Ingeborg Løstegaard Goverud

Low-potency glucocorticoid hydrocortisone has similar neurotoxic effects as high-potency glucocorticoid dexamethasone on neurons in the immature chicken cerebellum

High-potency glucocorticoids (GC) are used in the prophylaxis and treatment of neonatal bronchopulmonal dysplasia, but there is concern about side effects on the developing brain. Recently, the low-potency GC hydrocortisone (HC) has been suggested as an alternative to high-potency GC. We compared the neurotoxic effects of HC with the high-potency GC dexamethasone (DEX) in chicken cerebellum. A single dose of GC was injected into the egg at embryonic day 16 and the death of granule neurons in histologic sections of the cerebellar cortex was examined 24 h later. DEX and HC showed a similar dose-dependent induction of morphological apoptosis and caspase-3 activation in the internal granular layer. A doubling of the apoptosis rate compared to the basal rate was seen for the highest dose of DEX (5 mg/kg) and medium dose of HC (1 mg/kg). In cultures of embryonic chicken cerebellar granule cells, DEX and HC increased cell death and induced rapid caspase-3 activation in a similar dose-dependent manner. Transfection of granule cells with a luciferase reporter gene revealed that the dose needed for the activation of gene transcription (classical signalling pathway) with DEX was much lower than for HC. In conclusion, HC does not present itself as a safer drug than DEX in this model. In addition, it appears that DEX and HC induce apoptosis in immature granule neurons via a non-genomic (non-classical) mechanism.

Prenatal exposure to bisphenol A interferes with the development of cerebellar granule neurons in mice and chicken.

In mice, prenatal exposure to low doses of bisphenol A has been shown to affect neurogenesis and neuronal migration in cortex, resulting in disturbance of both neuronal positioning and the network formation between thalamus and cortex in the offspring brain. In the present study we investigated whether prenatal exposure to bisphenol A disturbs the neurodevelopment of the cerebellum. Two different model systems were used; offspring from two strains of mice from mothers receiving bisphenol A in the drinking water before mating, during gestation and lactation, and chicken embryos exposed to bisphenol A (in the egg) on embryonic day 16 for 24 h before preparation of cerebellar granule cell cultures. In the cerebellum, tight regulation of the level of transcription factor Pax6 is critical for correct development of granule neurons. During the development, the Pax6 level in granule neurons is high when these cells are located in the external granule layer and during their migration to the internal granule layer, and it is then reduced. We report that bisphenol A induced an increase in the thickness of the external granule layer and also an increase in the total cerebellar Pax6 level in 11 days old mice offspring. In cultured chicken cerebellar granule neurons from bisphenol A injected eggs the Pax6 level was increased day 6 in vitro. Together, these findings indicate that bisphenol A may affect the granule neurons in the developing cerebellum and thereby may disturb the correct development of the cerebellum.

Nicotine affects the expression of brain-derived neurotrophic factor mRNA and protein in the hippocampus of hypoxic newborn piglets

Abstract Brain-derived neurotrophic factor (BDNF) is highly expressed in the developing brain. It has anti-apoptotic abilities, and protects the neonatal brain. In experimental settings in adult animals, pre-treatment with nicotine has shown increased BDNF levels, indicating a possible contribution to nicotines anti-apoptotic effect. Apoptosis contributes to the development of brain damage in perinatal asphyxia. We examined the effects of nicotine on apoptosis-inducing factor (AIF), caspase-3 and BDNF in the hippocampus of a neonatal piglet model of global hypoxia. Forty-one anesthetized newborn piglets were randomized to one of four groups receiving different infusions after hypoxia (1) nicotine 130 μg/kg/h, 2) 260 μg/kg/h, 3) adrenaline, and 4) saline, all 2.6 mL/kg/h. Four hours after hypoxia they were euthanized. The left hemisphere/hippocampus was examined by histopathology and immunohistochemistry; the right hippocampus was analyzed using real time PCR. There was a significantly higher expression of BDNF mRNA and protein in the animals treated with nicotine 130 μg/kg/h vs. the saline treated group (mRNA P=0.038; protein P=0.009). There were no differences regarding AIF or caspase-3. We conclude that nicotine (130 μg/kg/h), infused over 1 h after global hypoxia in neonatal piglets, increases levels of both BDNF mRNA and protein in the hippocampus. This might imply neuroprotective effects of nicotine in asphyxiated neonates.

