Examination of How Vesicular 5-HT Release Is Affected by Age - Research Proposal Example

Name Course Institution Instructor Submission Date An optimization of a technique for the isolation of 5- HT positive enterochromaffin cells from the GI tract and examination of how vesicular 5-HT release is affected by age. Lay summary Serotonin is a chemical released from the cells in the lining of the bowel which has been shown to be important for normal movements of the contents of the gut. Observable changes with age in humans such as increased constipation suggest the involvement of these cells in this function and may change with age. In this study the role of the cells lining the internal part of the bowel in the movement of the gut contents and effect of aging on them is to be studied using mice. Stepwise process of isolation of the cells secreting the chemicals and quantifying the content will be done as well as performing other steps to demonstrate bowel action. Scientific summary Several studies have shown 5 hydroxytryptamine (5-HT, serotonin) to be essential for reflexes resulting from mucosal stimulation and also for normal propagation of colonic migrating motor complexes (CMMCs) as well as propulsion of faecal pellets in isolated colon. With age colonic dysmotility and constipation have been observed in humans and this raises questions about the effect of ageing on enterochromaffin (EC) cells which could explain the observed changes. This study uses a mouse model examine the role of EC cells and 5-HT in motility as well as their characterization and effects of age on these cells. Several steps will be used to obtain an enriched fraction of isolated EC cells by immunocytochemistry and serotonin content. These comprise a combination of enzymatic digestion, density gradient centrifugation and short-term culture. Background Among the enterocytes in the intestinal epithelium are the enteroendocrine (EE) cells which the enterochromaffin (EC) cells form a large portion. The EC cells produce and store about 95 % of the body’s pool of 5-Hydroxytryptamine (5-HT, serotonin) (Gershon and Tack, 2007). The stimuli from nutrients, chemical (acid or base), mechanical or pathologic sources in the gut lumen causes the release of 5-HT. The released 5-HT activates secretory and peristaltic reflexes via enteric neurons and vagus nerve (Monro et al., 2005). The EC cells take part in signal transduction of these signals by converting these stimuli to 5-HT production which acts via neurocrine, paracrine and endocrine manner (Gershon, 2013). This is suggestive that the stretch reflex is important for normal colonic propulsion and is triggered by mucosal 5-HT release. 5-HT released from these EC cells is involved in physiologic processes and in disease states (Grundy and Schemann, 2005, O'Hara et al, 2007). Alterations in serotonergic pathways occur in some disease states. For instance, a high amount of 5-HT release occurs in diarrhoea in vibrio cholera infection, inflammatory processes due to parasitic infections, nausea following chemo-or radiotherapy as well as symptoms of carcinoid syndrome (Betrand, 2006). The EC cells have also been thought to contribute to chronic gut conditions like inflammatory bowel disease (IBD) (Mawe et al., 2006 & Keating et al., 2008). These findings suggest that 5-HT is involved in disease states and these observations can be used to target therapeutic approaches for bowel diseases especially chronic conditions. Studies support that the peristaltic reflex is dependent on both 5-HT and mucosa (Heredia et al. 2009 & Dickson et al. 2010a, b), while others have contrary opinion (Keating & Spencer, 2010; Spencer et al.2005,). The colonic migrating motor complex (CMMC) is a sequence of movement that controls the transit of faecal matter by a sequence of rhythmic electrical activity and contractions in the colon. Presence of fecal matter causes the local mucosal reflexes to be activated with release of 5-HT. These stretch reflexes determine the point of origin of CMMC and progression hence causing forward movement of colonic contents (Heredia et al, 2009 & Dickson et al., 2010). There is no much detailed information about the changes in the motor functions of the gut with age which ought to be understood to provide insight to various physiological and pathological states. The gut has both intrinsic and extrinsic innervations which regulate its activity. The intrinsic (enteric) system consists of sensory neurons, interneurons, and motor neurons and organised into submucous (Meissner's plexus) and myenteric plexus (Auerbach's plexus) (Junqueira & Carnreiro , 2005). Many studies in rodents have revealed a loss of myenteric neurons whose physiological significance has not been clearly established (Saffrey, 2013). The reduced number of neurons has been associated with reduced migrating motor complexes in mice but ageing effects was not examined (Ro et al, 2006). Ageing does not influence transit time of the stomach and small intestines whereas among the elderly there is a reduction in propulsive capacity of the