- Open Access
Differential regulation of immune responses and macrophage/neuron interactions in the dorsal root ganglion in young and adult rats following nerve injury
Molecular Painvolume 5, Article number: 70 (2009)
Neuropathic pain is an apparently spontaneous experience triggered by abnormal physiology of the peripheral or central nervous system, which evolves with time. Neuropathic pain arising from peripheral nerve injury is characterized by a combination of spontaneous pain, hyperalgesia and allodynia. There is no evidence of this type of pain in human infants or rat pups; brachial plexus avulsion, which causes intense neuropathic pain in adults, is not painful when the injury is sustained at birth. Since infants are capable of nociception from before birth and display both acute and chronic inflammatory pain behaviour from an early neonatal age, it appears that the mechanisms underlying neuropathic pain are differentially regulated over a prolonged postnatal period.
We have performed a microarray analysis of the rat L4/L5 dorsal root ganglia (DRG), 7 days post spared nerve injury, a model of neuropathic pain. Genes that are regulated in adult rats displaying neuropathic behaviour were compared to those regulated in young rats (10 days old) that did not show the same neuropathic behaviour. The results show a set of genes, differentially regulated in the adult DRG, that are principally involved in immune system modulation. A functional consequence of this different immune response to injury is that resident macrophages cluster around the large A sensory neuron bodies in the adult DRG seven days post injury, whereas the macrophages in young DRG remain scattered evenly throughout the ganglion, as in controls.
The results show, for the first time, a major difference in the neuroimmune response to nerve injury in the dorsal root ganglion of young and adult rats. Differential analysis reveals a new set of immune related genes in the ganglia, that are differentially regulated in adult neuropathic pain, and that are consistent with the selective activation of macrophages around adult, but not young large A sensory neurons post injury. These differences may contribute to the reduced incidence of neuropathic pain in infants.
Physiological pain is an acute experience that results from the activation of peripheral nociceptors in injured and inflamed tissues and which normally passes when the stimulus ceases. In contrast, neuropathic pain is an apparently spontaneous experience triggered by abnormal physiology of the peripheral or central nervous system, which evolves with time. Neuropathic pain arising from peripheral nerve injury is characterized by a combination of spontaneous pain, hyperalgesia and allodynia and as it is poorly relieved by conventional analgesics, it is a significant clinical problem . Neuropathic pain is less common in children than in adults. Although it has been reported in very young paediatric patients , there is no evidence of this type of pain in infancy; brachial plexus avulsion, which causes intense neuropathic pain in adults, is not painful when the injury is sustained at birth . Consistent with this, persistent mechanical allodynia does not develop in spared nerve injury (SNI) and chronic constriction injury (CCI) rat models of neuropathic pain, if the injury is performed younger than 4 weeks of age . Furthermore, in the spinal nerve ligation model (SNL), neuropathic symptoms resolve faster in younger animals . Since infants are capable of nociception from before birth and display both acute and chronic inflammatory pain behaviour from an early neonatal age , it appears that the mechanisms underlying neuropathic pain are differentially regulated over a prolonged postnatal period.
The mechanisms underlying neuropathic pain in adults involve molecular and cellular changes in both the peripheral and central nervous system to produce the characteristic intensity and prolonged time course of the pain [7, 8]. Increased focus has been placed upon the role of neuroimmune interactions in animal models of persistent pain suggesting that the major stimulus for the underlying neuronal hypersensitivity in chronic pain states involves activation of glial cells, which when stimulated, increase production of a host of inflammatory/algesic mediators, such as cytokines and chemokines [9, 10]. In the peripheral nervous system, primary afferent neurons in the dorsal root ganglion (DRGs)  interact with two main types of non-neuronal cells in chronic pain states: satellite cells which surround neurons and control their chemical environment  and resident macrophages that are activated by nerve damage and inflammation [13–15].
Our overall aim is to discover why nerve injury induced neuropathic pain does not occur or is rapidly resolved in young animals, while it is intense and prolonged in adults. In the present study, we hypothesise that the postnatal maturation of the neuropathic pain response in young animals arises, at least in part, from developmental changes in the response of the dorsal root ganglion to nerve injury. To test our hypothesis, we analysed and compared transcriptional changes in young and adult DRGs after neuropathic nerve injury or sham controls, using high throughput screening with the Affymetrix® array rat203_v2. Our results reveal a new set of genes that implicate the peripheral immune system in the regulation of neuropathic pain. Immunohistochemical analysis of the dorsal root ganglia after nerve injury confirmed a selective pattern of macrophage activation that is specific for injured neurons in adult, but not young rats.
Materials and methods
Experiments were performed on adult (2 months old) male Sprague-Dawley rats and rat pups (10 days postnatal age, P10) and carried out in accordance with the United Kingdom Animal (Scientific Procedures) Act 1986. Spared nerve injury and sham surgery were performed under halothane general anaesthesia with antiseptic conditions as described previously . After skin preparation, the sciatic nerve and its three terminal branches were exposed in the upper lateral thigh. Axotomy and ligation of the common peroneal and tibial branches was performed under direct vision, leaving the sural nerve intact. Retrograde tracer (4 μL of 4% Fluorogold, Fluorochrome®, LLC) was injected into the cut end of the severed nerves to identify injured neurons . In adult rats after SNI surgery, 92.4% ± 1 (SE) of Fluorogold labelled neurons were also labelled with ATF-3 (which is known to mark damaged and regenerating neurons ) (Additional file 1). Muscle and skin were closed in two layers. In the sham operation, the procedures were the same but the nerves were only exposed and not cut or ligated. In all cases great care was taken not to stretch the nerve, or its branches, nor to damage the intact nerves.
After surgery, animals were returned to their cages and litters and maintained on a 12 hour light/dark cycle at constant ambient temperature with free access to food and water for 7 days post surgery until tissue collection.
Seven days post surgery, animals were sacrificed with 100 mg/kg of Euthanal®, fresh DRGs were collected, snap frozen on liquid nitrogen and stored at -80°C until RNA extraction. Both L4 and L5 DRGs ipsilateral to the lesion were collected and pooled from 3 animals per adult experimental group and 4 animals per P10 group, (i.e. n = 6 DRGs per adult sample, n = 8 per P10 sample) to minimise inter-animal differences. RNA was extracted using Trizol® reagent (Invitrogen®) and a QIAshredder column (Qiagen®) for homogenisation according to manufacturer's protocols. The RNA was cleaned by an "RNA purification column" (Qiagen®) and a final volume of 50 μL obtained. The amount of RNA was measured with a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Inc.) and its quality assessed with Experion RNA HighSens StdSens analysis kit (Bio-Rad®). Then, the RNA was precipitated and dissolved to obtain a final concentration of 1 μg/μL and stored at -80°C for cRNA synthesis or amplification by quantitative real-time polymerase chain reaction (qPCR).
