Matrix-assisted laser desorption/ionization imaging mass spectrometry revealed traces of dental problem associated with dental structure.

Periodontal disease is a serious dental problem because it does not heal naturally and leads to tooth loss. In periodontal disease, inflammation at periodontal tissue is thought as predominant, and its effect against tooth itself remains unclear. In this study, we applied matrix-assisted laser desorption/ionization imaging mass spectrometry (IMS) to teeth for the first time. By comparing anatomical structure of tooth affected with periodontal disease with normal ones, we analyzed traces of the disease on tooth. We found signals characteristic of enamel, dentin, and dental pulp, respectively, in mass spectra obtained from normal teeth. Ion images reconstructed using these signals showed anatomical structures of the tooth clearly. Next, we performed IMS upon teeth of periodontal disease. Overall characteristic of the mass spectrum appeared similar to normal ones. However, ion images reconstructed using signals from the tooth of periodontal disease revealed loss of periodontal ligament visualized together with dental pulp in normal teeth. Moreover, ion image clearly depicted an accumulation of signal at m/z 496.3 at root surface. Such an accumulation that cannot be examined only from mass spectrum was revealed by utilization of IMS. Recent studies about inflammation revealed that the signal at m/z 496.3 reflects lyso-phosphatidylcholine (LPC). Infiltration of the signal is statistically significant, and its intensity profile exhibited the influence has reached deeply into the tooth. This suggests that influence of periodontal disease is not only inflammation of periodontal tissue but also infiltration of LPC to root surface, and therefore, anti-inflammatory treatment is required besides conventional treatments.

Imaging mass spectrometry distinguished the cancer and stromal regions of oral squamous cell carcinoma by visualizing phosphatidylcholine (16:0/16:1) and phosphatidylcholine (18:1/20:4).

Most oral cancers are oral squamous cell carcinoma (OSCC). The anatomical features of OSCC have been histochemically evaluated with hematoxylin and eosin. However, the border between the cancer and stromal regions is unclear and large portions of the cancer and stromal regions are resected in surgery. To reduce the resected area and maintain oral function, a new method of diagnosis is needed. In this study, we tried to clearly distinguish the border on the basis of biomolecule distributions visualized by imaging mass spectrometry (IMS). In the IMS dataset, eleven signals were significantly different in intensity (p < 0.01) between the cancer and stromal regions. Two signals at m/z 770.5 and m/z 846.6 were distributed in each region, and a clear border was revealed. Tandem mass spectrometric (MS/MS) analysis identified these signals as phosphatidylcholine (PC) (16:0/16:1) at m/z 770.5 in the cancer region and PC (18:1/20:4) at m/z 846.6 in the stromal region. Moreover, the distribution of PC species containing arachidonic acid in the stromal region suggests that lymphocytes accumulated in response to the inflammation caused by cancer invasion. In conclusion, the cancer and stromal regions of OSCCs were clearly distinguished by use of these PC species and IMS analysis, and this molecular identification can provide important information to elucidate the mechanism of cancer invasion.

Dentinogenic ghost cell tumor treated with a combination of marsupialization and radical resection: a case report and review of the literature.

BACKGROUND: Dentinogenic ghost cell tumor is a rare benign tumor that accounts for less than 3% of all cases and consists of the stellate reticulum, which is made up of enamel epithelioid and basaloid cells. Although DGCT is a benign tumor, the local infiltration of the odontogenic epithelium or recurrences have been reported, and its detailed pathology and treatments remain unclear. CASE PRESENTATION: This report describes the case of a 60-year-old Japanese male diagnosed with a maxillary dentinogenic ghost cell tumor. Images showed well-circumscribed, multilocular cystic lesions with a calcified substance in the interior. Marsupialization was performed along with biopsy to prevent the expansion of the lesion, and a partial maxillectomy was performed 2 years after the initial examination. Histopathological findings showed ameloblastomatous proliferation containing clusters of ghost cells and dentinoid materials, resulting in the diagnosis of dentinogenic ghost cell tumor. This article also reviews recently reported cases of dentinogenic ghost cell tumor. CONCLUSION: It is important to perform marsupialization, proper resection, and postoperative follow-up because of possible recurrence.

Electron microscopic radioautographic study on mitochondrial DNA synthesis in adrenal cortical cells of developing and aging mice.

