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Figure 2. Number of L4 DRG neurones expressing 2A AR-IR in 8 unoperated animals (Control) or after injection into the ipsilateral hindpaw of the inflammatory agents Freund's complete adjuvant (n = 4), carrageenan (Carrag., n = 4) and formalin (n = 4). Data are means per 10 sections ± S.D.

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Figure 3. Number of L4 DRG neurones expressing 2C-AR-IR in unoperated animals (n = 8) or after partial (n = 7) or complete transection (n = 11) of the sciatic nerve Data are shown as means per 10 sections ± S.D. No change of 2C-AR expression was noted after injection of identical inflammatory agents as in Fig. 2.

Figure 4A and B and Table 1 show that after a partial transection of the sciatic nerve (n = 7) significantly more (>6-fold, P < 0·05) ipsilateral L₄ and L₅ DRG neurones demonstrated _2A-AR-IR than in normal or equivalent contralateral ganglia. Complete transection of the sciatic nerve resulted in similar marked increases in the number of neurones demonstrating _2A-positive IR in L₄ and L₅ ganglia of the lesioned side relative to normal ganglia (Fig. 5A and B and Table 1). Some ganglia contralateral to the sciatic lesions had apparent small increases in numbers of _2A-AR-IR neurones (Table 1) compared with normal animals but the variation from animal to animal was large enough for these differences to have occurred by chance (P > 0·05). To estimate the proportion of neurones in a ganglion expressing _2A-AR-IR, 500 L₄ neurones were counted in 10 sections spaced by 100 µm. After a partial sciatic transection, 14·8 % of this count exhibited _2A-AR-IR. After complete sciatic transection in another animal, a somewhat larger proportion, 21·1 % of 500 neurones, showed _2A-AR-IR. Considerable variation occurred from animal to animal as is documented by the range of values in the legend to Table 1, although even the smallest number of labelled neurones in the experimental group was substantially greater than was found in normal ganglia. Few _2A-labelled neurones (<20 neurones per 10 sections) were found in adjacent segments (L₁-L₃, L₆) after sciatic lesions. Seventy-two days after complete sciatic nerve transection (n = 2), the number of _2A-AR-IR neurones in ipsilateral L₄ and L₅ DRG did not significantly differ from findings in unoperated controls (<20 cells per 10 sections, P > 0·05).

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Figure 4. Diameter distribution of DRG neurones expressing 2A-AR-IR after partial transection of the sciatic nerve 7-14 days previously Data are means per 10 sections ± S.D. (n = 7). Filled bars, ipsilateral to lesion; hatched bars, contralateral to lesion. A, L4 DRG; B, L5 DRG. See Methods for additional details.

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Figure 5. Diameter distribution of DRG neurones expressing 2A-AR-IR after complete transection of the sciatic nerve 7-14 days previously Data are means per 10 sections ± S.D. (n = 11 animals). Filled bars, ipsilateral to the lesion; hatched bars, contralateral. A, L4 DRG; B, L5 DRG.

In contrast to the _2A observations, after partial or complete sciatic transection no increase appeared in the number of ipsilateral neurones labelled by _2C-AR-IR (Fig. 3 and Table 1).

Only limited and scattered _2A-AR-IR appeared in the control spinal dorsal horn. No obvious increase in _2A-AR-IR was noted with the antibody used in the ipsilateral L₄/L₅ spinal dorsal horn after nerve injury. Spinal cord _2C-AR-IR also did not increase after either complete or partial sciatic transections.

After the sciatic nerve lesions, all diameter ranges of ipsilateral L₄ DRG neurones expressing _2A-ARs increased in number, although the largest proportionate increase usually occurred in the medium-large diameter category (31-40 µm). After partial transection, 41·5 % of labelled L₄ neurones were in the 31-40 µm category, with a mean diameter of 34 µm. Following complete sciatic transection 40·3 % of the labelled L₄ cells were in the 31-40 µm range (mean diameter of 36 µm). This distribution contrasts with the diameter spectra of neuronal somata of comparable DRG from normal rat in which notably smaller cells predominate (Harper & Lawson, 1985; Tandrup, 1993). On the other hand, in mean values from L₅ ganglia after partial lesions, the greatest number of _2A-AR-IR appeared in the 21-30 µm group, although the size distribution of labelled neurones remained skewed toward the larger diameters relative to that of the DRG population as a whole (Fig. 4). Counting only neurones in histological sections with nuclei theoretically creates a bias in favour of large neurones (Harper & Lawson, 1985; Tandrup, 1993); however, this shift should account for only a small part of these distributions.

