Chapter 10

Animal Models of Early-Life Adversity

Hajar Benmhammed, Samer El Hayek, Inssaf Berkik,
Hicham Elmostafi, Rim Bousalham, Abdelhalem Mesfioui,
Ali Ouichou, and Aboubaker El Hessni

Abstract
From the prenatal period throughout the first years of life, the brain undergoes its most rapid development,
a period during which it is highly sensitive to external experiences. The timing of brain development differs
from one region to another, as it also differs between substrates, neurotransmitter systems, and central
endocrine circuitries. These discontinuities are part of the “critical periods of brain development.” Early-life
adversity (ELA), such as exposure to infection, maternal deprivation, and substance use, disrupts the
programmed brain development, yielding a myriad of deviations in brain circuitry, stress responsivity,
cognitive function, and general health. This is applicable to both humans and animal models.
In our laboratory, several experimental animal designs have been developed that allow investigating the
long-lasting consequences of ELA on brain function, cognitive and emotional development, and the risk to
develop stress-related psychopathology later in adulthood. This book chapter will provide a review of such
animal models, in particular, designs related to infections (LPS-induced), the quality of mother-infant
relationship (maternal deprivation and separation), and substance use (ethanol intoxication). The behavior
tests, biochemical, and immunohistochemistry assays applied after ELA will be explained. The behavioral
tests encompass the open-field, elevated plus maze, forced swim, sucrose preference, Y-maze, object
recognition, and Morris water maze tests. These experiments allow the assessment of several outcomes of
interest, pertaining to locomotor activity, anxiety-like symptoms, depressive-like symptoms, working
memory, recognition memory, spatial memory, and learning performance. The biochemical assays are
employed to measure the level of oxidative stress and inflammation in brain areas after application of
adversity. Immunohistochemistry puts into perspective the degree of immunoreactivity in the brain sub-
jected to adversity. The findings from our laboratory indicate that the nature and timing of exposure play a
critical role in sensitivity to develop neurodevelopmental disorders.

Key words Early-life adversity, Biochemical assays, Behavioral tests, Immunohistochemistry, Neuro-
developmental disorders

1 Introduction

From the prenatal period throughout the first years of life, the brain
undergoes its most rapid development, a period during which it is
highly sensitive to external experiences. Brain development starts at

Firas H. Kobeissy (ed.), Psychiatric Disorders: Methods and Protocols, Methods in Molecular Biology, vol. 2011,
https://doi.org/10.1007/978-1-4939-9554-7_10, © Springer Science+Business Media, LLC, part of Springer Nature 2019

143
144 Hajar Benmhammed et al.

gestation, peaks after birth through early postnatal stages, and

continues into adolescence until adult brain maturation is reached.
This development is not uniform, rather a discontinuous process
characterized by regional asynchrony [1]. The timing of brain
development differs from one region to another, as it also differs
between substrates, neurotransmitter systems, and central endo-
crine circuitries. These discontinuities are part of the so-called
critical periods of brain development, stages of increased vulnera-
bility to insults specific for each brain region or neurotransmitter
systems. Early-life adversity (ELA), such as exposure to infection,
maltreatment, maternal deprivation, family violence, parental insta-
bility, and substance use disrupts the programmed brain develop-
ment, yielding a myriad of deviations in brain circuitry, stress
responsivity, cognitive function, and general health [2–4].
In humans, exposure to early childhood stress and emotional
maltreatment has been associated with a higher prevalence of
depression, anxiety, substance use, eating disorders, suicidal symp-
tomatology, psychosis, and personality disorders in adulthood
[5–8], as well as diminished cognitive functioning [9, 10] and
poorer treatment response [8, 11]. At a neurological level, ELA
enhances the activation of the hypothalamus-pituitary-adrenal
(HPA)-axis and the neuronal sensitivity to stress hormones in a
long-lasting manner; it also has been correlated with smaller
volumes of the prefrontal cortex (PFC) [12], more specifically the
dorsal medial prefrontal cortex [13], and the hippocampus (HPC)
[12]. ELA has also been associated with later alterations in the
reward processing system of humans [14], the reactivity of the
nucleus accumbens [15], and other basal ganglia regions [16]
with a characteristic reduction in the response to emotions or
rewards. These effects on reward circuitries are likely secondary to
altered trajectories of connectivity within cortical regions, such as
the premature maturation of cortical amygdala functional
connectivity [17].
Animal models of ELA have provided further insight into the
role of external stimuli in reshaping the neurocircuitry of the brain
and subsequently contributing to the pathophysiology of neurop-
sychiatric diseases. Such experimental models have revealed that
stressful experiences can have functionally relevant effects on the
dendritic arbor, spine, and synapse number in many brain regions,
including the PFC, HPC, striatum, and amygdala. In particular, it
can induce the activity of immune and neuroendocrine systems
[18], reportedly causing neuronal damage [19]. In addition, ELA
in rodents has been linked to altered neurotransmission [20],
synaptogenesis, and neurogenesis [21, 22], with tremendous
effects on cognition and emotional functions [23]. These effects
can be mediated by various mechanisms, such as excitotoxicity
[24], inflammation [25], impairment of synaptic pruning, and
oxidative stress [26–28].
 Animal Models of Early-Life Adversity 145

