ABSTRACT
Our previous study showed that neuronal apoptosis was significantly increased upon treatment of conditioned medium (CM) from necroptotic astrocytes (NAS), leaving the underlying mechanism unclear. Considering the nutritive and supportive roles of astrocytes, we first examined the neurotrophic phenotype of necroptotic astrocytes with focus on glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF), two important neurotrophic factors, and it was unexpectedly found that the expression of GDNF and BDNF were up-regulated in necroptotic astrocytes in vitro. A question was raised as to whether the functional secreted forms of neurotrophic factors were increased. Considering that extracellular vesicles (EVs) were carriers of secreted substances and their roles in cellular interaction, we isolated EVs from astrocytes and found EVs from normal and necroptotic astrocytes (EVs-NAS) had characteristics of exosomes. We then examined GDNF and BDNF in EVs-NAS, and BDNF was interestingly found as an immature form of pro-BDNF. The expression of pro-BDNF was found to be increased in EVs-NAS, and EVs-NAS had a negative effect on neuronal survival. To verify that whether pro-BDNF was involved in the detrimental effect of EVs-NAS, anti-pro-BDNF antibody was applied, and we found that neuronal apoptosis-induced by EVs-NAS could be significantly attenuated by blocking pro-BDNF, which suggested that necroptotic astrocytes induced neuronal apoptosis partially through EVs-derived pro-BDNF. The data expand our understanding in neurotrophic phenotype of necroptotic astrocytes, and may provide us new strategies targeting on EVs-NAS in treatment of neurological diseases.
Subject(s)
Brain-Derived Neurotrophic Factor , Extracellular Vesicles , Apoptosis , Astrocytes/metabolism , Brain-Derived Neurotrophic Factor/metabolism , Cells, Cultured , Extracellular Vesicles/metabolism , Glial Cell Line-Derived Neurotrophic Factor/metabolism , Protein PrecursorsABSTRACT
In mature mammalian brains, the endocannabinoid system (ECS) plays an important role in the regulation of synaptic plasticity and the functioning of neural networks. Besides, the ECS also contributes to the neurodevelopment of the central nervous system. Due to the increase in the medical and recreational use of cannabis, it is inevitable and essential to elaborate the roles of the ECS on neurodevelopment. GABAergic interneurons represent a group of inhibitory neurons that are vital in controlling neural network activity. However, the role of the ECS in the neurodevelopment of GABAergic interneurons remains to be fully elucidated. In this review, we provide a brief introduction of the ECS and interneuron diversity. We focus on the process of interneuron development and the role of ECS in the modulation of interneuron development, from the expansion of the neural stem/progenitor cells to the migration, specification and maturation of interneurons. We further discuss the potential implications of the ECS and interneurons in the pathogenesis of neurological and psychiatric disorders, including epilepsy, schizophrenia, major depressive disorder and autism spectrum disorder.
Subject(s)
Autism Spectrum Disorder , Depressive Disorder, Major , Neural Stem Cells , Animals , Endocannabinoids , Humans , InterneuronsABSTRACT
Previous studies have shown that CCL2 may cause chronic pain, but the exact mechanism of central sensitization is unclear. In this article, we further explore the presynaptic role of CCL2. Behavioral experiments show that intervertebral foramen injection CCR2 antagonists into dorsal root ganglion (DRG) can inhibit the inflammatory pain caused by CCL2 in spinal cord. We raised the question of the role of presynaptic CCR2 in the spinal dorsal horn. Subsequent electron microscopy experiments showed that CCR2 was expressed in the presynaptic CGRP terminal in the spinal dorsal horn. CCL2 can enhance presynaptic calcium signal. Whole-cell patch-clamp recordings showed that CCL2 can enhance NMDAR-eEPSCs through presynaptic effects, and further application of glutamate sensor method proved that CCL2 can act on presynaptic CCR2 to increase the release of presynaptic glutamate. In conclusion, we suggest that CCL2 can directly act on the CCR2 on presynaptic terminals of sensory neurons in the spinal dorsal horn, leading to an increase in the release of presynaptic glutamate and participate in the formation of central sensitization.
