Nicotinamide Adenine Dinucleotide Diaphorase Research Articles

Distinct immunoreactions after a primary tumor microwave ablation using different heating parameters in a VX2 tumor model.

Thermal ablation of solid tumors in situ can activate the immune system and produce a specific immune response against the tumor. Microwave ablation (MWA) with different parameters can ablate tumors with similar sizes and cause different local inflammatory effects. Our aim was to determine the immunological effects induced by different energy modes of MWA for a primary tumor. Seventy rabbits with VX2 tumors that were implanted subcutaneously underneath the right second nipple were treated with high-power MWA (40 W for 1 min), low-power MWA (20 W for 2 min), or surgical resection or were left without treatment (control). Survival time was evaluated by log-rank test. On day 14 after ablation, immunohistochemistry and flow cytometry were used to evaluate the T-cell immune responses. In addition, the cytokine patterns were identified by enzyme-linked immunosorbent assay. Tumor eradication was achieved completely in the MWA groups, as proven by nicotinamide adenine dinucleotide diaphorase staining. Compared with the three treatment groups, the control group had a significantly higher number of pulmonary metastases and worse survival; however, no significant difference was observed among the three treatment groups. More intra-tumoral and systemic CD4+ and CD8+ T-cells were induced in the MWA groups than in the control group. Compared with operation, MWA induced more systemic CD4+ T-cells. More intra-tumoral CD4+ and CD8+ T-cells and systemic CD4+ T-cells were induced by high-power MWA than by low-power MWA. Moreover, MWA increased the interleukin 2 (IL2) and IL12 levels and decreased the IL4, IL6, and IL10 levels. Importantly, the serum IL12 level was significantly higher after high-power MWA than after low-power MWA. High-power MWA enhanced the type 1 T helper immune response and may be selected for the treatment of solid tumors. Future studies are needed to confirm our results.

Detection of Ablation Boundaries Using Different MR Sequences in a Swine Liver Model

PurposeTo determine the magnetic resonance (MR) sequences best suited for the assessment of ablation zones after radiofrequency ablation (RFA).MethodsThree percutaneous MR-guided RFA of the liver were performed on three swine. Four pre-contrast and two hepatobiliary post-contrast sequences were obtained after ablation. Tissue samples were extracted and stained for nicotinamide adenine dinucleotide diaphorase hydride (NADH) and with hematoxylin and eosin. Post-ablation MR images and NADH slides were segmented to determine the total ablation zone, their Dice similarity coefficient (DSC), and the contrast-to-noise ratio (CNR) of the visible ablation boundary to normal liver tissue.ResultsTwo distinct layers were combined to determine the ablation zone: an inner layer of coagulation necrosis and an outer layer defined as the peripheral transition zone. Corresponding zones could be found in the MR images as well. Compared to histology, the total area of the MR ablation zone was significantly smaller on the pre-contrast T1 images (p < 0.01) and significantly larger with T2 turbo spin-echo (p = 0.025). No significant difference in size of the ablation zone depiction could be found between histology, post-contrast T1 volumetric interpolated breath-hold examination (VIBE), and post-contrast T1 3D Turboflash (TFL) as well as T2 SPACE images. All sequences but the pre-contrast T1 VIBE sequence showed a DSC above 80% and a high CNR.ConclusionsPost-contrast T1 3DTFL performs best when assessing ablation zones after RFA. Since the sequence requires a long acquisition time, T1 VIBE post-contrast offers the best compromise between acquisition time and estimation accuracy.

Enzyme Histochemistry of Hemal Nodes of Indian Buffalo (Bubalus bubalis)

