Wide-field second harmonic generation (SHG) microscopy of rat lung tissue with induced pulmonary arterial hypertension (PAH) revealed characteristic changes in collagen morphology and content, suggesting it could be a novel tool for fast visualization of the fibrotic pathogenesis in PAH, according to a study recently published in Scientific Reports.

The authors induced PAH in 64 outbred white male rats using monocrotaline (MCT) injections at a dose of 60 mg/kg, revealing quantitative and qualitative changes in collagen content, texture, and individual fiber structure in the lung tissue, as reported by Padrez et al.

After 8 weeks of MCT administration, characteristic pathohistological features of PAH became noticeable, including plexiform lesions, which illustrate vascular changes; dilated lesions of the pulmonary arterioles; and classic arteritis with a transmural inflammatory reaction and signs of fibrinoid necrosis. 

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These results could inform future studies on SHG imaging-based diagnosis of PAH, which can implement machines that use accumulated data for algorithm training. Free from human error, wide-field SHG imaging was shown to be a fast, label-free, and nondestructive method, having the capacity to become the technique of choice for the investigation of PAH-associated progression of fibrosis. 

“Moreover, this method possesses the high potential to be used in a wider range of lung diseases or conditions, eg, for assessing pulmonary fibrosis in interstitial lung disease, after lung(s) transplantation, cancer, etc, if a biopsy is taken or the surgical material is available because clinically relevant methods for assessing fibrosis are currently limited,” the authors wrote.

This technique works as a 2-in-1 system, combining 2-photon excitation fluorescence (2PEF) and SHG microscopes. “This allows the simultaneous visualization of collagen structures and surrounding tissue as an endogenous 2PEF signal originates from such metabolic compounds” as nicotinamide adenine dinucleotides, flavins, lipopigments, and porphyrins, all components of lung tissue, the authors explained.

Additionally, this system obtains large SHG images in high resolution in less than 3 minutes without applying any labels or additional staining of the tissues, incorporating various methods of qualitative and quantitative data processing. Overall, this technique effectively analyzes collagen distribution/modification in lung tissue during fibrotic pathogenesis. 

PAH is a clinical syndrome with a grim prognosis. Previous studies show it causes damage to the endothelium, which leads to remodeling of the pulmonary vessels and increases in vascular resistance and blood pressure in the pulmonary artery. Remodeling of large arterial vessels involves fewer elastin fibers, increased collagen production, and calcium deposition, often accompanied by a violation of collagen degradation. This imbalance causes the progression of interstitial and perivascular reactive fibrosis, which could be assessed with wide-field SHG microscopy.


Padrez Y, Golubewa L, Kulahava T, et al. Quantitative and qualitative analysis of pulmonary arterial hypertension fibrosis using wide-field second harmonic generation microscopy. Sci Rep. 2022;12(1):7330. doi:10.1038/s41598-022-11473-5