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Wallerian degeneration after crush injury of rat sciatic nerve increases endo- and epineurial tumor necrosis factor-alpha protein
Neuroscience Letters 372 (2004) 215–219
Wallerian degeneration after crush injury of rat sciatic nerve increases endo- and epineurial tumor necrosis factor-alpha protein Annette George∗ , Achim Buehl, Claudia Sommer Neurologische Klinik der Universit¨at W¨urzburg, W¨urzburg, Germany Received 6 August 2004; received in revised form 16 September 2004; accepted 17 September 2004
Abstract The pro-inflammatory cytokine tumor necrosis factor-alpha (TNF) contributes to injury-induced peripheral nerve pathology and to the development of neuropathic pain. Here, we investigated whether TNF protein is altered at the site of crush injury of rat sciatic nerve using enzyme-linked immunoassay (ELISA) and immunohistochemistry (IHC). TNF protein levels determined by ELISA were low in nerve homogenates from naive rats. After crush injury, local TNF increased rapidly with a two-fold increase on day 0.5. TNF content remained elevated on day 3 and returned to baseline levels again by day 14 after crush. IHC revealed prominent TNF-immunoreactivity in many epineurial macrophages on days 0.5 to 3 after crush injury. These data indicate that TNF protein is early and transiently upregulated at the site of peripheral nerve trauma. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Peripheral nerve injury; Axonal damage; Macrophages; Neuropathic pain; Cytokines; Immunoassay
Peripheral nerve injury frequently leads to neuropathic pain. Recent animal studies revealed that pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF) contribute to injury-induced peripheral nerve pathology and to the development of neuropathic pain (for review see [11,13]). At the site of nerve lesion, local TNF has been consistently shown to be upregulated at the mRNA level [6,8,9,15,16]. Thus far, it is incompletely known how the injury-induced TNF mRNA changes translate to the protein level. TNF expression is regulated transcriptionally but also on a translational and posttranslational level [1]. Therefore, information on actual local tissue protein concentrations after nerve injury is essential in order to assess the role of TNF in the cytokine network during Wallerian degeneration [9]. In an in vitro study, TNF secretion increased in supernatants of injured mouse sciatic nerve segments [9]. An immunohistochemistry study (IHC) showed TNF∗ Corresponding author. Present address: Institute of Neurology, University College London, Queen Square, London WC1N 3BG, UK. Tel.: +44 20 7837 4186; fax: +44 20 7813 3107. E-mail address: [email protected] (A. George).
0304-3940/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.09.075
immunoreactivity (TNF-ir) in the rat sciatic nerve after nerve transection [14]. In chronic constriction injury (CCI), an injury model for neuropathic pain with nerve trauma plus local inflammatory reactions due to the ligatures tied around the nerve [2], local TNF protein is rapidly increased in rat sciatic nerves [3,10,12]. CCI-induced changes in TNF can be due to the local nerve trauma, or to the additional local inflammatory reactions caused by the ligatures tied around the nerve. Therefore, we asked in the present study whether local TNF protein is altered during Wallerian degeneration that is induced by crush injury. A crush-induced peripheral nerve lesion allows studying endoneurial TNF changes due to local nerve trauma only. We used enzyme-linked immunoassay (ELISA) and IHC to monitor the temporal course of local TNF protein changes during Wallerian degeneration after crush injury of rat sciatic nerve. Furthermore, we asked whether local TNF upregulation parallels the development of crush-induced neuropathic pain associated behavior. Female Sprague–Dawley rats (200–220 g; Charles-River, Germany) were used in procedures approved by the Bavarian State animal experimentation committee. Under deep barbiturate anaesthesia, crush injury was performed by pressing
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the sciatic nerve at the midthigh level with a jeweler’s forceps no. 5 for three successive periods of 10 s. Contralaterally, we performed a ‘sham-surgery’ where the sciatic nerve was mobilized but not injured. Crush-injured animals were monitored for the development of hyperalgesia to heat and of mechanical allodynia to calibrated von Frey hairs applied to the plantar surface of the operated hindpaw as previously described [5,12]. Analysis of variance with Tukey post hoc test
