Inflammation and nerve injury induce expression of pancreatitis-associated protein-II in primary sensory neurons

Increase in expression of PAP-I and -II in the rat DRG after peripheral tissue inflammation was suggested by our gene microarray analysis. We found that their expression was markedly increased in lumbar (L) 4 and L5 DRGs when peripheral tissue inflammation was induced by intraplantar CFA injection into the hindpaw of rats. The microarray signal for PAP-I mRNA was increased by 2394%, 1660% and 1128% at day 2, 4 and 7 after CFA injection, respectively, while the signal for PAP-II mRNA was increased by 87% at day 2, 134% at day 4 and 94% at day 7. These results suggest an inflammation-induced expression of PAP-I and -II in the DRG.

We then used RT-PCR to confirm the expression of PAP-I and -II in the rat DRG following peripheral inflammation. The level of PAP-I mRNA was markedly elevated in the DRG one day after inflammation (Fig. 1A). Such an increased expression reached the peak level at day 2 and remained at a reduced level, which was still higher than control, during 4-7 days after inflammation (Fig. 1A). In situ hybridization experiments showed that only ~2% of total neurons in control L4 and L5 DRGs contained PAP-I mRNA (Fig. 1B and 1C). The number of PAP-I mRNA-containing neurons was markedly increased to ~28% of total neurons at inflammation day 2 (Fig. 1B and 1C). Analysis of cell-size distribution showed that most of these neurons were small DRG neurons (Fig. 1B). Quantitative analysis showed that the PAP-I mRNA-containing neuron profiles had a cross-section area ranging from 100 to 1400 μm² with a peak area between 400 to 700 μm² (Fig. 1D). There were still ~6% of DRG neurons containing PAP-I mRNA at day 7 (Fig. 1C). These results indicate that the expression of PAP-I mRNA is induced in small DRG neurons after peripheral inflammation.

Similar to PAP-I, only a low level of PAP-II mRNA was found in L4 and L5 DRGs of normal rats. However, the expression level of PAP-II was markedly increased in the DRGs one day after intraplantar CFA injection (Fig. 2A). Such an elevated expression reached the peak level at day 2, and lasted at least for 7 days (Fig. 2A). In situ hybridization histochemistry was then employed to illustrate the cellular distribution of PAP-II mRNA. We found that less than ~3% of total neurons contained PAP-II mRNA in the DRG of control group (Fig. 2B and 2C). However, the percentage of PAP-II mRNA-containing DRG neurons was markedly increased, reaching ~23% of total neurons 2 days after inflammation (Fig. 2B and 2C). Most of these neurons are small DRG neurons (Fig. 2B and 2D). Seven days after peripheral inflammation, there were still ~10% of DRG neurons containing PAP-II mRNA (Fig. 2C). These results indicate that peripheral tissue inflammation triggers the expression of PAP-II mRNA in nociceptive small DRG neurons.

We next asked whether the increase in PAP-II mRNA resulted in an elevated protein level in small DRG neurons. Immunostaining showed that only ~3% of DRG neurons of normal rats contained PAP-II (Fig. 3A and 3B), consistent with the result of in situ hybridization. However, the number of PAP-II-immunoreactive neurons was markedly increased in L4 and L5 DRGs after peripheral inflammation induced by intraplantar CFA injection (Fig. 3A and 3B). Quantitative analysis showed that ~28% of total DRG neurons in the DRG at inflammation day 2 contained PAP-II (Fig. 3B). Most of PAP-II-immunoreactive neuron profiles (n = 220) were small DRG neurons which had a cross-section area less than 900 μm², and covered 45% of all small neurons. Although the number of PAP-II-positive neurons was decreased to ~15% of total neurons at day 4, PAP-II was still present in many DRG neurons (~16%) at day 7, suggesting a long-lasting up-regulation of this protein. Thus, peripheral inflammation induces the expression of PAP-II in small DRG neurons.

We further identified the subset of small DRG neurons that expressed PAP-II following peripheral inflammation. Small DRG neurons can be divided roughly into two subsets, namely the peptidergic subset containing neuropeptide substance P and calcitonin gene-related peptide (CGRP) and non-peptidergic subset positive for IB4, although there is considerable overlap between these two subsets of small neurons. Double-immunofluorescence staining showed that two days after peripheral tissue inflammation, most of PAP-II-immunoreactive small DRG neurons (76.7 ± 1.4%) were also IB4-positive, while 26.3 ± 3.0% of PAP-II-positive neurons contained CGRP (Fig. 4A and 4B). About 89% of PAP-II-positive DRG neurons were also immunostained for peripherin (Fig. 4A and 4B), a marker for small DRG neuron [35‐37]. In contrast, PAP-II was absent in Trk-B-immunoreactive DRG neurons (Fig. 4A), suggesting that inflammation-induced expression of PAP-II mainly occurs in small DRG neurons. Thus, the expression of PAP-II is induced mainly in IB4-positive subset of small DRG neurons.

It has been reported that the PAP-I expression in IB4-positive small DRG neurons is up-regulated and followed by a shift from small to large DRG neurons after peripheral nerve injury [33]. Therefore, we are interested to know whether peripheral nerve injury could induce the expression of PAP-II in DRG neurons. We found that the expression of PAP-II was also induced in DRG neurons after peripheral nerve injury. Immunostaining showed that only ~2% of DRG neurons were positive for PAP-II in L4 and L5 DRGs on the contralateral side after unilateral axotomy of the sciatic nerve (Fig. 5A and 5B), similar to that found in the DRGs of normal rats. However, the number of PAP-II-immunoreactive neurons was markedly increased in the ipsilateral L4 and L5 DRGs (Fig. 5A and 5B). Quantitative analysis showed that ~13% of total DRG neurons contained PAP-II in the ipsilateral DRGs 2 days after unilateral axotomy (Fig. 5B). Most of PAP-II-immunoreactive DRG neurons were small ones at this time interval (Fig. 5A and 5C).

Fourteen days after unilateral axotomy of the sciatic nerve, PAP-II was still present in many DRG neurons (~7% of total neurons), indicating a long-lasting up-regulation of this protein (Fig. 5A and 5B). However, there was a dynamic shift from small DRG neurons to large ones (Fig. 5A). In contrast to the presence of PAP-II-immunoreactivity in small DRG neurons 2 days after axotomy, the majority of PAP-II-positive DRG neurons was large ones (> 900 μm²) 14 days after axotomy (Fig. 5C). Therefore, the expression of PAP-II is rapidly up-regulated in many small DRG neurons 2 days after peripheral axotomy, followed by reduction in small DRG neurons but further up-regulation in large neurons.

Double-immunofluorescence staining showed that 2 days after peripheral nerve injury, most of PAP-II-immunoreactive small DRG neurons were also IB4 or CGRP-negative (Fig. 6A and 6B), while ~60% of PAP-II-positive DRG neurons were immunostained for peripherin (Fig. 6A and 6B). It is known that neuropeptide Y (NPY) is expressed at very low levels in DRG neurons of normal rats, but its expression is markedly up-regulated in large DRG neurons after peripheral nerve injury [38, 39]. Although the expression of NPY in DRG neurons remained at low levels 2 days after sciatic nerve axotomy, the expression of PAP-II had already been up-regulated in DRG neurons including some large neurons (Fig. 6A). Therefore, less than 10% of PAP-II-positive neurons contained NPY (Fig. 6A), suggesting that nerve injury-induced expression of PAP-II may represent an early response of DRG neurons to injury.

