In this investigation, DBTC-treated animals developed persistent pancreatic inflammation detected in pathological analysis and secondary pain-related hypersensitivity. The DRG and spinal cord from 10th-12th thoracic segments were selected for gene microarray analysis, as they portray the major source of sensory neuronal fibers in the pancreas. Amongst the 52 candidate genes upregulated in three animals in each experimental group (run in duplicate) was ET-1. ET-1 was upregulated greater than 2-fold in the animals with pancreatic inflammation and visceral pain-related behavior. This finding illustrates the benefits of gene array analysis in identifying relevant genes with possible direct roles in mediating pain in visceral inflammatory states. Visceral mechanical testing with von Frey microfilaments demonstrated functional significance showing increases in abdominal withdrawals on day 6-7 post DBTC insult in pancreatitis animals. In contrast, no changes were detected in normal rats receiving sham vehicle alone. Similarly, spontaneous pain related behaviors were unchanged in naïve rats treated with endothelin antagonists indicating that the agent had no effect on normal exploratory behaviors. Single highest dose treatment with ET-A antagonist (BQ123) provided significant protection against inflammatory pain related behaviors in animals with pancreatitis, while the ET-B antagonist (BQ788) had a short lasting effect.
In the current study, ET-A and ET-B receptors were both detected in DRG. ET-A receptors trigger vascular constriction and ET-B receptors act as vasodilators, but their role in neurons is not defined.
Although, ET-A and ET-B receptors are both present in the thoracic spinal segment, and DRG, ET-B receptors are primarily expressed in DRG satellite cells and ensheathing Schwann cells[27
] where they can stimulate the synthesis and release of prostaglandin E2, an active compound in inflammatory pain[16
]. In addition, ET-B receptors present on keratinocytes mediate the release of β-endorphin from these cells with a local analgesic effect[18
]. ET-1 has a mitogenic effect, promoting cancer cell growth in colon and pancreas in which there is also upregulation of ET-A receptors and moderate downregulation of ET-B receptors[28
In our hands, ET-A receptors appeared to be more localized around constricted vasculature in pancreatic tissues of animals with DBTC-induced pancreatitis. While, modest detectable differences were evident in the ET-B expression of the pancreatic tissues in comparisons of animals with pancreatitis to naïve controls.
Animals with pancreatitis demonstrated trend toward a reduction in active time and an increase in resting time. Immunohistological analysis revealed increased ET-A in DRG neurons of all sizes and ET-B in myelin sheath and satellite glia. The changes in spontaneous behaviors induced by pancreatitis were normalized by application of the ET-A antagonist (BQ123), suggesting that this receptor has a role in pancreatitis-induced behavioral changes.
Injection of ET-1 is reported to cause severe pain via
activation of ET-A receptors, in the sciatic nerve chronic constriction injury model[29
]. In the present study, both the endothelin-A (BQ-123) and B (BQ-788) receptor antagonists significantly reversed the thermal hypersensitivity assessed using the hot plate method at 20 min, 40 min after injection. However, the ET-B antagonist was ineffective at the 75 min time point while the efficacy for the endothelin-A receptor antagonist persisted. Neither agents affected vehicle injected animals. Additionally, we demonstrated that the thermal and mechanical hyperalgesia induced in pancreatitis animals were equally well normalized by the administration of morphine (10 mg/kg) with a dose dependent response, whereas a lower dose of BQ-123 and BQ-788 were required to reverse the nocifensive responses in the pancreatitic rats. Similarly, administration of the ET-A receptor antagonist reversed the attenuation of spontaneous exploratory behaviors observed in pancreatitic animals.
These data reveal that nocifensive responses invoked by the persistent pancreatitis in animals can be ameliorated by systemic post-treatment with endothelin-A receptor and to a lesser degree endothelin-B antagonist similar to conventional morphine administration. As has been suggested for neuropathic pain[29
], ET-A receptor antagonists deserve future study as potential novel therapy including against inflammatory pain.
Previous studies indicate that treatment with non-selective ET-A and ET-B (LU 302872) and selective ET-A (LU 302146) antagonists had no effect on the pancreatic pathological (edema and inflammatory infiltration) nor on trypsinogen activation 4h after caerulein-induced acute pancreatitis model. A slight increase in the pathological necrosis and vacuolization suggested the possible undesired effects of these compounds in the model[30
]. In contrast ET-1 at the high dose was found to be beneficial on morphological changes and trypsinogen activation in that model[31
]. In our hands we did not detect any undesirable pathological effects or any histological improvement in treated animals after single dose i.p. administration of endothelin-1 receptor antagonists (BQ123, BQ788).
In severe acute pancreatitis, microcirculatory disorders and increased capillary permeability contribute to multiple organ dysfunction syndrome, while, ET-A receptors stabilize capillary leakage and improved organ function[32
]. Of interest, upregulated genes reported here substantiate this finding. For example, multifunction pro-inflammatory cytokine IL-6 levels were significantly upregulated in DRG of rats with pancreatitis on day 6 after DBTC injection[20
]. Relative quantification of target cDNA levels in primary stellate cultures using real-time polymerase chain reaction revealed a dose-dependent reduction of endothelin-1 after treatment with inhibitors of histone deacetylases[33
]. This is relevant to anti-cancer activities and, therefore, of growing clinical interest[34-36
]. It is well established that diabetes can occur in acute pancreatitis as well as chronic pancreatitis. Insufficient pancreatic enzyme activity and dosing is treated in pancreatic steatorrhea with administration of lipase with meals in patients[37
]. In this model we detected modulation of genes for the inflammatory markers of pancreatitis, including upregulated lipase, α enolase and α tryptase, while insulin precursor, glucose transporter type 5, and glycogen phosphorylase were down regulated. In accordance with these findings the increased levels of the serum amylase and lipase were reported on day 3 and peaked on day 7 in this DBTC induced pancreatitis model, consistent with pancreatitis in patients[22
]. Finally, amongst those genes found to be downregulated were peripheral myelin protein 22, α platelet-derived growth factor receptor, dopamine receptor, and neuropeptide Y receptor. Further studies are warranted to investigate the role of these candidate genes as novel targeted therapeutic modalities in patients. ET-A receptor antagonists deserve future study as a potential novel therapy against inflammatory and neuropathic pain.
In conclusion, we report 8 different groups of genes modified in this model of pancreatitis. These candidate genes may be useful as future biomarkers for diagnostic and/or targeted gene therapy. As an example, endothelin-1 gene was upregulated and subsequently, ET-A and ET-B receptor antagonists were found to reverse inflammatory pain responses. These results demonstrate the potential utility of the gene microarray analysis to identify candidate genes for analgesic development.