First strand cDNA synthesis
2g (for qPCR) or 10 μg (microarray analysis) of total RNA and 100 pmol/μL of the T7(dT)24 primer (5'-GGCCAGTGAATTGTAATACGACTCACTATAGGGA GGCGGTTTTTTTTTTTTTTTTTTTTTTTT-3') were combined for annealing at 60°C for 10 mins and chilled on ice for 2 mins. Then, the SuperScript III kit (Invitrogen®) was added according to manufacturer instructions to synthesize the cDNA.
Second strand cDNA synthesis
the whole amount of the cDNA produced was used as template for the second strand synthesis plus 10 units of E. coli DNA ligase, 40 units of E. coli DNA polymerase (Invitrogen®), 3 μL of 10 mM dNTPs and 2 units of Rnase H (Invitrogen®). The reaction was stopped by addition of 10 μL of 0.5 M EDTA. The double stranded cDNA was then purified and precipitated and then resuspended in 10 μL of nuclease free water.
Synthesis of biotin-labelled cRNA
we used the Enzo-Bioarray kit (Enzo Life Sciences, Inc) for synthesis of the cRNA according to the manufacturer protocol. The resulting cRNA was purified with Rneasy kit (Qiagen®). The concentration was measured using a spectrophotometer and 20 μg of each sample were fragmented in 200 mM tris-acetate (pH= 8.1), 500 mM potassium acetate and 150 mM magnesium acetate for 35 mins at 95°C, then chilled on ice and stored at -80°C until microarray hybridisation.
For mRNA high-throughput screening we used GeneChip® Rat expression 230 version 2.0 arrays and GeneChip® Hybridization, Wash and stain kit from Affymetrix®. The hybridisation protocol was in 49/64 formats, appropriate for the selected array. The control oligonucleotides were: B2 and the Eukaryotic bioB, bioC, bioD and cre. Then, the samples were washed and stained on the Fluidics Station 450 (Affymetrix®) monitored with the GeneChip® Operating Software (Affymetrix®) (protocol FS450_003 appropriate for the rat 230 v2.0 array) and scanned with GeneChip Scanner 3000 (Affymetrix®).
Array statistical analysis
the ".CEL" files generated with GeneChip Scanner 3000 (Affymetrix®) (see additional file 2 for details) were acquired by "R environment for statistical computing and graphics language software" (the GNU project) using the BioConductor® package . The expression data was normalised by GC-Robust Multi-array Average (GC-RMA) background adjustment. Limma analysis  was used as a lineal method for contrasting the groups and identifying specific genes for each experimental group.
The quality of the arrays was assessed with Affymetrix Expression Console™ Software Versiowere combined for annealing n 1.0 (Affymetrix®) using an n = 3 per experimental group. Two samples out of 18 were outliers (Figure 1), one from P10 contralateral group and one from the P10 injured group. These two samples were removed from further analysis.
Quantitative Real-Time PCR
Specific primers for the gene candidates obtained from the microarray study were designed and synthesized by Sigma-Genosys (Table 1). An SYBR green-based system (Applied Biosystems) was used for amplification and detection. 1 μL of cDNA product per sample, 12.5 μl of Power SYBR green PCR Master Mix and 200 nM/primer was used for cocktail reaction. The amplification protocol was: step 1 at 95°C for 5', step 2 at 95°C for 45", step 3 at 60°C 1' step 4 at 70°C 1', step 5 plate reading, 39 cycles from step 2 to 4 and a final melting curve from 60°C to 90°C with reading every 0.5°C and 1" hold. The reaction plate was then placed in a DNA Engine Pelter thermal cycler with a Chromo 4 Real-Time PCR detector (Bio-Rad®). The amplification process was monitored with Opticon Monitor v3.1.32 software (MJ Genework Inc).
The DNA products from the qPCRs were sequenced on an AB PRISM 3100-Avant automated DNA capillary sequencer (AB Applied Biosystems, Lingley House, 120, Birchwood Boulevaid, Warrington) using BigDye Terminator v1.1 Cycle Sequencing chemistry (AB Applied Biosystems). Sequencing reactions were performed and precipitated in accordance with the manufacturer's instructions using the same sense or forward primer that was used for the qPCR amplification (Table 1). The reactions were resolved on a 50 cm standard sequencing capillary, running optimal 3100 POP-6TM polymer (AB Applied Biosystems). Sequence data was extracted and analysed using AB PRISM 3100-Avant Data Collection Software v2.0 and DNA Sequencing Analysis Software v5.1.1 respectively (AB Applied Biosystems).
Seven days post surgery rats were overdosed with 100 mg/kg of Euthanal® and perfused through the heart with 200 ml heparin-saline, followed by 200 mL 4% paraformaldehyde in 0.1 M phosphate buffer (PB). The lumbar ganglia L4 and L5 were removed and postfixed in 4% paraformaldehyde for 1 h at 4°C followed by immersion in 30% sucrose in 0.1 M of PB at 4°C for at least two days before sectioning. Serial, 20 μm cryostat sections were cut and stained after blocking in 10% normal goat serum, 0.3% Triton X in 1× PBS using primary antibodies: Rabbit anti-IBA-1 (1/1000, Dako®), mouse anti NF200 (1/1000, Chemicon®), rabbit anti ATF-3 (1/500, Santa Cruz®), mouse anti CD68/ED-1 (1/200. Serotec®), mouse anti NG2 (1/500, Millipore®) and rabbit-anti-CGRP (1/500 Chemicon®). The antibodies were incubated overnight at room temperature in 3% normal goat serum, 0.3% Triton X in 1× PBS. Secondary antibodies were goat anti-mouse (Alexa-593®) and goat anti-rabbit (Alexa-488®) at 1/200 dilution in 3% normal goat serum, 0.3% Triton X in 1× PBS. Antibody excess was removed by 3 washes in PBS for 10' at RT. The isolectin IB-4 (Bandeira simplicifolia) labelled with FITC (Sigma®) was used to identify the non-peptidergic neurons. After staining, the sections were kept for 16 h in the dark to normalize the background and then microscope images acquired at 10× magnification with OpenLab® software at a constant exposure time of 1.02".
Data Analysis for immunohistochemistry and qPCR
The images generated by immunohistochemistry were analysed with Image J 1.36 (NHS) software, n = 3 rats per experimental group, n = 4-6 sections per animal and n = 200 cells counted per section. The sections were from the centre of the ganglion and 160 μm apart to ensure that the same cell was not counted twice (large neurons may have a diameter up to 100 μm). Cells were counted as having macrophage 'ring-like' structures when the perimeter of the neuron was surrounded by macrophages which were closely adhered to the neuronal surface, as observed in merged images from IBA-1 and NF200 immunostaining. Data was pooled from animals in the same experimental group.
Pooled qPCR data from each experimental group (n = 3 samples per group) were plotted as "mean intensity normalized" (MIN) ± standard deviation (SD). The normalisation was calculated as the ratio with the housekeeping gene, Gapdh. All data was exported to SigmaStat, 2.03® (SPSS Inc®) for statistical analysis and plotted with SigmaPlot® (SPSS Inc®).
