Quantitative in Vivo Detection of Brain Cell Death after Hypoxia Ischemia Using the Lipid Peak at 1.3 ppm of Proton Magnetic Resonance Spectroscopy in Neonatal Rats

This study was performed to determine the accuracy of proton magnetic spectroscopy (1H-MRS) lipid peak as a noninvasive tool for quantitative in vivo detection of brain cell death. Seven day-old Sprague Dawley rats were subjected to 8% oxygen following a unilateral carotid artery ligation. For treatment, cycloheximide was given immediately after hypoxic ischemia (HI). Lipid peak was measured using 1H-MRS at 24 hr after HI, and then brains were harvested for fluorocytometric analyses with annexin V/propidium iodide (PI) and fluorescent probe JC-1, and for adenosine-5'-triphosphate (ATP) and lactate. Increased lipid peak at 1.3 ppm measured with 1H-MRS, apoptotic and necrotic cells, and loss of mitochondrial membrane potential (DeltaPsi) at 24 hr after HI were significantly improved with cycloheximide treatment. Significantly reduced brain ATP and increased lactate levels observed at 24 hr after HI showed a tendency to improve without statistical significance with cycloheximide treatment. Lipid peak at 1.3 ppm showed significant positive correlation with both apoptotic and necrotic cells and loss of DeltaPsi, and negative correlation with normal live cells. Lipid peak at 1.3 ppm measured by 1H-MRS might be a sensitive and reliable diagnostic tool for quantitative in vivo detection of brain cell death after HI.

Long-Term (Postnatal Day 70) Outcome and Safety of Intratracheal Transplantation of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells in Neonatal Hyperoxic Lung Injury

PURPOSE: This study was performed to evaluate the long-term effects and safety of intratracheal (IT) transplantation of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) in neonatal hyperoxic lung injury at postnatal day (P)70 in a rat model. MATERIALS AND METHODS: Newborn Sprague Dawley rat pups were subjected to 14 days of hyperoxia (90% oxygen) within 10 hours after birth and allowed to recover at room air until sacrificed at P70. In the transplantation groups, hUCB-MSCs (5x10(5)) were administered intratracheally at P5. At P70, various organs including the heart, lung, liver, and spleen were histologically examined, and the harvested lungs were assessed for morphometric analyses of alveolarization. ED-1, von Willebrand factor, and human-specific nuclear mitotic apparatus protein (NuMA) staining in the lungs and the hematologic profile of blood were evaluated. RESULTS: Impaired alveolar and vascular growth, which evidenced by an increased mean linear intercept and decreased amount of von Willebrand factor, respectively, and the hyperoxia-induced inflammatory responses, as evidenced by inflammatory foci and ED-1 positive alveolar macrophages, were attenuated in the P70 rat lungs by IT transplantation of hUCB-MSCs. Although rare, donor cells with human specific NuMA staining were persistently present in the P70 rat lungs. There were no gross or microscopic abnormal findings in the heart, liver, or spleen, related to the MSCs transplantation. CONCLUSION: The protective and beneficial effects of IT transplantation of hUCB-MSCs in neonatal hyperoxic lung injuries were sustained for a prolonged recovery period without any long-term adverse effects up to P70.

Neuroprotective effects of L-carnitine against oxygen-glucose deprivation in rat primary cortical neurons / 소아과