In order to study the aging changes of intramitochondrial DNA synthesis of mouse adrenal cortical cells, eight groups of developing mice, each consisting of three individuals (total 24), from fetal day 19 to postnatal newborn at days 1, 3, 9, 14, to adult at months 1, 2, and 6, were injected with 3H-thymidine, sacrificed 1 h later, and the adrenal tissues were fixed and processed for electron microscopic (EM) radioautography. On EM radioautograms obtained from each animal, the number of mitochondria and the mitochondrial labeling index labeled with 3H-thymidine showing DNA synthesis in each adrenal cortical cell, in three zones, were counted and the results in respective developing groups were compared. From the results, it was demonstrated that the numbers of mitochondria in the three zones, the zona glomerulosa, fasciculata, and reticularis, of mice at various ages increased from fetal day 19 to postnatal month 6 due to development and aging of animals, respectively, while the number of labeled mitochondria and the labeling index of intramitochondrial DNA syntheses incorporating 3H-thymidine increased from fetal day 19 to postnatal month 2, reaching the maxima, and decreased to month 6. It was shown that the activity of intramitochondrial DNA synthesis in the adrenal cortical cells in developing and aging mice changed due to aging.

Electron microscopic radioautographic study on protein synthesis in mitochondria of binucleate hepatocytes of aging mice.

In order to study the aging changes of intramitochondrial protein synthesis in mouse hepatocytes, 10 groups of aging mice, each consisting of three individuals, total 30, from fetal day 19 to postnatal year 2, were injected with 3H-leucine, a protein precursor, sacrificed 1 h later, and the liver tissues processed for electron microscopic radioautography. On electron microscopic radioautograms obtained from each animal, the numbers of mitochondria, the numbers of labeled mitochondria, and the mitochondrial labeling index labeled with 3H-leucine that showed protein synthesis in each hepatocyte, both mononucleate and binucleate cells, were counted and the averages in respective aging groups were compared. From the results, it was demonstrated that the numbers of mitochondria, the numbers of labeled mitochondria, and the labeling indices of intramitochondrial protein syntheses in both mononucleate and binucleate hepatocytes of mice at various ages increased due to development of animals. The numbers of mitochondria, the numbers of labeled mitochondria, and the labeling indices of intramitochondrial protein synthesis in binucleate hepatocytes were more than those of mononucleate hepatocytes at the same aging stages.

Electron microscopic radioautographic study on protein synthesis in hepatocyte mitochondria of aging mice.

For the purpose of studying the aging changes of intramitochondrial protein synthesis in mouse hepatocytes, 10 groups of aging mice, each consisting of 3 individuals (total 30), from fetal day 19 to postnatal month 24, were injected during development with 3H-leucine, a protein precursor, sacrificed 1 h later, and the liver tissues processed for electron microscopic (EM) radioautography. On EM radioautograms obtained from each animal, the number of mitochondria, the number of labeled mitochondria, and the mitochondrial labeling index labeled with silver grains due to 3H-leucine showing protein synthesis in each mononucleate hepatocytes were counted and the averages in respective aging groups were compared. From the results, it was demonstrated that the numbers of mitochondria, the numbers of labeled mitochondria, and the labeling indices of intramitochondrial protein syntheses in mononucleate hepatocytes of mice at various ages from embryonic day 19 to postnatal month 24 increased and decreased due to development and aging of animals.

Three-dimensional computed tomographic analysis of variations of the carotid artery.

BACKGROUND: Although the terms tortuous, coiling, and kinking have been used to describe the curvature of the carotid artery, the prevalence rates of these patterns have differed among studies. We morphologically evaluated the characteristics of the carotid artery by means of three-dimensional computed tomography (3DCT) to clarify the prevalence of tortuosity, coiling, and kinking. We present our results and discuss the clinical impact of our findings. METHODS: A total 148 patients underwent contrast-enhanced CT (including 55 patients who underwent dynamic CT), and anatomical variations were analyzed on the basis of 3DCT images. RESULTS: Among the 296 arteries, tortuosity was present in 254 (85.8%), coiling in 9 (3.0%), kinking in 3 (1.0%), and occlusion in 2 (0.7%). CONCLUSION: 3DCT image reconstruction is an effective means for classifying morphological variations of the ICA and detecting abnormalities of the carotid artery. It can thereby potentially reduce the risk of serious complications during neck surgery.

Electron microscopic radioautographic study of RNA synthesis in hepatocyte mitochondria of aging mouse.

