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Ibuprofen is an effective pharmacological intervention for closure of a patent ductus arteriosus in preterm infants, and is an alternative to surgical ligation; however it is not certain whether ibuprofen treatment is associated with adverse effects on the brain. Therefore, this study examined neuropathological outcomes of ibuprofen therapy for a patent ductus arteriosus. Fetal baboons were delivered at 125-days of gestation (dg, term ~185dg) by caesarean section, given surfactant and ventilated for 14-days with positive pressure ventilation. Baboons were randomly allocated to receive either ibuprofen (PPV + ibuprofen, n=8) or no therapy (PPV, n=5). Animals were euthanased on day 14 and brains assessed for cerebral growth, development and neuropathology. Body and brain weights, the total volume of the brain and the surface folding index (measure of brain growth) were not different (p>0.05) between PPV + ibuprofen-treated and PPV animals. There was no difference (p>0.05) in the number of myelin basic protein-immunoreactive oligodendrocytes, glial fibrillary acid protein-immunoreactive astrocytes or Iba1-immunoreactive macrophages/microglia in the forebrain. No overt cerebellar alterations were observed in either group. Ibuprofen treatment for patent ductus arteriosus closure in the preterm baboon neonate is not associated with any increased risk of neuropathology or alterations to brain growth and development.
Survival of prematurely-delivered and low birth weight infants has improved in recent years due to the advent of improved prenatal and neonatal care strategies. These infants are however at greater risk of poorer neurological outcomes than their full term counterparts with approximately 10% likely to develop cerebral palsy, while sensory and motor impairments and developmental delays are observed in 10-20% of infants (1-3). There is consequently considerable interest in the role that post-natal management and intervention strategies may play in neurological development.
Hemodynamic symptoms from a patent ductus arteriosus (PDA) are present in 55-70% of infants delivered below 1000g or prior to 28 weeks of gestation, with the PDA resulting in alterations in cerebral, renal, and mesenteric perfusion as well as producing pulmonary edema, impairing pulmonary mechanics, increasing the risk of pulmonary hemorrhage, and prolonging the need for mechanical ventilation (4). A persistent PDA is associated with the development of bronchopulmonary dysplasia (BPD); its direct role in causing BPD is not known however (5-7).
Current therapies for a PDA include pharmacological treatment with indomethacin or ibuprofen and/or surgical ligation. Surgical ligation produces definitive closure of a PDA but there is controversy as to its effects on subsequent neurodevelopmental outcome (5, 7). The trauma of surgery and the possible adverse effects of anaesthetics on the brain (8) are also factors to consider. Ibuprofen and indomethacin are effective treatments for closure however only a few studies have investigated whether there are any neuropathological or functional sequelae (9-13).
Assessment of such outcomes in humans is likely to be complicated by the unique and varying treatments that each infant experiences while in the neonatal intensive care unit (NICU) setting. We have the unique opportunity to examine neurological development in a baboon model, prematurely delivered at 125 days of gestation (dg), which has comparable brain (14) and cardiopulmonary (15, 16) development to human preterm infants at 26-27 weeks gestation. These baboons are maintained in a NICU setting which is similar to that used for premature human infants. We therefore have an appropriate model in which to investigate the influence of postnatal interventional strategies which have relevance to prematurely-delivered human infants.
Premature baboons have a similar neonatal course to human infants, developing respiratory distress and failure of PDA closure after birth. They develop histopathological changes in the lung similar to those described for human infants with BPD despite antenatal glucocorticoid treatment, early postnatal surfactant replacement, low tidal volume ventilation and low supplemental oxygen administration during the first 14 days post delivery (15, 16). Pharmacological closure with ibuprofen improves pulmonary mechanics, and decreases the detrimental effects of preterm birth on alveolarization (17). As the impact of ibuprofen on the immature non-human primate brain is not known, our objective in the present study was to examine brain growth and development in our premature baboon model of neonatal chronic lung disease (CLD) following pharmacological treatment of the PDA with ibuprofen commencing at 24 hours of age. We hypothesised that ibuprofen treatment will not increase the risk of neuropathology or impair brain growth compared to no treatment.
