Mohamed Abdelnaby1
Marwa Y. Sallam1Mai M. Helmy1Hanan M. El-Gowelli1Muddanna S. Rao2
Mahmoud M. El-Mas1,3*- 1Department of Pharmacology and Toxicology, Faculty of Pharmacy, Alexandria University, Alexandria, Egypt
- 2Department of Anatomy, College of Medicine, Kuwait University, Safat, Kuwait
- 3Department of Pharmacology and Toxicology, College of Medicine, Kuwait University, Safat, Kuwait
Introduction: The interplay between opioid analgesics and sepsis in intensive care units (ICUs) is multifaceted, often amplifying immune dysregulation and adversely affecting cardiovascular outcomes.
Aim: We investigated whether morphine, the prototypical opioid, influences sepsis-induced impairment of arterial baroreceptor function and the accompanying inflammation.
Methods: Rats were implanted with indwelling catheters in femoral vessels and intracisternal (i.c.) space, and sepsis was induced using the cecal ligation and puncture (CLP) technique. The baroreceptor-mediated control of chronotropic activity was assessed 24 h later in awake rats using the vasoactive method, which relates blood pressure changes caused by i.v. phenylephrine (PE) or sodium nitroprusside (SNP) to respective reciprocal changes in heart rate.
Results: The treatment of sham rats with morphine or induction of sepsis led to comparable attenuations of reflex decrements and increments in chronotropic responses and decreases in slopes of baroreflex curves (baroreflex sensitivity, BRS). The treatment of septic rats with morphine further amplified the decline in reflex bradycardic (BRSPE), but not tachycardic (BRSSNP), and this exaggerated bradycardia disappeared after (i) systemic blockade of opioid receptors by i.v. morphine or (ii) selective central inhibition of PI3K, MAPK-ERK, MAPK-JNK, NADPHox), or Rho-kinase (ROCK). These pharmacological interventions also attenuated the elevated protein expression of toll-like receptor 4 (TLR4) and monocyte chemoattractant protein-1 (MCP1) in the brainstem nucleus tractus solitarius (NTS) of morphine-treated CLP rats.
Conclusion: Overall, morphine augments the sepsis-induced depression of reflex cardiovagal activity through an opioid receptor sensitive mechanism that engages brainstem inflammatory and chemotactic circuits related to PI3K/MAPK/NADPHox/ROCK signaling.
1 Introduction
Sepsis is a life-threatening organ dysfunction that results from dysfunctional host reaction to infection (Singer et al., 2016). Annually, around 45 million patients suffer from sepsis with a 20% death rate (Chiu and Legrand, 2021). Sepsis is one of the most frequent fatal complications encountered in ICUs (Deutschman and Tracey, 2014). Sepsis is associated with dysregulated hemodynamic and cardiovascular functions (Greer, 2015; Sallam et al., 2016). The pathophysiology of sepsis involves activation of toll like receptor-4 (TLR4) (Anderberg et al., 2017) with subsequent release of proinflammatory mediators such as monocyte chemoattractant protein 1 (MCP1) (Zhu et al., 2017; Chen et al., 2023). These mediators cause profound vasodilation, hypotension, and other cardiovascular sequels of septic shock (Nguyen et al., 2006). TLR4 activation causes a consequent upregulation of the MAPK/NF-κB signaling pathway and MCP1 expression, which amplifies the inflammatory response (Chen et al., 2018). The increased expression levels of MCP1 and consequent inflammation disrupt central pathways that are pivotal for cardiovascular autonomic control (Fairchild et al., 2009; Mowry et al., 2021).
The arterial baroreflex pathway serves as a crucial homeostatic mechanism for short-term regulation of blood pressure and other cardiovascular functions (Karim et al., 2023; Salah et al., 2025). Attenuated baroreceptor activity increases the incidence of cardiovascular diseases like hypertension (Gordin et al., 2016) and cardiac arrhythmogenesis and sudden cardiac arrest (Shen and Zipes, 2014). Sepsis disrupts the baroreflex mechanism, resulting in reduced survival time and cardiovascular collapse (Sallam et al., 2021a; Merdji et al., 2022; El-Naggar et al., 2025). The reduction in baroreflex gain in sepsis is likely caused by elevated cytokines and excessive production of nitric oxide and reactive oxygen species (Ulleryd et al., 2017). The nucleus tractus solitarius (NTS) in the lower brainstem serves as the primary medullary site that receives afferent signals from peripheral arterial baroreceptors. The NTS integrates and relays this information to other cardiovascular-regulatory nuclei within the brainstem, as well as to hypothalamic and cortical centers of the central nervous system (Waki et al., 2008; Accorsi-Mendonça and Machado, 2013). Evidence also suggests that afferent vagal neurons are responsive to both pro-inflammatory and anti-inflammatory cytokines, transmitting these immune signals to neurons within the NTS (Amorim et al., 2019). This input triggers autonomic responses that modulate peripheral vascular resistance and elicit rapid adjustments in heart rate (Benarroch, 2008).
Opioids such as morphine and fentanyl are widely used in ICUs for pain control and sedation (Young and De Jong, 2021). The adverse effects of morphine in this context include respiratory depression, peripheral vasodilation, hypotension, and inhibition of arterial baroreceptor reflexes (Shanazari et al., 2011; Krantz et al., 2021; Watso et al., 2022). Although both morphine (Shanazari et al., 2011; Zeinivand et al., 2014) and sepsis (El-Naggar et al., 2025) are each known to impair baroreceptor responsiveness, to our knowledge, no study has examined their combined effects on reflex chronotropic responses to baroreceptor loading and unloading in conscious freely moving rats. Further, this study uniquely explores the role of TLR4 and associated chemotactic and inflammatory pathways within the cardiovascular-regulatory nuclei of the medullary solitary tract in mediating the sepsis-morphine interaction. Hemodynamic, pharmacologic, and protein expression analyses were also integrated to provide novel mechanistic insights into opioid-induced cardiovascular vulnerability during sepsis. The cecal ligation and puncture (CLP) procedure was used in our study to induce sepsis as it closely resembles sepsis in human patients (Gong and Wen, 2019). All the experiments were conducted in fully conscious and freely moving male Wistar rats, pre-instrumented with indwelling femoral and intracisternal catheters.
2 Materials and methods
2.1 Animals
A total of 60 adult male Wistar rats (200–250 g) were utilized, sourced from the Faculty of Pharmacy animal facility at Alexandria University in Alexandria, Egypt. Rats were kept at an ambient temperature and had a free access to standard rat chow with 19% protein and water. The sample size was estimated a priori using power analysis (G*Power 3.1.9.7) (Bate and Clark, 2014) and guided by our previous studies employing similar experimental models (El-Naggar et al., 2025). Before initiating the experiment, the analysis indicated that a minimum of 4 rats per group was required to detect an effect size of 0.90 at an α-error probability of 0.05 and a power (1–β) of 0.80. To ensure adequate statistical robustness, 5-6 rats per group were ultimately included. All experimental procedures and animal handling received approval from the Institutional Animal Care and Use Committee of Alexandria University (Approval No. AU/06.2021.2.6.1.93) and adhered to the ARRIVE guidelines (https://arriveguidelines.org/).
2.2 Drugs
The following substances were utilized: Morphine from Masr Pharmaceutical Co. in Cairo, Egypt; Heparin Sodium (5000 IU/mL ampoules) from Nile Pharmaceutical Co., also in Cairo; Naloxone from SERB Pharmaceutical Co. in Paris, France; and Thiopental, branded as Thiopental®, from Biochemie GmbH in Vienna, Austria. Further, wortmannin, PD98056 (PD), and SP600125 (SP) were obtained from Sigma-Aldrich in St. Louis, MO, United States. Diphenyleneiodonium (DPI) and fasudil were provided by Tocris Bioscience in Bristol, UK. PD98056, SP600125, wortmannin, fasudil, and DPI were dissolved in DMSO. Phenylephrine and sodium nitroprusside were also acquired from Sigma-Aldrich in St. Louis, MO, United States.
2.3 Sepsis induction by cecal ligation and puncture (CLP)
This procedure was performed as previously described (El-Naggar et al., 2025; Gong and Wen, 2019; El-Naggar et al., 2023). Under thiopental anesthesia (50 mg/kg, i.p.), the rat abdominal region was shaved and disinfected with a betadine. A midline laparotomy of ∼1 cm was made to expose the cecum. The distal one-third of the cecum was ligated, and 3 punctures were made on the same side using a 21-gauge needle. Gentle compression was applied to extrude a small amount of fecal material. The cecum was then returned to the abdominal cavity, and the incision was closed by suturing the abdominal muscle and skin layers.
