IDENTIFICACION DE PUNTOS DE MONITOREO AFLUENTE

The rapidity of the initial fall in heart rate and rise in peripheral and pulmonary vascular resistance at the onset of hypoxia provides the first indication of their being reflex in nature, i.e. with sensory information relayed to the brain stem via afferent pathways probably synapsing at the nucleus tractus solitarius (NTS. Felder and Mifflin, 1988 see 1.6.1) and then a command sent via efferent pathways to effector organs. Indeed early studies by Dawes et a l (1968) in the exteriorised anaesthetised fetus showed that if the arrival of low oxygenated blood at the hindlimb was delayed, a rapid vasoconstriction (1 0 - 2 0 sec) still occurred which suggested that the response was reflex rather than being

due to local blood gas changes. Also, the FHR response is reminiscent of the diving response seen in adults which functions to reduce cardiac output and increase MAP in order to maintain O2 supply to the vital organs.

The two prominent peripheral chemoreceptor locations are the carotid and aortic bodies, their afferent fibres running to the brain stem via the carotid sinus nerve (CSN: branch of the IXth nerve) and the aortic branch of the vagus (Xth) nerve, respectively. Other chemoreceptor sites exist but there is little information on them in the fetus and they will not be considered further in this thesis (see Hanson, 1988; Giussani, Spencer and Hanson, 1994c).

The carotid body is visible in early gestation, and fetal carotid (Blanco, Dawes, Hanson and McCooke, 1984) and aortic (Blanco, Dawes, Hanson and McCooke, 1982) chemoreceptors have been demonstrated to be functionally active from single fibre recordings in the last third of gestation in sheep. Basal chemoreceptor discharge, which is high compared to the adult, increases further when Pa0 2 is reduced (Blanco et a l,

1984).

Similarly, fetal sheep carotid and aortic baroreceptor discharge was detected from ca. 85 days gestation and found to be phasic and in synchrony with the arterial pulse (see

Hanson, 1988). Arterial baroreceptors have been implicated in the regulation of arterial pressure and heart rate in the fetus (Itskovitz, LaGamma and Rudolph, 1983; Yardley, Bowes, Wilkinson, Maloney, Ritchie and Walker, 1983).

Dawes et aL (1968) injected cyanide into the left atrium of the fetal lamb and produced a femoral vasoconstriction which led them to suggest that, while the aortic chemoreceptors are unlikely to be the only chemosensory sites in the fetus, they probably provide the first line of defence against alterations in arterial oxygenation. Since then extensive investigations of the contribution of aortic and carotid chemo- and baroreceptors to the fetal cardiovascular responses to hypoxia have been conducted. One approach has been to sinoaortic denervate sheep fetuses by cutting not only the CSN but also the aortic and superior laryngeal nerves (Itskovitz and Rudolph, 1982; Wood, 1989; Wood, Kane and Raff, 1990). This technique has certain disadvantages in that the aortic nerve is sometimes indistinguishable from the vagus and thus the vagus could be damaged, but also that the aortic nerve itself may contain some pulmonary mechanoreceptors (for review refer to Giussani, Spencer and Hanson, 1994c). This technique did however provide further evidence for peripheral arterial chemo- and baroreceptor involvement in fetal hypoxic responses since it abolished the rapid bradycardia, the rise in MAP and the peripheral vasoconstriction during hypoxia (Itskovitz, LaGamma, Bristow and Rudolph,

1991).

In the next phase of investigations attempts were made to delineate the relative contribution of aortic and carotid chemoreceptors. Initial studies compared carotid sinus denervation (CSD), alone or in combination with vagotomy, and found that both procedures altered CVO redistribution (Jansen, Belik, Ioffe and Chemick, 1989) and attenuated the rapid fall in pulmonary blood flow (Moore and Hanson, 1991) during hypoxia. Vagotomy is not ideal since the vagus carries both afferent and efferent fibres and so any effects observed could have been due to the removal of the influence of other pathways. The work of Giussani et a l (1993) addressed the specific contribution of the carotid chemo- and baroreflexes by cutting the CSN alone. They found that the rapid bradycardia and femoral vasoconstriction at the onset of hypoxia were primarily carotid chemoreflexes. This was confirmed by Bartelds et ah (1993) who compared carotid- with aortic-denervated fetuses and found that the carotid, but not the aortic, chemoreceptors contributed to the rapid bradycardia and peripheral vasoconstriction during hypoxia. Whether these effects are carotid chemo- or baroreflexes is hard to address from these studies. However the FHR response would not appear to be a baroreflex response since it occurs before the rise in MAP. Hypoxic carotid chemoreceptor stimulation produces a fall in RBF and an anti-diuresis in adult dogs (Al- Obaidi and Karim, 1992; Karim and Al-Obaidi, 1993) and rats (Behm, Mewes, DeMuinck Keizer, Unger and Rettig, 1993), and carotid chemodenervation in adults