All animal studies were performed at the Southwest Foundation for Biomedical Research in San Antonio, TX. All animal husbandry, animal handling, and procedures were reviewed and approved to conform to American Association for Accreditation of Laboratory Animal Care guidelines.
Pregnant baboon dams (Papio papio) with timed gestations were treated with 6mg of intramuscularly administered betamethasone 48 and 24 hours before elective delivery at 125±2 days of gestation (dg, term ~185 days). At birth animals were weighed, sedated, intubated and treated with 4ml/kg surfactant (Survanta, courtesy Ross Laboratories, Columbus, OH) prior to the initiation of ventilatory support.
Newborn baboons were mechanically ventilated for 14 days. A complete description of the details of the surgical procedures and animal care (including ventilator management, target goals for PaO2, PaCO2, tidal volume, and nutritional, fluid, transfusion, antibiotic and blood pressure management) have been described previously (17). Animals were randomized before delivery to either receive ibuprofen (PPV + ibuprofen, n=8) or no treatment (PPV, n=5). Ibuprofen was administered intravenously (over 20 minutes) according to the following schedule: 10mg/kg (24 hours), 5mg/kg (48 hours), 5mg/kg (72 hours), 5mg/kg (96 hours) and 5 mg/kg (120 hours).
At necropsy (at 14 days), brains were weighed and 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.
A section from each block was stained with haematoxylin and eosin (H&E) and assessed qualitatively for gross morphological changes including lesions and the presence of haemorrhages (scored: present - 1; absent - 0). Masson’s trichrome was used to assess for collagen deposition; Van Gieson’s stain for elastic fibres, reticulin for reticulated fibres; Perls stain to visualise hemosiderin deposition.
Rabbit anti-rat calbindin (1:500, Swant, Bellinzona, Switzerland) was used to identify cerebellar Purkinje cells; rabbit anti-cow glial fibrillary acid protein (GFAP; 1:500, Sigma, St Louis, MO, USA) to identify astrocytes; rat anti-bovine myelin basic protein (MBP, 1:100; Chemicon International, CA, USA) to assess myelination; rabbit anti-ionized calcium-binding adapter molecule 1 (Iba1, 1:1500, Wako Chemicals, Richmond, VA, USA) to identify microglia/macrophages, as described previously (18). Control and experimental material was stained simultaneously to avoid procedural variation. There was no staining when the primary antibody was omitted.
Analyses were performed on a section from each block for all brains of PPV + ibuprofen and PPV animals unless otherwise stated; measurements were made on coded slides. Areas and widths were assessed using a digitizing program (Sigma Scan Pro v4, SPSS Science, Chicago, IL, USA) and counts performed using an image analysis system (Image Pro Plus v4.1, Media Cybernetics, Maryland, USA). All measurements were performed on all animals; means were calculated for each animal and then a group mean determined.
A thorough qualitative assessment of each brain was carried out in each block including all regions (cerebral hemispheres including white matter, neocortex, basal ganglia, thalamus, hippocampus and the cerebellum) by two examiners. Sections were assessed for presence of lesions, neuronal death and focal gliosis ( astrogliosis or microgliosis) glia and microglial invasion.
In H&E-stained sections, the volumes of white matter, neocortex, deep grey matter (basal ganglia plus thalamus), hippocampus and ventricles were estimated by measuring the cross-sectional area of each region in a section from each block (10-12 per animal) and applying the Cavalieri principle (10).
In H&E-stained sections from each block, the surface folding index (SFI), which gives an estimation of the expansion of the surface area relative to volume, was determined (6).
Point counting (12) was used to determine the percentage of WM occupied by blood vessel profiles in deep and subcortical white matter (x660) as an indicator of vasodilation or vasculogenesis. Assessment was performed in GFAP-IR sections at each level (~12 sections, 2 regions in each section, 24 measurements/ region in total) as blood vessel profiles are clearly delineated (13).
