Il presente studio è stato sostenuto da sovvenzioni del Consiglio Nazionale della Scienza al Dott Bai-Chuang Shyu (NSC 99-2320-B-001-016-MY3, NSC 100-2311-B-001-003-MY3, e NSC 102-2320- B-001-026-MY3). Questo lavoro è stato condotto presso l&#39;Istituto di Scienze Biomediche, che ha ricevuto finanziamenti da Academia Sinica.

Il protocollo in questo studio ha ricevuto l&#39;approvazione dalla Sinica Istituzionali Animal Care Academia e del Comitato utilizzo a Taiwan. 1. preparati per animali <ol><li> Ottenere ratti maschi Sprague-Dawley (circa 300 - 400 g). Mantenere i ratti in una stanza con aria condizionata (21 - 22 ° C, 50% di umidità) sotto un / ciclo di 12 ore 12 ore di luce / buio (luce accesa alle 8:00 AM) con libero accesso a cibo e acqua. </li></ol> 2. Procedura sperimentale <ol><li> Consentire a tutti i ratti per adattarsi all&#39;ambiente nelle loro gabbie a casa per 1 settimana prima degli esperimenti. Durante l&#39;adattamento, eseguire la von Frey e prove di plantari per stabilire linee di base. </li><li> von Frey test <ol><li> Posizionare il ratto in una custodia acrilica (30 cm x 30 cm x 80 cm) per 30 min per abitazione. </li><li> Ottenere filamenti von Frey (filamento no 11 -. 20) che hanno la stessa lunghezza ma variando diametri di fornire una gammadelle forze di 2 - 100 g. </li><li> Per valutare la soglia paw recesso hindpaw del ratto, utilizzare filamenti von Frey per stimolare centro delle hindpaws tramite una porta a rete sulla piastra acrilica ad intervalli tra le prove 5 min. Per registrare la massima pressione applicata, utilizzare i filamenti in ordine crescente, dal basso verso l&#39;alto, fino a quando viene registrata la massima pressione applicata. 10 </li><li> Quando i topi mostrano una risposta zampa di ritiro alla stimolazione, registrare il numero filamento. Determinare la risposta recesso in questo processo secondo stimolo più basso. </li><li> Ripetere la stessa procedura immediatamente, per un totale di tre volte consecutive sullo stesso ratto. Convertire il numero filamento nel corrispondente forza (in grammi) e la media dei valori. </li></ol></li><li> plantare test <ol><li> Posizionare il topo in una scatola di plexiglas trasparente (diviso in quattro fotogrammi, 80 centimetri × 30 cm x 15 cm) per 30 minuti per l&#39;abitazione. </li><li> Utilizzare un inffascio NOMINALE per stimolare il centro della hindpaw attraverso una lastra di vetro. Regolare l&#39;intensità della luce infrarossa per ottenere una latenza media di risposta zampa il ritiro di circa 10 sec. Effettuare una prova premendo un tasto che accende la sorgente di luce infrarossa e avvia un timer a stato solido digitale. Manipolare la durata del fascio di luce infrarossa. </li><li> Registrare la durata della luce infrarossa quando i topi mostrano una risposta paw recesso. La più lunga durata non deve superare i 20 secondi in ogni prova al fine di evitare danni ai tessuti. Utilizzare un intervallo tra le prove di almeno 5 minuti per evitare la stimolazione successiva. </li><li> Ripetere il test con tre prove per le hindpaws destra e sinistra, e calcolare la media per ogni hindpaw per ogni ratto. </li></ol></li><li> Chirurgia Collagenasi Lesione <ol><li> Anestetizzare il topo con il 4% isoflurano fino alla perdita della risposta toe-pinch e le risposte somatiche a verificarsi stimoli chirurgici. Mantenere l&#39;anestesia con 1,5-2% isoflurane per la durata dell&#39;intervento. </li><li> Posizionare il topo in un dispositivo stereotassico con una semplice piastra elettrica per mantenere la temperatura corporea a 36,5-37,5 ° C. Applicare crema per gli occhi sugli occhi per prevenire la secchezza mentre sotto anestesia. </li><li> Accorciare il pelo con un rasoio elettrico sterilizzati, e fare una incisione liscio (circa 2-2,5 cm) con un bisturi lungo la linea mediana del cuoio capelluto. Pulire la pelle e il cranio con alternanza di provvidenza iodio e soluzione alcolica al 75% per la disinfezione. Durante la fase chirurgica, utilizzare il 75% di alcool per sterilizzare tutti i dispositivi, strumenti, e il banco di lavoro per mantenere condizioni di sterilità. Prima di allora, tutti i materiali chirurgici devono essere sterilizzati in autoclave a vapore. </li><li> Utilizzare i guanti e strumenti sterili, e praticare un piccolo foro (3 mm di diametro) nel cranio con un trapano elettrico su VB (compresi ventrale nucleo postero-talamo [VPM] e ventrale postero nucleo talamo [VPL]) del talamo (3.0 -3.5 mm posteriormente, 3,0-3,5 mm lateralial bregma, e 5,5-5,8 mm di profondità dalla superficie del cranio). </li><li> Microinject 0,5 ml di soluzione fisiologica o 0,125 U di tipo 4 soluzione di collagenasi nel controllo e gruppi sperimentali, rispettivamente. </li><li> Mantenere l&#39;ago di iniezione in posizione per altri 5 minuti per consentire la diffusione del farmaco. </li><li> Utilizzare cemento dentale per riempire il buco nel cranio, e suturare l&#39;incisione. Dopo l&#39;incisione di sutura, applicare le anestetici locali (lidocaina analgesici unguento) per la ferita, e restituirli alla loro gabbie casa. </li><li> Dopo l&#39;intervento chirurgico, singolarmente ospitare i ratti in gabbie di plastica fino a quando mantengono decubito sternale, e mantiene caldo fino recuperato da anestesia. </li></ol></li><li> Test comportamentali dopo il recupero chirurgico <ol><li> Dopo 7 giorni di recupero da un intervento chirurgico, ripetere le procedure di cui ai punti 2.2 e 2.3 per la fase di test. Eseguire i test comportamentali per 4 settimane