Fig. 1. (A) Thermal sensitivity to radiant heat after crush injury of rat sciatic nerve. The development of hyperalgesia to heat was monitored in crushinjured rats (triangles) and in naive controls (squares) (N = 7 rats per group). Withdrawal latencies (s) to a radiant heat stimulus applied to the plantar surface of the unoperated hindpaw were subtracted from the withdrawal latencies of the operated hindpaw. A positive ‘difference score’ indicated increase of the withdrawal latency after nerve injury. Thermal hypoalgesia is indicated by a significant increase of the ‘difference score’ compared to baseline values. A negative ‘difference score’ indicated reduction of the withdrawal latency after nerve injury. Thermal hyperalgesia was indicated by a significant reduction of the ‘difference score’ compared to baseline values (* P < 0.05; ANOVA with Tukey post hoc test). Note that crush injury induced a transient period of hypoalgesia to heat followed by the development of hyperalgesia to heat by day 10. (B) Mechanical sensitivity to von Frey hairs after crush injury of rat sciatic nerve. The development of mechanical allodynia to calibrated von Frey hairs was monitored in crush-injured rats (triangles) and in naive controls (squares) (N = 7 rats per group). The 50% withdrawal threshold gives the force of the von Frey hair to which an animal responds in 50% of the presentations. Mechanical allodynia was indicated by a significant reduction of the 50% withdrawal threshold of the injured hindpaw compared to baseline values. Mechanical hyposensitivity is indicated by a significant increase of the 50% withdrawal threshold of the injured hindpaw compared to baseline values (* P < 0.05; ANOVA with Tukey post hoc test). Note that crush injury induced a transient period of mechanical hyposensitivity followed by the development of mechanical allodynia by day 10.
was used to test for significant differences of the mean withdrawal thresholds or mean withdrawal latencies after crush injury when compared to baseline control values. To monitor crush injury-induced motor deficits we documented the animals’ spontaneous gait and the hindpaw posture. We tested for the ability to grasp by applying a small plastic rod to the plantar surface of the operated hindpaw in a gently lifted animal. For ELISA studies, animals were sacrificed on days 0, 0.5, 3 and 14 after surgery (n = 4 per time point; repeated twice on days 0, 0.5, 3 and 14). Sciatic nerves were removed by cutting the nerve shortly above the site of the crush lesion and 1 cm distally. Control nerves from naive rats (day 0) and nerves from the contralateral, sham-operated side were removed accordingly. Sciatic nerves were homogenized as described [3]. TNF levels were determined by the Cytoscreen Rat TNF Ultrasensitive ELISA kit (Biosourse, CA) according to the manufacturer’s instructions. This assay system detects rat TNF with a sensitivity of 0.7 pg/ml. TNF levels were expressed as pg/mg total protein. We used some aliquots from the nerve homogenates obtained in the present study for parallel measurements of interleukin-10 as reported recently [4]. For IHC studies, rat sciatic nerves were harvested on days 0, 0.5, 3 and 14 after crush (n = 3 per time point). Tissue from distal to the crush lesion was processed for IHC on 10 m frozen sections. Polyclonal antibodies (AB) against TNF (1:1000, Genzyme, R¨usselsheim, Germany) were used overnight as primary ABs; controls were performed by preabsorbing the ABs with the respective recombinant cytokine. An avidin–biotin complex (Vector) was used as a secondary system; staining was visualized with 3,3 -diaminobenzidine tetrahydrochloride. For immunofluorescence, primary ABs were the polyclonal AB to TNF and a monoclonal anti-mouse ED1 antibody (1:1000;
Fig. 2. Crush injury of rat sciatic nerve induces an early and transient increase of local TNF protein. Local content of TNF protein in rat sciatic nerve homogenates on days 0, 0.5, 3 and 14 after crush injury (filled bars) or shamsurgery (striped bars) determined by enzyme-linked immunoassay. Data are expressed as mean values (pg/mg protein ± S.D.; * P < 0.05 for crush vs. control values). Data for the control nerves (day 0) are indicated by open bars. Note that crush injury but not sham-surgery induced a significant increase of local TNF protein concentrations.
