Leardini-Tristão et al. Journal of Neuroinflammation
https://doi.org/10.1186/s12974-020-01771-y(2020) 17:117

RESEARCH

Open AccessPhysical exercise promotes astrocyte
coverage of microvessels in a model ofchronic cerebral hypoperfusion
Marina Leardini-Tristão1,2, Giulia Andrade1,2, Celina Garcia3, Patrícia A. Reis1, Millena Lourenço1, Emilio T. S. Moreira1,
Flavia R. S. Lima3, Hugo C. Castro-Faria-Neto1, Eduardo Tibirica2,4 and Vanessa Estato1,2*

Abstract
Background: Brain circulation disorders such as chronic cerebral hypoperfusion have been associated with a decline
in cognitive function during the development of dementia. Astrocytes together with microglia participate in the
immune response in the CNS and make them potential sentinels in the brain parenchyma. In addition, astrocytes
coverage integrity has been related to brain homeostasis. Currently, physical exercise has been proposed as aneffective intervention to promote brain function improvement. However, the neuroprotective effects of early physicalexercise on the astrocyte communication with the microcirculation and the microglial activation in a chronic cerebral
hypoperfusion model are still unclear. The aim of this study was to investigate the impact of early intervention withphysical exercise on cognition, brain microcirculatory, and inflammatory parameters in an experimental model ofchronic cerebral hypoperfusion induced by permanent bilateral occlusion of the common carotid arteries (2VO).
Methods: Wistar rats aged 12 weeks were randomly divided into four groups: Sham-sedentary group (Sham-Sed),
Sham-exercised group (Sham-Ex), 2VO-sedentary group (2VO-Sed), and 2VO-exercised group (2VO-Ex). The earlyintervention with physical exercise started 3 days after 2VO or Sham surgery during 12 weeks. Then, the brain
functional capillary density and endothelial-leukocyte interactions were evaluated by intravital microscopy; cognitive
function was evaluated by open-field test; hippocampus postsynaptic density protein 95 and synaptophysin were
evaluated by western blotting; astrocytic coverage of the capillaries, microglial activation, and structural capillarydensity were evaluated by immunohistochemistry.
Results: Early moderate physical exercise was able to normalize functional capillary density and reduce leukocyte
rolling in the brain of animals with chronic cerebral hypoperfusion. These effects were accompanied by restore
synaptic protein and the improvement of cognitive function. In addition, early moderate exercise improves astrocytes
coverage in blood vessels of the cerebral cortex and hippocampus, decreases microglial activation in the
hippocampus, and improves structural capillaries in the hippocampus.
(Continued on next page)* Correspondence: estato@ioc.fiocruz.br; vanessaestato@gmail.com
1
Laboratory of Immunopharmacology, Oswaldo Cruz Foundation, Av. Brasil,
4365, Manguinhos, Rio de Janeiro 21040-900, Brazil
2
Laboratory of Cardiovascular Investigation, Oswaldo Cruz Foundation, Rio deJaneiro, Brazil
Full list of author information is available at the end of the article© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
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Leardini-Tristão et al. Journal of Neuroinflammation(2020) 17:117

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Conclusions: Microcirculatory and inflammatory changes in the brain appear to be involved in triggering a cognitive
decline in animals with chronic cerebral ischemia. Therefore, early intervention with physical exercise may represent a
preventive approach to neurodegeneration caused by chronic cerebral hypoperfusion.
Keywords: Exercise, Cerebral hypoperfusion, Neuroinflammation, Glial cells, Microcirculation

Background
Dementia is a neurodegenerative disease with a progressive impairment of cognition and behavioral changes that
may be preceded or accompanied by loss of neurons, neuroinflammation, and oxidative stress [1, 2]. Brain circulation disorders, such as chronic cerebral hypoperfusion,
have been associated with a decline in cognitive function
in elderly people and during the development of dementia[3, 4]. Micro- and macrovascular changes are also related
to the neurodegenerative process and may occur before
the onset of dementia [5]. Vascular dementia highlights
the importance of vascular contributions to cognitive impairment and represents the second most common formof dementia following Alzheimer’s disease [6].
In the last few decades, animal models have been used
to study alterations and consequences of chronic reduction on cerebral blood flow (CBF) that may occur inneurodegenerative disease. The permanent bilateral occlusion of the common carotid arteries in rats (or 2vessel occlusion, 2VO) is a cerebral hypoperfusion modelthat impacts the regions of the brain associated withlearning and memory processes, such as the cortex andhippocampus [7]. The chronic phase of the 2VO model,
between the eighth and twelfth weeks after the procedure, more closely resembles the decrease in CBF in human aging and dementia. In this phase of the model, theneurodegenerative process is more extensive; therefore,
it is the most used phase for the study of dementia [4].
Astrocytes together with microglia participate in the immune response in the CNS. The location of these cells, in ornear the blood-brain barrier (BBB), makes them potential sentinels in the brain parenchyma. Astrocytes play an importantrole in communicating with blood vessels, being involved in
the maintenance of the BBB and CBF, providing for the nutrition of neurons, and promoting vasodilation in response toneuronal activation [8]. Microglia are the most common resident immune cells, which represent the first-line of inflammatory response in brain tissue and are generally activatedduring infections, lesions, or degenerative diseases [9]. Microglia activation in response to injury commonly involves morphological changes leading to transformation from a ramified
to amoeboid form [10]. Activation also induces the release ofproinflammatory cytokines and chemokines, which promotes
the infiltration of circulating leukocytes into the brain, leading
to the chronic inflammatory process present in neurodegenerative diseases [11].

