P7 Rats Research Articles

Developmental and sex differences in tetramethylenedisulfotetramine (TMDT)-induced syndrome in rats.

Tetramethylenedisulfotetramine (TMDT) is a synthetic neurotoxic rodenticide considered a chemical threat agent. Symptoms of intoxication include seizures leading to status epilepticus and death. While children and women have been often the victims, no studies exist investigating the neurotoxic effects of TMDT in developing individuals or females. Thus, we performed such an investigation in developing Sprague-Dawley rats of both sexes in order to identify potential age- or sex-dependent vulnerability to TMDT exposure. Subcutaneous injection was chosen as the preferred route of TMDT exposure. EEG recordings confirmed the seizure activity observed in both postnatal day 15 (P15) and adult rats. Additionally, P15 rats displayed greater sensitivity to TMDT than postnanatal day 25 or adult animals. Seizures were generally more severe in females compared to males. Barrel rotations accompanied convulsions in P25 and adult, but sparsely in P15 rats. Adults developed barrel rolling less frequently than P25 population. Neuronal cell death was not present in 24-h TMDT survivors at any age or sex tested. A seizure rechallenge with flurothyl 7 days following TMDT exposure demonstrated longer latencies to the first clonic seizure but a faster progression into the tonic-clonic seizure in P15 and adult survivors as compared to their vehicle-injected counterparts. In conclusion, the youngest age group represents the most vulnerable population to the TMDT-induced toxidrome. Females appear to be more vulnerable than males. TMDT exposure promotes seizure spread and progression in survivors. These findings will help to establish sex- and age-specific treatment strategies for TMDT-exposed individuals. © 2018 Wiley Periodicals, Inc. Develop Neurobiol 78: 403-416, 2018.

Poly-arginine R18 and R18D (D-enantiomer) peptides reduce infarct volume and improves behavioural outcomes following perinatal hypoxic-ischaemic encephalopathy in the P7 rat

We examined the neuroprotective efficacy of the poly-arginine peptide R18 and its D-enantiomer R18D in a perinatal hypoxic-ischaemic (HI) model in P7 Sprague-Dawley rats. R18 and R18D peptides were administered intraperitoneally at doses of 30, 100, 300 or 1000 nmol/kg immediately after HI (8% O2/92%N2 for 2.5 h). The previously characterised neuroprotective JNKI-1-TATD peptide at a dose of 1000 nmol/kg was used as a control. Infarct volume and behavioural outcomes were measured 48 h after HI. For the R18 and R18D doses examined, total infarct volume was reduced by 25.93% to 43.80% (P = 0.038 to < 0.001). By comparison, the JNKI-1-TATD reduced lesion volume by 25.27% (P = 0.073). Moreover, R18 and R18D treatment resulted in significant improvements in behavioural outcomes, while with JNKI-1-TATD there was a trend towards improvement. As an insight into the likely mechanism underlying the effects of R18, R18D and JNKI-1-TATD, the peptides were added to cortical neuronal cultures exposed to glutamic acid excitotoxicity, resulting in up to 89, 100 and 71% neuroprotection, respectively, and a dose dependent inhibition of neuronal calcium influx. The study further confirms the neuroprotective properties of poly-arginine peptides, and suggests a potential therapeutic role for R18 and R18D in the treatment of HIE.

A neurosteroid analogue with T-type calcium channel blocking properties is an effective hypnotic, but is not harmful to neonatal rat brain

BackgroundMore than 4 million children are exposed annually to sedatives and general anaesthetics (GAs) in the USA alone. Recent data suggest that common GAs can be detrimental to brain development causing neurodegeneration and long-term cognitive impairments. Challenged by a recent US Food and Drug Administration (FDA) warning about potentially neurotoxic effects of GAs in children, there is an urgent need to develop safer GAs. MethodsPostnatal Day 7 (P7) rat pups of both sexes were exposed to six (repeated every 2 h) injections of equipotent hypnotic doses of ketamine or the neuroactive steroid (3β,5β,17β)-3-hydroxyandrostane-17-carbonitrile (3β-OH) for 12 h. Loss of righting reflex was used to assess hypnotic properties and therapeutic index; quantitative caspase-3 immunohistochemistry was used to assess developmental neuroapoptosis; patch-clamp recordings in acute brain slices were used to assess the effects of 3β-OH on neuronal excitability and synaptic transmission. Cognitive abilities of rats exposed to ketamine, 3β-OH, or vehicle at P7 were assessed in young adulthood using the radial arm maze. ResultsThe neuroactive steroid 3β-OH has a therapeutic index similar to ketamine, a commonly used clinical GA. We report that 3β-OH is safe and, unlike ketamine, does not cause neuroapoptosis or impair cognitive development when administered to P7 rat pups. Interestingly, 3β-OH blocks T-type calcium channels and presynaptically dampens synaptic transmission at hypnotically-relevant brain concentrations, but it lacks a direct effect on γ-aminobutyric acid A or glutamate-gated ion channels. ConclusionsThe neurosteroid 3β-OH is a relatively safe hypnotic that warrants further consideration for paediatric anaesthesia.

