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. 2006 May 17;26(7-8):1203–1215. doi: 10.1007/s10571-006-9006-3

Intra- and Extraneuronal Changes of Immunofluorescence Staining for TNF- and TNFR1 in the Dorsal Root Ganglia of Rat Peripheral Neuropathic Pain Models

P Dubový 1,✉, R Jančálek 2, I Klusáková 1, I Svíženská 1, K Pejchalová 1
PMCID: PMC11520733  PMID: 16705482

Abstract

1. Several lines of evidence suggest that cytokines and their receptors are initiators of changes in the activity of dorsal root ganglia (DRG) neurons, but their cellular distribution is still very limited or controversial. Therefore, the goal of present study was to investigate immunohistochemical distribution of TNF-α and TNF receptor-1 (TNFR1) proteins in the rat DRG following three types of nerve injury.

2. The unilateral sciatic and spinal nerve ligation as well as the sciatic nerve transection were used to induce changes in the distribution of TNF-α and TNFR1 proteins. The TNF-α and TNFR1 immunofluorescence was assessed in the L4-L5 DRG affected by nerve injury for 1 and 2 weeks, and compared with the contralateral ones and those removed from naive or sham-operated rats. A part of the sections was incubated for simultaneous immunostaining for TNF-α and ED-1. The immunofluorescence brightness was measured by image analysis system (LUCIA-G v4.21) to quantify immunostaining for TNF-α and TNFR1 in the naive, ipsi- and contralateral DRG following nerve injury.

3. The ipsilateral L4-L5 DRG and their contralateral counterparts of the rats operated for nerve injury displayed an increased immunofluorescence (IF) for TNF-α and TNFR1 when compared with DRG harvested from naive or sham-operated rats. The TNFα IF was increased bilaterally in the satellite glial cells (SGC) and contralaterally in the neuronal nuclei following sciatic and spinal nerve ligature. The neuronal bodies and their SGC exhibited bilaterally enhanced IF for TNF-α after sciatic nerve transection for 1 and 2 weeks. In addition, the affected DRG were invaded by ED-1 positive macrophages which displayed simultaneously TNFα IF. The ED-1 positive macrophages were frequently located near the neuronal bodies to occupy a position of the satellites.

4. The sciatic and spinal nerve ligature resulted in an increased TNFR1 IF in the neuronal bodies of both ipsi- and contralateral DRG. The sciatic nerve ligature for 1 week induced a rise in TNFR1 IF in the contralateral DRG neurons and their SGC to a higher level than in the ipsilateral ones. In contrast, the sciatic nerve ligature for 2 weeks caused a similar increase of TNFR1 IF in the neurons and their SGC of both ipsi- and contralateral DRG. The spinal nerve ligature or sciatic nerve transection resulted in an increased TNFR1 IF located at the surface of the ipsilateral DRG neurons, but dispersed IF in the contralateral ones. In addition, the SGC of the contralateral in contrast to ipsilateral DRG displayed a higher TNFR1 IF.

5. Our results suggest more sources of TNF-α protein in the ipsilateral and contralateral DRG following unilateral nerve injury including macrophages, SGC and primary sensory neurons. In addition, the SGC and macrophages, which became to be satellites, are well positioned to regulate activity of the DRG neurons by production of TNF-α molecules. Moreover, the different cellular distribution of TNFR1 in the ipsi- and contralateral DRG may reflect different pathways by which TNF-α effect on the primary sensory neurons can be mediated following nerve injury.

KEY WORDS: proinflammatory cytokines, unilateral nerve injury, bilateral reaction, primary sensory neurons, satellite glial cells

INTRODUCTION

Neuropathic pain is defined as pain initiated or caused by primary lesions or dysfunction in the nervous system. The peripheral neuropathic pain is usually persistent, and nerve injury can produce sensory/motor deficits and other paradoxical sensations of a qualitative nature, such as hyperesthesias, paresthesias and dysesthesias (Zimmermann, 2001).

