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Analysis of Dose-Response and Analgesic Specificity of Locally Administered Morphine in the Carrageenan-Induced Inflammatory Model

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08 November 2024

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08 November 2024

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Abstract

Background/Objectives: This study investigates the role of peripheral opioid receptors in modulating pain outside the central nervous system. Methods: The first experiment aimed to establish a dose-effect relationship for intraplantar morphine administration in a rat model of carrageenan-induced inflammation by testing successive doses after the carrageenan injection. Additionally, the second experiment assessed whether a 5 mg/kg dose of morphine has central nervous system effects by comparing its effects with 5 mg/kg and 10 mg/kg doses given intraperitoneally. Results: In the first part we observed that lower doses (2.5 mg/kg) did not significantly affect paw withdrawal latency after thermal stimulation, while higher doses (5 mg/kg and 10 mg/kg) resulted in significant increases, suggesting that maximum receptor activation occurs at 5 mg/kg. However, the 20 mg/kg dose exhibited central nervous system effects, indicating systemic absorption. The second part compared intraplantarly and intraperitoneally administered morphine, finding that while the intraplantar 5 mg/kg dose provided a significant analgesic effect, the same dose given intraperitoneally did not produce similar results, reinforcing the hypothesis that local administration targets peripheral receptors. Conclusions: Overall, the findings indicate that morphine's analgesic effect at lower doses is primarily mediated by peripheral opioid receptors, with systemic effects becoming evident at higher doses. This study aligns with existing literature emphasizing the potential for peripheral opioid receptor activation to alleviate inflammatory pain with minimized central effects.

Keywords: 
morphine
;  
carrageenan
;  
analgesic effect
;  
intraplantar administration
;  
intraperitoneal administration 
;  
Subject: 
Medicine and Pharmacology  -   Anesthesiology and Pain Medicine

1. Introduction

Peripheral opioid receptors located in tissues such as the skin, muscles, and joints play a crucial role in modulating pain outside the central nervous system. These receptors include the μ-opioid receptor (MOR), δ-opioid receptor (DOR), and κ-opioid receptor (KOR) subtypes [1]. Their activation facilitates analgesia by locally inhibiting the transmission of pain signals, thereby minimizing the adverse effects on cognitive and respiratory functions typically associated with central opioids [2,3].
Recent research has increasingly focused on peripheral opioid receptors, particularly their potential to manage pain while minimizing side effects on the central nervous system (CNS). Primarily situated on sensory neurons and immune cells, peripheral opioid receptors are integral to localized pain management [4]. Their activity is heightened in inflamed tissues, where immune cells, including leukocytes that release endogenous opioid peptides such as β-endorphin. These peptides interact with opioid receptors on sensory neurons, resulting in a localized analgesic effect that avoids the CNS-related complications commonly associated with conventional opioid therapies [5,6].
Tissue injuries and inflammatory processes significantly enhance the synthesis and transport of opioid receptors to neurons in the dorsal root ganglia (DRG). During inflammatory conditions, these receptors become more responsive to peripheral opioids, thereby facilitating a localized analgesic effect [7,8].
Research indicates that opioid receptors are transported to peripheral nerve endings following inflammation, resulting in increased receptor density in affected areas and enhancing their accessibility to both endogenous and exogenous opioids. Inflammatory factors, including cytokines and nerve growth factor, promote the accumulation of receptors in these regions. When an opioid agonist binds to the peripheral receptor, it inhibits the excitability of sensory neurons and reduces the release of pronociceptive substances such as substance P, leading to a localized antinociceptive effect without affecting central functions [9].
Long-term administration of morphine leads to analgesic tolerance, requiring higher doses to maintain the analgesic effect, which can cause severe side effects such as respiratory depression and withdrawal symptoms [10,11]
Research published in literature examined morphine’s antinociceptive effects in carrageenan-induced inflammation in rats, comparing intraplantar and intravenous administration. Results indicate that intraplantar morphine raises vocalization thresholds under pressure in inflamed, but not in non-inflamed paws. With repeated carrageenan injections, intraplantar effects decrease while intravenous effects intensify, suggesting that morphine’s central action strengthens with recurrent inflammation. The findings conclude that enhanced systemic morphine effects are likely tied to central mechanisms, highlighting additional activation of the endogenous opioid system during inflammation [12].
Research from 2021 shows that opioids relieve pain by activating μ-opioid receptors (MOR) in the CNS, which inhibits pain-related neurotransmitters like substance P and glutamate. This activation also suppresses adenylyl cyclase, reducing cyclic AMP and lowering neuronal excitability [13] .Additionally, opioids seem to inhibit cyclooxygenase in the central nervous system similarly to paracetamol, which also appears capable of binding to certain opioid receptors [14,15,16].
Opioids activate both neuronal and non-neuronal pathways, particularly through Toll-like receptors (TLRs), affecting the central immune response. This interaction can impact opioid efficacy and contribute to hyperalgesia indicating that immune signaling is essential in understanding chronic pain and opioid tolerance [17].
Research offers insights into how opioids modulate excitatory and inhibitory neurotransmitter systems, impacting pain pathways [18].
In a previous research, it was found that morphine administered intraplantarly to rats with inflammation induced by carrageenan has an analgesic effect, with maximum intensity occurring at 3 hours after carrageenan administration, but this effect remains statistically significant for up to 48 hours after the onset of inflammation [19]. Therefore, we aimed to determine a dose-effect relationship through intraplantar administration of morphine in an experimental model of carrageenan-induced inflammation by administering successive doses of morphine 3 hours after the administration of carrageenan. Subsequently, we planned another experiment to verify whether a dose of 5 mg/kg body weight of morphine has central nervous system effects. To achieve this, we administered 5 mg/kg body weight of morphine intraplantarly and compared its effect with that of morphine at doses of 5 mg/kg body weight and 10 mg/kg body weight administered intraperitoneally to rats with inflammation induced by carrageenan.

