Sandra Dieni

Investigation of cerebral development and injury in the prematurely born primate by magnetic resonance imaging and histopathology

We summarize the preliminary results of brain histopathology and magnetic resonance imaging applied to a premature baboon model developed for evaluation of the pathogenesis and treatment of bronchopulmonary dysplasia. Cerebral development was assessed in 10 gestational control animals at time points of 125, 140 and 160 days of gestation (dg). On the basis of histopathology, conventional MRI and diffusion MRI, 125 dg is equivalent to 26–28 weeks of human gestation, 140 dg is equivalent to 30–32 weeks, and 160 dg is equivalent to term. Preliminary data are also presented for 33 experimental cases delivered at 125 dg, nursed for 2 weeks in an intensive care facility, and sacrificed at 139–140 dg. The commonest neuropathology in this cohort is white matter damage, manifest by reactive astrogliosis or activated microglia, and enlarged ventricular size. Subarachnoid, germinal matrix and intraventricular hemorrhages are also common. These preliminary results support the similarity of this model to the human preterm infant for both cerebral development and the pattern of cerebral injury. The prematurely born baboon appears an important model for the study of preterm human birth.

BDNF and TrkB protein expression is altered in the fetal hippocampus but not cerebellum after chronic prenatal compromise.

This study examines the effects of a chronic prenatal insult on both the expression of brain-derived neurotrophic factor (BDNF) and TrkB proteins and the structural development of the fetal hippocampus and cerebellum. Chronic placental insufficiency (CPI) was induced via unilateral ligation of the uterine artery from midgestation to near term in the pregnant guinea pig. Fetuses were delivered at 60 days of gestation (dg, term approximately 67 dg) and classified as control or growth-restricted (GR) according to established criteria. In hippocampi and cerebella from control (n = 7) and GR (n = 8) fetuses, immunohistochemistry was performed to detect the expression of BDNF and TrkB proteins, and the growth of neuropil and cellular layers was measured in each structure. The growth of neuropil layers was reduced in the dentate gyrus of GR fetuses compared to controls: hippocampi from severely GR fetuses showed a marked reduction in BDNF-IR and an increase in TrkB-IR. The most pronounced effects on neuropil growth were seen in the same fetuses that demonstrated marked alterations in BDNF-IR and TrkB-IR. In the cerebellum, there were significant reductions in the growth of the cellular and neuropil layers; however, BDNF-IR and TrkB-IR were not affected. These results demonstrate that CPI has a widespread effect in retarding process growth in the developing brain, but a differential effect on neurotrophin expression. Changes in BDNF and TrkB expression appear to be associated with the pronounced structural changes in the hippocampi of severely GR fetuses, however, structural abnormalities in the cerebellum were not associated with changes in these proteins; presumably, other factors are involved.

Distribution of brain-derived neurotrophic factor and TrkB receptor proteins in the fetal and postnatal hippocampus and cerebellum of the guinea pig

This study investigates the distribution of brain‐derived neurotrophic factor protein (BDNF) and its receptor, TrkB, during the development of hippocampus and cerebellum in a long‐gestation species, the guinea pig. In the granule cell populations of both structures, BDNF immunoreactivity (‐IR) was exclusive to postmigratory, mature neurons. In dentate granule cells, TrkB‐IR was coexpressed with BDNF‐IR, suggesting that the ligand‐receptor interaction could occur by means of an autocrine/paracrine mechanism. In cerebellar granule cells, TrkB‐IR was detected in both pre‐ and postmigratory cells, indicating that immature neurons are also BDNF‐responsive. With advancing gestational age an increase in the intensity of BDNF‐IR in granule cells was accompanied by concomitant increases in the staining and areal growth of the associated mossy fiber layer in the hippocampus, and the molecular layer in the cerebellum. The developmental increase in BDNF‐ and TrkB‐IR in the neuropil of both structures coincided with periods of significant growth in all strata, indicating a role for BDNF and TrkB in process outgrowth. In the hippocampus, CA2, CA3, and hilar, neurons demonstrated both BDNF‐ and TrkB‐IR during development and maturation, whereas CA1 neurons showed TrkB‐IR throughout this period but only transient BDNF‐IR in early gestation. In the fetal cerebellum, Purkinje cell bodies coexpressed BDNF‐IR and TrkB‐IR. In the postnatal period, BDNF‐IR was down‐regulated but TrkB‐IR persisted, indicating that mature Purkinje cells might retain their responsiveness to BDNF. Thus, we have demonstrated in both the hippocampus and cerebellum that the spatiotemporal distribution of BDNF‐IR and TrkB‐IR coincides with the maturation of granule cells prenatally and with significant periods of neuropil growth, both prenatally and in the immediate postnatal period. J. Comp. Neurol. 454:229–240, 2002.