Differential microarray screening of DRG following nerve injury reveals 'neuropathic pain' specific genes
We performed high throughput screening of the mRNA regulation in L4/L5 dorsal root ganglia following spared nerve injury (SNI) or sham injury in adult and young rats using the Affymetrix-rat genome 230 2.0 array. We first analysed the effect of postnatal age on mRNA regulation by comparing the two SNI groups: young, (PDI) and adult (ADI) rats. We chose seven days post surgery for this analysis as it is known that at this time point, neuropathic behaviour is present in adult but not in young rats [4, 16]. The term 'young rats' denotes animals that were 10 days old (P10) at the time of surgery, but whose DRGs were collected for microarray analysis 7 days later, at P17. We used two types of controls to extract specific information obtained from the SNI groups: 1) sham surgery groups in adults (AHI) and young rats (PHI) and 2) contralateral groups in adults (ADC) and young rats (PDC).
Limma analysis was performed to compare the experimental groups in two steps. First, the nerve injury specific gene regulation in adult or young rats was compared to their counterpart sham or contralateral groups to remove those genes that could be up or down-regulated by the surgery alone rather than specifically the nerve injury. The P-value was adjusted for false discovery rate and a threshold of p < 0.05 was set up to subtract those genes with statistically significant differential regulation (Additional file 2). Next, the resulting lists from both the adult and young comparisons were compared to obtain nerve injury specific genes in adult rats only (pain behaviour) or young rats only (no pain behaviour). The screening provided 206 Affymetrix identifications (Affy_IDs) associated with mRNAs specific to adult SNI rats and only 3 Affy_IDs specific to young (P10) SNI.
The 206 genes were sorted into basic functional groups according to the "Go biological process" . Figure 2A shows that the 206 Affy_IDs were distributed in approximately equal proportions in extracellular (23%), membrane (34%), nuclear (21%) and cytoplasmatic (21%) compartments. We also grouped the genes accordingly to "GO terms" (Figure 2B). Of the 206 genes, 39.6% (82 Affy_IDs) were related to Express Sequence tags ( EST) (for predicted genes or similarities to known genes) and unknown genes and the rest comprised: immune system/inflammation 18.4% (38 Affy_IDs), signal transduction 12.1% (25 Affy_IDs), nervous system 6.8% (14 Affy_IDs), RNA/DNA modulation 5.3% (10 Affy_IDs), apoptosis/cell cycle 4.8% (10 Affy_IDs), ion transport 1.9% (4 Affy_IDs), cell adhesion 1.9% (4 Affy_IDs) and 9.2% (19 Affy_IDs) related to other processes (additional file 3).
Neuropathic pain specific gene validation by quantitative Real-time PCR
From the 206 significantly regulated genes, we selected 16 that had some previous published association with pain processes and/or immune system, (Table 2). Using the "Go term" classification, 8 genes belonged to the 'Immune system': (Serine (or cysteine) proteinase inhibitor, clade A, member 3N (SERPINA3N), interleukin 6 (IL-6), colony-stimulating factor 1 (CSF-1), toll-like receptor 4 (TLR4), purinergic receptor P2Y, G-protein coupled, 14 (P2RY14), complement component 3 (C3), macrophage chemoattractant protein 1 (MCP-1/CCL2) and chloride intracellular channel 1 (CLIC-1), one gene belonged to the 'Nervous system': monoamino oxidase A (MaoA); one gene to 'Apoptosis': Caspase 3 (CASP3); two genes to 'Signal transduction': inositol 1,4,5-triphosphate 3-kinase C (ITPKC) and mitogen-activated kinase kinase 6 (MAP2K6); and a further four from other groups: cAMP responsive element modulator (CREM) to the RNA/DNA modulation group and serine (or cysteine) proteinase inhibitor, clade B, member 2 (SERPINB2), matrix metallopeptidase 3 (MMP3) and matrix metalloproteinase 16 (MMP16).
To validate the microarray results of these 16 genes, we used quantitative Real-time PCR (qPCR) and subsequent confirmation of the gene product by DNA sequencing. Figure 3 shows the microarray plot and its counterpart qPCR validation graph for each of the 16 selected Affy_IDs. The qPCR validation shows that the selected genes are differentially regulated in the adult SNI group but many were also regulated in the young SNI group. Overall, 15 genes were upregulated and only 1 gene was downregulated following SNI when compared to sham groups (ANOVA p < 0.001). The 15 upregulated genes were classified into four groups according to the relative expression of the adult versus the young SNI group (Tukey or SNK test p < 0.05) (Figure 3A-D, bar charts). These four groups were A: Genes preferentially expressed in adult SNI: SERPINA3N, SERPINB2, IL-6 and MMP3, B: Genes preferentially expressed in young SNI: CASPASE 3, TLR4, CLIC-1 and MMP16, C: Genes equally expressed in both adult and young SNI: CREM, MCP-1, MAOA, IPTKC and CSF1; and D: Genes specific to the young SNI group: P2RY14 and C3. The fifth group, E, comprises the only downregulated gene, MAP2K6, when compared to the sham groups (ANOVA p < 0.001) (Figure 3E). MAP2K6 was also down regulated in the adult SNI group when compared to the young SNI group (Tukey p < 0.05).
Immunohistochemical analysis highlights differences in the cellular immune response to nerve injury in young and adult in the DRG
Since many of the differentially regulated genes between young and adult SNI were related to the immune system, we compared the cellular neuroimmune reaction in the DRG following nerve injury at the two ages. We hypothesised that if the differing patterns of gene regulation had functional implications we might see differences in injury induced reactions of the major cellular component of the immune system in peripheral ganglia, the macrophages. To determine macrophage reactions in the DRG after nerve injury we immunostained them with the macrophage marker IBA-1 in combination with selective markers of subpopulations of DRG sensory neurons, NF200 (for A-neurons), CGRP and IB4 (for C-neurons), 7 days following SNI surgery in young and adult rats (Figures 4, 5 and 6).
Figure 4 shows the striking macrophage response in adult rats 7 days post SNI surgery. Macrophages can be seen (in red) clustering around neuronal cell bodies in characteristic 'ring-like' structures with processes extending from their enlarged cell bodies towards neurons (Figure 4A pink arrows). Figures 4B and 4C show that the macrophages are selectively clustered around the large A-neurons which are stained with the marker NF200 (in green) (Figure 4B, pink arrows) and not around the C-neurons (Figure 4, blue arrows). We have confirmed these results by double labelling IBA-1 +ve macrophages in these 'ring-like structures with CD68/ED-1, another specific marker of macrophages  (additional file 4). NG2, on the other hand, a specific marker of satellite cells , did not overlap with IBA-1(additional file 5). As C-neuron immunomarkers are down-regulated after nerve injury [24–26], these could not be used to quantify macrophage clustering, but instead neuronal diameter was used, The analysis showed that 'rings' of macrophages are preferentially (p < 0.01, t-test) located around large (> 25 μm) NF200 +ve A-neurons and that smaller (< 25 μm) C-neurons are relatively free of macrophages. Figure 4D shows that 37.6% of neurons from adult DRG have macrophage rings around them 7 days post SNI (n = 660 cells pooled from 3 animals). Of these, 31.2% surround large A-neurons while 6.4% surround small C-neurons.
