Use of Two Novel Dyes to Enhance Visualization of Cut Ends of the Vessel in Microvascular Anastomosis-An Experimental Study of 45 Rats.

BACKGROUND: Good visualization is a prerequisite for performing microvascular anastomosis. The most commonly used dye, methylene blue, has several limitations: it is washed off quickly and stains all the vessel layers. The objective of our study is to use 2 new novel dyes for improving visualization. METHODS: After ethical committee approval, 2 Dyes (2% cresyl violet, 1% eosin) were studied in 3 groups, 20 rats in each group and 5 rats in the combined group. End-to-side anastomosis was performed in the classic fashion in 45 rats. After venotomy, the dye was applied to the raw surface of the vessels and subsequently, anastomosis was performed. The improvement in visualization was judged by 3 blinded experts and nonexperts in 4 groups on a scale of 1-10. Scores were statistically analyzed. After 2 weeks, animals were re-explored to check the delayed patency, and segments were harvested for histopathologic analysis. RESULTS: The immediate and delayed patency rates were 100% (45/45) and 97% (33/34), respectively. In statistical analysis, the combined group (P = 0.005)was judged statistically significant because of the contrast in color. All the layers were stained by both dyes, staining lasted until the end of the surgery. Visibility of the cut ends was better in cresyl violet. All histopathologic findings suggested normal changes at the anastomotic site. CONCLUSIONS: This study showed that the use of these 2 dyes was not only feasible but highly efficacious. Even though all the layers were stained by both the dyes, the visibility of the cut ends was better. In both dyes, staining lasted until the end of surgery. To the best of our knowledge, this is the first study that has used these 2 novel dyes to improve visualization in microvascular anastomosis in an experimental setting.

Construction of a highly sensitive detection platform for heparin based on a "turn-off" cationic fluorescent dye.

A highly sensitive detection platform for heparin was constructed via the utilization of a commercially available cationic fluorescent dye (cresyl violet acetate, CV) as a fluorescence probe. The electrostatic binding between CV and heparin quenched the fluorescence in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic (HEPES) buffer solution (10 mM, pH 7.1). CV was highly selective towards heparin over other potential inferring substances. The detection limit of heparin detection was 5.19 ng/mL, and the linear working range was 0 ∼ 1 µg/mL in HEPES solution. In 1 % serum, the detection platform based on the fluorescence "turn-off" behavior of CV was also successfully constructed with a detection limit of 5.86 ng/mL in the linear range of 0 ∼ 0.8 µg/mL. Moreover, the CV-heparin complex was considered a potential sensor platform for the detection of protamine because of its stronger affinity for heparin and protamine.

Neuroprotective effects of Korean White ginseng and Red ginseng in an ischemic stroke mouse model.

Background: Stroke is a neurological disorder characterized by brain tissue damage following a decrease in oxygen supply to brain due to blocked blood vessels. Reportedly, 80% of all stroke cases are classified as cerebral infarction, and the incidence rate of this condition increases with age. Herein, we compared the efficacies of Korean White ginseng (WG) and Korean Red Ginseng (RG) extracts (WGex and RGex, respectively) in an ischemic stroke mouse model and confirmed the underlying mechanisms of action. Methods: Mice were orally administered WGex or RGex 1 h before middle cerebral artery occlusion (MCAO), for 2 h; the size of the infarct area was measured 24 h after MCAO induction. Then, the neurological deficit score was evaluated and the efficacies of the two extracts were compared. Finally, their mechanisms of action were confirmed with tissue staining and protein quantification. Results: In the MCAO-induced ischemic stroke mouse model, WGex and RGex showed neuroprotective effects in the cortical region, with RGex demonstrating superior efficacy than WGex. Ginsenoside Rg1, a representative indicator substance, was not involved in mediating the effects of WGex and RGex. Conclusion: WGex and RGex could alleviate the brain injury caused by ischemia/reperfusion, with RGex showing a more potent effect. At 1,000 mg/kg body weight, only RGex reduced cerebral infarction and edema, and both anti-inflammatory and anti-apoptotic pathways were involved in mediating these effects.

