Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry (LCM-LC/MS)

Summary

Here, we describe a protocol using laser capture microdissection coupled with LC/MS analysis to spatially-quantify drug distributions within pulmonary tuberculosis granulomas. The approach has broad applicability to quantifying drug concentrations within tissues at high spatial detail.

Abstract

Tuberculosis is still a leading cause of morbidity and mortality worldwide. Improvements to existing drug regimens and the development of novel therapeutics are urgently required. The ability of dosed TB drugs to reach and sterilize bacteria within poorly-vascularized necrotic regions (caseum) of pulmonary granulomas is crucial for successful therapeutic intervention. Effective therapeutic regimens must therefore contain drugs with favorable caseum penetration properties. Current LC/MS methods for quantifying drug levels in biological tissues have limited spatial resolution capabilities, making it difficult to accurately determine absolute drug concentrations within small tissue compartments such as those found within necrotic granulomas. Here we present a protocol combining laser capture microdissection (LCM) of pathologically-distinct tissue regions with LC/MS quantification. This technique provides absolute quantification of drugs within granuloma caseum, surrounding cellular lesion and uninvolved lung tissue and, therefore, accurately determines whether bactericidal concentrations are being achieved. In addition to tuberculosis research, the technique has many potential applications for spatially-resolved quantification of drugs in diseased tissues.

Introduction

The ability to spatially resolve and quantify drug levels is a crucial requirement for determining whether anti-tuberculosis drugs reach bacterial subpopulations within pulmonary lesions at sterilizing concentrations¹. Of particular importance is determining drug penetration into the necrotic core of the lesion (called caseum), which typically contains the highest number of bacilli and may be poorly accessible to drugs due to the absence of vascularization.

Traditional methods to assess lesion penetration, which involve homogenization of excised pulmonary lesions followed by solvent extraction and liquid chromatograp....

Protocol

All animal studies were carried out in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health with approval from the Institutional Animal Care and Use Committee of the NIAID (NIH), Bethesda, MD.

1. Animal Experiments and Tissue Collection

This section of the protocol describes animal procedures and sample collection under Biosafety Level 3 (BSL3) conditions. Detailed protocols of the Mycobacterium tuberculosis aerosol infection procedure and drug administration protocols in rabbits have been described previously^11,

Results

An overview of the LCM-LC/MS approach is shown in Figure 1. After sterilizing the tissue by gamma-irradiation, all subsequent steps (from tissue sectioning onwards) take place outside of BSL3 conditions. Figure 2 shows the lesion biopsy sections before and after tissue isolation by LCM. Necrotic and cellular areas of TB lesions can be easily identified and isolated by visual inspection of optical images alone (without the require.......

Discussion

Spatially-resolved quantification of drugs within pulmonary TB lesions is required to determine whether drug exposure reaches sterilizing concentrations to bacterial populations residing within the different lesion compartments. The LCM-LC/MS method described here enables absolute quantification of anti-TB drugs within all lesion compartments, including the bacteria-rich caseum, using only 1 - 3 tissue sections in total. Traditional tissue homogenization and LC/MS approaches for drug quantification in tissue often lack t.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
New Zealand White rabbits	Covance	N/A
HN878 Mycobacterium tuberculosis	BEI Resources	NR-13647
Ketathesia (Ketamine) 100 mg/mL C3N	Henry Schein Animal Health	56344
Anased (Xylazine) 100 mg/mL	Henry Schein Animal Health	33198
Euthasol (pentobarbital sodium and phenytoin sodium) Solution	Virbac	710101
Acetonitrile (LC-MS grade)	Fisher	A955-212
Methanol (LC-MS grade)	Fisher	A456-212
Formic Acid (LC-MS grade)	Fisher	A117-50
Water (LC-MS grade)	Fisher	W6212
0.2 mL flat-cap PCR tubes	Corning	07-200-392
Steel frames, PET-membrane	Leica	11505151
Premium Frosted Microscope Slides	Fisher	12-544-2
96 Deep well plate 2.0ML PP RB	Fisher	NC0363259
Zorbax SB-C8 column (4.6 by 50 mm; particle size, 3.5 μm)	Agilent	820631-001D
"Zipper” Seal Sample Bags	Fisher	01-816-1B
Name	Company	Catalog Number	Comments
Equipment
CM1850 cryostat	Leica	Discontinued	Leica CM1860 is the current model
Laser Microdissection System 6500	Leica	Discontinued	Leica LMD 6 is the current model
Agilent 1260 Infinity II HPLC	Agilent
API 4000 QTRAP Mass Spectrometer	Sciex