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

1. Preparazione di occhi bovini <ol><li> Ottenere occhi bovini subito dopo la macellazione. </li><li> Trasporto gli occhi protetti in &quot;Sickel soluzione&quot; un mezzo speciale contenente NaCl 120 mM, KCl 2 mM, 0,1 mM MgCl 2, 0,15 mM CaCl 2, 1.5 mM NaH 2 PO 4, 13,5 mM Na 2 HPO 4 e 5 mM di glucosio a RT. </li><li> Eseguire la preparazione della retina in condizioni adatte scuri con una luce rossa fioca. </li><li> Rimuovere la parte anteriore dell&#39;occhio. Eseguire un&#39;incisione ca. equatoriale 4 millimetri posteriormente al limbus. Successivamente rimuovere cornea, iride, corpo ciliare e obiettivo in un unico pezzo. Tenere le retine in Sickel-soluzione. </li><li> Meccanicamente allentare i collegamenti vetrose alla superficie retinica e rimuovere il vitreo dalla tazza occhio aperto. </li><li> Successivamente suddividere l&#39;occhio in quattro quadranti e pugni fuori turno aree di ca. 7 mm con un trapano. </li><li> Separare delicatamente la retinadal epitelio pigmentato e posizionarlo su un dispositivo di registrazione all&#39;interno di una scatola al riparo dalla luce. Il dispositivo di registrazione è costituito da un manutentore plastica con una maglia nel mezzo; posizionare la retina sulla maglia e poi fissare con un anello di plastica direttamente sugli elettrodi. NOTA: Il manutentore plastica ha due canali per permettere un flusso costante di fluido. </li></ol> 2. Registrazione del Elettroretinogramma (ERG) <ol><li> Per registrare l&#39;elettroretinogramma, utilizzare due elettrodi di argento / cloruro di argento-su entrambi i lati della retina e profumato retina ad una velocità costante di perfusione ca. 1 ml / min e temperatura costante di 37 ° C. Utilizzare il &quot;Sickel-soluzione&quot; satura di ossigeno. </li><li> Prima di avviare la misurazione, scuro adattare la retina (proteggerlo dalla luce durante tutte le misure) e utilizzare gli intervalli di stimolo di cinque minuti. Utilizzare un 1 Hz singolo flash allo xeno bianco per la stimolazione con un&#39;intensità impostato 6.3 mlx sulla superficie della retina. </li&gt; <li> Uso calibrato filtri di densità neutri e uno stimolo luce 10 usec controllato da un timer in modo da avere una risposta ottimale. </li><li> Per misurare ed elaborare i dati, filtrare l&#39;ERG e amplificarlo (100 Hz filtro passa alto, 50 filtro notch Hz, 100.000 x amplificazione) utilizzando un RPS312RM amplificatore Grass. Prova a filtrare le eventuali frequenze di disturbo che possono disturbare il segnale. Per elaborare i dati, utilizzare una scheda di acquisizione dati digitali da analogico a un computer desktop (PC compatibile). </li><li> Dopo il periodo di adattamento al buio sotto perfusione costante misurare le ampiezze del segnale elettrico fino sono registrati ampiezze b-onda stabili. NOTA: Le ampiezze sono considerate stabili se cinque misurazioni singole raggiungono un valore medio e discostano meno del 10%. Un buon esempio di singole misurazioni è riportata nella figura 1. </li><li> Per iniziare il test, sostituiscono l&#39;ossigeno puro o azoto puro (numero di singoli esperimenti, n = 5) per verificare ipossiao 250 micron glutammato (n = 5). </li><li> Registrare le risposte elettriche ogni 5 minuti per 45 min. </li><li> Dopo il periodo di prova, profumato le retine con medie standard satura di ossigeno per 75 minuti e guardare i cambiamenti di ampiezza b-wave. Questa è la fase di washout. Misurare l&#39;ampiezza dell&#39;onda b dal trogolo di un onda al picco del b-onda. </li><li> Per studiare l&#39;effetto di ipossia o glutammato sulle potenzialità fotorecettori in condizioni scotopiche, sopprimere la b-wave con l&#39;aggiunta di 1 mM di soluzione nutritiva. </li><li> Dopo aver registrato un potenziale fotorecettore stabile per 30 min, eseguire la procedura come prima, esponendo le retine 45 min alle diverse soluzioni di irrigazione con 1 mM aspartato. Utilizzare lo stesso periodo di washout (passo 2,8), come accennato in precedenza. </li></ol> Analisi 3. Dati <ol><li> Per valutare statisticamente i dati, garantire una distribuzione normale per tutti i dati, ad esempio utilizzando il Kolmogorov-SmirnovTest 15. </li><li> Calcolare la riduzione delle a- e b-onda ampiezze in percentuale dopo la fase di esposizione rispetto all&#39;ultima misura prima dell&#39;esposizione. Confrontare la riduzione delle ERG-ampiezze dopo 45 min - al termine del periodo di esposizione - ERG misurata prima dell&#39;applicazione. </li><li> Confrontare l&#39;onda a- e b- al termine della fase di washout all&#39;ampiezza corrispondente prima dell&#39;esposizione ad esaminare una possibile ripresa. </li><li> Per l&#39;analisi statistica, utilizzare il software statistico JMP software o software SPSS. Calcolare i dati tutto come media ± deviazione standard. Stimare significatività con il test statistico appropriato. NOTA: Questi test potrebbero essere diverse a seconda del campo di applicazione sperimentale. In questa impostazione, utilizzare paired t-test di Student. </li></ol>

