Cells-in-Touch: 3D Printing in Reconstruction and Modelling of Microscopic Biological Geometries for Education and Future Research Applications.

Additive manufacturing (3D printing) and computer-aided design (CAD) still have limited uptake in biomedical and bioengineering research and education, despite the significant potential of these technologies. The utility of organ-scale 3D-printed models of living structures is widely appreciated, while the workflows for microscopy data translation into tactile accessible replicas are not well developed yet. Here, we demonstrate an accessible and reproducible CAD-based methodology for generating 3D-printed scalable models of human cells cultured in vitro and imaged using conventional scanning confocal microscopy with fused deposition modeling (FDM) 3D printing. We termed this technology CiTo-3DP (Cells-in-Touch for 3D Printing). As a proof-of-concept, we created dismountable CiTo-3DP models of human epithelial, mesenchymal, and neural cells by using selectively stained nuclei and cytoskeletal components. We also provide educational and research context for the presented cellular models. In the future, the CiTo-3DP approach can be adapted to different imaging and 3D printing modalities and comprehensively present various cell types, subcellular structures, and extracellular matrices. The resulting CAD and 3D printed models could be used for a broad spectrum of education and research applications.

Treatment and outcome following substantial ketamine overdose in a dog.

A 9-year-old, 3.7 kg (8.14 lb) neutered male Yorkshire terrier mix was treated following a ketamine overdose after subcutaneous ureteral bypass surgery. Due to an error in communication and misinterpretation of an electronic treatment sheet, the dog was inadvertently placed on a continuous rate infusion (CRI) of ketamine at 67.6 mg/kg per hour, rather than the intended 0.2 mg/kg per hour rate. Four hours after initiation of the ketamine CRI, the dog developed signs indicative of a ketamine overdose including tachycardia, hyperthermia, anisocoria, and hypoglycemia. It was determined the dog had received an iatrogenic overdose of ketamine; the infusion had been running at 67.6 mg/kg per hour, resulting in 270 mg/kg of ketamine over 4 h. Aggressive supportive measures were undertaken, and the dog gradually recovered over an 18-hour period, without lasting consequences of the overdose. To the authors' knowledge, there are no current published reports of a ketamine overdose of this magnitude in a dog. This case report documents an iatrogenic 338 times intravenous ketamine overdose in a dog, which was successfully managed with supportive care. In addition, it highlights the importance of doctor-technician communication and the potential errors in using electronic treatment sheets.

Traitement et résultat à la suite d'une surdose importante de kétamine chez un chien. Un Yorkshire terrier mélangé mâle de 9 ans et pesant 3,7 kg (8,14 lb) a été traité à la suite d'une surdose de kétamine après un pontage urétéral sous-cutané. En raison d'une erreur de communication et d'une mauvaise interprétation d'une feuille de traitement électronique, le chien a été placé par inadvertance sous une perfusion à débit continu (IRC) de kétamine à 67,6 mg/kg par heure, au lieu du débit prévu de 0,2 mg/kg par heure. Quatre heures après le début de l'IRC de kétamine, le chien a développé des signes indiquant une surdose de kétamine, notamment de la tachycardie, de l'hyperthermie, de l'anisocorie et de l'hypoglycémie. Il a été déterminé que le chien avait reçu une surdose iatrogène de kétamine; la perfusion fonctionnait à 67,6 mg/kg par heure, entraînant 270 mg/kg de kétamine en 4 h. Des mesures de soutien agressives ont été mises en place et le chien s'est progressivement rétabli sur une période de 18 heures, sans conséquences durables du surdosage.À la connaissance des auteurs, il n'existe actuellement aucun rapport publié sur une surdose de kétamine de cette ampleur chez un chien. Ce rapport de cas documente une surdose iatrogène de kétamine de 338 fois par voie intraveineuse chez un chien, qui a été gérée avec succès avec des soins de soutien. De plus, il met en évidence l'importance de la communication médecin-technicien et les erreurs potentielles dans l'utilisation des fiches de traitement électroniques.(Traduit par Dr Serge Messier).

Pelvic limb anesthesia and analgesia in dogs undergoing tibial plateau leveling osteotomy (TPLO): A survey of board-certified anesthesiologists.

