Research Proposal:

N-ACETYL CYSTEINE (NAC) AS THERAPEUTIC INTERVENTION IN A

PRENATAL INFLAMMATION MODEL ON COGNITIVE MEMORY DEFICITS AND
CORTICO-STRIATAL OXIDATIVE STRESS.

By
Chanel van der Westhuizen (24295698),
Kylie le Roux (24101435) and
Sulette Kotze (24325880).

A dissertation submitted in partial fulfilment of the requirements for the degree of

Baccalaureus Pharmaciae

In the
School of Pharmacy
Faculty of Health Sciences
North–West University (Potchefstroom Campus)

Proposed study leader: Dr. Marisa Möller-Wolmarans

 1. INTRODUCTION:

Antenatal maternal exposure to various disturbances can directly alter the physiological
environment of the foetus, consequently associated with deviations regarding the normal
course of brain development during pregnancy (Rees & Harding, 2004).

This may have enduring effects on foetal brain and behavioural development, leading to the
occurrence of functional and structural brain abnormalities in the offspring (Rees & Inder,
2005).

Abnormal brain and development during foetal life is also implicated to contribute to the
aetiology of numerous neuropsychiatric disorders, including schizophrenia, manifesting
during the course of life (Weinberger, 1987; Rapoport et al., 2005).

1.1 Schizophrenia

Schizophrenia is a severe, chronic and disabling psychiatric disorder, affecting

approximately 1% of the population worldwide (Laruelle, 2014). It is characterized by
impaired thinking, emotions and behaviour (Ross et al., 2006). The symptoms can be
divided into three main categories namely positive, negative and cognitive symptoms
(Schults et al., 2007). Positive symptoms include delusions and paranoia, major thought
disorders and most commonly, visual and/or auditory hallucinations (Schults et al., 2007).
Negative symptoms include apathy, alogia, behavioural preservation, depression, social
withdrawal and loss of will, drive or pleasure (Schults et al., 2007). Cognitive symptoms
comprise disturbances in executive functions, memory impairment, attention disorders and
deficits in areas such as learning and behavioural flexibility (Meyer, 2013). Generally,
distinguishable symptoms to substantiate the diagnosis of schizophrenia does not present
until young adulthood. Notwithstanding the evidence, prodromal symptoms and certain
endophenotypic traits of cognitive and social deficits can precede the psychotic disorder
(Braff & Freedman, 2002).
1.2 The aetiology of schizophrenia

Research approaches have unfolded conflicting theories regarding the exact aetiology of
schizophrenia. In addition to the genetically aspects and neurotransmitter abnormalities of
the illness, the impact to environmental stimuli on early and late brain neurodevelopment has
also been implicated (Weiss & Feldon, 2001; Lewis & Levitt, 2002).The risk for
schizophrenia and related disorders, appears to increase with evidence implicating various
environmental factors including pre-, peri- and postnatal exposure to infections, stressors,
substance abuse and altered immune factors (Weiss & Feldon, 2001; Matheson et al.,
2001).
1.3 The pathophysiology of schizophrenia

The neuropathology of schizophrenia is associated with alterations in monoamine levels,

and oxidative systems of the frontal cortex and striatum of the brain (Meyer-lindenberg et al.,
2002) as well as anatomical neuropsychological abnormalities (Ross et al., 2006:139).

1.3.1 Neuropsychology

Schizophrenia is depicted by a disruption in fundamental circuitry in the brain, this disruption

lead to a generalized deficit in a basic cognitive process, which results to impairment in all
cognitive systems and subsystems, including attention, memory, language and executive
functions (Rapoport et al., 2005).

Schizophrenia is mainly characterized by intellectual decline that usually starts during

childhood. Although anti-psychotic treatment may have an effect on neuropsychology, it is
shown that medication-naïve schizophrenia patients have impairments in cognitive domains
(Mohamed et al., 1999). A greater decline in cognitive impairment originates after the first
episode of schizophrenia have taken place, although neuropsychological impairment already
existed before schizophrenia have been diagnosed (Kapur, 2003). There have been
suggested that poor verbal memory tasks could be a result of abnormalities in the left-
temporal hippocampus, thus playing an integral role in memory performance (Urakubo et al.,
2001).

Many studies have shown that animal models (for example the Logical Memory Test and the
Stroop Test) are useful in explaining one cognitive deficit, while it lacks the describing of
complex and diverse phenomena of schizophrenia (REFS). It is clear that there is a need for
new models and further research to understand the pathophysiological markers of the
neuropsychological impairments associated with schizophrenia.
1.3.2 Neuroanatomy
Structural magnetic resonance imaging (sMRI) of schizophrenia patients generally show
that they have dilated ventricles , a loss of brain tissue and volume abnormalities in the
temporal and prefrontal lobes especially in the medial and superior temporal lobes(Lawrie et
al., 2008).
Commented [CVdW1]: Dr, moet ons nog detail bysit?
Commented [MM2]: Julle kan dalk net nog ietsie se van
‘n studie of twee wat bewys het dat hierdie altered
neuroanatomy associated is met van die simptome?? ek
weet dit is functionalMRI studies (fMRI).

