Accepted Manuscript

Title: Effects of curcumin on short-term spatial and

recognition memory, adult neurogenesis and
neuroinflammation in a streptozotocin-induced rat model of
dementia of Alzheimer’s type

Authors: Taysa B. Bassani, Joelle M. Turnes, Eric L.R.

Moura, Jéssica M. Bonato, Valentı́n Cóppola-Segovia, Silvio
M. Zanata, Rúbia M.M.W. Oliveira, Maria A.B.F. Vital

PII: S0166-4328(17)31137-3
DOI: http://dx.doi.org/doi:10.1016/j.bbr.2017.08.014
Reference: BBR 11033

To appear in: Behavioural Brain Research

Received date: 11-7-2017

Revised date: 1-8-2017
Accepted date: 5-8-2017

Please cite this article as: Bassani Taysa B, Turnes Joelle M, Moura Eric
LR, Bonato Jéssica M, Cóppola-Segovia Valentı́n, Zanata Silvio M, Oliveira
Rúbia MMW, Vital Maria A.B.F.Effects of curcumin on short-term spatial and
recognition memory, adult neurogenesis and neuroinflammation in a streptozotocin-
induced rat model of dementia of Alzheimer’s type.Behavioural Brain Research
http://dx.doi.org/10.1016/j.bbr.2017.08.014

This is a PDF file of an unedited manuscript that has been accepted for publication.
As a service to our customers we are providing this early version of the manuscript.
The manuscript will undergo copyediting, typesetting, and review of the resulting proof
before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that
apply to the journal pertain.
Effects of curcumin on short-term spatial and recognition memory, adult

neurogenesis and neuroinflammation in a streptozotocin-induced rat

model of dementia of Alzheimer’s type

Taysa B. Bassani,1, Joelle M. Turnes,1 Eric L. R. Moura,1 Jéssica M. Bonato,2

Valentín Cóppola-Segovia,3 Silvio M. Zanata,3 Rúbia M. M. W. Oliveira,2 Maria

A. B. F. Vital1

1Department of Pharmacology, Federal University of Paraná, Curitiba, PR,

81531-980, Brazil.
2Department of Pharmacology and Therapeutics, State University of Maringá,

Maringá, PR, 87020-900, Brazil.

3Department of Basic Pathology, Federal University of Paraná, Curitiba, PR,

81531-990, Brazil.


Corresponding author:

Taysa Bervian Bassani

E-mail: taysa_bassani@yahoo.com.br

Phone: +55 41 3361-1693

Fax: +55 41 3266-2042

 2

Highlights

 Streptozotocin caused deficits in short-term spatial and recognition

memory

 Streptozotocin decreased adult neurogenesis and increased

neuroinflammation

 Curcumin prevented streptozotocin-induced impairments in recognition

memory

 Curcumin did not prevent streptozotocin-induced impairments in spatial

memory

 Curcumin did not improve adult neurogenesis and neuroinflammation

Abstract: Curcumin is a natural polyphenol with evidence of antioxidant, anti-

inflammatory and neuroprotective properties. Recent evidence also suggests

that curcumin increases cognitive performance in animal models of dementia,

and this effect would be related to its capacity to enhance adult neurogenesis.

The aim of this study was to test the hypothesis that curcumin treatment would

be able to preserve cognition by increasing neurogenesis and decreasing

neuroinflammation in the model of dementia of Alzheimer’s type induced by an

intracerebroventricular injection of streptozotocin (ICV-STZ) in Wistar rats. The

animals were injected with ICV–STZ or vehicle and curcumin treatments (25, 50

and 100 mg/kg, gavage) were performed for 30 days. Four weeks after surgery,

STZ-lesioned animals exhibited impairments in short-term spatial memory

(Object Location Test (OLT) and Y maze) and short-term recognition memory

(Object Recognition Test - ORT), decreased cell proliferation and immature

neurons (Ki-67- and doublecortin-positive cells, respectively) in the

 3

subventricular zone (SVZ) and dentate gyrus (DG) of hippocampus, and

increased immunoreactivity for the glial markers GFAP and Iba-1

(neuroinflammation). Curcumin treatment in the doses of 50 and 100 mg/kg

prevented the deficits in recognition memory in the ORT, but not in spatial

memory in the OLT and Y maze. Curcumin treatment exerted only slight

improvements in neuroinflammation, resulting in no improvements in

hippocampal and subventricular neurogenesis. These results suggest a positive

effect of curcumin in object recognition memory which was not related to

hippocampal neurogenesis.

Abbreviations: BDNF = brain-derived neurotrophic fator; CC = corpus

callosum; CNS = central nervous system; DCX = doublecortin; DG = dentate

gyrus; EPM = elevated plus maze; GFAP = glial fibrillary acidic protein; Iba-1 =

ionized calcium binding adaptor molecule-1; ICV = intracerebroventricular; LVs

= lateral ventricles; NSC = neural stem cells; NPC = neural progenitor cells;

OFT = open field test; OLT = object location test; ORT = object recognition test;

sAD = sporadic Alzheimer’s disease; SGZ = subgranular zone; STZ =

streptozotocin; SVZ = subventricular zone; TNF- = tumor necrosis factor alfa.

Keywords: Alzheimer’s disease; curcumin; adult neurogenesis; recognition

memory; spatial memory; neuroinflammation.

1. Introduction

Alzheimer’s disease (AD) is the most common progressive

neurodegenerative disorder of the elderly, characterized by a decline in

 4

cognitive functions. Sporadic AD (sAD; i.e., late-onset AD), the most frequent

form of AD with an unknown etiology, presents early abnormalities in brain

glucose metabolism and insulin signaling, thus resulting in an insulin-resistant

state in the brain, which has been suggested to be related to etiological events

in its pathogenesis [1,2]. Additionally, protein aggregates [3], mitochondrial

dysfunctions [4], oxidative stress [5], neuroinflammation [6], brain cholinergic

deficits [7], and dysfunctions in adult neurogenesis [8–10] are among the main

features of sAD. These features may be modeled by a intracerebroventricular

(ICV) injection of low, subdiabetogenic doses of streptozotocin (STZ) in rodents

[11,12] and non-human primates [13].

Adult neurogenesis is a multi-step process of the formation of new

neurons in the mammalian brain throughout life. Evidence suggests that newly

generated neurons play a crucial role in hippocampus-dependent learning and

memory (e.g., spatial memory and orientation) and recovery from neuronal

injury [14]. Neurogenesis starts from the proliferation of resident neural stem

cells (NSC) and neural progenitor cells (NPC) in two neurogenic niches in the

central nervous system (CNS): the subventricular zone (SVZ) of the lateral

ventricles (LVs), and the subgranular zone (SGZ) of the dentate gyrus (DG) of

the hippocampus. The process of adult neurogenesis can be influenced by

many intrinsic and extrinsic factors. Growth factors and neurotrophins, such as

brain-derived neurotrophic factor (BDNF), increase differentiation, maturation,

and survival of proliferating NSC/NPC, stimulating neurogenesis. In contrast,

microglial activation and release of proinflammatory cytokines (e.g., interleukin-

1 (IL-1), IL-6, and tumor necrosis factor- (TNF-)) have been demonstrated

to inhibit neurogenesis by reducing the proliferation of NSC/NPC. The

 5

consequence of inhibiting neurogenesis is cognitive deficit, reflected by

impairments in spatial and recognition learning and memory [15]. Several

studies have reported an increase in neuroinflammation in the STZ model of

sAD, reflected by reactive gliosis and an increase in proinflammatory markers

that are associated with cognitive decline [16–19]. Other studies reported a

decrease in neurogenesis associated with oxidative stress [20] or amyloid

pathology [21].

Current therapeutic options for AD are limited because most agents

provide only symptomatic relief from cognitive deficits. A neuroprotective agent

able to improve cognitive function along with slowing neuronal loss, decrease

neuroinflammation and improve neurogenesis is needed. Curcuma longa

(turmeric) is a rhizomatous native plant from South and Southeast Asia which

belongs to the ginger family Zingiberaceae [22]. Traditionally curcumin, the

main active ingredient in turmeric, has been used as a common food additive

and herbal medicine [23]. Epidemiological studies have suggested that societies

that widely use curcumin have a reduced incidence of AD and improved

cognitive function [24,25].

The natural polyphenol curcumin possesses pleiotropic properties. It has

been reported to provide neuroprotection in cellular and animal models of

neurodegenerative and neuropsychiatric disorders including AD, Parkinson’s

disease, Huntington’s disease, multiple sclerosis, depression, and

schizophrenia [26]. Neuroprotection provided by curcumin may be due to its

antioxidant properties, by upregulation of the transcription factor Nrf2 [27], and

anti-inflammatory properties, by suppression of NF-B activation [28]. Curcumin

also inhibits amyloid beta (A)-oligomerization and tau-phosphorylation [29] and

 6

increases BDNF levels in corticosterone-treated rats [30], chronically stressed

rats [31] and Wistar Kyoto rats [23].

In addition, curcumin potentiates spatial and non-spatial memory in aged

mice [32] and rats [33], in A-induced [34] and STZ-induced models of AD [35–

38]. The positive effects of curcumin on cognition may be related to its capacity

to enhance neurogenesis. Curcumin has shown to increase neurite outgrowth

and proliferation of NSC both in vitro and in vivo through the activation of the

ERK and MAP kinase pathways [39,40]. Curcumin treatment increases

neurogenesis in chronically stressed rats [41], aged rats [33] and in Aβ-induced

model of AD [26]. Nevertheless, the effects of prolonged curcumin treatment on

adult neurogenesis were not studied in the STZ-ICV model of sAD. Therefore,

we hypothesized that curcumin treatment would be able to preserve cognitive

functions of STZ-ICV injected rats by increasing adult neurogenesis and

decreasing neuroinflammation.

