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In addition, anastomotic tissues were collected and assayed by culture for the presence of collagenolytic Enterococcus. Expelled stool samples from each mouse were weighed daily. Luminal and tissue samples were obtained from groups of mice at the time of death. Tissue samples containing mucosa and overlying mucus were homogenized, and divided into two for DNA extraction and culture. Some tissue samples were analysed for collagenolytic Enterococcus. One faecal pellet was used for each stool sample, and 0.
The results of the quantification were used to normalize the amount of DNA for sequencing, and to ensure that each amplicon was represented evenly during sequencing. The final set of de-multiplexed sequences were then selected for exact sequence variant ESV picking using the DeBlur pipeline To improve downstream network analysis, ESVs present in ten or fewer samples were removed.
The data were also rarefied to a depth of reads per sample. To determine significantly different ESVs between groups of interest, the analyses of composition of microbiome pipeline was used, with a P value false discovery rate, FDR cut-off of less than 0.
Enterococcus collagenolytic species producing black colonies surrounded by a clearing halo were counted, and colony-forming unit counts were normalized to the tissue weight.
Data are reported as mean s. Consuming a WD resulted in a significant increase in bodyweight Fig. WD mice showed increased weight loss after antibiotics compared with SD mice.
By POD 3, both groups had experienced similar weight loss. By POD 5, weights in both groups had normalized to preoperative values. The WD resulted in a significant decrease in preoperative and postoperative stool mass Fig. The stool mass in SD mice decreased significantly after surgery, but recovered to preoperative levels by POD 7. Abx, after antibiotics; POD, postoperative day. AHS and individual values. Shaded area indicates anastomotic leak.
Loss of diversity in both luminal content and colonic tissue was observed in WD mice Fig. WD mice had a significant shift in microbiota composition compared with SD mice, characterized by a significant decrease in Bacteroidetes in luminal content and expelled stool, and emergence of Proteobacteria in colonic tissue Fig. Enterococcus abundance was low in both groups: 0. Mean values bold lines , i. Three compartments were analysed using 16S rRNA: expelled stool, colonic luminal content and anastomotic tissue.
To determine whether expelled stool could serve as a trackable biomarker in this model, all three compartments were compared and analysed.
In the expelled stool and luminal content of WD mice, there was a dominance of Enterococcus which comprised 65—90 per cent of the microbiota within the first week after surgery Fig. At the tissue level, differences in the relative abundance of Enterococcus were not as pronounced, but Enterococcus was still significantly more abundant in WD mice Fig. Enterococcus could be detected up to 1 month in expelled stool and anastomotic tissues in WD mice, but was completely absent from SD mice after POD 5 Fig.
In summary, WD mice had a spike in Enterococcus within the first week after surgery and the increase persisted for up to 1 month after surgery.
These changes were consistent between tissue and expelled stool, thus validating expelled stool as a potential marker of microbiota status following colonic surgery. Red arrows indicate disruption of microbiota in the early postoperative phase postoperative day POD 2,3. Green arrows depict reversal in the microbiota toward that of the preoperative preop. Taken that previous studies demonstrated a role for collagenolytic Enterococcus in the pathogenesis of anastomotic leak, culture analysis for collagenolytic Enterococcus was undertaken.
Fold increase in collagenolytic Enterococcus relative to preoperative level in mice fed a standard chow SD and b Western WD diet. To determine whether short-term preoperative switching from a WD to a SD affects anastomotic healing, mice fed a WD for 6 weeks were switched to SD for 2, 4 or 6 days before antibiotic treatment and colonic surgery Fig.
To account for the effect of the chow-based postoperative diet, a separate group of mice received the WD after surgery, resulting in an equally protective effect on anastomotic healing. Taken together, these results indicate that short-term consumption of a SD in the preoperative phase had a significant and durable effect on anastomotic healing independent of the postoperative diet. Prehabilitation with SD had two main effects on mouse weight: weight loss Fig. Short-term dietary prehabilitation was also able to minimize the loss of diversity in the colonic microbiota seen in WD mice Fig.
Box plots in b,c and e show mean values bold line , i. Historically, preoperative dietary modulation to reduce postoperative complications has been a major consideration among surgeons, but mostly involves the use of chemically defined, liquid sterile supplements.
