Questo lavoro è stato sostenuto in parte da una sovvenzione NIH concedere R01 GM087609 (a HPL), un regalo da Ingrid Lai e Bill Shu in onore di Anthony Shu (a HPL), NIH concedere U01 HL099776 (a MTL), il Laboratorio per Hagey Pediatrica Medicina Rigenerativa e La Fondazione Oak (a MTL e HPL). GGW è stato sostenuto dalla Stanford School of Medicine, il Programma di Formazione Stanford Medical Scientist, e borsa di formazione NIGMS GM07365. MSH è stato sostenuto da CIRM Clinical Fellow formazione di Grant TG2-01159. WXH è stato sostenuto da un finanziamento della Cardiovascular Foundation Sarnoff.

NOTA: Tutte le procedure descritte in questo documento vengono eseguite secondo le linee guida stabilite dal Pannello di Amministrazione Stanford on Laboratory Animal Care (APLAC). 1. Timed Gravidanze - Tecnica Superovulazione (Figura 1) NOTA: Proprio tempi l&#39;età gestazionale di embrioni di topo per la chirurgia fetale a E16.5 e E18.5 è di importanza fondamentale. In questa sezione abbiamo dettaglio il nostro protocollo di sincronizzazione gravidanze del mouse utilizzando mares siero incinta (PMS) e gonadotropina corionica umana (HCG) iniezioni per indurre superovulazione. <ol><li> Iniettare topi femmina (&lt;5 per gabbia) per via intraperitoneale (IP) con 3,0-5,0 unità internazionali (UI) di PMS in un volume di 100 microlitri PBS 1:00-15:00 per il giorno 1. </li><li> Tra le 12:00 e le 14:00 del giorno 3 (quarantacinque a 47 ore dopo l&#39;iniezione PMS), iniettare topi femmina IP con 3,0-5,0 UI di HCG in un volume di 100 microlitri PBS. NOTA: Le induce iniezione HCG<html> ovulazione circa 12 ore dopo l&#39;iniezione. </li><li> Subito dopo iniezioni di HCG, accoppiarsi con le femmine i maschi di età compresa tra 8-16 settimane. NOTA BENE: Noi di solito poniamo due femmine in una gabbia di singoli uomini. </li><li> Femmine separati da uomini la mattina del giorno 4 (6:00-10:00) e record come fetale E0.5 età. NOTA: tappi vaginali possono essere controllati in questo momento; Tuttavia, l&#39;osservazione di un tappo vaginale non garantisce gravidanza e femmine può diventare incinta quando non si osserva la spina. Dato che la gravidanza è in genere osservabile mediante ispezione visiva e / o la palpazione in età gestazionale E16.5 e E18.5, controllando tappi vaginali la mattina del giorno 4 non è strettamente necessario. Nella nostra esperienza, e seconda del ceppo, circa il 30-50% delle femmine super ovulato diventa incinta utilizzando la tecnica qui descritta. </li></ol>pg &quot;/&gt; Figura 1. Schema di Tecnica Superovulazione. Procedura mostra Schema superovulazione cronometrato gravidanze nei topi. <a href="https://www.jove.com/files/ftp_upload/52297/52297fig1large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> 2. murino fetale Surgery (Dorsale Ferimento) su E16.5 e E18.5 embrioni (Figura 2) <ol><li> Prima della procedura, pulire tutte le superfici della sala operatoria e attrezzature con il 70% di alcol isopropilico. Inoltre, sterilizzare tutte le forniture chirurgiche e gli strumenti che saranno utilizzati nella procedura da essi autoclave. Alcuni istituti possono consentire il successivo utilizzo di caldo tallone sterilizzazione. Per il funzionamento, utilizzare confezioni sterili che includono la garza e strumenti chirurgici. </li><li> Indurre l&#39;anestesia in donne in gravidanza (fetale E16.5 età o E18.5) inferiore al 2,5% della miscela isoflurano / ossigeno a 2 L al min seguita da anestesia di mantenimento a 1 L per min. </li><li> Per conimpresa adeguata anestesia, assicurano i riflessi pedale profondi del mouse vengono soppressi e posizionare il mouse in posizione prona. </li><li> Applicare una pomata oftalmica veterinario come Puralube prevenire l&#39;irritazione degli occhi o secchezza durante la procedura. </li><li> Preparare addome somministrando una leggera applicazione di crema depilatoria per non più di 30 secondi (Figura 2A). </li><li> Preparare addome per la chirurgia asettica con povidone-iodio e di alcol (figura 2B e 2C). </li><li> Eseguire linea mediana laparotomia sotto il microscopio con le forbici microchirurgia (Figura 2D e 2E). </li><li> Esporre delicatamente utero e feto selezionato per la chirurgia (Figura 2F). </li><li> Irrigare campo chirurgico con il caldo (38 ° C) PBS (PBS) con un ago smussato punta