Protective Effect of Curcumin on CA1 Region of Hippocampus in Rat Model of Ischemia/ Reperfusion Injury:.

BACKGROUND: The brain is the most complex and vital organ of the human body. It requires 20-25 % of the total oxygen supply. Because of the limited oxygen and glucose reserves, brain tissue is sensitive to ischemic injury. Indeed, the tolerance of brain tissue for ischemic injury is fragile. Currently, few therapeutic strategies could provide complete neuroprotection. Despite decades of intense research, the beneficial treatment of stroke remains limited. Hence, we aimed to investigate the effect of curcumin on the CA1 region of the hippocampus in a rat model of ischemia/reperfusion (I/R) injury. MATERIALS AND METHODS: In this experimental research, 24 male Wistar rats were randomly divided into three groups (n=8 per group) as control, I/R, and I/R plus curcumin. All rats underwent bilateral common carotid artery ligation followed by reperfusion. In the treatment group, curcumin (300 mg/kg) was injected 30 minutes before ischemia. Morphological changes of the hippocampus were assessed using Nissl staining, and apoptosis was determined via TUNEL immunohistochemical assays. RESULTS: Nissl staining data showed that the administration of curcumin significantly ameliorated the CA1 pyramidal cell loss due to transient global I/R injury. TUNEL immunohistochemical assays demonstrated that the number of apoptotic cells was significantly lower in the curcumin group than in the I/R groups. CONCLUSION: Our study demonstrates that curcumin had beneficial activity against ischemia and played a neuroprotective role in the pathogenesis of I/R injury.

Evaluation of the Effects of Human Bone Marrow Mesenchymal Stem Cells Conditioned Medium on Growth and Maturation of Mouse Ovarian Follicle after Vitrification.

The aim of this research study is to evaluate the effect of human bone marrow mesenchymal stem cells conditioned medium (hBMSCs-CM) on growth and maturation of mouse ovarian follicle, and embryonic development after vitrification. The hBMSCs were cultured, and the derived CM was collected, concentrated, and stored. 14-day-old mice ovaries were collected and randomly divided into vitrified and non-vitrified groups. Then their isolated preantral follicles were cultured for 12 days in α-MEM supplemented with different concentrations of CM (2.5, 5, and 7.5%). Finally, the growth and diameter of follicles, maturation of oocytes, hormone level, and embryo developmental rate were assessed. The results showed the antrum formation, oocyte maturation, and hormone secretion were significantly higher in the presence of 7.5% CM (p < 0.001). In the vitrified group, the developmental rate of follicles was lower than the non-vitrified group, and the subgroup containing 7.5% CM showed better results than the 5%, and 2.5% CM subgroups. However, no changes in fertilization and embryonic development rates were observed. Supplementing follicle culture media with 7.5% CM could enhance follicle growth and oocyte maturation of follicles after vitrification.

Atorvastatin Prevents the Neuron Loss in the Hippocampal Dentate Gyrus Region through its Anti-Oxidant and Anti-Apoptotic Activities.

BACKGROUND: Atorvastatin is a member of statins, which has shown positive vascular effects, anti-oxidant, anti-platelet, and anti-apoptotic properties. OBJECTIVE: In this study, we hypothesized that atorvastatin could prevent the neurons lost in the hippocampal dentate gyrus region after transient global Ischemia/Reperfusion (I/R) through its anti- oxidant and anti-apoptotic activities. METHOD: Twenty-four male Wistar rats, 12-13 weeks old and weighing 250-300 g, were divided randomly into four groups: control, I/R, vehicle (I/R treated with NaCl) and experiment (I/R treated with atorvastatin, 10 mg/kg); rats were sacrificed 96 hours after I/R. Quantitative expression of genes (caspase 8, p53, bax, bcl2, cytochrome c) was studied. The MDA level, SOD, CAT, and GPx activities were measured with biochemical tests. To detect apoptotic cells, TUNEL and Nissl staining were performed. Mitochondria were prepared from the hippocampus rats and used for the quantification of mitochondrial ROS, ATP level, GSH content, membrane potential, cytochrome c release, and determination of mitochondrial swelling. RESULTS: Atorvastatin attenuated the overexpression of bax, cytochrome C, p53, and caspase8 mRNAs and induced expression of bcl-2 mRNA (P<0.001). Atorvastatin treatment increased anti-oxidant enzyme levels (P<0.01). Treatment with atorvastatin reduced the number of TUNEL-positive cells. It could decrease the cytochrome c release (P<0.01), inhibit the decrease of MMP (P<0.001) and increase the ATP level (P<0.001) in hippocampal mitochondria compared with the I/R group. CONCLUSION: Atorvastatin treatment in I/R rats decreases oxidative stress, production of ROS, apoptosis rate in neuronal cells, and improves the mitochondrial function. Hence, atorvastatin has a proper neuronal protective effect against the I/R injury in the brain.

