Picrosirius Red Staining for Semiquantitative Histopathologic Evaluation of Collagen Deposition in Murine Models of Chronic Lung Allograft Rejection

Summary

Picrosirius Red staining is a semiquantitative method for objectively assessing collagen deposition in murine pulmonary fibrotic remodeling in a spatially resolved manner. Apart from evaluating total collagen deposition, Picrosirius Red staining allows for differentiating collagen fibers of different thicknesses.

Abstract

Fibrosis is the pathophysiologic hallmark of chronic rejection after lung transplantation and the foremost hurdle to long-term recipient survival. Several murine lung transplantation models are available for the study of chronic rejection. However, they display heterogeneous results regarding fibrotic changes of the graft, and the histologic extent of fibrosis is mostly reported qualitatively. Therefore, a spatially resolved approach that allows for statistical analysis can aid in evaluating fibrosis in these models. This study presents Picrosirius Red staining for a semiquantitative evaluation of collagen organization in murine lung allografts and compares it to standard Hematoxylin and Eosin, Masson's Trichrome, and Herovici's stains. Staining was performed on sections from two different murine transplantation models based on minor and major histocompatibility complex (MHC) mismatches. The method was established for semiquantitative analysis of collagen organization in whole lung sections. Thus, it can serve as a tool for murine experimental models of fibrotic lung diseases.

Introduction

Lung transplantation is the definite therapeutic option for patients suffering from end-stage lung disease. However, long-term survival is hampered by chronic rejection, affecting 50% of recipients within the first five postoperative years¹. Fibrotic remodeling of the small airways and the lung parenchyma is the histologic hallmark underlying the progressive loss of pulmonary function in chronic lung allograft rejection². Experimentally, chronic lung allograft rejection can be modeled with murine orthotopic lung transplantation between MHC-mismatched mouse strains. Different strain combinations have been proposed to achi....

Protocol

All animal protocols comply with the ethical principles of the 3Rs for humane animal research and were approved by the local veterinary ethical committee (Veterinäramt Kanton Zürich, Switzerland, Study number 45/2014). Likewise, readers should obtain permission from the relevant institutions before performing any procedures on laboratory animals.

1. Sample acquisition

1. Conduct animal experiments according to individual needs and respective ethical approval. A single microsurgeon performs all surgeries without any additional prolonged ischemia times.
   NOTE: For a reproduction of our representative results, obt....

Results

The protocol described above allows for an objective semiquantitative evaluation of collagen deposition in murine lung tissue. Fibrotic remodeling is the pathophysiological hallmark of chronic rejection after lung transplantation. Therefore, Picrosirius Red staining was applied in chronic lung allograft rejection models using left-sided murine orthotopic lung transplantation. The major MHC-mismatched lung transplantation from a BALB/c donor to a C57BL/6 recipient under mild immunosuppression results in fibrotic changes c.......

Discussion

Standard histologic methods such as Hematoxylin and Eosin, and Masson's Trichrome staining are widely used to detect fibrotic changes in murine lungs in a spatially resolved manner^16,19. However, additional methods are often necessary to quantify these changes and evaluate the tissue's collagen composition.

Picrosirius Red staining was first described in 1964 to identify collagen: under transmitted light microscopy, collagen ap.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Acetic acid	Honeywell, Charlotte, USA	33209
Axio Observer	Zeiss, Oberkochen, Germany	4633000956 (serial number)
Coverslip 1.5	Roth, Karlsruhe, Germany	KCY5.1
Formaldehyde 37%	Fisher Scientific, Leicestershire, UK	F/1501/PB15
Meyer's hemalum solution	Merck, Darmstadt, Germany	109249
Picrosirius Red Solution	Morphisto, Offenbach am Main, Germany	13422	alternatives that can be used: ab150681, abcam, Cambridge, UK; SRS250 ScyTek Laboratories, Logan City US
Polarizing filter	Zeiss, Oberkochen, Germany	000000-1121-813
Rotary microtome, HistoCore AUTOCUT	Leica, Wetzlar, Germany	149AUTO00C1, 14051956472
ROTI Histokitt mounting medium	Roth, Karlsruhe, Germany	6638.1
ROTI Plast Paraffin	Roth, Karlsruhe, Germany	6642.5
Rotilabo-embedding cassettes, POM	Roth, Karlsruhe, Germany	K113.1
Superfrost Plus Adhesion Microscope slide	epredia, Portsmouth, UK	J1800AMNZ
Tissue Processor	Leica, Wetzlar, Germany	TP 1020
Software
Fiji software version 2.14.0/1.54f	Open Source
Imaging Software ZEN 3.4.91	Zeiss, Oberkochen, Germany