Nicotinic Acetylcholine Receptors are Associated with Ketamine-induced Neuronal Apoptosis in the Developing Rat Retina

Early synchronized spontaneous network activity is a hallmark of the brain growth spurt period, during which general anesthetics cause widespread neuronal apoptosis and subsequent cognitive dysfunction. However, the relationship of such activity to anesthetic-induced neuronal apoptosis remains to be determined. In this study, we utilized patch-clamp electrophysiological recording, immunohistochemistry, and TUNEL assays to investigate the potential roles of spontaneous network activity in ketamine-induced neuronal apoptosis during early development. All experiments were performed using acutely dissected whole-mount Sprague–Dawley rat retinas (0–14 postnatal days [P0–P14]). Ketamine reversibly blocked spontaneous network activity in the rat retina from P0 to P9 and irreversibly blocked such activity from P10 to P12. The peak of physiological and ketamine-induced neuronal apoptosis mainly occurred from P7 to P9. Blockade of nicotinic acetylcholine receptors (nAChRs) also induced reversible inhibition of spontaneous network activity from P0 to P7 and extensive neuronal apoptosis in the P7 rat retina, while activation of nAChRs or increases in endogenous ACh levels attenuated ketamine-induced apoptotic responses. Furthermore, blockade of α7-nAChR and ß2-nAChR subtypes induced neuronal apoptosis in the developing retina, while activation of the α7-nAChR subtype attenuated ketamine-induced apoptotic responses. These results demonstrate that ketamine may inhibit early synchronized spontaneous network activity by blocking nAChRs, and that such inhibition may contribute to ketamine-induced neuronal apoptosis in the developing rat retina.

Targeting Germinal Matrix Hemorrhage-Induced Overexpression of Sodium-Coupled Bicarbonate Exchanger Reduces Posthemorrhagic Hydrocephalus Formation in Neonatal Rats.

BackgroundGerminal matrix hemorrhage (GMH) is a leading cause of mortality and lifelong morbidity in preterm infants. Posthemorrhagic hydrocephalus (PHH) is a common complication of GMH. A sodium‐coupled bicarbonate exchanger (NCBE) encoded by solute carrier family 4 member 10 gene is expressed on the choroid plexus basolateral membrane and may play a role in cerebrospinal fluid production and the development of PHH. Following GMH, iron degraded from hemoglobin has been linked to PHH. Choroid plexus epithelial cells also contain iron‐responsive element‐binding proteins (IRPs), IRP1, and IRP2 that bind to mRNA iron‐responsive elements. The present study aims to resolve the following issues: (1) whether the expression of NCBE is regulated by IRPs; (2) whether NCBE regulates the formation of GMH‐induced hydrocephalus; and (3) whether inhibition of NCBE reduces PHH development.Methods and Results GMH model was established in P7 rat pups by injecting bacterial collagenase into the right ganglionic eminence. Another group received iron trichloride injections instead of collagenase. Deferoxamine was administered intraperitoneally for 3 consecutive days after GMH/iron trichloride. Solute carrier family 4 member 10 small interfering RNA or scrambled small interfering RNA was administered by intracerebroventricular injection 24 hours before GMH and followed with an injection every 7 days over 21 days. NCBE expression increased while IRP2 expression decreased after GMH/iron trichloride. Deferoxamine ameliorated both the GMH‐induced and iron trichloride–induced decrease of IRP2 and decreased NCBE expressions. Deferoxamine and solute carrier family 4 member 10 small interfering RNA improved cognitive and motor functions at 21 to 28 days post GMH and reduced cerebrospinal fluid production as well as the degree of hydrocephalus at 28 days after GMH.ConclusionsTargeting iron‐induced overexpression of NCBE may be a translatable therapeutic strategy for the treatment of PHH following GMH.