OBJECTIVE: To report the locoregional anesthesia and analgesia preferences of veterinary anesthesiologists for use in dogs undergoing a TPLO and determine any association with specialty college, time from board-certification, or employment sector. STUDY DESIGN: Cross sectional study. SAMPLE POPULATION: Diplomates of the American (ACVAA) and European (ECVAA) Colleges of Veterinary Anesthesia and Analgesia. METHODS: An electronic survey was distributed to diplomates and responses were used to determine associations between preferred methods. RESULTS: The survey response rate was 28% (141/500) with 69% (97/141) of ACVAA diplomates and 31% of diplomates with ECVAA (44/141) certification. Peripheral nerve block (PNB) was preferred by 79% (111/141) of all diplomates, lumbosacral epidural (LE) by 21% (29/141), and peri-incisional infiltration (PI) by <1% (1/141). There was no association (p = .283) with specialty college. There was an association (p < .001) with time from board-certification with increased preference for LE when >10-years from certification and PI preferred by only those board-certified >20-years ago. There was an association with employment sector (p = .003) with more academic diplomates preferring LE. Anesthesiologists reported that treatment decisions were affected by various factors including time pressure and surgeon influence. CONCLUSION: Diplomates of ACVAA and ECVAA prefer PNB as the locoregional method of pelvic limb anesthesia in dogs undergoing TPLO. A greater percentage of newer and private practice diplomates prefer PNB while a larger percentage of senior and academic diplomates prefer LE. Decision making is multifactorial and includes perceived time pressure and surgeon influence. CLINICAL SIGNIFICANCE: Veterinary anesthesiologists prefer and frequently use PNB in dogs undergoing TPLO and surgeon influence may affect their chosen treatment.

Transient Postoperative Hemorrhage from Elbow Arthroscopy Portals following Intra-Articular Pentosan Polysulfate Sodium Injection.

Four adult, client owned dogs with diagnosed bilateral elbow dysplasia undergoing elbow arthroscopy for removal of fragmented medial coronoid process were identified via a retrospective database search, who also received intra-articular administration of pentosan polysulfate sodium (PPS) (Cartrophen Vet, Biopharm Australia Pty Ltd., Bondi Junction, New South Wales). Dogs had postoperative administration of 5 ml PPS injected into each elbow joint following elbow arthroscopy. Within 1-3 hours of administration, each dog experienced hemorrhage from arthroscopy incisions that was determined to be independent of surgical trauma given lack of hemorrhage intraoperatively. Pressure bandages were placed, and the hemorrhage and elevated coagulation parameters resolved 12-18 hours following intra-articular injection. No further intervention was required, and the dogs were discharged 20-26 hours postoperatively. The purpose of this case series is to describe 4 dogs who experienced transient and focal hemorrhage following off-label intra-articular administration of pentosan polysulfate sodium (PPS). While this case series is limited due to small number of cases, results following bilateral, intra-articular injection of PPS support a transient systemic coagulopathy. Though this report represents administration of PSS via a route and at doses beyond that recommended on the label, results suggest that administration of PSS in the manner described in this report should be avoided.

Targeting cell surface glycans with lectin-coated fluorescent nanodiamonds.

Glycosylation is arguably the most important functional post-translational modification in brain cells and abnormal cell surface glycan expression has been associated with neurological diseases and brain cancers. In this study we developed a novel method for uptake of fluorescent nanodiamonds (FND), carbon-based nanoparticles with low toxicity and easily modifiable surfaces, into brain cell subtypes by targeting their glycan receptors with carbohydrate-binding lectins. Lectins facilitated uptake of 120 nm FND with nitrogen-vacancy centers in three types of brain cells - U87-MG astrocytes, PC12 neurons and BV-2 microglia cells. The nanodiamond/lectin complexes used in this study target glycans that have been described to be altered in brain diseases including sialic acid glycans via wheat (Triticum aestivum) germ agglutinin (WGA), high mannose glycans via tomato (Lycopersicon esculentum) lectin (TL) and core fucosylated glycans via Aleuria aurantia lectin (AAL). The lectin conjugated nanodiamonds were taken up differently by the various brain cell types with fucose binding AAL/FNDs taken up preferentially by glioblastoma phenotype astrocyte cells (U87-MG), sialic acid binding WGA/FNDs by neuronal phenotype cells (PC12) and high mannose binding TL/FNDs by microglial cells (BV-2). With increasing recognition of glycans having a role in many diseases, the lectin bioconjugated nanodiamonds developed here are well suited for further investigation into theranostic applications.

