Here we describe a protocol to determine pulmonary fungal burden in mice with invasive aspergillosis by quantification of Gomori's modified methanamine silver staining in histological sections. Use of this method resulted in comparable results with less animals compared to assessment of fungal burden by quantitative PCR of lung fungal DNA.
The quantification of lung fungal burden is critical for the determination of the relative levels of immune protection and fungal virulence in mouse models of pulmonary fungal infection. Although multiple methods are used to assess fungal burden, quantitative polymerase chain reaction (qPCR) of fungal DNA has emerged as a technique with several advantages over previous culture-based methods. Currently, a comprehensive assessment of lung pathology, leukocyte recruitment, fungal burden, and gene expression in mice with invasive aspergillosis (IA) necessitates the use of a significant number of experimental and control animals. Here the quantification of lung histological staining to determine fungal burden using a reduced number of animals was examined in detail. Lung sections were stained to identify fungal structures with Gomori's modified methanamine silver (GMS) staining. Images were taken from the GMS-stained sections from 4 discrete fields of each formalin-fixed paraffin-embedded lung. The GMS stained areas within each image were quantified using an image analysis program, and from this quantification, the mean percentage of stained area was determined for each sample. Using this strategy, eosinophil-deficient mice exhibited decreased fungal burden and disease with caspofungin therapy, while wild-type mice with IA did not improve with caspofungin. Similarly, fungal burden in mice lacking γδ T cells were also improved by caspofungin, as measured by qPCR and GMS quantification. GMS quantification is therefore introduced as a method for the determination of relative lung fungal burden that may ultimately reduce the quantity of experimental animals required for comprehensive studies of invasive aspergillosis.
IA is an opportunistic infection that may develop in susceptible individuals with congenital or acquired immune deficiencies due to immune suppressive therapy or chronic infection1,2. Primary infection often occurs in the lungs, although in some instances dissemination of Aspergillus fumigatus to the liver, kidneys, heart, and brain may occur, resulting in extensive tissue invasion of hyphae accompanied by severe disease and high rates of mortality1,2. Furthermore, the efficacy of existing pharmacotherapies is limited, and may be further weakened by the emergence of antifungal-resistant strains in the environment3. It is therefore important to understand the mechanisms of fungal virulence and host pathology that promote development or exacerbation of invasive fungal disease.
Murine models remain important for mechanistic IA studies, as they allow researchers to assess the roles of fungal virulence genes and host immune effectors for the establishment and growth of A. fumigatus in vivo4,5. Consequently, multiple strategies have been devised in order to effectively quantify or compare fungal burden in groups of experimental animals6,7. These strategies involve culture-based, biochemical, immunoassay, or qPCR methods, each with distinct advantages and disadvantages. Furthermore, each of these methods involve the dedication of a subset of animals in addition to those sacrificed for assessments of immune effector function, gene expression analysis, and comparative histopathology7. Thus, comprehensive IA studies often require significant numbers of research animals at a significant cost. Effective strategies that reduce experimental time, animal costs, and ethical considerations by utilizing animal tissues for multiple analyses are therefore extremely valuable7.
In this report, a method describing the quantification of GMS staining in histological sections for comparison of the relative fungal burden between experimental groups of mice with IA is introduced. Each step from fungal culture to infection, tissue harvest and processing, and image acquisition and data analysis, is described in detail. Fungal burdens obtained by GMS quantification were compared with qPCR in neutropenic models of IA, and in caspofungin-treated wild-type or eosinophil-deficient mice with IA8. The results show similarity with GMS quantification and qPCR of fungal DNA. This suggests that GMS quantification may be useful to researchers engaged in histological analyses as a supplemental or alternative method of comparison of relative fungal burden in mice with IA, and may ultimately reduce the cost and use of research animals in complex, mechanistic studies.
All animal procedures were approved by the Animal Care and Use Committee of Indiana State University, the host campus of Indiana University School of Medicine — Terre Haute.
1. Preparation of A. fumigatus Conidia for Infection
Figure 1: Hemocytometer representative area used for conidia counting (box, arrow).