Verapamil Inhibits Mitochondria-Induced Reactive Oxygen Species and Dependent Apoptosis Pathways in Cerebral Transient Global Ischemia/Reperfusion.

The prefrontal cortex is the largest lobe of the brain and is consequently involved in stroke. There is no comprehensive practical pharmacological strategy for ameliorating prefrontal cortex injury induced by cerebral ischemia. Therefore, we studied the neuroprotective properties of verapamil (Ver) on mitochondrial dysfunction and morphological features of apoptosis in transient global ischemia/reperfusion (I/R). Ninety-six Wistar rats were allocated into four groups: control, I/R, I/R+Ver (10 mg/kg twice 1 hour prior to ischemia and 1 hour after reperfusion phase), and I/R+NaCl (vehicle). Animals were sacrificed, and mitochondrial dysfunction parameters (i.e., mitochondrial swelling, mitochondrial membrane potential, ATP concentration, ROS production, and cytochrome c release), antioxidant defense (i.e., superoxide dismutase, malondialdehyde, glutathione peroxidase, catalase, and caspase-3 activation), and morphological features of apoptosis were determined. The results showed that mitochondrial damage, impairment of antioxidant defense system, and apoptosis were significantly more prevalent in the I/R group in comparison with the other groups. Ver decreased mitochondrial damage by reducing oxidative stress, augmented the activity of antioxidant enzymes in the brain, and decreased apoptosis in the I/R neurons. The current study confirmed the role of oxidative stress and mitochondrial dysfunction in I/R progression and indicated the possible antioxidative mechanism of the neuroprotective activities of Ver.

Extracellular Vesicles Derived from Human Umbilical Cord Perivascular Cells Improve Functional Recovery in Brain Ischemic Rat via the Inhibition of Apoptosis.

Background: Ischemic stroke, as a health problem caused by the reduced blood supply to the brain, can lead to the neuronal death. The number of reliable therapies for stroke is limited. Mesenchymal stem cells (MSCs) exhibit therapeutic achievement. A major limitation of MSC application in cell therapy is the short survival span. MSCs affect target tissues through the secretion of many paracrine agents including extracellular vesicles (EVs). This study aimed to investigate the effect of human umbilical cord perivascular cells (HUCPVCs)-derived EVs on apoptosis, functional recovery, and neuroprotection. Methods: Ischemia was induced by middle cerebral artery occlusion (MCAO) in male Wistar rats. Animals were classified into sham, MCAO, MCAO + HUCPVC, and MCAO + EV groups. Treatments began at two hours after ischemia. Expressions of apoptotic-related proteins (BAX/BCl-2 [B-cell lymphoma-2] and caspase-3 and -9), the amount of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells, neuronal density (microtubule-associated protein 2 [MAP2]), and dead neurons (Nissl staining) were assessed on day seven post MCAO. Results: Administration of EVs improved the sensorimotor function (p < 0.001) and reduced the apoptotic rate of Bax/Bcl-2 ratio (p < 0.001), as well as caspases and TUNEL-positive cells (p < 0.001) in comparison to the MCAO group. EV treatment also reduced the number of dead neurons and increased the number of MAP2+ cells in the ischemic boundary zone (p < 0.001), as compared to the MCAO group. Conclusion: Our findings showed that HUCPVCs-derived EVs are more effective than their mother's cells in improving neural function, possibly via the regulation of apoptosis in the ischemic rats. The strategy of cell-free extracts is, thus, helpful in removing the predicaments surrounding cell therapy in targeting brain diseases.

Evaluating the Effects of Different Concentrations of Human Follicular Fluid on Growth, Development, and PCNA Gene Expression of Mouse Ovarian Follicles.

