Damjan Osredkar

Hypothermia is not neuroprotective after infection-sensitized neonatal hypoxic-ischemic brain injury

BACKGROUND Therapeutic hypothermia (HT) is the standard treatment after perinatal hypoxic-ischemic (HI) injury. Infection increases vulnerability to HI injury, but the effect of HT on lipopolysaccharide (LPS) sensitized HI brain injury is unknown. DESIGN/METHODS P7 rat pups were injected either with vehicle or LPS, and after a 4h delay they were exposed to left carotid ligation followed by global hypoxia inducing a unilateral stroke-like HI injury. Pups were randomized to the following treatments: (1) vehicle treated HI-pups receiving normothermia treatment (NT) (Veh-NT; n=30); (2) LPS treated HI-pups receiving NT treatment (LPS-NT; n=35); (3) vehicle treated HI-pups receiving HT treatment (Veh-HT; n=29); or (4) LPS treated HI-pups receiving HT treatment (LPS-HT; n=46). Relative area loss of the left/right hemisphere and the areas of hippocampi were measured at P14. RESULTS Mean brain area loss in the Veh-NT group was 11.2±14%. The brain area loss in LPS-NT pups was 29.8±17%, which was significantly higher than in the Veh-NT group (p=0.002). The Veh-HT group had a significantly smaller brain area loss (5.4±6%), when compared to Veh-NT group (p=0.043). The LPS-HT group showed a brain area loss of 32.5±16%, which was significantly higher than in the Veh-HT group (p<0.001). LPS-HT group also had significantly smaller size of the left hippocampus, which was not found in other groups. LPS-sensitization significantly decreased the sizes of the right, unligated-hemispheres, independent of post-HI treatment. CONCLUSIONS Therapeutic hypothermia is not neuroprotective in this LPS-sensitized unilateral stroke-like HI brain injury model in newborn rats. Lack of neuroprotection was particularly seen in the hippocampus. Pre-insult exposure to LPS also induced brain area loss in the unligated hemisphere, which is normally not affected in this model.

HIF-1α-Deficient Mice Have Increased Brain Injury after Neonatal Hypoxia-Ischemia

Evidence suggests that the activation of the transcription factor hypoxia-inducible factor 1α (HIF-1α) may promote cell survival in hypoxic or ischemic brain. To help understand the role of HIF-1α in neonatal hypoxic-ischemic brain injury, mice with conditional neuron-specific inactivation of HIF-1α underwent hypoxia-ischemia (HI). Mice heterozygous for Cre recombinase under the control of the calcium/calmodulin-dependent kinase II promoter were bred with homozygous ‘floxed’ HIF-1α transgenic mice. The resulting litters produced mice with a forebrain predominant neuronal deletion of HIF-1α (HIF-1αΔ/Δ), as well as littermates without the deletion. In order to verify reduction of HIF-1α at postnatal day 7, HIF-1αΔ/Δ and wild-type mice were exposed to a hypoxic stimulus (8% oxygen) or room air for 1 h, followed by immediate collection of brain cortices for determination of HIF-1α expression. Results of Western blotting of mouse cortices exposed to hypoxia stimulus or room air confirmed that HIF-1αΔ/Δ cortex expressed a minimal amount of HIF-1α protein compared to wild-type cortex with the same hypoxic stimulus. Subsequently, pups underwent the Vannucci procedure of HI at postnatal day 7: unilateral ligation of the right common carotid artery followed by 30 min of hypoxia (8% oxygen). Immunofluorescent staining of brains 24 h after HI confirmed a relative lack of HIF-1α in the HIF-1αΔ/Δ cortex compared to the wild type, and that HIF-1α in the wild type is located in neurons. HIF-1α expression was determined in mouse cortex 24 h after HI. Histological analysis for the degree of injury was performed 5 days after HI. HIF-1α protein expression 24 h after HI showed a large increase of HIF-1α in the hypoxic-ischemic cortex of the wild-type compared to the hypoxic only cortex. Histological analysis revealed that HI injury was increased in the neuronally deficient HIF-1αΔ/Δ mouse brain (p < 0.05) and was more severe in the cortex. Genetic reduction of neuronal HIF-1α results in a worsening of injury after neonatal HI, with a region-specific role for HIF-1α in the setting of neonatal brain injury.