An effective approach to develop targetable and responsive fluorescent probes for imaging of organelles based on cresyl violet scaffold.

Fluorescent probes combined with confocal microscopy are recognized as a powerful tool for imaging living cells and even organelles due to their high sensitivity and resolution. However, many of analyte-activatable and organelle-targetable fluorescent probes are developed via tedious attempts, and a relatively predictable method to design such probes is still lacking. Herein, we put forward an effective synthetic strategy to construct both targetable and responsive probes for organelles based on the cresyl violet scaffold. The approach allows access to a variety of organelle-targeting fluorescent probes for an analyte of interest via introducing the corresponding targeting and recognition groups to the 5- and 9-positions of cresyl violet, respectively. The potency of the approach is exemplified by its application to develop four cresyl violet-based fluorophores with different organelle-targeting groups, and a mitochondrion-targeting ratiometric probe capable of imaging Pd0 in living cells.

Multiscale imaging of the rat brain using an integrated diceCT and histology workflow.

Advancements in tissue visualization techniques have spurred significant gains in the biomedical sciences by enabling researchers to integrate their datasets across anatomical scales. Of particular import are techniques that enable the interpolation of multiple hierarchical scales in samples taken from the same individuals. In this study, we demonstrate that two-dimensional histology techniques can be employed on neural tissues following three-dimensional diffusible iodine-based contrast-enhanced computed tomography (diceCT) without causing tissue degradation. This represents the first step toward a multiscale pipeline for brain visualization. We studied brains from adolescent male Sprague-Dawley rats, comparing experimental (diceCT-stained then de-stained) to control (without diceCT) brains to examine neural tissues for immunolabeling integrity, compare somata sizes, and distinguish neurons from glial cells within the telencephalon and diencephalon. We hypothesized that if experimental and control samples do not differ significantly in morphological cell analysis, then brain tissues are robust to the chemical, temperature, and radiation environments required for these multiple, successive imaging protocols. Visualizations for experimental brains were first captured via micro-computed tomography scanning of isolated, iodine-infused specimens. Samples were then cleared of iodine, serially sectioned, and prepared again using immunofluorescent, fluorescent, and cresyl violet labeling, followed by imaging with confocal and light microscopy, respectively. Our results show that many neural targets are resilient to diceCT imaging and compatible with downstream histological staining as part of a low-cost, multiscale brain imaging pipeline.

Administering crocin ameliorates anxiety-like behaviours and reduces the inflammatory response in amyloid-beta induced neurotoxicity in rat.

Anxiety, hippocampus synaptic plasticity deficit, as well as pro-inflammatory cytokines, are involved in Alzheimer's disease (AD). The present study is designed to evaluate the possible therapeutic effect of crocin on anxiety-like behaviours, hippocampal synaptic plasticity and neuronal shape, as well as pro-inflammatory cytokines in the hippocampus using in vivo amyloid-beta (Aß) models of AD. The Aß peptide (1-42) was bilaterally injected into the frontal-cortex. Five hours after the surgery, the rats were given intraperitoneal (IP) crocin (30 mg/kg) daily up to 12 days. Elevated plus maze results showed that crocin treatment after bilateral Aß injection significantly increased the percentage of spent time into open arms, frequency of entries, and percentage of entries into open arms as compared with the Aß group. In the open field test, the Aß+crocin group showed a higher percentage of spent time in the centre and frequency of entries into central zone as compare with the Aß treated animals. Administering crocin increased the number of soma, dendrites and axonal arbores in the CA1 neurons among the rats with Aß neurotoxicity. Cresyl violet (CV) staining showed that crocin increased the number of CV-positive cells in the CA1 region of the hippocampus compared with the Aß group. Silver-nitrate staining indicated that crocin reduced neurofibrillary tangle formation induced by Aß. Crocin treatment attenuated the expression of TNF-α and IL-1ß mRNA in the hippocampus compared with the Aß group. Our results suggest that crocin attenuated Aß-induced anxiety-like behaviours and neuronal damage, and synaptic plasticity loss in hippocampal CA1 neurons may via its anti-inflammatory effects.

