Sigma receptor ligands haloperidol and ifenprodil attenuate hypoxia induced dopamine release in rat striatum.

OBJECTIVE: We aimed to investigate the hypothesis that sigma receptor ligands, haloperidol and ifenprodil, attenuate hypoxia-induced striatal dopamine release in vitro and determine the possible mechanisms. METHODS: Extracellular concentrations of dopamine were measured using acute brain slices method under hypoxic, aglycemic and ischemic conditions. Sigma receptor ligands haloperidol and ifenprodil attenuate striatal dopamine release induced by hypoxia in contrast to aglycemia and ischemia. To determine the possible contribution of glutamatergic system on this effect, we compared the effect of NMDA receptor antagonist MK-801 and haloperidol in hypoxia induced by Na-K-ATPaz enzyme inhibitor ouabain. Also, we compared the effect of dopamine uptake blocker nomifensine and haloperidol to determine the role of dopamine transporter on this effect. RESULTS: Haloperidol and nomifensine almost completely abolish ouabain-induced dopamine release unlike MK-801. Different effects of sigma ligands and glutamate receptor antagonists on the hypoxia and ouabain induced dopamine release show that glutamate receptor blockade is partial involved in inhibitory effect of sigma ligand on dopamine release under hypoxic conditions. Similar effect of dopamine uptake blocker nomifensine and sigma receptor ligand haloperidol on ouabain induced dopamine release supports the possibility that inhibition of reverse dopamine transport by sigma ligands might be involved in their protective effect. CONCLUSIONS: Data in this study suggest that sigma ligands may be a new therapeutic intervention for the management of hypoxic conditions.

The role of syringic acid as a neuroprotective agent for neurodegenerative disorders and future expectations.

Hundreds of millions of people are influenced by neurodegenerative disorders such as Alzheimer's disease (AD) and Parkinson's disease (PD), traumatic disorders of the nervous system, dementia, and various neurological disorders. Syringic acid (SA) is a natural phenolic compound that is found in medicinal herbs and dietary plants. The therapeutic potential of SA is due to its anti-oxidative, chemoprotective, anti-angiogenic, anti-glycating, anti-proliferative, anti-hyperglycaemic, anti-endotoxic, anti-microbial, anti-inflammatory, anti-diabetic and anti-depressant properties. However, in recent studies, its neuroprotective effect has drawn attention. The current review focuses on the neuroprotective bioactivities of SA and putative mechanisms of action. An electronic data search was performed using different search engines, and the relevant articles (with or without meta-analysis) with any language were selected. In the central and peripheral nervous system, SA has been shown a significant role in excitatory neurotransmitters and alleviate behavioral dysfunctions. The consensus of the literature search was that SA treatment may help neurological dysfunction or behavioral impairments management with antioxidant, anti-inflammatory properties. Furthermore, administration and proper dose of SA could be crucial factors for the effective treatment of neurological diseases.

(E)-3-furan-2-yl-N-phenylacrylamide (PAM-4) decreases nociception and emotional manifestations of neuropathic pain in mice by α7 nicotinic acetylcholine receptor potentiation.

