An improved method for avulsion of lumbar nerve roots as an experimental model of nitric oxide-mediated neuronal degeneration

doi:10.1016/S1385-299X(00)00017-9

Brain Research Protocols

Volume 5, Issue 3, July 2000, Pages 223-230

https://doi.org/10.1016/S1385-299X(00)00017-9 Get rights and content

Abstract

A root avulsion lesion on the spinal nerve of adult animals is a useful technique to make a model for axotomy-induced motoneuronal degeneration, which is thought to be mediated by nitric oxide (NO). Here, we show a simplified version of extravertebral avulsion in the young adult rat. The L4 nerve always runs under the transverse process of the L5 vertebra, which is located just rostral to the delineation of the iliac crest. We used the iliac crest as a clue for the identification of the L4 nerve during surgery, including before skin incision. In almost all animals the L4 nerve was successfully avulsed at the exit point from the spinal cord. This experimental result was similar to that shown in the previous literature; the number of either Nissl-stained or ChAT-immunoreactive (-ir) motoneurons (MN) gradually decreased, while NOS immunoreactivity was induced in the MN after avulsion. Furthermore, a combined method of confocal laser scanning microscopy and double fluorescent procedures carried out in this model suggested the existence of cellular interaction between NOS-ir MN and OX42-ir or ED1-ir microglia. It is concluded that this simple and fast method of spinal root avulsion is very useful for making a reproducible model of NO-mediated MN cell death, with which the mechanism of neuronal cell death, including neuron–glia interaction, can be further explored.

Section snippets

Types of research

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Animal models of motoneuronal cell death.
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Double immunofluorescent histochemistry.
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Confocal laser scanning microscopic analysis.

Time required

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Surgical procedure for the avulsion, including presurgical anesthesia: 10 min.
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Transcardial perfusion with 4% paraformaldehyde in PB and postfixation: 3 h.
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Immersion in 25% sucrose in PB: 1 day.
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Sectioning with a cryostat microtome: 30 min.
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Nissl staining: 1 h.
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Immunofluorescent histochemistry: 5 days.
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Confocal laser scanning microscopy: 2 h.

Materials

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Wistar rats of both sexes, weighing between 120 and 140 g (aged 4–6 weeks old) were used.
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Special equipment:
- dissecting microscope (Leica M420, Germany)
- CCD camera (FUJIX HC-2000, Japan)
- confocal laser scanning imaging system (LSM-GB200) with a microscope (Olympus, Japan)
- fluorescence microscope (Leica DMRXA, Germany).
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Chemicals and reagents:
- pentobarbital sodium salt (20 mg/kg)
- xylocaine spray (surface anesthesia) (Fujisawa-yakuhin, #4390, Japan)
- kanamysin sulfate (Meiji-seika, #ksp117, Japan)
- 0.01 M

Preliminary examination

A preliminary examination was performed to identify the exact spinal level on the iliac crest, which was expected to be an available clue to help locate one of the spinal nerves. Some animals were transcardially perfused with a fixative (10% formalin) and then bones and nerves in the lumber section were dissected out. The most rostral delineation of the iliac crest was invariably located on a line through the connection between the L5 and L6 vertebrae (Fig. 1A). The L4 nerve, which passed

Results

Nissl staining (Fig. 2) and the double immunofluorescent procedure for NOS and ChAT (Fig. 3) clearly demonstrated the effect of spinal root avulsion on the MN, that is, gradual loss of MN and induction of NOS in MN. The number of ChAT-ir MN and Nissl-stained MN dramatically decreased in the first week (50.6% and 56.2%, respectively, compared to the control side). Subsequently, each number gradually decreased for 7 weeks. After that time, both the number of surviving Nissl-stained MN and ChAT-ir

Discussion

Many studies have focused on the effects of peripheral axotomy, a useful injury model, on NO synthesis in spinal MN. The involvement of nitric oxide (NO) in the process of neuronal degeneration and subsequent death, as well as in protection against neuronal injury, seems of major importance, as seen in the morphological and biochemical changes (cf. review for Iadecola [2]). Expression of nitric oxide synthase (NOS) in MN and cell death of MN via cutting of spinal motor axons are induced only

Quick procedure

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Surgical procedure for L4 nerve avulsion.
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Fixation and tissue preparation.
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Double immunofluorescent histochemistry.
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Confocal laser scanning microscopic analysis.

Essential literature references

Original paper: Ref. [4].

Acknowledgements

We thank Mr. Takaaki Kanemaru for his help in preparing photomicrographs.

