Main

Hypoxic-ischemic cerebral damage is an important contributor to perinatal mortality and morbidity including long-term neurologic sequelae in term and preterm fetuses(1). Over the last decade many therapeutic strategies have been developed to reduce neuronal damage caused by ischemic insults in neonatal and adult animals. These have included application of calcium antagonists, glutamate antagonists, oxygen radical scavengers, and nitric oxide synthase blockers [for review, see Krieglstein et al.(2)]. Perinatal use of neuroprotective strategies may be particularly successful, because pretreatment of individuals at risk of hypoxic-ischemic encephalopathy might be possible. There is evidence that flunarizine, a class IV calcium channel blocker, is more effective as a neuroprotective drug in pretreatment(3–7) than in posttreatment protocols(8). However, the high dose regimen (30-45 mg/kg of body weight) used in those studies had severe cardiovascular side effects(7,9). The present study was therefore designed to test whether ischemic insults in fetal sheep near term could also be reduced by a low dose flunarizine protocol. To account for drug-related changes in cerebral blood flow that might affect neuronal cell damage we measured cerebral blood flow by the microsphere method.

METHODS

The experimental model and measurement of the associated variables have been described in detail previously(10).

Animal preparation. Twelve fetal sheep were chronically prepared at a gestational age of 125 d (term is at 147 d). All ewes were anesthetized by subarachnoid injection of 8 mL of 0.75% (wt/vol) bupivacaine at the lower spine, and were operated under sterile conditions. Polyvinyl catheters were placed in a maternal iliac artery and vein through tibial vessels. The ewe's abdominal wall was opened in the midline, and through a small uterine incision, the fetal hindlimbs were exposed. Using local anesthesia with 1.0% (wt/vol) prilocaine HCI, polyvinyl catheters were inserted via the pedal vein of each hindlimb into the inferior vena cava. The uterine incision was closed and a second uterine incision was made over the fetal snout. Head and neck of the fetus were exteriorized. To prevent the fetus from breathing, its head was covered by a water-filled rubber glove. Catheters were inserted into both fetal brachial arteries. Furthermore, both fetal common carotid arteries were prepared. Cerebral ischemia was induced by occluding the carotid arteries on both sides simultaneously below the thyroid and above the lingual artery for 30 min as described recently(10). Thus, blood flow to the cerebrum via anastomoses between the carotid and vertebral arteries was arrested. After cerebral ischemia a catheter was placed into the amniotic cavity, and the second intrauterine incision was closed. All catheters were filled with heparin(1000 IU/mL), plugged, and passed s.c. to the ewe's flank, where they were exteriorized and protected by a pouch sewn to the skin. On the day of surgery and each day thereafter, the ewe received 2 million units of penicillin G(Grünenthal, Germany) and 80 mg of gentamicin sulfate (Merck, Germany), half i.v. and half into the amniotic cavity.

Experimental protocol. About 30 min before preparation of the carotid arteries, six fetuses received a bolus of flunarizine (1 mg/kg of estimated body weight) i.v., whereas an equal volume of the solvent was administered to the remaining fetuses. The drugs were donated by Janssen Pharmaceutica, Beerse, Belgium. The dosage applied was within the therapeutic range(11). The experiments were done in a randomized fashion. The operators were not blind to the nature of injection. Sixty minutes after flunarizine injection cerebral ischemia was started. To determine the time course of changes in fetal cerebral blood flow before (-15 min), during (3 and 27 min), and after (10 min, 3 h, and 72 h) 30 min of cerebral ischemia, six batches of microspheres labeled with different isotopes (113Sn, 103Ru, 46Sc, 114In,141 Ce, and 95Nb, 16-µm diameter, New England Nuclear) were injected into the inferior vena cava, whereas reference samples from the brachial artery were withdrawn at a rate of 2.5 mL/min for 90 s(12). During and shortly after cerebral ischemia, fetal heart rate and ascending aortic and intrauterine pressure were recorded simultaneously. After having injected the fourth batch of microspheres (at 10 min) all catheters were closed and secured as described above, the abdominal wall was closed, and the ewe was brought back to the metabolic cage, where the fifth injection (at 3 h) was given. For organization reasons there were no pressure measurements at this point in time. Before each injection blood samples were obtained from the brachial artery to measure blood gases, oxygen saturation of Hb, and acid-based balance. At the end of the experiment (at 72 h) the ewe was given a lethal dose of sodium pentobarbitone and saturated potassium chloride i.v., and the fetus was perfused with 300 mL of formalin(15%, wt/vol, saline).

