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Selective opening of the blood-brain barrier in newborn piglets with experimental pneumothorax
Neuroscience Letters, 93 (1988) 3 8 43 Elsevier Scientific Publishers Ireland Ltd
38
NSL 05617
Selective opening of the blood-brain barrier in newborn piglets with experimental pneumothorax P6ter Temesvfiri and J6zsef Kovfics Department of Pediatrics, Szent-Gy6rgyi Albert University Medical School, Szeged (Hungary) (Received 25 April 1988; Revised version received 20 June 1988; Accepted 24 June 1988)
Key words: Piglet; Pneumothorax; Brain microvessel; Intravital fluorescence microscopy Pial-arachnoidal microvessels (40-210 pm) 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 ( < 80 pm); 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.
Perinatal brain damage occurs frequently in cardiovascularly compromised distressed newborn infants. Both circulatory and metabolic changes seriously affect the brain microvasculature, the blood-brain barrier (BBB), playing a fundamental role in the pathogenesis of secondary oedema and bleeding [10, 16, 17]. We have previously described an animal model of severe cardiovascular collapse in the neonatal period, which resembles shock in human infants in many aspects. In piglets with bilateral experimental pneumothorax (BEP) we found that in the course of severe cardiovascular (hypertension, hypotension, bradycardia) and metabolic (acidosis, hypoxaemia, hypercapnia) shock, at the critical phase of the disease, a cytotoxic type of brain oedema developed accompanied by water and sodium accumulation in the surrounding tissues [18, 19]. To reveal the source of the secondary brain damage we have further studied BBB intravitally. Open cranial window technique was used to visualize the piglets' pialarachnoidal microvessels by fluorescein dyes of different molecular weight. These Correspondence: P. Temesv~ri, Department of Pediatrics, Szent-Gy6rgyi Albert University, Medical School, P.O. Box 471, H-6701 Szeged, Hungary. 0304-3940/88/$ 03.50 O 1988 Elsevier Scientific Publishers Ireland Ltd.
39 microvessels correspond in many respects (permeability and vasoreactivity) to the intraparenchymal ones [10 17], and recent evidences indicate that the microvasculature of piglets' brain is an excellent object for examining microvascular changes during the newborn period [4, 5, 8, 10, 18, 19]. Moreover, bilateral pneumothorax remains the commonest clinical complication of neonatal respiratory distress, leading even to death in spite of the most modern therapeutic interventions [14]. In the present study 14 piglets of either sex aged between 3 and 8 h at the time of the experiments, weighing 1.0-l.4 kg were included. Under general (ketaminehydrochloride, Ketanest, Parke-Davis, 10 mg/kg b.wt. ; i.m.) and local (lidocainehydrochloride, EGYT, Budapest, 1.0 ml, 1.0 vol. %; s.c.) anaesthesia the animals were tracheotomized (Portex 2.5-3.0 tubes), paralyzed (pipecuronium bromatum, Arduan, Richter Gedeon, Budapest, 0.2 mg/kg b.wt./h; i.v.) and the anaesthesia was completed with ~-chloralose (Sigma, 30 mg/kg b.wt. as a bolus, thereafter 10 mg/kg b.wt./h; i.v.). Artificial ventilation was applied (Kutesz Infant vent., Hungary): tidal volumes 8 14 ml (room air), frequency 30-50/min to reach physiological arterial blood gases and cardiovascular parameters (mean blood pressure, heart rate). An open cranial window was performed above the right parietal cortex by the technique of Levasseur et al. [12], with the modification according to Wahl et al. [20]. The fluid reservoir of the window was filled under paraffin oil with artificial cerebrospinal fluid corresponding to the neonatal pig [15]. The animals were divided into 3 experimental groups. Group 1 piglets." 