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The receptor tyrosine kinase (RTK) KIT is a major focus of current research into canine mast cell tumors (MCTs). Little is known about the role of other RTKs, such as vascular endothelial growth factor receptors (VEGFRs) and platelet-derived growth factor receptors (PDGFRs). These RTKs are dysregulated in many human and animal cancers and are key regulators of tumor angiogenesis. The aims of this study were to assess the expression and activation (phosphorylation) status of KIT, VEGFR2, and PDGFR (α and β) in canine MCTs and to examine associations with various clinical outcomes. c-KIT mutational status and KIT cellular localization pattern were also evaluated for these tumors. Twenty-seven MCTs, consisting of 5 subcutaneous and 22 cutaneous tumors, from 25 dogs were evaluated. MCT biopsies, cultured mast cells, and skin from the surgical margin were analyzed through Western blotting. MCT biopsies were also used for KIT immunohistochemical labeling and polymerase chain reaction for c-KIT mutational analysis. MCT had heterogeneous expression profiles for all 3 RTKs, which varied in intensity and activation status. Statistical analyses showed phosphorylated KIT, VEGFR2, and KIT cellular localization to be predictive of decreased survival time, disease-free interval, and increased metastatic rate. Expression of VEGFR2 and KIT diffuse cytoplasmic labeling were also significantly associated with increased rate of local recurrence. The results of the study show that phosphorylated KIT, KIT, VEGFR2, and PDGFRβ, in addition to KIT localization, may be valuable prognostic determinants in MCTs and should be further studied to improve diagnostic and therapeutic modalities. [ABSTRACT FROM AUTHOR]

The article evaluates the pathology and clinical outcome of a large number of canine subcutaneous mast cell tumors (MCTs) to characterize their behavior, histology and clinical prognosis. It describes the clinical outcome for 306 dogs obtained from veterinarians and correlated with histologic features. Analysis reportedly indicates mitotic index to be predictive of survival, local reoccurrence and metastasis, and that the majority of subcutaneous MCTs have a favorable prognosis with extended survival times and low rates of reoccurrence and metastasis.

The article evaluates the prognostic utility of KIT immunohistochemical labeling pattern, c-KIT mutational status, and proliferation markers including mitotic index and Ki67 as independent prognosis markers for local recurrence and/or metastasis in canine subcutaneous mast cell tumors (MCT). It demonstrates that KIT diffuse cytoplasmic localization is a statistically significant risk factor for aggressive subcutaneous MCT disease. It suggests that a prospective analysis or larger retrospective study would be needed for stricter inclusion criteria.

AIM: Clinical measurements such as temperature are commonly used for screening and diagnosis. However, little is known about the reliability of specific thermometers or measurement techniques. METHODS: This study sought to define normal tympanic temperature and to assess the reliability of tympanic thermometry. Using a cross-sectional survey design, 244 children aged between one and six years at a paediatric outpatients department had their temperature recorded three times in each ear using a Braun Thermoscan thermometer. RESULTS: Mean temperature was 36.65 degrees C with an overall repeatability statistic of 0.78 degrees C, 0.55 degrees C in the left ear and 0.64 degrees C in the right. The level of measurement error did not increase with temperature, or differ between ages. Environmental temperature had only a moderate effect on temperature, and there was no effect from environmental humidity. CONCLUSIONS: Tympanic thermometry using the Braun ThermoScan is a reliable method of temperature measurement, with a temperature change of approximately 0.6 degrees C being significant. Although the effects of environmental conditions were slight, these were in temperate conditions. This study should be repeated for other models of thermometer. [ABSTRACT FROM AUTHOR]

The decline in mammary epithelial cell number as lactation progresses may be due, in part, to oxidative stress. Selenium is an integral component of several antioxidant enzymes. The present study was conducted to examine the effect of oxidative stress and selenomethionine (SeMet) on morphology, viability, apoptosis, and proliferation of bovine mammary epithelial cells (BMEC) in primary culture. Cells were isolated from mammary glands of lactating dairy cows and grown for 3 d in a low-serum gel system containing lactogenic hormones and 0 or 100 μM H2O2 with 0, 10, 20, or 50 nM SeMet. Hydrogen peroxide stress increased intracellular H2O2 to 3 times control concentrations and induced a loss of cuboidal morphology, cell–cell contact, and viability of BMEC by 25%. Apoptotic cell number more than doubled during oxidative stress, but proliferating cell number was not affected. Supplementation with SeMet increased glutathione peroxidase activity 2-fold and restored intracellular H2O2 to control levels with a concomitant return of morphology and viability to normal. Apoptotic BMEC number was decreased 76% below control levels by SeMet and proliferating cell number was increased 4.2-fold. These findings suggest that SeMet modulated apoptosis and proliferation independently of a selenoprotein-mediated reduction of H2O2. In conclusion, SeMet supplementation protects BMEC from H2O2-induced apoptosis and increased proliferation and cell viability under conditions of oxidative stress. [ABSTRACT FROM AUTHOR]