In order to study the aging changes of intramitochondrial RNA synthesis in mouse hepatocytes, 10 groups of aging mice, each consisting of three individuals (total 30) from fetal day 19 to postnatal month 24 were injected with 3H-uridine, an RNA precursor, sacrificed 1 hour later, and the liver tissues processed for electron microscopic radioautography. On EM radioautograms obtained from each animal the number of mitochondria, the number of labeled mitochondria, and the mitochondrial labeling index labeled with 3H-uridine showing RNA synthesis in each hepatocytes, both mononucleate and binucleate cells, were counted and the averages in respective aging groups were compared. From the results it was demonstrated that the numbers of mitochondria, the numbers of labeled mitochondria, and the labeling indices of intramitochondrial RNA syntheses in both mononucleate and binucleate hepatocytes of mice at various ages increased and decreased according to the age of the animals.

X-ray microanalysis of biological specimens by high voltage electron microscopy.

For the purpose of analyzing and imaging chemical components of cells and tissues at the electron microscopic level, 3 fundamental methods are available, chemical, physical and biological. Among the physical methods, two methods qualifying and quantifying the elements in the structural components are very often employed. The first method is radioautography which can demonstrate the localization of radiolabeled compounds which were incorporated into cells and tissues after the administration of radiolabeled compounds. The second method is X-ray microanalysis which can qualitatively analyze and quantify the total amounts of elements present in cells and tissues. We have developed the two methodologies in combination with intermediate high or high voltage transmission electron microscopy (200-400 kV) and applied them to various kinds of organic and inorganic compounds present in biological materials. As for the first method, radioautography, I had already contributed a chapter to PHC (37/2). To the contrary, this review deals with another method, X-ray microanalysis, using semi-thin sections and intermediate high voltage electron microscopy developed in our laboratory. X-ray microanalysis is a useful method to qualify and quantify basic elements in biological specimens. We first quantified the end-products of histochemical reactions such as Ag in radioautographs, Ce in phosphatase reaction and Au in colloidal gold immunostaining using semithin sections and quantified the reaction products observing by intermediate high voltage transmission electron microscopy at accelerating voltages from 100 to 400 kV. The P/B ratios of all the end products Ag, Ce and Au increased with the increase of the accelerating voltages from 100 to 400 kV. Then we analyzed various trace elements such as Zn, Ca, S and Cl which originally existed in cytoplasmic matrix or cell organelles of various cells, or such elements as Al which was absorbed into cells and tissues after oral administration, using both conventional chemical fixation and cryo-fixation followed by cryo-sectioning and freeze-drying, or freeze-substitution and dry-sectioning, or freeze-drying and dry-sectioning producing semithin sections similarly to radioautography. As the results, some trace elements which originally existed in cytoplasmic matrix or cell organelles of various cells in different organs such as Zn, Ca, S and Cl, were effectively detected. Zn was demonstrated in Paneth cell granules of mouse intestines and its P/B ratios showed a peak at 300 kV. Ca was found in human ligaments and rat mast cells with a maximum of P/B ratios at 350 kV. S and Cl were detected in mouse colonic goblet cells with maxima of P/B ratios at 300 kV. On the other hand, some elements which were absorbed by experimental administration into various cells and tissues in various organs, such as Al in lysosomes of hepatocytes and uriniferous tubule cells in mice was detected with a maximum of P/B ratios at 300 kV. From the results, it was shown that X-ray microanalysis using semi-thin sections observed by intermediate high voltage transmission electron microscopy at 300-400 kV was very useful resulting in high P/B ratios for quantifying some trace elements in biological specimens. These methodologies should be utilized in microanalysis of various compounds and elements in various cells and tissues in various organs.

Radioautographology general and special.

