Pre-clinical toxicology is a statutory requirement of drug development and plays a significant role in reducing attrition in drug discovery. Histopathology and indirect methods such as measurement of toxicity-associated systemic markers in blood or urine samples are the state-of-the-art techniques for toxicity evaluation. Further improvements over these conventional techniques are needed to detect signs of drug-induced toxicity at earlier stages with higher sensitivity and specificity. Multiphoton nonlinear imaging techniques such as two-/three-photon microscopy (2PF/3PF), fluorescence lifetime imaging microscopy (FLIM), second/third harmonic generation (SHG/THG) and coherent anti-Stokes Raman scattering (CARS) microscopy can extract complimentary structural and metabolic information of the target tissue in a label-free manner. In this study, we investigated the capability of a multimodal multiphoton microscopy technique (2PF/3PF/SHG/THG/FLIM/CARS) for detecting both functional and structural changes associated with drug-induced toxicity. Cisplatin, a platinum-based chemotherapy drug, is a cytotoxic agent used to treat many types of cancers. Common side effects of Cisplatin include nephrotoxicity and gonadal dysfunction. We obtained multimodal optical images of organs such as kidney, liver, and testis harvested from mice treated with a single dose of Cisplatin (3mg/kg) by intraperitoneal injection. A control group was treated with 0.9% saline. Structural and metabolic biomarkers related to Cisplatin-induced toxicity were identified and characterized from these multimodal optical images obtained ex vivo. The preliminary results suggest that it may be possible to develop a novel platform for drug toxicity identification and assessment based on multimodal nonlinear optical imaging techniques.
Toxicology of the male reproductive system has received increased interest in recent years partly fueled by the growing reports of falling sperm counts and rising reproductive disorders in the human population. Testicular toxicity (TT) in pharmaceutical development is a challenging issue due to the lack of simple and robust screening methods. Currently, histopathologic examination and hormonal evaluation are the commonly used methods to assess TT. Improved biomarker or screening platforms that would allow identification of TT at an earlier stage can have a significant impact on the safety evaluation of pharmaceutical candidates. We investigated the potential of label-free optical nonlinear imaging technologies such as fluorescence lifetime imaging microscopy (FLIM), multi-photon microscopy (MPM) and coherent anti-Stokes Raman scattering (CARS) microscopy to identify novel biomarkers for effective detection of TT. In this study, testicular damage was induced in rats by intraperitoneal injection with 3 mg/kg cisplatin, a chemotherapy drug. Multimodal optical images were obtained from the fixed, unstained testicular tissue sections of untreated and treated rats using a custom-built near-infrared multiphoton imaging system. Structural and biochemical parameters extracted from these images were compared between both groups to identify abnormal features associated with TT in the treated group. By analyzing the complimentary information obtained using these label-free optical imaging technologies, it may be possible to develop a novel platform for evaluation of TT in safety assessment of pharmaceuticals on reproduction and fertility, which reveal these changes at the molecular level and allow observation of these changes at an earlier time point than available today.
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