inhibits hypoxic renal vasoconstriction (see Honig, 1989). To date it has not been investigated whether the fall in RBF during hypoxia in the fetus is a carotid chemoreflex response. Conversely, the rise in MAP and CBF duringlare not chemoreflexly mediated (Giussani, Spencer, Moore, Bennet and Hanson, 1993).

Two autonomic efferent pathways have been identified: sympathetic a - and p- adrenergic effects and vagal cholinergic effects. The sympathetic efferent pathways will be considered in section 1.5.2 (catecholamines), however it does not seem that their activity is fully developed by birth, particularly since sympathectomy of the fetus does not alter basal vascular tone, FHR or MAP (Iwamoto, Rudolph, Mirkin and Keil, 1983; Jensen and Lang, 1992) and because renal haemodynamic responses to renal nerve stimulation are not as pronounced in the fetus as in the adult (see Robillard and Nakamura, 1988). In the fetus, the renal sympathetic nerves do not appear to contribute to the regulation of basal renal haemodynamics or function (Smith, Sato, McWeeny, Klinkefus and Robillard, 1990) but they have been implicated in mediating the RBF responses to hypoxia (Robillard, Nakamura and DiBona, 1986), probably via a - adrenoreceptor stimulation (see Robillard, Smith, Segar, Merrill and Jose, 1992). It is interesting that following renal denervation, hypoxia produces a transient, prostaglandin (PG)-mediated initial rise followed by a progressive fall in fetal RBF (Robillard et a l, 1986). Adult studies have already identified these renal nerves to form the efferent limb of a carotid chemoreflex which controls RBF during hypoxia (see Honig, 1989 and DiBona, 1989), but this has yet to be shown in the fetus.

The initial rapid bradycardia during hypoxia is blocked by cholinergic receptor, but not a-adrenoceptor, antagonism which suggests a vagal cholinergic efferent limb to this reflex response (Lewis, Donovan and Platzner, 1980; Giussani, Spencer, Moore, Bennet and Hanson, 1993). Furthermore transection of the spinal cord, to remove sympathetic efferents while keeping the vagal efferents intact, shows that the MAP response to hypoxia is abolished but the rapid bradycardia remains intact (Blanco, Dawes and Walker, 1983).

1.5.2 Endocrine mechanisms

Hormones such as angiotensin II (All), AVP, atrial natriuretic factor (ANF) and catecholamines have been implicated in a number of fetal studies of circulatory control under basal and hypoxic conditions. This section will review their varied contribution to cardiovascular control but with particular emphasis on the part played by All. I will also review the current knowledge on the interaction between Pa0 2 and these hormonal

Catecholamines

N orm oxia

Resting plasma [catecholamine] in the fetal circulation have been found to range between 0.5 and 1 ng.ml’l (Jones and Robinson, 1975; Schuijers, Walker, Browne and Thorbum, 1986). The calculated clearance rates for the fetal and maternal circulations are 840 and 4200 ml.mim^, respectively (Jones and Robinson, 1975).

c

The adrenal medulla contributes no more than 30% to basal plasma [cateholamine] (Jones, Roebuck, Walker, Lagercrantz and Johnston, 1987). Sympathectomy under normoxic conditions increases plasma [adrenaline] which suggests a compensatory secretion by the adrenal medulla in the absence of sympathoneuronal function (Jensen and Lang, 1992). On the other hand, it is a rise in plasma [catecholamines] from increased activity of the sympathetic nervous system, rather than from the adrenal medulla, which is thought to account for the rise in MAP in fetal sheep between 120 and 135 days gestation (Schiujers, Walker, Browne and Thorbum, 1986).