References (10)

C. Iadecola
Bright and dark sides of nitric oxide in ischemic brain injury
Trends Neurosci.
(1997)
A. Kishino et al.
BDNF prevents and reverses adult rat motor neuron degeneration and induces axonal outgrowth
Exp. Neurol.
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G.W. Kreutzberg
Microglia: a sensor for pathological events in the CNS
Trends Neurosci.
(1996)
L. Novikov et al.
Brain-derived neurotrophic factor promotes survival and blocks nitric oxide synthase expression in adult rat spinal motoneurons after ventral root avulsion
Neurosci. Lett.
(1995)
W. Wu et al.
Inhibition of nitric oxide synthase reduces motoneuron death due to spinal root avulsion
Neurosci. Lett.
(1993)

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Ventral root avulsion exclusively affects motor fibers leading to the retrograde degeneration of axotomized spinal motoneurons. Experimental VRA is an efficient model for studying degenerative changes of motoneurons and their underlying mechanisms (He et al., 2000; Koliatsos et al., 1994); as well as a reliable model for testing various therapeutic agents, including trophic factors (Koliatsos et al., 1994; Watabe et al., 2005). In addition to the general loss of cells, any surviving motoneurons suffer a substantial decrease of presynaptic terminals, which further reduces or even abolishes synaptic transmission (Carlstedt, 2009).
Ventral root avulsion (VRA) triggers a strong glial reaction which contributes to neuronal loss, as well as to synaptic detachment. To overcome the degenerative effects of VRA, treatments with neurotrophic factors and stem cells have been proposed. Thus, we investigated neuroprotection elicited by human embryonic stem cells (hESC), modified to overexpress a human fibroblast growth factor 2 (FGF-2), on motoneurons subjected to VRA. Lewis rats were submitted to VRA (L4-L6) and hESC/FGF-2 were applied to the injury site using a fibrin scaffold. The spinal cords were processed to evaluate neuronal survival, synaptic stability, and glial reactivity two weeks post lesion. Then, qRT-PCR was used to assess gene expression of β2-microglobulin (β2m), TNFα, IL1β, IL6 and IL10 in the spinal cord in vivo and FGF2 mRNA levels in hESC in vitro. The results indicate that hESC overexpressing FGF2 significantly rescued avulsed motoneurons, preserving synaptic covering and reducing astroglial reactivity. The cells were also shown to express BDNF and GDNF at the site of injury. Additionally, engraftment of hESC led to a significant reduction in mRNA levels of TNFα at the spinal cord ventral horn, indicating their immunomodulatory properties. Overall, the present data suggest that hESC overexpressing FGF2 are neuroprotective and can shift gene expression towards an anti-inflammatory environment.
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Reimplantation of avulsed rat lumbar spinal ventral roots results in poor recovery of function of the denervated hind limb muscles. In contrast, reimplantation of cervical or sacral ventral roots is a successful repair strategy that results in a significant degree of regeneration. A possible explanation for this difference could be that following lumbar root avulsion, axons have to travel longer distances towards their target muscles, resulting in prolonged denervation of the distal nerve and a diminished capacity to support regeneration. Here we present a detailed spatio-temporal analysis of motoneuron survival, axonal regeneration and neurotrophic factor expression following unilateral avulsion and implantation of lumbar ventral roots L3, L4, and L5. Reimplantation prolongs the survival of motoneurons up to one month post-lesion. The first regenerating motor axons entered the reimplanted ventral roots during the first week and large numbers of fibers gradually enter the lumbar plexus between 2 and 4 weeks, indicating that axons enter the reimplanted roots and plexus over an extended period of time. However, motor axon counts show that relatively few axons reach the distal sciatic nerve in the 16 week post-lesion period. The observed initial increase and subsequent decline in expression of glial cell line-derived neurotrophic factor and brain-derived neurotrophic factor correlate with the apparent spatio-temporal decline in the regenerative capacity of motor axons, indicating that the distal nerve is losing its capacity to support regenerating motor axons following prolonged denervation. These findings have important implications for future strategies to promote long-distance regeneration through distal, chronically denervated peripheral nerves.
Neuronal NOS deficiency promotes apoptotic cell death of spinal cord neurons after peripheral nerve transection
2004, Nitric Oxide - Biology and Chemistry
To study the role of endogenous NO in survival and recovery of spinal cord neurons after nerve lesions, wild type mice were compared to knock-out mice lacking neuronal, endothelial or inducible NO synthase (NOS) after sciatic nerve transection. The NO-generating capacities were assessed by NOS immunohistochemistry and NADPH-diaphorase staining. The feature of affected neurons was evaluated following Nissl- and TUNEL-staining, by immunocytochemical demonstration of cytochrome c-translocation, and by ultrastructural examination. Time point of cell loss was found to be independent of the mice type and occurred only at later post-axotomy states. The extent of neuronal degeneration, however, depended on the NO supply. Whereas a lack of endothelial or inducible NOS was well tolerated, deficiency of neuronal NOS enhanced the competence-to-die and led to a substantial apoptotic cell death of spinal cord neurons. Thus, NO supply turned out to be essential for cell survival and recovery with reference to the neuronal NOS isoform.