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Fig. 3. Crush injury of rat sciatic nerve increases TNF-immunoreactivity (TNF-ir) in the epi- and the endoneurial compartment. We show four representative photographs of frozen sections of rat sciatic nerves immunoreacted for TNF. After crush injury, we observed prominent TNF-ir in the epineurial (A, C) and in the endoneurial (B, D) compartment as shown for day 0.5 (A, B) and day 3 (C, D) after nerve injury. The dotted line indicates the border between the epi- and the endoneurial space. In the epineurium, TNF-ir cells are indicated by red arrows. In the endoneurium, TNF-ir cells are indicated by blue arrows. TNF-ir cells in the subperineurial space are indicated by pink arrows. Scale bar = 20 m. Note that many epineurial cells with the morphology of macrophages were TNF-ir on days 0.5 and 3 (A, C). Morphometric analysis of TNF-ir in the epineurial (E) and the endoneurial (F) compartment on days 0, 0.5, 3, and 14 after crush. The number of epineurial TNF-ir cells (E) is shown for control (open bar) and crush-injured (filled bars) nerves and expressed as mean values (number/section ± S.D.; * P < 0.05). The endoneurial TNF-ir area (F) is shown for control (open bar) and crush-injured (filled bars) nerves and expressed as mean values (m2 /nerve section ± S.D.; * P < 0.05).
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Serotec, UK). Cy3-conjugated or Cy2-conjugated secondary ABs (1:100; 2 h, Amersham Biosciences, Freiburg) were used for visualization. Nerve lesions were verified by toluidine blue staining on 1-m sections after plastic embedding. After crush injury we observed prominent TNF-immunoreactivity in both the epineurial and endoneurial compartment. This finding prompted us to perform concurrent morphometric analysis of TNF-ir after crush injury. Using morphometric analysis we asked whether endoneurial and epineurial TNFir are differentially regulated after crush injury. The nerve section area was digitized at a 1400-fold magnification with a Zeiss Axiophot 2 microscope and analyzed using Image ProPlus software. A density threshold was set above background level and the area of positively stained structures was measured on three nerves per time point by a blinded investigator and expressed as m2 /nerve section. The number of epineurial TNF-ir cells was counted per nerve section. Data represent mean values ± S.D. Analysis of variance was used to test for differences between groups and subsequent Scheff´e test for comparison of individual means throughout the time course. Statistical significance was assumed with P < 0.05. Crush injury significantly changed the thermal (Fig. 1A) and mechanical (Fig. 1B) sensitivity of the operated hindpaw.
Transient hypoalgesia to heat on day 3 was followed by the development of hyperalgesia to heat by day 10 after crush injury (Fig. 1A). Similarly, a transient period of mechanical hypoesthesia was followed by the development of mechanical allodynia by day 10 (Fig. 1B). Motor deficits after sciatic nerve crush were present from day 1 until day 10 (last time point investigated). TNF protein levels were determined by ELISA in sciatic nerve homogenates from the crush-injured (Fig. 2, filled bars) or the sham-operated (Fig. 2, striped bars) side. After crush but not sham-surgery, local TNF increased significantly on day 0.5 and day 3 by two-fold of baseline. On day 14, TNF protein levels returned to baseline values again. Concurrent IHC studies revealed that TNF-ir was prominent in many epineurial cells with the morphology of macrophages on day 0.5 and day 3 after crush (Fig. 3A and C). By day 3, TNF-ir cells with the morphology of macrophages were also observed in the perineurial and subperineurial space (Fig. 3C). Endoneurially, TNF-ir was localized in Schwann cell and macrophage like structures after crush (Fig. 3B and D). Colocalization studies on day 3 after crush injury confirmed that many epineurial ED1-positive cells indicating macrophages were immunoreactive for TNF (Fig. 4A and B),
Fig. 4. Macrophages in the epineurial and the endoneurial compartment colocalize with TNF-immunoreactivity (TNF-ir) on day 3 after crush injury of rat sciatic nerve. We show examples of the immunofluorescence colocalization study for ED-1 (A, C) and TNF-immunoreactive (B, D) cells in the epineurial (A, B) and the endoneurial (C, D) compartment on frozen sections of rat sciatic nerve harvested on day 3 after crush injury. ED-1 positive cells appear green (A, C). TNF-ir structures appear red (B, D). Structures that colocalize are indicated by an arrow. In the epineurium (A, B), colocalizing cells are indicated by red arrows. In the endoneurium (C, D), colocalizing cells are indicated by blue arrows. Colocalizing cells in the subperineurial space are indicated by a pink arrow. The dotted line indicates the border between the epi- and the endoneurial space. Scale bar = 15 m. Note that many of the epineurial ED-1 positive cells (indicating macrophages; A) colocalize with TNF-ir (B). Note that many but not all of the endoneurial TNF-ir (D) structures colocalize with ED-1 positive cells (C).