In our laboratory, several experimental animal models have

been developed that allow investigating the long-lasting conse-
quences of ELA on brain function, cognitive and emotional devel-
opment, and the risk to develop stress-related psychopathology
later in adulthood. This book chapter will provide a review of
such animal models, in particular, designs related to infections
(LPS-induced), the quality of the mother-infant relationship
(maternal deprivation and separation), and substance use (ethanol
intoxication). The behavior tests applied after ELA will be
explained; they encompass the open-field test, elevated plus maze
test, forced swim test, sucrose preference test, Y-maze test, object
recognition test, and Morris water maze. These experiments allow
assessment of several outcomes of interest, pertaining to locomotor
activity, anxiety-like symptoms, depressive-like symptoms, working
memory, recognition memory, spatial memory, and learning per-
formance. A summary of the biochemical assays used after ELA will
be also provided. These assays are employed to measure the level of
oxidative stress and inflammation in brain areas after application of
adversity. We also applied immunohistochemistry to put into per-
spective the degree of immunoreactivity in the brain subjected to
adversity. The findings from our laboratory indicate that nature and
timing of exposure play a critical role in sensitivity to develop
neurodevelopmental disorders.

2 Materials

2.1 Models of Early- 1. Pregnant Wistar rats (provided by the laboratory of Genetic,
Life Adversity Neuroendocrinology, and Biotechnology Laboratory located
at Ibn Tofail University, Kenitra, Morocco).
2. Standard pellet diet (provided by ALF SAHEL Society, El
Jadida, Morocco): a balanced diet containing protein
(20.1%), fat (4.1%), carbohydrates (60.0%), fibers (5.8%),
minerals (8%), and vitamins (2.0%).
3. Plexiglass cages (430
290
210 mm).
4. Intraperitoneal (IP) injection of phosphate buffer saline (PBS)
(1 mg/kg).
5. IP injection of LPS (250 μg/kg, Escherichia coli, serotype 026:
B6, L-3755, Sigma, St. Louis MO).
6. IP injection of ethanol 3 g/kg, concentration of 98% (v/v)
(SIGMA): diluted in 0.9% saline, final concentration of 20%
(v/v).
7. IP injection of 0.9% saline solution.
8. Separation pots, which bottom is covered with a paper towel.
146 Hajar Benmhammed et al.

9. Intragastric oral gavage (1 mL of oral gavage/100 g of body

weight/day): extracted from fresh seeds by artisanal methods
without any preliminary treatment.
10. Water.