2. Materials and Methods

The protocol for this study was approved by the Ethics Committee for Non-Clinical Studies conducted on laboratory animals at the 'Carol Davila' University of Medicine and Pharmacy Bucharest and received project authorization from the Bucharest Sanitary Veterinary and Food Safety Authority, no. 33/12.09.2022, in accordance with Law 43/2014 regarding the protection of animals used for scientific purposes, with subsequent amendments, and Directive EEC 86/609 of November 24, 1986, concerning acts with the power of law and administrative acts of member states for the protection of animals used in experimental and other scientific purposes.

2.1. Animals

In our research, we conducted two experiments using 100 mature male albino Wistar rats, each with an approximate average weight of 250 grams. The animals were obtained from the bio-base of Carol Davila University of Medicine and Pharmacy in Bucharest. Upon arrival, the rats were 6 weeks old, and they were allowed a one-week period for acclimatization and adaptation to their new location before the experiments began. The experiments took place over a two-day period, and upon completion, the animals were euthanized under general anesthesia. The number of animals per group was calculated based on the expected variance so that the test power would be at leats 80%. We specify that the experiments were conducted in accordance with the ethical guidelines for research on laboratory animals and with the approval of the Ethics Committee within the institution.

2.2. Inflamation

Inflammation was induced by 0.15 ml of 1% solution of lambda carrageenan in saline injected into the plantar surface of the right hind paw. Carrageenan was prepared 24 h before each experiment.

