Assessment of colorectal anastomosis perfusion with confocal laser endomicroscopy − an experimental study

Introduction: Early diagnosis of complicated healing of colorectal anastomosis can increase the chance for salvage surgery and thus reduce overall morbidity. Confocal laser endomicroscopy (CLE) enables in vivo assessment of tissue perfusion without disturbing its integrity. This experimental study evaluates the potential of CLE for postoperative monitoring of colorectal anastomosis. Methods: A hand-sewn colorectal anastomosis was performed in 9 pigs. The animals were subsequently divided into groups with normal (N=3) and ischemic anastomosis (N=6). Microscopic signs of hypoperfusion were evaluated postoperatively at regular intervals using CLE. Results: Uneven saturation of the images was evident in the group with ischemic anastomosis. The epithelium had inhomogeneous edges and more numerous crypt branching was visible. Tissue oedema quantified as the number of crypts per visual field was already more extensive at the first measurement after induction of ischemia. There was also a significant difference between the values measured before and 10 minutes after ischemia ­ 8.7±1.9 vs. 6.0±1.1 (p=0.013). Conclusion: Postoperative monitoring of the colorectal anastomosis using CLE enables prompt detection of perfusion disorders.

Randomized experimental study of two novel techniques for transanal repair of dehiscent low rectal anastomosis.

BACKGROUND: Anastomotic leak after low anterior rectal resection is a dreadful complication. Early diagnosis, prompt management of sepsis followed by closure of anastomotic defect may increase chances of anastomotic salvage. In this randomized experimental study, we evaluated two different methods of trans-anal anastomotic repair. METHODS: A model of anastomotic leak was created in 42 male pigs. Laparoscopic low anterior resection was performed with anastomosis created using a circular stapler with half of the staples removed. Two days later, animals were randomized into a TAMIS (trans-anal minimally invasive surgery) repair, endoscopic suture (ENDO) or control group with no treatment (CONTROL). Signs of intraabdominal infection (IAI), macroscopic anastomotic healing and burst tests were evaluated to assess closure quality after animals were sacrificed on the ninth postoperative day. RESULTS: Closure was technically feasible in all 28 animals. Two animals had to be euthanized due to progressive sepsis at four and five days after endoscopic closure. Healed anastomosis with no visible defect was observed in 10/14 and 11/14 animals in TAMIS and ENDO groups, respectively, versus 2/14 in CONTROL (p < 0.05). Overall IAI rate was significantly lower in TAMIS (4/14; p = 0.006) and ENDO (5/14; p = 0.018) compared to CONTROL (12/14). Burst tests confirmed sealed closure in healed anastomosis with a median failure pressure of 190 (110-300) mmHg in TAMIS and 200 (100-300) mmHg in ENDO group (p = 0.644). CONCLUSION: In this randomized experimental study, we found that both evaluated techniques are effective in early repair of dehiscent colorectal anastomosis with a high healing rate.

Autologous transplantation of mesenchymal stem cells into the portal vein of the miniature pig; a preliminary experiment for NOTES approach.

INTRODUCTION: There is evidence that mesenchymal stem cells (MSCs) could trans-differentiate into the liver cells in vitro and in vivo and thus may be used as an unfailing source for stem cell therapy of liver disease. Combination of MSCs (with or without their differentiation in vitro) and minimally invasive procedures as laparoscopy or Natural Orifice Transluminal Endoscopic Surgery (NOTES) represents a chance for many patients waiting for liver transplantation in vain. METHODS: Over 30 millions of autologous MSCs at passage 3 were transplanted via the portal vein in an eight months old miniature pig. The deposition of transplanted cells in liver parenchyma was evaluated histologically and the trans-differential potential of CM-DiI labeled cells was assessed by expression of pig albumin using immunofluorescence. RESULTS: Three weeks after transplantation we detected the labeled cells (solitary, small clusters) in all 10 samples (2 samples from each lobe) but no diffuse distribution in the samples. The localization of CM-DiI+ cells was predominantly observed around the portal triads. We also detected the localization of albumin signal in CM-DiI labeled cells. CONCLUSION: The study results showed that the autologous MSCs (without additional hepatic differentiation in vitro) transplantation through the portal vein led to successful infiltration of intact miniature pig liver parenchyma with detectable in vivo trans-differentiation. NOTES as well as other newly developed surgical approaches in combination with cell therapy seem to be very promising for the treatment of hepatic diseases in near future.

