This study was supported by NeuralStem, Inc., and Johns Hopkins Project RESTORE.

NOTA: Tutte le procedure sugli animali sono stati approvati dalla cura degli animali e del Comitato uso Johns Hopkins. Macchine per anestesia richiedono ispezione annuale e la calibrazione, se necessario. 1. Anestesia e Posizionamento <ol><li> Anestesia. <ol><li> Eseguire tutte le procedure chirurgiche in anestesia con 2-3% isofluorane. Confermare adeguato livello di anestesia da un pizzico punta e tasso di respirazione. Verificare che il topo non si muove in risposta ad un pizzico piedi. NOTA: Un sussulto indica anestesia che è troppo tardi e può richiedere l&#39;anestesia più lunghi prima di iniziare o una maggiore concentrazione isofluorane. Un tasso di respirazione inferiore 1 soffio ogni 2 secondi è troppo lento indica l&#39;anestesia è troppo elevata e può richiedere alleggerimento della concentrazione isofluorano. </li><li> Fissare l&#39;animale come necessario per evitare il movimento della testa durante la procedura. Mettere una goccia di lidocaina sull&#39;occhio chirurgica. Utilizzare lacrime artificiali ogni 10 minuti per evitare la secchezza mentre sotto anestesia. Dareuna iniezione di buprenorfina di 0,01 mg / kg SC pre-operatorio e poi ogni 6-8 ore, se necessario. </li></ol></li><li> Posizionamento. <ol><li> Mettere i ratti in un telaio stereotassico e tenere in caldo con una piastra elettrica. Bagnare il pelo cuoio capelluto con alcool avendo cura di evitare l&#39;esposizione sugli occhi. Utilizzare strumenti sterili e tecnica sterile per ridurre al minimo il rischio di infezioni post-operatorie. </li></ol></li></ol> 2. Controllo Eye <ol><li> Posizionare un 4-0 sutura nella congiuntiva laterale e legarlo con abbastanza sutura per consentire dolce trazione. </li></ol> 3. Dissection <ol><li> Dissezione iniziale. <ol><li> Fare un pollice un&#39;incisione ~ 1 nella pelle sovrastante cresta orbitale utilizzando una dimensione di 10 bisturi come mostrato in Figura 1. Ritrarre la pelle e la fascia sottostante e sezionare accuratamente via la fascia. Per evitare un eccessivo sanguinamento nel chirurgica, evitare di tagliare i vasi sanguigni, mentre sezionare la fascia. Utilizzare strategicamente plpunte di cotone ACED per fornire emostasi. </li></ol></li><li> Deeper dissezione. NOTA: Con una leggera trazione sulla congiuntiva tirando l&#39;occhio giù e dalla presa, il muscolo orbitale superiore entrerà in vista. Al fine di esporre il nervo ottico, questo muscolo deve essere tagliato e il grasso retro-orbitale rimosso. Il grasso può essere scartato e non deve essere sostituito dopo l&#39;iniezione. Da questo punto, la fascia nervo ottico deve essere visibile come un fascio del nervo ottico stesso, insieme con i vasi sanguigni avvolte in dura (Figura 1). <ol><li> Fai una piccola incisione nella dura con il bisturi o con un ago smussato 31 calibro di rendere penetrante meno traumatico. </li></ol></li></ol> 4. Pipetta Injector <ol><li> Estrarre una micropipetta di vetro ad un diametro di 50-100 micron. Per fornire la stabilità, montare la micropipetta su un micromanipolatore e legarsi a una siringa Hamilton collegato ad una pompa di infusione. </li><li> Tirare le perline (o cellule staminali)ricostituito in un volume 0,5-1,0 microlitri nella micropipetta retrogrado insieme con 0,5 ml di volume di soluzione di blu di metile prima e dopo. NOTA: Una velocità di infusione impostata 0,5-2 microlitri al minuto evita traumi al nervo ottico. </li></ol> 5. Iniezione <ol><li> Abbassare la punta della micropipetta sul nervo ottico appena sopra la dura intaccate. NOTA: Un piccolo, ma vivace movimento della punta di vetro nei risultati del nervo ottico nel minor danno. All&#39;inizio della pompa di infusione, il colorante blu di metile dovrebbe evidenziare l&#39;area del nervo ottico iniettato. Il colorante deve rimanere localizzata all&#39;interno del nervo ottico senza fuoriuscire nello spazio subaracnoideo. </li><li> Seguire la seconda banda di blu di metile per determinare quando l&#39;iniezione di cellule staminali è completa e ruotare la pompa di infusione fuori come la seconda dose di blu di metile viene iniettato. Mantenere la micropipetta ancora all&#39;interno del nervo ottico per 2 minuti per ogni volume 1 microlitri iniettato per prevenire altopressione espulsione al momento della revoca della micropipetta. NOTA: Un approccio alternativo è quello di collegare la punta della pipetta ad una siringa riempita di aria che può essere manipolato manualmente. In entrambi i casi, evitare una forza eccessiva per ridurre al minimo i danni ai nervi ottici. Si consiglia di iniettare più di 2 ml di volume in ogni nervo ottico. </li></ol> 6. Follow-up <ol><li> Immediato. <ol><li> Quando l&#39;iniezione, rimuovere la micropipetta insieme a qualche consiglio di cotone usati per fornire emostasi. Suturare la pelle con 3-0 seta e rimuovere la sutura congiuntivale. </li><li> Tenere ratti caldo su una piastra elettrica fino a quando non emergono dall&#39;anestesia. Non lasciare un animale incustodito fino a quando non ha ripreso conoscenza sufficiente a mantenere decubito sternale. Non restituire un animale che ha subito un intervento chirurgico per la compagnia di altri animali fino alla completa guarigione. </li><li> Se l&#39;animale sta esibendo i segni del dolore, letargia eccessiva, atassia, o Breathin affannosog, amministrare l&#39;analgesia appropriata con buprenorfina per via intramuscolare (0,05 mg / kg) fino a tre volte al giorno. </li></ol></li><li> Lungo termine. <ol><li> Per il prossimo 24-48 ore, osservare il topo per le complicazioni di un intervento chirurgico, tra cui gonfiore o scarico dalla ferita o altri segni di dolore, come vocalizzi, aspetto curvo, non governare, o non mangiare. Considerare consultato un veterinario o l&#39;eutanasia etico, se necessario. </li></ol></li></ol>