However, their effect size on postoperative complications seems to be diminishing, given the advances in anaesthesia and pain control, and use of minimally invasive techniques and enhanced recovery programmes. Therefore, it may be time to consider the consumption of customary unprocessed foodstuffs beyond their effect on BMI, serum protein markers and other standard nutritional parameters 15 , Although obesity increases the probability of postoperative healing complications, especially infections, it is important to recognize that the majority of obese patients do not develop complications and non-obese patients can experience serious complications including infections That this effect may be mediated, in part, by alterations to the microbiome bears further consideration.
First, Enterococcus faecalis and Pseudomonas aeruginosa were identified as two highly collagenolytic bacteria capable of causing leaks in both rats and mice 8 , The discovery of E. Although E. The present study has demonstrated an important link between diet, antibiotic exposure and anastomotic surgery in terms of their aggregate effect on the microbiome and their potential role in anastomotic healing. An important finding of the present study was the fidelity in the 16S rRNA analysis community structure and membership between expelled stool, luminal content and anastomotic tissues.
This study has some limitations, including that the WD used was completely lacking fibre, in contrast to a standard low-fibre WD. This was done to amplify the effects of fibre in the model. It is important to note that Western dietary fibre typically consists of non-fermentable sources of little benefit to the microbiota As with any murine study, translation to humans requires validation as there are major variations between mouse and human microbiomes; however, the global changes of the microbiota to diet and antibiotics in human and mice tend to be similar All data generated or analysed during this study are included in this published article and in the supporting information.
Disclosure : The authors declare no conflict of interest. Read article at publisher's site DOI : Foods , 10 9 , 19 Sep Biomedicines , 9 10 , 24 Sep Surg Endosc , 04 Oct Cited by: 0 articles PMID: Front Nutr , , 10 May J Gastrointest Surg , 25 11 , 07 Jun This data has been text mined from the article, or deposited into data resources.
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Br J Surg , 10 , 16 Apr In Chen, H. Springer Babaian, T. Springer Chircu, A. Cegielski, Eds. E-Commerce In , Eds. Wiley Encyclopedia of Management. Digital Payments In , Eds. The Handbook of Technology Management. Hoboken, N. J, Hunt, B, Eds. E-Commerce and V-Business. E-commerce Overview In Davis, G. B, Eds. Oxford, U. K: Blackwell Publishing Chircu, A. Lectures in E-Commerce. Duane, J. Jung, A. Blomenhofer, M. Masson, P. Kononchuk, N. Using the classical cecum ligation and puncture model to mimic sepsis [10—12], we found that cecum ligation and puncture led to learning and memory disorders, accompanied by the release of a large number of inflammatory factors.
Metformin has anti-anxiety and anti-depression effects in patients with type 2 diabetes [13, 14]. A large body of literature has shown that the metformin was effective in regulating neurodegeneration because of its anti-apoptotic and antioxidant properties [15, 16].
Metformin could potentially combat certain neurotoxins and malign effects of certain neurodegenerative diseases [17, 18]. Therefore, we hypothesized that metformin has a certain therapeutic potential for cognitive impairment, and might alleviate sepsis-induced cognitive impairment. In the current study, behavioral tests showed that metformin alleviated sepsis-induced learning and memory dysfunction in rats. In addition, metformin reduced inflammation in the hippocampus, restored synaptic protein expression, and improved synaptic damage.
These results suggest that intervention with metformin may be helpful improving learning and memory function following sepsis induced neuronal injury and cognitive impairment, and provide a basis for clinical management of sepsis. The rats were fed a normal rat diet and kept in a h cycle of light and 12 h of darkness, and drank water freely. The rats were randomly divided into different experimental groups and treated according to the experimental requirements.
After anesthesia, the rats could breathe smoothly without turning over, and the depth of anesthesia was assessed via toe pinch. The skin was well cleaned and the area was draped. Sterile gloves were then worn when performing this procedure. A longitudinal incision of about 1 cm was made along the middle of the abdomen, sterile forceps was used to explore the abdomen and the cecum was pulled out.
In sham operation group, only the cecum was pulled out of the abdominal cavity, and then the abdominal cavity was reduced. The cecum was then perforated twice with 18G trocar, with taking care not to damage the mesentery and cecum blood vessels.
After sending out little feces, the cecum was inserted back to the abdominal cavity and the abdominal wall incision was sutured layer by layer. All rats were given subcutaneous normal saline 10 ml and analgesic buprenorphine 0. Rats were placed on hot pads until they recovered from anesthesia and returned to the feeding room for normal feeding.