Clinical intervention of pain is often accompanied by changes in affective behaviors, so both assays of affective and sensorial aspects of nociception play an important role in the development of novel analgesics. Although positive allosteric modulation (PAM) of α7 nicotinic acetylcholine receptors (nAChRs) has been recognized as a novel approach for the relief of sensorial aspects of pain, their effects on affective components of pain remain unclear. Therefore, we investigated whether PAM-4, a highly selective α7-nAChR PAM, attenuates inflammatory and neuropathic pain, as well as the concomitant depressive/anxiety comorbidities. The anti-nociceptive activity of PAM-4 was assessed in mice using the formalin test and chronic constriction injury (CCI)-induced neuropathic pain model. The anxiolytic- and antidepressant-like activity of PAM-4 was evaluated using the marble burying test and forced swimming test. Acute systemic administration of PAM-4 dose-dependently reversed formalin-induced paw licking behavior and CCI-induced mechanical allodynia without development of any motor impairment. PAM-4 reversed the decreased swimming time and number of buried marbles in CCI-treated mice, suggesting that this ligand attenuates chronic pain-induced depression-like behavior and anxiogenic-like effects. The effects of PAM-4 were inhibited by the α7-selective antagonist methyllycaconitine, indicating molecular mechanism mediated by α7-nAChRs. Indeed, electrophysiological recordings showed the PAM-4 enhances human α7 nAChRs with higher potency and efficacy compared to rat α7 nAChRs. These findings suggest that PAM-4 reduces both sensorial and affective behaviors induced by chronic pain in mice by α7-nAChR potentiation. PAM-4 deserves further investigations for the management of chronic painful conditions with comorbidities.

Glutamate-induced modulation in energy metabolism contributes to protection of rat cortical slices against ischemia-induced damage.

OBJECTIVES: Glutamate excitotoxicity contributes to neurodegeneration during cerebral ischemia. Recent studies in the protective effect of glutamate against ischemia and hypoxia have shown the need for questioning the role of glutamate in energy metabolism during ischemia. Current study investigates the effect of glutamate on energy substrate metabolites such as alpha-ketoglutarate, lactate, and pyruvate release during control, oxygen-glucose deprivation (OGD), and reoxygenation (REO) conditions. METHODS: The effects of 0.5 and 2 mM glutamate on spontaneous alpha-ketoglutarate, lactate, and pyruvate release were tested in vitro, on acute rat cortical slices. Alpha-ketoglutarate, lactate, and pyruvate levels were determined by HPLC with UV detector. RESULTS: We observed that glutamate added into medium significantly increased alpha-ketogluarate release under control conditions. Although OGD and REO also had a glutamate-like effect, only REO-induced rise further enhanced by glutamate. In contrast to alpha-ketoglutarate, both OGD and REO conditions caused significant declines in pyruvate and lactate outputs. While OGD and REO-induced declines in pyruvate outputs were further potentiated, lactate output was not altered by glutamate added into the medium. Glutamate and alpha-ketoglutarate, moreover, also ameliorated OGD- and REO-induced losses in 2,3,5-triphenyltetrazolium chloride staining with a similar degree. CONCLUSION: These results indicate that glutamate probably increases alpha-ketoglutarate production as an alternative energy source for use in the TCA cycle under energy-depleted conditions. Thus, increasing the alpha-ketoglutarate production may represent a new therapeutic intervention for neurodegenerative disorders, including cerebral ischemia.

K channel blockage with 3,4-diaminopyridine potentiates the effect of L-DOPA on dopamine release in striatal slices prepared from 6-OHDA pre-treated rats.

Although L-DOPA revolutionized in the treatment of Parkinson's disease, most patients developed motor complications after several years of treatment. Adjunctive therapy to L-DOPA with drugs related to dopaminergic signaling may reduce its dose without decreasing the therapeutic efficiency and thus ameliorates its adverse effects. It has been shown that 3,4-diaminopyridine (3,4-DAP), a K channel blocker, increased dopamine release from striatal slices by increasing neuronal firing in striatal dopaminergic terminals. The current study investigates whether 3,4-DAP may enhance L-DOPA-induced dopamine (DA) release from striatal slices by increasing neuronal firing in striatal dopaminergic terminals. The effects of L-DOPA and 3,4-DAP on spontaneous DA and DOPAC release were tested in vitro, on acute rat striatal slices prepared from non-treated and 6-hydroxydopamine-pre-treated rats. DA and DOPAC levels were determined by HPLC methods. When 3,4-diaminopyridine was combined with L-DOPA, the observed effect was considerably greater than the increases induced by L-DOPA or 3,4-DAP alone in normoxic and neurodegenerative conditions produced by FeSO4 and 6-hydroxydopamine. Furthermore, L-DOPA plus 3,4-DAP also ameliorated DOPAC levels in neurodegenerative conditions. These data indicate that 3,4 DAP plus L-DOPA activates striatal dopaminergic terminals by increasing the DA release and, thus, could be considered as a promising finding in treatment of acute and chronic injury in dopaminergic neurons.

