Matthew Derrick

Developmental Changes in Diffusion Anisotropy Coincide with Immature Oligodendrocyte Progression and Maturation of Compound Action Potential

Disruption of oligodendrocyte lineage progression is implicated in the white-matter injury that occurs in cerebral palsy. We have previously published a model in rabbits consistent with cerebral palsy. Little is known of normal white-matter development in perinatal rabbits. Using a multidimensional approach, we defined the relationship of oligodendrocyte lineage progression and functional maturation of axons to structural development of selected cerebral white-matter tracts as determined by diffusion tensor imaging (DTI). Immunohistochemical studies showed that late oligodendrocyte progenitors appear at gestational age 22 [embryonic day 22 (E22)], whereas immature oligodendrocytes appear at E25, and both increase rapidly with time (∼13 cells/mm2/d) until the onset of myelination. Myelination began at postnatal day 5 (P5) (E36) in the internal capsule (IC) and at P11 in the medial corpus callosum (CC), as determined by localization of sodium channels and myelin basic protein. DTI of the CC and IC showed that fractional anisotropy (FA) increased rapidly between E25 and P1 (E32) (∼11% per day) and plateaued (<5% per day) after the onset of myelination. Postnatal maturation of the compound action potential (CAP) showed a developmental pattern similar to FA, with a rapid rise between E29 and P5 (in the CC, 18% per day) and a slower rise from P5 to P11 (in the CC, <5% per day). The development of immature oligodendrocytes after E29 coincides with changes in FA and CAP area in both the CC and IC. These findings suggest that developmental expansion of immature oligodendrocytes during the premyelination period may be important in defining structural and functional maturation of the white matter.

Preterm Fetal Hypoxia-Ischemia Causes Hypertonia and Motor Deficits in the Neonatal Rabbit: A Model for Human Cerebral Palsy?

Prenatal hypoxia-ischemia to the developing brain has been strongly implicated in the subsequent development of the hypertonic motor deficits of cerebral palsy (CP) in premature and full-term infants who present with neonatal encephalopathy. Despite the enormous impact of CP, there is no animal model that reproduces the hypertonia and motor disturbances of this disorder. We report a rabbit model of in utero placental insufficiency, in which hypertonia is accompanied by marked abnormalities in motor control. Preterm fetuses (67-70% gestation) were subjected to sustained global hypoxia. The dams survived and gave spontaneous birth. At postnatal day 1, the pups that survived were subjected to a battery of neurobehavioral tests developed specifically for these animals, and the tests were videotaped and scored in a masked manner. Newborn pups of hypoxic groups displayed significant impairment in multiple tests of spontaneous locomotion, reflex motor activity, and the coordination of suck and swallow. Increased tone of the limbs at rest and with active flexion and extension were observed in the survivors of the preterm insult. Histopathological studies identified a distinct pattern of acute injury to subcortical motor pathways that involved the basal ganglia and thalamus. Persistent injury to the caudate putamen and thalamus at P1 was significantly correlated with hypertonic motor deficits in the hypoxic group. Antenatal hypoxia-ischemia at preterm gestation results in hypertonia and abnormalities in motor control. These findings provide a unique behavioral model to define mechanisms and sequelae of perinatal brain injury from antenatal hypoxia-ischemia.

Which Neuroprotective Agents are Ready for Bench to Bedside Translation in the Newborn Infant

