Yazarlar Hong Jiang, doktora teşekkür etmek istiyorum ve Deepa Parathasarthy, MPH, teknik yardım için BDS.

1. Tam Kan Toplama <ol><li> İnsanlarda veya NEM / EDTA içeren tüplerde deney hayvanları gelen venöz kan toplayın. </li><li> Hemen 7 dakika rcf (göreceli merkezkaç kuvveti) plazma ve kırmızı kan hücre pelletini hazırlamak için 14.300 bir tezgah üstü santrifüj kan aşağı doğru döndürün. </li><li> Yüksek performanslı sıvı kromatografi (HPLC) ve chemilüminesan algılama (CLD) analizi için plazma örnekleri hazırlayın. HPLC: Plazma, çökelti plazma proteinlerine 10 dakika 13,200 rpm&#39;de vorteks ve santrifüj soğuk metanol 1:1 hacim ekleyin. HPLC analizi için supernatant toplamak. CLD: kısım özellikle tahlil nitrozo için Sulfanilamide ve cıva klorid ile örnek ve preincubate. </li><li> HPLC ve CLD analizi için kırmızı hücre pelletini hazırlayın. HPLC: 10 mM Milli Ekonomi Modeli&#39;nde, 2.5 mM EDTA ve ferrisiyanür 10 mM içeren bir hipotonik lizis çözüm ekle 01:04 kırmızı hücre pelet. Vortex iyice ve sonra 1:1 metanol, girdap ve centrif ekleyinÇökelti proteine ​​10 dakika süreyle 13.200 rpm uge. HPLC analizi için supernatant toplamak. CLD. 10 mM EDTA, 2.5 mM EDTA ve 10 mM ferrisiyanür ihtiva eden bir çözeltiye hipotonik lizis 01:04 kırmızı hücre peleti ekleyin. Tablet örnekleri nitrozo belirli tespiti için Sulfanilamide ve cıva klorid içeren tüplere. </li></ol> 2. Doku Ekstraksiyon ve Hazırlık <ol><li> NO metabolitlerinin doku seviyelerini belirlemek için, yukarıda tarif edildiği gibi örneği hazırlamak için kan serbest doku hasat etmek için gerekli olan ilk. Tam kan değişimi sol ventrikül apeks yoluyla fizyolojik tampon beslerken tarafından gerçekleştirilecektir. Tüm kan kaldırıldıktan sonra, ilgi dokular olabilir. </li><li> Doku örnekleri homojenize ve HPLC ile analiz CLD için yukarıda tarif edildiği gibi örnekler hazırlanır. </li></ol> 3. Endotel Fonksiyon için Aort Yüzük İzolasyonu Doku Organ Banyosu <ol><li> Fareler Anest olacaktırartık duyarlı tutam dizilmek ve cerrahi öncesi servikal dislokasyon geçmesi kadar dietil eter ile hetized. Bir torakotomi torasik ve abdominal aorta göstermek için yapılır. Bir 25 gauge şırınga sol ventrikül apeksi içine yerleştirilir ve oksijenli Krebs Henseleit tamponu ile kan serbest perfüze edilmiştir. </li><li> Sağ atrium kan için bir çıkış sağlamak için kesilir. Abdominal aort kaldırıldı ve adventisyanın temizlenecektir. </li><li> Halkalar 2 mm uzunluğunda parçalara kesilmiş ve fizyolojik bir tampon çözeltisi içinde kalmış bir dört-kanallı doku organ banyosu (DMT 720MO, AD Araçlar) üzerine monte edilecektir. </li><li> Damar halkaları 37 ° C&#39;de oksijenli Krebs tampon (95% O 2 ve% 5 CO2) ile 10 ml organ banyosu içinde tutulur Bir gram öngermeli her aort halkası (önceki deneylerde belirlendiği gibi uygun vazomotor işlev için uygun başlangıç ​​gerilimi) üzerine yerleştirilir. Sekiz kanallı sekizlik köprü (Powerlab) ve veri toplama yazılımı (Grafik sürüm 5.2.2) birBütün kuvvet ölçümleri kaydetmek için kullanılır yeniden. </li><li> Yüzüklerin her organ banyosunda tampon 80 dakika her 20 dakikada değişti için dengelenmesi için izin verilir. 