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Macrophages, Nitric oxide synthases and Heme oxygenases: possible roles during peripheral nerve regeneration

Magnusson, Sofia LU (2000)
Abstract (Swedish)
Popular Abstract in Swedish

Syftet med den här studien var att undersöka det strukturella underlaget för förslagen att makrofager och gaserna kvävemonooxid (NO) och kolmonoxid (CO), som bildas av kvävemonooxidsyntaser (NOS) respektive hemeoxygenaser (HO), är viktiga för att en perifer nerv ska regenerera. Det finns stöd för dessa hypoteser eftersom andra har visat att förhållanden som stör den invasion av makrofager (inflammatoriska celler) som sker vid nervskador också påverkar utväxten av nervtrådar. Det är dessutom så att produktionen av de enzymer som bildar NO, speciellt den neuronala formen av NOS (nNOS) ökar efter nervskada. En annan isoform , det inducerbara NOS (iNOS) finns i aktiverade makrofager.

... (More)
Popular Abstract in Swedish

Syftet med den här studien var att undersöka det strukturella underlaget för förslagen att makrofager och gaserna kvävemonooxid (NO) och kolmonoxid (CO), som bildas av kvävemonooxidsyntaser (NOS) respektive hemeoxygenaser (HO), är viktiga för att en perifer nerv ska regenerera. Det finns stöd för dessa hypoteser eftersom andra har visat att förhållanden som stör den invasion av makrofager (inflammatoriska celler) som sker vid nervskador också påverkar utväxten av nervtrådar. Det är dessutom så att produktionen av de enzymer som bildar NO, speciellt den neuronala formen av NOS (nNOS) ökar efter nervskada. En annan isoform , det inducerbara NOS (iNOS) finns i aktiverade makrofager.



När jag började mina undersökningar fanns det nästan ingen information om andra isoformer av NOS än nNOS i regenererande nervceller och ännu mindre informationom om HO. NO och CO stimulerar ett enzym (guanylyl cycklas) som genererar cGMP, en viktig intracellulär budbärare. När HO bryter ned hemgrupper bildas CO men också biliverdin (en pro-antioxidant). Biliverdin omvandlas i sin tur till bilirubin som är en viktig antioxidant. Det var därför naturligt att inkludera cGMP och biliverdin reduktas (BVR) i mina studier av de system som producerar CO och NO.



Jag har främst utnyttjat immuncytokemiska samt histokemiska metoder, men också in situ hybridisering, för att studera makrofager och olika isoformer av NOS, HO, c-GMP och BVR . Studierna utfördes på olika perifera ganglier från råtta, dels sensoriska (DRG och nodos gangliet) och dels på ganglier från det autonoma nervsystemet (övre cervikal gangliet (SCG), otic gangliet och sphenopalatin gangliet). Regenerativa förändringar inducerades antingen genom krosskada eller avskärning av nerver i råttan (in vivo), eller genom att dissekera ut ganglierna och odla dem (in vitro).



Resultaten visade att efter en nervskada in vivo sker en snabb aktivering av residenta makrofager och en massiv rekrytering av invaderande makrofager till nerven och dess ganglier. Aktiveringen och invasionen sker tidsmässigt före många andra viktiga regenerationsrelaterade förändringar i nervcellerna. Tanken att makrofager eller ämnen som frisätts från sådana celler t.ex. interleukiner, kan inducera eller stimulera regenerations-processen i nervcellerna ligger därför nära till hands. Däremot visade mina resultat att invaderande makrofager inte var nödvändiga för själva igångsättandet av utväxt av nervcellsutskott, eftersom sådan utväxt kan ske även då invaderande makrofager saknas. Utväxten/regenerationen är emellertid långsammare under sådana förhållanden.



Den inducerbara formen av NOS (iNOS) hittade jag i regenererande sensoriska och SCG nervceller samt hos aktiverade makrofager i SCG. Jag fann också att den neuronala formen av NOS (nNOS) uttrycktes i nervceller hos normala och odlade sensoriska ganglier, men inte i SCG.