<ol><li>Dahlmann-Noor, A., Vijay, S., Jayaram, H., Limb, A., Khaw, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Current+approaches+and+future+prospects+for+stem+cell+rescue+and+regeneration+of+the+retina+and+optic+nerve%5BTitle%5D)+AND+%22Canadian+journal+of+ophthalmology+Journal+canadien+d%27ophtalmologie%22%5BJournal%5D)">Current approaches and future prospects for stem cell rescue and regeneration of the retina and optic nerve.</a> Canadian journal of ophthalmology Journal canadien d'ophtalmologie. 45 (4), 333-341 (2010).</li>
<li>Quigley, H. A., Iglesia, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stem+cells+to+replace+the+optic+nerve%5BTitle%5D)+AND+%22Eye%22%5BJournal%5D)">Stem cells to replace the optic nerve.</a> Eye. 18 (11), 1085-1088 (2004).</li>
<li>Ghaffarieh, A., Levin, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optic+nerve+disease+and+axon+pathophysiology%5BTitle%5D)+AND+%22International+review+of+neurobiology%22%5BJournal%5D)">Optic nerve disease and axon pathophysiology.</a> International review of neurobiology. 105, 1-17 (2012).</li>
<li>Fukui, Y., Hayasaka, S., Bedi, K. S., Ozaki, H. S., Takeuchi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+study+of+the+development+of+the+optic+nerve+in+rats+reared+in+the+dark+during+early+postnatal+life%5BTitle%5D)+AND+%22Journal+of+anatomy%22%5BJournal%5D)">Quantitative study of the development of the optic nerve in rats reared in the dark during early postnatal life.</a> Journal of anatomy. 174, 37-47 (1991).</li>
<li>Celestre, P. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Minimally+invasive+approaches+to+the+cervical+spine%5BTitle%5D)+AND+%22The+Orthopedic+clinics+of+North+America%22%5BJournal%5D)">Minimally invasive approaches to the cervical spine.</a> The Orthopedic clinics of North America. 43 (1), 137-147 (2012).</li>
<li>Hallas, B. H., Wells, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Novel+Technique+for+Multiple+Injections+into+the+Mammalian+Optic+Nerve%5BTitle%5D)+AND+%22Kopf+Carrier%22%5BJournal%5D)">A Novel Technique for Multiple Injections into the Mammalian Optic Nerve.</a> Kopf Carrier. 54, (2001).</li>
<li>Harvey, A. R., Hellstrom, M., Rodger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gene+therapy+and+transplantation+in+the+retinofugal+pathway%5BTitle%5D)+AND+%22Progress+in+brain+research%22%5BJournal%5D)">Gene therapy and transplantation in the retinofugal pathway.</a> Progress in brain research. 175, 151-161 (2009).</li>
<li>Adachi-Usami, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optic+neuritis--from+diagnosis+to+optic+nerve+transplantation%5BTitle%5D)+AND+%22Nippon+Ganka+Gakkai+zasshi%22%5BJournal%5D)">Optic neuritis--from diagnosis to optic nerve transplantation.</a> Nippon Ganka Gakkai zasshi. 104 (12), 841-857 (2000).</li>
<li>Slater, B. J., Vilson, F. L., Guo, Y., Weinreich, D., Hwang, S., Bernstein, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optic+nerve+inflammation+and+demyelination+in+a+rodent+model+of+nonarteritic+anterior+ischemic+optic+neuropathy%5BTitle%5D)+AND+%22Investigative+ophthalmology+%26+visual+science%22%5BJournal%5D)">Optic nerve inflammation and demyelination in a rodent model of nonarteritic anterior ischemic optic neuropathy.</a> Investigative ophthalmology & visual science. 54 (13), 7952-7961 (2013).</li>
<li>Zarbin, M. A., Arlow, T., Ritch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regenerative+nanomedicine+for+vision+restoration%5BTitle%5D)+AND+%22Mayo+Clinic+proceedings%22%5BJournal%5D)">Regenerative nanomedicine for vision restoration.</a> Mayo Clinic proceedings. 88 (12), 1480-1490 (2013).</li>
</ol>

