Michelle E. Schober

Early and Sustained Increase in the Expression of Hippocampal IGF-1, But Not EPO, in a Developmental Rodent Model of Traumatic Brain Injury

Pediatric traumatic brain injury (pTBI) is the leading cause of traumatic death and disability in children in the United States. Impaired learning and memory in these young survivors imposes a heavy toll on society. In adult TBI (aTBI) models, cognitive outcome improved after administration of erythropoietin (EPO) or insulin-like growth factor-1 (IGF-1). Little is known about the production of these agents in the hippocampus, a brain region critical for learning and memory, after pTBI. Our objective was to describe hippocampal expression of EPO and IGF-1, together with their receptors (EPOR and IGF-1R, respectively), over time after pTBI in 17-day-old rats. We used the controlled cortical impact (CCI) model and measured hippocampal mRNA levels of EPO, IGF-1, EPOR, IGF-1R, and markers of caspase-dependent apoptosis (bcl2, bax, and p53) at post-injury days (PID) 1, 2, 3, 7, and 14. CCI rats performed poorly on Morris water maze testing of spatial working memory, a hippocampally-based cognitive function. Apoptotic markers were present early and persisted for the duration of the study. EPO in our pTBI model increased much later (PID7) than in aTBI models (12 h), while EPOR and IGF-1 increased at PID1 and PID2, respectively, similar to data from aTBI models. Our data indicate that EPO expression showed a delayed upregulation post-pTBI, while EPOR increased early. We speculate that administration of EPO in the first 1-2 days after pTBI would increase hippocampal neuronal survival and function.

Erythropoietin Improved Cognitive Function and Decreased Hippocampal Caspase Activity in Rat Pups after Traumatic Brain Injury

UNLABELLED Traumatic brain injury (TBI) is a leading cause of acquired neurologic disability in children. Erythropoietin (EPO), an anti-apoptotic cytokine, improved cognitive outcome in adult rats after TBI. To our knowledge, EPO has not been studied in a developmental TBI model. HYPOTHESIS We hypothesized that EPO would improve cognitive outcome and increase neuron fraction in the hippocampus in 17-day-old (P17) rat pups after controlled cortical impact (CCI). METHODS EPO or vehicle was given at 1, 24, and 48 h after CCI and at post injury day (PID) 7. Cognitive outcome at PID14 was assessed using Novel Object Recognition (NOR). Hippocampal EPO levels, caspase activity, and mRNA levels of the apoptosis factors Bcl2, Bax, Bcl-xL, and Bad were measured during the first 14 days after injury. Neuron fraction and caspase activation in CA1, CA3, and DG were studied at PID2. RESULTS EPO normalized recognition memory after CCI. EPO blunted the increased hippocampal caspase activity induced by CCI at PID1, but not at PID2. EPO increased neuron fraction in CA3 at PID2. Brain levels of exogenous EPO appeared low relative to endogenous. Timing of EPO administration was associated with temporal changes in hippocampal mRNA levels of EPO and pro-apoptotic factors. Conclusion/Speculation: EPO improved recognition memory, increased regional hippocampal neuron fraction, and decreased caspase activity in P17 rats after CCI. We speculate that EPO improved cognitive outcome in rat pups after CCI as a result of improved neuronal survival via inhibition of caspase-dependent apoptosis early after injury.

Traumatic Brain Injury Increased IGF-1B mRNA and Altered IGF-1 Exon 5 and Promoter Region Epigenetic Characteristics in the Rat Pup Hippocampus