The present study was conducted on hemal nodes of buffalo to study the histoenzymic distribution of phosphatases and oxidoreductases. The fresh unfixed buffalo hemal nodes were immediately collected after sacrifice and were subjected to cryostat sectioning at -200 C with cryostat microtome. The sections were incubated with substrates for demonstration of various enzymes. The study revealed that in both the groups, weak alkaline phosphatase (AKPase) activity was observed in capsule of the hemal node. The AKPase activity was moderate in subcapsular sinus, periphery of lymphoid follicles and diffused lymphocytes. Uniform weak Glucose 6-phosphate activity was observed in entire hemal node. Succinic dehydrogenase (SDH) activity was moderate in capsule and lymphoid follicles and weak in subcapsular sinuses. Lactic dehydrogenase activity was moderate to strong in capsule, sub-capsular sinus and peripheral area of lymphoid follicles. Nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-D) and reduced Nicotinamide adenine dinucleotide diaphorase (NADH-D) was weak to moderate in the capsule and moderate to strong in lymphoid follicles. The presence of variable activity of different enzymes in hemal nodes was correlated with maturation of lymphocytes and development of different metabolic pathways.

The effect of temperature-control microwave on HELA and MG-63 cells.

Hyperthermia has now been used to treat many kinds of solid malignancies. However, the applied thermal parameters about heat temperature and time varied all over the world, and no consensus about the optimal formula had been reached. Microwave ablation, as one of thermal ablation methods, is usually applied based on the fixed parameters of power and duration. As a result, too high temperature or overheating might not be avoided and excessive heating might cause some additional side effects to normal tissues. To explore the optimal parameters of power and duration for the HELA and MG-63 cells in vitro. With a temperature-controlled microwave workstation, a microwave thermal ablation experiment was performed in vitro. The HELA and MG-63 cells were heated with 40°C, 45°C, 50°C, 55°C, and 60°C lasting for 5-30 min, respectively. Then, the cell viability was detected using four methods: Flow cytometer assay, nicotinamide adenine dinucleotide-diaphorase staining, Calcein-acetoxymethyl ester staining immediately after treatment, and CCK-8 assay 24 h later. The temperature-controlled microwave has an excellent ablation effect on both cell lines. Furthermore, when the thermal stimulation reached 55°C 25 min and 55°C 20 min for the HELA and MG-63 cells, respectively, or 60°C 5 min for both, all the viability indexes indicated immediately devitalization. It presented a preliminary minimum lethal dose of heat was validated on the cellular level in vitro, which should be verified and corrected further in vivo.

Metabolic and vascular pattern in medial pterygoid muscle is altered by chronic stress in an animal model of hypodontia

Psychological stress is an important perpetuating, worsening and risk factor for temporomandibular disorders of muscular or articular origin. Occlusion instability, by the way, is considered a risk factor of this pathology and can be reproduced in some experimental animal models. The exact physiologic mechanism underlying these relations however, remains unclear. Our purpose was to test the hypothesis that chronic stress and unilateral exodontia induce metabolic and vascular changes in the medial pterygoid muscle of rats. Adult Wistar rats were submitted to chronic unpredictable stress and/or unilateral exodontia and their plasma and medial pterygoid muscle were removed for analysis. The parameters evaluated included plasma levels of corticosterone, metabolic activity by succinate dehydrogenase, oxidative capacity by nicotinamide adenine dinucleotide diaphorase, capillary density by laminin and alfa-CD staining and reactive oxidative species production. Chronic unpredictable stress as an isolated factor, increased oxidative metabolism, capillary density and reactive oxygen species production at medial pterygoid muscle. Conversely, exodontia has a main effect in metabolism, promoting glycolytic transformation of muscle fibers. Association of both factors induced a major glycolytic pattern in muscle and vascular changes. Our findings provide insights into the mechanisms, possibly inducing metabolic and vascular alterations on medial pterygoid muscle of rats, by which chronic stress and occlusal instabilities might be involved as risk factors in the pathophysiology of temporomandibular disorders with muscular components.

The Stomatogastric and Enteric Nervous System of the Pulmonate Snail Megalobulimus abbreviatus: A Neurochemical Analysis.