2. Methods

2.1. Animals

In this study, 3-4 months old male Wistar rats weighing 300-340 g at the

beginning of the experiment were used. All animals were obtained from our

breeding colony. The animals had free access to food and water, were

maintained in a temperature-controlled room (22C ± 2°C) on a 12 h/12 h

light/dark cycle (lights on at 7:00 AM), and were randomly housed in groups of

3-4 in polypropylene cages with wood shavings as bedding. The studies were
 7

performed in accordance with the guidelines of the Federal University of Paraná

and were approved by the University Ethics Committee (protocol no. 735).

2.2. Drugs

Streptozotocin (N-[methylnitrosocarbamoyl]-α-D-glucosamine) was

purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and

curcumin (from Curcuma longa) was purchased from Sigma-Aldrich (St. Louis,

MO, USA).

2.3. Experimental design

The rats were randomly divided into five groups: sham (n = 7), STZ (n =

7), STZ+curcumin 25 mg/kg (STZ+cur 25; n = 6), STZ+curcumin 50 mg/kg

(STZ+cur 50; n = 8), and STZ+curcumin 100 mg/kg (STZ+cur 100; n = 7). The

STZ groups received a single bilateral ICV injection of STZ (3 mg/kg total dose)

dissolved in sterile 0.9% saline (4.5 l per injection site). The sham group

received a single bilateral ICV injection of sterile 0.9% saline (4.5 l per injection

site; Fig. 1). Treatment with curcumin (25, 50 and 100 mg/kg, per os) or its

vehicle (0.5% carboxymethylcelulose in distilled water with 1% Tween 80) was

performed over 30 days, once a day in the afternoon, and started 1 h before the

beginning of stereotaxic surgeries.

The animals were allowed to recover for 3 weeks following surgery

before behavioral evaluations. These animals were assessed in the open field

test (OFT) to evaluate spontaneous locomotor activity on day 21 after surgery

and in the elevated plus maze (EPM) on day 22 to assess anxiety-like behavior.

Cognitive performance was evaluated in the object location test (OLT) on day
 8

28, in the object recognition test (ORT) on day 29, and in the spatial version of

the Y maze on day 30. Right after the last behavioral analysis, blood glucose

levels were measured using a G-Tech Free device using blood samples that

were collected by tail prick.

Afterward, animals were deeply anesthetized with chloral hydrate (400

mg/kg, i.p.) and intracardially perfused for posterior immunohistochemical

evaluation of Doublecortin (DCX; a marker of newborn and migrating neurons),

Ki-67 (a marker of cell proliferation), ionized calcium binding adaptor molecule

(Iba-1; a marker of microglia), and glial fibrillary acidic protein (GFAP; a marker

of astrocytes).

2.4. Stereotaxic surgery

The animals were deeply anesthetized with sodium thiopental (30 mg/kg,

i.p.) and chloral hydrate (150 mg/kg, i.p.) and placed in a stereotaxic frame

(David Kopf, USA). The skull was exposed, and the following stereotaxic

coordinates for the LVs, according to Paxinos and Watson [42], were

calculated: anterior/posterior, -0.8 mm from bregma; medial/lateral, ±1.5 mm

from the midline; dorsal/ventral, -3.8 mm from the skull. A hole was drilled

through the skull, and a 28-gauge stainless steel needle was manually lowered

into each LV. An electronic pump (Insight, Ribeirão Preto, SP, Brazil) was used

to control the flow of the injections at a rate of 1.0 μl/min over 4.5 min. At the

end of the injections, the needle was left in place for an additional 2 min to avoid

reflux. The lesioned group received bilateral ICV injections of STZ (3 mg/kg total

dose) dissolved in sterile saline (4.5 μl per injection site) into the LVs. Sham

surgery followed the same procedure, but the same volume of sterile saline was
 9

injected instead of STZ [35]. Streptozotocin was dissolved in cold sterile saline,

protected from light, and kept on ice until the time of the injection to prevent

degradation. After surgery, all the rats received Pentabiotic (0.1 ml,

intramuscular) to prevent infection and were allowed to recover from anesthesia

for 2-4 h in a heated and well-ventilated room. Food and water were placed

inside the cage for 10-15 days so that the animals could easily access it without

physical trauma caused by head surgery.

2.5. Open field test

The OFT was performed 21 days after surgery to evaluate spontaneous

locomotor activity of the animals. The open field apparatus was placed in a

moderately lit room (20 lux) and consisted of a circular arena (97 cm diameter,

42 cm height) which was divided into three concentric circles and subdivided

into 19 quadrants. A video camera was placed on the ceiling right above the

arena in order to record animal’s behavior for posterior analysis. The animals

were individually placed in the center of the apparatus and allowed to freely

explore it for 5 min. The following parameters were measured: the total number

of crossings from one quadrant to another, crossings in periphery, crossing in

the center, time spent in periphery, time spent in the center, and rearing

frequency (i.e., the number of times the animals stood on their hindpaws). A

crossing was considered only when the animal entered another quadrant with

its four paws. The apparatus was cleaned with a 10% ethanol-water solution

between each test to eliminate possible odors left by other rats.

2.6. Elevated plus maze

 10

The EPM test was performed to evaluate anxiety-like behavior 22 days

after surgery, according to previous studies [43,44]. The EPM apparatus was

constructed of wood and painted black. It consisted of two opposite open arms

(50 cm  10 cm) without sidewalls and two closed arms (50 cm  10 cm  30

cm) with sides and end walls extending from a central area (10 cm  10 cm).

The maze was elevated 60 cm above the floor and placed in a moderately lit

room (20 lux). A video camera was placed on the ceiling right above the arena

in order to record the animals’ behavior for posterior analysis. The animals were

individually placed in the central area of the EPM, facing an open arm. Their

behavior was recorded for 5 min and the following parameters were analyzed:

time spent on the open arms, percentage of time spent on the open arms, time

spent on the closed arms, percentage of time spent on the closed arms, number

of open arm entries, number of closed arm entries, total entries, risk

assessment (i.e., the number of times the rat left a closed arm with the

forepaws and head only and investigated the surroundings, not necessarily

accompanied by body stretching), and head dipping (i.e., the animal is in one of

the open arms with its head advanced to the edge of the platform). The EPM

was cleaned between each test with a 10% ethanol solution to eliminate odors

left by other rats.

2.7. Object Location Test (OLT) and Object Recognition Test (ORT)

The OLT and ORT were performed between day 27 and 29 following

surgery to evaluate short-term spatial and short-term recognition memory,

respectively. Both tests took place in a squared arena, 100 cm x 100 cm x 40

cm, made of wood and painted black. It was placed in a moderately lit room (20
 11

lux). A video camera was positioned over the arena, and the animals’ behavior

was recorded for later evaluation.

The first procedure consisted of habituation of the animals in the box.

Each animal was placed in the empty apparatus for 5 min for free exploration.

Twenty four hours later, a novel habituation session of 5 min was performed.

After a 1 h delay, on the training session, two identical objects were placed in

the apparatus in a symmetrical position about 10 cm away from the wall. The

animals were allowed to explore them freely for 5 min and were then returned to

their home cages. In the OLT (which was performed on day 28), after a 1 h

delay, during the test session, each rat was put back into the box with one of

the objects displaced 15 cm away from the original position (novel position);

animals were allowed to freely explore the objects for 3 min. In the ORT (which

was performed on day 29), a new training session was done with two other

identical objects placed in a symmetrical position about 10 cm away from the

wall, and the animals were allowed to explore them freely for 5 min and were

then returned to their home cages. After a 1 h interval, the rats were put back

into the arena for the test session; but now with two dissimilar objects, a familiar

one (the sample) and a new one, and the animals were allowed to freely

explore the objects for 3 min [45,46].

The objects were made of plastic, glass, or ceramic. In order to avoid an

olfactory bias, before each trial, the objects were cleaned with a 20% ethanol

solution. All objects and locations were balanced to reduce potential biases due

to preferences for particular locations or objects. A rat could not displace the

objects and the subjects were always placed into the box facing the same wall

[45]. Exploration was defined as sniffing at a distance of no more than 2 cm or

 12

touching the objects with the nose and/or forepaws. Sitting on or turning around

the objects was not considered as exploratory behavior [47]. The animals that

spent less than 5 seconds exploring the objects were excluded from the test.

The measures for both the OLT and ORT were the time spent by the rats

while exploring each object during the test session. The time spent exploring

the familiar and the new object (ORT)/displaced object (OLT) was represented

by ‘a’ and ‘b’, respectively. The following variables were calculated: e = a + b,

and d = (b - a)/e. The ‘e’ variable is a measure of the total exploration time of

both objects during the test session. ‘d’ is considered a discrimination index

between the new and the familiar objects/locations, and also a relative measure

of discrimination that corrects for the exploratory activity (e) [45].

2.8. Spatial version of Y maze

A symmetrical Y maze was constructed of wood and painted black. It

consisted of three arms (50 cm length, 27 cm height, 12 cm width) and

arranged at a 120° angle relative to each other. The spatial version of this test

was performed on day 30 after surgery. It consisted of a training session and a

test session that were performed at a 1 h interval. In the training session, one

arm was made inaccessible by a removable door that was placed in front of it.

