Tutti i ratti sono stati utilizzati secondo i protocolli approvati dal Comitato sperimentale sugli animali del Beijing Friendship Hospital, Capital Medical University.
1. Preparazione chirurgica, anestetizzazione e tecniche chirurgiche
NOTA: Un totale di 40 femmine di ratto Wistar, del peso di 200-220 g, sono stati ottenuti commercialmente per il presente studio. Dei 40 ratti, 10 sono stati selezionati casualmente come gruppo di controllo e il resto è stato trattato come gruppo di test. Tutti gli animali sono stati ospitati in un ambiente sterile nelle strutture per animali del Beijing Friendship Hospital, Capital Medical University.
<ol><li>Eseguire l'anestesia generale somministrando pentobarbital di sodio per via intraperitoneale (40 mg/kg). In alternativa, indurre l'anestesia usando isoflurano al 3% -4% e mantenerla all'1% -3% (inalato). Applicare unguento oftalmico sugli occhi per prevenire la secchezza. Quindi, posizionare il ratto sulla piattaforma chirurgica e fornire supporto termico. NOTA: Somministrare analgesici come buprenorfina, 0,05 mg/kg, SC, 0,1-0,2 mL all'inizio della procedura.</li>
	<li>Controllare la profondità dell'anestesia dalla mancanza di risposta al pizzico della punta. Rasare la pelliccia da tutta la zona posteriore con un rasoio.</li>
	<li>Sterilizzare il sito chirurgico con almeno 3 cicli di uno scrub a due stadi come clorexidina o iodio povidone seguito da alcool isopropilico. Fissare gli arti con nastro chirurgico e fare un'incisione mediana di circa 3 cm sulla schiena con forbici chirurgiche.</li>
	<li>Approfondisci l'incisione attraverso i tessuti sottocutanei usando le forbici chirurgiche e taglia i muscoli attaccati alla colonna vertebrale.</li>
	<li>Identificare ed esporre visivamente la 13a costola (lo spazio intervertebrale collegato aquella costola è l'intervallo T13\u2012L1). Segna la 13acostola usando una sutura.</li>
	<li>Dopo l'identificazione, resecare attentamente i muscoli attaccati alla colonna vertebrale ed esporre la colonna vertebrale. Resecare il legamento sopraspinoso e il legamento interspinoso per un'accurata identificazione della colonna vertebrale. Esporre il livello di L4\u2012L5 con forbici chirurgiche e pinze. NOTA: Il legamento sopraspinoso può essere identificato facilmente a causa della presenza di tessuto sottocutaneo sottile. Dopo la resezione del legamento sopraspinoso, il legamento tra il processo spinoso è il legamento interspinoso.</li>
	<li>Sezionare attentamente il processo spinoso vertebrale L4\u2012L5 e parti del processo trasversale usando una pinza Kelly per esporre il midollo spinale (Figura 1).</li>
	<li>Esporre completamente il cono midollare al livello L4\u2012L5 e transectare il midollare del cono totalmente con le forbici per iridectomia. Inserire un imballaggio di tessuto per bloccare il recupero del midollo spinale.</li>
	<li>Chiudere il muscolo e la pelle sovrastanti sullo strato esterno della pelle utilizzando una sutura non assorbibile 4-0.</li>
