This paper is dedicated to my beloved wife Maren and our little Karl.

1. Préparation des yeux de bovins <ol><li> Obtenir yeux bovins directement après que l&#39;animal est abattu. </li><li> Transporter les yeux protégées &quot;Sickel-solution&quot; un milieu spécial contenant 120 mM de NaCl, 2 mM de KCl, 0,1 mM de MgCl2, 0,15 mM de CaCl2, 1,5 mM de NaH 2 PO 4, 13,5 mM de Na 2 HPO 4 et 5 mM de glucose à RT. </li><li> Effectuer la préparation de la rétine dans des conditions adaptées sombres avec une lumière rouge sombre. </li><li> Retirer la partie antérieure de l&#39;œil. Effectuer une incision ca. équatoriale 4 mm en arrière du limbe. Par la suite retirer cornée, l&#39;iris, le corps ciliaire et la lentille en un seul morceau. Gardez les rétines dans Sickel-solution. </li><li> Desserrer mécaniquement les pièces jointes vitreux à la surface de la rétine et du vitré retirer de la coupe de l&#39;oeil ouvert. </li><li> Ensuite diviser l&#39;œil en quatre quadrants et punch out ronde domaines de ca. 7 mm de diamètre en utilisant un trépan. </li><li> Séparez délicatement la rétineà partir de l&#39;épithélium pigmentaire et la placer sur un dispositif d&#39;enregistrement à l&#39;intérieur d&#39;une boîte à l&#39;abri de la lumière. Le dispositif d&#39;enregistrement est constitué d&#39;un mainteneur plastique à ouverture de maille dans le milieu; placer la rétine sur le maillage, puis fixer avec un anneau en plastique directement sur les électrodes. NOTE: Le mainteneur plastique a deux canaux pour permettre un flux constant du milieu. </li></ol> 2. Enregistrement du Électrorétinogramme (ERG) <ol><li> Afin d&#39;enregistrer l&#39;électrorétinogramme, utiliser deux électrodes argent / chlorure d&#39;argent de chaque côté de la rétine et perfuser la rétine à une vitesse de perfusion constante de ca. 1 ml / min et la température constante de 37 ° C. Utilisez le &quot;Sickel-solution&quot; saturée en oxygène. </li><li> Avant de commencer la mesure, adapter sombre la rétine (la protéger de la lumière pendant toutes les mesures) et utiliser des intervalles de relance de cinq minutes. Utilisez un 1 Hz seul flash au xénon blanc pour la stimulation avec une intensité réglée sur 6,3 mlx à la surface de la rétine. </li&gt; <li> Utilisez calibré filtres de densité neutre et un stimulus lumineux de 10 microsecondes contrôlée par une minuterie afin d&#39;avoir des réponses optimales. </li><li> Pour mesurer et traiter les données, filtrer l&#39;ERG et l&#39;amplifier (100 Hz filtre passe-haut, 50 Hz filtre notch, 100 000 x amplification) en utilisant un amplificateur RPS312RM Herbe. Essayez de filtrer les fréquences perturbatrices possibles qui peuvent perturber le signal. Afin de traiter les données, utiliser une carte d&#39;acquisition analogique-numérique des données sur un ordinateur de bureau (PC compatible). </li><li> Après la période d&#39;adaptation à l&#39;obscurité sous perfusion constante, mesurer les amplitudes du signal électrique jusqu&#39;à ce amplitudes b-ondes stables sont enregistrées. NOTE: Les amplitudes sont considérés comme stable si cinq mesures simples atteignent une valeur moyenne et écartent moins de 10%. Un bon exemple de mesures individuelles est donnée à la figure 1. </li><li> Pour commencer le test, remplacer l&#39;oxygène pur soit par de l&#39;azote pur (nombre d&#39;expériences simples, n = 5) pour tester l&#39;hypoxieou glutamate 250 um (n = 5). </li><li> Notez les réponses électriques toutes les 5 min pendant 45 min. </li><li> Après la période d&#39;essai, perfuser les rétines avec un milieu standard saturé avec l&#39;oxygène pendant 75 minutes et regarder les changements de l&#39;amplitude de l&#39;onde b. Ce est la phase lavage. Mesurer l&#39;amplitude de l&#39;onde b de l&#39;auge de l&#39;un vague à l&#39;apogée de la b-ondes. </li><li> Pour étudier l&#39;effet de l&#39;hypoxie ou glutamate sur le potentiel de photorécepteur dans des conditions scotopiques, supprimer l&#39;onde b en ajoutant 1 mM à la solution nutritive. </li><li> Après l&#39;enregistrement d&#39;un potentiel de photorécepteur stable pendant 30 min, effectuer la procédure comme avant, exposant les rétines 45 min aux différentes solutions d&#39;irrigation avec 1 mM aspartate. Utilisez la même période de lavage (étape 2.8) tel que mentionné précédemment. </li></ol> 3. Analyse des données <ol><li> Afin d&#39;évaluer statistiquement les données, assurer une distribution normale pour toutes les données, par exemple en utilisant le Kolmogorov-Smirnovessai de 15. </li><li> Calculer la réduction des a- et B-ondes amplitudes en pourcentages après la phase d&#39;exposition par rapport à la dernière mesure avant l&#39;exposition. Comparer la réduction de l&#39;Erg-amplitudes après 45 min - à la fin de la période d&#39;exposition - à ERG mesurée avant l&#39;application. </li><li> Comparer l&#39;onde a- et b- à la fin de la phase de lavage à l&#39;amplitude correspondante avant exposition à examiner une récupération possible. </li><li> Pour l&#39;analyse statistique, utilisez le logiciel JMP logiciel statistique ou le logiciel SPSS. Calculer des données dans l&#39;ensemble en tant que moyenne ± écart-type. Estimer la signification par le test statistique approprié. NOTE: Ces tests peuvent être différents en fonction de la portée expérimentale. Dans ce cadre, utilisez t-test apparié de Student. </li></ol>