Follicle culture in vitro provides a method for investigating stages of folliculogenesis that can lead to preserving fertility through cryopreservation techniques. This study aims to assess the effects of various concentrations of human follicular fluid (hFF) on growth, development, and expression of the proliferating cell nuclear antigen (PCNA) gene in mouse ovarian follicles in vitro. Preantral follicles were isolated from 14-day NMRI mouse ovaries. The follicles were cultured in basic media enriched with FBS, FSH, and insulin-transferrin-selenium, and supplemented with different concentrations of hFF (10, 20, and 30%) for 12 days. During the culture period, survival rate and follicular maturation, follicular diameter, levels of estrogen and progesterone secretion, and PCNA gene expression rate were evaluated. Survival rate, maturation, and antrum formation were significantly higher in the 10% hFF group than in the 20 and 30% hFF groups. On day 4, follicle diameter in the 10% hFF group was also higher than in the 20 and the 30% hFF group. In comparison with other groups, significantly higher estrogen and progesterone production levels were measured in the 10% hFF group. PCNA gene expression was also higher with 10 than 20 and 30% hFF concentrations. The present study suggests that addition of 10% hFF to mice ovarian preantral follicle culture media enhances follicle growth and oocyte maturation.

Effects of Maternal Exposure to Lamotrigine during Pregnancy on Gonadal Toxicity in Fetus Rats.

BACKGROUND: Lamotrigine is one of the newest antiepileptic drugs that is used as one of the most common treatments in pregnancy. Since the investigation of the teratogenic effects of lamotrigine is very limited and there is no report of its teratogenic effects on fetal gonads, we aimed to investigate the teratogenic effects of lamotrigine on embryonic gonads. MATERIALS AND METHODS: This study was performed on nine female Wistar female rats (8 weeks, weighing 180-200 mg). At first, the animals were inspected regularly by the preparation of vaginal smear and in the estrus phase in separate cages of mating, and after observing the vaginal plaque, were randomly divided into three groups (n=3). Control group did not receive any treatment. In the lamotrigine group (20mg/kg), and the vehicle group (same volume of normal saline) were injected intraperitoneally from days 8 to 13 of pregnancy. On day 20, animals were anesthetized by sodium pentobarbital (40 mg/kg), and embryos were extracted through laparotomy. First, fetuses were weighed, and their height (crown-rump length) was measured. Then the gonads of the fetuses were removed and, stained with H & E, and examined by optical microscope. RESULTS: Our results showed that in the lamotrigine group, the number of seminiferous tubules and Sertoli cells in the male embryos and the number of oocytes in the female embryos decreased significantly compared to the control and vehicle groups (P≤0.05). CONCLUSION: The results of this study showed that treatment with 20 mg/kg lamotrigine in mothers during pregnancy could cause damage to fetal gonads.

Effects of neural stem cell-derived extracellular vesicles on neuronal protection and functional recovery in the rat model of middle cerebral artery occlusion.

Stroke imposes a long-term neurological disability with limited effective treatments available for neuronal recovery. Transplantation of neural stem cells (NSCs) is reported to improve functional outcomes in the animal models of brain ischemia. However, the use of cell therapy is accompanied by adverse effects, so research is growing to use cell-free extracts such as extracellular vesicles (EVs) for targeting brain diseases. In the current study, male Wistar albino rats (20 months old) were subjected to middle cerebral artery occlusion (MCAO). Then, EVs (30 µg) were injected at 2 hours after stroke onset via an intracerebroventricular (ICV) route. Measurements were done at day 7 post-MCAO. EVs administration reduced lesion volume and steadily improved spontaneous locomotor activity. EVs administration also reduced microgliosis (ionized calcium-binding adaptor molecule 1 (Iba1)+ cells) and apoptotic (terminal-deoxynucleotidyl transferase mediated nick end labelling [TUNEL]) positive cells and increased neuronal survival (neuronal nuclear (NeuN)+ cells) in the ischemic boundary zone (IBZ). However, it had no effect on neurogenesis within the sub-ventricular zone (SVZ) but decreased cellular migration toward the IBZ (doublecortin (DCX)+ cells). The results of this study showed neuroprotective and restorative mechanisms of NSC-EVs administration, which may offer new avenues for therapeutic intervention of brain ischemia. SIGNIFICANCE OF THE STUDY: Based on our results, EVs administration can effectively reduce microglial density and neuronal apoptosis, thereby steadily improves functional recovery after MCAO. These findings provide the beneficial effect of NSC-EVs as a new biological treatment for stroke.

