Korean J Pain 2022; 35(3): 271-279
Published online July 1, 2022 https://doi.org/10.3344/kjp.2022.35.3.271
Copyright © The Korean Pain Society.
1Neuroscience and Inflammation Unit, Department of Physiology, Faculty of Basic Medical Sciences, University of Ilorin, Ilorin, Kwara State, Nigeria
2Neuroscience and Inflammation Unit, Department of Physiology, Adeleke University, Ede, Osun State, Nigeria
3Department of Nursing, Faculty of Social Welfare and Health Sciences, University of Haifa, Haifa, Israel
Correspondence to:Bamidele Victor Owoyele
Neuroscience and Inflammation Unit, Department of Physiology, Faculty of Basic Medical Sciences, University of Ilorin, Ilorin, Kwara State 240003, Nigeria
Handling Editor: Jong Yeon Park
Previous presentation at conference: This article was presented at the Sociey for the Study of Pain Annual National Conference on July 22–23, 2021, in Nigeria.
Author contributions: Bamidele Victor Owoyele: Study conception; Ahmed Olalekan Bakare: Supervision; Olutayo Folajimi Olaseinde: Resources; Mohammed Jelil Ochu: Investigation; Akorede Munirdeen Yusuff: Investigation; Favour Ekebafe: Investigation; Oluwadamilare Lanre Fogabi: Formal analysis; Treister Roi: Study conception.
Received: February 3, 2022; Revised: April 17, 2022; Accepted: April 18, 2022
This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background: Inflammation is known to underlie the pathogenesis in neuropathic pain. This study investigated the anti-inflammatory and neuroprotective mechanisms involved in antinociceptive effects of co-administration of acetaminophen and L-carnosine in chronic constriction injury (CCI)-induced peripheral neuropathy in male Wistar rats.
Methods: Fifty-six male Wistar rats were randomly divided into seven experimental groups (n = 8) treated with normal saline/acetaminophen/acetaminophen + L-carnosine. CCI was used to induce neuropathic pain in rats. Hyperalgesia and allodynia were assessed using hotplate and von Frey tests, respectively. Investigation of spinal proinflammatory cytokines and antioxidant system were carried out after twenty-one days of treatment.
Results: The results showed that the co-administration of acetaminophen and L-carnosine significantly (P < 0.001) increased the paw withdrawal threshold to thermal and mechanical stimuli in ligated rats compared to the ligated naïve group. There was a significant (P < 0.001) decrease in the levels of nuclear factor kappa light chain enhancer B cell inhibitor, calcium ion, interleukin-1-beta, and tumour necrotic factor-alpha in the spinal cord of the group coadministered with acetaminophen and L-carnosine compared to the ligated control group. Co-administration with acetaminophen and L-carnosine increased the antioxidant enzymatic activities and reduced the lipid peroxidation in the spinal cord.
Conclusions: Co-administration of acetaminophen and L-carnosine has anti-inflammatory effects as a mechanism that mediate its antinociceptive effects in CCI-induced peripheral neuropathy in Wistar rat.
Keywords: Acetaminophen, Anti-Inflammatory Agents, Antioxidants, Carnosine, Cytokines, Hyperalgesia, Lipid Peroxidation, Neuralgia, NF-kappa B, Spinal Cord.
Neuropathic pain (NP) is an intractable consequence of lesion or disease to the somatosensory system that results in detrimental psychosocial life of the affected patients. It has been estimated that NP affects 7 to 10% of the world population . NP is characterised by hyperalgesia, allodynia, and anxiodepresive cormobidities [2,3]. Treatment of NP has proved difficult, as only about 2% of current drugs used yielded 50% pain reduction in addition to adverse side effects [1,3]. The multifaceted mechanisms involved in the development and maintenance of NP make it challenging to diagnose and treat successfully.