Quantification of Canine Apocrine Gland Anal Sac Adenocarcinoma (AGASACA) Tumor Specimen Shrinkage after Formalin Fixation.

The aim was to prospectively measure the shrinkage of primary apocrine gland anal sac adenocarcinoma (AGASACA) tumors after 24 and 48 h of formalin fixation. Dogs that were diagnosed with AGASACA pre-operatively by aspiration cytology were prospectively enrolled in the study. Tumor extirpation was performed in a closed technique. The tumor and associated tissues were examined on the back table away from the patient and the widest dimension of the tumor was measured using a sterile ruler (Medline®; Northfield, IL, USA). This measurement was recorded in mm (t0). The tissue was placed in 10% buffered formalin and stored at room temperature. Two further measurements were taken after 24 (t24) and 48 (t48) hours of formalin fixation. Once the 48 h measurement was taken, the tissue was submitted for histopathology. The percentage of shrinkage between time points was calculated by using the following equation: (1 - [time b/time a]) × 100. Overall, 23 dogs with 23 tumors were enrolled. The mean percentage of shrinkage after 24 and 48 h of formalin fixation was 4.8% and 7.2%, respectively. The median diameter of the tumors reduced by 1 mm over 48 h and was not significantly different at any time point. These data will aid clinicians in interpreting measurements of AGASACA tumors following formalin fixation and shows that minimal change in tumor size is expected following 48 h.

Simultaneous super-linear excitation-emission and emission depletion allows imaging of upconversion nanoparticles with higher sub-diffraction resolution.

Upconversion nanoparticles (UCNPs) are becoming increasingly popular as biological markers as they offer photo-stable imaging in the near-infrared (NIR) biological transparency window. Imaging at NIR wavelengths benefits from low auto-fluorescence background and minimal photo-damage. However, as the diffraction limit increases with the wavelength, the imaging resolution deteriorates. To address this limitation, recently two independent approaches have been proposed for imaging UCNPs with sub-diffraction resolution, namely stimulated emission-depletion (STED) microscopy and super linear excitation-emission (uSEE) microscopy. Both methods are very sensitive to the UCNP composition and the imaging conditions, i.e. to the excitation and depletion power. Here, we demonstrate that the imaging conditions can be chosen in a way that activates both super-resolution regimes simultaneously when imaging NaYF4:Yb,Tm UCNPs. The combined uSEE-STED mode benefits from the advantages of both techniques, allowing for imaging with lateral resolution about six times better than the diffraction limit due to STED and simultaneous improvement of the axial resolution about twice over the diffraction limit due to uSEE. Conveniently, at certain imaging conditions, the uSEE-STED modality can achieve better resolution at four times lower laser power compared to STED mode, making the method appealing for biological applications. We illustrate this by imaging UCNPs functionalized by colominic acid in fixed neuronal phenotype cells.

Adherence to follow-up recommendations for dogs with apocrine gland anal sac adenocarcinoma: A multicentre retrospective study.

Progressive disease is common following anal sacculectomy for apocrine gland anal sac adenocarcinoma (AGASACA); additional therapy may prolong survival. Adherence to medical recommendations influences therapeutic success in humans. The purpose of this study was to assess the adherence to follow-up recommendations in dogs with AGASACA. Medical records of patients that underwent anal sacculectomy for AGASACA, with or without iliosacral lymphadenectomy, between July 2015 and July 2018, were reviewed at eight referral institutions to assess post-operative recommendations and owner adherence to recommendations. One hundred and seventy-four dogs were included, of which 162 underwent unilateral anal sacculectomy, 12 underwent bilateral anal sacculectomy and 39 underwent concurrent iliosacral lymphadenectomy. Seventy-six owners (44%) received recommendations for staging at the time of discharge, histopathology results or at the first follow-up visit. One hundred and forty owners (80%) received recommendations for treatment following the initial surgery. Fifty of seventy-six (66%) owners pursued at least one staging recommendation and 69 of 140 (49%) owners pursued some kind of adjuvant treatment recommendation. Overall, 16 of 76 (21%) were adherent to staging recommendations with 20 adherent for the first year following surgery (26%). Forty-seven of 140 (34%) were adherent to treatment recommendations with 54 (39%) adherent for the first year. Owners that were adherent to restaging recommendations at 1 year following surgery were significantly more likely to pursue treatment for progressive disease (P = .014). Further work is required to assess owner motivation and evaluate strategies to improve adherence, given the potential impact on patient treatment.