This study examined the localization of cellular glutathione peroxidase (GPx1) and extracellular glutathione peroxidase (GPx3) in lactating mammary tissue and in primary cultures of bovine mammary epithelial cells (BMEC). The effect of selenium as selenomethionine (SeMet) on the growth and viability of BMEC and GPx protein expression and activity were also studied. Single mammary epithelial cells were recovered by serial collagenase/hyaluronidase digestion from lactating bovine mammary tissue and cultured in a low-serum collagen gel system enriched with lactogenic hormones and 0, 10, 20, or 50 nM SeMet. Positive immunostaining with anti-cytokeratin and bovine anti-casein confirmed the epithelial nature and differentiated state of BMEC. Addition of SeMet to media facilitated rapid confluence of BMEC and formation of dome structures. Immunohistochemical and immunocytochemical staining revealed that both GPx1 and GPx3 are synthesized by BMEC and localized in the cytoplasm and nucleus. Up to 50 nM SeMet linearly increased BMEC number and viability over 5 d of culture. Bovine mammary epithelial cells cultured in SeMet-supplemented medium also exhibited markedly elevated GPx activity and linear increases in abundance of GPx1 and GPx3 proteins. It is apparent that SeMet degradation to release Se for synthesis of selenoproteins is carried out by BMEC. Results indicate that bovine mammary epithelial cells express GPx1 and GPx3 in vivo and in vitro; SeMet enhances expression of these selenoproteins in vitro and the growth and viability of BMEC. [ABSTRACT FROM AUTHOR]

Rapamycin has been reported to reduce cancer cell survival in certain tumors following radiation therapy, but the mechanisms driving this phenomenon are unclear. Rapamycin inhibits mTOR signaling, a pathway responsible for several essential cell functions. The objective of this study was to investigate the effects of rapamycin and radiation on the activation and inhibition of mTOR signaling and the relationship between mTOR signaling and DNA damage response in vitro using canine mast cell tumor (MCT) cancer cell lines. Rapamycin rapidly inhibited S6K phosphorylation in a dose-dependent manner. Ionizing radiation (3, 6, or 10 Gy) was able to activate mTOR signalling, but the combination of radiation and rapamycin maintained mTOR inhibition. The comet assay revealed that co-treatment with rapamycin induced modest increases in the severity of DNA damage to MCT cells, but that these differences were not statistically significant. Although the relationship between mTOR and DNA damage response in MCT cancer cell lines remains unclear, our findings suggest the possibility of interaction, leading to enhancement of radiation response., (Copyright and/or publishing rights held by the Canadian Veterinary Medical Association.)

