Name: stereological estimation of dopaminergic neuron number in the mouse substantia nigra using the optical fractionator and standard microscopy equipment
Uploaded: 2017-09-01T14:00:00
Description: Watch this Scientific Journal Video about Stereological Estimation of Dopaminergic Neuron Number in the Mouse Substantia Nigra Using the Optical Fractionator and Standard Microscopy Equipment at JoVE.

Forfatterne er takknemlig Keali Röhm, Louisa Frieß og Heike Menzel for deres ekspert teknisk assistanse; til Helga Brünner for dyr omsorg; og Chistopher S. Ward for generasjon og distribusjon av optisk disector rutenett plugin for ImageJ programvaren.

alle gjeldende internasjonale, nasjonale eller institusjonelle retningslinjer og bruk av dyr ble fulgt. Protokollen ble godkjent av lokale myndigheter på Regierung von Unterfranken, Würzburg, Tyskland.
 1. vev behandling og Immunohistochemistry <ol><li>Euthanize mus med CO 2 eller andre godkjent metode.</li>
	<li>Perfuse seks 12-uke-gamle C56Bl/6N mannlige mus transcardially med 10 mL 0.1 M fosfat-bufret saltoppløsning (PBS) bruker en 25-G nål og en infusjon pumpe, etterfulgt...

<ol>
	<li>Ip, C. W., 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=AAV1/2-induced+overexpression+of+A53T-alpha-synuclein+in+the+substantia+nigra+results+in+degeneration+of+the+nigrostriatal+system+with+Lewy-like+pathology+and+motor+impairment:+a+new+mouse+model+for+Parkinson's+disease.">AAV1/2-induced overexpression of A53T-alpha-synuclein in the substantia nigra results in degeneration of the nigrostriatal system with Lewy-like pathology and motor impairment: a new mouse model for Parkinson's disease.</a> Acta Neuropathol Commun. 5 (1), 11 (2017).</li><li>Ip, C. W., Beck, S. 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Stereology starter med vev behandling. Seriell skjæring av SN vev må utføres med forsiktighet for å hindre tap av delene under stereological analyse. Dessuten er en viktig skritt å merke en halvkule for å skille rett fra venstre SN når du utfører stereology. Plassere et lite hull i den øvre delen av hjernestammen generert beste resultatene presentert studien. Videre siden jobbet med optisk fractionator metoden krav som vevet skjær i tykke snitt ca 30-40 µm, i stedet for 5-10 µm mer vanlig i immunohistochemist...

Kvantifisering av neuronal celle nummer er sentral i pre-klinisk Parkinsons sykdom forskning for å bestemme nivået av neurodegeneration innen substantia nigra (SN)1,2. Upartiske stereological estimering av celle nummer i et område av interesse anses gullstandarden3,4,5.
Før objektiv stereology, ble antall nevroner i delene vurdert ved å manipulere telt celle profiler korrigere variabel sannsynligheten at nervecellene kommer til syne i en inndeling. En av de mest brukte metodene var korreksjon av kvantifisert celletall beskrevet av Abercrombie6. Denne metoden forsøkte å ta hensyn til at cellene kan kvantifiseres mer enn én gang hvis fragmenter av samme celle finnes i tilstøtende tynne snitt. Derfor generert Abercrombie og andre forfattere ligninger som nødvendige forutsetninger om form, størrelse og retning av telles cellene7,8. Men var dette vanligvis ikke realisert og førte til systematisk feil og avvik fra hvor selve cellen (dvs. bias). Videre kunne bias ikke bli redusert med ytterligere prøvetaking3.
For stereological estimering celle tall ved hjelp av den optiske fractionator, brukes matematiske prinsipper for å anslå direkte celle tall direkte i et definert, 3-dimensjonale volum. Fordelen med denne metoden er at det ikke involverer antagelser om form, størrelse og retning av cellene blir regnet. Dermed anslagsvis cellen tallene er nærmere den sanne verdiene og komme nærmere som utvalgsstørrelsen (dvs. objektiv)3. Fordi mange regler må følges ved stereology for å holde metoden upartisk, klar til bruk kommersielle stereology systemer er utviklet (for vurdering, se Schmitz og Hof, 20054). Spesialisert stereology systemer implementere design-baserte stereological metoder med en priori definert sonder og prøvetaking ordninger for stereological vurderinger som fører til uavhengighet fra form, størrelse, romlige fordelingen og retningen på de cellene skal analysert4,9. Tilsvarede stereology systemer er imidlertid dyrt; Dette kan begrense implementering i ny forskning.
Målet med denne studien var å utvikle en brukervennlig teknikk for design-baserte stereological estimering dopaminergic celletall musen SN, sysselsetter metoden optiske fractionator og bruker standard mikroskopi utstyr (dvs. lys mikroskopet, standard mikroskop programvare og motoriserte x, y, z scenen). For dette vises en trinnvis veiledning på hvordan å behandle musen hjernevevet og beregne SN celle tall ved hjelp av design-baserte upartisk stereology. Dessuten tilbys maler for beregning av beregnet celle tall og koeffisientene til feil (CE).
Metoden beskrevet her er ikke begrenset til analyse av SN, men kan tilpasses for bruk i andre anatomisk definerte områder av mus eller rotte hjernen. For eksempel har upartisk stereology blitt brukt til å beregne neuronal cellen tallene i hippocampus10 og locus coeruleus11. I tillegg kan celletyper enn neurons, som astrocyttene12 og microglia13, vurderes også. Derfor kan denne metoden være nyttig for forskere som ønsker å implementere upartisk stereology i sin forskning, men er ikke villig til å bruke mye penger å kjøpe en stereology system.

