Background
Chronic Pancreatitis (CP) is a complex and multifactorial syndrome. Many contributing factors can result in abnormal pain etiology, including both peripheral and central pain processing and structural abnormalities of the pancreatic gland [
1]. The higher incidence in men reported nationally is attributed not only to alcohol and tobacco abuse as risk factors, but a genetic prevalence in men to have the CLDN2 risk factor DNA variant with alcohol associated chronic pancreatitis (47%) [
2‐
5]. Pain management in CP is challenging and often leads to time-consuming and unsatisfactory approaches to treatment with an unpredictable outcome. Despite the availability of analgesics approved for chronic or neuropathic pain, current analgesics only provide partial pain relief and/or produce significant side effects. Recently, availability of selective pharmacological tools has enabled great advance of our knowledge of the role of cannabinoid receptor 2 (CB2) in pathophysiology. The cannabinoid receptors are a class of cell membrane receptors under the G protein-coupled receptor superfamily. There are currently two known subtypes, termed CB1 and CB2. The CB1 receptors are expressed mainly in the brain (central nervous system or "CNS"), but also in the lungs, liver and kidneys. CB2 receptors are largely restricted to immune and hematopoetic cells, although functionally relevant expression has been found in specific regions of the brain and in myocardium, gut, endothelial, vascular smooth muscle and Kupffer cells, exocrine and endocrine pancreas, bone, reproductive organs/cells, and in various tumors [
6‐
9]. Evidence suggests that there are novel cannabinoid receptors, that is, non-CB1 and non-CB2, which are expressed in endothelial cells and in the CNS [
10].
Initially the interest in cannabinoid receptors as potential targets for chronic pain was limited to cannabinoid receptor 1 (CB1), with the aim of identifying an agonist suitable for drug development. Unfortunately analgesia induced by CB1 agonists is associated with undesirable central nervous system side effects which have hampered their progress for therapeutic development [
11]. More recently, the type 2 cannabinoid receptor (CB2) has emerged as an interesting target alternative to CB1. Its expression is upregulated in response to tissue or nerve injury, and in preclinical pain models the activation of this receptor induces significant analgesia without overt side effects [
12,
13].
The most likely cellular targets and executors of the CB2 receptor-mediated effects of endocannabinoids or synthetic agonists are proposed to be the immune and immune-derived cells (e.g. leukocytes, various populations of T and B lymphocytes, monocytes/macrophages, dendritic cells, mast cells, microglia in the brain, Kupffer cells in the liver, etc.). However, the number of other potential cellular targets is expanding, now including epithelial, endothelial and smooth muscle cells, fibroblasts of various origins, cardiomyocytes, and certain neuronal elements of the peripheral or central nervous systems [
7,
14‐
16]. In the brain, CB2 receptors are predominately expressed by microglial cells, where their role remains unclear [
7,
12,
14,
15,
17,
18] but is suggestive of an anti-inflammatory role. In particular, CB2 for chronic pain treatment has been successfully demonstrated by several studies with inflammatory and neuropathic preclinical pain models [
19,
20]. While the CB2 receptors are involved in mediating analgesic effects in the peripheral nervous system, these receptors are not expressed by nociceptive sensory neurons [
7]. At present CB2 receptors are believed to exist on an undetermined, non-neuronal cell in the vicinity of peripheral nerve terminals. Possible candidates in the pancreas include mast cells and innate immune stellate cells which are known to facilitate the inflammatory response. Cannabinoid mediated inhibition of these responses may be caused by a decrease in the receptor perception of noxious stimuli [
10,
21,
22]. Recent studies showed that CB1 and CB2 are also expressed on pancreatic acinar cells, and the relatively nonselective CB1/CB2 agonist HU210 ameliorates acute experimental pancreatitis by systemic administration [
23]. During acute pancreatitis, an upregulation especially of CB2 on apoptotic cells has been shown, and activation of cannabinoid receptor 2 attenuated the acute pancreatitis [
24]. Little is known about the effects of CB2 that impact inflammation and pain in chronic pancreatitis.
