ORIGINAL ARTICLE

Protective Effects of Cannabidiol Against Hippocampal CellDeath and Cognitive Impairment Induced by Bilateral CommonCarotid Artery Occlusion in Mice

Angelica Pupin Schiavon • Lıgia Mendes Soares • Jessica Mendes Bonato •

Humberto Milani • Francisco Silveira Guimaraes • Rubia Maria Weffort de Oliveira

Received: 29 October 2013 / Revised: 13 January 2014 / Accepted: 2 February 2014

� Springer Science+Business Media New York 2014

Abstract The present study investigated whether canna-

bidiol (CBD), a major non-psychoactive constituent of

marijuana, protects against hippocampal neurodegenera-

tion and cognitive deficits induced by brain ischemia in

adult mice. Male Swiss mice were subjected to a 17 min of

bilateral common carotid artery occlusion (BCCAO) and

tested in the Morris water maze 7 days later. CBD (3, 10,

and 30 mg/kg) was administered 30 min before and 3, 24,

and 48 h after BCCAO. After behavioral testing, the brains

were removed and processed to evaluate hippocampal cell

survival and degeneration using Nissl staining and Flu-

oroJade C histochemistry, respectively. Astroglial response

was examined using immunohistochemistry for glial

fibrillary acidic protein (GFAP). CBD (3–30 mg/kg)

improved spatial learning performance in BCCAO mice.

The Nissl and FJC staining results showed a decrease in

hippocampal neurodegeneration after CBD (10 and 30 mg/

kg) treatment. GFAP immunoreactivity was also decreased

in ischemic mice treated with CBD (30 mg/kg). These

findings suggest a protective effect of CBD on neuronal

death induced by ischemia and indicate that CBD might

exert beneficial therapeutic effects in brain ischemia. The

mechanisms that underlie the neuroprotective effects of

CBD in BCCAO mice might involve the inhibition of

reactive astrogliosis.

Keywords Cannabidiol � Morris water maze �Hippocampus � Bilateral common carotid artery occlusion �Mice

Introduction

Ischemic injury that results from global circulatory arrest in

the brain is one of the major causes of death and disability

in the adult population (Nikonenko et al. 2009; Neumann

et al. 2013). Prolonged circulatory deficits induce irre-

versible changes in brain tissue. The resulting neuronal

damage and death lead to a broad range of neurological and

behavioral dysfunctions (Anderson and Arciniegas 2010;

Peskine et al. 2010). Cognitive impairments are among the

better characterized sequelae following brain ischemia,

with memory being the most affected domain, followed by

attention and executive function (Zola-Morgan et al. 1986;

Kartsounis et al. 1995; Moulaert et al. 2010; Mateen et al.

2011). Post mortem studies have suggested that hippo-

campal damage may be a key factor associated with cog-

nitive impairment after ischemic events (Zola-Morgan

et al. 1986; Bachevalier and Meunier 1996; Blum et al.

2012). Despite intense efforts, few pharmacological treat-

ments have effectively minimized the functional impair-

ments following cerebral ischemia (Auriel and Bornstein

2010). Therefore, studies that seek to identify safe and

effective strategies for the treatment of ischemic brain

disease are needed.

A considerable number of preclinical studies have

demonstrated that cannabidiol (CBD), a major non-psy-

choactive constituent of marijuana, may provide neuro-

protection against acute or chronic brain damage

(Mechoulam and Shohami 2007; Garcia et al. 2011; Sag-

redo et al. 2011; Fernandez-Ruiz et al. 2013; Harvey et al.

A. P. Schiavon � L. M. Soares � J. M. Bonato � H. Milani �R. M. Weffort de Oliveira (&)

Department of Pharmacology and Therapeutics, State University

of Maringa, Av. Colombo, 5790, Maringa, PR 87020-900, Brazil

e-mail: rmmwoliveira@uem.br

F. S. Guimaraes

Department of Pharmacology, School of Medicine, USP, Av.

Bandeirantes, Ribeirao Preto, SP 14015-000, Brazil

123

Neurotox Res

DOI 10.1007/s12640-014-9457-0

2012; Pazos et al. 2012, 2013; Valdeolivas et al. 2012).