2.4 Intracisternal cannulation (i.c.)
Five days prior to the start of the experiment, i.e., three days before intravascular cannulation and CLP, a stainless steel guide cannula (23 G) was surgically implanted into the cisterna magna of the rats under thiopental anesthesia (50 mg/kg, i.p.), following procedures described in our previous studies (El-Mas et al., 2009; El-Mas et al., 2012). The cannula was carefully advanced between the occipital bone and cerebellum, with its tip positioned in the cisterna magna. It was then secured in place using dental acrylic cement. Successful placement was confirmed by the spontaneous outflow of cerebrospinal fluid. After the surgery, rats were housed individually for recovery.
2.5 Intravascular cannulation
The detailed methodology has been previously described (El-Mas et al., 2009; El-Mas et al., 2012). Following CLP or sham surgery, vascular catheters were implanted using a catheter consisting of a 5 cm segment of polyethylene-10 tubing fused to a 15 cm segment of polyethylene-50 tubing. These catheters were inserted into the inferior vena cava and abdominal aorta via the femoral vein and artery for intravenous drug administration and arterial blood pressure monitoring, respectively. The intravascular portion was composed of the polyethylene-10 segment. The catheters were then tunneled subcutaneously and exteriorized at the back of the neck between the scapulae. They were then flushed with heparinized saline (0.2 mL; 100 U/ml) and secured using stainless steel pins. On the following day, the arterial catheter was connected to a pressure transducer (model P23XL; Astro-Med, West Warwick, RI, United States), linked via an MLAC11 Grass adapter cable to a computerized data acquisition system (LabChart-7 Pro software, PowerLab 4/35, model ML866/P; AD Instruments Pty Ltd., Castle Hill, Australia) for continuous recording and analysis of blood pressure and heart rate. It is noteworthy that thiopental, the anesthetic drug used in this study, is an ultra-short-acting barbiturate that permits rapid recovery, typically within 30–40 min after i.p. administration. Normal spontaneous behavior and body reflexes are generally restored within 2 h after anesthesia (Tanaka et al., 2008; Kushikata et al., 2011). In our study, baroreflex assessment was conducted 24 h after intravascular cannulation, when the rats were fully awake, freely moving in their home cages, and had resumed normal eating and drinking habits. No differences were observed between the CLP and sham-operated groups in their recovery from anesthesia.
2.6 Measurement of baroreflex gain (Oxford method)
Baroreflex sensitivity (BRS) was assessed at the end of the experiment using the vasoactive (Oxford) method, as previously described (El-Mas and Abdel-Rahman, 1993; El-Mas et al., 2011; El-Naggar et al., 2024; El-Naggar et al., 2025). This approach evaluates reflex bradycardic and tachycardic responses to peripheral blood pressure changes induced by randomized intravenous bolus injections of PE or SNP (1–16 μg/kg, administered every 5 min), respectively. Baroreflex response curves were generated by measuring mean arterial pressure (MAP) against heart rate (HR) values before and after drug administration. Peak changes in MAP (ΔMAP) and HR (ΔHR) were determined, and the slope of the resulting linear regression curve was used to quantify BRS.
2.7 Immunohistochemistry
This was used to measure the expression of TLR4 and MCP1 in the brain medullary neurons of the nucleus tractus solitarius (NTS) (Sallam et al., 2019; Sallam et al., 2021a). Tissues were fixed in 10% formalin solution for 1 day, dehydrated in a graded series of ethanol (70, 95% and 100%), then embedded in paraffin. Tissue sections of 5 µm thickness of the rostral NTS (−12.3 mm relative to bregma) (Paxinos and Watson, 1986; Abuiessa et al., 2020) were placed on positively charged adhesion microscope slides, deparaffinized in xylene and rehydrated in a graded series of ethanol (100, 95% and 70%). Slides were then rinsed gently with PBS and drained. Heat-induced epitope retrieval was done by immersing slides in Coplin jars containing 10 mM citrate buffer solution and incubation in a microwave at power100 for 1min then power 30 for 9 min. The sections were subsequently rinsed with 1× TBST (50 mM Tris/HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20). To block endogenous peroxidases, a 3% hydrogen peroxide solution was applied, followed by washing with 1× TBST. Following that, a universal protein blocking solution was applied for 20 min. The appropriate primary monoclonal antibodies (rabbit anti-TLR4 & rabbit anti-MCP1) were diluted (1:300) as instructed by the manufacturer and applied to the slides then slides were incubated over night at 4 °C. Following this incubation, the slides were washed with 1× TBST, rinsed, and subsequently incubated for 30 min with the secondary antibody (HRP conjugate). The chromogen 3,3′-diaminobenzidine was prepared and applied in accordance with the manufacturer’s instructions for protein visualization. Each slide was counterstained with haematoxylin and dipped in ascending concentrations of alcohol and then xylene. OptikamB9 digital camera was used to take images (Optika® microscopes, Italy) followed by quantification of the immunohistochemical signals of expressed proteins in the NTS area of the brainstem using Fiji ImageJ software version 1.51n (National Institutes of Health, Bethesda, Maryland, United States). The quantification procedure involved measuring the stain intensity in 6–9 distinct rNTS sections from each hemisphere. The mean intensity values from both hemispheres were then averaged to obtain a representative value for each animal, ensuring consistency and minimizing regional variability.
2.8 Protocols and experimental groups
This study investigated the dose-dependent effects of systemic morphine administration on baroreflex activity in septic and sham operated rats. Male Wistar rats were randomly divided into seven experimental groups (n = 5-6 per group): (i) sham/saline, (ii) sham/morphine (3 mg/kg), (iii) sham/morphine (10 mg/kg), (iv) CLP/saline, (v) CLP/morphine (3 mg/kg), (vi) CLP/morphine (10 mg/kg), and (vii) CLP/naloxone (1 mg/mL)/morphine (10 mg/kg). Following a stabilization period of at least 45 min, either saline or morphine was administered intravenously. Two hours later, baroreflex response curves to PE and SNP were generated as previously described. These treatments were randomly assigned to the respective rat groups using the simple randomization sequence, which is based on a single sequence of random assignments. Rats were excluded from the experiment if any of the arterial or venous catheters were blocked or disconnected. At the end of the experiment, animals were euthanized with an overdose of thiopental (100 mg/kg). The brainstem was then collected, fixed in 10% formaldehyde, and processed for immunohistochemical analysis of MCP1 and TLR4 protein expression. A schematic diagram of the experimental timeline, including surgical procedures and drug administration protocols, is shown in Figure 1. Blinding of the experimental procedures was not feasible, as the same researcher conducted both the surgical interventions and drug administrations.
Figure 1. Diagrammatic representation of the timeline of surgical procedures, drug regimens and experimental groups.
To further examine the involvement of inflammatory PI3K/MAPK/NADPHox/ROCK pathway in the interaction between sepsis and morphine, five additional CLP rat groups (n = 5-6 per group) were included randomly assigned to receive one of the following i.c. regimens followed by morphine (10 mg/kg, i.v.): (i) PD98056 (10 μg/5 μL/rat, a MAPK/ERK1/2 inhibitor), (ii) SP600125 (30 μg/5 μL/rat, a MAPK/JNK inhibitor), (iii) wortmannin (0.5 μg/5 μL/rat, a PI3K inhibitor), (iv) fasudil (70 μg/5 μL/rat, a ROCK inhibitor), and (v) diphenyleneiodonium (DPI; 150 μg/5 μL/rat, a NADPHox inhibitor). The dosing regimens were selected based on previously published studies (Fujita et al., 2007; Sallam et al., 2016; Kumar and Bansal, 2018). A 10-min interval was maintained between the administration of the inhibitor and morphine in each treatment protocol. Following drug administration, baroreflex response curves to PE and SNP were constructed as previously described. At the end of hemodynamic monitoring, rats were euthanized via thiopental overdose (100 mg/kg), and brainstems were collected, fixed in 10% formaldehyde, and processed for immunohistochemical analysis of MCP1 and TLR4 protein expression, as detailed earlier.
2.9 Statistical analysis
Data are presented as means ± standard error of the mean (S.E.M.). BRS was evaluated by assessing the relationship between changes in MAP and corresponding changes in HR using linear regression analysis for each animal, as previously described (Paxinos and Watson, 1986; El-Mas and Abdel-Rahman, 1992; El-Mas et al., 2001; Abuiessa et al., 2020; Sallam et al., 2021a). The slope of the regression line, expressed in beats per minute per mm Hg, was used as an index of BRS. Statistical comparisons among groups were performed using one-way or repeated-measures ANOVA, followed by Tukey’s post hoc test for multiple comparisons. The analysis was performed using GraphPad Prism software, version. 10.6.1. Probability levels less than 0.05 were considered significant.
