Il lavoro contenuto in questo documento è stata sostenuta dal Dr. Douglas H. Johnson Award per il Glaucoma Research dalla American Health Assistance Foundation (XL), la William &amp; Mary Greve Scholar Award speciale della ricerca per prevenire la cecità (XL), il Illinois Società per la Prevenzione della Cecità (HC) e NIH concedere R01EY019034 (XL).

Procedure C57BL/6J topi (Jackson Laboratory, Bar Harbor, ME) sono allevati presso la Northwestern University Animal Care strumento. Tutti gli animali sono utilizzati secondo protocolli approvati dalla Northwestern University Institutional Animal Care e del Comitato uso e conforme alle linee guida sull&#39;uso degli animali in Neuroscience Research dal NIH. 1. Fotocoagulazione laser La procedura di fotocoagulazione laser viene modificata dai protocolli precedentemente pubblicati 5-7. <ol><li> Anestetizzare un 40-60 giorni vecchio mouse con un&#39;iniezione intraperitoneale di ketamina (100 mg / kg, Butler Schein la salute degli animali, OH) e xilazina (10 mg / kg, Lloyd Inc. di Iowa, Shenandoah, IA). </li><li> Dilatare la pupilla dell&#39;occhio destro dell&#39;animale sperimentale per il trattamento topico con una o due gocce di soluzione di solfato di atropina 1% (Alcon Labs, Inc., Fort Worth, TX). </li><li> Dopo midriasi, appiattire l&#39;unacamera di nterior per migliorare l&#39;induzione laser 6. Inserire una micropipetta di vetro con punta acuminata (World Precision Instruments Inc, Sarasota, FL) nello spazio anteriore sotto la lampada a fessura (SL-3E, Topcon, Oakland, NJ) per drenare il liquido nella camera anteriore. </li><li> Frena il mouse in un supporto cono di plastica (Braintree Sci Inc., MA) e legato su una piattaforma fatta in casa (vedere Figura 1A). Tenere il mouse con dispositivo di immobilizzazione ed espone l&#39;occhio destro del mouse per la fonte di luce dietro la lampada a fessura. Allineare l&#39;occhio destro del mouse anestetizzato sotto la lampada a fessura. </li><li> Tenendo premuto il dispositivo di immobilizzazione del mouse con entrambe le mani, applicare l&#39;illuminazione laser per il limbus corneale utilizzando un laser ad Argon (Ultima 2000SE, Coherent, Santa Clara, CA). Trasporti circa 80-100 punti laser (514 nm, 100 mW, 50 msec impulsi e 200 pronti micron) perpendicolarmente intorno alla circonferenza del trabecolato. I topi C57BL / 6 hanno iride pigmentata che funge da barriera per ogni pPotenziali energia randagio 7. </li><li> Instillare topico 0,5% moxifloxacina (Alcon Labs, Inc., Fort Worth, TX) sulla superficie oculare per disinfettare la zona trattata con laser e 0,5% Proparacaine (Bausch &amp; Lomb, Rochester, NY) per alleviare il dolore. </li><li> Tenere l&#39;animale su una piastra elettrica (Sunbeam Products Inc, Boca Raton, FL) per il recupero per circa un&#39;ora fino a quando non è completamente sveglio. </li><li> L&#39;occhio sinistro è trattata per servire come controllo. </li></ol> 2. IOP Misure <ol><li> Posizionare il mouse svegli in un tubo di caricare nel porta cono di plastica e poi frenare sulla piattaforma (vedi Figura 2A). </li><li> Consentire 9:55 minuti per lasciare il mouse ottenere adattato alla posizione di titolare. Avvicinatevi al tonometro rimbalzo (TonoLab, coloniale Medical Supply, Franconia, NH) per l&#39;occhio del mouse fino a quando la punta della sonda è di 2-3 mm di distanza dalla superficie della cornea 14. </li><li> Premere il tasto di misurazione per lasciare la punta della sonda ha colpito la superficie del centrodella cornea con delicatezza. Tre set consecutivi di sei misure di IOP dello stesso occhio sono acquisite e mediate come la IOP dell&#39;occhio. L&#39;occhio controllo non trattato è sempre misurato prima di ottenere una lettura di base per l&#39;occhio laser-trattato che viene misurato dopo. </li></ol> 3. Prova Optomotor Acuità visiva e la sensibilità al contrasto sono testati 14,15. Due occhi del singoli topi sono esaminati separatamente invertendo il senso reticolo deriva, cioè un reticolo deriva in senso orario viene utilizzato per identificare la funzione visiva dell&#39;occhio sinistro e una grata alla deriva in senso antiorario per l&#39;occhio destro 16. Ogni test richiede circa 15 minuti e si ripete da due osservatori in modo indipendente. <ol><li> Posizionare il mouse e lasciare il mouse per muoversi liberamente su una piattaforma elevata, circondato da quattro monitor per computer (Figura 3A-B). </li><li> Impostare i monitor in modo da visualizzare alla deriva orizzontalmente sinusoidalegrate come stimoli visivi con luminanza media di 39 cd / m 2. La direzione di spostamento del reticolo deve alternare consecutivamente tra senso orario e antiorario. </li><li> Analizzare i movimenti dell&#39;animale. Movimenti degli animali in-concerto con le grate alla deriva sono considerati &quot;positivi&quot; entro 15 secondi dopo lo stimolo visivo è accesa e poi gradualmente aumentata. La risposta di elicitare massima stimolo visivo è definita come acuità visiva dell&#39;animale 17. </li><li> Esaminare la sensibilità al contrasto a tre pre-selezionati frequenze spaziali: 0.075, 0.16, e 0,3 cicli per grado (CPD). La soglia di contrasto per ciascun occhio è definita come la più bassa contrasto che suscita risposte visive alla frequenza prefissata. La sensibilità al contrasto è il reciproco della soglia 17. </li></ol>

