A novel paper MAP method for rapid high resolution histological analysis

Human brain tumor samples

All study procedures were conducted in accordance with the Declaration of Helsinki. This study was approved by the Institutional Review Board (IRB) of Gangnam Severance Hospital, Yonsei University College of Medicine (IRB number: 3-2017-0232 Date of approval: 20 October 2017). The requirement for informed consent was waived by the IRB of Gangnam Severance Hospital because of the retrospective nature of this study. Fresh human brain tumor tissue samples were biopsied from neurosurgical patients operated on at the Gangnam Severance Hospital’s Department of Neurosurgery.

Animal experiments

This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Ministry of Agriculture, Food and Rural Affairs (MAFRA) and approved by the Institutional Animal Care and Use Committee (IACUC) of the Yonsei University College of Medicine (#2017–0230, Date of approval: 10 March 2020). All animal procedures were conducted under veterinarian supervision according to the guidelines imposed by the Ethical Committee. Mice used in these studies were cared for in accordance to the National Institutes of Health “Guide for the Care and Use of Laboratory Animals” and ARRIVE guidelines.

1. 1.

   5xFAD mouse model

2 week-old male 5xFAD (C57BL6) mice were at purchased from Central Lab. Animal Inc. (Seoul, Korea) and were raised in a specific pathogen free (SPF) environment.

1. 2.

   Spinal Cord Injury (SCI) mouse model

8 week-old adult male ICR (Institute of Cancer Research) mice were purchased from Koatech Inc. (Gyeonggi-do, Korea). To induce spinal cord injury, mice were anesthetized with 2% isoflurane, and a dorsal laminectomy was performed at the C5 region of the spinal cord. The dura was removed using microscissors and forceps. In this study, we used two models of spinal cord injury. The lateral hemi-section model involved the use of a microblade in the C5 spinal cord region. In the deletion model, we used a microspatula to remove regions C4-C6 along the dorsal midline blood vessel. In both models, injuries were applied only to the right side of the C5 spinal cord region. Wounds were closed using a 4–0 black silk suture. All mice received 500 μL of sterile saline, cefazolin (25 mg/kg; Bristol Myers Squibb, New York, NY, USA), and buprenorphine (0.05 mg/kg; Reckitt and Colman Pharmaceuticals Inc., VA, USA) for 3 days after surgery. The workflow of for subsequent behavioral tests used to assess the two SCI models is in Supplementary Figs. S1 and S2 online. Mice exhibiting hand cannibalism and/or sudden death, and those that otherwise could not be evaluated with these behavioral tests, were excluded from the study.

1. 3.

   Mouse orthotopic xenograft model for brain glioma tumor

6 to 8-week old male athymic nude mice were purchased from Central Lab. Animal Inc. (Seoul, Korea). 2 × 10⁵ dissociated U87MG cells (ATCC® HTB-14™; American Type Culture Collection, VA, USA) were implanted into the right frontal lobe of mice at a depth of 4.5 mm using a guide-screw system and Hamilton syringe. Gossypol (40 mg/kg; Sigma‐Aldrich Inc., St. Louis, MO, USA) and phenformin (100 mg/kg; Sigma‐Aldrich Inc., St. Louis, MO, USA) were orally administered daily. At either 9 or 26 days post-treatment, mice were euthanized and their brain tissues were harvested. If body weight decreased by more than 15% relative to baseline throughout the duration of the experiment, mice were euthanized according to the guidelines of the approved animal protocol. All experiments were performed as previously described²¹.

Behavioral tests of SCI mouse models

Behavioral tests were first performed 2 days post-surgery, and tests were conducted weekly from 1 to 4 weeks after surgery. Forelimb recovery was assessed using the forelimb locomotor rating scale (FLRS)²⁹, grip strength measurement (GSM)³⁰, forelimb foot fault scoring (FFS)³¹, and the Irvine, Beatties, and Bresnahan (IBB)³² forelimb recovery scale. Hindlimb recovery was assessed using the Basso mouse locomotor scale (BMS), and hindlimb FFS^29,32,33. In addition, body weight was measured 1 h before testing.