Chemical coding of stomatogastric nervous system (STNS) and enteric nervous system (ENS) of midgut and hindgut in the snail Megalobulimus abbreviatus was investigated using histochemistry, histofluorescence, and immunohistochemistry. The gastrointestinal plexuses, constituted by intrinsic neurons and fibers originating from the subesophageal ganglia and/or STNS, showed intense acetylcholinesterase (AChE) and nicotinamide adenine dinucleotide diaphorase (NADPHd) activity. The enteric neurons and fibers with AChE activity are scattered in the submucosa and between both muscular layers of gastrointestinal tract, whereas NADPHd neurons and fibers are more abundant between muscular layers than in the submucosa. Catecholaminergic nerve fibers and varicosities are found mainly within the submucosa across the mid- and hindgut. Serotoninand FMRFamide-immunoreactive neurons and fibers originating from the STNS are distributed in the submucosa of the intestine and rectum. FMRFamide-immunoreactive neurons and fibers are present in the mucosa, submucosa, and muscular layers of mid- and hindgut. The neuron-like intraepithelial cells exhibited AChE activity, a few NADPHd activity, and immunoreactivity for serotonin and FMRFamide. Intense glial fibrillary acidic protein (GFAP) immunoreaction is found throughout the intestine plexuses and in the STNS ganglia. The GFAP immunoreaction in intramural plexuses suggests the presence of glial cells as an important component of ENS in this pulmonate snail.

Phase II study on radiofrequency ablation in stage 0 and I breast cancer without extensive intraductal components.

e12094 Background: We reported about a multi-center registration study on radiofrequency ablation (RFA) in early breast cancer (BC) (ASCO2012 #1119). Although various devices and ablation procedure were used, 5-year’s recurrence-free survival was 96% in 425 cases of T1BC treated with RFA alone. To validate complete pathological ablation in BC, we started a non-randomized phase II study in 2013 (UMIN000013836). Methods: Unilateral BC patients with stage 0 (TisN0M0) or I (T1N0M0 or T1N1miM0) were eligible. Tumor diameter of 2cm or less should be diagnosed by ultrasound and MR mammography. RFA was performed by Cool-tip RF ablation system (Covidien, USA). One month later, ablated tissue was collected by core needle biopsy or vacuum-assisted breast biopsy. Cell viability was determined by central review of independent pathologists using tumor specimens stained with hematoxylin–eosin and nicotinamide adenine dinucleotide (NADH) diaphorase. In case of complete ablation, breast irradiation and adjuvant therapy was planned. In case of incomplete ablation, partial mastectomy was recommended. The primary endpoint is complete ablation rate. The secondary endpoints are breast deformity after RFA, relapse-free survival and overall survival for 10 years. Two-step design consisting of 9 cases at 1st step and 20 cases at 2nd step was used for statistical evaluation. We expected complete ablation rate of 95% and 29 cases were needed to reject the null hypothesis that complete ablation rate would be less than 80% at lower threshold. Thus, 32 cases were required. Results: Thirty-six patients were enrolled between February 2013 and May 2016. The mean tumor size was 1.3 cm measured by MR mammography. Two patients were ineligible because of macrometastasis in a sentinel node. Of 34 eligible patients, one patient had viable cancer cells with NADH diaphorase staining in ablated specimens. Finally, complete ablation rate was 97%. As of January 2017, one patient, who refused adjuvant therapy and breast irradiation, had in-breast tumor recurrence adjacent to previously ablated lesion. Conclusions: Long-term follow-up should be needed, but RFA in early BC is a promising strategy instead of breast-conserving surgery. Clinical trial information: UMIN000013836.

Chronological changes of radiofrequency ablation zone in rabbit liver: an in vivo correlation between gross pathology and histopathology.

To examine the gross pathology and histopathology of ablation zones created from radiofrequency (RF) ablation and to correlate their chronological changes. A total of 48 in vivo ablation zones (16 rabbit livers) were obtained immediately after and also 30 min, 1 h and 2 h after RF ablation and were subjected to haematoxylin and eosin (H&E) staining, nicotinamide adenine dinucleotide (NADH) diaphorase staining, terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining. Chronological changes in gross pathology and histopathology were evaluated and correlated with each other. Peripheral red zones on gross pathology correlated with peripheral zones on H&E staining, lightly stained peripheral zones on NADH staining and peripheral positive zones on TUNEL staining. Central white zones on gross pathology correlated with combined central and border zones on H&E staining, central negative zones on NADH staining and combined central-positive and middle-negative zones on TUNEL staining. Boundary visibility between central white and peripheral red zones on gross pathology was significantly higher at 1 and 2 h than immediately after RF ablation. As time increased after RF ablation, visibility of the border zone on H&E staining and the grade of positively stained hepatocytes in the peripheral zone on TUNEL staining increased. Chronological changes in gross pathology of RF ablation zones correlated well with histopathology. The boundary between the central white and peripheral red zones tended to become clear at 1 h after RF ablation. Advances in knowledge: (1) RF ablation zones show chronological changes on gross pathology and histopathology. (2) Gross pathology and histopathology correlate well with each other.