1.3.3 Neurochemistry

The dopamine (DA) hypothesis of schizophrenia postulates hyperactive DA transmission in

the striatum which has been shown to inversely correlate with hypoactive DA transmission in
the prefrontal cortex, resulting in elevated and reduced levels of DA, respectively (Walter et
al., 2009). Over-activity of striatum DA are believed to mediate the occurrence of positive
symptoms in schizophrenia, while under-activity of DA in the frontal cortex is associated with
negative symptoms observed in schizophrenia (Holcomb et al., 2004).

In contempt with dopaminergic abnormalities which have been in the frontline of research
targets for schizophrenia hitherto, other major neurotransmitter systems, including serotonin
(5-HT), noradrenaline (NA), γ-aminobutyric acid (GABA), acetylcholine (Ach) and glutamate
have also been implicated (Bloch, 2009).

The glutamate hypothesis postulates a dysfunction in the N-Methyl-D-aspartate (NMDA)

glutamate receptor. In schizophrenia neurochemistry, NMDA receptors appear to be the
most relevant glutamate receptors (Kalia et al., 2008). It plays an essential role in both
cognition and neurotoxicity (Lipton & Rosenberg, 1994). In order to support important brain
functions, the NMDA receptor can also adapt to numerous exogenous signals (Lin et al.,
2012). Both the striatum and cortex contains glutamatergic neurons (Schwartz et al.,
2012:195). In conjunction with positive symptoms, these glutamatergic neurons fire upon a
GABA interneuron, but in virtue of the dysfunctional NMDA receptors located on it, hypo
functioning of the GABA interneurons occurs (Schwartz et al., 2012:195). Following the loss
of GABA function, firing rates of secondary glutamate neuron, known as the DA neuron,
increase substantially leading to excessive DA activity (Schwartz et al., 2012:195). Negative
symptoms, on the contrary, involves a similar pathway, however, hyperactivity of
glutamatergic neurons occurs. The increased glutamate tone causes an extra stimulation on
the GABA interneurons and subsequently leading to higher levels of GABA. In respect to
elevated levels of GABA, DA neurons projecting to the frontal cortex are inhibited (Schwartz
et al., 2012:195).

Nonetheless, the resultant abnormal cortico-striatal reduction-oxidation (redox) control

occurring in recent animal models exposed to oxidative stress, has also elicited interest in
several studies for its partial role in the pathophysiology of schizophrenia (Zhang et al.,
2010).
1.3.4 Mitochondrial function

The mitochondria, satisfying the brain’s high oxygen and glucose demand, are the foremost
generators of high energy intermediates. In addition to this, it also serves meaningful
functions such as calcium homeostasis, apoptotic cell death, redox signalling, and
contributes to a significant role in neurodevelopment and neuronal activity modulation
(Rajasekaran et al., 2015; Robicsek et al., 2013). Contingent on reduced energy production
of the mitochondria, neuro-degenerative diseases, inclusive of schizophrenia (Gonçalves et
al., 2015) will be implicated.

In addition to the development of schizophrenia evident by failure to meet these high energy
demands of the brain, the mitochondria also enact in inflammatory and oxidative stress
(Rajasekaran et al., 2015). Reactive oxygen species (ROS) are formed as a natural toxic by-
product of aerobic (requiring oxygen) metabolism in the mitochondria (Devasagayam et al.,
2004). Given that nitric oxide synthase (NOS) may give way to DNA, protein and lipid
damage, the extent to which adversity in the mitochondrial pathway could contribute to the
increased formation of ROS and consequently neurodevelopmental changes, is clear
(Rajasekaran et al., 2015).

The NMDA glutamate transmitter system in particular, appears to play a pivotal role in
enabling excessive amounts of glutamate found in a variety of neurological disorders,
including schizophrenia (Krystal et al., 2005). In short, impaired NMDA receptors are
constantly stimulated by excessive glutamate levels. Glutamate, the central nervous system
utmost excitatory neurotransmitter, is notorious for its excitotoxicity effect at high levels
(Platt, 2007). Excessive amounts of glutamate cause activation of Ca²⁺ permeable NMDA
receptors, causing an influx of calcium ions to enter cells, which results in the further release
of glutamate. Excess calcium could potentially mediate the opening of the mitochondrial
permeability transition pore. The mitochondria are the major intracellular source of free
radicals and the pore usually opens as a result of unusual large amounts of calcium
absorbed by the organelles (Stavrovskaya & Kristal, 2005). This increase in mitochondrial
calcium concentration will enhance free radical generation which causes the mitochondria to
swell and release ROS and proteins consequently (Ankarcrona, 1995). Providing that
insufficient anti-oxidant levels, essential for neutralizing the produced ROS, occurs, elevated
ROS can induce structural damage to neurons (McQuillen et al., 2004). These findings
directly link excessive glutamate levels, in conjunction with the NMDA receptor
malfunctioning, to the redox status observed in schizophrenia patients (Smythies, 1999).
1.3.5. Oxidative stress and an altered anti-oxidant system

Oxidative stress reflects an imbalance between the production and elimination of ROS and
reactive nitrogen species (RNS) and the body’s anti-oxidant chemical system’s ability to
counterbalance and detoxify the reactive intermediates harmful effects (Kohen & Nyska,
2002).