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			<td height="25" style="height:25px;width:216px;">Paxinos mouse atlas</td>
			<td style="width:217px;"></td>
			<td style="width:119px;"></td>
			<td style="width:467px;">The Mouse Brain George Paxinos Keith B.J.FranklinCopyright @2001 by Academic Press CD Rom designet &#38; created by Paul Halasz</td>
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			<td height="21" style="height:21px;width:216px;">brain matrix slicer mouse</td>
			<td style="width:217px;">Zivic Instruments&#160;</td>
			<td style="width:119px;">BSMAS 001-1</td>
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			<td height="21" style="height:21px;width:216px;">paraformaldehyde</td>
			<td style="width:217px;">Merck</td>
			<td style="width:119px;">1040051000</td>
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			<td height="21" style="height:21px;width:216px;">sucrose /D(+) Saccharose&#160;</td>
			<td style="width:217px;">Roth</td>
			<td style="width:119px;">4621.1</td>
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			<td height="21" style="height:21px;width:216px;">isopentane</td>
			<td style="width:217px;">Roth</td>
			<td style="width:119px;">3927.1</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">glycerol</td>
			<td style="width:217px;">Merck</td>
			<td style="width:119px;">1040931000</td>
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			<td height="21" style="height:21px;width:216px;">Ethanol</td>
			<td style="width:217px;">Sigma Aldrich</td>
			<td style="width:119px;">32205-1L</td>
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			<td height="21" style="height:21px;width:216px;">Name</td>
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			<td>Comments</td>
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			<td height="42" style="height:42px;width:216px;">phosphate buffered saline ingredients:</td>
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			<td height="21" style="height:21px;width:216px;">sodium chloride</td>
			<td style="width:217px;">Sigma Aldrich</td>
			<td style="width:119px;">31434-1KG-R</td>
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			<td height="42" style="height:42px;width:216px;">potassium dihydrogen phosphate</td>
			<td style="width:217px;">Merck</td>
			<td style="width:119px;">1048731000</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">di-sodium hydrogen phosphate dihydrate</td>
			<td style="width:217px;">Merck</td>
			<td style="width:119px;">1065801000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">potassium chloride</td>
			<td style="width:217px;">Merck</td>
			<td style="width:119px;">1049360500</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">normal goat serum</td>
			<td style="width:217px;">Dako</td>
			<td style="width:119px;">X0907</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">bovine serum albumin</td>
			<td style="width:217px;">Sigma&#160;</td>
			<td style="width:119px;">A4503-100G</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton X-100</td>
			<td style="width:217px;">Sigma Aldrich</td>
			<td style="width:119px;">X100-100ml</td>
			<td>detergent</td>
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			<td height="63" style="height:63px;width:216px;">3,3-Diaminobenzidine-tetrahydrochlorid/DAB tablets 10mg pH 7.0</td>
			<td style="width:217px;">Kem En Tec</td>
			<td style="width:119px;">4170</td>
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			<td height="26" style="height:26px;width:216px;">H2O2/ Hydrogen peroxide 30%</td>
			<td style="width:217px;">Merck</td>
			<td style="width:119px;">1072090250</td>
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			<td height="21" style="height:21px;width:216px;">avidin/biotin reagent</td>
			<td style="width:217px;">Thermo Scientific</td>
			<td style="width:119px;">32050</td>
			<td>Standard Ultra Sensitive ABC Staining Kit, 1:100</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">rabbit anti mouse tyrosine hydroxylase antibody</td>
			<td style="width:217px;">abcam</td>
			<td style="width:119px;">Ab112</td>
			<td>1:1000</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">biotinylated goat-anti-rabbit IgG H+L</td>
			<td style="width:217px;">vector laboratories</td>
			<td style="width:119px;">BA-1000</td>
			<td>1:100</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">StereoInvestigator version 11.07</td>
			<td style="width:217px;">MBF</td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BX53 microscope</td>
			<td style="width:217px;">Olympus</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Visiview</td>
			<td style="width:217px;">Visitron Systems GmbH</td>
			<td style="width:119px;">3.3.0.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Axiophot2</td>
			<td style="width:217px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ImageJ software</td>
			<td style="width:217px;">NIH</td>
			<td style="width:119px;">Version 4.7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tissue-TEK OCT</td>
			<td style="width:217px;">Sakura</td>
			<td style="width:119px;">4583</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dry ice</td>
			<td style="width:217px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">grid overlay plugin</td>
			<td style="width:217px;">Wayne Rasband</td>
			<td style="width:119px;"></td>
			<td>https://imagej.nih.gov/ij/plugins/graphic-overlay.html</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">cell counter plugin</td>
			<td style="width:217px;">Kurt de Vos</td>
			<td style="width:119px;"></td>
			<td>https://imagej.nih.gov/ij/plugins/cell-counter.html).&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">optical disector macro</td>
			<td style="width:217px;">Christopher Ward</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">C57Bl/6N male mice</td>
			<td style="width:217px;">Charles River, Germany</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">SuperFrost Plus coated object slides</td>
			<td style="width:217px;">Langenbrinck, Germany</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">25G needle Microlance 3</td>
			<td style="width:217px;">BD&#160;</td>
			<td align="right" style="width:119px;">300400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">REGLO Analog Infusion pump</td>
			<td style="width:217px;">Ismatec</td>
			<td style="width:119px;">ISM 829</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">StereoInvestigator system</td>
			<td style="width:217px;"></td>
			<td style="width:119px;"></td>
			<td style="width:467px;">StereoInvestigator version 11.07</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BX53 microscope</td>
			<td style="width:217px;"></td>
			<td style="width:119px;"></td>
			<td style="width:467px;">BX53 microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">self-assorted stereology</td>
			<td style="width:217px;"></td>
			<td style="width:119px;"></td>
			<td style="width:467px;">Visiview</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Axiophot2</td>
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stereological estimation of dopaminergic neuron number in the mouse substantia nigra using the optical fractionator and standard microscopy equipment