In vitro, CBD produces a significant reduction of b-amy-

loid-induced neuronal death because of its ability to scav-

enge reactive oxygen species, reduce lipid peroxidation

(Iuvone et al. 2004), and decrease the expression of the

inducible form of nitric oxide synthase (iNOS) and inter-

leukin-1b (IL-1b) expression (Castillo et al. 2010). CBD

also reduces necrotic and apoptotic damage in forebrain

slices in newborn mice exposed to oxygen-glucose depri-

vation (Castillo et al. 2010). Moreover, CBD has been

shown to protect the immature brain from hypoxic-ische-

mic (HI) injury. The administration of CBD 30 min after

HI insult in newborn piglets (Lafuente et al. 2011), mice

(Castillo et al. 2010), and rats (Pazos et al. 2012) resulted

in histological, functional, and biochemical improvements.

Repeated treatment with CBD (3 mg/kg) also decreased

glial activation and improved survival rates after middle

cerebral artery occlusion (MCAO) in mice (Hayakawa

et al. 2008, 2009). From a functional perspective, Pazos

et al. (2012) showed that CBD improved the neurological

score (i.e., cycling, rolling, and leaning to the ipsilateral

side of the lesion) and motor coordination in MCAO mice.

Moreover, CBD (5 mg/kg) administered 5 min after bilateral

common carotid artery occlusion (BCCAO) antagonized

electroencephalographic flattening and hyperlocomotion in

gerbils (Braida et al. 2003).

To date, only one study has evaluated the effects of

CBD on cognitive deficits induced by experimental brain

ischemia. Pazos et al. (2012) showed that CBD (1 mg/kg)

administration in newborn rats after HI injury led to long-

lasting neuroprotective effects, reflected by improved

neurobehavioral performance in both sensorimotor tests

and the novel object recognition test 30 days after the

insult. The latter is considered a non-spatial memory test.

Remaining to be determined, however, the effects of CBD

on spatial memory deficits induced by brain ischemia in

adult animals. Therefore, the aim of this study was to

investigate the effects of CBD on the ability to perform a

spatial learning and memory task (the Morris water maze)

in adult mice after BCCAO. Because astrocytes play a

fundamental role in the pathogenesis of neuronal death

(Takuma et al. 2004; Szydlowska et al. 2010; Duan et al.

2011), we also examined the effects of CBD on hippo-

campal cell survival and astroglial response to experi-

mental brain ischemia.

Materials and Methods

Animals

Male Swiss albino mice (30–40 g, 35–45 days old) were

obtained from the central vivarium of the State University

of Maringa, Maringa, Brazil. Prior to and throughout the

experiments, the animals were maintained under conditions

of controlled temperature (22 ± 1 �C) with a 12/12 h light/

dark cycle (lights on at 7:00 AM). The animals were

housed in groups (n = 3–5) and given standard commer-

cial chow and tap water ad libitum. A total of 81 mice were

included in the experiments. Of these, 15 mice were

assigned to sham operation, and 66 were subjected to brain

ischemia. They were then, randomly distributed into the

independent experimental groups. The experimental pro-

cedures were approved by the Ethics Committee on Animal

Experimentation of the State University of Maringa (CEEA

004/2011) and are in accordance with the guidelines of

NIH and Brazilian College for Animal Experimentation.

Surgery

Transient global cerebral ischemia was induced by

BCCAO as previously described (Soares et al. 2013). The

mice were anesthetized with a mixture of isoflurane

(Isoforine, Cristalia, SP, Brazil) and oxygen delivered

through a universal vaporizer (Oxigel, SP, Brazil) con-

nected to a plastic mask adapted to the animal’s nose. The

vaporizer was regulated to release the minimal burble flow

(2.0 l/min). The mixture delivered to the animal was

monitored to maintain the minimal isoflurane concentration

required for an efficient anesthesia (evaluated by pinching

the animal’s tail). Under these conditions, the animal was

fixed in a stereotaxic frame and the anesthesia maintained

with 1.3-1.5 % isoflurane in 100 % oxygen during

*6 min, time necessary to make an incision in the ventral

neck to expose the common carotid arteries. Rectal tem-

perature was carefully monitored during surgery and

maintained at *37.5 �C using a heating blanket. Brain

ischemia was induced by 17 min of BCCAO using aneu-

rysm clips (ADCA, MG, Brazil). Throughout the occlusion

procedure, the mice were maintained in a warming box

(inner temperature, 30 ± 1 �C) to avoid ischemia-induced

brain hypothermia (Seif el Nasr et al. 1992). At the end of

each occlusion, the aneurism clips were removed, and the

carotid arteries were visually inspected for reperfusion.