Superoxide radical, hydroxyl radical, lipid peroxide and hydrogen peroxide constitute for the Commented [MM3]: Sit hulle chemiese afkortings ook in
most important ROS in humans (Kohen & Nyska, 2002), while comprise nitric oxide (NO)
and peroxynitrite (ONOO¯) amount for RNS (Do et al., 2009). Regardless of the detrimental
response to elevated RNS/ROS levels, normal levels are of great significance in the
functioning of cellular target gene expression and modulating signalling pathways (Sun &
Oberley, 1996). Moreover, the RNS and NO concentrations is notorious for vital
cardiovascular and central nervous system neurotransmitter and neuromodulator properties
(Garthwaite, 2010; Prast & Philippu, 2001).

Nonetheless, “normal” levels are key and failure to provide adequate protection against
elevated ROS/RNS levels increases the risk to lipid, protein and DNA damage (Kohen &
Nyska, 2002). The brain, compared to other organs, is especially vulnerable to the damaging
effects of oxidative stress (McQuillen et al., 2004). This susceptibility to oxidative damage
can be attributed by several aspects. The brain has relatively low levels of antioxidant
enzymes, but contains a high metal content of elevated iron, copper, zinc and manganese
levels, which is able to catalyse the formation of ROS (Bains & Shaw, 1997). Furthermore,
it’s high oxygen and glucose consumption satisfying its supreme energy demand plus high
concentration of peroxidizable polyunsaturated fatty acids, also renders the brain’s
vulnerability to oxidative stress (Bains & Shaw, 1997). Even though neuronal cells present
with protective mechanisms against changes in the redox balance, this additional oxidative
load occurring in the brain may overwhelm these mechanisms (Bains & Shaw, 1997).
Consequently, a lack of sufficient protective mechanisms may lead to cell damage via
endogenous oxidative processes and result in the pathophysiology of a neurological disease
(McQuillen et al., 2004).

Biological systems have developed complex strategies to protect the body and, especially
the brain, against free radical toxicity (Nordberg & Arnér, 2001).The antioxidant defence
system, containing both enzymatic and non-enzymatic components, maintains the normal
physiological homeostasis by neutralizing the free radicals and protecting the brain from the
potential toxicity of ROS and oxidative damage accordingly (Forman et al.,
2004).Glutathione peroxidase, catalase and superoxide dismutase are all examples of
antioxidant enzymes (Forman et al., 2004), while non-enzymatic anti-oxidants consist of α-
tocopherol (vitamin E), ascorbic acid (vitamin C), glutathione (GSH), carotenoids, and
flavonoids (Do et al., 2009). As in figure 1, Superoxide dismutase (SOD) converts
superoxide radicals to hydrogen peroxide(H₂O₂)., which in turn forms the highly reactive
hydroxyl radicals (OH¯). Supplementary to superoxide dismutaseSOD, glutathione
peroxidase and catalase catalyse the conversion of hydrogen peroxide to water (Dringen et
al., 2005). Glutathione peroxidase, in relation to the transmutation of hydroxyl and peroxide
radicals in to nontoxic forms, often acts via the oxidation of reduced glutathione (GSH), a
radical scavenger and the body’s most powerful anti-oxidant, into glutathione disulphide
(GSSG), the oxidized form of glutathione. GSSG can convert back to GSH by glutathione
reductase (Meister & Tate, 1976). Commented [MM4]: Cool figure maar dis bietjie
onduidelik. Wil julle dit nie dalk oor teken in powerpoint en
save as ‘n jpg nie?

Figure 1: The production and elimination of ROS and RNS and the body’s anti-oxidant
chemical system.
The molar oxidized/reduced glutathione ratio (GSSG: GSH) exceeds 1:100 in a resting cell
(Chai et al., 1994). An increased GSSG: GSH ratio to values of 1:10 and even 1:1, is
considered indicative of oxidative stress (Potesil et al., 2005). In relation to this, glutathione
(GSH) is perceived to modulate glutamatergic activity and reduction in GSH may contribute
to a dysfunction in glutamatergic pathways (Oja et al., 2000). This is substantiated by an
increased in both the striatum and frontal cortex’s glutamate activity during oxidative stress
(Haroutunian et al., 2003). Concomitantly in schizophrenia patients, a decreased in both
reduced and oxidized glutathione ratio is established (Mukherjee et al., 1996) along with
altered glutamatergic activity linked to oxidative stress (Prabakaran et al., 2004).

Growing evidence is supports the fact that alterations in the antioxidant system can lead to
cognitive impairment and most importantly, monoaminergic changes relevant to
schizophrenia (Garcia-Cazorla et al., 2008). In schizophrenia an altered anti-oxidant/redox
status are certain, proven by high lipid peroxidation levels and a decrease in endogenous
antioxidant enzymes including superoxide dismutaseSOD, catalase and glutathione
peroxidase, as indicated in figure 2 (Mukherjee et al., 1996; Kuloglu et al., 2002; Kunz et al.,
2008; Ng et al., 2008). However, other studies have observed contradicting results with
elevated levels of the anti-oxidant system in patients with schizophrenia (Dakhale et al.,
2004; Kunz et al., 2008), possibly in virtue of a reactive up-regulation of mentioned
protective mechanisms in contempt of redox irregularities (Raffa et al., 2009; Singh et al.,
2008). These changes correspond unmistakably to the hypothesis involving an impaired
antioxidant defence system in the pathophysiology of schizophrenia, but fluctuations in anti-
oxidant status, contingent to the time of measurement (during initial occurrence of
distinguishable symptoms akin to schizophrenia or progressed disease status), together with
the current antipsychotic treatment (Dakhale et al., 2004). This has led to antioxidants such
as N-Acetyl Cysteine (NAC) receiving considerable attention as a novel treatment option for
the interrelated anti-oxidant changes, either acting by means of anti-oxidant or pro-oxidant
pathways in the treatment of psychotic disorders (Möller et al., 2015). Commented [MM5]: Ref hier vir michael berk en olivia
dean se werk oor NAC ook
Figure 2: The role of redox balance disturbances and subsequent oxidative stress in
schizophrenia pathology
1.3.6 Inflammation