I odlade sensoriska och autonoma ganglier, ökade den inducerbara formen av HO (HO-1) kraftigt i de celler som omger nervcellerna (satellitceller). Även halten av cGMP ökade i satellitceller i de odlade sensoriska ganglierna. Sammantaget visar resultaten att NO och CO utnyttjas i signaleringen mellan nervceller och dess satellitceller, och att signaleringen ökar i samband med en nervskada. Den funktionella betydelsen av denna ökade aktivitet är okänd. Det finns emellertid undersökningar som antyder att NO och CO via cGMP kan förbättra överlevnaden av nervceller vars utskott blivit skadade.



Jag kunde också visa att BVR, enzymet som reglerar produktionen av antioxidanten bilirubin, inducerades i nervceller och fanns i deras kärnor efter en nervskada. Detta antyder att de regenererande nervcellerna behöver skydda sig mot oxidativa skador.



Jag har alltså främst med immunhistokemiska metoder kunnat visa att de enzymer som genererar NO och CO ökar i regenererande perifera ganglier, och att denna ökning sker i olika typer av celler (makrofager, nervceller och satellitceller) i olika ganglier. Resultaten ger stöd åt förslagen att makrofager, men också signalmolekylerna NO och CO är viktiga för regenerationsprocessen. (Less)
Abstract
The purpose of this study was to investigate the suggestions that macrophages, and the systems generating nitric oxide (NO) and carbon monoxide (CO), i.e. nitric oxide synthases (NOS) and heme oxygenases (HO), could be important for peripheral nerve regeneration.



Immunocytochemistry was used to visualise invading and resident macrophages, inducible NOS (iNOS), two isoforms of HO (HO-1 and HO-2), biliverdin reductase, and cGMP in regenerating peripheral ganglia in vitro and in vivo. HO-1 was also localised by in situ hybridisation and neuronal NOS (nNOS) by histochemistry. Two types of sensory ganglia (dorsal root ganglia (DRG) and nodose ganglia) and three autonomic ganglia (superior cervical ganglia (SCG), otic ganglia... (More)
The purpose of this study was to investigate the suggestions that macrophages, and the systems generating nitric oxide (NO) and carbon monoxide (CO), i.e. nitric oxide synthases (NOS) and heme oxygenases (HO), could be important for peripheral nerve regeneration.



Immunocytochemistry was used to visualise invading and resident macrophages, inducible NOS (iNOS), two isoforms of HO (HO-1 and HO-2), biliverdin reductase, and cGMP in regenerating peripheral ganglia in vitro and in vivo. HO-1 was also localised by in situ hybridisation and neuronal NOS (nNOS) by histochemistry. Two types of sensory ganglia (dorsal root ganglia (DRG) and nodose ganglia) and three autonomic ganglia (superior cervical ganglia (SCG), otic ganglia and sphenopalatine ganglia) were studied. Regeneration changes in the ganglia were induced by crush lesions or axotomy in vivo, or by excision and culturing.



Principal findings were that following nerve injury there is a rapid activation of resident and a massive invasion of macrophages to the site of an injury but also in the ganglia. The time course of this activation and invasion precedes certain regeneration-induced changes in the neurones of the ganglia, including neuropeptide synthesis. The results are consistent with the suggestion that products released by macrophages could be important for the regenerative response. However, invading macrophages do not seem to be crucial for initiation of axonal outgrowth of sensory axons.



iNOS was found in neurones of nodose ganglia and increased after injury both in these ganglia and SCG. An increase in iNOS was also observed in macrophages of regenerating SCG. nNOS was expressed in neurones of both intact and cultured sensory ganglia, although there was a marked difference in the number of nNOS positive cells in the DRG along the spinal cord. In satellite cells of sensory ganglia an increase of cGMP immunoreactivity was found. In cultured sensory and autonomic ganglia the inducible form of HO (HO-1) increased dramatically in satellite cells. HO-2 immunoreactivity was observed in neurones of all types of ganglia, but did not change in response to an injury. These findings suggests that both NO and CO may be important for intercellular signalling during regeneration.



Biliverdin reductase, the enzyme generating the antioxidant bilirubin, was observed in axons, nerve cell nuclei and nerve cell bodies of regenerating sensory and autonomic ganglia, implying an increased demand for antioxidants following injury.