durante il monitoraggio della salute e lo stato di sviluppo degli animali. </li><li&gt; Controllare le condizioni sanitarie degli animali (ad esempio, il peso corporeo, quantità di alimentazione, e libera circolazione) tra il controllo e gruppi sperimentali per la fase post-intervento chirurgico. </li></ol></li><li> Eseguire la cannula Impianto con PE-50 Tubing <ol><li> Anestetizzare ratto con isoflurano 4%, e mantenere la temperatura corporea 36,5 - 37,5 ° C con una semplice piastra elettrica. </li><li> Con un bisturi, tagliare due fori (cm diametro 2 ciascuno) sulla linea mediana della parte dorsale arti anteriori e intersezione della parte ventrale della spalla sinistra e la cavità toracica, rispettivamente. </li><li> Dissociare la pelle e il muscolo con un paio di forbici tra i due fori. </li><li> Collegare un&#39;estremità del PE 50 tubi (20 cm di lunghezza) alla vena giugulare esterna attraverso il foro ventrale. Collegare l&#39;estremità terminale dello stesso PE 50 tubi al foro dorsale, e apporre alla pelle. </li><li> Utilizzare una siringa per iniettare soluzione fisiologica nella vena giugulare per verificare che il tubo PE 50non sia ostruito. </li><li> Suturare l&#39;incisione, e iniettare i topi con 6 mg / kg di gentamicina (per via intraperitoneale). </li><li> Lavare il tubo a giorni dopo l&#39;intervento con 0,3 ml di soluzione salina allo 0,9%, seguiti da 0,1 ml di soluzione salina con 20 U / ml di eparina. </li></ol></li><li> Finali della prova comportamentale <ol><li> Una settimana dopo il punto 2.6, ripetere i passaggi 2,2-2,3 per confermare che il comportamento è stabile rispetto al punto 2.5 dopo il recupero chirurgico. </li></ol></li><li> iniezione radiotracer <ol><li> Posizionare il ratto in una gabbia di riposo per 5 - 10 min di adattamento. </li><li> Utilizzando uno splitter, il collegamento PE 50 tubi a due 1 ml siringhe. Riempire una siringa con soluzione fisiologica e riempire l&#39;altra con [14 C] -IAP soluzione (125 pCi / kg in un volume di 0,3 - 0,5 ml). </li><li> Iniettare il radiofarmaco nella vena giugulare esterna, e sostituire la siringa con un&#39;altra siringa piena di cloruro di 3 M di potassio. </li><li> Dieci secondi dopo la inj radiofarmacoessione, iniettare cloruro di 3 M di potassio sotto una dose eccessiva di isoflurano, e sacrificare gli animali secondo metodi eutanasia standard (ad esempio, isoflurano da un vaporizzatore per 50 min) sulla base delle linee guida del Sinica Istituzionale Animal Care mondo accademico e Comitato utilizzo in Taiwan. </li><li> Dopo 1 minuto, esporre il cranio, e tagliare il muscolo restante. Utilizzando rongeurs, staccarsi la superficie dorsale del cranio dal cervello. Tagliare via i lati del cranio con Rongeurs. Successivamente, utilizzando una spatola, tagliare i bulbi olfattivi e connessioni nervose lungo la superficie ventrale del cervello, e rimuovere il cervello. </li><li> Utilizzare mescola ottimale temperatura di taglio (OCT) per congelare il cervello in ghiaccio secco e metilbutano (circa -55 ° C). Conservare il tessuto cerebrale in un congelatore. 11, 12 </li></ol></li><li> cervello affettare <ol><li> Orientare il tessuto in un microtomo, con hindbrain rivolto verso il basso. Utilizzare un criostato per tagliare il cervello a 20 &amp;# 181; m sezioni di spessore. </li><li> Mettere le fette di cervello su vetrini da microscopio in un criostato a -20 ° C, con un intervallo di 240 micron tra ogni fetta. </li><li> Porre i vetrini da microscopio e cinque carte da filtro standard con radioattività classificato in cassette di esposizione per 3 giorni a -20 ° C. Secondo la sequenza delle sezioni di cervello, disporre i vetrini per microscopio da cima a fondo. Infine, posizionare le carte da filtro nel fondo delle cassette. 12 </li><li> Rimuovere lo schermo di fosfori dalle cassette di esposizione, e utilizzare un imager-mode variabile per leggere lo schermo fosforo e generare immagini per mostrare [14 C] -IAP assorbimento per le fette di cervello. </li></ol></li></ol> Analisi 3. Dati <ol><li> Dopo il punto 2.9.4, regolare le immagini utilizzando Statistical Parametric Mapping (SPM) e il software ImageJ. Ricostruire tutte le immagini utilizzando sezioni coronali di serie. Liscio e normalizzare le immagini secondo un modello di cervello di ratto di riferimento.12, 13 </li><li> Per valutazioni quantitative, misurare la regione-di-interesse (ROI) delle immagini del cervello utilizzando il software ImageJ per determinare l&#39;intensità del segnale dei pixel, e utilizzare il software statistico per l&#39;analisi. 12, 13 </li><li> Per indagare le connessioni tra i diversi nuclei cerebrali, usare MATLAB software di analisi di correlazione per visualizzare il rapporto radioattività in una matrice di correlazione interregionale, e visualizzare le matrici come mappe di colore. Infine, utilizzare il software Pajek per l&#39;analisi di rete. 12, 13 </li><li> Utilizzare 2 × 5 bidirezionale analisi mista della varianza (ANOVA), con il gruppo e settimane come fattori di confrontare la durata della tolleranza al calore nella prova plantare e forza meccanica nel test von Frey nei gruppi sham e cpsp al basale e settimane 1 - 5. Utilizzare un 2 × 31 ANOVA a due vie per misurare il rapporto di radioattività in base al gruppo e la zona del cervello. Se del caso, condurre una differenza significative di Tukey (HSD) test post hoc. Calla correlazione di Pearson colare coefficienti per valutare le correlazioni tra tutte le aree cerebrali selezionate nella farsa e gruppi cpsp. </li></ol>

