ITVT-04. Management of intraoperative technological advances [intraoperative MRI, neuronavigation system using PET, and 5-aminolevulinic acid (5-ALA)–induced fluorescence image-guided surgery] for glioblastoma

OBJECTIVE The maximum resection of Glioblastoma (GBM) is the standard therapy and is expected to improve prognosis. Image-guided surgery using a neuronavigation system is the standard technique for glioma. However, due to the brain shift during surgery, intraoperative technologies, such as 5-ALA fluorescence and intraoperative MRI (IoMRI), are employed. Radiotracers are used during positron emission tomography (PET) for metabolic imaging and assist the evaluation of glioma metabolism. We compared the effectiveness of these intraoperative technologies. METHODS Between January 2016 and May 2021, 52 patients with gliomas underwent IoMRI. 21 patients were selected for 5-ALA fluorescence-guided resection of GBM and underwent multiple PET studies (MET, FLT, and FMISO). We graded fluorescence level as strong, vague, or none. Following tumor resection, we identified the fluorescence level and evaluated the residual volume of gadolinium-enhanced T1WI (T1-Gd) on IoMRI and at each PET study. After calculating the extent of resection (EOR) for T1-Gd, we compared the residual volume on T1-Gd for IoMRI and each PET study, between EOR ≥ 93% and EOR < 93%. RESULTS We detected strong 5-ALA fluorescence during induction and before tumor resection in all 21 (100%) patients with a newly-diagnosed and histopathologically-confirmed GBM. Following tumor resection, we noted an EOR ≥ 93% for T1-Gd in 12 cases (vague, 4; none, 8) and an EOR < 93% for T1-Gd in 9 cases (vague, 5; none, 4). The compared median residual volume (mL) with no fluorescence between EOR ≥ 93% and EOR < 93% for T1-Gd were T1-Gd (0.22, 0.74), MET (0.29, 3.31), FLT (0.24, 1.77), and FMISO (0.22, 1.02). CONCLUSIONS GBM cells are difficult to distinguish in cases without 5-ALA fluorescence. For cases without 5-ALA fluorescence, we were able to maximize the resection of GBM by extracting the area of MET accumulation.

Age determination of the adult blow fly Lucilia sericata (Diptera: Calliphoridae) through quantitative pteridine fluorescence analysis

Determination of a minimal postmortem interval via age estimation of necrophagous diptera has been restricted to the juvenile stages and the time until emergence of the adult fly, i.e. up until 2–6 weeks depending on species and temperature. Age estimation of adult flies could extend this period by adding the age of the fly to the time needed for complete development. In this context pteridines are promising metabolites, as they accumulate in the eyes of flies with increasing age. We studied adults of the blow fly Lucilia sericata at constant temperatures of 16 °C and 25 °C up to an age of 25 days and estimated their pteridine levels by fluorescence spectroscopy. Age was given in accumulated degree days (ADD) across temperatures. Additionally, a mock case was set up to test the applicability of the method. Pteridine increases logarithmically with increasing ADD, but after 70–80 ADD the increase slows down and the curve approaches a maximum. Sex had a significant impact (p < 4.09 × 10−6) on pteridine fluorescence level, while body-size and head-width did not. The mock case demonstrated that a slight overestimation of the real age (in ADD) only occurred in two out of 30 samples. Age determination of L. sericata on the basis of pteridine levels seems to be limited to an age of about 70 ADD, but depending on the ambient temperature this could cover an extra amount of time of about 5–7 days after completion of the metamorphosis.

A Method for Precision Surgical Implantation and Probe Insertion in Sprague-Dawley Rat Brain

Motorized stereotaxic is an advanced tool for surgery and implantation of cannula and electrodes in neuroscience. Stereotaxic surgery and implantation has become increasingly important tool, applied in many experiments. The goal in this present study to determine the surgical implantation and probe insertion at the target location accurately and precisely using motorized stereotaxic. In this study, the method allowed to evaluate the DHEAS fluorescence level through in vivo imaging approach at the target region in the hippocampus rat brain. This present study also described the surgical implantation and probe insertion by motorized stereotaxic as the precise and accurate techniques than conventional stereotaxic procedures. With the rat brain atlas by Paxinos and Watson (2004), the imaging approach can be evaluated precisely at the target location corresponding surgical implantation and probe insertion techniques.

