Involvement of neuronal nitric oxide synthase in ongoing fetal brain injury following near-term rabbit hypoxia-ischemia.

Neuronal nitric oxide synthase (nNOS) and nitric oxide (NO) are implicated in neuronal injury following acute hypoxia-ischemia (HI). Our hypothesis was that NO from nNOS is responsible for ongoing mitochondrial dysfunction in near-term fetal HI. Recently, we synthesized new selective nNOS inhibitors that prevent the cerebral palsy phenotype in our animal model. We tested the efficacy of a selective nNOS inhibitor (JI-8) in fetal brains after in utero HI in our rabbit model. Brain slices at 29 days gestation were obtained after in utero HI, and immediately cultured in medium containing JI-8 or saline for 3-6 days. Mitochondrial membrane integrity and function were determined by flow cytometry using rhodamine 123 and JC-1, and cell death by using propidium iodide. JI-8 decreased NO production in brain slices and also showed significant preservation of mitochondrial function at both 3 and 6 days (p < 0.05) when compared with saline and inducible NOS inhibitor 1400W. There was no difference in cell death. In conclusion, nNOS is involved in ongoing mitochondrial dysfunction after in utero HI. The subacute brain slice model could be a tool for studying the mechanisms involved in ongoing neuronal injury, and for rapidly assessing potential neuroprotectants.

Tetrahydrobiopterin in the prevention of hypertonia in hypoxic fetal brain.

OBJECTIVE: Tetrahydrobiopterin (BH(4)) deficiency is a cause of dystonia at birth. We hypothesized that BH(4) is a developmental factor determining vulnerability of the immature fetal brain to hypoxic-ischemic injury and subsequent motor deficits in newborns. METHODS: Pregnant rabbits were subjected to 40-minute uterine ischemia, and fetal brains were investigated for global and focal changes in BH(4). Newborn kits were assessed by neurobehavioral tests following vehicle and sepiapterin (BH(4) analog) treatment of dams. RESULTS: Naive fetal brains at 70% gestation (E22) were severely deficient for BH(4) compared with maternal and other fetal tissues. BH(4) concentration rapidly increased normally in the perinatal period, with the highest concentrations found in the thalamus compared with basal ganglia, frontal, occipital, hippocampus, and parietal cortex. Global sustained 40-minute hypoxia-ischemia depleted BH(4) in E22 thalamus and to a lesser extent in basal ganglia, but not in the frontal, occipital, and parietal regions. Maternal supplementation prior to hypoxia-ischemia with sepiapterin increased BH(4) in all brain regions and especially in the thalamus, but did not increase the intermediary metabolite, 7,8-BH(2). Sepiapterin treatment also reduced incidence of severe motor deficits and perinatal death following E22 hypoxia-ischemia. INTERPRETATION: We conclude that early developmental BH(4) deficiency plays a critical role in hypoxic-ischemic brain injury. Increasing brain BH(4) via maternal supplementation may be an effective strategy in preventing motor deficits from antenatal hypoxia-ischemia.

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.

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 (approximately 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.

Potentiation of alkylating agents by NLCQ-1 or TPZ in vitro and in vivo.

PURPOSE: To investigate potential synergistic interactions between bioreductive agents, either NLCQ-1 or tirapazamine (TPZ) and two alkylating chemotherapeutic drugs, and how such interactions compare in vitro and in vivo. MATERIALS AND METHODS: V79 cells (in vitro studies) and the SCCVII/C3H murine tumor model (in vivo studies) were used. The alkylating chemotherapeutic agents examined were cisplatin (cisDDP) and melphalan (L-PAM). In vivo, all agents were administered by i.p. injection wherein NLCQ-1 and TPZ were given at equitoxic doses of 10 and 23 mg/kg, respectively. Optimal administration schedules and dose modification factors (DMF) were determined in vivo for the antitumor effect or bone marrow toxicity by using the in vivo-in vitro clonogenic assay as the endpoint. RESULTS: A schedule-dependent synergistic interaction was observed between NLCQ-1/TPZ and each alkylating agent, both in vitro and in vivo, and an optimal potentiation was obtained when each bioreductive agent was administered prior to each chemotherapeutic drug. However, significant DMF values and an in vivo therapeutic index (TI) was obtained only with NLCQ-1. Limited mechanistic studies in V79 cells by using the alkaline comet assay demonstrated that hypoxic preincubation with NLCQ-1 increases the cross-links induced by subsequent aerobic exposure to cisDDP. CONCLUSIONS: These results verify our previous observations in EMT6 tumors and suggest a potential clinical use of NLCQ-1 as a synergistic adjuvant to chemotherapy with alkylating agents against solid tumors possessing hypoxic regions.