Objective: preHEAT was a randomised controlled feasibility trial to determine how best to measure skin necrosis in breast reconstruction to inform the design of a larger multicentre trial., Background: Mastectomy skin flap necrosis (MSFN) is a serious complication resulting in prolonged wound healing. Local heat preconditioning of the MSF before surgery has been shown to reduce skin necrosis in immediate breast reconstruction patients (IBR)., Method: preHEAT was a single-centre, randomised control two-arm single-blind parallel arm feasibility trial of local heat preconditioning in breast cancer patients undergoing SSM and NSM at Guy's and St Thomas' Hospital, London, UK. All patients undergoing IBR above the age of 18 were included. Intervention patients heated breast skin to 43 °C in three, 30-min cycles interrupted by spontaneous cooling using hot water bottles. The primary aim was to compare measurement of skin necrosis using binary 'yes/no' assessment, the SKIN score, and wound area., Results: One hundred forty-one patients were randomised over a 2-year period (71 heated group, 70 controls). There was near perfect agreement between assessors using the "yes/no" measurement of necrosis. The proportion of patients experiencing necrosis in controls was 35% ( n  = 23/66) in the heated 26% ( n  = 18/68]). In the control group, 17% ( n  = 4/23) patients experiencing necrosis required surgical intervention for necrosis compared to 11% ( n  = 2/18) in the heated group., Conclusion: The binary outcome of MSFN "yes/no" is a suitable and reliable primary outcome measure of necrosis and was superior to the SKIN Score or necrosis area. The trial study design is feasible for a larger definitive trial., Trial Registration: ISRCTN15744669. Date of registration: 25/02/2018., Competing Interests: Full ethical approval for this study was granted from Health Research Authority NRES Committee South Central – Hampshire B (ethics number 14/SC/1334). This is mentioned in the text. All patients willingly consented to be included in this trial.This is not applicable.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Background: Essential strategies are needed to help reduce the number of post-operative complications and associated costs for breast cancer patients undergoing reconstructive breast surgery. Evidence suggests that local heat preconditioning could help improve the provision of this procedure by reducing skin necrosis. Before testing the effectiveness of heat preconditioning in a definitive randomised controlled trial (RCT), we must first establish the best way to measure skin necrosis and estimate the event rate using this definition., Methods: PREHEAT is a single-blind randomised controlled feasibility trial comparing local heat preconditioning, using a hot water bottle, against standard care on skin necrosis among breast cancer patients undergoing reconstructive breast surgery. The primary objective of this study is to determine the best way to measure skin necrosis and to estimate the event rate using this definition in each trial arm. Secondary feasibility objectives include estimating recruitment and 30 day follow-up retention rates, levels of compliance with the heating protocol, length of stay in hospital and the rates of surgical versus conservative management of skin necrosis. The information from these objectives will inform the design of a larger definitive effectiveness and cost-effectiveness RCT., Discussion: This article describes the PREHEAT trial protocol and detailed statistical analysis plan, which includes the pre-specified criteria and process for establishing the best way to measure necrosis. This study will provide the evidence needed to establish the best way to measure skin necrosis, to use as the primary outcome in a future RCT to definitively test the effectiveness of local heat preconditioning. The pre-specified statistical analysis plan, developed prior to unblinded data extraction, sets out the analysis strategy and a comparative framework to support a committee evaluation of skin necrosis measurements. It will increase the transparency of the data analysis for the PREHEAT trial., Trial Registration: ISRCTN ISRCTN15744669. Registered 25 February 2015., Competing Interests: Ethical approval was granted from the NRES Committee South Central–Hampshire B (14/SC/1334). Patients receive an information sheet and provide informed consent prior to screening to participate in study specific procedures.Not applicable.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Animal models of tinnitus are essential for determining the underlying mechanisms and testing pharmacotherapies. However, there is doubt over the validity of current behavioural methods for detecting tinnitus. Here, we applied a stimulus paradigm widely used in a behavioural test (gap-induced inhibition of the acoustic startle reflex GPIAS) whilst recording from the auditory cortex, and showed neural response changes that mirror those found in the behavioural tests. We implanted guinea pigs (GPs) with electrocorticographic (ECoG) arrays and recorded baseline auditory cortical responses to a startling stimulus. When a gap was inserted in otherwise continuous background noise prior to the startling stimulus, there was a clear reduction in the subsequent evoked response (termed gap-induced reductions in evoked potentials; GIREP), suggestive of a neural analogue of the GPIAS test. We then unilaterally exposed guinea pigs to narrowband noise (left ear; 8-10 kHz; 1 h) at one of two different sound levels - either 105 dB SPL or 120 dB SPL - and recorded the same responses seven-to-ten weeks following the noise exposure. Significant deficits in GIREP were observed for all areas of the auditory cortex (AC) in the 120 dB-exposed GPs, but not in the 105 dB-exposed GPs. These deficits could not simply be accounted for by changes in response amplitudes. Furthermore, in the contralateral (right) caudal AC we observed a significant increase in evoked potential amplitudes across narrowband background frequencies in both 105 dB and 120 dB-exposed GPs. Taken in the context of the large body of literature that has used the behavioural test as a demonstration of the presence of tinnitus, these results are suggestive of objective neural correlates of the presence of noise-induced tinnitus and hyperacusis., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Cannabinoids have been suggested as a therapeutic target for a variety of brain disorders. Despite the presence of their receptors throughout the auditory system, little is known about how cannabinoids affect auditory function. We sought to determine whether administration of arachidonyl-2'-chloroethylamide (ACEA), a highly-selective CB 1 agonist, could attenuate a variety of auditory effects caused by prior administration of salicylate, and potentially treat tinnitus. We recorded cortical resting-state activity, auditory-evoked cortical activity and auditory brainstem responses (ABRs), from chronically-implanted awake guinea pigs, before and after salicylate + ACEA. Salicylate-induced reductions in click-evoked ABR amplitudes were smaller in the presence of ACEA, suggesting that the ototoxic effects of salicylate were less severe. ACEA also abolished salicylate-induced changes in cortical alpha band (6-10 Hz) oscillatory activity. However, salicylate-induced increases in cortical evoked activity (suggestive of the presence of hyperacusis) were still present with salicylate + ACEA. ACEA administered alone did not induce significant changes in either ABR amplitudes or oscillatory activity, but did increase cortical evoked potentials. Furthermore, in two separate groups of non-implanted animals, we found no evidence that ACEA could reverse behavioural identification of salicylate- or noise-induced tinnitus. Together, these data suggest that while ACEA may be potentially otoprotective, selective CB 1 agonists are not effective in diminishing the presence of tinnitus or hyperacusis., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Tinnitus chronically affects between 10-15% of the population but, despite its prevalence, the underlying mechanisms are still not properly understood. One experimental model involves administration of high doses of sodium salicylate, as this is known to reliably induce tinnitus in both humans and animals. Guinea pigs were implanted with chronic electrocorticography (ECoG) electrode arrays, with silver-ball electrodes placed on the dura over left and right auditory cortex. Two more electrodes were positioned over the cerebellum to monitor auditory brainstem responses (ABRs). We recorded resting-state and auditory evoked neural activity from awake animals before and 2 h following salicylate administration (350 mg/kg; i.p.). Large increases in click-evoked responses (> 100%) were evident across the whole auditory cortex, despite significant reductions in wave I ABR amplitudes (in response to 20 kHz tones), which are indicative of auditory nerve activity. In the same animals, significant decreases in 6-10 Hz spontaneous oscillations (alpha waves) were evident over dorsocaudal auditory cortex. We were also able to demonstrate for the first time that cortical evoked potentials can be inhibited by a preceding gap in background noise [gap-induced pre-pulse inhibition (PPI)], in a similar fashion to the gap-induced inhibition of the acoustic startle reflex that is used as a behavioural test for tinnitus. Furthermore, 2 h following salicylate administration, we observed significant deficits in PPI of cortical responses that were closely aligned with significant deficits in behavioural responses to the same stimuli. Together, these data are suggestive of neural correlates of tinnitus and oversensitivity to sound (hyperacusis)., (© 2016 The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Companion animals may serve as valuable models for studying human cancers. Although KRAS is the most commonly mutated gene in human ductal pancreatic cancers (57%), with mutations frequently occurring at codons 12, 13 and 61, human pancreatic acinar cell carcinomas (ACCs) lack activating KRAS mutations. In the present study, 32 pancreatic ACC samples obtained from 14 dogs and 18 cats, including seven metastases, were analyzed for six common activating KRAS mutations located in codons 12 (n = 5) and 13 (n = 1) using Sequenom MassARRAY. No KRAS mutations were found, suggesting that, similar to human pancreatic ACC, KRAS mutations do not play a critical role in feline or canine pancreatic ACC. Due to the similarity of the clinical disease in dogs and cats to that of man, this study confirms that companion animals offer potential as a suitable model for investigating this rare subtype of pancreatic carcinoma., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Tinnitus is highly complex, diverse, and difficult to treat, in part due to the fact that the underlying causes and mechanisms remain elusive. Tinnitus is generated within the auditory brain; however, consolidating our understanding of tinnitus pathophysiology is difficult due to the diversity of reported effects and the variety of implicated brain nuclei. Here, we focus on the inferior colliculus (IC), a midbrain structure that integrates the vast majority of ascending auditory information and projects via the thalamus to the auditory cortex. The IC is also a point of convergence for corticofugal input and input originating outside the auditory pathway. We review the evidence, from both studies with human subjects and from animal models, for the contribution the IC makes to tinnitus. Changes in the IC, caused by either noise exposure or drug administration, involve fundamental, heterogeneous alterations in the balance of excitation and inhibition. However, differences between hearing loss-induced pathology and tinnitus-related pathology are not well understood. Moreover, variability in tinnitus induction methodology has a significant impact on subsequent neural and behavioral changes, which could explain some of the seemingly contradictory data. Nonetheless, the IC is likely involved in the generation and persistence of tinnitus perception.

