- Email: [email protected]
Role of the peripheral benzodiazepine receptor in sensory neuron regeneration
www.elsevier.com/locate/ymcne Mol. Cell. Neurosci. 30 (2005) 228 – 237
Role of the peripheral benzodiazepine receptor in sensory neuron regeneration Charles D. Mills,* Jaquelyn L. Bitler, and Clifford J. Woolf Neural Plasticity Research Group, Massachusetts General Hospital and Harvard Medical School, 149 13th Street, Charlestown, MA 02129, USA Received 4 May 2005; revised 13 July 2005; accepted 15 July 2005 Available online 15 August 2005
Peripheral benzodiazepine receptor (PBR) expression increases in small dorsal root ganglion (DRG) sensory neurons after peripheral nerve injury. To determine the functional significance of this induction, we evaluated the effects of PBR ligands on rodent sensory axon outgrowth. In vitro, Ro5-4864, a PBR agonist, enhanced outgrowth only of small peripherin-positive DRG neurons. When DRG cells were preconditioned into an active growth state by a prior peripheral nerve injury Ro5-4864 augmented and PK 11195, a PBR antagonist, blocked the injury-induced increased outgrowth. In vivo, Ro5-4864 increased the initiation of regeneration after a sciatic nerve crush injury and the number of GAP-43-positive axons in the distal nerve while PK 11195 inhibited the enhanced growth produced by a preconditioning lesion. These results show that PBR has a role in the early regenerative response of small caliber sensory axons, the preconditioning effect, and that PBR agonists enhance sensory axon regeneration. D 2005 Elsevier Inc. All rights reserved.
Introduction Injury to the peripheral axon of dorsal root ganglion (DRG) neurons induces changes in gene expression that initiate regeneration. Microarray studies of changes in gene expression profiles in these neurons have helped to elucidated many potential survival and regeneration-associated genes (Bonilla et al., 2002; Costigan et al., 2002; Xiao et al., 2002). One identified injury-induced gene is the peripheral benzodiazepine receptor (PBR), an 18 kDa mitochondrial membrane protein that forms heteromeric complexes with the mitochondrial permeability transition pore, and plays key roles in steroidogenesis and apoptosis (Casellas et al., 2002; Gavish et al., 1999). However, the functional significance of PBR upregulation in injured DRG neurons remains unclear.
* Corresponding author. Fax: +1 617 724 3632. E-mail address: [email protected] (C.D. Mills). Available online on ScienceDirect (www.sciencedirect.com). 1044-7431/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2005.07.010
In the central nervous system, PBR expression is usually associated with microglia (Casellas et al., 2002; Gavish et al., 1999). However, PBR is also expressed in cultured cortical neurons (Jayakumar et al., 2002) and DRG neurons in vivo after peripheral nerve injury (Karchewski et al., 2004; Xiao et al., 2002). PBR is involved in the mitochondrial intermembrane transport of cholesterol through interactions with the steroidogenic acute regulatory protein (StAR) (Krueger and Papadopoulos, 1990; Papadopoulos et al., 1990; Wang et al., 1998; West et al., 2001) thereby regulating synthesis of the neurosteroids pregnenolone (PREG), progesterone (PROG), and dehydroepiandrosterone (DHEA) (Ferzaz et al., 2002; Korneyev et al., 1993; Lacor et al., 1999; Papadopoulos et al., 1997). Both cholesterol and neurosteroids are important for neuronal regeneration (Schumacher et al., 1996). For example, cholesterol deficiency inhibits axonal branching and promotes axonal degeneration (Fan et al., 2001, 2002), DHEA enhances functional recovery and increases the number of nerve fibers in the sciatic nerve after a crush injury (Gudemez et al., 2002), and PROG increases neurite outgrowth in vitro and in vivo (Koenig et al., 1995, 2000). Two synthetic ligands are widely used to study PBR, Ro5-4864 (a reported agonist) and PK 11195 (an antagonist), each having nanomolar affinity for PBR (Awad and Gavish, 1987; Krueger, 1995; Le Fur et al., 1983a,b,c; Schoemaker et al., 1983; Syapin and Skolnick, 1979). The putative endogenous ligand for PBR, diazepam binding inhibitor (DBI), is a 10 kDa peptide that is cleaved into several active fragments: eicosaneuropeptide (ENP), octadecaneuropeptide (ODN), and triakontatetraneuropeptide (TTN) (Alho et al., 1990; Costa and Guidotti, 1991; Guidotti et al., 1983; Shoyab et al., 1986; Slobodyansky et al., 1989). Schwann cells and fibroblasts in the sciatic nerve express ODN and its expression increases in the distal nerve segment after peripheral nerve injury (Lacor et al., 1996, 1999). DBI is constitutively expressed at high levels in non-neuronal satellite cells of the DRG but its expression does not change after a peripheral nerve injury (Karchewski et al., 2004). We have now investigated whether PBR has a role in peripheral nerve regeneration and found that Ro5-4864 enhanced neurite outgrowth in rat small caliber sensory neurons in vitro and also
C.D. Mills et al. / Mol. Cell. Neurosci. 30 (2005) 228 – 237
enhanced mouse sensory axon regeneration in vivo, while PK 11195 inhibited nerve injury-induced outgrowth and blocked the effects of Ro5-4864. These data demonstrate a role for PBR in sensory axon regeneration after peripheral nerve injury.
Results Localization of PBR in rat DRG neurons PBR is upregulated in the rat DRG following sciatic nerve axotomy (Costigan et al., 2002; Karchewski et al., 2004; Xiao et
229
al., 2002). Here, we show by in situ hybridization that PBR mRNA is detected only in a subset of adult rat DRG neurons, those that are peripherin-positive (i.e. small DRG neurons with unmyelinated axons) following a sciatic nerve axotomy (Figs. 1A – F). Approximately 30% of neuronal profiles express PBR mRNA after peripheral nerve injury and of those, 98% have cell diameters less than 45 AM (Karchewski et al., 2004). PBR mRNA was not detected after nerve injury in large NF 200-positive neurons (with myelinated axons), Schwann cells, or satellite cells. When adult rat DRG neurons are dissociated and grown in vitro, PBR-immunoreactivity (IR) was localized throughout the cell, including the soma, neurites, and growth cones (Figs. 1G – I).
Fig. 1. Induction of PBR in rat DRG neurons in vivo and in vitro. PBR mRNA expression was low/undetectable in uninjured DRGs (A,B), but increased in peripherin-positive cells of L4 DRGs 7 days after a sciatic nerve axotomy (C,D). PBR mRNA expression was never seen in large NF 200-positive neurons (*) or in the non-neuronal satellite cells (r) that surround DRG neurons (E,F). Photomicrograph of a dissociated DRG neuron grown in culture for 24 h showing PBR-IR in the cell body, neurites, and growth cones (G). Confocal image of cultured DRG neuron growth cones labeled with h-tublin (H) and PBR (I). Scale bars = 50 Am (A – F) and 20 Am (G).
230
C.D. Mills et al. / Mol. Cell. Neurosci. 30 (2005) 228 – 237
Effects of PBR ligands on neurite outgrowth and survival in adult rat cultured DRG neurons The effects of two PBR ligands, Ro5-4864 and PK 11195, on neurite growth were measured in three different adult rat DRG neuron culture assays. In the first assay, designed to look at the actions of PBR ligands on neurite initiation, the ligands were added to the media 2 h after culturing and neurite outgrowth
measured 16 h later. Whereas PK 11195 had no effect on the length or the number of neurons with neurites, Ro5-4864, in a dose-dependent manner, significantly increased mean neurite length (Fig. 2D). In the second assay, looking at action of the PBR ligands on established growth, neurons were grown for 12 h before treatment and then grown for a further 24 h. In this assay, Ro5-4864 also significantly increased neurite outgrowth, while the highest dose of PK 11195 tested (1 AM) reduced outgrowth and
Fig. 2. Effects of PBR ligands on rat DRG neurite outgrowth in vitro. Sixteen hour treatment with Ro5-4864 dose-dependently increased neurite outgrowth when administered 2 h after culturing (A,B,D) without affecting the number of cells growing (F). Twenty-four hours of Ro5-4864 treatment, initiated 12 h after culturing, also dose-dependently increased neurite outgrowth (C,E) without affecting the number of cells growing (G). At the highest dose tested, PK 11195 had a slight inhibitory effect on neurite outgrowth and, when co-administered with Ro5-4864, PK 11195 completely blocked the growth promoting effects of Ro54864 (1 AM each). Outgrowth is reported as percent of naı¨ve, defined as 100%. *P < 0.05, **P < 0.01, and ***P < 0.001 compared to vehicle treatment groups.
C.D. Mills et al. / Mol. Cell. Neurosci. 30 (2005) 228 – 237
completely blocked the growth promoting effects of Ro5-4864 (Fig. 2E). Sciatic nerve axotomy 1 week prior to culturing preconditions DRG cells into an active growth state. This paradigm increases neurite initiation and the extent of neurite outgrowth of
231
cultured DRG neurons (Fig. 3) (Hu-Tsai et al., 1994; Lankford et al., 1998). The third assay was designed to look at action of PBR ligands on cells preconditioned by a prior nerve injury. Ro5-4864 treatment further enhanced neurite growth in the preconditioned cells, while PK 11195, at the highest dose tested (1 AM), inhibited the preconditioning effect (Fig. 3). Since PBR is expressed only in small cells after injury, the differential effect of Ro5-4864 on neurite outgrowth in peripherinand NF 200-positive cells was studied. In our cultures, 25% of neurons are NF 200-positive (Figs. 4A, B). Ro5-4864 increased neurite outgrowth in the peripherin-positive cells, but not NF-200positive cells (Figs. 4C, D). To rule out possible cell survival effects of the PBR ligands on axon outgrowth, we tested the effects of PK 11195 and Ro5-4864 on the survival of cultured adult rat DRGs. Neither PK 11195 nor Ro5-4864 at any of the doses tested (10 nM – 1 AM) affected the survival of adult DRG neurons in culture (Table 1). Effects of PBR ligands on the rate of peripheral nerve regeneration after injury in the adult mouse
Fig. 3. Effects of PBR ligands on preconditioned DRG rat neurons. A preconditioning sciatic nerve axotomy 7 days before dissociation and culturing increased neurite outgrowth (A – C) and the percentage of cells with neurites (D). PK 11195 (1 AM) partially inhibited the injury-induced increase in growth (C), while Ro5-4864 dose-dependently enhanced the preconditioning effect without affecting the number of cells growing (D). Asterisks indicate significance compared to the vehicle treatment group.
To avoid large injection volumes resulting from the amount of ligand needed and the poor solubility of the ligands in solutions other that ethanol, adult mice were used instead of adult rats. The rate of peripheral nerve regeneration in the sciatic nerve was determined using the nerve pinch test 2 and 4 days after a crush injury. The nerve pinch test determines the extent of regrowth by measuring the distance from the injury site to the most distal point on the nerve that produces a reflexive withdrawal when pinched with smooth forceps (Bajrovic et al., 2001; Kovacic et al., 2004). Ro5-4864 treated mice had higher rates of regeneration than crush alone, vehicle, and PK 11195 treated animals (Figs. 5A, B). Moreover, PK 11195 completely blocked the growth promoting effects of Ro5-4864. The absolute differences in mean growth rates remained constant between groups at both 2 and 4 days after injury, indicating that Ro54864 treatment specifically accelerated the initiation of axonal regeneration without increasing the maximum rate of growth. As seen in vitro, a preconditioning sciatic nerve injury enhanced the extent of regrowth 2 days after a second injury (Fig. 5C). Treatment with Ro5-4864 was as effective as a preconditioning lesion at initiating regrowth (Figs. 5A vs. C). Similar to the in vitro results (Figs. 2, 3), PK 11195 treatment blocked the preconditioning effect and the growth promoting effects of Ro54864 in the nerve pinch test. The extent of regeneration was confirmed by histological examination of the sciatic nerve. Markers for subpopulations of cells within the DRG and their associated fibers change in distribution and intensity after peripheral nerve injury. For example, NF-H (a large cell marker), CGRP, and IB4 (both markers for small and medium cells) are downregulated after peripheral nerve injury (Verge et al., 1995; Hammond et al., 2004; Wang et al., 2003). In contrast, GAP-43 is expressed in all growing axons and is widely used as a marker of regenerating fibers (Skenen, 1989; Sommervaille et al., 1991; Gaete et al., 1998). Therefore, we used the number of GAP-43 fibers to identify regenerating axons. Two days after injury, Ro5-4864 treated mice had greater numbers of GAP-43-positive fibers compared to other treatment groups 1.5 mm distal to the crush site (Fig. 6). However, since GAP-43 labels all growing fibers and does not differentiate between subpopulations of fiber types,
232
C.D. Mills et al. / Mol. Cell. Neurosci. 30 (2005) 228 – 237
Fig. 4. Effects of Ro5-4864 on rat DRG neurite outgrowth in peripherin- and NF 200-positive cells. Dissociated DRGs neurons (A) stained for peripherin (green) and NF 200 (red). When DRG cells were dissociated and allowed to grow for 36 h, there were three times as many peripherin-positive cells as NF 200positive (B). Twenty-four hour treatment with Ro5-4864 (1 AM) increased neurite outgrowth only of the peripherin-positive cells (C), without affecting the percentage growing of either cell type (D). Asterisks indicate significance between groups (unpaired, two-tailed t test).
the increase in outgrowth cannot be assigned to a specific subpopulation of fibers.
