Pulmonary Disease

**Non-invasive, in vivo imaging in an asthma animal model**

ProSense and analysis by Quantitative Tomography provides:

			Visual discrimination, measurement and quantification of disease progression and therapeutic response in vivo

			Specific protease activation of ProSense by cell subsets (eosinophils)

			*Correlation with conventional, invasive techniques and ex vivo* measurements**

Identification and measurement of heterogeneous areas of fluorescence in 3-D region of lungs

Images:
Reflectance image shows broad distribution fluorescence (protease-activated ProSense) with no apparent upper chest signal to be attributed to lung in the asthmatic mouse.
Quantitative Tomography (FMT 2500) identifies and measures heterogeneous regions of fluorescence.
Investigation: Non-invasive, in vivo imaging and quantification of lung inflammation in an ovalbumin (OVA)-induced asthma BALB/c mouse model.

Measurement of therapeutic response

Localized tomographic images quantified in terms of total lung fluorescence (equivalent to picomoles of activated ProSense) and the volume involved (equivalent to mm³of activated ProSense), comparing asthmatic, treated and negative controls.

A. Total Lung Fluorescence

Total quantified lung fluorescence = protease activity (A)

			Asthmatic mice: >65 pmols of lung fluorescence
			Steroid-treated mice: total lung fluorescence reduced 67%
			Negative controls: <5 pmols of lung fluorescence

B. Fluorescence Volume

Relative magnitude of affected lung tissue determined by fluorescence volumes (B)

			Asthmatic mice: 500 mm3 of lung tissue involved.
			Steroid-treated mice: volume of lung tissue showing disease-related protease activity reduced
			Negative controls: minimal fluorescence volume (~50 mm3) attributed to normal basal protease activity

Eosinophils and macrophages specifically activate ProSense in vivo

Investigation: Bronchoalveolar lavage cells from asthmatic mice were characterized by flow cytometry, using markers for eosinophil, macrophage, neutrophil, and lymphocyte sub-populations to identify the populations responsible for ProSense activation. As expected, the lungs showed a clear and robust influx of inflammatory cells after OVA immunization and intranasal challenge and it was readily established that eosinophils and macrophages are the cell populations that activate ProSense.

Correlation of Quantitative Tomography with conventional, invasive approaches

Bronchoalveolar lavage was performed on asthmatic, treated, and control mice to isolate, characterize and count the different cell sub-populations recruited into the lung.

A. BAL cells

			Eosinophil influx predominates in asthmatic mice
			Dexamethasone-treated and control mice show lower cellular influx, macrophages predominate

BAL/ProSense correlation in individual animals

B. BAL/ProSence correlation in individual animals

Quantitative Tomography data (ProSense fluorescence) correlates with BAL eosinophil numbers

Asthmatic mice: high fluorescence
levels in major and minor airways

Fluorescence reduced in dexamethasone-treated mice

Negligible fluorescence in controls

Image: Ex vivo fluorescence reflectance imaging of lung tissue. Lung tissue was excised from asthmatic, treated, and control mice and individual lung lobes were imaged using a reflectance imaging system.

Background information

Pulmonary diseases, including asthma, form one of today’s largest areas of clinical and therapeutic research, elevating the need for clinically relevant in vivo animal models that can be used to improve understanding of the disease biology and help to develop effective therapeutics. Changes in protease activity are known to be involved in the lung inflammatory response. Conventional assessments of mouse models of lung inflammation rely primarily on invasive measures to determine pulmonary function and terminal characterization of cells infiltrating into the lung. The capacity to non-invasively visualize, measure and quantify the underlying specific biological processes in mouse models in real time would provide a significant advance in characterizing disease processes and the effects of targeted therapeutics.
Detecting protease activity: ProSense is a fluorescence agent activated predominantly by cathepsin B in vivo (with some ability to activate by K, L and S, plasmin, plasma kallikrein, uPA and CD10 in vitro).
Asthma mouse model: The ovalbumin (OVA)-induced asthma BALB/c mouse model is used in research as a clinically relevant facsimile of the human disease, showing cellular influx into the lungs and demonstrating most of the cellular and mechanistic hallmarks of human asthma.