Two types of autologous cells in stricture development prevention after complete circular endoscopic dissection in minipig.

INTRODUCTION: Complete circular endoscopic dissection (CED) is frequently accompanied with post-operative strictures formation in the esophagus. Various types of therapeutic approaches have recently been tested to prevent these strictures, e.g. cell therapy or stenting. METHODS: Miniature pigs of Gottingen/Minnesota origin (n=10) were used in the study. First, we made the complete CED in the mid esophagus; next, the defect was left untreated or covered with mesenchymal stem cells (MSCs) or a mixture of MSCs and primary oral keratinocytes (pOKs) suspension without/with fully covered self-expandable metallic stent (SEMS). Consequently, we performed a control endoscopy with a stent removal, and necropsy was performed 17-36 days after cells application. RESULTS: All CED procedures were completed successfully without serious complications. Although we were able to detect MSCs or pOKs in the post-CED defects up to the 36th day after transplantation, the combination of MSCs or MSCs/pOKs with or without SEMS application did not prevent post-CED strictures development. The mixture of MSCs and pOKs resulted in the formation of cellular aggregates, which were mainly observed in submucosa, and the post-CED defect was covered with collagen fibers containing a thin scarred epithelium, accompanied by various degrees of reconstruction and integrity. CONCLUSION: Suspension application of autologous MSCs alone or in combination with pOKs with or without SEMS was ineffective in the prevention of strictures formation after complete CED. Nevertheless, the presence of MSCs or pOKs in the post-CED defect was confirmed even 5 weeks after transplantation.

Postoperative monitoring of colorectal anastomosis - experimental study.

INTRODUCTION: Inadequate blood supply is one of the major risk factors for colorectal anastomotic leak. Early postoperative detection of local ischemic changes can predict complicated healing and lead to better outcome. Microdialysis (MD) offers real-time evaluation of adequate bowel perfusion through monitoring of tissue metabolism. The aim of this study was to assess the feasibility of MD for early detection of ischemic changes in colorectal anastomosis. METHOD: Five pigs with end-to-end colorectal anastomosis were included. MD catheter was placed intramurally 5mm from anastomotic edge. Occlusive ischemia was induced after 3 measurements and followed by another 3 hours of monitoring. Tissue levels of different metabolites were measured every 60 minutes before and after ischemia induction. Mann-Whitney test was used to compare pre and post ischemic changes. RESULTS: The monitoring of colorectal anastomosis using MD was technically feasible and associated with no complications. Significant changes caused by local ischemia were observed in decreased levels of glucose or pyruvate and increased levels of lactate and glycerol. All metabolic changes were detectable already in first samples 60 minutes after ischemia induction. CONCLUSION: Postoperative ischemic changes in colorectal anastomosis can be detected by means of microdialysis.Key words: colorectal anastomosis anastomotic leak microdialysis.

Fixation of biomaterial to metallic stent and fixation of stents after circular endoscopic dissection in the esophagus on an animal model.

