Label-free Assessment of the Nascent State of Rat Non-alcoholic Fatty Liver Disease Using Spontaneous Raman Microscopy

Abstract

Spontaneous Raman microscopy, which can detect molecular vibrations in cells and tissues, could be a useful tool for the label-free assessment of non-alcoholic fatty liver disease (NAFLD). However, it is unclear whether it can be used to evaluate the nascent state of NAFLD. To address this, we analyzed the Raman spectra of rat liver tissues in the nascent state of NAFLD upon excitation at 532 nm. Raman and histochemical analyses were performed of liver tissues from rats fed a high-fat, high-cholesterol diet (HFHCD). Raman microscopic imaging analysis of formalin-fixed thin tissue slices showed hepatic steatosis, as revealed by the Raman band at 2,854 cm −1 , whereas lipid droplets were not detectable by hematoxylin-eosin staining of images until 3 days after feeding a HFHCD. Raman signals of retinol at 1,588 cm −1 emitted from hepatic stellate cells were distributed alongside hepatic cords; the retinol content rapidly decreased after feeding a HFHCD, whereas hepatic lipid content increased inversely. Raman microscopic analysis of the surface of fresh ex vivo livers enabled early detection of lipid accumulation after a 1-day feeding a HFHCD. In conclusion, spontaneous Raman microscopy can be applied to the label-free evaluation of the nascent state of NAFLD liver tissues.

Raman imaging of rat nonalcoholic fatty liver tissues reveals distinct biomolecular states

“…In the present results, we quantitatively differentiated NAFLD biochemical status by using Raman signals generated from the microchemical environment of the rat livers. Reports on NAFLD tissue analysis by Raman spectroscopy have increased in recent years [10,39–47]. Kochan et al.…”

“…The animal models were created according to previous reports with minor modifications [10,[25][26][27][28][29]. Adult Slc: Sprague Dawley male rats (Shimizu Laboratory Supplies, Japan) at 8 weeks old were maintained under 12-h light/ dark cycles with ad libitum access to food and water.…”

“…In the present results, we quantitatively differentiated NAFLD biochemical status by using Raman signals generated from the microchemical environment of the rat livers. Reports on NAFLD tissue analysis by Raman spectroscopy have increased in recent years [10,[39][40][41][42][43][44][45][46][47]. Kochan et al [48] studied two NAFLD models induced by a HFD (60 kcal %) and a low-carbohydrate high-protein diet by Raman spectroscopy and observed a significant decrease in lipid unsaturation in NAFLD.…”

“…Raman spectroscopy, widely used for the label-free identification of molecules in biological specimens [1][2][3][4], is an emerging tool having the potential to provide biomolecular information for the clinical diagnosis of various human diseases [5][6][7][8]. As Raman spectroscopic measurement is nondestructive and does not necessitate any pretreatment, fresh pathological specimens can be reused after the measurement for the subsequent histological assessments [9,10]. Raman hyperspectral imaging techniques [3] provide structural and chemical information about many molecules, and differences among the states of cells or tissues, for example, diseased or nondiseased, are expected to lead to significant Raman spectral differences.…”

Discrimination of lipid composition and cellular localization in human liver tissues by stimulated Raman scattering microscopy

“…[1][2][3][4][5] Raman microscopy is a technique that has been applied to NAFLD only recently and has great potential in further clarifying the role of lipotoxicity in NASH because it can characterize both the type and spatial localization of relevant lipid molecules. [6][7][8][9][10] Specifically, Minamikawa et al 6 applied Raman microscopy to visualize the distribution of lipid droplets in hepatocytes in NASH model mice liver tissues and demonstrated its capability of characterizing the molecular features of accumulated microvesicular and macrovesicular lipid droplets. Takemura et al 7 utilized spontaneous Raman microscopy to evaluate steatosis and retinol content in rat liver tissues at the nascent state of NAFLD.…”