An injury of the peripheral nerves is related with cellular and molecular changes in the peripheral nervous system, spinal cord and brain. There is compelling evidence demonstrating that hyperalgesia, allodynia, and ongoing pain associated with peripheral nerve injury reflect, at least in part, changes in the excitability of primary afferent neurons (Suzuki and Dickenson, 2000; Woolf and Salter, 2000; Zimmermann, 2001). It means that the primary sensory neurons of the dorsal root ganglia (DRG) play a key role in neuropathic hypersensibility (Woolf and Mannion, 1999).

Several lines of evidence suggest that pro- and anti-inflammatory cytokines contribute to both the induction and maintenance of neuropathic pain derived from cellular and molecular changes in the DRG including the activity of the primary sensory neurons (Wagner and Myers, 1996a,b; Sommer and Schafers, 1998; Sommer et al., 1998; Schafers et al., 2003a; Svensson et al., 2005). A damage of the peripheral nerve or lipopolysaccharide (LPS) injection may stimulate an increase of tumor necrosis factor-α (TNF-α) mRNA and TNF-α protein in the DRG (Murphy et al., 1995; Schafers et al., 2003c; Li et al., 2004), but the cellular distribution of the protein is still very limited or controversial.

Therefore, the goal of present study was to demonstrate immunohistochemical distribution of TNF-α and TNF receptor-1 (TNFR1) proteins in the DRG following three types of nerve injury. The changes of TNF-α and TNFR1 immunofluorescence in the DRG affected by unilateral nerve injury were compared with the contralateral ones removed from operated animals as well as with those removed from naive rats.

MATERIAL AND METHODS

Animals and Surgical Procedures

Thirty two female Wistar rats weighing 200–250 g at the time of testing were maintained in a climate-controlled room on a 12-h light/dark cycle with food and water available ad libitum. All of the handling of the animals and testing was performed in accordance with the policies and recommendations of the International Association for the Study of Pain (Zimmermann, 1983) and received approval from the Ethical Committee of the Faculty of Medicine, Brno, Czech Republic.

The rats were randomly divided into three experimental groups (n = 8), four rats without surgery were used for naive DRG control, and four sham control rats underwent the same operation and handling as the experimental animals but without nerve ligature or transection.

For surgical procedures, rats were routinely anaesthetized with 0.3 ml of a mixture of equal volumes of ketamine (100 mg/ml) and xylazine (20 mg/ml) administered intraperitoneally.

All surgical procedures were performed aseptically under an operating microscope. The right sciatic nerve was exposed in the midthigh and four loose ligatures (2-0, Ethicon) were applied at a 40 mm distance from corresponding L4-L5 DRG (group I, n = 8). At the same position the right sciatic nerve (group II) was simply ligated (2-0, Ethicon), cut distally by sharp scissors and the proximal stump was turned to prevent a reinnervation.

The right spinal nerves (L4-L5) were simply ligated (6-0, Ethicon) at a 4–6 mm distance from corresponding DRG in the rats of the experimental group III (n = 8).

All experimental and sham-operated animals were left to survive for 7 and 14 days.

Sections and Immunohistochemical Staining

For DRG harvesting, the rats were euthanized with a lethal dose of sodium pentobarbital (80 mg/kg body weight) and perfused transcardially, first with heparinized (1000 units/500 ml) phosphate-buffered saline (PBS, 10 mM sodium phosphate buffer, pH 7.4, containing 0.15 M NaCl) followed by 500 ml of Zamboni's fixative (Zamboni and deMartino, 1967).

The L4-L5 DRG were detected within their intervertebral foramina following total laminectomy and foraminotomy. Both ipsilateral and contralateral L4-L5 DRG were removed and immersed separately in Zamboni's fixative at 4°C overnight. The same pairs of DRG were also extracted from sham-operated and unoperated (naïve) rats.

