Name: quantitative measurement of intrathecally synthesized proteins in mice
Uploaded: 2019-11-29T13:00:00
Description: Watch this Scientific Journal Video about Quantitative Measurement of Intrathecally Synthesized Proteins in Mice at JoVE.com

The authors thank the staff of the Center for Comparative Medicine and Research (CCMR) at Dartmouth for their expert care of the mice used for these studies. The Bornstein Research Fund funded this research.

All animal work utilizes protocols reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at Geisel School of Medicine at Dartmouth.
1. Collection of fluids
NOTE: Both serum and CSF are required. Two protocols for each fluid collection are needed for survival and necropsy.
<ol>
	<li>Serum and CSF collection using survival procedures 
	NOTE: For survival fluid collection, serum collection should precede CSF collection as it is a less ...

<ol>
	<li>Whedon, J. M., Glassey, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebrospinal+fluid+stasis+and+its+clinical+significance.">Cerebrospinal fluid stasis and its clinical significance.</a> Alternative Therapies in Health and Medicine. 15 (3), 54-60 (2009).</li><li>Kang, J. H., Vanderstichele, H., Trojanowski, J. Q., Shaw, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+analysis+of+cerebrospinal+fluid+biomarkers+using+microsphere-based+xMAP+multiplex+technology+for+early+detection+of+Alzheimer's+disease.">Simultaneous analysis of cerebrospinal fluid biomarkers using microsphere-based xMAP multiplex technology for early detection of Alzheimer's disease.</a> Methods. 56 (4), 484-493 (2012).</li><li>Barro, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluid+biomarker+and+electrophysiological+outcome+measures+for+progressive+MS+trials.">Fluid biomarker and electrophysiological outcome measures for progressive MS trials.</a> Multiple Sclerosis. 23 (12), 1600-1613 (2017).</li><li>Tourtellotte, W. 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=Multiple+sclerosis:+measurement+and+validation+of+central+nervous+system+IgG+synthesis+rate.">Multiple sclerosis: measurement and validation of central nervous system IgG synthesis rate.</a> Neurology. 30 (3), 240-244 (1980).</li><li>Bonnan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrathecal+IgG+synthesis:+a+resistant+and+valuable+target+for+future+multiple+sclerosis+treatments.">Intrathecal IgG synthesis: a resistant and valuable target for future multiple sclerosis treatments.</a> Multiple Sclerosis International. 2015, 296184 (2015).</li><li>Reiber, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebrospinal+fluid--physiology,+analysis+and+interpretation+of+protein+patterns+for+diagnosis+of+neurological+diseases.">Cerebrospinal fluid--physiology, analysis and interpretation of protein patterns for diagnosis of neurological diseases.</a> Multiple Sclerosis. 4 (3), 99-107 (1998).</li><li>DiSano, K. D., Linzey, M. R., Royce, D. B., Pachner, A. R., Gilli, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+neuro-immune+patterns+in+two+clinically+relevant+murine+models+of+multiple+sclerosis.">Differential neuro-immune patterns in two clinically relevant murine models of multiple sclerosis.</a> Journal of Neuroinflammation. 16 (1), 109 (2019).</li><li>Pachner, A. R., Li, L., Lagunoff, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+cells+in+the+central+nervous+system+in+the+Theiler's+virus+model+of+multiple+sclerosis.">Plasma cells in the central nervous system in the Theiler's virus model of multiple sclerosis.</a> Journal of Neuroimmunology. 232 (1-2), 35-40 (2011).</li><li>Reiber, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+rate+of+cerebrospinal+fluid+(CSF)--a+concept+common+to+normal+blood-CSF+barrier+function+and+to+dysfunction+in+neurological+diseases.">Flow rate of cerebrospinal fluid (CSF)--a concept common to normal blood-CSF barrier function and to dysfunction in neurological diseases.</a> Journal of Neurological Sciences. 122 (2), 189-203 (1994).