4 animals without BEP. The pial-arachnoidal microvessels were intermittently observed through 1 h with a Wild Fluorescence Photomacroscope (M 400, Heerbrugg, Switzerland) after giving 2.0 ml/kg b.wt. 1.0 vol. % Na+-fluorescein (Aldrich Chemicals, Gillingham, mol.wt. 376) into the umbilical vein at the beginning and every 15 min thereafter. Filtered light for fluorescence excitation was provided by a 50-W mercury lamp and by an I2 filter (Leitz, Wetzlar, F.R.G.). Parallel with the fluorescence boluses microphotographs of the cortex were taken by a Wild MPS 55 photoautomat. At the given time intervals blood gas and acid base analyses were done. Group 2 piglets: 6 animals with BEP through the indwelling intrapleural drains. The intrapleural pressure applied was 0.1~).7 kPa in an increasing manner for inducing the critical phase of the experimental disease [18, 19]. The parameters of the artificial respiration remained the same as before the induction of BEP. As in group 1~ the animals were monitored, and Na +-fluorescein boluses were used at the same time intervals accompanied with the photodocumentation of the microvessels. At the critical phase of the disease (severe arterial hypotension following hypertension, bradycardia, hypoxaemia, hypercapnia, acidosis) an additional bolus was applied with photodocumentation. Group 3 piglets: these 4 piglets had BEP, too. The details of the experimental procedure were the same as in group 2, except for the fluorescein dye applied. At the given time scale, FITC-dextran (1.0 ml/kg b.wt., 5.0 vol. %, mol.wt. 40,000 in 2 cases, and 70,000 in the other two cases, Pharmacia, Uppsala, Sweden) was used as microvascular indicator. The photodocumentation was the same as in groups 1 and 2. After the
4(]
TABLE I LABORATORY DATA OF NEWBORN PIGLETS Values are mean+-S.D. BEP, bilateral experimental pneumothorax; BEP-~zritical phase interval, time elapsed from the induction of BEP till the blood~rain barrier (BBB) opening. Group I: animals without BEP; Group 2: animals with BEP Na ~-fluorescein given as BBB indicator; Group 3: animals with BEP FITC-dextran and Na ~-fluorescein given as BBB indicators; n = number of animals. Animal group 1 (n=4)
BEP-critical phase interval (min) Arterial blood gases and acid-base status Before BEP pH [HCOJ(mM) pCO2(kPa) pO2(kPa) At the critical phase pH [HCOJ(mM) pCO2(kPa) pO2(kPa) Mean arterial blood pressure (mmHg) BeforeBEP Maximal At the critical phase
7.45+ 22.4_+ 3.9+7.7+-
0.1 4.5 0.6 0.7
60:0+- 3.8 -
2 (n=6)
3(n=4)
55.5 + 10.5
53.8 + 8.5
7.39_+ 0.1 22.3+- 3.7 4.8+- 1.3 7.6+- 0.6
7.44+ 0.1 23.1+- 2.9 4.2_+ 0.9 7.5+- 0.8
6.94+13.8+ 14.8+2.4+-
7.0__+ 0.1 14.2+- 2.2 14.1_+ 3.0 2.7+- 0.6
0.1 2.9 4.6 0.7
63.0+- 6.0 95.3-+ 9.8 21.0+ 6.0
58.5+- 6.8 97.5+-11.3 19.5+- 5.3
last bolus o f F I T C - d e x t r a n , an additional dose (2.0 ml/kg b.wt., 1.0 vot. %) o f N a ÷fluorescein was given with p h o t o d o c u m e n t a t i o n at the critical phase. In group 1 the cardiovascular, the blood gas parameters and the acid-base status remained within the physiological range (Table I, g r o u p 1). There were no significant changes in the vascular reactivity (resting diameter 40-210/am) and permeability. The fluorescein dye remained inside the vessels in each animal. In group 2 the pial microvessels a p p e a r e d to be n o r m a l before the induction o f BEP (Fig. 1A). Significant vasodilation b o t h o f the venal and arterial vessels was observed during the developmental period o f the disease, without permeability changes for the N a +-fluorescein. At the critical phase (Table I, g r o u p 2) a significant vasoconstriction was found on the observed vessels o f both types, with a progressive sludging and m i c r o t h r o m b i f o r m a t i o n in the venules, a c c o m p a n i e d by fluorescein extravasation from the smallest venules (resting diameter < 80/Lm), beginning with intraparenchymal fluorescent spots (Fig. 1B), progressing to diffuse leakage (Fig. 1C). In group 3 during BEP t h e l a b o r a t o r y parameters (Table I, g r o u p 3) and the vasom o t o r alterations were almost the same as in g r o u p 2. We could observe no F I T C dextran extravasation either during the course, or at the critical phase o f the disease
41
Fig. 1. Fluorescence microphotographs of the cerebral parietal pial vessels (one field) of a newborn piglet with bilateral experimental p n e u m o t h o r a x (blood brain barrier marker Na+-fluorescein). A: before the induction of pneumothorax. The indicator remained confined to the intravascular space. B: fluorescein extravasation starts as a fluorescent spot into the brain parenchyma around a small venula (arrow) critical phase. C: fluorescein extravasation aggregates as diffuse fluorescence in the brain tissue (asterisk) accompanied with vasoconstriction and microthrombi formation critical phase. Bar = 1000 ibm
