Perinatal HI-induced braininjury is one of major causes of CP. There is increasing evidence showing that early DEX administration in preterm infants may increase the incidence of CP [4-6]. In accordance with these clinical findings, the current results show that early DEX exposure is able to increase the vulnerability of the neonatalbrain to subsequent HI damage. Although there are some stud- ies indicating that DEX pretreatment can protect neonatalbrain against subsequent HI injury [15,16,34], our results do not support a neuroprotective role for neonatal DEX treatment in cerebral HI. One possible explanation of these seemingly discrepant observations is the different doses and regimens of DEX used among studies. These findings reinforce the long-held view that the concentra- tion and duration of glucocorticoid treatment are major factors determining the beneficial or detrimental effects of glucocorticoids in the brain . In our model, DEX was administered over a long period (P1-3) in tapering doses in an attempt to mimic the longer treatment regimens commonly used in the neonatal intensive care setting [8,9,14,17,18]. While much caution is required when extrapolating data from animal models to the human con- dition, our findings highlight the risk for heightened devel- oping brain vulnerability to HI injury associated with neonatal DEX treatment.
Fig. 2. (A) Representative flow cytogram of an annexin V binding (abscissa) versus PI uptake (ordinate) in the ipsilateral cerebral hemisphere of newborn rat brain cells at 24 hours after HI. The numbers in the upper left quadrant (Q1), upper right quadrant (Q2), lower left quadrant (Q3), and lower right quadrant (Q4) represent the percentage of damaged (annexin V-/PI+), necrotic (annexin V+/PI+), live (annexin V-/PI-) and apoptotic cells (annexin V+/PI-), respectively. (B) Representative flow cytometric analyses of mitochondrial membrane potential using JC-1. The shift of JC-1 fluorescence from orange (FL2) to green (FL1) indicates a collapse of mitochondrial membrane potential. NC, normoxia control group; HC, HI control group; HG, HI with G-CSF treatment group.
physiological and pharmacological properties, including anti-aging, anti-carcinogenic, anti-inflammatory, anti- oxidant and anti-apoptotic properties [8–10]. Studies have identified a neuroprotective role for resveratrol in animal models of central nervous system diseases such as Alzheimer’s disease, and cerebral ischemia/reperfusion injury [9, 11–15]. The experimental studies in vivo shown that resveratrol is well tolerated and has low toxicity [16, 17]. Thus the rapid adoption of resveratrol has been tested in clinical trials, including for overweight/obesity, diabetic/metabolic syndrome, cancer and cardiovascular disease [18–21]. In addition, several studies have investigated its effect on the central nervous system in human adults [22, 23]. And no adverse effects of resveratrol were reported in the majority of human studies . The characteristics of being well-tolerated and low toxicity are important criteria for the application of a drug in medicine, particularly in neonates. Pretreatment with resveratrol has been reported to attenuate perinatal hypoxic-ischemicbraininjury [24–26]. However, few studies have reported whether post-treatment with resveratrol can protect against neonatalhypoxic-ischemicbraininjury.
Estetrol (E4), estradiol (E2) and progesterone (P4) have important antioxidative and neuroprotective effects in neuronal system. We aimed to study the consequence of combined steroid therapy in neonatalhypoxic-ischemic encephalopathy (HIE). In vitro the effect of E4 combined with other steroids on oxidative stress and the cell viability in primary hippocampal cultures was evaluated by lactate dehydrogenase and cell survival assays. In vivo neuroprotective and therapeutic efficacy of E4 combined with other steroids was studied in HIE model of immature rats. The rat pups rectal temperature, body and brain weights were evaluated. The hippocampus and the cortex were investigated by histo/immunohistochemistry: intact cell number counting, expressions of markers for early gray matter lose, neuro- and angiogenesis were studied. Glial fibrillary acidic protein was evaluated by ELISA in blood samples. In vitro E4 and combinations of high doses of E4 with P4 and/or E2 significantly diminished the LDH activity and upregulated the cell survival. In vivo pretreatment or treatment by different combinations of E4 with other steroids had unalike effects on body and brain weight, neuro- and angiogenesis, and GFAP expression in blood. The combined use of E4 with other steroids has no benefit over the single use of E4.