Aging protects rat cortical slices against to oxygen-glucose deprivation induced damage.

Objective: In present study, we aimed to clarify effect of aging on the susceptibility of brain tissue to neurodegeneration induced by ischemia.Methods: Damage induced by oxygen-glucose deprivation (OGD) followed by reoxygenation (REO) were compared in cortical slices prepared from young (3 months of age) and aged (22-24 months of age) male Sprague Dawley rats.Results: After incubation of the slices in an oxygen and glucose containing control condition, 2,3,5-triphenyl tetrazolium chloride (TTC) staining intensity was found significantly high in aged cortical slices. Although thirty minutes incubation of the slices in OGD medium followed by REO (OGD-REO) caused similar decline in TTC staining in young and aged cortical slices, staining intensity was still significantly higher in the slices prepared from aged animals. Thirty minutes of OGD-REO, on the other hand, also caused more increase in lactate dehydrogenase (LDH) leakage from young slices. While water contents of the slices were almost equal under control condition, it was significantly high in young cortical slices after OGD-REO incubations. In contrary to these findings, OGD and REO caused more increases in S100B output from aged rat cortical slices. S100B levels in brain regions including the cerebral cortex were also found higher in aged rats.Conclusion: All these results indicate that, cortical slices prepared from aged male rats are significantly less responsive to in vitro OGD-REO induced alterations. Since protein S100B outputs were almost doubled from aged cortical slices, a possible involvement of this enhanced S100B output seems to be likely.

Brain slice viability determined under normoxic and oxidative stress conditions: involvement of slice quantity in the medium.

Objective: In vitro acute adult brain slice methods are instruments in developing our knowledge of the nervous system. Optimization of this method for obtaining high-quality brain slices is extremely important in terms of consistency and reliability of the experimental results. Although some important topics such as slice thickness, temperature, and composition of the physiological medium have been studied for optimization, involvement of slice quantity in medium on tissue viability has not been investigated yet.Methods: Different number of slices (1, 3, or 6 slices) were incubated under normoxic or some prooxidant stress conditions induced by oxygen-glucose deprivation (OGD), H2O2, FeSO4+ ascorbic acid, or menadione to evaluate the effect of slice density on tissue viability.Results:Slice quantity in the normoxic incubation medium caused a significant increase in 2,3,5-triphenyltetrazolium chloride (TTC) staining intensity of the slices. Similarly, increase in the slice quantity in the medium also protected the slices against either OGD, H2O2, FeSO4, or menadione-induced decrease in TTC staining. In addition to TTC staining, lactate dehydrogenase leakage or malondialdehyde and reactive oxygen species production under normoxic or ischemia-like conditions were also attenuated by increasing slice quantity in the medium.Conclusion: These results show that when using brain slices method for investigating the structural and functional features of brain at the molecular and cellular levels, both slice quantity in the medium and incubation volume should be considered first. Increasing slice quantity or decreasing incubation volume probably causes an increase in the concentration of endogenous substance(s) involved in neuroprotection.

Impact of Menthol on Oral Nicotine Consumption in Female and Male Sprague Dawley Rats.