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Nei test comportamentali, il gruppo CPSP esibito una riduzione della soglia di ritiro zampa nella prova dolore termico e forza meccanica nella prova di von Frey al basale e le settimane 1 -. 5. I risultati erano coerenti con uno studio precedente 14 Il [14 C] metodo -IAP basa sull&#39;intensità del pixel di immagini del cervello per l&#39;analisi quantitativa di diverse sezioni di cervello. Per valutare i dati nelle immagini cerebrali, l&#39;intensità del segnale del ...

Corsa emorragia ha dimostrato di verificarsi in oltre l&#39;8% dei pazienti che soffrono di dolore neuropatico, di cui il dolore post-ictus come centrale (CPSP). 1-3 CPSP può derivare da una disfunzione somatosensoriale, inducendo in tal modo ipersensibilità e allodinia. 4 Tuttavia , i meccanismi fisiopatologici della disfunzione somatosensoriale in CPSP rimangono incerte. Per esempio, la perdita delle sensazioni somatiche risulta da deafferentazione neuronale nella zona emorragica cerebrale. Iperalgesia può essere causato dal ipereccitabilità dei neuroni centrali nocicettivi o disinibizione centrale, 5, 6, ma i substrati neurali che sono coinvolti nei sintomi cpsp rimangono sconosciute. Alcuni studi hanno suggerito che la corteccia dorsolaterale prefrontale (dPFC), rostrale della corteccia cingolata anteriore (ACC), amigdala, ippocampo, grigio periacqueduttale (PAG), midollo rostrale ventromediale, e le loro connessioni con l&#39;altro mediano di elaborazione nocicettivo. 7 Ulterioricircuiti -amygdala Ly, corteccia prefrontale mediale (mPFC) hanno dimostrato di essere coinvolto nella percezione del dolore legati. 8 I dati sui meccanismi fisiopatologici della CPSP sono diverse, e l&#39;attivazione di substrati neurali in CPSP ha bisogno di un ulteriore esame. [14 C] -Iodoantipyrine (IAP) si usa un assorbimento di osservare indirettamente regionale del flusso ematico cerebrale (rCBF), ipotizzando una relazione tra attività cerebrale e CBF. Anche se [14C] -IAP non può valutare l&#39;attività cerebrale in tempo reale, come ad esempio con la risonanza magnetica funzionale (fMRI), che ha diversi vantaggi. Ad esempio, [14C] -IAP è adatto per misurare spontaneamente si verificano eventi cerebrali durante gli stati patologici. 9, inoltre, [14C] -IAP assorbimento è misurata senza anestesia. Costa anche meno di altri metodi di imaging, tra cui fMRI e la tomografia ad emissione di positroni (PET). Il metodo [14 C] -IAP è stato suggerito in modo appropriato per measuring dolore spontaneo (ad esempio, CPSP) che è indotta da lesioni del nucleo basale ventrale (VB) del talamo. 9 Il presente Protocollo descrive come eseguire il -IAP metodo di [14C] per valutare il coinvolgimento dei substrati neurali di CPSP che è indotta da lesioni della VB del talamo in un modello animale. La tecnica offre un modo di determinare i meccanismi fisiopatologici che sono alla base dei sintomi cpsp a livello comportamentali e neuronali.

