AIM To establish a rat model of anal sphincter injury and test different systems to provide stem cells to injured area.METHODS Adipose-derived stem cells(ASCs) were isolated from BDIX rats and were transfected with gr...AIM To establish a rat model of anal sphincter injury and test different systems to provide stem cells to injured area.METHODS Adipose-derived stem cells(ASCs) were isolated from BDIX rats and were transfected with green fluorescent protein(GFP) for cell tracking. Biosutures(sutures covered with ASCs) were prepared with 1.5 × 10~6 GFPASCs, and solutions of 10~6 GFP-ASCs in normal saline were prepared for injection. Anorectal normal anatomy was studied on Wistar and BDIX female rats. Then, we designed an anal sphincter injury model consisting of a 1-cm extra-mucosal miotomy beginning at the anal verge in the anterior middle line. The sphincter lesion was confirmed with conventional histology(hematoxylin and eosin) and immunofluorescence with 4', 6-diamidino-2-phenylindole(commonly known as DAPI), GFP and α-actin. Functional effect was assessed with basal anal manometry, prior to and after injury. After sphincter damage, 36 BDIX rats were randomized to three groups for:(1) Cell injection without repair;(2) biosuture repair; and(3) conventional suture repair and cell injection. Functional and safety studies were conducted on all the animals. Rats were sacrificed after 1, 4 or 7 d. Then, histological and immunofluorescence studies were performed on the surgical area.RESULTS With the described protocol, biosutures had been covered with at least 820000-860000 ASCs, with 100% viability. Our studies demonstrated that some ASCs remained adhered after suture passage through the muscle. Morphological assessment showed that the rat anal anatomy is comparable with human anatomy; two sphincters are present, but the external sphincter is poorly developed. Anal sphincter pressure data showed spontaneous, consistent, rhythmic anal contractions, taking the form of "plateaus" with multiple twitches(peaks) in each pressure wave. These basal contractions were very heterogeneous; their frequency was 0.91-4.17 per min(mean 1.6980, SD 0.57698), their mean duration was 26.67 s and mean number of peaks was 12.53. Our morphological asses展开更多
AIM To efficiently replicate the biology and pathogenesis of human esophageal adenocarcinoma(EAC) using the modified Levrat model of end-to-side esophagojejunostomy. METHODS End-to-side esophagojejunostomy was perform...AIM To efficiently replicate the biology and pathogenesis of human esophageal adenocarcinoma(EAC) using the modified Levrat model of end-to-side esophagojejunostomy. METHODS End-to-side esophagojejunostomy was performed on rats to induce gastroduodenoesophageal reflux to develop EAC. Animals were randomly selected and serially euthanized at 10(n = 6),17(n = 8),24(n = 9),31(n = 6),38(n = 6),and 40(n = 6) wk postoperatively. The esophagi were harvested for downstream histopathology and gene expression. Histological evaluation wascompleted to determine respective rates of carcinogenic development. Quantitative reverse transcriptionpolymerase chain reaction was performed to determine gene expression levels of MUC2,CK19,and CK20,and results were compared to determine significant differences throughout disease progression stages.RESULTS The overall study mortality was 15%. Causes of mortality included anastomotic leak,gastrointestinal hemorrhage,stomach ulcer perforation,respiratory infection secondary to aspiration,and obstruction due to tumor or late anastomotic stricture. 10 wk following surgery,100% of animals presented with esophagitis. Barrett's esophagus(BE) was first observed at 10 wk,and was present in 100% of animals by 17 wk. Dysplasia was confirmed in 87.5% of animals at 17 wk,and increased to 100% by 31 wk. EAC was first observed in 44.4% of animals at 24 wk and increased to 100% by 40 wk. In addition,two animals at 38-40 wk post-surgery had confirmed macro-metastases in the lung/liver and small intestine,respectively. MUC2 gene expression was progressively down-regulated from BE to dysplasia to EAC. Both CK19 and CK20 gene expression significantly increased in a stepwise manner from esophagitis to EAC. CONCLUSION Esophagojejunostomy was successfully replicated in rats with low mortality and a high tumor burden,which may facilitate broader adoption to study EAC development,progression,and therapeutics.展开更多