INTRODUCTION: One of the preferable flavors in oral nicotine delivery systems is menthol which masks the harshness of tobacco. However, possible interactions between oral menthol and nicotine on intake and preference remain unclear. Therefore, we aimed to determine the impact of menthol on oral nicotine consumption. METHODS: Adult Sprague Dawley female and male rats (n = 8 per group) were given a choice of water or drug solution by using two-bottle free choice paradigm for 2 weeks: vehicle (5% ethanol), nicotine (20 mg/L), menthol (1 g/L) and mentholated nicotine groups. At the end of the study, plasma nicotine levels were determined. RESULTS: When rats were given a choice of nicotine or water, nicotine intake was similar between female and male rats. Menthol addition to nicotine solution significantly increased nicotine intake and preference in male but not female rats without a considerable effect on total fluid intake and body weight change in either sex. The average nicotine intake in male rats was 0.5 ± 0.05 and 1.4 ± 0.12 mg/kg/day for nicotine and menthol-nicotine combination (p < .05), respectively. The average nicotine intake in female rats was 0.6 ± 0.05 and 0.6 ± 0.03 mg/kg/day for nicotine and menthol-nicotine combination (p > .05), respectively. Plasma nicotine levels were not significantly different between the groups in either male (nicotine group: 20.8 ± 4.9, mentholated nicotine group: 31.9 ± 3.2 ng/mL) or female (nicotine group: 24.0 ± 3.3, mentholated nicotine group: 17.8 ± 2.9 ng/mL) rats (p > .05). CONCLUSIONS: Menthol increases oral nicotine consumption in male, but not female, rats. IMPLICATIONS: This study may provide data on the co-use of menthol and nicotine in smokeless tobacco, particularly oral dissolvable tobacco products.

Pharmacologic Overview of Chlorogenic Acid and its Metabolites in Chronic Pain and Inflammation.

BACKGROUND: Natural phenolic compounds in medicinal herbs and dietary plants are antioxidants which play therapeutic or preventive roles in different pathological situations, such as oxidative stress and inflammation. One of the most studied phenolic compounds in the last decade is chlorogenic acid (CGA), which is a potent antioxidant found in certain foods and drinks. OBJECTIVE: This review focuses on the anti-inflammatory and antinociceptive bioactivities of CGA, and the putative mechanisms of action are described. Ethnopharmacological reports related to these bioactivities are also reviewed. MATERIALS AND METHODS: An electronic literature search was conducted by authors up to October 2019. Original articles were selected. RESULTS: CGA has been shown to reduce inflammation and modulate inflammatory and neuropathic pain in animal models. CONCLUSION: The consensus of the literature search was that systemic CGA may facilitate pain management via bolstering antioxidant defenses against inflammatory insults.

Assessment of neuron-specific enolase, S100B and malondialdehyde levels in serum and vitreous of patients with proliferative diabetic retinopathy.

PURPOSE: To assess the vitreous and serum levels of neuron-specific enolase (NSE), S100B and malondialdehyde (MDA) in proliferative diabetic retinopathy (PDR) cases and investigate the correlation between preoperative and postoperative anatomical and clinical features. MATERIALS AND METHODS: The study group included patients who had pars plana vitrectomy (PPV) for PDR. The control group included non-diabetic individuals who underwent PPV surgery for vitreoretinal interface disorders. Samples of serum were taken from all participants preoperatively, while vitreous samples were taken during the PPV. Vitreous and serum levels of NSE, S100B and MDA were measured, and comparisons were made between the groups. RESULTS: The study group consisted of 56 eyes of 56 cases with PDR. The control group consisted of 20 eyes of 20 cases. The concentrations of vitreous NSE, S100B and MDA were significantly higher than the control group (p < 0.0001, p < 0.05, p < 0.001, respectively). Serum levels were statistically different for NSE and S100B (p < 0.05). CONCLUSION: Our results clearly show that vitreous levels of S100B, NSE and MDA and serum concentrations of NSE and S100B increased significantly in patients with PDR. The findings may possibly indicate neurodegeneration and oxidative stress; therefore, these markers may have a diagnostic value in patients with PDR.

The Association Between Olfaction and Taste Functions with Serum Ghrelin and Leptin Levels in Obese Women.