1. 1.

   Forelimb locomotor rating scale (FLRS)

Mice were placed inside a transparent acrylic glass path, and mouse movements were recorded twice from the front and the back of the path by two experienced observers. Forelimb function recovery was assessed on a 17-point scale consisting of joint movement, weight support, stepping, predominant paw position, and toe clearance. The average time mice took to pass through the acrylic glass path was approximately 15 s.

1. 2.

   Grip strength measurement (GSM)

Grip strength was measured using a grip strength meter (GSM, TSE Systems; SciPro Inc., London, United Kingdom). Mice were pre-trained three times a week for two weeks. Paper tape was placed on a single forepaw, and the maximum force of grip strength was measured by pulling the tail after the free paw without paper tape caught the GSM bar. Grip strength was tested three times per week for 6 weeks post-injury.

1. 3.

   Foot fault scoring (FFS)

The FFS test was performed using a video-recorded ladder rung walking task. The rung walking apparatus was consisted of two Plexiglas walls (70 cm × 15 cm) with 0.12 cm diameter holes at 5 cm intervals. The holes were filled with 8-cm metal bars (diameter 0.1 cm). Foot fault was scored using the following scale: 0 point (total miss), 1 point (deep slip), 2 points (slight slip), 3 points (replacement), 4 points (correction), 5 points (partial placement) and 6 points (correct placement).

1. 4.

   Irvine, Beatties, and Bresnahan (IBB) forelimb recovery scale

During the test, mice were given two minutes to eat circle-shaped cereal in a transparent acrylic glass cylinder (10-cm diameter) with glass mirrors on each side. Forelimb recovery was assessed via the following: predominant elbow position, proximal forelimb movement, contact non-volar support, predominant forepaw position, contact volar support, cereal adjustments, wrist movement, contact digit movements, and grasping method. Mice were also scored based on the original IBB scale, which consists of predominant elbow position, forepaw position, cereal adjustments, digit movements, and grasping method. Mice were acclimated to the testing environment 2 weeks prior to surgery, during which they were given a cereal diet within the cylinder used in the test.

1. 5.

   Basso mouse locomotor scale (BMS)

Basso mouse locomotor scale (BMS) testing was performed simultaneously with FLRS under similar conditions. After taking two video recordings per animal, FLRS and BMS were verified by video evaluation. In BMS, the scores were evaluated based on 9-point scale consisting of ankle movement, plantar stepping, coordination, paws parallel, trunk stability, and tail movement.

Injured lesion preparation and quantification

At each experimental endpoint, mice were anesthetized with an overdose of zoletil (Virbac, Carros, France) and rompun (Bayer HealthCare, Leverkusen, Germany). The thorax was exposed, and an incision was made in the right atrium of the heart. Trans-cardiac perfusion was performed with equal volumes of ice-cold 0.1 M PBS and 4% paraformaldehyde (PFA) using a 50 mL syringe. After fixation for 1 day in 4% PFA, the solution was replaced with 30% sucrose in 0.1 M PBS. A 3-mm sample of the spinal cord was then harvested without damage to the injury site, which was further processed into 20 µm-thick sections using a cryostat. To analyze the volume of the spinal cord lesion, sections were stained with hematoxylin (Sigma‐Aldrich Inc., St. Louis, MO, USA) and eosin (Sigma‐Aldrich Inc., St. Louis, MO, USA), and the volume was quantified using ImageJ software (National Institutes of Health, MD, USA).

Cell culture

U87 spheres were generated from the U87MG cell line (ATCC® HTB-14™; American Type Culture Collection, VA, USA). Cells were cultured in media, composed of DMEM/F12 (Life Technologies Co., Carlsbad, CA, USA), 10% fetal bovine serum (FBS; Life Technologies Co., Carlsbad, CA, USA), 1 × B27 (Invitrogen Inc., Carlsbad, CA, USA), 20 ng/mL basic fibroblast growth factor, and 20 ng/mL epidermal growth factor (Sigma-Aldrich Inc., St. Louis, MO, USA).