Hypoxia

The adrenal gland is thought to be the major source of the rise in arterial [adrenaline] and [noradrenaline] during hypoxia in the fetal sheep (Cohen, Piasecki and Jackson, 1982; Cohen, Piasecki, Cohn, Young and Jackson, 1984), although plasm a [noradrenaline] may be contributed to by paraganglia and sympathetic overflow (see Jones and Robinson, 1975; Jones, Roebuck, Walker, Lagercrantz and Johnston, 1987). The catecholamine response to hypoxia is functional in both early- (95-112 days) and late- (125-140 days) gestation sheep fetuses (Cohen, Piasecki and Jackson, 1982).

During hypoxia, after the initial rapid vagally-mediated bradycardia (Lewis, Donovan and Platzker, 1980), FHR retums to or above control levels. While this phenomenon could be due to increased sympathetic efferent activity, it is also associated with high circulating [catecholamines] which might exert a direct action on the heart (Jones and Robinson, 1975; Iwamoto, Rudolph, Mirkin and Keil, 1983), and is prevented by p- adrenergic blockade (Jones and Ritchie, 1983). Furthermore increased p-adrenergic activity has been implicated in limiting the vagally mediated negative chronotropic effects at the onset of hypoxia (Court, Parer, Block and Llanos, 1984).

After 1 min asphyxia [adrenaline] was higher in sympathectomised than intact fetuses, but similar to intact fetuses after 2 min asphyxia. This suggests that a massive adrenaline release from the adrenal medulla during asphyxia may have overridden any subtle difference between intact and sympathectomised fetuses (Jensen and Lang, 1992).

Furthermore hypoxia was tolerated to the same extent in sympathectomised and intact fetuses which also adds to the notion of the adrenal gland being an important source of catecholamines (Iwamoto, Rudolph, Mirkin and Keil, 1983). This is likely to be both via splanchnic nerve stimulation (Comline, Silver and Silver, 1965) and a direct action of hypoxia on the adrenal gland (Cohen, Piasecki, Cohn, Young and Jackson, 1984).

Alpha-adrenergic activity is implicated in the hypoxic vasoconstriction of hepatic, gut, spleen and carcass blood flow and the rise in blood pressure but is not responsible for renal vasoconstriction during hypoxia (Reuss, Parer, Harris and Krueger, 1982; Paulick, Meyers, Rudolph and Rudolph, 1991a; Giussani, Spencer, Moore, Bennet and Hanson, 1993). Maintenance of placental blood flow during hypoxia is due in part to increased p- adrenergic activity, either by a direct vasodilatation of placental vessels or via inotropic/chronotropic actions on the heart. Increased p-adrenergic activity may contribute to the rise in myocardial blood flow seen during hypoxia (Court, Parer, Block and Llanos, 1984). Furthermore hypoxia-induced constriction of umbilical veins is reversed by a-adrenergic blockade (Paulick et a l, 1991a). The variability in sensitivity of vascular beds to a-adrenergic stimulation may be due in part to differences in receptor numbers, for example the ai-receptor density is less in intrapulmonary than in aortic smooth muscle of the late-gestation sheep fetus (Shaul, Magness, Muntz, DeBeltz and Buja, 1990).

Despite the fact that the catecholamine response is maintained in the face of chemical sympathectomy, some of the circulatory responses to hypoxia are not, for example MAP did not rise, probably due to less vasoconstriction in the periphery (Iwamoto, Rudolph, Mirkin and Keil, 1983). Thus other neuronally mediated vasoconstrictors may be involved. More recently Giussani et a l (1993) observed that the initial rapid cardiovascular responses to hypoxia are attenuated by section of the CSNs suggesting that carotid chemoreceptors provide the afferent limb of these reflex responses [1.5.1]. The efferent limb of the reflex, at least with regards to the vasoconstriction in the periphery (Figure 1.2), is mediated by a-adrenergic mechanisms since the rise in femoral vascular resistance (FVR) was blocked by the administration of the a i / a2-receptor

antagonist, phentolamine. To date the effect of CSD on circulating catecholamines has not been investigated. In the study of Giussani et a l (1993), all fetuses in which CSD was combined with phentolamine treatment died. In contrast, the majority of intact fetuses treated with phentolamine survived. It was suggested that a component of the vasoconstriction during hypoxia is therefore due to carotid chemoreflexly-released vasoconstrictors that do not act via a-adrenergic mechanisms.