Physiological and induced neuronal death are not affected in NSE‐bax transgenic mice

Bax, a family member of the survival protein Bcl‐2, is expressed in the nervous system during development and throughout adulthood. Bax deficiency has been demonstrated to prevent developmental and trophic factor deprivation‐induced neuronal death. To further clarify the role of Bax in naturally occurring neuronal death and in neuronal death following apoptotic stimuli, we generated several lines of transgenic mice expressing the human Bax protein specifically in neurons, under the control of the neuron‐specific enolase promoter. Transgene expression was first detected around E10.5 and E12.5, depending on the transgenic line. The total number of ganglion cells in the retina and of pyramidal cells in the hippocampus, both expressing the transgene, was similar in control and transgenic mice. In addition, in our model system, Bax overexpression did not appear to influence the in vitro survival of sensory neurons isolated from dorsal root ganglia after nerve grwoth factor (NFG) deprivation or the apoptotic death of motor neurons following axotomy. J. Neurosci. Res. 52:247–259, 1998.

Chronic placental insufficiency has long-term effects on auditory function in the guinea pig.

Very low birth weight and growth-restricted infants have an increased risk of auditory impairments. It is uncertain whether these impairments are related to adverse pre-, peri- or postnatal events. We aimed to determine whether a period of chronic placental insufficiency (CPI) in the guinea pig results in long-term alterations to auditory function. Near mid-gestation, CPI was induced via unilateral ligation of the uterine artery. At 8 weeks of age, auditory brainstem responses (ABRs) were recorded in response to unilateral acoustic stimulation in prenatally-compromised (PC, n=8) and control animals (n=8). Stimuli consisted of 100 micros clicks, presented at 33 pulses per second (pps) and tone pip stimuli at frequencies of 2, 4, 8, 16 and 32 kHz. To examine temporal response properties, click stimuli were also presented at rates of 66, 132 and 200 pps. Normal ABR waveforms were elicited by both click and tone pip stimuli in all animals. Moreover, there was no difference between control and PC animals in stimulus detection thresholds across the frequencies examined. Using high rate click stimuli, PC animals demonstrated a significant increase in both the latency of wave III (normalised to 33 pps) and the wave I-III inter-peak interval compared to the controls. We hypothesise that these functional changes reflect alterations in myelination of the auditory brainstem and/or changes in synaptic efficacy. The results suggest subtle deficits in neural conduction in the PC guinea pig at maturity, and may have implications for speech perception abilities of low birth weight or prenatally affected infants.

Inhaled nitric oxide: Effects on cerebral growth and injury in a baboon model of premature delivery

Inhaled nitric oxide (iNO) enhances ventilation in very preterm infants, but the effects on the brain remain uncertain. We evaluated the impact of iNO on brain growth and cerebral injury in a premature baboon model. Baboons were delivered at 125 d of gestation (term 185 d of gestation) and ventilated for 14 d with either positive pressure ventilation (PPV) (n = 7) or PPV + iNO (n = 8). Brains were assessed histologically for parameters of development and injury. Compared with gestational controls (n = 7), brain and body weights were reduced but brain-to-body weight ratios were increased in all prematurely delivered (PD) animals; the surface folding index (SFI), was reduced in PPV but not PPV + iNO animals. Compared with controls, the brain damage index was increased (p < 0.05) in both cohorts of PD animals. There was no difference between ventilatory regimens, however, in 25% of animals with iNO therapy, there were organized hematomas in the subarachnoid space. Overall, iNO did not alter the extent of brain damage but did result in the presence of hematomas. These results do not confirm any protective or major injurious effect of nitric oxide therapy on the developing brain.

Cerebellar Development in a Baboon Model of Preterm Delivery: Impact of Specific Ventilatory Regimes

Premature infants now have an improved chance of survival, but the impact of respiratory therapies on the brain, particularly the cerebellum, remains unclear. We examined the effects of early nasal continuous positive airway pressure (EnCPAP) ventilation and delayed (Dn) CPAP on the development of the cerebellum in prematurely delivered baboons. The baboons were delivered at 125 ± 2days of gestation and ventilated for 28 days with either EnCPAP commencing at 24 hours (n = 5) or DnCPAP commencing at 5 days (n = 5). Gestational controls (n = 4) were delivered at 153 days. Cerebella were assessed histologically, and an ontogeny study (90 days to term) was performed to establish values for key cerebellar developmental indicators. Cerebellar weight was reduced in DnCPAP but not EnCPAP animals versus controls; cerebellar/total brain weight ratio was increased in EnCPAP (p < 0.05) versus control and DnCPAP animals. There was no overt damage in the cerebella of any animals, but a microstructural alteration index based on morphological developmental parameters and microglial immunoreactivity was increased in both prematurely delivered cohorts versus controls (p < 0.001) and was higher in DnCPAP than EnCPAP animals (p < 0.05). These results indicate that respiratory regimens can influence cerebellar development and that early compared with delayed extubation to nCPAP seems to be beneficial.

VIABLE PRIMARY NEURONAL CULTURES FROM GUINEA PIG EMBRYOS

Thirty 7-day-old guinea pig embryos are at the optimal and earliest developmental stage for a simultaneous culturing of viable neurons from hippocampus, cortex, and cerebellum. High-density primary neuronal cultures obtained from the three brain regions were successfully maintained using a chemically defined medium. The neurons, which quickly developed extensive neurite out growth and interneuronal connections, could be sustained for at least 3 weeks. The sensitivity of guinea pig neuronal cultures to a model toxicant like paraquat is reported.