Longitudinal frozen sections (12 μm) of naive, ipsilateral and contralateral L4-L5 DRG were cut in a crystat (Leica 1800), collected on gelatin-chromalum-coated slides, and washed with PBS containing 0.1% TWEEN 20 and 1% bovine serum albumin (PBS- TWEEN) for 10 min. After being treated with 5% normal donkey serum, the sections were incubated with rabbit polyclonal anti-TNF-α (Chemicon, 1:200) or mouse monoclonal anti-TNFR1 (H-5, Santa Cruz, 1:100) antibodies in humid chamber at room temperature (21–23°C) for 4 h. The specificity of antibodies was tested by preabsorption with recombinant rat TNF-α (Chemicon GF046, data not shown) or it was well characterized (TNFR1; Wesemann and Benveniste, 2003).

The immunoreaction was visualized by treatment with TRITC-conjugated and affinity purified donkey anti-mouse (-rabbit) secondary antibodies (1:100) for 90 min at room temperature. The control sections for immunofluorescence were incubated with omission of the primary antibody.

A part of the sections was incubated with a mixture (1:1) of mouse monoclonal anti-ED-1 (1:100; Serotec MCA341R) and rabbit polyclonal anti-TNF-α (1:200; Chemicon) antibodies with final dilution of single immunostaining. A mixture (1:1) of affinity purified TRITC-conjugated donkey anti-rabbit and FITC-conjugated donkey anti-mouse secondary antibodies were applied at a final dilution of 1:100 at room temperature for 90 min. The control sections were incubated with omission of the primary antibodies.

Immunostained sections were rinsed in PBS-TWEEN, counterstained with Hoechst 33342 to detect position of the satellite cell nuclei and mounted in a Vectashield aqueous mounting medium (Vector Laboratories Inc, Burlingame, CA).

Light Microscopy and Computer-Assisted Image Analysis

The immunostained sections were observed and analyzed by a Leica DMLB epifluorescence microscope equipped with appropriate filter combinations and HCX PL Fluotar objectives (Leica Microsystems Wetzlar GmbH, Germany), and a stabilized power supply of the lamp house. The images were captured under the same magnification with a Leica DFC-480 camera in the RGB mode. All images were acquired with identical camera settings, optics, lamp and the same exposure time, and stored in BMP format.

The measurement of immunofluorescence brightness was performed in ten randomly selected sections of the naive, ipsi- and contralateral DRG using a LUCIA-G v 4.21 image analysis system (Laboratory Imaging Ltd., Prague, Czech Republic) according to our protocol (Dubovy et al., 2002). The binary mask was obtained by thresholding technique and manual editing to select the structure for measurement. All image analysis was performed in duplicate by two investigators who were unaware of the samples; there was a high level of agreement between their reports.

The immunofluorescence intensity for investigated molecules was expressed as mean of brightness±SD. To verify differences a Mann–Whitney U-test was applied using STATISTICA 5.5 software (StatSoft, Tulsa, OK, USA) with p < 0.05 as level of significant differences between the tested samples.

RESULTS

Immunostaining for TNF-α

The intact (naive) L4-L5 DRG or those from sham-operated rats exhibited a very low level of immunofluorescence for TNF-α in the neuronal bodies and their satellite glial cells (Fig. 1A). Control sections of both ipsi- (Fig. 1B) and contralateral DRG treated for complete immunohistochemical procedure, but with omission of primary antibody displayed no immunofluorescence.

Fig. 1.

Fig. 1.

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Cryostat sections through L4 DRG from naive rat (A), from rat operated for unilateral spinal nerve ligature for 2 weeks (C, D), for unilateral sciatic nerve transection for 1 week (B, E, F) and 2 weeks (G, H). The sections were incubated for detection of TNF-α using indirect immunofluorescence method under the same conditions. The control section was incubated with omission of the primary antibody (B). Arrows indicate immunofluorescence staining in the satellite cells (C–H); arrowheads indicate position of the neuronal nuclei with or without TNF-α immunofluorescence (C, D). Scale bars = 60 μm for A–H; 30 μm for insertions.

