Exploring the Analgesic Potential of Cytidine, Uridine, and Gabapentin either alone or in combination: Insights from Formalin-Induced Pain and Diabetic Neuropathy Models

Exploring the Analgesic Potential of Cytidine, Uridine, and Gabapentin either alone or in combination: Insights from Formalin-Induced Pain and Diabetic Neuropathy Models | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Exploring the Analgesic Potential of Cytidine, Uridine, and Gabapentin either alone or in combination: Insights from Formalin-Induced Pain and Diabetic Neuropathy Models Esam Qnais, Muna Barakat, Rabaa Y. Athamneh, Mohammad A. A Al-Najjar, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4586386/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Diabetic neuropathic pain is a usual consequence of diabetes. In this study, the formalin-induced pain and the diabetic neuropathy models were used in mice to investigate if the cytidine, uridine, and gabapentin treatments either alone or in combination can reduce nephropathic pain or not. To achieve our goals, cytidine, uridine, and gabapentin, either alone or in combination were orally administered to mice at a dose of 100 mg/kg. The formalin test was used to examine pain-related behaviors throughout both the primary and secondary phases. Further, the potential pain-relieving efficacy of these therapies was assessed in a diabetic neuropathy model produced by streptozotocin injection. Oral administration of the combination (cytidine + uridine + gabapentin) reduces formalin-induced pain-associated behavior in the first and second phases more than in each treatment alone. In the diabetic neuropathy model, administering the cytidine + uridine + gabapentin combination significantly reversed the pain threshold detected. The combination of cytidine, uridine, and gabapentin decreased as well as the elevated spinal p-CREB levels caused by formalin, which was reversed by pre-treatment with naloxone, yohimbine, and methysergide. This study reveals that the cytidine, uridine, and gabapentin combination have strong synergistic pain-relieving properties in both formalin-induced pain and diabetic neuropathy models more than each treatment alone. Diabetic neuropathy Uridine Cytidine Gabapentin p-CREB Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Diabetic neuropathy is a common neurological condition that often arises as a result of diabetes mellitus 1 , 2 . It affects more than half of people with a history of diabetes for more than 25 years 3 . At least 20% of diabetic patients have neuropathic problems that might include pain, sensory irritation, foot ulcers, or weakness, resulting in a significant clinical burden in any diabetes services 4 . The cAMP element-binding protein (CREB) is a transcription factor that affects critical neurological processes 5 , 6 . CREB promotes inter-synaptic nociceptive interactions in the cell by efficiently modulating gene transcription, leading to a quick to long-lasting transition in nociceptive signaling 7 , 8 . A significant rise in the transcriptional rate of phosphorylated CREB (p-CREB), which migrates into the nucleus and induces the transcription of targeted genes containing a cyclic adenosine monophosphate response element, such as brain-derived neurotrophic factor, can contribute to pain development 9 , 10 . Analgesics such as nonsteroidal anti-inflammatory medications (NSAID) and opioids are commonly used to treat acute pain, but they are ineffective for neuropathic pain 11 – 13 . Regardless of their lack of effectiveness, traditional analgesics are still widely used among individuals with neuropathic pain, raising questions regarding opiate dependency 14 , 15 . Previous studies have shown the effect of using NSAIDs in patients with type 2 diabetes on the heart, which can increase the risk of heart failure 16 , 17 . Currently, anti-depressant including amitriptyline, mirtazapine, duloxetine, and fluoxetine, which are known for their anti-nociception effect, are also used to treat neuropathic pain 18 , 19 . These therapies are associated with significant adverse impacts that decrease patient compliance, such as dry mouth, dizziness, nausea, headache, and constipation 20 , 21 . Therefore, finding an effective treatment for neuropathic pain with minimal side effects is essential. Gabapentin (1-[aminomethyl]-cyclohexaneacetic acid) is an anticonvulsant that received approval in the United States in 1994 for usage among adult patients with partial seizures 22 . It has been indicated by personal experience and in off-label clinical trials to be efficient in the management of pain syndromes, including painful diabetic neuropathy 23 , 24 . A previous study has shown that gabapentin exhibited analgesic efficacy in the chronic constriction injury model of neuropathic pain in rats 25 . In parallel, uridine and cytidine belong to the pyrimidine nucleotide family, one of four major molecular biology groups (together with carbohydrates, lipids, and proteins) that have crucial functions in cellular metabolism 26 . Besides being components of DNA and engaging in genome transcription and translation, these nucleotides provide energy transfer for chemical bonds, intracellular communication, neurotransmission, and the transfer of biochemical radicals from intermediary metabolism 27 . Pharmacological research indicates that these drugs improve neuromuscular recovery in animals with sciatic nerve injuries and reduce the transmission of pain in the spinal cord 28 , 29 . New evidence demonstrates that nucleotide receptor agonists can effectively alleviate neuropathic pain 30 . Moreover, In vitro studies showed they may enhance neural fiber regeneration 31 . Currently, analgesics often fall short in efficacy or are associated with significant side effects, highlighting the necessity for alternative or adjunctive therapies. Cytidine and uridine, as nucleosides involved in cellular processes and neuroprotection, along with gabapentin, a well-established anticonvulsant with analgesic properties, present promising candidates for pain relief. This study seeks to explore the effects of this combination in relieving pain disorders using formalin-induced pain and diabetic neuropathy mice model. Results Effect of uridine, cytidine, and gabapentin alone or in combination on pain response in the formalin test The intraplantar injection of 5% formalin-induced acute nociceptive reactions lasted roughly 5 minutes, known as the first phase reaction. This first phase was followed by a persistent inflammatory response for about 20 to 40 minutes following formalin injection, known as the secondary phase reaction. In the formalin test, the control group, which was given a vehicle, showed an immediate nociceptive response in both phases. In contrast, the treated group, including cytidine, uridine, gabapentin, cytidine + uridine + gabapentin groups showed a significant decrease in the nociception reactions in both phases compared to the control group (* p < 0.05; *** p < 0.001, respectively), as shown in Fig. 1 A, and Fig. 1 B. Impact of cytidine, uridine, and gabapentin alone or in combination on pain behavior in a model of diabetic-induced neuropathy Following five weeks of a single intraperitoneal injection of streptozotocin, the mechanical stimulation threshold declined, as demonstrated by the von Frey filament test. 100 mg/kg of cytidine, uridine, and gabapentin alone or in combination were given orally 30 minutes before the mechanical pain evaluation. As shown in Fig. 2 , the combined treatment causes a reversal drop in the pain threshold compared to intact after 30, 60, and 120 minutes (* p < 0.05). In addition, there was a significant reversal drop in the pain threshold in the combined treatment compared to the gabapentin group after 120 minutes (* p < 0.05). Effect of cytidine, uridine, and gabapentin alone or in combination in p-CREB Protein in the Spinal Cord in Models of Formalin-Induced Pain and Diabetic-Induced Neuropathy Protein extractions from dissected lumbar spinal cords were collected 30 minutes after formalin injection to assess possible changes in the expression of phosphorylated CREB protein in the spinal cord. As seen in Fig. 3 A, formalin administration caused an increase in p-CREB expression through the spinal cord. Oral gabapentin or cytidine + uridine + gabapentin treatment administration reduced the increased levels of p-CREB caused by formalin injection. We continued our study by exploring alterations in p-CREB expression in the spinal cord of streptozotocin-induced neuropathy mice. Diabetic mice showed a considerable increase in CREB phosphorylation compared to normal mice. However, this increase was inhibited by administering gabapentin or cytidine + uridine + gabapentin treatment. The inhibition of p-CREB production by cytidine + uridine + gabapentin treatment was greater than when gabapentin was given alone, as illustrated in Fig. 3 B. Effect of naloxone, yohimbine, or methysergide on pain response in the formalin test This experiment evaluated the role of opioid, serotonergic, and adrenergic systems in the antinociception generated by cytidine, uridine, and gabapentin, either alone or in combination. As demonstrated in Fig. 4 A, giving naloxone (5 mg/kg, i.p.), which inhibits opioid receptors, counteract the antinociceptive effect caused by cytidine, uridine, and cytidine + uridine + gabapentin groups, while pretreatment with yohimbine (5 mg/kg, i.p.), which inhibits α2-adrenergic receptors, counteract the antinociceptive effect caused by uridine, gabapentin, and cytidine + uridine + gabapentin groups, as shown in Fig. 4 B. However, pretreatment of methysergide (5 mg/kg, i.p.), which inhibits serotonergic receptors, counteracts the antinociceptive effect generated by gabapentin and cytidine + uridine + gabapentin groups, as shown in Fig. 4 C. Notably, administering naloxone, methysergide, or yohimbine alone did not affect the pain response. Impact of naloxone, yohimbine, or methysergide on cytidine, uridine, and gabapentin alone or in combination in Modulating of Spinal p-CREB Expression in the Formalin Test Since the administration of naloxone reverses the antinociceptive effect induced by cytidine, uridine, and cytidine + uridine + gabapentin groups, pretreatment with yohimbine counteracts the antinociceptive effect caused by uridine, gabapentin, and cytidine + uridine + gabapentin groups, and methysergide counteracts the antinociceptive effect generated by gabapentin, and cytidine + uridine + gabapentin groups in the formalin test, Our study also aimed to determine if naloxone, yohimbine, and methysergide can reverse