Each animal was placed in one of the other arms (i.e., “start arm”), which was

randomized between groups. The animal was allowed to explore these two

arms for 5 min and was then returned to its home cage. After a 1 h interval, the

rat was returned to its corresponding start arm, but the blockade that prevented

access to the third arm (i.e., “novel arm”) was removed, thus providing access

to all three arms. The rat was allowed to explore the three arms for 3 min. A
 13

video camera was positioned over the Y maze to record the animals’ behavior

for later evaluation. An arm entry was considered only when both hindpaws

were placed completely inside an arm. Short-term spatial memory was

assessed as the time spent on the novel arm, which had to be significantly

greater than 33.3% of the total time in the maze (corrected for the latency to

move from the start arm to another arm and the time spent in the center of the

maze). The Y maze apparatus was cleaned between sessions with a 10%

ethanol solution to prevent an olfactory bias [46,48].

2.9. Immunohistochemistry

After the last behavioral assessment, 30 days after surgery, the animals

were deeply anesthetized with an overdose of chloral hydrate and transcardially

perfused with cold 0.01 M phosphate-buffered saline (PBS; pH 7.4) followed by

a cold fixative solution (4% paraformaldehyde in 0.1 M PBS, pH 7.4), using a

peristaltic pump (Insight, Ribeirão Preto, SP, Brazil). The brains were removed

and postfixed in the same fixative solution for 24 h at 4ºC. Cryoprotection was

made by immersion in a solution of 30% sucrose in 0.1 M PBS (pH 7.4) at 4ºC

until the block sank. After, the brains were snap-frozen in dry ice and ethanol

and stored at -80ºC until sectioning in a cryostat (Leica Biosystems, Nussloch,

Germany).

Frozen tissue was sliced into coronal semi-serial 30 m thick sections of

the dorsal hippocampus and LVs. For the dorsal hippocampus, brain slices

spaced 240 m apart were collected in 8 wells, corresponding to coronal

coordinates from bregma -2.56 to -4.52 mm [42], totaling 8-10 sections per well.

For the LVs, brain slices spaced 240 m apart were collected in 8 wells,
 14

corresponding to coronal coordinates from bregma +1.00 to -0.92 mm [42],

totaling 8-10 sections per well. The sections were stored at -20ºC in a

cryoprotectant solution that contained 30% ethylene glycol and 15% sucrose in

a 0.05 M phosphate buffer until processing. All sections of each well were used

in an immunohistochemistry reaction for a different marker.

Free-floating immunohistochemistry for DCX, Ki-67, GFAP, and Iba-1

was performed with a first step of antigen retrieval. Briefly, the brain slices were

washed with 0.1 M PBS with 0.5% Triton X-100, incubated with 0.1 M citrate

buffer (pH 6.0) for 30 min at 50ºC, washed again, incubated with 0.5% H2O2 for

30 min at room temperature and protected from light. The brain sections were

washed, and incubated with a 2% solution of bovine serum albumin (BSA) in

0.1 M PBS with 0.5% Triton X-100 for 1 h. The brain slices were incubated

overnight at 4o C with the following primary antibodies: goat polyclonal anti-DCX

(1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit polyclonal anti-

Ki-67 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit

polyclonal anti-GFAP (1:500; Dako, Carpinteria, CA, USA), and goat polyclonal

anti-Iba-1 (1:500; Abcam, Cambridge, MA, USA). The sections were then

incubated with the respective biotinylated secondary antibodies (1:200) for 2 h

at 4ºC, washed, and further incubated in ABC reagent (Vectastain Elite ABC Kit,

Vector Laboratories, Burlingame, CA, USA) for 2 h at room temperature. The

reaction was revealed by incubating the brain slices with 3,3’-diaminobenzidine

(DAB; Vector Laboratories, Burlingame, CA, USA) for 8 min in room

temperature [46].

Ki-67-stained slices of the DG were counterstained with Nissl staining

and coverslipped according to our standard protocol to facilitate visualization

 15

and counting of Ki-67-positive cells in the SGZ of the DG. The brain slices for

detection of the other markers and areas were mounted on microscope slides,

dehydrated in solutions of ascending ethanol concentrations, cleared in xylene,

and cover slipped. Ki-67-positive cells only in the SGZ, and DCX-positive cells

in both the SGZ and GCL of the DG of the dorsal hippocampus were manually

counted using a BX50 optical microscope (Olympus Optical, Center Valley, PA,

USA) at 400 x magnification. The periventricular areas (LVs, septum, and

corpus callosum), and the DG, CA1 area, and CA3 area of the dorsal

hippocampus were photographed using the same optical microscope at 200 x

magnification. The immunorreactivity of DCX, Ki-67, GFAP, and Iba-1 was

assessed as the optical density using Image J software (National Institutes of

Health, Bethesda, MD, USA). The data are expressed as the total number of

DCX- or Ki-67-positive cells in the DG of the hippocampus, and GFAP- or Iba-1-

immunorreactive (IR) cells relative to the sham group in DG, CA1 area and CA3

area of hippocampus. In the SVZ, the data are expressed as the percentage of

DCX, Ki-67-, GFAP-, and Iba-1-IR cells relative to the sham group.

2.10. Statistical analysis

The data are presented as mean ± standard error of the mean (SEM).

The data were tested for a Gaussian distribution using the Kolmogorov Smirnov

test. Group differences were analyzed using one-way analysis of variance

(ANOVA) with Tukey’s post-hoc test. Values of p < 0.05 were considered

statistically significant.

3. Results
 16

3.1. Non cognitive behavioral characterization

The OFT was performed 21 days after the STZ infusion to assess

spontaneous locomotion and exploratory behavior. The EPM was performed 22

days after the STZ infusion to assess anxiety-like behavior. Both behaviors

could interfere with the animals’ performance in the cognitive tests. The analysis

of spontaneous locomotor activity in the OFT did not reveal any changes in the

locomotion parameters analyzed in all of the groups compared to the sham

group (Table 1).

In the EPM, a reduction in the time spent in closed arms and percentage

of time spent in closed arms was observed in the STZ and STZ+cur 100 groups

compared to the sham group (Table 2), which is an indication of decreased

anxiety-like behavior in those STZ-lesioned animals. Anxiety-like behavior can

reduce the exploration of novelty in the OLT, ORT and Y maze. None of the

groups exhibited more anxiety-like behavior than the sham group in all of the

EPM parameters tested (Table 2). In addition, elevated glycemia is another

factor that could interfere with cognitive performance [49]. Therefore, blood

glucose levels were measured 30 days after surgery after the last behavioral

test, to ensure that the animals did not become diabetic. There were no

significant alterations in glycemia in all of the groups compared with the sham

group (p > 0.05; data not shown).

3.2. STZ-ICV caused cognitive impairments that were partially prevented by

curcumin prolonged treatment

 17

Short-term spatial memory was assessed in the OLT and Y maze on

days 28 and 30 after surgery, respectively, with a 1 h interval between the

training and test sessions. Short-term recognition memory was assessed in the

ORT on day 29 after surgery, with a 1 h interval between the training and test

sessions. STZ-lesioned animals presented deficits in short-term spatial

memory, reflected by a decrease in the discrimination index in the OLT (Fig.

2A), and a decrease in the time spent on the new arm in the Y maze (Fig. 2C)

when compared with the sham group. In the OLT, none of the curcumin-treated

groups showed an increased discrimination index compared with the STZ group

(Fig. 2A). In the Y maze, none of the curcumin-treated groups spent more time

in the new arm than the STZ group; nor was the time spent on the new arm

significantly higher than 33% (Fig. 2C). Therefore, the treatment of STZ-

lesioned animals with curcumin in different doses did not prevent the

impairment in short-term spatial memory. In the ORT, STZ-lesioned animals

presented deficits in short-term recognition memory, reflected by a decrease in

the discrimination index compared with the sham group (Fig. 2B). The STZ+cur

50 and STZ+cur 100 groups exhibited an increased discrimination index

compared with the STZ group, which suggests a possible beneficial effect of

curcumin in short-term recognition memory in these doses.

3.3. Curcumin effects on adult neurogenesis in STZ-lesioned animals

The STZ-injected animals exhibited decreased cell proliferation, reflected

by the reduction in Ki-67-immunoreactivity (IR) in the SVZ of the LVs (Fig. 3A,

4A), and a reduction in Ki-67-positive cells in the SGZ of the DG of the

hippocampus (Fig. 3B, 4A). However, treatment with curcumin in any dose did
 18

not restore Ki-67-IR in the SVZ (Fig. 3A, 4A) and Ki-67-positive cells in the SGZ

of the DG (Fig. 3B, 4A) when compared with the STZ group.

Similarly, the STZ-injected animals exhibited decreased newborn

neurons in both neurogenic niches, reflected by decreased DCX-IR in the SVZ

of the LVs (Fig. 3C, 4B), and decreased DCX-positive cells in the DG of the

hippocampus compared with the sham group (Fig. 3D, 4B). However, treatment

with curcumin in any dose did not restore DCX-IR in the SVZ (Fig. 3C, 4B) and

DCX-positive cells in the DG (Fig. 3D, 4B) when compared with the STZ group.