In the present study, a novel CB2 receptor agonist LY3038404 HCl was evaluated for therapeutic efficacy in the AHF chronic pancreatitis rat model. The major finding of the present study is that the potent CB2 receptor agonist LY3038404 HCl possesses tissue protection and analgesic properties. No side effects on higher brain function were observed. Thus, activation of CB2 receptors is suggested as a potential therapeutic target for visceral inflammation and pain management.
Conclusion
The major finding of the present study is that LY3038404 HCl, a potent CB2 receptor agonist, possesses tissue protective and analgesic properties. No effects on higher brain functions were observed including the diminished fear responses induced by the alcohol diet. Thus, activation of CB2 receptors is suggested as a potential therapeutic target for pancreas protection and pain management.
Methods
The studies were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health and were approved by the University of Kentucky Institutional Animal Care and Use Committee.
Experimental design
These studies were performed in order to evaluate the therapeutic efficiency of a novel CB2 receptor agonist LY3038404 HCl in the AHF chronic pancreatitis rat model. After the nocifensive behaviors were established, i.e. six weeks after the start of the alcohol/high fat liquid diet feeding, the LY3038404 HCl was given twice a day for 7 days (10 mg/kg, p.o.). The pain related behavioral tests were performed one hour after the second drug dosing each day. Higher order preference tests were performed on two additional days of treatment. At the conclusion of the study, animal pancreatic tissues were harvested to evaluate the severity of the pancreatitis. The therapeutic efficiencies of LY3038404 HCl were evaluated by analyzing 1) pancreatic tissue HSS and fibrosis for tissue protection; 2) evoked, place preference, and open field behavioral data for determination of nociceptive control and side effects.
Induction of chronic pancreatitis
Male Fischer 344 rats weighing between 240 – 250 g (Harlan Sprague–Dawley, Indiana) were used for this study. Rats were randomly divided into two groups: alcohol/high fat liquid diet (AHF) fed - (n = 12) and control chow fed- group (n = 6). Animals were single caged and kept in a temperature constant (23° ± 2°C) room on a 12/12 hour reversed dark–light cycle. Chronic pancreatitis was induced in animals fed an alcohol/high fat liquid diet (AHF) made from micro-stabilized rodent alcohol liquid diet mix (LD 101A; Test-Diet, Richmond, IN), composed of 3.9% fat from corn and safflower oil, 30.3% protein, 5% fiber, vitamins and minerals added as a dry powder to water and alcohol (w/v, 95% ethyl alcohol). The dose of alcohol was progressively increased from 4% to 6% as follows: 4% alcohol for the first week, 5% for second week, and 6% for the third to eighth week. Lard (8 g/per rat/per day) was added starting from the 4% alcohol week. Rats were allowed free access to water. Each rat consumed between 50–70 grams of liquid diet with alcohol per day for 12 weeks. Animals also received a daily lard supplement (8 g/day/per rat) bringing the total fat content of their diet to ~20% total daily dietary fat consumption. Control group was fed standard rodent chow (Teklab 8626, Harlan, Indiana) and they had access to food and water ad libitum. Animals were observed closely and no evidence of alcohol intoxication (no ataxia or lethargy) was noted. Body weight was monitored weekly and food consumption was monitored daily.
Drug administration
LY3038404 HCl and dosages were provided by Eli Lilly and Company, Indianapolis IN. LY3038404 HCl powder was freshly mixed with control rat chow powder and drinking water to form a small drug pellet (≈1 g/each) one day before use. The small pellets were dried in a 50°C-oven for 2 hours.
The LY3038404 HCl treatment started after the 6th week when pain related behavior testing detected a maximum hypersensitivity in the AHF fed animals. Rats were fed LY3038404 HCl (10 mg/kg) orally twice a day for 9 consecutive days. On the testing day one pellet was given at 8:30 - 9 am, and one at 12 - 1 pm. The evoked mechanical and heat tests were performed on Days 1–7, one hour after the second dosing of the day. Place preference and open field tests were performed on Days 8 and 9, respectively.