I pre-klinisk Parkinsons sykdom forskning er analyse av nigrostriatal-område, inkludert kvantifisering av dopaminergic Nevron tap i substantia nigra, avgjørende. For å beregne totalt dopaminergic Nevron nummeret, anses for tiden upartisk stereology med metoden optisk fractionator gull standard. Fordi teorien bak optisk fractionator metoden er komplekse og fordi stereology er vanskelig å oppnå uten spesialisert utstyr, eksisterer flere kommersielt tilgjengelig komplett stereology systemer som inkluderer nødvendig programvare, rent teller grunner for cellen. Siden kjøpe en spesialisert stereology oppsett ikke er alltid mulig, for mange grunner, denne rapporten beskriver en metode for dopaminergic neuronal celle stereological estimering teller bruker standard mikroskopi utstyr, inkludert en lett mikroskop, en motorisert objekttabellen (x, y, z-fly) med bildebehandlingsprogramvare og en datamaskin for analyse. Gis en trinnvis forklaring på hvordan du utfører stereological kvantifisering med metoden optiske fractionator og pre-programmerte filene for beregning av beregnet celletall tilbys. For å vurdere nøyaktigheten av denne metoden, ble en sammenligning til data fra et kommersielt tilgjengelig stereology apparatet utført. Sammenlignbare cellen tallene ble funnet ved hjelp av denne protokollen og stereology enheten, dermed demonstrere presisjonen for denne protokollen for upartisk stereology.