PURPOSE: Hypoxic-ischemic encephalopathy is an important cause of neonatal mortality, as this brain injury disrupts normal mitochondrial respiratory activity. Carnitine plays an essential role in mitochondrial fatty acid transport and modulates excess acyl coenzyme A levels. In this study, we investigated whether treatment of primary cultures of rat cortical neurons with L-carnitine was able to prevent neurotoxicity resulting from oxygen-glucose deprivation (OGD). METHODS: Cortical neurons were prepared from Sprague-Dawley rat embryos. L-Carnitine was applied to cultures just prior to OGD and subsequent reoxygenation. The numbers of cells that stained with acridine orange (AO) and propidium iodide (PI) were counted, and lactate dehydrogenase (LDH) activity and reactive oxygen species (ROS) levels were measured. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and the terminal uridine deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay were performed to evaluate the effect of L-carnitine (1 microM, 10 microM, and 100 microM) on OGD-induced neurotoxicity. RESULTS: Treatment of primary cultures of rat cortical neurons with L-carnitine significantly reduced cell necrosis and prevented apoptosis after OGD. L-Carnitine application significantly reduced the number of cells that died, as assessed by the PI/AO ratio, and also reduced ROS release in the OGD groups treated with 10 microM and 100 microM of L-carnitine compared with the untreated OGD group (P<0.05). The application of L-carnitine at 100 microM significantly decreased cytotoxicity, LDH release, and inhibited apoptosis compared to the untreated OGD group (P<0.05). CONCLUSION: L-Carnitine has neuroprotective benefits against OGD in rat primary cortical neurons in vitro.

Granulocyte Colony Stimulating Factor Attenuates Hyperoxia-Induced Lung Injury by Down-Modulating Inflammatory Responses in Neonatal Rats

PURPOSE: Granulocyte colony stimulating factor (G-CSF) has been known to increase neutrophil production and have anti-inflammatory properties, but the effect of G-CSF on pulmonary system is in controversy. We investigated whether G-CSF treatment could attenuate hyperoxia-induced lung injury, and whether this protective effect is mediated by the down-modulation of inflammatory responses in a neonatal rat model. MATERIALS AND METHODS: Newborn Sprague-Dawley rats (Orient Co., Seoul, Korea) were subjected to 14 days of hyperoxia (90% oxygen) beginning within 10 h after birth. G-CSF (20 microg/kg) was administered intraperitoneally on the fourth, fifth, and sixth postnatal days. RESULTS: This treatment significantly improved hyperoxia-induced reduction in body weight gain and lung pathology such as increased mean linear intercept, mean alveolar volume, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling positive cells. Hyperoxia-induced activation of nicotinamide adenine dinucleotide phosphate oxidase, which is responsible for superoxide anion production, as evidenced by upregulation and membrane translocation of p67phox was significantly attenuated after G-CSF treatment, as were inflammatory responses such as increased myeloperoxidase activity and mRNA expression of transforming growth factor-beta. However, the attenuation of other proinflammatory cytokines such as tumor necrosis factor-alpha and interleukin-6 was not significant. CONCLUSION: In sum, G-CSF treatment significantly attenuated hyperoxia-induced lung injury by down-modulating the inflammatory responses in neonatal rats.

Periventricular leukomalacia induced by in utero clamping of pregnant rat aorta in fetal rats / 소아과

PURPOSE: This study was undertaken to develop an animal model of periventricular leukomalacia (PVL) induced by in utero clamping of pregnant rat aorta in fetal rats. METHODS: A timed pregnanct Sprague-Dawley rat on embryonic day 21 just prior to delivery was sedated and anesthetized, and a Harvard ventilator for small animals was applied. Following laparotomy, the maternal aorta was clamped reversibly for 40 minutes using a surgical clip. The fetal rats were then delivered by Cesarean section, resuscitated if necessary, and reared by a surrogate mother rat until postnatal day 21 to obtain the brain specimen. After systemic perfusion and fixation, 10 microm thick serial brain sections were obtained and stained for pathologic examination and assessment of ventriculomegaly. Ventriculomegaly was assessed by the measured ventricle to total brain volume ratio. RESULTS: Eight out of eleven fetal rats (73%) survived in the ischemia group after induction of in utero ischemia by clamping maternal rat aorta, and all ten survived in the control group. Body and brain weights measured at postnatal day 21 were significantly lower in the ischemia group compared to the control group. In pathologic findings, significant ventriculomagaly (3.67+/-1.21% vs. 0.23+/-0.06%) was observed in the ischemia group compared to the control group; although cystic lesion was not observed, mild (n=6) and moderate (n=2) rerefaction of the brain tissue was observed. CONCLUSION: A fetal rat model of PVL induced by in utero clamping of pregnant rat aorta was developed.