3 Results
3.1 Morphine dose dependently aggravates CLP- induced baroreflex dysfunction
Table 1 shows that prior to saline or morphine administration, all CLP groups demonstrated significant decreases and increases in baseline MAP and HR values compared to respective sham values. Figures 2–4 illustrate the effect of morphine on baroreflex curves constructed by the vasoactive method in sham and CLP rats. In all rat groups, intravenous administration of graded doses of PE and SNP (1–16 μg/kg each) elicited dose-dependent increases and decreases in MAP (Figures 2A,B, respectively, accompanied by corresponding reciprocal changes in HR (Figures 2C,D). Compared with sham rats, the induction of sepsis by CLP did not affect MAP responses to PE or SNP but significantly blunted the corresponding reflex decreases and increases in HR, respectively (Figure 2), indicating impaired baroreflex-mediated heart rate regulation in septic rats. Comparisons of the slopes of the regression lines, which represent BRS, showed that the induction of sepsis by CLP caused significant decreases in baroreflex sensitivity measured by PE (BRSPE, −2.06 ± 0.08 vs. −2.88 ± 0.18 beats/min/mmHg, 95% CI of diff. 0.193 to 1.455, p = 0.004, Figure 3C) and SNP (BRSSNP, −2.15 ± 0.11 vs. −3.21 ± 0.24 beats/min/mmHg, 95% CI of diff. 0.1252 to 1.986, p = 0.018, Figure 4C) compared with control (sham-operated) values.
Table 1. Baseline measurements of mean arterial pressure (MAP, mmHg) and heart rate (HR, beats/min) recorded in conscious sham or CLP rats before the administration of any saline or drug treatments.
Figure 2. Effect of i.v. morphine (3 or 10 mg/kg) on increases and decrease in MAP elicited by phenylephrine (panel (A)) and sodium nitroprusside (panel (B)) (1–16 μg/kg each), respectively (upper panels) and reciprocal changes in HR (panels (C, D), respectively) in conscious sham operated rats and CLP rats. The effect of opioid receptor antagonism by naloxone (1 mg/kg i.v.) on morphine responses in CLP rats is also shown. Values are means ± SEM of five to six observations. aP < 0.05 vs. “sham/saline”, bP < 0.05 vs. “CLP/saline”, cP < 0.05 vs. “sham/morphine-10”, dP < 0.05 vs. “CLP/Morphine-10”.
Figure 3. Effect of i.v. morphine (3 or 10 mg/kg) on baroreflex curves generated by phenylephrine (1–16 μg/kg) and baroreflex sensitivity (BRSPE, slopes of the curves) in conscious sham operated rats and CLP rats. The effect of opioid receptor antagonism by naloxone (1 mg/kg i.v.) on morphine responses in CLP rats is also shown. In (A), the regression lines represent the following groups: sham/saline (solid line), sham/morphine-3 (dashed line), sham/morphine-10 (dotted line). In (B), the regression lines represent the following groups: CLP/saline (solid line), CLP/morphine-3 (dashed line), CLP/morphine-10 (dotted line), and CLP/naloxone/morphine-10 (dash-dotted line). In (C), BRSPE. Values are means ± SEM of five to six observations. The repeated measures ANOVA followed by the Tukey’s post hoc test were employed to test for statistical significance. aP < 0.05 vs. “sham/saline”, bP < 0.05 vs. “CLP/saline”, cP < 0.05 vs. “sham/morphine-10”, dP < 0.05 vs. “CLP/Morphine-10”.
Figure 4. Effect of i.v. morphine (3 or 10 mg/kg) on baroreflex curves generated by sodium nitroprusside (1–16 μg/kg) and baroreflex sensitivity (BRSSNP, slopes of the curves) in conscious sham operated rats and CLP rats. The effect of opioid receptor antagonism by naloxone (1 mg/kg i.v.) on morphine responses in CLP rats is also shown. In (A), the regression lines represent the following groups: sham/saline (solid line), sham/morphine-3 (dashed line), sham/morphine-10 (dotted line). In (B), the regression lines represent the following groups: CLP/saline (solid line), CLP/morphine-3 (dashed line), CLP/morphine-10 (dotted line), and CLP/naloxone/morphine-10 (dash-dotted line). In (C), BRSSNP. Values are means ± SEM of five to six observations. The repeated measures ANOVA followed by the Tukey’s post hoc test were employed to test for statistical significance. aP < 0.05 vs. “sham/saline”.
Figure 3 demonstrates that the administration of morphine at 10 mg/kg to CLP rats further worsened the sepsis-associated impairment in reflex bradycardia, leading to an additional reduction in BRSPE compared with CLP alone (95% CI of diff. 0.2731 to 1.536, p = 0.001, Figure 3C). The amplifying effect of morphine appears to be specific to the reflex bradycardic response, as the same dose did not result in any further decrease in BRSSNP in septic rats (95% CI of diff. −1.047 to 0.8132, p = 0.999, Figure 4C). Notably, the morphine effects were evaluated relative to the corresponding post-saline measurements, as baroreflex activity was not assessed before administration of morphine or saline. The depressant effect of morphine on BRSPE in CLP rats was abolished by prior co-administration of the opioid receptor antagonist naloxone (Figure 3).
Pharmacological antagonist studies were undertaken to investigate the role of central inflammatory and oxidative pathways in the morphine-BRSPE interaction in septic rats. Figure 5 shows that prior i.c. administration of the pharmacologic inhibitor of MAPK-ERK (PD98056, 10 μg/rat, Figure 5A), MAPK-JNK (SP600125, 30 μg/rat, Figure 5B), or ROCK (fasudil, 70 μg/rat, Figure 5D) caused downward shifts in the baroreflex curves generated by PE in morphine-treated CLP rats and significant increases in BRSPE (Figure 5F). No such effect was demonstrated after central inhibition of PI3K (wortmannin, 0.5 μg/rat, Figures 5C–F) or NADPHox (DPI, 150 μg/rat, Figures 5E,F). Further, none of these inhibitors affected BRSSNP in CLP-morphine-treated rats, except for a significant reduction caused by the MAPK-JNK inhibitor SP600125 (Figure 6).
Figure 5. Effect of i.c. pretreatment with PD98056 (10 μg/5 μL/rat, MAPKERK1/2 inhibitor, panel (A)), SP600125 (30 μg/5 μL/rat, MAPKJNK inhibitor, panel (B)), wortmannin (0.5 μg/5 μL/rat, PI3K inhibitor, panel (C)), fasudil (70 μg/5μL/rat, ROCK inhibitor, panel (D)) or DPI (150 μg/5 μL/rat NADPHox inhibitor, panel (E)) on baroreflex curves generated by phenylephrine (1–16 μg/kg) and baroreflex sensitivity (BRS curves) in conscious CLP rats treated with morphine (10 mg/kg). In (F), BRSPE. Values are means ± SEM of five to six observations. The repeated measures ANOVA followed by the Tukey’s post hoc test were employed to test for statistical significance. aP < 0.05 vs. “CLP/morphine”.
Figure 6. Effect of i.c. pretreatment with PD98056 (10 μg/5 μL/rat, MAPKERK1/2 inhibitor, panel (A)), SP600125 (30 μg/5 μL/rat, MAPKJNK inhibitor, panel (B)), wortmannin (0.5 μg/5 μL/rat, PI3K inhibitor, panel (C)), fasudil (70 μg/5μL/rat, ROCK inhibitor, panel (D))or DPI (150 μg/5 μL/rat NADPHox inhibitor, panel (E)) on baroreflex curves generated by sodium nitroprusside (1–16 μg/kg) and baroreflex sensitivity (BRS curves) in conscious CLP rats treated with morphine-10. In (F), BRSSNP. Values are means ± SEM of five to six observations. The repeated measures ANOVA followed by the Tukey’s post hoc test were employed to test for statistical significance. aP < 0.05 vs. “CLP/morphine”.
3.2 Modulation of sepsis-morphine baroreflex interaction by central inflammation and chemotaxis
The effect of morphine on the immunohistochemical expression of inflammatory (TLR4) and chemotactic (MCP1) signals in the NTS neuronal pools of septic rats are shown in Figures 7, 8. The induction of sepsis resulted in significant increases in the NTS expression of MCP1 (Figure 7) and TLR4 (Figure 8) and these effects were further augmented in NTS tissues of morphine-treated CLP rats. The enhancing effect of morphine on MCP1/TLR4 expression was abolished following the systemic administration of naloxone, suggesting the importance of µ-opioid receptors in mediating the morphine response. Moreover, the chemotactic and inflammatory actions of morphine in the solitary tract were ameliorated after i.c. administration of PD98056, SP600125, wortmannin, fasudil or DPI (Figures 7, 8). Representative images of the NTS expression of MCP1 and TLR4 are presented in Figures 7, 8, respectively. Figure 9 shows a cresyl violet–stained coronal section of the medulla oblongata (Plate 136, −12.36 mm from bregma) (Paxinos and Watson, 1986; Nakano et al., 2015; Abuiessa et al., 2020; Sallam et al., 2021b).