A new concept, termed "radioautographology" is advocated and its contents are reviewed. This term is the coinage synthesized from "radioautography" and "(o)logy", expressing a new science derived from radioautography. The concept of radioautographology (RAGology) is a science to localize the radioactive substances in the biological structure of the objects and to analyze and to study the significance of these substances in the biological structure. On the other hand, the old term radioautography (RAG) or autoradiography (ARG) is the technique to demonstrate the pattern of localization of various radiolabeled compounds in biological specimens. The specimens used in biology and medicine are cells and tissues. They are fixed, sectioned and made contact with the radioautographic emulsions, exposed and developed to produce metallic silver grains. Such specimens are designated as radioautographs (or autoradiographs) and the patterns of pictures made of silver grains are named radioautograms. Those people who produced radioautographs were formerly named radioautographers (or autoradiographers) who were only technicians, while those who study RAGology are not technicians but scientists and should be called as radioautographologists. The science of radioautographology was developed in the 20th century and can be divided into two parts, general radioautographology and special radioautographology, as most natural sciences usually can. The general radioautographology is the technology of RAG which consists of 3 fields of sciences, physics concerning radioactivity, histochemistry treating the cells and tissues and photochemistry dealing with the photographic emulsions. The special radioautographology, on the other hand, consists of applications of general radioautographology to various biological and medical sciences. The applications can be classified into several scientific fields, i.e., cellular molecular biology, anatomy, histology, embryology, pathology and pharmacology. Studies carried out in our laboratory were summarized and reviewed. The results obtained from the technology includes 4-dimensional structures of the organs taking the time dimension into account by labeling cells and localizing the sites of incorporation, synthesis, discharge of the labeled compounds in connection with the time lapse and aging of animals. All the results obtained from such applications should be systematized as a new filed of science in the future in the 21st century.

CELL POPULATION KINETICS OF THE SCLEROBLAST DURING ETHISTERONE-INDUCED ANAL-FIN PROCESS FORMATION IN ADULT FEMALES OF THE MEDAKA, ORYZIAS LATIPES.

Proliferation and migration of the scleroblasts and their precursor cells during ethisterone-induced anal-fin process formation in adult females of the medaka, Oryzias latipes, were investigated using a radioautographic technique with 3 H-thymidine. In the posterior margin of the joint plate, rapid increases in labeling indexes and scleroblast population began simultaneously in the central portion on the third day of ethisterone treatment, and the population became approximately ten times that in the control fish on the fifth day. However, nomarked increase in scleroblast population occurred in the peripheral portion, although the scleroblasts showed nearly the same labeling index as those in the central portion throughout the experiment. These observations indicate that the scleroblasts and their precursor cells migrated from the peripheral to the central portion along the proxi-distal axis of the joint plate. Some cells in the inner and hind spaces also migrated to the posterior margins and subsequently developed into scleroblasts. It may be concluded, therefore, that the scleroblasts are derived from precursor cells situated not only along the posterior margins but also in the adjacent spaces. These precursor cells proliferate and/or migrate to the central portion of the posterior margins and aggregate to form the scleroblast mass.

[A study of the feasibility and efficacy of fetal surgical closure of spontaneous cleft lip in CL/fraser mice].

Cleft lip with or without cleft palate is one of the most common human craniofacial congenital malformations. Failure to form a continuous tissue in the upper lip during the early embryonic period results in cleft lip. There has been an increasing interest in the possibility of in-utero correction of life threatening fetal abnormalities. In the present study, the feasibility and efficacy of surgical suturing of the fetal mouse lip was examined by using CL/Fraser mouse fetuses (spontaneous of cleft lip) at embryonic 15, 16 or 17 days. The obtained results were as follows. #1. Abortion rate of pregnant dam applied inhalation of Ethrane was less than that of intraperitoneal injection of Nembutal. #2. Successful delivery rate of fetal mouse surgically treated at the embryonic 16 day or 17 day is higher than that on the embryonic 15 day. #3. Fusion rate of mouse fetal lip surgically treated on the embryonic 16 day was higher after 60 hours in dam's uteri than that after 36 hours. These results suggest that inhalation of Ethrane is more recommendable, and that the optimal timing of fetal surgery of spontaneous cleft lip in CL/Fr. mouse fetus is an embryonic 16 day, and that the average time of 60 hours after surgical operation is enough for sutured lip to fuse in dam's uteri.

Cell Aging of Mouse Gastrointestinal Tract Observed by Light and Electron Microscopic Radioautography.

The term "cell aging" initially means how the cells change due to their aging. There are two meanings, i.e. how a cell changes when it is isolated from original animals such as in vitro cells in cell culture, otherwise how all the cells of an animal change in vivo due to the aging of the individual animal. We have been studying the latter changes from the viewpoint of the cell nutrients, the precursors for the macromolecular synthesis such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA), proteins, glucides and lipids, which are incorporated and synthesized into various cells of individual animals. Therefore, this article deals with only the cell aging of animal cells in vivo, how the metabolism, i.e. incorporations and syntheses of respective nutrient precursors in various kinds of cells change due to the aging of individual experimental animals such as mice by means of microscopic radioautography to localize the RI-labeled precursors. The incorporations and syntheses of various precursors for macromolecules such as DNA, RNA, proteins, glucides, lipids and others in various kinds of cells of various organs in the gastrointestinal tract such as the mouth, esophagus, stomach and intestines are reviewed referring many original papers already published from our laboratory during these 60 years since the late 20th century.