Experimental and clinical studies have shown that theacute phase after cerebral ischemia is a critical window for
the impairment of neuronal plasticity. Treatment during
this period can trigger and promote neuroprotective
mechanisms that will assist in the recovery of neuronal
functions [12–15]. Among new therapeutic strategies being pursued to minimize cognitive damage, physical exercise has been shown to support brain health and function,with an impact on neurogenesis [16, 17], reducing neuroinflamation [14, 18] and oxidative stress [14, 19]. Moreover, regular physical exercise is able to improve cognitive
function in animals [20, 21], and in adult and elderly populations [22–24].
This paper describes a new approach to the neuroprotective role of physical exercise in chronic hypoperfusion, a common feature of dementia. It is possible thatexercise modulates the interaction between glial cells
and brain microcirculation, but this hypothesis has not
yet been fully investigated. Thus, using the 2VO model,
we investigated the impact of early moderate exercise asa therapeutic strategy on brain microcirculation, neuroinflammation, and astrocytic coverage of brain vessels.

Methods
Animals and ethics statement

Fifty-eight male Wistar rats (Oswaldo Cruz Foundation
Animal Facilities, Rio de Janeiro, Brazil), 12 weeks old,
were housed under controlled temperature (21 ± 2 °C)
and light (12 h light/dark cycles) conditions and wereallowed free access to water and standard rat chow. All
experiments were conducted in accordance with internationally accepted principles for the care and use of laboratory animals and were approved by the Animal
Ethics Committee of the Oswaldo Cruz Foundation(protocol number L-002/2016).
Experimental design

We analyzed the effects of physical exercise on cerebral
hypoperfusion in the two-vessel occlusion (2VO) experimental model. We chose the cerebral hypoperfusion
model because it is a validated and representative modelof changes in the cerebral blood flow of aging without,therefore, including the bias of the physical limitations ofaging in the performance of physical exercise [4]. Animals
were randomly assigned to the following four experimental groups: (1) subjected to cerebral hypoperfusion and(2020) 17:117

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exercise (2VO-Ex; n = 15); (2) subjected to cerebral hypoperfusion and non-exercised (sedentary) (2VO-Sed; n =15); (3) subjected a sham procedure and exercise (ShamEx; n = 12); and (4) subjected a sham procedure and nonexercised (sedentary) (Sham-Sed; n = 11). The animals inthe sham groups were subjected to the same surgical procedure without ligation of the left and right common carotid arteries.
Three days after surgery, the exercised groups performed maximal exercise testing (MET). The exercise
protocol then started 24 h following MET. After completing 12 weeks of a moderate physical exercise protocol, the animals underwent cognitive, hemodynamic,
microcirculatory, and inflammatory assessments.

animals underwent additional procedures for cognitive
function, hemodynamic parameter levels, microcirculatory, and neuroinflammatory alterations.

Two-vessel occlusion (2VO) surgical procedure

Open-field test

In order to promote a global and chronic cerebral hypoperfusion, rats were anesthetized intraperitoneally (i.p.)with ketamine (90 mg/kg) and xylazine (10 mg/kg), andunderwent 2VO surgery as previously described (Farkas
et al. 2004). Briefly, a ventral incision was made in the
midline of the neck using a scalpel to expose the left andright common carotid arteries, which were gently separated from the vagus nerve. Animals in the 2VO group
had both vessels permanently occluded with 6-0 silk suture. The sham groups were subjected to the same procedure without occlusion of the carotid arteries. After
surgery, the animals received subcutaneous injections ofan anti-inflammatory drug, ketoprofen (1 mg/kg/day for 3days) and an antibiotic, meropenem (10 mg/kg, single
dose). The animals were kept in individual cages until theend of the experimental protocol to avoid stress [14, 25].

At the end of 12 weeks of exercise, cognitive function
was investigated by an open-field test. Habituation to an
open field was carried out as described by Vianna andcoworkers [27], and consists of quantitatively assessing
the exploratory and locomotor activities, as well as the
habituation memory of the animals. Briefly, animals were
gently placed in the center of an open box (50 cm high,
50 cm wide, 39 cm deep) with the floor divided into agrid and allowed to explore the arena for 5 min (training
session). After 24 h the animals were submitted to asimilar open-field session (test session). Crossing of thegridlines and rearing (rearing up on the hind legs) performed in both sessions were counted.

Leardini-Tristão et al. Journal of Neuroinflammation

Aerobic exercise training protocol

Before surgery, all the animals were adapted to a treadmill for rats (Model HT 2.0 Hectron Fitness Equipment,
Rio de Janeiro, Brazil) by running at a speed of 10 m/
min for 10 min over a period of 3 days. Then, the 3 days
after surgery the MET was performed to establish theappropriate training intensity [26]. The MET began at a
speed of 10 m/min; the speed was then increased by 3
m/min every 3 min until the animals reached exhaustion
and remained on the shock grid for more than 5 s. For
each MET trial, time to exhaustion, maximal speed, andmaximal distance were recorded. Maximal speed wasused to determine the training intensity for the durationof the investigation, which was 60% of the MET. The
MET was also performed on the sixth week of exercise
to adjust the intensity of the protocol.
The physical exercise consisted of 30-min sessions, 3times a week over 12 weeks at 60% of the maximal
speed, which occurred between 8:00 and 10:00 am.
Twenty-four hours after the last exercise for the exercised group or sedentarism for the sedentary group, theHemodynamic assessment

In order to monitor blood pressure, systolic blood pressure (SBP) was assessed in conscious animals one day
before 2VO/sham surgery (baseline measurement), 48 h
after 2VO/sham surgery and after 12 weeks of exercise or
sedentarism using a computerized tail-cuff plethysmography system (BP-2000; Visitech Blood Pressure Analysis
System, Apex, NC, USA). The animals were adapted tothe apparatus for three consecutive days prior to undergoing their baseline measurements.