Med metoden presentert, beregnet antall TH + dopaminergic neurons SN varierte mellom 7,363 og 7,987 celler til høyre og venstre SN, mellom 7,446 og 7,904 celler. Dermed var gjennomsnittlig antall dopaminergic nevroner (± SEM) 7,647 ± 83 celler for høyre SN og 7,675 ± 66 for venstre SN. Beregnet CE for hvert dyr var lavere enn 0,08 (område: 0.073-0.079) (figur 7). For å konstatere sammenlignbarheten metoden med kommersielt tilgjengelig og spesialisert s...

Watch this Scientific Journal Video about Stereological Estimation of Dopaminergic Neuron Number in the Mouse Substantia Nigra Using the Optical Fractionator and Standard Microscopy Equipment at JoVE.

Stereological Estimation of Dopaminergic Neuron Number in the Mouse Substantia Nigra Using the Optical Fractionator and Standard Microscopy Equipment

Dette arbeidet presenterer en trinnvis protokoll for upartisk stereological estimering dopaminergic neuronal cellen tallene i musen substantia nigra bruker standard mikroskopi utstyr (dvs. en lys mikroskop, en motorisert objekttabellen (x, y, z fly), og offentlige programvare for digital bildeanalyser.

Stereological estimering av Dopaminergic Neuron nummer i musen Substantia Nigra optiske Fractionator og Standard mikroskopi utstyr

In pre-clinical Parkinson&#39;s disease research, analysis of the nigrostriatal tract, including quantification of dopaminergic neuron loss within the ...

Research

JoVE Journal

Neuroscience

Imaging Odor-Evoked Activities in the Mouse Olfactory Bulb using Optical Reflectance and Autofluorescence Signals

Imaging Lukt-evoked Aktiviteter i Mus luktelappen bruker optisk Refleksjon og Autofluorescence Signaler

In the brain, sensory stimulation activates distributed populations of neurons among functional modules which participate to the coding of the stimulus. ...

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

I ovo electroporation i Chick midbrain for å studere gen-funksjon i dopaminerg Neuron Development

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain ...

Comprehensive Profiling of Dopamine Regulation in Substantia Nigra and Ventral Tegmental Area

Omfattende profilering av Dopamin forordning i substantia nigra og Ventral tegmentale området

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five ...

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Regissert dopaminerge Neuron Differensiering fra Menneskelig Pluripotent stamceller

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

Nevromuskulære krysset: Måling Synapse Størrelse, fragmentering og endringer i Synaptic Protein Tetthet Bruke Confocal Fluorescensmikroskopi

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

Optisk Kontroll av en neuronal Protein Ved hjelp av en genetisk kodet Unaturlig Amino Acid i nevroner

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to ...

Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells

Analyse for å måle nukleocytoplasmisk transport i sanntid innen Motor Neuron-lignende NSC-34-celler

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a ...

The Optical Fractionator Technique to Estimate Cell Numbers in a Rat Model of Electroconvulsive Therapy

Den optiske fraksjonatorteknikken til å beregne celletal i en rottemodel for elektrokonvulsiv terapi

Stereological methods are designed to describe quantitative parameters without making assumptions about size, shape, orientation and distribution of cells ...

Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

Live bilder av GLUT4 Protein menneskehandel musen primære hypothalamus nevroner bruker Deconvolution mikroskopi

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral ...

A Microbiomechanical System for Studying Varicosity Formation and Recovery in Central Neuron Axons

Et Microbiomechanical System for å studere Varicosity formasjon og utvinning i sentrale Nevron Axons

Axonal varicosities are enlarged structures along the shafts of axons with a high degree of heterogeneity. They are present not only in brains with ...