2.2 Behavioral Tests 1. Open-field test apparatus (Fig. 1): wooden-made apparatus
(100
100 cm), enclosed with walls of 40 cm in height, placed
under strong illumination (100 W, 2 m above the
apparatus) [29].
2. Elevated plus maze test apparatus (Fig. 2): wooden-made plus-
shaped apparatus, 70 cm elevated above the floor. The oppos-
ing arms (50
10 cm) are enclosed with walls of 40 cm in
height. At the intersection, the four ends of the two arms have a
central platform (10
10 cm), over which a 100 W lamp is
placed.
3. Forced swim test apparatus: cylinder (height ¼ 50 cm; diame-
ter ¼ 30 cm) containing 27 cm of water (22  C).
4. Sucrose preference test apparatus: two-bottle choice apparatus,
one containing sucrose solution in drinking water whereas the
other containing only water.
5. Y-maze test apparatus (Fig. 3): three-arm Y-shaped wooden-
made apparatus.
6. Object recognition test apparatus (Fig. 4): open-field box
(100
100 cm), containing familiar and novel objects.
7. Morris water maze apparatus (Fig. 5): white circular tank
(1.5 m in diameter), equipped with a retractable platform
(20 cm in diameter), and filled with opaque water (21  1  C).
8. Seventy percent ethyl alcohol.

Fig. 1 Open-field test apparatus. The wooden-made apparatus (100
100 cm)
is enclosed within walls of 40 cm in height
 Animal Models of Early-Life Adversity 147

Fig. 2 Elevated plus maze test apparatus. The wooden-made apparatus is plus-
shaped. Two opposing arms (50
10 cm) are 70 cm elevated above the floor
and enclosed within walls of 40 cm in height. At the intersection, the four ends of
the two arms have a central platform (10
10 cm)

Fig. 3 Y-maze test apparatus. The wooden-made apparatus is a three-arm

Y-shaped apparatus

2.3 Biochemical 1. Chloral hydrate (100 mg/kg).

Assays 2. Saline solution (0.9% NaCl).
3. Fixative solution (4% paraformaldehyde in 0.1 M phosphate
buffer, pH 7.4).
148 Hajar Benmhammed et al.

Fig. 4 Object recognition test apparatus. The wooden-made apparatus is an open-field box (100
100 cm).
(a) The apparatus contains two familiar objects. (b) The apparatus contains one familiar and one new object

Fig. 5 Morris water maze apparatus. The white circular tank (1.5 m in diameter)
is equipped with a retractable platform (20 cm in diameter) and filled with
opaque water

4. PBS (1 mg/kg).
5. Tris–HCl lysis buffer (RIPA lysis buffer + 1 mM PMSF).
6. Dounce homogenizer.
7. TNF-α ELISA kit (Invitrogen KRC3011).
8. Griess reagent (0.1% N-(1-naphthyl)ethylenediamine dihy-
drochloride; 1% sulfanilamide in 5% phosphoric acid; 1:1).
9. Lipid peroxidation mixture: 10% trichloroacetic acid, 0.67%
thiobarbituric acid, and butanol (2:1, v/v).
10. Catalase activity measurement kit: 1.95 mL of 50 mM phos-
phate buffer and 1 mL of H2O2 (0.019 M).
11. Riboflavin/methionine/nitroblue tetrazolium (NBT)
mixture.
 Animal Models of Early-Life Adversity 149

2.4 Immuno- 1. Brain coronal sections from Wister pups (50 μm thickness):
histochemistry obtained using a vibratome (VT 1000 S, Leica Microsystems).
2. Immunohistochemistry buffer solution: 0.1 M phosphate
buffer (pH 7.4) containing 0.3% bovine serum albumin and
0.3% triton X-100. The endogenous peroxidase activity was
quenched for 10 min at room temperature in a solution of 3%
hydrogen peroxide in 30% methanol.
3. Monoclonal antibody for ionized calcium-binding adapter
molecule 1 (Iba1) (diluted 1:2000): a marker of microglia.
4. Monoclonal antibody for glial fibril acid protein (GFAP)
(diluted 1:1000; Clone GA5, Sigma-Aldrich): a marker of
astrocytes.
5. Biotinylated goat anti-mouse immunoglobulin G (diluted
1:300; Pierce).
6. Avidin-biotin-peroxidase complex (diluted 1:250; Immuno-
Pure ABC peroxidase staining kit).
7. Final mix buffer: 2 mg/mL 3,3-diaminobenzidine, 0.01%
hydrogen peroxide, and 0.1 M phosphate buffer.
8. Leica DMRB-E microscope.
9. Digital length gauge device (Heidenhain-Metro MT
12/ND221): attached to the stage of the Leica microscope,
used to measure section thickness.
10. Adrenal tissue solutions: 10% buffered formalin, ethanol solu-
tion, paraffin, and hematoxylin-phloxine-saffron (HPS).