Human salivary gland organoid culture

Human single clonal stem cells were used to establish salivary gland organoids, as previously described²⁶. Cells were seeded in a petri dish coated with 1% of Pluronic F127 (Sigma-Aldrich Inc., St. Louis, MO, USA) in Phosphate-buffered saline (PBS) at a density of 40,000 cells/cm². Suspended cells were cultured in low glucose DMEM media (Life Technologies Co., Carlsbad, CA, USA) supplemented with 10% FBS and 100 U/ml streptomycin-penicillin (Invitrogen Inc., Carlsbad, CA, USA). After 7 days of culture, the established salivary gland organoid was post-treated to suit the purpose of the experiment.

MAP technique

Tissue clearing, denaturation, and expansion were performed according to previously established protocols⁹, which were specifically optimized for the clearing and expansion of whole intact tissues.

1. 1.

   Perfusion of experimental mouse

Mice were anesthetized with 2% isoflurane. Upon opening the thorax, an incision was made in the right atrium of the heart. Trans-cardiac perfusion was performed with equal volumes of ice-cold 0.1 M PBS and 4% PFA solution or either Tissue-MAP solution (30% acrylamide (Sigma‐Aldrich Inc., St. Louis, MO, USA), 0.1% bis-acrylamide (Bio-Rad Laboratories Inc, Hercules, CA, USA), 10% sodium acrylate (Sigma‐Aldrich Inc., St. Louis, MO, USA), 0.05% photoinitiator V-50 (Wako Chemicals, Richmond, VA, USA) in 0.1 M PBS), using a 50 mL syringe. The tissue was harvested following standard protocol^5,6,34.

1. 2.

   Tissue-MAP

Samples fixed in Tissue-MAP were incubated in low acrylamide (A.A) solution (4% acrylamide and 4% PFA in 0.1 M PBS) at 37 °C for 1 day under light protection. The tissue was then washed for 3 h with 0.1 M PBS, then incubated in inactivation solution (1% acetamide (Sigma‐Aldrich Inc., St. Louis, MO, USA), 1% glycine (Sigma‐Aldrich Inc., St. Louis, MO, USA), 0.02% sodium azide (Sigma‐Aldrich Inc., St. Louis, MO, USA) in dH₂O, pH 9.0) at 37 °C for 3 days. After inactivation, the sample was immersed in Tissue-MAP solution at room temperature for 3 days in a shaking incubator. The sample was embedded with nitrogen gas using Easy-Gel (LifeCanvas Technologies, Boston, MA, USA) at 45 °C for 2–4 h, and then transferred to denaturation solution (200 mM sodium dodecyl sulfate [SDS; Affymetrix Inc., Santa Clara, CA, USA], 200 mM sodium chloride [NaCl; Sigma‐Aldrich Inc., St. Louis, MO, USA], 50 mM Tris [Affymetrix Inc., Santa Clara, CA, USA], in dH₂O, pH 9.0). Subsequently, the tissue was incubated at 90 °C in denaturation solution for 2–3 days using EasyClear (LifeCanvas Technology, MA, USA). The denatured tissue was washed three times for 3 h in 0.1 M PBS. The sample was transferred to dH₂O and incubated at room temperature until it was expanded four-fold. Further details are provided in Fig. 1a.

1. 3.

   Paper-MAP

Samples fixed in 4% PFA were sliced into 100 μm thick sections using a Leica VT1000-S vibratome (Leica biosystems, Wetzlar, Germany). Sections were transferred to 0.1 M PBS and washed at room temperature for 30 min. Brain sections were washed three times in 0.1 M PBS for 3 h. Samples were transferred onto a 24 × 60 mm coverslip and embedded into a hybrid polymer by adding Paper-MAP solution (20% acrylamide, 10% sodium acrylate, 0.1% bis-acrylamide, 0.65% TEMED [Sigma‐Aldrich Inc., St. Louis, MO, USA], in 0.1 M PBS), which was left to polymerize for 5 min. Samples were then treated with freshly prepared 5% ammonium persulfate (APS; Sigma‐Aldrich Inc., St. Louis, MO, USA), incubated in denaturation solution at 90 °C for 9–12 h, and washed in 0.1 M PBS. Finally, samples were transferred to dH₂O until they expanded fourfold in size. Further details are provided in Fig. 1b.

