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Research report
Intracerebroventricular met-enkephalin administration modulates
adjuvant arthritis
a ,
*
b c a bAdlan M. Elhassan
, Nikos Papadogiannakis , Abdu Adem , Isam Suliman , Adel Gad ,
a
J. Urban Lindgren
a
Department of Orthopedic Surgery, Karolinska Institute, Huddinge Hospital, S-141 86 Huddinge, Sweden
b
Department of Pathology, Karolinska Institute, Huddinge Hospital, S-141 86 Huddinge, Sweden
c
Department of Pharmacology, Faculty of Medicine and Health Sciences, United Arab Emirates University, P.O. Box 17666, Al Am, United Arab Emirates
Accepted 5 July 2000
This work is dedicated to our late colleague, Associate Professor Adel Gad
Abstract
The purpose of this study was to investigate the anti-inflammatory properties of intracerebroventricular met-enkephalin (met-enk) administration in an animal model of arthritis. Adjuvant arthritis was induced in rats by intradermal inoculation of mycobacterium butyricum and the effects of intraventricular met-enk1thiorphan (enkephalinase inhibitor) were studied. Treatment was initiated either simultaneously with the bacterial inoculation (preventive group) or on post-inoculation day 17 after the appearance of inflammation (treatment group). The degree of inflammation was evaluated by measuring the diameter and the circumference of the ankle joint immediately before the sacrifice (day 31) and by histologic examination of ankle joint sections. The results of this study revealed that combined intraventricular injections of met-enk1thiorphan reduced the arthritic-like inflammation in the preventive group as well as in the treatment group. These findings suggest that centrally applied met-enk1thiorphan may suppress the development adjuvant arthritis as well as the symptoms of manifest arthritis. Thus central met-enk may be involved in both hypothalamic pituitary adrenal axis and immune forms of stress-induced modulation. 2000 Elsevier Science B.V. All rights reserved.
Theme: Neurotransmitters, modulators, transporters, and receptors
Topic: Other neurotransmitters
Keywords: Met-enk; Synovial cells; Ankle; Mycobacteria; Arthritis
1. Introduction peptide synthesis has been observed in activated T-cells
[37,39]. In vitro studies have revealed that met-enk modu-Adjuvant arthritis is an experimentally inducible form of lates a number of immune functions such as natural killer arthritis in the rat that shares many features with human cell activity, antibody production, lymphocyte proliferation rheumatoid arthritis [25]. Although the immune system has and macrophage activity [30]. In vivo studies revealed that long been thought to be involved in its pathogenesis met-enk administered intraperitoneally in the rat either [14,15], the nervous system may also play a role in the increased or decreased the plaque-forming cell response development of this disease [18]. Methionine-enkephalin and IgM production depending upon the dose used [16]. (met-enk) is an endogenous opioid that is present both in Moreover, peripherally administered met-enk has also been the central and peripheral nervous systems [33]. Receptors shown to affect IgG antibody production and hyper-for met-enk have been detected in immune cells and sensitivity skin reactions [21]. That met-enk may act through central mechanisms to modulate immune re-sponses is indicated by the following observations.
In-*Corresponding author. Tel.:146-8-5858-3869; fax:146-8-7114-292.
E-mail address: adlan.elhassan@kfcmail.hs.sll.se (A.M. Elhassan). tracerebroventricular (ICV) administration of met-enk
modulates the cutaneous immune response in rats [35], 2.3. Induction of arthritis decreases the clinical manifestations of experimental
aller-gic encephalomyelitis and markedly attenuates the mag- Arthritis was induced in 21 rats by intradermal injection nitude of inflammatory brain lesions [34]. The purpose of of a suspension (50 ml) of heat-killed Mycobacterium the present investigation was to examine the effects of butyricum in paraffin oil (10 mg / ml) into the base of the
centrally administered met-enk on adjuvant induced arth- tail [25]. The sham group (seven animals), serving as ritis in the rat. For these experiments, rats were given controls, received 50 ml paraffin oil via the same route. met-enk ICV in a concentration of 20mg which has been
shown to reduce the inflammatory response to peripherally 2.4. Drugs administration administered carrageenin [5]. In addition, the
enkephalin-ase inhibitor, thiorphan was given in some animals to The animals were randomly assigned to five treatment potentiate the effects of exogenously administered met- groups of seven rats each (except for naive), as follows: (1)
enk. The rats of the naive group (five rats) were unoperated and
received no intradermal injections; (2) The rats of the sham group were fitted with an ICV cannula but received
2. Materials and methods no intradermal injection; (3) The rats of the vehicle group
were cannulated and received a suspension of heat-killed The study included thirty-three 10-week-old female M. butyricum in paraffin oil (50ml) and an ICV injection Lewis rats with an initial body weight between 160 and of 40ml of ACSF 30 min before inoculation daily for 14 180 g. The animals were housed singly at 218C on a 12 h days; (4) The rats of the preventive study group received light / dark cycle and fed ad libitum with a standard pellet daily injections of met-enk (20 mg)1thiorphan (200 mg) diet and water. The experiments were approved by the into the right lateral ventricle 30 min before inoculation of regional ethical committee, Stockholm, Sweden. mycobacteria over the 14 day administration period; (5) The rats of the treatment group which received
myco-2.1. ICV cannulation bacteria inoculations for 14 days, received injections of
met-enk (20mg)1thiorphan (200 mg), in the right lateral The surgical procedure of cannulation was performed as ventricle on day 17 through day 30.