Subject(s)
Chemokine CCL2/metabolism , Nociceptors/metabolism , Pain/metabolism , Pain/physiopathology , Presynaptic Terminals/metabolism , Receptors, CCR2/metabolism , Spinal Cord/physiopathology , Synaptic Transmission/physiology , Animals , Benzoxazines/pharmacology , Calcitonin Gene-Related Peptide/metabolism , Calcium Signaling/drug effects , Ganglia, Spinal/drug effects , Ganglia, Spinal/metabolism , Glutamic Acid/metabolism , Hyperalgesia/complications , Inflammation/pathology , Injections, Spinal , Mice, Inbred C57BL , Neurons/drug effects , Neurons/metabolism , Pain/complications , Presynaptic Terminals/drug effects , Protein Binding/drug effects , Spinal Cord/drug effects , Spinal Cord/ultrastructure , Spinal Cord Dorsal Horn/drug effects , Spinal Cord Dorsal Horn/ultrastructure , Spiro Compounds/pharmacology , Synaptic Transmission/drug effects , Up-Regulation/drug effectsABSTRACT
The pre-Bötzinger complex (pre-BötC) residing in the ventrolateral medulla oblongata, is thought to be the kernel of respiratory rhythmogenesis. Episodic hypoxia exerts respiratory long-term facilitation, being recognized as electrophysiological characteristic of respiratory motor neuroplasticity. Our previous study demonstrated up-regulated expression of phospho-protein kinase C θ (P-PKCθ) in the pre-BötC of rats receiving chronic intermittent hypoxic (CIH) challenge. The present study was aimed to examine subcellular distribution of P-PKC substrates (P-PKCsub) and explore PKC down-stream targeting proteins in the pre-BötC in normoxic and CIH rats. Using neurokinin-1 receptor (NK1R) as a marker of the pre-BötC, P-PKCsub immunoreactivity was revealed by immunofluorescence and immuno-electron microscopic double-labeling in the pre-BötC. Western blot was applied to analyze P-PKCsub proteins in ventrolateral medulla, containing the pre-BötC. The results showed that NK1R immunoreactivity (NK1R-ir) was expressed mainly along plasma membranes of somata and processes, outlining pre-BötC neurons under the light microscope. P-PKCsub immunoreactive (P-PKCsub-ir) fluorophores in dot-like appearance appeared in somata and processes. Some were in close apposition to plasma membranes. A majority of P-PKCsub-ir neurons was found with NK1R-ir. CIH challenge up-regulated the expression of P-PKCsub proteins in the ventrolateral medulla. Under the electron microscope, NK1R-ir product was found to distribute along the inner membrane surfaces of somata and dendrites. P-PKCsub-ir gold particles were located in somata and dendrites, and some were distributed along the inner membrane surfaces, as well as in the endoplasmic reticulum and postsynaptic dense body. These results suggest that CIH challenge up-regulates the expression of P-PKCsub proteins, probably including some receptor proteins in the postsynaptic membrane, which may contribute to respiratory neuroplasticity via activation of PKCθ in the pre-BötC.
Subject(s)
Medulla Oblongata , Receptors, Neurokinin-1 , Animals , Hypoxia , Medulla Oblongata/metabolism , Neurons/metabolism , Rats , Rats, Sprague-Dawley , Receptors, Neurokinin-1/metabolismABSTRACT
Mitochondrial bioenergetics is dynamically coupled with neuronal activities, which are altered by hypoxia-induced respiratory neuroplasticity. Here we report structural features of postsynaptic mitochondria in the pre-Bötzinger complex (pre-BötC) of rats treated with chronic intermittent hypoxia (CIH) simulating a severe condition of obstructive sleep apnea. The subcellular changes in dendritic mitochondria and histochemistry of cytochrome c oxidase (CO) activity were examined in pre-BötC neurons localized by immunoreactivity of neurokinin 1 receptors. Assays of mitochondrial electron transport chain (ETC) complex I, IV, V activities, and membrane potential were performed in the ventrolateral medulla containing the pre-BötC region. We found significant decreases in the mean length and area of dendritic mitochondria in the pre-BötC of CIH rats, when compared to the normoxic control and hypoxic group with daily acute intermittent hypoxia (dAIH) that evokes robust synaptic plasticity. Notably, these morphological alterations were mainly observed in the mitochondria in close proximity to the synapses. In addition, the proportion of mitochondria presented with enlarged compartments and filamentous cytoskeletal elements in the CIH group was less than the control and dAIH groups. Intriguingly, these distinct characteristics of structural adaptability were observed in the mitochondria within spatially restricted dendritic spines. Furthermore, the proportion of moderately to darkly CO-reactive mitochondria was reduced in the CIH group, indicating reduced mitochondrial activity. Consistently, mitochondrial ETC enzyme activities and membrane potential were lowered in the CIH group. These findings suggest that hypoxia-induced respiratory plasticity was characterized by spatially confined mitochondrial alterations within postsynaptic spines in the pre-BötC neurons. In contrast to the robust plasticity evoked by dAIH preconditioning, a severe CIH challenge may weaken the local mitochondrial bioenergetics that the fuel postsynaptic activities of the respiratory motor drive.