80 dk için dengeleme sonra, 1 uM fenilefrin submaksimal kasılması için, her bir halka eklenir. </li><li> Stabilize edildikten sonra, bu tür asetilkolin olarak endotelial agonist damar gevşemesi NO üretimini ve derecesi belirlemek üzere eklenir. Asetilkolin için doz yanıt sonra, hamamlar nitrik oksit, düz kas yanıt belirlemek için ve% 100 rahatlama için bir değer almak için nitroprussid dışsal kaynağı ile yıkanır ve recontracted ve daha sonra ele alınacaktır. </li><li> NO temizleyiciler açıkça damar rahatlama ve NO inhibisyonu serbest göstermek için banyo eklenebilir. </li></ol> 4. Temsilcisi Sonuçlar ENO-20 özel HPLC kullanımı, nitrit ve nitrat spesifik ve duyarlı, yüksek verimlilik yöntemi kullanımı kolay sağlarbiyolojik matrisler. Algılama ve orijinal bir kromatogramıdır ilkesine Şekil 1 &#39;de gösterilmiştir. Bu yöntem, nitrit ve nitrat belirlenmesi için herhangi bir biyolojik numune için kullanılabilir. NO metabolitlerinin CLD tabanlı algılama NO moleküler kaynağını belirlemek için bir kimyasal derivitization adım gerektirir. Kemilüminesans detektörü için eşzamanlı oksidatif denitrosation ve indirgeyici denitrosation için deney düzeneği Şekil 2 &#39;de gösterilmiştir. Sağda Reaksiyon kabı PBS pH 7.4 &#39;de 800mm ferrisiyanür ile doldurulur ve sol tepkime kabına redüksiyon denitrosation için asetik asit (Şekil 2A) potasyum iyodür / iyot karışımı ile doldurulur. Tüm kurulum Şekil 2B &#39;de gösterilmiştir. Şekil 3 grubunun spesifik denitrosation nitrit algılamak ve sayısal olarak deneyler, nitrozo, nitrozaminler gibi nitrosil heme ürünler göstermektedir. Bu yöntem, daha önce tarif olmuşturyatak ve 4,5 doğrulandı. Bu önemli biyokimyasal analizler kolayca endotel deney hayvanlarında NO üretimini belirlemek için izole aort halkaları üzerinde fonksiyonel çalışmalar ile korele edilebilir. Bu klasik farmakolojik deney kolayca ve doğru endotel fonksiyonu ve üretim NO değerlendirebilir. Gibi asetilkolin gibi endotelyal agonistlere damar reaktivitesi Ölçme doğrudan sonra deney hayvanlarının kan ve doku tespit biyokimyasal belirteçler ile korele edilebilir endotelyal NO üretimini belirleyebilirsiniz. Asetilkolin için bir doz-yanıt tipik bir temsilini Şekil 4 &#39;de gösterilmiştir. Normal endotel fonksiyonu ile sağlıklı kontrol fareleri rahatlatarak asetilkolin yanıt. Endotel disfonksiyonu ile Fare (hiperkolesterolemik fareler) aynı uyarana NO üretimi azalır dolayı daha az rahatlama gösterir. <img alt="Şekil 1" src="/files/ftp_upload/3722/3722fig1.jpg" /> Incirure 1. ENO-20 ve örnek kromatogram tarafından nitrit ve nitrat saptama ilkesi. Nitrit ve nitrat için algılama ENO-20 yöntemi (Üst) Şematik. (Alt) 10 pmol nitrit ve nitrat standart chromotogram ENO-20 (nitrit ve nitrat 100 nM çözümü 100 ul) enjekte edildi. 100 ul her bir enjeksiyon hacmi ile anyon için 1 nM duyarlılığı. Proteini veya renkli türleri ile hiçbir müdahale. <img alt="Şekil 2" src="/files/ftp_upload/3722/3722fig2.jpg" /> Tasfiye nitrik oksit gaz gaz faz algılama ile iyodür / iyot testi kullanılarak ferrisiyanür ve indirgeyici denitrosation tarafından hem oksidatif denitrosation kullanarak Şekil 2. CLD deney düzeneği. <img alt="Şekil 3" src="/files/ftp_upload/3722/3722fig3.jpg" /> Şekil 3. (Panel A) grubu belirli Chemic ile örnek preinkübasyon tarafından indirgeyici denitrosation tayininde nitrit Kemilüminesans algılama, RSNO, RNNOal reaktifler. Pik alanlarının Çıkarma nitrit ve RSNOs algılanmasını sağlar. Ferrisiyanür oksidatif denitrosation çözeltisi kullanılarak nitrosil heme türlerinin (panel B) Chemiluminescent tespiti. Bu yöntem, RSNOs (GSNO ya SNO-albümini), ya da RNNO (NO-pirolidin ve N-nitrozo-albümini) ile bir çapraz reaktivitesi olan NO-heme ürünler için özel. <img alt="Şekil 4" src="/files/ftp_upload/3722/3722fig4.jpg" /> Şekil 4. Izole aort halkaları üzerinde ex vivo organ banyosu sonra HPLC ve CLD tarafından tespit biyokimyasal belirteçler ile korele edilebilir endotelyal NO üretimini doğrudan bir ölçü sağlar. Bu rakam NO üretiminin azalması nedeniyle endotel disfonksiyonu ile farelerde azalmış rahatlama gösterir.