in spite of the obvious sludging and microthrombi formation, but in each animal Na+-fluorescein quickly passed the walls of the smallest venules at the end of the experiments. For the first time in the literature we demonstrate here that at the severe phase of BEP brain microvessels lose their ability to retain sodium ftuorescein intravasally. This phenomenon is accompanied by visible microcirculatory disturbances, like sludging and microthrombi formation. However, at the same period of the disease BBB permeability remained undisturbed for larger molecules. Thus, the opening phenomenon seemed to be selective in our experimental model and may mainly be of cardiovascular origin. The increasing intrapleural pressure could seriously compromise venous outflow from the CNS. So far this factor is most likely being underestimated in the development of brain lesions in the neonatal period [13]. There are evidences that the hypertension we observed before the critical phase of the disease could damage the brain microvasculature too [4, 17]. Moreover, all BEP animals had mean arterial blood pressure values outside the autoregulatory range (35-90 mmHg) published for piglets [4, 5]. The deleterious effects of artificial ventilation on the BBB
42
-
[15] could be neglected in our model, because we did not notice any significant microcirculatory changes in animals without BEP. The metabolic changes (acidosis, hypoxaemia, hypercapnia) accompanying the severe hypotensive cardiovascular collapse also affect the BBB. In this respect the microvascular neurohumoral imbalance and the reaction of the damaged endothelial cells - like enlargement or shrinkage all leading to enhanced leakage, should be mentioned. The absence of the dextran penetration is not surprising, because we did not find any in vitro extravasation for large molecules at this stage of the disease [18]. One can assume that other molecules of similar physico-chemical properties as sodium could have passed the BBB in our model, taking part in the oedema formation in the CNS by a compressive effect through the glial elements on the microvasculature [11]. The observed microthrombi formation could be due to the release of catecholamines during the piglets' asphyxia [8]. In this respect the artificial light effects [6] and some properties of the fluorescent dyes [20] may be possible sources of alterations. These could be neglected in view of the control data. Otherwise, products with fluorescent characteristics could be generated during the lipid peroxidation process [9], especially in the brain of piglets with respiratory failure and bradycardia [7]. Our present in vivo data explaining the brain oedema formation processes give experimental evidence of the early (seizures, apnea, hypo- and hyperthermia) and the late (parietal necroses and atrophia, cerebral palsy, epilepsia, mental retardation) clinical complications occurring frequently after severe cardiovascular and metabolic shocks in the neonatal period. The most obvious accompanying morphological disturbances are also seen at the level of the smallest veins at necropsy [16]. Since agents, like furosemide [2] blocking the transport of sodium across the BBB are available, further studies are necessary to try to minimalise the enhanced leaking of the brain microvessels. Furthermore, patients with clinical picture resembling our model must be selected and large doses of sodium bicarbonate, which could aggrevate the obvious microcirculatory disturbances (salt loading, CO2 production, left shifted 02 dissociation curve) should be avoided [1]. In the clinical practice similar cases can more effectively be treated with peritoneal dialysis [3]. Supported by the Alexander van Humboldt Foundation, Bonn, F.R.G.
1 Bersin, R.M. and Arieff, A.I., Improved hemodynamic function during hypoxia with carbicarb, a new agent for the management of acidosis, Circulation, 77 (1988) 227-233. 2 Betz, A.L., Sodium transport from blood to brain: inhibition by furosemide and amiloride, J. Neurochem., 41 (1983) 1158-1164. 3 Boda, D., Mur~nyi, L., Altorjay, I. and Veress, I., Peritoneal dialysis in the treatment of hyaline membrane disease of newborn premature infants. Results of a controlled thai, Acta Paediatr. Scand., 60 (1971) 90-92. 4 Brubakk, A.M. Bratild, D., Oh, W., Yao, A.C. and Stonestreet, B.S., Atropine prevents increases in brain blood flow during hypertension in newborn piglets, Pediatr. Res., 18 (1984) 1121-1126. 5 Busija, D.W. and Leffler, C.W., Hypothermia reduces cerebral metabolic rate and cerebral blood flow in newborn pigs, Am. J. Physiol., 253 (Heart Circ. Physiol., 22) (1987) H86%H873.