Effect of Different Doses and Times of FK506 on Different Areas of the Hippocampus in the Rat Model of Transient Global Cerebral Ischemia-Reperfusion.

BACKGROUND: Stroke is a major worldwide problem that is leading to a high mortality rate in humans. Ischemia, as the most common type of stroke, is characterized by tissue damage that can occur due to insufficient blood flow to the brain even for a brief duration, leading to the release of inflammatory factors, cytokines, and free radicals. In this study, we investigated the effective dose and injection time of FK506 as an immunophilin ligand for providing a suitable effect on cells of CA2, CA3, and dentate gyrus of the hippocampus. METHODS: In this in vivo study, a total of 48 male Wistar rats were divided into nine groups. The ischemia model was induced by the ligation of bilateral common carotid arteries. The doses of FK506 (3, 6, and 10 mg/kg) were administered intravenously (IV) at the beginning of reperfusion, followed by repeated injections (10 mg/kg) at 6, 24, 48, and 72 hours after ischemia, respectively. Brains were removed and prepared for Nissl staining and the TdT-mediated dUTP Nick End Labeling method. RESULTS: Data showed that global ischemia did not decrease the number of viable pyramidal cells in CA2 and CA3 regions, but significant differences were observed in the number of viable granular cells and apoptotic bodies in the dentate gyrus between the control and ischemia groups. Repeated doses of 6 mg/kg of FK506 at an interval of 48 hours were deemed to be the suitable dose and best time of injection. CONCLUSIONS: It seems that FK506 can ameliorate the extent of apoptosis and may be a good candidate for the treatment of ischemia-induced brain damage.

Neuroprotective Effect of Pentoxifylline on 3,4-Methylenedioxymethamphetamine-Induced Apoptosis in CA1 Cells of Wistar Rat Hippocampus.

BACKGROUND: 3,4-Methylenedioxymethamphetamine is psychoactive and hallucinogenic and has been shown to produce neurotoxicity both in animals and in humans. Recently, vasodilator drugs such as pentoxifylline (PTX) have been introduced as an alternative with neuroprotective effects. There is no study about the protective effect of PTX on hippocampal apoptosis due to high-dose administration of 3,4-Methylenedioxymethamphetamine (MDMA), so in this study, the protective effect of PTX on the hippocampus of male Wistar rats following high-dose of the drug has been investigated. MATERIALS AND METHODS: Twenty-four male Wistar rats weighing 250-300 g were randomly divided into four groups: control, sham (MDMA injection), experimental (MDMA+PTX injection), and vehicle (MDMA+saline) groups. Two weeks later, the brains were removed and prepared for TUNEL and western blot techniques. Concomitantly the hippocampus was removed to study the change in Bcl-2 and BAX mRNA expression with quantitative real-time polymerase chain reaction. RESULTS: Data showed that the number of apoptotic bodies significantly decreased in the experimental group compared to the other groups, except for in control. Also, further investigation revealed that BAX reduced considerably, while Bcl-2 mRNA expression increased dramatically after PTX treatment. CONCLUSIONS: Our results suggest that PTX may be a neuroprotective agent, and its neuroprotective potential may contribute to reducing the severity of lesions in the hippocampus following a high dose administration of MDMA.

Effect of Honey/PVA Hydrogel Loaded by Erythromycin on Full-Thickness Skin Wound Healing in Rats; Stereological Study.

