doi: 10.1161/01.STR.0000251445.94697.64
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(Stroke. 2007;38:731.)
© 2007 American Heart Association, Inc.
Cerebral Ischemia and the Developing Brain: Introduction |
A Model of Cerebral Palsy From Fetal Hypoxia-Ischemia
From Department of Pediatrics, Northwestern University and Evanston Northwestern Healthcare, Evanston, Ill.
Correspondence to Sidhartha Tan, Department of Pediatrics, 2650 Ridge Avenue, Evanston, IL 60201. E-mail sidtan{at}northwestern.edu
Abstract
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.
Key Words: behavior • brain • dystonia • fetal anoxia • hypoxia • infant • ischemia • magnetic resonance imaging • newborn • placental insufficiency • premature • uterus
Cerebral palsy is a nonprogressive disorder of the developing brain principally affecting the motor system. Cerebral palsy affects 2 to 3 per 1000 newborns, with a conservative estimate of its impact on society being 
$5 billion per year. Cerebral palsy can be associated with epilepsy and abnormalities of speech, vision, and intellect. The impact of diseases affecting the newborn are much higher than diseases that affect the elderly because of the burden of disease when one considers mortality, years of life lost, and years of productive life lost. If one compares the economic impact of disease of an elderly person at the end of life compared with that of disease of a fetus or baby, the impact of the latter is far more. Lifetime costs for all persons of cerebral palsy are estimated to total $11.5 billion.1 These costs underscore the need to urgently develop preventive and secondary therapeutic measures for the fetus and newborn. Little progress has been made over the past few decades on the prevalence of cerebral palsy.2–4 The lack of development of new therapies has been partly because of the malpractice climate in United States.5 Progress is also slow because of the unique obstacles facing investigators studying brain injury in the perinatal period, principally related to the issue of timing.6 During the progression of neurological development, the timing of the insult and of end points becomes crucial in any study. The investigator has to take into account not only the plasticity of the stage of development but also the pace of enhanced recovery and the normal process of apoptosis7 that varies with different time periods of development. Another major obstacle has been the paucity of perinatal animal models manifesting neurobehavioral deficits.
Dynamic In Vivo Measurement of the Developing Brain Requires Noninvasive MRI
The developing brain undergoes rapid changes and the dynamic changes in brain structure and function is unique to this age. This necessitates the use of repeated noninvasive measurements. MRI is being increasingly used in premature newborn human infants and supplements the information obtained from the traditional mode of brain imaging, ultrasound. A specific technique about diffusion weighted imaging gives additional information about white matter development, myelination, and acute injury from H-I. This is also important for animal studies because these studies ultimately have to be clinically relevant. We used MRI as a noninvasive imaging tool to better characterize the dynamic changes after brain injury.
Methods
Recently, we described a model in preterm rabbits that resulted in hypertonia and neurobehavioral findings in postnatal day 1 rabbits (P1) mimicking those found in cerebral palsy.8,9 Briefly, in vivo uterine ischemia was induced in pregnant dams (Myrtle’s Rabbits; Thompson Station, TN) by inflation of an aortic balloon at a level proximal to the uterine arteries.8,10–12 This protocol resulted in global hypoxia to the fetus that was accompanied by immediate fetal bradycardia (from 180 to 80 bpm) and an immediate decrease in microvascular blood flow (laser Doppler measurements) to the fetal cerebral cortex which remained down for the duration of uterine ischemia. Preterm fetuses (21 and 22 days’ gestation) were subjected to sustained global hypoxia, and near-term fetuses (29 days’ gestation) to repetitive global hypoxia from uterine ischemia. The dams survived and gave birth spontaneously at term gestation (31.5 days).
Because severely affected kits were not able to survive if left with the rabbit dam, we hand-fed these kits by the orogastric route using a soft silicone catheter.9 We found the best survival in those rabbit kits fed rabbit milk.