Investigating the influence of spore maturation and sporulation conditions on MalS expression during Bacillus subtilis spore germination

Spore forming bacteria of the orders Bacillales and Clostridiales play a major role in food spoilage and food-borne diseases. The spores remain in a dormant state for extended periods due to their highly resistant features. When environmental conditions become favourable, they can germinate as the germinant receptors located on the spore’s inner membrane get activated via germinant binding. This leads to the formation of vegetative cells via germination and subsequent outgrowth. The present study focuses on the synthesis of protein MalS during B. subtilis spore germination by investigating the dynamics of the fluorescence level of a MalS-GFP fusion protein using time-lapse fluorescence microscopy. Our results show an initial increase within the first 15 minutes of germination, followed by a drop and stabilization of the fluorescence throughout the spore ripening period. Western blot analyses, however, indicate no increase in the levels of the MalS-GFP fusion protein during the first 15 minutes after the addition of the germinants. Thus the instantaneous increase in fluorescence of MalS-GFP may likely due to a change in the physical environment as the spore germination is triggered. Our findings also show that the different sporulation conditions and the maturation time of spores affect the expression of MalS-GFP and the germination behaviour of the spores.

Novel Approaches for Detection Fluorescent-Labeled by Cellvizio Lab System on Hippocampal CA1 Region

Neurosteroids have been identified in the 1981. Dehydroepiandrosterone sulphate (DHEAS) is one of the vital neurosteroids that de novo synthesized in the nervous system from cholesterol precursor (Baulieu & Robel, 1998). The aim of the study is to develop a method for fluorescence labelling. Alexa Fluor 488 dye with DHEAS antibody can binds the DHEAS antibody in the rat brain monitored by Cellvizio Lab System. DHEAS antibody (IgG isotype antibodies) was fluorescently conjugated by an amine-reactive compound, Alexa Fluor 5-SDP ester 488 dye. The resultant Alexa Fluor 488-conjugated antibodies were collected and analyzed by UV-Vis spectrophotometer instrument. The absorbance of the protein-dye conjugate at 280 nm and 494 nm were measured. Then, the degree of labeling (DOL) was calculated to achieve the desired results. Fluorescence labelling were carried out into the CA1 region of hippocampus Sprague-Dawley rat. We reported that the conjugation was successful. Optimal labeling depending on degree of labeling (DOL) needs some necessity to achieve and effective binding to the target neurosteroid, DHEAS. Cellvizio Lab system connected with Fiber Fluorescence Microscopy (FFM) probe is presented as a new approach in real-time imaging of DHEAS. In conclusion, we have developed a new method of DHEAS-Alexa Fluor fluorescence labelling to visualize and evaluate the changes of DHEAS fluorescence level in the rat hippocampus. This novel approach as a diagnostic tool and can be used to better understand the mechanisms and functions of DHEAS and other neurosteroids in future research.

Effect of Lactamase Inhibitors on the Biosensor Penp during the Measurement of Lactam Antibiotics Concentration

PenP is a fluorescent biosensor of lactam antibiotics (LA). It is structurally derived from the mutant lactamase TEM-1 comprising the substitution E166C, where fluorescein is covalently linked to cysteine. The presence of LA in the medium produces a change in the intrinsic fluorescence level of the biosensor, and the integral of the fluorescence level over time correlates directly with the LA concentration. Previously, we have successfully used PenP to determine the concentration of lactam antibiotics in clinical samples. The use of lactamase inhibitors (LI) is a common strategy to enhance the effect of LA due to the inhibition of an important resistance mechanism of pathogenic microorganisms. Structurally, LI and LA share the common element of recognition of lactamases (the lactam ring), but they differ in the reversibility of the mechanism of interaction with said enzyme. Because the biological recognition domain of PenP is derived from a lactamase, LI is expected to interfere with the PenP detection capabilities. Surprisingly, this work provides evidence that the effect of LI is marginal in the determination of LA concentration mediated by PenP.