the drop in spinal p-CREB expression produced by previous treatments. As shown in Fig. 5 A, pre-administration of naloxone (5 mg/kg, i.p.) reversed the reduction in p-CREB expression in cytidine, uridine, and cytidine + uridine + gabapentin groups. In comparison, pre-administration of yohimbine (5 mg/kg, i.p.) reversed the reduction in p-CREB expression in uridine, gabapentin, and cytidine + uridine + gabapentin groups, as shown in Fig. 5 B. Moreover, methysergide pretreatment reversed the reduction in p-CREB expression in gabapentin and cytidine + uridine + gabapentin groups, as shown in Fig. 5 C. Discussion This study explores for the first time the effect of cytidine, uridine, and gabapentin either alone or in combination in a formalin-induced pain model. Oral administration of cytidine + uridine + gabapentin combination significantly decreased pain behavior more than cytidine, uridine, and gabapentin alone throughout the formalin test's first and second phases. It has been widely recognized that intraplantar injection leads to a biphasic response 32 . The pain behavior seen throughout the first phase is primarily caused by the immediate stimulation of sensory neurons, while the pain behavior seen through the second phase includes both inflammatory mechanisms and central sensation inside the dorsal horn. These findings imply that pain behaviors observed during the first and second phases are significantly controlled 33 . Our key findings showed that the combination of cytidine, uridine, and gabapentin has a potential synergistic effect in reducing pain behavior caused by formalin injection into the plantar of mice's hid paws. This synergistic effect suggests that the combined therapeutic approach may enhance analgesic efficacy beyond what is achievable with each compound alone. The observed reduction in pain behavior indicates that these compounds might interact at multiple levels of pain modulation pathways, offering a multiple approaches to pain management 34 , 35 . Cytidine and uridine, known for their roles in neuroprotection and cellular repair 36 , 37 . They are involved in the synthesis of phospholipids and nucleotides, which are essential for maintaining neuronal integrity and function. By promoting neuronal health and repair, cytidine and uridine may reduce neuronal hyperexcitability and inflammation associated with pain. Gabapentin, on the other hand, primarily works by inhibiting voltage-gated calcium channels in the central nervous system, which reduces the release of excitatory neurotransmitters such as glutamate 38 , 39 . This inhibition leads to decreased neuronal excitability and reduced pain signaling. Therefore, combing cytidine and uridine may enhance the neuroprotective environment, reducing inflammation and supporting neuronal recovery, while gabapentin modulates pain transmission by inhibiting excitatory neurotransmitter release. This dual action likely results in a more effective reduction of pain behavior compared to the individual effects of each compound. Furthermore, The CREB protein is strongly linked to pain conduction, with its transcription in the spinal cord or dorsal root ganglia increasing in various chronic pain models, including neuropathic pain and neuropathy 34 , 35 . In acute and chronic inflammation models, such as the formalin pain model, p-CREB transcription in the spinal cord or brain areas is also elevated 40 – 42 . In our study, we observed that formalin-induced pain significantly increased p-CREB transcription in the spinal cord. Treatment with cytidine, uridine, and gabapentin, both alone and in combination, reduced this up-regulation, with the combination treatment showing the greatest reduction in p-CREB levels. Additionally, in the diabetic neuropathy model, gabapentin alone and the combination treatment also reduced the up-regulation of spinal p-CREB, with the combination treatment being more effective. These findings suggest that gabapentin, particularly when combined with cytidine and uridine, reduces nociception by lowering p-CREB levels in the spinal cord in both the formalin-induced pain and diabetic neuropathy models. The involvement of opioid, serotonergic, and adrenergic receptors in modulating nociceptive processes further supports this mechanism. Opioid receptors are known to reduce nociception 43 , and blocking spinal serotonergic and noradrenergic receptors can counteract the anti-nociceptive effects of supraspinal morphine administration 43 . In our study, the anti-nociceptive effect of the orally administered combination treatment of cytidine, uridine, and gabapentin was linked to the activation of opioid, α-2 adrenergic, and serotonergic receptors 44 , 45 . That also may contribute to the observed activity. Finally, this combination therapy could potentially address the limitations of current pain treatments, providing a more comprehensive analgesic strategy. Further research is warranted to explore the underlying mechanisms of this synergy and to assess the clinical relevance and potential applications in human pain management, especially for chronic and neuropathic pain conditions. These findings pave the way for novel pain management protocols that leverage the benefits of multi-compound therapies. Conclusion This study highlights the significant analgesic potential of combining cytidine, uridine, and gabapentin in reducing pain behavior in both formalin-induced pain and diabetic neuropathy models. The combination therapy not only demonstrated a synergistic effect in alleviating pain but also effectively reduced the up-regulation of p-CREB expression in the spinal cord, a key player in pain conduction. These findings pave the way for developing more effective and comprehensive pain management strategies, offering hope for better therapeutic outcomes in chronic and neuropathic pain conditions. Further research is warranted to fully elucidate the mechanisms and clinical applicability of these promising results. Materials and Methods Experimental animals Thirty adult Swiss albino mice weighing 24 to 28 g were housed at the Hashemite University's Animal House Center. All the experiments were approved by the institutional review board at the Hashemite University, Zarqa, Jordan (IRB #: 8/5/2019/2020). The mice were maintained under controlled temperature (23 ± 2°C) and humidity (50% ± 5%) conditions and kept on a 12 h light/dark cycle (lights on at 07:00 AM). They had free access to food and water throughout the experiment. Before the experiment started, mice had free access to the apparatus for 30 minutes for two days to habituate them to the testing apparatus and to minimize mice stress. Throughout the behavioral testing, a video camera was placed in front of the apparatus using a tripod. Each behavioral test was recorded for future reference. Two observers, blinded to the experiment conditions, evaluated the antinociception and mechanical allodynia parameters to prevent any potential bias. The mice were divided randomly into five groups: Control group ( Control , n = 6), Cytidine group ( Cyt , n = 6), Uridine group ( Uri , n = 6), Gabapentin group ( Gaba , n = 6), and Cytidine + Uridine + Gabapentin group ( Cty + Uri + Gaba , n = 6). Drugs Streptozotocin, Naloxone, Yohimbine, and Methysergide were obtained from Sigma-Aldrich (St. Louis, MO, USA), while Citicoline (250 mg Cytidine − 5’-diphosphocholine, Cognizin®, Kyowa Hakko Bio Co., Ltd.), and Uridine (300 mg 5’-monophosphate disodium salt, Amazing Nutrition®, USA) were obtained from local pharmacy. Cytidine and uridine were dissolved in distilled water associated with gentel heating. However, gababentine was dissolved in water with PH adjusted to 7.2. Production of streptozotocin-induced diabetic neuropathy model. Diabetic neuropathy was induced through the administration of a single dose of streptozotocin (0.1 mg/kg) intraperitoneally (i.p.). One week later, diabetes was confirmed. The experiment took place five weeks after diabetes induction. Pretreatment of antagonists Mice were initially treated intraperitoneally with saline, naloxone (5 mg/kg), methysergide (5 mg/kg), or yohimbine (5 mg/kg). This pretreatment was performed 10 minutes before the oral administration of the control vehicle, uridine, cytidine, gabapentin, or uridine + cytidine + gabapentine treatment (100 mg/kg from each). The observed formalin-induced nociceptive behavior was next monitored and analyzed. Von-Frey test Antinociception and mechanical allodynia were assessed using calibrated von-Frey filaments as described in Bonin et al. 2014 study 32 . Mechanosensitivity assessment is an essential tool for investigating pain in animal models. This is frequently performed using von Frey filaments (North Coast Medical, Inc., Gilroy, CA, USA) in an up-down testing method. After adapting the mice to their surroundings for 30 minutes, they were transported to separate Plexiglas chambers on top of a mesh of wires to stimulate the plantar surface of their rear paws. The paw withdrawal threshold was measured by increasing and decreasing the strength of the stimulus and computed using Dixon's up-down method 46 . Formalin test The formalin test in mice is a realistic and reliable nociception model that detects various analgesic medications 47 . The test involves a chemical nociceptive stimulation that causes a spontaneous pain sensation. According to Hunskaar et al.'s 1985 formalin test protocol 48 , the formalin test was conducted on mice that were independently subjected to a macrolone cage (30 cm x 12 cm x 13 cm) for 2 h. Using a micro-syringe with a 26-gauge needle, ten µl of 1% formalin in 0.9% normal saline was subcutaneously injected into the mice's dorsal hind paw with little restrictions. The mice were returned to the cage, and the duration of the animals licking the injected paw was precisely measured using a stopwatch to indicate nociception. The first phase of the test was defined as the time between zero and five minutes following the formalin injection, whereas the late or second phase lasted from twenty to forty minutes. These periods corresponded to the direct influence on nociceptors and the inflammatory nociceptive responses, as clarified by Hunskaar and Hole 1987 47 . Protein extraction and western blot The lumbar part of the mice's spinal cord was extracted. The tissue was washed twice with cold Tris-buffered saline (pH 7.5) containing 20 mmol/L Trizma base, and 137 mmol/L NaCl. During the washing, the tissue was lysed with sodium dodecyl sulfate lysis buffer consisting of 62.5 mmol/L Trizma base, 2% w/v sodium dodecyl sulfate, 10% glycerol, 0.1 mmol/L Na3VO4, 3 mg/mL