A significant challenge in tinnitus research lies in explaining how acoustic insult leads to tinnitus in some individuals, but not others. One possibility is genetic variability in the expression and function of neuromodulators - components of neural signaling that alter the balance of excitation and inhibition in neural circuits. An example is nitric oxide (NO) - a free radical and potent neuromodulator in the mammalian brain - that regulates plasticity via both pre-synaptic and postsynaptic mechanisms. Changes in NO have previously been implicated in tinnitus generation, specifically in the ventral cochlear nucleus (VCN). Here, we examined nitric oxide synthase (NOS) - the enzyme responsible for NO production - in the guinea pig VCN following acoustic trauma. NOS was present in most cell types - including spherical and globular bushy cells, small, medium, and large multipolar cells, and octopus cells - spanning the entire extent of the VCN. The staining pattern was symmetrical in control animals. Unilateral acoustic over-exposure (AOE) resulted in marked asymmetries between ipsilateral and contralateral sides of the VCN in terms of the distribution of NOS across the cochlear nuclei in animals with behavioral evidence of tinnitus: fewer NOS-positive cells and a reduced level of NOS staining was present across the whole extent of the contralateral VCN, relative to the ipsilateral VCN. The asymmetric pattern of NOS-containing cells was observed as early as 1 day after AOE and was also present in some animals at 3, 7, and 21 days after AOE. However, it was not until 8 weeks after AOE, when tinnitus had developed, that asymmetries were significant overall, compared with control animals. Asymmetrical NOS expression was not correlated with shifts in the threshold hearing levels. Variability in NOS expression between animals may represent one underlying difference that can be linked to whether or not tinnitus develops after noise exposure.