The sciatic and spinal nerve ligature for 1 or 2 weeks induced a very similar pattern of TNF-α immunoreactivity, therefore it is described together. The peripheral nerve ligature resulted in an increased TNF-α immunofluorescence in the satellite glial cells of both ipsi- and contralateral DRG (Fig. 1C and D; Table I). A slight elevation of immunostaining was detected in the cytoplasm of the neuronal bodies, but with no significance when compared with the naive DRG (Table I). In addition, the neuronal nuclei were immunostained for TNF-α in the contralateral in contrast to ipsilateral DRG (Fig. 1C and D and their insertions). The nuclear TNF-α immunoreaction was found in the large- and medium-sized DRG neurons (from 42–32 μm in diameter). No other distinct differences of the immunofluorescence intensity were found between ipsi-and contralateral DRG sections (Table I).

Table I.

Mean Brightness±SD of Immunofluorescence Labeling for TNF-α in the Neurons (N), Neuronal Nuclei (Ncl) and Satellite Glial Cells (SGC) of the Naive DRG as well as ipsi- and Contralateral DRG Following Sciatic and Spinal Nerve Ligature (ScN-lig, SpN-lig) and Sciatic Nerve Transection (ScN-T) for 1 and 2 Weeks (1W, 2W)

Ipsilateral DRG Contralateral DRG
N Ncl SGC N Ncl SGC
Naive DRG 48.8±5.1 38.4±3.1 49.2±3.8 48.8±5.1 38.4±3.1 49.2±3.8
ScN-lig-1W 49.4±5.2 40.3±4.1 61.3±3.6a 49.9±3.8 59.3±4.1a ,b 63.1±3.6a
ScN-lig-2W 50.9±4.5 40.8±4.2 63.5±4.1a 51.3±3.7 58.8±4.2a ,b 62.5±4.5a
SpN-lig-1W 50.4±3.8 39.3±3.8 68.5±4.2a 51.5±3.6 58.3±3.8a ,b 65.1±3.2a
SpN-lig-2W 51.8±6.4 41.2±3.0 64.4±3.8a 50.4±4.7 62.2±5.8a ,b 64.1±4.8a
ScN-T-1W 62.4±9.0a – 65.0±5.7a 78.6±7.9a ,b – 86.9±6.2a ,b
ScN-T-2W 53.4±6.5 – 57.8±4.7a 66.3±3.9a ,b – 76.5±5.4a ,b
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aIndicates significant differences (p < 0.05) when compared the naive DRG and ipsilateral or contralateral DRG following nerve ligature or transection.

bIndicates significant differences (p < 0.05) when compared the ipsilateral and contralateral DRG following nerve ligature or transection.

In comparison with naive DRG, the immunofluorescence staining was significantly increased in the neuronal bodies and satellite glial cells of both ipsilateral and contralateral L4-L5 DRG following sciatic nerve transection. After one week sciatic nerve transection, the TNF-α immunofluorescence was increased in the neuronal bodies of all-sized categories in both ipsi- and contralateral DRG, but a higher intensity was found in the DRG neurons and their satellite glial cells from contralateral than ipsilateral side (Fig. 1E and F; Table I). Two weeks after sciatic nerve transection, a lower level of TNF-α immunofluorescence was observed in the neuronal bodies and their satellite glial cells in comparison with one week transection. Generally, a higher intensity of immunostaining for TNF-α remained in the neuronal bodies and their satellite glial cells from contralateral than ipsilateral DRG (Fig. 1G and H; Table I).

A high amount of ED1+ macrophages invaded the perineuronal spaces of the ipsilateral DRG following both nerve ligature and transection. The ED-1 macrophages and their cytoplasmic processes were also frequently located in a close relation to neuronal bodies to become their satellites (Fig. 2A). Most of perineuronal ED1+ macrophages displayed simultaneously immunoreaction for TNF-α (Fig. 2B).

Fig. 2.

Fig. 2.

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A cryostat section through L4 DRG from rat operated for unilateral sciatic nerve ligature for 2 weeks simultaneously immunostained for ED-1 (TRITC) and TNF-α (FITC). The ED-1 positive macrophages located near the neuronal body to become its satellites displayed simultaneously TNF-α immunoreaction. Scale bars = 15 μm.