Discussion Given the induction of PBR in small DRG sensory neurons after peripheral nerve injury (Karchewski et al., 2004; Xiao et al., 2002), the regulation of steroid production by PBR, and the role that steroids play in axon growth, we investigated if PBR has a role in peripheral nerve regeneration. Although PBR is predominantly expressed on the outer mitochondrial membrane, it can also be found on the plasma membrane (Garnier et al., 1993; Oke et al., 1992), the inner mitochondrial membrane (Mukherjee and Das, 1989), and in nuclei (Hardwick et al., 1999; Kuhlmann and Guilarte, 2000). These different localizations may reflect different isoforms of PBR (Berkovich et al., 1993; Cahard et al., 1994; Tanimoto et al., 1999). We find that dissociated DRG neurons show PBR expression throughout the cell body (including the nucleus), neurites, and growth cones (Fig. 1). Nuclear PBR localization is reported in activated astrocytes, microglia (Kuhlmann and Gui-
larte, 2000), and invasive breast tumor cells where it regulates entry of cholesterol into the nucleus (Hardwick et al., 1999). While mitochondrial PBRs are involved in cellular respiration and cholesterol transport for steroidogenesis, the function of extramitochondrial and plasma membrane PBRs remains unclear. Intracellular cholesterol plays an essential role in the maintenance of microtubule stability (Fan et al., 2001), and axonal regeneration is dependent on a local supply (reutilization) of cholesterol (Goodrum et al., 2000). It is possible that PBR expressed in neurites and growth cones sequesters cholesterol for microtubule stability and membrane synthesis needed during regeneration. The PBR may also affect neurite outgrowth by controlling the rate of neurosteroid formation. Mitochondrial PBRs regulate the transport of cholesterol from the outer to inner membrane, which is the rate-limiting step in steroid production. The neurosteroids, PREG, PROG, and DHEA play major roles in neuronal regeneration. PROG increases neurite outgrowth of DRG explants, promotes regeneration in cryolesioned sciatic nerves, and promotes remyelination of regenerated nerve fibers (Koenig et al., 1995, 2000). Treatment with Ro5-4864 increases PREG production in the regenerating sciatic nerve, an effect blocked by PK 11195, which alone was found to have no effect (Lacor et al., 1999). PREG and
Table 1 Effect of PBR ligands on survival of adult dissociated DRG neurons Treatment duration
Control
Vehicle
PK 11195
Ro5-4864
10 nM
100 nM
1 AM
10 nM
100 nM
1 AM
24 h 48 h 96 h
89.9 T 8.9 90.0 T 2.5 74.8 T 10.0
83.2 T 3.1 81.5 T 1.3 62.8 T 2.6
91.5 T 3.1 87.3 T 6.2 66.4 T 5.8
94.5 T 2.3 90.7 T 4.0 69.7 T 7.6
91.3 T 13.2 88.6 T 9.7 67.7 T 9.3
90.9 T 1.7 81.2 T 5.5 55.1 T 0.6
92.1 T 1.5 85.7 T 6.3 64.0 T 4.3
95.3 T 3.0 90.9 T 6.2 77.7 T 10.4
Survival of adult DRG neurons treated with PBR ligands expressed as a percentage of baseline.
C.D. Mills et al. / Mol. Cell. Neurosci. 30 (2005) 228 – 237
233
returns to basal levels with reinnervation (Lacor et al., 1996, 1999). It is plausible that Ro5-4864 acts on Schwann cells to produce PREG, providing trophic support for regeneration. Ro5-4864 also increases cytokine synthesis (Taupin et al., 1993) and cytokines stimulate the synthesis of NGF by macrophages and Schwann cells, which would also provide trophic support enhancing regeneration (Ferzaz et al., 2002; Lacor et al., 1999). Another way that PBR could regulate outgrowth is from an increase in availability of ATP, which is an essential factor for neurite growth (Behrsing and Vulliet, 1999, 2004; D’Ambrosi et al., 2001). PBR associates with the mitochondrial permeability transition pore (MPTP), which allows the respiratory chain to create the transmembrane electrochemical gradient (DCm) that drives ATP synthesis. Specifically, PBR associates with the voltage-dependent anion channel (VDAC) and the adenine nucleotide carrier (ANC) of the MPTP. The VDAC is the main pathway for metabolite diffusion across the outer membrane and the ANC is an antiporter exchanging ATP generated in the matrix for ADP. Opening the MPTP dissipates the DCm and decreases ATP production. Reports show that PBR ligands regulate the DCm differently depending on the system being examined (Akao et al.,
Fig. 5. Effects of PBR ligands on peripheral nerve regeneration in the adult mouse. Mean distances of sensory axon regrowth 2 days (A) and 4 days (B) following a crush injury of the sciatic nerve as determined by the nerve pinch test. Ro5-4864 (10 mg/kg) increased the rate of peripheral nerve regeneration, an effect blocked by co-administration of PK 11195 (10 mg/kg each). Mean distances of sensory axon regrowth 2 days following crush injury in preconditioned sciatic nerves (C). PK 11195 blocked the preconditioning effect and the growth promoting effects of Ro5-4864. Asterisks indicate significance compared to the vehicle treatment group.
PROG levels increase in the injured sciatic nerve and have neurotrophic effects (Akwa et al., 1993; Koenig et al., 1995). PBR expression increases markedly in non-neuronal Schwann cells and macrophages in the regenerating sciatic nerve after injury and
Fig 6. Ro5-4864 treatment increased the numbers of regenerating fibers in the adult mouse sciatic nerve. Photomicrographs of GAP-43 IR fibers 1.5 mm from the injury site at 2 days post-injury: crush only (A), PK 11195 (B), Ro54864 (C), and PK 11195 + Ro5-4864 treated mice (10 mg/kg each; D). Mean number of GAP-43 IR fibers/section (E). Asterisks indicate significance compared to the vehicle treatment group.
234
C.D. Mills et al. / Mol. Cell. Neurosci. 30 (2005) 228 – 237
2004; Chelli et al., 2001, 2004; Fischer et al., 2001; Ravagnan et al., 1999; Tanimoto et al., 1999); however, the specificity of these ligands in the control of cellular respiration has been questioned (Casellas et al., 2002; Gavish et al., 1999). Although PBR regulates DCm and apoptosis, we find that PBR ligands have no effect on the survival of adult dissociated DRG cells in short-term cultures or the total percentage of cells growing. These results demonstrate that the effects of PBR ligands on growth are independent of any effects on survival. After peripheral nerve crush, there is an initial delay in the initiation of regrowth (McQuarrie et al., 1977; Pan et al., 2003). Ro5-4864 averts this initial stalling as evident by the increased growth detected 2 days after injury. Once peripheral regeneration begins, the rate increases to reach a maximum around 3 days after injury (Danielsen et al., 1986; McQuarrie et al., 1977; Pan et al., 2003). Our data suggest that Ro5-4864 primarily targets the initiation phase of growth rather than increasing the maximum rate of regeneration. An initial delay in growth is also seen when DRG cells are grown in vitro (Kocsis et al., 1994; Lankford et al., 1998). Ro5-4864 overcame this delay as shown by the 40% increase in growth after 18 h in culture. PK 11195 blocked the effects of Ro54864 on neurite outgrowth in vitro and peripheral regeneration in vivo, demonstrating that PK 11195 is antagonistic to the effects of Ro5-4864. DBI, the endogenous ligand for PBR, is expressed in nonneuronal cells of the sciatic nerve and DRG. Following sciatic nerve injury, there is an upregulation of ODN, a biologically active fragment of DBI, in Schwann cells of the distal segment where it is implicated in the reutilization of cholesterol for myelin membrane formation (Lacor et al., 1996, 1999). ODN levels in the sciatic nerve return to basal levels once regeneration is complete (Lacor et al., 1999) However, an injury-induced upregulaton of DBI is not seen in the DRG (Karchewski et al., 2004). The high level of DBI expressed in the DRG may represent a source of ligand for the injury-induced upregulation of PBR after injury. Axonal injury induces growth-associated gene expression within the cell body that transforms the injured cell into an active growth state. If axons are injured a second time during this growth state, regeneration is enhanced (Forman et al., 1980; Hu-Tsai et al., 1994; Lankford et al., 1998; McQuarrie et al., 1977; Neumann and Woolf, 1999; Qiu et al., 2002; Sjoberg and Kanje, 1990). The first lesion, termed the preconditioning lesion, overcomes the delay in initiating regrowth, and increases the speed of regeneration such that the maximum growth rate is reach sooner (Pan et al., 2003). In DRG neurons grown in culture, Ro5-4864 enhanced, while PK 11195 inhibited the preconditioning effect, demonstrating that pharmacological activation of PBR by Ro5-4864 contributes to the preconditioning effect. In vivo, PK 11195, presumably by antagonizing an endogenous PBR ligand, blocks the preconditioning effect indicating that activation of PBR contributes physiologically to the preconditioning effect. Conclusion PBR is induced in small peripherin-positive cells of the DRG following a peripheral nerve injury. The PBR ligand Ro5-4864, but not PK 11195, increased neurite outgrowth of peripherin-positive dissociated DRG neurons, with no effect on large NF 200-positive cells. Ro5-4864 treatment also increased the rate of peripheral nerve regeneration and the numbers of GAP-43 fibers in the sciatic
nerve following a crush injury in the adult mouse, an effect blocked by PK 11195. Ro5-4864 treatment overcame the initial delay in regeneration without affecting the speed of regenerative axonal growth and PK 11195, in addition to blocking the action of Ro54864, inhibited the conditioning effect of a prior nerve lesion. These data demonstrate a specific role for the PBR in initiating sensory neuron regeneration.
Experimental methods Experimental animals and injury production Adult male Sprague – Dawley rats, 190 – 210 g, and adult C57 BL/6 mice, 25 – 30 g, were obtained from Charles Rivers Laboratories and housed with a light/dark cycle of 12 h/12 h. All procedures were performed in accordance with Massachusetts General Hospital animal care regulations. Axotomy in the adult rat was produced by exposing the left sciatic nerve at the mid-thigh level, ligating with 3 – 0 suture and then transecting the nerve distal to the ligature. For crush injuries in adult mice, the left sciatic nerve was crushed with smooth forceps for 15 s at the proximal thigh level and the injury site marked with a 10 – 0 epineural suture. After injury, muscle and skin were closed in layers and the animals allowed to recover. Preconditioning axotomies in adult rats were performed 7 days prior to collecting DRGs for culturing. Adult mouse preconditioning crush injuries were performed 3 days prior to a second crush injury. Ligand preparation and dosing The PBR ligands PK 11195 and Ro5-4864 were obtained from Sigma and 1 mM stock solutions were prepared in 100% EtOH. For cell culture experiments, stock solutions were diluted with 100% EtOH so that 2 Al of ligand was added to 300 Al of cell culture media. For peripheral nerve regeneration experiments, 10 mg/kg/day of each ligand prepared in 100% EtOH was given intraperitoneally beginning at the time of injury (Torres et al., 2000; Wala et al., 2000). Cell culture, cell survival, and neurite outgrowth assays Primary adult dissociated DRG neuron-enriched cultures were prepared by dissecting DRGs into HBSS (Cellgro) and 10 mM HEPES, followed by digestion with 5 mg/ml collagenase A and 1 mg/ml dispase II (Roche) prior to treatment with 0.25% trypsin (GibcoBRL). Triturated cells were centrifuged through a Percoll (Sigma) gradient prior to plating on laminin in Neurobasal media (GibcoBRL) containing 2% (vol/vol) B27 supplement (GibcoBRL), 50 Ag/ml Pen-Strep, 10 AM Ara-C, 50 ng/ml NGF, 200 mM l-glutamine, and 2 ng/ml GDNF. Neurite outgrowth was determined by measuring the length of the longest neurite per cell using Image J software (NIH). A cell was defined as growing if it extended a neurite twice its diameter. For cell survival assays, adult DRG neurons were cultured and allowed to grow for 48 h, baseline counts were obtained and then cultures were treated with PBR ligands and neurons counted at 24, 48, and 96 h. The number of cells surviving is reported as a percent of baseline. Values reported for both survival and outgrowth assays are from three experiments repeated in triplicate. All measures were done blinded to condition.