Fluorescence Assay for Evaluating Microbicidal Activity of Hand Antiseptics

ABSTRACTWe developed a fluorescent β-d-glucuronidase activity (BGA)-based assay for detecting and quantifyingEscherichia coliin samples to assess the biocide efficacy of hand antiseptics. The fluorescence level is proportional to the number of viableE. coliorganisms present. We compared our assay results to those of theE. coliplate count method specified by the European standard for testing hygienic hand rub disinfectant products (EN1500). The plate count method requires excessive handling and materials and is not valid if the number of organisms per plate is too low or high for counting in many of the samples. We optimized the fluorescent assay based on the cleavage of 4-methylumbelliferyl-β-d-glucuronide by adding 4-nitrophenyl-β-d-glucuronide, a nonfluorogenic BGA substrate, to induce glucuronidase activity and reduce assay time. Furthermore, our method can be automated and eliminates the need for multiple dilutions. Fluorescence was temporally monitored, and the time required to reach a specific value of fluorescence was correlated with the initial number of viableE. coliorganisms on the samples. There was a positive correlation (P< 0.05) with a high correlation coefficient (R2= 0.82) between theE. colicounts by plate count and fluorescence methods. Reported effects in fluorescent BGA were compared to the EN1500 plate count method with five hand disinfectants. We found our method more advantageous, because it was as sensitive as the EN1500 method, requires less time to complete, and is less expensive and less laborious than conventional plating techniques.

Fluorescence Level of Composites assessed by Computer Processing of Digital Images: ScanWhite©

ABSTRACT The human dentition is naturally translucent, opalescent and fluorescent. Differences between the level of fluorescence of tooth structure and restorative materials may result in distinct metameric properties and consequently perceptible disparate esthetic behavior, which impairs the esthetic result of the restorations, frustrating both patients and staff. In this study, we evaluated the level of fluorescence of different composites (Durafill in tones A2 (Du), Charisma in tones A2 (Ch), Venus in tone A2 (Ve), Opallis enamel and dentin in tones A2 (OPD and OPE), Point 4 in tones A2 (P4), Z100 in tones A2 (Z1), Z250 in tones A2 (Z2), Te-Econom in tones A2 (TE), Tetric Ceram in tones A2 (TC), Tetric Ceram N in tones A1, A2, A4 (TN1, TN2, TN4), Four seasons enamel and dentin in tones A2 (and 4SD 4SE), Empress Direct enamel and dentin in tones A2 (EDE and EDD) and Brilliant in tones A2 (Br)). Cylindrical specimens were prepared, coded and photographed in a standardized manner with a Canon EOS digital camera (400 ISO, 2.8 aperture and 1/ 30 speed), in a dark environment under the action of UV light (25 W). The images were analyzed with the software ScanWhite©-DMC/Darwin systems. The results showed statistical differences between the groups (p < 0.05), and between these same groups and the average fluorescence of the dentition of young (18 to 25 years) and adults (40 to 45 years) taken as control. It can be concluded that: Composites Z100, Z250 (3M ESPE) and Point 4 (Kerr) do not match with the fluorescence of human dentition and the fluorescence of the materials was found to be affected by their own tone. How to cite this article Tonetto MR, de Oliveira Junior OB, de Campos EA, Saad JRC, Dantas AAR, de Souza Rastelli AN, de Toledo Porto Neto S, de Andrade MF. Fluorescence Level of Composites assessed by Computer Processing of Digital Images: ScanWhite©. World J Dent 2012;3(2):141-144.

