Pilocarpine mediated excessive calcium accumulation leads to ciliary muscle cell senescence and apoptosis.

The ciliary muscle constitutes a crucial element in refractive regulation. Investigating the pathophysiological mechanisms within the ciliary muscle during excessive contraction holds significance in treating ciliary muscle dysfunction. A guinea pig model of excessive contraction of the ciliary muscle induced by drops pilocarpine was employed, alongside the primary ciliary muscle cells was employed in in vitro experiments. The results of the ophthalmic examination showed that pilocarpine did not significantly change refraction and axial length during the experiment, but had adverse effects on the regulatory power of the ciliary muscle. The current data reveal notable alterations in the expression profiles of hypoxia inducible factor 1 (HIF-1α), ATP2A2, P53, α-SMA, Caspase-3, and BAX within the ciliary muscle of animals subjected to pilocarpine exposure, alongside corresponding changes observed in cultured cells treated with pilocarpine. Augmented levels of ROS were detected in both tissue specimens and cells, culminating in a significant increase in cell apoptosis in in vivo and in vitro experiments. Further examination revealed that pilocarpine induced an increase in intracellular Ca2+ levels and disrupted MMP, as evidenced by mitochondrial swelling and diminished cristae density compared to control conditions, concomitant with a noteworthy decline in antioxidant enzyme activity. However, subsequent blockade of Ca2+ channels in cells resulted in downregulation of HIF-1α, ATP2A2, P53, α-SMA, Caspase-3, and BAX expression, alongside ameliorated mitochondrial function and morphology. The inhibition of Ca2+ channels presents a viable approach to mitigate ciliary cells damage and sustain proper ciliary muscle function by curtailing the mitochondrial damage induced by excessive contractions.

Cholinergic and behavior-dependent beta and gamma waves are coupled between olfactory bulb and hippocampus.

Olfactory oscillations may enhance cognitive processing through coupling with beta (ß, 15-30 Hz) and gamma (γ, 30-160 Hz) activity in the hippocampus (HPC). We hypothesize that coupling between olfactory bulb (OB) and HPC oscillations is increased by cholinergic activation in control rats and is reduced in kainic-acid-treated epileptic rats, a model of temporal lobe epilepsy. OB γ2 (63-100 Hz) power was higher during walking and immobility-awake (IMM) compared to sleep, while γ1 (30-57 Hz) power was higher during grooming than other behavioral states. Muscarinic cholinergic agonist pilocarpine (25 mg/kg ip) with peripheral muscarinic blockade increased OB power and OB-HPC coherence at ß and γ1 frequency bands. A similar effect was found after physostigmine (0.5 mg/kg ip) but not scopolamine (10 mg/kg ip). Pilocarpine increased bicoherence and cross-frequency coherence (CFC) between OB slow waves (SW, 1-5 Hz) and hippocampal ß, γ1 and γ2 waves, with stronger coherence at CA1 alveus and CA3c than CA1 stratum radiatum. Bicoherence further revealed a nonlinear interaction of ß waves in OB with ß waves at the CA1-alveus. Beta and γ1 waves in OB or HPC were segregated at one phase of the OB-SW, opposite to the phase of γ2 and γ3 (100-160 Hz) waves, suggesting independent temporal processing of ß/γ1 versus γ2/γ3 waves. At CA1 radiatum, kainic-acid-treated epileptic rats compared to control rats showed decreased theta power, theta-ß and theta-γ2 CFC during baseline walking, decreased CFC of HPC SW with γ2 and γ3 waves during baseline IMM, and decreased coupling of OB SW with ß and γ2 waves at CA1 alveus after pilocarpine. It is concluded that ß and γ waves in the OB and HPC are modulated by a slow respiratory rhythm, in a cholinergic and behavior-dependent manner, and OB-HPC functional connectivity at ß and γ frequencies may enhance cognitive functions.

PPARß/δ Agonist GW0742 Modulates Microglial and Astroglial Gene Expression in a Rat Model of Temporal Lobe Epilepsy.

