Raul Chavez-Valdez

Necrostatin-1 attenuates mitochondrial dysfunction in neurons and astrocytes following neonatal hypoxia–ischemia

Receptor interacting protein (RIP)-1 kinase activity mediates a novel pathway that signals for regulated necrosis, a form of cell death prominent in traumatic and ischemic brain injury. Recently, we showed that an allosteric inhibitor of RIP-1 kinase activity, necrostatin-1 (Nec-1), provides neuroprotection in the forebrain following neonatal hypoxia-ischemia (HI). Because Nec-1 also prevents early oxidative injury, we hypothesized that mechanisms involved in this neuroprotection may involve preservation of mitochondrial function and prevention of secondary energy failure. Therefore, our objective was to determine if Nec-1 treatment following neonatal HI attenuates oxidative stress and mitochondrial injury. Postnatal day (p) 7 mice exposed to HI were injected intracerebroventricularly with 0.1 μL (80 μmol) of Nec-1 or vehicle. Nec-1 treatment prevented nitric oxide (NO•), inducible nitric oxide synthase (iNOS) and 3-nitrotyrosine increase, and attenuated glutathione oxidation that was found in vehicle-treated mice at 3h following HI. Similarly, Nec-1 following HI prevented: (i) up-regulation of hypoxia inducible factor-1 alpha (HIF-1α) and BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) expression, (ii) decline in mitochondrial complex-I activity, (iii) decrease in ATP levels, and (iv) mitochondrial structural pathology in astrocytes and in neurons. Up-regulation of glial fibrillary acidic protein (GFAP) following HI was also prevented by Nec-1 treatment. No differences by gender were observed. We conclude that Nec-1 immediately after HI, is strongly mitoprotective and prevents secondary energy failure by blocking early NO• accumulation, glutathione oxidation and attenuating mitochondrial dysfunction.

Caffeine Modulates TNF-α Production by Cord Blood Monocytes: The Role of Adenosine Receptors

Caffeine, a nonspecific adenosine receptor (AR) antagonist is widely used to treat apnea of prematurity. Because adenosine modulates multiple biologic processes including inflammation, we hypothesized that AR blockade by caffeine would increase cytokine release from neonatal monocytes. Using cord blood monocytes (CBM), we investigated 1) the changes in AR mRNA profile by real time quantitative reverse-transcription polymerase-chain-reaction (qRT-PCR) and protein expression (western blot) after in vitro culture, caffeine or lipopolysaccharide (LPS) exposure, and 2) the modulation of cytokine release and cyclic adenosine monophosphate (cAMP) production by enzyme-linked immunosorbent assay (ELISA) induced by caffeine and specific AR antagonists: DPCPX(A1R), ZM241385(A2aR), MRS1754(A2bR), and MRS1220(A3R). After 48 h in culture, A2aR and A2bR gene expression increased 1.9 (p = 0.04) and 2.5-fold (p = 0.003), respectively. A1R protein expression directly correlated with increasing LPS concentrations (p = 0.01), with minimal expression preexposure. Only caffeine (50 μM) and DPCPX (10 nM) decreased tumor necrosis factor-alpha (TNF-α) release from LPS activated-CBM by 20 and 25% (p = 0.01) and TNF-α gene expression by 30 and 50%, respectively, in conjunction with a ≥2-fold increase in cAMP (p < 0.05). AR blockade did not modulate other measured cytokines. The induction of A1R after LPS exposure suggests an important role of this receptor in the control of inflammation in neonates. Our findings also suggest that caffeine, via A1R blockade, increases cAMP production and inhibits pretranscriptional TNF-α production by CBM.