Inflammation is seen as the body’s first defence reaction to injury or infection (Kirkpatrick &
Miller, 2013). Leukocytes get activated and migrate to the injured area. Consequently, blood
vessels dilate and become more penetrable, permitting molecules and cells to depart from
blood vessels and infiltrate the damaged tissue (Müller et al., 2012).The inflammatory
response system also eliminates bacteria and parasites through the activation of a group of
proteins which forms a complicated molecular structure (Kirkpatrick & Miller, 2013). The
immune system initiates a response with cytokines (Gibney & Drexhage, 2013). Cytokines
act as chemical messengers that aid cell to cell communication in immune responses in
order to regulate the host response to inflammation, trauma or infection (Boksa, 2010). They
also play a key factor in the development of neurons in the brain (Urakubo et al., 2001). The
body consist of several types of cytokines with distinctive roles: Anti-inflammatory cytokines
act to encourage healing while pro-inflammatory cytokines aggravate diseases (Dinarello,
2000).The latter includes interleukin (IL)-1b, IL-6, and tumour necrosis factor (TNF)
produced by the immune system (Graciarena et al., 2010).

These complex cytokines molecules binds to the receptors on both the glial cells and
neurons (Hopkins & Rothwell, 1995). This bond to receptors generates the development of
additional cytokines, free radicals and chemokines, resulting into an even bigger neuro-
inflammatory response, causing enormous activation of cytokines in the brain (Sawada et
al., 1993). Inappropriate regulation of the immune system results in a negative impact on the
neurobiological system, causing the initiation of neurodegenerative diseases (Gibney &
Drexhage, 2013). Neuro-inflammation is one of the vital factors that contributes to the
development of schizophrenia (Swanepoel, 2015).

Moreover, higher or greater than normal levels of pro-inflammatory cytokines and reduced
levels of anti-inflammatory cytokines is noticed in patients with schizophrenia and their first-
degree family members (Martinez-Gras et al., 2012). Patients experiencing their first
schizophrenia incident has elevated pro-inflammatory cytokines (Miller et al., 2011) and
there is positive interaction between interleukin-6 and the continuance period of the disease
(Moller et al., 2015).

Patients with schizophrenia persist with reduced levels of the interferon(IFN)-y gene
(Freudenreich et al., 2010). Interleukin-2, the type one immune response pro-inflammatory
cytokine inhibits the release of dopamine at higher concentrations and increases it at lower
concentrations thus it has a concentration-dependent effect on dopamine (Mansur et al.,
2012). The interleukin-2 plasma levels and the production of interleukin-2 through
lymphocytes decreases in schizophrenia (Kim et al., 1998, Mansur et al., 2012).
Another pro-inflammatory cytokine, Tumour necrosis factor-α is elevated in schizophrenia
patients (Kim et al., 2009). Interleukin-4 and 10 activities also decreased in patients with
schizophrenia (Mansur et al., 2012).

Cytokines play an important role in schizophrenia, in the brain cytokines are responsible to
manage neurotransmitter activity such as dopamine ,glutamate, noradrenalin and serotonin
(Feleder et al., 2010). Dopamine and glutamate is exceptionally relevant to schizophrenia as
discussed earlier in section ????(Mansur et al., 2012).
Commented [MM6]: Watter van hierdie het julle al klaar
vroeer afgekort? gebruik die afkortings regdeur.
Commented [MM7]: Noem die section waar julle dit
reeds bespreek het.
1.3.7 Prenatal inflammation:

Schizophrenia-related changes have been reported in offspring’s prenatally exposed to

immune activation in both epidemiological as well as preclinical studies (Brown & Derkits,
2010; Moller et al., 2015). This had led to the hypothesis that critical mediators of the
association between altered early brain development and an increased risk of schizophrenia,
is attributable to the induction of pro-inflammatory cytokines by the maternal immune system
(Gilmore & Jarskog, 1997). In other words, prenatal/maternal cytokine-associated
inflammatory responses may play a key role in the epidemiological relationship between in
utero inflammation and schizophrenia. The suspected mechanism is as follows: An
increased production of inflammatory cytokines following maternal immune activation,
crosses the placenta, causing subsequent activation of foetal cells as well as the production
of interleukin (IL)-1b, IL-6, and tumour necrosis factor (TNF) and (Interferon) IFN-β amongst
others (Bitanihirwe & Woo, 2011).