The role of astroglial and microglial cells in the pathogenesis of epilepsy is currently under active investigation. It has been proposed that the activity of these cells may be regulated by the agonists of peroxisome proliferator-activated nuclear receptors (PPARs). This study investigated the effects of a seven-day treatment with the PPAR ß/δ agonist GW0742 (Fitorine, 5 mg/kg/day) on the behavior and gene expression of the astroglial and microglial proteins involved in the regulation of epileptogenesis in the rat brain within a lithium-pilocarpine model of temporal lobe epilepsy (TLE). TLE resulted in decreased social and increased locomotor activity in the rats, increased expression of astro- and microglial activation marker genes (Gfap, Aif1), pro- and anti-inflammatory cytokine genes (Tnfa, Il1b, Il1rn), and altered expression of other microglial (Nlrp3, Arg1) and astroglial (Lcn2, S100a10) genes in the dorsal hippocampus and cerebral cortex. GW0742 attenuated, but did not completely block, some of these impairments. Specifically, the treatment affected Gfap gene expression in the dorsal hippocampus and Aif1 gene expression in the cortex. The GW0742 injections attenuated the TLE-specific enhancement of Nlrp3 and Il1rn gene expression in the cortex. These results suggest that GW0742 may affect the expression of some genes involved in the regulation of epileptogenesis.

Transdermal pilocarpine on the skin over salivary glands to increase salivation: an in vivo study.

BACKGROUND: Hyposalivation is treated using oral cholinergic drugs; however, systemic side effects occasionally lead to discontinuation of treatment. We aimed to investigate the effects of transdermal pilocarpine on the salivary gland skin on saliva secretion and safety in rats. METHODS: Pilocarpine was administered to rats orally (0.5 mg/kg) or topically on the salivary gland skin (5 mg/body). Saliva volume, the number of sweat dots, and fecal weight were measured along with pilocarpine concentration in plasma and submandibular gland tissues. RESULTS: Saliva volume significantly increased 0.5 h after oral administration and 0.5, 3, and 12 h after topical administration. Fecal weight and sweat dots increased significantly 1 h after oral administration; however, no changes were observed after topical application. The pilocarpine concentration in the submandibular gland tissues of the topical group was higher than that in the oral group at 0.5, 3, and 12 h of administration. CONCLUSIONS: Pilocarpine application to salivary gland skin persistently increased salivary volume in rats without inducing sweating or diarrhea. Transdermal pilocarpine applied to the skin over the salivary glands may be an effective and safe treatment option for hyposalivation.

Dose-Dependent Induction of Differential Seizure Phenotypes by Pilocarpine in Rats: Considerations for Translational Potential.

Background and Objectives: Pilocarpine is used in experimental studies for testing antiepileptic drugs, but further characterization of this model is essential for its usage in testing novel drugs. The aim of our study was to study the behavioral and EEG characteristics of acute seizures caused by different doses of pilocarpine in rats. Materials and Methods: Male Wistar rats were treated with a single intraperitoneal dose of 100 mg/kg (P100), 200 mg/kg (P200), or 300 mg/kg (P300) of pilocarpine, and epileptiform behavior and EEG changes followed within 4 h. Results: The intensity and the duration of seizures were significantly higher in P300 vs. the P200 and P100 groups, with status epilepticus dominating in P300 and self-limiting tonic-clonic seizures in the P200 group. The seizure grade was significantly higher in P200 vs. the P100 group only during the first hour after pilocarpine application. The latency of seizures was significantly shorter in P300 and P200 compared with P100 group. Conclusions: Pilocarpine (200 mg/kg) can be used as a suitable model for the initial screening of potential anti-seizure medications, while at a dose of 300 mg/kg, it can be used for study of the mechanisms of epileptogenesis.

Effect of reflex and mechanical decreases in skin perfusion on thermal- and agonist-induced eccrine sweating in humans.

In humans, skin blood flux (SkBF) and eccrine sweating are tightly coupled, suggesting common neural control and regulation. This study was designed to separate these two sympathetic nervous system end-organ responses via nonadrenergic SkBF-decreasing mechanical perturbations during heightened sudomotor drive. We induced sweating physiologically via whole body heat stress using a high-density tube-lined suit (protocol 1; 2 women, 4 men), and pharmacologically via forearm intradermal microdialysis of two steady-state doses of a cholinergic agonist, pilocarpine (protocol 2; 4 women, 3 men). During sweating induction, we decreased SkBF via three mechanical perturbations: arm and leg dependency to engage the cutaneous venoarteriolar response (CVAR), limb venous occlusion to engage the CVAR and decrease perfusion pressure, and limb arterial occlusion to cause ischemia. In protocol 1, heat stress increased arm cutaneous vascular conductance and forearm sweat rate (capacitance hygrometry). During heat stress, despite decreases in SkBF during each of the acute (3 min) mechanical perturbations, eccrine sweat rate was unaffected. During heat stress with extended (10 min) ischemia, sweat rate decreased. In protocol 2, both pilocarpine doses (ED50 and EMAX) increased SkBF and sweat rate. Each mechanical perturbation resulted in decreased SkBF but minimal changes in eccrine sweat rate. Taken together, these data indicate that a wide range of acute decreases in SkBF do not appear to proportionally decrease either physiologically- or pharmacologically induced eccrine sweating in peripheral skin. This preservation of evaporative cooling despite acutely decreased SkBF could have consequential impacts for heat storage and balance during changes in body posture, limb position, or blood flow restrictive conditions.

