Postnatal Developmental Changes of Orexin in Spontaneously Hypertensive Rats

Orexins are involved in the regulation of cardiorespiratory functions and neurogenic hypertension. Using spontaneously hypertensive rats (SHRs), we have reported that 1) SHRs have more orexin neurons in the hypothalamus at young (postnatal days (P) 30–58) and adult ages than age‐matched, normotensive Wistar‐Kyoto (WKY) rats, 2) blocking orexin receptors (OXRs) with a dual OXR antagonist, almorexant, can normalize high blood pressure (BP) in young SHRs and significantly lower high BP in adult SHRs, and 3) hypercapnia (stress) activates more orexin neurons in the hypothalamus of SHRs than in normotensive WKY rats. Our study objective is to establish a developmental pattern of the orexinergic system in SHRs and WKYs and to better understand the role of the orexin system in hypertension. Specifically, we aim to define at which postnatal age the total number of orexin neurons starts to diverge between SHRs and normotensive rats, and to determine whether the increased number of orexin neurons in SHRs is due to an increase in the number of newly proliferated orexin neurons in the hypothalamus postnatally. To evaluate the total number and dispersion of orexin neurons in SHRs and WKY rats at P7–P25, we used immunohistochemical (IHC) staining of orexin A peptide. To evaluate new postnatal proliferation of orexin neurons, bromodeoxyuridine (BrdU) was injected (50 mg kg −1 , i.p.) in SHR and WKY pups for 5 consecutive days beginning at P5, P10, P15, or P25. Up to 7 days after the final injection, brains were harvested and the hypothalamus isolated. To assess the proliferation and maturity of orexinergic neurons in the hypothalamus, orexin positive neurons were double labelled with antibodies for BrdU, which incorporates as a thymidine into the genome of dividing cells and their progeny, doublecortin (DCX), which labels the cytoplasm of immature neurons (< 2 weeks old), or neuronal nuclear antigen (NeuN), which labels the nucleus of mature neurons (> 2 weeks old). Our early results show that 1) SHRs have significantly more orexin A‐immunoreactive (ir) neurons in the hypothalamus than age‐matched WKYs at P10–20, but not at P7, 2) SHRs have more BrdU/orexin‐ir neurons in the hypothalamus at P7–11 than at P25–29, and 3) SHRs have more DCX/orexin‐ir neurons in the hypothalamus at P14–20 than at P35 and later. Our data suggest that the divergence in number of orexin neurons between SHRs and normotensive rats can be seen as early as P10 and that this difference may be mediated by increased postnatal proliferation in the orexin system. This early divergence may imply a causal or upstream role in the development of neurogenic hypertension. These studies are essential in the determination of the mechanistic development of hypertension and thereby treatment of the origin of disease in the future. Support or Funding Information NIH HL28066