Kazim Tugyan

Erythropoietin improves long-term spatial memory deficits and brain injury following neonatal hypoxia-ischemia in rats

It is well known that neonatal hypoxic-ischemic brain injury leads to mental retardation and deficits in cognitive abilities such as learning and memory in human beings. The ameliorative effect of erythropoietin (Epo) on experimental hypoxic-ischemic brain injury in neonatal rats has been recently reported. However, the effect of Epo on cognitive abilities in the hypoxic-ischemic brain injury model is unknown. The aim of this study is to investigate the effects of Epo on learning-memory, behavior and neurodegeneration induced by hypoxia-ischemia. Seven days old Wistar Albino rat pups have been used in the study (n = 28). Experimental groups in the study were: (1) saline-treated hypoxia-ischemia group, (2) Epo-treated (i.p., 1000 U/kg) hypoxia-ischemia group, (3) sham-operated group, (4) control group. In hypoxia-ischemia groups, left common carotid artery was ligated permanently on the seventh postnatal day. Two hours after the procedure, hypoxia (92% nitrogen and 8% oxygen) was induced for 2.5 h. Epo was administered as a single dose immediately after the hypoxia period. When pups were 22 days old, learning experiments were performed using Morris water maze. On the 20th week, when brain development is accepted to be complete, learning experiments were repeated. Rats were then perfused and brains removed for macroscopic and microscopic evaluation. Epo treatment immediately after hypoxic-ischemic insult significantly improved long-term neurobehavioral achievements when tested during the subsequent phase of brain maturation and even into adulthood. Histopathological evaluation demonstrated that Epo also significantly diminished brain injury and spared hippocampal CA1 neurons. In conclusion, Epo administrated as a single dose immediately after neonatal hypoxic-ischemic insult provides benefit over a prolonged period in the still developing rat brain. Since the wide use of Epo in premature newborns, this agent may be potentially beneficial in treating asphyxial brain damage in the perinatal period.

The effects of regular aerobic exercise in adolescent period on hippocampal neuron density, apoptosis and spatial memory

It is known that positive effects of regular aerobic exercise on cognitive functions in humans and also animals; but how to the effects of aerobic exercise in adolescent period is unknown. The present study examined the effects of regular aerobic exercise on spatial memory using the Morris water maze, cell density and apoptosis of hippocampus in adolescent rats. Twenty-two days of age male rats were run on a treadmill for 30 min/session at a speed of 8m/min and 0 degrees slope, five times a week for 8 weeks. The present study showed that exercise induced significant cognitive improvement throughout brain maturation in rats. The number of hippocampal CA1 and CA3 neurons, and gyrus dentatus neurons were significantly increased in the exercised rats. There was no significant difference of CA2 neuron density between exercise and control groups. There was no significantly differences in any groups according to the results of apoptosis that account of TUNEL positive cells. The present results suggest that regular moderate aerobic treadmill exercise benefit in cognitive functions. This result may derive from treadmill exercise-induced increase cell density without altering of apoptosis in the hippocampus and dentate gyrus of adolescent rats.

Effect of Melatonin on Testicular Damage in Streptozotocin-Induced Diabetes Rats

Background: It is well known that diabetes mellitus is associated with impairment of testicular function. In the present study, we aimed to demonstrate the effect of melatonin on testicular damage in male rats with streptozotocin (STZ)-induced diabetes. Methods: Male Wistar rats were divided into 4 groups: (1) control group, (2) melatonin-treated nondiabetic group, (3) diabetic group and (4) melatonin-treated diabetic group. Diabetes was induced by STZ injection. Melatonin was administered intraperitoneally at the dose of 10 mg/kg for 5 days. Testicular damage was examined by using hematoxylin and eosin staining and periodic acid-Schiff staining, and apoptosis was determined by terminal-deoxynucleotidyl-transferase-mediated dUTP nick end labeling (TUNEL). Potential disorders associated with seminiferous tubular sperm formation were evaluated using the Johnsen score. Results: Diabetic rats showed a reduction in seminiferous tubule diameter, increased thickening of the basement membrane in seminiferous tubules and degenerated germ cells. TUNEL-positive cells were significantly more numerous in diabetic rats than in control rats. Melatonin significantly attenuated the diabetes-induced morphological changes and germ cell apoptosis in the diabetic rat testis. The number of polymorphonuclear leukocytes was significantly decreased in group 4 when compared to group 3. Conclusions: These results suggest that intraperitoneal administration of melatonin for 5 days is a potentially beneficial agent to reduce testicular damage in adult diabetic rats, probably by decreasing oxidative stress.

Erythropoietin attenuates lipopolysaccharide-induced white matter injury in the neonatal rat brain.