Neonatal Hypoxia-Ischemia Causes Functional Circuit Changes in Subplate Neurons.

Neonatal hypoxia-ischemia (HI) in the preterm human results in damage to subcortical developing white matter and cognitive impairments. Subplate neurons (SPNs) are among the first-born cortical neurons and are necessary for normal cerebral development. While moderate or severe HI at P1 in rats leads to SPN loss, it is unclear if HI, esp. forms not associated with overt cell loss lead to altered SPN circuits. Thus, we used two HI models with different severities in P1 rats. Cauterization of the common carotid artery (CCA) causes a largely transient and thus milder ischemia (HI-Caut) while CCA ligation causes more severe ischemia (HI-Lig). While HI-Lig caused subplate damage, HI-Caut did not cause overt histological damage on the light microscopic level. We used laser-scanning photostimulation (LSPS) in acute thalamocortical slices of auditory cortex during P5-10 to study the functional connectivity of SPNs. Both HI categories resulted in hyperconnectivity of excitatory and inhibitory circuits to SPNs. Thus, alterations on the circuit level are present in the absence of cell loss. Our results show that SPN circuits are uniquely susceptible to HI. Given the key developmental role of SPNs, our results suggest that altered SPN circuits might underlie the abnormal development of cortical function after HI.

Oxcarbazepine causes neurocyte apoptosis and developing brain damage by triggering Bax/Bcl-2 signaling pathway mediated caspase 3 activation in neonatal rats.

Anti-epileptic drugs (AEDs) are the main methods for treatment of neonatal seizures; however, a few AEDs may cause developing brain damage of neonate. This study aims to investigate effects of oxcarbazepine (OXC) on developing brain damage of neonatal rats. Both of neonatal and adult rats were divided into 6 groups, including Control, OXC 187.5 mg/kg, OXC 281.25 mg/kg, OXC 375 mg/kg group, LEV and PHT group. Body weight and brain weight were evaluated. Hematoxylin and eosin (HE) and Nissl staining were used to observe neurocyte morphology and Nissl bodies, respectively. Apoptosis was examined using TUNEL assay, and caspase 8 activity was evaluated using spectrophotometer method. Cytochrome C-release was evaluated using flow cytometry. Western blot was used to examine Bax and Bcl-2 expression. OXC 375 mg/kg treatment significantly decreased brain weight compared to Control group in neonatal rats (P5 rats) (p<0.05). OXC administration causes histological changes of neurocytes. OXC 281.25 mg/kg or more concentration significantly decreased neurocytes counts and increased TUNEL-staining positive neurocytes compared to Control group (p<0.05). OXC 281.25 mg/kg and OXC 375 mg/kg significantly increased caspase 3 activity compared to Control group in P5 rats (p<0.05). OXC 281.25 mg/kg and OXC 375 mg/kg significantly increased Bax, Bax/Bcl-2 ratio and cytochrome C release in frontal lobes compared to Control group in P5 rats (p<0.05). Oxcarbazepine at a concentration of 281.25 mg/kg or more causes neurocyte apoptosis and developing brain damage by triggering Bax/Bcl-2 signaling pathway mediated caspase 3 activation in neonatal rats.

EFEK HIPOGLIKEMIK INFUSA DAUN TANAMAN MAKASAR, &lt;em&gt;Brucea javanica&lt;/em&gt; (L) Merr, PADA TIKUS HIPERGLIKEMIK YANG DIINDUKSI ALOKSAN