C.D. Mills et al. / Mol. Cell. Neurosci. 30 (2005) 228 – 237
235
Immunochemistry
Statistical analysis
Cultured cells were fixed with 4% paraformaldehyde for 20 min, washed with PBS, incubated with polyclonal antibodies to PBR (a gift from Dr. Vassilios Papadopoulos; Delavoie et al., 2003), and visualized using Alex Fluor 488 (Molecular Probes). Confocal images were obtained using a Leica TCS NTD4D confocal laser microscopy imaging system (Leica, Heidelberg, Germany). For neurite outgrowth assays, cultured cells were incubated with GAP-43 (Chemicon) and Hsp 25 (Stressgen), with peripherin (Chemicon) and NF 200 (Sigma), or h-tubulin III (Sigma).
A one-way ANOVA was performed to assess changes in neurite outgrowth, peripheral nerve regeneration lengths, and the numbers of GAP-43-positive fibers. Tukey’s multiple comparison test was used for post hoc pairwise comparisons. An unpaired, two-tailed t test was used for pairwise comparisons of outgrowth between peripherin- and NF 200-positive cells. All data are reported as means T standard error of the mean (SEM).
In situ hybridization
This work was supported by NINDS grants NS45459 and NS038253.
For in situ hybridization, fresh DRGs were dissected, frozen in OCT, and stored at 80-C until sectioning at 10 Am. Fresh sections were fixed with 4% paraformaldehyde for 15 min, washed with PBS, acetylation solution [1.35% triethanolamine (TEA), 0.25% acetic anhydride] for 10 min, and hybridization buffer [50% formamide, 5 Denhardt’s solution, 5 saline sodium citrate (SSC), 0.25 mg/ml yeast RNA, 0.5 mg/ml denatured Salmon sperm DNA] for 2 h. Sections were hybridized with a digoxigenin labeled antisense probe to PBR (accession number NM 012515) corresponding to base pairs 3 – 671 (669 bps total) at 72-C overnight. Slides were washed with 0.2 SSC at 65-C, blocked, and then incubated with anti-digoxigenin Fab fragments (Roche Diagnostics) at 4-C overnight. Visualization was done using 3.5 Al/ ml 5-bromo-4-cholor-3-indolyl-phosphate (BCIP; Roche Diagnostics) and 4.5 Al/ml 4-nitro blue tetrazolium chloride (NBT; Roche Diagnostics) in 0.1 M Tris pH 9.5, 0.1 M NaCl, 50 mM MgCl2. Peripheral nerve regeneration The nerve pinch test was used to determine the rate of in vivo peripheral nerve regeneration (Bajrovic et al., 2001; Kovacic et al., 2004). To avoid large injection volumes resulting from the amount of ligand needed and the poor solubility of the ligands in solutions other that ethanol, adult mice were used instead of adult rats. On post-injury days 2 and 4 following sciatic crush, anesthesia was induced with 2.5% isoflurane and the left sciatic nerve exposed. The animals were brought to a light plane of anesthesia by lowering the isoflurane concentration to 1%—a level where pinching the skin of the contralateral uninjured paw elicited a reflex withdrawal. Starting distally, a series of pinches using fine smooth forceps was delivered to the sciatic nerve moving proximally toward the injury site. The rate of regeneration was determined by measuring the distance from the injury site to the most distal point on the nerve that produced a reflexive withdrawal when pinched (n = 5 – 6 per group). All measures were done blinded to condition. Since markers for subpopulations of cells within the DRG and their associated fibers change in distribution and intensity after peripheral nerve injury, we used GAP-43 that is expressed in all regenerating fibers to histologically characterize regeneration in the injured sciatic nerve 2 days post-injury. For quantification, sciatic nerves were serially sectioned at 10 Am and the mean number of GAP-43 immunoreactive fibers/section was determined in three sections (at least 30 Am apart) per animal, 1.5 mm beyond the injury site. The mean number of fibers/section per animal was determined for three animals in each treatment group.
Acknowledgments
References Akao, Y., Seki, N., Nakagawa, Y., Yi, H., Matsumoto, K., Ito, Y., Ito, K., Funaoka, M., Maruyama, W., Naoi, M., Nozawa, Y., 2004. A highly bioactive lignophenol derivative from bamboo lignin exhibits a potent activity to suppress apoptosis induced by oxidative stress in human neuroblastoma SH-SY5Y cells. Bioorg. Med. Chem. 12, 4791 – 4801. Akwa, Y., Schumacher, M., Jung-Testas, I., Baulieu, E.E., 1993. Neurosteroids in rat sciatic nerves and Schwann cells. C. R. Acad. Sci., Ser. III 316, 410 – 414. Alho, H., Bovolin, P., Slobodyansky, E., 1990. Diazepam binding inhibitor (DBI) processing: immunohistochemical studies in the rat brain. Neurochem. Res. 15, 209 – 216. Awad, M., Gavish, M., 1987. Binding of [3H]Ro 5-4864 and [3H]PK 11195 to cerebral cortex and peripheral tissues of various species: species differences and heterogeneity in peripheral benzodiazepine binding sites. J. Neurochem. 49, 1407 – 1414. Bajrovic, F., Srpcic, M., Sketelj, J., 2001. Schwann cell dependence of regenerating rat sensory neurons is inversely related to the quality of axon growth substratum. J. Neuropathol. Exp. Neurol. 60, 65 – 74. Behrsing, H.P., Vulliet, P.R., 1999. Purinergic and calcium-mediated enhancement of NGF-induced neurite expression in PC12 cells. Proc. West. Pharmacol. Soc. 42, 59 – 62. Behrsing, H.P., Vulliet, P.R., 2004. Mitogen-activated protein kinase mediates purinergic-enhanced nerve growth factor-induced neurite outgrowth in PC12 cells. J. Neurosci. Res. 78, 64 – 74. Berkovich, A., Ferrarese, C., Cavaletti, G., Alho, H., Marzorati, C., Bianchi, G., Guidotti, A., Costa, E., 1993. Topology of two DBI receptors in human lymphocytes. Life Sci. 52, 1265 – 1277. Bonilla, I.E., Tanabe, K., Strittmatter, S.M., 2002. Small proline-rich repeat protein 1A is expressed by axotomized neurons and promotes axonal outgrowth. J. Neurosci. 22, 1303 – 1315. Cahard, D., Canat, X., Carayon, P., Roque, C., Casellas, P., Le Fur, G., 1994. Subcellular localization of peripheral benzodiazepine receptors on human leukocytes. Lab. Invest. 70, 23 – 28. Casellas, P., Galiegue, S., Basile, A.S., 2002. Peripheral benzodiazepine receptors and mitochondrial function. Neurochem. Int. 40, 475 – 486. Chelli, B., Falleni, A., Salvetti, F., Gremigni, V., Lucacchini, A., Martini, C., 2001. Peripheral-type benzodiazepine receptor ligands: mitochondrial permeability transition induction in rat cardiac tissue. Biochem. Pharmacol. 61, 695 – 705. Chelli, B., Lena, A., Vanacore, R., Pozzo, E.D., Costa, B., Rossi, L., Salvetti, A., Scatena, F., Ceruti, S., Abbracchio, M.P., Gremigni, V., Martini, C., 2004. Peripheral benzodiazepine receptor ligands: mitochondrial transmembrane potential depolarization and apoptosis induction in rat C6 glioma cells. Biochem. Pharmacol. 68, 125 – 134. Costa, E., Guidotti, A., 1991. Diazepam binding inhibitor (DBI): a peptide with multiple biological actions. Life Sci. 49, 325 – 344.
236
C.D. Mills et al. / Mol. Cell. Neurosci. 30 (2005) 228 – 237
Costigan, M., Befort, K., Karchewski, L., Griffin, R.S., D’Urso, D., Allchorne, A., Sitarski, J., Mannion, J.W., Pratt, R.E., Woolf, C.J., 2002. Replicate high-density rat genome oligonucleotide microarrays reveal hundreds of regulated genes in the dorsal root ganglion after peripheral nerve injury. BMC Neurosci. 3, 16. D’Ambrosi, N., Murra, B., Cavaliere, F., Amadio, S., Bernardi, G., Burnstock, G., Volonte, C., 2001. Interaction between ATP and nerve growth factor signalling in the survival and neuritic outgrowth from PC12 cells. Neuroscience 108, 527 – 534. Danielsen, N., Lundborg, G., Frizell, M., 1986. Nerve repair and axonal transport: outgrowth delay and regeneration rate after transection and repair of rabbit hypoglossal nerve. Brain Res. 376, 125 – 132. Delavoie, F., Li, H., Hardwick, M., Robert, J.C., Giatzakis, C., Peranzi, G., Yao, Z.X., Maccario, J., Lacapere, J.J., Papadopoulos, V., 2003. In vivo and in vitro peripheral-type benzodiazepine receptor polymerization: functional significance in drug ligand and cholesterol binding. Biochemistry 42, 4506 – 4519. Fan, Q.W., Yu, W., Senda, T., Yanagisawa, K., Michikawa, M., 2001. Cholesterol-dependent modulation of tau phosphorylation in cultured neurons. J. Neurochem. 76, 391 – 400. Fan, Q.W., Yu, W., Gong, J.S., Zou, K., Sawamura, N., Senda, T., Yanagisawa, K., Michikawa, M., 2002. Cholesterol-dependent modulation of dendrite outgrowth and microtubule stability in cultured neurons. J. Neurochem. 80, 178 – 190. Ferzaz, B., Brault, E., Bourliaud, G., Robert, J.P., Poughon, G., Claustre, Y., Marguet, F., Liere, P., Schumacher, M., Nowicki, J.P., Fournier, J., Marabout, B., Sevrin, M., George, P., Soubrie, P., Benavides, J., Scatton, B., 2002. SSR180575 (7-chloro-N,N,5-trimethyl-4-oxo-3phenyl-3,5-dihydro-4H-pyridazino[4,5-b]i ndole-1-acetamide), a peripheral benzodiazepine receptor ligand, promotes neuronal survival and repair. J. Pharmacol. Exp. Ther. 301, 1067 – 1078. Fischer, R., Schmitt, M., Bode, J.G., Haussinger, D., 2001. Expression of the peripheral-type benzodiazepine receptor and apoptosis induction in hepatic stellate cells. Gastroenterology 120, 1212 – 1226. Forman, D.S., McQuarrie, I.G., Labore, F.W., Wood, D.K., Stone, L.S., Braddock, C.H., Fuchs, D.A., 1980. Time course of the conditioning lesion effect on axonal regeneration. Brain Res. 182, 180 – 185. Gaete, J., Kameid, G., Alvarez, J., 1998. Regenerating axons of the rat require a local source of proteins. Neurosci. Lett. 251, 197 – 200. Garnier, M., Boujrad, N., Oke, B.O., Brown, A.S., Riond, J., Ferrara, P., Shoyab, M., Suarez-Quian, C.A., Papadopoulos, V., 1993. Diazepam binding inhibitor is a paracrine/autocrine regulator of Leydig cell proliferation and steroidogenesis: action via peripheral-type benzodiazepine receptor and independent mechanisms. Endocrinology 132, 444 – 458. Gavish, M., Bachman, I., Shoukrun, R., Katz, Y., Veenman, L., Weisinger, G., Weizman, A., 1999. Enigma of the peripheral benzodiazepine receptor. Pharmacol. Rev. 51, 629 – 650. Goodrum, J.F., Brown, J.C., Fowler, K.A., Bouldin, T.W., 2000. Axonal regeneration, but not myelination, is partially dependent on local cholesterol reutilization in regenerating nerve. J. Neuropathol. Exp. Neurol. 59, 1002 – 1010. Gudemez, E., Ozer, K., Cunningham, B., Siemionow, K., Browne, E., Siemionow, M., 2002. Dehydroepiandrosterone as an enhancer of functional recovery following crush injury to rat sciatic nerve. Microsurgery 22, 234 – 241. Guidotti, A., Forchetti, C.M., Corda, M.G., Konkel, D., Bennett, C.D., Costa, E., 1983. Isolation, characterization, and purification to homogeneity of an endogenous polypeptide with agonistic action on benzodiazepine receptors. Proc. Natl. Acad. Sci. U. S. A. 80, 3531 – 3535. Hammond, D.L., Ackerman, L., Holdsworth, R., Elzey, B., 2004. Effects of spinal nerve ligation on immunohistochemically identified neurons in the L4 and L5 dorsal root ganglia of the rat. J. Comp. Neurol. 475, 575 – 589. Hardwick, M., Fertikh, D., Culty, M., Li, H., Vidic, B., Papadopoulos, V., 1999. Peripheral-type benzodiazepine receptor (PBR) in human breast cancer: correlation of breast cancer cell aggressive phenotype with PBR