Periventricular leukomalacia (PVL), a common neonatal brain white matter (WM) lesion, is frequently associated with cerebral palsy. Growing evidence has indicated that in addition to ischemia/reperfusion injury, cytokine-induced brain injury associated with maternal or fetal infection may also play an important role in the pathogenesis of PVL. Recent studies have shown that administration of lipopolysaccharide (LPS) to pregnant rats causes enhanced expression of the cytokines, i.e., IL-1β, TNF-α, and IL-6, in fetal brains. In recent years, it has been shown that erythropoietin (EPO) has a critical role in the development, maintenance, protection and repair of the nervous system. In the present study we investigated the effect of EPO on LPS-induced WM injury in Sprague-Dawley rats. LPS (500 µg/kg) suspension in pyrogen-free saline was administered intraperitoneally to pregnant rats at 18 and 19 days of gestation. The control group was treated with pyrogen-free saline. They were given 5,000 U/kg recombinant human EPO. Seven-day-old Sprague-Dawley rat pups were divided into four groups: control group, LPS-treated group, prenatal maternal EPO-treated group (5,000 U/kg, intraperitoneally given to pregnant rats at 18 and 19 days of gestation), and postnatal EPO-treated group (5,000 U/kg, intraperitoneally given to 1-day-old rat pups). Cytokine induction in the postnatal 7-day-old (P7) rat brain after maternal administration of LPS was determined by the ELISA method. The proinflammatory cytokine levels (IL-1β, TNF-α, and IL-6) in P7 rat pup brains were significantly increased in the LPS-treated group as compared with the control group. Prenatal maternal EPO treatment significantly reduced the concentration of TNF-α and IL-6 in the newborn rat brain following LPS injection. The concentration of IL-1β was decreased in the intrauterine EPO treatment group. Postnatal EPO treatment significantly decreased only the IL-6 concentration in the newborn rat brain following LPS injection. The concentration of cytokines, IL-1β and TNF-α, was reduced in the postnatal EPO treatment group. We demonstrated here that LPS administration in pregnant rats at gestational day 18 and 19 induced WM injury in P7 progeny characterized by apoptosis. Prenatal maternal and postnatal EPO treatment significantly reduced the number of apoptotic cells in the periventricular WM. Using immunohistochemistry techniques, we investigated the effects of maternal administration of LPS on myelin basic protein (MBP) staining, as a marker of myelination in the periventricular area in the neonatal rat brain. MBP staining was significantly less and weaker in the brains of the LPS-treated group as compared with the prenatal maternal EPO-treated group. However, the postnatal EPO treatment did not prevent LPS-stimulated loss of MBP-positive staining. In conclusion, especially prenatal maternal EPO treatment attenuates LPS-induced injury by reducing the expression of inflammatory cytokines and sparing MBP in the neonatal rat brain. While the postnatal EPO treatment prevented LPS-induced brain injury this effect was partial. To our knowledge, this is the first study that demonstrates a protective effect of EPO on LPS-induced WM injury in the developing brain. Regarding the wide use of EPO in premature newborns, this agent may be potentially beneficial in treating LPS-induced brain injury in the perinatal period.

The effects of selenium against cerebral ischemia-reperfusion injury in rats

It is known that the brain tissue is extremely sensitive to ischemia-reperfusion (IR) injury and therefore, brain ischemia and consecutive reperfusion result in neural damage and apoptosis. The proinflammatory cytokines such as tumor necrosis factor alfa (TNF-alpha) and interleukin-1 beta (IL-1beta) are produced during neurological disorders including cerebral ischemia. On the other hand, nerve growth factor (NGF), which is essential for the differentiation, survival and functions of neuronal cells in the central nervous system, regulate neuronal development through cell survival and cell death signaling. In the present study, we aimed to investigate the effect of selenium (Se) on prefrontal cortex and hippocampal damage in rats subjected to cerebral IR injury. Selenium was injected intraperitoneally at the doses of 0.625 mg/(kg day) after induction of IR injury. Prefrontal cortex and hippocampal damage was examined by cresyl-violet staining. Apostain and caspase-3 immune staining were used to detect apoptosis. TNF-alpha, IL-1beta and NGF levels were also evaluated. Histopathological evaluation showed that treatment with selenium after ischemia significantly attenuated IR-induced neuronal death in prefrontal cortex and hippocampal CA1 regions of rats. Apoptotic cells stained with apostain and caspase-3 were significantly decreased in treatment group when compared with the IR group. Additionally, treatment with selenium decreased the TNF-alpha and IL-1beta levels and increased the NGF levels in prefrontal cortex and hippocampal tissue of animals subjected to IR. The present results suggest that selenium is potentially a beneficial agent in treating IR-induced brain injury in rats.