3.4. Curcumin effects on glial markers in STZ-lesioned animals

Immunoreactivity for Iba-1 and GFAP was used to evaluate the effects of

prolonged curcumin treatment on glial markers and neuroinflammation. Thirty

days after surgery, the STZ group showed a marked increase in Iba-1-IR in all

periventricular areas analyzed (i.e., LVs, septum and corpus callosum) (Fig. 5A-

C, 6A), and also in the CA1 and CA3 areas of the dorsal hippocampus, but not

in the DG, when compared with the sham group (Fig. 5D-F, 6B). Curcumin

treatment in the doses of 25 and 50 mg/kg exerted an improvement in Iba-1-IR

in corpus callosum (Fig. 5C, 6A), but not in the LVs (Fig. 5A, 6A) and septum

(Fig. 5B, 6A), despite an important but non-significant reduction in the LVs and

septum. In the hippocampus, no improvement in Iba-1-IR was observed in the

DG, CA1 and CA3 areas (Fig. 5D-F, 6B) for all of the curcumin doses tested.

Similarly, the STZ-injected animals showed a marked increase in GFAP-

IR in all periventricular areas analyzed (i.e., LVs, septum and corpus callosum)

(Fig. 7A-C, 8A) and also in the DG, CA1 and CA3 areas of the dorsal

hippocampus when compared with the sham group (Fig. 7D-F, 8B). Curcumin
 19

treatment did not improve GFAP-IR in the periventricular areas (Fig. 7A-C, 8A)

and the hippocampus (Fig. 7D-F, 8B) in all of the doses tested.

4. Discussion

The present study sought to evaluate the effects of prolonged curcumin

treatment in short-term spatial and recognition memory, neuroinflammation and

adult neurogenesis in the STZ-ICV model of sAD. We also performed non-

cognitive behavioral evaluations of the animals in the OFT and EPM to assess

spontaneous locomotor activity and anxiety-like behavior, respectively. Motor

impairments and anxiety could reduce the animals’ performance in the cognitive

tests. No differences among the groups were observed in the OFT (Table 1),

suggesting that there were no motor impairments in the STZ-injected and

curcumin-treated animals. Additionally, the STZ and STZ+cur 100 groups spent

less time in the closed arms of the EPM and exhibited a reduced percentage of

time in the closed arms (Table 2). Thus, suggesting a decrease in anxiety-like

behavior compared with the sham animals. None of the groups showed more

anxiety-like behavior compared with the sham group in all of the EPM

parameters tested. Previous studies showed increased anxiety-like behavior in

STZ-ICV female mice [17]. The discrepancies reported could be attributable to

the different species used, the animals’ age and gender and the time interval

between the STZ injections and behavioral analysis.

The STZ-lesioned animals exhibited impairments in short-term spatial

memory in the OLT and Y maze, and impairments in short-term recognition

memory in the ORT (Fig. 2). Cognitive impairment related to the hippocampus-

dependent functions, such as a decline in spatial learning and memory, has

 20

been extensively reported in animals that receive STZ-ICV [17,19,46,50–53]. In

addition, impairments in object recognition memory were demonstrated in STZ-

ICV rats [54]. These features are relevant for animal models of AD because

spatial and temporal disorientation [55] and impairments in episodic memory

(i.e., memory that integrates the ‘what’, ‘when’ and ‘where’ components of an

episode or event) are the earliest features of the progressive cognitive decline

that is observed in AD patients [56]. Oral prolonged curcumin treatment (30

days) was not able to prevent the decline in short-term spatial memory in the

OLT and Y maze (Fig. 2A, C) in any of the three doses tested (25, 50 and 100

mg/kg). However, in the ORT, both the STZ+cur 50 and STZ+cur 100 groups

exhibited an increased discrimination index compared with the STZ group (Fig.

2B), suggesting a possible positive effect of curcumin in short-term recognition

memory.

Recognition and spatial memory are encoded in similar but not totally

overlapping brain structures. Evidence suggests that the parahippocampal

regions of the temporal lobe (namely the perirhinal, entorhinal, and inferior

temporal cortices) are very important for visual object recognition memory in

rodents and primates [57]. The perirhinal cortex is the site of several sensory

inputs such as visual, olfactory and somatosensory stimulus involved in object

recognition, and it sends outputs to the hippocampus. The perirhinal cortex is

involved in object recognition after short retention intervals (short-term

memory), while the hippocampus is responsible for long-term object recognition.

The dorsal hippocampus plays an important role, especially when spatial or

contextual information is a relevant factor [58]. Hippocampal lesions produce

deficits in acquisition and retention of spatial memory [59]. The different

 21

structures encoding spatial and recognition memory may be involved with the

discrepancies in curcumin effects in these different memories.

Several studies have shown positive effects of curcumin in cognitive

processes. Curcumin has been reported to improve spatial learning and

memory in cognitive tasks such as the Morris water maze and the passive

avoidance in aged rats [33] and mice [60], in the A peptide-infused rat model

of AD [34], and in the STZ-ICV model of sAD in mice [38] and rats [35–37].

Curcumin also improved object recognition memory in aged mice [60].

However, this is the first report of an improvement in short-term object

recognition memory for curcumin in the STZ-ICV model of sAD.

The relationship between the hippocampus-dependent learning and

memory and adult neurogenesis, especially in the DG, has been extensively

investigated in recent years. Several studies reported a positive correlation

between these two processes (i.e., increased hippocampal neurogenesis

facilitates hippocampus-dependent cognitive functions, such as pattern

separation, spatial learning and memory). Conversely, decreased hippocampal

neurogenesis is associated with impairments in pattern separation, spatial

learning and memory [14,61,62]. Recent reports also indicate a role of adult-

born neurons in acquisition and consolidation of object recognition memory

[63,64]. Our results are consistent with these previous reports. Cognitive deficits

in the STZ-injected animals, reflected by impairments in short-term spatial and

recognition memory, were associated with a reduction of adult neurogenesis in

the SVZ and DG of the hippocampus in the present study (Fig. 3). Impairments

in short- and long-term spatial memory associated with decreased cell

proliferation, survival and differentiation/maturation of the newborn neurons in

 22

STZ-ICV rats was already demonstrated by our group [46]. Qu et al. [20] have

reported a reduction in the number of new neurons (double-labeled BrdU- and

Tuj1-positive cells) in the DG after ICV-STZ administration, which was

associated with severe oxidative stress. Sun et al. [21] have reported a

decrease in the generation of immature and mature neurons in the

hippocampus in rats three months after the administration of STZ, which was

correlated with the development of Apathology. Prolonged treatment with

curcumin was expected to improve the impairments in adult neurogenesis

elicited by STZ-ICV because of previous reports of curcumin effects in

neurogenesis in vitro and in vivo [26,33,39–41,65]. Nevertheless, curcumin

treatment in all of the three doses tested did not improve proliferation of

NSCs/NPCs and neurogenesis, reflected by Ki-67- and DCX-positive cells,

respectively, both in the SVZ of the LVs and the DG of the hippocampus (Fig. 3,

4).

The mild beneficial effects of curcumin on cognition and neurogenesis

could be attributed to its limited capacity to widely avoid the neuroinflammatory

process in STZ-lesioned animals in the doses and administration regimen

tested in this study. Acute and chronic neuroinflammation can negatively impact

many steps of adult neurogenesis in the mammalian brain such as proliferation,

differentiation and survival of newborn neurons. Adult neurogenesis has been

found to be altered in several neurodegenerative disorders, including AD, and

neuroinflammation is a common feature of these pathologies [66]. In the present

study, we observed a severe inflammatory response in the periventricular areas

and the hippocampus of the STZ-ICV rats, reflected by an increase in the

immunorreactivity for Iba-1 (marker of microglia – Fig. 5, 6) and GFAP (marker

 23

of astrocytes – Fig. 7, 8) 30 days after surgery. The STZ+cur 25 and STZ+cur

50 groups exhibited a partial reduction in Iba-1-IR in corpus callosum (Fig. 5C),

but not in the LVs (Fig. 5A), septum (Fig. 5B) and hippocampus (Fig. 5D-F).

Curcumin treatment in any dose did not decrease GFAP-IR in the

periventricular areas and the hippocampus (Fig. 7, 8). The increase in both glial

markers occurred along with a decrease in the proliferation marker Ki-67 and

immature neurons marker DCX in the SVZ and DG, 30 days after STZ

administration (Fig. 3, 4). This suggests that the strong inflammatory response

that was triggered by STZ is involved with the inhibition of the proliferation of

NSC/NPC and differentiation in newborn neurons in both neurogenic niches.

Additionally, the limited effects of curcumin on inhibiting glial cells activation

seem to be related to its limited effects on neurogenesis and cognition. Clinical

trials of curcumin treatment in AD patients also did not find significant

improvements on cognition and biomarkers in blood and cerebrospinal fluid

[29]. However, these results are in contrast with other studies which

demonstrated anti-inflammatory effects of curcumin by suppressing NF-B

activation in vitro [28], reducing GFAP expression in the hippocampus of A-

induced AD model in rats [67] and reducing Iba-1- and GFAP-IR in the striatum

of 6-hydroxidopamine-lesioned mice [68].

The outcome of curcumin treatment may be influenced by its poor oral

bioavailability, despite of the doses tested being within the range of doses

reported to have positive effects in previous works. Studies have shown that

curcumin is poorly absorbed from the intestine after oral administration of

different doses of 3H-curcumin in rats. It is estimated that approximately 75% of

an oral dose of curcumin is excreted in the feces. Thus, suggesting low

 24

absorption of curcumin from the intestine [69]. The discrepancies between our

results and previous reports may be due to a wide range of curcumin doses

tested in these studies (10 - 300 mg/kg), administration pathways (oral gavage,

food supplementation or intraperitoneal), different vehicles, and treatment

duration (10 days to 12 weeks). Also, the STZ dose (3 mg/kg) used in this study

is considered to be high because of the intense oxidative stress and

neuroinflammation triggered by STZ, which would be comparable to a moderate

to advanced phase of AD [18]. Treatment with curcumin in animals lesioned

with lower doses of STZ, mimicking an early stage of AD, would probably result

in better therapeutic outcomes.