Erythropoietin Attenuates Brain Injury, Subventricular Zone Expansion, and Sensorimotor Deficits in Hypoxic-Ischemic Neonatal Rats

The aim of this study was to investigate the effect of erythropoietin (EPO) on histological brain injury, subventricular zone (SVZ) expansion, and sensorimotor function deficits induced by hypoxia-ischemia (HI) in newborn rat pups. Seven-day-old male rat pups were divided into six groups: normoxia control, normoxia EPO, hypoxia control, hypoxia EPO, HI control, and HI EPO group. Sham surgery or HI was performed in all animals. HI was induced by ligation of the right common carotid artery followed by 90 min of hypoxia with 8% oxygen. Recombinant human EPO 3 U/g or saline was administered intraperitoneally, immediately, at 24- and 48-hr after insult. At two weeks after insult, animals were challenged with cylinder-rearing test for evaluating forelimb asymmetry to determine sensorimotor function. All animals were then sacrificed for volumetric analysis of the cerebral hemispheres and the SVZ. The saline-treated HI rats showed marked asymmetry by preferential use of the non-impaired, ipsilateral paw in the cylinder-rearing test. Volumetric analysis of brains revealed significantly decreased preserved ipsilateral hemispheric volume and increased ipsilateral SVZ volume compared with the sham-operated animals. Treatment of EPO significantly improved forelimb asymmetry and preserved ipsilateral hemispheric volume along with decreased expansion of ipsilateral SVZ following HI compared to the saline-treated HI rats. These results support the use of EPO as a candidate drug for treatment of neonatal hypoxic-ischemic brain damage.

Granulocyte Stimulating Factor Attenuates Hypoxic-ischemic Brain Injury by Inhibiting Apoptosis in Neonatal Rats

PURPOSE: This study was undertaken to determine the neuroprotective effect of granulocyte stimulating factor (G-CSF) on neonatal hypoxic-ischemic brain injury. MATERIALS AND METHODS: Seven-day-old male newborn rat pups were subjected to 110 minutes of 8% oxygen following a unilateral carotid artery ligation. Apoptosis was identified by performing terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining and flow cytometry with a combination of fluorescinated annexin V and propidium iodide (PI) and JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide). The extent of cerebral infarction was evaluated at 2 weeks after recovery. RESULTS: With a single dose (50microgram/kg) of G-CSF treatment immediately after hypoxic-ischemic insult, hypoxia-ischemia induced increase in TUNEL-positive cells, annexinV+/PI- and JC-1 positive apoptotic cells in the ipsilateral cerebral cortex was significantly reduced at 24 hours, measured by flow cytometry, and the extent of cerebral infarction at 2 weeks after recovery was also significantly attenuated compared to the hypoxia-ischemia control group. CONCLUSION: Our data suggest that G-CSF is neuroprotective by inhibiting apoptosis, thereby reducing the ensuing cerebral infarction in a newborn rat pup model of cerebral hypoxia-ischemia (HI).

Erythropoietin Attenuates Hyperoxia-Induced Lung Injury by Down-modulating Inflammation in Neonatal Rats

This study was done to determine whether recombinant human erythropoietin (rhEPO) treatment could attenuate hyperoxia-induced lung injury, and if so, whether this protective effect is mediated by the down-modulation of inflammation in neonatal rats. Newborn Sprague Dawley rat pups were subjected to 14 days of hyperoxia (>95% oxygen) within 10 hr after birth. Treatment with rhEPO significantly attenuated the mortality and reduced body weight gain caused by hyperoxia. With rhEPO treatment, given 3 unit/gm intraperitoneally at 4th, 5th, and 6th postnatal day, hyperoxia- induced alterations in lung pathology such as decreased radial alveolar count, increased mean linear intercept, and fibrosis were significantly improved, and the inflammatory changes such as myeloperoxidase activity and tumor necrosis factor-alpha expression were also significantly attenuated. In summary, rhEPO treatment significantly attenuated hyperoxia-induced lung injury by down-modulating the inflammatory responses in neonatal rats.