Brucea javanica (L) Merr is a local plant used traditionally for diabetic medication in Lombok Island. The B. javanica seed has been recognized to have an antidiabetic effect, while the impact of the leave extract was understudied. The purpose of this research was to determine the effect of B. javanica leaves infusion on the blood glucose level of alloxan-induced diabetic rats. B. javanica leaves extract were applied by soaking leaves into hot water (95°C) to obtain the extract concentration of 5% (w/v). This experiment provides four treatments to rats within 15 days with five replicates for each treatment. The treatments for rats were K1 a positive control, K2 a negative control, K3 a healthy control, and P1 treated with leave infused water. The diabetic condition for K1, K2, and P1 was induced with an intravenous injection of 125 mg/kgBW alloxan monohydrate to the rats. After stable diabetic condition, K1 rats were treated with Glibenclamide (0,25 mg/kgBW), K2 rats were untreated, P1 rats were administered with leaves infused water of B. javanica (L) Merr 3 ml/day. K3 rats were uninduced and untreated. The hypoglycemic effect was observed by estimating the fasting blood glucose level of the rats every three days. After 15 days of treatments, P1 rats blood glucose level reduced by the 13,30%, while K1 rats 22,04%. The decrease in blood glucose level of both K1 and P1 rats have not reached the average blood glucose level of healthy rats. The result of the statistical analysis showed the infused extract effect was not significantly differed compared to positive control (P < 0.05).

Effect of VEGF on Inflammatory Regulation, Neural Survival, and Functional Improvement in Rats following a Complete Spinal Cord Transection.

After complete transection of the thoracic spinal segment, neonatal rats exhibit spontaneous locomotor recovery of hindlimbs, but this recovery is not found in adult rats after similar injury. The potential mechanism related to the difference in recovery of neonatal and adult rats remains unknown. In this study, 342 animals were analyzed. The vascular endothelial growth factor (VEGF) level in spinal segments below injury sites was significantly higher in postnatal day 1 rats (P1) compared with 28-day-old adult rats (P28) following a complete T9 transection. VEGF administration in P28 rats with T9 transection significantly improved the functional recovery; by contrast, treatment with VEGF receptor inhibitors in P1 rats with T9 transection slowed down the spontaneous functional recovery. Results showed more neurons reduced in the lumbar spinal cord and worse local neural network reorganization below injury sites in P28 rats than those in P1 rats. Transynaptic tracing with pseudorabies virus and double immunofluorescence analysis indicated that VEGF treatment in P28 rats alleviated the reduced number of neurons and improved their network reorganization. VEGF inhibition in neonates resulted in high neuronal death rate and deteriorated network reorganization. In in vivo studies, T9 transection induced less increase in the number of microglia in the spinal cord in P1 animals than P28 animals. VEGF treatment reduced the increase in microglial cells in P28 animals. VEGF administration in cultured spinal motoneurons prevented lipopolysaccharide (LPS)-induced neuronal death and facilitated neurite growth. Western blots of the samples of lumbar spinal cord after spinal transection and cultured spinal motoneurons showed a lower level of Erk1/2 phosphorylation after the injury or LPS induction compared with that in the control. The phosphorylation level increased after VEGF treatment. In conclusion, VEGF is a critical mediator involved in functional recovery after spinal transection and can be considered a potential target for clinical therapy.

Prenatal alcohol exposure enhances the susceptibility to NMDA-induced generalized tonic-clonic seizures in developing rats.

Prenatal alcohol exposure (PAE) is associated with a higher likelihood of developing generalized tonic-clonic seizures (GTCS) in infants and children. However, experimental studies of PAE-related seizures have yielded conflicting results. Here, we investigated the effect of acute PAE on N-methyl-D-aspartate (NMDA)-induced seizures in developing rats. Pregnant Sprague Dawley rats were given an oral dose of either ethanol (5g/kg body weight) or vehicle on embryonic day 18. The offspring were tested for susceptibility to NMDA-induced seizures on postnatal day 7 (P7), 21 (P21), 35 (P35), and 42 (P42). Specifically, the prevalence and latency of NMDA-induced continuous wild running-like behaviors (CWR), flexion seizures (FS), wild running seizures (WRS), GTCS, and tonic seizures (TS) were recorded and analyzed. N-methyl-D-aspartate-induced seizures consisted of CWR, FS, GTCS, and TS in <P21 rats, while WRS, GTCS, and TS were observed in >P21 rats. Thus, GTCS were consistently observed during development. PAE significantly increases the prevalence of GTCS in female and male P7-P21 rats and P7-P35 rats, respectively, but not in older rats. PAE also increases the prevalence of TS in male, but not female P21-P35 rats. The PAE animal model of GTCS may provide a new opportunity to investigate the mechanisms that underlie neuronal hyperexcitability in developing animals prenatally-exposed to alcohol.

Anti-ictogenic and antiepileptogenic properties of perampanel in mature and immature rats.

