Overexpression of met-ENK observable both in spinal cord and pancreas significantly improved pancreatic inflammatory and histological outcomes, as well as reversed hotplate hypersensitivity in a chronic pancreatitis model induced with a high fat and alcohol diet in young rats. Histologic signs of chronic pancreatic inflammation evident in controls were not observed at ten weeks after HSV-ENK treatment. Diet induced pancreatitis in controls (with or without the HSV-β-gal control vector treatment) was evident as fibrosis, edema, acinar necrosis, steatosis, ductal widening, and inflammatory cell infiltration, with no hepatic or Langerhans involvement. Hotplate sensitivity apparent after 3 weeks on the diet in all animals persisted though the ten week study in controls, but was attenuated for 4–6 weeks after HSV-ENK treatment without tolerance or other adverse opiate symptomatology.
HSV-ENK-Treated animals demonstrated normal histology and decreased inflammation
The most intriguing finding in these studies was the relative preservation of pancreatic tissue in HSV-ENK-treated rats fed the same high-fat and alcohol diet, compared to the significant inflammatory tissue damage noted in the vehicle and control vector-treated animals. The histological preservation and/or restoration of pancreatic tissue is coupled with decreased RANTES staining and inflammatory cell invasion in HSV-ENK-treated animals relative to vehicle and vector controls. Marked improvements in inflammatory cell infiltrates and decreased inflammatory mediator content (RANTES and COX-2) are seen after HSV-ENK treatment in two additional animal models in use in our laboratory (CFA arthritis and acute pancreatitis ). Radiographic improvement after inflammatory damage has been reported previously in arthritic animals treated with this enkephalin-encoding vector in addition to decreased periarticlar osteopenia . There is abundant evidence that opioid peptides delivered by infiltrating inflammatory cells contribute to tissue enkephalin content with modest anti-inflammatory effects [23–28]. However, unlike synthetic opiates, met-enkephalin is quickly degraded by endopeptidases and is not measureable in blood. It is important to note that over-expressed met-enkephalin is delivered directly to target tissues by nociceptive afferents affected by the HSV vector [19, 29]. Previous experiments, using antisera against human preproenkephalin, which does not cross-react with rodent preproenkephalin have demonstrated gene product expression in sensory neurons containing HSV protein after peripheral administration of this human preproenkephalin vector . The impact of opiates delivered to the site of inflammation by the affected neuronal endings has not previously been appreciated. Delivery of significant quantities of exogenous met-enkephalin by HSV vector-driven overexpression provides a means of getting at this issue and by all appearances provides significant tissue protection. Enkephalin has been shown to reduce release of substance P from dorsal root ganglia cells in culture by blocking voltage gated Ca++ channels , which would abrogate both nociceptive and neurogenic edema actions of substance P. We propose that it is the continuous release by the neuronal endings of significant levels of met-ENK, directly into pancreatic tissue in a physiologically relevant manner that provides additive effectiveness for tissue protection from inflammatory responses imposed by the diet. Follow-up studies will be required to assess the relevance of neuronally-released met-enkephalin to the inflammatory response.
A protocol with accelerated alcohol associated pancreatitis
Pancreatitis is defined by the presence of inflammatory mediators and inflammatory cells in the pancreas in histological samples. The animal model provided here has histopathology consistent with severe chronic pancreatitis without mortality. The rapidly induced chronic pancreatitis, complete with fibrosis in three weeks, is likely accelerated at this developmental stage since the pancreas of young animals is ill equipped for this diet. Many previously reported pancreatitis models employing high fat and alcohol diets in older rats take much longer to develop, and pancreatitis is not consistently observed in all animals. Animals given alcohol alone as adults can take up to eight months to develop pancreatitis [1, 2]. Modifications in this study from previously published models [31, 32] included initiation of high fat and alcohol diet at an earlier age, incrementally increasing alcohol concentration each week in the nutritionally balance commercial diet to assure animals are able to maintain increase in body weight and good health.
The chronic pancreatitis model described here was devised specifically to test the full time course of HSV viral vector overexpression. Our accelerated diet-based chronic pancreatitis model in young animals yields consistent inflammatory pathology, does not require surgical manipulation, is cost efficient and importantly does not have the associated high mortality rate we observe with acute chemical/surgically induced pancreatitis models. Furthermore, the animals show no outward signs of discomfort or weight loss, and thus studies can be blinded through the three month time course of study. The diet induced pancreatitis was mild enough to maintain a healthy appearance in young animals for the study duration, did not produce spontaneous behavioral signs of visceral pain or altered pain thresholds as in other more severe visceral pain models, and allowed blinded behavioral testing. These data further support the observation made previously that high fat diet causes pancreatic inflammation in animals though longer times are required for adult animals .