Intravital microscopy imaging

Brain microcirculation of all groups of animals was observed via cranial window imaging, and the functional
capillary density and leukocyte-endothelium interactions
were evaluated by fluorescence intravital video microscopy as previously described [28, 29]. Briefly, the animals
were anesthetized with ketamine (90 mg/kg, i.p.) andxylazine (10 mg/kg, i.p.) and fixed in a stereotaxic frame.
Then, their left parietal bones were exposed via midline
skin incisions, and a cranial window on the right parietal
bone (5 mm diameter, between the coronal and lambdoid sutures) was created with a high-speed drill. The
dura mater and arachnoid membranes were subsequently excised and withdrawn to expose the cerebral
microcirculation, after which the cranial windows were
suffused with artificial cerebrospinal fluid (132 mM
NaCl, 2.95 mM KCl, 1.71 mM CaCl2, 0.64 mM MgCl2,
24.6 mM NaHCO3, 3.71 mM dextrose, and 6.7 mMurea, pH 7.4, at 37 °C).
The visualization of the brain microvascular surface
was facilitated by intravenous administration of 0.1 mL
of 2% fluorescein isothiocyanate (FITC)-labeled dextran
using a microscope and epi-illumination (Olympus
BX150WI, NY, USA) outfitted with a fluorescent light(2020) 17:117

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source (460 to 490 nm using a 520 nm emission filter).
Microscopic images were subsequently acquired, and capillaries were counted using Cell Sens Standard 1.9 software. The functional capillary density, defined as the total
number of spontaneously perfused capillaries per square
millimeter of surface area, was determined in the random
microscopic field of view over a 4-min period [29].
Leukocytes were fluorescently labeled by intravenous
administration of rhodamine 6G (0.3 mg/kg) and observed through the cranial window. Adherent leukocytes
were defined as the number of leukocytes adhered to thevenular endothelium for a period of 30 s or longer andexpressed as cell number/min/100 μm. Leukocytes wereconsidered rolling on the vessel wall if they moved at a
slower rate than circulating erythrocytes, and expressedin cells/min. Brain surface venules with diameters ranging from 50 to 100 μm were analyzed [29].

data were obtained by Odyssey software (Li-Cor Biosciences, Lincoln, NE, USA).

Leardini-Tristão et al. Journal of Neuroinflammation

Tissue processing

After intravital microscopy, rats were euthanized bypentobarbital overdose (150 mg/kg, i.p.). The brain was
dissected, and the hippocampus and left hemisphere cortex regions were stored in the freezer at – 80 °C for subsequent western blot analysis. For immunohistochemical
analysis, 5 animals per group were anesthetized withketamine and xylazine (90 and 10 mg/kg i.p., respectively) and transcardially perfused with 0.9% saline
followed by 4% paraformaldehyde and the brains werethen removed and maintained in paraformaldehyde
solution.
Western blot analysis

Protein levels of synaptophysin and postsynaptic density
protein 95 (PSD-95) in the hippocampus were determined by western blot analysis. The hippocampus was
homogenized in 50 mM Tris, pH 8.0, containing 150
mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.5% sodium
deoxycholate, 0.1% SDS, and protease inhibitor cocktail(Complete Mini Protease Inhibitor Cocktail; Roche Diagnostics, IN, USA). Homogenates were incubated for 30
min on ice, sonicated for 3 min, and centrifuged at
10000×g at 4 °C. The supernatant was collected, and theprotein concentration was measured using bicinchoninicacid method, with bovine serum albumin (BSA) as thestandard (BCA Protein Assay Kit, Thermo Scientific,
Waltham, MA, USA). Then, 50 μg of protein per lane
was separated on a 12% SDS-PAGE gel and transferred
to a nitrocellulose membrane (BioRad Laboratories,
Munich, Germany). After blocking with 5% BSA (Sigma
Aldrich, St. Louis, MO, USA), the membranes were incubated overnight at 4 °C with antibodies to synaptophysin and PSD-95 (1:500 and 1:5000 respectively; Abcam)
followed by a secondary antibody (1:20000). Beta actin(1:10000; Abcam) was used as a loading control. The

Immunohistochemistry

For immunohistochemistry analysis, brains were quickly
excised after perfusion-fixation as described above andserially sectioned at 50 μm. The sections were washedwith PBS and incubated with 10% NGS diluted in PBS
with 0.3% Triton X-100 for 90 min. They were then incubated with antibodies to the glial fibrillary acidic protein(GFAP, Dako; 1:400) and the ionized calcium-binding
adaptor molecule 1 (Iba1, Wako; 1:200) or with biotinylated IB4 (Vector; 1:100) overnight at 4 °C, then washedagain with PBS and incubated with secondary antibodies
conjugated with Alexa Fluor 488 or 546 (1:400) or
streptavidin-Cy3 (1:400) for 2 h. The sections were counterstained with DAPI and coverslips were mounted withfluoromount. Negative controls were performed with
non-immune rabbit IgG. Slices were imaged using a confocal microscope (Leica TCS-SP5) equipped with a 63X
NA 1.40 oil-immersion objective. We were careful toapply the same selection criteria to all cuts. Initially, we located the CA1 region of the hippocampus and then we located the parietal cortex. Image processing was performed
using Fiji, ImageJ software.
For microglial morphology analysis, the ImageJ ShollAnalysis tool was used. This is a quantitative analysis
method commonly used in neuronal studies tocharacterize the morphological characteristics of an imaged neuron. Using Sholl analysis, a mathematical algorithm of the program called “branching index” is used toanalyze neuronal morphology. This index compares thedifference in the number of intersections make consecutive circles of the Sholl analysis in relation to the distance of the neuronal sum. In this study, microglial
processes were quantified as a number of intersections
from 2 to 40 μm from the sum and total number of intersections within the analysis circles, representing thetotal branch of the microglia.
For the analysis of the cerebral microvascular angioarchitecture, the AngioTool program was used (https://ccrod.cancer.gov/confluence/display/ROB2/Downloads) to determine
the total length of the brain capillaries on each image obtained by confocal microscopy. AngioTool is a validated
source for measuring vascular networks [30] and has alreadybeen described in analyses of murine brain and retinal angiogenesis [31, 32].
Statistical analysis