Metabolic Changes in Masseter Muscle of Rats Submitted to Acute Stress Associated with Exodontia

Clinical evidence has shown that stress may be associated with alterations in masticatory muscle functions. Morphological changes in masticatory muscles induced by occlusal alterations and associated with emotional stress are still lacking in the literature. The objective of this study was to evaluate the influence of acute stress on metabolic activity and oxidative stress of masseter muscles of rats subjected to occlusal modification through morphological and histochemical analyses. In this study, adult Wistar rats were divided into 4 groups: a group with extraction and acute stress (E+A); group with extraction and without stress (E+C); group without extraction and with acute stress (NO+A); and control group without both extraction and stress (NO+C). Masseter muscles were analyzed by Succinate Dehydrogenase (SDH), Nicotinamide Adenine Dinucleotide Diaphorase (NADH) and Reactive Oxygen Species (ROS) techniques. Statistical analyses and two-way ANOVA were applied, followed by Tukey-Kramer tests. In the SDH test, the E+C, E+A and NO+A groups showed a decrease in high desidrogenase activities fibers (P < 0.05), compared to the NO+C group. In the NADH test, there was no difference among the different groups. In the ROS test, in contrast, E+A, E+C and NO+A groups showed a decrease in ROS expression, compared to NO+C groups (P < 0.05). Modified dental occlusion and acute stress - which are important and prevalent problems that affect the general population - are important etiologic factors in metabolic plasticity and ROS levels of masseter muscles.

29 Attitude and behavior of veterinarians surrounding the use of complementary and alternative veterinary medicine (CAVM) in the treatment of equine musculoskeletal pain

The skeletal muscle has several types of fibers that differ in structure, metabolism, and function. Fiber types determine the ability of horses to perform in various sports or work activities. Gluteus medius muscles of Colombian creole horses (CCHs) were sampled to determine their fiber types. Samples were processed using myosin adenosine triphosphatase (mATPase) and immunohistochemistry for fiber typing, myofibril electrophoresis for myosin heavy chain composition, and nicotinamide adenine dinucleotide diaphorase activity for oxidative capacity. The following fiber proportions were observed by immunohistochemistry (n = 7): 22.5 ± 3.7% type I, 33.6 ± 2.3% type IIA, 43.6 ± 4.4% type IIX, and 0.3 ± 0.2% hybrid I/IIA fibers. The results obtained by mATPase were comparable. Regarding the oxidative capacity, 20.1 ± 4.0% had a high oxidative reaction, 32.7 ± 6.1% an intermediate reaction, and 47.2 ± 7.4% a low oxidative reaction. Of these, the high and low capacity correlated with type I and IIX fibers, respectively. Myosin heavy chain composition experiments (n = 7) showed 22.2 ± 1.6% of type I, 30.4 ± 2.7% of type IIA, and 47.4 ± 2.3% of type IIX. These values are close to those published for Andalusian horses but differ from some reported for other breeds. Considering the high percentage of type IIX fibers and low to intermediate oxidative capacity, we conclude that the CCH is adapted to perform power exercise and moderate- to high-intensity anaerobic work with short to average duration. Therefore, training should be aimed at increasing aerobic capacity and endurance if these animals are required to perform long-length exercise.