In conclusion, the ICV administration of STZ elicited marked cognitive

impairments on short-term spatial and recognition memory, reduction in adult

neurogenesis and increased neuroinflammation, which were only partially

improved by curcumin. Oral prolonged curcumin treatment in the doses of 50

and 100 mg/kg preserved short-term object recognition memory, but not short-

term spatial memory. All of the curcumin doses tested exerted only slight

improvements in neuroinflammation, resulting in no restoration in hippocampal

and subventricular neurogenesis. These results suggest a positive effect of

curcumin in object recognition memory which was not related with hippocampal

neurogenesis. More investigations are needed to assess the possible beneficial

effects of curcumin treatment in AD, especially in models relating to the early

stage of this disease.

Acknowledgements
 25

We thank Conselho Nacional de Desenvolvimento Científico e

Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de

Ensino Superior (CAPES) for the financial support. RMMW Oliveira, SM Zanata

and MABF Vital are recipients of CNPq fellowships.

Disclosures

The authors have no conflict of interest to declare.

 26

References

[1] S.C. Correia, R.X. Santos, G. Perry, X. Zhu, P.I. IMoreira, M.A. Smith,

Insulin-resistant brain state: The culprit in sporadic Alzheimer’s disease?,

Ageing Res. Rev. 10 (2011) 264–273. doi:10.1016/j.arr.2011.01.001.

[2] S.C. Correia, R.X. Santos, C. Carvalho, S. Cardoso, E. Candeias, M.S.

Santos, C.R. Oliveira, P.I. Moreira, Insulin signaling, glucose metabolism

and mitochondria: Major players in Alzheimer’s disease and diabetes

interrelation, Brain Res. 1441 (2012) 64–78.

doi:10.1016/j.brainres.2011.12.063.

[3] Y.-H. Suh, F. Checler, Amyloid precursor protein, presenilins, and -

synuclein: molecular pathogenesis and pharmacological applications in

Alzheimer’s disease., Pharmacol. Rev. 54 (2002) 469–525.

doi:10.1124/pr.54.3.469.

[4] P.I. Moreira, C. Carvalho, X. Zhu, M.A. Smith, G. Perry, Mitochondrial

dysfunction is a trigger of Alzheimer’s disease pathophysiology, Biochim.

Biophys. Acta - Mol. Basis Dis. 1802 (2010) 2–10.

doi:10.1016/j.bbadis.2009.10.006.

[5] D.J. Bonda, X. Wang, G. Perry, A. Nunomura, M. Tabaton, X. Zhu, M.A.

Smith, Oxidative stress in Alzheimer disease: A possibility for prevention,

Neuropharmacology. 59 (2010) 290–294.

doi:10.1016/j.neuropharm.2010.04.005.

[6] B. Cameron, G.E. Landreth, Inflammation, microglia, and alzheimer’s

disease, Neurobiol. Dis. 37 (2010) 503–509.

doi:10.1016/j.nbd.2009.10.006.

[7] D.S. Auld, T.J. Kornecook, S. Bastianetto, R. Quirion, Alzheimer’s

 27

disease and the basal forebrain cholinergic system: relations to -amyloid

peptides, cognition, and treatment strategies, Prog. Neurobiol. 68 (2002)

209–245.

[8] K. Jin, A.L. Peel, X.O. Mao, L. Xie, B. a Cottrell, D.C. Henshall, D. a

Greenberg, Increased hippocampal neurogenesis in Alzheimer ’ s

disease, Proc. Natl. Acad. Sci. U. S. A. 101 (2003) 343–347.

doi:10.1073_pnas.2634794100.

[9] E.K. Perry, M. Johnson, A. Ekonomou, R.H. Perry, C. Ballard, J. Attems,

Neurogenic abnormalities in Alzheimer’s disease differ between stages of

neurogenesis and are partly related to cholinergic pathology, Neurobiol.

Dis. 47 (2012) 155–162. doi:10.1016/j.nbd.2012.03.033.

[10] A. Ekonomou, G.M. Savva, C. Brayne, G. Forster, P.T. Francis, M.

Johnson, E.K. Perry, J. Attems, A. Somani, S.L. Minger, C.G. Ballard,

Stage-specific changes in neurogenic and glial markers in Alzheimer’s

disease, Biol. Psychiatry. 77 (2015) 711–719.

doi:10.1016/j.biopsych.2014.05.021.

[11] M. Ejaz Ahmed, M.M. Khan, H. Javed, K. Vaibhav, A. Khan, R.

Tabassum, M. Ashafaq, F. Islam, F. Safhi, Mohammed M., Islam,

Amelioration of cognitive impairment and neurodegeneration by catechin

hydrate in rat model of streptozotocin-induced experimental dementia of

Alzheimer’s type, Neurochem. Int. 62 (2013) 492–501.

doi:10.1016/j.neuint.2013.02.006.

[12] M. Salkovic-Petrisic, A. Knezovic, S. Hoyer, P. Riederer, What have we

learned from the streptozotocin-induced animal model of sporadic

Alzheimer’s disease, about the therapeutic strategies in Alzheimer’s

 28

research, J. Neural Transm. 120 (2013) 233–252. doi:10.1007/s00702-

012-0877-9.

[13] H.G. Yeo, Y. Lee, C.Y. Jeon, K.J. Jeong, Y.B. Jin, P. Kang, S.U. Kim, J.S.

Kim, J.W. Huh, Y.H. Kim, B.W. Sim, B.S. Song, Y.H. Park, Y. Hong, S.R.

Lee, K.T. Chang, Characterization of Cerebral Damage in a Monkey

Model of Alzheimer’s Disease Induced by Intracerebroventricular Injection

of Streptozotocin, J. Alzheimer’s Dis. 46 (2015) 989–1005.

doi:10.3233/JAD-143222.

[14] N.D. Hanson, M.J. Owens, C.B. Nemeroff, Depression, Antidepressants,

and Neurogenesis: A Critical Reappraisal, Neuropsychopharmacology. 36

(2011) 2589–2602. doi:10.1038/npp.2011.220.

[15] K.C. Vadodaria, F.H. Gage, SnapShot: Adult hippocampal neurogenesis,

Cell. 156 (2014) 1114–1114.e1. doi:10.1016/j.cell.2014.02.029.

[16] N. Rajasekar, C. Nath, K. Hanif, R. Shukla, Intranasal Insulin

Administration Ameliorates Streptozotocin (ICV)-Induced Insulin Receptor

Dysfunction, Neuroinflammation, Amyloidogenesis, and Memory

Impairment in Rats, Mol. Neurobiol. (2016). doi:10.1007/s12035-016-

0169-8.

[17] Y. Chen, Z. Liang, J. Blanchard, C.L. Dai, S. Sun, M.H. Lee, I. Grundke-

Iqbal, K. Iqbal, F. Liu, C.X. Gong, A Non-transgenic Mouse Model (icv-

STZ Mouse) of Alzheimer’s Disease: Similarities to and Differences from

the Transgenic Model (3xTg-AD Mouse), Mol. Neurobiol. (2012) 1–15.

doi:10.1007/s12035-012-8375-5.

[18] A. Kraska, M.D. Santin, O. Dorieux, N. Joseph-Mathurin, E. Bourrin, F.

Petit, C. Jan, M. Chaigneau, P. Hantraye, P. Lestage, M. Dhenain, In Vivo

 29

Cross-sectional Characterization of Cerebral Alterations Induced by

Intracerebroventricular Administration of Streptozotocin, PLoS One. 7

(2012) 1–9. doi:10.1371/journal.pone.0046196.

[19] H. Javed, M.M. Khan, A. Ahmad, K. Vaibhav, M.E. Ahmad, A. Khan, M.

Ashafaq, F. Islam, M.S. Siddiqui, M.M. Safhi, F. Islam, Rutin prevents

cognitive impairments by ameliorating oxidative stress and

neuroinflammation in rat model of sporadic dementia of Alzheimer type,

Neuroscience. 210 (2012) 340–352.

doi:10.1016/j.neuroscience.2012.02.046.

[20] Z. qiang Qu, Y. Zhou, Y. shan Zeng, Y. kun Lin, Y. Li, Z. qiang Zhong,

W.Y. Chan, Protective effects of a rhodiola crenulata extract and

salidroside on hippocampal neurogenesis against streptozotocin-induced

neural injury in the rat, PLoS One. 7 (2012).

doi:10.1371/journal.pone.0029641.

[21] P. Sun, A. Knezovic, M. Parlak, J. Cuber, M. Karabeg, J. Deckert, P.

Riederer, Q. Hua, M. Salkovic-Petrisic, A. Schmitt, Long-Term Effects of

Intracerebroventricular Streptozotocin Treatment on Adult Neurogenesis

in the Rat Hippocampus, Curr. Alzheimer Res. 12 (2015) 772–784.

doi:10.2174/1567205012666150710112147.

[22] H. Hatcher, R. Planalp, J. Cho, F.M. Torti, S. V. Torti, Curcumin: From

ancient medicine to current clinical trials, Cell. Mol. Life Sci. 65 (2008)

1631–1652. doi:10.1007/s00018-008-7452-4.