43 6 Dietrich, W.D., Busto, R., Watson, B.D., Scheinberg, P. and Ginsberg, M.D., Photochemically induced cerebral infarction. II. Edema and blood brain barrier disruption, Acta Neuropathol. 72 (1987) 326,334. 7 Goplerud, J.M., Mishra, O.P., Wagerle, L.C. and Delivoria-Papadopoulos, M., Brain cell membrane dysfunction following single and repeated apnea in newborn piglets, Pediatr. Res., 21 (1987) 362A. 8 Gree, R.S., Leffler, C.W., Busija, D.W., Fletcher, A.M. and Beasley, D.G., lndomethacin does not alter the circulating catecholamine response to asphyxia in the neonatal piglet, Pediatr. Res., 21 (1987) 534 537. 9 lio, T. and Yoden, K., Fluorescence formation and heine degradation at different stages of lipid peroxidation, Life Sci., 40 (1987) 2297 2302 10 Jo6, F., A unifying concept on the pathogenesis of brain oedemas, Neuropathol. Appl. Neurobiol., 13 (1987) 161 176. 11 Kempski, O., Chaussy, L., Gross, U., Zimmer, M. and Baethmann, A., Volume regulation and metabolism of suspended C6 glioma cells: an in vitro model to study cytotoxic brain edema, Brain Res.. 279 (1983) 217 288. 12 Levasseur, J.E., Wei, E.P., Raper, A.J., Kontos, H.A. and Patterson, J.L., Detailed description of a cranial window technique for acute and chronic experiments, Stroke, 6 (1975) 308 317. 13 Mayhan, W.G., Faraci, F.M. and Heistad, D.D., Disruption of the blood brain barrier in cerebrum and brain stem during acute hypertension, Am. J. Physiol., 251 (Heart Circ. Physiol., 20) (1986) HlI71 H1175. 14 McCord, F.B., Cursted, T., Halliday, H.L., McClure, G., Reid, M.M. and Robertson, B., Surfactant treatment and incidence of intraventricular haemorrhage in severe respiratory distress syndrome, Arch. Dis. Child., 63 (1988) I0 16. 15 Mirro, R., Armstead, W., Busija, D.W., Green, R. and Leffler, C.W., Increasing ventilation pressurc increases cortical subarachnoid cerebrospinal fluid prostanoids in newborn pigs, Pediatr. Res., 22 (1987) 647 650. 16 Miyata, H. and Itoh, H., CNS changes in the meconium aspiration syndrome, Kobe J. Med. Sci., 32 (1986) 179 195. 17 Rapoport, S.I., Blood Brain Barrier in Physiology and Medicine, Raven, New York, 1076. 18 Temesv~ri, P., Hencz, P., Jo6, F., Eck, E., Szerdahelyi, P. and Boda, D., Modulation of the blood brain barrier permeability in neonatal cytotoxic brain edema: laboratory and morphological findings obtained on newborn piglets with experimental pneumothorax, Biol. Neonat., 46 (1984) 198 208. 19 Temesvfiri, P., Jo6, F., Koltai, M., Eck, E., ,/~d~m, G., Sikl6s, L. and Boda, D., Cerebroprotectivc effect of dexamethasone by increasing the tolerance to hypoxia and preventing brain oedema in newborn piglets with experimental pneumothorax, Neurosci. Lett., 49 (1984) 87 92. 20 Wahl, M., Unterberg, A. and Baethmann, A., Intravital fluoresence microscopy for the study of blood brain barrier function. Int. J. Microcirc. Clin. Exp., 4 (1985) 3 18.
