Transient immunohistochemical labelling of rat retinal axons during Wallerian degeneration by a monoclonal antibody to neurofilaments

doi:10.1016/0006-8993(94)91917-8

Brain Research

Volume 648, Issue 1, 13 June 1994, Pages 162-166

https://doi.org/10.1016/0006-8993(94)91917-8 Get rights and content

Abstract

Immunohistochemical labelling with the monoclonal antibody SMI32 to non-phosphorylated epitopes on neurofilament proteins of high molecular weight class was low in rat central optic fibers of controls. After unilateral transection of optic nerve, a strong, transient increase of labelling with SMI32 occurred in degenerating fibers of optic tract at 2 and 4 days, which then declined at 8 and remained low at 21 days. Consequently, immunostaining with SMI32 may serve as a positive marker for degenerating fibers in rat optic system.

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Cited by (26)

Late onset neuropathy with spontaneous clinical remission in mice lacking the POZ domain of the transcription factor Myc-interacting zinc finger protein 1 (Miz1) in Schwann cells
2015, Journal of Biological Chemistry
Citation Excerpt :
The corresponding axon appeared darker and less granular, most likely due to changes of the neurofilament and microtubule cytoskeleton. Therefore, we co-stained sciatic nerves from P90 Miz1ΔPOZ and control mice with an antibody against unphosphorylated neurofilament-H from the SMI-32 clone and against neurofilament-M. SMI-32 positivity is a marker for degenerating axons, whereas the neurofilament-M antibody recognizes neurofilaments independently of the phosphorylation status (41, 42). Morphometric quantifications uncovered no significant increase in SMI-32 signal intensity in Miz1ΔPOZ compared with control mice (Fig. 8A).
The transcription factor Miz1 (Myc-interacting zinc finger 1) is a known regulator of the cell cycle but also has cell cycle-independent functions. Here we analyzed the role of Miz1 in the peripheral nervous system, using an early embryonic conditional knock-out model in which the Miz1 POZ domain is ablated in Schwann cells. Although the development of myelinated nerve fibers was not impaired, Miz1ΔPOZ mice acquired behavioral signs of a peripheral neuropathy at the age of 3 months. At this time, ultrastructural analysis of the sciatic nerve showed de- and dysmyelination of fibers, with massive outfoldings and a focal infiltration of macrophages. Although the expression of genes encoding structural myelin proteins, such as periaxin, myelin basic protein, and myelin protein zero, was decreased, genes associated with a negative regulation of myelination, including c-Jun, Sox2, and Id2, were up-regulated in Miz1ΔPOZ mice compared with controls. In animals older than 4 months, the motor disabilities vanished, and the ultrastructure of the sciatic nerve exhibited numerous tomacula and remyelinated fibers, as indicated by thinner myelin. No second acute attack was observed up to the age of 1 year. Thus, the deletion of the Miz1 POZ domain in Schwann cells induces an acute neuropathy with a subsequent regeneration in which there is ongoing balancing between de- and remyelination. Miz1ΔPOZ mice are impaired in the maintenance of myelinated fibers and are a promising model for studying remyelination in adult peripheral nerves.The Myc-binding transcription factor Miz1 has multiple functions in different cells and tissues.
Ablation of the Miz1 POZ domain in Schwann cells causes a late onset peripheral neuropathy with spontaneous remission.
Miz1 plays an essential role in myelin homeostasis of peripheral nerves.
Ablation of Miz1 function in Schwann cells leads to a new model for remitting peripheral neuropathies.
The mechanism of axonal degeneration after perikaryal excitotoxic injury to the retina
2012, Experimental Neurology
Citation Excerpt :
Early axonal injury in Wlds and wild-type SD rats was quantitatively assessed by immunolabeling for SMI-32, an antibody that recognizes the non-phosphorylated chain of neurofilament heavy. SMI-32 has been consistently demonstrated to be a highly sensitive, marker of axonal cytoskeleton disruption, (Chidlow et al., 2011; Meller et al., 1994) owing to the labile nature of non-phosphorylated neurofilament heavy chain and its susceptibility to degradation by calpain. Microglial status in Wlds and wild-type SD rats was quantitatively assessed by immunolabeling for Iba1, ED1 and OX-6.
We investigated the mechanism of secondary axonal degeneration after perikaryal excitotoxic injury to retinal ganglion cells (RGCs) by comparing pathological responses in wild-type rats and Wld(s) rats, which display delayed Wallerian degeneration. After perikaryal excitotoxic RGC injury, both types of rats exhibited a spatio-temporal pattern of axonal cytoskeletal degeneration consistent with Wallerian degeneration, which was delayed by up to 4 weeks in Wld(s) rats. Furthermore, RGC somal loss was greater in Wld(s) rats. Microglial response in the anterior visual pathway to injury was attenuated in the Wld(s) rats with lymphocytic infiltration that was relatively reduced; however, immunostaining for major histocompatibility complex class II antigens (OX6) was more pronounced in Wld(s) rats. These data indicate that perikaryal excitotoxic RGC injury causes a secondary Wallerian axonal degeneration, and support the notion of a labile, soma-derived axonal survival factor.