2.3. Experimental Procedures

In the first experiment, we used 50 Wistar rats, divided into 5 groups of 10 rats each.The environmental conditions of the workspace where the rats were kept remained unchanged (light, temperature, humidity). Under these conditions, we aimed to determine a dose-effect relationship through intraplantar administration of morphine in a carrageenan-induced inflammation experimental model. Successive doses of morphine were administered 3 hours after carrageenan administration.
  • Group 1 (control) received a 0.9% saline solution intraplanarly in the right hind paw 3 hours after carrageenan administration.
  • Group 2 received morphine at 2.5 mg/kg body weight intraplanarly in the right hind paw 3 hours after carrageenan administration.
  • Group 3 received morphine at 5 mg/kg body weight intraplanarly in the right hind paw 3 hours after carrageenan administration.
  • Group 4 received morphine at 10 mg/kg body weight intraplanarly in the right hind paw 3 hours after carrageenan administration.
  • Group 5 received morphine at 20 mg/kg body weight intraplanarly in the right hind paw 3 hours after carrageenan administration.
All substances were administered intraplantarly 3 hours after carrageenan administration. For each rat, pain sensitivity of the paw was measured using the Ugo Basile - Plantar Test for Thermal Stimulation - Hargreaves Apparatus, 10 minutes after morphine or 0.9% saline solution administration. The Hargreaves Method assesses thermal hyperalgesia in rodents by applying a precise thermal stimulus to the hind paw, which triggers a withdrawal response at increased temperatures. The apparatus employs a focused infrared light source that gradually heats the paw through a glass pane, with the withdrawal time automatically recorded to indicate the thermal pain threshold. The procedure involves placing the animal in a transparent enclosure, applying the thermal stimulus to the hind paw, and measuring the latency period to avoid discomfort [20]. If the latency reached a maximum value of 30 seconds, the measurement was stopped. The withdrawal response time is then analyzed to evaluate thermal sensitivity thresholds and the effects of analgesic agents. For each subject, the asessment began 10 minutes after morphine or saline injection and 5 asessments were conducted at the 5 minute intervals.
In the second experiment, we also used 50 Wistar rats, divided into 5 groups of 10 rats each.
  • Group 1 (intraplantar control) received a 0.9% saline solution intraplanarly in the right hind paw 3 hours after carrageenan administration.
  • Group 2 (intraperitoneal control) received a 0.9% saline solution intraperitoneally 3 hours after carrageenan administration.
  • Group 3 received morphine at 5 mg/kg body weight intraplanarly in the right hind paw 3 hours after carrageenan administration.
  • Group 4 received morphine at 5 mg/kg body weight intraperitoneally 3 hours after carrageenan administration.
  • Group 5 received morphine at 10 mg/kg body weight intraperitoneally 3 hours after carrageenan administration.
All substances were administered intraplantarly 3 hours after carrageenan administration. Also, for each rat, pain sensitivity of the paw was measured using the Ugo Basile - Plantar Test for Thermal Stimulation - Hargreaves Apparatus, 10 minutes after morphine or 0.9% saline solution administration. Five measurements were made at intervals of 5 minutes each.

2.4. Statistical Analysis

For statistical processing of the study data, IBM SPSS Statistics for Windows, Version 29.0 (30-day trial version) Armonk, NY: IBM Corp was used. Continuous variables were analyzed for normality and then expressed as mean ± standard deviation, median, minimum, and maximum. The Mann-Whitney U test was used to compare the mean values of the Right Paw Withdrawal Time variable between groups. The Kruskal-Wallis H test was used to compare the mean values of parameters between groups, considering that the variables have a non-normal distribution. A p-value <0,05 was considered statistically significant.