Protective effect of melatonin against head trauma-induced hippocampal damage and spatial memory deficits in immature rats

It is well known that head trauma induces the cognitive dysfunction resulted from hippocampal damage. In the present study, we aimed to demonstrate the effect of melatonin on hippocampal damage and spatial memory deficits in 7-day-old rat pups subjected to contusion injury. Melatonin was injected intraperitoneally at the doses of 5 or 20 mg/kg of body weight immediately after induction of traumatic injury. Hippocampal damage was examined by cresyl violet staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Spatial memory performance was assessed in the Morris water maze. Melatonin significantly attenuated trauma-induced neuronal death in hippocampal CA1, CA3 regions and dentate gyrus, and improved spatial memory deficits, which was equally effective at doses of 5-20 mg/kg. The present results suggest that melatonin is a highly promising agent for preventing the unfavorable outcomes of traumatic brain injury in young children.

Hyperoxic exposure leads to cell death in the developing brain

Premature infants have high incidence of motor and cognitive impairment in later life. Supraphysiological oxygen concentrations are routinely used in neonatal intensive care units and elicit injury to premature lungs and retina. Since the effects of hyperoxia on the developing brain are scarce, we studied the effects of high oxygen on this tissue. Wistar rat pups were exposed from birth until day 5 to 21% or 80% oxygen. The neuronal density and apoptosis in CA1 and dentate gyrus of hippocampus, prefrontal cortex, parietal cortex, subiculum, and retrosplenial cortex were assessed by immunohistochemistry and ELISA cell death assay. Neuronal density of the investigated brain areas were significantly decreased in the hyperoxia group. Furthermore, using ELISA cell death and TUNEL assays, we observed increased cell death in the developing brain. Our results show that hyperoxia induces cell death in the developing rat brain. This may be one of the important mechanisms that cause motor and cognitive impairment in later life of premature infants.

Effect of erythropoietin on oxygen-induced brain injury in the newborn rat.

The developing nervous system is sensitive to supraphysiological oxygen concentrations. Recent studies showed that exposure to hyperoxia in infant rats leads to extensive apoptotic degeneration in the cortex and white matter of the developing brain. A wide variety of experimental studies have shown that erythropoietin exerts a remarkable neuroprotection in both cell cultures and in animal models of nervous system disorders. In the present study, we investigated the effect of erythropoietin against hyperoxia-induced neurodegeneration in the developing brain. Eighteen Wistar rat pups were divided into three groups: control group, hyperoxia+saline-treated group and hyperoxia+erythropoietin-treated group. Hyperoxia groups were exposed to 80% oxygen (n=12) in a plexiglas chamber in which the oxygen concentration was monitored twice daily from birth until postnatal day 5. Hyperoxia exposure was 24h/day for 5 days. The hyperoxia+erythropoietin group received an intraperitoneal injection of recombinant human erythropoietin at a dose of 1000U/(kgday). At postnatal day 5, all animals were sacrificed. Neuronal cell death and apoptosis were evaluated. Histopathological examination showed that erythropoietin significantly diminished apoptosis in the CA1 region and dentate gyrus of hippocampus and parietal cortex in hyperoxia+erythropoietin-treated group. Regarding the safety profile of erythropoietin in premature and mature infants, this agent may be potentially beneficial in preventing hyperoxic brain injury.

Viability of crushed and diced cartilage grafts wrapped in oxidized regenerated cellulose and esterified hyaluronic acid: an experimental study.

Objectives: The objective of this study was to investigate the viability of diced/crushed cartilage grafts wrapped in esterified hyaluronic acid (HYAFF) and oxidized regenerated cellulose (Surgicel) with respect to macroscopic and microscopic parameters.

CARNOSINE ATTENUATES OXIDATIVE STRESS AND APOPTOSIS IN TRANSIENT CEREBRAL ISCHEMIA IN RATS

Cerebral ischemia leads to cognitive decline and neuronal damage in the hippocampus. Reactive oxygen species (ROS) play an important role in the neuronal loss after cerebral ischemia and reperfusion injury. Carnosine has both antioxidant and neuroprotective effects against ROS. In the present study, the effects of carnosine on oxidative stress, apoptotic neuronal cell death and spatial memory following transient cerebral ischemia in rats were investigated. Transient ischemia was induced by occlusion of right common carotid artery of rats for 30 min and reperfusion for 24 h or 1 week. Rats received intraperitoneal injection of 250 mg/kg carnosine or saline 30 min prior to experiment. Determination of antioxidant enzyme activities was performed spectrophotometrically. To detect apoptotic cells, TUNEL staining was performed using an In Situ Cell Death Detection Kit. Carnosine treatment elicited a significant decrease in lipid peroxidation and increase in antioxidant enzyme activities in ischemic rat brains. The number of TUNEL-positive cells was decreased significantly in carnosine-treated group when compared with the ischemia-induction group. Carnosine treatment did not provide significant protection from ischemia induced deficits in spatial learning. The results show that carnosine is effective as a prophylactic treatment for brain tissue when it is administered before ischemia without affecting spatial memory.