expression, nuclear localization, and PBR-mediated cell proliferation and nuclear transport of cholesterol. Cancer Res. 59, 831 – 842. Hu-Tsai, M., Winter, J., Emson, P.C., Woolf, C.J., 1994. Neurite outgrowth and GAP-43 mRNA expression in cultured adult rat dorsal root ganglion neurons: effects of NGF or prior peripheral axotomy. J. Neurosci. Res. 39, 634 – 645. Jayakumar, A.R., Panickar, K.S., Norenberg, M.D., 2002. Effects on free radical generation by ligands of the peripheral benzodiazepine receptor in cultured neural cells. J. Neurochem. 83, 1226 – 1234. Karchewski, L.A., Bloechlinger, S., Woolf, C.J., 2004. Axonal injurydependent induction of the peripheral benzodiazepine receptor in small-diameter adult rat primary sensory neurons. Eur. J. Neurosci. 20, 671 – 683. Kocsis, J.D., Rand, M.N., Lankford, K.L., Waxman, S.G., 1994. Intracellular calcium mobilization and neurite outgrowth in mammalian neurons. J. Neurobiol. 25, 252 – 264. Koenig, H.L., Schumacher, M., Ferzaz, B., Thi, A.N., Ressouches, A., Guennoun, R., Jung-Testas, I., Robel, P., Akwa, Y., Baulieu, E.E., 1995. Progesterone synthesis and myelin formation by Schwann cells. Science 268, 1500 – 1503. Koenig, H.L., Gong, W.H., Pelissier, P., 2000. Role of progesterone in peripheral nerve repair. Rev. Reprod. 5, 189 – 199. Korneyev, A., Pan, B.S., Polo, A., Romeo, E., Guidotti, A., Costa, E., 1993. Stimulation of brain pregnenolone synthesis by mitochondrial diazepam binding inhibitor receptor ligands in vivo. J. Neurochem. 61, 1515 – 1524. Kovacic, U., Zele, T., Osredkar, J., Sketelj, J., Bajrovic, F.F., 2004. Sexrelated differences in the regeneration of sensory axons and recovery of nociception after peripheral nerve crush in the rat. Exp. Neurol. 189, 94 – 104. Krueger, K.E., 1995. Molecular and functional properties of mitochondrial benzodiazepine receptors. Biochim. Biophys. Acta 1241, 453 – 470. Krueger, K.E., Papadopoulos, V., 1990. Peripheral-type benzodiazepine receptors mediate translocation of cholesterol from outer to inner mitochondrial membranes in adrenocortical cells. J. Biol. Chem. 265, 15015 – 15022. Kuhlmann, A.C., Guilarte, T.R., 2000. Cellular and subcellular localization of peripheral benzodiazepine receptors after trimethyltin neurotoxicity. J. Neurochem. 74, 1694 – 1704. Lacor, P., Benavides, J., Ferzaz, B., 1996. Enhanced expression of the peripheral benzodiazepine receptor (PBR) and its endogenous ligand octadecaneuropeptide (ODN) in the regenerating adult rat sciatic nerve. Neurosci. Lett. 220, 61 – 65. Lacor, P., Gandolfo, P., Tonon, M.C., Brault, E., Dalibert, I., Schumacher, M., Benavides, J., Ferzaz, B., 1999. Regulation of the expression of peripheral benzodiazepine receptors and their endogenous ligands during rat sciatic nerve degeneration and regeneration: a role for PBR in neurosteroidogenesis. Brain Res. 815, 70 – 80. Lankford, K.L., Waxman, S.G., Kocsis, J.D., 1998. Mechanisms of enhancement of neurite regeneration in vitro following a conditioning sciatic nerve lesion. J. Comp. Neurol. 391, 11 – 29. Le Fur, G., Vaucher, N., Perrier, M.L., Flamier, A., Benavides, J., Renault, C., Dubroeucq, M.C., Gueremy, C., Uzan, A., 1983a. Differentiation between two ligands for peripheral benzodiazepine binding sites, [3H]RO5-4864 and [3H]PK 11195, by thermodynamic studies. Life Sci. 33, 449 – 457. Le Fur, G., Perrier, M.L., Vaucher, N., Imbault, F., Flamier, A., Benavides, J., Uzan, A., Renault, C., Dubroeucq, M.C., Gueremy, C., 1983b. Peripheral benzodiazepine binding sites: effect of PK 11195, 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide: I. In vitro studies. Life Sci. 32, 1839 – 1847. Le Fur, G., Guilloux, F., Rufat, P., Benavides, J., Uzan, A., Renault, C., Dubroeucq, M.C., Gueremy, C., 1983c. Peripheral benzodiazepine binding sites: effect of PK 11195, 1-(2-chlorophenyl)-N-methyl-(1methylpropyl)-3 isoquinolinecarboxamide: II. In vivo studies. Life Sci. 32, 1849 – 1856.
C.D. Mills et al. / Mol. Cell. Neurosci. 30 (2005) 228 – 237 McQuarrie, I.G., Grafstein, B., Gershon, M.D., 1977. Axonal regeneration in the rat sciatic nerve: effect of a conditioning lesion and of dbcAMP. Brain Res. 132, 443 – 453. Mukherjee, S., Das, S.K., 1989. Subcellular distribution of ‘‘peripheral type’’ binding sites for [3H]Ro5-4864 in guinea pig lung. Localization to the mitochondrial inner membrane. J. Biol. Chem. 264, 16713 – 16718. Neumann, S., Woolf, C.J., 1999. Regeneration of dorsal column fibers into and beyond the lesion site following adult spinal cord injury. Neuron 23, 83 – 91. Oke, B.O., Suarez-Quian, C.A., Riond, J., Ferrara, P., Papadopoulos, V., 1992. Cell surface localization of the peripheral-type benzodiazepine receptor (PBR) in adrenal cortex. Mol. Cell. Endocrinol. 87, R1 – R6. Pan, Y.A., Misgeld, T., Lichtman, J.W., Sanes, J.R., 2003. Effects of neurotoxic and neuroprotective agents on peripheral nerve regeneration assayed by time-lapse imaging in vivo. J. Neurosci. 23, 11479 – 11488. Papadopoulos, V., Mukhin, A.G., Costa, E., Krueger, K.E., 1990. The peripheral-type benzodiazepine receptor is functionally linked to Leydig cell steroidogenesis. J. Biol. Chem. 265, 3772 – 3779. Papadopoulos, V., Amri, H., Boujrad, N., Cascio, C., Culty, M., Garnier, M., Hardwick, M., Li, H., Vidic, B., Brown, A.S., Reversa, J.L., Bernassau, J.M., Drieu, K., 1997. Peripheral benzodiazepine receptor in cholesterol transport and steroidogenesis. Steroids 62, 21 – 28. Qiu, J., Cai, D., Dai, H., McAtee, M., Hoffman, P.N., Bregman, B.S., Filbin, M.T., 2002. Spinal axon regeneration induced by elevation of cyclic AMP. Neuron 34, 895 – 903. Ravagnan, L., Marzo, I., Costantini, P., Susin, S.A., Zamzami, N., Petit, P.X., Hirsch, F., Goulbern, M., Poupon, M.F., Miccoli, L., Xie, Z., Reed, J.C., Kroemer, G., 1999. Lonidamine triggers apoptosis via a direct, Bcl-2-inhibited effect on the mitochondrial permeability transition pore. Oncogene 18, 2537 – 2546. Schoemaker, H., Boles, R.G., Horst, W.D., Yamamura, H.I., 1983. Specific high-affinity binding sites for [3H]Ro 5-4864 in rat brain and kidney. J. Pharmacol. Exp. Ther. 225, 61 – 69. Schumacher, M., Robel, P., Baulieu, E.E., 1996. Development and regeneration of the nervous system: a role for neurosteroids. Dev. Neurosci. 18, 6 – 21. Shoyab, M., Gentry, L.E., Marquardt, H., Todaro, G.J., 1986. Isolation and characterization of a putative endogenous benzodiazepineoid (endozepine) from bovine and human brain. J. Biol. Chem. 261, 11968 – 11973. Sjoberg, J., Kanje, M., 1990. The initial period of peripheral nerve regeneration and the importance of the local environment for the conditioning lesion effect. Brain Res. 529, 79 – 84. Skenen, J.H., 1989. Axonal growth-associated proteins. Annu. Rev. Neurosci. 12, 127 – 156. Slobodyansky, E., Guidotti, A., Wambebe, C., Berkovich, A., Costa, E., 1989. Isolation and characterization of a rat brain triakontatetraneuro-
237
peptide, a posttranslational product of diazepam binding inhibitor: specific action at the Ro 5-4864 recognition site. J. Neurochem. 53, 1276 – 1284. Sommervaille, T., Reynolds, M.L., Woolf, C.J., 1991. Time-dependent differences in the increase in GAP-43 expression in dorsal root ganglion cells after peripheral axotomy. Neuroscience 45, 213 – 220. Syapin, P.J., Skolnick, P., 1979. Characterization of benzodiazepine binding sites in cultured cells of neural origin. J. Neurochem. 32, 1047 – 1051. Tanimoto, Y., Onishi, Y., Sato, Y., Kizaki, H., 1999. Benzodiazepine receptor agonists modulate thymocyte apoptosis through reduction of the mitochondrial transmembrane potential. Jpn. J. Pharmacol. 79, 177 – 183. Taupin, V., Toulmond, S., Serrano, A., Benavides, J., Zavala, F., 1993. Increase in IL-6, IL-1 and TNF levels in rat brain following traumatic lesion. Influence of pre- and post-traumatic treatment with Ro5 4864, a peripheral-type (p site) benzodiazepine ligand. J. Neuroimmunol. 42, 177 – 185. Torres, S.R., Frode, T.S., Nardi, G.M., Vita, N., Reeb, R., Ferrara, P., Ribeiro-do-Valle, R.M., Farges, R.C., 2000. Anti-inflammatory effects of peripheral benzodiazepine receptor ligands in two mouse models of inflammation. Eur. J. Pharmacol. 408, 199 – 211. Verge, V.M., Richardson, P.M., Wiesenfeld-Hallin, Z., Hokfelt, T., 1995. Differential influence of nerve growth factor on neuropeptide expression in vivo: a novel role in peptide suppression in adult sensory neurons. J. Neurosci. 15, 2081 – 2096. Wala, E.P., Sloan, J.W., Jing, X., 2000. Pharmacokinetics of the peripheral benzodiazepine receptor antagonist, PK 11195, in rats. The effect of dose and gender. Pharmacol. Res. 41, 461 – 468. Wang, X., Liu, Z., Eimerl, S., Timberg, R., Weiss, A.M., Orly, J., Stocco, D.M., 1998. Effect of truncated forms of the steroidogenic acute regulatory protein on intramitochondrial cholesterol transfer. Endocrinology 139, 3903 – 3912. Wang, R., Guo, W., Ossipov, M.H., Vanderah, T.W., Porreca, F., Lai, J., 2003. Glial cell line-derived neurotrophic factor normalizes neurochemical changes in injured dorsal root ganglion neurons and prevents the expression of experimental neuropathic pain. Neuroscience 121, 815 – 824. West, L.A., Horvat, R.D., Roess, D.A., Barisas, B.G., Juengel, J.L., Niswender, G.D., 2001. Steroidogenic acute regulatory protein and peripheral-type benzodiazepine receptor associate at the mitochondrial membrane. Endocrinology 142, 502 – 505. Xiao, H.S., Huang, Q.H., Zhang, F.X., Bao, L., Lu, Y.J., Guo, C., Yang, L., Huang, W.J., Fu, G., Xu, S.H., Cheng, X.P., Yan, Q., Zhu, Z.D., Zhang, X., Chen, Z., Han, Z.G., Zhang, X., 2002. Identification of gene expression profile of dorsal root ganglion in the rat peripheral axotomy model of neuropathic pain. Proc. Natl. Acad. Sci. U. S. A. 99, 8360 – 8365.