INTRODUCTION: Complete circular endoscopic (submucosal) resection (CER) performed using the endoscopic submucosal dissection (ESD) technique is burdened with a high incidence of post-operative strictures in the esophagus. The most effective method of preventing them is not known so far; one of the possible methods is to prevent these strictures directly at the site of their formation by covering the defect with a stent. The aim of the study was to find a way to fix a selected biomaterial to a stent, and subsequently, to fix the stent at the CER site to prevent esophageal strictures in an animal model. METHOD: Miniature piglets from the Czech Academy of Sciences breeding unit in Libechov (N=10) were used. Endoscopy was performed using a single-channel endoscope. First, we made two circular mucosal cuts spaced 5 cm apart in the middle esophagus and we performed the CER between them using the endoscopic submucosal dissection technique. After that, we covered the defect with a stent coated with biomaterial (Xe-Derma®) while we tried to prevent stent migration into the stomach. For stent fixation, we tested endo-clips (N=3), the Apollo endoscopic system (N=1) and the suspension technique using two polyamide threads (N=6) anchored in the nasal septum. We performed a control endoscopy, stent removal and subsequent autopsy after 12 weeks. RESULTS: All procedures were completed successfully without serious complications or deaths. Although stents covered with Xe-Derma® were applied to the entire resection area, one case of mediastinitis and one paraesophageal abscess were found during autopsy, most likely due to microperforations caused during the procedure. Histological analysis showed that after contact with the biomaterial, re-epithelisation took place within one week of application to the resection area. Stent migration occurred in each case when the stent was attached to the esophageal wall by endo-clips or with the endoscopic suture system (Apollo). There was no stent dislocation in the cases where the stent was suspended by two polyamide threads. CONCLUSION: We developed a technique of fixing biomaterial to the surface of metallic stents which we used to prevent the formation of esophageal strictures after CER. Distal suspension fixation of the stent with a polyamide thread proved to be the most effective, while fixations by endo-clips or with the endoscopic suture system were ineffective.Key words: benign esophageal strictures circular endoscopic resection endoscopic submucosal dissection complication prevention.

A comparison of two endoscopic closures: over-the-scope clip (OTSC) versus KING closure (endoloop + clips) in a randomized long-term experimental study.

BACKGROUND: Both over-the-scope clip (OTSC) and KING (endoloop + clips) closures provide reliable and safe full-thickness endoscopic closure. Nevertheless, OTSC clip demonstrated significantly inferior histological healing in the short-term follow-up. AIM: To compare OTSC versus KING closure of a perforation with regard to long-term effectiveness and macroscopic and histological quality of healing. METHODS: We performed a randomized experimental study with 16 mini-pigs (mean weight 43.2 ± 11.2 kg). A standardized perforation was performed on the anterior sigmoid wall. KING closure (n = 8) was attained by approximation of an endoloop fixed to the margins of a perforation with endoclips. OTSC closure (n = 8) was performed by deploying OTSC (OVESCO) over the defect. Pigs underwent a control sigmoidoscopy 8 months after the closure to assess the macroscopic quality of healing. Then, autopsy was performed and the rectosigmoid was sent for histopathological assessment. RESULTS: All closures were completed successfully without air leaks. The duration of closure was similar in both techniques (OTSC 17.8 ± 7.6 min vs. KING 19.6 ± 8.8 min). At autopsy, all KING closures (100 %) were healed with a flat scar without signs of leakage. Microscopically, no inflammatory changes were observed after KING closure. In the OTSC group, microscopic ulcers were present in two pigs (25 %), cryptal abscesses in three pigs (38 %) and significant neutrophil accumulation in all eight pigs (P < 0.01). Giant cell granulomas, dysplasia or abundant scarification was not observed in either group. CONCLUSIONS: Both OTSC and KING closures offer a long-term reliable seal of a gastrointestinal perforation without stenosis or fistulas. KING closure provides long-term histologically superior healing.

Time course of spinal doublecortin expression in developing rat and porcine spinal cord: implication in in vivo neural precursor grafting studies.