A. George et al. / Neuroscience Letters 372 (2004) 215–219
whereas many but not all macrophages in the endoneurial compartment were TNF-ir (Fig. 4C and D). In control sciatic nerve sections from naive rats, TNF-ir was occasionally found in Schwann cell like structures endoneurially but was completely absent in the epineurial and perineurial compartment (data not shown). These data indicate that TNF protein is upregulated early and transiently at the site of a crush injury. Thus, a peripheral nerve trauma with axonal damage increases the proinflammatory cytokine TNF at the protein level. An increase of local TNF protein can be due to (1) an increase of endoneurial TNF production, (2) a reduced axonal transport of TNF with local accumulation of TNF at the site of crush and (3) an invasion of cells that contain TNF protein. In the present study, we observed TNF-ir macrophages in the epineurial and perineurial space early after nerve injury (day 0.5 to day 3). This suggests that macrophages that invade the peripheral nerve at the site of lesion by day 3 are highly loaded with TNF protein. At the same time, endoneurial Schwann cells are positive for TNF. Thus, our data suggest that macrophages that are recruited during the first days of Wallerian degeneration as well as endoneurial Schwann cells contribute markedly to the early increase of local TNF levels after crush injury. The effects of TNF protein at the site of nerve lesion are incompletely understood. Our data showing an early and transient increase of TNF protein further support the assumption that TNF is crucial in the early process of Wallerian degeneration and may act as an initiator of local inflammatory responses as described recently [9]. Furthermore, the finding of many TNF-ir epineurial macrophages suggests that TNF may be essential in nerve injury-induced macrophage recruitment. Indeed, a delay of nerve injury-induced macrophage recruitment was described in TNF-deficient animals [7]. Behavioral tests in the present study revealed that crush injury of the rat sciatic nerve induces a transient hypoalgesia to heat and a transient hypoesthesia to mechanical stimuli whereas heat hyperalgesia and mechanical allodynia develop by day 10 after crush. Thus, the early and transient increase of local TNF protein may precede and parallel the development of pain-associated behavior in the crush injury model. This finding further supports the assumption that TNF is essential in the early process of neuropathic pain generation and suggests that TNF-antagonizing treatments should be given early in nerve injury to prevent the development of neuropathic pain.
Acknowledgements The expert technical help of Lydia Biko, Barbara Dekant and Helga Br¨unner is greatly appreciated. This study forms
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part of the doctoral thesis of Achim B¨uhl. This work was supported by Volkswagenstiftung.
References [1] B.B. Aggarwal, K. Natarajan, Tumor necrosis factors: developments during the last decade, Eur. Cytokine Netw. 7 (1996) 93–124. [2] G.J. Bennett, Y.K. Xie, A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man, Pain 33 (1988) 87–107. [3] A. George, C. Kleinschnitz, M. Zelenka, J. Brinkhoff, G. Stoll, C. Sommer, Wallerian degeneration after crush or chronic constriction injury of rodent sciatic nerve is associated with a depletion of endoneurial interleukin-10 protein, Exp. Neurol. 188 (2004) 187– 191. [4] A. George, C. Schmidt, A. Weishaupt, K.V. Toyka, C. Sommer, Serial determination of tumor necrosis factor alpha content in rat sciatic nerve after chronic constriction injury, Exp. Neurol. 160 (1999) 124–132. [5] K. Hargreaves, R. Dubner, F. Brown, C. Flores, J. Joris, A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia, Pain 32 (1988) 77–88. [6] M. La Fleur, J.L. Underwood, D.A. Rappolee, Z. Werb, Basement membrane and repair of injury to peripheral nerve: defining a potential role for macrophages, matrix metalloproteinases, and tissue inhibitor of metalloproteinases-1, J. Exp. Med. 184 (1996) 2311–2326. [7] M. Liefner, H. Siebert, T. Sachse, U. Michel, G. Kolllias, W. Br¨uck, The role of TNF-alpha during Wallerian degeneration, J. Neuroimmunol. 