The experimental protocols were approved by the appropriate institutional review committee and met the guidelines of the governmental agency responsible.

Measurements. Ascending aortic and intrauterine pressure and fetal heart rate were recorded on a polygraph (Hellige, Germany). Blood gases and pH were measured in an automatic blood gas analyzer (278 blood gas system, Ciba Corning, Frankfurt, Germany), and base excess was calculated. Hb concentration and oxygen saturation of Hb were measured photometrically (OSM 2 Hemoximeter, Radiometer, Copenhagen, Denmark) in duplicate.

Fetal cerebral blood flow and the extent of neuronal cell damage were assessed in identical brain specimens. To determine fetal cerebral blood flow using the microsphere method, the fetal brain was removed and fixed in formalin for at least 7 d. Afterward the cerebrum was separated from the basal ganglia and divided in four frontal sections (rostral, pericentral, postcentral, occipital) with a thickness of about 1.5 cm. The right and left parts of these four sections were further subdivided into four equally sized segments (sagittal 1 and 2, lateral 1 and 2) each weighing 1-2 g. In addition to these 32 cerebral specimens, the caudate nucleus, thalamus, hippocampus, tegmentum-colliculi-pons, cerebellum, and medulla oblongata were separated. These brain structures were placed into vials, which were filled to the same height to reduce variations in geometry during gamma counting. The solid state semiconductor germanium gamma counter used had a high energy resolution of about 3 keV and was connected to a multichannel (2048) pulse height analyzer (ND 62; Nuclear Data Inc., Schaumburg, IL). The results were normalized with respect to time and sample weight.

After cerebral blood flow analysis the specimens of the fetal brain were removed from the counting vials and embedded in paraffin. Coronal subserial sections of 10 µm were obtained and then stained with cresyl violet/fuchsin. Every 40th section was mounted to evaluate the extent of neuronal cell damage. Neuronal cell damage was assessed at a magnification of 250×. Neurons with ischemic cell damage were identified according to the criteria of Brown and Brierley(13). Neuronal cell damage in each microscopical visual field was quantified as follows: 0-5% damage (score 1), 5-50% damage (score 2), 50-95% damage (score 3), 95-99% damage (score 4), and 100% damage (score 5).

Calculations. Fetal cerebral blood flow was calculated from counts of the injected nuclide recovered in the fetal cerebrum and the appropriate reference sample, and from the withdrawal rate of the reference sample(12). The histologic score of each cerebral specimen was calculated by averaging the scores of all visual fields analyzed from three sections of that specimen. The number of scored visual fields per specimen ranged between 400 and 500 each. The scores from corresponding specimens from the right and left hemisphere were averaged.

Effects of low dose flunarizine on fetal circulatory centralization. Because flunarizine, a class IV calcium antagonist, is a potent vasodilator, the neuroprotective effects of this drug may be counteracted by its cardiovascular side effects. To clarify this point we subjected 10 chronically instrumented sheep fetuses (gestational age, 132± 1 d) to acute intrauterine asphyxia(14). Fetuses from the study group received flunarizine (1 mg/kg of estimated body weight) i.v. 60 min before asphyxia, whereas solvent was administered to the fetuses of the control group. Organ blood flows, physiologic variables, and plasma concentrations of catecholamines were measured before, during, and after arrest of uterine blood flow for 2 min, i.e. at 0,1,2,3,4, and 30 min. Before asphyxia, the distribution of combined ventricular output, physiologic variables and concentrations of catecholamines in fetuses from the control group were within the normal range for chronically prepared fetal sheep near term. During acute asphyxia there was a redistribution of cardiac output toward the central organs (Fig. 1) accompanied by a pronounced bradycardia and a progressive increase in arterial blood pressure. Fetuses of the study group had higher plasma catecholamine levels than the control group. After asphyxia, circulatory centralization did not resolve quite as rapidly as it had developed, but was almost completely recovered at 30 min after the insult. There were hardly any differences between treated animals and controls in the time course of physiologic and cardiovascular variables measured before, during and after acute intrauterine asphyxia (Fig. 1). Low dose flunarizine does not, therefore, seem to affect fetal circulatory responses to acute asphyxia in sheep near term.