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The authors have nothing to disclose.

In questo studio, è stato trovato un significativo rilievo l&#39;ampiezza dell&#39;onda b dopo 45 min di ipossia. Tale riduzione è ancora significativo dopo la fase di lavaggio. Potrebbe essere osservato un effetto simile sul potenziale fotorecettori. I risultati sono supportati da altri dati pubblicati 16 e ci danno l&#39;opportunità di studiare i possibili effetti neuroprotettivi dopo ipossia. Dopo 45 min di esposizione 250 mM glutammato, abbiamo ...

Neuroprotezione è stato un forte campo di indagine nella ricerca oculistico negli ultimi decenni. La retina è una rete neuronale altamente sensibile che dipende sensibilmente l&#39;ossigenazione ed è fortemente influenzato dal metabolismo delle sue cellule circostanti. Le principali patologie oculari legate a danni delle cellule nervose sono occlusioni vascolari retiniche, glaucoma e distacco della retina. Occlusione dell&#39;arteria retinica, come esempio per occlusione vascolare retinica, porta ad una improvvisa perdita della vista a causa di ipossia della retina interna 1. E &#39;spesso associata a patologie vascolari generali 2 e porta ad una perdita visiva permanente 1, con solo l&#39;8% dei pazienti sottoposti acuità visiva significativamente 1. Sebbene fibrinolisi arteriosa è stato suggerito come opzione di trattamento, il beneficio non può essere mostrato in uno studio clinico randomizzato 3. Glaucoma e distacco della retinaentrambi hanno un aumento della concentrazione di glutammato 4-6. Glutammato in condizioni fisiologiche è rilevato come un trasmettitore eccitatorio tutto il sistema nervoso centrale e della retina interna 7,8. Elevati livelli di glutammato sono stati trovati non solo in glaucoma e distacco della retina 5,6 ma anche in retinopatia diabetica proliferativa 9. Un aumento glutammato eventualmente porta alla eccitotossicità e, di conseguenza, il danno delle cellule nervose 10. Nella maggior parte dei casi di distacco della retina e in alcuni casi di chirurgia proliferative retinopatia diabetica sulla retina (pars plana vitrectomia) sono necessari. Durante pars plana vitrectomia manipolazione meccanica, luce brillante della fibra ottica o sollecitazione di taglio esercitata da elevate portate di soluzioni per l&#39;irrigazione durante lunghi operazioni esercitare un ulteriore stress sulla retina 11,12. Tutte le malattie di cui hanno in comune che la patologia è localizzato al Retinun solo e posa la comunità oftalmologico con la sfida di trovare il modo di proteggere la retina come un sistema neurosensoriale. L&#39;elettroretinogramma (ERG) è il metodo standard per la valutazione della funzione in vivo fotorecettore (a-onda) e la funzione della retina interna (b-onda). L&#39;ERG è misurata con argento-elettrodi introdotti nella cornea e gli occhi sono stimolata da un crescente livello di luce per rilevare i difetti di barre o coni o nella retina interna. Diversi difetti nella retina possono essere rilevate da cambiamenti di ampiezza (la forza della risposta) o la latenza (la risposta-intervallo-to-) della ERG. Diverse ERG protocollo e metodi di misurazione (pattern-ERG, multifocale-ERG o campo chiaro ERG) permettono un&#39;ulteriore differenziazione dei difetti. La tecnica della retina isolato è stato introdotto recentemente, consentendo di valutare gli effetti sulla retina senza interferenze da esempio un animale di studioreazioni generali 13,14. E &#39;stato lo scopo di questo studio per valutare e introdurre un modello di stress definito e standardizzato per ipossia e lo stress glutammato sulla retina isolata superfused. Quindi, speriamo di gettare le basi per futuri studi sugli effetti neuroprotettivi di alcuni agenti o soluzioni di irrigazione intraoculari.