The roles played by elevated levels of proinflammatory mediators released by the traumatised nerve and non-neuronal cells (glial cells, infiltrating macrophages, monocytes, and T-lymphocytes) increase the neuronal sensitisation and potentiations [4,5]. Tumour necrotic factor-alpha (TNF-α) and interleukin-1-beta (IL-1β) play an active role in the advent of NP by mediating mechanical allodynia and thermal hyperalgesia [6,7]. The involvement of the reactive oxygen species (ROS) in NP has been well established. ROS and reactive nitrogen species (RNS) released from increased activities of the damaged sensory neurons and non-neuronal cells result in synaptic remodelling and functions that mediate allodynia and hyperalgesia. ROS and RNS increase the expression of sodium ion channels, excitatory receptors, and neuronal disinhibition. All this mediates increased sensitisation and a reduced neuronal threshold to nociceptive stimuli .
Acetaminophen (N-Acetyl-para-aminophenol) is a derivative of p-aminophenol from the metabolite of acetanilide and phenacetin. It is a multimodal analgesic agent with reduced opioid-related side effects  and an antipyretic function . L-carnosine is a histidine-containing dipeptide molecule synthesised naturally in the liver from β-alanine and L-histidine . It is widely distributed in various tissues of the body, including the nervous tissue. It is has been reported that L-carnosine possesses a proton buffering effect  and acted as an antioxidant via scavenging of free radicals and singlet oxygen and chelating with heavy metals [11,13].
There is limited information on the role of acetaminophen in NP. Likewise, the combined therapeutic effect of both acetaminophen and L-carnosine in painful neuropathy is yet to be investigated. Hence, this study is undertaken to investigate the combined potential antinociceptive effects of co-administration of acetaminophen and L-carnosine in chronic constriction injury (CCI)-induced peripheral neuropathy. The authors further assessed the anti-inflammatory properties and neuroprotective benefits of this combined treatment with acetaminophen and L-carnosine.
This study was consonant with the ethical guideline in animal experimentation outlined in the 2019 updated ARRIVE (Animal Research: Reporting of
Fifty-six male Wistar rats weighing 150–200 g were used for the study. The sample size was estimated with the aid of SigmaPlot version 12 software (Systat software, Inc., Chicago, IL). The statistical power of 80% at an alpha level of 0.05 was used for the estimation. Based on a previous study on the paw-withdrawal latency to the hotplate, a sample size of eight per group was derived. The animals were randomly hand-picked and grouped into seven. They were housed and acclimatised in the animal facilities of the Faculty of Basic Medical Sciences with free access to food and water.
Peripheral neuropathy was induced using the CCI model as described by Bennett and Xie . Briefly, the animal was completely anaesthetised with xylazine and ketamine hydrochloric (100 mg/kg intraperitoneal injection [i.p]). The proximal back of the left hind limb was shaved, sterilised, and dissected to expose the main sciatic nerve. Four loose ligatures (4-0 ligating silk) were placed around the sciatic nerve, approximately 0.5 mm apart before the trifurcation. The muscle and skin were subsequently sutured back in layers, and penicillin powder was applied to the wound after sutured to prevent infection. The rats were returned to their respective home cage after recovery with free access to food and water. The animals were only dissected and sutured back without ligatures on the sciatic nerve in the sham group operation.
Animals were treated with oral administration of either normal saline/acetaminophen/acetaminophen + L-carnosine once daily for twenty-one consecutive days. Post-treated groups were administered their respective treatment three days after surgical induction of NP and continued for the next twenty-one days. Pretreated groups were treated for seven consecutive days before induction of NP and continued subsequently. The study groups include:
Group A (Control): 10 mL/kg of body weight of normal saline
Group B (Sham): 10 mL/kg of body weight of normal saline
Group C (Ligated control): 10 mL/kg of body weight of normal saline
Group D (Pre-treated acetaminophen): 200 mg/kg of body weight
Group E (Post-treated acetaminophen): 200 mg/kg of body weight
Group F (Pre-treated co-treatment): acetaminophen (200 mg/kg of body weight) + L-carnosine (100 mg/kg of body weight)
Group G (Post-treated co-treatment): acetaminophen (200 mg/kg of body weight) + L-carnosine (100 mg/kg of body weight)
L-carnosine was a product of Hubei Hongpeptide Biotechnology Co., Ltd. China while acetaminophen was a product of Anhui BBCA pharmaceutical Co., Ltd. China. The dosage of 200 mg/kg of body weight of L-carnosine  and 400 mg/kg of body weight of acetaminophen  were used based on previously study.
