Alexander Drobyshevsky

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.

A rapid fMRI task battery for mapping of visual, motor, cognitive, and emotional function.

A set of sensory, motor, cognitive, and emotional tasks were combined in a simple, rapid-presentation task battery and tested on a group of 31, normal, healthy subjects aged 22 to 76. Five tasks were selected on the basis of widespread use in fMRI and their ability to produce robust and reliable regional activations. They were (1) a visual task designed to activate the occipital cortex; (2) a bimanual motor task designed to activate motor areas; (3) a verb generation task designed to activate speech processing areas; (4) an n-back task designed to activate areas associated with working memory and executive function; and (5) an emotional pictures task designed to provoke strong emotional responses that typically activate limbic structures. Most of the tasks produced reliable activations in individual subjects, and assessments of the distribution and reliability of individual subject activations in each targeted area are provided. The emotional pictures task did not demonstrate adequate sensitivity in a priori target regions, only in the a posteriori defined inferior temporal region. Age- and gender-specific differences were found in the activation patterns for both the cognitive and emotional tasks. The battery provides a prescribed means for researchers to obtain reliable functional localizers within 20-25 min of scanning, which can be used to support more elaborate mapping studies of brain function. The dataset can also serve as a reliability metric for new fMRI laboratories and novice investigators seeking to test their acquisition and analysis techniques with minimal time investment and expense.

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.

Motor Deficits Are Triggered by Reperfusion-Reoxygenation Injury as Diagnosed by MRI and by a Mechanism Involving Oxidants

The early antecedents of cerebral palsy (CP) are unknown but are suspected to be due to hypoxia-ischemia (H-I). In our rabbit model of CP, the MRI biomarker, apparent diffusion coefficient (ADC) on diffusion-weighted imaging, predicted which fetuses will develop postnatal hypertonia. Surviving H-I fetuses experience reperfusion-reoxygenation but a subpopulation manifested a continued decline of ADC during early reperfusion-reoxygenation, which possibly represented greater brain injury (RepReOx). We hypothesized that oxidative stress in reperfusion-reoxygenation is a critical trigger for postnatal hypertonia. We investigated whether RepReOx predicted postnatal neurobehavior, indicated oxidative stress, and whether targeting antioxidants at RepReOx ameliorated motor deficits, which included testing of a new superoxide dismutase mimic (MnTnHex-2-PyP). Rabbit dams, 79% gestation (E25), were subjected to 40 min uterine ischemia. Fetal brain ADC was followed during H-I, immediate reperfusion-reoxygenation, and 4–72 h after H-I. Endpoints were postnatal neurological outcome at E32, ADC at end of H-I, ADC nadir during H-I and reperfusion-reoxygenation, and area under ADC curve during the first 20 min of reperfusion-reoxygenation. Antioxidants targeting RepReOx were administered before and/or after uterine ischemia. The new MRI-ADC biomarker for RepReOx improved prediction of postnatal hypertonia. Greater superoxide production, mitochondrial injury, and oligodendroglial loss occurred in fetal brains exhibiting RepReOx than in those without. The antioxidants, MnTnHex-2-PyP and Ascorbate and Trolox combination, significantly decreased postnatal motor deficits and extent of RepReOx. The etiological link between early injury and later motor deficits can thus be investigated by MRI, and allows us to distinguish between critical oxidative stress that causes motor deficits and noncritical oxidative stress that does not.

Human Umbilical Cord Blood Cells Ameliorate Motor Deficits in Rabbits in a Cerebral Palsy Model

Cerebral palsy (CP) has a significant impact on both patients and society, but therapy is limited. Human umbilical cord blood cells (HUCBC), containing various stem and progenitor cells, have been used to treat various brain genetic conditions. In small animal experiments, HUCBC have improved outcomes after hypoxic-ischemic (HI) injury. Clinical trials using HUCBC are underway, testing feasibility, safety and efficacy for neonatal injury as well as CP. We tested HUCBC therapy in a validated rabbit model of CP after acute changes secondary to HI injury had subsided. Following uterine ischemia at 70% gestation, we infused HUCBC into newborn rabbit kits with either mild or severe neurobehavioral changes. Infusion of high-dose HUCBC (5 × 106 cells) dramatically altered the natural history of the injury, alleviating the abnormal phenotype including posture, righting reflex, locomotion, tone, and dystonia. Half the high dose showed lesser but still significant improvement. The swimming test, however, showed that joint function did not restore to naïve control function in either group. Tracing HUCBC with either MRI biomarkers or PCR for human DNA found little penetration of HUCBC in the newborn brain in the immediate newborn period, suggesting that the beneficial effects were not due to cellular integration or direct proliferative effects but rather to paracrine signaling. This is the first study to show that HUCBC improve motor performance in a dose-dependent manner, perhaps by improving compensatory repair processes.