Histopathological assessment of pancreatitis
At the end of study, rats were anesthetized with pentobarbital (100 mg/kg, i.p.) and perfused transcardially with warm heparinized saline followed by 4% ice-cold paraformaldehyde in 0.1 M phosphate buffer solution (pH 7.4). The pancreas was post-fixed in the buffered paraformaldehyde for 48 hours, carefully dissected into 2 parts: duodenal (pancreatic head) and splenic lobes (pancreatic tail) were put into 70% alcohol. After dehydration through graded ethanol (95, 100%), the pancreas was embedded in paraffin. Pancreatic tissue sections were cut at 5 μm thickness, mounted onto gelatin coated glass slides (Super Frost Plus, VWR, Radnor, PA), and stored at room temperature for histological staining. Analysis was performed blindly on serial sections from each rat.
Hematoxylin and Eosin (H&E) staining
Slides were de-paraffinized with Citrosolv (Fisher, Pittsburgh, PA), hydrated with graded ethanol, rinsed in tap water, immersed in 0.1% hematoxylin (Fisher) for 1–3 min, washed in tap water for 1 min, dehydrated through in graded ethanol (50%, 70%), immersed in 0.1% eosin (Fisher) for 1 min, and then dehydrated in 90% ethanol. Finally, sections were dehydrated in 100% ethanol and coverslipped with Permount (Fisher, Pittsburgh, PA).
Sirius Red staining for collagen
De-waxed and hydrated paraffin sections were stained with haematoxylin for 8 minutes and then washed for 10 minutes in running tap water. Sections were stained in picro-sirius red for one hour. After two washes in acidified water, sections were dehydrated in three changes of 100% ethanol, cleared in Citrosolv (Fisher, Pittsburgh, PA), and coverslipped with toluene based mounting medium (Fisher, Pittsburgh, PA).
Immunostaining for Ki67 cell proliferation biomarker
Pancreatic tissue proliferation in response to injury was monitored by immunostaining of the nuclear proliferation marker Ki67. Pancreatic paraffin tissue sections were exposed to antigen-retrieval in boiling Tris-EDTA Buffer (10 mM Tris Base, 1 mM EDTA Solution, 0.05% Tween 20, pH 9.0), followed by antibody incubation and detection using the ABC-Vectastain system with the Vector DAB substrate kit (Vector Laboratories, INC, Burlingame, CA). Sections were counterstained with Eosin. Primary antibody was rabbit anti-Ki67 (Abcam, Cambridge, UK). Quantification was performed by counting Ki67 positive cells in an entire tissue section using a 20× objective. The image of the whole tissue section was captured with a digital camera (Canon, PowerShot, ELPH 300HS). The total area (mm2) of the entire tissue sections was measured with the Image J program (v1.46r; NIH), and the number of Ki67 positive cells per mm2 area was determined.
Pancreas Histological Severity Scoring (HSS)
Hematoxylin-eosin (H&E) staining was performed on 5 μm paraffin embedded pancreatic sections. Severity of pancreatic tissue damage was graded by a semi-quantitative scoring system (HSS). Within pancreatic sections, areas of abnormal pancreatic tissue architecture were graded: global glandular degeneration (acini and islets), vacuolization (fat deposition), acinar and islet cell atrophy including focal atrophy, area atrophy; and fibrosis: periductal, interlobular and intralobular fibrosis. These parameters were graded as follows: 0, absent; 1, minimal (<10%); 2, moderate (30 - 50%); and 3, major (>50 - 70%) percentage of the entire pancreatic section examined, as described by others previously [
25‐
27,
45,
46]. Sections were observed and graded by two examiners blinded to animal treatment. A total score was calculated for both pancreatic head and tail.
Quantitative analysis of pancreas fibrosis with image J
The fibrous collagen in 5 μm-thick pancreas sections was stained with Sirius red. With this type of histological staining, the fibrous collagen deposition was stained red. Bright-field, polarized, TIFF images from the pancreas head and tail were acquired from consecutive non-overlapping fields using a Nikon E1000 microscope equipped with a Nikon DXM1200F digital camera and ACT-1 Program (Nikon Instruments, Inc., Melville, NY). Five images of each section were taken with 20× magnification at full resolution with a single image dimension setting of 3600 × 2880 pixels. The percentage of fibrotic tissue area was compared to the total area of tissue within an image. Mean values were obtained from 5 images of pancreas sections for each animal for comparison of groups.