Together these results show that systems with the potential to generate NO and CO are upregulated following a peripheral nerve injury, although the increase occurs in various cell types in different ganglia. I suggest that signalling between neurones and satellite cells occurs through NO/CO-cGMP, and that this signalling is enhanced during regeneration. (Less)
Please use this url to cite or link to this publication:
author
opponent
  • Dr deVente, Jan, Universiteit Maastricht, Dept. of Psychiatry and Neuropsychology, Maastricht, The Netherlands
organization
publishing date
type
Thesis
publication status
published
subject
keywords
satellite cells, peripheral ganglia, cell signalling, nerve regeneration, heme oxygenase, Macrophages, nitric oxide synthase, Animal physiology, Djurfysiologi
pages
112 pages
publisher
Department of Animal Physiology, Lund University
defense location
Lecture hall at the Dept. of Animal Physiology, Lund University
defense date
2000-03-24 09:00
external identifiers
  • Other:ISRN: LUNBDS/(NBZF/1050)/1-62(2000)
ISBN
ISBN 91-628-4050-9
language
English
LU publication?
yes
id
34ebd69c-f8f4-4fa9-9371-ad1a72026309 (old id 40342)
date added to LUP
2007-07-31 13:35:22
date last changed
2016-09-19 08:45:07
@phdthesis{34ebd69c-f8f4-4fa9-9371-ad1a72026309,
  abstract     = {The purpose of this study was to investigate the suggestions that macrophages, and the systems generating nitric oxide (NO) and carbon monoxide (CO), i.e. nitric oxide synthases (NOS) and heme oxygenases (HO), could be important for peripheral nerve regeneration.<br/><br>
<br/><br>
Immunocytochemistry was used to visualise invading and resident macrophages, inducible NOS (iNOS), two isoforms of HO (HO-1 and HO-2), biliverdin reductase, and cGMP in regenerating peripheral ganglia in vitro and in vivo. HO-1 was also localised by in situ hybridisation and neuronal NOS (nNOS) by histochemistry. Two types of sensory ganglia (dorsal root ganglia (DRG) and nodose ganglia) and three autonomic ganglia (superior cervical ganglia (SCG), otic ganglia and sphenopalatine ganglia) were studied. Regeneration changes in the ganglia were induced by crush lesions or axotomy in vivo, or by excision and culturing.<br/><br>
<br/><br>
Principal findings were that following nerve injury there is a rapid activation of resident and a massive invasion of macrophages to the site of an injury but also in the ganglia. The time course of this activation and invasion precedes certain regeneration-induced changes in the neurones of the ganglia, including neuropeptide synthesis. The results are consistent with the suggestion that products released by macrophages could be important for the regenerative response. However, invading macrophages do not seem to be crucial for initiation of axonal outgrowth of sensory axons.<br/><br>
<br/><br>
iNOS was found in neurones of nodose ganglia and increased after injury both in these ganglia and SCG. An increase in iNOS was also observed in macrophages of regenerating SCG. nNOS was expressed in neurones of both intact and cultured sensory ganglia, although there was a marked difference in the number of nNOS positive cells in the DRG along the spinal cord. In satellite cells of sensory ganglia an increase of cGMP immunoreactivity was found. In cultured sensory and autonomic ganglia the inducible form of HO (HO-1) increased dramatically in satellite cells. HO-2 immunoreactivity was observed in neurones of all types of ganglia, but did not change in response to an injury. These findings suggests that both NO and CO may be important for intercellular signalling during regeneration.<br/><br>
<br/><br>
Biliverdin reductase, the enzyme generating the antioxidant bilirubin, was observed in axons, nerve cell nuclei and nerve cell bodies of regenerating sensory and autonomic ganglia, implying an increased demand for antioxidants following injury.<br/><br>
<br/><br>
Together these results show that systems with the potential to generate NO and CO are upregulated following a peripheral nerve injury, although the increase occurs in various cell types in different ganglia. I suggest that signalling between neurones and satellite cells occurs through NO/CO-cGMP, and that this signalling is enhanced during regeneration.},
  author       = {Magnusson, Sofia},
  isbn         = {ISBN 91-628-4050-9},
  keyword      = {satellite cells,peripheral ganglia,cell signalling,nerve regeneration,heme oxygenase,Macrophages,nitric oxide synthase,Animal physiology,Djurfysiologi},
  language     = {eng},
  pages        = {112},
  publisher    = {Department of Animal Physiology, Lund University},
  school       = {Lund University},
  title        = {Macrophages, Nitric oxide synthases and Heme oxygenases: possible roles during peripheral nerve regeneration},
  year         = {2000},
}