Subject(s)
Dendritic Spines/metabolism , Hypoxia/metabolism , Medulla Oblongata/metabolism , Mitochondria/ultrastructure , Animals , Dendritic Spines/ultrastructure , Electron Transport Chain Complex Proteins/metabolism , Hypoxia/pathology , Medulla Oblongata/ultrastructure , Membrane Potential, Mitochondrial , Mitochondria/metabolism , Rats , Rats, Sprague-Dawley , Synapses/metabolism , Synapses/ultrastructureABSTRACT
Understanding the precise intracellular localization of lead (Pb) is a key in deciphering processes in Pb-induced toxicology. However, it is a great challenge to trace Pb in vitro, especially in cultured cells. We aimed to find an innovative and efficient approach to investigate distribution of Pb in cells and to validate it through determining the subcellular Pb content. We identified its ultra-structural distribution with autometallography under electron microscopy in a choroidal epithelial Z310 cell line. Electron microscopy in combination with energy-dispersive X-ray spectroscope (EDS) was employed to provide further evidence of Pb location. In addition, Pb content was determined in the cytosol, membrane/organelle, nucleus and cytoskeleton fractions with atomic absorption spectroscopy. Pb was found predominantly inside the nuclear membranes and some was distributed in the cytoplasm under low-concentration exposure. Nuclear existence of Pb was verified by EDS under electron microscopy. Once standardized for protein content, Pb percentage in the nucleus fraction reached the highest level (76%). Our results indicate that Pb is accumulated mainly in the nucleus of choroid plexus. This method is sensitive and precise in providing optimal means to study the ultra-structural localization of Pb for in vitro models. In addition, it offers the possibility of localization of other metals in cultured cells. Some procedures may also be adopted to detect target proteins via immunoreactions.
ABSTRACT
Mitochondria, as primary energy generators and Ca2+ biosensor, are dynamically coupled to neuronal activities, and thus play a role in neuroplasticity. Here we report that respiratory neuroplasticity induced by daily acute intermittent hypoxia (dAIH) evoked adaptive changes in the ultrastructure and postsynaptic distribution of mitochondria in the pre-Bötzinger complex (pre-BötC). The metabolic marker of neuronal activity, cytochrome c oxidase (CO), and dendritic mitochondria were examined in pre-BötC neurons of adult Sprague-Dawley rats preconditioned with dAIH, which is known to induce long-term facilitation (LTF) in respiratory neural activities. We performed neurokinin 1 receptor (NK1R) pre-embedding immunocytochemistry to define pre-BötC neurons, in combination with CO histochemistry, to depict ultrastructural alterations and CO activity in dendritic mitochondria. We found that the dAIH challenge significantly increased CO activity in pre-BötC neurons. Darkly CO-reactive mitochondria at postsynaptic sites in the dAIH group were much more prevalent than those in the normoxic control. In addition, the length and area of mitochondria were significantly increased in the dAIH group, implying a larger surface area of cristae for ATP generation. There was a fine, structural remodeling, notably enlarged and branching mitochondria or tapered mitochondria extending into dendritic spines. Mitochondrial cristae were mainly in parallel-lamellar arrangement, indicating a high efficiency of energy generation. Moreover, flocculent or filament-like elements were noted between the mitochondria and the postsynaptic membrane. These morphological evidences, together with increased CO activity, demonstrate that dendritic mitochondria in the pre-BötC responded dynamically to respiratory plasticity. Hence, plastic neuronal changes are closely coupled to active mitochondrial bioenergetics, leading to enhanced energy production and Ca2+ buffering that may drive the LTF expression.