[23] L.L. Hurley, L. Akinfiresoye, E. Nwulia, A. Kamiya, A.A. Kulkarni, Y.

Tizabi, Antidepressant-like effects of curcumin in WKY rat model of

depression is associated with an increase in hippocampal BDNF, Behav.

 30

Brain Res. 239 (2013) 27–30. doi:10.1016/j.bbr.2012.10.049.

[24] V. Chandra, R. Pandav, H.H. Dodge, J.M. Johnston, S.H. Belle, S.T.

DeKosky, M. Ganguli, Incidence of Alzheimer’s disease in a rural

community in India: the Indo-US study., Neurology. 57 (2001) 985–989.

doi:10.1212/WNL.57.6.985.

[25] T.P. Ng, P.C. Chiam, T. Lee, H.C. Chua, L. Lim, E.H. Kua, Curry

consumption and cognitive function in the elderly, Am. J. Epidemiol. 164

(2006) 898–906. doi:10.1093/aje/kwj267.

[26] S.K. Tiwari, S. Agarwal, B. Seth, A. Yadav, S. Nair, P. Bhatnagar, M.

Karmakar, M. Kumari, L.K.S. Chauhan, D.K. Patel, V. Srivastava, D.

Singh, S.K. Gupta, A. Tripathi, R.K. Chaturvedi, K.C. Gupta, Curcumin-

loaded nanoparticles potently induce adult neurogenesis and reverse

cognitive deficits in Alzheimer’s disease model via canonical Wnt/β-

catenin pathway, ACS Nano. 8 (2014) 76–103. doi:10.1021/nn405077y.

[27] C. Yang, X. Zhang, H. Fan, Y. Liu, Curcumin upregulates transcription

factor Nrf2 , HO-1 expression and protects rat brains against focal

ischemia, Brain Res. 1282 (2009) 133–141.

doi:10.1016/j.brainres.2009.05.009.

[28] B. Singh, S; Aggarwal, Activation of Transcription Factor NF-kB Is

Suppressed by Curcumin ( Diferulolylmethane )*, J. Biol. Chem. 270

(1995) 24995–25000.

[29] T. Hamaguchi, K. Ono, M. Yamada, Curcumin and Alzheimer’s disease,

CNS Neurosci. Ther. 16 (2010) 285–297. doi:10.1111/j.1755-

5949.2010.00147.x.

[30] Z. Huang, X. Zhong, Z. Li, C. Feng, A. Pan, Q. Mao, Neuroscience Letters

 31

Curcumin reverses corticosterone-induced depressive-like behavior and

decrease in brain BDNF levels in rats, Neurosci. Lett. 493 (2011) 145–

148. doi:10.1016/j.neulet.2011.02.030.

[31] Y. Xu, B. Ku, L. Tie, H. Yao, W. Jiang, X. Ma, X. Li, Curcumin reverses

the effects of chronic stress on behavior, the HPA axis, BDNF expression

and phosphorylation of CREB, Brain Res. 1122 (2006) 56–64.

doi:10.1016/j.brainres.2006.09.009.

[32] S.Y. Yu, M. Zhang, J. Luo, L. Zhang, Y. Shao, G. Li, Progress in Neuro-

Psychopharmacology & Biological Psychiatry Curcumin ameliorates

memory de fi cits via neuronal nitric oxide synthase in aged mice, Prog.

Neuropsychopharmacol. Biol. Psychiatry. 45 (2013) 47–53.

doi:10.1016/j.pnpbp.2013.05.001.

[33] S. Dong, Q. Zeng, E.S. Mitchell, J. Xiu, Y. Duan, C. Li, J.K. Tiwari, Y. Hu,

X. Cao, Z. Zhao, Curcumin enhances neurogenesis and cognition in aged

rats: Implications for transcriptional interactions related to growth and

synaptic plasticity, PLoS One. 7 (2012) 1–12.

doi:10.1371/journal.pone.0031211.

[34] T. Ahmed, S.A. Enam, A.H. Gilani, Curcuminoids enhance memory in an

amyloid-infused rat model of Alzheimer’s disease, Neuroscience. 169

(2010) 1296–1306. doi:10.1016/j.neuroscience.2010.05.078.

[35] T. Ishrat, M.N. Hoda, M.B. Khan, S. Yousuf, M. Ahmad, M.M. Khan, A.

Ahmad, F. Islam, Amelioration of cognitive deficits and neurodegeneration

by curcumin in rat model of sporadic dementia of Alzheimer’s type

(SDAT), Eur. Neuropsychopharmacol. 19 (2009) 636–647.

doi:10.1016/j.euroneuro.2009.02.002.
 32

[36] A.T. Isik, T. Celik, G. Ulusoy, O. Ongoru, B. Elibol, H. Doruk, E. Bozoglu,

H. Kayir, M.R. Mas, S. Akman, Curcumin ameliorates impaired insulin/IGF

signalling and memory deficit in a streptozotocin-treated rat model, Age

(Omaha). 31 (2009) 39–49. doi:10.1007/s11357-008-9078-8.

[37] R. Agrawal, B. Mishra, E. Tyagi, C. Nath, R. Shukla, Effect of curcumin on

brain insulin receptors and memory functions in STZ (ICV) induced

dementia model of rat, Pharmacol. Res. 61 (2010) 247–252.

doi:10.1016/j.phrs.2009.12.008.

[38] H. Awasthi, S. Tota, K. Hanif, C. Nath, R. Shukla, Protective effect of

curcumin against intracerebral streptozotocin induced impairment in

memory and cerebral blood fl ow, Life Sci. 86 (2010) 87–94.

doi:10.1016/j.lfs.2009.11.007.

[39] K.K. Liao, M.J. Wu, P.Y. Chen, S.W. Huang, S.J. Chiu, C.T. Ho, J.H. Yen,

Curcuminoids promote neurite outgrowth in PC12 cells through

MAPK/ERK- and PKC-dependent pathways, J. Agric. Food Chem. 60

(2012) 433–443. doi:10.1021/jf203290r.

[40] S.J. Kim, T.G. Son, H.R. Park, M. Park, M.-S. Kim, H.S. Kim, H.Y. Chung,

M.P. Mattson, J. Lee, Curcumin Stimulates Proliferation of Embryonic

Neural Progenitor Cells and Neurogenesis in the Adult Hippocampus, J.

Biol. Chem. 283 (2008) 14497–14505. doi:10.1074/jbc.M708373200.

[41] Y. Xu, B. Ku, L. Cui, X. Li, P.A. Barish, T.C. Foster, W.O. Ogle, Curcumin

reverses impaired hippocampal neurogenesis and increases serotonin

receptor 1A mRNA and brain-derived neurotrophic factor expression in

chronically stressed rats, Brain Res. 1162 (2007) 9–18.

doi:10.1016/j.brainres.2007.05.071.
 33

[42] G. Paxinos, C. Watson, The rat brain in stereotaxic coordinates, 5th

edition, Academic Press, San Diego, 1997. doi:10.1016/0143-

4179(83)90049-5.

[43] S. Pellow, P. Chopin, S.E. File, M. Briley, Validation of open: closed arm

entries in an elevated plus-maze as a measure of anxiety in the rat, J.

Neurosci. Methods. 14 (1985) 149–167. doi:10.1016/0165-

0270(85)90031-7.

[44] J.M. Gray, H.A. Vecchiarelli, M. Morena, T.T. Lee, D.J. Hermanson, A.B.

Kim, R.J. McLaughlin, K.I. Hassan, C. Kuhne, C.T. Wotjak, J.M.

Deussing, S. Patel, M.N. Hill, Corticotropin-releasing hormone drives

anandamide hydrolysis in the amygdala to promote anxiety, J. Neurosci.

35 (2015) 3879–3892. doi:10.1523/JNEUROSCI.2737-14.2015.

[45] N.M.W.J. de Bruin, J. Prickaerts, A. van Loevezijn, J. Venhorst, L. de

Groote, P. Houba, O. Reneerkens, S. Akkerman, C.G. Kruse, Two novel

5-HT6 receptor antagonists ameliorate scopolamine-induced memory

deficits in the object recognition and object location tasks in Wistar rats,

Neurobiol. Learn. Mem. 96 (2011) 392–402.

doi:10.1016/j.nlm.2011.06.015.

[46] T.B. Bassani, J.M. Bonato, M.M.F. Machado, V. Cóppola-Segovia, E.L.R.

Moura, S.M. Zanata, R.M.M.W. Oliveira, M.A.B.F. Vital, Decrease in Adult

Neurogenesis and Neuroinflammation Are Involved in Spatial Memory

Impairment in the Streptozotocin-Induced Model of Sporadic Alzheimer’s

Disease in Rats, Mol. Neurobiol. (2017). doi:10.1007/s12035-017-0645-9.

[47] P.B. Mello-Carpes, I. Izquierdo, The nucleus of the solitary tract→nucleus

paragigantocellularis→locus coeruleus→CA1 region of dorsal

 34

hippocampus pathway is important for consolidation of object recognition

memory, Neurobiol. Learn. Mem. 100 (2013) 56–63.

doi:10.1016/j.nlm.2012.12.002.

[48] A.S.R. Sierksma, J. Prickaerts, L. Chouliaras, S. Rostamian, L. Delbroek,

B.P.F. Rutten, H.W.M. Steinbusch, D.L.A. van den Hove, Behavioral and

neurobiological effects of prenatal stress exposure in male and female

APPswe/PS1dE9 mice, Neurobiol. Aging. 34 (2013) 319–337.

doi:10.1016/j.neurobiolaging.2012.05.012.