NOTA: Si può essere fatto piegando con cautela la punta di un ago di grosso calibro. (Figura 2G e 2H). </li><li> Posizionare il feto in modo da consentires pieno accesso a dorso. </li><li> Passare un punto stringa di borsa utilizzando 7-0 nylon sutura attraverso l&#39;utero che insiste sul sito di ferimento dorsale previsto (Figura 2I). Posizione stringa di borsa su una regione del dorso a sinistra oa destra del midollo spinale, e in una regione di parete uterina privo di grandi vasi sanguigni. </li><li> Eseguire un&#39;incisione 3 millimetri attraverso la parete uterina e sacco amniotico nel centro della stringa borsa (Figura 2J). </li><li> Irrigare sito di incisione con acqua calda (38 ° C) PBS. </li><li> Utilizzando forbici microchirurgiche, tagliare una sola ferita excisional tutto spessore, di circa 1 mm di lunghezza, nel dorso del feto. </li><li> Delicatamente sito di incisione asciugare con cotone applicatore. </li><li> Iniettare 3 ml di volume India sottocutanea inchiostro nel sito della ferita per contrassegnare posizione di ferita (Figura 2K). </li><li> Irrigare con acqua calda (38 ° C) PBS per assicurare l&#39;inchiostro è stato mantenuto all&#39;interno del sito della ferita. </li><li> Avere chirurgica assistant inject caldo (38 ° C) PBS attraverso blunt punta siringa da 10 G nel sacco amniotico, come la stringa di borsa è chiusa (Figura 2L). Ritrarre siringa come chiusura stringa di borsa si avvicina al completamento (Figura 2M). </li><li> Ritorno delicatamente dell&#39;utero nella cavità addominale (Figura 2N). </li><li> Evert pelle e del peritoneo. </li><li> Hanno assistente chirurgica irrigare cavità addominale con acqua calda (38 ° C) PBS. </li><li> Chiudi addome rapidamente pinzatura pelle e del peritoneo chiuso (Figura 2O). La chiusura standard viene eseguita in due strati; peritoneo e muscoli addominali in uno strato, tessuto sottocutaneo e la pelle nel secondo strato. Per sacrificio immediato e la raccolta del feto, la nostra manifestazione dimostra la chiusura in un solo strato. </li><li> Posto l&#39;animale sotto osservazione in un incubatore calda a 37 ° C per 30 minuti o fino a quando l&#39;animale riprende conoscenza sufficiente per mantenere decubito sternale. </li><li> Non returna l&#39;animale per la compagnia di altri animali fino a quando non ha pienamente recuperato dalla procedura. </li><li> Al risveglio dall&#39;anestesia e durante il successivo 48 hr, amministrare l&#39;iniezione sottocutanea di buprenorfina (0,05 mg / kg) ogni 12 ore di analgesia come necessario sulla base della valutazione del dolore. Somministrare carprofen (5 mg / kg) per via sottocutanea per il sollievo del dolore post-operatorio supplementare se necessario. </li><li> Animali Ritorno alla gabbia e di fornire loro cibo e acqua ad libitum. </li><li> Monitorare attentamente per manifestazioni di dolore. </li><li> Post-operatorio 48 ore, sacrificio madre incinta con una dose eccessiva di isoflurano e del raccolto feriti feto. Per fare questo, regolare la concentrazione isoflurano al 5% o superiore e mantenere l&#39;esposizione per 1 min dopo la cessazione della respirazione. Confermare l&#39;eutanasia con dislocazione cervicale. Raccolto un embrione illeso per il controllo di pari età. Embrioni tardivi dovrebbero avere un sistema diverso di eutanasia in linea con le raccomandazioni IACUC, come decaptazione, dislocazione cervicale, o iniezione chimica. </li></ol><img alt="Figura 2" src="/files/ftp_upload/52297/52297fig2highres.jpg" /> Figura 2. Schema di Murine fetale Surgery. Passi generali per la dorsale ferendo in E16.5 e E18.5 embrioni di topo. (A) Depilazione di topo dell&#39;addome. (B e C) Preparazione di topo dell&#39;addome. (D) microscopio utilizzato per chirurgica procedura. (E) Midline laparotomia. (F) L&#39;esposizione dell&#39;utero. (G) Creazione di blunt-punta dell&#39;ago. (H) Irrigazione dell&#39;utero con soluzione salina calda. (I) Creazione di stringa di borsa sutura. (J) incisione attraverso uterina muro e 1 mm completa generazione ferita excisional spessore. (K) L&#39;iniezione sottocutanea di inchiostro di china. (L e M) Chiusura della stringa di borsa sutura. (N e O)Chiusura dell&#39;addome. <a href="https://www.jove.com/files/ftp_upload/52297/52297fig2large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a>