3 Methods

3.1 Models of Early- See Notes 1–4 for applicable rules.

Life Adversity
1. Pregnant Wistar rats are maintained at a constant temperature
3.1.1 Animal Models (24  C) with a relative humidity of 50–60%.
2. A 12-h dark-light cycle is applied with lights on between
7:00 pm and 7:00 am.
3. The standard pellet diet and water are available ad libitum.
4. The rats are checked for litters daily at 9:30 am. If litters are
present, the day of birth is defined as post-natal day (PND)
0. On the day after parturition, each litter is culled to ten
healthy pups with a 1:1 male to female ratio.
5. The pups are weaned at PND21; males and females are then
separated.
6. Animals are housed in groups of four per plexiglass cages until
90–97 days of age when the behavioral tests and biochemical
assays are carried out.
150 Hajar Benmhammed et al.

3.1.2 LPS-Induced The immune system and its cytokines can strongly shape the devel-
Infection (LII) opment and activity of the central nervous system. Systemic inflam-
mation induced by neonatal infection may result in long-term
hyperactivation of microglial cells and consequently an increase in
pro-inflammatory cytokines (i.e., tumor necrosis factor TNF-α,
interleukin 1 (IL-1), and IL-6), free radicals (i.e., neuronal cyto-
toxic nitric oxide (NO)), and lipid peroxidation [30]. These inflam-
matory mediators can affect neuronal migration, neurogenesis,
synaptogenesis, and synaptic pruning, contributing to the patho-
physiology of neuropsychiatric affective and cognitive disorders
[31–33]. In animal models of LPS-induced infection, our labora-
tory uses four separate groups:
1. A control group with 7-day-old pups who receive an IP injec-
tion of PBS.
2. A LII group with 7-day-old pups who receive an IP injection
of LPS.
3. A LII group with 9-day-old pups who receive an IP injection
of LPS.
4. A LII group with 14-day-old pups who receive an IP injection
of LPS.

3.1.3 Maternal Defined as the lack of maternal care during the first 24 postnatal
Deprivation (MD) hours, MD seems to play a pivotal role. This stems from a combi-
nation of three different factors: the decrease in tactile stimulation
and licking-grooming frequency, the lack of nursing behaviors and
nutrients, and the decrease in body temperature or hypothermia
due to the absence of mature thermal regulatory system in the
neonates [34]. Taken together, each stressor acts as a crucial factor
for this animal model of early-life stress.
In our laboratory, maternal deprivation is applied to animal
models with and without LII. This combination of two stressors
is novel as it allows the assessment of cumulative stress on neuropa-
thology. Our laboratory uses four separate groups:
1. A control group with pups given an IP injection of PBS
at PND1.
2. A LII group with pups given an IP injection of LPS at PND1.
3. An isolation group with pups exposed to a prolonged period
(24 h) of MD on PND9.
4. An isolation plus LII group with pups given an IP injection of
LPS on PND1 then exposed to a prolonged period (24 h) of
MD on PND9.
 Animal Models of Early-Life Adversity 151

3.1.4 Maternal Another design pertaining to the quality of the mother-infant

Separation relationship is maternal separation (MS). This design follows a
two-step approach.
1. At the primary level, first-generation offspring of female rats,
3 months of age, are divided into two groups. The first group
includes male and female pups reared in normal condition, i.e.,
they have been hatched by mothers until weaning. The second
group is exposed to MS, i.e., pups have been separated from
their mothers (removed from the dam cage and disposed of in a
separation pot) for 3 h on a daily basis from PND1 to PND22.
2. At the secondary level, the same mothers, at 4 months of age,
mate again. All rats of this second-generation offspring are
reared in normal condition. The litters are then divided into
two groups: male and female descendants of control mothers
(i.e., mothers not exposed to MS at the primary level) and male
and female descendants of mothers previously submitted to MS
(i.e., mothers separated from their first offspring at the primary
level).