Purpose: To investigate the olfaction and taste functions in obese female patients and the association between serum ghrelin and leptin levels compared with healthy controls. Methods: Fifty-two obese women, who have a body mass index >30 kg/m2, and 15 healthy women were included in the study. After 8 hrs fasting, blood samples were taken for serum biochemical parameters, ghrelin, and leptin level measurement. For the quantitative assessment of olfactory function, all participants underwent an N-butanol threshold test and odor identification test using 12 Sniffin' Sticks® fragrance sticks. The gustatory function was tested by administering a whole-mouth above threshold test using sucrose solutions. Results: The sucrose taste threshold score in obese women was significantly higher than the controls (P = 0.004). We found positively significant correlation between serum ghrelin levels and n-butanol threshold scores in obese women (r = 0.300, P = 0.031). N-butanol smell threshold was not significantly different between the two groups (P = 0.149), while the Sniffin' Sticks smell test scores were significantly lower in obese women compared with the controls (P = 0.007). Serum leptin levels were also significantly higher in obese women (P < 0.001) although there was no significant difference in serum ghrelin levels between the two groups (P = 0.768). There was no correlation between serum leptin levels and Sniffin' Sticks scores, n-butanol, and sucrose taste threshold scores in obese women. Conclusions: These results might suggest that leptin, which is an anorexigenic peptide, may have a negative effect on taste and smell functions. More studies are warranted to elucidate the exact role of ghrelin secretion on olfaction and taste functions.

Exenatide treatment causes suppression of serum fasting ghrelin levels in patients with type 2 diabetes mellitus.

AIM: In the present study, we investigated the long-term effects of exenatide treatment on serum fasting ghrelin levels in patients with type 2 diabetes mellitus. METHODS: Type 2 diabetic patients, who were using metformin with and without the other antihyperglycemic drugs on a stable dose for at least 3 months, were enrolled in the study. BMI>35 kg/m2 and HbA1c>7.0% were the additional inclusion criteria. Oral antihyperglycemic drugs, other than metformin, were stopped, and metformin treatment was continued at 2000 mg per day. Exenatide treatment was initiated at 5 µg per dose subcutaneously (sc) twice daily, and after one month, the dose of exenatide was increased to 10 µg twice daily. Changes in anthropometric variables, glycemic control, lipid parameters and total ghrelin levels were evaluated at baseline and following 12 weeks of treatment. RESULTS: Thirty-eight patients (male/female = 7/31) entered the study. The mean age of patients was 50.5 ± 8.8 years with a mean diabetes duration of 8.5 ± 4.9 years. The mean BMI was 41.6 ± 6.3 kg/m2 and the mean HbA1c of patients was 8.9 ± 1.4%. The mean change in the weight of patients was -5.6 kg and the percentage change in weight was -5.2 ± 3.7% following 12 weeks of treatment. BMI, fasting plasma glucose and HbA1c levels of patients were decreased significantly (P < 0.001 and P < 0.001; respectively), while there was no change in lipid parameters. Serum fasting ghrelin levels were significantly suppressed following 12 weeks of exenatide treatment compared with baseline values (328.4 ± 166.8 vs 245.3 ± 164.8 pg/mL) (P = 0.024). CONCLUSION: These results suggest that the effects of exenatide on weight loss may be related with the suppression of serum fasting ghrelin levels, which is an orexigenic peptide.

The α7 nicotinic receptor dual allosteric agonist and positive allosteric modulator GAT107 reverses nociception in mouse models of inflammatory and neuropathic pain.