To investigate whether macrophage reactivity was restricted to neurons whose axons were directly damaged in the SNI surgery or whether neurons with 'spared' intact axons also display 'rings' of macrophages, we distinguished damaged from non-damaged neurons in adult L4 and L5 DRG using a retrograde tracer. Fluorogold was injected into the injured nerves (tibial and common peroneal) at the time of surgery and subsequently counterstained with the neuronal markers NF200, IB4 or CGRP (Figure 5A). We then counted 'ring-like' structures around damaged and non-damaged neurons and correlated the data with cell body diameter. Of the total population of large A-neurons in adult L4 and L5 DRGs, 7 days post SNI, 52.8% were damaged (Fluorogold +ve) and 47.2% were non-damaged (Fluorogold -ve) (n = 559 A-neurons form n = 3 animals). The data shows that the proportion of 'ring-like' structures in damaged A-neurons is significantly greater than in undamaged A-neurons: 33.8% of damaged A-neurons have 'ring-like' structures compared to 9.9% of the non-damaged A-neurons (Figure 5B) (ANOVA p < 0.001). In contrast, only 10% of the damaged C-neurons have 'ring-like' structures, while the non-damaged C-neurons have none (Figure 5C) (n = 284 C-neurons pooled form from 3 animals).
In young DRGs, however, the macrophages rarely formed 'rings' around neurons but remained scattered throughout the DRG (Figure 6A, white arrows). Thus, the distribution of macrophages in DRGs from young rats subjected to SNI surgery resemble that of adult and young sham groups with small cell bodies and few processes (Figure 6A white arrows). Figure 6B shows that only 11.7% of large A-neurons have macrophage 'ring-like' formations in the young SNI group, which although significantly greater than sham (1-2%), is significantly less than the adult SNI group, where 43.7% of the large A-neurons display 'rings' of reactive macrophages (ANOVA, p < 0.001; SNK p < 0.05, n = 2219 A-neurons from 3 animals, Figure 6B). Figure 6C compares the damaged and non-damaged neuronal groups in adult and young rats with SNI surgery. While in adult DRGs the macrophage 'ring-like' structures occur in 33.8% of damaged large A-neurons and 9.9% of non-damaged A-neurons, in young DRGs, significantly fewer A-neurons have macrophage 'ring-like' structures (10.6% of damaged, 1.1% of non-damaged) (Figure 6C). (n = 559 A-neurons from 3 animals)
In this study, we have used differential Affymetrix screening to examine age-related differences in transcriptional changes in the dorsal root ganglion (DRG) of young and adult rats after partial nerve damage "spared nerve injury" model ). The study was triggered by the absence of neuropathic behaviour following nerve injury in young rats and humans in contrast to the hypersensitivity and spontaneous pain that develops after adult nerve injury . Our results show a very high level of transcriptional activity in DRGs following nerve injury in adulthood compared to nerve injury in early life. Validation experiments provided a list of genes, normally implicated in the regulation of the immune system (Table 2), which have a specific and different pattern of expression in the DRGs of adults and young rats. We have also used immunohistochemical analysis to reveal a major differential reactivity in DRGs macrophages, the cellular component of immune responses. Reactive macrophages cluster in a characteristic display around the large A-neurons in adult neuropathic DRGs while little difference is found in the pattern of macrophages in young rats. Together, our results suggest a strong correlation between the regulation of the peripheral nervous system immune system and the progression towards neuropathic pain.
The microarray analysis results provided 206 candidate genes that were statistically significantly regulated in adult rats only. Intriguingly, only 3 genes were differentially regulated in young animals after nerve injury and no overlap was observed between the two age groups. The absence of neuropathic pain behaviour at 7 days post nerve injury in young rats [4, 5] correlates with this low level of differentially modulated genes in the young rat DRGs at the same time point.
On validating those Affy_IDs related to adult neuropathic pain, we found that most genes were closely related to the immune system/inflammation, followed by genes related to signal transduction, the nervous system and RNA/DNA modulation. It is important to note that the highest proportion (39%) of Affy_IDs (82 entries) belong to a group of unknown "transcribed loci" or "EST" (Expressed Sequence Tags). Other microarray analyses of the DRG transcriptome have highlighted the differential regulation between different experimental neuropathic models using total or partial axotomy [27–30]. Here we present a whole new set of "molecular tools" as new targets to dissect out the peripheral mechanisms in the 'spared nerve injury' model of neuropathic pain.
Several (16 Affy_IDs) of the top gene candidates from the microarray analysis were validated using quantitative real-time PCR (qPCR). Interestingly, the majority of these genes (15 Affy_IDs) were also differentially regulated in young animals after nerve injury. The difference between the initial microarray results and the qPCR data arises because microarray studies show widespread variations, even under the same experimental conditions and it is essential that the results are confirmed by other independent methods . Thus, qPCR highlights small differences not recognised by microarray technology. In addition, our gene lists were subjected to a stringent p-value correction for "false discovery rate" in the Limma test, designed to minimize false positive candidates . Overall these results suggest that the young DRG is not simply regulating the same genes less efficiently than in the adult but rather there is a different pattern of regulated genes in early life.
Our data reveals an interesting set of genes which are implicated in the modulation of the immune system. Thus, we have found a set of chemokines which are upregulated in the dorsal root ganglia after SNI nerve injury such as interleukin-6 (IL-6), macrophage chemoattractant protein-1 (MCP-1/CCL-2), colony-stimulating factor 1 (CSF-1) and complement component 3 (C3). Proinflammatory cytokines have a recognized role in neuropathic pain . Particularly well-studied is IL-6, which is upregulated in the DRG following chronic constriction injury and when blocked by minocycline reverses hyperalgesia and allodynia . IL-6 is upregulated in several microarray analyses of other models of axotomy [27, 30] and is associated with macrophage invasion in peripheral nerve . Spinal administration of IL-6 has anti-nociceptive effects in the spinal nerve ligation neuropathic pain model measured by a dose-related inhibition of electrically evoked C-fibre activity . MCP-1 is a chemokine that plays a role in the recruitment of monocytes to sites of injury and infection  and a role in pain processes . Exogenous administration of MCP-1 is known to depolarize and evoke action potential activity in DRG  and to excite cultured neonatal DRG neurons and to promote the secretion of the neuropeptides, substance P and CGRP [40, 41]. Moreover, MCP-1 receptor CCR2 knockout mice fail to develop mechanical allodynia after nerve injury . As for immune system activation and recruitment, MCP-1 is associated with both resident spinal microglia and infiltrating macrophages  and CCR2 null mice do not develop microglial activation . Nerve injury promotes the expression of MCP-1 in DRG cells  and it is possible that it is released centrally in neuropathic pain states. CSF-1 also has a role in microglia activation . The upregulation of these three genes observed here in the DRG suggests a similar role as described in the spinal cord. Since macrophage activation in the DRG after nerve injury is greater in adult than in young SNI rats, chemokine modulation could be a key factor underlying the different neuropathic pain behaviour at the two ages.