<ol>
	<li>Gupta, N., Yucel, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glaucoma+as+a+neurodegenerative+disease.">Glaucoma as a neurodegenerative disease.</a> Curr. Opin. Ophthalmol. 18, 110-114 (2007).</li><li>Quigley, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+death+in+glaucoma.">Neuronal death in glaucoma.</a> Prog. Retin. Eye Res. 18, 39-57 (1999).</li><li>McKinnon, S. J., Schlamp, C. L., Nickells, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+retinal+ganglion+cell+death+and+glaucoma.">Mouse models of retinal ganglion cell death and glaucoma.</a> Experimental Eye Research. 88, 816-824 (2009).</li><li>Pang, I. H., Clark, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+models+for+glaucoma+retinopathy+and+optic+neuropathy.">Rodent models for glaucoma retinopathy and optic neuropathy.</a> J. Glaucoma. 16, 483-505 (2007).</li><li>Levkovitch-Verbin, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translimbal+laser+photocoagulation+to+the+trabecular+meshwork+as+a+model+of+glaucoma+in+rats.">Translimbal laser photocoagulation to the trabecular meshwork as a model of glaucoma in rats.</a> Investigative Ophthalmology &amp; Visual Science. 43, 402-410 (2002).</li><li>Aihara, M., Lindsey, J. D., Weinreb, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+mouse+ocular+hypertension:+establishment+of+the+model.">Experimental mouse ocular hypertension: establishment of the model.</a> Investigative Ophthalmology &amp; Visual Science. 44, 4314-4320 (2003).</li><li>Grozdanic, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-induced+mouse+model+of+chronic+ocular+hypertension.">Laser-induced mouse model of chronic ocular hypertension.</a> Investigative ophthalmology &amp; visual science. 44, 4337-4346 (2003).</li><li>Sappington, R. M., Carlson, B. J., Crish, S. D., Calkins, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microbead+occlusion+model:+a+paradigm+for+induced+ocular+hypertension+in+rats+and+mice.">The microbead occlusion model: a paradigm for induced ocular hypertension in rats and mice.</a> Investigative ophthalmology &amp; visual science. 51, 207-216 (2010).</li><li>Ding, C., Wang, P., Tian, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+general+anesthetics+on+IOP+in+elevated+IOP+mouse+model.">Effect of general anesthetics on IOP in elevated IOP mouse model.</a> Experimental Eye Research. 92, 512-520 (2011).</li><li>Kalesnykas, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+ganglion+cell+morphology+after+optic+nerve+crush+and+experimental+glaucoma.">Retinal ganglion cell morphology after optic nerve crush and experimental glaucoma.</a> Investigative Ophthalmology &amp; Visual Science. 53, 3847-3857 (2012).</li><li>Shareef, S. R., Garcia-Valenzuela, E., Salierno, A., Walsh, J., Sharma, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+ocular+hypertension+following+episcleral+venous+occlusion+in+rats.">Chronic ocular hypertension following episcleral venous occlusion in rats.</a> Experimental Eye Research. 61, 379-382 (1995).</li><li>Chiu, K., Chang, R., So, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-induced+chronic+ocular+hypertension+model+on+SD+rats.">Laser-induced chronic ocular hypertension model on SD rats.</a> J. Vis. Exp. (10), e549 (2007).</li><li>Fu, C. T., Sretavan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-induced+ocular+hypertension+in+albino+CD-1+mice.">Laser-induced ocular hypertension in albino CD-1 mice.