Figure 7. (A) illustrates the effects of naloxone (1 mg/kg i.v.) or i.c. administration of wortmannin (0.5 μg/5 μL/rat, PI3K inhibitor), PD98056 (10 μg/5 μL/rat, MAPKERK1/2 inhibitor), SP600125 (30 μg/5 μL/rat, MAPKJNK inhibitor), DPI (150 μg/5 μL/rat NADPHox inhibitor) or fasudil (70 μg/5μL/rat, ROCK inhibitor) on morphine (10 mg/kg)-evoked rises in immunohistochemical protein expressions of MCP1 in the nucleus tractus solitarius (NTS) of septic rats. Representative images of the NTS expression of MCP1 are shown in (B). Values are means ± SEM of five observations. aP < 0.05 vs. “sham/saline”, bP < 0.05 vs. “CLP/saline”, cP < 0.05 vs. “CLP/morphine-10”.
Figure 8. (A) illustrates the effects of naloxone (1 mg/kg i.v.) or i.c. administration of wortmannin (0.5 μg/5 μL/rat, PI3K inhibitor), PD98056 (10 μg/5 μL/rat, MAPKERK1/2 inhibitor), SP600125 (30 μg/5 μL/rat, MAPKJNK inhibitor), DPI (150 μg/5 μL/rat NADPHox inhibitor) or fasudil (70 μg/5μL/rat, ROCK inhibitor) on morphine (10 mg/kg)-evoked rises in immunohistochemical protein expressions of TLR4 in the nucleus tractus solitarius (NTS) of septic rats. Representative images of the NTS expression of TLR4 are shown in (B). Values are means ± SEM of five observations. aP < 0.05 vs. “sham/saline”, bP < 0.05 vs. “CLP/saline”, cP < 0.05 vs. “CLP/morphine-10”.
Figure 9. (A) is a photomicrograph of a coronal section of the medulla oblongata corresponding to Plate no 136 (−12.36 mm from bregma) in the rat brain atlas of Paxinos and Watson (Paxinos and Watson, 1986; Nakano et al., 2015; Abuiessa et al., 2020; Sallam et al., 2021b), stained with cresyl violet. The nucleus tractus solitarius (NTS) is outlined on the left side with a dotted red line. On the right side, the area enclosed within the square box represents the region selected for further analysis, which is illustrated in Figures 8 and 9 and shown at a higher magnification in (B) (scale bar represents 500 µm). (B) shows a magnified view of the NTS, highlighting the morphology of its neurons as revealed by cresyl violet staining (scale bar corresponds to 40 µm). (C) depicts an even higher magnification of the area enclosed by the square box in panel B, allowing a more detailed visualization of the neuronal features (scale bar represents 20 µm).
4 Discussion
This study reports on the interaction between morphine and sepsis induced by CLP on arterial baroreceptor control in awake rats and possible underlying molecular mechanisms. The data demonstrates that both morphine treatment and sepsis independently reduce baroreceptor-mediated control of heart rate, and that morphine further amplifies the sepsis-induced impairment of cardiovagal reflex control. Immunohistochemical studies reveal that this effect of morphine is mediated via the upregulation of central TLR4 and MCP1 expressions. Pharmacological inhibition of opioid receptors or key signaling inflammatory (MAPK-ERK or MAPK-JNK) or oxidative molecules (Rho-kinase) reversed both the exaggerated reflex bradycardic impairment and upregulated inflammatory signals. These findings highlight that morphine worsens sepsis-induced baroreflex dysfunction via opioid receptor-dependent activation of brainstem inflammatory and chemotactic signaling pathways.
Physiologically, arterial baroreceptors function as a key homeostatic mechanism that buffers abrupt changes in BP chiefly through modulating central sympathetic and vagal outflows to cardiovascular structures (El-Mas et al., 2001; Stocker et al., 2019). Baroreceptor dysfunction is commonly observed in cardiovascular diseases such as hypertension and myocardial infarction and is frequently correlated with poor prognosis (Gerritsen et al., 2001; Shen et al., 2004; Shi et al., 2007). The impairment of baroreflex activity is also provoked by the septic challenge in both clinical (Sharshar et al., 2003) and experimental studies (Milanez et al., 2022). The diminished baroreflex activity has been blamed for hemodynamic instability and increased incidence of morbidity and mortality in septic shock (Carrara et al., 2020; Merdji et al., 2022). A restraining influence of arterial baroreceptors against septic sequels is also supported by the observation that the septic hypotension and cardiovascular collapse are heightened after surgical interruption of arterial baroreceptor afferents (Amorim et al., 2020). Consistent with these earlier reports, the assessment of baroreceptor function in the present study using the vasoactive method revealed clear evidence of blunted reflex bradycardic and tachycardic responses compared to sham-operated controls.
The present study builds upon our earlier work (Abdelnaby et al., 2025) by extending the investigation of opioid–sepsis interactions to a distinct and more refined experimental framework. Unlike the previous study (Abdelnaby et al., 2025), which focused on systemic cardiovascular parameters like blood pressure, cardiac contractility, and heart rate variability, the current work specifically aims to examine the influence of morphine on arterial baroreflex dysfunction induced by sepsis. By combining direct baroreflex sensitivity assessment in awake animals with intracisternal pharmacological manipulation and molecular analyses within the NTS, this study provides novel, site-specific mechanistic insight into central pathways linking opioid exposure to reflex cardiovascular regulation during sepsis. Addressing this question is especially important as opioids are commonly administered in ICUs to control pain and mitigate stress in critically ill patients, including those suffering from sepsis (Molina, 2006; Wang et al., 2025). Conflicting findings have been reported concerning the effect of opioids on baroreflexes, with studies reporting both inhibitory (Maley and Elde, 1982; Wang and Li, 1988; Zeinivand et al., 2014) and stimulatory effects (Shanazari et al., 2011). These discrepancies are thought to arise from variations in the subject under investigation and dose and duration of opioids. Our finding that morphine inhibited baroreflex-mediated bradycardia, but not tachycardia, in septic rats likely reflects a differential influence of morphine on vagal versus sympathetic arms of the autonomic reflex. This premise is based on reports that baroreflex-induced bradycardia is predominantly mediated by activation of cardiac vagal efferents, whereas reflex tachycardia largely depends on sympathetic excitation (El-Mas et al., 2001; El-Mas et al., 2002; Moore et al., 2022). The presumed selective interaction of morphine with central vagal pools receives support from the observations that μ-opioid receptors are expressed in brainstem vagal nuclei, e.g., nucleus ambiguous and dorsal motor nucleus of the vagus, and that morphine reduces excitatory input to these cardiac vagal neurons (Mansour et al., 1995; Nomura et al., 1996; Venkatesan et al., 2003). Moreover, the present finding that the BRSPE depressant effect of morphine in septic rats was attenuated in rats pretreated with the μ-opioid receptor antagonist naloxone reinforces the critical involvement of μ-opioid receptors in mediating the morphine-induced reduction of vagally driven baroreflex responses.
Pharmacological interventions were employed to explore the roles of central PI3K/MAPKs and NADPHox/ROCK pathways in the amplifying effect of morphine on sepsis-induced impairment of BRSPE. PI3K and MAPK play pivotal roles in the pathophysiology of the inflammatory response to sepsis and associated cardiovascular, autonomic and baroreceptor dysfunctions (El-Naggar et al., 2024; El-Naggar et al., 2025). NADPHox and ROCK are oxidative signals that cross talk with the inflammatory pathway to enhance microglial activation, impair cardiovascular functions and disrupt autonomic reflexes (Liu et al., 2025). In the present study, the inhibitors of these molecular targets were administered into the cisterna magna, a cerebellomedullary area through which cerebrospinal fluid bathes key autonomic nuclei of the brainstem including (Miura and Reis, 1969; Paxinos and Watson, 1986; Chalmers and Pilowsky, 1991; Misu et al., 1996; Abuiessa et al., 2020). Our findings demonstrated that the central inhibition of MAPK-ERK, MAPK-JNK, or ROCK by i.c. PD98056, SP600125 and fasudil, respectively, abolished the depressant effect of morphine on baroreceptor-mediated bradycardia in CLP rats, inferring the involvement of these central pathways in the morphine-BRSPE interaction. By contrast, PI3K or Rho-kinase appear to serve no role in this setting because the inhibition of these molecules by wortmannin and DPI, respectively, failed to reverse the BRSPE depressant effect of morphine. Notably, although MAPKs are frequently activated downstream of PI3K signaling, they can also be triggered via PI3K-independent pathways (Wang et al., 2025). Evidence also exists that ROCK can be activated through mechanisms independent of Rho GTPases (Croft and Olson, 2006).