</li><li>Reiber, H., Zeman, D., Kusnierova, P., Mundwiler, E., Bernasconi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+relevance+of+free+light+chains+in+cerebrospinal+fluid+-+The+hyperbolic+reference+range+for+reliable+data+interpretation+in+quotient+diagrams.">Diagnostic relevance of free light chains in cerebrospinal fluid - The hyperbolic reference range for reliable data interpretation in quotient diagrams.</a> Clinica Chimica Acta. 497, 153-162 (2019).</li><li>Reiber, H., Felgenhauer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+transfer+at+the+blood+cerebrospinal+fluid+barrier+and+the+quantitation+of+the+humoral+immune+response+within+the+central+nervous+system.">Protein transfer at the blood cerebrospinal fluid barrier and the quantitation of the humoral immune response within the central nervous system.</a> Clinica Chimica Acta. 163 (3), 319-328 (1987).</li><li>Dorta-Contreras, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reibergrams:+essential+element+in+cerebrospinal+fluid+immunological+analysis.">Reibergrams: essential element in cerebrospinal fluid immunological analysis.</a> Revista de Neurologia. 28 (10), 996-998 (1999).</li><li>Metzger, F., Mischek, D., Stoffers, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Connected+Steady+State+Model+and+the+Interdependence+of+the+CSF+Proteome+and+CSF+Flow+Characteristics.">The Connected Steady State Model and the Interdependence of the CSF Proteome and CSF Flow Characteristics.</a> Frontiers Neuroscience. 11, 241 (2017).</li><li>Wolforth, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+of+blood+collection+in+the+mouse.">Methods of blood collection in the mouse.</a> Laboratory Animals. 29, 47-53 (2000).</li><li>Liu, L., Duff, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+technique+for+serial+collection+of+cerebrospinal+fluid+from+the+cisterna+magna+in+mouse.">A technique for serial collection of cerebrospinal fluid from the cisterna magna in mouse.</a> Journal of Visualized Experiments. (21), e960 (2008).</li><li>Machholz, E., Mulder, G., Ruiz, C., Corning, B. F., Pritchett-Corning, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manual+restraint+and+common+compound+administration+routes+in+mice+and+rats.">Manual restraint and common compound administration routes in mice and rats.</a> Journal of Visualized Experiments. (67), e2771 (2012).</li><li>Johnston, S. A., Tobias, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Veterinary+Surgery:+Small+Animal+Expert+Consult+-+E-Book.">Veterinary Surgery: Small Animal Expert Consult - E-Book.</a> Elsevier Health Sciences. , (2017).</li><li>Nigrovic, L. E., Shah, S. S., Neuman, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correction+of+cerebrospinal+fluid+protein+for+the+presence+of+red+blood+cells+in+children+with+a+traumatic+lumbar+puncture.">Correction of cerebrospinal fluid protein for the presence of red blood cells in children with a traumatic lumbar puncture.</a> Journal of Pediatrics. 159 (1), 158-159 (2011).</li><li>McCarthy, D. P., Richards, M. H., Miller, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+multiple+sclerosis:+experimental+autoimmune+encephalomyelitis+and+Theiler's+virus-induced+demyelinating+disease.">Mouse models of multiple sclerosis: experimental autoimmune encephalomyelitis and Theiler's virus-induced demyelinating disease.</a> Methods in Molecular Biology. 900, 381-401 (2012).</li><li>Link, H., Tibbling, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+albumin+and+IgG+analyses+in+neurological+disorders.+II.+Relation+of+the+concentration+of+the+proteins+in+serum+and+cerebrospinal+fluid.">Principles of albumin and IgG analyses in neurological disorders. II. Relation of the concentration of the proteins in serum and cerebrospinal fluid.</a> Scandinavian Journal of Clinical Laboratory Investigation. 37 (5), 391-396 (1977).</li><li>Tibbling, G., Link, H., Ohman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+albumin+and+IgG+analyses+in+neurological+disorders.