_2A-AR-IR and evidence of injury

An indication of which DRG neurones in the L₄/L₅ ganglia had divided peripheral fibres after complete sciatic transections was gained from experiments with Fluoro-Gold (FG). Seven to 14 days after complete sciatic nerve transection, the number of DRG neurones that were labelled by both _2A-AR-IR and FG (72 neurones per 10 sections) was significantly greater (P < 0·05) than the number of _2A-AR-IR-positive cells in control or contralateral ganglia (Fig. 6A); however, more neurones showed only _2A-AR-IR than those that were double-labelled (P < 0·05, Fig. 6A). About 25 % of FG-containing neurones were _2A-AR-IR positive. For both FG-positive and FG-negative subsets most of the _2A-AR-IR-positive neurones were of the medium- large diameter category (31-40 µm).

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Figure 6. Diameter distribution of DRG neurones expressing 2A-AR-IR in relation to markers for transection or injury of peripheral fibres A, mean number (± S.D.) of 2A-AR-IR neurones in 10 sections of the ipsilateral L4 DRG from 5 animals after complete sciatic nerve transection 7-14 days previously. Filled bars, 2A-AR-IR only; hatched bars, 2A-AR-IR and retrogradely transported Fluoro-Gold (FG). B, mean number (± S.D.) of 2A-AR-IR neurones in 10 sections of the ipsilateral L4 DRG from 6 animals after partial transection of the ipsilateral sciatic nerve. Filled bars, 2A-AR-IR only; hatched bars, 2A-AR-IR and c-jun-IR. See Methods and Results for additional details.

Transection of a peripheral nerve results in a massive increase in the number of DRG neurones expressing the c-jun proto-oncogene from the very low level present in the absence of injury (Jenkins & Hunt, 1991). The c-jun expression may be due to alterations in growth or neurotropic factors (Jenkins et al. 1993). The increase in c-jun labelling continues until regeneration of the nerve is complete (Herdegen et al. 1993). We used immunoreactivity to detect the presence of c-jun protein and thereby to identify neurones with peripheral fibres putatively transected or otherwise injured by the nerve lesion. The proportion of neurones in an L₄ DRG exhibiting c-jun-IR after the complete sciatic nerve transections (36 %) was roughly comparable to those marked by FG in similar experiments (28 %; difference P > 0·05). The number of neurones in the ipsilateral L₄ DRG expressing the _2A-AR-IR alone or in combination with c-jun-IR increased significantly relative to the contralateral side (P < 0·05); however, as with the FG marker, the profiles with only _2A-AR-IR (presumably uninjured) were more numerous (Fig. 6B). As depicted in Fig. 7, after partial or complete sciatic transection, the c-jun protein appeared in more small diameter than large diameter DRG neurones (44 % in the smallest diameter category, 10-20 µm), as might be anticipated from the relative proportion of small neurones in DRG. The distribution of injury marked cells in Fig. 7 contrasts with the size distribution of the largest category staining with both labels (e.g. Fig. 6), those of medium-large diameter (31-40 µm). Nonetheless, some smaller diameter neurones (21-30 µm) had both _2A-AR-IR and c-jun-IR. It is noteworthy that not all FG or c-jun-labelled neurones showed _2A-AR-IR. While there is no certainty that either the retrograde and the c-jun tags label all and only injured neurones, the similarity of results with the two techniques suggests that after sciatic lesions more uninjured neurones than injured ones develop _2A-AR-IR.

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Figure 7. Diameter distribution of neurones displaying c-jun-IR after nerve injury Note that the ordinate is the proportion (percentage) of the total counted in L4 ganglia (± S.D.). A, 788 counted positive neurones from 2 animals following complete ipsilateral sciatic transection. B, 658 counted positive neurones from 2 animals after partial ipsilateral sciatic transection.

Effects of local inflammation

The three agents injected into the plantar footpad to induce local inflammation - carrageenan, formalin and Freund's adjuvant - produced variable degrees of oedema and guarding of the hindlimb. Formalin and Freund's adjuvant caused the most marked local reactions. After induction of the local inflammation, the number of neurones labelled by either the _2A or the _2C antibodies in the L₄/L₅ ganglia did not significantly differ from controls (P > 0·05) either ipsilaterally or contralaterally to the injected paw (Table 1 and Fig. 2).

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

Following partial and complete transection of the sciatic nerve, the antibody we used against the _2A adrenergic receptor fragmant labels a sharply increased number of DRG neurones. The increase is confined to ipsilateral ganglia supplying large numbers of afferent fibres to the injured nerve (L₄-L₅). The proportion of _2A-AR-IR neurones in ipsilateral ganglia adjacent to L₄ and L₅ remains comparable to that of control animals or contralateral ganglia. On the other hand, we noted no increase in the number of neurones expressing _2C-AR-IR after the sciatic injuries, an indication that the _2A-AR-IR changes were specific. Increases in _2A-AR-IR also did not appear in the inflammatory models.