GFAP-IR cells were counted (x660) in two randomly selected areas (0.02mm2) in each of the deep and subcortical white matter regions (~12 sections/animal, 24 measurements/region in total); the cerebral neocortex (three sites in blocks from frontal/temporal, parietal/temporal and occipital lobes in layers 5 and 6); and the hippocampus (stratum radiatum in the CA1 region, 2 sections/ animals, 4 measurement in total).
MBP-IR oligodendrocytes were counted (x300) in two sections per animal. Two regions (0.42mm2) in each section were randomly selected in both the deep and subcortical white matter from the parietal/temporal lobe (4 measurements/region in total).
Iba1-IR cells were counted (x660) in randomly selected areas (0.02mm2) in each of the deep and subcortical white matter regions (three sites in blocks from each of the frontal/temporal, parietal/temporal and occipital lobes, 6 measurements/region in total).
In H&E-stained sections the width of the external granule layer (EGL) was assessed in the cerebellum (19) in 10 randomly selected regions in each of lobules 1 and 8 (3 measurements/ region, 60 measurements/animal in total).
GFAP- immunoreactive (IR) radial glial fibres in the forebrain were scored on a scale of 0-3 (18).
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 (HR), 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 (20). The interval flux for a specific physiological variable was the difference between the maximum and minimum values of the variable during a specified time interval (15). For each animal we then: 1) identified the maximum flux; and 2) calculated the mean of the interval fluxes over the entire experimental period. In previous studies a greater degree of flux particularly in FiO2 has been associated with an increased incidence of neuropathology (18, 20-22).
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 PPV + ibuprofen and PPV groups was tested using t-tests; 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).
The ductus in the PPV + ibuprofen group either closed (and remained closed, n=5), or closed (and then intermittently reopened, n=3) after PPV + ibuprofen treatment. In contrast, all of the animals in the PPV group had a patent ductus arteriosus that remained open throughout the entire 14 days experiment (mean pulmonary to systemic blood flow (Qp/Qs) ratio from 24 hrs through 14 days PPV, 1.8±0.2 vs PPV + ibuprofen, 1.1±0.1; p<0.001). The PPV + ibuprofen group had a significantly higher (p<0.05) mean (as well as systolic and diastolic) systemic blood pressure following the start of treatment. There were no differences (p>0.05) between the two groups in base deficit, serum bicarbonate or need for dopamine/dobutamine administration during the 14-day treatment course. There were also no differences (p>0.05) in the fluid intake and urine output between the two groups (17).
There was no difference (p>0.05) between groups in the body, brain, cerebellar weights or brain/body weight ratio (Table 1).
There was no difference (p>0.05) between groups in the total volume of the forebrain (right), white matter, neocortical, deep grey matter or hippocampal volumes (Table 1). There was no difference (p>0.05) between the groups in the ratios of white matter, neocortical or deep grey matter volumes to the total forebrain volume nor in the ratio of white matter/neocortex (Table 1).
The overall SFI of the forebrain was not different (p>0.05) between groups (Table 1).
There was no evidence of cerebral infarction or haemorrhages (intraventricular, intracerebral or subventricular) in any animal in either group. There was also no evidence of cystic or non-cystic lesions, neuronal loss (pyknosis or apoptosis), hypertrophic astrocytes or focal aggregates of astroglia or microglia in either group in any region of the forebrain including the white matter, neocortex, hippocampus and deep grey matter (basal ganglia and thalamus) or the cerebellum.
There was no difference (p>0.05) between groups in either the deep or subcortical white matter, cerebral neocortex or the stratum radiatum of the hippocampus (Table 2).
The overall density in both the deep and subcortical white matter was not different (p>0.05) between groups (Table 2).
There was no difference (p>0.05) between ventilated groups in the number of ramified Iba1-IR cells in the deep and subcortical white matter or within the neocortex (Table 2). Activated microglia (round morphology and attenuated processes) were observed infrequently in both groups.
Intensely GFAP-IR radial glial fibres were present at the ventricular surface projecting into the deep white matter in all animals. There was no difference (p>0.05) in the occurrence of GFAP-IR radial glial between groups (Table 2).
