Chronic fetal hypoxia produces selective brain injury associated with altered nitric oxide synthases

Abstract

Objective: The purpose of this study was to investigate the impact of chronic hypoxia on the nitric oxide synthase isoenzymes in specific brain structures.

Study design: Time-mated pregnant guinea pigs were exposed to 10.5% molecular oxygen for 14 days (animals with chronic fetal hypoxia; HPX) or room air (control animals; NMX); L-N6-(1-iminoethyl)-lysine (L-NIL; an inducible nitric oxide synthase inhibitor, 1 mg/kg/d) was administered to HPX group for 14 days (L-NIL + HPX). Fetal brains were harvested at term. Multilabeled immunofluorescence was used to generate a brain injury map. Laser capture microdissection and quantitative polymerase chain reaction were applied; cell injury markers, apoptosis activation, neuron loss, total nitric oxide, and the levels of individual nitric oxide synthase isoenzymes were quantified.

Results: Chronic hypoxia causes selective fetal brain injury rather than global. Injury is associated with differentially affected nitric oxide synthases in both neurons and glial cells, with inducible macrophage-type nitric oxide synthase up-regulated at all injury sites. L-NIL attenuated the injury, despite continued hypoxia.

Conclusion: These studies demonstrate that chronic hypoxia selectively injures the fetal brain in part by the differential regulation of nitric oxide synthase isoenzymes in an anatomic- and cell-specific manner.

Figure 1. Chronic Fetal Hypoxia Results in Selective Anatomic Brain Injury

Sample photomicrographs from mirror image coronal sections at the interaural level of 6.72mm to 5.40 mm (Bregma: from −2.28 mm to −3.60mm) including cerebral cortex, hippocampus, and thalamic nuclei from hypoxic (n=6) and control fetuses (n=6) stained for ATF3 (10x), GFAB (10x), TUNEL (10x), and Nissl (20x). Chronic hypoxia (10.5% O₂ over the last 30% of gestation) visually increases ATF3, GFAB and TUNEL staining with an accompanying decrease in neuronal density.

Figure 2. MAP of the Fetal Brain Injury Associated with Chronic Hypoxia

Injured areas in the mirror sections were selected at the interaural level of 6.72mm to 5.40 mm (Bregma: from −2.28 mm to −3.60mm) were reconstructed based on quantification of immunostaining and TUNEL staining. The quantification of ATF3, GFAP and TUNEL were projected onto the standard coronal section. Chronic fetal hypoxia increased ATF3 (red), GFAP (green), and TUNEL (blue) staining specifically in cerebral cortex, hippocampus, and thalamic / hypothalamic nuclei. In cortex, the damage was widely distributed, while in hippocampus, the injury was greatest in cingulum, orices layer, and CA1, CA2, CA3 layers. In thalamic nuclei, the injury was greatest in the reuniens, central, ventral, and laterodorsal thalamic nuclei. The injury markers were also up regulated in the dorsomedial and ventromedial hypothalamic nuclei and lateral hypothalamus.

Figure 3. Neuronal Loss is Associated with Chronic Fetal Hypoxia

Neural cell density expressed as the total Nissl staining cells/ mm². Five areas were randomly selected from each slide, digitally photographed, and neuronal density quantified using the Stereo Investigator 8.0 system. Chronic hypoxia over the latter 30% of gestation reduced neuronal density by some 20% in fetal cerebral cortex, hippocampus and thalamus nuclei.

Figure 4. mRNA Expression of Brain Injury Indices is Increased by Chronic Fetal Hypoxia

Cerebral cortex, hippocampus and thalamus nuclei were selectively biopsied at the sites of injury discovered in the brain slices using laser capture microdissection (LCM) and ATF3, GFAP and Bax mRNA expressions measured by Q-rtPCR. The mRNA expression for each injury marker was up regulated by chronic hypoxia (n=6, p<0.05) in both whole brain samples and at each of the LCM biopsied sites. These results, combined with the immunostaining findings demonstrate that chronic hypoxia selectively injures the fetal brain and that both mRNA and protein levels are increased.