Subject(s)
Dendrites/pathology , Electron Transport Complex IV/metabolism , Hypoxia/enzymology , Hypoxia/pathology , Mitochondria/pathology , Respiratory Center/enzymology , Adenosine Triphosphate/biosynthesis , Animals , Dendrites/ultrastructure , Dendritic Spines/pathology , Dendritic Spines/ultrastructure , Energy Metabolism , Long-Term Potentiation , Mitochondria/ultrastructure , Neuronal Plasticity , Rats , Rats, Sprague-Dawley , Receptors, Neurokinin-1/biosynthesisABSTRACT
The rostral ventrolateral medulla (RVLM) contains cardiovascular-related catecholaminergic neurons and respiratory-related pre-Bötzinger complex (pre-BötC) neurons, which are intermingled and functionally connected for coordinating cardiorespiratory activities. Daily acute intermittent hypoxia (dAIH) is known to elicit respiratory plasticity. However, it is unclear if the catecholaminergic neurons directly synapse onto pre-BötC neurons, and if the local circuitry exhibits plasticity when exposed to dAIH. The present study was aimed to determine the synaptic phenotypes between dopamine-ß-hydroxylase (DßH)-immunoreactive (ir) catecholaminergic neurons and neurokinin 1 receptor (NK1R)-ir pre-BötC neurons, and the effect of dAIH on the neuronal network. Immunofluorescence histochemistry was used to reveal immunoreactivities of DßH and NK1R in the RVLM of normoxic and dAIH rats. Synaptic phenotypes were examined with double-labeling immunoelectron microscopy. We found that DßH immunoreactivity was expressed in somata and processes, some of which were in close apposition to NK1R-ir pre-BötC neurons. DßH-ir gold particles were localized to somata, dendrites, and axonal terminals. DßH-ir terminals formed asymmetric synapses, and occasionally, symmetric synapses in the pre-BötC, featuring the local circuitry. Of the synapses, 28% in normoxic and 29.6% in dAIH groups were apposed to NK1R-ir dendrites. Significant increases in DßH expression and NK1R-ir processes were found in the dAIH group. Moreover, the area and number of processes in close appositions were significantly elevated, strongly suggesting that dAIH induced plasticity with increased connections and interactions between the cardiovascular- and respiratory-related neurons in the local circuitry. In conclusion, asymmetric synapses are predominant in the crosstalk between catecholaminergic and pre-BötC neurons in the RVLM, elaborating excitatory transmission driving the coupling of cardiorespiratory activities. The neural network manifests plasticity in response to dAIH challenge.
Subject(s)
Catecholamines/metabolism , Hypoxia/pathology , Medulla Oblongata/pathology , Neurons/physiology , Respiratory Center/pathology , Synapses/metabolism , Animals , Dopamine beta-Hydroxylase/metabolism , Dopamine beta-Hydroxylase/ultrastructure , Male , Microscopy, Immunoelectron , Neurons/ultrastructure , Rats , Rats, Sprague-Dawley , Receptors, Neurokinin-1/metabolism , Receptors, Neurokinin-1/ultrastructure , Respiratory Center/ultrastructure , Synapses/ultrastructureABSTRACT
Cannabinoid type 1 receptor (CB1R), which is traditionally located on axon terminals, plays an important role in the pathology of epilepsy and neurodegenerative diseases by modulating synaptic transmission. Using the pilocarpine model of chronic spontaneous recurrent seizures, which mimics the main features of mesial temporal lobe epilepsy (TLE) with hippocampal sclerosis (HS) in humans, we examined the expression of CB1R in hippocampal astrocytes of epileptic rats. Furthermore, we also examined the expression of astrocytic CB1R in the resected hippocampi from patients with medically refractory mesial TLE. Using immunofluorescent double labeling, we found increased expression of astrocytic CB1R in hippocampi of epileptic rats, whereas expression of astrocytic CB1R was not detectable in hippocampi of saline treated animals. Furthermore, CB1R was also found in some astrocytes in sclerotic hippocampi in a subset of patients with intractable mesial TLE. Detection with immune electron microscopy showed that the expression of CB1R was increased in astrocytes of epileptic rats and modest levels of CB1R were also found on the astrocytic membrane of sclerotic hippocampi. These results suggest that increased expression of astrocytic CB1R in sclerotic hippocampi might be involved in the cellular basis of the effects of cannabinoids on epilepsy.