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The authors have nothing to disclose.

Dans cette étude, un impact significatif sur l&#39;amplitude de l&#39;onde b après 45 min de l&#39;hypoxie a été trouvé. Cette réduction était encore significative après la phase de lavage. Un effet similaire sur le potentiel de photorécepteur a pu être observée. Les résultats sont corroborés par d&#39;autres données publiées et de 16 nous donner la possibilité d&#39;étudier des effets neuroprotecteurs possibles après l&#39;hypoxie. A...

Neuroprotection a été un fort champ d&#39;investigation dans la recherche ophtalmologique dans les dernières décennies. La rétine est un réseau neuronal très sensible qui dépend de manière significative sur l&#39;oxygénation et est fortement influencé par le métabolisme de ses cellules environnantes. Pathologies oculaires majeurs liés aux dommages de cellules nerveuses rétiniennes sont occlusions vasculaires, le glaucome, et décollement de la rétine. Occlusion de l&#39;artère rétinienne, comme un exemple pour occlusion vasculaire rétinienne, conduit à une perte soudaine de la vision due à l&#39;hypoxie de la rétine interne 1. Elle est souvent associée à des pathologies vasculaires générales 2 et conduit à une perte visuelle persistante 1, avec seulement 8% des patients qui se remettent de façon significative une acuité visuelle. Bien que la fibrinolyse artérielle a été suggéré comme une option de traitement, le bénéfice n&#39;a pas pu être montré dans un essai clinique randomisé 3. Le glaucome et décollement de la rétineont tous deux une augmentation de la concentration de glutamate 6.4. Glutamate dans des conditions physiologiques est rencontré comme un transmetteur excitateur dans tout le système nerveux central et le 7,8 rétine interne. Niveaux élevés de glutamate ont été trouvés non seulement dans le glaucome et décollement de la rétine 5,6 mais aussi dans la rétinopathie diabétique proliférante neuf. Une augmentation de glutamate conduit éventuellement à l&#39;excitotoxicité et, par conséquent, des lésions des cellules nerveuses 10. Dans la plupart des cas de décollement de la rétine et dans certains cas de la chirurgie de la rétinopathie diabétique proliférante sur la rétine (vitrectomie pars plana) sont nécessaires. Pendant pars plana vitrectomie manipulation mécanique, la lumière vive de la fibre optique ou contrainte de cisaillement exercée par des débits élevés de solutions d&#39;irrigation pendant de longues opérations exercent un stress supplémentaire sur la rétine 11,12. Toutes les maladies mentionnées ont en commun que la pathologie est localisée à la rétineun seul et pose la communauté ophtalmologique au défi de trouver des moyens pour protéger la rétine comme un système neurosensoriel. L&#39;électrorétinogramme (ERG) est la méthode standard pour l&#39;évaluation in vivo la fonction des photorécepteurs (a-ondes) et la fonction de la rétine interne (b-ondes). L&#39;ERG est mesurée par Silver-électrodes introduites dans la cornée et les yeux sont stimulées par une augmentation du niveau de la lumière pour détecter des défauts dans des tiges ou des cônes ou dans la rétine interne. Différents défauts de la rétine peuvent être détectées par des changements dans l&#39;amplitude (la force de la réponse) ou la latence (la réponse-à-intervalle de temps-) de l&#39;ERG. Protocole et méthodes de mesure différentes ERG (pattern-ERG, multifocale-ERG ou champ clair ERG) permettent une différenciation plus