	<li>Per il gruppo di controllo, eseguire i passaggi 1.1\u20121.7 e lasciare intatto il cono midollare. Chiudere l'incisione secondo il punto 1.9.</li>
</ol>2. Recupero degli animali
<ol><li>Tenere i ratti in un'incubatrice a temperatura controllata (37 °C) durante la prima ora dopo l'operazione e monitorarli fino a quando non sono sternali o in movimento attivo. NOTA: Ci vuole circa mezz'ora per il recupero totale.</li>
	<li>Trasferire l'animale in una gabbia pulita con cibo e acqua sufficienti. Tieni i ratti in gabbie separate. NOTA: Il successo della transezione è indicato quando i ratti nel gruppo di prova si muovono solo con l'aiuto delle zampe anteriori, mentre i ratti nel gruppo di controllo potrebbero camminare normalmente.</li>
</ol>3. Gestione post-operatoria
<ol><li>Iniettare penicillina G, un antibiotico (50.000 U/ml per animale) per via intraperitoneale. Somministrare analgesici come buprenorfina, 0,05 mg/kg, SC, 0,1-0,2 ml ogni 6-12 ore per 48 ore dopo l'intervento.</li>
	<li>Comprimere la vescica urinaria nell'ipogastrio per aiutare con lo svuotamento. Eseguilo due volte al giorno alla stessa ora (8 am e 8 pm) per sei settimane. NOTA: La perdita della normale costrizione del detrusore è il simbolo dell'uranio impoverito.</li>
	<li>Ospitare tutti i ratti in gabbie metaboliche, ciascuna contenente un imbuto di raccolta delle urine posto su una carta assorbente precedentemente pesata per monitorare la minzione e l'incontinenza.</li>
	<li>Raccogliere e annotare separatamente la variazione di peso della carta assorbente, che indica separatamente il volume vuoto (VV) e il volume residuo di urina.</li>
</ol>4. Test urodinamici
<ol><li>A sei settimane dopo l'operazione, eseguire un cistometrogramma, utilizzando apparecchiature di misurazione urodinamica come segue.<ol><li>Anestetizzare i ratti iniettando il 10% di cloralio idrato nella cavità peritoneale (3 ml/kg).</li>
		<li>Comprimere la vescica per lo svuotamento, quindi fissare il ratto alla piattaforma chirurgica usando un nastro.</li>
		<li>Inserire il catetere epidurale (3F) nella vescica e collegare l'apparecchiatura di misurazione urodinamica, il catetere epidurale e la pompa per infusione tramite il tubo a tre arti.</li>
		<li>Pompare soluzione fisiologica salina ad una velocità di 0,2 ml/min per la misurazione urodinamica (vedi Tabella dei materiali). Registrare l'MCC e il DOP e la conformità della vescica (calcolata dividendo δ volume della vescica con δ pressione del detrusore).</li>
	</ol></li>
</ol>5. Analisi statistica
<ol><li>Eseguire analisi statistiche utilizzando software disponibili in commercio.</li>
	<li>Utilizzare il test di Kolmogorov-Smirnov per testare la normalità dei dati.</li>
	<li>Esprimere le variabili normalmente distribuite come valori medi con deviazioni standard. Utilizzare i test t di Student accoppiati a due code per confrontare i parametri del cistometrogramma in entrambi i gruppi. NOTA: p < 0,05 indica che la differenza aveva rilevanza statistica.</li>
</ol>