The increase in these inflammatory cytokines, primarily interferon-β, initiates the production
of free radicals that develop DNA fragmentation. As a result of this effect, harm to the
nuclear an mitochondrial DNA inside the neuron could be more drastic because of the
absence of cell turnover and the elevated neuronal energy consumption rate (Bitanihirwe &
Woo, 2011).
1.3.8Lipopolysaccharide (LPS)

Various experimental approaches has been done to evaluate the hypothesis that the
unfavourable long-term effects caused by prenatal infections on the offspring’s behavioural
and brain development, may be caused by the effects related with the stimulation of the
foetal/maternal inflammatory response system (Meyer, 2013).

The result of experimental studies on pregnant rodents has indicated that infection with the
bacterial endotoxin, LPS during different phases of pregnancy, causes significant changes
related to schizophrenia (Shi et al., 2003), revealing both behavioural and neurochemical
uniformity (Moller et al., 2015).

LPS, a gram negative bacteria cell wall constituent, is a frequented model of bacterial
infection (Urakubo et al., 2001). LPS activates the immune response of the foetus by
causing immunological effects of Gram-negative infections (Boksa, 2010). LPS binds to toll-
like receptor 4(TLR-4) on macrophages, provoking a signal transduction which leads to the
initializing of the transcription factor, nuclear factor kappa B (NFjB) (Boksa, 2010).
Subsequent transcription of genes encoding pro-inflammatory and anti-inflammatory
mediators, follows the activation of NFjB (Boksa, 2010). Accordingly, the administration of
LPS evokes the synthesis and release of pro-inflammatory cytokines, interleukin-1 (IL-1), IL-
6 and tumour necrosis factor-αa (TNF-αa) (Ashdown et al., 2006).

A study done by Lanté and colleagues on the effects of LPS initiating a bacterial infection
and activating oxidative stress during the development of the foetal brain had found that LPS
therapy activates the oxidative stress reaction in the hippocampus of the foetuses brain
(Lanté et al., 2007). This was confirmed by the a rapid increase in protein carboxylation,
indicating protein damage and the reduction in α-tocopherol levels as well as in the
relationship between reduced and oxidised forms of glutathione (GSH/GSSG) (Lanté et al.,
2007). LPS encourages phagocytes to generate ROS, this causes a disparity in both
antioxidants/oxidants and oxidative stress (Li et al., 2007).
1.4 Treatment

1.4.1 Current treatment

The pharmacological treatment of schizophrenia was originally established by the regulation

of DA systems through the antagonism of the Dopamine-2 (D2) receptors (Swanepoel,
2015). These drugs are known as first generation drugs. Examples of typical first generation
drugs includes Droperidol, Haloperidol, Chlorpromazine and Flupentixol, while drugs such as
Clozapine, Risperidone, Sulpride and Qlanzapine are classified as atypical first generation
drugs (Swanepoel, 2015). Schizophrenia patients treated with these first generation drugs
are generally present with dominant side effects including extrapyramidal syndrome,
neuroleptic malignant syndrome as well as tardive dyskinesia (Swanepoel, 2015).
New discoveries of atypical antipsychotics is based on a broader mechanism which
incorporates 5-HT, Ach, GABA and NA neurotransmitter pathways (Kinon & Lieberman,
1996). These drugs either has a small affinity for 1) D2 receptors vs. serotonin5-HT-2A
receptors or 2) combination of reuptake sites or multiple receptors (Swanepoel, 2015).
Serotonin-1A receptor agonists or antagonists ,Serotonin-2A receptor antagonists,
serotonin-2c receptor inverse / partial agonists or neutral antagonists and serotonin-6-and-7 Commented [MM8]: Afkortings???
receptor or antagonists are the combination mechanisms that tend to be more successful
and generally better tolerated against cognitive of negative symptoms causing less side
effects than the other drugs (first generation drugs)(Geddes et al., 2000) however these
drugs has their own side effects which includes impaired glucose tolerance and weight
gaining (Kapur, 2003).

Negative and cognitive symptoms does not generally respond so successfully to atypical and
typical antipsychotics in contrast to positive symptoms that generally respond successfully to
both the atypical and the typical antipsychotics (Kapur, 2003). Therefore, the need to
investigate new avenues of treatment for schizophrenia such as a glutathione precursor,
anti-oxidant and glutamate modulator, namely NAC (Möller et al., 2011, Lavoie et al., 2008).
This leave us to the interesting question whether NAC may offer clinical utility in treating
schizophrenia.
1.4.2 N-acetyl cysteine (NAC) treatment:

Based on the above mentioned role of oxidative stress in schizophrenia, numerous

hypotheses have suggested anti-oxidative treatment as being beneficial. Indeed, therapy
using antioxidants have an ability to delay, prevent or ameliorate many neurologic disorders,
inclusive of schizophrenia (Dodd et al., 2008).

Acetylcysteine, also known as N-acetylcysteine or N-Acetyl-L-cysteine (NAC), a thiol, is a

membrane-permeable precursor of the endogenous antioxidant GSH, which has antioxidant,
anti-inflammatory, and neuroprotective properties (Dean et al., 2011, Atkuri et al., 2007).
NAC is also known to be a free radical scavenger and an intracellular source of sulfhydryl
groups, which interacts with ROS (Zafarullah et al., 2003; Dean et al., 2011).