[49] E.O. Alvarez, J. Beauquis, Y. Revsin, A.M. Banzan, P. Roig, A.F. De

Nicola, F. Saravia, Cognitive dysfunction and hippocampal changes in

experimental type 1 diabetes, Behav. Brain Res. 198 (2009) 224–230.

doi:10.1016/j.bbr.2008.11.001.

[50] G. Mayer, R. Nitsch, S. Hoyer, Effects of changes in peripheral and

cerebral glucose metabolism on locomotor activity, learning and memory

in adult male rats, Brain Res. 532 (1990) 95–100. doi:10.1016/0006-

8993(90)91747-5.

[51] Lannert H; Hoyer S, Intracerebroventricular administration of

streptozotocin causes long-term diminutions in learning and memory

abilities and in cerebral energy metabolism in adult rats, Behav. Neurosci.

112 (1998) 1199–1208.

[52] R. Biasibetti, A.C. Tramontina, A.P. Costa, M.F. Dutra, A. Quincozes-

Santos, P. Nardin, C.L. Bernardi, K.M. Wartchow, P.S. Lunardi, C.A.

Gonçalves, Green tea (-)epigallocatechin-3-gallate reverses oxidative

stress and reduces acetylcholinesterase activity in a streptozotocin-

induced model of dementia, Behav. Brain Res. 236 (2013) 186–193.

 35

doi:10.1016/j.bbr.2012.08.039.

[53] S. Hosseinzadeh, M. Zahmatkesh, M.R. Zarrindast, G.R. Hassanzadeh,

M. Karimian, A. Sarrafnejad, Elevated CSF and plasma microparticles in

a rat model of streptozotocin-induced cognitive impairment, Behav. Brain

Res. 256 (2013) 503–511. doi:10.1016/j.bbr.2013.09.019.

[54] K.G. Ravelli, B. dos A. Rosário, R. Camarini, M.S. Hernandes, L.R. Britto,

Intracerebroventricular Streptozotocin as a Model of Alzheimer’s Disease:

Neurochemical and Behavioral Characterization in Mice, Neurotox. Res.

31 (2017) 327–333. doi:10.1007/s12640-016-9684-7.

[55] S. Tu, S. Wong, J.R. Hodges, M. Irish, O. Piguet, M. Hornberger, Lost in

spatial translation - A novel tool to objectively assess spatial disorientation

in Alzheimer’s disease and frontotemporal dementia, Cortex. 67 (2015)

83–94. doi:10.1016/j.cortex.2015.03.016.

[56] E. Dere, B.M. Pause, R. Pietrowsky, Emotion and episodic memory in

neuropsychiatric disorders, Behav. Brain Res. 215 (2010) 162–171.

doi:10.1016/j.bbr.2010.03.017.

[57] R.S. Hammond, L.E. Tull, R.W. Stackman, On the delay-dependent

involvement of the hippocampus in object recognition memory, Neurobiol.

Learn. Mem. 82 (2004) 26–34. doi:10.1016/j.nlm.2004.03.005.

[58] M. Antunes, G. Biala, The novel object recognition memory:

Neurobiology, test procedure, and its modifications, Cogn. Process. 13

(2012) 93–110. doi:10.1007/s10339-011-0430-z.

[59] J.M.J. Ramos, Differential contribution of hippocampus, perirhinal cortex

and postrhinal cortex to allocentric spatial memory in the radial maze,

Behav. Brain Res. 247 (2013) 59–64. doi:10.1016/j.bbr.2013.03.017.

 36

[60] S.Y. Yu, M. Zhang, J. Luo, L. Zhang, Y. Shao, G. Li, Curcumin

ameliorates memory deficits via neuronal nitric oxide synthase in aged

mice, Prog. Neuro-Psychopharmacology Biol. Psychiatry. 45 (2013) 47–

53. doi:10.1016/j.pnpbp.2013.05.001.

[61] S.Y. Yau, A. Li, K.F. So, Involvement of Adult Hippocampal Neurogenesis

in Learning and Forgetting, Neural Plast. 2015 (2015).

doi:10.1155/2015/717958.

[62] A. Sahay, K.N. Scobie, A.S. Hill, C.M. O’Carroll, M.A. Kheirbek, N.S.

Burghardt, A.A. Fenton, A. Dranovsky, R. Hen, Increasing adult

hippocampal neurogenesis is sufficient to improve pattern separation,

Nature. 472 (2011) 466–470. doi:10.1038/nature09817.

[63] I. Suárez-Pereira, S. Canals, Á.M. Carrión, Adult newborn neurons are

involved in learning acquisition and long-term memory formation: The

distinct demands on temporal neurogenesis of different cognitive tasks,

Hippocampus. 25 (2015) 51–61. doi:10.1002/hipo.22349.

[64] S. Jessberger, R.E. Clark, N.J. Broadbent, G.D. Clemenson, A. Consiglio,

D.C. Lie, L.R. Squire, F.H. Gage, Dentate gyrus-specific knockdown of

adult neurogenesis impairs spatial and object recognition memory in adult

rats, Learn. Mem. 16 (2009) 147–154. doi:10.1101/lm.1172609.

[65] S.M. Nam, J.H. Choi, D.Y. Yoo, W. Kim, H.Y. Jung, J.W. Kim, M. Yoo, S.

Lee, C.J. Kim, Y.S. Yoon, I.K. Hwang, Effects of Curcumin ( Curcuma

longa ) on Learning and Spatial Memory as Well as Cell Proliferation and

Neuroblast Differentiation in Adult and Aged Mice by Upregulating Brain-

Derived Neurotrophic Factor and CREB Signaling, J. Med. Food. 17

(2014) 641–649. doi:10.1089/jmf.2013.2965.

 37

[66] B. Wang, K. Jin, Current perspectives on the link between

neuroinflammation and neurogenesis, Metab. Brain Dis. 30 (2015) 355–

365. doi:10.1007/s11011-014-9523-6.

[67] Y. Wang, H. Yin, L. Wang, A. Shuboy, J. Lou, B. Han, X. Zhang, J. Li,

Curcumin as a Potential Treatment for Alzheimer’s Disease: A Study of

the Effects of Curcumin on Hippocampal Expression of Glial Fibrillary

Acidic Protein, Am. J. Chin. Med. 41 (2013) 59–70.

doi:10.1142/S0192415X13500055.

[68] W. Tripanichkul, E.O. Jaroensuppaperch, Ameliorating effects of

curcumin on 6-OHDA-induced dopaminergic denervation, glial response,

and SOD1 reduction in the striatum of hemiparkinsonian mice, Eur. Rev.

Med. Pharmacol. Sci. 17 (2013) 1360–1368.

[69] R.K. Maheshwari, A.K. Singh, J. Gaddipati, R.C. Srimal, Multiple

biological activities of curcumin: A short review, Life Sci. 78 (2006) 2081–

2087. doi:10.1016/j.lfs.2005.12.007.
 38

Figures’ captions

Figure 1. Experimental design. The present study was designed to assess the

effects of prolonged curcumin treatment in three different doses (25, 50 and 100

mg/kg) on short-term spatial and recognition memory, neuroinflammation and

adult neurogenesis in rats that received a single bilateral ICV injection of STZ.

IHC, immunohistochemistry; OFT, open field test; EPM, elevated plus maze;

OLT, object location test; ORT, object recognition test.

Figure 1
 39

Figure 2. Behavioral evaluation of curcumin effects in STZ-injected animals

compared with sham animals. Cognitive performance was evaluated in the

object location test (A), object recognition test (B) and spatial version of the Y

maze (C). The data are expressed as mean ± SEM. n = 6-8 per group. *p <

0.05, **p < 0.01, vs. sham group, #p < 0.05, vs. STZ group (one-way ANOVA

with Tukey’s multiple comparison test).

Figure 2

O b je c t L o c a t io n T e s t
0 .8

0 .6
D is c r im in a tio n in d e x

0 .4

0 .2

0 .0

- 0 .2

- 0 .4

- 0 .6
*
- 0 .8
0
5

0
m

0
2

5
T
a

1
S

r
h

r
S

u
c

c
+

+
Z

Z
T

T
S

O b je c t R e c o g n i t io n T e s t
0 .8 #
#
D is c r im in a tio n in d e x

0 .6

0 .4

0 .2

*
0 .0

- 0 .2
0
5

0
m

0
2

5
T
a

1
S

r
h

r
S

u
c

c
+

+
Z

Z
T

T
S

Y m aze
% t im e s p e n t in t h e n e w a r m

60

50

40

**
30

20

10

0
0
5

0
m

0
2

5
T
a

1
S

r
h

r
S

u
c

c
+

+
Z

Z
T

T
S

S
 40

Figure 3. Immunohistochemical analysis of Ki-67- and DCX-positive cells to

detect proliferating cells and immature neurons, respectively. The ICV injection

of STZ reduced proliferating NSC/NPC (Ki-67-positive cells) in the SVZ (A) and

DG (B) and reduced immature neurons (DCX-positive cells) in the SVZ (C) and

DG (D) 30 days after surgery. Curcumin treatment did not restore the Ki-67- and

DCX-positive cells both in the SVZ and DG. The data are expressed as mean ±

SEM. n = 6 per group. *p < 0.05, **p < 0.01, ***p < 0.001, vs. sham group (one-

way ANOVA with Tukey’s multiple comparison test).