Treatment temperature and insult severity influence the neuroprotective effects of therapeutic hypothermia

Therapeutic hypothermia (HT) is standard care for moderate and severe neonatal hypoxic-ischaemic encephalopathy (HIE), the leading cause of permanent brain injury in term newborns. However, the optimal temperature for HT is still unknown, and few preclinical studies have compared multiple HT treatment temperatures. Additionally, HT may not benefit infants with severe encephalopathy. In a neonatal rat model of unilateral hypoxia-ischaemia (HI), the effect of five different HT temperatures was investigated after either moderate or severe injury. At postnatal-day seven, rat pups underwent moderate or severe HI followed by 5 h at normothermia (37 °C), or one of five HT temperatures: 33.5 °C, 32 °C, 30 °C, 26 °C, and 18 °C. One week after treatment, neuropathological analysis of hemispheric and hippocampal area loss, and CA1 hippocampal pyramidal neuron count, was performed. After moderate injury, a significant reduction in hemispheric and hippocampal loss on the injured side, and preservation of CA1 pyramidal neurons, was seen in the 33.5 °C, 32 °C, and 30 °C groups. Cooling below 33.5 °C did not provide additional neuroprotection. Regardless of treatment temperature, HT was not neuroprotective in the severe HI model. Based on these findings, and previous experience translating preclinical studies into clinical application, we propose that milder cooling should be considered for future clinical trials.

Early onset West syndrome with severe hypomyelination and coloboma-like optic discs in a girl with SPTAN1 mutation

Recent study has shown that mutations in the alpha‐II‐spectrin (SPTAN1) gene cause early onset intractable seizures, severe developmental delay, diffuse hypomyelination, and widespread brain atrophy. We report a Slovene girl with hypotonia, lack of visual attention, early onset epileptic encephalopathy, and severe developmental delay. The patient presented with segmental myoclonic jerks at the age of 6 weeks, and infantile spasms at the age of 3.5 months. Her seizures were resistant to treatment. Multiple electroencephalography recordings showed deterioration of the background activity, followed by multifocal abnormalities before progressing to hypsarrhythmia. Ophthalmologic examination revealed bilateral dysplastic, coloboma‐like optic discs. Brain magnetic resonance imaging showed diffusely reduced white matter and brainstem volumes with hypomyelination. A de novo heterozygous in‐frame deletion was detected in SPTAN1: c.6619_6621delGAG (p.E2270del). This report supports the causative relationship between SPTAN1 mutations and early onset intractable seizures with severe hypomyelination and widespread brain volume reduction. Coloboma‐like optic discs might be an additional feature observed in patients with SPTAN1 mutations.

A Comparison of Respiratory Patterns in Healthy Term Infants Placed in Car Safety Seats and Beds

OBJECTIVE: The purpose of this work was to compare the incidence of apnea, hypopnea, bradycardia, or oxygen desaturation in healthy term newborns placed in hospital cribs, infant car safety beds, or infant car safety seats. METHODS: A consecutive series of 200 newborns was recruited on the second day of life. Each subject was studied while placed in the hospital crib (30 minutes), car bed (60 minutes), and car seat (60 minutes). Physiologic data, including oxygen saturation, frequency, and type of apnea, hypopnea, and bradycardia were obtained and analyzed in a blinded manner. RESULTS: The mean oxygen saturation level was significantly different among all of the positions (97.9% for the hospital crib, 96.3% for the car bed, and 95.7% for the car seat; P < .001). The mean minimal oxygen saturation level was lower while in both safety devices (83.7% for the car bed and 83.6% for the car seat) compared with in the hospital crib (87.4%) (P < .001). The mean total time spent with an oxygen saturation level of <95% was significantly higher (P = .003) in both safety devices (car seat: 23.9%; car bed: 17.2%) when compared with the hospital crib (6.5%). A second study of 50 subjects in which each infant was placed in each position for 120 minutes yielded similar results. CONCLUSIONS: In healthy term newborns, significant desaturations were observed in both car beds and car seats as compared with hospital cribs. This study was limited by lack of documentation of sleep stage. Therefore, these safety devices should only be used for protection during travel and not as replacements for cribs.