Traumatic brain injury (TBI) is a major cause of acquired cognitive disability in childhood. Such disability may be blunted by enhancing the brains endogenous neuroprotective response. An important endogenous neuroprotective response is the insulin-like growth factor-1 (IGF-1) mRNA variant, IGF-1B. IGF-1B mRNA, characterized by exon 5 inclusion, encodes the IGF-1 and Eb peptides. IGF-1A mRNA excludes exon 5 and encodes the IGF-1 and Ea peptides. A region in the human IGF-1B homologue acts as an exon-splicing enhancer (ESE) to increase IGF-1B mRNA. It is not known if TBI is associated with increased brain IGF-1B mRNA. Epigenetic modifications may underlie altered gene expression in the brain after TBI. We hypothesized that TBI would increase hippocampal IGF-1B mRNA in 17-day-old rats, associated with DNA methylation and/or histone modifications at the promoter site 1 (P1) or exon 5/ESE region. Hippocampi from rat pups after controlled cortical impact (CCI) were used to measure IGF-1B mRNA, DNA methylation, and histone modifications at the P1, P2, and exon5/ESE regions. In CCI hippocampi, IGF-1B mRNA peaked at post-injury day (PID) 2 (1700±320% sham), but normalized by PID 14. IGF-1A peaked at PID 3 (280±52% sham), and remained elevated at PID 14. Increased IGF-1B mRNA was associated with increased methylation at P1, and increased histone modifications associated with gene activation at P2 and exon5/ESE, together with differential methylation in the exon 5/ESE regions. We report for the first time that hippocampal IGF-1B mRNA increased after developmental TBI. We speculate that epigenetic modifications at the P2 and exon 5/ESE regions are important in the regulation of IGF-1B mRNA expression. The exon 5/ESE region may present a means for future therapies to target IGF-1B transcription after TBI.

Alpha II Spectrin breakdown products in immature Sprague Dawley rat hippocampus and cortex after traumatic brain injury

After traumatic brain injury (TBI), proteolysis of Alpha II Spectrin by Calpain 1 produces 145 Spectrin breakdown products (SBDPs) while proteolysis by Caspase 3 produces 120 SBDPs. 145 and 120 SBDP immunoblotting reflects the relative importance of caspase-dependent apoptosis or calpain-dependent excitotoxic/necrotoxic cell death in brain regions over time. In the adult rat, controlled cortical impact (CCI) increased 120 SBDPs in the first hours, lasting a few days, and increased 145 SBDPs within the first few days lasting up to 14 days after injury. Little is known about SBDPs in the immature brain after TBI. Since development affects susceptibility to apoptosis after TBI, we hypothesized that CCI would increase 145 and 120 SBDPs in the immature rat brain relative to SHAM during the first 3 and 5 days, respectively. SBDPs were measured in hippocampi and cortices at post injury days (PID) 1, 2, 3, 5, 7 and 14 after CCI or SHAM surgery in the 17 day old Sprague Dawley rat. 145 SBDPs increased in both brain tissues ipsilateral to injury during the first 3 days, while changes in contralateral tissues were limited to PID2 cortex. 145 SBDPs elevations were more marked and enduring in hippocampus than in cortex. Against expectations, 120 SBDPs only increased in PID1 hippocampus and PID2 cortex. 145 SBDPs elevations occurred early after CCI, similar to previous studies in the adult rat, but resolved more quickly. The minimal changes in 120 SBDPs suggest that calpain-dependent, but not caspase-dependent, cell death predominates in the 17 day old rat after CCI.

Altered expression and chromatin structure of the hippocampal IGF1r gene is associated with impaired hippocampal function in the adult IUGR male rat.

Exposure to intrauterine growth restriction (IUGR) is an important risk factor for impaired learning and memory, particularly in males. Although the basis of IUGR-associated learning and memory dysfunction is unknown, potential molecular participants may be insulin-like growth factor 1 (Igf1) and its receptor, IGF1r. We hypothesized that transcript levels and protein abundance of Igf1 and IGF1r in the hippocampus, a brain region critical for learning and memory, would be lower in IUGR male rats than in age-matched male controls at birth (postnatal day 0, P0), at weaning (P21) and adulthood (P120). We also hypothesized that changes in messenger Ribonucleic acid (mRNA) transcript levels and protein abundance would be associated with specific histone marks in IUGR male rats. Lastly, we hypothesized that IUGR male rats would perform poorer on tests of hippocampal function at P120. IUGR was induced by bilateral ligation of the uterine arteries in pregnant dams at embryonic day 19 (term is 21 days). Hippocampal Igf1 mRNA transcript levels and protein abundance were unchanged in IUGR male rats at P0, P21 or P120. At P0 and P120, IGF1r expression was increased in IUGR male rats. At P21, IGF1r expression was decreased in IUGR male rats. Increased IGF1r expression was associated with more histone 3 lysine 4 dimethylation (H3K4Me2) in the promoter region. In addition, IUGR male rats performed poorer on intermediate-term spatial working memory testing at P120. We speculate that altered IGF1r expression in the hippocampus of IUGR male rats may play a role in learning and memory dysfunction later in life.

