Funktionel og Morfologisk vurdering af Membran innervation af phrenic Motor neuroner
Summary
Compound muscle action potential recording quantitatively assesses functional diaphragm innervation by phrenic motor neurons. Whole-mount diaphragm immunohistochemistry assesses morphological innervation at individual neuromuscular junctions. The goal of this protocol is to demonstrate how these two powerful methodologies can be used in various rodent models of spinal cord disease.
Abstract
This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and morphological levels. Compound muscle action potential (CMAP) recording following phrenic nerve stimulation can be used to quantitatively assess functional diaphragm innervation by PhMNs of the cervical spinal cord in vivo in anesthetized rats and mice. Because CMAPs represent simultaneous recording of all myofibers of the whole hemi-diaphragm, it is useful to also examine the phenotypes of individual motor axons and myofibers at the diaphragm NMJ in order to track disease- and therapy-relevant morphological changes such as partial and complete denervation, regenerative sprouting and reinnervation. This can be accomplished via whole-mount immunohistochemistry (IHC) of the diaphragm, followed by detailed morphological assessment of individual NMJs throughout the muscle. Combining CMAPs and NMJ analysis provides a powerful approach for quantitatively studying diaphragmatic innervation in rodent models of CNS and PNS disease.
Introduction
Amyotrofisk lateral sklerose (ALS) er en invaliderende motorisk neuron sygdom forbundet med tabet af både øvre og nedre motorneuroner og deraf følgende muskel lammelse. Efter diagnosen, patientoverlevelse er i gennemsnit kun 2-5 år 1. Phrenic motor neuron (PhMN) tab er en kritisk komponent i patogenesen af ALS. Patienter i sidste ende dør på grund af tab af PhMN innervation af membranen, den primære muskel til inspiration 2,3. Traumatisk rygmarvsskade (SCI) er også et alvorligt problem med tilknyttede åndedrætsbesvær. Ca. 12.000 nye tilfælde af SCI opstår hvert år 4 på grund traumatisk skade på rygmarven. Trods sygdom heterogenitet med hensyn til placering, type og sværhedsgrad, de fleste SCI sager vedrører traumer til cervikal rygmarv, hvilket ofte resulterer i invaliderende og vedvarende respiratorisk kompromis. Foruden ALS og SCI, andre centralnervesystem (CNS) sygdomme kan være forbundet wed diafragma respiratorisk dysfunktion 5,6.
Phrenic nerve er en efferente motoriske nerve, der innerverer den ipsilaterale hemi-membran og som stammer fra PhMN cellelegemer placeret i C3-C5 niveauer af den ipsilaterale cervikale rygmarv. PhMN udgang styres ved faldende bulbospinal input fra hjernestammen i et område kendt som den rostrale ventrale respiratoriske gruppe (rVRG) 7. Den rVRG-PhMN-membran kredsløb er central for kontrol af inspiratorisk vejrtrækning, samt andre ikke-ventilatoriske membran adfærd. Forskellige traumatiske skader og neurodegenerative lidelser, som påvirker dette kredsløb kan føre til en dyb nedgang i respiratorisk funktion og patientens livskvalitet. Faldende input til PhMNs fra rVRG, PhMN overlevelse, phrenic nerve integritet og korrekt innervation ved mellemgulvet neuromuskulære junction (NMJ) er alle nødvendige for normal membran funktion. Det er derfor vigtigt at ansætte teknikker,kan kvantitativt vurdere dette kredsløb in vivo i gnaver modeller af ALS, SCI og andre CNS-sygdomme.
Med denne protokol, målet er at beskrive eksperimentelle værktøjer til vurdering PhMN innervation af membranen på både elektrofysiologiske og morfologiske niveauer. Forbindelse muskel aktionspotentialer (CMAP'er) registreres ved at stimulere alle efferente motor neuron axoner af en given motor nerve og derefter analysere fremkaldte depolarisering respons målet myofibre. Denne teknik kan anvendes in vivo i bedøvede rotter og mus til at kvantificere funktionel innervation af hemi-membran ved PhMNs 8. På grund af det faktum, at CMAP'er repræsenterer samtidig optagelse af alle (eller i det mindste mange / de fleste) myofibre i hele hemi-membran, er det nyttigt at også undersøge de fænotyper enkelte motoriske axoner og myofibre på mellemgulvet NMJ for at spore sygdomme – og terapi-relevant morfologiske ændringer såsom delvis og komplete denervering, regenerativ spiring og reinnervation. Dette kan opnås via hel-mount immunhistokemi (IHC) af membranen, efterfulgt af detaljeret morfologisk vurdering af individuelle NMJs hele musklen 9. Kombination CMAP'er og NMJ analyse giver en kraftfuld metode til kvantitativ studere diafragma innervation i gnaver modeller af CNS og PNS sygdom.
Protocol
Representative Results
Discussion
Som åndedrætsfunktionen er kompromitteret i både traumatisk SCI og ALS, udvikle behandlinger, der er målrettet vejrtrækning og specifikt membran innervation er klinisk relevant 5,6. For at omfattende undersøgelse respiratoriske funktion, bør en kombineret fremgangsmåde metode anvendes. CMAP'er måle graden af funktionel innervation af membranen ved hjælp af ekstern phrenic nerve stimulation, men ikke endogent bulbospinal respiratorisk drive 8. Hertil kommer, at disse optagelser ikke mu…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by the NINDS (grant #1R01NS079702 to A.C.L.) and the SURP Program at Thomas Jefferson University (M.M.).
Materials
| Paraformaldehyde | Fisher | T353-500 | Make 10% solution first in de-ionized distilled water; make 4% with 1X PBS, adjust pH to 7.4 |
| 1X Phosphate Buffered Saline, pH 7.4 | Invitrogen | 10010049 | |
| 2% Bovine serum albumin (2% BSA) | Sigma-Aldrich | A3059-100g | Dissolve 2g BSA into 100mL of 1X PBS |
| 0.2% Triton X100 in 2% BSA/PBS (Blocking Buffer) | Sigma-Aldrich | T9284-100mL | Dissolve 0.2ml/100mL 2% BSA/PBS |
| 0.1M Glycine | Sigma-Aldrich | G-7126 | Add 0.185g to 25mL of 2% BSA/PBS |
| α-bungarotoxin | Invitrogen | T1175 | Concentration 1:400 |
| SMI-312 | Sternberger Monoclonals | SMI312 | Concentration 1:1,000 |
| SV2 | Developmental Studies Hybridoma Bank | SV2-Supernatant | Concentration 1:10 |
| FITC goat anti-mouse IgG1 | Roche | 3117731001 | Concentration 1:100 |
| Silicone rubber | Sylgard, Dow Corning | Part # 184 | Follow instructions that come with kit: can use multiple sized culture dish (30mm, 60mm, 100mm) depending on needs |
| Vectashield fluorescent mounting medium | Vector laboratories | H-1000 | This is not a hard-set medium. You will need to secure the cover slip with clear nail polish. |
| Small Spring Scissors | Fine Science Tools | 15002-08 | |
| Dissection forceps | Fine Science Tools | 11295-51 | |
| Software for CMAP recordings | Scope 3.5.6; ADI | ||
| Disk surface electrodes | Natus neurology | 019-409000 | |
| Subdermal needle electrodes | Natus neurology | 019-453100 | |
| Conductive gel | Aquasonic | 122-73720 | |
| Stimulator/recording system for CMAP recordings | ADI Powerlab 8SP stimulator | ||
| Amplifier for CMAP recordings | BioAMP |
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