</a> Investigative Ophthalmology &amp; Visual Science. 51, 980-990 (2010).</li><li>Rangarajan, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+visual+deficits+in+aging+DBA/2J+mice+by+two+behavioral+assays.">Detection of visual deficits in aging DBA/2J mice by two behavioral assays.</a> Curr. Eye Res. 36, 481-491 (2011).</li><li>Wang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direction-specific+disruption+of+subcortical+visual+behavior+and+receptive+fields+in+mice+lacking+the+beta2+subunit+of+nicotinic+acetylcholine+receptor.">Direction-specific disruption of subcortical visual behavior and receptive fields in mice lacking the beta2 subunit of nicotinic acetylcholine receptor.</a> J. Neurosci. 29, 12909-12918 (2009).</li><li>Douglas, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Independent+visual+threshold+measurements+in+the+two+eyes+of+freely+moving+rats+and+mice+using+a+virtual-reality+optokinetic+system.">Independent visual threshold measurements in the two eyes of freely moving rats and mice using a virtual-reality optokinetic system.</a> Visual Neuroscience. 22, 677-684 (2005).</li><li>Prusky, G. T., Alam, N. M., Beekman, S., Douglas, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+quantification+of+adult+and+developing+mouse+spatial+vision+using+a+virtual+optomotor+system.">Rapid quantification of adult and developing mouse spatial vision using a virtual optomotor system.</a> Investigative Ophthalmology &amp; Visual Science. 45, 4611-4616 (2004).</li><li>Morrison, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rat+model+of+chronic+pressure-induced+optic+nerve+damage.">A rat model of chronic pressure-induced optic nerve damage.</a> Experimental Eye Research. 64, 85-96 (1997).</li><li>Cone, F. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+anesthesia,+mouse+strain+and+age+on+intraocular+pressure+and+an+improved+murine+model+of+experimental+glaucoma.">The effects of anesthesia, mouse strain and age on intraocular pressure and an improved murine model of experimental glaucoma.</a> Experimental Eye Research. 99, 27-35 (2012).</li><li>Liu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain-derived+neurotrophic+factor+and+TrkB+modulate+visual+experience-dependent+refinement+of+neuronal+pathways+in+retina.">Brain-derived neurotrophic factor and TrkB modulate visual experience-dependent refinement of neuronal pathways in retina.</a> J. Neurosci. 27, 7256-7267 (2007).</li><li>Liu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+neonatal+development+of+retinal+ganglion+cell+dendrites+by+neurotrophin-3+overexpression.">Regulation of neonatal development of retinal ganglion cell dendrites by neurotrophin-3 overexpression.</a> The Journal of Comparative Neurology. 514, 449-458 (2009).</li><li>Sun, W., Li, N., He, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+morphological+survey+of+mouse+retinal+ganglion+cells.">Large-scale morphological survey of mouse retinal ganglion cells.</a> The Journal of Comparative Neurology. 451, 115-126 (2002).</li><li>Feng, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained+Ocular+Hypertension+Induces+Dendritic+Degeneration+of+Mouse+Retinal+Ganglion+Cells+that+Depends+on+Cell-type+and+Location.">Sustained Ocular Hypertension Induces Dendritic Degeneration of Mouse Retinal Ganglion Cells that Depends on Cell-type and Location.</a> Investigative Ophthalmology &amp; Visual Science. , (2013).</li></ol>