<table><tbody><tr><td>&lt;strong&gt;Anesthetic:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Isoflurane</td><td>Halocarbon Products Corporation&amp;nbsp;</td><td>NDC 12164-002-25</td><td>4%</td></tr><tr><td>&lt;strong&gt;Surgery&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>homeothermic blanket system</td><td>Harvard Apparatus</td><td>Model 50&amp;ndash;7079</td><td>body temperature were maintained at 36.5 - 37.5 &amp;deg;C.</td></tr><tr><td>10 &amp;micro;l micro syringe</td><td>Hamilton</td><td>80008, Model 1701SN</td><td>injected with collagenase</td></tr><tr><td>polyethylene-50 tubing</td><td>Becton, Dickinson and Company</td><td>427411</td><td>catheterized into external jugular vein</td></tr><tr><td>1 c.c syringe</td><td>Terumo Medical Products</td><td>SS-01T</td><td>injected with &lt;sup&gt;14&lt;/sup&gt;C-IAP and saline.</td></tr><tr><td>saline (Sodium Chloride 0.9 gm)</td><td>Taiwan Biotech Co., LTD.</td><td>100-130-0201</td><td>To flush the tube</td></tr><tr><td>&lt;strong&gt;Drugs&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>type 4 collagenase</td><td>Sigma</td><td>C5138-500MG</td><td>0.125 U</td></tr><tr><td>Gentamicin</td><td>Sigma</td><td>G1264-250MG</td><td>6 mg/kg</td></tr><tr><td>Heparin</td><td>Sigma</td><td>H9399</td><td>20 U/ml; 0.1 ml/day</td></tr><tr><td>&lt;sup&gt;14&lt;/sup&gt;C-iodoantipyrine (IAP)</td><td>PerkinElmer</td><td>NEC712</td><td>125 mCi/kg in 300 ml of 0.9% saline</td></tr><tr><td>Potassium chloride</td><td>Merck</td><td>1.04936.1000</td><td>3 M</td></tr><tr><td>&lt;strong&gt;Behavior system:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>von Frey esthesiometer</td><td>Fabrication Enterprises, Inc.</td><td>Baseline Tactile Monofilaments 12-1666</td><td>mechanical hyperalgesia was assessed by measuring the withdrawal response to a mechanical stimulus</td></tr><tr><td>plantar test apparatus</td><td>IITC Life Science</td><td>IITC 390G Plantar Test</td><td>Thermal hyperalgesia was assessed by measuring the hind paw withdrawal latency in response to radiant heat.</td></tr><tr><td>&lt;strong&gt;Brain slice:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Optimal Cutting Temperature compound</td><td>Sakura Fintek Inc</td><td>4583</td><td>embedded the brain</td></tr><tr><td>dry ice</td><td></td><td></td><td>frozen in dry ice/methylbutane (approximately &amp;minus;55 &amp;deg;C)</td></tr><tr><td>methylbutane</td><td>Sigma</td><td>M32631-1L</td><td>frozen in dry ice/methylbutane (approximately &amp;minus;55 &amp;deg;C)</td></tr><tr><td>Cryostat</td><td>&amp;nbsp;Leica Biosystems Nussloch GmbH, Germany</td><td>Leica CM1850</td><td>Coronal brain slice were sectioned on this machine.</td></tr><tr><td>&lt;strong&gt;Data analyze&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>exposure cassettes with a phosphor screen</td><td>Amersham Biosciences&amp;nbsp;</td><td>20 cm x 25 cm</td><td>The slices were dried on glass slides and placed alongside five standard filter papers with graded radioactivity. All of the slides were exposed to the cassettes at &amp;minus;20 &amp;deg;C.</td></tr><tr><td>&amp;gamma;-counter</td><td>Beckman Coulter</td><td>Beckman LS 6500 Liquid Scintillation Counter</td><td>To measure the radioactivity count of the filter papers.</td></tr><tr><td>Typhoon 9410 Variable Mode Imager</td><td>&amp;nbsp;GMI, Inc.</td><td>WS-S9410</td><td>To read&amp;nbsp; phosphor screen which was exposed by brain slice</td></tr><tr><td>Statistical Parametric Mapping (SPM)</td><td>Wellcome Centre for Neuroimaging</td><td>version 8</td><td>all of the brains were averaged to create the final brain template. To determine significant differences between the images in these two groups, the images were derived by subtracting the sham group from the CPSP group.</td></tr><tr><td>ImageJ</td><td>http://imagej.nih.gov/ij</td><td>version 1.46</td><td>Adjacent sections were aligned both manually and using Stack- Reg, an automated pixel-based registration algorithm in ImageJ software. All of the original three-dimensionally reconstructed brains were smoothed and normalized to the reference rat brain model.</td></tr><tr><td>Matlab</td><td>MathWorks</td><td>version 2009b</td><td>used Pearson correlation coefficients to examine the relationships between the CPSP and sham groups. An inter-regional correlation matrix was calculated across animals from each group.</td></tr><tr><td>Pajek</td><td>http://Pajek.imfm.si/</td><td>version 3.06</td><td>Graphical theoretical analysis was performed on networks defined by the above correlation matrices using Pajek software.</td></tr></tbody></table>

autoradiographic measurements of [14c]-iodoantipyrine in rat brain following central post-stroke pain