<ol>
	<li>Kelm, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide+metabolism+and+breakdown.">Nitric oxide metabolism and breakdown.</a> Biochimica et Biophysica Acta. 1411, 273-289 (1999).</li><li>Furchgott, R. F., Zawadzki, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+obligatory+role+of+endothelial+cells+in+the+relaxation+of+arterial+smooth+muscle+by+acetycholine.">The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetycholine.</a> Nature. 288, 373-376 (1980).</li><li>Ignarro, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelium-derived+relaxing+factor+produced+and+released+from+artery+and+vein+is+nitric+oxide.">Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide.</a> Proc. Natl. Acad Sci. U.S.A. 84, 9265-9269 (1987).</li><li>Feelisch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concomitant+S-,+N-,+and+heme-nitrosylation+in+biological+tissues+and+fluids:+implications+for+the+fate+of+NO+in+vivo.">Concomitant S-, N-, and heme-nitrosylation in biological tissues and fluids: implications for the fate of NO in vivo.</a> FASEB J. 16, 1775-1785 (2002).</li><li>Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+nitric+oxide+levels+in+the+red+cell:+validation+of+tri-iodide-based+chemiluminescence+with+acid-sulfanilamide+pretreatment.">Measurement of nitric oxide levels in the red cell: validation of tri-iodide-based chemiluminescence with acid-sulfanilamide pretreatment.</a> J. Biol. Chem. 281, 26994-27002 (2006).</li><li>Angelo, M., Singel, D. J., Stamler, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+S-nitrosothiol+(SNO)+synthase+function+of+hemoglobin+that+utilizes+nitrite+as+a+substrate.">An S-nitrosothiol (SNO) synthase function of hemoglobin that utilizes nitrite as a substrate.</a> Proc. Natl. Acad. Sci. U.S.A. 103, 8366-8371 (2006).</li><li>Bryan, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bound+NO+in+human+red+blood+cells:+fact+or+artifact.">Bound NO in human red blood cells: fact or artifact.</a> Nitric Oxide. 10, 221-228 (2004).</li><li>Feelisch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+Processing+of+Nitrite+in+Hypoxia:+An+Intricate+Interplay+of+Nitric+Oxide-Generating+and+-Scavenging+Systems.">Tissue Processing of Nitrite in Hypoxia: An Intricate Interplay of Nitric Oxide-Generating and -Scavenging Systems.</a> J. Biol. Chem. 283, 33927-33934 (2008).</li><li>Bryan, N. S., Grisham, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+detect+nitric+oxide+and+its+metabolites+in+biological+samples.">Methods to detect nitric oxide and its metabolites in biological samples.</a> Free Radic. Biol. Med. 43, 645-657 (2007).</li><li>Lundberg, J. O., Weitzberg, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NO+generation+from+inorganic+nitrate+and+nitrite:+Role+in+physiology,+nutrition+and+therapeutics.">NO generation from inorganic nitrate and nitrite: Role in physiology, nutrition and therapeutics.</a> Arch. Pharm. Res. 32, 1119-1126 (2009).</li><li>Lundberg, J. O., Weitzberg, E., Gladwin, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nitrate-nitrite-nitric+oxide+pathway+in+physiology+and+therapeutics.">The nitrate-nitrite-nitric oxide pathway in physiology and therapeutics.</a> Nat. Rev. Drug. Discov. 7, 156-157 (2008).</li></ol>

Çıkar çatışması ilan etti.

