Erdener Özer

Neuroprotective Effect of Erythropoietin on Hypoxic-Ischemic Brain Injury in Neonatal Rats

Erythropoietin (Epo) prevents ischemia and hypoxia-induced neuronal death in vitro. Recent studies have shown that this cytokine also has in vivo neuroprotective effects in cerebral and spinal ischemia in adult rodents. In this study, we aimed to investigate the effect of systemically administered recombinant human Epo on infarct volume and apoptotic neuronal death in a newborn rat hypoxic-ischemic brain injury model. Our results showed that a single dose of intraperitoneal Epo treatment (1,000 U/kg) significantly decreased the mean infarct volume as compared to the control group. In contrast to the Epo-treated group, histopathological examination by positive terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling of the affected brain in control animals revealed widespread neuronal injury associated with numerous apoptotic cells. Morphometric analysis to determine the extent of damage quantitatively ascertained that the mean infarct volume was significantly lower in the Epo-treated group (p < 0.003). These results suggest the beneficial neuroprotective effect of Epo in this model of neonatal hypoxic-ischemic brain injury. To our knowledge, this is the first study that demonstrates a protective effect of Epo against hypoxia-ischemia in the developing brain.

Selective Inhibition of Nitric Oxide in Hypoxic-Ischemic Brain Model in Newborn Rats: Is It an Explanation for the Protective Role of Erythropoietin?

Erythropoietin (Epo) exerts neuroprotection against neuronal death induced by ischemia and hypoxia in vitro and in vivo. Recent studies suggest that the neuroprotective effects of Epo may depend upon different mechanisms, including the inhibition of nitric oxide (NO). We recently demonstrated that Epo exerts neuroprotection in a model of neonatal hypoxic-ischemic brain damage. In the present study, we directly determined whether systemic administration of recombinant Epo modulates cerebral NO production in a neonatal rat model of hypoxic-ischemic brain injury. Seven-day-old Wistar rat pups were subjected to left carotid artery occlusion followed by 2.5 h of hypoxic exposure. Brain nitrite levels were evaluated in both hemispheres (carotid ligated or nonligated) by Griess reagent 72 h after the hypoxic-ischemic insult. Our results show that hypoxic-ischemic insult results a significant increase in NO production as compared with NO levels in hypoxic hemispheres and control animals. A single dose of Epo treatment (1,000 U/kg i.p.) significantly decreased NO overproduction in the hypoxic-ischemic hemisphere, whereas no significant change appeared in hypoxia alone or in controls. These data suggest that the selective inhibitory effect of Epo on NO overproduction could have a neuroprotective effect in neonatal hypoxic-ischemic brain injury.

Erythropoietin Downregulates Bax and DP5 ProApoptotic Gene Expression in Neonatal Hypoxic-Ischemic Brain Injury

Background: Perinatal asphyxia is an important cause of neonatal mortality and subsequent serious sequelae such as motor and cognitive deficits and seizures. The ameliorative effect of erythropoietin (Epo) on experimental hypoxic-ischemic brain injury in neonatal rats has been recently reported. Recent studies also confirm the antiapoptotic effect of Epo in a variety of in vitro and in vivo neuronal injury models including hypoxic-ischemic brain injury. However, molecular mechanisms of Epo protection and antiapoptotic effect in this model are unclear. Epo may exert its antiapoptotic effect via the differential regulation of the expression of genes involved in the apoptotic process. Objectives: Thus, in the present study, we studied the effects of systemically administered Epo on antiapoptotic (bcl-2, bcl-XL), proapoptotic (bax and DP5) gene expression following hypoxic-ischemic brain injury in neonatal rats. Methods: Seven- day-old Wistar rat pups were divided into three groups: control group (n = 15), saline-treated group (n = 17), and Epo-treated group (n = 18). Rat pups were subjected to left carotid artery occlusion followed by 2.5 h of hypoxic exposure. Epo-treated group received an intraperitoneal injection of recombinant human Epo at a dose of 1,000 units/kg, saline-treated group received an intraperitoneal injection of saline at the same volume of Epo. Forty-eight hours after hypoxia, 3 animals in each group were killed for histopathological evaluation. To detect DNA fragmentation in cell nuclei, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling reaction was applied. Bcl-2 and bax protein expression were also analyzed with immunohistochemistry. For reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, rats were sacrificed 4, 12, and 24 h after hypoxia. Bcl-2, bcl-XL, bax, and DP5 mRNA expression were analyzed by RT-PCR. Results: Epo significantly prevented hypoxia-ischemia-induced bax and DP5 mRNA upregulation in brain tissue. Epo did not show any effect on bcl-XL transcription altered by injury. However, Epo reversed injury-induced downregulation in bcl-2 transcription. Modulating effects of Epo on bcl-2 and bax protein expression were also revealed by immunohistochemistry. Conclusions: These results suggest that Epo exerts a neuroprotective effect against hypoxic-ischemic brain injury, at least partially, via the differential regulation of the expression of genes involved in apoptotic process.