BACKGROUND AND PURPOSE: Orthosteric agonists and positive allosteric modulators (PAMs) of the α7 nicotinic ACh receptor (nAChR) represent novel therapeutic approaches for pain modulation. Moreover, compounds with dual function as allosteric agonists and PAMs, known as ago-PAMs, add further regulation of receptor function. EXPERIMENTAL APPROACH: Initial studies examined the α7 ago-PAM, GAT107, in the formalin, complete Freund's adjuvant (CFA), LPS inflammatory pain models, the chronic constriction injury neuropathic pain model and the tail flick and hot plate acute thermal nociceptive assays. Additional studies examined the locus of action of GAT107 and immunohistochemical markers in the dorsal horn of the spinal cord in the CFA model. KEY RESULTS: Complementary pharmacological and genetic approaches confirmed that the dose-dependent antinociceptive effects of GAT107 were mediated through α7 nAChR. However, GAT107 was inactive in the tail flick and hot plate assays. In addition, GAT107 blocked conditioned place aversion elicited by acetic acid injection. Furthermore, intrathecal, but not intraplantar, injections of GAT107 reversed nociception in the CFA model, suggesting a spinal component of action. Immunohistochemical evaluation revealed an increase in the expression of astrocyte-specific glial fibrillary acidic protein and phosphorylated p38MAPK within the spinal cords of mice treated with CFA, which was attenuated by intrathecal GAT107 treatment. Importantly, GAT107 did not elicit motor impairment and continued to produce antinociceptive effects after subchronic administration in both phases of the formalin test. CONCLUSIONS AND IMPLICATIONS: Collectively, these results provide the first proof of principle that α7 ago-PAMs represent an effective pharmacological strategy for treating inflammatory and neuropathic pain.

Protective Effects of Chlorogenic Acid and its Metabolites on Hydrogen Peroxide-Induced Alterations in Rat Brain Slices: A Comparative Study with Resveratrol.

The effectiveness of chlorogenic acid and its main metabolites, caffeic and quinic acids, against oxidative stress was investigated. Resveratrol, another natural phenolic compound, was also tested for comparison. Rat cortical slices were incubated with 200 µM H2O2 for 1 h, and alterations in oxidative stress parameters, such as 2, 3, 5-triphenyltetrazolium chloride (TTC) staining and the production of both malondialdehyde (MDA) and reactive oxygen species (ROS), were assayed in the absence or presence of phenolic compounds. Additionally, the effectiveness of chlorogenic acid and other compounds on H2O2-induced increases in fluorescence intensities were also compared in slice-free incubation medium. Although quinic acid failed, chlorogenic and caffeic acids significantly ameliorated the H2O2-induced decline in TTC staining intensities. Although resveratrol also caused an increase in staining intensity, its effect was not dose-dependent; the high concentrations of resveratrol tested in the present study (10 and 100 µM) further lessened the staining of the slices. Additionally, all phenolic compounds significantly attenuated the H2O2-induced increases in MDA and ROS levels in cortical slices. When the IC50 values were compared to H2O2-induced alterations, chlorogenic acid was more potent than either its metabolites or resveratrol for all parameters studied under these experimental conditions. In slice-free experimental conditions, on the other hand, chlorogenic and caffeic acids significantly attenuated the fluorescence emission enhanced by H2O2 with a similar order of potency to that obtained in slice-containing physiological medium. These results indicate that chlorogenic acid is a more potent phenolic compound than resveratrol and its main metabolites caffeic and quinic acids against H2O2-induced alterations in oxidative stress parameters in rat cortical slices.

Freeze-dried egg yolk based extenders containing various antioxidants improve post-thawing quality and incubation resilience of goat spermatozoa.

The aim of this study was to evaluate different antioxidants-supplemented freeze-dried egg yolk based extenders for the post-thawing quality and incubation resilience of goat spermatozoa. Pooled semen were diluted in a two-step dilution method to a final concentration of 1/5 (semen/extender) in control and antoxidant supplemented freeze-dried extenders (methionine, cysteamine and butylated hydroxytoluene). Semen samples were assessed for sperm motility, plasma membrane functional integrity using hypoosmotic swelling test (HOST), damaged acrosome using FITC-Pisum sativum agglutinin (PSA-FITC) and DNA integrity using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). Membrane lipid peroxidation status was also analyzed using the malondialdehyde (MDA) concentration. In the study, antioxidant supplemented freeze-dried egg yolk based extenders have beneficial effect on goat sperm parameters. In addition, we achieved a higher quality in post thawed goat semen even after 6 h incubation when the extender was supplemented by 5 mM BHT or cysteamine.

