Trigeminal neuroplasticity after pulpitis and oral administration of paracetamol in rats
Neuroplasticidad trigeminal luego de una pulpitis yadministración oral de paracetamol en ratas
1 https://ror.org/0081fs513 Universidad de Buenos Aires Facultad de Odontología Cátedra de Histología y Embriología Buenos Aires Argentina
2 UBA-CONICET Instituto de Biología Celular y Neurociencia “Prof. E. De Robertis” Buenos Aires Argentina
Corresponding Author:Mariela Celeste Canzobre mariela.canzobre@odontologia.uba.arABSTRACT
Experimental stimulation of dental pulp nociceptors induces plastic changes in the trigeminal subnucleus caudalis (Vc) through increased intracellular calcium levels and discharge of nociceptive afferent fibers. Acetaminophen (paracetamol) is a potent and selective inhibitor of COX-2 within the central nervous system (CNS) and may act in association with the cannabinoid system and the descending serotonergic pathway.
Aim
In this context, the main objective of this study is to analyze the plastic changes induced by paracetamol in the nociceptive circuit of the Vc following unilateral dental pulp inflammation (pulpitis) in rats.
Material and Method
Twenty-one female Wistar rats, 50 days old, were divided into three groups. In the experimental group 1 (EG1), a unilateral pulpitis was induced in the left mandibular first molar, and animals received no further treatment. In group 2 (EG2), the procedure was the same as in EG1, but 48 h later, animals received paracetamol in 3 daily doses (300 mg/kg each) every 4 h for 2 days. The control group (CG) received no treatment. After euthanasia, liver, hemimandibles and brainstem samples were analyzed. The results were analyzed using Student’s t-test or ANOVA (p<0.05).
Results
5-hydroxytryptamine transporter (5HT-T) analysis in the ipsilateral Vc demonstrated that paracetamol administration increased plexus length and varicosity density compared with EG1 and CG (p<0.05). Paracetamol also increased cannabinoid CB1 receptor (CB1r) expression (p<0.05), and decreased the number of calbindin D28K positive projection neurons (p<0.05).
Conclusion
Following experimental pulpitis, oral administration of paracetamol induces plastic changes in the serotonergic and endocannabinoid systems within the Vc. This plasticity is reflected by a reduced number of fusiform calbindin D28K projection neurons in lamina I, associated with modulation of orofacial nociceptive processing.
RESUMEN
La estimulación experimental de los nociceptores de la pulpa dental induce cambios plásticos en el subnúcleo caudal del trigémino (Vc) generando un aumento de los niveles de calcio intracelular y la descarga de las fibras aferentes nociceptivas. El paracetamol es un potente inhibidor selectivo de la COX-2 en el sistema nervioso central (SNC) y puede actuar en asociación con el sistema cannabinoide y la vía serotoninérgica descendente.
Objetivo
el objetivo principal de este estudio es analizar los cambios plásticos inducidos por el paracetamol en el circuito nociceptivo del Vc tras una inflamación unilateral de la pulpa dental (pulpitis) en ratas.
Materiales y métodos
21 ratas Wistar hembras, de 50 días de edad, se dividieron en tres grupos. En el grupo experimental 1 (GE1), se indujo una pulpitis unilateral en el primer molar mandibular izquierdo, y los animales no recibieron ningún tratamiento adicional. En el grupo 2 (GE2), el procedimiento fue el mismo que en GE1, pero 48 h después, los animales recibieron paracetamol en 3 dosis diarias (300 mg/kg cada una) cada 4 h durante 2 días. El grupo control (GC) no recibió ningún procedimiento. Tras la eutanasia, se analizaron muestras de hígado, hemimandíbulas y tronco encefálico. Los resultados se analizaron mediante la prueba t de Student o ANOVA (p < 0,05).
Resultados
En el Vc ipsilateral del GE2 se demostró que la administración de paracetamol aumentó la longitud del plexo y la densidad de varicosidades del transportador de 5-hidroxitriptamina (5HT-T) en comparación con el EG1 y el CG (p < 0,05). También se demostró mayor expresión del receptor cannabinoide CB1 (CB1r) y menor número de neuronas de proyección positivas para calbindina D28K (p < 0,05).
Conclusión
Tras una pulpitis experimental, la administración oral de paracetamol induce cambios plásticos en los sistemas serotoninérgico y endocannabinoide dentro del Vc. Esta neuroplasticidad se refleja en una reducción del número de neuronas de proyección fusiformes positivas para calbindina D28K en la lámina I, asociada a la modulación del procesamiento nociceptivo orofacial.