The results are expressed as the mean ± standard error of
the mean (SEM) for each group. Between-group statisticalcomparisons were performed using one-way analysis of variance (ANOVA), followed by Bonferroni’s post hoc test.
When appropriate, the results were analyzed using unpairedLeardini-Tristão et al. Journal of Neuroinflammation(2020) 17:117

Student’s t test. MET and open-field parameters were analyzed using paired Student’s t tests. Systolic blood pressure
before and 48 h after surgery were analyzed using Student’s ttests. Differences with p values less than 0.05 were considered significant. All calculations were performed using commercially available statistical software (GraphPad Prism
Software, CA, USA).

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Table 2 Physical performance in the maximal exercise test
Parameter

Sham-Ex2VO-ExBaseline

14.52 ± 1.74

14.63 ± 2.14

Readjustment

20.89 ± 1.21 **19.57 ± 1.89§

Time to exhaustion (min)Maximal speed (m/min)Baseline

25.00 ± 0.91

25.75 ± 0.84

Results

Readjustment

29.10 ± 1.23 *30.00 ± 0.77§

Early moderate physical exercise reduced systolic bloodpressure in animals with chronic cerebral hypoperfusion

Maximal distance (m)Baseline

283.00 ± 23.38

322.00 ± 23.64

Readjustment

405.00 ± 31.64*

429.70 ± 23.03§§

The SBP of all animal groups was assessed before 2VOor sham surgery, 48h after 2VO or sham surgery and at
the end of 12 weeks of physical exercise (2VO-Ex andSham-Ex) or physical inactivity (2VO-Sed and ShamSed). At baseline, all animals exhibited similar SBP (datanot shown). As expected, 48 h after 2VO surgery an increase in SBP was observed, compared to those submitted to sham surgery (Table 1). At 12 weeks the SBP was
higher in the 2VO-Sed group compared to the respective
sham groups. Moreover, physical exercise was able tobring SBP back to basal levels in the 2VO-Ex group.
Physical performance was not altered in animals withchronic cerebral hypoperfusion

Prior to the physical exercise protocol, animals wereadapted to the treadmill for 3 days before the surgicalprocedure, and then submitted to the MET for the correct prescription of exercise intensity for the course ofthis investigation. Table 2 shows that both exercisedgroups presented improvement at the readjustment
MET when compared with pre-training values, regarding
time to exhaustion, maximal speed, maximal distanceand training velocity. No differences were found between
Table 1 Systolic blood pressure (SBP) during chronic cerebral
hypoperfusion
48 h after surgery

after 12 weeks with or without
physical exercise

GroupsSBP

Sham

130.4 ± 2.24

2VO

147 ± 4.30 §§Sham-Sed

131 ± 2.3

Sham-Ex

125 ± 4.44

2VO-Sed140.7 ± 1.75*

2VO-Ex130 ± 2.06 #Systolic blood pressure (SBP) during chronic cerebral hypoperfusion. Results
are expressed as the mean ± SEM of 9 animals per group. SBP at 48 h after
sham or 2VO surgery. Systolic blood pressure before and 48 h after surgery
were analyzed using Student’s t tests and after 12 weeks with or without
physical exercise was analyzed using ANOVA. Sham sham surgery group, 2VO
chronic cerebral hypoperfusion, Sham-Sed sham surgery non-exercised group,
Sham-Ex sham surgery exercised group, 2VO-Sed chronic cerebral
hypoperfusion non-exercised group, 2VO-Ex chronic cerebral hypoperfusion
exercised group§§p<0.01 vs. Sham#p < 0.05 vs. 2VO-Sed##
p < 0.01 vs. 2VO-Sed

Training velocity (m/min)Baseline

15.00 ± 0.54

15.45 ± 0.50

Readjustment

17.45 ± 0.74 *18.00 ± 0.46§§

MET was performed 3 days after 2VO or sham surgery and on the sixth weekof exercise to determine the appropriate exercise training for the duration of
the investigation. Sham-Ex, sham surgery exercised group (n = 12); 2VO-Ex,
chronic cerebral hypoperfusion exercised group (n = 15). Values represent themean ± SEM*p < 0.05 vs. Sham-Ex baseline**p < 0.01 vs. Sham-Ex baseline§p < 0.05 vs. 2VO-Ex baseline§§p < 0.01 vs. 2VO-Ex baseline (Student’s t test)

the groups that received either the 2VO or shamsurgery.
Early moderate physical exercise was able to improve
memory habituation in animals with chronic cerebral
hypoperfusion

