Péter Temesvári

Recombinant human tumor necrosis factor α constricts pial arterioles and increases blood-brain barrier permeability in newborn piglets

Tumor necrosis factor alpha (TNF alpha) plays a significant role in the pathogenesis of central nervous system infections. We investigated the effect of intracisternal injection of recombinant human TNF alpha (50-50,000 IU) on pial vasoreactivity and blood-brain barrier permeability in newborn piglets. The cytokine administration resulted in arterial vasoconstrictions, blood-brain barrier opening for Na-fluorescein (mol. wt. 376 Da) and increased Na-fluorescein uptake in brain regions examined (parietal and occipital cortex, cerebellum, pons/medulla, periventricular white matter) in a dose-dependent manner. TNF alpha may be involved in the pathophysiology of neonatal brain injuries.

Regulation of the macromolecular transport in the brain microvessels: The role of cyclic GMP

The possible effects of dibutyryl cyclic GMP (db-cGMP), the lipid-soluble derivative of cyclic GMP, on brain microvessels were studied by light microscopic detection of albumin and quantitative electron microscopy. Different concentrations (25, 50, 100 and 200 micrograms) of db-cGMP were given to adult rats by intracarotid infusion, while in the controls, the animals were infused either with Krebs-Ringer solution or with the same solution containing butyrate or cyclic GMP. In contrast to the controls, db-cGMP was found to be able to increase the permeability of brain microvessels to albumin in a dose-dependent manner. At the same time, the number of transport vesicles being indicative of the pinocytotic activity of the endothelial cells was also increased. These results indicate that, similarly to the cyclic AMP whose effects have been revealed earlier, the cyclic GMP-system may also be involved in the regulation of opening mechanisms of the blood-brain barrier.

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.

Modulation of the Blood-Brain Barrier Permeability in Neonatal Cytotoxic Brain Edema: Laboratory and Morphological Findings Obtained on Newborn Piglets with Experimental Pneumothorax

Acute, bilateral pneumothorax (PT) was produced in 14 newborn piglets. The clinical status of the operated and 14 control animals was monitored by measuring the arterial blood gases, acid-base balance and mean arterial blood pressure. Different brain regions were processed for electron microscopy and albumin immunohistochemistry; water and electrolyte contents were also determined at the end stage of experimental intervention. Electron microscopy showed more intense pinocytotic activity in the endothelium of brain capillaries from PT animals evaluated by morphometry. Statistically significant (p less than 0.01) differences were found in the distribution of pinocytotic vesicles in different brain areas of PT animals. The blood-brain barrier seemed to be impermeable to albumin in all brain regions both in the controls and in the PT group. Parallel with the changes observed in pinocytosis, the water and sodium contents were also increased in the PT group in the parietal cortex (water content 85.18 +/- SD 0.81% vs. 84.10 +/- SD 0.52%, p less than 0.01; sodium content in wet brain tissue 70.94 +/- SD 8.44 mmol/kg vs. 65.09 +/- SD 4.43 mmol/kg, p less than 0.05, in dry brain tissue 481.70 +/- 75.70 mmol/kg vs. 410.15 +/- SD 35.45 mmol/kg, p less than 0.05) and in the cerebellum (water content 83.95 +/- SD 1.08% vs. 83.02 +/- SD 0.89%, p less than 0.05; sodium content in wet brain tissue 60.67 +/- SD 3.16 mmol/kg vs. 55.90 +/- 6.26 mmol/kg, p less than 0.01). However, in other brain regions--especially in the water-shed area--there was no correlation between the changes of pinocytosis and water-electrolyte contents of the tissues. It is suggested that the type of edema developing in this severe cardiovascular/hypoxic collapse is cytotoxic of origin and this fact should be more seriously taken into account in the treatment of the disease.

Effects of acute hypoxia on the adenylate cyclase and evans blue transport of brain microvessels

Kinetic parameters of the albumin transport were measured during and after an acute hypoxic insult evoked in newborn piglets by experimental bilateral pneumothorax. Adenylate cyclase activity was determined in the cerebral microvessels isolated by ultracentrifugation from different stages of brain damage. A decrease of the adenylate cyclase activity was observed in the cerebral microvessels of animals with acute hypoxic condition. However, the adenylate cyclase activity was found to be increased significantly in the microvessels during recirculation. The activation of adenylate cyclase in the microvessel wall may be of pathogenetic importance in the development of vasogenic brain oedema.

