ubd),}interleukin 23 receptor \emph{(Il23r),} toll like receptor
8\emph{(Tlr8),} tripartite motif containing 38 \emph{(Trim38),} C-C
motif chemokine ligand 11 (\emph{Ccl11} ) and lymphocyte antigen 6
family member K (\emph{Ly6k} ) (Figure 6D). The top downregulated genes
by angiotensin II + hydralazine compared to angiotensin II in the brain
included aquaporin-2 (\emph{Aqp2} ), uromodulin (\emph{Umod} ),
polymeric immunoglobulin receptor (\emph{Pigr} ), sialic acid binding Ig
like lectin 10 (\emph{Siglecg} ), Sh2 domain containing 2a
(\emph{Sh2d2a} ), C-C motif chemokine ligand 8 (\emph{Ccl8} ),
lymphocyte antigen 6 family member G (\emph{Ly6g)} , interleukin 22
receptor subunit alpha1 (\emph{Il22ra} ) and C-type lectin domain family
4 member D (\emph{Clec4d} ) (Figure 6D).

When comparing mice infused with angiotensin II alone versus vehicle
alone, there were 633 differentially expressed genes (324 upregulated
and 309 downregulated) specifically in the hippocampus (Figure 7A).
There were 921 differentially expressed genes (347 upregulated and 574
downregulated) in the hippocampus when comparing mice infused with
angiotensin II alone versus angiotensin II + hydralazine (Figure 7B).

Further analysis revealed that the genes most upregulated by angiotensin
II compared to vehicle in the hippocampus included interleukin 12
receptor subunit beta 2 (\emph{Il12rb2} ), protocadherin gamma subfamily
b4 (\emph{Pcdhgb4} ), interferon induced transmembrane protein 7
(\emph{Ifitm7} ), hyaluronidase 1 (\emph{Hyal1} ), immunoglobulin
superfamily member 6 (\emph{Igsf6} ), intercellular adhesion molecule 1
(\emph{Icam1} ), interleukin 2 receptor subunit beta (\emph{Il2rb} ),
annexin A11 (\emph{Anxa11} ), lysine demethylase 4a (\emph{Kdm4a} ), TNF
receptor superfamily member 25 (\emph{Tnfrsf25} ), docking protein 3
(\emph{Dok3} ) and B cell CLL/lymphoma 9 protein (\emph{Bcl9} ) (Figure
7C). The top downregulated genes following infusion with angiotensin II
compared to vehicle included pterin-4 alpha-carbinolamine dehydratase
(\emph{Pcbd1} ), angiotensinogen (\emph{Agt} ), odd-skipped related
transcription factor 1 (\emph{Osr1} ), formyl peptide receptor 1
(\emph{Fpr1} ), WD repeat domain 62 (\emph{Wdr62} ), FKBP prolyl
isomerase 11 (\emph{Fkbp11} ), interleukin 1 receptor accessory protein
2 (\emph{Il1rapl2} ), macrophage stimulating 1 receptor (\emph{Mst1r} ),
tachykinin receptor 1 (\emph{Tacr1} ), natriuretic peptide A
(\emph{Nppa} ) and BarH like homeobox 1 (\emph{Barhl1} ).

The genes most upregulated by angiotensin + hydralazine compared to
angiotensin II in the hippocampus included ninjurin 2 (\emph{Ninj2} ),
wnt family member 4 (\emph{Wnt4} ), prostaglandin-endoperoxide synthase
1 (\emph{Ptgs1} ), interleukin-18 binding protein (\emph{Il18bp} ), cd74
molecule (\emph{Cd74} ), tumour necrosis factor alpha-induced protein 2
(\emph{Tnfaip2} ), cathepsin k (\emph{Ctsk} ), dual specificity
phosphatase 10 (\emph{Dusp10} ), platelet factor 4 (\emph{Pf4} ),
tripartite motif containing 4 (\emph{Trim4i} ) and interleukin 1
receptor associated kinase 4 (\emph{Irak4} ) (Figure 7D). The top
downregulated genes by angiotensin II + hydralazine compared to
angiotensin II in the hippocampus included raf-1 proto-oncogene,
serine/threonine kinase (\emph{Raf1} ), KLF transcription factor 9
(\emph{Klf9} ), protocadherin gamma subfamily A, 2 (\emph{Pcdhga2} ),
C-X-C motif chemokine ligand 10 (\emph{Cxcl10} ), aquaporin 1
(\emph{Aqp1} ), ubiquitin specific peptidase 33 (\emph{Usp33} ),
hyaluronidase 1 (\emph{Hyal1} ), midline 1 (\emph{Mid1} ), protocadherin
gamma subfamily b4 (\emph{Pcdhgb4} ), diacylglycerol kinase gamma
(\emph{Dgkg} ), C-type lectin domain containing 10a (\emph{Clec10a} ),
interleukin 12 receptor subunit beta 2 (\emph{Il12rb2} ) and collagen
type XIX alpha 1 chain (\emph{Col19a1} ).