INTRODUCTION
Prostaglandins play an essential role in processing inflammatory pain, not only by sensitizing the peripheral terminals of primary afferent fibers, but also by increasing input of nociceptive information in the central nervous system (CNS)1. The sensory information of orofacial structures is relayed by the trigeminal system, which comprises principal, mesencephalic and spinal nuclei. Nociceptive information is processed in the spinal subnucleus oralis (Vo), interpolaris (Vi) and caudalis (Vc), depending on the orofacial region involved. Experimental stimulation of orofacial nociceptors has been shown to induce plastic changes (neuroplasticity) at various levels of nociceptive pathways2,3. Afferent projections from mandibular tooth pulp terminate in the dorsomedial region of the Vc, which has been identified as the first center involved in orofacial nociceptive modulation4. Glutamatergic trigeminal ganglion neurons carry the nociceptive information at the superficial laminae I and II and activate glutamatergic receptors. From laminae I (LI), those nociceptive messages are transmitted by fusiform projection neurons to other regions of the brainstem and to the thalamus4,5. The majority of fusiform neurons in LI project to supraspinal targets and more than half co-express glutamate and calbindin immunoreactivity. Calbindin may therefore serve as a marker of projection neurons and contribute to buffering intracellular calcium increases induced by discharge of nociceptive afferent fibers5. These transient changes in local Vc circuits are critical for maintaining balance between excitatory and inhibitory neurons in nociceptive pathways6,7.
Neurons in substantia gelatinosa or lamina II (LII) are considered interneurons that process sensory information and do not project out of Vc. In addition to local modulation by the Vc, the descending system presents components functionally related with those of the opioid, endocannabinoid, noradrenergic and serotonergic systems, and play an important role in the orofacial nociceptive pathway8,9.
Dental clinical evidence shows that patients with pulpitis frequently undergo analgesic therapy to mitigate treatment-associated pain. Acetaminophen (paracetamol) is an effective analgesic medication which is metabolized in the liver and can thus cause liver injury at high doses10. In terms of functional effects, paracetamol exerts weak inhibition of peripheral COX-1 and COX-2 linked to the inflammatory response following injury11,12, but acts predominantly at the CNS.
It also produces antinociceptive effects through interactions with serotonergic and endocannabinoid pathways13-15. Descending facilitatory and inhibitory serotoninergic fibers originated in the midbrain periaqueductal gray (PAG) and ventromedial medulla (VMM) descend and make contacts with primary afferents and interneurons at laminae of Vc8,9. The 5-hydroxytryptamine transporter (5HTT) modulates neurotransmission by facilitating the removal of serotonin from synapses through neurotransmitter reuptake by presynaptic terminals16. In turn, the effects of endocannabinoids are mediated by receptors CB1 or CB2 and are responsible of physiological responses as analgesia and synaptic plasticity, among others17. Recent research has demonstrated that cannabinoid CB1 receptor (CB1r) is the most abundant in CNS and is expressed in glutamatergic, GABAergic, serotonergic, cholinergic, noradrenergic and dopaminergic neurons18. The activation of these receptors present in both the plasmatic membrane and on the outer mitochondrial membrane, decreases the release of glutamate and GABA14,18,19. Given the limited evidence available of the action of serotonin and CB1r on Vc cells, this study analyzed paracetamol-induced plastic changes in the trigeminal Vc nociceptive circuit after unilateral pulpitis (inflammation of the dental pulp) in rats. We hypothesize that oral administration of paracetamol after pulpitis induces neuroplastic changes in the Vc through serotonergic and endocannabinoid mechanisms. These changes are reflected by alterations in 5HT-T expression, CB1 receptor distribution, and in the number of calbindin D28K-positive neurons. These mechanisms are likely interconnected rather than independent, as evidence suggests that paracetamol metabolites (e.g., AM404) simultaneously engage cannabinoid receptors and serotonergic pathways within central pain circuits14, 15.
Current evidence indicates that paracetamol analgesia involves a multi-target central mechanism, including indirect activation of CB1 receptors and facilitation of descending serotonergic pathways, rather than isolated actions on COX enzymes.