<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

Neuroprotezione è stato un forte campo di indagine nella ricerca oculistico negli ultimi decenni e colpisce malattie come il glaucoma, occlusione vascolare retinica, distacco della retina, e la retinopatia diabetica. E &#39;stato l&#39;oggetto di questo studio di introdurre un modello di stress test standardizzato per il futuro terapeutico preclinica. Retine bovini sono stati preparati e perfusi con una soluzione standard di ossigeno satura, e l&#39;ERG è stato registrato. Dopo la registrazione di b-onde stabili, ipossia (puro N 2) o di stress glutammato (250 micron glutammato) è stata esercitata per 45 min. Per studiare gli effetti sulla funzione dei fotorecettori da solo, 1 aspartato mM è stato aggiunto per ottenere un-onde. ERG-recupero è stato monitorato per 75 min. Per ipossia, una diminuzione in onda ampiezza del 87,0% è stato osservato (p &lt;0.01) dopo un tempo di esposizione di 45 min (diminuzione del 36,5% dopo la fine del washout p = 0,03). Inoltre, un dim inizialefacilità di ampiezze b-onda di 87.23% è stato registrato, che ha raggiunto la significatività statistica (p &lt;0.01, diminuzione del 25,5% alla fine del lavaggio, p = 0,03). Per 250 micron glutammato, una prima riduzione di 7,8% ampiezze un onda (p&gt; 0.05) seguita da una riduzione di 1,9% (p&gt; 0.05). Una riduzione del 83,7% delle ampiezze b-onda (p &lt;0,01) è stato osservato; dopo un lavaggio di 75 min la riduzione è stata del 2,3% (p = 0.62). In questo studio, un modello standardizzato di stress è presentato che può essere utile per identificare i possibili effetti neuroprotettivi in ​​futuro.

Dopo 1 ora di perfusione dei preparati della retina con una soluzione standard di ossigeno-saturi (Figure 1A e B) ERG-ampiezze mostravano stabilizzazione e meno variazione ampiezze tra singole misurazioni. pH, pressione osmotica, la temperatura, e pO 2 (tranne per il test ipossia) sono state mantenute costanti per tutte le prove. Per isolare il segnale fotorecettore dal segnale della retina interna, 1 mM aspartato è stato aggiunto alla soluzione standard per sopprim...

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

Con questo studio, si introduce un modello di stress standardizzato per l&#39;isolato retina superfused bovina per futuri test terapeutici preclinica. L&#39;effetto di una ipossia (puro N 2) o di stress glutammato (250 uM glutammato) sulla funzione della retina rappresentato da a- e ampiezze b-onda è stata valutata.

Glutammato e ipossia come modello di stress per l&#39;isolato perfuso vertebrati 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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7.8K Views.  University Eye Hospital Tübingen.  Con questo studio, si introduce un modello di stress standardizzato per l'isolato retina superfused bovina per futuri test terapeutici preclinica. L'effetto di una ipossia (puro N 2) o di stress glutammato (250 uM glutammato) sulla funzione della retina rappresentato da a- e ampiezze b-onda è stata valutata. 

Video: 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.

Sviluppo di un unilateralmente lesionata 6-OHDA modello murino della malattia di Parkinson

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

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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.

The Social Trier Protocollo di prova dello stress per indurre stress psicologico

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.

Biomarcatori in un modello animale per Rivelare correlati neurali, ematologiche, e comportamentali di PTSD

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.

Gli<em&gt; In ovo</em&gt; CAM-saggio come Xenograftmodel per 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 ...

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.

Distacco della retina Modello in Roditori da iniezione sottoretinico di ialuronato di sodio

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.

Eccitotossica stimolazione del cervello Microslices come<em&gt; In vitro</em&gt; Modello di Corsa

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.

Isolata epatica perfusione come Trattamento di metastasi epatiche di melanoma uveale

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 trombotica Stroke modello basato sulla transitoria cerebrale ipossia-ischemia

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 modello per simulare ipossia clinicamente rilevanti negli esseri umani

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.

Misurazione di contrattilità isometrica dell'arteria mesenterica Mouse con filo Myography

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