2. Immunosuppression and Infection of Murine Model
Figure 2: Experimental schedule. This figure was modified from a previous publication8.
3. Harvest and Preservation of Mouse Lungs for Histological Analysis
4. Harvest and Preservation of Mouse Lungs for DNA Fungal Burden
5. Microscopy and Imaging
6. Using an Image Processing Program to Calculate the Fungal Burden (Figure 3)
Figure 3: Representative screenshots for each step of GMS histological field quantification. (A) Select File > folder with samples to quantify > select image. (B) Select Image > Type > convert to "RGB Stack". Use the right arrow key to select the second of the three given images. (C) Hit "control + shift + T" to bring up the "Threshold" menu settings adjustor. Locate the original .tiff or .jpg image as a reference and open with photo viewer program. Pull the top slider all the way left and adjust the bottom slider until the area selected (in red) is representative of the microscopy image (typically ranging from 130-160 as indicated to the right of the slider. (D) Once selected press "Set" > "OK". Select "Analyze" > "Set Measurements" > check "Area, Area Fraction, Limit to Threshold, Display Label" and leave the bottom settings (the drop-down menu and decimal places) as default > "OK". (E) Finally select "Analyze" > "Measure" (a window with data should appear, or a tab on the windows taskbar will appear labeled "Results"). Please click here to view a larger version of this figure.
7. DNA Fungal Burden
Figure 4 includes a graph of survival of wild type or eosinophil-deficient mice with IA treated with caspofungin. The results show that eosinophil-deficient mice exhibited increased survival when compared to wild-type mice (50% mortality versus 100% mortality, respectively). Figure 5 shows representative GMS staining from neutropenic wild-type and eosinophil-deficient mice with IA treated or untreated with caspofungin. Caspofungin treatment resulted in relative fungal clearance in eosinophil-deficient, but not wild-type mice (right panels), while both groups that did not receive caspofungin were similar (left panels). Figure 6 shows the comparison of fungal burden in caspofungin-treated and control untreated wild-type or eosinophil-deficient mice, using both qPCR of fungal DNA (Figure 6A) and representative GMS quantification of 4 (10X objective) fields (Figure 6B). The results generated from both techniques show that caspofungin treatment results in the most significant fungal burden decrease between wild-type and eosinophil-deficient mice (Figure 6A). However, in untreated mice, only GMS quantification resulted in a significant decrease in mice lacking eosinophils (Figure 6B). When the mean area of whole-lung GMS-stained sections were calculated (4X objective), the differences were similar to the results obtained with 4 representative 10X fields, though with less statistical significance (Figure 6C). Figure 7 shows survival, images of representative GMS staining (4 10X fields), and quantification of fungal burden by both methods in γδ T cell-deficient (TCRδKO) mice that were treated with caspofungin in comparison to control, untreated mice. Caspofungin treatment improved survival (Figure 7A) and fungal burden (Figure 7B-D) in TCRδKO mice. Similar to the results of Figure 6, the results of fungal burden as measured by qPCR (Figure 7B) and representative GMS quantification (Figure 7C) were comparable.
Figure 4: Increased survival in eosinophil-deficient (ΔdblGATA) mice with IA after treatment with caspofungin. 15-30 mice/group. Summary of 3-6 experiments. **** p < 0.0001. This figure was modified from a previous publication8. Please click here to view a larger version of this figure.
Figure 5: Representative images of GMS-stained lung sections from wild-type, eosinophil deficient, and caspofungin-treated or control untreated mice with IA. Representative of 3-4 mice/group. Scale bar is equivalent to 100 µm. This figure was modified from a previous publication8. Please click here to view a larger version of this figure.
Figure 6: Fungal burden in wild-type, eosinophil-deficient mice with IA treated or control treated with caspofungin. (A) qPCR of fungal DNA. 15-30 mice/group, summary of 3-6 experiments. (B) GMS quantification of histological sections, mean% of GMS staining from 4 fields (10x objective). (C) GMS quantification, mean% of whole lung section (4X objective). B and C are a summary of 5 mice/group. * p < 0.05. *** p < 0.001. **** p < 0.0001. This figure was generated using data that were reported in a previous publication8. Please click here to view a larger version of this figure.