108 (2000) 147–152. [8] K. Okamoto, D.P. Martin, J.D. Schmelzer, Y. Mitsui, P.A. Low, Pro- and anti-inflammatory cytokine gene expression in rat sciatic nerve constriction injury model of neuropathic pain, Exp. Neurol. 169 (2001) 386–391. [9] S. Shamash, F. Reichert, S. Rotshenker, The cytokine network of Wallerian degeneration: tumor necrosis factor-alpha, interleukin1alpha, and interleukin-1beta, J. Neurosci. 22 (2002) 3052–3060. [10] V.I. Shubayev, R.R. Myers, Upregulation and interaction of TNFalpha and gelatinase A and B in painful peripheral nerve injury, Brain Res. 855 (2000) 83–89. [11] C. Sommer, Cytokines and neuropathic pain, in: P. Hansson, H. Fields, R. Hill, P. Marchettini (Eds.), Neuropathic Pain: Pathophysiology and Treatment, IASP Press, Seattle, 2001, pp. 37–62. [12] C. Sommer, M. Marziniak, R.R. Myers, The effect of thalidomide treatment on vascular pathology and hyperalgesia caused by chronic constrictive injury of rat sciatic nerve, Pain 74 (1998) 83–91. [13] G. Stoll, S. Jander, R.R. Myers, Degeneration and regeneration of the peripheral nervous system: from Augustus Waller’s observations to neuroinflammation, J. Periph. Nervous Syst. 7 (2002) 13–27. [14] G. Stoll, S. Jung, S. Jander, P. van der Meide, H.P. Hartung, Tumor necrosis factor-alpha in immune-mediated demyelination and Wallerian degeneration of the rat peripheral nervous system, J. Neuroimmunol. 45 (1993) 175–182. [15] H.S. Taskinen, T. Olsson, A. Bucht, M. Khademi, L. Svelander, M. Roytta, Peripheral nerve injury induces endoneurial expression of IFN-gamma, IL-10 and TNF-alpha mRNA, J. Neuroimmunol. 102 (2000) 17–25. [16] R. Wagner, R. Myers, Schwann cells produce tumor necrosis factor alpha: expression in injured and non-injured nerves, Neuroscience 73 (1996) 625–629.
Wallerian degeneration after crush injury of rat sciatic nerve increases endo- and epineurial tumor necrosis factor-alpha protein Annette George∗ , Achim Buehl, Claudia Sommer Neurologische Klinik der Universit¨at W¨urzburg, W¨urzburg, Germany Received 6 August 2004; received in revised form 16 September 2004; accepted 17 September 2004
Abstract The pro-inflammatory cytokine tumor necrosis factor-alpha (TNF) contributes to injury-induced peripheral nerve pathology and to the development of neuropathic pain. Here, we investigated whether TNF protein is altered at the site of crush injury of rat sciatic nerve using enzyme-linked immunoassay (ELISA) and immunohistochemistry (IHC). TNF protein levels determined by ELISA were low in nerve homogenates from naive rats. After crush injury, local TNF increased rapidly with a two-fold increase on day 0.5. TNF content remained elevated on day 3 and returned to baseline levels again by day 14 after crush. IHC revealed prominent TNF-immunoreactivity in many epineurial macrophages on days 0.5 to 3 after crush injury. These data indicate that TNF protein is early and transiently upregulated at the site of peripheral nerve trauma. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Peripheral nerve injury; Axonal damage; Macrophages; Neuropathic pain; Cytokines; Immunoassay
Peripheral nerve injury frequently leads to neuropathic pain. Recent animal studies revealed that pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF) contribute to injury-induced peripheral nerve pathology and to the development of neuropathic pain (for review see [11,13]). At the site of nerve lesion, local TNF has been consistently shown to be upregulated at the mRNA level [6,8,9,15,16]. Thus far, it is incompletely known how the injury-induced TNF mRNA changes translate to the protein level. TNF expression is regulated transcriptionally but also on a translational and posttranslational level [1]. Therefore, information on actual local tissue protein concentrations after nerve injury is essential in order to assess the role of TNF in the cytokine network during Wallerian degeneration [9]. In an in vitro study, TNF secretion increased in supernatants of injured mouse sciatic nerve segments [9]. An immunohistochemistry study (IHC) showed TNF∗ Corresponding author. Present address: Institute of Neurology, University College London, Queen Square, London WC1N 3BG, UK. Tel.: +44 20 7837 4186; fax: +44 20 7813 3107. E-mail address: [email protected] (A. George).