aprotinin, and 20 mmol/L NaF. Following a brief sonication period, the protein concentration was determined using a detergent-compatible protein assay reagent (Bio-Rad Laboratories, Hercules, CA, USA). Equal quantities of extracted proteins have been blended with 5X Laemmle loading dye and separated on polyacrylamide gels (SDS-PAGE) ranging from 6–10%. Proteins were then transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). Membranes were blocked with 3% nonfat dry milk in TBST (10 mM Trizma base, pH 8.0, 150 mM NaCl, and 20% Tween 20) for 30 minutes at 4°C and rinsed with TBST. Membranes were incubated overnight at 4°C with one of the following primary antibodies: p-CREB (Abcam, 1:1000) and β-actin (Cell Signaling Technology, 1:1000). Mouse Anti-β-Tubulin antibody was used as a loading control (1:5000). After incubating overnight, the primary antibody was eliminated. The membrane was washed four times in TBST for 5 minutes each before adding the secondary antibody (anti-rabbit IgG-horseradish peroxidase conjugate (1:4000)) for 1 hour. After 1 hour, the secondary antibody was removed and the membrane was incubated with ECL-plus solution (Millipore, Billerica, MA, USA) for imaging using a Luminescent Image Analyzer (LAS-4000, Fuji Film Co., Japan). A Multi-Gauge Version 3.1 (Fuji Film, Japan) system was used to quantify the detected bands and expressed them as a percentage relative to the control. Statistical analysis All data is presented as mean ± SEM. One-way ANOVA followed by Tukey's post-hoc test was used in the statistical analysis, using GraphPad Prism 8.0.1 software (GraphPad Software Inc, CA, USA). P < 0.05 was considered statistically significant. Declarations Data availability Data available within the article and its supplementary materials. Ethics statement This study is reported in accordance with the ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments). All animal experiments were conducted following the ethical standards and guidelines set forth by our institution and approved by the Animal Research Ethics Committee at Hashemite University (IRB number: HU 8/5/2019/2020), and all procedures were performed in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Author contributions Conceptualization, A.A., and M.B.; methodology, E.Q.; software, R.Y.A.; validation E.Q. and A.A.; formal analysis, A.A.; investigation, M.A.A.; resources, M.B.; data curation, L.A.; writing—original draft preparation, M.B, D.K.CH and L.A.; writing—review and editing, A.A. and M.B, D.K.CH; visualization, A.A.; supervision, E.Q.; project administration, A.A.; funding acquisition, A.A. All authors have read and agreed to the published version of the manuscript. Funding This research was supported by the Deanship of Scientific Research at the Hashemite University, Zarqa, Jordan (Grant number: 49/2020). Competing interests No potential conflict of interest relevant to this article was reported. References Goldberg, H. et al. A double-blind, randomized, comparative study of the use of a combination of uridine triphosphate trisodium, cytidine monophosphate disodium, and hydroxocobalamin, versus isolated treatment with hydroxocobalamin, in patients presenting with compressive neuralgias. Journal of pain research , 397-404 (2017). Russell, J. W. & Zilliox, L. A. Diabetic neuropathies. Continuum: lifelong learning in neurology 20 , 1226-1240 (2014). Pirart, J. Diabetes mellitus and its degenerative complications: a prospective study of 4,400 patients observed between 1947 and 1973. Diabetes care 1 , 252-263 (1978). Ward, J. The cost of diabetic neuropathy. Pharmacoeconomics 8 , 52-54 (1995). Sheng, M. & Greenberg, M. E. The regulation and function of c-fos and other immediate early genes in the nervous system. Neuron 4 , 477-485 (1990). Carlezon Jr, W. A. et al. Regulation of cocaine reward by CREB. Science 282 , 2272-2275 (1998). Carrano, N. et al. The synaptonuclear messenger RNF10 acts as an architect of neuronal morphology. Molecular Neurobiology 56 , 7583-7593 (2019). Di Benedetto, B. The role of the ERK/MAPK pathway in the adult mouse brain for fear potentiated startle learning: a model to study fear-based anxiety disorders , Technische Universität München, (2008). Navabpour, S., Rezayof, A. & Ghasemzadeh, Z. Activation of VTA/CeA/mPFC cannabinoid CB1 receptors induced conditioned drug effects via interacting with hippocampal CAMKII-CREB-BDNF signaling pathway in rats. European journal of pharmacology 909 , 174417 (2021). Wen, J. et al. The cAMP response element-binding protein/brain-derived neurotrophic factor pathway in anterior cingulate cortex regulates neuropathic pain and anxiodepression like behaviors in rats. Frontiers in Molecular Neuroscience 15 , 831151 (2022). Arner, S. & Meyerson, B. Lack of analgesic effect of opioids on neuropathic and idiopathic forms of pain. Pain 33 , 11-23 (1988). Gilron, I. & Coderre, T. J. Emerging drugs in neuropathic pain. Expert opinion on emerging drugs 12 , 113-126 (2007). Max, M. B., Schafer, S. C., Culnane, M., Dubner, R. & Gracely, R. H. Association of pain relief with drug side effects in postherpetic neuralgia: a single‐dose study of clonidine, codeine, ibuprofen, and placebo. Clinical Pharmacology & Therapeutics 43 , 363-371 (1988). Berger, A., Dukes, E. M. & Oster, G. Clinical characteristics and economic costs of patients with painful neuropathic disorders. The journal of pain 5 , 143-149 (2004). Vo, T., Rice, A. S. & Dworkin, R. H. Non-steroidal anti-inflammatory drugs for neuropathic pain: how do we explain continued widespread use? PAIN® 143 , 169-171 (2009). Bhala, N. et al. Vascular and upper gastrointestinal effects of non-steroidal anti-inflammatory drugs: meta-analyses of individual participant data from randomised trials. Lancet (London, England) 382 , 769-779 (2013). Holt, A. et al. Heart failure following anti-inflammatory medications in patients with type 2 diabetes mellitus. Journal of the American College of Cardiology 81 , 1459-1470 (2023). Gilron, I., Watson, C. P. N., Cahill, C. M. & Moulin, D. E. Neuropathic pain: a practical guide for the clinician. Cmaj 175 , 265-275 (2006). McCleane, G. Pharmacological strategies in relieving neuropathic pain. Expert Opinion on Pharmacotherapy 5 , 1299-1312 (2004). Moulin, D. et al. Pharmacological management of chronic neuropathic pain–consensus statement and guidelines from the Canadian Pain Society. Pain Research and Management 12 , 13-21 (2007). Riediger, C. et al. Adverse effects of antidepressants for chronic pain: a systematic review and meta-analysis. Frontiers in neurology 8 , 307 (2017). Morris, G. L. Gabapentin. Epilepsia 40 , s63-s70 (1999). Rosner, H., Rubin, L. & Kestenbaum, A. Gabapentin adjunctive therapy in neuropathic pain states. The Clinical journal of pain 12 , 56-58 (1996). Stacey, B., Tipton, K., Owen, G., Sinclair, J. & Glick, R. Gabapentin and neuropathic pain states: a case series report. Regional Anesthesia and Pain Medicine 21 , 65-65 (1996). Bennett, G. J. & Xie, Y.-K. A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man. Pain 33 , 87-107 (1988). Vincenzetti, S., Polzonetti, V., Micozzi, D. & Pucciarelli, S. Enzymology of pyrimidine metabolism and neurodegeneration. Current medicinal chemistry 23 , 1408-1431 (2016). Voet, D., Voet, J. G. & Pratt, C. W. Fundamentals of biochemistry: life at the molecular level . (John Wiley & Sons, 2013). Wattig, B., Schalow, G., Heydenreich, F., Warzok, R. & Cervós-Navarro, J. Enhancement of nerve fibre regeneration by nucleotides after peripheral nerve crush damage. Electrophysiologic and morphometric investigations. Arzneimittel-forschung 42 , 1075-1078 (1992). Wattig, B. et al. Acceleration of nerve and muscle regeneration by administration of nucleotides--electroneurophysiological and morphometrical investigations. Acta histochemica. Supplementband 42 , 333-339 (1992). Ando, R., Mehesz, B., Gyires, K., Illes, P. & Sperlagh, B. A comparative analysis of the activity of ligands acting at P2X and P2Y receptor subtypes in models of neuropathic, acute and inflammatory pain. British journal of pharmacology 159 , 1106-1117 (2010). Verdugo, R. J., Cea, J. G., Castillo, J. L., Stuardo, A. & Contreras, C. PS-51-7 Cytidine monophosphate (CMP), uridine triphosphate (UTP) and hydroxycobalamin in the treatment of diabetic polyneuropathy: preliminary study. Electroencephalography and Clinical Neurophysiology/Electromyography and Motor Control 97 , S219-S220 (1995). Bonin, R. P., Bories, C. & De Koninck, Y. A simplified up-down method (SUDO) for measuring mechanical nociception in rodents using von Frey filaments. Molecular pain 10 , 1744-8069-1710-1726 (2014). Tjølsen, A., Berge, O.-G., Hunskaar, S., Rosland, J. H. & Hole, K. The formalin test: an evaluation of the method. Pain 51 , 5-17 (1992). Miyabe, T. & Miletic, V. Multiple kinase pathways mediate the early sciatic ligation-associated activation of CREB in the rat spinal dorsal horn. Neuroscience letters 381 , 80-85 (2005). SONG, X. s. et al. Activation of ERK/CREB pathway in spinal cord contributes to chronic constrictive injury‐induced neuropathic pain in rats 1. Acta Pharmacologica Sinica 26 , 789-798 (2005). Cansev, M. Uridine and cytidine in the brain: their transport and utilization. Brain research reviews 52 , 389-397 (2006). Piermartiri, T. C. & Tasca, C. I. in Natural Molecules in Neuroprotection and Neurotoxicity 661-676 (Elsevier, 2024). Cheng, J.-K. & Chiou, L.-C. Mechanisms of the antinociceptive action of gabapentin. Journal of pharmacological sciences 100 , 471-486 (2006). McGivern, J. G. & McDonough, S. I. Voltage-gated calcium channels as targets for the treatment of chronic pain. Current Drug Targets-CNS Neurological Disorders 3 , 457-478 (2004). Hermanson, O. & Blomqvist, A. Differential expression of the AP-1/CRE-binding proteins FOS and CREB in preproenkephalin mRNA-expressing neurons of the rat parabrachial nucleus after nociceptive stimulation. Molecular brain research 51 , 188-196 (1997). Seo, Y.