One of suspect environmental endocrine disruptors that affect mouse male reproduction by altering the morphology of Sertoli cells and spermatogenic cells is phthalate. The effects of mono(2-ethylhexyl)phthalate (MEHP), one of metabolites of di(2-ethylhexyl)phthalate , on immature mouse testes in vivo were examined. We have recently shown
that MEHP induced Sertoli cells necrosis and spermatogenic cells apoptosis in mice by TUNEL method, F-actin staining,
and ultrastructural study, but there is no data for biochemical changing of testes due to those methods could not explore.
To verify in detail of it, we conducted Raman spectroscopy study with 785 nm wavelength laser line, 50mW of laser power and 3 minutes of exposure time to analysis the MEHP-treated testicular tissue, which has been fixatived by 4% paraformaldehyde (PFA). Five weeks old (5 w.o) male mice were used in this experiment. As the results, the alterations were observed by Raman spectroscopy that there are significantly differences of DNA, actin filament, type IV collagen and amide I between control group (0 μM MEHP) and treatment group (100 μM MEHP). These results significantly support histology staining observation (such as the apoptotic spermatogenic cells which is associated with DNA fragmentation and F-actin disruption) and ultrastructural observation (such as mitochondria rupture and disintegration of nucleus membrane). Raman spectroscopy can be used for 4% PFA-fixatived tissue observation. However, we recommend that Raman spectroscopy may be able to be expanded as an armamentarium not just for the clarification of histology staining and ultrastructural study, but furthermore, it may be as a non-invasion assessment for screening animal tissue toxicity of chemical in future.
An optical biopsy system which equips miniaturized Raman probes, a miniaturized endoscope and a fluorescent image
probe has been developed for in vivo studies of live experimental animals. The present report describes basic optical
properties of the system and its application studies for in vivo cancer model animals and ex vivo human cancer tissues. It
was developed two types of miniaturized Raman probes, micro Raman probe (MRP) made of optical fibers and ball lens hollow optical fiber Raman probe (BHRP) made of single hollow optical fiber (HOF) with a ball lens. The former has rather large working distance (WD), up to one millimeter. The latter has small WD (~300μm) which depends on the focal length of the ball lens. Use of multiple probes with different WD allows one to obtain detailed information of subsurface tissues in the totally noninvasive manner. The probe is enough narrow to be inserted into a biopsy needle (~19G), for observations of the lesion at deeper inside bodies. The miniaturized endoscope has been applied to observe progression of a stomach cancer in the same rat lesion. It was succeeded to visualize structure of non-stained cancer tissue in live model animals by the fluorescent image technique. The system was also applied to ex vivo studies of human breast and stomach cancers.
Living pancreatic cancer tissues grown subcutaneously in nude mice are studied by in vivo Raman spectroscopy and
autofluorescence imaging. Comparing the same point spectra of alive pancreatic cancer tissue to that of the dead tissue, it
is found that they are different each other. The results suggest that the spectral changes reflect the protein conformational
changes in the tumor tissue with death of the host animal. From the result of autofluorescence study, in vivo
autofluorescence imaging has potential as a method to assign the histological elements of the pancreatic cancer tissue
without any staining. These results strongly suggest that combination of these techniques is very important to study
biological tissue.
Living pancreatic cancer tissues grown subcutaneously in nude mice are studied by in vivo microscope Raman
spectroscopy. Comparing the spectra of living pancreatic cancer tissue to that of the dead same tissue, it is found that
they are different each other. In the subtraction spectrum, Raman bands observed at 937, 1251, 1447 and 1671 cm-1 are
appeared in negative direction and those observed at 966 and 1045 cm-1 are appeared in positive direction. The results
strongly suggest that the spectral changes reflect the protein conformational changes in the tumor tissue with death of the
host animal. The present result demonstrates the importance of in vivo, real time studies of biomedical tissues using
Raman spectroscopy.
An optical biopsy system for small experimental animals has been developed. The system includes endoscope probe, portable probe and two kinds of miniaturized Raman probes. The micro Raman probe (MRP) is made of optical fibers and the ball lens hollow optical fiber Raman probe (BHRP) is made of hollow fiber. The former has large focal depth and suitable to measure average spectra of subsurface tissue. The latter has rather small focal depth and it is possible to control focal length by selecting ball lens attached at the probe head. It is suitable to survey materials at the fixed depth in the tissue. The system is applied to study various small animal cancer models, such as esophagus and stomach rat models and subcutaneous mouse models of pancreatic cancers. In the studies of subcutaneous tumor model mouse, it is suggested that protein conformational changes occur in the tumor tissue within few minutes after euthanasia of the mouse. No more change is observed for the following ten minutes. Any alterations in the molecular level are not observed in normal skin, muscle tissues. Since the change completes in such a short time, it is suggested that this phenomenon caused by termination of blood circulation.
Two types of hollow fiber-optic probes are developed to measure the in vivo Raman spectra of small animals. One is
the minimized probe which is end-sealed with the micro-ball lens. The measured spectra reflect the information of the
sample's sub-surface. This probe is used for the measurement of the esophagus and the stomach via an endoscope.
The other probe is a confocal Raman probe which consists of a single fiber and a lens system. It is integrated into the
handheld microscope. A simple and small multimodal probe is realized because the hollow optical fiber requires no
optical filters. The performance of each probe is examined and the effectiveness of these probes for in vivo Raman
spectroscopy is shown by animal tests.
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