Abstract OT1-3-01: Phase II study on radiofrequency ablation in stage 0 and I breast cancer without extensive intraductal components

Abstract Background: Screening mammography makes it possible to identify small size of breast cancer (BC) and minimize surgical management. Previously we reported about a multi-center cohort study on radiofrequency ablation (RFA) in early breast cancer (ASCO2012 #1119). Although various devices and ablation procedure were attempted, 5-year’s recurrence-free survival of ipsilateral breast tumor was 96% in 425 cases of T1BC treated with RFA alone (the mean follow-up, 50 months). To validate complete pathological ablation in BC, we started a non-randomized phase II study in 2013 (UMIN000013836). Eligibility criteria: Unilateral BC patients with stage 0 (TisN0M0) or I (T1N0M0 or T1N1miM0) are eligible. Written informed consent is obtained. Tumor diameter of 2cm or less should be diagnosed by ultrasound and MR mammography. BC with diffuse calcification, extensive intraductal components, or multiple tumors is excluded. Methods: RFA is performed by Cool-tip RF ablation system (Covidien, USA). The needle electrode is inserted into the center of referent tumor under ultrasound guidance. The first ablation is started at an initial electrical power level of 5W for 1 min and the power is increased at 10W for 1 min. After then, it is increased in steps of 10W from every minute until rapid elevation of tissue impedance. The second ablation is allowed by physician’s discretion. One month later, ablated tissue is collected by core needle biopsy or vacuum-assisted breast biopsy. Cell viability is examined by central review of independent pathologists using tumor specimens stained with hematoxylin–eosin and nicotinamide adenine dinucleotide (NADH) diaphorase. In case of complete ablation, breast irradiation and adjuvant therapy will be performed. In case of incomplete ablation, partial mastectomy should be recommended. Aims: The primary endpoint is complete ablation rate. The secondary endpoints are deformity after RFA, relapse-free survival and overall survival for 10 years. Two-step design is used for statistical evaluation. Finally, 32 patients will be needed. Present accrual: As of April 2014, 16 patients were enrolled. One patient was ineligible because of macrometastasis in a sentinel node. Of 13 eligible patients who underwent pathological examination, one patient had viable cancer cells with NADH diaphorase staining. We also investigate optimal histological examination of cell viability and adequate image diagnosis after RFA. Citation Format: Shigeru Imoto, Shinji Nagamine, Shunichi Ito, Hitoshi Tsuda, Masayuki Yoshida, Mitsuhiro Tozaki, Satoshi Morita, Takayuki Ueno. Phase II study on radiofrequency ablation in stage 0 and I breast cancer without extensive intraductal components [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr OT1-3-01.

Muscle Fiber Types of Gluteus Medius in the Colombian Creole Horse

The skeletal muscle has several types of fibers that differ in structure, metabolism, and function. Fiber types determine the ability of horses to perform in various sports or work activities. Gluteus medius muscles of Colombian creole horses (CCHs) were sampled to determine their fiber types. Samples were processed using myosin adenosine triphosphatase (mATPase) and immunohistochemistry for fiber typing, myofibril electrophoresis for myosin heavy chain composition, and nicotinamide adenine dinucleotide diaphorase activity for oxidative capacity. The following fiber proportions were observed by immunohistochemistry (n = 7): 22.5 ± 3.7% type I, 33.6 ± 2.3% type IIA, 43.6 ± 4.4% type IIX, and 0.3 ± 0.2% hybrid I/IIA fibers. The results obtained by mATPase were comparable. Regarding the oxidative capacity, 20.1 ± 4.0% had a high oxidative reaction, 32.7 ± 6.1% an intermediate reaction, and 47.2 ± 7.4% a low oxidative reaction. Of these, the high and low capacity correlated with type I and IIX fibers, respectively. Myosin heavy chain composition experiments (n = 7) showed 22.2 ± 1.6% of type I, 30.4 ± 2.7% of type IIA, and 47.4 ± 2.3% of type IIX. These values are close to those published for Andalusian horses but differ from some reported for other breeds. Considering the high percentage of type IIX fibers and low to intermediate oxidative capacity, we conclude that the CCH is adapted to perform power exercise and moderate- to high-intensity anaerobic work with short to average duration. Therefore, training should be aimed at increasing aerobic capacity and endurance if these animals are required to perform long-length exercise.

