Effects of neonatal hyperoxia on the critical period of postnatal development of neurochemical expressions in brain stem respiratory-related nuclei in the rat

Abstract

We have identified a critical period of respiratory development in rats at postnatal days P12-13, when inhibitory influence dominates and when the response to hypoxia is at its weakest. This critical period has significant implications for Sudden Infant Death Syndrome (SIDS), the cause of which remains elusive. One of the known risk factors for SIDS is prematurity. A common intervention used in premature infants is hyperoxic therapy, which, if prolonged, can alter the ventilatory response to hypoxia and induce sustained inhibition of lung alveolar growth and pulmonary remodeling. The goal of this study was to test our hypothesis that neonatal hyperoxia from postnatal day (P) 0 to P10 in rat pups perturbs the critical period by altering the normal progression of neurochemical development in brain stem respiratory-related nuclei. An in-depth, semiquantitative immunohistochemical study was undertaken at P10 (immediately after hyperoxia and before the critical period), P12 (during the critical period), P14 (immediately after the critical period), and P17 (a week after the cessation of hyperoxia). In agreement with our previous findings, levels of cytochrome oxidase, brain-derived neurotrophic factor (BDNF), TrkB (BDNF receptor), and several serotonergic proteins (5-HT_1A and _2A receptors, 5-HT synthesizing enzyme tryptophan hydroxylase [TPH], and serotonin transporter [SERT]) all fell in several brain stem respiratory-related nuclei during the critical period (P12) in control animals. However, in hyperoxic animals, these neurochemicals exhibited a significant fall at P14 instead. Thus, neonatal hyperoxia delayed but did not eliminate the critical period of postnatal development in multiple brain stem respiratory-related nuclei, with little effect on the nonrespiratory cuneate nucleus.

Keywords: BDNF; brain stem; critical period; cytochrome oxidase; respiratory development; serotonin.

Figure 1

Optical densitometric values of cytochrome oxidase at the 4 developmental time points were plotted separately for the normoxic and the hyperoxic groups. A significant fall in CO reactivity at P12 and a significant rise at P14 were found in XII (A), PBC (B), and NTS_VL (C), but not in CN (D) of normoxic animals (left graph of each panel). In hyperoxic animals, however, the pattern was shifted to the right, such that the P10‐12‐14 pattern of normoxia became that of P12‐14‐17 of hyperoxia with a distinct fall at P14 in the latter group (right graph of each panel). N = 5 for each group for this and all subsequent figures. *P < 0.05; **P < 0.01 (Tukey's test comparing one age group with its immediately younger age group).

Figure 2

Optical densitometric measurements of immunoreaction product of BDNF in neurons of XII (A), PBC (B), NTS_VL (C), and CN (D) of normoxic and hyperoxic animals at P10, P12, P14, and P17. In the first three nuclei, the P10‐12‐14 pattern of normoxic animals with a distinct fall at P12 is similar to that of P12‐14‐17 in hyperoxic animals, in which the significant fall is at P14. No significant differences were found between the age groups in either normoxic or hyperoxic animals in the CN. N = 5 for each group. *P < 0.05; **P < 0.01 (Tukey's test).

Figure 3

Optical densitometric measurements of immunoreaction product of TrkB in neurons of XII (A), PBC (B), NTS_VL (C), and CN (D) of normoxic and hyperoxic animals at P10, P12, P14, and P17. In the first three nuclei, the P10‐12‐14 pattern of normoxic animals with a distinct fall at P12 is comparable to that of P12‐14‐17 in hyperoxic animals, in which the significant fall is at P14. No significant differences were found between the age groups in either normoxic or hyperoxic animals in the CN. N = 5 for each group. *P < 0.05; **P < 0.01 (Tukey's test).

Figure 4

Optical densitometric measurements of immunoreaction product of 5‐HT _1A receptors in neurons of XII (A), PBC (B), NTS_VL (C), and CN (D) of normoxic and hyperoxic animals at P10, P12, P14, and P17. In the first three nuclei, the significant fall in immunoreactivity from P10 to P12 in normoxic animals is mimicked in hyperoxic animals but with a distinct fall from P12 to P14. No significant differences were found between the age groups in either normoxic or hyperoxic animals in the CN. N = 5 for each group. *P < 0.05; **P < 0.01 (Tukey's test).

Figure 5

Optical densitometric measurements of immunoreaction product of 5‐HT _2A receptors in neurons of XII (A), PBC (B), NTS_VL (C), and CN (D) of normoxic and hyperoxic animals at P10, P12, P14, and P17. In the first two nuclei, a significant fall in immunoreactivity from P10 to P12 in normoxic animals is mimicked by a distinct fall from P12 to P14 in hyperoxic animals. No significant differences were found between the age groups in either normoxic or hyperoxic animals in the CN. N = 5 for each group. *P < 0.05; **P < 0.01 (Tukey's test).

Figure 6

Optical densitometric measurements of immunoreaction product of SERT in the neuropil of XII (A), PBC (B), NTS_VL (C), and CN (D) of normoxic and hyperoxic animals at P10, P12, P14, and P17. In the first two nuclei, a significant fall in immunoreactivity from P10 to P12 in normoxic animals is paralleled by a distinct fall from P12 to P14 in hyperoxic animals. In NTS_VL, a distinct rise in immunoreactivity was noted from P10 to P12 in hyperoxic animals. Otherwise, there were no significant differences between the age groups in NTS_VL of normoxic animals, nor in both groups in the CN. N = 5 for each group. *P < 0.05; **P < 0.01 (Tukey's test).

Figure 7

Optical densitometric measurements of immunoreaction product of tryptophan hydroxylase in neurons of RM (A), ROb (B), RP (C), and VLMS (D) of normoxic and hyperoxic animals at P10, P12, P14, and P17. In these nuclei, a distinct fall in immunoreactivity from P10 to P12 is noted in normoxic animals (though not reaching significance in the RP). This fall is shifted in hyperoxic animals to occur from P12 to P14, reaching significance in RM and RP. No significant differences were found between the age groups in ROb and VLMS neurons of hyperoxic animals. N = 5 for each group. *P < 0.05; **P < 0.01 (Tukey's test).