Other validated genes support the role of macrophage activation in adult DRGs, such as toll-like receptor 4 (TLR4) [46, 47] and the chloride intracellular channel 1 (CLIC-1) . Interestingly, in young injured DRGs we have found other markers related to macrophage activity such as the chemotactic purinergic receptor P2Y, G-protein coupled 14  and complement component 3 [50–52] suggesting a differential macrophage activity, rather than simple absence of activity through the postnatal development.
A further group of regulated genes in DRGs after nerve injury have a general role in regulating the extracellular matrix environment. Thus, matrix metallopeptidases 3 and 16, (MMP3 and MMP16) are proteoglycanases capable of degrading the major components of the extracellular matrix [53, 54] and are related to macrophage invasion . Recent studies have shown that other metalloproteinases, MMP2 and MMP9 contribute to the allodynia that follows neuropathy . MMP3 has recently been implicated in the inflammatory response  and the immune reaction in some neuropathies . Possibly counteracting the metalloproteinases, we found two genes: serine (or cysteine) inhibitor, clade A, member 3N (Serpin A3) and serine (or cysteine) inhibitor, clade B, member 2 (Serpin B2) that are implicated in protease inhibition and inflammation [59–61] which are specific for the adult SNI group. This data suggest that the balance of these particular MMPs and Serpins may be essential for macrophage invasion in the DRGs after nerve injury.
The final group of validated genes was related to intracellular signalling pathways such as Mitogen-activated protein kinase kinase 6 (MAP2K6) and Monoamino oxidase A (MAOA) which are related to the modulation of the p38 pathway [62, 63]. The p38 pathway has a role in microglia activation in the spinal cord [33, 64] and so this data suggests two new putative candidate targets for macrophage modulation as part of the p38 cascade of activation. Caspase 3, known to be involved in apoptosis  and cell loss  and TLR4 modulation  are upregulated in both adult and young SNI group, which is consistent with the stress produced by the nerve injury. cAMP responsive element modulator (CREM) isoform (ICER) has a demonstrated role in c-fos regulation after thermal stimulation and its overexpression represses the c-fos and c-jun promoters and attenuates early gene induction following noxious stimulation . Inositol 1,4,5-trisphosphate 3-kinase C (ITPKC) has been implicated in Ca2+ homeostasis and T lymphocytes activity , its upregulation after the nerve injury suggests a possible role in the lymphocyte signalling.
Our microarray analysis highlights the importance of the immune system in the SNI neuropathic model and corroborates the relationship between the activation of both microglia and macrophages and neuropathic pain behaviour [9, 14]. Nevertheless, it is clear that neuropathic pain behaviour is not simply a result of macrophage activation: some genes related to macrophage/microglia reactivity are upregulated in young rats after nerve injury despite the fact that they have no neuropathic pain response . Immunohistochemical analysis suggests that equally important is the pattern of macrophage response: in the adult rat post injury, activated macrophages take on a characteristic [70, 14] distribution throughout the DRGs, selectively surrounding the large A-neurons in a 'ring-like' structures. In contrast, macrophages in young rats post injury do not form these 'ring-like' structures and are not selectively recruited to the injured A-neurons. This observation suggests that the failure of macrophages in young animals to be attracted toward damaged A-neurons could be a key factor responsible for the lack of neuropathic behaviour. This could be due to the absence of released macrophage activation/chemoattractant factors. Our results are consistent with those of, Murphy et al, 1996 who described the presence of IL-6 in large and medium size neurons in axotomized adult DRGs . Hence, the immature neuroimmune response to nerve injury and the consequent lack of neuropathic pain in young mammal may not be due to a deficient immunological capacity per se but to antigenic inexperience  which leads to macrophages failing to specifically target neurons in the DRGs.
Differential microarray analysis has revealed clear differences in the response in the dorsal root ganglia to peripheral nerve injury in adult and young rats which are likely to underlie the known age related differences in the onset of neuropathic pain. Genes involved in the immune system, many of which play a role in macrophage activity, are the most differentially regulated group following adult nerve injury relative to the younger animals. Consistent with this, macrophages are activated in the adult DRGs, in a characteristic pattern around large A sensory neurons following nerve injury but this pattern of activation does not occur in young rats. This study highlights the important contribution of the maturity of the immune system and neuron/macrophage interactions to the postnatal development of neuropathic pain.
Spared Nerve Injury
Adult SNI group, ipsilateral side
Adult SNI group, contralateral side
Adult sham group, ipsilateral side
Young SNI group, ipsilateral side
Young SNI group, contralateral side
Young sham group, ipsilateral side
Dorsal Root Ganglia.
Baron R: Mechanisms of disease: neuropathic pain--a clinical perspective. Nat Clin Pract Neurol 2006, 2: 95–106. 10.1038/ncpneuro0113
Howard RF: Current status of pain management in children. JAMA 2003, 290: 2464–2469. 10.1001/jama.290.18.2464
Anand P, Birch R: Restoration of sensory function and lack of long-term chronic pain syndromes after brachial plexus injury in human neonates. Brain 2002, 125: 113–122. 10.1093/brain/awf017
Howard RF, Walker SM, Mota PM, Fitzgerald M: The ontogeny of neuropathic pain: postnatal onset of mechanical allodynia in rat spared nerve injury (SNI) and chronic constriction injury (CCI) models. Pain 2005, 115: 382–389. 10.1016/j.pain.2005.03.016
Ririe DG, Eisenach JC: Age-dependent responses to nerve injury-induced mechanical allodynia. Anesthesiology 2006, 104: 344–350. 10.1097/00000542-200602000-00021
Fitzgerald M: The development of nociceptive circuits. Nat Rev Neurosci 2005, 6: 507–520. 10.1038/nrn1701
Ji RR, Strichartz G: Cell signaling and the genesis of neuropathic pain. Sci STKE 2004, 2004: reE14. 10.1126/stke.2522004re14
Woolf CJ, Salter MW: Neuronal plasticity: increasing the gain in pain. Science 2000, 288: 1765–1769. 10.1126/science.288.5472.1765
De Leo JA, Tawfik VL, LaCroix-Fralish ML: The tetrapartite synapse: path to CNS sensitization and chronic pain. Pain 2006, 122: 17–21. 10.1016/j.pain.2006.02.034
DeLeo JA, Yezierski RP: The role of neuroinflammation and neuroimmune activation in persistent pain. Pain 2001, 90: 1–6. 10.1016/S0304-3959(00)00490-5
Hunt S, Koltzenburg M: The Neurobiology of Pain. Oxford University Press; 2005.