The MRI methods and results have been described before.13 Briefly, rabbit kits at postnatal day 1 (10 days after uterine ischemia) and 5 were imaged under sedation with intramuscular injection of a mixture of ketamine (35 mg/kg), xylazine (5 mg/kg), and acepromazine (1.0 mg/kg). We used a 4.7 T Bruker Biospec system (Bruker), with custom-made surface coils used for excitation and reception, and inner diameter of 28 mm. Diffusion weighted imaging in 3 orthogonal directions (diffusion trace-weighted) were performed on 10 oblique coronal brain slices with diffusion weighted spin echo sequence, repetition time/echo time was 2000/35 ms, with b=780 s/mm2 and one reference image with b=0 s/mm2; matrix size was 128x64, and 8 averages taken. Slice thickness/in-plane resolution were 1/0.156 mm.13
Results
Cerebral Palsy Model in Rabbits
These results have been described extensively in previous publications.8,9 Newborn kits that survived (58%) the hypoxic- ischemic insult, at 22 days’ gestation (E22), displayed significant impairment in multiple tests of spontaneous locomotion, reflex motor activity, motor responses to olfactory stimuli, and the coordination of suck and swallow.8,9 Increased tone of the limbs at rest and with active flexion and extension were observed in the survivors of the preterm (Figure 1A and 1B) but not in the survivors of the near-term insult.
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Surviving Rabbit Kits Continue to Show Hypertonia
The motor deficits remained unchanged over the first 11 days in the survivors (Figure 1C). The finding of persistent and unchanging hypertonia fulfils one of the requirements for the diagnosis of cerebral palsy.14
What Determines Hypertonia
The central features of cerebral palsy are hypertonia and postural changes. In our animal model we have attempted to explain why some kits became hypertonic and some did not.13 We hypothesized that white matter injury was responsible for the hypertonia. After in vivo global fetal hypoxia-ischemia in pregnant rabbits at E22 and subsequent birth at term, newborn kits at P1 with and without hypertonia were compared. The aim was to examine white matter injury by diffusion tensor MRI indices, including fractional anisotropy (FA).13
At P1, FA and area of white matter were significantly lower in corpus callosum, internal capsule and corona radiata of the hypertonic kits than controls, whereas nonhypertonic kits were not different from controls (Figure 2). The decrease in FA correlated with decrease in area only in hypertonic kits, whereas nonhypertonic and control kits showed no correlation. More importantly, values below a threshold of FA combined with area could identify a sub-population of kits that only contained hypertonic kits. (Figure 3).13 A reduction in volume and loss of phosphorylated neurofilaments in corpus callosum and internal capsule were observed, which suggests an axonal pathology as an explanation for the rabbit hypertonia.13 In humans, abnormal signal intensity in the internal capsule is an accurate predictor of neurodevelopmental outcome in term hypoxic-ischemic infants.15 Rabbit gray matter structures showed loss in area in most structures (Table), and the combination of gray and white matter lesions is consistent with human studies.16
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Discussion
The development of an animal model that incorporates etiology and structural and functional features17 provides future investigators with a valuable tool to investigate mechanisms of how prenatal H-I causes the motor deficits of cerebral palsy and develop diagnostic markers of events that result in cerebral palsy and test both prenatal and postnatal therapeutic interventions. This study also shows that axonopathy in the white matter may be an important pathogenetic pathway in the development of motor deficits.
Maternal-Placental-Fetal and Postnatal Models
The clinically applicable models of perinatal hypoxia-ischemia have centered on the maternal-placental-fetal unit. Disorders of any of the components of the maternal-placental-fetal unit can injure the fetal brain. Thus, the various animal models can be categorized according to the subunit of the maternal-placenta-fetal unit studied. For example, maternal and fetal models have been used in rats, mice, guinea pigs, and sheep. Examples of placental models can be found in rats, mice, sheep and our rabbit model. However, the most common animal models are postnatal in origin and use stroke pathophysiology; examples are found in rats, mice, pigs, sheep, and baboons. Carotid artery occlusion alone does not cause behavior changes in rodents. Combined with hypoxia, behavioral changes are elicited only with sophisticated tests,18–20 but no overt motor deficits21,22 or transient motor deficits23,24 are observed, even with bilateral carotid occlusion.25 There have been very few studies investigating motor and tone outcomes in higher animals, such as sheep, piglets or nonhuman primates.
