C UESTIONES ABIERTAS PARA EL DEBATE

Anaesthetics/sedatives

In earlier pilot studies designed to develop a method for repeated collection of CSF samples in sheep, it was observed that CNP peptide concentration was lower in the initial samples which were obtained — whilst the animals were under the influence of ketamine/diazepam — than in subsequent samples (n = 5, Lincoln University 2009–2010, unpublished). This suggested that brain regions sensitive to the depressive effects of anaesthetics and sedatives were the possible

sources of CNP peptides appearing in CSF. Consequently, samples of CSF were collected before, during and after anaesthesia to map any changes in CSF concentrations of CNP peptides which could be linked to the decrease and subsequent increase in arousal level that accompanies anaesthesia and recovery. The compounds investigated were ketamine (on its own or in

conjunction with the sedative, diazepam) and isoflurane. The sedative, diazepam, was also tested on its own.

Lipopolysaccharide

There are many studies linking CNP to inflammation described in the literature, however the nature of CNP’s involvement is unclear. Whereas higher plasma concentrations of CNP are associated with severe inflammation in adults (Koch et al. 2011), acutely ill children were shown to have lower plasma concentrations of the CNP peptides (Prickett et al. 2013). Evidence for an anti-inflammatory effect of CNP was published by Qian et al. (2002) where it was shown that overexpression of CNP reduced macrophage infiltration in injured arteries, and suppressed the expression of adhesion molecules that were stimulated by the injury in vitro. Elevated plasma concentrations of CNP have been observed in patients diagnosed with Crimean-Congo fever (Turkdogan et al. 2012) although it is unlikely that this is related to CNP having an active role in this setting — instead CNP may be released from endothelial cells which are damaged by the virus (Ergonul et al. 2004).

Suga et al. (1993) reported that lipopolysaccharide (LPS), a pyrogenic endotoxin, induces CNP secretion from bovine endothelial cells in vitro. Furthermore, Osterbur et al. (2013) found that LPS, tumour necrosis factor-α and interleukin-1β stimulated NTproCNP secretion from canine aortic vascular endothelium in vitro. Interleukin 1-β, which can act as an endogenous pyrogen in the rat brain (Dascombe et al. 1989), was the strongest stimulant of NTproCNP secretion of all the stimulants tested (Osterbur et al. 2013). CNP may also be involved in the central control of

44 thermoregulation, because it was shown that intracerebroventricular administration of CNP acutely elevated colon temperature in rats (Pataki et al. 1999).

LPS, when administered to sheep at a dose of 700 ng/kg live weight, reliably produces a fever in our laboratory. It was used here to provide a model inflammatory response to determine the possible role of this stimulus on the concentration of CNP peptides in CSF.

Progesterone

There are several studies linking sex steroids to the regulation of CNP synthesis. Exogenous oestrogen has been shown to increase plasma concentrations of CNP peptides in pre-pubertal ewes (Prickett et al. 2008). Testosterone treatment in children with growth hormone deficiency and idiopathic short stature is accompanied by markedly increased plasma concentrations of NTproCNP (Olney et al. 2007). However, evidence against a direct effect of testosterone on CNP synthesis was provided in later studies by Prickett et al. (2008), where it was shown that

exogenous testosterone had no effect on plasma concentrations of CNP peptides in pre-pubertal lambs. In contrast to testosterone and oestrogens, the effects of progesterone on CNP peptides have not been studied. Controlled internal drug release devices (CIDRs) are silicon coated devices containing progesterone which are inserted intra-vaginally to large animals such as sheep and cattle (Wheaton et al. 1993). After a period of time whereby the progesterone is slowly released, the CIDRs are removed, allowing oestrus to be synchronised throughout a herd of animals. CIDRs were used here as a convenient progesterone delivery source to investigate its possible role in physiology of the CNP peptides in the CNS.

Morphine

Opioid receptors are widely distributed in the brain and are bound by endogenous ligands including β-endorphin and various forms of encephalin; their actions possibly providing analgesic effects under normal physiological conditions (Wolozin & Pasternak 1981). Morphine is a

selective µ opioid receptor agonist which is commonly prescribed to relieve acute and chronic pain (reviewed by Mellon & Bayer 1998) and targets a specific set of brain pathways.

Furthermore, Babarczy et al. (1995) reported that central administration of CNP prior to a morphine injection depressed the antinociceptive properties of the opiate in mice. The effect of morphine on the affective state, locomotor activity and other behavioural aspects have been well characterised in sheep (Verbeek et al. 2012). A dose was selected that has been shown to be safe

45 but sufficient to induce behavioural changes and affect arousal status, as measured by an

increase in locomotor activity and number of vocalisations (Verbeek et al. 2012). L-deprenyl

Espiner et al. (2014) showed that CSF concentrations of NTproCNP were reduced in Parkinson’s disease patients — compared with CSF from individuals without a neurological disorder — and that the decline seen with disease progression was prevented in patients who received L- deprenyl. L-deprenyl (Selegiline) was developed in the 1960s for possible use as an anti- depressant, but was approved by the FDA (Food and Drug Administration, USA) in 1989 for treatment of Parkinson’s disease because it is a potent and selective inhibitor of monoamine oxidase B enzyme (Knoll 1983). Monoamine oxidase B enzyme degrades dopamine, and loss of dopamine (due to neurodegeneration in the substantia nigra) is one of the hallmarks of

Parkinson’s disease (Grosch et al. 2016). Whether changes in dopamine levels would affect CSF concentrations of CNP peptides is unknown, however several studies have reported the ability of CNP to influence dopamine pathways. Thiriet et al. (2001) reported that microinjections of CNP into the rat brain prevented cocaine-induced dopamine release and altered transcription factors that coordinated changes in gene expression underlying neuronal plasticity. Jouvert et al. (2004) later confirmed the site of action of CNP and showed that these changes were mediated via cGMP-dependent protein kinase I. The most common therapeutic dose that is prescribed for Parkinson’s disease patients is 10 mg l-deprenyl, taken daily through the form of two 5 mg tablets. Hypothesising that changes in dopamine levels may alter CSF concentrations of CNP peptides, the effect of a single dose of 10 mg l-deprenyl was investigated here.

Dexamethasone

Several studies have shown that glucocorticoids have the potential to regulate CNP peptide pathways, however whether they act at the level of the CNS is not known. Prickett et al. (2009) reported that plasma concentrations of CNP and NTproCNP in growing lambs were lower 24 and 48 h after repeated (125 µg/kg live weight for 2 days) administration of dexamethasone, a

synthetic glucocorticoid. In contrast, it has been shown that dexamethasone increases expression of NPPC in chondrocytes in vivo (Agoston et al. 2002). On this background of contradictory results, the present study investigated the effect of a single peripherally-administered bolus of dexamethasone on central concentrations of CNP peptides.

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