Programmed Necrosis: A Prominent Mechanism of Cell Death following Neonatal Brain Injury

Despite the introduction of therapeutic hypothermia, neonatal hypoxic ischemic (HI) brain injury remains a common cause of developmental disability. Development of rational adjuvant therapies to hypothermia requires understanding of the pathways of cell death and survival modulated by HI. The conceptualization of the apoptosis-necrosis “continuum” in neonatal brain injury predicts mechanistic interactions between cell death and hydrid forms of cell death such as programmed or regulated necrosis. Many of the components of the signaling pathway regulating programmed necrosis have been studied previously in models of neonatal HI. In some of these investigations, they participate as part of the apoptotic pathways demonstrating clear overlap of programmed death pathways. Receptor interacting protein (RIP)-1 is at the crossroads between types of cellular death and survival and RIP-1 kinase activity triggers formation of the necrosome (in complex with RIP-3) leading to programmed necrosis. Neuroprotection afforded by the blockade of RIP-1 kinase following neonatal HI suggests a role for programmed necrosis in the HI injury to the developing brain. Here, we briefly review the state of the knowledge about the mechanisms behind programmed necrosis in neonatal brain injury recognizing that a significant proportion of these data derive from experiments in cultured cell and some from in vivo adult animal models. There are still more questions than answers, yet the fascinating new perspectives provided by the understanding of programmed necrosis in the developing brain may lay the foundation for new therapies for neonatal HI.

Inflammation in the carotid body during development and its contribution to apnea of prematurity

Breathing is a complex function that is dynamic, responsive, automatic and often unstable during early development. The carotid body senses dynamic changes in arterial oxygen and carbon dioxide tension and reflexly alters ventilation and plays an essential role in terminating apnea. The carotid body contributes 10-40% to baseline ventilation in newborns and has the greatest influence on breathing in premature infants who characteristically have unstable breathing leading to apnea of prematurity. In this review, we will discuss how both excessive and minimal contributions from the carotid body destabilizes breathing in premature infants and how exposures to hypoxia or infection can lead to changes in the sensitivity of the carotid body. We propose that inflammation/infection during a critical period of carotid body development causes acute and chronic changes in the carotid body contributing to a protracted course of intractable and severe apnea known to occur in a subset of premature infants.

Contribution of early glycemic status in the development of severe retinopathy of prematurity in a cohort of ELBW infants

Objective:The objective of this study is to investigate the relationship between glycemic status and severe retinopathy of prematurity (ROP).Study Design:This is a retrospective cohort study of 114 infants <1000 g admitted to a level IV neonatal intensive care unit within 48 h of life. A cumulative, time-weighted glucose level (TWGL) derived from plotting glucose values over time was included in logistic regression analysis to identify predictors for severe ROP.Result:Infants had 26.6±2 weeks gestational age and had a birth weight of 782±136 g. TWGL during first 10 and 30 days of life were greater in the severe ROP group (P<0.01). Unlike single events of glucose levels ⩾150 mg dl−1, 10 days TWGL ⩾100 mg dl−1 (odds ratio (OR) 5.2, P<0.02) and 30 days TWGL ⩾118 mg dl−1 (OR 5.7, P<0.02) were predictors for severe ROP (univariate). Multivariate regression confirmed 30 days TWGL ⩾118 mg dl−1 (OR 9.4 to 10) and gram-positive sepsis (OR 4.1 to 5) as predictors for severe ROP (P<0.05).Conclusion:High overall glycemic status is associated with the development of severe ROP.

Effect of hyperoxic exposure during early development on neurotrophin expression in the carotid body and nucleus tractus solitarii

Synaptic activity can modify expression of neurotrophins, which influence the development of neuronal circuits. In the newborn rat, early hyperoxia silences the synaptic activity and input from the carotid body, impairing the development and function of chemoreceptors. The purpose of this study was to determine whether early hyperoxic exposure, sufficient to induce hypoplasia of the carotid body and decrease the number of chemoafferents, would also modify neurotrophin expression within the nucleus tractus solitarii (nTS). Rat pups were exposed to hyperoxia (fraction of inspired oxygen 0.60) or normoxia until 7 or 14 days of postnatal development (PND). In the carotid body, hyperoxia decreased brain-derived neurotrophic factor (BDNF) protein expression by 93% (P = 0.04) after a 7-day exposure, followed by a decrease in retrogradely labeled chemoafferents by 55% (P = 0.004) within the petrosal ganglion at 14 days. Return to normoxia for 1 wk after a 14-day hyperoxic exposure did not reverse this effect. In the nTS, hyperoxia for 7 days: 1) decreased BDNF gene expression by 67% and protein expression by 18%; 2) attenuated upregulation of BDNF mRNA levels in response to acute hypoxia; and 3) upregulated p75 neurotrophic receptor, truncated tropomyosin kinase B (inactive receptor), and cleaved caspase-3. These effects were not observed in the locus coeruleus (LC). Hyperoxia for 14 days also decreased tyrosine hydroxylase levels by 18% (P = 0.04) in nTS but not in the LC. In conclusion, hyperoxic exposure during early PND reduces neurotrophin levels in the carotid body and the nTS and shifts the balance of neurotrophic support from prosurvival to proapoptotic in the nTS, the primary brain stem site for central integration of sensory and autonomic inputs.

