Farelerde Ağrıya Bağlı Yüz İfadesinin Otomatik Değerlendirilmesi (FARE-MİMİK)

TÜBİTAK EEEAG Proje01.05.2018Moleküler agrı mekanizmalarının temellerinin anlasılmasındaki önemli ilerlemelere veendüstrideki büyük yatırımlara karsın, agrı kesici ilaç gelistirme için yapılan temel arastırma ileklinik uygulama arasındaki translasyonel çalısmalardaki basarılar oldukça sınırlı kalmıstır. Buarayısın önündeki önemli engellerden biri mevcut hayvan modellerindeki sezinlenebilirlik(specifity) basarısının düsük olması, dolayısı ile gelistirilecek ilaçlar için hızlı ve güvenilir birdeneysel tarama testinin bulunmamasıdır. Bu ihtiyacı karsılamaya yönelik olarak bu projede, fare deneylerinde agrı paradigmalarıuygulanarak farelerin yüzlerinde olusan agrı mimiklerinin video kayıtlarından otomatik olarakderecelendirilmesini saglayacak hesaplamalı yöntemlerin gelistirilmesi hedeflenmistir.Literatürde Langford ve ekibi (2010) tarafından gelistirilen Fare YüzburusturmasıDerecelendirmesi (Mouse Grimace Scaling, MGS) isimli manuel bir yöntem mevcuttur.Farelerde agrı mimiklerinin otomatik derecelendirilmesi, hız ve daha tarafsız bir etiketlemebasarısının saglanmasının yanısıra etiketlemeyi manuel olarak yapabilecek uzmanlarınyetistirilmesi zorunlulugunu ortadan kaldırmasından dolayı da önem tasımaktadır. Proje, ortak doktora programı bulunan Hacettepe Üniversitesi (HÜ) ile Orta Dogu TeknikÜniversitesi'nin (ODTÜ) nörolojik bilimler ve teknoloji alanlarındaki deneyimleri bir arayagetirilerek yürütülmüstür. Projede HÜ Nörolojik Bilimler ve Psikiyatri Enstitüsü (NBPE)tarafında yapılan çalısmalarda, iki degisik agrı paradigması kullanılarak farelerde basagrısı vekarın agrısı yaratılmıs, farelerde olusan agrıya baglı yüz ifadeleriyle ilgili video kayıtlarıtoplanmıs, agrı miktarı uzmanlar tarafından manuel derecelendirilerek veri etiketlemesiyapılmıstır. ODTÜ Nörobilim ve Nöroteknoloji (NSNT) - Elektrik ve Elektronik Mühendisligi(EEMB) tarafındaki çalısmalarda ise toplanan video verilerinde fare yüzünün tespit ve takipedilmesini ve yüz ifadelerinden agrının otomatik derecelendirilmesini saglamak üzerebilgisayarla görme ve derin ögrenme adı verilen makine ögrenmesine dayalı hesaplamalıyöntemlerin gelistirilmesi yoluna gidilmistir. Serbest dolasan farelerde otomatik agrı derecelendirmek üzere, bu projede önerilerekgelistirilen 6 kameralı ODTÜ-HÜ gözlem kutusu, farenin hareketlerinin kısıtlandıgı 2 kameralıLangford kutusuna göre daha kullanıslı ve basarılı olmustur. Bu projeyle gelistirilen yöntem,farklı agrıların fare yüz ifadelerinden otomatik tespitinde ve potansiyel agrı kesicilerin hızlıtaranması için tranlasyonel tıp alanında nesnel, kolay uygulanır ve güvenilir bir yaklasımgetirmesi açısından önem tasımaktadır.Despite important improvements in understanding of molecular pain mechanisms and biginvestments in pain industry, translational success of basic science findings into clinicalpractice remained limited. One important limitation is the low specifity of the present animalmodels, and thus, a lack of rapid and reliable experimental tests for screening potentialanalgesic molecules.In order to meet this need, this project aims to develop computational methods thatautomatically grade pain mimics on mouse from video recordings of pain induced mice. Inthe literature there is a manual method named Mouse Grimace Scaling (MGS) developed byLangford et al. (2010). Automatic scoring of pain mimics in mice is also important because itspeeds up and achieves a more neutral labeling success, as well as eliminates the need totrain specialists who can do the labeling manually.The project was carried out by bringing together the expertise of Hacettepe University(HU) and Middle East Technical University (METU) in neurological sciences and technology.Headache and abdominal pain were induced in mice using two different pain paradigms bythe HU Institute of Neurological Sciences and Psychiatry (NBPE) group. Video recordings ofpain-related face expressions in mice were collected and manually scored by trainedexperimenters for data labeling. METU Neuroscience and Neurotechnology (NSNT) groupdeveloped computational methods based on computer vision and machine learning (deeplearning) for mice face detection, tracking and automatic scoring of pain from mice mimics inthe video recordings collected.The 6-camera METU-HU observation box, which was proposed and developed in thisproject for automatic pain rating in freely moving mice, has been more useful and successfulthan the 2-camera Langford box, which restricts the movement of mice. The methodologydeveloped by this project provides an objective, easy-to-implement and reliable approach inthe field of transranial medicine for automatic detection of various painful mouse facialexpressions and rapid screening of potential painkillers.Keywords: pain, headache, migraine, pain paradigms, mouse grimace, automaticfacial expression recognition, computer vision, video processing, machine learning,pattern recognition, biomedical signal processin