MATERIALS AND METHOD
Animals
Animals were housed in galvanized steel cages, with three animals per cage, in a temperature-controlled room (21-24°C) under 12-hour light/dark cycle. Food (Standard diet rat-mouse chow – Cooperación) and water were provided ad libitum. Our protocol followed the internationally accepted standards of the Guidelines for the Care and Use of Laboratory Animals (NIH Publication 1985, and the National Academy of Science, US) and was approved by the Ethics Committee of the School of Dentistry at Universidad de Buenos Aires (Cicual-Odon/ FOUBA 006/2023). Twenty-one female Wistar rats, 50 days old, weighing 120-140 g were randomly assigned to one of three groups of seven animals each (n=7), using a simple randomization method. In experimental group 1 (EG1), a unilateral dental pulp injury was induced in the left mandibular first molar, and animals received no further treatment. In experimental group 2 (EG2), the procedure was the same as in EG1, but 48 h later animals received paracetamol antinociceptive treatment, similar to the analgesia used when the nociceptive pathway is activated by inflammatory tissue injury in humans. The control group (CG) received no treatment.
Surgical procedure of molar access cavity
Animals were anesthetized by intraperitoneal injection of ketamine (50 mg/kg, Holliday Laboratory) and xylazine (10 mg/kg König Laboratory). In the pulpitis groups, i.e., EG1 and EG2, enamel and dentin were removed from the left mandibular first molar using a 1/4 round bur at low speed to expose the mesial horn of the pulp chamber (ipsilateral). The cavity was then sealed with phosphate cement (Harvard; Richter and Hoffmann, Berlin, Germany) to prevent bacterial contamination and stimulate pulp inflammation through direct contact with cement. After the procedure, animals were monitored for discomfort or pain, and body weight was followed up until euthanasia. Only in the first 24 h after surgery, EG1 and EG2 animals showed decreased activity and ate less than the previous day. In EG2, 900 mg/kg/day paracetamol (Termofren™ drops, Roemmers, Buenos Aires, Argentina) for 2d 10,20.21. Paracetamol was administrated orally starting 48 h after pulp injury to simulate delayed clinical analgesic intervention. Animals received three doses (300 mg/kg each) per day (every 4 h starting at 8 am), resulting in a 12-hour drug-free interval. On day 4, EG2 and CG animals were anesthetized and 500 μl blood was collected from the rat tail vessels for analysis of serum transaminase levels (GPT: alanine transaminase and GOT: aspartate transaminase) and to evaluate the potential hepatotoxic effect of paracetamol. Serum transaminase levels in control animals were used as reference values.
Fixation and dissection
On day 4 after molar pulp injury, all animal groups were euthanized with anesthesia overdose and transcardially perfused through the left ventricle with cold saline solution (Ringer’s sodium chloride - potassium chloride - calcium chloride) plus 50 IU heparin and 60 ml cold paraformaldehyde (Merck, Rahway, NJ, US) 4% (w/v) in 0.1 M phosphate buffer, pH 7.3-7.5. After euthanasia, animals were dissected and liver, hemimandibles and brainstem samples collected. Livers were removed and processed using routine histologic techniques. Paraplast (Sigma-Aldrich, Burlington, MA, US) sections of approximately 8-μm thickness were prepared and stained with hematoxylin and eosin (H&E). To detect glycogen deposition in hepatocytes some sections were processed through periodic acid- Schiff (PAS) histochemistry. The hemimandibles were decalcified in 10% EDTA/phosphate-buffered saline (PBS) 1X for 5 wk. and embedded in Paraplast. Mesiodistal longitudinal sections of approximately 8-μm thickness were obtained from the mandibular first molars, and sections were stained with H&E. After brainstem dissection, the right ventral side was marked with a blade to identify the contralateral side. Brainstems were cryoprotected in sucrose/PBS 30% (w/v), and frozen coronal sections (30 μm) from the C4 to -5.6 interaural level22 were collected as floating sections into PBS/azide.
Immunofluorescence
Sections were incubated in blocking solution (3% normal sheep serum, Tris Buffer saline (TBS), 0.3% Triton X-100) for 2-4 hours at room temperature. At that time, sections were incubated for 48 hours at 4°C with combined primary antibodies: mouse monoclonal anti-5-hydroxytryptamine transporter (RRID Millipore Cat# MAB1564, RRID:AB_94220 1:1000), rabbit anti-calbindin D28K (1:5000; RRID Lot Nº 03, Swant Cat# CB38, RRID:AB_2721225) and rabbit anti-CB1r (1:3000; RRID Cayman Chemical Cat#10006590, RRID:AB_10098690) in 1% normal sheep serum, 0.1% Triton X-100, TBS. Negative controls were performed by replacing primary antibodies with goat serum. After 40 min washing with TBS, rhodamine red (TRITC 1:2000 Jackson Immuno Research) or fluorescein isothiocyanate (FITC 1:2000 Jackson Immuno Research) conjugated secondary antibodies were used. Sections were incubated for 3 hours, and then washed with TBS for 30 min. Cell nuclei were labeled with the fluorescent DNA marker Hoechst 33342. Finally, sections were mounted on glass slides previously dipped in 1% gelatin, air dried and slides were coverslipped using TBS:glycerol (1:3) for UV microscopic observation.