Lipopolysaccharide and Morphine-3-Glucuronide-Induced Immune Signalling Increases the Expression of Polysialic Acid in PC12 Cells.

Polysialic acid (polySia), a long homopolymer of 2,8-linked sialic acids, is abundant in the embryonic brain and is restricted largely in adult brain to regions that exhibit neurogenesis and structural plasticity. In the central nervous system (CNS), polySia is highly important for cell-cell interactions, differentiation, migration and cytokine responses, which are critical neuronal functions regulating intercellular interactions that underlie immune signalling in the CNS. In recent reports, a metabolite of morphine, morphine-3-glucuronide (M3G), has been shown to cause immune signalling in the CNS. In this study, we compared the effects of neurite growth factor (NGF), lipopolysaccharide (LPS) and M3G exposure on the expression of polySia in PC12 cells using immunocytochemistry and Western blot analysis. PolySia was also extracted from stimulated cell proteins by endo-neuraminidase digestion and quantitated using fluorescent labelling followed by HPLC analysis. PolySia expression was significantly increased following NGF, M3G or LPS stimulation when compared with unstimulated cells or cells exposed to the TLR4 antagonist LPS-RS. Additionally, we analyzed the effects of test agent exposure on cell migration and the oxidative stress response of these cells in the presence and absence of polySia expression on their cell surface. We observed an increase in oxidative stress in cells without polySia as well as following M3G or LPS stimulation. Our study provides evidence that polySia expression in neuronal-like PC12 cells is influenced by M3G and LPS exposure alike, suggestive of a role of TLR4 in triggering these events.

A Nanoparticle-Based Affinity Sensor that Identifies and Selects Highly Cytokine-Secreting Cells.

We developed a universal method termed OnCELISA to detect cytokine secretion from individual cells by applying a capture technology on the cell membrane. OnCELISA uses fluorescent magnetic nanoparticles as assay reporters that enable detection on a single-cell level in microscopy and flow cytometry and fluorimetry in cell ensembles. This system is flexible and can be modified to detect different cytokines from a broad range of cytokine-secreting cells. Using OnCELISA we have been able to select and sort highly cytokine-secreting cells and identify cytokine-secreting expression profiles of different cell populations in vitro and ex vivo. We show that this system can be used for ultrasensitive monitoring of cytokines in the complex biological environment of atherosclerosis that contains multiple cell types. The ability to identify and select cell populations based on their cytokine expression characteristics is valuable in a host of applications that require the monitoring of disease progression.

Complication of an artificial urethral sphincter in the treatment of a urethral sphincter mechanism incompetence.

A 5-year-old, 39-kg spayed female mixed breed dog was treated for urinary sphincter mechanism incompetence with an artificial urethral sphincter (AUS). The dog was continent for 2 months, but then dramatic incontinence abruptly recurred. Imaging indicated that there was a leak in the cuff of the AUS. The AUS was replaced and continence was re-established.

Complication d'un sphincter urétral artificiel dans le traitement d'incontinence due au mécanisme du sphincter urétral. Une chienne de race croisée âgée de 5 ans et pesant 39 kg a été traitée pour de l'incontinence due au mécanisme du sphincter urinaire avec un sphincter urétral artificiel (SUA). La chienne était continente pour 2 mois, mais de l'incontinence dramatique est réapparue soudainement. Un examen par imagerie a montré qu'il y avait une fuite dans le manchon du SAU. Le SAU fut replacé et la continence ré-établie.(Traduit par Dr Serge Messier).

3D sub-diffraction imaging in a conventional confocal configuration by exploiting super-linear emitters.