+I.+Establishment+of+reference+values.">Principles of albumin and IgG analyses in neurological disorders. I. Establishment of reference values.</a> Scandinavian Journal of Clinical Laboratory Investigation. 37 (5), 385-390 (1977).</li><li>Deisenhammer, F., 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=Guidelines+on+routine+cerebrospinal+fluid+analysis.+Report+from+an+EFNS+task+force.">Guidelines on routine cerebrospinal fluid analysis. Report from an EFNS task force.</a> European Journal of Neurology. 13 (9), 913-922 (2006).</li><li>Johanson, C. E., Stopa, E. G., McMillan, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+blood-cerebrospinal+fluid+barrier:+structure+and+functional+significance.">The blood-cerebrospinal fluid barrier: structure and functional significance.</a> Methods in Molecular Biology. 686, 101-131 (2011).</li><li>Zaias, J., Mineau, M., Cray, C., Yoon, D., Altman, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reference+values+for+serum+proteins+of+common+laboratory+rodent+strains.">Reference values for serum proteins of common laboratory rodent strains.</a> Journal of the American Association for Laboratory Animal Science. 48 (4), 387-390 (2009).</li><li>Felgenhauer, K., Renner, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamic+radii+versus+molecular+weights+in+clearance+studies+of+urine+and+cerebrospinal+fluid.">Hydrodynamic radii versus molecular weights in clearance studies of urine and cerebrospinal fluid.</a> Annals of Clinical Biochemistry. 14 (2), 100-104 (1977).</li><li>Pachner, A. R., DiSano, K., Royce, D. B., Gilli, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+utility+of+a+molecular+signature+in+inflammatory+demyelinating+disease.">Clinical utility of a molecular signature in inflammatory demyelinating disease.</a> Neurology, Neuroimmunology &amp; Neuroinflammation. 6 (1), 520 (2019).</li><li>Pachner, A. R., Li, L., Gilli, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokine+biomarkers+in+central+nervous+system+tissue+and+cerebrospinal+fluid+in+the+Theiler's+virus+model+mirror+those+in+multiple+sclerosis.">Chemokine biomarkers in central nervous system tissue and cerebrospinal fluid in the Theiler's virus model mirror those in multiple sclerosis.</a> Cytokine. 76 (2), 577-580 (2015).</li><li>Gerbi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+concentration+in+the+arterial+and+venous+renal+blood+serum+of+the+rabbit.">Protein concentration in the arterial and venous renal blood serum of the rabbit.</a> Archives of Biochemistry and Biophysics. 31 (1), 49-61 (1951).</li><li>Abbott, N. J., Patabendige, A. A., Dolman, D. E., Yusof, S. R., Begley, D. 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Essentials in neurology and psychiatry.</a> Arquivos de Neuropsiquiatria. 74 (6), 501-512 (2016).</li><li>Kuehne, L. K., Reiber, H., Bechter, K., Hagberg, L., Fuchs, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebrospinal+fluid+neopterin+is+brain-derived+and+not+associated+with+blood-CSF+barrier+dysfunction+in+non-inflammatory+affective+and+schizophrenic+spectrum+disorders.">Cerebrospinal fluid neopterin is brain-derived and not associated with blood-CSF barrier dysfunction in non-inflammatory affective and schizophrenic spectrum disorders.</a> Journal of Psychiatric Research. 47 (10), 1417-1422 (2013).</li><li>Bromader, S., 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=Changes+in+serum+and+cerebrospinal+fluif+cytokines+in+response+to+non-neurological+surgery:+an+observational+study.">Changes in serum and cerebrospinal fluif cytokines in response to non-neurological surgery: an observational study.</a> Journal of Neuroinflammation. 9, 242 (2012).</li><li>Starhof, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebrospinal+fluid+pro-inflammatory+cytokines+differentiate+parkinsonian+syndromes.">Cerebrospinal fluid pro-inflammatory cytokines differentiate parkinsonian syndromes.</a> Journal of Neuroinflammation. 15 (1), 305 (2018).</li></ol>

