Name: generating a reproducible model of mid-gestational maternal immune activation using poly(i:c) to study susceptibility and resilience in offspring
Uploaded: 2022-08-17T13:40:00
Description: Watch this Scientific Journal Video about Generating a Reproducible Model of Mid-Gestational Maternal Immune Activation using PolyI:C to Study Susceptibility and Resilience in Offspring at JoVE.com

हम माउस एमआईए मॉडल में परिवर्तनशीलता को संबोधित करने में उनकी दृढ़ता के लिए डॉ मायका एस्टेस और एस्टेस एट अल .8 में सभी योगदानकर्ताओं को उनके काम के लिए धन्यवाद देते हैं, जिससे यहां वर्णित विधियों के प्रोटोकॉल का विकास हुआ। यहां रिपोर्ट किए गए शोध को एनआईएमएच 2पी 50 एमएच 106438-06 (ए.के.एम.) और एनआईएमएच टी32एमएच112507 (के.पी.) द्वारा समर्थित किया गया था।

सभी प्रोटोकॉल कैलिफोर्निया विश्वविद्यालय-डेविस संस्थागत पशु देखभाल और उपयोग समिति (आईएसीयूसी) के अनुमोदन के तहत किए जाते हैं।
1. जानवरों की तैयारी
<ol><li>जानवरों को प्राप्त करते समय, अधि...

मातृ संक्रमण मनुष्यों में और कृन्तकों और गैर-मानव प्राइमेट्स 4,5,7 दोनों में मस्तिष्क के विकास के पाठ्यक्रम को बदल देता है। यहां, पॉली (आई: सी) का उपयोग करके मध्य-गर्भकाल...