Sub-diffraction microscopy enables bio-imaging with unprecedented clarity. However, most super-resolution methods require complex, costly purpose-built systems, involve image post-processing and struggle with sub-diffraction imaging in 3D. Here, we realize a conceptually different super-resolution approach which circumvents these limitations and enables 3D sub-diffraction imaging on conventional confocal microscopes. We refer to it as super-linear excitation-emission (SEE) microscopy, as it relies on markers with super-linear dependence of the emission on the excitation power. Super-linear markers proposed here are upconversion nanoparticles of NaYF4, doped with 20% Yb and unconventionally high 8% Tm, which are conveniently excited in the near-infrared biological window. We develop a computational framework calculating the 3D resolution for any viable scanning beam shape and excitation-emission probe profile. Imaging of colominic acid-coated upconversion nanoparticles endocytosed by neuronal cells, at resolutions twice better than the diffraction limit both in lateral and axial directions, illustrates the applicability of SEE microscopy for sub-cellular biology.

The effect of particle size on nanodiamond fluorescence and colloidal properties in biological media.

Fluorescent nanodiamonds (FNDs) are extremely photostable markers and nanoscale sensors, which are increasingly used in biomedical applications. Nanoparticle size is a critical parameter in the majority of these applications. Yet, the effect of particle size on FND's fluorescence and colloidal properties is not well understood today. Here, we investigate the fluorescence and colloidal stability of commercially available high-pressure high-temperature FNDs containing nitrogen-vacancy (NV) centers in biological media. Unconjugated FNDs in sizes ranging between 10 nm and 140 nm with an oxidized surface are studied using dynamic light scattering and fluorescence spectroscopy. We determine their colloidal stability in water, fetal bovine serum, Dulbecco's Modified Eagle Medium and complete media. The FNDs' relative fluorescence brightness, the NV charge-state, and the FND fluorescence against media autofluorescence are analyzed as a function of FND size. Our results will enable researchers in biology and beyond to identify the most promising FND particle size for their application.

Visualizing neuroinflammation with fluorescence and luminescent lanthanide-based in situ hybridization.

BACKGROUND: Neurokine signaling via the release of neurally active cytokines arises from glial reactivity and is mechanistically implicated in central nervous system (CNS) pathologies such as chronic pain, trauma, neurodegenerative diseases, and complex psychiatric illnesses. Despite significant advancements in the methodologies used to conjugate, incorporate, and visualize fluorescent molecules, imaging of rare yet high potency events within the CNS is restricted by the low signal to noise ratio experienced within the CNS. The brain and spinal cord have high cellular autofluorescence, making the imaging of critical neurokine signaling and permissive transcriptional cellular events unreliable and difficult in many cases. METHODS: In this manuscript, we developed a method for background-free imaging of the transcriptional events that precede neurokine signaling using targeted mRNA transcripts labeled with luminescent lanthanide chelates and imaged via time-gated microscopy. To provide examples of the usefulness this method can offer to the field, the mRNA expression of toll-like receptor 4 (TLR4) was visualized with traditional fluorescent in situ hybridization (FISH) or luminescent lanthanide chelate-based in situ hybridization (LISH) in mouse BV2 microglia or J774 macrophage phenotype cells following lipopolysaccharide stimulation. TLR4 mRNA staining using LISH- and FISH-based methods was also visualized in fixed spinal cord tissues from BALB/c mice with a chronic constriction model of neuropathic pain or a surgical sham model in order to demonstrate the application of this new methodology in CNS tissue samples. RESULTS: Significant increases in TLR4 mRNA expression and autofluorescence were visualized over time in mouse BV2 microglia or mouse J774 macrophage phenotype cells following lipopolysaccharide (LPS) stimulation. When imaged in a background-free environment with LISH-based detection and time-gated microscopy, increased TLR4 mRNA was observed in BV2 microglia cells 4 h following LPS stimulation, which returned to near baseline levels by 24 h. Background-free imaging of mouse spinal cord tissues with LISH-based detection and time-gated microscopy demonstrated a high degree of regional TLR4 mRNA expression in BALB/c mice with a chronic constriction model of neuropathic pain compared to the surgical sham model. CONCLUSIONS: Advantages offered by adopting this novel methodology for visualizing neurokine signaling with time-gated microscopy compared to traditional fluorescent microscopy are provided.

Understanding cellular glycan surfaces in the central nervous system.