HYPOXIA

cholinergic

CSN

3 r a in s t e m

'carotid ■

body p itu ita ry _{f catecholam ine]}t plasm a _{m e d u l l a}a d r e n a l

?splanchnic? a-^ adrenergic ACTH / | \ p e r i p h e r a l v a s c u l a r r e s i s t a n c e a d r e n a l c o r te x c o r tis o l

Figure 1.2 Diagram to show the carotid chem oreflexes and the question o f the unidentified hormonal factor (?) as w ell as the question o f whether the renal nerve-m ediated rise in renal vascular resistance (R V R ) during hypoxia is a chem oreflex response. C SN , carotid sinus nerve; FHR, fetal heart rate. The black arrows represent known pathw ays, whereas the grey arrow s denote th ose that have yet to be identified (Adapted from Giussani, 1992).

Arginine vasopressin

N o rm o xia

A rginine vasopressin is present in fetal sheep hypothalam ic extracts as early as 70 days gestation and increases betw een 100 and 130 days gestation (Currie and B rooks, 1992). N orm al plasm a [AVP] is betw een 0.5 and 3.5 p U .m l'k F ollow ing A V P adm inistration, there is an initial rapid phase o f clearance from the fetal circulation with a half-life of 2.8 min and clearance o f 60.5±8.72m l.m in F k g 'F C learance is contributed to by the fetal kidney, but not by the placenta or by fetal-m aternal transport (W iriyathian, Porter, Naden and Rosenfeld, 1983), and is reported to be three tim es higher than that from the maternal system (R urak, 1978).

Tw o subtypes o f A V P receptors have been proposed, a V i vascular receptor and V% renal receptor (Ervin, Ross, Leake and Fisher, 1992). A V P infusion produces a gradual rise in M AP, a fall in FHR, possibly via a direct action on the heart as well as a baroreflex response (R urak, 1978; Iw am oto, R udolph, Keil and H eym ann, 1979; W iriy ath ian , Porter, N aden and R osenfeld, 1983), and redistribution o f blood flow (Iw am oto et aL,

1979). H ow ever under norm oxic conditions AV P V i receptor antagonism does not have any effect on fetal cardiovascular param eters (Perez, E spinoza, R iquelm e, P arer and Llanos, 1989).

H ypoxia

It is now well established that plasma [AVP] rises in response to hypoxia in mid­ gestation (ca. 80-100 days: Iwamoto, Kaufman, Keil and Rudolph, 1989) and late- gestation (125-141 days: Robillard, Nakamura and DiBona, 1986; 132 days: Piacquadio, Brace and Cheung, 1990; 123-144 days: Raff, Kane and Wood, 1991; 119-125 days: Giussani, McGarrigle, Spencer, Moore, Bennet and Hanson, 1994b) fetal sheep. Hypoxia and acidosis during hypoxic insults were both found to be potent stimuli for the release of A VP (Daniel, Stark, Zubrow, Fox, Husain and James, 1983), and plasma [AVP] was inversely related to arterial pH (Rurak, 1978 Wood and Chen, 1989). Hypercapnia alone however had no effect on plasma [AVP], but hypercapnie acidaemia did augment the AVP response to hypoxia (Raff, Kane and Wood, 1991; Chen and Wood, 1993).

The observation that AVP infusion caused a redistribution of blood flow similar to that seen during fetal hypoxia led investigators to suggest that AVP may be an important mediator in fetal hypoxic responses (Iwamoto, Rudolph, Keil and Heymann, 1979). Indeed later studies showed that an AVP V%-receptor antagonist reversed the hypoxia- elevated resistance of placenta, brain, gut and liver vascular beds as well as the hypoxic bradycardia and hypertension response. Thus AVP is implicated in CVO redistribution in hypoxia (Perez, Espinoza, Riquelme, Parer and Llanos, 1989) and may play a part in the reduction of hindlimb O2 consumption during hypoxia in the sheep fetus (Rurak,

Stobbs, Kwan and Hall, 1995).