an in vitro hypoxia model . However, Jou et al. demonstrated that melatonin decreased reactive oxygen species and reduced mitochon- drial swelling caused by H 2 O 2 in the cell, inter- rupting early apoptotic events such as expo- sure of the serous phosphate ester serine, calcium overload, nuclear apoptosis, etc. . Thus, they suggested that the anti-apoptotic effect of melatonin was mainly due to its inhibi- tion of mitochondrial calcium overload and depolarization of the mitochondrial membrane, which decreased membrane permeability . Moreover, melatonin may play a protective role by blocking the release of cytochrome C and by activating the caspase-3 downstream motif, preventing the occurrence of nuclear enrich- ment . The mechanism of the protective and nerve regenerative effects of melatonin against hypoxia and ischemia braininjury are not the focus of this study, and the specific reg- ulatory mechanism of melatonin in the apop- totic pathway remains to be further elucidated. Treatment of neonatalhypoxic-ischemicbraininjury can be achieved through the blockade or reversal of the neuron apoptotic process, which would decrease cell loss. Given that the devel- oping neonatalbrain has very strong plasticity, nerve regeneration in the injured area should greatly improve the long-term prognosis. Takagi et al. found the proliferation of neural stem cells (NSCs) in the dentate gyrus of the hippo- campus after transient forebrain ischemia in mice . Furthermore, the BrdU-positive cells in the dentate gyrus and ventricular zone were increased at days 3, 7, and 10 after reperfusion . Studies have also found that cerebral ischemia can promote the proliferation of NSCs in the hippocampal dentate gyrus, and NSCs in the SVZ and ischemic hemisphere were also increased . Jin et al. also showed that, in the gerbil brain, the number of cells in the den- tate gyrus increased 12-fold at 1-2 weeks after cerebral ischemia . However, in the case of ischemic-hypoxicbrain damage, it is difficult for these endogenously regenerated nerve cells to reach the damaged area and replace the apop- totic neurons, which is the main obstacle for self-repair and neurological function recovery . In this study, DCX/BrdU and NeuN/BrdU double-positive cells were found in the braininjury region in melatonin-treated rats, suggest- ing that these endogenously regenerated nerve cells could reach the damaged area and facili- tate nerve regeneration.
The molecular mechanisms underlying miR-210-mediated braininjury may be due to an alteration of its target genes. From the gene bank of rodent BDNF mRNA sequence and the database on target miRNAs (www.microrna.org), we have identified that miR-210 has a potential binding site at 3’ UTR of gene encoding BDNF mRNA region (see the diagram in figure 6). Furthermore, previous studies have experimentally validated the binding sites of miR-210 at BDNF 3’UTR sequence and demonstrated that miR-210 directly regulates BDNF expression . These observations suggest that the BDNF gene may be one of the important target genes of miR-210. BDNF, a member of the neurotrophin family, plays crucial roles in the developing central nervous system . BDNF, binding with its receptor TrkB, controls the neuronal development, differentiation, proliferation and survival . Altered BDNF/TrkB expression impacts not only the morphological development of neurons as well as their functional activities during brain development . For example, BDNF involves in the formation of appropriate synaptic connections in the developing brain. Deletion of BDNF contributes to disorders of cognitive function . In the present study, in addition to decreased brain weight (Figure 1), maternal nicotine treatment also significantly decreased BDNF and TrkB protein expressions in the developing brains of neonates as compared with the saline control groups, suggesting that the reduced expression of brain BDNF/TrkB may significantly contribute to the nicotine-mediated aberrant brain development and neurological dysfunction. In addition, we observed that miR-210-LNA treatment countered nicotine-induced reduction of BDNF/TrkB expression and eliminated the differences of BDNF/TrkB expression between the nicotine-treated and saline control groups. These findings suggests that miR-210 could directly regulate BDNF/TrkB expression, and the molecular mechanisms underlying miR-210-mediated braininjury in response to perinatal nicotine exposure may be due to decreased BDNF/TrkB expression in neonatal brains. Indeed, previous studies have reported that intravenous treatment with BDNF significantly reduced infarct size and neurological outcome after focal cerebral ischemia, further supporting the notion that BDNF plays a significant role neuroprotection against ischemicinjury [46, 47]. Therefore, it’s
In neonatal H-I models, there is previous evidence that ce- rebral ischemia leads to delayed cell death with DNA damage (13–15). For example, 12 h after bilateral carotid ligation and exposure to 15 min of hypoxia in P8 rats, TUNEL labeling and karyorrhectic cells can be identified (13). Extensive DNA lad- dering was observed by others (14) at 18 h after H-I in the neo- natal model we used; however, only this one time point was ex- amined. We used both biochemical and anatomical criteria to look for the occurrence and time course of DNA damage and caspase activation in this model. Both TUNEL labeling and DNA laddering revealed evidence for DNA damage begin- ning at 6 h after H-I. These methods are sensitive but not al- ways specific for apoptosis. For example, it has been shown in cultured cells that while most cells with DNA laddering are dy- ing via apoptosis, there are exceptions in which DNA ladder- ing is present in the setting of necrosis (42). EM analysis, how- ever, also appears to confirm that at least some of the cell death occurring in this model is consistent with apoptosis. The potential importance of our findings of DNA damage along with delayed caspase activation is that cell death with these features can be under active cell control. For example, func- tional inhibition of ced-3 in Caenorhabditis elegans (43) or the mammalian homologues of ced-3 (caspases) can prevent cell death in diverse cell types, including neurons (18, 19, 44). Whether caspase inhibitors would be protective after acute nervous system injury is just beginning to be studied. Recent findings using IL-1 b –like protease inhibitors including z-VAD- DCB in adult rats (45) and z-VAD.FMK in adult mice (46) showed decreases in ischemic damage after ICV administra- tion in the setting of middle cerebral artery occlusion. In one study, z-VAD-DCB was given just before ischemia (45), and in the other, z-VAD.FMK was protective when administered at the end of arterial occlusion but not when treatment was de- layed by 1 h. Whether delayed treatment was not as effective as BAF in our model may be due to differences in the drugs, the models, or developmental age at time of injury.