BACKGROUND: Skin wounds are a significant public health risk, and treatment of wound remains a challenging clinical problem for medical teams and researchers. MATERIALS AND METHODS: In the present study, we aimed to investigate the healing effects of honey/polyvinyl alcohol (PVA) hydrogel loaded with erythromycin as wound dressing on skin wounds in rats, based on histological studies. In this study, 60 male Wistar rats, with a 1.5 ×1.5 cm2 diameter full-thickness wounds on the backs were divided into four groups: honey/PVA with the erythromycin hydrogel group, honey group, PVA group, and the control group, with no treatment. Skin biopsies were prepared at days 4, 7, and 14 for microscopic analyses. The stereological analysis, including the mean area of the wound, length of vessels, numerical density of fibroblast, macrophage, basal cell and volume of the epidermis, dermis, and fibrous tissue were performed. RESULTS: Wounds area in the honey/PVA hydrogel with the erythromycin group were significantly (P<0.05) smaller than in the other group. The numerical density of fibroblast, macrophage, basal cell and volume of the epidermis in the honey/PVA hydrogel with the erythromycin group were significantly higher than other groups. CONCLUSION: According to our results, honey/PVA hydrogel with erythromycin may promote early wound healing and has a positive influence on fibroblast proliferation and re-epithelialization, and its administration is recommended after further validation of clinical data.

Protective effect of pentoxifylline on male Wistar rat testicular germ cell apoptosis induced by 3,4-methylenedioxymeth amphetamine.

OBJECTIVES: 3, 4-methylenedioxymethamphetamine (MDMA) one of the methamphetamine derivatives that affect the reproductive system, has not been well understood. Many young people are consumers of drugs such as MDMA that can affect their reproductive capability. Apoptosis is the main mechanism for male infertility. Pentoxifylline (PTX) increases cAMP intracellularly and reduces tumor necrosis factor-α. This study aimed to investigate the protective effect of PTX administration in MDMA-induced apoptosis in testes of male Wistar rats. MATERIALS AND METHODS: Thirty male Wistar rats weighing 250-300 g were randomly divided into five groups: control group (without any intervention), group receiving 7.5 mg/kg MDMA three times every two hours for one day, first experimental group receiving 100 mg/kg PTX just at the time of third injection of MDMA, second experimental group receiving 100 mg/kg PTX a week before MDMA administration, and the vehicle group, which received MDMA+saline. Two weeks later, testes were removed and prepared for H&E staining, TUNEL and Western blot techniques. RESULTS: There was a significant decrease of the score in the MDMA group compared with the control group. In first and second experimental groups, the quality of seminiferous epithelium was improved compared with the MDMA group. The number of TUNEL-positive cells/tubule increased in MDMA and vehicle groups, which is decreased by administration of PTX before MDMA. Expression of active caspase-3 significantly increased in MDMA group, which is significantly decreased by administration of PTX before MDMA. CONCLUSION: PTX can significantly reduce the severity of lesions in the testes following administration of MDMA.

Reduction in ischemic brain injury following the administration of pentoxifylline after transient global ischemia/ reperfusion in a rat model.

BACKGROUND: It is well known that the hippocampus, the CA1 Pyramidal cells in particular, is selectively vulnerable during global cerebral ischemia. Recently, it is observed that pentoxifylline has a neuroprotective effect. This study explored the pharmacological relationship between ischemiainduced cell death of the hippocampus and the efficacy of a vasodilator agent (pentoxifylline) in the prevention of delayed neuronal death. METHODS: This experimental study was performed on 4 groups: control, ischemia, experimental (200mg/kg pentoxifylline injection one hour prior to and one hour following ischemia) and vehicle (normal saline). Transient global ischemia was induced by bilateral common carotid arteries occlusion. To investigate the apoptotic bodies and caspase-3 activities as a central role in the execution phase of apoptosis, the brains were prepared for the TUNEL technique. RESULTS: Pentoxifylline administration limited apoptosis and caspase-3 activities in rats' hippocampi. Our data showed no significant difference between the number of apoptotic bodies in the CA1 region of the hippocampus in the control and pentoxifylline -treated groups (p= 0.994). The results of one- way ANOVA revealed that that ischemia significantly increased caspase-3 levels in the hippocampus (p< 0.05); however, the level of caspase-3 in pentoxifylline -treated rats was less than the ischemic group. CONCLUSION: These results suggest that the neuroprotective effect of pentoxifylline (200mg/kg) may be accompanied by a reduction in ischemic damage within the CA1 region of the hippocampus in rats subjected to transient global cerebral ischemia.

Effect of Cyperus rotundus on ischemia-induced brain damage and memory dysfunction in rats.

