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An exploration of the mechanisms behind peripheral nerve injury
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.
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Despite surgical innovation, the sensory and motor outcome after peripheral nerve injury is incomplete. In this thesis, the biological pathways potentially responsible for the poor functional recoveries were investigated in both the distal nerve stump/target organ, spinal motoneurons and dorsal root ganglia (DRG). The effect of delayed nerve repair was determined in a rat sciatic nerve transection model. There was a dramatic decline in the number of regenerating motoneurons and myelinated axons found in the distal nerve stumps of animals undergoing nerve repair after a delay of 3 and 6 months. RT-PCR of the distal nerve stumps showed a decline in expression of Schwann cells (SC) markers, with a progressive increase in fibrotic and proteoglycan scar markers, with increased delayed repair time. Furthermore, the yield of SC which could be isolated from the distal nerve segments progressively fell with increased delay in repair time. Consistent with the impaired distal nerve stumps the target medial gastrocnemius (MG) muscles at 3- and 6-months delayed repair were atrophied with significant declines in wet weights (61% and 27% compared with contralateral sides). The role of myogenic transcription factors, muscle specific microRNAs and musclespecific E3 ubiquitin ligases in the muscle atrophy was investigated in both gastrocnemius and soleus muscles following either crush or nerve transection injury. In the crush injury model, the soleus muscle showed significantly increased recovery in wet weight at days 14 and 28 (compared with day 7) which was not the case for the gastrocnemius muscle which continued to atrophy. There was a significantly more pronounced up-regulation of MyoD expression in the denervated soleus muscle compared with the gastrocnemius muscle. Conversely, myogenin was more markedly elevated in the gastrocnemius versus soleus muscles. The muscles also showed significantly contrasting transcriptional regulation of the microRNAs miR-1 and miR-206. MuRF1 and Atrogin-1 showed the highest levels of expression in the denervated gastrocnemius muscle. Morphological and molecular changes in spinal motoneurons were compared after L4-L5 ventral root avulsion (VRA) and distal peripheral nerve axotomy (PNA). Neuronal degeneration was indicated by decreased immunostaining for microtubule-associated protein-2 in dendrites and synaptophysin in presynaptic boutons after both VRA and PNA. Immunostaining for ED1-reactive microglia and GFAPpositive astrocytes was significantly elevated in all experimental groups. qRT-PCR analysis and Western blotting of the ventral horn from L4-L5 spinal cord segments revealed a significant upregulation of apoptotic cell death mediators including caspases-3 and -8 and a range of related death receptors following VRA. In contrast, following PNA, only caspase-8 was moderately upregulated. The mechanisms of primary sensory neuron degeneration were also investigated in the DRG following peripheral nerve axotomy, where several apoptotic pathways including those involving the endoplasmic reticulum were shown to be upregulated. In summary, these results show that the critical time point after which the outcome of regeneration becomes too poor appears to be 3-months. Both proximal and distal injury affect spinal motoneurons morphologically, but VRA induces motoneuron degeneration mediated through both intrinsic and extrinsic apoptotic pathways. Primary sensory neuron degeneration involves several different apoptotic pathways, including the endoplasmic reticulum.

Place, publisher, year, edition, pages
Umeå: Umeå University , 2016. , 53 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1853
Keyword [en]
Peripheral nerve injury, target organ, spinal motoneurons, primary sensory neurons, degeneration
National Category
Cell and Molecular Biology
Research subject
Human Anatomy
Identifiers
URN: urn:nbn:se:umu:diva-127357ISBN: 978-91-7601-591-9OAI: oai:DiVA.org:umu-127357DiVA: diva2:1045445
Public defence
2016-12-02, Sal KB3A9, KBC-huset, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2016-11-11 Created: 2016-11-09 Last updated: 2016-11-21Bibliographically approved
List of papers
1. Effect of delayed peripheral nerve repair on nerve regeneration, Schwann cell function and target muscle recovery
Open this publication in new window or tab >>Effect of delayed peripheral nerve repair on nerve regeneration, Schwann cell function and target muscle recovery
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2013 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 2, e56484Article in journal (Refereed) Published
Abstract [en]

Despite advances in surgical techniques for peripheral nerve repair, functional restitution remains incomplete. The timing of surgery is one factor influencing the extent of recovery but it is not yet clearly defined how long a delay may be tolerated before repair becomes futile. In this study, rats underwent sciatic nerve transection before immediate (0) or 1, 3, or 6 months delayed repair with a nerve graft. Regeneration of spinal motoneurons, 13 weeks after nerve repair, was assessed using retrograde labeling. Nerve tissue was also collected from the proximal and distal stumps and from the nerve graft, together with the medial gastrocnemius (MG) muscles. A dramatic decline in the number of regenerating motoneurons and myelinated axons in the distal nerve stump was observed in the 3- and 6-months delayed groups. After 3 months delay, the axonal number in the proximal stump increased 2-3 folds, accompanied by a smaller axonal area. RT-PCR of distal nerve segments revealed a decline in Schwann cells (SC) markers, most notably in the 3 and 6 month delayed repair samples. There was also a progressive increase in fibrosis and proteoglycan scar markers in the distal nerve with increased delayed repair time. The yield of SC isolated from the distal nerve segments progressively fell with increased delay in repair time but cultured SC from all groups proliferated at similar rates. MG muscle at 3- and 6-months delay repair showed a significant decline in weight (61% and 27% compared with contra-lateral side). Muscle fiber atrophy and changes to neuromuscular junctions were observed with increased delayed repair time suggestive of progressively impaired reinnervation. This study demonstrates that one of the main limiting factors for nerve regeneration after delayed repair is the distal stump. The critical time point after which the outcome of regeneration becomes too poor appears to be 3-months.