Quantitative analysis of pancreatic fibrosis was performed using the Image J program (v1.46r; NIH) with the color thresholding plug-in. The threshold is set specifically for the Sirius red stained component (fibrosis). After setting the scale, the percentage of fibrosis was calculated by determining the tissue areas occupied by collagen fibers versus the total pancreas tissue areas, excluding the lumen (i.e., empty spaces) [
47,
48] (Reinking L, 2007
Image J Basics
http://rsbweb.nih.gov/ij/docs/pdfs/ImageJ.pdf;
Examples of Image Analysis Using ImageJ
http://rsb.info.nih.gov/ij/docs/pdfs/examples.pdf). The fibrosis % (of total tissue) = (area of fibrosis /area of total tissue) x100, i.e. = (area of entire image- non threshold area) / (area of entire image- area of empty space) × 100.
Weekly behavioral testing was performed during the animal’s dark cycle active period (i.e. 0900 h – 1500 h). Investigators performing the behavioral studies were blinded to animal treatment.
Assessment of hindpaw secondary mechanical allodynia
Secondary mechanical allodynia on the hindpaws was assessed by measuring the mechanical withdrawal threshold using von Frey filaments with the “up - down” method described by Chaplan et al. [
49]. Animals were placed on a raised wire mesh table (76 ×38 cm), under a clear plastic ventilated rat restrainer (18 ×13 ×15 cm) for a 30 min acclimatization period. The mechanical withdrawal threshold testing was done on the plantar surface of both hindpaws using a set 8 of von Frey monofilaments. The von Frey filaments were applied perpendicularly to the plantar surface with sufficient force to bend the monofilament slightly and held for about 5 seconds. A positive response was defined as an abrupt withdrawal (flick response) of the foot during stimulation or immediately after the removal of stimulus. Whenever there was a negative or positive response, the next stronger or weaker filament was applied, respectively. The pattern of positive and negative responses was converted into a 50% threshold value (in grams) using a curve-fitting algorithm [
50]. The 50% decreased paw withdrawal threshold (PWT) indicated secondary mechanical allodynia.
Assessment of abdominal secondary heat hyperalgesia
Nocifensive responses to heat stimuli were tested by measuring the abdomen withdrawal latency with the modified Hargreaves test [
51]. The radiant heat source was shone onto the abdominal skin of the reclining rats. Rats’ abdominal skin was shaved one day before the test each week. Rats were placed in separate clear plastic ventilated restrainers (18 ×13 ×15 cm) on a glass-top table (approximately 2 mm thick glass) and allowed to acclimate to their new environment for 30 min before testing. A high-intensity light beam was applied to the shaved abdominal skin surface through the glass and the latency of reflexive withdrawal responses timed (seconds). The cutoff time for abdominal withdrawal latency was set at 40 s to avoid any damage to the skin. A withdrawal event to radiant heat was defined as a weight shifting (either abdominal musculature contraction or lifting of the abdomen through postural adjustment) accompanied by head turning toward the stimuli and licking of the abdominal area. The abdomen withdrawal latency was tested in 3 trials for averages. The 5-min intervals between trials and a cooling fan under the table assured the temperature of the glass surface returned to room temperature (22 - 24°C) prior to the next trial. Shortened abdomen withdrawal latency values indicated a secondary heat hyperalgesia.