Transdermal iontophoretic application of l-NAME is available in sweating research induced by heat stress in young healthy adults.

Iontophoretic transdermal administration of NG-nitro-l-arginine methyl ester hydrochloride [l-NAME, a nitric oxide synthase (NOS) inhibitor] has been used as a non-invasive evaluation of NOS-dependent mechanisms in human skin. However, the availability has yet to be investigated in sweating research. Prior observations using invasive techniques (e.g., intradermal microdialysis technique) to administer l-NAME have implicated that NOS reduces sweating induced by heat stress but rarely influences the response induced by the administration of cholinergic muscarinic receptor agonists. Therefore, we investigated whether the transdermal iontophoretic administration of l-NAME modulates sweating similar to those prior observations. Twenty young healthy adults (10 males, 10 females) participated in two experimental protocols on separate days. Before each protocol, saline (control) and 1% l-NAME were bilaterally administered to the forearm skin via transdermal iontophoresis. In protocol 1, 0.001% and 1% pilocarpine were iontophoretically administered at l-NAME-treated and untreated sites. In protocol 2, passive heating was applied by immersing the lower limbs in hot water (43 °C) until the rectal temperature increased by 0.8 °C above baseline. The sweat rate was continuously measured throughout both protocols. Pilocarpine-induced sweat rate was not significantly different between the control and l-NAME-treated sites in both pilocarpine concentrations (P ≥ 0.316 for the treatment effect and interaction of treatment and pilocarpine concentration). The sweat rate during passive heating was attenuated at the l-NAME-treated site relative to the control (treatment effect, P = 0.020). Notably, these observations are consistent with prior sweating studies administrating l-NAME into human skin using intradermal microdialysis techniques. Based on the similarity of our results with already known observations, we conclude that transdermal iontophoresis of l-NAME is a valid non-invasive technique for the investigation of the mechanisms of sweating related to NOS during heat stress.

Hybridization into a Bitopic Ligand Increased Muscarinic Receptor Activation for Isopilocarpine but Not for Pilocarpine Derivatives.

Pilocarpine (1), a secondary metabolite of several Pilocarpus species, is a therapeutically used partial agonist of muscarinic acetylcholine receptors (mAChRs). The available pharmacological data and structure-activity relationships do not provide comparable data for all five receptor subtypes. In this study, pilocarpine (1), its epimer isopilocarpine (2), racemic analogues pilosinine (3) and desmethyl pilosinine (4), and the respective hybrid ligands with a naphmethonium fragment (5-C6 to 8-C6) were synthesized and analyzed in mini-G nano-BRET assays at the five mAChRs. In line with earlier studies, pilocarpine was the most active compound among the orthosteric ligands 1-4. Computational docking of pilocarpine and isopilocarpine to the active M2 receptor suggests that the trans-configuration of isopilocarpine leads to a loss of the hydrogen bond from the lactone carbonyl to N6.52, explaining the lower activity of isopilocarpine. Hybrid formation of pilocarpine (1) and isopilocarpine (2) led to an inverted activity rank, with the trans-configured isopilocarpine hybrid (6-C6) being more active. The hydrogen bond of interest is formed by the isopilocarpine hybrid (6-C6) but not by the pilocarpine hybrid (5-C6). Hybridization thus leads to a modified binding mode of the orthosteric moiety, as the binding mode of the hybrid is dominated by the high-affinity allosteric moiety.

PET Imaging and Neurohistochemistry Reveal that Curcumin Attenuates Brain Hypometabolism and Hippocampal Damage Induced by Status Epilepticus in Rats.