Effects of erythropoietin on hyperoxic lung injury in neonatal rats

Pulmonary oxygen toxicity is believed to play a prominent role in the lung injury that leads to the development of bronchopulmonary dysplasia (BPD). To determine whether human recombinant erythropoietin (rhEPO) treatment reduces the risk of developing BPD, we investigated the effect of rhEPO treatment on the histopathologic changes seen in hyperoxia-induced lung injury of BPD. Twenty-five rat pups were divided into four groups: air-exposed control group (n = 5), hyperoxia-exposed placebo group (n = 7), hyperoxia-exposed rhEPO-treated group (n = 6), and air-exposed rhEPO-treated group (n = 7). Measurement of alveolar surface area, quantification of secondary crest formation, microvessel count, evaluation of alveolar septal fibrosis, and smooth muscle actin immunostaining were performed to assess hyperoxia-induced changes in lung morphology. Treatment of hyperoxia-exposed animals with rhEPO resulted in a significant increase in the mean alveolar area, number of secondary crests formed, and the microvessel count in comparison with hyperoxia-exposed placebo-treated animals. There was significantly less fibrosis in rhEPO-treated animals. However, treatment of hyperoxia-exposed animals with rhEPO did not result in a significant change in smooth muscle content compared with hyperoxia-exposed placebo treated animals. Our results suggest treatment with rhEPO during hyperoxia exposure is associated with improved alveolar structure, enhanced vascularity, and decreased fibrosis. Therefore, we conclude that treatment of preterm infants with EPO might reduce the risk of developing BPD.

The effects of intraamniotic human neonatal urine and meconium on the intestines of the chick embryo with gastroschisis

BACKGROUND/PURPOSE Urinary waste products in the amniotic fluid has been implicated as a cause of intestinal damage (ID) in gastroschisis based on the fact that fetus urinates physiologically into the amniotic cavity. However, experimental and clinical data suggest that intrauterine defecation is a physiological event, thus gastrointestinal waste products also may be responsible for ID in gastroschisis. An experimental study was performed to investigate the effects of intraamniotic human neonatal urine and diluted meconium on the intestines of chick embryo with gastroschisis. METHODS Five-day-old fertilized chick eggs (Gallus domesticus) were used. Gastroschisis was created through amniotic cavity without opening the allantoic cavity. Sterile urine and meconium were obtained from newborn humans, and 1% meconium suspension was prepared. The eggs were divided in to 3 groups. In the first group, gastroschisis was created, and amniotic fluid was reinstilled without changing its composition (control group). Equal amounts of amniotic fluid and urine mixture was instilled into the amniotic cavity in second group (urine group) and 1% meconium suspension was instilled in similar fashion in the third group after creation of gastroschisis (meconium group). RESULTS Histopathologic features of the intestines of the urine group did not differ from the intestines of the control group. The meconium groups bowel showed serosal thickening, inflammation, focal fibrin, and collagen deposits. Histopathologic changes of intestines induced by intraamniotic diluted meconium are consistent with the ones described for human gastroschisis specimens. CONCLUSION Gastrointestinal waste products seem responsible for the ID in gastroschisis rather than urinary waste products.

Deleterious effects of local corticosteroid injections on the Achilles tendon of rats.

Abstract The purpose of this study was to examine the pathological changes in the Achilles tendon and its paratenon after intratendinous corticosteroid injections and to reveal the effects of this drug on healthy tendon. We also sought for the effects of these injections compared with compression with a clamp on the Achilles tendons of the rats. Fifty-two Achilles tendons in 26 male Wistar rats were included in the study. Betamethasone injections were applied to the left tendons at different intervals, while the right tendons served for compression with mosquito clamps for varied periods. At the end of 30 days, all of the tendons were excised and examined histopathologically according to a semiquantitative scoring system. Histopathologic evaluation demonstrated some degree of degeneration in both groups. Statistical analysis showed no significant difference among the two groups, but in macroscopic evaluation, the tendons in the betamethasone group demonstrated enlargement and strong adhesion to the subcutaneous tissue. We conclude that intratendinous betamethasone injections are as harmful as compression with a clamp and can be used as a degeneration-producing model in further studies. Enlargement of the tendon mass and strong adhesion to the subcutaneous tissue can be due to injection of the betamethasone partly outside the tendon.

Amniotic fluid exchange for the prevention of neural tissue damage in myelomeningocele: an alternative minimally invasive method to open in utero surgery.

