In this study, treatment with a GHRH analog (JI-34) during IH exposures mimicking OSA was accompanied by significant attenuation of neurocognitive deficits and depression, the latter being assessed by the forced swim test. Conversely, we found no evidence for further deterioration in any of the outcome measures when the GHRH antagonist MIA602 was administered during IH exposures, even if adverse effects were detected in normoxic mice treated with MIA602. Furthermore, JI-34 administration led to decreases in oxidative stress markers, such as MDA and 8-OHDG, as well as increased gene expression of two recognized endogenous cellular survival pathways, namely IGF-1 and EPO, suggesting that exogenous GHRH analog administration may serve as an effective therapeutic intervention aiming to mitigate the extent and severity of OSA-associated cognitive dysfunction. These results suggest that peripheral GHRH administration stimulated either directly through GHRH receptor binding or by alternatively inducing a downstream response to increase cellular GH and downstream pathways in the brain that may ameliorate the hypoxia-induced injury of OSA.
Before we address the potential significance of our findings, some technical and methodological issues deserve comment. First, IH exposures do not constitute the full complement of OSA-associated physiological disturbances, but rather serve as a corollary to one of its major characteristics, namely the recurring nature of hypoxia and re-oxygenation cycles that are characteristically present in the vast majority of patients. In addition, IH exposures do induce some degree of sleep perturbation, particularly centered around fragmentation of sleep (Polotsky et al. 2006; Kaushal et al. 2012), even though the pattern of such sleep disruption is not as prominent as in OSA itself, most likely because of the absence of concomitant hypercapnia and chest wall afferent inputs induced by increased respiratory efforts during airway occlusion (Wilcox et al. 1990; Szollosi et al. 2004; Calero et al. 2006; Buchanan and Richerson 2010). It is likely that the interactions between IH alone and IH-associated sleep perturbations may lead to reduced harnessing of GHRH synthesis and bioavailability in regions such as the hippocampus and cortex, thereby increasing their susceptibility to injury. In summary, our results show that exogenous administration of the GHRH analog J1-34 during the course of IH exposures attenuates behavioral impairments associated with IH and decreases oxidative stress markers (i.e., MDA and 8-OHDG), indicating that the GHRH analog J1-34 confers neuronal protection against IH-induced oxidative stress. Indeed, GHRH appears to play a significant role in the regulation of sleep, particularly NREM sleep (Obal and Krueger 2004; Szentirmai et al. 2007; Liao et al. 2010), such that the alterations in sleep architecture induced by IH could be mediated, at least in part, by IH-induced reductions in GHRH gene transcription and bioavailability. The mechanisms underlying the putative effects on GHRH and potentially other downstream related peptides (i.e., GH and IGF-1) remain unknown. Preliminary evidence using GHRH antagonists would suggest the presence of reciprocal interactions between hypoxia-inducible factor-1α (HIF-1α) and GHRH (Munoz-Moreno et al. 2013). As HIF-1α is an important transcriptional regulator of both IGF-1 and EPO in CNS (Acker and Acker 2004; Freeman and Barone 2005), it is possible that the relatively reduced recruitment of HIF-1α during chronic IH exposures may result in a disproportionately lower expression of neuroprotective elements, such as IGF-1, and EPO (Dayyat et al. 2012), and that exogenous administration of GHRH agonists may restore the desirable protective effects of these genes. Furthermore, our data also show that the GHRH antagonist MIA602 reduces cognitive performance in room air-exposed control animals. It is possible that GHRH-related pathways may play an intrinsic role in maintenance of specific aspects of cognition, and the present observation should prompt further study in this direction. However, when IH exposures were added, MIA602 did not induce any further discernible deleterious effects on performance in the water maze. One thing that needs to be taken into account is that the treatment for IH was for 21 days, a period during which most of the adverse effects of IH have already taken place. Evidence supporting such possibility derives from previous studies in aging, whereby class II G-protein coupled receptors, which play major roles in regulating the function and plasticity of neuronal circuits in CNS, can be activated by GHRH, and increase the resistance of neuronal cells to oxidative, metabolic, and