Development of a double-hit model of schizophrenia in male sprague dawley rats.

Abstract

Schizophrenia is a debilitating neuropsychiatric disorder that affects approximately 1% of the world’s population. Schizophrenia symptoms are classified as positive, negative, and cognitive. Patients with schizophrenia may present with a combination of symptoms, which causes complications in the diagnosis and treatment. Available drugs used to treat schizophrenia have shown high efficacy against the positive symptoms, while the negative and cognitive symptoms proved to be more resistant against the available treatment. There is an urgent need to develop an animal model of schizophrenia that will assist researchers in investigating the pathophysiology of schizophrenia. In this study we aim to develop a double hit animal model of schizophrenia that will be able to mimic behavioural and molecular changes similar to those observed from positive, negative, and cognitive symptoms of schizophrenic patients. The study objectives are divided into three manuscripts, manuscript 1 will characterise behavioural and molecular changes associated with the negative and cognitive symptoms of schizophrenia using our double hit model. Furthermore, manuscript 2 will evaluate neurotransmitter changes and receptor changes associated with the positive symptoms of schizophrenia using our double hit model. Lastly, manuscript 3 will examine or explore mechanisms or pathway involved the pathophysiology of schizophrenia. Animal models of schizophrenia provide a platform to screen the progression of the disease. They further offer researchers the opportunity to conduct invasive monitoring of molecular and structural changes involved in the disease. These models are divided into four categories viz: neurodevelopmental, pharmaceutical, lesion, and genetical. In this study we will focus on the neurodevelopmental (post-weaning social isolation) and pharmacological (ketamine) animal models of schizophrenia. Social isolation is not always a reliable model of schizophrenia as the behavioural impairments induced can be easily reversed when the animals are handled frequently during experimentation. Researchers have shown that ketamine causes psychotic behaviour in normal human volunteers and exacerbates symptoms in individuals with schizophrenia. Even though ketamine model is considered reliable, there are weaknesses associated with the model. Researchers used different dosages, and this may result in divergent or contradictory results. Due to weaknesses associated with the social isolation and ketamine models, researchers have tried combining the two models to produce what is known as the double hit model. We conducted a systematic review to evaluate the effectiveness of the rodent “double hit” (post-weaning social isolation and N-methyl-D-aspartate (NMDA) receptor antagonist, ketamine) model in producing symptoms of schizophrenia. This Review aimed to evaluate the effectiveness of the rodent “double hit” (post-weaning social isolation and N-methyl-D-aspartate (NMDA) receptor antagonist) model to produce symptoms of schizophrenia. The MEDLINE (PubMed) and Ebscohost databases were used to search for studies. The systematic review is based on quantitative animal studies. Studies in languages other than English that can be translated sufficiently using Google translate were also included. Data extraction was performed individually by two independent reviewers and discrepancies between them were resolved by a third reviewer. SYRCLE’s risk of bias tool was used to test the quality and biases of included studies. Our primary search yielded a total of 47 articles. Seventeen articles met the inclusion criteria for this systematic review. Ten of the seventeen studies found that the “double hit” model was more effective in developing various symptoms of schizophrenia. Five studies showed that the “double hit” model was robust and capable of inducing cognitive impairments and positive symptoms of schizophrenia than either treatment alone. In our lab we designed a double hit model of schizophrenia combining post-weaning social isolation and ketamine (SIK) in order to induce negative and cognitive symptoms of schizophrenia. A total of 32 male Sprague Dawley rats (21 days old) were acquired from the breeding unit of the University of KwaZulu-Natal. The animals were collected on PND21 because at that age they are independent not depending on dams for survival. On postnatal day (PND) 23 the animals were