Yöntemleri çoklu biyolojik bölümlerinde ilgili NO metabolitlerinin ölçümü için burada açıklanan endotelde NO fonksiyonel ölçümleri ile korele edilebilir sağlık ve hastalık NO biyoloji parmak izi için izin verecektir. Bu yöntemler, yüksek verimlilik için uyum potansiyeli olan basit örnek hazırlama gerektirir. Bu moleküllerin miktarı nispi hastalığın deneysel modellerinde ve insan hastalarda bile seri örnekleri arasında bir sayı, NO üretimini ve metabolik akıbeti anlamak yardımcı olabili...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td></tr><tr><td> N-etilmaleimid </td><td> Thermo Scientific </td><td> 23030 </td></tr><tr><td> EDTA </td><td> Sigma-Aldrich </td><td> E7889 </td></tr><tr><td> Potasyum ferrisiyanürle </td><td> Fluka </td><td> 60299 </td></tr><tr><td> HPLC </td><td> EICOM Corp </td><td> ENO-20 </td></tr><tr><td> Otomatik Numune </td><td> Alcott </td><td></td></tr><tr><td> DMT Myograph </td><td> AD Aletleri </td><td></td></tr><tr><td> PowerLab </td><td> AD Aletleri </td><td></td></tr><tr><td> Chemiluminescent </td><td> EcoPhysics </td><td> CLD 88Y </td></tr><tr><td> Santrifüj </td><td> Eppendorf </td><td> 5415D </td></tr><tr><td> Asetilkolin </td><td> Sigma-Aldrich</td><td> A6625 </td></tr><tr><td> R-(-) fenilefrin </td><td> Sigma-Aldrich </td><td> P6126 </td></tr></tbody></table>

analytical techniques for assaying nitric oxide bioactivity

Nitrik oksit (NO) biyolojik sistemler (kan dolaşımında, 1 saniyeden az) 1 derece kısa ömürlü bir iki atomlu serbest radikaldir. NO dahil olmak üzere temel işlevleri düzenleyen, vücudumuzda üretilen en önemli sinyal molekülleri biri olarak ancak kan basıncı, bağışıklık yanıtı ve sinirsel iletişim düzenleme ile sınırlı değildir olabilir. Bu nedenle biyolojik matrisler onun doğru tespiti ve miktar tayini sağlık ve hastalık NO rolünü anlamak için önemlidir. NO gibi kısa bir fizyolojik yarı ömrü ile, NO biyokimya reaksiyon ürünlerinin tespiti için alternatif stratejiler geliştirilmiştir. Çoklu biyolojik bölümlerinde ilgili NO metabolitleri miktarının in vivo açısından NO üretimini, biyoyararlanım ve metabolizması ile değerli bilgiler sağlar. Sadece kan veya plazma gibi tek bir bölme örnekleme her zaman tüm bo doğru bir değerlendirme sağlamak olmayabilirözellikle dokularda, NO durumu dy. Deney hayvanlarında seçin dokuları ile kan karşılaştırmak için yeteneği kadar sağlık ve hastalık NO biyobelirteçlerin tanısal ve prognostik programı gibi temel bilim ve klinik tıp arasındaki uçurumu yardımcı olacaktır. Bu nedenle, belirli ilgi dokulara plazma ya da kan NO durumu ekstrapolasyon artık geçerli bir yaklaşımdır. Sonuç olarak, metotları geliştirmiş ve in vivo deneysel hayvan birden bölümlerinde NO ve NO ile ilgili ürünler / metabolitlerinin saptanması ve miktarının izin hangi geçerliliği devam etmektedir. Ve nitrat (NO 3 -) - nitrit (NO 2) nihai oksidasyona çözünür guanil siklaz (SGC) aktivasyonu NO sentaz tarafından üretim NO biyokimya kurulan paradigması sadece in vivo NO etkilerini bir parçasını temsil edebilir. S-nitrosothi oluşturmak üzere proteini tiyoller, ikincil aminler, ve metaller ile NO ve NO-türevi metabolitlerinin etkileşimiols (RSNOs), N-nitrozaminler (RNNOs) ve nitrosil heme-sırasıyla NO cGMP bağımsız etkilerini temsil eder ve NO SGC aktivasyonu gibi fizyolojik olasılığı kadar önemlidir. Fizyolojisi NO gerçek bir anlayış aynı anda birden fazla bölmeli örnekleme in vivo deneyler türetilmiştir. Nitrik oksit (NO) metodolojisi YOK biyokimya ile ilgili pek çok tartışma ve tartışma karmaşık ve çoğu zaman kafa karıştırıcı bilim ve odak noktasıdır. Yeteneğimizi özellikle üzerine NO menteşeleri içeren yeni mekanizmalar ve sinyal yollarının aydınlatılması, seçici ve hassas tespit ve NO ile ilgili tüm kompleks biyolojik matrisler NO ürünleri ve metabolitleri ölçmek. Burada, bir HPLC ile nitrit ve nitrat hızlı ve duyarlı analiz yöntemi gibi NO moleküler kaynak yanı sıra ile ex vivo olarak belirlemek için kimyasal derivitazation ile in vitro ozon bazlı chemiluminescence kullanarak biyolojik örnekler serbest NO algılanmasıorgan banyosu kasılmaların kâğıda çizilmesi.

