Open Field Test

1.3.1 Overview of the previous behavioural experiments

Many studies in the literature has indicated a significant increase in emotional changes in patients suffering from MS (1–3) as well as from other autoimmune diseases (4). Major depression (5–7), bipolar depression (2, 8), anxiety (9–11), alcohol abuse (12, 13), and other substance abuses (14) are all common in MS patients. These emotional dysfunctions are not simply a reactive psychological response to the impact of this pathology on the life style of the patient, but it has been correlated with the development of MS and other autoimmune diseases. Studies show that depression and anxiety rates are higher in MS than in those patients experiencing other chronic diseases (15). It has been estimated that between 40-50% of patients with MS will experience a type of depression within their lifetime. As a consequence of this, MS patients show a higher rate of suicides when compared to a normal population with most occurring within 5 years of diagnosis (16, 17).

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Previous studies addressed the behavioural modifications occurring in EAE mouse models. These studies reported either no changes (24) or an inverse correlation between social exploration and the rise of inflammatory mediators including IL-1, TNF-α, and prostaglandin E2 (25). It has been demonstrated that mice immunized with a suboptimal dose of myelin oligodendrocyte glycoprotein35-55 (MOG35–55) showed the manifestation of motor impairment at day 60 after immunization and had an increase anxiety-like behaviour that correlated with an increase in the level of TNF-α and with neuronal loss in the hippocampus (26). This was also associated with a doubled depressive-like behaviour in the learned helplessness paradigm. In a more recent study, the behaviour of mice subjected to EAE were assessed before locomotor defects started to show (27). Their results suggested firstly that high anxiety indexes in EAE mice precede the appearance of motor defects and secondly that TNF-α has a pivotal role in the high anxiety response because of the ability of this cytokine to cause striatum inflammation and activation of microglia. In addition, intracerebroventricular administration of etanercept, an inhibitor of TNF-α signalling, resulted in anxiolytic-like effects in EAE mice.

All these studies focused on the biochemical and cellular changes occurring in the CNS while very little has been explored in terms of possible changes occurring in the periphery such as in the blood. Indeed, a great deal of studies have shown that T-cells play a pivotal role in regulating emotion in mice (31–34) as well as in humans (35, 36) besides being the main drive of autoimmune diseases.

1.3.2 Experiment results

1.3.2.1Phases of Disease in MOG35–55-Induced EAE

Immunization of C57 black 6 (C57BL/6) mice with MOG35–55/complete Freund’s adjuvant (CFA) causes a neurodegenerative inflammatory disease that resembles the primary progressive form of MS (42, 43). Although the expressions of the disease are not always synchronous among all the mice, it is possible to separate three main phases: the pre-onset, the onset, and the disease phase. During the pre-onset (day 0–10), mice do not show any visible motor defects while behavioural changes are already visible since day 2 post immunization. At the onset of the disease (day 10–12), mice develop a weak or soft tail and start to show signs of motor dysfunction. During the disease phase (day 12–22) mice progressively loose the ability to move the hind legs and a significant weight loss occurs. Mice were tested starting from day 2 [before pertussis toxin (PTX) injection] and every other day till day 10, before the occurrence of any motor defect.

1.3.2.2Changes in Behaviour in the Open Field Test

The open field test has been previously used in the majority of studies assessing anxiety behaviour during EAE. This test was used as read-out system for the behavioural changes at the early stages of the EAE. The convenience of this test is that it provides easy and simultaneous measure of multiple parameters including locomotion, exploration, and anxiety.

Both MOG35–55/CFA immunized mice and PBS-treated control group showed a gradual and time-dependent decrease in locomotor activity (between 50 and 35%) that became almost stable from day 4 onward. The number of passages through the central square is considered a measure of anxiety and exploratory activity. Control mice showed a variable but overall stable number of central square visits throughout the 8-days of testing. In contrast, immunized mice showed a significant reduction by day 2 and a further decrease at day 4. This value also remained constant for the next 4 days.

Rearing in mice and other rodents is an emotional and protective response to the stress of a new environment; this is a typical vertical activity that consists in the standing completely erect on the hind legs. This “risk-assessment” behaviour indicates that the animal is hesitant to move from its present location to a new position. In the open field test, the latency to the first rear is considered a measure of depression and associated anxiety (40, 44). Control mice did not show any significant changes in the number of rearing or in the inactivity to the first rear throughout the time of the experiment. Conversely, immunized mice showed a steep increase in latency to rearing until day 4 and then a decline to almost basal level from day 8. When the fold changes were compared to baseline values of all the parameters we have measured, the latency to rearing showed the highest fold change. Most interestingly, this followed a linear correlation from day 0 to 4 with a slope that was significantly different from zero.