Figure 1. Oxygen-glucose deprivation (OGD) alters mitochondrial membrane potential, morphology and function in primary cortical neurons. (a) Primary mouse cortical neurons were loaded with JC-1 dye and Hoechst before exposure to OGD. Cells were imaged live before (Con) and at 15, 45 and 90 min during OGD. Both mitochondrial morphology (as observed in red, top row) and membrane potential (increased green signal, second row) are altered during exposure to OGD. Scale bar represents 100 µm; Figures are representative of three individual experiments: (b) Enlargement (3×) of regions defined by white boxes in (a). As the experiment progresses, mitochondria morphology appears to alter from tubular structures to round punctate or larger aggregations; (c) Primary neurons loaded with JC-1 were imaged every minute during OGD followed by analyses of mitochondrial length. Data shown are an average of 360 mitochondria per time point, and mitochondria from the first and last time points analyzed by student’s t-test in the panel below, *** p < 0.001; (d) Primary neurons were subjected to 90 min of OGD followed by up to 24 h incubation in normal growth medium. Lysates were assayed for citrate synthase activity at time points shown following the insult. Data is shown ± SD, n = 4–6 independent litters, determined by two-way ANOVA followed by a Bonferroni post-hoc test, ** p < 0.01 for interaction and treatment.
blocked using autophagy inhibitors. The term necroptosis is used to describe this alternative non-apoptotic cell death pathway. A specific necroptosis inhibitor, Nec-1 has been found through chemical library screening. Nec-1 efficiently inhibits TNFα-induced cell death, without the requiring caspase inhibition. Necroptosis has also been shown to occur in the injured mouse brain after middle cerebral artery occlusion (MCAO), with Nec-1 treatment significantly reducing infarct size 27 . Nec-1 has also been shown to
Despite these encouraging data, it will be critical to pro- ceed with caution when developing novel therapeutics. A hallmark of the neonatalbrain is the unique potential for plasticity. Although advantageous from a treatment per- spective, it is also likely that administration of exogenous compounds could alter critical developmental processes and/or mechanisms associated with neuroplasticity. It is now known that neurogenesis is prominent after ischemic insult in the neonatal rat brain. The Levison laboratory found that neural progenitors from the subventricular zone (SVZ), and possibly glial progenitors, migrate to and populate columnar regions of the neocortex that show prominent cell death in response to H-I . Another study in mouse showed that treatment with the broad- spectrum MMP inhibitor GM6001 once daily for 10 days inhibited the migration of neural progenitors from the SVZ to the corpus striatum after transient MCAO . Although it is presently unknown whether the migration of neural progenitors results in synapse formation that restores function, prolonged MMP inhibition could pro- hibit proteolytic cleavage of ECM proteins that is neces- sary for migration of progenitors, subsequent neuroplasticity and repair. Similarly, inhibition of MMPs could reduce chemokine signaling through reduced processing of MCP chemokines and SDF-1α, thereby reducing progenitor cell migration. Additional concerns need to be considered regarding the inhibition of MMPs, particularly MMP-9, and the potential effects on myelina- tion during a critical period of oligodendrocyte prolifera- tion and maturation. Nonetheless, experimental research has provided strong and convincing evidence that MMP inhibitors are excellent candidate therapeutics if adminis- tered selectively at the appropriate time points.