Approximately 8% of stroke patients present symptoms of central post-stroke pain (CPSP). CPSP is associated with allodynia and hypersensitivity to nociceptive stimuli. Although some studies have shown that neuropathic pain may involve the dorsolateral prefrontal cortex, rostral anterior cingulate cortex, amygdala, hippocampus, periaqueductal gray, rostral ventromedial medulla, and medial thalamus, the neural substrates and their connections that mediate CPSP remain unclear. [14C]-Iodoantipyrine (IAP) uptake can be measured to evaluate spontaneously active pain. It can be used to assess the activation of neural substrates that may be involved in CPSP in an animal model. The [14C]-IAP method in rats is less expensive to perform compared with other brain mapping techniques. The present [14C]-IAP protocol is used to measure the activation of neural substrates that are involved in CPSP that is induced by lesions of the ventral basal nucleus (VB) of the thalamus in a rodent model.

La figura 1A illustra la timeline sperimentale. I ratti sono stati assegnati alla farsa e gruppi cpsp per i test comportamentali (ad esempio, von Frey prova e plantare test). Il primo giorno dell&#39;esperimento servito come base di riferimento, e le prove sono state ripetute alle settimane 1 - 5. PE-50 cateterizzazione è stata eseguita nella vena giugulare esterna alla settimana 4. L&#39;eparina (20 U / ml, 0,1 ml / giorno) è stato iniettato durante settimane...

Watch this Scientific Journal Video about Autoradiographic Measurements of [14C]-Iodoantipyrine in Rat Brain Following Central Post-Stroke Pain at JoVE.com

Autoradiographic Measurements of [14C]-Iodoantipyrine in Rat Brain Following Central Post-Stroke Pain

We present a protocol to measure [14C]-iodoantipyrine (IAP) uptake and assess the activation of neural substrates that are involved in central post-stroke pain (CPSP) in a rodent model.

Misure autoradiografici di [<sup&gt; 14</sup&gt; C] -Iodoantipyrine nel cervello di ratto Dopo centrale post-ictus Pain

Approximately 8% of stroke patients present symptoms of central post-stroke pain (CPSP). CPSP is associated with allodynia and hypersensitivity to ...

autoradiographic-measurements-14c-iodoantipyrine-rat-brain-following

Research

JoVE Journal

Medicine

Autoradiographic Measurements of [14C]-Iodoantipyrine in Rat Brain Following Central Post-Stroke Pain

8.7K Views.  Fo Guang University. We present a protocol to measure [14C]-iodoantipyrine (IAP) uptake and assess the activation of neural substrates that are involved in central post-stroke pain (CPSP) in a rodent model.

Video: Autoradiographic Measurements of [14C]-Iodoantipyrine in Rat Brain Following Central Post-Stroke Pain

2-Vessel Occlusion/Hypotension: A Rat Model of Global Brain Ischemia

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ischemia produces damage to specific brain regions shown to be highly sensitive to ischemia 1. Hippocampal neurons have higher sensitivity to ischemic insults compared to other cell populations, and specifically, the CA1 region of the hippocampus is particularly vulnerable to ischemia/reperfusion 2.
The design of therapeutic interventions, or study of mechanisms involved in cerebral damage, requires a model that produces damage similar to the clinical condition and in a reproducible manner. Bilateral carotid vessel occlusion with hypotension (2VOH) is a model that produces reversible forebrain ischemia, emulating the cerebral events that can occur during cardiac arrest and resuscitation. We describe a model modified from Smith et al. (1984) 2, as first presented in its current form in Sanderson, et al. (2008) 3, which produces reproducible injury to selectively vulnerable brain regions 3-6. The reliability of this model is dictated by precise control of systemic blood pressure during applied hypotension, the duration of ischemia, close temperature control, a specific anesthesia regimen, and diligent post-operative care. An 8-minute ischemic insult produces cell death of CA1 hippocampal neurons that progresses over the course of 6 to 24 hr of reperfusion, while less vulnerable brain regions are spared. This progressive cell death is easily quantified after 7-14 days of reperfusion, as a near complete loss of CA1 neurons is evident at this time.
In addition to this brain injury model, we present a method for CA1 damage quantification using a simple, yet thorough, methodology. Importantly, quantification can be accomplished using a simple camera-mounted microscope, and a free ImageJ (NIH) software plugin, obviating the need for cost-prohibitive stereology software programs and a motorized microscopic stage for damage assessment.

Bilateral carotid occlusion coupled with systemic hypotension produces global brain ischemia in the rat, resulting in damage to the hippocampus with reproducible severity. Animal subjects are impaired with predictable patterns of brain damage, they recover expediently, and mortality rates are comparatively low.

2-occlusione di vasi / Ipotensione: un modello di ratto di Global Ischemia cerebrale

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ...