Distribution of Phosphatases, Oxidoreductases and Esterases in Sublingual Salivary Gland of Buffalo During Neonatal Development

The present study was aimed to elucidate the distribution of various enzymes in sublingual salivary gland of neonatal buffalo (n = 10), ranging from 1 to 3 months age, and correlate these enzyme systems with the secretory activity of gland. At 1 month age, a uniform moderate to strong positive diffused granular alkaline phosphatase activity was observed in serous acini and ductal epithelium. A moderate positive glucose-6-phosphatase and succinic dehydrogenase activity was seen close to the basement membrane. At 2 months age, glutamic dehydrogenase showed moderate activity in basal portions of serous acinar cells, while succinic dehydrogenase activity was strong, granular and evenly distributed in basement membrane of serous cells. Weak activity of monoamine oxidase and moderate non-specific esterase activity was seen. At 3 months age, alkaline phosphatase showed strong positive perinuclear activity in acinar cells, while glucose-6-phosphatase showed a moderate to strong activity in acinar cells and in ductal epithelium. Moderate to strong positive non-specific esterase activity was localized in nerve fibres of intralobular and interlobular connective tissues. Glucose-6-phosphate dehydrogenase and nicotinamide adenine dinucleotide diaphorase showed strong positive granular activity in basement membrane of serous acinar cells and ducts. The high basal activities of oxidoreductases were because of basal mitochondria and were associated with high energy production to meet the energy requirement for active and rapid transport of ions and water.

146. Positive predictive value of breast lesions of uncertain malignant potential (B3): Implications in the surgical treatment and value of a multidisciplinary team

result of radiofrequency ablation (RFA) immediately followed by breastconserving surgery (BCS) in 30 patients with T1N0 BC (Breast, 2009). Complete pathological ablation was found in 26 patients (87%) from the pathological diagnosis of tumor specimens stained with hematoxylineeosin and nicotinamide adenine dinucleotide diaphorase. Thus, we conducted a phase II study to evaluate the safety and reliability of RFA alone in BC. Material and methods: T1 and sentinel node-negative BC patients were eligible. BC with diffuse calcification or extensive intraductal component (EIC) was excluded. RFAwas performed using a LeVeen needle electrode system (Boston Scientific Corporation, USA). Primary endpoint was breast deformity after RFA, which was evaluated by calculating absolute difference of measurement from nipple to several points of the breast. Results: Twenty patients were enrolled from Apr. 2009 to Feb. 2013. There were no adverse events like skin burn, bleeding and infection. As of Jan. 2014, all patients were free of recurrence at the median follow-up of 34 months. Breast deformity after RFA was very limited in most patients. Conclusion: RFA is a promising alternative to BCS in stage I BC without EIC. No conflict of interest.

Histological and Biochemical Analysis of Mechanical and Thermal Bioeffects in Boiling Histotripsy Lesions Induced by High Intensity Focused Ultrasound

Recent studies have shown that shockwave heating and millisecond boiling in high-intensity focused ultrasound fields can result in mechanical fractionation or emulsification of tissue, termed boiling histotripsy. Visual observations of the change in color and contents indicated that the degree of thermal damage in the emulsified lesions can be controlled by varying the parameters of the exposure. The goal of this work was to examine thermal and mechanical effects in boiling histotripsy lesions using histologic and biochemical analysis. The lesions were induced in ex vivo bovine heart and liver using a 2-MHz single-element transducer operating at duty factors of 0.005–0.01, pulse durations of 5–500 ms and in situ shock amplitude of 73 MPa. Mechanical and thermal damage to tissue was evaluated histologically using conventional staining techniques (hematoxylin and eosin, and nicotinamide adenine dinucleotide-diaphorase). Thermal effects were quantified by measuring denaturation of salt soluble proteins in the treated region. According to histologic analysis, the lesions that visually appeared as a liquid contained no cellular structures larger than a cell nucleus and had a sharp border of one to two cells. Both histologic and protein analysis showed that lesions obtained with short pulses (<10 ms) did not contain any thermal damage. Increasing the pulse duration resulted in an increase in thermal damage. However, both protein analysis and nicotinamide adenine dinucleotide-diaphorase staining showed less denaturation than visually observed as whitening of tissue. The number of high-intensity focused ultrasound pulses delivered per exposure did not change the lesion shape or the degree of thermal denaturation, whereas the size of the lesion showed a saturating behavior suggesting optimal exposure duration. This study confirmed that boiling histotripsy offers an effective, predictable way to non-invasively fractionate tissue into sub-cellular fragments with or without inducing thermal damage.