Improved technique for evaluating oral free flaps by pinprick testing assisted by indocyanine green near-infrared fluorescence angiography.

In head and neck surgery, free-flap reconstruction using a microvascular anastomosis is an indispensable option after tumor ablation. Because the success of free-flap reconstruction is enhanced by rapid identification and salvage of failing flaps, postoperative monitoring of free flaps is essential. We describe a new technique using indocyanine green (ICG) near-infrared angiography and pinprick testing to monitor intraoral free flaps. A solution of ICG (Diagnogreen, 5 ml) was intravenously injected, and scanning was performed with a near-infrared video camera system. Thirty seconds after ICG injection, a pinprick test was performed by placing a 24-gage needle through the dermis to the subcutaneous fat of the flap. Pinprick testing during ICG fluorescence imaging was performed in 30 patients. Flap perfusion was confirmed in all patients, and all flaps survived postoperatively. ICG fluorescence imaging demonstrated that flap perfusion was maintained.

RNA synthesis in the pancreatic acinar cells of aging mice as revealed by electron microscopic radioautography.

For the purpose of studying the aging changes of macromolecular synthesis in the pancreatic acinar cells of experimental animals, we studied 10 groups of aging mice during development and aging from fetal day 19 to postnatal month 24. They were injected with 3H-uridine, a precursor for RNA synthesis, sacrificed and the pancreatic tissues were taken out, fixed and processed for light and electron microscopic radioautography. On many radioautograms the localization of silver grains demonstrating RNA synthesis in pancreatic acinar cells in respective aging groups were analyzed qualitatively. The number of mitochondria per cell, the number of labeled mitochondria with silver grains and the number of silver grains in each cell in respective aging groups were analyzed quantitatively in relation to the aging of animals. The results revealed that the RNA synthetic activity as expressed by the incorporations of RNA precursor, i.e., the number of silver grains in cell nuclei, cell organelles, changed due to the aging of animals. The number of mitochondria, the number of labeled mitochondria and the mitochondrial labeling index labeled with silver grains were counted in each pancreatic acinar cell. It was demonstrated that the number of mitochondria, the number of labeled mitochondria and the labeling indices showing RNA synthesis at various ages increased from embryonic day 19 to postnatal newborn day 1, 3, 9, 14, adult month 1, 2 and 6, reaching the maxima, then decreased to senile stage at postnatal year 1 to 2, indicating the aging changes. Based upon our findings, available literature on macromolecular synthesis in mitochondria of various cells are reviewed.

Clinical significance of combined assessment of the maximum standardized uptake value of F-18 FDG PET with nodal size in the diagnosis of cervical lymph node metastasis of oral squamous cell carcinoma.

RATIONALE AND OBJECTIVES: This study aimed to elucidate the diagnostic accuracy of F-18 fluorodeoxyglucose (FDG) positron emission tomography (PET)/computed tomography (CT) for nodal involvement in oral squamous cell carcinoma (OSCC), and to reveal clinically useful factors to distinguish between true-positive (TP) and false-positive (FP) nodes. MATERIALS AND METHODS: Thirty-eight patients with primary OSCC who underwent neck dissection were assessed. The diagnostic accuracy of F-18 FDG PET/CT was evaluated, and then compared with that of CT/ultrasonography (US). Furthermore, the association of the maximum standardized uptake value (SUVmax) and nodal size with the histopathologic findings was examined. RESULTS: Sensitivity and specificity using F-18 FDG PET/CT were 77.1% and 97.3%, and those using CT/US were 72.9% and 98.9%, respectively. The SUVmax of TP nodes was significantly higher than that of FP nodes. Nodes with SUVmax >4.5 were pathologically confirmed as metastasis. Nodes with SUVmax ≤4.5 were further discriminated between TP and FP nodes by using the long axis diameters or the ratios of long to short axis diameter as clinical parameters. Positive correlation between the SUVmax and the short-axis diameter was found in TP nodes. The AUC obtained from the ROC curves of the SUVmax alone (AUC, 0.804) was improved by combination with the long-axis diameter (AUC, 0.867) or the short-axis diameter (AUC, 0.846), although no significant difference was found. CONCLUSIONS: These results indicated that F-18 FDG PET/CT was potentially useful in diagnosing preoperative nodal state. Furthermore, combined assessment of SUVmax with nodal size could be significant in the identification of metastatic lymph nodes in OSCC patients.