Then, each animal was again anesthetized for 2 min and

the incision closed with sutures. For 3 h after reperfusion,

the mice were maintained in a warming box at 30 �C.

Sham-operated animals were subjected to the same anes-

thetic and surgical interventions, with the exception that

the carotid arteries remained intact.

Treatment

Vehicle (0.9 % NaCl with 1 % Tween 80) or CBD (THC

Pharma, Germany) was prepared immediately before use

and injected intraperitoneally (i.p.) in a 1 ml/kg volume.

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123

The animals were randomly assigned to receive vehicle or

CBD (3, 10, or 30 mg/kg) 30 min before and 3, 24, and

48 h after surgery. The treatment regimen and doses of

CBD were based on Hayakawa et al. (2008) and Campos

et al. (2012).

Morris Water Maze

The behavioral testing occurred during the light phase

between 8:00 AM and 2:00 PM under identical conditions.

The experiments were video-recorded, and the behavioral

scores were later analyzed using ANY-maze image ana-

lyzer software (Stoelting, Wood Dale, IL, USA). All of the

behavioral tests were conducted 7 days after BCCAO

because the neuropathological and neurobehavioral con-

sequences of BCCAO in Swiss mice are well established

over that time interval so the neuroprotective effect of a

given strategy can be properly assessed (Soares et al.

2013).

The Morris water maze apparatus consisted of a swim-

ming pool made of black painted fiberglass (90 cm diam-

eter, 35 cm height). For the tests, the tank was filled with

water maintained at 24 ± 1 �C. The target platform

(10 cm2) was made of transparent acrylic and submerged

1 cm beneath the surface of the water. The starting points

for the animals were marked on the outside of the pool as

north (N), south (S), east (E), and west (W). Four distant

visual cues were placed on the walls of the experimental

room.

The mice were subjected to the Morris water maze using

a procedure that was similar to one described previously

(Prediger et al. 2008; Soares et al. 2013). In brief, the

training session consisted of ten consecutive trials, during

which the animals were left in the tank facing the wall, and

then allowed to swim freely to the submerged platform.

The platform was located in a constant position (middle of

the southwest quadrant), equidistant from the center and

wall of the pool. If the animal did not find the platform

during a 60 s trial period, then it was gently guided to it.

The animal was allowed to remain on the platform for 10 s.

This procedure was repeated ten times, with the starting

point (i.e., the axis of one imaginary quadrant) varying in a

pseudo-random manner. The latency to find the platform

was recorded for each animal. 24 h after the last training

trial (i.e., acquisition phase), the probe trial test (i.e.,

retention phase) began and consisted of a single trial in

which the platform was removed from the pool. The mouse

was allowed to swim for 60 s, and the time spent in the

correct quadrant (i.e., where the platform was located

during the training session) was recorded as an index of

memory strength.

Histology

Two histological groups were generated by random group

assignment. One group (n = 6–15 subjects/experimental

group) underwent histological assessment of hippocampal

cell survival using Nissl staining. A second group (n = 5–8

subjects/experimental group) underwent Fluorojade-C

(FJC) staining and immunohistochemical assays. Coronal

brain sections were obtained at a stereotaxic level between

-1.70 and -2.70 mm posterior to bregma (Franklin and

Paxinos 1997). The histological analysis was performed

blind to the treatment groups.