Paper-MAP for human SMG organoids

Human submandibular gland (SMG) organoids were fixed with 4% PFA for 1 h and washed three times for 3 h with 0.1 M PBS. Organoids were embedded into a MAP hybrid polymer using Paper-MAP solution, followed by treatment with freshly prepared 5% APS. Gels were sliced into 100 µm sections and incubated in denaturation solution at 95 °C for 30 min. Sections were washed three times for 3 h in 0.1 M PBS, and incubated at room temperature in dH₂O until they expanded fourfold in size.

Paper-MAP for human brain tumor samples

4% PFA fixed human brain tumor samples were embedded in O.C.T. compound (Sakura Finetek USA Inc., CA, USA) at –20 °C. Samples were processed into 60 µm sections using a Leica CM1850 cryostat microtome (Leica biosystems, Wetzlar, Germany). Sections were treated with Paper-MAP solution and 5% APS, then incubated in denaturation solution at 90 °C for 9–12 h. Upon washing in 0.1 M PBS, sections were transferred to dH₂O until they expanded fourfold in size. Further details are provided in Fig. 7d.

Immunostaining and imaging

After tissue clearance and expansion, samples were blocked with 2% bovine serum albumin (BSA; Sigma‐Aldrich Inc., St. Louis, MO, USA) in 0.1 M PBS for 6 h and treated with primary antibodies for 1–4 days at room temperature. Samples were washed 3 times in PBST (0.1% Triton X‐100 [Sigma‐Aldrich Inc., St. Louis, MO, USA] in 0.1 M PBS) for 3–24 h and incubated with conjugated secondary antibodies for 1–4 days. All antibodies and dyes used in this study are listed in Supplementary Table S1 online.

Prior to imaging, tissues were re-expanded in dH₂O at room temperature for 1–24 h. Intact whole tissue samples were placed on a slide inside a U-shaped Blu-Tack adhesive (Bostik, WI, USA), and covered with a glass-bottom Wilco or confocal dish filled with dH₂O. Samples of Paper-MAP can long term storage in dH₂O for until a month at room temperature. Fluorescence-labeled samples of Paper-MAP can long term storage for 1–2 months with shrinkage form in refractive index matching solutions (EasyIndex; LifeCanvas Technologies, Cambridge, MA, USA). All clear images were captured using iPhone-X camera (Apple Inc., Cupertino, CA, USA). Neural fiber and blood vessel images of whole spinal cord tissue were obtained with a LaVision Light-sheet Ultramicroscope (LaVision BioTec GmbH, Bielefeld, Germany) at 2.0 × (0.5 NA) magnification. All images of Paper-MAP were captured using a confocal microscope (LSM780 and LSM980; Carl Zeiss, Oberkochen, Germany) at a magnification of 10 × (0.45 NA, 2.0 mm working distance), 20 × (0.8 NA), and 40 × (0.8 NA). Images were processed and analyzed using Zeiss ZEN-2 software (Carl Zeiss, Oberkochen, Germany), and results were processed into three-dimensional images and videos using Imaris v8.0.1 software (Bitplane, Belfast, United Kingdom).

Quantification of vascular architecture

We compared the lengths, density, and volume fraction of the hippocampal vessels between several sub-regions including the three cornu ammonis (CA1, CA2, CA3) regions and dentate gyrus (DG) in wild type and 5xFAD brains. Upon performing immunostaining for lectin, blood vessel length and volume were quantified using Imaris v8.0.1 software. Images were reconstructed with “Surpass view” on the “Surface” icon in the objects toolbar. To analyze the Region of Interest (ROI), 10 ROIs were selected from each sub-region. “Channel” was used as the source channel, and “Absolute Intensity” was used to adjust thresholds. Results were filtered by the number of voxels using the “Classify Surfaces” tab, according to default settings. Quantification was performed using the “Statistics” tool.