Perampanel (PER) is a selective noncompetitive antagonist at α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, the first of its class approved for the adjunctive treatment of partial onset seizures and generalized seizures. This study explored anti-ictogenic and antiepileptogenic effects of PER in rats at different stages of development. Using a rapid kindling model in postnatal day 14 (P14), P21, P28, and P60 rats, we studied two doses of PER: 1 and 2mg/kg injected intraperitoneally 30min before afterdischarge assessment. We also assessed blood and brain concentrations of PER 30min after the injection. PER 2mg/kg significantly increased the afterdischarge threshold (ADT) at all ages, whereas PER at 1mg/kg increased ADT only in P21 rats. PER 2mg/kg also shortened the afterdischarge duration in P14 and P28 rats. PER increased the number of stimulations required to achieve a stage 4-5 seizure in a dose-dependent manner in P14 and P21 rats, with almost complete elimination of stage 4-5 seizures. At P28, only PER 2mg/kg increased the number of stimulations required to develop a stage 4-5 seizure. In contrast, PER had no effect on the number of stage 4-5 seizures at P60. We did not observed any age-dependent significant difference in the serum and brain levels of PER 30min after the injection. PER exerted anti-ictogenic effects from P14 to P60 independent of brain maturation. PER also exhibited antiepileptogenic effects with a stronger effect in the younger animals.

Retinal VEGF levels correlate with ocular circulation measured by a laser speckle-micro system in an oxygen-induced retinopathy rat model.

We used a Laser speckle flowgraphy (LSFG)-micro system to examine the relationship between ocular blood flow and retinal vascular endothelial growth factor (VEGF) at retinopathy onset in oxygen-induced ischemic retinopathy (OIR) model rats. Sixteen 50/10 OIR rats were compared with 17 control rats reared in room air. In postnatal day 14 (P14) and P18 rats, we measured and analyzed the left eye's mean blur rate (MBR) by setting a rubber band on the optic nerve head center, using the LSFG-Micro. At P18, the rats were sacrificed and their left-eye retinas were fixed, flat-mounted and stained with adenosine diphosphatase (ADPase). The right-eye retinas were homogenized; the lysate was centrifuged for an enzyme-linked immunosorbent assay (ELISA). The avascular area was measured as the percentage (%AVA) of the total retinal area. Retinal VEGF was measured by an ELISA. The examination's reproducibility was good. Our multivariate linear mixed model analysis revealed significantly high MBRs in the OIR rats (p=0.0017). In the P18 OIR rats, significant correlations were seen between the MBR and %AVA (r=0.80, p=0.0002) and between the MBR and VEGF (r=0.76, p=0.0006). The LSFG-Micro provided reproducible blood flow measurements in neonatal rats. Because of the vitreous blood vessels, measurement of only the retinal vessels was not possible. However, the MBR was higher in the OIR rats than in the control rats, and the MBR and %AVA were correlated, as were the MBR and retinal VEGF. The MBR may thus serve as an indicator of OIR severity.

Neuroprotective Effects of Intranasal IGF-1 against Neonatal Lipopolysaccharide-Induced Neurobehavioral Deficits and Neuronal Inflammation in the Substantia Nigra and Locus Coeruleus of Juvenile Rats

Neonatal lipopolysaccharide (LPS) exposure-induced brain inflammation resulted in motor dysfunction and brain dopaminergic neuronal injury, and increased the risks of neurodegenerative disorders in adult rats. Our previous studies showed that intranasal administration of insulin-like growth factor-1 (IGF-1) protects against LPS-induced white matter injury in the developing rat brain. To further examine whether IGF-1 protects against LPS-induced brain neuronal injury and neurobehavioral dysfunction, recombinant human IGF-1 (rhIGF-1) at a dose of 50 µg/pup was administered intranasally 1 h following intracerebral injection of LPS (1 mg/kg) in postnatal day 5 (P5) Sprague-Dawley rat pups. Neurobehavioral tests were carried out from P7 to P21, and brain neuronal injury was examined at P21. Our results showed that LPS exposure resulted in disturbances of motor behaviors in juvenile rats. Moreover, LPS exposure caused injury to central catecholaminergic neurons, as indicated by a reduction of tyrosine hydroxylase (TH) immunoreactivity in the substantia nigra (SN), ventral tegmental area (VTA) and olfactory bulb (OB), and brain noradrenergic neurons, as indicated by a reduction of TH immunoreactivity in the locus coeruleus (LC) of the P21 rat brain. The LPS-induced reduction of TH+ cells was observed at a greater degree in the SN and LC of the P21 rat brain. Intranasal rhIGF-1 treatment attenuated LPS-induced central catecholaminergic neuronal injury and motor behavioral disturbances, including locomotion, beam walking test and gait analysis. Intranasal rhIGF-1 administration also attenuated LPS-induced elevation of IL-1β levels and numbers of activated microglia, and cyclooxygenase-2+ cells, which were double labeled with TH+ cells in the SN, VTA, OB and LC of the P21 rat brain. These results suggest that IGF-1 may provide protection against neonatal LPS exposure-induced central catecholaminergic neuronal injury and motor behavioral disturbances, and that the protective effects are associated with the inhibition of microglia activation and the reduction of neuronal oxidative stress by the suppression of the neuronal cyclooxygenase-2 expression.