There are considerable data favouring the concept that immunoreactivity to the antibody we employed marks an _2A-type of adrenergic receptor. The antibody has been extensively characterized in other studies and shown to label selectively neurones established to express the _2A-type of adrenergic receptor (Rosin et al. 1993; Talley et al. 1996). This characterization includes in situ hybridization studies demonstrating the presence of _2A-AR-like mRNA (Zeng & Lynch, 1991; Nicholas et al. 1993). However, other immunocytochemical studies analysing the presence of ₂-adrenergic receptors in DRG neurones have reported varying results, including some differing quantitatively from ours (e.g. Roberts & Connick, 1997; Gold et al. 1997). We can only speculate about the basis of these differences. In part, it could reflect differences in experimental protocols (e.g. Gold et al. 1997). These could represent different experimental procedures, varying immunocytochemical methods, cross-reactivity with other proteins and/or problems in interpretation due to high levels of background reactivity. From their published illustration, the latter could contribute to the larger number of _2A- and _2C-immunoreactive DRG neurones reported by Gold et al. (1997). We took special care to keep the background in our analyses very low. Our judgements were based on the presence of the punctate reaction product, at least partially distributed around profiles containing a nucleus. These criteria made for ready identification of positive labelling. In our experiments, the appearance of the reaction product and _2A-AR-IR staining of neurones was regularly blocked by preabsorbance with the fusion protein or omission of the primary antibody.

Uninjured as well as injured neurones participate in the increase in _2A-AR-IR, as evidenced by the absence of labels for transection/injury (presence of Fluoro-Gold and c-jun expression). Admittedly, the completeness of marking of injured neurones can be questioned. The FG marker was used only for complete transection and in this situation, it has been argued to label retrogradely most, if not all, neurones whose fibres are transected (Baranowski et al. 1992). Similarly, the c-jun protein or mRNA is reported to appear in many, if not all, injured and regrowing neurones after axotomy (Jenkins et al. 1993; Herdegren et al. 1993). While it is possible that some neurones with injured fibres were unmarked by either the FG or the c-jun technique, it is notable that substantial numbers of neurones with _2A-AR-IR but lacking the other marker appeared in both variations of the double-labelling experiments. Moreover, the distribution of sizes of neurones labelled for the c-jun protein compares well with the established distribution of neuronal size in normal ganglia but differs distinctly from the size distribution of the DRG neurones exhibiting the _2A-IR after the partial or complete transections (cf. Fig 4, Fig. 5, Fig. 6 and Fig. 7). Therefore, the conclusion that substantial numbers of uninjured neurones develop increased _2A-immunoreactivity after nerve injury seems tenable.

The proportion of neurones in a DRG exhibiting the increased _2A-AR-IR presence after sciatic transection is considerable. We estimate it to be about 15 % or more. That many neurones changing phenotype after nerve injury have the capability to produce significant changes in afferent signalling and sensory function. It is important to note that not every injured neurone exhibited _2A-AR-IR, implying a form of selectivity in the process.

A particularly puzzling part of our data is that the number of L₅ DRG neurones in seven animals showing the _2A-AR-IR after partial sciatic lesions was greater than either the mean for L₄ or L₅ after complete transection. It is possible that this apparent anomaly is the result of the large proportion of apparently uninjured neurones that develop the immunoreactivity. Whatever combination of factors links the lesion of the nerve to the change in adrenergic receptor expression, the partial lesions in our sample may have selected more of those triggering the change in the L₅ distribution. A possibility is the relative proportion of sympathetic efferent fibres divided by the lesion (Bossut et al. 1996). Perhaps interuption of both sets of fibres, primary sensory and sympathetic postganglionic, combines to influence the increased _2A-AR-IR. In any case, the number of peripheral fibres divided does not appear to be directly related to the number of DRG neurones showing increased appearance of _2A-AR-IR.

It is also unclear how the increase in adrenergic receptor expression after nerve lesions relates to reports of inflammation-induced appearance of adrenergic excitation of nociceptive afferent units. The absence of an increase in the number of DRG neurones exhibiting _2A-AR-IR (or _2C-AR-IR) in association with inflammation emphasizes differences in the factors mediating its actions and those of nerve injury.

