• Development of 'In vitro' intestinal models to study the pharmacology of drugs affecting the gastrointestinal tract in normal and diseased conditions. Development of a cell culture model for intestinal pharmacology.

      Javid, Farideh A.; Denyer, Morgan C.T.; Britland, Stephen T.; Batista Lobo, Samira (University of BradfordSchool of Pharmacy, 2010-05-11)
      Studies investigating the effect of 5-HT receptors mediating a response in the neonatal intestine have been limited. There are evidences that the development of new neurones continues past postnatal term and this suggests that receptors expression may differ during maturation. Thus, `in vitro¿ experiments were carried out to investigate the effects of ACh, atropine, 5-HT and its related drugs on intact intestinal segments taken from the ileum of adult and neonate rats. The application of ACh (3nM-1mM) and 5-HT (3nM-1mM) induced contractions in a concentration dependent manner in all tissues examined. The 5-HT induced contractions were only sensitive to antagonism by atropine (1¿M) in segments taken from the neonates but not adults. The pre-treatment with methysergide (5-HT1/2/5-7 receptor antagonist), ritanserin (5-HT2 receptor antagonist), granisetron (5-HT3 receptor antagonist) and RS 23597 (5-HT4 receptor antagonist) at 1¿M or a combination of ritanserin, granisetron, plus RS 23597 at 1¿M significantly reduced or abolished contractile responses induced by 5-HT. SB 269970A (5-HT7 receptor antagonist) and WAY 100635 (5-HT1A receptor antagonist) at 1¿M failed to influence contractile responses induced by 5-HT or the challenges to 5-HT receptor agonists, 5-CT (5-HT1A/7 receptor agonist) and 8-OH-DPAT (5-HT1A receptor agonist) at a concentration range of 10nM-0.1mM, indicating the unlikely involvement of 5-HT1A and 5-HT7 receptors in the mediation of contractile responses in the neonatal rat ileum. Results indicate differences in cholinergic receptor involvement during postnatal maturation and suggest the involvement of 5-HT2, 5-HT3 and 5-HT4 receptors in the mediation of contractile responses to 5-HT in the neonatal rat ileum. There is a growing need to decrease animal usage in pharmacological experiments. This may be achieved by the development of `in vitro¿ cell culture models. Thus attempts were also made to develop a cell culture model of neonatal intestine to further investigate the action of pharmacologically active agents. The isolation of individual cell populations from segments taken from the intestine of rat neonates were achieved by ligation of both ends of the intestine prior to incubation in trypsin so that a gradual dissociation could be monitored. This was supported by histological procedures, determining the time required to extract large numbers of cells from different intestinal layers. Differential adhesion and selective cytotoxicity techniques were used for further purification of intestinal smooth muscle cells (ISMC), neuronal cells, and a coculture of ISMC and neuronal cells, and these were characterised through immunostaining with antibodies to ¿-smooth muscle actin, ¿-actinin and the 5-HT3 receptor. A protocol for cryopreservation of ISMC was designed in order to protect cells against genetic instability, enhance cell availability and reduce animal usage. Results showed that cells extracted from the intestine are viable for up to 4-months. ISMC functionality was analysed via the application of known pharmacologically active drugs on ISMC, which were plated onto glass and silicone elastomer substrate. The cultured ISMC responded to the application of drugs such as potassium chloride (KCl), carbachol, 5-HT and noradrenaline (NA). Large population of cocultures seeded onto silicone elastomers or cholesteric liquid crystal substrates (LC) were assessed for their ability to produce a collective response to KCl application. Attempts were made to detect any deformations of the substrate surface due to the exposure to KCl and NA. Cholesteric LC substrates seemed to be the most suitable material for investigating the cellular tensions. The availability of cell cultures allowed the development of an intestinal model of inflammation. This was achieved through the use of lipopolysaccharide (LPS)-induced inflammation and was confirmed by assessing the levels pro-inflammatory mediators interleukin (IL-8) and nitric oxide (NO), which were significantly elevated. Reduction of IL-8 ad NO was also examined using granisetron and L-NAME and Chaga mushroom extract. Granisetron and L-NAME reduced the NO production during short incubation times. However, an elevated level of NO was observed when longer treatment times were examined. The Chaga mushroom extract caused a significant reduction in NO production in the model of inflammation. This indicates that this model may be a valuable tool for the investigation of other pro-inflammatory mediators and may contribute for the investigation of more selective drugs in the management of intestinal inflammation in neonates.