The authors have no meaningful disclosures to report.

Direct injection into the optic nerve of stem cells or other products intended to facilitate regeneration provides a convenient model compared to other means of injections into the CNS. This technique takes less time, requires less total anesthesia, avoids drilling or removing skull or bone tissue, reduces complications rates and allows for more rapid recovery following surgery. 
The most critical steps in this protocol include: 1. Adequate hemostasis in the surgical field to allow clear visua...

Il nervo ottico offre una posizione ideale per il sistema nervoso centrale (SNC) ricerca rigenerativa comprese le condizioni oftalmologiche quali neurite ottica, glaucoma e traumi. Iniezioni di una varietà di cellule staminali hanno né dimostrato l&#39;efficacia o mostrato risultati promettenti nel sostituire persi mielina, aumentando conta assonale e / o prevenire le malattie degenerative. 1,2 Il nervo ottico umano contiene circa 1,2 milioni di assoni parallele viaggiano dalla retina al chiasma con un diametro di circa 3,0-3,5 mm. 3 Per modellare malattie umane in laboratorio, il ratto è stato utilizzato di frequente. Il nervo ottico ratto adulto contiene circa 100.000 assoni in un diametro di circa 0,5 mm. 4 Una delle maggiori limitazioni nella CNS ricerca rigenerativa è l&#39;accesso senza osso diretto. Complicanze e rischi chirurgici per l&#39;animale più elevati, se il cranio o vertebre vengono rimossi. Simile ai benefici dellaapprocci minimamente invasivi nella colonna vertebrale, 5 iniezioni del nervo ottico diretti senza aprire il cranio offrono ridotto complicazioni e un recupero più rapido. Questa tecnica è stata utilizzata in studi precedenti. 6 In questo manoscritto e video che accompagna, dimostriamo una procedura minimamente invasiva per iniettare le cellule staminali nel nervo ottico topo.