A number of functional changes take place in primary afferent neurones after a peripheral nerve injury including the appearance of abnormal spontaneous activity (Wall & Gutnick, 1974; Devor et al. 1994) and adrenergic excitation of nociceptors (Sato & Perl, 1991; Bossut & Perl, 1995; O'Halloran & Perl, 1997). It seems reasonable to hypothesize that the increased number of DRG somata expressing an ₂-adrenergic receptor marker is related to the induction of adrenergic excitation of these afferent neurones at their peripheral terminals (Sato & Perl, 1991; O'Halloran & Perl, 1997; Abdulla & Smith, 1997). This latter is consistent with the apparent peripheral origin of some symptoms in human cases of sympathetically related neuralgias (Nathan, 1947; Wallin et al. 1976; Torebjork et al. 1995). A moderate increase was detected in the number of small diameter neurones with _2A-AR-IR. This size range of somata is likely to be associated with C fibre receptors and therefore with nociceptors (Harper & Lawson, 1985). However, the greatest increase in _2A-AR-IR appeared in the medium-large diameter group of DRG neurones, a category primarily related to myelinated fibre, low threshold mechanoreceptors (Harper & Lawson, 1985). Some types of low threshold mechanoreceptors, muscle spindles, cutaneous slowly adapting type I and Pacinian corpuscles in the normal animal manifest enhanced activity after sympathetic stimulation (e.g. Hunt, 1960; Akoev, 1981; Roberts et al. 1985; Barasi & Lynn, 1986). Our observations suggest that the number of the latter types of sensory neurones with the potential for excitement by adrenergic agents markedly increases after partial denervation and that this increase takes place in both injured and uninjured neurones. The cellular mechanisms by which activation of an ₂-adrenergic receptor leads to excitation of a neurone is not well understood. Abdulla & Smith (1997) have provided evidence indicating that after sciatic transection, nor-adrenaline acting through an ₂-adrenergic receptor suppresses N-type Ca²⁺ channel current in dissociated DRG neurones and possibly thereby reduces Ca²⁺-mediated K⁺ conductances. The latter could be expected to increase excitability of neurones. In their work noradrenaline actions were particularly evident in the smaller DRG somata. The ₂-AR-mediated effect was not found for DRG neurones dissociated from normal animals.

The development of sympathetic adrenergic excitation of nociceptors conceptually is readily linked to neuropathic pain (Sato & Perl, 1991; O'Halloran & Perl, 1997). On the other hand, a connection between a possible sympathetic excitatory action on low threshold mechanoreceptors and the symptom of pain requires a more involved explanation (e.g. Roberts, 1986). At this stage, the relationship of the increased _2A-AR presence to neuropathic states is a matter of conjecture (Devor, 1983; Sato & Perl, 1991; Perl, 1994).

Removal or loss of the peripheral sympathetic innervation has been reported to be associated with increased adrenergic receptor binding by some DRG somata (Nishiyama et al. 1993) as well as by cells in other tissues (Davies et al. 1982). The present increased expression of ₂-AR-IR corresponds in time (1-3 weeks) to expected expression of adrenergic denervation supersensitivity (Cannon & Rosenblueth, 1949). The suggestion has been made that sympathetically associated neuropathies may be related to mechanisms underlying sympathetic denervation supersensitivity (Perl, 1994). In this concept the presence of sympathetic innervation and mediators would suppress expression of the ₂-adrenergic receptors by DRG neurones.

The augmented _2A-adrenergic receptor-like expression we observed may be a factor in the increase reported in adrenergic connections from sympathetic postganglionic fibres to dorsal root ganglia after peripheral nerve injury (McLachlan et al. 1993; Chung et al. 1996). The ingrowth of fibres staining for sympathetic markers is described by McLachlan et al. (1993) to surround medium and large DRG neurones at a time (2-3 months) when our observations on two animals showed the _2A-AR-IR no longer to be elevated. Although there may be a causal relationship between these two phenomena, the nature of that linkage and the significance of the increased sympathetic innervation remains unclear.

The large number of apparently uninjured neurones developing the _2A-receptor expression suggests a possible basis could be alteration in the environment of DRG neurones. The increased adrenergic receptor expression conceivably could be triggered by processes related to growth of transected fibres or expansion of the peripheral terminal field (of uninjured fibres) following division of other peripheral nerve fibres, afferent or sympathetic. Peripheral nerve injury has been shown to result in a number of changes in DRG neurones including induction of immediate-early genes (e.g. c-jun) and trophic factors (Jenkins et al. 1993; Zhou et al. 1996). It is possible that whatever factors (e.g. leukemia inhibitory factor, Thompson & Majithia, 1998) and processes stimulate DRG neurones with transected fibres to respond with growth may spread to adjacent uninjured neurones and cause them to undergo similar alterations.

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Acknowledgements

This study was supported by grants NS10321 and NS14899 from the NINDS of the NIH. The authors gratefully acknowledge Drs D. Rosin and K. R. Lynch (USPHS grant no. DA07216; K. R. L.) for their generous gift of antibodies directed against the ₂-adrenoreceptors. We thank K. McNaughton and C. Connor for their expert technical assistance and S. Derr for her substantial help in preparation of this manuscript.

Corresponding author

E. R. Perl: Department of Cell and Molecular Physiology, CB no. 7545, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7545, USA.

Email: erp{at}med.unc.edu

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