Expression of doublecortin (DCX), a 43-53 kDa microtubule binding protein, is frequently used as (i) an early neuronal marker to identify the stage of neuronal maturation of in vivo grafted neuronal precursors (NSCs), and (ii) a neuronal fate marker transiently expressed by immature neurons during development. Reliable identification of the origin of DCX-immunoreactive cells (i.e., host vs. graft) requires detailed spatial and temporal mapping of endogenous DCX expression at graft-targeted brain or spinal cord regions. Accordingly, in the present study, we analyzed (i) the time course of DCX expression in pre- and postnatal rat and porcine spinal cord, and (ii) the DCX expression in spinally grafted porcine-induced pluripotent stem cells (iPS)-derived NSCs and human embryonic stem cell (ES)-derived NSCs. In addition, complementary temporospatial GFAP expression study in porcine spinal cord was also performed. In 21-day-old rat fetuses, an intense DCX immunoreactivity distributed between the dorsal horn (DH) and ventral horn was seen and was still present in the DH neurons on postnatal day 20. In animals older than 8 weeks, no DCX immunoreactivity was seen at any spinal cord laminae. In contrast to rat, in porcine spinal cord (gestational period 113-114 days), DCX was only expressed during the pre-natal period (up to 100 days) but was no longer present in newborn piglets or in adult animals. Immunohistochemical analysis was confirmed with a comparable expression profile by western blot analysis. Contrary, the expression of porcine GFAP started within 70-80 days of the pre-natal period. Spinally grafted porcine iPS-NSCs and human ES-NSCs showed clear DCX expression at 3-4 weeks postgrafting. These data indicate that in spinal grafting studies which employ postnatal or adult porcine models, the expression of DCX can be used as a reliable marker of grafted neurons. In contrast, if grafted neurons are to be analyzed during the first 4 postnatal weeks in the rat spinal cord, additional markers or grafted cell-specific labeling techniques need to be employed to reliably identify grafted early postmitotic neurons and to differentiate the DCX expression from the neurons of the host.

Survival and differentiation of human embryonic stem cell-derived neural precursors grafted spinally in spinal ischemia-injured rats or in naive immunosuppressed minipigs: a qualitative and quantitative study.

In previous studies, we have demonstrated that spinal grafting of human or rat fetal spinal neural precursors leads to amelioration of spasticity and improvement in ambulatory function in rats with spinal ischemic injury. In the current study, we characterize the survival and maturation of three different human embryonic stem (ES) cell line-derived neural precursors (hNPCs) once grafted into ischemia-injured lumbar spinal cord in rats or in naive immunosuppressed minipigs. Proliferating HUES-2, HUES-7, or HUES-9 colonies were induced to form embryoid bodies. During the nestin-positive stage, the rosettes were removed and CD184(+)/CD271(-)/CD44(-)/CD24(+) population of ES-hNPCs FAC-sorted and expanded. Male Sprague-Dawley rats with spinal ischemic injury or naive immunosuppressed Gottingen-Minnesota minipigs received 10 bilateral injections of ES-NPCs into the L2-L5 gray matter. After cell grafting, animals survived for 2 weeks to 4.5 months, and the presence of grafted cells was confirmed after staining spinal cord sections with a combination of human-specific (hNUMA, HO14, hNSE, hSYN) or nonspecific (DCX, MAP2, CHAT, GFAP, APC) antibodies. In the majority of grafted animals, hNUMA-positive grafted cells were identified. At 2-4 weeks after grafting, double-labeled hNUMA/DCX-immunoreactive neurons were seen with extensive DCX(+) processes. At survival intervals of 4-8 weeks, hNSE(+) neurons and expression of hSYN was identified. Some hSYN-positive terminals formed putative synapses with the host neurons. Quantitative analysis of hNUMA(+) cells at 2 months after grafting showed comparable cell survival for all three cell lines. In the presence of low-level immunosuppression, no grafted cell survival was seen at 4.5 months after grafting. Spinal grafting of proliferating pluripotent HUES-7 cells led to consistent teratoma formation at 2-6 weeks after cell transplantation. These data show that ES-derived, FAC-sorted NPCs can represent an effective source of human NPCs to be used in CNS cell replacement therapies.

Optimized conditions for mesenchymal stem cells to differentiate into osteoblasts on a collagen/hydroxyapatite matrix.

Collagen/hydroxyapatite (HA) composite scaffolds are known to be suitable scaffolds for seeding with mesenchymal stem cells (MSCs) differentiated into osteoblasts and for the in vitro production of artificial bones. However, the optimal collagen/HA ratio remains unclear. Our study confirmed that a higher collagen content increased scaffold stiffness but that a greater stiffness was not sufficient for bone tissue formation, a complex process evidently also dependent on scaffold porosity. We found that the scaffold pore diameter was dependent on the concentration of collagen and HA and that it could play a key role in cell seeding. In conclusion, the optimal scaffold for new bone formation and cell proliferation was found to be a composite scaffold formed from 50 wt % HA in 0.5 wt % collagen I solution.