Gli autori dichiarano di non avere interessi finanziari in competizione. Gli autori sono dipendenti a tempo pieno della Northwestern University. Gli autori hanno ricevuto alcun finanziamento che è stato fornito da aziende che producono reagenti e strumenti utilizzati in questo articolo.

Segnaliamo soprattutto che l&#39;ipertensione oculare sostenuta può essere indotta da illuminazione laser negli occhi del mouse. Rispetto al modello iniezione salina 18 e il modello cauterizzazione vena 11 entrambi richiedono estese competenze microchirurgia, l&#39;illuminazione laser è relativamente semplice e facile da eseguire. Di solito siamo in grado di eseguire l&#39;illuminazione laser per 4-6 topi in 2-3 ore. I passi fondamentali per raggiungere sostenuta IOP elevazione sono la camera ant...

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent</td>
 </tr>
 <tr>
 <td>moxifloxacin</td>
 <td>Alcon Labs, Inc.</td>
 <td>NDC 0065-4013-03</td>
 <td>0.5 %, Rx only</td>
 </tr>
 <tr>
 <td>Proparacaine Hydrochloride</td>
 <td>Bausch &amp; Lomb</td>
 <td>NDC 24208-730-06</td>
 <td>0.5 %, Rx only</td>
 </tr>
 <tr>
 <td>Ophthalmic Solution USP</td>
 <td>Bausch &amp; Lomb</td>
 <td>NDC 24208-730-06</td>
 <td>.5 %, Rx only</td>
 </tr>
 <tr>
 <td>ketamine</td>
 <td>Butler Schein Animal Health</td>
 <td>NDC 11695-0550-1</td>
 <td>100 mg / kg </td>
 </tr>
 <tr>
 <td>xylazine</td>
 <td>LLOYD Inc. of Iowa</td>
 <td>NADA 139-236</td>
 <td>10 mg / kg </td>
 </tr>
 <tr>
 <td>atropine sulfate solution</td>
 <td>Alcon Labs, Inc.</td>
 <td>NDC 61314-303-02</td>
 <td>1 %, Rx only</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment </td>
 </tr>
 <tr>
 <td>Slit Lamp, TOPCON </td>
 <td>Visual Systems Inc</td>
 <td>SL-3E</td>
 <td>powered by PS-30A</td>
 </tr>
 <tr>
 <td>OptoMotry 1.8.0 virtual</td>
 <td>CerebralMechanics Inc.</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>opto-kinetic testing system</td>
 <td>CerebralMechanics Inc.</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Tonometer, TonoLab, for mice</td>
 <td>Colonial Medical Supply</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Heating pad</td>
 <td>Sunbeam Products Inc</td>
 <td>722-810</td>
 <td></td>
 </tr>
 <tr>
 <td>Argon laser </td>
 <td>Coherent Inc</td>
 <td>Ultima 2000SE</td>
 <td></td>
 </tr>
 <tr>
 <td>DECAPICONE Plastic cone holder </td>
 <td>Braintree Sci Inc.</td>
 <td>MDC-200</td>
 <td>for mouse</td>
 </tr>
 </tbody>
 </table>

a laser-induced mouse model of chronic ocular hypertension to characterize visual defects

Glaucoma, frequentemente associata a elevata pressione intraoculare (IOP), è una delle principali cause di cecità. Abbiamo cercato di stabilire un modello murino di ipertensione oculare per imitare ad alta tensione umana glaucoma. Qui illuminazione laser è applicato al limbus corneale per fotocoagulare il deflusso acquosa, inducendo chiusura dell&#39;angolo. Le variazioni di IOP sono controllati mediante un tonometro rimbalzo prima e dopo il trattamento laser. Un test comportamentale optomotor viene utilizzato per misurare corrispondenti variazioni della capacità visiva. Il risultato rappresentante di un topo che si è sviluppato subito dopo IOP elevazione illuminazione laser è indicato. Un acuità visiva diminuita e la sensibilità al contrasto sono osservati in questo oculare ipertensiva mouse. Insieme, il nostro studio introduce un sistema modello prezioso per studiare la degenerazione neuronale e dei meccanismi molecolari sottostanti nei topi glaucomatosi.

Come descritto nelle Procedure, illuminazione laser è finalizzata alla trabecolato nella regione limbare fotocoagulare per il deflusso acquosa, inducendo chiusura di angolo (Figura 1). Occhi più esposte al laser senza significativi danni fisici, distacco pigmento o infezione, in linea con i risultati precedenti 6. Quando un piccolo gruppo di topi (meno del 5% di tutti gli animali al laser) espone i segni fisici di gravi danni, come palle sgonfie occhio, grave cataratta, distacco significati...

Watch this Scientific Journal Video about A Laser-induced Mouse Model of Chronic Ocular Hypertension to Characterize Visual Defects at JoVE.com

A Laser-induced Mouse Model of Chronic Ocular Hypertension to Characterize Visual Defects

Ipertensione oculare cronica è indotta mediante fotocoagulazione laser del trabecolato negli occhi del mouse. La pressione intraoculare (IOP) è elevato per diversi mesi dopo il trattamento laser. La diminuzione di acuità visiva e la sensibilità al contrasto di animali da esperimento sono monitorati utilizzando il test optomotor.

Un modello di topo indotta da laser di cronica ipertensione oculare per caratterizzare difetti visivi

Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model ...

a-laser-induced-mouse-model-chronic-ocular-hypertension-to

Research

JoVE Journal

Medicine

12.9K Views.  Northwestern University. Ipertensione oculare cronica è indotta mediante fotocoagulazione laser del trabecolato negli occhi del mouse. La pressione intraoculare (IOP) è elevato per diversi mesi dopo il trattamento laser. La diminuzione di acuità visiva e la sensibilità al contrasto di animali da esperimento sono monitorati utilizzando il test optomotor.