Numerous preclinical studies provide evidence that curcumin, a polyphenolic phytochemical extracted from Curcuma longa (turmeric) has neuroprotective, anti-inflammatory and antioxidant properties against various neurological disorders. Curcumin neuroprotective effects have been reported in different animal models of epilepsy, but its potential effect attenuating brain glucose hypometabolism, considered as an early marker of epileptogenesis that occurs during the silent period following status epilepticus (SE), still has not been addressed. To this end, we used the lithium-pilocarpine rat model to induce SE. Curcumin was administered orally (300 mg/kg/day, for 17 days). Brain glucose metabolism was evaluated in vivo by 2-deoxy-2-[18F]Fluoro-D-Glucose ([18F]FDG) positron emission tomography (PET). In addition, hippocampal integrity, neurodegeneration, microglia-mediated neuroinflammation, and reactive astrogliosis were evaluated as markers of brain damage. SE resulted in brain glucose hypometabolism accompanied by body weight (BW) loss, hippocampal neuronal damage, and neuroinflammation. Curcumin did not reduce the latency time to the SE onset, nor the mortality rate associated with SE. Nevertheless, it reduced the number of seizures, and in the surviving rats, curcumin protected BW and attenuated the short-term glucose brain hypometabolism as well as the signs of neuronal damage and neuroinflammation induced by the SE. Overall, our results support the potential adaptogen-like effects of curcumin attenuating key features of SE-induced brain damage.

Modulation of Membrane Trafficking of AQP5 in the Lens in Response to Changes in Zonular Tension Is Mediated by the Mechanosensitive Channel TRPV1.

In mice, the contraction of the ciliary muscle via the administration of pilocarpine reduces the zonular tension applied to the lens and activates the TRPV1-mediated arm of a dual feedback system that regulates the lens' hydrostatic pressure gradient. In the rat lens, this pilocarpine-induced reduction in zonular tension also causes the water channel AQP5 to be removed from the membranes of fiber cells located in the anterior influx and equatorial efflux zones. Here, we determined whether this pilocarpine-induced membrane trafficking of AQP5 is also regulated by the activation of TRPV1. Using microelectrode-based methods to measure surface pressure, we found that pilocarpine also increased pressure in the rat lenses via the activation of TRPV1, while pilocarpine-induced removal of AQP5 from the membrane observed using immunolabelling was abolished by pre-incubation of the lenses with a TRPV1 inhibitor. In contrast, mimicking the actions of pilocarpine by blocking TRPV4 and then activating TRPV1 resulted in sustained increase in pressure and the removal of AQP5 from the anterior influx and equatorial efflux zones. These results show that the removal of AQP5 in response to a decrease in zonular tension is mediated by TRPV1 and suggest that regional changes to PH2O contribute to lens hydrostatic pressure gradient regulation.

Structural Insights into M1 Muscarinic Acetylcholine Receptor Signaling Bias between Gαq and ß-Arrestin through BRET Assays and Molecular Docking.

The selectivity of drugs for G protein-coupled receptor (GPCR) signaling pathways is crucial for their therapeutic efficacy. Different agonists can cause receptors to recruit effector proteins at varying levels, thus inducing different signaling responses, called signaling bias. Although several GPCR-biased drugs are currently being developed, only a limited number of biased ligands have been identified regarding their signaling bias for the M1 muscarinic acetylcholine receptor (M1mAChR), and the mechanism is not yet well understood. In this study, we utilized bioluminescence resonance energy transfer (BRET) assays to compare the efficacy of six agonists in inducing Gαq and ß-arrestin2 binding to M1mAChR. Our findings reveal notable variations in agonist efficacy in the recruitment of Gαq and ß-arrestin2. Pilocarpine preferentially promoted the recruitment of ß-arrestin2 (∆∆RAi = -0.5), while McN-A-343 (∆∆RAi = 1.5), Xanomeline (∆∆RAi = 0.6), and Iperoxo (∆∆RAi = 0.3) exhibited a preference for the recruitment of Gαq. We also used commercial methods to verify the agonists and obtained consistent results. Molecular docking revealed that certain residues (e.g., Y404, located in TM7 of M1mAChR) could play crucial roles in Gαq signaling bias by interacting with McN-A-343, Xanomeline, and Iperoxo, whereas other residues (e.g., W378 and Y381, located in TM6) contributed to ß-arrestin recruitment by interacting with Pilocarpine. The preference of activated M1mAChR for different effectors may be due to significant conformational changes induced by biased agonists. By characterizing bias towards Gαq and ß-arrestin2 recruitment, our study provides insights into M1mAChR signaling bias.