Reventilation with room air or 100% oxygen after asphyxia differentially affects cerebral neuropathology in newborn pigs

Aim: To test if reventilation with room air (RA) or 100% oxygen (O2) after asphyxia would differentially affect neuronal damage in different brain areas of newborn pigs. Methods: Anaesthetized piglets were subjected to 10 min asphyxia (n= 27) or served as time controls (n= 7). Reventilation started with either RA or O2 for 1 h, and was continued with RA for an additional 1 – 3 h. Cortical or cerebellar blood flow was assessed with laser‐Doppler flowmetry (LDF). Haematoxylin/eosin‐stained sections from six brain regions were prepared for blinded neuropathological examination and scoring. Results: Asphyxia resulted in significant neuronal damage compared to time controls in all areas examined except the pons. O2 ventilation elicited greater neuronal lesions in the hippocampus and the cerebellum but smaller damage in the basal ganglia compared to RA. The assessed physiological parameters including the LDF signals were similar in both ventilation groups, except for PaO2 in the first hour of reventilation (RA 75±5 mmHg, O2 348±57 mmHg; p <0.05). Interestingly, however, reactive hyperaemia was much higher in the O2‐sensitive cerebellum as compared with the cortex (1101±227 vs 571±73; p <0.05, area under the curve).

The role of cerebral microvessels in the elimination of histamine released during postasphyxial reperfusion in newborn piglets

Histamine, released from intracerebral sources during hypoxic-ischemic conditions, may take part in the pathogenesis of neonatal brain injuries. In order to elucidate the possible role of cerebral microvessels in the elimination of histamine from the extracellular space, we determined the concentration of histamine using a modified radioenzymatic method in plasma taken from the internal jugular vein, in cerebrospinal fluid, and in capillary-rich fraction of cerebral microvessels prepared from cortex in 12 sham-operated piglets. Then, bilateral pneumothorax was induced in 20 piglets, samples were taken from the same compartments as from the controls before and during asphyxia, as well as 15 and 180 min thereafter, respectively. Plasma histamine level was significantly (P < 0.05) elevated in animals during hypoxic cardiovascular and metabolic failure (13.5 +/- 1.9 nM l-1) compared to value measured in the control group (2.2 +/- 0.5 nM l-1), preceding any detectable change of histamine concentration in cerebrospinal fluid (5.2 +/- 1.9 versus 3.8 +/- 1.1 nM l-1, respectively) or in cerebral microvessels (8.4 +/- 0.8 versus 7.1 +/- 0.6 pM (mg protein)-1). After resuscitation, histamine levels in plasma samples remained high during the early (15 min, 16.2 +/- 4.3 nM x l-1) and late (180 min, 15.3 +/- 2.9 nM l-1) reperfusion period. By contrast, histamine concentration was increased considerably (P < 0.05) in cerebrospinal fluid samples obtained 15 min (12.8 +/- 6.5 nM l-1), but not 180 min (5.2 +/- 1.9 nM l-1) after resuscitation.(ABSTRACT TRUNCATED AT 250 WORDS)

Selective opening of the blood-brain barrier in newborn piglets with experimental pneumothorax

Pial-arachnoidal microvessels (40-210 micron) were studied by fluorescent microscopy in anaesthetized, immobilized and ventilated newborn piglets in the course of bilateral experimental pneumothorax (BEP; n = 10) using the open cranial window technique. Na+-fluorescein and fluorescein isothiocyanate (FITC)-dextran (mol.wt. 40,000 and 70,000 Da) administered i.v. served as blood-brain barrier (BBB) indicators. After gradual exhaustion of compensatory mechanisms a critical phase, characterized by severe acidosis, bradycardia, arterial hypotension following hypertension and arterial hypoxaemia ensued, with vasoconstriction following vasodilation. Moreover, progressive circulation disturbances, sludging and microthrombi formation occurred in small venules. Concomitantly, diffuse BBB opening for Na+-fluorescein ensued in all piglets with BEP as shown by extended fluorescence in the brain tissue around the small venules (less than 80 micron); never observed for FITC-dextran and in the control animals (n = 4) without BEP. In the acute phase of pneumothorax a selective opening of the BBB should be considered.

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.

Basic Molecular Events Underlying Transendothelial Transport in Brain Capillaries

Endothelial cells which are joined by tight junctions in the brain form a peculiar barrier restricting the movements of macro-molecules and many other solutes between blood and brain. So far. several enzymes including cholinesterases6,7, DOPA decarboxylase2 γ -glutamyl transpeptidase17. Na-K-activated adenosine triphospha-tase4,1, and other ATPases8,12 have been reported not only to be present in brain capillaries, but also to be involved in the regulation of blood-brain barrier permeability. Since the discovery of the importance of cyclic nucleotides in the mediation of the hormonal influences on different cellular functions, we have been trying to elucidate the possible role of cAMP and cGMP in the regulation of permeability of brain microvessels. Up to now, the following data have been collected: dibutyryl (dibu) cAMP increases permeability and pinocytosis in brain capillaries9, adenylate cyclase could be found in brain microvessels histochemically11, histamine receptors — mainly of H2-type — are linked to the capillary adenylate cyclase as detected by biochemical measurements14, the presence of guanylate cyclase was found in brain capillaries by means of histochemistry and biochemical measurements15.