GSH, known as the primary endogenous antioxidant, is responsible for maintaining the
oxidative balance in the cell (Dean et al., 2011). This balance is being established by
removal of the reactive species via glutathione peroxidase in a nicotinamide adenine
dinucleotide phosphate (NADPH) – dependent reaction. Glutathione reductase is
responsible for reducing the remaining oxidized glutathione, known as glutathione di-sulfide,
to begin the cycle again (Dean et al., 2011, Lavoie et al., 2008).

NAC can freely penetrate cells, including the blood-brain barrier without requiring active
transport mechanisms (Zhu et al., 2007), resulting in increased GSH levels, especially in
animal models (Polydoro et al., 2004). Therefore, systematic administration of NAC is
responsible for correcting low levels of GSH, as seen in schizophrenia patients (Rushworth
& Megson, 2014). The changes in brain GSH levels, makes it relevant to psychiatry.
(Buhimschi et al., 2003). Thus, this increase in GSH levels will protect the brain from the
potential toxicity of ROS and oxidative damage, and will improve the symptoms of
schizophrenia patients (Padurariu et al., 2010; Dean et al., 2011).

Several studies have shown that GSH deficits, as well as oxidative stress in rodents can be
reversed by NAC (Dean et al., 2011; Moller et al., 2013). As mentioned previously, NAC has
anti-inflammatory properties that are able to inhibit the activation of microglia and
macrophages thus preventing the production of oxidative species and cytokine (Beloosesky
et al., 2012). Fetal brains of rats prenatally treated with LPS, leading to an increase in IL-6
and IL-10 levels, could be reversed by NAC administration (Beloosesky et al., 2012). These Commented [MM9]: Hierdie sin klink nie reg nie??? Stel
dit net anders.
results suggest that NAC can provide protection against prenatal inflammation-associated
brain damage and possible long term pathology that are associated with schizophrenia
(Beloosesky et al., 2012; Pedrini et al., 2012).
NAC also has a direct influence on neurotransmission, especially glutamate and DA levels Commented [MM10]: Afkorting nie regdeur gebruik nie
(Dodd et al., 2008). NAC are responsible for regulating the neuronal intra- and extracellular
glutamate levels via the cysteine- glutamate antiporter (Dean et al., 2011). The amount of
glutamate in the extracellular space is directly regulated via the GSH production by the
neurons (Atkuri et al., 2007; Dodd et al., 2008). GSH, has been shown to potentiate brain
NMDA receptor response to glutamate in rats (Dean et al., 2011). Thus, NAC treatment will
increase and recover normal GSH levels, while also improving NMDA receptor functioning
(Heresco-Levy & Javitt, 1998).

According to several studies, NAC has very potent anti-oxidant properties which can protect
against LPS-induced adverse developmental outcomes including intrauterine fetal death and
preterm labor (Buhimschi et al., 2003). Maternal NAC administration in rats prevents
prenatal LPS-induced impairments in spatial memory and hippocampal long-term
potentiation in the offspring (Meyer, 2013). Thus, the administration of NAC can neutralize
the produced ROS, which will have a positive effect on neurons (Smythies, 1999). The
imbalance between the production and elimination of ROS, as being characterized by
oxidative stress, will also be eliminated by NAC. In result, the glutamate levels will decrease
and the NMDA receptor will function normally (Smythies, 1999). These findings positsuggest
that treatments with antioxidants to bemight be an effective therapeutic strategy in
schizophrenia patients.
 2. RESEARCH PROBLEM

Prenatal inflammation, is a major risk factor responsible for the development of psychiatric
conditions, including schizophrenia, in infants. Mimicking maternal inflammation by exposing Commented [MM11]: ????? schizophreni in infants?
Skryf sin oor.
pregnant rodents to bacterial endotoxin LPS, may therefore lead to major brain disorders in
the offspring.

Based on the reported associations, we have pioneered two an experimental animal model
of schizophrenias: a prenatal inflammation model (via antenatal maternal administration of
LPS, a bacterial endotoxin) and ancompared to an experimental control model (via antenatal
administration of saline). In view of oxidative stress prevailed in schizophrenia, we postulate
that an anti-oxidant will be of advantage value to these animal modelsin the prenatal
inflammation model. In our study, we will be testing this hypothesis by the incorporation of
NAC as novel treatment for managing reversing schizophrenia-like symptoms behaviours
and alterations.oxidative stress alterations

Hypothesis:

We propose that rats prenatally exposed to immune activation with LPS, will present with
various behavioural and neurochemical alterations as seen in patients with schizophrenia.
We also hypothesize that treatment with NAC will reverse the above mentioned bio-
behavioural alterations observed in the LPS exposed offspring.

Aims:

Our primary aim is to determine whether gestational exposure to immune activation, via
prenatal administration of LPS, is associated with redox irregularities, akin to schizophrenia.
Our secondary aim is to establish whether schizophrenia related cognitive changes occur in
a prenatal LPS model. Together with this, we aim to establish whether LPS-induced
biochemical and behavioural changes can be reversed or improved with chronic treatment of
NAC, an anti-oxidant.