TLR4 and MCP1 are pivotal mediators of the innate immune response and sepsis pathogenesis. TLR4 causes downstream activation of MAPKs through MyD88-and TRIF-dependent pathways, that leads to the production of cytokine storm triggered by sepsis (Wang et al., 2025). In parallel, MCP1 is a chemokine that recruits monocytes, memory T cells, and dendritic cells to sites of infection and inflammation, thereby amplifying the immune response (Chen et al., 2023). TLR4 activation during sepsis acts through the MAPK/NF-κB pathway to upregulate MCP1 production, which serves as a positive feedback loop that promotes inflammation (Chen et al., 2018). Our results demonstrate that sepsis induced an upregulation of TLR4 and MCP1 immunoreactivity in neuroanatomical areas of the rostral NTS, an effect that was further exacerbated by morphine. The observed parallel between the morphine-induced enhancement of TLR4/MCP1 expression in the NTS and the concurrent suppression of BRSPE suggests a potential causal link between the two effects. Moreover, the ability of naloxone and pharmacological MAPK/ROCK inhibitors to prevent the morphine-induced upregulation of TLR4 and MCP1 points to the involvement of these molecular pathways in mediating the µ-opioid receptor-mediated baroreceptor dysfunction induced by morphine during sepsis. It should be noted that the NTS was specifically targeted due to its major role in receiving and central processing peripheral inflammatory (Waki et al., 2008) and arterial baroreceptor signals (Accorsi-Mendonça and Machado, 2013). Importantly, both TLR4 and MCP1 are expressed in the NTS, and their upregulation has been shown to contribute to cardiovascular dysfunction associated with endotoxemia (Paxinos and Watson, 1986; Nakano et al., 2015; Abuiessa et al., 2020; Sallam et al., 2021b). Furthermore, the identification of opioid receptors and neurons within the NTS (Reeves et al., 2022) supports the potential neuromodulatory role of opioid signaling in modulating the baroreflex-inflammatory axis. It is noteworthy that we focused on the rostral NTS in the present study because our previous findings reported that this region plays a central role in integrating cardiovascular and autonomic responses during septic inflammation (Paxinos and Watson, 1986; Nakano et al., 2015; Abuiessa et al., 2020; Sallam et al., 2021b). Given that both the caudal and rostral NTS are involved in the central regulation of cardiac and neuroinflammatory processes (Phillis et al., 1997; Pereyra et al., 2025), The role of caudal NTS neurons in the CLP/morphine interaction warrants further study to determine potential region-specific differences in inflammatory signaling.
Our finding that morphine increased TLR4 and MCP1 expression in the NTS appears contradictory to the widely recognized immunosuppressive effects of opioids, including morphine (Bettinger and Friedman, 2024). Nonetheless, it is notable that the immunomodulatory effects of morphine are multifaceted and vary depending on the physiological context. In peripheral tissues, morphine is generally immunosuppressive, reducing natural killer (NK) cell activity, inhibiting T and B lymphocyte proliferation, and decreasing cytokine production (Sacerdote, 2006; Liang et al., 2016). In contrast, morphine enhances central neuroinflammation through the binding to TLR4 on microglia, leading to the release of proinflammatory cytokines, and disrupting glutamate transporters (Wang et al., 2012; Eidson et al., 2017).
It is important to comment on two potential limitations of the current study. First, the arterial baroreflex activity was not assessed before individual drug treatments. The absence of baseline baroreflex measurements prevented each rat from serving as its own control, which could have helped minimize the influence of inter-animal variability stemming from differences in basal baroreflex activity. This approach was not feasible in our study because generating two full dose-response curves for phenylephrine and sodium nitroprusside (i.e., before and after individual drug treatments) would necessitate i.v. administration of relatively large volumes of the drug solutions, along with additional flushing between doses. This may potentially cause acute volume overload, a condition that may act in itself to alter autonomic and arterial baroreflex activities (Wong and Johns, 1999; Raimundo Fernades et al., 2020) and perhaps confound data interpretation. Remarkably, spontaneous baroreceptor sensitivity measurement is an alternative to the traditional Oxford vasoactive method. The spontaneous approach exploits natural beat-to-beat fluctuations in systolic blood pressure and the corresponding reflex changes in heart rate or RR intervals observed at rest (Pinna et al., 2015). This method avoids pharmacological interventions, provides insight into real-time physiological baroreflex function, and allows for long-term baroreflex monitoring. However, unlike the vasoactive method, which assesses the full range of baroreflex function, the spontaneous method is limited by its dependence on adequate intrinsic BP variability and its focus on responses around the resting pressure range. Such narrower dynamic range may lead to underestimation of baroreflex sensitivity (Laude et al., 2008; Milic et al., 2009).
The second limitation of the present study concerns the possibility that the analgesia induced by morphine upon i.v. administration may have contributed to its interaction with arterial baroreflex activity. The postoperative pain induced by invasive surgical procedures such as femoral catheterization and laparotomy are believed to be coupled with elevated sympathetic drive, which typically persists for 24–48 h postoperatively and can interfere with cardiovascular function and reflexes (Roughan and Flecknell, 2001; Leach et al., 2011; Zubrzycki et al., 2018). Considering that morphine administration relieves the nociceptive pain and reduces the sympathetic drive (Pertovaara and Wei, 2001; Baby et al., 2021), the possibility cannot be overlooked that the attenuation of baroreflex activity observed in our study could reflect both direct pharmacological effects of morphine on cardiovascular autonomic regulation, and indirect effects mediated via analgesia and reduced nociceptive input. Further studies are warranted to unravel these contributions.
Together, we demonstrate that acute morphine exposure selectively amplifies the sepsis-induced impairment of baroreceptor-mediated bradycardic, but not tachycardic, responses. Pharmacological interventions and protein expression analyses implicate brainstem inflammatory and chemotactic pathways within the neuroanatomical area of the NTS, along with downstream MAPK and ROCK signaling, in mediating the baroreflex effects of morphine. Accordingly, the interruption of these signaling pathways may offer a therapeutic strategy to maintain cardiovascular reflex integrity in patients receiving opioids, particularly in pathological states where baroreflex function is already impaired like sepsis.
Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.
Ethics statement
The animal study was approved by Animal Care and Use Committee of Alexandria University (Approval No. AU/06.2021.2.6.1.93). The study was conducted in accordance with the local legislation and institutional requirements.
Author contributions
MA: Data curation, Formal analysis, Methodology, Writing – original draft. MS: Data curation, Formal analysis, Writing – review and editing. MH: Conceptualization, Formal analysis, Writing – review and editing. HE-G: Conceptualization, Supervision, Writing – review and editing. MR: Conceptualization, Formal analysis, Writing – review and editing. ME-M: Conceptualization, Supervision, Writing – review and editing.
Funding
The author(s) declared that financial support was not received for this work and/or its publication.
Conflict of interest
The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.
Generative AI statement
The author(s) declared that generative AI was not used in the creation of this manuscript.
Any alternative text (alt text) provided alongside figures in this article has been generated by Frontiers with the support of artificial intelligence and reasonable efforts have been made to ensure accuracy, including review by the authors wherever possible. If you identify any issues, please contact us.
Publisher’s note
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
Supplementary material
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphar.2025.1669284/full#supplementary-material
Footnotes
Abbreviations:BRS, baroreflex sensitivity; CLP, cecal ligation and puncture; ERK, extracellular signal-regulated kinase; HR, heart rate; i.c., intracisternal cannulation; ICUs, intensive care units; JNK, c-Jun N-terminal Kinase; MAPKs, mitogen-activated protein kinases; MAP, mean arterial pressure; MCP1, monocyte chemoattractant protein-1; NADPHox, nicotinamide adenine dinucleotide phosphate oxidase; NTS, nucleus tractus solitarius; PE, phenylephrine; PI3K, phosphoinositide-3 kinases; ROCK, rho-associated coiled-coil kinase; RVLM, rostral ventrolateral medullary; SNP, sodium nitroprusside; TLR4, toll-like receptor 4.