Role of the peripheral benzodiazepine receptor in sensory neuron regeneration Charles D. Mills,* Jaquelyn L. Bitler, and Clifford J. Woolf Neural Plasticity Research Group, Massachusetts General Hospital and Harvard Medical School, 149 13th Street, Charlestown, MA 02129, USA Received 4 May 2005; revised 13 July 2005; accepted 15 July 2005 Available online 15 August 2005
Peripheral benzodiazepine receptor (PBR) expression increases in small dorsal root ganglion (DRG) sensory neurons after peripheral nerve injury. To determine the functional significance of this induction, we evaluated the effects of PBR ligands on rodent sensory axon outgrowth. In vitro, Ro5-4864, a PBR agonist, enhanced outgrowth only of small peripherin-positive DRG neurons. When DRG cells were preconditioned into an active growth state by a prior peripheral nerve injury Ro5-4864 augmented and PK 11195, a PBR antagonist, blocked the injury-induced increased outgrowth. In vivo, Ro5-4864 increased the initiation of regeneration after a sciatic nerve crush injury and the number of GAP-43-positive axons in the distal nerve while PK 11195 inhibited the enhanced growth produced by a preconditioning lesion. These results show that PBR has a role in the early regenerative response of small caliber sensory axons, the preconditioning effect, and that PBR agonists enhance sensory axon regeneration. D 2005 Elsevier Inc. All rights reserved.
Introduction Injury to the peripheral axon of dorsal root ganglion (DRG) neurons induces changes in gene expression that initiate regeneration. Microarray studies of changes in gene expression profiles in these neurons have helped to elucidated many potential survival and regeneration-associated genes (Bonilla et al., 2002; Costigan et al., 2002; Xiao et al., 2002). One identified injury-induced gene is the peripheral benzodiazepine receptor (PBR), an 18 kDa mitochondrial membrane protein that forms heteromeric complexes with the mitochondrial permeability transition pore, and plays key roles in steroidogenesis and apoptosis (Casellas et al., 2002; Gavish et al., 1999). However, the functional significance of PBR upregulation in injured DRG neurons remains unclear.
* Corresponding author. Fax: +1 617 724 3632. E-mail address: [email protected] (C.D. Mills). Available online on ScienceDirect (www.sciencedirect.com). 1044-7431/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2005.07.010
In the central nervous system, PBR expression is usually associated with microglia (Casellas et al., 2002; Gavish et al., 1999). However, PBR is also expressed in cultured cortical neurons (Jayakumar et al., 2002) and DRG neurons in vivo after peripheral nerve injury (Karchewski et al., 2004; Xiao et al., 2002). PBR is involved in the mitochondrial intermembrane transport of cholesterol through interactions with the steroidogenic acute regulatory protein (StAR) (Krueger and Papadopoulos, 1990; Papadopoulos et al., 1990; Wang et al., 1998; West et al., 2001) thereby regulating synthesis of the neurosteroids pregnenolone (PREG), progesterone (PROG), and dehydroepiandrosterone (DHEA) (Ferzaz et al., 2002; Korneyev et al., 1993; Lacor et al., 1999; Papadopoulos et al., 1997). Both cholesterol and neurosteroids are important for neuronal regeneration (Schumacher et al., 1996). For example, cholesterol deficiency inhibits axonal branching and promotes axonal degeneration (Fan et al., 2001, 2002), DHEA enhances functional recovery and increases the number of nerve fibers in the sciatic nerve after a crush injury (Gudemez et al., 2002), and PROG increases neurite outgrowth in vitro and in vivo (Koenig et al., 1995, 2000). Two synthetic ligands are widely used to study PBR, Ro5-4864 (a reported agonist) and PK 11195 (an antagonist), each having nanomolar affinity for PBR (Awad and Gavish, 1987; Krueger, 1995; Le Fur et al., 1983a,b,c; Schoemaker et al., 1983; Syapin and Skolnick, 1979). The putative endogenous ligand for PBR, diazepam binding inhibitor (DBI), is a 10 kDa peptide that is cleaved into several active fragments: eicosaneuropeptide (ENP), octadecaneuropeptide (ODN), and triakontatetraneuropeptide (TTN) (Alho et al., 1990; Costa and Guidotti, 1991; Guidotti et al., 1983; Shoyab et al., 1986; Slobodyansky et al., 1989). Schwann cells and fibroblasts in the sciatic nerve express ODN and its expression increases in the distal nerve segment after peripheral nerve injury (Lacor et al., 1996, 1999). DBI is constitutively expressed at high levels in non-neuronal satellite cells of the DRG but its expression does not change after a peripheral nerve injury (Karchewski et al., 2004). We have now investigated whether PBR has a role in peripheral nerve regeneration and found that Ro5-4864 enhanced neurite outgrowth in rat small caliber sensory neurons in vitro and also
C.D. Mills et al. / Mol. Cell. Neurosci. 30 (2005) 228 – 237
enhanced mouse sensory axon regeneration in vivo, while PK 11195 inhibited nerve injury-induced outgrowth and blocked the effects of Ro5-4864. These data demonstrate a role for PBR in sensory axon regeneration after peripheral nerve injury.
Results Localization of PBR in rat DRG neurons PBR is upregulated in the rat DRG following sciatic nerve axotomy (Costigan et al., 2002; Karchewski et al., 2004; Xiao et
229
al., 2002). Here, we show by in situ hybridization that PBR mRNA is detected only in a subset of adult rat DRG neurons, those that are peripherin-positive (i.e. small DRG neurons with unmyelinated axons) following a sciatic nerve axotomy (Figs. 1A – F). Approximately 30% of neuronal profiles express PBR mRNA after peripheral nerve injury and of those, 98% have cell diameters less than 45 AM (Karchewski et al., 2004). PBR mRNA was not detected after nerve injury in large NF 200-positive neurons (with myelinated axons), Schwann cells, or satellite cells. When adult rat DRG neurons are dissociated and grown in vitro, PBR-immunoreactivity (IR) was localized throughout the cell, including the soma, neurites, and growth cones (Figs. 1G – I).
Fig. 1. Induction of PBR in rat DRG neurons in vivo and in vitro. PBR mRNA expression was low/undetectable in uninjured DRGs (A,B), but increased in peripherin-positive cells of L4 DRGs 7 days after a sciatic nerve axotomy (C,D). PBR mRNA expression was never seen in large NF 200-positive neurons (*) or in the non-neuronal satellite cells (r) that surround DRG neurons (E,F). Photomicrograph of a dissociated DRG neuron grown in culture for 24 h showing PBR-IR in the cell body, neurites, and growth cones (G). Confocal image of cultured DRG neuron growth cones labeled with h-tublin (H) and PBR (I). Scale bars = 50 Am (A – F) and 20 Am (G).
230
C.D. Mills et al. / Mol. Cell. Neurosci. 30 (2005) 228 – 237
Effects of PBR ligands on neurite outgrowth and survival in adult rat cultured DRG neurons The effects of two PBR ligands, Ro5-4864 and PK 11195, on neurite growth were measured in three different adult rat DRG neuron culture assays. In the first assay, designed to look at the actions of PBR ligands on neurite initiation, the ligands were added to the media 2 h after culturing and neurite outgrowth
measured 16 h later. Whereas PK 11195 had no effect on the length or the number of neurons with neurites, Ro5-4864, in a dose-dependent manner, significantly increased mean neurite length (Fig. 2D). In the second assay, looking at action of the PBR ligands on established growth, neurons were grown for 12 h before treatment and then grown for a further 24 h. In this assay, Ro5-4864 also significantly increased neurite outgrowth, while the highest dose of PK 11195 tested (1 AM) reduced outgrowth and
Fig. 2. Effects of PBR ligands on rat DRG neurite outgrowth in vitro. Sixteen hour treatment with Ro5-4864 dose-dependently increased neurite outgrowth when administered 2 h after culturing (A,B,D) without affecting the number of cells growing (F). Twenty-four hours of Ro5-4864 treatment, initiated 12 h after culturing, also dose-dependently increased neurite outgrowth (C,E) without affecting the number of cells growing (G). At the highest dose tested, PK 11195 had a slight inhibitory effect on neurite outgrowth and, when co-administered with Ro5-4864, PK 11195 completely blocked the growth promoting effects of Ro54864 (1 AM each). Outgrowth is reported as percent of naı¨ve, defined as 100%. *P < 0.05, **P < 0.01, and ***P < 0.001 compared to vehicle treatment groups.
C.D. Mills et al. / Mol. Cell. Neurosci. 30 (2005) 228 – 237
completely blocked the growth promoting effects of Ro5-4864 (Fig. 2E). Sciatic nerve axotomy 1 week prior to culturing preconditions DRG cells into an active growth state. This paradigm increases neurite initiation and the extent of neurite outgrowth of
231
cultured DRG neurons (Fig. 3) (Hu-Tsai et al., 1994; Lankford et al., 1998). The third assay was designed to look at action of PBR ligands on cells preconditioned by a prior nerve injury. Ro5-4864 treatment further enhanced neurite growth in the preconditioned cells, while PK 11195, at the highest dose tested (1 AM), inhibited the preconditioning effect (Fig. 3). Since PBR is expressed only in small cells after injury, the differential effect of Ro5-4864 on neurite outgrowth in peripherinand NF 200-positive cells was studied. In our cultures, 25% of neurons are NF 200-positive (Figs. 4A, B). Ro5-4864 increased neurite outgrowth in the peripherin-positive cells, but not NF-200positive cells (Figs. 4C, D). To rule out possible cell survival effects of the PBR ligands on axon outgrowth, we tested the effects of PK 11195 and Ro5-4864 on the survival of cultured adult rat DRGs. Neither PK 11195 nor Ro5-4864 at any of the doses tested (10 nM – 1 AM) affected the survival of adult DRG neurons in culture (Table 1). Effects of PBR ligands on the rate of peripheral nerve regeneration after injury in the adult mouse
Fig. 3. Effects of PBR ligands on preconditioned DRG rat neurons. A preconditioning sciatic nerve axotomy 7 days before dissociation and culturing increased neurite outgrowth (A – C) and the percentage of cells with neurites (D). PK 11195 (1 AM) partially inhibited the injury-induced increase in growth (C), while Ro5-4864 dose-dependently enhanced the preconditioning effect without affecting the number of cells growing (D). Asterisks indicate significance compared to the vehicle treatment group.
To avoid large injection volumes resulting from the amount of ligand needed and the poor solubility of the ligands in solutions other that ethanol, adult mice were used instead of adult rats. The rate of peripheral nerve regeneration in the sciatic nerve was determined using the nerve pinch test 2 and 4 days after a crush injury. The nerve pinch test determines the extent of regrowth by measuring the distance from the injury site to the most distal point on the nerve that produces a reflexive withdrawal when pinched with smooth forceps (Bajrovic et al., 2001; Kovacic et al., 2004). Ro5-4864 treated mice had higher rates of regeneration than crush alone, vehicle, and PK 11195 treated animals (Figs. 5A, B). Moreover, PK 11195 completely blocked the growth promoting effects of Ro5-4864. The absolute differences in mean growth rates remained constant between groups at both 2 and 4 days after injury, indicating that Ro54864 treatment specifically accelerated the initiation of axonal regeneration without increasing the maximum rate of growth. As seen in vitro, a preconditioning sciatic nerve injury enhanced the extent of regrowth 2 days after a second injury (Fig. 5C). Treatment with Ro5-4864 was as effective as a preconditioning lesion at initiating regrowth (Figs. 5A vs. C). Similar to the in vitro results (Figs. 2, 3), PK 11195 treatment blocked the preconditioning effect and the growth promoting effects of Ro54864 in the nerve pinch test. The extent of regeneration was confirmed by histological examination of the sciatic nerve. Markers for subpopulations of cells within the DRG and their associated fibers change in distribution and intensity after peripheral nerve injury. For example, NF-H (a large cell marker), CGRP, and IB4 (both markers for small and medium cells) are downregulated after peripheral nerve injury (Verge et al., 1995; Hammond et al., 2004; Wang et al., 2003). In contrast, GAP-43 is expressed in all growing axons and is widely used as a marker of regenerating fibers (Skenen, 1989; Sommervaille et al., 1991; Gaete et al., 1998). Therefore, we used the number of GAP-43 fibers to identify regenerating axons. Two days after injury, Ro5-4864 treated mice had greater numbers of GAP-43-positive fibers compared to other treatment groups 1.5 mm distal to the crush site (Fig. 6). However, since GAP-43 labels all growing fibers and does not differentiate between subpopulations of fiber types,
232
C.D. Mills et al. / Mol. Cell. Neurosci. 30 (2005) 228 – 237
Fig. 4. Effects of Ro5-4864 on rat DRG neurite outgrowth in peripherin- and NF 200-positive cells. Dissociated DRGs neurons (A) stained for peripherin (green) and NF 200 (red). When DRG cells were dissociated and allowed to grow for 36 h, there were three times as many peripherin-positive cells as NF 200positive (B). Twenty-four hour treatment with Ro5-4864 (1 AM) increased neurite outgrowth only of the peripherin-positive cells (C), without affecting the percentage growing of either cell type (D). Asterisks indicate significance between groups (unpaired, two-tailed t test).
the increase in outgrowth cannot be assigned to a specific subpopulation of fibers.