Watch this Scientific Journal Video about Analytical Techniques for Assaying Nitric Oxide Bioactivity at JoVE.com

Analytical Techniques for Assaying Nitric Oxide Bioactivity

Nitrik oksit (NO) endojen üretimi biyolojik fonksiyonları çok çeşitli düzenler. Bu NO dayalı sinyal bozulması veya düzensizliği birçok insan hastalıkları dahil olduğu giderek daha açık hale gelmektedir. Yöntem NO metabolitleri insan hastalığı için yeni tanısal veya prognostik biyolojik sağlayabilir ilgili ölçmek için.

Nitrik Oksit Biyoaktivite içlin Analitik Teknikler

Nitric oxide (NO) is a diatomic free radical that is extremely short lived in biological systems (less than 1 second in circulating blood)1. NO may be ...

analytical-techniques-for-assaying-nitric-oxide-bioactivity

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Medicine

17.9K Görüntüleme.  University of Texas Health Science Center at Houston. Nitrik oksit (NO) endojen üretimi biyolojik fonksiyonları çok çeşitli düzenler. Bu NO dayalı sinyal bozulması veya düzensizliği birçok insan hastalıkları dahil olduğu giderek daha açık hale gelmektedir. Yöntem NO metabolitleri insan hastalığı için yeni tanısal veya prognostik biyolojik sağlayabilir ilgili ölçmek için.

Video: Analytical Techniques for Assaying Nitric Oxide Bioactivity

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.

Yüksek Tuz Diyet ve Lewis Rat Kronik Böbrek Hastalığı tetikleyebilecek Nitrik Oksit Sentaz inhibisyonu ile bağlantılı 5/6th nefrektomi

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

Tıp

Techniques for Processing Eyes Implanted With a Retinal Prosthesis for Localized Histopathological Analysis

With the recent development of retinal prostheses, it is important to develop reliable techniques for assessing the safety of these devices in preclinical studies. However, the standard fixation, preparation, and automated histology procedures are not ideal. Here we describe new procedures for evaluating the health of the retina directly adjacent to an implant. Retinal prostheses feature electrode arrays in contact with eye tissue. Previous methods have not been able to spatially localize the ocular tissue adjacent to individual electrodes within the array. In addition, standard histological processing often results in gross artifactual detachment of the retinal layers when assessing implanted eyes. Consequently, it has been difficult to assess localized damage, if present, caused by implantation and stimulation of an implanted electrode array. Therefore, we developed a method for identifying and localizing the ocular tissue adjacent to implanted electrodes using a (color-coded) dye marking scheme, and we modified an eye fixation technique to minimize artifactual retinal detachment. This method also rendered the sclera translucent, enabling localization of individual electrodes and specific parts of an implant. Finally, we used a matched control to increase the power of the histopathological assessments. In summary, this method enables reliable and efficient discrimination and assessment of the retinal cytoarchitecture in an implanted eye.

Techniques for visualizing retinal cytoarchitecture directly adjacent to individual electrodes within a retinal stimulator.

Lokalize histopatolojik analizi için bir Retina Protez ile implant İşleme Gözler Teknikleri

With  the recent development of retinal prostheses, it is important to develop  reliable techniques for assessing the safety of these devices in ...

Techniques for Processing Eyes Implanted with a Retinal Prosthesis for Localized Histopathological Analysis: Part 2 Epiretinal Implants with Retinal Tacks