No haemorrhages, regions of infarction or overt structural anomalies were observed in either group. There was no difference (p>0.05) in the width of the EGL between groups (PPV + ibuprofen, 31.8±1.6mm vs PPV, 33.1±2.5mm).
There was an increase (p<0.05) in the mean interval flux of PaO2 in PPV compared to PPV + ibuprofen-treated animals. There was no difference (p>0.05) in the mean interval flux of pH, PaCO2, FiO2, MAP or HR between groups during the 14-day study period (Table 3).
The major finding of this study is that pharmacological closure of a PDA with ibuprofen does not pose an increased risk of brain injury and/or impairment in brain development compared to no treatment in the first days of life. There was no difference between treated or untreated animals in brain weight, gyral formation or relative growths of white and grey matter. Nor was there a difference in astrocyte, oligodendrocyte or microglial densities in any region of the brain suggesting that ibuprofen treatment does not cause astrogliosis, affect the oligodendrocyte lineage and myelination or have an impact on the cerebral inflammatory response. Data from gestational control animals has not been included here as we have already established in previous studies using this model (18, 20-22), that premature delivery per se reduces the normal trajectory of brain growth and increases the incidence of subtle neuropathologies; the purpose of the present study was to examine the difference between treatment groups. We cannot directly compare the neuropathological outcomes from the present study where animals received antenatal steroids, with our earlier study on ductal ligation where steroids were not administered (20); however, we note that neither regimen was associated with overt damage such as haemorrhage or cystic infarction.
It is possible that our findings could be viewed from a different perspective. In the study alluded to above (20) we found that animals exposed to a persistent PDA had subtle increases in neuropathology and a trend towards poorer brain growth compared with animals with ductal ligation If the patent ductus arteriosus does contribute to adverse effects on the premature brain, then one might anticipate that its effective closure with ibuprofen should have resulted in fewer astrocytes, increased density of oligodendrocytes, and better cerebral growth. This was not observed in our study.
It is possible that the use of antenatal glucocorticoids, in our present study, minimizes the differences between the two treatment groups. It is also possible that the lack of difference indicates that any benefit derived from PDA closure was negated by ibuprofen. We think the second alternative is less likely because pharmacological treatment of PDA with cyclo-oxygenase inhibitors has been associated with better, not worse, neurological outcomes in preterm infants. Prophylactic indomethacin has been associated with a decreased risk for periventricular and intraventricular haemorrhage (IVH) (11, 13) and appears to have no adverse effects on later neurodevelopmental outcome (11-13). Furthermore, early ibuprofen treatment has been associated with a reduction in periventricular leukomalacia (9). Thus our findings concur with data from these preterm human infant studies demonstrating that early closure of a patent PDA compared to no treatment is not associated with any increase in significant neuropathology nor does it increase the risk of altered brain growth and development. It is reassuring that no novel form of brain injury or alteration in brain development was noted in the setting of ibuprofen treatment.
In translating our findings to the human preterm infant, we acknowledge that there are limitations in our study including the small number of animals and the relatively short duration of the study. We also note that the preterm baboon model was electively delivered without any pre-existing complications such as infection, hypoxemia or growth restriction. While the long-term effects of ibuprofen treatment on the brain cannot be established in the present study, it is likely that pharmacological closure may provide benefits if the PDA is contributing to persistent pulmonary and hemodynamic instability as has been shown in other organ systems (23).
Our present findings show that ibuprofen treatment for PDA closure in the preterm baboon neonate is not associated with any increased risk of neuropathology and/or alterations to brain growth and development. Furthermore this therapy has been shown to improve pulmonary outcomes (17).
We thank Dr Jacqueline Coalson, Ms Vicki Winter and staff at the Bronchopulmonary Dysplasia Resource Centre, San Antonio, Texas for provision of baboon tissue.
Statement of financial support: This work was supported by NIH Grant R01 HL074942 and in part by NIH grants HL52636, HL56061; HL46691, HL77395, and HL52646.
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