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Sensory neuronal protection & improving regeneration after peripheral nerve injury
Umeå University, Faculty of Medicine, Surgical and Perioperative Sciences, Hand Surgery. Umeå University, Faculty of Medicine, Integrative Medical Biology, Anatomy.
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.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 679
Keyword [en]
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
Keyword [sv]
Neurovetenskap
National Category
Neurology
Research subject
Neurosurgery
Identifiers
URN: urn:nbn:se:umu:diva-52ISBN: 91-7305-370-8 (print)OAI: oai:DiVA.org:umu-52DiVA: diva2:143683
Public defence
2003-05-28, Stora föreläsningssalen, Biologihuset, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2003-06-19 Created: 2003-06-19 Last updated: 2009-10-13Bibliographically approved
List of papers
1. Primary sensory neurons and satellite cells after peripheral axotomy in the adult rat: timecourse of cell death & elimination
Open this publication in new window or tab >>Primary sensory neurons and satellite cells after peripheral axotomy in the adult rat: timecourse of cell death & elimination
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
Keyword
TUNEL, stereology, optical dissection, ; apoptosis, dorsal root ganglion, rat
National Category
Neurology Neurosciences
Identifiers
urn:nbn:se:umu:diva-4535 (URN)10.1007/s00221-001-0929-0 (DOI)000174227300002 ()11819038 (PubMedID)
Available from: 2003-06-19 Created: 2003-06-19 Last updated: 2017-12-14Bibliographically approved
2. Systemic acetyl-L-carnitine eliminates sensory neuronal loss after peripheral axotomy: a new clinical approach in the management of peripheral nerve trauma
Open this publication in new window or tab >>Systemic acetyl-L-carnitine eliminates sensory neuronal loss after peripheral axotomy: a new clinical approach in the management of peripheral nerve trauma
2002 (English)In: Experimental Brain Research, ISSN 0014-4819, E-ISSN 1432-1106, Vol. 145, no 2, 182-189 p.Article in journal (Refereed) Published
Abstract [en]

Several hundred thousand peripheral nerve injuries occur each year in Europe alone. Largely due to the death of around 40% of primary sensory neurons, sensory outcome remains disappointingly poor despite considerable advances in surgical technique; yet no clinical therapies currently exist to prevent this neuronal death. Acetyl-L-carnitine (ALCAR) is a physiological peptide with roles in mitochondrial bioenergetic function, which may also increase binding of nerve growth factor by sensory neurons. Following unilateral sciatic nerve transection, adult rats received either one of two doses of ALCAR or sham, or no treatment. Either 2 weeks or 2 months later, L4 and L5 dorsal root ganglia were harvested bilaterally, in accordance with the Animal (Scientific Procedures) Act 1986. Neuronal death was quantified with a combination of TUNEL [TdT (terminal deoxyribonucleotidyl transferase) uptake nick end labelling] and neuron counts obtained using the optical disector technique. Sham treatment had no effect upon neuronal death. ALCAR treatment caused a large reduction in the number of TUNEL-positive neurons 2 weeks after axotomy (sham treatment 33/group; low-dose ALCAR 6/group, P=0.132; high-dose ALCAR 3/group, P<0.05), and almost eliminated neuron loss (sham treatment 21%; low-dose ALCAR 0%, P=0.007; high-dose ALCAR 2%, P<0.013). Two months after axotomy the neuroprotective effect of high-dose ALCAR treatment was preserved for both TUNEL counts (no treatment five/group; high-dose ALCAR one/group) and neuron loss (no treatment 35%; high-dose ALCAR -4%, P<0.001). These results provide further evidence for the role of mitochondrial bioenergetic dysfunction in post-traumatic sensory neuronal death, and also suggest that acetyl-L-carnitine may be the first agent suitable for clinical use in the prevention of neuronal death after peripheral nerve trauma.