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The authors have nothing to disclose.

Il protocollo chirurgico presentato qui descrive un modello escissionale fetale guarigione scarless murino pubblicato nel 2006 dal nostro laboratorio 10. Oltre agli altri modelli consolidati di ferimento excisional 11, esistono modelli incisionali di fetale murino scarless guarigione così 12,13. Indagini di fetale guarigione scarless nella scimmia, agnello, coniglio, opossum, e ratti sono stati segnalati 14-17. Tuttavia, i topi rappresentano un modello ideale per esplorare fe...

Ferite cutanee fetali guariscono rapidamente e scarlessly fino a tardi in gestazione 1. Fetal riparazione della ferita scarless è caratterizzata dalla rigenerazione di architettura tessuto normale e la funzione. Il passaggio da un scarless a cicatrici fenotipo avviene nel terzo trimestre di gravidanza negli esseri umani e in tutto il giorno embrionale 18 (E18) in topi 2,3. In confronto ad adulto, riparazione della ferita fetale è caratterizzata da una rapida riepitelizzazione, la deposizione di tessuto connettivo, e la migrazione dei fibroblasti. Molti studi hanno offerto possibili spiegazioni per il fenomeno di scarless guarigione delle ferite durante le prime fasi dello sviluppo fetale. L&#39;infiammazione è una componente fondamentale di riparazione adulti ferita; Tuttavia, ferite fetali sono caratterizzati da una mancanza di infiammazione acuta 4. Se questa è una conseguenza della immaturità funzionale del sistema immunitario durante fasi fetale rimane chiaro. Uno studio recente ha suggerito che le differenze di abbondanza, tappetourity, e la funzione dei mastociti in E15 vs. E18 cute fetale possono essere responsabili per il passaggio da un fenotipo scarless, almeno nel topo 3. Altri studi ipotizzano che le differenze nelle proprietà e abbondanza di macrofagi ferite fetali e adulti sono responsabili per la riforma della matrice extracellulare normale (ECM) durante ferita fetale riparazione 5. Le differenze nei fattori ambientali durante lo sviluppo fetale e adulti possono anche interessare la riparazione delle ferite. Longaker e colleghi hanno dimostrato che il fluido ferita dal feto possiede alti livelli di attività acido ialuronico stimolante rispetto a nessuno in adulti ferita fluido 6. Di conseguenza, i livelli elevati di acido ialuronico, un glicosaminoglicano che promuove un microambiente favorevole alla motilità e proliferazione cellulare, nell&#39;ambiente della ferita fetale possono essere responsabili del fenotipo scarless visto durante precoce sviluppo fetale. Altre linee di punto prova al fatto che la fetaambiente l ferita è relativamente ipossiemico e sommersa in sterile liquido amniotico ricco di fattori di crescita 7. Tuttavia, nessuna risposta definitiva è stata fornita per un evento critico o fattore durante l&#39;embriogenesi che innesca la transizione dalla rigenerazione scarless a riparazione fibrotica. La comprensione dei meccanismi responsabili per la guarigione scarless nel feto richiede un modello preciso e riproducibile. Qui i dettagli di un modello riproducibile di fetale scarless guarigione delle ferite nel dorso di E16.5 (scarless) ed embrioni E18.5 (cicatrici) del mouse. Inoltre, variazioni minori di questo modello può essere utilizzato per eseguire una serie di ulteriori studi, come l&#39;analisi di espressione genica di ferite fetali e 8,9 pelle. Dato che le gravidanze a tempo proprio sono fondamentali per il successo della ricapitolazione questo scarless ferita modello guarigione fetale, abbiamo anche dettagliato il nostro protocollo superovulazione cronometrato gravidanze.