Nissl Staining

The mice were deeply anesthetized with 50 mg/kg sodium

thiopental (Thiopentax; Cristalia, SP, Brazil) and tran-

scardially perfused with 0.9 % saline followed by Bouin’s

fixative. Following decapitation, the head was immersed in

crushed ice (1–2 �C) for 2 h to avoid the appearance of

dark neurons, which could confound the actual extent of

neurodegeneration. The brain was carefully removed and

postfixed in Bouin’s solution for 3 days. Using a rotating

microtome (RM2445, Leica, Goettingen, Germany), 7 lm

paraffin-embedded coronal sections were cut and distrib-

uted into four sets of slides that contained four coronal

sections, each 28 lm apart. After standard dehydration and

diaphanization procedures, slides that contained adjacent

sections were immersed in distilled water and submerged

in 0.2 % Cresyl violet solution (Nissl staining) for 5 min.

The slides were then rinsed in distilled water, dehydrated in

a graded series of ethanol (70, 80, 90, and 100 %), cleared

in xylene, and coverslipped with xylene using Permount

(Fisher Scientific, Sao Paulo, Brazil). In each hemisphere,

the number of cells that presented a well delimited,

spherical form with a distinct nucleus and nucleolus was

counted throughout the CA1, CA2, CA3, and CA4 sub-

fields of the hippocampus (4009 magnification; Olympus

BX-41 microscope). Neurons that had shrunken cell bodies

or surrounding empty spaces were considered destined to

die and excluded from the counting. The number of intact-

appearing cells is expressed as the mean ± SEM.

Fluorojade-C and Immunohistochemistry

The animals were deeply anesthetized and transcardially

perfused with saline followed by 4 % paraformaldehyde in

0.2 M phosphate buffer (PB). The brains were removed,

postfixed in the same fixative for 2 h, and cryoprotected by

immersion in 30 % sucrose. Frozen tissue was serially

sectioned on a cryostat (Criocut 1800, Reichert-Jung,

Heidelberg, Germany) into 30 lm coronal sections that

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123

were collected in quadruplicate in Eppendorf tubes that

contained antifreeze solution.

FJC is an anionic fluorochrome that detects neurons that

are undergoing the death process (Schmued et al. 2005).

Slides containing frozen sections were incubated in citrate

buffer for 30 min at 95 �C. The slides were then rinsed in

distilled water for 1 min and incubated in a 0.06 %

potassium permanganate solution for 15 min. The slides

were washed again in distilled water and incubated in

0.0001 % FJC (Histo-Chem, Jefferson, AR, USA) dis-

solved in a solution that contained 0.1 % acetic acid for

30 min in a dark room at 25 �C. They were then washed in

water, dried at room temperature, dehydrated, cleared in

xylene, and coverslipped after the addition of Permount

media. Using a fluorescence microscope (4009 magnifi-

cation, Zeiss microscope, Axiostar Plus, Jena, Germany),

FJC-positive cells were counted throughout the CA1-CA4

subfields in four coronal sections per animal from both

brain hemispheres. The number of FJC-positive cells is

expressed as the mean ± SEM.

Immunohistochemistry was performed for glial fibrillary

acidic protein (GFAP). The free-floating sections were

quenched in 1 % H2O2 for 30 min, and then blocked with

2 % bovine serum albumin in 0.1 M phosphate-buffered

saline (PBS) for 60 min. The sections were incubated

overnight with rabbit polyclonal anti-GFAP antibody

(1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA)

in PBS that contained 0.3 % Triton X-100 at room tem-

perature. The sections were then incubated with the

respective biotinylated secondary antibodies (1:500; Santa

Cruz Biotechnology, Santa Cruz, CA, USA) for 2 h and

further incubated in ABC solution (Vectastain Elite ABC

Kit, Vector Laboratories, Burlingame, CA, USA) for 2 h.

The peroxidase reaction was performed by incubating the

sections with 3-30-diaminobenzidine (DAB; Sigma) and

0.05 % H2O2. The sections were mounted on gelatin-

coated slides, air dried, cleared with xylene, and covers-

lipped with Permount.