colon (Marsden and Graff, 2004). Constipation is a common problem in the elderly group, however no clear implications of colonic dysmotility or any other demonstrable change in colonic transit physiology has been associated. Hypothesis EC cells are involved in gut motility and the properties of these cells change with age, contributing to constipation. Aim of the investigation: To optimize EC cells isolation from mouse colon To characterize the effects of age on 5-HT release properties from murine enterochromaffin cells using electrochemical-sensing Materials and Methods of Research C57BL/6 male mice (90–730 days old) will be killed humanely by stunning with a blow to the skull continued by severing the carotid arteries and then dissecting the animal as per ethical standards. A ventral midline incision will be made and the whole colon removed and placed in ice cold Krebs solution. All dissections will be performed in 4% Krebs solution {contains (in mmol/L) 120.35 NaCl, 5.9 KCl, 15.5 NaHCO3, 1.2 NaH2PO4, 1.2 MgSO4, 2.5 CaCl2, and 11.5 glucose, gassed continuously with a mixture of 3% CO2–97% O2 (vol/vol), to give a final pH of 7.3–7.4} to reduce the uptake of 5-HT released from the mucosa into the serotonergic interneurons. The EC cells will be isolated by dissecting the 4-6 centimeters of distal colon and scraping off and mincing the mucosal layer of the distal colon washed in buffer X (in mM 140 NaCl, 10 Hepes, 5KCl, 1MgCl2, 2 CaCl2, 5 D-glucose, pH 7.4). Then the tissue is digested in trypsin-EDTA (0.05%) with collagenase A (1mg ml-1) at 37 for approximately 20 min with continuous agitation. Later an equal volume of growth medium (DMEM containing 10% FBS, 1% penicillin- streptomycin and 1% L-glutamine) is used to stop the enzyme reaction. The suspension is filtered through a 40 nylon filter mesh and the cells will be centrifuged at 1000g for 3min. The resultant pellet will be subsequently resuspend in 2ml of growth medium and layered onto Percoll density gradients load into the tubes filling the layer from the bottom in order of 1.03 g/ml density, 1.08g/ml density and topped with cells. Afterwards, the tubes will be centrifuged at 1100g for 8min with slow breaking to be able to collect the cells at 1.07g/ml from the tubes. The harvest EC cells will be washed once in growth medium and plated onto pretreated cell culture dishes on the cover slips containing polylycin. The cover slips containing cell will then be incubated for 24hrs at 37 to allow the cells to grow and increase in numbers. The immunocytochemical analysis of EC cell purification will be preceded by fixing the cell for 18-20h in Zamboni’s fixative at 4 followed by serial 5 min washes as follows: ([4 x 80% EtOH], [2 x 100% EtOH], [3 x DMSO] and [4 x PBS]). Later fixed cells will be incubated for 24 hrs in a humid chamber with rabbit monoclonal antibody against 5-HT (Anti rabbit 1gG 1:400). Subsequently the cells will be washed 3 times with PBS, and then incubated secondary antibody (Anti mouse against Cy3 1:500) for 2h in humid chamber. Cells will then be washed once more again 3 times with PBS and then the cover slips will mounted onto glass slides to be observed under microscope for certain properties. For the next stage of this research the entire emptied colon would be placed in the organ bath and fixed with two pins in the oral opening to allow the artificial faecal pellets to be inserted and also fixed at the anal opening. Faecal pellets of different diameters will be used. Tension recordings of colonic migrating motor complexes (CMMCs) will be achieved by inserting a glass micropipette glued to an epoxy-coated faecal pellet and inserted into the lumen fixed to the organ bath floor using U-shaped pins held the two ends. Two isometric tension transducers will be attached at regular lengths to the wall of the colon obtain the tension of the circular muscle. The movements will be recorded with a video camera linked to a computer and analyzed with in-house volumetry software to generate spatiotemporal maps illustrating the movement over time. Balloon distention might as well be used to show effects of distention as well as. Mucosal reflexes will be tested by brush stimulation applied to the mucosa of the colon opened along a 10-mm length in the middle with micropipette electrodes to record the relevant electrophysiogical parameters. Recording from electrical field stimulation of segments of intact colon with and without mucosa will also be done. Electrochemical techniques will be used to record 5-HT release selectively and quantitatively. Measurement of steady state 5-HT levels from the isolated cells will be obtained and as well as the 5-HT produced by mechanical stimulation. 5-HT can also be selectively detected by electrochemical techniques by amperometry. 5-HT oxidises at certain voltage and when this voltage is reached the 5-HT oxidizes causing a peak in the oxidation current. Data analysis Statistical comparison of data will carried out using Student’s (paired or unpaired) t-tests and a minimum level of significance was reached at P Read More