References
Abdelnaby, M., Sallam, M. Y., Helmy, M. M., El-Gowelli, H. M., and El-Mas, M. M. (2025). Role of central inflammatory and oxidative pathways in the morphine exacerbation of cardiovascular effects of sepsis in rats. Pharmaceuticals 18 (6), 882. doi:10.3390/ph18060882
Abuiessa, S. A., Wedn, A. M., El-Gowilly, S. M., Helmy, M. M., and El-Mas, M. M. (2020). Pre-eclamptic fetal programming alters neuroinflammatory and cardiovascular consequences of endotoxemia in sex-specific manners. J. Pharmacol. Exp. Ther. 373 (2), 325–336. doi:10.1124/jpet.119.264192
Accorsi-Mendonça, D., and Machado, B. H. (2013). Synaptic transmission of baro- and chemoreceptors afferents in the NTS second order neurons. Aut. Neurosci. 175 (1), 3–8. doi:10.1016/j.autneu.2012.12.002
Amorim, M. R., de Deus, J. L., Cazuza, R. A., Mota, C. M. D., da Silva, L. E. V., Borges, G. S., et al. (2019). Neuroinflammation in the NTS is associated with changes in cardiovascular reflexes during systemic inflammation. J. Neuroinflammation 16 (1), 125. doi:10.1186/s12974-019-1512-6
Amorim, M. R., de Deus, J. L., Pereira, C. A., da Silva, L. E. V., Borges, G. S., Ferreira, N. S., et al. (2020). Baroreceptor denervation reduces inflammatory status but worsens cardiovascular collapse during systemic inflammation. Sci. Rep. 10 (1), 6990. doi:10.1038/s41598-020-63949-x
Anderberg, S. B., Luther, T., and Frithiof, R. (2017). Physiological aspects of toll-like receptor 4 activation in sepsis-induced acute kidney injury. Acta Physiol. (Oxf) 219 (3), 573–588. doi:10.1111/apha.12798
Baby, S. M., Discala, J. F., Gruber, R., Getsy, P. M., Cheng, F., Damron, D. S., et al. (2021). Tempol reverses the negative effects of morphine on arterial blood-gas chemistry and tissue oxygen saturation in freely-moving rats. Front. Pharmacol. 12, 749084. doi:10.3389/fphar.2021.749084
Bate, S. T., and Clark, R. A. (2014). The design and statistical analysis of animal experiments. Cambridge: Cambridge University Press. doi:10.1017/CBO9781139344319
Benarroch, E. E. (2008). The arterial baroreflex. Neurology 71 (21), 1733–1738. doi:10.1212/01.wnl.0000335246.93495.92
Bettinger, J. J., and Friedman, B. C. (2024). Opioids and immunosuppression: clinical evidence, mechanisms of action, and potential therapies. Palliat. Med. Rep. 5 (1), 70–80. doi:10.1089/pmr.2023.0049
Carrara, M., Herpain, A., Baselli, G., and Ferrario, M. (2020). Vascular decoupling in septic shock: the combined role of autonomic nervous system, arterial stiffness, and peripheral vascular tone. Front. Physiol. 11, 594. doi:10.3389/fphys.2020.00594
Chalmers, J., and Pilowsky, P. (1991). Brainstem and bulbospinal neurotransmitter systems in the control of blood pressure. J. Hypertens. 9 (8), 675–694. doi:10.1097/00004872-199108000-00001
Chen, S., Lyu, C., Zhou, J., Huang, S., Zhang, Y., Liu, G., et al. (2018). TLR4 signaling pathway mediates the LPS/ischemia-induced expression of monocytechemotactic protein-induced protein 1 in microglia. Neurosci. Lett. 686, 33–40. doi:10.1016/j.neulet.2018.08.052
Chen, Z., Li, C., and Yu, J. (2023). Monocyte chemoattractant protein-1 as a potential marker for patients with sepsis: a systematic review and meta-analysis. Front. Med. (Lausanne) 10, 1217784. doi:10.3389/fmed.2023.1217784
Chiu, C., and Legrand, M. (2021). Epidemiology of sepsis and septic shock. Curr. Opin. Anaesthesiol. 34, 71–76. doi:10.1097/ACO.0000000000000958
Croft, D. R., and Olson, M. F. (2006). The Rho GTPase effector ROCK regulates cyclin A, cyclin D1, and p27Kip1 levels by distinct mechanisms. Mol. Cell Biol. 26 (12), 4612–4627. doi:10.1128/mcb.02061-05
Deutschman, C. S., and Tracey, K. J. (2014). Sepsis: current dogma and new perspectives. Immunity 40 (4), 463–475. doi:10.1016/j.immuni.2014.04.001
Eidson, L. N., Inoue, K., Young, L. J., Tansey, M. G., and Murphy, A. Z. (2017). Toll-like receptor 4 mediates morphine-induced neuroinflammation and tolerance via soluble tumor necrosis factor signaling. Neuropsychopharmacology 42 (3), 661–670. doi:10.1038/npp.2016.131
El-Mas, M. M., and Abdel-Rahman, A. A. (1992). Role of aortic baroreceptors in ethanol-induced impairment of baroreflex control of heart rate in conscious rats. J. Pharmacol. Exp. Ther. 262 (1), 157–165. doi:10.1016/s0022-3565(25)10729-5
El-Mas, M. M., and Abdel-Rahman, A. A. (1993). Direct evidence for selective involvement of aortic baroreceptors in ethanol-induced impairment of baroreflex control of heart rate. J. Pharmacol. Exp. Ther. 264 (3), 1198–1205. doi:10.1016/s0022-3565(25)10131-6
El-Mas, M. M., Afify, E. A., Mohy El-Din, M. M., Omar, A. G., and Sharabi, F. M. (2001). Testosterone facilitates the baroreceptor control of reflex bradycardia: role of cardiac sympathetic and parasympathetic components. J. Cardiovasc. Pharmacol. 38, 754–763. doi:10.1097/00005344-200111000-00012
El-Mas, M. M., Afify, E. A., Omar, A. G., and Sharabi, F. M. (2002). Cyclosporine adversely affects baroreflexes via inhibition of testosterone modulation of cardiac vagal control. J. Pharmacol. Exp. Ther. 301 (1), 346–354. doi:10.1124/jpet.301.1.346
El-Mas, M. M., El-Gowelli, H. M., Ghazal, A. R., Harraz, O. F., and Mohy El-Din, M. M. (2009). Facilitation of central imidazoline I(1)-site/extracellular signal-regulated kinase/p38 mitogen-activated protein kinase signalling mediates the hypotensive effect of ethanol in rats with acute renal failure. Br. J. Pharmacol. 158 (6), 1629–1640. doi:10.1111/j.1476-5381.2009.00444.x
El-Mas, M. M., El-Gowilly, S. M., Fouda, M. A., and Saad, E. I. (2011). Role of adenosine A2A receptor signaling in the nicotine-evoked attenuation of reflex cardiac sympathetic control. Toxicol. Appl. Pharmacol. 254 (3), 229–237. doi:10.1016/j.taap.2011.04.014
El-Mas, M. M., Omar, A. G., Helmy, M. M., and Mohy El-Din, M. M. (2012). Crosstalk between central pathways of nitric oxide and carbon monoxide in the hypertensive action of cyclosporine. Neuropharmacology 62 (4), 1890–1896. doi:10.1016/j.neuropharm.2011.12.017
El-Naggar, A. E., Helmy, M. M., El-Gowilly, S. M., and El-Mas, M. M. (2023). Adenosine A1 receptors of the medullary solitary tract arbitrate the nicotine counteraction of neuroinflammation and cardiovascular dysfunction in septic rats. Sci. Rep. 13 (1), 17818. doi:10.1038/s41598-023-44601-w
El-Naggar, A. E., Helmy, M. M., El-Gowilly, S. M., and El-Mas, M. M. (2024). Suppression by central adenosine A3 receptors of the cholinergic defense against cardiovascular aberrations of sepsis: role of PI3K/MAPKs/NFκB signaling. Front. Pharmacol. 15, 1418981. doi:10.3389/fphar.2024.1418981
El-Naggar, A. E., Helmy, M. M., El-Gowilly, S. M., and El-Mas, M. M. (2025). The cholinergic amelioration of sepsis-induced baroreflex dysfunction and brainstem inflammation is negated by central adenosine A3 receptors. Pharm. (Basel) 18 (3), 388. doi:10.3390/ph18030388
Fairchild, K. D., Saucerman, J. J., Raynor, L. L., Sivak, J. A., Xiao, Y., Lake, D. E., et al. (2009). Endotoxin depresses heart rate variability in mice: cytokine and steroid effects. Am. J. Physiol. Regul. Integr. Comp. Physiol. 297 (4), R1019–R1027. doi:10.1152/ajpregu.00132.2009
Fujita, M., Ando, K., Nagae, A., and Fujita, T. (2007). Sympathoexcitation by oxidative stress in the brain mediates arterial pressure elevation in salt-sensitive hypertension. Hypertension 50 (2), 360–367. doi:10.1161/HYPERTENSIONAHA.107.091009
Gerritsen, J., Dekker, J. M., TenVoorde, B. J., Kostense, P. J., Heine, R. J., Bouter, L. M., et al. (2001). Impaired autonomic function is associated with increased mortality, especially in subjects with diabetes, hypertension, or a history of cardiovascular disease: the Hoorn study. Diabetes Care 24 (10), 1793–1798. doi:10.2337/diacare.24.10.1793
Gong, W., and Wen, H. (2019). Sepsis induced by cecal ligation and puncture. Methods Mol. Biol. 1960, 249–255. doi:10.1007/978-1-4939-9167-9_22
Gordin, D., Vikatmaa, P., Vikatmaa, L., Groop, P. H., Albäck, A., and Tikkanen, I. (2016). Baroreflex activation therapy in the treatment of resistant hypertension. Duodecim 132 (20), 1874–1881.