\textbf{Intervention with hydralazine during established hypertension
reduces blood pressure but not brain inflammation or cognitive
impairment}

Angiotensin II infusion increased systolic blood pressure which was
sustained for 28 days (Figure 8A). In some mice, intervention with
hydralazine from day 14 to 28 of angiotensin II infusion reduced blood
pressure to almost baseline (Figure 8A). However, at day 28 there was no
significant difference between groups in either immune cell numbers in
the brain (Figure 8C) or cognitive function (Figure 8E).

\textbf{DISCUSSION}

There are several novel findings of this study. First,
neuroinflammation, IgG deposition, transcriptomic changes and cognitive
impairment during hypertension are dependent on the degree of elevated
blood pressure. Second, treatment of hypertensive mice with hydralazine
for two weeks does not reverse brain inflammation or cognitive
impairment. Our findings are consistent with the concept that chronic
hypertension promotes brain inflammation and memory impairment, but
these effects may not be readily reversible by lowering blood pressure.

Chronic hypertension is known to cause brain injury, and indeed infusion
with angiotensin II or aldosterone can promote cerebrovascular
dysfunction (Chrissobolis, Drummond, Faraci, \& Sobey, 2014) and
oxidative stress (Dinh et al., 2016). Hypertension may also promote
leukocyte infiltration in blood pressure-regulating organs including the
kidneys (Dinh et al., 2021; Krishnan et al., 2019) and blood vessels
(Chan et al., 2015; Wei et al., 2014). We and others have reported that
angiotensin II infusion promotes T cell infiltration in the brain
(Don-Doncow, Vanherle, Zhang, \& Meissner, 2019; Wei et al., 2014), and
here we show that there are increases in several immune cell subsets in
additional to T cells in the brain during two models of experimental
hypertension. Under normotensive conditions, circulating cells and
numerous molecules are prevented from entering the brain by the
blood-brain barrier, but chronic hypertension can cause blood-brain
barrier breakdown, allowing entry to the brain of a variety of factors
such as immune cells. IgG is one such class of circulating proteins that
do not normally penetrate the blood-brain barrier, but our finding of
significant IgG deposition in the hippocampus and cortex during
hypertension is indicative of hypertension-induced blood-brain barrier
dysfunction. These findings are analogous to reports that peripheral
immune infiltrate the brain stroke (Qian et al., 2018) and in
Alzheimer's disease (Zenaro et al., 2015). In our study, oral
administration of the anti-hypertensive agent hydralazine prevented
angiotensin II-induced hypertension as well as IgG deposition and immune
cell infiltration, suggesting that these latter changes were also blood
pressure-dependent. Hydralazine also prevented angiotensin II-induced
expression of \emph{Ccr2} , and its ligands \emph{Ccl2} and \emph{Ccl8}
, consistent with \emph{Ccr2} -mediated immune cell infiltration as a
key aspect of brain inflammation during hypertension.

Hypertension is a major risk factor for cognitive impairment in humans,
and our data are consistent with such a relationship in mouse models
(Csiszar et al., 2013; Foulquier et al., 2018). Importantly, our study
provides direct evidence that angiotensin II-induced cognitive
impairment is indeed blood pressure-dependent. There are a number of
ways in which hypertension could cause brain injury and cognitive
impairment, such as through disruption of cerebral artery structure and
function which could ultimately result in cerebral small-vessel disease,
ischemic white matter lesions and vascular dementia (Iadecola et al.,
2016; Verhaaren et al., 2013). Indeed, higher systolic blood pressure is
associated with faster decline in cognition (Levine et al., 2019), and
poorer cognition is associated with more severe hypertension (Muela et
al., 2017). While results from the Systolic Blood Pressure Intervention
Trial (SPRINT) substudy Memory and Cognition in Decreased Hypertension
(SPRINT MIND) suggest that intensive reduction in systolic blood
pressure (\textless{}120 mmHg) decreases the risk of developing mild
cognitive impairment but not dementia in patients compared to standard
reduction in systolic blood pressure (\textless{}140 mmHg) (Group et
al., 2019), there were no apparent effects on brain inflammation and
cognitive deficit after just two weeks of hypertension reversal in our
study in mice.