Lack of CCR7 induces pulmonary hypertension involving perivascular leukocyte infiltration and inflammation

The chemokine receptor CCR7 regulates lymphocyte trafficking, and CCR7 deficiency induces infiltration of T and B cells adjacent to vessels in mouse lungs. Perivascular infiltration of T and B cells has also been found in human pulmonary arterial hypertension, and downregulation of the CCR7 receptor in circulating leukocytes of such patients has been observed. To investigate whether changes in the CCR7 system contribute to the pathogenesis of pulmonary hypertension, we utilized mice deficient of the CCR7 receptor. The cardiopulmonary and inflammatory responses of CCR7 depletion were evaluated in CCR7-deficient and wild-type mice. Measurements of cytokines upregulated in the animal model were also performed in patients with pulmonary hypertension and controls and in vascular smooth muscle cells. We found that mice lacking CCR7 had increased right ventricular systolic pressure, reduced pulmonary artery acceleration time, increased right ventricular/tibial length ratio, Rho kinase-mediated pulmonary vasoconstriction, and increased muscularization of distal arteries, indicating pulmonary hypertension. These mice also showed increased perivascular infiltration of leukocytes, consisting mainly of T and B cells, and increased mRNA levels of the inflammatory cytokines interleukin-12 and CX3CL1 within pulmonary tissue. Increased serum levels of interleukin-12 and CX3CL1 were also observed in patients with pulmonary hypertension, particularly in those with pulmonary hypertension associated with connective tissue disorder. In smooth muscle cells, interleukin-12 induced secretion of the angiogenic cytokine interleukin-8. We conclude that these results suggest a role for CCR7 in the development of pulmonary arterial hypertension, at least in some subgroups, possibly via pulmonary infiltration of lymphocytes and secretion of interleukin-12 and CX3CL1.

Glucocorticoids dexamethasone and hydrocortisone inhibit proliferation and accelerate maturation of chicken cerebellar granule neurons.

Glucocorticoid (GC) treatment in premature infants may have detrimental effects on the immature brain. Here we show that GCs dexamethasone (Dex) and hydrocortisone (HC) reduce proliferation and induce differentiation of chicken embryo cerebellar neurons in vivo and in vitro. Granule neurons incorporating bromodeoxyuridine were reduced in the internal granular layer (IGL) after 24-h exposure to both substances on embryonic day 17, with Dex about 100-fold more potent than HC. The effects were blocked by GR antagonist RU 38486. Both GCs also increased the expression of neuronal differentiation markers microtubule-associated protein 2 (Map2) and neuronal nuclei protein (NeuN), measured by western blotting of whole cerebellar lysates and immunohistochemistry, respectively. Treatment of cerebellar granule neuron cultures with both GCs significantly reduced the percentage of proliferating-cell nuclear antigen (PCNA) positive neurons and increased NeuN positive neurons, with similar dose-response relationship as in vivo. The cytostatic agent cytosine arabinoside showed comparable effects both on proliferation and differentiation. In conclusion, the effects of Dex and HC on chicken cerebellar granule neuron proliferation are GR mediated and reflect their pharmacological potency. In addition, the effects on differentiation may be related to a cell cycle block per se, since cytosine arabinoside mimicked the effect of the GCs.

Newborn piglets exposed to hypoxia after nicotine or saline pretreatment: Long-term effects on brain and heart

Objective. We wished to assess the effect of global hypoxia and the effect of nicotine pretreatment on the brain and heart of newborn pigs. Hypothesising that nicotine might give a better outcome because of its anti-apoptotic and anti-inflammatory effects. Methods. Twenty-two anaesthetised piglets were randomised to pretreatment with saline or nicotine (130 μg/kg/h) before 45 min global hypoxia. They were observed for 27 h. The brain and heart were assessed with histopathological methods. Serum for Troponin t (TnT) analyses was collected at baseline and at the end of the experiment. Results. There were no significant differences between the groups. At the end of hypoxia, BE was −14.8 ± 4.9 mmol/l and MABP was 25 ± 9 mmHg. Seven animals had autolysis of the cerebrum/cerebellum, their BE after hypoxia was −19 ± 1.8 mmol/l and MABP 23 ± 3 mmHg. The remaining 15 animals had a BE of −13 ± 4.7mmol/l (p = 0.0004) and a MABP of 26 ± 11 mmHg (ns). Eleven animals presented myocardial damage. A significant increase in TnT occurred in both groups. TnT increase and myocardial damage correlated (p = 0.001; r = 0.67). Animals with severe increase in TnT presented severe brain damage. Conclusions. Severe increase in serum TnT levels was linked to severe cerebral damage. Nicotine pretreatment had no impact on cerebral or cardiac histopathology.

