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High frequency oscillatory ventilation (HFOV) may improve pulmonary outcome in very preterm infants but the effects on the brain are largely unknown. We hypothesized that early prolonged HFOV compared to low volume positive pressure ventilation (LV-PPV) would not increase the risk of delayed brain growth or injury in a primate model of neonatal chronic lung disease. Baboons were delivered at 127±1 days` gestation (dg; term ~185dg), ventilated for 22–29 days with either: LV-PPV (n=6) or HFOV (n=5). Gestational controls were delivered at 153dg (n=4). Brains were assessed using quantitative histology. Body, brain and cerebellar weights were lower in both groups of prematurely delivered animals compared to controls; the brain to body weight ratio was higher in HFOV compared to LV-PPV and the surface folding index was lower in LV-PPV compared to controls. In both ventilated groups compared to controls, there was an increase in astrocytes and microglia and a decrease in oligodendrocytes (p<0.05) in the forebrain and a decrease in cerebellar granule cell proliferation (p<0.01); there was no difference between ventilated groups. LV-PPV and HFOV ventilation in prematurely delivered animals is associated with decreased brain growth and an increase in subtle neuropathologies; HFOV may minimize adverse effects on brain growth.
Advances in prenatal and neonatal care including respiratory support have significantly improved survival rates for prematurely delivered infants in recent years. These infants however still have a higher incidence of poor neurodevelopmental outcome than full term infants with approximately 10% at risk of developing cerebral palsy and 10–20% likely to have some form of developmental delay or sensory or motor impairment (1,2). Thus, it is critical to understand the effects of postnatal intervention on the developing brain and, in particular, to determine if specific modes of respiratory support and other postnatal treatments influence the nature and severity of cerebral injury.
High frequency oscillatory ventilation (HFOV) is used in the neonatal setting as both primary and rescue therapy as it is believed to cause less trauma to the immature lungs compared to other forms of mechanical ventilation. In models of neonatal lung injury HFOV has been shown to improve pulmonary outcome (3,4). Recent meta-analyses in human studies have cast some doubt on whether there is a better prognosis for chronic lung disease (CLD) after HFOV compared to an optimised protocol of conventional mechanical ventilation (5,6). The effects on the brain are also controversial with some studies suggesting an increased risk of both intracranial hemorrhage and periventricular leucomalacia (7,8) and others reporting that there is no difference in neonatal associated morbidity, including hemorrhage (9). Long-term neurodevelopmental (10) and neuromotor (11) outcomes do not appear to be worse in infants ventilated with HFOV compared to conventional ventilation.
Neuropathological analysis of the effects that specific respiratory regimens have on the human preterm brain are likely to be complicated by the processes which contributed to the infant’s death. An animal model with brain and cardiopulmonary development comparable to the human preterm neonate, managed in a neonatal intensive care environment closely mimicking the current approach for humans, may provide a better understanding of the effects of various interventional support strategies. We have established that the prematurely-delivered baboon at 125 days of gestation (dg) has similar brain (12) and cardio-respiratory (13) development to the very preterm human infant at about 26–28 weeks of gestation and is thus a highly appropriate model for such investigations. Our aim here was to deliver baboon neonates at 125dg and study brain growth and the pattern of cerebral injury in this model of neonatal CLD following early, prolonged HFOV compared to low tidal volume positive pressure ventilation (LV-PPV). We hypothesized that early sustained HFOV compared to LV-PPV would not increase the risk of brain injury and/or altered brain development and growth associated with prematurity.
Studies were performed at the Southwest Foundation for Biomedical Research, San Antonio, TX. All animal husbandry, handling, and procedures were approved to conform to American Association for Accreditation of Laboratory Animal Care guidelines.
Pregnant baboon dams (Papio papio) were treated with 6 mg of intramuscular betamethasone 48 and 24 hours prior to elective hysterotomy under general anesthesia. Study animals were delivered at 127±1 dg (term ~185 dg). At birth, animals were weighed, sedated, intubated, and treated with 4ml/kg bolus of exogenous surfactant (Survanta, courtesy Ross Laboratories, Columbus, OH) through the endotracheal tube. For gestational control brains, additional pregnant baboon dams were treated with 6mg of intramuscular betamethasone at 123 and 124 dg and animals (n=4) delivered by elective hysterotomy at 153dg; animals were euthanized immediately.