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			<td height="20" style="height:20px;width:219px;">Name of Material/Equipment</td>
			<td style="width:184px;">Company</td>
			<td style="width:108px;">Catalog Number</td>
			<td style="width:155px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">7-O MONOSOF Suture</td>
			<td>eSuture</td>
			<td>SN-1647G</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Surgical Forceps</td>
			<td>Kent Scientific</td>
			<td>INS650916</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Micro-scissors</td>
			<td>Kent Scientific</td>
			<td>INS600127</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Autoclip 9mm</td>
			<td>Texas Scientific Instruments</td>
			<td>205060</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Insulin Syringe</td>
			<td>Thermo Fisher Scientific</td>
			<td>22-272-382</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Black Pigment</td>
			<td>AIMS</td>
			<td>242</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BD Safety-Lok 3ml Syringe</td>
			<td>BD Biosciences</td>
			<td>309596</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate Buffered Saline</td>
			<td>Life Technologies</td>
			<td>10010-049</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">OPMI-MD Surgical Microscope</td>
			<td>Carl Zeiss Surgical Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pregnant Mares Serum (PMS)</td>
			<td>Millipore</td>
			<td>367222</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Human Chorionic Gonadotropin (HCG)</td>
			<td>Sigma-Aldrich</td>
			<td>CG10</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Povidone Iodine Prep Solution</td>
			<td>Dynarex</td>
			<td>1415</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Nair (depilatory cream)</td>
			<td>Church and Dwight Co.</td>
			<td>22600267058</td>
			<td></td>
		</tr>
	</tbody>
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a mouse fetal skin model of scarless wound repair

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs across species but is age dependent. The transition from a scarless to scarring phenotype occurs in the third trimester of pregnancy in humans and around embryonic day 18 (E18) in mice. However, this varies with the size of the wound with larger defects generating a scar at an earlier gestational age. The emergence of lineage tracing and other genetic tools in the mouse has opened promising new avenues for investigation of fetal scarless wound healing. However, given the inherently high rates of morbidity and premature uterine contraction associated with fetal surgery, investigations of fetal scarless wound healing in vivo require a precise and reproducible surgical model. Here we detail a reliable model of fetal scarless wound healing in the dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

Per l&#39;analisi istologica, ferite cutanee della pelle dorsale E16.5 e E18.5 embrioni di topo dovrebbero essere raccolti 48 ore dopo il ferimento, fissati in 4% PFA, e inclusi in paraffina. Nei modelli transgenici fluorescenti, la crioconservazione di ottobre può essere appropriato. Ci sono diverse macchie che possono essere utilizzati per visualizzare architettura tessuto cellulare e connettivo. Ematossilina e eosina è una macchia a due colori che macchia nuclei blu e strutture eosinofili (es., Citoplasma ...

Watch this Scientific Journal Video about A Mouse Fetal Skin Model of Scarless Wound Repair at JoVE.com

A Mouse Fetal Skin Model of Scarless Wound Repair

During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

Un Mouse fetale Modello Skin of Wound Repair scarless

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs ...

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Medicine

12.2K Views.  Stanford University School of Medicine. During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

Video: A Mouse Fetal Skin Model of Scarless Wound Repair

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&beta; using a Whole Blood Stimulation Assay

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues as well as altered cytokine homeostasis. The innate immune system plays a crucial role in recognizing pathogens and other endogenous danger stimuli. One of the major cytokines released by innate immune cells is Interleukin (IL)-1. Therefore, we utilize a whole blood stimulation assay in order to measure the secretion of inflammatory cytokines and specifically of the pro-inflammatory cytokine IL-1&beta; 1, 2, 3.
Patients with genetic dysfunctions of the innate immune system causing autoinflammatory syndromes show an exaggerated release of mature IL-1&beta; upon stimulation with LPS alone. In order to evaluate the innate immune component of patients who present with inflammatory-associated pathologies, we use a specific immunoassay to detect cellular immune responses to pathogen-associated molecular patterns (PAMPs), such as the gram-negative bacterial endotoxin, lipopolysaccharide (LPS). These PAMPs are recognized by pathogen recognition receptors (PRRs), which are found on the cells of the innate immune system 4, 5, 6, 7. A primary signal, LPS, in conjunction with a secondary signal, ATP, is necessary for the activation of the inflammasome, a multiprotein complex that processes pro-IL-1&beta; to its mature, bioactive form 4, 5, 6, 8, 9, 10.
The whole blood assay requires minimal sample manipulation to assess cytokine production when compared to other methods that require labor intensive isolation and culturing of specific cell populations. This method differs from other whole blood stimulation assays; rather than diluting samples with a ratio of RPMI media, we perform a white blood cell count directly from diluted whole blood and therefore, stimulate a known number of white blood cells in culture 2. The results of this particular whole blood assay demonstrate a novel technique useful in elucidating patient cohorts presenting with autoinflammatory pathophysiologies.