38
NSL 05617
Selective opening of the blood-brain barrier in newborn piglets with experimental pneumothorax P6ter Temesvfiri and J6zsef Kovfics Department of Pediatrics, Szent-Gy6rgyi Albert University Medical School, Szeged (Hungary) (Received 25 April 1988; Revised version received 20 June 1988; Accepted 24 June 1988)
Key words: Piglet; Pneumothorax; Brain microvessel; Intravital fluorescence microscopy Pial-arachnoidal microvessels (40-210 pm) 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 ( < 80 pm); 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.
Perinatal brain damage occurs frequently in cardiovascularly compromised distressed newborn infants. Both circulatory and metabolic changes seriously affect the brain microvasculature, the blood-brain barrier (BBB), playing a fundamental role in the pathogenesis of secondary oedema and bleeding [10, 16, 17]. We have previously described an animal model of severe cardiovascular collapse in the neonatal period, which resembles shock in human infants in many aspects. In piglets with bilateral experimental pneumothorax (BEP) we found that in the course of severe cardiovascular (hypertension, hypotension, bradycardia) and metabolic (acidosis, hypoxaemia, hypercapnia) shock, at the critical phase of the disease, a cytotoxic type of brain oedema developed accompanied by water and sodium accumulation in the surrounding tissues [18, 19]. To reveal the source of the secondary brain damage we have further studied BBB intravitally. Open cranial window technique was used to visualize the piglets' pialarachnoidal microvessels by fluorescein dyes of different molecular weight. These Correspondence: P. Temesv~ri, Department of Pediatrics, Szent-Gy6rgyi Albert University, Medical School, P.O. Box 471, H-6701 Szeged, Hungary. 0304-3940/88/$ 03.50 O 1988 Elsevier Scientific Publishers Ireland Ltd.
39 microvessels correspond in many respects (permeability and vasoreactivity) to the intraparenchymal ones [10 17], and recent evidences indicate that the microvasculature of piglets' brain is an excellent object for examining microvascular changes during the newborn period [4, 5, 8, 10, 18, 19]. Moreover, bilateral pneumothorax remains the commonest clinical complication of neonatal respiratory distress, leading even to death in spite of the most modern therapeutic interventions [14]. In the present study 14 piglets of either sex aged between 3 and 8 h at the time of the experiments, weighing 1.0-l.4 kg were included. Under general (ketaminehydrochloride, Ketanest, Parke-Davis, 10 mg/kg b.wt. ; i.m.) and local (lidocainehydrochloride, EGYT, Budapest, 1.0 ml, 1.0 vol. %; s.c.) anaesthesia the animals were tracheotomized (Portex 2.5-3.0 tubes), paralyzed (pipecuronium bromatum, Arduan, Richter Gedeon, Budapest, 0.2 mg/kg b.wt./h; i.v.) and the anaesthesia was completed with ~-chloralose (Sigma, 30 mg/kg b.wt. as a bolus, thereafter 10 mg/kg b.wt./h; i.v.). Artificial ventilation was applied (Kutesz Infant vent., Hungary): tidal volumes 8 14 ml (room air), frequency 30-50/min to reach physiological arterial blood gases and cardiovascular parameters (mean blood pressure, heart rate). An open cranial window was performed above the right parietal cortex by the technique of Levasseur et al. [12], with the modification according to Wahl et al. [20]. The fluid reservoir of the window was filled under paraffin oil with artificial cerebrospinal fluid corresponding to the neonatal pig [15]. The animals were divided into 3 experimental groups. Group 1 piglets." 4 animals without BEP. The pial-arachnoidal microvessels were intermittently observed through 1 h with a Wild Fluorescence Photomacroscope (M 400, Heerbrugg, Switzerland) after giving 2.0 ml/kg b.wt. 1.0 vol. % Na+-fluorescein (Aldrich Chemicals, Gillingham, mol.wt. 376) into the umbilical vein at the beginning and every 15 min thereafter. Filtered light for fluorescence excitation was provided by a 50-W mercury lamp and by an I2 filter (Leitz, Wetzlar, F.R.G.). Parallel with the fluorescence boluses microphotographs of the cortex were taken by a Wild MPS 55 photoautomat. At the given time intervals blood gas and acid base analyses were done. Group 2 piglets: 6 animals with BEP through the indwelling intrapleural drains. The intrapleural pressure applied was 0.1~).7 kPa in an increasing manner for inducing the critical phase