Hanani M: Satellite glial cells in sensory ganglia: from form to function. Brain Res Brain Res Rev 2005, 48: 457–476. 10.1016/j.brainresrev.2004.09.001
Dublin P, Hanani M: Satellite glial cells in sensory ganglia: their possible contribution to inflammatory pain. Brain Behav Immun 2007, 21: 592–598. 10.1016/j.bbi.2006.11.011
Hu P, Bembrick AL, Keay KA, McLachlan EM: Immune cell involvement in dorsal root ganglia and spinal cord after chronic constriction or transection of the rat sciatic nerve. Brain Behav Immun 2007, 21: 599–616. 10.1016/j.bbi.2006.10.013
Scholz J, Woolf CJ: The neuropathic pain triad: neurons, immune cells and glia. Nat Neurosci 2007, 10: 1361–1368. 10.1038/nn1992
Decosterd I, Woolf CJ: Spared nerve injury: an animal model of persistent peripheral neuropathic pain. Pain 2000, 87: 149–158. 10.1016/S0304-3959(00)00276-1
Schmued L, Kyriakidis K, Heimer L: In vivo anterograde and retrograde axonal transport of the fluorescent rhodamine-dextran-amine, Fluoro-Ruby, within the CNS. Brain Res 1990, 526: 127–134. 10.1016/0006-8993(90)90258-D
Shortland PJ, Baytug B, Krzyzanowska A, McMahon SB, Priestley JV, Averill S: ATF3 expression in L4 dorsal root ganglion neurons after L5 spinal nerve transection. Eur J Neurosci 2006, 23: 365–373. 10.1111/j.1460-9568.2005.04568.x
Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M, Dudoit S, Ellis B, Gautier L, Ge Y, Gentry J, Hornik K, Hothorn T, Huber W, Iacus S, Irizarry R, Leisch F, Li C, Maechler M, Rossini AJ, Sawitzki G, Smith C, Smyth G, Tierney L, Yang JY, Zhang J: Bioconductor: open software development for computational biology and bioinformatics. Genome Biol 2004, 5: R80. 10.1186/gb-2004-5-10-r80
Wettenhall JM, Smyth GK: Limma GUI: a graphical user interface for linear modeling of microarray data. Bioinformatics 2004, 20: 3705–3706. 10.1093/bioinformatics/bth449
Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, Harris MA, Hill DP, Issel-Tarver L, Kasarskis A, Lewis S, Matese JC, Richardson JE, Ringwald M, Rubin GM, Sherlock G: Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet 2000, 25: 25–29. 10.1038/75556
Holness CL, Simmons DL: Molecular cloning of CD68, a human macrophage marker related to lysosomal glycoproteins. Blood 1993, 81: 1607–1613.
Rezajooi K, Pavlides M, Winterbottom J, Stallcup WB, Hamlyn PJ, Lieberman AR, Anderson PN: NG2 proteoglycan expression in the peripheral nervous system: upregulation following injury and comparison with CNS lesions. Mol Cell Neurosci 2004, 25: 572–584. 10.1016/j.mcn.2003.10.009
Bennett DL, Michael GJ, Ramachandran N, Munson JB, Averill S, Yan Q, McMahon SB, Priestley JV: A distinct subgroup of small DRG cells express GDNF receptor components and GDNF is protective for these neurons after nerve injury. J Neurosci 1998, 18: 3059–3072.
Hokfelt T, Zhang X, Wiesenfeld-Hallin Z: Messenger plasticity in primary sensory neurons following axotomy and its functional implications. Trends Neurosci 1994, 17: 22–30. 10.1016/0166-2236(94)90031-0
Verge VM, Richardson PM, Wiesenfeld-Hallin Z, Hokfelt T: Differential influence of nerve growth factor on neuropeptide expression in vivo: a novel role in peptide suppression in adult sensory neurons. J Neurosci 1995, 15: 2081–2096.
Costigan M, Befort K, Karchewski L, Griffin RS, D'Urso D, Allchorne A, Sitarski J, Mannion JW, Pratt RE, Woolf CJ: Replicate high-density rat genome oligonucleotide microarrays reveal hundreds of regulated genes in the dorsal root ganglion after peripheral nerve injury. BMC Neurosci 2002, 3: 16. 10.1186/1471-2202-3-16
Levin ME, Jin JG, Ji RR, Tong J, Pomonis JD, Lavery DJ, Miller SW, Chiang LW: Complement activation in the peripheral nervous system following the spinal nerve ligation model of neuropathic pain. Pain 2008, 137: 182–201. 10.1016/j.pain.2007.11.005
Maratou K, Wallace VC, Hasnie FS, Okuse K, Hosseini R, Jina N, Blackbeard J, Pheby T, Orengo C, Dickenson AH, McMahon SB, Rice AS: Comparison of dorsal root ganglion gene expression in rat models of traumatic and HIV-associated neuropathic pain. Eur J Pain 2009, 13: 387–398. 10.1016/j.ejpain.2008.05.011
Nilsson A, Moller K, Dahlin L, Lundborg G, Kanje M: Early changes in gene expression in the dorsal root ganglia after transection of the sciatic nerve; effects of amphiregulin and PAI-1 on regeneration. Brain Res Mol Brain Res 2005, 136: 65–74. 10.1016/j.molbrainres.2005.01.008
Jayapal M, Melendez AJ: DNA microarray technology for target identification and validation. Clin Exp Pharmacol Physiol 2006, 33: 496–503. 10.1111/j.1440-1681.2006.04398.x
Benjamini Y, Hochberg Y: Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc 1995, 57: 289–300.
DeLeo JA, Sorkin LS, Watkins LR: Immune and glial regulation of pain. 1st edition. IASP Press; 2007:67–106.
Zanjani TM, Sabetkasaei M, Mosaffa N, Manaheji H, Labibi F, Farokhi B: Suppression of interleukin-6 by minocycline in a rat model of neuropathic pain. Eur J Pharmacol 2006, 538: 66–72. 10.1016/j.ejphar.2006.03.063
Ma W, Quirion R: Targeting invading macrophage-derived PGE2, IL-6 and calcitonin gene-related peptide in injured nerve to treat neuropathic pain. Expert Opin Ther Targets 2006, 10: 533–546. 10.1517/1472818.104.22.1683
Flatters SJ, Fox AJ, Dickenson AH: Spinal interleukin-6 (IL-6) inhibits nociceptive transmission following neuropathy. Brain Res 2003, 984: 54–62. 10.1016/S0006-8993(03)03092-0
Lu B, Rutledge BJ, Gu L, Fiorillo J, Lukacs NW, Kunkel SL, North R, Gerard C, Rollins BJ: Abnormalities in monocyte recruitment and cytokine expression in monocyte chemoattractant protein 1-deficient mice. J Exp Med 1998, 187: 601–608. 10.1084/jem.187.4.601
Abbadie C, Lindia JA, Cumiskey AM, Peterson LB, Mudgett JS, Bayne EK, DeMartino JA, MacIntyre DE, Forrest MJ: Impaired neuropathic pain responses in mice lacking the chemokine receptor CCR2. Proc Natl Acad Sci USA 2003, 100: 7947–7952. 10.1073/pnas.1331358100
Sun JH, Yang B, Donnelly DF, Ma C, LaMotte RH: MCP-1 enhances excitability of nociceptive neurons in chronically compressed dorsal root ganglia. J Neurophysiol 2006, 96: 2189–2199. 10.1152/jn.00222.2006
Oh SB, Tran PB, Gillard SE, Hurley RW, Hammond DL, Miller RJ: Chemokines and glycoprotein120 produce pain hypersensitivity by directly exciting primary nociceptive neurons. J Neurosci 2001, 21: 5027–5035.