Quantitative methods for the evaluation of increased CSF protein concentrations are useful tools in the characterization of the physiological and pathological state of the CNS. However, beyond reliable quantification of CSF protein levels, the detection of CSF proteins requires an expression of results that discriminates between blood- and CNS-derived fractions in the CSF. However, to date, the commonly used protein quantification assays do not allow discrimination between the two protein components, even with the aid of...

Cerebrospinal fluid (CSF), a clear and colorless liquid surrounding the brain and the spinal cord, holds great clinical and basic scientific importance. The CSF preserves the electrolytic environment of the central nervous system (CNS), balances the systemic acid-base status, supplies nutrients to neuronal and glial cells, functions as a lymphatic system for the CNS, and transports hormones, neurotransmitters, cytokines and other neuropeptides throughout the CNS1. Thus, as the CSF composition reflects the activity of the CNS, this fluid offers a valuable, though indirect, access to characterize the physiological and pathological state of the CNS.
CSF has been used to diagnose conditions that affect the CNS for over a hundred years, and for most of this time, it was primarily studied by clinicians as a diagnostic tool. However, in recent years neurobiologists have recognized the potential of CSF for studying the pathophysiology of the CNS. In particular, several high-throughput protein analysis tools have been introduced in the neuroscience realm allowing a detailed study of the protein composition of the CSF, with the expectation that this analysis may help provide insight into the dynamic changes occurring within the CNS.
Technological developments in multiplex immunoassay techniques such as Luminex and Simoa technologies2,3, provide researchers today with the ability to detect hundreds of proteins at very low concentrations. Moreover, these same technologies allow the use of small sample volumes, thereby promoting studies in small animals, including mice, in which limited sample volumes of CSF has precluded detailed characterizations of the fluid until recently.
Nevertheless, one caveat is that proteins measured in CSF may derive from intrathecal synthesis and/or transudation from serum due to a damaged blood-brain interface (BBI). Unfortunately, protein analysis of CSF alone can only determine the sum of these two components. To discriminate between transudate and intrathecally produced proteins, CSF protein measurements using any available protein analysis tool must be adjusted for individual variability in serum concentrations as well as barrier integrity. However, although this adjustment is commonly used in clinical practice, e.g., the CSF IgG index, which has high sensitivity for detecting intrathecal IgG synthesis4,5,6, to date very few research studies have corrected CSF protein concentrations for serum concentration and barrier integrity7,8.
Currently, the Reibergram approach is the best way to determine the barrier function and intrathecal synthesis of proteins. It is a graphical evaluation in CSF/serum quotient diagrams which analyzes, in an integrated way, both the barrier (dys)function and intrathecal protein synthesis, referring to an exclusively blood-derived protein9,10. The highly abundant protein albumin is usually chosen as reference protein because it is produced only in the liver and because its size, approximately 70 kDa, is intermediate between small and large proteins11. The analysis diagram was first defined by Reiber and Felgenhauer in 1987 for the major classes of immunoglobulins (Igs)11, being empirically based on the results obtained from the analysis of thousands of human samples9. The approach was subsequently confirmed by the application of the two Fick&#8217;s laws of diffusion in the theory of molecular diffusion/flow rate12. Such a theory demonstrates the diffusion of a protein through the barrier has a hyperbolic distribution and can quantitatively explain the dynamics of proteins in the CNS9,13. Overall, the advantage of using the Reibergram for demonstrating intrathecal protein synthesis is that it concurrently identifies the protein fraction that enters the CSF from serum as well as the amount of protein found in the CSF because of local production.
The present article and the related protocol describe the entire procedure, from CSF and blood collection to the final calculations correcting CSF protein levels, for the quantitative measurement of intrathecal protein synthesis in mouse models of neurological disorders. This procedure provides a baseline against which to assess (1) the pathophysiological origin of any CSF protein and (2) the stability and functional significance of the barrier integrity. This procedure and protocol are not only useful for assessing mouse CSF samples but are also useful in analyzing CSF in a multitude of animal models of neurological diseases and human patients.