महामारी विज्ञान के साक्ष्य मातृ संक्रमण को मनोरोग और एनडीडी के बढ़ते जोखिम से जोड़ते हैं, जिसमें स्किज़ोफ्रेनिया (एसजेड) और ऑटिज्म स्पेक्ट्रम डिसऑर्डर (एएसडी) 1,2,3,4,5,6,7 शामिल हैं। एमआईए माउस मॉडल को इन विकारों के एटियलजि में कार्य-कारण और एमआईए की यांत्रिक भूमिका का परीक्षण करने के साथ-साथ आणविक बायोमाकर्स की पहचान करने और नैदानिक और चिकित्सीय उपकरण 4,6 दोनों विकसित करनेके लिए विकसित किया गया था। इस मॉडल की उपयोगिता और इसकी बढ़ती लोकप्रियता के बावजूद, क्षेत्र के भीतर एमआईए प्रेरण प्रोटोकॉल में काफी परिवर्तनशीलता है, जिससे अध्ययनों में परिणामों की तुलना करना औरनिष्कर्षों को दोहराना मुश्किल हो जाता है। इसके अलावा, मॉडल के अधिकांश पुनरावृत्तियां एमआईए के दो महत्वपूर्ण ट्रांसलेशनल पहलुओं की जांच नहीं करती हैं: (i) कई संतान एमआईए के लिए लचीला हैं, और (ii) अतिसंवेदनशील संतान फेनोटाइप8 के अलग-अलग संयोजन प्रदर्शित कर सकती हैं।
प्रतिलिपि प्रस्तुत करने योग्य एमआईए मॉडल उत्पन्न करने के लिए, जांचकर्ताओं को बांधों में प्रेरित एमआईए के परिमाण के कम से कम एक मात्रात्मक माप की रिपोर्ट करनी चाहिए। गर्भावस्था के दौरान एमआईए को प्रेरित करने के लिए, हमारी प्रयोगशाला डबल फंसे हुए आरएनए वायरल मिमिक पॉलीइनोसिटिक के इंट्रापरिटोनियल (आई.पी.) इंजेक्शन करती है: पॉलीसाइटिडिलिक एसिड [पॉली (आई: सी)]। पॉली (आई: सी) इन्फ्लूएंजा वायरस के समान एक प्रतिरक्षा कैस्केड को प्रेरित करता है क्योंकि यह टोल-जैसे रिसेप्टर 3 (टीएलआर 3)10 द्वारा पहचाना जाता है। नतीजतन, पॉली (आई: सी) तीव्र चरण प्रतिक्रिया को सक्रिय करता है जिसके परिणामस्वरूप प्रोइंफ्लेमेटरी साइटोकिन्स 8,11,12 की तेजी से ऊंचाई होती है। पिछले अध्ययनों से पता चला है कि इंटरल्यूकिन -6 (आईएल -6) सहित प्रोइंफ्लेमेटरी साइटोकिन्स की ऊंचाई, एमआईए11,12,13 के परिणामस्वरूप संतानों में व्यवहार संबंधी असामान्यताएं और न्यूरोपैथोलॉजी पैदा करने के लिए आवश्यक है। इस प्रकार, पॉली (आई: सी) इंजेक्शन के बाद 2.5 घंटे पर अपने चरम के दौरान एकत्र किए गए मातृ सीरम में आईएल -6 का स्तर एमआईए का एक सम्मोहक मात्रात्मक उपाय है जिसका उपयोग क्षेत्र के भीतर प्रयोगशालाओं में परिणामों की तुलना करने के लिए किया जा सकता है।
एक एमआईए मॉडल उत्पन्न करने के लिए जो एकल प्रेरण प्रोटोकॉल 8,14 के साथ लचीलापन और संवेदनशीलता के ट्रांसलेशनल रूप से आवश्यक तत्वों को संबोधित करता है, शोधकर्ता गर्भावस्था8 से पहले बांध की बेसलाइन विकृति (बीआईआर) के लक्षण वर्णन के साथ विशिष्ट प्रेरण दृष्टिकोणको जोड़ सकते हैं। हाल ही में, यह पता चला कि कुंवारी मादा C57BL/6 चूहे गर्भावस्था8 से पहले पॉली (I: C) के कम खुराक वाले जोखिम के लिए IL-6 प्रतिक्रियाओं की एक विस्तृत श्रृंखला दिखाते हैं। यह केवल इन मादाओं का एक उप-समूह है जो अतिसंवेदनशील संतान पैदा करता है, और केवल प्रतिरक्षा सक्रियण के कुछ परिमाणों पर जैसा कि बीआईआर और पॉली (आई: सी) खुराक8 के संयोजन से निर्धारित होता है। एमआईए एक उल्टे यू पैटर्न में फेनोटाइप को प्रेरित करता है; संतान सबसे बड़ा व्यवहार और आणविक विपथन दिखाती है जब बांध मामूली रूप से इम्यूनोरिएक्टिव होते हैं, और मातृ सूजन का परिमाण पहुंचता है, लेकिन एक महत्वपूर्ण सीमा8 से अधिक नहीं होता है। यहां, पॉली (आई: सी) के मध्य-गर्भकालीन इंजेक्शन के परिणामस्वरूप अलग-अलग व्यवहार फेनोटाइप के साथ लचीला और अतिसंवेदनशील संतान दोनों को मज़बूती से बनाने का एक विस्तृत तरीका प्रदान किया गया है।