Management of ventilated animals has been described in detail previously (3). At 5 min of age, based on prior antenatal assignment the animals were converted to HFOV or maintained on low tidal volume (4–6 ml/kg) PPV. Similar goals were set and achieved for oxygenation (55–70 torr) and ventilation (45–55 torr). Surgical ductal ligation was performed on 3 animals in the HFOV group and 1 animal in the LV-PPV group. Three other animals in the LV-PPV group were treated with indomethacin for patent ductus arteriosus. More detailed methods related to management of nutrition, ventilation, blood pressure, and infection can be found in a prior publication (3).
Brains were weighed, immersed in 4% paraformaldehyde in 0.1M phosphate buffer and coronal blocks from the right forebrain (at 5mm intervals) and a mid-sagittal block from the cerebellar vermis of each brain were processed to paraffin. Ten 8µm sections were cut from the rostral surface of each forebrain block (10–12 per animal) and in the sagittal plane for the cerebellum.
Analyses were performed on sections from each block for all brains of LV-PPV, HFOV, and gestational control animals; measurements were made on coded slides. Areas and widths were assessed using a digitizing program (Sigma Scan Pro v4, SPSS Science, Chicago, IL, USA); optical density (OD) estimates and counts performed using an image analysis system (Image Pro Plus v4.1, Media Cybernetics, Maryland, USA). Means were calculated for each animal and then a group mean determined.
Sections were stained with hematoxylin and eosin (H&E) and assessed qualitatively for gross morphological changes including lesions and the presence of hemorrhages (scored: present - 1; absent - 0).
Rabbit anti-rat calbindin (1:500, Swant, Bellinzona, Switz) was used to identify Purkinje cells; rabbit anti-cow glial fibrillary acid protein (GFAP; 1:500, Sigma, St Louis, MO, USA) to identify astrocytes: mouse anti-human Ki-67 clone MIB-1 (1:100; DakoCytomation, Denmark) to identify mitotic cells; rat anti-bovine myelin basic protein (MBP, 1:100; Chemicon, USA) to assess the extent of myelination; rabbit anti-ionized calcium-binding adapter molecule 1 (Iba1, 1:1500, Wako Chemicals, Osaka, Japan) to identify microglia/macrophages, and rabbit anti-human p27 (Kip1) cyclin-dependent kinase inhibitor (1:1000, Millipore, Billerica, MA USA), to identify post-mitotic cells, as previously described (14). Control and experimental material was stained simultaneously to avoid procedural variation. Control experiments were performed omitting the primary antibodies whereupon staining failed to occur.
The following measurements were made in the forebrain: volumes of the white and grey matter components (12); surface folding index (SFI; 12); areal densities of astrocytes in the neocortex, white matter and hippocampus (15); somal areas and areal densities of oligodendrocytes in the white matter (15); areal densities of microglia in the neocortex and white matter. The following measurements were made in the cerebellum: areas of cerebellar layers (14); Purkinje cell (14) and granule cell somal areas; Ki67-IR cell counts in the EGL and WM (14); areal densities of oligodendrocytes and microglia in the deep WM (14); GFAP-IR optical density in the deep WM (14) (Supplemental materials link).
GFAP-IR radial glial fibers in the forebrain were scored on a scale of 0–3 (15).
Physiological data, including arterial blood gases (PaO2, partial pressure of oxygen, PaCO2, partial pressure of carbon dioxide, pH, fraction of inspired oxygen (FiO2)), mean arterial blood pressure (MAP) and heart rate, were monitored throughout the experimental period and mean values calculated. The “interval flux” of physiological parameters was calculated as a surrogate measure of instability as described previously (14). For each animal we then: 1) identified the maximum flux; and 2) calculated the mean of the interval fluxes over the experimental period.
Linear regression analysis was carried out to determine if there was a correlation between: a) physiological variables and quantitative parameters; b) quantitative parameters and volumetric measurements. Significance of differences between ventilated and control groups was tested using a one-way ANOVA with post-hoc analysis (Tukey’s test) for histological parameters; t-tests were used to compare between ventilated groups for physiological parameters; a probability of p<0.05 was considered to be significant. Results are expressed as mean ± SEM (weights and areas) and mean of means ± SEM (histological parameters).
