Orsolya Oláh

Evaluation of laser-speckle contrast image analysis techniques in the cortical microcirculation of piglets

A new laser speckle-contrast analysis (LASCA) technique based on multi-exposure imaging was employed to simultaneously study pial arteriolar responses with cerebrocortical perfusion changes to various vasodilator (5-10% CO(2) ventilation, bradykinin (1-10 μM), N-methyl-D-aspartate (100 μM)) vasoconstrictor (10-100 μM noradrenaline, 1M KCl), or neutral (2.1% H(2) ventilation) stimuli as well as to asphyxia in the newborn piglet. Anesthetized, ventilated animals (n=20) were fitted with closed cranial windows. Multiple exposure laser-speckle image series (1-100 ms) were obtained using a near infrared diode laser (λ=808 nm). The autocorrelation decay time (τ) of speckle fluctuations was determined over pial arterioles and parenchymal areas to express 1/τ being proportional to blood flow velocity by two different LASCA techniques: our novel multi-exposure or a single exposure (2 and 20 ms) approach. 1/τ values yielded by different LASCA techniques were not significantly different at most points. LASCA easily detected both increases and decreases in cortical blood flow (CoBF). Cortical 1/τ changes to hypercapnia closely matched quantitative CoBF data determined previously, and were also in accordance with increases of pial arteriolar blood flow, calculated from arteriolar flow velocity and cross sectional area changes. In summary, LASCA emerges as an appealing method to simultaneously study microvascular reactivity and cortical perfusion changes in the piglet.

Hydrogen is Neuroprotective and Preserves Cerebrovascular Reactivity in Asphyxiated Newborn Pigs

Hydrogen (H2) has been reported to neutralize toxic reactive oxygen species. Oxidative stress is an important mechanism of neuronal damage after perinatal asphyxia. We examined whether 2.1% H2-supplemented room air (H2-RA) ventilation would preserve cerebrovascular reactivity (CR) and brain morphology after asphyxia/reventilation (A/R) in newborn pigs. Anesthetized, ventilated piglets were assigned to one of the following groups: A/R with RA or H2-RA ventilation (A/R-RA and A/R-H2-RA; n = 8 and 7, respectively) and respective time control groups (n = 9 and 7). Asphyxia was induced by suspending ventilation for 10 min, followed by reventilation with the respective gases for 4 h. After euthanasia, the brains were processed for neuropathological examination. Pial arteriolar diameter changes to graded hypercapnia (5–10% CO2 inhalation), and NMDA (10−4 M) were determined using the closed cranial window/intravital microscopy before and 1 h after asphyxia. Neuropathology revealed that H2-RA ventilation significantly reduced neuronal injury induced by A/R in virtually all examined brain regions including the cerebral cortex, the hippocampus, basal ganglia, cerebellum, and the brainstem. Furthermore, H2-RA ventilation significantly increased CR to hypercapnia after A/R (% vasodilation was 23 ± 4% versus 41 ± 9%, p < 0.05). H2-RA ventilation did not affect reactive oxygen species-dependent CR to NMDA. In summary, H2-RA could be a promising approach to reduce the neurologic deficits after perinatal asphyxia.

PACAP and VIP differentially preserve neurovascular reactivity after global cerebral ischemia in newborn pigs

Pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are neuroprotective in numerous models. Impairment of cerebrovascular reactivity (CR) contributes to ischemia/reperfusion (I/R)-induced neuronal damage. We tested whether PACAP and/or VIP preserve CR to I/R-sensitive dilator responses dependent on endothelial and/or neuronal function. Accordingly, changes in pial arteriolar diameters in response to hypercapnia (5-10% CO(2) ventilation) or topical N-methyl-d-aspartate (NMDA, 10(-4) M) were determined before and after I/R via intravital microscopy in anesthetized/ventilated piglets. Local pretreatment with non-vasoactive doses of PACAP (10(-8) M) and VIP (10(-9) M) prevented the attenuation of postischemic CR to hypercapnia; to 10% CO(2), the CR values were 27+/-8% vs 92+/-5% vs 88+/-13% (vehicle vs PACAP38 vs VIP, CR expressed as a percentage of the response before I/R, mean+/-SEM, n=8-8, p<0.05). PACAP, but not VIP, preserved CR to NMDA after I/R, with CR values of 31+/-10% vs 87+/-8% vs 35+/-12% (vehicle vs PACAP38 vs VIP, n=6-6). Unlike PACAP, VIP-induced vasodilation has not yet been investigated in the piglet. We tested whether VIP-induced arteriolar dilation was sensitive to inhibitors of cyclooxygenase (COX)-1 (SC-560, 1 mg/kg), COX-2 (NS-398, 1 mg/kg), indomethacin (5 mg/kg), and nitric oxide synthase (L-NAME, 15 mg/kg). VIP (10(-8)-10(-7)-10(-6) M, n=8) induced reproducible, dose-dependent vasodilation of 16+/-3%, 33+/-6%, and 70+/-8%. The response was unaffected by all drugs, except that the vasodilation to 10(-8) M VIP was abolished by SC-560 and indomethacin. In conclusion, PACAP and VIP differentially preserve postischemic CR; independent of their vasodilatory effect.