follows. The rats were anaesthetized by intraperitoneal Prior to sacrifice, the extent of the inflammatory reaction injection of sodium pentobarbitone (60 mg / kg body was evaluated by tape measuring the circumference of the weight i.p., Mebumal, Nord vacc, Stockholm, Sweden). ankle joint in all animals. The diameter of the right and left The head was fixed in a stereotaxic frame and a mid-line ankles were measured in the frontal plane at the level of skin incision was made from bregma to lambda and the the maleoli using vernier calipers. All the animals were connective tissue over the periosteum was removed. A killed on day 31. Animals were anaesthetized by intra-single guide cannula (28-gauge stainless-steel, 9.5 mm peritoneal injection of sodium pentobarbitone (60 mg / kg long) was implanted just above the right lateral ventricle body weight) and perfused intra-arterially with phosphate (coordinates: 1.5 cm caudally, 1.5 laterally and 3.5 ventral- buffer saline (PBS), followed by Zamboni’s buffered ly from the bregma). Three stainless steel screws fixed to paraformaldehyde solution containing 0.2% picric acid the skull and cannulae were fixed in place by dental [19]. The ankles were excised and immersed in Zamboni’s cement. The drugs were injected slowly through a 28- fixative for 2 days at 148C. The specimens were
de-
gauge cannula (Plastic Product Inc., Roanoke, NJ, USA). mineralized in a 4% ethylene-diamino-tetraacetic acid Animals were allowed at least 7 days to recover prior to (EDTA) solution at pH 7.3 for 3 weeks on average [6]. the initiation of experiments. During the recovery period, Subsequently, the bones were rinsed for at least 2 days in
¨
the rats were handled for about 10 mm / day for at least 5 10% sucrose in 0.1 M Sodrensen phosphate buffer, con-days to familiarize them with infusion procedures. taining sodium azide and bacitracin (Sigma Chemicals, St. Louis). The ankles were blocked in paraffin and cut with a
2.2. Substances microtome. Multiple 5-mm sections were obtained and
(mono-nuclear cells and polymorpho(mono-nuclear leukocytes), the presence of follicles, detection of granuloma and others factors such as the presence of abscess and necrosis.
2.4.1. Statistical analysis
Comparisons of different treatment conditions on ankle circumference and diameter were performed by one-way ANOVA followed by Fisher’s protected LSD analysis. For all comparisons, P,0.05 was considered as significant.
3. Results
3.1. Clinical evaluation
Fig. 2. Measurement of ankle joint diameter was made between the
Clinical signs of arthritis were evident on average by
naive, control and the other groups. Each value (cm) corresponds to the
day 10 post inoculation of mycobacteria. The signs
per-mean6S.E.M., (n57, except for the naive55). Differences are expressed
sisted until the end of the experiment. X-ray films of the by asterisks according to level of significance: *** P,0.001 when [
ankle joints from vehicle-treated rats on day 30 confirm the compared to both the control and the naive groups. P,0.05 when compared to vehicle group.
occurrence of arthritis. No sign of inflammation was noted in the naive and sham groups while the other groups showed variable degrees of inflammation.
The mean diameter of the ankle joints of the vehicle difference between the vehicle and the treatment groups group was significantly greater than that found in the (Fig. 2). There was no significant difference between the control and naive groups (Fig. 1). However, there was no circumference and diameter of the right versus left ankle significant difference between the mean diameter of the joints in the individual rat.
ankle joints of the preventive and the treatment conditions
compared to the naive or the sham groups. A significant 3.2. Histologic findings difference was observed between the vehicle group and the
preventive and the treatment groups (Fig. 1). Histological examination performed on ankle joints The mean ankle joint circumference of the vehicle, collected from the animals on post inoculation day 31 preventive and treatment groups were significantly greater showed that the injection of mycobacteria was capable of compared to the naive and sham groups (Fig. 2). More- inducing arthritis. Arthritic-like symptoms included syno-over, there was a significant difference between the vial hypertrophy, pannus formation, neovascularization and preventive and the vehicle groups, but no significant synovial infiltrates comprised predominantly of mononu-clear inflammatory cells, some polymorphonuclear leukocytes, and a the presence of a few eosinophils.