Neonatal encephalopathy caused by perinatal hypoxiaischemia in term newborn infants occurs in 1 to 3 per 1000 births1 and leads to high mortality and morbidity rates with life-long chronic disabilities.2,3 Although therapeutic hypothermia is a significant advance in the developed world and improves outcome,4,5 hypothermia offers just 11% reduction in risk of death or disability, from 58% to 47%. Therefore, there still is an urgent need for other treatment options. Further, there are currently no clinically established interventions that can be given antenatally to ameliorate brain injury after fetal distress. One of the major limitations to progress is what may be called “the curse of choice.” A large number of possible neuroprotective therapies have shown promise in pre-clinical studies.6,7 How should we select from them? There is no consensus at present on which drugs have a high chance of success for either antenatal or postnatal treatment. There are insufficient societal resources available to test them all. Thus, it is imperative to marshal finite resources and prioritize potential therapies for investigation. The authors believe that facilitating discussion of strategy and findings in “competing” laboratories is critical to facilitate efficient progress toward optimizing neuroprotection after hypoxia-ischemia. Few studies have examined possible interactions of medications with hypothermia and whether combination therapies augment neuroprotection. The timing of the administration of medications may be critical to optimize benefit and avoid neurotoxicity (eg, early acute treatments targeted at amelioration of the neurotoxic cascade compared with subacute treatment that can promote regeneration and repair). Intervention early on in the cascade of neural injury is likely to achieve more optimal neuroprotection8,9; however, there is frequently little warning of impending perinatal hypoxia-ischemia episodes. Sensitizing factors such as maternal pyrexia,10 maternal/fetal infection,11,12 and poor fetal growth13 are well recognized and contribute to the heterogeneity of the fetal response and outcome in neonatal encephalopathy. We include potential antenatal therapy medications in the scoring process; however, electronic fetal monitoring has a low positive predictive value (3%–18%) for identifying intrapartum asphyxia.14–18 At present, therefore, any antenatal intervention potentially involves treatment of many cases that do not need treatment in order to benefit a few at risk of brain injury. In January 2008, investigators from research institutions with a special interest in neuroprotection of the newborn appraised published evidence about medications that have been used in pre-clinical animal models, pilot clinical studies, or both as treatments for: (1) antenatal therapy for fetuses with a diagnosis of antenatal fetal distress at term; and (2) postnatal therapy of infants with moderate to severe neonatal encephalopathy. The aims of this study were to: (1) prioritize potential treatments for antenatal and postnatal therapy; and (2) provide a balanced reference for further discussions in the perinatal neuroscience community for future research and clinical translation of novel neuroprotective treatments of the newborn.

Cell therapy for neonatal hypoxia–ischemia and cerebral palsy

Perinatal hypoxic–ischemic brain injury remains a major cause of cerebral palsy. Although therapeutic hypothermia is now established to improve recovery from hypoxia–ischemia (HI) at term, many infants continue to survive with disability, and hypothermia has not yet been tested in preterm infants. There is increasing evidence from in vitro and in vivo preclinical studies that stem/progenitor cells may have multiple beneficial effects on outcome after hypoxic–ischemic injury. Stem/progenitor cells have shown great promise in animal studies in decreasing neurological impairment; however, the mechanisms of action of stem cells, and the optimal type, dose, and method of administration remain surprisingly unclear, and some studies have found no benefit. Although cell‐based interventions after completion of the majority of secondary cell death appear to have potential to improve functional outcome for neonates after HI, further rigorous testing in translational animal models is required before randomized controlled trials should be considered. ANN NEUROL 2012;

A model of cerebral palsy from fetal hypoxia-ischemia.

Disorders of the maternal-placental-fetal unit often results in fetal brain injury, which in turn results in one of the highest burdens of disease, because of the lifelong consequences and cost to society. Investigating hypoxia-ischemia in the perinatal period requires the factoring of timing of the insult, determination of end-points, taking into account the innate development, plasticity, and enhanced recovery. Prenatal hypoxia-ischemia is believed to account for a majority of cerebral palsy cases. We have modeled sustained and repetitive hypoxia-ischemia in the pregnant rabbit in utero to mimic the insults of abruptio placenta and labor, respectively. Rabbits have many advantages over other animal species; principally, their motor development is in the perinatal period, akin to humans. Sustained hypoxia-ischemia at 70% (E22) and 79% (E25) caused stillbirths and multiple deficits in the postnatal survivors. The deficits included impairment in multiple tests of spontaneous locomotion, reflex motor activity, motor responses to olfactory stimuli, and the coordination of suck and swallow. Hypertonia was observed in the E22 and E25 survivors and persisted for at least 11 days. Noninvasive imaging using MRI suggests that white matter injury in the internal capsule could explain some of the hypertonia. Further investigation is underway in other vulnerable regions such as the basal ganglia, thalamus and brain stem, and development of other noninvasive determinants of motor deficits. For the first time critical mechanistic pathways can be tested in a clinically relevant animal model of cerebral palsy.