<ol>
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L'uranio impoverito è una causa comune di sintomi del tratto urinario inferiore sia negli uomini che nelle donne. È una costellazione complessa di sintomi con poche opzioni di trattamento che possono ridurre significativamente la qualità della vita (Qol) delle persone colpite18. Sebbene si ritenga che l'uranio impoverito sia multifattoriale, la comprensione della sua patogenesi rimane rudimentale. Gli studi hanno dimostrato che la patogenesi dell'uranio impoverito potrebbe essere correlata a fa...

L'underactivity del detrusore (DU) è una tipica disfunzione del tratto urinario inferiore che è rimasta sotto studiata. Anche se l'uranio impoverito è stato definito dall'International Continence Society (ICS)1, vengono utilizzate numerose terminologie diverse per riferirsi a questa malattia, ad esempio "insufficienza del detrusore", "vescica acontrattile", "areflessia del detrusore"2. L'uranio impoverito, come definito dall'International Continence Society (ICS) nel 2002, è una contrazione di forza e durata ridotte, che si traduce in un aumento prolungato del tempo per lo svuotamento della vescica, con conseguente incapacità di raggiungere lo svuotamento completo della vescica entro un periodo normale.
L'uranio impoverito può colpire il 48% degli uomini e il 12% delle donne (di età compresa >70 anni)3 con sintomi del tratto urinario inferiore. Sembra essere multifattoriale e non esiste un trattamento efficace. È stato riportato che l'uranio impoverito è onnipresente nei pazienti con disfunzione neurogena della vescica, come la sclerosi multipla4, il diabete mellito5, il morbo di Parkinson6 o l'ictus cerebrale7. L'uranio impoverito può anche essere causato da danni ai nervi iatrogeni, come l'isterectomia laparoscopica, la prostatectomia o altri interventi chirurgici nella piccola pelvi8. I cambiamenti fisiopatologici e i trattamenti disponibili dell'uranio impoverito sono ancora confusi a causa della mancanza di un modello animale appropriato per lo studio.
Il riflesso della minzione è controllato da vie spino-bulbospinali che combinano il centro di minzione pontina, il nucleo parasimpatico sacrale e i centri della corteccia più anziani9. L'attivazione e il mantenimento del riflesso della minzione dipendono principalmente dal trasporto regolare dei segnali sensoriali dalla vescica ai centri della corteccia più anziani. Si può postulare che la disfunzione sensoriale contribuisca all'uranio impoverito.
La maggior parte degli studi sperimentali sugli animali relativi alle disfunzioni del tratto urinario inferiore si sono concentrati su modelli di vescica iperattiva (OAB)10. Questi modelli forniscono una ragionevole comprensione della fisiopatologia e della prognosi dell'OAB. Tuttavia, sono stati riportati solo pochi modelli di uranio impoverito, ad esempio lesioni sopraspinali (lesioni locali, decerebrazione e occlusione dell'arteria cerebrale media), trassezione del midollo spinale o lesione da contusione, somministrazione sistemica (ad es. ciclofosfamide) o intravescicale di agenti irritanti o infiammatori (ad es. acido, acroleina e lipopolisaccaride)11,12,13,14 . Tra questi metodi, solo il metodo di transezione del midollo spinale o lesione da contusione può essere utilizzato per stabilire un modello animale di DU13. I tentativi di lesione del centro di minzione pontino e dei centri della corteccia superiore sono stati abbandonati a causa del grave trauma. Quindi, viene prestata maggiore attenzione per trovare una posizione accurata nel centro del riflesso della minzione per indurre l'uranio impoverito con effetti collaterali minimi.
Come accennato in precedenza, uno dei meccanismi di induzione dell'uranio impoverito è quello di danneggiare il midollo spinale per danneggiare la via di segnalazione del riflesso della minzione. Il metodo di caduta del peso di Allen è stato sviluppato per stabilire animali da laboratorio con midollo spinale ferito15. Tuttavia, non sono disponibili ulteriori dati sperimentali su questo metodo. Inoltre, poiché parti degli animali hanno recuperato la funzione spinale dopo l'ictus senza DU, non può essere considerato un metodo perfetto per generare un modello animale di uranio impoverito16.
Nel 1987, Bregman ha escogitato un processo di transetto del midollo spinale per generare il modello animale all'uranio impoverito e ha acquisito dati sperimentali17. Tuttavia, questo metodo non è stato applicato per stabilire il modello animale all'uranio impoverito. A quel tempo, i ricercatori erano ancora confusi sulla patogenesi dell'uranio impoverito. Poiché le posizioni nel midollo spinale associate all'induzione di OAB o DU sono adiacenti l'una all'altra, non sono state in grado di trovare il sito preciso del danno al midollo spinale per indurre DU17. OAB e DU sono stati introdotti insieme o separatamente con questo metodo. Quindi, sebbene questo metodo abbia introdotto l'uranio impoverito, era impreciso e non poteva essere utilizzato per la comprensione dell'occorrenza e dell'elaborazione dell'uranio impoverito.
Come detto sopra, la mancanza di un modello animale adatto di uranio impoverito è uno dei principali ostacoli per lo studio dell'uranio impoverito. I ricercatori sono continuamente alla ricerca di un modello accurato e gestibile in grado di simulare la patologia dell'uranio impoverito. Anche le opzioni di trattamento per l'uranio impoverito non sono migliorate significativamente negli ultimi 20 anni. Collettivamente, c'è un grande bisogno di descrivere un protocollo standard per stabilire un modello animale di uranio impoverito.
Quindi, in questo articolo, descriviamo un metodo per stabilire con successo un modello di ratto di uranio impoverito mediante transezione del cono midollare. La transezione è stata eseguita a livello L4\u2012L5 per separare il cono midollare. La capacità cistometrica massima (MCC), la pressione di apertura del detrusore (DOP) e la conformità della vescica sono state registrate e analizzate per convalidare il protocollo. Il protocollo indicato di seguito combina fattibilità e affidabilità in modo standardizzato per stabilire il modello animale di uranio impoverito, simulando la presenza e l'elaborazione dell'uranio impoverito. Il protocollo può essere utilizzato come tecnica per ulteriori studi sull'uranio impoverito.