A pilot investigation on the effect of induced saliva flow on digestive parameters in sheep, and a comparison with cattle.

Sheep with a relatively low methane yield were observed to have shorter fluid and particle mean retention times (MRT). Because the application of pilocarpine, a saliva stimulant, was successful in reducing retention times in ruminants in previous studies, we applied this substance to sheep, expecting a reduction in MRT and methane yield. Three non-pregnant sheep (74 ± 10 kg) were fed a hay-only diet in a 3 × 3 Latin square design with oral doses of 0, 2.5 and 5 mg pilocarpine/kg body weight and day. Measurements included feed and water intake, MRT of liquid and particulate phases in the reticulorumen (RR) and total gastrointestinal tract (GIT), ruminal microbial yield (via urinary purine bases and metabolic faecal nitrogen), total tract methane emission, apparent nutrient digestibility and rumen fluid parameters. Data were investigated for linear and quadratic effects using orthogonal polynomial contrasts. The MRT of liquid and small particles in the RR and total GIT, and the short-chain fatty acid concentration in rumen fluid, linearly declined with increasing pilocarpine dosage, while no quadratic relationship was detected. Intake of feed DM and water, apparent nutrient digestibility, methane yield and microbial yield were not affected by pilocarpine. When combining the sheep data with that of a similar experiment in cattle, we found that the MRT of the liquid phase was positively associated with estimated NDF digestibility and with methane production per digested NDF, but was not associated with microbial yield or the ratio of acetate to propionate. The ratio between MRT of the particulate and the liquid phase was smaller for sheep than that for cattle, and was not affected by treatment. Differences in this ratio might explain why species reacted differently to the saliva-inducing agent, which might help to explain the discrepancy between species in the effect of induced saliva flow on digestive parameters.

Effect of induced saliva flow on fluid retention time, ruminal microbial yield and methane emission in cattle.

Both in vitro and animal studies indicated that a higher dilution rate is related to a more efficient microbial synthesis and a lower methane (CH4 ) yield. The latter could be a consequence of the former, as an increase in microbial cell synthesis offers an alternative hydrogen sink competing with methanogenesis. To test this assumption in live animals, we applied a saliva stimulant, pilocarpine, to modify liquid flow rate in cattle. Four non-lactating cows (750 ± 71 kg) were fed forage only (restricted to constant intake) in a 4 × 4 Latin square design with oral doses of 0, 1, 2.5 and 5mg pilocarpine/kg body weight and day. We quantified feed and water intake, ruminal and total tract mean retention time (MRT) of solute and particle markers, ruminal microbial yield (via urinary purine bases or metabolic faecal nitrogen), CH4 emission, digestibility, chewing behaviour, reticular motility and rumen fluid parameters. The effect of induced saliva flow was evident by visibly increased salivation and water intake. Increasing the pilocarpine dosages resulted in a linearly decreased MRT of fluid and small particles (p < 0.001 and p< 0.05, respectively) and methane yield as related to digested DM (p < 0.05), the latter at a magnitude of 5%. No effect of treatment was found on ruminal microbial yield estimated via purine derivates. Metabolic faecal N as an indicator of microbial growth linearly correlated with pilocarpine dosages (p < 0.05). No significant relationship was found between pilocarpine dosages and large particle MRT, nutrient digestibility, ruminal pH and short-chain fatty acids. In conclusion, different from some in vitro studies, there was little indication of a reciprocal effect of CH4 and microbial biomass production in cows fed a forage-only diet.

Effects of tetraethylammonium-sensitive K+ channel blockade on cholinergic and thermal sweating in endurance-trained and untrained men.