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.9% NaCl physiological endotoxin free saline</td>
			<td>Sigma-Aldrich</td>
			<td>7647-14-5</td>
			<td>Control and vehicle for Poly(I:C)</td>
		</tr>
		<tr>
			<td>35mm petri dish</td>
			<td>Thomas Scientific</td>
			<td>1219Z45</td>
			<td>Used to hold oil during tail bleed</td>
		</tr>
		<tr>
			<td>7.5% TGX gels</td>
			<td>Bio-rad</td>
			<td>4561084</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Ancare Nestlets</td>
			<td>Fisher Scientific</td>
			<td>NC9365966</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>anti-&#946;-tubulin</td>
			<td>Millipore</td>
			<td>MAB3408</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Bio-Plex Pro Mouse Cytokine Standards Group I</td>
			<td>Bio-rad</td>
			<td>171I50001</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Plex Pro Reagent Kit with Flat Plate</td>
			<td>Bio-rad</td>
			<td>171304070M</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>ThermoFisher</td>
			<td>23209</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Covidien Monoject 1/2 mL Insulin Syringe with 28G x 1/2 in. Needle</td>
			<td>Spectrum</td>
			<td>552-58457-083</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol</td>
			<td>Sigma-Aldrich</td>
			<td>D9779-10G</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Environmental enrichment</td>
			<td>Bio-serv</td>
			<td>K3327 and K3322</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Ethovision</td>
			<td>Noldus</td>
			<td>Ethovision</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Fluorsecent-tagged seondary ntibodies</td>
			<td>Li-cor</td>
			<td>925-32213 and 925-68072</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Food-grade edible oil (like olive, canola or grapeseed)</td>
			<td>Various vendors</td>
			<td></td>
			<td>Use to lubricate tail during tail bleeds</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>ThermoFisher</td>
			<td>14060040</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>High molecular weight polyinositic:polycytidilic acid</td>
			<td>Invivogen</td>
			<td>#tlrl-pic-5</td>
			<td>Used to establish females&#39; BIR</td>
		</tr>
		<tr>
			<td>Humane Mouse Restrainer</td>
			<td>AIMS</td>
			<td>1000</td>
			<td>Used to restrain mouse during tail bleeds</td>
		</tr>
		<tr>
			<td>Image Studio Software</td>
			<td>Licor</td>
			<td>5.2</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Laemmli buffer</td>
			<td>Bio-rad</td>
			<td>1610737EDU</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Luminex200</td>
			<td>ThermoFisher</td>
			<td>APX10031</td>
			<td></td>
		</tr>
		<tr>
			<td>Microvette CB300 300&#956;l Serum capillary tube</td>
			<td>Sarstedt</td>
			<td>16.440.100</td>
			<td></td>
		</tr>
		<tr>
			<td>Mixed molecular weight polyinositic:polycytidilic acid</td>
			<td>Sigma-Aldrich</td>
			<td>#P0913</td>
			<td>Gestational induction of MIA</td>
		</tr>
		<tr>
			<td>monoclonal anti-MEF2A</td>
			<td>AbCam</td>
			<td>ab76063</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>monoclonal anti-STAT3</td>
			<td>Cell signaling</td>
			<td>12640S</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Observer</td>
			<td>Noldus</td>
			<td>Observer</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Odyssey blocking buffer (TBS)</td>
			<td>Li-cor</td>
			<td>927-50003</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Odyssey CLx imaging system</td>
			<td>Li-cor</td>
			<td>9140</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Omnipure PBS</td>
			<td>Millipore</td>
			<td>65054L</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit</td>
			<td>ThermoFisher</td>
			<td>23227</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>polyclonal anti_TH</td>
			<td>Pel-Freez</td>
			<td>P4101-150</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>PVDF membrane</td>
			<td>Bio-rad</td>
			<td>162-0177</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Qsonica Sonicator Q500</td>
			<td>Fisher Scientific</td>
			<td>15-338-282</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Quick blood stopper</td>
			<td>Petco</td>
			<td>17140</td>
			<td></td>
		</tr>
		<tr>
			<td>Seal-Rite 1.5 ml microcentrifuge tube, natural non-sterile</td>
			<td>USA Scientific</td>
			<td>1615-5500</td>
			<td></td>
		</tr>
		<tr>
			<td>Soldering stand</td>
			<td>Amazon</td>
			<td>B08Y12QC73</td>
			<td>Used to hold capillary tube during tail bleeds</td>
		</tr>
		<tr>
			<td>Sunflower seeds</td>
			<td>Bio-serv</td>
			<td>S5137-1</td>
			<td>Use to increase breeding efficiency</td>
		</tr>
		<tr>
			<td>The Bio-Plex Pro Mouse IL-6 set,</td>
			<td>Bio-rad</td>
			<td>171G5007M</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Fisher Scientific</td>
			<td>BP152-1</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Bio-rad</td>
			<td>23209</td>
			<td>Optional</td>
		</tr>
	</tbody>
</table>

generating a reproducible model of mid-gestational maternal immune activation using poly(i:c) to study susceptibility and resilience in offspring