Video: A Laser-induced Mouse Model of Chronic Ocular Hypertension to Characterize Visual Defects

Driving Simulation in the Clinic: Testing Visual Exploratory Behavior in Daily Life Activities in Patients with Visual Field Defects

Patients suffering from homonymous hemianopia after infarction of the posterior cerebral artery (PCA) report different degrees of constraint in daily life, despite similar visual deficits. We assume this could be due to variable development of compensatory strategies such as altered visual scanning behavior. Scanning compensatory therapy (SCT) is studied as part of the visual training after infarction next to vision restoration therapy. SCT consists of learning to make larger eye movements into the blind field enlarging the visual field of search, which has been proven to be the most useful strategy1, not only in natural search tasks but also in mastering daily life activities2. Nevertheless, in clinical routine it is difficult to identify individual levels and training effects of compensatory behavior, since it requires measurement of eye movements in a head unrestrained condition. Studies demonstrated that unrestrained head movements alter the visual exploratory behavior compared to a head-restrained laboratory condition3. Martin et al.4 and Hayhoe et al.5 showed that behavior demonstrated in a laboratory setting cannot be assigned easily to a natural condition. Hence, our goal was to develop a study set-up which uncovers different compensatory oculomotor strategies quickly in a realistic testing situation: Patients are tested in the clinical environment in a driving simulator. SILAB software (Wuerzburg Institute for Traffic Sciences GmbH (WIVW)) was used to program driving scenarios of varying complexity and recording the driver's performance. The software was combined with a head mounted infrared video pupil tracker, recording head- and eye-movements (EyeSeeCam, University of Munich Hospital, Clinical Neurosciences).
The positioning of the patient in the driving simulator and the positioning, adjustment and calibration of the camera is demonstrated. Typical performances of a patient with and without compensatory strategy and a healthy control are illustrated in this pilot study. Different oculomotor behaviors (frequency and amplitude of eye- and head-movements) are evaluated very quickly during the drive itself by dynamic overlay pictures indicating where the subjects gaze is located on the screen, and by analyzing the data. Compensatory gaze behavior in a patient leads to a driving performance comparable to a healthy control, while the performance of a patient without compensatory behavior is significantly worse. The data of eye- and head-movement-behavior as well as driving performance are discussed with respect to different oculomotor strategies and in a broader context with respect to possible training effects throughout the testing session and implications on rehabilitation potential.

Patients with visual deficits after stroke report about different constraints in daily life most likely due to variable compensatory strategies, which are difficult to differentiate in clinical routine. We present a clinical set-up which allows measurement of different compensatory head- and eye-movement-strategies and evaluating their effects on driving performance.

Simulazione di guida presso la Clinica: Test di Visual comportamento esplorativo in attività della vita quotidiana in pazienti con difetti del campo visivo

Patients suffering from homonymous hemianopia after infarction of the posterior cerebral artery (PCA) report different degrees of constraint in daily ...

Ricerca

Medicina

5/6th Nephrectomy in Combination with High Salt Diet and Nitric Oxide Synthase Inhibition to Induce Chronic Kidney Disease in the Lewis Rat

Chronic kidney disease (CKD) is a global problem. Slowing CKD progression is a major health priority. Since CKD is characterized by complex derangements of homeostasis, integrative animal models are necessary to study development and progression of CKD. To study development of CKD and novel therapeutic interventions in CKD, we use the 5/6th nephrectomy ablation model, a well known experimental model of progressive renal disease, resembling several aspects of human CKD. The gross reduction in renal mass causes progressive glomerular and tubulo-interstitial injury, loss of remnant nephrons and development of systemic and glomerular hypertension. It is also associated with progressive intrarenal capillary loss, inflammation and glomerulosclerosis. Risk factors for CKD invariably impact on endothelial function. To mimic this, we combine removal of 5/6th of renal mass with nitric oxide (NO) depletion and a high salt diet. After arrival and acclimatization, animals receive a NO synthase inhibitor (NG-nitro-L-Arginine) (L-NNA) supplemented to drinking water (20 mg/L) for a period of 4 weeks, followed by right sided uninephrectomy. One week later, a subtotal nephrectomy (SNX) is performed on the left side. After SNX, animals are allowed to recover for two days followed by LNNA in drinking water (20 mg/L) for a further period of 4 weeks. A high salt diet (6%), supplemented in ground chow (see time line Figure 1), is continued throughout the experiment. Progression of renal failure is followed over time by measuring plasma urea, systolic blood pressure and proteinuria. By six weeks after SNX, renal failure has developed. Renal function is measured using 'gold standard' inulin and para-amino hippuric acid (PAH) clearance technology. This model of CKD is characterized by a reduction in glomerular filtration rate (GFR) and effective renal plasma flow (ERPF), hypertension (systolic blood pressure&gt;150 mmHg), proteinuria (&gt; 50 mg/24 hr) and mild uremia (&gt;10 mM). Histological features include tubulo-interstitial damage reflected by inflammation, tubular atrophy and fibrosis and focal glomerulosclerosis leading to massive reduction of healthy glomeruli within the remnant population (&lt;10%). Follow-up until 12 weeks after SNX shows further progression of CKD.