<table><tbody><tr><td>Lewis rat</td><td>Charles River</td><td>4</td><td>Any rat strain will work.</td></tr><tr><td>Anesthesia machine</td><td>Surgivet</td><td>CDS9000</td><td>CDS 9000 Small Animal Anesthesia Machine - Pole Mount</td></tr><tr><td>Infusion pump</td><td>Stoelting</td><td>53129</td><td></td></tr><tr><td>Dissection microscope</td><td>National Optical</td><td>409-411-1105</td><td></td></tr><tr><td>Fiber-optic light source</td><td>Fisher Scientific</td><td>12-562-21</td><td></td></tr><tr><td>Dissection and Stereotaxic Instrument</td><td>Stoelting</td><td>51400</td><td></td></tr><tr><td>Pipette Puller</td><td>Kopf</td><td>750</td><td></td></tr><tr><td>Pipettes</td><td>World Precision Instruments</td><td>18150-6</td><td></td></tr><tr><td>Disposable scalpel blades</td><td>Harvard Apparatus</td><td>810-15-021</td><td></td></tr><tr><td>Iridectomy scissors</td><td>Electron Microscopy Sciences</td><td>Uniband LA-4XF</td><td></td></tr></tbody></table>

minimally-invasive technique for injection into rat optic nerve

The rat optic nerve is a useful model for stem cell regeneration research. Direct injection into the rat optic nerve allows delivery into the central nervous system in a minimally-invasive surgery without bone removal. This technique describes an approach to visualization and direct injection of the optic nerve following minor fascial dissection from the orbital ridge, using a conjunctival traction suture to gently pull the eye down and out. Representative examples of an injected optic nerve show successful injection of dyed beads.

Al termine dell&#39;esperimento, i ratti sono stati sacrificati e perfusi con paraformaldeide al 4%. I nervi ottici sono stati accuratamente sezionati e montate per criostato sezionamento. La Figura 2 mostra un esempio di un ratto intera nervo ottico a bassa potenza in cui Evans colorante blu è stato iniettato per visualizzare il sito. La freccia indica la posizione precisa di iniezione. Questo dissezione è stato fatto entro pochi minuti dell&#39;iniezione come indicato dalla diffusione ristretta del ...

Watch this Scientific Journal Video about Minimally-invasive Technique for Injection into Rat Optic Nerve at JoVE.com

Minimally-invasive Technique for Injection into Rat Optic Nerve

Direct injection into the rat optic nerve is useful for regenerative research. We demonstrate a minimally-invasive technique for direct injection into a rat optic nerve that does not involve opening the skull. Using this method, surgical complications are minimized and recovery is more rapid.

Tecnica mini-invasiva per iniezione in Rat nervo ottico

The rat optic nerve is a useful model for stem cell regeneration research. Direct injection into the rat optic nerve allows delivery into the central ...

minimally-invasive-technique-for-injection-into-rat-optic-nerve

Research

JoVE Journal

Neuroscience

9.6K Views.  Johns Hopkins University. Direct injection into the rat optic nerve is useful for regenerative research. We demonstrate a minimally-invasive technique for direct injection into a rat optic nerve that does not involve opening the skull. Using this method, surgical complications are minimized and recovery is more rapid. 

Video: Minimally-invasive Technique for Injection into Rat Optic Nerve

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 ...

Ricerca

Neuroscienze

Intravitreous Injection for Establishing Ocular Diseases Model

Intravitreous injection is a widely used technique in visual sciences research. It can be used to establish animal models with ocular diseases or as direct application of local treatment. This video introduces how to use simple and inexpensive tools to finish the intravitreous injection procedure. Use of a 1 ml syringe, instead of a hemilton syringe, is used. Practical tips for how to make appropriate injection needles using glass pipettes with perfect tips, and how to easily connect the syringe needle with the glass pipette tightly together, are given.
To conduct a good intravitreous injection, there are three aspects to be observed: 1) injection site should not disrupt retina structure; 2) bleeding should be avoided to reduce the risk of infection; 3) lens should be untouched to avoid traumatic cataract. In brief, the most important point is to reduce the interruption of normal ocular structure. To avoid interruption of retina, the superior nasal region of rat eye was chosen. Also, the puncture point of the needle was at the par planar, which was about 1.5 mm from the limbal region of the rat eye. A small amount of vitreous is gently pushed out through the puncture hole to reduce the intraocular pressure before injection. With the 45&deg; injection angle, it is less likely to cause traumatic cataract in the rat eye, thus avoiding related complications and influence from lenticular factors. In this operation, there was no cutting of the conjunctiva and ocular muscle, no bleeding. With quick and minor injury, a successful intravitreous injection can be done in minutes. 
The injection set outlined in this particular protocol is specific for intravitreous injection. However, the methods and materials presented here can also be used for other injection procedures in drug delivery to the brain, spinal cord or other organs in small mammals.