गर्भावस्था के दौरान मातृ प्रतिरक्षा सक्रियण (एमआईए) लगातार संतानों में न्यूरोडेवलपमेंटल और न्यूरोसाइकियाट्रिक विकारों के बढ़ते जोखिम से जुड़ा हुआ है। एमआईए के पशु मॉडल का उपयोग कार्य-कारण का परीक्षण करने, तंत्र की जांच करने और इन विकारों के लिए निदान और उपचार विकसित करने के लिए किया जाता है। उनके व्यापक उपयोग के बावजूद, कई एमआईए मॉडल प्रजनन क्षमता की कमी से पीड़ित हैं और लगभग सभी इस जोखिम कारक के दो महत्वपूर्ण पहलुओं को अनदेखा करते हैं: (i) कई संतान एमआईए के लिए लचीला हैं, और (ii) अतिसंवेदनशील संतान फेनोटाइप के अलग-अलग संयोजन प्रदर्शित कर सकती हैं। प्रजनन क्षमता बढ़ाने और एमआईए के लिए संवेदनशीलता और लचीलापन दोनों को मॉडल करने के लिए, गर्भावस्था से पहले महिला चूहों की बेसलाइन विकृति (बीआईआर) का उपयोग यह अनुमान लगाने के लिए किया जाता है कि एमआईए के संपर्क में आने के बाद कौन सी गर्भावस्था के परिणामस्वरूप या तो लचीला वंश या परिभाषित व्यवहार और आणविक असामान्यताओं के साथ संतान होगी। यहां, गर्भावस्था के 12.5 दिनों में डबल फंसे आरएनए (डीएसआरएनए) वायरल मिमिक पॉली (आई: सी) के इंट्रापरिटोनियल (आईपी) इंजेक्शन के माध्यम से एमआईए को प्रेरित करने की एक विस्तृत विधि प्रदान की गई है। यह विधि बांध में एक तीव्र भड़काऊ प्रतिक्रिया को प्रेरित करती है, जिसके परिणामस्वरूप चूहों में मस्तिष्क के विकास में गड़बड़ी होती है जो मानव मनोरोग और न्यूरोडेवलपमेंटल विकारों (एनडीडी) में समान रूप से प्रभावित डोमेन पर मैप करती है।

ई 12.5 पर 30 मिलीग्राम / किलोग्राम पॉली (आई: सी) के संपर्क में आने वाले सभी जानवर लगातार व्यवहार संबंधी असामान्यताओं के साथ संतान पैदा नहीं करते हैं। हालांकि 30 मिलीग्राम / किग्रा और 40 मिल...

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Generating a Reproducible Model of Mid-Gestational Maternal Immune Activation using PolyI:C to Study Susceptibility and Resilience in Offspring

मातृ संक्रमण न्यूरोडेवलपमेंटल विकारों के लिए एक जोखिम कारक है। मातृ प्रतिरक्षा सक्रियण (एमआईए) के माउस मॉडल मस्तिष्क के विकास और कार्य पर संक्रमण के प्रभाव को स्पष्ट कर सकते हैं। यहां, एमआईए के संपर्क में मज़बूती से लचीला और अतिसंवेदनशील संतान पैदा करने के लिए सामान्य दिशानिर्देश और एक प्रक्रिया प्रदान की जाती है।

संतानों में संवेदनशीलता और लचीलापन का अध्ययन करने के लिए पॉली (आई: सी) का उपयोग करके मध्य-गर्भकालीन मातृ प्रतिरक्षा सक्रियण का एक प्रतिलिपि प्रस्तुत करने योग्य मॉडल तैयार करना

Maternal immune activation (MIA) during pregnancy is consistently linked to increased risk of neurodevelopmental and neuropsychiatric disorders in ...

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Generating a Reproducible Model of Mid-Gestational Maternal Immune Activation using Poly(I:C) to Study Susceptibility and Resilience in Offspring

Using MazeSuite and Functional Near Infrared Spectroscopy to Study Learning in Spatial Navigation

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments1. MazeSuite can be used to design and edit adapted virtual 3D environments, track a participants' behavioral performance within the virtual environment and synchronize with external devices for physiological and neuroimaging measures, including electroencephalogram and eye tracking.
Functional near-infrared spectroscopy (fNIR) is an optical brain imaging technique that enables continuous, noninvasive, and portable monitoring of changes in cerebral blood oxygenation related to human brain functions2-7. Over the last decade fNIR is used to effectively monitor cognitive tasks such as attention, working memory and problem solving7-11. fNIR can be implemented in the form of a wearable and minimally intrusive device; it has the capacity to monitor brain activity in ecologically valid environments. 
Cognitive functions assessed through task performance involve patterns of brain activation of the prefrontal cortex (PFC) that vary from the initial novel task performance, after practice and during retention12. Using positron emission tomography (PET), Van Horn and colleagues found that regional cerebral blood flow was activated in the right frontal lobe during the encoding (i.e., initial na&iuml;ve performance) of spatial navigation of virtual mazes while there was little to no activation of the frontal regions after practice and during retention tests. Furthermore, the effects of contextual interference, a learning phenomenon related to organization of practice, are evident when individuals acquire multiple tasks under different practice schedules13,14. High contextual interference (random practice schedule) is created when the tasks to be learned are presented in a non-sequential, unpredictable order. Low contextual interference (blocked practice schedule) is created when the tasks to be learned are presented in a predictable order.
Our goal here is twofold: first to illustrate the experimental protocol design process and the use of MazeSuite, and second, to demonstrate the setup and deployment of the fNIR brain activity monitoring system using Cognitive Optical Brain Imaging (COBI) Studio software15. To illustrate our goals, a subsample from a study is reported to show the use of both MazeSuite and COBI Studio in a single experiment. The study involves the assessment of cognitive activity of the PFC during the acquisition and learning of computer maze tasks for blocked and random orders. Two right-handed adults (one male, one female) performed 315 acquisition, 30 retention and 20 transfer trials across four days. Design, implementation, data acquisition and analysis phases of the study were explained with the intention to provide a guideline for future studies.