Ricerca

Medicina

A Middle Cerebral Artery Occlusion Technique for Inducing Post-stroke Depression in Rats

Post-stroke depression (PSD) is the most recurrent of all psychiatric complications resulting from an ischemic stroke. A greater majority (about 60%) of all ischemic stroke patients suffer from PSD, a disorder considered to be an ischemic stroke-related precursor for increased death and degradation in health. The pathophysiology of PSD is still obscure. To study the mechanism of development and occurrence of PSD further, and to find out a therapy, we attempted to develop a new protocol that requires occluding the middle cerebral artery (MCA) via the internal carotid artery (ICA) in rats. This protocol describes a model of PSD induced in rats through the middle cerebral artery occlusion (MCAO). Also used in the experiment are the Porsolt forced swim test and the sucrose preference test to confirm and evaluate the depressive mood of the rats under investigation. Rather than inserting the catheter through the external carotid artery (ECA), as stipulated for the original procedure, this MCAO technique has the monofilament passing directly through the ICA. This MCAO technique was developed a few years ago and leads to a reduction in mortality and variability. It is generally accepted that the criteria used are preferred in the selection of biological models. The data obtained with this protocol show that this model of MCAO could be a way of inducing PSD in rats and could potentially lead to the understanding of the pathophysiology and the future development of new drugs and other neuroprotective agents.

Here we present a protocol to induce post-stroke depression in rats by occluding the middle cerebral artery via the internal carotid artery. We use the Porsolt forced swim test and the sucrose preference test to confirm and evaluate induced depressive moods.

Una tecnica di occlusione dell'arteria cerebrale media per indurre la depressione post-ictus nei ratti

Post-stroke depression (PSD) is the most recurrent of all psychiatric complications resulting from an ischemic stroke. A greater majority (about 60%) of ...

Comportamento

A Dual Tracer PET-MRI Protocol for the Quantitative Measure of Regional Brain Energy Substrates Uptake in the Rat

We present a method for comparing the uptake of the brain&#39;s two key energy substrates: glucose and ketones (acetoacetate [AcAc] in this case) in the rat. The developed method is a small-animal positron emission tomography (PET) protocol, in which&#160;11C-AcAc and 18F-fluorodeoxyglucose (18F-FDG) are injected sequentially in each animal. This dual tracer PET acquisition is possible because of the short half-life of 11C (20.4 min). The rats also undergo a magnetic resonance imaging (MRI) acquisition seven days before the PET protocol. Prior to image analysis, PET and MRI images are coregistered to allow the measurement of regional cerebral uptake (cortex, hippocampus, striatum, and cerebellum). A quantitative measure of 11C-AcAc and 18F-FDG brain uptake (cerebral metabolic rate; &#956;mol/100 g/min) is determined by kinetic modeling using the image-derived input function (IDIF) method. Our new dual tracer PET protocol is robust and flexible; the two tracers used can be replaced by different radiotracers to evaluate other processes in the brain. Moreover, our protocol is applicable to the study of brain fuel supply in multiple conditions such as normal aging and neurodegenerative pathologies such as Alzheimer&#39;s and Parkinson&#39;s diseases.

Small-animal positron emission tomography enables the assessment of the brain&#39;s two main energy substrates:&#160;glucose and ketones. In the present method, 11C-acetoacetate and 18F-fluorodeoxyglucose are injected sequentially in each animal, and their uptake is measured quantitatively in specific brain regions determined from the magnetic resonance images.

We present a method for comparing the uptake of the brain&#39;s two key energy substrates: glucose and ketones (acetoacetate [AcAc] in this case) in the ...

Neuroscienze

Measurement of Glutamate Uptake using Radiolabeled L-[3H]-Glutamate in Acute Transverse Slices Obtained from Rodent Resected Hippocampus

Glutamate removal from the extracellular space by high-affinity Na+-dependent transporters is essential to ensure that the brain&#39;s intrinsic connectivity mechanisms work properly and homeostasis is maintained. The hippocampus is a unique brain structure that manages higher cognitive functions, and is the subject of several studies regarding neurologic diseases. The investigation of physiological and pathological mechanisms in rodent models can benefit from acute hippocampal slice (AHS) preparations. AHS has the advantage of providing reliable information on cell function since the cytoarchitecture and synaptic circuits are preserved. Although AHS preparations are commonly used in neurochemistry laboratories, it is possible to find some methodological differences in the literature. Considering that distinctive slice preparation protocols might change the hippocampal regions analyzed, this current protocol proposes a standard technique for obtaining transverse AHS from resected hippocampus. This simple-to-perform protocol may be used in mice and rats&#39; experimental models and allow several ex vivo approaches investigating neurochemical dynamics (in dorsal, intermediate and ventral hippocampus) in different backgrounds (e.g., transgenic manipulations) or after in vivo manipulations (e.g., pharmacological treatments or suitable rodent models to study clinical disorders). After dissecting the hippocampus from the rodent brain, transverse slices along the septo-temporal axis (300 &#181;m thick) were obtained. These AHS contain distinct parts of the hippocampus and were subjected to an individual neurochemical investigation (as an example: neurotransmitter transporters using their respective substrates). As the hippocampus presents a high density of excitatory synapses, and glutamate is the most important neurotransmitter in the brain, the glutamatergic system is an interesting target for in vivo observed phenomena. Thus, the current protocol provides detailed steps to explore glutamate uptake in ex vivo AHS using L-[3H]-Glutamate. Using this protocol to investigate hippocampal function may help to better understand the influence of glutamate metabolism on mechanisms of neuroprotection or neurotoxicity.