Glycosylation, the enzymatic process by which glycans are attached to proteins and lipids, is the most abundant and functionally important type of post-translational modification associated with brain development, neurodegenerative disorders, psychopathologies and brain cancers. Glycan structures are diverse and complex; however, they have been detected and targeted in the central nervous system (CNS) by various immunohistochemical detection methods using glycan-binding proteins such as anti-glycan antibodies or lectins and/or characterized with analytical techniques such as chromatography and mass spectrometry. The glycan structures on glycoproteins and glycolipids expressed in neural stem cells play key roles in neural development, biological processes and CNS maintenance, such as cell adhesion, signal transduction, molecular trafficking and differentiation. This brief review will highlight some of the important findings on differential glycan expression across stages of CNS cell differentiation and in pathological disorders and diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, amyotrophic lateral sclerosis, schizophrenia and brain cancer.

The effect of streptozotocin-induced hyperglycemia on N-and O-linked protein glycosylation in mouse ovary.

Post-translational modification of proteins namely glycosylation influences cellular behavior, structural properties and interactions including during ovarian follicle development and atresia. However, little is known about protein glycosylation changes occurring in diabetes mellitus in ovarian tissues despite the well-known influence of diabetes on the outcome of successful embryo implantation. In our study, the use of PGC chromatography-ESI mass spectrometry in negative ion mode enabled the identification of 138 N-glycans and 6 O-glycans on the proteins of Streptozotocin-induced (STZ) diabetic mouse ovarian tissues (n = 3). Diabetic mouse ovaries exhibited a relative decrease in sialylation, fucosylation and, to a lesser extent, branched N-linked glycan structures, as well as an increase in oligomannose structures on their proteins, compared with nondiabetic mouse ovaries. Changes in N-glycans occurred in the diabetic liver tissue but were more evident in diabetic ovarian tissue of the same mouse, suggesting an organ-specific effect of diabetes mellitus on protein glycosylation. Although at a very low amount, O-GalNAc glycans of mice ovaries were present as core type 1 and core type 2 glycans; with a relative increase in the NeuGc:NeuAc ratio as the most significant difference between control and diabetic ovarian tissues. STZ-treated mice also showed a trend towards an increase in TNF-α and IL1-B inflammatory cytokines, which have previously been shown to influence protein glycosylation.

Microwave pretreatment of paramylon enhances the enzymatic production of soluble ß-1,3-glucans with immunostimulatory activity.

A hydrothermal microwave pretreatment was established to facilitate the enzymatic production of soluble bioactive ß-1,3-glucans from the recalcitrant substrate paramylon. The efficacy of this pretreatment was monitored with a newly developed direct Congo Red dye-based assay over a range of temperatures. Microwave pretreatment at 170 °C for 2 min resulted in a significantly enhanced enzymatic hydrolysis of paramylon. The action of endo-ß-1,3- and exo- ß-1,3-glucanases on the microwave-pretreated paramylon produced soluble ß-1,3-glucans with degrees of polymerisation (DP) ranging from 2-59 and 2-7, respectively. In comparison, acid-mediated hydrolysis of untreated paramylon resulted in ß-1,3-glucans with a DP range of 2-38. The hydrolysates were assayed on their immunostimulatory effect on murine macrophages by measuring the production of the inflammation-linked marker tumour necrosis factor alpha (TNFα) using immunofluorescence. All of the tested hydrolysis products were shown to induce TNFα production, with the most significant immunostimulatory effect observed with the hydrolysate from the exo-ß-1,3-glucanase treatment.

Reduced background autofluorescence for cell imaging using nanodiamonds and lanthanide chelates.

Bio-imaging is a key technique in tracking and monitoring important biological processes and fundamental biomolecular interactions, however the interference of background autofluorescence with targeted fluorophores is problematic for many bio-imaging applications. This study reports on two novel methods for reducing interference with cellular autofluorescence for bio-imaging. The first method uses fluorescent nanodiamonds (FNDs), containing nitrogen vacancy centers. FNDs emit at near-infrared wavelengths typically higher than most cellular autofluorescence; and when appropriately functionalized, can be used for background-free imaging of targeted biomolecules. The second method uses europium-chelating tags with long fluorescence lifetimes. These europium-chelating tags enhance background-free imaging due to the short fluorescent lifetimes of cellular autofluorescence. In this study, we used both methods to target E-selectin, a transmembrane glycoprotein that is activated by inflammation, to demonstrate background-free fluorescent staining in fixed endothelial cells. Our findings indicate that both FND and Europium based staining can improve fluorescent bio-imaging capabilities by reducing competition with cellular autofluorescence. 30 nm nanodiamonds coated with the E-selectin antibody was found to enable the most sensitive detective of E-selectin in inflamed cells, with a 40-fold increase in intensity detected.