In 70% to 80% of cases, cerebral palsy is antepartum in origin, and most of the cases of antenatal hypoxia-ischemia are considered idiopathic.26 One of the leading causes of cerebral palsy from antenatal hypoxia-ischemia is abruptio placentae.27 We have been interested in developing suitable animal models of hypoxia-ischemic brain injury that mimic clinical perinatal brain injury. Thus, we focused on a rabbit model of acute placental insufficiency to investigate hypoxia-ischemia-mediated brain injury.10–12
Advantages of Rabbit as an Animal Model
The principal advantage of using rabbits is that motor development and white matter development are close to that of humans. Rodents are postnatal brain developers, with most of myelin formation occurring after the first week.28 Almost all other mammals are prenatal brain developers. This is true even for primate species that are considered the closest to humans where most of the myelin development is over before birth. This makes sense because these mammals have to have enough motor function at birth to escape predators. Rabbits, however, are burrow animals and are perinatal brain developers.29,30 Myelination begins soon after birth in our rabbits at approximately P5. Interestingly, humans, in their evolutionary leap from the rest of the primates, have become perinatal brain developers, with prolonged development of myelination. Myelination starts in the prenatal period,31 and that of some sensory and motor fibers begin at birth,32,33 but the process continues during the first year of life.
Another advantage of rabbits is that rabbits are similar to humans in that both have low circulating levels of xanthine oxidase, a free radical generator,34,35 and are unlike rodents that have high levels.36,37 If this enzyme is crucial in the pathophysiology of free radical-mediated injury in humans, then the rodent could be unsuitable for investigating this mechanism. Further advantages of our model are that it is a true fetal model, involves global hypoxia-ischemia, the dam is not significantly affected by the uterine ischemia, rabbits are relatively inexpensive with an average of 8 fetuses per dam, and dams fully recover from survival surgeries and can give birth normally.
Survival of Postnatal Kits Is Directly Linked to Intensive Care
The survival of postnatal kits after antenatal hypoxia-ischemia could be certainly improved with improvements in rabbit neonatal intensive care. In human hypoxic-ischemic encephalopathy, a high percentage of sick infants would also die if they did not have the benefit of neonatal intensive care that includes ventilator care, parenteral nutrition, and artificial feeds. We are trying to reach the same level of sophistication for postnatal rabbit kits as for humans, but this involves a lot of time and money.
Conclusion
In conclusion, a clinically relevant model was generated from antenatal H-I that mimics the insult of acute placental insufficiency in humans, and resulted in a cerebral palsy phenotype. This model strongly supports the concept that antenatal factors are predominantly related to the subsequent development of cerebral palsy. The behavioral end points have become accessible to study to investigators. It is our hope that this model will further the investigations of cellular and molecular mechanisms that precede cerebral palsy and to assess whether these deficits can be ameliorated by therapeutic interventions.
Acknowledgments
Sources of Funding
This study received grant support from NIH, NS43285, and March of Dimes Birth Defects Foundation 294.
Disclosures
None.
Received September 11, 2006; revision received October 20, 2006; accepted October 24, 2006.
References
1. Centers for Disease Control and Prevention (CDC). Economic costs associated with mental retardation, cerebral palsy, hearing loss, and vision impairment–United States, 2003. MMWR-Mob Mort Wkly Rep. 2004; 53: 57–59.
2. Meberg A, Broch H. Etiology of cerebral palsy. J Perinat Med. 2004; 32: 434–439.[CrossRef][Medline] [Order article via Infotrieve]
3. Bhasin TK, Brocksen S, Avchen RN, Van Naarden BK. Prevalence of four developmental disabilities among children aged 8 years–Metropolitan Atlanta Developmental Disabilities Surveillance Program, 1996 and 2000. MMWR-Mob Mort Wkly Rep. 2006; Surveillance Summaries 55: 1–9.
4. Himmelmann K, Hagberg G, Beckung E, Hagberg B, Uvebrant P. The changing panorama of cerebral palsy in Sweden. IX. Prevalence and origin in the birth-year period 1995–1998. Acta Paediatr. 2005; 94: 287–294.[Medline] [Order article via Infotrieve]
5. Towbin A. Obstetric malpractice litigation: the pathologist’s view. Am J Obstet Gynecol. 1986; 155: 927–935.[Medline] [Order article via Infotrieve]
6. Ferriero DM. Timing is everything–delaying therapy for delayed cell death. Dev Neurosci. 2002; 24: 349–351.[CrossRef][Medline] [Order article via Infotrieve]
7. Nijhawan D, Honarpour N, Wang X. Apoptosis in neural development and disease. Annu Rev Neurosci. 2000; 23: 73–87.[Medline] [Order article via Infotrieve]
8. Derrick M, Luo NL, Bregman JC, Jilling T, Ji X, Fisher K, Gladson CL, Beardsley DJ, Murdoch G, Back SA, Tan S. Preterm fetal hypoxia-ischemia causes hypertonia and motor deficits in the neonatal rabbit: a model for human cerebral palsy? J Neurosci. 2004; 24: 24–34.