Delayed neurovascular dysfunction is alleviated by hydrogen in asphyxiated newborn pigs.

Background: The neurovascular unit encompasses the functional interactions of cerebrovascular and brain parenchymal cells necessary for the metabolic homeostasis of neurons. Previous studies indicated marked but only transient (1-4 h) reactive oxygen species-dependent neurovascular dysfunction in newborn pigs after severe hypoxic/ischemic (H/I) stress contributing to the neuronal injury after birth asphyxia. Objectives: Our major purpose was to determine if neurovascular dysfunction would also occur later, at 24 h after a milder H/I stress. We also tested if the putative hydroxyl radical scavenger hydrogen (H2) exerted neurovascular protection. Methods: Anesthetized, ventilated piglets were assigned to three groups of 9 animals: time control, asphyxia/reventilation with air, and asphyxia/reventilation with air +2.1% H2 for 4 h. Asphyxia was induced by suspending ventilation for 8 min. Cerebrovascular reactivity (CR) of pial arterioles was determined using closed cranial window/intravital microscopy 24 h after asphyxia to the endothelium-dependent cerebrovascular stimulus hypercapnia, the neuronal function-dependent stimulus N-methyl-D-aspartate (NMDA), norepinephrine, and sodium nitroprusside. The brains were subjected to histopathology. Results: Hemodynamic parameters, blood gases, and core temperature did not differ significantly among the experimental groups. In the early reventilation period, the recovery of electroencephalographic activity was significantly better in H2-treated animals. Asphyxia/reventilation severely attenuated CR to hypercapnia and NMDA; however, reactivity to norepinephrine and sodium nitroprusside were unaltered. H2 fully or partially preserved CR to hypercapnia or NMDA, respectively. Histopathology revealed modest neuroprotection afforded by H2. Conclusions: Severe stimulus-selective delayed neurovascular dysfunction develops and persists even after mild H/I stress. H2 alleviates this delayed neurovascular dysfunction that can contribute to its neuroprotective effect.

Regional Differences in the Neuronal Expression of Cyclooxygenase-2 (COX-2) in the Newborn Pig Brain

Cyclooxygenase (COX)-2 is the major constitutively expressed COX isoform in the newborn brain. COX-2 derived prostanoids and reactive oxygen species appear to play a major role in the mechanism of perinatal hypoxic-ischemic injury in the newborn piglet, an accepted animal model of the human term neonate. The study aimed to quantitatively determine COX-2 immunopositive neurons in different brain regions in piglets under normoxic conditions (n=15), and 4 hours after 10 min asphyxia (n=11). Asphyxia did not induce significant changes in neuronal COX-2 expression of any studied brain areas. In contrast, there was a marked regional difference in all experimental groups. Thus, significant difference was observed between fronto-parietal and temporo-occipital regions: 59±4% and 67±3% versus 41±2%* and 31±3%* respectively (mean±SEM, data are pooled from all subjects, n=26, *p<0.05, vs. fronto-parietal region). In the hippocampus, COX-2 immunopositivity was rare (highest expression in CA1 region: 14±2%). The studied subcortical areas showed negligible COX-2 staining. Our findings suggest that asphyxia does not significantly alter the pattern of neuronal COX-2 expression in the early reventilation period. Furthermore, based on the striking differences observed in cortical neuronal COX-2 distribution, the contribution of COX-2 mediated neuronal injury after asphyxia may also show region-specific differences.

Neuronal tumour necrosis factor-α and interleukin-1β expression in a porcine model of intracerebral haemorrhage: Modulation by U-74389G

Tumour necrosis factor α (TNF-α) and interleukin 1β (IL-1β) are important mediators of intracerebral haemorrhage (ICH) inflammatory response. Lazaroids, established antioxidants and neuroprotectants, have been studied in several brain pathologies. The present study was designed to investigate: a) TNF-α and IL-1β changes, in neurons and b) U-74389G effects, 4 and 24h after haematoma induction in a porcine model of intracerebral haemorrhage. In twenty male landrace pigs (swines) aged 135-150 days old, autologous whole blood was injected around the right basal ganglia territory; in ten of the pigs the lazaroid compound U-74389G was administered. Brain TNF-α and IL-1β immunopositive neurons were determined by immunoarray techniques at 4 and 24h timepoints. After the haematoma induction the number of TNF-α immunopositive neurons ipsilateral to the haematoma was significantly higher compared to the contralateral site at 4h (p<0.0005), while U-74389G significantly reduced the number of TNF-α immunopositive neurons, ipsilateral to the haematoma, at 4h (p=0.002); at 24h, TNF-α immunopositive neurons were found significantly lower in the control group ipsilateral to the haematoma in comparison to 4h timepoint(p<0.0005). The number of IL-1β immunopositive neurons at 4h after the hematoma induction was significantly higher ipsilateral to the haematoma site (p<0.0005). U-74389G had no statistical significant effect. TNF-α and IL-1β, increase in neurons, 4h after the haematoma induction, ipsilateral to the haematoma site. The administration of the antioxidant compound U-74389G, results in early (at 4h) decrease of TNF-α immunopositive neurons but shows no statistical significant effect to IL-1β immunopossitive neurons.