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Robert Koch Institute, Nordufer 20, D-13353 Berlin, Germany
Correspondence
Christine Kratzel
kratzelc{at}rki.de
Michael Beekes
beekesm{at}rki.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Beekes & McBride, 2007), can be detected in affected animals and humans by analytical methods such as Western blotting (Beekes et al., 1996; Wadsworth et al., 2001), immunohistochemistry (McBride et al., 2001; Ironside et al., 2002) and paraffin-embedded tissue (PET) blotting (Schulz-Schaeffer et al., 2000), and have been established in many studies as a reliable biochemical marker for infectious TSE agents or ‘prions’ (McKinley et al., 1983; Gabizon et al., 1987; Bolton et al., 1991; Jendroska et al., 1991; Rubenstein et al., 1991; Beekes et al., 1996; Baldauf et al., 1997). The prion hypothesis holds that the causative agents of TSEs are proteinaceous infectious particles (prions) that consist essentially – if not entirely – of abnormal protease-resistant prion protein (Prusiner, 1991).
During the past few years, comprehensive research on the pathogenesis of peripherally acquired TSE infections has achieved substantial progress in dissecting the pathways by which prions spread through the body. These studies imply that prions spread from peripheral sites of infection along synaptically linked neural conduits to target areas in the brain and spinal cord (Beekes et al., 1998; Groschup et al., 1999; McBride et al., 2001; Bartz et al., 2002; Beekes & McBride, 2007). Although such defined dissemination along neuroanatomical projections is suggestive of axons as a track for prions, hard data on the cellular components and molecular mechanisms that mediate the spread of TSE agents in the nervous system are scant. Several studies found that propagation of infection in various components of the nervous system takes place at a net rate of 0.7–2 mm day–1, which would be consistent with transport by slow axonal flow (Kimberlin et al. 1983; Glatzel & Aguzzi, 2000a). PrPTSE deposits have been detected in nerve fibres of parenterally infected scrapie hamsters between the myelin sheath and axon (Groschup et al., 1999) and described to be present within axons in brain samples from CJD patients (Kovacs et al., 2005). However, transgenic mice overexpressing four-repeat tau with a reported impairment of axonal transport displayed comparable incubation times to control mice following intraneural infection with scrapie (Künzi et al., 2002). Also, the incubation time after peripheral prion infection was not found to be altered in mice with a heterozygous mutation of dynein, a motor protein involved in the transport of axonal cargo along the cytoskeleton (Hafezparast et al., 2005). In addition, pharmacological segregation of axonal neurofilaments and microtubuli by 
,'-iminodiproprionitrile did not influence the observed rate of spread of infection along peripheral nerves after administration of scrapie agent into the footpad of hamsters (Kratzel et al., 2007a). Thus, apparently conflicting findings argue in favour or against axons and their transport mechanisms as key mediators of nerve-associated prion dissemination. In contrast, the axonal movement of cellular PrP has been well established (Borchelt et al., 1994; Butowt et al., 2006).
Here, we used sciatic neurectomy as an experimental tool to generate in vivo incomplete nerve conduits containing segments depleted or devoid of axons, allowing further scrutinization of the host components necessary for the propagation of prions along neural pathways. Surprisingly, examination of the model conduits produced by our approach revealed that prion propagation seems to be possible along nerve structures that do not contain detectable axonal components.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Mock-operated control animals were anaesthetized and skin suture was carried out at the femoral level on the right hind leg.
Animal inoculation.
Adult male and female Syrian hamsters were sedated with isoflurane and infected with scrapie by inoculation into the footpad. For infection with two different concentrations of prions, 20 µl of a 0.001 % (w/v, ‘low dose’) or 2 % (w/v, ‘high dose’) scrapie 263K strain hamster brain homogenate from terminally ill donor animals were injected into the sole of the right foot using a 27-gauge cannula. The animals were regularly monitored for clinical signs of scrapie (Kratzel et al., 2007a). When terminally affected with scrapie, or when the experiment was terminated at 314 days post-infection (p.i.), the hamsters were sacrificed by CO2 asphyxiation.
Experimental groups and tissue collection.
Footpad infections with low or high doses of scrapie agent were performed at 4 weeks after mock operation (n=7 for each of the low- and high-dose groups) or neurectomy (n=8 for each of the low- and high-dose groups). Non-neurectomized negative-control animals (n=4) were similarly mock infected with a 2 % (w/v) normal hamster brain homogenate. One animal in the mock-operated high-dose group died before termination of the experiment for reasons unrelated to TSE disease. For PET blot detection of cerebral PrPTSE, brains were removed from animals, and for immunohistological examinations of axonal cytoskeleton components (i.e. tubulin and neurofilaments), sciatic nerve samples were obtained from two neurectomized hamsters that succumbed to terminal scrapie at 155 days after infection with a high dose of scrapie agent. These nerve specimens, and the brains for PET blotting, were fixed overnight immediately after collection by immersion in 4 % (v/v) formaldehyde. After incubation in PBS (pH 7.2), tissue samples were transferred to 70 % ethanol, processed in a tissue processor and subsequently embedded in paraffin wax. Nerve specimens from four neurectomized animals that developed terminal scrapie after footpad infection with a high dose of scrapie agent were collected for Western blot testing for PrPTSE. These specimens were frozen at –80 °C immediately after preparation. For the Western blots, sciatic nerve specimens were further dissected into proximal nerve stumps, medial nerve tubes and distal nerve stumps. Every precaution was taken to avoid cross-contamination.