9. Tan S, Drobyshevsky A, Jilling T, Ji X, Ullman LM, Englof I, Derrick M. Model of cerebral palsy in the perinatal rabbit. J Child Neurol. 2005; 20: 972–979.[Medline] [Order article via Infotrieve]
10. Tan S, Liu YY, Nielsen VG, Skinner K, Kirk KA, Baldwin ST, Parks DA. Maternal infusion of antioxidants (Trolox and ascorbic acid) protects the fetal heart in rabbit fetal hypoxia. Pediatr Res. 1996; 39: 499–503.[Medline] [Order article via Infotrieve]
11. Tan S, Zhou F, Nielsen VG, Wang Z, Gladson CL, Parks DA. Sustained hypoxia-ischemia results in reactive nitrogen and oxygen species production and injury in the premature fetal rabbit brain. J Neuropathol Exp Neurol. 1998; 57: 544–553.[Medline] [Order article via Infotrieve]
12. Tan S, Zhou F, Nielsen VG, Wang Z, Gladson CL, Parks DA. Increased injury following intermittent fetal hypoxia-reoxygenation is associated with increased free radical production in fetal rabbit brain. J Neuropathol Exp Neurol. 1999; 58: 972–981.[Medline] [Order article via Infotrieve]
13. Drobyshevsky A, Derrick M, Wyrwicz AM, Ji X, Englof I, Ullman LM, Zelaya ME, Northington FJ, Tan S. White Matter Injury Correlates with Hypertonia in an Animal Model of Cerebral Palsy. J Cereb Blood Flow Metab. 2006: advance online publication, 17 May. 2006.
14. Koman LA, Smith BP, Shilt JS. Cerebral palsy. Lancet. 2004; 363: 1619–1631.[CrossRef][Medline] [Order article via Infotrieve]
15. Rutherford MA, Pennock JM, Counsell SJ, Mercuri E, Cowan FM, Dubowitz LMS, Edwards AD. Abnormal magnetic resonance signal in the internal capsule predicts poor neurodevelopmental outcome in infants with hypoxic-ischemic encephalopathy. Pediatrics. 1998; 102: 323–328.
16. Woodward LJ, Anderson PJ, Austin NC, Howard K, Inder TE. Neonatal MRI to predict neurodevelopmental outcomes in preterm infants. N Engl J Med. 2006; 355: 685–694.
17. Petersen MC, Kube DA, Palmer FB. Classification of developmental delays. Semin Pediatr Neurol. 1998; 5: 2–14.[CrossRef][Medline] [Order article via Infotrieve]
18. McQuillen PS, Sheldon RA, Shatz CJ, Ferriero DM. Selective vulnerability of subplate neurons after early neonatal hypoxia-ischemia. J Neurosci. 2003; 23: 3308–3315.
19. Bona E, Johansson BB, Hagberg H. Sensorimotor function and neuropathology five to six weeks after hypoxia-ischemia in seven-day-old rats. Pediatr Res. 1997; 42: 678–683.[Medline] [Order article via Infotrieve]
20. Follett PL, Deng W, Dai W, Talos DM, Massillon LJ, Rosenberg PA, Volpe JJ, Jensen FE. Glutamate receptor-mediated oligodendrocyte toxicity in periventricular leukomalacia: a protective role for topiramate. J Neurosci. 2004; 24: 4412–4420.
21. Arteni NS, Salgueiro J, Torres I, Achaval M, Netto CA. Neonatal cerebral hypoxia-ischemia causes lateralized memory impairments in the adult rat. Brain Res. 2003; 973: 171–178.[CrossRef][Medline] [Order article via Infotrieve]
22. Liu Y, Barks JD, Xu G, Silverstein FS. Topiramate extends the therapeutic window for hypothermia-mediated neuroprotection after stroke in neonatal rats. Stroke. 2004; 35: 1460–1465.