Computational Study of Cresyl Violet Covalently Attached to the Silane Coupling Agents: Application to TiO2-Based Photocatalysts and Dye-Sensitized Solar Cells.

The covalent attachment of photosensitizing dyes to TiO2 using silane coupling agents (SCAs) is a promising strategy for enhancing the photocatalytic activity of TiO2-based photocatalysts and the photovoltaic conversion of dye-sensitized solar cells (DSSCs). This approach can control the geometry and orientation of the photosensitizing dye on the TiO2 surface. In this study, a density functional theory (DFT) and time-dependent DFT (TD-DFT) investigation was carried out on cresyl violet (CV) covalently attached to SCAs with a terminal oxirane group (OTES-Cn) to reveal the influence of OTES-Cn on the geometry of the photosensitizing dyes. The potential of CV covalently attached to OTES-Cn (CV-OTES-Cn) to act as a photosensitizing dye was also analyzed. The hydroxyl group formed by the epoxy-opening reaction between CV and OTES-Cn strongly influenced the geometry of CV-OTES-Cn, which was attributed to a CH-O interaction. Additionally, TD-DFT, frontier molecular orbital and molecular electrostatic potential calculations revealed that CV-OTES-Cn has excellent optical properties and electron injection ability. In particular, the characteristics of the unbent conformation of CV-OTES-Cn are expected to contribute significantly to the photocurrent in TiO2-based photocatalysts and DSSCs. These findings enhance the understanding of the covalent attachment strategy using SCAs and contribute to improving TiO2-based photocatalysts and DSSCs.

Cresyl violet as a new contrast agent in probe-based confocal laser endomicroscopy for in vivo diagnosis of gastric intestinal metaplasia.

BACKGROUND AND AIM: Cresyl violet (CV) is a topical dye that allows simultaneous chromoendoscopy and in vivo confocal laser endomicroscopy in identification of neoplastic changes of the lower gastrointestinal tract without intravenous injection of fluorescein, but as yet no investigation has reported its application in the diagnosis of gastric intestinal metaplasia (GIM). This study aims to assess the feasibility as well as diagnosis accuracy of topical CV for in vivo diagnosis of GIM by using probe-based confocal laser endomicroscopy (pCLE). METHODS: In this prospective, open-label, feasibility study, 129 confocal videos from 22 patients with known GIM were analyzed and compared with corresponding histological images to establish the CV staining characteristics. In addition, 47 patients with known or suspected GIM were prospectively enrolled to evaluate the accuracy of this topical CV endomicroscopic imaging. RESULTS: Probe-based confocal laser endomicroscopy with topical CV enabled clear visualization of the goblet cells, absorptive cells, and intestinal villi of GIM. The accuracy, sensitivity, specificity, positive predictive value, and negative predictive value of pCLE diagnosis of GIM on a per-location analysis was 93.01%, 91.95%, 93.51%, 86.96%, and 96.11%, respectively. The intraclass correlation coefficient for inter-observer agreement and mean kappa value for intra-observer agreement for the diagnosis of GIM was 0.82 and 0.87, respectively. CONCLUSIONS: Topical CV enables real-time chromoendoscopy in conjunction with pCLE examination of the stomach and warrants accurate diagnosis of GIM. It may be an acceptable and potentially alternative dye for confocal imaging in the future.

Labeling Acidic Compartments of Neutrophils with Cresyl Violet.

We introduce the acidotropic marker cresyl violet to stain acidic granules in live neutrophils. Cresyl violet is less phototoxic, more photostable, and more cost-effective than other commercially available acidotropic markers. Additionally, it does not photoconvert to fluorescent species of a different color, a limitation of other commonly used acidotropic markers. Staining can be readily detected by fluorescence microscopy or by flow cytometry, and can be used as a readout of degranulation in activated neutrophils.