GFAP immunoreactivity was evaluated by measuring

the integrated optical density (IOD) using ImageJ software

(National Institutes of Health, Bethesda, MD, USA). Color

images of the CA1 region from both brain hemispheres

were captured at 409 objective using a camera (QColor,

Olympus, America Inc.) and an Olympus BX 41 research

microscope. The region of interest was selected and a

previously defined area located between the CA1 pyrami-

dal cell layer and lacunosum molecular lay of the hippo-

campus (Franklin and Paxinos 1997) in both brain

hemispheres. The image was then converted to gray scale,

the background subtracted and, the IOD obtained. Results

were presented as the mean ± SEM of three sections/

animal.

Statistical Analysis

The SAS package (version 9.3) was used. Both behavioral and

histological data were examined for the assumptions of nor-

mality, homocedasticity, and sphericity (repeated measures).

In the Morris water maze test, learning performance was

measured across ten consecutive trials. The values obtained

for each mouse across two consecutive trials were aver-

aged, yielding five blocks with two trials per block. These

individual values (in blocks) were used to compute the

mean ± SEM for each group. A two-way repeated-mea-

sures analysis of variance (ANOVA), with experimental

group as the independent factor and test block (five blocks)

as the repeated factor, was performed. The sham- and

CBD-treated animals were compared with vehicle-treated

ischemic mice using a simple-contrast analysis. One-way

ANOVA was used to quantify the total latency (summed

over the 10 trials) and time spent in the correct quadrant.

The histological data were normalized to the mean

values of the sham- (Nissl and GFAP) or BCCAO-operated

(FJC) groups. The data were analyzed using the general-

ized linear model using the Poisson distribution for mod-

eling the count data (number of cells; Nissl and FJC) and

the Gamma distribution for the continuing measurements

(IOD; GFAP), respectively.

Differences with a probability value of p \ 0.05 were

considered statistically significant.

Results

Survival

Overall, 81 animals entered the experiment. Fifteen ani-

mals underwent sham surgery and were namely vehi-

cle ? sham group (veh sham; n = 15). Of the 66 mice

subjected to BCCAO, seven (10.6 %) died after complete

recovery from anesthesia, likely reflecting a severe and

fatal effect of brain ischemia. The remaining 59 ischemic

animals were randomly distributed into the following

experimental groups: vehicle ? ischemia (veh isch;

n = 23), CBD 3 mg/kg ? ischemia (CBD3 isch; n = 11),

CBD 10 mg/kg ? ischemia (CBD10 isch; n = 14), and

CBD 30 mg/kg ? ischemia (CBD30 isch; n = 11). 7 days

after sham or BCCAO surgery the animals were tested in

the Morris water maze task.

Morris Water Maze Task

Learning and memory performance in the Morris water maze

are shown in Fig. 1. The latency to find the platform was

measured in a single session (day) across 10 consecutive

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123

trials and is expressed as the learning curve during five

blocks with two trials each (latency in blocks) and total

latency (summed across the 10 trials). The latency to find the

platform decreased regularly across the trials for all of the

experimental groups (F4,276 = 20.5, p \ 0.001). A signifi-

cant effect of treatment was found (F4,73 = 16.7,

p \ 0.001). The sham and ischemic animals treated with

CBD (3–30 mg/kg) exhibited a significant decrease in the

latency to find the platform compared with vehicle-treated

ischemic mice (Fig. 1a; p \ 0.05). No significant difference

was found in the time spent in the correct quadrant (Fig. 1c).

Nissl Staining

Representative photomicrographs of Nissl staining for the

hippocampus are shown in Fig. 2. BCCAO resulted in

severe neurodegeneration in the hippocampus as seen by

the presence of shrunken and dark-stained neurons (Fig. 2).

Compared with sham surgery, BCCAO caused significant

cell loss over the entire CA1-CA4 hippocampal fields

(v2 = 6,337.54, p \ 0.0001). Compared with vehicle,

CBD treatment at 10 and 30 mg/kg significantly increased

the number of intact hippocampal cells compared to veh

isch group (p \ 0.0001). There was also a significant dif-

ference between ischemic animals treated with CBD 10

and 30 mg/kg (p \ 0.05), indicating that the dose of

10 mg/kg was more effective in attenuating the neurode-

generative effect of brain ischemia.