Intravitreous injection is a widely used technique in visual sciences research for ocular diseases or as direct application of local treatment. This video demonstrated a protocol for intravitreous injection using a 1ml syringe with glass pipette. Useful tips about avoiding massive bleeding and lens damage are given.

Iniezione intravitreale per stabilire Modello malattie oculari

Intravitreous injection is a widely used technique in visual sciences research. It can be used to establish animal models with ocular diseases or as ...

Biologia

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

Subretinal injections have been successfully used in both humans and rodents to deliver therapeutic interventions of proteins, viral agents, and cells to the interphotoreceptor/subretinal compartment that has direct exposure to photoreceptors and the retinal pigment epithelium (RPE). Subretinal injections of plasminogen as well as recent preclinical and clinical trials have demonstrated safety and/or efficacy of delivering viral vectors and stem cells to individuals with advanced retinal disease. Mouse models of retinal disease, particularly hereditary retinal dystrophies, are essential for testing these therapies. The most common injection procedure in rodents is to use small transcorneal or transcleral incisions with an anterior approach to the retina. With this approach, the injection needle penetrates the neurosensory retina disrupting the underlying RPE and on insertion can easily nick the lens, causing lens opacification and impairment of noninvasive imaging. Accessing the subretinal space via a transcleral, posterior approach avoids these problems: the needle crosses the sclera approximately 0.5 mm from the optic nerve, without retinal penetration and avoids disrupting the vitreous. Collateral damage is limited to that associated with the focal sclerotomy and the effects of a transient, serous retinal detachment. The simplicity of the method minimizes ocular injury, ensures rapid retinal reattachment and recovery, and has a low failure rate. The minimal damage to the retina and RPE allows for clear assessment of the efficacy and direct effects of the therapeutic agents themselves. This manuscript describes a novel subretinal injection technique that can be used to target viral vectors, pharmacological agents, stem cells or induced pluripotent stem (iPS) cells to the subretinal space in mice with high efficacy, minimal damage, and fast recovery.

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

Subretinal injections are the most common technique for delivering large therapeutic agents such as proteins and viral vectors to photoreceptors and the retinal pigment epithelium. An alternative method in mice that successfully targets the subretinal space with minimal collateral damage and fast recovery times is described here.

Un metodo alternativo e validate iniezione per l&#39;accesso ai sottoretinico Spazio<i&gt; via</i&gt; Un approccio Transcleral posteriore

Subretinal injections have been successfully used in both humans and rodents to deliver therapeutic interventions of proteins, viral agents, and cells to ...

Ultrahigh Resolution Mouse Optical Coherence Tomography to Aid Intraocular Injection in Retinal Gene Therapy Research

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the subretinal space, and to evaluate toxicity and efficacy in vivo. HR-SD-OCT uses near infrared light (800-880 nm) and has optics specifically designed for the unique optics of the mouse eye with sub-2-micron axial resolution. Transgenic mouse models of outer retinal (photoreceptor) degeneration and controls were imaged to assess the disease progression. Pulled glass microneedles were used to deliver sub retinal injections of adeno-associated virus (AAV) or nanoparticles (NP) via a trans-scleral and trans-choroidal approach. Careful positioning of the needle into the subretinal space was required prior to a calibrated pressure injection, which delivers fluid into the sub retinal space. Real time subretinal surgery was conducted on our retinal imaging system (RIS). HR-SD-OCT demonstrated progressive uniform retinal degeneration due to expression of a toxic mutant human mutant rhodopsin (P347S) (RHOP347S) transgene in mice. HR-SD-OCT allows rigorous quantification of all the retinal layers. Outer nuclear layer (ONL) thickness and photoreceptor outer segment length (OSL) measurements correlate with photoreceptor vitality, degeneration, or rescue. The RIS delivery system allows real-time visualization of subretinal injections in neonatal (~P10-14) or adult mice, and HR-SD-OCT immediately determines success of delivery and maps areal extent. HR-SD-OCT is a powerful tool that can evaluate the success of subretinal surgery in mice, in addition to measuring vitality of photoreceptors in vivo. HR-SD-OCT can also be used to identify uniform animal cohorts to evaluate the extent of retinal degeneration, toxicity, and therapeutic rescue in preclinical gene therapy research studies.