3. Results

In the first experiment conducted to highlight the analgesic effect, we compared the average withdrawal latency of the paw in the control group with the average withdrawal latency of the paw in the groups that received morphine at different doses. The average withdrawal latency in the control group was 2.48 ± 0.76 seconds, in the group that received morphine at 2.5 mg/kg body weight, the average latency was 3.39 ± 0.96 seconds, in the group that received morphine at 5 mg/kg body weight, the average latency was 4.18 ± 1.08 seconds, in the group that received morphine at 10 mg/kg body weight, the average latency was 4.28 ± 2.15 seconds, and in the group that received morphine at 20 mg/kg body weight, the average latency was 27.10 ± 4.48 seconds. These results are also presented in the Table 1 and Figure 1 below.
We compared the control group with each group that received morphine at different doses, and the results were statistically analyzed using the Mann-Whitney U test. Specifically, in Group 2, where the average withdrawal time was 3.39 ± 0.96 seconds, no statistically significant differences were observed compared to the control (p > 0.05). In Group 3, where the average latency was 4.18 ± 1.08 seconds, significant statistical differences were highlighted compared to the control group (p = 0.003). In Group 4, where the average latency was 4.28 ± 2.15 seconds, significant statistical differences (p = 0.009) were also evident compared to the control regarding the withdrawal time of the paw. Finally, in Group 5, where the average withdrawal latency was by far the highest at 27.10 ± 4.48 seconds, significant statistical differences were noted compared to the control group.
In the second experiment, we compared the average withdrawal time of the paw in the intraplantar control group with the group that received morphine at 5 mg/kg body weight intraplantar. Thus, in the intraplantar control group, the average withdrawal latency of the paw was 6.04 ± 1.67 seconds, while in the group that received morphine at 5 mg/kg body weight intraplantar, the average withdrawal latency was 8.76 ± 1.93 seconds. To compare the average withdrawal times between the two groups, I applied the Mann Whitney U test. Since p = 0.03, we find that there are statistically significant differences between the two groups. These data are also presented in Table 2 and Figure 2.
Furthermore, we compared the average withdrawal values of the paw between the intraperitoneal control group and the group that received morphine at 5 mg/kg body weight intraperitoneal, as well as the group that received morphine at 10 mg/kg body weight intraperitoneal. Thus, the average latency for the intraperitoneal control group was 5.86 ± 2.1 seconds, while in the group that received morphine at 5 mg/kg body weight intraperitoneal, the average latency was 5.91 ± 1.44 seconds (Figure 3). By applying the Mann Whitney U test between the two groups, we observed that p = 0.912, indicating that there are no statistically significant differences. In the case of the group that received morphine at 10 mg/kg body weight, the average withdrawal latency was 26.4 ± 3.40 seconds, showing a statistical significance compared to the intraperitoneal control group (p < 0.05). These data can be seen in Table 3.