White Matter Injury Correlates with Hypertonia in an Animal Model of Cerebral Palsy

Hypertonia and postural deficits are observed in cerebral palsy and similar abnormalities are observed in postnatal rabbits after antenatal hypoxia–ischemia. To explain why some kits become hypertonic, we hypothesized that white matter injury was responsible for the hypertonia. We compared newborn kits at postnatal day 1 (P1) with and without hypertonia after in vivo global fetal hypoxia–ischemia in pregnant rabbits at 70% gestation. The aim was to examine white matter injury by diffusion tensor magnetic resonance imaging indices, including fractional anisotropy (FA). At P1, FA and area of white matter were significantly lower in corpus callosum, internal capsule, and corona radiata of the hypertonic kits (n = 32) than that of controls (n = 19) while nonhypertonic kits (n = 20) were not different from controls. The decrease in FA correlated with decrease in area only in hypertonia. A threshold of FA combined with area identified only hypertonic kits. A reduction in volume and loss of phosphorylated neurofilaments in corpus callosum and internal capsule were observed on immunostaining. Concomitant hypertonia with ventriculomegaly resulted in a further decrease of FA from P1 to P5 while those without ventriculomegaly had a similar increase of FA as controls. Thus, hypertonia is associated with white matter injury, and a population of hypertonia can be identified by magnetic resonance imaging variables. The white matter injury manifests as a decrease in the number and density of fiber tracts causing the decrease in FA and volume. Furthermore, the dynamic response of FA may be a good indicator of the plasticity and repair of the postnatal developing brain.

Serial Diffusion Tensor Imaging Detects White Matter Changes That Correlate with Motor Outcome in Premature Infants

The objective of the study was to assess predictive value of serial diffusion tensor MRI (DTI) for the white matter injury and neurodevelopmental outcome in a cohort of premature infants. Twenty-four infants less than 32 weeks’ gestation were stratified to a control group (n = 11), mild brain injury with grades 1–2 of intraventricular hemorrhage (n = 6) and severe brain injury with grades 3–4 intraventricular hemorrhage (n = 4). Serial DTI studies were performed at around 30 and 36 weeks’ gestation. Fractional anisotropy (FA) and apparent diffusion coefficient were calculated. Twelve infants were followed up for developmental outcome. Developmental testing was performed with the Bayley Scales of Infant Development to obtain psychomotor index (Performance Developmental Index). Apparent diffusion coefficient was higher in the severe injury group at the second MRI in the central and occipital white matter, and corona radiata; FA was lower in optic radiation compared to controls. Performance Developmental Indexscore correlated with FA on the scan taken at the 30th week and inversely with the change of FA between scans in internal capsule and occipital white matter. A low value of FA at 30 weeks and a higher change of FA predicted less favorable motor outcome at 2 years and suggests that early subtle white matter injury can be detected in premature infants even without obvious signs of injury.

Selective neuronal nitric oxide synthase inhibitors and the prevention of cerebral palsy

To design a new class of selective neuronal nitric oxide synthase (NOS) inhibitors, and demonstrate that administration in a rabbit model for cerebral palsy (CP) prevents hypoxia‐ischemia–induced deaths and reduces the number of newborn kits exhibiting signs of CP.

Timing of appearance of late oligodendrocyte progenitors coincides with enhanced susceptibility of preterm rabbit cerebral white matter to hypoxia-ischemia

Emerging evidence supports that premature infants are susceptible to both cerebral white and gray matter injury. In a fetal rabbit model of placental insufficiency, preterm rabbits at embryonic day 22 (E22) exhibited histologic evidence of gray matter injury but minimal white matter injury after global hypoxia-ischemia (H-I). We hypothesized that the dissociation between susceptibility to gray and white matter injury at E22 was related to the timing of appearance of late oligodendrocyte progenitors (preOLs) that are particularly vulnerable in preterm human white matter lesions. During normal rabbit oligodendrocyte (OL) lineage progression, early OL progenitors predominated at E22. PreOL density increased between E24 and E25 in major forebrain white matter tracts. After H-I at E22 and E25, we observed a similar magnitude of cerebral H-I, assessed by cortical microvascular blood flow, and gray matter injury, assessed by caspase activation. However, the increased preOL density at E25 was accompanied by a significant increase in acute white matter injury after H-I that coincided with enhanced preOL degeneration. At E29, significant white matter atrophy developed after H-I at E25 but not E22. Thus, the timing of appearance of preOLs coincided with onset of a developmental window of enhanced white but not gray matter susceptibility to H-I.

Fetal Brain Magnetic Resonance Imaging Response Acutely to Hypoxia-Ischemia Predicts Postnatal Outcome

Cerebral palsy (CP) is caused by either hypoxia‐ischemia (H‐I) or long‐standing causative factors such as inflammation or genetics. Multiple pathophysiological events over time are thought to contribute eventually to cerebral palsy. Our objective was to examine whether the immediate response of the fetus to an acute H‐I event determined the motor deficits associated with cerebral palsy.