Accumulating evidence has suggested inflammation to be a mediator of
cognitive impairment (Skelly et al., 2018). Vascular inflammation is one
mechanism by which hypertension may alter the structure and function of
cerebral blood vessels through processes such as vascular remodelling
(Shenoy et al., 2018) and fibrosis (Aldrich \& Kielian, 2011).
Furthermore, inflammation and immunity are features of cerebral small
vessel disease (Fu \& Yan, 2018). Since anti-hypertensive therapy with
hydralazine prevented both angiotensin II-induced brain inflammation and
cognitive impairment, our data are consistent with the concept that
hypertension-induced brain inflammation leads to cognitive impairment.
Indeed, targeting inflammation is reported to reduce cognitive
impairment during hypertension (Faraco et al., 2016; Jalal, Yang,
Thompson, Roitbak, \& Rosenberg, 2015) and other cognitive disorders (Fu
et al., 2019; Ni et al., 2018).

Bulk RNA sequencing showed that angiotensin II infusion upregulated
genes in the brain involved in biological processes such as inflammation
(\emph{Gdf7, Elf3} ), immune cell migration (\emph{Ccl8, Ccr1} ),
macrophage activity (\emph{Cd163l1, Cd209} ), T cell signalling and
proliferation (\emph{Cd3d, Trbjj2-3, H2-q6, Hsh2d, Il6} ), PANoptosis
(\emph{Zbp1} ), fibrosis (\emph{Fgf23} ) and thrombosis (\emph{F12} ).
This supports our flow cytometry data that angiotensin II activated
pathways that promote immune cell migration and neuroinflammation.
Angiotensin II downregulated genes involved in biological processes such
as memory consolidation (\emph{Arc} ), anti-inflammatory (\emph{Dusp1}
), T regulatory cell development (\emph{Junb} ), macrophage
differentiation (\emph{Egr2} ), negative regulation of microglia
proliferation (\emph{Btg2} ), inhibition of apoptosis (\emph{Nr4a1} )
and the complement pathway (\emph{C7, Cr2} ). \emph{Arc} is a master
regulator of long-term memory formation (Plath et al., 2006), hence,
downregulation of\emph{Arc} may have contributed to the cognitive
decline observed in angiotensin II-infused mice.

In the hippocampus, a key brain region for learning and memory,
angiotensin II promoted upregulation of genes involved in inflammation
(\emph{Igsf6, Hyal1} ), T cell differentiation and activity
(\emph{Il12rb2, Ifitm7, Il2rb, Tnfrsf25} ), microglia regulation
(\emph{Kdm4a, Dok3} ), cell adhesion (\emph{Pcdhgb4, Icam1} ) and
apoptosis (\emph{Anxa11, Bcl9} ). Immune cells specifically in the
hippocampus were not analysed in our study but the data are suggestive
of increased immune cell infiltration as there was upregulation of the
cell adhesion molecule, \emph{Icam1,} and evidence of BBB injury\emph{.
Il12rb2, Ifitm7, Il2rb} and \emph{Tnfrsf25} are involved in T cell
differentiation and proliferation, consistent with a T cell response in
the hippocampus. We showed an increased brain infiltration of T cells,
which have been reported to promote cognitive impairment in a mouse
model of Alzheimer's disease (Machhi et al., 2021). Hence, the presence
of T cells in the hippocampus may have contributed to the development of
cognitive impairment.