Objectives:

Primary objectives:

 Determine whether prenatal administration of LPS on gestational day 15-16 can

induce cortico-striatal oxidative stress, in accordance to redox changes observed in
schizophrenia, in the offspring.
 Establish whether a prenatal LPS model can cause cognitive memory deficits, akin to
schizophrenia symptomology.
Secondary objectives:

 Investigate if chronic NAC treatment from post-natal day 51-64 could improve
prenatal LPS induced bio-behavioural alterations.
 3. EKSPERIMENTAL PROSEDURES – METHODS AND MATERIALS

3.1 Animals

Pregnant Sprague-Dawley rats will be used for all experiments. After weaning, male Commented [MM12]: Hierdie is nie reg nie want hulle
word net vir die eerste deel gebruik. Se dus hoeveel van
offspring will be randomly allocated into different groups. These groups will be consisting out hulle gebruik gaan word.
of either saline-receiving or NAC (N-acetyl cysteine)-receiving groups, with 12rats in each
group. The experimental rats will be kept in the vivarium of the Preclinical Drug
Development Platform Research Centre at the North West University of Potchefstroom,
South Africa. The conditions of the rats will be equal: cages (230(h)x380(w)x380(l)mm)) will
be filled with corncob (weekly replaced), room temperature (21±0.5 °C) and humidity
(50±10%) will be constant with an artificial 12-hour light/dark cycle (white light (350-400 lux))
and free access to food and water. The rats will be minimally handled and will receive no
environmental enrichment. Behavioural analyses will be done between 15:00-21:00. This
study will be ethically approved before any experimental work will start.

3.2 Drugs and drug treatment protocol

LPS from E. coli (Sigma-Aldrich, Johannesburg, South Africa) will be dissolved in saline and
administered subcutaneously (SC) to pregnant dams at a dose of 100 μg/kg at GD 15-16
(Fortier et al., 2007; Baharnoori et al., 2013). This administrated dose is based on previous
studies performed at our laboratory, as well as after consultation with other studies (Kinon
and Lieberman, 1996). The control group (containing 12 rats) will receive saline injections
(pH 5). The other group (containing 12 rats) will receive NAC (Sigma-Aldrich, Johannesburg,
South Africa), dissolved in saline and buffered with 1 M glacial acetic acid and NaOH
(pH=6.0), at a dose of 150 mg/kg SC (Möller et al., 2013a). All control animals will receive
an equivalent volume of saline at coinciding times.
 3.3 Study design
Pregnant Sprague-Dawley rats will receive saline or 100 μg/kg of the gram-negative
bacterial endotoxin, LPS on gestational day (GD) 15-16. Offspring will be divided into either
saline-receiving or NAC-receiving treatment groups with 12 rats in each group. NAC
treatment (150 mg/kg s.c) and saline (0.5ml/rat s.c.) will be administered from post-natal day
(PND) 51-64. Behavioural analyses of memory assessment via the Novel object recognition
test (nORT) will be done on PND 64, with rats sacrificed on PND 65 and the frontal cortex
and striatum dissected, as indicated in figure 3. The following oxidative stress analyses will
be done in both the frontal cortex and striatum of all treatment groups: oxidized and reduced
glutathione, lipid peroxidation and SOD.
Figure 3: Graphic presentation of the study design and timeline of all treatments and
analysed. Throughout the study we will use a control group and a maternal immune
activated group treated with saline and LPS, respectively. Each group will consist of
pregnant females, with 12 rats / individual group. The study will therefore use a total of 24
rats. Treatment will consist of NAC or saline in both the control and immune activated
group’s offspring’s. All offspring’s, including male and female offspring’s, will be treated
accordingly and used in behavioural testing, as well as neurochemical analysis.
Commented [MM13]: Die study design moet net na die
“Animals” opskryf bespreek word. Maak die prentjie in
kleur, dis moeilik om te sien wat daar staan. Dui ook aan
watter rotte is die male offspring, anders lyk dit asof die
prenant rats alles gaan kry. Julle moet ook aandui presies
hoeveel rotte in ‘n groep is (n=12) ens. Of Julle moet die
aandui in die Figure legend.
Commented [MM14]: Dis study design is nogsteeds nie
reg nie. Julle moet duidelik aandui waar die pregnant rotte
is en dan waar die male offspring gebore word. Net die male
offspring gaan dan Saline / NAC kry ens. Die prentjie moet
ook nog duideliker, die grys is baie moeilik om te lees teen
die wit en alles is te klein geskryf.
Commented [CVdW15]: Hoe weet ons watter is die male
offspring?
Commented [MM16]: Julle moet ook aandui hoeveel
swanger rotte in elke groep gaan wees. As daar 24 male
offspring is en elke swanger rot het vir jou 4 mannetjies
gegee kan julle uitwerk hoeveel hulle is. Noem dit in die teks
en in die fig legend.
3.4 Behavioural analyses:

3.4.1 Novel object recognition test (nORT):

This test will be carried out to analyse the memory of the rats and whether they
would explore new objects. The nORT will be carried out as described previously
(Grayson et al., 2007).The rats will be situated in a 70x70x40 cm pexiglas box. The
test will be carried out at 19:00. At first the box contains two identical objects for the
acquisition trial where the rats will then be put in the box for 5 minutes. After 5
minutes they will be put back into their home cages for 90 minutes for the inter-trail
interval. For the retention trail one of the two original objects will be substituted with a
new novel object, for this interval the rats will be placed back into the pexiglas box
where they will be able to investigate/explore the familiar and the novel objects for a
total time of 5 minutes (Moller et al., 2015).