Figure 5. Chronic Hypoxia Differentially Impacts NO Synthases in a Manner that Reflects the Anatomic Location

NOS isoform mRNA was measure by Q-rtPCR in whole brain and at sites of injury in cerebral cortex, hippocampus and thalamus biopsied using LCM. In fetal whole brain (panel A), both nNOS and iNOS expressions were increased, but eNOS decreased. However, the impact of chronic hypoxia on NOS isoform expression varied by site. iNOS was increased, eNOS was decreased, and nNOS unchanged in hippocampus (panel B). In cerebral cortex, only iNOS was increased (panel C). In thalamus, nNOS and iNOS were both increased by chronic hypoxia but eNOS was unchanged (panel D).

Figure 6. iNOS Co-localizes with Markers of Brain Injury

Co-localization of iNOS and injury markers was sought using double fluorescent staining techniques (cross section hippocampus, ATF3 and GFAP, 10x). Chronic hypoxia increased iNOS (panels A1 vs. B1 and C1 vs. D1), ATF3 (panels A2 vs. B2) and GFAP panels (C2 vs. D2). iNOS co-localized with both ATF3 and GFAP positive cells (panels B3 and D3).

Figure 7. nNOS Co-localizes with Markers of Brain Injury

Co-localization of nNOS and injury markers was sought using double fluorescent staining techniques (cross section hippocampus, ATF3 and GFAP, 10x). Chronic hypoxia increased nNOS (panels A1 vs. B1 and C1 vs. D1), ATF3 (panels A2 vs. B2), GFAP (panels C2 vs. D2). nNOS co-localized with both ATF3 and GFAP positive cells (panels B3 and D3).

Figure 8. ATF3 is Present in Injured Fetal Neuronal and Glial Cells During Chronic Hypoxia

Neuronal and glial cells were selectively stained for Tubulin III and GFAP, respectively (cross section hippocampus, 10x). Chronic fetal hypoxia increased ATF3 (panels A2 vs. B2, C2 vs. D2). Chronic hypoxia decreased neuronal density (panels A1 vs. B1). GFAP was activated by chronic hypoxia indicating the development of gliosis (panels C1 vs. D1). However, and in contrast to adult studies, ATF3 was not unique to neurons (A3 vs. B3), but also co-localized to glial cell after chronic hypoxia (panels C3 vs. D3).

Figure 9. L-NIL Selectively Inhibits iNOS in Fetal Brain During Chronic Hypoxia

NOS isoforms mRNA expression was quantified by Real-time PCR using the fetal whole brain tissue (n=6). nNOS and iNOS but eNOS was up-regulated significantly by chronic hypoxia and unaffected by L-NIL; only iNOS was attenuated by L-NIL.

Figure 10. L-NIL Blunts the Increase in Total NO in Fetal Brain Secondary to Chronic Hypoxia

Whole fetal brain total NO (NO₂⁻ and NO⁻) was increased by chronic fetal hypoxia (HPX, n=6) vs. control animals (NMX, n=6), but significantly decreased in L-NIL treatment group vs. HPX.

Figure 11. L-NIL Reduces Indices of Brain Injury During Chronic Hypoxia

ATF3, GFAP, and Bax were applied as indices of brain injury (n=6). Chronic hypoxia up regulated the expression of each; L-NIL attenuates the effect of unopposed chronic hypoxia.

Figure 12. L-NIL Prevents Neuronal Loss in Fetal Hippocampus During Chronic Hypoxia

The impact of L-NIL on neuronal loss during chronic fetal hypoxia is illustrated here for hippocampus. Neural cell density is expressed as the total Nissl staining cells/ mm². L-NIL essentially eliminated neuronal loss secondary to chronic hypoxia.