Modified 44°C hotplate test
Because C-fiber nociceptors are active under sustained, low-intensity heat stimulation, the modified hotplate test assessed responses to moderately noxious stimuli [
52,
53]. In this study, two temperature controlled hotplates were topped with vented Plexiglas enclosures (26 cm × 26 cm × 28 cm) (Columbus Instruments, OH). One hotplate temperature was set at 38°C for pre-warming to normalize all animal paw temperatures, and the other one was set at 44°C. After a 30 min acclimation to the environment, rats were placed on the 38°C warm-up plate for 10 min. Then the rats were placed on the 44°C test plate for a 10 min test period. The latency to the first hindlimmb withdrawal response, the duration, and the frequency of hindlimb withdrawal events during heat stimulation were recorded. Event duration with hindpaws withdrawn from the plate began when the limb was lifted and finished when the limb made contact with the plate again. The events were plotted against time at 1 min intervals and a leftward shift of the event/time curve was an index of heat hyperalgesia. Erect leaning posture with forelegs against the wall of the enclosure (rearing event) was recorded. Rearing events were considered a noxious-evoked escape response during the hotplate test [
54,
55].
Exploratory activity testing
Open field exploratory activity of the animals was monitored using the automated Flexfield Animal Activity System (San Diego Instruments, San Diego, CA) with Photobeam Activity System software (PAS) coupled to a HP computer (Hewlett Packard, Palo Alto, CA). The activity enclosure included a transparent Plexiglas chamber (40 × 40 ×40 cm) equipped with infrared photobeam sensors, 16 beams on each axis ( X and Y, total 32), arranged 4 cm above the chamber floor. Obstruction of these photobeams constitutes movements in the x and y planes. Another set of 16 beams is located 12 cm above the chamber floor to record movements in the z-plane (rearing events and duration). Data were collected for 45 minutes in nine 5-minute intervals.
The experiment was carried out in an isolated, temperature controlled (21 - 22°C) room, with consistent background white noise. When the tests started, the observer exits the room leaving the subjects undisturbed. Animals were tested at the same time of the day (from 9 am to 3 pm). To avoid acclimation of animals to the environment, repeat testing of the same animal occurred at least 24 h after the last test [
56]. Six main parameters were measured in the nine 5 min intervals: rearing events and rearing duration; total activities, i.e., number of photobeams broken (included the number of different zone entries and stationary fine movement, e.g., grooming) and distance traveled; active time and resting time. Resting time is defined as a period when the animal remained in place for 1 second or longer. This test is based on the natural exploratory or investigatory behaviors of rodents in a novel “open field” environment. All of the six parameters are important for evaluation of spontaneous pain and for comparison of the effects of drug treatment. Changes in activity may not be reflected by a single parameter; therefore, each of the six parameters was evaluated in the comparisons [
57,
58]. The test chambers were cleaned with Vindicator disinfectant cleaner (Hillyard, Inc) between tests to eliminate urine and other olfactory cues from previous subjects.
Light/dark preference test
A light/dark box with two equally divided compartments (26x13x28 cm) was used to assess anxiety-like behaviors of the rats [
42,
59]. The two chambers were connected by a 10 × 10 cm doorway in the center of the partition to allow free access to the adjacent chamber. Rats were moved into the test room with the test apparatus 30 min prior to the test. All tests are conducted between 9 am and 11 am. Animals were placed initially in the dark chamber facing away from the door leading to the adjacent light chamber; and behaviors were recorded for 10 min (600 s). The following behavioral parameters were documented: the number of chamber crossings (light/dark chamber transition), defined as at least three of the animal’s paws stepping through the doorway from one chamber to the next; and total time (sec) spent in the light chamber.
Statistical analysis
The data are expressed as means ±S.E.M. Comparisons among groups were performed with a One-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons post-tests. The time course comparison among groups was performed with a Two-way ANOVA with Bonferroni post-tests using GraphPad Prism version 6.0 (GraphPad Software, San Diego California USA). Two-tailed t-tests were also used where appropriate. A p ≤ 0.05 was considered significant. Calculation of the correlation coefficient between tissue HSS and pain related behaviors was done with the Microsoft Excel program.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
All authors approved the final version of the manuscript. LPZ performed all experiments including feeding the animals; performing the behavioral testing, histology, and immunostaining; analyzed the data, prepared the figures, and wrote the manuscript. RHK helped feed the animals, performed the 44°C hotplate and light /dark box testing, assisted with the histopathology, produced several of the figures, and edited the manuscript. TAM and MAJ designed the drug dosing, supplied the drug, and edited the manuscript. KNW designed the experiments and edited the manuscript and the figures.