-J. et al. Differential expression of phosphorylated Ca2+/calmodulin-dependent protein kinase II and phosphorylated extracellular signal-regulated protein in the mouse hippocampus induced by various nociceptive stimuli. Neuroscience 156 , 436-449 (2008). Mao, Q. et al. Antinociceptive effects of analgesic-antitumor peptide (AGAP), a neurotoxin from the scorpion Buthus martensii Karsch, on formalin-induced inflammatory pain through a mitogen-activated protein kinases–dependent mechanism in mice. PLoS One 8 , e78239 (2013). Yaksh, T. L. et al. An automated flinch detecting system for use in the formalin nociceptive bioassay. Journal of applied physiology 90 , 2386-2402 (2001). Spinella, M. The role of medullary cholinergic and excitatory amino acid receptors mediating mesencephalic opioid antinociception . (City University of New York, 1999). Dirksen, R. The clinical relevance of endorphin receptors: the antinociceptive effectiveness of epidurally or intrathecally injected endorphinomimetics , [Sl: sn], (1983). Chaplan, S. R., Bach, F. W., Pogrel, J., Chung, J. & Yaksh, T. Quantitative assessment of tactile allodynia in the rat paw. Journal of neuroscience methods 53 , 55-63 (1994). Hunskaar, S. & Hole, K. The formalin test in mice: dissociation between inflammatory and non-inflammatory pain. Pain 30 , 103-114 (1987). Hunskaar, S., Fasmer, O. B. & Hole, K. Formalin test in mice, a useful technique for evaluating mild analgesics. Journal of neuroscience methods 14 , 69-76 (1985). Additional Declarations No competing interests reported. Supplementary Files AnalysisNew.pzfx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4586386","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":320921387,"identity":"91abdee0-ef84-449a-9ab8-a00b0fc59797","order_by":0,"name":"Esam Qnais","email":"","orcid":"","institution":"The Hashemite University","correspondingAuthor":false,"prefix":"","firstName":"Esam","middleName":"","lastName":"Qnais","suffix":""},{"id":320921388,"identity":"eafa8594-9c28-46b9-a546-3a69104aace6","order_by":1,"name":"Muna Barakat","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA20lEQVRIiWNgGAWjYFACHhCRwMDA3gDmMjaAecRo4eE5QLIWiQS4FvxAt4H34OeKmjR5e8nHjz/zMNjIbjjA8PABPi1mB/iSJc8cyzHskU4zk+ZhSDMGakk2wK+Fx0Cyga2CsUc6wYyZh+FwIlBLmgQBLcY/G/5V2PdIHv8MdNh/orSYSTa25ST2SPAYAB12gBgtfGmWjX1pyT1ncsok5xgkG888TNAvvIdvNnxLtm1vP775w5sKO9m+4z2JD/BpYZBHkQYZz8yTgFcHNsB+gGQto2AUjIJRMKwBAJxhSTIV3BalAAAAAElFTkSuQmCC","orcid":"","institution":"Applied Science Private University","correspondingAuthor":true,"prefix":"","firstName":"Muna","middleName":"","lastName":"Barakat","suffix":""},{"id":320921389,"identity":"39420907-580e-4503-a9b7-4c45a8d6e927","order_by":2,"name":"Rabaa Y. Athamneh","email":"","orcid":"","institution":"Zarqa University","correspondingAuthor":false,"prefix":"","firstName":"Rabaa","middleName":"Y.","lastName":"Athamneh","suffix":""},{"id":320921390,"identity":"7bc69d1d-7b1d-4d9a-a4ba-efb184d65215","order_by":3,"name":"Mohammad A. A Al-Najjar","email":"","orcid":"","institution":"Applied Science Private University","correspondingAuthor":false,"prefix":"","firstName":"Mohammad","middleName":"A. A","lastName":"Al-Najjar","suffix":""},{"id":320921391,"identity":"f729677a-e717-46f0-bff0-3d45f9bb7e34","order_by":4,"name":"Lujain F. Alzaghari","email":"","orcid":"","institution":"Applied Science Private University","correspondingAuthor":false,"prefix":"","firstName":"Lujain","middleName":"F.","lastName":"Alzaghari","suffix":""},{"id":320921393,"identity":"efee3a96-05f7-4185-84d1-468eea03848c","order_by":5,"name":"Dinesh Kumar Chellappan","email":"","orcid":"","institution":"International Medical University","correspondingAuthor":false,"prefix":"","firstName":"Dinesh","middleName":"Kumar","lastName":"Chellappan","suffix":""},{"id":320921395,"identity":"ffc8584e-765c-43d9-a8a9-a5d999d4f3ac","order_by":6,"name":"Abdelrahim Alqudah","email":"","orcid":"","institution":"The Hashemite University","correspondingAuthor":false,"prefix":"","firstName":"Abdelrahim","middleName":"","lastName":"Alqudah","suffix":""}],"badges":[],"createdAt":"2024-06-15 11:53:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4586386/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4586386/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":59604999,"identity":"ebcd1370-5c5c-40d7-a3e7-cb99f9548077","added_by":"auto","created_at":"2024-07-03 18:27:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":28043,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCytidine, Uridine and Gabapentin’s Impact on Formalin-Induced Nociceptive Response.\u003c/strong\u003eMice were orally administered with \u003cstrong\u003eCytidine, Uridine and Gabapentin\u003c/strong\u003e (100 mg/kg from each) for a duration of 30 minutes prior to subcutaneous injection of formalin (5%, 10 μL) into the plantar region of the left hind paw. The combined duration of licking, biting, and shaking of the injected paw was assessed in two-time intervals: 0–5 minutes (first phase) (A) and 20–40 minutes (second phase) (B). The vertical bars on the graph represent the standard error of the mean. Each experimental group consisted of 6 animals (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, compared to the control group).\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-4586386/v1/816346af5d55ec38a3e08f7d.png"},{"id":59605130,"identity":"812a0062-f9e0-4e92-bc8b-9e2548944c2f","added_by":"auto","created_at":"2024-07-03 18:35:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":29270,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe Analgesic Effect of Cytidine, Uridine and Gabapentin in a Diabetic Neuropathy Model\u003c/strong\u003e. An animal model of diabetic neuropathy was generated by administering a single intraperitoneal injection of 0.1 mg/kg streptozotocin. The experiment was conducted five weeks post streptozotocin injection. The vertical lines on the graph represent the standard error of the mean. Each group consisted of 6 animals (*\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, compared to the intact group).\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-4586386/v1/4272546ba6a0f3eb4cb20cb6.png"},{"id":59605001,"identity":"92b47c25-5979-4fd9-bddb-0db8ea99d179","added_by":"auto","created_at":"2024-07-03 18:27:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":86235,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAlterations in phosphorylated \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCREB\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e protein levels within the spinal cord due to Cytidine, Uridine and Gabapentin treatment in pain models induced by formalin and diabetic neuropathy.\u003c/strong\u003e A. After the injection of formalin, proteins were extracted from dissected lumbar spinal cord tissue 30 minutes later for Western blot analysis. Each experimental group consisted of six animals. B. The experiment was conducted 5 weeks following streptozotocin injection to induce diabetes. Proteins were extracted from dissected lumbar spinal cord tissue 1 hour after oral Cytidine, Uridine and Gabapentin administration for Western blot analysis. An internal loading control, \u003cem\u003eβ-Actin\u003c/em\u003e (diluted 1:1000), was employed. Signal quantification was performed using laser scanning densitometry, and results were presented as a percentage relative to the control. Mean values ± SEM are displayed (###\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, compared to the Control group; *\u003cem\u003e P\u003c/em\u003e\u0026lt;0.05, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, compared to the formalin-treated group; ###\u003cem\u003eP\u0026lt;0.001\u003c/em\u003ecompared to the Normal group; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, **\u0026lt;0.001, compared to the Diabetic group).\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-4586386/v1/865873ef94093eb83c8bfd01.png"},{"id":59605000,"identity":"545712bb-0a67-4f54-8fd4-149167fcb302","added_by":"auto","created_at":"2024-07-03 18:27:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":32155,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpact of naloxone (A), \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eyohimbine (B)\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, or \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003emethysergide\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e (C) on the analgesic response triggered by Cytidine, Uridine and Gabapentin in the formalin test.\u003c/strong\u003e Following intraperitoneal (i.p.) pretreatment with \u003cem\u003enaloxone\u003c/em\u003e, \u003cem\u003eyohimbine\u003c/em\u003e, or \u003cem\u003emethysergide\u003c/em\u003e (at a dose of 5 mg/kg) for a duration of 10 minutes, mice were orally administered with Cytidine, Uridine and Gabapentin (at a dose of 100 mg/kg) for a period of 30 minutes before subcutaneous injection of formalin (5%, 10 μL) into the plantar region of the left hind-paw. Each experimental group consisted of six animals. The vertical bars on the graph represent the standard error of the mean. (*\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, \u0026lt;**\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01, when compared to the formalin-treated group).\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-4586386/v1/2c7a6c87067a17d2d0cfa5b7.png"},{"id":59605002,"identity":"3add9290-0045-4746-9337-7f42ed57e31d","added_by":"auto","created_at":"2024-07-03 18:27:56","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":104455,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpact of naloxone (A), \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eyohimbine (B)\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, or \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003emethysergide\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e (C) spinal \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ep-CREB\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eexpression in the formalin test.\u003c/strong\u003e Following a 10-minute intraperitoneal (i.p.) pretreatment with naloxone, \u003cem\u003eyohimbine\u003c/em\u003e, or \u003cem\u003emethysergide\u003c/em\u003e(5 mg/kg), mice were orally administered Cytidine, Uridine and Gabapentin (100 mg/kg) for 30 minutes prior to subcutaneous injection of formalin (5%, 10 μL) into the plantar region of the left hind-paw. Proteins were extracted from dissected lumbar spinal cord tissue 30 minutes after formalin injection for subsequent Western blot analysis. Each experimental group consisted of six animals. An internal loading control, \u003cem\u003eβ-Actin\u003c/em\u003e (diluted 1:1000), was utilized. The vertical bars on the graph represent the standard error of the mean. (*\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, compared to the formalin group).