<table>
	<tbody>
		<tr>
			<td>0.9% saline</td>
			<td>Wuhan Prosai Company</td>
			<td>EY-C1178</td>
			<td>pump for urodynamic measurement</td>
		</tr>
		<tr>
			<td>10% chloral hydrate</td>
			<td>Shandong Yulong Co., Ltd</td>
			<td>H37022673</td>
			<td>3mL/kg&#65292;administered intraperitoneally</td>
		</tr>
		<tr>
			<td>Buprenorphine Hydrochloride Injection</td>
			<td>Tianjin Pharmaceutical Research Institute Pharmaceutical Co. LTD</td>
			<td>H12020275</td>
			<td>0.05mg/kg subcutaneously 24h and 48h postoperation</td>
		</tr>
		<tr>
			<td>Epidural Catheter</td>
			<td>Shandong Xinghua Co, Ltd</td>
			<td>VABR3L</td>
			<td>for urodynamic measurement</td>
		</tr>
		<tr>
			<td>Penicillin G</td>
			<td>Alta Technology Co., Ltd</td>
			<td>1ST5637</td>
			<td>50,000 unit/ml per animal</td>
		</tr>
		<tr>
			<td>pentobarbital</td>
			<td>Beijing solabo Technology Co., Ltd</td>
			<td>NK-WF0001</td>
			<td>40 mg/kg, administered intraperitoneally</td>
		</tr>
		<tr>
			<td>Suture line(4-0)</td>
			<td>ETHICON</td>
			<td>VCP422H</td>
			<td>suture the injury</td>
		</tr>
		<tr>
			<td>Three-limb tube</td>
			<td>Shandong Xinghua Co, Ltd</td>
			<td>VAB3T</td>
			<td>for urodynamic measurement</td>
		</tr>
		<tr>
			<td>Trace infusion pump</td>
			<td>Zhejiang Smith Medical Instrument Co., Ltd</td>
			<td>20162540335</td>
			<td>Pump the saline at a speed of 0.2ml/min for urodynamic measurement</td>
		</tr>
		<tr>
			<td>Urodynamic measurement equipment</td>
			<td>Medical Measurement SystemsB.V.</td>
			<td>08-0467</td>
			<td>urodynamic measurement equipment can not only help the diagnosis of dysuria, but also provide objective materials for treatment and therapeutic effect. It is the most commonly used examination method in clinical diagnosis and treatment of lower urinary tract functional diseases</td>
		</tr>
		<tr>
			<td>Wistar Rats</td>
			<td>HFK Biotechnology Co.Ltd,Beijing ,China</td>
			<td>SCXK2012-0023</td>
			<td>200-220g</td>
		</tr>
	</tbody>
</table>