3.1.5 Substance Use The ethanol intoxication procedure is conducted as previously

(Ethanol Intoxication described [35–37]. The oral gavage has a similar composition to
and Oral Gavage that used in human food, originating in southwestern Morocco
Procedures) (Agadir-Inezgane) [38]. In the substance use animal model, our
laboratory uses four separate groups (see Notes 5–7):
1. A control group: Adolescent rats (PND30 to PND43) are
treated with IP injections of PBS over 2 consecutive days, at
48-h intervals (total of eight PBS injections).
2. A group supplemented with ethanol: Adolescent rats (PND30
to PND43) are treated with IP injections of 3 g/kg ethanol
over 2 consecutive days, at 48-h intervals (total of eight ethanol
injections).
3. A group receiving oral gavage procedure without ethanol
supplementation.
4. A group receiving oral gavage procedure after ethanol
supplementation.

3.2 Behavioral Tests The behavioral tests are grouped into three categories, according to
their outcome of interest: measurement of anxiety, depression, or
cognition (see Notes 8–11 for applicable rules).
152 Hajar Benmhammed et al.

3.2.1 Measurement The central area of a novel environment is anxiogenic and aversive.
of Anxiety Therefore, behavioral inhibition appears as an avoidance of the
central zone [39].
Open-Field Test (OFT)
(Fig. 1) 1. The area of the OFT is divided into 25 squares (20
20 cm),
defined as 9 central and 16 peripheral squares.
2. The quantified parameters are the time spent in the center of
the area and the number of returns to the central squares.
3. Central perimeter residence time is used as a measure of
anxiety [29].
4. The number of returns to the central area is an indicator of
emotional reactivity [40].

Elevated Plus Maze Test The EPM test is an ethological model of anxiety in rodents, pro-
(EPM) (Fig. 2) voked by novelty and repulsion as a result of elevation and illumi-
nation of the maze [41]. This test is based on the creation of a
conflict between the exploratory drive of the rat and its innate fear
of open and exposed areas. Thus, increased open-arm exploration
indicates reduced anxiety-related behavior.
1. Rats are placed in the central area of the maze facing an
open arm.
2. In the 5-min test, the following parameters of anxiety-related
behavior are measured: (1) entries into the open arms, (2) time
spent in the open arms, and (3) number of full entries into
the arms.
3. A decrease in anxiety-like behaviors is illustrated by a statisti-
cally significant increase of parameters in the open arms (time
and/or entries).

3.2.2 Measurement See Notes 12 and 13 for applicable rules.

of Depression
1. As previously described [42], rats are individually placed in the
Forced Swim Test cylinder containing water for a duration of 5 min.
2. The duration of the animal immobility is measured.
3. The latency to the first bout of immobility is also recorded,
starting after placing the rats in the cylinders.
4. A rat is judged immobile when it ceases all active behaviors (i.e.,
struggling, swimming, and jumping) and remains passively
floating or making minimal movements necessary to maintain
its nostrils above water.
5. A longer time of floating is interpreted as an increase in
depressive-like response.
 Animal Models of Early-Life Adversity 153

Sucrose Preference Test Sucrose preference is attenuated by a diversity of chronic stressors,

(SPT) including chronic mild stress, unpredictable stress [43–45], and
social defeat stress [46].
1. The SPT is a useful test to investigate anhedonia (i.e., inability
to feel pleasure), particularly in stress-based models of
depression.
2. The two-bottle choice procedure allows assessment of
behavioral preference for sucrose solution rather than water
(see Note 14).
3. Preference is measured by volume and/or weight of liquid
consumed on a daily basis.
4. The score is then converted to a percent preference compared
to a water-only baseline period.