Pain behavioral assessment was done to investigate the antinociceptive effects of the treatment on thermal hyperalgesia and mechanical allodynia. Baseline tests were carried out before CCI and three days after CCI. Pain behaviour was further monitored on the 3rd, 7th, 14th, and 21st day of treatments.
A hotplate test  was used to evaluate the thermal hyperalgesia in each treatment group. Briefly, a hotplate with a restricting pyrex cage, 30 cm high, with the temperature set and maintained at 55°C ± 0.5°C was used. The rats were dropped gently on the surface of the hotplate. The time taken by each rat to either flinch, lick, or jump out of the pyrex enclosure was recorded chronologically with a stopwatch, and taken as paw withdrawal latency (PWL).
Responses to the mechanical bending force of various strengths of von Frey filaments were used to assess mechanical allodynia as described in the author’s previous study . Animals were placed in a transparent Perspex cage with a wire mesh floor and allowed to rest for 15 minutes. Von Frey filaments grading 1.4 g, 2 g, 4 g, 6 g, 8 g, 10 g, 15 g, 26 g, 60 g, and 100 g bending forces were applied individually to the plantar surface of each ligated hind paws of rats in ascending orders. The paw withdrawal threshold was defined by the mechanical force (in gram) that produced ligated hind limb withdrawal, licking, or flinching in three consecutive trials. Von Frey filaments that produced a lifting of the whole hind limb without sudden withdrawal, licking or flinching were taken as a positive response.
After twenty-one days of post-treatment with respective drugs, the rats were euthanised with a high dose of ketamine hydrochloride (180 mg/kg i.p.). The lumbar segment of the spinal cord (L4-L6) was collected via the hydraulic extrusion method. The sample was homogenised in Tris-buffer (1 M, pH 7.4) and centrifuged at 16,000 RPM for fifteen minutes. The supernatants collected were used for the assessment of inflammatory markers, calcium ions, antioxidant parameters, and lipid peroxidation. IL-1β (Abcam, Cambridge, UK), TNF-α (Abcam), nuclear factor kappa light chain enhancer B cell inhibitor (NF-κB) (Cayman Chemical, Ann Arbor, MI), were analysed with microplate reader using their ELISA manufacturer’s instructional manual. Superoxide dismutase (SOD) activities , reduced glutathione (GSH) concentration , malondialdehyde (MDA) concentration , and calcium ion level were estimated spectrophotometrically.
Haematoxylin and eosin (H&E) stain as described by Muthuraman et al.  was used to investigate the neuronal architecture of the sciatic nerve. Briefly, the sciatic nerve was excised from the rat following trans-cardiac perfusion with phosphate buffer and formalin. The tissues were fixed in 10% formalin solution and blocked. They were sectioned longitudinally into 5 μm before staining with H&E and observed under a high-power light microscope. Images were acquired from the microscope by using Leica ICC50 E Digital Camera (Leica Microsystems, Wetzlar, Germany) connected to a computer and analysed with Armscope software version 3.7 (Amscope, Irvine, CA).
Graphpad prism software version 5 (GraphPad Software, San Diego, CA) was used for the analysis of data. All data were expressed as mean ± standard error of the mean. Two-way analysis of variance (ANOVA) was used to analyse behavioural pain tests, while one-way ANOVA was used for the analysis of biochemical parameters. The Bonferroni post hoc multiple comparison test with (
The hotplate test showed that CCI significantly (
CCI significantly (
CCI significantly (
There was a significant (
Fig. 5A and 5B shows the normal cytoarchitecture of the sciatic nervein the unligated naïve group, and sham group groups respectively. CCI induces derangement in the cytoarchitecture of the sciatic nerve, as indicated in Fig. 5C. There is wide demyelination and degeneration of the neuron fibres as well as shrinkage and extinction of the nuclei of Schwann cell. CCI results in pronounced vacuolisation of the sciatic nerves. Post-treatment with acetaminophen showed preservation of myelinated and unmyelinated neurons. However, there is the presence of vacuolation and a significant reduction in Schwann cells (Fig. 5D). Animals pretreated with acetaminophen have improved myelinated neurons, Schwann cell, and reduced vacuolation, as indicated in Fig. 5E. The combined treatment with acetaminophen + L-carnosine showed a well improved normal cytoarchitecture of the sciatic nerve. There is increased Schwann cells, myelinated and unmyelinated neurons, as well as drastically reduced vacuolation (Fig. 5F, G).