A two-stage method to establish chronic kidney disease (CKD) in the Lewis rat by surgically removing 5/6th of renal mass is described. Combination of the surgical procedure, NOS-inhibition and a high-salt diet leads to a model resembling human CKD, allowing study of causal mechanisms and development of novel therapeutic interventions.

5/6th nefrectomia in combinazione con dieta di sale e di ossido nitrico sintasi Inibizione di indurre la malattia renale cronica nel ratto Lewis

Chronic kidney disease (CKD) is a global problem. Slowing CKD progression is a major health priority. Since CKD is characterized by complex derangements ...

Adult Mouse Venous Hypertension Model: Common Carotid Artery to External Jugular Vein Anastomosis.

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model creating an artificial fistula between the common carotid artery (CCA) and the external jugular vein (EJV) has been widely described and proved technically feasible. This construct provokes a consistent cerebral venous hypertension (CVH), and therefore has helped studying the contribution of venous hypertension to formation, clinical symptoms, and prognosis of brain AVMs and dural AVFs. Equivalent mice models have been only scarcely described and have shown trouble with stenosis of the fistula. An established murine model would allow the study of not only pathophysiology but also potential genetic therapies for these cerebrovascular diseases.
We present a model of arteriovenous fistula that produces a durable intracranial venous hypertension in the mouse. Microsurgical anastomosis of the murine CCA and EJV can be difficult due to diminutive anatomy and frequently result in a non-patent fistula. In this step-by-step protocol we address all the important challenges encountered during this procedure. Avoiding excessive retraction of the vein during the exposure, using 11-0 sutures instead of 10-0, and making a carefully planned end-to-side anastomosis are some of the critical steps. Although this method requires advanced microsurgical skills and a longer learning curve that the equivalent in the rat, it can be consistently developed.
This novel model has been designed to integrate transgenic mouse techniques with a previously well-established experimental system that has proved useful to study brain AVMs and dural AVFs. By opening the possibility of using transgenic mice, a broader spectrum of valid models can be achieved and genetic treatments can also be tested. The experimental construct could also be further adapted to the study of other cerebrovascular diseases related with venous hypertension such as migraine, transient global amnesia, transient monocular blindness, etc.

We describe a method for creating a reliable model of cerebral venous hypertension in the adult mouse. This model has been widely described and tested in the rat. This new counterpart in the mice opens the possibility of using genetic modified animals and thereby broadens the applications of the model.

Topo adulto venosa Ipertensione Modello: carotide comune di External Vena giugulare anastomosi.

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model ...

Assessment of Vascular Function in Patients With Chronic Kidney Disease

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this is only partially explained by traditional CVD risk factors. Vascular dysfunction is an important non-traditional risk factor, characterized by vascular endothelial dysfunction (most commonly assessed as impaired endothelium-dependent dilation [EDD]) and stiffening of the large elastic arteries. While various techniques exist to assess EDD and large elastic artery stiffness, the most commonly used are brachial artery flow-mediated dilation (FMDBA) and aortic pulse-wave velocity (aPWV), respectively. Both of these noninvasive measures of vascular dysfunction are independent predictors of future cardiovascular events in patients with and without kidney disease. Patients with CKD demonstrate both impaired FMDBA, and increased aPWV. While the exact mechanisms by which vascular dysfunction develops in CKD are incompletely understood, increased oxidative stress and a subsequent reduction in nitric oxide (NO) bioavailability are important contributors. Cellular changes in oxidative stress can be assessed by collecting vascular endothelial cells from the antecubital vein and measuring protein expression of markers of oxidative stress using immunofluorescence. We provide here a discussion of these methods to measure FMDBA, aPWV, and vascular endothelial cell protein expression.

The degree of vascular dysfunction and contributing physiological mechanisms can be assessed in patients with chronic kidney disease by measuring brachial artery flow-mediated dilation, aortic pulse-wave velocity, and vascular endothelial cell protein expression.

Valutazione della funzione vascolare nei pazienti con malattia renale cronica

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this ...