3.2.3 Measurement Spatial working memory is assessed using the Y-maze spontaneous
of Cognition alternation test [47].
Y-Maze Test (Fig. 3) 1. Each mouse is placed in the center of the Y-maze and allowed
to explore the arena for 8 min.
2. The number of entries into the maze is counted: an entry
requires that both hind paws of the animal are completely
placed inside the arm.
3. A rat would make a triad when it visits the three arms
consecutively.
4. As a measure of working memory, the percentage of alterna-
tions that a rat makes is calculated (see Note 15). If a rat scores
significantly above 50% alternations (the chance level of choos-
ing the unfamiliar arm), this is indicative of functional working
memory.

Object Recognition Test The ORT is useful for studying rodent recognition memory as it
(ORT) (Fig. 4) uses its natural preference for a new object in relation to a familiar
one. The ORT is performed in our laboratory according to the
method previously described [48] but with certain modifications.
1. In our design, the experiment is carried in a black open-field
box and is divided into three sessions: familiarization, training,
and test sessions.
2. On the test day, the animal is placed in the box and is free to
explore the space with no objects present (familiarization ses-
sion). The considered habituation time is 5 min (the time varies
between 3 and 10 min).
3. Two objects are introduced in two corners (Fig. 4a), approxi-
mately 30 cm apart from each other. During the subsequent
5-min period, the time spent exploring each object (training
session) is measured. Then, rats return to their home cage.
154 Hajar Benmhammed et al.

4. After a waiting period of 120 min, the rats are again placed in
the test box.
5. After another 5 min, they are reintroduced to one of the
familiar objects used in the previous training session and to a
novel object (Fig. 4b). During the subsequent 5-min period,
the time spent exploring each object (test session) is measured.
6. Rats are considered to be exploring when they were facing,
sniffing, or biting the object.
7. The same test is repeated again after 24 h.
8. A recognition index (RI) is used to measure memory prefer-
ence (see Note 16).

Morris Water Maze (MWM) The MWM test evaluates spatial learning and memory: rats are
(Fig. 5) trained to escape from water by swimming to a hidden platform
whose location can be identified using only distal extra-maze cues
[49–51].
1. The pool is virtually subdivided into four equal quadrants: the
target quadrant, adjacent right and left quadrants, and opposite
quadrant. The procedure involves a learning phase in which rats
receive five blocks of training trials over 5 consecutive days.
2. Each rat is placed in the pool at one of four randomized start
positions (N, O, S, and E) and is then allowed to locate the
hidden platform.
3. Trials last for 120 s and are separated by 15–20 min interval.
4. In each trial, the latency to reach the platform is measured.
5. Spatial learning performance is assessed in a probe trial done on
the fifth day, 1 h after training, during which the target plat-
form is removed from the pool. The performance is measured
via preference to the target quadrant, as well as by the number
of times the rat crosses the theoretical platform location (see
Notes 13 and 17).
6. The longer the rat spends time in the target quadrant and cuts
off the theoretical location of the platform, the more it exhibits
good memory of the spatial location of the platform.

3.3 Biochemical 1. After the completion of the behavioral analysis, animals are
Assays deeply anesthetized with chloral hydrate.
2. They are perfused through the left cardiac ventricle, first with
50 mL of saline solution and then with 250 mL of fixative
solution.
3. Animals are then sacrificed via cervical dislocation.
4. Brains are immediately removed, immersed overnight at 4  C
in the same fixative solution, and then rinsed with PBS.
 Animal Models of Early-Life Adversity 155

5. The PFC and HPC of each hemisphere are dissected out and
separated from the rest of the encephalon and then homoge-
nized in ice-cold 20 mM Tris–HCl lysis buffer using a Dounce
homogenizer.
6. The homogenates are used for determination of TNF-α level,
nitrite content, lipid peroxidation degree, and catalase and
superoxide dismutase activities.

3.3.1 TNF-α Assay 1. The homogenates are centrifuged (15 min at 14,000
g) and
then stored at 80  C.
2. The content of the samples is assayed using TNF-α ELISA kit
assay 3 months after LPS injection.
3. TNF-α content is expressed as picogram of cytokine per millili-
ter of homogenate.