Exenatide Treatment Causes Suppression of Serum Ghrelin Levels following Mixed Meal Test in Obese Diabetic Women.

AIM: To investigate the effect of exenatide treatment on serum ghrelin levels in obese female patients with type 2 diabetes mellitus. METHODS: Fourteen female patients with type 2 diabetes mellitus being treated with metformin and exenatide were enrolled. A mixed meal test was applied to the patients while continuing with their daily medications. Blood samples were taken before and at 60, 120, and 180 minutes following mixed meal test to measure serum total ghrelin, glucose, and insulin levels. The following week, exenatide treatment of the patients was paused for 24 hours and the same experimental procedures were repeated. RESULTS: Serum ghrelin levels were suppressed significantly at 180 minutes with exenatide treatment compared with baseline (294.4 ± 57.5 versus 234.5 ± 59.4 pg/mL) (p < 0.001). Serum ghrelin levels at 180 minutes were statistically different when percentage change in serum ghrelin levels after mixed meal tests with and without exenatide usage were compared (p = 0.001). Estimated total area under the curve values for serum ghrelin concentrations was also significantly lower with exenatide compared with omitted treatment (p = 0.035). CONCLUSION: These results suggest that the effect of exenatide on weight loss may be related with the suppression of serum ghrelin levels, which is an orexigenic peptide.

Effect of rainbow trout (Oncorhynchus mykiss) seminal plasma on the post-thaw quality of ram semen cryopreserved in a soybean lecithin-based or egg yolk-based extender.

The aim of the current study was to evaluate the effects of different concentrations of rainbow trout seminal plasma (RTSP) (0.1%, 1% and 10%) in extenders containing either egg yolk or lecithin for use in Awassi ram semen cryopreservation. Pooled sperm were diluted in a two-step dilution method to a final concentration of 1/5 (semen/extender) in egg yolk or lecithin extender containing no RTSP, 0.1%, 1% or 10% RTSP (v/v). Semen samples were assessed for sperm motility, plasma membrane integrity [hypoosmotic swelling test (HOST) and Hoechst 33258] and defective acrosomes [FITC-conjugated Pisum sativum agglutinin (PSA-FITC)] at the following five time points: after dilution with extender A; after equilibration; and post-thaw at 0h, 3h and 5h. Malondialdehyde (MDA) was examined only after thawing. Freezing and thawing procedures (dilution, equilibration and post-thaw incubation at 0h, 3h and 5h) negatively affected the motility (P<0.001) and acrosome integrity (P<0.001). Additionally, freezing and thawing negatively affected the plasma membrane integrity, as determined by the HOST and Hoechst 33258 (P<0.001). The extender group affected the motility (P<0.001) and the HOST results (P<0.001). Levels of MDA in the egg yolk extender with 1% RTSP group were significantly lower than in the lecithin control group (P<0.05). In conclusion, the egg yolk extender groups that were supplemented with 10% and 1% RTSP provided greater cryoprotective effects for semen survivability during 5h incubation than the other extender groups.

Chlorogenic Acid Enhances Abdominal Skin Flap Survival Based on Epigastric Artery in Nondiabetic and Diabetic Rats.