Retinal prostheses for the treatment of certain forms of blindness are gaining traction in clinical trials around the world with commercial devices currently entering the market. In order to evaluate the safety of these devices, in preclinical studies, reliable techniques are needed. However, the hard metal components utilised in some retinal implants are not compatible with traditional histological processes, particularly in consideration for the delicate nature of the surrounding tissue. Here we describe techniques for assessing the health of the eye directly adjacent to a retinal implant secured epiretinally with a metal tack.
Retinal prostheses feature electrode arrays in contact with eye tissue. The most commonly used location for implantation is the epiretinal location (posterior chamber of the eye), where the implant is secured to the retina with a metal tack that penetrates all the layers of the eye. Previous methods have not been able to assess the proximal ocular tissue with the tack in situ, due to the inability of traditional histological techniques to cut metal objects. Consequently, it has been difficult to assess localized damage, if present, caused by tack insertion.
Therefore, we developed a technique for visualizing the tissue around a retinal tack and implant. We have modified an established technique, used for processing and visualizing hard bony tissue around a cochlear implant, for the soft delicate tissues of the eye. We orientated and embedded the fixed eye tissue, including the implant and retinal tack, in epoxy resin, to stabilise and protect the structure of the sample. Embedded samples were then ground, polished, stained, and imaged under various magnifications at incremental depths through the sample. This technique allowed the reliable assessment of eye tissue integrity and cytoarchitecture adjacent to the metal tack.

Here we describe histological techniques for visualising ocular tissue directly adjacent to a metal epiretinal tack and retinal prosthesis.

Lokalize Histopatolojik Analiz için bir Retina Protez ile implant İşleme Teknikleri Gözler için: Bölüm Retina Tacks ile 2 Epiretinal İmplantlar

Retinal prostheses for the treatment of certain forms of blindness are gaining traction in clinical trials around the world with commercial devices ...

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic study in the liver. Here, numerous assays are described to comprehensively measure the phenotype and parameters of hepatic steatosis in mouse and hepatocyte models. 
Combining the physiological, histological, and biochemical methods, this system can be used to assess the progress of hepatic steatosis. In vivo, the measurements of body weight and nuclear magnetic resonance (NMR) provide a general understanding of mice in a non-invasive manner. Hematoxylin and Eosin (H&amp;E) and Oil Red O staining determine the histological morphology and lipid deposition of liver tissue under nutrient overload conditions, such as high-fat diet feeding. Next, the total lipid contents are isolated by chloroform/methanol extraction, which are followed by a biochemical analysis for triglyceride and cholesterol. Moreover, mouse primary hepatocytes are treated with high glucose plus insulin to stimulate lipid accumulation, an efficient in vitro model to mimic diet-induced hyperglycemia and hyperinsulinemia in vivo. Then, the lipid deposition is measured by Oil Red O staining and chloroform/methanol extraction. Oil Red O staining determines intuitive hepatic steatotic phenotypes, while the lipid extraction analysis determines the parameters that can be analyzed statistically. The present protocols are of interest to scientists in the fields of fatty liver diseases, insulin resistance, and type 2 diabetes.

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described.

Optimize Analizi<em&gt; İn Vivo</em&gt; ve<em&gt; İn Vitro</em&gt; Karaciğer Steatoz

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic ...

Synthesis, Characterization, and Application of Superparamagnetic Iron Oxide Nanoprobes for Extrapulmonary Tuberculosis Detection

A molecular imaging probe comprising superparamagnetic iron oxide (SPIO) nanoparticles and Mycobacterium tuberculosis surface antibody (MtbsAb) was synthesized to enhance imaging sensitivity for extrapulmonary tuberculosis (ETB). An SPIO nanoprobe was synthesized and conjugated with MtbsAb. The purified SPIO-MtbsAb nanoprobe was characterized using TEM and NMR. To determine the targeting ability of the probe, SPIO-MtbsAb nanoprobes were incubated with Mtb for in vitro imaging assays and injected into Mtb-inoculated mice for in vivo investigation with magnetic resonance (MR). The contrast enhancement reduction on magnetic resonance imaging (MRI) of Mtb and THP1 cells showed proportional to the SPIO-MtbsAb nanoprobe concentration. After 30 min of intravenous SPIO-MtbsAb nanoprobe injection into Mtb-infected mice, the signal intensity of the granulomatous site was enhanced by 14-fold in the T2-weighted MR images compared with that in mice receiving PBS injection. The MtbsAb nanoprobes can be used as a novel modality for ETB detection.

To improve serological diagnostic tests for Mycobacterium tuberculosis antigens, we developed superparamagnetic iron oxide nanoprobes to detect extrapulmonary tuberculosis.

Ekstrapulmoner Tüberküloz Tespiti için Süperparamanyetik Demir Oksit Nanoprobukların Sentezi, Karakterizasyonu ve Uygulanması

A molecular imaging probe comprising superparamagnetic iron oxide (SPIO) nanoparticles and Mycobacterium tuberculosis surface antibody (MtbsAb) was ...