Nerolidol ameliorates cyclophosphamide-induced oxidative stress, neuroinflammation and cognitive dysfunction: Plausible role of Nrf2 and NF- κB.

AIM: Cyclophosphamide (CP) is a potent anticancer and immunosuppressant drug. Studies have shown significant oxidative stress and cognitive impairment but neuroinflammatory and histological aberrations with its administration is underexplored. Nerolidol (NER) is a lipophilic bioactive molecule with antioxidant and anti-inflammatory properties but it has not been explored for neuroprotective potential in CP-induced neurotoxic manifestations. Therefore, in the present study, we aimed to evaluate the neuroprotective potential of NER in CP-induced neuroinflammation and associated comorbid conditions like depression and cognitive dysfunctions. MATERIALS AND METHOD: In-silico study using Schrödinger software was used to assess the binding affinity of NER with Nrf2. In the In vivo study, NER 200 and 400 mg/kg p.o. were given from 1st day to 14th day. CP 200 mg/kg, i.p., was administered on the 7th day. After 24 h of the last dosing, neurobehavioral tests like spontaneous body alternation, passive avoidance and forced swim test were performed. On completion of study, mice were sacrificed, hippocampus and cortex were removed for biochemical estimations, histopathology and immunohistochemistry of p65 NF- κB and Nrf2. KEY FINDINGS: In-silico study showed significant binding of NER into the pocket domain of Nrf2. In-vivo study showed protective effect of NER against CP-induced neuroinflammation, oxidative stress, cognitive impairment and structural abnormalities in the hippocampus and cortex regions. SIGNIFICANCE: Findings of the study suggested that NER is a potential therapeutic molecule which can mitigate CP-induced neurotoxic manifestations via Nrf2 and NF-κB pathway. However, more detailed studies are needed to explicate the mechanism underlying its neuroprotective effect.

1-triacontanol cerotate isolated from Marsilea quadrifolia Linn. safeguards hippocampal CA3 neurons and augments special memory deficit in chronic epileptic rats / Cerotato de 1-triacontanol aislado de Marsilea quadrifolia Linn. protege las neuronas CA3 del hipocampo y mejora el déficit de memoria especial en ratas epilépticas crónicas

SUMMARY: Currently many people with epilepsy do not have seizure control even with the best available medications. Moreover various antiepileptics have adverse cognitive impact with other side effect. Thus, need for new antiepileptic drugs still remains challenge. However, many of the natural components have antiepileptic action and this fact remains scientifically unexplored. This study was designed to check the behavioral and neuro-pathological outcome of 1-Triacontanol cerotate (1TAC), isolated from Marsilea quadrifolia Linn. (MQ) on chronic Pentylenetetrazol (PTZ) kindling model of epilepsy in rats. Two-month-old adult male Wistar rats (n=60) were randomly divided into six groups; Group I (Cage Control), II (Vehicle Control), III (Positive Control), IV (Standard drug treated), V (1TAC: 40 mg/kg) & VI (1TAC: 80 mg/kg). To induce kindling a 35 mg/kg dose of PTZ was injected i.p. in every 48 hrs for 30 days in Group III to VI. Spatial memory performance was tested using Morris water maze, following which brains were further processed for histopathological investigations. Interestingly, 1TAC was able to minimize the loss of pyramidal cells in hippocampal CA3 region. These cellular changes were behaviorally responded as improved special learning and memory, a better spatial navigation and object place configuration. The current study strongly implicates that 1TAC from MQ has potent neuroprotective role and augments special memory deficit in chronic epileptic rats. The isolated component which attenuates spatial memory performance could be beneficial outcome to retain cognitive blunting in chronic epilepsy.