Place, publisher, year, edition, pages
Springer-Verlag New York, 2002
Keyword
TUNEL, stereology, nerve injury, pharmacotherapy, dorsal root ganglion
National Category
Neurology Neurosciences
Identifiers
urn:nbn:se:umu:diva-4536 (URN)10.1007/s00221-002-1100-2 (DOI)000177074300007 ()12110958 (PubMedID)
Available from: 2003-06-19 Created: 2003-06-19 Last updated: 2017-12-14Bibliographically approved
3. Pharmacological enhancement of peripheral nerve regeneration in the rat by systemic acetyl-L-carnitine treatment
Open this publication in new window or tab >>Pharmacological enhancement of peripheral nerve regeneration in the rat by systemic acetyl-L-carnitine treatment
2002 (English)In: Neuroscience Letters, ISSN 0304-3940, E-ISSN 1872-7972, Vol. 334, no 3, 181-185 p.Article in journal (Refereed) Published
Abstract [en]

Peripheral nerve trauma remains a major cause of morbidity, largely due to the death of similar to40% of innervating sensory neurons, and to slow regeneration after repair. Acetyl-L-carnitine (ALCAR) is a physiological peptide that virtually eliminates sensory neuronal death, and may improve regeneration after primary nerve repair. This study determines the effect of ALCAR upon regeneration after secondary nerve repair, thereby isolating its effect upon neuronal regenerative capacity. Two months after unilateral sciatic nerve division 1 cm nerve graft repairs were performed (n = 5), and treatment with 50 mg/kg/day ALCAR was commenced for 6 weeks until harvest. Regeneration area and distance were determined by quantitative immunohistochemistry. ALCAR treatment significant increased immunostaining for both nerve fibres (total area 264% increase, P < 0.001; percentage area 229% increase, P < 0.001), and Schwann cells (total area 264% increase, P < 0.05; percentage area 86% increase, P < 0.05), when compared to no treatment. Regeneration into the distal stump was greatly enhanced (total area 2242% increase, P = 0.008; percentage area 3034% increase, P = 0.008). ALCAR significantly enhances the regenerative capacity of neurons that survive peripheral nerve trauma, in addition to its known neuroprotective effects.

Place, publisher, year, edition, pages
Elsevier, 2002
Keyword
peripheral nerve, regeneration, acetyl-L-carnitine, mitochondria, nerve repair, quantitative immunohistochemistry
National Category
Neurology
Identifiers
urn:nbn:se:umu:diva-4537 (URN)10.1016/S0304-3940(02)00982-5 (DOI)000179763300010 ()12453625 (PubMedID)
Available from: 2003-06-19 Created: 2003-06-19 Last updated: 2017-12-14Bibliographically approved
4. Sensory neuroprotection, mitochondrial preservation, and therapeutic potential of N-acetyl-cysteine after nerve injury.
Open this publication in new window or tab >>Sensory neuroprotection, mitochondrial preservation, and therapeutic potential of N-acetyl-cysteine after nerve injury.
2004 (English)In: Neuroscience, ISSN 0306-4522, E-ISSN 1873-7544, Vol. 125, no 1, 91-101 p.Article in journal (Refereed) Published
Abstract [en]

Neuronal death is a major factor in many neuropathologies, particularly traumatic, and yet no neuroprotective therapies are currently available clinically, although antioxidants and mitochondrial protection appear to be fruitful avenues of research. The simplest system involving neuronal death is that of the dorsal root ganglion after peripheral nerve trauma, where the loss of approximately 40% of primary sensory neurons is a major factor in the overwhelmingly poor clinical outcome of the several million nerve injuries that occur each year worldwide. N-acetyl-cysteine (NAC) is a glutathione substrate which is neuroprotective in a variety of in vitro models of neuronal death, and which may enhance mitochondrial protection. Using TdT uptake nick-end labelling (TUNEL), optical disection, and morphological studies, the effect of systemic NAC treatment upon L4 and 5 primary sensory neuronal death after sciatic nerve transection was investigated. NAC (150 mg/kg/day) almost totally eliminated the extensive neuronal loss found in controls both 2 weeks (no treatment 21% loss, NAC 3%, P=0.03) and 2 months after axotomy (no treatment 35% loss, NAC 3%, P=0.002). Glial cell death was reduced (mean number TUNEL positive cells 2 months after axotomy: no treatment 51/ganglion pair, NAC 16/ganglion pair), and mitochondrial architecture was preserved. The effects were less profound when a lower dose was examined (30 mg/kg/day), although significant neuroprotection still occurred. This provides evidence of the importance of mitochondrial dysregulation in axotomy-induced neuronal death in the peripheral nervous system, and suggests that NAC merits investigation in CNS trauma. NAC is already in widespread clinical use for applications outside the nervous system; it therefore has immediate clinical potential in the prevention of primary sensory neuronal death, and has therapeutic potential in other neuropathological systems.