Birth weight was lower (p<0.03) in HFOV (353±18g) compared to LV-PPV (406±11g) animals. The gestational age at birth (LV-PPV, 126±1dg vs HFOV, 127±1dg), age at post-mortem (LV-PPV, 26±1d vs HFOV, 27±1d), and male/female ratios (LV-PPV, 4/2 vs HFOV, 2/3) were not different between groups.
At necropsy body, brain and cerebellar weights were lower in both groups of prematurely delivered animals compared to gestational controls (p<0.01; Table 1). The body weight was lower (p<0.05) and the brain to body weight ratio higher (p<0.01) in HFOV compared to LV-PPV. The cerebellar/body weight ratio was lower in LV-PPV compared to control (p<0.05) and HFOV (p<0.01) animals (Table 1). The cerebellar/brain weight was not different between groups (p>0.05). The failure of HFOV animals to gain body weight over the 28 days of the study might be due to increased caloric use during periods of extubation.
Compared to gestational controls the total, neocortical, WM, and deep grey matter volumes were reduced (p<0.001) in both prematurely delivered groups (Table 2). Although ventricular volume in absolute terms was not different (p>0.05) from controls in either group, when expressed as a percentage of the total hemispheric volume, it was increased in both LV-PPV (p<0.01) and HFOV (p<0.001) animals. The SFI was reduced in LV-PPV (p<0.05) but not HFOV compared to controls.
No focal necrotic lesions or hemorrhages were observed in any animal. Examination of Ki-67-IR- sections showed that cell proliferation was minimal in the subventricular and subgranular zones at 153dg in all animals; no differences were observed between groups.
There was an increase in density in the deep (p<0.001) and subcortical WM (p<0.01; Figures 1A–C), neocortex (p<0.05) and the stratum radiatum of the hippocampus (p<0.01) in LV-PPV and HFOV animals compared to controls but no difference between ventilated groups (Table 3).
There was a reduction (p<0.05) in density in deep (Figures 1D–F) and subcortical WM in LV-PPV and HFOV animals compared to controls (Table 3). There was a reduction in the somal areas of MBP-IR oligodendrocytes in the deep (p<0.05; control, 84.3±2.6µm2; LV-PPV, 70.8±3.3 µm2; HFOV, 66.5±2.8 µm2) but not subcortical WM (p>0.05) in ventilated animals compared to controls. There was no difference between ventilated groups in any of these parameters.
Ramified Iba1-IR cells were increased in subcortical WM (Figures 1G–I) and neocortex in the LV-PPV and HFOV groups compared to controls (p<0.05); there was no difference between ventilated groups (Table 3). Activated microglia (round morphology and attenuated processes) were observed infrequently with no greater incidence in prematurely delivered groups compared to gestational controls.
GFAP-IR radial glia were rarely observed in control brains at 153dg but evident at the ventricular surface projecting into the deep and occasionally the subcortical WM in LV-PPV (p<0.05) and HFOV (p<0.01) animals (Table 3).
There was no evidence of hemorrhages or overt damage.
The cross-sectional area of the vermis was reduced in LV-PPV (p<0.01) and HFOV (p<0.05) animals compared to controls, as was the volume of the IGL (p<0.001), the widths of the EGL (p<0.001; Figures 2A–C)) and the ML (p<0.01; Figures 2A–C; Table 4). The WM volume was not different between ventilated groups (p>0.05).
For the following parameters, data from lobules 1 and 8 has been pooled, as there was no difference between lobules.
The number of Ki-67-IR cells in the EGL was reduced in LV-PPV and HFOV animals compared to controls (p<0.001; Table 5; Figs 2D–F). In control and HFOV animals, approximately 50% of cells in the EGL were Ki-67-IR; in LV-PPV animals, this was higher (64%; p<0.05; Table 5) suggesting abnormalities in the cell cycle. In all animals, EGL cells not Ki-67-IR were P27-IR (data not shown). There was a decrease in Ki67-IR Bergmann glial cells in ventilated animals compared to controls (p<0.001). Ki67-IR cells were seen throughout the deep WM; there was no difference (p>0.05) between groups.