of the experimental disease [18, 19]. The parameters of the artificial respiration remained the same as before the induction of BEP. As in group 1~ the animals were monitored, and Na +-fluorescein boluses were used at the same time intervals accompanied with the photodocumentation of the microvessels. At the critical phase of the disease (severe arterial hypotension following hypertension, bradycardia, hypoxaemia, hypercapnia, acidosis) an additional bolus was applied with photodocumentation. Group 3 piglets: these 4 piglets had BEP, too. The details of the experimental procedure were the same as in group 2, except for the fluorescein dye applied. At the given time scale, FITC-dextran (1.0 ml/kg b.wt., 5.0 vol. %, mol.wt. 40,000 in 2 cases, and 70,000 in the other two cases, Pharmacia, Uppsala, Sweden) was used as microvascular indicator. The photodocumentation was the same as in groups 1 and 2. After the
4(]
TABLE I LABORATORY DATA OF NEWBORN PIGLETS Values are mean+-S.D. BEP, bilateral experimental pneumothorax; BEP-~zritical phase interval, time elapsed from the induction of BEP till the blood~rain barrier (BBB) opening. Group I: animals without BEP; Group 2: animals with BEP Na ~-fluorescein given as BBB indicator; Group 3: animals with BEP FITC-dextran and Na ~-fluorescein given as BBB indicators; n = number of animals. Animal group 1 (n=4)
BEP-critical phase interval (min) Arterial blood gases and acid-base status Before BEP pH [HCOJ(mM) pCO2(kPa) pO2(kPa) At the critical phase pH [HCOJ(mM) pCO2(kPa) pO2(kPa) Mean arterial blood pressure (mmHg) BeforeBEP Maximal At the critical phase
7.45+ 22.4_+ 3.9+7.7+-
0.1 4.5 0.6 0.7
60:0+- 3.8 -
2 (n=6)
3(n=4)
55.5 + 10.5
53.8 + 8.5
7.39_+ 0.1 22.3+- 3.7 4.8+- 1.3 7.6+- 0.6
7.44+ 0.1 23.1+- 2.9 4.2_+ 0.9 7.5+- 0.8
6.94+13.8+ 14.8+2.4+-
7.0__+ 0.1 14.2+- 2.2 14.1_+ 3.0 2.7+- 0.6
0.1 2.9 4.6 0.7
63.0+- 6.0 95.3-+ 9.8 21.0+ 6.0
58.5+- 6.8 97.5+-11.3 19.5+- 5.3
last bolus o f F I T C - d e x t r a n , an additional dose (2.0 ml/kg b.wt., 1.0 vot. %) o f N a ÷fluorescein was given with p h o t o d o c u m e n t a t i o n at the critical phase. In group 1 the cardiovascular, the blood gas parameters and the acid-base status remained within the physiological range (Table I, g r o u p 1). There were no significant changes in the vascular reactivity (resting diameter 40-210/am) and permeability. The fluorescein dye remained inside the vessels in each animal. In group 2 the pial microvessels a p p e a r e d to be n o r m a l before the induction o f BEP (Fig. 1A). Significant vasodilation b o t h o f the venal and arterial vessels was observed during the developmental period o f the disease, without permeability changes for the N a +-fluorescein. At the critical phase (Table I, g r o u p 2) a significant vasoconstriction was found on the observed vessels o f both types, with a progressive sludging and m i c r o t h r o m b i f o r m a t i o n in the venules, a c c o m p a n i e d by fluorescein extravasation from the smallest venules (resting diameter < 80/Lm), beginning with intraparenchymal fluorescent spots (Fig. 1B), progressing to diffuse leakage (Fig. 1C). In group 3 during BEP t h e l a b o r a t o r y parameters (Table I, g r o u p 3) and the vasom o t o r alterations were almost the same as in g r o u p 2. We could observe no F I T C dextran extravasation either during the course, or at the critical phase o f the disease
41
Fig. 1. Fluorescence microphotographs of the cerebral parietal pial vessels (one field) of a newborn piglet with bilateral experimental p n e u m o t h o r a x (blood brain barrier marker Na+-fluorescein). A: before the induction of pneumothorax. The indicator remained confined to the intravascular space. B: fluorescein extravasation starts as a fluorescent spot into the brain parenchyma around a small venula (arrow) critical phase. C: fluorescein extravasation aggregates as diffuse fluorescence in the brain tissue (asterisk) accompanied with vasoconstriction and microthrombi formation critical phase. Bar = 1000 ibm