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments. Functional near-infrared spectroscopy (fNIR) is an optical brain imaging technique that enables noninvasive and portable monitoring of cerebral blood oxygenation changes. This paper summarizes collective use of MazeSuite and fNIR within a cognitive processing learning paradigm.

इन्फ्रारेड स्पेक्ट्रोस्कोपी के पास MazeSuite और कार्यात्मक का उपयोग करने के लिए सीखने के स्थानिक नेविगेशन में अध्ययन

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments1. MazeSuite can be used to design and edit adapted ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

प्रतिलिपि प्रस्तुत करने योग्य माउस sciatic तंत्रिका और पूरे पर्वत स्नायु विश्लेषण द्वारा पुनर्जनन के बाद आकलन क्रश

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

एक मॉडल के रूप घ्राण प्रणाली axonal विकास पैटर्न और आकृति विज्ञान का अध्ययन करने के लिए<em&gt; Vivo</em

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

M&#252;ller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after severe damage. Investigating the regeneration pathways in zebrafish might bring new insights to develop innovative strategies for the treatment of retinal degenerative diseases in mammals. Herein, we focused on the induction of a focal lesion to the outer retina in adult zebrafish by means of a 532 nm diode laser. A localized injury allows investigating biological processes that take place during retinal degeneration and regeneration directly at the area of damage. Using non-invasive optical coherence tomography (OCT), we were able to define the location of the damaged area and monitor subsequent regeneration in vivo. Indeed, OCT imaging produces high-resolution, cross-sectional images of the zebrafish retina, providing information which was previously only available with histological analyses. In order to confirm the data from real-time OCT, histological sections were performed and regenerative response after the induction of the retinal injury was investigated by immunohistochemistry.

The zebrafish is a popular animal model to study mechanisms of retinal degeneration/regeneration in vertebrates. This protocol describes a method to induce localized injury disrupting the outer retina with minimal damage to the inner retina. Subsequently, we monitor in vivo the retinal morphology and the M&#252;ller glia response throughout retinal regeneration.

म्यूलर Glia सेल सक्रियकरण में एक लेजर प्रेरित रेटिना अध-पतन और Zebrafish में मॉडल पुनर्जनन

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after ...

An Objective and Reproducible Test of Olfactory Learning and Discrimination in Mice

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and parenting. Importantly, mice can be trained to associate novel odors with specific behavioral responses to provide insight into olfactory circuit function. This protocol details the procedure for training mice on a Go/No-Go operant learning task. In this approach, mice are trained on hundreds of automated trials daily for 2&#8211;4 weeks and can then be tested on novel Go/No-Go odor pairs to assess olfactory discrimination, or be used for studies on how odor learning alters the structure or function of the olfactory circuit. Additionally, the mouse olfactory bulb (OB) features ongoing integration of adult-born neurons. Interestingly, olfactory learning increases both the survival and synaptic connections of these adult-born neurons. Therefore, this protocol can be combined with other biochemical, electrophysiological, and imaging techniques to study learning and activity-dependent factors that mediate neuronal survival and plasticity.

Here, we train mice on an associative learning task to test odor discrimination. This protocol also allows for studies on learning-induced structural changes in the brain.

घ्राण सीखने और चूहों में भेदभाव का एक उद्देश्य और Reproducible परीक्षण

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and ...