MiR-30d-5p Plays an Important Role in Autophagy and Apoptosis in Developing Rat Brains After Hypoxic-Ischemic Injury.

Increasing evidence has demonstrated a vital role of microRNAs (miRNAs) in diverse biological processes. However, their functions in developing brain with hypoxia-ischemia (HI) remain largely unknown. Through a miRNA microarray analysis in a P10 rat model of cerebral HI, we found that miR-30d-5p was one of the most deregulated miRNAs in neonatal brains in response to HI. MiR-30d-5p was downregulated in a time-dependent manner in brain cortex after HI, which was accompanied by increased expression of Beclin1 both at transcript and protein levels. Increase of miR-30d-5p by agomir (AG) resulted in reduction of autophagy and increase of apoptosis, whereas inhibition of miR-30d-5p by antagomir (AT) enhanced autophagy and inhibited apoptosis in rat brains after HI. Moreover, miR-30d-5p AG increased infarct volume, delayed recovery of neurological function, and impaired improvement of spatial memory ability. MiR-30d-5p AT decreased infarct volume, promoted neurological recovery, and improved behavior performance of rats subjected to HI. Collectively, these results indicated that miR-30d-5p modulated survival programs of neural cell by regulating autophagy and apoptosis.

Controlled arterial reflow after ischemia induces better outcomes in the juvenile rat brain

Our objective was to determine whether controlled reflow on one side and/or the other side after bilateral carotid occlusion release could reduce cell death in focal ischemic P14 rats. Arterial blood flow was measured using ultrasonography. Cell death, inflammation and nitrotyrosine were measured using immunofluorescence. When reflow was first induced in the contralateral side, we observed improved outcome markers compared with those when reflow was first induced in the ipsilateral side and/or simultaneous reflow was induced in both sides. Our data suggest that progressive rerouting of arterial flow through the circle of Willis toward the ischemic site reduced cell death.

Modification to the Rice-Vannucci perinatal hypoxic-ischaemic encephalopathy model in the P7 rat improves the reliability of cerebral infarct development after 48 hours

BackgroundThe Rice-Vannucci model of hypoxic-ischaemic encephalopathy (HIE) has been associated with a high degree of variability with respect to the development of cerebral infarction and infarct lesion volume. For this reason, we examined the occurrence of communicational blood flow within the common carotid (CCA), internal (ICA), and external (ECA) carotid arteries following CCA occlusion as a source of variability in the model. New methodWe propose a novel modification to the Rice-Vannucci model, whereby both the CCA and ECA are permanently ligated; mitigating communicational blood flow. ResultsUsing magnetic resonance angiography, phase-contrast velocity encoding, and pulsed arterial spin labelling, the modified Rice-Vannucci model (CCA/ECA occlusion) was demonstrated to mitigate communicational blood flow, whilst significantly reducing ipsilateral hemispherical cerebral blood flow (CBF). Comparatively, the original Rice-Vannucci model (CCA occlusion) demonstrated anterograde and retrograde blood flow within the ICA and CCA, respectively, with a non-significant reduction in ipsilateral CBF. Furthermore, CCA/ECA occlusion plus hypoxia (8% O2/92% N2; 2.5h) resulted in 100% of animals presenting with an infarct (vs 87%), significantly larger infarct volume at 48h (18.5% versus 10.0%; p<0.01), reduced standard deviation (±10% versus ±15%), and significantly worsened functional outcomes when compared to CCA occlusion plus hypoxia. Comparison with existing methodWe compared a modified Rice-Vannucci model (CCA/ECA occlusion±hypoxia) to the commonly used Rice-Vannucci model (CCA occlusion±hypoxia). ConclusionThis study demonstrates that CCA/ECA occlusion in the Rice-Vannucci model of HIE reduces infarct volume variability by limiting communicational blood flow.