3.3.2 Nitrite Oxide Assay 1. An aliquot of crude homogenate (10%) from the PFC and
HPC is centrifuged at 4  C (10 min at 800
g).
2. The supernatant is used to analyze nitrite levels, as previously
described [52].
3. Briefly, samples are incubated at room temperature for 20 min
with Griess reagent.
4. The absorbance is measured at 550 nm and compared to that of
standard solutions of sodium nitrite.

3.3.3 Lipid Peroxidation 1. Lipid peroxidation is evaluated by measuring the thiobarbituric

Assay acid reacting substances (TBARS) in homogenates, as previ-
ously described [53].
2. Briefly, an aliquot of crude homogenate from the PFC and
HPC is centrifuged at 4  C (10 min at 1000
g).
3. The supernatant is mixed with 1 mL 10% trichloroacetic acid
and 1 mL 0.67% thiobarbituric acid. The mixture is then
heated in a boiling water bath for 15 min; butanol (2:1, v/v)
is subsequently added to the solution.
4. After another centrifugation (5 min at 800
g), TBARS
absorbance is measured at 535 nm. The results are expressed
as nmol of malondialdehyde per gram of wet tissue.

3.3.4 Determination 1. Catalase activity is measured by the technique that converts

of Catalase and Superoxide H2O2 to H2O and O2 [54].
Dismutase Activities 2. The catalase measurement kit and 0.05 mL of the homogenate
are mixed together [55].
3. The absorbance is measured at 240 nm at time 0 (T0) and after
every 30 s for a duration of 2 min.
4. Results are expressed as mmol/min/mg of protein.
156 Hajar Benmhammed et al.

5. The superoxide dismutase activity is evaluated by measuring its

ability to inhibit the photoreduction of NBT [56].
6. In aerobic media, the riboflavin/methionine/NBT mixture
gives a blue coloration, the optical density of which is measured
at 580 nm.
7. One unit of superoxide dismutase corresponds to the amount
of protein necessary to inhibit photoreduction by 50%.

3.4 Immuno- 1. Immunohistochemistry of brain coronal sections is carried out

histochemistry on free-floating sections under moderate shaking.
2. After several washes in immunohistochemistry buffer solution,
sections are incubated overnight with monoclonal antibodies
for either Iba1 or GFAP at 4  C.
3. Sections are then rinsed in buffer and incubated for 2 h at room
temperature with anti-mouse immunoglobulin G.
4. After several washes in buffer, sections are again incubated for
90 min at room temperature with the avidin-biotin-peroxidase
complex.
5. The reaction product is revealed by incubating the sections
with the final mix buffer.
6. Finally, the sections are dehydrated, mounted on gelatinized
slides, cover-slipped, and examined under a microscope.

3.4.1 Glial Cell Count 1. The number of immunoreactive cells is estimated by the optical
dissector method [57] using total section thickness for dissec-
tor height and a counting frame of 55-3-55 lm.
2. A total of 78 counting frames is assessed per animal and per
each zone analyzed.
3. Cell nuclei from immunoreactive cells that came into focus
while focusing down through the dissector height are counted.
4. As an alternative method to evaluate glial reactivity, the surface
density of Iba1 and GFAP immunoreactive cell bodies and cell
processes is assessed in three zones of the HPC.
5. The ratio of the surface of immunoreactive profiles to the
reference volume (surface density SY) is calculated via the
formula SY ¼ 2I/L (see Notes 18 and 19).
6. All immunoreactive profiles, whether heavily or less heavily
labeled, are considered for quantification. For each animal, a
minimum of two sections is evaluated.

3.4.2 Adrenal Histology 1. Adrenal tissues are initially fixed in 10% buffered formalin and
then gradually dried in ethanol solution.
2. Tissue specimen is embedded in paraffin, and then 5 μm-thick
serial histologic sections are obtained from the paraffin blocks.
 Animal Models of Early-Life Adversity 157

3. Three slices of each block are carried out and stained by HPS.
4. To highlight any differences in the size of morphological elements,
slices are observed at several magnifications (5, 10, and 20).