Keyword
Acetylcysteine/*therapeutic use, Animals, Axotomy, Dose-Response Relationship; Drug, Ganglia; Spinal/drug effects/pathology, In Situ Nick-End Labeling, Lumbosacral Region, Male, Microscopy; Electron, Mitochondria/*drug effects/ultrastructure, Nerve Degeneration/*drug therapy, Neurons; Afferent/drug effects/*pathology, Neuroprotective Agents/*therapeutic use, Rats, Sciatic Nerve/pathology/surgery
Identifiers
urn:nbn:se:umu:diva-12710 (URN)doi:10.1016/j.neuroscience.2003.12.040 (DOI)15051148 (PubMedID)
Available from: 2007-12-11 Created: 2007-12-11 Last updated: 2017-12-14Bibliographically approved
5. Exogenous leukaemia inhibitory factor enhances nerve regeneration after late secondary repair using a bioartificial nerve conduit
Open this publication in new window or tab >>Exogenous leukaemia inhibitory factor enhances nerve regeneration after late secondary repair using a bioartificial nerve conduit
2003 (English)In: British Journal of Plastic Surgery, ISSN 0007-1226, E-ISSN 1465-3087, Vol. 56, no 5, 444-450 p.Article in journal (Refereed) Published
Abstract [en]

The clinical outcome of peripheral nerve injuries remains disappointing, even in the ideal situation of a primary repair performed with optimal microsurgical techniques. Primary repair is appropriate for only about 85% of injuries, and outcome is worse following secondarynerverepair, partly owing to the reduced regenerative potential of chronically axotomised neurons. Leukaemiainhibitoryfactor (LIF) is a gp-130 neurocytokine that is thought to act as an ‘injury factor’, triggering the early-injury phenotype within neurons and potentially boosting their regenerative potential aftersecondarynerverepair. At 2–4 months after sciatic nerve axotomy in the rat, 1 cm gaps were repaired using either nerve isografts or poly-3-hydroxybutyrate conduits containing a calcium alginate and fibronectin hydrogel.

Regeneration was determined by quantitative immunohistochemistry 6 weeks afterrepair, and the effect of incorporating recombinant LIF (100 ng/ml) into the conduits was assessed. LIF increased the regeneration distance in repairs performed after both 2 months (69%, P=0.019) and 4 months (123%, P=0.021), and was statistically comparable to nerve graft. The total area of axonal immunostaining increased by 21% (P>0.05) and 63% (P>0.05), respectively. Percentage immunostaining area was not increased in the 2 months group, but increased by 93% in the repairs performed 4 months after axotomy. Exogenous LIF, therefore, has a potential role in promoting peripheral nerveregenerationaftersecondaryrepair, and can be effectively delivered within poly-3-hydroxybutyrate bioartificialconduits used for nerverepair.

Place, publisher, year, edition, pages
Elsevier, 2003
Keyword
Peripheral nerveregeneration, Peripheral nerverepair, Leukaemiainhibitoryfactor, Nerveconduit, Growth factors
National Category
Surgery Neurology
Identifiers
urn:nbn:se:umu:diva-4539 (URN)10.1016/S0007-1226(03)00134-6 (DOI)000184829700004 ()12890457 (PubMedID)
Conference
Conference: Winter Scientific Meeting of the British-Association-of-Plastic-Surgeons, Location: London, England, Date: November, 2001, Sponsor(s):British Assoc Plast Surg
Available from: 2003-06-19 Created: 2003-06-19 Last updated: 2017-12-14Bibliographically approved
6. Interaction of glial growth factor & cultured Schwann cells in regeneration after chronic peripheral nerve injury
Open this publication in new window or tab >>Interaction of glial growth factor & cultured Schwann cells in regeneration after chronic peripheral nerve injury
In: Journal of the peripheral nervous system : JPNSArticle in journal (Refereed) Submitted
Identifiers
urn:nbn:se:umu:diva-4540 (URN)
Available from: 2003-06-19 Created: 2003-06-19Bibliographically approved

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