Rats in each group were dynamically observed in 24 h after operation. In sham operation group, rats could move and eat normally after waking up, there was no significant change in urination and defecation, and the fatality rate was 0 in 24 h. The rats were anesthetized with isoflurane and placed on stereotactic devices. The position of the stereodirectional drilling of the bilateral skull is as follows: 1.
After injection, the needle was left in place to stay for 2 min and then slowly pulled out. The experimental test was carried out after the rats were bought to the laboratory and stayed for 2 day. The distance travelled and the number of times they crossed the intermediate area were tracked and measured to assess their motor ability.
The animals were placed in the laboratory and allowed to stay for 1 day, before the experimental test was carried out. Objects A and B were placed on two corners of the box, and the rat was placed in the center of the box, and allowed to explore freely for 5 minutes. Two hours later, object B was replaced by a new object C, and the rats were subsequently placed in the box for another 5 minutes. Twenty-four hours later, object C was replaced with object D and the rats were again placed in the box for 5 minutes.
The exploration time of object A, B, C and D were recorded respectively. The MWM test was used to measure spatial learning and memory. The rats were put into the test room a day earlier. The rats found a hidden platform in a water maze within 60 s for 5 days, and if the rats did not find the platform within 60 s, they were guided to it and allowed to stay there for 20 s. On day 6, the platform was removed and spatial memory was tested by recording the times of crossing the platform position and the time spent by the rats in the target quadrant.
The rats were quickly sacrificed and their brain tissue was quickly removed, and the brain slices were cut on a concussive sectioning machine. The slices were incubated in artificial cerebrospinal fluid CSF for 30 min, followed by three groups of high-frequency stimulation HFS of Hz, lasting for 1 s. The field excitatory post synaptic potentials fEPSPs were recorded.
After 3 times wash with washing buffer, the second antibody was incubated at room temperature for 1 h followed by another 3 washes. The Odyssey system was used to observe the protein expression level. These kits use a double-antibody sandwich ELISA method to measure inflammatory factors in hippocampus tissues. Color substrate TMB is added. This TMB becomes blue under the catalysis of horseradish peroxidase, and becomes yellow with the addition of termination solution.
To investigate the effect of CLP on cognitive function, 20 healthy SD rats were randomly divided into two groups. The CLP model group was established according to the classic model of sepsis, and the control group underwent sham operation as stated in the methodology.
Rats were placed on hot pads until they recovered from anesthesia and returned to the feeding room for normal feeding, followed by behavioral, electrophysiological, and biochemical tests Fig. Behavioural tests were then carried out to assess cognitive function. The results from open field test showed that there was no significant difference in distance covered and zone crossing between the 2 groups Fig.
Morris Water Maze MWM was used to test memory and learning function, and the result showed that the CLP group have a significantly increase in the latency of finding hidden platform Fig. On day 6, the platform was removed for spatial memory test. Compared with the control group, both the time in the target quadrant Fig.
But there was no significant difference in swimming speed Fig. Taken together, these results suggest that CLP causes learning and memory impairments. Cecum ligation and puncture triggers cognitive deficits in rats. A Experimental design sketch.
The open field test results showed the total distance covered B and zone crossing C. Novel object recognition test showed the time spent exploringthe new object at 2 hours D and 24 h E. Morris water maze showed the latency to find the hidden platform F. On day 6, with the platform removed, the time in the target quadrant G , the number of target platform crossing H and swimming speed were analyzed I.
It is known that the hippocampus HIP plays an important role in learning and memory function [19—22]. In order to investigate the underlying mechanisms how CLP induces learning and memory deficits, we explored the long-term potentiation LTP of the hippocampus which reflects synaptic plasticity [23, 24].
In addition, Western blotting was used to test synaptic associated proteins in the hippocampus. The results Fig. Thus, we concluded that CLP leads to cognitive impairment probably via synaptic dysfunction which is reflected as a decrease in synaptic proteins. Cecum ligation and puncture induces synaptic dysfunction in rats. C , D Brain tissues from hippocampi were homogenized, and synaptic protein levels were detected by immunoblotting. CLP causes a systemic inflammatory response that releases various immune factors and cytokines [11, 25].
To explore whether CLP could induce the release of these hippocampal immune factors, we used an enzyme-linked immunosorbent assay ELISA kit to detect these factors in hippocampal tissue lysates. These results suggest that CLP could activate or trigger the release of inflammatory factors in the hippocampus. Cecum ligation and puncture upregulates the release of inflammatory factors. Hiram Polk and Dr. William Cheadle. The biochemistry major prepared Bobby for a career in medicine.
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