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&#946; using a Whole Blood Stimulation Assay

We describe a simple immunoassay to measure the production of pro-inflammatory cytokines, such as IL-1 beta production, in patients presenting with autoinflammatory phenotypes. By activating cells in whole blood cultures with pathogen-associated molecular patterns, specifically with lipopolysaccharide, cytokine secretion can be conveniently evaluated in whole blood supernatants.

Misurazione accurata e semplice della citochina pro-infiammatoria IL-1β utilizzando un intero saggio stimolazione del sangue

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues ...

Ricerca

Medicina

A Contusive Model of Unilateral Cervical Spinal Cord Injury Using the Infinite Horizon Impactor

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of spinal cord injury (SCI) in which the thoracic spinal cord is injured. Additionally, because most human cord injuries occur as the result of blunt, non-penetrating trauma (e.g. motor vehicle accident, sporting injury) where the spinal cord is violently struck by displaced bone or soft tissues, the majority of SCI researchers are of the opinion that the most clinically relevant injury models are those in which the spinal cord is rapidly contused.1 Therefore, an important step in the preclinical evaluation of novel treatments on their way to human translation is an assessment of their efficacy in a model of contusion SCI within the cervical spinal cord. Here, we describe the technical aspects and resultant anatomical and behavioral outcomes of an unilateral contusive model of cervical SCI that employs the Infinite Horizon spinal cord injury impactor.
Sprague Dawley rats underwent a left-sided unilateral laminectomy at C5. To optimize the reproducibility of the biomechanical, functional, and histological outcomes of the injury model, we contused the spinal cords using an impact force of 150 kdyn, an impact trajectory of 22.5&deg; (animals rotated at 22.5&deg;), and an impact location off of midline of 1.4 mm. Functional recovery was assessed using the cylinder rearing test, horizontal ladder test, grooming test and modified Montoya's staircase test for up to 6 weeks, after which the spinal cords were evaluated histologically for white and grey matter sparing.
The injury model presented here imparts consistent and reproducible biomechanical forces to the spinal cord, an important feature of any experimental SCI model. This results in discrete histological damage to the lateral half of the spinal cord which is largely contained to the ipsilateral side of injury. The injury is well tolerated by the animals, but does result in functional deficits of the forelimb that are significant and sustained in the weeks following injury. The cervical unilateral injury model presented here may be a resource to researchers who wish to evaluate potentially promising therapies prior to human translation.

A reliable and repeatable way to produce a cervical unilateral spinal cord injury using the Infinite Horizon impactor is described. The method takes advantage of a custom designed frame and clamp to stabilize the spine. The standardized procedure and biomechanical injury parameters result in sufficient and sustained injuries.

Un modello contusivo unilaterale di lesioni del midollo spinale cervicale Uso della Impactor Infinite Horizon

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of ...