Subject(s)
Astrocytes/metabolism , Epilepsy, Temporal Lobe/pathology , Hippocampus/pathology , Receptor, Cannabinoid, CB1/biosynthesis , Adult , Animals , Convulsants/toxicity , Disease Models, Animal , Epilepsy, Temporal Lobe/metabolism , Female , Fluorescent Antibody Technique , Hippocampus/metabolism , Humans , Immunohistochemistry , Male , Microscopy, Immunoelectron , Pilocarpine/toxicity , Rats , Rats, Sprague-Dawley , Sclerosis/metabolism , Sclerosis/pathology , Seizures/metabolism , Seizures/pathology , Young AdultABSTRACT
Relapse is a major clinical problem and remains a major challenge in the treatment of drug addiction. There is strong evidence that the endocannabinoid system of the nucleus accumben core (NAcc) is involved in drug-seeking behavior, as well as in the mechanisms that underlie relapse to drug use. To reveal the mechanism that underlies this finding, we examined the expression pattern of the cannabinoid receptor 1 (CB1-R) in the NAcc of SD rats that had been undergoing morphine withdrawal (MW) for 1 day, 3 days and 3 weeks (acute, latent and chronic phases, respectively). Morphine exposure induced conditioned place preference (CPP) in rats. Significant increase of CB1-R expression in NAcc was observed in animals in the 1 day, 3 days and 3 weeks morphine withdrawal compare to the control group. Immunofluorescence labeling showed axonal fibers or terminals by fluorescence microscope observation. Immunoelectron microscopy detection showed silver-gold particles located in the presynaptic membranes that mainly give rise to symmetrical synapses. Quantitative electron microscopy showed an increase in number of CB1-R-positive terminals in the morphine withdrawal groups and the number of immunogold particles was significantly increased at these inhibitory terminals. We also confirmed that infusions of the CB1-R antagonist rimonabant into the NAcc attenuated the CPP during morphine withdrawal. Our present data have thus indicated that increasing pattern of CB1-R expression in the NAcc during above morphine withdrawal phases, which might underlie the relapse associated drug seeking behavior after morphine withdrawal.
Subject(s)
Disease Models, Animal , Morphine Dependence/metabolism , Nucleus Accumbens/metabolism , Receptor, Cannabinoid, CB1/biosynthesis , Substance Withdrawal Syndrome/metabolism , Up-Regulation/physiology , Animals , Male , Rats , Rats, Sprague-Dawley , Rats, WistarABSTRACT
OBJECTIVES: This study examined the use of methacryloxylethyl cetyl dimethyl ammonium chloride (DMAE-CB) as a potential matrix metalloproteinases (MMPs) inhibitor on both soluble recombinant and dentine matrix-bound endogenous MMPs, meanwhile attempted to determine the effective anti-MMP group of quaternary ammonium methacrylates (QAMs). METHODS: The possible inhibitory effects of five DMAE-CB mass concentrations (0.1%, 0.5%, 1%, 3%, 5%) on soluble rhMMP-9 were measured using a colorimetic assay kit. Methyl methacrylate (MMA) and [2-(methacryloyloxy)ethyl] trimethylammonium chloride (METMAC) were also screened against rhMMP-9 to compare the inhibitory effect with DMAE-CB. Matrix-bound endogenous MMP-activity was evaluated in completely demineralized dentine beams. Thirty beams were randomly divided into three groups (N=10) and respectively placed into 500µL of calcium- and zinc-containing media (CM; control), 0.2% chlorhexidine or 3% DMAE-CB in CM aged for 30 days. The changes in modulus of elasticity, loss of dry mass and solubilization of collagen peptides were measured via three-point bending, precision weighing and hydroxyproline assay, respectively. RESULTS: 0.5-5% mass concentrations of DMAE-CB were highly effective (P<0.05) in inhibiting rhMMP-9 varied between 76.56±6.44% and 97.06±3.24%, the inhibitory effect of MMA was much lower than that of METMAC and DMAE-CB at the same concentration (P<0.05). Dentine beams incubated in 3% DMAE-CB showed only a 26.34% decrease in the modulus of elasticity (75.74% decrease in control), a 1.72±1.56% loss of dry mass (29.70±9.12% loss in control), and significantly less solubilized hydroxyproline when compared with the control (P<0.05). CONCLUSIONS: DMAE-CB is effective in inhibiting both soluble recombinant MMPs and matrix-bound dentine MMPs. Quaternary ammonium group is the effective anti-MMP group of QAMs. CLINICAL SIGNIFICANCE: The incorporation of DMAE-CB into dental adhesives has the potential to enhance the durability of dentine bonding and thus increases the longevity of restorations.