Brain infection with Staphylococcus aureus leads to high extracellular levels of glutamate, aspartate, γ‐aminobutyric acid, and zinc

Staphylococcal brain infections may cause mental deterioration and epileptic seizures, suggesting interference with normal neurotransmission in the brain. We injected Staphylococcus aureus into rat striatum and found an initial 76% reduction in the extracellular level of glutamate as detected by microdialysis at 2 hr after staphylococcal infection. At 8 hr after staphylococcal infection, however, the extracellular level of glutamate had increased 12‐fold, and at 20 hr it had increased >30‐fold. The extracellular level of aspartate and γ‐aminobutyric acid (GABA) also increased greatly. Extracellular Zn2+, which was estimated at ∼2.6 µmol/liter in the control situation, was increased by 330% 1–2.5 hr after staphylococcal infection and by 100% at 8 and 20 hr. The increase in extracellular glutamate, aspartate, and GABA appeared to reflect the degree of tissue damage. The area of tissue damage greatly exceeded the area of staphylococcal infiltration, pointing to soluble factors being responsible for cell death. However, the N‐methyl‐D‐aspartate receptor antagonist MK‐801 ameliorated neither tissue damage nor the increase in extracellular neuroactive amino acids, suggesting the presence of neurotoxic factors other than glutamate and aspartate. In vitro staphylococci incubated with glutamine and glucose formed glutamate, so bacteria could be an additional source of infection‐related glutamate. We conclude that the dramatic increase in the extracellular concentration of neuroactive amino acids and zinc could interfere with neurotransmission in the surrounding brain tissue, contributing to mental deterioration and a predisposition to epileptic seizures, which are often seen in brain abscess patients.© 2014 Wiley Periodicals, Inc.

Staphylococcal α-hemolysin is neurotoxic and causes lysis of brain cells in vivo and in vitro

Formation of a bacterial brain abscess entails loss of brain cells and formation of pus. The mechanisms behind the cell loss are not fully understood. Staphylococcus aureus, a common cause of brain abscesses, produces various exotoxins, including α-hemolysin, which is an important factor in brain abscess formation. α-Hemolysin may cause cytolysis by forming pores in the plasma membrane of various eukaryotic cells. However, whether α-hemolysin causes lysis of brain cells is not known. Nor is it known whether α-hemolysin in the brain causes cell death through pore formation or by acting as a chemoattractant, recruiting leukocytes and causing inflammation. Here we show that α-hemolysin injected into rat brain causes cell damage and edema formation within 30 min. Cell damage was accompanied by an increase in extracellular concentrations of zinc, GABA, glutamate, and other amino acids, indicating plasma membrane damage, but leukocytic infiltration was not seen 0.5-12h after α-hemolysin injection. This was in contrast to injection of S. aureus, which triggered extensive infiltration with neutrophils within 8h. In vitro, α-hemolysin caused concentration-dependent lysis of isolated nerve endings and cultured astrocytes. We conclude that α-hemolysin contributes to the cell death inherent in staphylococcal brain abscess formation as a pore-forming neurotoxin.

Transventricular non-transmural laser treatment of hypoperfused porcine myocardium acutely reduces left ventricular contractile function.

OBJECTIVE Creation of non-transmural myocardial channels by lasers transmitted through endovascular fiberoptics is a novel therapeutic option in the management of patients with coronary artery disease. The acute effect of transventricular laser treatment (TvL) on coronary blood flow, myocardial metabolism and left ventricular function are not well established. METHODS In five anesthetized pigs, flow in the proximal left anterior descending coronary artery (LAD) was reduced and maintained at 70% of baseline. A venous shunt had previously been established draining the hypoperfused region. At 30 min of ischemia, non-transmural myocardial channels were created through the endocardium using a Ho:YAG laser. We measured (a) left ventricular, central venous and arterial pressures, (b) ascending aortic, LAD and coronary venous blood flows, as well as (c) lactate concentration and blood gases in arterial and coronary venous blood, prior to ischemia (baseline), before and 30 min after TvL. Data (given as mean +/- SD) were analyzed with repeated measures ANOVA. RESULTS Reduction of LAD blood flow resulted in reduced regional coronary venous blood flow and myocardial oxygen consumption, conversion of regional myocardial lactate uptake to release and adaptation of left ventricular contractility to a lower level. Following transventricular laser, the peak left ventricular systolic pressure declined from 86 +/- 12 to 77 +/- 11 mmHg (P < 0.05), its maximal first positive derivative (LV dP/dt) declined from 900 +/- 221 to 763 +/- 127 mmHg/s (P < 0.05) and the stroke volume decreased from 19.2 +/- 4.1 to 16.4 +/- 5.4 ml (P < 0.05). The changes in regional coronary venous flow, myocardial oxygen consumption and myocardial lactate release after TvL were not significant compared to before TvL. Significant intramural hematomas and tissue destruction were found around the channels at autopsy and by histologic examination. CONCLUSION Transventricular laser treatment of hypoperfused myocardium decreased left ventricular contractility in the acute phase, possibly due to development of perichannel hematomas and disruption of the wall architecture. In addition, TvL did not alter the regional myocardial oxygen supply/demand balance. These results call for caution in the treatment of patients with coronary artery disease by transventricular Ho-YAG laser when there is significant impairment of the left ventricular contractile function.