Coregistered fluorescence-enhanced tumor resection of malignant glioma: relationships between δ-aminolevulinic acid–induced protoporphyrin IX fluorescence, magnetic resonance imaging enhancement, and neuropathological parameters

Object The aim of this study was to investigate the relationships between intraoperative fluorescence, features on MR imaging, and neuropathological parameters in 11 cases of newly diagnosed glioblastoma multiforme (GBM) treated using protoporphyrin IX (PpIX) fluorescence-guided resection. Methods In 11 patients with a newly diagnosed GBM, δ-aminolevulinic acid (ALA) was administered to enhance endogenous synthesis of the fluorophore PpIX. The patients then underwent fluorescence-guided resection, coregistered with conventional neuronavigational image guidance. Biopsy specimens were collected at different times during surgery and assigned a fluorescence level of 0–3 (0, no fluorescence; 1, low fluorescence; 2, moderate fluorescence; or 3, high fluorescence). Contrast enhancement on MR imaging was quantified using two image metrics: 1) Gd-enhanced signal intensity (GdE) on T1-weighted subtraction MR image volumes, and 2) normalized contrast ratios (nCRs) in T1-weighted, postGd-injection MR image volumes for each biopsy specimen, using the biopsy-specific image-space coordinate transformation provided by the navigation system. Subsequently, each GdE and nCR value was grouped into one of two fluorescence categories, defined by its corresponding biopsy specimen fluorescence assessment as negative fluorescence (fluorescence level 0) or positive fluorescence (fluorescence level 1, 2, or 3). A single neuropathologist analyzed the H & E–stained tissue slides of each biopsy specimen and measured three neuropathological parameters: 1) histopathological score (0–IV); 2) tumor burden score (0–III); and 3) necrotic burden score (0–III). Results Mixed-model analyses with random effects for individuals show a highly statistically significant difference between fluorescing and nonfluorescing tissue in GdE (mean difference 8.33, p = 0.018) and nCRs (mean difference 5.15, p < 0.001). An analysis of association demonstrated a significant relationship between the levels of intraoperative fluorescence and histopathological score (χ2 = 58.8, p < 0.001), between fluorescence levels and tumor burden (χ2 = 42.7, p < 0.001), and between fluorescence levels and necrotic burden (χ2 = 30.9, p < 0.001). The corresponding Spearman rank correlation coefficients were 0.51 (p < 0.001) for fluorescence and histopathological score, and 0.49 (p < 0.001) for fluorescence and tumor burden, suggesting a strongly positive relationship for each of these variables. Conclusions These results demonstrate a significant relationship between contrast enhancement on preoperative MR imaging and observable intraoperative PpIX fluorescence. The finding that preoperative MR image signatures are predictive of intraoperative PpIX fluorescence is of practical importance for identifying candidates for the procedure. Furthermore, this study provides evidence that a strong relationship exists between tumor aggressiveness and the degree of tissue fluorescence that is observable intraoperatively, and that observable fluorescence has an excellent positive predictive value but a low negative predictive value.

Using Rhodamine 123 Accumulation in CD8+Cells as a Surrogate Indicator to Study the P-Glycoprotein Modulating Effect of Cepharanthine Hydrochloride In Vivo

The purpose of this study was the use of rhodamine 123 (Rho123) accumulation in peripheral bloodCD8+cells as a surrogate indicator to evaluate the modulating effect of P-glycoprotein (P-gp) inhibitors in the multidrug resistance (MDR) tumor-bearing mouse model. Rho123 was administered to mice, and the fluorescence level in CD8+cells was measured. Cepharanthine hydrochloride (CH) and verapamil (VER), two P-gp inhibitors, were administered to mice 1 hour prior to Rho123 administrationin vivoor added to peripheral blood 1 hour prior to Rho123 additionex vivo. The tumor inhibition effect of 5-fluorouracil/adriamycin/cisplatin (FAP) protocol plus CH was also investigated. A concentration- or dose-response relationship was shown between the concentration and dose of CH and Rho123 accumulation or the antitumor activity. In conclusion, the measurement of Rho123 accumulation in CD8+cells provides a surrogate assay for the screening of candidate P-gp inhibitors in preclinical trials, and CH is effective in modulating P-gp-mediated MDRin vivo.