This experiment will be recorded digitally and touching of the objects with the rats
forepaws as well as the sniffing and licking of these objects will be classified as
observation and examination which indicates exploration of these objects by the rats
(Grayson et al., 2007). The more time the rats spend exploring/examining the novel
foreign object indicated an accurate memory of the familiar object that they had seen
before in the acquisition trail period.

The discrimination index will be used to calculate nORT scores in the following
formula:

DI = (Time spend exploring novel object – time spend exploring familiar object)
divided by (time spend exploring the novel object + time spend exploring familiar
object)(Antunes and Biala, 2012, Moller et al., 2015).
3.5 Neurochemical (redox) analysis:

3.5.1 Preparation of brain tissue:

After the rats are euthanized, the brains will be dissected. The frontal cortex and
striatum will be removed and immediately placed on an ice-cooled glass slab as
previously described (REFS). The brain regions will then be snap frozen in liquid
nitrogen and stored at -80°C until the day of analysis. On the day of assay, the
sections of the brain will be removed from the freezer, weighed and allowed to thaw
on ice.

3.5.2 Oxidized and reduced glutathione:

GSSG and GSH will be analysed by using a liquid/mass spectrometry (LC/MS)

method as described previously (Möller et al., 2011).
3.5.3 Lipid peroxidation:

Thiobarbituruc acid reactive agents (TBARS) is often used to analyse lipid

peroxidation in brain tissue (Pillai et al., 2007). We will be using the Parameter™
TBARS assay from R&D Systems (Minneapolis, USA) to analyse lipid peroxidation in
brain tissue (Swanepoel, 2005). Oxidizing agents can adjust lipid structures, which
result in lipid peroxidation of the membranes. This leads to accumulation of
malondialdehyde (MDA) in tissues and will be used as index for oxidative stress
induced cellular damage (Padurariu et al., 2010). MDA is measured as TBARS and
determined by using a microplate reader measuring at 530-532 nm. The level of lipid
peroxidation in the sample will be represented by the intensity of the colour at
532nm. The standard curve of MDA will be used for the unknown samples.

3.5.4 Superoxide dismutase (SOD) activity:

The percentage Superoxide dismutase activity in the brain homogenates will be

measured with the available colometric assay kit (SOD assay kit – WST). The
amount of SOD necessary to cause 50% inhibition of the absorbance change per
minute of the blank reaction (diluents rate) expressed in U/mg protein is defined as
one-unit U of the enzyme activity (Möller et al., 2011).
 4. STATISTICAL ANALYSIS

All statistical analyses will be conducted, in subject to the Statistical Consultation Service of
the North-West University guidance.

To model the behavioural and immune-neurochemical measurements respectively, a two-

way factorial ANOVA and Bonferroni post-hoc test will be applied for the respective
treatments (Saline and NAC) and behavioural/neurochemical parameters. In all cases, data
will be expressed as the mean ± standard error of the mean (SEM), with a p value of <0.05
deemed statistically significant (Graphpad Prism 5; SAS/STAT® Software.)

5.3 Ethical consideration Commented [MM17]: Hier moet julle bespreek oor die
ethical consideration en julle moet kies watter kategorie
julle dink julle studie is volgends die etiese kategoriee. Sien
die doc wat ek vir julle aanheg, skryf dit net oor in julle eie
woorde en se dat julle die “monitoring sheet” sal aanheg
saam met die proposal.
 5. CONCLUSION AND EXPECTED OUTCOME

The study will elucidate on the underlying immune activation action of LPSacquainted
withSchizophrenia-related immune response changes. Furthermore, this dissertation will
disclose evidence of NAC as a novel treatment using a neurodevelopmental animal model of
relevance for Schizophrenia.

We propose the following outcomes:

 LPS will induce cortico-striatal oxidative stress in the offspring, subjected to

neurochemical (redox) analysis, compared to control animals treated with saline.
 LPS will lead to an impaired antioxidant defence system, correlated with alterations in
endogenous antioxidant enzymes with an increase in SOD in the striatum and a
decrease in the frontal cortex, determined by SOD activity, in comparison to saline-
treated controlled animals.
 LPS will evoke elevated MDA levels, a product of lipid peroxidation, in both the
striatum and frontal cortex, in comparison to saline-treated control animals.
 LPS will inflict an altered GSSH/GSH ratio in both the frontal and the striatum by
reduced GSSH and elevated GSH levels, verses saline-treated controlled animals.
 LPS will induce cognitive memory deficits, as determined in the nORT, in comparison
to the controlled animals treated with saline.
 The bio-behavioural changes induced by LPS will be reversed by sub-chronic
treatment with NAC.
 This reversal, supplemented by NAC will only be beneficial granted that bio-
behavioural changes does occur, and will not appear to saline-treated controlled
animals.
6. TIMELINE OF THE STUDY
 7. BUDGET AND FINANCES

This study will be financed by money won due to one very successful gambling
evening. All rats were sponsored from Dickson University, while Dr Moller-
Wolmarans has willingly agreed to sponsor all the drugs for her favourite three
students
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Kyles en sulette - Sit hierdie nog bo in alfabetiese volgorde! Sulette let asb op op jou verwysings. Die
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Ek het al van dit reggemaak.alles wat hier onder is moet nog bo ingesit word. Kyk waar hy
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