Measurement of Glutamate Uptake using Radiolabeled L-[3H]-Glutamate in Acute Transverse Slices Obtained from Rodent Resected Hippocampus

This article describes a reliable and simple way to obtain ex vivo acute hippocampal transverse slices from mice and rats using a tissue chopper. Slices obtained from resected hippocampi can be submitted for functional glutamate uptake analysis to investigate glutamatergic system homeostasis.

Glutamate removal from the extracellular space by high-affinity Na+-dependent transporters is essential to ensure that the brain&#39;s intrinsic ...

Biochimica

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to return to normal in a large majority of patients2, which is mainly due to current lack of clinical treatment for acute stroke. This necessitates understanding the physiological effects of cerebral ischemia on brain tissue over time and is a major area of active research. Towards this end, experimental progress has been made using rats as a preclinical model for stroke, particularly, using non-invasive methods such as 18F-fluorodeoxyglucose (FDG) coupled with Positron Emission Tomography (PET) imaging3,10,17. Here we present a strategy for inducing cerebral ischemia in rats by middle cerebral artery occlusion (MCAO) that mimics focal cerebral ischemia in humans, and imaging its effects over 24 hr using FDG-PET coupled with X-ray computed tomography (CT) with an Albira PET-CT instrument. A VOI template atlas was subsequently fused to the cerebral rat data to enable a unbiased analysis of the brain and its sub-regions4. In addition, a method for 3D visualization of the FDG-PET-CT time course is presented. In summary, we present a detailed protocol for initiating, quantifying, and visualizing an induced ischemic stroke event in a living Sprague-Dawley rat in three dimensions using FDG-PET.

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Brain damage resulting from cerebral ischemia may be non-invasively imaged and studied in rats using pre-clinical positron emission tomography coupled with the injectable radioactive probe, 18F-fluorodeoxyglucose. Further, the use of modern software tools that include volume of interest (VOI) brain templates dramatically increase the quantitative information gleaned from these studies.

Imaging non invasiva e analisi di ischemia cerebrale in ratti Living Utilizzando Positron Emission Tomography con<sup&gt; 18</sup&gt; F-FDG

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to ...

Intracranial Pharmacotherapy and Pain Assays in Rodents

Pain is a salient sensory experience with affective and cognitive dimensions. However, central mechanisms for pain remain poorly understood, hindering the development of effective therapeutics. Intracranial pharmacology presents an important tool for understanding the molecular and cellular mechanisms of pain in the brain, as well as for novel treatments. Here we present a protocol that integrates intracranial pharmacology with pain behavior testing. Specifically, we show how to infuse analgesic drugs into a select brain region, which may be responsible for pain modulation. Furthermore, to determine the effect of the candidate drug in the central nerve system, pain assays are performed after intracranial treatment. Our results demonstrate that intracranial administration of analgesic drugs in a targeted region can provide relief of pain in rodents. Thus, our protocol successfully demonstrates that intracranial pharmacology, combined with pain behavior testing, can be a powerful tool for the study of pain mechanisms in the brain.

Here we present a protocol to perform intracranial pharmacological experiments followed by pain behavior assays in rodents. This protocol allows researchers to deliver molecular and cellular targets in the brain, for pharmacologic agents in the treatment of pain.

Dosaggi di dolore nei roditori e farmacoterapia intracranica

Pain is a salient sensory experience with affective and cognitive dimensions. However, central mechanisms for pain remain poorly understood, hindering the ...

Quantitative Autoradiographic Method for Determination of Regional Rates of Cerebral Protein Synthesis In Vivo

Protein synthesis is required for development and maintenance of neuronal function and is involved in adaptive changes in the nervous system. Moreover, it is thought that dysregulation of protein synthesis in the nervous system may be a core phenotype in some developmental disorders. Accurate measurement of rates of cerebral protein synthesis in animal models is important for understanding these disorders. The method that we have developed was designed to be applied to the study of awake, behaving animals. It is a quantitative autoradiographic method, so it can yield rates in all regions of the brain simultaneously. The method is based on the use of a tracer amino acid, L-[1-14C]-leucine, and a kinetic model of the behavior of L-leucine in the brain. We chose L-[1-14C]-leucine as the tracer because it does not lead to extraneous labeled metabolic products. It is either incorporated into protein or rapidly metabolized to yield 14CO2 which is diluted in a large pool of unlabeled CO2 in the brain. The method and the model also allow for the contribution of unlabeled leucine derived from tissue proteolysis to the tissue precursor pool for protein synthesis. The method has the spatial resolution to determine protein synthesis rates in cell and neuropil layers, as well as hypothalamic and cranial nerve nuclei. To obtain reliable and reproducible quantitative data, it is important to adhere to procedural details. Here we present the detailed procedures of the quantitative autoradiographic L-[1-14C]-leucine method for the determination of regional rates of protein synthesis in vivo.