Angiotensin II downregulated genes involved in anti-inflammatory
pathways such as \emph{Dusp1} and \emph{Btg2} . Overexpression
of\emph{Dusp1} attenuates inflammation in a mouse model of
cardiomyopathy (Tan et al., 2022) while deficiency of \emph{Dusp1} has
been shown to exacerbate inflammation in a mouse model of septic
peritonitis (Hammer et al., 2010). Deficiency of \emph{Btg2} has been
found to increase activation of microglia and impair spatial learning
ability in a mouse model of chronic cerebral hypoperfusion (Suzuki et
al., 2021). Downregulation of these genes thus likely contributed to a
pro-inflammatory response to angiotensin II. Co-treatment with
angiotensin II + hydralazine caused upregulation of \emph{Dusp1}
and\emph{Btg2} suggesting that hydralazine reduced neuroinflammation
partly through these pathways. Co-administration of angiotensin II and
hydralazine also promoted downregulation of markers of immune cells and
their activity (\emph{Sh2d2a, Ly6g} ), immune cell migration
(\emph{Ccl8} ) and immunoglobulin binding (\emph{Umod, Pigr)} consistent
with protective effects of hydralazine on neuroinflammation.
Interestingly, administration of hydralazine with angiotensin II
reversed the upregulation of \emph{Il12rb2} in the hippocampus caused by
angiotensin II alone. As mentioned, \emph{Il12rb2} is involved in T cell
differentiation and proliferation and so the resulting reduction in
hippocampal neuroinflammation by hydralazine may have contributed to the
preservation of cognitive function during angiotensin II infusion,
potentially as an indirect effect of blood pressure reduction.

It is noteworthy that, while we found that development of brain
inflammation and cognitive impairment during hypertension were blood
pressure-dependent, intervention with hydralazine to lower blood
pressure in established hypertension did not readily reverse immune cell
infiltration in the brain or cognitive impairment. However, it is
plausible that a period of just 2 weeks of hydralazine administration is
insufficient to resolve the brain inflammation that develops in response
to angiotensin II-induced hypertension which may persist after the
initial hypertensive stimulus has abated, consistent with several
clinical effects of anti-hypertensive therapy on cognitive dysfunction
(Stuhec, Keuschler, Serra-Mestres, \& Isetta, 2017). New strategies that
target both blood pressure and inflammation may be a more effective
approach to treat cognitive impairment.

It seems likely that breakdown of the blood-brain barrier was an
important factor in hypertension-induced cognitive impairment.
Blood-brain barrier dysfunction is associated with early cognitive
decline in vascular cognitive impairment (Li, Li, Zuo, Hu, \& Jiang,
2021) and Alzheimer's disease patients (Bowman et al., 2018) even in the
absence of amyloid-\selectlanguage{greek}β \selectlanguage{english}and tau changes (Nation et al., 2019). Our data
reveal that in hypertensive mice the blood-brain barrier was compromised
in the hippocampus and cortex, key brain regions involved in cognition.
Besides facilitating immune cell infiltration, a leaky blood-brain
barrier is also associated with pathological events such as white matter
hyperintensities (Zhang et al., 2019) which can contribute to the
development of cognitive impairment.

The present findings indicate that hypertensive stimuli can promote
brain inflammation, cognitive impairment and changes to the brain
transcriptome in a blood pressure-dependent manner. The processes
involved in neuroinflammation during hypertension are complex and our
findings suggest that blood-brain barrier breakdown and CCR2 expression
are likely to be involved, and may contribute to the development of
cognitive impairment. As we found no evidence that drug-induced
attenuation of established hypertension could reduce brain inflammation
or cognitive dysfunction, our study highlights the importance of
managing hypertension to reduce the risk of developing neuroinflammation
and cognitive impairment.



\textbf{KEY POINT SUMMARY}

\textbf{What is already known:}

Hypertension promotes inflammation and is associated with cognitive
impairment.

\textbf{What this study adds:}

\begin{itemize}
\tightlist
\item
  Hypertension results in infiltration of several immune cell subsets
  into the brain.
\item
  Hypertension promotes unique transcriptomic changes in the hippocampus
  and the whole brain.
\item
  Reversal of established hypertension does not ameliorate
  neuroinflammation and cognitive impairment.
\end{itemize}

\textbf{Clinical significance:}

Cognitive impairment and neuroinflammation caused by hypertension are
not reversible by blood pressure lowering alone.

\textbf{SOURCES OF FUNDING}

This work was supported by Project Grants to CGS from the National
Health and Medical Research Council of Australia (NHMRC; GNT1064686 and
GNT1085323) and a Senior Research Fellowship to CGS (GNT1079467).