RESUMEN: Actualmente, muchas personas con epilepsia no cuentan con un control adecuado de las convulsiones, incluso con los mejores medicamentos disponibles. Además, varios antiepilépticos tienen un impacto cognitivo adverso además de efectos secundarios. Por lo tanto, la necesidad de nuevos fármacos antiepilépticos sigue siendo un desafío. Sin embargo, muchos de los componentes naturales tienen acción antiepiléptica y este hecho permanece científicamente inexplorado. Este estudio se diseñó para verificar el resultado conductual y neuro-patológico del cerotato de 1-triacontanol (1TAC), aislado de Marsilea quadrifolia Linn. (MQ) en el modelo de epilepsia en ratas del pentilenetetrazol (PTZ) crónico (PTZ). Ratas Wistar adultas de dos meses de edad (n = 60) se dividieron aleatoriamente en seis grupos; Grupo I (Control de jaula), II (Control de vehículo), III (Control positivo), IV (Medicamento estándar de tratamiento), V (1TAC: 40 mg / kg) y VI (1TAC: 80 mg / kg). Para inducir la inflamación se inyectó una dosis de 35 mg / kg de PTZ i.p. en cada 48 horas durante 30 días en los grupos III a VI. El rendimiento de la memoria espacial se probó utilizando el laberinto de agua de Morris, después de lo cual se procesaron los cerebros para investigaciones histopatológicas. Curiosamente, 1TAC pudo minimizar la pérdida de células piramidales en la región CA3 del hipocampo. Estos cambios celulares respondieron de manera conductual como una mejora del aprendizaje especial y la memoria, una mejor navegación espacial y la configuración del lugar del objeto. El estudio actual implica fuertemente que 1TAC de MQ tiene un potente papel neuroprotector y mejora el déficit de memoria especial en ratas epilépticas crónicas. El componente aislado que atenúa el rendimiento de la memoria espacial podría ser un resultado beneficioso para retener la reducción cognitiva en la epilepsia crónica.

Asymmetric Changes in Limbic Cortex and Planum Temporale in Patients with Alzheimer Disease.

BACKGROUND: There are several cortical areas related to the limbic system that form the output from the hippocampal formation whose cellular and morphological features are important for the onset and progression of AD. We hypothesized that there would be a significant difference in the size of cortical pyramidal neurons and that there would also be a hemispheric asymmetry between Alzheimer disease patients and controls. These differences would potentially be accompanied by an increase in the numbers of Fluoro-Jade B-positive degenerating cortical neurons and a corresponding decrease in the numbers of DAPI-stained cortical neuronal nuclei in subjects with AD compared to controls. Such changes could potentially be used as another marker in postmortem neuropathological diagnosis of AD. METHODS: We measured absolute numbers of DAPI and Fluoro-Jade B stained cells in five cortical areas of the limbic system and four subareas of planum temporale in the post-mortem brains of subjects with Alzheimer disease. We also measured the size of pyramidal neurons in layer III in the five cortical areas of the limbic system in these subjects. All measurements were performed separately for the left and right hemisphere in order to identify asymmetries between the two hemispheres. RESULTS: We observed a significant decrease in numbers of DAPI stained cells in layers IV-VI of the anterior cingulate gyrus on the right side, in layers I-III of the posterior cingulate gyrus on the left side, in layers IV-VI in the transition region from superior temporal gyrus into planum temporale on the right and in layers IV-VI in the transition from planum temporale to insular cortex on the left. We also observed a significant increase in the numbers of Fluoro-Jade stained cells in layers I-III of the anterior cingulate gyrus and in layers I-III on the left and layers IV-VI of the right gyrus of Heschl. Shortening of the size of layer III pyramidal neurons in subjects with Alzheimer´s disease was found in the anterior cingulate gyrus on the right, in the posterior cingulate gyrus and entorhinal cortex on the left and on the right in the parahippocampal gyrus. CONCLUSION: Our study demonstrates asymmetries in different cortical regions of the temporal lobe that can be used as another marker in the postmortem diagnosis of AD.