Somal areas were reduced (p<0.001) and the areal density of Purkinje cells increased in lobule 8 (p<0.05) in ventilated animals compared to controls (Table 5).
Ramified Iba-IR cells were observed throughout the cerebella in all groups; the density was higher (p<0.05) in the ML in ventilated animals compared to controls; there was no difference between groups in the IGL (Table 5; p>0.05).
There was no difference (p>0.05) between groups in either parameter (Table 5).
There was no difference (p>0.05) in the mean interval flux of pH, PaO2, PaCO2, FiO2, MAP or heart rate between LV-PPV and HFOV animals during the 28d study period (Table S1,Supplemental materials link).
There were negative correlations between: the mean interval flux in FiO2 and both the density of deep WM oligodendrocytes (r2=0.42; p<0.03) and forebrain WM volume (r2=0.45; p<0.02); density of astrocytes and density of oligodendrocytes in the deep WM (r2=0.47, p<0.005); forebrain WM volume and density of astrocytes (r2=0.79, p<0.0001). There were positive correlations between the mean interval flux in FiO2 and the density of hippocampal astrocytes (r2=0.37; p<0.05) and between the density of astrocytes and density of microglia in both the deep (r2=0.50; p<0.03) and subcortical (r2=0.54; p<0.002) WM; there were no correlations between any physiological parameters and structural alterations in the cerebellum.
We have demonstrated that long-term ventilation of the prematurely–delivered baboon neonate with both LV-PPV and HFOV is associated with reduced brain growth and an increase in subtle brain injury compared to gestational controls. Animals ventilated with HFOV were not at a greater risk of injury or reduced brain growth than those ventilated with LV-PPV; neither regimen was associated with hemorrhage or overt GM or WM damage. Our findings therefore concur with human studies which have shown that HFOV is not associated with an increased risk of neonatal morbidity including intracranial hemorrhage (1,2). In translating our findings to the human neonate, we acknowledge that they are based on small animal numbers and that preterm baboons were delivered without any pre-existing complications such as infection, hypoxemia, or growth restriction.
Premature delivery and prolonged ventilation with either HFOV or LV-PPV was associated with a reduction in brain weights and volumes compared to controls however the brain to body weight ratio was higher in animals ventilated with HFOV than with LV-PPV. The SFI, a measure of cortical gyrification was reduced in LV-PPV but not HFOV animals compared to controls. Gyrification of the cerebral hemispheres is thought to reflect development of cerebral and subcortical connectivity (16) hence connectivity might be reduced or delayed in LV-PPV animals. Taken together, these data suggest that HFOV ventilation might minimize the adverse effects of very preterm birth on brain growth.
We observed subtle neuropathological alterations in GM and WM in both groups of prematurely delivered animals however there was no difference between groups in any of the quantitative parameters. In both groups we identified mild ventriculomegaly which could result from cell loss or a lack of growth of the neuropile; ventriculomegaly is associated with an increased risk of mild to moderate learning difficulties (17). Astrogliosis was observed throughout the forebrain in both prematurely delivered groups. Reactive astrocytes produce IGF-1 and FGF (18) to support neuronal survival but they also produce cytokines (19) and reactive oxygen species which could exacerbate any underlying injury via oxidative and inflammatory pathways. Microgliosis was observed in the cortex and subcortical WM; these microglia were predominantly in a ramified state. Microglia might be playing either a protective role by releasing growth factors or, when activated, a harmful role through the release of cytokines (20) which will exacerbate any neuronal injury.
Radial glial fibers, which were not present in control animals at 153dg, were evident in the forebrain of both groups of prematurely delivered animals. It is possible that the normal maturation of radial glia into astrocytes has been inhibited by cerebral hypoxia leading to their prolonged presence in the developing brain (18). Alternatively, hypoxia might have caused astrocytes to revert to radial glia (18) in an attempt to repair damage as radial glia are neuronal precursors. Although every effort is made in the NICU to keep blood oxygen saturation at optimal levels, immaturity of respiratory control means that periods of mild hypoxemia might occur.