The pancreatitis model provided accelerated development of hotplate sensitization within three weeks in all animals tested. The hotplate test was well tolerated and allowed determination of the full time course of the HSV mediated met-enkephalin overexpression effect. This has not been possible with other pancreatitis models which either resolve within weeks or produce more severe pancreatitis with multi-organ failure possible if allowed to persist. Outright spontaneous pain behaviors were not evident in these animals, nor were results positive for other conventional pain-related behavioral tests, such as Hargreaves, von Frey and open field tests, although our preliminary tests indicate that these measures will be useful in extended studies (over three months). Although there have been few studies assessing hyperalgesia or allodynic responses in humans, both chronic pancreatitis and pancreatic cancer produce secondary thermal hyperalgesia in humans [34, 35]. Chronic pancreatitis produces generalized deep hyperalgesia consistent with central sensitization [36, 37]. Most pain testing in patients using mechanical and electrical stimulation, report heightened responses, VAS scores and increased descriptive words for the pain. There are at least 3 quality of life instruments that have been used in pancreatitis related pathologies. These include the SF-36, which is validated across countries, ethnicities and is non-organ specific. This test reflects the physical and mental restrictions placed on the patient secondary to generalized pain. There are also modified SF-36 instruments focused for pancreatic cancer. The standard SF-36 includes a domain for bodily pain (BP), which assesses general body pain. The normative value for BP is >85 for most studies. Pancreatic pain patients usually score around 40–50, which is significantly less, but improvement in BP is due to therapeutic interventions.
The met-enkephalin-induced analgesia suggests that spinal cord opiate receptors modulate transmission of visceral pain information that is being provided to higher centers involved in the processing affective responses to pain. Assessments of affective pain due to visceral pain are considerably more difficult in preclinical testing. The hotplate test provokes complex higher level pain induced responses (foot flick, paw licking, etc) mediated by supraspinal as well as reflexive motor commands . Tests relying primarily on reflexive spinal cord and brainstem responses to stimulation of cutaneous regions of the abdomen or footpad were unaffected in this model, as was noted in a tail amputation model previously . Despite measurable blood alcohol levels, open field activity testing in the San Diego apparatus revealed no differences between groups through the ten weeks of testing. This is reflective of the mild nature of the model which we have chosen for these chronic studies. It is likely that continuation of the high fat and alcohol diet would initiate referred and secondary nociceptive sensitization. Erichsen (see ) stated that pain from urinary bladder is sometimes referred to soles of the feet. Ness and colleagues  have reported sensitization as far as the knees in rats after repeated bladder distensions. The pancreas of rat is innervated bilaterally by axons from dorsal root ganglia T6-L2 (primarily T9–T13, . Kuo and De Groat  determined that splanchnic nerve innervating pancreas (and other visceral organs) is composed of 90% unmyelinated fibers. Nociceptive foot withdrawal in response to applied heat occurs at about the same skin temperature as activation of nociceptors, thus response latency is an accurate measure of changes in nociceptive threshold produced by drug treatments [43–45]. Mechanosensitivity was not evident in this model, implying there was no recruitment of peripheral Aδ or Aβ fibers or sensitization of central neurons to this type of input. Previous studies have reported differential hyperresponsivity for mechanical without thermal sensitization [40, 46–48]. An example of thermal hyperalgesia alone was shown after gene transfer for overproduction of nerve growth factor (NGF). The NGF produced thermal and mechanical hyperalgesia in the injected paw, but only thermal hyperalgesia in the uninjected paw . Spinal sensitization mechanisms are likely responsible, while higher level sensitization is implied for the hotplate test. Meller and Gebhardt  propose that differential responses indicate that different mechanisms underlie thermal and mechanical hypersensitivity. Thermal hyperalgesia occurs with N-methyl-D-aspartate (NMDA) receptor mediated calcium-dependent production of nitric oxide, while mechanical hyperalgesia results from coactivation of alpha-amino-3-hydroxy-5-methylisoxazole-5-propionate (AMPA) and metabotropic glutamate receptor mediated cyclo-oxygenase products of arachidonic acid metabolism.
