Respiratory dysfunction following neonatal sustained hypoxia exposure during a critical window of brain stem extracellular matrix formation

Abstract

The extracellular matrix (ECM) modulates brain maturation and plays a major role in regulating neuronal plasticity during critical periods of development. We examined 1) whether there is a critical postnatal period of ECM expression in brain stem cardiorespiratory control regions and 2) whether the attenuated hypoxic ventilatory response (HVR) following neonatal sustained (5 days) hypoxia [SH (11% O₂, 24 h/day)] exposure is associated with altered ECM formation. The nucleus tractus solitarius (nTS), dorsal motor nucleus of the vagus, hypoglossal motor nucleus, cuneate nucleus, and area postrema were immunofluorescently processed for aggrecan and Wisteria floribunda agglutinin (WFA), a key proteoglycan of the ECM and the perineuronal net. From postnatal day ( P) 5 ( P5), aggrecan and WFA expression increased postnatally in all regions. We observed an abrupt increase in aggrecan expression in the nTS, a region that integrates and receives afferent inputs from the carotid body, between P10 and P15 followed by a distinct and transient plateau between P15 and P20. WFA expression in the nTS exhibited an analogous transient plateau, but it occurred earlier (between P10 and P15). SH between P11 and P15 attenuated the HVR (assessed at P16) and increased aggrecan (but not WFA) expression in the nTS, dorsal motor nucleus of the vagus, and area postrema. An intracisternal microinjection of chondroitinase ABC, an enzyme that digests chondroitin sulfate proteoglycans, rescued the HVR and the increased aggrecan expression. These data indicate that important stages of ECM formation take place in key brain stem respiratory neural control regions and appear to be associated with a heightened vulnerability to hypoxia.

Keywords: brain stem extracellular matrix; breathing; development; hypoxia.

Fig. 1.

Timeline and experimental setup used to expose rats to sustained hypoxia [SH (11% O₂, 24 h/day) between postnatal days (P) 11 (P11) and P15]. At the end of P15, rats were removed from the SH chamber, and the acute hypoxic ventilatory response (HVR) was assessed by whole body plethysmography on the following day (P16). Open star designates time points at which semiquantification of Wisteria floribunda agglutinin and aggrecan was carried out within brain stem regions (see Figs. 2 and 3). B, birth.

Fig. 2.

Aggrecan immunoreactivity in various brain stem regions at various postnatal ages. A: densitometry. Brain regions include nucleus tractus solitarius (nTS), hypoglossal motor nucleus (XII), dorsal motor nucleus of the vagus (DMNV), cuneate nucleus (CN), and area postrema (AP). CC, central canal. Values are means ± SE; n = 6 at P5, 9 at P10, 8 at P15, 7 at P20 , and 5 at P90. *Statistically different from preceding (younger) age group (P < 0.05, by One-way ANOVA). B: representative images of brain stem regions at P5, P10, P15, P20, and P90 (adult). Scale bar = 100 μm.

Fig. 3.

Wisteria floribunda agglutinin (WFA) immunoreactivity in various brain stem regions at various postnatal ages. A: densitometry. Brain regions include nTS XII, DMNV, CN, and AP. Values are means ± SE; n = 6 at P5, 9 at P10, 8 at P15, 7 at P20 , and 5 at P90. *Statistically different from preceding (younger) age group (P < 0.05, by One-way ANOVA). B: representative images of brain stem regions at P5, P10, P15, P20, and P90 (adult). Scale bar = 100 μm.

Fig. 4.

HVR at P16 in rats following SH exposure between P11 and P15 with or without microinjection of chondroitinase ABC (ChABC) into the 4th ventricle. A: changes in ventilation (V̇e, i.e., HVR), tidal volume (Vt), and respiratory frequency (f_R) of rats exposed to SH between P11 and P15 after microinjection of saline [SH + S (○)] or ChABC [SH + ChABC (Δ)]. Control groups include rats exposed to normoxia between P11 and P15 following microinjection of saline [NX + S (●)] or ChABC [NX + ChABC (▼)].Values are means ± SE; n = 12 NX + S, 11 NX + ChABC, 12 SH + S, and 11 SH + ChABC. *Significantly different from NX + S for a given time point (P < 0.05, by 2-way, repeated-measures ANOVA). B: representative breathing traces from animals at baseline and minutes 1 and 5 of acute hypoxia for each treatment group.

Fig. 5.

Baseline (normoxia) V̇e, f_R, and Vt of rats raised in normoxia or exposed to SH between P11 and P15 after prior (at P14) intracisternal microinjection of saline or ChABC. Statistical significance (P < 0.05, by Two-way ANOVA) between groups as indicated.

Fig. 6.

Changes in aggrecan immunoreactivity in various brain stem regions following SH and ChABC microinjection. A: increase in aggrecan expression in nTS, DMNV, and AP, but no effect in XII, following SH. Increased expression in nTS and DMNV was reduced by microinjection of ChABC into the 4th ventricle. Values are means ± SE; n = 9 NX + S,  9 NX + ChABC, 8 SH + S, and 9 SH + ChABC. Statistical significance (P < 0.05, by Two-way ANOVA) between groups as indicated. B: representative images of brain stem regions. Scale bar = 100 μm.

Fig. 7.

WFA immunoreactivity in various brain stem regions following SH exposure and microinjection of ChABC into the 4th ventricle. A: no effect of SH or ChABC on WFA immunoreactivity in nTS, DMNV, AP, or XII. Values are means ± SE; n = 9 NX + S, 9 NX + ChABC, 8 SH + S, and 9 SH + ChABC. B: representative images of brain stem regions. Ctrl (NX), normoxia treated rats. Scale bar = 100 μm.

Fig. 8.

A and C: aggrecan and WFA immunoreactivity showing relative expression across various brain regions in P15 normoxic rats. Values are means ± SE; n = 8. *P < 0.05 vs. AP; @P < 0.05 vs. DMNV; #P < 0.05 vs. nTS; $P < 0.05 vs. CN (by 1-way ANOVA). B and D: representative images from single rats. AP, area postrema; DMNV, dorsal motor nucleus of the vagus; nTS, nucleus tractus solitarius; CN, cuneate nucleus; XII, hypoglossal motor nucleus; IO, inferior olive; sp5, spinal trigeminal tract; sp5I, spinal 5 interpolar. Scale bar = 200 μm.

Fig. 9.

Rostral-caudal images of dorsal brain stem 45 min following a 3-μl microinjection of pontamine sky blue dye into the 4th ventricle of a 14-day-old rat. Note localized penetration of dye, especially into the nTS and DMNV. Scale bar = 200 μm.