excitotoxic injury (Thornton et al. 2000; Martin et al. 2005). To further confirm the potential beneficial effect of a GHRH agonist in IH-induced cognitive impairments, we sought to explore whether JI-34 modified any of the previously identified markers of either injury or protection. We found that animals treated daily with the GHRH agonist while undergoing IH exposures exhibited not only attenuated increases in oxidative stress markers but also displayed increased IGF-1 mRNA and EPO mRNA expression in the hippocampus, suggesting that the somatotropic axis in the brain was effectively activated by exogenous subcutaneous administration of an agonist of GHRH. Some of these findings appear to be in contradiction with previously published study on IH and HIF-1α expression, which most likely represent differences in HIF-1α regulation in different tissues or when using different IH profiles (Nanduri et al. 2008; Yuan et al. 2008, 2011; Belaidi et al. 2009). Indeed, our previous study (Dayyat et al. 2012) and the study by Aviles-Reyes et al. (2010) clearly showed that long-term exposures to IH are associated with initial increases in HIF-1α transcriptional activity, but are followed by progressive reductions of such activity to baseline normoxic levels despite ongoing IH exposures. We now show that administration of the GHRH analog JI-34, but not the GHRH antagonist MIA602, elicited increases in HIF-1α DNA binding activity in hippocampus and cortex, in addition to increased expression of the HIF-1α target genes EPO and IGF-1. Secondly, our behavioral phenotype reporter assays explored only selective aspects of cognition, that is, spatial task acquisition and retention using the Morris water maze, and of mood, that is, forced swim test. It is possible that different aspects of cognition and behavioral functioning may be differentially modulated by OSA-associated perturbations, and that GHRH-related cognitive and behavioral effects may differ under normal conditions and conditions such as IH during sleep (Telegdy et al. 2011; Telegdy and Schally 2012a, b). Indeed, the dual beneficial cognitive and mood effects of both GHRH antagonists (Telegdy et al. 2011; Klukovits et al. 2012), and of agonists (Vitiello et al. 2006) may reflect the divergent interactions of these compounds on the endogenous regulation of the somatotropic axis and IH-effects. The GHRH analogs can cross the blood–brain barrier (Jaeger et al. 2005) and exert their effects in CNS, or whether such effects are because of modulation of alternative peripheral mechanisms. Peptides such as GHRH can cross the blood–brain barrier (Dogrukol-Ak et al. 2004), and previously reported effects of GHRH-related compounds on several neurotransmitters would suggest that the beneficial effects of JI-34 were likely centrally mediated.
Of note, IH and SF are both associated with increased oxidative stress, the magnitude of which is a critical determinant of end-organ injury in general, and CNS dysfunction in particular (Row et al. 2003, 2007; Xu et al. 2004; Shan et al. 2007; Douglas et al. 2010; Nair et al. 2011a). GHRH has been postulated to modulate oxidative stress in specific cancer lines, as well as other settings (Barabutis and Schally 2008; Banks et al. 2010; Wang et al. 2012), such that the beneficial effects of JI-34 in our IH-exposure murine model could be ascribable to the attenuation of free-radical induced neuronal injury. A substantial component of the neuroprotective effect conferred by the administration of the GHRH analog JI-34 may be because of the downstream effects of this compound on GH-IGF-1 dependent cellular pro-survival pathways. This possibility was suggested by the increased expression of IGF-1 after treatment with JI-34, and further confirmation of this possibility will require further study, particularly considering the previously reported favorable effects of exogenous GH treatment in rodent OSA models (Li et al. 2011). Notably, the beneficial effects of GHRH agonists on hypoxia-induced phenomena are in agreement with their effects on pancreatic β-islet survival (Ludwig et al. 2010, 2012). A recent study from our lab further supports the concept that exogenous administration of recombinant human EPO will attenuate IH-induced NADPH oxidase mediated hippocampal oxidative stress injury and cognitive and behavioral deficits (Dayyat et al. 2012), indicating the protective effects of GHRH analog JI-34 may be via the EPO pathway.
In summary, treatment with a selective GHRH agonist reduced markers of oxidative stress in the cortex and hippocampus, promoted enhanced expression of the neuroprotective genes IGF-1 and EPO, and markedly attenuated IH-induced cognitive and behavioral deficits.