    • Exploration of cognitive and neurochemical deficits in an animal model of schizophrenia. Investigation into sub-chronic PCP-induced cognitive deficits using behavioural, neurochemical and electrophysiological techniques; and use of receptor-selective agents to study the pharmacology of antipsychotics in female rats.

      Neill, Joanna C.; Woolley-Roberts, Marie; McLean, Samantha L. (University of BradfordSchool of Pharmacy, 2010-10-19)
      Cognitive dysfunction is a core characteristic of schizophrenia, which can often persist when other symptoms, particularly positive symptoms, may be improved with drug treatment. The non-competitive NMDA receptor antagonist, phencyclidine (PCP), is a psychomotor stimulant drug that has been shown to induce symptoms characteristic of schizophrenia in humans and animals. The aim of these studies was to use the sub-chronic PCP model in rats to investigate cognitive dysfunction in behavioural tests which have been highlighted as relevance by the MATRICS initiative (MATRICS.ucla.edu). The main tests used were attentional set-shifting, operant reversal learning, and novel object recognition tasks. The pharmacology of antipsychotics was studied in the reversal learning task using receptor selective compounds. Following this, experiments were carried out using in vitro electrophysiology and in vivo microdialysis in an attempt to investigate the mechanisms underpinning the PCP-induced cognitive deficits. The attentional set-shifting task is a test of executive function, the extra-dimensional shift (EDS) phase relates to the ability to shift attention to a different stimulus dimension; this is impaired in patients with schizophrenia. The studies presented in chapter 2 showed that sub-chronic PCP administration impaired attentional set-shifting performance selectively in the EDS phase, a deficit which was significantly attenuated by sub-chronic administration of clozapine and risperidone, but not haloperidol. The effect of PCP was also shown to be more robust in female rats compared to males. A deficit in set-shifting ability was also observed in isolation reared rats. However, the deficits produced by PCP were more robust than the deficit produced by isolation rearing. The reversal learning task is another test of executive function. Chapter 3 reported that sub-chronic PCP administration impairs reversal learning ability in an operant task, as demonstrated by reduced percent correct responding in the reversal phase of the reversal learning task. It was found that a D1 agonist (SKF-38398), a 5-HT1A partial agonist (buspirone), a 5-HT2C antagonist (SB-243213A) and an agonist and positive allosteric modulator of the alpha 7 nACh receptor (PNU-282987 and PheTQS respectively) are able to reverse the sub-chronic PCP-induced deficit in reversal learning. Although many antipsychotics have affinity for muscarinic M1 and histamine H1 receptors, selective agents at these receptors were not able to improve the PCP-induced deficit. In chapter 4, the atypical antipsychotics, clozapine and risperidone, when given alone to naïve rats had no effect on reversal learning. Haloperidol when given to naïve rats impaired performance at the highest dose. Sub-chronic PCP was again found to impair reversal learning performance. Investigative experiments revealed that the 2 min time-out could be important as a cue. Following a double reversal, olanzapine-treated rats lost the ability to switch between the rules, whereas clozapine and risperidone-treated rats could perform the double reversal. Experiments with the extended (15 min) reversal phase could allow the investigation of the time-course effects of antipsychotics or selective compounds. The studies presented in chapter 5 found a reduction in gamma oscillations in the CA3 region of the hippocampus, following sub-chronic PCP treatment (2-5 weeks post treatment) that was paralleled by a deficit in parvalbumin immunoreactive (IR) cell density, at a similar time point (2 weeks post treatment). In contrast, a time-dependent increase in gamma oscillations was observed (6-8 weeks post treatment), at which point parvalbumin IR cell density was unchanged (8 weeks post treatment). Gamma oscillations were unchanged in the prefrontal cortex (PFC) following the PCP treatment regime. Locomotor activity tests were also carried out to ensure that the sub-chronic PCP treatment was successful. In-vivo microdialysis revealed that vehicle-treated rats show an increase in dopamine in the PFC which is selective for the retention trial of the novel object recognition task. PCP-treated rats were unable to distinguish between the novel and familiar objects and the increase in dopamine observed in vehicle rats was absent. As a control experiment it was also shown that sub-chronic PCP did not induce anxiety-like symptoms in the elevated plus maze and open field tests. These studies suggest that sub-chronic PCP induces cognitive deficits in behavioural tasks, and these deficits may be due to GABAergic mediated processes in the hippocampus and dopaminergic dysfunction in the PFC. These behavioural and neurochemical results are concurrent to findings observed in schizophrenia.