Histological examination of synovial lining, subsynovial cells, leucocytic infiltration, follicular, granuloma, necrosis and abscess formation revealed that groups were affected differentially. With regard to synovial lining hypertrophy, the vehicle group was most affected while the treatment group and the preventive group were affected the least (Table 1). Subsynovial cell infiltrates (Table 2) and
Table 1
Ankle joint histologic sections from different treatment groups assigned
a
synovial lining score
Group Normal Mild Moderate Severe
Synovial lining
Naive 5 (100%) 0 0 0
Sham 7 (100%) 0 0 0
Fig. 1. Comparison of ankle joint circumference was made between the
Vehicle 0 3 (43%) 3 (43%) 1 (14%)
naive, control, enk preventive and enk treatment groups. Each value (cm)
Enk preventive 5 (71%) 2 (2 9%) 0 0 corresponds to the mean6S.E.M., (n57, except for the naive55).
Enk treatment 3 (43%) 4 (57%) 0 0 Differences are expressed by asterisks according to level of significance:
[ a
Table 2 4. Discussion Ankle joint histologic sections from different treatment groups assigned
a
subsynovial cells score
The results of this study illustrate two important points
Group Normal Mild Moderate Severe concerning the effects of intraventricular met-enk in the
Subsynovial cells development and treatment of adjuvant arthritis. Firstly,
Naive 5 (100%) 0 0 0 exogenous met-enk modulates the development of adjuvant
Sham 7 (100%) 0 0 0 arthritis. All rats developed less severe clinical and
histo-Vehicle 0 1 (14%) 3 (43%) 3 (43%)
pathological signs of adjuvant arthritis when the peptides
Enk preventive 5 (71%) 2 (29%) 0 0
were given as a preventive drugs. Secondly, ICV injection
Enk treatment 3 (43%) 3 (43%) 0 1 (14%)
of met-enk attenuates the development of adjuvant arthritis
a
Each value corresponds to the number of animals and the percentage in
and reduces the appearance of both clinical and
histo-(brackets) showing the specific score for each group.
pathological signs of the disease.
Met-enk in the brain is rapidly degraded by multiple
Table 3
Ankle joint histologic sections from different treatment group assigned enkephalin-degrading enzymes [17]. Thiorphan is known
a
leucocytes score as an enkephalinase inhibitor and extends the half-life of
enkephalins [27]. At the concentration used in this study,
Group Normal Mild Moderate Severe
no other effect of thiorphan has been described on the
Leucocytes
literature. Roques et al. observed that met-enk
adminis-Naive 5 (100%) 0 0 0
Sham 7 (100%) 0 0 0 tered intracerebro-ventricularly which alone had no
signifi-Vehicle 2 (29%) 2 (29%) 3 (42%) 0 cant effect on the tail-withdrawal test, resulted in strong Enk preventive 5 (71%) 2 (2 9%) 0 0 and long lasting analgesia when administered along with Enk treatment 5 (72%) 1 (14%) 0 1 (14%)
thiorphan. Thiorphan administered alone elicited a clear
a
Each value corresponds to the number of animals and the percentage in antinociceptive activity [7]. (brackets) showing the specific score for each group.
Using a range of concentrations of thiorphan, Yaksh and Harty showed that thiorphan (200 mg) potentiated the leucocytic infiltration (Table 3) were most apparent in the effects of met-enk [38]. In agreement with this finding, vehicle group followed by the treatment group and the Malin et al. used 250 mg thiorphan ICV to augment
preventive groups. transcranial electrostimulation analgesia [20]. In a related
Tissues from animals in the preventive or treatment model, Bhattacharya and co-workers ICV administration of conditions did not contain follicles, granulomas or areas of met-enk triggers a variety of neurohormonal effects, some necrosis. However, follicles were observed in tissues of them related to the pathogenesis of inflammation [5]. derived from five rats of the vehicle group. Three vehicle The median eminence is rich in opioid receptors [1]. Many animals also displayed granulomas and one showed necro- studies showed that met-enk has major effects on the sis (results not shown). The total mean scores from the release of many pituitary hormones including adrenocorti-total evaluation of the ankle joints histopathology parame- cotropic hormone (ACTH) [9,11], growth hormone [8], ters for each treatment group is summarized in Fig. 3. prolactin [8], thyrotropin [23] and luteinizing hormone [8]. In the hypothalamus enkephalin immunoreactivity has been localized in the parvocellular neurons of the hypo-thalamic paraventricular nucleus which projects on to the median eminence [13], the site where hypothalamic releas-ing factors are secreted into the portal blood, hence the anterior pituitary gland.
The female Lewis rat is susceptible to adjuvant arthritis after inoculation of mycobacteria. In contrast, the his-tocompatible female Fisher rat develops only a mild transient acute arthritis. Steinberg and colleagues showed that compared to Fisher rats, the female Lewis rat displays a reduction in glucocorticoid release in response to in-jections of streptococcal cell wall peptidoglycan and that these deficits are due to reductions in hypothalamic CRH synthesis and release [31,32]. It is interesting to note that deficits in CRH production occur together with the reduc-tions in the expression of the hypothalamic enkephalin gene [32]. It is possible that the administration of met-enk
Fig. 3. Total score assigned to the histologic sections of the ankle joints
in Lewis rats may restore the hypothalamic function in
removed at the end of the experiment. This is the mean total score for
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