Figure 3

150
K i- 6 7 - IR / S V Z ( % o f s h a m )

100

*** *** ***

***
50

0
0
5

0
Z
m

0
2

5
T
a

1
S

r
h

r
S

u
c

c
+

+
Z

Z
T

T
S

6
K i- 6 7 - p o s itiv e c e lls / D G

*
** **
2

0
0
5

0
Z
m

0
2

5
T
a

1
S

r
h

r
S

u
c

c
+

+
Z

Z
T

T
S

C
 D
D C X - p o s it iv e c e lls / D G D C X - IR / S V Z ( % o f s h a m )

0
10
20
30
40
50
0
25
50
75
100
125
150

S
h S
a h
m a
m

S
T S
Z T

*
S Z
T S
***

Z T
+ Z
c +
u c
r u

Figure 3 (continuation)
2 r
5 2

**
S 5
T S
***

Z T
+ Z
c +
u c
r u
5 r
S 0 5

*
T S 0
Z T
***

+ Z
c +
u c
r u
1 r
0 1
0 0
0
***
41
 42

Figure 4. Representative photomicrographs of Ki-67- (A) and DCX-positive cells

(B) in the DG and in the SVZ. Ki-67 in DG is counterstained with Nissl staining.

Scale bar = 200 m.

Figure 4

Figure 4 (continuation)

B
43
 44

Figure 5. Curcumin effects on microglial cells (i.e., Iba-1-immunorreactivity) in

STZ-ICV rats. The Iba-1-IR significantly increased in the periventricular areas

(i.e., lateral ventricles (LVs), septum, and corpus callosum (CC)) (A-C) and in

the CA1 and CA3 areas of the hippocampus (E, F) in STZ-ICV rats, 30 days

after surgery. The STZ+cur 25 and STZ+cur 50 groups showed a partial

decrease in Iba-1-IR in the corpus callosum. The data are expressed as mean ±

SEM. n = 6 per group. *p < 0.05, **p < 0.01, ***p < 0.001, vs. sham group, #p <

0.05, vs. STZ group (one-way ANOVA with Tukey’s multiple comparison test).

Figure 5

300
Ib a - 1 - IR / L V s ( % o f s h a m )

**

200

100

0
0
5

0
Z
m

0
2

5
T
a

1
S

r
h

r
S

u
c

c
+

+
Z

Z
T

T
S

B
Ib a - 1 - IR / s e p t u m ( % o f s h a m )

400

***
300 ***
**
*
200

100

0
0
5

0
Z
m

0
2

5
T
a

1
S

r
h

r
S

u
c

c
+

+
Z

Z
T

T
S

C
F
E
D
Ib a - 1 - IR / C A 1 ( % o f s h a m ) Ib a - 1 - IR / D G ( % o f s h a m ) Ib a - 1 - IR / C C ( % o f s h a m )

0
50
100
150
200
250
0
50
100
150
200
0
200
400
600
800

S S S
h h h
a a a
m m m

S S S
T T T
Z Z Z

*
S S S
***

T T T
Z Z Z
+ + +
c c c
u u u

Figure 5 (continuation)
r r r
2 2 2
5 5 5

*
#

**

S S S
T T T
Z Z Z
+ + +
c c c
u u u
r r r
5 5 5
S 0 S 0 S 0
#
**

T T T
Z Z Z
+ + +
c c c
u u u
r r r
1 1 1
0 0 0
0 0 0

**
***
45
 Ib a - 1 - IR / C A 3 ( % o f s h a m )

0
50
100
150
200

S
h
a
m

S
T
Z
*

S
T
Z
+
c
u
r
2
5
**

S
T
Z
+
c
u
r
5
S 0
T
Z
+
c
u
r
1
0
0
*
46
 47

Figure 6. Representative photomicrographs of Iba-1-IR cells in the

periventricular areas (i.e., lateral ventricle (LV), septum, and corpus callosum -

A) and in the DG, CA1 area, and CA3 area of the hippocampus (B). Scale bar =

200 m.

Figure 6

A
 48

Figure 6 (continuation)

B
 49

Figure 7. Curcumin effects on astrocytes (i.e., GFAP-immunorreactivity) in STZ-

ICV rats. The GFAP-IR significantly increased in the periventricular areas (i.e.,

lateral ventricles (LVs), septum, and corpus callosum (CC) - A-C) and in the

DG, CA1 and CA3 areas of the hippocampus (D-F), 30 days after the ICV

injection of STZ. Curcumin treatment did not reduce GFAP-IR in all of the

hippocampus and periventricular areas analyzed. The data are expressed as

mean ± SEM. n = 6 per group. *p < 0.05, **p < 0.01, ***p < 0.001, vs. sham

group (one-way ANOVA with Tukey’s multiple comparison test).

Figure 7

300
G F A P - IR / L V s ( % o f s h a m )

***
**
* *
200

100

0
0
5

0
Z
m

0
2

5
T
a

1
S

r
h

r
S

u
c

c
+

+
Z

Z
T

T
S

B
G F A P - IR / s e p t u m ( % o f s h a m )

500
*** ***
*** ***
400

300

200

100

0
0
5

0
Z
m

0
2

5
T
a

1
S

r
h

r
S

u
c

c
+

+
Z

Z
T

T
S

C
F
E
D
G F A P - IR / C A 1 ( % o f s h a m ) G F A P - IR / D G ( % o f s h a m ) G F A P - IR / C C ( % o f s h a m )

0
100
200
300
0
50
100
150
200
250
0
100
200
300
400

S S S
h h h
a a a
m m m

S S S
T T T
Z Z Z

*
*
S S S
***

T T T
Z Z Z
+ + +
c c c
u u u

Figure 7 (continuation)
r r r
2 2 2
5 5 5

*
*
S S S
***

T T T
Z Z Z
+ + +
c c c
u u u
r r r
5 5 5
S 0 S 0 S 0

*
*
T T T
***

Z Z Z
+ + +
c c c
u u u
r r r
1 1 1
0 0 0
0 0 0

***
***
50
 G F A P - IR / C A 3 ( % o f s h a m )

0
100
200
300

S
h
a
m

S
T
Z
S
***

T
Z
+
c
u
r
2
5
S
***

T
Z
+
c
u
r
5
S 0
**

T
Z
+
c
u
r
1
0
0
***
51
 52

Figure 8. Representative photomicrographs of GFAP-IR cells in the

periventricular areas (i.e., lateral ventricle (LV), septum, and corpus callosum –

A) and in the DG, CA1 area, and CA3 area of the hippocampus (B). Scale bar =

200 m.

Figure 8

A
 53

Figure 8 (continuation)

B
 54

Table 1. Open Field Test

STZ+cur STZ+cur STZ+cur
Sham STZ
25 50 100
Total number of
91.86 ± 100.00 ± 85.67 ± 60.57 ± 82.83 ±
crossings 8.97 16.85 13.11 15.91 12.02
Crossings in
76.43 ± 79.50 ± 74.17 ± 48.86 ± 67.00 ±
periphery 5.972 15.41 12.32 13.14 7.66
Crossings in
6.86 ± 9.25 ± 5.83 ± 5.43 ± 7.67 ±
center 2.09 2.02 0.83 1.90 1.89
Time in
272.40 ± 247.50 ± 278.50 ± 204.90 ± 248.30 ±
periphery (s) 1.70 12.45 6.04 45.62 27.15
Time in center
27.61 ± 52.51 ± 21.43 ± 95.08 ± 51.62 ±
(s) 1.70 12.45 6.04 45.62 27.15

Rearing 32.43 ± 31.25 ± 28.17 ± 22.57 ± 18.33 ±

3.05 4.80 4.18 6.66 4.06
The data are mean ± SEM. One-way ANOVA with Tukey’s multiple
comparisons test.
 55

Table 2. Elevated plus maze test

Sham STZ STZ+cur STZ+cur STZ+cur

25 50 100
Time in Open
44.87 ± 148.00 ± 82.46 ± 101.50 ± 109.80 ±
Arms (s)
15.17 27.06 12.80 21.73 40.20
Time in Closed
210.80 ± 106.80 ± 178.50 ± 131.00 ± 114.30 ±
Arms (s)
14.89 19.56** 9.90 22.38 28.38*
% of Time in
14.96 ± 49.32 ± 27.49 ± 33.82 ± 36.60 ±
Open Arms
5.06 9.02 4.27 7.25 13.40
% of Time in
70.25 ± 35.58 ± 59.49 ± 43.67 ± 38.09 ±
Closed Arms
4.97 6.52** 3.30 7.46 9.46*
Open Arm
4.57 ± 9.13 ± 7.00 ± 6.43 ± 5.00 ±
Entries
1.27 2.37 1.00 1.45 1.44
Closed Arm
11.29 ± 7.38 ± 9.50 ± 6.43 ± 9.17 ±
Entries
0.75 1.72 1.26 1.66 3.09
Total Entries 15.86 ± 16.50 ± 16.50 ± 12.86 ± 14.17 ±
1.50 2.41 1.26 2.96 4.49
Risk
10.14 ± 6.13 ± 6.33 ± 6.00 ± 6.33 ±
Assessment
1.18 1.83 1.73 1.66 2.25
Head Dipping 7.00 ± 20.88 ± 15.50 ± 14.71 ± 12.83 ±
2.38 4.89 2.45 4.03 2.64
The data are mean ± SEM. *p < 0.05, **p < 0.01 vs. sham group (One-
way ANOVA with Tukey’s multiple comparisons test)