0304-3940/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.09.075
immunoreactivity (TNF-ir) in the rat sciatic nerve after nerve transection [14]. In chronic constriction injury (CCI), an injury model for neuropathic pain with nerve trauma plus local inflammatory reactions due to the ligatures tied around the nerve [2], local TNF protein is rapidly increased in rat sciatic nerves [3,10,12]. CCI-induced changes in TNF can be due to the local nerve trauma, or to the additional local inflammatory reactions caused by the ligatures tied around the nerve. Therefore, we asked in the present study whether local TNF protein is altered during Wallerian degeneration that is induced by crush injury. A crush-induced peripheral nerve lesion allows studying endoneurial TNF changes due to local nerve trauma only. We used enzyme-linked immunoassay (ELISA) and IHC to monitor the temporal course of local TNF protein changes during Wallerian degeneration after crush injury of rat sciatic nerve. Furthermore, we asked whether local TNF upregulation parallels the development of crush-induced neuropathic pain associated behavior. Female Sprague–Dawley rats (200–220 g; Charles-River, Germany) were used in procedures approved by the Bavarian State animal experimentation committee. Under deep barbiturate anaesthesia, crush injury was performed by pressing
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the sciatic nerve at the midthigh level with a jeweler’s forceps no. 5 for three successive periods of 10 s. Contralaterally, we performed a ‘sham-surgery’ where the sciatic nerve was mobilized but not injured. Crush-injured animals were monitored for the development of hyperalgesia to heat and of mechanical allodynia to calibrated von Frey hairs applied to the plantar surface of the operated hindpaw as previously described [5,12]. Analysis of variance with Tukey post hoc test
Fig. 1. (A) Thermal sensitivity to radiant heat after crush injury of rat sciatic nerve. The development of hyperalgesia to heat was monitored in crushinjured rats (triangles) and in naive controls (squares) (N = 7 rats per group). Withdrawal latencies (s) to a radiant heat stimulus applied to the plantar surface of the unoperated hindpaw were subtracted from the withdrawal latencies of the operated hindpaw. A positive ‘difference score’ indicated increase of the withdrawal latency after nerve injury. Thermal hypoalgesia is indicated by a significant increase of the ‘difference score’ compared to baseline values. A negative ‘difference score’ indicated reduction of the withdrawal latency after nerve injury. Thermal hyperalgesia was indicated by a significant reduction of the ‘difference score’ compared to baseline values (* P < 0.05; ANOVA with Tukey post hoc test). Note that crush injury induced a transient period of hypoalgesia to heat followed by the development of hyperalgesia to heat by day 10. (B) Mechanical sensitivity to von Frey hairs after crush injury of rat sciatic nerve. The development of mechanical allodynia to calibrated von Frey hairs was monitored in crush-injured rats (triangles) and in naive controls (squares) (N = 7 rats per group). The 50% withdrawal threshold gives the force of the von Frey hair to which an animal responds in 50% of the presentations. Mechanical allodynia was indicated by a significant reduction of the 50% withdrawal threshold of the injured hindpaw compared to baseline values. Mechanical hyposensitivity is indicated by a significant increase of the 50% withdrawal threshold of the injured hindpaw compared to baseline values (* P < 0.05; ANOVA with Tukey post hoc test). Note that crush injury induced a transient period of mechanical hyposensitivity followed by the development of mechanical allodynia by day 10.
