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Primary sensory neurons and satellite cells after peripheral axotomy in the adult rat: timecourse of cell death & elimination
Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Hand Surgery. Blond-McIndoe Laboratories, Royal Free and University College Medical School, University Department of Surgery, Royal Free Campus, Rowland Hill Street, London, UK.
Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy. Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Hand Surgery.
Blond-McIndoe Laboratories, Royal Free and University College Medical School, University Department of Surgery, Royal Free Campus, Rowland Hill Street, London, UK.
2002 (English)In: Experimental Brain Research, ISSN 0014-4819, E-ISSN 1432-1106, Vol. 142, no 3, 308-318 p.Article in journal (Refereed) Published
Abstract [en]

The timecourse of cell death in adult dorsal root ganglia after peripheral axotomy has not been fully characterised. It is not clear whether neuronal death begins within I week of axotomy or continues beyond 2 months after axotomy. Similarly, neither the timecourse of satellite cell death in the adult, nor the effect of nerve repair has been described. L4 and L5 dorsal root ganglia were harvested at 1-14 days, 1-6 months after sciatic nerve division in the adult rat, in accordance with the Animals (Scientific Procedures) Act 1986. In separate groups the nerve was repaired either immediately or following a 1-week delay, and the ganglia were harvested 2 weeks after the initial transection. Microwave permeabilisation and triple staining enabled combined TUNEL staining, morphological examination and neuron counting by the stereological optical dissector technique. TUNEL-positive neurons, exhibiting a range of morphologies, were seen at all timepoints (peak 25 cells/group 2 weeks after axotomy) in axotomised ganglia only. TUNEL-positive satellite cell numbers peaked 2 months after axotomy and were more numerous in axotomised than control ganglia. L4 control ganglia contained 13,983 (SD 568) neurons and L5, 16,285 (SD 1,313). Neuron loss was greater in L5 than L4 axotomised ganglia, began at I week (15%, P=0.045) post-axotomy, reached 35% at 2 months (P<0.001) and was not significantly greater at 4 months or 6 months. Volume of axotomised ganglia fell to 19% of control by 6 months (P<0.001). In animals that underwent nerve repair, both the number of TUNEL-positive neurons and neuron loss were reduced. Immediate repair was more protective than repair after a 1-week delay. Thus TUNEL positivity precedes actual neuron loss, reflecting the time taken to complete cell death and elimination. Neuronal death begins within I day of peripheral axotomy, the majority occurs within the first 2 months, and limited death is still occurring at 6 months. Neuronal death is modulated by peripheral nerve repair and by its timing after axotomy. Secondary satellite cell death also occurs, peaking 2 months after axotomy. These results provide a logical framework for future research into neuronal and satellite cell death within the dorsal root ganglia and provide further insight into the process of axotomy induced neuronal death.

Place, publisher, year, edition, pages
Springer-Verlag New York, 2002. Vol. 142, no 3, 308-318 p.
Keyword [en]
TUNEL, stereology, optical dissection, ; apoptosis, dorsal root ganglion, rat
National Category
Neurology Neurosciences
URN: urn:nbn:se:umu:diva-4535DOI: 10.1007/s00221-001-0929-0ISI: 000174227300002PubMedID: 11819038OAI: diva2:143677
Available from: 2003-06-19 Created: 2003-06-19 Last updated: 2014-04-15Bibliographically approved
In thesis
1. Sensory neuronal protection & improving regeneration after peripheral nerve injury
Open this publication in new window or tab >>Sensory neuronal protection & improving regeneration after peripheral nerve injury
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Peripheral nerve trauma is a common cause of considerable functional morbidity, and healthcare expenditure. Particularly in the ~15% of injuries unsuitable for primary repair, standard clinical management results in inadequate sensory restitution in the majority of cases, despite the rigorous application of complex microsurgical techniques. This can largely be explained by the failure of surgical management to adequately address the neurobiological hurdles to optimal regeneration. Most significant of these is the extensive sensory neuronal death that follows injury, and which is accompanied by a reduction in the regenerative potential of axotomised neurons, and in the supportive capacity of the Schwann cell population if nerve repair is delayed.

The present study aimed to accurately delineate the timecourse of neuronal death, in order to identify a therapeutic window during which clinically applicable neuroprotective strategies might be adopted. It then proceeded to investigate means to increase the regenerative capacity of chronically axotomised neurons, and to augment the Schwann cells’ ability to promote that regenerative effort.

Unilateral sciatic nerve transection in the rat was the model used, initially assessing neuronal death within the L4&5 dorsal root ganglia by a combination of morphology, TdT uptake nick-end labelling (TUNEL), and statistically unbiased estimation of neuronal loss using the stereological optical disector technique. Having identified 2 weeks, and 2 months post-axotomy as the most biologically relevant timepoints to study, the effect upon neuronal death of systemic treatment with acetyl-L-carnitine (ALCAR 10, or 50mg/kg/day) or N-acetyl-cysteine (NAC 30, or 150mg/kg/day) was determined. A model of secondary nerve repair was then adopted; either 2 or 4 months after unilateral sciatic nerve division, 1cm gap repairs were performed using either reversed isografts, or poly-3-hydroxybutyrate (PHB) conduits containing an alginate-fibronectin hydrogel. Six weeks later nerve regeneration and the Schwann cell population were quantified by digital image analysis of frozen section immunohistochemistry.

Sensory neuronal death begins within 24 hours of injury, but takes 1 week to translate into significant neuronal loss. The rate of neuronal death peaks 2 weeks after injury, and neuronal loss is essentially complete by 2 months post-axotomy. Nerve repair is incompletely neuroprotective, but the earlier it is performed the greater the benefit. Two clinically safe pharmaceutical agents, ALCAR & NAC, were found to virtually eliminate sensory neuronal death after peripheral nerve transection. ALCAR also enhanced nerve regeneration independently of its neuroprotective role. Plain PHB conduits were found to be technically simple to use, and supported some regeneration, but were not adequate in themselves. Leukaemia inhibitory factor enhanced nerve regeneration, though cultured autologous Schwann cells (SC’s) were somewhat more effective. Both were relatively more efficacious after a 4 month delay in nerve repair. The most profuse regeneration was found with recombinant glial growth factor (rhGGF-2) in repairs performed 2 months after axotomy, with results that were arguably better than were obtained with nerve grafts. A similar conclusion can be drawn from the result found using both rhGGF-2 and SC’s in PHB conduits 4 months after axotomy.

In summary, these findings reinforce the significance of sensory neuronal death in peripheral nerve trauma, and the possibility of its` limitation by early nerve repair. Two agents for the adjuvant therapy of such injuries were identified, that can virtually eliminate neuronal death, and enhance regeneration. Elements in the creation of a bioartificial nerve conduit to replace, or surpass autologous nerve graft for secondary nerve repair are presented.

Place, publisher, year, edition, pages
Umeå: Kirurgisk och perioperativ vetenskap, 2003. 61 p.
Umeå University medical dissertations, ISSN 0346-6612 ; 679
Neurosciences, cell death, TUNEL, optical disection, dorsal root ganglion, peripheral nerve, nerve conduit, Schwann cell, glial growth factor, leukaemia inhibitory factor, acetyl-L-carnitine, N-acetyl-cysteine, Neurovetenskap
National Category
Research subject
urn:nbn:se:umu:diva-52 (URN)91-7305-370-8 (ISBN)
Public defence
2003-05-28, Stora föreläsningssalen, Biologihuset, Umeå, 09:00 (English)
Available from: 2003-06-19 Created: 2003-06-19 Last updated: 2009-10-13Bibliographically approved

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