Jyothi Lakshmi, R., Kartha, V. B., Murali Krishna, C., Solomon, J. G. R., Ullas, G. and Uma Devi, P. Tissue Raman Spectroscopy for the Study of Radiation Damage: Brain Irradiation of Mice. Radiat. Res. 157, 175–182 (2002).Radiotherapy is routinely employed in the treatment of head and neck cancers. Acute cell death, radiation-activated chemical cascades, and the induction of genes coding for protective factors like cytokines are considered to be the major processes involved in radiation damage and repair. It should be possible to follow these processes by monitoring the biochemical interactions initiated by radiation. We have carried out Raman spectroscopy studies on tissue from mice subjected to brain irradiation to identify the biochemical changes occurring in tissue and brain as a result of radiation injury. These studies show that brain irradiation produces drastic spectral changes even in tissue far removed from the irradiation site. The changes are very similar to those produced by the stress of inoculation and restraint and the administration of an anesthetic drug. While the changes produced by stress or anesthetics last for only a short time (a few hours to 1 or 2 days), radiation-induced changes persist even after 1 week. The spectral changes can be interpreted in terms of the observation of new spectra that are dominated by bands due to proteins. The results thus support the hypothesis that various protective factors are released throughout the body when the central nervous system (CNS) is exposed to radiation.

Early diagnosis of oral cancers, one of the major cancers, is of utmost importance as 5-year disease-free survival rates are some of the lowest, despite advances in treatment and surgical modalities. In vivo Raman spectroscopy has shown efficacy in the detection of normal, premalignant and malignant lesions and even of early changes such as cancer-field-effects/malignancy-associated-changes. However, the need for a dedicated instrument and stringent laboratory conditions, at all diagnostic centers, limits wide screening applications of this method. In light of this, it is pertinent to explore ex vivo samples like serum due to its ease of collection, storage, transport and analysis at a centralized facility. Hence, Raman studies were carried out on serum from 14 buccal mucosa and 40 tongue cancers as well as 16 healthy control samples. Spectral features indicate differential contributions of proteins, DNA, and amino acids like Phe, Trp and Tyr and β-carotene in the analyzed groups. Highly intense Raman bands assigned to β-carotene could be due to resonance Raman, and were observed in all sera with the highest relative intensity in normal samples. Higher DNA and protein content were observed in the mean cancer spectra. Principal component-linear discriminant analysis (PC-LDA) followed by cross-validation using leave-one-out cross-validation (LOOCV) were employed for data analysis which was carried out both spectra- and patient-wise. Findings indicate the possibility of classifying normal and oral cancer sera in both these approaches; however, the patient-wise approach could be the preferred mode for prospective studies. Besides, a tendency of classification for buccal mucosa and tongue cancers was also observed. Prospective validation of these results on a large sample size may help in the translation of this methodology to clinics.

Cancers of oral cavities are one of the most common malignancies in India and other south-Asian countries. Tobacco habits are the main etiological factors for oral cancer. Identification of premalignant lesions is required for improving survival rates related to oral cancer. Optical spectroscopy methods are projected as alternative/adjunct for cancer diagnosis. Earlier studies have demonstrated the feasibility of classifying normal, premalignant, and malignant oral ex-vivo tissues. We intend to evaluate potentials of Raman spectroscopy in detecting premalignant conditions. Spectra were recorded from premalignant patches, contralateral normal (opposite to tumor site), and cancerous sites of subjects with oral cancers and also from age-matched healthy subjects with and without tobacco habits. A total of 861 spectra from 104 subjects were recorded using a fiber-optic probe-coupled HE-785 Raman spectrometer. Spectral differences in the 1200- to $1800-cm−1$ region were subjected to unsupervised principal component analysis and supervised linear discriminant analysis followed by validation with leave-one-out and an independent test data set. Results suggest that premalignant conditions can be objectively discriminated with both normal and cancerous sites as well as from healthy controls with and without tobacco habits. Findings of the study further support efficacy of Raman spectroscopic approaches in oral-cancer applications.

Micro-Raman spectra of formalin-fixed oral squamous normal and carcinoma tissues, stored at room temperature for 2 months, have been recorded. Spectra were recorded both in the epithelial and subepithelial regions of the tissues. No noticeable spectral contamination due to formalin was observed. Very significant differences between spectra of normal epithelial and malignant epithelial samples were found. No such differences in spectra of subepithelial malignant and subepithelial normal samples could be observed. This study shows that spectra from the epithelial region changes drastically because of malignancy-induced biochemical changes in this region. Major differences between normal and malignant spectra seem to arise from the protein composition, conformational/structural changes, and possible increase in protein content in malignant epithelia. The differences between normal epithelial and subepithelial spectra, as expected, arise mainly from the collagen in subepithelial tissue. Principal component analysis of the combined sets of spectra—epithelial and subepithelial, normal and malignant— showed that very good discrimination can be achieved by Raman microspectroscopy. This study thus validates the suitability of formalin-fixed tissues for optical pathology in oral malignancy.