A Mouse Model for Laser-induced Choroidal Neovascularization

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) research. By administering targeted laser injury to the RPE and Bruch&#8217;s membrane, the procedure induces angiogenesis, modeling the hallmark pathology observed in neovascular AMD. 
First developed in non-human primates, the laser-induced CNV model has come to be implemented into many other species, the most recent of which being the mouse. Mouse experiments are advantageously more cost-effective, experiments can be executed on a much faster timeline, and they allow the use of various transgenic models. The miniature size of the mouse eye, however, poses a particular challenge when performing the procedure. Manipulation of the eye to visualize the retina requires practice of fine dexterity skills as well as simultaneous hand-eye-foot coordination to operate the laser. However, once mastered, the model can be applied to study many aspects of neovascular AMD such as molecular mechanisms, the effect of genetic manipulations, and drug treatment effects. 
The laser-induced CNV model, though useful, is not a perfect model of the disease. The wild-type mouse eye is otherwise healthy, and the chorio-retinal environment does not mimic the pathologic changes in human AMD. Furthermore, injury-induced angiogenesis does not reflect the same pathways as angiogenesis occurring in an age-related and chronic disease state as in AMD.
Despite its shortcomings, the laser-induced CNV model is one of the best methods currently available to study the debilitating pathology of neovascular AMD. Its implementation has led to a deeper understanding of the pathogenesis of AMD, as well as contributing to the development of many of the AMD therapies currently available.

Here, we present the mouse laser-induced choroidal neovascularization (CNV) protocol, an experimental model that re-creates the vascular hallmarks of neovascular age-related macular degeneration (AMD). Once mastered, it can reliably and effectively induce CNV as a model system to test various experimental measures.

Un modello di topo per Laser-indotta della coroide neovascolarizzazione

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) ...

A Magnetic Microbead Occlusion Model to Induce Ocular Hypertension-Dependent Glaucoma in Mice

The use of rodent models of glaucoma has been essential to understand the molecular mechanisms that underlie the pathophysiology of this multifactorial neurodegenerative disease. With the advent of numerous transgenic mouse lines, there is increasing interest in inducible murine models of ocular hypertension. Here, we present an occlusion model of glaucoma based on the injection of magnetic microbeads into the anterior chamber of the eye using a modified microneedle with a facetted bevel. The magnetic microbeads are attracted to the iridocorneal angle using a handheld magnet to block the drainage of aqueous humour from the anterior chamber. This disruption in aqueous dynamics results in a steady elevation of intraocular pressure, which subsequently leads to the loss of retinal ganglion cells, as observed in human glaucoma patients. The microbead occlusion model presented in this manuscript is simple compared to other inducible models of glaucoma and also highly effective and reproducible. Importantly, the modifications presented here minimize common issues that often arise in occlusion models. First, the use of a bevelled glass microneedle prevents backflow of microbeads and ensures that minimal damage occurs to the cornea during the injection, thus reducing injury-related effects. Second, the use of magnetic microbeads ensures the ability to attract most beads to the iridocorneal angle, effectively reducing the number of beads floating in the anterior chamber avoiding contact with other structures (e.g., iris, lens). Lastly, the use of a handheld magnet allows flexibility when handling the small mouse eye to efficiently direct the magnetic microbeads and ensure that there is little reflux of the microbeads from the eye when the microneedle is withdrawn. In summary, the microbead occlusion mouse model presented here is a powerful investigative tool to study neurodegenerative changes that occur during the onset and progression of glaucoma.

Here, we present a protocol to induce ocular hypertension in the murine eye that results in the loss of retinal ganglion cells as observed in glaucoma. Magnetic microbeads are injected into the anterior chamber and attracted to the iridocorneal angle using a magnet to block the outflow of aqueous humour.

Un magnetica microperla occlusione modello per indurre ipertensione oculare-Dependent Glaucoma nei topi

The use of rodent models of glaucoma has been essential to understand the molecular mechanisms that underlie the pathophysiology of this multifactorial ...

A Simple Mechanical Procedure to Create Limbal Stem Cell Deficiency in Mouse

Limbal stem cell deficiency (LSCD) is a state of malfunction or loss of limbal epithelial stem cells, after which the corneal epithelium is replaced with conjunctiva. Patients suffer from recurrent corneal defects, pain, inflammation, and loss of vision.
Previously, a murine model of LSCD was described and compared to two other models. The goal was to produce a consistent mouse model of LSCD that both mimics the phenotype in humans and lasts long enough to make it possible to study the disease pathophysiology and to evaluate new treatments. Here, the technique is described in more detail.
A motorized tool with a rotating burr has been designed to remove the rust rings from the corneal surface or to smooth the pterygium bed in patients. It is a suitable device to create the desired LSCD model. It is a readily available, easy-to-use tool with a fine tip that makes it appropriate for working on small eyes, as in mice. Its application prevents unnecessary trauma to the eye and it does not result in unwanted injuries, as often is the case with chemical injury models. As opposed to a blunt scraper, it removes the epithelium with the basement membrane. In this protocol, the limbal area was abraded two times, and then the whole corneal epithelium was shaved from limbus to limbus. To avoid stroma injury, care was taken not to brush the corneal surface once the epithelium was already removed.