randomly separated to 4 different groups as follows: 8 group housed male rats + saline (0.9% NaCl) injected subcutaneously (GH), 8 grouped housed male rats + ketamine (16 mg/kg) injected subcutaneously (GHK), 8 socially isolated male rats + saline (0.9% NaCl) injected subcutaneously (SI), and 8 socially isolated male rats + ketamine (16 mg/kg) injected subcutaneously (SIK). The Shapiro-Wilk test was used to test for normality. Two-way ANOVA, followed by Bonferroni’s multiple comparisons test was used to analyse the data. Multiple comparisons will be conducted even in the absent of significant interaction effects because we want to compare the effect of the double hit model when compared to other groups. Our SIK animals group showed high anxiety like behaviour, with increased adrenocorticotropic hormone (ACTH) concentration (GH vs. SIK, p<0.0001; GHK vs. SIK, p<0.0001; SI vs. SIK, p<0.0001), corticosterone concentration (GH vs. SIK, p<0.0001; GHK vs. SIK, p<0.0001; SI vs. SIK, p<0.0001) and norepinephrine concentration (GH vs. SIK, p<0.0001; GHK vs. SIK, p<0.0001; SI vs. SIK, p=0.0021) when compared to the other groups. SIK animals showed reduced social interaction (GH vs. SIK, p<0.0001; GHK vs. SIK, p=0.0022; SI vs. SIK, p<0.0001) and decreased oxytocin concentration (GH vs. SIK, p<0.0001; GHK vs. SIK, p<0.0001; SI vs. SIK, p<0.0001) in the amygdala. The SIK group of animals were more aggressive toward a juvenile intruder (GH vs. SIK, p<0.0001; GHK vs. SIK, p<0.0001; SI vs. SIK, p=0.0401) but had less testosterone concentration (GH vs. SIK, p<0.0001; GHK vs. SIK, p=0.0004; SI vs. SIK, p=0.0009). The SIK group showed impaired visual learning and memory and increased expression of proinflammatory cytokines IL 6 (GH vs. SIK, p<0.0001; GHK vs. SIK, p<0.0001; SI vs. SIK; p<0.0001), TNFα (GH vs. SIK, p<0.0001; GHK vs. SIK, p<0.0001; SI vs SIK, p<0.0001). We suggest that our double hit model, SIK, is more robust in inducing negative and cognitive symptoms of schizophrenia than each treatment alone. We further investigated the behavioural and neurochemical changes associated with locomotor activity in schizophrenia using the double-hit model. The SIK group showed high locomotor hyperactivity (GH vs. SIK, p<0.0001; GHK vs. SIK, p<0.0001; SI vs. SIK, p=0.0003), which was accompanied by high dopamine D2 mRNA expression (GH vs. SIK, p<0.0001; GHK vs. SIK, p<0.0001; SI vs. SIK, p=0.028), high acetylcholine concentration (GH vs. SIK, p<0.0001; GHK vs. SIK, p=0.0004; SI vs. SIK, p<0.0001), lower glutamate concentration (GH vs. SIK, p<0.0001; GHK vs. SIK, p=0.0001; SI vs. SIK, p<0.0001), and GABA concentration (GH vs. SIK, p<0.0001; GHK vs. SIK, p=0.0006; SI vs. SIK, p=0.0001). We proposed that our double hit model is more robust in inducing behavioural and molecular changes associated with positive symptoms of schizophrenia. To improve our knowledge on the mechanisms involved in the pathophysiology of schizophrenia, we investigated the effect of the model on GABAergic function and oxidative stress. Group housed animal injected with ketamine (GHK) and social isolated animals injected with saline (SI) showed reduced mRNA expression of genes such as glutamate decarboxylate 1 (GAD1) (GH vs. GHK, p<0.0001; GH vs. SI, p<0.0001), brain derived neurotrophic factor (BDNF) (GH vs. GHK, p<0.0001; GH vs. SI, p=0.0002), and parvalbumin (PVALB) (GH vs. GHK, p<0.0001; GH vs. SI, p<0.0001), which are involved in GABAergic neurotransmission. The expression of these genes was reduced even further in the SIK group (GH vs. SIK, p<0.0001) in both the prefrontal cortex (PFC) and the hippocampus. The concentration of the antioxidants superoxide dismutase (SOD) (GH vs. SIK, p<0.0001; GHK vs. SIK, p=0.0005; SI vs. SIK, p<0.0001) and glutathione peroxidase (GPx1) (GH vs. SIK, p<0.0001; GHK vs. SIK, p=0.0003; SI vs. SIK, p<0.0001) was reduced in the SIK group while malondialdehyde (MDA) concentration (GH vs. SIK, p<0.0001; GHK vs. SIK, p<0.0001; SI vs. SIK, p<0.0001) was increased. In conclusion SIK showed that oxidative stress and dysfunction in the GABAergic pathway are involved in the pathophysiology of schizophrenia. Our double hit model was able to successfully induce behavioural and molecular changes similar to those observed on positive, negative, and cognitive symptoms of schizophrenia. This double hit model will assist researchers to better understand the pathophysiology of schizophrenia, and this will assist researchers to develop better therapeutic drugs to treat schizophrenia.