A refined model of chronic cerebral hypoperfusion resulting in cognitive impairment and a low mortality rate in rats.

OBJECTIVE: The cognitive deficits of vascular dementia and the vasoocclusive state of moyamoya disease have often been mimicked with bilateral stenosis/occlusion of the common carotid artery (CCA) or internal carotid artery. However, the cerebral blood flow (CBF) declines abruptly in these models after ligation of the CCA, which differs from "chronic" cerebral hypoperfusion. While some modified but time-consuming techniques have used staged occlusion of both CCAs, others used microcoils for CCA stenosis, producing an adverse effect on the arterial endothelium. Thus, the authors developed a new chronic cerebral hypoperfusion (CCH) model with cognitive impairment and a low mortality rate in rats. METHODS: Male Sprague-Dawley rats were subjected to unilateral CCA occlusion and contralateral induction of CCA stenosis (modified CCA occlusion [mCCAO]) or a sham operation. Cortical regional CBF (rCBF) was measured using laser speckle flowmetry. Cognitive function was assessed using a Barnes circular maze (BCM). MRI studies were performed 4 weeks after the operation to evaluate cervical and intracranial arteries and parenchymal injury. Behavioral and histological studies were performed at 4 and 8 weeks after surgery. RESULTS: The mCCAO group revealed a gradual CBF reduction with a low mortality rate (2.3%). White matter degeneration was evident in the corpus callosum and corpus striatum. Although the cellular density declined in the hippocampus, MRI revealed no cerebral infarctions after mCCAO. Immunohistochemistry revealed upregulated inflammatory cells and angiogenesis in the hippocampus and cerebral cortex. Results of the BCM assessment indicated significant impairment in spatial learning and memory in the mCCAO group. Although some resolution of white matter injury was observed at 8 weeks, the animals still had cognitive impairment. CONCLUSIONS: The mCCAO is a straightforward method of producing a CCH model in rats. It is associated with a low mortality rate and could potentially be used to investigate vascular disease, moyamoya disease, and CCH. This model was verified for an extended time point of 8 weeks after surgery.

CYP1A gene expression as a basic factor for fipronil toxicity in Caspian kutum fish.

The aim of this study was to assess the effects of fipronil insecticide on the Caspian kutum fish at different levels of biological organizations and to find possible relationship between these biomarkers. Different doses of fipronil (65, 130 and 200 mg/kg) were intraperitoneally administered to the fish for 2 weeks. After 7 and 14 days of exposure, alterations in organ-somatic index, tissue and DNA structure, oxidative stress and CYP1A gene expression in gill, liver, brain and kidney were studied. Determination of these parameters in the liver showed that the degree of tissue change (DTC), comet tail, superoxide dismutase (SOD) and relative CYP1A mRNA expression increased mostly in a time dependent manner whereas in the kidney increased mostly in a dose dependent manner. These parameters in the gill increased more in time and dose dependent manner. Apart from the changes in CYP1A expression and oxidative stress, no alterations was observed in the brain. Multiple regression analysis showed that the CYP1A had the most correlation with the organ-somatic index (R2 = 0.76) and comet tail (R2 = 0.89) in the liver, and with DTC (R2 = 0.93) and oxidative stress (R2 = 0.87) in the kidney. Generally, this study showed that CYP1A gene expression can be considered as one basic factor for fipronil toxicity in this fish. However, other possible factors also should be considered for future research.

[Diagnosis of Helicobacter pylori infection on gastric biopsies: Standard stain, special stain or immunohistochemistry?] / Diagnostic d'infection à Helicobacter pylori sur biopsies gastriques : coloration standard, coloration spéciale ou immunohistochimie ?