poussée des défauts. La technique de la rétine isolée a été introduit récemment, ce qui permet d&#39;évaluer les effets sur la rétine sans interférences d&#39;un animal, par exemple de l&#39;étude de13,14 réactions générales. Ce était le but de cette étude pour évaluer et mettre en place un modèle de stress défini et normalisé pour l&#39;hypoxie et le stress de glutamate sur la rétine isolée superfusées. Ainsi, nous espérons jeter les bases pour de futures études sur les effets neuroprotecteurs de certains agents ou des solutions d&#39;irrigation intraoculaires.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">120 mM NaCl&#160;</td>
			<td style="width:71px;">Merck Pharma, Germany</td>
			<td align="right" style="width:119px;">1,064,041,000</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">2 mM KCl,&#160;&#160;</td>
			<td style="width:71px;">Merck Pharma, Germany</td>
			<td align="right" style="width:119px;">1,050,010,250</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">0.1 mM MgCl2,&#160;</td>
			<td style="width:71px;">Merck Pharma, Germany</td>
			<td align="right" style="width:119px;">58,330,250</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">0.15 mM CaCl2</td>
			<td style="width:71px;">Merck Pharma, Germany</td>
			<td style="width:119px;">111 TA106282</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">1.5 mM NaH2PO4/13.5 mM Na2HPO4&#160;&#160;</td>
			<td style="width:71px;">Merck Pharma, Germany</td>
			<td align="right" style="width:119px;">1,065,860,500</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">5 mM glucose</td>
			<td style="width:71px;">Merck Pharma, Germany</td>
			<td align="right" style="width:119px;">40,741,000</td>
			<td></td>
		</tr>
	</tbody>
</table>

glutamate and hypoxia as a stress model for the isolated perfused vertebrate retina

Neuroprotection a été un fort champ d&#39;investigation dans la recherche ophtalmologique dans les dernières décennies et affecte maladies comme le glaucome, une occlusion vasculaire rétinienne, décollement de la rétine, et la rétinopathie diabétique. Ce était l&#39;objet de cette étude d&#39;introduire un modèle de stress normalisé pour les futurs essais thérapeutiques précliniques. Rétines bovins ont été préparés et perfusés avec une solution standard d&#39;oxygène saturé, et l&#39;ERG a été enregistré. Après l&#39;enregistrement B-ondes stables, l&#39;hypoxie (N 2) pur ou un stress du glutamate (250 um de glutamate) a été exercée pendant 45 min. Pour étudier les effets sur la fonction des photorécepteurs seul, 1 mM aspartate a été ajouté pour obtenir un-ondes. ERG recouvrement a été suivie pendant 75 min. Pour une hypoxie, une diminution de l&#39;amplitude d&#39;une onde de 87,0% a été notée (p &lt;0,01) après un temps d&#39;exposition de 45 min (diminution de 36,5% après la fin du lavage p = 0,03). En outre, un dim initialefacilité dans amplitudes b-ondes de 87,23% a été enregistrée, qui a atteint la signification statistique (p &lt;0,01, baisse de 25,5% à la fin de l&#39;affouillement, p = 0,03). Pour 250 um glutamate, une première réduction de l&#39;amplitude d&#39;onde un (p&gt; 0,05), suivie par une réduction de 1,9% (p&gt; 0,05) de 7,8%. Une réduction de 83,7% des amplitudes d&#39;onde (b p &lt;0,01) a été notée; après un lavage de 75 min, la réduction a été de 2,3% (p = 0,62). Dans cette étude, un modèle de stress standardisé est présenté qui peut être utile pour identifier les effets neuroprotecteurs possibles à l&#39;avenir.