Ultrasound-guided radiofrequency ablation of early breast cancer in a resection specimen: Lessons for further research

PurposeTo assess the feasibility and effectiveness of radiofrequency ablation (RFA) in breast cancer, using different histopathologic staining methods to evaluate tissue viability. Materials and methodsIn twenty patients with unifocal small (≤1, 5 cm) invasive ductal carcinoma, ultrasound-guided RFA was performed immediately after surgery. Cell viability was assessed using cytokeratin 8 (CK 8) and nicotinamide adenine dinucleotide diaphorase (NADHD) in addition to hematoxylin–eosin (HE). ResultsAt histopathological examination, ex vivo RFA resulted in complete cell death of the target lesion in 17/20 patients. In two cases viable ductal carcinoma in situ (DCIS) was found just outside the completely ablated lesion. ConclusionRFA of small invasive breast cancer seems to be a feasible treatment option. Both NADHD and CK 8 demonstrate a clear and comparable demarcation between viable and non-viable tissue. A high level of accuracy is required in proper positioning of the needle electrode and a “hot retraction” is mandatory.

Effect of retinoic acid on the nitrergic innervation of meibomian glands in rats

The purpose of this study was to investigate the effect of prenatal administration of retinoic acid (RA) on the development of nicotinamide adenine dinucleotide diaphorase (NADPH-d) positive structures in the rat Meibomian glands. One mg/kg of RA was applied to pregnant Wistar rats intraperitonaelly during the gestational period in each of the 12th–14th embryonic days (totally 3 mg/kg). Sections of the central upper eyelids were investigated in rat pups on the 14th postnatal day. They were processed histochemically for NADPH-d, to study the presence and distribution of nitric oxide synthase (NOS) positive nerve structures. NADPH-d staining of Meibomian glands was compared in two groups of rat pups. In the control group, eyelids of 14 day-old rats were studied with no experimental intervention. The second group consisted of rat pups which were prenatally administered the excess of RA. Histochemical analysis of control eyelids revealed numerous NADPH-d well-stained acini of Meibomian glands arranged tightly into groups. Intensively stained vessels and NADPH-d/NOS-positive nerve fibers bordered acini of Meibomian glands. These structures were present in the submucosal layer as well. The analysis of RA group showed less numerous, shrunken acini of Meibomian glands that were seen not only smaller in size, but also in density of their staining and the amount of nitrergic nerve fibers around acini were considerably lowered. In the submucosa differences were noticed compared to the control group, there were numerous NADPH-d stained vessels accompanied by NADPH-d/NOS-positive nerve fibers. The excess of RA during the prenatal period may influence on the development and morphology of NADPH-d positive structures of rat's Meibomian glands.

Image and Pathological Changes after Radiofrequency Ablation of Invasive Breast Cancer: A Pilot Study of Nonsurgical Therapy of Early Breast Cancer

The surgical treatment of early breast cancer has proceeded to less invasive approaches with better cosmetic results. The current study was undertaken to evaluate the clinical and pathological findings after radiofrequency ablation (RFA) without resection for a longer period of time. A total of 14 patients with breast cancer were enrolled. All patients were diagnosed to have invasive ductal carcinoma, and the median breast tumor size was 12 mm (range, 6-20 mm). Six patients received RFA treatment followed by immediate resection and eight patients without resection. The patients without resection were evaluated by ultrasound, MRI, and the pathological findings of a core needle biopsy after RFA. The removed specimens were examined by hematoxylin-eosin (HE) staining and nicotinamide adenine dinucleotide (NADH) diaphorase staining. The median follow-up of the patients was 39.9 months. NADH staining was necessary to diagnose complete tumor cell death in the tissue for 3 months after RFA. However, HE staining alone could confirm the effect without NADH staining more than 6 months after RFA. Post-RFA, MRI scans clearly demonstrated the area as a complete ablated lesion in all patients without resection. The ablated area detected by MRI or ultrasound became gradually smaller. All patients that underwent RFA with no resection were alive without relapse. RFA therefore could be an effective alternative to partial mastectomy for early breast cancer. Further research will be necessary to establish the standardization of the indications, as well as the optimal techniques and post treatment evaluation modalities.