The following article provides an easy, reproducible technique to effectively create a sustainable mouse model of limbal stem cell deficiency (LSCD). This animal model is useful in testing and comparing the efficacy of treatments for limbal stem cell diseases.

Una semplice procedura meccanica per creare Limbal carenza Stem Cell in mouse

Limbal stem cell deficiency (LSCD) is a state of malfunction or loss of limbal epithelial stem cells, after which the corneal epithelium is replaced with ...

Induction and Phenotyping of Acute Right Heart Failure in a Large Animal Model of Chronic Thromboembolic Pulmonary Hypertension

The development of acute right heart failure (ARHF) in the context of chronic pulmonary hypertension (PH) is associated with poor short-term outcomes. The morphological and functional phenotyping of the right ventricle is of particular importance in the context of hemodynamic compromise in patients with ARHF. Here, we describe a method to induce ARHF in a previously described large animal model of chronic PH, and to phenotype, dynamically, right ventricular function using the gold standard method (i.e., pressure-volume PV loops) and with a non-invasive clinically available method (i.e., echocardiography). Chronic PH is first induced in pigs by left pulmonary artery ligation and right lower lobe embolism with biological glue once a week for 5 weeks. After 16 weeks, ARHF is induced by successive volume loading using saline followed by iterative pulmonary embolism until the ratio of the systolic pulmonary pressure over systemic pressure reaches 0.9 or until the systolic systemic pressure decreases below 90 mmHg. Hemodynamics are restored with dobutamine infusion (from 2.5 &#181;g/kg/min to 7.5 &#181;g/kg/min). PV-loops and echocardiography are performed during each condition. Each condition requires around 40 minutes for induction, hemodynamic stabilization and data acquisition. Out of 9 animals, 2 died immediately after pulmonary embolism and 7 completed the protocol, which illustrates the learning curve of the model. The model induced a 3-fold increase in mean pulmonary artery pressure. The PV-loop analysis showed that ventriculo-arterial coupling was preserved after volume loading, decreased after acute pulmonary embolism and was restored with dobutamine. Echocardiographic acquisitions allowed to quantify right ventricular parameters of morphology and function with good quality. We identified right ventricular ischemic lesions in the model. The model can be used to compare different treatments or to validate non-invasive parameters of right ventricular morphology and function in the context of ARHF.

We present a protocol to induce and phenotype an acute right heart failure in a large animal model with chronic pulmonary hypertension. This model can be used to test therapeutic interventions, to develop right heart metrics&#160;or to improve the understanding of acute right heart failure pathophysiology.

Induzione e fenotipizzazione dell'insufficienza cardiaca destra acuta in un modello animale di ipertensione polmonare tromboembolica cronica

The development of acute right heart failure (ARHF) in the context of chronic pulmonary hypertension (PH) is associated with poor short-term outcomes. The ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

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

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

A Reversible Silicon Oil-Induced Ocular Hypertension Model in Mice

Elevated intraocular pressure (IOP) is a well-documented risk factor for glaucoma. Here we describe a novel, effective method for consistently inducing stable IOP elevation in mice that mimics the post-operative complication of using silicone oil (SO) as a tamponade agent in human vitreoretinal surgery. In this protocol, SO is injected into the anterior chamber of the mouse eye to block the pupil and prevent inflow of aqueous humor. The posterior chamber accumulates aqueous humor and this in turn increases the IOP of the posterior segment. A single SO injection produces reliable, sufficient, and stable IOP elevation, which induces significant glaucomatous neurodegeneration. This model is a true replicate of secondary glaucoma in the eye clinic. To further mimic the clinical setting, SO can be removed from the anterior chamber to reopen the drainage pathway and allow inflow of aqueous humor, which is drained through the trabecular meshwork (TM) at the angle of the anterior chamber. Because IOP quickly returns to normal, the model can be used to test the effect of lowering IOP on glaucomatous retinal ganglion cells. This method is straightforward, does not require special equipment or repeat procedures, closely simulates clinical situations, and may be applicable to diverse animal species. However, minor modifications may be required.

Here, we present a protocol to induce ocular hypertension and glaucomatous neurodegeneration in mouse eyes by intracameral injection of silicone oil and the procedure for silicone oil removal from the anterior chamber to return elevated intraocular pressure to normal.

Un modello di ipertensione oculare indotta da olio di silicone reversibile nei topi

Elevated intraocular pressure (IOP) is a well-documented risk factor for glaucoma. Here we describe a novel, effective method for consistently inducing ...