INTRODUCTION: There is no consensus on the benefit of performing a systematic complementary technique for the diagnosis of Helicobacter pylori infection. In our laboratory, a cresyl violet was carried out systematically until July 2014; since that date, a cresyl violet or immunohistochemistry is only made on request. We evaluated the value of cresyl violet staining of gastric biopsies to diagnose H. pylori infection by comparing a period of systematic staining to a time when it was made on demand. MATERIAL AND METHODS: We retrospectively studied the gastric biopsy of 786 consecutive patients from April to November 2014, taken in the absence of focal endoscopic lesion. During the first period, hematoxylin-eosin and cresyl violet were performed on all biopsies. During the second period, hematoxylin-eosin was performed and then, if necessary, cresyl violet or immunohistochemistry. All hematoxylin-eosin stained slides were revised to identify H. pylori. We performed immunohistochemistry in cases of active chronic gastritis without H. pylori identified on hematoxylin-eosin or cresyl violet. RESULTS: We have shown that gastric biopsy performed in the absence of focal mucosal lesion are normal in 55% of cases. The percentage of H. pylori infection was similar in both groups. In cases of active chronic gastritis, H. pylori infection is visible, in most cases, on hematoxylin-eosin (94%). Immunohistochemistry should be prescribed only in case of chronic active gastritis without H. pylori identified on standard staining, with bacteria rare or atypically located. CONCLUSION: In our experiment, H. pylori is present only in case of active gastritis (33% of the biopsies in our series) and being almost always identifiable on the standard staining with H-E (in 94% of the cases), it is not It is not necessary to systematically perform, on all gastric biopsies, a complementary histo- or immunohistochemical technique.

Confocal scanning microscopy provides rapid, detailed intraoperative histological assessment of brain neoplasms: Experience with 106 cases.

OBJECTIVES: Frozen section histological analysis is currently the mainstay for intraprocedural tissue diagnosis during the resection of intracranial neoplasms and for evaluating tumor margins. However, frozen sections are time-consuming and often do not reveal the histological features needed for final diagnosis when compared with permanent sections. Confocal scanning microscopy (CSM) with certain stains may be a valuable technology that can add rapid and detailed histological assessment advantage for the neurosurgical operating room. This study describes potential advantages of CSM imaging of fresh human brain tumor tissues labeled with acriflavine (AF), acridine orange (AO), cresyl violet (CV), methylene blue (MB), and indocyanine green (ICG) within the neurosurgical operating room facility. PATIENTS AND METHODS: Acute slices from orthotopic human intracranial neoplasms were incubated with AF/AO and CV solutions for 10 s and 1 min respectively. Staining was also attempted with MB and ICG. Samples were imaged using a bench-top CSM system. Histopathologic features of corresponding CSM and permanent hematoxylin and eosin images were reviewed for each case. RESULTS: Of 106 cases, 30 were meningiomas, 19 gliomas, 13 pituitary adenomas, 9 metastases, 6 schwannomas, 4 ependymomas, and 25 other pathologies. CSM using rapid fluorophores (AF, AO, CV) revealed striking microvascular, cellular and subcellular structures that correlated with conventional histology. By rapidly staining and optically sectioning freshly resected tissue, images were generated for intraoperative consultations in less than one minute. With this technique, an entire resected tissue sample was imaged and digitally stored for tele-pathology and archiving. CONCLUSION: CSM of fresh human brain tumor tissue provides clinically meaningful and rapid histopathological assessment much faster than frozen section. With appropriate stains, including specific cellular structure or antibody staining, CSM could improve the timeliness of intraoperative decision-making, and the neurosurgical-pathology workflow during resection of human brain tumors, ultimately improving patient care.

Laser Capture Microdissection and Isolation of High-Quality RNA from Frozen Endometrial Tissue.

Laser capture microdissection (LCM) allows expression profiling of specific cell populations within tissues. However, isolation of high-quality RNA from laser capture microdissected frozen tissue is beset by problems arising from intrinsic tissue RNase activity. Herein, we describe an optimized staining/LCM/RNA extraction protocol developed for the isolation of epithelial RNA from frozen tissue sections using human endometrial cancer as a model tissue. This method combines excellent, reproducible visualization of tissue morphology with the isolation of high-integrity RNA suitable for downstream applications such as expression microarray analysis. We present quantitative and qualitative RNA data obtained from >200 endometrial epithelial samples (normal, hyperplastic, and cancerous), where 92% of samples had RIN values of 7 and above and highlight common pitfalls faced by investigators. This method should also be broadly applicable to a range of other tissue types.