Immunostaining for TNFR1

No distinct immunofluorescence for TNFR1 was found in the DRG neurons of the naive and sham-operated rats. A nuclear localization of a faint immunoreaction was only observed in a very few neurons (Fig. 3A). No immunofluorescence for TNFR1 was found in control sections of both ipsi- and contralateral DRG incubated with omission of primary antibody (Fig. 3B).

Fig. 3.

Fig. 3.

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Cryostat sections through L4 DRG from naive rat (A), from rat operated for unilateral sciatic nerve ligature for 1 (C, D) and 2 weeks (B, E, F), and from rat with unilateral spinal nerve ligature for 2 weeks (G, H) and sciatic nerve transection for 2 weeks (I, J). The sections were incubated for detection of TNFR1 using indirect immunofluorescence method under the same conditions. A nuclear localization of a faint TNFR1 immunoreaction (opened arrow) was only observed in a very few neurons of the naive DRG (A). The control section was incubated with omission of the primary antibody (B). Arrows indicate immunopositivity in the satellite cells (D–F, H, J); arrowheads show TNFR1 immunostaining at the surface of neurons (G–I). Scale bars = 60 μm; 38 μm for insertions.

The sciatic nerve ligature increased the immunofluorescence in the neuronal bodies and their satellite glial cells. A higher intensity of diffused immunofluorescence was observed in all-sized neuronal bodies of the contralateral than ipsilateral DRG after one week ligature. In addition, the satellite glial cells of contralateral DRG displayed a distinct immunostaining for TNFR1 than those of ipsilateral DRG (Fig. 3C and D; Table II). A strong immunofluorescence for TNFR1 of similar intensity was found in the neuronal bodies of both ipsi- and contralateral DRG 2 weeks after sciatic nerve ligature. The TNFR1 immunofluorescence of a higher intensity was observed in the satellite glial cells of the contralateral than ipsilateral DRG (Fig. 3E and F; Table II).

Table II.

Mean Brightness±SD of Immunofluorescence Labeling for TNFR1 in the Neurons (N) and Satellite Glial Cells (SGC) of the Naive DRG as well as ipsi- and Contralateral DRG 1 and 2 Weeks (1W, 2W) after Sciatic Nerve Ligature (ScN-lig)

Ipsilateral DRG Contralateral DRG
N SGC N SGC
Naive DRG 46.2±3.8 45.8±3.1 46.2±3.8 45.8±3.1
ScN-lig-1W 47.4±3.9 45.5±4.6 59.3±3.6a ,b 78.1±3.2a ,b
ScN-lig-2W 59.8±4.4a 57.4±4.7a 60.2±5.8a ,b 76.3±4.8a ,b
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aIndicates significant differences (p < 0.05) when compared the naive DRG and ipsilateral or contralateral DRG following sciatic nerve ligature.

bIndicates significant differences (p < 0.05) when compared the ipsilateral and contralateral DRG following sciatic nerve ligature.

The unilateral spinal nerve ligature resulted in an increased immunostaining for TNFR1 dispersed in the neurons of both ipsilateral and contralateral DRG. In addition, ipsilateral DRG displayed a strong TNFR1 immunoreaction at the surface of neuronal bodies (Fig. 3G) that could be identified by position of the satellite glial cell nuclei counterstained with Hoechst. In contrast to the distinct immunofluorescence located at the surface of ipsilateral DRG neurons, the TNFR1 immunostaining was only dispersed in the contralateral ones (Fig. 3H). One or two weeks following sciatic nerve transection, an immunofluorescence for TNFR1 was dispersed in the neurons and located at the neuronal surface of the ipsilateral DRG (Fig. 3I) as was indicated by the nuclei of satellite cells. In contrast to DRG removed from ipsilateral side, the DRG contralateral to the spinal nerve ligature or sciatic nerve transection displayed a strong immunofluorescence for TNFR1 in the satellite glial cells (Fig. 3H and J).

