Supplemental material for Environmental enrichment prevents pup mortality in laboratory mice

<p>Supplemental material for Environmental enrichment prevents pup mortality in laboratory mice by Charlotte S Leidinger, Christa Thöne-Reineke, Nadine Baumgart and Jan Baumgart in Laboratory Animals</p

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

Questionnaire for the in utero electroporation simulation model.</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

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

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

Quality control analysis.

<p>(<b>A</b>) Monitoring of lock mass offset and lock mass ion intensity as function of sample injection. Notice that the lock mass and internal standard TAG 17:1/17:1/17:1 is not detected in injection 07 and 08. Manual inspection of FT MS spectra revealed that the particular sample had not been spiked with internal standards. (<b>B</b>) Assessing the specificity of the PI species profile and intensity across all samples from wild-type mice and the negative control blank samples. Note that in the negative control blank sample (red) a low abundant background ion is detected and falsely identified as PI 40:3. Dubious lipid species can be removed using background subtraction and filtering during subsequent processing in Orange.</p

Screenshots of the ALEX target list generator and ALEX extractor.

<p>(<b>A</b>) The ALEX target list generator allows users to select lipid classes and species to be identified using criteria such as lipid class, adduction, C index, db index and OH index. Individual lipid species including internal standards can also be selected. The ALEX target list generator output is a .txt file with a shortlist of selected lipid species, respective <i>m/z</i> values and accessory lipid features. The ALEX target list generator also supports inclusion of isotope information that can be used for deisotoping and isotope correction [20] by applying algorithms within the auxiliary workflow. (<b>B</b>) The ALEX extractor identifies lipid species, exports intensity data and incorporates accessory lipid features. As input the ALEX extractor requires the location of spectral peak lists generated by the ALEX converter, a target list compiled by the ALEX target list generator and a location to deposit output files. The ALEX extractor features options to specify an <i>m/z</i> tolerance window for lipid identification, to apply a constant <i>m/z</i> offset to correct lipid searches for a constant FT MS calibration offset or to apply a lock mass adjustment routine that automatically corrects lipid searches for drifts in FT MS calibration. The automated lock mass adjustment routine requires specification of well-characterized and ubiquitous lock mass ions in order to estimate the FT MS calibration offset.</p