Xenon combined with therapeutic hypothermia is not neuroprotective after severe hypoxia-ischemia in neonatal rats

Background Therapeutic hypothermia (TH) is standard treatment following perinatal asphyxia in newborn infants. Experimentally, TH is neuroprotective after moderate hypoxia-ischemia (HI) in seven-day-old (P7) rats. However, TH is not neuroprotective after severe HI. After a moderate HI insult in newborn brain injury models, the anesthetic gas xenon (Xe) doubles TH neuroprotection. The aim of this study was to examine whether combining Xe and TH is neuroprotective as applied in a P7 rat model of severe HI. Design/Methods 120 P7 rat pups underwent a severe HI insult; unilateral carotid artery ligation followed by hypoxia (8% O2 for 150min at experimental normothermia (NT-37: Trectal 37°C). Surviving pups were randomised to immediate NT-37 for 5h (n = 36), immediate TH-32: Trectal 32°C for 5h (n = 25) or immediate TH-32 plus 50% inhaled Xe for 5h (n = 24). Pups were sacrificed after one week of survival. Relative area loss of the ligated hemisphere was measured, and neurons in the subventricular zone of this injured hemisphere were counted, to quantify brain damage. Results Following the HI insult, median (interquartile range, IQR) hemispheric brain area loss was similar in all groups: 63.5% (55.5–75.0) for NT-37 group, 65.0% (57.0–65.0) for TH-32 group, and 66.5% (59.0–72.0) for TH-32+Xe50% group (not significant). Correspondingly, there was no difference in neuronal cell count (NeuN marker) in the subventricular zone across the three treatment groups. Conclusions Immediate therapeutic hypothermia with or without additional 50% inhaled Xe, does not provide neuroprotection one week after severe HI brain injury in the P7 neonatal rat. This model aims to mimic the clinical situation in severely asphyxiated neonates and treatment these newborns remains an ongoing challenge.

Hypothermia Does Not Reverse Cellular Responses Caused by Lipopolysaccharide in Neonatal Hypoxic-Ischaemic Brain Injury

Introduction: Bacterial lipopolysaccharide (LPS) injection prior to hypoxia-ischaemia significantly increases hypoxia-ischaemic brain injury in 7-day-old (P7) rats. In addition, therapeutic hypothermia (HT) is not neuroprotective in this setting. However, the mechanistic aspects of this therapeutic failure have yet to be elucidated. This study was designed to investigate the underlying cellular mechanisms in this double-hit model of infection-sensitised hypoxia-ischaemic brain injury. Material and Methods: P7 rat pups were injected with either vehicle or LPS, and after a 4-hour delay were exposed to left carotid ligation followed by global hypoxia inducing a unilateral stroke-like hypoxia-ischaemic injury. Pups were randomised to the following treatments: (1) vehicle-treated pups receiving normothermia treatment (NT) (Veh-NT; n = 40), (2) LPS-treated pups receiving NT treatment (LPS-NT; n = 40), (3) vehicle-treated pups receiving HT treatment (Veh-HT; n = 38) and (4) LPS-treated pups receiving HT treatment (LPS-HT; n = 35). On postnatal day 8 or 14, Western blot analysis or immunohistochemistry was performed to examine neuronal death, apoptosis, astrogliosis and microglial activation. Results: LPS sensitisation prior to hypoxia-ischaemia significantly exacerbated apoptotic neuronal loss. NeuN, a neuronal biomarker, was significantly reduced in the LPS-NT and LPS-HT groups (p = 0.008). Caspase-3 activation was significantly increased in the LPS-sensitised groups (p < 0.001). Additionally, a significant increase in astrogliosis (glial fibrillary acidic expression, p < 0.001) was seen, as well as a trend towards increased microglial activation (Iba 1 expression, p = 0.051) in LPS-sensitised animals. Treatment with HT did not counteract these changes. Conclusion: LPS-sensitised hypoxia-ischaemic brain injury in newborn rats is mediated through neuronal death, apoptosis, astrogliosis and microglial activation. In this double-hit model, treatment with HT does not ameliorate these changes.