After 12 weeks of physical exercise or sedentarism, animals
were evaluated for cognitive function by the open-field test.
This test is based on the tendency of rodents to explore a
new environment more than a familiar one. During a training day, animals are allowed to explore and familiarize themselves with an open-field arena, which has a grid marked.
The following test day, the animals are expected to recognize
and explore the environment less. For the Sham-Sed andSham-Ex groups, there were a reduced number of gridline
crossings and hind leg rearings in the test day suggesting familiarity with the environment (Fig. 1a, b). Whereas the
2VO-Sed animals showed no difference in the exploratory
profile on training and test days. This trend was absent in
the 2VO-Ex animals which demonstrated a reduction in environmental exploration on the test day indicating intact cognitive skills.
Early moderate physical exercise was able to normalizefunctional capillary density and reduce leukocyte rollingin the brain of animals with chronic cerebral
hypoperfusion

We next analyzed brain microcirculation and inflammatory cell dynamics by intravital microscopy. A reduced
number of perfused capillaries was observed in theLeardini-Tristão et al. Journal of Neuroinflammation(2020) 17:117

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Fig. 1 Analysis of cognitive function by open-field test. Bars represent the mean ± S.E.M. (n = 12–15 animals per group). a The number of gridline
crossings, and b the number of hind leg rearing of the animals on the training day (white bars) and 24 h later on the test day (black bars). Sham-Sed,sham surgery non-exercised group; Sham-Ex, sham surgery exercised group; 2VO-Sed, chronic cerebral hypoperfusion non-exercised group; 2VO-Ex,
chronic cerebral hypoperfusion exercised group. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. training day (paired Student’s t test)

cortical area of the brains of the 2VO-Sed group whencompared to Sham-Sed group (Fig. 2a). The reduction in
the number of perfused capillaries was prevented in the
2VO-Ex group, as capillary density in the 2VO-Ex was
not significantly different from the sham groups.
We also evaluated the rolling of leukocytes on postcapillary venules. Changes in leukocyte-endothelial interactions were observed in the 2VO-Sed animals, which
exhibited increased leukocyte rolling in brain vessels
when compared to the Sham-Sed group, while physicalexercise prevented this inflammatory event in the 2VOEx group (Fig. 2b).

exercise presented a significant reduction in the amountof IB4+ vessels surrounded by GFAP+ astrocytes in thecortex and hippocampus (Fig. 4). Physical exercise recovered astrocytes vessel coverage in both the cortex
and hippocampus in the 2VO animals (Fig. 4a, b). Although there were no differences between the Sham-Sed
and Sham-Ex groups compared to the 2VO-Ex in the
hippocampus, there was a reduction in the Sham-Sed
group in the cerebral cortex.

Early moderate physical exercise restored synaptic
proteins expression in the brain of animals with chronic
cerebral hypoperfusion

We next investigated the effects of physical exercise on
microglial morphology following the 2VO protocol. Although isolectin IB4 stains both microglia and vessels,labeling with the microglia-specific IBA1 enabled themorphology of these cells to be observed more clearly.
Analysis of parenchymal Iba1+ cells in the 2VO-Ex
group revealed an increase in the number of microglia
compared to respective sedentary group (Fig. 5a). In the
2VO-Sed group, microglia displayed an amoeboid
phenotype and increased soma when compared to
Sham-Sed group, whilst both the 2VO-Ex and theSham-Ex groups presented microglia with more ramified
morphology (Fig. 5b). This was supported by quantitative data obtained by Sholl analysis showing that the
maximum number of microglia process interactions andthe distance from the soma where these interactions occurred was significantly decreased in the 2VO-Sed
group, but not in the exercised groups, that had levels
similar to sham animals (Fig. 5c).

In order to investigate the effects of chronic hypoperfusion on the expression of hippocampal plasticity-related
proteins, we performed western blots for the detectionof synaptophysin and PSD-95 in the hippocampus of animals. Early physical exercise was able to increase synaptophysin expression in the brain of both exercisedgroups (Fig. 3a). Similarly, increased expression of PSD95 was detected in the 2VO-Ex group with statistical significance of p = 0.556 (Fig. 3b).
Early moderate physical exercise improves astrocytes
coverage in blood vessels of the cerebral cortex andhippocampus of animals with chronic cerebral
hypoperfusion

We also investigated the interaction of astrocytes withthe vasculature in the cortex and hippocampus (Fig. 4a).
Immunohistochemical analysis allowed the evaluation ofvessel coverage by astrocytes on the cerebral microcirculation, which is an important feature of the neurovascular unit and BBB integrity. Animals that were submitted
to chronic cerebral hypoperfusion but not to physicalPhysical exercise decreases microglial activation in the
hippocampus from animals submitted to chronic cerebral
hypoperfusion

Early moderate physical exercise improves structural
capillaries in hippocampus

Further investigation of the structural capillaries in thecortex and hippocampus was performed by the AngioTool

Leardini-Tristão et al. Journal of Neuroinflammation(2020) 17:117

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Fig. 2 Representative images of cerebral intravital microscopy and microcirculation analysis of the cerebral cortex. The values represent the mean
± S.E.M (n = 6–8 per group). Bar graphs represent a the functional capillary density, and b the number of rolling leukocytes in venules after 12
weeks of physical exercise or sedentarism. Sham-Sed, sham surgery non-exercised group; Sham-Ex, sham surgery exercised group; 2VO-Sed,chronic cerebral hypoperfusion non-exercised group; 2VO-Ex, chronic cerebral hypoperfusion exercised group. In a *p < 0.05 vs. Sham-Sed and##p < 0.01 vs. 2VO-Sed (unpaired Student’s t test); in b **p < 0.01 vs. Sham-Sed and #p < 0.05 vs. 2VO-Sed (ANOVA). Scale bar 100μm,
magnification100X in a and 200X in b

technique. There were no significant differences in thelength of the capillaries of the cortex (Fig. 6a). However,
exercise was able to increase capillary length in the hippocampus in the 2VO-Ex group compared to the sedentary
2VO-Sed group (Fig. 6b).