It is well known that somatic and visceral pain differ substantially in perception. In particular, a classic feature of visceral pain is referral to another part of the body , often following a dermatomal pattern . A previous study reports thermal sensitivity of hindpaw after acute bladder inflammation (50% turpentine for 1 hr)  and provides review of two primary theories to explain referred pain. The axon reflex theory proposed by Sinclair, Weddell and Feindel in 1948  stated that axons with collateral branches innervating both somatic and visceral targets may become sensitized sending erroneous messages to spinal cord. A second theory by Hardy and others proposes convergence at a central site that becomes an irritable focus [49, 53–55]. The data presented in the present study provide support for central viscerosomatic convergence. Convergence of both visceral and somatic input has been shown onto spinal neurons located in the visceral processing region near the central canal (lamina X)  that projects to regions of the brain involved in processing of affective pain . Thus, the hotplate test is able to distinguish spinal and supraspinal (higher order decision making) responses specific to visceral pain and analgesia in this model. Mutant enk-/- mice are rendered highly sensitive to the hotplate test while their baseline responses to other nociceptive tests, such as tail-flick and force swim, were unaffected . When the knockout animals were challenged with acute pain as in the formalin test or when normal animals are treated with naloxone, nociceptive hyper-responsivity indicated a direct response to the lack of enkephalin. Konig and colleagues  have also speculated that opiates affect an anatomical pathway for subjective pain rather than discriminative pain. Studies in humans show that opiates do not alter baseline responses to pain (pain thresholds) but only the subjective experience of pain . The present studies provide support for a role by met-enkephalin in visceral pain transmission and ultimately involvement in affective responses to pain implying that subjective pain is not entirely mediated by the many opiate receptors distributed at higher brain levels, including the limbic system.
HSV-ENK significantly reduced behavioral sensitization without tolerance for 4 weeks
Noxious thermal stimulation has been used to assess central sensitization induced by various experimental models of clinical pain syndromes [60–63]. In this study, the hot plate test indicated hypersensitivity after three weeks on the diet. Response latencies recovered within 2 weeks to near-baseline levels in HSV-ENK-treated animals, but not in animals treated with control HSV-β-gal virus or the vehicle. The single HSV-ENK treatment provided an anti-nociceptive effect persisting for at least 4 weeks (week 5–9) without tolerance normally observed in rats with opiate therapy. This overexpression time course and efficacy profile is consistent with other HSV viral vector studies in animal models of ongoing central sensitization [13, 64, 65]. Thus, with further molecular manipulations, these vectors have potential for treatment of chronic pain, including visceral pain.
In a site-directed manner, pancreatic surface application selectively and effectively provides met-enkephalin to the same receptors on nerve ending receiving information from the inflamed pancreas, thus potently contributing to anti-nociception and tissue preservation/restoration. Standard pain therapies relying on higher and higher levels of circulating opiates, on the other hand, can result in intolerable side-effects in patients and rapid development of tolerance in rats within days. Differential effects are noted with mu opiate treatments, however, when an inflammatory model is used in rats [66, 67]. Enkephalin administration has also been shown to attenuate morphine tolerance [68, 69], though it is rapidly inactivated by endopeptidases if administered by conventional routes. The present study indicates that site specific overexpression of met-enkephalin is a suitable opiate replacement therapy or adjunct to low dose morphine, and would avoid intolerable systemic side-effects.
The gradual return of hyperalgesic responses (decreased latency thresholds) was noted between weeks 8 and 10 to the same levels seen in the control animals with pancreatitis, despite significantly high levels of met-enkephalin in the dorsal horn and pancreas. This mismatch may be either reflective of the development of tolerance by week 10 and/or diminishing synthesis of the transgene product with increasing HSV latency. Alternatively, other plastic changes may develop by ten weeks in the spinal cord of the now adult rats related to descending facilitation pathways and/or the diverse population of receptors and transcription factors known to have a role in the development of tolerance [70–72]. The literature provides few clues for the extended time course of the present study, so additional studies are warranted.
Fos expression in animals with pancreatitis
Expression of Fos protein in the nucleus is another established indicator of cell activation, especially after noxious stimulation [73–78], including visceral pain [79–82]. With the exception of the HSV-ENK-treated animals, Fos expression was present at ten weeks in spinal cord, ventrolateral periaqueductal gray (PAG) and dorsal raphe. These regions are major central nervous system sites involved in modulation of nociception and concomitant behavioral and autonomic responses [83–85]. Noxious stimulation of viscera has been shown to evoke significant increases in Fos expression in the ventrolateral column of the PAG in acute pain models [86–89, 80, 86–88]. Typically, enhanced expression of Fos is reported for chronic pain models only after another acute noxious stimulation is given . The presence of Fos at ten weeks in the control animals with pancreatitis was unexpected and likely related to the persisting inflammation that was providing continuous noxious stimulation of pancreatic afferents. While significant Fos expression was observed in hypersensitive control rats fed high fat and alcohol diet (vehicle and HSV-β-gal controls), little Fos was evident in animals treated with HSV-ENK, even though the hotplate sensitivity was re-established at ten weeks. It is likely that Fos protein detected by the polyclonal antibody is a chronic Fos-related antigen (FRAs; Ex. deltaFosB), a stabile protein that can persist for months which served here as a biomarker for activation only in groups with chronic ongoing pancreatic inflammation and hypersensitivity as reported previously in other inflammatory and restraint stress models [90–92]. This supports the role for met-enkephalin in long-term visceral nociceptive processing and/or autonomic control as previously speculated .