Platelet-based Detection of Nitric Oxide in Blood by Measuring VASP Phosphorylation

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent vasoactive agents, nitric oxide (NO), can also act as a powerful inhibitor of platelet aggregation. Direct NO detection in blood is very challenging due to its high reactivity with cell-free hemoglobin that limits NO half-life to the millisecond range. Currently, NO changes after interventions are only estimated based on measured changes of nitrite and nitrate (members of the nitrate-nitrite-NO metabolic pathway). However precise, these measurements are rather difficult to interpret vis a vis actual NO changes, due to the naturally high baseline nitrite and nitrate levels that are several orders of magnitude higher than expected changes of NO itself. Therefore, the development of direct and simple methods that would allow one to detect NO directly is long overdue. This protocol addresses a potential use of platelets as a highly sensitive NO sensor in blood. It describes initial platelet rich plasma (PRP) and washed platelet preparations and the use of nitrite and deoxygenated red blood cells as NO generators. Phosphorylation of VASP at serine 239 (P-VASPSer239) is used to detect the presence of NO. The fact that VASP protein is highly expressed in platelets and that it is rapidly phosphorylated when NO is present leads to a unique opportunity to use this pathway to directly detect NO presence in blood.

Here, we present a protocol to address the potential use of platelets as a highly sensitive nitric oxide sensor in blood. It describes initial platelet preparation and the use of nitrite and deoxygenated red blood cells as nitric oxide generators.

Nitrik oksit VASP fosforilasyon ölçme tarafından kan trombosit tabanlı algılama

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent ...

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a promising therapeutic target in many disorders, such as autoimmune diseases, atherosclerosis, and cancer. RNAi-mediated gene silencing is a valuable approach of choice to probe and manipulate macrophage function; however, the transfection of macrophages with siRNA is often considered to be technically challenging, and, at present, few methodologies dedicated to the siRNA transfer to macrophages are available. Here, we present a protocol of using polyethyleneimine-coated superparamagnetic iron oxide nanoparticles (PEI-SPIONs) as a vehicle for the targeted delivery of siRNA to macrophages. PEI-SPIONs are capable of binding and completely condensing siRNA when the Fe:siRNA weight ratio reaches 4 and above. In vitro, these nanoparticles can efficiently deliver siRNA into primary macrophages, as well as into the macrophage-like RAW 264.7 cell line, without compromising cell viability at the optimal dose for transfection, and, ultimately, they induce siRNA-mediated target gene silencing. Apart from being used for in vitro siRNA transfection, PEI-SPIONs are also a promising tool for delivering siRNA to macrophages in vivo. In view of its combined features of magnetic property and gene-silencing ability, systemically administered PEI-SPION/siRNA particles are expected not only to modulate macrophage function but also to enable macrophages to be imaged and tracked. In essence, PEI-SPIONs represent a simple, safe, and effective nonviral platform for siRNA delivery to macrophages both in vitro and in vivo.

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

We describe a method of using polyethyleneimine (PEI)-coated superparamagnetic iron oxide nanoparticles for transfecting macrophages with siRNA. These nanoparticles can efficiently deliver siRNA to macrophages in vitro and in vivo and silence target gene expression.

Polyethyleneimine kaplı demir oksit nano tanecikleri küçük müdahale RNA makrofajlar için teslim için bir araç olarak Vitro ve In Vivo

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a ...

Tracking Superparamagnetic Iron Oxide-labeled Mesenchymal Stem Cells using MRI after Intranasal Delivery in a Traumatic Brain Injury Murine Model

Stem cell-based therapies for brain injuries, such as traumatic brain injury (TBI), are a promising approach for clinical trials. However, technical hurdles such as invasive cell delivery and tracking with low transplantation efficiency remain challenges in translational stem-based therapy. This article describes an emerging technique for stem cell labeling and tracking based on the labeling of the mesenchymal stem cells (MSCs) with superparamagnetic iron oxide (SPIO)&#160;nanoparticles, as well as intranasal delivery of the labeled MSCs. These nanoparticles are fluorescein isothiocyanate (FITC)-embedded and safe to label the MSCs, which are subsequently delivered to the brains of TBI-induced mice by the intranasal route. They are then tracked non-invasively in vivo by real-time magnetic resonance imaging (MRI). Important advantages of this technique that combines SPIO for cell labeling and intranasal delivery include (1) non-invasive, in vivo MSC tracking after delivery for long tracking periods, (2) the possibility of multiple dosing regimens due to the non-invasive route of MSC delivery, and (3) possible applications to humans, owing to the safety of SPIO, non-invasive nature of the cell-tracking method by MRI, and route of administration.