Effect of development on [Ca2+]i transients to ATP in petrosal ganglion neurons: a pharmacological approach using optical recording

ATP, acting through P2X(2)/P2X(3) receptor-channel complexes, plays an important role in carotid body chemoexcitation in response to natural stimuli in the rat. Since the channels are permeable to calcium, P2X activation by ATP should induce changes in intracellular calcium ([Ca(2+)](i)). Here, we describe a novel ex vivo approach using fluorescence [Ca(2+)](i) imaging that allows screening of retrogradely labeled chemoafferent neurons in the petrosal ganglion of the rat. ATP-induced [Ca(2+)](i) responses were characterized at postnatal days (P) 5-8 and P19-25. While all labeled cells showed a brisk increase in [Ca(2+)](i) in response to depolarization by high KCl (60 mM), only a subpopulation exhibited [Ca(2+)](i) responses to ATP. ATP (250-1,000 μM) elicited one of three temporal response patterns: fast (R1), slow (R2), and intermediate (R3). At P5-8, R2 predominated and its magnitude was attenuated 44% by the P2X(1) antagonist, NF449 (10 μM), and 95% by the P2X(1)/P2X(3)/P2X(2/3) antagonist, TNP-ATP (10 μM). At P19-25, R1 and R3 predominated and their magnitudes were attenuated 15% by NF449, 66% by TNP-ATP, and 100% by suramin (100 μM), a nonspecific P2 purinergic receptor antagonist. P2X(1) and P2X(2) protein levels in the petrosal ganglion decreased with development, while P2X(3) protein levels did not change significantly. We conclude that the profile of ATP-induced P2X-mediated [Ca(2+)](i) responses changes in the postnatal period, corresponding with changes in receptor isoform expression. We speculate that these changes may participate in the postnatal maturation of chemosensitivity.

Optimizing Cerebral Autoregulation May Decrease Neonatal Regional Hypoxic-Ischemic Brain Injury.

Background: Therapeutic hypothermia provides incomplete neuroprotection for neonatal hypoxic-ischemic encephalopathy (HIE). We examined whether hemodynamic goals that support autoregulation are associated with decreased brain injury and whether these relationships are affected by birth asphyxia or vary by anatomic region. Methods: Neonates cooled for HIE received near-infrared spectroscopy autoregulation monitoring to identify the mean arterial blood pressure with optimized autoregulatory function (MAPOPT). Blood pressure deviation from MAPOPT was correlated with brain injury on MRI after adjusting for the effects of arterial carbon dioxide, vasopressors, seizures, and birth asphyxia severity. Results: Blood pressure deviation from MAPOPT related to neurologic injury in several regions independent of birth asphyxia severity. Greater duration and deviation of blood pressure below MAPOPT were associated with greater injury in the paracentral gyri and white matter. Blood pressure within MAPOPT related to lesser injury in the white matter, putamen and globus pallidus, and brain stem. Finally, blood pressures that exceeded MAPOPT were associated with reduced injury in the paracentral gyri. Conclusions: Blood pressure deviation from optimal autoregulatory vasoreactivity was associated with MRI markers of brain injury that, in many regions, were independent of the initial birth asphyxia. Targeting hemodynamic ranges to optimize autoregulation has potential as an adjunctive therapy to hypothermia for HIE.