Fluorojade-C Staining

FJC mainly stained cell bodies of degenerating neurons and

occasionally their processes (Schmued et al. 2005). The

majority of FJC-positive neurons were detected in the CA1

pyramidal layer of the hippocampus (Fig. 2c, e), which

paralleled the reduction of Nissl-stained neurons (Fig. 2b,

d). No FJC-positive cells were observed in sham-operated

animals. The statistical analysis revealed a significant

decrease in the number of FJC-positive cells in the hip-

pocampus in ischemic animals treated with CBD (3, 10 and

30 mg/kg) compared with vehicle-treated ischemic animals

(v2 = 1,656.90, p \ 0.0001).

Immunohistochemistry

GFAP-immunoreactive cells are shown in Fig. 3. BCCAO

significantly increased the IOD in ischemic animals treated

with vehicle or CBD 3 mg/kg compared with the sham

group (v2 = 17.63, p = 0.001). Compared with vehicle,

CBD 30 mg/kg significantly decreased the IOD of GFAP-

positive cells in vehicle (p = 0.0003) and CBD 3 mg/kg

(p = 0.0013) treated animals.

Discussion

We found that CBD reduced behavioral impairment and

hippocampal neurodegeneration in response to brain

ischemia in mice. Treatment with CBD (3-30 mg/kg)

30 min before and 3, 24, and 48 h after BCCAO improved

spatial learning performance in mice tested in the Morris

water maze 7 days after the ischemic insult. The Nissl

staining results showed an increase in the hippocampal cell

survival in ischemic animals after CBD treatment, sug-

gesting a protective effect of CBD on neuronal death

induced by ischemia. On the other hand, the number of

cells undergoing degeneration (FJC-positive) was reduced

by CBD treatment. GFAP immunoreactivity was also

decreased in ischemic mice treated with CBD (10 and

30 mg/kg). These findings indicate that CBD might exert

Fig. 1 Effects of CBD on learning deficits induced by BCCAO in

mice. The animals received vehicle or CBD (3, 10 or 30 mg/kg)

30 min before and 3, 24 and 48 h after surgery. Learning performance

was assessed 7 days after surgery and was expressed by the latency to

find the platform along 5 blocks of 2 trials each, i.e., ‘‘latency in

blocks’’ (a) and ‘‘total latency’’ (b). Memory retention was given by

the ‘‘time spent in the correct quadrant’’ during the probe trial (c).

Values are mean ± SEM of the groups: sham mice treated with

vehicle (sham veh, n = 15), ischemic mice treated with vehicle (isch

veh, n = 23), ischemic mice treated with CBD 3 (isch CBD3,

n = 11), 10 (isch CBD10, n = 14), or 30 (isch CBD 30, n = 11) mg/

kg. *p \ 0.05, **p \ 0.001 in comparison with isch veh group

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beneficial therapeutic effects in global brain ischemia. The

protective effects of CBD on hippocampal cell survival and

functional recovery after BCCAO may involve the inhibi-

tion of reactive astrogliosis.

BCCAO induces cognitive impairments in passive

avoidance and spatial tasks in mice (Kumaran et al.

2008; Zhang et al. 2010; Bora et al. 2011; Soares et al.

2013). In the present study, mice subjected to BCCAO

exhibited an increase in the latency to find the platform

in the Morris water maze, indicating spatial learning

impairment. These spatial deficits were counteracted by

CBD administration (3–30 mg/kg) 30 min before and 3,

24, and 48 h after the ischemic insult. Our results are

consistent with previous studies that reported protective

effects of CBD on functional recovery after MCAO

(Hayakawa et al. 2009) and HI (Pazos et al. 2012) and

extend the CBD effects to experimental global brain

ischemia in mice.

Fig. 2 Effects of CBD on hippocampal cell survival (Nissl staning)

and degeneration (FJC staining) after BCCAO in mice. Histological

analysis was performed along the CA1–CA4 hippocampal subfields

7 days after sham or BCCAO surgery. a Representative diagram

illustrating a coronal brain section obtained at a stereotaxic level

between -1.70 and -2.70 mm posterior to bregma (Franklin and

Paxinos, 1997). In b, c columns represent the mean ± SEM of

neurons stained for Nissl (n = 6–15) or FJC (n = 5–8), respectively.