Objective: Experimental studies have shown that neural tissue damage in myelomeningocele (MMC) is acquired, resulting from exposure of neural tissue to amniotic fluid (AF). Similar to neural tissue damage in MMC, in gastroschisis, intestines exposed to AF are damaged. In gastroschisis, intestinal damage can be prevented by changing the composition of the AF with partial AF exchanges. An experimental study was performed to investigate whether the neural tissue damage in MMC can be prevented by AF exchange. Methods: Thirteen-day-old fertilized chick eggs were used. In group 1, the amnio-allantoic membrane was opened to create a common cavity, and MMC was created (MMC-only group). In group 2, after creation of MMC, amnio-allantoic fluid exchange was performed (MMC-plus-exchange group). Chicks were extirpated for histopathologic examination 5 days later. Results: While edema, focal calcification, fibrosis, capillary cell proliferation and scattered mononuclear cells were observed in the MMC-only group, histopathologic changes were mild in the exchange group. The number of neuron-specific enolase stainings (+) neural cell count was significantly higher in the exchange group compared to the MMC-only group (p < 0.01). Conclusion: Exposure of MMC to AF causes structural neural tissue damage that can be prevented by AF exchange. AF exchange is minimally invasive compared to open in utero surgery for the closure of MMC. By AF exchange, neural tissue damage that occurs during the gestational period may be prevented.

Comparison of different surgical repairs in the treatment of experimental duodenal injuries

BACKGROUND In this experimental study, we aimed to investigate the results of different surgical repair methods for delayed reconstruction of severe experimental duodenal defects. METHODS A large duodenal defect with irregular and tagged margins covering about 50% of the circumference was created in the second part of duodenum of male Wistar rats. The effectiveness of primary repair, jejunal serosal patch, Roux-en-Y duodenojejunostomy, or expanded polytetrafluoroethylene patch repair techniques were investigated on the basis of survival and histologic assessment. RESULTS No significant survival benefit was observed between jejunal serosal patch, Roux-en-Y duodenojejunostomy, or expanded polytetrafluoroethylene patch repair techniques. But these repair modalities were associated with better survival rates than no-treatment or primary repair techniques. Complete coverage of the expanded polytetrafluoroethylene grafts by neomucosa consisting of columnar epithelium with villus formation was observed in surviving rats about 16 weeks after surgery. CONCLUSIONS Expanded polytetrafluoroethylene patch can be used in the repair of experimental large duodenal defects, which can not be repaired primarily.

Prognostic significance of angiogenesis and immunoreactivity of cathepsin d and type IV collagen in high-grade stage T1 primary bladder cancer

OBJECTIVES To assess the prognostic significance of biologic parameters such as angiogenesis, expression of cathepsin D (a lysosomal protease), and degradation of type IV collagen (a basement membrane protein), we studied 20 patients with primary grade III Stage T1 transitional cell carcinoma of the bladder. METHODS Endothelial cells were labeled immunohistochemically using factor VIII-related antigen. The vascular surface density (VSD) and the microvessel number (NVES) were assessed by means of stereology. The tumor tissues were also analyzed by immunohistochemical methods for the expression of cathepsin D and the staining pattern of type IV collagen. RESULTS Eight patients (40%) having either recurrence or progressive disease showed greater NVES and VSD values (P = 0.002 and P = 0.01, respectively) than patients without. The significance of vascular parameters was found to be statistically independent from coexisting carcinoma in situ, bacille Calmette-Guerin (BCG) treatment, tumor size, and number. Additionally, these parameters did not show statistical significance between progressive and recurrent tumors. However, tumors with solid morphologic features had higher VSD values and a significantly greater rate of recurrence or progression (P = 0.01 and P = 0.07, respectively). Tissue from 17 (85%) of 20 tumors showed absent or patchy basement membrane staining for type IV collagen, and 12 (60%) showed strong immunoreactivity for cathepsin D antibody. There were no differences for either molecule with either BCG treatment or other parameters related to prognosis. CONCLUSIONS Angiogenesis may have an independent role in predicting prognosis in grade III Stage T1 bladder carcinoma. Grade III Stage pT1 tumors with solid morphologic features have higher angiogenetic activity and a worse prognosis. Cathepsin D and type IV collagen do not seem to play a role in predicting biologic behavior.

Hepatic glycogenosis: a rare cause of hepatomegaly in Type 1 diabetes mellitus

Hepatomegaly, with or without abnormal liver function tests, was a common feature of both pediatric and adult patients with diabetes mellitus. We are reporting a case of a 16-year-old diabetic boy in whom we found hepatomegaly, mildly elevated transaminases and elevated serum lipids never noticed before. Abdominal ultrasound confirmed hepatomegaly; liver biopsy pointed out a picture compatible with glycogenosis. The patients abnormal liver function tests, elevated serum lipids and hepatomegaly decreased over a period of 4 weeks with tight metabolic control. This situation was due to overinsulinization because the patient assumed an excessive quantity of food and therefore took an excessive quantity of insulin. In conclusion, hepatomegaly may be seen in diabetic patients due to hepatic glycogen accumulation as a result of excessive food and insulin consumption. In hepatic glycogenosis, the pathological findings improve in 4 weeks when good metabolic control is provided. Therefore, the other reasons must be investigated when hepatomegaly persists for a longer period.