\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-4586386/v1/3fc03c5e8b1fb1998d8dfd3f.png"},{"id":64206698,"identity":"7c079c70-a1b5-4253-9b16-d58f2ac99ea5","added_by":"auto","created_at":"2024-09-10 05:33:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1106290,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4586386/v1/77addfdd-8ecb-4543-a5bb-367b5b412fd2.pdf"},{"id":59605004,"identity":"136c50f2-e4cd-486b-b630-ad17665e355d","added_by":"auto","created_at":"2024-07-03 18:27:56","extension":"pzfx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":179696,"visible":true,"origin":"","legend":"","description":"","filename":"AnalysisNew.pzfx","url":"https://assets-eu.researchsquare.com/files/rs-4586386/v1/83164db42aa2e4751f05356a.pzfx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Exploring the Analgesic Potential of Cytidine, Uridine, and Gabapentin either alone or in combination: Insights from Formalin-Induced Pain and Diabetic Neuropathy Models","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDiabetic neuropathy is a common neurological condition that often arises as a result of diabetes mellitus \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. It affects more than half of people with a history of diabetes for more than 25 years \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. At least 20% of diabetic patients have neuropathic problems that might include pain, sensory irritation, foot ulcers, or weakness, resulting in a significant clinical burden in any diabetes services \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe cAMP element-binding protein (CREB) is a transcription factor that affects critical neurological processes \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. CREB promotes inter-synaptic nociceptive interactions in the cell by efficiently modulating gene transcription, leading to a quick to long-lasting transition in nociceptive signaling \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. A significant rise in the transcriptional rate of phosphorylated CREB (p-CREB), which migrates into the nucleus and induces the transcription of targeted genes containing a cyclic adenosine monophosphate response element, such as brain-derived neurotrophic factor, can contribute to pain development \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Analgesics such as nonsteroidal anti-inflammatory medications (NSAID) and opioids are commonly used to treat acute pain, but they are ineffective for neuropathic pain \u003csup\u003e\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Regardless of their lack of effectiveness, traditional analgesics are still widely used among individuals with neuropathic pain, raising questions regarding opiate dependency \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Previous studies have shown the effect of using NSAIDs in patients with type 2 diabetes on the heart, which can increase the risk of heart failure \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Currently, anti-depressant including amitriptyline, mirtazapine, duloxetine, and fluoxetine, which are known for their anti-nociception effect, are also used to treat neuropathic pain \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. These therapies are associated with significant adverse impacts that decrease patient compliance, such as dry mouth, dizziness, nausea, headache, and constipation \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Therefore, finding an effective treatment for neuropathic pain with minimal side effects is essential.\u003c/p\u003e \u003cp\u003eGabapentin (1-[aminomethyl]-cyclohexaneacetic acid) is an anticonvulsant that received approval in the United States in 1994 for usage among adult patients with partial seizures \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. It has been indicated by personal experience and in off-label clinical trials to be efficient in the management of pain syndromes, including painful diabetic neuropathy \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. A previous study has shown that gabapentin exhibited analgesic efficacy in the chronic constriction injury model of neuropathic pain in rats \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. In parallel, uridine and cytidine belong to the pyrimidine nucleotide family, one of four major molecular biology groups (together with carbohydrates, lipids, and proteins) that have crucial functions in cellular metabolism \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Besides being components of DNA and engaging in genome transcription and translation, these nucleotides provide energy transfer for chemical bonds, intracellular communication, neurotransmission, and the transfer of biochemical radicals from intermediary metabolism \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Pharmacological research indicates that these drugs improve neuromuscular recovery in animals with sciatic nerve injuries and reduce the transmission of pain in the spinal cord \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. New evidence demonstrates that nucleotide receptor agonists can effectively alleviate neuropathic pain \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Moreover, In vitro studies showed they may enhance neural fiber regeneration \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCurrently, analgesics often fall short in efficacy or are associated with significant side effects, highlighting the necessity for alternative or adjunctive therapies. Cytidine and uridine, as nucleosides involved in cellular processes and neuroprotection, along with gabapentin, a well-established anticonvulsant with analgesic properties, present promising candidates for pain relief. This study seeks to explore the effects of this combination in relieving pain disorders using formalin-induced pain and diabetic neuropathy mice model.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eEffect of uridine, cytidine, and gabapentin alone or in combination on pain response in the formalin test\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe intraplantar injection of 5% formalin-induced acute nociceptive reactions lasted roughly 5 minutes, known as the first phase reaction. This first phase was followed by a persistent inflammatory response for about 20 to 40 minutes following formalin injection, known as the secondary phase reaction. In the formalin test, the control group, which was given a vehicle, showed an immediate nociceptive response in both phases. In contrast, the treated group, including cytidine, uridine, gabapentin, cytidine\u0026thinsp;+\u0026thinsp;uridine\u0026thinsp;+\u0026thinsp;gabapentin groups showed a significant decrease in the nociception reactions in both phases compared to the control group (*\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; ***\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, respectively), as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eImpact of cytidine, uridine, and gabapentin alone or in combination on pain behavior in a model of diabetic-induced neuropathy\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFollowing five weeks of a single intraperitoneal injection of streptozotocin, the mechanical stimulation threshold declined, as demonstrated by the von Frey filament test. 100 mg/kg of cytidine, uridine, and gabapentin alone or in combination were given orally 30 minutes before the mechanical pain evaluation. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the combined treatment causes a reversal drop in the pain threshold compared to intact after 30, 60, and 120 minutes (*\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In addition, there was a significant reversal drop in the pain threshold in the combined treatment compared to the gabapentin group after 120 minutes (*\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of cytidine, uridine, and gabapentin alone or in combination in p-CREB Protein in the Spinal Cord in Models of Formalin-Induced Pain and Diabetic-Induced Neuropathy\u003c/b\u003e \u003c/p\u003e \u003cp\u003eProtein extractions from dissected lumbar spinal cords were collected 30 minutes after formalin injection to assess possible changes in the expression of phosphorylated CREB protein in the spinal cord. As seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, formalin administration caused an increase in p-CREB expression through the spinal cord. Oral gabapentin or cytidine\u0026thinsp;+\u0026thinsp;uridine\u0026thinsp;+\u0026thinsp;gabapentin treatment administration reduced the increased levels of p-CREB caused by formalin injection. We continued our study by exploring alterations in p-CREB expression in the spinal cord of streptozotocin-induced neuropathy mice. Diabetic mice showed a considerable increase in CREB phosphorylation compared to normal mice. However, this increase was inhibited by administering gabapentin or cytidine\u0026thinsp;+\u0026thinsp;uridine\u0026thinsp;+\u0026thinsp;gabapentin treatment. The inhibition of p-CREB production by cytidine\u0026thinsp;+\u0026thinsp;uridine\u0026thinsp;+\u0026thinsp;gabapentin treatment was greater than when gabapentin was given alone, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEffect of naloxone, yohimbine, or methysergide on pain response in the formalin test\u003c/h2\u003e \u003cp\u003eThis experiment evaluated the role of opioid, serotonergic, and adrenergic systems in the antinociception generated by cytidine, uridine, and gabapentin, either alone or in combination. As demonstrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, giving naloxone (5 mg/kg, i.p.), which inhibits opioid receptors, counteract the antinociceptive effect caused by cytidine, uridine, and cytidine\u0026thinsp;+\u0026thinsp;uridine\u0026thinsp;+\u0026thinsp;gabapentin groups, while pretreatment with yohimbine (5 mg/kg, i.p.), which inhibits α2-adrenergic receptors, counteract the antinociceptive effect caused by uridine, gabapentin, and cytidine\u0026thinsp;+\u0026thinsp;uridine\u0026thinsp;+\u0026thinsp;gabapentin groups, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB. However, pretreatment of methysergide (5 mg/kg, i.p.), which inhibits serotonergic receptors, counteracts the antinociceptive effect generated by gabapentin and cytidine\u0026thinsp;+\u0026thinsp;uridine\u0026thinsp;+\u0026thinsp;gabapentin groups, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC. Notably, administering naloxone, methysergide, or yohimbine alone did not affect the pain response.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eImpact of naloxone, yohimbine, or methysergide on cytidine, uridine, and gabapentin alone or in combination in Modulating of Spinal p-CREB Expression in the Formalin Test\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSince the administration of naloxone reverses the antinociceptive effect induced by cytidine, uridine, and cytidine\u0026thinsp;+\u0026thinsp;uridine\u0026thinsp;+\u0026thinsp;gabapentin groups, pretreatment with yohimbine counteracts the antinociceptive effect caused by uridine, gabapentin, and cytidine\u0026thinsp;+\u0026thinsp;uridine\u0026thinsp;+\u0026thinsp;gabapentin groups, and methysergide counteracts the antinociceptive effect generated by gabapentin, and cytidine\u0026thinsp;+\u0026thinsp;uridine\u0026thinsp;+\u0026thinsp;gabapentin groups in the formalin test, Our study also aimed to determine if naloxone, yohimbine, and methysergide can reverse the drop in spinal p-CREB expression produced by previous treatments. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, pre-administration of naloxone (5 mg/kg, i.p.) reversed the reduction in p-CREB expression in cytidine, uridine, and cytidine\u0026thinsp;+\u0026thinsp;uridine\u0026thinsp;+\u0026thinsp;gabapentin groups. In comparison, pre-administration of yohimbine (5 mg/kg, i.p.) reversed the reduction in p-CREB expression in uridine, gabapentin, and cytidine\u0026thinsp;+\u0026thinsp;uridine\u0026thinsp;+\u0026thinsp;gabapentin groups, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB. Moreover, methysergide pretreatment reversed the reduction in p-CREB expression in gabapentin and cytidine\u0026thinsp;+\u0026thinsp;uridine\u0026thinsp;+\u0026thinsp;gabapentin groups, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study explores for the first time the effect of cytidine, uridine, and gabapentin either alone or in combination in a formalin-induced pain model. Oral administration of cytidine\u0026thinsp;+\u0026thinsp;uridine\u0026thinsp;+\u0026thinsp;gabapentin combination significantly decreased pain behavior more than cytidine, uridine, and gabapentin alone throughout the formalin test's first and second phases. It has been widely recognized that intraplantar injection leads to a biphasic response \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. The pain behavior seen throughout the first phase is primarily caused by the immediate stimulation of sensory neurons, while the pain behavior seen through the second phase includes both inflammatory mechanisms and central sensation inside the dorsal horn. These findings imply that pain behaviors observed during the first and second phases are significantly controlled \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Our key findings showed that the combination of cytidine, uridine, and gabapentin has a potential synergistic effect in reducing pain behavior caused by formalin injection into the plantar of mice's hid paws.\u003c/p\u003e \u003cp\u003eThis synergistic effect suggests that the combined therapeutic approach may enhance analgesic efficacy beyond what is achievable with each compound alone. The observed reduction in pain behavior indicates that these compounds might interact at multiple levels of pain modulation pathways, offering a multiple approaches to pain management \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Cytidine and uridine, known for their roles in neuroprotection and cellular repair \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. They are involved in the synthesis of phospholipids and nucleotides, which are essential for maintaining neuronal integrity and function. By promoting neuronal health and repair, cytidine and uridine may reduce neuronal hyperexcitability and inflammation associated with pain. Gabapentin, on the other hand, primarily works by inhibiting voltage-gated calcium channels in the central nervous system, which reduces the release of excitatory neurotransmitters such as glutamate \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. This inhibition leads to decreased neuronal excitability and reduced pain signaling. Therefore, combing cytidine and uridine may enhance the neuroprotective environment, reducing inflammation and supporting neuronal recovery, while gabapentin modulates pain transmission by inhibiting excitatory neurotransmitter release. This dual action likely results in a more effective reduction of pain behavior compared to the individual effects of each compound.\u003c/p\u003e \u003cp\u003eFurthermore, The CREB protein is strongly linked to pain conduction, with its transcription in the spinal cord or dorsal root ganglia increasing in various chronic pain models, including neuropathic pain and neuropathy \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. In acute and chronic inflammation models, such as the formalin pain model, p-CREB transcription in the spinal cord or brain areas is also elevated \u003csup\u003e\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. In our study, we observed that formalin-induced pain significantly increased p-CREB transcription in the spinal cord. Treatment with cytidine, uridine, and gabapentin, both alone and in combination, reduced this up-regulation, with the combination treatment showing the greatest reduction in p-CREB levels. Additionally, in the diabetic neuropathy model, gabapentin alone and the combination treatment also reduced the up-regulation of spinal p-CREB, with the combination treatment being more effective. These findings suggest that gabapentin, particularly when combined with cytidine and uridine, reduces nociception by lowering p-CREB levels in the spinal cord in both the formalin-induced pain and diabetic neuropathy models. The involvement of opioid, serotonergic, and adrenergic receptors in modulating nociceptive processes further supports this mechanism. Opioid receptors are known to reduce nociception \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, and blocking spinal serotonergic and noradrenergic receptors can counteract the anti-nociceptive effects of supraspinal morphine administration \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. In our study, the anti-nociceptive effect of the orally administered combination treatment of cytidine, uridine, and gabapentin was linked to the activation of opioid, α-2 adrenergic, and serotonergic receptors \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. That also may contribute to the observed activity.\u003c/p\u003e \u003cp\u003eFinally, this combination therapy could potentially address the limitations of current pain treatments, providing a more comprehensive analgesic strategy. Further research is warranted to explore the underlying mechanisms of this synergy and to assess the clinical relevance and potential applications in human pain management, especially for chronic and neuropathic pain conditions. These findings pave the way for novel pain management protocols that leverage the benefits of multi-compound therapies.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study highlights the significant analgesic potential of combining cytidine, uridine, and gabapentin in reducing pain behavior in both formalin-induced pain and diabetic neuropathy models. The combination therapy not only demonstrated a synergistic effect in alleviating pain but also effectively reduced the up-regulation of p-CREB expression in the spinal cord, a key player in pain conduction. These findings pave the way for developing more effective and comprehensive pain management strategies, offering hope for better therapeutic outcomes in chronic and neuropathic pain conditions. Further research is warranted to fully elucidate the mechanisms and clinical applicability of these promising results.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eExperimental animals\u003c/h2\u003e \u003cp\u003eThirty adult Swiss albino mice weighing 24 to 28 g were housed at the Hashemite University's Animal House Center. All the experiments were approved by the institutional review board at the Hashemite University, Zarqa, Jordan (IRB #: 8/5/2019/2020). The mice were maintained under controlled temperature (23 ± 2°C) and humidity (50% ± 5%) conditions and kept on a 12 h light/dark cycle (lights on at 07:00 AM). They had free access to food and water throughout the experiment. Before the experiment started, mice had free access to the apparatus for 30 minutes for two days to habituate them to the testing apparatus and to minimize mice stress. Throughout the behavioral testing, a video camera was placed in front of the apparatus using a tripod. Each behavioral test was recorded for future reference. Two observers, blinded to the experiment conditions, evaluated the antinociception and mechanical allodynia parameters to prevent any potential bias. The mice were divided randomly into five groups: Control group (\u003cb\u003eControl\u003c/b\u003e, n = 6), Cytidine group (\u003cb\u003eCyt\u003c/b\u003e, n = 6), Uridine group (\u003cb\u003eUri\u003c/b\u003e, n = 6), Gabapentin group (\u003cb\u003eGaba\u003c/b\u003e, n = 6), and Cytidine + Uridine + Gabapentin group (\u003cb\u003eCty + Uri + Gaba\u003c/b\u003e, n = 6).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDrugs\u003c/h3\u003e\n\u003cp\u003eStreptozotocin, Naloxone, Yohimbine, and Methysergide were obtained from Sigma-Aldrich (St. Louis, MO, USA), while Citicoline (250 mg Cytidine − 5’-diphosphocholine, Cognizin®, Kyowa Hakko Bio Co., Ltd.), and Uridine (300 mg 5’-monophosphate disodium salt, Amazing Nutrition®, USA) were obtained from local pharmacy. Cytidine and uridine were dissolved in distilled water associated with gentel heating. However, gababentine was dissolved in water with PH adjusted to 7.2.\u003c/p\u003e \u003cp\u003e \u003cb\u003eProduction of streptozotocin-induced diabetic neuropathy model.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eDiabetic neuropathy was induced through the administration of a single dose of \u003cem\u003estreptozotocin\u003c/em\u003e (0.1 mg/kg) intraperitoneally (i.p.). One week later, diabetes was confirmed. The experiment took place five weeks after diabetes induction.