Inhibition of Acetylcholinesterase and NO Synthase Activity in the Mice Brain: Effect of a Withania Somnifera Leaf Juice

Inhibitory effects of a fresh leaf juice of the Withania somnifera (W.s.) on the enzymes acetylcholinesterase (AChE; EC 3.1.17) and nicotinamide adenine dinucleotide diaphorase (NADPH-d) were studied in the mice brain using histochemical and biochemical approaches. The results obtained show significant reduction in the number of and reaction intensity in AChE- and NADPH-d-positive neurons within selected areas of the brains of W.s.-treated mice. Colocalization of AChE and NADPH-d activity also showed significant reduction in the number of cells positive for both enzymes after W.s. treatment, as compared to cells with isolated localization of AChE or NADPH-d. Biochemical estimation of the AChE and NADPH-d levels in brain tissue also showed significant dose-dependent inhibition of the activity of both enzymes after W.s. treatment. In conclusion, our study allows us to suggest that W.s. constituents significantly inhibit both AChE and NADPH-d activity. Inhibition of AChE is direct, while NADPH-d is inhibited indirectly. This study might be important for possible therapeutic use of W.s. in neurodegenerative disorders, in particular in Alzheimer’s disease where inhibition of both AChE and nNOS is important.

Expression of the P2X2receptor in different classes of ileum myenteric neurons in the female obese ob/ob mouse

To examine whether the ob/ob mouse model of obesity is accompanied by enteric nervous system abnormalities such as altered motility. The study examined the distribution of the P2X₂ receptor (P2X₂R) in myenteric neurons of female ob/ob mice. Specifically, we used immunohistochemistry to analyze the co-expression of the P2X₂R with neuronal nitric oxide synthase (nNOS), choline acetyltransferase (ChAT), and calretinin (CalR) in neurons of the small intestine myenteric plexus in ob/ob and control female mice. In these sections, we used scanning confocal microscopy to analyze the co-localization of these markers as well as the neuronal density (cm²) and area profile (μm²) of P2X₂R-positive neurons. In addition, enteric neurons were labeled using the nicotinamide adenine dinucleotide (NADH) diaphorase method and analyzed with light microscopy as an alternate means by which to analyze neuronal density and area. In the present study, we observed a 29.6% increase in the body weight of the ob/ob animals (OG) compared to the control group (CG). In addition, the average small intestine area was increased by approximately 29.6% in the OG compared to the CG. Immunoreactivity (IR) for the P2X₂R, nNOS, ChAT and CalR was detectable in the myenteric plexus, as well as in the smooth muscle, in both groups. This IR appeared to be mainly cytoplasmic and was also associated with the cell membrane of the myenteric plexus neurons, where it outlined the neuronal cell bodies and their processes. P2X₂R-IR was observed to co-localize 100% with that for nNOS, ChAT and CalR in neurons of both groups. In the ob/ob group, however, we observed that the neuronal density (neuron/cm²) of P2X₂R-IR cells was increased by 62% compared to CG, while that of NOS-IR and ChAT-IR neurons was reduced by 49% and 57%, respectively, compared to control mice. The neuronal density of CalR-IR neurons was not different between the groups. Morphometric studies further demonstrated that the cell body profile area (μm²) of nNOS-IR, ChAT-IR and CalR-IR neurons was increased by 34%, 20% and 55%, respectively, in the OG compared to controls. Staining for NADH diaphorase activity is widely used to detect alterations in the enteric nervous system; however, our qualitative examination of NADH-diaphorase positive neurons in the myenteric ganglia revealed an overall similarity between the two groups. We demonstrate increases in P2X₂R expression and alterations in nNOS, ChAT and CalR IR in ileal myenteric neurons of female ob/ob mice compared to wild-type controls.