was used to test for significant differences of the mean withdrawal thresholds or mean withdrawal latencies after crush injury when compared to baseline control values. To monitor crush injury-induced motor deficits we documented the animals’ spontaneous gait and the hindpaw posture. We tested for the ability to grasp by applying a small plastic rod to the plantar surface of the operated hindpaw in a gently lifted animal. For ELISA studies, animals were sacrificed on days 0, 0.5, 3 and 14 after surgery (n = 4 per time point; repeated twice on days 0, 0.5, 3 and 14). Sciatic nerves were removed by cutting the nerve shortly above the site of the crush lesion and 1 cm distally. Control nerves from naive rats (day 0) and nerves from the contralateral, sham-operated side were removed accordingly. Sciatic nerves were homogenized as described [3]. TNF levels were determined by the Cytoscreen Rat TNF Ultrasensitive ELISA kit (Biosourse, CA) according to the manufacturer’s instructions. This assay system detects rat TNF with a sensitivity of 0.7 pg/ml. TNF levels were expressed as pg/mg total protein. We used some aliquots from the nerve homogenates obtained in the present study for parallel measurements of interleukin-10 as reported recently [4]. For IHC studies, rat sciatic nerves were harvested on days 0, 0.5, 3 and 14 after crush (n = 3 per time point). Tissue from distal to the crush lesion was processed for IHC on 10 m frozen sections. Polyclonal antibodies (AB) against TNF (1:1000, Genzyme, R¨usselsheim, Germany) were used overnight as primary ABs; controls were performed by preabsorbing the ABs with the respective recombinant cytokine. An avidin–biotin complex (Vector) was used as a secondary system; staining was visualized with 3,3 -diaminobenzidine tetrahydrochloride. For immunofluorescence, primary ABs were the polyclonal AB to TNF and a monoclonal anti-mouse ED1 antibody (1:1000;
Fig. 2. Crush injury of rat sciatic nerve induces an early and transient increase of local TNF protein. Local content of TNF protein in rat sciatic nerve homogenates on days 0, 0.5, 3 and 14 after crush injury (filled bars) or shamsurgery (striped bars) determined by enzyme-linked immunoassay. Data are expressed as mean values (pg/mg protein ± S.D.; * P < 0.05 for crush vs. control values). Data for the control nerves (day 0) are indicated by open bars. Note that crush injury but not sham-surgery induced a significant increase of local TNF protein concentrations.
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Fig. 3. Crush injury of rat sciatic nerve increases TNF-immunoreactivity (TNF-ir) in the epi- and the endoneurial compartment. We show four representative photographs of frozen sections of rat sciatic nerves immunoreacted for TNF. After crush injury, we observed prominent TNF-ir in the epineurial (A, C) and in the endoneurial (B, D) compartment as shown for day 0.5 (A, B) and day 3 (C, D) after nerve injury. The dotted line indicates the border between the epi- and the endoneurial space. In the epineurium, TNF-ir cells are indicated by red arrows. In the endoneurium, TNF-ir cells are indicated by blue arrows. TNF-ir cells in the subperineurial space are indicated by pink arrows. Scale bar = 20 m. Note that many epineurial cells with the morphology of macrophages were TNF-ir on days 0.5 and 3 (A, C). Morphometric analysis of TNF-ir in the epineurial (E) and the endoneurial (F) compartment on days 0, 0.5, 3, and 14 after crush. The number of epineurial TNF-ir cells (E) is shown for control (open bar) and crush-injured (filled bars) nerves and expressed as mean values (number/section ± S.D.; * P < 0.05). The endoneurial TNF-ir area (F) is shown for control (open bar) and crush-injured (filled bars) nerves and expressed as mean values (m2 /nerve section ± S.D.; * P < 0.05).
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Serotec, UK). Cy3-conjugated or Cy2-conjugated secondary ABs (1:100; 2 h, Amersham Biosciences, Freiburg) were used for visualization. Nerve lesions were verified by toluidine blue staining on 1-m sections after plastic embedding. After crush injury we observed prominent TNF-immunoreactivity in both the epineurial and endoneurial compartment. This finding prompted us to perform concurrent morphometric analysis of TNF-ir after crush injury. Using morphometric analysis we asked whether endoneurial and epineurial TNFir are differentially regulated after crush injury. The nerve section area was digitized at a 1400-fold magnification with a Zeiss Axiophot 2 microscope and analyzed using Image ProPlus software. A density threshold was set above background level and the area of positively stained structures was measured on three nerves per time point by a blinded investigator and expressed as m2 /nerve section. The number of epineurial TNF-ir cells was counted per nerve section. Data represent mean values ± S.D. Analysis of variance was used to test for differences between groups and subsequent Scheff´e test for comparison of individual means throughout the time course. Statistical significance was assumed with P < 0.05. Crush injury significantly changed the thermal (Fig. 1A) and mechanical (Fig. 1B) sensitivity of the operated hindpaw.