4. Discussion

Opioid receptors are predominantly distributed throughout the central nervous system, with a more limited presence in the peripheral nervous system [21]. Findings from various studies indicate that both central and peripheral effects may play a role in enhancing opioid efficacy during inflammatory conditions [22] .
As can be seen from the data above, in the first experiment, morphine did not significantly increase the paw withdrawal latency at a dose of 2.5 mg/kg body weight. At doses of 5 mg/kg and 10 mg/kg body weight, morphine significantly increased the withdrawal latency, with the averages for these two groups being very close, at 4.18±1.08 seconds and 4.28±2.15 seconds, respectively. In Group 5, the paw withdrawal latency increased substantially, reaching an average value of 27.1±4.48 seconds; however, unlike the other groups, these rats showed clear signs of central nervous activity from morphine, such as immobility and Straub tail (it refers to a specific response observed in rats due to central nervous system effects of opioids [23] ), In this group, the withdrawal latency exceeded the maximum acceptable time of 30 seconds.
The 5 mg/kg and 10 mg/kg doses of morphine produced similarly intense increases in paw withdrawal latency, suggesting that the maximum number of opioid receptors in the carrageenan-inflamed rat paw likely corresponds to a 5 mg/kg dose of morphine. Additionally, results from the 20 mg/kg dose, which demonstrated clear central nervous effects of morphine, indicate that morphine can be absorbed following intraplantar injection, though this occurs only at very high doses. The fact that the doses of 5 mg/kg and 10 mg/kg of morphine administered intraplantarly increased the paw withdrawal latency to around 4 seconds, while the 20 mg/kg dose of morphine administered intraplantarly, which also had central nervous effects, increased the paw withdrawal latency to around 27 seconds, leads us to assume that the analgesic effect produced by stimulating peripheral opioid receptors is less intense than that produced by stimulating central nervous opioid receptors. These data led us to believe that morphine administered intraplantarly at a lower dose of 10 mg/kg acts only locally, as it is likely absorbed very little from the injection site. To further investigate this hypothesis, we conducted a second experiment in which we compared the analgesic effect of morphine administered intraplantarly with that of morphine administered intraperitoneally. In this second experiment, it was observed that the 5 mg/kg dose administered intraplantarly had a statistically significant analgesic effect compared to the control, while the same dose administered intraperitoneally did not produce a statistically significant analgesic effect. Since intraperitoneal administration always implies very good and rapid absorption, it follows that the 5 mg/kg dose administered intraperitoneally is not capable of producing central nervous effects, as it did not produce a statistically significant analgesic effect.
If the analgesic effect of morphine administered intraplantarly had occurred through the stimulation of central nervous opioid receptors, the same dose administered intraperitoneally, a route of administration that ensures much better absorption, should have had a much more intense analgesic effect, which was not the case. This leads us to assume that the analgesic effect of intraplantar morphine administration occurred exclusively through the stimulation of peripheral opioid receptors. This specificity of action likely depends heavily on the amount of morphine administered locally. The 10 mg/kg dose, which has been shown to have an analgesic effect of the same intensity as the 5 mg/kg dose, when administered intraperitoneally, had a much greater analgesic effect accompanied by clear signs of central nervous action. Practically, the 10 mg/kg body weight morphine administered intraperitoneally had effects similar to those of the 20 mg/kg body weight morphine administered intraplantarly, as demonstrated in the previous experiment, certainly because intraperitoneal administration implies much better absorption than intraplantar administration. Our research is in accordance with other studies in the literature that indicate that peripheral opioid receptors can be activated without unwanted central effects at lower doses, as seen with intraplantar administration, where absorption is more localized and the analgesic effect relies on both dose and injection site [24,25].
To support the analysis of differences between intraplantar and intraperitoneal morphine effects, studies highlight that peripheral opioid receptor stimulation can effectively alleviate inflammatory pain with minimal systemic impact. Specifically, smaller intraplantar doses generally avoid central effects due to localized absorption at the inflammation site. In contrast, higher doses (above 10 mg/kg) can exceed the threshold for systemic absorption, leading to central activation and observable effects such as immobility and Straub tail reflex [26].
Research has shown that morphine, when administered via systemic and central routes, exhibits increased antinociceptive effects in inflammatory conditions [27,28]. It is widely accepted that opioids primarily exert their pain-relieving effects through actions within the central nervous system [29,30]. However, there remains uncertainty regarding the role of both peripheral and central opioid receptors in modulating pain during inflammation [31,32].
Despite the significant findings, this study has several limitations that should be considered: the use of carrageenan as an inducer of inflammation may not represent all types of inflammatory responses, the measurement period may not capture long-term effects, methods of assessing pain may introduce variability affecting result reliability, statistically significant findings may not equate to clinically meaningful outcomes.

5. Conclusions

Morphine administered intraplantarly in a rat model of carrageenan-induced inflammation has a dose-dependent analgesic effect. The maximum number of peripheral opioid receptors in the inflamed paw is likely equivalent to a dose of 5 mg/kg body weight of morphine. Doses less than or equal to 10 mg/kg body weight of morphine administered intraplantarly do not have detectable central nervous effects, while a dose of 20 mg/kg body weight of morphine administered intraplantarly produces a significantly greater analgesic effect, accompanied by evident central nervous manifestations. Given that the dose of 5 mg/kg body weight of morphine administered intraperitoneally does not produce a statistically significant analgesic effect compared to the control, and the analgesic effect of morphine administered intraplantarly at 5 mg/kg body weight is statistically significant compared to the control, this effect is likely due to the stimulation of peripheral opioid receptors.

Author Contributions

N.M.J. Developed the experimental design, conducted the protocol, collected the specimens and data, and analyzed them. S.S. Supervised the experiment, data collection, and analyzed the final manuscript. C.O. Supervised the final manuscript. M.P. Supervised the statistical analysis. A.Z. Developed the experimental design, supervised the experiment. O.C.D. Supervised the final manuscript. E.P. Supervised the statistical analysis. A.M.J contributed to the manuscript preparation. I.G.F. Supervised the experiment and analyzed the final manuscript. The published version of the manuscript has been read and approved by all authors.