Figure 1
figure 1

Blood flow to the cerebrum, heart, and carcass (mL/min × 100 g) in fetal sheep near term before, during, and after arrest of uterine blood flow for 2 min, i.e. at 0, 1, 2, 3, 4, and 30 min. Fetuses from the study group (n = 5) received flunarizine (1 mg/kg of estimated body weight) i.v. 60 min before asphyxia, whereas solvent was administered to the fetuses of the control group(n = 5).

Full size image

Statistics. Values are given as means ± SD. Statistical differences in physiologic variables, cerebral blood flow, and neuronal cell damage within and between groups were evaluated using a two-way ANOVA followed by Games-Howell post hoc test. Owing to the problem of multiple test performing, the resulting p values have to be regarded as descriptive.

RESULTS

The physiologic variables of the control and the study group before, during, and after 30 min of cerebral ischemia are shown in Table 1. There were no significant differences in these variables between groups. At control the arterial blood pH, the plasma concentrations of both glucose and lactate, and the mean arterial blood pressure were slightly above the normal range for chronically prepared fetal sheep(15). However, after ischemia these variables normalized. Furthermore, there were no time-dependent differences in the remaining physiologic variables (Table 1).

Table 1 Physiologic variables before, during, and after 30 min of cerebral ischemia with (F) and without (C) pretreatment with flunarizine (1 mg/kg of estimated body weight)
Full size table

No differences in blood flow to the 32 specimens from the fetal cerebrum could be observed between treated and control groups.Table 2 shows the mean blood flows to these specimens. During ischemia cerebral blood flow was reduced to less than 20 mL·100 g-1 min-1. In the immediate recovery period there was hyperperfusion (at 10 min) followed by a tendency toward hypoperfusion later on (at 3 h). Finally, at 72 h after ischemia, cerebral blood flow was higher than control rates. This time course of changes in cerebral blood flow could be observed in almost all brain structures.

Table 2 Regional cerebral blood flow (ml/100 g tissue/min) before, during and after 30 min of cerebral ischemia with (F) and without (C) pretreatment with flunarizine (1 mg/kg of estimated body weight)
Full size table

In Table 3 the histologic scores for the 16 corresponding areas from both sides of the cerebrum are shown. The most pronounced neuronal cell damage was detected in the parasagittal regions, whereas in the more lateral part of the cortex only minor neuronal damage occurred. Ischemic injury could be observed in 172 out of 192 cerebral specimens of the control group, but in only 69 of 192 study group samples. Damage to deeper brain structures could be detected only in a few areas, mainly in the hippocampus and cerebellum (Table 4). Neuronal cell damage was found more often in controls than in treated animals in the hippocampus (10/12versus 4/12) and cerebellum (6/12versus 1/12). Interestingly, there was a tremendous reduction in neuronal cell damage in almost all parts of the cerebrum after pretreatment with flunarizine (Table 3).

Table 3 Neuronal cell damage in the cerebrum after 30 min of ischemia with (F) and without (C) pretreatment with flunarizine (1 mg/kg of estimated body weight)
Full size table
Table 4 Neuronal cell damage in various areas of the diencephalon, mesencephalon, and brain stem after 30 min of ischemia with (F) and without (C) pretreatment with flunarizine (1 mg/kg of estimated body weight)
Full size table

DISCUSSION

As shown in previous studies pretreatment of immature animals with flunarizine seems to reduce ischemic neuronal cell damage dramatically(3–7). In some of these studies neonatal rats were subjected to hypoxia after ligation of one carotid artery(3–6). Flunarizine was applied at a relatively high concentration (30 mg/kg of body weight) before hypoxia. Ipsilateral infarction of the brain could be almost totally prevented in animals receiving such treatment(3–6). Similar results have been described by Gunn et al.(7) using a fetal sheep model. However, the high dose regimens used by these authors had severe cardiovascular side effects(7,9). In the present study we were able to show that pretreatment of fetal sheep near term with low dose flunarizine significantly reduces cerebral damage after ischemia. Because we have also provided evidence that low dose flunarizine does not alter the response of the fetal cardiovascular system to intrauterine asphyxia, this therapeutic regimen appears to be safe and may qualify for clinical trials. Because the transport of flunarizine across the placental membrane barrier is very fast due to its lipophilic properties(11), direct application to the fetus by chordocentesis would not be necessary. Thus, women carrying babies at risk of hypoxic-ischemic encephalopathy, i.e. intrauterine growth retarded or preterm fetuses, could be given low dose flunarizine i.v. before birth.