Discussion Given the induction of PBR in small DRG sensory neurons after peripheral nerve injury (Karchewski et al., 2004; Xiao et al., 2002), the regulation of steroid production by PBR, and the role that steroids play in axon growth, we investigated if PBR has a role in peripheral nerve regeneration. Although PBR is predominantly expressed on the outer mitochondrial membrane, it can also be found on the plasma membrane (Garnier et al., 1993; Oke et al., 1992), the inner mitochondrial membrane (Mukherjee and Das, 1989), and in nuclei (Hardwick et al., 1999; Kuhlmann and Guilarte, 2000). These different localizations may reflect different isoforms of PBR (Berkovich et al., 1993; Cahard et al., 1994; Tanimoto et al., 1999). We find that dissociated DRG neurons show PBR expression throughout the cell body (including the nucleus), neurites, and growth cones (Fig. 1). Nuclear PBR localization is reported in activated astrocytes, microglia (Kuhlmann and Gui-
larte, 2000), and invasive breast tumor cells where it regulates entry of cholesterol into the nucleus (Hardwick et al., 1999). While mitochondrial PBRs are involved in cellular respiration and cholesterol transport for steroidogenesis, the function of extramitochondrial and plasma membrane PBRs remains unclear. Intracellular cholesterol plays an essential role in the maintenance of microtubule stability (Fan et al., 2001), and axonal regeneration is dependent on a local supply (reutilization) of cholesterol (Goodrum et al., 2000). It is possible that PBR expressed in neurites and growth cones sequesters cholesterol for microtubule stability and membrane synthesis needed during regeneration. The PBR may also affect neurite outgrowth by controlling the rate of neurosteroid formation. Mitochondrial PBRs regulate the transport of cholesterol from the outer to inner membrane, which is the rate-limiting step in steroid production. The neurosteroids, PREG, PROG, and DHEA play major roles in neuronal regeneration. PROG increases neurite outgrowth of DRG explants, promotes regeneration in cryolesioned sciatic nerves, and promotes remyelination of regenerated nerve fibers (Koenig et al., 1995, 2000). Treatment with Ro5-4864 increases PREG production in the regenerating sciatic nerve, an effect blocked by PK 11195, which alone was found to have no effect (Lacor et al., 1999). PREG and
Table 1 Effect of PBR ligands on survival of adult dissociated DRG neurons Treatment duration
Control
Vehicle
PK 11195
Ro5-4864
10 nM
100 nM
1 AM
10 nM
100 nM
1 AM
24 h 48 h 96 h
89.9 T 8.9 90.0 T 2.5 74.8 T 10.0
83.2 T 3.1 81.5 T 1.3 62.8 T 2.6
91.5 T 3.1 87.3 T 6.2 66.4 T 5.8
94.5 T 2.3 90.7 T 4.0 69.7 T 7.6
91.3 T 13.2 88.6 T 9.7 67.7 T 9.3
90.9 T 1.7 81.2 T 5.5 55.1 T 0.6
92.1 T 1.5 85.7 T 6.3 64.0 T 4.3
95.3 T 3.0 90.9 T 6.2 77.7 T 10.4
Survival of adult DRG neurons treated with PBR ligands expressed as a percentage of baseline.
C.D. Mills et al. / Mol. Cell. Neurosci. 30 (2005) 228 – 237
233
returns to basal levels with reinnervation (Lacor et al., 1996, 1999). It is plausible that Ro5-4864 acts on Schwann cells to produce PREG, providing trophic support for regeneration. Ro5-4864 also increases cytokine synthesis (Taupin et al., 1993) and cytokines stimulate the synthesis of NGF by macrophages and Schwann cells, which would also provide trophic support enhancing regeneration (Ferzaz et al., 2002; Lacor et al., 1999). Another way that PBR could regulate outgrowth is from an increase in availability of ATP, which is an essential factor for neurite growth (Behrsing and Vulliet, 1999, 2004; D’Ambrosi et al., 2001). PBR associates with the mitochondrial permeability transition pore (MPTP), which allows the respiratory chain to create the transmembrane electrochemical gradient (DCm) that drives ATP synthesis. Specifically, PBR associates with the voltage-dependent anion channel (VDAC) and the adenine nucleotide carrier (ANC) of the MPTP. The VDAC is the main pathway for metabolite diffusion across the outer membrane and the ANC is an antiporter exchanging ATP generated in the matrix for ADP. Opening the MPTP dissipates the DCm and decreases ATP production. Reports show that PBR ligands regulate the DCm differently depending on the system being examined (Akao et al.,
Fig. 5. Effects of PBR ligands on peripheral nerve regeneration in the adult mouse. Mean distances of sensory axon regrowth 2 days (A) and 4 days (B) following a crush injury of the sciatic nerve as determined by the nerve pinch test. Ro5-4864 (10 mg/kg) increased the rate of peripheral nerve regeneration, an effect blocked by co-administration of PK 11195 (10 mg/kg each). Mean distances of sensory axon regrowth 2 days following crush injury in preconditioned sciatic nerves (C). PK 11195 blocked the preconditioning effect and the growth promoting effects of Ro5-4864. Asterisks indicate significance compared to the vehicle treatment group.
PROG levels increase in the injured sciatic nerve and have neurotrophic effects (Akwa et al., 1993; Koenig et al., 1995). PBR expression increases markedly in non-neuronal Schwann cells and macrophages in the regenerating sciatic nerve after injury and
Fig 6. Ro5-4864 treatment increased the numbers of regenerating fibers in the adult mouse sciatic nerve. Photomicrographs of GAP-43 IR fibers 1.5 mm from the injury site at 2 days post-injury: crush only (A), PK 11195 (B), Ro54864 (C), and PK 11195 + Ro5-4864 treated mice (10 mg/kg each; D). Mean number of GAP-43 IR fibers/section (E). Asterisks indicate significance compared to the vehicle treatment group.
234
C.D. Mills et al. / Mol. Cell. Neurosci. 30 (2005) 228 – 237
2004; Chelli et al., 2001, 2004; Fischer et al., 2001; Ravagnan et al., 1999; Tanimoto et al., 1999); however, the specificity of these ligands in the control of cellular respiration has been questioned (Casellas et al., 2002; Gavish et al., 1999). Although PBR regulates DCm and apoptosis, we find that PBR ligands have no effect on the survival of adult dissociated DRG cells in short-term cultures or the total percentage of cells growing. These results demonstrate that the effects of PBR ligands on growth are independent of any effects on survival. After peripheral nerve crush, there is an initial delay in the initiation of regrowth (McQuarrie et al., 1977; Pan et al., 2003). Ro5-4864 averts this initial stalling as evident by the increased growth detected 2 days after injury. Once peripheral regeneration begins, the rate increases to reach a maximum around 3 days after injury (Danielsen et al., 1986; McQuarrie et al., 1977; Pan et al., 2003). Our data suggest that Ro5-4864 primarily targets the initiation phase of growth rather than increasing the maximum rate of regeneration. An initial delay in growth is also seen when DRG cells are grown in vitro (Kocsis et al., 1994; Lankford et al., 1998). Ro5-4864 overcame this delay as shown by the 40% increase in growth after 18 h in culture. PK 11195 blocked the effects of Ro54864 on neurite outgrowth in vitro and peripheral regeneration in vivo, demonstrating that PK 11195 is antagonistic to the effects of Ro5-4864. DBI, the endogenous ligand for PBR, is expressed in nonneuronal cells of the sciatic nerve and DRG. Following sciatic nerve injury, there is an upregulation of ODN, a biologically active fragment of DBI, in Schwann cells of the distal segment where it is implicated in the reutilization of cholesterol for myelin membrane formation (Lacor et al., 1996, 1999). ODN levels in the sciatic nerve return to basal levels once regeneration is complete (Lacor et al., 1999) However, an injury-induced upregulaton of DBI is not seen in the DRG (Karchewski et al., 2004). The high level of DBI expressed in the DRG may represent a source of ligand for the injury-induced upregulation of PBR after injury. Axonal injury induces growth-associated gene expression within the cell body that transforms the injured cell into an active growth state. If axons are injured a second time during this growth state, regeneration is enhanced (Forman et al., 1980; Hu-Tsai et al., 1994; Lankford et al., 1998; McQuarrie et al., 1977; Neumann and Woolf, 1999; Qiu et al., 2002; Sjoberg and Kanje, 1990). The first lesion, termed the preconditioning lesion, overcomes the delay in initiating regrowth, and increases the speed of regeneration such that the maximum growth rate is reach sooner (Pan et al., 2003). In DRG neurons grown in culture, Ro5-4864 enhanced, while PK 11195 inhibited the preconditioning effect, demonstrating that pharmacological activation of PBR by Ro5-4864 contributes to the preconditioning effect. In vivo, PK 11195, presumably by antagonizing an endogenous PBR ligand, blocks the preconditioning effect indicating that activation of PBR contributes physiologically to the preconditioning effect. Conclusion PBR is induced in small peripherin-positive cells of the DRG following a peripheral nerve injury. The PBR ligand Ro5-4864, but not PK 11195, increased neurite outgrowth of peripherin-positive dissociated DRG neurons, with no effect on large NF 200-positive cells. Ro5-4864 treatment also increased the rate of peripheral nerve regeneration and the numbers of GAP-43 fibers in the sciatic
nerve following a crush injury in the adult mouse, an effect blocked by PK 11195. Ro5-4864 treatment overcame the initial delay in regeneration without affecting the speed of regenerative axonal growth and PK 11195, in addition to blocking the action of Ro54864, inhibited the conditioning effect of a prior nerve lesion. These data demonstrate a specific role for the PBR in initiating sensory neuron regeneration.
Experimental methods Experimental animals and injury production Adult male Sprague – Dawley rats, 190 – 210 g, and adult C57 BL/6 mice, 25 – 30 g, were obtained from Charles Rivers Laboratories and housed with a light/dark cycle of 12 h/12 h. All procedures were performed in accordance with Massachusetts General Hospital animal care regulations. Axotomy in the adult rat was produced by exposing the left sciatic nerve at the mid-thigh level, ligating with 3 – 0 suture and then transecting the nerve distal to the ligature. For crush injuries in adult mice, the left sciatic nerve was crushed with smooth forceps for 15 s at the proximal thigh level and the injury site marked with a 10 – 0 epineural suture. After injury, muscle and skin were closed in layers and the animals allowed to recover. Preconditioning axotomies in adult rats were performed 7 days prior to collecting DRGs for culturing. Adult mouse preconditioning crush injuries were performed 3 days prior to a second crush injury. Ligand preparation and dosing The PBR ligands PK 11195 and Ro5-4864 were obtained from Sigma and 1 mM stock solutions were prepared in 100% EtOH. For cell culture experiments, stock solutions were diluted with 100% EtOH so that 2 Al of ligand was added to 300 Al of cell culture media. For peripheral nerve regeneration experiments, 10 mg/kg/day of each ligand prepared in 100% EtOH was given intraperitoneally beginning at the time of injury (Torres et al., 2000; Wala et al., 2000). Cell culture, cell survival, and neurite outgrowth assays Primary adult dissociated DRG neuron-enriched cultures were prepared by dissecting DRGs into HBSS (Cellgro) and 10 mM HEPES, followed by digestion with 5 mg/ml collagenase A and 1 mg/ml dispase II (Roche) prior to treatment with 0.25% trypsin (GibcoBRL). Triturated cells were centrifuged through a Percoll (Sigma) gradient prior to plating on laminin in Neurobasal media (GibcoBRL) containing 2% (vol/vol) B27 supplement (GibcoBRL), 50 Ag/ml Pen-Strep, 10 AM Ara-C, 50 ng/ml NGF, 200 mM l-glutamine, and 2 ng/ml GDNF. Neurite outgrowth was determined by measuring the length of the longest neurite per cell using Image J software (NIH). A cell was defined as growing if it extended a neurite twice its diameter. For cell survival assays, adult DRG neurons were cultured and allowed to grow for 48 h, baseline counts were obtained and then cultures were treated with PBR ligands and neurons counted at 24, 48, and 96 h. The number of cells surviving is reported as a percent of baseline. Values reported for both survival and outgrowth assays are from three experiments repeated in triplicate. All measures were done blinded to condition.