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		<tr height="21">
			<td height="21" style="height:21px;width:327px;">1 mL insulin syringe</td>
			<td style="width:184px;">BD</td>
			<td style="width:127px;">329650</td>
			<td style="width:847px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 mL syringe</td>
			<td style="width:184px;">BD</td>
			<td style="width:127px;">329622</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">25 gauge needle</td>
			<td style="width:184px;">BD</td>
			<td style="width:127px;">305122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">3 mL syringe</td>
			<td style="width:184px;">BD</td>
			<td style="width:127px;">309582</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">30 gauge insulin needle</td>
			<td style="width:184px;">BD</td>
			<td style="width:127px;">305106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Absorbent pads</td>
			<td style="width:184px;">Any suitable brand</td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Acepromazine</td>
			<td style="width:184px;">Patterson Vet Supply Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">BioPlex Handheld Magnetic Washer</td>
			<td style="width:184px;">BioRad</td>
			<td style="width:127px;">171020100</td>
			<td>Magnet</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">BioPlex MAGPIX Multiplex Reader</td>
			<td style="width:184px;">BioRad</td>
			<td style="width:127px;">171015001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">BioPlex Pro Flat Bottom Plates</td>
			<td style="width:184px;">BioRad</td>
			<td style="width:127px;">171025001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Biotinilated detection antibody</td>
			<td style="width:184px;">Any suitable source</td>
			<td style="width:127px;"></td>
			<td>The antibody has to be directed against the species of the protein of interest.</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:184px;">Sigma</td>
			<td>A4503</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Buprenorphine hydrochloride</td>
			<td style="width:184px;">PAR Pharmaceutical</td>
			<td style="width:127px;">NDC 42023-179-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Capillary Tubes</td>
			<td style="width:184px;">Sutter Instrument</td>
			<td style="width:127px;">B100-75-10</td>
			<td>OD: 1.0 mm, ID: 0.75 mm Borosilicate glass 10 cm; drawn over Bunsen to make ID smaller.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Centrifuge tube, 0.2 mL</td>
			<td style="width:184px;">VWR</td>
			<td style="width:127px;">20170-012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Centrifuge tube, 0.5 mL</td>
			<td style="width:184px;">VWR</td>
			<td style="width:127px;">87003-290</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Centrifuge tube, 1.5 mL</td>
			<td style="width:184px;">VWR</td>
			<td style="width:127px;">87003-294</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Chlorhexidine diacetate</td>
			<td style="width:184px;">Nolvasan</td>
			<td style="width:127px;">E004272</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Disposable pipettes tips</td>
			<td style="width:184px;">Any suitable brand</td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Ear bars</td>
			<td style="width:184px;">KOPF Instruments</td>
			<td style="width:127px;">1921 or 1922</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Ethanol</td>
			<td style="width:184px;">Kopter</td>
			<td style="width:127px;">V1001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Freezer</td>
			<td style="width:184px;">VWR</td>
			<td style="width:127px;">VWR32086A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Gauze</td>
			<td style="width:184px;">Medline</td>
			<td style="width:127px;">NON25212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Heating pad</td>
			<td style="width:184px;">Sunbeam</td>
			<td style="width:127px;"></td>
			<td>XL King Size SoftTouch, 4 Heat Settings with Auto-Off, Teal, 12-Inch x 24-Inch</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Induction Chamber</td>
			<td style="width:184px;">VETEQUIP</td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Isoflurane</td>
			<td style="width:184px;">Patterson Vet Supply Inc</td>
			<td style="width:127px;">NDC 14043-704-06</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Ketamine (KetaVed)</td>
			<td style="width:184px;">Patterson Vet Supply Inc</td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MagPlex Microspheres (antibody-coupled)</td>
			<td style="width:184px;">BioRad</td>
			<td style="width:127px;"></td>
			<td style="width:847px;">Antibody-coupled magnetic bead</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Microplate Shaker</td>
			<td style="width:184px;">Southwest Scientific</td>