Electron Microscopic Radioautographic Study on the Protein Synthesis in the Pancreas of Aging Mice With Special Reference to Mitochondria.

BACKGROUND: The purpose of this study was to investigate the aging changes of macromolecular synthesis in animal cells. METHODS: We studied 10 groups of mice during development aged from fetal day 19 to postnatal month 24. They were injected with 3H-leucine, a precursor for protein synthesis, sacrificed and the pancreatic tissues were taken out, fixed and processed for light and electron microscopic radioautography. On many radioautograms the localization of silver grains demonstrating protein synthesis in pancreatic acinar cells in respective aging groups were first analyzed qualitatively. Then the number of silver grains and the number of cell organelles in each cell in respective aging groups were analyzed quantitatively in relation to the aging of animals. The number of mitochondria, the number of labeled mitochondria and the mitochondrial labeling index labeled with silver grains were counted in each pancreatic acinar cell. RESULTS AND CONCLUSIONS: The number of silver grains in cell nuclei and cell organelles changed with the aging of animals. The number of mitochondria, the number of labeled mitochondria and the labeling indices showing protein synthesis at various ages increased from embryonic day 19 to postnatal newborn day 1, 3, 7, 14, to young adult month 1, and 2, reaching the maxima, then decreased at old adult month 6 and senile year 1 to 2, indicating the aging changes.

Macromolecular synthesis in the livers of aging mice as revealed by electron microscopic radioautography.

For the purpose of studying the aging changes of macromolecular synthesis in animal cells, we studied many groups of aging mice during development and aging from fetal day 19 to postnatal newborn, juvenile, young adults, aged and senescent adults up to 12 and month 24 (2 years). They were injected with (3)H-thymidine, (3)H-uridine or (3)H-leucine, precursors for DNA, RNA and proteins, as well as (3)H-glucose, (3)H-glucosamine, (35)S-sulfuric acid, or (3)H-glycerol for glucide and lipid precursors, respectively, then sacrificed and the liver tissues were taken out, fixed and processed for light and electron microscopic radioautography. On many radioautograms the localization of silver grains demonstrating DNA, RNA and proteins in hepatocytes in respective aging groups were analyzed qualitatively. The number of silver grains and the number of cell organelles in each cell of each animal in respective aging groups were analyzed quantitatively in relation to the aging of individual animals. The results revealed that the localization of respective precursors as well as the number of silver grains in cell nuclei, cell organelles, changed with the aging of animals. The numbers of labeled nuclei and cell organelles, as well as the numbers of silver grains in nuclei and cell organelles changed due to aging of individual animals. The number of mitochondria, the number of labeled mitochondria and the mitochondrial labeling index labeled with silver grains were counted in each hepatocyte. It was demonstrated that the numbers of mitochondria, the numbers of labeled mitochondria and the labeling indices showing DNA, RNA and protein synthesis at various ages from embryonic day 19 to postnatal newborn day 1, 3, 9, 14, adult month 1, 2 and 6, reaching the maxima, then decreased to senile year 1 to 2, indicating the aging changes. The results indicated that mitochondria in hepatocytes synthesized nucleic acids and proteins independently from the nuclei, but their synthetic activities were affected from the aging of the individual animals.

Submucosal glycerol injection-assisted laser surgical treatment of oral lesions.

Recently, we modified laser surgery for superficial lesions in the oral cavity by using submucosal glycerol injection. This procedure was based on a technique for endoscopic mucosal resection (EMR) in the gastrointestinal tract. The aim of this study was to evaluate the effectiveness of the modified laser surgery assisted by a submucosal glycerol injection. Eleven superficial oral lesions in ten patients were treated with diode laser (continuous wave mode, 3 W) after a submucosal injection of glycerol solution. Injection of glycerol solution created mucosal expansion, which enabled the procedures to be done without bleeding, over cutting, over coagulation and unintended irradiation. The surface of the wounds showed little carbonization, resulting in good healing. Submucosal glycerol injection for laser treatment in the oral cavity is a promising technique for treating superficial oral lesions by virtue of less invasion and good results.