Après 1 heure de la perfusion de la préparation de la rétine avec la solution étalon saturée en oxygène (figure 1A et B) ERG-amplitudes ont montré stabilisation et moins de variations d&#39;amplitudes entre les mesures simples. le pH, la pression osmotique, la température et la pO 2 (sauf pour l&#39;essai de l&#39;hypoxie) ont été maintenues constantes pour tous les tests. Pour isoler le signal de photorécepteur à partir du signal de la rétine interne, 1...

Watch this Scientific Journal Video about Glutamate and Hypoxia as a Stress Model for the Isolated Perfused Vertebrate Retina at JoVE.com

Glutamate and Hypoxia as a Stress Model for the Isolated Perfused Vertebrate Retina

Avec cette étude, nous présentons un modèle de stress standardisé pour la rétine bovine superfusé isolée pour l&#39;avenir essais thérapeutiques précliniques. L&#39;effet de l&#39;hypoxie soit (N 2 pur) ou le stress de glutamate (250 uM de glutamate) sur la fonction rétinienne représenté par a et b amplitudes d&#39;onde a été évaluée.

Glutamate et hypoxie comme un modèle de stress pour la isolé perfusé vertébrés Retina

Neuroprotection has been a strong field of investigation in ophthalmological research in the past decades and affects diseases such as glaucoma, retinal ...

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Medicine

7.8K vues.  University Eye Hospital Tübingen.  Avec cette étude, nous présentons un modèle de stress standardisé pour la rétine bovine superfusé isolée pour l'avenir essais thérapeutiques précliniques. L'effet de l'hypoxie soit (N 2 pur) ou le stress de glutamate (250 uM de glutamate) sur la fonction rétinienne représenté par a et b amplitudes d'onde a été évaluée. 

Vidéo: Glutamate and Hypoxia as a Stress Model for the Isolated Perfused Vertebrate Retina

Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson's Disease

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our understanding of the mechanisms underlying parkinsonian symptoms, since it recapitulates the changes in basal ganglia circuitry and pharmacology observed in parkinsonian patients1-4. However, the precise cellular and molecular changes occurring at cortico-striatal synapses of the output pathways within the striatum, which is the major input region of the basal ganglia remain elusive, and this is believed to be site where pathological abnormalities underlying parkinsonian symptoms arise3,5.
In PD, understanding the mechanisms underlying changes in basal ganglia circuitry following degeneration of the nigro-striatal pathway has been greatly advanced by the development of bacterial artificial chromosome (BAC) mice over-expressing green fluorescent proteins driven by promoters specific for the two striatal output pathways (direct pathway: eGFP-D1; indirect pathway: eGFP-D2 and eGFP-A2a)8, allowing them to be studied in isolation. For example, recent studies have suggested that there are pathological changes in synaptic plasticity in parkinsonian mice9,10. However, these studies utilised juvenile mice and acute models of parkinsonism. It is unclear whether the changes described in adult rats with stable 6-OHDA lesions also occur in these models. Other groups have attempted to generate a stable unilaterally-lesioned 6-OHDA adult mouse model of PD by lesioning the medial forebrain bundle (MFB), unfortunately, the mortality rate in this study was extremely high, with only 14% surviving the surgery for 21 days or longer11. More recent studies have generated intra-nigral lesions with both a low mortality rate &gt;80% loss of dopaminergic neurons, however expression of L-DOPA induced dyskinesia11,12,13,14 was variable in these studies. Another well established mouse model of PD is the MPTP-lesioned mouse15. Whilst this model has proven useful in the assessment of potential neuroprotective agents16, it is less suitable for understanding mechanisms underlying symptoms of PD, as this model often fails to induce motor deficits, and shows a wide variability in the extent of lesion17, 18.
Here we have developed a stable unilateral 6-OHDA-lesioned mouse model of PD by direct administration of 6-OHDA into the MFB, which consistently causes &gt;95% loss of striatal dopamine (as measured by HPLC), as well as producing the behavioural imbalances observed in the well characterised unilateral 6-OHDA-lesioned rat model of PD. This newly developed mouse model of PD will prove a valuable tool in understanding the mechanisms underlying generation of parkinsonian symptoms.

Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson&#039;s Disease

A protocol for performing unilateral 6-OHDA lesions of the medial forebrain bundle in mice is described. This method has a low mortality rate (13.3 %) with 89% of the surviving animals showing &#62;95% loss of striatal dopamine and 90.63&#177;-4.02 % ipsiversive rotational bias towards the side of the lesion.