Figure 7: Decreased severity of IA in γδ T cell-deficient mice after caspofungin treatment. (A) Survival. (B) qPCR of fungal DNA. (C) GMS quantification of histology. (D) Representative images of GMS-stained sections from γδ T cells with IA, treated or untreated with caspofungin. A and B are a summary of two experiments with 7-10 mice/group. C and D are a summary of 4 mice/group. * p < 0.05. ** p < 0.01. *** p < 0.001. This figure was modified from a previous publication8. Please click here to view a larger version of this figure.
The purpose of this article was to introduce a method for determination of lung fungal burden in mice with IA by utilizing GMS-stained lung histological sections for image analysis and quantification. In this study, treatment with the β-glucan-synthesis-targeting antifungal drug caspofungin14 did not improve survival or fungal burden in neutropenic wild-type mice with IA8. However, in the absence of eosinophils or γδ T cells, survival and fungal burden improved. The results of our study also demonstrated that comparable results may be obtained by GMS fungal burden in comparison to the widely-used fungal DNA qPCR method6.
There are several benefits to utilizing GMS fungal burden quantification. First, the process may utilize existing histological samples, thus potentially reducing the number of experiments required to determine significant differences. Second, in this study, less animals were required for GMS fungal burden quantification to achieve significant differences in comparison to qPCR of fungal DNA (Figure 6, Figure 7). Third, comparison of fungal burden in different isolates by qPCR may be affected by isolate-dependent differences in ribosomal DNA copy number15. In contrast, GMS quantification is not affected by copy number, as fungal burden is determined by relative levels of lung fungal growth. Thus, the use of GMS quantification for fungal burden reduces the use of vertebrate animals and does not require pre-determination of rDNA copy number. Finally, in addition to modifying fungal morphology, caspofungin therapy increases fungal fragmentation, and thus may artificially increase fungal burden when measured by isolation of colony forming units from lung homogenates12,16,17. Thus, GMS quantification of fungal burden avoids several limitations inherent with other commonly used methods.
However, the limitations of GMS quantification and/or this study are important to note. First, the authors assumed a comparable distribution of hyphal growth throughout the lungs of each experimental group, and thus used quantification from 4 representative 10x objective fields as a representative measurement for the fungal burden in the entire lung (Figure 6B). It is possible that in some instances the relative distribution, size, and density of hyphal foci would sufficiently differ so that the fungal burden would appear different with this method and equivalent by the qPCR method. However, our additional results with whole lung section quantification using the 4X objective fields showed similar, albeit less statistically significant, differences between groups (Figure 6C). The standard error of this quantification was increased with this strategy, likely due to decreased hyphal resolution with the 4X objective and increased background in suboptimal fields. Therefore, a representative quantification of fewer fields at higher magnification is preferred. Second, only a single, central section was used for each sample. It is possible, based on the fungal isolates or mice strains used, that some studies may result in an uneven distribution of hyphal growth. In those instances, additional sections throughout each paraffin block should be quantified to obtain a more representative burden. Third, in experiments that induce substantial production of mucins (i.e., quantifying airway fungal growth in allergic bronchopulmonary aspergillosis (ABPA)18 or cystic fibrosis (CF)18), GMS reactivity with polysaccharide-rich mucins19 could yield non-specific GMS+ results and thus skew some samples in favor of higher fungal burden. Since only the neutropenic model of IA was used in this study, it is possible that the use of other immune competent or suppressive models could results in less comparable results. Despite these caveats, GMS quantification provides a comparable technique to determine fungal burden, and its continued use in additional studies may further validate the utility of this method as consistent, reliable, and cost-effective.
The authors have nothing to disclose.