23. Young RS, Kolonich J, Woods CL, Yagel SK. Behavioral performance of rats following neonatal hypoxia-ischemia. Stroke. 1986; 17: 1313–1316.
24. Silverstein F, Johnston MV. Effects of hypoxia-ischemia on monoamine metabolism in the immature brain. Ann Neurol. 1984; 15: 342–347.[CrossRef][Medline] [Order article via Infotrieve]
25. Fan LW, Lin S, Pang Y, Lei M, Zhang F, Rhodes PG, Cai Z. Hypoxia-ischemia induced neurological dysfunction and brain injury in the neonatal rat. Behav Brain Res. 2005; 165: 80–90.[CrossRef][Medline] [Order article via Infotrieve]
26. Perlman JM. Intrapartum hypoxic-ischemic cerebral injury and subsequent cerebral palsy: medicolegal issues. Pediatrics. 1997; 99: 851–859.
27. Matsuda Y, Maeda T, Kouno S. Comparison of neonatal outcome including cerebral palsy between abruptio placentae and placenta previa. Eur J Obstet Gynecol Reprod Biol. 2003; 106: 125–129.[CrossRef][Medline] [Order article via Infotrieve]
28. Rasband MN, Peles E, Trimmer JS, Levinson SR, Lux SE, Shrager P. Dependence of nodal sodium channel clustering on paranodal axoglial contact in the developing CNS. J Neurosci. 1999; 19: 7516–7528.
29. van Marthens E, Harel S, Zamenshof S. Experimental intrauterine growth retardation. Biol Neonate. 1975; 26: 221–231.[CrossRef][Medline] [Order article via Infotrieve]
30. Harel S, Shapira Y, Tomer A, Donahue MJ, Quilligan E. Vascular-induced intrauterine growth retardation: relations between birth weight and the development of biochemical parameters in young rabbits. Israel J Med Sci. 1985; 21: 829–832.[Medline] [Order article via Infotrieve]
31. Hasegawa M, Houdou S, Mito T, Takashima S, Asanuma K, Ohno T. Development of myelination in the human fetal and infant cerebrum: a myelin basic protein immunohistochemical study. Brain Dev. 1992; 14: 1–6.[Medline] [Order article via Infotrieve]
32. Kinney HC, Brody BA, Kloman AS, Gilles FH. Sequence of central nervous system myelination in human infancy. II. Patterns of myelination in autopsied infants. J Neuropathol Exp Neurol. 1988; 47: 217–234.[Medline] [Order article via Infotrieve]
33. Paus T, Collins DL, Evans AC, Leonard G, Pike B, Zijdenbos A. Maturation of white matter in the human brain: a review of magnetic resonance studies. Brain Res Bull. 2001; 54: 255–266.[CrossRef][Medline] [Order article via Infotrieve]
34. Tan S, Radi R, Gaudier F, Evans RA, Rivera A, Kirk KA, Parks DA. Physiologic levels of uric acid inhibit xanthine oxidase in human plasma. Pediatr Res. 1993; 34: 303–307.[Medline] [Order article via Infotrieve]
35. White CR, Darley-Usmar V, Berrington WR, McAdams M, Gore JZ, Thompson JA, Parks DA, Tarpey MM, Freeman BA. Circulating plasma xanthine oxidase contributes to vascular dysfunction in hypercholesterolemic rabbits. Proc Natl Acad Sci U S A. 1996; 93: 8745–8749.
36. Parks DA, Skinner KA, Tan S, Skinner HB. Xanthine oxidase in biology and medicine. In: Gilbert DL, Colton CA, editors. Reactive Oxygen Species in Biological Systems: Selected Topics. New York: Kluwer Academic/Plenum Publishers, 1999: 397–420.
37. Yokoyama Y, Beckman JS, Beckman TK, Wheat JK, Cash TG, Freeman BA, Parks DA. Circulating xanthine oxidase: potential mediator of ischemic injury. Am J Physiol. 1990; 258: G564–G570.[Medline] [Order article via Infotrieve]
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