The observed neuroprotective effect of flunarizine in fetal sheep appears to be at variance with some studies in adult animals(16–19). These discrepancies may arise from differences in experimental design. The anticonvulsive properties of flunarizine should also be taken into consideration here. Unlike adult studies, the fetuses in intrauterine experiments are subjected not only to the ischemic insult itself, but also to postischemic seizure activity(20,21). Flunarizine is in part an anticonvulsant(11). Therefore, in studies in which seizure activity is part of the experiment, the neuroprotective effects may be more pronounced. This may explain the overt differences between experiments in adult and fetal animals.

An interesting finding of this study is that flunarizine increases plasma catecholamine levels during acute asphyxia. Because elevated catecholamine levels seem to reduce ischemic cerebral injury(22), this observation may reveal one of the mechanisms by which flunarizine protects the fetal brain from neuronal cell damage.

In the present study there was little or no damage to deeper brain structures. This may be related to minor degrees of the ischemic insult; neuroprotection could not be demonstrated, for example, in diencephalic and brain stem areas. The fact that deeper brain structures were not damaged by the ischemic model used here may constitute an important difference to the clinical situation, because basal ganglia seem to be selectively vulnerable in hypoxic-ischemic encephalopathy(1). However, to mimic a clinical situation asphyxia models are required, which have the disadvantage that the degree and distribution of neuronal injury varies immensely. Using such models it is, therefore, very difficult to test the neuroprotective properties of different drugs. Because this was the main aim of the present study, we chose to use the ischemic model described.

We observed a more pronounced neuronal cell damage in this study than in a previous one(10), a fact that might be related to differences in methods. We now used a double occlusion of the carotid arteries as described in "Methods," because double occlusion of the carotid arteries was necessary to account for residual blood flow via various anastomoses between carotid and vertebral arteries. This resulted in a more severe reduction in cerebral blood flow.

The differences in the severity of neuronal cell damage between the control group in the present study and that in a study performed by Gunn et al.(7) may be explained by strain and vendor differences. In fact, the volume of cerebral infarcts, e.g. in adult rats, depends largely on these variables, a fact that is probably due to differences in the cerebrovascular system(23,24).

After cerebral ischemia there was reactive hyperemia in almost all structures of the fetal brain studied. This reactive hyperemia is largely caused by a reduction in vascular tone on the basis of tissue acidosis during and a decrease in blood viscosity after ischemia(25,26). The hyperemic period was followed by a tendency toward hypoperfusion of the brain. Similar changes in cerebral blood flow could be observed in adult animals after ischemia. However, despite extensive research, the biologic significance of this phenomenon is not fully understood(27). In the control group, postischemic brain blood flow at 72 h was higher than the preischemic blood flow. This phenomenon has also been described in severely asphyxiated human infants(28). Whether this is related to a loss in CO2 reactivity and autoregulation of the cerebral vascular bed due to the ischemic insult is unknown.

As discussed previously the decrease in arterial blood pH as well as the increase in mean arterial blood pressure and plasma concentrations of both glucose and lactate are related to the acute experimental animal procedure(10). However, in spite of a few disadvantages of this acute animal model its clear advantage is that total arrest of carotid arterial blood flow could be ensured and controlled visibly.

We conclude that pretreatment by low dose flunarizine (1 mg/kg of estimated body weight) protects the brain of fetal sheep near term from ischemic injury. We further conclude that this neuroprotective effect is not mediated by drug-dependent changes in cerebral blood flow. Because low dose flunarizine does not alter the response of the fetal cardiovascular system to asphyxia, its clinical use should be reconsidered, particularly in fetuses who are at risk of hypoxic-ischemic encephalopathy.