Développement d&#39;une manière unilatérale-lésés 6-OHDA modèle murin de la maladie de Parkinson

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our ...

Recherche

Médecine

The Trier Social Stress Test Protocol for Inducing Psychological Stress

This article demonstrates a psychological stress protocol for use in a laboratory setting. Protocols that allow researchers to study the biological pathways of the stress response in health and disease are fundamental to the progress of research in stress and anxiety.1 Although numerous protocols exist for inducing stress response in the laboratory, many neglect to provide a naturalistic context or to incorporate aspects of social and psychological stress. Of psychological stress protocols, meta-analysis suggests that the Trier Social Stress Test (TSST) is the most useful and appropriate standardized protocol for studies of stress hormone reactivity.2 In the original description of the TSST, researchers sought to design and evaluate a procedure capable of inducing a reliable stress response in the majority of healthy volunteers.3 These researchers found elevations in heart rate, blood pressure and several endocrine stress markers in response to the TSST (a psychological stressor) compared to a saline injection (a physical stressor).3 Although the TSST has been modified to meet the needs of various research groups, it generally consists of a waiting period upon arrival, anticipatory speech preparation, speech performance, and verbal arithmetic performance periods, followed by one or more recovery periods. The TSST requires participants to prepare and deliver a speech, and verbally respond to a challenging arithmetic problem in the presence of a socially evaluative audience.3 Social evaluation and uncontrollability have been identified as key components of stress induction by the TSST.4 In use for over a decade, the goal of the TSST is to systematically induce a stress response in order to measure differences in reactivity, anxiety and activation of the hypothalamic-pituitary-adrenal (HPA) or sympathetic-adrenal-medullary (SAM) axis during the task.1 Researchers generally assess changes in self-reported anxiety, physiological measures (e.g. heart rate), and/or neuroendocrine indices (e.g. the stress hormone cortisol) in response to the TSST. Many investigators have adopted salivary sampling for stress markers such as cortisol and alpha-amylase (a marker of autonomic nervous system activation) as an alternative to blood sampling to reduce the confounding stress of blood-collection techniques. In addition to changes experienced by an individual completing the TSST, researchers can compare changes between different treatment groups (e.g. clinical versus healthy control samples) or the effectiveness of stress-reducing interventions.1

This article describes a protocol for inducing psychological stress in participants, which enables researchers to measure psychological, physiological and neuroendocrine responses to stress within single participants or between groups.

Le protocole social Trèves Stress Test pour induire un stress psychologique

This article demonstrates a psychological stress protocol for use in a laboratory setting.  Protocols that allow researchers to study the biological ...

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Biomarqueurs dans un modèle animal pour Révéler Corrélats neuronaux, hématologiques et du comportement de stress post-traumatique

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

The In ovo CAM-assay as a Xenograft Model for Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions.
This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.

The In ovo CAM-assay as a Xenograft Model for Sarcoma

The in ovo chorioallantoic membrane (CAM) is grafted with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior.

Le<em&gt; En ovo</em&gt; CAM-test comme un Xenograftmodel pour le sarcome

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for ...

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD).

Creating experimental retinal detachments with a reproducible and sustained height of detachment, and without subretinal hemorrhage, is important for studying the pathophysiology of photoreceptor cell loss in retinal disease and evaluating potential therapeutic interventions. Here, we report such a method in detail.

Décollement de la rétine modèle chez les rongeurs par injection sous-rétinienne de hyaluronate de sodium

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Excitotoxiques stimulation de Microslices cerveau comme un<em&gt; In vitro</em&gt; Modèle de l&#39;AVC

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Isolated Hepatic Perfusion as a Treatment for Liver Metastases of Uveal Melanoma

Isolated hepatic perfusion (IHP) is a procedure where the liver is surgically isolated and perfused with a high concentration of the chemotherapeutic agent melphalan. Briefly, the procedure starts with the setup of a percutaneous veno-venous bypass from the femoral vein to the external jugular vein. Via a laparotomy, catheters are then inserted into the proper hepatic artery and the caval vein. The portal vein and the caval vein, both supra- and infrahepatically, are then clamped. The arterial and venous catheters are connected to a heart lung machine and the liver is perfused with melphalan (1 mg/kg body weight) for 60 min. This way it is possible to locally perfuse the liver with a high dose of a chemotherapeutic agent, without leakage to the systemic circulation.
In previous studies including patients with isolated liver metastases of uveal melanoma, an overall response rate of 33-100% and a median survival between 9 and 13 months, have been reported. The aim of this protocol is to give a clear description of how to perform the procedure and to discuss IHP as a treatment option for liver metastases of uveal melanoma.