detrusor underactivity model in rats by conus medullaris transection

L'obiettivo del protocollo presentato era quello di stabilire un modello di sottoattività del detrusore (DU) nel ratto attraverso la transezione del cono midollare. La laminectomia è stata eseguita in un totale di 40 ratti Wistar femmina (gruppo di controllo: 10 ratti; gruppo di test: 30 ratti) del peso di 200-220 g, e il cono midollare è stato transettato al livello L4-L5 nel gruppo di test. Tutti i ratti sono stati alloggiati e nutriti nelle stesse condizioni ambientali per sei settimane. Nel gruppo di test, lo svuotamento delle urine è stato eseguito due volte al giorno per sei settimane ed è stato registrato il volume medio residuo di urina. Un cistometrogramma è stato eseguito in entrambi i gruppi. Sono stati registrati e calcolati la capacità cistometrica massima (MCC), la pressione di apertura del detrusore (DOP) e la conformità della vescica. Il gruppo di test ha mostrato una significativa ritenzione urinaria dopo l'intervento chirurgico, sia durante che dopo lo shock spinale. Tuttavia, nessuna anomalia è stata osservata nel gruppo di controllo. Rispetto al gruppo di controllo, l'MCC e la conformità della vescica nel gruppo di prova erano significativamente superiori a quelli del gruppo di prova (3,24 ± 2,261 ml contro 1,04 ± 0,571 ml; 0,43 ± 0,578 ml / cmH 2 O contro 0,032 ± 0,016 ml / cmH 2 O), mentre il DOP nel gruppo di prova era inferiore al controllo (20,28 ± 14,022 cmH 2 O contro 35 ± 13,258 cmH2 O). Questo metodo per stabilire un modello animale di uranio impoverito mediante la transezione del cono midollare offre un'eccellente opportunità per comprendere meglio la fisiopatologia dell'uranio impoverito.

L'intera procedura della transezione del cono midollare può essere completata entro 45 minuti da chirurghi esperti. Il nostro laboratorio ha eseguito oltre 100 casi di interventi chirurgici di transezione del cono midollare. Il tasso di successo è superiore al 95%, come definito dalla sopravvivenza dei ratti e dall'induzione riuscita dell'uranio impoverito. Il test urodinamico ha confermato l'induzione dell'uranio impoverito.
Sulla base della nostra esperienza, l'induzione dell'uranio impove...

Watch this Scientific Journal Video about Detrusor Underactivity Model in Rats by Conus Medullaris Transection at JoVE.com

Detrusor Underactivity Model in Rats by Conus Medullaris Transection

Presentiamo un metodo per stabilire un modello di sottoattività del detrusore mediante transezione del conus medullaris nei ratti. La sottoattività del detrusore è stata stimolata con successo in questi animali. Il modello può essere utilizzato per studiare la funzione del tratto urinario.

Modello di sottoattività del detrusore nei ratti mediante transezione di Conus medullaris

The goal of the presented protocol was to establish a detrusor underactivity (DU) model in the rat through conus medullaris transection. Laminectomy was ...

detrusor-underactivity-model-in-rats-by-conus-medullaris-transection

Research

JoVE Journal

Neuroscience

2.0K Views.  Capital Medical University. Presentiamo un metodo per stabilire un modello di sottoattività del detrusore mediante transezione del conus medullaris nei ratti. La sottoattività del detrusore è stata stimolata con successo in questi animali. Il modello può essere utilizzato per studiare la funzione del tratto urinario.

Video: Detrusor Underactivity Model in Rats by Conus Medullaris Transection

Optic Nerve Transection: A Model of Adult Neuron Apoptosis in the Central Nervous System

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve 
transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous
 chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals 
through the vitreous fluid ensures that they act upon the entire RGC population. Moreover, RGCs can be selectively transfected by applying short interfering RNAs 
(siRNAs), plasmids, or viral vectors to the cut end of the optic nerve 5-7 or injecting vectors into their target, the superior colliculus 8. 
This allows researchers to study apoptotic mechanisms in the desired neuronal population without confounding effects on other bystander neurons or surrounding glia. 
An additional benefit is the ease and accuracy with which cell survival can be quantified after injury. The retina is a flat, layered tissue and RGCs are localized in 
the innermost layer, the ganglion cell layer. The survival of RGCs can be tracked over time by applying a fluorescent tracer (3% Fluorogold) to the cut end of the 
optic nerve at the time of axotomy, or by injecting the tracer into the superior colliculus (RGC target) one week prior to axotomy. The tracer is retrogradely transported, labeling 
the entire RGC population. Because the ganglion cell layer is a monolayer (one cell thick), RGC densities can be quantified in flat-mounted tissue, without the need 
for stereology. Optic nerve transection leads to the apoptotic death of 90% of injured RGCs within 14 days postaxotomy 9-11. RGC apoptosis has a 
characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a time window for 
experimental manipulations directed against pathways involved in apoptosis.