Tinnitus is often identified in animal models by using the gap prepulse inhibition of acoustic startle. Impaired gap detection following acoustic over-exposure (AOE) is thought to be caused by tinnitus "filling in" the gap, thus, reducing its salience. This presumably involves altered perception, and could conceivably be caused by changes at the level of the neocortex, i.e., cortical reorganization. Alternatively, reduced gap detection ability might reflect poorer temporal processing in the brainstem, caused by AOE; in which case, impaired gap detection would not be a reliable indicator of tinnitus. We tested the latter hypothesis by examining gap detection in inferior colliculus (IC) neurons following AOE. Seven of nine unilaterally noise-exposed guinea pigs exhibited behavioral evidence of tinnitus. In these tinnitus animals, neural gap detection thresholds (GDTs) in the IC significantly increased in response to broadband noise stimuli, but not to pure tones or narrow-band noise. In addition, when IC neurons were sub-divided according to temporal response profile (onset vs. sustained firing patterns), we found a significant increase in the proportion of onset-type responses after AOE. Importantly, however, GDTs were still considerably shorter than gap durations commonly used in objective behavioral tests for tinnitus. These data indicate that the neural changes observed in the IC are insufficient to explain deficits in behavioral gap detection that are commonly attributed to tinnitus. The subtle changes in IC neuron response profiles following AOE warrant further investigation.

Animal models of tinnitus allow us to study the relationship between changes in neural activity and the tinnitus percept. Here, guinea pigs were subjected to unilateral noise trauma and tested behaviourally for tinnitus 8 weeks later. By comparing animals with tinnitus with those without, all of which were noise-exposed, we were able to identify changes unique to the tinnitus group. Three physiological markers known to change following noise exposure were examined: spontaneous firing rates (SFRs) and burst firing in the inferior colliculus (IC), evoked auditory brainstem responses (ABRs), and the number of neurons in the cochlear nucleus containing nitric oxide synthase (NOS). We obtained behavioural evidence of tinnitus in 12 of 16 (75%) animals. Both SFRs and incidences of burst firing were elevated in the IC of all noise-exposed animals, but there were no differences between tinnitus and no-tinnitus animals. There were significant decreases in ipsilateral ABR latencies in tinnitus animals, contrary to what might be expected with a small hearing loss. Furthermore, there was an ipsilateral-contralateral asymmetry in NOS staining in the ventral cochlear nucleus (VCN) that was only apparent in tinnitus animals. Tinnitus animals had a significantly greater number of NOS-containing neurons on the noise-exposed side, whereas no-tinnitus animals did not. These data suggest that measuring NOS in the VCN and recording ABRs supplement behavioural methods for confirming tinnitus in animals, and that nitric oxide is involved in plastic neural changes associated with tinnitus., (© 2014 The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)