Abstract: Objective: To investigate the neuroprotective effects of grape seed proanthocyanidin extract （ GSPE ） on a model of neonatalhypoxicischemic encephalopathy (HIE), we investigated the changes in neuronal cells and astrocytes after pre-treatment with GSPE. Methods: Seven-day-old pups were randomly divided into sham, HI, and GSPE+HI groups. The HIE model was established using a modified Rice-Vannucci method. GSPE was injected intraperitoneally 20 min before surgery. The change in markers of neuronal cells and astrocytes (NeuN/GFAP) were detected by immunofluorescence and Western blot. Results: Compared with the sham group, the expression of NeuN in the HI group was significantly reduced, and the expression of NeuN was significantly increased after GSPE pre-treatment. The expression of GFAP was opposite to NeuN. Conclusion: Our study showed that GSPE pre-treatment significantly protected neurons and inhibited astrocyte over-proliferation. Therefore, we believe that GSPE is a potential drug for the treatment of HIE and can prevent brain damage caused by hypoxia and ischemia.
Our results showed that the number of Nestin positive cells was significantly larger in HI and HBO group than that in sham group. It is report- ed that the HIBD can induce the proliferation of NSCs in situ [17, 18]. We speculated that the proliferation of NSCs in situ was involved with the Nestin expression after HIBD. Figure 2 indi- cated that the shape of Nestin positive cells was similar with astrocyte. A previous report revealed that a majority of Nestin expression was observed in astrocytes following brain inju- ry . The Nestin expression in astrocytes consisted with the embryonal protein played key role in brain remodeling and recovery [20, 21]. In this study the Nestin positive cells observed may be reactive hyperplasia astro- cytes differentiated by SNC after HIBD. Our results also showed that HBO treatment pre- dominately increased the BrdU labeled cells in DG region of pups, which suggested that HBO promoted the proliferation and differentiation in HI rats. Besides, reports documented that the optimum time of HBO treatment was at 6h after injury  and in contrast, HBO treatment aggravated the injury after 12 h [23, 24]. We found that the both Nestin and BrdU positive cells peaked 4 days after surgery and then decreased significantly. The most effect of HBO on HIBD treatment may be for 4 days. Thus, there is a problem which is the best treatment of HBO for HIBD that needs to be further investigated.
Cerebral hypoxia – ischemia (HI) is a principal risk factor of perinatal brain injuries in both full-term and preterm neonates worldwide leading to acute mortality and chronic disability [1 – 4]. Despite current therapeutic mo- dalities, HI still accounts for 23% of all neonatal deaths globally . Survivors of perinatal asphyxia suffer life- long disabilities such as cerebral palsy, epilepsy, and cog- nitive, behavioral, attentional, socialization, and learning difficulties [6 – 10]. Although numerous neuroprotective treatments have appeared promising in animal experi- ments, most of them were not reliable or effective in human patients with hypoxicischemic encephalopathy (HIE). Thus, there is an urgent need for the identifica- tion of new therapeutic targets for salvaging brain to address treatment of HIE.
Abstract: Perinatal hypoxic-ischemic encephalopathy (HIE) affects one to three per 1,000 live full-term births and can lead to severe and permanent neuropsychological sequelae, such as cerebral palsy, epilepsy, mental retardation, and visual motor or visual perceptive dysfunction. Melatonin has begun to be contemplated as a good choice in order to diminish the neurological sequelae from hypoxic-ischemicbraininjury. Melatonin emerges as a very interesting medication, because of its capacity to cross all physiological barriers extending to subcellular compartments and its safety and effectiveness. The purpose of this commentary is to detail the evidence on the use of melatonin as a neuroprotection agent. The pharmacologic aspects of the drug as well as its potential neuroprotective characteristics in human and animal studies are described in this study. Melatonin seems to be safe and beneficial in protecting neonatal brains from perinatal HIE. Larger randomized controlled trials in humans are required, to implement a long-awaited feasible treatment in order to avoid the dreaded sequelae of HIE.