DISCUSSION

A role of inflammation in initiating and augmenting neuropathic pain has been appreciated by clinical and experimental results (Shafer et al., 1994; Watkins et al., 1995; Michaelis et al., 1998; Cui et al., 2000; DeLeo and Yezierski, 2001). The peripheral neuropathic pain has been investigated using rat peripheral nerve injury models such as chronic constriction injury (CCI) of the sciatic nerve (Bennett and Xie, 1988), partial sciatic nerve ligation (PSNL) (Seltzer et al., 1990), and L4 and L5 spinal nerve ligation (SNL) (Kim and Chung, 1992). A periaxonal inflammation by chromic gut itself used for the CCI model of neuropathic pain was clearly demonstrated by Clatworthy et al. (1995). Therefore, we used only a model of partial unilateral sciatic and spinal nerve ligation by 2-0 or 6-0 sterilized thread (Ethicon) applied under aseptic conditions to study changes of immunofluorescence staining for TNF-α and TNFR1 proteins in the ipsilateral and contralateral DRG induced by direct traumatic injury to the nerve trunk. The sciatic nerve transection was used to compare the distribution of TNF-α and TNFR1 immunoreaction following partial axonal injury with total axotomy.

TNF-α is a pleiotropic proinflammatory cytokine that is a principal modulator of the early degenerative changes in peripheral nerve injury (Myers et al., 1999). The TNF-α molecules are produced during Wallerian degeneration (Wagner and Myers, 1996b; Sommer and Schafers, 1998) and contribute to both inflammatory (Woolf et al., 1997) and neuropathic hyperalgesia (Watkins et al., 1994; Wagner and Myers, 1996a; Sorkin and Doom, 2000; Sweitzer et al., 2001).

A very low level of immunofluorescence for TNF-α was found in the L4-L5 DRG removed from naive and sham-operated rats, but increased immunoreactivity was observed in both ipsilateral and contralateral L4-L5 DRG of the rats operated for experimental nerve injury. This is in accordance with results of Sommer and Schafers (1998) demonstrating very low level of TNF-α protein in the peripheral nerve and DRG of the intact (naive) rats. However, in contrast to results of Schafers and co-workers (Schafers et al., 2002, 2003a), we did not find a significant increase of TNF-α immunofluorescence in the DRG neurons following the nerve ligature. The sciatic and spinal nerve ligature of our experiments induced a distinct elevation of TNF-α immunofluorescence only in the satellite cells of both ipsi- and contralateral DRG as well as in some neuronal nuclei of contralateral DRG. In addition to the satellite cells, the DRG neurons, mainly those in contralateral side, displayed an increased immunofluorescence for TNF-α after the sciatic nerve transection. An increased immunostaining for TNF-α in the neurons of both ipsi- and contralateral L4-L5 DRG induced by the nerve transection is consistent with up-regulation of TNF-α in the injured and contralateral intact sciatic nerve (Ruohonen et al., 2002). Moreover, the sciatic nerve transection and subsequent repair enhanced cytokine expression (IL-1ß, TGF-ß1) in the contralateral DRG and promoted contralateral nerve regeneration in vivo by shortening the initial delay (Ryoke et al., 2000).

The results suggest that the primary sensory neurons are capable of TNF-α synthesis themselves. This is supported by a possibility of anterograde transport of the molecules from rat DRG to spinal cord and injured sciatic nerve (Schafers et al., 2002; Shubayev and Myers, 2002). In contrast to the neuronal localization of TNF-α, only non-neuronal immunostaining for TNF-α was observed in the rat DRG paraffin sections within 6 h after intraperitoneal injection of LPS (Li et al., 2004). However, usefulness of the paraffin sections for immunofluorescence detection of the molecules is not generally accepted. Therefore, the use of paraffin sections is probably reason for the discrepancy between no or background immunostaining for TNF-α in the neuronal bodies (Li et al., 2004) and our findings of intraneuronal TNF-α immunofluorescence following unilateral nerve ligature and mainly transection. In addition, no in situ hybridization signals suggested that TNF-α molecules are not synthesized in the DRG neurons following injection of LPS (Li et al., 2004), but the results did not exclude an axon injury-induced synthesis of the molecules in the DRG neurons.

