Glycopyrrolate

All procedures were approved by the Institutional Animal Care and Use Committees at the University of Pennsylvania and the Michael J. Crescenz Veterans Affairs Medical Center and adhered to the guidelines set forth in the NIH Public Health Service Policy on Humane Care and Use of Laboratory Animals (2015).
For the current study, specimens were obtained from a tissue archive of castrated male pigs subjected to a single mild TBI. This tissue archive was also used in Grovola et al. (25 (link)). All pigs were 5 to 6 months old, sexually mature (considered to be young adult), Yucatan miniature pigs at a mean weight of 34 ± 4 kg (total n = 29, mean ± SD). Pigs were fasted for 16 hours then anesthesia was induced with 20 mg/kg of ketamine and 0.5 mg/kg of midazolam. Following induction, 0.1 mg/kg of glycopyrrolate was subcutaneously administered and 50 mg/kg of acetaminophen was administered per rectum. All animals were intubated with an endotracheal tube and anesthesia was maintained with 2% isoflurane per 2 liters O₂. Heart rate, respiratory rate, arterial oxygen saturation, and temperature were continuously monitored throughout the experiment. A forced‐air temperature management system was used to maintain normothermia throughout the procedure.
In order to attain closed‐head diffuse mild TBI in animals, we used a previously described model of head rotational acceleration in pigs (26 (link), 27 (link)). Similar methods were described in Grovola et al. (25 (link)). Briefly, each animal's head was secured to a bite plate, which itself was attached to a pneumatic actuator and a custom assembly that converts linear motion into angular momentum. The pneumatic actuator rotated each animal's head in the coronal plane, reaching an angular velocity between 165 and 185 radians per second (rad/s) for the lower‐level injured group (n = 4) and 230–270 rad/s for the higher‐level injured group (n = 15). Any presence of apnea was recorded (maximum apnea time = 45 s), and animals were hemodynamically stabilized if necessary. No animals were excluded from the study due to apnea or hemodynamic instability. Sham animals (n = 10) underwent identical protocols, including being secured to the bite plate, however, the pneumatic actuator was not initiated. All animals were transported back to their housing facility, monitored acutely for 3 hours, and given access to food and water. Afterward, animals were monitored daily for 3 days by veterinary staff.
At 3 days post‐injury (DPI) (n = 4), 7 DPI (n = 4 at 165–185 rad/s; n = 5 at 230–270 rad/s), 30 DPI (n = 3), or 1‐year post‐injury (YPI) (n = 3), animals were induced and intubated as described above. Sham animals survived for 7 days (n = 4), 30 days (n = 1), or 1 year (n = 5). While under anesthesia, animals were transcardially perfused with 0.9% heparinized saline followed by 10% neutral buffered formalin (NBF). Animals were then decapitated, and tissue was stored overnight in 10% NBF at 4°C. The following day, the brain was extracted, weighed, and post‐fixed in 10% NBF at 4°C for 1 week. To block the tissue, an initial coronal slice was made immediately rostral to the optic chiasm. The brain was then blocked into 5 mm thick coronal sections from that point by the same investigator. This allowed for consistent blocking and section coordinates across animals. All blocks of tissue were paraffin‐embedded and 8 µm thick sections were obtained using a rotary microtome.
Four sections from each specimen––one containing striatal tissue (approximately 10 mm anterior to the optic chiasm), one containing anterior aspects of hippocampal tissue (approximately 10 mm posterior to the optic chiasm), one containing posterior aspects of hippocampal tissue (approximately 15 mm posterior to the optic chiasm), and one containing cerebellar tissue (approximately 35 mm posterior to the optic chiasm)––were used for the ensuing Amyloid Precursor Protein (APP) histological analysis. Additional histological analysis examined only two sections from each specimen––one containing anterior aspects of hippocampal tissue and one containing posterior aspects of hippocampal tissue, as these sections displayed increased APP pathology in specific neuroanatomical regions. Histological analysis of the corpus callosum only included sections with anterior hippocampal tissue, as sections with posterior hippocampal tissue did not contain corpus callosum.
For 3,3′‐Diaminobenzidine (DAB) immunohistochemical labeling, we used a protocol outlined in Johnson et al. (5 (link)). Briefly, slides were dewaxed in xylene, rehydrated in ethanol and de‐ionized water. Antigen retrieval was completed in Tris‐EDTA buffer pH 8.0 using a microwave pressure cooker then blocked with normal horse serum. Slides were incubated overnight at 4°C using either an anti‐mouse APP (22C11) (Millipore, MAB348, 1:80,000), an anti‐mouse GFAP (SMI‐22) (Millipore, NE1015, 1:12,000), or an anti‐rabbit Iba‐1 (Wako, 019‐19741, 1:4000) primary antibody. The following day, slides were rinsed in PBS and incubated in a horse anti‐mouse/rabbit biotinylated IgG secondary antibody (VECTASTAIN Elite ABC Kit, Vector Labs, PK‐6200). Sections were rinsed again, then incubated with an avidin/biotinylated enzyme complex (VECTASTAIN Elite ABC Kit), rinsed again, and incubated with the DAB enzyme substrate (Vector Labs, SK‐4100) for 7 min. Sections were counterstained with hematoxylin, dehydrated in ethanol, cleared in xylene, and finally coverslipped using cytoseal. All sections were stained in the same histological sample run. All sections were imaged and analyzed at 20× optical zoom using an Aperio CS2 digital slide scanner (Leica Biosystems Inc., Buffalo Grove, IL).