Exposure to Enriched Environment Restores Altered Passive Avoidance Learning and Ameliorates Hippocampal Injury in Male Albino Wistar Rats Subjected to Chronic Restraint Stress.

AIMS: The aim of the study was to investigate the effects of exposure to enriched environment (EE) on passive avoidance learning and hippocampal cellular morphology in rats exposed to chronic restraint stress. MATERIALS AND METHODS: Adult male albino Wistar rats were assigned into the following groups: normal control (NC) remained undisturbed in their home cages; stressed group (S) subjected to restrained stress (6 h/day) followed by housing in standard housing for 21 days; And stressed + EE (S + EE) subjected to restrained stress followed by housing in EE for 21 days. On 22nd day, six animals from each of the three groups were exposed to passive avoidance test. The remaining animals were sacrificed. Hippocampus was isolated and processed for cellular morphology using cresyl violet staining. STATISTICAL ANALYSIS USED: Data were analyzed using one-way analysis of variance followed by Tukey's multiple comparison test (post hoc). RESULTS: Stressed rats exposed to EE showed significant improvement in passive avoidance learning test compared to NC. Quantification of the surviving neurons in the hippocampal subfields and their cellular morphology revealed significant neuroprotection in S + EE in cornu ammonis-2 (CA2) neurons and CA3 hippocampal neurons. No significant changes were found in CA1 hippocampal subfield. CONCLUSIONS: The outcome of this study makes us to think the possibilities of adopting EE as an alternative strategy in brain diseases where there is chronic stress and to minimize the impairment in learning and memory.

LCM-Seq: A Method for Spatial Transcriptomic Profiling Using Laser Capture Microdissection Coupled with PolyA-Based RNA Sequencing.

LCM-seq couples laser capture microdissection of cells from frozen tissues with polyA-based RNA sequencing and is applicable to single neurons. The method utilizes off-the-shelf reagents and direct lysis of the cells without RNA purification, making it a simple and relatively cheap method with high reproducibility and sensitivity compared to previous methods. The advantage with LCM-seq is also that tissue sections are kept intact and thus the positional information of each cell is preserved.

Comparison of unbiased stereological estimation of total number of cresyl violet stained neurons and parvalbumin positive neurons in the adult human spiral ganglion.

Estimation of total number of neurons in the spiral ganglion (SG) at various ages and their functional status is important as these neurons are constantly exposed to noise and other environmental factors that may lead to neuronal loss with aging due to excitotoxic damage. Parvalbumin (PV) is a calcium-binding protein (CBP), found in highly metabolically active neurons. It helps in buffering cytosolic calcium, which is essential for neurotransmitter release. The neurons in the adult human SG express PV more strongly than other CBPs like calbindin and calretinin. These CBPs can be used as signatures to recognise neurons. In the present study, we quantified the number of neurons expressing PV by unbiased stereology and compared it to the number of neurons stained by cresyl violet (CV), which is a Nissl stain, in the adult human SG. Five adult human cadaveric temporal bones were obtained from the forensic science mortuary, after due clearance from the institute ethics committee. Independent CV stained and PV immunostained sections were used to estimate the total number of neurons (optical fractionator), with StereoInvestigator (SI) software. The estimated total number of SG neurons was 27,485±3251 and 26,705±1823 in the PV and CV stained sections, respectively. There was no significant difference between the estimates (p=0.552). Therefore, CV staining is simpler and more cost effective when estimating neuronal number. Although PV stains spiral ganglion neurons (SGNs) with a greater intensity and provides a functional status, its tedious protocol limits its use for quantification.