Greer, J. R. (2015). Pathophysiology of cardiovascular dysfunction in sepsis. BJA Educ. 15 (6), 316–321. doi:10.1093/bjaceaccp/mkv003
Karim, S., Chahal, A., Khanji, M. Y., Petersen, S. E., and Somers, V. K. (2023). Autonomic cardiovascular control in health and disease. Compr. Physiol. 13 (2), 4493–4511. doi:10.1002/cphy.c210037
Krantz, M. J., Palmer, R. B., and Haigney, M. C. P. (2021). Cardiovascular complications of opioid use. JACC 77(2), 205–223. doi:10.1016/j.jacc.2020.11.002
Kumar, M., and Bansal, N. (2018). Fasudil hydrochloride ameliorates memory deficits in rat model of streptozotocin-induced Alzheimer's disease: involvement of PI3-kinase, eNOS and NFkappaB. Behav. Brain Res. 351, 4–16. doi:10.1016/j.bbr.2018.05.024
Kushikata, T., Yoshida, H., Kudo, M., Kudo, T., Kudo, T., and Hirota, K. (2011). Role of coerulean noradrenergic neurones in general anaesthesia in rats. Br. J. Anaesth. 107 (6), 924–929. doi:10.1093/bja/aer303
Laude, D., Baudrie, V., and Elghozi, J. L. (2008). Applicability of recent methods used to estimate spontaneous baroreflex sensitivity to resting mice. Am. J. Physiol. Regul. Integr. Comp. Physiol. 294 (1), R142–R150. doi:10.1152/ajpregu.00319.2007
Leach, M. C., Coulter, C. A., Richardson, C. A., and Flecknell, P. A. (2011). Are we looking in the wrong place? Implications for behavioural-based pain assessment in rabbits (Oryctolagus cuniculi) and beyond? PLoS One 6 (3), e13347. doi:10.1371/journal.pone.0013347
Liang, X., Liu, R., Chen, C., Ji, F., and Li, T. (2016). Opioid system modulates the immune function: a review. Transl. Perioper. Pain Med. 1 (1), 5–13.
Liu, Z., Li, F., Li, N., Chen, Y., and Chen, Z. (2025). MicroRNAs as regulators of cardiac dysfunction in sepsis: pathogenesis and diagnostic potential. Front. Cardiovasc. Med. 12, 1517323. doi:10.3389/fcvm.2025.1517323
Maley, B., and Elde, R. (1982). Immunohistochemical localization of putative neurotransmitters within the feline nucleus tractus solitarii. Neuroscience 7 (10), 2469–2490. doi:10.1016/0306-4522(82)90208-1
Mansour, A., Fox, C. A., Burke, S., Akil, H., and Watson, S. J. (1995). Immunohistochemical localization of the cloned mu opioid receptor in the rat CNS. J. Chem. Neuroanat. 8 (4), 283–305. doi:10.1016/0891-0618(95)00055-c
Merdji, H., Siegemund, M., and Meziani, F. (2022). Acute and long-term cardiovascular complications among patients with sepsis and septic shock. J. Clin. Med. 11 (24), 7362. doi:10.3390/jcm11247362
Milanez, M. I. O., Liberatore, A. M. A., Nishi, E. E., Bergamaschi, C. T., Campos, R. R., and Koh, I. H. J. (2022). Patterns of renal and splanchnic sympathetic vasomotor activity in an animal model of survival to experimental sepsis. Braz J. Med. Biol. Res. 55, e11873. doi:10.1590/1414-431X2021e11873
Milic, M., Sun, P., Liu, F., Fainman, C., Dimsdale, J., Mills, P. J., et al. (2009). A comparison of pharmacologic and spontaneous baroreflex methods in aging and hypertension. J. Hypertens. 27 (6), 1243–1251. doi:10.1097/HJH.0b013e32832a6e1b
Misu, Y., Goshima, Y., Ueda, H., and Okamura, H. (1996). Neurobiology of L-DOPAergic systems. Prog. Neurobiol. 49 (5), 415–454. doi:10.1016/0301-0082(96)00025-1
Miura, M., and Reis, D. J. (1969). Termination and secondary projections of carotid sinus nerve in the cat brain stem. Am. J. Physiol. 217 (1), 142–153. doi:10.1152/ajplegacy.1969.217.1.142
Molina, P. E. (2006). Opioids and opiates: analgesia with cardiovascular, haemodynamic and immune implications in critical illness. J. Intern. Med. 259 (2), 138–154. doi:10.1111/j.1365-2796.2005.01569.x
Moore, J. P., Simpson, L. L., and Drinkhill, M. J. (2022). Differential contributions of cardiac, coronary and pulmonary artery vagal mechanoreceptors to reflex control of the circulation. J. Physiol. 600 (18), 4069–4087. doi:10.1113/jp282305
Mowry, F. E., Peaden, S. C., Stern, J. E., and Biancardi, V. C. (2021). TLR4 and AT1R mediate blood-brain barrier disruption, neuroinflammation, and autonomic dysfunction in spontaneously hypertensive rats. Pharmacol. Res. 174, 105877. doi:10.1016/j.phrs.2021.105877
Nakano, Y., Furube, E., Morita, S., Wanaka, A., Nakashima, T., and Miyata, S. (2015). Astrocytic TLR4 expression and LPS-induced nuclear translocation of STAT3 in the sensory circumventricular organs of adult mouse brain. J. Neuroimmunol. 278, 144–158. doi:10.1016/j.jneuroim.2014.12.013
Nguyen, H. B., Rivers, E. P., Abrahamian, F. M., Moran, G. J., Abraham, E., Trzeciak, S., et al. (2006). Severe sepsis and septic shock: review of the literature and emergency department management guidelines. Ann. Emerg. Med. 48 (1), 28–54. doi:10.1016/j.annemergmed.2006.02.015
Nomura, S., Ding, Y. Q., Kaneko, T., Li, J. L., and Mizuno, N. (1996). Localization of mu-opioid receptor-like immunoreactivity in the central components of the vagus nerve: a light and electron microscope study in the rat. Neuroscience 73 (1), 277–286. doi:10.1016/0306-4522(96)00027-9
Paxinos, G., and Watson, C. (1986). Atlas of the rat brain in stereotaxic coordinates. New York: Academic.
Pereyra, K., Diaz-Jara, E., Bernal-Santander, I., Vicencio, S., Del Rio, R., and Iturriaga, R. (2025). Carotid bodies mediate glial cell activation and neuroinflammation in the NTS following long-term intermittent hypoxia: role in cardiorespiratory dysfunction. Am. J. Physiol. Lung Cell Mol. Physiol. 328 (3), L357–l371. doi:10.1152/ajplung.00280.2024
Pertovaara, A., and Wei, H. (2001). Peripheral effects of morphine in neuropathic rats: role of sympathetic postganglionic nerve fibers. Eur. J. Pharmacol. 429 (1-3), 139–145. doi:10.1016/s0014-2999(01)01315-2
Phillis, J. W., Scislo, T. J., and O'Leary, D. S. (1997). Purines and the nucleus tractus solitarius: effects on cardiovascular and respiratory function. Clin. Exp. Pharmacol. Physiol. 24 (9-10), 738–742. doi:10.1111/j.1440-1681.1997.tb02124.x
Pinna, G. D., Maestri, R., and La Rovere, M. T. (2015). Assessment of baroreflex sensitivity from spontaneous oscillations of blood pressure and heart rate: proven clinical value? Physiol. Meas. 36 (4), 741–753. doi:10.1088/0967-3334/36/4/741
Raimundo Fernades, E. M., de Moura, S. S., Silva, R. O., Totou, N. L., Baleeiro, R. D. S., de Oliveira, E. C., et al. (2020). Acute volume expansion decreased baroreflex response after swimming but not after running exercise training in hypertensive rats. Clin. Exp. Hypertens. 42 (5), 460–468. doi:10.1080/10641963.2019.1693588
Reeves, K. C., Shah, N., Muñoz, B., and Atwood, B. K. (2022). Opioid receptor-mediated regulation of neurotransmission in the brain. Front. Mol. Neurosci. 15, 919773. doi:10.3389/fnmol.2022.919773
Roughan, J. V., and Flecknell, P. A. (2001). Behavioural effects of laparotomy and analgesic effects of ketoprofen and carprofen in rats. Pain 90 (1-2), 65–74. doi:10.1016/s0304-3959(00)00387-0
Sacerdote, P. (2006). Opioids and the immune system. Palliat. Med. 20 (Suppl. 1), s9–s15. doi:10.1191/0269216306pm1124oa
Salah, H. M., Gupta, R., Hicks, A. J., Mahmood, K., Haglund, N. A., Bindra, A. S., et al. (2025). Baroreflex function in cardiovascular disease. J. Cardiac Fail. 31 (1), 117–126. doi:10.1016/j.cardfail.2024.08.062
Sallam, M. Y., El-Gowilly, S. M., Abdel-Galil, A. G., and El-Mas, M. M. (2016). Modulation by central MAPKs/PI3K/sGc of the TNF-α/iNOS-dependent hypotension and compromised cardiac autonomic control in endotoxic rats. J. Cardiovasc Pharmacol. 68 (2), 171–181. doi:10.1097/fjc.0000000000000400
Sallam, M. Y., El-Gowilly, S. M., Fouda, M. A., Abd-Alhaseeb, M. M., and El-Mas, M. M. (2019). Brainstem cholinergic pathways diminish cardiovascular and neuroinflammatory actions of endotoxemia in rats: role of NFκB/α7/α4β2AChRs signaling. Neuropharmacology 157, 107683. doi:10.1016/j.neuropharm.2019.107683
Sallam, M. Y., El-Gowilly, S. M., and El-Mas, M. M. (2021a). Androgenic modulation of arterial baroreceptor dysfunction and neuroinflammation in endotoxic male rats. Brain Res. 1756, 147330. doi:10.1016/j.brainres.2021.147330
Sallam, M. Y., El-Gowilly, S. M., and El-Mas, M. M. (2021b). Cardiac and brainstem neuroinflammatory pathways account for androgenic incitement of cardiovascular and autonomic manifestations in endotoxic Male rats. J. Cardiovasc Pharmacol. 77 (5), 632–641. doi:10.1097/fjc.0000000000000993
Shanazari, A. A., Aslani, Z., Ramshini, E., and Alaei, H. (2011). Acute and chronic effects of morphine on cardiovascular system and the baroreflexes sensitivity during severe increase in blood pressure in rats. ARYA Atheroscler. 7 (3), 111–117.