Effects of xenon and hypothermia on cerebrovascular pressure reactivity in newborn global hypoxic-ischemic pig model

Autoregulation of cerebral perfusion is impaired in hypoxic-ischemic encephalopathy. We investigated whether cerebrovascular pressure reactivity (PRx), an element of cerebral autoregulation that is calculated as a moving correlation coefficient between averages of intracranial and mean arterial blood pressure (MABP) with values between −1 and +1, is impaired during and after a hypoxic-ischemic insult (HI) in newborn pigs. Associations between end-tidal CO2, seizures, neuropathology, and PRx were investigated. The effect of hypothermia (HT) and Xenon (Xe) on PRx was studied. Pigs were randomized to Sham, and after HI to normothermia (NT), HT, Xe or xenon hypothermia (XeHT). We defined PRx >0.2 as peak and negative PRx as preserved. Neuropathology scores after 72 hours of survival was grouped as ‘severe’ or ‘mild.’ Secondary PRx peak during recovery, predictive of severe neuropathology and associated with insult severity (P = 0.05), was delayed in HT (11.5 hours) than in NT (6.5 hours) groups. Seizures were associated with impaired PRx in NT pigs (P = 0.0002), but not in the HT/XeHT pigs. PRx was preserved during normocapnia and impaired during hypocapnia. Xenon abolished the secondary PRx peak, increased (mean (95% confidence interval (CI)) MABP (6.5 (3.8, 9.4) mm Hg) and cerebral perfusion pressure (5.9 (2.9, 8.9) mm Hg) and preserved the PRx (regression coefficient, −0.098 (95% CI (−0.18, −0.01)), independent of the insult severity.

Recording conventional and amplitude-integrated EEG in neonatal intensive care unit.

Neonatal electroencephalography (EEG) presents a challenge due to its difficult interpretation that differs significantly from interpretation in older children and adolescents. Also, from the technological point of view, it is more difficult to perform and is not a standard procedure in all neonatal intensive care units (NICUs). During recent years, long-term cerebral function monitoring by the means of amplitude-integrated EEG (aEEG) has become popular in NICUs because it is easy to apply, allows real-time interpretation by the neonatologist treating the newborn, and has predictive value for outcome. On the other side, to record conventional EEG (cEEG), which is still considered the gold standard of neonatal EEG, the EEG technician should not only be well trained in performing neonatal EEG but also has to adapt to suboptimal working conditions. These issues need to be understood when approaching the neonatal cEEG in NICU and the main structure of the article is dedicated to this technique. The authors discuss the benefits of the digitalization and its positive effects on the improvement of NICU recording. The technical aspects as well as the standards for cEEG recording are described, and a section is dedicated to possible artifacts. Thereafter, alternative and concomitant use of aEEG and its benefits are briefly discussed. At the end there is a section that presents a review of our own cEEG and aEEG recordings that were chosen as the most frequently encountered patterns according to Consensus statement on the use of EEG in the intensive care unit.

Infant botulism: first two confirmed cases in Slovenia and literature review.

In Europe, infant botulism is a rare but probably under-diagnosed disease. With the intent to spread the awareness of this potentially life-threatening disease, we present a review of the literature with the emphasis on European epidemiology and a practical approach to diagnosis. We also report the first two confirmed cases of infant botulism in Slovenia and describe our way to the final diagnosis in a clinical setting where all appropriate diagnostic tests and treatment options are not readily available. The second case is particularly interesting, presenting with profound diarrhea following initial constipation, an unlikely symptom for an infant with botulism and possibly caused by Bacteroides fragilis.