Western blotting.
Tissue extraction of PrPTSE in the form of protease-resistant PrP27–30 was initiated by collagenase digestion of samples for 1 h and subsequently performed as described previously (Thomzig et al., 2003). The extracts were subjected to PAGE and Western blotting using the anti-PrP monoclonal antibody (mAb) 3F4 (Kascsak et al., 1987) as described in detail elsewhere (Thomzig et al., 2003). Samples from uninfected hamsters spiked with 263K scrapie brain homogenate from terminally ill donors were used as positive controls for the extraction procedure, as specified in the legend to Fig. 5.
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PET blotting of brain sections was performed according to the method of Schulz-Schaeffer et al. (2000) with slight modifications. In brief, 6 µm thick sections were dried on nitrocellulose membranes overnight at 55 °C. After deparaffinization, sections were digested overnight with 15 µg proteinase K ml–1 in digestion buffer containing 10 mM Tris/HCl (pH 7.8), 30 mM NaCl and 0.1 % (w/v) Brij-35 detergent. Sections were denatured in 3 M guanidine thiocyanate and blocked with 0.2 % (w/v) casein before labelling with the primary mAb 3F4 diluted 1 : 2500. Binding of the secondary antibody diluted 1 : 2000 was visualized using NBT/BCIP (AppliChem).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Kratzel et al., 2007b) and to ensure axonal degeneration distal to the transection side.
After footpad infection of hamsters with the low dose of scrapie agent, six out of seven mock-operated animals developed clinical signs of scrapie and reached the terminal stage of disease between 219 and 308 days p.i. (Fig. 1a). Spot-checked animals from this group including the non-diseased hamster (n=5) showed PrPTSE accumulation in the brain as evidenced by PET blotting (Fig. 2a). In contrast, none of the eight neurectomized animals developed clinical signs of scrapie after the low-dose challenge up to termination of the experiment at 314 days p.i. (Fig. 1a). Only one of these animals showed the start of cerebral PrPTSE deposition, whereas the other seven animals produced negative results following PET blotting for PrPTSE (Fig. 2b).
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). Hamsters mock-challenged with normal brain homogenate remained free of scrapie symptoms until termination of the experiment.
Status of nerve regeneration
Post-mortem macroscopic examination of sciatic nerves from all neurectomized animals revealed the process of nerve regeneration. Nerve-like tubes between proximal and distal nerve stumps could be observed and proximal stumps often showed a neuroma-like formation (Fig. 3).
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). Control nerve segments from the unmanipulated contralateral side of these animals showed regular immunofluorescent staining of axonal key components such as tubulin and neurofilaments (Fig. 4a and e). Staining of nerve segments from the neurectomized ipsilateral side revealed strong but disordered signals for tubulin and neurofilament in the proximal nerve stumps (Fig. 4b and f), weak signals in the medial nerve tubes (Fig. 4c and g) and no signals for the tested axonal components in the distal stump (Fig. 4d and h).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Beekes et al., 1998; Groschup et al., 1999; McBride et al., 2001; Bartz et al., 2002; Beekes & McBride, 2007). This, and other findings, has led to the suggestion that axons are essentially involved in the neural spread of prions and, more specifically, that axonal transport mechanisms may mediate this process. However, several experimental approaches have produced apparently contradictory results on the role of axons (Künzi et al., 2002; Hafezparast et al., 2005; Kovacs et al., 2005; Kratzel et al., 2007a), and other modes of spread of prions along neural projections are conceivable. Among these might be cell-free or cell-associated dissemination in perineural lymphatics as well as in neural interspaces (Mims & White, 1984) or sequential infection of Schwann cells (Follet et al., 2002). Alternatively, exosomal vehicles for transmission of the infectious prion protein (Février et al., 2005) or internalization of secreted forms of PrP by neighbouring cells (Butowt et al., 2006) have been discussed, and Glatzel & Aguzzi (2000b) suggested a domino-like conversion of cellular PrP into PrPTSE.
In order to establish whether prion propagation in peripheral neural tissue is feasible without an intact axonal anatomy or any detectable axonal structures at all, we used a functional sciatic nerve model, which constituted a neural conduit containing segments depleted or devoid of axons. Neurectomy of regional nerves was carried out previously for studies on the pathogenesis of neurotropic infectious diseases (Bassant et al., 1986; Carbone et al., 1987). However, to the best of our knowledge, nerve segments devoid of axons purposefully induced by experimental surgery have not been used to date to probe the necessity of neurites in the spread of prions through the body.
