Sciatic nerve regeneration in mice and rats: recovery of sensory innervation is followed by a slowly retreating neuropathic pain-like syndrome

doi:10.1016/j.brainres.2004.08.036

Brain Research

Volume 1027, Issues 1–2, 19 November 2004, Pages 67-72

https://doi.org/10.1016/j.brainres.2004.08.036 Get rights and content

Abstract

Peripheral nerve regeneration has been studied extensively in the sciatic nerve crush model, at the level of both function and gene expression. The crush injury allows full recovery of sensory and motor function in about 3 weeks as assessed by the foot reflex withdrawal test and De Medinacelli walking patterns. We used the recently developed CatWalk paradigm to study walking patterns in more detail in mice and rats. We found that, following the recovery of sensory function, the animals developed a state of mechanical allodynia, which retreated slowly over time. The motor function, although fully recovered with the conventional methods, was revealed to be still impaired because the animals did not put weight on their previously injured paw. The development of neuropathic pain following successful sensory recovery has not been described before in crush-lesioned animals and may provide an important new parameter to assess full sensory recovery.

Introduction

The sciatic nerve crush model is a well-characterized model for peripheral nerve regeneration. After a crush lesion, nerve fibers in the distal stump degenerate. Myelin and axon debris are removed by the process of Wallerian degeneration. The endoneurial tubes remain intact, enabling fast and qualitatively good anatomical and functional recovery [2], [7], [10], [23]. Fibers in the proximal nerve stump start to sprout and new axons extend through the distal endoneurial tube to reinnervate their target organs. During this process of axonal regeneration, numerous changes in morphology (chromatolysis) and gene expression occur in the neuronal cell bodies [1], [10], [11], [14].

Recently, a new method for gait analysis was described by one of us, the CatWalk [13]. This technique gives more detailed information on how the animal uses its paw during locomotion than the conventional techniques, e.g., the De Medinacelli method, which essentially measures the ability to use the muscles in the lower paw and foot [9]. The CatWalk has, for example, been successful in showing that animals suffering from neuropathic pain do not support weight upon their paw, although they do place it [26]. Neuropathic pain is a well-known phenomenon in nerve injury models with impaired regeneration, e.g., sciatic nerve transection, in models of neural inflammation, and in models with chronic nerve damage, e.g., chronic constriction injury (CCI) [5], [15], [22], [30].

We performed the sciatic nerve crush in mice and rats, and measured functional recovery of sensory and motor functions using the foot reflex withdrawal test and the De Medinacelli method. The CatWalk paradigm was used to investigate walking performance in more detail. In rats, tactile allodynia was also scored using von Frey filament testing. We demonstrate that, although full functional recovery of sensory and motor function occurred in both species, the animals developed a state of neuropathic pain which only became apparent after full sensory recovery as measured with the foot reflex withdrawal test was present. As such, recovery as assessed by the De Medinacelli method or the foot reflex withdrawal test indicates successful regeneration of damaged axons to their previous target area, but does not indicate renormalization of function.

Section snippets

Animals and surgery

All procedures in this study were performed in accordance with the Ethical Guidelines of the International Association for the Study of Pain [31] and were approved by the Ethical Committee on Animal Experiments of the University of Utrecht. A crush lesion was placed in both rats and mice in the sciatic nerve of the right paw, at mid-thigh level, as described by de Koning et al. [8]. In short, Wistar rats (Charles River) were anesthetized with Hypnorm® (Janssen Pharmaceutics, Tilburg, the

Results

We investigated recovery of sensory and motor functions in C57BL/6J mice and Wistar rats. Recovery of sensory function was assessed using the foot reflex withdrawal test. At 18 dpo, 5 of the 6 mice (86%) and at 20 dpo, 6/6 mice reacted to the 0.1 mA electric current, indicating recovery of sensory function (Fig. 1). At 20 dpo, 7 of the 10 rats, and at 21 dpo, 10/10 rats reacted to the 0.1 mA electric current. These observations indicate that the time course of sensory recovery was roughly