Short-Hairpin Rna Silencing Of Endogenous Fibroblast Growth Factor 2 In Rat Hippocampus Increases Anxiety Behavior

Background: The fibroblast growth factor system has been implicated in the pathophysiology of mood disorders in humans and in affective behavior in animal models. However, the studies have been either correlative or involved exogenous administration of fibroblast growth factor 2 (FGF2). None of them have directly linked endogenous FGF2 to changes in emotional responses. Therefore, we began a series of studies to knockdown FGF2 by RNA interference to examine the role of brain FGF2 in emotional responsiveness. Methods: We assessed the efficacy of short-hairpin RNA (shRNA) sequences targeted to FGF2 in COS7 cells transfected with a plasmid vector containing the full-length FGF2 sequence. We then sought to assess the effects of knocking down FGF2 gene expression in vivo on behavior. We microinjected a lentiviral vector containing either a shRNA targeting FGF2 or a nonsilencing sequence bilaterally into the dentate gyrus of the rat. Results: In a reporter assay system, three different shRNA sequences resulted in significant FGF2 knockdown in vitro. Five weeks following a single microinjection of one of those sequences in vivo, we observed a significant decrease in FGF2 gene expression by messenger RNA in situ hybridization in the hippocampus. The FGF2 knockdown increased the time spent in the closed arms of the elevated-plus maze, a test of anxiety behavior. Conclusions: The FGF2 knockdown in the hippocampus resulted in an anxiogenic effect. Together with our findings of an inverse correlation between anxiety and FGF2 expression levels, these results implicate FGF2 in the genesis and expression of anxiety disorders.Wo

Assesment of Pain in Mouse Facial Images

Analysing mouse behavior in medical experiments to determine adverse effects of medical drugs requires special expertise and it is a time consuming tedious task. Automatic scaling of facial pain mimics in mice are important for a fast and objective labeling. Although there exists a manual procedure for scaling mouse facial pain expression, a full automatic method does not exist yet. In this paper, a computational method is proposed for assesment of pain through facial exressions of mouses in experiments where pain paradigms are applied. For this purpose, mouse face regions in videos were extracted manually frame by frame and also their pain scales were labeled by experts in order to construct a data set. Then, this data set were used for training a neural network using deep learning. The results obtained in this preliminary study, where a limited dataset of still images was used, are quite encouraging. Our studies in order to make our results more reliable and to devolope a fully automatic approach for scaling mouse grimace in videos are still going on

Nuclear Expansion And Pore Opening Are Instant Signs Of Neuronal Hypoxia And Can Identify Poorly Fixed Brains

The initial phase of neuronal death is not well characterized. Here, we show that expansion of the nuclear membrane without losing its integrity along with peripheralization of chromatin are immediate signs of neuronal injury. Importantly, these changes can be identified with commonly used nuclear stains and used as markers of poor perfusion-fixation. Although frozen sections are widely used, no markers are available to ensure that the observed changes were not confounded by perfusion-induced hypoxia/ischemia. Moreover, HMGB1 was immediately released and p53 translocated to mitochondria in hypoxic/ischemic neurons, whereas nuclear pore complex inhibitors prevented the nuclear changes, identifying novel neuroprotection targets.WoSScopu

Vesicular HMGB1 release from neurons stressed with spreading depolarization enables confined inflammatory signaling to astrocytes

The role of high mobility group box 1 (HMGB1) in inflammation is well characterized in the immune system and in response to tissue injury. More recently, HMGB1 was also shown to initiate an “inflammatory signaling cascade” in the brain parenchyma after a mild and brief disturbance, such as cortical spreading depolarization (CSD), leading to headache. Despite substantial evidence implying a role for inflammatory signaling in prevalent neuropsychiatric disorders such as migraine and depression, how HMGB1 is released from healthy neurons and how inflammatory signaling is initiated in the absence of apparent cell injury are not well characterized. We triggered a single cortical spreading depolarization by optogenetic stimulation or pinprick in naïve Swiss albino or transgenic Thy1-ChR2-YFP and hGFAP-GFP adult mice. We evaluated HMGB1 release in brain tissue sections prepared from these mice by immunofluorescent labeling and immunoelectron microscopy. EzColocalization and Costes thresholding algorithms were used to assess the colocalization of small extracellular vesicles (sEVs) carrying HMGB1 with astrocyte or microglia processes. sEVs were also isolated from the brain after CSD, and neuron-derived sEVs were captured by CD171 (L1CAM). sEVs were characterized with flow cytometry, scanning electron microscopy, nanoparticle tracking analysis, and Western blotting. We found that HMGB1 is released mainly within sEVs from the soma of stressed neurons, which are taken up by surrounding astrocyte processes. This creates conditions for selective communication between neurons and astrocytes bypassing microglia, as evidenced by activation of the proinflammatory transcription factor NF-ĸB p65 in astrocytes but not in microglia. Transmission immunoelectron microscopy data illustrated that HMGB1 was incorporated into sEVs through endosomal mechanisms. In conclusion, proinflammatory mediators released within sEVs can induce cell-specific inflammatory signaling in the brain without activating transmembrane receptors on other cells and causing overt inflammation