Sciatic neurectomy as performed in our hamster model is known to induce Wallerian degeneration in the distal nerve stumps (Waller, 1850; Burnett & Zager, 2004). After a latency period of a few days, physical fragmentation of both axons and myelin occurs in the disconnected distal stump. Axon and myelin debris are phagocytosed by Schwann cells and macrophages. Finally, only nerve fibre remnants consisting of Schwann cells within an endoneurial sheet remain in the distal stump. This degenerative process is usually completed within a few weeks. Nerve regeneration may be initiated by Schwann cell proliferation starting from the proximal stump, leading to continuous columns of cells known as the bands of Büngner (Büngner, 1891). These bands provide a guide for axons that can sprout from the proximal stump into surrounding tissue and eventually grow into the distal stump (Ide, 1996; Burnett & Zager, 2004). However, after transection, such regenerating axons are no longer guided by their original sheaths and are thus likely to fail functionally correct reinnervation.
In our study, the effect of sciatic neurectomy observed upon footpad infection of hamsters with a low dose of scrapie agent was pronounced (Fig. 1a). Six out of seven mock-operated hamsters developed terminal scrapie between 219 and 308 days p.i. In contrast, all neurectomized animals remained free of symptoms until 314 days p.i. when the experiment for this group was terminated, and only one of these animals showed weak deposition of PrPTSE in the brain. These effects of neurectomy functionally demonstrated that the nerve innervating the site of infection provided the prime conduit for prions to the CNS after footpad inoculation. For hamsters subjected to neurectomy of the sciatic nerve before high-dose scrapie footpad infection, we observed a prolongation of the survival time of about 60 % compared with mock-operated control animals (Fig. 1b). The difference between the observed incubation times without and with neurectomy (112 vs 182 days) approximately corresponded to that observed in incubation time-interval assays after intracerebral inoculation of 1.5x102 LD50 and a 100-fold lower dose, respectively (not shown).
However, despite substantially prolonged survival after sciatic neurectomy, all animals of the high-dose group succumbed to terminal disease. How did the infection spread to the CNS in these animals? Centripetal spreading pathways via non-sciatic neural projections, lymph or blood may provide an explanation. However, excision of the draining lymph node 4 weeks before footpad inoculation of non-neurectomized hamsters with 2 % 263K scrapie brain homogenate did not alter survival time (Kratzel et al., 2007b). Alternatively, delayed invasion of the brain could be accounted for by prion propagation from the distal nerve stump after partial sciatic regeneration. This prompted us to examine the disrupted parts of the sciatic nerve in more detail. An inspection of the operation site revealed that all neurectomized hamsters developed macroscopically visible tube-like structures connecting the proximal and distal stumps of the transected nerves (Fig. 3), although functional recovery was not observed in any of the neurectomized animals. Immunohistochemical examination with antibodies specific to key components of the axonal cytoskeleton such as tubulin and neurofilaments indicated sprouting axons in the proximal neuroma, growing distally to the level of the former nerve transection (Fig. 4b–d and f–h). No evidence of axons could be detected in the examined distal stumps of terminally ill hamsters from the high-dose group sacrificed at 155 days p.i. (Fig. 4d and h). In contrast, when we tested regenerated nerve samples from scrapie hamsters between 150 and 212 days p.i. for PrPTSE by highly sensitive Western blotting (Fig. 5), PrPTSE was consistently found in the proximal and medial portion of the injured nerve, and also in the distal nerve stumps.
The footpad inoculations of scrapie agents were performed 4 weeks after the neurectomy when Wallerian degeneration in the distal nerve stump was fairly advanced, and previous studies have shown that, even in intact sciatic nerves, PrPTSE becomes detectable only at 60 days following footpad infection (Kratzel et al., 2007a). Thus, the accumulation of PrPTSE in the distal nerve stumps cannot represent a remnant from prion propagation in the distal nerve stumps occurring prior to Wallerian degeneration, but must have been sustained by neural components other than axons.
Schwann cells have been shown to express cellular PrP in murine nerves and displayed PrPTSE formation after exposure to scrapie agent in culture (Follet et al., 2002). Furthermore, Herzog et al. (2004) reported the detection of PrPTSE in Schwann cells of the sciatic nerve from primates peripherally infected with BSE agent. In the peripheral nervous system (PNS), these glial cells ensheath axonal projections as well as synaptic junctions (Fields & Stevens-Graham, 2002). Against this background, the prion propagation we observed in axon-free neural repair tissue of distal nerve stumps can plausibly be explained by an involvement of Schwann cells. However, other modes of non-axonal spread cannot be ruled out as yet and also need to be addressed in future studies. Additionally, it must be emphasized that our findings do not necessarily argue against a key role for axonal conduits for the spread of prions along neural projections. Rather, they hint at an alternative or additional mode of propagation that may operate in parts of the PNS and possibly involves Schwann cells. Evidently, the role of glial cells and other non-neuronal components in the spread of TSE agents along PNS projections requires further elucidation – for example, by studies using transgenic mice, by pharmacological approaches or by using the model system of neural conduits devoid of axons described in this report.
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ACKNOWLEDGEMENTS |
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Received 25 May 2007;
accepted 9 August 2007.
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