			<td>SBT1500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Microretractors</td>
			<td style="width:184px;">Carfill Quality</td>
			<td style="width:127px;">ACD-010</td>
			<td>Blunt - 1 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Microsoft Office (Excel)</td>
			<td style="width:184px;">Microsoft</td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">MilliPlex MAP Mouse Immunoglobulin Isotyping Magnetic Bead Panel</td>
			<td style="width:184px;">EMD Millipore</td>
			<td style="width:127px;">MGAMMAG-300K</td>
			<td>Commercial kit for the quantification through Luminex of a panel of immunoglobulin isotypes and subclasses in mouse fluids.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Mouse Albumin capture ELISA kit</td>
			<td style="width:184px;">Novus Biological</td>
			<td style="width:127px;">NBP2-60484</td>
			<td>Commercial kit for the quantification through ELISA of albumin in mouse fluids.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Multichannel pipette</td>
			<td style="width:184px;">Eppendorf</td>
			<td>3125000060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Non-Sterile swabs</td>
			<td style="width:184px;">MediChoice</td>
			<td style="width:127px;">WOD1002</td>
			<td>Need to be autoclaved for sterility</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Oxygen</td>
			<td style="width:184px;">AIRGAS</td>
			<td style="width:127px;">OX USPEA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Pasteur Pippettes</td>
			<td style="width:184px;">Fisher</td>
			<td style="width:127px;">13-678-20A</td>
			<td>5 &#38; 3/4&#34;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">PDS suture with disposable needle, 6-0 Prolen</td>
			<td style="width:184px;">Patterson Vet</td>
			<td style="width:127px;">8695G</td>
			<td>P-3 Reverse Cutting, 18&#34;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">PE-Streptavidin</td>
			<td style="width:184px;">BD Biosciences</td>
			<td style="width:127px;">554061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Pipetters</td>
			<td style="width:184px;">Eppendorf</td>
			<td style="width:127px;">Research seriers</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polyethylene tubing</td>
			<td style="width:184px;"></td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Refrigerated Centrifuge</td>
			<td style="width:184px;">Beckman Coulter</td>
			<td style="width:127px;">ALLEGRA X-12R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Scale</td>
			<td style="width:184px;">Uline</td>
			<td style="width:127px;">H2716</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Scalpel</td>
			<td style="width:184px;">Feather</td>
			<td style="width:127px;">EF7281</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Shaver</td>
			<td style="width:184px;">Harvard Apparatus</td>
			<td style="width:127px;">52-5204</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Standard proteins</td>
			<td>Any suitable source</td>
			<td></td>
			<td>The best choice for a reference standard is a purified, known concentration of the protein of interest.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Stereotaxic instrument</td>
			<td style="width:184px;">KOPF Instruments</td>
			<td style="width:127px;">Model 900LS</td>
			<td>Standard Accessories</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Sterile 1 x PBS</td>
			<td style="width:184px;">Corning Cellgro</td>
			<td style="width:127px;">21-040-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Sterile saline</td>
			<td style="width:184px;">Baxter</td>
			<td style="width:127px;">0338-0048-02</td>
			<td>0.9 % Sodium Chloride Irrigation USP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Surgical Forceps Curved, 7 (2)</td>
			<td style="width:184px;">Fine Science Tools</td>
			<td style="width:127px;">11271-30</td>
			<td>Dumont</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Surgical Scissors</td>
			<td style="width:184px;">Fine Science Tools</td>
			<td style="width:127px;">14094-11</td>
			<td>Stainless 25x</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Vaporizer + Flow meter</td>
			<td style="width:184px;">Moduflex Anhestesia Instruments</td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Vortex</td>
			<td style="width:184px;">Fisher</td>
			<td>02-215-414</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Warming pad</td>
			<td style="width:184px;">Kent Scientific Corporation</td>
			<td style="width:127px;">RT-JR-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Water Sonicator</td>
			<td style="width:184px;">Cole Parmer</td>
			<td>EW-08895-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Xylazine</td>
			<td style="width:184px;">Patterson Vet Supply Inc</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