Transient Middle Cerebral Artery Occlusion Model of Neonatal Stroke in P10 Rats.

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature and mature brain. Stroke occurs commonly in the perinatal period in humans, and transient ischemia-reperfusion is the most common form of stroke in neonates. The reperfusion phase is a critical component of injury progression, which occurs over a period of days to weeks, and of the endogenous response to injury. This postnatal day 10 (p10) rat model of transient middle cerebral artery occlusion (tMCAO) creates a unilateral, non-hemorrhagic focal ischemia-reperfusion injury that can be utilized to study the mechanisms of focal injury and repair in the full-term-equivalent brain. The injury pattern that is produced by tMCAO is consistent and highly reproducible and can be confirmed with MRI or histological analyses. The severity of injury can be manipulated through changes in occlusion time and other methods that will be discussed.

Transient Middle Cerebral Artery Occlusion Model of Neonatal Stroke in P10 Rats

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature and mature brain. Stroke occurs commonly in the perinatal period in humans, and transient ischemia-reperfusion is the most common form of stroke in neonates. The reperfusion phase is a critical component of injury progression, which occurs over a period of days to weeks, and of the endogenous response to injury. This postnatal day 10 (p10) rat model of transient middle cerebral artery occlusion (tMCAO) creates a unilateral, non-hemorrhagic focal ischemia-reperfusion injury that can be utilized to study the mechanisms of focal injury and repair in the full-term-equivalent brain. The injury pattern that is produced by tMCAO is consistent and highly reproducible and can be confirmed with MRI or histological analyses. The severity of injury can be manipulated through changes in occlusion time and other methods that will be discussed.

A dissociation between renewal and contextual fear conditioning in juvenile rats.

We investigated whether juvenile rats do not express renewal following extinction of conditioned fear due to their inability to form a long-term contextual fear memory. In experiment 1, postnatal day (P) 18 and 25 rats received 3 white-noise and footshock pairings, followed by 60 white-noise alone presentations the next day. When tested in a different context to extinction, P25 rats displayed renewal whereas P18 rats did not. Experiments 2A and 2B surprisingly showed that P18 and P25 rats do not show differences in contextual and cued fear, regardless of the conditioning-test intervals and the number of white-noise-footshock pairings received. Finally, we observed age differences in contextual fear when P25 rats were weaned at P21 in experiment 3. These results indicate that the developmental dissociation observed in renewal of extinguished fear is not related to the widely believed late emergence of contextual fear learning.

Does reduced carotid body BDNF contribute to developmental hyperoxia‐induced respiratory plasticity?

Chronic exposure to moderate hyperoxia (60% O2) reduces carotid body expression of brain‐derived neurotrophic factor (BDNF) in neonatal rats. Reduced BDNF expression correlates with abnormal carotid body development and blunted hypoxic ventilatory responses, but a causal relationship has not been established. First, we used ELISA to confirm that both sustained and intermittent exposures to 60% O2 reduced carotid body BDNF levels; intermittent hypercapnic hypoxia had the opposite effect. Next, we attempted to block the effects of developmental hyperoxia by administering LM22A4 (50 mg kg−1, i.p., daily), an agonist for BDNF's TrkB receptor. After 7 days of treatment with 60% O2, carotid body volume and the hypoxic ventilatory response were significantly reduced in P7 rats that received either LM22A4 or vehicle (saline) from birth; the drug treatment did not attenuate this effect. Similar results were obtained using an alternate TrkB agonist, 7,8‐dihydroxyflavone (5 mg kg−1, i.p., daily). Although carotid body BDNF expression appears to be regulated by environmental O2, our data do not support the hypothesis that reduced BDNF is directly responsible for hyperoxia‐induced developmental plasticity in respiratory control.Support or Funding InformationSupported by NIH grant R15 HL‐114001.