Previous studies showed that chlorogenic acid (CGA) accelerates wound healing via its antioxidant activity. We aimed to investigate the effect of CGA in an experimental epigastric abdominal skin flap model in nondiabetic and diabetic rats. Rats were firstly divided into 2 groups: nondiabetic and diabetic. Diabetes was induced by streptozotocin. Then, 4 subgroups were created for each group: vehicle as well as 0.2 mg/0.5 mL, 1 mg/0.5 mL, and 5 mg/0.5 mL CGA treatments. Right epigastric artery-based abdominal skin flaps were elevated and sutured back into their original position. Chlorogenic acid or vehicle was injected once into the femoral arteries by leaving the epigastric artery as the single artery feeding the flaps during the injection. On postoperative day 7, flap survivals were evaluated, and the rats were killed. Distal flap tissues were collected for histopathological and biochemical assays. Chlorogenic acid showed greater flap survival in both nondiabetic and diabetic rats. Capillary density was increased, and necrosis was reduced in the CGA-treated rats. Chlorogenic acid decreased malondialdehyde levels as well as increased reduced glutathione and superoxide dismutase levels in the flap tissues. This study showed that CGA significantly improved flap survival by its antioxidant activities with intra-arterial local injections.

In vivo systemic chlorogenic acid therapy under diabetic conditions: Wound healing effects and cytotoxicity/genotoxicity profile.

Oxidative stress occurs following the impairment of pro-oxidant/antioxidant balance in chronic wounds and leads to harmful delays in healing progress. A fine balance between oxidative stress and endogenous antioxidant defense system may be beneficial for wound healing under redox control. This study tested the hypothesis that oxidative stress in wound area can be controlled with systemic antioxidant therapy and therefore wound healing can be accelerated. We used chlorogenic acid (CGA), a dietary antioxidant, in experimental diabetic wounds that are characterized by delayed healing. Additionally, we aimed to understand possible side effects of CGA on pivotal organs and bone marrow during therapy. Wounds were created on backs of streptozotocin-induced diabetic rats. CGA (50 mg/kg/day) was injected intraperitoneally. Animals were sacrificed on different days. Biochemical and histopathological examinations were performed. Side effects of chronic antioxidant treatment were tested. CGA accelerated wound healing, enhanced hydroxyproline content, decreased malondialdehyde/nitric oxide levels, elevated reduced-glutathione, and did not affect superoxide dismutase/catalase levels in wound bed. While CGA induced side effects such as cyto/genotoxicity, 15 days of treatment attenuated blood glucose levels. CGA decreased lipid peroxidation levels of main organs. This study provides a better understanding for antioxidant intake on diabetic wound repair and possible pro-oxidative effects.

Pharmacologic overview of systemic chlorogenic acid therapy on experimental wound healing.

Chlorogenic acid (CGA) is a well-known natural antioxidant in human diet. To understand the effects of CGA on wound healing by enhancing antioxidant defense in the body, the present study sought to investigate the potential role of systemic CGA therapy on wound healing and oxidative stress markers of the skin. We also aimed to understand whether chronic CGA treatment has side effects on pivotal organs or rat bone marrow during therapy. Full-thickness experimental wounds were created on the backs of rats. CGA (25, 50, 100, 200 mg/kg) or vehicle was administered intraperitoneally for 15 days. All rats were sacrificed on the 16th day. Biochemical, histopathological, and immunohistochemical examinations were performed. Possible side effects were also investigated. The results suggested that CGA accelerated wound healing in a dose-dependent manner. CGA enhanced hydroxyproline content, decreased malondialdehyde and nitric oxide levels. and elevated reduced glutathione, superoxide dismutase, and catalase levels in wound tissues. Epithelialization, angiogenesis, fibroblast proliferation, and collagen formation increased by CGA while polymorph nuclear leukocytes infiltration decreased. CGA modulated matrix metalloproteinase-9 and tissue inhibitor-2 expression in biopsies. Otherwise, high dose of CGA increased lipid peroxidation of liver and kidney without affecting the heart and muscle samples. Chronic CGA increased micronuclei formation and induced cytotoxicity in the bone marrow. In conclusion, systemic CGA has beneficial effects in improving wound repair. Antioxidant, free radical scavenger, angiogenesis, and anti-inflammatory effects of CGA may ameliorate wound healing. High dose of CGA may induce side effects. In light of these observations, CGA supplementation or dietary CGA may have benefit on wound healing.