quantitative measurement of intrathecally synthesized proteins in mice

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the CSF protein composition delivers crucial information in basic neuroscience research as well as neurological diseases. One caveat is that proteins measured in CSF may derive from both intrathecal synthesis and transudation from serum, and protein analysis of CSF can only determine the sum of these two components. To discriminate between protein transudation from the blood and intrathecally produced proteins in animal models as well as in humans, CSF protein profiling measurements using conventional protein analysis tools must include the calculation of the albumin CSF/serum quotient (Qalbumin), a marker of the integrity of the blood-brain interface (BBI), and the protein index (Qprotein/Qalbumin), an estimate of intrathecal protein synthesis. This protocol illustrates the entire procedure, from CSF and blood collection to quotients and indices calculations, for the quantitative measurement of intrathecal protein synthesis and BBI impairment in mouse models of neurological disorders.

This representative experiment aimed to compare the intrathecal synthesis of IgG in two clinically relevant rodent models of multiple sclerosis (MS): the PLP139-151-induced relapsing experimental autoimmune encephalomyelitis (R-EAE) and the chronic progressive, Theiler&#8217;s murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD). R-EAE is a useful model for understanding relapsing-remitting MS, whereas the TMEV-IDD model features chronic progressive MS19.
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Watch this Scientific Journal Video about Quantitative Measurement of Intrathecally Synthesized Proteins in Mice at JoVE.com

Quantitative Measurement of Intrathecally Synthesized Proteins in Mice

Elevated spinal fluid protein levels can either be the result of diffusion of plasma protein across an altered blood-brain barrier or intrathecal synthesis. An optimized testing protocol is presented in this article that helps to discriminate both cases and provides quantitative measurements of intrathecally synthesized proteins.

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the ...