NEW FINDINGS: What is the central question of this study? Does inhibition of K+ channels modulate the exercise-training-induced augmentation in cholinergic and thermal sweating? What is the main finding and its importance? Iontophoretic administration of tetraethylammonium, a K+ channel blocker, blunted sweating induced by a low dose (0.001%) of the cholinergic agent pilocarpine, but not heat-induced sweating. However, no differences in the cholinergic sweating were observed between young endurance-trained and untrained men. Thus, while K+ channels play a role in the regulation of eccrine sweating, they do not contribute to the increase in sweating commonly observed in endurance-trained adults. Our findings provide important new insights into the mechanisms underlying the regulation of sweating by endurance conditioning. ABSTRACT: We evaluated the hypothesis that the activation of K+ channels mediates the exercise-training-induced augmentation of cholinergic and thermal sweating. On separate days, 11 endurance-trained and 10 untrained men participated in two experimental protocols. Prior to each protocol, we administered 2% tetraethylammonium (TEA, K+ channels blocker) and saline (Control) at forearm skin sites on both arms via transdermal iontophoresis. In protocol 1, low (0.001%) and high (1%) doses of pilocarpine were administered at the TEA-treated and Control sites over a 60-min period. In protocol 2, participants were passively heated by immersing their lower limbs in hot water (43°C) until core (rectal) temperature (Tc ) increased by 0.8°C above resting levels. Administration of TEA attenuated cholinergic sweating (P = 0.001) during the initial 20 min after the treatment of low dose of pilocarpine only whilst the response was similar between the groups (P = 0.163). Cholinergic and thermal sweating were higher in the trained relative to the untrained men (all P ≤ 0.033). Thermal sweating reached ∼90% of the response at a Tc elevation of 0.8°C during the initial 20 min of passive heating, which corresponds to the period wherein TEA attenuated cholinergic sweating in protocol 1. However, sweating did not differ between the Control and TEA sites in either group (P = 0.704). We showed that activation of K+ channels does not appear to mediate the elevated sweating response induced by a low dose of pilocarpine in trained men. We also demonstrated that K+ channels do not contribute to sweating during heat stress in either group.

Effects of GABAB receptor positive allosteric modulator BHF177 and IRS-1 on apoptosis of hippocampal neurons in rats with refractory epilepsy via the PI3K/Akt pathway.

The present study was conducted to determine the effects of the γ-aminobutyric acid B (GABAB ) receptor positive allosteric modulator BHF177 on refractory epilepsy (RE). An RE rat model was initially established via treatment with lithium-pilocarpine. The RE rats were then treated with BHF177 or the GABAB receptor antagonist CGP46381, followed by recording of their seizure rate and assessment of their spatial learning in the Morris water maze test. Treatment of BHF177 reduced the seizure intensity, whereas this effect was revered upoj treatment with CGP46381. Immunohistochemistry revealed that BHF177 treatment diminished P-glycoprotein (P-gp) expression in the hippocampal tissues of RE rats. Next, we found that BHF177 activated GABAB receptor, resulting in upregulated expression of insulin receptor substrate 1 (IRS-1) and PI3K, as well as antiapoptotic factors (Bcl-2 and mTOR), along with suppression of the apoptosis factors Bax and cleaved caspase-3 in the hippocampal tissues. Further, activation of GABAB receptors by BHF177 alleviated the inflammatory response in hippocampal tissues of RE rats, as evidenced by reduced VCAM-1, ICAM-1, and tumor necrosis factor-α levels. Next, we treated primary cultured rat hippocampal neurons with BHF177 and the IRS-1 selective inhibitor NT157. BHF177 inhibited hippocampal apoptosis in rat hippocampal neurons by regulating the IRS-1/PI3K/Akt axis through crosstalk between GABAB and insulin-like growth factor-1 receptors. Collectively, our findings indicate that the BHF177 inhibited neuron apoptosis, thus protecting against RE through the IRS-1/PI3K/Akt axis, which may present a new therapeutic channel for RE.

Ellagic acid and allopurinol decrease H2O2 concentrations, epileptiform activity and astrogliosis after status epilepticus in the hippocampus of adult rats.

Status epilepticus (SE) can result in an overproduction of hydrogen peroxide (H2O2), which contributes to oxidative stress and brain injury during different phases of epileptogenesis and seizures. The purpose of this study was to evaluate the effects of ellagic acid and allopurinol administered after SE on H2O2 concentrations, electrical activity and GFAP immunoreactivity in the hippocampus of rats evaluated on Day 18 after SE. H2O2 levels were measured using an online technique with high temporal resolution and simultaneous electrical activity recording. For this purpose, the lateral ventricles of male Wistar rats (200-250 g) were injected with pilocarpine (2.4 mg/2 µl) to induce SE. After SE, rats were injected with ellagic acid (50 mg/kg i.p., and two additional doses at 24 and 48 h) or allopurinol (50 mg/kg i.p., single dose). Administration of ellagic acid or allopurinol after SE significantly reduced the H2O2 concentrations and decreased the presence of epileptiform activity and GFAP immunoreactivity in the hippocampus 18 days after SE. In conclusion, the administration of antioxidants potentially reduces oxidative stress, which indicates the possible attenuation of the neurobiological consequences after SE.