Therapeutic Hypothermia Provides Variable Protection against Behavioral Deficits after Neonatal Hypoxia-Ischemia: A Potential Role for Brain-Derived Neurotrophic Factor

Background: Despite treatment with therapeutic hypothermia (TH), infants who survive hypoxic ischemic (HI) encephalopathy (HIE) have persistent neurological abnormalities at school age. Protection by TH against HI brain injury is variable in both humans and animal models. Our current preclinical model of hypoxia-ischemia (HI) and TH displays this variability of outcomes in neuropathological and neuroimaging end points with some sexual dimorphism. The detailed behavioral phenotype of this model is unknown. Whether there is sexual dimorphism in certain behavioral domains is also not known. Brain-derived neurotrophic factor (BDNF) supports neuronal cell survival and repair but may also be a marker of injury. Here, we characterize the behavioral deficits after HI and TH stratified by sex, as well as late changes in BDNF and its correlation with memory impairment. Methods: HI was induced in C57BL6 mice on postnatal day 10 (p10) (modified Vannucci model). Mice were randomized to TH (31°C) or normothermia (NT, 36°C) for 4 h after HI. Controls were anesthesia-exposed, age- and sex-matched littermates. Between p16 and p39, growth was followed, and behavioral testing was performed including reflexes (air righting, forelimb grasp and negative geotaxis) and sensorimotor, learning, and memory skills (open field, balance beam, adhesive removal, Y-maze tests, and object location task [OLT]). Correlations between mature BDNF levels in the forebrain and p42 memory outcomes were studied. Results: Both male and female HI mice had an approximately 8-12% lower growth rate (g/day) than shams (p ≤ 0.01) by p39. TH ameliorated this growth failure in females but not in males. In female mice, HI injury prolonged the time spent at the periphery (open field) at p36 (p = 0.004), regardless of treatment. TH prevented motor impairments in the balance beam and adhesive removal tests in male and female mice, respectively (p ≤ 0.05). Male and female HI mice visited the new arm of the Y-maze 12.5% (p = 0.05) and 10% (p = 0.03) less often than shams, respectively. Male HI mice also had 35% lower exploratory preference score than sham (p ≤ 0.001) in the OLT. TH did not prevent memory impairments found with Y-maze testing or OLT in either sex (p ≤ 0.01) at p26. At p42, BDNF levels in the forebrain ipsilateral to the HI insult were 1.7- to 2-fold higher than BDNF levels in the sham forebrain, and TH did not prevent this increase. Higher BDNF levels in the forebrain ipsilateral to the insult correlated with worse performance in the Y-maze in both sexes and in OLT in male mice (p = 0.01). Conclusions: TH provides benefit in specific domains of behavior following neonatal HI. In general, these benefits accrued to both males and females, but not in all areas. In some domains, such as memory, no benefit of TH was found. Late differences in individual BDNF levels may explain some of these findings.

Lipopolysaccharide exposure during the early postnatal period adversely affects the structure and function of the developing rat carotid body

The carotid body (CB) substantially influences breathing in premature infants by affecting the frequency of apnea and periodic breathing. In adult animals, inflammation alters the structure and chemosensitivity of the CB, yet it is not known if this pertains to neonates. We hypothesized that early postnatal inflammation leads to morphological and functional changes in the developing rat CB, which persists for 1 wk after the initial provoking insult. To test our hypothesis, we exposed rat pups at postnatal day 2 (P2) to lipopolysaccharide (LPS; 100 μg/kg) or saline (SAL) intraperitoneally. At P9-10 (1 wk after treatment), LPS-exposed animals had significantly more spontaneous intermittent hypoxic (IH) events, attenuated ventilatory responses to changes in oxygen tension (measured by whole body plethysmography), and attenuated hypoxic chemosensitivity of the carotid sinus nerve (measured in vitro), compared with SAL-exposed controls. These functional changes were associated with the following: 1) increased inflammatory cytokine mRNA levels; 2) decreased volume of supportive type II cells; and 3) elevated dopamine levels (a major inhibitory neuromodulator) within the CB. These findings suggest that early postnatal inflammation in newborn rats adversely affects the structure and function of the CB and is associated with increased frequency of intermittent desaturations, similar to the phenomenon observed in premature infants. Furthermore, this is the first newborn model of spontaneous intermittent desaturations that may be used to understand the mechanisms contributing to IH events in newborns.