Here we demonstrate a novel approach to using high resolution spectral-domain optical coherence tomography (HR-SD-OCT) to assist delivery of gene therapy agents into the subretinal space, assess its areal coverage, and characterize photoreceptor vitality.

Tomografia ottica di coerenza di risoluzione ultraelevata Mouse per aiutare l'iniezione intraoculare nella ricerca retinica terapia genica

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the ...

Medicina

Sheath-Preserving Optic Nerve Transection in Rats to Assess Axon Regeneration and Interventions Targeting the Retinal Ganglion Cell Axon

Retinal ganglion cell (RGC) axons converge at the optic nerve head to convey visual information from the retina to the brain. Pathologies such as glaucoma, trauma, and ischemic optic neuropathies injure RGC axons, disrupt transmission of visual stimuli, and cause vision loss. Animal models simulating RGC axon injury include optic nerve crush and transection paradigms. Each of these models has inherent advantages and disadvantages. An optic nerve crush is generally less severe than a transection and can be used to assay axon regeneration across the lesion site. However, differences in crush force and duration can affect tissue responses, resulting in variable reproducibility and lesion completeness. With optic nerve transection, there is a severe and reproducible injury that completely lesions all axons. However, transecting the optic nerve dramatically alters the blood brain barrier by violating the optic nerve sheath, exposing the optic nerve to the peripheral environment. Moreover, regeneration beyond a transection site cannot be assessed without reapposing the cut nerve ends. Furthermore, distinct degenerative changes and cellular pathways are activated by either a crush or transection injury.
The method described here incorporates the advantages of both optic nerve crush and transection models while mitigating the disadvantages. Hydrostatic pressure delivered into the optic nerve by microinjection completely transects the optic nerve while maintaining the integrity of the optic nerve sheath. The transected optic nerve ends are reapposed to allow for axon regeneration assays. A potential limitation of this method is the inability to visualize the complete transection, a potential source of variability. However, visual confirmation that the visible portion of the optic nerve has been transected is indicative of a complete optic nerve transection with 90-95% success. This method could be applied to assess axon regeneration promoting strategies in a transection model or investigate interventions that target the axonal compartments.

<meta charset="utf8"></head><body>Transezione del nervo ottico con conservazione della guaina nei ratti per valutare la rigenerazione degli assoni e gli interventi mirati all'assone delle cellule g

This protocol describes an optic nerve transection that preserves the optic nerve sheath in rats. Hydrostatic pressure from microinjections into the optic nerve generate a complete transection, allowing for sutureless reapposition of the transected optic nerve ends and direct targeting of the axonal compartment in a transection model.

Transezione del nervo ottico con conservazione della guaina nei ratti per valutare la rigenerazione degli assoni e gli interventi mirati all'assone delle cellule gangliari retiniche

Retinal ganglion cell (RGC) axons converge at the optic nerve head to convey visual information from the retina to the brain. Pathologies such as ...

A Neonatal Rodent Model of Retroorbital Vein Injection

Intravenous (iv) injection is the most used route of drug administration in neonates in the clinical setting. Therefore, retroorbital vein injection is an important method for compound administration in research, where successful proof-of-concept studies can progress into much-needed neonatal clinical trials. Most intravenous studies in neonatal rodents use the superficial temporal/facial vein. However, retroorbital injection becomes unreliable in neonatal rodents older than 2 days after the skin darkens and the vein is no longer visible. In the present protocol, we describe the retroorbital injection of the venous sinus in both the neonatal mouse and rat at ages when the superficial temporal vein is no longer visible, but the eyes have not opened yet. Eye-opening facilitates retro-orbital injection by enabling the researcher to clearly see that they are not perforating the eye when inserting the needle. We demonstrate that this technique can be performed in a reliable and reproducible manner without adverse effects. Additionally, we show that it can be used in many studies, such as administering compounds to study neonatal brain injury.

This protocol aims to demonstrate a reproducible venous administration route that can be used in rats and mice throughout the neonatal period. This procedure is important for preclinical rodent studies that wish to mirror drug administration in neonatal care units primarily using intravenous administration.

Un modello di roditore neonatale di iniezione venosa retroorbitaria

Intravenous (iv) injection is the most used route of drug administration in neonates in the clinical setting. Therefore, retroorbital vein injection is an ...