Therapeutic Window for Cycloheximide Treatment after Hypoxic-Ischemic Brain Injury in Neonatal Rats

We have previously shown that cycloheximide significantly inhibited apoptosis, and reduced ensuing cerebral infarction in a newborn rat model of cerebral hypoxiaischemia. This study was performed to determine the therapeutic window for cycloheximide therapy. Seven day-old newborn rat pups were subjected to 100 min of 8% oxygen following a unilateral carotid artery ligation, and cycloheximide was given at 0, 6, 12 and 24 hr after hypoxia-ischemia (HI). Apoptosis or necrosis was identified by performing flow cytometry with a combination of fluorescinated annexin V and propidium iodide, and the extent of cerebral infarction was evaluated with triphenyl tetrazolium chloride (TTC) at 48 hr and 72 hr after HI, respectively. With cycloheximide treatment at 0 hr after HI, both apoptotic and necrotic cells by flow cytometry were significantly reduced, only necrotic cells were significantly reduced at 6 and 12 hr, and no protective effect was seen if administration was delayed until 24 hr after HI compared to the HI control group. Infarct volume, measured by TTC, was significantly reduced by 92% and 61% when cycloheximide was given at 0 or 6 hr after HI respectively; however, there was an insignificant trend in infarct reduction if cycloheximide was administered 12 hr after HI, and no protective effect was observed when administration was delayed until 24 hr after HI. In summary, cycloheximide was neuroprotective when given within 6 hr after HI in the developing newborn rat brain.

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

This study was done to determine the neuroprotective effect of cycloheximide on neonatal hypoxic-ischemic brain injury. Seven day-old newborn rat pups were subjected to 90 min of 8% oxygen following a unilateral carotid artery ligation. The extent of cerebral infarction was evaluated at 1 and 4 week of recovery. Apoptosis was identified by performing terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining and flow cytometry with a combination of fluoresceinated annexin V and propidium iodide. Brain infarction area was significantly increased at 4 week compared to 1 week after hypoxia-ischemia in the control group. With cycloheximide treatment, the number of TUNEL positive cells in the ipsilateral cerebral cortex at 48 hr and peri-infarct area at 1 and 4 week of recovery was significantly reduced, both apoptotic and necrotic cells by flow cytometry 48 hr after the injury were significantly reduced, and the extent of cerebral infarction at 1 and 4 week of recovery was also significantly attenuated compared to the hypoxia-ischemia control group. In summary, our data suggest that apoptosis plays an important role in the development of delayed infarction, and inhibition of apoptosis with cycloheximide significantly reduces the ensuing cerebral infarction in a newborn rat pup model of cerebral hypoxia-ischemia.

Single Cell Dissociation Methods for Flow Cytometric Cell Death Analysis of Hypoxia-Ischemia Injured Newborn Rat Pup Brain / 소아과

PURPOSE: Newborn brain tissue has to be dissociated into a single cell suspension for flow cytometric analysis of cell death during hypoxia-ischemia. Thus the development of a method to dissociate cells from the brain tissue with least damage and maintenance of membrane and antigen integrity remains the challenge for the in vivo application of this technique. We evaluated the efficacy of mechanical or enzymatic (collagenase or tryspin) methods of brain tissue disaggregation. METHODS: The extent of the damage to the plasma membrane and loss of the characteristics of the membrane induced with each dissociation method was determined by comparing the flow cytometric results labeled with both fluorescent annexin V and propidium iodide of the newborn rat pup brain tissue in the control group (n=10) and in the 48-hour after hypoxia-ischemia group (n=10). RESULTS: In the control group, the cell percentage of damaged, apoptotic and necrotic cells of both hemispheres with the mechanical dissociation method was significantly increased compared to the trypsin or collagenase method. In the 48-hour after hypoxia-ischemia group, the cell percentage of apoptotic and necrotic cells of the right hemisphere with the collagenase method significantly increased, and live cells significantly decreased compared to the left hemisphere, control group. Although the same trend was observed, the extent of alterations made with the trypsin method was significantly less compared to the collagenase method. CONCLUSION: The dissociation of neonatal brain tissue for flow cytometric analysis with collagenase was most efficacious with the least cell damage and preservation of the plasma membrane characteristics.