Discussion
Using a two-vessel occlusion model, 2VO, in rats wefound that chronic cerebral hypoperfusion (i) caused a
reduction in habituation memory; (ii) induced brain
functional capillary rarefaction, as well as an increase inrolling leukocytes to the microvascular endothelium; (iii)induced alterations in astrocyte coverage of brain microvessel; and (iv) induced microglia activation phenotype.
In addition, early intervention with moderate physicalexercise over a period of 12 weeks effectively reduced
systolic blood pressure and vascular inflammation in the
post-capillary venules; increased the total length ofcapillaries in the hippocampus; improved coverage of astrocytes on cerebral capillaries; improved microglia activation phenotype; and improved habituation memory
accompanied by an increase in the expression of synaptophysin and PSD-95 proteins.
Following the 2VO surgery, the systolic blood pressure
was increased, which likely occurred due to a reduction
in carotid blood flow after occlusion. This in turn reduces the stretching of the arterial walls resulting in reduced baroreceptor stimulation, which responds to thefall in blood pressure by increasing sympathetic tone,
consequently increasing the systemic blood pressure [4].
Previous studies that analyzed the mean blood pressure
found significant increases after 2VO in both the shortterm [14, 33] and the long-term [34]. This was reported
by Farkas and colleagues who described a 10 to 20
mmHg increase in systolic blood pressure at 24 h after
2VO, persisting for up to 9 weeks [4]. Physical training,Leardini-Tristão et al. Journal of Neuroinflammation(2020) 17:117

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Fig. 3 Western blot analysis for the expression of synaptophysin (a) and PSD-95 (b) in the hippocampus. Values represent the mean ± S.E.M. of
4–7 animals per group. Quantitative protein expression of synaptophysin and PSD-95 bands detected by Western blot using β-actin as a loading
control (c). Sham-Sed, sham surgery non-exercised group; Sham-Ex, sham surgery exercised group; 2VO-Sed, chronic cerebral hypoperfusion nonexercised group; 2VO-Ex, chronic cerebral hypoperfusion exercised group. In a *p < 0.05 vs. Sham-Sed and #p < 0.05 vs. 2VO-Sed (unpaired
Student’s t test); in b #p = 0.0556 vs. 2VO-Sed (unpaired Student’s t test).

particularly aerobic exercise, has been shown to be aneffective non-pharmacological therapeutic approach in
the reduction of blood pressure in models of metabolic
syndrome [35] and hypertension [36]. A normalizationin systolic blood pressure was observed in our 2VOmodel after 12 weeks of moderate exercise compared toanimals that were sedentary for this period. Endothelial
dysfunction is considered one of the central mechanismsof structural and functional vascular changes in neurodegenerative diseases. In the 2VO model, it has been observed that endothelial activation markers increase 1 day
post-occlusion to 28 days, with a peak at 3 days [37].
Yata et al. found increased leukocyte rolling and adhesion in pial surface venules after 24 h, 1, and 2 weeks of
cerebral hypoperfusion suggesting that leukocyte activation may be the first step in the whole neurovascular
unit stress response to chronic cerebral hypoperfusion[38]. Previous results from our group have shown thatphysical exercise initiated early following 2VO andmaintained for 1 week was able to reduce adherent leukocytes in brain venules and to reduce NADPH-oxidase
expression in the brain [14]. Here, we observed that 12
weeks of physical exercise was able to reduce the rollingof leukocytes on brain venules.

Further positive effects of physical exercise following
2VO were observed, such as the ability to normalize thefunctional capillary density in the cerebral cortex and anincrease in the total length capillaries in the hippocampus. Our observations are in agreement with previous
studies showing that physical training can induce an increase in cortical capillary density and lead to the growthof new capillaries in the brain, especially in the motor
cortex [39–41]. Although we have not quantified the influence of angiogenic factors on the response of physicalexercise to cerebral microcirculation, several studies suggest the participation of VEGF as an important factor inincreasing the structural capillary density in central andperipheral vascular beds after physical exercise [42, 43].
Physical exercise for 4 months in aged rats was able toincrease the volume, capillary length, and total surface
area of capillaries in the cerebral cortex [44]. De Jonget al. observed that the cognitive performance of rats
submitted to 2VO correlated closely with capillarymorphology in the hippocampal CA1 area, suggestingthat capillary integrity is one of the important determinants of brain function [45]. Furthermore, a positive correlation between cognition and brain nutrition has been
demonstrated in several studies [45, 46].

Leardini-Tristão et al. Journal of Neuroinflammation(2020) 17:117

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Fig. 4 Immunohistochemical assessment of vessels covered by astrocytes in the cerebral parietal cortex and in the CA1 region of hippocampus.
Representative confocal images of astrocyte coverage (GFAP, green) in vessels of the cerebral cortex and hippocampus (IB4, red) of animals whichwere exercised or sedentary for 12 weeks after cerebral hypoperfusion (2VO-Ex and 2VO-Sed, respectively) or after sham surgery (Sham-Ex and ShamSed, respectively). DAPI-labeled nuclei, blue. Quantification of the a cerebral cortex and b hippocampus. Data represent the mean ± SEM of up to 10
vessels per image of 3-4 animals per group. *p < 0.05 vs. Sham-Sed and #p < 0.05 vs. 2VO-Sed (ANOVA). Scale bar 50 μm, magnification 200X