\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003ePretreatment of antagonists\u003c/h2\u003e \u003cp\u003eMice were initially treated intraperitoneally with saline, naloxone (5 mg/kg), methysergide (5 mg/kg), or yohimbine (5 mg/kg). This pretreatment was performed 10 minutes before the oral administration of the control vehicle, uridine, cytidine, gabapentin, or uridine + cytidine + gabapentine treatment (100 mg/kg from each). The observed formalin-induced nociceptive behavior was next monitored and analyzed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eVon-Frey test\u003c/h2\u003e \u003cp\u003eAntinociception and mechanical allodynia were assessed using calibrated von-Frey filaments as described in \u003cem\u003eBonin et al. 2014\u003c/em\u003e study \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Mechanosensitivity assessment is an essential tool for investigating pain in animal models. This is frequently performed using von Frey filaments (North Coast Medical, Inc., Gilroy, CA, USA) in an up-down testing method. After adapting the mice to their surroundings for 30 minutes, they were transported to separate Plexiglas chambers on top of a mesh of wires to stimulate the plantar surface of their rear paws. The paw withdrawal threshold was measured by increasing and decreasing the strength of the stimulus and computed using Dixon's up-down method \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eFormalin test\u003c/h2\u003e \u003cp\u003eThe formalin test in mice is a realistic and reliable nociception model that detects various analgesic medications \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. The test involves a chemical nociceptive stimulation that causes a spontaneous pain sensation. According to Hunskaar et al.'s 1985 formalin test protocol \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e, the formalin test was conducted on mice that were independently subjected to a macrolone cage (30 cm x 12 cm x 13 cm) for 2 h. Using a micro-syringe with a 26-gauge needle, ten µl of 1% formalin in 0.9% normal saline was subcutaneously injected into the mice's dorsal hind paw with little restrictions. The mice were returned to the cage, and the duration of the animals licking the injected paw was precisely measured using a stopwatch to indicate nociception. The first phase of the test was defined as the time between zero and five minutes following the formalin injection, whereas the late or second phase lasted from twenty to forty minutes. These periods corresponded to the direct influence on nociceptors and the inflammatory nociceptive responses, as clarified by Hunskaar and Hole 1987 \u003csup\u003e47\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eProtein extraction and western blot\u003c/h2\u003e \u003cp\u003eThe lumbar part of the mice's spinal cord was extracted. The tissue was washed twice with cold Tris-buffered saline (pH 7.5) containing 20 mmol/L Trizma base, and 137 mmol/L NaCl. During the washing, the tissue was lysed with sodium dodecyl sulfate lysis buffer consisting of 62.5 mmol/L Trizma base, 2% w/v sodium dodecyl sulfate, 10% glycerol, 0.1 mmol/L Na3VO4, 3 mg/mL aprotinin, and 20 mmol/L NaF. Following a brief sonication period, the protein concentration was determined using a detergent-compatible protein assay reagent (Bio-Rad Laboratories, Hercules, CA, USA). Equal quantities of extracted proteins have been blended with 5X Laemmle loading dye and separated on polyacrylamide gels (SDS-PAGE) ranging from 6–10%. Proteins were then transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). Membranes were blocked with 3% nonfat dry milk in TBST (10 mM Trizma base, pH 8.0, 150 mM NaCl, and 20% Tween 20) for 30 minutes at 4°C and rinsed with TBST. Membranes were incubated overnight at 4°C with one of the following primary antibodies: p-CREB (Abcam, 1:1000) and β-actin (Cell Signaling Technology, 1:1000). Mouse Anti-β-Tubulin antibody was used as a loading control (1:5000). After incubating overnight, the primary antibody was eliminated. The membrane was washed four times in TBST for 5 minutes each before adding the secondary antibody (anti-rabbit IgG-horseradish peroxidase conjugate (1:4000)) for 1 hour. After 1 hour, the secondary antibody was removed and the membrane was incubated with ECL-plus solution (Millipore, Billerica, MA, USA) for imaging using a Luminescent Image Analyzer (LAS-4000, Fuji Film Co., Japan). A Multi-Gauge Version 3.1 (Fuji Film, Japan) system was used to quantify the detected bands and expressed them as a percentage relative to the control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll data is presented as mean ± SEM. One-way ANOVA followed by Tukey's post-hoc test was used in the statistical analysis, using GraphPad Prism 8.0.1 software (GraphPad Software Inc, CA, USA). P \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData available within the article and its supplementary materials.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study is reported in accordance with the ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments). All animal experiments were conducted following the ethical standards and guidelines set forth by our institution and approved by the Animal Research Ethics Committee at Hashemite University (IRB number: HU 8/5/2019/2020), and all procedures were performed in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, A.A., and M.B.; methodology, E.Q.; software, R.Y.A.; validation\u0026nbsp;E.Q. and A.A.; formal analysis, A.A.; investigation, M.A.A.; resources, M.B.; data curation, L.A.; writing\u0026mdash;original draft preparation, M.B, D.K.CH and L.A.; writing\u0026mdash;review and editing, A.A. and M.B, D.K.CH; visualization, A.A.; supervision, E.Q.; project administration, A.A.; funding acquisition, A.A. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the Deanship of Scientific Research at the Hashemite University, Zarqa, Jordan (Grant number: 49/2020).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo potential conflict of interest relevant to this article was reported.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGoldberg, H.\u003cem\u003e et al.\u003c/em\u003e A double-blind, randomized, comparative study of the use of a combination of uridine triphosphate trisodium, cytidine monophosphate disodium, and hydroxocobalamin, versus isolated treatment with hydroxocobalamin, in patients presenting with compressive neuralgias. \u003cem\u003eJournal of pain research\u003c/em\u003e, 397-404 (2017).\u003c/li\u003e\n\u003cli\u003eRussell, J. W. \u0026amp; Zilliox, L. A. Diabetic neuropathies. \u003cem\u003eContinuum: lifelong learning in neurology\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, 1226-1240 (2014).\u003c/li\u003e\n\u003cli\u003ePirart, J. Diabetes mellitus and its degenerative complications: a prospective study of 4,400 patients observed between 1947 and 1973. \u003cem\u003eDiabetes care\u003c/em\u003e \u003cstrong\u003e1\u003c/strong\u003e, 252-263 (1978).\u003c/li\u003e\n\u003cli\u003eWard, J. The cost of diabetic neuropathy. \u003cem\u003ePharmacoeconomics\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 52-54 (1995).\u003c/li\u003e\n\u003cli\u003eSheng, M. \u0026amp; Greenberg, M. E. The regulation and function of c-fos and other immediate early genes in the nervous system. \u003cem\u003eNeuron\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 477-485 (1990).\u003c/li\u003e\n\u003cli\u003eCarlezon Jr, W. A.\u003cem\u003e et al.\u003c/em\u003e Regulation of cocaine reward by CREB. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e282\u003c/strong\u003e, 2272-2275 (1998).\u003c/li\u003e\n\u003cli\u003eCarrano, N.\u003cem\u003e et al.\u003c/em\u003e The synaptonuclear messenger RNF10 acts as an architect of neuronal morphology. \u003cem\u003eMolecular Neurobiology\u003c/em\u003e \u003cstrong\u003e56\u003c/strong\u003e, 7583-7593 (2019).\u003c/li\u003e\n\u003cli\u003eDi Benedetto, B. \u003cem\u003eThe role of the ERK/MAPK pathway in the adult mouse brain for fear potentiated startle learning: a model to study fear-based anxiety disorders\u003c/em\u003e, Technische Universit\u0026auml;t M\u0026uuml;nchen, (2008).\u003c/li\u003e\n\u003cli\u003eNavabpour, S., Rezayof, A. \u0026amp; Ghasemzadeh, Z. Activation of VTA/CeA/mPFC cannabinoid CB1 receptors induced conditioned drug effects via interacting with hippocampal CAMKII-CREB-BDNF signaling pathway in rats. \u003cem\u003eEuropean journal of pharmacology\u003c/em\u003e \u003cstrong\u003e909\u003c/strong\u003e, 174417 (2021).\u003c/li\u003e\n\u003cli\u003eWen, J.\u003cem\u003e et al.\u003c/em\u003e The cAMP response element-binding protein/brain-derived neurotrophic factor pathway in anterior cingulate cortex regulates neuropathic pain and anxiodepression like behaviors in rats. \u003cem\u003eFrontiers in Molecular Neuroscience\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 831151 (2022).\u003c/li\u003e\n\u003cli\u003eArner, S. \u0026amp; Meyerson, B. Lack of analgesic effect of opioids on neuropathic and idiopathic forms of pain. \u003cem\u003ePain\u003c/em\u003e \u003cstrong\u003e33\u003c/strong\u003e, 11-23 (1988).\u003c/li\u003e\n\u003cli\u003eGilron, I. \u0026amp; Coderre, T. J. Emerging drugs in neuropathic pain. \u003cem\u003eExpert opinion on emerging drugs\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 113-126 (2007).\u003c/li\u003e\n\u003cli\u003eMax, M. B., Schafer, S. C., Culnane, M., Dubner, R. \u0026amp; Gracely, R. H. Association of pain relief with drug side effects in postherpetic neuralgia: a single‐dose study of clonidine, codeine, ibuprofen, and placebo. \u003cem\u003eClinical Pharmacology \u0026amp; Therapeutics\u003c/em\u003e \u003cstrong\u003e43\u003c/strong\u003e, 363-371 (1988).\u003c/li\u003e\n\u003cli\u003eBerger, A., Dukes, E. M. \u0026amp; Oster, G. Clinical characteristics and economic costs of patients with painful neuropathic disorders. \u003cem\u003eThe journal of pain\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 143-149 (2004).\u003c/li\u003e\n\u003cli\u003eVo, T., Rice, A. S. \u0026amp; Dworkin, R. H. Non-steroidal anti-inflammatory drugs for neuropathic pain: how do we explain continued widespread use? \u003cem\u003ePAIN\u0026reg;\u003c/em\u003e \u003cstrong\u003e143\u003c/strong\u003e, 169-171 (2009).\u003c/li\u003e\n\u003cli\u003eBhala, N.\u003cem\u003e et al.\u003c/em\u003e Vascular and upper gastrointestinal effects of non-steroidal anti-inflammatory drugs: meta-analyses of individual participant data from randomised trials. \u003cem\u003eLancet (London, England)\u003c/em\u003e \u003cstrong\u003e382\u003c/strong\u003e, 769-779 (2013).\u003c/li\u003e\n\u003cli\u003eHolt, A.\u003cem\u003e et al.\u003c/em\u003e Heart failure following anti-inflammatory medications in patients with type 2 diabetes mellitus. \u003cem\u003eJournal of the American College of Cardiology\u003c/em\u003e \u003cstrong\u003e81\u003c/strong\u003e, 1459-1470 (2023).\u003c/li\u003e\n\u003cli\u003eGilron, I., Watson, C. P. N., Cahill, C. M. \u0026amp; Moulin, D. E. Neuropathic pain: a practical guide for the clinician. \u003cem\u003eCmaj\u003c/em\u003e \u003cstrong\u003e175\u003c/strong\u003e, 265-275 (2006).\u003c/li\u003e\n\u003cli\u003eMcCleane, G. Pharmacological strategies in relieving neuropathic pain. \u003cem\u003eExpert Opinion on Pharmacotherapy\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 1299-1312 (2004).\u003c/li\u003e\n\u003cli\u003eMoulin, D.\u003cem\u003e et al.