Next, we asked whether carvacrol can reduce brain dam- age in vivo using a mouse neonatalhypoxic-ischemicbraininjury model. We observed that carvacrol pre- treatment (30 and 50 mg/kg i.p., 30 min before HI) sig- nificantly reduced infarct volume 24 hours after HI. TTC staining of coronal sections of mouse brains was used for evaluating the infarct volume. Representative images of TTC staining were shown in Figure 2A, where white areas indicated brain damage. There was no detect- able infarction in the sham group (data not shown). Infarct volume in the vehicle-treated HI group (Vehicle) was 57.83 ± 5.18% (n = 24 pups). Carvacrol pre-treatment (30 and 50 mg/kg) significantly reduced the infarct volume to 31.11 ± 7.63% (n = 11 pups) and 6.18 ± 3.73% (n = 17 pups), respectively, compared to the vehicle-treated group (*, p < 0.05). The reduction of infarct volume was dose- dependent (#, p < 0.05) (Figure 2A). Furthermore, 7 days after HI, whole brains were fixed, imaged, and then sec- tioned for Nissl staining. Carvacrol pre-treatment (50 mg/ kg) also resulted in significantly less brain damage (both in whole brains and coronal sections, n = 15 pups) 7 days after HI (Figure 2B) compared to vehicle treatment (with larger brain tissue loss, n = 12 pups). Brain imaging at 7 days further supported TTC staining at one day with re- spect to the neuroprotective effects of carvacrol in HI.
difference was detected in the primitive reflexes (cliff avoidance and negative geotaxis test) and motor func- tion test among treatment, vehicle, and sham groups. On the other hand, significant improvement in step- down passive avoidance test was found after ceftriaxone treatment. The difference of behavior between HIE group and normal control group included long-lasting sensorimotor and locomotor deficits . But, unlike human, rats exposed to HIE injury did not exhibit gross motor function deficit in some studies although some permanent deficit has also been observed . This may be due to a higher degree of plasticity of neonatal rat brain compared with that of human brain. Step-down passive avoidance reflects learning and memory func- tion. In our studies, ceftriaxone rescued hippocampal cells from apoptosis which may contribute to improved step-down passive avoidance results.
In the brain, there are 2 major isoforms of NO syn- thase (NOS) expressed constitutively, the neuronal iso- form (nNOS) present in neurons and the endothelial isoform (eNOS) present primarily within the vascular endothelium. NO derived from nNOS in the ischemicneonatalbrain is cytotoxic, contributing to neurodegen- eration after hypoxic-ischemicinjury because of its re- action with the superoxide radical with the formation of the potent oxidant peroxynitrite, but NO generated from eNOS within the vascular endothelium may be protec- tive through induction of vasodilation, which, in turn, decreases the severity of ischemia. 24 A third isoform,
calculated for statistical analysis. A paired t test was used to detect statistical differences in the DTI indices and histologic evaluations between the injury and control ECs. After correction for multiple comparisons (5 times the repeated measurement of DTI indices), P ⬍ .01 was regarded as a significant difference in the comparison of in- jury/control DTI indices. Because DTI indices are repeated-measures data, longitudinal changes of DTI indices were analyzed by using a linear mixed model, followed by a least significant difference post hoc pair-wise comparison test. The ratios and absolute values of DTI in- dices were the dependent variables, whereas the independent vari- ables were subjects (a random factor) and time points (a categoric variable). Because the histologic results are cross-sectional data, the changes of absolutes and ratios of injury/control histologic evalua- tions between different time points were evaluated by the 1-way anal- ysis of variance (ANOVA) test, followed by the Tukey test. The Pear- son correlation test was used to evaluate correlations between DTI indices and histologic staining intensity. To evaluate the intraobserver reliability of region-of-interest measurement of DTI indices, we ran- domly selected 4 rats in every time point (total of 20 rats) for remea- surement. To determine the consistency of DTI indices between dif- ferent FOVs, we repeated measurements of DTI indices in 3 randomly selected rats, which were scanned by using the 2 different FOVs (3.2 cm 2 and 4.0 cm 2 ). Intraobserver reliability and consistency of differ-
infant, the therapeutic window would be short and possibly no longer than 1 to 2 hours. In this regard, no drug has been found efficacious in reducing the severity of hypoxic–ischemicbrain damage in imma- ture animals when given later than 2 hours after termination of the hypoxic–ischemic insult (see below). An additional issue that constantly arises regard- ing therapeutic intervention of the asphyxiated full- term newborn infant is the identification of those infants at highest risk for permanent brain damage. Given the presumed short therapeutic window, such infants must be identified as soon after birth as pos- sible so that an appropriate drug is administered on arrival in the neonatal intensive care unit. Clinical investigations suggest that infants at highest risk for hypoxic–ischemicbrain damage include those who have exhibited progressive fetal heart rate abnormal- ities shortly before birth, are severely depressed at birth (very low Apgar scores), exhibit an acidosis with a pH , 7.0 on umbilical cord blood oxygen and acid-base analysis, and require major resuscitation in the delivery room, including cardiac massage and intubation. 11–14 An additional dilemma relates to the