Discussion

These data indicate that although sensory and motor innervation of the paw were fully recovered at 24 dpo, the animals did not yet support their weight on the lesioned paw. The print area, although recovered on the photographic films (De Medinacelli method), where one does not need much pressure to get a print, did not return to normal in either rats or mice during the follow-up period when visualized with the CatWalk system. This reduction in pressure applied during stance was also shown by

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Injuries of the peripheral nervous system are common among the population affecting around 3% of all trauma patients. This high clinical need in the field of peripheral nerve injury and regeneration has been steadily driving experimental and epidemiological research. Thereby, it is crucial to determine the exact degree of recovery of end-organ function. Regeneration after nerve injuries is assessed by a wide variety of techniques and pre-clinical model systems, where rodent models are among the most widely used. However, results from rodents are difficult to translate to human patients in general, and reproducible and comparable assessment of functional recovery is of highest importance. Computerized gait analysis allows comprehensive acquisition of locomotor function. As the animals cross the recording device voluntarily, functional recovery is assessable with a minimum degree of human interference on their behavior. This article aims to give a detailed overview on the existing literature on CatWalk gait analysis in rodent models of peripheral nerve injuries of upper and lower extremities, e.g. axonotmesis, neurotmesis or fibrosis, with special emphasis on differences between models. Researchers interested in assessment of locomotor function in such models will especially benefit from this work as it will provide them with an overview of the various experimental setups and expected outcomes. This work also addresses potential pitfalls and hurdles in order to promote well designed, comparable studies allowing for accelerated development of therapeutic strategies in peripheral repair and regeneration.
Transcriptional Reprogramming of Distinct Peripheral Sensory Neuron Subtypes after Axonal Injury
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Citation Excerpt :
We observed that peripheral axotomy results in profound transcriptional reprogramming of DRG neurons, involving both induction of a common set of injury-response genes across neuronal subtypes and the coincident downregulation of genes that define transcriptional identity. This transcriptional reprogramming is reversible, as the transcriptional states of injured neuronal nuclei return to their naive states within weeks (Figures 5A, 5B, S3E, and S3F), coinciding with target reinnervation (Figure S5B) (Navarro et al., 1994; Vogelaar et al., 2004). Injury-induced transcriptional reprogramming leads to a new transcriptional state in which neuronal subtypes are difficult to distinguish by their gene expression profiles.
Primary somatosensory neurons are specialized to transmit specific types of sensory information through differences in cell size, myelination, and the expression of distinct receptors and ion channels, which together define their transcriptional and functional identity. By profiling sensory ganglia at single-cell resolution, we find that all somatosensory neuronal subtypes undergo a similar transcriptional response to peripheral nerve injury that both promotes axonal regeneration and suppresses cell identity. This transcriptional reprogramming, which is not observed in non-neuronal cells, resolves over a similar time course as target reinnervation and is associated with the restoration of original cell identity. Injury-induced transcriptional reprogramming requires ATF3, a transcription factor that is induced rapidly after injury and necessary for axonal regeneration and functional recovery. Our findings suggest that transcription factors induced early after peripheral nerve injury confer the cellular plasticity required for sensory neurons to transform into a regenerative state.
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The experimental results displayed that BMSCs improve STZ-induced diabetes symptoms in rats by decreasing blood glucose and urinary protein. Functionally, BMSCs ameliorate oxidative stress, painful diabetic neuropathy, neurotrophic status and angiogenesis in STZ-induced rats. Moreover, BMSCs participate in the regulation of sciatic neuro morphology in diabetic neuropathy rat model. In mechanism, BMSCs alleviate diabetic peripheral neuropathy via activating GSK-3β/β-catenin signaling pathway in rats and improve Schwann's cells viability by activating GSK-3β/β-catenin signaling pathway under high glucose.