The data were normalized to the mean values of the sham veh (Nissl

staining) and isch veh (FJC staining) groups, respectively.

**p \ 0.001 compared to sham veh group; ##p \ 0.001 compared

to isch veh group and ?p \ 0.05 compared to CBD3. Representative

photomicrographies of CA1 hippocampal field containing Nissl- (d–

h) and FJC- (i–m) positive neurons of each experimental group. White

arrows show CA1 intact-appearing neurons and black arrows show

shrunken and dark-stained neurons, indicating neurodegeneration

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123

CBD 10 and 30 mg/kg significantly increased the

number of intact-appearing, Nissl-stained cells and

decreased the number of FJC-positive cells in the hippo-

campus of ischemic animals. These results indicate that

CBD not only promoted functional recovery but also

decreased the delayed hippocampal cell loss and mitigated

the undergoing neurodegenerative processes induced by

global brain ischemia in mice. Despite the hippocampal

damage observed, the animals treated with CBD 3 mg/kg

were able to learn the task and perform at level similar to

controls. This finding suggests that the effect of CBD in

cognition was distinct from the structural damage in the

hippocampus. Therefore, one issue is how CBD 3 mg/kg

could have facilitated learning despite the hippocampal

lesion. Notably, dysfunction of complex behaviors and

recovery of function may reflect alterations at the subcel-

lular, synaptic, or electrophysiologic levels, or even

widespread morphological changes that would not be

restricted to a given brain structure, such as the hippo-

campus (de la Tremblaye and Plamondon 2011). In fact,

existing studies rarely assess neuronal damage outside the

hippocampus in relationship to behavioral impairments

following global ischemia. Only few authors have dem-

onstrated ischemia-induced alterations of cellular activity

in brain regions outside hippocampus, which could have a

significant impact on behavioral recovery following

ischemia. For example, Caruana et al. (2008), reported

epileptiform EEG activity in the CA1/subcular region,

perirhinal cortex and prefrontal cortex following 15 min of

global ischemia in rats. Moreover, significant losses of

CRH-positive neurons have been demonstrated in amyg-

dala 6 weeks following hypoxia–ischemia (Carty et al.

2010) a phenomenon associated with hyperactivity in

response to novel open-field exposure in ischemic rats. In

humans, reduced amygdalar volume has been correlated in

stroke and cardiac arrest survivors with cognitive

Fig. 3 Effects of CBD on

GFAP expression after BCCAO

in mice. a Representative

diagram illustrating a coronal

brain section containing the

hippocampus with a selected

area where the IOD of GFAP

immunostaining was obtained.

In b columns represent the

mean ± SEM of IOD of GFAP-

immunoreactive cells in the

different experimental groups

(n = 5–8). The data were

normalized to the mean values

of the isch veh group.

**p \ 0.001 and *p \ 0.05

compared to sham veh group;##p \ 0.001 compared to isch

veh group; ?p \ 0.05 compared

to CBD3 group. c–

g Representative

photomicrographies of GFAP

immunostaining showing

morphological changes in

GFAP expression of ischemic

compared to sham animals.

Details in a, c–g showing the

selected area where GFAP

immunolabeling was evaluated.

Py pyramidal cell lay, Lmol

lacunosum moleculare layer,

DG dentate gyrus

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impairments (Sachdev et al. 2007). These findings support

the notion that factors other than hippocampal lesion might

influence behavioral output after brain ischemia. However,

whether or not the CBD treatment would provide transient

or enduring protective effects on neurohistological out-

comes of BCCAO in mice, is still unclear. The degree of

neuroprotection afforded by other drugs, such as the a-

amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

receptor antagonist NBQX, N-channel calcium blocker

SNX, and noncompetitive N-methyl-D-aspartate antagonist

MK801, has been shown to decline when the postischemic

time of the analysis is extended from the first 7–30 days

overall (Corbett and Crooks 1997; Corbett and Nurse

1998).