Institutional Review Board Statement

The animal study was approved by the Ethics Committee for Non-Clinical Studies conducted on laboratory animals at the 'Carol Davila' University of Medicine and Pharmacy Bucharest and received project authorization from the Bucharest Sanitary Veterinary and Food Safety Authority, no. 33/12.09.2022, in accordance with Law 43/2014 regarding the protection of animals used for scientific purposes, with subsequent amendments, and Directive EEC 86/609 of November 24, 1986, concerning acts with the power of law and administrative acts of member states for the protection of animals used in experimental and other scientific purposes.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Withdrawal latency in seconds of the inflamed paw with carrageenan after the application of a painful stimulus in groups that received morphine at different doses. The average values and 95% confidence intervals are presented.
Figure 1. Withdrawal latency in seconds of the inflamed paw with carrageenan after the application of a painful stimulus in groups that received morphine at different doses. The average values and 95% confidence intervals are presented.
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Figure 2. Withdrawal latency in seconds of the inflamed paw with carrageenan after applying a painful stimulus in the groups that received morphine 5 mg/kg body weight intraplantarly. The average values and 95% confidence intervals are presented.
Figure 2. Withdrawal latency in seconds of the inflamed paw with carrageenan after applying a painful stimulus in the groups that received morphine 5 mg/kg body weight intraplantarly. The average values and 95% confidence intervals are presented.
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Figure 3. Withdrawal latency in seconds of the inflamed paw with carrageenan after applying a painful stimulus in the groups that received morphine 5 mg/kg body weight intraperitoneally. The average values and 95% confidence intervals are presented.
Figure 3. Withdrawal latency in seconds of the inflamed paw with carrageenan after applying a painful stimulus in the groups that received morphine 5 mg/kg body weight intraperitoneally. The average values and 95% confidence intervals are presented.
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Table 1. Withdrawal latency in seconds of the inflamed paw with carrageenan after applying a painful stimulus in the groups that received morphine at different doses. * Statistical significance compared to the control group (p<0.05).
Table 1. Withdrawal latency in seconds of the inflamed paw with carrageenan after applying a painful stimulus in the groups that received morphine at different doses. * Statistical significance compared to the control group (p<0.05).
Average Mean Standard
deviation		 P value
Control group 2.48 2.23 0.763
Morphine group 2.5 mg/kg body weight 3.39 3.30 0.960 .058
Morphine group 5 mg/kg body weight 4.18 4.38 1.087 .003 *
Morphine group 10mg/kg body weight 4.25 4.23 2.152 .009 *
Morphine group 20mg/kg body weight 27.10 30.00 4.483 .000 *
Table 2. Withdrawal latency in seconds of the inflamed paw with carrageenan after applying a painful stimulus in the groups that received morphine intraplantarly. * Statistical significance compared to the control group (p<0.05).
Table 2. Withdrawal latency in seconds of the inflamed paw with carrageenan after applying a painful stimulus in the groups that received morphine intraplantarly. * Statistical significance compared to the control group (p<0.05).
Average Mean Standard
deviation		 P value
Control group (intraplantar) 6,04 5,7 1,67
Morphine group 5 mg/kg body weight (intraplantar) 8,76 8,60 1,93 0,003*
Table 3. Withdrawal latency in seconds of the inflamed paw with carrageenan after applying a painful stimulus in the groups that received morphine intraperitoneally. * Statistical significance compared to the control group (p<0.05).
Table 3. Withdrawal latency in seconds of the inflamed paw with carrageenan after applying a painful stimulus in the groups that received morphine intraperitoneally. * Statistical significance compared to the control group (p<0.05).
Average Mean Standard
deviation		 P value
Control group (intraperitoneal) 5,86 5,70 2,10
Morphine group 5 mg/kg body weight (intraperitoneal) 5,91 5,90 1,44 0,912
Morphine group 10 mg/kg body weight (intraperitoneal) 26,40 27,50 3,40 0,000*
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