A Simplified Technique for Producing an Ischemic Wound Model

One major obstacle in current diabetic wound research is a lack of an ischemic wound model that can be safely used in diabetic animals. Drugs that work well in non-ischemic wounds may not work in human diabetic wounds because vasculopathy is one major factor that hinders healing of these wounds. We published an article in 2007 describing a rabbit ear ischemic wound model created by a minimally invasive surgical technique. Since then, we have further simplified the procedure for easier operation. On one ear, three small skin incisions were made on the vascular pedicles, 1-2 cm from the ear base. The central artery was ligated and cut along with the nerve. The whole cranial bundle was cut and ligated, leaving only the caudal branch intact. A circumferential subcutaneous tunnel was made through the incisions, to cut subcutaneous tissues, muscles, nerves, and small vessels. The other ear was used as a non-ischemic control. Four wounds were made on the ventral side of each ear. This technique produces 4 ischemic wounds and 4 non-ischemic wounds in one animal for paired comparisons. After surgery, the ischemic ear was cool and cyanotic, and showed reduced movement and a lack of pulse in the ear artery. Skin temperature of the ischemic ear was 1-10 &deg;C lower than that on the normal ear and this difference was maintained for more than one month. Ear tissue high-energy phosphate contents were lower in the ischemic ear than the control ear. Wound healing times were longer in the ischemic ear than in the non-ischemic ear when the same treatment was used. The technique has now been used on more than 80 rabbits in which 23 were diabetic (diabetes time ranging from 2 weeks to 2 years). No single rabbit has developed any surgical complications such as bleeding, infection, or rupture in the skin incisions. The model has many advantages, such as little skin disruption, longer ischemic time, and higher success rate, when compared to many other models. It can be safely used in animals with reduced resistance, and can also be modified to meet different testing requirements.

We have developed a minimally invasive technique to create a rabbit ischemic ear wound model by dividing the central artery and nerve and the cranial neurovascular bundle. A subcutaneous tunnel then cuts all subcutaneous tissues. This procedure causes minimal skin disruption and can be safely used in diabetic animals.

Una tecnica semplificata per produrre un modello di lesione ischemica

One major obstacle in current diabetic wound research is a lack of an ischemic wound model that can be safely used in diabetic animals. Drugs that work ...

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

Valutare i cambiamenti teratogeni in un modello di Zebrafish di esposizione alcolica fetale

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

Un nuovo approccio chirurgico per somministrazione endotracheale di agenti bioattivi in ​​un modello murino fetale

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

Modello murino di guarigione delle ferite

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

Fetal Echocardiography and Pulsed-wave Doppler Ultrasound in a Rabbit Model of Intrauterine Growth Restriction

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ultrasound and fetal echocardiography. Increasingly, these imaging modalities are being employed clinically to examine cardiac function and assess wellbeing in utero, thereby guiding timing of birth decisions. Here, we used a rabbit model of IUGR that allows analysis of cardiac function in a clinically relevant way. Using isoflurane induced anesthesia, IUGR is surgically created at gestational age day 25 by performing a laparotomy, exposing the bicornuate uterus and then ligating 40-50% of uteroplacental vessels supplying each gestational sac in a single uterine horn. The other horn in the rabbit bicornuate uterus serves as internal control fetuses. Then, after recovery at gestational age day 30 (full term), the same rabbit undergoes examination of fetal cardiac function. Anesthesia is induced with ketamine and xylazine intramuscularly, then maintained by a continuous intravenous infusion of ketamine and xylazine to minimize iatrogenic effects on fetal cardiac function. A repeat laparotomy is performed to expose each gestational sac and a microultrasound examination (VisualSonics VEVO 2100) of fetal cardiac function is performed. Placental insufficiency is evident by a raised pulsatility index or an absent or reversed end diastolic flow of the umbilical artery Doppler waveform. The ductus venosus and middle cerebral artery Doppler is then examined. Fetal echocardiography is performed by recording B mode, M mode and flow velocity waveforms in lateral and apical views. Offline calculations determine standard M-mode cardiac variables, tricuspid and mitral annular plane systolic excursion, speckle tracking and strain analysis, modified myocardial performance index and vascular flow velocity waveforms of interest. This small animal model of IUGR therefore affords examination of in utero cardiac function that is consistent with current clinical practice and is therefore useful in a translational research setting.

We describe examination of fetal cardiac function with contemporary functional fetal echocardiography and fetoplacental Doppler ultrasound using the VisualSonics VEVO 2100 microultrasound in a surgically induced model of intrauterine fetal growth restriction in a rabbit.

L&#39;ecocardiografia fetale e Pulsed-Doppler ad ultrasuoni in un modello di coniglio di ritardo di crescita intrauterino

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ...