The protective effect of CBD was not limited to neurons

but extended to astrocytes. The administration of 30 mg/kg

CBD decreased reactive astrogliosis induced by BCCAO,

suggesting that astrocytes might be involved in the

observed neuroprotective effect of CBD. Astrocytes are the

most numerous cell type in the brain and dynamically

involved in synaptic transmission, metabolic and ionic

homeostasis, the anti-inflammatory response, and antioxi-

dant defense (Robel et al. 2011). An increase in astrocyte

expression, characterized by increased cell proliferation,

cell hypertrophy, and upregulation of the intermediate fil-

aments GFAP and vimentin (Robel et al. 2011), has been

demonstrated in nearly every type of central nervous sys-

tem injury, including focal (Cheung et al. 1999; Mao et al.

2011) and transient global (Kindy et al. 1992; Norenberg

1996; Ridet et al. 1997) brain ischemia. Our data are

consistent with other reports. However, astrocytes can exert

both beneficial and detrimental effects during repair pro-

cesses in the injured brain (Stoll et al. 1998; Robel et al.

2011). Astrocytes secrete neurotrophins and may provide a

permissive substratum to support axonal regrowth during

injury, but sustained reactive astrogliosis may be a major

impediment to axonal regeneration and protection (Lee

et al. 1996; Ridet et al. 1997). For example, when GFAP

filaments are genetically ablated in mice, they develop less

dense scars and show improved posttraumatic regeneration

after hemisection of the spinal cord and improved survival

and differentiation of transplanted stem cells (Pekny and

Pkna 2004; Pekny and Nilsson 2005). Moreover, increased

GFAP immunoreactivity in the CA1 hippocampal region

after brain ischemia has been associated with the extent and

maturation of neuronal necrosis (Petito and Halaby 1993;

Stoll et al. 1998). Therefore, decreased astrogliosis in

CBD-treated mice might have influenced neuronal death

induced by the ischemic insult. However, the inhibition of

astrogliosis by CBD may be a consequence, not the cause,

of the reduced neuronal damage provoked by CBD.

Additional experimentation addressing the signaling pro-

tective mechanism of CBD need be conducted.

A limitation of the present study is that the behavior

improvements observed in the Morris test were not dose-

dependent and did not correlate directly with the histo-

logical findings. The reason for this discrepancy is

unknown. However, CBD usually produces bell-shaped

dose–response curves, an effect not completely understood

but could involve the different pharmacological effects

triggered by this drug (for review, see Campos et al. 2012).

Although CBD has been reported to increase hippocampal

cell proliferation, this effect disappears at high concentra-

tions of the drug (Campos et al. 2013). CBD-mediated

neuroprotection after experimental brain ischemia has

generally been associated with the modulation of neuro-

toxicity, inflammation, and oxidative stress (Castillo et al.

2010; Pazos et al. 2012). Pharmacological studies have

shown that serotonin-1A, cannabinoid CB2, and adenosine

receptors are also involved in the neuroprotective effect of

CBD in the immature ischemic brain (Castillo et al. 2010;

Lafuente et al. 2011; Pazos et al. 2012). Recently, it was

proposed that CBD exerts its effects on brain injury by

inhibiting the astroglial differentiation of neural stem pre-

cursor cells (Shinjyo and Di Marzo 2013), an effect that

could be detrimental to neurogenesis (Robel et al. 2011;

Colangelo et al. 2012). CBD markedly downregulated

reactive astrogliosis by activating peroxisome proliferator-

activated receptors following b-amyloid neurotoxicity,

which in turn resulted in neuroprotection (Esposito et al.

2011). Therefore, the protective effects of CBD on hip-

pocampal cell death and functional recovery after BCCAO

may involve the inhibition of reactive astrogliosis.

Remaining to be investigated is whether similar mecha-

nisms are involved in CBD-induced neuroprotection after

global brain ischemia.

Acknowledgments The authors thank Marco Alberto Trombelli for

his technical support. This study was supported by Conselho Nacional

de Desenvolvimento Cientıfico e Tecnologico (CNPq), FAPESP,

State University of Maringa and Fundacao Araucaria.

Conflict of interest The authors have no conflict of interest.

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