\u003c/em\u003e Pharmacological management of chronic neuropathic pain\u0026ndash;consensus statement and guidelines from the Canadian Pain Society. \u003cem\u003ePain Research and Management\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 13-21 (2007).\u003c/li\u003e\n\u003cli\u003eRiediger, C.\u003cem\u003e et al.\u003c/em\u003e Adverse effects of antidepressants for chronic pain: a systematic review and meta-analysis. \u003cem\u003eFrontiers in neurology\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 307 (2017).\u003c/li\u003e\n\u003cli\u003eMorris, G. L. Gabapentin. \u003cem\u003eEpilepsia\u003c/em\u003e \u003cstrong\u003e40\u003c/strong\u003e, s63-s70 (1999).\u003c/li\u003e\n\u003cli\u003eRosner, H., Rubin, L. \u0026amp; Kestenbaum, A. Gabapentin adjunctive therapy in neuropathic pain states. \u003cem\u003eThe Clinical journal of pain\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 56-58 (1996).\u003c/li\u003e\n\u003cli\u003eStacey, B., Tipton, K., Owen, G., Sinclair, J. \u0026amp; Glick, R. Gabapentin and neuropathic pain states: a case series report. \u003cem\u003eRegional Anesthesia and Pain Medicine\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 65-65 (1996).\u003c/li\u003e\n\u003cli\u003eBennett, G. J. \u0026amp; Xie, Y.-K. A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man. \u003cem\u003ePain\u003c/em\u003e \u003cstrong\u003e33\u003c/strong\u003e, 87-107 (1988).\u003c/li\u003e\n\u003cli\u003eVincenzetti, S., Polzonetti, V., Micozzi, D. \u0026amp; Pucciarelli, S. Enzymology of pyrimidine metabolism and neurodegeneration. \u003cem\u003eCurrent medicinal chemistry\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 1408-1431 (2016).\u003c/li\u003e\n\u003cli\u003eVoet, D., Voet, J. G. \u0026amp; Pratt, C. W. \u003cem\u003eFundamentals of biochemistry: life at the molecular level\u003c/em\u003e. (John Wiley \u0026amp; Sons, 2013).\u003c/li\u003e\n\u003cli\u003eWattig, B., Schalow, G., Heydenreich, F., Warzok, R. \u0026amp; Cerv\u0026oacute;s-Navarro, J. Enhancement of nerve fibre regeneration by nucleotides after peripheral nerve crush damage. Electrophysiologic and morphometric investigations. \u003cem\u003eArzneimittel-forschung\u003c/em\u003e \u003cstrong\u003e42\u003c/strong\u003e, 1075-1078 (1992).\u003c/li\u003e\n\u003cli\u003eWattig, B.\u003cem\u003e et al.\u003c/em\u003e Acceleration of nerve and muscle regeneration by administration of nucleotides--electroneurophysiological and morphometrical investigations. \u003cem\u003eActa histochemica. Supplementband\u003c/em\u003e \u003cstrong\u003e42\u003c/strong\u003e, 333-339 (1992).\u003c/li\u003e\n\u003cli\u003eAndo, R., Mehesz, B., Gyires, K., Illes, P. \u0026amp; Sperlagh, B. A comparative analysis of the activity of ligands acting at P2X and P2Y receptor subtypes in models of neuropathic, acute and inflammatory pain. \u003cem\u003eBritish journal of pharmacology\u003c/em\u003e \u003cstrong\u003e159\u003c/strong\u003e, 1106-1117 (2010).\u003c/li\u003e\n\u003cli\u003eVerdugo, R. J., Cea, J. G., Castillo, J. L., Stuardo, A. \u0026amp; Contreras, C. PS-51-7 Cytidine monophosphate (CMP), uridine triphosphate (UTP) and hydroxycobalamin in the treatment of diabetic polyneuropathy: preliminary study. \u003cem\u003eElectroencephalography and Clinical Neurophysiology/Electromyography and Motor Control\u003c/em\u003e \u003cstrong\u003e97\u003c/strong\u003e, S219-S220 (1995).\u003c/li\u003e\n\u003cli\u003eBonin, R. P., Bories, C. \u0026amp; De Koninck, Y. A simplified up-down method (SUDO) for measuring mechanical nociception in rodents using von Frey filaments. \u003cem\u003eMolecular pain\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 1744-8069-1710-1726 (2014).\u003c/li\u003e\n\u003cli\u003eTj\u0026oslash;lsen, A., Berge, O.-G., Hunskaar, S., Rosland, J. H. \u0026amp; Hole, K. The formalin test: an evaluation of the method. \u003cem\u003ePain\u003c/em\u003e \u003cstrong\u003e51\u003c/strong\u003e, 5-17 (1992).\u003c/li\u003e\n\u003cli\u003eMiyabe, T. \u0026amp; Miletic, V. Multiple kinase pathways mediate the early sciatic ligation-associated activation of CREB in the rat spinal dorsal horn. \u003cem\u003eNeuroscience letters\u003c/em\u003e \u003cstrong\u003e381\u003c/strong\u003e, 80-85 (2005).\u003c/li\u003e\n\u003cli\u003eSONG, X. s.\u003cem\u003e et al.\u003c/em\u003e Activation of ERK/CREB pathway in spinal cord contributes to chronic constrictive injury‐induced neuropathic pain in rats 1. \u003cem\u003eActa Pharmacologica Sinica\u003c/em\u003e \u003cstrong\u003e26\u003c/strong\u003e, 789-798 (2005).\u003c/li\u003e\n\u003cli\u003eCansev, M. Uridine and cytidine in the brain: their transport and utilization. \u003cem\u003eBrain research reviews\u003c/em\u003e \u003cstrong\u003e52\u003c/strong\u003e, 389-397 (2006).\u003c/li\u003e\n\u003cli\u003ePiermartiri, T. C. \u0026amp; Tasca, C. I. in \u003cem\u003eNatural Molecules in Neuroprotection and Neurotoxicity\u003c/em\u003e 661-676 (Elsevier, 2024).\u003c/li\u003e\n\u003cli\u003eCheng, J.-K. \u0026amp; Chiou, L.-C. Mechanisms of the antinociceptive action of gabapentin. \u003cem\u003eJournal of pharmacological sciences\u003c/em\u003e \u003cstrong\u003e100\u003c/strong\u003e, 471-486 (2006).\u003c/li\u003e\n\u003cli\u003eMcGivern, J. G. \u0026amp; McDonough, S. I. Voltage-gated calcium channels as targets for the treatment of chronic pain. \u003cem\u003eCurrent Drug Targets-CNS Neurological Disorders\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, 457-478 (2004).\u003c/li\u003e\n\u003cli\u003eHermanson, O. \u0026amp; Blomqvist, A. Differential expression of the AP-1/CRE-binding proteins FOS and CREB in preproenkephalin mRNA-expressing neurons of the rat parabrachial nucleus after nociceptive stimulation. \u003cem\u003eMolecular brain research\u003c/em\u003e \u003cstrong\u003e51\u003c/strong\u003e, 188-196 (1997).\u003c/li\u003e\n\u003cli\u003eSeo, Y.-J.\u003cem\u003e et al.\u003c/em\u003e Differential expression of phosphorylated Ca2+/calmodulin-dependent protein kinase II and phosphorylated extracellular signal-regulated protein in the mouse hippocampus induced by various nociceptive stimuli. \u003cem\u003eNeuroscience\u003c/em\u003e \u003cstrong\u003e156\u003c/strong\u003e, 436-449 (2008).\u003c/li\u003e\n\u003cli\u003eMao, Q.\u003cem\u003e et al.\u003c/em\u003e Antinociceptive effects of analgesic-antitumor peptide (AGAP), a neurotoxin from the scorpion Buthus martensii Karsch, on formalin-induced inflammatory pain through a mitogen-activated protein kinases\u0026ndash;dependent mechanism in mice. \u003cem\u003ePLoS One\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, e78239 (2013).\u003c/li\u003e\n\u003cli\u003eYaksh, T. L.\u003cem\u003e et al.\u003c/em\u003e An automated flinch detecting system for use in the formalin nociceptive bioassay. \u003cem\u003eJournal of applied physiology\u003c/em\u003e \u003cstrong\u003e90\u003c/strong\u003e, 2386-2402 (2001).\u003c/li\u003e\n\u003cli\u003eSpinella, M. \u003cem\u003eThe role of medullary cholinergic and excitatory amino acid receptors mediating mesencephalic opioid antinociception\u003c/em\u003e. (City University of New York, 1999).\u003c/li\u003e\n\u003cli\u003eDirksen, R. \u003cem\u003eThe clinical relevance of endorphin receptors: the antinociceptive effectiveness of epidurally or intrathecally injected endorphinomimetics\u003c/em\u003e, [Sl: sn], (1983).\u003c/li\u003e\n\u003cli\u003eChaplan, S. R., Bach, F. W., Pogrel, J., Chung, J. \u0026amp; Yaksh, T. Quantitative assessment of tactile allodynia in the rat paw. \u003cem\u003eJournal of neuroscience methods\u003c/em\u003e \u003cstrong\u003e53\u003c/strong\u003e, 55-63 (1994).\u003c/li\u003e\n\u003cli\u003eHunskaar, S. \u0026amp; Hole, K. The formalin test in mice: dissociation between inflammatory and non-inflammatory pain. \u003cem\u003ePain\u003c/em\u003e \u003cstrong\u003e30\u003c/strong\u003e, 103-114 (1987).\u003c/li\u003e\n\u003cli\u003eHunskaar, S., Fasmer, O. B. \u0026amp; Hole, K. Formalin test in mice, a useful technique for evaluating mild analgesics. \u003cem\u003eJournal of neuroscience methods\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 69-76 (1985).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Diabetic neuropathy, Uridine, Cytidine, Gabapentin, p-CREB","lastPublishedDoi":"10.21203/rs.3.rs-4586386/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4586386/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDiabetic neuropathic pain is a usual consequence of diabetes. In this study, the formalin-induced pain and the diabetic neuropathy models were used in mice to investigate if the cytidine, uridine, and gabapentin treatments either alone or in combination can reduce nephropathic pain or not. To achieve our goals, cytidine, uridine, and gabapentin, either alone or in combination were orally administered to mice at a dose of 100 mg/kg. The formalin test was used to examine pain-related behaviors throughout both the primary and secondary phases. Further, the potential pain-relieving efficacy of these therapies was assessed in a diabetic neuropathy model produced by streptozotocin injection. Oral administration of the combination (cytidine\u0026thinsp;+\u0026thinsp;uridine\u0026thinsp;+\u0026thinsp;gabapentin) reduces formalin-induced pain-associated behavior in the first and second phases more than in each treatment alone. In the diabetic neuropathy model, administering the cytidine\u0026thinsp;+\u0026thinsp;uridine\u0026thinsp;+\u0026thinsp;gabapentin combination significantly reversed the pain threshold detected. The combination of cytidine, uridine, and gabapentin decreased as well as the elevated spinal p-CREB levels caused by formalin, which was reversed by pre-treatment with naloxone, yohimbine, and methysergide. This study reveals that the cytidine, uridine, and gabapentin combination have strong synergistic pain-relieving properties in both formalin-induced pain and diabetic neuropathy models more than each treatment alone.\u003c/p\u003e","manuscriptTitle":"Exploring the Analgesic Potential of Cytidine, Uridine, and Gabapentin either alone or in combination: Insights from Formalin-Induced Pain and Diabetic Neuropathy Models","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-03 18:27:51","doi":"10.21203/rs.3.rs-4586386/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d51d0668-2f48-4771-af3d-ed737833c2c1","owner":[],"postedDate":"July 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-09-10T05:24:59+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-03 18:27:51","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4586386","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4586386","identity":"rs-4586386","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}