Osteogenic differentiation of miniature pig mesenchymal stem cells in 2D and 3D environment.

Mesenchymal stem cells (MSCs) have been repeatedly shown to be able to repair bone defects. The aim of this study was to characterize the osteogenic differentiation of miniature pig MSCs and markers of this differentiation in vitro. Flow-cytometrically characterized MSCs were seeded on cultivation plastic (collagen I and vitronectin coated/uncoated) or plasma clot (PC)/plasma-alginate clot (PAC) scaffolds and differentiated in osteogenic medium. During three weeks of differentiation, the formation of nodules and deposition of calcium were visualized by Alizarin Red Staining. In addition, the production of alkaline phosphatase (ALP) activity was quantitatively detected by fluorescence. The expression of osteopontin, osteonectin and osteocalcin were assayed by immunohistochemistry and Western Blot analysis. We revealed a decrease of osteopontin expression in 2D and 3D environment during differentiation. The weak initial osteonectin signal, culminating on 7(th) or 14(th) day of differentiation, depends on collagen I and vitronectin coating in 2D system. The highest activity of ALP was detected on 21(th) day of osteogenic differentiation. The PC scaffolds provided better conditions for osteogenic differentiation of MSCs than PAC scaffolds in vitro. We also observed expected effects of collagen I and vitronectin on the acceleration of osteogenic differentiation of miniature pig MSC. Our results indicate similar ability of miniature pig MSCs osteogenic differentiation in 2D and 3D environment, but the expression of osteogenic markers in scaffolds and ECM coated monolayers started earlier than in the monolayers without ECM.

[Transrectal hybrid NOTES versus laparoscopic cholecystectomy--a randomized prospective study in a large laboratory animal]. / Transrektální hybridní NOTES versus laparoskopická cholecystektomie--randomizovaná prospektivní studie na velkém laboratorním zvíreti.

INTRODUCTION: NOTES (Natural Orifice Transluminal Endoscopic Surgery) technique was developed to achieve less invasive surgery with the aim to lower frequency of postoperative complications. Cholecystectomy is one of the most frequent elective surgical procedures and is relevant for evaluation of NOTES. The aim of the experimental study was to compare hybrid transrectal and laparoscopic cholecystectomy regarding feasibility and inflammatory response. MATERIAL AND METHODS: A total of 20 pigs weighing 26-56 kg were randomized to laparoscopic or NOTES group. Transrectal approach (15-18 cm from anal edge) was created by needle knife, followed by balloon dilatation and two-channel endoscope was introduced into the abdominal cavity. Cystic artery and duct were clipped and dissected. After extirpation of the gall bladder the colostomy was closed by occlusion loop-and-clip (King' closure) technique. In the laparoscopic group, cholecystectomy was performed by three-port access. Blood samples for evaluation of inflammatory response markers (leukocytes, CRP, interleukin 6) were taken 0, 2nd, 7th and 30th postoperative day, when the experiment ended and pig was euthanized. RESULTS: Two pigs were excluded (1 died early postoperatively for pneumonia, 1 for the rectal closure impossibility after it's laceration during of the gall bladder extirpation). Other pigs survived without complications. Procedure time was significantly longer in NOTES group (134 +/- 27 minutes versus 60 +/- 22 minutes, p < 0.05). White blood cells count and CRP level increased significantly in both groups 2nd and 7th postoperative day and then normalized. Differences between groups were not significant in any of the measured laboratory markers. Sectional finding of exudate and adhesions was comparable in both groups and all transrectal closures were healed. Small subhepatal abscess was found in one pig from NOTES group. CONCLUSION: Transrectal hybrid cholecystectomy is a safe and feasible method with comparable inflammatory responses and longer operating time compared to laparoscopy. A novel loop-and-clip technique was verified as a safe and simple rectal closure.