in spite of the obvious sludging and microthrombi formation, but in each animal Na+-fluorescein quickly passed the walls of the smallest venules at the end of the experiments. For the first time in the literature we demonstrate here that at the severe phase of BEP brain microvessels lose their ability to retain sodium ftuorescein intravasally. This phenomenon is accompanied by visible microcirculatory disturbances, like sludging and microthrombi formation. However, at the same period of the disease BBB permeability remained undisturbed for larger molecules. Thus, the opening phenomenon seemed to be selective in our experimental model and may mainly be of cardiovascular origin. The increasing intrapleural pressure could seriously compromise venous outflow from the CNS. So far this factor is most likely being underestimated in the development of brain lesions in the neonatal period [13]. There are evidences that the hypertension we observed before the critical phase of the disease could damage the brain microvasculature too [4, 17]. Moreover, all BEP animals had mean arterial blood pressure values outside the autoregulatory range (35-90 mmHg) published for piglets [4, 5]. The deleterious effects of artificial ventilation on the BBB
42
-
[15] could be neglected in our model, because we did not notice any significant microcirculatory changes in animals without BEP. The metabolic changes (acidosis, hypoxaemia, hypercapnia) accompanying the severe hypotensive cardiovascular collapse also affect the BBB. In this respect the microvascular neurohumoral imbalance and the reaction of the damaged endothelial cells - like enlargement or shrinkage all leading to enhanced leakage, should be mentioned. The absence of the dextran penetration is not surprising, because we did not find any in vitro extravasation for large molecules at this stage of the disease [18]. One can assume that other molecules of similar physico-chemical properties as sodium could have passed the BBB in our model, taking part in the oedema formation in the CNS by a compressive effect through the glial elements on the microvasculature [11]. The observed microthrombi formation could be due to the release of catecholamines during the piglets' asphyxia [8]. In this respect the artificial light effects [6] and some properties of the fluorescent dyes [20] may be possible sources of alterations. These could be neglected in view of the control data. Otherwise, products with fluorescent characteristics could be generated during the lipid peroxidation process [9], especially in the brain of piglets with respiratory failure and bradycardia [7]. Our present in vivo data explaining the brain oedema formation processes give experimental evidence of the early (seizures, apnea, hypo- and hyperthermia) and the late (parietal necroses and atrophia, cerebral palsy, epilepsia, mental retardation) clinical complications occurring frequently after severe cardiovascular and metabolic shocks in the neonatal period. The most obvious accompanying morphological disturbances are also seen at the level of the smallest veins at necropsy [16]. Since agents, like furosemide [2] blocking the transport of sodium across the BBB are available, further studies are necessary to try to minimalise the enhanced leaking of the brain microvessels. Furthermore, patients with clinical picture resembling our model must be selected and large doses of sodium bicarbonate, which could aggrevate the obvious microcirculatory disturbances (salt loading, CO2 production, left shifted 02 dissociation curve) should be avoided [1]. In the clinical practice similar cases can more effectively be treated with peritoneal dialysis [3]. Supported by the Alexander van Humboldt Foundation, Bonn, F.R.G.
1 Bersin, R.M. and Arieff, A.I., Improved hemodynamic function during hypoxia with carbicarb, a new agent for the management of acidosis, Circulation, 77 (1988) 227-233. 2 Betz, A.L., Sodium transport from blood to brain: inhibition by furosemide and amiloride, J. Neurochem., 41 (1983) 1158-1164. 3 Boda, D., Mur~nyi, L., Altorjay, I. and Veress, I., Peritoneal dialysis in the treatment of hyaline membrane disease of newborn premature infants. Results of a controlled thai, Acta Paediatr. Scand., 60 (1971) 90-92. 4 Brubakk, A.M. Bratild, D., Oh, W., Yao, A.C. and Stonestreet, B.S., Atropine prevents increases in brain blood flow during hypertension in newborn piglets, Pediatr. Res., 18 (1984) 1121-1126. 5 Busija, D.W. and Leffler, C.W., Hypothermia reduces cerebral metabolic rate and cerebral blood flow in newborn pigs, Am. J. Physiol., 253 (Heart Circ. Physiol., 22) (1987) H86%H873.
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