Optic Nerve transection is a widely used model of adult CNS injury. Ninety percent of retinal ganglion cells (RGCs) whose axons are completely transected (axotomy) die within 14 days after axotomy. This model is easily amenable to experimental manipulations and highly reproducible.

Resezione del nervo ottico: un modello di apoptosi Neuron adulti nel Sistema Nervoso Centrale

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be ...

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Neuroscienze

Methods for Experimental Manipulations after Optic Nerve Transection in the Mammalian CNS

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals through the vitreous fluid ensures that they act upon the entire RGC population. Viral vectors, plasmids or short interfering RNAs (siRNAs) can also be delivered to the vitreous chamber in order to infect or transfect retinal cells 5-12. The high tropism of Adeno-Associated Virus (AAV) vectors is beneficial to target RGCs, with an infection rate approaching 90% of cells near the injection site 6, 7, 13-15. Moreover, RGCs can be selectively transfected by applying siRNAs, plasmids, or viral vectors to the cut end of the optic nerve 16-19 or injecting vectors into their target the superior colliculus 10. This allows researchers to study apoptotic mechanisms in the injured neuronal population without confounding effects on other bystander neurons or surrounding glia. RGC apoptosis has a characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a window for experimental manipulations directed against pathways involved in apoptosis. Manipulations that directly target RGCs from the transected optic nerve stump are performed at the time of axotomy, immediately after cutting the nerve. In contrast, when substances are delivered via an intraocular route, they can be injected prior to surgery or within the first 3 days after surgery, preceding the initiation of apoptosis in axotomized RGCs. In the present article, we demonstrate several methods for experimental manipulations after optic nerve transection.

Optic Nerve transection is a widely used model of adult CNS injury. This model is ideal for performing a number of experimental manipulations that target the retina globally or directly target the injured neuronal population of retinal ganglion cells.

Metodi per manipolazioni sperimentali dopo resezione del nervo ottico nel sistema nervoso centrale dei mammiferi

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be ...

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

Un metodo generale per la valutazione di incubazione della brama di saccarosio in ratti

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the use of IUE for investigating behavior or neuropathology in the adult brain has been limited by insufficient methods for monitoring of IUE transfection success by non-invasive techniques in postnatal animals. 
 For the present study, E16 rats were used for IUE. After intraventricular injection of the nucleic acids into the embryos, positioning of the tweezer electrodes was critical for targeting either the developing cortex or the hippocampus. 
 Ventricular co-injection and electroporation of a luciferase gene allowed monitoring of the transfected cells postnatally after intraperitoneal luciferin injection in the anesthetized live P7 pup by in vivo bioluminescence, using an IVIS Spectrum device with 3D quantification software. 
 Area definition by bioluminescence could clearly differentiate between cortical and hippocampal electroporations and detect a signal longitudinally over time up to 5 weeks after birth. This imaging technique allowed us to select pups with a sufficient number of transfected cells assumed necessary for triggering biological effects and, subsequently, to perform behavioral investigations at 3 month of age. As an example, this study demonstrates that IUE with the human full length DISC1 gene into the rat cortex led to amphetamine hypersensitivity. Co-transfected GFP could be detected in neurons by post mortem fluorescence microscopy in cryosections indicating gene expression present at &ge;6 months after birth.
 We conclude that postnatal bioluminescence imaging allows evaluating the success of transient transfections with IUE in rats. Investigations on the influence of topical gene manipulations during neurodevelopment on the adult brain and its connectivity are greatly facilitated. For many scientific questions, this technique can supplement or even replace the use of transgenic rats and provide a novel technology for behavioral neuroscience.

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

Genes can be manipulated during development of the cortex or the hippocampus of the rat via in utero electroporation (IUE) at E16, to enable fast and targeted modifications in neuronal connectivity for later studies of behavior or neuropathology in adult animals. Postnatal in vivo imaging for control of IUE success is performed by bioluminescence of activating co-transfected luciferase.

Generazione di topi transgenici per uso topico<em&gt; In utero</em&gt; Elettroporazione e<em&gt; In vivo</em&gt; Screening bioluminescenza

 In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the ...