Direct Vagus Nerve Injection Protocol For Rats

There is a relative abundance of strategies and methodologies to facilitate drug delivery to the central nervous system. However, drug delivery directly to the peripheral nervous system is less common, with fewer detailed methods publications available to aid researchers. Here, we describe a direct nerve injection method for peripheral nervous system drug delivery, using the vagus nerve as a model nerve. This method can be used in the treatment of autonomic nervous system disorders through targeting of the left vagus nerve, although this general injection method can be extrapolated to injection of other nerves with minor modification. This method explains all critical steps involved in the procedure involving microsurgery in anesthetized adult rats under a dissecting microscope. The use of a tracking dye is described to facilitate the monitoring of injection fidelity in real time. Illustrations of successful and failed injections are provided. If carried out properly, direct vagus nerve injections can be conducted in a safe manner that is well-tolerated by the rat without post-delivery complications. For example, once the surgeons were trained in this method, six out of six rats were successfully injected without any complications. This method of direct nerve injection for preclinical rat studies is capable of delivering agents (including but not limited to gene therapy) to peripheral nerves.

We present a protocol for direct vagus nerve injection in rats, enabling drug delivery directly into the nerve without post-injection complications. This method applies to preclinical neurological studies involving autonomic nervous system manipulation. It can be used for direct nerve injection for other nerves in rats and other species, with necessary modifications.

Protocollo di iniezione diretta del nervo vago per i ratti

There is a relative abundance of strategies and methodologies to facilitate drug delivery to the central nervous system. However, drug delivery directly ...

Ocular Therapeutic Delivery and Advanced Tissue Retrieval in Adult Rats

Therapeutic delivery to the posterior segment of the eye, including the retina and optic nerve, is complicated by the presence of blood-brain and blood-retinal barriers. Small animal models, such as rats, are utilized for studying various ocular pathologies. While therapeutic delivery to the posterior eye is challenging, achieving it is essential for treating ocular disorders, many of which require validation in small animal models for translational relevance. Therefore, two posterior therapeutic delivery techniques are presented: intravitreal injection (IVI) and retrobulbar injection (RBI) for use in adult rats. Additionally, a method for the en bloc removal of the eyes and optic nerves is introduced for various histological and molecular analysis techniques. The dissection protocol enables full observation of the neuro-visual system while minimizing post-mortem injury to retinal and optic nerve tissues. Successful delivery of the therapeutic cyclosporine to the retina and optic nerve was achieved, with detectable concentrations observed twenty-four hours after injection using both IVI and RBI. Furthermore, en bloc retina and nerve samples were successfully extracted for full eye histological tissue analysis, facilitating comprehensive observation of the retina and the wider neuro-visual system.

This study presents a methodology for the delivery of therapeutics into the retina and optic nerves of the adult rat. Additionally, a unique tissue retrieval method is introduced for a top-down en bloc collection of the optic nerve and retina in an adult rat.

Somministrazione terapeutica oculare e recupero avanzato dei tessuti nei ratti adulti

Therapeutic delivery to the posterior segment of the eye, including the retina and optic nerve, is complicated by the presence of blood-brain and ...

Transconjunctival Approach for Injection into the Rat Optic Nerve

The rat serves as an important model for studying disorders of the optic nerve, including heritable, traumatic, neoplastic, and autoimmune conditions. Accessing the rat optic nerve for experimental manipulations and injections is challenging due to the small size of the orbit and the vascularity of the surrounding orbital tissues. Previous techniques have involved a cutaneous incision, which carries a higher risk of infection and requires wound closure. This study aims to describe a unique, less invasive, and potentially more efficient approach to accessing the rat optic nerve. A transconjunctival incision, along with a rat spinal cord hook, is used to isolate the optic nerve for experimental injections. In these experiments, an adenoviral vector is injected into the optic nerve with the goal of using this technique for direct gene therapy delivery in the future. This technique can also be applied by investigators requiring access to the rat optic nerve for other experimental studies, including drug delivery and optic nerve injury research.

This protocol describes a unique technique using a transconjunctival approach to access the rat optic nerve for injections.

Approccio transcongiuntivale per l'iniezione nel nervo ottico del ratto

The rat serves as an important model for studying disorders of the optic nerve, including heritable, traumatic, neoplastic, and autoimmune conditions. ...

Tecnica mini-invasiva per iniezione in Rat nervo ottico

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