Image analysis
Photomicrographs were acquired with a Zeiss Axiophot light microscope equipped with epifluorescence, alternating between FITC and rhodamine filter sets. Images were captured at 24-bit resolution (8 bpp × 3 channels) using an Olympus Q-color 5 camera. Histomorphometric analysis was performed on digital photomicrograph from histological sections using Image Pro Plus 6 software (Media Cybernetics, Silver Springs, MD, US). Image acquisition and analyses were performed by an observer blinded to group allocation. The following parameters were studied: On livers: histological features on H&E stained and PAS histochemistry sections.
On left mandibular first molars: percentage of pulp inflammation volume per total cameral pulp volume (2D) (IV/PV2D%) and histological features.
On dorsomedial area and two levels of ipsilateral Vc per animal:
- 5HT-T plexus: determined as the total length of fibers (in μm) and quantification of 5HTT varicosities (in number of varicosities) / area of 0.01 mm2 in laminae I, II and III).
- Calb+ 5HT-T: percentage of calbindin D28k positive neurons with 5HT-T varicosities around the soma / total calbindin D28k positive neurons per area of 0.01 mm2 in laminae I, II and III, in %.
- CB1r or Calbindin D28k positive cells: quantification of fusiform somas compatible with the morphology of projection neurons in LI and quantification of smaller somas compatible with modulatory interneurons in LII / area of 0.05 mm2
Statistical analysis
Data are shown as means ± SEM. Data were statistically compared by Student t-test or two-way analyses of variance (ANOVA), followed by the post hoc Bonferroni test. Statistical differences were considered significant for p-values below 0.05. All statistical analyses were performed with Graph Pad Prism (version 5.03; GraphPad Software, Williston, VT, UD).
RESULTS
Analysis of hepatotoxicity
To evaluate hepatotoxicity associated with paracetamol, the livers of CG and EG2 were processed using routine histological techniques with H&E (Fig. 1A - C) and examined for glycogen deposits using PAS (Fig. 1D - F) staining. After paracetamol administration, the hepatic tissue and cell morphology showed normal histological appearance. Hepatocytes showed positive PAS staining for intracellular glycogen deposits. In the portal space, the morphology was preserved and exhibited no evidence of inflammatory infiltrate. Biochemical serum final transaminase levels (UI/L) exhibited no significant differences between EG2 and CG (final GPT EG2: 68.5 ± 12.0; CG: 78.5 ± 27.6; final GOT EG2: 292 ± 11.3; CG: 250 ± 19.8; p>0.05).
Analysis of left mandibular first molar in situ
In CG (Fig. 2 A), the molars showed normal histological features, with no signs of inflammatory process in the pulp or periodontal ligament. Sections of left mandibular first molar in EG1 and EG2 revealed an acute inflammatory infiltrate in the coronal pulp in direct contact with the sealing material 4 d post injury. Inflammatory reactions observed in both groups were similar in terms of the percentage of volume pulp inflammation in relation to the total volume cameral pulp (IV/PV2D%) (EG1: 29% ± 2.92; EG2: 28% ± 2.41; p>0.05) and histological features (Fig. 2 B and C). This histopathological reaction may be compatible with a pulpitis diagnosis and the consequent nociceptive stimulation.
Analysis of 5HT-T expression in the Vc
Descending serotonergic fibers showed varicosities along their path and 5HT-T was expressed in these varicosities. 5HT-T analysis revealed that the descending fibers were distributed as a plexus in the Vc, primarily in LI and III, with a minimal presence in LII (Fig. 3 A and D). Comparisons of plexus length in each lamina of the ipsilateral Vc rendered significantly greater length in LI, II and III of EG2 compared to CG (Fig. 3 G). Paracetamol administration significantly increased the number of 5HT-T positive varicosities in LI (CG: 88.13 ± 24.40; EG1: 95.36 ± 14.47; EG2: 200.36 ± 28.99; p<0.05) and II (CG: 57.92 ± 10.78; EG1: 76.79 ± 9.43; EG2: 118.39 ± 12.48; p<0.05) (Fig. 3 A, D and H).