C.D. Mills et al. / Mol. Cell. Neurosci. 30 (2005) 228 – 237
235
Immunochemistry
Statistical analysis
Cultured cells were fixed with 4% paraformaldehyde for 20 min, washed with PBS, incubated with polyclonal antibodies to PBR (a gift from Dr. Vassilios Papadopoulos; Delavoie et al., 2003), and visualized using Alex Fluor 488 (Molecular Probes). Confocal images were obtained using a Leica TCS NTD4D confocal laser microscopy imaging system (Leica, Heidelberg, Germany). For neurite outgrowth assays, cultured cells were incubated with GAP-43 (Chemicon) and Hsp 25 (Stressgen), with peripherin (Chemicon) and NF 200 (Sigma), or h-tubulin III (Sigma).
A one-way ANOVA was performed to assess changes in neurite outgrowth, peripheral nerve regeneration lengths, and the numbers of GAP-43-positive fibers. Tukey’s multiple comparison test was used for post hoc pairwise comparisons. An unpaired, two-tailed t test was used for pairwise comparisons of outgrowth between peripherin- and NF 200-positive cells. All data are reported as means T standard error of the mean (SEM).
In situ hybridization
This work was supported by NINDS grants NS45459 and NS038253.
For in situ hybridization, fresh DRGs were dissected, frozen in OCT, and stored at 80-C until sectioning at 10 Am. Fresh sections were fixed with 4% paraformaldehyde for 15 min, washed with PBS, acetylation solution [1.35% triethanolamine (TEA), 0.25% acetic anhydride] for 10 min, and hybridization buffer [50% formamide, 5 Denhardt’s solution, 5 saline sodium citrate (SSC), 0.25 mg/ml yeast RNA, 0.5 mg/ml denatured Salmon sperm DNA] for 2 h. Sections were hybridized with a digoxigenin labeled antisense probe to PBR (accession number NM 012515) corresponding to base pairs 3 – 671 (669 bps total) at 72-C overnight. Slides were washed with 0.2 SSC at 65-C, blocked, and then incubated with anti-digoxigenin Fab fragments (Roche Diagnostics) at 4-C overnight. Visualization was done using 3.5 Al/ ml 5-bromo-4-cholor-3-indolyl-phosphate (BCIP; Roche Diagnostics) and 4.5 Al/ml 4-nitro blue tetrazolium chloride (NBT; Roche Diagnostics) in 0.1 M Tris pH 9.5, 0.1 M NaCl, 50 mM MgCl2. Peripheral nerve regeneration The nerve pinch test was used to determine the rate of in vivo peripheral nerve regeneration (Bajrovic et al., 2001; Kovacic et al., 2004). To avoid large injection volumes resulting from the amount of ligand needed and the poor solubility of the ligands in solutions other that ethanol, adult mice were used instead of adult rats. On post-injury days 2 and 4 following sciatic crush, anesthesia was induced with 2.5% isoflurane and the left sciatic nerve exposed. The animals were brought to a light plane of anesthesia by lowering the isoflurane concentration to 1%—a level where pinching the skin of the contralateral uninjured paw elicited a reflex withdrawal. Starting distally, a series of pinches using fine smooth forceps was delivered to the sciatic nerve moving proximally toward the injury site. The rate of regeneration was determined by measuring the distance from the injury site to the most distal point on the nerve that produced a reflexive withdrawal when pinched (n = 5 – 6 per group). All measures were done blinded to condition. Since markers for subpopulations of cells within the DRG and their associated fibers change in distribution and intensity after peripheral nerve injury, we used GAP-43 that is expressed in all regenerating fibers to histologically characterize regeneration in the injured sciatic nerve 2 days post-injury. For quantification, sciatic nerves were serially sectioned at 10 Am and the mean number of GAP-43 immunoreactive fibers/section was determined in three sections (at least 30 Am apart) per animal, 1.5 mm beyond the injury site. The mean number of fibers/section per animal was determined for three animals in each treatment group.
Acknowledgments
References Akao, Y., Seki, N., Nakagawa, Y., Yi, H., Matsumoto, K., Ito, Y., Ito, K., Funaoka, M., Maruyama, W., Naoi, M., Nozawa, Y., 2004. A highly bioactive lignophenol derivative from bamboo lignin exhibits a potent activity to suppress apoptosis induced by oxidative stress in human neuroblastoma SH-SY5Y cells. Bioorg. Med. Chem. 12, 4791 – 4801. Akwa, Y., Schumacher, M., Jung-Testas, I., Baulieu, E.E., 1993. Neurosteroids in rat sciatic nerves and Schwann cells. C. R. Acad. Sci., Ser. III 316, 410 – 414. Alho, H., Bovolin, P., Slobodyansky, E., 1990. Diazepam binding inhibitor (DBI) processing: immunohistochemical studies in the rat brain. Neurochem. Res. 15, 209 – 216. Awad, M., Gavish, M., 1987. Binding of [3H]Ro 5-4864 and [3H]PK 11195 to cerebral cortex and peripheral tissues of various species: species differences and heterogeneity in peripheral benzodiazepine binding sites. J. Neurochem. 49, 1407 – 1414. Bajrovic, F., Srpcic, M., Sketelj, J., 2001. Schwann cell dependence of regenerating rat sensory neurons is inversely related to the quality of axon growth substratum. J. Neuropathol. Exp. Neurol. 60, 65 – 74. Behrsing, H.P., Vulliet, P.R., 1999. Purinergic and calcium-mediated enhancement of NGF-induced neurite expression in PC12 cells. Proc. West. Pharmacol. Soc. 42, 59 – 62. Behrsing, H.P., Vulliet, P.R., 2004. Mitogen-activated protein kinase mediates purinergic-enhanced nerve growth factor-induced neurite outgrowth in PC12 cells. J. Neurosci. Res. 78, 64 – 74. Berkovich, A., Ferrarese, C., Cavaletti, G., Alho, H., Marzorati, C., Bianchi, G., Guidotti, A., Costa, E., 1993. Topology of two DBI receptors in human lymphocytes. Life Sci. 52, 1265 – 1277. Bonilla, I.E., Tanabe, K., Strittmatter, S.M., 2002. Small proline-rich repeat protein 1A is expressed by axotomized neurons and promotes axonal outgrowth. J. Neurosci. 22, 1303 – 1315. Cahard, D., Canat, X., Carayon, P., Roque, C., Casellas, P., Le Fur, G., 1994. Subcellular localization of peripheral benzodiazepine receptors on human leukocytes. Lab. Invest. 70, 23 – 28. Casellas, P., Galiegue, S., Basile, A.S., 2002. Peripheral benzodiazepine receptors and mitochondrial function. Neurochem. Int. 40, 475 – 486. Chelli, B., Falleni, A., Salvetti, F., Gremigni, V., Lucacchini, A., Martini, C., 2001. Peripheral-type benzodiazepine receptor ligands: mitochondrial permeability transition induction in rat cardiac tissue. Biochem. Pharmacol. 61, 695 – 705. Chelli, B., Lena, A., Vanacore, R., Pozzo, E.D., Costa, B., Rossi, L., Salvetti, A., Scatena, F., Ceruti, S., Abbracchio, M.P., Gremigni, V., Martini, C., 2004. Peripheral benzodiazepine receptor ligands: mitochondrial transmembrane potential depolarization and apoptosis induction in rat C6 glioma cells. Biochem. Pharmacol. 68, 125 – 134. Costa, E., Guidotti, A., 1991. Diazepam binding inhibitor (DBI): a peptide with multiple biological actions. Life Sci. 49, 325 – 344.
236
C.D. Mills et al. / Mol. Cell. Neurosci. 30 (2005) 228 – 237
Costigan, M., Befort, K., Karchewski, L., Griffin, R.S., D’Urso, D., Allchorne, A., Sitarski, J., Mannion, J.W., Pratt, R.E., Woolf, C.J., 2002. Replicate high-density rat genome oligonucleotide microarrays reveal hundreds of regulated genes in the dorsal root ganglion after peripheral nerve injury. BMC Neurosci. 3, 16. D’Ambrosi, N., Murra, B., Cavaliere, F., Amadio, S., Bernardi, G., Burnstock, G., Volonte, C., 2001. Interaction between ATP and nerve growth factor signalling in the survival and neuritic outgrowth from PC12 cells. Neuroscience 108, 527 – 534. Danielsen, N., Lundborg, G., Frizell, M., 1986. Nerve repair and axonal transport: outgrowth delay and regeneration rate after transection and repair of rabbit hypoglossal nerve. Brain Res. 376, 125 – 132. Delavoie, F., Li, H., Hardwick, M., Robert, J.C., Giatzakis, C., Peranzi, G., Yao, Z.X., Maccario, J., Lacapere, J.J., Papadopoulos, V., 2003. In vivo and in vitro peripheral-type benzodiazepine receptor polymerization: functional significance in drug ligand and cholesterol binding. Biochemistry 42, 4506 – 4519. Fan, Q.W., Yu, W., Senda, T., Yanagisawa, K., Michikawa, M., 2001. Cholesterol-dependent modulation of tau phosphorylation in cultured neurons. J. Neurochem. 76, 391 – 400. Fan, Q.W., Yu, W., Gong, J.S., Zou, K., Sawamura, N., Senda, T., Yanagisawa, K., Michikawa, M., 2002. Cholesterol-dependent modulation of dendrite outgrowth and microtubule stability in cultured neurons. J. Neurochem. 80, 178 – 190. Ferzaz, B., Brault, E., Bourliaud, G., Robert, J.P., Poughon, G., Claustre, Y., Marguet, F., Liere, P., Schumacher, M., Nowicki, J.P., Fournier, J., Marabout, B., Sevrin, M., George, P., Soubrie, P., Benavides, J., Scatton, B., 2002. SSR180575 (7-chloro-N,N,5-trimethyl-4-oxo-3phenyl-3,5-dihydro-4H-pyridazino[4,5-b]i ndole-1-acetamide), a peripheral benzodiazepine receptor ligand, promotes neuronal survival and repair. J. Pharmacol. Exp. Ther. 301, 1067 – 1078. Fischer, R., Schmitt, M., Bode, J.G., Haussinger, D., 2001. Expression of the peripheral-type benzodiazepine receptor and apoptosis induction in hepatic stellate cells. Gastroenterology 120, 1212 – 1226. Forman, D.S., McQuarrie, I.G., Labore, F.W., Wood, D.K., Stone, L.S., Braddock, C.H., Fuchs, D.A., 1980. Time course of the conditioning lesion effect on axonal regeneration. Brain Res. 182, 180 – 185. Gaete, J., Kameid, G., Alvarez, J., 1998. Regenerating axons of the rat require a local source of proteins. Neurosci. Lett. 251, 197 – 200. Garnier, M., Boujrad, N., Oke, B.O., Brown, A.S., Riond, J., Ferrara, P., Shoyab, M., Suarez-Quian, C.A., Papadopoulos, V., 1993. Diazepam binding inhibitor is a paracrine/autocrine regulator of Leydig cell proliferation and steroidogenesis: action via peripheral-type benzodiazepine receptor and independent mechanisms. Endocrinology 132, 444 – 458. Gavish, M., Bachman, I., Shoukrun, R., Katz, Y., Veenman, L., Weisinger, G., Weizman, A., 1999. Enigma of the peripheral benzodiazepine receptor. Pharmacol. Rev. 51, 629 – 650. Goodrum, J.F., Brown, J.C., Fowler, K.A., Bouldin, T.W., 2000. Axonal regeneration, but not myelination, is partially dependent on local cholesterol reutilization in regenerating nerve. J. Neuropathol. Exp. Neurol. 59, 1002 – 1010. Gudemez, E., Ozer, K., Cunningham, B., Siemionow, K., Browne, E., Siemionow, M., 2002. Dehydroepiandrosterone as an enhancer of functional recovery following crush injury to rat sciatic nerve. Microsurgery 22, 234 – 241. Guidotti, A., Forchetti, C.M., Corda, M.G., Konkel, D., Bennett, C.D., Costa, E., 1983. Isolation, characterization, and purification to homogeneity of an endogenous polypeptide with agonistic action on benzodiazepine receptors. Proc. Natl. Acad. Sci. U. S. A. 80, 3531 – 3535. Hammond, D.L., Ackerman, L., Holdsworth, R., Elzey, B., 2004. Effects of spinal nerve ligation on immunohistochemically identified neurons in the L4 and L5 dorsal root ganglia of the rat. J. Comp. Neurol. 475, 575 – 589. Hardwick, M., Fertikh, D., Culty, M., Li, H., Vidic, B., Papadopoulos, V., 1999. Peripheral-type benzodiazepine receptor (PBR) in human breast cancer: correlation of breast cancer cell aggressive phenotype with PBR