Interestingly, the Fos labeled nuclei in spinal cord of control animals with diet-induced pancreatitis were localized in deeper laminae of the dorsal horn rather than superficial laminae as in some pain models, i.e. cutaneous and neuropathic pain. Localization in deeper lamina has been noted previously with bladder stimulation . Abbadie and Besson  speculated that deep laminar distribution of Fos seen in their early CFA studies might denote chronic versus acute noxious activation. We further speculate that deep tissue, whole body insults such as their CFA injections at the base of the tail or pancreatitis in the current study, activate cells deep in the dorsal horn and around lamina X, in contrast to superficial cutaneous insults which activate cells and inducing Fos in the superficial dorsal horn. The deeper distribution of activated cells is also consistent with visceral pain transmission by (1) lateral spinothalamic tract cells in laminae IV, V and VII and (2) post-synaptic dorsal column cells in laminae III and X .
The role of opiate receptors in inflammation
No evidence of inflammation was seen at ten weeks in HSV-ENK-treated animals, i.e. no inflammatory cell infiltration or RANTES, despite apparent decrease in antinociceptive effect of the vector at this time point. This apparent contradiction may be related to clinical observations that analgesic effectiveness of opiates can diminish due to decrease of opiate receptors while other physiological effects of opiates are sustained. Adverse sympathetic effects occur in response to use of synthetic opioids in patients even after development of tolerance to the analgesic effects . Therefore, anti-inflammatory and analgesic effects of opioids may each have uniquely dedicated mechanisms and time courses.
The improved histological findings for pancreatic tissues after HSV-ENK in the present study were coincident with decreased RANTES and COX-2 and increased met-enkephalin staining. The anti-inflammatory effects of opioids, including met-enkephalin, have been previously reported, and met-enkephalin is believed to be the major anti-inflammatory peptide of the preproenkephalin gene products (for review see ). Studies have shown that peripheral opioid receptor effects provided by blood borne inflammatory cells are increased in efficacy and potency during active inflammatory conditions, and a role for neuronal opioid peptides in reduction of inflammatory mediators has also been reported [21, 28, 98–104]. The increase in efficacy during inflammation has been shown to be true for reduction of RANTES in particular [69, 105]. A previous study has shown that met-enkephalin is also protective against stress ulcers in the gastrointestinal tract of rats .
The literature suggests the opioid-induced immunosuppression in peripheral target cells include both opioid receptor-dependent and receptor-independent mechanisms, although the mechanisms are not fully elucidated at this time. While delta opiate receptors are abundant in the pancreas, their usual role is as an indirect mediator of the acinar secretory functions [107, 108]. Delta-opioid receptor agonists such as met-enkephalin do not affect the stimulant effect of KCl on isolated pancreatic lobules or acinar cells in vitro, rather they decrease pancreatic enzyme secretion by inhibiting cholinergic transmission. No labeled nerves were observed in the fragile samples from the pancreas, nor were delta opiate receptor antibodies available at the time of these studies. Mu opiate receptors and met-enkephalin protein are found in the pancreas in amounts equal to the brain homogenate , and since they are normally involved in glucose metabolism their localization is typically in the glucagon cells of the islets of Langerhans. While mu opiate receptors were unchanged at the end of the present long term study after HSV-ENK administration, mu opiate receptors were shown to be increased at one week in our previous study in all animals with acutely inflamed pancreata . The mu opiate localization evident in the pancreatic acinar cells one week after HSV-ENK is thus far unexplained. The significance of the abundance of met-enkephalin in the acini of the pancreas after HSV-ENK treatment in the absence of any remaining evidence of HSV protein, however, suggest that the met-enkephalin is of neuronal origin in agreement with previous studies with this vector or a similar one where stimulated release product was measured peripherally or stained proximally in ligated peripheral nerves [19, 29].