A Simple and Reproducible Method to Prepare Membrane Samples from Freshly Isolated Rat Brain Microvessels

The blood-brain barrier (BBB) is a dynamic barrier tissue that responds to various pathophysiological and pharmacological stimuli. Such changes resulting from these stimuli can greatly modulate drug delivery to the brain and, by extension, cause considerable challenges in the treatment of central nervous system (CNS) diseases. Many BBB changes that affect pharmacotherapy, involve proteins that are localized and expressed at the level of endothelial cells. Indeed, such knowledge on BBB physiology in health and disease has sparked considerable interest in the study of these membrane proteins. From a basic science research standpoint, this implies a requirement for a simple but robust and reproducible method for isolation of microvessels from brain tissue harvested from experimental animals. In order to prepare membrane samples from freshly isolated microvessels, it is essential that sample preparations be enriched in endothelial cells but limited in the presence of other cell types of the neurovascular unit (i.e., astrocytes, microglia, neurons, pericytes). An added benefit is the ability to prepare samples from individual animals in order to capture the true variability of protein expression in an experimental population. In this manuscript, details regarding a method that is utilized for isolation of rat brain microvessels and preparation of membrane samples are provided. Microvessel enrichment, from samples derived, is achieved by using four centrifugation steps where dextran is included in the sample buffer. This protocol can easily be adapted by other laboratories for their own specific applications. Samples generated from this protocol have been shown to yield robust experimental data from protein analysis experiments that can greatly aid the understanding of BBB responses to physiological, pathophysiological, and pharmacological stimuli.

Here, a method for isolation of rat brain microvessels and for the preparation of membrane samples is described. This protocol has the clear advantage of producing enriched microvessel samples with acceptable protein yield from individual animals. Samples can then be used for robust protein analyses at the brain microvascular endothelium.

एक सरल और Reproducible विधि हौसले से अलग चूहे मस्तिष्क Microvessels से झिल्ली के नमूने तैयार करने के लिए

The blood-brain barrier (BBB) is a dynamic barrier tissue that responds to various pathophysiological and pharmacological stimuli. Such changes resulting ...

A Neurosphere Assay to Evaluate Endogenous Neural Stem Cell Activation in a Mouse Model of Minimal Spinal Cord Injury

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied in vitro using the neurosphere assay. This colony-forming assay is a powerful tool to study the response of NSCs to exogenous factors in a dish; however, this can also be used to study the effect of in vivo manipulations with the proper understanding of the strengths and limitations of the assay. One manipulation of the clinical interest is the effect of injury on endogenous NSC activation. Current models of spinal cord injury provide a challenge to study this as the severity of common contusion, compression, and transection models cause the destruction of the NSC niche at the site of the injury where the stem cells reside. Here, we describe a minimal injury model that causes localized damage at the superficial dorsolateral surface of the lower thoracic level (T7/8) of the adult mouse spinal cord. This injury model spares the central canal at the level of injury and permits analysis of the NSCs that reside at the level of the lesion at various time points following injury. Here, we show how the neurosphere assay can be utilized to study the activation of the two distinct, lineally-related, populations of NSCs that reside in the spinal cord periventricular region - primitive and definitive NSCs (pNSCs and dNSCs, respectively). We demonstrate how to isolate and culture these NSCs from the periventricular region at the level of injury and the white matter injury site. Our post-surgical spinal cord dissections show increased numbers of pNSC and dNSC-derived neurospheres from the periventricular region of injured cords compared to controls, speaking to their activation via injury. Furthermore, following injury, dNSC-derived neurospheres can be isolated from the injury site &#8212; demonstrating the ability of NSCs to migrate from their periventricular niche to sites of injury.

Here, we demonstrate the performance of a minimal spinal cord injury model in an adult mouse that spares the central canal niche housing endogenous neural stem cells (NSCs). We show how the neurosphere assay can be used to quantify activation and migration of definitive and primitive NSCs following injury.

एक Neurosphere परख का मूल्यांकन करने के लिए अंतर्जात तंत्रिका कोशिका सक्रियकरण कम रीढ़ की हड्डी की चोट के एक माउस मॉडल में स्टेम

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied ...