Presented here is a protocol for non-invasive mesenchymal stem cell (MSC) delivery and tracking in a mouse model of traumatic brain injury. Superparamagnetic iron oxide&#160;nanoparticles are employed as a magnetic resonance imaging (MRI) probe for MSC labeling and non-invasive in vivo tracking following intranasal delivery using real-time MRI.

Travmatik Beyin Hasarı Murine Modelinde İntranazal Doğum sonrası MRG kullanarak Süperparamanyetik Demir Oksit etiketli Mezenkimal Kök Hücrelerin Takibi

Stem cell-based therapies for brain injuries, such as traumatic brain injury (TBI), are a promising approach for clinical trials. However, technical ...

Near Infrared Photoimmunotherapy for Mouse Models of Pleural Dissemination

The efficacy of photoimmunotherapy can be evaluated more accurately with an orthotopic mouse model than with a subcutaneous one. A pleural dissemination model can be used for the evaluation of treatment methods for intrathoracic diseases such as lung cancer or malignant pleural mesothelioma.
Near-infrared photoimmunotherapy (NIR-PIT) is a recently developed cancer treatment strategy that combines the specificity of tumor-targeting antibodies with toxicity caused by a photoabsorber (IR700Dye) after exposure to NIR light. The efficacy of NIR-PIT has been reported using various antibodies; however, only a few reports have shown the therapeutic effect of this strategy in an orthotopic model. In the present study, we demonstrate an example of efficacy evaluation of the pleural disseminated lung cancer model, which was treated using NIR-PIT.

Near-infrared photoimmunotherapy (NIR-PIT) is an emerging cancer therapeutic strategy that utilizes an antibody-photoabsorber (IR700Dye) conjugate and NIR light to destroy cancer cells. Here, we present a method to evaluate the antitumor effect of NIR-PIT in a mouse model of pleural disseminated lung cancer and malignant pleural mesothelioma using bioluminescence imaging.

Plevral Yayılım Fare Modelleri için Yakın Kızılötesi Fotoimmünoterapi

The efficacy of photoimmunotherapy can be evaluated more accurately with an orthotopic mouse model than with a subcutaneous one. A pleural dissemination ...

A Novel Inhalation Mask System to Deliver High Concentrations of Nitric Oxide Gas in Spontaneously Breathing Subjects

Nitric Oxide (NO) is administered as gas for inhalation to induce selective pulmonary vasodilation. It is a safe therapy, with few potential risks even if administered at high concentration. Inhaled NO gas is routinely used to increase systemic oxygenation in different disease conditions. The administration of high concentrations of NO also exerts a virucidal effect in vitro. Owing to its favorable pharmacodynamic and safety profiles, the familiarity in its use by critical care providers, and the potential for a direct virucidal effect, NO is clinically used in patients with coronavirus disease-2019 (COVID-19). Nevertheless, no device is currently available to easily administer inhaled NO at concentrations higher than 80 parts per million (ppm) at various inspired oxygen fractions, without the need for dedicated, heavy, and costly equipment. The development of a reliable, safe, inexpensive, lightweight, and ventilator-free solution is crucial, particularly for the early treatment of non-intubated patients outside of the intensive care unit (ICU) and in a limited-resource scenario. To overcome such a barrier, a simple system for the non-invasive NO gas administration up to 250 ppm was developed using standard consumables and a scavenging chamber. The method has been proven safe and reliable in delivering a specified NO concentration while limiting nitrogen dioxide levels. This paper aims to provide clinicians and researchers with the necessary information on how to assemble or adapt such a system for research purposes or clinical use in COVID-19 or other diseases in which NO administration might be beneficial.

This simple and highly adaptable system device for the inhalation of high-concentration nitric oxide (NO) gas does not require mechanical ventilators, positive pressure, or high gas flows. Standard medical consumables and a snug-fitting mask are used to safely deliver NO gas to spontaneously breathing subjects.

Kendiliğinden Solunan Deneklerde Yüksek Konsantrasyonlarda Nitrik Oksit Gazı Sağlamak için Yeni Bir Soluma Maske Sistemi

Nitric Oxide (NO) is administered as gas for inhalation to induce selective pulmonary vasodilation. It is a safe therapy, with few potential risks even if ...