Motor Nerve Transection and Time-lapse Imaging of Glial Cell Behaviors in Live Zebrafish

The nervous system is often described as a hard-wired component of the body even though it is a considerably fluid organ system that reacts to external stimuli in a consistent, stereotyped manner, while maintaining incredible flexibility and plasticity. Unlike the central nervous system (CNS), the peripheral nervous system (PNS) is capable of significant repair, but we have only just begun to understand the cellular and molecular mechanisms that govern this phenomenon. Using zebrafish as a model system, we have the unprecedented opportunity to couple regenerative studies with in vivo imaging and genetic manipulation. Peripheral nerves are composed of axons surrounded by layers of glia and connective tissue. Axons are ensheathed by myelinating or non-myelinating Schwann cells, which are in turn wrapped into a fascicle by a cellular sheath called the perineurium. Following an injury, adult peripheral nerves have the remarkable capacity to remove damaged axonal debris and re-innervate targets. To investigate the roles of all peripheral glia in PNS regeneration, we describe here an axon transection assay that uses a commercially available nitrogen-pumped dye laser to axotomize motor nerves in live transgenic zebrafish. We further describe the methods to couple these experiments to time-lapse imaging of injured and control nerves. This experimental paradigm can be used to not only assess the role that glia play in nerve regeneration, but can also be the platform for elucidating the molecular mechanisms that govern nervous system repair.

Although the peripheral nervous system (PNS) is capable of significant repair after injury, little is known about the cellular and molecular mechanisms that govern this phenomenon. Using live, transgenic zebrafish and a reproducible nerve transection assay, we can study dynamic glial cell behaviors during nerve degeneration and regeneration.

Motor Nerve transezione e Time-lapse imaging di gliali comportamenti delle cellule in vivo Zebrafish

The  nervous system is often described as a hard-wired component of the body even  though it is a considerably fluid organ system that reacts to external ...

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6&#160;weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2&#160;days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days post-injury.

Spinal Cord transezione nel larvale Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

A Lateralized Odor Learning Model in Neonatal Rats for Dissecting Neural Circuitry Underpinning Memory Formation

Rat pups during a critical postnatal period (&#8804; 10 days) readily form a preference for an odor that is associated with stimuli mimicking maternal care. Such a preference memory can last from hours, to days, even life-long, depending on training parameters. Early odor preference learning provides us with a model in which the critical changes for a natural form of learning occur in the olfactory circuitry. An additional feature that makes it a powerful tool for the analysis of memory processes is that early odor preference learning can be lateralized via single naris occlusion within the critical period. This is due to the lack of mature anterior commissural connections of the olfactory hemispheres at this early age. This work outlines behavioral protocols for lateralized odor learning using nose plugs. Acute, reversible naris occlusion minimizes tissue and neuronal damages associated with long-term occlusion and more aggressive methods such as cauterization. The lateralized odor learning model permits within-animal comparison, therefore greatly reducing variance compared to between-animal designs. This method has been used successfully to probe the circuit changes in the olfactory system produced by training. Future directions include exploring molecular underpinnings of odor memory using this lateralized learning model; and correlating physiological change with memory strength and durations.

This protocol introduces lateralized early odor preference learning in rats using acute single naris occlusion. Lateralized learning permits the examination of behavioral outcomes and underpinning biological mechanisms within the same animals, reducing variance induced by between-animal designs. This protocol can be used to investigate molecular mechanisms underpinning early odor learning.

Un laterizzazione Odore Learning Modello in ratti neonati per dissezione Formazione Neural circuiti Sostenere la memoria

Rat pups during a critical postnatal period (&#8804; 10 days) readily form a preference for an odor that is associated with stimuli mimicking maternal ...