Behavioral sensitization to methamphetamine induces specific interneuronal mRNA pathology across the prelimbic and orbitofrontal cortices.

Schizophrenia is associated with significant pathophysiological changes to interneurons within the prefrontal cortex (PFC), with mRNA and protein changes associated with the GABA network localized to specific interneuron subtypes. Methamphetamine is a commonly abused psychostimulant that can induce chronic psychosis and symptoms that are similar to schizophrenia, suggesting that chronic METH induced psychosis may be associated with similar brain pathology to schizophrenia in the PFC. The aim of this study, therefore, was to examine mRNA expression of interneuron markers across two regions of the PFC (prelimbic (PRL) and orbitofrontal cortices (OFC)) following METH sensitization, an animal model of METH psychosis. We also studied the association between GABA mRNA expression and interneuronal mRNA expression to identify whether particular changes to the GABA network could be localized to a specific inhibitory cellular phenotype. METH sensitization increased the transcriptional expression of calbindin, calretinin, somatostatin, cholecyctokinin and vasoactive intestinal peptide in the PRL while parvalbumin, calbindin, cholectokinin and vasoactive intestinal peptide were upregulated in the OFC. Based on our previous findings, we also found significant correlations between GAD67, GAT1 and parvalbumin while GAD67, GAD65 and GAT1 were positively correlated with cholecystokinin in the PRL of METH sensitized rats. Within the OFC, the expression of GABAAα1 was positively correlated with somatostatin while GABAAα5 was negatively associated with somatostatin and calbindin. These findings suggest that METH sensitization differentially changes the expression of mRNAs encoding for multiple peptides and calcium binding proteins across the PRL and the OFC. Furthermore, these findings support that changes to the GABA network may also occur within specific cell types. These results, therefore, provide the first evidence that METH sensitization mediates differential interneuronal pathology across the PRL and OFC and such changes could have profound consequences on behavior and cognitive output.

Neurochemistry of neurons in the ventrolateral medulla activated by hypotension: Are the same neurons activated by glucoprivation?

Previous studies have demonstrated that a range of stimuli activate neurons, including catecholaminergic neurons, in the ventrolateral medulla. Not all catecholaminergic neurons are activated and other neurochemical content is largely unknown hence whether stimulus specific populations exist is unclear. Here we determine the neurochemistry (using in situ hybridization) of catecholaminergic and noncatecholaminergic neurons which express c-Fos immunoreactivity throughout the rostrocaudal extent of the ventrolateral medulla, in Sprague Dawley rats treated with hydralazine or saline. Distinct neuronal populations containing PPCART, PPPACAP, and PPNPY mRNAs, which were largely catecholaminergic, were activated by hydralazine but not saline. Both catecholaminergic and noncatecholaminergic neurons containing preprotachykinin and prepro-enkephalin (PPE) mRNAs were also activated, with the noncatecholaminergic population located in the rostral C1 region. Few GlyT2 neurons were activated. A subset of these data was then used to compare the neuronal populations activated by 2-deoxyglucose evoked glucoprivation (Brain Structure and Function (2015) 220:117). Hydralazine activated more neurons than 2-deoxyglucose but similar numbers of catecholaminergic neurons. Commonly activated populations expressing PPNPY and PPE mRNAs were defined. These likely include PPNPY expressing catecholaminergic neurons projecting to vasopressinergic and corticotrophin releasing factor neurons in the paraventricular nucleus, which when activated result in elevated plasma vasopressin and corticosterone. Stimulus specific neurons included noncatecholaminergic neurons and a few PPE positive catecholaminergic neuron but neurochemical codes were largely unidentified. Reasons for the lack of identification of stimulus specific neurons, readily detectable using electrophysiology in anaesthetized preparations and for which neural circuits can be defined, are discussed.