This protocol allows CSF and blood collection in the quantitative correction of CSF protein levels to measure into a thecal protein synthesis in mouse models of neurological disorders. This procedure provides a baseline, against which the pathophysiological origin of CSF proteins of interest, and the stability and functional significance of the blood CSF favor integrity can be assessed. The analysis of interthecal protein synthesis and barrier integrity can be applied to other animal models and human studies.For example, to check for diseases of the central nervous system. Collecting significant volumes of clean CSF can be technically challenging in mice, so practicing the technique until large volumes of uncontaminated sample can be obtained is advised. Demonstrating the procedures will be Micheal Linzey, a grad student from the program in experimental and molecular medicine at Dartmouth, in our new immunology research group.For serum collection via retro orbital bleeding. After confirming a lack of response to pedal reflex in a greater than 15 gram anesthetized mouse, grasp the loose skin behind the ears with the thumb and index finger of the non-dominant hand, and use the index finger to draw back the skin above the eyes. Using the thumb to draw back the skin below the eyes, place the tip of a Pasteur pipette, held at an approximately 45 degree angle, into the eye socket underneath the eyeball, directed toward the middle of the eye socket, while rotating the pipette between the fingers.Then apply brief, gentle pressure and release to allow blood to enter the pipette. When the blood sample has been collected, gently remove the capillary without injuring the eye, and transfer the blood to a 1.5 milliliter centrifuge tube. After closing the eyelid, apply mild pressure with gauze to prevent further bleeding.Allow the blood to clot for 30 to 60 minutes at room temperature before spinning down the sample by centrifugation. Using a clean pipette technique, collect the separated serum into a new, labeled 500 microliter vial, and immediately freeze the serum at 80 degrees Celsius. After confirming the lack of response to pedal reflex, remove a large enough area of hair to allow collection of the cerebral spinal fluid immediately at the caudal end of the skull of the anesthetized mouse.Place the mouse in a prone position on a stereotaxic device under sterile conditions, and steady the head with ear bars. Swab the surgical site with 30%chlorhexidine diacetate, and make a sagittal skin incision inferior to the occiput to expose the muscles overlying the cisterna magna. Using forceps, blunt dissect the subcutaneous tissue and muscles, and use micro retractors to hold the muscles apart to expose the dura mater meningeal layer over the cisterna magna.Gently wash the tissue with sterile PBS to remove any possible blood contamination, and use a sterile cotton swab to blot the dura mater dry. Positioning the initial puncture at the cisterna magna is essential for obtaining abundant, non-contaminated CSF. Using a 30 gauge needle, gently puncture the membrane covering the cisterna magna, and quickly and gently insert a small glass capillary tube into the puncture.When five to 12 microliters of CSF have been collected, carefully remove the tube from the membrane and use a piece of polyethylene tubing to connect the tube to a three milliliter syringe. Inject the collected CSF into a labeled 500 microliter tube on ice, and use a disposable needle and varied polydioxanone sutures to close the incision. Clean the area of any dried blood or tissue, and place the mouse in a clean, warm cage with monitoring until full recumbency.Collect the CSF by centrifugation, and visually inspect the pellet and supernate it for any signs of blood contamination. Then using clean pipette technique, transfer the CSF-containing supernate into a new 200 microliter tube, and dilute the CSF at a one to three ratio with PBS for immediate storage at 80 degrees Celsius. For serum collection by cardiac puncture, immediately after CSF collection, as just demonstrated, place the mouse in the supine position.Swab the abdominal skin with 70%alcohol. Use scissors to open the thoracic cavity, exposing the heart, and insert a 25 gauge needle attached to a 3 milliliter syringe into the left ventricle. Then gently apply negative pressure to the syringe plunger, withdrawing the needle after the blood has been collected.Depress the plunger to eject the collected blood into a 1.5 milliliter vial. Now, allow the blood to clot for 30 to 60 minutes at room temperature before separating the serum by centrifugation. Then, using a clean pipette technique, transfer the serum into a new labeled 500 microliter vial for immediate storage at 80 degrees Celsius.To quantify the target proteins and albumen in matched serum and CSF specimens, use a standard protein quantification assay, using referenced standard proteins to prepare a standard curve for each protein of interest. After the analysis, export the raw data to an appropriate software graphing program, and graph the detection signal fluorescence intensity versus the standard protein concentrations to create a standard curve for each protein of interest. Then, use the standard curves to calculate the concentrations of each analyte of interest in the samples.Finally, use albumen and target analyte concentrations to calculate Q values and intrathecal index. The actual levels of total IgG are significantly increased in the CSF of two tested rodent models of multiple sclerosis, compared to the corresponding age matched sham controls. R-EAE mice show significantly enhanced albumen quotient values, indicating an increased permeability of the blood brain barrier in these mice.Nonversely, no differences in albumen quotient exist between TMEV-IDD and sham mice, corroborating previous findings of an intact barrier in TMEV-IDD mice. In addition, in TMEV-IDD animals, significantly higher IgG index values and therefore, intrathecal IgG production are observed. The calculation of a protein index facilitates the identification of novel protein biomarkers useful for early diagnosis, outcome prediction, and disease course monitoring for both neuroinflammatory and neurodegenerative diseases.

Research

JoVE Journal

Neuroscience

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Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

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