Qin X, Wan Y, Wang X: CCL2 and CXCL1 trigger calcitonin gene-related peptide release by exciting primary nociceptive neurons. J Neurosci Res 2005, 82: 51–62. 10.1002/jnr.20612
Zhang J, De Koninck Y: Spatial and temporal relationship between monocyte chemoattractant protein-1 expression and spinal glial activation following peripheral nerve injury. J Neurochem 2006, 97: 772–783. 10.1111/j.1471-4159.2006.03746.x
Zhang J, Shi XQ, Echeverry S, Mogil JS, De Koninck Y, Rivest S: Expression of CCR2 in both resident and bone marrow-derived microglia plays a critical role in neuropathic pain. J Neurosci 2007, 27: 12396–12406. 10.1523/JNEUROSCI.3016-07.2007
Thacker MA, Clark AK, Bishop T, Grist J, Yip PK, Moon LD, Thompson SW, Marchand F, McMahon SB: CCL2 is a key mediator of microglia activation in neuropathic pain states. Eur J Pain 2009, 13: 263–272. 10.1016/j.ejpain.2008.04.017
Mitrasinovic OM, Grattan A, Robinson CC, Lapustea NB, Poon C, Ryan H, Phong C, Murphy GM Jr: Microglia overexpressing the macrophage colony-stimulating factor receptor are neuroprotective in a microglial-hippocampal organotypic coculture system. J Neurosci 2005, 25: 4442–4451. 10.1523/JNEUROSCI.0514-05.2005
Tanga FY, Nutile-McMenemy N, DeLeo JA: The CNS role of Toll-like receptor 4 in innate neuroimmunity and painful neuropathy. Proc Natl Acad Sci USA 2005, 102: 5856–5861. 10.1073/pnas.0501634102
Wadachi R, Hargreaves KM: Trigeminal nociceptors express TLR-4 and CD14: a mechanism for pain due to infection. J Dent Res 2006, 85: 49–53. 10.1177/154405910608500108
Novarino G, Fabrizi C, Tonini R, Denti MA, Malchiodi-Albedi F, Lauro GM, Sacchetti B, Paradisi S, Ferroni A, Curmi PM, Breit SN, Mazzanti M: Involvement of the intracellular ion channel CLIC1 in microglia-mediated beta-amyloid-induced neurotoxicity. J Neurosci 2004, 24: 5322–5330. 10.1523/JNEUROSCI.1170-04.2004
Lee BC, Cheng T, Adams GB, Attar EC, Miura N, Lee SB, Saito Y, Olszak I, Dombkowski D, Olson DP, Hancock J, Choi PS, Haber DA, Luster AD, Scadden DT: P2Y-like receptor, GPR105 (P2Y14), identifies and mediates chemotaxis of bone-marrow hematopoietic stem cells. Genes Dev 2003, 17: 1592–1604. 10.1101/gad.1071503
de Bruijn MH, Fey GH: Human complement component C3: cDNA coding sequence and derived primary structure. Proc Natl Acad Sci USA 1985, 82: 708–712. 10.1073/pnas.82.3.708
Fischetti F, Durigutto P, Macor P, Marzari R, Carretta R, Tedesco F: Selective therapeutic control of C5a and the terminal complement complex by anti-C5 single-chain Fv in an experimental model of antigen-induced arthritis in rats. Arthritis Rheum 2007, 56: 1187–1197. 10.1002/art.22492
Griffin RS, Costigan M, Brenner GJ, Ma CH, Scholz J, Moss A, Allchorne AJ, Stahl GL, Woolf CJ: Complement induction in spinal cord microglia results in anaphylatoxin C5a-mediated pain hypersensitivity. J Neurosci 2007, 27: 8699–8708. 10.1523/JNEUROSCI.2018-07.2007
Sellers A, Murphy G: Collagenolytic enzymes and their naturally occurring inhibitors. Int Rev Connect Tissue Res 1981, 9: 151–190.
Takino T, Sato H, Shinagawa A, Seiki M: Identification of the second membrane-type matrix metalloproteinase (MT-MMP-2) gene from a human placenta cDNA library. MT-MMPs form a unique membrane-type subclass in the MMP family. J Biol Chem 1995, 270: 23013–23020. 10.1074/jbc.270.39.23013
Haro H, Crawford HC, Fingleton B, MacDougall JR, Shinomiya K, Spengler DM, Matrisian LM: Matrix metalloproteinase-3-dependent generation of a macrophage chemoattractant in a model of herniated disc resorption. J Clin Invest 2000, 105: 133–141. 10.1172/JCI7090
Kawasaki Y, Xu ZZ, Wang X, Park JY, Zhuang ZY, Tan PH, Gao YJ, Roy K, Corfas G, Lo EH, Ji RR: Distinct roles of matrix metalloproteases in the early- and late-phase development of neuropathic pain. Nat Med 2008, 14: 331–336. 10.1038/nm1723
Nerusu KC, Warner RL, Bhagavathula N, McClintock SD, Johnson KJ, Varani J: Matrix metalloproteinase-3 (stromelysin-1) in acute inflammatory tissue injury. Exp Mol Pathol 2007, 83: 169–176. 10.1016/j.yexmp.2007.04.003
Nishida K, Kuchiiwa S, Oiso S, Futagawa T, Masuda S, Takeda Y, Yamada K: Up-regulation of matrix metalloproteinase-3 in the dorsal root ganglion of rats with paclitaxel-induced neuropathy. Cancer Sci 2008, 99: 1618–1625. 10.1111/j.1349-7006.2008.00877.x
Horvath AJ, Irving JA, Rossjohn J, Law RH, Bottomley SP, Quinsey NS, Pike RN, Coughlin PB, Whisstock JC: The murine orthologue of human antichymotrypsin: a structural paradigm for clade A3 serpins. J Biol Chem 2005, 280: 43168–43178. 10.1074/jbc.M505598200
Takamiya A, Takeda M, Yoshida A, Kiyama H: Expression of serine protease inhibitor 3 in ocular tissues in endotoxin-induced uveitis in rat. Invest Ophthalmol Vis Sci 2001, 42: 2427–2433.