Our results showed that when animals are sedentary
following chronic cerebral hypoperfusion their exploratory response was unchanged when presented a second
time to a same environment, suggesting they did notconsolidate memory. Cognitive dysfunction analyzed byother tests, such as the Morris water maze or radial arm
maze tasks, has been widely reported in this model during the chronic phase [47, 48]. However, upon physicalexercise the animals explored the environment less inthe test session when compared to the training session,suggesting familiarity with the environment and thus
normal cognitive function. A study by Choi et al. corroborates these findings by showing that aerobic exercise,started 3 weeks after 2VO and maintained for 4 weeks,
was able to reduce cognitive impairment and increasehippocampal neurogenesis in a murine model of dementia [49]. Cechetti et al. also demonstrated that forced
treadmill exercise prevents oxidative stress and memory
deficits following chronic cerebral hypoperfusion [50].
Although the exact molecular mechanisms by which
physical training affects brain function are unclear, it issuggested that inhibition of cellular and molecular pathways involved in neuroinflammation contributes to neuroprotection. Positive regulation of the expression ofneurotrophic factors, such as synaptophysin, a presynaptic marker, and PSD-95, a postsynaptic marker may help

Leardini-Tristão et al. Journal of Neuroinflammation(2020) 17:117

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Fig. 5 Exercise increases the number of microglia process in the CA1 region of hippocampus following cerebral hypoperfusion. Representative
confocal images of microglia morphology (Iba1+, yellow) in vessels of the hippocampus (IB4, red) of animals that were exercised or sedentary for 12
weeks after cerebral hypoperfusion (2VO-Ex and 2VO-Sed, respectively) or after sham surgery (Sham-Ex and Sham-Sed, respectively). Data represent
the mean ± SEM of up to 40 microglial cells per group of 3 animals. In a number of microglia in the hippocampus, b and c number of microglia
branches. *p < 0.05 vs. Sham-Sed and **p < 0.01 vs. Sham-Sed; #p < 0.05 and ##p < 0.01 vs. 2VO-Sed (ANOVA). Scale bar 50 μm, magnification 200X

in understanding the protective effects induced by physical exercise. We analyzed expression of these proteinsin the hippocampus, which is the memory storage centerand an important region for the habituation memory to

a new environment. We observed that moderate physicalexercise for 12 weeks was able to increase protein content of synaptophysin and PSD-95 in animals submitted
to 2VO model. Although not significant, the decrease inLeardini-Tristão et al. Journal of Neuroinflammation(2020) 17:117

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Fig. 6 Structural brain capillaries assessed by AngioTool analysis. Data represent the mean ± SEM of 4 images per group of 3 animals. Total length ofthe vessels in a the cortex and b the hippocampus of rats submitted to 2VO and sedentary (2VO-Sed) or exercised (2VO-Ex) for 12 weeks, and theirrespective controls that had been submitted to sham surgery (Sham-Sed and Sham-Ex). ##p < 0.01 vs. 2VO-Sed (ANOVA).Scale bar 50 μm,
magnification 200X

blood pressure observed in the Sham-Ex group may be related to the increase in synaptic proteins synaptophysin,
therefore, it can contribute to the improvement of neuronalplasticity, even in the absence of cerebral hypoperfusion.
Corroborating our results, other experimental studies havealso shown that aerobic exercise was able to increase synaptophysin and PSD-95 levels in the hippocampus and prefrontal cortex of healthy, ischemic, and obese rats [51–53].
Microvascular inflammation and endothelial activation
markers are associated with progressive changes in neurovascular unit. It is still unknown whether inflammation is the main cause of the cognitive impact in
cerebrovascular diseases and whether this is triggered by
cerebral or systemic processes. However, interruption of
the interaction between glia cells and vessels probably
contributes to the disruption of homeostasis [54].
Microglia are CNS-resident immune cells that actively participate in neuronal homeostasis and adult neural pathology.
Under normal physiological conditions, the microglia monitors the microenvironment and communicates bidirectionally with the neurons [55]. During their native resting state,microglia have small cell bodies with numerous long andhighly branching processes, displaying high ramification.

However, during pathological conditions such as infections,trauma, or cerebral ischemia, microglia rapidly change theirmorphology from branched cells to reactive cells with thickened and retracted processes, acquiring a more roundedshape, referred to as amoeboid [55, 56]. Chronic microglial
activation is an important feature of many neurodegenerative
diseases contributing significantly to disease progression [10].
Thus, the modulation of microglial activation has been thesubject of intensive research efforts as a potential therapeutictarget.
Physical exercise as a therapeutic approach has beenshown to significantly attenuate microglia activation in
aging [57–59]. The improvement in cognitive function
was associated with changes in modulation of the microglial phenotype from proinflammatory M1 to antiinflammatory M2 in the corpus callosum, after 28 daysof exercise in rats with chronic cerebral hypoperfusion[60]. These studies support our data showing that moderate physical training reduced microglial activation andincreased the number of its branches in rats with brain
chronic hypoperfusion. Branched microglia are constantly “monitoring” the microenvironment in order to
respond promptly to a pathological event. Results from(2020) 17:117