Place, publisher, year, edition, pages
Public Library of Science, 2013
National Category
Other Basic Medicine
Identifiers
urn:nbn:se:umu:diva-68439 (URN)10.1371/journal.pone.0056484 (DOI)000315157200136 ()23409189 (PubMedID)
Available from: 2013-04-19 Created: 2013-04-19 Last updated: 2016-11-10Bibliographically approved
2. Investigation of the Expression of Myogenic Transcription Factors, microRNAs and Muscle-Specific E3 Ubiquitin Ligases in the Medial Gastrocnemius and Soleus Muscles following Peripheral Nerve Injury
Open this publication in new window or tab >>Investigation of the Expression of Myogenic Transcription Factors, microRNAs and Muscle-Specific E3 Ubiquitin Ligases in the Medial Gastrocnemius and Soleus Muscles following Peripheral Nerve Injury
2015 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 12, e0142699Article in journal (Refereed) Published
Abstract [en]

Despite surgical innovation, the sensory and motor outcome after a peripheral nerve injury remains incomplete. One contributing factor to the poor outcome is prolonged denervation of the target organ, leading to apoptosis of both mature myofibres and satellite cells with subsequent replacement of the muscle tissue with fibrotic scar and adipose tissue. In this study, we investigated the expression of myogenic transcription factors, muscle specific microRNAs and muscle-specific E3 ubiquitin ligases at several time points following denervation in two different muscles, the gastrocnemius (containing predominantly fast type fibres) and soleus (slow type) muscles, since these molecules may influence the degree of atrophy following denervation. Both muscles exhibited significant atrophy (compared with the contra-lateral sides) at 7 days following either a nerve transection or crush injury. In the crush model, the soleus muscle showed significantly increased muscle weights at days 14 and 28 which was not the case for the gastrocnemius muscle which continued to atrophy. There was a significantly more pronounced up-regulation of MyoD expression in the denervated soleus muscle compared with the gastrocnemius muscle. Conversely, myogenin was more markedly elevated in the gastrocnemius versus soleus muscles. The muscles also showed significantly contrasting transcriptional regulation of the microRNAs miR-1 and miR-206. MuRF1 and Atrogin-1 showed the highest levels of expression in the denervated gastrocnemius muscle. This study provides further insights regarding the intracellular regulatory molecules that generate and maintain distinct patterns of gene expression in different fibre types following peripheral nerve injury.

Place, publisher, year, edition, pages
San Francisco: Public Library Science, 2015
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-114579 (URN)10.1371/journal.pone.0142699 (DOI)000367092300002 ()26691660 (PubMedID)
Available from: 2016-02-16 Created: 2016-01-25 Last updated: 2016-11-10Bibliographically approved
3. A Morphological and Molecular Characterization of the Spinal Cord after Ventral Root Avulsion or Distal Peripheral Nerve Axotomy Injuries in Adult Rats
Open this publication in new window or tab >>A Morphological and Molecular Characterization of the Spinal Cord after Ventral Root Avulsion or Distal Peripheral Nerve Axotomy Injuries in Adult Rats
2016 (English)In: Journal of Neurotrauma, ISSN 0897-7151, E-ISSN 1557-9042Article in journal (Refereed) Epub ahead of print
Abstract [en]

Retrograde cell death in sensory dorsal root ganglion cells following peripheral nerve injury is well established. However, available data regarding the underlying mechanism behind injury induced motoneuron death are conflicting. By comparing morphological and molecular changes in spinal motoneurons after L4-L5 ventral root avulsion (VRA) and distal peripheral nerve axotomy (PNA) 7 and 14 days postoperatively, we aimed to gain more insight about the mechanism behind injury-induced motoneuron degeneration. Morphological changes in spinal cord were assessed by using quantitative immunohistochemistry. Neuronal degeneration was revealed by decreased immunostaining for microtubuleassociated protein-2 in dendrites and synaptophysin in presynaptic boutons after both VRA and PNA. Significant motoneuron atrophy was already observed at 7 days post-injury, independently of injury type. Immunostaining for ED1 reactive microglia was significantly elevated in all experimental groups, as well as the astroglial marker glial fibrillary acidic protein (GFAP). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of the ventral horn from L4-L5 spinal cord segments revealed a significant upregulation of genes involved in programmed cell death including caspase-3, caspase-8, and related death receptors TRAIL-R, tumor necrosis factor (TNF)-R, and Fas following VRA. In contrast, following PNA, caspase-3 and the death receptor gene expression levels did not differ from the control, and there was only a modest increased expression of caspase-8. Moreover, the altered gene expression correlated with protein changes. These results show that the spinal motoneurons reacted in a similar fashion with respect to morphological changes after both proximal and distal injury. However, the increased expression of caspase-3, caspase-8, and related death receptors after VRA suggest that injury- induced motoneuron degeneration is mediated through an apoptotic mechanism, which might involve both the intrinsic and the extrinsic pathways.

Keyword
apoptosis, motoneurons, PNA, VRA
National Category
Surgery
Identifiers
urn:nbn:se:umu:diva-127355 (URN)10.1089/neu.2015.4378 (DOI)
Available from: 2016-11-09 Created: 2016-11-09 Last updated: 2016-11-10
4. Evaluation of apoptotic pathways in dorsal root ganglion neurons following peripheral nerve injury
Open this publication in new window or tab >>Evaluation of apoptotic pathways in dorsal root ganglion neurons following peripheral nerve injury
(English)Manuscript (preprint) (Other academic)
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-127356 (URN)
Available from: 2016-11-09 Created: 2016-11-09 Last updated: 2016-11-10

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