Partial Optic Nerve Transection in Rats: A Model Established with a New Operative Approach to Assess Secondary Degeneration of Retinal Ganglion Cells

Previous studies have shown that the secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. Partial optic nerve transection is considered a useful and reproducible model. Compared with other optic nerve injury models used commonly for assessing secondary degeneration, e.g. complete optic nerve transection and optic nerve crush models, the partial optic nerve transection model is superior as it distinguishes primary from secondary degeneration in situ. Therefore, it serves as an excellent tool for evaluating secondary degeneration. This study describes a novel operative approach of partial optic nerve transection by directly accessing the area of the retrobulbar optic nerve through the orbital lateral wall of the eyeball. Moreover, we present a newly designed, low cost surgical instrument to assist with transection. As demonstrated by the representative results in distinguishing the boundary of primary and secondary injury areas, the new approach and instrument ensures high efficiency and stability of the model by providing adequate space for surgical operation. This in turn makes it easy to separate the meningeal sheath and ophthalmic vessels from the optic nerve before transection. An additional benefit is that this space-saving operative approach improves the investigators&#39; ability to administer drugs, carriers, or selective RGC tracers to the stump of the partially transected optic nerve, allowing the exploration of mechanisms behind secondary injury in RGCs, in a new way.

Secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. This study describes an innovative operative approach for partial optic nerve transection. The use of this space-saving operative approach extends the model's application range, and allows exploration of secondary injury mechanisms in RGCs in a new way.

Transection parziale del nervo ottico in ratti: un modello stabilito con un nuovo approccio operativo per valutare la degenerazione secondaria delle cellule ganglionari retiniche

Previous studies have shown that the secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. Partial optic nerve transection ...

Sub-acute Cerebral Microhemorrhages Induced by Lipopolysaccharide Injection in Rats

Cerebral microhemorrhages (CMHs) are common in aged patients and are correlated to various neuropsychiatric disorders. The etiology of CMHs is complex, and neuroinflammation is often observed as a co-occurrence. Here, we describe a sub-acute CMHs rat model induced by lipopolysaccharide (LPS) injection, as well as a method for detecting CMHs. Systemic LPS injection is relatively simple, economical, and cost-effective. One major advantage of LPS injection is its stability to induce inflammation. CMHs caused by LPS injection could be detected by gross observation, hematoxylin and eosin (HE) staining, Perl&#39;s Prussian staining, Evans blue (EB) double-labeling, and magnetic resonance imaging-susceptibility weighted imaging (MRI-SWI) technology. Finally, other methods of developing CMHs animal models, including their advantages and/or disadvantages, are also discussed in this report.

We present a protocol to induce and detect CMHs caused by LPS injection in Sprague-Dawley rats, which may be utilized in future research investigations on the pathogenesis of CMHs.

Sub-acuta microemorragie cerebrale indotta tramite l'iniezione del Lipopolysaccharide in ratti

Cerebral microhemorrhages (CMHs) are common in aged patients and are correlated to various neuropsychiatric disorders. The etiology of CMHs is complex, ...

A Preclinical Model to Assess Brain Recovery After Acute Stroke in Rats

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery occlusion/reperfusion (MCAO/R) model in male Sprague-Dawley rats was chosen. By changing the rat&#39;s weight (260&#8722;330 g), the thread bolt type (2636/2838/3040/3043) and the brain infarct time (2-3 h), a higher Longa&#39;s score, a larger infarct volume and a greater model success ratio were screened using the Longa&#39;s score and TTC staining. The optimum model condition (300 g, 3040 thread bolt, 3 h brain infarct time) was acquired and used in a 1-90 day observation period after reperfusion via assessment of sensorimotor functions and infarct volume. At these conditions, the bilateral asymmetry test had a significant difference from 1 to 90 days, and the grid-walking test had a significant difference from 1 to 60 days; both differences could be a suitable sensorimotor functional test. Thus, the most appropriate condition of a novel rat model in the recovery and sequela stages of brain ischemia was found: 300 g rats that underwent MCAO with a 3040 thread bolt for a 3 h brain infarct and then reperfused. The appropriate sensorimotor functional tests were a bilateral asymmetry test and a grid-walking test.

The&#160;purpose of this study is to establish and validate an animal model for research in the recovery and sequela stages of brain ischemia by testing brain infarction and sensorimotor function after middle cerebral artery occlusion/reperfusion (MCAO/R) after 1-90 days in rats.

Un modello preclinico per valutare il recupero del cervello dopo l'ictus acuto nei ratti

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery ...