The Role Of The Peripheral System In The Development Of Pain In Chronic Pancreatitis

AimsChronic pancreatitis (CP) is one of the most important pancreatic diseases, the most frustrating and challenging aspect of caring for patients with CP is pain management. The current approach remains empirical and unsatisfactory for several reasons, the most crucial of which may be an incomplete knowledge of the underlying neurobiology. The role of neurogenic inflammation in pain of CP has been focused in recent years. Neuronal sensitization induced by damage to the perineurium and subsequent exposure of the nerves to mediators and products of the inflammation plays a key role in pain of CP. Inflammatory and immune cells, various kinds of cytokines, neuropeptides, neurotrophins are likely to result in sensitization of pancreatic nociceptors, which mediates the development of pancreatic hyperalgesia.As one of the best-known members of the proteinase-activated receptors family, proteinase-activated receptor 2 (PAR2) is a promising target in anti-inflammatory and chronic pain. It is functionally expressed in primary afferent neurons. PAR2 activation could convey nociceptive messages directly or sensitize transient receptor potential vanilloid 1 (TRPV1) receptor indirectly, which leads to the development of visceral hyperalgesia and neurogenic inflammation. It has been demonstrated that the presence of PAR2 expression in adult rat pancreas and dorsal root ganglia (DRGs). Functions of PAR2 in pancreatic inflammation and the exocrine process were well studied. However, the role of PAR2 in pancreatic pain and somatic hyperalgesia in the setting of CP remains to be established. So, in this study, we assessed the expression of PAR2 and TRPV1 in DRGs and validated its role of thermal hyperalgesia in trinitrobenzene sulfonic acid (TNBS)-induced CP rat model.Nonsteroidal anti-inflammatory durgs (NSAIDs) are widely prescribed for the treatment of pain in CP. But its long-term effects on pancreatic pain and fibrosis of CP has been unknown. In WBN/Kob rats, chronic inflammatory changes and subsequent fibrosis can be inhibited by specific Cyclooxygenase 2 (COX-2) inhibitor rofecoxib. NSAIDs, COX-2 activity can be inhibited as well, but there are other matters to consider in the setting of CP. NSAIDs induced the development of oxidative stress (OS) and the intestinal damage, OS and bacterial endotoxins were one of the most important mechanisms in fibrosis progression of CP. Considering nonselective COX inhibitors'pathogenic and anti-pathogenic roles in CP, their integrated effect on CP should be identified. Therefore we analyzed the effect of naproxen on thermal hyperalgesia and fibrosis in TNBS-induced CP rats.MethodsMale SD rats (250-300g) were randomly divided into CP and control groups. CP was induced by TNBS infusion into rat pancreatic ducts. While in the control group, pancreases were just dragged lightly. Abdominal hyperalgesia was measured by thermal withdrawal latencies. The expression of PAR2 and TRPV1 in T8-T10 DRGs were analyzed by immunofluorescence and Western blot. We also made simple correlation between the averaged withdrawal latencies of each rat and its Western blot data for PAR2. Trypsin inhibitor ulinastatin (50000u/Kg & 25000u/Kg, i.p.) was used to inhibit PAR2 activity, the effect of acute and chronic ulinastatin treatment (5 days) on the abdominal thermal latencies was evaluated.Naproxen treatment (20mg/Kg & 40mg/Kg, p.o.& i.p.) started 2 weeks after the induction of CP for 3 weeks. Histological analysis of the pancreas, Van-Gieson & Sirus-red staining and contents of hydroxyproline were used to evaluate pancreatic damage and fibrosis. The effect of naproxen on nociceptive reflective behaviors and serum tumor necrosis factor-a (TNF-a) concentration were also performed. The plasma endotoxins and diamine oxidase (DAO) levels were analyzed for the evaluation of intestinal barrier functions.Data were analyzed using SPSS PC version 13.0. P<0.05 was considered statistically significant.Results1. The CP rat model with abdominal thermal hyperalgesia was established.All rats injected with TNBS displayed histopathological features of CP at the time of sacrifice, as reflected by sublobular and lobular atrophy, fibrosis, edema, inflammatory cells infiltration and pancreatic duct dilation. However, the pancreatic islet was intact. While the control group showed no histopathological changes. Increased collagen deposition was found in CP rats compared with the control group. Positive areas of sirus-red staining sections in CP rats were higher than the control group (5.73±1.45% vs. 2.23±0.42%, P=0.001). And hydroxyproline content was higher in the CP group (519.34±168.52ug/g vs.328.02±59.08ug/g, P=0.003).CP rats demonstrated increased sensitization to thermal stimuli on the abdominal area, i.e., decreased withdrawal latencies (6.52±0.68s vs.10.68±1.28s; P=0.000).2. PAR2 was up-regulated in DRGs of CP rats.The expression of PAR2 relative to GAPDH was dramatically increased in CP rats compared with the control rats (1.00,0.80-1.27 vs.0.45,0.40-0.49; P=0.004). The distribution of PAR-2 in the DRGs was similar to previously reported results, with prominent localization in the medium and small-diameter neurons. When counted at×200 magnification, the percentage of PAR-2-positive neurons was increased in CP rats compared with the control group ((61.98%,54.43%-69.23%; 896 PAR2 labeled neurons of the 1457 ganglion neurons) vs. (49.23%,35.70%-56.20%; 710 PAR2 labeled neurons of the 1448 ganglion neurons); P=0.000). When