The down-regulated expression of CGRP and IB4 in small DRG neurons, and the up-regulated expression of NPY in large DRG neurons became predominant 14 days after sciatic nerve axotomy (Fig. 7A), consistent with previous studies [38, 39]. We found that only a few PAP-II-containing DRG neurons were immunostained for CGRP but none for IB4 (Fig. 7A and 7B). Although the percentage of PAP-II-positive neurons expressing peripherin was slightly decreased (from 54.6 ± 10.3% to 48.7 ± 8.7%), co-expression of PAP-II and peripherin was still present in small DRG neurons (Fig. 7A and 7B). The number of large DRG neurons that co-expressed PAP-II and NPY was markedly increased, contributing to 61.1 ± 2.8% of total PAP-II-containing DRG neurons (Fig. 7A and 7B). Thus, the expression of PAP-II can be induced in large DRG neurons that express NPY 14 days after peripheral nerve injury.

Although PAP-II-immunoreactivity was found in the cell bodies of small DRG neurons, we failed to detect its presence in sensory afferent fibers in lamina I-II of the rat spinal cord. This might be due to either a lack of PAP-II transport in axons or a rapid release of PAP-II at afferent terminals. To test these possibilities, we examined whether PAP-II protein could be transported to nerve terminals. The sciatic nerve and dorsal root of rats were ligated for 1 day to attenuate the axonal transport of proteins, resulting in an accumulation of transported protein near the ligation site. Using immunostaining, we found that PAP-II was accumulated in the nerve fibers in the portion of the ligated sciatic nerve and dorsal root that was proximal to L4 and L5 DRGs but not in the distal portion (Fig. 8A and 8B), suggesting that PAP-II can be anterogradely transported to the afferent terminals. After peripheral tissue inflammation, the accumulation of PAP-II in the proximal side of the dorsal root and the sciatic nerve was significantly increased (Fig. 8B and 8C). As a control, an accumulation of CGRP was also found in the proximal portion of the ligated sciatic nerve and dorsal root (data not shown). These results suggest that newly synthesized PAP-II can be transported to sensory afferent terminals in the spinal cord and peripheral tissues.

All interventions and animal care were performed in accordance with the policy of the Society for Neuroscience (USA) on the use of animals in neuroscience research and the guidelines of the Committee for Research and Ethic Issues of International Association for the Study of Pain. The experiments were approved by the Committee of Use of Laboratory Animals and Common facility, Institute of Neuroscience, CAS. All efforts have been made to minimize the number of animals used and their discomfort after peripheral inflammation. Sprague-Dawley male rats (200~250 g, Shanghai Center of Experimental Animals, CAS, Shanghai, China) were anesthetized with sodium pentobarbital (60 mg/kg). Paw inflammation was induced by injection of complete Freund's adjuvant (CFA, Sigma) into the rat plantar subcutaneous space of a hindpaw (200 μl/paw). For peripheral nerve injury model, 5 mm portion of the left sciatic nerve of rats was transected at mid-thigh level. The rats were allowed to survive for 1, 2, 4 and 7 days for inflammation model and 2, 7, 14 and 28 days for nerve injury model (15 rats for each time point for RT-PCR). Axonal transport was studied by ligating the L4 and L5 dorsal roots and proximal portion of sciatic nerve (5 rats) at mid-thigh level 1 day before perfusion fixation. L4 and L5 DRGs of these rats and of normal rats were then dissected and frozen on dry ice. For in situ hybridization or immunohistochemistry, the same time course was chosen (6 rats for each time point; 3 rats for in situ hybridization and immunohistochemistry respectively), and three normal rats were used as control. The rats were anesthetized and perfused via the ascending aorta with warm (37°C) saline followed by warm solution composed of 4% paraformaldehyde and 0.2% picric acid in 0.1 M phosphate buffer at pH 6.9. The perfusion was then followed by 200 ml of the same fixative (4°C) for another 5 min. L4 and L5 DRGs and the lumbar spinal cord were then dissected out. The tissues were post-fixed in the same fixative for 90 min at 4°C, and were then immersed in 10% sucrose in 0.1 M phosphate buffer for at least 2 days.