OBJECTIVES: Global cerebral ischemia-reperfusion injury causes loss of pyramidal cells in CA1 region of hippocampus. In this study, we investigated the possible neuroprotective effects of the ethanol extract of Cyperus rotundus (EECR) on a model of global transient ischemia in rat, by evaluating the pathophysiology of the hippocampal tissue and spatial memory. MATERIALS AND METHODS: Treatment group (EECR, 100 mg/kg/day) was gavaged from 4 days before, to 3 days after ischemia. Morris water maze test was performed 1 week after ischemia for 4 days. Brain tissue was prepared for Nissl staining. RESULTS: Our data showed no statistical difference between the treatment and ischemia groups in water maze task. So, treatment of ischemia with EECR cannot improve spatial learning and memory. On the contrary EECR ameliorated the CA1 pyramidal cell loss due to transient global ischemia/reperfusion injury. CONCLUSION: These results suggest that EECR cannot reduce the ischemia-induced, cognitive impairments seen after transient, global cerebral ischemia but can prevent pyramidal cell loss in CA1 region of hippocampus.

Pentoxifylline Protects the Rat Liver Against Fibrosis and Apoptosis Induced by Acute Administration of 3,4-Methylenedioxymethamphetamine (MDMA or Ecstasy).

OBJECTIVE(S): 3,4-Methylenedioxymethamphetamine (MDMA) is one of the most popular drugs of abuse in the world with hallucinogenic properties that has been shown to induce apoptosis in liver cells. The present study aimed to investigate the effects of pentoxifylline (PTX) on liver damage induced by acute administration of MDMA in Wistar rat. MATERIALS AND METHODS: Animals were administered with saline or MDMA (7.5 mg/kg, IP) 3 times with 2 hr intervals. PTX (200 mg kg, IP), was administered simultaneously with last injection of MDMA in experimental group. RESULTS: The concomitant administration of pentoxifylline and MDMA decreased liver injury including apoptosis, fibrosis and hepatocytes damages. CONCLUSION: Our results showed for the first time that PTX treatment diminishes the extent of apoptosis and fibrosis caused by MDMA in rat liver.

Effects of FK506 on Hippocampal CA1 Cells Following Transient Global Ischemia/Reperfusion in Wistar Rat.

Transient global cerebral ischemia causes loss of pyramidal cells in CA1 region of hippocampus. In this study, we investigated the neurotrophic effect of the immunosuppressant agent FK506 in rat after global cerebral ischemia. Both common carotid arteries were occluded for 20 minutes followed by reperfusion. In experimental group 1, FK506 (6 mg/kg) was given as a single dose exactly at the time of reperfusion. In the second group, FK506 was administered at the beginning of reperfusion, followed by its administration intraperitoneally (IP) 6, 24, 48, and 72 hours after reperfusion. FK506 failed to show neurotrophic effects on CA1 region when applied as a single dose of 6 mg/kg. The cell number and size of the CA1 pyramidal cells were increased, also the number of cell death decreased in this region when FK506 was administrated 48 h after reperfusion. This work supports the possible use of FK506 in treatment of ischemic brain damage.

Effect of Pentoxifylline on Ischemia- induced Brain Damage and Spatial Memory Impairment in Rat.

OBJECTIVES: The brief interruption of cerebral blood flow causes permanent brain damage and behavioral dysfunction. The hippocampus is highly vulnerable to ischemic insults, particularly the CA1 pyramidal cell layer. There is no effective pharmacological strategy for improving brain tissue damage induced by cerebral ischemia. Previous studies reported that pentoxifylline (PTX) has a neuroprotective effect on brain trauma. The possible neuroprotector effects of PTX on behavioral deficit were studied in male Wistar rats subjected to a model of transient global brain ischemia. MATERIALS AND METHODS: Animals (n= 32) were assigned to control, sham-operated, vehicle, and PTX- treated (200 mg/kg IP) groups. PTX administered at 1hr before and 3 hr after ischemia. Global cerebral ischemia was induced by bilateral common carotid artery occlusion, followed by reperfusion. RESULTS: Morris Water maze testing revealed that PTX administration in cerebral ischemia significantly improved hippocampal-dependent memory and cognitive spatial abilities after reperfusion as compared to sham-operated and vehicle-treated animals. After the behavioral test, the rats were sacrificed and brain sections were stained with Nissl staining. There were no significant differences between number of pyramidal cells in both control and PTX groups. CONCLUSION: Our study demonstrated that pentoxifylline had a protective effect on rats with transient global ischemia and could reduce cognitive impairment.