Here, we present a protocol how to perform an isolated liver perfusion (IHP) with melphalan and we also discuss IHP as a treatment option for liver metastases of uveal melanoma.

Perfusion hépatique isolée comme un traitement pour les métastases hépatiques de mélanome uvéal

Isolated hepatic perfusion (IHP) is a procedure where the liver is surgically isolated and perfused with a high concentration of the chemotherapeutic ...

A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA thrombolysis) rarely produces benefits in mechanical occlusion-based experimental models, which dominate preclinical stroke research. This split between the bench and bedside suggests the need to employ tPA-responsive models in preclinical stroke research. To this end, a simple and tPA-reactive thrombotic stroke model is invented and described here. This model consists of transient occlusion of the unilateral common carotid artery and delivery of 7.5% oxygen through a face mask in adult mice for 30 min, while maintaining the animal rectal temperature at 37.5 &#177; 0.5 &#176;C. Although reversible ligation of the unilateral carotid artery or hypoxia each suppressed cerebral blood flow only transiently, the combination of both insults caused lasting reperfusion deficits, fibrin and platelet deposition, and large infarct in the middle cerebral artery-supplied territory. Importantly, tail-vein injection of recombinant tPA at 0.5, 1, or 4 hr post-tHI (10 mg/kg) provided time-dependent reduction of the mortality rate and infarct size. This new stroke model is simple and can be standardized across laboratories to compare experimental results. Further, it induces thrombosis without craniectomy or introducing pre-formed emboli. Given these unique merits, the tHI model is a useful addition to the repertoire of preclinical stroke research.

Thromboembolic stroke models are vital tools for optimizing the recanalization therapy. Here we report a murine thrombotic stroke model based on transient cerebral hypoxic-ischemic (tHI) insult, which triggers thrombosis and infarction, and responds favorably to tissue plasminogen activator (tPA)-mediated fibrinolysis in a therapeutic window similar to those in stroke patients.

Un modèle de course thrombotique Basé sur cérébrale transitoire hypoxie-ischémie

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA ...

A Model to Simulate Clinically Relevant Hypoxia in Humans

In case of apnea, arterial partial pressure of oxygen (pO2) decreases, while partial pressure of carbon dioxide (pCO2) increases. To avoid damage to hypoxia sensitive organs such as the brain, compensatory circulatory mechanisms help to maintain an adequate oxygen supply. This is mainly achieved by increased cerebral blood flow. Intermittent hypoxia is a commonly seen phenomenon in patients with obstructive sleep apnea. Acute airway obstruction can also result in hypoxia and hypercapnia. Until now, no adequate model has been established to simulate these dynamics in humans. Previous investigations focusing on human hypoxia used inhaled hypoxic gas mixtures. However, the resulting hypoxia was combined with hyperventilation and is therefore more representative of high altitude environments than of apnea. Furthermore, the transferability of previously performed animal experiments to humans is limited and the pathophysiological background of apnea induced physiological changes is poorly understood. In this study, healthy human apneic divers were utilized to mimic clinically relevant hypoxia and hypercapnia during apnea. Additionally, pulse-oximetry and Near Infrared Spectroscopy (NIRS) were used to evaluate changes in cerebral and peripheral oxygen saturation before, during, and after apnea.

Hypoxia simulation in humans has usually been performed by inhaling hypoxic gas mixtures. For this study, apneic divers were used to simulate dynamic hypoxia in humans. Additionally, physiological changes in desaturation and re-saturation kinetics were evaluated with non-invasive tools such as Near-Infrared-Spectroscopy (NIRS) and peripheral oxygenation saturation (SpO2).

Un modèle pour Simuler Hypoxie cliniquement importants chez les humains

In case of apnea, arterial partial pressure of oxygen (pO2) decreases, while partial pressure of carbon dioxide (pCO2) increases. To avoid damage to ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Mesure de la contractilité isométrique de l’artère mésentérique de souris à l’aide de fil myographie

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...