Tracheal bifurcation located at proximal third of oesophageal length in Sprague Dawley rats of all ages

Levrat’s rat model is often the first choice for basic studies of oesophageal adenocarcinoma. The position of the tracheal bifurcation represents the preferred location for the high-intrathoracic anastomosis following oesophagectomy for cancer and is thus of importance in basic research of oesophageal adenocarcinoma. In addition, it is also the typical location for trachea-oesophageal fistulae in congenital oesophageal atresia and its rat model. We thus analysed whether the position of the tracheal bifurcation would be affected by a rat’s growth throughout life. We analysed absolute and relative carinal position of the tracheal bifurcation and its relationship to oesophageal length in two cohorts of Sprague Dawley rats (RjHan:SD) of both sexes: one consisted of 30 eight-week old rats and the other of 20 rats aged between 15 and 444 days. We analysed their relationship by Pearson’s r and univariate linear regression. Bootstrap confidence intervals were calculated for all calculated coefficients. Absolute carinal position correlated with oesophageal length in the eight-week old cohort (r=0.4, 95% CI: 0.08-0.71, p=0.015) and those of different ages (r=0.92, 95% CI: 0.77-0.96, p=0.0066). Absolute carinal position increased with oesophageal length in both cohorts (F(1,28)=5.56; p=0.0256 and F(1,18)=94.93; p<0.0001 respectively). Consequently, relative tracheal bifurcation position was not influenced by oesophageal length in both cohorts (F(1,28)=2.49; p=0.1257 and F(1,18)=1.92; p=0.183). Absolute carinal position increased with oesophageal length, but relative position remained constant at around 30% of proximal oesophageal length throughout life

Development of a 3D simulator for training the mouse in utero electroporation.

In utero electroporation (IUE) requires high-level training in microinjection through the mouse uterine wall into the lateral ventricle of the mouse brain. Training for IUE is currently being performed in live mice as no artificial models allow simulations yet. This study aimed to develop an anatomically realistic 3D printed simulator to train IUE in mice. To this end, we created embryo models containing lateral ventricles. We coupled them to uterus models in six steps: (1) computed tomography imaging, (2) 3D model segmentation, (3) 3D model refinement, (4) mold creation to cast the actual model, (5) 3D mold printing, and (6) mold casting the molds with a mix of soft silicones to ensure the hardness and consistency of the uterus and embryo. The results showed that the simulator assembly successfully recreated the IUE. The compression test did not differ in the mechanical properties of the real embryo or in the required load for uterus displacement. Furthermore, more than 90% of the users approved the simulator as an introduction to IUE and considered that the simulator could help reduce the number of animals for training. Despite current limitations, our 3D simulator enabled a realistic experience for initial approximations to the IUE and is a real alternative for implementing the 3Rs. We are currently working on refining the model

Evaluation of the IUE simulator.

(A-E) The user responses to each of our questions to evaluate the IUE simulator. The X-axis indicates percentage points, while the Y-axis indicates the level of user experience. Statistical comparisons were made according to the ’ ’user’s level of expertise (Expert in white and beginner/intermediate in black). Student’s t-test was used for statistical comparison (n = 3). Significance was set as P-value less than 0.05. Bars in the graph represent SD. (F) indicates the statistical comparison between the questions. The X axes indicate percentage points, whereas the Y axis indicates the respective question. The one-way ANOVA followed by Tukey’s HSD (honestly significant difference) test was used for statistical comparison. Significance was set as P-value less than 0.05. Different letters indicate significant differences. Data are depicted in percentage units.</p

Steps of mold creation from the digital embryo model.

(A) Importation of embryo model into Netfabb. (B) Importation of the posterior ventricle into Netfabb. (C) Creation of a box from the part library; it was scaled to cover the embryo model. (D) The plane cut through the box, the embryo model, and the lateral ventricle model. (E) Merging of the upper and lower parts of the lateral ventricle after the plane cut. (F) Subtraction of the head part of the embryo after the plane was cut from the surrounding box with the "Boolean difference" function. (G) Subtraction of the embryo body model after the plane is cut from w the surrounding box with the "Boolean difference" function. (H) Separation of the embryo’s body mold into two halves to create the final silicon embryo.</p

Simulation of <i>in utero</i> electroporation (IUE).

(A) Opening of the mouse abdominal cavity. (B) Handling of embryos outside of the abdominal cavity. (C) Injection of the DNA-containing solution in the lateral ventricle. (D) Application of voltage through forceps-type platinum electrodes.</p

Assembly of the silicone embryo model.

(A) The upper part of the head of the embryo model. The silicone was poured into the mold and hardened overnight. Due to the partial shape of the lateral ventricle on the mold cover on the right, the silicone contains two cavities representing the respective part of the lateral ventricle. (B) The model of the embryo body as seen above. Using a cover, the remaining shape of the lateral ventricle is represented as two cavities in the embryo model. (C) The upper and lower parts of the embryo model are assembled so that the two parts of the lateral ventricle meet each other. (D) The silicone embryo model was assembled with the upper and lower parts glued. The two parts of the lateral ventricle meet each other due to automatic registration in the design process, forming the complete shape of the lateral ventricle.</p

Questionnaire for the <i>in utero</i> electroporation simulation model.

Questionnaire for the in utero electroporation simulation model.</p

Comparison of the mechanical properties from biosamples and their models.

(A) Statistical comparison between actual embryos and the simulator to evaluate the load [N] per displacement [mm] as a measure of hardness. The two-way ANOVA with Sidak multiple comparison was used to compare the statistics (Embryo’s (E15): n = 19, Embryo model: n = 3). The bars in the graph represent the mean and the SD. For displacement of 1 mm: mean difference = -0.1981, 95% CI -0.3992 to 0.002982. For 1.5 mm displacement: mean difference = -0.3966, 95% CI: -0.5977 to -0.1956, P-valueex vivo uteri as well (uterus(mean = 0.02574) vs. simulator Sh00-20(mean = 0.09674): P-value = 0.0079; uterus versus simulator Sh00-30(mean = 0.1349): P-value<0.0001). Significance was set as P-value less than 0.05. Bars in the graph represent the SD.</p

Steps of mold creation from the digital uterus model.

The mold creation for the uterus model followed the same steps as for the embryo model, but used a three-part design to accommodate the anatomical tube shape of a single uterus horn. (A) and (B) Import of the uterus model into Netfabb and Meshmixer to clean up the model. (C) An additional uterus shape was created within the original, with an offset of 0.5 mm. (D)The offset uterus shape was equipped with cylinders at both ends and a cube at the lower end. (E) The original uterus model and the cube were subtracted from a surrounding box. The printed model consists of the two outer parts that outline the original uterus shape and the merged offset uterus model combined with the cylinders and the box to sit inside the two outer parts.</p