4 Conclusion

As reviewed via experimental animal models of life adversity, we can

acknowledge that early stress occurs at a time when the brain is
learning how to adapt to its lifelong environment. Once an envi-
ronmental exposure occurs, particularly a stressful one, this modi-
fies the architecture of the neural circuit in such a way that certain
patterns of future activity are preferred [58]. As these modifications
develop, the central nervous system encounters developmental
time points that require particular functionality. Neuropsychiatric
disorders may arise if the trajectory of the brain has left it unpre-
pared for transient changes during early and throughout late
adolescence.
Using animal designs of ELA, the results from our laboratory
put into perspective that exposure to early-life stressors such as
LPS-induced infection, maternal deprivation, maternal separation,
or ethanol intoxication can evoke a cascade of neuroimmune, neu-
rohumoral, and neurotransmitter events. This cascade may alter
neuronal migration and differentiation, axons outgrowth, synapto-
genesis, synaptic pruning, and myelination. All these development
disturbances can produce enduring deleterious alterations in brain
structure and function, which subsequently triggers anxiety-like or
depression-like symptoms and cognitive decline. In humans, such
disturbances in concert with genetic predisposition could result in
neuropsychiatric disorders, including schizophrenia, autism spec-
trum disease, bipolar disorder, and others.

5 Notes

1. The researcher should wear a lab coat and gloves throughout all
experiments and procedures. Perfume is not allowed as it might
interfere with olfactory cues.
2. To properly conduct the experiments, the researcher should
avoid talking or producing any noise.
3. All injections, when applicable, should be done at 10:00 am.
4. Control animals should undergo the same handling and stress
procedures as other groups.
5. In the substance use animal model, daily intragastric gavage is
done between 8:00 am and 10:00 am starting 1 day after
weaning (PND22) [59–61] for either 5 (PND63) or
158 Hajar Benmhammed et al.

20 weeks (PND163). During the same period, the control

group should receive an equivalent amount of water.
6. In the substance use animal model, body weights are measured
on a weekly basis.
7. In the substance use animal model, the behavioral assessment is
characteristically done at two different time points depending
on the length of the oral gavage procedure: at PND63 and
PND163.
8. All behavioral tests, when applicable, should be done from
9:00 am till 12:00 pm.
9. If the animal is incapable of undergoing a specific behavioral
test, it should be removed from the experimental setting.
10. In all behavioral tests, the animal should be monitored through
a camera videotape to avoid disrupting its focus and to limit
stress.
11. In the OFT, EPM test, Y-maze test, and ORT, the apparatus
should be regularly cleaned in between sessions. The device is
cleaned using 70% ethyl alcohol to disinfect the area and
remove the smell of the previous animal model.
12. For the forced swim test, the rim of the cylinder should be high
enough to prevent the animal from escaping.
13. For behavioral tests using water (forced swim test and MWM
test), the pool should be regularly cleaned, and water changed
throughout the period of testing.
14. For the SPT, animals should be initially trained to consume a
sucrose solution. The basic sucrose intake should be tested
3 days prior to the initiation of the test.
15. For the Y-maze test, the percentage of alterations if calculated
as is the number of triads divided by the total number of
possible alternations, i.e., [total number of entries  2]
100.
16. RI ¼ [TN/(TN + TF)]
100 with TN ¼ time spent on the
novel object and TF ¼ time spent on the familiar object.
17. For the MWM test, the researcher has to decrease the intensity
of stress by providing appropriate water temperature, physical
handling, and swimming duration for the animals.
18. In the formula of SY ¼ 2I/L, “I” is the number of points at
which the immunoreactive profiles (vimentin or GFAP immu-
noreactive astroglial cell bodies and processes) cross the test
grid lane, whereas “L” is the test line length in the tissue [62].
19. The test grid used is based on the C16 grid of Weibel [62] and
has 10-3-10 lines of a total length of 2000 lm (L ¼ 2 mm).
Magnification was calculated with a calibrated slide (100 lines/
mm).

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