For Luxol Fast Blue (LFB) staining, slides were dewaxed in xylene, and rehydrated in ethanol and deionized water. Slides were placed in a solution of 0.1% Solvent Blue 38 (Sigma, S‐3382) and 95% ethanol warmed to 60°C for 4 h, then differentiated in a lithium carbonate solution followed by 70% ethanol. Slides were counterstained in cresyl violet solution (Sigma, C5042), dehydrated in ethanol, cleared in xylene, and finally coverslipped using cytoseal. All slides were stained for LFB in the same histological sample run.
To assess brain atrophy, we examined the size of the lateral ventricle during gross pathological evaluation at the level of our initial coronal slice made immediately rostral to the optic chiasm. To measure the ventricle‐to‐brain ratio, we drew a region of interest to contain all brain parenchyma followed by regions of interest containing the lateral ventricles to determine area using ImageJ software. Ventricle‐to‐brain ratio was calculated as the total ventricular area divided by total brain area, multiplied by 100 so that the ratio is reported in whole numbers.
For APP semi‐quantitative analysis, we initially characterized four specimens (three 7 DPI and one sham) and stained sections every 5 mm throughout the brain and brainstem for APP. Based on these slides, we identified six anatomical regions that contained APP pathology: periventricular white matter, striatum, ventral thalamus, dorsal thalamus, fimbria/fornix, and cerebellum. These regions were assessed by two blinded observers in the four previously described tissue sections for every specimen and given a 0–3 pathological burden score based on the amount of APP+ axons in the region (Figure 1A–C). The scores were summed then divided by the number of anatomical regions to provide a single, averaged pathological score for each specimen.
For astrocyte semi‐quantitative analysis, hippocampus and periventricular white matter were assessed in two sections per specimen, as well as inferior temporal gyrus and cingulate gyrus––two anatomical regions without APP pathology. We have adapted a semi‐quantitative scale from Sofroniew et al. to histologically classify the progressive severity of reactive astrocytes (28 (link)). Each region was given a 0–3 glial fibrillary acidic protein (GFAP) reactivity score based on cell body size and density of GFAP+ cells in the region (Figure 2A–D). The scores were summed then divided by the number of anatomical regions to provide a single, averaged reactivity score for each specimen.
For microglia cell density, Fiji software (National Institute of Health) was used to convert the ionized calcium‐binding adapter protein‐1 (Iba‐1)‐stained images to grayscale and perform color deconvolution, and then the “Analyze Particles” plugin was used to count cells in an automated fashion using an objective set of exclusion parameters (29 (link)). Particles less than 20 µm² were excluded as these tended to be DAB‐stained microglial processes in the field of view, detached from a microglial cell body.
For Iba‐1 skeletal analysis, we employed methods similar to Morrison et al., who imaged three coronal brain sections per animal twice, once in each left and right hemisphere, for analysis (23 (link)). The current study imaged five 40× images per anatomical region for analysis. The number of images were determined by power analysis of pilot skeletal analysis data. Specifically for the pilot study, we analyzed one image in the hippocampal molecular layer from one animal at each time point post‐injury and calculated the effect sizes. A moderate effect size (Cohen's d = 0.50) was observed between several groups. Using this effect size, an a priori power analysis required a sample size of five images per animal to achieve a power of 0.80. To conduct skeletal analysis, all Iba‐1 positive cells in each 40× field were manually selected, and the image was deconvoluted using Fiji software. Bandpass filters, unsharp mask, and close plugins were applied before converting the image to binary, skeletonizing, and removing skeletons not overlaid with the manually selected cells (Figure S1). The Analyze Skeleton plugin was then applied to quantify the skeletal features such as number of process branches, junctions, process endpoints, and slab voxels in order to measure changes in microglia ramification (30 (link)). For each image, each feature was summed then divided by the total number of cells, thus providing a single field average normalized per cell. Therefore, we examined five values from five images in the same histological slide for each animal in each anatomical region, which serves as a repeated measure, regional analysis. Slab voxels were then multiplied by the volume of the voxel to calculate the summed process length per cell.
For LFB analysis, measurements of the superior to inferior extent of the corpus callosum were obtained at five points along the mediolateral extent: at its juncture with the cingulate gyrus in both hemispheres as the lateral boundaries, at the midline of the corpus callosum, and midway between the corpus callosum midline and these lateral boundaries. These five measurements were averaged for each specimen. To measure the color intensity of the staining, the RGB color components were measured in ImageJ on a 0–255 AU scale. The scale for the blue color component was then inverted so that a zero value would indicate the whitest color while a 255 value would indicate the bluest color.
Statistical analysis was performed using GraphPad Prism statistical software (GraphPad Software Inc. La Jolla, CA). Due to low sample size, the 230–270 rad/s injured group's APP, GFAP, Iba‐1 cell density, corpus callosum thickness, and LFB color intensity data were analyzed with a Kruskal–Wallis test and Dunn's multiple comparisons. Kruskal–Wallis test results are reported as (H (degrees of freedom) = H test statistic, p value). The 165–185 rad/s injured group's APP data were analyzed via a two‐tailed Mann–Whitney test. Mann–Whitney results are reported as (U = U test statistic, p value). Nonlinear regression lines were created via an exponential growth equation. Goodness of fit is quantified using the standard deviation of the residuals (Se), the vertical distance (in Y units) of the experimental data point from the regression line, with a lower Se score indicative of a better predictive model. The skeletal analysis was statistically assessed via one‐way analysis of variance (ANOVA) and Tukey's multiple comparisons test. One‐way ANOVA results are reported as (F (degrees of freedom numerator/degrees of freedom denominator) = F value, p value). Mean, standard deviation, and 95% confidence intervals were reported. Differences were considered significant if p < 0.05. As this was an archival study, power calculations were not used to determine the number of specimens for each experimental group; the current study used all available specimens exposed to a single mild TBI. The number of images chosen for skeletal analysis was determined by power analysis from a pilot study.