Furthermore, 5HT-T varicosities associated with calbindin D28K positive neurons were quantified. In LI, projection neurons showed a fusiform soma and two long primary extensions emerging from each end of the soma. LII showed smaller neurons compatible with the morphology of modulatory interneurons (Fig. 3 B-C, E-F). EG2 demonstrated a significant increase of varicosities associated with calbindin D28K positive somas in LI (CG: 20% ± 7; EG1: 29% ± 6; EG2: 49% ± 4.5; p<0.05) and LII (CG: 10% ± 1.6; EG1: 13% ± 2.3; EG2: 22% ± 4; p<0.05) (Fig. 3 I).
Analysis of CB1r expression in the Vc
CB1r expression was observed in the perinuclear cytoplasm and at the beginning of the neuronal processes in both projection neurons (P) and interneurons (M). Furthermore, the distribution of CB1r in nociceptive LI-II and V was studied (Fig. 4 A - C). Expression analysis of CB1r in the superficial laminae demonstrated that the number of projection neurons located in LI on the ipsilateral side was significantly greater in EG2 than in EG1 or CG (CG: 3.67 ± 0.33; EG1: 2.6 ± 0.24; EG2: 6.0 ± 0.58; p< 0.05). In LII, no significant changes were observed in CB1r expression (CG: 26 ± 3.06; EG1: 18 ± 2.10; EG2: 20 ± 2.16; p > 0.05) (Fig 4 D, E and H, I).
Analysis of calbindin D28K expression in the Vc
The number of calbindin D28K positive fusiform neurons per area of LI in the ipsilateral Vc was significantly larger in EG1 than in EG2 or CG at 4 d post-injury (CG: 3.3 ± 0.63; EG1: 4.29 ± 0.47; EG2:3.0 ± 0.19; p<0.05). In contrast, calbindin D28K expression in smaller modulatory interneurons of LII showed non-significant differences (CG: 45.5 ± 6.19; EG1: 34.4 ± 10.34; EG2: 44.38 ± 7.85; p >0.05) (Fig. 4 F - I).
DISCUSSION
Paracetamol is occasionally used to manage pain before and after dental procedures. In EG2, paracetamol was administrated orally starting 48 h after pulp injury to simulate delayed clinical analgesic intervention, once pulp inflammation (pulpitis) had developed. To maximize antinociceptive efficacy, the EG2 animals received three doses (300 mg/kg each) per day (every 4 h), resulting in a 12-hour drugfree interval. The dosing used in this study is higher than typical clinical doses in humans, however, such doses are commonly employed in experimental models20,21 to ensure central pharmacological effects. Because 90–95% of paracetamol undergoes hepatic biotransformation and its toxicity increases with dose12, we examined liver histology and serum transaminases in both control and paracetamol treated rats to check for possible liver toxicity. Importantly, no hepatotoxicity was detected, consistent with previous studies showing that toxicity depends on metabolic saturation rather than dose alone under controlled conditions10,12,21. Histological assessment showed preserved lobular architecture and portal tracts in paracetamoltreated animals. Hepatocytes contained cytoplasmic glycogen, and there were no signs of necrosis or inflammation. Serum transaminase levels in EG2 remained within CG ranges. These findings confirm that, under our dosing regimen, rats do not develop paracetamol-induced liver injury10. Nevertheless, caution is warranted when extrapolating these findings to clinical settings.
Different tooth injuries induce varying degrees of neuroplastic change at the trigeminal nucleus, depending on severity. In this study we used a type II intermediate molar tooth injury, which caused partial pulp loss, controlled infection with an inflammatory reaction, and occasionally necrosis of the pulp, but did not spread to adjacent territories such as periapical tissues23. Pulp inflammation was induced in the left mandibular first molar by exposing the mesial pulp chamber, and sealing with restorative material was then performed to mimic what may happen in the dental clinic procedures. Histopathological examination of the exposed pulp confirmed the development of pulpitis. This inflammatory response generates nociceptive signals that are conveyed predominantly via Aδ and C primary afferent fibers originated from the trigeminal ganglion and carry nociceptive information to the Vc4.