Nicotinamide mitigates radiation injury in submandibular gland by protecting mitochondrial structure and functions.

BACKGROUND: Radiation damage to salivary gland is inevitable in head and neck cancer patients receiving radiotherapy. Safe and effective treatments for protecting salivary glands from radiation are still unavailable. Mitochondrial damage is a critical mechanism in irradiated salivary gland; however, treatment targeting mitochondria has not received much attention. Nicotinamide is a key component of the mitochondrial metabolism. Here, we investigated the effects and underlying mechanisms of nicotinamide on protecting irradiated submandibular gland. METHODS: Submandibular gland cells and tissues were randomly divided into four groups: control, nicotinamide alone, radiation alone, and radiation with nicotinamide pretreatment. Cell viability was detected by PrestoBlue cell viability reagent. Histopathological alterations were observed with HE staining. Pilocarpine-stimulated saliva was measured from Wharton's duct. Cell apoptosis was determined by flow cytometry and terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. Nicotinamide phosphoribosyl transferase was examined with immunofluorescence. The levels of nicotinamide adenine dinucleotide, mitochondrial membrane potential, and adenosine triphosphate were measured with the relevant kits. The mitochondrial ultrastructure was observed under transmission electron microscopy. RESULTS: Nicotinamide significantly mitigated radiation damage both in vitro and in vivo. Also, nicotinamide improved saliva secretion and reduced radiation-induced apoptosis in irradiated submandibular glands. Moreover, nicotinamide improved nicotinamide phosphoribosyl transferase and the levels of nicotinamide adenine dinucleotide/adenosine triphosphate and mitochondrial membrane potential, all of which were decreased by radiation in submandibular gland cells. Importantly, nicotinamide protected the mitochondrial ultrastructure from radiation. CONCLUSION: These findings demonstrate that nicotinamide alleviates radiation damage in submandibular gland by replenishing nicotinamide adenine dinucleotide and maintaining mitochondrial function and ultrastructure, suggesting that nicotinamide could be used as a prospective radioprotectant for preventing radiation sialadenitis.

Modafinil exerts anticonvulsive effects against lithium-pilocarpine-induced status epilepticus in rats: A role for tumor necrosis factor-α and nitric oxide signaling.

BACKGROUND: Status epilepticus (SE) is a continuous episode of seizures which leads to hippocampal neurodegeneration, severe systemic inflammation, and extreme damage to the brain. Modafinil, a psychostimulant and wake-promoting agent, has exerted neuroprotective and anti-inflammatory effects in previous preclinical studies. The aim of this study was to assess effects of modafinil on the lithium-pilocarpine-induced SE rat model and to explore possible involvement of tumor necrosis factor-α (TNF-α) and nitric oxide (NO) pathways in this regard. METHODS: Status epilepticus was provoked by injection of lithium chloride (127 mg/kg, intraperitoneally [i.p]) and pilocarpine (60 mg/kg, i.p.) in rats. Animals received different modafinil doses (50, 75, 100, and 150 mg/kg, i.p.) and SE scores were documented over 3 hours of duration. Moreover, the role of the nitrergic pathway in the effects of modafinil was evaluated by injection of the non-selective NO synthase (NOS) inhibitor L-NG-Nitro arginine methyl ester (L-NAME, 10 mg/kg, i.p.), the selective neuronal NOS inhibitor 7-nitroindazole (30 mg/kg, i.p.), and the selective inducible NOS inhibitor aminoguanidine (100 mg/kg, i.p.) 15 min before saline/vehicle or modafinil. The ELISA method was used to quantify TNF-α and NO metabolite levels in the isolated hippocampus. RESULTS: Modafinil at 100 mg/kg significantly decreased SE scores (P < 0.01). Pre-treatment with L-NAME, 7-nitroindazole, and aminoguanidine significantly reversed the anticonvulsive effects of modafinil. Status epilepticus-induced animals showed significantly higher NO metabolite and TNF-α levels in their hippocampal tissues, an effect that was reversed by modafinil (100 mg/kg, i.p.) treatment. Administration of NOS inhibitors resulted in excessive NO level reduction but an escalation of TNF-α level in modafinil-treated SE-animals. CONCLUSION: Our study revealed anticonvulsive effects of modafinil in the lithium-pilocarpine-induced SE rat model via possible involvement of TNF-α and nitrergic pathways.