Total RNA of normal rat DRGs and that of inflamed or axotomized rats at different time points were extracted with TRIzol reagent (Life Technologies). The mRNA was purified with an Oligotex mRNA kit (Qiagen). The mRNA (200 ng) of DRGs from normal and inflammatory or axotomized rats was reverse transcribed to cDNA. The PCR products were analyzed on 1.5% agarose gel. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as control. The primers for detection of mRNA expression were as following: for PAP-I, 5'-TCCTGCCTGATGCTCTTA-3' and 5'-TCATTGTTACTCCACTCCC-3'; for PAP-II, 5'-TGCCCTCTACACGAACCA-3' and 5'-ACTCCACTCCCATCCACC-3'; for GAPDH, 5'-ATCTCCGCCCCTTCCGCT-3' and 5'-TTGAAGTCACAGGAGACAACCT-3'.

A 600 bp digoxigenin-labeled antisense cRNA riboprobe spanning the entire PAP-II or PAP-I coding sequence and 3' UTR was generated from PAP-II cDNA as described previously [52]. DRG sections were fixed in 4% paraformaldehyde for 20 min, treated with proteinase K (10 μg/ml in DEPC water containing 50 mM Tris-HCl, pH 7.5 and 5 mM EDTA) for 20 min, acetylated in 0.25% acetic anhydride/0.1 M triethanolamine (pH 8.0) and prehybridized in hybridization buffer (50% formamide, 5 × SSC, 0.3 mg/ml yeast tRNA, 0.1 mg/ml heparin, 1 × Denhardt's solution, 0.1% Tween-20, 5 mM EDTA in DEPC water) for 4 h at 65°C. The prehybridization buffer was substituted by hybridization buffer with 1 μg/ml of the antisense probe in which the sections were incubated for 14 h at 65°C. After hybridization, excess probe was removed by washing three times with 2 × SSC at 67°C and once with RNase A (1 μg/ml) for 10 min. Sections were then incubated in alkaline phosphatase-conjugated sheep anti-digoxigenin antibodies (1:5,000; Roche Molecular Biochemicals), and then in 1 μl/ml NBT and 3.5 μl/ml BCIP substrates in alkaline phosphatase buffer (100 mM Tris-HCl, pH 9.5, 50 mM MgCl₂, 100 mM NaCl, 0.1% Tween-20 in distilled water). Control experiments were carried out using a digoxigenin-labelled sense riboprobe for PAP-I or PAP-II.

For quantitative analysis, 2~3 sections (14 μm-thick) from each DRG were representative for each rat and the data were collected from at least three animals at each time point. To determine the percentage of labelled neuron profiles, the number of positive neuron profiles was divided by the total number of neuron profiles. To determine the distribution of labelled neuron within a subset of DRG neurons, the cross-section area from the neuron profiles with a clear nucleus was examined and indexed with 100-μm² interval. Since the quantitative analysis on the percentage of the labelled neurons was based on profile counts and, therefore, only provides approximate estimates.

For all groups, 12 μm-thick sections of the fixed L4 and L5 DRGs, and L4-5 spinal cord segments were cut in series in a cryostat and mounted on same gelatin-coated slides. The sections were processed with indirect immunofluorescence histochemistry. The antibodies were diluted in phosphate-buffered saline with 1% bovine serum albumin and 0.3% Triton X-100. Briefly, the sections were incubated with a mixture of goat anti-PAP-II antibodies (1:200; Lifespan Biosciences) and goat anti-CGRP antibodies (1:5,000; DiaSorin), or rabbit anti-Trk B antibodies (1:5,000; Santa Cruz), or rabbit anti-peripherin (1:5,000; Chemicon) overnight at 4°C. To label IB4-positive small DRG neurons, sections were incubated with fluorescein-labelled IB4 (1:100; Vector Laboratories). After several rinses in PBS, the sections were incubated with fluorescence-conjugated donkey anti-rabbit and rhodamine-conjugated donkey anti-goat IgG (1:100; Jackson ImmunoResearch) for 45 min at 37°C. The sections were rinsed and mounted with a mixture of glycerol/PBS (3:1) containing 0.1% paraphenylenediamine and examined under a Leica SP2 confocal microscope.

The data were evaluated by unpaired Student's t-test. All data are shown as mean ± S.E.M. P value < 0.05 was considered to be significant.