Transient hypoalgesia to heat on day 3 was followed by the development of hyperalgesia to heat by day 10 after crush injury (Fig. 1A). Similarly, a transient period of mechanical hypoesthesia was followed by the development of mechanical allodynia by day 10 (Fig. 1B). Motor deficits after sciatic nerve crush were present from day 1 until day 10 (last time point investigated). TNF protein levels were determined by ELISA in sciatic nerve homogenates from the crush-injured (Fig. 2, filled bars) or the sham-operated (Fig. 2, striped bars) side. After crush but not sham-surgery, local TNF increased significantly on day 0.5 and day 3 by two-fold of baseline. On day 14, TNF protein levels returned to baseline values again. Concurrent IHC studies revealed that TNF-ir was prominent in many epineurial cells with the morphology of macrophages on day 0.5 and day 3 after crush (Fig. 3A and C). By day 3, TNF-ir cells with the morphology of macrophages were also observed in the perineurial and subperineurial space (Fig. 3C). Endoneurially, TNF-ir was localized in Schwann cell and macrophage like structures after crush (Fig. 3B and D). Colocalization studies on day 3 after crush injury confirmed that many epineurial ED1-positive cells indicating macrophages were immunoreactive for TNF (Fig. 4A and B),
Fig. 4. Macrophages in the epineurial and the endoneurial compartment colocalize with TNF-immunoreactivity (TNF-ir) on day 3 after crush injury of rat sciatic nerve. We show examples of the immunofluorescence colocalization study for ED-1 (A, C) and TNF-immunoreactive (B, D) cells in the epineurial (A, B) and the endoneurial (C, D) compartment on frozen sections of rat sciatic nerve harvested on day 3 after crush injury. ED-1 positive cells appear green (A, C). TNF-ir structures appear red (B, D). Structures that colocalize are indicated by an arrow. In the epineurium (A, B), colocalizing cells are indicated by red arrows. In the endoneurium (C, D), colocalizing cells are indicated by blue arrows. Colocalizing cells in the subperineurial space are indicated by a pink arrow. The dotted line indicates the border between the epi- and the endoneurial space. Scale bar = 15 m. Note that many of the epineurial ED-1 positive cells (indicating macrophages; A) colocalize with TNF-ir (B). Note that many but not all of the endoneurial TNF-ir (D) structures colocalize with ED-1 positive cells (C).
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whereas many but not all macrophages in the endoneurial compartment were TNF-ir (Fig. 4C and D). In control sciatic nerve sections from naive rats, TNF-ir was occasionally found in Schwann cell like structures endoneurially but was completely absent in the epineurial and perineurial compartment (data not shown). These data indicate that TNF protein is upregulated early and transiently at the site of a crush injury. Thus, a peripheral nerve trauma with axonal damage increases the proinflammatory cytokine TNF at the protein level. An increase of local TNF protein can be due to (1) an increase of endoneurial TNF production, (2) a reduced axonal transport of TNF with local accumulation of TNF at the site of crush and (3) an invasion of cells that contain TNF protein. In the present study, we observed TNF-ir macrophages in the epineurial and perineurial space early after nerve injury (day 0.5 to day 3). This suggests that macrophages that invade the peripheral nerve at the site of lesion by day 3 are highly loaded with TNF protein. At the same time, endoneurial Schwann cells are positive for TNF. Thus, our data suggest that macrophages that are recruited during the first days of Wallerian degeneration as well as endoneurial Schwann cells contribute markedly to the early increase of local TNF levels after crush injury. The effects of TNF protein at the site of nerve lesion are incompletely understood. Our data showing an early and transient increase of TNF protein further support the assumption that TNF is crucial in the early process of Wallerian degeneration and may act as an initiator of local inflammatory responses as described recently [9]. Furthermore, the finding of many TNF-ir epineurial macrophages suggests that TNF may be essential in nerve injury-induced macrophage recruitment. Indeed, a delay of nerve injury-induced macrophage recruitment was described in TNF-deficient animals [7]. Behavioral tests in the present study revealed that crush injury of the rat sciatic nerve induces a transient hypoalgesia to heat and a transient hypoesthesia to mechanical stimuli whereas heat hyperalgesia and mechanical allodynia develop by day 10 after crush. Thus, the early and transient increase of local TNF protein may precede and parallel the development of pain-associated behavior in the crush injury model. This finding further supports the assumption that TNF is essential in the early process of neuropathic pain generation and suggests that TNF-antagonizing treatments should be given early in nerve injury to prevent the development of neuropathic pain.
Acknowledgements The expert technical help of Lydia Biko, Barbara Dekant and Helga Br¨unner is greatly appreciated. This study forms
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part of the doctoral thesis of Achim B¨uhl. This work was supported by Volkswagenstiftung.
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