This study was supported in part by an Indiana University School of Medicine Research Enhancement Grant and by NIH-NIAID 1R03AI122127-01. N.A. was partly supported during this period by a Careers in Immunology Fellowship from the American Association of Immunologists.
Aspergillus fumigatus 293 Stock Solution | Fungal Genetics Stock Center | FGSC #A1100 | |
HyPure Cell Culture Grade Water | Thermo Fisher Scientific | SH30529.03 | |
Malt Extract | MP Biomedicals | 2155315 | White Powder |
BD BBL Acidicase Dehydrated Culture Media: Peptone | Fisher Scientific | L11843 | |
Dextrose (D-Glucose) Anhydrous (Granular Powder/Certified ACS) | Fisher Scientific | D16-3 | |
Fisher BioReagents Agar, Powder / Flakes, Fisher BioReagents | Fisher Scientific | BP1423-500 | |
Auto Dry Cabinet | Shanghai Hasuc Instrument Manufacture Co.,LTD | HSFC160FD | |
1300 Series Class II, Type A2 Biological Safety Cabinet | Thermo Fisher Scientific | 1375 | |
0.5 mm Glass Beads | BioSpec Products | 11079105 | |
15 ml Conical Tubes | Thermo Fisher Scientific | 339650 | |
Hemacytometer | Fisher Scientific | # 0267110 | |
Leica Model DME Microscope | Leica | 13595XXX | |
Dulbecco's Phosphate Buffered Saline | Sigma-Aldrich | D8662-500 ml | |
1.5 ml tubes | Fisher Scientific | # 05-408-129 | |
BD Precisionglide syringe needles, gauge 27, L 1/2 in. | Sigma-Aldrich | Z192384 | |
Anti-mouse-Ly-6G antibody | BioXCell | BP0075-1 | Clone 1A8 |
Caspofungin diacetate | Sigma-Aldrich | SML0425 | |
Isoflurane | Henry Schein Animal Health | 1169567762 | |
Non-Rebreathing Table Top Veterinary Anesthesia Machine | Supera Anesthesia Innovations | M3000 | |
Pureline Oxygen Concentrator | Supera Anesthesia Innovations | OC8000 | |
Slant Board Restraint | Indiana State University Facilities Management | Custom made | |
Gilson PIPETMAN Classic 200 ml Pipets | Fisher Scientific | F123601G | |
Pentobarbital Sodium (Fatal-Plus) | Vortech Pharmaceuticals | 0298-9373-68 | |
General-Purpose Broad-Tipped Forceps | Fisher Scientific | 10-300 | |
Fisherbrand Standard Dissecting Scissors | Fisher Scientific | 08-951-20 | |
Fisherbrand General-Purpose Curved Forceps | Fisher Scientific | 10-275 | |
Ethyl Alcohol-200 Proof | PHARMCO-AAPER | 111000200 | |
Fisherbrand Absorbent Underpads | Fisher Scientific | 14-206-63 | |
BD Precisionglide syringe needles, gauge 25, L 1 in. | Fisher Scientific | Z192406 | |
All-Plastic Norm-Ject Syringes | Fisher Scientific | 14-817-30 | |
IV CATH ANGIOCATH 22GX1GIN 50B | Fisher Scientific | NC9742754 | |
Formalin Solution, neutral buffered, 10% | Sigma-Aldrich | HT501128-4L | |
50 ml Conical Tubes | Thermo Fisher Scientific | 339652 | |
Thermo Scientific Shandon Embedding Cassettes II in Tube Packs | Fisher Scientific | B1000729 | |
Fisherfinest Histoplast Paraffin Wax | Fisher Scientific | 22-900-700 | |
Disposable Base Molds | Fisher Scientific | 22-363-554 | |
Reichert Jung Histocut 820 Microtome | Labequip.com | 31930 | |
Water Bath | Precision Scientific | 66630-23 | |
CO2 Incubator | Fisher Scientific | 116875H | |