In comparison to naive DRG, an increased TNF-α immunoreactivity was also found in the satellite glial cells of both ipsilateral and contralateral DRG following unilateral nerve ligation or transection. Many of the TNF-α positive cells were dispersed in the interstitial DRG spaces as well as located near the neuronal bodies to occupy a certain position of satellite cells. The peripheral nerve injury induces cellular and molecular changes in the affected DRG including a massive invasion of macrophages (Hu and McLachlan, 2002) which are able to produce TNF-α and other interleukins (Schnell et al., 1999). Many perineuronal cells exhibiting TNF-α immunoreactivity are suggested to be ED-1 positive macrophages (see Fig. 2A and B) that occupy a position of the satellites. Thus, the original satellite glial cells and macrophages which became to be satellites of the primary sensory neurons are well positioned to regulate activity of the DRG neurons by production of TNF-α molecules. There is conclusive evidence that TNF-α is involved in ectopic changes of DRG neurons inducing the neuropathic pain (Sorkin et al., 1997; Zhang et al., 2002).

The TNF-α molecules are synthesized and released by many cells found in the peripheral nervous system including macrophages, mast cells, Schwann cells, fibroblasts and endothelial cells in response to inflammation, tissue injury and immunological reactions (Dinarello, 1994; Wagner and Myers, 1996b). The results of our immunohistochemical detection support more sources of TNF-α protein in the ipsilateral and contralateral DRG following unilateral nerve injury. However, it remains uncertain whether the critical source of TNF-α increasing the excitability of DRG neurons is released by activated satellite glial cells, macrophages recruited to the affected DRG or by the neurons themselves.

TNF-α exerts its effect through two known receptors, the TNF receptor 1 (TNFR1) and the TNF receptor 2 (TNFR2). These receptors are present in both neurons and glia and have distinct ligand-binding affinities and elicit different physiological functions (Vandenabeele et al., 1995). The effects associated with experimental hyperalgesia have been shown to be dependent on TNFR1 (Sommer et al., 1998), which is in line with an up-regulation of TNFR1 following experimental nerve lesion (Schafers et al., 2003b).

The spinal nerve ligature induces transient TNFR1 increase of immunoreactivity in the DRG neurons 6 and 120 h after operation (Schafers et al., 2003b). Our results indicate that increased immunoreactivity for TNFR1 is present in the DRG neurons up to 2 weeks following the sciatic nerve ligature. In contrast to the sciatic nerve ligature, the ligation of spinal nerve, i.e., near to DRG as well as the sciatic nerve transection induced an intense TNFR1 immunofluorescence at the surface of ipsilateral DRG neurons. In contrast, the contralateral DRG displayed an increased TNFR1 immunostaining only in the satellite glial cells. The results suggest that cellular distribution of TNFR1 in the DRG is altered in relation to type of nerve injury and time of survival. The different cellular distribution of TNFR1 in the ipsi- (neurons) and contralateral DRG (satellite glial cells) is very similar to localization of EGF receptor (Xian and Zhou, 1999) and may reflect various pathways by which TNF-α influence on the primary sensory neurons can be mediated.

In contrast to naive DRG, an increased TNF-α and TNFR1 immunofluorescence was observed in both ipsi- and contralateral DRG at least 2 weeks after unilateral nerve ligature or transection. There is conclusive evidence that unilateral peripheral nerve lesions also affect the contralateral DRG (Koltzenburg et al., 1999; Kleinschnitz et al., 2005; Dubovy et al., 2005). Our results of immunostaining for TNF-α and TNFR1 proteins suggest that cytokines and their receptors may play significant roles in molecular and cellular events in the contralateral DRG following unilateral nerve injury. However, cues to mechanisms triggering the molecular changes in the DRG contralateral to nerve injury remain to be elucidated.

ACKNOWLEDGMENTS

We thank Ms. Dana Kutějová, Mrs. Jitka Pokorná, and Ms. Stana Bartová for their skilful technical assistance. This work was supported by grants GAČR 309/03/1199 and MSM0021622404.

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