Family clusters of variant X-linked chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a rare inherited immunodeficiency disorder. The clinical presentation is varied depending on the degree of involvement of the NADPH oxidase system responsible for the oxidative burst of neutrophils. We present 3 cases of variant X-linked CGD in an effort to introduce the disease and highlight the importance and limitations of CGD screening. The variant X-linked form of CGD results in a less severe phenotype and frequently presents later in life. Variant X-linked CGD is difficult to diagnose, but is becoming more readily recognized based on improved testing methods. A high index of suspicion in the setting of unusual infections such as Burkholderia cepacia pneumonia is essential to make the diagnosis. Family screening can lead to early intervention, prophylaxis, and appropriate genetic counseling.

Isoflurane Exposure did not Adversely Affect Recognition Memory or Decrease Hippocampal Brain Derived Neurotrophic Factor Expression in the 17 Day Old Rat Pup

A previous study showed that hippocampal BDNF mRNA decreased in 17 day old (D17) rats, relative to agematched naive rats, at day 1, 3, 7 and 14 after sham surgery in a traumatic brain injury model. The anesthetic isoflurane activates GABA and inhibits NMDA receptor currents, both of which are known to decrease Brain-Derived Neurotrophic Factor (BDNF) mRNA in rat hippocampi. Hippocampal BDNF is necessary for normal cognitive function. Effects of isoflurane alone on hippocampal BDNF are not known. We hypothesized that, in D17 rat pups, isoflurane would decrease hippocampal mRNA/protein levels of BDNF and Synapsin I (a downstream target of BDNF important for cognitive function) and impair performance on the Novel Object Recognition Test (NOR). ISOF BDNF and Synapsin I mRNA decreased relative to Naive at day 1 and 8 after exposure, but not at day 14. Isoflurane exposure did not decrease hippocampal protein levels of BDNF or Synapsin I and did not impair NOR performance. In contrast to the neonatal rat pup, anesthetic exposure did not impair cognitive function. We speculate that adverse effects of anesthetics on rat pup cognitive function and BDNF expression are highly dependent on age at exposure.

EPO improved neurologic outcome in rat pups late after traumatic brain injury

In adult rats, erythropoietin improved outcomes early and late after traumatic brain injury, associated with increased levels of Brain Derived Neurotrophic Factor. Using our model of pediatric traumatic brain injury, controlled cortical impact in 17-day old rats, we previously showed that erythropoietin increased hippocampal neuronal fraction in the first two days after injury. Erythropoietin also decreased activation of caspase3, an apoptotic enzyme modulated by Brain Derived Neurotrophic Factor, and improved Novel Object Recognition testing 14 days after injury. Data on long-term effects of erythropoietin on Brain Derived Neurotrophic Factor expression, histology and cognitive function after developmental traumatic brain injury are lacking. We hypothesized that erythropoietin would increase Brain Derived Neurotrophic Factor and improve long-term object recognition in rat pups after controlled cortical impact, associated with increased neuronal fraction in the hippocampus. METHODS Rats pups received erythropoietin or vehicle at 1, 24, and 48 h and 7 days after injury or sham surgery followed by histology at 35 days, Novel Object Recognition testing at adulthood, and Brain Derived Neurotrophic Factor measurements early and late after injury. RESULTS Erythropoietin improved Novel Object Recognition performance and preserved hippocampal volume, but not neuronal fraction, late after injury. CONCLUSIONS Improved object recognition in erythropoietin treated rats was associated with preserved hippocampal volume late after traumatic brain injury. Erythropoietin is approved to treat various pediatric conditions. Coupled with exciting experimental and clinical studies suggesting it is beneficial after neonatal hypoxic ischemic brain injury, our preliminary findings support further study of erythropoietin use after developmental traumatic brain injury.