Facial Nerve Surgery in the Rat Model to Study Axonal Inhibition and Regeneration

This protocol describes consistent and reproducible methods to study axonal regeneration and inhibition in a rat facial nerve injury model. The facial nerve can be manipulated along its entire length, from its intracranial segment to its extratemporal course. There are three primary types of nerve injury used for the experimental study of regenerative properties: nerve crush, transection, and nerve gap. The range of possible interventions is vast, including surgical manipulation of the nerve, delivery of neuroactive reagents or cells, and either central or end-organ manipulations. Advantages of this model for studying nerve regeneration include simplicity, reproducibility, interspecies consistency, reliable survival rates of the rat, and an increased anatomic size relative to murine models. Its limitations involve a more limited genetic manipulation versus the mouse model and the superlative regenerative capability of the rat, such that the facial nerve scientist must carefully assess time points for recovery and whether to translate results to higher animals and human studies. The rat model for facial nerve injury allows for functional, electrophysiological, and histomorphometric parameters for the interpretation and comparison of nerve regeneration. It thereby boasts tremendous potential toward furthering the understanding and treatment of the devastating consequences of facial nerve injury in human patients.

This protocol describes a reproducible approach to facial nerve surgery in the rat model, including descriptions of various inducible patterns of injury.

एक्सोनल अवरोध और उत्थान का अध्ययन करने के लिए चूहा मॉडल में चेहरे तंत्रिका सर्जरी

This protocol describes consistent and reproducible methods to study axonal regeneration and inhibition in a rat facial nerve injury model. The facial ...

Combining Behavior and EEG to Study the Effects of Mindfulness Meditation on Episodic Memory

Although there has been recent interest in how mindfulness meditation can affect episodic memory as well as brain structure and function, no study has examined the behavioral and neural effects of mindfulness meditation on episodic memory. Here we present a protocol that combines mindfulness meditation training, an episodic memory task, and EEG to examine how mindfulness meditation changes behavioral performance and the neural correlates of episodic memory. Subjects in a mindfulness meditation experimental group were compared to a waitlist control group. Subjects in the mindfulness meditation experimental group spent four weeks training and practicing mindfulness meditation. Mindfulness was measured before and after training using the Five Facet Mindfulness Questionnaire (FFMQ). Episodic memory was measured before and after training using a source recognition task. During the retrieval phase of the source recognition task, EEG was recorded. The results showed that mindfulness, source recognition behavioral performance, and EEG theta power in right frontal and left parietal channels increased following mindfulness meditation training. In addition, increases in mindfulness correlated with increases in theta power in right frontal channels. Therefore, results obtained from combining mindfulness meditation training, an episodic memory task, and EEG reveal the behavioral and neural effects of mindfulness meditation on episodic memory.

Here we present a protocol for combining mindfulness meditation training, an episodic memory task, and EEG to understand the behavioral and neural effects of mindfulness meditation on episodic memory.

एपिसोडिक मेमोरी पर माइंडफुलनेस मेडिटेशन के प्रभावों का अध्ययन करने के लिए व्यवहार और ईईजी का संयोजन

Although there has been recent interest in how mindfulness meditation can affect episodic memory as well as brain structure and function, no study has ...

A Scalable Model to Study the Effects of Blunt-Force Injury in Adult Zebrafish

Blunt-force traumatic brain injuries (TBI) are the most common form of head trauma, which spans a range of severities and results in complex and heterogenous secondary effects. While there is no mechanism to replace or regenerate the lost neurons following a TBI in humans, zebrafish possess the ability to regenerate neurons throughout their body, including the brain. To examine the breadth of pathologies exhibited in zebrafish following a blunt-force TBI and to study the mechanisms underlying the subsequent neuronal regenerative response, we modified the commonly used rodent Marmarou weight drop for the use in adult zebrafish. Our simple blunt-force TBI model is scalable, inducing a mild, moderate, or severe TBI, and recapitulates many of the phenotypes observed following human TBI, such as contact- and post-traumatic seizures, edema, subdural and intracerebral hematomas, and cognitive impairments, each displayed in an injury severity-dependent manner. TBI sequelae, which begin to appear within minutes of the injury, subside and return to near undamaged control levels within 7 days post-injury. The regenerative process begins as early as 48 hours post-injury (hpi), with the peak cell proliferation observed by 60 hpi. Thus, our zebrafish blunt-force TBI model produces characteristic primary and secondary injury TBI pathologies similar to human TBI, which allows for investigating disease onset and progression, along with the mechanisms of neuronal regeneration that is unique to zebrafish.

We modified the Marmarou weight drop model for adult zebrafish to examine a breadth of pathologies following blunt-force traumatic brain injury (TBI) and the mechanisms underlying subsequent neuronal regeneration. This blunt-force TBI model is scalable, induces a mild, moderate, or severe TBI, and recapitulates injury heterogeneity observed in human TBI.