基金Supported by Spanish Ministry of Health and Consumer Affairs,No.PI060305
文摘AIM To establish a rat model of anal sphincter injury and test different systems to provide stem cells to injured area.METHODS Adipose-derived stem cells(ASCs) were isolated from BDIX rats and were transfected with green fluorescent protein(GFP) for cell tracking. Biosutures(sutures covered with ASCs) were prepared with 1.5 × 10~6 GFPASCs, and solutions of 10~6 GFP-ASCs in normal saline were prepared for injection. Anorectal normal anatomy was studied on Wistar and BDIX female rats. Then, we designed an anal sphincter injury model consisting of a 1-cm extra-mucosal miotomy beginning at the anal verge in the anterior middle line. The sphincter lesion was confirmed with conventional histology(hematoxylin and eosin) and immunofluorescence with 4', 6-diamidino-2-phenylindole(commonly known as DAPI), GFP and α-actin. Functional effect was assessed with basal anal manometry, prior to and after injury. After sphincter damage, 36 BDIX rats were randomized to three groups for:(1) Cell injection without repair;(2) biosuture repair; and(3) conventional suture repair and cell injection. Functional and safety studies were conducted on all the animals. Rats were sacrificed after 1, 4 or 7 d. Then, histological and immunofluorescence studies were performed on the surgical area.RESULTS With the described protocol, biosutures had been covered with at least 820000-860000 ASCs, with 100% viability. Our studies demonstrated that some ASCs remained adhered after suture passage through the muscle. Morphological assessment showed that the rat anal anatomy is comparable with human anatomy; two sphincters are present, but the external sphincter is poorly developed. Anal sphincter pressure data showed spontaneous, consistent, rhythmic anal contractions, taking the form of "plateaus" with multiple twitches(peaks) in each pressure wave. These basal contractions were very heterogeneous; their frequency was 0.91-4.17 per min(mean 1.6980, SD 0.57698), their mean duration was 26.67 s and mean number of peaks was 12.53. Our morphological asses
基金Samantha Martin for providing statistical support
文摘AIM To efficiently replicate the biology and pathogenesis of human esophageal adenocarcinoma(EAC) using the modified Levrat model of end-to-side esophagojejunostomy. METHODS End-to-side esophagojejunostomy was performed on rats to induce gastroduodenoesophageal reflux to develop EAC. Animals were randomly selected and serially euthanized at 10(n = 6),17(n = 8),24(n = 9),31(n = 6),38(n = 6),and 40(n = 6) wk postoperatively. The esophagi were harvested for downstream histopathology and gene expression. Histological evaluation wascompleted to determine respective rates of carcinogenic development. Quantitative reverse transcriptionpolymerase chain reaction was performed to determine gene expression levels of MUC2,CK19,and CK20,and results were compared to determine significant differences throughout disease progression stages.RESULTS The overall study mortality was 15%. Causes of mortality included anastomotic leak,gastrointestinal hemorrhage,stomach ulcer perforation,respiratory infection secondary to aspiration,and obstruction due to tumor or late anastomotic stricture. 10 wk following surgery,100% of animals presented with esophagitis. Barrett's esophagus(BE) was first observed at 10 wk,and was present in 100% of animals by 17 wk. Dysplasia was confirmed in 87.5% of animals at 17 wk,and increased to 100% by 31 wk. EAC was first observed in 44.4% of animals at 24 wk and increased to 100% by 40 wk. In addition,two animals at 38-40 wk post-surgery had confirmed macro-metastases in the lung/liver and small intestine,respectively. MUC2 gene expression was progressively down-regulated from BE to dysplasia to EAC. Both CK19 and CK20 gene expression significantly increased in a stepwise manner from esophagitis to EAC. CONCLUSION Esophagojejunostomy was successfully replicated in rats with low mortality and a high tumor burden,which may facilitate broader adoption to study EAC development,progression,and therapeutics.