In recent years, we have studied neurons in LI, II, and V of the rat Vc and their modulatory role in the orofacial nociceptive pathway after pulpitis7. Our analysis focused on female rats, testing the hypothesis of sex-based differences in nociceptive thresholds. The literature indicates that these responses are highly genotype-dependent; for instance, while Sprague-Dawley and Long-Evans strains exhibit dimorphic sensitivity, Wistar-Kyoto rats often show no significant difference between sexes24. Consequently, the present study evaluates the potential antinociceptive effect of paracetamol in female Wistar rats. Within this framework, both EG1 and EG2 groups displayed a similar degree of pulp inflammation, indicating that paracetamol exerts a weak inhibitory effect on peripheral pulp inflammation—an observation consistent with its limited efficacy in other inflamed tissues11,12. In contrast, recent evidence indicates that the central analgesic effects of paracetamol are mediated by its metabolite AM404, which activates CB1 receptors and interacts with serotonergic pathways within central nociceptive circuits14,15,25. Nevertheless, how oral paracetamol modulates synaptic and cellular plasticity within the Vc in an odontogenic pulpitis model remains to be determined.
Paracetamol administration is associated with remodeling of the serotonergic pathways in the superficial dorsal laminae of the Vc. EG2 exhibited a significant increase in both the length of 5HT-T positive plexus and the density of 5HT-T–immunoreactive varicosities, indicative of enhanced serotonin reuptake capacity16. Colocalization analysis further revealed that these 5HT-T varicosities formed close appositions nearby calbindin-D28k–positive neurons. These findings suggest modulation of nociceptive processing rather than direct evidence of functional inhibition.
Moreover, the cannabinoid system modulates synaptic transmission within SNC17. Activation of CB1r in neurons in the trigeminal complex inhibits neurotransmitter release, leading to a decrease in nociceptive responses, or pain signaling26. Therefore, CB1r activation contributes to antinociception by blocking the progression of the pain pathway.
In line with Farquhar-Smith et al.27, our immunohistochemical analysis revealed CB1r localization in the perinuclear cytoplasm of neurons within the superficial laminae of the Vc. EG2 showed a notable increase in CB1r immunoreactivity, specifically in ipsilateral LI projection neurons, that receive central projections from trigeminal primary afferent neurons innervating the mandibular division. Animal studies have demonstrated that calbindin D28K, a calcium-binding protein, is expressed by distinct neuronal populations in the Vc, suggesting different functional properties among these neurons. Small multipolar calbindin-positive neurons in LI and II modulate nociceptive input, whereas fusiform calbindin-positive neurons in LI constitute ascending projection cells targeting the medial thalamus and other pain-related subnuclei5,28. Multiple Ca2+ channels and Ca2+ influx are involved in the glutamatergic synaptic transmission29. Consistent with our preceding study7, enhanced calbindin D28K expression in LI fusiform neurons, supports the activation of the ascending orofacial nociceptive pathway after pulpitis. In contrast, the present study demonstrates that oral paracetamol administration produces a significant reduction in the number of calbindin D28K–positive projection neurons in LI of the Vc. This decrease likely reflects enhanced presynaptic inhibition mediated by the concomitant increase of 5HT-T and CB1r expression in EG2, resulting in reduced calcium-dependent neurotransmitter release. In agreement with these findings, previous studies of excitatory synaptic transmission have indicated that presynaptic action of CB1r and 5HTr reduce directly or indirectly Ca2+ influx and glutamate release29,30.
CONCLUSIONS
In conclusion, our findings indicate that after experimental activation of dental pulp nociceptors (pulpitis), oral administration of paracetamol produces plastic changes that can be observed at the level of the serotonergic and endocannabinoid systems of the Vc. This plasticity is expressed as a lower number of fusiform calbindin D28K projection neurons in lamina I. The reduction in the number of calbindin D28K-positive neurons in lamina I may reflect adaptive neuroplasticity associated with the modulation of orofacial nociceptive processing.
From a clinical perspective, these findings support the concept that paracetamol may exert part of its analgesic effect through central modulation of trigeminal nociceptive circuits, which is particularly relevant in odontogenic pain conditions such as pulpitis.
A major limitation of this study is the absence of behavioral nociceptive assessment. Further research including behavioral and electrophysiological approaches, as well as co-expression analyses of fusiform neurons with glutamate, CB1r or 5HT receptor subtypes, are needed to validate the functional relevance of these findings. Elucidating these mechanisms may contribute to optimizing the central antinociceptive effects of paracetamol and analgesic strategies in dental practice.
ACKNOWLEDGMENTS
We would like to thank Prof. Osvaldo Zmener and Dr. Carlota Alicia Gamba for their helpful comments; and Mariela Lacave, Andrea Pecile and Carlos Bárcenas for their skillful technical assistance.