Chemical sympathectomy in fetal sheep reduces the rise in [AVP] during hypoxia (Iwamoto, Rudolph, Mirkin and Keil, 1983) and asphyxia (Jensen and Lang, 1992). This led investigators to suggest that the sympathetic nervous system is involved in stimulating AVP secretion either via peripheral chemoreceptors or central neural pathways. Indeed in adult dogs the peripheral chemoreceptors are implicated in the neurohypophysial (but not other brain region) vasodilatation during hypoxia and perhaps in the neurosecretion of AVP (Hanley, Wilson, Feldman and Traystman, 1988). However fetal sheep studies where sinoaortic-denervation (Raff, Kane and Wood, 1991), bilateral section of the CSNs and cervical vagosympathetic trunks (Chen and Wood, 1993), or bilateral CSD alone (Giussani, McGarrigle, Spencer, Moore, Bennet and Hanson, 1994b) was carried out do not implicate peripheral chemoreceptors in the AVP response to hypercapnia, normoxic hypoxia or hypercapnie hypoxia. Furthermore vagal nerve section did not block the rise in [AVP] during hypoxia which suggests that, unlike the adult, chemoreceptors other than those located in the carotid body may play a role in this response (Rurak, 1978). Thus despite the large stimulation of AVP production during hypoxia in the fetus, it does not appear to be reflexly released and therefore is not

likely to contribute to the rapid components of the fetal cardiovascular responses to hypoxia (see Giussani e ta l, 1994b).

Atrial natriuretic fa cto r

N orm oxia

Fetal plasma [ANF] is greater than in the adult (Cheung, Gibbs and Brace, 1987). Fetal ANF clearance rates (ca 120ml.min.kg-^: Brace and Cheung, 1987; Brace, Bayer and Cheung, 1989) are double than those of the maternal circulation (Ervin et a l, 1988), although others have reported no difference between the fetus, newborn and adult (Robillard, Nakamura, Varille, Matherne and McWeeny, 1988a). Low urine [ANF] suggest either that the fetal kidney contributes minimally to ANF clearance (Robillard et a l, 1988a; Brace et a l, 1989), or that circulating ANF is metabolised by the kidney so that little is excreted (Cheung, Gibbs and Brace, 1987). There is reported to be no fetal placental clearance of ANF (Rosenfeld, Samson, Roy, Faucher and Magness, 1992). Instead, kallikrein or other proteases may contribute to ANF degradation.

Basal ANF production from atrial muscle cells, is suggested to be primarily dependent on FHR. Basal plasma [ANF] is high in the immature fetus (110-119 days) and decreases with advancing gestation, concurrent with a fall in FHR (Cheung, 1992). Atrial natriuretic factor secretion is stimulated by endothelin-1 (ET-1), perhaps via an inotropic action on the heart and increased atrial muscle tension (Cheung, 1994), and by the infusion of All and the a-agonist phenylephrine, which produce a concomitant rise in right atrial pressure (Rosenfeld et a l, 1992).

Administration of ANF antiserum, to immunoneutralise endogenous ANF, causes a delayed but sustained rise in MAP for the duration of infusion (Cheung, 1991). This is in agreement with studies in which infusion of ANF caused hypotension (Brace, Bayer and Cheung, 1989). Changes in FHR appear to be reciprocal to changes in MAP and do not suggest a direct action of ANF on the heart. In contrast, Robillard et a l (1988b) did not find the changes in MAP and FHR to be of significance. In some studies fetal sheep kidneys (103-128 days) are reported to be as responsive as those of adults to ANF (Shine, McDougall, Towstoless and Wintour, 1987), while others suggest that the cardiovascular, renal vasoconstriction and renal functional responses increase during maturation so that the overall response is larger in the adult (Robillard, Nakamura, Varille, Anderesen, Matherne and VanOrden, 198^.

H ypoxia

Hypoxia is a potent stimulus for elevating plasma [ANF], probably via a direct action on the fetal heart (Cheung and Brace, 1988) but there may also be contributions from the

action of AVP (Cheung, 1992), catecholamines (Ervin et a l, 1991) and the autonomic nervous system (Cheung, 1992). The rise in [ANF] may contribute to the reduction in blood volume (Rosenfeld, Samson, Roy, Faucher and Magness, 1992) seen during hypoxia (Cheung and Brace, 1988), probably independent of changes in urine flow (Brace, Bayer and Cheung, 1989). It also appears that the immature fetal sheep (110-119 days) displays a greater ANF response to hypoxia than the mature fetal sheep (130-135 days), probably due to the combination of a higher release and lower clearance rate of