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.

Mechanisms involved in the potentiation of paclitaxel or 5-fU by the hypoxic cytotoxin NLCQ-1 (NSC 709257) in vitro.

BACKGROUND: 4-[3- (2-Nitro-1-imidazolyl)-propylamino]-7-chloroquinoline hydrochloride (NLCQ-1) is a novel, weakly DNA-intercalating hypoxia selective cytotoxin, which significantly potentiates the antitumor effect, but not the systemic toxicity, of commonly used chemotherapeutic agents. In the present study, we investigated if the same schedule-dependent synergism seen in vivo exists in vitro as well, between NLCQ-1 and paclitaxel or 5-fluorouracil (5FU), and some of the mechanisms involved in such interactions. MATERIALS AND METHODS: V79 cells, while in monolayers, were exposed to paclitaxel or 5FU under aerobic conditions at various time-intervals before or after their hypoxic exposure to NLCQ-1, while in suspension. Survival was assessed by the clonogenic assay and synergistic interactions were evaluated by using the fractional product analysis. Cells treated as above were examined for apoptosis, DNA damage and repair, as well as DNA, RNA and protein syntheses inhibition. RESULTS: A schedule-dependent synergistic interaction existed between NLCQ-1 and paclitaxel/5FU, similar to that seen in transplanted EMT6 murine tumors. Optimal potentiation was observed when NLCQ-1 was administered 2 or 4 h after the antimitotic paclitaxel or the thymidylate synthase inhibitor 5FU. Apoptosis induced by paclitaxel or 5FU alone was enhanced by NLCQ-1 up to 2.5- and 1.7-fold, respectively. DNA damage was detected as single-strand breaks (ssbs) in the combination treatments and was unrepairable at least up to 24 h post treatment. DNA, RNA and protein syntheses were inhibited by paclitaxel/5FU alone ca. 50% immediately post treatment and this inhibition was persistent up to 24 h post treatment. In combination with NLCQ-1, a slight synergistic and persistent inhibition was observed in all three syntheses. CONCLUSION: Enhancement in apoptosis, unrepairable DNA damage and inhibition of DNA, RNA and protein syntheses are some of the mechanisms involved in the potentiation of paclitaxel or 5FU by NLCQ-1.

Model of cerebral palsy in the perinatal rabbit.

Perinatal brain injury results in one of the highest burdens of disease in view of the lifelong consequences and is of enormous cost to society. This makes it imperative to develop better animal models that mimic the human condition. Many neurodevelopmental deficits, such as cerebral palsy, are believed to be a result of prenatal hypoxia-ischemia in humans. Fetal global hypoxia-ischemia is most commonly a consequence of acute placental insufficiency. Our laboratory has modeled in utero sustained and repetitive hypoxia-ischemia in the pregnant rabbit to mimic the insults of abruptio placenta and labor, respectively. Sustained hypoxia-ischemia at 70% (22 days' gestation) and 79% (25 days' gestation) and repetitive hypoxia-ischemia at 90% gestation (28 days' gestation) 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 22 and 25 days' gestation survivors but not in the 28 days' gestation group. Hypertonic survivors were artificially fed and found to have the motor deficits persist for at least 11 postnatal days. A spectrum of brain abnormalities is found on magnetic resonance imaging. This is the first animal model to mimic cerebral palsy. The findings also suggest a window of vulnerability during brain development when the injury results in hypertonia in newborn pups.