Temporal response profiles of serum ubiquitin C-terminal hydrolase-L1 and the 145-kDa alpha II-spectrin breakdown product after severe traumatic brain injury in children

OBJECTIVE Traumatic brain injury (TBI) is the leading cause of acquired disability among children. Brain injury biomarkers may serve as useful diagnostic and prognostic indicators for TBI. Levels of ubiquitin C-terminal hydrolase-L1 (UCH-L1) and the 145-kDa alpha II-spectrin breakdown product (SBDP-145) correlate with outcome in adults after severe TBI. The authors conducted a pilot study of these biomarkers in children after severe TBI to inform future research exploring their utility in this population. METHODS The levels of UCH-L1 and SBDP-145 were measured in serum, and UCH-L1 in CSF from pediatric patients after severe TBI over 5 days after injury. Both biomarkers were also measured in age-matched control serum and CSF. RESULTS Adequate numbers of samples were obtained in serum, but not CSF, to assess biomarker temporal response profiles. Using patients with samples from all time points, UCH-L1 levels increased rapidly and transiently, peaking at 12 hours after injury. SBDP-145 levels showed a more gradual and sustained response, peaking at 48 hours. The median serum UCH-L1 concentration was greater in patients with TBI than in controls (median [IQR] = 361 [187, 1330] vs 147 [50, 241] pg/ml, respectively; p < 0.001). Receiver operating characteristic (ROC) analysis revealed an AUC of 0.77. Similarly, serum SBDP-145 was greater in children with TBI than in controls (median [IQR] = 172 [124, 257] vs 69 [40, 99] pg/ml, respectively; p < 0.001), with an ROC AUC of 0.85. When only time points of peak levels were used for ROC analysis, the discriminability of each serum biomarker increased (AUC for UCH-L1 at 12 hours = 1.0 and for SBDP-145 at 48 hours = 0.91). Serum and CSF UCH-L1 levels correlated well in patients with TBI (r = 0.70, p < 0.001). CONCLUSIONS Findings from this exploratory study reveal robust increases of UCH-L1 and SBDP-145 in serum and UCH-L1 in CSF obtained from children after severe TBI. In addition, important temporal profile differences were found between these biomarkers that can help guide optimal time point selection for future investigations of their potential to characterize injury or predict outcomes after pediatric TBI.

UTEROPLACENTAL INSUFFICIENCY AFFECTS HIPPOCAMPAL INSULIN-LIKE GROWTH FACTOR 1 MESSENGER RIBONUCLEIC ACID LEVELS IN JUVENILE RATS.: 140

Background Intrauterine growth retardation (IUGR) predisposes affected human and rat newborns toward persistent hippocampal dysfunction. This often occurs in association with low circulating insulin-like growth factor 1 (IGF-1) levels. Both circulating IGF-1 and locally produced IGF-1 are required for normal hippocampal development and adult cognitive function. However, the effects of IUGR upon hippocampal IGF-1 expression are not known. Objective We hypothesized that uteroplacental insufficiency (UPI) and subsequent IUGR would decrease postnatal hippocampal IGF-1 mRNA levels. IGF-1 expression involves multiple promoters (P1, P2) and multiple exon variants (IGF-1A, IGF-1B). Each exon variant produces a different E peptide. Design/Methods We used a well-characterized rat model of UPI-induced IUGR: briefly, bilateral uterine artery ligation was performed on day 19 of gestation (term-21.5 d). Litters were culled to six after birth, and brain was harvested and dissected at day 21 of postnatal life (D21). In this model, we have shown that IUGR animals perform poorly on tests of hippocampal function at adulthood. They also have abnormal anatomic and functional hippocampal markers, as well as low serum IGF-1 levels. IGF-1 mRNA variants and total IGF-1 mRNA were quantified by real-time RT-PCR (n = 6-8 litters). Results Data are expressed as % of control. IUGR significantly decreased D21 hippocampal P1 IGF-1 mRNA levels in both male and female rats to 87% of control values (p = .02). As expected, male rats were more severely affected than the female rats. Expression of the IGF-1B mRNA was also significantly decreased in D21 IUGR hippocampal samples to 77.3% of control values (p = .01). Surprisingly, the IGF-1B mRNA levels were more severely in the female IUGR rats versus the male rats. Conclusion Despite low serum levels of IGF-1, hippocampal IGF-1 expression is not up-regulated in P21 IUGR rats. In addition, P1, an IGF-1 mRNA that predominates in the brain and is believed to be the paracrine form of IGF-1, is decreased by IUGR. Finally, IGF-1B, an mRNA variant thought to encode the EB peptide, a peptide that may be important in neuronal differentiation, is also decreased by IUGR. These findings are interesting in the up-regulation of cerebral IGF-1 normally occurs in response to acute hypoxia-ischemia. We speculate that the failure of the IUGR hippocampus to increase IGF-1 mRNA levels contributes to the neurocognitive dysfunction observed in both the IUGR human and rat. (CHRC)