Sharshar, T., Blanchard, A., Paillard, M., Raphael, J. C., Gajdos, P., and Annane, D. (2003). Circulating vasopressin levels in septic shock. Crit. Care Med. 31 (6), 1752–1758. doi:10.1097/01.Ccm.0000063046.82359.4a
Shen, M. J., and Zipes, D. P. (2014). Role of the autonomic nervous system in modulating cardiac arrhythmias. Circ. Res. 114 (6), 1004–1021. doi:10.1161/circresaha.113.302549
Shen, F. M., Guan, Y. F., Xie, H. H., and Su, D. F. (2004). Arterial baroreflex function determines the survival time in lipopolysaccharide-induced shock in rats. Shock 21 (6), 556–560. doi:10.1097/01.shk.0000126647.51109.5c
Shi, K. Y., Shen, F. M., Liu, A. J., Chu, Z. X., Cao, Y. L., and Su, D. F. (2007). The survival time post-cecal ligation and puncture in sinoaortic denervated rats. J. Cardiovasc Pharmacol. 50 (2), 162–167. doi:10.1097/FJC.0b013e31805c942d
Singer, M., Deutschman, C. S., Seymour, C. W., Shankar-Hari, M., Annane, D., Bauer, M., et al. (2016). The third international consensus definitions for sepsis and septic shock (Sepsis-3). JAMA 315 (8), 801–810. doi:10.1001/jama.2016.0287
Stocker, S. D., Sved, A. F., and Andresen, M. C. (2019). Missing pieces of the Piezo1/Piezo2 baroreceptor hypothesis: an autonomic perspective. J. Neurophysiol. 122 (3), 1207–1212. doi:10.1152/jn.00315.2019
Tanaka, M., Suemaru, K., Watanabe, S., Cui, R., Li, B., and Araki, H. (2008). Comparison of Short- and long-acting benzodiazepine-receptor agonists with different receptor selectivity on motor coordination and muscle relaxation following thiopental-induced Anesthesia in mice. J. Pharmacol. Sci. 107 (3), 277–284. doi:10.1254/jphs.FP0071991
Ulleryd, M. A., Prahl, U., Börsbo, J., Schmidt, C., Nilsson, S., Bergström, G., et al. (2017). The association between autonomic dysfunction, inflammation and atherosclerosis in men under investigation for carotid plaques. PLoS One 12 (4), e0174974. doi:10.1371/journal.pone.0174974
Venkatesan, P., Baxi, S., Evans, C., Neff, R., Wang, X., and Mendelowitz, D. (2003). Glycinergic inputs to cardiac vagal neurons in the nucleus ambiguus are inhibited by nociceptin and mu-selective opioids. J. Neurophysiol. 90 (3), 1581–1588. doi:10.1152/jn.01117.2002
Waki, H., Gouraud, S. S., Maeda, M., and Paton, J. F. R. (2008). Specific inflammatory condition in nucleus tractus solitarii of the SHR: novel insight for neurogenic hypertension? Aut. Neurosci. 142 (1), 25–31. doi:10.1016/j.autneu.2008.07.003
Wang, Q., and Li, P. (1988). Inhibition of baroreflex following microinjection of GABA or morphine into the nucleus tractus solitarii in rabbits. J. Aut. Nerv. Syst. 25 (2), 165–172. doi:10.1016/0165-1838(88)90021-5
Wang, X., Loram, L. C., Ramos, K., de Jesus, A. J., Thomas, J., Cheng, K., et al. (2012). Morphine activates neuroinflammation in a manner parallel to endotoxin. Proc. Natl. Acad. Sci. U. S. A. 109 (16), 6325–6330. doi:10.1073/pnas.1200130109
Wang, W., Mu, S., Yan, D., Qin, H., and Zheng, Z. (2025). Comprehending toll-like receptors: pivotal element in the pathogenesis of sepsis and its complications. Front. Immunol. 16, 1591011. doi:10.3389/fimmu.2025.1591011
Watso, J. C., Belval, L. N., Cimino, F. A., 3rd, Orth, B. D., Hendrix, J. M., Huang, M., et al. (2022). Low-dose morphine reduces pain perception and blood pressure, but not muscle sympathetic outflow, responses during the cold pressor test. Am. J. Physiol. Heart Circ. Physiol. 323 (1), H223–h234. doi:10.1152/ajpheart.00092.2022
Wong, P. S., and Johns, E. J. (1999). Effect of acute saline volume loading on renal sympathetic nerve activity in anaesthetised fructose-fed and fat-fed rats. J. Auton. Nerv. Syst. 75 (1), 60–69. doi:10.1016/s0165-1838(98)00180-5
Young, P. J., and De Jong, A. (2021). New insights into the comparative effectiveness of fentanyl and morphine infusions in ICU patients. Am. J. Respir. Crit. Care Med. 204 (11), 1243–1245. doi:10.1164/rccm.202109-2112ED
Zeinivand, M., Shamsizadeh, A., Pourshanazari, A. A., Hassanshahi, G., and Allatavakoli, M. (2014). Morphine and nicotine addiction and withdrawal influence baroreflex sensitivity and blood pressure in two-kidney one clip hypertensive (2K1C) rats. Bratisl. Lek. Listy 115 (12), 743–748. doi:10.4149/bll_2014_143
Zhu, T., Liao, X., Feng, T., Wu, Q., Zhang, J., Cao, X., et al. (2017). Plasma monocyte chemoattractant protein 1 as a predictive marker for sepsis prognosis: a prospective cohort study. Tohoku J. Exp. Med. 241 (2), 139–147. doi:10.1620/tjem.241.139
Keywords: arterial baroreceptors, brainstem, inflammation, morphine, oxidative stress, sepsis
Citation: Abdelnaby M, Sallam MY, Helmy MM, El-Gowelli HM, Rao MS and El-Mas MM (2026) Inflammatory and chemotactic signals of the brainstem solitary tract mediate the morphine exacerbation of impaired reflex chronotropism in septic rats. Front. Pharmacol. 16:1669284. doi: 10.3389/fphar.2025.1669284
Received: 19 July 2025; Accepted: 29 December 2025;
Published: 16 January 2026.
Edited by:
Narasaiah Kolliputi, University of South Florida, United StatesReviewed by:
Roberto Luisetto, University of Padua, ItalyYasmin Hisham, Konkuk University, Republic of Korea
Copyright © 2026 Abdelnaby, Sallam, Helmy, El-Gowelli, Rao and El-Mas. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Mahmoud M. El-Mas, bWFobW91ZC5lbG1hc0BrdS5lZHUua3c=, bWFobW91ZC5lbG1hc0BhbGV4dS5lZHUuZWc=