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Taken together, the present results indicate the surviving effects of AxCAhGDNF on the injury and loss of adult vagal motoneurons after an injury to either the vagal nerve or recurrent laryngeal nerve. The ChAT immunoreactivity is known to decrease rapidly 1 week after facial or spinal nerve avulsion, and it gradually decreases thereafter up to 7 weeks [12,18,26,30,33]. The local or subcutaneous administration of GDNF after facial nerve transection has been reported to attenuate the decrease in ChAT immunoreactivity in adult rats [33].
We examined neuroprotective effects of an adenoviral vector encoding glial cell line-derived neurotrophic factor (AxCAhGDNF) on the lesioned adult rat motoneurons in the nucleus ambiguus. After vagal nerve avulsion, AxCAhGDNF, AxCALacZ (adenovirus encoding β-galactosidase gene) or PBS was inoculated into the jugular foramen. Four days after the avulsion and treatment with AxCALacZ, the animals expressed β-galactosidase activity in the lesioned motoneurons in the nucleus ambiguus. The animals avulsed and inoculated with AxCAhGDNF showed immunolabeling for GDNF in the nucleus ambiguus on the treated side and expression of virus-induced human GDNF mRNA transcripts in the brainstem tissue that contained the nucleus ambiguus of the treated side. The treatment with AxCAhGDNF after avulsion prevented the loss of lesioned motoneurons in the nucleus ambiguus, ameliorated the choline acetyltransferase immunoreactivity, and also suppressed the activity of nitric oxide synthase in these neurons. These results indicate that adenovirus-mediated GDNF gene transfer may prevent the degeneration of motoneurons in humans after either vagal nerve injury or recurrent laryngeal nerve injury.
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A technique has been developed for cutting single nerve fibers in mammalian spinal cord. In the presence of diaminobenzidine (DAB), a laser microbeam was applied to carbocyanine (Dil) stained sensory fibers in cultured spinal cords of the newly born opossum Monodelphis domestica. Digital images of fluorescent fibers were acquired with an intensified video CCD-camera coupled to an image processor. Laser illumination of two spots on a fiber in the presence of 3 mg/ml DAB cut it, so that following DAB wash out, Dil fluorescence did not return after the intermediate segment was bleached. In contrast, when a similar procedure was carried out without DAB, fluorescence of the bleached segment was recovered within minutes in darkness, by dye diffusion from adjacent regions of the uncut fiber. After exposure to DAB, through-conduction of compound action potentials continued in undamaged fibers. The DAB reaction product remained as a dark precipitate, helping to localize the lesion sites. By illuminating a continuous series of spots it was possible to cut whole nerve roots. Fluorescent fibers extended across the cut segment 24 h later. With minor modifications, the procedure described here allows a precise lesioning of single fibers within an intact nervous system.
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2001, Brain Research
Citation Excerpt :
Here, we show temporal, yet distinct, molecular changes in reactive astrocytes, microglia and synapses, and these alterations are compared with the peripheral nerve transection model. Our methods of nerve avulsion are based on the protocol of He et al. [21]. A total of 21 female Wistar rats (28–35 days old, 60–100 g) were deeply anesthetized with intraperitoneal injections of 20 mg/kg of sodium pentobarbitol.
The aim of this study was to demonstrate acute to subacute molecular episodes in the dorsal horn following root avulsion using immunohistochemical methods with the markers for synapses, astrocytes and such stress-responsive molecules as heat shock proteins (Hsps) and p38 MAP kinase (p38). Among them, Hsp27 was accumulated selectively in the injured substantia gelatinosa 24 h after avulsion injury. The localization of Hsp27 in astrocytes within the substantia gelatinosa was confirmed by the double immunofluorescence method using anti-Hsp27 antibody and either anti-synaptophysin antibody or anti-glutamine synthetase antibody and by immunoelectron microscopy for Hsp27. The pattern of Hsp27 expression subsequently changed from glial pattern to punctate pattern by 7 days. Immunoelectron microscopy revealed that the punctate pattern in the subacute stage corresponded to distal parts of the astrocytic processes. Hsp27 immunoreaction was decreased 21 days after root avulsion. In the distal axotomy model, Hsp27 was accumulated later in the ipsilateral dorsal horn in a punctate pattern from 7 days after the axotomy. Phosphorylation of p38 was detected in microglia in the dorsal horn following both avulsion and axotomy. Substance P was slightly decreased in the injured substantia gelatinosa in both the avulsion and axotomy models around 14–21 days. We conclude that Hsp27 is a useful marker for demonstrating dorsal horn lesions following avulsion injury and that avulsion injury may induce Hsp27 in the dorsal horn more rapidly than distal axotomy.

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ProtocolAn improved method for avulsion of lumbar nerve roots as an experimental model of nitric oxide-mediated neuronal degeneration