Immunoelectron microscopy reveals the presence of neurofilament proteins in retinal terminals undergoing dark degeneration
2008, Brain Research
Citation Excerpt :
Excessive activation of calpains after injury has been proposed as a contributing factor in axonal degeneration, myelin breakdown, death of cells, and apoptosis (Couto et al., 2004; Paquet-Durand, 2007). In intact terminals, the continuous influx of calcium during depolarization is thought to stimulate calpains, disassembling NFs (Roots, 1983; Meller et al., 1994), whereas the pathophysiological mechanisms of TD probably involve a decreased enzymatic breakdown, due to decreased depolarization by the disconnection of the axon from the cell body, and increased influx of NF proteins from the distal segments of the axons by continuous axonal transport. Another interesting point addressed here is the comparison of the temporal pattern of optic nerve degeneration and retinal fiber terminal degeneration.
After nerve crushing or section, the distal stump undergoes morphological changes described as Wallerian degeneration (WD). Immediately after nerve injury, early ultrastructural alterations occur in the terminal boutons, a process known as terminal degeneration (TD), which occurs before degeneration of the axon and leads to electrophysiological impairment. In this study we investigated the presence of neurofilament (NF) proteins in TD and compared the results with degeneration in the optic nerve. Young adult Wistar rats were submitted to bilateral enucleation and perfused after 24 h, 48 h and 1 week. Optic nerves (ON) and superior colliculus (SC) segments were processed for electron microscopy (EM) and immunoelectron microscopy (IEM) for NF subunits. Analysis of ultrathin sections of SC, at 24 h, revealed terminals undergoing TD. At 48 h and 1 week after enucleation, there was a clear increase in the number of degenerating terminals. The cytoarchitecture of the optic nerve did not change considerably at 24 h, but it was progressively altered at 48 h and 1 week after enucleation, when we observed intense astrogliosis, and most fibers exhibited dark degeneration (DD). The IEM for the NF subunits of normal ON showed gold particles located along the filaments, but we did not observe labeling for neurofilament proteins in normal retinal terminals. However, 48 h after lesion, we observed immunogold particles for the NF proteins in fibers undergoing DD and on terminals undergoing TD. Therefore, we can conclude that NF proteins participate in the process of TD, and this event occurs before complete axonal degeneration, suggesting different mechanisms for TD and DD.
Hypoxic injury of isolated axons is independent of ionotropic glutamate receptors
2007, Neurobiology of Disease
Citation Excerpt :
SMI31 recognizes phosphorylated neurofilaments. An increase in non-phosphorylated neurofilaments may indicate the early stages of injury (Meller et al., 1994). Immunoreactivity for non-phosphorylated neurofilaments recognized by SMI32 was not detected in OGD treated axons at 1, 6 and 24 h after the initial injury (data not shown).
Axonal injury in white matter is an important consequence of many acute neurological diseases including ischemia. A role for glutamate-mediated excitotoxicity is suggested by observations from in vitro and in situ models that AMPA/kainate blockers can reduce axonal injury. We assessed axonal vulnerability in primary murine neuronal cultures, with axons isolated from their cell bodies using a compartmented chamber design. Transient removal of oxygen and glucose in the axon compartment resulted in irreversible loss of axon length and neurofilament labeling. This injury was not prevented by addition of ionotropic glutamate receptor blockers and could not be reproduced by glutamate receptor agonists. However, hypoxic injury was prevented by blockade of voltage-gated sodium channels, inhibition of calpain and removal of extracellular calcium. These results suggest that isolated, unmyelinated axons are vulnerable to hypoxic injury which is mediated by influx of sodium and calcium but is independent of glutamate receptor activation.
Injury to axons and oligodendrocytes following endothelin-1-induced middle cerebral artery occlusion in conscious rats
2006, Brain Research
Citation Excerpt :
Assessments of axonal injury using APP antiserum are however limited as immunostaining is restricted mainly to the margins of the ischemic lesion, and hence is largely absent from the ischemic core (Yam et al., 1997). SMI32 immunohistochemistry has been used to characterize axonal injury in several pathologies including optic nerve transection in rats (Meller et al., 1994), experimental autoimmune encephalomyelitis in mice (Jackson et al., 2005), and focal thrombotic cortical lesions in mice (Bidmon et al., 1997). To our knowledge, however, SMI32 immunohistochemistry has not been used to characterize axonal injury following middle cerebral artery occlusion (MCAO) in rats.