\textbf{CONFLICTS OF INTEREST/ DISCLOSURES}

None

\textbf{FIGURE LEGENDS}

\textbf{Figure 1}

\textbf{Angiotensin II infusion promotes immune cell infiltration in the
brain. A:} Angiotensin II-induced hypertension (n=6-8). All data are
mean \selectlanguage{ngerman}± S.E.M. *P\textless{}0.05. Two-way ANOVA with Tukey's
test.\textbf{B:} Representative flow cytometry dot plots showing gating
strategy for microglia (CD45+ low) and total leukocytes (CD45+ high)
from the brains of mice infused with vehicle or angiotensin II. The
effect of angiotensin II infusion on
\textbf{C:}CD45+\textsuperscript{low} microglia, \textbf{D:} CD45+
leukocytes,\textbf{E:} CD3+ T cells, \textbf{F:} CD4+ T cells,
\textbf{G:} CD11b+ myeloid cells, \textbf{H:} Ly6G+ neutrophils,
\textbf{I:}Ly6C+\textsuperscript{low} monocytes,
\textbf{J:}Ly6C+\textsuperscript{high} monocytes, \textbf{K:} F4/80+
macrophages and \textbf{L:} CD19+ B cells in the brain (n=6-8). All data
are mean ± S.E.M. *P\textless{}0.05. Student's unpaired t-test.

\textbf{Figure 2}

\textbf{Aldosterone infusion promotes immune cell infiltration in the
brain. A:} Aldosterone-induced hypertension (n=8). All data are mean ±
S.E.M. *P\textless{}0.05. Two-way ANOVA with Tukey's test.
\textbf{B:}Representative flow cytometry dot plots showing gating
strategy for microglia (CD45+ low) and total leukocytes (CD45+ high)
from the brains of mice infused with vehicle or aldosterone. The effect
of aldosterone infusion on \textbf{C:} CD45+\textsuperscript{low}
microglia,\textbf{D:} CD45+ leukocytes, \textbf{E:} CD3+ T cells,
\textbf{F:} CD4+ T cells, \textbf{G:} CD11b+ myeloid cells and
\textbf{H:} F4/80+ macrophages in the brain (n=8). All data are mean ±
S.E.M. *P\textless{}0.05. Student's unpaired t-test.

\textbf{Figure 3}

\textbf{Angiotensin II infusion promotes cognitive impairment and
blood-brain barrier breakdown which is prevented by co-treatment with
hydralazine. A:} The effect of hydralazine on blood pressure in mice
infused with vehicle or angiotensin II (n=7-8). All data are mean ±
S.E.M. *P\textless{}0.05 vs vehicle. Two-way ANOVA with Tukey's
test.\textbf{B:} Representative heatmap plots showing interaction
between familiar and novel objects in mice infused with vehicle,
angiotensin II, vehicle + hydralazine and angiotensin II + hydralazine.
\textbf{C:}Effect of angiotensin II infusion and co-treatment of
hydralazine on recognition memory (n=9-12). All data are mean ± S.E.M.
*P\textless{}0.05. One-sample t-test vs. 50\%. \textbf{D:}Representative
images showing IgG deposition (Alexa Flour 555, red) in hippocampus of
mice infused with vehicle, angiotensin II, vehicle + hydralazine and
angiotensin II + hydralazine. Nuclei are identified by DAPI
counter-stain (blue). Scale bar = 200 \selectlanguage{greek}µ\selectlanguage{english}m. \textbf{E:} Effect of
angiotensin II infusion and co-treatment of hydralazine on IgG
deposition in the hippocampus (n=7-8). All data are mean \selectlanguage{ngerman}± S.E.M.
*P\textless{}0.05. Two-way ANOVA with Sidak's test.
\textbf{F:}Correlation between IgG deposition in hippocampus and
systolic blood pressure (R\textsuperscript{2}=0.13, P=0.05). \textbf{G:}
Representative images showing IgG deposition (Alexa Flour 555, red) in
cortex of mice infused with vehicle, angiotensin II, vehicle +
hydralazine and angiotensin II + hydralazine. Nuclei are identified by
DAPI counter-stain (blue). Scale bar = 200 \selectlanguage{greek}µ\selectlanguage{english}m. \textbf{H:} Effect of
angiotensin II infusion and co-treatment of hydralazine on IgG
deposition in the cortex (n=7-8). All data are mean \selectlanguage{ngerman}± S.E.M.
*P\textless{}0.05. Two-way ANOVA with Sidak's test.
\textbf{I:}Correlation between IgG deposition in cortex and systolic
blood pressure (R\textsuperscript{2}=0.26, P\textless{}0.05).