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other groups indicated that exercise for 8 weeks before
the 2VO was able prevent the activation of microglia
and astrocytes, accompanied by reduction of neuronal
apoptosis [61].
In addition to microglial analysis, we examined theprotective effects of moderate physical exercise on vessel
coverage by astrocytes in animals with chronic cerebral
hypoperfusion. These cells are part of the neurovascular
unit and BBB, playing a key role in the structure of the
BBB, in the communication with neurons, glutamate
homeostasis [62], and also stimulating the release ofvasoactive compounds in microcirculation.
Previous experimental work has shown that physicalexercise was able to increase the levels of the astrocyte
marker, GFAP, in the hippocampus [63] and inducechanges in the astrocyte morphology, suggesting beneficial effects on neuronal activity and plasticity [63]. In a
recent study, an increase in the number of astrocytic
processes during the exercise protocol was observed inother brain areas, such as the prefrontal cortex, striatum,and entorhinal cortex of mice [64].
In the present work, we used immunohistochemistry
analysis to evaluate the coverage of astrocytes on brain
microvessels. Our results showed that early and longterm physical exercise after cerebral hypoperfusion increased the astrocytic coverage of cerebral blood vessels
in parietal cortex and CA1 region of the hippocampus,
regions of the brain associated with learning and memory processes. Astrocyte coverage is important for BBB
integrity and for the cross-talk between neurons andvessels. This result highlights the important impact ofphysical exercise on the neurovascular unit.
Until recently, reactive astrogliosis was a reliable andsensitive marker of diseased tissue. However, there is a
growing body of evidence that points towards the potential protective contributions of astrocytes during topathological mechanisms. Astrocytes are considered toprovide a suitable environment for neuron function. It is
known that the density of GFAP differs between brain
regions; however, our results suggest that the differences
observed in the distribution of processes of astrocytes
are part of the response to ischemic injury induced by
2VO and improved by physical exercise. Substantial increases in astroglial metabolism and synaptic protein
synthesis, consistent with healthy cellular hypertrophy inresponse to increased physiological demands, indicatesthe importance of these cells in neuronal plasticity andregulation of cerebral microcirculation [65, 66].
Our results are in accordance with this data, as the increase in astrocytic processes found in the brains of theexercised animals was accompanied by a reduction in
microglia activation, an increase in the synaptic proteins,synaptophysin and PSD-95, normalization of brain
microcirculation and improvement in cognitive function.

Limitations of this study should be considered. It is
well known that differences in brain regions, such ascortex and hippocampus, as well as variations in behavior between males and females. Therefore, in this study
we used only male rats to avoid any hormonal interference that may occur in the results [67, 68]. However,most studies are moving away from this paradigm andin our future studies, we will make efforts to perform onheterogeneous samples that comprise male and female
animals.
In conclusion, to our knowledge, this is the first study
that has examined the effect of physical exercise on achronic cerebral hypoperfusion model, reporting differences in the astrocytic coverage of cerebral blood vessels
associated with microcirculation and cognitive function.

Leardini-Tristão et al. Journal of Neuroinflammation

Conclusions
Cerebral microcirculatory and inflammatory changes important in the neurodegenerative process are triggered in
the initial phase of cerebral hypoperfusion and maintained
during the chronic phase. We suggest that early and longterm moderate exercise induce significant improvements
in brain inflammation and cognition, highlighting the neuroprotective potential of physical exercise on astrocytes
coverage on brain microcirculation.
The present findings have important implications for improving care of neurodegenerative diseases. Therefore, further evaluation of physical exercise as a non-pharmacologicalapproach may assist in the discovery of neuroprotective
mechanisms. Recommending physical exercise could preventor reduce cognitive changes and their evolution to dementia,
thus improving the quality of life of these patients.
Abbreviations
BBB: Blood-brain barrier; BSA: Bovine serum albumin; CBF: Cerebral blood
flow; 2VO: 2-vessel occlusion; Iba-1: Ionized calcium-binding adaptor-1;
GFAP: Glial fibrillary acidic protein; MET: Maximal exercise testing;
SBP: Systolic blood pressure; PSD-95: Postsynaptic density protein 95
Acknowledgements
We would like to thank the Confocal Platform from Oswald Cruz Foundationand the Confocal Microscopy Unit from the Graduate Program inMorphological Sciences at the Federal University of Rio de Janeiro for theirsupport with the immunohistochemistry images.
Authors’ contributions
ML-T, VE, and ET conceived the study and design; ML-T and GA conducted
the experiments; CG and FL executed immunohistochemistry experiments;
VE analyzed immunofluorescence; ML-T and EM conducted cognitive tests;
ML-T and PR supervised cognitive tests; ML-T, ML, and GA performed western blotting; ML-T and PR supervised western blotting; ML-T, VE, ET, and HN
assisted with funding, experimental design, analyzed the data, and edited
the manuscript. All authors read, revised, and approved the final manuscript.
Funding
This work was sponsored by Conselho Nacional de Desenvolvimento
Científico e Tecnológico- CNPq and Fundação de Amparo à Pesquisa do
Estado do Rio de Janeiro- FAPERJ.

Leardini-Tristão et al. Journal of Neuroinflammation(2020) 17:117

Availability of data and materials
The datasets used and/or analyzed during the current study are available
from the corresponding author on reasonable request.
Ethics approval and consent to participate
All experiments were conducted in accordance with internationally accepted
principles for the care and use of laboratory animals and were approved by
the Animal Ethics Committee of the Oswaldo Cruz Foundation (protocol
number L-002/2016).

Page 13 of 14

16.

17.
18.
19.

Consent for publication
Not applicable

20.

Competing interests
The authors declare no competing interests

21.

Author details
1
Laboratory of Immunopharmacology, Oswaldo Cruz Foundation, Av. Brasil,
4365, Manguinhos, Rio de Janeiro 21040-900, Brazil. 2Laboratory of
Cardiovascular Investigation, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil.
3
Laboratory of Glial Cell Biology, Biomedical Sciences Institute, Federal
University of Rio de Janeiro, Rio de Janeiro, Brazil. 4National Institute ofCardiology, Rio de Janeiro, Brazil.

22.

23.

24.
Received: 6 January 2020 Accepted: 12 March 2020
25.
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