Subject(s)
Dentin/enzymology , Matrix Metalloproteinase Inhibitors/pharmacology , Methacrylates/pharmacology , Quaternary Ammonium Compounds/pharmacology , Chlorhexidine/pharmacology , Collagen/drug effects , Colorimetry , Dentin/chemistry , Dipeptides/pharmacology , Elastic Modulus , Humans , Hydroxyproline/analysis , Materials Testing , Matrix Metalloproteinase 9/drug effects , Methylmethacrylate/pharmacology , Pliability , Solubility , Time Factors , Tooth Demineralization/enzymology , Tooth Demineralization/metabolismABSTRACT
The pre-Bötzinger complex (pre-BötC) in the ventrolateral medulla oblongata is a presumed kernel of respiratory rhythmogenesis. Ca(2+) -activated non-selective cationic current is an essential cellular mechanism for shaping inspiratory drive potentials. Ca(2+) /calmodulin-dependent protein kinase II (CaMKII), an ideal 'interpreter' of diverse Ca(2+) signals, is highly expressed in neurons in mediating various physiological processes. Yet, less is known about CaMKII activity in the pre-BötC. Using neurokinin-1 receptor as a marker of the pre-BötC, we examined phospho (P)-CaMKII subcellular distribution, and found that P-CaMKII was extensively expressed in the region. P-CaMKII-ir neurons were usually oval, fusiform, or pyramidal in shape. P-CaMKII immunoreactivity was distributed within somas and dendrites, and specifically in association with the post-synaptic density. In dendrites, most synapses (93.1%) examined with P-CaMKII expression were of asymmetric type, occasionally with symmetric type (6.9%), whereas in somas, 38.1% were of symmetric type. P-CaMKII asymmetric synaptic identification implicates that CaMKII may sense and monitor Ca(2+) activity, and phosphorylate post-synaptic proteins to modulate excitatory synaptic transmission, which may contribute to respiratory modulation and plasticity. In somas, CaMKII acts on both symmetric and asymmetric synapses, mediating excitatory and inhibitory synaptic transmission. P-CaMKII was also localized to the perisynaptic and extrasynaptic regions in the pre-BötC.
Subject(s)
Calcium-Calmodulin-Dependent Protein Kinase Type 2/biosynthesis , Medulla Oblongata/enzymology , Synapses/enzymology , Synaptic Transmission/physiology , Animals , Calcium-Calmodulin-Dependent Protein Kinase Type 2/analysis , Immunohistochemistry , Medulla Oblongata/ultrastructure , Microscopy, Electron, Transmission , Neurons/enzymology , Neurons/ultrastructure , Rats , Rats, Sprague-DawleyABSTRACT
PURPOSE: To determine whether bonds of contemporary etch-and-rinse adhesives made with ethanol-wet bonding are stronger and more durable than those made with water-wet bonding, and to explore the possible reasons for the bonding results. MATERIALS AND METHODS: Flat surfaces of midcoronal dentin were made in extracted human third molars. The dentin surfaces were randomized into 6 groups according to bonding techniques (water- vs ethanol-wet bonding) and dental adhesives [Single Bond 2 (SB), Prime Bond NT (PB), and Gluma Comfort Bond (GB)]. After etching and rinsing, dentin surfaces were either left water-moist or immersed in ethanol. Following adhesive application and composite buildups, the bonded teeth were sectioned into beams for microtensile bond strength evaluation with or without NaOCl challenge. The morphology of the hybrid layer was analyzed with SEM. The wettability of water- vs. ethanol-saturated dentin was evaluated. The concentrations of non-volatile ingredients in the adhesives were compared. RESULTS: Compared to water-wet bonding, ethanol-wet bonding yielded similar (p > 0.05 for PB and GB) or higher (p < 0.05 for SB) 24-h bond strength, displayed significantly higher bond strength after chemical challenge (p < 0.05, for all three adhesives), and produced more even hybrid layers. Moreover, ethanol-saturated dentin exhibited a lower contact angle than water-saturated specimens, and the concentrations of non-volatile ingredients of the adhesives decreased in the order of SB > GB > PB. CONCLUSION: Ethanol-wet bonding could improve the bonding efficacy of contemporary etch-and-rinse adhesives, probably due to the good wettability of ethanol-saturated dentin and the structure of the hybrid layer. Moreover, this positive effect of ethanol-wet bonding might be influenced by the composition of adhesives.