Takamiya A, Takeda M, Yoshida A, Kiyama H: Inflammation induces serine protease inhibitor 3 expression in the rat pineal gland. Neuroscience 2002, 113: 387–394. 10.1016/S0306-4522(02)00198-7
Alonso G, Ambrosino C, Jones M, Nebreda AR: Differential activation of p38 mitogen-activated protein kinase isoforms depending on signal strength. J Biol Chem 2000, 275: 40641–40648. 10.1074/jbc.M007835200
Ou XM, Chen K, Shih JC: Monoamine oxidase A and repressor R1 are involved in apoptotic signaling pathway. Proc Natl Acad Sci USA 2006, 103: 10923–10928. 10.1073/pnas.0601515103
Ji RR, Suter MR: p38 MAPK, microglial signaling, and neuropathic pain. Mol Pain 2007, 3: 33. 10.1186/1744-8069-3-33
Murata Y, Nannmark U, Rydevik B, Takahashi K, Olmarker K: Nucleus pulposus-induced apoptosis in dorsal root ganglion following experimental disc herniation in rats. Spine 2006, 31: 382–390. 10.1097/01.brs.0000199618.85326.17
Lekan HA, Chung K, Yoon YW, Chung JM, Coggeshall RE: Loss of dorsal root ganglion cells concomitant with dorsal root axon sprouting following segmental nerve lesions. Neuroscience 1997, 81: 527–534. 10.1016/S0306-4522(97)00173-5
Gilchrist M, Thorsson V, Li B, Rust AG, Korb M, Kennedy K, Hai T, Bolouri H, Aderem A: Systems biology approaches identify ATF3 as a negative regulator of Toll-like receptor 4. Nature 2006, 441: 173–178. 10.1038/nature04768
Naranjo JR, Mellstrom B, Carrion AM, Lucas JJ, Foulkes NS, Sassone-Corsi P: Peripheral noxious stimulation induces CREM expression in dorsal horn: involvement of glutamate. Eur J Neurosci 1997, 9: 2778–2783. 10.1111/j.1460-9568.1997.tb01707.x
Pouillon V, Hascakova-Bartova R, Pajak B, Adam E, Bex F, Dewaste V, Van Lint C, Leo O, Erneux C, Schurmans S: Inositol 1,3,4,5-tetrakisphosphate is essential for T lymphocyte development. Nat Immunol 2003, 4: 1136–1143. 10.1038/ni980
Hu P, McLachlan EM: Macrophage and lymphocyte invasion of dorsal root ganglia after peripheral nerve lesions in the rat. Neuroscience 2002, 112: 23–38. 10.1016/S0306-4522(02)00065-9
Murphy P, Grondin J, Altares M, Richardson P: Induction of interleukin-6 in axotomized sensory neurons. J Neurosci. 1995,15(7 Pt 2):5130–5138.
Fadel S, Sarzotti M: Cellular immune responses in neonates. Int Rev Immunol 2000, 19: 173–193. 10.3109/08830180009088504
Gilchrist RB, Rowe DB, Ritter LJ, Robertson SA, Norman RJ, Armstrong DT: Effect of granulocyte-macrophage colony-stimulating factor deficiency on ovarian follicular cell function. J Reprod Fertil 2000, 120: 283–292. 10.1530/reprod/120.2.283
Hojo Y, Ikeda U, Takahashi M, Shimada K: Increased levels of monocyte-related cytokines in patients with unstable angina. Atherosclerosis 2002, 161: 403–408. 10.1016/S0021-9150(01)00636-0
Sasmono RT, Ehrnsperger A, Cronau SL, Ravasi T, Kandane R, Hickey MJ, Cook AD, Himes SR, Hamilton JA, Hume DA: Mouse neutrophilic granulocytes express mRNA encoding the macrophage colony-stimulating factor receptor (CSF-1R) as well as many other macrophage-specific transcripts and can transdifferentiate into macrophages in vitro in response to CSF-1. J Leukoc Biol 2007, 1: 111–23. 10.1189/jlb.1206713
Gradl G, Gaida S, Gierer P, Mittlmeier T, Vollmar B: In vivo evidence for apoptosis, but not inflammation in the hindlimb muscle of neuropathic rats. Pain 2004, 112: 121–130. 10.1016/j.pain.2004.08.007
Jin HW, Ichikawa H, Nomura K, Mukae K, Terayama R, Yamaai T, Sugimoto T: Activation of the caspase cascade underlies the rat trigeminal primary neuronal apoptosis induced by neonatal capsaicin administration. Arch Histol Cytol 2005, 68: 301–310. 10.1679/aohc.68.301
Jin HW, Ichikawa H, Fujita M, Yamaai T, Mukae K, Nomura K, Sugimoto T: Involvement of caspase cascade in capsaicin-induced apoptosis of dorsal root ganglion neurons. Brain Res 2005, 1056: 139–144. 10.1016/j.brainres.2005.07.025
Dewaste V, Pouillon V, Moreau C, Shears S, Takazawa K, Erneux C: Cloning and expression of a cDNA encoding human inositol 1,4,5-trisphosphate 3-kinase C. Biochem J 2000,352(Pt 2):343–351. 10.1042/0264-6021:3520343
Li CK, Pender SL, Pickard KM, Chance V, Holloway JA, Huett A, Goncalves NS, Mudgett JS, Dougan G, Frankel G, MacDonald TT: Impaired immunity to intestinal bacterial infection in stromelysin-1 (matrix metalloproteinase-3)-deficient mice. J Immunol 2004, 173: 5171–5179.
Van Bilsen JH, Wagenaar-Hilbers JP, Cammen MJ, van Dijk ME, van Eden W, Wauben MH: Successful immunotherapy with matrix metalloproteinase-derived peptides in adjuvant arthritis depends on the timing of peptide administration. Arthritis Res 2002, 4: R2. 10.1186/ar421
Shofuda KI, Hasenstab D, Kenagy RD, Shofuda T, Li ZY, Lieber A, Clowes AW: Membrane-type matrix metalloproteinase-1 and -3 activity in primate smooth muscle cells. FASEB J 2001, 15: 2010–2012.
Yamanaka H, Obata K, Fukuoka T, Dai Y, Kobayashi K, Tokunaga A, Noguchi K: Induction of plasminogen activator inhibitor-1 and -2 in dorsal root ganglion neurons after peripheral nerve injury. Neuroscience 2005, 132: 183–191. 10.1016/j.neuroscience.2004.12.003
The authors would like to acknowledge the Wellcome Trust and Medical Research Council for their grant funding. We also like to thank Mrs Mary Rahmann (Department of Neurosciences Physiology and Pharmacology, University College of London) for support in using the Affymetrix platform and Mrs Jacqueta Meredith-Middleton for the care of animals.
The authors declare that they have no competing interests.
DVA designed the study, carried out all the experiments, performed the statistical analysis and wrote the manuscript. SMG assisted with the Affymetrix data analysis. MF is the director of the research and wrote the manuscript. All authors have read and approved the final manuscript.