Hypoxia-ischemia causes persistent movement deficits in a perinatal rabbit model of cerebral palsy: assessed by a new swim test.

The relationship of movement between different muscle groups has not been quantified before in the newborn period. Cerebral palsy (CP), which often occurs as a result of perinatal hypoxia–ischemia (H–I), is categorized depending on clinical presentation, brain region involvement and extent of involvement. In order to test different brain region involvement, this study investigates individual and multi‐joint involvement in a rabbit model of CP. Pregnant rabbits at 70% gestation were subjected to 40‐min uterine ischemia. Newborn rabbit kits were subjected to a swim test at 5 time points over the first 11 days of life. H–I kits were divided into hypertonic and non‐hypertonic groups based on muscle tone at birth. The ranges and velocity of angular movement of the forelimb and hind limb joints (wrist, elbow, shoulder, ankle, knee and hip) during supported swimming were determined. Severely impaired (hypertonic) animals have significantly reduced range and angular velocity of joint motion, which do not improve over time. The non‐hypertonic group showed deficits in wrist and hind limb movements that were not evident on prolonged observation. Preventive treatment with an inhibitor of neuronal nitric oxide synthase decreased the incidence of severely impaired kits; the non‐hypertonic kits showed a different pattern of swimming. Supported swimming allows quantification of limb and joint motion in the principal plane of movement in the absence of weight bearing and decreases the need for balance control. Identification and quantification of milder deficits allows mechanistic studies in the causation of H–I injury as well as estimation of recovery with therapeutic agents.

Sensory deficits and olfactory system injury detected by novel application of MEMRI in newborn rabbit after antenatal hypoxia–ischemia

Sensory deficits are frequently observed in cerebral palsy patients. The motor response to smell was found to be abnormal in an animal model of cerebral palsy following fetal hypoxia-ischemia. We hypothesized that fetal hypoxia-ischemia causes long-lasting and selective olfactory tract injury. A population of newborn rabbits with motor deficits was selected after spontaneous delivery following uterine ischemia at 22 days gestation (E22, 70% term). MnCl(2), 20 mg/kg, was administered in both nostrils at postnatal day 1 (E32). One nostril was occluded to control for smell augmentation through the other open nostril by intermittent amyl acetate stimulation for 6 h. T1-weighted MRI images were obtained on newborn rabbits. Amyl acetate exposure increased augmentation of Mn(2+) uptake in olfactory epithelium on the open side in control group but the augmentation was decreased after hypoxia. The proportion of animals with a greater enhancement in the open side increased in controls after amyl acetate, but not in hypoxia. Mn(2+) took longer to arrive at the olfactory bulbs and the rate of subsequent increase was slower in hypoxia. Concomitantly, the thickness of olfactory epithelium and the number of mature olfactory neurons, detected on olfactory marker protein immunostaining, were significantly less in the hypoxic group. Functional MRI studies are superior to neurobehavioral smell testing in the rabbit kits as they are more sensitive and quantifiable measures and do not depend upon the motor response. Antenatal hypoxia-ischemia causes long-lasting injury to neuronal tracts of the olfactory system including olfactory epithelium.

Near-Term Fetal Hypoxia–Ischemia in Rabbits MRI Can Predict Muscle Tone Abnormalities and Deep Brain Injury

Background and Purpose— The pattern of antenatal brain injury varies with gestational age at the time of insult. Deep brain nuclei are often injured at older gestational ages. Having previously shown postnatal hypertonia after preterm fetal rabbit hypoxia–ischemia, the objective of this study was to investigate the causal relationship between the dynamic regional pattern of brain injury on MRI and the evolution of muscle tone in the near-term rabbit fetus. Methods— Serial MRI was performed on New Zealand white rabbit fetuses to determine equipotency of fetal hypoxia–ischemia during uterine ischemia comparing 29 days gestation (E29, 92% gestation) with E22 and E25. E29 postnatal kits at 4, 24, and 72 hours after hypoxia–ischemia underwent T2- and diffusion-weighted imaging. Quantitative assessments of tone were made serially using a torque apparatus in addition to clinical assessments. Results— Based on the brain apparent diffusion coefficient, 32 minutes of uterine ischemia was selected for E29 fetuses. At E30, 58% of the survivors manifested hind limb hypotonia. By E32, 71% of the hypotonic kits developed dystonic hypertonia. Marked and persistent apparent diffusion coefficient reduction in the basal ganglia, thalamus, and brain stem was predictive of these motor deficits. Conclusions— MRI observation of deep brain injury 6 to 24 hours after near-term hypoxia–ischemia predicts dystonic hypertonia postnatally. Torque-displacement measurements indicate that motor deficits in rabbits progressed from initial hypotonia to hypertonia, similar to human cerebral palsy, but in a compressed timeframe. The presence of deep brain injury and quantitative shift from hypo- to hypertonia may identify patients at risk for developing cerebral palsy.