The reduction in both somal size and density of MBP-IR myelinating oligodendrocytes in the WM of LV-PPV and HFOV animals compared to controls suggests a significant effect on myelination with consequences for the normal functioning of axonal pathways. No cystic infarction in the white matter was noted in any animal. The diffuse pattern and more subtle nature of injury in the preterm baboon brain is consistent with that which is reported in recent neuroimaging studies in the preterm infant (21).
We observed microstructural alterations within the vermis in both groups of prematurely delivered animals. Cerebellar weights and morphological parameters were comparable to those seen between 125 and 140dg (14) suggesting developmental delay. We posit that specific alterations in developmental events are also involved, particularly reduced proliferation of granule cells; the EGL, was significantly thinner than it would be at any stage of normal development (14). This effect on neurogenesis was the most profound that we have observed with any of the postnatal intervention strategies investigated in the preterm baboon (12,14,15,22). The reduction in Bergmann glial cell density in ventilated animals could affect granule cell migration and influence Purkinje cell dendritic alignment (23). The reduction in Purkinje cell somal size is likely to be associated with reduced dendritic growth (24) and account for the thinner molecular layer and increased Purkinje cell density. These alterations could have a long lasting effect on the complex pattern of cerebellar connectivity and hence function.
Microgliosis in the molecular layer suggests an underlying inflammatory response with possible consequences similar to those mentioned above for the forebrain. Unlike the forebrain, myelination did not appear to be affected in the cerebellum.
The mechanisms underlying reduced growth and injury in both HFOV and LV-PPV groups compared to gestational controls are likely to be multi-factorial and could include altered nutritional status, fluctuations in blood gases, length of time intubated and subsequent activation of inflammatory cascades. The finding that HFOV appeared to minimize adverse outcomes on brain growth might relate to more favorable oxygenation; HFOV animals have better lung function with sustained improvement in pulmonary mechanics compared to animals managed with LV-PPV (3). In all ventilated animals, higher interval fluxes in FiO2 were associated with increased astrocyte densities in the hippocampus and reduced oligodendrocyte densities and forebrain white matter volume, suggesting that greater variation in oxygen delivery appears to result in a greater level of brain injury.
In both LV-PPV and HFOV groups cerebellar growth was more markedly affected than forebrain growth possibly as a consequence of the rapid growth of this structure during the premature period (14, 25) and hence its enhanced vulnerability.
HFOV and LV-PPV have poorer outcomes for brain growth and more injury and structural alterations than previously observed after 28 days of a more passive ventilatory regimen, nasal continuous positive airway pressure (nCPAP) ventilation, particularly early nCPAP (14,22). This could be due to longer periods of mechanical ventilation, less stable oxygen delivery, increased stress and increased inflammation amongst other factors. For example, there was a greater degree of fluctuation in FiO2 in both LV-PPV and HFOV animals compared to early (but not delayed) nCPAP (22). The density of astrocytes in the cerebral hemispheres of animals ventilated with HFOV and LV-PPV was 40% higher than with nCPAP (22) suggesting that there might be a greater level of underlying neuronal stress. In the cerebellum, both LV-PPV and HFOV ventilation appeared to have an adverse affect on granule cell neurogenesis not observed with either early or delayed nCPAP (14). We suggest that this might be a consequence of the sensitivity of cell division to fluctuations in oxygen and/or inflammation.
In conclusion, early sustained HFOV compared to LV-PPV did not increase the risk of brain injury and/or altered brain development in prematurely delivered baboons. Both ventilatory regimens were associated with an overall decrease in brain growth; there was a tendency for HFOV to minimize adverse effects. In all prematurely delivered animals, subtle neuropathologies were observed in the forebrain and cerebellum. Such adverse effects on brain morphology could contribute to neurodevelopmental delay or sensory and motor impairments in postnatal life.
We are grateful to Dr Jaqueline Coalson, Ms Vicki Winter, and staff at the Bronchopulmonary Dysplasia Resource Centre, San Antonio, Texas for provision of baboon tissue and Ms Kathryn Munro for histological assistance.
Financial Support: NIH Grant R01 HL074942. Supported in part by NIH grants HL52636 and HL52646.
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