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“Stem cell therapy to promote limb function recovery in peripheral nerve damage in a rat model” – Experimental research
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Optimizing nerve regeneration and mitigating muscle atrophy are the keys to successful outcomes in peripheral nerve damage. We investigated whether mesenchymal stem cell (MSC) therapy can improve limb function recovery in peripheral nerve damage.
We used sciatic nerve transection/repair (SNR) and individual nerve transection/repair (INR; branches of sciatic nerve - tibial, peroneal, sural) models to study the effect of MSCs on proximal and distal peripheral nerve damages, respectively, in male Lewis rats. Syngeneic MSCs (5 × 10⁶; passage≤6) or saline were administered locally and intravenously. Sensory/motor functions (SF/MF) of the limb were assessed.
Rat MSCs (>90%) were CD29⁺, CD90⁺, CD34⁻, CD31⁻ and multipotent. Total SF at two weeks post-SNR & INR with or without MSC therapy was ∼1.2 on a 0–3 grading scale (0 = No function; 3 = Normal); by 12 weeks it was 2.6–2.8 in all groups (n ≥ 9/group). MSCs accelerated SF onset. At eight weeks post-INR, sciatic function index (SFI), a measure of MF (0 = Normal; −100 = Nonfunctional) was −34 and −77 in MSC and vehicle groups, respectively (n ≥ 9); post-SNR it was −72 and −92 in MSC and vehicle groups, respectively. Long-term MF (24 weeks) was apparent in MSC treated INR (SFI -63) but not in SNR (SFI -100). Gastrocnemius muscle atrophy was significantly reduced (P < 0.05) in INR. Nerve histomorphometry revealed reduced axonal area (P < 0.01) but no difference in myelination (P > 0.05) in MSC treated INR compared to the naive contralateral nerve.
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Promoting axon growth after peripheral nerve injury may support recovery. Soluble laminin polymers formed at pH 4 (aLam) accelerate axon growth from adult dorsal root ganglion neurons in vitro. We used an adult rat model of a peripheral (peroneal) nerve crush to investigate whether an injection of aLam enhances axon growth and functional recovery in vivo. Rats that received an injection of aLam into the crush at 2 days post-injury show significant improvements in hind limb motor function at 2 and 5 weeks after injury compared with control rats that received phosphate-buffered saline. Functional improvement was not associated with changes in sensitivity to thermal or mechanical stimuli. Treatment with aLam decreased the occurrence of autophagia and abolished non-compliance with treadmill walking. Rats treated with aLam showed increased axon presence in the crush site at 2 weeks post-injury and larger axon diameter at 10 weeks post-injury compared with controls. Treatment with aLam did not affect Schwann cell presence or axon myelination. Our results demonstrated that aLam accelerates axon growth and maturity in a crushed peroneal nerve associated with expedited hind limb motor function recovery. Our data support the therapeutic potential of injectable aLam polymers for treatment of peripheral nerve crush injuries.
Incidence of peripheral nerve injury has been estimated to be as high as 5% of all cases entering a Level 1 trauma center and the majority of cases are young males. Peripheral nerves have some endogenous repair capabilities, but overall recovery of function remains limited, which typically has devastating effects on the individual, family, and society, as wages are lost and rehabilitation is extended until the nerves can repair. We report here that laminin polymers injected into a crush accelerated repair and recovery, had no adverse effects on sensory function, obliterated non-compliance for walking tests, and decreased the occurrence of autophagia. These data support the use of laminin polymers for safe and effective recovery after peripheral nerve injury.

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¹: Currently working at MRC Cambridge University Centre for Brain Repair, Robinson Way, Cambridge, CB2 2PY, United Kingdom.

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Research reportSciatic nerve regeneration in mice and rats: recovery of sensory innervation is followed by a slowly retreating neuropathic pain-like syndrome

Abstract

Introduction

Section snippets

Animals and surgery

Results

Discussion

Pain

J. Neurosci. Methods

Peptides

J. Neurol. Sci.

Prog. Brain Res.

Trends Neurosci.

Neuroscience

Pain

Pain

Neurosci. Lett.

Exp. Neurol.

Pain

Axotomy-induced changes in primary sensory neurons

Pathology of the peripheral nerve

A device for measuring plantar pressures under the sole of the foot

Eng. Med.

Research report
Sciatic nerve regeneration in mice and rats: recovery of sensory innervation is followed by a slowly retreating neuropathic pain-like syndrome