Protein synthesis is&#160;a critical biological process for cells. In brain, it is required for adaptive changes. Measurement of rates of protein synthesis in the intact brain requires careful methodological considerations. Here we present the L-[1-14C]-leucine quantitative autoradiographic method for determination of regional rates of cerebral protein synthesis in vivo.

Metodo autoradiografico quantitativo per la determinazione dei tassi regionali di sintesi proteica cerebrale in vivo

Protein synthesis is required for development and maintenance of neuronal function and is involved in adaptive changes in the nervous system. Moreover, it ...

PET Imaging of Neuroinflammation Using [11C]DPA-713 in a Mouse Model of Ischemic Stroke

Neuroinflammation is central to the pathological cascade following ischemic stroke. Non-invasive molecular imaging methods have the potential to provide critical insights into the temporal dynamics and role of certain neuroimmune interactions in stroke. Specifically, Positron Emission Tomography (PET) imaging of translocator protein 18 kDa (TSPO), a marker of activated microglia and peripheral myeloid-lineage cells, provides a means to detect and track neuroinflammation in vivo. Here, we present a method to accurately quantify neuroinflammation using [11C]N,N-Diethyl-2-[2-(4-methoxyphenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl]acetamide ([11C]DPA-713), a promising second generation TSPO-PET radiotracer, in distal middle cerebral artery occlusion (dMCAO) compared to sham-operated mice. MRI was performed 2 days post-dMCAO surgery to confirm stroke and define the infarct location and volume. PET/Computed Tomography (CT) imaging was carried out 6 days post-dMCAO to capture the peak increase in TSPO levels following stroke. Quantitation of PET images was conducted to assess the uptake of [11C]DPA-713 in the brain and spleen of dMCAO and sham mice to assess central and peripheral levels of inflammation. In vivo [11C]DPA-713 brain uptake was confirmed using ex vivo autoradiography.

PET Imaging of Neuroinflammation Using [11C]DPA-713 in a Mouse Model of Ischemic Stroke

Positron Emission Tomography (PET) imaging of translocator protein 18 kDa (TSPO) provides a non-invasive means to visualize the dynamic role of neuroinflammation in the development and progression of brain diseases. This protocol describes TSPO-PET and ex vivo autoradiography to detect neuroinflammation in a mouse model of ischemic stroke.

Imaging del Neuroinflammation utilizzando [11C] DPA-713 in un modello murino di ictus ischemico per animali

Neuroinflammation is central to the pathological cascade following ischemic stroke. Non-invasive molecular imaging methods have the potential to provide ...

Autoradiography as a Simple and Powerful Method for Visualization and Characterization of Pharmacological Targets

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The method is based on the specific binding of a radioligand to its biological target. Therefore, frozen tissue sections are incubated with radioligand solution, and the binding to the target is subsequently localized by the detection of radioactive decay, for example, by using photosensitive film or phosphor imaging plates. Resulting digital autoradiograms display remarkable spatial resolution, which enables quantification and localization of radioligand binding in distinct anatomical structures. Moreover, quantification allows for the pharmacological characterization of ligand affinity by means of dissociation constants (Kd), inhibition constants (Ki) as well as the density of binding sites (Bmax) in selected tissues. Thus, the method provides information about both target localization and ligand selectivity. Here, the technique is exemplified with autoradiographic characterization of the high-affinity &#947;-hydroxybutyric acid (GHB) binding sites in mammalian brain tissue, with special emphasis on methodological considerations regarding the binding assay parameters, the choice of the radioligand and the detection method.

The method of autoradiography is routinely used to study binding of radioligands to tissue sections for determination of qualitative or quantitative pharmacology.

Autoradiografia come un metodo semplice e potente per la visualizzazione e caratterizzazione di bersagli farmacologici

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The ...

Measuring Post-Stroke Cerebral Edema, Infarct Zone and Blood-Brain Barrier Breakdown in a Single Set of Rodent Brain Samples

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke involves middle cerebral artery occlusion (MCAO). Infarct zone, brain edema and blood-brain barrier (BBB) breakdown are measured as parameters that reflect the extent of brain injury after MCAO. A significant limitation to this method is that these measurements are normally obtained in different rat brain samples, leading to ethical and financial burdens due to the large number of rats that need to be euthanized for an appropriate sample size. Here we present a method to accurately assess brain injury following MCAO by measuring infarct zone, brain edema and BBB permeability in the same set of rat brains. This novel technique provides a more efficient way to evaluate the pathophysiology of stroke.

This protocol describes a novel technique of measuring the three most important parameters of ischemic brain injury on the same set of rodent brain samples. Using only one brain sample is highly advantageous in terms of ethical and economic costs.

Misurazione dell'edema cerebrale post-ictus, della zona infarct e della rottura della barriera ematica-encefalica in un singolo set di campioni cerebrali dei roditori

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke ...

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