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, Dribbling of saliva during the daytime
Loss or change in your ability to taste or smell ,
, Difficulty swallowing food or drink or problems with choking
Vomiting or feelings of sickness (nausea) ,
Constipation (less than 3 bowel movements a week) or having to strain to pass a stool (faeces) ,
, Bowel (fecal) incontinence
, Feeling that your bowel emptying is incomplete after having been to the toilet
, A sense of urgency to pass urine makes you rush to the toilet
Getting up regularly at night to pass urine ,
, Unexplained pains (not due to known conditions such as arthritis)
, Problems remembering things that have happened recently or forgetting to do things
, Loss of interest in what is happening around you or doing things
, Seeing or hearing things that you know or are told are not there
Difficulty concentrating or staying focussed ,
, Feeling anxious, frightened or panicky
, Feeling less interested in sex or more interested in sex
, Feeling light headed, dizzy or weak standing from sitting or lying, p.20
, Finding it difficult to stay awake during activities such as working, driving or eating, p.22
Intense, vivid dreams or frightening dreams ,
, Unpleasant sensations in your legs at night or while resting, and a feeling that you need to move, Talking or moving about in your sleep as if you are 'acting' out a
, Believing things are happening to you that other people say are not true, p.30
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The optimal EFS parameters were determined after using several values of voltage (5, 10 and 15V) and frequencies (10, 16 and 80 Hz) on test samples. We decided to stimulate the ENS from 2 months treated mice using three different pulse durations (200, 400 and 800 µs) and reduced the number to two different pulse durations (200 and 400 µs) for the 4 months treated mice. In all cases a train duration of 30 seconds with a voltage of 10V, a pulse delay of 0 seconds and a pulse rate of 16Hz were used. The reaction of the sample to EFS was determined after the last change of bath solution and after adding each of the following substances, atropine (ATR, 10 -5 M), guanethidine (GUA, 10 -5 M), serotonin (SHT, 10 -4 M) and tetrodotoxin, vol.297, pp.10-16, 2009. ,
, Additionally, to characterize the sudden increases and decreases in tension caused by the addition of the different substances resulting in an excitation or relaxation curve (see Figure?), an interface was created to manually determine the beginning of the curve, the maximum or minimum of the curve and the end of the curve. The program created with Matlab then automatically determined the slope, the amplitude of the increase/decrease, the e-Factor and the e-Exponent of the curve. The effect for each substance concentration on the different an ice-cold eppendorf holder using ice cold protein extraction buffer (60 mM TrisHCl (pH 6,8), 1mM Na3VO4, 1% SDS) (300µl) in the presence of protease and phosphatase inhibitors (phenylmethylsulphonyl fluoride (PMSF, Sigma Aldrich 78830-5g), leupeptin (Roche, 11017101001) and aprotinin (Roche, 10236624001), sodium fluoride, Contractility data for each time-point was translated into a curve by the data acquisition system. Parameters such as maximal force, minimal force, amplitude and frequency before and after each treatment were automatically extracted using a self-made program written with Matlab (version R2014b
, Samples were then diluted in each case to have the same end protein concentration of 30µg and 20 µl of protein extract were loaded together with 5 µl of loading buffer (Invitrogen, Germany, EU) into a 4-containing 5% methanol, at 10V during 3 hours. To verify the efficiency of the transfer, membranes were incubated in a Red Ponceau solution during 2 min and washed 3 times (3*5min) in deionised water. The aspecific signal was then blocked by incubation of the membranes in Tris-buffered saline + 0.1% Tween 20 (TBST 1X) and either 5% (w/v) non-fat dry milk for total protein study or 5% (w/v) bovine serum albumine for phosphorylated-antigens, SDS-PAGE and Western Blotting Protein concentration from intestine extracts was measured using the Nanodrop X100, pp.40101-150
, On the following day, blots were washed 3 x 5 min. with TBST 1X and incubated with horseradish peroxidase-conjugated anti-rabbit and anti-mouse (1:5000, provider) at room temperature for 1 in TBST 1X and developed with the Prime Western Blotting detection Reagent (GE Healthcare Amersham RPN2232) using the LAS3000 bioimager (Fujifilm
, dopamine and electric field stimulation before and after atropine, guanethidine and serotonin addition on the motility of duodenum, jejunum, ileum and colon from vehicle and rotenone treated mice. Our results show that rotenone induces changes in the dose-response curves to carbachol or dopamine in all tested regions. These alterations are region and treatment-time dependent. We did not observe difference in the basal frequency or the basal mean amplitude (max-min) (i.e. after stabilizing the samples and before adding any substances) between the samples coming from rotenone-treated or control mice (Supplementary Figure 1). contractility of the intestine. We compared the GC dose-response curves to carbachol in all different segments from 2 months and 4 months treated control and rotenone-treated mice
, After 4 months of treatment, differences are observed in the slope and in the amplitude of increase of the curve generated by carbachol in rotenone treated mice when compared to controls (slope: p< 0.01;amplitude of increase: p=0.0038, see Figure 2A and 2B). No differences between control and rotenone treated samples were, After two months of treatment, we observed an increased reaction to carbachol in the duodenum of rotenone treated samples when compared to control. Specifically the ?maximum (p<0.001)
, 001) in rotenone-treated samples when compared to control after 2 months (Figure 1D and 1E). Interestingly, the post-pre differences in the frequency (p<0.05), the slope (p<0.01) and the amplitude of increase (p<0.05) were higher in samples coming from control mice (Figure 1F-H), the jejunum, we observed an increase reaction to carbachol in the ?maximum (p<0.05) and the ?mean amplitude (p<0
, Whereas the mean amplitude was higher on rotenone treated samples (p<0.01), the slope (p<0.001), the amplitude of increase (p<0.001) and the e-factor (p<0.05) were higher on control mice (Figure 1I-L). Interestingly, after 4 months of rotenone treatment carbachol induced a higher reaction on samples coming from vehicle treated animals when compared to rotenone treated samples (Figure 2G)(p<0.01). No significant difference was observed in any of the other measured parameters, Interestingly the effect of rotenone on the ileum was more heterogeneous after 2 months of treatment
, After 4 months, rotenone treatment increased the mean amplitude (p<0.05) and decreased the frequency (p<0.05) in rotenone-treated mice when compared to control (Figure 2H-I)
, The differences observed on the effect of AT on the C-Off phase between rotenone treated and control samples was opposite to the one observed in the duodenum, the ileum and the colon. AT induced a significant decrease in the contractility of control mice, the jejunum, we observed no differences in the effect of AT on the R1 or C-Off phases of EFS 200 or EFS 400 after 2 months of treatment (Figure 6E-H), vol.87
, the R1 phase with higher relaxations during EFS 400 in rotenone treated mice (EFS 400: 136.076±36.44, p>0.05) when compared to control samples (EFS 400: 30.042±21.168, p>0.01) (Figure 6K)
, The effect of AT on the C-Off contractility was higher on treated samples, Further 2 months of treatment induced a non-significant higher R1-relaxation in the presence of AT on control mice
, After 4 months of treatment, AT had no effect on the C-Off contractility in control or treated mice (Figure 7N and P). Interestingly, the effect of AT on the R1 EFS-induced relaxation was opposite to all other intestinal regions and to the effect after 2 months of treatment on treated samples. AT diminished the relaxation induced by EFS in both control, p>0.05) and rotenone treated (EFS 200: 230.193%±98.75, p>0.05) samples in the R1 phase during the EFS 200 (Figure 6M), vol.87
, Altogether these results suggest that rotenone treatment affects cholinergic innervation in the intestine. The quality and degree of dysfunction depends on the region affected
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