The treatment of NP remains a challenge worldwide. This study reports the antinociceptive effects of combined administration of acetaminophen and L-carnosine in chronic constricted injury-induced peripheral neuropathy.
CCI is a well-established procedure in modelling peripheral neuropathy in laboratory animals. This study showed that animals developed thermal hyperalgesia and mechanical allodynia following CCI. This is evidenced by a widely reduced threshold to both mechanical and thermal stimuli. Treatment with either acetaminophen or its combination with L-carnosine effectively mitigated both thermal hyperalgesia and mechanical allodynia. Pretreatment of rats with acetaminophen only is more beneficial in preventing the development of allodynia than thermal hyperalgesia. However, combined pretreatment with acetaminophen with L-carnosine effectively slowed down the onset of thermal hyperalgesia. It ameliorated mechanical allodynia, as evidenced by increased paw response threshold to thermal and mechanical stimuli. Post-treatment with a combined dose of acetaminophen and L-carnosine has delayed therapeutic onset, unlike post-treatment with acetaminophen alone. Hence, it was deduced that the antinociceptive properties of acetaminophen are strengthened by co-treatment with L-carnosine.
Hyperalgesia and allodynia have been reported to be mediated by the increased advent of proinflammatory cytokines [4,7]. Excercabation of NF-κB promotes the production of TNF-α and IL-β. Coadministered acetaminophen and L-carnosine effectively reversed increased TNF-α and IL-β in the ligated animals. This may be due to their effects on NF-κB and calcium ion concentration. TNF-α and IL-β have been widely reported to mediate hypersensitivities by increasing neuronal excitation and lowering the neuronal threshold [6,7]. They are involved in synaptic remodelling leading to increased neuronal hyperexcitation [6,23]. A single treatment with acetaminophen alone led to reduced TNF-α and IL-β but not via the NF-κB pathway. Enhanced NF-κB is associated with increased excitatory synapses, neuronal hyperexcitability, and disinhhibition [24,25]. Hence the improved antinociceptive effects of combined acetaminophen and L-carnosine are mediated via anti-inflammatory activities in mitigating NF-κB.
ROS play a pivotal role in the development of hyperalgesia and allodynia [8,26]. Agents that possess antioxidant properties have been reported to be effective in treating NP. Therapeutic agents targeting oxidative stress improve NP via scavenging the ROS [27,28]. This study was in consonant with a previous study that showed that CCI is characterised by increased oxidative stress . A combined dose of acetaminophen and L-carnosine ameliorated increased oxidative stress as indicated by a marked reduction in lipid peroxidation in the spinal cord. Combined acetaminophen and L-carnosine also stimulated the production of antioxidant enzyme (SOD) and reduced GSH that mopped up ROS. This further explains the combined potential of the two therapeutic agents that mediated their antinociceptive properties. Antioxidant properties of the combined treatment are evidence of the structural integrity of the sciatic nerves. Improved structural architecture of the sciatic nerve with combined acetaminophen and L-carnosine indicated a high degree of neuroprotection that manifested the observed antinociceptive properties. Increased lipid peroxidation results in distortion in neuronal integrity and derangement in nerve conduction. Pretreatment with combined acetaminophen and L-carnosine showed a better neuroprotective effect which may be due to increased antioxidant properties via increased production of reduced GSH.
In conclusion, the antinociceptive effects of combined acetaminophen and L-carnosine are mediated by its neuroprotective effect via reduced lipid peroxidation and NF-κB. Additive antinociceptive properties of acetaminophen and L-carnosine are mediated by the inhibition of IL-1β via reductions in NF-κB and stimulation of GSH and SOD synthesis. This serves as an effective mechanism that underpins the combined antinociceptive activities of acetaminophen and L-carnosine.
Data files are available from Harvard Dataverse: https://doi.org/10.7910/DVN/4IFYKM.
No potential conflict of interest relevant to this article was reported.
No funding to declare.