Injury to axons and oligodendrocytes has been poorly characterized in most animal models of stroke, and hence has been difficult to target therapeutically. It is therefore necessary to characterize axonal and oligodendroglial injury in these models, in order to rationally design putative protective compounds that minimize this injury. This study aims to characterize injury to axons and oligodendrocytes in the endothelin-1 (ET-1) model of middle cerebral artery occlusion (MCAO) in conscious rats. Transient forebrain ischemia was induced in conscious adult male Long Evans rats by the perivascular microinjection of ET-1. Quantitative histopathology was performed on forebrain sections at 6, 24, 48 and 72 h after ET-1 administration, using ballistic light analyses and immunohistochemistry for amyloid precursor protein (APP), SMI32, and Tau-1. Ballistic light analyses of cortical and striatal lesions revealed that the infarct volume was maximal in these regions by 6 h. APP and SMI32 immunohistochemistry demonstrated that axonal injury was maximal by 6 h in this model; however, some injured axons appeared to maintain good structural integrity up to 72 h after insult. Density measurements for Tau-1-immunopositive oligodendrocytes were significantly elevated within the corpus callosum from 48 h, but reductions in total oligodendrocyte numbers were not apparent up 72 h after ET-1 injection. These results indicate that axonal and oligodendroglial injury should be investigated as potential targets for delayed therapeutic intervention after MCAO.
Neurensin-1 expression in the mouse retina during postnatal development and in cultured retinal neurons
2006, Brain Research
Neurensin-1/Neuro-p24 (previously named Neuro-p24) is a neuron-specific membrane protein that is localized particularly in neurites. Neurensin-1 is considered to play an essential role in neurite extension during nervous development, regeneration and plasticity. To understand what role Neurensin-1 plays in retinal differentiation, we examined Neurensin-1 distribution and gene expression pattern in the postnatally developing retina of the mouse, because the retina is an excellent model for nervous development. In the postnatal day (PD) 1 retina, intense Neurensin-1 immunoreactivity was found in the optic nerve fiber layer. Faint staining was seen in the ganglion cells, presumptive amacrine and horizontal cells. As the postnatal development proceeded, the optic fibers became more intensely stained in addition to other parts of the retina such as the ganglion cells, inner plexiform layer and horizontal cells. In PD 10 retinas, the horizontal cell processes showed a prominently stained configuration. As the retina developed further to attain maturity, the staining in the retina became less pronounced, although the optic nerves remained positively stained. The distribution of Neurensin-1 mRNA was consistent with these results and confirmed that the ganglion, amacrine and horizontal cells actively synthesize Neurensin-1 in the developing retina. In the retinal cell culture from newborn mice, two types of neural cells were stained for Neurensin-1, one of which showed long processes and appeared presumptive ganglion cells. These results suggest that Neurensin-1 plays a role in the fiber extension of the retinal neurons, as has been observed in other central nervous tissues, and indicate that the developing retina is a suitable experimental model for the analysis of Neurensin function, both in vivo and in vitro.

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: This study was supported by grants of the Deutsche Forschungsgemeinschaft (DFG EY 8/13-2; Schm 776/4-1, Heisenberg-Programm).

^*: The authors are indebted to Ms. U. Neubacher for excellent help in the laboratory, and to Ms. D. Strehler for photographic work.

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Transient immunohistochemical labelling of rat retinal axons during Wallerian degeneration by a monoclonal antibody to neurofilaments

Abstract

Neurosci. Lett.

Exp. Neurol.

Exp. Cell. Res.

Brain Res.

J. Neurol. Sci.

Brain Res.

Trends Neurosci.

Exp. Neurol.

Int. J. Dev. Neurosci.

Brain Res.

Brain Res.

The fate of axonal debris in Wallerian degeneration of rat optic and sciatic nerves

J. Neuropathol. Exp. Neurol.

Neurofilament phosphorylation in axons and perikarya: immunofluorescence study of the rat spinal cord and dorsal root ganglia with monoclonal antibodies

J. Comp. Neurol.

Monoclonal antibodies specific for glial fibrillary acidic (GFA) protein and for each of the neurofilament triplet polypeptides

Differentiation

Antibodies to heavy neurofilament subunit detect a subpopulation of damaged ganglion cells in retina

Nature