\textbf{Figure 4}

\textbf{Angiotensin II-induced increase in brain immune cell
infiltration is blood pressure-dependent. A:} Representative flow
cytometry dot plots showing gating strategy for total leukocytes (CD45+
high) from the brains of mice infused with vehicle, angiotensin II,
vehicle + hydralazine and angiotensin II + hydralazine. The effect of
angiotensin infusion and co-treatment with hydralazine on
\textbf{B:}CD45+ leukocytes, \textbf{C:} CD3+ T cells, \textbf{D:} CD4+
T cells,\textbf{E:} CD11b+ myeloid cells, \textbf{F:} Ly6G+
neutrophils,\textbf{G:} Ly6C+\textsuperscript{low} monocytes,
\textbf{H:}Ly6C+\textsuperscript{high} monocytes, \textbf{I:} F4/80+
macrophages and \textbf{J:} CD19+ B cells in the brain (n=7-8). All data
are mean ± S.E.M. *P\textless{}0.05. Two-way ANOVA with Tukey's test.

\textbf{Figure 5}

\textbf{Angiotensin II-induced increase in neuroinflammation is blood
pressure-dependent.} The effect of angiotensin infusion and co-treatment
with hydralazine on mRNA expression of \textbf{A:} chemokine (C-C motif)
receptor 2 (CCR2), \textbf{B:} chemokine (C-C motif) ligand (CCL)
2,\textbf{C:} CCL7, \textbf{D:} CCL8, \textbf{E:} tumour necrosis
factor-\selectlanguage{greek}α, \selectlanguage{english}and \textbf{F:} interleukin-1\selectlanguage{greek}β \selectlanguage{english}in the brain (n=7-8). All data
are mean \selectlanguage{ngerman}± S.E.M. *P\textless{}0.05. Two-way ANOVA with Tukey's test.

\textbf{Figure 6}

\textbf{Angiotensin II infusion promotes transcriptomic changes in the
brain which is modulated by treatment with hydralazine.} Volcano plot of
differentially expressed genes in the brains of mice infused
with\textbf{A:} vehicle vs angiotensin II or \textbf{B:} angiotensin II
vs angiotensin II + hydralazine. The threshold of differential
expression is p-value \textless{}0.05. The horizontal axis is the log2
fold change of genes. The vertical axis is statistical significance
scaled as -log 10 p-value. Each dot represents an individual gene (blue:
no significant difference; red: upregulated expression; green:
downregulated expression). The top upregulated and downregulated genes
in brains of mice infused with \textbf{C:} vehicle vs angiotensin II or
\textbf{D:}angiotensin II vs angiotensin II + hydralazine. Upregulated
genes in red and downregulated genes in blue. The colour scale
represents the log10 (average FPKM + 1) value.

\textbf{Figure 7}

\textbf{Angiotensin II infusion promotes transcriptomic changes in the
hippocampus which is modulated by treatment with hydralazine.} Volcano
plot of differentially expressed genes in the hippocampus of mice
infused with \textbf{A:} vehicle vs angiotensin II or
\textbf{B:}angiotensin II vs angiotensin II + hydralazine. The threshold
of differential expression is p-value \textless{}0.05. The horizontal
axis is the log2 fold change of genes. The vertical axis is statistical
significance scaled as -log 10 p-value. Each dot represents an
individual gene (blue: no significant difference; red: upregulated
expression; green: downregulated expression). The top upregulated and
downregulated genes in hippocampus of mice infused with
\textbf{C:}vehicle vs angiotensin II or \textbf{D:} angiotensin II vs
angiotensin II + hydralazine. Upregulated genes in red and downregulated
genes in blue. The colour scale represents the log10 (average FPKM + 1)
value.

\textbf{Figure 8}

\textbf{Intervention with hydralazine does not reverse angiotensin
II-induced immune cell infiltration in the brain and cognitive
impairment. A:} Angiotensin II-induced hypertension and effect of
intervention with hydralazine (n=7-8). All data are mean ± S.E.M.
*P\textless{}0.05. Two-way ANOVA with Tukey's test.
\textbf{B:}Representative flow cytometry dot plots showing gating
strategy for total leukocytes (CD45+ high) from the brains of mice
infused with angiotensin II and angiotensin II + hydralazine.
\textbf{C:} The effect of intervention with hydralazine on angiotensin
II-induced leukocyte infiltration in the brain (n=7-8). All data are
mean ± S.E.M.\textbf{D:} Representative heatmaps showing interaction
between familiar and novel objects in mice infused with angiotensin II
and angiotensin II + hydralazine. \textbf{E:} The effect of intervention
with hydralazine on angiotensin II-induced cognitive impairment (n=7-8).
All data are mean ± S.E.M.



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