A New Murine Model of Endovascular Aortic Aneurysm Repair

Endovascular aneurysm exclusion is a validated technique to prevent aneurysm rupture. Long-term results highlight technique limitations and new aspects of Abdominal aortic aneurysm (AAA) pathophysiology. There is no abdominal aortic aneurysm endograft exclusion model cheap and reproducible, which would allow deep investigations of AAA before and after treatment. We hereby describe how to induce, and then to exclude with a covered coronary stentgraft an abdominal aortic aneurysm in a rat. The well known elastase induced AAA model was first reported in 19901 in a rat, then described in mice2. Elastin degradation leads to dilation of the aorta with inflammatory infiltration of the abdominal wall and intra luminal thrombus, matching with human AAA. Endovascular exclusion with small covered stentgraft is then performed, excluding any interactions between circulating blood and the aneurysm thrombus. Appropriate exclusion and stentgraft patency is confirmed before euthanasia by an angiography thought the left carotid artery. Partial control of elastase diffusion makes aneurysm shape different for each animal. It is difficult to create an aneurysm, which will allow an appropriate length of aorta below the aneurysm for an easy stentgraft introduction, and with adequate proximal and distal neck to prevent endoleaks. Lots of failure can result to stentgraft introduction which sometimes lead to aorta tear with pain and troubles to stitch it, and endothelial damage with post op aorta thrombosis. Giving aspirin to rats before stentgraft implantation decreases failure rate without major hemorrhage. Clamping time activates neutrophils, endothelium and platelets, and may interfere with biological analysis.

Histological and biochemical modifications after aneurysm endograft exclusion are still unclear. We describe a new model of endograft implantation on a murine aneurysm. Through thrombus analysis with and without persistant circulating blood stream interface, and new endovascular biomaterials evaluation, this model will help to better understand endovascular aneurysm exclusion pathobiology.

Un nuovo modello murino di endovascolare aneurisma dell&#39;aorta riparazione

Endovascular aneurysm exclusion is a validated technique to prevent aneurysm rupture. Long-term results highlight technique limitations and new aspects of ...

A Surgical Procedure for Resecting the Mouse Rib: A Model for Large-Scale Long Bone Repair

This protocol introduces researchers to a new model for large-scale bone repair utilizing the mouse rib. The procedure details the following: preparation of the animal for surgery, opening the thoracic body wall, exposing the desired rib from the surrounding intercostal muscles, excising the desired section of rib without inducing a pneumothorax, and closing the incisions. Compared to the bones of the appendicular skeleton, the ribs are highly accessible. In addition, no internal or external fixator is necessary since the adjacent ribs provide a natural fixation. The surgery uses commercially available supplies, is straightforward to learn, and well-tolerated by the animal. The procedure can be carried out with or without removing the surrounding periosteum, and therefore the contribution of the periosteum to repair can be assessed. Results indicate that if the periosteum is retained, robust repair occurs in 1 - 2 months. We expect that use of this protocol will stimulate research into rib repair and that the findings will facilitate the development of new ways to stimulate bone repair in other locations around the body.

The overall goal of this procedure is to successfully resect a portion of bone from the rib of a mouse. The procedure was developed as a model to study large-scale long bone repair.

Una procedura chirurgica per la resezione Rib Mouse: Un modello per le grandi ossa lunghe Repair

This protocol introduces researchers to a new model for large-scale bone repair utilizing the mouse rib. The procedure details the following: preparation ...

Demonstration of the Rat Ischemic Skin Wound Model

The propensity for chronic wounds in humans increases with ageing, disease conditions such as diabetes and impaired cardiovascular function, and unrelieved pressure due to immobility. Animal models have been developed that attempt to mimic these conditions for the purpose of furthering our understanding of the complexity of chronic wounds. The model described herein is a rat ischemic skin flap model that permits a prolonged reduction of blood flow resulting in wounds that become ischemic and resemble a chronic wound phenotype (reduced vascularization, increased inflammation and delayed wound closure). It consists of a bipedicled dorsal flap with 2 ischemic wounds placed centrally and 2 non-ischemic wounds lateral to the flap as controls. A novel addition to this ischemic skin flap model is the placement of a silicone sheet beneath the flap that functions as a barrier and a splint to prevent revascularization and reduce contraction as the wounds heal. Despite the debate of using rats for wound healing studies due to their quite distinct anatomic and physiologic differences compared to humans (i.e., the presence of a panniculus carnosus muscle, short life-span, increased number of hair follicles, and their ability to heal infected wounds) the modifications employed in this model make it a valuable alternative to previously developed ischemic skin flap models.

The rat, due to its size, availability, and rather docile behavior, has been utilized as a research model for many years. The goal of this protocol is to utilize the rat as an ischemic skin wound healing model to provide valuable insight into the pathophysiology of chronic wounds.