Silver Stain (Modified GMS) Kit | Sigma-Aldrich | HT100A-1KT | |
Fast Green FCF | Fisher Scientific | AC410530250 | |
Frosted Microscope Slides | Fisher Scientific | 12-550-343 | |
Fisherfinest Superslip Cover Glass | Fisher Scientific | 12-545-89 | |
Cytoseal XYL | Fisher Scientific | 22-050-262 | |
Olympus Provis AX70 Microscope | Olympus | OLYMPUS-AX70 | |
U-PHOTO Universal Photo System | Olympus | OLYMPUS-U-PHOTO | |
U-MCB-2 MULTI CONTROL BOX | Olympus | OLYMPUS-U-MCB-2 | |
U-PS POWER SUPPLY UNIT | Olympus | OLYMPUS-U-PS | |
FreeZone 1 Liter Benchtop Freeze Dry System | LABCONCO | 7740020 | |
Maxima C Plus Vacuum Pump | Fisher Scientific | 01-257-80 | Displacement- 6.1 cfm |
1-Butanol | Fisher Scientific | A383-4 | |
AMRESCO PHENOL-CHLORFORM-OSOAMYL 100ML DFS | Fisher Scientific | NC9573988 | |
Free-Standing Microcentrifuge Tubes with Screw Caps | Fisher Scientific | # 02-682-557 | |
Mini-Beadbeater-24 | BioSpec Products | 112011 | |
BioSpec ProductsSupplier Diversity Partner 2.3 MM ZIRCONIA BEADS | Fisher Scientific | NC0451999 | |
Tris Base (White Crystals or Crystalline Powder/Molecular Biology) | Fisher Scientific | BP152-1 | |
Hydrochloric Acid, Certified ACS Plus | Fisher Scientific | A144S | |
Ethylenediaminetetraacetic Acid, Disodium Salt Dihydrate | Fisher Scientific | S311 | |
Sodium Dodecyl Sulfate (SDS), White Powder, Electrophoresis | Fisher Scientific | BP166 | |
Chloroform/isoamyl alcohol 24:1 | Fisher Scientific | AC327155000 | |
Biotek Epoch Microplate Spectrophotometer | Fisher Scientific | 11-120-570 | |
Thermo Scientific™ ABsolute Blue qPCR Mixes | Fisher Scientific | AB4137A | |
Hybridization Probe, 5′-FAM-AGCCAGCGGCCCGCAAATG-TAMRA-3′ | Integrated DNA Technologies | N/A | |
Sense Amplification Primer, 5′-GGCCCTTAAATAGCCCGGT-3′ | Integrated DNA Technologies | N/A | |
Antisense Amplification Primer, 5′-TGAGCCGATAGTCCCCCTAA-3′ | Integrated DNA Technologies | N/A | |
Applied Biosystems™ MicroAmp™ Optical 8-Tube Strip, 0.2mL | Fisher Scientific | 43-165-67 | |
Thermo Scientific Domed and Flat PCR Cap Strips | Fisher Scientific | AB-0386 | |
Mx3005P QPCR System, 110 Volt | Agilent | 401443 | Stratagene is now owned by Agilent |
BALB/c mice | The Jackson Laboratory | 000651 | |
C57BL/6 (B6) mice | The Jackson Laboratory | 000664 | |
Eosinophil-deficient (ΔdblGATA, BALB/c background) mice | The Jackson Laboratory | 005653 | |
γδ T cell-deficient (TCRδ-/-, B6 background) mice | The Jackson Laboratory | 002120 | |
ImageJ Software | National Institutes of Health (NIH) | N/A | https://imagej.nih.gov/ij/ |
Spot Advanced Software | Spot Imaging | SPOT53A | http://www.spotimaging.com/software/spot-advanced/ |
GraphPad Prism 6 | GraphPad Software | N/A | https://www.graphpad.com/scientific-software/prism/ |
Fisherbrand Absorbent Underpads | Fisher Scientific | 14-206-62 | |
Step 4.5 | http://www.bdbiosciences.com/sg/resources/protocols/paraffin_sections.jsp ; http://www.jove.com/science-education/5039 ; http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/General_Information/1/ht100.pdf |