Therapeutic advantage from combining paclitaxel with the hypoxia-selective cytotoxin NLCQ-1 in murine tumor- or human xenograft-bearing mice.

PURPOSE: The antitumor effect of paclitaxel was investigated against murine tumors and human xenografts in combination with the hypoxia-selective cytotoxin NLCQ-1. METHODS: The tumor regrowth assay was used as the endpoint and an optimal administration schedule was followed, based on previous studies. In certain cases the hypoxia-selective cytotoxin tirapazamine (TPZ) was included for comparison. NLCQ-1 was given i.p. in saline, whereas paclitaxel was given i.p. (C3H) or i.v. (athymic mice) in an appropriately formulated vehicle. RESULTS: In the SCCVII/C3H model, when NLCQ-1 (10 mg/kg) was given 90 min after paclitaxel (8 mg/kg) twice a day 4 h apart on days 0 and 9, tumor regrowth delay was increased by 10.3 days compared to paclitaxel alone, at fivefold the original tumor size. This corresponds to 1.51 log cell kill. In the same study, TPZ resulted in 4.6 days of extra delay compared to paclitaxel alone, which corresponds to 0.91 log cell kill. Paclitaxel alone resulted in 3.9 days of tumor growth delay compared to control, or 0.42 log cell kill, but this delay was not statistically significant ( P<0.2). In the FSaIIC/C3H model, when NLCQ-1 (10 mg/kg) was given 90 min after paclitaxel (12 mg/kg) on day 0, tumor regrowth delay was increased by 5.8 days compared to paclitaxel alone, at 20-fold the original tumor size. In athymic nude mice bearing PC-3 prostate xenografts, NLCQ-1 (10 mg/kg) given 90 min before paclitaxel (8 mg/kg) for five consecutive days, increased tumor regrowth delay by 5.6 days compared to paclitaxel alone, at threefold the original tumor size. This corresponds to 0.95 log cell kill whereas the log cell kill for paclitaxel alone was 0.52. No improvement was observed in the tumor regrowth delay at any lower paclitaxel doses given in combination with NLCQ-1. No concurrent enhancement in paclitaxel-induced toxicity was observed in any of the combination treatments or in any of the models tested. NLCQ-1 alone was ineffective at the doses given. CONCLUSIONS: These results suggest that an enhancement in tumor growth delay can be achieved both in murine tumors and in human xenografts due to a synergistic interaction between NLCQ-1 and paclitaxel.

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.

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.

Fetal brain magnetic resonance imaging response acutely to hypoxia-ischemia predicts postnatal outcome.

OBJECTIVE: 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. METHODS: Serial diffusion-weighted imaging were performed on 79% gestation New Zealand white rabbits using a 3-Tesla magnetic resonance scanner during 40 minutes of uterine ischemia, 20 minutes of reperfusion, and at 4, 24, and 72 hours. Individual fetuses were tracked to near term, and the delivered kits were divided into hypertonic H-I (n = 18), nonhypertonic H-I (n = 9), stillbirth H-I (n = 4), and control groups (n = 16). RESULTS: The hypertonia group had significantly less of a nadir in apparent diffusion coefficient (ADC) during H-I (71.6 +/- 23.8% vs 84.5 +/- 9.3% baseline) and slower and incomplete recovery of ADC during reperfusion compared with the nonhypertonic group. All fetuses in the hypertonic and stillbirth groups had an ADC nadir of less than 0.83 microm(2)/msec (70.3% decrease from baseline), whereas 94% of control animals had an ADC nadir greater than this value. The difference between outcome groups was the largest at 4 hours reperfusion and persisted for 24 hours. INTERPRETATION: Serial fetal brain scans indicate that the immediate response of a fetus to H-I is crucial to the development of hypertonia. If the fetal brain can be scanned at the time of insult, ADC changes can predict which fetuses will have an unfavorable outcome.

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.