analyzed at×100 magnification, the integrated optical density of PAR2-positive neurons was also increased in CP rats compared with the control rats (336.31±73.00 vs.155.47±51.42; P=0.000).3. TRPV1 was up-regulated in DRGs of CP rats.The expression of TRPV1 relative to GAPDH was increased compared with the control rats (0.85,0.57-0.87 vs.0.38,0.36-0.55; P=0.036). The distribution of TRPV1 in the DRGs was similar to previously reported results, with prominent localization in small-diameter neurons. When counted at x200 magnification, the percentage of TRPV1-positive neurons was increased in CP rats compared with the control group ((61.47%,55.82%-66.81%; 881 TRPV1 labeled neurons of the 1432 ganglion neurons) vs. (46.11%,37.44%-51.39%,577 TRPV1 labeled neurons of the 1358 ganglion neurons), P=0.000). When analyzed at×100 magnification, the integrated optical density of TRPV1-positive neurons was also increased in CP rats compared with the control group (255.58±75.52 vs.162.89±50.99, P=0.003).4. PAR2 mediates the development of thermal hyperalgeisa of CP rats.We made simple correlation between the averaged withdrawal latencies of each rat and its Western blot data for PAR2. The increased PAR2 protein expression was tightly correlated with thermal withdrawal latencies (r=-0.821, P=0.001). Acute ulinastatin treatment could inhibit the thermal hyperalgesia of the CP rats in a dose-dependent manner. High-dose ulinastatin treatment increased the abdominal thermal latencies of the CP rats compared to the control ones (8.67±1.76s vs.6.17±1.05s, P=0.001). And low-dose ulinastatin treatment also increased the abdominal thermal latencies of the CP rats compared to the control ones (7.74±1.00s vs.6.17±1.05s, P=0.013). While chronic ulinastatin treatment didn't affect the abdominal thermal latencies of the CP rats (P>0.05).5. Effect of naproxen treatment on CP rats and systemic inflammation.20% percent of the animals died in vechile-treated CP rats.25% percent of the animals died in CP rats treated with low-dose naproxen (20mg/Kg).27.5% percent of the animals died in CP rats treated with high-dose naproxen (40mg/Kg) by intraperitoneal injection. About 50% rats died in the CP group treated with high-dose naproxen administered orally. And high-dose naproxen-treated animals had dark stools but maintained their dietary intake in the first week of naproxen administration, significant weight reduction was observed in this group (24±14.75g).Only jaundiced CP rats showed a higher level of serum TNF-a compared to control groups (P＜0.05,169.61±39.95pg/ml vs.47.62±14.72 pg/ml). Naproxen treatment did not affect serum TNF-a level, irrespective of its doses or routes of administration.6. High-dose naproxen administered orally aggravated pancreatic damage and fibrosis.CP rats that were treated with high-dose naproxen (40mg/Kg, p.o.) showed more severe pancreatic damage compared to vechile-treated CP rats, especially fibrosis and inflammation. The fibrosis scores were 2.5±0.5 and 1.2±0.4 separately in the above two groups (P＜0.05). The inflammation scores were 2.2±0.8 and 1.0±0.0 separately in the above two groups (P＜0.05). Low-dose naproxen (20mg/Kg) treatment and high-dose naproxen administered by intraperitoneal injections did not affect pancreatic damage in CP rats (P>0.05).High-dose naproxen administered orally (group A2) increased the positive areas of VG staining sections compared to vechile-treated CP rats ((8.89±0.78)% vs. (5.77±1.65)%, P<0.05). Hydroxyproline content was also higher in the CP group treated with high-dose naproxen administered orally (821.67±274.96ug/g vs.534.83±234.34 ug/g, P＜0.05). The routes of administration affected pancreatic collagen content. The positive area of VG-staining sections in group A2 was higher than the CP groups treated with naproxen (20mg/Kg and 40mg/Kg) by intraperitoneal injection ((8.89±0.78)% vs. (4.51±1.07)%, (6.43±1.94)%, P＜0.05). Hydroxyproline content was also higher in group A2 compared to the CP groups treated with naproxen (20mg/Kg and 40mg/Kg) by intraperitoneal injection (821.67±274.96ug/g vs 557.04±166.99ug/g,559.38±178.38ug/g, P＜0.05).However, we didn't find naproxen treatment affected plasma DAO or endotoxin levels, irrespective of its doses or routes of administration.7. High-dose naproxen treatment decreased the thermal thresholds of CP rats.High-dose naproxen treatment (40mg/Kg, p.o and i.p.) decreased thermal withdrawal latencies in CP rats (5.02±1.54s vs.6.52±1.49s; 5.17±1.31s vs.6.67±1.61s; P＜0.05). No change in the withdrawal latencies was observed in CP rats treated with low-dose naproxen (P>0.05).Conclusions 1. The CP rat model with abdominal thermal hyperalgesia could be established successfully by TNBS-injection into pancreatic ducts.2. We found clear evidence for the up-regulation of PAR2 and TRPV1 in CP rats, at the protein levels. The increased PAR2 protein expression was tightly correlated with decreased thermal withdrawal latencies of CP rats. Acute ulinastatin treatment could inhibit the thermal hyperalgesia of the CP rats in a dose-dependent manner. It hinted that the up-regulation of PAR2 was involved in the development of thermal hyperalgesia in CP. The above effect may be dependent on TRPV1 sensitization by PAR2.3. Instead of relieving pain, high-dose naproxen treatment reduced the withdrawal latencies to heat stimuli in CP rats. High-dose naproxen administered orally aggravated pancreatic damage and fibrosis in TNBS-induced CP rats. Intestinal damage and oxidative stress induced by high-dose naproxen may be involved in the above effects.