expression, nuclear localization, and PBR-mediated cell proliferation and nuclear transport of cholesterol. Cancer Res. 59, 831 – 842. Hu-Tsai, M., Winter, J., Emson, P.C., Woolf, C.J., 1994. Neurite outgrowth and GAP-43 mRNA expression in cultured adult rat dorsal root ganglion neurons: effects of NGF or prior peripheral axotomy. J. Neurosci. Res. 39, 634 – 645. Jayakumar, A.R., Panickar, K.S., Norenberg, M.D., 2002. Effects on free radical generation by ligands of the peripheral benzodiazepine receptor in cultured neural cells. J. Neurochem. 83, 1226 – 1234. Karchewski, L.A., Bloechlinger, S., Woolf, C.J., 2004. Axonal injurydependent induction of the peripheral benzodiazepine receptor in small-diameter adult rat primary sensory neurons. Eur. J. Neurosci. 20, 671 – 683. Kocsis, J.D., Rand, M.N., Lankford, K.L., Waxman, S.G., 1994. Intracellular calcium mobilization and neurite outgrowth in mammalian neurons. J. Neurobiol. 25, 252 – 264. Koenig, H.L., Schumacher, M., Ferzaz, B., Thi, A.N., Ressouches, A., Guennoun, R., Jung-Testas, I., Robel, P., Akwa, Y., Baulieu, E.E., 1995. Progesterone synthesis and myelin formation by Schwann cells. Science 268, 1500 – 1503. Koenig, H.L., Gong, W.H., Pelissier, P., 2000. Role of progesterone in peripheral nerve repair. Rev. Reprod. 5, 189 – 199. Korneyev, A., Pan, B.S., Polo, A., Romeo, E., Guidotti, A., Costa, E., 1993. Stimulation of brain pregnenolone synthesis by mitochondrial diazepam binding inhibitor receptor ligands in vivo. J. Neurochem. 61, 1515 – 1524. Kovacic, U., Zele, T., Osredkar, J., Sketelj, J., Bajrovic, F.F., 2004. Sexrelated differences in the regeneration of sensory axons and recovery of nociception after peripheral nerve crush in the rat. Exp. Neurol. 189, 94 – 104. Krueger, K.E., 1995. Molecular and functional properties of mitochondrial benzodiazepine receptors. Biochim. Biophys. Acta 1241, 453 – 470. Krueger, K.E., Papadopoulos, V., 1990. Peripheral-type benzodiazepine receptors mediate translocation of cholesterol from outer to inner mitochondrial membranes in adrenocortical cells. J. Biol. Chem. 265, 15015 – 15022. Kuhlmann, A.C., Guilarte, T.R., 2000. Cellular and subcellular localization of peripheral benzodiazepine receptors after trimethyltin neurotoxicity. J. Neurochem. 74, 1694 – 1704. Lacor, P., Benavides, J., Ferzaz, B., 1996. Enhanced expression of the peripheral benzodiazepine receptor (PBR) and its endogenous ligand octadecaneuropeptide (ODN) in the regenerating adult rat sciatic nerve. Neurosci. Lett. 220, 61 – 65. Lacor, P., Gandolfo, P., Tonon, M.C., Brault, E., Dalibert, I., Schumacher, M., Benavides, J., Ferzaz, B., 1999. Regulation of the expression of peripheral benzodiazepine receptors and their endogenous ligands during rat sciatic nerve degeneration and regeneration: a role for PBR in neurosteroidogenesis. Brain Res. 815, 70 – 80. Lankford, K.L., Waxman, S.G., Kocsis, J.D., 1998. Mechanisms of enhancement of neurite regeneration in vitro following a conditioning sciatic nerve lesion. J. Comp. Neurol. 391, 11 – 29. Le Fur, G., Vaucher, N., Perrier, M.L., Flamier, A., Benavides, J., Renault, C., Dubroeucq, M.C., Gueremy, C., Uzan, A., 1983a. Differentiation between two ligands for peripheral benzodiazepine binding sites, [3H]RO5-4864 and [3H]PK 11195, by thermodynamic studies. Life Sci. 33, 449 – 457. Le Fur, G., Perrier, M.L., Vaucher, N., Imbault, F., Flamier, A., Benavides, J., Uzan, A., Renault, C., Dubroeucq, M.C., Gueremy, C., 1983b. Peripheral benzodiazepine binding sites: effect of PK 11195, 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide: I. In vitro studies. Life Sci. 32, 1839 – 1847. Le Fur, G., Guilloux, F., Rufat, P., Benavides, J., Uzan, A., Renault, C., Dubroeucq, M.C., Gueremy, C., 1983c. Peripheral benzodiazepine binding sites: effect of PK 11195, 1-(2-chlorophenyl)-N-methyl-(1methylpropyl)-3 isoquinolinecarboxamide: II. In vivo studies. Life Sci. 32, 1849 – 1856.
C.D. Mills et al. / Mol. Cell. Neurosci. 30 (2005) 228 – 237 McQuarrie, I.G., Grafstein, B., Gershon, M.D., 1977. Axonal regeneration in the rat sciatic nerve: effect of a conditioning lesion and of dbcAMP. Brain Res. 132, 443 – 453. Mukherjee, S., Das, S.K., 1989. Subcellular distribution of ‘‘peripheral type’’ binding sites for [3H]Ro5-4864 in guinea pig lung. Localization to the mitochondrial inner membrane. J. Biol. Chem. 264, 16713 – 16718. Neumann, S., Woolf, C.J., 1999. Regeneration of dorsal column fibers into and beyond the lesion site following adult spinal cord injury. Neuron 23, 83 – 91. Oke, B.O., Suarez-Quian, C.A., Riond, J., Ferrara, P., Papadopoulos, V., 1992. Cell surface localization of the peripheral-type benzodiazepine receptor (PBR) in adrenal cortex. Mol. Cell. Endocrinol. 87, R1 – R6. Pan, Y.A., Misgeld, T., Lichtman, J.W., Sanes, J.R., 2003. Effects of neurotoxic and neuroprotective agents on peripheral nerve regeneration assayed by time-lapse imaging in vivo. J. Neurosci. 23, 11479 – 11488. Papadopoulos, V., Mukhin, A.G., Costa, E., Krueger, K.E., 1990. The peripheral-type benzodiazepine receptor is functionally linked to Leydig cell steroidogenesis. J. Biol. Chem. 265, 3772 – 3779. Papadopoulos, V., Amri, H., Boujrad, N., Cascio, C., Culty, M., Garnier, M., Hardwick, M., Li, H., Vidic, B., Brown, A.S., Reversa, J.L., Bernassau, J.M., Drieu, K., 1997. Peripheral benzodiazepine receptor in cholesterol transport and steroidogenesis. Steroids 62, 21 – 28. Qiu, J., Cai, D., Dai, H., McAtee, M., Hoffman, P.N., Bregman, B.S., Filbin, M.T., 2002. Spinal axon regeneration induced by elevation of cyclic AMP. Neuron 34, 895 – 903. Ravagnan, L., Marzo, I., Costantini, P., Susin, S.A., Zamzami, N., Petit, P.X., Hirsch, F., Goulbern, M., Poupon, M.F., Miccoli, L., Xie, Z., Reed, J.C., Kroemer, G., 1999. Lonidamine triggers apoptosis via a direct, Bcl-2-inhibited effect on the mitochondrial permeability transition pore. Oncogene 18, 2537 – 2546. Schoemaker, H., Boles, R.G., Horst, W.D., Yamamura, H.I., 1983. Specific high-affinity binding sites for [3H]Ro 5-4864 in rat brain and kidney. J. Pharmacol. Exp. Ther. 225, 61 – 69. Schumacher, M., Robel, P., Baulieu, E.E., 1996. Development and regeneration of the nervous system: a role for neurosteroids. Dev. Neurosci. 18, 6 – 21. Shoyab, M., Gentry, L.E., Marquardt, H., Todaro, G.J., 1986. Isolation and characterization of a putative endogenous benzodiazepineoid (endozepine) from bovine and human brain. J. Biol. Chem. 261, 11968 – 11973. Sjoberg, J., Kanje, M., 1990. The initial period of peripheral nerve regeneration and the importance of the local environment for the conditioning lesion effect. Brain Res. 529, 79 – 84. Skenen, J.H., 1989. Axonal growth-associated proteins. Annu. Rev. Neurosci. 12, 127 – 156. Slobodyansky, E., Guidotti, A., Wambebe, C., Berkovich, A., Costa, E., 1989. Isolation and characterization of a rat brain triakontatetraneuro-
237
peptide, a posttranslational product of diazepam binding inhibitor: specific action at the Ro 5-4864 recognition site. J. Neurochem. 53, 1276 – 1284. Sommervaille, T., Reynolds, M.L., Woolf, C.J., 1991. Time-dependent differences in the increase in GAP-43 expression in dorsal root ganglion cells after peripheral axotomy. Neuroscience 45, 213 – 220. Syapin, P.J., Skolnick, P., 1979. Characterization of benzodiazepine binding sites in cultured cells of neural origin. J. Neurochem. 32, 1047 – 1051. Tanimoto, Y., Onishi, Y., Sato, Y., Kizaki, H., 1999. Benzodiazepine receptor agonists modulate thymocyte apoptosis through reduction of the mitochondrial transmembrane potential. Jpn. J. Pharmacol. 79, 177 – 183. Taupin, V., Toulmond, S., Serrano, A., Benavides, J., Zavala, F., 1993. Increase in IL-6, IL-1 and TNF levels in rat brain following traumatic lesion. Influence of pre- and post-traumatic treatment with Ro5 4864, a peripheral-type (p site) benzodiazepine ligand. J. Neuroimmunol. 42, 177 – 185. Torres, S.R., Frode, T.S., Nardi, G.M., Vita, N., Reeb, R., Ferrara, P., Ribeiro-do-Valle, R.M., Farges, R.C., 2000. Anti-inflammatory effects of peripheral benzodiazepine receptor ligands in two mouse models of inflammation. Eur. J. Pharmacol. 408, 199 – 211. Verge, V.M., Richardson, P.M., Wiesenfeld-Hallin, Z., Hokfelt, T., 1995. Differential influence of nerve growth factor on neuropeptide expression in vivo: a novel role in peptide suppression in adult sensory neurons. J. Neurosci. 15, 2081 – 2096. Wala, E.P., Sloan, J.W., Jing, X., 2000. Pharmacokinetics of the peripheral benzodiazepine receptor antagonist, PK 11195, in rats. The effect of dose and gender. Pharmacol. Res. 41, 461 – 468. Wang, X., Liu, Z., Eimerl, S., Timberg, R., Weiss, A.M., Orly, J., Stocco, D.M., 1998. Effect of truncated forms of the steroidogenic acute regulatory protein on intramitochondrial cholesterol transfer. Endocrinology 139, 3903 – 3912. Wang, R., Guo, W., Ossipov, M.H., Vanderah, T.W., Porreca, F., Lai, J., 2003. Glial cell line-derived neurotrophic factor normalizes neurochemical changes in injured dorsal root ganglion neurons and prevents the expression of experimental neuropathic pain. Neuroscience 121, 815 – 824. West, L.A., Horvat, R.D., Roess, D.A., Barisas, B.G., Juengel, J.L., Niswender, G.D., 2001. Steroidogenic acute regulatory protein and peripheral-type benzodiazepine receptor associate at the mitochondrial membrane. Endocrinology 142, 502 – 505. Xiao, H.S., Huang, Q.H., Zhang, F.X., Bao, L., Lu, Y.J., Guo, C., Yang, L., Huang, W.J., Fu, G., Xu, S.H., Cheng, X.P., Yan, Q., Zhu, Z.D., Zhang, X., Chen, Z., Han, Z.G., Zhang, X., 2002. Identification of gene expression profile of dorsal root ganglion in the rat peripheral axotomy model of neuropathic pain. Proc. Natl. Acad. Sci. U. S. A. 99, 8360 – 8365.