Comparison study between pilocarpine and tropicamide drops on corneal topography and their effect on IL-6 and TNF-α levels in tear.

Corneal stability is essential for contact lenses and refractive surgery. It seems that paralyzing eye drops or expansion of the ciliary muscle affect the radius of curvature and the strength of the cornea, and this effect is to increase the strength of the cornea during muscle spasm and decrease it in the relaxed state of the muscle. On the other hand, different factors (such as contact lens wear, ocular surface disorders, trauma, dry eye, and immunosuppression) could alter the immune defense mechanisms of the outer eye and permit microorganisms to invade the cornea. Therefore, the present study compared Pilocarpine and tropicamide drop on corneal topography and their effect on IL-6 and TNF-α levels in tear. This prospective study was performed on sixty normal and healthy eyes of sixty volunteers with a mean age of 38.19 years and without any ocular pathology. Volunteers were divided into two groups of thirty. In the first group, corneal topography of both eyes was measured before and 30 minutes after instillation of topical tropicamide 1% in only one eye. The other eye was the control eye, and no drop was given. The same routine was performed in the second group, except that subject received one drop of Pilocarpine 2% in one eye. Statistical comparison between groups for the central corneal power, corneal radius, and corneal astigmatism was performed using paired t-test. IL-6 and TNF-α levels in tear were analyzed using two Luminex commercial assays with Bio-Plex 200TM System (Bio-Rad, Hercules, California, USA). In group 1, no significant changes were found in corneal radius, power, and astigmatism. However, in group 2 subjects who received pilocarpine eye drops, the mean corneal radius value decreased significantly by 0.05 mm. The mean corneal power increased by +0.32 D. There was no significant difference change in corneal astigmatism in both groups. Evaluation of IL-6 levels in tears showed a significant difference between the control and treatment groups (P = 0.041). But no significant difference was observed between the Pilocarpine and the Tropicamide groups (P = 0.761). Evaluation of TNF-α level in tears also showed no significant difference between these groups (P = 0.088). Pilocarpine induced ciliary muscle contraction, which may cause pressure on the corneal limbus and scleral spur, resulting in changes in corneal curvature. But tropicamide eye drop did not affect corneal radius and other corneal parameters, and corneal topography can be carried out after the installation of tropicamide eye drop.

Lactation in large litters influences anxiety, memory, and spreading depression in adult male rats that were chronically subjected to a non-convulsive pilocarpine dose.

Objectives: Unfavorable lactation influences brain excitability and behavioral reactions in adults. Administration early in life of the cholinergic agonist, pilocarpine, even at non-convulsive doses, alters the brain excitability-related phenomenon known as cortical spreading depression (CSD), and produce anxiogenic-like behavior. However, the influence of unfavorable lactation on the CSD- and memory-effects of pilocarpine administration late in life has not been investigated. Herein, we analyzed the ponderal, electrophysiological (CSD), and behavioral effects of chronic treatment with a non-convulsive dose of pilocarpine, in adult rats suckled under favorable and unfavorable conditions.Methods: Wistar rats were suckled in litters with 9 or 15 pups (groups L9 and L15, respectively). A very low dose of pilocarpine (45/mg/kg/day) was chronically administered in mature rats from postnatal day (PND) 69-90. Behavioral tests occurred at PND91 [elevated plus maze (EPM)], PND93 [open field (OF)], and PND94-95 [object recognition memory (ORM)]. CSD was recorded between PND96-120.Results: Pilocarpine-treated rats performed worse in the anxiety and memory tests, and displayed lower CSD propagation velocity when compared with saline-treated controls. In addition, L15 rats showed an increase in the distance traveled and a decrease in the immobility time in the EPM, impaired ORM, and accelerated CSD propagation when compared with L9 rats (p ≤ 0.05).Discussion: These data suggest that sub-convulsive pilocarpine treatment in adult rats can affect behavioral and excitability-related reactions. In addition, unfavorable lactation increases the ambulatory effects of pilocarpine. Further studies should investigate the possible cholinergic molecular mechanisms involved in these effects.